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Diamond‐Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by erythroid aplasia. Pathogenic variants in ribosomal protein (RP) genes, GATA1, TSR2, and EPO, are considered to be the etiology of DBA. Variants in 5′‐untranslated regions (UTRs) of these genes are poorly studied and can complicate the variant interpretation. We investigated the functional consequences NM_001011.4:c.‐19 + 1G > T variant in the donor splice‐site of the RPS7 5′‐UTR. This variant was found in a family where two sons with DBA were carriers. Father, who also had this variant, developed myelodysplastic syndrome, which caused his death. Search for candidate causal variants and copy number variations in DBA‐associated genes left RPS7 variant as the best candidate. Trio whole exome sequencing analysis revealed no pathogenic variants in other genes. Functional analysis using luciferase expression system revealed that this variant leads to disruption of splicing. Also, a decrease in the levels of mRNA and protein expression was detected. In conclusion, the established consequences of 5′‐UTR splice‐site variant c.‐19 + 1G > T in the RPS7 gene provide evidence that it is likely pathogenic. [ABSTRACT FROM AUTHOR]

Molecular diagnostic laboratories screen for mutations in disease-causing genes in order to confirm a clinical diagnosis. The classification of DNA variants as ‘pathogenic’ or ‘likely pathogenic’ mutations creates a workflow bottleneck, which becomes increasingly challenging as greater number of genes are screened. The classification challenge is also acute if there are conflicting reports regarding pathogenicity and differing classification criteria between laboratories. This study aimed to compare two procedures for the classification of variants in the breast cancer (BRCA)1 gene. Methods: This bioinformatic study was conducted at LabPLUS, Auckland, New Zealand, from February to June 2017. DNA was extracted from peripheral blood samples of 30 patients and gene library construction was carried out using a commercially available targeted panel for the BRCA1 and BRCA2 genes. The genes were subsequently sequenced and the sequence data analysed. The guidelines published by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/ AMP) provides a comprehensive framework for the interpretation of variants in genes that are associated with Mendelian disorders. The use of these guidelines were compared to the variant classifications that were achieved by reference to those reported in the BRCA Exchange database. Results: The results showed concordance between the two classification protocols for a panel of 30 BRCA1 gene variants, although the transparency in following the ACMG/AMP guidelines provides a diagnostic laboratory with a generalisable approach that allows laboratorydirected revisions to be undertaken in light of new information. Conclusion: The ACMG/AMP-based guidelines were applied to a cohort of patients with BRCA1 gene variants. The use of these guidelines provides a system which creates consistency in variant interpretation and supports subsequent clinical management. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To elucidate the pathogenesis of isolated growth hormone (GH) deficiency in a Japanese girl without consanguinity. Design: A 2-year-old girl of height 77.2cm (−3.0 SD for Japanese girls) was found to have an insulin-like growth factor (IGF)-1 level of 7ng/mL and IGF binding protein-3 (IGFBP-3) level of 0.41μg/mL. GH responded modestly to a series of pharmacological stimulants, increasing to 2.81ng/mL with insulin-induced hypoglycemia, 3.78ng/mL with arginine, and 3.93 with GH-releasing hormone (GHRH). Following direct sequencing of the GHRH receptor (GHRHR) gene, evaluation by the luciferase reporter assay, immunofluorescence study, and in vitro splicing assay with minigene constructs was conducted. Results: Novel compound heterozygous GHRHR gene mutations were identified in the patient. A p.G136V substitution elicited no luciferase activity increment in response to GHRH stimulation, with normal membranous expression. Splicing assay demonstrated that the IVS2+3a>g mutation would lead to aberrant splicing. Conclusions: A case of isolated GH deficiency due to novel GHRHR gene mutations was identified. [Copyright &y& Elsevier]

The proband is an elderly woman (79 years of age) of Surinamese–Hindustani origin, suspected of being a carrier of a nondeletional α-thalassemia (thal) because of a moderate microcytic hypochromic anemia at normal ferritin levels and in the absence of any other α-thal deletions. Sequence analysis revealed a silent mutation (GGC→GGT) at codon 22 of the α2-globin gene. This mutation generates a splice donor site consensus sequence (GGTGAG) between codons 22 and 23. The abnormally spliced mRNA leads to a premature termination between codons 48 and 49. The presence of a downstream intron may induce the intracellular degradation of the affected mRNA, a pathway known as nonsense mediated decay (NMD), and this explains the α+-thal phenotype observed in the patient. The codon 22 (GGC→GGT) transition described in this report is the first mutation creating a splice donor site in one of the α-globin genes. [ABSTRACT FROM AUTHOR]

Retroviruses encapsidate two copies of full-length viral RNA molecules linked together as a dimeric genome. RNA stem loop structures harboring palindromic (or “kissing”) loop sequences constitute important cis-elements for viral dimerization known as dimer initiation sites (DIS). In murine leukemia virus (MLV), a 10-mer and a 16-mer palindrome (DIS-1 and DIS-2, respectively) located in the viral leader region mediate dimerization in vitro and affect dimer stability of vector RNA in vivo. We have investigated the effect on viral replication of introducing deletions or nucleotide substitutions within these palindromes in a full-length MLV genome. Our results demonstrate that viruses modified at the dimer initiation site regions are viable and show wild-type levels of RNA encapsidation. One mutant lacking the DIS-1 palindrome was severely impaired and displayed an increased cellular ratio of spliced versus genomic RNA that most likely contributes to the inefficient replication. The implications for development of DIS-modified retrovirus-based vectors are discussed. [Copyright &y& Elsevier]