Single substitution in H3.3G34 alters DNMT3A recruitment to cause progressive neurodegeneration.

Germline histone H3.3 amino acid substitutions, including H3.3G34R/V, cause severe neurodevelopmental syndromes. To understand how these mutations impact brain development, we generated H3.3G34R/V/W knock-in mice and identified strikingly distinct developmental defects for each mutation. H3.3G34R-mutants exhibited progressive microcephaly and neurodegeneration, with abnormal accumulation of disease-associated microglia and concurrent neuronal depletion. G34R severely decreased H3K36me2 on the mutant H3.3 tail, impairing recruitment of DNA methyltransferase DNMT3A and its redistribution on chromatin. These changes were concurrent with sustained expression of complement and other innate immune genes possibly through loss of non-CG (CH) methylation and silencing of neuronal gene promoters through aberrant CG methylation. Complement expression in G34R brains may lead to neuroinflammation possibly accounting for progressive neurodegeneration. Our study reveals that H3.3G34-substitutions have differential impact on the epigenome, which underlie the diverse phenotypes observed, and uncovers potential roles for H3K36me2 and DNMT3A-dependent CH-methylation in modulating synaptic pruning and neuroinflammation in post-natal brains.

AXIN1 bi-allelic variants disrupting the C-terminal DIX domain cause craniometadiaphyseal osteosclerosis with hip dysplasia.

Sclerosing skeletal dysplasias result from an imbalance between bone formation and resorption. We identified three homozygous, C-terminally truncating AXIN1 variants in seven individuals from four families affected by macrocephaly, cranial hyperostosis, and vertebral endplate sclerosis. Other frequent findings included hip dysplasia, heart malformations, variable developmental delay, and hematological anomalies. In line with AXIN1 being a central component of the ß-catenin destruction complex, analyses of primary and genome-edited cells harboring the truncating variants revealed enhanced basal canonical Wnt pathway activity. All three AXIN1-truncating variants resulted in reduced protein levels and impaired AXIN1 polymerization mediated by its C-terminal DIX domain but partially retained Wnt-inhibitory function upon overexpression. Addition of a tankyrase inhibitor attenuated Wnt overactivity in the AXIN1-mutant model systems. Our data suggest that AXIN1 coordinates the action of osteoblasts and osteoclasts and that tankyrase inhibitors can attenuate the effects of AXIN1 hypomorphic variants.

Truncating SRCAP variants outside the Floating-Harbor syndrome locus cause a distinct neurodevelopmental disorder with a specific DNA methylation signature.

Truncating variants in exons 33 and 34 of the SNF2-related CREBBP activator protein (SRCAP) gene cause the neurodevelopmental disorder (NDD) Floating-Harbor syndrome (FLHS), characterized by short stature, speech delay, and facial dysmorphism. Here, we present a cohort of 33 individuals with clinical features distinct from FLHS and truncating (mostly de novo) SRCAP variants either proximal (n = 28) or distal (n = 5) to the FLHS locus. Detailed clinical characterization of the proximal SRCAP individuals identified shared characteristics: developmental delay with or without intellectual disability, behavioral and psychiatric problems, non-specific facial features, musculoskeletal issues, and hypotonia. Because FLHS is known to be associated with a unique set of DNA methylation (DNAm) changes in blood, a DNAm signature, we investigated whether there was a distinct signature associated with our affected individuals. A machine-learning model, based on the FLHS DNAm signature, negatively classified all our tested subjects. Comparing proximal variants with typically developing controls, we identified a DNAm signature distinct from the FLHS signature. Based on the DNAm and clinical data, we refer to the condition as "non-FLHS SRCAP-related NDD." All five distal variants classified negatively using the FLHS DNAm model while two classified positively using the proximal model. This suggests divergent pathogenicity of these variants, though clinically the distal group presented with NDD, similar to the proximal SRCAP group. In summary, for SRCAP, there is a clear relationship between variant location, DNAm profile, and clinical phenotype. These results highlight the power of combined epigenetic, molecular, and clinical studies to identify and characterize genotype-epigenotype-phenotype correlations.

Biallelic loss-of-function variants of SLC12A9 cause lysosome dysfunction and a syndromic neurodevelopmental disorder.

PURPOSE: Pathogenic variants of FIG4 generate enlarged lysosomes and neurological and developmental disorders. To identify additional genes regulating lysosomal volume, we carried out a genome-wide activation screen to detect suppression of enlarged lysosomes in FIG4-/- cells. METHODS: The CRISPR-a gene activation screen utilized sgRNAs from the promoters of protein-coding genes. Fluorescence-activated cell sorting separated cells with correction of the enlarged lysosomes from uncorrected cells. Patient variants of SLC12A9 were identified by exome or genome sequencing and studied by segregation analysis and clinical characterization. RESULTS: Overexpression of SLC12A9, a solute co-transporter, corrected lysosomal swelling in FIG4-/- cells. SLC12A9 (NP_064631.2) colocalized with LAMP2 at the lysosome membrane. Biallelic variants of SLC12A9 were identified in 3 unrelated probands with neurodevelopmental disorders. Common features included intellectual disability, skeletal and brain structural abnormalities, congenital heart defects, and hypopigmented hair. Patient 1 was homozygous for nonsense variant p.(Arg615∗), patient 2 was compound heterozygous for p.(Ser109Lysfs∗20) and a large deletion, and proband 3 was compound heterozygous for p.(Glu290Glyfs∗36) and p.(Asn552Lys). Fibroblasts from proband 1 contained enlarged lysosomes that were corrected by wild-type SLC12A9 cDNA. Patient variant p.(Asn552Lys) failed to correct the lysosomal defect. CONCLUSION: Impaired function of SLC12A9 results in enlarged lysosomes and a recessive disorder with a recognizable neurodevelopmental phenotype.

A recurrent homozygous LMNA missense variant p.Thr528Met causes atypical progeroid syndrome characterized by mandibuloacral dysostosis, severe muscular dystrophy, and skeletal deformities.

Atypical progeroid syndromes (APS) are premature aging syndromes caused by pathogenic LMNA missense variants, associated with unaltered expression levels of lamins A and C, without accumulation of wild-type or deleted prelamin A isoforms, as observed in Hutchinson-Gilford progeria syndrome (HGPS) or HGPS-like syndromes. A specific LMNA missense variant, (p.Thr528Met), was previously identified in a compound heterozygous state in patients affected by APS and severe familial partial lipodystrophy, whereas heterozygosity was recently identified in patients affected by Type 2 familial partial lipodystrophy. Here, we report four unrelated boys harboring homozygosity for the p.Thr528Met, variant who presented with strikingly homogeneous APS clinical features, including osteolysis of mandibles, distal clavicles and phalanges, congenital muscular dystrophy with elevated creatine kinase levels, and major skeletal deformities. Immunofluorescence analyses of patient-derived primary fibroblasts showed a high percentage of dysmorphic nuclei with nuclear blebs and typical honeycomb patterns devoid of lamin B1. Interestingly, in some protrusions emerin or LAP2α formed aberrant aggregates, suggesting pathophysiology-associated clues. These four cases further confirm that a specific LMNA variant can lead to the development of strikingly homogeneous clinical phenotypes, in these particular cases a premature aging phenotype with major musculoskeletal involvement linked to the homozygous p.Thr528Met variant.

Biallelic CACNA2D1 loss-of-function variants cause early-onset developmental epileptic encephalopathy.

Voltage-gated calcium (CaV) channels form three subfamilies (CaV1-3). The CaV1 and CaV2 channels are heteromeric, consisting of an α1 pore-forming subunit, associated with auxiliary CaVß and α2δ subunits. The α2δ subunits are encoded in mammals by four genes, CACNA2D1-4. They play important roles in trafficking and function of the CaV channel complexes. Here we report biallelic variants in CACNA2D1, encoding the α2δ-1 protein, in two unrelated individuals showing a developmental and epileptic encephalopathy. Patient 1 has a homozygous frameshift variant c.818_821dup/p.(Ser275Asnfs*13) resulting in nonsense-mediated mRNA decay of the CACNA2D1 transcripts, and absence of α2δ-1 protein detected in patient-derived fibroblasts. Patient 2 is compound heterozygous for an early frameshift variant c.13_23dup/p.(Leu9Alafs*5), highly probably representing a null allele and a missense variant c.626G>A/p.(Gly209Asp). Our functional studies show that this amino-acid change severely impairs the function of α2δ-1 as a calcium channel subunit, with strongly reduced trafficking of α2δ-1G209D to the cell surface and a complete inability of α2δ-1G209D to increase the trafficking and function of CaV2 channels. Thus, biallelic loss-of-function variants in CACNA2D1 underlie the severe neurodevelopmental disorder in these two patients. Our results demonstrate the critical importance and non-interchangeability of α2δ-1 and other α2δ proteins for normal human neuronal development.

Variant-specific effects define the phenotypic spectrum of HNRNPH2-associated neurodevelopmental disorders in males.

Bain type of X-linked syndromic intellectual developmental disorder, caused by pathogenic missense variants in HRNRPH2, was initially described in six female individuals affected by moderate-to-severe neurodevelopmental delay. Although it was initially postulated that the condition would not be compatible with life in males, several affected male individuals harboring pathogenic variants in HNRNPH2 have since been documented. However, functional in-vitro analyses of identified variants have not been performed and, therefore, possible genotype-phenotype correlations remain elusive. Here, we present eight male individuals, including a pair of monozygotic twins, harboring pathogenic or likely pathogenic HNRNPH2 variants. Notably, we present the first individuals harboring nonsense or frameshift variants who, similarly to an individual harboring a de novo p.(Arg29Cys) variant within the first quasi-RNA-recognition motif (qRRM), displayed mild developmental delay, and developed mostly autistic features and/or psychiatric co-morbidities. Additionally, we present two individuals harboring a recurrent de novo p.(Arg114Trp), within the second qRRM, who had a severe neurodevelopmental delay with seizures. Functional characterization of the three most common HNRNPH2 missense variants revealed dysfunctional nucleocytoplasmic shuttling of proteins harboring the p.(Arg206Gln) and p.(Pro209Leu) variants, located within the nuclear localization signal, whereas proteins with p.(Arg114Trp) showed reduced interaction with members of the large assembly of splicing regulators (LASR). Moreover, RNA-sequencing of primary fibroblasts of the individual harboring the p.(Arg114Trp) revealed substantial alterations in the regulation of alternative splicing along with global transcriptome changes. Thus, we further expand the clinical and variant spectrum in HNRNPH2-associated disease in males and provide novel molecular insights suggesting the disorder to be a spliceopathy on the molecular level.

The Discovery of a LEMD2-Associated Nuclear Envelopathy with Early Progeroid Appearance Suggests Advanced Applications for AI-Driven Facial Phenotyping.

Over a relatively short period of time, the clinical geneticist's "toolbox" has been expanded by machine-learning algorithms for image analysis, which can be applied to the task of syndrome identification on the basis of facial photographs, but these technologies harbor potential beyond the recognition of established phenotypes. Here, we comprehensively characterized two individuals with a hitherto unknown genetic disorder caused by the same de novo mutation in LEMD2 (c.1436C>T;p.Ser479Phe), the gene which encodes the nuclear envelope protein LEM domain-containing protein 2 (LEMD2). Despite different ages and ethnic backgrounds, both individuals share a progeria-like facial phenotype and a distinct combination of physical and neurologic anomalies, such as growth retardation; hypoplastic jaws crowded with multiple supernumerary, yet unerupted, teeth; and cerebellar intention tremor. Immunofluorescence analyses of patient fibroblasts revealed mutation-induced disturbance of nuclear architecture, recapitulating previously published data in LEMD2-deficient cell lines, and additional experiments suggested mislocalization of mutant LEMD2 protein within the nuclear lamina. Computational analysis of facial features with two different deep neural networks showed phenotypic proximity to other nuclear envelopathies. One of the algorithms, when trained to recognize syndromic similarity (rather than specific syndromes) in an unsupervised approach, clustered both individuals closely together, providing hypothesis-free hints for a common genetic etiology. We show that a recurrent de novo mutation in LEMD2 causes a nuclear envelopathy whose prognosis in adolescence is relatively good in comparison to that of classical Hutchinson-Gilford progeria syndrome, and we suggest that the application of artificial intelligence to the analysis of patient images can facilitate the discovery of new genetic disorders.

Paralog Studies Augment Gene Discovery: DDX and DHX Genes.

Members of a paralogous gene family in which variation in one gene is known to cause disease are eight times more likely to also be associated with human disease. Recent studies have elucidated DHX30 and DDX3X as genes for which pathogenic variant alleles are involved in neurodevelopmental disorders. We hypothesized that variants in paralogous genes encoding members of the DExD/H-box RNA helicase superfamily might also underlie developmental delay and/or intellectual disability (DD and/or ID) disease phenotypes. Here we describe 15 unrelated individuals who have DD and/or ID, central nervous system (CNS) dysfunction, vertebral anomalies, and dysmorphic features and were found to have probably damaging variants in DExD/H-box RNA helicase genes. In addition, these individuals exhibit a variety of other tissue and organ system involvement including ocular, outer ear, hearing, cardiac, and kidney tissues. Five individuals with homozygous (one), compound-heterozygous (two), or de novo (two) missense variants in DHX37 were identified by exome sequencing. We identified ten total individuals with missense variants in three other DDX/DHX paralogs: DHX16 (four individuals), DDX54 (three individuals), and DHX34 (three individuals). Most identified variants are rare, predicted to be damaging, and occur at conserved amino acid residues. Taken together, these 15 individuals implicate the DExD/H-box helicases in both dominantly and recessively inherited neurodevelopmental phenotypes and highlight the potential for more than one disease mechanism underlying these disorders.

Werner syndrome in a Lebanese family.

Werner syndrome (WS) is an extremely rare, autosomal recessive segmental progeroid disorder caused by biallelic pathogenic variants in the WRN, which encodes a multifunctional nuclear protein that belongs to the RecQ family of DNA helicases. Despite extensive research on WS in the last years, the population-specific mutational spectrum still needs to be elucidated. Moreover, there is an evident lack of detailed clinical descriptions accompanied with photographs of affected individuals. Here, we report a consanguineous Lebanese family in whom we identified a pathogenic homozygous nonsense variant c.1111G>T, p.Glu371* in the WRN. The index individual, at the age of 54 years, was suspected to have WS due to a history of early-onset cataracts, premature hair loss and graying, chronic nonhealing leg ulcers, Achilles' tendon calcifications, type 2 diabetes mellitus, dyslipidemia, hypothyroidism, and premature coronary artery disease. His four sisters, three of which deceased in the fifth decade, had clinical signs suggestive of WS. Moreover, his daughter, aged 23 years, had short stature, hair loss and flat feet. Taken together, we report a detailed clinical course of disease in several affected members of a consanguineous family, which is additionally documented by photographs.

A novel homozygous synonymous variant further expands the phenotypic spectrum of POLR3A-related pathologies.

Pathogenic biallelic variants in POL3RA have been associated with different disorders characterized by progressive neurological deterioration. These include the 4H leukodystrophy syndrome (hypomyelination, hypogonadotropic hypogonadism, and hypodontia) and adolescent-onset progressive spastic ataxia, as well as Wiedemann-Rautenstrauch syndrome (WRS), a recognizable neonatal progeroid syndrome. The phenotypic differences between these disorders are thought to occur mainly due to different functional effects of underlying POLR3A variants. Here we present the detailed clinical course of a 37-year-old woman in whom we identified a homozygous synonymous POLR3A variant c.3336G>A resulting in leaky splicing r.[3336ins192, =, 3243_3336del94]. She presented at birth with intrauterine growth retardation, lipodystrophy, muscular hypotonia, and several WRS-like facial features, albeit without sparse hair and prominent scalp veins. She had no signs of developmental delay or intellectual disability. Over the years, above characteristic facial features, she showed severe postnatal growth retardation, global lipodystrophy, joint contractures, thoracic hypoplasia, scoliosis, anodontia, spastic quadriplegia, bilateral hearing loss, aphonia, hypogonadotropic hypogonadism, and cerebellar peduncles hyperintensities in brain imaging. These manifestations partially overlap the clinical features of the previously reported POLR3A-associated disorders, mostly mimicking the WRS. Thus, our study expands the POLR3A-mediated phenotypic spectrum and suggests existence of a phenotypic continuum underlying biallelic POLR3A variants.

ANK3 related neurodevelopmental disorders: expanding the spectrum of heterozygous loss-of-function variants.

ANK3 encodes multiple isoforms of ankyrin-G, resulting in variegated tissue expression and function, especially regarding its role in neuronal development. Based on the zygosity, location, and type, ANK3 variants result in different neurodevelopmental phenotypes. Autism spectrum disorder has been associated with heterozygous missense variants in ANK3, whereas a more severe neurodevelopmental phenotype is caused by isoform-dependent, autosomal-dominant, or autosomal-recessive loss-of-function variants. Here, we present four individuals affected by a variable neurodevelopmental phenotype harboring a heterozygous frameshift or nonsense variant affecting all ANK3 transcripts. Thus, we provide further evidence of an isoform-based phenotypic continuum underlying ANK3-associated pathologies and expand its phenotypic spectrum.

Activating Mutations in PAK1, Encoding p21-Activated Kinase 1, Cause a Neurodevelopmental Disorder.

p21-activated kinases (PAKs) are serine/threonine protein kinases acting as effectors of CDC42 and RAC, which are members of the RHO family of small GTPases. PAK1's kinase activity is autoinhibited by homodimerization, whereas CDC42 or RAC1 binding causes PAK1 activation by dimer dissociation. Major functions of the PAKs include actin cytoskeleton reorganization, for example regulation of the cellular protruding activity during cell spreading. We report the de novo PAK1 mutations c.392A>G (p.Tyr131Cys) and c.1286A>G (p.Tyr429Cys) in two unrelated subjects with developmental delay, secondary macrocephaly, seizures, and ataxic gait. We identified enhanced phosphorylation of the PAK1 targets JNK and AKT in fibroblasts of one subject and of c-JUN in those of both subjects compared with control subjects. In fibroblasts of the two affected individuals, we observed a trend toward enhanced PAK1 kinase activity. By using co-immunoprecipitation and size-exclusion chromatography, we observed a significantly reduced dimerization for both PAK1 mutants compared with wild-type PAK1. These data demonstrate that the two PAK1 variants function as activating alleles. In a cell spreading assay, subject-derived fibroblasts showed significant enrichment in cells occupied by filopodia. Interestingly, application of the PAK1 inhibitor FRAX486 completely reversed this cellular phenotype. Together, our data reveal that dominantly acting, gain-of-function PAK1 mutations cause a neurodevelopmental phenotype with increased head circumference, possibly by a combined effect of defective homodimerization and enhanced kinase activity of PAK1. This condition, along with the developmental disorders associated with RAC1 and CDC42 missense mutations, highlight the importance of RHO GTPase members and effectors in neuronal development.

Prevalence and clinical prediction of mitochondrial disorders in a large neuropediatric cohort.

Neurological symptoms are frequent and often a leading feature of childhood-onset mitochondrial disorders (MD) but the exact incidence of MD in unselected neuropediatric patients is unknown. Their early detection is desirable due to a potentially rapid clinical decline and the availability of management options. In 491 children with neurological symptoms, a comprehensive diagnostic work-up including exome sequencing was performed. The success rate in terms of a molecular genetic diagnosis within our cohort was 51%. Disease-causing variants in a mitochondria-associated gene were detected in 12% of solved cases. In order to facilitate the clinical identification of MDs within neuropediatric cohorts, we have created an easy-to-use bedside-tool, the MDC-NP. In our cohort, the MDC-NP predicted disease conditions related to MDs with a sensitivity of 0.83, and a specificity of 0.96.

MN1 C-terminal truncation syndrome is a novel neurodevelopmental and craniofacial disorder with partial rhombencephalosynapsis.

MN1 encodes a transcriptional co-regulator without homology to other proteins, previously implicated in acute myeloid leukaemia and development of the palate. Large deletions encompassing MN1 have been reported in individuals with variable neurodevelopmental anomalies and non-specific facial features. We identified a cluster of de novo truncating mutations in MN1 in a cohort of 23 individuals with strikingly similar dysmorphic facial features, especially midface hypoplasia, and intellectual disability with severe expressive language delay. Imaging revealed an atypical form of rhombencephalosynapsis, a distinctive brain malformation characterized by partial or complete loss of the cerebellar vermis with fusion of the cerebellar hemispheres, in 8/10 individuals. Rhombencephalosynapsis has no previously known definitive genetic or environmental causes. Other frequent features included perisylvian polymicrogyria, abnormal posterior clinoid processes and persistent trigeminal artery. MN1 is encoded by only two exons. All mutations, including the recurrent variant p.Arg1295* observed in 8/21 probands, fall in the terminal exon or the extreme 3' region of exon 1, and are therefore predicted to result in escape from nonsense-mediated mRNA decay. This was confirmed in fibroblasts from three individuals. We propose that the condition described here, MN1 C-terminal truncation (MCTT) syndrome, is not due to MN1 haploinsufficiency but rather is the result of dominantly acting C-terminally truncated MN1 protein. Our data show that MN1 plays a critical role in human craniofacial and brain development, and opens the door to understanding the biological mechanisms underlying rhombencephalosynapsis.

Two novel cases further expand the phenotype of TOR1AIP1-associated nuclear envelopathies.

Biallelic variants in TOR1AIP1, encoding the integral nuclear membrane protein LAP1 (lamina-associated polypeptide 1) with two functional isoforms LAP1B and LAP1C, have initially been linked to muscular dystrophies with variable cardiac and neurological impairment. Furthermore, a recurrent homozygous nonsense alteration, resulting in loss of both LAP1 isoforms, was identified in seven likely related individuals affected by multisystem anomalies with progeroid-like appearance and lethality within the 1st decade of life. Here, we have identified compound heterozygosity in TOR1AIP1 affecting both LAP1 isoforms in two unrelated individuals affected by congenital bilateral hearing loss, ventricular septal defect, bilateral cataracts, mild to moderate developmental delay, microcephaly, mandibular hypoplasia, short stature, progressive muscular atrophy, joint contractures and severe chronic heart failure, with much longer survival. Cellular characterization of primary fibroblasts of one affected individual revealed absence of both LAP1B and LAP1C, constitutively low lamin A/C levels, aberrant nuclear morphology including nuclear cytoplasmic channels, and premature senescence, comparable to findings in other progeroid forms of nuclear envelopathies. We additionally observed an abnormal activation of the extracellular signal-regulated kinase 1/2 (ERK 1/2). Ectopic expression of wild-type TOR1AIP1 mitigated these cellular phenotypes, providing further evidence for the causal role of identified genetic variants. Altogether, we thus further expand the TOR1AIP1-associated phenotype by identifying individuals with biallelic loss-of-function variants who survived beyond the 1st decade of life and reveal novel molecular consequences underlying the TOR1AIP1-associated disorders.

De Novo Variants in GRIA4 Lead to Intellectual Disability with or without Seizures and Gait Abnormalities.

Using trio whole-exome sequencing, we have identified de novo heterozygous pathogenic variants in GRIA4 in five unrelated individuals with intellectual disability and other symptoms. GRIA4 encodes an AMPA receptor subunit known as GluR4, which is found on excitatory glutamatergic synapses and is important for learning and memory. Four of the variants are located in the highly conserved SYTANLAAF motif in the transmembrane protein M3, and the fifth is in an extra-cellular domain. Molecular modeling of the altered protein showed that three of the variants in the SYTANLAAF motif orient toward the center of the pore region and most likely lead to disturbance of the gating mechanism. The fourth variant in the SYTANLAAF motif most likely results in reduced permeability. The variant in the extracellular domain potentially interferes with the binding between the monomers. On the basis of clinical information and genetic results, and the fact that other subunits of the AMPA receptor have already been associated with neurodevelopmental disorders, we suggest that pathogenic de novo variants in GRIA4 lead to intellectual disability with or without seizures, gait abnormalities, problems of social behavior, and other variable features.

De Novo Missense Mutations in DHX30 Impair Global Translation and Cause a Neurodevelopmental Disorder.

DHX30 is a member of the family of DExH-box helicases, which use ATP hydrolysis to unwind RNA secondary structures. Here we identified six different de novo missense mutations in DHX30 in twelve unrelated individuals affected by global developmental delay (GDD), intellectual disability (ID), severe speech impairment and gait abnormalities. While four mutations are recurrent, two are unique with one affecting the codon of one recurrent mutation. All amino acid changes are located within highly conserved helicase motifs and were found to either impair ATPase activity or RNA recognition in different in vitro assays. Moreover, protein variants exhibit an increased propensity to trigger stress granule (SG) formation resulting in global translation inhibition. Thus, our findings highlight the prominent role of translation control in development and function of the central nervous system and also provide molecular insight into how DHX30 dysfunction might cause a neurodevelopmental disorder.

Mutations in EXTL3 Cause Neuro-immuno-skeletal Dysplasia Syndrome.

EXTL3 regulates the biosynthesis of heparan sulfate (HS), important for both skeletal development and hematopoiesis, through the formation of HS proteoglycans (HSPGs). By whole-exome sequencing, we identified homozygous missense mutations c.1382C>T, c.1537C>T, c.1970A>G, and c.2008T>G in EXTL3 in nine affected individuals from five unrelated families. Notably, we found the identical homozygous missense mutation c.1382C>T (p.Pro461Leu) in four affected individuals from two unrelated families. Affected individuals presented with variable skeletal abnormalities and neurodevelopmental defects. Severe combined immunodeficiency (SCID) with a complete absence of T cells was observed in three families. EXTL3 was most abundant in hematopoietic stem cells and early progenitor T cells, which is in line with a SCID phenotype at the level of early T cell development in the thymus. To provide further support for the hypothesis that mutations in EXTL3 cause a neuro-immuno-skeletal dysplasia syndrome, and to gain insight into the pathogenesis of the disorder, we analyzed the localization of EXTL3 in fibroblasts derived from affected individuals and determined glycosaminoglycan concentrations in these cells as well as in urine and blood. We observed abnormal glycosaminoglycan concentrations and increased concentrations of the non-sulfated chondroitin disaccharide D0a0 and the disaccharide D0a4 in serum and urine of all analyzed affected individuals. In summary, we show that biallelic mutations in EXTL3 disturb glycosaminoglycan synthesis and thus lead to a recognizable syndrome characterized by variable expression of skeletal, neurological, and immunological abnormalities.

Mutations in genes encoding regulators of mRNA decapping and translation initiation: links to intellectual disability.

Intellectual disability (ID) affects at least 1% of the population, and typically presents in the first few years of life. ID is characterized by impairments in cognition and adaptive behavior and is often accompanied by further delays in language and motor skills, as seen in many neurodevelopmental disorders (NDD). Recent widespread high-throughput approaches that utilize whole-exome sequencing or whole-genome sequencing have allowed for a considerable increase in the identification of these pathogenic variants in monogenic forms of ID. Notwithstanding this progress, the molecular and cellular consequences of the identified mutations remain mostly unknown. This is particularly important as the associated protein dysfunctions are the prerequisite to the identification of targets for novel drugs of these rare disorders. Recent Next-Generation sequencing-based studies have further established that mutations in genes encoding proteins involved in RNA metabolism are a major cause of NDD. Here, we review recent studies linking germline mutations in genes encoding factors mediating mRNA decay and regulators of translation, namely DCPS, EDC3, DDX6 helicase and ID. These RNA-binding proteins have well-established roles in mRNA decapping and/or translational repression, and the mutations abrogate their ability to remove 5' caps from mRNA, diminish their interactions with cofactors and stabilize sub-sets of transcripts. Additional genes encoding RNA helicases with roles in translation including DDX3X and DHX30 have also been linked to NDD. Given the speed in the acquisition, analysis and sharing of sequencing data, and the importance of post-transcriptional regulation for brain development, we anticipate mutations in more such factors being identified and functionally characterized.