Chromosomal microarray in clinical diagnosis: a study of 337 patients with congenital anomalies and developmental delays or intellectual disability.

FORENSIC SCIENCE 231 Croat Med J. 2017;58:231-8 https://doi.org/10.3325/cmj.2017.58.231 Chromosomal microarray in clinical diagnosis: a study of 337 patients with congenital anomalies and developmental delays or intellectual disability Ivona Sansović1, Ana-Maria Ivankov1, Adriana Bobinec1, Mijana Kero1, Ingeborg Barišić1 Department of Medical Genetics and Reproductive Health, Children’s Hospital Zagreb, University of Zagreb, School of Medicine, Zagreb, Croatia 1 Aim To determine the diagnostic yield and criteria that could help to classify and interpret the copy number variations (CNVs) detected by chromosomal microarray (CMA) technique in patients with congenital and developmental abnormalities including dysmorphia, developmental delay (DD) or intellectual disability (ID), autism spectrum disorders (ASD) and congenital anomalies (CA). Method CMA analysis was performed in 337 patients with DD/ID with or without dysmorphism, ASD, and/or CA. In 30 of 337 patients, chromosomal imbalances had previously been detected by classical cytogenetic and molecular cytogenetic methods. Results In 73 of 337 patients, clinically relevant variants were detected and better characterized. Most of them were >1 Mb. Variants of unknown clinical significance (VOUS) were discovered in 35 patients. The most common VOUS size category was <300 kb (40.5%). Deletions and de novo imbalances were more frequent in pathogenic CNV than in VOUS category. CMA had a high diagnostic yield of 43/307, excluding patients previously detected by other methods. Conclusion CMA was valuable in establishing the diagnosis in a high proportion of patients. Criteria for classification and interpretation of CNVs include CNV size and type, mode of inheritance, and genotype-phenotype correlation. Agilent ISCA v2 Human Genome 8x60 K oligonucleotide microarray format proved to be reasonable resolution for clinical use, particularly in the regions that are recommended by the International Standard Cytogenomic Array (ISCA) Consortium and associated with well-established syndromes. Received: April 24, 2017 Accepted: June 02, 2017 Correspondence to: Ivona Sansović Department of Medical Genetics and Reproductive Health Children’s Hospital Zagreb Klaićeva 16 10000 Zagreb, Croatia www.cmj.hr 232 FORENSIC SCIENCE Patients with developmental delay or intellectual disability (DD/ID), autism spectrum disorders (ASD), and congenital anomalies (CA) account for the largest proportion of cytogenetic testing due to their high prevalence in the population. More than 80% of the chromosomal aberrations found in ID/DD or ASD are submicroscopic and not detected by classical cytogenetic methods. Chromosomal microarray (CMA) is used as the first test to detect copy number variations (CNVs) that are major cause of these disorders. Large cohort studies of patients with DD/ID, CA, or ASD screened by microarray found a diagnostic yield of 14–20% compared with ~ 3% for the standard G-banded karyotype (1,2). The resolution level of the CMA has no limit, ie, it depends on the size and distance between the test probes on the array. Today, there are a number of commercially available array platforms, which differ in genome coverage, resolution, and application. A higher array resolution can mean not only an increased yield of clinical genetic diagnosis, but also a higher level of detection of benign CNVs or variants of unknown clinical significance (VOUS). Therefore, the correct choice of the resolution array platform that balances sensitivity and specificity is very important for clinical practice. Defining the pathogenicity of CNVs is the major difficulty in the interpretation of the array results. The usual criteria used in the interpretation of the clinical relevance of a CNV are inheritance, size, type, and gene content. In this study, we present the CMA analysis of 337 patients with DD/ID with or without dysmorphic features, ASD and/ or CA. In 30 patients, chromosomal imbalances had been previously detected by classical cytogenetic and molecular cytogenetic methods. These patients were analyzed by CMA to define more precisely the breakpoints and the gene content of the rearrangements and to clarify if there were any additional CNVs. The aim of the study was to determine the diagnostic yield of the CMA analysis on the remaining 307 patients and criteria that could help in classification and interpretation of the CNVs detected. Patients and methods Patients The analysis included 337 unrelated patients from Croatia, referred to the Department of Medical Genetics and Reproductive Health, Children’s Hospital Zagreb, University of Zagreb, School of Medicine, who were diag- www.cmj.hr Croat Med J. 2017;58:231-8 nosed by clinical geneticists or pediatricians to have DD/ ID, ASD, CA, or a combination of those features. We divided the patients according to the main clinical features into six groups as follows: ID/DD, ASD, CA with/without dysmorphism; ID/DD, CA with/without dysmorphism; ID/DD, ASD with or without dysmorphism; ID/DD with/without dysmorphism; CA with/without dysmorphism; and ASD with/ without dysmorphism. The median age of patients at examination was 7 years (range: one months to 25 years). Samples were analyzed by CMA in the period between January 2016 and April 2017. In 30 patients, chromosomal imbalances had already been detected by high resolution conventional cytogenetics, Multiplex Ligation-dependent Probe Amplification (MLPA) or Fluorescence In Situ Hybridization (FISH). To determine the origin of the imbalances, parental blood samples were requested and those available were tested by CMA, conventional cytogenetics or FISH, depending of the size of the imbalances. Informed consent was obtained from all patients or their parents/ guardians. METHODS DNA extraction Genomic DNA was extracted from peripheral blood lymphocytes using Illustra blood genomicPrep Mini Spin (GE Healthcare Life Sciences, Chalfont St Giles, Buckinghamshire, UK) or NucleoSpin Blood (Macherey-Nagel, Düren, Germany) kit according to the manufacturers’ instructions. DNA concentration and purity were measured using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and BioDrop uLite Spectrophotometer (Isogen Life Science, De Meern, The Netherlands). Chromosomal microarray High-resolution whole genome analysis was performed using Agilent SurePrint G3 Unrestricted CGH ISCA v2 Human Genome microarrays according to the manufacturers’ instructions (Agilent Technologies, Santa Clara, CA, USA). The 8x60 K oligonucleotide microarray format that contains approximately 60 000 sixty-mer probes with 60 kb average probe spacing, with a higher resolution within the region recommended by International Standard Cytogenomic Array (ISCA) Consortium, was used. Slides were scanned on a SureScan Dx Microarray scanner (Agilent Technologies, USA) and processed with Feature Extraction software (v12.0). Results were analyzed using Agilent CytoGenomics (v3.0 and (...truncated)