Analysis of common genetic mutations in a cohort of children with salt wasting form of Congenital Adrenal Hyperplasia.

INTRODUCTION: Steroid hydroxylase deficiency due to CYP21A2 gene mutation is the most common cause of Congenital Adrenal Hyperplasia (CAH). Mutation spectrum in Sri Lankan CAH patients has not been investigated adequately. OBJECTIVES: This study attempted to study the spectrum of mutations in CYP21A2 gene in 30 patients with salt wasting form of CAH in Sri Lanka. METHODS: Allele specific polymerase chain reaction was carried out using mutation site specific primers for eight mutations (P30L, I2G, 8bp deletion, I172N, E6 cluster, V281L, Q318X and R356W) reported as frequently occurring in other populations. RESULTS: Fourteen patients had homozygous mutations; six patients were compound heterozygotes as determined by investigating parents of the patients, one patient had a large gene deletion which was previously reported and the remaining patients had at least one heterozygous mutation. The following allele frequencies were observed for each mutation P30L-10%, I2G- 40%, 8bp-18.33%, I172N-3.33%, E6 cluster- 5%, Q318X-40% and R356W-3.33%. V281L mutation was not observed in the study cohort. DNA sequencing revealed a novel mutation G292S in one patient. CONCLUSION: This is the first report describing a broad spectrum of mutations in CYP21A2 gene in Sri Lankan patients with CAH. Mutation frequencies did not vary from other ethnic groups reported around the world.

Novel gross deletion at the GHRHR gene locus possibly mediated by Alu specific microhomology identified in a Sri Lankan patient with isolated growth hormone deficiency.

OBJECTIVE: Characterization of a deletion in the exon 1 and 5' regulatory region of the GHRHR gene in a proband with isolated growth hormone deficiency. METHODS: Multiple ligation dependent probe amplification (MLPA) assay was carried out to confirm the homozygous deletion which was suspected during screening of the GHRHR gene by single strand conformation polymorphism. A series of short range PCR amplifications were carried out to map the approximate location of the break points of the deletion. Sanger sequencing was carried out to locate the break points and to identify the length of the deletion. Long range PCR amplification was carried out to confirm the length of the deletion and to screen the parents of the proband for the deletion. RESULTS: A homozygous deletion was confirmed via MLPA assay. Zones of sequence similarity between upstream intergenic region and intron 1 of the GHRHR gene were identified. Break points of the deletion were identified within perfectly matching 32 bp repeat sequences ie: microhomologies in the specified zones. The novel deletion may have arisen via Alu specific microhomology mediated non-recurrent rearrangement in the maternal lineage of the proband. The deletion being reported in this study include, last 3118 bp from the upstream intergenic region and complete exon 1 and first 2620 bp from intron 1 and one of the 32 bp microhomologies. The total length of the deleted segment was 5875 bp. As the deleted region contained significant elements essential for gene expression, the identified deletion is being reported as likely pathogenic. The same deletion was identified in the mother in heterozygous state. CONCLUSION: We have characterized a novel deletion that seems to have arisen via Alu specific microhomology mediated non-recurrent rearrangement at GHRHR gene locus. HGVS nomenclature of the deletion is c.-3166_58-2057del. This novel structural variant was identified to be the cause of IGHD of the affected proband.