Ambry Genetics Research Articles

Enhanced Detection of Longer Insertions and Deletions in Clinical Exome Sequencing Improves Diagnostic Yield

Whole exome sequencing (WES) has been remarkably successful as both a diagnostic and novel gene discovery tool since its introduction to the clinical laboratory in 2011. Where traditional diagnostic methods have been uninformative in discovering the pathogenic etiology in patients, diagnostic exome sequencing (DES) has provided answers for roughly one-third of patients tested, thus contributing to the management of patients' overall healthcare. Single nucleotide variants are generally efficiently identified by DES in well-covered exonic regions. However, accurate mapping of insertions and deletions, especially those larger than 20 nucleotides, is challenging due to gapped alignment and paired- end sequence inference. We have customized and validated a robust exome analysis pipeline that accurately and efficiently calls insertions or deletions ranging from 20 to 200 base pairs from next generation sequencing data and contributes to one of the highest diagnostic yields reported for clinical exome analysis. Out of 284 positive/likely positive cases in the first 1000 unselected DES cases referred to Ambry Genetics, causative mutations in 9 (3.2%) were associated with insertions, deletions or indels between 20 and 200 bp in length. Our data highlight the importance of an optimized clinical exome workflow for the detection of longer insertions and deletions to improve clinical sensitivity and diagnostic yield.

Abstract P4-12-08: Utilizing next generation sequencing technologies for hereditary breast cancer risk assessments in a private oncology practice

Abstract Background: The recent introduction of clinical Next Generation Sequencing (NGS) technologies has transformed the ability of health care providers to provide personalized hereditary breast cancer risk assessments to patients and their family members. However, along with the plethora of data derived from NGS technologies come challenges regarding how to interpret this data in a clinically meaning way to guide medical management decisions. Methods: A retrospective analysis was performed on a population of 242 patients with a personal or family history of breast and/or ovarian cancer and completed NGS genetic testing between March 2012 and May 2014 at Ambry Genetics. Data points included age, gender, primary diagnosis, type of NGS genetic test and result. Patients with prior genetic testing were not excluded from the analysis. Results: Of the 242 patients who completed NGS genetic testing 199 (82%) had a personal history of cancer and 43 (18%) had a family history of cancer. Ten (5%) clinically significant deleterious mutations were found in the affected population; 3 MUTYH mutations were consider not clinically significant and excluded from the analysis. Four (40%) were CHEK2 mutations, 2(20%) BRCA1 mutations, 1(10%) NBN mutation, 1(10%) ATM mutation, 1(10%) MSH6 mutation and 1(10%) PTEN mutation. One patient with a history of ovarian cancer had two deleterious mutations in the BRCA1 and NBN genes, and three affected patients had a deleterious mutation in addition to a variant of uncertain significance (VUS); a patient with synchronous breast and ovarian cancers had a MSH6 mutation and a BRCA2 VUS, a patient with breast cancer had a CHEK2 mutation and a CHEK2 VUS, and a patient with breast cancer had a CHEK2 mutation and a MSH6 VUS. A total of 37 (19%) VUS were found in the affected population, including one patient with a history of breast cancer and a VUS in both ATM and MRE11A. A single (2%) clinically significant BRCA1 deleterious mutation and 6(14%) VUS were detected in the unaffected population. Conclusions: NGS technologies allow for health care providers to complete more comprehensive hereditary breast cancer risk assessments for patients and their families. However, the high VUS rate (14-19%) and the possibility for multiple mutations and/or variants in a single patient create unique challenges when interpreting NGS genetic test results. Furthermore, the paucity of prospective studies and consensus guidelines limits the ability of the health care provider to interpret NGS genetic test results in a clinically meaningful way to guide patient management. This is highlighted by the case example above regarding the patient with synchronous breast and ovarian cancers who was found to have a MSH6 mutation and a BRCA2 VUS. Given that the patient does not meet Amsterdam criteria, the MSH6 mutations would have been missed had it not been for the NGS technology utilized for this patient. However, challenges still exist about how to interpret this data for both the patient and her family in a clinically meaningful way given that the family does not look like a classic Lynch Syndrome family. Citation Format: Barbara Hamlington, Scot Sedlacek, Lucy Langer, Brittney Goetsch, Katie Lemas, Sami Diab. Utilizing next generation sequencing technologies for hereditary breast cancer risk assessments in a private oncology practice [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-12-08.

In the Aftermath of the “Myriad Case” – Myriad Is Denied Preliminary Injunction Against Ambry Genetics

On March 10, 2014, the U.S. District Court of Utah, Central Division, decided in University of Utah Research Foundation, et al., v. Ambry Genetics Corporation, holding that “Plaintiffs are not entitled to a preliminary injunction”, as they “are unable to establish that they are likely to succeed on the merits of the claims” nor “that the equitable factors support issuance of the requested injunction”.This case note gives an overview of the U.S. District Court's of Utah memorandum decision and order denying plaintiffs’ motion for preliminary injunction and discusses its implications for the implementation of the criterion of isolation to “synthetic” DNA sequences, such as primers and probes.

Patients and families generally welcome secondary genomic findings

Patients and families generally welcome secondary genomic findings

Clinical exome sequencing leads to the diagnosis of mitochondrial complex I deficiency in a family with global developmental delays, ataxia, and cerebellar and pons hypoplasia

Exome sequencing was performed on a 14 year-old female with familial ataxia, global developmental delays, and cerebellar and pons hypoplasia. The family history was remarkable for a 3 yearold sister with a similar phenotype. Nearly a decade of molecular, cytogenetic, and biochemical testing was uninformative. Exome sequencing revealed compound heterozygous alterations of the NUBPL gene (c.311T>C; p.L104P & c.815-27T>C). The c.311T>C missense alteration is located at a highly conserved amino acid. The c.815-27T>C alteration is located at a highly conserved nucleotide and previous in vitro analyses demonstrated splicing defects. The affected sister manifested both alterations; each parent carried one alteration. Alterations within the NUBPL gene occur in an autosomal recessive fashion in association with mitochondrial complex I deficiency syndrome (CI deficiency) (MIM_252010). The NUBPL gene was first discovered in association with disease in 2010 and has only been reported in two other families, both of which displayed remarkable clinical overlap with the family herein. Exome sequencing is an especially powerful tool to aid in the diagnosis of CI deficiency given the extreme clinical and genetic heterogeneity making establishing a clinical diagnosis exceedingly difficult. Further, the underlying mutation has not been discovered in about half of patients with CI deficiency, thought to be due to yet undiscovered associated genes. Diagnostic exome sequencing led to the successful identification of the NUBPL alterations and, after years of unsuccessful analyses, led to a molecular diagnosis for the family. METHODS  Genomic deoxyribonucleic acid (gDNA) was isolated from whole blood from the patients and sister. Samples were prepared using the SureSelect Target Enrichment System (Agilent Technologies, Santa Clara, CA). The enriched exome libraries were sequenced using paired-end, 100-cycle chemistry on the Illumina HiSeq 2000 (Illumina, San Diego, CA).  The Human Gene Mutation Database (HGMD; Stenson, 2009), OMIM, and several other databases were used to search for previously described gene mutations and polymorphisms. A molecular geneticist performed interpretive filtering based on the deleterious nature of the candidate alterations literature search and analysis of the relevance of the candidate genes’ function in relation to the patient’s phenotype.  Each candidate variant was analyzed by Sanger sequencing for mutation confirmation and co-segregation studies were performed for the family. Figure 2. c. 815-27C>T is a common branch point mutation Table 1: Variant Filtering Based on Inheritance Model & Interpretation P. Gray1, W. Zeng1, S. Tang1, J.J. Wei1, K. Gonzalez1, X. Li1, HM Lu1, H. Lu1, E.C. Chao1,2, V. Kimonis2 1) Ambry Genetics, Aliso Viejo, CA 2) Department of Pediatrics, University of California, Irvine, CA

Patient decisions for disclosure of secondary findings among the first 200 individuals undergoing clinical diagnostic exome sequencing

Purpose:Exome sequencing of a single individual for a clinical indication may result in the identification of incidental deleterious variants unrelated to the indication for testing (secondary findings). Given the recent availability of clinical exome testing, there is a limited knowledge regarding the disclosure preferences and impact of secondary findings in a clinical diagnostic setting. In this article, we provide preliminary data regarding the preferences for secondary findings results disclosure based on the first 200 families referred to Ambry Genetics for diagnostic exome sequencing.Methods:Secondary findings were categorized into four groups in the diagnostic exome sequencing consent: carrier status of recessive disorders, predisposition to later-onset disease, predisposition to increased cancer risk, and early-onset disease. In this study, we performed a retrospective analysis of patient responses regarding the preferences for secondary findings disclosure.Results:The majority of patients (187/200; 93.5%) chose to receive secondary results for one or more available categories. Adult probands were more likely than children to opt for blinding of secondary data (16 vs. 4%, respectively). Among responses for blinding, preferences were evenly scattered among categories.Conclusion:These data represent the unprecedented results of a large reference laboratory providing clinical exome sequencing. We report, for the first time, the preferences of patients and families for the receipt of secondary findings based on clinical genome sequencing. Overwhelmingly, families undergoing exome sequencing opt for the disclosure of secondary findings. The data may have implications regarding the development of guidelines for secondary findings reporting among patients with severe and/or life-threatening disease undergoing clinical genomic sequencing.

Prometheus bound, but myriad loose ends

Prometheus bound, but myriad loose ends

Myriad Genetics Sues BRCA Diagnostic Rivals

Abstract Myriad Genetics sued Ambry Genetics and Gene by Gene, alleging infringement on patent claims covering synthetic DNA and other methods related to testing BRCA1 and BRCA2 genes to aid in assessing cancer risk.

U.S. supreme court decision paves way for better genetic testing

U.S. supreme court decision paves way for better genetic testing

Pleuropulmonary blastoma: The causative role of germ-line DICER1 mutations.

10024 Background: Pleuropulmonary blastoma (PPB) is a rare, aggressive childhood lung cancer that is often the first manifestation of the PPB-DICER1 familial tumor predisposition which includes other benign and malignant conditions. The initial genetic mutation is inherited by a germ-line loss of function of DICER1 gene which was discovered by Hill et al. It is proposed that loss of DICER1 expression alters miRNA regulation of key regulatory cellular mechanisms which promote tumor growth. PPB can progress from the purely cystic, curable Type I to a high-grade Type III having a dismal prognosis. We sought to determine the frequency of DICER1mutation in the largest cohort of PPB patients to date. Methods: We obtained germ-line DNA from saliva or blood samples from 113 PPB patients collected from 2005-2012. DNA was extracted using the Maxwell 16 Research System (Promega Corporation, Madison WI). Sample quality and quantity was checked using the Nanodrop 2000 (Thermo Scientific, Wilmington, DE). Sequencing was performed using the Sanger sequencer. For a subset of cases we used targeted sequencing services or full gene sequence analysis (Ambry Genetics, Aliso Viejo, CA). Results: Seventy-four (65.5%) PPB patients were found to have deleterious DICER1germ-line mutations. The most common mutation type found was small insertion/deletions. These 74 samples were composed of 7 (9.5%)Type Ir PPBs, 22 (29.7%)Type I PPBs, 24 (32.4%) Type II PPBs, and 21 (28.4%)Type III PPBs. The following Table shows a summary of mutation types identified. Conclusions: Our results confirm that DICER1 germline mutations are the most common genetic alterations in PPB making it critical for genetic testing to be performed at diagnosis. Additionally, this underscores the need for important correlative studies to describe the genotype-phenotype relationship in order for appropriate screening guidelines to be developed and implemented to allow for early detection. While a majority of cases are explained by germline DICER1 mutations using current sequencing methods, further investigation is warranted to elucidate other possible mechanisms in the development of PPB. [Table: see text]

The Tip of the Iceberg

In February 2012, Ambry Genetics, a testing company based in Aliso Viejo, California, began to offer the BreastNext genetic test to women with earlyonset or familial breast cancer [...]

Abstract 2096: Variants of uncertain clinical significance in cancer predisposition genes: Development of a family studies program for reclassification

Abstract The identification of genetic variants of uncertain significance is a fairly common, albeit unwelcomed result of gene sequence analyses. The frequency at which these variants are identified will likely continue to increase with the introduction of next-generation sequencing panels and the recent availability of clinical exome sequencing. When applied to testing for cancer predisposition syndromes, accurate variant assessment and classification helps to guide screening, treatment, family planning, and testing of at-risk family members. Multiple resources are available to assess genetic variants: population-based cohort allele frequencies, published literature, in silico prediction tools, gene-specific databases, evolutionary conservation data, and co-segregation studies. Ambry Genetics, a CAP-accredited and CLIA-certified commercial clinical laboratory providing genetic services focused on clinical diagnostics and genomic services, has developed and implemented a complementary family studies program to collect co-segregation data for variants of unknown significance identified through diagnostic testing. The Ambry family studies program is available to all specialties, but is especially active for genes associated with cancer predisposition and intellectual disability. Data collected from the cancer family studies program is combined with published literature, database information, and data compiled with Ambry Variant Analyzer (AVA©), a novel bioinformatics tool, to allow for a comprehensive interpretation of all variants of uncertain clinical significance in cancer predisposition genes. In less than one year, the program has grown to 60 active cancer family studies at the time of submission and a current rate of >5 new applications per week. Variants successfully reclassified due to family studies data include the p.Y327D missense alteration in MEN1 and in-frame deletions in both TP53 and MLH1. Variant reclassification leads to the automatic generation of amended reports for any individual previously identified by our laboratory as a carrier. Immediately distributed to health care professionals, these reports offer the most up-to-date clinical interpretation possible improving medical decision making and clarifying risk of disease. Here we provide an overview of the family studies program and discuss successful variant reclassification via the Ambry model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2096. doi:1538-7445.AM2012-2096