Preimplantation Genetic Testing (PGT)

Preimplantation Genetic Testing (PGT) is the earliest form of prenatal diagnosis, done on embryos. During the IVF cycle, a single cell or a few cells are removed (biopsied) from the embryo and are checked for specific genetic abnormalities. The embryos that are reported normal are transferred into the mother’s uterus. PGT offers a new alternative to chorionic villus sampling or amniocentesis. For any PGT testing, one needs to have multiple embryos developed by ICSI.

 

The steps involved are:

 

1. Medical evaluation of the couple to check the suitability for an IVF/ICSI cycle.

2. Genetic evaluation of the couple

3. The ICSI cycle.

4. Embryo biopsy which is followed by embryo cryopreservation.

5. Genetic testing of embryos (PGT)

6. Transfer of unaffected embryos

7. A Pregnancy test 14 days later.

 

Utility of PGT:

 

Improves pregnancy rates and decreases spontaneous abortion rates in:

1. Older age women

2. Previous unsuccessful IUI/IVF attempts

3. History of recurrent spontaneous miscarriages

4. Male factor infertility

5. Past history of repeated terminations because of an abnormality diagnosed during pregnancy

6. Known carriers of a genetic defect (e.g. Beta-Thalassemia, Sickle cell anemia, Cystic Fibrosis, Spinal Muscular Atrophy, Duchenne Muscular Dystrophy, Fragile X Syndrome, Huntington Chorea or a chromosomal translocation

 

Objectives:

 

• Prevents chromosomally or genetically abnormal births in couples having a history of failed fertilization after IVF-ICSI, missed abortions or carriers of balanced translocations.

• Increases the chances of implantation.

• Reduces the incidence of spontaneous miscarriages.

• Reduces the incidence of multiple offspring.

• Eliminates the risk of a child being affected with a familial gene disorder.

• A previously affected child can be cured by a Saviour Sibling (HLA matched sibling) who can then donate the stem cells to the affected child. eg. Thalassemia major.

 

Different techniques offered at our Centre:

 

  1. PGT (PGS or PGT-SR) by Fluorescence In Situ Hybridization (FISH) for telomeric/cryptic translocations and inversions

PGT by the FISH technique is now mainly carried out in cases where the husband or wife is a carrier of a balanced translocation or inversion. This is required to select embryos free of related unbalanced rearrangements. Pre-PGT work up of the couple using specific probes for the rearrangements is necessary to check for an additional tiny or cryptic rearrangement which may not have been noticed earlier. The embryo biopsy is done on Day-5. About 4-8 trophectoderm cells are biopsied from each embryo and are fixed on slides and subjected to the FISH procedure using probes specific for each couple, as per the chromosomes involved. The fluorescent signals are observed using fluorescent microscopy technique. A normal cell will show two signals of each colour for each chromosome. If there are 3 signals or 1 signal present, it indicates abnormal embryos which are not transferred to the mother.

 

Our team has >20 years of experience in such cases and has reported the first livebirths for Robertsonian /Reciprocal translocations and inversions in India.

 

  1. PGT for all 24 chromosome aneuploidies (PGS or PGT-A) using the array Comparative Genomic Hybridization (aCGH) technique: 

For 24 chromosome PGT, the biopsied cells are “tubed” and the DNA is amplified and tested for chromosomal aneuploidies using the microarray technique. Detection of all 24 chromosomes is possible on Day 5 of embryonic development. The results are available within 24 hours. The technique can also be used for detection of unbalanced translocations except those involving the telomeres (tips) of the chromosomes.

 

  1. PGT for all 24 chromosomal aneuploidies (PGS or PGT-A) using Next Generation Sequencing (NGS):

For PGT, the biopsied cells are “tubed”. The embryonic DNA is further subjected to a series of procedures and is finally sequenced using NGS technology to check for chromosomal aneuploidies. Detection of all 24 chromosomes is possible on Day 5 of embryonic development. The results are available within 24 hours.

 

4.  PGT for single gene disorders (PGT-M or PGD):

Whole genome amplification is carried out to increase the quantity of DNA externally in a PCR machine. Then the amplified product is subjected to further testing for the single gene disorder. The parental genetic markers are matched with the embryonic markers in order to make a diagnosis.

Prenatal Diagnosis (PND)

This involves genetic analysis of cells of fetal origin in various disorders by chorionic villus sampling, amniocentesis, or cord blood sampling, by karyotyping and FISH or molecular analysis such as chromosome microarray or DNA sequencing.

 

 

Indications:

 

  • Women with advanced maternal age
  • Previous child with a chromosome abnormality.
  • Women who have had previous still births or early neonatal deaths
  • Parents with balanced translocations
  • Cases in which the Biochemical Marker tests or NIPT (Non Invasive Prenatal Test) shows a high risk for Trisomy
  • Abnormalities seen on ultrasonography
  • Both husband and wife are carriers of a single gene autosomal recessive disorder such as Beta- Thalassemia

 

The FISH technique is very useful for rapidly detecting aneuploidies in high-risk pregnancies. A ‘high risk’ for Trisomy 21 by the Double marker / Quadruple Marker test or Ultrasonography causes a lot of anxiety to the expectant couple. This can be relieved in a day by FISH on uncultured CVS or amniotic fluid cells. Karyotyping is simultaneously done to rule out many structural and numerical chromosome abnormalities. If the quantity of chorionic villi obtained in a biopsy is insufficient for karyotyping, FISH is still possible from interphase nuclei.

 

 

Sample:

 

  • Amniotic fluid (8 ml for FISH; 15 ml for karyotyping; in 2-4 plain – red top – vacutainers). In case of a sanguineous tap, collect in heparin vacutainers.
  • Cord blood (1ml in sodium heparin – green top – vacutainer).
  • CVS in sterile normal saline with 2 drops of Gentamycin or in media tubes supplied by the laboratory.

Products of Conception (POC)

Missed abortions and still-births can be studied by karyotyping and FISH to detect chromosomal abnormalities. Spontaneous abortion samples show a high frequency of aneuploidy. Aneuploidy detection by FISH can include combinations of chromosomes 13, 21 / 18, X, Y / 16 / 22.

Karyotyping from ‘products of conception’ (POC) of a spontaneous or missed abortion is done by fibroblast culture to rule out a chromosome abnormality, which is a likely cause of the abortion.

 

Sample:

To avoid contamination of tissue, the sample from early abortions should be taken directly in a sterile Ovum Forceps or Menstrual Regulation (MR) syringe and transported in a sterile container with normal saline to which 2-3 drops of Gentamycin are added. The sac surrounded by chorionic villi is the tissue of choice in early abortions while a 1” piece of placenta and fetal skin could be collected from 2nd and 3rd trimester samples. Cord blood (0.5-1ml) could also be collected in a sodium heparin vaccutainer from stillborn fetuses for karyotyping.

The samples can be stored in the refrigerator overnight if required. Rapid transportation is required with a cool pack.

 

We have a high success rate of karyotyping from POC.

Postnatal Diagnosis

This involves analysis of samples such as blood, bone marrow, buccal cells, urine, formalin fixed paraffin embedded (FFPE) tumor tissue for various genetic disorders using different cytogenetic and molecular techniques like karyotyping, FISH, PCR and Sanger Sequencing.

 

Indications:

  1. Primary or Secondary Infertility
  2. Primary amenorrhea
  3. Ambiguous genitalia
  4. Developmental delay
  5. Dysmorphic features
  6. Microdeletion Syndromes
  7. Chromosomal breakage disorders like Fanconi anemia, Bloom Syndrome, Ataxia telangiectasia
  8. Hematological malignancies
  9. Breast / oesophageal / bladder Cancer and Lymphomas
  10. Hematological disorders like Beta thalassemia, MTHFR mutations, Prothrombin gene mutation
  11. Periodic Fever Syndromes / Auto-inflammatory Disorders

Genetic Counseling

Genetic counseling helps parents to understand a genetic disorder which may be picked up on prenatal diagnosis, or during investigation of an affected child and reach decisions about what to do next.

 

The family history is evaluated and the risks of recurrence in a subsequent pregnancy are explained, with information on available prenatal diagnostic tests. The possible cause of the condition is explained in order to reduce the feeling of guilt experienced by parents. Options of assisted reproductive techniques to bypass an inherited condition are discussed. Other family members needing prenatal diagnosis are identified and awareness given to try and prevent the birth of another affected child in the extended family.

 

Genetic counseling is given to the couple and family members wherever needed. The couples are guided on tests to be carried out to identify the disorder, and the availability of prenatal or preimplantation genetic testing (PGT) to prevent the birth of an affected child. They are also briefed about the consequences of genetic disorders and given information about support groups. A Pedigree Chart (Family Tree) is drawn based on the information given, to determine the pattern of inheritance and the risk of recurrence. eg. In Thalassemia, if there is an affected child, there is a 25% risk in every pregnancy that another child can also be affected. Therefore, prenatal diagnosis has to be carried out at 11-12 weeks from CVS, in every pregnancy in such cases, or they can opt for PGT before the next pregnancy.

 

If there is a history of recurrent miscarriages, karyotyping of the couple may show a balanced translocation in one of the partners, who is only a carrier. PGT (Preimplantation Genetic Testing) is available at our Centre for Robertsonian or Reciprocal translocations and Inversions, using the couple’s own gametes. About 5-6 trophectoderm cells from each embryo are tested and the abnormal embryos (unbalanced) are not transferred.

 

In case of a child suffering from a suspected genetic disorder, it is always advisable to test the DNA and identify the mutation, as it will help for prenatal diagnosis in the next pregnancy. This also applies to products of conception (POC) of recurrent miscarriages. In case the testing is not so affordable immediately, blood in an EDTA tube or a little fetal tissue in saline could be sent only for DNA extraction and storage. In an emergency, a little blood in EDTA could even be refrigerated for a few days prior to DNA extraction. This stored DNA of an affected child which may have expired later, comes in use when couples want to prevent recurrence of the same disorder in a subsequent pregnancy. Very often, couples come for genetic testing when the wife is already pregnant again and the affected child’s DNA was not tested nor stored. For accurate prenatal diagnosis, it is necessary to know the exact DNA mutation or variant in the earlier affected child, and then test the couple to determine the risk of recurrence. Ideally, genetic testing such as next generation sequencing should be carried out before planning a subsequent pregnancy, as the tests take time. Further targeted testing of other family members may also be necessary. Hence everyone needs to be aware of the importance of genetic testing.

 

Couple carrier screening is also available now. Some couples opt for this test prior to the 1st pregnancy itself even if there is no history of a genetic disorder in the family. This is because most of us are probable carriers of small changes in DNA called variants or mutations which we are not aware of, since we are apparently normal. As these changes are present on only one copy of a pair of chromosomes while the corresponding chromosome has the normal gene, we are carriers. A common example is Thalassemia. The aim of these genetic screening tests on our blood is to see that both the husband and wife are not carriers of a variant in the same gene, or different genes of a similar disease group such as blindness or deafness. In case both partners happen to be such carriers, there is a 25% chance in every pregnancy that their child will inherit 2 copies of this variant, one from each parent, and will be affected. Previously, the only option available was to carry out Prenatal diagnosis for that disorder around 11 weeks gestation at the earliest. If the fetus was affected, the couple could consider a medical termination of pregnancy.

 

Now-a-days, such couples have the option of Preimplantation Genetic Testing (PGT) of embryos during an IVF-ICSI cycle, at our centre. Though they do not have any issues of infertility, they can opt for IVF where a single sperm is injected into each ovum/egg to obtain embryos in the laboratory.  Those embryos which are growing well up to day 5 are biopsied and tested, while the embryos are frozen. Unaffected embryos can then be selected for transfer. This eliminates the trauma of a medical termination of an affected pregnancy in the second trimester, if there was a natural conception. Before implantation, the normal/unaffected embryos are further screened to check for abnormalities of all the 23 pairs of chromosomes, including Trisomy 21 or Down syndrome. Single embryo transfer of the normal embryos is preferred. The other normal embryos are kept frozen for later use, when required. We have had success with PGT for many disorders, both rare and common.

 

 

Genetic Counseling OPD: Monday – Friday 2.00-5.00pm; Saturday 1.00-3.00pm.

 

We have 2 well experienced genetic counsellers who guide the couples with a history of known genetic disorders.

 

Honorary Consultant Geneticist: Dr. Prochi F. Madon, Ph.D. (>40 years of experience in the field of Genetics)

Molecular cytogeneticist: Dr. Arundhati S. Athalye, Ph.D. (>20 years of experience in the field of Genetics)

 

Contact for appointments: +919820006336 (Direct) / +91-22-66573343

Image module
Image module

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) tumor tissues 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 workstation        Normal Male Karyotype            Translocation between chr 6 and 19

Fluorescence in situ hybridization (FISH) is a rapid procedure to detect certain 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 certain cases of 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

Infertility Management

Indications for Cytogenetic Analysis:

 

  • Couples with repeated spontaneous miscarriages
  • Couples with primary infertility
  • Males with oligozoospermia and azoospermia
  • Males with hypogonadism, breast development and lack of facial hair
  • Women with primary amenorrhoea, short stature, and streak gonads.

 

Sample: 2 ml Blood in sodium heparin vaccutainer (Green top tube), transported at room temperature. No fasting is required.

 

KARYOTYPING

.

Indication for Cytogenetics Analysis

Fresh semen sampleRoutine analysis of sperm chromosomes is not feasible in clinical practice. However, interphase FISH on spermatozoa offers an accurate and reliable method of analysis even in the presence of a low sperm count.

The percentage of sperm with hypo/hyperhaploidy (less chromosomes / more chromosomes) is calculated to counsel the couple.

 

Sample : Fresh / Frozen-thawed semen sample.

 

Outstation samples are also accepted.

 

Note: All samples should be accompanied with a copy of semen analysis report at the time of sample collection which should include the name of the patient, age, count, motility and morphology of the semen sample.

Y chromosome micro-deletions like DAZ (deletion in azoospermia) and RBM (RNA binding motif gene) cause spermatogenic defects. In the Yq arm (long arm of the Y chromosome), intervals V and VI comprise an Azoospermia Factor (AZF) which contains the DAZ and RBM genes. Several genes have been identified within this region and have been proposed candidates for infertility. We can check for 29 different micro-deletions including 5 partial deletions in the AZF region by multiplex PCR.

 

If Y chromosome microdeletions are seen only in the AZFc region, there is a good chance that TESA will be successful. However, if microdeletions are seen in the AZFa and AZFb regions, the chances of success after TESA are extremely low.

 

 

Multiplex PCR for Y microdeletion detection

The Sperm DNA Fragmentation Index (DFI) test is used to determine whether the male partner of an infertile couple has a high, fair, or poor fertility potential by studying fragmentation of his Sperm DNA.

 

SPERM APOPTOSIS BY TUNEL METHOD

Apoptosis is a mode of programmed cell death based on a genetic mechanism that induces a series of cellular, morphological and biochemical alterations, resulting in fragmentation of the genomic DNA. The sensitive TUNEL method is based on the detection of single and double stranded DNA breaks occurring at early stages in apoptosis.

 

After an enzymatic reaction, the fluorescein label incorporated at the damaged sites of DNA is visualized by fluorescence microscopy. The apoptotic sperm appear greener, while the normal non-apoptotic sperm take up more of the DAPI stain and appear blue. Partially apoptotic sperm will appear partially blue and green. This test allows the detection of apoptosis at the single cell level and may serve as an indicator for use of antioxidants prior to IVF.

 

The DFI is a ratio expressed as a percentage of sperm which has fragmented DNA due to apoptosis divided by the total number of sperm analyzed.

 

The sperm apoptosis test provides a reliable analysis of sperm DNA integrity that may help to identify men who are at a risk of failing to initiate a healthy ongoing pregnancy. It may help in the clinical diagnosis, management and treatment of male infertility and could be of prognostic value in assessing the outcome of various methods of Assisted Conception.

 

Apoptosis may not completely eliminate the cell’s competence for fertilization. Hence, an apoptotic spermatozoon, possessing high levels of DNA fragmentation, can fertilize the oocyte using ICSI. Such a pregnancy with DNA damage in the sperm may lead to miscarriage.

 

FOLLICULAR FLUID FISH

Follicular fluid is a novel source of gonadal cells for detection of low-grade mosaicism in the ovaries. Study of gonadal cells present in the follicular fluid of women undergoing IVF can determine the chances of a successful pregnancy. If there is evidence of gonadal mosaicism, the chances of a live birth could be low. Mosaicism for the X chromosome can be detected rapidly by FISH on follicular fluid cells and can be one more indication for Preimplantation Genetic Testing (PGT) in the same and subsequent cycles.

FISH ON BLOOD

Mosaicism is the presence of chromosomally normal and abnormal cells in the same individual. The percentage of mosaicism is known to vary in different tissues. We can easily study buccal, urine and blood cells by FISH. When more than 5% aneuploidy for the X chromosome is detected in the follicular fluid cells derived from the ovary, we check for aneuploidy in these other tissues as well.

MTHFR Mutation testing:

 

Methylenetetrahydrofolate Reductase (MTHFR) Deficiency is the most common genetic cause of elevated levels of homocysteine in plasma (Hyperhomocysteinemia).

 

Clinical Significance and Utility:

Hyperhomocysteinemia is a risk factor for arterial disease and venous thrombosis. Homocysteine levels are affected by nutritional and genetic factors.

  • Two mutations (C677T and A1298C) in the MTHFR gene have been associated with increased levels of circulating homocysteine. Homozygosity for the C677T mutation or compound heterozygosity for C677T and A1298C is associated with reduced MTHFR activity. Decreased MTHFR activity leads to hyperhomocysteinemia and lowers plasma folate levels. Homozygosity for 677C>T may increase risk for venous thrombosis and pregnancy complications (pre-eclampsia, placental abruption, intrauterine growth restriction).
  • In case of homozygosity for the C677T mutation or compound heterozygosity for C677T and A1298C mutations and homocysteine results, physicians can develop dietary and medical recommendations – increased intake of methylfolate alone or in combination with vitamins B6 and  B12 are recommended.

 

 

Prothrombin Mutation testing:

Prothrombin or Factor II is a protein in the blood that is required for the formation of fibrin in blood clotting process.

 

Clinical significance and utility:

  • Low levels of prothrombin causes frequent bleeding whereas high levels cause unwanted blood clots.
  • A history of recurrent pregnancy loss or stillbirth, may be indicative of an underlying thrombophilia (the blood has an increased tendency to clot).
  • Mutation G20210A in the prothrombin gene leads to increased levels of prothrombin which ultimately leads to increased risk of blood clotting. Individual with this mutation have an increased risk of developing Deep Vein thrombosis (DVT).

 

Indications for Testing:

Indications for testing as part of the screening for thrombophilia include: venous thromboembolism (especially if in an unusual site), deep vein thrombosis during pregnancy, venous thromboembolism while on oral contraceptives, fetal death after 10 weeks gestation, fetal growth restriction and/or preclampsia, and/or family history of stroke, pulmonary embolus, deep vein thrombosis in first degree relatives under the age 50.

Other Genetic Disorders

Karyotyping from blood can be carried out for all age groups.

 

Indications for cytogenetic analysis:

 

Children with-

  • Dysmorphic features
  • Developmental delay
  • Congenital anomalies
  • Ambiguous genitalia
  • Hypogonadism / undescended testes / hypospadias
  • Delayed menarche and short stature

 

Sample: 2-3 ml blood in sodium heparin vaccutainer (green top tube) transported at room temperature

In addition to G-banding, specific staining techniques such as C-banding, DA-DAPI staining, Quinacrine fluorescence are also offered on request. Sequential FISH on G banded slides is carried out to characterize complex translocations.

The diagnosis of chromosome breakage disorders such as Fanconi anemia, Ataxia Telangiectasia, Sister Chromatid exchange and Blooms syndrome involves addition of chromosome breakage inducing agents specific for each disorder. The test is run simultaneously with blood of a matched control. Multiple cultures of the patient and control samples are set up. A positive sample will show a higher frequency of breaks, fragile sites, radial figures or sister chromatid exchanges, compared to the control. The diagnosis of Fragile X by molecular methods has replaced cytogenetic analysis for Fragile X.

CHROMOSOMAL BREAKAGE STUDIESCHROMOSOMAL BREAKAGE STUDIES

Sample: Blood (3 ml in sodium heparin) from patient and age/sex matched control transported at room temperature.

For Fanconi Anemia, 6-8ml blood should be sent in sodium heparin.

Patients with low-grade mosaicism can be detected with FISH techniques. This technique is very useful to rule out common numerical chromosome abnormalities like Down syndrome, Turner syndrome, Klinefelter Syndrome, monosomy and trisomy. Different varieties of tissues can be used such as lymphocytes from direct or cultured blood, buccal cells, urine cells, chorionic villi, placenta, cultured fibroblasts and products of conception (POC). The report is available within 2-3 days.

Microdeletions are often missed by karyotyping, but are easily detected by FISH on cultured lymphocytes.

Prader Willi Syndrome and Angelman Syndrome are two clinical conditions caused by a microdeletion on chromosome 15. Occasionally, these syndromes may be caused by a mutation or uniparental disomy (only one parent contributes the genetic material to the child of a particular chromosome) instead of a deletion. FISH using specific probes can pick up such cases caused by deletions.

FISH_A

.
Certain cases of Autism are caused by duplication of the same region on chromosome 15, instead of a deletion.

DiGeorge syndrome is caused by a microdeletion in chromosome 22 and is associated mainly with cardiac defects in children, together with cleft palate and learning problems. Seizures, hypocalcemia and hypoplasia of parathyroid glands is also known to occur (CATCH 22).

Williams syndrome is caused by a microdeletion on chromosome 7. The main clinical features are supravalvular aortic stenosis, elfin face and mental retardation.

Sample: 2-3 ml blood in sodium heparin vaccutainer (green top tube) transported at room temperature.

Beta Thalassemia is one of the commonest blood disorders in India caused by a defect in the hemoglobin (Hb) molecule which in turn leads to severe anemia. The condition necessitates frequent blood transfusion for survival. Every year 10,000 children with thalassemia major are born in India, which constitutes 10% of the total number in the world, and one out of every 8 carriers of thalassemia worldwide lives in India. There are pockets of high incidence and areas of lower incidence in our country. The specific communities with a high incidence are Kutchi Lohanas, Gujaratis, Sindhis, Punjabis, Khojas and Marwaris. Thalassemia is an autosomal recessive condition where if the abnormal Hb molecules from both the carrier parents are transmitted to the baby, then the baby is affected. If only 1 abnormal Hb molecule is present then the baby is not affected. If both the parents are carrier of 1 abnormal Hb molecule, then there are 25% chances of having an affected baby, 50% chance of having a carrier baby and 25% chance of having a totally normal baby.

 

Hence mutation identification is very important in cases of carrier couples before going for pregnancy. Once the mutations are identified. the couple can avail the PGT-M technology to get pregnant with an embryo which is free of thalassemia major condition. Alternatively after conceiving naturally, the fetus can be tested prenatally for the affected condition.

Periodic fever syndromes are a set of disorders characterized by recurrent episodes of systemic and organ-specific inflammation.

Periodic fever syndromes refer to diseases that cause periodic (episodic) fever that do not have an infectious (virus, bacteria) cause. In general, children with these syndromes are well between episodes. Many of these syndromes are hereditary (passed down from parents) and result from a mutation (defect or mistake) in a gene (this is the code that determines the structure of our proteins). The syndromes are defined by several factors, including:

  • The gene defect
  • The clinical features of the syndrome
  • The parts of the body affected in addition to the fever
  • The age of the child when the syndrome starts
  • The ethnicity (the area of the world where the child or parents come from) of the child and parents

Many of these syndromes have a specific treatment, often based on understanding the problem caused by the genetic defect.

Heamatological and other Malignacies

Several specific rearrangements occur in the chromosomes in different types of leukemias like CML, CLL, AML, ALL, MPD and MDS. The treatment varies in certain abnormalities. Hence karyotyping is essential to find out the type of abnormality present in individual cases. A bone marrow sample (which has actively dividing cells) is aspirated from the patient and cultured under various conditions to obtain chromosomes. These are G-banded and analyzed for aneuploidy, translocations, deletions and other abnormalities. Multiple cultures are set up, as the abnormality may not be detected in all cultures.  Acquired chromosome abnormalities are occasionally seen in only a few poor quality cells, hence detailed analysis is carried out. However, subtle translocations cannot always be ruled out by karyotyping. Hence FISH is preferable.

 

bone-marrow

.
A bone marrow karyotype of a patient with CLL (chronic lymphocytic leukemia) showing various chromosome abnormalities.

 

Sample : The latest WBC count should be sent along with the sample. The quantity of bone marrow required is inversely proportional to the WBC count. For example,  2 ml bone marrow in a sodium heparin vaccutainer is required for a normal WBC count. If the WBC count is low, proportionately more bone marrow may be required. The sample should be transported at room temperature.

The FISH technique is very useful in diagnosis, prognosis, and management of leukemia patients and  in the detection of minimal residual disease.

 

In order to periodically monitor the progress of therapy in BCR/ABL fusion (Philadelphia +ve) and PML/RARA fusion (AML-M3) cases, semi-quantitative FISH can be carried out on heparinized blood instead of bone-marrow. In case a relapse is suspected, karyotyping from bone-marrow can be repeated to look for clonal evolution.

 

The double-fusion BCR/ABL and PML/RARA probes reduce the chances of false positive results. FISH is also useful in complex translocations leading to a masked Philadelphia chromosome, which may be missed on karyotyping.

positivecell

.In Multiple Myeloma, FISH is carried out on immunomagnetically separated CD138 positive plasma cells.

 

List of FISH Probes in Leukemia used by our lab

  • BCR-ABL/Philadelphia or Ph chromosome/t(9;22) for CML, AML, ALL
  • PML-RARA / t(15;17) for APML and RARA breakapart rearrangement for APML
  • AML1-ETO  / RUNX1T1-RUNX1 / t(8;21) for AML
  • Inversion 16 (CBFB)/t(16;16) for AML
  • t(3;3)/inversion 3/RPN1-MECOM for AML
  • MLL (11q23) rearrangement for AML / MDS / ALL / Lymphoma
  • Del 5q (EGR1) for MDS / AML
  • Del 7q (D7S486) for MDS / AML
  • Del 20q (20qter) for MDS / AML
  • Trisomy 8 for MDS / AML
  • TEL-AML /ETV6-RUNX1 / t(12;21) for ALL
  • t(1;19) or TCF3/PBX1 for ALL
  • TRAD (14q11.2) rearrangement for T-ALL
  • Chr. 12, Del 13q, Del p53 and Del ATM for CLL / Multiple Myeloma
  • IGH (14q32) rearrangement for Multiple Myeloma / ALL / Lymphoma
  • RB1 (13q) deletion for CLL
  • Deletion MYB or deletion 6q for CLL
  • t(4;14)/ IGH-FGFR3 for Multiple Myeloma
  • t(14;16) / IGH-MAF for Multiple Myeloma
  • 1p loss/deletion and 1q gain /amplification for Multiple Myeloma
  • PDGFRA and PDGFRB rearrangement for myeloid neoplasm with hyper eosinophilia.
  • BCL2 and BCL6 for Lymphoma
  • t(14;18)/ IGH-BCL2 for Lymphoma
  • Burkitt’s Lymphoma / (8q24) /(c-Myc) rearrangement

 

Sample: Based on the diagnosis and depending on WBC count 2-3 ml peripheral bone marrow sample (or blood in some cases)  in heparin tube (Green top) transported at room temperature. No fasting is needed.

 

Note: Not all FISH tests will show positive results if set up on a blood sample. Hence, bone marrow is the preferred sample for testing.

In casesof sex-mismatched bone-marrow transplantation, determination of the percentage of XX and XY chimerism by FISH gives an indication of the success of a transplant.

 

mismatched_bone

Sample: 2-3 ml Bone marrow / blood in heparin vaccutainer (Green top) with latest WBC count transported at room temperature.

We offer the Vysis UroVysion FISH test to monitor bladder cancer. The test is designed to detect aneuploidy for chromosomes 3, 7, 17, and loss of the 9p21 locus (containing the p16 tumor suppressor gene), which is one of the most common alterations in urothelial carcinoma.  Results from the UroVysion Kit are intended for use in conjunction with current standard diagnostic procedures, as an aid for initial diagnosis of bladder carcinoma in patients with hematuria, and subsequent monitoring for tumor recurrence in patients previously diagnosed with bladder cancer.

 

  • UroVysion detects chromosomal abnormalities associated with the development and progression of bladder cancer.
  • UroVysion in conjunction with cystoscopy delivers the best balance of sensitivity (97%) and specificity (95%).
  • It allows for more accurate patient monitoring, by detecting bladder cancer recurrence up to 6 months sooner than current diagnostic methods.
  • It is more sensitive than cytology and reduces false negative results.
  • The UroVysion kit detects all stages and grades of bladder cancer.  It is highly sensitive for higher grades and stages of tumours.
  • The test is not affected by BCG immunotherapy.

Early detection of high grade disease is critical to improve survival.

FISH is carried out in our Centre on formalin fixed paraffin embedded (FFPE) tumor tissues to check for

  • Her2/neu amplification in breast, oesophageal and other cancers.
  • BCL2, BCL6, IGH and MYC rearrangements in Lymphomas.

This helps in prognostication and choice of therapy.

FAQs

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

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.

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.

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.

Chromosome abnormalities are mainly of two types.

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

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.

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, with 47 chromosomes in each cell instead of 46, is the most common cause 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.

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 very important.

  • 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.

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).

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 gaining importance in a variety of cases ranging from prenatal diagnosis to cancer. The test result is available in 1-2 days as tissue culture to obtain chromosomes is not required. There is no risk of a culture failure due to contamination or inadequate sample size.

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 prognosis.

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.

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.

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.

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

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.

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

Prenatal diagnosis is recommended in the following cases:

  • Advanced maternal age
  • High risk on screening tests
  • 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 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.

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.

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.

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 their male offspring.

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 allowing only normal embryos to be transferred.

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 which need specific tests and some which may not be picked up on testing.

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.