MOLECULAR GENETICS
Introduction
In recent years, the field of medical genetics has been expanding our knowledge in the
involvement of genes and chromosomes with certain diseases and disorders. It is now
well established that genetic aspects are responsible for a variety of birth defects,
chronic diseases and a high percentage of mental retardation.
The Molecular Genetics Laboratory at Biolab provides comprehensive DNA-based
diagnostic testing for a variety of genetic conditions and diseases, as well as prenatal
diagnosis, presymptomatic, predictive and carrier testing.
A new test, the Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR), has been
introduced to perform rapid prenatal diagnoses of common chromosome aneuploidies.
The clinical utility of this assay has repeatedly been confirmed together with its high
sensitivity and specificity in detecting major chromosome abnormalities. One of the
advantages of QF-PCR is the automation of part of the procedure that allows high
throughput of samples; this makes rapid prenatal diagnosis available to all pregnancies
either as a tool to reduce parental anxiety while waiting for completion of fetal
karyotype or to improve pregnancy management in case of abnormal result.
The Molecular Genetics Laboratory combines state-of-the art genetic testing run by a
team of qualified consultants, skilled specialized scientists working in a professional
environment dedicated to provide quality diagnostic services constantly aligned with
the rapidly growing field of laboratory medicine.
Indications for Molecular Genetics Testing
Common reasons for referral of molecular genetic tests
Thrombosis Screen
 For the evaluation of inherited thrombotic risk within families.
 Transient ischemic attack or premature stroke
 Hyperhomocysteinemia, low plasma folate levels.
 Prior to pregnancy, oral contraceptive prescription, estrogen therapy or major
surgery if there is a family history of thrombosis
 Recurrent abortion.
Single Gene Disorders
1. Recessive Disorders
e.g. Cystic Fibrosis, Familial Mediterranean Fever, Hereditary Hemochromatosis,
Spinal Muscular Atrophy
 Carrier testing and risk assessment in families
2. Dominant Disorders
e.g. Osteogenesis Imperfecta
 Confirmation or exclusion of a diagnosis
3. X – Linked Disorders
e.g. Duchenne Muscular Dystrophy, Fragile X Syndrome
 Carrier testing and risk assessment in families
Fertility Testing
e.g. Y- Chromosome Microdeletions, Cystic Fibrosis
 Azoospermia
 Confirmation or exclusion of a diagnosis
 Carrier testing and risk assessment in families
Hemoglobinopathies
e.g. alpha-Thalassemia, beta-Thalassemia, Sickle Cell Anemia
 Carrier testing and risk assessment in families
Tissue Typing and Rh Genotyping
 Organ transplant
 Maternal-fetal Rh incompatability which can cause Alloimmune Hemolytic
Disease in the fetus or newborn.
Oncology
e.g. Breast Cancer Genes 1 and 2 (BRCA1 and BRCA 2),
Philadelphia
Chromosome bcr/abl Translocation
 Carrier testing and risk assessment in families
 Presymptomatic testing in individuals at risk of late-onset genetic disorder (patient
may present positive family history or positive preliminary testing).
Prenatal Testing
Prenatal diagnosis is available for the following genetic disorders:
 Cystic Fibrosis
 α-Thalassemia
 β-Thalassemia
 Sickle Cell Anemia
 Duchenne and Becker Muscular Dystrophies
 Fragile X Syndrome
 Spinal Muscular Atrophy
 AmnioPCR by QF-PCR for aneuploidy screening of chromosomes 13,18, 21, X, and
Y
AmnioPCR by QF-PCR (QUANTITATIVE FLUORESCENT POLYMERASE CHAIN REACTION)

AmnioPCR by QF-PCR is a rapid screening for the detection of aberrant copy
numbers of human chromosomes 13, 18, 21, X and Y in small uncultured amniotic
fluid samples. This is not equivalent to a full chromosome analysis and is always
followed by a full karyotype.

AmnioPCR by QF-PCR can be undertaken for rapid sexing on amniotic fluid. Useful
when suspecting Turner syndrome, or where there is a risk of sex linked single gene
disorders.
Limitations of Molecular Genetics Testing
 Reliable results are dependent on adequate sample collection, transport, storage
and processing procedures.

Prenatal diagnosis is affected by maternal cell contamination in amniotic fluid and
may therefore complicate the interpretation of test results.

On rare instances, technical limitations associated with some of the technologies
used may be present.

The diagnostic efficiency of the test can be impeded by drug interference.

Heparin inhibits the PCR reaction, therefore samples collected in heparin tubes will
be rejected and a repeat assay requested.
Molecular Genetics Tests
Genetic Tests
Amnio PCR for aneuploidy screening of
chromosomes 13, 18, 21, X and Y, QF-PCR
Cardio Vascular Disease Risk Factors, 12 Mutations
Profile, PCR
Cystic Fibrosis CFTR Gene Mutations (36
mutations), Amniotic fluid
Cystic Fibrosis CFTR Gene Mutations (36
mutations), Blood
Cystic Fibrosis CFTR Gene Mutations (36
mutations), CVS
Duchenne and Becker Muscular Dystrophy
(DMD,BMD), Genetic Analysis
Duchenne and Becker Muscular Dystrophy
(DMD,BMD), Genetic Analysis
Duchenne and Becker Muscular Dystrophy
(DMD,BMD), Genetic Analysis
Factor II G20210A Prothrombin Gene Mutation (for
CVD and Recurrent Abortion)
Factor V G1691A (Leiden) Gene Mutation ( for
CVD and Recurrent Abortion)
Familial Mediterranean Fever (12 mutations)
Hereditary Hemochromatosis HFE Gene Mutations
(H63D, S65C, C282Y), PCR
HLA ABC, Blood, PCR
HLA B27, Blood, PCR
HLA B5, Blood, PCR
HLA DR, Blood, PCR
Human Oncogenes, Breast Cancer genes (BRCA1,
BRCA2), Gene Analysis, PCR
MTHFR C677T (for CVD and Recurrent Abortion)
Spinal Muscular Atrophy (SMA), Genetic Analysis
Spinal Muscular Atrophy (SMA), Genetic Analysis
Specimen
sample
Turn
Around
Time
Amniotic fluid
2 – 3 days
120
EDTA whole
blood
7 days
60
Amniotic fluid
17 days
190
7 days
90
17 days
190
18 – 20
days
150
30 days
250
30 days
250
4 days
25
4 days
25
4 days
68
4 days
50
7 days
110
4 days
30
4 days
30
4 days
50
3 weeks
350
4 days
25
EDTA whole
blood
Chorionic Villus
Sampling (CVS)
EDTA whole
blood
Amniotic fluid
Chorionic Villus
Sampling (CVS)
EDTA whole
blood
EDTA whole
blood
EDTA whole
blood
EDTA whole
blood
EDTA whole
blood
EDTA whole
blood
EDTA whole
blood
EDTA whole
blood
EDTA whole
blood
EDTA whole
blood
EDTA whole
blood
Amniotic fluid
Referral
Price/JDs
18 – 20
days
150
30 days
250
Spinal Muscular Atrophy (SMA), Genetic Analysis
Chorionic Villus
Sampling (CVS)
Y Chromosome Microdeletions (6 deletions), PCR
EDTA whole
blood
7 days
60
Amniotic fluid
7 days
170
7 days
170
α-Globin Gene, α-Thalassemia,
PCR, Amniotic Fluid
α-Globin Gene, α-Thalassemia,
PCR, Blood
α-Globin Gene, α-Thalassemia,
PCR, CVS
β-Globin Gene, β-Thalassemia,
PCR, Amniotic Fluid
β-Globin Gene, β-Thalassemia,
PCR, Blood
(21 mutations),
(21 mutations),
(21 mutations),
(22 mutations),
(22 mutations),
β-Globin Gene, β-Thalassemia, (22 mutations),
PCR, CVS
EDTA whole
blood
Chorionic Villus
Sampling (CVS)
30 days
250
7 days
170
Amniotic fluid
17 days
175
EDTA whole
blood
7 days
75
Chorionic Villus
Sampling (CVS)
17 days
175
MICROBIAL GENETICS
Introduction
Emerging infections are attracting greater attention from the public health and medical
communities. Pathologists and other physicians are increasingly aware of the importance
of the subspecialty of infectious disease pathology as a tool for diagnosis, monitoring
therapy and prognosis as well as research of emerging infections.
In view of the progress in the science of molecular biology, the applications of diagnostic
microbial genetics has taken a major role in the way clinicians manage their patients
utilizing an array of technologies such as PCR, RT-PCR, and Gene sequencing.
The microbial genetics laboratory provides a list of essential molecular bacteriological
and viral tests that can detect, quantify and genotype these pathogenic organisms.
Indications for Microbial Genetics Testing
Common reasons for referral of Microbial Genetic tests
 Confirm or exclude a diagnosis of an infectious disease
 Monitor viral load in established infected individuals and evaluate the efficacy of
antiviral treatment
 Assess reactivation of infection in transplant patients and evaluate potential
disease transmission from prospective donors to tissue transplant recipients
 Diagnosis of sexually transmitted diseases
Limitations of Microbial Genetics Testing
 Reliable results are dependent on adequate sample collection, transport, storage
and processing procedures.
 Heparin inhibits the PCR reaction, therefore samples collected in heparin tubes will
be rejected and a repeat assay requested.
 Any diagnostic test has its limits with regards to sensitivity and detection limit.
 In rare instances, the diagnostic efficiency of some tests can be impeded by drug
interference.
Turn
Around
Time
Referral
Price/JDs
Nasal swabs,
nasopharynge
al swabs,
serum, plasma
4 days
50
Mycobacterium tuberculosis
Body fluids
4 days
25
Herpes Simplex Virus I &II DNA
Body fluids,
swabs
4 days
30
Body fluids,
swabs, warts
4 days
70
Chlamydia trachomatis
Body fluids,
swabs
4 days
35
Neisseria gonorrhea
Body fluids,
swabs
4 days
25
Specimen
sample
Genetic Tests
Qualitative Assays
H1N1 Influenza Virus RNA
Human Papilloma Virus DNA Genotyping
Quantitative Assays
Hepatitis C Virus RNA, Quantitative
Serum
4 days
60
Hepatitis C Virus RNA, Genotyping
Serum
4 days
80
Hepatitis B Virus DNA, Quantitative
Serum
4 days
60
CYTOGENETICS
Introduction
Cytogenetic techniques allow for the unambiguous identification of each human
chromosome
and
the
detection
of
aneuploidy
and
many
large
structural
rearrangements that cause physical and / or mental retardation, congenital anomalies
related to inherited conditions, abnormal sexual development, and other defects. New
techniques allow for increased resolution of chromosome banding patterns, permitting
differentiation of a greater number of abnormalities. Fluorescent in situ Hybridization
(FISH) technique (using Chromosome- Gene- or Translocation- specific DNA probes) is
used in the detection of genetically important trisomies, diagnosis and classification of
various neoplasia and hematological malignancies as well as prenatal and preimplantation diagnosis.
The Department's activities fall into three main sectors, namely prenatal diagnosis
(amniotic fluid, fetal blood, chorionic villus samples, product of conception, fetal
biopsies), constitutional cytogenetics (peripheral blood) and onco-hematological
cytogenetics (bone marrow, lymph nodes, solid tumors, and pleural effusions).
Indications for Cytogenetics Testing
CHROMOSOMAL KARYOTYPING
Post-Natal Blood Cytogenetics Testing
 Dysmorphology/multiple congenital abnormalities suggestive of a chromosome
abnormality
 Ambiguous genitalia/indeterminate gender
 Unexplained learning difficulties/developmental delay
 Delayed puberty, gynecomastia or inappropriate secondary sexual development
 Short stature, amenorrhea in females
 Oligospermia or azoospermia in males
 Parental karyotyping after two or more unexplained pregnancy loss.
 Parental karyotyping after pregnancy loss of an unkaryotyped fetus with multiple
congenital abnormalities or intrauterine growth restriction (IUGR)
 Family history (first degree relatives) of a known chromosome abnormality other
than simple aneuploidy due to non-disjunction
 Suspected family history of chromosome abnormality where the karyotype of the
affected individual is not known
 Conditions such as growth failure, microcephaly, neurological abnormalities,
immunodeficiency, and an increased incidence of malignancy
Postnatal Cytogenetic Testing on Skin Biopsies and Other Tissues
 Karyotyping of skin biopsy or other types of tissues from a live patient is usually
performed to investigate the possibility of tissue-specific chromosomal mosaicism.
This is usually undertaken once a karyotyping has been obtained on a blood
sample.
Prenatal Cytogenetic Testing (Amniotic Fluid, Chorionic Villus Sampling)
 High risk of carrying fetus with chromosomal abnormality that may have been
detected as a result of a screening test (first or second trimester screen).
 Intracytoplasmic sperm injection (ICSI) or other medical intervention that increases
the probability of chromosome abnormalities.
 Abnormal ultrasonographic findings indicative of chromosome abnormality.
 History of chromosome abnormality indicative of increased risk for future
pregnancies (Chromosome abnormalities in either parent, a previous pregnancy
or the family history)
 Augmented nuchal translucency
Cytogenetic Testing of Fetal/Placental Tissue after Pregnancy Loss
 Any fetus, stillbirth or neonatal death with congenital abnormality suggestive of a
chromosome anomaly or with neural tube defect or with intrauterine growth
restriction (IUGR).
 Unexplained stillbirth or spontaneous abortion.
 Determine recurrence risk for future pregnancy losses.
FLUORESCENT IN SITU HYBRIDISATION (FISH)
Rapid Neonatal Aneuploidy Screening by FISH

Rapid neonatal screening by FISH for trisomies 13, 18 and 21 and for sex. This is not
equivalent to a full chromosome analysis and is always followed by a full
karyotyping.
Cytogenetic Testing of Bone Marrow and Unstimulated Blood

Chronic Lymphocytic Leukemia (CLL)

Acute Lymphocytic Leukemia (ALL)

Acute Myeloid Leukemia (AML)

Myelodysplastic Disorder (MSD)

Multiple Myeloma (MM)

Lymphoma

Cytopenia
Cytogenetic Testing of Solid Tumors

Ewing Sarcoma

Neuroblastoma

Lymphoma

Hepatoblastoma

Germ Cell Tumor

Rhabdomyosarcoma

Synovial Sarcoma

Breast Carcinoma
Limitations of Cytogenetics Testing
Small Subtle Chromosome Abnormalities
 Cytogenetic analysis relies on G-band quality and resolution. In general, blood
samples give the best quality chromosomes and therefore provide the best
chance of detecting small subtle chromosome abnormalities. Chromosomes from
other tissues (e.g. amniotic fluid, chorionic villus and products of conception) give
poorer quality chromosomes; hence the risk of missing a subtle abnormality is
increased.
 It should be noted that on rare occasions a subtle abnormality may be missed at
prenatal diagnosis, only to be diagnosed later on a postnatal blood sample.
 It should also be understood that even a G-band blood karyotype can never
exclude extremely subtle chromosome abnormalities that are at the limit of
resolution of light microscopy.
Detection of Mosaicism
 It is well established that although mosaicism may be detected by routine
karyotyping it can never be 100% excluded. However, if there is an indication of
suspected mosaicism, additional cells will be examined to exclude 10% mosaicism
at a 95% confidence level.
Interpretation of Mosaicism in Prenatal Diagnosis
 True mosaicism, when detected prenatally, can be difficult to interpret and a
further invasive diagnostic test may be required. Mosaic cell lines may be
unevenly distributed between the fetus and extra-fetal tissues leading to false
positive and false negative results in the most extreme cases. ‘Confined placental
mosaicism' (CPM) is observed in approximately 1-2% of CVS samples.
 Pseudomosaicism can arise as an artifact of culture and is not representative of
the fetal karyotype. This is normally present in only one of two or three
independently established cultures and can therefore be interpreted accordingly.
In most cases, no further invasive testing is required.
Maternal Cell Contamination in Prenatal Diagnosis
 Maternal cell contamination of chorionic villus and amniotic fluid occurs in
approximately 1/250 samples and may occasionally complicate the interpretation
of results.
Normal Variation
 Each chromosome pair has a specific and identical G-banding pattern in all
individuals. However, variation may occur around the centromeric regions and
short arms of some chromosomes that is of no clinical significance. These
variations are known as ‘polymorphic variants’, ‘polymorphisms’ or ‘normal
variants’.
 In a few cases, it is necessary to distinguish between such variation and a true
abnormality. It may therefore be necessary to karyotype the parents or to carry
out further tests on a repeat sample.
On rare instances, technical limitations associated with some of the technologies used
may be present.
Cytogenetics Tests
Karyotyping Requirements
Specimen
Quantity
Container
Maximum Transit
Time
Turnaround Time
Referral
Price
Peripheral
blood
2 – 4 ml
Li-Heparin bottle (sterile,
vacutainer, plastic)
48 hours
7 days
43
Fetal
blood/Cord
0.5 – 1 ml
Li-Heparin bottle (sterile,
plastic)
48 hours
7 days
43
Bone marrow
and
unstimulated
blood
2 - 5 ml
Li-Heparin bottle (sterile,
plastic)
24 hours
5 days
55
Amniotic Fluid
15 – 20 ml
Universal container or 16
ml conical tube (sterile,
plastic)
24 hours
14 days
75
Chorionic
Villus
Sampling
2 - 25 mg
Universal container
(sterile, plastic). In sterile
media.
24 hours
14 days
80
Products Of
Conception
Variable
Universal container
(sterile, plastic). In sterile
saline.
24 hours
14 days
80
Fetal
tissue/skin
1 cm3
Universal container
(sterile, plastic). In sterile
saline
48 hours
14 days
80
Fragile X
2 - 4 ml
Li-Heparin bottle (sterile,
vacutainer, plastic)
48 hours
7 days
60
Chromosomal
Breakage
(Fanconi's
anemia)
2 - 4 ml
Li-Heparin bottle (sterile,
vacutainer, plastic)
48 hours
7 days
70
FISH Requirements
Tests
Quantity
Container
Maximum
Transit
Time
Average
Turnaround
Time
Referral
Price
Amniotic Fluid, Blood, Chorionic Villus Sampling (CVS)
Trisomy 13 [Patau Syndrome]
55
Trisomy 18 [Edward’s Syndrome]
55
Amniotic
Fluid in
Universal
container
or conical
tube,
(sterile,
plastic)
Trisomy 21 [Down’s Syndrome]
Rapid sexing ( X and Y )
Prader Willi
3 ml
Digeorge
Miller Dieker
Li-Heparin
bottle /
EDTA
whole
blood
(sterile,
plastic)
55
55
Within
24 hours
1-2
working
days
200
400
CVS in
media
(sterile,
plastic)
Trisomy 21, XY
200
95
Trisomy 13, 18 and 21
130
Trisomy 13, 18, 21 and XY
155
Preimplantation Genetic Diagnosis (PGD)
PGD
Fixated
Blastomere
Salinated
Slide
N/A
Within 24
hours
50 For each
embryo
Bone Marrow and Unstimulated Blood
BCR/ABL: t(9;22) [CML/ALL]
70
GLI: (12q13) [CLL]
100
100
ATM: (11q22) [CLL]
p53: (17p13) [CLL]
DLEU: 13q14 [CLL]
ETO/AML: t(8;21) [AML/M2]
1 - 2 ml
Li-Heparin
bottle
(sterile,
plastic)
100
24 hours
2 - 3 days
100
100
CBFB: inv(16) [AML/M4]
100
PML/RARA: t(15;17) [AML/M3]
100
TEL/AML: t(12;21) [ALL]
100
MLL: 11q23 [ALL/AML]
100
Deleted q5: [MDS]/[AML]
100
Deleted q7: [MDS]/ [AML]
100
LSI/MYC: t(8q24) [Burkitt lymphoma]
(screening)
100
MYC/IGH: t(8;14) [Burkitt lymphoma]
100
BCL1/IGH: t(11;14) [Mantle cell lymphoma]
100
IGH/BCL2: t(14;18) [Follicular lymphoma]
100
LSI/ALK: t(2p23) [Anaplastic large cell
lymphoma]
100
Solid Tumors
270
HER-2: [Breast carcinoma]
EWSR: [Ewing sarcoma]
N-MYC: [Neuroblastoma]
FKHR: 13q14 [Alveolar rhabdomyosarcoma]
SYT: 18q11.2 [Synovial sarcoma]
N/A
Paraffin
embedded
block
270
N/A
3 – 5 days
270
270
270