Polymorphic inversions are ubiquitous in humans and they have been linked to both adaptation and disease. Following their discovery in Drosophila more than a century ago, inversions have proved to be more elusive than other structural variants. A wide variety of methods for the detection and genotyping of inversions have recently been developed: multiple techniques based on selective amplification by PCR, short- and long-read sequencing approaches, principal component analysis of small variant haplotypes, template strand sequencing, optical mapping, and various genome assembly methods. Many methods apply complex wet lab protocols or increasingly refined bioinformatic analyses. This review is an attempt to provide a practical summary and comparison of the methods that are in current use, with a focus on metrics such as the maximum size of segmental duplications at inversion breakpoints that each method can tolerate, the size range of inversions that they recover, their throughput, and whether the locations of putative inversions must be known beforehand. <<<

Long-read sequencing (LRS) has been around for more than a decade, but widespread adoption of the technology has been slow due to the perceived high error rates and high sequencing cost. This is changing due to the recent advancements to produce highly accurate sequences and the reducing costs. LRS promises significant improvement over short read sequencing in four major areas: (1) better detection of structural variation (2) better resolution of highly repetitive or nonunique regions (3) accurate long-range haplotype phasing and (4) the detection of base modifications natively from the sequencing data. Several successful applications of LRS have demonstrated its ability to resolve molecular diagnoses where short-read sequencing fails to identify a cause. However, the argument for increased diagnostic yield from LRS remains to be validated. Larger cohort studies may be required to establish the realistic boundaries of LRS's clinical utility and analytical validity, as well as the development of standards for clinical applications. We discuss the limitations of the current standard of care, and contrast with the applications and advantages of two major LRS platforms, PacBio and Oxford Nanopore, for molecular diagnostics of constitutional disorders, and present a critical argument about the potential of LRS in diagnostic settings. <<<

Jeffrey K Ng, Pankaj Vats, Elyn Fritz-Waters, Stephanie Sarkar, Eleanor I Sams, Evin M Padhi, Zachary L Payne, Shawn Leonard, Marc A West, Chandler Prince, Lee Trani, Marshall Jansen, George Vacek, Mehrzad Samadi, Timothy T Harkins, Craig Pohl, Tychele N Turner <<<

Detection of de novo variants (DNVs) is critical for studies of disease-related variation and mutation rates. To accelerate DNV calling, we developed a graphics processing units-based workflow. We applied our workflow to whole-genome sequencing data from three parent-child sequenced cohorts including the Simons Simplex Collection (SSC), Simons Foundation Powering Autism Research (SPARK), and the 1000 Genomes Project (1000G) that were sequenced using DNA from blood, saliva, and lymphoblastoid cell lines (LCLs), respectively. The SSC and SPARK DNV callsets were within expectations for number of DNVs, percent at CpG sites, phasing to the paternal chromosome of origin, and average allele balance. However, the 1000G DNV callset was not within expectations and contained excessive DNVs that are likely cell line artifacts. Mutation signature analysis revealed 30% of 1000G DNV signatures matched B-cell lymphoma. Furthermore, we found variants in DNA repair genes and at Clinvar pathogenic or likely-pathogenic sites and significant excess of protein-coding DNVs in IGLL5; a gene known to be involved in B-cell lymphomas. Our study provides a new rapid DNV caller for the field and elucidates important implications of using sequencing data from LCLs for reference building and disease-related projects. <<<

Xiao Han, Xuanye Cao, Vanessa Aguiar-Pulido, Wei Yang, Menuka Karki, Paula Andrea Pimienta Ramirez, Robert M Cabrera, Ying Linda Lin, Bogdan J Wlodarczyk, Gary M Shaw, M Elizabeth Ross, Cuilian Zhang, Richard H Finnell, Yunping Lei <<<

Neural tube defects (NTDs) are congenital malformations resulting from abnormal embryonic development of the brain, spine, or spinal column. The genetic etiology of human NTDs remains poorly understood despite intensive investigation. CIC, homolog of the Capicua transcription repressor, has been reported to interact with ataxin-1 (ATXN1) and participate in the pathogenesis of spinocerebellar ataxia type 1. Our previous study demonstrated that CIC loss of function (LoF) variants contributed to the cerebral folate deficiency syndrome by downregulating folate receptor 1 (FOLR1) expression. Given the importance of folate transport in neural tube formation, we hypothesized that CIC variants could contribute to increased risk for NTDs by depressing embryonic folate concentrations. In this study, we examined CIC variants from whole-genome sequencing (WGS) data of 140 isolated spina bifida cases and identified eight missense variants of CIC gene. We tested the pathogenicity of the observed variants through multiple in vitro experiments. We determined that CIC variants decreased the FOLR1 protein level and planar cell polarity (PCP) pathway signaling in a human cell line (HeLa). In a murine cell line (NIH3T3), CIC loss of function variants downregulated PCP signaling. Taken together, this study provides evidence supporting CIC as a risk gene for human NTD. <<<

Prader-Willi syndrome (PWS; MIM# 176270) is a neurodevelopmental disorder caused by the loss of expression of paternally imprinted genes within the PWS region located on 15q11.2. It is usually caused by either maternal uniparental disomy of chromosome 15 (UPD15) or 15q11.2 recurrent deletion(s). Here, we report a healthy carrier of a balanced X;15 translocation and her two daughters, both with the karyotype 45,X,der(X)t(X;15)(p22;q11.2),-15. Both daughters display symptoms consistent with haploinsufficiency of the SHOX gene and PWS. We explored the architecture of the derivative chromosomes and investigated effects on gene expression in patient-derived neural cells. First, a multiplex ligation-dependent probe amplification methylation assay was used to determine the methylation status of the PWS-region revealing maternal UPD15 in daughter 2, explaining her clinical symptoms. Next, short read whole genome sequencing and 10X genomics linked read sequencing was used to pinpoint the exact breakpoints of the translocation. Finally, we performed transcriptome sequencing on neuroepithelial stem cells from the mother and from daughter 1 and observed biallelic expression of genes in the PWS region (including SNRPN) in daughter 1. In summary, our multi-omics analysis highlights two different PWS mechanisms in one family and provide an example of how structural variation can affect imprinting through long-range interactions. <<<

Cristina Perez-Becerril, Andrew J Wallace, Helene Schlecht, Naomi L Bowers, Philip T Smith, Carolyn Gokhale, Helen Eaton, Chris Charlton, Rachel Robinson, Ruth S Charlton, D Gareth Evans, Miriam J Smith <<<

Schwannomatosis comprises a group of hereditary tumor predisposition syndromes characterized by, usually benign, multiple nerve sheath tumors, which frequently cause severe pain that does not typically respond to drug treatments. The most common schwannomatosis-associated gene is NF2, but SMARCB1 and LZTR1 are also associated. There are still many cases in which no pathogenic variants (PVs) have been identified, suggesting the existence of as yet unidentified genetic risk factors. In this study, we performed extended genetic screening of 75 unrelated schwannomatosis patients without identified germline PVs in NF2, LZTR1, or SMARCB1. Screening of the coding region of DGCR8, COQ6, CDKN2A, and CDKN2B was carried out, based on previous reports that point to these genes as potential candidate genes for schwannomatosis. Deletions or duplications in CDKN2A, CDKN2B, and adjacent chromosome 9 region were assessed by multiplex ligation-dependent probe amplification analysis. Sequencing analysis of a patient with multiple schwannomas and melanomas identified a novel duplication in the coding region of CDKN2A, disrupting both p14ARF and p16INK4a. Our results suggest that none of these genes are major contributors to schwannomatosis risk but the possibility remains that they may have a role in more complex mechanisms for tumor predisposition. <<<

Htoo A Wai, Matthew Constable, Cosima Drewes, Ian C Davies, Eliska Svobodova, Esther Dempsey, Anand Saggar, Tessa Homfray, Sahar Mansour, Sofia Douzgou, Kate Barr, Catherine Mercer, David Hunt, Andrew G L Douglas, Diana Baralle <<<

Use of blood RNA sequencing (RNA-seq) as a splicing analysis tool for clinical interpretation of variants of uncertain significance (VUSs) found via whole-genome and exome sequencing can be difficult for genes that have low expression in the blood due to insufficient read count coverage aligned to specific genes of interest. Here, we present a short amplicon reverse transcription-polymerase chain reaction(RT-PCR) for the detection of genes with low blood expression. Short amplicon RT-PCR, is designed to span three exons where an exon harboring a variant is flanked by one upstream and one downstream exon. We tested short amplicon RT-PCRs for genes that have median transcripts per million (TPM) values less than one according to the genotype-tissue expression database. Median TPM values of genes analyzed in this study are SYN1 = 0.8549, COL1A1 = 0.6275, TCF4 = 0.4009, DSP = .2894, TTN = 0.2851, COL5A2 = 0.1036, TERT = 0.04452, NTRK2 = 0.0344, ABCA4 = 0.00744, PRPH = 0, and WT1 = 0. All these genes show insufficient exon-spanning read coverage in our RNA-seq data to allow splicing analysis. We successfully detected all genes tested except PRPH and WT1. Aberrant splicing was detected in SYN1, TCF4, NTRK2, TTN, and TERT VUSs. Therefore, our results show short amplicon RT-PCR is a useful alternative for the analysis of splicing events in genes with low TPM in blood RNA for clinical diagnostics. <<<