Biotinidase biochemical and molecular analyses: Experience at a large reference laboratory.

BACKGROUND: Biotinidase deficiency is caused by absent activity of the biotinidase, encoded by the biotinidase gene (BTD). Affected individuals cannot recycle the biotin, leading to heterogeneous symptoms that are primarily neurological and cutaneous. Early treatment with biotin supplementation can prevent irreversible neurological damage and is recommended for patients with profound deficiency, defined as enzyme activity <10% mean normal (MN). Molecular testing has been utilized along with biochemical analysis for diagnosis and management. In this study, our objective was to correlate biochemical phenotype/enzyme activity to BTD genotype in patients for whom both enzyme and molecular testing were performed at our lab, and to review how the correlations inform on variant severity. METHODS: We analyzed results of biotinidase enzyme analysis and BTD gene sequencing in 407 patients where samples were submitted to our laboratory from 2008 to 2020. RESULTS: We identified 84 BTD variants; the most common was c.1330G>C, and 19/84 were novel BTD variants. A total of 36 patients had enzyme activity <10% of MN and the most common variant found in this group was c.528G>T. No variant was reported in one patient in the profound deficiency group. The most common variant found in patients with enzyme activity more than 10% MN was c.1330G>C. CONCLUSIONS: Although enzyme activity alone may be adequate for diagnosing profound biotinidase deficiency, molecular testing is necessary for accurate carrier screening and in cases where the enzyme activity falls in the range where partial deficiency and carrier status cannot be discriminated.

BRCA Share: A Collection of Clinical BRCA Gene Variants.

As next-generation sequencing increases access to human genetic variation, the challenge of determining clinical significance of variants becomes ever more acute. Germline variants in the BRCA1 and BRCA2 genes can confer substantial lifetime risk of breast and ovarian cancer. Assessment of variant pathogenicity is a vital part of clinical genetic testing for these genes. A database of clinical observations of BRCA variants is a critical resource in that process. This article describes BRCA Share™, a database created by a unique international alliance of academic centers and commercial testing laboratories. By integrating the content of the Universal Mutation Database generated by the French Unicancer Genetic Group with the testing results of two large commercial laboratories, Quest Diagnostics and Laboratory Corporation of America (LabCorp), BRCA Share™ has assembled one of the largest publicly accessible collections of BRCA variants currently available. Although access is available to academic researchers without charge, commercial participants in the project are required to pay a support fee and contribute their data. The fees fund the ongoing curation effort, as well as planned experiments to functionally characterize variants of uncertain significance. BRCA Share™ databases can therefore be considered as models of successful data sharing between private companies and the academic world.

Cryptic transcription mediates repression of subtelomeric metal homeostasis genes.

Nonsense-mediated mRNA decay (NMD) prevents the accumulation of transcripts bearing premature termination codons. Here we show that Saccharomyces cerevisiae NMD mutants accumulate 5'-extended RNAs (CD-CUTs) of many subtelomeric genes. Using the subtelomeric ZRT1 and FIT3 genes activated in response to zinc and iron deficiency, respectively, we show that transcription of these CD-CUTs mediates repression at the bona fide promoters, by preventing premature binding of RNA polymerase II in conditions of metal repletion. Expression of the main ZRT1 CD-CUT is controlled by the histone deacetylase Rpd3p, showing that histone deacetylases can regulate expression of genes through modulation of the level of CD-CUTs. Analysis of binding of the transcriptional activator Zap1p and insertion of transcriptional terminators upstream from the Zap1p binding sites show that CD-CUT transcription or accumulation also interferes with binding of the transcriptional activator Zap1p. Consistent with this model, overexpressing Zap1p or using a constitutively active version of the Aft1p transcriptional activator rescues the induction defect of ZRT1 and FIT3 in NMD mutants. These results show that cryptic upstream sense transcription resulting in unstable transcripts degraded by NMD controls repression of a large number of genes located in subtelomeric regions, and in particular of many metal homeostasis genes.