Ferber, Matthew

AbstractBackgroundWe describe a patient presenting with pachygyria, epilepsy, developmental delay, short stature, failure to thrive, facial dysmorphisms, and multiple osteochondromas.MethodsThe patient underwent extensive genetic testing and analysis in an attempt to diagnose the cause of his condition. Clinical testing included metaphase karyotyping, array comparative genomic hybridization, direct sequencing and multiplex ligation‐dependent probe amplification and trio‐based exome sequencing. Subsequently, research‐based whole transcriptome sequencing was conducted to determine whether it might shed light on the undiagnosed phenotype.ResultsClinical exome sequencing of patient and parent samples revealed a maternally inherited splice‐site variant in the doublecortin (DCX) gene that was classified as likely pathogenic and diagnostic of the patient's neurological phenotype. Clinical array comparative genome hybridization analysis revealed a 16p13.3 deletion that could not be linked to the patient phenotype based on affected genes. Further clinical testing to determine the cause of the patient's multiple osteochondromas was unrevealing despite extensive profiling of the most likely causative genes, EXT1 and EXT2, including mutation screening by direct sequence analysis and multiplex ligation‐dependent probe amplification. Whole transcriptome sequencing identified a SAMD12‐EXT1 fusion transcript that could have resulted from a chromosomal deletion, leading to the loss of EXT1 function. Re‐review of the clinical array comparative genomic hybridization results indicated a possible unreported mosaic deletion affecting the SAMD12 and EXT1 genes that corresponded precisely to the introns predicted to be affected by a fusion‐causing deletion. The existence of the mosaic deletion was subsequently confirmed clinically by an increased density copy number array and orthogonal methodologiesConclusionsWhile mosaic mutations and deletions of EXT1 and EXT2 have been reported in the context of multiple osteochondromas, to our knowledge, this is the first time that transcriptomics technologies have been used to diagnose a patient via fusion transcript analysis in the congenital disease setting.

Abstract Background: Established laboratory methods, including next-generation sequencing (NGS), can accurately and inexpensively detect certain classes of clinically important mutations in readily accessible protein coding regions of a patient's DNA. These events include single nucleotide variants (SNVs), small insertions/deletions (indels), and sometimes medium sized copy-number deletions or duplications (del/dups). However, other mutation types with clinical relevance are known, and indeed data on over 80,000 patients show that pathogenic, medically important variants of other, more technically challenging types are prevalent in genes related to hereditary breast and ovarian cancer (see associated abstract by Lincoln et al.). We sought to develop and evaluate a reference standard by which laboratories may be able to assess and improve their performance on representative examples of complex, technically “hard” mutations in medically important genes associated with breast, ovarian, and other cancers. Methods: We selected a diverse set of 23 challenging alterations, uncovered by the aforementioned clinical study, all of which are considered pathogenic and potentially actionable under ACMG and NCCN guidelines when uncovered in the germline DNA of a patient. These mutations are in the BRCA1, BRCA2, MLH1, MSH2, MSH6, and PMS2 genes. We generated a single synthetic DNA specimen with all 23 mutations introduced into a known genomic background. Following presentation of this general strategy at a 2016 meeting, 7 laboratories volunteered to collaboratively evaluate this approach. This DNA was created, validated, blinded and provided to these laboratories who sequenced it using a total of 9 different NGS based workflows, including 5 validated clinical tests with customized methods and two sequencing technology vendor (Illumina, Ion Torrent) default pipelines. Multiple target capture biochemical methods were used, as was whole genome sequencing. Results: Twelve of the 23 variants were detected by all 9 laboratory workflows, but just 2 workflows detected all 23. Many, but not all, of these test limitations were previously known. Importantly, evidence of each variant was present in the raw data, suggesting that this strategy is compatible with the diverse biochemical methods used in NGS laboratories today. Raw data for the synthetic variants mimicked that of the endogenous ones (including presenting similar technical artifacts which make these variants “hard”) demonstrating that controls such as these may be useful in the development of methods with improved sensitivity. The vendor-supplied bioinformatics pipelines fared the worst, reinforcing the importance of carefully selecting bioinformatics algorithms and parameters in any laboratory developed test. Discussion: Medically important but technically challenging mutations are prevalent in genes involved in hereditary breast, ovarian, and other cancers. Although patient specimens are also critical, synthetic controls may help efficiently assess the analytic range of any clinical test, highlighting certain strengths and limitations, and can help laboratories develop new methods to improve sensitivity for these challenging variants. Citation Format: Lincoln S, Zook J, Truty R, Chowdhury S, Fellowes A, Mahamdallie S, Ferber M, Cleveland M, Huang C, Tomson F, Klee E, DeSilva W, Seal S, Aradhya S, Nussbaum R, Garlick R, Kingsmore S, Rahman N, Salit M, Shirts B. An interlaboratory study of complex mutation detection in genes associated with hereditary breast and ovarian cancer highlights both successes and current challenges [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-06-08.