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3.  Duchenne’s muscular dystrophy 

 

Background:  One form of inherited muscular dystrophy, Duchenne’s, is X-linked and therefore affects primarily males.  The symptoms of Duchenne's muscular dystrophy (DMD) include progressive and severe skeletal muscle weakness.  A common mutation associated with DMD is a deletion of one or more exons in the dystrophin gene.  These deletions can be detected by restriction enzyme digestion and Southern blotting using a combination of probes that will bind to multiple dystrophin exons.

 

Case A:   Jean and Bill have three sons, ages 12, 8, and 7, and a daughter, age 6.  The oldest son and daughter are healthy, but the two younger sons are exhibiting symptoms of muscle weakness consistent with early muscular dystrophy.   Jean knows that  she has a family history of muscular dystrophy, but she does not know whether she is a carrier of the disease gene.  She seeks DNA testing to determine whether her younger sons may have inherited the form of the dystrophin gene associated with Duchenne's muscular dystrophy (DMD).

 

DNA samples:      Jean (mother)                                       

                                oldest son (unaffected)                                      

                                daughter

                                8-year-old son (possibly affected)

                                7-year-old son (possibly affected)                                                   

 

Digest each DNA sample with HindIII, then perform a Southern blot with the dystrophin gene probe (DMD probe).  Based on the number and sizes of the fragments bound by the probe, determine the status of each of the individuals tested.  (Hint:  Some fragments are small, so you may need to use shorter run times to see them all.)

 

a.        What conclusions can you draw from these results? 

b.       What is the molecular basis of this disease, and why does this result in the observed gel patterns? 

        c.     What issues are raised by this type of testing?

 

 

Case B:  Tabitha walked slowly to the kitchen table and sat down.  She gazed out the window where her two daughters (aged 10 and 12) were playing in the sprinkler and chuckled at their antics.  She thought of the son she and her husband lost nearly 2 years ago.  He would have been 14 this year.  And she just found out she was pregnant again - unplanned and unexpected.  As she sipped her coffee she knew she could not endure a pregnancy wondering if the baby would turn out healthy, nor could she bear to lose another child to muscular dystrophy.  She called the clinic to set up an appointment for DNA testing.  She knew now that she was the source of the mutated gene and she wondered if she had passed it to her new baby.  She also wondered if either of her daughters were carrying it,  and hoped fervently they were not.

 

DNA samples:                      Tabitha (mother)
fetus
10 yr old daughter
12 yr old daughter

 

Digest each DNA sample with HindIII, then perform a Southern blot with the dystrophin gene probe (DMD probe).  Based on the number and sizes of the fragments bound by the probe, determine the status of each of the individuals tested.  (Hint:  Some fragments are small, so you may need to use shorter run times to see them all.)

 

        a.     What conclusions can you draw from the results?

        b.     What is the molecular basis of this disease, and why does this result in the observed   gel patterns?

        c.     What options are available to the family?

        d.     What issues are raised by this type of testing?

        e.     Is the fetus healthy?
f.      Is the fetus male or female?

 

 

Case C:  Scott and Mary met as teenagers at the local MD telethon.  Each was there volunteering their time in support of brothers they watched slowly suffer from progressive muscle degeneration.  Now, years later, they were married to each other and ready to start a family of their own.  Mary’s pregnancy test came back positive and the news filled them with both joy and dread.  What if their child had muscular dystrophy?  Mary decides to go in for DNA testing to find out is she is a carrier, and if the baby is affected. 

 

DNA samples:                      Mary (mother)

Scott (father)
fetus

 

Digest each DNA sample with HindIII, then perform a Southern blot with the dystrophin gene probe (DMD probe).  Based on the number and sizes of the fragments bound by the probe, determine the status of each of the individuals tested.  (Hint:  Some fragments are small, so you may need to use shorter run times to see them all.)

 

        a.     What conclusions can you draw from the results?

        b.     What is the molecular basis of this disease, and why does this result in the observed   gel patterns?

        c.     What options are available to the family?

        d.     What issues are raised by this type of testing?

        e.     Is the fetus healthy?
f.      Is the fetus male or female?  How do you know?

 

 

 

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4.  Alzheimer disease

 

Background:  Alzheimer disease is by far the most common cause of dementia in aging persons.  The disease symptoms are identical to other forms of senile dementia, and diagnosis had been possible only at autopsy by the detection of protein clusters called amyloid plaques in the cerebrum.  The disease is multifactorial and inheritance patterns are complex.  Some forms of familial Alzheimer disease appear to be inherited as autosomal dominant traits, while others are recessive.  Spontaneous Alzheimer disease also can occur in the absence of inherited factors.

 

Mutations in at least four genes have been linked to Alzheimer disease.  One of these is the amyloid precursor protein (APP) gene, which encodes the b-amyloid peptide found in the cerebral plaques of Alzheimer patients.  The function of APP is not yet known, but certain APP point mutations are associated with inheritance of late-onset Alzheimer disease in some families.  Two examples which can be detected by RFLP analysis are the codon 693 Glutamic acid to Glycine mutation and the codon 717 Valine to Isoleucine mutation.  The 693 mutation results in the loss of a MboII site, while the 717 mutation results in the gain of a BclI site.

 

Case A:   Martha, age 71, has been exhibiting increasingly severe symptoms of senile dementia and has been hospitalized for testing.  She is in good health otherwise.  Her three children - Sam (age 43), Joan (age 41) and Robert (age 38) - want to find out the cause of the dementia and determine the prognosis for Martha's future condition.  They are also concerned that Martha may have a form of familial Alzheimer disease and want to know if they are at risk.    The physician decides initially to test Martha for two mutations, 693 Gly and 717 Ile, in the amyloid precursor protein (APP) gene which are associated with inherited Alzheimer disease.  

 

DNA samples:      Martha (mother)

                                Sam (son)

                                Joan (daughter)

                                Robert (son)

                                Control normal APP gene

                                Control with 693 mutation

                                Control with 717 mutation

 

To test for the 693 Gly mutation, digest the DNA with MboII and perform a Southern blot using the APP probe.  To test for the 717 Ile mutation, digest the DNA with BclI and then use the APP probe.  Compare the test samples to the control samples, and use the results to determine the genotype of each individual.  [Note:  Small fragments are generated with the MboII digestion - use 1.2% agarose and short run times.]

 

Case B:   Lisa, age 17, and her cousin Jen age 18, were half-listening to music in the den and half-listening to their mothers discuss Grandma Eloise and her older sister Florence.  Lisa and Jen loved Eli and Flo dearly but even they could tell something wasn’t quite right about their increasingly odd behavior.   The teens moved into the kitchen to join the conversation.  “Is Grandma’s erratic behavior and forgetfulness Alzheimer’s or just senile dementia commonly associated with old age?”  They decide to talk to Eloise and Florence about DNA testing.  The  mothers also wonder about their risk for Alzheimer disease and decide to be tested.

 

DNA samples:              Eloise (grandmother)
Florence (Eloise’s older sister)

Lisa’s mother   
Jen’s mother
Control with 693 mutation

                                        Control with 717 mutation

                                        Control normal APP gene

 

To test for the 693 Gly mutation, digest the DNA with MboII and perform a Southern blot using the APP probe.  To test for the 717 Ile mutation, digest the DNA with BclI and then use the APP probe.  Compare the test samples to the control samples, and use the results to determine the genotype of each individual.  [Note:  Small fragments are generated with the MboII digestion - use 1.2% agarose and short run times.]

 

        a.     What conclusions can you draw from the results?

        b.     What is the molecular basis of this disease, and why does this result in the observed   gel patterns?

        c.     What options are available to the family?

        d.     What issues are raised by this type of testing?

 

 

 

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5. Breast Cancer Susceptibility

 

Background:  Breast cancer is the most common malignancy among women.  Current estimates are that one in eight women born in 1990 will contract breast cancer by age 85.  Many factors contribute to breast cancer risk.  Inheritance of breast cancer susceptibility genes contribute to approximately 5-10% of all breast cancers.   The breast/ovarian cancer susceptibility gene BRCA1 has been identified on chromosome 17.  Women who inherit certain BRCA1 mutations have an 80% risk of breast cancer. 

 

BRCA1 appears to encode a tumor suppressor protein.  Mutations that affect the function of this protein cause increased rates of cell division and a predisposition towards the development of malignancy.  Several BRCA1 mutations, including point mutations, deletions, and insertions, have been identified that may contribute to loss of tumor suppressor function.  These mutations can be identified by amplifying portions of the BRCA1 gene by PCR and then using RFLP analysis, direct sequencing, or hybridization with specific probes to detect the presence of mutations.  Large scale screening trials are underway to gain more information about the nature of the mutations responsible for increased cancer risk.  One deletion mutation in exon 2, 185delAG, is highly prevalent among women of Eastern European Jewish descent, and screening efforts have targeted this population of women for further study.

 

For the screening, a small amount of blood is drawn.  DNA is isolated from the blood, and part of the BRCA1 gene is amplified by PCR.  The amplified DNA is run on a dot blot with specific probes corresponding to mutations known to be linked to increased breast cancer susceptibility.  The probe will only bind to the DNA if that mutation is present.   Probes corresponding to the normal sequence for that mutation site will help you determine whether the individual is homozygous or heterozygous for the mutation.  Control DNA samples known to have specific mutation also are included.  To analyze  these cases, use the PCR function on the Data Screen, rather than the 96-well PCR, to generate DNA samples for the dot blot.  Load probes in the spots and load the DNA samples into the corresponding wells.

 

Case A:  While Elizabeth is reading the morning newspaper, she notices an ad for a free genetic screening for breast cancer at the clinic next week.    The ad specifically invites women of Ashkenazi Jewish ancestry to participate.   According to the newspaper ad, subjects will be tested to see whether they have mutations in the BRCA1 gene which would predispose them to breast cancer.  Elizabeth, age 27, had heard about the discovery of the gene and about the mutation linked to Jewish women.  Her paternal grandmother had been diagnosed with breast cancer at age 51 and died two years later, and Elizabeth worried that she had inherited the disease.   She also worried about her mother, age 52 and apparently cancer-free so far, and her 7-year old daughter.  Her daughter is not allowed to participate in the screening, but Elizabeth convinces her mother to go with her to get tested.

 

DNA samples:      Elizabeth

                                Mother

                                185delAG (DNA containing this mutation)

                                4184delTCAA (DNA containing this mutation)

                                5382insC  (DNA containing this mutation)

                                Normal BRCA1 (no mutations)

 

 

Probes:                   185delAG  (AG deletion in exon 2)

                                Normal 185 (no mutation at this site)

                                4184delTCAA (TCAA deletion in exon 11)

                                Normal 4184

                                5382insC (C insertion in exon 13)

                                Normal 5382

 

Primers:                  Forward and reverse PCR primers for the BRCA1 gene

 

Questions

        a.     What conclusions can you draw from the results of the DNA analysis?

        b.     How would you counsel Elizabeth and her mother based on the results of the test?

        c.     Who should have access to the test results?

        d.     What other issues does this type of testing raise, and how should these issues be addressed?

 

Case B:  The time passes slowly as Deborah waits for Aunt Millie to come out of surgery.  It had come as no surprise when Aunt Millie was diagnosed with breast cancer.  After all, for as long as Deborah could remember, her mother had talked about how breast cancer “ran in the family.”  Deborah has already read the literature the doctor gave them about genetic testing for breast cancer susceptibility genes.  It is one thing to know that several women in her mother’s family had developed breast cancer; it is quite another to learn that Aunt Millie has tested positive for such a gene and therefore, Deborah and her mother are at higher risk.  Her mother has made it clear that she has no intention of being tested but as Deborah sits in the surgery waiting room, she comes to the inevitable conclusion that she has to find out if she carries the gene.

 

DNA Samples:      Aunt Millie

                                Deborah

                                185delAG (DNA containing this mutation)

                                4184delTCAA (DNA containing this mutation)

                                5382insC  (DNA containing this mutation)

                                Control BRCA1 (no mutations)

 

Probes:                   185delAG  (AG deletion in exon 2)

                                Normal 185 (no mutation at this site)

                                4184delTCAA (TCAA deletion in exon 11)

                                Normal 4184

                                5382insC (C insertion in exon 13)

                                Normal 5382

 

Primers:                  Forward and reverse PCR primers for the BRCA1 gene

 

Questions

 

Case C:  Cindy took the news very hard when her mother was diagnosed with breast cancer.  The surgery and chemotherapy her mother has gone through have taken a significant toll on the whole family.  Discovering that the breast cancer is related to the presence of a breast cancer susceptibility gene has only added to the concern.  Cindy’s sister, Ellen, decided to have genetic testing done to determine if she carried the gene, but Cindy chose not to go with her for testing.  The news that Ellen tested positive was devastating to Cindy and it has been even more difficult to accept Ellen’s subsequent decision to have a prophylactic double mastectomy.  These events have caused Cindy to reevaluate her decision not to be tested; she almost feels an obligation to go through this experience for her sister.  She schedules the appointment for testing, still undecided how she will react if the test is positive.

 

DNA samples:      Mother

                                Ellen

                                Cindy

                                185delAG (DNA containing this mutation)

                                4184delTCAA (DNA containing this mutation)

                                5382insC  (DNA containing this mutation)

                                Control BRCA1 (no mutations)

 

Probes:                   185delAG  (AG deletion in exon 2)

                                Normal 185 (no mutation at this site)

                                4184delTCAA (TCAA deletion in exon 11)

                                Normal 4184

                                5382insC (C insertion in exon 13)

                                Normal 5382

 

Primers:                  Forward and reverse PCR primers for the BRCA1 gene

                                               

Questions

 

 

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6.  Cystic Fibrosis

 

Background:  Cystic fibrosis (CF) is generally considered the most common severe autosomal recessive disorder in the Caucasian population, with a disease frequency of 1 in 2,000 and a carrier frequency of 1 in 20.  The major clinical symptoms include chronic pulmonary disease, pancreatic insufficiency, and an increase in sweat electrolyte concentrations.  The cause of the disease appears to be a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane protein involved in transporting ions across epithelial surfaces, such as the linings of the lungs and intestines.  Several mutations have been identified as being associated with a non-functional CFTR protein.  The most common mutation, accounting for about 50% of CF cases, is called delta F508;  it is a three-base deletion resulting in the loss of a phenylalanine at position 508, in the ATP-binding portion of the protein.  This mutation is detected by sequence analysis of PCR-amplified DNA, or by hybridization with mutation-specific probes (the latter method is illustrated in Case B).

 

Rapid screening for cystic fibrosis is also done using RFLP markers linked to the CF gene on chromosome 7 (illustrated in Case A).  Several RFLP analyses can be performed relatively quickly on PCR-amplified DNA from a blood sample or chorionic villus sample.  Then, if a positive result is obtained with an RFLP marker, sequence analysis or mutation-specific probe hybridization can be done to confirm the CFTR mutation.  An example of a linked RFLP marker is in the locus Mp6d.9, in which a point mutation linked to CF results in the loss of an MspI site.

 

Case A:  As Sharon Brown browsed the local newspaper, she noticed the story about the five-year old boy with cystic fibrosis who lives on the next block.  The article was mainly a human interest story about how the family was coping.  There also was some background information about the disease and its inheritance patterns, including the statistics indicating that approximately 1 in 18 people in this part of Minnesota carried a cystic fibrosis mutation.

 

Sharon is two months pregnant.  She realizes that she and her husband, Bob,  should have been tested for the cystic fibrosis (CF) mutation since they each have some family history of the disease, but they really hadn’t expected to have a child so soon.  She discusses this with her physician during her check-up the next day, and together they decide to test Sharon and Bob for a mutation in linked to the CF gene.  They also decide to test the developing fetus.  Two other families in the same town who also are in the first trimester of a pregnancy, Jill and Mike Jones and Carol and Ron Smith, also decide to be tested after reading the article.

 

Blood is drawn from the parents, and a chorionic villus sample is taken from each fetus.  DNA is isolated from the samples, and a small portion of chromosome 7 near the CF gene, a locus called Mp6d.9, is amplified by PCR. (Use the PCR function on the Data Screen, rather than 96-well PCR.)  Control DNA samples with and without the CF mutation are also included.  Digestion of the PCR fragments with the enzyme MspI is used to detect the RFLP linked to the mutated CF gene, which results in the loss of a MspI site.  [Note:  Small fragments are generated, so use shorter run times to see all of the fragments.]

 

        a.     What conclusions can you draw from the gel results?

        b.     What options are available to the parents?

        c.     What issues are raised by this type of testing?

 

 

Case B:  (Contributed Stephanie Dahlby, Dan Tally, and Janelle Veerkamp, Biol 305 Students, Spring 1997, UW-River Falls)

 

Lynda and Jim are expecting their first child.  Recently, however, they learn that Lynda’s aunt died of CF and Jim’s uncle died of CF.  They are worried that they might be carriers for the disease and pass cystic fibrosis on to their unborn child.  They learn about a procedure which can determine whether they are carriers. They also learn about a procedure called amniocentesis which can detect if their unborn child has CF or is a carrier.  However, amniocentesis is a very risky procedure.  Jim and Lynda ultimately decide that they first want to be tested to see if they are carriers for the disease.  If they learn that they both are carriers, they would like to go through with the amniocentesis to see if their child is affected.

 

DNA Samples:      Lynda                                    

                                Fetus                                     

                                Jim

                                Control DNA with F508 mutation

                                Control normal DNA, without mutation

 

Procedure:  Run PCR on each of the DNA samples using the CF primers (NOTE:  Use the PCR function on the Data Screen rather than the 96-well PCR).  Then, using the dot blot screen, load the probes into the dots.   Load the DNA samples into the corresponding wells.  By comparing the dot blot patterns of Jim, Lynda, and the fetus to those of the two controls, determine whether these DNA samples are homozygous positive for the CF mutation, homozygous negative for CF, or heterozygous carriers for CF.

 

a.     What conclusions can you draw from the gel results?

            b.     What options are available to the parents?

            c.     Should large-scale screening for CF carriers be done?

d.     How has the prognosis for children with CF changed and how might it change in the future?

            e.     What other issues are raised by this type of testing?

 

 

Case C:  The pre-marriage counseling session Carl and Maggie are having with Pastor Frank is not going at all as they had expected it to.  After some of the anticipated discussion of relationship issues, the conversation turns to family planning.  When both Carl and Maggie say they want to have children, Pastor Frank, instead of giving advise on how to properly rear children, begins to talk about genetic testing for Cystic Fibrosis!  It turns out that Pastor Frank and his wife had two children affected with CF who died in their early teens.  Because of the relatively high frequency of CF carriers and his opposition to abortion, Pastor Frank believes that all couples should be tested for the CF gene before getting married.  Carl and Maggie are not sure they share Pastor Frank’s beliefs but decide to go along with being tested.

 

 DNA Samples:                     Carl

                                                Maggie

                                                Control DNA with F508 mutation

                                                Control normal DNA without mutation

 

Procedure: Run PCR on each of the DNA samples using the CF primers. (NOTE:  Use the PCR function on the Data Screen rather than the 96-well PCR.)  Then, using the dot blot screen, load the probes into the spots.   Load the DNA samples into the corresponding wells.  By comparing the dot blot patterns of Carl and Maggie to those of the two controls, determine whether these DNA samples are homozygous positive for the CF mutation, homozygous negative for CF, or heterozygous carriers for CF.

 

             

            a.     What conclusions can you draw from the gel results?

            b.     What options are available to the parents?

            c.     Should large-scale screening for CF carriers be done?

d.     How has the prognosis for children with CF changed and how might it change in the future?

            e.     What other issues are raised by this type of testing?

 

 

 

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7.  Phenylketonuria (PKU)

        (Contributed by Duane Zimmerman, Biol 451 Student, UWRF, Fall 1996)

 

Background:  PKU is a genetic metabolic disease caused by a mutation in the phenylalanine hydroxylase enzyme.  In the most common form of PKU, a C to T point mutation causes an arginine to be replaced by tryptophan at amino acid position 408, resulting in an inactive enzyme and incomplete metabolism of phenylalanine-containing compounds such as proteins.  The resulting buildup of phenylalanine can cause mental retardation, eczema, loss of skin pigmentation, and other disorders.  If detected early, the disease is treatable by excluding foods high in phenylalanine form the diet.

 

PKU is typically tested by measuring the blood level of phenylalanine in a blood sample taken at birth.  The molecular test would be valuable as a follow-up to confirm the cause of high phenylalanine levels and to be better able to predict treatment outcomes. The mutation can be detected by dot blot analysis of PCR-amplified DNA from the blood sample.   Probes that will bind either the mutated or non-mutated sequence are used in the dot blot to determine which form of the gene is present.

 

To analyze these cases, use PCR with the PKU primers to amplify a portion of the phenylalanine hydroxylase gene from blood DNA samples.  (NOTE:  Use the PCR function on the Data Screen, rather than the 96-well PCR.)  Then, using the dot blot, load the probes into the spots and add the PCR-amplified DNA samples into the corresponding wells.

 

Case A:  Peter and Pam just had their first child.  The PKU blood test performed at birth indicated a high level of phenylalanine in the blood.  The physician suggests a follow-up DNA test immediately to confirm the PKU diagnosis and to determine the most appropriate treatment.  She also suggests that Peter and Pam be tested to confirm their carrier status and predict the risk of PKU in subsequent offspring.

 

DNA samples:      Peter                                      

                                Pam                                        

                                Infant

                                Control DNA containing the PKU mutation

                                Control normal DNA without mutation

                                                                                               

        a.     What conclusions can you draw from the results of the DNA test?

        b.     What is the molecular basis for the test, and how does this explain the test results?

c.        What issues does this type of testing raise?

 

Case B:  Angie watched her little brother, Alan, grow up with PKU.  She knows how wonderful it is that the dietary treatment that he has undertaken since being diagnosed by neonatal screening has prevented development of the worst PKU symptoms.  But she has also seen that his life has not been an easy one.  It is never easy being different and Alan’s strict dietary regimen has significantly affected his social interactions at school.  Angie has always said that if she ever decides to have a child, she will be tested before she gets pregnant to see if she carries the PKU gene.  This makes her current situation, an unplanned pregnancy by a man who was out of her life before either of them even knew, especially difficult.  There are so many unknowns.  Does she want to continue the pregnancy under these circumstances, even if she isn’t a carrier?  If she is a carrier and the fetus is unaffected, is this her best chance to have child unaffected by PKU?  Angie decides that the starting point for her difficult decisions must be to find out if she is a carrier for PKU.

 

DNA Samples:      Angie

                                Control DNA containing the PKU mutation

                                Control normal DNA without mutation

 

Case C:  When Richard and Kathy’s first child, Robert, was tentatively diagnosed with Phenylketonuria on the basis of neonatal screening for high levels of phenylalanine, they were relieved to learn that appropriate dietary restrictions are an effective treatment for PKU.  After some experience with maintaining the strict diet and the constant medical monitoring, they make some decisions about family planning.  They know they still want to have a large family, but feel that they cannot handle the rigors of more children with PKU.  When Kathy becomes pregnant again, they seek genetic testing to confirm the diagnosis and test the fetus for PKU.

 

DNA Samples:                      Robert

                                                Richard

                                                Kathy

                                                Fetus

                                                Control DNA containing the PKU mutation

                                                Control normal DNA without mutation

 

 

 

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8.  Fragile X Syndrome

        (Contributed by Gretchen Hessler, Melissa LeFebvre, and Jenni Swanson, Biol 305 Students, Spring 1997,              UWRF)

 

Background:  Fragile X syndrome is the leading cause of inherited mental retardation.  The mutated gene that causes the disorder is called fmr1 and is located on the long arm of the X-chromosome.  It is currently unclear whether this trait is dominant or recessive, because both types of expression have been demonstrated.

 

The mutation involves exaggerated repetition of the CGG triplet in a portion of the fmr1 gene near the 5' end.  Those with a functional gene have 6 to 50 CGG repeats, whereas those with a full mutation have 200 or more such repeats.  Between 50 and 200 repeats of the codon constitute a premutation.  An individual with a premutation is considered a carrier, but does not display any symptoms of fragile X.  A premutation may undergo additional repetition to generate a full mutation.

 

The fmr1 gene was discovered in 1991, and therefore DNA testing for the disorder is relatively new.  In the past, those with this disorder were often diagnosed as being learning disabled, autistic, or hyperactive.  With the advent of DNA testing, accuracy of diagnosis has increased tremendously.

 

Case A:  Doug and Grace are expecting their third child.  They have recently learned of fragile X syndrome and strongly suspect that their son, Brad, might have this disorder.  For this reason, they would like their family to undergo genetic testing.  Their daughter, Katie, shows no symptoms of fragile X.  They also decide at this time to test the fetus for the same disorder.

 

DNA Samples:      Doug     

                                Grace

                                Brad

                                Katie      

                                Fetus

                                Control normal DNA

                                Control DNA with premutation

                                Control DNA with full mutation

 

Digest each of these DNA samples with EcoR1.  Then use the probe corresponding to the region of the fmr1  mutation to determine the genotype of each individual.

 

a.     What conclusions can you draw from these results?

b.     What options are available to the parents?

c.     What issues may be raised by the results of the testing?

 

 

Case B:  Melissa has always found dealing with her brother, David, very difficult.  His developmental disability and behavior problems were an embarrassment to her as she was growing up.  When she married Paul and moved away, she was delighted finally to be free of her “problem brother.”  However, her freedom for her “problem” is to be very short lived.   She and Paul want to start their family as soon as possible and Melissa gets pregnant soon after their marriage.  When Melissa calls home to tell her family the good news, her mother, Emma, bursts into tears.  Melissa listens in shock as her mother tells her that their family physician has learned of new research on a genetic condition called Fragile X Syndrome and has suggested that this might be the cause of David’s problems.  Emma and David have already made an appointment for genetic testing and Melissa quickly decides that she and her fetus should also be tested.

 

DNA Samples:                      David

                                                Emma

                                                Melissa

                                                Fetus

                                                Control normal DNA

                                                Control DNA with premutation

                                                Control DNA with full mutation

 

Digest each of these DNA samples with EcoR1.  Then use the probe corresponding to the region of the fmr1  mutation to determine the genotype of each individual.

   

 

Case C:  As Janet, Beth and Alison sit in the reception area of the genetics clinic, they discuss their anxiety about the upcoming test results and their anger at their mother.  Ever since Uncle Al, their mother’s brother, was diagnosed with Fragile X Syndrome, their family has discussed genetic testing for the disorder.  Their mother has steadfastly refused to consider being tested and even her unexpected pregnancy at age 42 has had no effect on her decision.  The daughters have decided that they will be tested because they want to know their status and also because they intend to use any positive results as leverage to convince their mother to change her mind about testing for her and the fetus.

 

DNA Samples:                      Janet

                                                Beth

                                                Alison

                                                Control normal DNA

                                                Control DNA with premutation

                                                Control DNA with full mutation

 

Digest each of these DNA samples with EcoR1.  Then use the probe corresponding to the region of the fmr1  mutation to determine the genotype of each individual.

 

 

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9.  Tay-Sachs Disease

         

Background:  Tay-Sachs Disease (TSD) is an autosomal recessive inborn error of metabolism whose basic biochemical defect is a deficiency of a lysosomal enzyme known as hexosaminidase A  (hex A), which normally catalyzes a step in the degradation of a membrane glycolipid called ganglioside GM2.  In the absence of hex A activity, GM2 accumulates in central nervous system cells, eventually compromising their function.  In the classical form of TSD, infantile TSD, clinical symptoms typically appear at three to six months of age and progress rapidly to blindness, deafness, uncontrollable seizures and death before age five years.  The disease occurs with increased frequency in the Ashkenazi Jewish population, with frequencies of heterozygotes ranging from 1 in 25 to 1 in 45.  An adult form of TSD, resulting from a partial deficiency of hex A activity, is associated with an age of onset in the twenties or thirties and is characterized by an unsteady gait followed by progressive central nervous system deterioration.  There are no effective therapies currently available for either form of TSD.  Characterization of the enzyme defect in infantile TSD in the 1960's resulted in development of a test for hex A activity that allowed for identification of heterozygotes and prenatal diagnosis of affected fetuses through amniocentesis.  The availability of these tests combined with the relatively  high frequency of heterozygotes in a well defined population led to TSD carrier screening programs being instituted in most major cities in the United States.  The Tay-Sachs gene has now been identified on chromosome 15 and three mutations that result in TSD have been characterized, allowing for more accurate diagnosis.  Studies of TSD carriers have shown that 78% have a four-nucleotide insertion mutation in exon 11.

 

To analyze these cases, use PCR with the TSD primers to amplify a portion of the hexosaminidase A  (hex A) gene from blood DNA samples.  (NOTE:  Use the PCR function on the Data Screen, rather than the 96-well PCR.)  Then, using the dot blot, load the probes into the spots and add the PCR-amplified DNA samples into the corresponding wells.

 

Case A:  When Megan and Greg announce their plans to get married, Megan's mother, Rachel, finally explains why Megan never got the baby brother or sister that she always asked for when she was younger.  Shortly after Megan was born, her parents learned that a Tay-Sachs Disease carrier screening program was being organized in their area.  Since they were planning to have more children, they decided to be tested.  The news they received was not what they had hoped for; they both tested positive for carrier status.  Because they did not want to risk having a child with TSD and their religious beliefs did not permit aborting an affected fetus, they chose not to have any more children.  When Megan tells Greg this news, he questions his parents and learns that they had chosen not to be tested because of fear of stigmatization and discrimination.  Greg and Megan decide that they must be tested before they get married.

 

DNA samples:                      Megan

                                                Greg

                                                Control DNA, with the TSD mutation

                                                Control normal DNA without the TSD mutation

Case B:  Lisa had always wondered about the results of her first Tay-Sachs Disease carrier test.  She had been tested at age 18 when large-scale screening was done in her hometown of Minneapolis.  The test used then measured levels of the Tay-Sachs enzyme and Lisa’s test results were in a range that made the diagnosis uncertain.  Even repeat testing could not resolve the question.  Since she was not planning to have children right away, Lisa had put her concern aside and gone on with her life.  With a busy career and an active social life, she had never married.  Now, at age 40, she suddenly found herself with an unplanned pregnancy and facing some difficult decisions.  Although the thought of being a single parent is daunting, Lisa decides that she wants to have a child.  The father is not interested in being involved in the child’s future and also refuses to undergo genetic testing.  Lisa decides to have DNA testing done to resolve her carrier status and to determine the genotype of the fetus.

 

DNA samples:                      Lisa

                                                Fetus

                                                Control DNA, containing the TSD mutation

                                                Control normal DNA without the TSD  mutation

(Note:  These represent DNA samples already amplified by PCR.)

 

        a.     What conclusions can you draw from the results of the DNA analysis?

        b.     How would you counsel Lisa based on the results of her tests?

c.     What issues are raised by this case?

 

 

Case C:  When Lisa (see Case B) tells her younger sister, Rose, about her decision to be tested for Tay-Sachs, Rose informs Lisa that she and her husband, Frank, are also expecting a child.  Rose is now concerned about her own Tay-Sachs status and decides that she and Frank should be tested.  They also decide to test their unborn child, while she is in the early stages of the pregnancy.

 

DNA samples:      Rose

                                Frank

                                Fetus

                                Control DNA, containing the TSD mutation

                                Control normal DNA without the TSD  mutation

 

        a.     What conclusions can you draw from the results of the DNA analysis?

        b.     How would you counsel Rose based on the results of her tests?

c.     What issues are raised by this case?

 

 

 

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B.  Infectious Diseases

 

Infectious diseases are caused by bacteria, viruses, or other pathogenic agents.  Diagnosis may involve detecting the presence of proteins or nucleic acid from the suspected pathogen using ELISA or PCR, respectively.  In some cases, the patient's blood will be tested for the presence of antibodies specific for a pathogen, as an indication that the person was previously infected with that agent.

 

 

1.  HIV/AIDS

 

Background:  Human immunodeficiency virus (HIV) causes the disease Acquired Immunodeficiency Syndrome (AIDS).  AIDS is characterized by the inability to mount an immune response to bacteria and other pathogens, resulting in a variety of life-threatening infections.  The virus is spread when bodily fluids, such as blood and semen, from an infected person directly enter the bloodstream of an uninfected person.  For example, unprotected sexual intercourse and sharing needles during injected drug use can spread the virus.  Once in the body, HIV infects and destroys certain white blood cells (called CD4 cells) and impairs the immune system.  It may take years after HIV infection for the symptoms of AIDS to appear.  HIV infection is routinely detected indirectly, via tests which measure whether a person's blood contains antibodies against HIV; if so, they must have been previously infected with the virus.  Recently, a viral load test was developed which directly measures the amount of HIV in a person's blood using the PCR technique.

 

Video introduction to HIV (from "AIDS: A Changing Epidemic", copyright 2002 Discovery Education - used by permission)

 

 

Note:  An ELISA test is considered positive if the color production (absorbance) for a sample is at least twice the negative control.  PCR results are recorded as viral load values, i.e. how many copies of the virus were detected.  When running a Western blot to detect antibodies to HIV proteins, the HIV proteins visible (from largest to smallest, running left to right), include gp160, gp120, p55, p41, p31 and p24.  The positive control antibody will bind to all of the proteins.  To be considered HIV positive, a sample must bind to two of these three proteins: gp120, gp41, and p24.  Any other binding pattern is considered "indeterminate".  A result can only be called negative if there is no binding to any of the HIV proteins.

 

As you analyze the HIV cases, here are some general questions you might consider:

• How did this person become infected with HIV?
• Have others also become infected?  Who else should be tested?
• How reliable are the tests?
• How often should someone be tested?
• Why do people engage in risky behaviors?
• What impact is the infection having on this person's life?
• How can this person prevent further transmission of the virus?
• What other ethical decisions does this person face?
• How common is this case?
• Are there cultural differences regarding attitudes about HIV and prevention?

 

 

 

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U.S. HIV cases

 

Case A.   Anna is a 27-year-old women from Guatemala, who is living with her boyfriend and is pregnant with her first child.  A blood test during her second trimester revealed that she was HIV positive.  Anna is surprised, because her first trimester test was negative, and she did not have sexual contact with anyone other than her boyfriend.  She is very concerned about the fate of her child, who may contract the virus from Anna.

Video version of Anna case (from "AIDS: A Changing Epidemic", copyright 2002 Discovery Education - used by permission)

To analyze this case, run an ELISA on the following blood samples (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with proteins, use antibodies as samples” option).  Then perform a Western blot on each sample. 

 

Anna, first trimester

Anna, second trimester

Anna's baby, 6 weeks after birth

Anna's baby, 3 months after birth

Anna's baby, 6 months after birth

Anna's boyfriend

        Positive and negative controls

 

· What is the status of each person tested?  How would you explain these results to Anna?

· How would explain the difference between Anna's first and second trimester results?

·         What recommendations would you give Anna as she cares for herself and her baby

 

After her baby is born, Anna begins taking antiretroviral medications.  A viral load test is performed one, three, and six months after she begins this drug treatment.  After running the PCR analysis on these samples, what would you conclude about the effectiveness of the treatment?

 

 

 

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Case B.   Katrice grew up in rural Alabama, where there was not much discussion about HIV and AIDS.  At seventeen, she had sex with a popular boy, who she later learned was very promiscuous and a drug user.  She tested HIV positive during a routine blood test several months later.  Katrice went untreated for four years, living in denial about her HIV infection.  She became involved with another man and had a daughter.  She finally sought medical treatment when she thought her daughter might have been exposed to the virus.

Video version of Katrice case Part 1 and Part 2 (from "AIDS: A Changing Epidemic", copyright 2002 Discovery Education - used by permission)

 

To analyze this case, run an ELISA on following blood samples (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with proteins, use antibodies as samples” option).  Then perform a Western blot on each sample. 

 

Katrice

Latranya's father

Latranya, 3 months old

Latranya, 6 months old

Positive and negative controls

 

·            What is the status of each person tested?  How would you explain these results to Katrice?

·            What recommendations would you give Katrice as she cares for herself and her baby?

 

On the advice of her physician, Katrice begins taking antiretroviral medications.  A viral load test is performed one, three, and six months after she begins this drug treatment.  After running the PCR analysis on these samples, what would you conclude about the effectiveness of the treatment?

 

 

 

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Case C.   Laverne is a 31-year-old African-American woman who is pregnant and HIV positive.  When she found out she was pregnant, she and her partner, Henry, decided that they could not terminate the pregnancy.  She already has named her baby Marcus, and she is trying to do everything possible to prevent him from becoming infected with the virus.  She is taking medications and eating healthy foods.  She will undergo a Caesarian section, and Marcus will take medications for his first six weeks until he is tested for HIV.

Video version of Laverne case (from "AIDS: A Changing Epidemic", copyright 2002 Discovery Education - used by permission)

 

To analyze this case, run an ELISA on the following blood samples (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with proteins, use antibodies as samples” option).  Then perform a Western blot on each sample. 

 

Laverne

Marcus, 6 weeks old

Marcus, 3 months old

Positive and negative controls

 

·            What is the status of each person tested?  How would you explain these results to Laverne?

·            What recommendations would you give Laverne as she cares for herself and her baby?

 

After her baby is born, Laverne continues taking antiretroviral medications.  A viral load test is performed one, three, and six months after later.  After running the PCR analysis on these samples, what would you conclude about the effectiveness of the treatment?

 

 

 

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Case D.  Doug grew up in southern California, an all-American boy who surfed and played volleyball.  When he was 20 years old and a junior in college, he revealed that he was gay and moved to San Francisco.  He had a hard time adjusting, and he felt that, in order to fit in, he had to engage in the same risky behaviors as everyone else.  Even though he knew the risks, he had unprotected sex.  Four days before his 24th birthday, he tested positive for HIV.

Video version of Doug case Part 1 and Part 2 (from "AIDS: A Changing Epidemic", copyright 2002 Discovery Education - used by permission)

 

To analyze this case, run an ELISA on the following blood samples (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with proteins, use antibodies as samples” option). Then perform a Western blot on each sample.

 

Doug

Doug's partner the night he believes he was infected (partner 1)

An earlier partner of Doug's (partner 2)

Positive and negative controls

 

·            What is the status of each person tested?  How would you explain these results to Doug?

·            What recommendations would you give Doug?

 

Doug and his partner begin antiretroviral drug treatments.  A viral load test is performed one, three, and six months after they begin this drug treatment.  After running the PCR analysis on these samples, what would you conclude about the effectiveness of the treatments?

 

 

 

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Case E.  Lisa grew up in a wealthy neighborhood, and the kids she grew up with didn't think they needed to worry about HIV and AIDS.  However, she believes she was infected with while on an island vacation during her college years.  Her father, a physician, helped her find the best medical care, and she immediately began taking medications which seemed to keep her healthy.  A few years later, she married David and wanted to start a family.  They decided to have unprotected sex during times when her viral load was low.  She continued to take medications during her pregnancy, and had C-section deliveries to reduce the risk of passing the virus to her baby.  Lisa and David now have three children.

Video version of Lisa case Part 1 and Part 2 (from "AIDS: A Changing Epidemic", copyright 2002 Discovery Education - used by permission)

 

To analyze this case, run an ELISA on the following blood samples (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with proteins, use antibodies as samples” option).  Then perform a Western blot on each sample. 

 

Lisa

David

3-year old child

6 1/2-year old child

9-year old child

Positive and negative controls

 

·            What is the status of each person tested?  How would you explain these results to Lisa?

·            What recommendations would you give Lisa as she cares for herself and her family?

 

In order to become pregnant, David and Lisa chose to have sexual intercourse during times when her viral load was low.  Analyze the DNA samples provided by PCR.   Based on the PCR results, when do you think they should have tried to conceive?

 

 

 

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Case F.   Jennifer is a white female who grew up, in her words, “with old-fashioned parents and old-fashioned values.”  She got good grades throughout high school and did not date until her late teens.  But after she graduated from high school and went away to college, she was eager to change her lifestyle.  She started to party a lot and dated several different people.  However, after a drunken sexual encounter with a man she met at a party that left them both feeling horrible the next morning, she decided to take better care of herself and stopped having sex.  Several months later she underwent a series of routing medical tests, including a blood test for HIV.  The HIV test came back positive.  Shocked, Jennifer decided to be tested again at a different clinic.  She contacted two of the boys with whom she had sexual contact (including Jeff, the boy from the party), and suggested they also get tested.

Video version of Jennifer case Part 1 and Part 2 (from "AIDS: A Changing Epidemic", copyright 2002 Discovery Education - used by permission)

 

To analyze this case, run an ELISA on the following blood samples (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with proteins, use antibodies as samples” option).  Then perform a Western blot on each sample. 

 

        Jennifer

        Jeff

        Paul

        Positive and negative controls

 

·         What is the HIV status of each person tested?

·         Do the results provide any information about how Jennifer may have been infected?

·         What recommendations would you give to Jennifer as she deals with her HIV diagnosis?

 

Jennifer begins antiretroviral drug treatments.  A viral load test is performed one, three, and six months after she begins this drug treatment.  After running the PCR analysis on these samples, what would you conclude about the effectiveness of the treatments?

 

 

 

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Case G.  In the early 1990’s Steve was an avid long distance runner and the picture of health.  But he suddenly started getting a lot of colds and unusual infections.   Eventually a blood test determined that he was HIV positive.  By the time he was tested he was already exhibiting full-blown AIDS and his prognosis was poor.  He started antiretroviral drug treatments, but they did not seem to help.  When a new class of drugs, protease inhibitors, was approved, Steve changed his medications and immediately began to show improvement.  Amazingly, his immune system seemed to return to normal and he regained much of the weight he lost.  He began running again and finished a marathon.  Two of his running partners were inspired by his situation and decided to get tested.  They both had reason to believe they have been exposed to HIV, but were reluctant to get tested because they were afraid that a positive test would be a “death sentence”. 

Video version of Steve case Part 1 and Part 2 (from "AIDS: A Changing Epidemic", copyright 2002 Discovery Education - used by permission)

To analyze this case, run an ELISA on the following blood samples (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with proteins, use antibodies as samples” option).  Then perform a Western blot on each sample. 

               

        Steve

        Runner 1

        Runner 2

        Positive and negative controls

 

·         What is the HIV status of each person tested?

·         What recommendations would you give each of them based on their results?

 

Viral load tests were run three, six, and nine months after Steve started each of his drug treatments. After running the PCR analysis on these samples, what would you conclude about the effectiveness of the treatments?

 

 

 

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Case H.  Marie (U.S.) thought she had found her perfect match when she started dating Rick, a divorced man who seemed to live a healthy lifestyle and treated her well.  They traveled together frequently and became very close.  But suddenly, Rick's behavior changed and he became unpredictable,  His mood swings eventually caused them to split up.  Several months later, Marie learned through a mutual friend that Rick was dying of AIDS.  Marie was shocked, and she immediately got tested for HIV.   She also began a program of exercise and nutritional supplements to try to improve her chances of staying healthy.

 

Video version of Marie (U.S) case Part 1 ,Part 2 and Part 3 (from "AIDS: A Changing Epidemic", copyright 2002 Discovery Education - used by permission)

To analyze this case, run an ELISA on the following blood samples (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with proteins, use antibodies as samples” option).  Then perform a Western blot on each sample. 

 

        Marie

        Rick

        Positive and negative controls

 

·         What is the HIV status of each person tested?

·         What recommendations would you give to Marie as she deals with her HIV diagnosis?

·         What role do you think Marie's exercise and nutrition program will play in her health?

 

Marie begins antiretroviral drug treatments.  A viral load test is performed one, three, and six months after she begins this drug treatment.  After running the PCR analysis on these samples, what would you conclude about the effectiveness of the treatments?

 

 

 

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African HIV cases

Video introduction to African cases (from "The Faces of AIDS", Media for Development International - used by permission)

 

Case I.   Nicole is a 25-year old woman living in Cameroon.  She has been diagnosed with AIDS, and is staying in a hospital because she is unable to care for herself.  Her father has refused to help her because he believes she contracted the disease by "sleeping around".  Her father also forbids her brothers and sisters from seeing her.  She believes she may have been infected by a boy she was seeing for a while, but she has not heard from him in months. 

 

Video version of Nicole case (from "The Faces of AIDS", Media for Development International - used by permission)

 

To analyze this case, run an ELISA on the following blood samples (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with proteins, use antibodies as samples” option).  Then perform a Western blot on each sample.

 

Nicole

Nicole's former boyfriend

Positive and negative controls

 

·         What is the status of each person tested?  How would you explain these results to Nicole?

·         What options does Nicole have?

·         What do you think about Nicole's father's decision not to help her?

 

Because her father will not support her financially, she is given only supportive care at the hospital.  As part of a UNAIDS study, blood samples are taken every few months and sent to a hospital in the capital city, Yaounde, so a viral load test can be performed. 

 

Run PCR on the following DNA samples isolated from Nicole's blood:

 

        3 months after diagnosis

        6 months after diagnosis

        12 months after diagnosis

 

·         Based on these PCR results, what is Nicole's prognosis?

 

 

 

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Case J.     Auxilia, who has just been diagnosed with AIDS, lives with her five children on a small plot of land in Zimbabwe.  Her husband died several years ago.  Auxilia is worried about who will look after her children if she dies.  She knows that there is a lot of stigma associated with AIDS and that people are afraid to interact with her.

Video version of Auxilia case (from "The Faces of AIDS", Media for Development International - used by permission)

 

To analyze this case, run an ELISA on the following blood samples (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with proteins, use antibodies as samples” option).  Then perform a Western blot on each sample. 

 

Auxilia

Auxilia's oldest child

Auxilia's youngest child

Positive and negative controls

 

·            What is the status of each person tested?  How would you explain these results to Auxilia?

·            What recommendations would you give Auxilia as she cares for herself and her baby?

·            What can Auxilia tell people to help them understand that they should not be afraid of her?

 

Auxilia is selected to enroll in a program that allows her to receive antiretroviral medications.  A viral load test is performed one, three, and six months after she begins this drug treatment.  After running the PCR analysis on these samples, what would you conclude about the effectiveness of the treatment?

 

 

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Case K.   Marie (African) lives on a small plot of land in Cameroon with her five children.  Her husband died two years ago, and Marie has just been diagnosed with AIDS.  Before she was diagnosed, she did not believe that the disease existed in her country.  Fortunately, Marie's brother is very supportive and willing to look after her and her children.  He is not afraid of catching the disease from her.

Video version of Marie (African) case(from "The Faces of AIDS", Media for Development International - used by permission)

 

To analyze this case, run an ELISA on the blood samples provided (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with proteins, use antibodies as samples” option).  Then perform a Western blot on each sample. 

 

Marie

Marie's brother

Marie's husband

Marie's oldest child

Marie's youngest child

Positive and negative controls

 

·            What is the status of each person tested?  How would you explain these results to Marie?

·            What recommendations would you give Marie as she cares for herself and her baby?

·            What precautions does her brother need to take to keep from getting infected?

 

Marie is selected to enroll in a program that allows her to receive antiretroviral medications.  A viral load test is performed one, three, and six months after she begins this drug treatment.  After running the PCR analysis on these samples, what would you conclude about the effectiveness of the treatment?

 

 

 

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Case L.   Tendayi and her husband, Farayi, a married couple in Zimbabwe, learned that they were both HIV positive when their baby died two years ago.  They are supporting each other and planning to stay together; neither blames the other for what happened.  They are focused on finding a way to live with AIDS and to educate others about it.

Video version of Tendayi case (from "The Faces of AIDS", Media for Development International - used by permission)

 

To analyze this case, run an ELISA on the following blood samples (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with proteins, use antibodies as samples” option).  Then perform a Western blot on each sample. 

 

Tendayi

Farayi

Their 3-year old child

Positive and negative controls

 

·            What is the status of each person tested?  How would you explain these results to Tendayi?

·            What recommendations would you give Tendayi and Farayi as they care for themselves and their remaining child?

·            What can they do to help others understand the disease?

 

Tendayi and Farayi are selected to enroll in a program that allows them to receive antiretroviral medications.  A viral load test is performed one and six months after they begin this drug treatment.  After running the PCR analysis on these samples, what would you conclude about the effectiveness of the treatment?

 

 

 

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Case M.   Safari grew up in a rural village in Kenya and owns a small plot of land.  After he marries, he decides that the land won't support his family, so he goes to the city to find work.   Eventually, he finds a job and is able to send money home to his wife.  He is only able to visit his home once in a while, and he spends most of his time in the city.  Lonely, he turns to other women for companionship.  His wife, meanwhile, is left to take care of the house and the land by herself.  She becomes pregnant and gives birth to a child, and Safari continues to work in the city.  Safari becomes chronically sick and starts to miss a work frequently.  Safari's doctor eventually tests him for HIV, and he is positive.  Are his wife and child also infected?

Video version of Safari case Part 1 , Part 2 , Part 3 , and Part 4 (from "AIDS - Life at Stake", Media for Development International - used by permission)

 

To analyze this case, run an ELISA on the following blood samples (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with proteins, use antibodies as samples” option).  Then perform a Western blot on each sample.  

 

Safari

Safari's wife

Their baby

Positive and negative controls

 

·            What is the status of each person tested?  How would you explain these results to Safari and to his wife?

·            What recommendations would you give to the couple as they care for Safari?

 

Safari is selected to enroll in a program that allows him to receive antiretroviral medications.  A viral load test is performed one and six months after they begin this drug treatment.  After running the PCR analysis on these samples, what would you conclude about the effectiveness of the treatment?

 

 

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2.  Influenza

 

Human Influenza

 

Background:  Influenza virus causes respiratory infections ("the flu") that can be quite severe.  About 36,000 people die from flu complications every year in the U.S.  The symptoms of the flu include fever, headache, sore throat, cough, and muscle aches.  These symptoms are difficult to distinguish from other respiratory infections.   A more definitive diagnosis can be made by testing respiratory fluids, often obtained via a throat or nasopharyngeal swab, for the presence of influenza proteins (using ELISA or similar techniques) or nucleic acid (using RT-PCR) (Since the virus genetic material is RNA, reverse transcription (RT) is used to copy the RNA into cDNA for the PCR test).  Since it takes a few weeks for antibodies to influenza to be detected in a patient's blood, testing the patient's blood for influenza antibodies is usually used to confirm an infection after the illness has subsided, e.g. to monitor the extent of an epidemic.  Influenza virus is highly contagious and is easily spread via respiratory droplets.  There are drugs available to treat influenza virus infections, but they are generally only used when an infected person is at risk for serious complications, or to control an epidemic.  The influenza vaccine ("flu shot") can protect individuals from getting infected, and it is highly recommended for individuals most at risk for the serious complications from flu, such as people older than 65, children under 2 years old, and anyone with chronic heart or lung conditions.  The vaccine usually contains three different strains of influenza, and the antibodies generated to the vaccine can protect against these strains and related strains.  Influenza virus has a high mutation rate and there are new strains of the virus appearing every year.  There are two main strains of influenza, A and B, that cause the annual outbreaks of flu.  Strain A viruses are further distinguished based on their surface proteins, abbreviated H and N.  The strain names also include the location and year where they were isolate.  For example, strain A/Fujian/02 (H3N2) was isolated in the Fijian province in China in 2002 and its surface proteins are designated H3N2.

 

Note:  An ELISA test is considered positive if the color production (absorbance) for a sample is at least twice the negative control value.   PCR results are recorded as viral load values, i.e. how many copies of the virus were detected.

 

Case A.   This fall, for the first time in several years, Sheila did not get a flu shot.  She has been very busy, especially since she started babysitting her grandchildren (ages 1 and 3) on weekdays.  She also does not like needles and shots, so it was easy for her to come up with excuses not to go get the shot.  Sheila is 67 years old, but she has been in good health and does not have any chronic health conditions.  Two days ago, she came down with a fever (102^o F), sore throat, and a bad cough.  She has been taking ibuprofen, but it does not seem to be helping.  Sheila feels just awful, but she drags herself to the clinic.  The physician is concerned that the fever has not subsided, and because Sheila's age places her at some risk for serious complications from influenza, she decides to test Sheila for influenza and takes a throat swab sample.  Although Sheila's grandchildren have not been with her the past two days, they did stay at her house the day before she got sick.  The physician suggests that both children be tested, even though they have not yet shown any symptoms. 

 

To analyze this case, first run an ELISA on the throat swab samples from the following sources to test for the presence of influenza virus proteins, using antibodies specific for influenza A and B viruses. (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with antibodies, use proteins as samples” option).  Then perform a Western blot on each sample.  Then perform a PCR test on the cDNA isolated from the swabs, using primers specific for influenza A and B, to see if influenza virus genetic material can be detected. 

 

        Positive control for influenza virus

        Negative control

        Sheila

        1-year old

        3-year old

 

• Do Sheila's symptoms appear to be caused by infection with influenza virus?
• Are either of the grandchildren infected?
• What should the physician recommend for treatment for these patients?
• What should Sheila do to avoid spreading her illness to others?

 

 

Case B.   Shannon, a college junior, was really looking forward to playing in her first big basketball tournament.  The whole team had flow to Hawaii for one week during the semester break to play against teams from all over the country.  Unfortunately, Shannon's team was not in top form.  Three team members had to stay home due to illness.  They had headaches, fever, and muscle aches that prevented them from getting out of bed, let alone play basketball.   Four other teammates had similar symptoms the week before but had recovered enough to join the team on the trip, although they were somewhat out of practice.  On the morning of their first game, Shannon woke up in the hotel room she was sharing with three other teammates feeling terrible.  She was distressed to realize she was experiencing the same symptoms as the sick team members, and she did not want to tell her coach because she did not want to miss the game.  But soon after getting up she knew she was too sick to play and reluctantly told her coach.  The coach was anxious to find out what was sweeping through her team, so she brought Shannon to a clinic in Honolulu.  The physician there took a throat swab and tested for influenza virus.  She also recommended that the other team members rooming with Shannon be tested.  In addition, she tested blood from the team members who had recovered from a similar illness, to see if they had antibodies against the same virus.

 

To analyze this case, run an ELISA on the throat swab samples from the following sources to test for the presence of influenza virus proteins, using antibodies specific for influenza A and B viruses. (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with antibodies, use proteins as samples” option).  Then perform a PCR test on the cDNA isolated from the swabs, using primers specific for influenza A and B, to see if influenza virus genetic material can be detected. 

 

        Positive control for influenza virus

        Negative control

        Shannon

        Roommate 1

        Roommate 2

        Roommate 3

 

Test the blood samples from the four players who recovered from the illness using an ELISA to see if they have antibodies to influenza A or B proteins.  (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with proteins, use antibodies as samples” option).

 

·         Is Shannon infected with Influenza virus?

·         What are Shannon's prospects for playing in any of the games this week?

·         What should the physician recommend for Shannon?

·         Are any of her roommates infected?

·         Does is appear that the recovered players had the same virus infection?

·         What should be done to prevent the rest of the team from getting sick?

 

 

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Avian influenza

 

Background:   Certain strains of influenza virus preferentially infect birds rather than humans.  These viruses occur naturally in wild bird populations, particularly waterfowl, and can be spread to domestic birds.  Like human influenza, avian influenza is highly contagious.  The virus is shed in the saliva, nasal secretions, and feces of infected birds.  Wild birds generally do not get sick from the infection, but some forms are highly pathogenic for domestic poultry and can be over 90% fatal.  In 1997, one type of avian influenza strain A with surface proteins designated H5N1 (see the description of human influenza virus above) was found to be able to infect humans who came into contact with infected birds.   Humans infected with avian influenza have a high mortality rate, but the virus does not seem to be able to spread from person to person.  If the virus mutates such that it can spread more easily between people, it has the potential to cause a global pandemic, because it is different enough from the human influenza viruses that few people will have developed immunity from previous exposures. 

 

 

Case C.   An outbreak of avian influenza recently swept through several farms in a rural area outside Ho Chi Minh City, Viet Nam.  The government has ordered that all farms in the area destroy their chickens to prevent the virus from spreading any further.  World Health Organization (WHO) officials are collecting samples and testing them for influenza.  The Vanh family's farm is located just outside this area and they are hoping that they do not have to destroy their flock.  But a few of their chickens have recently died, so the WHO workers take samples for testing. Their daughter, age 8, has a respiratory infection that the family worries may be related to the avian flu.  WHO officials arrange for her to be tested for influenza, and recommend that other family members be tested even though they are not showing symptoms.  Throat swab samples are taken from the father, mother, and two children (the sick daughter and her brother).

 

To analyze this case, test the five samples of respiratory fluids from three of the dead chickens on the Vanh farm, and from the four family members, by ELISA for the presence of influenza H5N1 proteins (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with antibodies, use proteins as samples” option).  Then perform a PCR test on the cDNA isolated from the samples to see if influenza virus genetic material can be detected.

 

• Did the Vanh's chickens die from influenza infection?
• Should the rest of their chickens be destroyed?  What are the alternatives?
• Is the Vanh's daughter infected with avian flu?  What are the options for treating her illness?
• Do any other family members appear to be infected?
• What can be done to reduce the risk of avian influenza infection?

 

 

Case D.    Turkey was one of the first European countries to report avian flu H5N1 infections in domestic poultry.   Dozens of birds have died from the infection, and thousands more have been destroyed in order to stopped the spread of the virus.   Several people in the same regions have come down with serious respiratory infections, and two people have died.  Two of the patients are sisters in a family whose chickens tested positive for H5N1 influenza.  Local health official want to test the two girls to see if their illness is caused by influenza H5N1 infection.  The also want to test the rest of the family members, so throat swabs samples are taken from the mother, father and brother in addition the two girls. The family is adamant that only the older sister had direct contact with sick chickens so they do not understand why both girls would be ill if this virus was causing the symptoms.

 

To analyze this case, run an ELISA to test the throat swab samples from each of the family members for the presence of influenza H5N1 proteins  (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with antibodies, use proteins as samples” option).  The test cDNA isolated from each sample for influenza genetic material using PCR.

 

• Are the two sisters infected with avian influenza?
• Do any of the other family members appear to be infected?
• What are the implications if the second sister was infected without having had direct contact with infected chickens?  How else might she have become infected?
• Is it possible to determine whether the two children were infected from the same source?
• What is the risk to the other family members?
• What can be done to reduce the further spread of the virus?

 

 

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3.  Severe Acute Respiratory Syndrome (SARS)

 

Background:   SARS is caused by infection with a coronavirus called SARS-associated coronavirus (SARS-CoV).  The disease was first reported in Asia in 2003, and SARS-CoV was unrecognized prior to this outbreak.  It infected over 8,000 people and killed over 700.   There have been no new cases since April 2004.  SARS-CoV is spread person-to-person via respirtory droplets produced when a person coughs or sneezes.  Symptoms include high fever, headache, and body aches, with a rapid progression to pneumonia. Laboratory diagnosis is made using RT-PCR to detect the presence of SARS-CoV genetic material in respiratory samples, or the testing the blood for antibodies to SARS-CoV by ELISA.  Antibodies may not be detectable until a week or longer after infection, so RT-PCR is preferred early in an infection. (Since the virus genetic material is RNA, reverse transcription (RT) is used to copy the RNA into cDNA for the PCR test).  There is no specific treatment for SARS, nor is there a vaccine for SARS-CoV.

 

Note:  An ELISA test is considered positive if the color production (absorbance) for a sample is at least twice the negative control value. PCR results are recorded as viral load values, i.e. how many copies of the virus were detected.

 

 

Case A.  Dr. Smith, a physician at a hospital in Hamilton, Ontario observed four cases of what appeared to be a viral pneumonia within the last week.  The symptoms resemble those associated with SARS.   The patients all had high fevers and difficulty breathing, and their x-rays showed signs of pneumonia.  Bacterial cultures were negative.  None of the patients reported traveling out of the country recently.  The first patient who was admitted seems to be recovering, but the second patient has taken a turn for the worse, and may not last the night.  Dr. Smith remembers very well the SARS outbreak in Toronto, just one hour north of Hamilton.  Most of the more than 300 people infected were exposed to the virus while in a Toronto hospital, as a patient or visitor.  Some of the casualties were health care workers who contracted the virus while treating patients.  He and the rest of the clinical staff have been taking every possible precaution, and the patients are in an isolation ward in the hospital. Dr. Smith is anxiously awaiting the results of the laboratory tests. 

 

To analyze this case, two types of tests are necessary.  Run an ELISA test on blood samples to test for the presence of virus proteins (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with antibodies, use proteins as samples” option).  Also, run RT-PCR on the samples to try to detect the virus genetic material; DNA from these samples is used in the PCR test.  Samples include:

 

        Positive control for SARS-CoV

        Negative control

        Patient 1 (57-year-old male)

        Patient 2 (51-year old female, wife of patient 1)

        Patient 3 (43-year-old female)

        Patient 4 (69-year old male)

 

·         Are any of the patients infected with the SARS coronavirus?

·         If so, what should be done to treat them?

·         What are the risks of spreading the virus in a hospital setting? 

·         Where/how might these patients have been exposed to the virus?

·         If any of the patients are negative for SARS CoV, what else could be causing their symptoms?

·         How risky is it for the health care workers who treat SARS patients?

 

 

Case B.  Shi Jiao-hui has lived in the New York City almost all of this life.  His parents moved there when he was two years old.  He is a U.S. citizen and considers himself a New Yorker, but the rest of his extended family still lives in Guangdong province in China.  Finally, at age 30, he and his wife, Ming, and their son and daughter traveled to China to visit his relatives and see his homeland.  Their 3-week trip took them through Hong Kong and several cities in China, and they spent the last week with relatives near the city of Guangzhou.  Unfortunately, Ming was very sick most of that week.  She had a high fever and difficulty breathing.  She was taken to a hospital in Guangzhou where she was treated for pneumonia.  She recovered enough to fly back home and is doing fine now.  However, Jiao felt ill soon after returning home and now has the same symptoms as Ming.  When he goes to the clinic the physician is alarmed by the description of the symptoms and the fact that the family had recently visited the area where SARS was first reported.  He recommends that Jiao be tested for SARS coronavirus infection and collects a throat swab.  He also recommends that the two children be brought in for testing.  In the meantime, Jiao is hospitalized and placed in an isolation ward.

 

To analyze this case, run an ELISA on the proteins in throat swab samples from Jiao and the two children (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with antibodies, use proteins as samples” option), as well as a PCR test on cDNA isolated from the sample.  Also, test Ming's blood for antibodies to SARS-CoV proteins (in this case, select the “coat wells with proteins, use antibodies as samples” option).

 

        Protein and DNA samples:

        Positive control for SARS CoV

        negative control

        Jiao

        Son

        Daughter

 

        Blood (antibody) sample:

        Ming

 

·         Is Jiao infected with SARS-CoV?

·         Do either of the children appear to be infected?

·         What should the recommended treatment be?

·         Was the physician right to place Jiao in isolation?

·         Was Ming infected with SARS-CoV while in China?  If so, why didn't the doctors there tell her?

·         What should be done to minimize the risk that Jiao will infect anyone else?

 

 

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4.  West Nile virus

 

Background:  West Nile virus (WNV) was first reported in the U.S. in 1997.  It is spread by mosquitoes that bite an infected animal (usually a bird), and then bite another animal, transmitting the virus.  In addition to birds, the virus can be spread to humans and other mammals including dogs, cats, and horses.  It can also be transmitted via blood transfusion or organ transplant from an infected person.  Many humans experience no symptoms, but about 20% will contract "West Nile fever", with fever, headache, body aches, nausea, and rash that can last for weeks.  In a few cases (less than1%), the illness will become more serious, leading to permanent neurological effects such as muscle weakness, vision loss, and coma.  There is no specific treatment for WNV illness.  Diagnosis of WNV infection is accomplished by detecting virus proteins (by ELISA) or genetic material (by PCR) in blood or cerebrospinal fluid samples. (Since the virus genetic material is RNA, reverse transcription (RT) is used to copy the RNA into cDNA for the PCR test).  Antibodies to WNV can also be detected in the patient's blood by ELISA, but these may not be detectable until later in the infection process.

 

Note:  An ELISA test is considered positive if the color production (absorbance) for a sample is at least twice the negative control value. PCR results are recorded as viral load values, i.e. how many copies of the virus were detected.

 

 

Case A.  The annual blood drive in Mitchell, SD is usually held during September each year.  However, this year there was an outbreak of West Nile infections in mid-August.  No one died, but at least 30 people were diagnosed with West Nile fever, and five cases were severe enough to require hospitalization.  It is likely that many more people were infected with WNV but did not have symptoms, so all of the donated blood will be screened for antibodies to WNV.  Any samples testing positive for antibodies will be tested for WNV genetic material by PCR.  The presence of WNV cDNA would indicate an active infection, otherwise the individual has probably recovered from the infection.

 

To analyze this case, run an ELISA on the set of 10 donated blood samples provided, testing them for antibodies to WNV proteins (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with proteins, use antibodies as samples” option).  If any of the samples test positive for antibodies, test those samples for WNV genetic material by PCR.

 

• How many blood samples, if any, tested positive for WNV antibodies?
• Did any of the samples test positive for WNV genetic material?
• What is the significance of finding WNV antibodies or genetic material in blood?
• Is the number of positives what you would expect, based on the number of cases of West Nile fever?
• Should the individuals who donated the blood that tested positive be notified?
• What can be done to reduce the number of WNV infections next season?

 

 

Case B. Rachel has lived in Anaheim, CA, all of her life, and had never noticed anything like this.  For the past several weeks she had been finding dead birds, mostly crows and a few sparrows, in her yard.  She has also seen them in the neighbor's yards when she walked her dog.  She estimates that she has seen at least 35 dead birds.   Rachel finally contacts the California Department of Health, which collects some of the birds for West Nile virus testing.  Lab technicians take samples from brain tissue and test them for the presence of WNV proteins by ELISA.  Although no humans have reported symptoms of West Nile disease, health officials decide to test individuals for the presence of antibodies to WNV in their blood, to determine the extent of human infections.  Rachel, her husband, and two children are tested.

 

To analyze this case, run an ELISA on the six samples of brain tissue from dead birds (four crows and two sparrows) for the presence of WNV protein (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with antibodies, use proteins as samples” option).  Also, test the blood samples from Rachel's family members for antibodies to WNV proteins (in this case, select the “coat wells with proteins, use antibodies as samples” option).

 

• Did the birds die from WNV infection?
• Were any members of Rachel's family infected with WNV?
• Are there any consequences if someone is infected with WNV but exhibits no symptoms?
• Should more people in the area be tested?
• What can the residents do to protect themselves from WNV infection?
• What impact will that many dead birds have on the local crow population?
• What is the potential environmental impact reducing crow numbers?

 

 

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5.  Ebola

 

Background:  Ebola virus causes a hemorrhagic fever that is often fatal.  The disease was first reported in 1976 in the Democratic Republic of the Congo (formerly Zaire), and outbreaks have appeared sporadically since then.  The disease progresses quickly upon infection, usually in 2-10 days.  The symptoms begin with fever, headache, and muscle aches, followed by diarrhea and vomiting.  In severe cases, internal and external bleeding occur.  The virus is spread from person to person via contact with infected blood or secretions. The source of the initial infection in an outbreak is not known, but it is hypothesized to be transmitted from an infected animal.  The infection can be diagnosed within a few days of the onset of symptoms, using an ELISA to test for Ebola virus proteins or RT-PCR to test for virus genetic material.  (Since the virus genetic material is RNA, reverse transcriptase (RT) is used to copy the RNA into cDNA for the PCR test).  After recovery, patients can be tested for antibodies to Ebola virus proteins in their blood to confirm infection.  There is no treatment for Ebola hemorrhagic fever other than supportive care.  The death rate from infection is typically 70-80%.  There is no vaccine available for Ebola virus.

 

Note:  An ELISA test is considered positive if the color production (absorbance) for a sample is at least twice the negative control value. PCR results are recorded as viral load values, i.e. how many copies of the virus were detected.

 

Case A.  An outbreak of a disease that resembles Ebola hemorrhagic fever has been reported in a village outside of Kinshasa, the capital of the Democratic Republic of Congo.  Local health officials are concerned about the outbreak spreading to such a large population.  Health workers are sent to the village to determine whether the disease is caused by Ebola virus.  Most of the victims so far belong to one family.  Blood samples are collected from the family members showing symptoms of hemorrhagic fever and tested for Ebola virus proteins and genetic material.

 

To analyze this case, run an ELISA on the proteins in throat blood samples from the various family members (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with antibodies, use proteins as samples” option), as well as a PCR test on cDNA isolated from the samples.

 

                Protein and DNA samples:

                Positive control for Ebola proteins

                Mother

                Father

                Grandmother

                Son

                Daughter

 

• Are these family members infected with Ebola virus?
• What should be done for these individuals if they are infected?
• What precautions should be taken when handling these blood samples?
• What should be done to prevent the virus from spreading to the city of Kinshasa?

               

 

Case B.  One of the animals suspected of being a reservoir for Ebola virus is the fruit bat.  After an outbreak of Ebola hemorrhagic fever in Gabon, scientists captured dozens of bats in the forests near the village and tested their blood for antibodies to Ebola virus.  The presence of Ebola antibodies would indicate that the bats had been infected with Ebola at some time and survived.  None of the bats were showing symptoms of hemorrhagic fever at the time they were captured.

 

To analyze this case, run an ELISA on the 10 bat blood samples provided, testing them for antibodies to Ebola proteins (Click the Method button on the 96-well plate and choose ELISA, then select the “coat wells with proteins, use antibodies as samples” option).  If any of the samples test positive for antibodies, test those samples for Ebola cDNA by PCR.

 

• How many bats, if any, tested positive for Ebola antibodies?
• Did any samples test positive for Ebola genetic material? What does a positive result mean?
• Does a positive result mean that bats started the Ebola outbreak in Gabon?
• If bats test positive for Ebola, should efforts be made to eliminate bats in areas near human populations?

 

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6. Salmonella

[Note: This case is included to demonstrate new features in v5.03, including the ability to open very large DNA files (e.g. the entire 5 Mb Salmonella genome) and to run multiplex PCR. Additional cases using these features will be developed and made available on the Case It! home page. Although included here in the online version, the Word document describing this case must be downloaded separately.]

Background

“Salmonellosis is an infection with a bacteria called Salmonella. Most persons infected with Salmonella develop diarrhea, fever, and abdominal cramps 12 to 72 hours after infection. The illness usually lasts 4 to 7 days, and most persons recover without treatment. However, in some persons the diarrhea may be so severe that the patient needs to be hospitalized. In these patients, the Salmonella infection may spread from the intestines to the blood stream, and then to other body sites and can cause death unless the person is treated promptly with antibiotics. The elderly, infants, and those with impaired immune systems are more likely to have a severe illness.”

Source: Center for Disease Control website

Case A . Sarah, a 6-year-old girl, was admitted to the hospital with a 4-day history of fever, vomiting, abdominal pain, and diarrhea. Lab tests indicated that her white blood cell count is elevated and that she has mild liver dysfunction. A stool culture taken on admission yielded a Gram negative rod that appeared to be Salmonella. Blood, nasopharyngeal, and urine cultures were negative. Sarah had no history of overseas travel, and there was no indication that she had ingested any suspect foods. No other family members were ill. Sarah has a pet turtle, a red-eared slider, for which she is the sole caregiver. A water specimen from the turtle’s tank also yielded a culture of Salmonella. To determine whether the bacteria isolated from the turtle’s tank and from Sarah were the same, genomic DNA was isolated from each of the bacteria samples.

To analyze this case, digest each of the DNA samples with XbaI. Then digest each of the original DNA samples (not the XbaI-digested samples) with BlnI. Run the digested samples on a gel, using a 0.5% agarose gel. (Note that this would normally be done using pulse-field gel electrophoresis, due to the large sizes of the DNA fragments, but here the fragments will run correctly using the standard agarose gel procedure). Do the band patterns for the DNA isolated from Sarah and from the turtle match?

Multiplex PCR can be used to determine which strain of Salmonella Sarah has. To perform this procedure, use the Salmonella primers to run PCR on the DNA isolated from the turtle’s tank, the DNA isolated from Sarah, and from control DNA samples isolated from S. typhimurium and S. paratyphi. The primers file contains two sets of primers, one which identifies all strains of Salmonella and amplifies a 204 bp DNA fragment; the other is specific for S. typhimurium and amplifies a 402 bp fragment. Use a 1.0% agarose gel to run the PCR products.

• Which strain of Salmonella is Sarah infected with?
• Do the results indicate that Sara contracted the Salmonella infection by handling the Turtle?
• How should Sarah be treated so that she can recover from the infection?
• What is the level of risk associated with reptile pets?
• What other pets are associated with possible bacterial infections?

References

Nagano, N. et al. Jpn. J. Infect. Dis. 59, 132-134, 2006
Alvarsez, J. et al., J. Clin. Microbiol. 42, 1734-1738, 2004
http://www.cdc.gov/

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7. Sexually transmitted diseases

 

This case was developed by C. Dinitra White, North Carolina A & T State University, as part of the 2006 BioQUEST summer workshop held at Beloit College.

 

Background:  Sexually transmitted diseases are a major public health problem in the U.S.  Several organisms, including bacteria and viruses, can be transmitted through sexual contact. Neisseria gonorrheae (which causes gonorrhea) and Chlamydia trachomatis can cause urethritis inflammation of the urethra) in males and females, and pelvic inflammatory disease (PID) in women.  PID is infection of the uterus and fallopian tubes that can lead to chronic pelvic pain, infertility, ectopic pregnancy, abscess formation, and internal bleeding.  Organisms that cause genital ulcer disease (GUD) include the bacteria Haemophilus ducreyi (which causes chancroid), Treponema pallidum (syphilis), and herpes simplex virus type 2 (genital herpes).  GUD results in painful genital lesions in both males and females.  These organisms can be detected by culture methods, antibody-based tests such as ELISA, and nucleic acid amplification tests such as PCR.  Multiplex PCR can test for several organisms simultaneously.  Treatment is primarily antibiotics (or anti-viral drugs for Herpes simplex virus).

 

Case A, The Soldier’s Unexpected Gift

 

Robert Jr. is a 22 year old soldier who very recently returned to his hometown in the Midwestern region of the United States. Lucky for Robert, his assignment in Asia ended just in time to return to Old School University to complete a degree in Science and Mathematics Education. To celebrate his return, Robert’s girlfriend Jenna, a foreign exchange student, gathered lots of food, alcohol, and party favors for a night of celebration with friends. Several days later, the director of Old School University’s student health department issued an alert to the university president and the local department of health to report a dramatic increase in the number of genital ulcer disease (GUD) cases on campus. There were lots of rumors about an outbreak of HIV or gonorrhea on campus, however no official warnings were released by the university.  Three days after the party, Robert became very worried upon noticing an unusual, inflamed ‘bump’ on his penis during a shower. However, he resisted a trip to student health… he hoped it would simply go away. Two days later, the bump began bleeding. Immediately, Robert called Jenna and urged her to go with him to the student health facility to be tested for what he thought may be HIV.  But rather than test them for HIV, the student health nurse took an endocervical swab sample from Jenna and a urethral swab sample from Robert.  DNA was extracted from the swab samples and multiplex PCR was performed to test for five different sexually-transmitted diseases.

 

 

OPTIONS FOR CASE ANALYSIS

 

Option A:  Perform multiplex PCR on the DNA samples listed below, using multiple primer sets (in a single combined file) that recognize five sexually transmitted organisms.

 

For a tutorial on multiplex PCR using a different organism (Salmonella), see the ‘multiplex PCR’ link on the Case It home page at http://caseit.uwrf.edu/tutorialv5/Multiplex/1.htm

 

 

 

Option B:  Perform 96-well PCR on the DNA samples listed below, testing each sample for the five sexually transmitted organisms.  Each sample should be tested separately for each the five sexually transmitted organisms (the Option B folder includes five primer sets, in five separate files).  Option B allows for the use of a negative control.

 

For a tutorial on 96-well PCR using a different disease (AIDS), see the ’96-well PCR’ link on the Case It home page at http://caseit.uwrf.edu/tutorialv5/Protein/3.htm.

 

 

DNA samples:

Robert

Jenna

Another male student at the same college (student 1)

Another female student at the same college (student 2)

Negative control (Option B only)

Positive control, which includes:

Haemophilus ducreyi (chancroid)

Herpes simplex virus type 2 (herpes)

Neisseria gonorrheae (gonorrhea)

Chlamydia trachomatis serovar D (chamydia)

Treponema pallidum (syphilis)

 

If the target DNA is present, the primers will amplify the following sizes of DNA fragments (in kilobases):

Haemophilus ducreyi, 1.24

Herpes simplex, 0.34

Neisseria gonorrheae, 0.55

Chlamydia trachomatis, 1.93

Treponema pallidum, 0.40

 

a.     What organism is most likely causing Robert’s genital ulcer?

b.    Was his girlfriend Jenna also infected?

c.     Were the other college students tested infected with the same organisms?  Do these results give you any information about how the diseases are being spread?

d.    What would you recommend as a treatment for Robert?

e.     How would you discuss with Robert the importance of preventing the spread of these infections?

 

 

References

 

BioQUEST 2006 summer faculty workshop resources, http://www.bioquest.org/summer2006/resources.php

Centers for Disease Control and Prevention, Sexually transmitted disease information, http://www.cdc.gov/std/default.htm

 

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C.        Forensics

 

1. Murder case

 

A woman has been brutally stabbed to death outside of her home.  Two suspects have been arrested - 1) her ex-husband, whom the deceased woman claimed had been stalking her in the two months prior to her death, and 2) an acquaintance of her ex-husband who had been living in the ex-husband’s house for about six months and who could not provide an alibi for the time of the murder.  Blood samples are taken from the crime seen - one spot found near the victim’s body and one taken from a glove found near the crime scene. 

 

DNA is isolated from these blood spots, as well as from blood samples taken from the victim and the two suspects.  Each DNA sample is subjected to PCR analysis, amplifying a polymorphic region of chromosome 1. (NOTE:  Use the PCR function of the Data Screen, rather than 96-well PCR.)  Digesting this amplified DNA with HindIII will yield distinctive banding patterns that should help identify the source of the blood spots from the crime scene.

 

DNA samples:      blood spot 1 (from sidewalk)                             

                                            blood spot 2 (from glove)                                      

                                            victim’s blood

                                            suspect 1 (ex-husband)

                                            suspect 2 (acquaintance)

 

(Note:  There are three versions of this scenario, Case A, B, and C, each with a different outcome.)

 

        a.     What conclusions can you draw from these results? 

        b.     Do you think these data are sufficient to convict someone?

        c.     What additional issues are raised by this type of testing?

 

 

2.  Thomas Jefferson / Sally Hemings case

 

Background

 

There has long been controversy regarding whether Thomas Jefferson fathered any children with Sally Hemings, one of his slaves.  Jefferson was accused of fathering two of Hemings’ sons: Thomas Woodson, who was born in 1802 shortly after Jefferson and Hemings returned from an extended stay in France, and Eston Hemings Jefferson (born 1808), who bore a striking resemblance to Jefferson and took his name as an adult.  No known documentation either directly supports or refutes these claims.  Recently, researchers in the United Kingdom have attempted to address these questions scientifically by analyzing DNA from the Y chromosome of male descendants of Jefferson’s uncle, Jefferson’s sister, and Hemings.  Thomas Jefferson himself had no undisputed, surviving sons.

 

Most of the Y chromosome passes unchanged from father to son, except for occasional mutations.  Several Y chromosome genetic markers, some of which are genes while others are non-coding, can be used for this analysis since they can be inherited in one of two allelic forms (called bi-allelic).  The alleles are detected by dot blot analysis using probes that will bind to one allele or the other.  It is possible to determine whether two individuals are closely related by comparing how frequently their alleles match.  Another type of marker that can be used in this analysis is microsatellite short tandem repeats (microsatellite STRs).  These are regions where short (2-3 base pairs) sequences are repeated.  The number of repeats is inherited like an allele, and can be determined by the size of the band detected on a gel after PCR amplification of that region.

 

The Case

 

DNA was isolated from the following individuals (numbers correspond to reference numbers used in the study):

 

H21 - Eston Hemings Jefferson’s great-great grandson

 

W55, W70 - Thomas Woodson’s great-great grandson

 

J41, J47, J49 - Descendants of Field Jefferson, Thomas Jefferson’s uncle

 

C27, C31 - Descendants of John Carr, Thomas Jefferson’s nephew

 

To determine which allele each individual has at each of the Y chromosome bi-allelic markers, use the appropriate primers to amplify the DNA by PCR (use the PCR function on the Data Screen rather than 96-well PCR) and then perform a dot blot using the probes for that marker.    Load the probes into the spots and the amplified DNA into the corresponding wells.  To detect microsatellite STRs, use the appropriate primers to amplify the DNA and then run the PCR products on a gel to determine the relative sizes of the fragments.

 

                Bi-allelic markers                                  Microsatellite STRs

                                YAP                                                        19

                                sY81                                                       389A

                                92R7                                                       389D

                                SRY                                                        392

 

a.        What can you conclude from the dot blot and gel results?  Which individuals appear to be the most closely related?

b.       Are these results consistent with Thomas Jefferson having fathered either Easton Hemings Jefferson or Thomas Woodson?

c.        Are there other ways to interpret these results?

 

 

 

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C.  Phylogenetic studies

 

1.   Primate relationships

        (suggested by Rick Berken, East High School, Green Bay, WI)

 

Compare hemoglobin genes from human, chimpanzee, and gorilla to determine how closely related these species are.  Two types of analyses can be performed:

 

a.     Digest each DNA sample with restriction enzyme(s) (choose one or a combination)  and compare the fragment patterns generated.  Are the patterns for one pair of species more similar than another pair  (e.g. is gorilla more similar to chimp or to human)?   How many different enzymes do you need to use in order to yield reliable data?

 

b.     After digestion, perform a Southern blot with one of the hemoglobin probes from chimp.  With the probe stringency (match) set at 100%, does the probe hybridize to DNA from either of the other species?  If not, how much do you have to reduce the stringency before the probe hybridizes to the other samples?

 

       

2.   Squirrel taxonomy

        (contributed by Steven Rice, Wake Forest University, Winston-Salem, NC)

 

In this example you will compare mitochondrial cytochrome b sequences from various squirrel populations.  Cytochrome b is an integral part of the mitochondrial electron transport system.  One DNA sample is from Sciurus aberti aberti, the tassel-eared squirrel that resides in Arizona, extending to the southern rim of the Grand Canyon.  DNA samples also are available for individuals from a different subspecies, Sciurus aberti ferreus, and also from another species in the genus, Sciurus niger.  The former is an individual of the Kaibab squirrel that has been isolated on the north rim of the Grand Canyon.  The latter is a fox squirrel that is common in the midwest. 

 

Open each DNA sample, digest the DNA fragments with the AluI enzyme, load each into a different well and run the gel.  Use a short run time (10 minutes).

 

•       Which of the types had similar restriction fragments? 

•       How do these differences compare with what you would expect based on the taxonomic differences among the individuals? 

 

 

 

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D.     Simulation of wet labs

 

These were developed to be used along with electrophoresis labs, to prepare students for the lab and/or to allow extensions of the lab activity.  They also could be used in place of the lab if time or equipment is not available.

 

1.   Digestion of Lambda DNA

        (contributed by Brack Gillespie, Ashwaubenon High School, Ashwaubenon, WI and Rick Berken,          

        East High School, Green Bay, WI)

 

This is a standard lab activity to illustrate the basic features of restriction enzyme digestions.  DNA isolated from bacteriophage Lambda is digested with common restriction enzymes - EcoRI, BamHI, HindIII - to demonstrate that enzymes with different recognition sites will yield different band patterns on a gel. 

 

Activity extensions: