Srikanth Jammulapati

A mutation in PCSK9 causing autosomal-dominant hypercholesterolemia in a Utah pedigree

Familial hypercholesterolemia results from mutations in the low-density lipoprotein (LDL) receptor or apolipoproteinxa0B genes. We have previously reported the identification of a Utah autosomal-dominant hypercholesterolemia pedigree (kindred 1173) that did not show linkage to either of these loci (Hunt et al. 2000). Expansion of the pedigree and increased marker density within the region of interest have resulted in a multipoint LOD score of 9.6 and enabled us to decrease the size of the linked region to approximately 7.5xa0Mbp. In addition, we were able to identify additional families sharing the same microsatellite haplotype. While all haplotype carriers in kindred 1173 (K1173) are affected, the haplotype carriers within the newly identified families are unaffected, suggesting that the causal mutation in K1173 had occurred after divergence of these pedigrees from a common ancestor. Mutation screening of genes in the region identified a single nucleotide variant (G→T) present on the K1173 haplotype that was not present on the same haplotype in the other kindreds. This variant results in a D374Y missense change in the gene PCSK9.

Patterns of genomic loss of heterozygosity predict homologous recombination repair defects in epithelial ovarian cancer

Background:Defects in BRCA1, BRCA2, and other members of the homologous recombination pathway have potential therapeutic relevance when used to support agents that introduce or exploit double-stranded DNA breaks. This study examines the association between homologous recombination defects and genomic patterns of loss of heterozygosity (LOH).Methods:Ovarian tumours from two independent data sets were characterised for defects in BRCA1, BRCA2, and RAD51C, and LOH profiles were generated. Publically available data were downloaded for a third independent data set. The same analyses were performed on 57 cancer cell lines.Results:Loss of heterozygosity regions of intermediate size were observed more frequently in tumours with defective BRCA1 or BRCA2 (P=10−11). The homologous recombination deficiency (HRD) score was defined as the number of these regions observed in a tumour sample. The association between HRD score and BRCA deficiency was validated in two independent ovarian cancer data sets (P=10−5 and 10−29), and identified breast and pancreatic cell lines with BRCA defects.Conclusion:The HRD score appears capable of detecting homologous recombination defects regardless of aetiology or mechanism. This score could facilitate the use of PARP inhibitors and platinum in breast, ovarian, and other cancers.

A major predisposition locus for severe obesity, at 4p15-p14.

Although the predisposition to morbid obesity is heritable, the identities of the disease-causing genes are largely unknown. Therefore, we have conducted a genomewide search with 628 markers, using multigenerational Utah pedigrees to identify genes involved in predisposition to obesity. In the genomewide search, we identified a highly significant linkage to high body-mass index in female patients, at D4S2632, with a multipoint heterogeneity LOD (HLOD) score of 6.1 and a nonparametric linkage (NPL) score of 5.3. To further delineate the linkage, we increased both the marker density around D4S2632 and the size of our pedigree data set. As a result, the linkage evidence increased to a multipoint HLOD score of 9.2 (at D4S3350) and an NPL score of 11.3. Evidence from almost half of the families in this analysis support this linkage, and therefore the gene in this region might account for a significant percentage of the genetic predisposition to severe obesity in females. However, further studies are necessary to clarify the effect that this gene has in males and in the general population.

Linkage of body mass index to chromosome 20 in Utah pedigrees

Several linkage studies have hinted at the existence of an obesity predisposition locus on chromosome 20, but none of these studies has produced conclusive results. Therefore, we analyzed 48 genetic markers on chromosome 20 for linkage to severe obesity (BMI≥35) in 103 extended Utah pedigrees (1,711 individuals), all of which had strong aggregation of severe obesity. A simple dominant model produced a maximum multipoint heterogeneity LOD score of 3.5 at D20S438 (55.1xa0cM). Two additional analyses were performed. First, a one-gene, two-mutation model (with one dominant mutation and one recessive mutation) increased the LOD score to 4.2. Second, a two-locus model (with one locus dominant and one recessive) generated a multipoint LOD score of 4.9. We conclude that one or more severe obesity predisposing genes lie within an interval of approx. 10xa0cM on chromosome 20. This study generated significant LOD scores which confirm suggestive linkage reports from previous studies. In addition, our analyses suggest that the predisposing gene(s) is localized very near the chromosome 20 centromere.

Development and analytical validation of a 25-gene next generation sequencing panel that includes the BRCA1 and BRCA2 genes to assess hereditary cancer risk

BackgroundGermline DNA mutations that increase the susceptibility of a patient to certain cancers have been identified in various genes, and patients can be screened for mutations in these genes to assess their level of risk for developing cancer. Traditional methods using Sanger sequencing focus on small groups of genes and therefore are unable to screen for numerous genes from several patients simultaneously. The goal of the present study was to validate a 25-gene panel to assess genetic risk for cancer in 8 different tissues using next generation sequencing (NGS) techniques.MethodsTwenty-five genes associated with hereditary cancer syndromes were selected for development of a panel to screen for risk of these cancers using NGS. In an initial technical assessment, NGS results for BRCA1 and BRCA2 were compared with Sanger sequencing in 1864 anonymized DNA samples from patients who had undergone previous clinical testing. Next, the entire gene panel was validated using parallel NGS and Sanger sequencing in 100 anonymized DNA samples. Large rearrangement analysis was validated using NGS, microarray comparative genomic hybridization (CGH), and multiplex ligation-dependent probe amplification analyses (MLPA).ResultsNGS identified 15,877 sequence variants, while Sanger sequencing identified 15,878 in the BRCA1 and BRCA2 comparison study of the same regions. Based on these results, the NGS process was refined prior to the validation of the full gene panel. In the validation study, NGS and Sanger sequencing were 100% concordant for the 3,923 collective variants across all genes for an analytical sensitivity of the NGS assay of >99.92% (lower limit of 95% confidence interval). NGS, microarray CGH and MLPA correctly identified all expected positive and negative large rearrangement results for the 25-gene panel.ConclusionThis study provides a thorough validation of the 25-gene NGS panel and indicates that this analysis tool can be used to collect clinically significant information related to risk of developing hereditary cancers.

Abstract 3116: Patterns of genomic loss of heterozygosity predict homologous recombination repair defects in ovarian cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnIntroduction: BRCA1 and BRCA2 are key members of the homologous recombination (HR) pathway. Mutations in these genes and other HR pathway defects have potential therapeutic relevance when used to support agents that introduce or exploit double-strand DNA breaks. This study examines the association between HR deficiency and genomic patterns of loss of heterozygosity (LOH). Methods: Ovarian tumors from two independent datasets were characterized for germline and somatic defects in BRCA1 and BRCA2. Whole genome LOH profiles were generated using Affymetrix SNP arrays. Publically available data was downloaded from the TCGA website for a third independent ovarian cancer dataset. RAD51C promoter methylation was assayed in two of the datasets. Comprehensive profiling of BRCA1 and BRCA2 defects, and genome wide LOH was also performed on approximately 70 breast, ovarian, colon and pancreatic cell lines. Results: Examination of the pattern of LOH within ovarian tumors with BRCA1, BRCA2, or RAD51C defects compared to tumors without defects in these genes has resulted in the development of a homologous recombination deficiency (HRD) score that has highly significant association with HRD (p=9*10-11). An intermediate class of LOH sizes (>15 Mb but less than a whole chromosome) is highly positive correlated with defective HR, suggesting this class of LOH exists due to double strand DNA break formation and requires repair by HR. The HRD score was validated in two independent ovarian cancer datasets (p=2*10-7 and 9*10-29), and successfully identified breast and pancreatic cell lines with BRCA defects, suggesting it will be effective across multiple tumor types. Conclusions: BRCA1 or BRCA2 mutation carriers have improved outcomes following treatment with DNA damaging agents such as platinum salts, and preclinical studies have demonstrated PARP inhibitor efficacy in BRCA1 or BRCA2 deficient cells. HR deficiency in ovarian cancer is not solely due to germline BRCA1 and BRCA2 mutations, and HR deficiency is not unique to ovarian tumors. Each type of cancer is likely to have a unique spectrum of genetic variants resulting in HRD. The HRD score appears capable of detecting HRD regardless of etiology or mechanism. This score could have clinical utility in breast and ovarian cancer, and could be used to target the use of PARP inhibitors and platinum salts in other cancers.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3116. doi:1538-7445.AM2012-3116