A de novo missense mutation in synaptotagmin-1 associated with neurodevelopmental disorder desynchronizes neurotransmitter release.

Synaptotagmin-1 (Syt1) is a presynaptic calcium sensor with two calcium binding domains, C2A and C2B, that triggers action potential-induced synchronous neurotransmitter release, while suppressing asynchronous and spontaneous release. We identified a de novo missense mutation (P401L) in the C2B domain in a patient with developmental delay and autistic symptoms. Expressing the orthologous mouse mutant (P400L) in cultured Syt1 null mutant neurons revealed a reduction in dendrite outgrowth with a proportional reduction in synapses. This was not observed in single Syt1PL-rescued neurons that received normal synaptic input when cultured in a control network. Patch-clamp recordings showed that spontaneous miniature release events per synapse were increased more than 500% in Syt1PL-rescued neurons, even beyond the increased rates in Syt1 KO neurons. Furthermore, action potential-induced asynchronous release was increased more than 100%, while synchronous release was unaffected. A similar shift to more asynchronous release was observed during train stimulations. These cellular phenotypes were also observed when Syt1PL was overexpressed in wild type neurons. Our findings show that Syt1PL desynchronizes neurotransmission by increasing the readily releasable pool for asynchronous release and reducing the suppression of spontaneous and asynchronous release. Neurons respond to this by shortening their dendrites, possibly to counteract the increased synaptic input. Syt1PL acts in a dominant-negative manner supporting a causative role for the mutation in the heterozygous patient. We propose that the substitution of a rigid proline to a more flexible leucine at the bottom of the C2B domain impairs clamping of release by interfering with Syt1's primary interface with the SNARE complex. This is a novel cellular phenotype, distinct from what was previously found for other SYT1 disease variants, and points to a role for spontaneous and asynchronous release in SYT1-associated neurodevelopmental disorder.

DNA methylation episignature, extension of the clinical features, and comparative epigenomic profiling of Hao-Fountain syndrome caused by variants in USP7.

PURPOSE: Hao-Fountain syndrome (HAFOUS) is a neurodevelopmental disorder caused by pathogenic variants in USP7. HAFOUS is characterized by developmental delay, intellectual disability, speech delay, behavioral abnormalities, autism spectrum disorder, seizures, hypogonadism, and mild dysmorphic features. We investigated the phenotype of 18 participants with HAFOUS and performed DNA methylation (DNAm) analysis, aiming to generate a diagnostic biomarker. Furthermore, we performed comparative analysis with known episignatures to gain more insight into the molecular pathophysiology of HAFOUS. METHODS: We assessed genomic DNAm profiles of 18 individuals with pathogenic variants and variants of uncertain significance (VUS) in USP7 to map and validate a specific episignature. The comparison between the USP7 cohort and 56 rare genetic disorders with earlier reported DNAm episignatures was performed with statistical and functional correlation. RESULTS: We mapped a sensitive and specific DNAm episignature for pathogenic variants in USP7 and utilized this to reclassify the VUS. Comparative epigenomic analysis showed evidence of HAFOUS similarity to a number of other rare genetic episignature disorders. CONCLUSION: We discovered a sensitive and specific DNAm episignature as a robust diagnostic biomarker for HAFOUS that enables VUS reclassification in USP7. We also expand the phenotypic spectrum of 9 new and 5 previously reported individuals with HAFOUS.

The Importance of Screening for Additional Anomalies in Patients with Anorectal Malformations: A Retrospective Cohort Study.

BACKGROUND: In children with anorectal malformations (ARM), additional anomalies can occur within the VACTERL-association. Routine screening is of great importance for early identification and potential treatment. However, uniformity in screening protocols is lacking and only small cohorts have been described in literature. The aim of this study was to assess and describe a unique large cohort of ARM patients who underwent VACTERL screening in the neonatal period. METHODS: A retrospective mono-center cohort study was performed. Included were all neonates born between January 2000 and December 2020 who were diagnosed with ARM and screened for additional anomalies. Full screening consisted of x-ray and ultrasound of the spine, cardiac and renal ultrasound, and physical examination for limb deformities, esophageal atresia, and ARM. Criteria for VACTERL-classification were predefined according to the EUROCAT-definitions. RESULTS: In total, 216 patients were included, of whom 167 (77.3%) underwent full VACTERL-screening (66% in 2000-2006 vs. 82% in 2007-2013 vs. 86% in 2014-2020). Median age at follow-up was 7.0 years (IQR 3.0-12.8). In 103/167 patients (61.7%), additional anomalies were identified. Some 35/216 patients (16.2%) fulfilled the criteria of a form of VACTERL-association. In 37/216 patients (17.1%), a genetic cause or syndrome was found. CONCLUSIONS: The majority of ARM patients underwent full screening to detect additional anomalies (77%), which improved over time to 86%. Yet, approximately a quarter of patients was not screened, with the potential of missing important additional anomalies that might have severe consequences in the future. Forms of VACTERL-association or genetic causes were found in 16% and 17% respectively. This study emphasizes the importance of routine screening. LEVEL OF EVIDENCE: III.

The Incidence of Associated Anomalies in Children with Congenital Duodenal Obstruction-A Retrospective Cohort Study of 112 Patients.

BACKGROUND: Duodenal obstruction (DO) is a congenital anomaly that is highly associated with other anomalies, such as cardiac anomalies and trisomy 21. However, an overview of additional anomalies and patient-specific risk factors for cardiac anomalies is lacking. Potential association with the vertebral, anorectal, cardiac, trachea-esophageal, renal and limb anomalies (VACTERL) spectrum remains unknown. Therefore, we aim to examine the incidence of associated anomalies, a VACTERL-spectrum association and patient-specific risk factors for cardiac anomalies in patients with DO. METHODS: A retrospective cohort study was performed between 1996 and 2021. Outcomes were the presence of any additional anomalies. Risk factors for cardiac anomalies were analyzed using multivariate logistic regression. RESULTS: Of 112 neonates with DO, 47% (N = 53/112) had one associated anomaly and 38% (N = 20/53) had multiple anomalies. Cardiac anomalies (N = 35/112) and trisomy 21 (N = 35/112) were present in 31%. In four patients, VACTERL-spectrum was discovered, all with cardiac anomalies. Trisomy 21 was found to be a risk factor for cardiac anomalies (OR:6.5; CI-95%2.6-16.1). CONCLUSION: Associated anomalies were present in half of patients with DO, of which cardiac anomalies and trisomy 21 occurred most often, and the VACTERL-spectrum was present in four patients. Trisomy 21 was a significant risk factor for cardiac anomalies. Therefore, we recommend a preoperative echocardiogram in patients with DO. In case a cardiac anomaly is found without trisomy 21, VACTERL-screening should be performed.

5q11.2 deletion syndrome revisited-Further narrowing of the smallest region of overlap for the main clinical characteristics of the syndrome.

Microdeletions at 5q11.2 are rare. Subjects show a phenotypic spectrum that overlaps CHARGE syndrome and 22q11.2 deletion syndrome. A growing number of subjects present with learning difficulty and/or intellectual disability, immune deficiency, congenital heart malformation, and dysmorphism. DHX29 and IL6ST have been proposed as candidate genes for the development of the major clinical manifestations. We present a new case and narrow down the shortest region of overlap to evaluate possible candidate genes. Our case does not present developmental delay or immune deficiency indicating a reduced penetrance for some of the main clinical manifestations. The shortest region of overlap between subjects with deletions at 5q11.2 is approximately 450 kb (position 54.3-54.7 Mb). The narrowed region comprises 10 protein coding genes, including DHX29. DHX29 is a strong candidate gene for the main features of 5q11.2-microdeletion syndrome; however, our findings suggest a joined impact of several genes as the cause of the syndrome.

An autosomal dominant neurological disorder caused by de novo variants in FAR1 resulting in uncontrolled synthesis of ether lipids.

PURPOSE: In this study we investigate the disease etiology in 12 patients with de novo variants in FAR1 all resulting in an amino acid change at position 480 (p.Arg480Cys/His/Leu). METHODS: Following next-generation sequencing and clinical phenotyping, functional characterization was performed in patients' fibroblasts using FAR1 enzyme analysis, FAR1 immunoblotting/immunofluorescence, and lipidomics. RESULTS: All patients had spastic paraparesis and bilateral congenital/juvenile cataracts, in most combined with speech and gross motor developmental delay and truncal hypotonia. FAR1 deficiency caused by biallelic variants results in defective ether lipid synthesis and plasmalogen deficiency. In contrast, patients' fibroblasts with the de novo FAR1 variants showed elevated plasmalogen levels. Further functional studies in fibroblasts showed that these variants cause a disruption of the plasmalogen-dependent feedback regulation of FAR1 protein levels leading to uncontrolled ether lipid production. CONCLUSION: Heterozygous de novo variants affecting the Arg480 residue of FAR1 lead to an autosomal dominant disorder with a different disease mechanism than that of recessive FAR1 deficiency and a diametrically opposed biochemical phenotype. Our findings show that for patients with spastic paraparesis and bilateral cataracts, FAR1 should be considered as a candidate gene and added to gene panels for hereditary spastic paraplegia, cerebral palsy, and juvenile cataracts.

Bi-allelic Pathogenic Variants in HS2ST1 Cause a Syndrome Characterized by Developmental Delay and Corpus Callosum, Skeletal, and Renal Abnormalities.

Heparan sulfate belongs to the group of glycosaminoglycans (GAGs), highly sulfated linear polysaccharides. Heparan sulfate 2-O-sulfotransferase 1 (HS2ST1) is one of several specialized enzymes required for heparan sulfate synthesis and catalyzes the transfer of the sulfate groups to the sugar moiety of heparan sulfate. We report bi-allelic pathogenic variants in HS2ST1 in four individuals from three unrelated families. Affected individuals showed facial dysmorphism with coarse face, upslanted palpebral fissures, broad nasal tip, and wide mouth, developmental delay and/or intellectual disability, corpus callosum agenesis or hypoplasia, flexion contractures, brachydactyly of hands and feet with broad fingertips and toes, and uni- or bilateral renal agenesis in three individuals. HS2ST1 variants cause a reduction in HS2ST1 mRNA and decreased or absent heparan sulfate 2-O-sulfotransferase 1 in two of three fibroblast cell lines derived from affected individuals. The heparan sulfate synthesized by the individual 1 cell line lacks 2-O-sulfated domains but had an increase in N- and 6-O-sulfated domains demonstrating functional impairment of the HS2ST1. As heparan sulfate modulates FGF-mediated signaling, we found a significantly decreased activation of the MAP kinases ERK1/2 in FGF-2-stimulated cell lines of affected individuals that could be restored by addition of heparin, a GAG similar to heparan sulfate. Focal adhesions in FGF-2-stimulated fibroblasts of affected individuals concentrated at the cell periphery. Our data demonstrate that a heparan sulfate synthesis deficit causes a recognizable syndrome and emphasize a role for 2-O-sulfated heparan sulfate in human neuronal, skeletal, and renal development.

Mutations in the KIF21B kinesin gene cause neurodevelopmental disorders through imbalanced canonical motor activity.

KIF21B is a kinesin protein that promotes intracellular transport and controls microtubule dynamics. We report three missense variants and one duplication in KIF21B in individuals with neurodevelopmental disorders associated with brain malformations, including corpus callosum agenesis (ACC) and microcephaly. We demonstrate, in vivo, that the expression of KIF21B missense variants specifically recapitulates patients' neurodevelopmental abnormalities, including microcephaly and reduced intra- and inter-hemispheric connectivity. We establish that missense KIF21B variants impede neuronal migration through attenuation of kinesin autoinhibition leading to aberrant KIF21B motility activity. We also show that the ACC-related KIF21B variant independently perturbs axonal growth and ipsilateral axon branching through two distinct mechanisms, both leading to deregulation of canonical kinesin motor activity. The duplication introduces a premature termination codon leading to nonsense-mediated mRNA decay. Although we demonstrate that Kif21b haploinsufficiency leads to an impaired neuronal positioning, the duplication variant might not be pathogenic. Altogether, our data indicate that impaired KIF21B autoregulation and function play a critical role in the pathogenicity of human neurodevelopmental disorder.

Genetic variants in the KDM6B gene are associated with neurodevelopmental delays and dysmorphic features.

Lysine-specific demethylase 6B (KDM6B) demethylates trimethylated lysine-27 on histone H3. The methylation and demethylation of histone proteins affects gene expression during development. Pathogenic alterations in histone lysine methylation and demethylation genes have been associated with multiple neurodevelopmental disorders. We have identified a number of de novo alterations in the KDM6B gene via whole exome sequencing (WES) in a cohort of 12 unrelated patients with developmental delay, intellectual disability, dysmorphic facial features, and other clinical findings. Our findings will allow for further investigation in to the role of the KDM6B gene in human neurodevelopmental disorders.

Results of next-generation sequencing gene panel diagnostics including copy-number variation analysis in 810 patients suspected of heritable thoracic aortic disorders.

Simultaneous analysis of multiple genes using next-generation sequencing (NGS) technology has become widely available. Copy-number variations (CNVs) in disease-associated genes have emerged as a cause for several hereditary disorders. CNVs are, however, not routinely detected using NGS analysis. The aim of this study was to assess the diagnostic yield and the prevalence of CNVs using our panel of Hereditary Thoracic Aortic Disease (H-TAD)-associated genes. Eight hundred ten patients suspected of H-TAD were analyzed by targeted NGS analysis of 21 H-TAD associated genes. In addition, the eXome hidden Markov model (XHMM; an algorithm to identify CNVs in targeted NGS data) was used to detect CNVs in these genes. A pathogenic or likely pathogenic variant was found in 66 of 810 patients (8.1%). Of these 66 pathogenic or likely pathogenic variants, six (9.1%) were CNVs not detectable by routine NGS analysis. These CNVs were four intragenic (multi-)exon deletions in MYLK, TGFB2, SMAD3, and PRKG1, respectively. In addition, a large duplication including NOTCH1 and a large deletion encompassing SCARF2 were detected. As confirmed by additional analyses, both CNVs indicated larger chromosomal abnormalities, which could explain the phenotype in both patients. Given the clinical relevance of the identification of a genetic cause, CNV analysis using a method such as XHMM should be incorporated into the clinical diagnostic care for H-TAD patients.

Copy number variation analysis identifies novel CAKUT candidate genes in children with a solitary functioning kidney.

Copy number variations associate with different developmental phenotypes and represent a major cause of congenital anomalies of the kidney and urinary tract (CAKUT). Because rare pathogenic copy number variations are often large and contain multiple genes, identification of the underlying genetic drivers has proven to be difficult. Here we studied the role of rare copy number variations in 80 patients from the KIMONO study cohort for which pathogenic mutations in three genes commonly implicated in CAKUT were excluded. In total, 13 known or novel genomic imbalances in 11 of 80 patients were absent or extremely rare in 23,362 population controls. To identify the most likely genetic drivers for the CAKUT phenotype underlying these rare copy number variations, we used a systematic in silico approach based on frequency in a large data set of controls, annotation with publicly available databases for developmental diseases, tolerance and haploinsufficiency scores, and gene expression profile in the developing kidney and urinary tract. Five novel candidate genes for CAKUT were identified that showed specific expression in the human and mouse developing urinary tract. Among these genes, DLG1 and KIF12 are likely novel susceptibility genes for CAKUT in humans. Thus, there is a significant role of genomic imbalance in the determination of kidney developmental phenotypes. Additionally, we defined a systematic strategy to identify genetic drivers underlying rare copy number variations.

First steps in exploring prospective exome sequencing of consanguineous couples.

Consanguinity is one of the most frequent risk factors for congenital disorders. In theory, prospective exome sequencing of consanguineous couples could identify couples who both are carriers of autosomal recessive diseases, and empower such couples to make informed reproductive decisions. To investigate this, we sent blood samples to our laboratory of four pairs of consanguineous parents having one or more children affected by an autosomal recessive disorder, without revealing any diagnostic information. The study was restricted to find identical, previously described, or evidently pathogenic mutations in both parents of each couple, in over 400 genes known to result in severe autosomal recessive disorders. Out of the six autosomal recessive disorders known to the four couples studied, two were correctly identified. Carrier status of one not previously known autosomal recessive disorder was discovered. As expected, given the pipeline used, large deletions, mutations in genes not present in the gene list, mutations outside the exons and consensus splice sites, and mutations that were not evidently pathogenic and previously not reported, were not identified. The restriction to detecting only couples with identical mutations diminishes the risk of revealing unsolicited findings and shortens the time needed for analysis, but also results in missing couples with different mutations in the same gene. In addition to the proposed pipeline, couples should be offered testing for carrier status of frequent disorders that can present themselves by large deletions, non-exonic mutations or compound heterozygous mutations (e.g. thalassemia, spinal muscular atrophy, cystic fibrosis). Even though sensitivity is reduced, offering exome sequencing prospectively will increase reproductive options for consanguineous couples.

Mutations in genes encoding the cadherin receptor-ligand pair DCHS1 and FAT4 disrupt cerebral cortical development.

The regulated proliferation and differentiation of neural stem cells before the generation and migration of neurons in the cerebral cortex are central aspects of mammalian development. Periventricular neuronal heterotopia, a specific form of mislocalization of cortical neurons, can arise from neuronal progenitors that fail to negotiate aspects of these developmental processes. Here we show that mutations in genes encoding the receptor-ligand cadherin pair DCHS1 and FAT4 lead to a recessive syndrome in humans that includes periventricular neuronal heterotopia. Reducing the expression of Dchs1 or Fat4 within mouse embryonic neuroepithelium increased progenitor cell numbers and reduced their differentiation into neurons, resulting in the heterotopic accumulation of cells below the neuronal layers in the neocortex, reminiscent of the human phenotype. These effects were countered by concurrent knockdown of Yap, a transcriptional effector of the Hippo signaling pathway. These findings implicate Dchs1 and Fat4 upstream of Yap as key regulators of mammalian neurogenesis.

Novel COL4A1 mutations cause cerebral small vessel disease by haploinsufficiency.

Mutations in COL4A1 have been identified in families with hereditary small vessel disease of the brain presumably due to a dominant-negative mechanism. Here, we report on two novel mutations in COL4A1 in two families with porencephaly, intracerebral hemorrhage and severe white matter disease caused by haploinsufficiency. Two families with various clinical presentations of cerebral microangiopathy and autosomal dominant inheritance were examined. Clinical, neuroradiological and genetic investigations were performed. Electron microscopy of the skin was also performed. In one of the families, sequence analysis revealed a one base deletion, c.2085del, leading to a frameshift and a premature stopcodon, p.(Gly696fs). In the other family, a splice site mutation was identified, c.2194-1G>A, which most likely leads to skipping of an exon with a frameshift and premature termination as a result. In fibroblasts of affected individuals from both the families, nonsense-mediated decay (NMD) of the mutant COL4A1 messenger RNAs (mRNAs) and a clear reduction of COL4A1 protein expression were demonstrated, indicating haploinsufficiency of COL4A1. Moreover, thickening of the capillary basement membrane in the skin was documented, similar to reports in patients with COL4A1 missense mutations. These findings suggest haploinsufficiency, a different mechanism from the commonly assumed dominant-negative effect, for COL4A1 mutations as a cause of (antenatal) intracerebral hemorrhage and white matter disease.

PITX2 and FOXC1 spectrum of mutations in ocular syndromes.

Anterior segment dysgenesis (ASD) encompasses a broad spectrum of developmental conditions affecting anterior ocular structures and associated with an increased risk for glaucoma. Various systemic anomalies are often observed in ASD conditions such as Axenfeld-Rieger syndrome (ARS) and De Hauwere syndrome. We report DNA sequencing and copy number analysis of PITX2 and FOXC1 in 76 patients with syndromic or isolated ASD and related conditions. PITX2 mutations and deletions were found in 24 patients with dental and/or umbilical anomalies seen in all. Seven PITX2-mutant alleles were novel including c.708_730del, the most C-terminal mutation reported to date. A second case of deletion of the distant upstream but not coding region of PITX2 was identified, highlighting the importance of this recently discovered mechanism for ARS. FOXC1 deletions were observed in four cases, three of which demonstrated hearing and/or heart defects, including a patient with De Hauwere syndrome; no nucleotide mutations in FOXC1 were identified. Review of the literature identified several other patients with 6p25 deletions and features of De Hauwere syndrome. The 1.3-Mb deletion of 6p25 presented here defines the critical region for this phenotype and includes the FOXC1, FOXF2, and FOXQ1 genes. In summary, PITX2 or FOXC1 disruptions explained 63% of ARS and 6% of other ASD in our cohort; all affected patients demonstrated additional systemic defects with PITX2 mutations showing a strong association with dental and/or umbilical anomalies and FOXC1 with heart and hearing defects. FOXC1 deletion was also found to be associated with De Hauwere syndrome.

Van Maldergem syndrome: further characterisation and evidence for neuronal migration abnormalities and autosomal recessive inheritance.

We present six patients from five unrelated families with a condition originally described by Van Maldergem et al and provide follow-up studies of the original patient. The phenotype comprises a distinctive facial appearance that includes blepharophimosis, maxillary hypoplasia, telecanthus, microtia and atresia of the external auditory meatus, intellectual disability, digital contractures and skeletal anomalies together with subependymal and subcortical neuronal heterotopia. Affected patients typically have neonatal hypotonia, chronic feeding difficulties and respiratory problems. In our cohort, we have observed one instance of sibling recurrence and parental consanguinity in three of the families, indicating that autosomal recessive inheritance is likely.

[A neonate with a congenital hand defect]. / Een neonaat met een aangeboren handafwijking.

A newborn presented with a birth defect of his left hand and unilateral hypoplasia of his left musculus pectoralis major and left nipple. He was diagnosed with Poland syndrome.

Identical but not the same: the value of discordant monozygotic twins in genetic research.

Monozygotic (MZ) twins show remarkable resemblance in many aspects of behavior, health, and disease. Until recently, MZ twins were usually called "genetically identical"; however, evidence for genetic and epigenetic differences within rare MZ twin pairs has accumulated. Here, we summarize the literature on MZ twins discordant for Mendelian inherited disorders and chromosomal abnormalities. A systematic literature search for English articles on discordant MZ twin pairs was performed in Web of Science and PubMed. A total number of 2,016 publications were retrieved and reviewed and 439 reports were retained. Discordant MZ twin pairs are informative in respect to variability of phenotypic expression, pathogenetic mechanisms, epigenetics, and post-zygotic mutagenesis and may serve as a model for research on genetic defects. The analysis of single discordant MZ twin pairs may represent an elegant approach to identify genes in inherited disorders.