Elsevier

European Journal of Medical Genetics

Volume 52, Issue 6, November–December 2009, Pages 398-403
European Journal of Medical Genetics

Original article
Challenges for CNV interpretation in clinical molecular karyotyping: Lessons learned from a 1001 sample experience

https://doi.org/10.1016/j.ejmg.2009.09.002Get rights and content

Abstract

Molecular karyotyping has moved from bench to bedside for the genetic screening of patients with mental retardation and/or congenital anomalies. The commercial availability of high-resolution microarray platforms has significantly facilitated this process. However, the notion that copy number variants are also abundantly present in the general population challenges the interpretation of the clinical significance of detected copy number variants (CNVs) in these patients. Moreover, the awareness of incomplete penetrance and variable expression, exemplified by the inheritance of causal CNVs from apparently unaffected parents, has further blurred the boundary between benign and pathogenic variation. We analyzed 1001 patients using a large insert clone array (298 patients) and an oligonucleotide-based (703 patients) platform. In this cohort we encountered several examples of causal imbalances that could have been easily interpreted as benign variants when relying on established paradigms. Based on our experience and the pitfalls we encountered, we suggest a decision tree that can be used as a guideline in clinical diagnostics. Using this workflow, we detected 106 clinically significant CNVs in 100 patients, giving a diagnostic yield of at least 10%. Of these imbalances, 58 occurred de novo, 22 were inherited and 26 of unknown inheritance. This underscores that inherited CNVs should not be automatically disregarded as benign variants. Among the clinically relevant CNVs were 11 single-gene aberrations, highlighting the power of high-resolution molecular karyotyping to identify causal genes.

Introduction

Following the introduction of array comparative genomic hybridization (array CGH) in 1997 [1], [2], it took several years before methodology and platforms allowed robust analysis of DNA copy number changes in cancer and constitutional genetics. Preliminary studies showed that this technique sensitively detects submicroscopic genomic imbalances (or copy number variations, CNVs) in patients with mental retardation and/or multiple congenital anomalies (MR/MCA) [3], [4]. The term “molecular karyotyping” was introduced for these high-resolution whole genome screens to distinguish them from conventional karyotyping [5]. First generation whole genome arrays used large insert clones (BACs/PACs) as reporters, with an average probe spacing of approximately 1 Mb across the genome. Early BAC arrays were difficult to produce and hence, accessibility to these arrays was limited to specialized labs [6]. Technological advances made it possible to cover the genome with a tiling-path, allowing the identification of aberrations down to ∼300 kb [7]. Nowadays BAC, oligonucleotide and SNP arrays have become commercially available and produce reliable and robust results. Moreover, increased probe density leads to ultra high genome-wide resolutions up to 5 kb.

The application of array CGH for genome-wide screening had a major impact on clinical genetic diagnostics. First, array CGH made it possible to identify submicroscopic aberrations in at least 9% of patients with idiopathic mental retardation [8]. Second, it facilitated the identification of disease genes of some well-known clinical syndromes [9], [10] and led to the characterization of many novel microdeletion and microduplication syndromes such as the 12q14 microdeletion syndrome [11], [12], the 17q21.31 microdeletion syndrome, the 15q13.3 microdeletion syndrome and the 16p11p12.1 deletion syndrome (reviewed by Slavotinek [13]). Finally, it has contributed to the elucidation of the mechanisms causing genomic disorders [14]. Array CGH technology also facilitated the discovery of a new important source of human genetic variation. Analysis of normal control individuals revealed that copy number variations are abundantly present in the human genome [15], [16], [17]. A catalogue of this structural variation is provided by the Database of Genomic Variants (DGV, http://projects.tcag.ca/variation/) and currently covers about 29% of the human genome. This notion has complicated the interpretation of results, as the clinical significance of these CNVs remains largely unknown [18], [19]. Although guidelines for the use of molecular karyotyping in clinical diagnostics have been suggested [20], [21], [22], the clinical interpretation of detected copy number variants is still challenging. In this paper, we present the results obtained by screening a cohort of 1001 patients with idiopathic mental retardation and/or congenital anomalies for submicroscopic aberrations. We discuss the challenges that accompany the use of high-resolution platforms in terms of determining the clinical significance of the detected aberrations and – based on our experience – propose a decision tree that can be used as a guideline in a clinical setting.

Section snippets

Patients

Patients included in the study had mental retardation and/or (multiple) congenital anomalies of unknown etiology. The total cohort consisted of 1001 patients, 298 of who were analyzed on in-house produced ∼1 Mb BAC arrays and for the remaining 703 patients, copy number analysis was performed on the Agilent Human Genome CGH Microarray 44 K (AMADID#014950). DNA was isolated from total blood using the Puregene Genomic DNA Purification Kit (Qiagen, Belgium) or QIAamp DNA Blood Mini Kit (Qiagen,

General pick-up rate and assessment of clinical relevance

Our cohort consisted of a total of 1001 patients. Of these, 298 patients were analyzed on a ∼1 Mb BAC array and 703 were profiled on a 44 K oligonucleotide array (Agilent, AMADID#014950). Array CGH analysis revealed 1596 copy number variants (CNVs) in 650 patients. Using the ∼1 Mb BAC array, we identified 58 CNVs (33 deletions and 25 duplications) in 47 patients. Using the 44 K oligonucleotide array, we identified 1538 CNVs (829 deletions and 709 duplications) in 603 patients. This means that

Assessment of clinical significance

After a slow start due to technical complications, molecular karyotyping is now emerging in every molecular cytogenetic lab. The advent of commercially available microarrays has facilitated the implementation of this technique in clinical diagnostic laboratories. The increased resolution offered by these arrays allows the detection of larger (>1 Mb) aberrations with high confidence, but at the same time leads to the detection of a high number of smaller CNVs for which causality often remains to

Acknowledgements

We are grateful to all patients, their families and the clinicians involved for their cooperation. We would like to thank Lies Vantomme and Shalina Baute for expert technical assistance. Karen Buysse is supported by a Ph.D. fellowship of the Research Foundation - Flanders (FWO). Bart Loeys and Geert Mortier are senior clinical investigators of the Research Foundation - Flanders (FWO). This work was supported by grant SBO60848 from the Institute for the Promotion of Innovation by Science and

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