Genetic Diagnosis

Disease Diagnosis
Prenatal diagnosis
Carrier testing
Preimplantation
Population screening program
diagnosis
Neonatal
Testing healthy members of
screening/diagnosis
the families with genetic
Diagnosis in patients diseases
Presymptomatic
diagnosis for genetic
disorders with late onset
Diagnostic tools:
Biochemical tests
Enzymatic assay
Ultrasound
DNA analysis

DNA analysis
Direct testing for mutation
Linkage analysis in family

A first case of genetic disease in a family

DNA analysis:
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

in family
disorders, e.g. cystic Linkage
fibrosis, analysis
PKU, Tay-Sachs
disease etc.

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

A polymorphic marker and a gene are linked when the
marker is located inside the gene or in close proximity
The chance for crossing over is very low
STR

STR

NF1
NF1

If the marker is located far from the gene the

chance of crossing over is increasing
STR

NF1

Linkage analysis in a family with

Neurofibromatosis type I (autosomal dominant)
Disease-causing gene is known: NF1
Is fetus II-4
affected ?

II-1

II-2

II-3

II-4
11

8
5
3

II
?

STR closely
located (linked)
to the NF1 gene
is genotyped

Genotype
Father

3/8

Mother

5/11

II-1

8/11

II-2

8/5

II-3

3/5

II-4

3/11

Paternal
chromosomes

al3

NF1

al8

NF1

II-1

II-2

II-3

II-4
11

II
?

5
3

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 8
Therefore the mutant NF1 gene is on the chromosome with
allele 3
Fetus II-4 inherited the paternal chromosome with the
allele 3 and therefore is affected

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. affected
B. a heterozygous carrier
C. a normal homozygote

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 carrier
Sibs 5 is a normal homozygote
Sibs 6 is affected

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?

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

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?

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%.

The marker is not informative in this family.

How might diagnostic accuracy be

improved in this case?

The marker is not informative in this family.

How might diagnostic accuracy be

improved in this case?
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.

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 diseaseproducing 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 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?

Lesch Nyhan is X-linked recessive disease

Healthy
(A) Carrier of the diseaseproducing 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 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?
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

Kaplan

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?
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

Kaplan

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?

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 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 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

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
Q 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 diseaseproducing allele
(B) Hemizygous for the diseaseproducing allele
(C) Homozygous for the normal
allele
(D) Homozygous for the diseaseproducing allele
(E) Manifesting heterozygote

A
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

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 114and 36-bp fragments. The PCR product(s) is visualized directly by
gel electrophoresis. Based on the results shown below in Figure 116-3 using this assay on DNA samples from this family, what is the
most likely conclusion about sibling 2?

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 114and 36-bp fragments. The PCR product(s) is visualized directly by
gel electrophoresis. Based on the results shown below in Figure 116-3 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

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) 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) 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

If marker is located far from the gene the

chance of crossing over is increasing
RFLP

RFLP+

RFLP+

RFLP-

Normal PAH gene

Mutant PAH gene

Normal PAH gene

Mutant PAH gene

Optional
slide

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 dominant
extended CAG repeat
anticipation (earlier onset in subsequent
generation)
late onset
100% penetrance
dementia
no cure

Optional
slide

Huntington disease:
prenatal or preimplantation fetus selection (slide1)
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
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

1,2

3,4
?
4,4

STR or other
markers close
to the HD gene

Optional
slide

Huntington disease:
prenatal or preimplantation fetus selection (slide 2)
Genotypes
1,4 and 2,4
include STR
alleles inherited
from
grandfather and
therefore there
is a risk for HD

1,2

3,4

4,4

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