KAPLAN_USMLE_STEP_1_LECTURE_NOTES_2018_BIOCHEMISTRY_and_GENETICS

Chapter 6 ● Genetic Diagnosis

Indirect genetic diagnosis using STRPs

Suppose there is a 3-generation family in which Marfan syndrome is being transmitted. Each family member has been typed for a 4-allele STRP that is closely linked to the disease locus. The affected father in generation I transmitted the disease-causing mutation to his daughter, and he also transmitted allele 3 of the marker. This allows us to establish linkage phase in this family.

Because of the close linkage between the marker and the disease locus, we can predict accurately that the offspring in generation III who receive allele 3 from their mother will also receive the disease-causing mutation. Thus, the risk for each child, instead of being the standard 50% recurrence risk for an autosomal dominant disease, is much more definitive: nearly 100% or nearly 0%.

The genotype of a closely linked marker locus is shown below each individual.

Figure II-6-4. Three-Generation Family in Which

Marfan Syndrome Is Being Transmitted

Recurrence risks may have to take into account the small chance of recombination between the marker allele and the disease-causing gene. If the STR and the disease-causing gene used in this case show 1% recombination, then the recurrence risk for a fetus in generation III whose marker genotype is 2,2 would be 1% rather than 0%. If a fetus in generation III had the marker genotype 2,3, the recurrence risk for that child would be 99%.

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Indirect genetic testing using RFLPs

If an RFLP is used as a marker for a disease-causing gene, the data may be analyzed by using Southern blotting and a probe for the gene region.

A man and a woman seek genetic counseling because the woman is 8 weeks pregnant, and they had a previous child who died in the perinatal period. A retrospective diagnosis of long-chain acyl-CoA dehydrogenase (LCAD) deficiency was made based on the results of mass spectrometry performed on a blood sample. The couple also has an unaffected 4-year-old daughter with a normal level of LCAD activity consistent with homozygosity for the normal LCAD allele. The parents wish to know whether the current pregnancy will result in a child with the same rare condition as the previous child who died. DNA samples from both parents and their unaffected 4-year-old daughter are tested for mutations in the LCAD gene. All test negative for the common mutations. The family is then tested for polymorphism at a BamII site within exon 3 of the LCAD gene by using a probe for the relevant region of this exon. The RFLP marker proves informative. Fetal DNA obtained by amniocentesis is also tested in the same way. The results of the Southern blot are shown below. What is the best conclusion about the fetus?

Figure II-6-5. RFLP Analysis for LCAD

(Answer: Fetus is homozygous for LCAD mutation and should be clinically affected)

Although RFLP analysis can be used as both an indirect test and a direct test, there is a significant difference between the two situations.

In the direct test, the mutation causing the disease is the same as the one that alters the restriction site. There is no distance separating the mutations and no chance for recombination to occur, which might lead to an incorrect conclusion.

In the indirect assay, the mutation in the restriction site (a marker) has occurred independently of the mutation causing the disease. Because the mutations are close together on the chromosome, the RFLP can be used as a surrogate marker for the disease-producing mutation. Linkage phase in each family must be established. Because the RFLP and the locus of the disease-producing mutation are some distance apart, there is a small chance for recombination and incorrect conclusions.

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RFLP analysis for an X-linked disease

Individual II-2 in the family shown below has Lesch Nyhan disease. His sister, II-4, is pregnant and wants to know the likelihood that her child will be affected. The mutation in this family is uncharacterized, but is mapped to within 0.05 cM of an EcoR1 site that is informative in this family. DNA from all family members is obtained. Fetal DNA is obtained by chorionic villus sampling. What is the best conclusion about the fetus?

I

II

III

Figure II-6-6. RFLP Analysis of HGPRT Deficiency in a Family

(Answer: Fetus (a girl) will not be affected; nor will she be a carrier because her mother, II-4, is not a carrier)

Direct versus Indirect Genetic Diagnosis

Direct genetic diagnosis is used whenever possible. Its major limitation is that the disease-producing mutation(s) must be known if one is to test for them.

If a family carries a mutation not currently documented, as in the family above with LCAD deficiency, it will not be detected by direct mutation testing. In these cases, indirect genetic testing can be used.

Table II-6-1. Features of Indirect and Direct Genetic Diagnosis

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APPLICATIONS OF GENETIC DIAGNOSIS

Genetic diagnosis is used in a variety of settings, including the ones listed below.

•Carrier diagnosis in recessive diseases

•Presymptomatic diagnosis for late-onset diseases

•Asymptomatic diagnosis for diseases with reduced penetrance

•Prenatal diagnosis

•Preimplantation testing

Prenatal Genetic Diagnosis

Prenatal diagnosis is one of the most common applications of genetic diagnosis. Diagnosis of a genetic disease in a fetus may assist parents in making an informed decision regarding pregnancy termination and in preparing them emotionally and medically for the birth of an affected child. There are various types of prenatal diagnosis.

Amniocentesis

With amniocentesis, a small sample of amniotic fluid (10–20 mL) is collected at approximately 16 weeks’ gestation. Fetal cells are present in the amniotic fluid and can be used to diagnose single-gene disorders, chromosome abnormalities, and some biochemical disorders. Elevated α-fetoprotein levels indicate a fetus with a neural tube defect. The risk of fetal demise due to amniocentesis is estimated to be approximately 1/200.

Chorionic villus sampling

This technique, typically performed at 10–12 weeks’ gestation, involves the removal of a small sample of chorionic villus material (either a transcervical or a transabdominal approach may be used). The villi are of fetal origin and thus provide a large sample of actively dividing fetal cells for diagnosis. This technique has the advantage of providing a diagnosis earlier in the pregnancy. There is a small possibility of diagnostic error because of placental mosaicism (i.e., multiple cell types in the villi). The risk of fetal demise is higher than with amniocentesis (about 1/100).

Preimplantation diagnosis

Embryos derived from in vitro fertilization can be diagnosed by removing a single cell, typically from the eight-cell stage (this does not harm the embryo). DNA is PCR amplified and is used to make a genetic diagnosis. The advantage of this technique is that pregnancy termination need not be considered: only embryos without the mutation are implanted. There is a possibility of diagnostic error as a result of PCR amplification from a single cell.

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Review Questions

Select the ONE best answer.

1.The pedigree below shows a family in which hemophilia A, an X-linked disorder, is segregating. PCR products for each member of the family are also shown for a short tandem repeat polymorphism located within an intron of the factor VIII gene. What is the best explanation for the phenotype of individual II-1?

I

II

A.Heterozygous for the disease-producing allele

B.Homozygous for the disease-producing allele

C.Homozygous for the normal allele

D.Incomplete penetrance

E.Manifesting heterozygote

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2.A 22-year-old woman with Marfan syndrome, a dominant genetic disorder, is referred to a prenatal genetics clinic during her tenth week of pregnancy. Her family pedigree is shown below (the arrow indicates the pregnant woman). PCR amplification of a short tandem repeat (STR) located in an intron of the fibrillin gene is carried out on DNA from each family member. What is the best conclusion about the fetus (III-1)?

I

II

III

A.Has a 25% change of having Marfan syndrome

B.Has a 50% chance of having Marfan syndrome

C.Will develop Marfan syndrome

D.Will not develop Marfan syndrome

E.Will not develop Marfan syndrome, but will be a carrier of the disease allele

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3.The pedigree below represents a family in which phenylketonuria (PKU), an autosomal recessive disease, is segregating. Southern blots for each family member are also shown for an RFLP that maps 10 million bp upstream from the phenylalanine hydroxylase gene. What is the most likely explanation for the phenotype of II-3?

I

II

A.A large percentage of her cells have the paternal X chromosome carrying the PKU allele active

B.Heteroplasmy

C.Male I-2 is not the biologic father

D.PKU shows incomplete penetrance

E.Recombination has occurred

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4.A 14-year-old boy has Becker muscular dystrophy (BMD), an X-linked recessive disease. A maternal uncle is also affected. His sisters, aged 20 and 18, wish to know their genetic status with respect to the BMD. Neither the boy nor his affected uncle has any of the known mutations in the dystrophin gene associated with BMD. Family members are typed for a HindII restriction site polymorphism that maps to the 5′ end of intron 12 of the dystrophin gene. The region around the restriction site is amplified with a PCR. The amplified product is treated with the restriction enzyme HindII and the fragments separated by agarose gel electrophoresis. The results are shown below. What is the most likely status of individual III-2?

I

II

III

115 bp

75 bp

40bp

A.Carrier of the disease-producing allele

B.Hemizygous for the disease-producing allele

C.Homozygous for the normal allele

D.Homozygous for the disease-producing allele

E.Manifesting heterozygote

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5.Two phenotypically normal second cousins marry and would like to have a child. They are aware that one ancestor (great-grandfather) had PKU and are concerned about having an affected offspring. They request ASO testing and get the following results. What is the probability that their child will be affected?

Man Woman

ASO Normal allele

ASO Mutant allele

A.1.0

B.0.75

C.0.67

D.0.50

E.0.25

6.A 66-year-old man (I-2) has recently been diagnosed with Huntington disease, a late-onset, autosomal dominant condition. His granddaughter (III-1) wishes to know whether she has inherited the disease-producing allele, but her 48-year-old father (II-1) does not wish to be tested or to have his status known. The grandfather, his unaffected wife, the granddaughter, and her mother (II-2) are tested for alleles of a marker closely linked to the huntingtin gene on 4p16.3. The pedigree and the results of testing are shown below. What is the best information that can be given to the granddaughter (III-1) about her risk for developing Huntington disease?

A.50%

B.25%

C.Marker is not informative

D.Nearly 100%

E.Nearly 0%

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