Trilochan Sahoo

Prader-Willi phenotype caused by paternal deficiency for the HBII-85 C/D box small nucleolar RNA cluster.

Prader-Willi syndrome (PWS) is caused by deficiency for one or more paternally expressed imprinted transcripts within chromosome 15q11-q13, including SNURF-SNRPN and multiple small nucleolar RNAs (snoRNAs). Balanced chromosomal translocations that preserve expression of SNURF-SNRPN and centromeric genes but separate the snoRNA HBII-85 cluster from its promoter cause PWS. A microdeletion of the HBII-85 snoRNAs in a child with PWS provides, in combination with previous data, effectively conclusive evidence that deficiency of HBII-85 snoRNAs causes the key characteristics of the PWS phenotype, although some atypical features suggest that other genes in the region may make more subtle phenotypic contributions.

Recurrent reciprocal 1q21.1 deletions and duplications associated with microcephaly or macrocephaly and developmental and behavioral abnormalities

Chromosome region 1q21.1 contains extensive and complex low-copy repeats, and copy number variants (CNVs) in this region have recently been reported in association with congenital heart defects, developmental delay, schizophrenia and related psychoses. We describe 21 probands with the 1q21.1 microdeletion and 15 probands with the 1q21.1 microduplication. These CNVs were inherited in most of the cases in which parental studies were available. Consistent and statistically significant features of microcephaly and macrocephaly were found in individuals with microdeletion and microduplication, respectively. Notably, a paralog of the HYDIN gene located on 16q22.2 and implicated in autosomal recessive hydrocephalus was inserted into the 1q21.1 region during the evolution of Homo sapiens; we found this locus to be deleted or duplicated in the individuals we studied, making it a probable candidate for the head size abnormalities observed. We propose that recurrent reciprocal microdeletions and microduplications within 1q21.1 represent previously unknown genomic disorders characterized by abnormal head size along with a spectrum of developmental delay, neuropsychiatric abnormalities, dysmorphic features and congenital anomalies. These phenotypes are subject to incomplete penetrance and variable expressivity.

Paternally inherited microdeletion at 15q11.2 confirms a significant role for the SNORD116 C/D box snoRNA cluster in Prader–Willi syndrome

Prader–Willi syndrome (PWS) is a neurobehavioral disorder manifested by infantile hypotonia and feeding difficulties in infancy, followed by morbid obesity secondary to hyperphagia. It is caused by deficiency of paternally expressed transcript(s) within the human chromosome region 15q11.2. PWS patients harboring balanced chromosomal translocations with breakpoints within small nuclear ribonucleoprotein polypeptide N (SNRPN) have provided indirect evidence for a role for the imprinted C/D box containing small nucleolar RNA (snoRNA) genes encoded downstream of SNRPN. In addition, recently published data provide strong evidence in support of a role for the snoRNA SNORD116 cluster (HBII-85) in PWS etiology. In this study, we performed detailed phenotypic, cytogenetic, and molecular analyses including chromosome analysis, array comparative genomic hybridization (array CGH), expression studies, and single-nucleotide polymorphism (SNP) genotyping for parent-of-origin determination of the 15q11.2 microdeletion on an 11-year-old child expressing the major components of the PWS phenotype. This child had an ∼236.29 kb microdeletion at 15q11.2 within the larger Prader–Willi/Angelman syndrome critical region that included the SNORD116 cluster of snoRNAs. Analysis of SNP genotypes in proband and mother provided evidence in support of the deletion being on the paternal chromosome 15. This child also met most of the major PWS diagnostic criteria including infantile hypotonia, early-onset morbid obesity, and hypogonadism. Identification and characterization of this case provide unequivocal evidence for a critical role for the SNORD116 snoRNA molecules in PWS pathogenesis. Array CGH testing for genomic copy-number changes in cases with complex phenotypes is proving to be invaluable in detecting novel alterations and enabling better genotype–phenotype correlations.

Clinical implementation of chromosomal microarray analysis: summary of 2513 postnatal cases.

Background Array 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 Findings CMA 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. Conclusions This 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.

Microdeletion 15q13.3: a locus with incomplete penetrance for autism, mental retardation, and psychiatric disorders

Background: Microdeletions within chromosome 15q13.3 are associated both with a recently recognised syndrome of mental retardation, seizures, and dysmorphic features, and with schizophrenia. Methods and results: Based on routine diagnostic testing of ∼8200 samples using array comparative genomic hybridisation, we identified 20 individuals (14 children and six parents in 12 families) with microdeletions of 15q13.3. Phenotypes in the children included developmental delay, mental retardation, or borderline IQ in most and autistic spectrum disorder (6/14), speech delay, aggressiveness, attention deficit hyperactivity disorder, and other behavioural problems. Both parents were available in seven families, and the deletion was de novo in one, inherited from an apparently normal parent in four, and inherited from a parent with learning disability and bipolar disorder in two families. Of the 14 children, six in five families were adopted, and DNA was available for only one of these 10 biological parents; the deletion was very likely inherited for one of these families with two affected children. Among the unavailable parents, two mothers were described as having mental retardation, another mother as having “mental illness”, and one father as having schizophrenia. We hypothesise that some of the unavailable parents have the deletion. Conclusions: The occurrence of increased adoption, frequent autism, bipolar disorder, and lack of penetrance are noteworthy findings in individuals with deletion 15q13.3. A high rate of adoption may be related to the presence of the deletion in biological parents. Unconfirmed histories of antisocial behaviours in unavailable biological parents raise the concern that future research may show that deletion 15q13.3 is associated with such behaviours.

Prenatal diagnosis of chromosomal abnormalities using array-based comparative genomic hybridization

Purpose: This study was designed to evaluate the feasibility of using a targeted array-CGH strategy for prenatal diagnosis of genomic imbalances in a clinical setting of current pregnancies.Methods: Women undergoing prenatal diagnosis were counseled and offered array-CGH (BCM V4.0) in addition to routine chromosome analysis. Array-CGH was performed with DNA directly from amniotic fluid cells with whole genome amplification, on chorionic villus samples with amplification as necessary, and on cultured cells without amplification.Results: Ninety-eight pregnancies (56 amniotic fluid and 42 CVS specimens) were studied with complete concordance between karyotype and array results, including 5 positive cases with chromosomal abnormalities. There was complete concordance of array results for direct and cultured cell analysis in 57 cases tested by both methods. In 12 cases, the array detected copy number variation requiring testing of parental samples for optimal interpretation. Array-CGH results were available in an average of 6 and 16 days for direct and cultured cells, respectively. Patient acceptance of array-CGH testing was 74%.Conclusion: This study demonstrates the feasibility of using array-CGH for prenatal diagnosis, including reliance on direct analysis without culturing cells. Use of array-CGH should increase the detection of abnormalities relative to the risk, and is an option for an enhanced level of screening for chromosomal abnormalities in high risk pregnancies.

Speech delay and autism spectrum behaviors are frequently associated with duplication of the 7q11.23 Williams-Beuren syndrome region

Purpose: Williams-Beuren syndrome is among the most well-characterized microdeletion syndromes, caused by recurrent de novo microdeletions at 7q11.23 mediated by nonallelic homologous recombination between low copy repeats flanking this critical region. However, the clinical phenotype associated with reciprocal microduplication of this genomic region is less well described. We investigated the molecular, clinical, neurodevelopmental, and behavioral features of seven patients with dup(7)(q11.23), including two children who inherited the microduplication from one of their parents, to more fully characterize this emerging microduplication syndrome.Methods: Patients were identified by array-based comparative genomic hybridization. Clinical examinations were performed on seven affected probands, and detailed cognitive and behavioral evaluations were carried out on four of the affected probands.Results: Our findings confirm initial reports of speech delay seen in patients with dup(7)(q11.23) and further delineate and expand the phenotypic spectrum of this condition to include communication, social interactions, and repetitive interests that are often observed in individuals diagnosed with autism spectrum disorders.Conclusions: Array-based comparative genomic hybridization is a powerful means of detecting genomic imbalances and identifying molecular etiologies in the clinic setting, including genomic disorders such as Williams-Beuren syndrome and dup(7)(q11.23). We propose that dup(7)(q11.23) syndrome may be as frequent as Williams-Beuren syndrome and a previously unrecognized cause of language delay and behavioral abnormalities. Indeed, these individuals may first be referred for evaluation of autism, even if they do not ultimately meet diagnostic criteria for an autism spectrum disorder.

Microarray-based CGH detects chromosomal mosaicism not revealed by conventional cytogenetics†

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.

Complex rearrangements in patients with duplications of MECP2 can occur by fork stalling and template switching

Duplication at the Xq28 band including the MECP2 gene is one of the most common genomic rearrangements identified in neurodevelopmentally delayed males. Such duplications are non-recurrent and can be generated by a non-homologous end joining (NHEJ) mechanism. We investigated the potential mechanisms for MECP2 duplication and examined whether genomic architectural features may play a role in their origin using a custom designed 4-Mb tiling-path oligonucleotide array CGH assay. Each of the 30 patients analyzed showed a unique duplication varying in size from approximately 250 kb to approximately 2.6 Mb. Interestingly, in 77% of these non-recurrent duplications, the distal breakpoints grouped within a 215 kb genomic interval, located 47 kb telomeric to the MECP2 gene. The genomic architecture of this region contains both direct and inverted low-copy repeat (LCR) sequences; this same region undergoes polymorphic structural variation in the general population. Array CGH revealed complex rearrangements in eight patients; in six patients the duplication contained an embedded triplicated segment, and in the other two, stretches of non-duplicated sequences occurred within the duplicated region. Breakpoint junction sequencing was achieved in four duplications and identified an inversion in one patient, demonstrating further complexity. We propose that the presence of LCRs in the vicinity of the MECP2 gene may generate an unstable DNA structure that can induce DNA strand lesions, such as a collapsed fork, and facilitate a Fork Stalling and Template Switching event producing the complex rearrangements involving MECP2.

Genomic Imbalances in Neonates With Birth Defects: High Detection Rates by Using Chromosomal Microarray Analysis

OBJECTIVES. Our aim was to determine the frequency of genomic imbalances in neonates with birth defects by using targeted array-based comparative genomic hybridization, also known as chromosomal microarray analysis. METHODS. Between March 2006 and September 2007, 638 neonates with various birth defects were referred for chromosomal microarray analysis. Three consecutive chromosomal microarray analysis versions were used: bacterial artificial chromosome–based versions V5 and V6 and bacterial artificial chromosome emulated oligonucleotide–based version V6 Oligo. Each version had targeted but increasingly extensive genomic coverage and interrogated >150 disease loci with enhanced coverage in genomic rearrangement–prone pericentromeric and subtelomeric regions. RESULTS. Overall, 109 (17.1%) patients were identified with clinically significant abnormalities with detection rates of 13.7%, 16.6%, and 19.9% on V5, V6, and V6 Oligo, respectively. The majority of these abnormalities would not be defined by using karyotype analysis. The clinically significant detection rates by use of chromosomal microarray analysis for various clinical indications were 66.7% for “possible chromosomal abnormality” ± “others” (other clinical indications), 33.3% for ambiguous genitalia ± others, 27.1% for dysmorphic features + multiple congenital anomalies ± others, 24.6% for dysmorphic features ± others, 21.8% for congenital heart disease ± others, 17.9% for multiple congenital anomalies ± others, and 9.5% for the patients referred for others that were different from the groups defined. In all, 16 (2.5%) patients had chromosomal aneuploidies, and 81 (12.7%) patients had segmental aneusomies including common microdeletion or microduplication syndromes and other genomic disorders. Chromosomal mosaicism was found in 12 (1.9%) neonates. CONCLUSIONS. Chromosomal microarray analysis is a valuable clinical diagnostic tool that allows precise and rapid identification of genomic imbalances and mosaic abnormalities as the cause of birth defects in neonates. Chromosomal microarray analysis allows for timely molecular diagnoses and detects many more clinically relevant genomic abnormalities than conventional cytogenetic studies, enabling more informed decision-making and management and appropriate assessment of recurrence risk.