Matthew DeFelice

Genome-Wide Association Analysis Identifies Loci for Type 2 Diabetes and Triglyceride Levels

New strategies for prevention and treatment of type 2 diabetes (T2D) require improved insight into disease etiology. We analyzed 386,731 common single-nucleotide polymorphisms (SNPs) in 1464 patients with T2D and 1467 matched controls, each characterized for measures of glucose metabolism, lipids, obesity, and blood pressure. With collaborators (FUSION and WTCCC/UKT2D), we identified and confirmed three loci associated with T2D—in a noncoding region near CDKN2A and CDKN2B, in an intron of IGF2BP2, and an intron of CDKAL1—and replicated associations near HHEX and in SLC30A8 found by a recent whole-genome association study. We identified and confirmed association of a SNP in an intron of glucokinase regulatory protein (GCKR) with serum triglycerides. The discovery of associated variants in unsuspected genes and outside coding regions illustrates the ability of genome-wide association studies to provide potentially important clues to the pathogenesis of common diseases.

Collaborative genome-wide association analysis supports a role for ANK3 and CACNA1C in bipolar disorder

To identify susceptibility loci for bipolar disorder, we tested 1.8 million variants in 4,387 cases and 6,209 controls and identified a region of strong association (rs10994336, P = 9.1 × 10−9) in ANK3 (ankyrin G). We also found further support for the previously reported CACNA1C (alpha 1C subunit of the L-type voltage-gated calcium channel; combined P = 7.0 × 10−8, rs1006737). Our results suggest that ion channelopathies may be involved in the pathogenesis of bipolar disorder.

Whole-genome association study of bipolar disorder

We performed a genome-wide association scan in 1461 patients with bipolar (BP) 1 disorder, 2008 controls drawn from the Systematic Treatment Enhancement Program for Bipolar Disorder and the University College London sample collections with successful genotyping for 372 193 single nucleotide polymorphisms (SNPs). Our strongest single SNP results are found in myosin5B (MYO5B; P=1.66 × 10−7) and tetraspanin-8 (TSPAN8; P=6.11 × 10−7). Haplotype analysis further supported single SNP results highlighting MYO5B, TSPAN8 and the epidermal growth factor receptor (MYO5B; P=2.04 × 10−8, TSPAN8; P=7.57 × 10−7 and EGFR; P=8.36 × 10−8). For replication, we genotyped 304 SNPs in family-based NIMH samples (n=409 trios) and University of Edinburgh case–control samples (n=365 cases, 351 controls) that did not provide independent replication after correction for multiple testing. A comparison of our strongest associations with the genome-wide scan of 1868 patients with BP disorder and 2938 controls who completed the scan as part of the Wellcome Trust Case–Control Consortium indicates concordant signals for SNPs within the voltage-dependent calcium channel, L-type, alpha 1C subunit (CACNA1C) gene. Given the heritability of BP disorder, the lack of agreement between studies emphasizes that susceptibility alleles are likely to be modest in effect size and require even larger samples for detection.

The Genomic Landscape of Pediatric Ewing Sarcoma

UNLABELLED Pediatric Ewing sarcoma is characterized by the expression of chimeric fusions of EWS and ETS family transcription factors, representing a paradigm for studying cancers driven by transcription factor rearrangements. In this study, we describe the somatic landscape of pediatric Ewing sarcoma. These tumors are among the most genetically normal cancers characterized to date, with only EWS-ETS rearrangements identified in the majority of tumors. STAG2 loss, however, is present in more than 15% of Ewing sarcoma tumors; occurs by point mutation, rearrangement, and likely nongenetic mechanisms; and is associated with disease dissemination. Perhaps the most striking finding is the paucity of mutations in immediately targetable signal transduction pathways, highlighting the need for new therapeutic approaches to target EWS-ETS fusions in this disease. SIGNIFICANCE We performed next-generation sequencing of Ewing sarcoma, a pediatric cancer involving bone, characterized by expression of EWS-ETS fusions. We found remarkably few mutations. However, we discovered that loss of STAG2 expression occurs in 15% of tumors and is associated with metastatic disease, suggesting a potential genetic vulnerability in Ewing sarcoma.

Mutations causing medullary cystic kidney disease type 1 lie in a large VNTR in MUC1 missed by massively parallel sequencing

Although genetic lesions responsible for some mendelian disorders can be rapidly discovered through massively parallel sequencing of whole genomes or exomes, not all diseases readily yield to such efforts. We describe the illustrative case of the simple mendelian disorder medullary cystic kidney disease type 1 (MCKD1), mapped more than a decade ago to a 2-Mb region on chromosome 1. Ultimately, only by cloning, capillary sequencing and de novo assembly did we find that each of six families with MCKD1 harbors an equivalent but apparently independently arising mutation in sequence markedly under-represented in massively parallel sequencing data: the insertion of a single cytosine in one copy (but a different copy in each family) of the repeat unit comprising the extremely long (∼1.5–5 kb), GC-rich (>80%) coding variable-number tandem repeat (VNTR) sequence in the MUC1 gene encoding mucin 1. These results provide a cautionary tale about the challenges in identifying the genes responsible for mendelian, let alone more complex, disorders through massively parallel sequencing.

DGAT1 mutation is linked to a congenital diarrheal disorder

Congenital diarrheal disorders (CDDs) are a collection of rare, heterogeneous enteropathies with early onset and often severe outcomes. Here, we report a family of Ashkenazi Jewish descent, with 2 out of 3 children affected by CDD. Both affected children presented 3 days after birth with severe, intractable diarrhea. One child died from complications at age 17 months. The second child showed marked improvement, with resolution of most symptoms at 10 to 12 months of age. Using exome sequencing, we identified a rare splice site mutation in the DGAT1 gene and found that both affected children were homozygous carriers. Molecular analysis of the mutant allele indicated a total loss of function, with no detectable DGAT1 protein or activity produced. The precise cause of diarrhea is unknown, but we speculate that it relates to abnormal fat absorption and buildup of DGAT substrates in the intestinal mucosa. Our results identify DGAT1 loss-of-function mutations as a rare cause of CDDs. These findings prompt concern for DGAT1 inhibition in humans, which is being assessed for treating metabolic and other diseases.

Targeted Exon Sequencing by In‐Solution Hybrid Selection

This unit describes a protocol for the targeted enrichment of exons from randomly sheared genomic DNA libraries using an in‐solution hybrid selection approach for sequencing on an Illumina Genome Analyzer II. The steps for designing and ordering a hybrid selection oligo pool are reviewed, as are critical steps for performing the preparation and hybrid selection of an Illumina paired‐end library. Critical parameters, performance metrics, and analysis workflow are discussed. Curr. Protoc. Hum. Genet. 66:18.4.1‐18.4.24

Development and Validation of a Mass Spectrometry–Based Assay for the Molecular Diagnosis of Mucin-1 Kidney Disease

Mucin-1 kidney disease, previously described as medullary cystic kidney disease type 1 (MCKD1, OMIM 174000), is an autosomal dominant tubulointerstitial kidney disease recently shown to be caused by a single-base insertion within the variable number tandem repeat region of the MUC1 gene. Because of variable age of disease onset and often subtle signs and symptoms, clinical diagnosis of mucin-1 kidney disease and differentiation from other forms of hereditary kidney disease have been difficult. The causal insertion resides in a variable number tandem repeat region with high GC content, which has made detection by standard next-generation sequencing impossible to date. The inherently difficult nature of this mutation required an alternative method for routine detection and clinical diagnosis of the disease. We therefore developed and validated a mass spectrometry-based probe extension assay with a series of internal controls to detect the insertion event using 24 previously characterized positive samples from patients with mucin-1 kidney disease and 24 control samples known to be wild type for the variant. Validation results indicate an accurate and reliable test for clinically establishing the molecular diagnosis of mucin-1 kidney disease with 100% sensitivity and specificity across 275 tests called.

Abstract LB-122: High-throughput tumor genomic profiling by massively parallel sequencing

The use of tumor genetic and molecular information to accurately predict a patient9s response to therapy is critical to personalized cancer medicine. Knowledge of somatic genetic alterations in tumors - including mutations, copy number alterations, and pharmacogenomic polymorphisms - should ultimately facilitate individualized approaches to cancer treatment. However, the development and real-world implementation of comprehensive genomic profiling has not yet been achieved. Here, we describe the development of a high-throughput, massively parallel sequencing platform to detect tumor genomic alterations that may provide an efficient and cost-effective means to address this challenge. Our approach combines several recent innovations - solution phase hybrid selection, DNA barcoding and pooling, and massively parallel sequencing - to achieve a high-throughput genomic profiling platform. We designed and synthesized ∼7000 biotinylated RNA baits corresponding to the coding sequence of the top 150 “druggable” or potentially “actionable” genes known to undergo somatic genomic alterations in cancer. We obtained genomic DNA from cell lines with known genetic alterations and used this DNA to generate barcoded sequencing libraries. Following quantification of libraries, equimolar pools were generated consisting of up to 12 barcoded tumor DNAs and normal diploid control DNAs. These DNA pools were subjected to solution-phase hybrid capture with the biotinylated RNA baits followed by massively parallel sequencing. The sequencing data were deconvoluted to match all high-quality reads with the corresponding tumor samples and call base mutations and copy number alterations. In all samples tested, sequence reads were highly specific to the targeted exonic regions. In addition to detecting mutations, exon capture detected high-level amplifications and deletions, two types of “actionable” genomic alterations that are poorly detectable by mass spectrometric approaches to mutation profiling. Comparison with copy number data previously obtained using high-density SNP array data demonstrated a robust correlation. As a proof-of-principle, we performed the entire profiling approach on clinical tumor specimens from patients with refractory, aggressive cancers to assess the use of genomic profiling in a real-world situation where knowledge of “driver” genetic alterations might prove valuable. Altogether, our data endorse this sequencing-based approach as a promising method to detect critical “actionable” genetic alterations in a large panel of cancer genes. The platform is scalable, refractory to contaminating stromal DNA or hyperploidy, and completed at a fraction of the cost of comparable sequencing approaches. By providing a rapid, sensitive, cost-effective means to sequence >100 cancer genes deeply and simultaneously, this approach may ultimately empower the rational selection of specific drugs targeting the genetic alterations in each patient9s tumor - clearly an unmet need in oncology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-122.