De novo variants in the non-coding spliceosomal snRNA gene RNU4-2 are a frequent cause of syndromic neurodevelopmental disorders.

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Increasingly, large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here, we identify the non-coding RNA RNU4-2 as a novel syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 bp region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and Stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 119 individuals with NDD. The vast majority of individuals (77.3%) have the same highly recurrent single base-pair insertion (n.64_65insT). We estimate that variants in this region explain 0.41% of individuals with NDD. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to its contiguous counterpart RNU4-1 and other U4 homologs, supporting RNU4-2's role as the primary U4 transcript in the brain. Overall, this work underscores the importance of non-coding genes in rare disorders. It will provide a diagnosis to thousands of individuals with NDD worldwide and pave the way for the development of effective treatments for these individuals.

Cholic acid increases plasma cholesterol in Smith-Lemli-Opitz syndrome: A pilot study.

Background: Smith-Lemli-Opitz syndrome (SLOS) is an inherited disorder of cholesterol biosynthesis associated with congenital malformations, growth delay, intellectual disability and behavior problems. SLOS is caused by bi-allelic mutations in DHCR7, which lead to reduced activity of 7-dehydrocholesterol reductase that catalyzes the last step in cholesterol biosynthesis. Symptoms of SLOS are thought to be due to cholesterol deficiency and accumulation of its precursor 7-dehydrocholesterol (7-DHC) and 8-dehydrocholesterol (8-DHC), and toxic oxysterols. Therapy for SLOS often includes dietary cholesterol supplementation, but lipids are poorly absorbed from the diet, possibly due to impaired bile acid synthesis. We hypothesized that bile acid supplementation with cholic acid would improve dietary cholesterol absorption and raise plasma cholesterol levels. Methods: Twelve SLOS subjects (10 M, 2F, ages 2-27 years) who had plasma cholesterol ≤125 mg/dL were treated with cholic acid (10 mg/kg/day) divided twice daily for 2 months. Plasma cholesterol, 7-DHC and 8-DHC were measured by GC-MS. Oxysterols were measured by ultra-high-performance LC-MS/MS. Data were analyzed using paired t-tests. Results: At baseline, plasma cholesterol was 75 ± 24 mg/dL (mean ± SD; range 43-125, n = 12). After 2 months on cholic acid, mean plasma cholesterol increased to 97 ± 29 mg/dL (p = 0.011). Eleven of 12 subjects showed an increase in plasma cholesterol that varied from 3.8% to 85.7% (mean 38.7 ± 23.3%). 7-Hydroxycholesterol decreased by 20.6% on average (p = 0.013) but no significant changes were seen in 7-DHC or 8-DHC. Mean body weight tended to increase (3.6% p = 0.069). Subjects tolerated cholic acid well and experienced no drug-related adverse events. Conclusions: In this pilot study, cholic acid supplementation was well tolerated and safe and resulted in an increase in plasma cholesterol in most SLOS subjects. Further controlled longitudinal studies are needed to look for the sustainability of the biochemical effect and possible clinical benefits.

Five siblings expand the spectrum of GPC6-related skeletal dysplasia.

Skeletal dysplasias broadly include disorders of cartilage or bone. Omodysplasia-1 is a type of skeletal dysplasia caused by biallelic loss of function variants in the GPC6 gene. GPC6 codes for the protein glypican 6 (GPC6) (OMIM *604404), which stimulates bone growth. We report a family in which five out of nine children were presented with a skeletal dysplasia characterized phenotypically by mild short stature and rhizomelia. All affected individuals were found to have homozygous missense variants in GPC6: c.511 C>T (p.Arg171Trp). Radiograph findings included rhizomelic foreshortening of all four extremities, coxa breva, and ulna minus deformity. Using a Hedgehog (Hh) reporter assay, we demonstrate that the variant found in this family results in significantly reduced stimulation of Hh activity when compared to the wild-type GPC6 protein, however protein function is still present. Thus, the milder phenotype seen in the family presented is hypothesized due to decreased GPC6 protein activity versus complete loss of function as seen in omodysplasia-1. Given the unique phenotype and molecular mechanism, we propose that this family's findings widen the phenotypic spectrum of GPC6-related skeletal dysplasias.

KDM5A mutations identified in autism spectrum disorder using forward genetics.

Autism spectrum disorder (ASD) is a constellation of neurodevelopmental disorders with high phenotypic and genetic heterogeneity, complicating the discovery of causative genes. Through a forward genetics approach selecting for defective vocalization in mice, we identified Kdm5a as a candidate ASD gene. To validate our discovery, we generated a Kdm5a knockout mouse model (Kdm5a-/-) and confirmed that inactivating Kdm5a disrupts vocalization. In addition, Kdm5a-/- mice displayed repetitive behaviors, sociability deficits, cognitive dysfunction, and abnormal dendritic morphogenesis. Loss of KDM5A also resulted in dysregulation of the hippocampal transcriptome. To determine if KDM5A mutations cause ASD in humans, we screened whole exome sequencing and microarray data from a clinical cohort. We identified pathogenic KDM5A variants in nine patients with ASD and lack of speech. Our findings illustrate the power and efficacy of forward genetics in identifying ASD genes and highlight the importance of KDM5A in normal brain development and function.

Automated syndrome diagnosis by three-dimensional facial imaging.

PURPOSE: Deep phenotyping is an emerging trend in precision medicine for genetic disease. The shape of the face is affected in 30-40% of known genetic syndromes. Here, we determine whether syndromes can be diagnosed from 3D images of human faces. METHODS: We analyzed variation in three-dimensional (3D) facial images of 7057 subjects: 3327 with 396 different syndromes, 727 of their relatives, and 3003 unrelated, unaffected subjects. We developed and tested machine learning and parametric approaches to automated syndrome diagnosis using 3D facial images. RESULTS: Unrelated, unaffected subjects were correctly classified with 96% accuracy. Considering both syndromic and unrelated, unaffected subjects together, balanced accuracy was 73% and mean sensitivity 49%. Excluding unrelated, unaffected subjects substantially improved both balanced accuracy (78.1%) and sensitivity (56.9%) of syndrome diagnosis. The best predictors of classification accuracy were phenotypic severity and facial distinctiveness of syndromes. Surprisingly, unaffected relatives of syndromic subjects were frequently classified as syndromic, often to the syndrome of their affected relative. CONCLUSION: Deep phenotyping by quantitative 3D facial imaging has considerable potential to facilitate syndrome diagnosis. Furthermore, 3D facial imaging of "unaffected" relatives may identify unrecognized cases or may reveal novel examples of semidominant inheritance.

Mapping the Relationship between Dysmorphology and Cognitive, Behavioral, and Developmental Outcomes in Children with Autism Spectrum Disorder.

Previous studies investigating the association between dysmorphology and cognitive, behavioral, and developmental outcomes among individuals with autism spectrum disorder (ASD) have been limited by the binary classification of dysmorphology and lack of comparison groups. We assessed the association using a continuous measure of dysmorphology severity (DS) in preschool children aged 2-5 years (322 with ASD and intellectual disability [ID], 188 with ASD without ID, and 371 without ASD from the general population [POP]). In bivariate analyses, an inverse association between DS and expressive language, receptive language, fine motor, and visual reception skills was observed in children with ASD and ID. An inverse association of DS with fine motor and visual reception skills, but not expressive language and receptive language, was found in children with ASD without ID. No associations were observed in POP children. These results persisted after exclusion of children with known genetic syndromes or major morphologic anomalies. Quantile regression models showed that the inverse relationships remained significant after adjustment for sex, race/ethnicity, maternal education, family income, study site, and preterm birth. DS was not associated with autistic traits or autism symptom severity, behaviors, or regression among children with ASD with or without ID. Thus, DS was associated with a global impairment of cognitive functioning in children with ASD and ID, but only with fine motor and visual reception deficits in children with ASD without ID. A better understanding is needed for mechanisms that explain the association between DS and cognitive impairment in children with different disorders. Autism Res 2020, 13: 1227-1238. © 2020 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: We examined whether having more dysmorphic features (DFs) was related to developmental problems among children with autism spectrum disorder (ASD) with or without intellectual disability (ID), and children without ASD from the general population (POP). Children with ASD and ID had more language, movement, and learning issues as the number of DFs increased. Children with ASD without ID had more movement and learning issues as the number of DFs increased. These relationships were not observed in the POP group. Implications are discussed.

IRF2BPL gene mutation: Expanding on neurologic phenotypes.

Heterozygous loss of function variants in the IRF2BPL are a newly described cause of neurodevelopmental disabilities and epilepsy. As of 2019, fewer than 20 patients have been described in the published literature. This article reports an additional case of a patient with a pathogenic IRF2BPL variant and offers a comprehensive review of the published cases of individuals with IRF2BPL variants, in order to help expand the phenotype. The patient has a history of infantile spasms evolving into drug-resistant epilepsy with underlying epileptic encephalopathy consistent with Lennox-Gastaut syndrome. While at the extreme end of the spectrum, his phenotype is consistent with those previously described. Our literature review highlights the wide range of phenotypes exhibited by those with diseases related to IRF2BPL gene variants. This article also briefly discusses other comorbidities seen in the patient and those previously reported. While the molecular underpinnings of the role of IRF2BPL gene in the central nervous system are newly established, the specifics of its effects elsewhere have yet to be delineated. Furthermore, its pathogenesis in other organ systems is not yet understood and could be of importance from a management perspective.

A Novel Approach to Dysmorphology to Enhance the Phenotypic Classification of Autism Spectrum Disorder in the Study to Explore Early Development.

The presence of multiple dysmorphic features in some children with autism spectrum disorder (ASD) might identify distinct ASD phenotypes and serve as potential markers for understanding causes and prognoses. To evaluate dysmorphology in ASD, children aged 3-6 years with ASD and non-ASD population controls (POP) from the Study to Explore Early Development were evaluated using a novel, systematic dysmorphology review approach. Separate analyses were conducted for non-Hispanic White, non-Hispanic Black, and Hispanic children. In each racial/ethnic group, ~ 17% of ASD cases were Dysmorphic compared with ~ 5% of POP controls. The ASD-POP differential was not explained by known genetic disorders or birth defects. In future epidemiologic studies, subgrouping ASD cases as Dysmorphic vs. Non-dysmorphic might help delineate risk factors for ASD.

Correction: The ARID1B spectrum in 143 patients: from nonsyndromic intellectual disability to Coffin-Siris syndrome.

The original version of this Article contained an error in the spelling of the author Pleuntje J. van der Sluijs, which was incorrectly given as Eline (P. J.) van der Sluijs. This has now been corrected in both the PDF and HTML versions of the Article.

The ARID1B spectrum in 143 patients: from nonsyndromic intellectual disability to Coffin-Siris syndrome.

PURPOSE: Pathogenic variants in ARID1B are one of the most frequent causes of intellectual disability (ID) as determined by large-scale exome sequencing studies. Most studies published thus far describe clinically diagnosed Coffin-Siris patients (ARID1B-CSS) and it is unclear whether these data are representative for patients identified through sequencing of unbiased ID cohorts (ARID1B-ID). We therefore sought to determine genotypic and phenotypic differences between ARID1B-ID and ARID1B-CSS. In parallel, we investigated the effect of different methods of phenotype reporting. METHODS: Clinicians entered clinical data in an extensive web-based survey. RESULTS: 79 ARID1B-CSS and 64 ARID1B-ID patients were included. CSS-associated dysmorphic features, such as thick eyebrows, long eyelashes, thick alae nasi, long and/or broad philtrum, small nails and small or absent fifth distal phalanx and hypertrichosis, were observed significantly more often (p < 0.001) in ARID1B-CSS patients. No other significant differences were identified. CONCLUSION: There are only minor differences between ARID1B-ID and ARID1B-CSS patients. ARID1B-related disorders seem to consist of a spectrum, and patients should be managed similarly. We demonstrated that data collection methods without an explicit option to report the absence of a feature (such as most Human Phenotype Ontology-based methods) tended to underestimate gene-related features.

The co-occurrence of Down syndrome and autism spectrum disorder: is it because of additional genetic variations?

Individuals with Down syndrome (DS) are diagnosed with autism spectrum disorder (ASD) at a significantly higher frequency than the typical population. The differentiation of ASD symptoms from those of severe intellectual disability presents diagnostic challenges, which have led to more refined methods in the clinical evaluation of ASD in DS. These improved phenotypic characterization methods not only provide better diagnosis of ASD in DS, but may also be useful in elucidating the etiology of the increased prevalence of ASD in DS. Because all individuals with the classic presentation of DS have trisomy 21, it is possible that those with co-occurring DS and ASD may have additional genetic variants which can act as modifiers of the phenotype, leading to the development of ASD. © 2016 John Wiley & Sons, Ltd.

Discovery of a potentially deleterious variant in TMEM87B in a patient with a hemizygous 2q13 microdeletion suggests a recessive condition characterized by congenital heart disease and restrictive cardiomyopathy.

Restrictive cardiomyopathy (RCM) is a rare cause of heart muscle disease with the highest mortality rate among cardiomyopathy types. The etiology of RCM is poorly understood, although genetic causes have been implicated, and syndromic associations have been described. Here, we describe a patient with an atrial septal defect and restrictive cardiomyopathy along with craniofacial anomalies and intellectual disabilities. Initial screening using chromosomal microarray analysis (CMA) identified a maternally inherited 2q13 microdeletion. The patient had many of the features reported in previous cases with the recurrent 2q13 microdeletion syndrome. However, the inheritance of the microdeletion from an unaffected mother combined with the low incidence (10%) and milder forms of cardiac defects in previously reported cases made the clinical significance of the CMA results unclear. Whole-exome sequencing (WES) with trio-based analysis was performed and identified a paternally inherited TMEM87B mutation (c.1366A>G, p.Asn456Asp) in the patient. TMEM87B, a highly conserved, transmembrane protein of currently unknown function, lies within the critical region of the recurrent 2q13 microdeletion syndrome. Furthermore, a recent study had demonstrated that depletion of TMEM87B in zebrafish embryos affected cardiac development and led to cardiac hypoplasia. Thus, by combining CMA and WES, we potentially uncover an autosomal-recessive disorder characterized by a severe cardiac phenotype caused by mutations in TMEM87B. This study expands the spectrum of phenotypes associated with the recurrent 2q13 microdeletion syndrome and also further suggests the role of TMEM87B in its etiology, especially the cardiac pathology.

Assays of plasma dehydrocholesteryl esters and oxysterols from Smith-Lemli-Opitz syndrome patients.

Smith-Lemli-Opitz syndrome (SLOS) is caused by mutations in the gene encoding 3ß-hydroxysterol-Δ(7)-reductase and as a result of this defect, 7-dehydrocholesterol (7-DHC) and 8-dehydrocholesterol (8-DHC) accumulate in the fluids and tissues of patients with this syndrome. Both 7- and 8-DHC are susceptible to peroxidation reactions, and several biologically active DHC oxysterols are found in cell and animal models of SLOS. Ex vivo oxidation of DHCs can be a confounding factor in the analysis of these sterols and their esters, and we developed HPLC/MS methods that permit the direct analysis of cholesterol, 7-DHC, 8-DHC, and their esters in human plasma, thus avoiding ex vivo oxidation. In addition, three oxysterols were classified as endogenously formed products by the use of an isotopically-labeled 7-DHC (d(7)-7-DHC) added to the sample before workup, followed by MS analysis of products formed. Analysis of 17 SLOS plasma samples shows that 8-DHC linoleate correlates better with the SLOS severity score of the patients than other sterols or metabolites, including cholesterol and 7-DHC. Levels of 7-ketocholesterol also correlate with the SLOS severity score. 8-DHC esters should have utility as surrogate markers of severity in SLOS for prognostication and as endpoints in clinical trials.

Cone ERG responses in patients with Smith-Lemli-Opitz Syndrome (SLOS).

PURPOSE: To evaluate cone and cone-driven retinal function in patients with Smith-Lemli-Opitz syndrome (SLOS), a condition characterized by low cholesterol. Rod and rod-driven function in patients with SLOS are known to be abnormal. METHODS: Electroretinographic (ERG) responses to full-field stimuli presented on a steady, rod suppressing background were recorded in 13 patients who had received long-term cholesterol supplementation. Cone photoresponse sensitivity (S(CONE)) and saturated amplitude (R(CONE)) parameters were estimated using a model of the activation of phototransduction, and post-receptor b-wave and 30 Hz flicker responses were analyzed. The responses of the patients were compared to those of control subjects (N = 13). RESULTS: Although average values of both S(CONE) and R(CONE) were lower than in controls, the differences were not statistically significant. Post-receptor b-wave amplitude and implicit time and flicker responses were normal. CONCLUSIONS: The normal cone function contrasts with the significant abnormalities in rod function that were found previously in these same patients. Possibly, cholesterol supplementation has a greater protective effect on cones than on rods as has been demonstrated in the rat model of SLOS.

Rod photoreceptor responses in children with Smith-Lemli-Opitz syndrome.

OBJECTIVE: To test the hypothesis that the kinetics of activation and deactivation of rod phototransduction are altered in children with Smith-Lemli-Optiz syndrome (SLOS), a common genetic disorder caused by an inborn error in cholesterol biosynthesis. METHODS: Thirteen patients with SLOS (median age, 4 years) were studied by means of scotopic full-field electroretinography. The kinetics of activation and deactivation of rod phototransduction were derived from the electroretinographic a-wave. Postreceptoral electroretinographic components were also evaluated. RESULTS: The kinetics of activation were below normal limits in all but 3 of the 13 patients. Rod cell recovery (deactivation) in SLOS was slower than normal in all 8 patients in whom it was studied. Postreceptoral sensitivity was below normal limits in all but 1 of the 13 patients. CONCLUSIONS: The kinetics of phototransduction are slow in children with SLOS. This is likely a consequence of altered sterol composition in the cell membranes of the rod photoreceptors. To our knowledge, this is the first demonstration of altered kinetics of a membrane-bound signaling system in SLOS. Investigation of other membrane-bound signaling systems may be warranted in the quest to understand development and phenotype of individuals with SLOS.