Detection of pathogenic gene copy number variations in patients with mental retardation by genomewide oligonucleotide array comparative genomic hybridization

Genomic imbalance is a major cause of developmental disorders. Microarray-based comparative genomic hybridization (aCGH) has revealed frequent imbalances associated with clinical syndromes, but also a large number of copy number variations (CNVs), which have complicated the interpretation of results. We studied 100 consecutive patients with unexplained mental retardation and a normal karyotype using several platforms of CGH arrays. A genomewide array with 44,290 oligonucleotide probes (OaCGH44K) detected imbalances in 15% of cases studied with sizes ranged from 459 kb to 19 Mb while revealing a small number of CNVs (0.72/individual). Another platform with approximately 240,000 oligonucleotide probes (OaCGH244K) revealed a large number of CNVs (20/individual) in selected cases and their normal parents. We used a comprehensive approach for interpreting the results of aCGH, including consideration of the size, inheritance and gene content of CNVs, and consultation with an online Database of Genomic Variants (DGV) and Online Mendelian Inheritance in Men (OMIM) for information on the genes involved. Our study suggests that genomewide oligonucleotide arrays such as the OaCGH44K platform can be used as a powerful diagnostic tool for detection of genomic imbalances associated with unexplained mental retardation or syndromic autism spectrum disorders. It is interesting to note that a small number of common variants were revealed by OaCGH244K in some study subjects but not in their parents and that some inherited CNVs had altered breakpoints. Further investigations on these alterations may provide useful information for understanding the mechanism of CNVs.     

Molecular karyotyping of patients with unexplained mental retardation by SNP arrays: A multicenter study

Genomic microarrays have been implemented in the diagnosis of patients with unexplained mental retardation. This method, although revolutionizing cytogenetics, is still limited to the detection of rare de novo copy number variants (CNVs). Genome‐wide single nucleotide polymorphism (SNP) microarrays provide high‐resolution genotype as well as CNV information in a single experiment. We hypothesize that the widespread use of these microarray platforms can be exploited to greatly improve our understanding of the genetic causes of mental retardation and many other common disorders, while already providing a robust platform for routine diagnostics. Here we report a detailed validation of Affymetrix 500k SNP microarrays for the detection of CNVs associated to mental retardation. After this validation we applied the same platform in a multicenter study to test a total of 120 patients with unexplained mental retardation and their parents. Rare de novo CNVs were identified in 15% of cases, showing the importance of this approach in daily clinical practice. In addition, much more genomic variation was observed in these patients as well as their parents. We provide all of these data for the scientific community to jointly enhance our understanding of these genomic variants and their potential role in this common disorder. Hum Mutat 30:1–11, 2009. © 2009 Wiley‐Liss, Inc.     

X‐chromosome duplications in males with mental retardation: pathogenic or benign variants?

Gijsbers ACJ, den Hollander NS, Helderman‐van de Enden ATJM, Schuurs‐Hoeijmakers JHM, Vijfhuizen L, Bijlsma EK, van Haeringen A, Hansson KBM, Bakker E, Breuning MH, Ruivenkamp CAL. X‐chromosome duplications in males with mental retardation: pathogenic or benign variants? Studies to identify copy number variants (CNVs) on the X‐chromosome have revealed novel genes important in the causation of X‐linked mental retardation (XLMR). Still, for many CNVs it is unclear whether they are associated with disease or are benign variants. We describe six different CNVs on the X‐chromosome in five male patients with mental retardation that were identified by conventional karyotyping and single nucleotide polymorphism array analysis. One deletion and five duplications ranging in size from 325 kb to 12.5 Mb were observed. Five CNVs were maternally inherited and one occurred de novo. We discuss the involvement of potential candidate genes and focus on the complexity of X‐chromosomal duplications in males inherited from healthy mothers with different X‐inactivation patterns. Based on size and/or the presence of XLMR genes we were able to classify CNVs as pathogenic in two patients. However, it remains difficult to decide if the CNVs in the other three patients are pathogenic or benign.     

Application of array comparative genomic hybridization in 102 patients with epilepsy and additional neurodevelopmental disorders

Copy-number variants (CNVs) collectively represent an important cause of neurodevelopmental disorders such as developmental delay (DD)/intellectual disability (ID), autism, and epilepsy. In contrast to DD/ID, for which the application of microarray techniques enables detection of pathogenic CNVs in ～10-20% of patients, there are only few studies of the role of CNVs in epilepsy and genetic etiology in the vast majority of cases remains unknown. We have applied whole-genome exon-targeted oligonucleotide array comparative genomic hybridization (array CGH) to a cohort of 102 patients with various types of epilepsy with or without additional neurodevelopmental abnormalities. Chromosomal microarray analysis revealed 24 non-polymorphic CNVs in 23 patients, among which 10 CNVs are known to be clinically relevant. Two rare deletions in 2q24.1q24.3, including KCNJ3 and 9q21.13 are novel pathogenic genetic loci and 12 CNVs are of unknown clinical significance. Our results further support the notion that rare CNVs can cause different types of epilepsy, emphasize the efficiency of detecting novel candidate genes by whole-genome array CGH, and suggest that the clinical application of array CGH should be extended to patients with unexplained epilepsies. ? 2012 Wiley Periodicals, Inc.     

Array CGH in patients with developmental delay or intellectual disability: are there phenotypic clues to pathogenic copy number variants?

Shoukier M, Klein N, Auber B, Wickert J, Schröder J, Zoll B, Burfeind P, Bartels I, Alsat EA, Lingen M, Grzmil P, Schulze S, Keyser J, Weise D, Borchers M, Hobbiebrunken E, Röbl M, Gärtner J, Brockmann K, Zirn B. Array CGH in patients with developmental delay or intellectual disability: are there phenotypic clues to pathogenic copy number variants? Array comparative genomic hybridization (array CGH) is now widely adopted as a first‐tier clinical diagnostic test in individuals with unexplained developmental delay/intellectual disability (DD/ID) and congenital anomalies. Our study aimed at enlarging the phenotypic spectrum associated with clinically relevant copy number variants (CNVs) as well as delineating clinical criteria, which may help separating patients with pathogenic CNVs from those without pathogenic CNVs. We performed a retrospective review of clinical and array CGH data of 342 children with unexplained DD/ID. The phenotypic features of patients with clinically significant CNV were compared with those without pathogenic CNVs. Array CGH detected pathogenic CNVs in 13.2% of the patients. Congenital anomalies, especially heart defects, as well as primary microcephaly, short stature and failure to thrive were clearly more frequent in children with pathogenic CNVs compared with children with normal array CGH results. Thus, we assume that in patients with unexplained DD/ID, array CGH will more probably detect a significant CNV if any of these features is part of the patient's phenotype.     

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

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.     

Identification of gene copy number variations in patients with mental retardation using array-CGH: Novel syndromes in a large French series

Array-CGH has revealed a large number of copy number variations (CNVs) in patients with multiple congenital anomalies and/or mental retardation (MCA/MR). According to criteria recently listed, pathogenicity was clearly suspected for some CNVs but benign CNVs, considered as polymorphisms, have complicated the interpretation of the results. In this study, genomic DNAs from 132 French patients with unexplained mental retardation were analysed by genome wide high-resolution Agilent^® 44K oligonucleotide arrays. The results were in accordance with those observed in previous studies: the detection rate of pathogenic CNVs was 14.4%. A non-random involvement of several chromosomal regions was observed. Some of the microimbalances recurrently involved regions (1q21.1, 2q23.1, 2q32q33, 7p13, 17p13.3, 17p11.2, 17q21.31) corresponding to known or novel syndromes. For all the pathogenic CNVs, further cases are needed to allow more accurate genotype–phenotype correlations underscoring the importance of databases to group patients with similar molecular data.     

Genomic microarrays in mental retardation: A practical workflow for diagnostic applications

Microarray‐based copy number analysis has found its way into routine clinical practice, predominantly for the diagnosis of patients with unexplained mental retardation. However, the clinical interpretation of submicroscopic copy number variants (CNVs) is complicated by the fact that many CNVs are also present in the general population. Here we introduce and discuss a workflow that can be used in routine diagnostics to assess the clinical significance of the CNVs identified. We applied this scheme to our cohort of 386 individuals with unexplained mental retardation tested using a genome‐wide tiling‐resolution DNA microarray and to 978 additional patients with mental retardation reported in 15 genome‐wide microarray studies extracted from the literature. In our cohort of 386 patients we identified 25 clinically significant copy number losses (median size 2.6 Mb), nine copy number gains (median size 2.0 Mb), and one mosaic numerical chromosome aberration. Accordingly, the overall diagnostic yield of clinically significant CNVs was 9.1%. Taken together, our cohort and the patients described in the literature include a total of 1,364 analyses of DNA copy number in which a total of 11.2% (71.9% losses, 19.6% gains, 8.5% complex) could be identified, reflecting the overall diagnostic yield of clinically significant CNVs in individuals with unexplained mental retardation. Hum Mutat 0, 1–10, 2008. © 2008 Wiley‐Liss, Inc.     

A parallel SNP array study of genomic aberrations associated with mental retardation in patients and general population in Estonia

The increasing use of whole-genome array screening has revealed the important role of DNA copy-number variations in the pathogenesis of neurodevelopmental disorders and several recurrent genomic disorders have been defined during recent years. However, some variants considered to be pathogenic have also been observed in phenotypically normal individuals. This underlines the importance of further characterization of genomic variants with potentially variable expressivity in both patient and general population cohorts to clarify their phenotypic consequence. In this study whole-genome SNP arrays were used to investigate genomic rearrangements in 77 Estonian families with idiopathic mental retardation. In addition to this family-based approach, phenotype and genotype data from a cohort of 1000 individuals in the general population were used for accurate interpretation of aberrations found in mental retardation patients. Relevant structural aberrations were detected in 18 of the families analyzed (23%). Fifteen of those were in genomic regions where clinical significance has previously been established. In 3 families, 4 novel aberrations associated with intellectual disability were detected in chromosome regions 2p25.1-p24.3, 3p12.1-p11.2, 7p21.2-p21.1 and Xq28. Carriers of imbalances in 15q13.3, 16p11.2 and Xp22.31 were identified among reference individuals, affirming the variable phenotypic consequence of rare variants in some genomic regions considered as pathogenic.     

Whole-genome array CGH identifies pathogenic copy number variations in fetuses with major malformations and a normal karyotype

Despite a wide range of clinical tools, the etiology of mental retardation and multiple congenital malformations remains unknown for many patients. Array-based comparative genomic hybridization (aCGH) has proven to be a valuable tool in these cases, as its pangenomic coverage allows the identification of chromosomal aberrations that are undetectable by other genetic methods targeting specific genomic regions. Therefore, aCGH is increasingly used in clinical genetics, both in the postnatal and the prenatal settings. While the diagnostic yield in the postnatal population has been established at 10-12%, studies investigating fetuses have reported variable results. We used whole-genome aCGH to investigate fetuses presenting at least one major malformation detected on ultrasound, but for whom standard genetic analyses (including karyotype) failed to provide a diagnosis. We identified a clinically significant chromosomal aberration in 8.2% of tested fetuses (4/49), and a result of unclear clinical significance in 12.2% of tested fetuses (6/49). Our results document the value of whole-genome aCGH as a prenatal diagnostic tool and highlight the interpretation difficulties associated with copy number variations of unclear significance.     

A new diagnostic workflow for patients with mental retardation and/or multiple congenital abnormalities: test arrays first

High-density single-nucleotide polymorphism (SNP) genotyping technology enables extensive genotyping as well as the detection of increasingly smaller chromosomal aberrations. In this study, we assess molecular karyotyping as first-round analysis of patients with mental retardation and/or multiple congenital abnormalities (MR/MCA). We used different commercially available SNP array platforms, the Affymetrix GeneChip 262K NspI, the Genechip 238K StyI, the Illumina HumanHap 300 and HumanCNV 370 BeadChip, to detect copy number variants (CNVs) in 318 patients with unexplained MR/MCA. We found abnormalities in 22.6% of the patients, including six CNVs that overlap known microdeletion/duplication syndromes, eight CNVs that overlap recently described syndromes, 63 potentially pathogenic CNVs (in 52 patients), four large segments of homozygosity and two mosaic trisomies for an entire chromosome. This study shows that high-density SNP array analysis reveals a much higher diagnostic yield as that of conventional karyotyping. SNP arrays have the potential to detect CNVs, mosaics, uniparental disomies and loss of heterozygosity in one experiment. We, therefore, propose a novel diagnostic approach to all MR/MCA patients by first analyzing every patient with an SNP array instead of conventional karyotyping.     

Clinical application of array-based comparative genomic hybridization by two-stage screening for 536 patients with mental retardation and multiple congenital anomalies

Recent advances in the analysis of patients with congenital abnormalities using array-based comparative genome hybridization (aCGH) have uncovered two types of genomic copy-number variants (CNVs); pathogenic CNVs (pCNVs) relevant to congenital disorders and benign CNVs observed also in healthy populations, complicating the screening of disease-associated alterations by aCGH. To apply the aCGH technique to the diagnosis as well as investigation of multiple congenital anomalies and mental retardation (MCA/MR), we constructed a consortium with 23 medical institutes and hospitals in Japan, and recruited 536 patients with clinically uncharacterized MCA/MR, whose karyotypes were normal according to conventional cytogenetics, for two-stage screening using two types of bacterial artificial chromosome-based microarray. The first screening using a targeted array detected pCNV in 54 of 536 cases (10.1%), whereas the second screening of the 349 cases negative in the first screening using a genome-wide high-density array at intervals of approximately 0.7?Mb detected pCNVs in 48 cases (13.8%), including pCNVs relevant to recently established microdeletion or microduplication syndromes, CNVs containing pathogenic genes and recurrent CNVs containing the same region among different patients. The results show the efficient application of aCGH in the clinical setting.     

Valuing the benefit of diagnostic testing for genetic causes of idiopathic developmental disability: willingness to pay from families of affected children

Idiopathic developmental disability (DD) has been found to put significant psychological distress on families of children with DD. The cause of the disability, however, is unknown for up to one-half of the affected children. Chromosomal abnormalities identified by cytogenetic analysis are the most frequently recognized cause of DD, although they account for less than 10% of cases. Array genomic hybridization (AGH) is a new diagnostic tool that provides a much higher detection rate for chromosomal imbalance than conventional cytogenetic analysis. This increase in diagnostic capability comes at greater monetary costs, which provides an impetus for understanding how individuals value genetic testing for DD. This study estimated the willingness to pay (WTP) for diagnostic testing to find a genetic cause of DD from families of children with DD. A discrete choice experiment was used to obtain WTP values. When it was assumed that AGH resulted in twice as many diagnoses and a 1-week reduction in waiting time compared with conventional cytogenetic analysis, this study found that families were willing to pay up to CDN$1118 (95% confidence interval, $498–1788) for the expected benefit. These results support the conclusion that the introduction of AGH into the Canadian health care system may increase the perceived welfare of society, but future studies should examine the cost-benefit of AGH vs cytogenetic testing.     

Whole-genome array-CGH identifies novel contiguous gene deletions and duplications associated with developmental delay, mental retardation, and dysmorphic features

Cytogenetic imbalances are the most frequently identified cause of developmental delay or mental retardation, which affect 1-3% of children and are often seen in conjunction with growth retardation, dysmorphic features, and various congenital anomalies. A substantial number of patients with developmental delay or mental retardation are predicted to have cytogenetic imbalances, but conventional methods for identifying these imbalances yield positive results in only a small fraction of these patients. We used microarray-based comparative genomic hybridization (aCGH) to study a panel of 20 patients predicted to have chromosomal aberrations based on clinical presentation of developmental delay or mental retardation, growth delay, dysmorphic features, and/or congenital anomalies. Previous G-banded karyotypes and fluorescence in situ hybridization results were normal for all of these patients. Using both oligonucleotide-based and bacterial artificial chromosome (BAC)-based arrays on the same panel of patients, we identified 10 unique deletions and duplications ranging in size from 280 kb to 8.3 Mb. The whole-genome oligonucleotide arrays identified nearly twice as many imbalances as did the lower-resolution whole-genome BAC arrays. This has implications for using aCGH in a clinical setting. Analysis of parental DNA samples indicated that most of the imbalances had occurred de novo. Moreover, seven of the 10 imbalances represented novel disorders, adding to an increasing number of conditions caused by large-scale deletions or duplications. These results underscore the strength of high-resolution genomic arrays in diagnosing cases of unknown genetic etiology and suggest that contiguous genomic alterations are the underlying pathogenic cause of a significant number of cases of developmental delay.     

基因拷贝数变异与智力障碍病因学研究进展

基因拷贝数变异(copy number variations,CNVs)是指DNA片段大小从1 kb至Mb的亚微观结构变异,已成为探讨与疾病相关遗传变异的研究热点,特别是神经认知系统的遗传病,如精神分裂症、智力障碍等综合征、本文综述了基因拷贝数变异与智力障碍的关联关系和基因拷贝数变异检测技术进展在智力障碍诊断中的应用,这对揭示智力障碍发病机制有着重要意义.     

基因拷贝数变异与智力障碍病因学研究进展

基因拷贝数变异(copy number variations,CNVs)是指DNA片段大小从1 kb至Mb的亚微观结构变异,已成为探讨与疾病相关遗传变异的研究热点,特别是神经认知系统的遗传病,如精神分裂症、智力障碍等综合征、本文综述了基因拷贝数变异与智力障碍的关联关系和基因拷贝数变异检测技术进展在智力障碍诊断中的应用,这对揭示智力障碍发病机制有着重要意义.     

基因拷贝数变异与智力障碍病因学研究进展

基因拷贝数变异(copy number variations,CNVs)是指DNA片段大小从1 kb至Mb的亚微观结构变异,已成为探讨与疾病相关遗传变异的研究热点,特别是神经认知系统的遗传病,如精神分裂症、智力障碍等综合征、本文综述了基因拷贝数变异与智力障碍的关联关系和基因拷贝数变异检测技术进展在智力障碍诊断中的应用,这对揭示智力障碍发病机制有着重要意义.     

Genomic profile of copy number variants on the short arm of human chromosome 8

We evaluated 966 consecutive pediatric patients with various developmental disorders by high-resolution microarray-based comparative genomic hybridization and found 10 individuals with pathogenic copy number variants (CNVs) on the short arm of chromosome 8 (8p), representing approximately 1% of the patients analyzed. Two patients with 8p terminal deletion associated with interstitial inverted duplication (inv dup del(8p)) had different mechanisms leading to the formation of a dicentric intermediate during meiosis. Three probands carried an identical ∼5.0 Mb interstitial duplication of chromosome 8p23.1. Four possible hotspots within 8p were observed at nucleotide coordinates of ∼10.45, 24.32–24.82, 32.19–32.77, and 38.94–39.72 Mb involving the formation of recurrent genomic rearrangements. Other CNVs with deletion- or duplication-specific start or stop coordinates on the 8p provide useful information for exploring the basic mechanisms of complex structural rearrangements in the human genome.     

Additional cryptic CNVs in mentally retarded patients with apparently balanced karyotypes

Apparently balanced chromosome abnormalities are occasionally associated with mental retardation (MR). These balanced rearrangements may disrupt genes. However, the phenotype may also be caused by small abnormalities present at the breakpoints or elsewhere in the genome. Conventional karyotyping is not instrumental for detecting small abnormalities because it only identifies genomic imbalances larger than 5–10 Mb. In contrast, high-resolution whole-genome arrays enable the detection of submicroscopic abnormalities in patients with apparently balanced rearrangements.Here, we report on the whole-genome analysis of 13 MR patients with previously detected balanced chromosomal abnormalities, five de novo, four inherited, and four of unknown inheritance, using Single Nucleotide Polymorphism (SNP) arrays. In all the cases, the patient had an abnormal phenotype. In one familial case and one unknown inheritance case, one of the parents had a phenotype which appeared identical to the patient’s phenotype. Additional copy number variants (CNVs) were identified in eight patients. Three patients contained CNVs adjacent to one or either breakpoints. One of these patients showed four and two deletions near the breakpoints of a de novo pericentric inversion. In five patients we identified CNVs on chromosomes unrelated to the previously observed genomic imbalance.These data demonstrate that high-resolution array screening and conventional karyotyping is necessary to tie complex karyotypes to phenotypes of MR patients.     

Application of Array-Based Comparative Genomic Hybridization to Pediatric Neurologic Diseases

Purpose

Array comparative genomic hybridization (array-CGH) is a technique used to analyze quantitative increase or decrease of chromosomes by competitive DNA hybridization of patients and controls. This study aimed to evaluate the benefits and yield of array-CGH in comparison with conventional karyotyping in pediatric neurology patients.

Materials and Methods

We included 87 patients from the pediatric neurology clinic with at least one of the following features: developmental delay, mental retardation, dysmorphic face, or epilepsy. DNA extracted from patients and controls was hybridized on the Roche NimbleGen 135K oligonucleotide array and compared with G-band karyotyping. The results were analyzed with findings reported in recent publications and internet databases.

Results

Chromosome imbalances, including 9 cases detected also by G-band karyotyping, were found in 28 patients (32.2%), and at least 19 of them seemed to be causally related to the abnormal phenotypes. Regarding each clinical symptom, 26.2% of 42 developmental delay patients, 44.4% of 18 mental retardation patients, 42.9% of 28 dysmorphic face patients, and 34.6% of 26 epilepsy patients showed abnormal array results.

Conclusion

Although there were relatively small number of tests in patients with pediatric neurologic disease, this study demonstrated that array-CGH is a very useful tool for clinical diagnosis of unknown genome abnormalities performed in pediatric neurology clinics.