Variant Detection in 3' Exons of PMS2 Using Exome Sequencing Data.

PMS2 is one of the mismatch repair genes included in routine genetic testing for Lynch syndrome, colorectal, ovarian, and endometrial cancers. PMS2 is also included in the American College of Medical Genetics and Genomics (ACMG) secondary findings gene list in the context of clinical exome and genome sequencing. However, sequencing of PMS2 by short-read based next generation sequencing (NGS) technologies is complicated by the presence of the pseudogene PMS2CL and often supplemented by long-range based approaches such as long-range polymerase chain reaction (LR-PCR) or long-read based next generation sequencing, which increases the complexity and cost. Here, we described a bioinformatics homology triage workflow that can eliminate the need for long-read based testing for PMS2 for the vast majority of patients undergoing exome sequencing, thus simplifying PMS2 testing and reducing the associated cost.

Development and Validation of a Next-Generation Sequencing Panel for Syndromic and Nonsyndromic Hearing Loss.

BACKGROUND: Deafness and hearing loss are common conditions that can be seen independently or as part of a syndrome and are often mediated by genetic causes. We sought to develop and validate a hereditary hearing loss panel (HHLP) to detect single nucleotide variants (SNVs), insertions and deletions (indels), and copy number variants (CNVs) in 166 genes related to nonsyndromic and syndromic hearing loss. METHODS: We developed a custom-capture next-generation sequencing (NGS) reagent to detect all coding regions, ±10 flanking bp, for the 166 genes related to nonsyndromic and syndromic hearing loss. Our validation consisted of testing 52 samples to establish accuracy, reproducibility, and analytical sensitivity. In addition to NGS, supplementary methods, including multiplex ligation-dependent probe amplification, long-range PCR, and Sanger sequencing, were used to ensure coverage of regions that had high complexity or homology. RESULTS: We observed 100% positive and negative percentage agreement for detection of SNVs (n = 362), small indels (1-22 bp, n = 25), and CNVs (gains, n = 8; losses, n = 17). Finally, we showed that this assay was able to detect variants with a variant allele frequency ≥20% for SNVs and indels and ≥30% to 35% for CNVs. CONCLUSIONS: We validated an HHLP that detects SNVs, indels, and CNVs in 166 genes related to syndromic and nonsyndromic hearing loss. The results of this assay can be utilized to confirm a diagnosis of hearing loss and related syndromic disorders associated with known causal genes.

Generation of RORγt+ Antigen-Specific T Regulatory 17 Cells from Foxp3+ Precursors in Autoimmunity.

Th17 cells are potent mediators in autoimmune diseases, and RORγt is required for their development. Recent studies have shown that RORγt+ Treg cells in the gut regulate intestinal inflammation by inhibiting effector T cell function. In the current study, we report that RORγt+ Treg cells were also found in lymph nodes following immunization. Not only distinct from intestinal RORγt+ Treg cells in their transcriptomes, peripheral RORγt+ Treg cells were derived from Foxp3+ thymic Treg cells in an antigen-specific manner. Development of these RORγt+ Treg cells, coined T regulatory 17 (Tr17) cells, depended on IL-6/Stat3 signaling. Tr17 cells showed suppressive activity against antigen-specific effector T cells in vitro. In addition, Tr17 cells efficiently inhibited myelin-specific Th17-cell-mediated CNS auto-inflammation in a passive EAE model. Collectively, our study demonstrates that Tr17 cells are effector Treg cells that potentially restrict autoimmunity.

Mutational Profiles Reveal an Aberrant TGF-ß-CEA Regulated Pathway in Colon Adenomas.

Mutational processes and signatures that drive early tumorigenesis are centrally important for early cancer prevention. Yet, to date, biomarkers and risk factors for polyps (adenomas) that inordinately and rapidly develop into colon cancer remain poorly defined. Here, we describe surprisingly high mutational profiles through whole-genome sequence (WGS) analysis in 2 of 4 pairs of benign colorectal adenoma tissue samples. Unsupervised hierarchical clustered transcriptomic analysis of a further 7 pairs of adenomas reveals distinct mutational signatures regardless of adenoma size. Transitional single nucleotide substitutions of C:G>T:A predominate in the adenoma mutational spectrum. Strikingly, we observe mutations in the TGF-ß pathway and CEA-associated genes in 4 out of 11 adenomas, overlapping with the Wnt pathway. Immunohistochemical labeling reveals a nearly 5-fold increase in CEA levels in 23% of adenoma samples with a concomitant loss of TGF-ß signaling. We also define a functional role by which the CEA B3 domain interacts with TGFBR1, potentially inactivating the tumor suppressor function of TGF-ß signaling. Our study uncovers diverse mutational processes underlying the transition from early adenoma to cancer. This has broad implications for biomarker-driven targeting of CEA/TGF-ß in high-risk adenomas and may lead to early detection of aggressive adenoma to CRC progression.

TGF-ß/ß2-spectrin/CTCF-regulated tumor suppression in human stem cell disorder Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is a human stem cell disorder, and individuals with this disease have a substantially increased risk (~800-fold) of developing tumors. Epigenetic silencing of ß2-spectrin (ß2SP, encoded by SPTBN1), a SMAD adaptor for TGF-ß signaling, is causally associated with BWS; however, a role of TGF-ß deficiency in BWS-associated neoplastic transformation is unexplored. Here, we have reported that double-heterozygous Sptbn1+/- Smad3+/- mice, which have defective TGF-ß signaling, develop multiple tumors that are phenotypically similar to those of BWS patients. Moreover, tumorigenesis-associated genes IGF2 and telomerase reverse transcriptase (TERT) were overexpressed in fibroblasts from BWS patients and TGF-ß-defective mice. We further determined that chromatin insulator CCCTC-binding factor (CTCF) is TGF-ß inducible and facilitates TGF-ß-mediated repression of TERT transcription via interactions with ß2SP and SMAD3. This regulation was abrogated in TGF-ß-defective mice and BWS, resulting in TERT overexpression. Imprinting of the IGF2/H19 locus and the CDKN1C/KCNQ1 locus on chromosome 11p15.5 is mediated by CTCF, and this regulation is lost in BWS, leading to aberrant overexpression of growth-promoting genes. Therefore, we propose that loss of CTCF-dependent imprinting of tumor-promoting genes, such as IGF2 and TERT, results from a defective TGF-ß pathway and is responsible at least in part for BWS-associated tumorigenesis as well as sporadic human cancers that are frequently associated with SPTBN1 and SMAD3 mutations.

Draft Genome Sequence of New Leprosy Agent Mycobacterium lepromatosis.

Mycobacterium lepromatosis is a newly discovered cause of leprosy. Here, we present a near-complete genome of M. lepromatosis from strain FJ924 obtained from a patient who died of leprosy. The genome contained 3,215,823 nucleotides and matched ~87% with the Mycobacterium leprae genome. This genome is likely the smallest of all mycobacterial genomes known to date.

Telomere dysfunction drives aberrant hematopoietic differentiation and myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) risk correlates with advancing age, therapy-induced DNA damage, and/or shorter telomeres, but whether telomere erosion directly induces MDS is unknown. Here, we provide the genetic evidence that telomere dysfunction-induced DNA damage drives classical MDS phenotypes and alters common myeloid progenitor (CMP) differentiation by repressing the expression of mRNA splicing/processing genes, including SRSF2. RNA-seq analyses of telomere dysfunctional CMP identified aberrantly spliced transcripts linked to pathways relevant to MDS pathogenesis such as genome stability, DNA repair, chromatin remodeling, and histone modification, which are also enriched in mouse CMP haploinsufficient for SRSF2 and in CD34(+) CMML patient cells harboring SRSF2 mutation. Together, our studies establish an intimate link across telomere biology, aberrant RNA splicing, and myeloid progenitor differentiation.

RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention.

A hexanucleotide GGGGCC repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic abnormality in familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The function of the C9ORF72 protein is unknown, as is the mechanism by which the repeat expansion could cause disease. Induced pluripotent stem cell (iPSC)-differentiated neurons from C9ORF72 ALS patients revealed disease-specific (1) intranuclear GGGGCCexp RNA foci, (2) dysregulated gene expression, (3) sequestration of GGGGCCexp RNA binding protein ADARB2, and (4) susceptibility to excitotoxicity. These pathological and pathogenic characteristics were confirmed in ALS brain and were mitigated with antisense oligonucleotide (ASO) therapeutics to the C9ORF72 transcript or repeat expansion despite the presence of repeat-associated non-ATG translation (RAN) products. These data indicate a toxic RNA gain-of-function mechanism as a cause of C9ORF72 ALS and provide candidate antisense therapeutics and candidate human pharmacodynamic markers for therapy.