review September 2007 ⅐ Vol. 9 ⅐ No. 9 Structural variation in the human genome: the impact of copy number variants on clinical diagnosis Laia Rodriguez-Revenga, PhD1–3, Montserrat Mila, PhD3–5, Carla Rosenberg, PhD6, Allen Lamb, PhD, FACMG7,8*, Charles Lee, PhD, FACMG1,2* Over the past few years, the application of whole-genome scanning array technologies has catalyzed the appreciation of a new form of submicroscopic genomic imbalances, referred to as copy number variants. Copy number variants contribute substantially to genetic diversity and result from gains and losses of genomic regions that are 1000 base pairs in size or larger, sometimes encompassing millions of bases of contiguous DNA sequences. As genome-wide scanning techniques become more widely used in diagnostic laboratories, a major challenge is how to accurately interpret which submicroscopic genomic imbalances are pathogenic in nature and which are benign. Herein, we review the literature from the past 3 years on this new source of genomic variability and comment on factors that should be considered when trying to differentiate between a pathogenic and a benign copy number variant. Genet Med 2007:9(9):600–606. Key Words: copy number variant, CNV, genomic diversity, pathogenic variant, benign variant INTRODUCTION On the other side of the spectrum, genotyping technologies have allowed us to detect smaller and more abundant forms of Genomic variability in humans exists on widely different genomic variability (e.g., single nucleotide polymorphisms scales. Microscopic variants, 5 Mb or greater in size, have been [SNPs]). In fact, SNPs were long considered the largest source identified since 1959 by using standard cytogenetic analysis of genomic variation in humans, with estimates of at least 10 (e.g., G-banded karyotyping).1 At this level of analysis, it is million SNPs within the human population, averaging 1 SNP possible to survey the entire human genome for gains, losses, for every 300 nucleotides in an individual.4 or rearrangements of genetic material in a single test, but in With the development of array-based comparative genomic practice, imbalances smaller than 10 to 20 Mb are often not hybridization (CGH) technologies, a large number of sub- readily detected. Over the years, classical cytogenetic studies microscopic genomic imbalances have now also being have uncovered several heteromorphisms and euchromatic identified.5,6 These genomic imbalances are referred to as copy variants that do not seem to have clinical significance.2,3 number variants (CNVs) and are defined as deletions and du- With the advent of molecular cytogenetic techniques, such plications of DNA segments larger than 1000 bases (1 kb) and as fluorescence in situ hybridization (FISH), it became possible up to several Mb in size that are present in variable copy num- to more precisely define the extent and the actual DNA se- ber compared with a reference genome.7–9 quences involved in these chromosomal variants at a much Over the past few years, the term CNV has been broadly higher resolution. However, FISH uses probes specifically tar- used,10 going beyond the clinical definition of a variant, which geting a given chromosomal locus and assessment of genomic usually implies a benign genetic change that does not cause a imbalances at multiple chromosomal loci using this technique clinically recognizable phenotype.2,11 However, with increased rapidly becomes labor intensive. genotype-phenotype correlations, CNVs that were once thought to be benign or of unknown clinical significance are From the 1Department of Pathology, Brigham and Women’s Hospital, Boston, Massachu- setts; 2Harvard Medical School, Boston, Massachusetts; 3Centre for Biomedical Research on now known to be associated with and definitive of specific 12–14 Rare Diseases (CIBERER), Barcelona, Spain; 4Biochemistry and Molecular Genetics Depart- genomic syndromes. Such associations are appreciated ment, Hospital Clı´nic, Barcelona, Spain; 5Institut d’Investigacions Biome`diques August Pi i when a particular CNV is observed recurrently among unre- Sunyer (IDIBAPS), Barcelona, Spain; 6Department of Genetics and Evolutionary Biology, lated individuals with similar clinical presentations and/or 7 University of Sao Paulo, Sao Paulo, Brazil; Genzyme Genetics, Santa Fe, New Mexico; and when the genomic imbalance is found to cosegregate with the 8ARUP Laboratories, Cytogenetics, Salt Lake City, Utah. clinical presentation in families containing multiple affected Charles Lee, PhD, Department of Pathology, Brigham and Women’s Hospital, 221 Longwood Avenue, Boston, MA 02115 or Allen Lamb, PhD, ARUP Laboratories, Cytogenetics, 500 Chipeta individuals. Our limited understanding of the phenotypic im- Way, Salt Lake City, UT 84108; E-mail: [email protected] or [email protected] pact of the hundreds of CNVs that have already been discov- *AL and CL contributed equally to this work. ered warrants the use of qualifiers to minimize confusion (es- Disclosure: The authors report no conflict of interest. pecially in a diagnostic setting). Hence, in this perspective, we Submitted for publication April 24, 2007. refer to CNVs as being pathogenic, benign, or of unknown clinical Accepted for publication June 26, 2007. significance—definitions that are based on our current under- DOI: 10.1097/GIM.0b013e318149e1e3 standing of the structure and function of the human genome. 600 Genetics IN Medicine Structural variation in the human genome AN ABUNDANCE OF CNVS IN THE HUMAN GENOME ent array platforms: a high-density, genome-wide SNP array (the Affymetrix 500k EA genotyping chips)17 and a whole ge- In 2004, two independent studies screened the human genome nome tilepath (WGTP) BAC array containing clones that to- of healthy individuals by using array CGH and reported the wide- gether represented 94% of the euchromatic portion of the hu- spread presence of CNVs.5,6 Iafrate and coworkers5 used a bacte- man genome.18 Both methods were capable of detecting CNVs rial artificial chromosome (BAC)-based array, with clones chosen and, in many ways, were complementary to each other. The at approximately 1-Mb intervals throughout the human genome to identify more than 200 variable loci among 39 unrelated SNP arrays tended to detect smaller CNVs in regions that had healthy individuals. Sebat and colleagues6 used a microarray plat- good probe coverage and provided better definition of the form containing oligonucleotides spaced at 35-kb intervals and structure of CNVs at these regions. The WGTP platform detected 76 CNVs among 20 individuals. Although both studies seemed to be more useful in detecting larger and more com- used slightly different approaches to study the genome of unre- plex CNVs. Incidentally, these were often regions of the human lated individuals, they reached the same conclusion: phenotypi- genome that were overlapping segmental duplications (also cally normal individuals have an unexpectedly high number of known as low copy repeats), which have been found to be genomic imbalances throughout their genomes. However, be- regions sparsely covered by SNP genotyping probes.19 Over- cause of the small number of individuals examined and the lim- lapping and juxtaposed CNVs identified by both platforms ited resolution of both platforms, neither study provided a com- were merged together into 1447 discrete CNV regions prehensive evaluation of CNVs in the human genome. Indeed, (CNVRs) (discrete CNVRs can be seen for the 1p36.33 chro- the number of CNVs identified by these two studies seemed likely mosome region in Figure 1). The CNVRs identified in this to be an underestimation of the true number of CNVs in humans.15 study represented 12% (360 Mb) of the human genome. A A few years later, Redon and coworkers16 published a more whole-genome view of the distribution of CNVRs revealed that comprehensive CNV map for the human genome. In this they are ubiquitously distributed throughout the genome, with study, the DNAs of 270 healthy individuals from four popula- approximately 24% of the CNVRs located near previously tions (the HapMap collection) were analyzed using two differ- known segmental duplications. CNVs that are located in close Fig. 1. A schematic representation of a copy number variant region (CNVR) and CNVs for a part of chromosome region 1p36.33. CNVs called using two different array CGH platforms (i.e., a high-density genome-wide SNP array Affymetrix 500k EA chip and a whole-genome tilepath [WGTP] BAC array) in four different HapMap individuals are represented in colored boxes (Individuals A, B, C, and D). The relative size and position of BAC clones on this WGTP array and the relative position of SNP-detecting oligonucleotide on the Affymetrix 500k EA chip are shown above the CNV regions. Figure adapted from the Database of Genomic Variants (http://projects.tcag.ca/variation). September 2007 ⅐ Vol. 9 ⅐ No. 9 601 Rodriguez-Revenga et al. proximity to segmental duplications are thought to be gener- (e.g., duplication of a putative repressor element) led to decreased ated and maintained via nonallelic homologous recombina- expression levels of an overlapping or nearby gene. An example tion (NAHR) mechanisms that result from recombination for this is a small duplication (Ͻ150 kb) downstream of the pro- events between flanking segmental duplications.20 teolipid protein gene (PLP1) that silences PLP1 gene expression Based on current information, CNVs tend to be preferen- and results in a spastic paraplegia type 2 phenotype that is also tially located