Variants of uncertain significance in newborn screening disorders: implications for large-scale genomic sequencing.

PURPOSE: As exome and genome sequencing using high-throughput sequencing technologies move rapidly into the diagnostic process, laboratories and clinicians need to develop a strategy for dealing with uncertain findings. A commitment must be made to minimize these findings, and all parties may need to make adjustments to their processes. The information required to reclassify these variants is often available but not communicated to all relevant parties. METHODS: To illustrate these issues, we focused on three well-characterized monogenic, metabolic disorders included in newborn screens: classic galactosemia, caused by GALT variants; phenylketonuria, caused by PAH variants; and medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, caused by ACADM variants. In 10 years of clinical molecular testing, we have observed 134 unique GALT variants, 46 of which were variants of uncertain significance (VUS). In PAH, we observed 132 variants, including 17 VUS, and for ACADM, we observed 64 unique variants, of which 33 were uncertain. CONCLUSION: After this review, 17 VUS (37%; 7 in ACADM, 9 in GALT, and 1 in PAH) were reclassified from uncertain (6 to benign or likely benign and 11 to pathogenic or likely pathogenic). We identified common types of missing information that would have helped make a definitive classification and categorized this information by ease and cost to obtain.Genet Med 19 1, 77-82.

Detection limit of intragenic deletions with targeted array comparative genomic hybridization.

BACKGROUND: Pathogenic mutations range from single nucleotide changes to deletions or duplications that encompass a single exon to several genes. The use of gene-centric high-density array comparative genomic hybridization (aCGH) has revolutionized the detection of intragenic copy number variations. We implemented an exon-centric design of high-resolution aCGH to detect single- and multi-exon deletions and duplications in a large set of genes using the OGT 60 K and 180 K arrays. Here we describe the molecular characterization and breakpoint mapping of deletions at the smaller end of the detectable range in several genes using aCGH. RESULTS: The method initially implemented to detect single to multiple exon deletions, was able to detect deletions much smaller than anticipated. The selected deletions we describe vary in size, ranging from over 2 kb to as small as 12 base pairs. The smallest of these deletions are only detectable after careful manual review during data analysis. Suspected deletions smaller than the detection size for which the method was optimized, were rigorously followed up and confirmed with PCR-based investigations to uncover the true detection size limit of intragenic deletions with this technology. False-positive deletion calls often demonstrated single nucleotide changes or an insertion causing lower hybridization of probes demonstrating the sensitivity of aCGH. CONCLUSIONS: With optimizing aCGH design and careful review process, aCGH can uncover intragenic deletions as small as dozen bases. These data provide insight that will help optimize probe coverage in array design and illustrate the true assay sensitivity. Mapping of the breakpoints confirms smaller deletions and contributes to the understanding of the mechanism behind these events. Our knowledge of the mutation spectra of several genes can be expected to change as previously unrecognized intragenic deletions are uncovered.

Parent-of-origin testing for 15q11-q13 gains by quantitative DNA methylation analysis.

The Prader-Willi/Angelman syndrome critical region (PWS/ASCR), located at chromosome 15q11-q13, is associated with several diseases. Absence of paternally expressed genes in this region cause Prader-Willi syndrome (PWS), whereas absence of the maternally expressed UBE3A gene causes Angelman syndrome (AS). In addition, duplications and triplications of this region are also associated with distinct clinical features, indicating that the overexpression of genes within the PWS/ASCR can also lead to distinct phenotypes. Maternally inherited increases in copy number generally lead to a more severe phenotype do than paternally inherited increases. We describe a real-time methylation-sensitive PCR (Q-MSP) assay that quantifies methylation at the promoter of the differentially methylated SNRPN gene located within the PWS/ASCR. Q-MSP can detect both PWS and AS, as well as determine the parent of origin for the allele that carries the PWS/ASCR gains. In addition, Q-MSP requires only a small amount of DNA, is amenable to high-throughput analysis, and can be used in clinical testing as a reflex test to determine the parent of origin after identification of a gain of this region on chromosome 15.