Jenny C. Taylor

A genome-wide association study of global gene expression

We have created a global map of the effects of polymorphism on gene expression in 400 children from families recruited through a proband with asthma. We genotyped 408,273 SNPs and identified expression quantitative trait loci from measurements of 54,675 transcripts representing 20,599 genes in Epstein-Barr virus–transformed lymphoblastoid cell lines. We found that 15,084 transcripts (28%) representing 6,660 genes had narrow-sense heritabilities (H2) > 0.3. We executed genome-wide association scans for these traits and found peak lod scores between 3.68 and 59.1. The most highly heritable traits were markedly enriched in Gene Ontology descriptors for response to unfolded protein (chaperonins and heat shock proteins), regulation of progression through the cell cycle, RNA processing, DNA repair, immune responses and apoptosis. SNPs that regulate expression of these genes are candidates in the study of degenerative diseases, malignancy, infection and inflammation. We have created a downloadable database to facilitate use of our findings in the mapping of complex disease loci.

Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas

Many individuals with multiple or large colorectal adenomas or early-onset colorectal cancer (CRC) have no detectable germline mutations in the known cancer predisposition genes. Using whole-genome sequencing, supplemented by linkage and association analysis, we identified specific heterozygous POLE or POLD1 germline variants in several multiple-adenoma and/or CRC cases but in no controls. The variants associated with susceptibility, POLE p.Leu424Val and POLD1 p.Ser478Asn, have high penetrance, and POLD1 mutation was also associated with endometrial cancer predisposition. The mutations map to equivalent sites in the proofreading (exonuclease) domain of DNA polymerases ɛ and δ and are predicted to cause a defect in the correction of mispaired bases inserted during DNA replication. In agreement with this prediction, the tumors from mutation carriers were microsatellite stable but tended to acquire base substitution mutations, as confirmed by yeast functional assays. Further analysis of published data showed that the recently described group of hypermutant, microsatellite-stable CRCs is likely to be caused by somatic POLE mutations affecting the exonuclease domain.

Monitoring chronic lymphocytic leukemia progression by whole genome sequencing reveals heterogeneous clonal evolution patterns

Chronic lymphocytic leukemia is characterized by relapse after treatment and chemotherapy resistance. Similarly, in other malignancies leukemia cells accumulate mutations during growth, forming heterogeneous cell populations that are subject to Darwinian selection and may respond differentially to treatment. There is therefore a clinical need to monitor changes in the subclonal composition of cancers during disease progression. Here, we use whole-genome sequencing to track subclonal heterogeneity in 3 chronic lymphocytic leukemia patients subjected to repeated cycles of therapy. We reveal different somatic mutation profiles in each patient and use these to establish probable hierarchical patterns of subclonal evolution, to identify subclones that decline or expand over time, and to detect founder mutations. We show that clonal evolution patterns are heterogeneous in individual patients. We conclude that genome sequencing is a powerful and sensitive approach to monitor disease progression repeatedly at the molecular level. If applied to future clinical trials, this approach might eventually influence treatment strategies as a tool to individualize and direct cancer treatment.

Metabolic Modulator Perhexiline Corrects Energy Deficiency and Improves Exercise Capacity in Symptomatic Hypertrophic Cardiomyopathy

Background— Hypertrophic cardiomyopathy patients exhibit myocardial energetic impairment, but a causative role for this energy deficiency in the pathophysiology of hypertrophic cardiomyopathy remains unproven. We hypothesized that the metabolic modulator perhexiline would ameliorate myocardial energy deficiency and thereby improve diastolic function and exercise capacity. Methods and Results— Forty-six consecutive patients with symptomatic exercise limitation (peak &OV0312;o2 <75% of predicted) caused by nonobstructive hypertrophic cardiomyopathy (mean age, 55±0.26 years) were randomized to perhexiline 100 mg (n=24) or placebo (n=22). Myocardial ratio of phosphocreatine to adenosine triphosphate, an established marker of cardiac energetic status, as measured by 31P magnetic resonance spectroscopy, left ventricular diastolic filling (heart rate normalized time to peak filling) at rest and during exercise using radionuclide ventriculography, peak &OV0312;o2, symptoms, quality of life, and serum metabolites were assessed at baseline and study end (4.6±1.8 months). Perhexiline improved myocardial ratios of phosphocreatine to adenosine triphosphate (from 1.27±0.02 to 1.73±0.02 versus 1.29±0.01 to 1.23±0.01; P=0.003) and normalized the abnormal prolongation of heart rate normalized time to peak filling between rest and exercise (0.11±0.008 to −0.01±0.005 versus 0.15±0.007 to 0.11±0.008 second; P=0.03). These changes were accompanied by an improvement in primary end point (peak &OV0312;o2) (22.2±0.2 to 24.3±0.2 versus 23.6±0.3 to 22.3±0.2 mL · kg−1 · min−1; P=0.003) and New York Heart Association class (P<0.001) (all P values ANCOVA, perhexiline versus placebo). Conclusions— In symptomatic hypertrophic cardiomyopathy, perhexiline, a modulator of substrate metabolism, ameliorates cardiac energetic impairment, corrects diastolic dysfunction, and increases exercise capacity. This study supports the hypothesis that energy deficiency contributes to the pathophysiology and provides a rationale for further consideration of metabolic therapies in hypertrophic cardiomyopathy. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00500552.

Reassessment of Mendelian gene pathogenicity using 7,855 cardiomyopathy cases and 60,706 reference samples

Purpose:The accurate interpretation of variation in Mendelian disease genes has lagged behind data generation as sequencing has become increasingly accessible. Ongoing large sequencing efforts present huge interpretive challenges, but they also provide an invaluable opportunity to characterize the spectrum and importance of rare variation.Methods:We analyzed sequence data from 7,855 clinical cardiomyopathy cases and 60,706 Exome Aggregation Consortium (ExAC) reference samples to obtain a better understanding of genetic variation in a representative autosomal dominant disorder.Results:We found that in some genes previously reported as important causes of a given cardiomyopathy, rare variation is not clinically informative because there is an unacceptably high likelihood of false-positive interpretation. By contrast, in other genes, we find that diagnostic laboratories may be overly conservative when assessing variant pathogenicity.Conclusions:We outline improved analytical approaches that evaluate which genes and variant classes are interpretable and propose that these will increase the clinical utility of testing across a range of Mendelian diseases.Genet Med 19 2, 192–203.

DNA polymerase ɛ and δ exonuclease domain mutations in endometrial cancer

Accurate duplication of DNA prior to cell division is essential to suppress mutagenesis and tumour development. The high fidelity of eukaryotic DNA replication is due to a combination of accurate incorporation of nucleotides into the nascent DNA strand by DNA polymerases, the recognition and removal of mispaired nucleotides (proofreading) by the exonuclease activity of DNA polymerases δ and ɛ, and post-replication surveillance and repair of newly synthesized DNA by the mismatch repair (MMR) apparatus. While the contribution of defective MMR to neoplasia is well recognized, evidence that faulty DNA polymerase activity is important in cancer development has been limited. We have recently shown that germline POLE and POLD1 exonuclease domain mutations (EDMs) predispose to colorectal cancer (CRC) and, in the latter case, to endometrial cancer (EC). Somatic POLE mutations also occur in 5–10% of sporadic CRCs and underlie a hypermutator, microsatellite-stable molecular phenotype. We hypothesized that sporadic ECs might also acquire somatic POLE and/or POLD1 mutations. Here, we have found that missense POLE EDMs with good evidence of pathogenic effects are present in 7% of a set of 173 endometrial cancers, although POLD1 EDMs are uncommon. The POLE mutations localized to highly conserved residues and were strongly predicted to affect proofreading. Consistent with this, POLE-mutant tumours were hypermutated, with a high frequency of base substitutions, and an especially large relative excess of G:C>T:A transversions. All POLE EDM tumours were microsatellite stable, suggesting that defects in either DNA proofreading or MMR provide alternative mechanisms to achieve genomic instability and tumourigenesis.

Hereditary mixed polyposis syndrome is caused by a 40-kb upstream duplication that leads to increased and ectopic expression of the BMP antagonist GREM1

Hereditary mixed polyposis syndrome (HMPS) is characterized by apparent autosomal dominant inheritance of multiple types of colorectal polyp, with colorectal carcinoma occurring in a high proportion of affected individuals. Here, we use genetic mapping, copy-number analysis, exclusion of mutations by high-throughput sequencing, gene expression analysis and functional assays to show that HMPS is caused by a duplication spanning the 3′ end of the SCG5 gene and a region upstream of the GREM1 locus. This unusual mutation is associated with increased allele-specific GREM1 expression. Whereas GREM1 is expressed in intestinal subepithelial myofibroblasts in controls, GREM1 is predominantly expressed in the epithelium of the large bowel in individuals with HMPS. The HMPS duplication contains predicted enhancer elements; some of these interact with the GREM1 promoter and can drive gene expression in vitro. Increased GREM1 expression is predicted to cause reduced bone morphogenetic protein (BMP) pathway activity, a mechanism that also underlies tumorigenesis in juvenile polyposis of the large bowel.

Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation

Background The majority of human embryos created using in vitro fertilisation (IVF) techniques are aneuploid. Comprehensive chromosome screening methods, applicable to single cells biopsied from preimplantation embryos, allow reliable identification and transfer of euploid embryos. Recently, randomised trials using such methods have indicated that aneuploidy screening improves IVF success rates. However, the high cost of testing has restricted the availability of this potentially beneficial strategy. This study aimed to harness next-generation sequencing (NGS) technology, with the intention of lowering the costs of preimplantation aneuploidy screening. Methods Embryo biopsy, whole genome amplification and semiconductor sequencing. Results A rapid (<15 h) NGS protocol was developed, with consumable cost only two-thirds that of the most widely used method for embryo aneuploidy detection. Validation involved blinded analysis of 54 cells from cell lines or biopsies from human embryos. Sensitivity and specificity were 100%. The method was applied clinically, assisting in the selection of euploid embryos in two IVF cycles, producing healthy children in both cases. The NGS approach was also able to reveal specified mutations in the nuclear or mitochondrial genomes in parallel with chromosome assessment. Interestingly, elevated mitochondrial DNA content was associated with aneuploidy (p<0.05), a finding suggestive of a link between mitochondria and chromosomal malsegregation. Conclusions This study demonstrates that NGS provides highly accurate, low-cost diagnosis of aneuploidy in cells from human preimplantation embryos and is rapid enough to allow testing without embryo cryopreservation. The method described also has the potential to shed light on other aspects of embryo genetics of relevance to health and viability.

PDK-1 regulates lactate production in hypoxia and is associated with poor prognosis in head and neck squamous cancer.

Here we describe the expression and function of a HIF-1-regulated protein pyruvate dehydrogenase kinase-1 (PDK-1) in head and neck squamous cancer (HNSCC). Using RNAi to downregulate hypoxia-inducible PDK-1, we found that lactate and pyruvate excretion after 16–48 h of hypoxia was suppressed to normoxic levels. This indicates that PDK-1 plays an important role in maintaining glycolysis. Knockdown had no effect on proliferation or survival under hypoxia. The immunohistochemical expression of PDK-1 was assessed in 140 cases of HNSCC. PDK-1 expression was not expressed in normal tissues but was upregulated in HNSCC and found to be predominantly cytoplasmic with occasional strong focal nuclear expression. It was strongly related to poor outcome (P=0.005 split by median). These results indicate that HIF regulation of PDK-1 has a key role in maintaining lactate production in human cancer and that the investigation of PDK-1 inhibitors should be investigated for antitumour effects.

SAMHD1 is mutated recurrently in chronic lymphocytic leukemia and is involved in response to DNA damage

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase and a nuclease that restricts HIV-1 in noncycling cells. Germ-line mutations in SAMHD1 have been described in patients with Aicardi-Goutières syndrome (AGS), a congenital autoimmune disease. In a previous longitudinal whole genome sequencing study of chronic lymphocytic leukemia (CLL), we revealed a SAMHD1 mutation as a potential founding event. Here, we describe an AGS patient carrying a pathogenic germ-line SAMHD1 mutation who developed CLL at 24 years of age. Using clinical trial samples, we show that acquired SAMHD1 mutations are associated with high variant allele frequency and reduced SAMHD1 expression and occur in 11% of relapsed/refractory CLL patients. We provide evidence that SAMHD1 regulates cell proliferation and survival and engages in specific protein interactions in response to DNA damage. We propose that SAMHD1 may have a function in DNA repair and that the presence of SAMHD1 mutations in CLL promotes leukemia development.