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Recommendations from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) for interpreting sequence variants specify the use of computational predictors as "supporting" level of evidence for pathogenicity or benignity using criteria PP3 and BP4, respectively. However, score intervals defined by tool developers, and ACMG/AMP recommendations that require the consensus of multiple predictors, lack quantitative support. Previously, we described a probabilistic framework that quantified the strengths of evidence (supporting, moderate, strong, very strong) within ACMG/AMP recommendations. We have extended this framework to computational predictors and introduce a new standard that converts a tool's scores to PP3 and BP4 evidence strengths. Our approach is based on estimating the local positive predictive value and can calibrate any computational tool or other continuous-scale evidence on any variant type. We estimate thresholds (score intervals) corresponding to each strength of evidence for pathogenicity and benignity for thirteen missense variant interpretation tools, using carefully assembled independent data sets. Most tools achieved supporting evidence level for both pathogenic and benign classification using newly established thresholds. Multiple tools reached score thresholds justifying moderate and several reached strong evidence levels. One tool reached very strong evidence level for benign classification on some variants. Based on these findings, we provide recommendations for evidence-based revisions of the PP3 and BP4 ACMG/AMP criteria using individual tools and future assessment of computational methods for clinical interpretation.

PDF file 65K, Primers used in assessment of RINT1 transcripts

PDF file 199K, RINT1 c.1334-5delA, c.1334-1_1335delGTT minigene assay

PDF file 69K, Distribution of cases and controls included in the analysis, by study center and ethnic group

Urothelial carcinoma (UC) presents most frequently as bladder cancer and is the most common cancer of the urinary system in the United States. UC relapse and progression are common and impose a significant negative impact on the lives of patients and healthcare resources. To elucidate the biological mechanisms of UC and find novel biomarkers, we analyzed the clinical and genomic data in the Oncology Research Information Exchange Network (ORIEN). We conducted gene-based and gene-set based association tests on rare germline variants comparing the exome sequencing of 336 UC patients and genome sequencing of 366 healthy controls (42 unrelated individuals from the Centre d'Etudes du Polymorphisme Humain [CEPH] families and 324 from the University of Utah Heritage 1000 [H1K] Projects). The analysis of loss-of-function (LoF) variants revealed that the forkhead box (FOX) J2 gene set was significantly associated with UC at the genome-wide level (Bonferroni-corrected p-value=0.01). Genes in this gene set contain a motif that matches the FOXJ2 transcription factor binding site. Firth-penalized Cox proportional hazard regression on overall survival identified LoF variants in genes down-regulated in naive CD8 T cells to be associated with worse prognosis (Bonferroni-corrected p-value=0.032, hazard ratio=28.2, and 95% confidence interval 6.66 to 119.0). In exome sequencing of tumor tissues, we searched for driver genes and pathways by testing for higher variant allelic fractions than the genome average. The tests yielded eleven genes with genome-wide significance (Table 1). By gene set analysis using the MSigDB Hallmark database, significant pathways included the P53 pathway (p Table 1. Genes with high allelic fractions Gene P-value Number of Variants TP53 Citation Format: Bing-Jian Feng, Wendy Kohlmann, David A. Nix, Aaron Atkinson, Kenneth M. Boucher, Courtney Carroll, Jill Kolesar, Eric A. Singer, Stephen B. Edge, Kamal Sahu, Alejandro Sanchez, Mikaela Larson, Michelle L. Churchman, Laura Graham, John D. Carpten, Yousef Zakharia, Lindsey Byrne, Rohit K. Jain, Kenneth G. Nepple, Ahmad Shabsigh, Jad Chahoud, Sumati Gupta. Germline and somatic genomic profiling of urothelial carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6074.

Purpose The American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) have developed guidelines for classifying germline variants as pathogenic or benign to interpret genetic testing results. Cosegregation analysis is an important component of the guidelines. There are two main approaches for cosegregation analysis: meiosis counting and Bayes factor–based quantitative methods. Of these, the ACMG/AMP guidelines employ only meiosis counting. The accuracy of either approach has not been sufficiently addressed in previous works. Methods We analyzed hypothetical, simulated, and real-life data to evaluate the accuracy of each approach for cancer-associated genes. Results We demonstrate that meiosis counting can provide incorrect classifications when the underlying genetic basis of the disease departs from simple Mendelian situations. Some Bayes factor approaches are currently implemented with inappropriate penetrance. We propose an improved penetrance model and describe several critical considerations, including the accuracy of cosegregation for moderate-risk genes and the impact of pleiotropy, population, and birth year. We highlight a webserver, COOL (Co-segregation Online, http://BJFengLab.org/), that implements an accurate Bayes factor cosegregation analysis. Conclusion An appropriate penetrance model improves the accuracy of Bayes factor cosegregation analysis for high-penetrant variants, and is a better choice than meiosis counting whenever feasible.

Objective.Delays in the diagnosis and treatment of psoriatic arthritis (PsA) are common. These delays contribute to impairments in quality of life and joint damage. This study aims to calculate the incidence rate of PsA over time and identify clinical features that may be used for PsA prediction in patients with psoriasis (PsO).Methods.The study population for PsA incidence analysis included 1128 participants enrolled in the Utah Psoriasis Initiative between 2002 and 2014. Clinical evaluation and medical record review were performed to identify new cases of PsA after enrollment. To identify PsO features associated with PsA, the population was restricted to 627 participants who did not have PsA before PsO phenotyping and had been followed up for subsequent PsA diagnosis. We conducted Cox proportional hazard regressions to estimate the HR of PsA associated with PsO characteristics and other health-related features.Results.PsA incidence rate increased for > 60 years following PsO onset (trend P < 0.0001). There was a significant association between PsA and induration severity in untreated lesions (P < 0.001, HR 1.46), history of fingernail involvement (P < 0.001, HR 2.38), pustular PsO (P < 0.001, HR 3.32), fingernail involvement at enrollment (P < 0.001, HR 2.04), and Koebner phenomenon (P < 0.001, HR 1.90). Multivariate analysis yielded a model that included a history of fingernail involvement (P < 0.001, HR 2.16) and untreated induration (P < 0.001, HR 1.41).Conclusion.Risk of PsA increases steadily for > 60 years following PsO onset. Patient-reported history of PsO characteristics has greater predictive power than physician-measured features at enrollment visits. The characteristics identified in this study provide guidance for screening for PsA risk in patients with PsO.

The American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) have developed guidelines for classifying germline variants as pathogenic or benign to interpret genetic testing results. Cosegregation analysis is an important component of the guidelines. There are two main approaches for cosegregation analysis: meiosis counting and Bayes factor-based quantitative methods. Of these, the ACMG/AMP guidelines employ only meiosis counting. The accuracy of either approach has not been sufficiently addressed in previous works.We analyzed hypothetical, simulated, and real-life data to evaluate the accuracy of each approach for cancer-associated genes.We demonstrate that meiosis counting can provide incorrect classifications when the underlying genetic basis of the disease departs from simple Mendelian situations. Some Bayes factor approaches are currently implemented with inappropriate penetrance. We propose an improved penetrance model and describe several critical considerations, including the accuracy of cosegregation for moderate-risk genes and the impact of pleiotropy, population, and birth year. We highlight a webserver, COOL (Co-segregation Online, http://BJFengLab.org/ ), that implements an accurate Bayes factor cosegregation analysis.An appropriate penetrance model improves the accuracy of Bayes factor cosegregation analysis for high-penetrant variants, and is a better choice than meiosis counting whenever feasible.

PURPOSE Germline mutations in DNA repair (DR) genes and susceptibility genes CDKN2A and HOXB13 have previously been associated with prostate cancer (PC) incidence and/or progression. However, the role and prevalence of this class of mutations in metastatic PC (mPC) are not fully understood. PATIENTS AND METHODS To evaluate the frequency of pathogenic/likely pathogenic germline variants (PVs/LPVs) in men with mPC, this study sequenced 38 DR genes, CDKN2A, and HOXB13 in a predominantly white cohort of 317 patients with mPC. A PC registry at the University of Utah was used for patient sample acquisition and retrospective clinical data collection. Deep target sequencing allowed for germline and copy number variant analyses. Validated PVs/LPVs were integrated with clinical and demographic data for statistical correlation analyses. RESULTS All pathogenic variants were found in men self-reported as white, with a carrier frequency of 8.5% (DR genes, 7.3%; CDKN2A/ HOXB13, 1.2%). Consistent with previous reports, mutations were most frequently identified in the breast cancer susceptibility gene BRCA2. It was also found that 50% of identified PVs/LPVs were categorized as founder mutations with European origins. Correlation analyses did not support a trend toward more advanced or earlier-onset disease in comparisons between carriers and noncarriers of deleterious DR or HOXB13 G84E mutations. CONCLUSION These findings demonstrate a lower prevalence of germline PVs/LPVs in an unselected, predominantly white mPC cohort than previously reported, which may have implications for the design of clinical trials testing targeted therapies. Larger studies in broad and diverse populations are needed to more accurately define the prevalence of germline mutations in men with mPC.

BACKGROUND Few genes have germline mutations which predispose men to more aggressive prostate cancer (PCa). This study evaluated the contribution of germline loss of function (LOF) variants in PPFIBP2 to risk of lethal PCa. METHODS A case-case study of 1414 PCa patients with lethal PCa and low-risk localized PCa was performed. Germline DNA samples from these patients were sequenced for PPFIBP2. Mutation carrier rates and association with lethal PCa were analyzed using the Fisher exact test, logistic regression, and Kaplan-Meier survival analysis. RESULTS In the entire study population, eight patients, all of European ancestry, were identified as carrying PPFIBP2 pathogenic or likely pathogenic mutations. Seven (1.52%) of 462 lethal PCa patients were carriers compared with only one (0.12%) carrier in 810 low-risk PCa patients, P = 0.0029. The estimated Odds Ratio (OR) of carrying PPFIBP2 mutation for lethal PCa was 13.8 in European American population. The PPFIBP2 loss-of-function mutation carrier rate in lethal PCa cases was also higher than in 33 370 non-Finnish European individuals from the Exome Aggregation Consortium (ExAC) (carrier rate of 0.17%, P = 1.92 × 10-5 ) and in 498 men with localized PCa from The Cancer Genome Atlas cohort (TCGA) cohort (carrier rate of 0%, P = 0.0058). Survival analysis in European American lethal cases revealed PPFIBP2 mutation status as an independent predictor of shorter survival after adjusting for age at diagnosis, PSA at diagnosis, and genetic background (hazard ratio = 2.62, P = 0.034). CONCLUSIONS While larger studies are needed, germline mutations in a novel gene, PPFIBP2, differentiated risk for lethal PCa from low-risk cases and were associated with shorter survival times after diagnosis.

To interpret genetic variants discovered from next-generation sequencing, integration of heterogeneous information is vital for success. This article describes a framework named PERCH (Polymorphism Evaluation, Ranking, and Classification for a Heritable trait), available at http://BJFengLab.org/. It can prioritize disease genes by quantitatively unifying a new deleteriousness measure called BayesDel, an improved assessment of the biological relevance of genes to the disease, a modified linkage analysis, a novel rare-variant association test, and a converted variant call quality score. It supports data that contain various combinations of extended pedigrees, trios, and case-controls, and allows for a reduced penetrance, an elevated phenocopy rate, liability classes, and covariates. BayesDel is more accurate than PolyPhen2, SIFT, FATHMM, LRT, Mutation Taster, Mutation Assessor, PhyloP, GERP++, SiPhy, CADD, MetaLR, and MetaSVM. The overall approach is faster and more powerful than the existing quantitative method pVAAST, as shown by the simulations of challenging situations in finding the missing heritability of a complex disease. This framework can also classify variants of unknown significance (variants of uncertain significance) by quantitatively integrating allele frequencies, deleteriousness, association, and co-segregation. PERCH is a versatile tool for gene prioritization in gene discovery research and variant classification in clinical genetic testing.

Colorectal cancer (CRC) has a large hereditary component, which is only partially explained by known genetic causes. Recently, variants in ribosomal protein S20 (RPS20, [OMIM: 603682]) were identified in a family with familial CRC type X and in a CRC cancer case-control screen. This study describes a novel splice donor variant in RPS20, NM_001023.3:c.177+1G>A. It segregates with CRC [OMIM: 114500] and polyposis [HP: 0200063] within the proband’s family. Reverse transcription-polymerase chain reaction (RT-PCR) confirms the variant results in two aberrantly-spliced transcripts that are absent in controls. The location of the novel RPS20 variant is near two previously-reported truncating RPS20 variants associated with CRC. DNA from colon adenocarcinoma showed no evidence of loss-of-heterozygosity, supporting a haploinsufficiency or dominant negative disease mechanism. These findings support designation of RPS20 as a CRC predisposition gene, and expand the phenotypic spectrum of RPS20 truncating variants to include polyposis.

The American College of Medical Genetics and Genomics (ACMG) guidelines for sequence variant classification include two criteria, PP3 and BP4, for combining computational data with other evidence types contributing to sequence variant classification. PP3 and BP4 assert that computational modeling can provide “Supporting” evidence for or against pathogenicity within the ACMG framework. Here, leveraging a meta-analysis of ATM and CHEK2 breast cancer case-control mutation screening data, we evaluate the strength of evidence determined from the relatively simple computational tool Align-GVGD. Importantly, application of Align-GVGD to these ATM and CHEK2 data is free of logical circularities, hidden multiple testing, and use of other ACMG evidence types. For both genes, rare missense substitutions that are assigned the most severe Align-GVGD grade exceed a “Moderate pathogenic” evidence threshold when analyzed in a Bayesian framework; accordingly, we argue that the ACMG classification rules be updated for well-calibrated computational tools. Additionally, congruent with previous analyses of ATM and CHEK2 case-control mutation screening data, we find that both genes have a considerable burden of pathogenic missense substitutions, and that severe ATM rare missense have increased odds ratios compared to truncating and splice junction variants, indicative of a potential dominant-negative effect for those missense substitutions.

This study examined the utility of sets of single-nucleotide polymorphisms (SNPs) in familial but non-BRCA-associated breast cancer (BC). We derived a polygenic risk score (PRS) based on 24 known BC risk SNPs for 4,365 women from the Breast Cancer Family Registry and Kathleen Cuningham Consortium Foundation for Research into Familial Breast Cancer familial BC cohorts. We compared scores for women based on cancer status at baseline; 2,599 women unaffected at enrollment were followed-up for an average of 7.4 years. Cox proportional hazards regression was used to analyze the association of PRS with BC risk. The BOADICEA risk prediction algorithm was used to measure risk based on family history alone. The mean PRS at baseline was 2.25 (SD, 0.35) for affected women and was 2.17 (SD, 0.35) for unaffected women from combined cohorts (P < 10–6). During follow-up, 205 BC cases occurred. The hazard ratios for continuous PRS (per SD) and upper versus lower quintiles were 1.38 (95% confidence interval: 1.22–1.56) and 3.18 (95% confidence interval: 1.84–5.23) respectively. Based on their PRS-based predicted risk, management for up to 23% of women could be altered. Including BC-associated SNPs in risk assessment can provide more accurate risk prediction than family history alone and can influence recommendations for cancer screening and prevention modalities for high-risk women. Genet Med 19 1, 30–35.

Background Moderate-risk genes have not been extensively studied, and missense substitutions in them are generally returned to patients as variants of uncertain significance lacking clearly defined risk estimates. The fraction of early-onset breast cancer cases carrying moderate-risk genotypes and quantitative methods for flagging variants for further analysis have not been established. Methods We evaluated rare missense substitutions identified from a mutation screen of ATM , CHEK2 , MRE11A , RAD50 , NBN , RAD51 , RINT1 , XRCC2 and BARD1 in 1297 cases of early-onset breast cancer and 1121 controls via scores from Align-Grantham Variation Grantham Deviation (GVGD), combined annotation dependent depletion (CADD), multivariate analysis of protein polymorphism (MAPP) and PolyPhen-2. We also evaluated subjects by polygenotype from 18 breast cancer risk SNPs. From these analyses, we estimated the fraction of cases and controls that reach a breast cancer OR≥2.5 threshold. Results Analysis of mutation screening data from the nine genes revealed that 7.5% of cases and 2.4% of controls were carriers of at least one rare variant with an average OR≥2.5. 2.1% of cases and 1.2% of controls had a polygenotype with an average OR≥2.5. Conclusions Among early-onset breast cancer cases, 9.6% had a genotype associated with an increased risk sufficient to affect clinical management recommendations. Over two-thirds of variants conferring this level of risk were rare missense substitutions in moderate-risk genes. Placement in the estimated OR≥2.5 group by at least two of these missense analysis programs should be used to prioritise variants for further study. Panel testing often creates more heat than light; quantitative approaches to variant prioritisation and classification may facilitate more efficient clinical classification of variants.

Background\ud Germline genetic testing with hereditary cancer gene panels can identify women at increased risk of breast cancer. However, those at increased risk of triple-negative (estrogen receptor–negative, progesterone receptor–negative, human epidermal growth factor receptor–negative) breast cancer (TNBC) cannot be identified because predisposition genes for TNBC, other than BRCA1, have not been established. The aim of this study was to define the cancer panel genes associated with increased risk of TNBC.\ud \ud Methods\ud Multigene panel testing for 21 genes in 8753 TNBC patients was performed by a clinical testing laboratory, and testing for 17 genes in 2148 patients was conducted by a Triple Negative Breast Cancer Consortium (TNBCC) of research studies. Associations between deleterious mutations in cancer predisposition genes and TNBC were evaluated using results from TNBC patients and reference controls.\ud \ud Results\ud Germline pathogenic variants in BARD1, BRCA1, BRCA2, PALB2, and RAD51D were associated with high risk (odds ratio > 5.0) of TNBC and greater than 20% lifetime risk for overall breast cancer among Caucasians. Pathogenic variants in BRIP1, RAD51C, and TP53 were associated with moderate risk (odds ratio > 2) of TNBC. Similar trends were observed for the African American population. Pathogenic variants in these TNBC genes were detected in 12.0% (3.7% non-BRCA1/2) of all participants.\ud \ud Conclusions\ud Multigene hereditary cancer panel testing can identify women with elevated risk of TNBC due to mutations in BARD1, BRCA1, BRCA2, PALB2, and RAD51D. These women can potentially benefit from improved screening, risk management, and cancer prevention strategies. Patients with mutations may also benefit from specific targeted therapeutic strategies.

Gene panel testing for breast cancer susceptibility has become relatively cheap and accessible. However, the breast cancer risks associated with mutations in many genes included in these panels are unknown.We performed custom-designed targeted sequencing covering the coding exons of 17 known and putative breast cancer susceptibility genes in 660 non-BRCA1/2 women with familial breast cancer. Putative deleterious mutations were genotyped in relevant family members to assess co-segregation of each variant with disease. We used maximum likelihood models to estimate the breast cancer risks associated with mutations in each of the genes.We found 31 putative deleterious mutations in 7 known breast cancer susceptibility genes (TP53, PALB2, ATM, CHEK2, CDH1, PTEN and STK11) in 45 cases, and 22 potential deleterious mutations in 31 cases in 8 other genes (BARD1, BRIP1, MRE11, NBN, RAD50, RAD51C, RAD51D and CDK4). The relevant variants were then genotyped in 558 family members. Assuming a constant relative risk of breast cancer across age groups, only variants in CDH1, CHEK2, PALB2 and TP53 showed evidence of a significantly increased risk of breast cancer, with some supportive evidence that mutations in ATM confer moderate risk.Panel testing for these breast cancer families provided additional relevant clinical information for2% of families. We demonstrated that segregation analysis has some potential to help estimate the breast cancer risks associated with mutations in breast cancer susceptibility genes, but very large case-control sequencing studies and/or larger family-based studies will be needed to define the risks more accurately.

Missense variants of uncertain significance (VUS) in the BRCA1 and BRCA2 cancer predisposition genes confound clinical interpretation. To improve methods for classification of VUS, functional data for 248 BRCA1 and 207 BRCA2 variants were comprehensively compared to 40 in-silico prediction algorithms, using default and optimized thresholds. Integrated frameworks predicting the pathogenicity of BRCA1 and BRCA2 missense variants were also developed using a random forest model (RF) and a naive voting method (NVM). Both models yielded high area under the receiver operating characteristic curve (AUC) (>0.90) for pathogenic variants in these genes. When applied to other genes, the predictive ability of the models were superior to other algorithms. The quantitative prediction of functional impact of all possible variants in clinically relevant functional domains of BRCA1 and BRCA2 using these models provides the first high-throughput validated method for classification of VUS identified by clinical genetic testing.

A 51-year-old French Canadian man presented to his family physician owing to an extensive family history of prostate cancer in five brothers, his father and two paternal uncles. His serum PSA level was 4.9 ng/ml and a six-core biopsy revealed the presence of a prostate adenocarcinoma with a Gleason score of 7 (3+4). He was treated with radical prostatectomy. Repeat PSA tests revealed a gradual rise in PSA levels despite androgen deprivation therapy with bicalutamide and goserelin over the course of 3 years. Genetic evaluation was undertaken in view of his personal and family history. The proband died at the age of 58 years of widespread metastasis.PSA testing, six-core biopsy, genetic counselling and mutation analysis for French Canadian founder mutations in the BRCA1 and BRCA2 genes, histopathological review of tumour tissue from family members, examination of loss of heterozygosity at the BRCA2 gene locus, immunohistochemistry to determine the expression of the ERG nuclear oncoprotein in prostate tumours, genotyping with eight selected risk-associated single nucleotide polymorphisms, Doppler ultrasonography of the leg, CT of the abdomen and pelvis with intravenous and oral contrast, chest CT with intravenous contrast for the assessment of metastatic prostate cancer, genetic testing for the G84E variant in the HOXB13 gene.Early-onset and aggressive prostate cancer associated with a nonsense French Canadian BRCA2 founder mutation, c.5857GT (p.Glu1953(*)).Radical prostatectomy, hormone therapy with bicalutamide and goserelin, palliative chemotherapy initially with docetaxel plus prednisone then with mitoxantrone plus prednisone, as well as genetic counselling and testing for the proband and his family members.

To interpret genetic variants discovered from next-generation sequencing (NGS), integration of heterogeneous information is vital for success. This paper describes a framework named PERCH (Polymorphism Evaluation, Ranking, and Classification for a Heritable trait), available at http://BJFengLab.org/. It can prioritize disease genes by quantitatively unifying a new deleteriousness measure called BayesDel, an improved assessment of the biological relevance of genes to the disease, a modified linkage analysis, a novel rare-variant association test, and a converted variant call quality score. It supports data that contain a various combination of extended pedigrees, trios, and case-controls, and allows for a reduced penetrance, an elevated phenocopy rate, liability classes and covariates. BayesDel is more accurate than PolyPhen2, SIFT, FATHMM, LRT, Mutation Taster, Mutation Assessor, PhyloP, GERP++, SiPhy, CADD, MetaLR, and MetaSVM. The overall approach is faster and more powerful than the existing quantitative method pVAAST, as shown by the simulations of challenging situations in finding the missing heritability of a complex disease. This framework can also classify variants of unknown significance (VUS, or variants of uncertain significance) by quantitatively integrating allele frequencies, deleteriousness, association, and co-segregation. PERCH is a versatile tool for gene prioritization in gene discovery research and variant classification in clinical genetic testing.

// Lucie Lanikova 1, 2, * , N. Scott Reading 1, 3, 4, * , Hao Hu 5 , Tsewang Tashi 1 , Tatiana Burjanivova 6 , Anna Shestakova 1, 4 , Bhola Siwakoti 7 , Binay Kumar Thakur 7 , Chin Bahadur Pun 7 , Amir Sapkota 8 , Sarah Abdelaziz 9 , Bing-Jian Feng 10 , Chad D. Huff 5 , Mia Hashibe 9 , Josef T. Prchal 1, 11 1 Division of Hematology, University of Utah School of Medicine, Salt Lake City, Utah, USA 2 Institute of Molecular Genetics, Academy of Sciences, Prague, Czech Republic 3 Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, Utah, USA 4 Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah, USA 5 University of Texas, MD Anderson Cancer Center, Houston, Texas, USA 6 Department of Molecular Biology, Comenius University in Bratislava, Jessenius Faculty of Medicine in Martin, Slovak Republic 7 B.P. Koirala Memorial Cancer Hospital, Bharatpur, Chitwan, Nepal 8 Maryland Institute for Applied Environmental Health, and University of Maryland College Park School of Public Health, Maryland, USA 9 Division of Public Health, Department of Family & Preventive Medicine and Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, Utah, USA 10 Department of Dermatology, University of Utah School of Medicine, Salt Lake City, Utah, USA 11 Department of Pathophysiology and 1st Department of Medicine, 1st Faculty of Medicine, Charles University in Prague, Czech Republic * These authors have contributed equally to this work Correspondence to: Josef T. Prchal, email: josef.prchal@hsc.utah.edu Mia Hashibe, email: mia.hashibe@utah.edu Keywords: hypoxia, ENGL1/PHD2, EPAS1/HIF-2α, high-altitude adaptation, lung cancer Received: June 15, 2016 Accepted: December 16, 2016 Published: December 28, 2016 ABSTRACT Tibetans existed in high altitude for ~25 thousand years and have evolutionary selected unique haplotypes assumed to be beneficial to hypoxic adaptation. EGLN1 /PHD2 and EPAS1 /HIF-2α, both crucial components of hypoxia sensing, are the two best-established loci contributing to high altitude adaptation. The co-adapted Tibetan-specific haplotype encoding for PHD2:p.[D4E/C127S] promotes increased HIF degradation under hypoxic conditions. The Tibetan-specific 200 kb EPAS1 haplotype introgressed from an archaic human population related to Denisovans which underwent evolutionary decay; however, the functional variant(s) responsible for high-altitude adaptation at EPAS1 /HIF-2α have not yet been identified. Since HIF modulates the behavior of cancer cells, we hypothesized that these Tibetan selected genomic variants may modify cancer risk predisposition. Here, we ascertained the frequencies of EGLN1 D4E/C127S and EGLN1 C127S variants and ten EPAS1 /HIF-2α variants in lung cancer patients and controls in Nepal, whose population consists of people with Indo-Aryan origin and Tibetan-related Mongoloid origin. We observed a significant association between the selected Tibetan EGLN 1/PHD2 haplotype and lung cancer (p=0.0012 for D4E, p=0.0002 for C127S), corresponding to a two-fold increase in lung cancer risk. We also observed a two-fold or greater increased risk for two of the ten EPAS1 /HIF-2α variants, although the association was not significant after correcting for multiple comparisons (p=0.12). Although these data cannot address the role of these genetic variants on lung cancer initiation or progression, we conclude that some selected Tibetan variants are strongly associated with a modified risk of lung cancer.

Psoriasis has long been considered a genetic disorder. The first observation that psoriasis is an inherited, familial skin disorder was made in 1957. Extensive epidemiologic evidence and monozygotic twin concordance studies confirmed familial clustering of this chronic inflammatory disease and drove scientific investigation into the genetic basis of psoriasis. Since 1957, the list of psoriasis-associated genetic polymorphisms, including some rare causal mutations, has grown tremendously due to advanced high-throughput genotyping platforms and statistical methods. Nevertheless, the majority of psoriasis patients lack known psoriasis-associated susceptibility loci and the exact molecular mechanisms by which polymorphisms contribute to psoriasis remains poorly understood. Recent work points to a complex interplay between genetics, epigenetics, and the inflammatory signaling networks of skin and immune cell mediators. In contrast, other mutations appear to be causative, such as the recently identified mutations in IL36RN and CARD14, and shed light on new immunologic pathways driving pustular psoriasis. Evidence is also rapidly accumulating for the role of epigenetic changes in psoriasis heritability. The decreasing cost and rapid advancements in genetic technologies in combination with the formation of multi-institutional patient registries will likely result in a better understanding of the “missing heritability” in psoriasis and other complex, multigenic diseases.

The 185delAG* BRCA1 mutation is encountered primarily in Jewish Ashkenazi and Iraqi individuals, and sporadically in non-Jews. Previous studies estimated that this is a founder mutation in Jewish mutation carriers that arose before the dispersion of Jews in the Diaspora ~2500 years ago. The aim of this study was to assess the haplotype in ethnically diverse 185delAG* BRCA1 mutation carriers, and to estimate the age at which the mutation arose. Ethnically diverse Jewish and non-Jewish 185delAG*BRCA1 mutation carriers and their relatives were genotyped using 15 microsatellite markers and three SNPs spanning 12.5 MB, encompassing the BRCA1 gene locus. Estimation of mutation age was based on a subset of 11 markers spanning a region of ~5 MB, using a previously developed algorithm applying the maximum likelihood method. Overall, 188 participants (154 carriers and 34 noncarriers) from 115 families were included: Ashkenazi, Iraq, Kuchin-Indians, Syria, Turkey, Iran, Tunisia, Bulgaria, non-Jewish English, non-Jewish Malaysian, and Hispanics. Haplotype analysis indicated that the 185delAG mutation arose 750-1500 years ago. In Ashkenazim, it is a founder mutation that arose 61 generations ago, and with a small group of founder mutations was introduced into the Hispanic population (conversos) ~650 years ago, and into the Iraqi-Jewish community ~450 years ago. The 185delAG mutation in the non-Jewish populations in Malaysia and the UK arose at least twice independently. We conclude that the 185delAG* BRCA1 mutation resides on a common haplotype among Ashkenazi Jews, and arose about 61 generations ago and arose independently at least twice in non-Jews.

To identify genetic susceptibility loci for nasopharyngeal carcinoma (NPC), a genome-wide association study was performed using 464,328 autosomal SNPs in 1,583 NPC affected individuals (cases) and 1,894 controls of southern Chinese descent. The top 49 SNPs from the genome-wide association study were genotyped in 3,507 cases and 3,063 controls of southern Chinese descent from Guangdong and Guangxi. The seven supportive SNPs were further confirmed by transmission disequilibrium test analysis in 279 trios from Guangdong. We identified three new susceptibility loci, TNFRSF19 on 13q12 (rs9510787, Pcombined=1.53x10(-9), odds ratio (OR)=1.20), MDS1-EVI1 on 3q26 (rs6774494, Pcombined=1.34x10(-8), OR=0.84) and the CDKN2A-CDKN2B gene cluster on 9p21 (rs1412829, Pcombined=4.84x10(-7), OR=0.78). Furthermore, we confirmed the role of HLA by revealing independent associations at rs2860580 (Pcombined=4.88x10(-67), OR=0.58), rs2894207 (Pcombined=3.42x10(-33), OR=0.61) and rs28421666 (Pcombined=2.49x10(-18), OR=0.67). Our findings provide new insights into the pathogenesis of NPC by highlighting the involvement of pathways related to TNFRSF19 and MDS1-EVI1 in addition to HLA molecules.

Psoriasis is a common immune-mediated disorder that affects the skin, nails and joints. To identify psoriasis susceptibility loci, we genotyped 438,670 SNPs in 1,409 psoriasis cases and 1,436 controls of European ancestry. We followed up 21 promising SNPs in 5,048 psoriasis cases and 5,041 controls. Our results provide strong support for the association of at least seven genetic loci and psoriasis (each with combined P < 5 x 10(-8)). Loci with confirmed association include HLA-C, three genes involved in IL-23 signaling (IL23A, IL23R, IL12B), two genes that act downstream of TNF-alpha and regulate NF-kappaB signaling (TNIP1, TNFAIP3) and two genes involved in the modulation of Th2 immune responses (IL4, IL13). Although the proteins encoded in these loci are known to interact biologically, we found no evidence for epistasis between associated SNPs. Our results expand the catalog of genetic loci implicated in psoriasis susceptibility and suggest priority targets for study in other auto-immune disorders.

Contains fulltext : 52087.pdf (Publisher’s version ) (Closed access) Alzheimer disease (AD) is the most common cause of dementia. We conducted a genome screen of 103 patients with late-onset AD who were ascertained as part of the Genetic Research in Isolated Populations (GRIP) program that is conducted in a recently isolated population from the southwestern area of The Netherlands. All patients and their 170 closely related relatives were genotyped using 402 microsatellite markers. Extensive genealogy information was collected, which resulted in an extremely large and complex pedigree of 4,645 members. The pedigree was split into 35 subpedigrees, to reduce the computational burden of linkage analysis. Simulations aiming to evaluate the effect of pedigree splitting on false-positive probabilities showed that a LOD score of 3.64 corresponds to 5% genomewide type I error. Multipoint analysis revealed four significant and one suggestive linkage peaks. The strongest evidence of linkage was found for chromosome 1q21 (heterogeneity LOD [HLOD]=5.20 at marker D1S498). Approximately 30 cM upstream of this locus, we found another peak at 1q25 (HLOD=4.0 at marker D1S218). These two loci are in a previously established linkage region. We also confirmed the AD locus at 10q22-24 (HLOD=4.15 at marker D10S185). There was significant evidence of linkage of AD to chromosome 3q22-24 (HLOD=4.44 at marker D3S1569). For chromosome 11q24-25, there was suggestive evidence of linkage (HLOD=3.29 at marker D11S1320). We next tested for association between cognitive function and 4,173 single-nucleotide polymorphisms in the linked regions in an independent sample consisting of 197 individuals from the GRIP region. After adjusting for multiple testing, we were able to detect significant associations for cognitive function in four of five AD-linked regions, including the new region on chromosome 3q22-24 and regions 1q25, 10q22-24, and 11q25. With use of cognitive function as an endophenotype of AD, our study indicates the that the RGSL2, RALGPS2, and C1orf49 genes are the potential disease-causing genes at 1q25. Our analysis of chromosome 10q22-24 points to the HTR7, MPHOSPH1, and CYP2C cluster. This is the first genomewide screen that showed significant linkage to chromosome 3q23 markers. For this region, our analysis identified the NMNAT3 and CLSTN2 genes. Our findings confirm linkage to chromosome 11q25. We were unable to confirm SORL1; instead, our analysis points to the OPCML and HNT genes.

An elevated familial relative risk may indicate either an important genetic component in etiology or shared environmental exposures within the family. Incidence rates of kidney cancer are particularly high in Central Europe, although no data were available on the familial aggregation or genetic background of kidney cancer in this region. We have, therefore, investigated the role of family history in first-degree relatives in a large multicenter case-control study in Central Europe. A total number of 1,097 cases of kidney cancer and 1,555 controls were recruited from 2000 to 2003 from seven centers in Czech Republic, Poland, Romania, and Russia. The risk of kidney cancer increased with the increasing number of relatives with history of any cancer [odds ratio (OR), 1.15; 95% confidence interval (95% CI), 1.00-1.31 per affected relative], and this association seemed to be more prominent among subjects with young onset (OR, 1.55; 95% CI, 1.09-2.20 per affected relative). Overall, the OR was 1.40 (95% CI, 0.71- 2.76) for the subjects who had at least one first-degree relative with kidney cancer after adjusting for tobacco smoking, body mass index, and medical history of hypertension, and this association was most apparent among subjects with affected siblings (OR, 4.09; 95% CI, 1.09-15.4). Based on the relative risk to siblings in our study population, we estimated that 80% of the kidney cancer cases are likely to occur in 20% of the population with the highest genetic risk, which indicate the importance of further investigation of genetic factors in cancer prevention for kidney cancer. (Cancer Epidemiol Biomarkers Prev 2007;16(6):1287–90)

Contains fulltext : 53679.pdf (Publisher’s version ) (Closed access) Ubiquilin 1 (UBQLN1) is involved in the ubiquitination machinery, which has been implicated in Alzheimer's disease (AD) as well as Parkinson's disease (PD). A polymorphism in the gene encoding for UBQLN1 has been previously associated with a higher risk of AD. We studied the role of the SNP rs12344615 on the UBQLN 1 gene in AD, PD and cognitive function in a population-based study, the Rotterdam Study, and a family-based study embedded in the genetic research in isolated population (GRIP) program. The Rotterdam Study includes 549 patients with AD and 157 patients with PD. The GRIP program includes a series of 123 patients with AD and a study of 1049 persons who are characterized for cognitive function. Data were analysed using logistic and multiple regression analysis. We found no significant difference in risk of AD or PD by the UBQLN1 SNP rs12344615 in our overall and stratified analyses in the Rotterdam Study. In our family-based study, we did not find evidence for linkage of AD to the region including the UBQLN1 gene. In the family-based study we also failed to detect an effect of this polymorphism on cognitive function. Our results suggest that it is unlikely that the SNP rs12344615 of the UBQLN1 gene is related to the onset of AD, PD or cognitive function.

North Africa is one of the major Nasopharyngeal Carcinoma (NPC) endemic regions. Specific food items unique to this area were implicated to be associated with NPC risk, but results were inconsistent. Here we have performed a large-scale case-control study in the Maghrebian population from Tunisia, Algeria and Morocco. From 2002 to 2005, interviews were conducted on 636 cases and 615 controls. Controls were hospitalized individuals from 15 non-cancer hospital departments, or friends and family members of non-NPC cancer subjects, matched by center, childhood household type (rural or urban), age and sex. Conditional logistic regression is used to evaluate the risk of factors. In results, consumption of rancid butter, rancid sheep fat and preserved meat not spicy (mainly quaddid) were associated with significantly increased risk of NPC, while consumption of cooked vegetables and industrial preserved fish was associated with reduced risk. Other foods such as fresh citrus fruits and spicy preserved meat (mainly osban) in childhood, industrial made olive condiments in adulthood, were marginally associated. In multivariate analyses, only rancid butter, rancid sheep fat and cooked vegetables were significantly associated with NPC. In regard to possible causative substances, our results implicate the involvement of butyric acid, a potential Epstein-Barr virus (EBV) activator.

Background Inherited mutations in DNA mismatch repair genes predispose to different cancer syndromes depending on whether they are mono-allelic or bi-allelic. This supports a causal relationship between expression level in the germline and phenotype variation. As a model to study this relationship, our study aimed to define the pathogenic characteristics of a recurrent homozygous coding variant in PMS2 displaying an attenuated phenotype identified by clinical genetic testing in seven Inuit families from Northern Quebec. Methods Pathogenic characteristics of the PMS2 mutation NM_000535.5:c.2002A>G were studied using genotype–phenotype correlation, single-molecule expression detection and single genome microsatellite instability analysis. Results This PMS2 mutation generates a de novo splice site that competes with the authentic site. In homozygotes, expression of the full-length protein is reduced to a level barely detectable by conventional diagnostics. Median age at primary cancer diagnosis is 22 years among 13 NM_000535.5:c.2002A>G homozygotes, versus 8 years in individuals carrying bi-allelic truncating mutations. Residual expression of full-length PMS2 transcript was detected in normal tissues from homozygotes with cancers in their 20s. Conclusions Our genotype–phenotype study of c.2002A>G illustrates that an extremely low level of PMS2 expression likely delays cancer onset, a feature that could be exploited in cancer preventive intervention.

Marijuana use is legal in two states and additional states are considering legalization. Approximately 18 million Americans are current marijuana users. There is currently no consensus on whether marijuana use is associated with cancer risk. Our objective is to review the epidemiologic studies on this possible association. We identified 34 epidemiologic studies on upper aerodigestive tract cancers (n = 11), lung cancer (n = 6), testicular cancer (n = 3), childhood cancers (n = 6), all cancers (n = 1), anal cancer (n = 1), penile cancer (n = 1), non-Hodgkin lymphoma (n = 2), malignant primary gliomas (n = 1), bladder cancer (n = 1), and Kaposi sarcoma (n = 1). Studies on head and neck cancer reported increased and decreased risks, possibly because there is no association, or because risks differ by human papillomavirus status or geographic differences. The lung cancer studies largely appear not to support an association with marijuana use, possibly because of the smaller amounts of marijuana regularly smoked compared with tobacco. Three testicular cancer case–control studies reported increased risks with marijuana use [summary ORs, 1.56; 95% confidence interval (CI), 1.09–2.23 for higher frequency and 1.50 (95% CI, 1.08–2.09) for ≥10 years]. For other cancer sites, there is still insufficient data to make any conclusions. Considering that marijuana use may change due to legalization, well-designed studies on marijuana use and cancer are warranted. Cancer Epidemiol Biomarkers Prev; 24(1); 15–31. ©2015 AACR.

Gastric carcinoma is one of the most common malignancies in Asia. Although the allelic deletion of 7q has been reportedly associated with primary gastric carcinoma tumorigenesis, no predisposing genes in this region have been identified so far. Here, we report the results of genotype and loss of heterozygosity (LOH) analysis on 7q in this tumor. A panel of nine microsatellite markers distributed over the whole chromosome 7q was used for genotyping primary gastric carcinomas. Of 72 primary tumors LOH of D7S486 occurred in 24.0% (12/50) of cases. Fine mapping with 12 additional markers flanking D7S486 resulted in LOH of 30.36% (17/56) and defined one minimal deleted region in primary gastric carcinomas, a 90-kilobase region bounded by D7S2543 and D7S486 at 7q31.2. The allelic deletion correlates statistically with clinicopathologic variables. Our data suggest a possible link between putative tumor suppressor genes and gastric carcinoma in the 7q31 region.

Nasopharyngeal carcinoma is a common malignancy in Southeast Asian countries, and genetic background is a well-known component of the complexity underlying its tumorigenic process. We have mapped a nasopharyngeal carcinoma susceptibility locus to chromosome 4p15.1-q12 in a previous linkage study on nasopharyngeal carcinoma pedigrees. In this study provided in this communication, we screened all the genes in this region, with a focus on exons, promoters, and the exon-intron boundary to identify nasopharyngeal carcinoma–associated mutations or functional variants. Importantly, we found a novel gene (LOC344967) with a single nucleotide polymorphism −32G/A in the promoter region. This gene is a member of the acyl CoA thioesterase family that plays an important role in fatty acid metabolism and is involved in the progression of various types of tumors. The −32A variant was found cosegregated with the disease phenotype in the nasopharyngeal carcinoma pedigrees that we previously used for the linkage study. Moreover, this −32A variant creates an activator protein (AP-1)–binding site in the transcriptional regulatory region of LOC344967, which significantly enhanced the binding of AP-1 to the promoter region and the transcription activity of the promoter in vivo. Furthermore, the expression of LOC344967 was significantly up-regulated at both mRNA and protein levels in nasopharyngeal carcinoma cells sharing the −32G/A genotype compared with nasopharyngeal carcinoma cells with the −32G/G genotype. Collectively, these results provide evidence that the −32A variant is a functional sequence change and may be related to nasopharyngeal carcinoma susceptibility in the families studied. (Cancer Res 2006; 66(2): 693-700)

The striking geographical and ethnic distribution of nasopharyngeal carcinoma (NPC) suggests the involvement of genetic and environmental factors in NPC development. The purpose of this study is to investigate the fit of single gene, polygenic and multifactorial models to the observed pattern of transmission of NPC in a hospital-based family history study conducted by the Cancer Center of Sun Yat-Sen University (CCSYU) in Guangzhou, China. Complex segregation analysis of a total of 1903 Cantonese pedigrees ascertained at CCSYU was conducted using a unified mixed model after the pedigrees were partitioned into 3737 nuclear families. The mixed model assumes that a phenotype is influenced by the additive and independent effect of a major gene, together with multifactorial components (genetic and environmental) and a random environmental effect. The current results do not provide evidence for a major gene and the observed data are best explained by a multifactorial mode of inheritance for NPC.

BACKGROUND Previous studies have suggested that genetic susceptibility may play an important role in the etiology of nasopharyngeal cancer (NPC). However, to date, few large-scale studies have been conducted on familial risk and clustering of NPC in a high-risk area of China. METHODS In the current study, 2252 patients with NPC who were treated at the Cancer Center of Sun Yat-Sen University in Guangdong Province, China, were identified as probands. Family histories of NPC and other malignancies were observed in first-degree relatives (FDRs) and second-degree relatives, and other information was obtained through interviews. One thousand nine hundred and three Cantonese families were selected for further investigation. To assess familial aggregation, the authors used standardized incidence ratios (SIRs) to measure the risk of NPC for FDRs and compared the observed number of cases with the number predicted by population-based frequencies in the Cantonese population of Hong Kong. RESULTS The current analysis indicated that families with ≥ 3 relatives who had NPC were distributed predominantly among a high-risk subgroup of the Cantonese population in Guangdong Province and that the frequency of these families was 0.68%. An SIR of 2.09 (95% confidence interval [CI], 1.80–2.40) was observed among 13,833 FDRs in the high-risk subgroup, and a significantly elevated risk for NPC was observed in FDRs of probands with early age of onset (age < 40 years; SIR, 9.01 [95% CI, 6.10–13.30]). Furthermore, decreased risks of hepatic, lung, esophageal, gastric, and breast carcinoma, as well as malignancy of all sites, were observed among FDRs of probands with NPC when Hong Kong and Shanghai populations were used as reference groups. CONCLUSIONS NPC tends to aggregate in Cantonese families in Guangdong Province, and the malignancies in these families appear to be site specific, with no excess of any other malignancy. Cancer 2004. © 2004 American Cancer Society.

Nasopharyngeal carcinoma (NPC) is a malignancy with striking geographic and ethnic distribution, and familial clustering of NPC is frequently observed. Emigration and dialect studies also suggest the involvement of genetic factors in the development of NPC. Therefore, many investigations on susceptibility genes have been carried out and polymorphism or specific allele types of HLA, TCR, GSTM1, PIGR and cytochrome P450 were reported to be related to high risk of NPC. More importantly, a pedigree-based study provided evidence of linkage of NPC to chromosome 4p15.1-q12. Somatic genetic changes, such as allelic loss, chromosomal gains or losses, specific gene alterations, were reported to occur frequently in NPC. These findings mentioned above were summarized in this paper and a carcinogenesis model for NPC is proposed.

Development of nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. However, NPC occurs with a marked geographic and racial distribution, whereas EBV infection is ubiquitous in the world. This leads to a question whether certain subtypes of EBV have a greater potential to induce cell transformation. Latent membrane protein 1 (LMP1) is an EBV-encoded oncogenic protein and its 30-bp deleted variant (del-LMP1) has been reported to be predominant in biopsies of NPC. We have assessed the polymorphism of LMP1 in 47 biopsies of NPC, 107 cases of throat washings (TWs) from NPC patients, and 106 cases of TWs from non-NPC patients in Guangzhou, an endemic area of NPC in southern China, as well as 103 cases of TWs from healthy donors in Haerbin, a non-endemic area of NPC in northern China. Our results found a similar extent of the LMP1 polymorphism between NPC patients and non-NPC patients in Guangzhou, with the del-LMP1 being predominant in both Guangzhou and Haerbin. Sequence analyses showed identical substitutions in other coding regions of the del-LMP1 isolated from Guangzhou and Haerbin. These results indicate that del-LMP1 represents a geographic or race-associated polymorphism rather than an NPC disease phenotype-associated polymorphism.

Importance Germline pathogenic variants in BRCA1 and BRCA2 predispose to an increased lifetime risk of breast cancer. However, the relevance of germline variants in other genes from multigene hereditary cancer testing panels is not well defined. Objective To determine the risks of breast cancer associated with germline variants in cancer predisposition genes. Design, Setting, and Participants A study population of 65 057 patients with breast cancer receiving germline genetic testing of cancer predisposition genes with hereditary cancer multigene panels. Associations between pathogenic variants in non- BRCA1 and non- BRCA2 predisposition genes and breast cancer risk were estimated in a case-control analysis of patients with breast cancer and Exome Aggregation Consortium reference controls. The women underwent testing between March 15, 2012, and June 30, 2016. Main Outcomes and Measures Breast cancer risk conferred by pathogenic variants in non- BRCA1 and non- BRCA2 predisposition genes. Results The mean (SD) age at diagnosis for the 65 057 women included in the analysis was 48.5 (11.1) years. The frequency of pathogenic variants in 21 panel genes identified in 41 611 consecutively tested white women with breast cancer was estimated at 10.2%. After exclusion of BRCA1 , BRCA2 , and syndromic breast cancer genes ( CDH1 , PTEN , and TP53 ), observed pathogenic variants in 5 of 16 genes were associated with high or moderately increased risks of breast cancer: ATM (OR, 2.78; 95% CI, 2.22-3.62), BARD1 (OR, 2.16; 95% CI, 1.31-3.63), CHEK2 (OR, 1.48; 95% CI, 1.31-1.67), PALB2 (OR, 7.46; 95% CI, 5.12-11.19), and RAD51D (OR, 3.07; 95% CI, 1.21-7.88). Conversely, variants in the BRIP1 and RAD51C ovarian cancer risk genes; the MRE11A , RAD50 , and NBN MRN complex genes; the MLH1 and PMS2 mismatch repair genes; and NF1 were not associated with increased risks of breast cancer. Conclusions and Relevance This study establishes several panel genes as high- and moderate-risk breast cancer genes and provides estimates of breast cancer risk associated with pathogenic variants in these genes among individuals qualifying for clinical genetic testing.