Smith-Magenis syndrome (SMS), caused by del(17)p11.2, represents one of the most frequently observed human microdeletion syndromes. We have identified three copies of a low-copy-number repeat (SMS-REPs) located within and flanking the SMS common deletion region and show that SMS-REP represents a repeated gene cluster. We have isolated a corresponding cDNA clone that identifies a novel junction fragment from 29 unrelated SMS patients and a different-sized junction fragment from a patient with dup(17)p11.2. Our results suggest that homologous recombination of a flanking repeat gene cluster is a mechanism for this common microdeletion syndrome.

Studies of histone methylation have shown that H3 can be methylated at lysine 4 (Lys4) or lysine 9 (Lys9). Whereas H3-Lys4 methylation has been correlated with active gene expression, H3-Lys9 methylation has been linked to gene silencing and assembly of heterochromatin in mouse and Schizosaccharomyces pombe. The chromodomain of mouse HP1 (and Swi6 in S. pombe) binds H3 methylated at Lys9, and methylation at this site is thought to mark and promote heterochromatin assembly. We have used a well-studied model of mammalian epigenetic silencing, the human inactive X chromosome, to show that enrichment for H3 methylated at Lys9 is also a distinguishing mark of facultative heterochromatin. In contrast, H3 methylated at Lys4 is depleted in the inactive X chromosome, except in three 'hot spots' of enrichment along its length. Chromatin immunoprecipitation analyses further show that Lys9 methylation is associated with promoters of inactive genes, whereas Lys4 methylation is associated with active genes on the X chromosome. These data demonstrate that differential methylation at two distinct sites of the H3 amino terminus correlates with contrasting gene activities and may be part of a 'histone code' involved in establishing and maintaining facultative heterochromatin.

Purpose: We developed a microarray for clinical diagnosis of chromosomal disorders using large insert genomic DNA clones as targets for comparative genomic hybridization (CGH). Methods: The array contains 362 FISH-verified clones that span genomic regions implicated in over 40 known human genomic disorders and representative subtelomeric clones for each of the 41 clinically relevant human chromosome telomeres. Three or four clones from almost all deletion or duplication genomic regions and three or more clones for each subtelomeric region were included. We tested chromosome microarray analysis (CMA) in a masked fashion by examining genomic DNA from 25 patients who were previously ascertained in a genetic clinic and studied by conventional cytogenetics. A novel software package implemented in the R statistical programming language was developed for normalization, visualization, and inference. Results: The CMA results were entirely consistent with previous cytogenetic and FISH findings. For clone by clone analysis, the sensitivity was estimated to be 96.7% and the specificity was 99.1%.Major advantages of this selected human genome array include the following: interrogation of clinically relevant genomic regions, the ability to test for a wide range of duplication and deletion syndromes in a single analysis, the ability to detect duplications that would likely be undetected by metaphase FISH, and ease of confirmation of suspected genomic changes by conventional FISH testing currently available in the cytogenetics laboratory. Conclusion:The array is an attractive alternative to telomere FISH and locus-specific FISH, but it does not include uniform coverage across the arms of each chromosome and is not intended to substitute for a standard karyotype.Limitations of CMA include the inability to detect both balanced chromosome changes and low levels of mosaicism.Genet Med 2005:7(6):422-432.

We have constructed a 3.1 megabase (Mb) physical map of chromosome 17p11.2-p12, which contains a submicroscopic duplication in patients with Charcot-Marie-Tooth disease type 1A (CMT1A). We find that the CMT1A duplication is a tandem repeat of 1.5 Mb of DNA. A YAC contig encompassing the CMT1A duplication and spanning the endpoints was also developed. Several low copy repeats in 17p11.2-p12 were identified including the large (> 17 kb) CMT1A-REP unit which may be part of a mosaic repeat. CMT1A-REP flanks the 1.5 Mb CMT1A monomer unit on normal chromosome 17 and is present in an additional copy on the CMT1A duplicated chromosome. We propose that the de novo CMT1A duplication arises from unequal crossing over due to misalignment at these CMT1A-REP repeat sequences during meiosis.

BackgroundArray Comparative Genomic Hybridization (a-CGH) is a powerful molecular cytogenetic tool to detect genomic imbalances and study disease mechanism and pathogenesis. We report our experience with the clinical implementation of this high resolution human genome analysis, referred to as Chromosomal Microarray Analysis (CMA).Methods and FindingsCMA was performed clinically on 2513 postnatal samples from patients referred with a variety of clinical phenotypes. The initial 775 samples were studied using CMA array version 4 and the remaining 1738 samples were analyzed with CMA version 5 containing expanded genomic coverage. Overall, CMA identified clinically relevant genomic imbalances in 8.5% of patients: 7.6% using V4 and 8.9% using V5. Among 117 cases referred for additional investigation of a known cytogenetically detectable rearrangement, CMA identified the majority (92.5%) of the genomic imbalances. Importantly, abnormal CMA findings were observed in 5.2% of patients (98/1872) with normal karyotypes/FISH results, and V5, with expanded genomic coverage, enabled a higher detection rate in this category than V4. For cases without cytogenetic results available, 8.0% (42/524) abnormal CMA results were detected; again, V5 demonstrated an increased ability to detect abnormality. Improved diagnostic potential of CMA is illustrated by 90 cases identified with 51 cryptic microdeletions and 39 predicted apparent reciprocal microduplications in 13 specific chromosomal regions associated with 11 known genomic disorders. In addition, CMA identified copy number variations (CNVs) of uncertain significance in 262 probands; however, parental studies usually facilitated clinical interpretation. Of these, 217 were interpreted as familial variants and 11 were determined to be de novo; the remaining 34 await parental studies to resolve the clinical significance.ConclusionsThis large set of clinical results demonstrates the significantly improved sensitivity of CMA for the detection of clinically relevant genomic imbalances and highlights the need for comprehensive genetic counseling to facilitate accurate clinical correlation and interpretation.

Microdeletions within chromosome 22q11.2 cause a variable phenotype, including DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS). About 97% of patients with DGS/VCFS have either a common recurrent approximately 3 Mb deletion or a smaller, less common, approximately 1.5 Mb nested deletion. Both deletions apparently occur as a result of homologous recombination between nonallelic flanking low-copy repeat (LCR) sequences located in 22q11.2. Interestingly, although eight different LCRs are located in proximal 22q, only a few cases of atypical deletions utilizing alternative LCRs have been described. Using array-based comparative genomic hybridization (CGH) analysis, we have detected six unrelated cases of deletions that are within 22q11.2 and are located distal to the approximately 3 Mb common deletion region. Further analyses revealed that the rearrangements had clustered breakpoints and either a approximately 1.4 Mb or approximately 2.1 Mb recurrent deletion flanked proximally by LCR22-4 and distally by either LCR22-5 or LCR22-6, respectively. Parental fluorescence in situ hybridization (FISH) analyses revealed that none of the available parents (11 out of 12 were available) had the deletion, indicating de novo events. All patients presented with characteristic facial dysmorphic features. A history of prematurity, prenatal and postnatal growth delay, developmental delay, and mild skeletal abnormalities was prevalent among the patients. Two patients were found to have a cardiovascular malformation, one had truncus arteriosus, and another had a bicuspid aortic valve. A single patient had a cleft palate. We conclude that distal deletions of chromosome 22q11.2 between LCR22-4 and LCR22-6, although they share some characteristic features with DGS/VCFS, represent a novel genomic disorder distinct genomically and clinically from the well-known DGS/VCF deletion syndromes.

Purpose: Genomic rearrangements of chromosome 22q11.2, including the microdeletion associated with DiGeorge/velocardiofacial syndrome, are mediated by nonallelic homologous recombination between region-specific low-copy repeats. To date, only a small number of patients with 22q11.2 microduplication have been identified. Methods:We report the identification by array-comparative genomic hybridization of 14 individuals from eight families who harbor microduplications within the 22q11.2 region. Results: We have now observed a variety of microduplications, including the typical common ϳ3-Mb microduplication, ϳ1.5-Mb nested duplication, and smaller microduplications within and distal to the DiGeorge/velocardiofacial syndrome region, consistent with nonallelic homologous recombination using distinct low-copy repeats in the 22q11.2 DiGeorge/velocardiofacial syndrome region. These microduplications likely represent the predicted reciprocal rearrangements to the microdeletions characterized in the 22q11.2 region. The phenotypes seen in these individuals are generally mild and highly variable; familial transmission is frequently observed. Conclusions: These findings highlight the unbiased ability of array-comparative genomic hybridization to identify genomic imbalances and further define the molecular etiology and clinical phenotypes seen in microduplication 22q11.2 syndrome. Our findings also further support that the 22q11.2 region is highly dynamic with frequent rearrangements using alternative low-copy repeats as recombination substrates. Genet Med 2008:10(4):267-277.

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.