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190
Board Review Series Genetics
D.
Sickle Cell Disease.
1.
Neonatal genetic screening utilizes a small amount of blood obtained from a heel prick to
assay hemoglobin S (HbS) levels using HPLC or isoelectric focusing.
2.
Neonates with hemoglobins that suggest sickle cell disease or other hemoglobinopathies
are recalled for evaluation by six weeks of age.
3.
Molecular genetic testing is used clinically for: confirmatory diagnostic testing, carrier
testing, and prenatal diagnosis. Molecular genetic testing methods include:
a.
Targeted mutation analysis.
PCR-based and restriction fragment length polymorphism
(RLFP) methods can be used to detect point mutations in the HBB gene which include:
E6V mutation associated with HbS, E6K mutation associated with HbC, E121Q muta-
tion associated with HbD, and E121K mutation associated with HbO.
b.
Gene sequence analysis.
HBB gene sequence analysis is used if targeted mutation analy-
sis is uninformative or to identify HBB gene mutations associated with ß-thalassemia
hemoglobin variants.
4.
Sickle cell disease has a very high carrier frequency in African, Mediterranean, Middle
Eastern, Indian, Caribbean, and portions of Central and South American populations. In
particular, sickle cell disease has a 1 in 12 carrier frequency in the African American pop-
ulation and a 1 in 4 carrier frequency in the west central African population. Carrier test-
ing can be done using HPLC, isoelectric focusing, and DNA based assays.
5.
Prenatal genetic testing is available by DNA analysis of fetal cells obtained by amniocentesis
(at L15 to 18 weeks of gestation) or chorionic villus sampling (at L 12 weeks of gestation).
HBB gene mutations must be identified in both parents before prenatal testing can be done.
The indications for family genetic screening are high-risk individuals or couples due to a posi-
tive family history of:
■
An autosomal dominant genetic disorder with reduced penetrance or late onset (e.g.,
Huntington disease)
■
Familial adenomatous polyposis coli
■
Hereditary nonpolyposis colorectal cancer) where heterozygotes can be identified
■
An autosomal recessive genetic disorder (e.g., cystic fibrosis) where heterozygotes (or
carriers) can be identified
■
A X-linked recessive disorder (e.g., Duchenne muscular dystrophy) where female het-
erozygotes (or carriers) can be identified
■
Chromosomal rearrangement (e.g., translocation).
A. Huntington Disease (HD).
1.
Family genetic screening utilizes a commercially available kit to determine the CAG repeat
number
for each allele.
2.
Asymptomatic at-risk adults seek presymptomatic family genetic screening in order to
make personal decisions regarding careers, financial estates, reproductive decisions, or
just the “need to know.”
3.
Reproductive decisions may be aided by knowledge of disease status and can provide
assurance to those who do not have the mutation.
4.
Genetic testing of asymptomatic at-risk adults does not accurately predict the exact age of
onset, severity, type of symptoms, or the rate of progression. However, the size of the CAG
trinucleotide repeat does generally correlate with age of onset.
5.
Extensive pre- and posttesting counseling is required to address issues like: disability
insurance coverage, employment discrimination, educational discrimination, changes
family interactions, depression, and suicide ideation.
6.
Genetic testing of asymptomatic at-risk individuals
18 years of age is generally not rec-
ommended.
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7.
Prenatal genetic testing for pregnancies at 50% risk is available by DNA analysis of fetal
cells obtained by amniocentesis (at L15 to 18 weeks of gestation) or chorionic villus sam-
pling (at L12 weeks of gestation).
B. Familial Adenomatous Polyposis Coli (FAPC).
1.
Molecular genetic testing is used clinically for: confirmatory diagnostic testing, predictive
testing, and prenatal diagnosis. Molecular genetic testing methods include:
a.
Mutation scanning.
Mutation scanning identifies point mutations in the APC gene.
Mutation scanning is performed by denaturing HPLC, which is a fast and efficient
method to detect locus-specific point mutations.
b.
Gene sequence analysis.
APC gene sequence analysis is used to identify mutations.
c.
Protein truncation testing.
Protein truncation testing identifies premature truncation of
the APC protein.
d.
Duplication/deletion analysis.
MLPA identifies large duplications and deletions when no
APC gene mutations have been identified by mutation scanning or full gene sequence
analysis.
2.
Genetic testing of asymptomatic at-risk adults and children can be used with certainty
when a clinically diagnosed relative undergoes genetic testing and the specific mutation
in the APC gene is identified.
3.
When a clinically diagnosed relative is not available, failure to identify an APC gene dis-
ease-causing mutation in the at-risk adult or child does not eliminate the possibility that
an APC gene disease-causing mutation is present in the at-risk adult or child.
4.
Those with mutations in the APC gene can benefit from early diagnosis. With regular mon-
itoring by colonoscopy beginning at L10 years of age, polyps can be removed so they can-
not develop into malignancies.
5.
Prenatal genetic testing for pregnancies at 50% risk is available by DNA analysis of fetal
cells obtained by amniocentesis (at L15 to 18 weeks of gestation) or chorionic villus sam-
pling (at L12 weeks of gestation). The APC disease-causing mutation in an affected family
member must be identified before prenatal testing is performed.
C. Cystic Fibrosis (CF).
1.
Molecular genetic testing is used clinically for: diagnosis in symptomatic individuals, car-
rier testing, and prenatal diagnosis. Molecular genetic testing methods include:
a.
Targeted mutation analysis.
A targeted mutation analysis pan-ethnic panel is available for
23 common CFTR gene mutations.
b.
Gene sequence analysis.
CFTR gene sequence analysis is used to identify mutations.
c.
5T/TG tract analysis.
A poly T tract (a string of thymidine bases) and a TG tract (a string of
TG repeats) are associated with CFTR-related disorders.
d.
Duplication/deletion analysis.
MLPA identifies large duplications and deletions when no
CFTR gene mutations have been identified by targeted mutation analysis or full gene
sequence analysis.
2.
CF has a 1 in 28 carrier frequency in Caucasian populations, 1 in 61 in the African
American population, and 1 in 29 in the Ashkenazi Jewish population. Carrier testing can
be done using the pan-ethnic 23-mutation panel and 5T/TG tract analysis. CF carrier test-
ing is recommended for non-Jewish Caucasians and Ashkenazi Jews. CF carrier testing is
also offered as routine prenatal care in some centers.
3.
Prenatal genetic testing for pregnancies at 50% risk is available by DNA analysis of fetal
cells obtained by amniocentesis (at L15 to 18 weeks of gestation) or chorionic villus sam-
pling (at L12 weeks of gestation). CFTR gene mutations must be identified in both parents
before prenatal testing can be done.
4.
When CFTR gene mutations are identified in both parents, pregnancy can be achieved
through assisted reproductive technology. In this case, preimplantation genetic testing is
available using embryonic cells obtained from a multicell stage embryo during the in vitro
fertilization technique.
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D. Duchenne Muscular Dystrophy (DMD).
1.
Molecular genetic testing is used clinically for: diagnosis in symptomatic individuals, car-
rier testing, and prenatal diagnosis. Molecular genetic testing methods include:
a.
Targeted mutation analysis.
PCR-based, Southern blotting, and FISH methods can be
used to detect deletions in the DMD gene. Southern blotting and quantitative PCR can
be used to detect duplications.
b.
Mutation scanning.
Mutation scanning identifies small deletions, small insertions, point
mutations, or splicing mutations in the DMD gene.
c.
Gene sequence analysis.
DMD gene sequence analysis also identifies small deletions,
small insertions, point mutations, or splicing mutations.
d.
Duplication/deletion analysis.
MLPA identifies large duplications and deletions in probands
and carrier females.
2.
DMD has a 1 in 4,000 carrier frequency in the US population although it is difficult to cal-
culate because L33% of DMD cases are new mutations. When the DMD gene mutation of
the proband is known, carrier testing can be performed by real-time PCR, FISH, or gene
sequence analysis. When the DMD gene mutation of the proband is not known, linkage
analysis can be offered to at-risk female to determine carrier status in families with more
than one affected DMD male.
3.
Prenatal genetic testing for pregnancies of carrier mothers is available if the DMD gene
mutation has been identified in a family member or if linkage has been established. The
usual procedure is to determine fetal sex by karyotype by DNA analysis of fetal cells
obtained by amniocentesis (at L15 to 18 weeks of gestation) or chorionic villus sampling
(at L12 weeks of gestation). If the karyotype is 46, XY, the DNA can be analyzed for the
known DMD gene mutation.
VII. POPULATION GENETIC SCREENING
Population genetic screening is the systematic application of a test in a population to identify
high-risk individuals who have genotypes that may lead to genetic disorders in themselves or
their descendants and have not sought medical attention for the disorder. The implementation
of a population genetic screening program is a very involved logistical endeavor, which
requires:
■
Financial, staffing, and technological resources
■
A workable solution to introduce the program into the population
■
A method to monitor the outcomes
■
Quality control assurance
Some examples of population genetic screening are discussed below.
A.
Tay-Sachs Disease.
1.
Molecular genetic testing is used clinically for: confirmatory diagnosis, carrier testing,
and prenatal diagnosis. Molecular genetic testing methods include:
a.
Targeted mutation analysis.
A targeted mutation analysis panel is available for 6 common
HEXA gene mutations: 3 null alleles (
TATC1278, 1IVC12, 1IVS9), G269S allele, and
2 pseudodeficiency alleles (R247W and R249W).
b.
Mutation scanning.
Mutation scanning identifies HEXA gene mutations in individuals
who are affected or have carrier-level enzyme activity but do not have a mutation iden-
tified by the above-mentioned panel.
c.
Gene sequence analysis.
HEXA gene sequence analysis is used to identify HEXA gene
mutations in individuals who are affected or have carrier-level enzyme activity but do
not have a mutation identified by the above-mentioned panel.
2.
Tay-Sachs disease has a 1 in 30 carrier frequency in Ashkenazi Jewish population. Carrier
testing can be done by demonstrating the absence of hexosaminidase A activity (but normal
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hexosaminidase B activity) using serum or white blood cells or by direct detection of HEXA
gene mutations.
3.
Options available to carrier couples are pregnancy termination and artificial insemina-
tion with noncarrier donors. In some Orthodox Jewish groups, carriers are forbidden to
marry.
4.
Prenatal genetic testing is available by DNA analysis or hexosaminidase A assay of fetal
cells obtained by amniocentesis (at L15 to 18 weeks of gestation) or chorionic villus sam-
pling (at L12 weeks of gestation). HEXA gene mutations must be identified in both parents
before prenatal testing can be done.
5.
Tay-Sachs disease is especially prevalent in the Ashkenazi Jewish population. In this regard,
the implementation of genetic screening programs and education in the Ashkenazi Jewish
population have led to a 90% decline in Tay-Sachs disease births.
B.
Alpha-Thalassemia.
1.
When suspicion for
-thalassemia is high, the following screening tests can be used: red
blood cell indices, peripheral blood smear, red blood cell supravital staining of peripheral
blood, quantitative and qualitative hemoglobin analysis.
2.
Molecular genetic testing is used clinically for: diagnostic testing, carrier testing, predic-
tion of clinical severity, and prenatal diagnosis. Molecular genetic testing methods
include:
a. Targeted mutation analysis.
PCR-based methods can be used to detect deletions in the
HBA1 gene and HBA2 gene. PCR primer panels that are targeted to the most common
HBA1 gene and HBA2 gene mutations within the geographical location of the proband
are generally used. Southern blotting can be used to detect less common or novel dele-
tions.
b. Gene sequence analysis.
HBA1 gene and HBA2 gene sequence analysis is used to iden-
tify point mutations when suspicion for
-thalassemia is high and a deletion mutation
is not detected.
3.
-Thalassemia has a very high carrier frequency in the African, Mediterranean, Arabic,
Indian, and Southeast Asian populations. Carrier testing can be done initially by red blood
cell indices, peripheral blood smear, red blood cell supravital staining of peripheral blood,
quantitative and qualitative hemoglobin analysis. Suspicions can be confirmed by molec-
ular genetic testing to detect HBA1 gene and HBA2 gene mutations.
4.
Prenatal genetic testing is available by DNA analysis of fetal cells obtained by amniocentesis
(at L15 to 18 weeks of gestation) or chorionic villus sampling (at L12 weeks of gestation).
HBA1 gene and HBA2 gene mutations must be identified in both parents before prenatal
testing can be done. If the known HBA1 gene or HBA2 gene mutation is present in the fetus,
globin chain synthesis analysis can be performed by percutaneous umbilical blood sam-
pling (at L18 to 21 weeks) especially in indeterminate-risk pregnancies. Ultrasonography
demonstrating increased nuchal thickness or a large pleural effusion should lead to further
prenatal genetic testing. Prenatal genetic testing is usually influenced by factors such as reli-
gion, culture, education, and number of children in the family.
5.
-Thalassemia is especially prevalent in the African, Mediterranean, Arabic, Indian, and
Southeast Asian populations. In this regard, the implementation of genetic screening pro-
grams has been very effective in decreasing the incidence in
-thalassemia disease births.
Screening of children, pregnant women, and individuals visiting public health facilities,
premarital screening programs, and restrictions on issuance of marital certificates have
also been effective.
C.
Cystic Fibrosis (CF) Carrier Screening.
1.
The most pressing issue in CFTR gene carrier screening by target mutation analysis is not
only the
1,000 known mutations but also the differences in mutant alleles in different
ethnic populations.
2.
Carrier testing using the pan-ethnic panel for 23 common CFTR gene mutations identifies
80% of Caucasian carriers.
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3.
However, carrier testing using the pan-ethnic panel for 23 common CFTR gene mutations
only identifies 70% of Hispanic carriers, 65% of African American carriers, and 50% of
Asian American carriers.
4.
This means that screening panels cannot identify all CFTR gene mutations even though
many screening panels are designed for specific populations. Consequently, a negative
screening panel for one or both of the partners does not exclude the possibility of a CF-
affected offspring. This is known as the residual risk.
5.
Recently developed multiplex PCR-based testing can identify many different mutant alleles
in the CFTR gene simultaneously in a single procedure and makes it possible to establish
population carrier screening for cystic fibrosis. Multiplex PCR-based testing identifies
90% of all carriers.
VI. METHODS USED FOR GENETIC TESTING
A detailed description of the methodology employed in each of the techniques listed below is
beyond the scope of this book. The specific details and descriptions of each of these techniques
can be easily found online using various search engines.
A.
DNA Sequencing.
B.
Southern Blotting.
C.
Restriction Fragment Length Polymorphism (RFLP) Analysis.
D.
Polyacrylamide Gel Analysis of Polymerase Chain Reaction (PCR) Products.
E.
Dot Blot Hybridization Using Allele Specific Oligonucleotides.
F.
Oligonucleotide Ligation Assay (OLA).
G.
PCR.
H.
ARMS-PCR.
I.
Real Time PCR.
J.
Multiplex PCR
K.
Multiplex Ligation-Dependent Probe Amplification (MLPA).
L.
DNA Microarrays (Chips).
M.
Denaturing High Pressure Liquid Chromatography (dHPLC).
N.
Conformation-Sensitive Capillary Electrophoresis (CSCE).
O.
High Resolution Melt (HRM) Curve Assay.
P.
Mass Spectroscopy.
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