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PURPOSE Screening and prevention decisions for women at increased risk of developing breast cancer depend on genetic and clinical factors to estimate risk and select appropriate interventions. Integration of polygenic risk into clinical breast cancer risk estimators can improve discrimination. However, correlated genetic effects must be incorporated carefully to avoid overestimation of risk. MATERIALS AND METHODS A novel Fixed-Stratified method was developed that accounts for confounding when adding a new factor to an established risk model. A combined risk score (CRS) of an 86–single-nucleotide polymorphism polygenic risk score and the Tyrer-Cuzick v7.02 clinical risk estimator was generated with attenuation for confounding by family history. Calibration and discriminatory accuracy of the CRS were evaluated in two independent validation cohorts of women of European ancestry (N = 1,615 and N = 518). Discrimination for remaining lifetime risk was examined by age-adjusted logistic regression. Risk stratification with a 20% risk threshold was compared between CRS and Tyrer-Cuzick in an independent clinical cohort (N = 32,576). RESULTS Simulation studies confirmed that the Fixed-Stratified method produced accurate risk estimation across patients with different family history. In both validation studies, CRS and Tyrer-Cuzick were significantly associated with breast cancer. In an analysis with both CRS and Tyrer-Cuzick as predictors of breast cancer, CRS added significant discrimination independent of that captured by Tyrer-Cuzick ( P < 10−11 in validation 1; P < 10−7 in validation 2). In an independent cohort, 18% of women shifted breast cancer risk categories from their Tyrer-Cuzick–based risk compared with risk estimates by CRS. CONCLUSION Integrating clinical and polygenic factors into a risk model offers more effective risk stratification and supports a personalized genomic approach to breast cancer screening and prevention.

Background: Germline pathogenic variants (PVs) in the moderate penetrance ATM gene confer a roughly 2-fold increased risk for breast cancer. Currently, any woman with an ATM PV meets the >20% lifetime risk threshold for consideration of relatively aggressive screening recommendations, which include initiating screening at younger ages and consideration of breast MRI. We and others have previously shown that breast cancer risks for women with inherited PVs in many hereditary breast cancer genes can be adjusted using PRS data, breast cancer family history, and other clinical information. Herein we show the development of a comprehensive breast cancer risk model for ATM PV carriers incorporating a previously described 86-variant PRS along with family history/clinical information captured by Tyrer-Cuzick V7.02. Methods: This IRB-approved study included de-identified clinical records from 353,809 women of European ancestry who were tested clinically for hereditary cancer risk with a multi-gene panel. Model development analyzed ATM PV carriers (N=2,666) and women negative for breast cancer gene PVs (N=351,143) who were tested between September 2013 and November 2019 (July 2019 for non-carriers). Women with the unusually high risk ATM c.7271 allele were excluded from analysis. Risk estimates incorporating ATM, PRS, and Tyrer-Cuzick were calculated using a fixed-stratified method that accounted for correlations between risk factors in a manner equivalent to multivariable co-estimation. Risk stratification was assessed in an independent cohort of ATM carriers (N=272) who were tested after November 2019 and were not included in model development. Results: We detected significant positive correlation of ATM status with breast cancer family history (p=1.6×10−7). Within ATM PV carriers, we observed positive yet non-significant (at alpha 50%). Conclusions: In ATM PV carriers, our comprehensive model allowed for differentiation of ATM PV carriers into low, moderate, and high breast cancer risk categories. Precision breast cancer risk estimation may inform individualized clinical screening and prevention strategies. Citation Format: Shannon Gallagher, Elisha Hughes, Eric Rosenthal, Allison W. Kurian, Susan Domchek, Judy Garber, Braden Probst, Brian Morris, Placede Tshiaba, Benjamin Roa, Thomas P. Slavin, Susanne Wagner, Jeffrey N. Weitzel, Alexander Gutin, Jerry S. Lanchbury, Mark E. Robson. Development of a breast cancer risk assessment model for ATM mutation carriers incorporating tyrer-cuzick and a polygenic risk score (PRS) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD10-09.

A substantial proportion of pathogenic variants associated with an increased risk of hereditary cancer are sequence variants affecting RNA splicing. The classification of these variants can be complex when both non-functional and functional transcripts are produced from the variant allele. We present four BRCA2 splice site variants with complex variant interpretations (BRCA2 c.68-3T>G, c.68-2A>G, c.425G>T, c.8331+2T>C). Evidence supporting a pathogenic classification is available for each variant, including in silico models, absence in population databases, and published functional data. However, comprehensive RNA analysis showed that some functional transcript may be produced by each variant. BRCA2 c.68-3T>G results in a partial splice defect. For BRCA2 c.68-2A>G and c.425G>T, aberrant splicing was shown to produce a potentially functional, in-frame transcript. BRCA2 c.8331+2T>C may utilize a functional GC donor in place of the wild-type GT donor. The severity of cancer history for carriers of these variants was also assessed using a history weighting algorithm and was not consistent with pathogenic controls (carriers of known pathogenic variants in BRCA2). Due to the conflicting evidence, our laboratory classifies these BRCA2 variants as variants of uncertain significance. This highlights the importance of evaluating new and existing evidence to ensure accurate variant classification and appropriate patient care.

PURPOSE Women with a family history of breast cancer are frequently referred for hereditary cancer genetic testing, yet < 10% are found to have pathogenic variants in known breast cancer susceptibility genes. Large-scale genotyping studies have identified common variants (primarily single-nucleotide polymorphisms [SNPs]) with individually modest breast cancer risk that, in aggregate, account for considerable breast cancer susceptibility. Here, we describe the development and empirical validation of an SNP-based polygenic breast cancer risk score. METHODS A panel of 94 SNPs was examined for association with breast cancer in women of European ancestry undergoing hereditary cancer genetic testing and negative for pathogenic variants in breast cancer susceptibility genes. Candidate polygenic risk scores (PRSs) as predictors of personal breast cancer history were developed through multivariable logistic regression models adjusted for age, cancer history, and ancestry. An optimized PRS was validated in 2 independent cohorts (n = 13,174; n = 141,160). RESULTS Within the training cohort (n = 24,259), 4,291 women (18%) had a personal history of breast cancer and 8,725 women (36%) reported breast cancer in a first-degree relative. The optimized PRS included 86 variants and was highly predictive of breast cancer status in both validation cohorts ( P = 6.4 × 10−66; P < 10−325). The odds ratio (OR) per unit standard deviation was consistent between validations (OR, 1.45 [95% CI, 1.39 to 1.52]; OR 1.47 [95% CI, 1.45 to 1.49]). In a direct comparison, the 86-SNP PRS outperformed a previously described PRS of 77 SNPs. CONCLUSION The validation and implementation of a PRS for women without pathogenic variants in known breast cancer susceptibility genes offers potential for risk stratification to guide surveillance recommendations.

PURPOSE Breast cancer risks for CHEK2 and ATM pathogenic variant (PV) carriers are modified by an 86-single nucleotide polymorphism polygenic risk score (PRS) and individual clinical factors. Here, we describe comprehensive risk prediction models for women of European ancestry combining PV status, PRS, and individual clinical variables. MATERIALS AND METHODS This study included deidentified clinical records from 358,095 women of European ancestry who received testing with a multigene panel (September 2013 to November 2019). Model development included CHEK2 PV carriers (n = 4,286), ATM PV carriers (n = 2,666), and women negative for other breast cancer risk gene PVs (n = 351,143). Odds ratios (ORs) were calculated using multivariable logistic regression with adjustment for familial cancer history. Risk estimates incorporating PV status, PRS, and Tyrer-Cuzick v7.02 were calculated using a Fixed-Stratified method that accounts for correlations between risk factors. Stratification of PV carriers into risk categories on the basis of remaining lifetime risk (RLR) was assessed in independent cohorts of PV carriers. RESULTS ORs for association of PV status with breast cancer were 2.01 (95% CI, 1.88 to 2.16) and 1.83 (95% CI, 1.68 to 2.00) for CHEK2 and ATM PV carriers, respectively. ORs for PRS per one standard deviation were 1.51 (95% CI, 1.37 to 1.66) and 1.45 (95% CI, 1.30 to 1.64) in CHEK2 and ATM PV carriers, respectively. Using the combined model (PRS plus Tyrer-Cuzick plus PV status), RLR was low (≤ 20%) for 24.2% of CHEK2 PV carriers, medium (20%-50%) for 63.8%, and high (> 50%) for 12.0%. Among ATM PV carriers, RLR was low for 31.5% of patients, medium for 58.5%, and high for 9.7%. CONCLUSION In CHEK2 and ATM PV carriers, risk assessment including PRS, Tyrer-Cuzick, and PV status has the potential for more precise direction of screening and prevention strategies.

Background: The incorporation of polygenic scores into breast cancer risk assessment models has led to improved risk management for women of European ancestry. However, few studies have examined the extent to which polygenic scores modify risk for carriers of breast cancer susceptibility gene mutations. Current literature suggests that a 77-SNP polygenic score developed from large, mainly non-carrier general population studies can stratify breast cancer risk for carriers of the CHEK2 founder mutation 1100delC with the same effect size observed for non-carriers. Consequently, several groups have modified risk estimates for carriers of other pathogenic variants by applying published effect estimates from non-carriers. In a recent study, a similar 88-SNP polygenic score showed reduced effectiveness in BRCA1 and BRCA2 carriers compared to non-carriers. This highlights the need for more information on polygenic score performance among women who carry pathogenic variants in ATM or PALB2, or in CHEK2 other than the 1100delC founder mutation. We previously described an 86-SNP polygenic residual risk score (RRS) that characterizes genetic risk which is not due to high- or moderate-penetrance breast cancer gene mutations. Here, we present pre-specified independent validation of the RRS for BRCA1, BRCA2, CHEK2, ATM and PALB2 carriers.Methods: This IRB-approved study included 152,172 women of European ancestry who were tested clinically for hereditary cancer risk with a multi-gene panel. The RRS was evaluated separately for carriers of BRCA1 (N=2,249), BRCA2 (N=2,638), CHEK2 (N=2,564), ATM (N=1,445) and PALB2 (N=906), and for mutation-free women (N=141,160). Clinical information was obtained from test requisition forms. Multivariable logistic regression was used to examine the association of RRS with invasive breast cancer after accounting for age and family cancer history. Effect sizes, expressed as standardized odds ratios (ORs) with 95% confidence intervals (CIs), were assessed for carriers of each gene and for non-carriers. Results: RRS was strongly associated with breast cancer risk in each of the BRCA1, BRCA2, CHEK2, ATM and PALB2 carrier populations (p Citation Format: Shannon Gallagher, Elisha Hughes, Susanne Wagner, Placede Tshiaba, Eric Rosenthal, Benjamin B. Roa, Allison Kurian, Susan Domchek, Judy Garber, Johnathan M. Lancaster, Jeffrey Weitzel, Alexander Gutin, Jerry S. Lanchbury, Mark Robson. Polygenic breast cancer risk modification in carriers of high and intermediate risk gene mutations [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-08-07.

Previous analysis of next‐generation sequencing (NGS) hereditary pan‐cancer panel testing demonstrated that approximately 40% of TP53 pathogenic and likely pathogenic variants (PVs) detected have NGS allele frequencies between 10% and 30%, indicating that they likely are acquired somatically. These are seen more frequently in older adults, suggesting that most result from normal aging‐related clonal hematopoiesis. For this analysis, apparent heterozygous germline TP53 PV carriers (NGS allele frequency 30–70%) were offered follow‐up testing to confirm variant origin. Ninety‐eight probands had samples submitted for follow‐up family member testing, fibroblast testing, or both. The apparent heterozygous germline TP53 PV was not detected in 32.6% (15/46) of submitted fibroblast samples, indicating that it was acquired somatically, either through clonal hematopoiesis or via constitutional mosaicism. Notably, no individuals with confirmed germline or likely germline TP53 PVs met classic Li–Fraumeni syndrome (LFS) criteria, only 41% met Chompret LFS criteria, and 59% met neither criteria, based upon provider‐reported personal and family cancer history. Comprehensive reporting of TP53 PVs detected using NGS, combined with follow‐up analysis to confirm variant origin, is advised for clinical testing laboratories. These findings underscore the investment required to provide individuals and family members with clinically accurate genetic test results pertaining to their LFS risk., In next‐generation sequencing (NGS) hereditary cancer panel testing, TP53 pathogenic variants whose NGS allele frequencies might appear consistent with those expected for germline variants, but the variant might be acquired somatically. Confirming TP53 variant origin can markedly impact medical management for tested individuals and their family members. The report highlights the need for a conservative approach to initial reporting of TP53‐positive findings, along with investment in follow‐up testing to deliver clinically accurate results.

Expanded genetic test utilization to guide cancer management has driven the development of larger gene panels and greater diversity in the patient population pursuing testing, resulting in increased identification of atypical or technically challenging genetic findings. To ensure appropriate patient care, it is critical that genetic tests adequately identify and characterize these findings. We describe genetic testing challenges frequently encountered by our laboratory and the methodologies we employ to improve test accuracy for the identification and characterization of atypical genetic findings. While these findings may be individually rare, 15,745 (9%) individuals tested by our laboratory for hereditary cancer risk had an atypical genetic finding, highlighting the importance of employing highly accurate and comprehensive methods in clinical genetic testing.

IMPORTANCE: To date, few studies have examined the extent to which polygenic single-nucleotide variation (SNV) (formerly single-nucleotide polymorphism) scores modify risk for carriers of pathogenic variants (PVs) in breast cancer susceptibility genes. In previous reports, polygenic risk modification was reduced for BRCA1 and BRCA2 PV carriers compared with noncarriers, but limited information is available for carriers of CHEK2, ATM, or PALB2 PVs. OBJECTIVE: To examine an 86-SNV polygenic risk score (PRS) for BRCA1, BRCA2, CHEK2, ATM, and PALB2 PV carriers. DESIGN, SETTING, AND PARTICIPANTS: A retrospective case-control study using data on 150 962 women tested with a multigene hereditary cancer panel between July 19, 2016, and January 11, 2019, was conducted in a commercial testing laboratory. Participants included women of European ancestry between the ages of 18 and 84 years. MAIN OUTCOMES AND MEASURES: Multivariable logistic regression was used to examine the association of the 86-SNV score with invasive breast cancer after adjusting for age, ancestry, and personal and/or family cancer history. Effect sizes, expressed as standardized odds ratios (ORs) with 95% CIs, were assessed for carriers of PVs in each gene as well as for noncarriers. RESULTS: The median age at hereditary cancer testing of the population was 48 years (range, 18-84 years); there were 141 160 noncarriers in addition to carriers of BRCA1 (n = 2249), BRCA2 (n = 2638), CHEK2 (n = 2564), ATM (n = 1445), and PALB2 (n = 906) PVs included in the analysis. The 86-SNV score was associated with breast cancer risk in each of the carrier populations (P

Cancer risks have been previously reported for some retrotransposon element (RE) insertions; however, detection of these insertions is technically challenging and very few oncogenic RE insertions have been reported. Here we evaluate RE insertions identified during hereditary cancer genetic testing using a comprehensive testing strategy. Individuals who had single-syndrome or pan-cancer hereditary cancer genetic testing from February 2004 to March 2017 were included. RE insertions were identified using Sanger sequencing, Next Generation Sequencing, or multiplex quantitative PCR, and further characterized using targeted PCR and sequencing analysis. Personal cancer history, ancestry, and haplotype were evaluated. A total of 37 unique RE insertions were identified in 10 genes, affecting 211 individuals. BRCA2 accounted for 45.9% (17/37) of all unique RE insertions. Several RE insertions were detected with high frequency in populations of conserved ancestry wherein up to 100% of carriers shared a high degree of haplotype conservation, suggesting founder effects. Our comprehensive testing strategy resulted in a substantial increase in the number of reported oncogenic RE insertions, several of which may have possible founder effects. Collectively, these data show that the detection of RE insertions is an important component of hereditary cancer genetic testing and may be more prevalent than previously reported.

1504 Background: Women with pathogenic variants in the moderate penetrance CHEK2 gene have on average an estimated > 20% lifetime risk for breast cancer, thereby meeting an established threshold for more aggressive screening, including consideration of breast magnetic resonance imaging (MRI). However, we previously showed that CHEK2 penetrance is modified by an 86-SNP PRS. CHEK2 risk is further modified by family history (FH) and other TC model variables. Here, we describe development of a comprehensive risk prediction model for women of European ancestry to more precisely estimate risk by incorporating CHEK2, PRS and TC V7.02. The number of CHEK2 carriers with low ( < 20%), moderate (20%-50%) and high ( > 50%) remaining lifetime risk based on the combined model was examined in an independent study cohort. Methods: This IRB-approved study included de-identified clinical records from 358,471 women of European ancestry who were tested clinically for hereditary cancer risk with a multi-gene panel. Model development was based on analysis of CHEK2 PV carriers ( N= 4,331) and women negative for BC gene PV ( N =353,681) who were tested between September 2013 and July 2019. Risk estimates incorporating CHEK2, PRS and TC were calculated using a fixed-stratified (FS) method that accounts for correlations between risk factors in a manner equivalent to multivariable co-estimation. Risk stratification was assessed in an independent cohort of CHEK2 carriers ( N= 459) who were tested after July 2019 and not included in model development. Results: We detected significant correlation of CHEK2 status with FH ( p= 4.1 × 10−17) and of PRS with FH among CHEK2 carriers ( p= 1.7× 10−5). For these factors, joint effects were co-estimated using the FS method. In an independent cohort, 24.0% of CHEK2 carriers were categorized as low risk ( < 20%), and 62.6% were categorized as moderate risk (20-50%). For 13.4% of CHEK2 carriers, risk estimation incorporating PRS and TC generated BC risks of greater than 50%, consistent with genes recognized as highly penetrant. Conclusions: In CHEK2 PV carriers, comprehensive risk assessment could inform individualized decision-making and may lead to improved targeting of screening and prevention strategies.

Background Healthcare providers increasingly use information about pathogenic variants in cancer predisposition genes, including sequence variants and large rearrangements (LRs), in medical management decisions. While sequence variant detection is typically robust, LRs can be difficult to detect and characterize and may be underreported as a cause for hereditary cancer risk. This report describes the outcomes of hereditary cancer genetic testing using a comprehensive strategy that employs next-generation sequencing (NGS) for LR detection, coupled with LR confirmation using repeat hybrid capture NGS, microarray comparative genomic hybridization (microarray-CGH), and/or multiplex ligation-dependent probe amplification (MLPA). Methods Sequencing and LR analysis were conducted in a consecutive series of 376,159 individuals who received clinical testing with a hereditary pan-cancer gene panel from September 2013 through May 2017. NGS dosage analysis was used to evaluate potential deletions or duplications, with controls in place to exclude pseudogene reads. Samples positive for a putative LR based on NGS were confirmed using a comprehensive approach that included targeted microarray-CGH and/or MLPA analysis, with further examination as needed to ascertain the nature of the LR. Results A total of 3461 LRs were identified and classified as a deleterious mutation (DM), suspected deleterious mutation (SDM) or variant of uncertain significance. Pathogenic LRs (DM/SDM) accounted for the majority of LRs (67.7%), the largest proportion of which were deletions (86.1%), followed by duplications (11.3%), insertions (1.8%), triplications (0.5%), and inversions (0.3%). Several cases presented illustrate that the laboratory approach employed here can ensure consistent identification and accurate characterization of LRs. In the absence of this comprehensive testing strategy, 9% of LRs identified in this testing population might have been missed, potentially leading to inappropriate medical management in as many as 210 individuals referred for hereditary cancer testing. Conclusions These data show that copy number analysis using NGS coupled with confirmatory testing reliably detects and characterizes LRs. Further, LRs comprise a substantial proportion (7.2%) of pathogenic variants identified by the test. A robust and accurate LR identification strategy is an essential component of a high-quality genetic testing program, enabling clinicians to optimize patient medical management decisions.

Background Histopathologic examination is sometimes inadequate for accurate and reproducible diagnosis of certain melanocytic neoplasms. As a result, more sophisticated and objective methods have been sought. The goal of this study was to identify a gene expression signature that reliably differentiated benign and malignant melanocytic lesions and evaluate its potential clinical applicability. Herein, we describe the development of a gene expression signature and its clinical validation using multiple independent cohorts of melanocytic lesions representing a broad spectrum of histopathologic subtypes. Methods Using quantitative reverse-transcription polymerase chain reaction (PCR) on a selected set of 23 differentially expressed genes, and by applying a threshold value and weighting algorithm, we developed a gene expression signature that produced a score that differentiated benign nevi from malignant melanomas. Results The gene expression signature classified melanocytic lesions as benign or malignant with a sensitivity of 89% and a specificity of 93% in a training cohort of 464 samples. The signature was validated in an independent clinical cohort of 437 samples, with a sensitivity of 90% and specificity of 91%. Conclusions The performance, objectivity, reliability and minimal tissue requirements of this test suggest that it could have clinical application as an adjunct to histopathology in the diagnosis of melanocytic neoplasms.

Angelman syndrome is a neurodevelopmental disorder caused by a deficiency of the imprinted and maternally expressed UBE3A gene. Although de novo genetic and epigenetic imprinting defects of UBE3A genomic locus account for majority of Angelman diagnoses, approximately 10% of individuals affected with Angelman syndrome are a result of UBE3A loss-of-function mutations occurring on the expressed maternal chromosome. The variants described in this manuscript represent the analysis of 2,515 patients referred for UBE3A gene sequencing at our institution, along with a comprehensive review of the UBE3A mutation literature. Of these, 267 (10.62%) patients had a report issued for detection of a UBE3A gene nucleotide variant, which in many cases involved family studies resulting in reclassification of variants of unknown clinical significance (VUS). Overall, 111 (4.41%) probands had a nucleotide change classified as pathogenic or strongly favored to be pathogenic, 29 (1.15%) had a VUS, and 126 (5.0%) had a nucleotide change classified as benign or strongly favored to be benign. All variants and their clinical interpretations are submitted to NCBI ClinVar, a freely accessible human variation and phenotype database.

BACKGROUND Next-generation sequencing (NGS) allows for simultaneous sequencing of multiple cancer susceptibility genes and, for an individual, may be more efficient and less expensive than sequential testing. The authors assessed the frequency of deleterious germline mutations among individuals with breast cancer who were referred for BRCA1 and BRCA2 (BRCA1/2) gene testing using a panel of 25 genes associated with inherited cancer predisposition. METHODS This was a cross-sectional study using NGS in 2158 individuals, including 1781 who were referred for commercial BRCA1/2 gene testing (cohort 1) and 377 who had detailed personal and family history and had previously tested negative for BRCA1/2 mutations (cohort 2). RESULTS Mutations were identified in 16 genes, most frequently in BRCA1, BRCA2, CHEK2, ATM, and PALB2. Among the participants in cohort 1, 9.3% carried a BRCA1/2 mutation, 3.9% carried a mutation in another breast/ovarian cancer susceptibility gene, and 0.3% carried an incidental mutation in another cancer susceptibility gene unrelated to breast or ovarian cancer. In cohort 2, the frequency of mutations in breast/ovarian-associated genes other than BRCA1/2 was 2.9%, and an additional 0.8% had an incidental mutation. In cohort 1, Lynch syndrome-related mutations were identified in 7 individuals. In contrast to BRCA1/2 mutations, neither age at breast cancer diagnosis nor family history of ovarian or young breast cancer predicted for other mutations. The frequency of mutations in genes other than BRCA1/2 was lower in Ashkenazi Jews compared with non-Ashkenazi individuals (P=.026). CONCLUSIONS Using an NGS 25-gene panel, the frequency of mutations in genes other than BRCA1/2 was 4.3%, and most mutations (3.9%) were identified in genes associated with breast/ovarian cancer. Cancer 2015;121:25–33. © 2014 American Cancer Society.

BRCA1 and BRCA2 sequencing analysis detects variants of uncertain clinical significance in approximately 2 % of patients undergoing clinical diagnostic testing in our laboratory. The reclassification of these variants into either a pathogenic or benign clinical interpretation is critical for improved patient management. We developed a statistical variant reclassification tool based on the premise that probands with disease-causing mutations are expected to have more severe personal and family histories than those having benign variants. The algorithm was validated using simulated variants based on approximately 145,000 probands, as well as 286 BRCA1 and 303 BRCA2 true variants. Positive and negative predictive values of ≥99 % were obtained for each gene. Although the history weighting algorithm was not designed to detect alleles of lower penetrance, analysis of the hypomorphic mutations c.5096G>A (p.Arg1699Gln; BRCA1) and c.7878G>C (p.Trp2626Cys; BRCA2) indicated that the history weighting algorithm is able to identify some lower penetrance alleles. The history weighting algorithm is a powerful tool that accurately assigns actionable clinical classifications to variants of uncertain clinical significance. While being developed for reclassification of BRCA1 and BRCA2 variants, the history weighting algorithm is expected to be applicable to other cancer- and non-cancer-related genes.

BACKGROUND This study sought to determine the prevalence of PALB2 mutations in a cohort referred for diagnostic testing for hereditary breast cancer. METHODS Sanger sequencing was used to analyze the entire coding region and flanking introns of PALB2 in anonymized DNA samples from 1479 patients. Samples were stratified into a “high-risk” group, 955 samples from individuals predicted to have a high probability of carrying a mutation in BRCA1 or BRCA2 based on their personal and family history, and a “lower-risk” group consisting of 524 samples from patients with breast cancer, but fewer risk factors for being a BRCA1 or BRCA2 mutation carrier. All patients were known to be negative for deleterious sequence mutations and large rearrangements in BRCA1 and BRCA2. RESULTS We identified 12 disease-associated PALB2 mutations among the 1479 patients (0.8%). The PALB2 mutations included 8 nonsense, 3 frameshift mutations and a splice-site mutation. The mutation prevalence for the high-risk population was 1.05% (95% CI = 0.5-1.92), whereas that for the lower-risk population was 0.38% (95% CI = 0.05-1.37). We identified 59 PALB2 variants of uncertain significance (VUS) among 57 of the 1479 patients (3.9%). CONCLUSIONS These results suggest that PALB2 mutations occur at a frequency of ∼1% in patients with hereditary breast cancer. Cancer 2014;120:963–967. © 2013 American Cancer Society.

Identifying individuals at increased risk for hereditary cancer leads to early detection and prevention opportunities with the ability to reduce both cancer incidence and mortality. Hereditary cancer syndromes have genetic heterogeneity and new susceptibility genes have been recently identified. Next generation sequencing allows testing of multiple target genes simultaneously, can reduce the time and cost of sequential gene testing, and may improve mutation detection. To date, no large scale studies have reported the mutation prevalence of multiple cancer susceptibility genes among patients referred for BRCA1/BRCA2 testing. A study was performed to determine the mutation prevalence in 25 cancer susceptibility genes among a large U.S. patient population referred to a diagnostic laboratory for BRCA1/BRCA2 testing. DNA from 1955 prospectively accrued cases was anonymized after testing was complete. Patients with Ashkenazi Jewish heritage were excluded in order to determine the relative prevalence of mutations in a generalizable population. In addition, an independent external validation set of 405 patients, including those of Ashkenazi ancestry, with history consistent with hereditary breast and ovarian cancer (HBOC) syndrome and who had previously tested negative for BRCA1/BRCA2 mutations was assessed. Extracted genomic DNA from blood was PCR amplified with a custom amplicon library on a Raindance ThunderStorm instrument. The DNA products were sequenced on an Illumina HiSeq2500. Sequence variations and large rearrangements among the 25 genes were detected and classified for pathogenicity. Among the 1955 anonymized patients referred for BRCA1/BRCA2 testing, 275 (14.07%) patients were mutation carriers in at least one of the 25 genes. 182 (9.31%) patients had a mutation in BRCA1 or BRCA2, and 96 of 1955 (4.91%) patients had a mutation in other genes (Table 1). Table 1GenePatients with mutation (n = 96)%ATM1414.58%BARD177.29%BRIP177.29%CHEK23031.25%MSH222.08%MSH622.08%MUTYH11.04%NBN1414.58%PALB21313.54%PMS244.17%TP5322.08% No mutations were found in CDH1, PTEN, STK11, RAD51C, RAD51D, BMPR1A, SMAD4, MLH1, EPCAM, CDKN2A, CDK4, or APC. 1738 of 1955 patients had a personal history of breast cancer (BC), with 63% diagnosed prior to age 50, and 37% at or after age 50. Mutation prevalence for patients with BC, ovarian cancer (OC), both BC and OC, or other HBOC cancers is listed in Table 2. Table 2Patient Cancer HistoryPatients (n)BRCA1/2Other GeneBreast CA < 50 years1091116* (10.63%)51 (4.67%)Breast CA ≥ 50 years64740** (6.18%)30 (4.64%)Ovarian CA16217 (10.49%)6 (3.70%)Breast and Ovarian CA408 (20.00%)4 (10.00%)Other HBOC Cancer151 (6.67%)2 (13.33%)*2 and **1 patients had an additional mutation in a non-BRCA1/2 gene 1902 (97.29%) patients had a variant of uncertain significance in at least one of the genes tested and an average of three variants was found per patient. As of June 11, 2013 the independent external validation cases results are pending. Compared with BRCA1/BRCA2 testing alone, using the 25 gene panel increased the identification of mutations in cancer susceptibility genes by 4.76% (95% CI: 2.71% – 6.81%), which represents a 51.1% increase in mutation detection for this population with suspected HBOC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD4-8.

BACKGROUND Recently, a 23‐gene signature was developed to produce a melanoma diagnostic score capable of differentiating malignant and benign melanocytic lesions. The primary objective of this study was to independently assess the ability of the gene signature to differentiate melanoma from benign nevi in clinically relevant lesions. METHODS A set of 1400 melanocytic lesions was selected from samples prospectively submitted for gene expression testing at a clinical laboratory. Each sample was tested and subjected to an independent histopathologic evaluation by 3 experienced dermatopathologists. A primary diagnosis (benign or malignant) was assigned to each sample, and diagnostic concordance among the 3 dermatopathologists was required for inclusion in analyses. The sensitivity and specificity of the score in differentiating benign and malignant melanocytic lesions were calculated to assess the association between the score and the pathologic diagnosis. RESULTS The gene expression signature differentiated benign nevi from malignant melanoma with a sensitivity of 91.5% and a specificity of 92.5%. CONCLUSIONS These results reflect the performance of the gene signature in a diverse array of samples encountered in routine clinical practice. Cancer 2017;123:617–628. © 2016 American Cancer Society., A 23‐gene signature has recently been developed to differentiate malignant and benign melanocytic lesions. This study shows that the gene expression signature differentiates benign nevi from malignant melanoma with a sensitivity of 91.5% and a specificity of 92.5% in comparison with a triple‐concordant histopathologic review.

BACKGROUND: Current estimates of the contribution of large rearrangement (LR) mutations in the BRCA1 (breast cancer 1, early onset) and BRCA2 (breast cancer 2, early onset) genes responsible for hereditary breast and ovarian cancer are based on limited studies of relatively homogeneous patient populations. The prevalence of BRCA1/2 LRs was investigated in 48,456 patients with diverse clinical histories and ancestries, referred for clinical molecular testing for suspicion of hereditary breast and ovarian cancer. METHODS: Sanger sequencing analysis was performed for BRCA1/2 and LR testing for deletions and duplications using a quantitative multiplex polymerase chain reaction assay. Prevalence data were analyzed for patients from different risk and ethnic groups between July 2007 and April 2011. Patients were designated as “high-risk” if their clinical history predicted a high prior probability, wherein LR testing was performed automatically in conjunction with sequencing. “Elective” patients did not meet the high-risk criteria, but underwent LR testing as ordered by the referring health care provider. RESULTS: Overall BRCA1/2 mutation prevalence among high-risk patients was 23.8% versus 8.2% for the elective group. The mutation profile for high-risk patients was 90.1% sequencing mutations versus 9.9% LRs, and for elective patients, 94.1% sequencing versus 5.9% LRs. This difference may reflect the bias in high-risk patients to carry mutations in BRCA1, which has a higher penetrance and frequency of LRs compared with BRCA2. There were significant differences in the prevalence and types of LRs in patients of different ancestries. LR mutations were significantly more common in Latin American/Caribbean patients. CONCLUSIONS: Comprehensive LR testing in conjunction with full gene sequencing is an appropriate strategy for clinical BRCA1/2 analysis.

Aims: The aim of these studies was to validate the analytical performance of a cell cycle progression (CCP) gene signature that provides prognostic information for early stage lung adenocarcinomas. Materials & methods: Formalin-fixed paraffin-embedded (FFPE) lung resections were evaluated by quantitative RT-PCR for the expression of 31 target and 15 housekeeper genes comprising the CCP score. Results: The signature had a standard deviation (SD) of 0.06 score units and a dynamic range spanning CCP scores between -13 and 14. The average amplicon efficiencies for target and housekeeper genes were 107% and 105%, respectively. All but one amplicon had a SD T. Conclusion: These studies demonstrate that the gene signature is robust and reproducible, making it suitable for use in a clinical setting.

Objectives Aetiological assessment of 71 probands whose clinical presentation suggested a genetic syndrome or auditory neuropathy. Methods Sanger sequencing was performed on DNA isolated from peripheral blood or lymphoblastoid cell lines. Genes were selected for sequencing based on each patient9s clinical presentation and suspected diagnosis. Observed DNA sequence variations were assessed for pathogenicity by review of the scientific literature, and mutation and polymorphism databases, through the use of in silico tools including sorting intolerant from tolerant (SIFT) and polymorphism phenotyping (PolyPhen), and according to the recommendations of the American College of Medical Genetics and Genomics for the interpretation of DNA sequence variations. Novel DNA sequence variations were sought in controls. Results DNA sequencing of the coding and near-coding regions of genes relevant to each patient9s clinical presentation revealed 37 sequence variations of known or uncertain pathogenicity in 9 genes from 25 patients. 14 novel sequence variations were discovered. Assessment of phenotypes revealed notable findings in 9 patients. Conclusions DNA sequencing in patients whose clinical presentation suggested a genetic syndrome or auditory neuropathy provided opportunities for aetiological assessment and more precise genetic counselling of patients and families. The failure to identify a genetic aetiology in many patients in this study highlights the extreme heterogeneity of genetic hearing loss, the incompleteness of current knowledge of aetiologies of hearing loss, and the limitations of conventional DNA sequencing strategies that evaluate only coding and near-coding segments of genes.

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

Aim: These studies were to validate the analytical performance of a gene expression signature that differentiates melanoma and nevi, using RNA expression from 14 signature genes and nine normalization genes that generates a melanoma diagnostic score (MDS). Materials & Methods: Formalin-fixed paraffin-embedded melanocytic lesions were evaluated in these studies. Results: The overall SD of the assay was determined to be 0.69 MDS units. Individual amplicons within the signature had an average amplification efficiency of 92% and a SD less than 0.5 CT. The MDS was reproducible across a 2000-fold dilution range of input RNA. Melanin, an inhibitor of PCR, does not interfere with the signature. Conclusion: These studies indicate this signature is robust and reproducible and is analytically validated on formalin-fixed paraffin-embedded melanocytic lesions.

Background & Aims Multigene panels are commercially available tools for hereditary cancer risk assessment that allow for next-generation sequencing of numerous genes in parallel. However, it is not clear if these panels offer advantages over traditional genetic testing. We investigated the number of cancer predisposition gene mutations identified by parallel sequencing in individuals with suspected Lynch syndrome. Methods We performed germline analysis with a 25-gene, next-generation sequencing panel using DNA from 1260 individuals who underwent clinical genetic testing for Lynch syndrome from 2012 through 2013. All patients had a history of Lynch syndrome–associated cancer and/or polyps. We classified all identified germline alterations for pathogenicity and calculated the frequencies of pathogenic mutations and variants of uncertain clinical significance (VUS). We also analyzed data on patients' personal and family history of cancer, including fulfillment of clinical guidelines for genetic testing. Results Of the 1260 patients, 1112 met National Comprehensive Cancer Network (NCCN) criteria for Lynch syndrome testing (88%; 95% confidence interval [CI], 86%–90%). Multigene panel testing identified 114 probands with Lynch syndrome mutations (9.0%; 95% CI, 7.6%−10.8%) and 71 with mutations in other cancer predisposition genes (5.6%; 95% CI, 4.4%−7.1%). Fifteen individuals had mutations in BRCA1 or BRCA2 ; 93% of these met the NCCN criteria for Lynch syndrome testing and 33% met NCCN criteria for BRCA1 and BRCA2 analysis ( P = .0017). An additional 9 individuals carried mutations in other genes linked to high lifetime risks of cancer (5 had mutations in APC , 3 had bi-allelic mutations in MUTYH , and 1 had a mutation in STK11 ); all of these patients met NCCN criteria for Lynch syndrome testing. A total of 479 individuals had 1 or more VUS (38%; 95% CI, 35%–41%). Conclusions In individuals with suspected Lynch syndrome, multigene panel testing identified high-penetrance mutations in cancer predisposition genes, many of which were unexpected based on patients' histories. Parallel sequencing also detected a high number of potentially uninformative germline findings, including VUS.

Background: Prostate cancer is the most common cancer in men in the developed world. Appropriate clinical care requires accurate prognostic information to determine whether definitive treatment or conservative management is most appropriate for a given patient. We previously demonstrated that a gene expression signature, which measures the RNA expression of 31 cell cycle progression (CCP) genes and generates a CCP score, is a robust predictor of patient outcome in cohorts of conservatively managed patients diagnosed by needle biopsy or transurethral resection of the prostate. Methods: These current studies represent the analytical validation of this gene signature, for the testing of either formalin-fixed paraffin-embedded (FFPE) prostate resection tissue (radical prostatectomy, RP) or FFPE prostate needle biopsy samples. Results: The measured standard deviation (SD) of the signature was determined to be 0.1 score units, representing 1.6% of the range of scores observed within previous clinical validation studies. Individual amplicons for all genes within the signature had a SD

Mutations in GJB2 are associated with hereditary hearing loss. DNA sequencing of GJB2 in a cohort of hearing impaired patients and a multi-ethnic control group is reported. Among 610 hearing impaired cases, 43 DNA sequence variations were identified in the coding region of GJB2 including 24 mutations, 8 polymorphisms, 3 unclassi-fied variants (G4D, R127C, M163V), 1 controversial variant (V37I), and 7 novel variants (G12C, N14D, V63A, T86M, L132V, D159, 592_600delinsCAGTGTTCATGACATTC). Sixteen non-coding sequence variations were also identified among cases including the IVS1+1A>G mutation, 2 polymorphisms, and 13 novel variants. A diagnosis of GJB2-associated hearing loss was confirmed for 63 cases (10.3%). Heterozygous mutations were found in 39 cases (6.4%). Eleven cases carrying novel or unclassified variants (1.8 %) and 18 cases carrying the controversial V37I variant were identified (3%). In addition, 294 control subjects from 4 ethnic groups were sequenced for GJB2. Thirteen sequence variations in the coding region of GJB2 were identified among controls including 2 mutations, 6 polymorphisms, 2 unclassified variants (G4D, T123N), 1 controversial variant (V37I), and 2 novel variants (R127L, V207L). Nine sequence variations were identified among controls in the non-coding regions in and around GJB2 exon 2. Of particular interest among controls were the variability in carrier rates and ethnic stratification of alleles, and the complex genotypes among Asians, 47% of whom carried two to four sequence variations in the coding region of GJB2. These data provide new information about carrier rates for GJB2-based hearing loss in various ethnic groups and contribute to evaluation of the pathogenicity of the controversial V37I variant.

Purpose: To provide a summary of the outcomes of two working conferences organized by the Centers for Disease Control and Prevention (CDC), to develop recommendations for practical, sustainable mechanisms to make quality control (QC) materials available to the genetic testing community. Methods: Participants were selected to include experts in genetic testing and molecular diagnostics from professional organizations, government agencies, industry, laboratories, academic institutions, cell repositories, and proficiency testing (PT)/external Quality Assessment (EQA) programs. Current efforts to develop QC materials for genetic tests were reviewed; key issues and areas of need were identified; and workgroups were formed to address each area of need and to formulate recommendations and next steps. Results: Recommendations were developed toward establishing a sustainable process to improve the availability of appropriate QC materials for genetic testing, with an emphasis on molecular genetic testing as an initial step. Conclusions: Improving the availability of appropriate QC materials is of critical importance for assuring the quality of genetic testing, enhancing performance evaluation and PT/EQA programs, and facilitating new test development. To meet the needs of the rapidly expanding capacity of genetic testing in clinical and public health settings, a comprehensive, coordinated program should be developed. A Genetic Testing Quality Control Materials Program has therefore been established by CDC in March 2005 to serve these needs.

We report a 5(1/2)-year-old girl with a maternal family history of Huntington disease (HD), who presented clinically with unbalanced gait, impaired speech, and increasing difficulty with fine motor control. Onset of symptoms began at the age of 3(1/2) years. The suspected diagnosis of juvenile HD, based upon her family history, was confirmed by DNA analysis. At age 7, the patient died secondary to complications of her underlying disorder. Juvenile-onset Huntington disease is uncommon, predominantly transmitted by fathers and is always associated with very large expansions of the CAG repeat. Interestingly, this patient inherited a large CAG size expansion from her mother, who herself had symptoms of HD at the age of 18. Molecular analysis revealed that the mother had 70 CAG repeats whereas our patient had approximately 130 CAG repeats. This is the largest reported CAG expansion from a maternal transmission that has been confirmed molecularly and it demonstrates that very large expansions can also occur through the maternal lineage.

Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant disorder characterized by ataxia, seizures, and anticipation. It is caused by an expanded ATTCT pentanucleotide repeat in intron 9 of a novel gene, designated "SCA10." The ATTCT expansion in SCA10 represents a novel class of microsatellite repeat and is one of the largest found to cause human diseases. The expanded ATTCT repeat is unstably transmitted from generation to generation, and an inverse correlation has been observed between size of repeat and age at onset. In this multifamily study, we investigated the intergenerational instability, somatic and germline mosaicism, and age-dependent repeat-size changes of the expanded ATTCT repeat. Our results showed that (1) the expanded ATTCT repeats are highly unstable when paternally transmitted, whereas maternal transmission resulted in significantly smaller changes in repeat size; (2) blood leukocytes, lymphoblastoid cells, buccal cells, and sperm have a variable degree of mosaicism in ATTCT expansion; (3) the length of the expanded repeat was not observed to change in individuals over a 5-year period; and (4) clinically determined anticipation is sometimes associated with intergenerational contraction rather than expansion of the ATTCT repeat.

Aim: To validate the analytical performance of a 12-gene molecular assay that predicts distant recurrence for early-stage ER+/HER2- invasive breast cancer as run within a central reference laboratory. Materials & methods: Formalin-fixed paraffin-embedded breast resections were evaluated by quantitative reverse transcription polymerase chain reaction for the expression of eight target genes, three housekeeper genes and one control gene to assess for DNA contamination. Results: The assay results were highly correlated with a validated reference laboratory. The assay had a broad linear range for input RNA, with similar amplicon efficiencies for target and housekeeper genes. The assay test was highly reproducible, with comparable inter- and intrabatch precision to the reference laboratory. Conclusion: These studies demonstrate that the 12-gene molecular assay is highly robust and accurate., Lay abstract Several tests have been developed to aid in breast cancer treatment decisions by assessing the likelihood that an individual’s breast cancer will recur. One such test is a 12-gene molecular assay. This test provides a clinical score that combines molecular information from genes within the tumor with clinical information to determine whether a breast cancer is at high or low risk of recurrence. The data presented here show that this test is very reproducible and robust, making it appropriate and reliable for clinical use.

Background Conventional Sanger sequencing reliably detects the majority of genetic mutations associated with hereditary cancers, such as single-base changes and small insertions or deletions. However, detection of genomic rearrangements, such as large deletions and duplications, requires special technologies. Microarray analysis has been successfully used to detect large rearrangements (LRs) in genetic disorders. Methods We designed and validated a high-density oligonucleotide microarray for the detection of gene-level genomic rearrangements associated with hereditary breast and ovarian cancer (HBOC), Lynch, and polyposis syndromes. The microarray consisted of probes corresponding to the exons and flanking introns of BRCA1 and BRCA2 (≈1,700) and Lynch syndrome/polyposis genes MLH1, MSH2, MSH6, APC, MUTYH, and EPCAM (≈2,200). We validated the microarray with 990 samples previously tested for LR status in BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, MUTYH, or EPCAM. Microarray results were 100% concordant with previous results in the validation studies. Subsequently, clinical microarray analysis was performed on samples from patients with a high likelihood of HBOC mutations (13,124), Lynch syndrome mutations (18,498), and polyposis syndrome mutations (2,739) to determine the proportion of LRs. Results Our results demonstrate that LRs constitute a substantial proportion of genetic mutations found in patients referred for hereditary cancer genetic testing. Conclusion The use of microarray comparative genomic hybridization (CGH) for the detection of LRs is well-suited as an adjunct technology for both single syndrome (by Sanger sequencing analysis) and extended gene panel testing by next generation sequencing analysis. Genetic testing strategies using microarray analysis will help identify additional patients carrying LRs, who are predisposed to various hereditary cancers.

Objective: We sought to establish an association between preeclampsia and the methionine to threonine polymorphism at amino acid residue 235 (Met235Thr) in angiotensinogen in a Hispanic population. We looked for a relationship between this allele and the allele in the endothelial nitric oxide synthase gene (NOS3) that produces the A form (NOS3*A) with respect to preeclampsia. Study Design: Clinical data were collected from 87 patients with preeclampsia and 53 control subjects. Patients and controls were genotyped for the angiotensinogen polymorphism allele (AGT*T) and the NOS3*A polymorphism. We then compared patients with preeclampsia and control subjects and investigated disease severity within the preeclampsia group as a function of genotype. Results: The AGT*T allelic frequencies among patients with preeclampsia and control subjects were 0.72 and 0.70, respectively ( P = .84). The blood pressure of patients with an AGT*T allele who also carried a NOS3*A allele was higher at earlier gestational ages ( r = –0.052; P = .02). Analysis suggested that the systolic blood pressure differences were due to gestational age effects and the presence of a NOS3*A allele ( P Conclusion: The AGT*T allele was not associated with the development of preeclampsia. Independently of the presence of an AGT*T allele, the NOS3*A allele was associated with a higher blood pressure at an earlier gestational age. (Am J Obstet Gynecol 2001;184:1345-51.)

A 55-bp deletion in exon 9 of the glucocerebrosidase gene was identified in a 28-year-old male affected with Gaucher disease. The diagnosis was established during an evaluation for mild pancytopenia and was confirmed by bone marrow histology and biochemical studies. The patient is of German ancestry. Initial DNA testing indicated homozygosity for the N370S mutation. However, subsequent testing of the patient's parents suggested that the patient and his mother carried a null allele by our assay for N370S. Further molecular studies identified a 55-bp deletion in exon 9 of the glucocerebrosidase gene (g.6767_6822del55). This deletion has been previously reported in a patient with severe Gaucher disease (1), and is present in the glucocerebrosidase pseudogene. In the previously reported case, initial DNA testing also suggested the genotype N370S/N370S, but further mutation studies were undertaken because clinical severity was greater than expected for that genotype. In contrast, our patient has an unusually mild clinical course. Thus, clinical severity cannot be reliably used to determine when to test for the presence of the 55-bp deletion. While the 55-bp deletion is not reported to be common, its actual frequency may be underestimated since it eludes detection by many standard clinical assays for Gaucher disease. This report points out the need to consider this deletion mutation which may cause erroneous interpretation of results in existing assays for the common mutations N370S and L444P. Furthermore, the importance of recommending parental analysis for individuals who test homozygous for autosomal mutations is highlighted.

We report the prenatal diagnosis of a fetus with a de novo Robertsonian translocation: 45,XY,der(15;15)(q10;q10). Although Robertsonian translocations are common chromosomal rearrangements, those involving homologous chromosomes are infrequent. Since chromosome 15 is imprinted, uniparental disomy (UPD) is a concern when chromosomal rearrangements involving chromosome 15 are identified. In the present case, UPD studies showed normal biparental inheritance. In contrast to the fact that most homologous acrocentric rearrangements are isochromosomes, these results indicate postzygotic formation of a Robertsonian translocation between biparentally inherited chromosomes 15.

Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder affecting 1/10,000-15,000 girls. The disease-causing gene was identified as MECP2 on chromosome Xq28, and mutations have been found in approximately 80% of patients diagnosed with RTT. Numerous mutations have been identified in de novo and rare familial cases, and they occur primarily in the methyl-CpG-binding and transcriptional-repression domains of MeCP2. Our first diagnostic strategy used bidirectional sequencing of the entire MECP2 coding region. Subsequently, we implemented a two-tiered strategy that used denaturing high-performance liquid chromatography (DHPLC) for initial screening of nucleotide variants, followed by confirmatory sequencing analysis. If a definite mutation was not identified, then the entire MECP2 coding region was sequenced, to reduce the risk of false negatives. Collectively, we tested 228 unrelated female patients with a diagnosis of possible (209) or classic (19) RTT and found MECP2 mutations in 83 (40%) of 209 and 16 (84%) of 19 of the patients, respectively. Thirty-two different mutations were identified (8 missense, 9 nonsense, 1 splice site, and 14 frameshifts), of which 12 are novel and 9 recurrent in unrelated patients. Seven unclassified variants and eight polymorphisms were detected in 228 probands. Interestingly, we found that T203M, previously reported as a missense mutation in an autistic patient, is actually a benign polymorphism, according to parental analysis performed in a second case identified in this study. These findings highlight the complexities of missense variant interpretation and emphasize the importance of parental DNA analysis for establishing an etiologic relation between a possible mutation and disease. Overall, we found a 98.8% concordance rate between DHPLC and sequence analyses. One mutation initially missed by the DHPLC screening was identified by sequencing. Modified conditions subsequently enabled its detection, underscoring the need for multiple optimized conditions for DHPLC analysis. We conclude that this two-tiered approach provides a sensitive, robust, and efficient strategy for RTT molecular diagnosis.

Molecular laboratory techniques are increasingly important in the evaluation of fetuses at risk for a single gene disorder or chromosomal abnormality and for the detection of genetic or other conditions that can lead to an adverse fetal or maternal outcome. The localization and identification of novel disease genes allows for mutation analysis or linkage studies on fetuses at risk for these disorders. New assays or techniques for mutation detection in single gene disorders such as amplification refractory mutation system polymerase chain reaction, fluorescent polymerase chain reaction, heteroduplex analysis and the protein truncation test are now applied in prenatal diagnosis. Recent advances in molecular cytogenetics, such as comparative genomic hybridization, the primed in-situ labeling technique, the development of new telomeric probes and spectral karyotyping, are being evaluated for their role in the prenatal diagnosis of chromosomal abnormalities. These methods may greatly improve the accuracy and applicability of preimplantation genetic diagnosis or diagnosis on fetal cells isolated from maternal blood.

The authors describe a 6-year-old girl with developmental delay, psychomotor regression, seizures, mental retardation, and autistic features associated with low CSF levels of 5-methyltetrahydrofolate, the biologically active form of folates in CSF and blood. Folate and B12 levels were normal in peripheral tissues, suggesting cerebral folate deficiency. Treatment with folinic acid corrected CSF abnormalities and improved motor skills.

This study sought to determine the prevalence of PALB2 mutations in a cohort referred for diagnostic testing for hereditary breast cancer.Sanger sequencing was used to analyze the entire coding region and flanking introns of PALB2 in anonymized DNA samples from 1479 patients. Samples were stratified into a "high-risk" group, 955 samples from individuals predicted to have a high probability of carrying a mutation in BRCA1 or BRCA2 based on their personal and family history, and a "lower-risk" group consisting of 524 samples from patients with breast cancer, but fewer risk factors for being a BRCA1 or BRCA2 mutation carrier. All patients were known to be negative for deleterious sequence mutations and large rearrangements in BRCA1 and BRCA2.We identified 12 disease-associated PALB2 mutations among the 1479 patients (0.8%). The PALB2 mutations included 8 nonsense, 3 frameshift mutations and a splice-site mutation. The mutation prevalence for the high-risk population was 1.05% (95% CI = 0.5-1.92), whereas that for the lower-risk population was 0.38% (95% CI = 0.05-1.37). We identified 59 PALB2 variants of uncertain significance (VUS) among 57 of the 1479 patients (3.9%).These results suggest that PALB2 mutations occur at a frequency of ~1% in patients with hereditary breast cancer.

Autosomal dominant Charcot-Marie-Tooth type-1A neuropathy (CMT1A) is a demyelinating peripheral nerve disorder that is commonly associated with a submicroscopic tandem DNA duplication of a 1.5-Mb region of 17p11.2p12 that contains the peripheral myelin genePMP22. Clinical features of CMT1A include progressive distal muscle atrophy and weakness, foot and hand deformities, gait abnormalities, absent reflexes, and the completely penetrant electrophysiologic phenotype of symmetric reductions in motor nerve conduction velocities (NCVs). Molecular and fluorescence in situ hybridization (FISH) analyses were performed to determine the duplication status of thePMP22 gene in four patients with rare cytogenetic duplications of 17p. Neuropathologic features of CMT1A were seen in two of these four patients, in addition to the complex phenotype associated with 17p partial trisomy. Our findings show that the CMT1A phenotype of reduced NCV is specifically associated withPMP22 gene duplication, thus providing further support for thePMP22 gene dosage mechanism for CMT1A.

The myelin protein zero gene (MPZ) maps to chromosome 1q22-q23 and encodes the most abundant peripheral nerve myelin protein. The Po protein functions as a homophilic adhesion molecule in myelin compaction. Mutations in the MPZ gene are associated with the demyelinating peripheral neuropathies Charcot-Marie-Tooth disease type 1B (CMT1B), and the more severe Dejerine-Sottas syndrome (DSS). We have surveyed a cohort of 70 unrelated patients with demyelinating polyneuropathy for additional mutations in the MPZ gene. The 1.5-Mb DNA duplication on chromosome 17p11.2-p12 associated with CMT type 1A (CMT1A) was not present. By DNA heteroduplex analysis, four base mismatches were detected in three exons of MPZ. Nucleotide sequence analysis identified a de novo mutation in MPZ exon 3 that predicts an Ile(135)Thr substitution in a family with clinically severe early-onset CMT1, and an exon 3 mutation encoding a Gly(137)Ser substitution was identified in a second CMT1 family. Each predicted amino acid substitution resides in the extracellular domain of the Po protein. Heteroduplex analysis did not detect either base change in 104 unrelated controls, indicating that these substitutions are disease-associated mutations rather than common polymorphisms. In addition, two polymorphic mutations were identified in MPZ exon 5 and exon 6, which do not alter the codons for Gly(200) and Ser(228), respectively. These observations provide further confirmation of the role of MPZ in CMT1B and suggest that MPZ coding region mutations may account for a limited percentage of disease-causing mutations in nonduplication CMT1 patients. © 1996 Wiley-Liss, Inc.