Preimplantation Genetic Testing for Couples with Balanced Chromosomal Rearrangements.

Background: Chromosomal rearrangements play an important role in infertility. Carriers of chromosomal rearrangements have a lower chance of producing normal or balanced gametes due to abnormal segregation of chromosomes at meiosis, which leads to recurrent spontaneous abortions and infertility. Preimplantation genetic testing for structural chromosome rearrangements (PGT-SR) is offered to couples who have balanced chromosomal rearrangements in order to select embryos with a balanced karyotype prior to implantation, thereby increasing the chances of pregnancy. The purpose of the current study was to assess the outcomes of PGT-SR in patients carrying various balanced chromosomal rearrangements and to assess their clinical pregnancy outcome after in vitro fertilization (IVF). Methods: In this study, infertile couples with balanced chromosomal abnormalities undergoing PGT-SR were retrospectively analyzed at a single fertility center from January 2016 to December 2019. Results: PGT-SR was performed on 87 embryos from 22 couples in whom one partner carried a balanced translocation or an inversion. Fifty-seven (65.5%) of these embryos had unbalanced or sporadic aneuploidies, 30 (34.5%) embryos were normal or chromosomally balanced, which were then transferred in 18 couples. A higher rate of unbalanced translocations in comparison to sporadic aneuploidies was observed in couples with reciprocal translocation. The live birth rate per embryo transfer was found to be 66.6% (12/18). Conclusion: PGT-SR is a useful tool in selecting normal or balanced embryos for transfer in IVF, which could lead to a pregnancy by reducing the chance of miscarriages due to chromosome aneuploidy in couples with balanced chromosomal rearrangements.

A novel leaky splice variant in centromere protein J (CENPJ)-associated Seckel syndrome.

Primary microcephaly and Seckel syndrome are rare genetically and clinically heterogenous brain development disorders. Several exonic/splicing mutations are reported for these disorders to date, but ∼40% of all cases remain unexplained. We aimed to uncover the genetic correlate(s) in a family of multiple siblings with microcephaly. A novel homozygous intronic variant (NC_000013.10:g.25459823T>C) in CENPJ (13q12) segregating with all four affected male siblings was identified by exome sequencing and validated by targeted linkage approach (logarithm of the odds score 1.8 at θ 0.0). RT-PCR of CENPJ in affected siblings using their EBV derived cell lines showed aberrant transcripts suggestive of exon skipping confirmed by Sanger sequencing. Significantly reduced wild type transcript/protein in the affected siblings having the splice variant indicates a leaky gene expression of pathological relevance. Based on known CENPJ function, assessing for mitotic alterations revealed defect in centrosome duplication causing mono/multicentrosome(s) at prophase, delayed metaphase, and unequal chromosomal segregation in patient cells. Clinical features witnessed in this study expand the spectrum of CENPJ-associated primary microcephaly and Seckel syndrome. Furthermore, besides the importance of regulatory variants in classical monogenic disorders these findings provide new insights into splice site biology with possible implications for ASO-based therapies.

Y chromosome microdeletion and cytogenetic findings in male infertility: A cross-sectional descriptive study.

BACKGROUND: Infertility affects about 15% of couples worldwide, and the male factor alone is responsible for approximately 50% of the cases. Genetic factors have been found to play important roles in the etiology of azoospermia and severe oligospermia conditions that affect 30% of individuals seeking treatment at infertility clinics. OBJECTIVE: To determine the frequency of chromosomal abnormalities and Y chromosome microdeletion in infertile men. MATERIALS AND METHODS: A total of 100 infertile men with abnormal semen parameters were included in this study from 2014 to 2018. Chromosomal analysis was carried out using standard G-banding using Trypsin Giemsa protocol. Multiplex polymerase chain reaction was used to determine the Y microdeletion frequency. RESULTS: All participants were aged between 22 and 48 yr with a mean and standard deviation of 35.5 ± 5.1. Of the 100 subjects included in the study, three had Klinefelter syndrome-47,XXY, one had balanced carrier translocation-46,XY,t(2;7)(q21;p12), one with the balanced carrier translocation with inversion of Y chromosome 45,XY,der(13;14)(q10;q10),inv(Y), one had polymorphic variant of chromosome 15, one had Yqh-, and another had an inversion of chromosome 9. Y chromosome microdeletion of Azoospermia factor c region was observed in 2% of the cases. To the best of our knowledge, the current study is the first reported case with unique, balanced carrier translocation of chromosome 2q21 and 7p21. CONCLUSION: The present study emphasizes the importance of routine cytogenetic screening and Y microdeletion assessment for infertile men, which can provide specific and better treatment options before undergoing assisted reproductive technology during genetic counseling.

A de novo marker chromosome 15 in a child with isolated developmental delay.

We report a rare case of a 14-month-old male child who was referred for developmental delay. Clinical examination revealed a hypotonic infant with speech delay and no dysmorphic features. The banding cytogenetics revealed a small supernumerary marker chromosome. Upon silver staining, the marker showed the presence of satellite regions on either ends. Further, analysis using fluorescence in situ hybridization on marker chromosome revealed its origin from chromosome 15.

Association of Sperm Aneuploidy Frequency and DNA Fragmentation Index in Infertile Men.

BACKGROUND: For improving the evaluation of male infertility, many parameters were studied and reported in earlier literature. The aim of this study was to estimate the frequency of sperm aneuploidy and DNA fragmentation in infertile men and to assess the correlation between sperm aneuploidy and DNA fragmentation. METHODS: In this study 100 infertile men were included, cases with azoospermia were 68%, oligospermia 18%, severe oligospermia 6%, and oligoasthenoteratospermia (OAT) 8%. Ten normozoospermic men who had two normal children were included as a control. The sperm aneuploidy test by Fluorescence In Situ Hybridization (FISH) and sperm DNA fragmentation index by TdT (Terminal deoxynucleotidyl transferase)-mediated dUTP nick end labelling (TUNEL) were carried out. To determine the aneuploidy status and DNA fragmentation index, frequency was used. The correlation between sperm aneuploidy and sperm DNA fragmentation along with age was assessed by using Spearman's correlation coefficient. The p<0.05 was considered significant. RESULTS: The age of 100 subjects ranged between 22-48 years (35.5±5.1). Sperm aneuploidy frequency and DNA fragmentation rate were found to be higher in infertile men compared to control men (n=10). There was a significant relationship between age and sex chromosomal aneuploidy (p<0.05) and significant difference between sperm aneuploidy and damaged DNA (p<0.05). CONCLUSION: FISH and TUNEL assay results showed increased sperm aneuploidy frequency, and DNA fragmentation index in infertile men compared with the fertile men. There is significant relationship observed between sperm aneuploidy and DNA fragmentation. These two parameters are important and they must be investigated for clinical practice.

Localization of the SRY Gene on Chromosome 3 in a Patient with Azoospermia and a Complex Karyotype 45,X/46,X,i(Y)(q10)/46,XX/ 47,XX,i(Y)(q10).

This study aimed to identify the cause of azoospermia in a 38-year-old infertile man who was referred for genetic testing. Cytogenetic evaluation was performed by G-banding, C-banding, and FISH using centromeric probes for chromosomes X and Y and showed the presence of a monocentric isochromosome Y with a complex, mosaic karyotype 45,X/46,X,i(Y)(q10)/46,XX/47,XX,i(Y)(q10). Multiplex PCR for the commonly deleted genes in the AZFa, AZFb, and AZFc regions of the Y chromosome was performed and indicated the presence of all 3 regions. Further, PCR amplification followed by DNA sequencing of the SRY gene was done, which ruled out mutations in that gene. To identify the position of the SRY gene, FISH using a locus-specific probe was used and showed that the gene had been translocated to chromosome 3. Subtelomere FISH for 3q and Yp evidenced that the subtelomeric region of the Y chromosome was found on the terminal region of 3q. The clinical symptoms of the patient can be attributed to this abnormal genotype. The importance of genetic testing in infertile patients and the need for genetic counselling to prevent the transmission of the defect are emphasized.

Clinical and Molecular Characterization of Prader-Willi Syndrome.

OBJECTIVES: To describe the clinical presentations and molecular diagnosis to aid the clinicians in early diagnosis and appropriate management of Prader-Willi syndrome (PWS). METHODS: Thirty-four clinically diagnosed PWS cases were enrolled after obtaining informed consent/assent. Demographic details, clinical data and anthropometry were recorded using structured proforma. The facial dysmorphology was evaluated. Appropriate genetic testing was performed to confirm the diagnosis. RESULTS: At diagnosis, the most common clinical features included obesity (59%) and short stature (53%). Distinct dysmorphic features were observed in 67%. Neonatal hypotonia with feeding difficulty, delayed development in infancy and childhood behavioral problems were reported in 94%, 94% and 74% respectively. Food seeking behavior and hyperphagia was reported in 67%. Seizures were reported in 47%. All children had underdeveloped external genitalia. Growth hormone (GH) deficiency and impaired glucose tolerance were found in 56% and 50% respectively. Sleep related problems were seen in 67%. Skin and rectal picking were reported in 67%. FISH confirmed micro-deletion was found in 64.7% and abnormal methylation in 35%, of which uniparental disomy was confirmed in 14.7%. CONCLUSIONS: Clinical suspicion is vital for early detection of PWS. Confirmation of the diagnosis requires complex multi-tier molecular genetic testing.

Next-Generation Sequencing Reveals Novel Mutations in X-linked Intellectual Disability.

Robust diagnostics for many human genetic disorders are much needed in the pursuit of global personalized medicine. Next-generation sequencing now offers new promise for biomarker and diagnostic discovery, in developed as well as resource-limited countries. In this broader global health context, X-linked intellectual disability (XLID) is an inherited genetic disorder that is associated with a range of phenotypes impacting societies in both developed and developing countries. Although intellectual disability arises due to diverse causes, a substantial proportion is caused by genomic alterations. Studies have identified causal XLID genomic alterations in more than 100 protein-coding genes located on the X-chromosome. However, the causes for a substantial number of intellectual disability and associated phenotypes still remain unknown. Identification of causative genes and novel mutations will help in early diagnosis as well as genetic counseling of families. Advent of next-generation sequencing methods has accelerated the discovery of new genes involved in mental health disorders. In this study, we analyzed the exomes of three families from India with nonsyndromic XLID comprising seven affected individuals. The affected individuals had varying degrees of intellectual disability, microcephaly, and delayed motor and language milestones. We identified potential causal variants in three XLID genes, including PAK3 (V294M), CASK (complex structural variant), and MECP2 (P354T). Our findings reported in this study extend the spectrum of mutations and phenotypes associated with XLID, and calls for further studies of intellectual disability and mental health disorders with use of next-generation sequencing technologies.

Practice of Consanguinity and Unusual Cases of Inherited Familial Chromosome Abnormalities: A Case Report.

We present 2 cases of likely rare event. In case 1, 3(rd) degree consanguineous marriage revealed inv(6) with same break points in parents who were found to be phenotypically normal. The same inv(6) being inherited in progeny but presented with low AMH (anti Mullerian hormone) and high level of FSH (follicular stimulating hormone) leading to polycystic ovarian syndrome/premature ovarian failure. In case 2, a couple was presented with 2(nd) degree consanguineous marriage and referred for 2 recurrent/ missed abortions. The amounts of shared genes are suggestive of more lethal genetic outcomes and inferred endogamy is a major driver to reproductive fiascoes, the ancestries of which are deeply tied at the meiotic level.

Pre- and Postnatal Diagnosis of 10p14 Deletion and 22q11.2 Deletion Syndrome and Significance of Non-Cardiac Markers.

Congenital heart defect (CHD) is the most common form of birth defects. There is a high association between increased nuchal translucency and CHD in fetuses, and CHD in the antenatal period has a high incidence of 22q11.2 deletion syndrome (22q11.2DS). Apart from 22q11.2DS, the BRUNOL3 gene at 10p14 is also associated with DiGeorge-like features. We studied a total of 110 pre- and postnatal CHD cases with FISH probes. 22q11.2DS was detected in 5 cases and 10p14 deletion in 1 case. Antenatally diagnosed cases of CHD should be investigated by karyotyping and 22q11.2DS testing. Cases with increased nuchal translucency, intrauterine growth retardation, and other non-cardiac malformations because of 22q11.2DS should be screened carefully for thymus dysgenesis. It is also advisable to screen patients referred for 22q11.2DS for a 10p14 deletion, therefore enabling appropriate parental counseling.

Split Hand/Foot Malformation Associated with 7q21.3 Microdeletion: A Case Report.

Split hand/foot malformation (SHFM) or ectrodactyly is a rare genetic condition affecting limb development. SHFM shows clinical and genetic heterogeneity. It can present as an isolated form or in combination with additional anomalies affecting the long bones (nonsyndromic form) or other organ systems including the craniofacial, genitourinary and ectodermal structures (syndromic ectrodactyly). This study reports a girl with SHFM who also exhibited developmental delay, mild dysmorphic facial features and sensorineural hearing loss. High-resolution banding analysis indicated an interstitial deletion within the 7q21 band. FISH using locus-specific BAC probes confirmed the microdeletion of 7q21.3. Chromosomal microarray analysis also revealed a microdeletion of 1.856 Mb in 7q21.3. However, a larger 8.44-Mb deletion involving bands 7q21.11q21.2 was observed, and the breakpoints were refined. The phenotype and the candidate genes underlying the pathogenesis of this disorder are discussed.

Pre- and Postnatal Analysis of Chromosome 15q26.1 and 8p23.1 Deletions in Congenital Diaphragmatic Hernia.

Congenital diaphragmatic hernia (CDH) is defined as a protrusion of abdominal content into the thoracic cavity through an abnormal opening in the diaphragm present at birth. It is a common birth defect with high mortality and morbidity. Submicroscopic deletions of 15q26.1 and 8p23.1 have been reported in several cases of CDH. We studied a total of 17 cases with CDH in pre- and postnatal samples using FISH probes. Deletion 15q26.1 was seen in 1/17 prenatal samples. There was no deletion for 8p23.1 in all the samples analyzed. CDH has a genetic etiology, and deletion 15q26.1 increases the risk of CDH. Deletion 15q26.1 in a fetus with CDH is a predictor of poor prognosis. This deletion is also seen in a phenotype similar to Fryns syndrome. CDH identified pre- or postnatally should be investigated further to exclude a 15q26.1 deletion and enable appropriate parental counseling.

Pre- and Postnatal Analysis of Chromosome 1q44 Deletion in Agenesis of Corpus Callosum.

Agenesis of corpus callosum (ACC) is one of the common brain abnormalities and also a common finding in children with mental disability. ACC is heterogeneous and can occur as an isolated condition or as part of a syndrome. ACC can be accurately identified by the absence of the cavum septum pallucidum and tear drop effect of the lateral ventricle after 18 weeks of pregnancy in an ultrasound scan. Genetic causes have been attributed to 30-45% of cases with ACC. Submicroscopic deletions of 1q43q44 have been reported in several cases of ACC. The AKT3 gene, mapped to 1q44, is required for the development of the callosum and brain size. It is considered to be a candidate gene for ACC. We studied a total of 22 cases with ACC, in pre- and postnatal samples using FISH probes. None of the samples showed a deletion in 1q44, implying that the AKT3 gene may not be associated with ACC.

Splice, insertion-deletion and nonsense mutations that perturb the phenylalanine hydroxylase transcript cause phenylketonuria in India.

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by mutational inactivation of the phenylalanine hydroxylase (PAH) gene. Missense mutations are the most common PAH mutation type detected in PKU patients worldwide. We performed PAH mutation analysis in 27 suspected Indian PKU families (including 7 from our previous study) followed by structure and function analysis of specific missense and splice/insertion-deletion/nonsense mutations, respectively. Of the 27 families, disease-causing mutations were detected in 25. A total of 20 different mutations were identified of which 7 "unique" mutations accounted for 13 of 25 mutation positive families. The unique mutations detected exclusively in Indian PKU patients included three recurrent mutations detected in three families each. The 20 mutations included only 5 missense mutations in addition to 5 splice, 4 each nonsense and insertion-deletion mutations, a silent variant in coding region and a 3'UTR mutation. One deletion and two nonsense mutations were characterized to confirm significant reduction in mutant transcript levels possibly through activation of nonsense mediated decay. All missense mutations affected conserved amino acid residues and sequence and structure analysis suggested significant perturbations in the enzyme activity of respective mutant proteins. This is probably the first report of identification of a significantly low proportion of missense PAH mutations from PKU families and together with the presence of a high proportion of splice, insertion-deletion, and nonsense mutations, points to a unique PAH mutation profile in Indian PKU patients.

Variation at diabetes- and obesity-associated Loci may mirror neutral patterns of human population diversity and diabetes prevalence in India.

South Asian populations harbor a high degree of genetic diversity, due in part to demographic history. Two studies on genome-wide variation in Indian populations have shown that most Indian populations show varying degrees of admixture between ancestral north Indian and ancestral south Indian components. As a result of this structure, genetic variation in India appears to follow a geographic cline. Similarly, Indian populations seem to show detectable differences in diabetes and obesity prevalence between different geographic regions of the country. We tested the hypothesis that genetic variation at diabetes- and obesity-associated loci may be potentially related to different genetic ancestries. We genotyped 2977 individuals from 61 populations across India for 18 SNPs in genes implicated in T2D and obesity. We examined patterns of variation in allele frequency across different geographical gradients and considered state of origin and language affiliation. Our results show that most of the 18 SNPs show no significant correlation with latitude, the geographic cline reported in previous studies, or by language family. Exceptions include KCNQ1 with latitude and THADA and JAK1 with language, which suggests that genetic variation at previously ascertained diabetes-associated loci may only partly mirror geographic patterns of genome-wide diversity in Indian populations.

Characterization of TTAGG telomeric repeats, their interstitial occurrence and constitutively active telomerase in the mealybug Planococcus lilacinus (Homoptera; Coccoidea).

We confirmed the occurrence of the insect TTAGG telomeric repeats in the mealybug Planococcus lilacinus, a radiation-resistant coccid, by single primer polymerase chain reaction (PCR) and Southern hybridization. Analysis of Bal31 nuclease-digested DNA by Southern hybridization and chromosomes by FISH suggests that these repeats occur mainly at the ends of the chromosomes. However, sequence analysis of the PCR products of TTAGG-associated sequences from genomic DNA showed their interstitial occurrence and association with certain unrelated low-copy repeats. Because of their shorter length, the interstitial TTAGG sequences were detectable by primed in situ hybridizations but not by FISH. Analysis of chromosomes recovered after irradiation by fluorescent in situ hybridization suggested acquisition of TTAGG repeats at a majority of the healed ends. We also observed mild telomerase activity in unirradiated insects which was further enhanced after irradiation. Taken together, these results suggest that the mealybug has an efficient mechanism of formation of TTAGG repeats at radiation-induced chromosome ends and constitutively active telomerase may be a feature associated with rapid recovery of chromosome ends damaged by ionizing radiation.

Deletion of RBM and DAZ in azoospermia: evaluation by PRINS.

Molecular and cytogenetic studies from infertile men have shown that one or more genes controlling spermatogenesis are located in proximal Yq11.2 in interval 6 of the Y chromosome. Microdeletions within the azoospermia factor region (AZF) are often associated with azoospermia and severe oligospermia in men with idiopathic infertility. We evaluated cells from a normal-appearing 27-year-old man with infertility and initial karyotype of 45,der(X)t(X;Y)(p22.3;p11.2)[8]/46,t(X;Y)(p22.3;p11.2)[12]. By fluorescence in situ hybridization with dual-color whole chromosome paint probes for X and Y chromosomes, we confirmed the Xp-Yp interchange. By primed in situ labeling, we identified translocation of the SRY gene from its original location on Yp to the patient's X chromosome at band Xp22. We also obtained evidence that the apparent marker was a der(Y) (possibly a ring) containing X and Y domains, and observed that the patient's genome was deleted for RBM and DAZ, two candidate genes for AZF.

Prader Willi/Angelman and DiGeorge/velocardiofacial syndrome deletions: diagnosis by primed in situ labeling (PRINS).

A recently developed methodology-primed in situ labeling (PRINS)-can be used in place of fluorescence in situ hybridization (FISH) to diagnose microdeletions. To demonstrate the efficiency, sensitivity, and specificity of PRINS in the diagnosis of microdeletions, we studied groups of patients with Prader Willi/Angelman (PWS/AS) syndrome and DiGeorge/velocardiofacial syndrome (DGS/VCFS). Results obtained by PRINS were then confirmed with the results obtained with FISH. Oligonucleotide primers specific for SNRPN and GABRB3 were used for PWS/AS syndromes. For DGS/VCFS, the primers used were DGCR2/TUPLE1 loci. Labeling patterns obtained by PRINS and FISH were analyzed and scored under a fluorescence microscope. Five normal subjects served as controls and were used for standardization of the PRINS protocol. In all, 20 study patients were involved: 10 PWS/AS and 10 DGS/VCFS. Five of the 10 patients referred with the clinical diagnosis of PWS/AS showed absence of labeling for SNRPN and GABRB3 on one chromosome 15, confirming deletion of the two loci. Similarly, 6 of the 10 patients referred for DGS/VCFS showed deletion for the DGCR2/TUPLE1 loci on one chromosome 22. The remaining patients and controls had normal patterns for all the loci as indicated by FISH and PRINS. Concordant FISH and PRINS results were obtained in all patients and controls studied.

Primed in situ labeling (PRINS) for evaluation of gene deletions in cancer.

Rearrangements involving the 13q14 and 17p13 chromosomal regions are often observed in leukemias and lymphomas. These rearrangements are not always identifiable cytogenetically. In more than 50% of cases, deletions occur at the submicroscopic level and the karyotypes appear normal. Molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH) have accordingly contributed to the identification of a variety of subtle rearrangements such as those involving submicroscopic deletions. However, FISH is expensive, time consuming, technically burdensome, and requires cloned DNA probes. A newer technique, primed in situ labeling (PRINS), has been tested as a possible alternative to FISH. To assess the utility and efficiency of the PRINS method in the detection of RB1 and p53 deletions, we evaluated 10 patients with hematological disorders and known rearrangements, i.e., deletions involving 13q14 and 17p13 regions. The data in these cases were validated against data obtained with standard FISH probes. Our results indicate that PRINS could be used with relative ease in cytogenetics laboratories and could serve as an alternative to conventional FISH for defining deletions involving unique sequences.