Molecular Genetics

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 / Specimen sample / Turn Around Time / Referral Price/JDs
Amnio PCR for aneuploidy screening of chromosomes 13, 18, 21, X and Y, QF-PCR / Amniotic fluid / 2 – 3 days / 120
Cardio Vascular Disease Risk Factors, 12 Mutations Profile, PCR / EDTA whole blood / 7 days / 60
Cystic Fibrosis CFTR Gene Mutations (36 mutations), Amniotic fluid / Amniotic fluid / 17 days / 190
Cystic Fibrosis CFTR Gene Mutations (36 mutations), Blood / EDTA whole blood / 7 days / 90
Cystic Fibrosis CFTR Gene Mutations (36 mutations), CVS / Chorionic Villus Sampling (CVS) / 17 days / 190
Duchenne and Becker Muscular Dystrophy (DMD,BMD), Genetic Analysis / EDTA whole blood / 18 – 20 days / 150
Duchenne and Becker Muscular Dystrophy (DMD,BMD), Genetic Analysis / Amniotic fluid / 30 days / 250
Duchenne and Becker Muscular Dystrophy (DMD,BMD), Genetic Analysis / Chorionic Villus Sampling (CVS) / 30 days / 250
Factor II G20210A Prothrombin Gene Mutation (for CVD and Recurrent Abortion) / EDTA whole blood / 4 days / 25
Factor V G1691A (Leiden) Gene Mutation ( for CVD and Recurrent Abortion) / EDTA whole blood / 4 days / 25
Familial Mediterranean Fever (12 mutations) / EDTA whole blood / 4 days / 68
Hereditary Hemochromatosis HFE Gene Mutations (H63D, S65C, C282Y), PCR / EDTA whole blood / 4 days / 50
HLA ABC, Blood, PCR / EDTA whole blood / 7 days / 110
HLA B27, Blood, PCR / EDTA whole blood / 4 days / 30
HLA B5, Blood, PCR / EDTA whole blood / 4 days / 30
HLA DR, Blood, PCR / EDTA whole blood / 4 days / 50
Human Oncogenes, Breast Cancer genes (BRCA1, BRCA2), Gene Analysis, PCR / EDTA whole blood / 3 weeks / 350
MTHFR C677T (for CVD and Recurrent Abortion) / EDTA whole blood / 4 days / 25
Spinal Muscular Atrophy (SMA), Genetic Analysis / EDTA whole blood / 18 – 20 days / 150
Spinal Muscular Atrophy (SMA), Genetic Analysis / Amniotic fluid / 30 days / 250
Spinal Muscular Atrophy (SMA), Genetic Analysis / Chorionic Villus Sampling (CVS) / 30 days / 250
Y Chromosome Microdeletions (6 deletions), PCR / EDTA whole blood / 7 days / 60
α-Globin Gene, α-Thalassemia, (21 mutations), PCR, Amniotic Fluid / Amniotic fluid / 7 days / 170
α-Globin Gene, α-Thalassemia, (21 mutations), PCR, Blood / EDTA whole blood / 7 days / 170
α-Globin Gene, α-Thalassemia, (21 mutations), PCR, CVS / Chorionic Villus Sampling (CVS) / 7 days / 170
β-Globin Gene, β-Thalassemia, (22 mutations), PCR, Amniotic Fluid / Amniotic fluid / 17 days / 175
β-Globin Gene, β-Thalassemia, (22 mutations), PCR, Blood / EDTA whole blood / 7 days / 75
β-Globin Gene, β-Thalassemia, (22 mutations), PCR, CVS / 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.

Genetic Tests / Specimen sample / Turn Around Time / Referral Price/JDs
Qualitative Assays
H1N1 Influenza Virus RNA / Nasal swabs, nasopharyngeal 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
Human Papilloma Virus DNA Genotyping / Body fluids, swabs, warts / 4 days / 70
Chlamydia trachomatis / Body fluids, swabs / 4 days / 35
Neisseria gonorrhea / Body fluids, swabs / 4 days / 25
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 pre-implantation 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