Pathogenic variants in autism gene KATNAL2 cause hydrocephalus and disrupt neuronal connectivity by impairing ciliary microtubule dynamics.

Enlargement of the cerebrospinal fluid (CSF)-filled brain ventricles (cerebral ventriculomegaly), the cardinal feature of congenital hydrocephalus (CH), is increasingly recognized among patients with autism spectrum disorders (ASD). KATNAL2, a member of Katanin family microtubule-severing ATPases, is a known ASD risk gene, but its roles in human brain development remain unclear. Here, we show that nonsense truncation of Katnal2 (Katnal2Δ17) in mice results in classic ciliopathy phenotypes, including impaired spermatogenesis and cerebral ventriculomegaly. In both humans and mice, KATNAL2 is highly expressed in ciliated radial glia of the fetal ventricular-subventricular zone as well as in their postnatal ependymal and neuronal progeny. The ventriculomegaly observed in Katnal2Δ17 mice is associated with disrupted primary cilia and ependymal planar cell polarity that results in impaired cilia-generated CSF flow. Further, prefrontal pyramidal neurons in ventriculomegalic Katnal2Δ17 mice exhibit decreased excitatory drive and reduced high-frequency firing. Consistent with these findings in mice, we identified rare, damaging heterozygous germline variants in KATNAL2 in five unrelated patients with neurosurgically treated CH and comorbid ASD or other neurodevelopmental disorders. Mice engineered with the orthologous ASD-associated KATNAL2 F244L missense variant recapitulated the ventriculomegaly found in human patients. Together, these data suggest KATNAL2 pathogenic variants alter intraventricular CSF homeostasis and parenchymal neuronal connectivity by disrupting microtubule dynamics in fetal radial glia and their postnatal ependymal and neuronal descendants. The results identify a molecular mechanism underlying the development of ventriculomegaly in a genetic subset of patients with ASD and may explain persistence of neurodevelopmental phenotypes in some patients with CH despite neurosurgical CSF shunting.

De novo variants implicate chromatin modification, transcriptional regulation, and retinoic acid signaling in syndromic craniosynostosis.

Craniosynostosis (CS) is the most common congenital cranial anomaly. Several Mendelian forms of syndromic CS are well described, but a genetic etiology remains elusive in a substantial fraction of probands. Analysis of exome sequence data from 526 proband-parent trios with syndromic CS identified a marked excess (observed 98, expected 33, p = 4.83 × 10-20) of damaging de novo variants (DNVs) in genes highly intolerant to loss-of-function variation (probability of LoF intolerance > 0.9). 30 probands harbored damaging DNVs in 21 genes that were not previously implicated in CS but are involved in chromatin modification and remodeling (4.7-fold enrichment, p = 1.1 × 10-11). 17 genes had multiple damaging DNVs, and 13 genes (CDK13, NFIX, ADNP, KMT5B, SON, ARID1B, CASK, CHD7, MED13L, PSMD12, POLR2A, CHD3, and SETBP1) surpassed thresholds for genome-wide significance. A recurrent gain-of-function DNV in the retinoic acid receptor alpha (RARA; c.865G>A [p.Gly289Arg]) was identified in two probands with similar CS phenotypes. CS risk genes overlap with those identified for autism and other neurodevelopmental disorders, are highly expressed in cranial neural crest cells, and converge in networks that regulate chromatin modification, gene transcription, and osteoblast differentiation. Our results identify several CS loci and have major implications for genetic testing and counseling.

Pleiotropic role of TRAF7 in skull-base meningiomas and congenital heart disease.

While somatic variants of TRAF7 (Tumor necrosis factor receptor-associated factor 7) underlie anterior skull-base meningiomas, here we report the inherited mutations of TRAF7 that cause congenital heart defects. We show that TRAF7 mutants operate in a dominant manner, inhibiting protein function via heterodimerization with wild-type protein. Further, the shared genetics of the two disparate pathologies can be traced to the common origin of forebrain meninges and cardiac outflow tract from the TRAF7-expressing neural crest. Somatic and inherited mutations disrupt TRAF7-IFT57 interactions leading to cilia degradation. TRAF7-mutant meningioma primary cultures lack cilia, and TRAF7 knockdown causes cardiac, craniofacial, and ciliary defects in Xenopus and zebrafish, suggesting a mechanistic convergence for TRAF7-driven meningiomas and developmental heart defects.

Variants in autophagy genes MTMR12 and FAM134A are putative modifiers of the hepatic phenotype in α1-antitrypsin deficiency.

BACKGROUND AND AIMS: In the classical form of α1-antitrypsin deficiency, a misfolded variant α1-antitrypsin Z accumulates in the endoplasmic reticulum of liver cells and causes liver cell injury by gain-of-function proteotoxicity in a sub-group of affected homozygotes but relatively little is known about putative modifiers. Here, we carried out genomic sequencing in a uniquely affected family with an index case of liver failure and 2 homozygous siblings with minimal or no liver disease. Their sequences were compared to sequences in well-characterized cohorts of homozygotes with or without liver disease, and then candidate sequence variants were tested for changes in the kinetics of α1-antitrypsin variant Z degradation in iPS-derived hepatocyte-like cells derived from the affected siblings themselves. APPROACH AND RESULTS: Specific variants in autophagy genes MTMR12 and FAM134A could each accelerate the degradation of α1-antitrypsin variant Z in cells from the index patient, but both MTMR12 and FAM134A variants were needed to slow the degradation of α1-antitrypsin variant Z in cells from a protected sib, indicating that inheritance of both variants is needed to mediate the pathogenic effects of hepatic proteotoxicity at the cellular level. Analysis of homozygote cohorts showed that multiple patient-specific variants in proteostasis genes are likely to explain liver disease susceptibility at the population level. CONCLUSIONS: These results validate the concept that genetic variation in autophagy function can determine susceptibility to liver disease in α1-antitrypsin deficiency and provide evidence that polygenic mechanisms and multiple patient-specific variants are likely needed for proteotoxic pathology.

A novel SMARCC1 BAFopathy implicates neural progenitor epigenetic dysregulation in human hydrocephalus.

Hydrocephalus, characterized by cerebral ventriculomegaly, is the most common disorder requiring brain surgery in children. Recent studies have implicated SMARCC1, a component of the BRG1-associated factor (BAF) chromatin remodelling complex, as a candidate congenital hydrocephalus gene. However, SMARCC1 variants have not been systematically examined in a large patient cohort or conclusively linked with a human syndrome. Moreover, congenital hydrocephalus-associated SMARCC1 variants have not been functionally validated or mechanistically studied in vivo. Here, we aimed to assess the prevalence of SMARCC1 variants in an expanded patient cohort, describe associated clinical and radiographic phenotypes, and assess the impact of Smarcc1 depletion in a novel Xenopus tropicalis model of congenital hydrocephalus. To do this, we performed a genetic association study using whole-exome sequencing from a cohort consisting of 2697 total ventriculomegalic trios, including patients with neurosurgically-treated congenital hydrocephalus, that total 8091 exomes collected over 7 years (2016-23). A comparison control cohort consisted of 1798 exomes from unaffected siblings of patients with autism spectrum disorder and their unaffected parents were sourced from the Simons Simplex Collection. Enrichment and impact on protein structure were assessed in identified variants. Effects on the human fetal brain transcriptome were examined with RNA-sequencing and Smarcc1 knockdowns were generated in Xenopus and studied using optical coherence tomography imaging, in situ hybridization and immunofluorescence. SMARCC1 surpassed genome-wide significance thresholds, yielding six rare, protein-altering de novo variants localized to highly conserved residues in key functional domains. Patients exhibited hydrocephalus with aqueductal stenosis; corpus callosum abnormalities, developmental delay, and cardiac defects were also common. Xenopus knockdowns recapitulated both aqueductal stenosis and cardiac defects and were rescued by wild-type but not patient-specific variant SMARCC1. Hydrocephalic SMARCC1-variant human fetal brain and Smarcc1-variant Xenopus brain exhibited a similarly altered expression of key genes linked to midgestational neurogenesis, including the transcription factors NEUROD2 and MAB21L2. These results suggest de novo variants in SMARCC1 cause a novel human BAFopathy we term 'SMARCC1-associated developmental dysgenesis syndrome', characterized by variable presence of cerebral ventriculomegaly, aqueductal stenosis, developmental delay and a variety of structural brain or cardiac defects. These data underscore the importance of SMARCC1 and the BAF chromatin remodelling complex for human brain morphogenesis and provide evidence for a 'neural stem cell' paradigm of congenital hydrocephalus pathogenesis. These results highlight utility of trio-based whole-exome sequencing for identifying pathogenic variants in sporadic congenital structural brain disorders and suggest whole-exome sequencing may be a valuable adjunct in clinical management of congenital hydrocephalus patients.

TRIM71 mutations cause a neurodevelopmental syndrome featuring ventriculomegaly and hydrocephalus.

Congenital hydrocephalus (CH), characterized by cerebral ventriculomegaly, is one of the most common reasons for pediatric brain surgery. Recent studies have implicated lin-41 (lineage variant 41)/TRIM71 (tripartite motif 71) as a candidate CH risk gene, however, TRIM71 variants have not been systematically examined in a large patient cohort or conclusively linked with an OMIM syndrome. Through cross-sectional analysis of the largest assembled cohort of patients with cerebral ventriculomegaly, including neurosurgically-treated CH (totaling 2,697 parent-proband trios and 8,091 total exomes), we identified 13 protein-altering de novo variants (DNVs) in TRIM71 in unrelated children exhibiting variable ventriculomegaly, CH, developmental delay, dysmorphic features, and other structural brain defects including corpus callosum dysgenesis and white matter hypoplasia. Eight unrelated patients were found to harbor arginine variants, including two recurrent missense DNVs, at homologous positions in RPXGV motifs of different NHL domains. Seven additional patients with rare, damaging, unphased or transmitted variants of uncertain significance were also identified. NHL-domain variants of TRIM71 exhibited impaired binding to the canonical TRIM71 target CDKN1A; other variants failed to direct the subcellular localization of TRIM71 to processing bodies. Single-cell transcriptomic analysis of human embryos revealed expression of TRIM71 in early first-trimester neural stem cells of the brain. These data show TRIM71 is essential for human brain morphogenesis and that TRIM71 mutations cause a novel neurodevelopmental syndrome featuring ventriculomegaly and CH.

TRAPPC6B biallelic variants cause a neurodevelopmental disorder with TRAPP II and trafficking disruptions.

Highly conserved transport protein particle (TRAPP) complexes regulate subcellular trafficking pathways. Accurate protein trafficking has been increasingly recognized to be critically important for normal development, particularly in the nervous system. Variants in most TRAPP complex subunits have been found to lead to neurodevelopmental disorders with diverse but overlapping phenotypes. We expand on limited prior reports on TRAPPC6B with detailed clinical and neuroradiologic assessments, and studies on mechanisms of disease, and new types of variants. We describe 29 additional patients from 18 independent families with biallelic variants in TRAPPC6B. We identified seven homozygous nonsense (n = 12 patients) and eight canonical splice-site variants (n = 17 patients). In addition, we identified one patient with compound heterozygous splice-site/missense variants with a milder phenotype and one patient with homozygous missense variants. Patients displayed non-progressive microcephaly, global developmental delay/intellectual disability, epilepsy and absent expressive language. Movement disorders including stereotypies, spasticity and dystonia were also observed. Brain imaging revealed reductions in cortex, cerebellum and corpus callosum size with frequent white matter hyperintensity. Volumetric measurements indicated globally diminished volume rather than specific regional losses. We identified a reduced rate of trafficking into the Golgi apparatus and Golgi fragmentation in patient-derived fibroblasts that was rescued by wild-type TRAPPC6B. Molecular studies revealed a weakened interaction between mutant TRAPPC6B (c.454C>T, p.Q152*) and its TRAPP binding partner TRAPPC3. Patient-derived fibroblasts from the TRAPPC6B (c.454C>T, p.Q152*) variant displayed reduced levels of TRAPPC6B as well as other TRAPP II complex-specific members (TRAPPC9 and TRAPPC10). Interestingly, the levels of the TRAPPC6B homologue TRAPPC6A were found to be elevated. Moreover, co-immunoprecipitation experiments showed that TRAPPC6A co-precipitates equally with TRAPP II and TRAPP III, while TRAPPC6B co-precipitates significantly more with TRAPP II, suggesting enrichment of the protein in the TRAPP II complex. This implies that variants in TRAPPC6B may preferentially affect TRAPP II functions compared to TRAPP III functions. Finally, we assessed phenotypes in a Drosophila TRAPPC6B-deficiency model. Neuronal TRAPPC6B knockdown impaired locomotion and led to wing posture defects, supporting a role for TRAPPC6B in neuromotor function. Our findings confirm the association of damaging biallelic TRAPPC6B variants with microcephaly, intellectual disability, language impairments, and epilepsy. A subset of patients also exhibited dystonia and/or spasticity with impaired ambulation. These features overlap with disorders arising from pathogenic variants in other TRAPP subunits, particularly components of the TRAPP II complex. These findings suggest that TRAPPC6B is essential for brain development and function, and TRAPP II complex activity may be particularly relevant for mediating this function.

Network assisted analysis of de novo variants using protein-protein interaction information identified 46 candidate genes for congenital heart disease.

De novo variants (DNVs) with deleterious effects have proved informative in identifying risk genes for early-onset diseases such as congenital heart disease (CHD). A number of statistical methods have been proposed for family-based studies or case/control studies to identify risk genes by screening genes with more DNVs than expected by chance in Whole Exome Sequencing (WES) studies. However, the statistical power is still limited for cohorts with thousands of subjects. Under the hypothesis that connected genes in protein-protein interaction (PPI) networks are more likely to share similar disease association status, we developed a Markov Random Field model that can leverage information from publicly available PPI databases to increase power in identifying risk genes. We identified 46 candidate genes with at least 1 DNV in the CHD study cohort, including 18 known human CHD genes and 35 highly expressed genes in mouse developing heart. Our results may shed new insight on the shared protein functionality among risk genes for CHD.

Rare variants in ANO1, encoding a calcium-activated chloride channel, predispose to moyamoya disease.

Moyamoya disease, a cerebrovascular disease leading to strokes in children and young adults, is characterized by progressive occlusion of the distal internal carotid arteries and the formation of collateral vessels. Altered genes play a prominent role in the aetiology of moyamoya disease, but a causative gene is not identified in the majority of cases. Exome sequencing data from 151 individuals from 84 unsolved families were analysed to identify further genes for moyamoya disease, then candidate genes assessed in additional cases (150 probands). Two families had the same rare variant in ANO1, which encodes a calcium-activated chloride channel, anoctamin-1. Haplotype analyses found the families were related, and ANO1 p.Met658Val segregated with moyamoya disease in the family with an LOD score of 3.3. Six additional ANO1 rare variants were identified in moyamoya disease families. The ANO1 rare variants were assessed using patch-clamp recordings, and the majority of variants, including ANO1 p.Met658Val, displayed increased sensitivity to intracellular Ca2+. Patients harbouring these gain-of-function ANO1 variants had classic features of moyamoya disease, but also had aneurysm, stenosis and/or occlusion in the posterior circulation. Our studies support that ANO1 gain-of-function pathogenic variants predispose to moyamoya disease and are associated with unique involvement of the posterior circulation.

Familial and syndromic forms of arachnoid cyst implicate genetic factors in disease pathogenesis.

Arachnoid cysts (ACs) are the most common space-occupying lesions in the human brain and present significant challenges for clinical management. While most cases of ACs are sporadic, nearly 40 familial forms have been reported. Moreover, ACs are seen with increased frequency in multiple Mendelian syndromes, including Chudley-McCullough syndrome, acrocallosal syndrome, and autosomal recessive primary ciliary dyskinesia. These findings suggest that genetic factors contribute to AC pathogenesis. However, traditional linkage and segregation approaches have been limited in their ability to identify causative genes for ACs because the disease is genetically heterogeneous and often presents asymptomatically and sporadically. Here, we comprehensively review theories of AC pathogenesis, the genetic evidence for AC formation, and discuss a different approach to AC genomics that could help elucidate this perplexing lesion and shed light on the associated neurodevelopmental phenotypes seen in a significant subset of these patients.

A neural stem cell paradigm of pediatric hydrocephalus.

Pediatric hydrocephalus, the leading reason for brain surgery in children, is characterized by enlargement of the cerebral ventricles classically attributed to cerebrospinal fluid (CSF) overaccumulation. Neurosurgical shunting to reduce CSF volume is the default treatment that intends to reinstate normal CSF homeostasis, yet neurodevelopmental disability often persists in hydrocephalic children despite optimal surgical management. Here, we discuss recent human genetic and animal model studies that are shifting the view of pediatric hydrocephalus from an impaired fluid plumbing model to a new paradigm of dysregulated neural stem cell (NSC) fate. NSCs are neuroprogenitor cells that comprise the germinal neuroepithelium lining the prenatal brain ventricles. We propose that heterogenous defects in the development of these cells converge to disrupt cerebrocortical morphogenesis, leading to abnormal brain-CSF biomechanical interactions that facilitate passive pooling of CSF and secondary ventricular distention. A significant subset of pediatric hydrocephalus may thus in fact be due to a developmental brain malformation leading to secondary enlargement of the ventricles rather than a primary defect of CSF circulation. If hydrocephalus is indeed a neuroradiographic presentation of an inborn brain defect, it suggests the need to focus on optimizing neurodevelopment, rather than CSF diversion, as the primary treatment strategy for these children.

De novo mutations in the BMP signaling pathway in lambdoid craniosynostosis.

Lambdoid craniosynostosis (CS) is a congenital anomaly resulting from premature fusion of the cranial suture between the parietal and occipital bones. Predominantly sporadic, it is the rarest form of CS and its genetic etiology is largely unexplored. Exome sequencing of 25 kindreds, including 18 parent-offspring trios with sporadic lambdoid CS, revealed a marked excess of damaging (predominantly missense) de novo mutations that account for ~ 40% of sporadic cases. These mutations clustered in the BMP signaling cascade (P = 1.6 × 10-7), including mutations in genes encoding BMP receptors (ACVRL1 and ACVR2A), transcription factors (SOX11, FOXO1) and a transcriptional co-repressor (IFRD1), none of which have been implicated in other forms of CS. These missense mutations are at residues critical for substrate or target sequence recognition and many are inferred to cause genetic gain-of-function. Additionally, mutations in transcription factor NFIX were implicated in syndromic craniosynostosis affecting diverse sutures. Single cell RNA sequencing analysis of the mouse lambdoid suture identified enrichment of mutations in osteoblast precursors (P = 1.6 × 10-6), implicating perturbations in the balance between proliferation and differentiation of osteoprogenitor cells in lambdoid CS. The results contribute to the growing knowledge of the genetics of CS, have implications for genetic counseling, and further elucidate the molecular etiology of premature suture fusion.

Variants in Mitochondrial ATP Synthase Cause Variable Neurologic Phenotypes.

OBJECTIVE: ATP synthase (ATPase) is responsible for the majority of ATP production. Nevertheless, disease phenotypes associated with mutations in ATPase subunits are extremely rare. We aimed at expanding the spectrum of ATPase-related diseases. METHODS: Whole-exome sequencing in cohorts with 2,962 patients diagnosed with mitochondrial disease and/or dystonia and international collaboration were used to identify deleterious variants in ATPase-encoding genes. Findings were complemented by transcriptional and proteomic profiling of patient fibroblasts. ATPase integrity and activity were assayed using cells and tissues from 5 patients. RESULTS: We present 10 total individuals with biallelic or de novo monoallelic variants in nuclear ATPase subunit genes. Three unrelated patients showed the same homozygous missense ATP5F1E mutation (including one published case). An intronic splice-disrupting alteration in compound heterozygosity with a nonsense variant in ATP5PO was found in one patient. Three patients had de novo heterozygous missense variants in ATP5F1A, whereas another 3 were heterozygous for ATP5MC3 de novo missense changes. Bioinformatics methods and populational data supported the variants' pathogenicity. Immunohistochemistry, proteomics, and/or immunoblotting revealed significantly reduced ATPase amounts in association to ATP5F1E and ATP5PO mutations. Diminished activity and/or defective assembly of ATPase was demonstrated by enzymatic assays and/or immunoblotting in patient samples bearing ATP5F1A-p.Arg207His, ATP5MC3-p.Gly79Val, and ATP5MC3-p.Asn106Lys. The associated clinical profiles were heterogeneous, ranging from hypotonia with spontaneous resolution (1/10) to epilepsy with early death (1/10) or variable persistent abnormalities, including movement disorders, developmental delay, intellectual disability, hyperlactatemia, and other neurologic and systemic features. Although potentially reflecting an ascertainment bias, dystonia was common (7/10). INTERPRETATION: Our results establish evidence for a previously unrecognized role of ATPase nuclear-gene defects in phenotypes characterized by neurodevelopmental and neurodegenerative features. ANN NEUROL 2022;91:225-237.

Biallelic Variants in the Ectonucleotidase ENTPD1 Cause a Complex Neurodevelopmental Disorder with Intellectual Disability, Distinct White Matter Abnormalities, and Spastic Paraplegia.

OBJECTIVE: Human genomics established that pathogenic variation in diverse genes can underlie a single disorder. For example, hereditary spastic paraplegia is associated with >80 genes, with frequently only few affected individuals described for each gene. Herein, we characterize a large cohort of individuals with biallelic variation in ENTPD1, a gene previously linked to spastic paraplegia 64 (Mendelian Inheritance in Man # 615683). METHODS: Individuals with biallelic ENTPD1 variants were recruited worldwide. Deep phenotyping and molecular characterization were performed. RESULTS: A total of 27 individuals from 17 unrelated families were studied; additional phenotypic information was collected from published cases. Twelve novel pathogenic ENTPD1 variants are described (NM 001776.6): c.398_399delinsAA; p.(Gly133Glu), c.540del; p.(Thr181Leufs*18), c.640del; p.(Gly216Glufs*75), c.185 T > G; p.(Leu62*), c.1531 T > C; p.(*511Glnext*100), c.967C > T; p.(Gln323*), c.414-2_414-1del, and c.146 A > G; p.(Tyr49Cys) including 4 recurrent variants c.1109 T > A; p.(Leu370*), c.574-6_574-3del, c.770_771del; p.(Gly257Glufs*18), and c.1041del; p.(Ile348Phefs*19). Shared disease traits include childhood onset, progressive spastic paraplegia, intellectual disability (ID), dysarthria, and white matter abnormalities. In vitro assays demonstrate that ENTPD1 expression and function are impaired and that c.574-6_574-3del causes exon skipping. Global metabolomics demonstrate ENTPD1 deficiency leads to impaired nucleotide, lipid, and energy metabolism. INTERPRETATION: The ENTPD1 locus trait consists of childhood disease onset, ID, progressive spastic paraparesis, dysarthria, dysmorphisms, and white matter abnormalities, with some individuals showing neurocognitive regression. Investigation of an allelic series of ENTPD1 (1) expands previously described features of ENTPD1-related neurological disease, (2) highlights the importance of genotype-driven deep phenotyping, (3) documents the need for global collaborative efforts to characterize rare autosomal recessive disease traits, and (4) provides insights into disease trait neurobiology. ANN NEUROL 2022;92:304-321.

Whole-exome sequencing reveals damaging gene variants associated with hypoalphalipoproteinemia.

Low levels of high density lipoprotein-cholesterol (HDL-C) are associated with an elevated risk of arteriosclerotic coronary heart disease. Heritability of HDL-C levels is high. In this research discovery study, we used whole-exome sequencing to identify damaging gene variants that may play significant roles in determining HDL-C levels. We studied 204 individuals with a mean HDL-C level of 27.8 ± 6.4 mg/dl (range: 4-36 mg/dl). Data were analyzed by statistical gene burden testing and by filtering against candidate gene lists. We found 120 occurrences of probably damaging variants (116 heterozygous; four homozygous) among 45 of 104 recognized HDL candidate genes. Those with the highest prevalence of damaging variants were ABCA1 (n = 20), STAB1 (n = 9), OSBPL1A (n = 8), CPS1 (n = 8), CD36 (n = 7), LRP1 (n = 6), ABCA8 (n = 6), GOT2 (n = 5), AMPD3 (n = 5), WWOX (n = 4), and IRS1 (n = 4). Binomial analysis for damaging missense or loss-of-function variants identified the ABCA1 and LDLR genes at genome-wide significance. In conclusion, whole-exome sequencing of individuals with low HDL-C showed the burden of damaging rare variants in the ABCA1 and LDLR genes is particularly high and revealed numerous occurrences in HDL candidate genes, including many genes identified in genome-wide association study reports. Many of these genes are involved in cancer biology, which accords with epidemiologic findings of the association of HDL deficiency with increased risk of cancer, thus presenting a new area of interest in HDL genomics.

Biallelic AOPEP Loss-of-Function Variants Cause Progressive Dystonia with Prominent Limb Involvement.

BACKGROUND: Monogenic causes of isolated dystonia are heterogeneous. Assembling cohorts of affected individuals sufficiently large to establish new gene-disease relationships can be challenging. OBJECTIVE: We sought to expand the catalogue of monogenic etiologies for isolated dystonia. METHODS: After the discovery of a candidate variant in a multicenter exome-sequenced cohort of affected individuals with dystonia, we queried online platforms and genomic data repositories worldwide to identify subjects with matching genotypic profiles. RESULTS: Seven different biallelic loss-of-function variants in AOPEP were detected in five probands from four unrelated families with strongly overlapping phenotypes. In one proband, we observed a homozygous nonsense variant (c.1477C>T [p.Arg493*]). A second proband harbored compound heterozygous nonsense variants (c.763C>T [p.Arg255*]; c.777G>A [p.Trp259*]), whereas a third proband possessed a frameshift variant (c.696_697delAG [p.Ala234Serfs*5]) in trans with a splice-disrupting alteration (c.2041-1G>A). Two probands (siblings) from a fourth family shared compound heterozygous frameshift alleles (c.1215delT [p.Val406Cysfs*14]; c.1744delA [p.Met582Cysfs*6]). All variants were rare and expected to result in truncated proteins devoid of functionally important amino acid sequence. AOPEP, widely expressed in developing and adult human brain, encodes a zinc-dependent aminopeptidase, a member of a class of proteolytic enzymes implicated in synaptogenesis and neural maintenance. The probands presented with disabling progressive dystonia predominantly affecting upper and lower extremities, with variable involvement of craniocervical muscles. Dystonia was unaccompanied by any additional symptoms in three families, whereas the fourth family presented co-occurring late-onset parkinsonism. CONCLUSIONS: Our findings suggest a likely causative role of predicted inactivating biallelic AOPEP variants in cases of autosomal recessive dystonia. Additional studies are warranted to understand the pathophysiology associated with loss-of-function variation in AOPEP. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Biallelic variants in HPDL cause pure and complicated hereditary spastic paraplegia.

Human 4-hydroxyphenylpyruvate dioxygenase-like (HPDL) is a putative iron-containing non-heme oxygenase of unknown specificity and biological significance. We report 25 families containing 34 individuals with neurological disease associated with biallelic HPDL variants. Phenotypes ranged from juvenile-onset pure hereditary spastic paraplegia to infantile-onset spasticity and global developmental delays, sometimes complicated by episodes of neurological and respiratory decompensation. Variants included bona fide pathogenic truncating changes, although most were missense substitutions. Functionality of variants could not be determined directly as the enzymatic specificity of HPDL is unknown; however, when HPDL missense substitutions were introduced into 4-hydroxyphenylpyruvate dioxygenase (HPPD, an HPDL orthologue), they impaired the ability of HPPD to convert 4-hydroxyphenylpyruvate into homogentisate. Moreover, three additional sets of experiments provided evidence for a role of HPDL in the nervous system and further supported its link to neurological disease: (i) HPDL was expressed in the nervous system and expression increased during neural differentiation; (ii) knockdown of zebrafish hpdl led to abnormal motor behaviour, replicating aspects of the human disease; and (iii) HPDL localized to mitochondria, consistent with mitochondrial disease that is often associated with neurological manifestations. Our findings suggest that biallelic HPDL variants cause a syndrome varying from juvenile-onset pure hereditary spastic paraplegia to infantile-onset spastic tetraplegia associated with global developmental delays.

Histone H2B monoubiquitination regulates heart development via epigenetic control of cilia motility.

Genomic analyses of patients with congenital heart disease (CHD) have identified significant contribution from mutations affecting cilia genes and chromatin remodeling genes; however, the mechanism(s) connecting chromatin remodeling to CHD is unknown. Histone H2B monoubiquitination (H2Bub1) is catalyzed by the RNF20 complex consisting of RNF20, RNF40, and UBE2B. Here, we show significant enrichment of loss-of-function mutations affecting H2Bub1 in CHD patients (enrichment 6.01, P = 1.67 × 10-03), some of whom had abnormal laterality associated with ciliary dysfunction. In Xenopus, knockdown of rnf20 and rnf40 results in abnormal heart looping, defective development of left-right (LR) asymmetry, and impaired cilia motility. Rnf20, Rnf40, and Ube2b affect LR patterning and cilia synergistically. Examination of global H2Bub1 level in Xenopus embryos shows that H2Bub1 is developmentally regulated and requires Rnf20. To examine gene-specific H2Bub1, we performed ChIP-seq of mouse ciliated and nonciliated tissues and showed tissue-specific H2Bub1 marks significantly enriched at cilia genes including the transcription factor Rfx3 Rnf20 knockdown results in decreased levels of rfx3 mRNA in Xenopus, and exogenous rfx3 can rescue the Rnf20 depletion phenotype. These data suggest that Rnf20 functions at the Rfx3 locus regulating cilia motility and cardiac situs and identify H2Bub1 as an upstream transcriptional regulator controlling tissue-specific expression of cilia genes. Our findings mechanistically link the two functional gene ontologies that have been implicated in human CHD: chromatin remodeling and cilia function.

Mutations in TFAP2B and previously unimplicated genes of the BMP, Wnt, and Hedgehog pathways in syndromic craniosynostosis.

Craniosynostosis (CS) is a frequent congenital anomaly featuring the premature fusion of 1 or more sutures of the cranial vault. Syndromic cases, featuring additional congenital anomalies, make up 15% of CS. While many genes underlying syndromic CS have been identified, the cause of many syndromic cases remains unknown. We performed exome sequencing of 12 syndromic CS cases and their parents, in whom previous genetic evaluations were unrevealing. Damaging de novo or transmitted loss of function (LOF) mutations were found in 8 genes that are highly intolerant to LOF mutation (P = 4.0 × 10-8); additionally, a rare damaging mutation in SOX11, which has a lower level of intolerance, was identified. Four probands had rare damaging mutations (2 de novo) in TFAP2B, a transcription factor that orchestrates neural crest cell migration and differentiation; this mutation burden is highly significant (P = 8.2 × 10-12). Three probands had rare damaging mutations in GLI2, SOX11, or GPC4, which function in the Hedgehog, BMP, and Wnt signaling pathways; other genes in these pathways have previously been implicated in syndromic CS. Similarly, damaging de novo mutations were identified in genes encoding the chromatin modifier KAT6A, and CTNNA1, encoding catenin α-1. These findings establish TFAP2B as a CS gene, have implications for assessing risk to subsequent children in these families, and provide evidence implicating other genes in syndromic CS. This high yield indicates the value of performing exome sequencing of syndromic CS patients when sequencing of known disease loci is unrevealing.