© American College of Medical Genetics and Genomics ORIGINAL RESEARCH ARTICLE Clinical relevance of small copy-number variants in chromosomal microarray clinical testing Dana Hollenbeck, MPH, MS1, Crescenda L. Williams, MS1,2, Kathryn Drazba, MPH, MS1, Maria Descartes, MD1,3, Bruce R. Korf, MD, PhD1,3, S. Lane Rutledge, MD1,3, Edward J. Lose, MD1,3, Nathaniel H. Robin, MD1,3, Andrew J. Carroll, PhD1 and Fady M. Mikhail, MD, PhD1 Purpose: The 2010 consensus statement on diagnostic chromosomal (1.02%) had two or more small VUS CNVs, 20 (0.45%) had one small microarray (CMA) testing recommended an array resolution ≥400 kb VUS CNV and a recurrent CNV, 113 (2.56%) had one small patho- throughout the genome as a balance of analytical and clinical sensi- genic or likely pathogenic CNV, 17 (0.38%) had two or more small tivity. In spite of the clear evidence for pathogenicity of large copy- pathogenic or likely pathogenic CNVs, and 12 (0.27%) had one small number variants (CNVs) in neurodevelopmental disorders and/or pathogenic or likely pathogenic CNV and a recurrent CNV. Within congenital anomalies, the significance of small, nonrecurrent CNVs the pathogenic group, 80 of 142 patients (56% of all small pathogenic (<500 kb) has not been well established in a clinical setting. CNV cases) were found to have a single whole-gene or exonic dele- tion. The themes that emerged from our study are presented in the Methods: We investigated the clinical significance of all nonpoly- Discussion section. morphic small, nonrecurrent CNVs (<500 kb) in patients referred for CMA clinical testing over a period of 6 years, from 2009 to 2014 (a Conclusions: Our study demonstrates the diagnostic clinical rel- total of 4,417 patients). We excluded from our study patients with evance of small, nonrecurrent CNVs <500 kb during CMA clinical benign or likely benign CNVs and patients with only recurrent testing and underscores the need for careful clinical interpretation microdeletions/microduplications <500 kb. of these CNVs. Results: In total, 383 patients (8.67%) were found to carry at least Genet Med advance online publication 15 September 2016 one small, nonrecurrent CNV, of whom 176 patients (3.98%) had one Key Words: CMA clinical testing; intragenic deletion; single-gene small CNV classified as a variant of uncertain significance (VUS), 45 deletion; small CNV INTRODUCTION yield of the test. Higher resolution platforms, however, also Cytogenetic testing has long been used to identify the loss lead to the increased detection of CNVs with uncertain or gain of genetic material. High-resolution chromosomal clinical significance.2,4 Interpretation of these rare CNVs is microarray (CMA) has emerged as the gold standard for iden- further complicated by the presence of CNVs in healthy indi- tifying small losses or gains of genomic material, also called viduals (benign CNVs).5 In an effort to unify clinical inter- copy-number variants (CNVs), which could be implicated in pretation, the 2010 consensus statement on diagnostic CMA the pathogenesis of developmental delay, intellectual disabil- testing was released, which recommended an array resolu- ity, autism spectrum disorders, and/or congenital anomalies.1 tion ≥400 kb throughout the genome as a balance of analyti- In 2010 CMA was designated as a first-tier clinical diagnostic cal and clinical sensitivity; however, the American College test for individuals with neurodevelopmental disorders and of Medical Genetics and Genomics standards and guidelines congenital anomalies. This testing raised the diagnostic yield encouraged individual laboratories to determine their own of cytogenetic testing from 4 to 17–19%.2 The CMA technol- size restrictions.2,6 The clinical significance of small, nonre- ogy has evolved from utilizing targeted areas of known recur- current CNVs (<500 kb) that are not mediated by nonallelic rent microdeletions and microduplications to a platform that homologous recombination has not been well established in a also includes whole-genome coverage capable of identify- clinical setting. Recent efforts have been made to understand ing novel microdeletions and microduplications anywhere the role of these small, nonrecurrent CNVs in neurodevelop- in the human genome.3 CMA technology is able to identify mental disorders.7–9 abnormalities as small as 20–50 kb in targeted regions and In this study we evaluate the role of these small, nonrecur- 100–250 kb throughout the genome.2 This level of resolution rent CNVs (<500 kb) in a cohort of 4,417 patients referred to allows for the detection of abnormalities spanning single our lab to determine the overall significance of these CNVs in genes as well as intragenic CNVs, thus raising the diagnostic patients undergoing CMA clinical testing for developmental 1Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA; 2Current address: Children’s Health Hospital, Dallas,Texas, USA; 3Department of Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama, USA. Correspondence: Fady M. Mikhail ([email protected]) Submitted 16 February 2016; accepted 21 July 2016; advance online publication 15 September 2016. doi:10.1038/gim.2016.132 GEnEticS in mEdicinE | Volume 19 | Number 4 | April 2017 377 ORIGINAL RESEARCH ARTICLE HOLLENBECK et al | Clinical relevance of small CNVs in CMA clinical testing delay, intellectual disability, dysmorphic features, and/or con- four adjacent oligos (probes) with a mean log2 ratio of approxi- genital anomalies. mately +0.6 for duplications. The clinical significance of CNVs was interpreted and reported in accordance with the current MATERIALS AND METHODS American College of Medical Genetics and Genomics stan- Patients dards and guidelines for interpretation and reporting of post- Genotype and phenotype information were collected for all natal constitutional CNVs.6 All reported abnormalities did not patients who were referred for CMA clinical testing at the overlap with benign polymorphic CNVs. Databases used for University of Alabama at Birmingham Clinical Cytogenetics interpretation of the clinical significance of CNVs included the Laboratory during the period between 2009 and 2014, and Database of Genomic Variants, DECIPHER, ClinGen, OMIM, had an abnormal CMA result. Phenotypic information was and PubMed. The UCSC Genome Browser was used to examine collected using a standardized phenotypic checklist utilizing the gene content and to access all five databases. In this study a Human Phenotype Ontology codes (Supplementary Table S1 gene is considered a well-characterized haploinsufficient gene online). These data have been collected for all patients with if shown in multiple peer-reviewed publications to result in a abnormal CMA results as part of our ongoing effort to assem- phenotype if involved by a heterozygous deletion or if it harbors ble a comprehensive genotype–phenotype database. Inclusion a heterozygous loss-of-function mutation. Similarly, a gene is criteria in this study included identification of a small, non- considered a well-characterized triplosensitive gene if shown in recurrent CNV (<500 kb) that is not mediated by nonallelic multiple peer-reviewed publications to result in a phenotype if homologous recombination without the presence of a larger involved by a duplication. On the other hand, a gene considered (>500 kb) pathogenic abnormality. Individuals were excluded to have some evidence of dosage sensitivity is one described in from our study if the identified abnormalities were classified a single case report to result in a phenotype if involved by a as benign or likely benign CNVs. We also excluded recurrent CNV and has a gene function that is relevant to the reason for microdeletions/microduplications <500 kb that are mediated patient referral. The ClinGen dosage sensitivity scores (https:// by nonallelic homologous recombination, unless they coexisted www.ncbi.nlm.nih.gov/projects/dbvar/clingen/index.shtml) with a small, nonrecurrent CNV. This study was approved by and/or the DECIPHER haploinsufficiency index scores were the institutional review board for human subject research at the also included in the interpretation algorithm.40 University of Alabama at Birmingham. Confirmatory studies included metaphase and interphase fluorescence in situ hybridization (FISH) testing using bacte- Methods rial artificial chromosome clones from the RPCI-11 library High-resolution whole-genome array comparative genomic (Empire Genomics, Buffalo, NY) for deletions between 100 and hybridization (aCGH) was performed using three platforms. 500 kb in size and duplications between 400 and 500 kb. The The Agilent 4 × 44k aCGH, Agilent 8 × 60k aCGH, and Agilent clones’ identities were confirmed by end sequencing and were 4 × 180k aCGH+SNP platforms were used from January 2009 directly labeled by nick translation using either SpectrumGreen through December 2011, January 2012 through August 2013, or SpectrumOrange fluorescent dyes (Abbott, Abbott Park, IL). and September 2013 through December 2014, respectively Deletions <100 kb and duplications <400 kb were confirmed by (Agilent Technologies, Santa Clara, CA). All three platforms FISH using directly labeled oligonucleotide SureFISH probes are custom-designed arrays that are based on the International (Agilent Technologies). All FISH probes were validated on nor- Standard Cytogenomic Array Consortium design. Peripheral mal metaphase chromosomes. CNVs with sizes smaller than the blood samples were used for DNA extraction. A more FISH resolution were