Exonic deletions in AUTS2 cause a syndromic form of intellectual disability and suggest a critical role for the C terminus.

Genomic rearrangements involving AUTS2 (7q11.22) are associated with autism and intellectual disability (ID), although evidence for causality is limited. By combining the results of diagnostic testing of 49,684 individuals, we identified 24 microdeletions that affect at least one exon of AUTS2, as well as one translocation and one inversion each with a breakpoint within the AUTS2 locus. Comparison of 17 well-characterized individuals enabled identification of a variable syndromic phenotype including ID, autism, short stature, microcephaly, cerebral palsy, and facial dysmorphisms. The dysmorphic features were more pronounced in persons with 3'AUTS2 deletions. This part of the gene is shown to encode a C-terminal isoform (with an alternative transcription start site) expressed in the human brain. Consistent with our genetic data, suppression of auts2 in zebrafish embryos caused microcephaly that could be rescued by either the full-length or the C-terminal isoform of AUTS2. Our observations demonstrate a causal role of AUTS2 in neurocognitive disorders, establish a hitherto unappreciated syndromic phenotype at this locus, and show how transcriptional complexity can underpin human pathology. The zebrafish model provides a valuable tool for investigating the etiology of AUTS2 syndrome and facilitating gene-function analysis in the future.

TM4SF20 ancestral deletion and susceptibility to a pediatric disorder of early language delay and cerebral white matter hyperintensities.

White matter hyperintensities (WMHs) of the brain are important markers of aging and small-vessel disease. WMHs are rare in healthy children and, when observed, often occur with comorbid neuroinflammatory or vasculitic processes. Here, we describe a complex 4 kb deletion in 2q36.3 that segregates with early childhood communication disorders and WMH in 15 unrelated families predominantly from Southeast Asia. The premature brain aging phenotype with punctate and multifocal WMHs was observed in ~70% of young carrier parents who underwent brain MRI. The complex deletion removes the penultimate exon 3 of TM4SF20, a gene encoding a transmembrane protein of unknown function. Minigene analysis showed that the resultant net loss of an exon introduces a premature stop codon, which, in turn, leads to the generation of a stable protein that fails to target to the plasma membrane and accumulates in the cytoplasm. Finally, we report this deletion to be enriched in individuals of Vietnamese Kinh descent, with an allele frequency of about 1%, embedded in an ancestral haplotype. Our data point to a constellation of early language delay and WMH phenotypes, driven by a likely toxic mechanism of TM4SF20 truncation, and highlight the importance of understanding and managing population-specific low-frequency pathogenic alleles.

Copy number variants in patients with intellectual disability affect the regulation of ARX transcription factor gene.

Protein-coding mutations in the transcription factor-encoding gene ARX cause various forms of intellectual disability (ID) and epilepsy. In contrast, variations in surrounding non-coding sequences are correlated with milder forms of non-syndromic ID and autism and had suggested the importance of ARX gene regulation in the etiology of these disorders. We compile data on several novel and some already identified patients with or without ID that carry duplications of ARX genomic region and consider likely genetic mechanisms underlying the neurodevelopmental defects. We establish the long-range regulatory domain of ARX and identify its brain region-specific autoregulation. We conclude that neurodevelopmental disturbances in the patients may not simply arise from increased dosage due to ARX duplication. This is further exemplified by a small duplication involving a non-functional ARX copy, but with duplicated enhancers. ARX enhancers are located within a 504-kb region and regulate expression specifically in the forebrain in developing and adult zebrafish. Transgenic enhancer-reporter lines were used as in vivo tools to delineate a brain region-specific negative and positive autoregulation of ARX. We find autorepression of ARX in the telencephalon and autoactivation in the ventral thalamus. Fluorescently labeled brain regions in the transgenic lines facilitated the identification of neuronal outgrowth and pathfinding disturbances in the ventral thalamus and telencephalon that occur when arxa dosage is diminished. In summary, we have established a model for how breakpoints in long-range gene regulation alter the expression levels of a target gene brain region-specifically, and how this can cause subtle neuronal phenotypes relating to the etiology of associated neuropsychiatric disease.

Small rare recurrent deletions and reciprocal duplications in 2q21.1, including brain-specific ARHGEF4 and GPR148.

We have identified a rare small (~450 kb unique sequence) recurrent deletion in a previously linked attention-deficit hyperactivity disorder (ADHD) locus at 2q21.1 in five unrelated families with developmental delay (DD)/intellectual disability (ID), ADHD, epilepsy and other neurobehavioral abnormalities from 17 035 samples referred for clinical chromosomal microarray analysis. Additionally, a DECIPHER (http://decipher.sanger.ac.uk) patient 2311 was found to have the same deletion and presented with aggressive behavior. The deletion was not found in either six control groups consisting of 13 999 healthy individuals or in the DGV database. We have also identified reciprocal duplications in five unrelated families with autism, developmental delay (DD), seizures and ADHD. This genomic region is flanked by large, complex low-copy repeats (LCRs) with directly oriented subunits of ~109 kb in size that have 97.7% DNA sequence identity. We sequenced the deletion breakpoints within the directly oriented paralogous subunits of the flanking LCR clusters, demonstrating non-allelic homologous recombination as a mechanism of formation. The rearranged segment harbors five genes: GPR148, FAM123C, ARHGEF4, FAM168B and PLEKHB2. Expression of ARHGEF4 (Rho guanine nucleotide exchange factor 4) is restricted to the brain and may regulate the actin cytoskeletal network, cell morphology and migration, and neuronal function. GPR148 encodes a G-protein-coupled receptor protein expressed in the brain and testes. We suggest that small rare recurrent deletion of 2q21.1 is pathogenic for DD/ID, ADHD, epilepsy and other neurobehavioral abnormalities and, because of its small size, low frequency and more severe phenotype might have been missed in other previous genome-wide screening studies using single-nucleotide polymorphism analyses.

A novel dominant COL11A1 mutation resulting in a severe skeletal dysplasia.

Mutations in the type XI collagen alpha-1 chain gene (COL11A1) cause a change in protein structure that alters its interactions with collagens II and V, resulting in abnormalities in cartilage and ocular vitreous. The most common type XI collagenopathies are dominantly inherited Stickler or Marshall syndromes, while severe recessive skeletal dysplasias, such as fibrochondrogenesis, occur less frequently. We describe a family with a severe skeletal dysplasia caused by a novel dominantly inherited COL11A1 mutation. The siblings each presented with severe myopia, hearing loss, micromelia, metaphyseal widening of the long bones, micrognathia, and airway compromise requiring tracheostomy. The first child lived for over 2 years, while the second succumbed at 5 months of age. Their mother has mild rhizomelic shortening of the limbs, brachydactyly, and severe myopia. Sequencing of COL11A1 revealed a novel deleterious heterozygous mutation in COL11A1 involving the triple helical domain in both siblings, and a mosaic mutation in their mother, indicating germline mosaicism with subsequent dominant inheritance. These are the first reported individuals with a dominantly inherited mutation in COL11A1 associated with a severe skeletal dysplasia. The skeletal involvement is similar to, yet milder than fibrochondrogenesis and allowed for survival beyond the perinatal period. These cases highlight both a novel dominant COL11A1 mutation causing a significant skeletal dysplasia and the phenotypic heterogeneity of collagenopathies.

Recurrent distal 7q11.23 deletion including HIP1 and YWHAG identified in patients with intellectual disabilities, epilepsy, and neurobehavioral problems.

We report 26 individuals from ten unrelated families who exhibit variable expression and/or incomplete penetrance of epilepsy, learning difficulties, intellectual disabilities, and/or neurobehavioral abnormalities as a result of a heterozygous microdeletion distally adjacent to the Williams-Beuren syndrome region on chromosome 7q11.23. In six families with a common recurrent ∼1.2 Mb deletion that includes the Huntingtin-interacting protein 1 (HIP1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG) genes and that is flanked by large complex low-copy repeats, we identified sites for nonallelic homologous recombination in two patients. There were no cases of this ∼1.2 Mb distal 7q11.23 deletion copy number variant identified in over 20,000 control samples surveyed. Three individuals with smaller, nonrecurrent deletions (∼180-500 kb) that include HIP1 but not YWHAG suggest that deletion of HIP1 is sufficient to cause neurological disease. Mice with targeted mutation in the Hip1 gene (Hip1⁻(/)⁻) develop a neurological phenotype characterized by failure to thrive, tremor, and gait ataxia. Overall, our data characterize a neurodevelopmental and epilepsy syndrome that is likely caused by recurrent and nonrecurrent deletions, including HIP1. These data do not exclude the possibility that YWHAG loss of function is also sufficient to cause neurological phenotypes. Based on the current knowledge of Hip1 protein function and its proposed role in AMPA and NMDA ionotropic glutamate receptor trafficking, we believe that HIP1 haploinsufficiency in humans will be amenable to rational drug design for improved seizure control and cognitive and behavioral function.

Haploinsufficiency of SOX5 at 12p12.1 is associated with developmental delays with prominent language delay, behavior problems, and mild dysmorphic features.

SOX5 encodes a transcription factor involved in the regulation of chondrogenesis and the development of the nervous system. Despite its important developmental roles, SOX5 disruption has yet to be associated with human disease. We report one individual with a reciprocal translocation breakpoint within SOX5, eight individuals with intragenic SOX5 deletions (four are apparently de novo and one inherited from an affected parent), and seven individuals with larger 12p12 deletions encompassing SOX5. Common features in these subjects include prominent speech delay, intellectual disability, behavior abnormalities, and dysmorphic features. The phenotypic impact of the deletions may depend on the location of the deletion and, consequently, which of the three major SOX5 protein isoforms are affected. One intragenic deletion, involving only untranslated exons, was present in a more mildly affected subject, was inherited from a healthy parent and grandparent, and is similar to a deletion found in a control cohort. Therefore, some intragenic SOX5 deletions may have minimal phenotypic effect. Based on the location of the deletions in the subjects compared to the controls, the de novo nature of most of these deletions, and the phenotypic similarities among cases, SOX5 appears to be a dosage-sensitive, developmentally important gene.

High-resolution array CGH defines critical regions and candidate genes for microcephaly, abnormalities of the corpus callosum, and seizure phenotypes in patients with microdeletions of 1q43q44.

Microdeletions of 1q43q44 result in a recognizable clinical disorder characterized by moderate to severe intellectual disability (ID) with limited or no expressive speech, characteristic facial features, hand and foot anomalies, microcephaly (MIC), abnormalities (agenesis/hypogenesis) of the corpus callosum (ACC), and seizures (SZR). Critical regions have been proposed for some of the more prominent features of this disorder such as MIC and ACC, yet conflicting data have prevented precise determination of the causative genes. In this study, the largest of pure interstitial and terminal deletions of 1q43q44 to date, we characterized 22 individuals by high-resolution oligonucleotide microarray-based comparative genomic hybridization. We propose critical regions and candidate genes for the MIC, ACC, and SZR phenotypes associated with this microdeletion syndrome. Three cases with MIC had small overlapping or intragenic deletions of AKT3, an isoform of the protein kinase B family. The deletion of only AKT3 in two cases implicates haploinsufficiency of this gene in the MIC phenotype. Likewise, based on the smallest region of overlap among the affected individuals, we suggest a critical region for ACC that contains ZNF238, a transcriptional and chromatin regulator highly expressed in the developing and adult brain. Finally, we describe a critical region for the SZR phenotype which contains three genes (FAM36A, C1ORF199, and HNRNPU). Although ~90% of cases in this study and in the literature fit these proposed models, the existence of phenotypic variability suggests other mechanisms such as variable expressivity, incomplete penetrance, position effects, or multigenic factors could account for additional complexity in some cases.

Recurrent 200-kb deletions of 16p11.2 that include the SH2B1 gene are associated with developmental delay and obesity.

PURPOSE: The short arm of chromosome 16 is rich in segmental duplications, predisposing this region of the genome to a number of recurrent rearrangements. Genomic imbalances of an approximately 600-kb region in 16p11.2 (29.5-30.1 Mb) have been associated with autism, intellectual disability, congenital anomalies, and schizophrenia. However, a separate, distal 200-kb region in 16p11.2 (28.7-28.9 Mb) that includes the SH2B1 gene has been recently associated with isolated obesity. The purpose of this study was to better define the phenotype of this recurrent SH2B1-containing microdeletion in a cohort of phenotypically abnormal patients not selected for obesity. METHODS: Array comparative hybridization was performed on a total of 23,084 patients in a clinical setting for a variety of indications, most commonly developmental delay. RESULTS: Deletions of the SH2B1-containing region were identified in 31 patients. The deletion is enriched in the patient population when compared with controls (P = 0.003), with both inherited and de novo events. Detailed clinical information was available for six patients, who all had developmental delays of varying severity. Body mass index was ≥95th percentile in four of six patients, supporting the previously described association with obesity. The reciprocal duplication, found in 17 patients, does not seem to be significantly enriched in our patient population compared with controls. CONCLUSIONS: Deletions of the 16p11.2 SH2B1-containing region are pathogenic and are associated with developmental delay in addition to obesity.

Interchromosomal template-switching as a novel molecular mechanism for imprinting perturbations associated with Temple syndrome.

BACKGROUND: Intrachromosomal triplications (TRP) can contribute to disease etiology via gene dosage effects, gene disruption, position effects, or fusion gene formation. Recently, post-zygotic de novo triplications adjacent to copy-number neutral genomic intervals with runs of homozygosity (ROH) have been shown to result in uniparental isodisomy (UPD). The genomic structure of these complex genomic rearrangements (CGRs) shows a consistent pattern of an inverted triplication flanked by duplications (DUP-TRP/INV-DUP) formed by an iterative DNA replisome template-switching mechanism during replicative repair of a single-ended, double-stranded DNA (seDNA), the ROH results from an interhomolog or nonsister chromatid template switch. It has been postulated that these CGRs may lead to genetic abnormalities in carriers due to dosage-sensitive genes mapping within the copy-number variant regions, homozygosity for alleles at a locus causing an autosomal recessive (AR) disease trait within the ROH region, or imprinting-associated diseases. METHODS: Here, we report a family wherein the affected subject carries a de novo 2.2-Mb TRP followed by 42.2 Mb of ROH and manifests clinical features overlapping with those observed in association with chromosome 14 maternal UPD (UPD(14)mat). UPD(14)mat can cause clinical phenotypic features enabling a diagnosis of Temple syndrome. This CGR was then molecularly characterized by high-density custom aCGH, genome-wide single-nucleotide polymorphism (SNP) and methylation arrays, exome sequencing (ES), and the Oxford Nanopore long-read sequencing technology. RESULTS: We confirmed the postulated DUP-TRP/INV-DUP structure by multiple orthogonal genomic technologies in the proband. The methylation status of known differentially methylated regions (DMRs) on chromosome 14 revealed that the subject shows the typical methylation pattern of UPD(14)mat. Consistent with these molecular findings, the clinical features overlap with those observed in Temple syndrome, including speech delay. CONCLUSIONS: These data provide experimental evidence that, in humans, triplication can lead to segmental UPD and imprinting disease. Importantly, genotype/phenotype analyses further reveal how a post-zygotically generated complex structural variant, resulting from a replication-based mutational mechanism, contributes to expanding the clinical phenotype of known genetic syndromes. Mechanistically, such events can distort transmission genetics resulting in homozygosity at a locus for which only one parent is a carrier as well as cause imprinting diseases.

Identification of novel candidate disease genes from de novo exonic copy number variants.

BACKGROUND: Exon-targeted microarrays can detect small (<1000 bp) intragenic copy number variants (CNVs), including those that affect only a single exon. This genome-wide high-sensitivity approach increases the molecular diagnosis for conditions with known disease-associated genes, enables better genotype-phenotype correlations, and facilitates variant allele detection allowing novel disease gene discovery. METHODS: We retrospectively analyzed data from 63,127 patients referred for clinical chromosomal microarray analysis (CMA) at Baylor Genetics laboratories, including 46,755 individuals tested using exon-targeted arrays, from 2007 to 2017. Small CNVs harboring a single gene or two to five non-disease-associated genes were identified; the genes involved were evaluated for a potential disease association. RESULTS: In this clinical population, among rare CNVs involving any single gene reported in 7200 patients (11%), we identified 145 de novo autosomal CNVs (117 losses and 28 intragenic gains), 257 X-linked deletion CNVs in males, and 1049 inherited autosomal CNVs (878 losses and 171 intragenic gains); 111 known disease genes were potentially disrupted by de novo autosomal or X-linked (in males) single-gene CNVs. Ninety-one genes, either recently proposed as candidate disease genes or not yet associated with diseases, were disrupted by 147 single-gene CNVs, including 37 de novo deletions and ten de novo intragenic duplications on autosomes and 100 X-linked CNVs in males. Clinical features in individuals with de novo or X-linked CNVs encompassing at most five genes (224 bp to 1.6 Mb in size) were compared to those in individuals with larger-sized deletions (up to 5 Mb in size) in the internal CMA database or loss-of-function single nucleotide variants (SNVs) detected by clinical or research whole-exome sequencing (WES). This enabled the identification of recently published genes (BPTF, NONO, PSMD12, TANGO2, and TRIP12), novel candidate disease genes (ARGLU1 and STK3), and further confirmation of disease association for two recently proposed disease genes (MEIS2 and PTCHD1). Notably, exon-targeted CMA detected several pathogenic single-exon CNVs missed by clinical WES analyses. CONCLUSIONS: Together, these data document the efficacy of exon-targeted CMA for detection of genic and exonic CNVs, complementing and extending WES in clinical diagnostics, and the potential for discovery of novel disease genes by genome-wide assay.

Delineation of candidate genes responsible for structural brain abnormalities in patients with terminal deletions of chromosome 6q27.

Patients with terminal deletions of chromosome 6q present with structural brain abnormalities including agenesis of corpus callosum, hydrocephalus, periventricular nodular heterotopia, and cerebellar malformations. The 6q27 region harbors genes that are important for the normal development of brain and delineation of a critical deletion region for structural brain abnormalities may lead to a better genotype-phenotype correlation. We conducted a detailed clinical and molecular characterization of seven unrelated patients with deletions involving chromosome 6q27. All patients had structural brain abnormalities. Using array comparative genomic hybridization, we mapped the size, extent, and genomic content of these deletions. The smallest region of overlap spans 1.7 Mb and contains DLL1, THBS2, PHF10, and C6orf70 (ERMARD) that are plausible candidates for the causation of structural brain abnormalities. Our study reiterates the importance of 6q27 region in normal development of brain and helps identify putative genes in causation of structural brain anomalies.

New perspectives on the face in fetal alcohol syndrome: what anthropometry tells us.

Fetal alcohol syndrome (FAS) refers to the adverse effects to the fetus from prenatal exposure to alcohol. Originally, the diagnosis of FAS was given only to those individuals that were the most severely affected. Since that time, it has become apparent that the effects of prenatal alcohol exposure are broad-based, and those individuals diagnosed with FAS represent the severe end of the continuum in their phenotypic expression. This study utilized 21 craniofacial anthropometric measurements on 100 prenatally exposed individuals to quantify the elements of the FAS facial phenotype and to extend the quantitative phenotype to individuals who exhibited less severe or incomplete manifestations of prenatal alcohol exposure.

CHRNA7 triplication associated with cognitive impairment and neuropsychiatric phenotypes in a three-generation pedigree.

Although deletions of CHRNA7 have been associated with intellectual disability (ID), seizures and neuropsychiatric phenotypes, the pathogenicity of CHRNA7 duplications has been uncertain. We present the first report of CHRNA7 triplication. Three generations of a family affected with various neuropsychiatric phenotypes, including anxiety, bipolar disorder, developmental delay and ID, were studied with array comparative genomic hybridization (aCGH). High-resolution aCGH revealed a 650-kb triplication at chromosome 15q13.3 encompassing the CHRNA7 gene, which encodes the alpha7 subunit of the neuronal nicotinic acetylcholine receptor. A small duplication precedes the triplication at the proximal breakpoint junction, and analysis of the breakpoint indicates that the triplicated segment is in an inverted orientation with respect to the duplication. CHRNA7 triplication appears to occur by a replication-based mechanism that produces inverted triplications embedded within duplications. Co-segregation of the CHRNA7 triplication with neuropsychiatric and cognitive phenotypes provides further evidence for dosage sensitivity of CHRNA7.

Phenotypic spectrum and genotype-phenotype correlations of NRXN1 exon deletions.

Copy number variants (CNVs) and intragenic rearrangements of the NRXN1 (neurexin 1) gene are associated with a wide spectrum of developmental and neuropsychiatric disorders, including intellectual disability, speech delay, autism spectrum disorders (ASDs), hypotonia and schizophrenia. We performed a detailed clinical and molecular characterization of 24 patients who underwent clinical microarray analysis and had intragenic deletions of NRXN1. Seventeen of these deletions involved exons of NRXN1, whereas seven deleted intronic sequences only. The patients with exonic deletions manifested developmental delay/intellectual disability (93%), infantile hypotonia (59%) and ASDs (56%). Congenital malformations and dysmorphic features appeared infrequently and inconsistently among this population of patients with NRXN1 deletions. The more C-terminal deletions, including those affecting the ß isoform of neurexin 1, manifested increased head size and a high frequency of seizure disorder (88%) when compared with N-terminal deletions of NRXN1.

MECP2 duplications in six patients with complex sex chromosome rearrangements.

Duplications of the Xq28 chromosome region resulting in functional disomy are associated with a distinct clinical phenotype characterized by infantile hypotonia, severe developmental delay, progressive neurological impairment, absent speech, and proneness to infections. Increased expression of the dosage-sensitive MECP2 gene is considered responsible for the severe neurological impairments observed in affected individuals. Although cytogenetically visible duplications of Xq28 are well documented in the published literature, recent advances using array comparative genomic hybridization (CGH) led to the detection of an increasing number of microduplications spanning MECP2. In rare cases, duplication results from intrachromosomal rearrangement between the X and Y chromosomes. We report six cases with sex chromosome rearrangements involving duplication of MECP2. Cases 1-4 are unbalanced rearrangements between X and Y, resulting in MECP2 duplication. The additional Xq material was translocated to Yp in three cases (cases 1-3), and to the heterochromatic region of Yq12 in one case (case 4). Cases 5 and 6 were identified by array CGH to have a loss in copy number at Xp and a gain in copy number at Xq28 involving the MECP2 gene. In both cases, fluorescent in situ hybridization (FISH) analysis revealed a recombinant X chromosome containing the duplicated material from Xq28 on Xp, resulting from a maternal pericentric inversion. These cases add to a growing number of MECP2 duplications that have been detected by array CGH, while demonstrating the value of confirmatory chromosome and FISH studies for the localization of the duplicated material and the identification of complex rearrangements.

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.