Shweta Birla

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.

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.

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

Summary 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.

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

Abstract 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.

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

Summary 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.

Y Cell Line in a Turner Mosaic: A Case Report

Turner Syndrome is one of the most common chromosomal aneuploidy seen in humans with an incidence of about 1: 2500 newborn females. Approximately 60% patients with Turner syndrome have 45, X karyotype while others show X chromosome abnormalities like deletions of long arm or short arm, isochromosome or ring chromosome. About 6-9% cases also show presence of Y chromosome or Y derived sequences. Turner Syndrome patients with ovarian dysgenesis and Y Cellline / Y derived sequences have higher risk of developing gonadal tumors. In the present study we report on a patient with Turner Syndrome showing mosaicism with Y cell line. Our case is a 19 yrs old unmarried female who reported to Gyn OPD with primary Amenorrhoea. She was referred to our lab for cytogenetic analysis. Routine karyotype was done using standard protocol for Giemsa trypsin banding. The Cytogenetic results were confirmed by Flourescent In Situ Hybridisation (FISH) using probes for chromosomes X and Y. The patient was a Turner mosaic with karyotype 45, X in 65% and 46, XX in 10% of the cells; the remaining 25% of the cells showed presence of Ychromosome with karyotype 46, XY. The detection of Y-cell line is important in view of 10-30% higher risk of developing gonadal tumors. Prophylactic gonadectomy is recommended to patients of Turner syndrome with Ychromosome mosaicism and ovarian dysgenesis.