Clinical application of 2.7M Cytogenetics array for CNV detection in subjects with idiopathic autism and/or intellectual disability.

Higher resolution whole-genome arrays facilitate the identification of smaller copy number variations (CNVs) and their integral genes contributing to autism and/or intellectual disability (ASD/ID). Our study describes the use of one of the highest resolution arrays, the Affymetrix(®) Cytogenetics 2.7M array, coupled with quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments (QMPSF) for detection and validation of small CNVs. We studied 82 subjects with ASD and ID in total (30 in the validation and 52 in the application cohort) and detected putatively pathogenic CNVs in 6/52 cases from the application cohort. This included a 130-kb maternal duplication spanning exons 64-79 of the DMD gene which was found in a 3-year-old boy manifesting autism and mild neuromotor delays. Other pathogenic CNVs involved 4p14, 12q24.31, 14q32.31, 15q13.2-13.3, and 17p13.3. We established the optimal experimental conditions which, when applied to select small CNVs for QMPSF confirmation, reduced the false positive rate from 60% to 25%. Our work suggests that selection of small CNVs based on the function of integral genes, followed by review of array experimental parameters resulting in highest confirmation rate using multiplex PCR, may enhance the usefulness of higher resolution platforms for ASD and ID gene discovery.

Outcome of array CGH analysis for 255 subjects with intellectual disability and search for candidate genes using bioinformatics.

Array CGH enables the detection of pathogenic copy number variants (CNVs) in 5-15% of individuals with intellectual disability (ID), making it a promising tool for uncovering ID candidate genes. However, most CNVs encompass multiple genes, making it difficult to identify key disease gene(s) underlying ID etiology. Using array CGH we identified 47 previously unreported unique CNVs in 45/255 probands. We prioritized ID candidate genes using five bioinformatic gene prioritization web tools. Gene priority lists were created by comparing integral genes from each CNV from our ID cohort with sets of training genes specific either to ID or randomly selected. Our findings suggest that different training sets alter gene prioritization only moderately; however, only the ID gene training set resulted in significant enrichment of genes with nervous system function (19%) in prioritized versus non-prioritized genes from the same de novo CNVs (7%, p < 0.05). This enrichment further increased to 31% when the five web tools were used in concert and included genes within mitogen-activated protein kinase (MAPK) and neuroactive ligand-receptor interaction pathways. Gene prioritization web tools enrich for genes with relevant function in ID and more readily facilitate the selection of ID candidate genes for functional studies, particularly for large CNVs.

Genomic changes detected by array CGH in human embryos with developmental defects.

Developmental abnormalities of human embryos can be visualized in utero using embryoscopy. Our previous embryoscopic and genetic evaluations detected developmental abnormalities in the majority of both euploid (74%) and aneuploid or polyploid (90%) miscarriages. Since we found the pattern of morphological changes to be similar in euploid and non-euploid embryos, we proposed that lethal submicroscopic changes, not detected by standard chromosome testing, may be responsible for miscarriage of euploid embryos. Whole genome oligo and bacterial artificial chromosome array comparative genome hybridization (CGH) was used to screen for submicroscopic chromosomal changes (DNA copy number variants or CNVs) in 17 euploid embryonic miscarriages, with a range of developmental abnormalities documented by embryoscopy. The CNV breakpoints were refined using a custom array (Agilent) with high resolution coverage of the CNVs. Six unique CNVs, previously not reported, were identified in 5 of the 17 embryos (29% of all cases or 50% of cases studied with higher resolution arrays). All six unique CNVs were <250 kb in size. On the basis of parental array CGH analysis, a de novo origin of a CNV was determined for one embryo (at 13q32.1) and suspected for another (at 10p15.3). Three CNVs, at Xq28, 1q25.3 and 7p14.3, were inherited and a CNV at 17p13.1 was of unknown origin. The genes contained within these unique CNVs will be discussed, with specific reference to rearrangements of syntaxin and tryptophan-aspartic acid (WD) repeat genes. Our report describes for the first time, de novo and inherited unique CNVs in euploid human embryos with specific developmental defects.

Identification of copy number variants in miscarriages from couples with idiopathic recurrent pregnancy loss.

BACKGROUND: Recurrent pregnancy loss (RPL), defined as two or more miscarriages, affects 3-5% of couples trying to establish a family. Despite extensive evaluation, no factor is identified in ∼40% of cases. In this study, we investigated the possibility that submicroscopic chromosomal changes, not detectable by conventional cytogenetic analysis, exist in miscarriages with normal karyotypes (46,XY or 46,XX) from couples with idiopathic RPL. METHODS: Array comparative genomic hybridization (array-CGH) was used to assess for DNA copy number variants (CNVs) in 26 miscarriages with normal karyotypes. Parental array-CGH analysis was performed to determine if miscarriage CNVs were de novo or inherited. RESULTS: There were 11 unique (previously not described) CNVs, all inherited, identified in 13 miscarriages from 8 couples. The maternal origin of two CNVs was of interest as they involved the imprinted genes TIMP2 and CTNNA3, which are only normally expressed from the maternal copy in the placenta. Two additional cohorts, consisting of 282 women with recurrent miscarriage (RM) and 61 fertile women, were screened for these two CNVs using a Quantitative Multiplex Fluorescent PCR of Short Fragments assay. One woman with RM, but none of the fertile women, carried the CTNNA3-associated CNV. CONCLUSIONS: This preliminary study shows that array-CGH is useful for detecting CNVs in cases of RPL. Further investigations of CNVs, particularly those involving genes that are imprinted in placenta, in women with RPL could be worthwhile.

Molecular cytogenetic investigation of two patients with Y chromosome rearrangements and intellectual disability.

We describe two males with intellectual disability (ID) and facial dysmorphism, both of whom have non-mosaic Y chromosome rearrangements resulting in deletions of large portions of the Y chromosome. Patient A, with ID, mild dysmorphism, speech delay, Duane anomaly of the eye, hypermetropia and conductive hearing loss, had two structurally rearranged Y chromosomes resulting in both p and q arm deletions in addition to a Yp duplication. Patient B, also with speech and language delay, developmental delay and short stature, had an interstitial deletion of Yq11.21-11.23. Array-CGH excluded the presence of additional submicroscopic rearrangements at the 1 Mb resolution level. A review of males with Y chromosome rearrangements and ID was performed. Our study provides a more detailed molecular cytogenetic assessment of Y rearrangements in individuals with ID than has been previously possible, and facilitates assessment and comparison of other individuals with a Y chromosome rearrangement.

Putatively benign copy number variants in subjects with idiopathic autism spectrum disorder and/or intellectual disability.

Putatively benign copy number variants (bCNVs) can be broadly defined as DNA copy number gains or losses that do not lead to a recognizable clinical phenotype. Detection of bCNVs in genomes of clinically healthy individuals is increasing with the widespread use of whole genome arrays of different resolutions and the use of sequence comparison methods. However, the role of bCNVs in human disease susceptibility and phenotype diversity is mostly unknown. In order to explore a potential role of bCNVs in the susceptibility to and/or pathogenesis of human neurodevelopmental disorders we examined the frequency and type of common bCNVs (detected in >/=2 independent control studies) amongst 221 subjects with an autism spectrum disorder (ASD) and/or intellectual disability (ID) in comparison to 40 controls using three array platforms of increasing resolution (Spectral Genomics (1 Mb), Agilent (0.03 Mb) and NimbleGen (0.01 Mb)). We determined that the number of bCNVs/subject, type and frequency of most common bCNVs were similar for both the test and control cohorts when the same array platform was used. The comparison of the 'load' of bCNVs (i.e. number/subject) to a standardized metric of phenotypic features (see de Vries et al., 2001) in 91 ASD subjects revealed that a phenotype score >/=4 is significantly more common (P < 0.05) in persons with an ASD having one or more bCNVs via 1 Mb array-CGH, whereas individuals without any recognizable bCNVs are significantly more likely to have a less complex phenotype and a score

Autism-associated familial microdeletion of Xp11.22.

We describe two brothers with autistic disorder, intellectual disability (ID) and cleft lip/palate with a microdeletion of Xp11.22 detected through screening individuals with autism spectrum disorders (ASDs) for microdeletions and duplications using 1-Mb resolution array comparative genomic hybridization. The deletion was confirmed by fluorescence in situ hybridization/real-time quantitative polymerase chain reaction (RT-qPCR) and shown to be inherited from their unaffected mother who had skewed (100%) X inactivation of the aberrant chromosome. RT-qPCR characterization of the del(X)(p11.22) region ( approximately 53,887,000-54,359,000 bp) revealed complete deletion of the plant homeodomain finger protein 8 (PHF8) gene as well as deletions of the FAM120C and WNK lysine-deficient protein kinase 3 (WNK3) genes, for which a definitive phenotype has not been previously characterized. Xp11.2 is a gene-rich region within the critical linkage interval for several neurodevelopmental disorders. Rare interstitial microdeletions of Xp11.22 have been recognized with ID, craniofacial dysmorphism and/or cleft lip/palate and truncating mutations of the PHF8 gene within this region. Despite evidence implicating genes within Xp11.22 with language and cognitive development that could contribute to an ASD phenotype, their involvement with autism has not been systematically evaluated. Population screening of 481 (319 males/81 females) and 282 X chromosomes (90 males/96 females) in respective ASD and control cohorts did not identify additional subjects carrying this deletion. Our findings show that in addition to point mutations, a complete deletion of the PHF8 gene is associated with the X-linked mental retardation Siderius-Hamel syndrome (OMIM 300263) and further suggest that the larger size of the Xp11.22 deletion including genes FAM120C and WNK3 may be involved in the pathogenesis of autism.

Clinical and molecular cytogenetic characterisation of a newly recognised microdeletion syndrome involving 2p15-16.1.

BACKGROUND: During whole genome microarray-based comparative genomic hybridisation (array CGH) screening of subjects with idiopathic intellectual disability, we identified two unrelated individuals with a similar de novo interstitial microdeletion at 2p15-2p16.1. Both individuals share a similar clinical phenotype including moderate to severe intellectual disability, autism/autistic features, microcephaly, structural brain anomalies including cortical dysplasia/pachygyria, renal anomalies (multicystic kidney, hydronephrosis), digital camptodactyly, visual impairment, strabismus, neuromotor deficits, communication and attention impairments, and a distinctive pattern of craniofacial features. Dysmorphic craniofacial features include progressive microcephaly, flat occiput, widened inner canthal distance, small palpebral fissures, ptosis, long and straight eyelashes, broad and high nasal root extending to a widened, prominent nasal tip with elongated, smooth philtrum, rounding of the upper vermillion border and everted lower lips. METHODS: Clinical assessments, and cytogenetic, array CGH and fluorescence in situ hybridisation (FISH) analyses were performed. RESULTS: The microdeletions discovered in each individual measured 4.5 Mb and 5.7 Mb, spanning the chromosome 2p region from 57.2 to 61.7 Mb and from 56 to 61.7 Mb, respectively. Each deleted clone in this range demonstrated a dosage reduction from two to one copy in each proband except for clone RP11-79K21, which was present in three copies in each proband and in four copies in their respective parents (two per each chromosome 2 homologue). DISCUSSION: The common constellation of features found in the two affected subjects indicates that they have a newly recognised microdeletion syndrome involving haploinsufficiency of one or more genes deleted within at least a 4.5-Mb segment of the 2p15-16.1 region.

A double-blind crossover comparative study of the efficacy of single daily doses of conventional and slow-release metoprolol.

The antihypertensive effect of slow-release metoprolol was studied in 50 patients suffering from mild to moderate essential hypertension. In a double-blind crossover study, the effect of slow-release metoprolol was compared with conventional metoprolol for efficacy and tolerability in 40 patients. After a 2-week period on placebo, patients received either 200 mg slow-release metoprolol once daily or 200 mg conventional metoprolol once daily for 6 weeks before crossing over to the alternative dosage form for a further 6 weeks. The results showed that both slow-release and conventional metoprolol were equipotent and reduced significantly the pulse rate and also the supine, sitting and standing systolic and diastolic blood pressures. Slow-release metoprolol also reduced the exercise blood pressure in 10 patients following 2-weeks' administration of 200 mg daily in a single dose. Good patient compliance with the medication was observed. No adverse effect attributable to slow-release metoprolol or conventional metoprolol was detected throughout the study.

Assessment of the efficacy and acceptability of an acebutolol/hydrochlorothiazide combination in the treatment of mild to moderate essential hypertension.

The efficacy and acceptability of single daily doses of a fixed combination of 400 mg acebutolol and 25 mg hydrochlorothiazide were assessed in an open study of 30 patients suffering from mild to moderate essential hypertension. Results from 20 patients who completed the 6-months' active drug period, and 9 patients whose treatment was terminated at various times before the end of this period, indicated significant reduction in the systolic and diastolic blood pressure in supine, sitting and standing positions. This effect was apparent from the first month on drug therapy and was maintained throughout the 6-month trial. In no patient was withdrawal from this study attributable to side-effects of the drug. Results indicated good control of hypertension by a single dose of the combined drugs and good patient compliance was observed during the study. Potassium loss, frequently experienced during hydrochlorothiazide treatment, was not observed in the presence of acebutolol.

15q duplication associated with autism in a multiplex family with a familial cryptic translocation t(14;15)(q11.2;q13.3) detected using array-CGH.

Autism spectrum disorders (ASDs) are a group of neurodevelopmental disorders with a strong genetic aetiology. In approximately 1% of cases, duplication of the 15q11-13 region has been reported. We report the clinical, array-comparative genomic hybridization (CGH) and cytogenetic evaluation of two individuals from a multiplex family demonstrating autism due to a maternally inherited gain of 15q11-13. Our findings indicate that unlike most 15q11-13 gains, which are caused by interstitial duplication of this region or supernumerary marker chromosomes deriving from proximal 15q, the 15q gain in this family is the result of abnormal segregation of a cryptic familial translocation with breakpoints at 14q11.2 and 15q13.3. The affected members of this family were found to have a normal karyotype at >550 band resolution. This translocation was identified using the 1-Mb resolution whole genome array (Spectral Genomics). The affected individuals have a gain of seven clones from proximal 15q, a loss of two clones from proximal 14q and a gain of two clones from 6q. Fluorescent in situ hybridization (FISH) analysis with clones from chromosomes 14 and 15, combined with DAPI reverse banding, showed an abnormal karyotype with one normal chromosome 15 and the der(15) t(14;15)(q11.2.;q13.3), resulting in the gain of proximal 15q and the loss of proximal 14q in affected individuals. The duplication of two clones from 6q in the affected subjects was also found in unaffected members of the family. Our findings suggest that the gain of 15q in autism may in some cases be due to cryptic translocations with breakpoints in the pericentromic regions of chromosome 15 and a different acrocentric chromosome. Variation in the size of pericentromic regions of any acrocentric chromosome may justify karyotype and FISH studies of autistic probands and their parents using probes from the 15q proximal region to determine recurrence risk for autism in some families.

A variant Cri du Chat phenotype and autism spectrum disorder in a subject with de novo cryptic microdeletions involving 5p15.2 and 3p24.3-25 detected using whole genomic array CGH.

Cri du Chat syndrome (CdCs) is a well-defined clinical entity, with an incidence of 1/15,000 to 1/50,000. The critical region for CdCs has been mapped to 5p15, with the hallmark cat-like cry sublocalized to 5p15.3 and the remaining clinical features to 5p15.2. We report findings in a subject with a de novo t(5;7)(p15.2;p12.2) and an inv(3)(p24q24), who was found to have a cryptic microdeletion in the critical region for CdCs detected using a 1-Mb genomic microarray. In addition to 5p deletion, the proband had a de novo single clone loss at the 3p breakpoint of inv(3)(p24q24) and a familial single clone deletion at 18q12. Deletions were confirmed using microsatellite analysis and fluorescence in situ hybridization. The 5p deletion encompasses approximately 3 Mb, mapping to the border between bands 5p15.2 and 5p15.31. The single clone deletion on chromosome 3 maps to 3p24.3-3p25, for which there is no known phenotype. The clinical features of our proband differ from the characteristic CdC phenotype, which may reflect the combined effect of the two de novo microdeletions and/or may further refine the critical region for CdCs. Typical features of CdCs that are present in the proband include moderate intellectual disability, speech, and motor delay as well as dysmorphic features (e.g. broad and high nasal root, hypertelorism, and coarse facies). Expected CdCs features that are not present are growth delay, microcephaly, round facies, micrognathia, epicanthal folds, and the signature high-pitched cry. Behavioral traits in this subject included autism spectrum disorder, attention-deficit hyperactivity disorder, and unmanageable behavior including aggression, tantrums, irritability, and self-destructive behavior. Several of these behaviors have been previously reported in patients with 5p deletion syndrome. Although most agree on the cat-cry critical region (5p15.3), there is discrepancy in the precise location and size of the region associated with the more severe manifestations of CdCs. The clinical description of this proband and the characterization of his 5p deletion may help to further refine the phenotype-genotype associations in CdCs and autism spectrum disorder.

Submicroscopic deletions and duplications in individuals with intellectual disability detected by array-CGH.

Intellectual disability (ID) affects about 3% of the population (IQ < 70), and in about 40% of moderate (IQ 35-49) to severe ID (IQ < 34), and 70% of cases of mild ID (IQ 50-70), the etiology of the disease remains unknown. It has long been suspected that chromosomal gains and losses undetectable by routine cytogenetic analysis (i.e., less than 5-10 Mb in size) are implicated in ID of unknown etiology. Array CGH has recently been used to perform a genome-wide screen for submicroscopic gains and losses in individuals with a normal karyotype but with features suggestive of a chromosome abnormality. In two recent studies, the technique has demonstrated a approximately 15% detection rate for de novo copy number changes of individual clones or groups of clones. Here, we describe a study of 22 individuals with mild to moderate ID and nonsyndromic pattern of dysmorphic features suspicious of an underlying chromosome abnormality, using the 3 Mb and 1 Mb commercial arrays (Spectral Genomics). Deletions and duplications of 16 clones, previously described to show copy number variability in normal individuals [Iafrate et al., 2004; Lapierre et al., 2004; Schoumans et al., 2004; Vermeesch et al., 2005] were seen in 21/22 subjects and were considered polymorphisms. In addition, three subjects showed submicroscopic deletions and duplications not previously reported as normal variants. Two of these submicroscopic changes were of de novo origin (microdeletions at 7q36.3 and a microduplication at 11q12.3-13.1) and one was of unknown origin as parental testing of origin could not be performed (microduplication of Xp22.3). The clinical description of the three subjects with submicroscopic chromosomal changes at 7q36.3, 11q12.3-13.1, Xp22.3 is provided.