Lorraine Toji

Characterization of 107 Genomic DNA Reference Materials for CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1: A GeT-RM and Association for Molecular Pathology Collaborative Project

Pharmacogenetic testing is becoming more common; however, very few quality control and other reference materials that cover alleles commonly included in such assays are currently available. To address these needs, the Centers for Disease Control and Preventions Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, have characterized a panel of 107 genomic DNA reference materials for five loci (CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1) that are commonly included in pharmacogenetic testing panels and proficiency testing surveys. Genomic DNA from publicly available cell lines was sent to volunteer laboratories for genotyping. Each sample was tested in three to six laboratories using a variety of commercially available or laboratory-developed platforms. The results were consistent among laboratories, with differences in allele assignments largely related to the manufacturers assay design and variable nomenclature, especially for CYP2D6. The alleles included in the assay platforms varied, but most were identified in the set of 107 DNA samples. Nine additional pharmacogenetic loci (CYP4F2, EPHX1, ABCB1, HLAB, KIF6, CYP3A4, CYP3A5, TPMT, and DPD) were also tested. These samples are publicly available from Coriell and will be useful for quality assurance, proficiency testing, test development, and research.

Pharmacogenetic allele nomenclature: International workgroup recommendations for test result reporting

This article provides nomenclature recommendations developed by an international workgroup to increase transparency and standardization of pharmacogenetic (PGx) result reporting. Presently, sequence variants identified by PGx tests are described using different nomenclature systems. In addition, PGx analysis may detect different sets of variants for each gene, which can affect interpretation of results. This practice has caused confusion and may thereby impede the adoption of clinical PGx testing. Standardization is critical to move PGx forward.

Development of Genomic Reference Materials for Cystic Fibrosis Genetic Testing

The number of different laboratories that perform genetic testing for cystic fibrosis is increasing. However, there are a limited number of quality control and other reference materials available, none of which cover all of the alleles included in commercially available reagents or platforms. The alleles in many publicly available cell lines that could serve as reference materials have neither been confirmed nor characterized. The Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the genetic testing community as well as Coriell Cell Repositories, have characterized an extended panel of publicly available genomic DNA samples that could serve as reference materials for cystic fibrosis testing. Six cell lines [containing the following mutations: E60X (c.178G>T), 444delA (c.312delA), G178R (c.532G>C), 1812-1G>A (c.1680-1G>A), P574H (c.1721C>A), Y1092X (c.3277C>A), and M1101K (c.3302T>A)] were selected from those existing at Coriell, and seven [containing the following mutations: R75X (c.223C>T), R347H (c.1040G>A), 3876delA (c.3744delA), S549R (c.1646A>C), S549N (c.1647G>A), 3905insT (c.3773_3774insT), and I507V (c.1519A>G)] were created. The alleles in these materials were confirmed by testing in six different volunteer laboratories. These genomic DNA reference materials will be useful for quality assurance, proficiency testing, test development, and research and should help to assure the accuracy of cystic fibrosis genetic testing in the future. The reference materials described in this study are all currently available from Coriell Cell Repositories.

Quality assurance for Duchenne and Becker muscular dystrophy genetic testing: development of a genomic DNA reference material panel.

Duchenne and Becker muscular dystrophies (DMD/BMD) are allelic X-linked recessive disorders that affect approximately 1 in 3500 and 1 in 20,000 male individuals, respectively. Approximately 65% of patients with DMD have deletions, 7% to 10% have duplications, and 25% to 30% have point mutations in one or more of the 79 exons of the dystrophin gene. Most clinical genetics laboratories test for deletions, and some use technologies that can detect smaller mutations and duplications. Reference and quality control materials for DMD/BMD diagnostic and carrier genetic testing are not commercially available. To help address this need, the Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the genetic testing and the DMD/BMD patient communities and the Coriell Cell Repositories, have characterized new and existing cell lines to create a comprehensive DMD/BMD reference material panel. Samples from 31 Coriell DMD cell lines from male probands and female carriers were analyzed using the Affymetrix SNP Array 6.0 and Multiplex Ligation-Dependent Probe Amplification (MRC-Holland BV, Amsterdam, the Netherlands), a multiplex PCR assay, and DNA sequence analysis. Identified were 16 cell lines with deletions, 9 with duplications, and 4 with point mutations distributed throughout the dystrophin gene. There were no discordant results within assay limitations. These samples are publicly available from Coriell Institute for Medical Research (Camden, NJ) and can be used for quality assurance, proficiency testing, test development, and research, and should help improve the accuracy of DMD testing.

Development of genomic DNA reference materials for genetic testing of disorders common in people of ashkenazi jewish descent.

Many recessive genetic disorders are found at a higher incidence in people of Ashkenazi Jewish (AJ) descent than in the general population. The American College of Medical Genetics and the American College of Obstetricians and Gynecologists have recommended that individuals of AJ descent undergo carrier screening for Tay Sachs disease, Canavan disease, familial dysautonomia, mucolipidosis IV, Niemann-Pick disease type A, Fanconi anemia type C, Bloom syndrome, and Gaucher disease. Although these recommendations have led to increased test volumes and number of laboratories offering AJ screening, well-characterized genomic reference materials are not publicly available. The Centers for Disease Control and Prevention-based Genetic Testing Reference Materials Coordination Program, in collaboration with members of the genetic testing community and Coriell Cell Repositories, have developed a panel of characterized genomic reference materials for AJ genetic testing. DNA from 31 cell lines, representing many of the common alleles for Tay Sachs disease, Canavan disease, familial dysautonomia, mucolipidosis IV, Niemann-Pick disease type A, Fanconi anemia type C, Bloom syndrome, Gaucher disease, and glycogen storage disease, was prepared by the Repository and tested in six clinical laboratories using three different PCR-based assay platforms. A total of 33 disease alleles was assayed and 25 different alleles were identified. These characterized materials are publicly available from Coriell and may be used for quality control, proficiency testing, test development, and research.

Development and Characterization of Reference Materials for MTHFR, SERPINA1, RET, BRCA1, and BRCA2 Genetic Testing

Well-characterized reference materials (RMs) are integral in maintaining clinical laboratory quality assurance for genetic testing. These RMs can be used for quality control, monitoring of test performance, test validation, and proficiency testing of DNA-based genetic tests. To address the need for such materials, the Centers for Disease Control and Prevention established the Genetic Testing Reference Material Coordination Program (GeT-RM), which works with the genetics community to improve public availability of characterized RMs for genetic testing. To date, the GeT-RM program has coordinated the characterization of publicly available genomic DNA RMs for a number of disorders, including cystic fibrosis, Huntington disease, fragile X, and several genetic conditions with relatively high prevalence in the Ashkenazi Jewish population. Genotypic information about a number of other cell lines has been collected and is also available. The present study includes the development and commutability/genotype characterization of 10 DNA samples for clinically relevant mutations or sequence variants in the following genes: MTHFR; SERPINA1; RET; BRCA1; and BRCA2. DNA samples were analyzed by 19 clinical genetic laboratories using a variety of assays and technology platforms. Concordance was 100% for all samples, with no differences observed between laboratories using different methods. All DNA samples are available from Coriell Cell Repositories and characterization information can be found on the GeT-RM website.

Development of a Genomic DNA Reference Material Panel for Rett Syndrome (MECP2-Related Disorders) Genetic Testing

Rett syndrome is a dominant X-linked disorder caused by point mutations (approximately 80%) or by deletions or insertions (approximately 15% to 18%) in the MECP2 gene. It is most common in females but lethal in males, with a distinctly different phenotype. Rett syndrome patients have severe neurological and behavioral problems. Clinical genetic testing laboratories commonly use characterized genomic DNA reference materials to assure the quality of the testing process; however, none are commercially available for MECP2 genetic testing. The Centers for Disease Control and Preventions Genetic Testing Reference Material Coordination Program, in collaboration with the genetic testing community and the Coriell Cell Repositories, established 27 new cell lines and characterized the MECP2 mutations in these and in 8 previously available cell lines. DNA samples from the 35 cell lines were tested by eight clinical genetic testing laboratories using DNA sequence analysis and methods to assess copy number (multiplex ligation-dependent probe amplification, semiquantitative PCR, or array-based comparative genomic hybridization). The eight common point mutations known to cause approximately 60% of Rett syndrome cases were identified, as were other MECP2 variants, including deletions, duplications, and frame shift and splice-site mutations. Two of the 35 samples were from males with MECP2 duplications. These MECP2 and other characterized genomic DNA samples are publicly available from the NIGMS Repository at the Coriell Cell Repositories.

A Dynamic Database of Microarray-Characterized Cell Lines with Various Cytogenetic and Genomic Backgrounds

The Human Genetic Cell Repository sponsored by the National Institute of General Medical Sciences (NIGMS) contains more than 11,000 cell lines and DNA samples collected from numerous individuals. All of these cell lines and DNA samples are categorized into several collections representing a variety of disease states, chromosomal abnormalities, heritable diseases, distinct human populations, and apparently healthy individuals. Many of these cell lines have previously been studied with detailed conventional cytogenetic analyses, including G-banded karyotyping and fluorescence in situ hybridization. This work was conducted by investigators at submitting institutions and scientists at Coriell Institute for Medical Research, where the NIGMS Repository is hosted. Recently, approximately 900 cell lines, mostly chosen from the Chromosomal Aberrations and Heritable Diseases collections, have been further characterized in detail at the Coriell Institute using the Affymetrix Genome-Wide Human SNP Array 6.0 to detect copy number variations and copy number neutral loss of heterozygosity. A database containing detailed cytogenetic and genomic information for these cell lines has been constructed and is freely available through several sources, such as the NIGMS Repository website and the University of California at Santa Cruz Genome Browser. As additional cell lines are analyzed and subsequently added into it, the database will be maintained dynamically.

Allelic decomposition and exact genotyping of highly polymorphic and structurally variant genes

High-throughput sequencing provides the means to determine the allelic decomposition for any gene of interest—the number of copies and the exact sequence content of each copy of a gene. Although many clinically and functionally important genes are highly polymorphic and have undergone structural alterations, no high-throughput sequencing data analysis tool has yet been designed to effectively solve the full allelic decomposition problem. Here we introduce a combinatorial optimization framework that successfully resolves this challenging problem, including for genes with structural alterations. We provide an associated computational tool Aldy that performs allelic decomposition of highly polymorphic, multi-copy genes through using whole or targeted genome sequencing data. For a large diverse sequencing data set, Aldy identifies multiple rare and novel alleles for several important pharmacogenes, significantly improving upon the accuracy and utility of current genotyping assays. As more data sets become available, we expect Aldy to become an essential component of genotyping toolkits.Many genes of functional and clinical significance are highly polymorphic and experience structural alterations. Here, Numanagić et al. develop Aldy, a computational tool for resolving the copy number and the sequence content of each copy of a gene by analyzing whole or targeted genome sequencing data.

Brief Communication: Regional Mapping Panels for Human Chromosomes 1, 2, and 7

The NIGMS Human Genetic Cell Repository has assembled regional mapping panels for human chromosomes 1, 2, and 7 from human rodent somatic cell hybrids submitted to the collection by researchers from 14 different laboratories. All hybrids were characterized initially by the submitters and verified by the Repository. Each hybrid carries a stable defined human segment as a derivative or deletion chromosome. These panels define 8–10 intervals for each chromosome. The panel for chromosome 2 is a new resource. The panels for chromosomes 1 and 7 complement previously published panels. The Repository distributes these regional mapping panels as cell cultures or as DNA. Information about these panels as well as for panels for chromosomes 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 21, 22, and X may be viewed in the NIGMS Repository electronic catalog (http://locus.umdnj.edu/ptnigms).