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Figure S1. Summary of HRD testing in PrECOG and combined cisplatin cohorts; Figure S2. Distribution of HRD scores in PrECOG and combined cisplatin cohorts; Figure S3. Performance of HRD in predicting response in PrECOG and combined cisplatin cohorts; Figure S4. Logistic regression models to predict response in PrECOG and combined cisplatin cohorts; Table S1. Study cohorts for threshold training set; Table S2. Summary statistics of HRD scores in BRCA1/2 deficient samples by tissue in training; Table S3. Patient clinical and demographic data; Table S4. HRD score and association with therapy response in entire cohorts; Table S5. Univariate associations between clinical variables and RCB 0/1 or HR deficiency status; Table S6. Analysis of RCB 0/1 and pCR in combined PrECOG 0105 and Cisplatin Trials cohorts (logistic regression models adjusted for cohort)

Purpose:BRCA1/2-mutated and some sporadic triple-negative breast cancers (TNBC) have DNA repair defects and are sensitive to DNA-damaging therapeutics. Recently, three independent DNA-based measures of genomic instability were developed on the basis of loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transitions (LST).Experimental Design: We assessed a combined homologous recombination deficiency (HRD) score, an unweighted sum of LOH, TAI, and LST scores, in three neoadjuvant TNBC trials of platinum-containing therapy. We then tested the association of HR deficiency, defined as HRD score ≥42 or BRCA1/2 mutation, with response to platinum-based therapy.Results: In a trial of neoadjuvant platinum, gemcitabine, and iniparib, HR deficiency predicted residual cancer burden score of 0 or I (RCB 0/I) and pathologic complete response (pCR; OR = 4.96, P = 0.0036; OR = 6.52, P = 0.0058). HR deficiency remained a significant predictor of RCB 0/I when adjusted for clinical variables (OR = 5.86, P = 0.012). In two other trials of neoadjuvant cisplatin therapy, HR deficiency predicted RCB 0/I and pCR (OR = 10.18, P = 0.0011; OR = 17.00, P = 0.0066). In a multivariable model of RCB 0/I, HR deficiency retained significance when clinical variables were included (OR = 12.08, P = 0.0017). When restricted to BRCA1/2 nonmutated tumors, response was higher in patients with high HRD scores: RCB 0/I P = 0.062, pCR P = 0.063 in the neoadjuvant platinum, gemcitabine, and iniparib trial; RCB 0/I P = 0.0039, pCR P = 0.018 in the neoadjuvant cisplatin trials.Conclusions: HR deficiency identifies TNBC tumors, including BRCA1/2 nonmutated tumors more likely to respond to platinum-containing therapy. Clin Cancer Res; 22(15); 3764–73. ©2016 AACR.

PURPOSE Polygenic risk scores (PRSs) for breast cancer (BC) risk stratification have been developed primarily in women of European ancestry. Their application to women of non-European ancestry has lagged because of the lack of a formal approach to incorporate genetic ancestry and ancestry-dependent variant frequencies and effect sizes. Here, we propose a multiple-ancestry PRS (MA-PRS) that addresses these issues and may be useful in the development of equitable PRSs across other cancers and common diseases. MATERIALS AND METHODS Women referred for hereditary cancer testing were divided into consecutive cohorts for development (n = 189,230) and for independent validation (n = 89,126). Individual genetic composition as fractions of three reference ancestries (African, East Asian, and European) was determined from ancestry-informative single-nucleotide polymorphisms. The MA-PRS is a combination of three ancestry-specific PRSs on the basis of genetic ancestral composition. Stratification of risk was evaluated by multivariable logistic regression models controlling for family cancer history. Goodness-of-fit analysis compared expected with observed relative risks by quantiles of the MA-PRS distribution. RESULTS In independent validation, the MA-PRS was significantly associated with BC risk in the full cohort (odds ratio, 1.43; 95% CI, 1.40 to 1.46; P = 8.6 × 10–308) and within each major ancestry. The top decile of the MA-PRS consistently identified patients with two-fold increased risk of developing BC. Goodness-of-fit tests showed that the MA-PRS was well calibrated and predicted BC risk accurately in the tails of the distribution for both European and non-European women. CONCLUSION The MA-PRS uses genetic ancestral composition to expand the utility of polygenic risk prediction to non-European women. Inclusion of genetic ancestry in polygenic risk prediction presents an opportunity for more personalized treatment decisions for women of varying and mixed ancestries.

Purpose This study was designed to assess efficacy, safety, and predictors of response to iniparib in combination with gemcitabine and carboplatin in early-stage triple-negative and BRCA1/2 mutation–associated breast cancer. Patients and Methods This single-arm phase II study enrolled patients with stage I to IIIA (T ≥ 1 cm) estrogen receptor–negative (≤ 5%), progesterone receptor–negative (≤ 5%), and human epidermal growth factor receptor 2–negative or BRCA1/2 mutation–associated breast cancer. Neoadjuvant gemcitabine (1,000 mg/m2 intravenously [IV] on days 1 and 8), carboplatin (area under curve of 2 IV on days 1 and 8), and iniparib (5.6 mg/kg IV on days 1, 4, 8, and 11) were administered every 21 days for four cycles, until the protocol was amended to six cycles. The primary end point was pathologic complete response (no invasive carcinoma in breast or axilla). All patients underwent comprehensive BRCA1/2 genotyping, and homologous recombination deficiency was assessed by loss of heterozygosity (HRD-LOH) in pretreatment core breast biopsies. Results Among 80 patients, median age was 48 years; 19 patients (24%) had germline BRCA1 or BRCA2 mutations; clinical stage was I (13%), IIA (36%), IIB (36%), and IIIA (15%). Overall pathologic complete response rate in the intent-to-treat population (n = 80) was 36% (90% CI, 27 to 46). Mean HRD-LOH scores were higher in responders compared with nonresponders (P = .02) and remained significant when BRCA1/2 germline mutations carriers were excluded (P = .021). Conclusion Preoperative combination of gemcitabine, carboplatin, and iniparib is active in the treatment of early-stage triple-negative and BRCA1/2 mutation–associated breast cancer. The HRD-LOH assay was able to identify patients with sporadic triple-negative breast cancer lacking a BRCA1/2 mutation, but with an elevated HRD-LOH score, who achieved a favorable pathologic response. Confirmatory controlled trials are warranted.

Defects in the homologous recombination (HR) DNA repair pathway sensitize tumors to therapeutics that target this pathway. A significant proportion of triple-negative breast cancers (TNBC) carry HR defects. The HRD assay is highly associated with sensitivity to neoadjuvant platinum-based chemotherapy in TNBC. Standard chemotherapy consists of some combination of an anthracycline, cyclophosphamide, and taxane. This study assesses the association of HR deficiency status with response to standard neoadjuvant chemotherapy in TNBC or BRCA1/2 mutation-associated breast cancer. Tumor samples were retrospectively obtained from 45 TNBC patients and 2 BRCA1/2 mutant, hormone receptor-positive/HER2-negative breast cancer patients who received anthracycline- and/or taxane-based neoadjuvant chemotherapy at Stanford University or Cedars-Sinai Medical Centers. The HRD score and tumor BRCA1/2 mutation status were determined from baseline tumor biopsies. HR deficient tumors were those with a HRD score of ≥ 42 or a tumor BRCA1/2 mutation. Response was categorized by the residual cancer burden (RCB) index. HR deficient patients were more likely to achieve a pathologic complete response (pCR) compared with non-deficient patients (OR 13.06, CI 1.52–11.241, p = 0.0028). Among BRCA1/2 mutation wild-type patients, HR deficient patients were more likely to achieve a pCR (OR 16, 95% CI 1.65–160.41, p = 0.0041) compared with HR non-deficient patients. Further, HRD scores were highly concordant pre- and post-therapy (Spearman correlation > 99%). HR deficiency status is significantly associated with response to standard neoadjuvant chemotherapy in TNBC. This observation is consistent with the mechanisms of action of doxorubicin and cyclophosphamide as DNA damaging agents.

Purpose: BRCA1/2-mutated and some sporadic triple-negative breast cancers (TNBC) have DNA repair defects and are sensitive to DNA-damaging therapeutics. Recently, three independent DNA-based measures of genomic instability were developed on the basis of loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transitions (LST). Experimental Design: We assessed a combined homologous recombination deficiency (HRD) score, an unweighted sum of LOH, TAI, and LST scores, in three neoadjuvant TNBC trials of platinum-containing therapy. We then tested the association of HR deficiency, defined as HRD score ≥42 or BRCA1/2 mutation, with response to platinum-based therapy. Results: In a trial of neoadjuvant platinum, gemcitabine, and iniparib, HR deficiency predicted residual cancer burden score of 0 or I (RCB 0/I) and pathologic complete response (pCR; OR = 4.96, P = 0.0036; OR = 6.52, P = 0.0058). HR deficiency remained a significant predictor of RCB 0/I when adjusted for clinical variables (OR = 5.86, P = 0.012). In two other trials of neoadjuvant cisplatin therapy, HR deficiency predicted RCB 0/I and pCR (OR = 10.18, P = 0.0011; OR = 17.00, P = 0.0066). In a multivariable model of RCB 0/I, HR deficiency retained significance when clinical variables were included (OR = 12.08, P = 0.0017). When restricted to BRCA1/2 nonmutated tumors, response was higher in patients with high HRD scores: RCB 0/I P = 0.062, pCR P = 0.063 in the neoadjuvant platinum, gemcitabine, and iniparib trial; RCB 0/I P = 0.0039, pCR P = 0.018 in the neoadjuvant cisplatin trials. Conclusions: HR deficiency identifies TNBC tumors, including BRCA1/2 nonmutated tumors more likely to respond to platinum-containing therapy. Clin Cancer Res; 22(15); 3764–73. ©2016 AACR.

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.

Human esophageal adenocarcinoma (EAC) cell lines and xenografts are powerful tools in the search for genetic alterations because these models are composed of pure human cancer cell populations without admixture of normal human cells. In particular detection of homozygous deletions (HDs) is easier using these pure populations of cancer cells. Identification of HDs could potentially lead to the subsequent identification of new tumor suppressor genes (TSGs) involved in esophageal adenocarcinogenesis. Genome wide single nucleotide polymorphism (SNP) arrays were used to identify HDs in 10 verified EAC cell lines and nine EAC xenografts. In total, 61 HDs (range 16 per sample) were detected and confirmed by polymerase chain reaction. Besides HDs observed in common fragile genomic regions (n = 26), and gene deserts (n = 8), 27 HDs were located in gene-containing regions. HDs were noted for known TSGs, including CDKN2A, SMAD4 and CDH3/CDH1. Twenty-two new chromosomal regions were detected harboring potentially new TSGs involved in EAC carcinogenesis. Two of these regions of homozygous loss, encompassing the ITGAV and RUNX1 gene, were detected in multiple samples indicating a potential role in the carcinogenesis of EAC. To exclude culturing artifacts, these last two deletions were confirmed by fluorescent in situ hybridization in the primary tumors of which the involved cell lines and xenografts were derived. In summary, in this report we describe the identification of HDs in a series of verified EAC cell lines and xenografts. The deletions documented here are a step forward identifying the key genes involved in EAC development. (c) 2011 Wiley Periodicals, Inc.

12017Background: The PARP inhibitor olaparib is approved for maintenance treatment of platinum-sensitive relapsed ovarian cancer (PSR OC) in the USA based on pivotal studies (Study 19, SOLO2) that ...

5563Background: The PARP inhibitor olaparib is approved in the US for patients with mBRCA (germline) advanced ovarian cancer, and in the EU for platinum-sensitive relapsed mBRCA (germline or somati...

Psoriasis is an immune-mediated skin disorder that is inherited as a multifactorial trait. Linkage studies have clearly identified a primary disease susceptibility locus lying within the major histocompatibility complex (MHC), but have generated conflicting results for other genomic regions. To overcome this difficulty, we have carried out a genome-wide association scan, where we analyzed more than 408,000 SNPs in an initial sample of 318 cases and 288 controls. Outside of the MHC, we observed a single cluster of disease-associated markers, spanning 47 kb on chromosome 20q13. The analysis of two replication data sets confirmed this association, with SNP rs495337 yielding a combined P-value of 1.4 x 10(-8) in an overall sample of 2679 cases and 2215 controls. Rs495337 maps to the SPATA2 transcript and is in absolute linkage disequilibrium with five SNPs lying in the adjacent ZNF313 gene (also known as RNF114). Real-time PCR experiments showed that, unlike SPATA2, ZNF313 is abundantly expressed in skin, T-lymphocytes and dendritic cells. Furthermore, an analysis of the expression data available from the Genevar database indicated that rs495337 is associated with increased ZNF313 transcripts levels (P = 0.003), suggesting that the disease susceptibility allele may be a ZNF313 regulatory variant tagged by rs495337. Homology searches indicated that ZNF313 is a paralogue of TRAC-1, an ubiquitin ligase regulating T-cell activation. We performed cell-free assays and confirmed that like TRAC-1, ZNF313 binds ubiquitin via an ubiquitin-interaction motif (UIM). These findings collectively identify a novel psoriasis susceptibility gene, with a putative role in the regulation of immune responses.

Osteoarthritis (OA), the most prevalent form of arthritis in the elderly, is characterized by the degradation of articular cartilage and has a strong genetic component. Our aim was to identify genetic variants involved in risk of knee OA in women. A pooled genome-wide association scan with the Illumina550 Duo array was performed in 255 controls and 387 cases. Twenty-eight variants with p < 1 x 10(-5) were estimated to have probabilities of being false positives

Objective: The objective was to provide an overall assessment of genetic linkage data of BMI and BMI-defined obesity using a nonparametric genome scan meta-analysis. Research Methods and Procedures: We identified 37 published studies containing data on over 31,000 individuals from more than >10,000 families and obtained genome-wide logarithm of the odds (LOD) scores, non-parametric linkage (NPL) scores, or maximum likelihood scores (MLS). BMI was analyzed in a pooled set of all studies, as a subgroup of 10 studies that used BMI-defined obesity, and for subgroups ascertained through type 2 diabetes, hypertension, or subjects of European ancestry. Results: Bins at chromosome 13q13.2- q33.1, 12q23-q24.3 achieved suggestive evidence of linkage to BMI in the pooled analysis and samples ascertained for hypertension. Nominal evidence of linkage to these regions and suggestive evidence for 11q13.3-22.3 were also observed for BMI-defined obesity. The FTO obesity gene locus at 16q12.2 also showed nominal evidence for linkage. However, overall distribution of summed rank p values

The purpose of this study is to evaluate the efficacy and safety of neoadjuvant treatment with carboplatin and eribulin in patients with early-stage triple negative breast cancer (TNBC), and to explore biomarkers based on DNA and protein expression profiles as predictors of response. Patients with histologically confirmed early-stage TNBC received carboplatin AUC 6 iv every 21 days, and eribulin 1.4 mg/m(2) day 1 and day 8 every 21 days for four cycles. The primary endpoint of the study was pathologic complete response (pCR), with secondary endpoints including clinical response and safety of the combination. Exploratory studies assessed DNA-based biomarkers [homologous recombination deficiency (HRD) score, and HR deficiency status (HRD score + BRCA1/BRCA2 mutation status)], protein-based biomarkers (Ki67, TP53, androgen receptor, Cyclin E, CDK2, Cyclin D, CDK4, Pin1 and Smad3), and clinical pretreatment factors as predictors of pCR. 13/30 (43.3 %) patients enrolled in the study achieved pCR. 24 (80.0 %) had a clinical complete or partial response. The combination was safe with mostly grade 1 and 2 toxicities. HRD score (P = 0.0024) and HR deficiency status (P = 0.0012) significantly predicted pCR. Pretreatment cytoplasmic CDK2 was also associated with pCR (P = 0.021). Significant differences in pre- versus post-treatment expression levels of nuclear Cyclin D (P = 0.020), nuclear CDK4 (P = 0.0030), and nuclear Smad3 (P = 0.015) were detected. The combination of carboplatin and eribulin is safe and efficacious in the treatment of early-stage TNBC. HRD score, HR deficiency status, and cytoplasmic CDK2 predicted pCR in this patient population.

The molecular etiology of obesity predisposition is largely unknown. Here, we present evidence that genetic variation in TBC1D1 confers risk for severe obesity in females. We identified a coding variant (R125W) in TBC1D1 that segregated with the disease in 4p15-14-linked obesity pedigrees. In cases derived from pedigrees with the strongest linkage evidence, the variant was significantly associated with obesity (P = 0.000007) and chromosomes carrying R125W accounted for the majority of the evidence that originally linked 4p15-14 with the disease. In addition, by selecting families that segregated R125W with obesity, we were able to generate highly significant linkage evidence for an obesity predisposition locus at 4q34-35. This result provides additional and confirming evidence that R125W affects obesity susceptibility, delimits the location of an obesity gene at 4q34-35 and identifies a gene/gene interaction that influences the risk for obesity predisposition. Finally, although the function of TBC1D1 is unknown, the protein is structurally similar to a known regulator of insulin-mediated Glut4 translocation.

APAF1, encoding the protein apoptosis protease activating factor 1 (Apaf-1), has recently been established as a chromosome 12 gene conferring predisposition to major depression in humans. The molecular phenotypes of Apaf-1 variants were determined by in vitro reconstruction of the apoptosome complex in which Apaf-1 activates caspase 9 and thus initiates a cascade of proteolytic events leading to apoptotic destruction of the cell. Cellular phenotypes were measured using a yeast heterologous expression assay in which human Apaf-1 and other proteins necessary to constitute a functional apoptotic pathway were overexpressed. Apaf-1 variants encoded by APAF1 alleles that segregate with major depression in families linked to chromosome 12 shared a common gain-of-function phenotype in both assay systems. In contrast, other Apaf-1 variants showed neutral or loss-of-function phenotypes. The depression-associated alleles thus have a common phenotype that is distinct from that of non-associated variants. This result suggests an etiologic role for enhanced apoptosis in major depression.

Introduction: Interpretation of results from mutation screening of tumour suppressor genes known to harbour high risk susceptibility mutations, such as APC, BRCA1, BRCA2, MLH1, MSH2, TP53, and PTEN, is becoming an increasingly important part of clinical practice. Interpretation of truncating mutations, gene rearrangements, and obvious splice junction mutations, is generally straightforward. However, classification of missense variants often presents a difficult problem. From a series of 20 000 full sequence tests of BRCA1 carried out at Myriad Genetic Laboratories, a total of 314 different missense changes and eight in-frame deletions were observed. Before this study, only 21 of these missense changes were classified as deleterious or suspected deleterious and 14 as neutral or of little clinical significance. Methods: We have used a combination of a multiple sequence alignment of orthologous BRCA1 sequences and a measure of the chemical difference between the amino acids present at individual residues in the sequence alignment to classify missense variants and in-frame deletions detected during mutation screening of BRCA1. Results: In the present analysis we were able to classify an additional 50 missense variants and two in-frame deletions as probably deleterious and 92 missense variants as probably neutral. Thus we have tentatively classified about 50% of the unclassified missense variants observed during clinical testing of BRCA1. Discussion: An internal test of the analysis is consistent with our classification of the variants designated probably deleterious; however, we must stress that this classification is tentative and does not have sufficient independent confirmation to serve as a clinically applicable stand alone method.

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.

Defects in the lipoprotein lipase (LPL) gene are associated with dyslipidemia in the general population. Several rare mutations in the gene, as well as two common coding region polymorphisms, D9N and N291S, exhibit deleterious effects on circulating lipid levels. Using a linkage-based approach, we have identified a large Utah kindred segregating the D9N variant in the LPL gene. The kindred was ascertained for premature coronary heart disease and was expanded based on familial dyslipidemia. A genomic scan identified a region of linkage including LPL, and mutation screening identified the segregating variant. In the kindred, the variant shows high penetrance for a hypoalphalipoproteinemia phenotype, but is also associated with hypertriglyceridemia and elevated insulin levels. The strength of linkage was dependent on the combination of phenotype definition and model parameters, favoring the use of a MOD score approach. Most other studies of LPL have proceeded by mutation screening of randomly chosen individuals or selected affected probands; this is the first example identifying a segregating LPL mutation using direct linkage.

Coronary heart disease (CHD) accounts for half of the 1 million deaths annually ascribed to cardiovascular disease and for almost all of the 1.5 million acute myocardial infarctions. Within families affected by early and apparently heritable CHD, dyslipidemias have a much higher prevalence than in the general population; 20%-30% of early familial CHD has been ascribed to primary hypoalphalipoproteinemia (low HDL-C). This study assesses the evidence for linkage of low HDL-C to chromosomal region 11q23 in 105 large Utah pedigrees ascertained with closely related clusters of early CHD and expanded on the basis of dyslipidemia. Linkage analysis was performed by use of 22 STRP markers in a 55-cM region of chromosome 11. Two-point analysis based on a general, dominant-phenotype model yielded LODs of 2.9 for full pedigrees and 3.5 for 167 four-generation split pedigrees. To define a localization region, model optimization was performed using the heterogeneity, multipoint LOD score (mpHLOD). This linkage defines a region on 11q23.3 that is approximately 10 cM distal to-and apparently distinct from-the ApoAI/CIII/AIV gene cluster and thus represents a putative novel localization for the low HDL-C phenotype.

The problem of multilocus linkage analysis is expressed as a graphical model, making explicit a previously implicit connection, and recent developments in the field are described in this context. A novel application of blocked Gibbs sampling for Bayesian networks is developed to generate inheritance matrices from an irreducible Markov chain. This is used as the basis for reconstruction of historical meiotic states and approximate calculation of the likelihood function for the location of an unmapped genetic trait. We believe this to be the only approach that currently makes fully informative multilocus linkage analysis possible on large extended pedigrees.

Background: Defects in the homologous recombination (HR) DNA repair pathway sensitize tumors to therapeutics that target this pathway. A significant proportion of triple negative breast cancers (TNBC) carry HR defects. Recently three DNA-based measures (LOH, Abkevich et al.; TAI, Birkbak et al; LST, Popova et al.) have been developed and shown to be highly associated with BRCA1/2 mutation status and sensitivity to platinum-based chemotherapy in TNBC. Standard chemotherapy consists of some combination of an anthracycline and cyclophosphamide, with or without a taxane. This study assesses the association of LOH, TAI, LST and the sum of these measures, the HRD Score, with response to standard neoadjuvant chemotherapy in patients with TNBC. Methods: Tumor samples were retrospectively obtained from 45 TNBC patients and 2 BRCA1/2 mutation positive hormone receptor-positive/HER2-negative breast cancer patients who received anthracycline-based neoadjuvant chemotherapy at Stanford University Medical Center or Cedars-Sinai Medical Center under IRB approved protocols. Measures of LOH, TAI, LST were obtained; tumor BRCA1/2 mutation analysis was conducted; and BRCA1 promoter methylation status was determined. Response was categorized by the residual cancer burden (RCB) score with responders defined as RCB 0 or 1, and non-responders as RCB 2 or 3. Data were also analyzed using the outcome of pathologic complete response (RCB 0). BRCA1/2 deficiency was defined as either BRCA1/2 germline or somatic mutant, or BRCA1 methylated with loss of the second allele in the tumor confirmed by LOH at the affected gene. Associations of LOH, TAI, LST, HRD Score, BRCA1/2 deficiency and mutation status with response to neoadjuvant therapy were evaluated with univariate logistic regression models. Results: All three metrics showed significant association with response (LOH: p=0.0018; TAI: p=0.0057; LST: p=0.0043) and pCR (LOH: p=0.0061; TAI: p=0.0044; LST: p=0.047). The sum of the scores, HRD Score, was also significantly associated with response (p=0.0021) and pCR (0.011). Neither BRCA1/2 mutation status (p=0.78 and p=0.31 respectively) nor BRCA1/2 deficiency (p=0.12 and p=0.34 respectively) were significantly associated with response or pCR in this cohort. When the analysis was restricted to BRCA1/2 intact samples (n=29) all scores were still significantly associated with pCR (LOH: p=0.019; TAI: p=0.0046; LST: p=0.013; HRD: p=0.0067), while only the sum was significant for response (p=0.042). Conclusions:All three measures of HR deficiency, LOH, TAI and LST and the sum of these three metrics, the HRD Score, are significantly associated with response to standard neoadjuvant chemotherapy in TNBC. This observation is consistent with the mechanisms of action of doxorubicin and cyclophosphamide as DNA damaging agents, though we have yet to examine cohorts of patients treated with non-anthracycline-containing standard chemotherapy regimens, such as docetaxel and cyclophosphamide. The HRD Score could be used clinically to identify patients with increased sensitivity to DNA damaging therapeutics. Citation Format: Melinda L Telli, William Audeh, Kirstin C Jensen, Shikha Bose, Kirsten Timms, Alexander Gutin, Victor Abkevich, Jerry Lanchbury, Chris Neff, Elisha Hughes, Zaina Sangale, Joshua Jones, Richard Wenstrup, Anne-Renee Hartman, Pei-Jen Chang, Shaveta Vinayak, James M Ford. Homologous recombination deficiency (HRD) score predicts response to standard neoadjuvant chemotherapy in patients with triple negative or BRCA1/2 mutation-associated breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-06-01.

Background : Protein engineering has been used extensively to evaluate the properties of transition states in protein folding. Although the method has proved useful, its limitations and the details of interpretation of the obtained results remain largely unexplored. Results : Lattice model simulations are used to test and verify the protein engineering analysis of the transition state in protein folding. It is shown that in some cases – but not always – this method is able to determine the transition state with reasonable accuracy. Limitations of protein engineering are revealed and analyzed. In particular, the change in non-native interactions as a result of mutations is shown to influence the results of the protein engineering analysis. Furthermore, the temperature dependencies of ϕ values (which are a measure of the participation of a residue in the transition state) and the character of the transition state ensemble are studied. It is shown that as a general trend ϕ values decrease when the temperature decreases, a finding consistent with recent experimental results. Our analysis suggests that this trend results primarily from the formation of some contacts (native and non-native) in the unfolded state at a lower temperature, when the barrier for folding is energetic. Conclusions : Our analysis helps to interpret the results of protein engineering and allows observed ϕ values to be directly related to structural features of the unfolded state, the transition state and the native state.

This study focuses of the phenomenon of kinetic partitioning when a polypeptide chain has two ground-state conformations, one of which is kinetically more reachable than the other. We designed sequences for lattice model proteins with two different conformations of equal energy corresponding to the global energy minimum. Folding simulations revealed that one of these conformations was indeed much more kinetically accessible than the other. We found that the number and strength of local contacts in the ground-state conformation are the major factors that determine which conformation is reached faster; the greater the number of local contacts, the more kinetically reachable a conformation is. We present simple statistical-mechanical arguments to explain these findings. Our results may be relevant in explaining the phenomenology of such proteins as human plasminogen activator inhibitor-1 (PAI-1), photosystem II, and prions.

Sequences of fast-folding model proteins (48 residues long on a cubic lattice) were generated by an evolution-like selection toward fast folding. We find that fast-folding proteins exhibit a specific folding mechanism in which all transition state conformations share a smaller subset of common contacts (folding nucleus). Acceleration of folding was accompanied by dramatic strengthening of interactions in the folding nucleus whereas average energy of nonnucleus interactions remained largely unchanged. Furthermore, the residues involved in the nucleus are the most conserved ones within families of evolved sequences. Our results imply that for each protein structure there is a small number of conserved positions that are key determinants of fast folding into that structure. This conjecture was tested on two protein superfamilies: the first having the classical monophosphate binding fold (CMBF; 98 families) and the second having type-III repeat fold (47 families). For each superfamily, we discovered a few positions that exhibit very strong and statistically significant “conservatism of conservatism”—amino acids in those positions are conserved within every family whereas the actual types of amino acids varied from family to family. Those amino acids are in spatial contact with each other. The experimental data of Serrano and coworkers [Lopez-Hernandez, E. & Serrano, L. (1996) Fold. Des. (London) 1, 43–55]. for one of the proteins of the CMBF superfamily (CheY) show that residues identified this way indeed belong to the folding nucleus. Further analysis revealed deep connections between nucleation in CMBF proteins and their function.

Monte Carlo simulation of model proteins on a cubic lattice are used to study the thermodynamics and kinetics of protein folding over a wide range of temperatures. Both random sequences and sequences designed to have a pronounced minimum of energy are examined. There is no indication in the kinetics of a “glass” transition at low temperature, i.e., below the temperature of the equilibrium folding transition, the kinetics of folding is described by the Arrhenius law at all temperatures that were examined. The folding kinetics is single-exponential in the whole range of studied temperatures for random sequences. The general implications of the temperature dependence of the folding rate are discussed and related to certain properties of the energy spectrum. The results obtained in the simulations are in qualitative disagreement with the conclusions of a theoretical analysis of protein folding kinetics based on certain kinetics assumptions introduced in the Random Energy Model. The origins of the discrepancies are analyzed and a simple phenomenological theory is presented to describe the temperature dependence of the folding time for random sequences.

By means of Monte Carlo simulation, we investigated the equilibrium between folded and unfolded states of lattice model proteins. The amino acid sequences were designed to have pronounced energy minimum target conformations of different length and shape. For short fully compact (36-mer) proteins, the all-or-none transition from the unfolded state to the native state was observed. This was not always the case for longer proteins. Among 12 designed sequences with the native structure of a fully compact 48-mer, a simple all-or-none transition was observed in only three cases. For the other nine sequences, three states of behavior-the native, denatured, and intermediate states-were found. The contiguous part of the native structure (domain) was conserved in the intermediate state, whereas the remaining part was completely unfolded and structureless. These parts melted separately from each other.

Folding of the lattice model of proteins is studied using Monte Carlo simulation. The amino acid sequence is designed to have a pronounced energy minimum for a given target (native) conformation. Our simulations reveal two possible scenarios. When the overall attraction between residues dominates, we find that folding to the native conformation is preceded by a rapid collapse into a burst intermediate which is a compact but structureless globule. Then, after a much longer time, an all-or-none transition from the globule to the native conformation occurs. In contrast, when the overall attraction is not strong, we do not observe a burst collapse stage. Instead, we find an all-or-none transition directly from the coil to the native conformation. Both scenarios yield comparable rates of folding. On the basis of these findings we discuss the role of intermediates in thermodynamics and kinetics of protein folding.

We propose an algorithm providing sequences of model proteins with rapid folding into a given target (native) conformation. This algorithm is applied to a chain of 27 residues on a cubic lattice. It generates sequences with folding 2 orders of magnitude faster than that of the practically random starting sequence. Thermodynamic analysis shows that the increase in speed is matched by an increase in stability: the evolved sequences are much more stable in their native conformation than the initial random sequence. The unfolding temperature for evolved sequences is slightly higher than the simulation temperature, bearing direct correspondence to the relatively low stability of real proteins.

The implications of thermodynamics of heteropolymers for their folding kinetics are formulated and discussed. The predictions are tested by Monte Carlo simulation of folding of a lattice model 36‐monomer proteins at different temperatures. Using a simulated annealing procedure in sequence space, a number of sequences are designed which have sufficiently low energy in a given target conformation. This conformation plays a role of the native structure for the model proteins. The folding transition is found to be cooperative, and the nature of the free energy barrier is studied. At high temperature the barrier is entropic, and at low temperature it is mainly energetic. This can be explained by transformation of the early partly folded intermediate from the disordered state (belonging to the quasicontinuous part of the energy spectrum) at high temperature to the non‐native low‐energy frozen conformation at low temperature. The latter plays a role of an off‐pathway trap. A parallel folding process has been detected at low temperature where direct folding competes with relaxation of the intermediate. The frozen intermediate must unfold to make it possible to form a folding nucleus, which is the prerequisite of subsequent fast descent to the native state.

Purpose The prevalence of BRCA½ mutations in germline DNA from unselected ovarian cancer patients is 11% to 15.3%. It is important to determine the frequency of somatic BRCA½ changes, given the sensitivity of BRCA-mutated cancers to poly (ADP ribose) polymerase-1 (PARP1) inhibitors and platinum analogs. Patients and Methods In 235 unselected ovarian cancers, BRCA½ was sequenced in 235, assessed by copy number analysis in 95, and tiling arrays in 65. 113 tumors were sequenced for TP53. BRCA½ transcript levels were assessed by quantitative polymerase chain reaction in 220. When available for tumors with BRCA½ mutations, germline DNA was sequenced. Results Forty-four mutations (19%) in BRCA1 (n = 31)/BRCA2 (n = 13) were detected, including one homozygous BRCA1 intragenic deletion. BRCA½ mutations were particularly common (23%) in high-grade serous cancers. In 28 patients with available germline DNA, nine (42.9%) of 21 and two (28.6%) of seven BRCA1 and BRCA2 mutations were demonstrated to be somatic, respectively. Five mutations not previously identified in germline DNA were more commonly somatic than germline (four of 11 v one of 17; P = .062). There was a positive association between BRCA1 and TP53 mutations (P = .012). BRCA½ mutations were associated with improved progression-free survival (PFS) after platinum-based chemotherapy in univariate (P = .032; hazard ratio [HR] = 0.65; 95% CI, 0.43 to 0.98) and multivariate (P = .019) analyses. BRCA½ deficiency, defined as BRCA½ mutations or expression loss (in 24 [13.3%] BRCA½–wild-type cancers), was present in 67 ovarian cancers (30%) and was also significantly associated with PFS in univariate (P = .026; HR = 0.67; 95% CI, 0.47 to 0.96) and multivariate (P = .008) analyses. Conclusion BRCA½ somatic and germline mutations and expression loss are sufficiently common in ovarian cancer to warrant assessment for prediction of benefit in clinical trials of PARP1 inhibitors.

Genomic copy number variations are a typical feature of cancer. These variations may influence cancer outcomes as well as effectiveness of treatment. There are many computational methods developed to detect regions with deletions and amplifications without estimating actual copy numbers (CN) in these regions. We have developed a computational method capable of detecting regions with deletions and amplifications as well as estimating actual copy numbers in these regions. The method is based on determining how signal intensity from different probes is related to CN, taking into account changes in the total genome size, and incorporating into analysis contamination of the solid tumors with benign tissue. Hidden Markov Model is used to obtain the most likely CN solution. The method has been implemented for Affymetrix 500K GeneChip arrays and Agilent 244K oligonucleotide arrays. The results of CN analysis for normal cell lines, cancer cell lines, and tumor samples are presented. The method is capable of detecting copy number alterations in tumor samples with up to 80% contamination with benign tissue. Analysis of 178 cancer cell lines reveals multiple regions of common homozygous deletions and strong amplifications encompassing known tumor suppressor genes and oncogenes as well as novel cancer related genes.

Linkage analysis on Utah pedigrees with strong family histories of major depression including only cases with the SLC6A4 HTTLPR short allele revealed a linkage peak on chromosome 4 (maximum HLOD = 3.5). This evidence suggests epistasis between SLC6A4 and an unknown gene as risk factors for major depression.

http://jcem.endojournals.org/cgi/rapidpdf/jc.2006-2316v1; International audience; CONTEXT: The melanin-concentrating hormone receptor 2 (MCHR2) is a G protein-coupled receptor for melanin-concentrating hormone, a neuropeptide that plays an important role in feeding behaviors. MCHR2 maps on chromosome 6q16.3, in a susceptibility locus for childhood obesity. OBJECTIVE: The aim of this study was to investigate the association between MCHR2 variation and human obesity. DESIGN: Case control and family-based studies were performed. PARTICIPANTS: A total of 141 obese children and 24 nonobese adult subjects was sequenced, and case-control analyses were conducted using 628 severely obese children and 1,401 controls. RESULTS: There were 11 single nucleotide polymorphisms (SNPs) identified. We showed nominal association among -38,245 ATG A/G SNP (P = 0.03; 95% confidence interval 1.02-1.34; odds ratio 1.17), A76A T/C SNP (P = 0.03; 95% confidence interval 0.58-0.97; odds ratio 0.75), and childhood obesity. Analysis of 645 trios with childhood obesity supported further the A76A T/C association, showing an overtransmission to obese children of the at risk T allele (59.0%; P = 0.01), especially in children with most severe forms of obesity (Z score of body mass index > 4) (67.0%; P = 0.003). The A76A at risk T allele was also associated with overeating during meals (P = 0.02) in an additional group of 102 nonobese children. None of the MCHR2 variants, including the A76A SNP, showed association with adult severe obesity, although a trend for association of the T allele of this variant with food disinhibition (P = 0.06) and higher hunger (P = 0.09) was found. This variant was not associated with childhood obesity in an independent case-control study, including 1,573 subjects (P = 0.98). Moreover, the A76A SNP did not explain the linkage on the 6q locus. CONCLUSION: Our results altogether suggest that MCHR2 is not a major contributor to polygenic obesity and support a modest effect of the A76A SNP on food intake abnormalities in childhood.

Recently, separate genome-wide association analyses have identified nonsynonymous SNPs in IL23R and ATG16L1 (rs11209026; c1142GA, R381Q, and rs2241880; c1338AG, T300A, respectively) as strong candidate susceptibility factors for Crohn's disease (CD) in whites. The aim of our study was to test whether these SNPs are associated with CD in a population-based cohort of New Zealand Caucasian inflammatory bowel disease (IBD) patients.Allele frequencies of rs11209026 and rs2241880 were determined in 496 CD patients, 466 ulcerative colitis (UC) patients, and 591 controls. Distribution of the relevant alleles was compared between controls and IBD patients. rs11209026 and rs2241880 genotype distributions were examined both within IBD clinical subphenotypes and CARD15 genotypes.rs11209026 and rs2241880 were both associated with CD (P valuers11209026=0.0026, OR 0.54, 95% CI 0.36-0.81; P valuers2241880=0.0001, OR 1.41, 95% CI 1.18-1.67). In addition, there was evidence for association of rs11209026 with UC (P value=0.037, OR 0.66, 95% CI 0.45-0.98). No significant association was observed between IL23R genotype or ATG16L1 genotype and IBD subphenotypes. IL23R was associated with CD and UC only in the absence of CARD15 mutations, whereas ATG16L1 was associated with CD in the presence and absence of CARD15 mutations.We replicated the previously reported associations between CD and rs11209026 and rs2241880, confirming that IL23R and ATG16L1 are susceptibility loci for CD in the New Zealand population. We also provide further evidence for association of rs11209026 with UC and a report of an additive effect between IL23R and CARD15 genotypes in CD.

Background: Genome-wide analysis was conducted on tumors obtained from patients enrolled in the NOVA study, a Phase 3 clinical trial evaluating the PARP inhibitor niraparib as a maintenance treatment in patients with platinum-sensitive ovarian cancer. Homologous recombination deficiency (HRD) and mutations in DNA damage repair genes were evaluated. Material and methods: DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue and used to create libraries that were hybridized to a custom Agilent SureSelect capture array carrying probes for 54,091 single nucleotide polymorphism sites distributed across the human genome, as well as probes targeting 43 genes involved in DNA repair, including BRCA1 and BRCA2. The captured and enriched DNA was sequenced on an Illumina HiSeq 2500 sequencer. Sequences covering SNP positions were used to generate allelic imbalance profiles. Measures of genomic instability, including determination of an HRD score (integer value of 0-100), were calculated using allelic imbalance profiles and determination of loss of heterozygosity (LOH) by allele-specific copy number (ASCN). A previously identified HRD threshold score of 42 was used to define HRD positivity in the absence of a BRCA mutation. Results: The NOVA study is a Phase 3, multicenter, randomized, double-blind, placebo-controlled study of niraparib as maintenance therapy in ovarian cancer patients who have either gBRCAmut or a tumor with high-grade serous histology and who have responded to their most recent chemotherapy containing a platinum agent. Tumor BRCA mutational status, HRD score and genomic sequencing of 43 DNA repair genes were obtained from tumor samples from both gBRCAmut and non-gBRCAmut cohorts. In the gBRCAmut cohort, HRD analysis of the tumor confirmed the presence of a deleterious or suspected deleterious mutation in all cases. In addition, an HRD score ≥ 42 and the presence of a deleterious mutation in TP53 with loss of heterozygosity (LOH) were observed in nearly all tumors. In the non-gBRCA cohort, somatic BRCA mutations were observed in approximately 13% of tumors, and approximately half of tumors with no evidence of a BRCA mutation had a high HRD score. In both cohorts, the use of three scoring algorithms (LOH, telomeric allelic imbalance [TAI], large-scale state transitions [LST]), was more predictive of BRCA mutational status than LOH alone. Additional genomic sequencing identified deleterious mutations with LOH in DNA repair genes, such as BRIP1, CDK12, RAD51C, PTEN, and RAD51D, with many tumors exhibiting multiple deleterious mutations. Conclusions: High grade serous ovarian cancer is characterized by a high degree of genomic instability. Genomic analysis in the clinical setting is able to identify patients with both germline and somatic BRCA mutations, in addition to BRCAwt tumors with other genetic defects that may be sensitive to agents exploiting deficiencies in HR. Citation Format: Keith Wilcoxen, Christopher Neff, Victor Abkevich, Joshua Timothy Jones, Kathryn Kolquist, Michael Mirza, Jerry Lanchbury, Keith Mikule, Shefali Agarwal, Anne-Renee Hartman, Alexander Gutin, Kirsten Timms. Homologous recombination deficiency (HRD) of high grade serous ovarian tumors from the NOVA Phase III clinical study. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C53.

1018 Background: The HRD-LOH (loss of heterozygosity) score is significantly associated with favorable response to neoadjuvant platinum-based therapy in PrECOG 0105. We set out here to assess the c...

5532 Background: The therapeutic potential of PARP inhibitors is predicted to extend beyond patients with germline BRCA mutations (BRCAmut) to those with homologous recombination deficient (HRD) ca...

1017 Background: Platinum-based chemotherapy has been reported to have efficacy in patients with Triple Negative Breast Cancer (TNBC). Germline BRCA1/2 mutation status has been shown to be predicti...

Major depressive disorder (MDD) is a common, clinically heterogeneous disorder often found comorbid with other disorders. We studied recurrent, early-onset MDD (MDD-RE) and anxiety disorders in combination to define powerful phenotypes for genetic study. We used 87 large, extended Utah pedigrees to investigate linkage to 3 phenotypes: "MDD-RE;" "MDD-RE or anxiety;" and "MDD-RE and anxiety;" where in the latter definition the disorders must appear comorbid within an individual. Pedigrees ranged in size from 2 to 6 generations and contained 3 to 42 individuals affected with MDD or anxiety (718 total). In primary analyses, we identified three regions with at least suggestive genome-wide evidence for linkage on chromosomes 3centr, 7p, and 18q. Both 7p and 18q are replication findings for related phenotypes. The best linkage evidence was for a novel locus at 3p12.3-q12.3 (LOD = 3.88, "MDD-RE or anxiety") and 18q21.33-q22.2 (LOD = 3.75, "MDD-RE and anxiety"), a well-established susceptibility locus for bipolar disorder. In our secondary sex-specific analyses, we identified two further regions of interest on chromosomes 4q and 15q. Using linked pedigrees, we localized 3centr and 18q to 9.8 and 12.2 cM, respectively, with potential for further localization with the addition of markers in specific pedigrees. Our success in replication and novel locus identification illustrates the utility of large extended pedigrees for common disorders, such as MDD. Further, it supports the hypothesis that MDD and anxiety disorders have over-lapping genetic etiologies and suggests that comorbid diagnoses may be useful in defining more genetically homogeneous forms of MDD for linkage mapping.

Classical parametric two-point linkage analysis is a powerful analysis tool, however there are clear disadvantages too, including the sensitivity to allele frequency mis-specification. Conversely, multipoint linkage analysis is not sensitive to allele frequency mis-specification, but it is sensitive to genetic model mis-specification. Goring and Terwilliger [Am J Hum Genet 66:1095-106, 2000] proposed a new robust multipoint statistic that increased the robustness of multipoint analyses. In this paper we have referred to this new statistic as the tlod. We applied this new statistic to the Genetic Analysis Workshop (GAW) 12 data using affected status (AFF) as the phenotype of interest. The heterogeneity tlod and two-point hlod scores correlated highly across the genome (p < 0.0001), as expected, but the het-tlod had a lower number false positives. In addition, the tlod analysis handled missing data better, as would be expected for a multipoint method. When one-third of the genotype data was removed (dead people) the tlod analysis was less affected than the two-point analysis. When tlod scores were compared with multipoint lod scores in true gene locations, the robustness of the tlod to model mis-specification was clearly evident. When the "best" replicate from the general population was analyzed, a borderline genome-wide significant two-point hlod result (3.6) was found 4 cM from MG6 and MG7 on chromosome 6. The heterogeneity tlod score was lower than the two-point hlod score (1.8), but greater than the heterogeneity multipoint lod score (0.4). However, when replicate 1 of the isolated population was analyzed none of the true gene locations were identified with either statistic.

206 Background: Several clinical reports have indicated increased sensitivity to DNA damaging agents and PARP inhibitors in patients with PAC arising on a background of known BRCA1/2 or PALB2 germline mutation. While the mechanism of tumorigenesis in these cancer remains unclear, it appears likely that BRCA1/2germline mutations can predispose to PAC via 2 mechanisms, only one of which requires loss of the second allele. Determination as to whether biallelic inactivation of these genes is present in the tumor may have important therapeutic implications in predicting sensitivity to PARP inhibitors and other strategies targeting DNA repair. Methods: Following approval by the IRB and human bio specimen utilization committee at MSKCC, full exon sequencing of coding regions of 28 genes including BRCA1, 2 and PALB2 using exon capture by hybridization and next generation sequencing was performed on DNA extracted from 135 PAC samples. In addition, samples were analyzed for the presence of BRCA1 promotor methylation, KRAS hotspot mutations, and genome wide loss of heterozygosity (LOH) as an exploratory assessment of number and length of LOH as a marker of homologous repair deficiency / genomic instability. All patients underwent surgery for PAC at MSKCC after year 2000. Patients were selected on the basis of survival and included if they suffered a cancer-specific death within 1 year of resection or survived at least 30 months. Results: 7 tumors had mutations in BRCA2, 1 in BRCA1 and 1 in PALB2. Of 4 BRCA2 mutated samples evaluated for LOH at the BRCA2 locus, 2 demonstrated LOH while 2 did not. There was no LOH at PALB2 in the sample where a mutation was identified. Activating KRAS mutations were identified in BRCA2 mutated tumors with and without LOH. Methylation of the BRCA1promotor regions was not identified. Conclusions: The frequency of BRCA1/2 mutation carriers was as anticipated in this population. Biallelic loss of BRCA2 or PALB2 are not required for pancreatic tumorigenesis. The benefit of PARP inhibitors in BRCA1 / 2 mutated PAC may be limited to tumors with LOH of these genes. Inactivation of BRCA1 through promoter methylation is not identified in sporadic PAC.

Epidemiological studies have shown an association of decreased serum bilirubin levels with coronary artery disease. Two segregation analyses in large pedigrees have suggested a major gene responsible for high bilirubin levels occurring in about 12% of the population. Based on a recessive model from a previous segregation analysis, we performed a genome scan using 587 markers genotyped in 862 individuals from 48 Utah pedigrees to detect loci linked to high bilirubin levels. As a complementary approach, non-parametric linkage (NPL) analysis was performed. These two methods identified four regions showing evidence for linkage. The first region is on chromosome 2q34-37 with multipoint LOD and NPL scores of 3.01 and 3.22, respectively, for marker D2S1363. This region contains a previously described gene, uridine diphosphate glycosyltransferase 1, which has been associated with high bilirubin levels. A polymorphism in the promoter of this gene was recently shown to be responsible for Gilbert syndrome which is associated with mild hyperbilirubinemia. The other regions were found on chromosomes 9q21, 10q25-26, and 18q12 with maximum NPL scores of 2.39, 1.55, and 2.79, respectively. Furthermore, we investigated in these pedigrees the association between bilirubin levels and coronary artery disease. One-hundred and sixty-one male and 41 female subjects had already suffered a coronary artery disease event. Male patients showed significantly lower bilirubin concentrations than age-matched controls. This association, however, was not observed in females. These results provide evidence that loci influencing bilirubin variation exist on chromosomes 2q34-37, 9q21, 10q25-26, and 18q12 and confirms the association of low bilirubin levels with coronary artery disease in males.

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 (BMIor =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 (55.1 cM). 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. 10 cM 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.

We study the impact of disulfide bonds on protein stability and folding. Using lattice model simulations, we show that formation of a disulfide bond stabilizes a protein to an extent that depends on the distance along the chain between linked cysteine residues. However, the impact of disulfide bonds on folding kinetics varies broadly, from acceleration when disulfides are introduced in or close to the folding nucleus, to slowing when disulfides are introduced outside the nucleus. Having established the effect of disulfide bonds on stability, we study the correlation between the number of disulfide bonds and the composition of certain amino acid classes with the goal to use it as a statistical probe into factors that contribute to stability of proteins. We find that the number of disulfides is negatively correlated with aliphatic hydrophobic but not aromatic content. It is surprising that we observe a strong correlation of disulfide content with polar (Q,S,T,N) amino acid content and a strong negative correlation with charged (E,D,K,R) content. These findings provide insights into factors that determine protein stability and principles of protein design as well as possible relations of disulfide bonds and protein function.

The folding ability of a heteropolymer model for proteins subject to Monte Carlo dynamics on a simple cubic lattice is shown to be strongly correlated with the energy gap between the native state and the structurally dissimilar part of the spectrum. We consider a number of estimates of the energy gap that can be determined without simulation, including the gap in energy between the native and first excited fully compact states for sequences with fully compact native states. These estimates are found to be more robust predictors of folding ability than a parameter $\sigma$ that requires simulation for its evaluation: $\sigma = 1 - T_f/T_\theta$, where $T_f$ is the temperature at which the fluctuation of the order parameter is at its maximum and $T_\theta$ is the temperature at which the specific heat is at its maximum. We show that the interpretation of $T_\theta$ as the collapse transition temperature is not correct in general and that the correlation between $\sigma$ and the folding ability arises from the fact that $\sigma$ is essentially a measure of the energy gap., Comment: 12 pages, 3 tables, 3 figures