Mes Lewis

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.

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 ∼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.

Parental Perspectives on the Causes of an Autism Spectrum Disorder in their Children

Autism Spectrum Disorders (ASDs) are complex neurodevelopmental disorders with many biological causes, including genetic, syndromic and environmental. Such etiologic heterogeneity impacts considerably upon parents’ needs for understanding their childs diagnosis. A descriptive survey was designed to investigate parental views on the cause(s) of ASD in their child. Among the 41 parents who replied to the questionnaire, genetic influences (90.2%), perinatal factors (68.3%), diet (51.2%), prenatal factors (43.9%) and vaccines (40.0%) were considered to be the most significant contributory factors. Parents reported inaccurately high recurrence risks, misperceptions of the contribution of various putative factors, feelings of guilt and blame regarding their childs diagnosis, as well as a lack of advocacy for genetic counseling by non-geneticist professionals. This study offers clinicians and researchers further insight into what parents believe contributed to their childs diagnosis of ASD and will help facilitate genetic counseling for these families.

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 (∼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.

Autism severity is associated with child and maternal MAOA genotypes.

Cohen IL, Liu X, Lewis MES, Chudley A, Forster‐Gibson C, Gonzalez M, Jenkins EC, Brown WT, Holden JJA. Autism severity is associated with child and maternal MAOA genotypes.

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.

Phenomic determinants of genomic variation in autism spectrum disorders

Background: Autism spectrum disorders (ASDs) are common, heritable neurobiologic conditions of unknown aetiology confounded by significant clinical and genetic heterogeneity. Methods: This study evaluated a broad categorisation of phenotypic traits (or phenome) for 100 subjects with Autism Diagnostic Interview-Revised/Autism Diagnostic Observation Schedule-Generic (ADI-R/ADOS-G) confirmed idiopathic ASD undergoing 1 Mb bacterial artificial chromosome (BAC) array comparative genomic hybridisation (CGH). Results and conclusions: Array CGH uncovered nine different pathogenic copy number variants (pCNVs) in 9/100 ASD subjects having complex phenotypes (ASD± intellectual disability (ID; IQ<70)) and/or physical anomalies), normal karyotype, fragile X analysis, and comprehensive evaluation by a clinical geneticist. Unique pCNVs in our cohort included del(5)(p15.2p15.31) (2.4 Mb), del(3)(p24.3) (0.1 Mb) and dup(18)(p11.3)(0.9 Mb). Five pCNVs were recurrent in our cohort or were previously described in subjects with ASD±ID: (dup(7)(q11.23)(1.5 Mb); del(2)(p15p16.1) (6.1 Mb and 7.9 Mb); del(14)(q11.2) (0.7 Mb) and dup(15)(q11q13) (10 Mb), including del(X)(p11.22) (470 Kb) in two autistic brothers. Male: female distribution in subjects with pCNVs was reduced to 1.25:1 from 3.2:1 in the original cohort. The authors stratified the study population according to a broad spectrum of clinical features and correlated specific phenotypes with respect to CNV load and pathogenicity. The findings indicate increased prevalence of pCNVs in subjects with microcephaly (<2nd centile; n = 2 of 4 ASD subjects with microcephaly; p = 0.04), and ID (n = 9 of 64 subjects with ASD and ID; p = 0.02). Interestingly, in the absence of ID co-morbidity with an ASD, no pCNVs were found. The relationship between parental ages at delivery and CNV load and pathogenicity was also explored.

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.

Loud and clear evidence for gene silencing by epigenetic mechanisms in autism spectrum and related neurodevelopmental disorders

The human genome is a structurally and functionally complex structure comprising uniform genomic information for different cell functions within a complex organism. By contrast, the epigenome varies from tissue to tissue, controlling the differential expression of genes and providing a specific identity to each cell type. DNA and the genes it encodes are structurally integrated with a number of histone proteins forming chromatin; the DNA–protein scaffold for chromosomes. The histone proteins that support DNA are known to play a very important role in DNAmethylation and differential gene expression. Accordingly, there is increasing evidence to support the presence of a silent or hidden ‘histone code’; histone modifications, acting alone or in specific combinations that provide binding platforms for chromatin-associated proteins that initiate or block gene transcription.

Human artificial chromosomes: emerging from concept to reality in biomedicine.

The major challenge for mammalian artificial chromosomes (MACs) and human artificial chromosomes (HACs) as applied to gene transfer technology is to yield more stable, predictable and natural regulation and expression of single, large or multiple genes and proteins without disturbing the integrity and function of the host cell chromosomes. By definition, MACs/HACs are fully autonomous chromosomal vectors that do not integrate within the host cell genome, and are amenable to manipulation of copy number and contain sufficient genomic sequence flanking the therapeutic gene to recapitulate the correct chromosomal context for tissue-specific and temporal gene regulation. The development of HACs ultimately holds promise for our better understanding the intricacies of human chromosome structure and function for application in biotechnology and biomedicine and for advancing their utility in the fields of functional genomics, animal transgenics and human gene therapy. In general, MACs require a minimal structural array of three different types of cis-acting DNA sequence in order to function efficiently as autonomous elements within host cells. This includes the presence of multiple tandem telomeric sequences, specific origins of replication and the presence of alphoid satellite DNA conferring centromeric function for mitotic stability in dividing cells (Fig. 2). Co et al. (2000) have adopted a novel in 6i6o methodology, based upon the application of a mammalian (murine) satellite DNA-based artificial chromosome (SATAC), to investigate their utility in the generation of transgenic animals. This approach is based upon the induction of large-scale amplification and formation of de no6o centromeres and chromosomes in rodent cells and upon the integration of exogenous DNA sequences into the pericentric or ribosomal (rDNA) regions of mouse chromosomes. In their study, conventional microinjection techniques were modified to enable the delivery of 60-Mb SATACs into the pronuclei of fertilized murine and bovine oocytes rather than conventional ES cells. The 60-Mb prototype SATAC contains neutral non-coding heterochromatic sequences that serve as a core backbone interspersed with multiple copies of foreign DNA consisting of the lacZ (b-galactosidase) and hph (hygromycin phosphotransferase) marker genes. Surviving embryos were first studied in 6itro, scored for normal development, b-galactosidase expression and studied by FISH analysis. These findings confirmed an absence of adverse effect upon embryo development imposed by the SATAC methodology. In addition, no translocation events between SATAC marker sequences and native chromosomes were observed and, at the level of FISH analysis, the SATACs were found to be intact and present in 44% of murine embryos examined at various levels of mosaicism. Subsequently, SATAC-injected embryos were assessed for development beyond the preimplantation stage. This confirmed the presence of an intact SATAC in one female transgenic founder exam-