The clinical and molecular spectrum of the KDM6B-related neurodevelopmental disorder.

De novo variants are a leading cause of neurodevelopmental disorders (NDDs), but because every monogenic NDD is different and usually extremely rare, it remains a major challenge to understand the complete phenotype and genotype spectrum of any morbid gene. According to OMIM, heterozygous variants in KDM6B cause "neurodevelopmental disorder with coarse facies and mild distal skeletal abnormalities." Here, by examining the molecular and clinical spectrum of 85 reported individuals with mostly de novo (likely) pathogenic KDM6B variants, we demonstrate that this description is inaccurate and potentially misleading. Cognitive deficits are seen consistently in all individuals, but the overall phenotype is highly variable. Notably, coarse facies and distal skeletal anomalies, as defined by OMIM, are rare in this expanded cohort while other features are unexpectedly common (e.g., hypotonia, psychosis, etc.). Using 3D protein structure analysis and an innovative dual Drosophila gain-of-function assay, we demonstrated a disruptive effect of 11 missense/in-frame indels located in or near the enzymatic JmJC or Zn-containing domain of KDM6B. Consistent with the role of KDM6B in human cognition, we demonstrated a role for the Drosophila KDM6B ortholog in memory and behavior. Taken together, we accurately define the broad clinical spectrum of the KDM6B-related NDD, introduce an innovative functional testing paradigm for the assessment of KDM6B variants, and demonstrate a conserved role for KDM6B in cognition and behavior. Our study demonstrates the critical importance of international collaboration, sharing of clinical data, and rigorous functional analysis of genetic variants to ensure correct disease diagnosis for rare disorders.

Germline and Mosaic Variants in PRKACA and PRKACB Cause a Multiple Congenital Malformation Syndrome.

PRKACA and PRKACB code for two catalytic subunits (Cα and Cß) of cAMP-dependent protein kinase (PKA), a pleiotropic holoenzyme that regulates numerous fundamental biological processes such as metabolism, development, memory, and immune response. We report seven unrelated individuals presenting with a multiple congenital malformation syndrome in whom we identified heterozygous germline or mosaic missense variants in PRKACA or PRKACB. Three affected individuals were found with the same PRKACA variant, and the other four had different PRKACB mutations. In most cases, the mutations arose de novo, and two individuals had offspring with the same condition. Nearly all affected individuals and their affected offspring shared an atrioventricular septal defect or a common atrium along with postaxial polydactyly. Additional features included skeletal abnormalities and ectodermal defects of variable severity in five individuals, cognitive deficit in two individuals, and various unusual tumors in one individual. We investigated the structural and functional consequences of the variants identified in PRKACA and PRKACB through the use of several computational and experimental approaches, and we found that they lead to PKA holoenzymes which are more sensitive to activation by cAMP than are the wild-type proteins. Furthermore, expression of PRKACA or PRKACB variants detected in the affected individuals inhibited hedgehog signaling in NIH 3T3 fibroblasts, thereby providing an underlying mechanism for the developmental defects observed in these cases. Our findings highlight the importance of both Cα and Cß subunits of PKA during human development.

De Novo Heterozygous POLR2A Variants Cause a Neurodevelopmental Syndrome with Profound Infantile-Onset Hypotonia.

The RNA polymerase II complex (pol II) is responsible for transcription of all ∼21,000 human protein-encoding genes. Here, we describe sixteen individuals harboring de novo heterozygous variants in POLR2A, encoding RPB1, the largest subunit of pol II. An iterative approach combining structural evaluation and mass spectrometry analyses, the use of S. cerevisiae as a model system, and the assessment of cell viability in HeLa cells allowed us to classify eleven variants as probably disease-causing and four variants as possibly disease-causing. The significance of one variant remains unresolved. By quantification of phenotypic severity, we could distinguish mild and severe phenotypic consequences of the disease-causing variants. Missense variants expected to exert only mild structural effects led to a malfunctioning pol II enzyme, thereby inducing a dominant-negative effect on gene transcription. Intriguingly, individuals carrying these variants presented with a severe phenotype dominated by profound infantile-onset hypotonia and developmental delay. Conversely, individuals carrying variants expected to result in complete loss of function, thus reduced levels of functional pol II from the normal allele, exhibited the mildest phenotypes. We conclude that subtle variants that are central in functionally important domains of POLR2A cause a neurodevelopmental syndrome characterized by profound infantile-onset hypotonia and developmental delay through a dominant-negative effect on pol-II-mediated transcription of DNA.

De Novo Pathogenic Variants in CACNA1E Cause Developmental and Epileptic Encephalopathy with Contractures, Macrocephaly, and Dyskinesias.

Developmental and epileptic encephalopathies (DEEs) are severe neurodevelopmental disorders often beginning in infancy or early childhood that are characterized by intractable seizures, abundant epileptiform activity on EEG, and developmental impairment or regression. CACNA1E is highly expressed in the central nervous system and encodes the α1-subunit of the voltage-gated CaV2.3 channel, which conducts high voltage-activated R-type calcium currents that initiate synaptic transmission. Using next-generation sequencing techniques, we identified de novo CACNA1E variants in 30 individuals with DEE, characterized by refractory infantile-onset seizures, severe hypotonia, and profound developmental impairment, often with congenital contractures, macrocephaly, hyperkinetic movement disorders, and early death. Most of the 14, partially recurring, variants cluster within the cytoplasmic ends of all four S6 segments, which form the presumed CaV2.3 channel activation gate. Functional analysis of several S6 variants revealed consistent gain-of-function effects comprising facilitated voltage-dependent activation and slowed inactivation. Another variant located in the domain II S4-S5 linker results in facilitated activation and increased current density. Five participants achieved seizure freedom on the anti-epileptic drug topiramate, which blocks R-type calcium channels. We establish pathogenic variants in CACNA1E as a cause of DEEs and suggest facilitated R-type calcium currents as a disease mechanism for human epilepsy and developmental disorders.

Disruption of RFX family transcription factors causes autism, attention-deficit/hyperactivity disorder, intellectual disability, and dysregulated behavior.

PURPOSE: We describe a novel neurobehavioral phenotype of autism spectrum disorder (ASD), intellectual disability, and/or attention-deficit/hyperactivity disorder (ADHD) associated with de novo or inherited deleterious variants in members of the RFX family of genes. RFX genes are evolutionarily conserved transcription factors that act as master regulators of central nervous system development and ciliogenesis. METHODS: We assembled a cohort of 38 individuals (from 33 unrelated families) with de novo variants in RFX3, RFX4, and RFX7. We describe their common clinical phenotypes and present bioinformatic analyses of expression patterns and downstream targets of these genes as they relate to other neurodevelopmental risk genes. RESULTS: These individuals share neurobehavioral features including ASD, intellectual disability, and/or ADHD; other frequent features include hypersensitivity to sensory stimuli and sleep problems. RFX3, RFX4, and RFX7 are strongly expressed in developing and adult human brain, and X-box binding motifs as well as RFX ChIP-seq peaks are enriched in the cis-regulatory regions of known ASD risk genes. CONCLUSION: These results establish a likely role of deleterious variation in RFX3, RFX4, and RFX7 in cases of monogenic intellectual disability, ADHD and ASD, and position these genes as potentially critical transcriptional regulators of neurobiological pathways associated with neurodevelopmental disease pathogenesis.

Integrative approach to interpret DYRK1A variants, leading to a frequent neurodevelopmental disorder.

PURPOSE: DYRK1A syndrome is among the most frequent monogenic forms of intellectual disability (ID). We refined the molecular and clinical description of this disorder and developed tools to improve interpretation of missense variants, which remains a major challenge in human genetics. METHODS: We reported clinical and molecular data for 50 individuals with ID harboring DYRK1A variants and developed (1) a specific DYRK1A clinical score; (2) amino acid conservation data generated from 100 DYRK1A sequences across different taxa; (3) in vitro overexpression assays to study level, cellular localization, and kinase activity of DYRK1A mutant proteins; and (4) a specific blood DNA methylation signature. RESULTS: This integrative approach was successful to reclassify several variants as pathogenic. However, we questioned the involvement of some others, such as p.Thr588Asn, still reported as likely pathogenic, and showed it does not cause an obvious phenotype in mice. CONCLUSION: Our study demonstrated the need for caution when interpreting variants in DYRK1A, even those occurring de novo. The tools developed will be useful to interpret accurately the variants identified in the future in this gene.

Choline transporter-like 1 deficiency causes a new type of childhood-onset neurodegeneration.

Cerebral choline metabolism is crucial for normal brain function, and its homoeostasis depends on carrier-mediated transport. Here, we report on four individuals from three families with neurodegenerative disease and homozygous frameshift mutations (Asp517Metfs*19, Ser126Metfs*8, and Lys90Metfs*18) in the SLC44A1 gene encoding choline transporter-like protein 1. Clinical features included progressive ataxia, tremor, cognitive decline, dysphagia, optic atrophy, dysarthria, as well as urinary and bowel incontinence. Brain MRI demonstrated cerebellar atrophy and leukoencephalopathy. Moreover, low signal intensity in globus pallidus with hyperintensive streaking and low signal intensity in substantia nigra were seen in two individuals. The Asp517Metfs*19 and Ser126Metfs*8 fibroblasts were structurally and functionally indistinguishable. The most prominent ultrastructural changes of the mutant fibroblasts were reduced presence of free ribosomes, the appearance of elongated endoplasmic reticulum and strikingly increased number of mitochondria and small vesicles. When chronically treated with choline, those characteristics disappeared and mutant ultrastructure resembled healthy control cells. Functional analysis revealed diminished choline transport yet the membrane phosphatidylcholine content remained unchanged. As part of the mechanism to preserve choline and phosphatidylcholine, choline transporter deficiency was implicated in impaired membrane homeostasis of other phospholipids. Choline treatments could restore the membrane lipids, repair cellular organelles and protect mutant cells from acute iron overload. In conclusion, we describe a novel childhood-onset neurometabolic disease caused by choline transporter deficiency with autosomal recessive inheritance.

Compound heterozygous mutations in two different domains of ALDH18A1 do not affect the amino acid levels in a patient with hereditary spastic paraplegia.

Mutations in ALDH18A1 can cause autosomal recessive and dominant hereditary spastic paraplegia and autosomal recessive and dominant cutis laxa. ALDH18A1 encodes delta-1-pyrroline-5-carboxylate synthetase (P5CS), which consists of two domains, the glutamate 5-kinase (G5K) and the gamma-glutamyl phosphate reductase (GR5P) domain. The location of the mutations in the gene has influence on whether the amino acid levels are affected. Mutations affecting the G5K domain have previously been found to cause reduced plasma levels of proline, citrulline and arginine, whereas such effect is not seen with mutations affecting the GR5P domain. We present a 19-year old male patient with autosomal recessive spastic paraplegia and compound heterozygosity for two ALDH18A1 mutations, one in each of the P5CS domains. This young man has spastic paraplegia with onset in childhood and temporal lobe epilepsy, but normal levels of proline, ornithine and arginine. To our knowledge, this is the first case with compound heterozygous mutations affecting both P5CS domains, where levels of plasma amino acids have been reported.

ALG1-CDG: Clinical and Molecular Characterization of 39 Unreported Patients.

Congenital disorders of glycosylation (CDG) arise from pathogenic mutations in over 100 genes leading to impaired protein or lipid glycosylation. ALG1 encodes a ß1,4 mannosyltransferase that catalyzes the addition of the first of nine mannose moieties to form a dolichol-lipid linked oligosaccharide intermediate required for proper N-linked glycosylation. ALG1 mutations cause a rare autosomal recessive disorder termed ALG1-CDG. To date 13 mutations in 18 patients from 14 families have been described with varying degrees of clinical severity. We identified and characterized 39 previously unreported cases of ALG1-CDG from 32 families and add 26 new mutations. Pathogenicity of each mutation was confirmed based on its inability to rescue impaired growth or hypoglycosylation of a standard biomarker in an alg1-deficient yeast strain. Using this approach we could not establish a rank order comparison of biomarker glycosylation and patient phenotype, but we identified mutations with a lethal outcome in the first two years of life. The recently identified protein-linked xeno-tetrasaccharide biomarker, NeuAc-Gal-GlcNAc2 , was seen in all 27 patients tested. Our study triples the number of known patients and expands the molecular and clinical correlates of this disorder.

SCRIB and PUF60 are primary drivers of the multisystemic phenotypes of the 8q24.3 copy-number variant.

Copy-number variants (CNVs) represent a significant interpretative challenge, given that each CNV typically affects the dosage of multiple genes. Here we report on five individuals with coloboma, microcephaly, developmental delay, short stature, and craniofacial, cardiac, and renal defects who harbor overlapping microdeletions on 8q24.3. Fine mapping localized a commonly deleted 78 kb region that contains three genes: SCRIB, NRBP2, and PUF60. In vivo dissection of the CNV showed discrete contributions of the planar cell polarity effector SCRIB and the splicing factor PUF60 to the syndromic phenotype, and the combinatorial suppression of both genes exacerbated some, but not all, phenotypic components. Consistent with these findings, we identified an individual with microcephaly, short stature, intellectual disability, and heart defects with a de novo c.505C>T variant leading to a p.His169Tyr change in PUF60. Functional testing of this allele in vivo and in vitro showed that the mutation perturbs the relative dosage of two PUF60 isoforms and, subsequently, the splicing efficiency of downstream PUF60 targets. These data inform the functions of two genes not associated previously with human genetic disease and demonstrate how CNVs can exhibit complex genetic architecture, with the phenotype being the amalgam of both discrete dosage dysfunction of single transcripts and also of binary genetic interactions.

17q12 deletion and duplication syndrome in Denmark-A clinical cohort of 38 patients and review of the literature.

17q12 deletions and duplications are two distinct, recurrent chromosomal aberrations usually diagnosed by chromosomal microarray analysis (CMA). The aberrations encompass the genes, HNF1B, LHX1, and ACACA, among others. We here describe a large national cohort of 12 phenotyped patients with 17q12 deletions and 26 phenotyped patients with 17q12 duplications. The total cohort includes 19 index patients and 19 family members. We also reviewed the literature in order to further improve the basis for the counseling. We emphasize that renal disease, learning disability, behavioral abnormalities, epilepsy, autism, schizophrenia, structural brain abnormalities, facial dysmorphism, and joint laxity are features seen in both the 17q12 deletion syndrome and the reciprocal 17q12 duplication syndrome; and we extend the list of features seen in both patient categories to include strabismus, esophageal defects, and duodenal atresia. Delayed language development, learning disability, kidney involvement, and eye dysmorphism and strabismus were the most consistently shared features among patients with 17q12 deletion. Patients with 17q12 duplications were characterized by an extremely wide phenotypic spectrum, including a variable degree of learning disabilities, delayed language development, delayed motor milestones, and a broad range of psychiatric and neurological features. This patient group also included adults achieving an academic degree. Assessing index patients and non-index patients separately, our observations illustrate that an overall milder disease burden is seen, in particular in patients with 17q12 duplications who are ascertained on the duplication rather than the phenotype. This evidence may be useful in prenatal counseling. © 2016 Wiley Periodicals, Inc.

De novo heterozygous mutations in SMC3 cause a range of Cornelia de Lange syndrome-overlapping phenotypes.

Cornelia de Lange syndrome (CdLS) is characterized by facial dysmorphism, growth failure, intellectual disability, limb malformations, and multiple organ involvement. Mutations in five genes, encoding subunits of the cohesin complex (SMC1A, SMC3, RAD21) and its regulators (NIPBL, HDAC8), account for at least 70% of patients with CdLS or CdLS-like phenotypes. To date, only the clinical features from a single CdLS patient with SMC3 mutation has been published. Here, we report the efforts of an international research and clinical collaboration to provide clinical comparison of 16 patients with CdLS-like features caused by mutations in SMC3. Modeling of the mutation effects on protein structure suggests a dominant-negative effect on the multimeric cohesin complex. When compared with typical CdLS, many SMC3-associated phenotypes are also characterized by postnatal microcephaly but with a less distinctive craniofacial appearance, a milder prenatal growth retardation that worsens in childhood, few congenital heart defects, and an absence of limb deficiencies. While most mutations are unique, two unrelated affected individuals shared the same mutation but presented with different phenotypes. This work confirms that de novo SMC3 mutations account for ∼ 1%-2% of CdLS-like phenotypes.

Recurrent duplications of 17q12 associated with variable phenotypes.

The ability to identify the clinical nature of the recurrent duplication of chromosome 17q12 has been limited by its rarity and the diverse range of phenotypes associated with this genomic change. In order to further define the clinical features of affected patients, detailed clinical information was collected in the largest series to date (30 patients and 2 of their siblings) through a multi-institutional collaborative effort. The majority of patients presented with developmental delays varying from mild to severe. Though dysmorphic features were commonly reported, patients do not have consistent and recognizable features. Cardiac, ophthalmologic, growth, behavioral, and other abnormalities were each present in a subset of patients. The newly associated features potentially resulting from 17q12 duplication include height and weight above the 95th percentile, cataracts, microphthalmia, coloboma, astigmatism, tracheomalacia, cutaneous mosaicism, pectus excavatum, scoliosis, hypermobility, hypospadias, diverticulum of Kommerell, pyloric stenosis, and pseudohypoparathryoidism. The majority of duplications were inherited with some carrier parents reporting learning disabilities or microcephaly. We identified additional, potentially contributory copy number changes in a subset of patients, including one patient each with 16p11.2 deletion and 15q13.3 deletion. Our data further define and expand the clinical spectrum associated with duplications of 17q12 and provide support for the role of genomic modifiers contributing to phenotypic variability.

The role of SLC2A1 mutations in myoclonic astatic epilepsy and absence epilepsy, and the estimated frequency of GLUT1 deficiency syndrome.

The first mutations identified in SLC2A1, encoding the glucose transporter type 1 (GLUT1) protein of the blood-brain barrier, were associated with severe epileptic encephalopathy. Recently, dominant SLC2A1 mutations were found in rare autosomal dominant families with various forms of epilepsy including early onset absence epilepsy (EOAE), myoclonic astatic epilepsy (MAE), and genetic generalized epilepsy (GGE). Our study aimed to investigate the possible role of SLC2A1 in various forms of epilepsy including MAE and absence epilepsy with early onset. We also aimed to estimate the frequency of GLUT1 deficiency syndrome in the Danish population. One hundred twenty patients with MAE, 50 patients with absence epilepsy, and 37 patients with unselected epilepsies, intellectual disability (ID), and/or various movement disorders were screened for mutations in SLC2A1. Mutations in SLC2A1 were detected in 5 (10%) of 50 patients with absence epilepsy, and in one (2.7%) of 37 patient with unselected epilepsies, ID, and/or various movement disorders. None of the 120 MAE patients harbored SLC2A1 mutations. We estimated the frequency of SLC2A1 mutations in the Danish population to be approximately 1:83,000. Our study confirmed the role of SLC2A1 mutations in absence epilepsy with early onset. However, our study failed to support the notion that SLC2A1 aberrations are a cause of MAE without associated features such as movement disorders.

A newly recognized 13q12.3 microdeletion syndrome characterized by intellectual disability, microcephaly, and eczema/atopic dermatitis encompassing the HMGB1 and KATNAL1 genes.

Proximal deletions of the long arm of chromosome 13 have been reported only rarely. Here we present three unrelated patients with heterozygous, apparently de novo deletions encompassing 13q12.3. The patients present with moderate demonstrated or apparent intellectual disability, postnatal microcephaly, and eczema/atopic dermatitis as the predominant symptoms. In addition, they had pronounced feeding difficulties in early infancy. They displayed similar facial features such as malar flattening, a prominent nose with underdeveloped alae nasi, a smooth philtrum, and a thin vermillion of the upper lip. The proximal and distal breakpoints were clustered and the deletions spanned from 1.4 to 1.7 Mb, comprising at least 11 RefSeq genes. However, heterozygous deletions partially overlapping those observed in the present patients have been described in healthy parents of patients with Peters-Plus syndrome, an autosomal recessive disorder caused by inactivation of the B3GALTL gene. We therefore propose that the critical region of the 13q12.3 microdeletion syndrome contains only three genes, namely, KATNAL1, HMGB1, and LINC00426, a non-protein coding RNA. The KATNAL1 protein belongs to a family of microtubule severing enzymes that have been implicated in CNS plasticity in experimental models, but little is known about its function in humans. The HMGB1 protein is an evolutionarily conserved chromatin-associated protein involved in many biologically important processes. In summary, we propose that microdeletion 13q12.3 represents a novel clinically recognizable condition and that the microtubule severing gene KATNAL1 and the chromatin-associated gene HMGB1 are candidate genes for intellectual disability inherited in an autosomal dominant pattern.

Further clinical and molecular characterization of an XLID syndrome associated with BRWD3 variants, a gene implicated in the leukemia-related JAK-STAT pathway.

BACKGROUND: Since the first description of a BRWD3-associated nonsydromic intellectual disability (ID) disorder in 2007, 21 additional families have been reported in the literature. METHODS: Using exome sequencing (ES) and international data sharing, we identified 14 additional unrelated individuals with pathogenic BRWD3 variants (12 males and 2 females, including one with skewed X-inactivation). We reviewed the 31 previously published cases in the literature with clinical data available, and describe the collective phenotypes of 43 males and 2 females, with 33 different BRWD3 variants. RESULTS: The most common features in males (excluding one patient with a mosaic variant) included ID (39/39 males), speech delay (24/25 males), postnatal macrocephaly (28/35 males) with prominent forehead (18/25 males) and large ears (14/26 males), and obesity (12/27 males). Both females presented with macrocephaly, speech delay, and epilepsy, while epilepsy was only observed in 4/41 males. Among the 28 variants with available segregation reported, 19 were inherited from unaffected mothers and 9 were de novo. CONCLUSION: This study demonstrates that the BRWD3-related phenotypes are largely non-specific, leading to difficulty in clinical recognition of this disorder. A genotype-first approach, however, allows for the more efficient diagnosis of the BRWD3-related nonsyndromic ID. The refined clinical features presented here may provide additional diagnostic assistance for reverse phenotyping efforts.

The neurodevelopmental and facial phenotype in individuals with a TRIP12 variant.

Haploinsufficiency of TRIP12 causes a neurodevelopmental disorder characterized by intellectual disability associated with epilepsy, autism spectrum disorder and dysmorphic features, also named Clark-Baraitser syndrome. Only a limited number of cases have been reported to date. We aimed to further delineate the TRIP12-associated phenotype and objectify characteristic facial traits through GestaltMatcher image analysis based on deep-learning algorithms in order to establish a TRIP12 gestalt. 38 individuals between 3 and 66 years (F = 20, M = 18) - 1 previously published and 37 novel individuals - were recruited through an ERN ITHACA call for collaboration. 35 TRIP12 variants were identified, including frameshift (n = 15) and nonsense (n = 6) variants, as well as missense (n = 5) and splice (n = 3) variants, intragenic deletions (n = 4) and two multigene deletions disrupting TRIP12. Though variable in severity, global developmental delay was noted in all individuals, with language deficit most pronounced. About half showed autistic features and susceptibility to obesity seemed inherent to this disorder. A more severe expression was noted in individuals with a missense variant. Facial analysis showed a clear gestalt including deep-set eyes with narrow palpebral fissures and fullness of the upper eyelids, downturned corners of the mouth and large, often low-set ears with prominent earlobes. We report the largest cohort to date of individuals with TRIP12 variants, further delineating the associated phenotype and introducing a facial gestalt. These findings will improve future counseling and patient guidance.

Mutations affecting the N-terminal domains of SHANK3 point to different pathomechanisms in neurodevelopmental disorders.

Shank proteins are major scaffolds of the postsynaptic density of excitatory synapses. Mutations in SHANK genes are associated with autism and intellectual disability. The effects of missense mutations on Shank3 function, and therefore the pathomechanisms are unclear. Several missense mutations in SHANK3 affect the N-terminal region, consisting of the Shank/ProSAP N-terminal (SPN) domain and a set of Ankyrin (Ank) repeats. Here we identify a novel SHANK3 missense mutation (p.L270M) in the Ankyrin repeats in patients with an ADHD-like phenotype. We functionally analysed this and a series of other mutations, using biochemical and biophysical techniques. We observe two major effects: (1) a loss of binding to δ-catenin (e.g. in the p.L270M variant), and (2) interference with the intramolecular interaction between N-terminal SPN domain and the Ank repeats. This also interferes with binding to the α-subunit of the calcium-/calmodulin dependent kinase II (αCaMKII), and appears to be associated with a more severe neurodevelopmental pathology.