Teena Koshy

Cytogenetic Analysis of Patients with Primary Amenorrhea

Abstract Amenorrhea is a normal clinical feature in prepubertal, pregnant, and postmenopausal females. It also accounts for 20% of patients with infertility. The physiology of menstruation and reproduction has a strong correlation with the expression of the X chromosome. Thus, the role of genetics in terms of diagnosis, risk assessment, and genetic counseling is significant. The genetic contribution to amenorrhea is studied both at the cellular and molecular level aiming at abnormalities in chromosomes and mutations in genes. The present study aimed at performing chromosomal analysis in patients present with primary amenorrhea (n=140) employing GTG banding. The karyotype results revealed 71.2% (n=101) with normal chromosome composition and 27.8% (n=39) showed chromosomal abnormalities. In patients with abnormal chromosome constituents, 74% (n=29) exhibit numerical aberration and 26% (n=10) showed structural abnormalities. The X-chromosome abnormality was observed in 49% of the subject population which is consistent with results of studies conducted in the past. Also, the involvement of Y chromosome and origin of marker chromosome was confirmed by FISH in four patients.

Differential expression of microRNAs let-7a, miR-125b, miR-100, and miR-21 and interaction with NF-kB pathway genes in periodontitis pathogenesis

Periodontitis is a chronic inflammatory disease which is caused by destruction of the tissues that surrounds and supports the tooth. Deregulation of microRNAs has been reported to cause several inflammatory diseases such as autoimmune disease, chronic periodontitis, and cancer. In the present study, we have investigated the expression pattern of microRNAs let‐7a, miR‐125b, miR‐100, miR‐21, and RNA‐binding protein LIN‐28A among healthy individuals and chronic periodontitis patients. Total RNA was isolated from gingival tissue samples collected from 100 healthy individuals and 100 chronic periodontitis patients. The expression of microRNAs and LIN‐28 was performed by qPCR. Target prediction for the microRNAs was done using miRWalk and miRTarbase online databases and the experimentally validated targets were analyzed for their molecular function, biological processes, and related pathways using gProfiler software. The expression analysis revealed that let‐7a and miR‐21 were upregulated, whereas, miR‐100, miR‐125b, and LIN‐28 were down regulated. The age dependent expression analysis revealed that the expression levels of all the microRNAs and LIN‐28 were found to increase with age (more than 50 years), thereby suggesting an increased risk to chronic periodontitis. Among the various targets predicted using miRWalk and miRTarbase databases, NFKB was found to be a common target among all the four microRNAs. gProfiler revealed several functions such as NF‐ĸB signaling pathway, cytokine‐cytokine receptor interaction, osteoclast differentiation, etc., all of which specific to inflammation and periodontitis.

Class II Analphoid Chromosome in a Child with Aberrant Chromosome 7: A Rare Cytogenetic Association.

A neocentromere is a functional centromere that has arisen within a region not known to have a centromere. We present a case with a very rarely reported class II neocentromere formation in an aberrant chromosome 7. A 22-month-old male was referred because of dysmorphic features. Banding cytogenetics was performed, and a ring 7 and a supernumerary marker chromosome along with a normal chromosome 7 were found. In situ hybridization using a centromeric probe revealed 46 signals, of which 2 signals for chromosome 7 were observed, one on the normal and one on the ring chromosome. Further analysis using FISH revealed that the linear acentric fragment was part of the 7q region, which suggests that there could be a possible McClintock mechanism.

Recurrence of Angelman Syndrome in Siblings: Challenges in Genetic Counseling

The authors report a rare occurrence of two siblings with Angelman syndrome. Their karyotype revealed monosomy of chromosome 15 and a derivative chromosome 1 leading to Angelman syndrome. Their mother was a balanced translocation carrier involving chromosomes 1p and 15p. In her subsequent pregnancy, prenatal karyotype analysis was offered and the fetus was unaffected.

Reciprocal Microduplication of the Williams-Beuren Syndrome Chromosome Region in a 9-Year-Old Omani Boy

BACKGROUND Microdeletions of the 7q11.23 Williams-Beuren syndrome chromosome region (WBSCR) are reported with a frequency of 1 in 10,000, whereas microduplications of the region, although expected to occur at the same frequency, are not widely reported. METHOD We evaluated a 9-year old Omani boy for idiopathic intellectual disability using genetic methods, including multiplex ligation-dependent probe amplification (MLPA), for detection of microdeletions (P064-B3). RESULTS MLPA analysis revealed that the boy has a rare microduplication of the WBSCR. Prominent clinical features include global developmental delay with pronounced speech delay, dysmorphic facies, and autistic features. CONCLUSION Microduplications, in general, are reported at a lesser frequency, perhaps owing to their milder phenotype. Complete genetic assessment in children with idiopathic intellectual disability would help in identifying rare conditions such as duplication of the WBSCR.

Mutation Analysis of TBX1 in Children with Conotruncal Heart Anomalies

To the Editor: Conotruncal heart anomalies (CTA) are structural malformations involving the outflow tract. While the exact incidence of CTA in India is not known, it remains the most common type of structural birth defect with a major impact on pediatric morbidity and mortality. While most CTA are sporadic, a few are associated with genetic syndromes; the 22q11 deletion syndrome (22q11.2DS) being the predominate one. The CTA related to the 22q11.2DS are usually associated with a common 3 Mb or 1.5 Mb proximally deleted region, both of which include the TBX1 gene. However, mutations of the TBX1 gene have also been reported in patients who do not have the 22q11.2 deletion but present with CTA. The TBX1 gene encodes a transcription factor of the T-box family and mouse models have demonstrated that TBX1 haploinsufficiency cause cardiac outflow tract lesions. In a casecontrol study involving 96 cases of CTA and 100 control subjects, ranging in age from newborns to 18 y, fluorescence in situ hybridization (FISH) was performed on the cases to rule out the 22q11.2 μ-deletion. Further, screening for mutations or sequence variants in four exons of the Tbox region, which showed 98 % homology to mouse TBX1, was performed using Sanger sequencing. One out of the 96 cases with CTA (1 %) was found to have the 22q11.2 μ-deletion. However, no pathogenic mutations or sequence variants of TBX1 were detected in the patients and healthy controls. While this is in agreement with the report by Conti et al. [1], it is also in contrast with a few studies that have documented either mutations or polymorphisms in TBX1 associated with isolated CTA [2–5]. Our results, though negative, provide corroborative evidence that TBX1 mutations may not be associated with CTA in the selected pediatric population.

Subtelomeric rearrangements in Indian children with idiopathic intellectual disability/developmental delay: Frequency estimation & clinical correlation using fluorescence in situ hybridization (FISH)

Background & objectives: Subtelomeres are prone to deleterious rearrangements owing to their proximity to unique sequences on the one end and telomeric repetitive sequences, which increase their tendency to recombine, on the other end. These subtelomeric rearrangements resulting in segmental aneusomy are reported to contribute to the aetiology of idiopathic intellectual disability/developmental delay (ID/DD). We undertook this study to estimate the frequency of subtelomeric rearrangements in children with ID/DD. Methods: One hundred and twenty seven children with idiopathic ID/DD were tested for subtelomeric rearrangements using karyotyping and FISH. Blood samples were cultured, harvested, fixed and GTG-banded using the standard protocols. Results: Rearrangements involving the subtelomeres were observed in 7.8 per cent of the tested samples. Detection of rearrangements visible at the resolution of the karyotype constituted 2.3 per cent, while those rearrangements detected only with FISH constituted 5.5 per cent. Five deletions and five unbalanced translocations were detected. Analysis of parental samples wherever possible was informative regarding the inheritance of the rearrangement. Interpretation & conclusions: The frequency of subtelomeric rearrangements observed in this study was within the reported range of 0-35 per cent. All abnormal genotypes were clinically correlated. Further analysis with array technologies presents a future prospect. Our results suggest the need to test individuals with ID/DD for subtelomeric rearrangements using sensitive methods such as FISH.

Isolation and evaluation of cytogenetic effect of Brahmi saponins on cultured human lymphocytes exposed in vitro

Major saponins of Brahmi (Bacopa monniera, Fam: Scrophulariaceae) – bacosides A and B – were isolated from the total methanol extract and characterised based on melting point, TLC, IR, 1H NMR and 13C NMR. They were evaluated for their in vitro cytogenetic effects on human peripheral blood lymphocytes by chromosomal aberration (CA) assay and sister chromatid exchange (SCE) assay. The frequency of chromatid type aberrations and reciprocal interchanges between sister chromatids in the treated cells was scored in comparison to the untreated control. At 30 μg/mL dose, bacoside A showed a statistically significant increase in the frequency of both CA and SCE and bacoside B showed an increase only in SCE. Our report of the genotoxicity of the saponins is significant in view of the reports of anticancer activity of Brahmi extracts.

Association of estrogen, progesterone and follicle stimulating hormone receptor polymorphisms with in vitro fertilization outcomes

ABSTRACT Despite the advances in in vitro fertilization (IVF), the implantation success rate for infertile women remains approximately only 15%. In this study, we sought to determine whether implantation failure after repeated IVF treatments is influenced by the presence of common variants in estrogen α, progesterone and follicle stimulating hormone receptor genes. The study population included three groups of women: group 1 were 50 women who had the transfer of ≥3 high-quality embryos during the IVF procedure without ever having had a clinical pregnancy; group 2 were 50 women who achieved a clinical pregnancy after ≤3 high-quality embryos transfers and group 3 were 50 control subjects who achieved a clinical pregnancy without any fertility therapy that resulted in a one live-born infant. Genotype analysis was performed using polymerase chain reaction and Sanger sequencing for rs6165, rs6166, rs2234693, rs9340799. While progesterone receptor single nucleotide polymorphism (SNP) was genotyped based on the amplicon size, the repeats for the ESR1 TA-repeat polymorphism were calculated based on the fragment length. A higher frequency of the heterozygote AG genotype was observed in the infertile groups when compared to controls. Significantly, an allele combination of T of rs2234693, A of rs9340799; S of ESR1 (TA), A of rs6166, G of rs6165 and del of PROGINS had a higher frequency in women who had a successful IVF outcome compared to women who had an unsuccessful IVF outcome, indicating a possible protective combined genotype that could reduce a negative outcome during IVF. This study has demonstrated that combining several candidate genes is needed to assess which may play a role in fertility. Abbreviations: CI: confidence interval; COH: controlled ovarian hyperstimulation; DNA: deoxyribonucleic acid; ESR: estrogen receptors; FSH: follicle stimulating hormones; FSHR: FSH receptor; IVF: in vitro fertilization; PGR: progesterone receptors; SNP: single nucleotide polymorphism

A retrospective exploratory study of fetal genetic invasive procedures at a University Hospital

Abstract This is a retrospective analysis of the patient demographics and cytogenetic results of patients who underwent prenatal invasive testing for genetic analysis at the Foetal Medicine Division of the Department of Obstetrics and Gynecology, Sri Ramachandra Medical College and Research Institute. The main objective of this study was to characterise the changing trends in indications of pregnant women for foetal karyotyping in a 7-year period. A total of 257 procedures were performed in this period, and there was a significant change in the trend of indications for invasive prenatal diagnosis from an advanced maternal age in 2009 to a positive screen test by 2014. Chromosome abnormalities were observed in 9.8% of the cases, with trisomy 21 being the most frequent finding. The findings demonstrate the changing trends in screening and diagnostic testing in the tertiary care centre, with an acceptance of the first and second trimester maternal serum screening tests as a determinant for high-risk pregnancies. Impact statement What is already known on this subject? Despite the fact that India has one of the world’s highest birth rates, there is still no public health care policy for the application of cytogenetic prenatal diagnosis. Nevertheless, we have been offering this test in our university teaching hospital since 2008, allowing us to characterise the changing trends in indications of pregnant women who sought invasive diagnostic procedures for foetal genetic studies. What do the results of this study add? The results of our study show that there were major changes in the common indications for prenatal diagnosis during the study period. In 2009, the main indication was an advanced maternal age, referred to in 31% of the cases, which declined steadily to 5% by 2014. In 2014, 51% of cases opted for a prenatal diagnosis because of a first trimester screen positive result, increasing from 12% in 2009. What are the implications of these findings for clinical practice and/or further research? This data is relevant as it would encourage other tertiary hospitals in developing countries like India to consider extending first trimester screening for all women, regardless of age and educate them on the options of prenatal genetic diagnosis for reassurance.