Detection of chromosomal imbalances in children with idiopathic mental retardation by array based comparative genomic hybridisation (array-CGH)

Chromosomal aberrations are a common cause of multiple anomaly syndromes that include growth and developmental delay and dysmorphism. Novel high resolution, whole genome technologies, such as array based comparative genomic hybridisation (array-CGH), improve the detection rate of submicroscopic chromosomal abnormalities allowing re-investigation of cases where conventional cytogenetic techniques, Spectral karyotyping (SKY), and FISH failed to detect abnormalities. We performed a high resolution genome-wide screening for submicroscopic chromosomal rearrangements using array-CGH on 41 children with idiopathic mental retardation (MR) and dysmorphic features. The commercially available microarray from Spectral Genomics contained 2600 BAC clones spaced at approximately 1 Mb intervals across the genome. Standard chromosome analysis (>450 bands per haploid genome) revealed no chromosomal rearrangements. In addition, multi-subtelomeric FISH screening in 30 cases and SKY in 11 patients did not detect any abnormality. Using array-CGH we detected chromosomal imbalances in four patients (9.8%) ranging in size from 2 to 14 Mb. Large scale copy number variations were frequently observed. Array-CGH has become an important tool for the detection of chromosome aberrations and has the potential to identify genes involved in developmental delay and dysmorphism. Moreover, the detection of genomic imbalances of clinical significance will increase knowledge of the human genome by performing genotype-phenotype correlation.     

Identification of chromosome abnormalities in subtelomeric regions by microarray analysis: a study of 5,380 cases

Subtelomeric imbalances are a significant cause of congenital disorders. Screening for these abnormalities has traditionally utilized GTG-banding analysis, fluorescence in situ hybridization (FISH) assays, and multiplex ligation-dependent probe amplification. Microarray-based comparative genomic hybridization (array-CGH) is a relatively new technology that can identify microscopic and submicroscopic chromosomal imbalances. It has been proposed that an array with extended coverage at subtelomeric regions could characterize subtelomeric aberrations more efficiently in a single experiment. The targeted arrays for chromosome microarray analysis (CMA), developed by Baylor College of Medicine, have on average 12 BAC/PAC clones covering 10 Mb of each of the 41 subtelomeric regions. We screened 5,380 consecutive clinical patients using CMA. The most common reasons for referral included developmental delay (DD), and/or mental retardation (MR), dysmorphic features (DF), multiple congenital anomalies (MCA), seizure disorders (SD), and autistic, or other behavioral abnormalities. We found pathogenic rearrangements at subtelomeric regions in 236 patients (4.4%). Among these patients, 103 had a deletion, 58 had a duplication, 44 had an unbalanced translocation, and 31 had a complex rearrangement. The detection rates varied among patients with a normal karyotype analysis (2.98%), with an abnormal karyotype analysis (43.4%), and with an unavailable or no karyotype analysis (3.16%). Six patients out of 278 with a prior normal subtelomere-FISH analysis showed an abnormality including an interstitial deletion, two terminal deletions, two interstitial duplications, and a terminal duplication. In conclusion, genomic imbalances at subtelomeric regions contribute significantly to congenital disorders. Targeted array-CGH with extended coverage (up to 10 Mb) of subtelomeric regions will enhance the detection of subtelomeric imbalances, especially for submicroscopic imbalances.     

Oligonucleotide arrays for high-resolution analysis of copy number alteration in mental retardation/multiple congenital anomalies

Genetic diseases arising from microdeletions and microduplications lead to copy number alterations of genomic regions containing one or more genes. Clinically, these rearrangements may be detected by routine cytogenetic testing, which may include karyotype analysis, subtelomeric analysis with fluorescence in situ hybridization, and/or fluorescence in situ hybridization directed at known chromosomal rearrangement-based disorders. The major limitations of these tests are low resolution and limited coverage of the genome. Array-based comparative genomic hybridization has recently become a widely used approach in the genome-wide analysis of copy number alterations in children with mental retardation and/or multiple congenital anomalies. Oligonucleotide-based arrays provide a genome-wide coverage at a much higher resolution than microarrays currently used in clinical diagnostics, greatly improving the rate of detection of submicroscopic copy number alterations in children with mental retardation and/or multiple congenital anomalies.     

Detection of submicroscopic constitutional chromosome aberrations in clinical diagnostics: a validation of the practical performance of different array platforms

For several decades etiological diagnosis of patients with idiopathic mental retardation (MR) and multiple congenital anomalies (MCA) has relied on chromosome analysis by karyotyping. Conventional karyotyping allows a genome-wide detection of chromosomal abnormalities but has a limited resolution. Recently, array-based comparative genomic hybridization (array CGH) technologies have been developed to evaluate DNA copy-number alterations across the whole-genome at a much higher resolution. It has proven to be an effective tool for detection of submicroscopic chromosome abnormalities causing congenital disorders and has recently been adopted for clinical applications. Here, we investigated four high-density array platforms with a theoretical resolution < or =100 kb: 33K tiling path BAC array, 500K Affymetrix SNP array, 385K NimbleGen oligonucleotide array and 244K Agilent oligonucleotide array for their robustness and implementation in our diagnostic setting. We evaluated the practical performance based on the detection of 10 previously characterized abnormalities whose size ranged from 100 kb to 3 Mb. Furthermore, array data analysis was performed using four computer programs developed for each corresponding platform to test their effective ability of reliable copy-number detection and their user-friendliness. All tested platforms provided sensitive performances, but our experience showed that accurate and user-friendly computer programs are of crucial importance for reliable copy-number detection.     

Gene dosage methods as diagnostic tools for the identification of chromosome abnormalities

Cytogenetics is the part of genetics that deals with chromosomes, particularly with numerical and structural chromosome abnormalities, and their implications in congenital or acquired genetic disorders. Standard karyotyping, successfully used for the last 50 years in investigating the chromosome etiology in patients with infertility, fetal abnormalities and congenital disorders, is constrained by the limits of microscopic resolution and is not suited for the detection of subtle chromosome abnormalities. The ability to detect submicroscopic chromosomal rearrangements that lead to copy-number changes has escalated progressively in recent years with the advent of molecular cytogenetic techniques. Here, we review various gene dosage methods such as FISH, PCR-based approaches (MLPA, QF-PCR, QMPSF and real time PCR), CGH and array-CGH, that can be used for the identification and delineation of copy-number changes for diagnostic purposes. Besides comparing their relative strength and weakness, we will discuss the impact that these detection methods have on our understanding of copy number variations in the human genome and their implications in genetic counseling.     

Comparison of chromosome analysis and chromosomal microarray analysis: what is the value of chromosome analysis in today’s genomic array era?

PurposeChromosomal microarray analysis enables the detection of microdeletions/duplications and has become the standard in clinical diagnostic testing for individuals with congenital anomalies and developmental disabilities. In the era of genomic arrays, the value of traditional chromosome analysis needs to be reassessed.MethodsWe studied 3,710 unrelated patients by chromosomal microarray analysis and chromosome analysis simultaneously and compared the results.ResultsWe found that chromosomal microarray analysis detected the chromosomal imbalances that were identified by chromosome analysis with the exception of six cases (0.16%) that had mosaic abnormalities. Of note, one case showed mosaicism for two abnormal cell lines, resulting in a balanced net effect and a normal chromosomal microarray analysis. Further structural abnormalities such as unbalanced translocations, rings, and complex rearrangements were subsequently clarified by chromosome analysis in 18% of the cases with abnormal chromosomal microarray analysis results. Apparently balanced rearrangements were detected by chromosome analysis in 30 cases (0.8%).ConclusionOur data demonstrate that although chromosomal microarray analysis should be the first-tier test for clinical diagnosis of chromosome abnormalities, chromosome analysis remains valuable in the detection of mosaicism and delineation of chromosomal structural rearrangements.ConclusionGenet Med 2013:15(6):450–457     

Comparative genomic hybridisation in mentally retarded patients with dysmorphic features and a normal karyotype

Segmental aneusomy for small chromosomal regions has been shown to be a common cause of mental retardation and multiple congenital anomalies. A screening method for such chromosome aberrations that are not detected using standard cytogenetic techniques is needed. Recent studies have focused on detection of subtle terminal chromosome aberrations using subtelomeric probes. This approach however excludes significant regions of the genome where submicroscopic rearrangements are also liable to occur. The aim of the present study was to evaluate the efficiency of comparative genomic hybridisation (CGH) for screening of submicroscopic chromosomal rearrangements. CGH was performed in a cohort of 17 patients (14 families) with mental retardation, dysmorphic features and a normal karyotype. Five subtle unbalanced rearrangements were identified in 7 patients. Subsequent FISH studies confirmed these results. Although no interstitial submicroscopic rearrangement was detected in this small series, the study emphasises the value of CGH as a screening approach to detect subtle chromosome rearrangements in mentally retarded patients with dysmorphic features and a normal karyotype.     

Molecular cytogenetic studies of chromosomal abnormalities and disorders in nervous and mental diseases: search for biological markers for diagnosis

Molecular cytogenetic and cytogenetic studies of chromosomal disorders in patients with nervous and mental disorders were conducted using currently available approaches, including fluorescence in situ hybridization (FISH) with an original collection of centromeric, telomeric, region-specific DNA probes. A novel in situ hybridization protocol for rapid (15-30 min) chromosomal detection procedure and directly fluoresceinated DNA probes are recommended for use in mental retardation and congenital malformations. Chromosomal abnormalities could be detected in postnatal cases with chromosomal structural rearrangements, aneuplodies of gonosomes (including mosaicisms) and autosomes (including marker chromosomes). Molecular cytogenetics (or FISH diagnosis) can be used when classical cytogenetic methods are insufficient. The authors' experience shows that FISH should be utilized only as an adjunctive test for classical cytogenetic studies when banding techniques are ineffective; cytogenetic methods should be utilized for the preclinical diagnosis of Rett's syndrome. Detection by original probes gives an additional possibility in FISH analysis. Molecular cytogenetic methods are shown to provide a rapid accurate approach to studying and diagnosing chromosomal anomalies and disorders in mental retardation with congenital malformations and Rett's syndrome in children and to exploring aneuploidies in the postmortem cell samples from schizophrenic patients.     

Deletion at chromosomal band Xp22.12–Xp22.13 involving PDHA1 in a patient with congenital lactic acidosis

We present a patient with congenital lactic acidosis, agenesis of the corpus callosum, and profound developmental delay. Assays of pyruvate dehydrogenase complex function were normal in lymphocytes, but decreased in fibroblasts. Sequencing of the PDHA1 gene did not reveal deleterious mutations, and BAC based microarray analysis did not reveal any chromosomal abnormality. However, gene dosage analysis with oligonucleotide-based chromosomal microarray revealed a deletion of Xp22.12–Xp22.13 involving complete deletion of PDHA1. This is the first report of a whole gene deletion of PDHA1 detected by oligonucleotide-based microarray.     

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.     

Mate pair sequencing for the detection of chromosomal aberrations in patients with intellectual disability and congenital malformations

Recently, microarrays have replaced karyotyping as a first tier test in patients with idiopathic intellectual disability and/or multiple congenital abnormalities (ID/MCA) in many laboratories. Although in about 14–18% of such patients, DNA copy-number variants (CNVs) with clinical significance can be detected, microarrays have the disadvantage of missing balanced rearrangements, as well as providing no information about the genomic architecture of structural variants (SVs) like duplications and complex rearrangements. Such information could possibly lead to a better interpretation of the clinical significance of the SV. In this study, the clinical use of mate pair next-generation sequencing was evaluated for the detection and further characterization of structural variants within the genomes of 50 ID/MCA patients. Thirty of these patients carried a chromosomal aberration that was previously detected by array CGH or karyotyping and suspected to be pathogenic. In the remaining 20 patients no causal SVs were found and only benign aberrations were detected by conventional techniques. Combined cluster and coverage analysis of the mate pair data allowed precise breakpoint detection and further refinement of previously identified balanced and (complex) unbalanced aberrations, pinpointing the causal gene for some patients. We conclude that mate pair sequencing is a powerful technology that can provide rapid and unequivocal characterization of unbalanced and balanced SVs in patient genomes and can be essential for the clinical interpretation of some SVs.     

The impact of chromosomal microarray on clinical management: a retrospective analysis

PurposeChromosomal microarray has been widely adopted as the first-tier clinical test for individuals with multiple congenital anomalies, developmental delay, intellectual disability, and autism spectrum disorders. Although chromosomal microarray has been extensively shown to provide a higher diagnostic yield than conventional cytogenetic methods, some health insurers refuse to provide coverage for this test, claiming that it is experimental and does not affect patients’ clinical management.MethodsWe retrospectively reviewed the electronic medical records of all patients who had abnormal chromosomal microarray findings reported by our laboratory over a 3-year period and quantified the management recommendations made in response to these results.ResultsAbnormal chromosomal microarray findings were reported for 12.7% of patients (227/1,780). For patients with clinical follow-up notes available, these results had management implications for 54.5% of patients in the entire abnormal cohort (102/187) and for 42.1% of patients referred for isolated neurodevelopmental disorders (16/38). Recommendations included pharmacological treatment, cancer-related screening or exclusion of screening, contraindications, and referrals for further evaluation.ConclusionThese results empirically demonstrate the clinical utility of chromosomal microarray by providing evidence that management was directly affected for the majority of patients in our cohort with abnormal chromosomal microarray findings.     

Emerging patterns of cryptic chromosomal imbalance in patients with idiopathic mental retardation and multiple congenital anomalies: a new series of 140 patients and review of published reports

Background

Chromosomal abnormalities are a major cause of mental retardation and multiple congenital anomalies (MCA/MR). Screening for these chromosomal imbalances has mainly been done by standard karyotyping. Previous array CGH studies on selected patients with chromosomal phenotypes and normal karyotypes suggested an incidence of 10–15% of previously unnoticed de novo chromosomal imbalances.

Objective

To report array CGH screening of a series of 140 patients (the largest published so far) with idiopathic MCA/MR but normal karyotype.

Results

Submicroscopic chromosomal imbalances were detected in 28 of the 140 patients (20%) and included 18 deletions, seven duplications, and three unbalanced translocations. Seventeen of 24 imbalances were confirmed de novo and 19 were assumed to be causal. Excluding subtelomeric imbalances, our study identified 11 clinically relevant interstitial submicroscopic imbalances (8%). Taking this and previously reported studies into consideration, array CGH screening with a resolution of at least 1 Mb has been undertaken on 432 patients with MCA/MR. Most imbalances are non‐recurrent and spread across the genome. In at least 8.8% of these patients (38 of 432) de novo intrachromosomal alterations have been identified.

Conclusions

Array CGH should be considered an essential aspect of the genetic analysis of patients with MCA/MR. In addition, in the present study three patients were mosaic for a structural chromosome rearrangement. One of these patients had monosomy 7 in as few as 8% of the cells, showing that array CGH allows detection of low grade mosaicisims.     

Chromosomal microarray analysis,including constitutional and neoplastic disease applications, 2021 revision: a technical standard of the American College of Medical Genetics and Genomics (ACMG)

Chromosomal microarray technologies, including array comparative genomic hybridization and single-nucleotide polymorphism array, are widely applied in the diagnostic evaluation for both constitutional and neoplastic disorders. In a constitutional setting, this technology is accepted as the first-tier test for the evaluation of chromosomal imbalances associated with intellectual disability, autism, and/or multiple congenital anomalies. Furthermore, chromosomal microarray analysis is recommended for patients undergoing invasive prenatal diagnosis with one or more major fetal structural abnormalities identified by ultrasonographic examination, and in the evaluation of intrauterine fetal demise or stillbirth when further cytogenetic analysis is desired. This technology also provides important genomic data in the diagnosis, prognosis, and therapy of neoplastic disorders, including both hematologic malignancies and solid tumors. To assist clinical laboratories in the validation of chromosomal microarray methodologies for constitutional and neoplastic applications, the American College of Medical Genetics and Genomics (ACMG) Laboratory Quality Assurance Committee has developed these updated technical laboratory standards, which replace the ACMG technical standards and guidelines for microarray analysis in constitutional and neoplastic disorders previously published in 2013.     

Clinical phenotype and candidate genes for the 5q31.3 microdeletion syndrome

Array-based technologies have led to the identification of many novel microdeletion and microduplication syndromes demonstrating multiple congenital anomalies and intellectual disability (MCA/ID). We have used chromosomal microarray analysis for the evaluation of patients with MCA/ID and/or neonatal hypotonia. Three overlapping de novo microdeletions at 5q31.3 with the shortest region of overlap (SRO) of 370?kb were detected in three unrelated patients. These patients showed similar clinical features including severe neonatal hypotonia, neonatal feeding difficulties, respiratory distress, characteristic facial features, and severe developmental delay. These features are consistent with the 5q31.3 microdeletion syndrome originally proposed by Shimojima et al., providing further evidence that this syndrome is clinically discernible. The 370?kb SRO encompasses only four RefSeq genes including neuregulin 2 (NRG2) and purine-rich element binding protein A (PURA). NRG2 is one of the members of the neuregulin family related to neuronal and glial cell growth and differentiation, thus making NRG2 a good candidate for the observed phenotype. Moreover, PURA is also a good candidate because Pura-deficient mice demonstrate postnatal neurological manifestations.     

Clinical and genetic findings in Hungarian pediatric patients carrying chromosome 16p copy number variants and a review of the literature

The short arm of chromosome 16 (16p) is enriched for segmental duplications, making it susceptible to recurrent, reciprocal rearrangements implicated in the etiology of several phenotypes, including intellectual disability, speech disorders, developmental coordination disorder, autism spectrum disorders, attention deficit hyperactivity disorders, obesity and congenital skeletal disorders. In our clinical study 73 patients were analyzed by chromosomal microarray, and results were confirmed by fluorescence in situ hybridization or polymerase chain reaction. All patients underwent detailed clinical evaluation, with special emphasis on behavioral symptoms. 16p rearrangements were identified in 10 individuals. We found six pathogenic deletions and duplications of the recurrent regions within 16p11.2: one patient had a deletion of the distal 16p11.2 region associated with obesity, while four individuals had duplications, and one patient a deletion of the proximal 16p11.2 region. The other four patients carried 16p variations as second-site genomic alterations, acting as possible modifying genetic factors. We present the phenotypic and genotypic results of our patients and discuss our findings in relation to the available literature.     

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.     

Fontaine-Farriaux craniosynostosis: second report in the literature

Craniosynostosis is determined by the precocious fusion of one or more calvarial sutures leading to an abnormal skull shape. Additionally, nodular heterotopia is a disorder of neuronal migration and/or proliferation. We describe a very rare multiple congenital anomalies (MCA) syndrome in which craniosynostosis is associated with bilateral periventricular nodular heterotopia (BPNH) of the gray matter and other malformations involving hands, feet, and the gut. Clinical findings and further investigations suggest the diagnosis of craniosynostosis Fontaine-Farriaux type. To the best of our knowledge, this case is only the second report of this MCA syndrome. Based on the clinical and radiological data of the two cases reported, we hypothesize that this malformative complex may be considered a new BPNH/MCA syndrome and propose to classify it as BPNH/craniosynostosis. Previous studies demonstrated that at least two BPNH/MCA syndromes have been mapped to the Xq28 chromosomal region in which a causative gene for isolated BPNH is located. The same authors hypothesized that other BPNH syndromes could be due to microrearrangements at the same Xq28 region. Our case presents several overlapping features with some BPNH/MCA syndromes and it is possible that this new complex disorder may be caused by rearrangements at the same chromosomal region that could alter expression of different genes in Xq28.     

Smith-Magenis syndrome and Moyamoya disease in a patient with del(17)(p11.2p13.1)

Chromosomal rearrangements causing microdeletions and microduplications are a major cause of congenital malformation and mental retardation. Because they are not visible by routine chromosome analysis, high resolution whole-genome technologies are required for the detection and diagnosis of small chromosomal abnormalities. Recently, array-comparative genomic hybridization (aCGH) and multiplex ligation-dependent probe amplification (MLPA) have been useful tools for the identification and mapping of deletions and duplications at higher resolution and throughput. Smith-Magenis syndrome (SMS) is a multiple congenital anomalies/mental retardation syndrome caused by deletion or mutation of the retinoic acid induced 1 (RAI1) gene and is often associated with a chromosome 17p11.2 deletion. We report here on the clinical and molecular analysis of a 10-year-old girl with SMS and moyamoya disease (occlusion of the circle of Willis). We have employed a combination of aCGH, FISH, and MLPA to characterize an approximately 6.3 Mb deletion spanning chromosome region 17p11.2-p13.1 in this patient, with the proximal breakpoint within the RAI1 gene. Further, investigation of the genomic architecture at the breakpoint intervals of this large deletion documented the presence of palindromic repeat elements that could potentially form recombination substrates leading to unequal crossover.