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To develop a novel pharmacogenetic genotyping panel, a multidisciplinary team evaluated available evidence and selected 29 genes implicated in interindividual drug response variability, including 130 sequence variants and additional copy number variants (CNVs). Of the 29 genes, 11 had guidelines published by the Clinical Pharmacogenetics Implementation Consortium. Targeted genotyping and CNV interrogation were accomplished by multiplex single‐base extension using the MassARRAY platform (Agena Biosciences) and multiplex ligation‐dependent probe amplification (MRC Holland), respectively. Analytical validation of the panel was accomplished by a strategic combination of > 500 independent tests performed on 170 unique reference material DNA samples, which included sequence variant and CNV accuracy, reproducibility, and specimen (blood, saliva, and buccal swab) controls. Among the accuracy controls were 32 samples from the 1000 Genomes Project that were selected based on their enrichment of sequence variants included in the pharmacogenetic panel (VarCover.org). Coupled with publicly available samples from the Genetic Testing Reference Materials Coordination Program (GeT‐RM), accuracy validation material was available for the majority (77%) of interrogated sequence variants (100% with average allele frequencies > 0.1%), as well as additional structural alleles with unique copy number signatures (e.g., CYP2D6*5, *13, *36, *68; CYP2B6*29; and CYP2C19*36). Accuracy and reproducibility for both genotyping and copy number were > 99.9%, indicating that the optimized panel platforms were precise and robust. Importantly, multi‐ethnic allele frequencies of the interrogated variants indicate that the vast majority of the general population carries at least one of these clinically relevant pharmacogenetic variants, supporting the implementation of this panel for pharmacogenetic research and/or clinical implementation programs.

// Eirwen M. Miller 2 , Nicole E. Patterson 1 , Jenna Marcus Zechmeister 2 , Michal Bejerano-Sagie 1 , Maria Delio 1 , Kunjan Patel 1 , Nivedita Ravi 1 , Wilber Quispe-Tintaya 1 , Alexander Maslov 1 , Nichelle Simmons 1 , Maria Castaldi 1 , Jan Vijg 1 , Rouzan G. Karabakhtsian 3 , John M. Greally 1 , Dennis Y.S. Kuo 2 and Cristina Montagna 1 1 Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA 2 Department of Obstetrics & Gynecology and Women’s Health, Division of Gynecologic Oncology, Montefiore Medical Center, Bronx, NY 10461, USA 3 Department of Pathology, Montefiore Medical Center, Bronx, NY 10461, USA Correspondence to: Cristina Montagna, email: cristina.montagna@einstein.yu.edu Keywords: target sequencing; next generation sequencing; endometrial carcinoma; tumor recurrence Received: July 29, 2017 Accepted: September 08, 2017 Published: October 26, 2017 ABSTRACT Next generation sequencing (NGS) technologies have revolutionized our approach to genomic research. The use of whole genome sequencing (WGS), whole exome sequencing (WES), transcriptome profiling, and targeted DNA sequencing has exponentially improved our understanding of the human genome and the genetic complexities underlying malignancy. Yet, WGS and WES clinical applications remain limited due to high costs and the large volume of data generated. When utilized to address biological questions in basic science studies, targeted sequencing panels have proven extremely valuable due to reduced costs and higher sequencing depth. However, the routine application of targeted sequencing to the clinical setting is limited to a few cancer subtypes. Some highly aggressive tumor types, like type 2 endometrial cancer (EC), could greatly benefit from routine genomic analysis using targeted sequencing. To explore the potential utility of a mid size panel (~150 genes) in the clinical setting, we developed and validated a custom panel against WGS, WES, and another commercially available targeted panel. Our results indicate that a mid size custom designed panel is as efficient as WGS and WES in mapping variants of biological and clinical relevance, rendering higher coverage, at a lower cost, with fewer variants of uncertain significance. Because of the much higher sequencing depth that could be achieved, our results demonstrate that targeted sequencing outperformed WGS and WES in the mapping of pathogenic variants in a breast cancer case, as well as a case of mixed serous and high-grade endometrioid EC, the most aggressive EC subtype.

The Department of Genetics in collaboration with a group of Montefiore's clinicians initiated the development of clinical sequencing tests with the final goal to incrementally build clinical sequencing assays to improve clinical care in the Bronx. The use of next generation sequencing is particularly relevant for cancer biology since the heterogeneity of cancer requires deep sequencing and high read depth, which prior to recent advances in technology was expensive and labor intensive. We designed a custom gene panel for target sequencing using the Illumina HiSeq 2500 consisting of 650 genes for multiple disorders and diseases, including various cancers, in addition to rare and complex mendelian disorders. We sequenced 17 samples, including a tumor-normal breast cancer pair, where we identified a novel, pathogenic stop variant in the highly mutated TP53 tumor suppressor gene. In addition to our custom target panel, we sequenced this sample pair on two additional cancer panels (AmpliSeq Cancer Hotspot and the Comprehensive Cancer Panel using the Ion Torrent Technology) and performed Whole Exome Sequencing (WES) to ensure the sensitivity and specificity of our custom panel. This variant was detected on all platforms. We found targeted capture high-throughput sequencing to be a cost effective, time sensitive and efficient approach in detecting pathogenic variants to aid diagnosis of individuals affected by cancer or mendelian disorders. We are currently receiving samples to sequence and plan to expand our program to various groups within the Einstein-Montefiore community. Using a personalized medicine approach we expect to detect biomarkers and prospective targets to develop an accurate and effective treatment plan for these individuals. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-12-09.

Velo-cardio-facial syndrome/DiGeorge syndrome/22q11.2 deletion syndrome (22q11.2DS) is caused by meiotic non-allelic homologous recombination events between flanking low copy repeats termed LCR22A and LCR22D, resulting in a 3 million base pair (Mb) deletion. Due to their complex structure, large size and high sequence identity, genetic variation within LCR22s among different individuals has not been well characterized. In this study, we sequenced 13 BAC clones derived from LCR22A/D and aligned them with 15 previously available BAC sequences to create a new genetic variation map. The thousands of variants identified by this analysis were not uniformly distributed in the two LCR22s. Moreover, shared single nucleotide variants between LCR22A and LCR22D were enriched in the Breakpoint Cluster Region pseudogene (BCRP) block, suggesting the existence of a possible recombination hotspot there. Interestingly, breakpoints for atypical 22q11.2 rearrangements have previously been located to BCRPs. To further explore this finding, we carried out in-depth analyses of whole genome sequence (WGS) data from two unrelated probands harbouring a de novo 3Mb 22q11.2 deletion and their normal parents. By focusing primarily on WGS reads uniquely mapped to LCR22A, using the variation map from our BAC analysis to help resolve allele ambiguity, and by performing PCR analysis, we infer that the deletion breakpoints were most likely located near or within the BCRP module. In summary, we found a high degree of sequence variation in LCR22A and LCR22D and a potential recombination breakpoint near or within the BCRP block, providing a starting point for future breakpoint mapping using additional trios.

Primary hepatic small cell carcinoma (HSCC) is a rare malignancy that has previously been described in only few case reports. The clinicopathological course, natural history, molecular markers and ideal treatment strategy for this tumor have not been fully elucidated. Herein, we report on two cases of spontaneously arising, metastatic primary HSCC that were treated at our Institution. Both patients succumbed to their disease within two months of initial presentation. Both cases underwent postmortem examination and no evidence of a pulmonary or other non-hepatic small cell primary was found. Unlike pulmonary small cell tumors, these two hepatic primaries showed only locoregional spread and very few distant metastases. Formalin-fixed samples were obtained at autopsy and sequenced using single-nucleotide polymorphism arrays and whole-genome sequencing. Four mutations in the epidermal growth factor receptor (EGFR) gene known to be associated with response to tyrosine kinase inhibitors (TKIs) were detected in one of the two HSCC samples. A systematic review and pooled analysis of all previously reported cases of primary HSCCs was conducted. The median overall survival was estimated at 4 months. Surgical resection was significantly associated with longer overall survival (hazard ratio =0.13, 95% confidence interval=0.03-0.69). Although several case reports of primary HSCC have been reported prior to this publication, to our knowledge this is the first time that molecular and systematic analysis has been conducted in order to more fully characterize this rare disease. Our results indicate that surgical resection, when feasible, may be a valid option in primary HSCC, and that some tumors may respond to TKIs against EGFR.

Hearing loss is a complex disorder caused by both genetic and environmental factors. Previously, mutations in CIB2 have been identified as a common cause of genetic hearing loss in Pakistani and Turkish populations. Here we report a novel (c.556CT; p.(Arg186Trp)) transition mutation in the CIB2 gene identified through whole exome sequencing (WES) in a Caribbean Hispanic family with non-syndromic hearing loss. CIB2 belongs to the family of calcium-and integrin-binding (CIB) proteins. The carboxy-termini of CIB proteins are associated with calcium binding and intracellular signaling. The p.(Arg186Trp) mutation is localized within predicted type II PDZ binding ligand at the carboxy terminus. Our ex vivo studies revealed that the mutation did not alter the interactions of CIB2 with Whirlin, nor its targeting to the tips of hair cell stereocilia. However, we found that the mutation disrupts inhibition of ATP-induced Ca2+ responses by CIB2 in a heterologous expression system. Our findings support p.(Arg186Trp) mutation as a cause for hearing loss in this Hispanic family. In addition, it further highlights the necessity of the calcium binding property of CIB2 for normal hearing.

We explored potential genetic risk factors implicated in nonalcoholic fatty liver disease (NAFLD) within a Caribbean–Hispanic population in New York City. A total of 316 individuals including 40 subjects with biopsy‐proven NAFLD, 24 ethnically matched non‐NAFLD controls, and a 252 ethnically mixed random sampling of Bronx County, New York were analyzed. Genotype analysis was performed to determine allelic frequencies of 74 known single‐nucleotide polymorphisms (SNPs) associated with NAFLD risk based on previous genome‐wide association study (GWAS) and candidate gene studies. Additionally, the entire coding region of PNPLA3, a gene showing the strongest association to NAFLD was subjected to Sanger sequencing. Results suggest that both rare and common DNA variations in PNPLA3 and SAMM50 may be correlated with NAFLD in this small population study, while common DNA variations in CHUK and ERLIN1, may have a protective interaction. Common SNPs in ENPP1 and ABCC2 have suggestive association with fatty liver, but with less compelling significance. In conclusion, Hispanic patients of Caribbean ancestry may have different interactions with NAFLD genetic modifiers; therefore, further investigation with a larger sample size, into this Caribbean–Hispanic population is warranted.

Background While next generation sequencing (NGS) is a useful tool for the identification of genetic variants to aid diagnosis and support therapy decision, high sequencing costs have limited its application within routine clinical care, especially in economically depressed areas. To investigate the utility of a multi-disease NGS based genetic test, we designed a custom sequencing assay targeting over thirty disease-associated areas including cardiac disorders, intellectual disabilities, hearing loss, collagenopathies, muscular dystrophy, Ashkenazi Jewish genetic disorders, and complex Mendelian disorders. We focused on these specific areas based on the interest of our collaborative clinical team, suggesting these diseases being the ones in need for the development of a sequencing-screening assay. Results We targeted all coding, untranslated regions (UTR) and flanking intronic regions of 650 known disease-associated genes using the Roche-NimbleGen EZ SeqCapV3 capture system and sequenced on the Illumina HiSeq 2500 Rapid Run platform. Eight controls with known variants and one HapMap sample were first sequenced to assess the performance of the panel. Subsequently, as a proof of principle and to explore the possible utility of our test, we analyzed test disease subjects (n = 16). Eight had known Mendelian disorders and eight had complex pediatric diseases. In addition to assess whether copy number variation may be of utility as a companion assay relative to these specific disease areas, we used the Affymetrix Genome-Wide SNP Array 6.0 to analyze the same samples. Conclusion We identified potentially disease-associated variants: 22 missense, 4 nonsense, 1 frameshift, and 1 splice variants (16 previously identified, 12 novel among dbSNP and 15 novel among NHLBI Exome Variant Server). We found multi-disease targeted high-throughput sequencing to be a cost efficient approach in detecting disease-associated variants to aid diagnosis.

Hearing loss is a complex disorder caused by both genetic and environmental factors. Previously, mutations in CIB2 have been identified as a common cause of genetic hearing loss in Pakistani and Turkish populations. Here we report a novel (c.556C>T; p.(Arg186Trp)) transition mutation in the CIB2 gene identified through whole exome sequencing (WES) in a Caribbean Hispanic family with non-syndromic hearing loss. CIB2 belongs to the family of calcium-and integrin-binding (CIB) proteins. The carboxy-termini of CIB proteins are associated with calcium binding and intracellular signaling. The p.(Arg186Trp) mutation is localized within predicted type II PDZ binding ligand at the carboxy terminus. Our ex vivo studies revealed that the mutation did not alter the interactions of CIB2 with Whirlin, nor its targeting to the tips of hair cell stereocilia. However, we found that the mutation disrupts inhibition of ATP-induced Ca2+ responses by CIB2 in a heterologous expression system. Our findings support p.(Arg186Trp) mutation as a cause for hearing loss in this Hispanic family. In addition, it further highlights the necessity of the calcium binding property of CIB2 for normal hearing.

Velocardiofacial and DiGeorge syndromes, also known as 22q11.2 deletion syndrome (22q11DS), are congenital-anomaly disorders caused by a de novo hemizygous 22q11.2 deletion mediated by meiotic nonallelic homologous recombination events between low-copy repeats, also known as segmental duplications. Although previous studies exist, each was of small size, and it remains to be determined whether there are parent-of-origin biases for the de novo 22q11.2 deletion. To address this question, we genotyped a total of 389 DNA samples from 22q11DS-affected families. A total of 219 (56%) individuals with 22q11DS had maternal origin and 170 (44%) had paternal origin of the de novo deletion, which represents a statistically significant bias for maternal origin (p = 0.0151). Combined with many smaller, previous studies, 465 (57%) individuals had maternal origin and 345 (43%) had paternal origin, amounting to a ratio of 1.35 or a 35% increase in maternal compared to paternal origin (p = 0.000028). Among 1,892 probands with the de novo 22q11.2 deletion, the average maternal age at time of conception was 29.5, and this is similar to data for the general population in individual countries. Of interest, the female recombination rate in the 22q11.2 region was about 1.6-1.7 times greater than that for males, suggesting that for this region in the genome, enhanced meiotic recombination rates, as well as other as-of-yet undefined 22q11.2-specific features, could be responsible for the observed excess in maternal origin.

Haploinsufficiency of the elastin gene (ELN) on 7q11.23 is responsible for supravalvular aortic stenosis (SVAS) and other arteriopathies in patients with Williams–Beuren syndrome (WBS). These defects occur with variable penetrance and expressivity, but the basis of this is unknown. To determine whether DNA variations in ELN could serve as genetic modifiers, we sequenced the 33 exons and immediately surrounding sequence of the ELN gene (9,455 bp of sequence) in 49 DNAs from patients with WBS and compared cardiovascular phenotypes. Four missense, and four novel intronic variants were identified from a total of 24 mostly intronic single nucleotide variations and one indel. Two missense changes were present in one patient each, one published, p.Gly610Ser in exon 27 (MAF, 0.003) and one novel, p.Cys714Tyr, in exon 33 (MAF, 0.001), were rare in the general population. To identify a statistical association between the variants identified here and cardiovascular phenotypes a larger cohort would be needed. � 2013 Wiley Periodicals, Inc.

Background Prenatal carrier screening has traditionally focused on well-defined ethnic groups with an increased chance of being a carrier for specific genetic diseases. However, many people are of mixed ethnicity or cannot accurately identify their full ethnic. With the ability to test for hundreds of diseases at one time, expanded carrier screening panels are an attractive option. Objectives To build a comprehensive pan-ethnic carrier screening panel with a rapid turnaround time (TAT). Design/method Following literature review, internal research, and physician input, a panel of 281 autosomal recessive and X-linked diseases were selected. Next generation sequencing (NGS) was performed using Agilent’s SureSelect QXT target enrichment approach followed by Illumina HiSeq 2500 Rapid Run mode sequencing. Additional methodologies include MLPA, Asuragen CGG repeat analysis/Southern blotting, and Sequenom/Luminex genotyping assays to assay variants not amenable to NGS. Variant confirmation is performed simultaneously by genotyping, or subsequently by Sanger sequencing. Over 3,700 recurrent pathogenic variants with a well-established relationship to disease phenotype are guaranteed to be sequenced at a depth of > 20X. Results 524 validation samples were tested, including positive controls with various mutation types, and both prenatal and postnatal samples covering a variety of specimen types. A minimum average coverage of 240X was achieved, with > 20X coverage of 99% of bases. Conclusions This panel can be customizable to order, including a high-frequency panel of 10 diseases, a Comprehensive Jewish panel of 96 diseases, and the full 281 gene panel. Automation can handle thousands of samples per month. TAT currently averages 8–10 days.

Background Hearing and/or vision loss can result from both genetic and non-genetic etiologies. Up to 70% of prelingual hearing loss has a genetic basis, as does up to 60% of congenital blindness among infants. Next generation sequencing (NGS) technology is ideal for diagnostic testing for such disorders due to extreme locus heterogeneity and phenotypic overlap. Objectives The aim is to design and validate a comprehensive, customizable NGS panel for detection of pathogenic variants in genes involved in hearing and/or vision loss. Design/method 380 genes were selected for inclusion based on literature review and comparison with commercially available assays, including 245 genes for vision loss and 83 genes for hearing loss. Agilent SureSelect biotinylated RNA baits were designed to capture 1.5 Mb of DNA using SureSelect XT target enrichment followed by Illumina HiSeq 2500 sequencing. DNA from six HapMap cell lines and five Coriell repository samples with known pathogenic variants were used to assess intra-run and inter-run variability. Additionally, DNA from a family with a known hearing loss aetiology was included. Results An average yield of 6 Gb of sequence per sample was obtained, where 94% of bases were covered at a depth >20X. Genotype calls from HapMap samples were highly concordant across three runs. Eight known pathogenic variants were confirmed in GJB2, USH1C, MARVELD2, TRIOBP, and OAT. Conclusions This NGS panel targets an extensive list of genes implicated in diseases of the ear and the eye. Customizable testing allows ordering of an ear-specific or eye-specific gene panel, or both.