Machine learning-assisted prediction of trabeculectomy outcomes among patients of juvenile glaucoma by using 5-year follow-up data.

OBJECTIVE: To develop machine learning (ML) models, using pre and intraoperative surgical parameters, for predicting trabeculectomy outcomes in the eyes of patients with juvenile-onset primary open-angle glaucoma (JOAG) undergoing primary surgery. SUBJECTS: The study included 207 JOAG patients from a single center who met the following criteria: diagnosed between 10 and 40 years of age, with an IOP of >22 mmHg in the eyes on two or more occasions, open angle on gonioscopy in both eyes, with glaucomatous optic neuropathy, and requiring a trabeculectomy for IOP control. Only the patients with a minimum 5-year follow-up after surgery were included in the study. METHODS: A successful surgical outcome was defined as IOP ≤18 mmHg (criterion A) or 50% reduction in IOP from baseline (criterion B) 5 years after trabeculectomy. Feature selection techniques were used to select the most important contributory parameters, and tenfold cross-validation was used to evaluate model performance. The ML models were evaluated, compared, and prioritized based on their accuracy, sensitivity, specificity, Matthew correlation coefficient (MCC) index, and mean area under the receiver operating characteristic curve (AUROC). The prioritized models were further optimized by tuning the hyperparameters, and feature contributions were evaluated. In addition, an unbiased relationship analysis among the parameters was performed for clinical utility. RESULTS: Age at diagnosis, preoperative baseline IOP, duration of preoperative medical treatment, Tenon's thickness, scleral fistulation technique, and intraoperative mitomycin C (MMC) use, were identified as the main contributing parameters for developing efficient models. The three models developed for a consensus-based outcome to predict trabeculectomy success showed an accuracy of >86%, sensitivity of >90%, and specificity of >74%, using tenfold cross-validation. The use of intraoperative MMC and a punch for scleral fistulation compared to the traditional excision with scissors were significantly associated with long-term success of trabeculectomy. CONCLUSION: Optimizing surgical parameters by using these ML models might reduce surgical failures associated with trabeculectomy and provide more realistic expectations regarding surgical outcomes in young patients.

In vivo identification of angle dysgenesis and its relation to genetic markers associated with glaucoma using artificial intelligence.

PURPOSE: To predict the presence of angle dysgenesis on anterior-segment optical coherence tomography (ADoA) by using deep learning (DL) and to correlate ADoA with mutations in known glaucoma genes. PARTICIPANTS: In total, 800 high-definition anterior-segment optical coherence tomography (AS-OCT) images were included, of which 340 images were used to build the machine learning (ML) model. Images used to build the ML model included 170 scans of primary congenital glaucoma (16 patients), juvenile-onset open-angle glaucoma (62 patients), and adult-onset primary open-angle glaucoma eyes (37 patients); the rest were controls (n = 85). The genetic validation dataset consisted of another 393 images of patients with known mutations that were compared with 320 images of healthy controls. METHODS: ADoA was defined as the absence of Schlemm's canal, the presence of hyperreflectivity over the region of the trabecular meshwork, or a hyperreflective membrane. DL was used to classify a given AS-OCT image as either having angle dysgenesis or not. ADoA was then specifically looked for on AS-OCT images of patients with mutations in the known genes for glaucoma. RESULTS: The final prediction, which was a consensus-based outcome from the three optimized DL models, had an accuracy of >95%, a specificity of >97%, and a sensitivity of >96% in detecting ADoA in the internal test dataset. Among the patients with known gene mutations, ( MYOC, CYP1B1, FOXC1, and LTBP2 ) ADoA was observed among all the patients in the majority of the images, compared to only 5% of the healthy controls. CONCLUSION: ADoA can be objectively identified using models built with DL.

Latent Tuberculosis Infection Diagnosis among Household Contacts in a High Tuberculosis-Burden Area: a Comparison between Transcript Signature and Interferon Gamma Release Assay.

Diagnosis of latent tuberculosis infection (LTBI) using biomarkers in order to identify the risk of progressing to active TB and therefore predicting a preventive therapy has been the main bottleneck in eradication of tuberculosis. We compared two assays for the diagnosis of LTBI: transcript signatures and interferon gamma release assay (IGRA), among household contacts (HHCs) in a high tuberculosis-burden population. HHCs of active TB cases were recruited for our study; these were confirmed to be clinically negative for active TB disease. Eighty HHCs were screened by IGRA using QuantiFERON-TB Gold Plus (QFT-Plus) to identify LTBI and uninfected cohorts; further, quantitative levels of transcript for selected six genes (TNFRSF10C, ASUN, NEMF, FCGR1B, GBP1, and GBP5) were determined. Machine learning (ML) was used to construct models of different gene combinations, with a view to identify hidden but significant underlying patterns of their transcript levels. Forty-three HHCs were found to be IGRA positive (LTBI) and thirty-seven were IGRA negative (uninfected). FCGR1B, GBP1, and GBP5 transcripts differentiated LTBI from uninfected among HHCs using Livak method. ML and ROC (Receiver Operator Characteristic) analysis validated this transcript signature to have a specificity of 72.7%. In this study, we compared a quantitative transcript signature with IGRA to assess the diagnostic ability of the two, for detection of LTBI cases among HHCs of a high-TB burden population; we concluded that a three gene (FCGR1B, GBP1, and GBP5) transcript signature can be used as a biomarker for rapid screening. IMPORTANCE The study compares potential of transcript signature and IGRA to diagnose LTBI. It is first of its kind study to screen household contacts (HHCs) in high TB burden area of India. A transcript signature (FCGR1B, GBP1, & GBP5) is identified as potential biomarker for LTBI. These results can lead to development of point-of-care (POC) like device for LTBI screening in a high TB burdened area.

Screening Malaria-box compounds to identify potential inhibitors against SARS-CoV-2 Mpro, using molecular docking and dynamics simulation studies.

Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) Main protease (Mpro) is one of the vital drug targets amongst all the coronaviruses, as the protein is indispensable for virus replication. The study aimed to identify promising lead molecules against Mpro enzyme through virtual screening of Malaria Venture (MMV) Malaria Box (MB) comprising of 400 experimentally proven compounds. The binding affinities were studied using virtual screening based molecular docking, which revealed five molecules having the highest affinity scores compared to the reference molecules. Utilizing the established 3D structure of Mpro the binding affinity conformations of the docked complexes were studied by Molecular Dynamics (MD) simulations. The MD simulation trajectories were analysed to monitor protein deviation, relative fluctuation, atomic gyration, compactness covariance, residue-residue map and free energy landscapes. Based on the present study outcome, we propose three Malaria_box (MB) compounds, namely, MB_241, MB_250 and MB_266 to be the best lead compounds against Mpro activity. The compounds may be evaluated for their inhibitory activities using experimental techniques.

Dataset of mutational analysis, miRNAs targeting SARS-CoV-2 genes and host gene expression in SARS-CoV and SARS-CoV-2 infections.

The identification of host-miRNAs targeting mutated virus genes is crucial to understand the miRNA mediated host-defense mechanism in virus infections. To understand the mechanism in COVID-19 infections, we collected genome sequences of SARS-CoV-2 with its metadata from the GISAID database (submitted till April 2020) and identified mutational changes in the sequences. The dataset consists of genes with mutation event count and entropy scores. We predicted host-miRNAs targeting the genes in the genomes and compared it with that in related viral species. We have identified 2284 miRNAs targeting MERS genomes, 2074 miRNAs targeting SARS genomes, and 1599 miRNAs targeting SARS-CoV-2 genomes, identified using the miRNA target prediction software miRanda. The host miRNAs targeting SARS-CoV-2 genes were further validated to be anti-viral miRNAs and their role in respiratory diseases through a literature survey, which helped in the identification of 42 conserved antiviral miRNAs. The data could be used to validate the anti-viral role of the predicted miRNAs and design miRNA-based therapeutics against SARS-CoV-2.

Integrative analyses of SARS-CoV-2 genomes from different geographical locations reveal unique features potentially consequential to host-virus interaction, pathogenesis and clues for novel therapies.

We have performed an integrative analysis of SARS-CoV-2 genome sequences from different countries. Apart from mutational analysis, we have predicted host antiviral miRNAs targeting virus genes, PTMs in the virus proteins and antiviral peptides. A comparison of the analyses with other coronavirus genomes has been performed, wherever possible. Our analysis confirms unique features in the SARS-CoV-2 genomes absent in other evolutionarily related coronavirus family genomes, which presumably confer unique infection, transmission and virulence capabilities to the virus. For understanding the crucial factors involved in host-virus interactions, we have performed Bioinformatics aided analysis integrated with experimental data related to other corona viruses. We have identified 42 conserved miRNAs that can potentially target SARS-CoV-2 genomes. Interestingly, out of these, 3 are previously reported to exhibit antiviral activity against other respiratory viruses. Gene expression analysis of known host antiviral factors reveals significant over-expression of IFITM3 and down regulation of cathepsins during SARS-CoV-2 infection, suggesting its active role in pathogenesis and delayed immune response. We also predicted antiviral peptides which can be used in designing peptide based drugs against SARS-CoV-2. Our analysis explores the functional impact of the virus mutations on its proteins and interaction of its genes with host antiviral mechanisms.

Classifying juvenile onset primary open angle glaucoma using cluster analysis.

AIM: To classify unrelated patients with juvenile onset primary open angle glaucoma (JOAG) into clinically useful phenotypes using cluster analysis. METHODS: Out of the 527 unrelated patients with JOAG, the study included 414 patients who had all the phenotypic characteristics required for the study. A cluster analysis was performed to classify the patients based on their iris and angle morphology, age of onset, highest untreated intraocular pressure (IOP), worst mean deviation and greatest vertical cup disc ratio of the worst eye. The iris features were broadly classified into three groups: those with normal iris crypts (NIC), those with prominent iris crypts (PIC) and those with absence of iris crypts. The gonio photographs were graded as normal appearing angle or those with angle dysgenesis in the form of a featureless angle, one with a high iris insertion and an angle with prominent iris processes. Using a hierarchical clustering model and a two-way cluster analysis, the distribution of clusters of JOAG was analysed to obtain a classification of JOAG subtypes. RESULTS: The four major clusters identified were: Cluster 1 with NIC and normal angles had the lowest untreated IOP and higher age of onset among all clusters. Cluster 2 with NIC and featureless angle was found to be associated with earliest age of onset. Cluster 3 had NIC and either a high iris insertion or prominent iris processes. Cluster 4 was a heterogeneous cluster with maximum number of patients in a group comprising of those with PIC and high iris insertion. CONCLUSIONS: Cluster analysis extracted four subgroups of the JOAG phenotype that have clinical and prognostic significance and can potentially be helpful while evaluating these patients in the clinics.

Molecular characterization of a rare phenotype of X-linked retinoschisis with angle-closure glaucoma.

A 11-year-old boy presented with complaints of blurred vision and on evaluation was found to have X-linked retinoschisis (XLRS) with angle-closure glaucoma. Clinical and genetic evaluation of first-degree family members was done. His brother had a milder form of XLRS with shallow anterior chamber. Topical dorzolamide 2% and timolol 0.5% were used to control intraocular pressure. Genetic analysis revealed a novel three base pair deleterious mutation (c. 375_377 del AGA) in exon-5 of the RS1 gene in three members of the family.

Characterization of a Novel POU1F1 Mutation Identified on Screening 160 Growth Hormone Deficiency Patients.

The objective of the study is the functional characterization of a novel POU1F1 c.605delC mutation in combined pituitary hormone deficiency (CPHD) and to report the clinical and genetic details of 160 growth hormone deficiency patients. Screening of GH1, GHRHR, POU1F1, PROP1, and HESX1 genes by Sanger sequencing was carried out in 160 trios and 100 controls followed by characterization of the POU1F1 c.605delC mutation by expression studies including site directed mutagenesis, co-transfection, protein degradation, and luciferase assays to compare the wild type and mutant POU1F1. In vitro studies showed that the POU1F1 c.605delC mutation codes for a truncated protein with reduced transactivation capacity on its downstream effectors, viz., growth hormone (GH) and prolactin (PRL) causing severe CPHD. Experiments using different protease inhibitors reveal rescue of the protein upon blockage of the lysosomal pathway that might be useful in novel drug designing using targeted approach thereby maintaining the milieu and preventing/delaying the disease. The study provides an insight into the disease causing mechanism of POU1F1 c.605delC mutation identified in a CPHD child with severe short stature and failure to thrive. It also shows mutation effect on the expression, function and turnover of protein and highlights mechanistic details by which these potent regulators may operate.

Mutations in membrane cofactor protein (CD46) gene in Indian children with hemolytic uremic syndrome.

BACKGROUND: Mutations in the CD46 gene account for an important proportion of patients with atypical hemolytic uremic syndrome (aHUS) who characteristically show multiple relapses, no response to plasma exchange and low recurrence risk in allograft. We screened for mutations in CD46 in patients with and without circulating anti-factor H (FH) antibodies-associated aHUS. METHODS: We estimated CD46 surface expression by flow cytometry and sequenced the CD46 gene in 23 and 56 patients with and without circulating anti-FH antibodies, respectively. Human Splicing Finder and PolyPhen2 were used for in silico prediction of pathogenicity. RESULTS: Two novel and three known (c.286 +2T > G, c.104G > A and c.565T > G) mutations in CD46 were found in nine (11.4%) patients; one patient had a variant of unknown significance and two patients presented during the first year of life. Novel intronic (c.1127 + 46C > G) and exonic (c.911C > T) mutations are proposed to activate cryptic splicing sites or alter protein conformation. Markedly reduced CD46 surface expression was found in homozygous states in five patients. CONCLUSION: Patients with mutations in CD46 present at all ages, including the first year of life. Mutations in intron 2, (c.286 +2T > G) may be a potential hot spot in Indian children. Flow cytometry for CD46 expression is a satisfactory screening tool enabling early diagnosis.

Identification of novel GHRHR and GH1 mutations in patients with isolated growth hormone deficiency.

OBJECTIVE: Human growth is an elementary process which starts at conception and continues through different stages of development under the influence of growth hormone (GH) secreted by the anterior pituitary gland. Variation affecting the production, release and functional activity of GH leads to growth hormone deficiency (GHD), which is of two types: isolated growth hormone deficiency (IGHD) and combined pituitary hormone deficiency (CPHD). IGHD may result from mutations in GH1 and GHRHR while CPHD is associated with defects in transcription factor genes PROP1, POU1F1 and HESX1. The present study reports on the molecular screening of GHRHR and GH1 in IGHD patients. METHODS: A total of 116 clinically diagnosed IGHD patients and 100 controls were enrolled for the study after taking informed consent. Family history was noted and 5ml blood sample was drawn. Anatomical and/or morphological pituitary gland alterations were studied using magnetic resonance imaging (MRI). DNA from blood samples was processed for screening the GHRHR and GH1 by Sanger sequencing. RESULTS: Mean age at presentation of the 116 patients (67 males and 49 females) was 11.71±3.5years. Mean height standard deviation score (SDS) and weight SDS were -4.5 and -3.5 respectively. Nine (7.8%) were familial and parental consanguinity was present in 21 (19.8%) families. Eighty-three patients underwent MRI and morphological alterations of the pituitary were observed in 39 (46.9%). GH1 and GHRHR screening revealed eleven variations in 24 (21%) patients of which, four were novel deleterious, one novel non-pathogenic and six reported changes. CONCLUSIONS: GHRHR contributed more to IGHD in our patients which confirmed that GHRHR should be screened first before GH1 in our population. Identification of GH1 and GHRHR variations helped in defining our mutational spectrum which will play a crucial role in providing predictive and prenatal genetic testing to the patients.

Clinical profile and inheritance pattern of CYP21A2 gene mutations in patients with classical congenital adrenal hyperplasia from 10 families.

CONTEXT: Congenital adrenal hyperplasia (CAH) is an autosomal recessive metabolic disorder caused by mutations in the CYP21A2 gene. Genetic diagnosis of 21-OH deficiency causing CAH is more complicated than any other monogenic disorder due to high variability of the locus. The disease has a wide spectrum of clinical variants making it difficult to establish a genotyp-phenotype correlation. Therefore, family studies are necessary to ascertain parental genotype and segregation of the mutant allele among the offspring. AIM: The present study aimed to identify CYP21A2 gene mutations and analyze the segregation pattern in CAH trios (patients and their parents). MATERIALS AND METHODS: A total of ten families having at least one CAH child were recruited. RESULTS: Out of 31 children from ten families, 15 were affected with CAH and 13 of/them (12 females and 1 male) were available for genetic testing. One family had all the children affected with CAH. Compound heterozygous mutations were identified in seven patients (53.8%) whereas p.P30L, In2 and Δ8 bp mutations were present in homozygous state in three (23.1%), two (15.3 %) and one (7.6%) patient respectively. CONCLUSIONS: In majority of the families, mutant alleles observed in the patients were inherited from the parents whereas three families showed sporadic mutations without any paternal or maternal origin. This indicated their novel occurrence due to misalignment of the parental genes and/or large deletion of the gene. Female preponderance was noted in the CAH families and also among the patients raising the possibility of survival advantage among females.

Novel MEN 1 gene findings in rare sporadic insulinoma--a case control study.

BACKGROUND: Insulinomas, which are rare tumors causing hyperinsulinemic hypoglycemia are usually sporadic but may also occur in association with multiple endocrine neoplasia type 1 (MEN-1) syndrome an autosomal dominant disorder caused by MEN1 gene mutations. MEN1 encodes a nuclear protein Menin, a tumor suppressor which acts as an adapter and interacts with partner proteins involved in crucial activities like transcriptional regulation, cell division, proliferation and genome stability. This study reports on clinical findings and mutation screening in sporadic insulinoma patients. METHODS: Seventeen patients diagnosed with insulinoma were recruited along with 30 healthy volunteers who acted as controls for the present study. The patients presented with symptoms of sweating, tremors, drowsiness, palpitations, loss of consciousness, abnormal behavior, seizures and weight gain. Detailed clinical and family history was collected from all the participants along with 5 ml of blood sample after taking informed consent. Genomic DNA isolated from blood was subjected to MEN1 gene amplification followed by direct sequencing. Nucleotide sequences obtained were compared with published MEN1 cDNA sequences. Prediction of functional effects of novel changes was done using various bioinformatics algorithms. RESULTS: Molecular analysis revealed presence of three novel exonic mutations (M561K, Q192K and Q261Q), two novel intronic variations c.445-44G → A and c.913-42G → C in introns two and six respectively and three reported exon SNPs; H433H (rs540012), D418D (rs2071313), A541T (rs2959656) and one intronic SNP (rs669976). CONCLUSIONS: The study identified presence of novel pathogenic MEN1 mutations in sporadic cases of insulinoma. The new mutations identified were in regions involved in defective binding of menin to proteins implicated in genetic and epigenetic mechanisms. The outcome of the study extends the growing list of MEN1 pathogenic mutations even in sporadic cases providing consequential insight into phenotypic heterogeneity and in the expression of individual mutations.

Impact of a novel 14 bp MEN1 deletion in a patient with hyperparathyroidism and gastrinoma.

UNLABELLED: Multiple endocrine neoplasia type 1 (MEN-1) is a rare autosomal-dominant disease characterized by tumors in endocrine and/or non endocrine organs due to mutations in MEN1 encoding a nuclear scaffold protein'menin' involved in regulation of different cellular activities. We report a novel 14 bp MEN1 deletion mutation in a 35-year-old female with history of recurrent epigastric pain, vomiting, loose stools and weight loss. On evaluation she was diagnosed to have multifocal gastro-duodenal gastrinoma with paraduodenal lymph nodes and solitary liver metastasis. She was also found to have primary hyperparathyroidism with bilateral inferior parathyroid adenoma. Pancreatico-duodenectomy with truncalvagotomy was performed. Four months later, radiofrequency ablation (RFA) of segment 4 of the liver was done followed by three and a half parathyroidectomy. MEN1 screening was carried out for the patient and her family members. MEN-1 sequencing in the patient revealed a heterozygous 14 bp exon 8 deletion. Evaluation for pathogenicity and protein structure prediction showed that the mutation led to a frameshift thereby causing premature termination resulting in a truncated protein. To conclude, a novel pathogenic MEN1 deletion mutation affecting its function was identified in a patient with hyperparathyroidism and gastrinoma. The report highlights the clinical consequences of the novel mutation and its impact on the structure and function of the protein. It also provides evidence for co-existence of pancreatic and duodenal gastrinomas in MEN1 syndrome. MEN1 testing provides important clues regarding etiology and therefore should be essentially undertaken in asymptomatic first degree relatives who could be potential carriers of the disease. LEARNING POINTS: Identification of a novel pathogenic MEN1 deletion mutation.MEN1 mutation screening in patients with pituitary, parathyroid and pancreatic tumors, and their first degree relatives gives important clues about the etiology.Pancreatic and duodenal gastrinomas may co-exist simultaneously in MEN1 syndrome.

Rare association of acromegaly with left atrial myxoma in Carney's complex due to novel PRKAR1A mutation.

UNLABELLED: Carney complex (CNC) is a rare autosomal dominant syndrome characterized by pigmented lesions of the skin and mucosae along with cardiac, endocrine, cutaneous, and neural myxomatous tumors. Mutations in the PRKAR1A gene have been identified in ∼70% of the CNC cases reported worldwide. A 30-year-old male was referred to the endocrinology clinic with suspected acromegaly. He had a history of recurrent atrial myxoma for the past 8 years for which he underwent repeated surgeries. Presently, he complained of having headache, excessive snoring, sweating, and also noticed increase in his shoe size. Evaluation for acromegaly revealed elevated levels of GH in random as well as in suppressed condition. Magnetic resonance imaging scan revealed enlarged sella with microadenoma in the left anterior pituitary. Screening of PRKAR1A gene was carried out for the patient, his parents and siblings who were available and willing to undergo the test. The patient was diagnosed to have the rare CNC syndrome characterized by recurrent atrial myxoma and acromegaly due to a novel 22 bp insertion mutation in PRKAR1A which was predicted to be deleterious by in silico analysis. Screening the available family members revealed the absence of this mutation in them except the elder brother who also tested positive for this mutation. The present study reports on a novel PRKAR1A insertion mutation in a patient with acromegaly and left atrial myxoma in CNC. LEARNING POINTS: Identification of a novel deleterious PRKAR1A insertion mutation causing CNC.It is important that patients with cardiac myxoma be investigated for presence of endocrine overactivity suggestive of CNC.PRKAR1A mutation analysis should be undertaken in such cases to confirm the diagnosis in the patients as well as first degree relatives.This case highlights an important aspect of diagnosis, clinical course, and management of this rare condition.

Growth and hormonal profile from birth to adolescence of a girl with aromatase deficiency.

BACKGROUND: Aromatase deficiency is a rare autosomal recessive disorder caused by mutations in the CYP19A1 gene and characterized by lack of conversion of androgens to estrogens. It presents with virilization of pregnant mothers during the antenatal period, and virilization of female fetuses at birth. Affected subjects of either gender later manifest with features of estrogen deficiency and androgen excess. PATIENT AND METHODS: We describe the clinical course of an Indian girl with aromatase deficiency from birth to 16 years of age. Estrogen replacement was begun at age 13.5 years. The child's growth, hormonal, radiological, and metabolic parameters were monitored throughout the course of treatment. RESULTS: The child presented with obesity, tall stature, delayed bone age, osteoporosis, hyperinsulinemia with acanthosis nigricans, and hypergonadotropic hypogonadism with cystic ovaries. Estrogen replacement resulted in a plateauing of height, improvement of bone maturation, and pubertal progression with the disappearance of ovarian cysts. However, hyperinsulinemia and acanthosis nigricans persisted despite estrogen replacement and metformin. Genetic analysis revealed a homozygous arginine to cysteine substitution at codon 435 in exon 10 of CYP19A1. CONCLUSIONS: This is the first case of aromatase deficiency reported from India. This case highlights the role of estrogen in skeletal maturation and mineralization and the effect of estrogen deficiency and androgen excess over glucose metabolism in adolescent females.

Genetic characterization of growth hormone 1 gene in patients with isolated growth hormone deficiency.

INTRODUCTION: Growth hormone (GH) secretion and release is a complex and highly regulated process. Any alteration disturbing synthesis, secretion or biological action of GH, results into growth hormone deficiency (GHD). GHD is of two types-isolated growth hormone deficiency (IGHD) and combined pituitary hormone deficiency (CPHD), of which IGHDis more common. The genes implicated in its etiology are growth hormone 1(GH1) and receptor of growth hormone-releasing hormone (GHRHR). Mutations within the coding region and/or either entire or partial deletions of the GH1gene lead to IGHD. In addition, GH1 possesses upstream regulatory elements and a promoter with binding sites for various transcription factors, which control its expression. AIM: The study was planned with an aim to identify entire GH1 locus deletion, mutations in the GH1 coding region and sequence variations (polymorphisms) in the promoter region of the gene in patients with IGHD. MATERIALS AND METHODS: Thirty patients clinically diagnosed with IGHD and 30 healthy individuals who formed the controls were enrolled for the study. Genomic DNA was isolated from peripheral blood sample and processed for amplification of the desired regions followed by direct sequencing and/or restriction endonuclease digestion. RESULTS: Out of the 30 IGHD patients screened, 20% of the cases showed consanguinity and 16% had a positive family history. Seven percentage of the patients showed homozygous deletion of the GH1gene while rest of them had heterozygous deletion. Screening of the coding region of GH1 showed sequence variations in exon 1 in 20% of the patients whereas the promoter region showed the presence of polymorphisms-rs2005171 in 20%, rs2005172 in 15% and rs11568828 in 18% of the cases. The haplotype comprising rs2005171 and rs2005172 was observed in four patients. CONCLUSION: The present study is an attempt to characterize the GH1 locus in IGHD patients. To the best of our knowledge this is the first study of its kind where entire GH1locus, upstream regulatory elements and promoter region have been studied. Such an analysis would provide valuable information on the etiology of IGHD.

Identification of novel SRY mutations and SF1 (NR5A1) changes in patients with pure gonadal dysgenesis and 46,XY karyotype.

Primary amenorrhea due to 46,XY disorders of sexual development (DSD) is complex with the involvement of several genes. Karyotyping of such patients is important as they may develop dysgerminoma and molecular analysis is important to identify the underlying mechanism and explore the cascade of events occurring during sexual development. The present study was undertaken for the genetic analysis in seven patients from five families presenting with primary amenorrhea and diagnosed with pure gonadal dysgenesis. Karyotyping was done and the patients were screened for underlying changes in SRY, desert hedgehog (DHH), DAX1 (NR0B1) and SF1 (NR5A1) genes, mutations in which are implicated in DSD. All the patients had 46,XY karyotype and two novel SRY mutations were found. In Family 1 (Patient S1.1) a missense mutation c.294G>A was seen, which results in a stop codon at the corresponding amino acid (Trp98X) and in Family 2 (Patients S2.1, S2.2 and S2.3), a missense mutation c.334G>A (Glu112Leu) was identified in all affected sisters. Both mutations were seen to occur in the conserved high mobility group box of SRY gene. One heterozygous change c.427G>A resulting in Glu143Lys in DHH gene in one patient and two heterozygous changes in the intronic region of SF1 (NR5A1) gene (c.244+80G>A+ c.1068-20C>T) in another patient were noted. One individual did not show changes in any of the genes analyzed. These results reiterate the importance of SRY and others, such as SF1 (NR5A1) and DHH, that are involved in the cascade of events leading to sex determination and also their role in sex reversal.