Arundhati S. Athalye

A Study of Y Chromosome Microdeletions in Infertile Indian Males

Abstract Male partners of infertile couples are known to frequently have abnormal semen parameters. Some of these cases are due to underlying genetic factors such as Y chromosome microdeletions, an abnormal karyotype or cystic fibrosis mutations. Y chromosome microdeletions generally cannot be detected by karyotyping. At our clinic we undertook a study of male partners of infertile couples to determine the frequency and common loci of Y chromosome microdeletions in India, using the PCR technique. We studied 100 patients mainly having azoospermia (AZ) or oligoasthenoteratozoospermia (OAT). Multiplex PCR analysis for 18 loci on the Y chromosome was carried out using commercially available kit (Promega Version 1.1). Y chromosome microdeletions were observed in 12/100 (12%) patients including 8/27 (29.63%) with azoospermia, 3/56 (5.35%) with oligoasthenoteratozoospermia and 1/ 7 (14.28%) with only asthenoteratozoospermia. All loci of the DAZ gene were deleted along with DYS237 and DYS236 from AZFd in 5/27 (18.52%) azoospermic males studied. The most commonly deleted loci were DYS240 in 11/12 (91.67%) and DYS219 in 7/12 (58.33%) patients with microdeletions. The use of ICSI in such patients can lead to transmission of Y chromosome microdeletions and subsequent infertility from father to son. Hence screening for Y chromosome microdeletions will help in the proper counseling and management of couples with male factor infertility.

Microdeletion Syndromes Detected by FISH - 73 Positive from 374 Cases

Abstract Fluorescence in situ hybridization (FISH) has facilitated the detection of microdeletions seen in Prader-Willi/Angelman (PW/AS), Williams and DiGeorge syndromes. Out of 374 suspected cases tested at Jaslok Hospital inthe past 5 years, 73 were positive, including 29 cases of Angelman, 16 of Prader-Willi, 24 of Williams and 4 ofDiGeorge syndrome. Male preponderance was seen, mainly in Williams syndrome. The mechanisms causing Prader-Willi and Angelman syndrome include microdeletions, intragenic mutations, uniparental disomy and imprintingdefects, though FISH can only detect microdeletions. Metaphase FISH helped to detect 1 case each with deletion ofthe control (PML) signal and duplication of the critical PW/AS region, which are associated with autism. Onesuspected case of Prader-Willi syndrome had a Robertsonian translocation t(14;15)(q10;q10) which led to a deletionof a major part of the SNRPN region in 10% cells, resulting in low-grade mosaicism. Another FISH-positive case wasdue to a reciprocal translocation t(2;15)(q37;q11), where loss of critical genes at the breakpoint on chromosome 15caused the Prader-Willi phenotype. FISH in a child with an Angelman phenotype showed no microdeletion, thoughTrisomy 15 was seen in 1 metaphase suggesting uniparental disomy due to trisomy rescue. A known polymorphism inthe form of an additional tiny green signal on chromosome 14 was observed in 17 of 284 (6%) cases studied forPrader-Willi/Angelman syndrome. Another inherited polymorphism was seen in 5 cases, where one control signal wasvery small. Prenatal diagnosis was carried out with normal results, in 12 women with a previously affected child.

FLUORESCENCE IN-SITU HYBRIDIZATION (FISH)-A RAPID AND USEFUL TECHNIQUE FOR DIAGNOSIS AND MANAGEMENT IN LEUKEMIA

Abstract Fluorescence in situ hybridization (FISH) is a rapid reliable technique in molecular cytogenetics. It supplements conventional karyotyping by providing additional information in certain cases. A large number of cells are available for quantitative analysis by FISH, as even interphase nuclei can be studied. It helps in detection of minimal residual disease and disease recurrence, as a very small percentage of abnormal cells can also be identified. The FISH probes used in this study were for the detection of the BCR/ABL fusion or t(9;22) in chronic myeloid leukemia (CML), PML/RARA fusion or t(15;17) in acute promyelocytic leukemia (APML) and determination of the XX/XY ratio in sex mismatched bone marrow transplantation. One hundred and fifty eight heparinized bone marrow or leukemic blood samples referred by consultants were analysed using the above mentioned FISH probes. These included samples of patients who underwent bone marrow transplantation. Karyotyping was carried out where indicated. Cases where FISH provided additional information or a different interpretation to karyotype analysis have been described.

PGD by FISH for a Reciprocal Translocation: First Baby from India

A couple with a history of five early miscarriages due to a balanced reciprocal translocation t(6;19) (p22;q13.4) in the wife, was referred for preimplantation genetic diagnosis (PGD) by fluorescence in situ hybridization (FISH). After oocyte retrieval, nine embryos were obtained. One cell from each embryo was biopsied, fixed and subjected to FISH using centromere and subtelomere probes for chromosomes 6 and 19. Five embryos which had unbalanced translocations, were not transferred. A balanced translocation or absence of translocation was seen in three embryos, which were transferred. One embryo had an anucleate cell and subsequently arrested. A pregnancy was achieved in the first intracytoplasmic sperm injection cycle itself and resulted in the birth of a healthy baby. This is the first baby after PGD for a reciprocal translocation in India. In 2010, the team of the authors reported the first successful pregnancy after PGD for a Robertsonian translocation and a normal child was born.

First Successful Pregnancy After Pre-implantation Genetic Diagnosis by FISH for an Inversion Together with a Cryptic Translocation in India

The technique of pre-implantation genetic diagnosis (PGD) by fluorescence in situ hybridization (FISH) in cases of repeated miscarriages due to parental balanced inversions and translocations is relatively new in India. In a couple with a history of recurrent miscarriages and implantation failures, karyotyping done in three laboratories showed that the husband had an insertion or inversion of chromosome 12. Hence, they were referred to us for PGD. The anomaly turned out to be more complex. A pre-PGD workup using a series of FISH probes on metaphases accompanied by reflex FISH was required to characterize the anomaly. For subsequent PGD, single blastomeres were biopsied from seven embryos obtained by intracytoplasmic sperm injection. FISH analysis had to be carried out using ten probes in four rounds. On pre-PGD workup for inversion 12 by FISH, an additional anomaly of a cryptic translocation between 9qter and 12qter was detected in the husband. His complex karyotype according to the detailed ISCN nomenclature was therefore 46,XY,t(9;12)(9pter→9q34.1::12q24.2→12qter),der(12)inv(12)(12pter→12p11.2::12q24.2→12p11.2::9q34.1→9qter). After PGD, the normal and balanced embryos transferred, resulted in the birth of healthy twins conceived in the first cycle itself. Therefore, a pre-PGD workup is important and needs reflex FISH in the event of an unexpected cytogenetic anomaly. PGD will need the analysis of additional chromosomes on the same cell by FISH in such cases. An experienced in vitro fertilization and Genetics team is essential for success. This is the first report of PGD by FISH for an inversion coupled with a cryptic translocation from India.

Preimplantation Genetic Diagnosis for the Better Management of Couples During Assisted Reproduction

Abstract Intracytoplasmic Sperm Injection (ICSI) offers the real prospect of genetic parenthood for men with profound oligozoospermia and azoospermia. However, it may result in transgenerational transmission of genetic defect, which substantially increases the recurrence risk of infertility in the offspring of couples treated with ICSI. Recent developments in the field of genetics and assisted reproduction have led to emergence of preimplantation genetic diagnosis (PGD). PGD helps in the negative selection of the aneuploid and abnormal embryos. For PGD, the embryo is biopsied and 1-2 blastomeres are removed from the 6-10 cell embryo. For establishing the diagnosis, the genetic analysis is carried out using FISH (for structural and numerical chromosomal anomalies) or PCR (for single gene disorders). Only the normal embryos are transferred back to the uterus, thus ensuring a normal pregnancy. Spare or arrested embryos obtained following ICSI were used for the study. FISH was performed on 5 embryos using CEP X spectrum green probes. PCR was done for cystic fibrosis common mutation ΔF508 and for β-thalassaemia IVS1→5 and 619 bp deletion using blood as positive control. Mosaicism was noted in two of the five embryos using FISH. The blastomeres were found to be normal for cystic fibrosis and β-thalassaemia. PGD has an important role in helping patients to avoid the risk of transmission of genetic defect or abnormalities and also helps in avoiding repeated medical termination of pregnancy.

Preimplantation genetic testing for a complex chromosome rearrangement, case report of a cryptic translocation detected on pre-PGT workup

Pre-implantation genetic testing (PGT) is an established alternative to prenatal diagnosis of inherited disorders at the chromosome or gene level. Different molecular techniques are now available for detection of 24 chromosome aneuploidy and translocations. However, fluorescence in situ hybridization (FISH) is still used for certain telomeric and subtelomeric translocations and inversions. A non-consanguineous Indian couple had a son with an unbalanced translocation t (16;17)(p13.3;q25.3) detected after microarray showed deletion 16p13.3 and duplication 17q25.3. Prenatal diagnosis during the second pregnancy showed a similarly affected fetus. The husband was a carrier of the balanced translocation. We then recommended IVF-ICSI with PGT. During pre-PGD workup with FISH probes for telomeres 16p, 17q and centromere 16 on metaphases from the husband’s lymphocyte culture, it was found that one signal of 17q was present on a C group chromosome instead 16p. G banding of fresh metaphase spreads followed by reflex-FISH on the same metaphases showed that the signal of 17q was located on 9q. This 3-way translocation was further confirmed on other G banded metaphases using a 9q34 probe, which showed that this region was translocated to 16p. Thus the karyotype of the husband was revised as 46,XY,t(9;16;17)(q34;p13.3;q25.3) due to the cryptic translocation seen only by FISH. PGT by FISH was carried out for the couple during the IVF-ICSI cycle on three embryos which reached the blastocyst stage on day-5. A few trophectoderm cells were biopsied and fixed on slides. FISH was set up using probes for chromosomes 16 and 17 in the first round and for 9q in the second round. Only 1 of 3 embryos showed a normal or balanced status. The third round of FISH on the same cells showed no aneuploidy for chromosomes 13, 18, 21 X and Y. The lady got pregnant after a frozen embryo transfer in the next cycle.

Detection of t(14;16)(q32;q22) and Monosomy 13 by FISH Analysis in a Patient with Multiple Myeloma Associated with Sjögren's Syndrome: The First Case Report from India

Sjögrens syndrome (SS) is a chronic slowly progressive autoimmune disorder characterized by symptoms of oral and ocular dryness, exocrine dysfunction, and lymphocytic infiltration of exocrine glands. Multiple myeloma (MM) is a bone-marrow-based malignant neoplasm of plasma cells associated with serum/urine monoclonal paraproteins and lytic skeletal lesions. There have been very few reported cases of MM, who had SS as the first presentation. We report a case of a woman diagnosed with Sjögrens syndrome, who was later suspected to have multiple myeloma on serum protein electrophoresis. Fluorescence in situ hybridization (FISH) was carried out to check for deletions of loci 13q14.3, ATM, p53, and IGH (14q32) rearrangements on a bone marrow aspirate. Monosomy 13 was observed in 49% of cells, and a rearrangement at the IGH locus was seen in 42% of cells. To determine the partner chromosome associated with the IGH rearrangement, further FISH tests were set up for t(4;14)(p16;q32) followed by t(14;16)(q32;q22) on fresh slides. The test was negative for t(4;14) but positive for t(14;16) in 27% of cells. This confirmed the diagnosis of MM. We report the first case from India, having an association of Sjögrens syndrome with multiple myeloma, which showed t(14;16) and monosomy 13 by FISH analysis.

P15 PGD by FISH in India: first live-birth after PGD for a reciprocal translocation

expression pattern of specific genes important for fetal growth and nutrition. Materials and Methods: Blood samples from children conceived after ART (n = 54) and from spontaneously conceived children (n = 29) were collected after informed consent in PAXGene Blood RNA Tubes. The children were part of an existing cohort from the UK, followed since birth. Blood samples were stored at 20oC and transferred frozen to Belgium. RNA was extracted for reverse transcription and Real-Time PCR analysis using gene expression assays for DNA methyltransferase 1 (DNMT1), Glucocorticoid receptor (NR3C1) and Peroxisome Proliferator Activated Receptor Alpha (PPARA) and Hypoxanthine Phosphoribosyltransferase 1 (HPRT1) as an endogenous control. Statistical analysis was done using the Student T test. Results: Gene expression levels were normalised against the endogenous control HPRT1 and relatively quantified using the DDCt method. The mean blood mRNA levels of DNMT1, NR3C1 and PPARA in the ART children show a 0.95, 1.26 and 1.35 fold change respectively compared to those of the children from spontaneous conception (p = 0.95, p = 0.73 and p = 0.54 respectively). Discussion and Conclusion: No significant difference was observed in mRNA expression levels of DNMT1, NR3C1 and PPARA between children conceived after ART and children spontaneously conceived, reassuring the epigenetic safety of ART methods with respect to the studied genes. Nevertheless, no definitive conclusions can be drawn without testing other gene panels of imprinted and non-imprinted genes.

Fanconi Anemia with Triphalangeal Thumbs, Syndactyly and Contractures of the Fingers in a 2 Year Old Boy

Abstract Fanconi Anemia (FA) is a rare autosomal recessive disorder associated with pancytopenia, spontaneous chromosome instability and a variety of congenital anomalies. Hypersensitivity to bifunctional alkylating or DNA crosslinking agents like Mitomycin C (MMC), Diepoxybutane (DEB) and Nitrogen Mustard (HN2) is used as a differential diagnostic test. A variable phenotype and age of onset of anemia make diagnosis difficult in some cases. We report a case of Fanconi anemia detected by the MMC stress test in a 2 year old boy, operated for bilateral syndactyly and contractures of fingers. He had a bifid thumb on the left hand and bilateral triphalangeal thumbs. There was no history of consanguinity or malformations, though a maternal uncle had a bifid thumb. USG in a subsequent pregnancy showed bony anomalies like scoliosis, talipes, contractures and radial aplasia, consistent with FA. The parents opted for termination. An early diagnosis of FA in a non-manifesting child would provide more time to explore different treatment options, since a delay in diagnosis could have serious consequences.