Facilities at the Genetics and PGT Laboratory

Our state-of-the-art laboratory offers Cytogenetic tests such as Karyotyping and FISH (fluorescence in situ hybridization) as well as Molecular Genetic tests like PCR (Polymerase Chain Reaction) for many single gene disorders.

Cytogenetic tests are carried out on suspicion of a chromosome anomaly in newborns, children, adults, the unborn child in high-risk pregnancies or an aborted fetus (products of conception). They are also carried out in patients with blood cancer or leukemia and help in their diagnosis and management. Tissue culture is done in a majority of cytogenetic tests, hence strict aseptic precautions are necessary for sample collection. Karyotyping involves the analysis of all 23 pairs of chromosomes. The rapid FISH test is used when certain specific chromosome abnormalities are suspected. We also offer FISH testing on formalin-fixed paraffin-embedded (FFPE) tumour tissue sections and FISH for bladder cancer.

Molecular tests are carried out for different single gene disorders such as Y chromosome microdeletions (30 different loci), MTHFR and Prothrombin gene mutations, beta thalassemia mutations and Periodic Fever Syndromes.

Karyotyping is a detailed analysis of chromosomes to detect abnormalities of the number or structure of chromosomes. The chromosomes are obtained from peripheral blood, bone marrow aspirates, chorionic villi, amniotic fluid and different tissues from a miscarried fetus. These tissues are cultured for short and long term duration and chromosomes are obtained. The metaphases are fixed on slides, Geimsa banded, mapped and analyzed for abnormalities. We have the Zeiss/Metasystems automated karyotyping workstations where images of the metaphases are captured, processed and stored. Numerical and structural chromosome abnormalities such as trisomies, monosomies, deletions, duplications, inversions and translocations can be detected. Chromosomal breaks also can be analyzed using special techniques.

karyotype

Fluorescence in situ hybridization (FISH) is a rapid procedure to detect common chromosome abnormalities. We mainly use directly labeled commercially available FISH probes which are complementary to the region of interest on a particular chromosome. The analysis is usually done by counting coloured dots under a fluorescent microscope. The cells are pretreated and fixed on slides. The slides are dehydrated, co-denatured with an appropriate probe, hybridized, washed and mounted in antifade solution together with counterstain. The FISH signals are observed under a fluorescence microscope using appropriate filters and the images are captured and analyzed with the software.

FISH is a rapid technique to detect common aneuploidies, microdeletions and low-grade mosaicism mainly in infertility cases, prenatal diagnosis, products of conception and pediatric cases.

Common translocations, deletions, duplications in leukemia can be analysed for diagnosis and to determine response to therapy. Chromosome analysis by FISH is possible even on interphase nuclei.

Bladder cancer can be checked by FISH on urine cells.

We carry out FISH on FFPE Tumor tissue to check for HER2/neu amplification in case of breast cancer as well as for other cancers (various other genes).

Polymerase Chain Reaction (PCR) is an in vitro DNA amplification procedure that can quickly isolate and amplify a specific segment of DNA by as much as a billion fold. This involves several steps that allow a specific DNA sequence to be replicated several times and then rapidly analyzed by electrophoresis. We detect mutations like Y chromosome microdeletions by multiplex PCR in severe male factor infertility. PCR is also used in gene disorders like Beta Thalassemia, Periodic Fever Syndromes, mutation detection in the MTHFR and the Prothrombin gene.

Tests offered for infertility management:

  • Blood karyotyping for individuals with infertility / Poor Obstetric outcome / Repeated Spontaneous Abortions /Repeated Implantation Failure
  • Sperm DNA Fragmentation Index (DFI)
  • Sperm FISH for chromosomal aneuploidy (chromosomes 13,18, 21, X, Y or any other specific chromosome if required)
  • Y chromosome microdeletion detection (PCR for 30 mutations)
  • FISH for SRY (sex determining region Y) deletion/translocation
  • FISH on Follicular Fluid in case of IVF procedures
  • FISH for detection of low grade chromosomal mosaicism on different tissues such as blood, buccal cells, urine.
  • FISH for Sex Chromosome Mosaicism (Turner/Klinefelter Syndrome) from Blood /Buccal cells
  • FISH for presence or absence of SRY in case of XY female or XX male

Bad Obstetric History (BOH) Panel:

  • Blood karyotype with variants analysis
  • Products of conception (POC) karyotype
  • FISH for common chromosomal aneuploidies (13, 18, 21, X and Y) in POC
  • Low-grade mosaicism detection by FISH
  • FISH for Sex chromosome mosaicism (Turner/Klinefelter Syndrome)
  • FISH for presence or absence of SRY in case of XY female or XX male
  • MTHFR mutation analysis
  • Prothrombin mutation analysis

Post natal tests:

a) Karyotyping, Microdeletion Syndromes detection:

i) Blood karyotype

ii) FISH for aneuploidy and microdeletion syndromes

  • 13 & 21 and/or 18, X & Y
  • Down Syndrome/Trisomy 21
  • Edward Syndrome/Trisomy 18
  • Patau Syndrome/Trisomy 13
  • Prader Willi Syndrome
  • Angelman Syndrome
  • Deletion of SNRPN region
  • Williams Syndrome
  • DiGeorge Syndrome
  • Velocardiofacial Syndrome
  • Deletion 22q Syndrome /CATCH 22
  • Cri-du-chat Syndrome or 5p deletion
  • Wolf Hirschhorn Syndrome (WHS) or 4p deletion
  • SRY deletion/translocation
  • Low-grade mosaicism detection
  • Sex chromosome mosaicism (Turner/Klinefelter Syndrome)
  • Presence or absence of SRY in case of XY female or XX male

b) Chromosomal Breakage Studies:

Chromosome Breakage Study for

  • Ataxia telangiectasia
  • Fanconi’s Anemia /Chromosome stress test
  • Bloom’s/Cockayne Syndrome (Sister Chromatid Exchange Analysis)

c) Periodic Fever Syndrome / Autoinflammatory  Disorders testing:

  • FMF Testing (MEFV gene exon 10- 29 mutations)
  • FMF Testing (MEFV gene) exons 2, 5
  • Crohn’s Disease (NOD2/CARD15 genes) R702W, G908R, 1007fs
  • Crohn’s Disease (NOD2/CARD15 genes) exon 4 (parts 1-5)
  • Blau Syndrome (NOD2/CARD15) exon 4 (parts 1-5)
  • CAPS (CIAS1) exon 3 for MWS, FCAS and NOMID (parts 1 -4)
  • HIDS testing (MVK) exon 9 (I268T) and exon 11 (V377I)
  • TRAPS testing (TNFRSF1) exons 2, 3 (2+3), 4, 5 (4+5)
  • TRAPS Testing (TNFRSF1) exons 6, 7
  • DIRA (ILRN) sequencing exons 1-4
  • DIRA breakpoint assay 175kb deletion
  • DAD2 testing (CECR1) 9 exons

d) Other Molecular testing:

  • DNA extraction and storage from different tissues.
  • Beta thalassemia for 6 common Indian mutations
  • Beta thalassemia  for additional  14 common Indian mutations
  • Beta thalassemia for 20 common Indian mutations
  • HbS mutation detection for Sickle Cell Anemia
  • HbE mutation detection
  • MTHFR mutations C677T and A1298C
  • Prothrombin Gene mutation

Prenatal Genetic Diagnosis:

Karyotyping on

  • CVS
  • Amniotic Fluid
  • Cord Blood

FISH for

  • Aneuploidies (13, 18, 21, X and Y or 13, 21 or 18, X, Y and/or 16, 22)
  • Microdeletion syndrome (Uncultured cells or cultured metaphases)

Molecular testing for

  • Beta-thalassemia prenatal test with VNTR analysis for maternal cell contamination
  • HbS prenatal test with VNTR analysis for maternal cell contamination
  • CVS cleaning
  • DNA extraction with VNTR analysis for maternal cell contamination
  • Maternal cell contamination check for external DNA

Hematological Malignancies:

i) Bone Marrow Karyotyping

ii) FISH for

  • BCR-ABL/Philadelphia or Ph chromosome/t(9;22) for CML, AML
  • BCR-ABL/Philadelphia or Ph chromosome/t(9;22) for ALL
  • PML-RARA / t(15;17) / APML
  • RARA breakapart rearrangement
  • AML1-ETO  / RUNX1T1-RUNX1 / t(8;21)
  • Inversion 16 (CBFB)/t(16;16)
  • Burkitt’s Lymphoma / (8q24) /(c-Myc) rearrangement
  • MLL (11q23) rearrangement
  • IGH (14q32) rearrangement
  • Del 5q (EGR1)
  • Del 7q (D7S486)
  • Del 20q (20qter)
  • Trisomy 8
  • TEL-AML /ETV6-RUNX1 / t(12;21)
  • BCL2 (Non Hodgkins Lymphoma NHL)
  • RB1 (13q) deletion
  • Hyper-eosinophilic syndrome (4q12) (FIP1L1-PDGFRA)
  • Deletion MYB or deletion 6q
  • t(1;19) or TCF3/PBX1
  • t(3;3)/inversion 3/RPN1-MECOM
  • t(4;14)/ IGH-FGFR3
  • t(14;16) / IGH-MAF
  • t(14;18)/ IGH-BCL2
  • 1p loss/deletion and 1q gain /amplification
  • BCL6 breakapart rearrangement
  • PDGFRB breakapart rearrangement
  • TRAD/14q11
  • Multiple Myeloma on isolated CD138+ cells (Chr. 12, Del 13q, Del p53, Del ATM and IGH) / t(4;14) / t(14;16)
  • CLL (Chr. 12, Del 13q, Del p53 and Del ATM)
  • Post BMT XX/XY chimerism (Sex mismatched BMT)

Other malignancies:

FISH for

  • Bladder cancer/ hematuria by Urovysion panel
  • HER2/neu (ErbB2) on formalin fixed paraffin embedded (FFPE) tissue blocks for breast cancer / ovarian cancer / esophageal cancer
  • FFPE (formalin fixed paraffin embedded) tissues for other cancers

FAQs

What is Genetics?

Genetics is the study of heredity and genetic variation. It deals with hereditary diseases and birth defects.

What are the different types of Genetic Diagnostic Tests?

Genetic Diagnostic Tests can be broadly classified into:

  • Cytogenetic Tests – Karyotyping & FISH for chromosome analysis.
  • Molecular Genetic Tests – PCR, sequencing, microarray and Next Generation Sequencing for DNA analysis.
  • Biochemical Genetic Tests – To detect inborn errors of metabolism.

What are chromosomes?

Chromosomes are X shaped thread like structures made up of DNA, and carry the hereditary material of an individual. Different genes are located at specific points on chromosomes, which are visible under the microscope when the nucleus of a cell is dividing.

How many chromosomes are present in the cells of a human being?

Human beings have 46 chromosomes. These are present in pairs. This is termed as a diploid set of chromosomes. The sex chromosomes are XX in females and XY in males. The other chromosomes, besides the sex chromosomes are called autosomes. The ova and sperm however contain only 23 unpaired chromosomes each (a haploid set) so that when fertilization takes place, the cells of the embryo will again have 46 chromosomes.

What are the main types of chromosome abnormalities?

Chromosome abnormalities are mainly of two types.

  • Numerical – e.g. Trisomy, Monosomy, Triploidy, Tetraploidy, Mosaicism
  • Structural – e.g. Translocation, Deletion, Inversion, Duplication

What is trisomy?

Trisomy is the most common type of numerical chromosome abnormality. There is one extra chromosome in any pair e.g. Trisomy 21. These individuals suffer from Down syndrome. Thus the total number of chromosomes in each cell will be 47 instead of 46.

What is Down Syndrome?

Down Syndrome is a genetic disorder where the individual is usually mentally challenged. Such individuals have typical facial features like upslanting eyes, depressed nasal bridge, and an open mouth with a rough protruding tongue. They often have a single palmar crease or Simian crease on their palm. Trisomy 21, is the most common cause of Down syndrome.

What are the other causes of Down syndrome?

Down syndrome is occasionally caused by a translocation when 2 chromosomes have fused, so the total number of chromosomes remains 46 instead of 47. This can be inherited from a parent who is a carrier, having 45 chromosomes instead of 46 because of this fusion. In such cases, there is a risk that subsequent children may also be affected. Prenatal Diagnosis is important in these cases to determine if the fetus has Down syndrome. In the rare instance of a parent carrying a 21/21 translocation, all the children will have Down syndrome, so assisted reproduction with donor sperm or oocytes can be offered accordingly. This illustrates the importance of Karyotyping.

What is karyotyping?

Karyotyping is the process of chromosome analysis using banding techniques. The chromosomes seen under the microscope are arranged in pairs and scrutinized for any visible chromosome abnormalities. The method involves tissue culture to obtain dividing cells. Hence collection of appropriate samples under aseptic conditions is important.

What are the indications for Karyotyping?

  • Turner Syndrome (45,X and variants)
  • Klinefelter Syndrome (47,XXY and variants)
  • Down Syndrome (Trisomy 21, translocation and mosaicism)
  • Couples with infertility of unknown cause
  • Couples with recurrent spontaneous miscarriages
  • Children with ambiguous genitalia
  • Female children with inguinal hernia
  • Children with mental subnormality and dysmorphic features
  • Suspected cases of Fanconi anaemia, Ataxia Telangiectasia, Bloom syndrome
  • Bone marrow analysis in leukemias (blood cancer).
  • Prenatal Diagnosis of fetal chromosome disorders in high-risk pregnancies.

What is aneuploidy?

Aneuploidy is the presence of one extra chromosome (trisomy) or absence of one chromosome (monosomy) in each cell. This leads to different abnormalities such as:

  • Trisomy 21- Down syndrome or Mongolism
  • Trisomy 13 – Patau syndrome
  • Trisomy 18 – Edward syndrome.
  • Sex chromosome abnormalities- XXX, XXY, XO, XYY.

In products of conception (tissue from spontaneous abortions) trisomy 16 and trisomy 22 are quite common.

Common aneuploidies can be rapidly detected by a cytogenetic technique called Fluorescence in situ hybridization (FISH).

What is FISH?

FISH (Fluorescence in situ hybridization) is a rapid molecular cytogenetic technique. FISH is mainly used to detect common aneuploidies like trisomy or monosomy of chromosomes 21, 18 or 13, sex chromosome abnormalities like Turner (XO) or Klinefelter (XXY) syndrome and mosaicism where there is a mixture of normal and abnormal cells. This test is important in a variety of cases ranging from prenatal diagnosis to cancer. The test results are available in 1-2 days as tissue culture is not required. There is no risk of a culture failure due to contamination or inadequate sample size.

What are the benefits of FISH?

The FISH test has many benefits. It reduces parental anxiety especially in prenatal diagnosis when the Triple test shows a high-risk pregnancy, as the FISH report is available much earlier than the karyotype reports. A large number of interphase nuclei can be studied to detect low-grade mosaicism. The test can also be carried out on a wide range of samples like chorionic villi, amniotic fluid, cord/adult blood, placental biopsy, products of conception, buccal cells and sperm. FISH has wide applications in detecting the type of  cancer and in the prognosis of the disease.

What is mosaicism?

Mosaicism is the presence of chromosomally normal and abnormal cells in a person. In such cases, the clinical manifestation varies according to the percentage of normal and abnormal cells. Low-grade mosaicism, where the percentage of one of the cell-lines is very small, can be easily detected by FISH.

What are microdeletions?

Microdeletions are deletions of very small segments of chromosomes. They are often missed by karyotyping, but can be easily detected by FISH.  Prader-Willi /Angelman Syndromes may be caused by a microdeletion on chromosome 15.

How is FISH analysis done?

Commercially available DNA probes labeled with different fluorescent dyes are hybridized to the nuclei of cells, and analyzed under a fluorescence microscope. If the probe for chromosome 21 is labeled with an orange fluorescent dye, for example, we will see 2 orange signals under a fluorescence microscope in normal cells, and 3 orange signals in cells of an individual with Down syndrome (Trisomy 21). The diagnosis is thus made by counting the number of signals of different colours in each cell. The cells are counterstained with a blue dye DAPI, to differentiate the cells.

What are the limitations of FISH?

All chromosome abnormalities cannot be ruled out by FISH. Hence it cannot replace conventional karyotyping. FISH is useful to detect probe-specific abnormalities only.

Which are the common leukemias where FISH is useful?

FISH is commonly used in the rapid diagnosis, prognosis and management of chronic myeloid leukemia (CML) and acute promyelocytic leukemia (AML-M3), especially since specific treatment is available in each case. FISH is also used in many other hematological malignancies such as ALL, AML, MDS, Multiple Myeloma, CLL, Lymphomas and in solid tumors.

What is prenatal diagnosis?

Prenatal diagnosis is the detection of certain cytogenetic, molecular or biochemical genetic abnormalities in the unborn child.

When is prenatal diagnosis recommended?

Prenatal diagnosis is recommended in the following cases:

  • Advanced maternal age
  • Screening tests show high risk of abnormality
  • Abnormalities on ultrasonography
  • A parent with a balanced translocation
  • History of a previous abnormal child
  • X-linked genetic disorders
  • Couples with Thalassemia trait
  • Couples with family history of known monogenic disorders such as Beta Thalassemia
  • Cases in which NIPT (Non-Invasive Prenatal Testing) show a high risk for Trisomy 13, 18, 21.

Prenatal diagnosis can be carried out at what stage of pregnancy?

Prenatal diagnosis can be carried out in the 1st, 2nd or 3rd trimester depending on the stage at which an abnormality is detected or suspected. Different tissues are sampled according to the gestational age, e.g.

  • Chorionic villus (which forms the placenta) at 10-12 weeks
  • Amniotic fluid (which surrounds the fetus) at 16-18 weeks
  • Cord blood (from the umbilical cord) at greater than 19 weeks.

What are the requirements for pre-conception or prenatal diagnosis?

As per the PCPNDT (Prohibition of sex selection) Act 2003, the sample collection and analysis has to be carried out in registered clinics/ laboratories/centers as applicable after the mother has signed an informed consent form. The sex of the fetus is not revealed.

What is PCR?

PCR (Polymerase Chain Reaction) is a molecular diagnostic technique used to generate (amplify) large amounts of specific sequences of genes from DNA, for genetic analysis. It can be used to diagnose single gene disorders like beta-Thalassemia and Cystic Fibrosis, Sickle Cell Anaemia, Duchenne Muscular Dystrophy,  Metabolic Disorder in the new born such as Phenyl Ketonuria and Familial Hereditary Cancer Syndromes.

What are Y-Chromosome Microdeletions?

There are small regions on the Y chromosomes which are responsible for mature sperm formation. If a part or parts of the region is lost, it is noted as a deletion. These regions on the Y chromosome are too small to be detected through the microscope. These deletions can be detected by multiplex PCR technique. They are usually seen in males with azoospermia (No sperm found in the semen sample). Depending on which region is deleted, men with these deletions can father a child with Assisted Reproduction Techniques, but the couple has to be counselled that the male partner may transmit infertility to the male offspring.

What is PGT?

PGT is Preimplantation Genetic Testing. It is an additional step during the ICSI procedure, where trophectoderm cells from an embryo can be tested to rule out certain genetic conditions such as chromosomal aneuploidy, thus allowing only normal embryos to be transferred.

Can all genetic disorders be diagnosed or ruled out by these tests?

No. There are thousands of genetic disorders. Though recent technologies such as Next Generation Sequencing (NGS) and Chromosome Microarray (CMA) can pick up a large number of abnormalities, there are many others that need specific tests and some that may not be picked up on testing. You can visit our IVF center in Mumbai for consultation

What is Genetic Counseling?

During Genetic Counseling, the inheritance pattern of a particular genetic disorder in a family is studied. The chances of recurrence of the same abnormality in the family are explained. If genetic tests for diagnosis of that disorder are available, arrangements are made to send the required samples for testing. Prenatal diagnosis in a subsequent pregnancy in family members ‘at risk’ can be carried out. As most genetic disorders cannot be cured, genetic counseling helps to prevent the recurrence of the same disorder in the extended family.