Genetic Diagnosis

Disease Diagnosis Prenatal diagnosisPreimplantation

diagnosisNeonatal

screening/diagnosisDiagnosis in patientsPresymptomatic

diagnosis for genetic disorders with late onset

Carrier testingPopulation screening

program

Testing healthy members of the families with genetic diseases

Diagnostic tools:Biochemical

testsEnzymatic

assayUltrasoundDNA analysis

DNA analysisDirect testing for mutation

Linkage analysis in family

Linkage analysis in family

A first case of genetic disease in a family

Disease-causing gene is known:mutation analysis in the family (sequencing

coding region and splicing sites) to identify mutation

If a mutation is known then direct test for mutation

Such diagnostic service is available for many single-gene disorders, e.g. cystic fibrosis, PKU, Tay-Sachs disease etc.

DNA analysis:

However direct testing for mutation is not feasible for diseases with

very large genes and no common mutation and in such cases indirect

tests can be used

Linkage analysis in diagnosis

Linkage analysis in diagnosis

NF1STR

NF1STR

A polymorphic marker and a gene are linked when the marker is located inside the gene or in close

proximityThe chance for crossing over is very low

NF1STR

If the marker is located far from the gene the chance of crossing over is increasing

Linkage analysis in a family with Neurofibromatosis type I (autosomal

dominant) Disease-causing gene is known: NF1

F M II-1 II-2 II-3 II-4

?

11

8

53

GenotypeFather 3/8Mother 5/11II-1 8/11II-2 8/5II-3 3/5II-4 3/11

STR closely located (linked)

to the NF1 gene is

genotyped

I

II

Is fetus II-4 affected ?

NF1al3

NF1al8

?

F M II-1 II-2 II-3 II-4I

II

Affected father has the normal NF1 gene on one chromosome and the mutant NF1 on the other

The affected child (II-3) inherited from father the chromosome with the allele 3, while two healthy children (II-1 and II-2) inherited allele 8Therefore the mutant NF1 gene is on the chromosome

with allele 3Fetus II-4 inherited the paternal chromosome with the

allele 3 and therefore is affected

11

8

53

Paternal chromosomes

Linkage study in family which is used for diagnostic purpose

is not

Linkage analysis used for gene mapping

Individual 3 has PKU. A two-allele RFLP closely linked to the PKU locus has been assayed for each family member, and the figure shows the genotypes of each individual. The marker alleles are 5 kb and 3 kb in size. Based on the genotypes of the linked marker, what is correct for sibs 4, 5, 6? Answer the question separately for each sibs.

A. affectedB. a heterozygous carrierC. a normal homozygote

Q

Individual 3 has PKU. A two-allele RFLP closely linked to the PKU locus has been assayed for each family member, and the figure shows the genotypes of each individual. The marker alleles are 5 kb and 3 kb in size. Based on the genotypes of the linked marker, what is correct for sibs 4, 5, 6? Answer the question separately for each sibs.

Sibs 4 is a heterozygous carrierSibs 5 is a normal homozygoteSibs 6 is affected

A

The affected individuals have neurofibromatosis type 1 (NF1), an autosomal dominant condition. A four-allele microsatellite system closely linked to the NF1 locus has been typed for each family member. Based on the genotypes shown in the accompanying figure, will individual 6 develop NF1?

Q

The affected individuals have neurofibromatosis type 1 (NF1), an autosomal dominant condition. A four-allele microsatellite system closely linked to the NF1 locus has been typed for each family member. Based on the genotypes shown in the accompanying figure, will individual 6 develop NF1? Individual 6 is unaffected

A

In the pedigree for an autosomal dominant disorder shown above, a tightly linked two-allele RFLP has been typed in each family member. Based on this information, what can you tell the family about the risk that the offspring in generation III will develop the disorder?

Q

In the pedigree for an autosomal dominant disorder shown above, a tightly linked two-allele RFLP has been typed in each family member. Based on this information, what can you tell the family about the risk that the offspring in generation III will develop the disorder?

The marker is not informative in this family. The risk for a child is 50%.

A

How might diagnostic accuracy be improved in this case?

The marker is not informative in this family.

Q

How might diagnostic accuracy be improved in this case?

The marker is not informative in this family.

Use STR instead of RFLP marker. In contrast to two-allelic RFLP marker STR have many alleles

and therefore the higher chance to be informative.

A

Individual 11-2 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 EcoRl 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?

(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

Q

Individual 11-2 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 EcoRl 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?

HealthyLesch –Nyhan is X-linked recessive disease

(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

A

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 individuals II-1?

Kaplan

(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

I

II

Q

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 individuals II-1?

Kaplan

(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

I

II

A

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 BamW 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 in Figure 11-6-6. What is the best conclusion about the fetus?

Q

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 BamW 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 in Figure 11-6-6. What is the best conclusion about the fetus?

Fetus is affected

A

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)?

A. Has a 25% chance 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

Q

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)?

A. Has a 25% chance 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

A

14-year-old boy has Becker muscular dystrophy (BMD), an X-Iinked 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.

Is this linkage analysis or direct test for mutation?What is the most likely status of individuals III-1 ? What is the most likely status of individuals III-2 ?

(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

Q

Is this linkage analysis or direct test for mutation?It is linkage analysis

What is the most likely status of individuals III-1 ? Carrier of the disease-producing allele What is the most likely status of individuals III-2 ? Homozygous for the normal allele

A

A 14-year-old girl has been diagnosed with Gaucher disease (glucocerebrosidase A deficiency), an autosomal recessive disorder of sphingolipid catabolism. The mutation, TI448C, in this family also affects an HphI restriction site. PCR amplification of the area containing the mutation yields a 150-bp product. The PCR product from the normal allele of the gene is not cut by HphI. The PCR product of the mutant allele T1448C is cut by HphI to yield 114- and 36-bp fragments. The PCR product(s) is visualized directly by gel electrophoresis. Based on the results shown below in Figure 11-6-3 using this assay on DNA samples from this family, what is the most likely conclusion about sibling 2?

Q

A 14-year-old girl has been diagnosed with Gaucher disease (glucocerebrosidase A deficiency), an autosomal recessive disorder of sphingolipid catabolism. The mutation, TI448C, in this family also affects an HphI restriction site. PCR amplification of the area containing the mutation yields a 150-bp product. The PCR product from the normal allele of the gene is not cut by HphI. The PCR product of the mutant allele T1448C is cut by HphI to yield 114- and 36-bp fragments. The PCR product(s) is visualized directly by gel electrophoresis. Based on the results shown below in Figure 11-6-3 using this assay on DNA samples from this family, what is the most likely conclusion about sibling 2?

Sibling 2 is affected

In this question the direct test for

mutation is used

A

(A) A large percentage of her cells have the paternal X chromosome carrying the PKU allele active(B) Heteroplasmy(C) Male 1-2 is not the biologic father(D) PKU shows incomplete penetrance(E) Recombination has occurred

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?

Q

(A) A large percentage of her cells have the paternal X chromosome carrying the PKU allele active(B) Heteroplasmy(C) Male 1-2 is not the biologic father(D) PKU shows incomplete penetrance(E) Recombination has occurred

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?

A

Normal PAH geneRFLP-

Mutant PAH gene

If marker is located far from the gene the chance of crossing over is increasing

RFLP+ X

Normal PAH geneRFLP+

Mutant PAH geneRFLP-

Presymptomatic diagnosis in disorders with late onset

Huntington disease (HD)direct test for mutation ( sizing of CAG repeat)

Some family members want to know if they may later develop Huntington disease and ask for genetic test

Some family members do not want to know their mutation status

HD: autosomal dominantextended CAG repeatanticipation (earlier onset in subsequent

generation) late onset100% penetrance dementiano cure

Optional slide

A pregnant woman has a father with Huntington disease. She does not want to know her mutation status, but wants her future child be free of Huntington disease

Huntington disease: prenatal or preimplantation fetus selection

(slide1)

?

1,2 3,4

4,4

Direct test for the Huntington mutation can not be done

because finding the mutation in

a fetus automatically means that

the mother has the mutation

STR or other markers close to the HD gene

Optional slide

?

1,2 3,4

4,4

Genotypes 1,4 and 2,4 include STR alleles inherited from grandfather and therefore there is a risk for HD

Pregnancy is continued only if a fetus has STR allele from maternal grandmother

In case of in vitro fertilization only embryos with STR allele from maternal grandmother are used

Genotypes 3,4 and 4,4 include STR alleles inherited from grandmother and therefore there is no risk for HD

Huntington disease: prenatal or preimplantation fetus selection

(slide 2)

Optional slide