Prochi F. Madon

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.

Pure Cerebellar Ataxia with Homozygous Mutations in the PNPLA6 Gene

Autosomal-recessive cerebellar ataxias (ARCA) are clinically and genetically heterogeneous conditions primarily affecting the cerebellum. Mutations in the PNPLA6 gene have been identified as the cause of hereditary spastic paraplegia and complex forms of ataxia associated with retinal and endocrine manifestations in a field where the genotype-phenotype correlations are rapidly expanding. We identified two cousins from a consanguineous family belonging to a large Zoroastrian (Parsi) family residing in Mumbai, India, who presented with pure cerebellar ataxia without chorioretinal dystrophy or hypogonadotropic hypogonadism. We used a combined approach of clinical characterisation, homozygosity mapping, whole-exome and Sanger sequencing to identify the genetic defect in this family. The phenotype in the family was pure cerebellar ataxia. Homozygosity mapping revealed one large region of shared homozygosity at chromosome 19p13 between affected individuals. Within this region, whole-exome sequencing of the index case identified two novel homozygous missense variants in the PNPLA6 gene at c.3847G>A (p.V1283M) and c.3929A>T (p.D1310V) in exon 32. Both segregated perfectly with the disease in this large family, with only the two affected cousins being homozygous. We identified for the first time PNPLA6 mutations associated with pure cerebellar ataxia in a large autosomal-recessive Parsi kindred. Previous mutations in this gene have been associated with a more complex phenotype but the results here suggest an extension of the associated disease spectrum.

Epigenetic Regulatory Mechanisms Associated with Infertility

Infertility is a complex human condition and is known to be caused by numerous factors including genetic alterations and abnormalities. Increasing evidence from studies has associated perturbed epigenetic mechanisms with spermatogenesis and infertility. However, there has been no consensus on whether one or a collective of these altered states is responsible for the onset of infertility. Epigenetic alterations involve changes in factors that regulate gene expression without altering the physical sequence of DNA. Understanding these altered epigenetic states at the genomic level along with higher order organisation of chromatin in genes associated with infertility and pericentromeric regions of chromosomes, particularly 9 and Y, could further identify causes of idiopathic infertility. Determining the association between DNA methylation, chromatin state, and noncoding RNAs with the phenotype could further determine what possible mechanisms are involved. This paper reviews certain mechanisms of epigenetic regulation with particular emphasis on their possible role in 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.

Challenges in prenatal and pre-implantation genetic diagnosis studies

Prenatal diagnosis (PND) of chromosomal anomalies by karyotyping and fluorescence in situ hybridization (FISH) is a routine procedure in many cytogenetic laboratories. However, PND of single gene disorders is available in only a few centres specializing in molecular genetic testing in India. Preimplantation genetic diagnosis (PGD) is a state-of the art technique involving biopsy of a single cell from a cleavage stage embryo obtained after intra-cytoplasmic sperm injection (ICSI). This is an additional step during in-vitro fertilization (IVF). The biopsied cell is then fixed on a slide in a critical step where the cytoplasm is removed to expose the nucleus. Single cells from many embryos of the couple are fixed and then screened for common aneuploidies by FISH using multicolour DNA probes. A maximum of 5 chromosomes can be checked in 1 hybridization. Hence the probes are stripped and slides are rehybridized in 2-4 rounds to study more chromosomes within a limited time frame. Only the chromosomally normal embryos are transferred or cryopreserved for a subsequent transfer. Karyotyping of couples with recurrent early miscarriages helps to identify the cause if the husband or wife is a carrier of a balanced translocation. PGD can help such couples to have a healthy baby earlier than by normal chance. Specific FISH probes have to be ordered especially for reciprocal translocations and a pre-PGD work up is important. This helped to identify an additional cryptic translocation between chromosomes 9 and 12 in a case from a neighbouring country, where the husband had inversion 12. Hence PGD included testing of chromosome 9 in this case. The wife has an ongoing pregnancy in the first attempt. Currently, ours is the only centre in India which offers PGD even for reciprocal/ Robertsonian translocations and microdeletions. We have had success with the birth of healthy babies. PGD for single gene disorders is yet in its infancy in our country. It involves genetic testing from DNA extracted from a single cell. Pre-PGD work up is more demanding, especially in cases where a HLA matched normal savior sibling is desired to cure a child affected with a hematological disorder such as Thalassemia.

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.