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.

The A1298C Methylenetetrahydrofolate Reductase Gene Variant as a Susceptibility Gene for Non-Syndromic Conotruncal Heart Defects in an Indian Population.

Conotruncal heart defects (CTHDS) are a subgroup of congenital heart malformations that are considered to be a folate-sensitive birth defect. It has been hypothesized that polymorphisms in genes that code for key enzymes in the folate pathway may alter enzyme activity, leading to disruptions in folate metabolism and thus may influence the risk of such heart defects. This study was designed to investigate the association of six selected folate-metabolizing gene polymorphisms with the risk of non-syndromic CTHDs in an Indian population. This was a case-control study involving 96 cases of CTHDs and 100 control samples, ranging in age from birth to 18 years. Genotyping using Sanger sequencing was performed for six single nucleotide polymorphisms of genes involved in folate metabolism. Logistic regression analyses revealed that for the 5,10-methylenetetrahydrofolate (MTHFR) A1298C polymorphism, the CC variant homozygote genotype was associated with a significantly increased risk of CTHDs. The results of this study support an association between the inherited MTHFR A1298C genotype and the risk of CTHDs in an Indian population.

Generation of Induced Pluripotent Stem Cells from Turner Syndrome (45XO) Fetal Cells for Downstream Modelling of Neurological Deficits Associated with the Syndrome.

Chromosomal aneuploidies cause severe congenital malformations including central nervous system malformations and fetal death. Prenatal genetic screening is purely diagnostic and does not elucidate disease mechanism. Although cells from aneuploid fetuses are valuable biological material bearing the chromosomal aneuploidy, these cells are short lived, limiting their use for downstream research experiments. Generation of induced pluripotent stem cell (iPSC) models is an effective method of cell preparation for perpetual conservation of aneuploid traits. They are self-renewing and differentiate into specialized cells reminiscent of embryonic development. Thus, iPSCs serve as excellent tools to study early developmental events. Turner syndrome (TS) is a rare condition associated with a completely or partially missing X chromosome. The syndrome is characterized by infertility, short stature, endocrine, metabolic, autoimmune and cardiovascular disorders and neurocognitive defects. The following protocol describes isolation and culturing of fibroblasts from TS (45XO) fetal tissue, generation of integration free TSiPSCs through delivery of episomal reprogramming plasmids by nucleofection followed by characterization. The reprogramming TSiPSCs were initially screened by live cell alkaline phosphatase staining followed by extensive probing for pluripotency biomarkers. Selected colonies were mechanically dissected, passaged several times and stable self-renewing cells were used for further experiments. The cells expressed pluripotency transcription factors OCT4, NANOG, SOX2, cell surface markers SSEA 4 and TRA1-81 typical of pluripotent stem cells. The original 45XO karyotype was retained post reprogramming. The TSiPSCs were able to form embryoid bodies and differentiate into cells of endoderm, mesoderm and ectoderm expressing lineage specific biomarkers ((SRY BOX17), (MYOSIN VENTRICULAR HEAVY CHAINα/ß), (ßIII TUBULIN)). The exogenous episomal plasmids were lost spontaneously and not detected after passage 15 in cells. These TSiPSCs are a valuable cellular resource for modelling defective molecular and cellular neurodevelopment causing neurocognitive deficits associated with Turner syndrome.

Novel glioblastoma markers with diagnostic and prognostic value identified through transcriptome analysis.

PURPOSE: Current methods of classification of astrocytoma based on histopathologic methods are often subjective and less accurate. Although patients with glioblastoma have grave prognosis, significant variability in patient outcome is observed. Therefore, the aim of this study was to identify glioblastoma diagnostic and prognostic markers through microarray analysis. EXPERIMENTAL DESIGN: We carried out transcriptome analysis of 25 diffusely infiltrating astrocytoma samples [WHO grade II--diffuse astrocytoma, grade III--anaplastic astrocytoma, and grade IV--glioblastoma (GBM)] using cDNA microarrays containing 18,981 genes. Several of the markers identified were also validated by real-time reverse transcription quantitative PCR and immunohistochemical analysis on an independent set of tumor samples (n = 100). Survival analysis was carried out for two markers on another independent set of retrospective cases (n = 51). RESULTS: We identified several differentially regulated grade-specific genes. Independent validation by real-time reverse transcription quantitative PCR analysis found growth arrest and DNA-damage-inducible alpha (GADD45alpha) and follistatin-like 1 (FSTL1) to be up-regulated in most GBMs (both primary and secondary), whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 were up-regulated in the majority of primary GBM. Further, identification of the grade-specific expression of GADD45alpha and FSTL1 by immunohistochemical staining reinforced our findings. Analysis of retrospective GBM cases with known survival data revealed that cytoplasmic overexpression of GADD45alpha conferred better survival while the coexpression of FSTL1 with p53 was associated with poor survival. CONCLUSIONS: Our study reveals that GADD45alpha and FSTLI are GBM-specific whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 are primary GBM-specific diagnostic markers. Whereas GADD45alpha overexpression confers a favorable prognosis, FSTL1 overexpression is a hallmark of poor prognosis in GBM patients.

Analysis of telomere damage by fluorescence in situ hybridisation on micronuclei in lymphocytes of breast carcinoma patients after radiotherapy.

Radiotherapy has become an indispensable tool in the effective management of most of the cancers. There have been efforts earlier to study the differential radio-sensitivity patterns in patients undergoing radiation treatment to correlate with treatment induced complications such as tissue injury, cell death, and chromosomal aberration frequencies etc. The present study is an attempt to correlate the radiation-induced damage in the peripheral blood lymphocytes (PBLs) of breast cancer patients with the frequency of telomere mediated chromosomal damage. Blood samples from 55 patients with (Gr-II and Gr-III) CA-breast were obtained pre- and post-radiotherapy. The patients were treated with external beam radiotherapy of 50.4 Gy over a period of 6 weeks. Chromosome damage was measured by analysing micronucleus (MN) frequency in PBLs. The MN-frequency of the irradiated patients increased significantly compared to the patients being self-controls. The micronuclei were hybridized with telomere probes to study the extent of telomere damage. The fluorescence signals of the telomere regions in the first generation of the binucleated cells were significantly higher in the post-radiotherapy patients. There was also significant correlation observed in the patients with higher-grade tumours. Inter-individual variability was observed in the radiation-induced MN frequency in lymphocytes of patients after six weeks of radiotherapy. There was a significant correlation between functionally intact telomeres and the cellular response to ionising radiation. Our findings suggest that fluorescence in situ hybridisation on micronuclei could be effectively used as routine clinical application to determine the individual sensitivity to ionising radiation with respect to telomere damage.

Single Nucleotide Polymorphism-Based Noninvasive Prenatal Testing: Experience in India.

INTRODUCTION: Noninvasive prenatal testing (NIPT) has revolutionized prenatal screening for chromosomal aneuploidies in some countries. Its implementation has been sporadic in developing countries. Given the genetic variation of the people in different countries, we evaluated the performance of the SNP-based NIPT in India . MATERIALS AND METHODS: The Panorama™ NIPT was performed in 516 pregnancies, which had tested intermediate-to-high risk on conventional first and second trimester screening. Results were confirmed either by invasive diagnostic testing or by clinical evaluation after birth. RESULTS: Of 511 samples analyzed, results were obtained in 499 (97.7%). Of these, 480 (98.2%) were low risk and 19 were high risk. A sensitivity of 100% was obtained for detection of trisomies 21, 18, 13 and sex chromosomal abnormalities. The specificity ranged from 99.3 to 100% for abnormalities tested. Taken together, the positive predictive value for trisomies 21, 18, 13 and monosomy X was 85.7%. The average fetal fraction was 8.2%, which is lower than the average observed elsewhere. CONCLUSION: This is the first report of detailed experience with NIPT in India and demonstrates comparable performance in all aspects of testing to the results elsewhere.

Shift in AP-2alpha localization characterizes astrocytoma progression.

Activator protein 2alpha (AP-2alpha) has been shown to be lost in the advanced stages of many cancers, including gliomas. In this study, we wanted to analyze the expression of AP-2alpha in astrocytoma samples of different grades both at the RNA level, by real-time qPCR and at the protein level, by immunohistochemistry, and to examine its correlation, if any, with patient outcome. Five Grade I, 14 Grade II, 18 Grade III, 72 Grade IV samples and 13 normal brain controls were included. We did not find any clear pattern of regulation at the RNA level with tumor grade. The RNA expression levels however, correlated to a large extent with the nuclear AP-2alpha staining in these samples (72.09%; 31/43). Further, we did not find a complete loss of nuclear AP-2alpha expression in the higher grades, in contrast to previous reports. Interestingly, we found cytoplasmic AP-2alpha expression in a majority of higher grade astrocytomas (Grade IV-85%; 33/39 and Grade III-74%; 14/19) in comparison to lower grades (Grade I-0%; 0/5 and Grade II-37.5%; 3/8) suggesting that the translocation of this protein from the nucleus to the cytoplasm may be responsible for the increased malignancy. The nuclear expression in these grades was found to be concomitantly reduced. Within GBMs, we found that decreased nuclear expression was indicative of a better prognosis. The striking observation was the shift in localization of this protein from the nucleus to the cytoplasm with increasing tumor grade, pointing to a crucial role for this transcription factor in the progression of astrocytomas.

Effects of an integrated yoga program in modulating psychological stress and radiation-induced genotoxic stress in breast cancer patients undergoing radiotherapy.

Effects of an integrated yoga program in modulating perceived stress levels, anxiety, as well as depression levels and radiation-induced DNA damage were studied in 68 breast cancer patients undergoing radiotherapy. Two psychological questionnaires--Hospital Anxiety and Depression Scale (HADS) and Perceived Stress Scale (PSS)--and DNA damage assay were used in the study. There was a significant decrease in the HADS scores in the yoga intervention group, whereas the control group displayed an increase in these scores. Mean PSS was decreased in the yoga group, whereas the control group did not show any change pre- and postradiotherapy. Radiation-induced DNA damage was significantly elevated in both the yoga and control groups after radiotherapy, but the postradiotherapy DNA damage in the yoga group was slightly less when compared to the control group. An integrated approach of yoga intervention modulates the stress and DNA damage levels in breast cancer patients during radiotherapy.

A Homozygous Missense Variant in INPP5E Associated with Joubert Syndrome and Related Disorders.

Joubert syndrome and related disorders (JSRD; ORPHA 140874) is a complex set of neurodevelopmental disorders with multiple organ involvement. JSRD is a type of ciliopathy which is caused by the presence of defective primary cilia in an individual. JSRD is commonly inherited in an autosomal recessive pattern, and more than 23 genes are known to be associated with JSRD. We report a novel homozygous mutation identified in the INPP5E gene, c.1303C>T, which leads to a change of an amino acid from arginine to tryptophan at residue 435 in the protein chain. In silico analysis indicates that p.Arg435Trp substitution affects the functionality of the protein product of the gene. Our result adds to the growing body of evidences that underlines the clinical utility of next-generation sequencing in the diagnosis of a genetic disorder when clinical features are inconclusive.

Upregulation of ASCL1 and inhibition of Notch signaling pathway characterize progressive astrocytoma.

Astrocytoma is the most common type of brain cancer constituting more than half of all brain tumors. With an aim to identify markers describing astrocytoma progression, we have carried out microarray analysis of astrocytoma samples of different grades using cDNA microarray containing 1152 cancer-specific genes. Data analysis identified several differentially regulated genes between normal brain tissue and astrocytoma as well as between grades II/III astrocytoma and glioblastoma multiforme (GBM; grade IV). We found several genes known to be involved in malignancy including Achaete-scute complex-like 1 (Drosophila) (ASCL1; Hash 1). As ASCL has been implicated in neuroendocrine, medullary thyroid and small-cell lung cancers, we chose to examine the role of ASCL1 in the astrocytoma development. Our data revealed that ASCL1 is overexpressed in progressive astrocytoma as evidenced by increased levels of ASCL1 transcripts in 85.71% (6/7) of grade II diffuse astrocytoma (DA), 90% (9/10) of grade III anaplastic astrocytoma (AA) and 87.5% (7/8) of secondary GBMs, while the majority of primary de novo GBMs expressed similar to or less than normal brain levels (66.67%; 8/12). ASCL1 upregulation in progressive astrocytoma is accompanied by inhibition of Notch signaling as seen by uninduced levels of HES1, a transcriptional target of Notch1, increased levels of HES6, a dominant-negative inhibitor of HES1-mediated repression of ASCL1, and increased levels of Notch ligand Delta1, which is capable of inhibiting Notch signaling by forming intracellular Notch ligand autonomous complexes. Our results imply that inhibition of Notch signaling may be an important early event in the development of grade II DA and subsequent progression to grade III AA and secondary GBM. Furthermore, ASCL1 appears to be a putative marker to distinguish primary GBM from secondary GBM.

Exonic microdeletions in the X-linked PQBP1 gene in mentally retarded patients: a pathogenic mutation and in-frame deletions of uncertain effect.

Mutations in PQBP1 were recently identified in families with syndromic and non-syndromic X-linked mental retardation (XLMR). Clinical features frequently associated with MR were microcephaly and/or short stature. The predominant mutations detected so far affect a stretch of six AG dinucleotides in the polar-amino-acid-rich domain (PRD), causing frameshifts in the fourth coding exon. We searched for PQBP1 exon 4 frameshifts in 57 mentally retarded males in whom initial referral description indicated at least one of the following criteria: microcephaly, short stature, spastic paraplegia or family history compatible with XLMR, and in 772 mentally retarded males not selected for specific clinical features or family history. We identified a novel frameshift mutation (23 bp deletion) in two half-brothers with specific clinical features, and performed prenatal diagnosis in this family. We also found two different 21 bp in-frame deletions (c.334-354del(21 bp) and c.393-413del(21 bp)) in four unrelated probands from various ethnic origins, each deleting one of five copies of an imperfect seven amino-acid repeat. Although such deletions have not been detected in 1180 X chromosomes from European controls, the c. 334-354del(21 bp) was subsequently found in two of 477 Xs from Indian controls. We conclude that pathogenic frameshift mutations in PQBP1 are rare in mentally retarded patients lacking specific associated signs and that the 21 bp in-frame deletions may be non-pathogenic, or alternatively could act subtly on PQBP1 function. This touches upon a common dilemma in XLMR, that is, how to distinguish between mutations and variants that may be non-pathogenic or represent risk factors for cognitive impairment.

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.

Alterations in tumour suppressor gene p53 in human gliomas from Indian patients.

Alterations in tumour suppressor p53 gene are the most common defects seen in a variety of human cancers. In order to study the significance of the p53 gene in the genesis and development of human glioma from Indian patients, we checked 44 untreated primary gliomas for mutations in exons 5-9 of the p53 gene by PCR-SSCP and DNA sequencing. Sequencing analysis revealed six missense mutations. The incidence of p53 mutations was 13.6% (6 of 44). All the six mutations were found to be located in the central core domain of p53, which carries the sequence-specific DNA-binding domain. These results suggest a rather low incidence but a definite involvement of p53 mutations in the gliomas of Indian patients.

Freeman-Sheldon syndrome--prenatal and postnatal diagnosis.

A six-day-old girl, born to normal non-consanguineous parents presented with mask like facies with a small mouth giving a 'whistling' appearance. Other dysmorphic features include deep set eyes, broad nasal bridge, long philtrum and 'H' shaped cutaneous dimple on the chin. There was congenital windmill vane hand position and severe talipes equinovarus deformity. The above features are characteristic of Freeman-Sheldon syndrome also known as Whistling Face syndrome. Ultrasound scanning during 8(th) month of the pregnancy showed the fetus to have facial abnormality and bilateral clenched hand and talipes with extension contractures of knees. Provisional diagnosis of FSS was made which was confirmed after the birth. Thus all cases of Arthrogryposis during prenatal scan should be carefully looked for the facial abnormality in the fetus.

Differential protein expression in human gliomas and molecular insights.

Gliomas are the most common of the primary intracranial tumors with astrocytomas constituting about 40%. Using clinically and histologically assessed astrocytomas, we have studied their protein profiles using a two-dimensional gel electrophoresis-mass spectrometry approach and identified differentially expressed proteins which may be useful molecular indicators to understand these tumors. Examination of the protein profiles of 27 astrocytoma samples of different grades revealed 72 distinct, differentially expressed proteins belonging to various functional groups such as cytoskeleton and intermediate filament proteins, heat shock proteins (HSPs), enzymes and regulatory proteins. Based on the consistency of their differential expression, 29 distinct proteins could be short-listed and may have a role in the pathology of astrocytomas. Some were found to be differentially expressed in both Grade III and IV astrocytomas while others were associated with a particular grade. A notable observation was underexpression of Prohibitin, a potential tumor suppressor protein, Rho-GDP dissociation inhibitor, Rho-GDI, a regulator of Rho GTPases and HSPs as well as destabilization of glial fibrillary acidic protein, GFAP, major protein of the glial filaments, in Grade III malignant tumors. We attempt to explain glioma malignancy and progression in terms of their combined role.