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Insertional translocations (ITs) are rare events that require at least three breaks in the chromosomes involved and thus qualify as complex chromosomal rearrangements (CCR). In the current study, we identified 40 ITs from approximately 18,000 clinical cases (1:500) using array-comparative genomic hybridization (aCGH) in conjunction with fluorescence in situ hybridization (FISH) confirmation of the aCGH findings, and parental follow-up studies. Both submicroscopic and microscopically visible IT events were detected. They were divided into three major categories: (1) simple intrachromosomal and interchromosomal IT resulting in pure segmental trisomy, (2) complex IT involving more than one abnormality, (3) deletion inherited from a parent with a balanced IT resulting in pure segmental monosomy. Of the cases in which follow-up parental studies were available, over half showed inheritance from an apparently unaffected parent carrying the same unbalanced rearrangement detected in the propositi, thus decreasing the likelihood that these IT events are clinically relevant. Nevertheless, we identified six cases in which small submicroscopic events were detected involving known disease-associated genes/genomic segments and are likely to be pathogenic. We recommend that copy number gains detected by clinical aCGH analysis should be confirmed using FISH analysis whenever possible in order to determine the physical location of the duplicated segment. We hypothesize that the increased use of aCGH in the clinic will demonstrate that IT occurs more frequently than previously considered but can identify genomic rearrangements with unclear clinical significance., (Copyright 2010 Wiley-Liss, Inc.)

Dopamine beta-hydroxylase (DBH) deficiency is characterized by a lack of sympathetic noradrenergic function. Affected individuals exhibit profound deficits in autonomic regulation of cardiovascular function. The diagnosis of DBH deficiency is based on clinical findings, biochemical studies, and sequencing of DBH gene. We report here the characterization of a mosaic cytogenetic abnormality detected by array-CGH in a 16-year-old female with primary DBH deficiency together with dysmorphic features. These features could not be explained by DBH deficiency leading to further investigation. Karyotype was reported normal (46,XX), while a targeted genomic array-CGH revealed a mosaic loss for a segment of at least 1 Mb across 11p13. This segmental loss included the PAX6 and WT1 genes within the WAGR syndrome critical region. Interestingly, the derivative chromosome 11 was observed only in about 28% of cells analyzed. Utilizing a genome-wide oligonucleotide-based array, the deletion segment was estimated to encompass a segment of approximately 10 Mb. Mosaic deletions of 11p13 in WAGR are extremely uncommon. In this case it is distinctly possible that the patient's bilateral iris colobomata might be a manifestation, albeit abbreviated, of the haploinsufficiency for PAX6. This case highlights the importance of cytogenetic analysis when a mutation alone cannot account for the complete phenotype. It also emphasizes the enhanced ability of high-resolution array-CGH techniques in accurately detecting subtle rearrangements in a mosaic form. Finally, it demonstrates the possible phenotypic effects of low-level PAX6 haploinsufficiency in a dosage-sensitive manner., ((c) 2010 Wiley-Liss, Inc.)

We describe a patient with multiple congenital anomalies including deafness, lacrimal duct stenosis, strabismus, bilateral cervical sinuses, congenital cardiac defects, hypoplasia of the corpus callosum, and hypoplasia of the cerebellar vermis. Mutation analysis of EYA1, SIX1, and SIX5, genes that underlie otofaciocervical and/or branchio-oto-renal syndrome, was negative. Pathologic diagnosis of the excised cervical sinus tracts was revised on re-examination to heterotopic salivary gland tissue. Using high resolution chromosomal microarray analysis, we identified a novel 2.52 Mb deletion at 19p13.12, which was confirmed by fluorescent in situ hybridization and demonstrated to be a de novo mutation by testing of the parents. Overall, deletions of chromosome 19p13 are rare., (2009 Wiley-Liss, Inc.)

We report on a 26-month-old boy with developmental delay and multiple congenital anomalies, including many features suggestive of either branchiootorenal syndrome (BOR) or oculoauriculovertebral spectrum (OAVS). Chromosomal microarray analysis (CMA) initially revealed a copy-number gain with a single BAC clone (RP11-79M1) mapping to 14q23.1. FISH analysis showed that the third copy of this genomic region was inserted into the long arm of one chromosome 13. The same pattern was also seen in the chromosomes of the father, who has mental retardation, short stature, hypernasal speech, and minor craniofacial anomalies, including tall forehead, and crowded dentition. Subsequent whole genome oligonucleotide microarray analysis revealed an approximately 11.79 Mb duplication of chromosome 14q22.3-q23.3 and a loss of an approximately 4.38 Mb sequence in 13q21.31-q21.32 in both the propositus and his father and FISH supported the apparent association of the two events. Chromosome 14q22.3-q23.3 contains 51 genes, including SIX1, SIX6, and OTX2. A locus for branchiootic syndrome (BOS) has been mapped to 14q21.3-q24.3, and designated as branchiootic syndrome 3 (BOS3). Interestingly, mutations in SIX1 have been reported in patients with BOR/BOS3. We propose that the increased dosage of SIX1, SIX6, or OTX2 may be responsible for the BOR and OAVS-like features in this family., (Copyright 2008 Wiley-Liss, Inc.)

Recent advances in molecular cytogenetics enable identification of small chromosomal aberrations that are undetectable by routine chromosome banding in 5-20% of patients with mental retardation/developmental delay (MR/DD) and dysmorphism. The aim of this study was to compare the clinical usefulness of two molecular cytogenetic techniques, metaphase high-resolution comparative genomic hybridization (HR-CGH) and targeted array CGH, also known as Chromosomal Microarray Analysis (CMA). A total of 116 patients with unexplained mild to severe MR and other features suggestive of a chromosomal abnormality with apparently normal or balanced karyotypes were analyzed using HR-CGH (43 patients) and/or CMA (91 patients). Metaphase HR-CGH detected seven interstitial deletions (16.3%). Rare deletions of chromosomes 16 (16p11.2p12.1) and 8 (8q21.11q21.2) were identified. Targeted CMA revealed copy-number changes in 19 of 91 patients (20.8%), among which 11 (11.8%) were clinically relevant, 6 (6.5%) were interpreted as polymorphic variants and 2 (2.1%) were of uncertain significance. The changes varied in size from 0.5 to 12.9 Mb. In summary, our results show that metaphase HR-CGH and array CGH techniques have become important components in cytogenetic diagnostics, particularly for detecting cryptic constitutional chromosome imbalances in patients with MR, in whom the underlying genetic defect is unknown. Additionally, application of both methods together increased the detection rates of genomic imbalances in the tested groups., (Copyright 2008 Wiley-Liss, Inc.)

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., ((c) 2008 Wiley-Liss, Inc.)

Recurrent genomic alterations, mainly losses and gains of specific chromosomes and/or regions, in chronic lymphocytic leukemia (CLL) are recognized as important independent predictors of prognosis and disease progression. The current standard clinical practice for identifying these alterations is chromosome analysis and in situ hybridization with probes targeting 4-5 chromosome regions. We sought to apply array comparative genomic hybridization (array-CGH) technology for the simultaneous detection of genomic imbalances of all loci implicated in CLL. DNA from enriched B-cells from CLL patients were analyzed by array-CGH on a customized CLL BAC array. Copy number changes were detected in 87% of samples with a sensitivity of 100% in samples with clonal abnormalities present in at least 23% of the cells. Furthermore, in nine cases genomic alterations were observed that were undetectable by standard cytogenetic and/or FISH analyses. One of these patients had a 13q14 deletion that was missed by the clinical CLL FISH panel probe set. Our results suggest that a subset of potentially significant genomic alterations in CLL is being missed by the current available techniques. Furthermore, this pilot study clearly shows the robustness, high sensitivity, and high specificity for the targeted CLL microarray analysis as well as the potential for use in routine screening in CLL., ((c) 2007 Wiley-Liss, Inc.)

Noonan syndrome is an autosomal dominant disorder with an estimated incidence of 1 in 1,000 to 1 in 2,500 live births. It is characterized by postnatal-onset short stature, characteristic facial changes, webbed neck, pectus carinatum, or excavatum, congenital heart defects, and bleeding abnormalities. Gain-of-function mutations in the PTPN11, KRAS, SOS1, and RAF1 genes that are components of the RAS/MEPK signaling pathway are identified in about 70-85% of individuals with Noonan syndrome. We report here a case of duplication of chromosome region 12q24.11q24.23 identified by array comparative genomic hybridization (aCGH) that includes the PTPN11 gene in a 3-year-old girl with apparent Noonan syndrome. The patient presented with postnatal-onset failure-to-thrive, developmental delay, microcephaly, velopalatal incompetence, pectus excavatum, coarctation of aorta, atrial and ventricular septal defects, decreased muscle tone, and minor facial anomalies consistent with Noonan syndrome. At 3 years of age her speech, gross and fine motor development were at the level of a 12-18 month old child. This degree of developmental delay was atypical for an individual with Noonan syndrome, raising concerns for a chromosomal abnormality. Array-CGH showed an interstitial duplication of 10 Mb including the PTPN11 gene. Sequencing of PTPN11, KRAS, SOS1 and the coding region of RAF1 did not identify mutations. The increased gene dosage of the PTPN11 gene in the form of duplication is expected to have the same consequence as gain-of-function mutations seen in Noonan syndrome. We propose that at least some of the 15-30% of individuals with Noonan syndrome who do not have a mutation by sequencing may have a gain in copy number of PTPN11 and recommend that comprehensive testing for Noonan syndrome should include analysis for copy number changes of PTPN11., (Copyright 2008 Wiley-Liss, Inc.)

Somatic chromosomal mosaicism is a well-established cause for birth defects, mental retardation, and, in some instances, specific genetic syndromes. We have developed a clinically validated, targeted BAC clone array as a platform for comparative genomic hybridization (aCGH) to enable detection of a wide range of pathologic copy number changes in DNA. It is designed to provide high sensitivity to detect well-characterized submicroscopic micro-deletion and duplication disorders while at the same time minimizing detection of variation of uncertain clinical significance. In the course of studying 2,585 samples submitted to our clinical laboratory, chromosomal mosaicism was detected in 12 patient samples; 10 of these cases were reported to have had a normal blood chromosome analysis. This enhanced ability of aCGH to detect mosaicism missed by routine chromosome analysis may be due to some combination of testing multiple cell lineages and/or failure of cytogenetically abnormal T lymphocytes to respond to mitogens. This suggests that aCGH may detect somatic chromosomal mosaicism that would be missed by conventional cytogenetics., ((c) 2007 Wiley-Liss, Inc.)

Chromosomal microarray analysis (CMA) by array-based comparative genomic hybridization (CGH) is a new clinical test for the detection of well-characterized genomic disorders caused by chromosomal deletions and duplications that result in gene copy number variation (CNV). This powerful assay detects an abnormality in approximately 7-9% of patients with various clinical phenotypes, including mental retardation. We report here on the results found in a 6-year-old girl with mildly dysmorphic facies, obesity, and marked developmental delay. CMA was requested and showed a heterozygous loss in copy number with clones derived from the genomic region cytogenetically defined as Xq27.3-Xq28. This loss was not cytogenetically visible but was seen on FISH analysis with clones from the region. Further studies confirmed a loss of one copy each of the FMR1, FMR2, and IDS genes (which are mutated in Fragile X syndrome, FRAXE syndrome, and Hunter syndrome, respectively). Skewed X-inactivation has been previously reported in girls with deletions in this region and can lead to a combined Fragile X/Hunter syndrome phenotype in affected females. X-inactivation and iduronate 2-sulfatase (IDS) enzyme activity were therefore examined. X-inactivation was found to be random in the child's peripheral leukocytes, and IDS enzyme activity was approximately half of the normal value. This case demonstrates the utility of CMA both for detecting a submicroscopic chromosomal deletion and for suggesting further testing that could possibly lead to therapeutic options for patients with developmental delay.