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Mutations in the TMEM260 gene cause structural heart defects and renal anomalies syndrome, but the function of the encoded protein remains unknown. We previously reported wide occurrence of O-mannose glycans on extracellular immunoglobulin, plexin, transcription factor (IPT) domains found in the hepatocyte growth factor receptor (cMET), macrophage-stimulating protein receptor (RON), and plexin receptors, and further demonstrated that two known protein O-mannosylation systems orchestrated by the POMT1/2 and transmembrane and tetratricopeptide repeat-containing proteins 1-4 gene families were not required for glycosylation of these IPT domains. Here, we report that the TMEM260 gene encodes an ER-located protein O-mannosyltransferase that selectively glycosylates IPT domains. We demonstrate that disease-causing TMEM260 mutations impair O-mannosylation of IPT domains and that TMEM260 knockout in cells results in receptor maturation defects and abnormal growth of 3D cell models. Thus, our study identifies the third protein-specific O-mannosylation pathway in mammals and demonstrates that O-mannosylation of IPT domains serves critical functions during epithelial morphogenesis. Our findings add a new glycosylation pathway and gene to a growing group of congenital disorders of glycosylation.

Eukaryotic initiation factor-4A2 (EIF4A2) is an ATP-dependent RNA helicase and a member of the DEAD-box protein family that recognizes the 5' cap structure of mRNAs, allows mRNA to bind to the ribosome, and plays an important role in microRNA-regulated gene repression. Here, we report on 15 individuals from 14 families presenting with global developmental delay, intellectual disability, hypotonia, epilepsy, and structural brain anomalies, all of whom have extremely rare de novo mono-allelic or inherited bi-allelic variants in EIF4A2. Neurodegeneration was predominantly reported in individuals with bi-allelic variants. Molecular modeling predicts these variants would perturb structural interactions in key protein domains. To determine the pathogenicity of the EIF4A2 variants in vivo, we examined the mono-allelic variants in Drosophila melanogaster (fruit fly) and identified variant-specific behavioral and developmental defects. The fruit fly homolog of EIF4A2 is eIF4A, a negative regulator of decapentaplegic (dpp) signaling that regulates embryo patterning, eye and wing morphogenesis, and stem cell identity determination. Our loss-of-function (LOF) rescue assay demonstrated a pupal lethality phenotype induced by loss of eIF4A, which was fully rescued with human EIF4A2 wild-type (WT) cDNA expression. In comparison, the EIF4A2 variant cDNAs failed or incompletely rescued the lethality. Overall, our findings reveal that EIF4A2 variants cause a genetic neurodevelopmental syndrome with both LOF and gain of function as underlying mechanisms.

Transformer2 proteins (Tra2α and Tra2β) control splicing patterns in human cells, and no human phenotypes have been associated with germline variants in these genes. The aim of this work was to associate germline variants in the TRA2B gene to a novel neurodevelopmental disorder.Twelve individuals from 11 unrelated families who had predicted loss-of-function monoallelic variants, mostly de novo, were recruited. RNA-seq and Western blot analyses of Tra2β-1 and Tra2β-3 isoforms from patient-derived cells were performed. Tra2β1-GFP, Tra2β3-GFP and CHEK1 exon 3 plasmids were transfected into HEK-293 cells.All variants clustered in the 5' part of TRA2B, upstream of an alternative translation start site responsible for the expression of non-canonical Tra2β-3 isoform. All of affected individuals presented intellectual disability and/or developmental delay, frequently associated with infantile spasms, microcephaly, brain anomalies, autism spectrum disorder, feeding difficulties, and short stature. Experimental studies showed that these variants decreased the expression of the canonical Tra2β-1 isoform, while they increased the expression of the Tra2β-3 isoform, which is shorter and lacks the N-terminal RS1 domain. Increased expression of Tra2β-3-GFP were shown to interfere with the incorporation of CHEK1 exon 3 into its mature transcript, normally incorporated by Tra2β-1.Predicted loss-of-function variants clustered in the 5' portion of TRA2B cause a new neurodevelopmental syndrome through an apparently dominant negative disease mechanism involving the use of an alternative translation start site and the overexpression of a shorter, repressive Tra2β protein.

ZMYND8 encodes a multidomain protein that serves as a central interactive hub for coordinating critical roles in transcription regulation, chromatin remodeling, regulation of super-enhancers, DNA damage response and tumor suppression. We delineate a novel neurocognitive disorder caused by variants in the ZMYND8 gene.An international collaboration, exome sequencing, molecular modeling, yeast two-hybrid assays, analysis of available transcriptomic data and a knockdown Drosophila model were used to characterize the ZMYND8 variants.ZMYND8 variants were identified in 11 unrelated individuals; 10 occurred de novo and one suspected de novo; 2 were truncating, 9 were missense, of which one was recurrent. The disorder is characterized by intellectual disability with variable cardiovascular, ophthalmologic and minor skeletal anomalies. Missense variants in the PWWP domain of ZMYND8 abolish the interaction with Drebrin and missense variants in the MYND domain disrupt the interaction with GATAD2A. ZMYND8 is broadly expressed across cell types in all brain regions and shows highest expression in the early stages of brain development. Neuronal knockdown of the DrosophilaZMYND8 ortholog results in decreased habituation learning, consistent with a role in cognitive function.We present genomic and functional evidence for disruption of ZMYND8 as a novel etiology of syndromic intellectual disability.

MBD5-associated neurodevelopmental disorder (MAND) is an autism spectrum disorder (ASD) characterized by intellectual disability, motor delay, speech impairment and behavioral problems; however, the biological role of methyl-CpG-binding domain 5, MBD5, in neurodevelopment and ASD remains largely undefined. Hence, we created neural progenitor cells (NPC) derived from individuals with chromosome 2q23.1 deletion and conducted RNA-seq to identify differentially expressed genes (DEGs) and the biological processes and pathways altered in MAND. Primary skin fibroblasts from three unrelated individuals with MAND and four unrelated controls were converted into induced pluripotent stem cell (iPSC) lines, followed by directed differentiation of iPSC to NPC. Transcriptome analysis of MAND NPC revealed 468 DEGs (q

Craniofacial microsomia (CFM) is the second most common congenital facial anomaly, yet its genetic etiology remains unknown. We perform whole-exome or genome sequencing of 146 kindreds with sporadic (n = 138) or familial (n = 8) CFM, identifying a highly significant burden of loss of function variants in SF3B2 (P = 3.8 × 10−10), a component of the U2 small nuclear ribonucleoprotein complex, in probands. We describe twenty individuals from seven kindreds harboring de novo or transmitted haploinsufficient variants in SF3B2. Probands display mandibular hypoplasia, microtia, facial and preauricular tags, epibulbar dermoids, lateral oral clefts in addition to skeletal and cardiac abnormalities. Targeted morpholino knockdown of SF3B2 in Xenopus results in disruption of cranial neural crest precursor formation and subsequent craniofacial cartilage defects, supporting a link between spliceosome mutations and impaired neural crest development in congenital craniofacial disease. The results establish haploinsufficient variants in SF3B2 as the most prevalent genetic cause of CFM, explaining ~3% of sporadic and ~25% of familial cases., Despite being a common congenital facial anomaly, the genetic etiology of craniofacial microsomia (CFM) is not well understood. Here, the authors use exome and genome sequencing of 146 individuals with CFM to identify haploinsufficient variants in SF3B2 as a prevalent underlying cause.

International audience; In humans, de novo truncating variants in WASF1 (Wiskott–Aldrich syndrome protein family member 1) have been linked to presentations of moderate-to-profound intellectual disability (ID), autistic features, and epilepsy. Apart from one case series, there is limited information on the phenotypic spectrum and genetic landscape of WASF1-related neurodevelopmental disorder (NDD). In this report, we describe detailed clinical characteristics of six individuals with WASF1-related NDD. We demonstrate a broader spectrum of neurodevelopmental impairment including more mildly affected individuals. Further, we report new variant types, including a copy number variant (CNV), resulting in the partial deletion of WASF1 in monozygotic twins, and three missense variants, two of which alter the same residue, p.W161. This report adds further evidence that de novo variants in WASF1 cause an autosomal dominant NDD.

Key Clinical Message Clinicians should consider that clinical exome sequencing provides the unique potential to disentangle complex phenotypes into multiple genetic etiologies. Further, functional studies on variants of uncertain significance are necessary to arrive at an accurate diagnosis for the patient.

Nucleic acid isolation is often the starting point for all downstream experiments in biomedical research. It is therefore the most crucial step in any molecular technique. DNA and RNA extraction follow protocols with standardized reagents, many of which are available in quality-controlled commercial kits. Irrespective of the protocol, successful extraction of high-quality nucleic acid from biological tissues requires sufficient disruption of the tissue and cellular structures, denaturation of nucleoprotein complexes, inactivation of nucleases, and nucleic acid purification. These steps can be modified based on nucleic acid of interest and biological sample source. This chapter addresses DNA and RNA extraction from a variety of sample and tissue types, including saliva, and formalin-fixed, paraffin-embedded tissues, which are often archived in clinical pathology laboratories. Special considerations and common pitfalls of each protocol will also be discussed, as will nucleic acid quantitation techniques.

Smith-Magenis syndrome (SMS) is a neurodevelopmental disorder associated with intellectual disability, sleep disturbances, early onset obesity and vast behavioral deficits. We used the Behavior Problems Inventory-01 to categorize the frequency and severity of behavioral abnormalities in a SMS cohort relative to individuals with intellectual disability of heterogeneous etiology. Self-injurious, stereotyped, and aggressive/destructive behavioral scores indicated that both frequency and severity were significantly higher among individuals with SMS relative to those with intellectual disability. Next, we categorized food behaviors in our SMS cohort across age using the Food Related Problems Questionnaire (FRPQ) and found that problems began to occur in SMS children as early as 5-11 years old, but children 12-18 years old and adults manifested the most severe problems. Furthermore, we evaluated the similarities of SMS adult food-related behaviors to those with intellectual disability and found that SMS adults had more severe behavioral problems. Many neurodevelopmental disorders exhibit syndromic obesity including SMS. Prader-Willi syndrome (PWS) is the most frequent neurodevelopmental disorder with syndromic obesity and has a well-established management and treatment plan. Using the FRPQ we found that SMS adults had similar scores relative to PWS adults. Both syndromes manifest weight gain early in development, and the FRPQ scores highlight specific areas in which behavioral similarities exist, including preoccupation with food, impaired satiety, and negative behavioral responses. SMS food-related behavior treatment paradigms are not as refined as PWS, suggesting that current PWS treatments for prevention of obesity may be beneficial for individuals with SMS.

Roughly 20% of autism spectrum disorders (ASD) are syndromic with a well-established genetic cause. Studying the genes involved can provide insight into the molecular and cellular mechanisms of ASD. 2q23.1 deletion syndrome (causative gene, MBD5) is a recently identified genetic neurodevelopmental disorder associated with ASD. Mutations in MBD5 have been found in ASD cohorts. In this study, we provide a phenotypic update on the prevalent features of 2q23.1 deletion syndrome, which include severe intellectual disability, seizures, significant speech impairment, sleep disturbance, and autistic-like behavioral problems. Next, we examined the phenotypic, molecular, and network/pathway relationships between nine neurodevelopmental disorders associated with ASD: 2q23.1 deletion Rett, Angelman, Pitt-Hopkins, 2q23.1 duplication, 5q14.3 deletion, Kleefstra, Kabuki make-up, and Smith-Magenis syndromes. We show phenotypic overlaps consisting of intellectual disability, speech delay, seizures, sleep disturbance, hypotonia, and autistic-like behaviors. Molecularly, MBD5 possibly regulates the expression of UBE3A, TCF4, MEF2C, EHMT1 and RAI1. Network analysis reveals that there could be indirect protein interactions, further implicating function for these genes in common pathways. Further, we show that when MBD5 and RAI1 are haploinsufficient, they perturb several common pathways that are linked to neuronal and behavioral development. These findings support further investigations into the molecular and pathway relationships among genes linked to neurodevelopmental disorders and ASD, which will hopefully lead to common points of regulation that may be targeted toward therapeutic intervention.

Deformed epidermal autoregulatory factor-1 (DEAF1), a transcription factor essential for central nervous system and early embryonic development, has recently been implicated in a series of intellectual disability-related neurodevelopmental anomalies termed, in this study, as DEAF1-associated neurodevelopmental disorder (DAND). We identified six potentially deleterious DEAF1 variants in a cohort of individuals with DAND via clinical exome sequencing (CES) and in silico analysis, including two novel de novo variants: missense variant c.634G > A p.Gly212Ser in the SAND domain and deletion variant c.913_915del p.Lys305del in the NLS domain, as well as c.676C > T p.Arg226Trp, c.700T > A p.Trp234Arg, c.737G > C p.Arg246Thr, and c.791A > C p.Gln264Pro. Luciferase reporter, immunofluorescence staining, and electrophoretic mobility shift assays revealed that these variants had decreased transcriptional repression activity at the DEAF1 promoter and reduced affinity to consensus DEAF1 DNA binding sequences. In addition, c.913_915del p.K305del localized primarily to the cytoplasm and interacted with wild-type DEAF1. Our results demonstrate that variants located within the SAND or NLS domains significantly reduce DEAF1 transcriptional regulatory activities and are thus, likely to contribute to the underlying clinical concerns in DAND patients. These findings illustrate the importance of experimental characterization of variants with uncertain significance identified by CES to assess their potential clinical significance and possible use in diagnosis.

Retinoic acid induced 1 (RAI1) encodes a dosage-sensitive gene that when haploinsufficient results in Smith–Magenis syndrome (SMS) and when overexpressed results in Potocki–Lupski syndrome (PTLS). Phenotypic and molecular evidence illustrates that haploinsufficiency of RAI1 disrupts circadian rhythm through the dysregulation of the master circadian regulator, circadian locomotor output cycles kaput (CLOCK), and other core circadian components, contributing to prominent sleep disturbances in SMS. However, the phenotypic and molecular characterization of sleep features in PTLS has not been elucidated. Using the Pittsburgh Sleep Quality Index (PSQI), caregivers of 15 school-aged children with PTLS reported difficulties in initiating sleep. Indeed, more than 70% of individuals manifested moderate to severe sleep latency, as defined by the PSQI. Moreover, these individuals manifested difficulties in sleep maintenance, with middle of the night and early morning awakenings. When assessing daytime sleepiness through the Epworth Sleepiness Scale, approximately 21% of the individuals manifested excessive daytime somnolence. This indicates that mild dyssomnia characterizes the majority of the sleep phenotype, with occasionally problematic daytime somnolence, a phenotype different than that expressed by individuals with SMS, where daytime sleepiness is a chronic problem. Gene expression analysis of the core circadian machinery in the hypothalamus of the PTLS mouse model (Rai1-Tg) found significant dysregulation of the transcriptional activators, Clock and Arntl, and the transcriptional repressors, Per1–3 and Cry1/2, during both light and dark phases. These findings suggest a partial loss of circadian entrainment typically evoked by environmental photic cues. Examination of circadian clock gene expression in the Rai1-Tg mouse heart, liver, and kidney found unchanged expression of Clock and most of its downstream targets during both light and dark phases, suggesting an asynchronized circadian rhythm. Furthermore, examination of circadian gene expression in synchronized PTLS lymphoblasts revealed reduced transcripts of the Period (PER1–3) family and normal expression of CRY1/2. The finding that central circadian gene expression was altered while many peripheral circadian components were intact suggests a tissue-specific circadian uncoupling of the circadian machinery due to Rai1 overexpression. Overall, our results demonstrate that overexpression of RAI1 results in sleep deficiencies in individuals with PTLS due to a lack of properly regulated circadian machinery gene expression and highlight the importance of evaluating sleep concerns in individuals with PTLS.

Individuals with autism spectrum disorders (ASD) who have an identifiable single-gene neurodevelopmental disorder (NDD), such as fragile X syndrome (FXS, FMR1), Smith-Magenis syndrome (SMS, RAI1), or 2q23.1 deletion syndrome (del 2q23.1, MBD5) share phenotypic features, including a high prevalence of sleep disturbance. We describe the circadian deficits in del 2q23.1 through caregiver surveys in which we identify several frequent sleep anomalies, including night/early awakenings, coughing/snoring loudly, and difficulty falling asleep. We couple these findings with studies on the molecular analysis of the circadian deficits associated with haploinsufficiency of MBD5 in which circadian gene mRNA levels of NR1D2, PER1, PER2, and PER3 were altered in del 2q23.1 lymphoblastoid cell lines (LCLs), signifying that haploinsufficiency of MBD5 can result in dysregulation of circadian rhythm gene expression. These findings were further supported by expression microarrays of MBD5 siRNA knockdown cells that showed significantly altered expression of additional circadian rhythm signaling pathway genes. Based on the common sleep phenotypes observed in del 2q23.1, SMS, and FXS patients, we explored the possibility that MBD5, RAI1, and FMR1 function in overlapping circadian rhythm pathways. Bioinformatic analysis identified conserved putative E boxes in MBD5 and RAI1, and expression levels of NR1D2 and CRY2 were significantly reduced in patient LCLs. Circadian and mTOR signaling pathways, both associated with sleep disturbance, were altered in both MBD5 and RAI1 knockdown microarray data, overlapping with findings associated with FMR1. These data support phenotypic and molecular overlaps across these syndromes that may be exploited to provide therapeutic intervention for multiple disorders.

Item does not contain fulltext Copy number variations associated with abnormal gene dosage have an important role in the genetic etiology of many neurodevelopmental disorders, including intellectual disability (ID) and autism. We hypothesize that the chromosome 2q23.1 region encompassing MBD5 is a dosage-dependent region, wherein deletion or duplication results in altered gene dosage. We previously established the 2q23.1 microdeletion syndrome and report herein 23 individuals with 2q23.1 duplications, thus establishing a complementary duplication syndrome. The observed phenotype includes ID, language impairments, infantile hypotonia and gross motor delay, behavioral problems, autistic features, dysmorphic facial features (pinnae anomalies, arched eyebrows, prominent nose, small chin, thin upper lip), and minor digital anomalies (fifth finger clinodactyly and large broad first toe). The microduplication size varies among all cases and ranges from 68 kb to 53.7 Mb, encompassing a region that includes MBD5, an important factor in methylation patterning and epigenetic regulation. We previously reported that haploinsufficiency of MBD5 is the primary causal factor in 2q23.1 microdeletion syndrome and that mutations in MBD5 are associated with autism. In this study, we demonstrate that MBD5 is the only gene in common among all duplication cases and that overexpression of MBD5 is likely responsible for the core clinical features present in 2q23.1 microduplication syndrome. Phenotypic analyses suggest that 2q23.1 duplication results in a slightly less severe phenotype than the reciprocal deletion. The features associated with a deletion, mutation or duplication of MBD5 and the gene expression changes observed support MBD5 as a dosage-sensitive gene critical for normal development.

Is it time to retire fragile X testing as a first-tier test for developmental delay, intellectual disability, and autism spectrum disorder?

Microdeletions of chromosomal region 2q23.1 that disrupt MBD5 (methyl-CpG-binding domain protein 5) contribute to a spectrum of neurodevelopmental phenotypes; however, the impact of this locus on human psychopathology has not been fully explored. To characterize the structural variation landscape of MBD5 disruptions and the associated human psychopathology, 22 individuals with genomic disruption of MBD5 (translocation, point mutation and deletion) were identified through whole-genome sequencing or cytogenomic microarray at 11 molecular diagnostic centers. The genomic impact ranged from a single base pair to 5.4 Mb. Parents were available for 11 cases, all of which confirmed that the rearrangement arose de novo. Phenotypes were largely indistinguishable between patients with full-segment 2q23.1 deletions and those with intragenic MBD5 rearrangements, including alterations confined entirely to the 5'-untranslated region, confirming the critical impact of non-coding sequence at this locus. We identified heterogeneous, multisystem pathogenic effects of MBD5 disruption and characterized the associated spectrum of psychopathology, including the novel finding of anxiety and bipolar disorder in multiple patients. Importantly, one of the unique features of the oldest known patient was behavioral regression. Analyses also revealed phenotypes that distinguish MBD5 disruptions from seven well-established syndromes with significant diagnostic overlap. This study demonstrates that haploinsufficiency of MBD5 causes diverse phenotypes, yields insight into the spectrum of resulting neurodevelopmental and behavioral psychopathology and provides clinical context for interpretation of MBD5 structural variations. Empirical evidence also indicates that disruption of non-coding MBD5 regulatory regions is sufficient for clinical manifestation, highlighting the limitations of exon-focused assessments. These results suggest an ongoing perturbation of neurological function throughout the lifespan, including risks for neurobehavioral regression.

The cohesin complex is an evolutionarily conserved multi-subunit protein complex which regulates sister chromatid cohesion during mitosis and meiosis. Additionally, the cohesin complex regulates DNA replication, DNA repair, and transcription. The core of the complex consists of four subunits; SMC1A, SMC3, RAD21, and STAG1/2. Loss-of-function mutations in many of these proteins have been implicated in human developmental disorders collectively termed “cohesinopathies”. Through clinical exome sequencing of an 8-year-old girl with a clinical history of global developmental delay, microcephaly, microtia with hearing loss, language delay, ADHD, and dysmorphic features, we describe a heterozygous de novo variant (c.205C>T; p.(Arg69*)) in the integral cohesin structural protein, STAG2. This variant is associated with decreased STAG2 protein expression. Further, the analyses of metaphase spreads did not exhibit premature sister chromatid separation; however, delayed sister chromatid cohesion was observed. To further support the pathogenicity of STAG2 variants, we identified two additional female cases from the DECIPHER research database with mutations in STAG2 and phenotypes similar to our patient. Interestingly, the clinical features of these three cases are remarkably similar to those observed in other well-established cohesinopathies. Herein, we suggest that STAG2 is a dosage-sensitive gene and that heterozygous loss-of-function variants lead to a cohesinopathy.

Haploinsufficiency of RAI1 results in Smith-Magenis syndrome (SMS), a disorder characterized by intellectual disability, multiple congenital anomalies, obesity, neurobehavioral abnormalities, and a disrupted circadian sleep-wake pattern. An inverted melatonin rhythm (i.e., melatonin peaks during the day instead of at night) and associated sleep-phase disturbances in individuals with SMS, as well as a short-period circadian rhythm in mice with a chromosomal deletion of Rai1, support SMS as a circadian-rhythm-dysfunction disorder. However, the molecular cause of the circadian defect in SMS has not been described. The circadian oscillator temporally orchestrates metabolism, physiology, and behavior largely through transcriptional modulation. Data support RAI1 as a transcriptional regulator, but the genes it might regulate are largely unknown. Investigation into the role that RAI1 plays in the regulation of gene transcription and circadian maintenance revealed that RAI1 regulates the transcription of circadian locomotor output cycles kaput (CLOCK), a key component of the mammalian circadian oscillator that transcriptionally regulates many critical circadian genes. Data further show that haploinsufficiency of RAI1 and Rai1 in SMS fibroblasts and the mouse hypothalamus, respectively, results in the transcriptional dysregulation of the circadian clock and causes altered expression and regulation of multiple circadian genes, including PER2, PER3, CRY1, BMAL1, and others. These data suggest that heterozygous mutation of RAI1 and Rai1 leads to a disrupted circadian rhythm and thus results in an abnormal sleep-wake cycle, which can contribute to an abnormal feeding pattern and dependent cognitive performance. Finally, we conclude that RAI1 is a positive transcriptional regulator of CLOCK, pinpointing a novel and important role for this gene in the circadian oscillator.

Background The cohesin complex is a multi‐subunit protein complex which regulates sister chromatid cohesion and separation during cellular division. In addition, this evolutionarily conserved protein complex plays an integral role in DNA replication, DNA repair, and the regulation of transcription. The core complex is composed of four subunits: RAD21, SMC1A, SMC3, and STAG1/2. Mutations in these proteins have been implicated in human developmental disorders collectively termed “cohesinopathies.” Methods Using clinical exome sequencing, we have previously identified three female cases with heterozygous STAG2 mutations and overlapping syndromic phenotypes. Subsequently, a familial missense variant was identified in five male family members. Results We now present the case of a 4‐year‐old male with developmental delay, failure to thrive, short stature, and polydactyly with a likely pathogenic STAG2 de novo missense hemizygous variant, c.3027A>T, p.Lys1009Asn. Furthermore, we compare the phenotypes of the four previously reported STAG2 variants with our case. Conclusion We conclude that mutations in STAG2 cause a novel constellation of sex‐specific cohesinopathy‐related phenotypes and are furthermore, essential for neurodevelopment, human growth, and behavioral development.

The identification of structural variants of uncertain clinical significance is increasing; however, studies delineating the functional consequence of these variants in the pathogenicity of phenotypic features are lacking. Understanding the consequence of structural variants such as copy number alterations and their role in gene expression changes is paramount in order to perform a comprehensive analysis of genetic effects on phenotypic variation and disease. RAI1 is a dosage-sensitive essential neurodevelopmental gene. Copy number loss of RAI1 results in Smith-Magenis syndrome while copy number gain results in Potocki-Lupski syndrome. Here, we present a case of a six year old female with a newly identified maternally inherited copy number loss that lies within the Smith-Magenis syndrome common deletion region, but RAI1 copy number is normal. Integration of the Encyclopedia of DNA Elements (ENCODE) data at the affected region suggests that the deletion disrupts several cis-acting regulatory elements upstream of RAI1, such as multiple repressor sites and an insulator region. Gene expression studies revealed that both the proband and the mother have significantly elevated RAI1 mRNA levels suggesting that the structural variant alters gene expression regulation. The proband and the mother both have some features of Potocki-Lupski syndrome, while the child appears to be more affected with autistic-like features. Overall, our work demonstrates that the integration of ENCODE data with structural variants of uncertain significance aids in delineating a functional consequence to a genomic aberration and subsequent diagnosis.

Smith-Magenis syndrome (SMS) is a complex genetic disorder characterized by sleep disturbance, multiple developmental anomalies, psychiatric behavior, and obesity. It is caused by a heterozygous 17p11.2 microdeletion containing the retinoic acid-induced 1 (RAI1) gene or mutation within RAI1. Sleep disorder is one of the most penetrant features of SMS. Molecular genetic studies indicate that RAI1 regulates circadian rhythm genes and when haploinsucient, causes a distorted molecular circadian network that may be the cause of the sleep disturbance and the inverted rhythm of melatonin present in most individuals with SMS. RAI1 also regulates genes involved in development, neurobehavior, and lipid metabolism. Sleep debt, daytime melatonin secretion, and environmental stress often contribute to negative behavior in persons with SMS, and food entrained circadian rhythm also influences food intake behavior and humoral signals, which also affect development and neurobehavior. The cross-talk between circadian rhythm, development, metabolism and behaviors affect the multiple phenotypic outcomes in Smith-Magenis syndrome. These findings shed light on possible effective and personalized drug treatments for SMS patients in the future.

Background Smith-Magenis syndrome (SMS) is a complex neurobehavioral disorder associated with recurrent otitis. Most SMS cases result from heterozygous interstitial chromosome 17p11.2 deletions that encompass not only the intellectual disability gene retinoic acid-induced 1 but also other genes associated with immunodeficiency, autoimmunity, and/or malignancy. Objectives The goals of this study were to describe the immunological consequence of 17p11.2 deletions by determining the prevalence of immunological diseases in subjects with SMS and by assessing their immune systems via laboratory methods. Methods We assessed clinical histories of 76 subjects with SMS with heterozygous 17p11.2 deletions and performed in-depth immunological testing on 25 representative cohort members. Laboratory testing included determination of serum antibody concentrations, vaccine titers, and lymphocyte subset frequencies. Detailed reactivity profiles of SMS serum antibodies were performed using custom-made antigen microarrays. Results Of 76 subjects with SMS, 74 reported recurrent infections including otitis (88%), pneumonia (47%), sinusitis (42%), and gastroenteritis (34%). Infections were associated with worsening SMS-related neurobehavioral symptoms. The prevalence of autoimmune and atopic diseases was not increased. Malignancy was not reported. Laboratory evaluation revealed most subjects with SMS to be deficient of isotype-switched memory B cells and many to lack protective antipneumococcal antibodies. SMS antibodies were not more reactive than control antibodies to self-antigens. Conclusions Patients with SMS with heterozygous 17p.11.2 deletions display an increased susceptibility to sinopulmonary infections, but not to autoimmune, allergic, or malignant diseases. SMS sera display an antibody reactivity profile favoring neither recognition of pathogen-associated antigens nor self-antigens. Prophylactic strategies to prevent infections may also provide neurobehavioral benefits to selected patients with SMS.

2q23.1 deletion syndrome is characterized by intellectual disability, speech impairment, seizures, disturbed sleep pattern, behavioral problems, and hypotonia. Core features of this syndrome are due to haploinsufficiency of MBD5. Deletions that include coding and noncoding exons show reduced MBD5 mRNA expression. We report a patient with a neurological and behavioral phenotype similar to 2q23.1 deletion syndrome with an inherited intronic deletion in the 5-prime untranslated region of MBD5. Our data show that this patient has normal MBD5 mRNA expression; therefore, this deletion is likely not causative for 2q23.1 deletion syndrome. Overall, it is important to validate intronic deletions for pathogenicity.

MBD5-associated neurodevelopmental disorder (MAND) is an umbrella term that describes a group of disorders, 2q23.1 deletion syndrome, 2q23.1 duplication syndrome, and MBD5 variants, that affect the function of methyl-binding domain 5 (MBD5) and share a common set of neurodevelopmental, cognitive, and behavioral impairments. This review provides a comprehensive clinical and molecular synopsis of 2q23.1 deletion syndrome. Approaches to diagnosis, genetic counseling, and up-to-date management are summarized, followed by a discussion of the molecular and functional role of MBD5. Finally, we also include a brief summary of MBD5 variants that affect function of MBD5 and 2q23.1 duplication syndrome.

We report on two patients with developmental delay, hypotonia, and autistic features associated with duplications of chromosome region 2q23.1-2q23.2 detected by chromosome microarray analysis. The duplications include one OMIM Morbid Map gene, MBD5, as well as seven known RefSeq genes (ACVR2A, ORC4L, EPC2, KIF5C, MIR1978, LYPD6B, and LYPD6). MBD5 lies in the minimum area of overlap of the 2q23.1 microdeletion syndrome. This report provides the first detailed clinical examination of two individuals with a duplication of this region and suggests that brain development and cognitive function may be affected by an increased dosage of the genes involved. © 2012 Macmillan Publishers Limited All rights reserved., link_to_OA_fulltext

Item does not contain fulltext Persons with neurodevelopmental disorders or autism spectrum disorder (ASD) often harbor chromosomal microdeletions, yet the individual genetic contributors within these regions have not been systematically evaluated. We established a consortium of clinical diagnostic and research laboratories to accumulate a large cohort with genetic alterations of chromosomal region 2q23.1 and acquired 65 subjects with microdeletion or translocation. We sequenced translocation breakpoints; aligned microdeletions to determine the critical region; assessed effects on mRNA expression; and examined medical records, photos, and clinical evaluations. We identified a single gene, methyl-CpG-binding domain 5 (MBD5), as the only locus that defined the critical region. Partial or complete deletion of MBD5 was associated with haploinsufficiency of mRNA expression, intellectual disability, epilepsy, and autistic features. Fourteen alterations, including partial deletions of noncoding regions not typically captured or considered pathogenic by current diagnostic screening, disrupted MBD5 alone. Expression profiles and clinical characteristics were largely indistinguishable between MBD5-specific alteration and deletion of the entire 2q23.1 interval. No copy-number alterations of MBD5 were observed in 7878 controls, suggesting MBD5 alterations are highly penetrant. We surveyed MBD5 coding variations among 747 ASD subjects compared to 2043 non-ASD subjects analyzed by whole-exome sequencing and detected an association with a highly conserved methyl-CpG-binding domain missense variant, p.79Gly>Glu (c.236G>A) (p = 0.012). These results suggest that genetic alterations of MBD5 cause features of 2q23.1 microdeletion syndrome and that this epigenetic regulator significantly contributes to ASD risk, warranting further consideration in research and clinical diagnostic screening and highlighting the importance of chromatin remodeling in the etiology of these complex disorders.

Microdeletion of chromosome 2q23.1 results in a novel syndrome previously reported in five individuals. Many of the del(2)(q23.1) cases were thought to have other syndromes such as Angelman, Prader-Willi, or Smith-Magenis because of certain overlapping clinical features. We report two new cases of the 2q23.1 microdeletion syndrome, describe the syndrome phenotype, define the minimal critical region, and analyze the expression of critical region genes toward identification of the causative gene(s) for the disorder. Individuals with del(2)(q23.1) have severe developmental and cognitive delays, minimal speech, seizures, microcephaly, mild craniofacial dysmorphism, behavioral disorders, and short stature. The deletions encompassing 2q23.1 range from4 Mb to200 kb, as identified by oligonucleotide and BAC whole-genome array comparative hybridization. The minimal critical region includes a single gene, MBD5, deleted in all cases, whereas all but one case also include deletion of EPC2. Quantitative real-time PCR of patient lymphoblasts/lymphocytes showed an approximately 50% reduced expression of MBD5 and EPC2 compared with controls. With similar phenotypes among the 2q23.1 deletion patients, the idea of one or more common genes causing the pathological defect seen in these patients becomes evident. As all five previous cases and the two cases in this report share one common gene, MBD5, we strongly suspect that haploinsufficiency of MBD5 causes most of the features observed in this syndrome.

Smith-Magenis syndrome is a complex genomic disorder in which a majority of individuals are obese by adolescence. While an interstitial deletion of chromosome 17p11.2 is the leading cause, mutation or deletion of the RAI1 gene alone results in most features of the disorder. Previous studies have shown that heterozygous knockout of Rai1 results in an obese phenotype in mice and that Smith-Magenis syndrome mouse models have a significantly reduced fecundity and an altered transmission pattern of the mutant Rai1 allele, complicating large, extended studies in these models. In this study, we show that breeding C57Bl/6J Rai1+/− mice with FVB/NJ to create F1 Rai1+/− offspring in a mixed genetic background ameliorates both fecundity and Rai1 allele transmission phenotypes. These findings suggest that the mixed background provides a more robust platform for breeding and larger phenotypic studies. We also characterized the effect of dietary intake on Rai1+/− mouse growth during adolescent and early adulthood developmental stages. Animals fed a high carbohydrate or a high fat diet gained weight at a significantly faster rate than their wild type littermates. Both high fat and high carbohydrate fed Rai1+/− mice also had an increase in body fat and altered fat distribution patterns. Interestingly, Rai1+/− mice fed different diets did not display altered fasting blood glucose levels. These results suggest that dietary regimens are extremely important for individuals with Smith- Magenis syndrome and that food high in fat and carbohydrates may exacerbate obesity outcomes.