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We here report a case of a 23-year-old female from Mumbai, Maharashtra, India who was detected to carry the α chain variant Hb J-Norfolk [HBA2: c.173G>A (or HBA1]. She had no clinical symptoms and was referred to us for routine investigations and screening. An abnormal peak was detected on both high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) with a fast-moving band on cellulose acetate electrophoresis. There is no detailed study on the HPLC and CE pattern of this hemoglobin (Hb) variant, and therefore, this study will help in detecting and avoiding missing these variants during routine investigations and population screening. This is the first report of this variant in the Indian population.

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. Electronic supplementary material The online version of this article (doi:10.1007/s12311-016-0769-x) contains supplementary material, which is available to authorized users.

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.

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.

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

Preimplantation genetic testing (PGT) is an early form of prenatal genetic diagnosis where abnormal embryos are identified, thereby allowing transfer of genetically normal embryos. This technology has become an integral part of Assisted Reproductive Technology (ART) procedures. Initial experiments with animals as early as 1890 and those in the mid and later part of the last century paved the forward path of ART and PGT. This review article covers the evolution of PGT and is a pointer toward current and fast-evolving technology, allowing scientists and doctors to better comprehend human reproduction, and ensure healthy pregnancy outcomes.

Polymorphic variants on chromosomes are considered 'normal', as heterochromatin has no coding potential and nucleolar organizing regions (NOR) contain genes coding for rRNA. Variants have been reported in infertility and recurrent abortions. With refined molecular techniques, genes for fertility and viability are now thought to reside in heterochromatin. DNA sequence analysis of human chromosome 9 has shown that it is highly structurally polymorphic, with many intrachromosomal and interchromosomal duplications, and contains the largest autosomal block of heterochromatin. Transcriptional activation of constitutive heterochromatic domains of the human genome in response to environmental stress was reported recently. Heat shock triggers the assembly of nuclear stress bodies on the pericentromeric heterochromatin of human chromosomes including chromosome 9. These are characterized by an epigenetic status typical of euchromatic regions. On acrocentric chromosomes, NOR-associated protein count and morphology was reported to separate benign and malignant melanocytic lesions. Hence all variants may not be 'normal'. The present study of karyotyping 842 individuals attending an IVF clinic with primary infertility or repeated miscarriages, showed polymorphic variants in 28.82% of males and 17.19% of females, which was quite high. It is suggested that variants should not be ignored by cytogeneticists. Screening prospective gamete donors for chromosome variants may help enhance the success of IVF.

KEYWORDS Male infertility; ICSI; semen analysis; PGD; AZF deletions; azoospermia; OAT 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.

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.

Sjögren’s 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 Sjögren’s syndrome, who was later suspected to have multiple myeloma on serum protein electrophoresis. Fluorescencein situhybridization (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 Sjögren’s syndrome with multiple myeloma, which showed t(14;16) and monosomy 13 by FISH analysis.

Objective To study the association of chromosomal polymorphic variations with infertility and subfertility. Design A comparative case-controlled association study using cytogenetic techniques to compare the frequency of chromosomal variations in infertile individuals versus fertile controls. Setting Department of Infertility Management and Assisted Reproduction, Jaslok Hospital and Research Centre, Mumbai, India. Patient(s) 760 infertile individuals and 555 fertile controls. Intervention(s) ICSI, IUI, karyotyping, inverted 4′,6-diamidino-2-phenylindole (DAPI), CBG banding. Main Outcome Measure(s) Frequency of chromosomal polymorphic variations in infertile individuals undergoing infertility treatment versus fertile individuals. Result(s) A highly statistically significant increase in the frequency of total chromosomal variants in infertile women (28.31% vs. 15.16%) and infertile men (58.68% vs. 32.55%) was observed. The frequency of 9qh+ was statistically significantly increased in women with primary infertility (16.22% vs. 6.41%) and in men with severe male factor infertility (14.69% vs. 4.25%). A highly statistically significant increase in the frequency of Yqh+ was observed in men whose wives had a bad obstetric history (30.20% vs. 12.74%). Conclusion(s) The statistically significantly higher incidence of heterochromatic variations found in infertile individuals stresses on the need to evaluate their role in infertility and subfertility. Potential epigenetic, genetic, and chromosomal modifications could be associated with certain complex disorders such as infertility and bad obstetric history.

Fanconi Anemia (FA) is a rare autosomal recessive disorder associated with pancytopenia, sponta- neous 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 ex- plore different treatment options, since a delay in diagno- sis could have serious consequences.

Intracytoplasmic Sperm Injection (ICSI) of- fers the real prospect of genetic parenthood for men with profound oligozoospermia and azoospermia. However, it may result in transgenerational transmission of genetic de- fect, which substantially increases the recurrence risk of infertility in the offspring of couples treated with ICSI. Re- cent developments in the field of genetics and assisted re- production have led to emergence of preimplantation ge- netic diagnosis (PGD). PGD helps in the negative selection of the aneuploid and abnormal embryos. For PGD, the em- bryo is biopsied and 1-2 blastomeres are removed from the 6-10 cell embryo. For establishing the diagnosis, the ge- netic 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 ∆ F508 and for β-thalassaemia IVS1→ 5 and 619 bp deletion using blood as positive control. Mosa- icism was noted in two of the five embryos using FISH. The blastomeres were found to be normal for cystic fibro- sis and β-thalassaemia. PGD has an important role in help- ing patients to avoid the risk of transmission of genetic de- fect or abnormalities and also helps in avoiding repeated medical termination of pregnancy.

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.

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.