Dimitri J. Stavropoulos

Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies.

Chromosomal microarray (CMA) is increasingly utilized for genetic testing of individuals with unexplained developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD), or multiple congenital anomalies (MCA). Performing CMA and G-banded karyotyping on every patient substantially increases the total cost of genetic testing. The International Standard Cytogenomic Array (ISCA) Consortium held two international workshops and conducted a literature review of 33 studies, including 21,698 patients tested by CMA. We provide an evidence-based summary of clinical cytogenetic testing comparing CMA to G-banded karyotyping with respect to technical advantages and limitations, diagnostic yield for various types of chromosomal aberrations, and issues that affect test interpretation. CMA offers a much higher diagnostic yield (15%-20%) for genetic testing of individuals with unexplained DD/ID, ASD, or MCA than a G-banded karyotype ( approximately 3%, excluding Down syndrome and other recognizable chromosomal syndromes), primarily because of its higher sensitivity for submicroscopic deletions and duplications. Truly balanced rearrangements and low-level mosaicism are generally not detectable by arrays, but these are relatively infrequent causes of abnormal phenotypes in this population (<1%). Available evidence strongly supports the use of CMA in place of G-banded karyotyping as the first-tier cytogenetic diagnostic test for patients with DD/ID, ASD, or MCA. G-banded karyotype analysis should be reserved for patients with obvious chromosomal syndromes (e.g., Down syndrome), a family history of chromosomal rearrangement, or a history of multiple miscarriages.

SHANK1 Deletions in Males with Autism Spectrum Disorder

Recent studies have highlighted the involvement of rare (<1% frequency) copy-number variations and point mutations in the genetic etiology of autism spectrum disorder (ASD); these variants particularly affect genes involved in the neuronal synaptic complex. The SHANK gene family consists of three members (SHANK1, SHANK2, and SHANK3), which encode scaffolding proteins required for the proper formation and function of neuronal synapses. Although SHANK2 and SHANK3 mutations have been implicated in ASD and intellectual disability, the involvement of SHANK1 is unknown. Here, we assess microarray data from 1,158 Canadian and 456 European individuals with ASD to discover microdeletions at the SHANK1 locus on chromosome 19. We identify a hemizygous SHANK1 deletion that segregates in a four-generation family in which male carriers--but not female carriers--have ASD with higher functioning. A de novo SHANK1 deletion was also detected in an unrelated male individual with ASD with higher functioning, and no equivalent SHANK1 mutations were found in >15,000 controls (p = 0.009). The discovery of apparent reduced penetrance of ASD in females bearing inherited autosomal SHANK1 deletions provides a possible contributory model for the male gender bias in autism. The data are also informative for clinical-genetics interpretations of both inherited and sporadic forms of ASD involving SHANK1.

Rare deletions at the neurexin 3 locus in autism spectrum disorder.

The three members of the human neurexin gene family, neurexin 1 (NRXN1), neurexin 2 (NRXN2), and neurexin 3 (NRXN3), encode neuronal adhesion proteins that have important roles in synapse development and function. In autism spectrum disorder (ASD), as well as in other neurodevelopmental conditions, rare exonic copy-number variants and/or point mutations have been identified in the NRXN1 and NRXN2 loci. We present clinical characterization of four index cases who have been diagnosed with ASD and who possess rare inherited or de novo microdeletions at 14q24.3-31.1, a region that overlaps exons of the alpha and/or beta isoforms of NRXN3. NRXN3 deletions were found in one father with subclinical autism and in a carrier mother and father without formal ASD diagnoses, indicating issues of penetrance and expressivity at this locus. Notwithstanding these clinical complexities, this report on ASD-affected individuals who harbor NRXN3 exonic deletions advances the understanding of the genetic etiology of autism, further enabling molecular diagnoses.

PhenoTips: Patient Phenotyping Software for Clinical and Research Use

We have developed PhenoTips: open source software for collecting and analyzing phenotypic information for patients with genetic disorders. Our software combines an easy‐to‐use interface, compatible with any device that runs a Web browser, with a standardized database back end. The PhenoTips’ user interface closely mirrors clinician workflows so as to facilitate the recording of observations made during the patient encounter. Collected data include demographics, medical history, family history, physical and laboratory measurements, physical findings, and additional notes. Phenotypic information is represented using the Human Phenotype Ontology; however, the complexity of the ontology is hidden behind a user interface, which combines simple selection of common phenotypes with error‐tolerant, predictive search of the entire ontology. PhenoTips supports accurate diagnosis by analyzing the entered data, then suggesting additional clinical investigations and providing Online Mendelian Inheritance in Man (OMIM) links to likely disorders. By collecting, classifying, and analyzing phenotypic information during the patient encounter, PhenoTips allows for streamlining of clinic workflow, efficient data entry, improved diagnosis, standardization of collected patient phenotypes, and sharing of anonymized patient phenotype data for the study of rare disorders. Our source code and a demo version of PhenoTips are available at http://phenotips.org.

Molecular Diagnostic Yield of Chromosomal Microarray Analysis and Whole-Exome Sequencing in Children With Autism Spectrum Disorder

IMPORTANCE The use of genome-wide tests to provide molecular diagnosis for individuals with autism spectrum disorder (ASD) requires more study. OBJECTIVE To perform chromosomal microarray analysis (CMA) and whole-exome sequencing (WES) in a heterogeneous group of children with ASD to determine the molecular diagnostic yield of these tests in a sample typical of a developmental pediatric clinic. DESIGN, SETTING, AND PARTICIPANTS The sample consisted of 258 consecutively ascertained unrelated children with ASD who underwent detailed assessments to define morphology scores based on the presence of major congenital abnormalities and minor physical anomalies. The children were recruited between 2008 and 2013 in Newfoundland and Labrador, Canada. The probands were stratified into 3 groups of increasing morphological severity: essential, equivocal, and complex (scores of 0-3, 4-5, and ≥6). EXPOSURES All probands underwent CMA, with WES performed for 95 proband-parent trios. MAIN OUTCOMES AND MEASURES The overall molecular diagnostic yield for CMA and WES in a population-based ASD sample stratified in 3 phenotypic groups. RESULTS Of 258 probands, 24 (9.3%, 95%CI, 6.1%-13.5%) received a molecular diagnosis from CMA and 8 of 95 (8.4%, 95%CI, 3.7%-15.9%) from WES. The yields were statistically different between the morphological groups. Among the children who underwent both CMA and WES testing, the estimated proportion with an identifiable genetic etiology was 15.8% (95%CI, 9.1%-24.7%; 15/95 children). This included 2 children who received molecular diagnoses from both tests. The combined yield was significantly higher in the complex group when compared with the essential group (pairwise comparison, P = .002). [table: see text]. CONCLUSIONS AND RELEVANCE Among a heterogeneous sample of children with ASD, the molecular diagnostic yields of CMA and WES were comparable, and the combined molecular diagnostic yield was higher in children with more complex morphological phenotypes in comparison with the children in the essential category. If replicated in additional populations, these findings may inform appropriate selection of molecular diagnostic testing for children affected by ASD.

Whole-genome sequencing expands diagnostic utility and improves clinical management in paediatric medicine

The standard of care for first-tier clinical investigation of the aetiology of congenital malformations and neurodevelopmental disorders is chromosome microarray analysis (CMA) for copy-number variations (CNVs), often followed by gene(s)-specific sequencing searching for smaller insertion–deletions (indels) and single-nucleotide variant (SNV) mutations. Whole-genome sequencing (WGS) has the potential to capture all classes of genetic variation in one experiment; however, the diagnostic yield for mutation detection of WGS compared to CMA, and other tests, needs to be established. In a prospective study we utilised WGS and comprehensive medical annotation to assess 100 patients referred to a paediatric genetics service and compared the diagnostic yield versus standard genetic testing. WGS identified genetic variants meeting clinical diagnostic criteria in 34% of cases, representing a fourfold increase in diagnostic rate over CMA (8%; P value=1.42E−05) alone and more than twofold increase in CMA plus targeted gene sequencing (13%; P value=0.0009). WGS identified all rare clinically significant CNVs that were detected by CMA. In 26 patients, WGS revealed indel and missense mutations presenting in a dominant (63%) or a recessive (37%) manner. We found four subjects with mutations in at least two genes associated with distinct genetic disorders, including two cases harbouring a pathogenic CNV and SNV. When considering medically actionable secondary findings in addition to primary WGS findings, 38% of patients would benefit from genetic counselling. Clinical implementation of WGS as a primary test will provide a higher diagnostic yield than conventional genetic testing and potentially reduce the time required to reach a genetic diagnosis.

Pathogenic rare copy number variants in community-based schizophrenia suggest a potential role for clinical microarrays

Individually rare, large copy number variants (CNVs) contribute to genetic vulnerability for schizophrenia. Unresolved questions remain, however, regarding the anticipated yield of clinical microarray testing in schizophrenia. Using high-resolution genome-wide microarrays and rigorous methods, we investigated rare CNVs in a prospectively recruited community-based cohort of 459 unrelated adults with schizophrenia and estimated the minimum prevalence of clinically significant CNVs that would be detectable on a clinical microarray. A blinded review by two independent clinical cytogenetic laboratory directors of all large (>500 kb) rare CNVs in cases and well-matched controls showed that those deemed to be clinically significant were highly enriched in schizophrenia (16.4-fold increase, P < 0.0001). In a single community catchment area, the prevalence of individuals with these CNVs was 8.1%. Rare 1.7 Mb CNVs at 2q13 were found to be significantly associated with schizophrenia for the first time, compared with the prevalence in 23 838 population-based controls (42.9-fold increase, P = 0.0002). Additional novel findings that will facilitate the future clinical interpretation of smaller CNVs in schizophrenia include: (i) a greater proportion of individuals with two or more rare exonic CNVs >10 kb in size (1.5-fold increase, P = 0.0109) in schizophrenia; (ii) the systematic discovery of new candidate genes for schizophrenia; and, (iii) functional gene enrichment mapping highlighting a differential impact in schizophrenia of rare exonic deletions involving diverse functions, including neurodevelopmental and synaptic processes (4.7-fold increase, P = 0.0060). These findings suggest consideration of a potential role for clinical microarray testing in schizophrenia, as is now the suggested standard of care for related developmental disorders like autism.

1q21.1 Microduplication expression in adults

Purpose:Rare, recurrent chromosome 1q21.1 duplications have been associated with developmental delay, congenital anomalies, and macrocephaly in children. Data on adult clinical expression would help to inform genetic counseling.Methods:A systematic review of 22 studies reporting 107 individuals (59 children and 48 adults) with 1q21.1 duplications was conducted. We compiled the available phenotypic data to attempt to identify the most highly associated clinical features and to determine expression in adults. We also report on seven adult cases newly identified in the studies of schizophrenia and tetralogy of Fallot at our center.Results:Five cases were ascertained as controls, 32 as relatives of probands, and 70 as having clinical features: autism spectrum disorder (n = 15), congenital heart disease (n = 12), schizophrenia (n = 10), or other, mostly developmental, features (n = 33). The 1q21.1 duplication was significantly enriched in the cohorts with schizophrenia (P = 0.0155) and tetralogy of Fallot (P = 0.0040) at our center as compared with controls. There was a paucity of clinical data for adults; the most common features, other than those used for ascertainment, included macrocephaly and abnormalities of possible connective tissue origin (e.g., carpal tunnel syndrome).Conclusion:Further data are needed to characterize lifetime expression of 1q21.1 duplications. These initial results, however, suggest that anticipatory care should include attention to later-onset conditions such as schizophrenia.Genet Med 2013:15(4):282–289

Clinically relevant copy number variations detected in cerebral palsy

Cerebral palsy (CP) represents a group of non-progressive clinically heterogeneous disorders that are characterized by motor impairment and early age of onset, frequently accompanied by co-morbidities. The cause of CP has historically been attributed to environmental stressors resulting in brain damage. While genetic risk factors are also implicated, guidelines for diagnostic assessment of CP do not recommend for routine genetic testing. Given numerous reports of aetiologic copy number variations (CNVs) in other neurodevelopmental disorders, we used microarrays to genotype a population-based prospective cohort of children with CP and their parents. Here we identify de novo CNVs in 8/115 (7.0%) CP patients (∼1% rate in controls). In four children, large chromosomal abnormalities deemed likely pathogenic were found, and they were significantly more likely to have severe neuromotor impairments than those CP subjects without such alterations. Overall, the CNV data would have impacted our diagnosis or classification of CP in 11/115 (9.6%) families.

Delineating the 15q13.3 microdeletion phenotype: a case series and comprehensive review of the literature

Purpose:Recurrent 15q13.3 deletions are enriched in multiple neurodevelopmental conditions including intellectual disability, autism, epilepsy, and schizophrenia. However, the 15q13.3 microdeletion syndrome remains ill-defined.Methods:We systematically compiled all cases of 15q13.3 deletion published before 2014. We also examined three locally available cohorts to identify new adults with 15q13.3 deletions.Results:We identified a total of 246 cases (133 children, 113 adults) with deletions overlapping or within the 15q13.3 (breakpoint (BP)4–BP5) region, including seven novel adult cases from local cohorts. No BP4–BP5 deletions were identified in 23,838 adult controls. Where known, 15q13.3 deletions were typically inherited (85.4%) and disproportionately of maternal origin (P < 0.0001). Overall, 198 cases (121 children, 77 adults; 80.5%) had at least one neuropsychiatric diagnosis. Accounting for ascertainment, developmental disability/intellectual disability was present in 57.7%, epilepsy/seizures in 28.0%, speech problems in 15.9%, autism spectrum disorder in 10.9%, schizophrenia in 10.2%, mood disorder in 10.2%, and attention deficit hyperactivity disorder in 6.5%. By contrast, major congenital malformations, including congenital heart disease (2.4%), were uncommon. Placenta previa occurred in the pregnancies of four cases.Conclusion:The 15q13.3 microdeletion syndrome is predominantly characterized by neuropsychiatric expression. There are implications for pre- and postnatal detection, genetic counseling, and anticipatory care.Genet Med 17 2, 149–157.