Brendan Blumenstiel

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.

Two independent alleles at 6q23 associated with risk of rheumatoid arthritis

To identify susceptibility alleles associated with rheumatoid arthritis, we genotyped 397 individuals with rheumatoid arthritis for 116,204 SNPs and carried out an association analysis in comparison to publicly available genotype data for 1,211 related individuals from the Framingham Heart Study. After evaluating and adjusting for technical and population biases, we identified a SNP at 6q23 (rs10499194, ∼150 kb from TNFAIP3 and OLIG3) that was reproducibly associated with rheumatoid arthritis both in the genome-wide association (GWA) scan and in 5,541 additional case-control samples (P = 10−3, GWA scan; P < 10−6, replication; P = 10−9, combined). In a concurrent study, the Wellcome Trust Case Control Consortium (WTCCC) has reported strong association of rheumatoid arthritis susceptibility to a different SNP located 3.8 kb from rs10499194 (rs6920220; P = 5 × 10−6 in WTCCC). We show that these two SNP associations are statistically independent, are each reproducible in the comparison of our data and WTCCC data, and define risk and protective haplotypes for rheumatoid arthritis at 6q23.

Whole exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer

Comprehensive analyses of cancer genomes promise to inform prognoses and precise cancer treatments. A major barrier, however, is inaccessibility of metastatic tissue. A potential solution is to characterize circulating tumor cells (CTCs), but this requires overcoming the challenges of isolating rare cells and sequencing low-input material. Here we report an integrated process to isolate, qualify and sequence whole exomes of CTCs with high fidelity using a census-based sequencing strategy. Power calculations suggest that mapping of >99.995% of the standard exome is possible in CTCs. We validated our process in two patients with prostate cancer, including one for whom we sequenced CTCs, a lymph node metastasis and nine cores of the primary tumor. Fifty-one of 73 CTC mutations (70%) were present in matched tissue. Moreover, we identified 10 early trunk and 56 metastatic trunk mutations in the non-CTC tumor samples and found 90% and 73% of these mutations, respectively, in CTC exomes. This study establishes a foundation for CTC genomics in the clinic.

A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries

Genome targeting methods enable cost-effective capture of specific subsets of the genome for sequencing. We present here an automated, highly scalable method for carrying out the Solution Hybrid Selection capture approach that provides a dramatic increase in scale and throughput of sequence-ready libraries produced. Significant process improvements and a series of in-process quality control checkpoints are also added. These process improvements can also be used in a manual version of the protocol.

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.

EGFR variant heterogeneity in glioblastoma resolved through single-nucleus sequencing

UNLABELLED Glioblastomas (GBM) with EGFR amplification represent approximately 50% of newly diagnosed cases, and recent studies have revealed frequent coexistence of multiple EGFR aberrations within the same tumor, which has implications for mutation cooperation and treatment resistance. However, bulk tumor sequencing studies cannot resolve the patterns of how the multiple EGFR aberrations coexist with other mutations within single tumor cells. Here, we applied a population-based single-cell whole-genome sequencing methodology to characterize genomic heterogeneity in EGFR-amplified glioblastomas. Our analysis effectively identified clonal events, including a novel translocation of a super enhancer to the TERT promoter, as well as subclonal LOH and multiple EGFR mutational variants within tumors. Correlating the EGFR mutations onto the cellular hierarchy revealed that EGFR truncation variants (EGFRvII and EGFR carboxyl-terminal deletions) identified in the bulk tumor segregate into nonoverlapping subclonal populations. In vitro and in vivo functional studies show that EGFRvII is oncogenic and sensitive to EGFR inhibitors currently in clinical trials. Thus, the association between diverse activating mutations in EGFR and other subclonal mutations within a single tumor supports an intrinsic mechanism for proliferative and clonal diversification with broad implications in resistance to treatment. SIGNIFICANCE We developed a novel single-cell sequencing methodology capable of identifying unique, nonoverlapping subclonal alterations from archived frozen clinical specimens. Using GBM as an example, we validated our method to successfully define tumor cell subpopulations containing distinct genetic and treatment resistance profiles and potentially mutually cooperative combinations of alterations in EGFR and other genes.

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.