Further delineation of the rare GDACCF (global developmental delay, absent or hypoplastic corpus callosum, dysmorphic facies syndrome): genotype and phenotype of 22 patients with ZNF148 mutations.

BACKGROUND: Pathogenic variants in the zinc finger protein coding genes are rare causes of intellectual disability and congenital malformations. Mutations in the ZNF148 gene causing GDACCF syndrome (global developmental delay, absent or hypoplastic corpus callosum, dysmorphic facies; MIM #617260) have been reported in five individuals so far. METHODS: As a result of an international collaboration using GeneMatcher Phenome Central Repository and personal communications, here we describe the clinical and molecular genetic characteristics of 22 previously unreported individuals. RESULTS: The core clinical phenotype is characterised by developmental delay particularly in the domain of speech development, postnatal growth retardation, microcephaly and facial dysmorphism. Corpus callosum abnormalities appear less frequently than suggested by previous observations. The identified mutations concerned nonsense or frameshift variants that were mainly located in the last exon of the ZNF148 gene. Heterozygous deletion including the entire ZNF148 gene was found in only one case. Most mutations occurred de novo, but were inherited from an affected parent in two families. CONCLUSION: The GDACCF syndrome is clinically diverse, and a genotype-first approach, that is, exome sequencing is recommended for establishing a genetic diagnosis rather than a phenotype-first approach. However, the syndrome may be suspected based on some recurrent, recognisable features. Corpus callosum anomalies were not as constant as previously suggested, we therefore recommend to replace the term 'GDACCF syndrome' with 'ZNF148-related neurodevelopmental disorder'.

Identification of DNA methylation episignature for the intellectual developmental disorder, autosomal dominant 21 syndrome, caused by variants in the CTCF gene.

PURPOSE: The main objective of this study was to assess clinical features and genome-wide DNA methylation profiles in individuals affected by intellectual developmental disorder, autosomal dominant 21 (IDD21) syndrome, caused by variants in the CCCTC-binding factor (CTCF) gene. METHODS: DNA samples were extracted from peripheral blood of 16 individuals with clinical features and genetic findings consistent with IDD21. DNA methylation analysis was performed using the Illumina Infinium Methylation EPIC Bead Chip microarrays. The methylation levels were fitted in a multivariate linear regression model to identify the differentially methylated probes. A binary support vector machine classification model was constructed to differentiate IDD21 samples from controls. RESULTS: We identified a highly specific, reproducible, and sensitive episignature associated with CTCF variants. Six variants of uncertain significance were tested, of which 2 mapped to the IDD21 episignature and clustered alongside IDD21 cases in both heatmap and multidimensional scaling plots. Comparison of the genomic DNA methylation profile of IDD21 with that of 56 other neurodevelopmental disorders provided insights into the underlying molecular pathophysiology of this disorder. CONCLUSION: The robust and specific CTCF/IDD21 episignature expands the growing list of neurodevelopmental disorders with distinct DNA methylation profiles, which can be applied as supporting evidence in variant classification.

Amniotic band syndrome and limb body wall complex in Europe 1980-2019.

Amniotic band syndrome (ABS) and limb body wall complex (LBWC) have an overlapping phenotype of multiple congenital anomalies and their etiology is unknown. We aimed to determine the prevalence of ABS and LBWC in Europe from 1980 to 2019 and to describe the spectrum of congenital anomalies. In addition, we investigated maternal age and multiple birth as possible risk factors for the occurrence of ABS and LBWC. We used data from the European surveillance of congenital anomalies (EUROCAT) network including data from 30 registries over 1980-2019. We included all pregnancy outcomes, including live births, stillbirths, and terminations of pregnancy for fetal anomalies. ABS and LBWC cases were extracted from the central EUROCAT database using coding information responses from the registries. In total, 866 ABS cases and 451 LBWC cases were included in this study. The mean prevalence was 0.53/10,000 births for ABS and 0.34/10,000 births for LBWC during the 40 years. Prevalence of both ABS and LBWC was lower in the 1980s and higher in the United Kingdom. Limb anomalies and neural tube defects were commonly seen in ABS, whereas in LBWC abdominal and thoracic wall defects and limb anomalies were most prevalent. Twinning was confirmed as a risk factor for both ABS and LBWC. This study includes the largest cohort of ABS and LBWC cases ever reported over a large time period using standardized EUROCAT data. Prevalence, clinical characteristics, and the phenotypic spectrum are described, and twinning is confirmed as a risk factor.

Further delineation of auriculocondylar syndrome based on 14 novel cases and reassessment of 25 published cases.

Auriculocondylar syndrome (ACS) is a rare craniofacial disorder characterized by mandibular hypoplasia and an auricular defect at the junction between the lobe and helix, known as a "Question Mark Ear" (QME). Several additional features, originating from the first and second branchial arches and other tissues, have also been reported. ACS is genetically heterogeneous with autosomal dominant and recessive modes of inheritance. The mutations identified to date are presumed to dysregulate the endothelin 1 signaling pathway. Here we describe 14 novel cases and reassess 25 published cases of ACS through a questionnaire for systematic data collection. All patients harbor mutation(s) in PLCB4, GNAI3, or EDN1. This series of patients contributes to the characterization of additional features occasionally associated with ACS such as respiratory, costal, neurodevelopmental, and genital anomalies, and provides management and monitoring recommendations.

Missense Variants in the Histone Acetyltransferase Complex Component Gene TRRAP Cause Autism and Syndromic Intellectual Disability.

Acetylation of the lysine residues in histones and other DNA-binding proteins plays a major role in regulation of eukaryotic gene expression. This process is controlled by histone acetyltransferases (HATs/KATs) found in multiprotein complexes that are recruited to chromatin by the scaffolding subunit transformation/transcription domain-associated protein (TRRAP). TRRAP is evolutionarily conserved and is among the top five genes intolerant to missense variation. Through an international collaboration, 17 distinct de novo or apparently de novo variants were identified in TRRAP in 24 individuals. A strong genotype-phenotype correlation was observed with two distinct clinical spectra. The first is a complex, multi-systemic syndrome associated with various malformations of the brain, heart, kidneys, and genitourinary system and characterized by a wide range of intellectual functioning; a number of affected individuals have intellectual disability (ID) and markedly impaired basic life functions. Individuals with this phenotype had missense variants clustering around the c.3127G>A p.(Ala1043Thr) variant identified in five individuals. The second spectrum manifested with autism spectrum disorder (ASD) and/or ID and epilepsy. Facial dysmorphism was seen in both groups and included upslanted palpebral fissures, epicanthus, telecanthus, a wide nasal bridge and ridge, a broad and smooth philtrum, and a thin upper lip. RNA sequencing analysis of skin fibroblasts derived from affected individuals skin fibroblasts showed significant changes in the expression of several genes implicated in neuronal function and ion transport. Thus, we describe here the clinical spectrum associated with TRRAP pathogenic missense variants, and we suggest a genotype-phenotype correlation useful for clinical evaluation of the pathogenicity of the variants.

Discovering a new part of the phenotypic spectrum of Coffin-Siris syndrome in a fetal cohort.

PURPOSE: Genome-wide sequencing is increasingly being performed during pregnancy to identify the genetic cause of congenital anomalies. The interpretation of prenatally identified variants can be challenging and is hampered by our often limited knowledge of prenatal phenotypes. To better delineate the prenatal phenotype of Coffin-Siris syndrome (CSS), we collected clinical data from patients with a prenatal phenotype and a pathogenic variant in one of the CSS-associated genes. METHODS: Clinical data was collected through an extensive web-based survey. RESULTS: We included 44 patients with a variant in a CSS-associated gene and a prenatal phenotype; 9 of these patients have been reported before. Prenatal anomalies that were frequently observed in our cohort include hydrocephalus, agenesis of the corpus callosum, hypoplastic left heart syndrome, persistent left vena cava, diaphragmatic hernia, renal agenesis, and intrauterine growth restriction. Anal anomalies were frequently identified after birth in patients with ARID1A variants (6/14, 43%). Interestingly, pathogenic ARID1A variants were much more frequently identified in the current prenatal cohort (16/44, 36%) than in postnatal CSS cohorts (5%-9%). CONCLUSION: Our data shed new light on the prenatal phenotype of patients with pathogenic variants in CSS genes.

Loss-of-function variants in SRRM2 cause a neurodevelopmental disorder.

PURPOSE: SRRM2 encodes the SRm300 protein, a splicing factor of the SR-related protein family characterized by its serine- and arginine-enriched domains. It promotes interactions between messenger RNA and the spliceosome catalytic machinery. This gene, predicted to be highly intolerant to loss of function (LoF) and very conserved through evolution, has not been previously reported in constitutive human disease. METHODS: Among the 1000 probands studied with developmental delay and intellectual disability in our database, we found 2 patients with de novo LoF variants in SRRM2. Additional families were identified through GeneMatcher. RESULTS: Here, we report on 22 patients with LoF variants in SRRM2 and provide a description of the phenotype. Molecular analysis identified 12 frameshift variants, 8 nonsense variants, and 2 microdeletions of 66 kb and 270 kb. The patients presented with a mild developmental delay, predominant speech delay, autistic or attention-deficit/hyperactivity disorder features, overfriendliness, generalized hypotonia, overweight, and dysmorphic facial features. Intellectual disability was variable and mild when present. CONCLUSION: We established SRRM2 as a gene responsible for a rare neurodevelopmental disease.

Genome sequencing in families with congenital limb malformations.

The extensive clinical and genetic heterogeneity of congenital limb malformation calls for comprehensive genome-wide analysis of genetic variation. Genome sequencing (GS) has the potential to identify all genetic variants. Here we aim to determine the diagnostic potential of GS as a comprehensive one-test-for-all strategy in a cohort of undiagnosed patients with congenital limb malformations. We collected 69 cases (64 trios, 1 duo, 5 singletons) with congenital limb malformations with no molecular diagnosis after standard clinical genetic testing and performed genome sequencing. We also developed a framework to identify potential noncoding pathogenic variants. We identified likely pathogenic/disease-associated variants in 12 cases (17.4%) including four in known disease genes, and one repeat expansion in HOXD13. In three unrelated cases with ectrodactyly, we identified likely pathogenic variants in UBA2, establishing it as a novel disease gene. In addition, we found two complex structural variants (3%). We also identified likely causative variants in three novel high confidence candidate genes. We were not able to identify any noncoding variants. GS is a powerful strategy to identify all types of genomic variants associated with congenital limb malformation, including repeat expansions and complex structural variants missed by standard diagnostic approaches. In this cohort, no causative noncoding SNVs could be identified.

Missense Mutations of the Pro65 Residue of PCGF2 Cause a Recognizable Syndrome Associated with Craniofacial, Neurological, Cardiovascular, and Skeletal Features.

PCGF2 encodes the polycomb group ring finger 2 protein, a transcriptional repressor involved in cell proliferation, differentiation, and embryogenesis. PCGF2 is a component of the polycomb repressive complex 1 (PRC1), a multiprotein complex which controls gene silencing through histone modification and chromatin remodelling. We report the phenotypic characterization of 13 patients (11 unrelated individuals and a pair of monozygotic twins) with missense mutations in PCGF2. All the mutations affected the same highly conserved proline in PCGF2 and were de novo, excepting maternal mosaicism in one. The patients demonstrated a recognizable facial gestalt, intellectual disability, feeding problems, impaired growth, and a range of brain, cardiovascular, and skeletal abnormalities. Computer structural modeling suggests the substitutions alter an N-terminal loop of PCGF2 critical for histone biding. Mutant PCGF2 may have dominant-negative effects, sequestering PRC1 components into complexes that lack the ability to interact efficiently with histones. These findings demonstrate the important role of PCGF2 in human development and confirm that heterozygous substitutions of the Pro65 residue of PCGF2 cause a recognizable syndrome characterized by distinctive craniofacial, neurological, cardiovascular, and skeletal features.

ZMYND11-related syndromic intellectual disability: 16 patients delineating and expanding the phenotypic spectrum.

Pathogenic variants in ZMYND11, which acts as a transcriptional repressor, have been associated with intellectual disability, behavioral abnormalities, and seizures. Only 11 affected individuals have been reported to date, and the phenotype associated with pathogenic variants in this gene have not been fully defined. Here, we present 16 additional patients with predicted pathogenic heterozygous variants in including four individuals from the same family, to further delineate and expand the genotypic and phenotypic spectrum of ZMYND11-related syndromic intellectual disability. The associated phenotype includes developmental delay, particularly affecting speech, mild-moderate intellectual disability, significant behavioral abnormalities, seizures, and hypotonia. There are subtle shared dysmorphic features, including prominent eyelashes and eyebrows, a depressed nasal bridge with bulbous nasal tip, anteverted nares, thin vermilion of the upper lip, and wide mouth. Novel features include brachydactyly and tooth enamel hypoplasia. Most identified variants are likely to result in premature truncation and/or nonsense-mediated decay. Two ZMYND11 variants located in the final exon-p.(Gln586*) (likely escaping nonsense-mediated decay) and p.(Cys574Arg)-are predicted to disrupt the MYND-type zinc-finger motif and likely interfere with binding to its interaction partners. Hence, the homogeneous phenotype likely results from a common mechanism of loss-of-function.

High Rate of Recurrent De Novo Mutations in Developmental and Epileptic Encephalopathies.

Developmental and epileptic encephalopathy (DEE) is a group of conditions characterized by the co-occurrence of epilepsy and intellectual disability (ID), typically with developmental plateauing or regression associated with frequent epileptiform activity. The cause of DEE remains unknown in the majority of cases. We performed whole-genome sequencing (WGS) in 197 individuals with unexplained DEE and pharmaco-resistant seizures and in their unaffected parents. We focused our attention on de novo mutations (DNMs) and identified candidate genes containing such variants. We sought to identify additional subjects with DNMs in these genes by performing targeted sequencing in another series of individuals with DEE and by mining various sequencing datasets. We also performed meta-analyses to document enrichment of DNMs in candidate genes by leveraging our WGS dataset with those of several DEE and ID series. By combining these strategies, we were able to provide a causal link between DEE and the following genes: NTRK2, GABRB2, CLTC, DHDDS, NUS1, RAB11A, GABBR2, and SNAP25. Overall, we established a molecular diagnosis in 63/197 (32%) individuals in our WGS series. The main cause of DEE in these individuals was de novo point mutations (53/63 solved cases), followed by inherited mutations (6/63 solved cases) and de novo CNVs (4/63 solved cases). De novo missense variants explained a larger proportion of individuals in our series than in other series that were primarily ascertained because of ID. Moreover, these DNMs were more frequently recurrent than those identified in ID series. These observations indicate that the genetic landscape of DEE might be different from that of ID without epilepsy.

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.

Widening of the genetic and clinical spectrum of Lamb-Shaffer syndrome, a neurodevelopmental disorder due to SOX5 haploinsufficiency.

PURPOSE: Lamb-Shaffer syndrome (LAMSHF) is a neurodevelopmental disorder described in just over two dozen patients with heterozygous genetic alterations involving SOX5, a gene encoding a transcription factor regulating cell fate and differentiation in neurogenesis and other discrete developmental processes. The genetic alterations described so far are mainly microdeletions. The present study was aimed at increasing our understanding of LAMSHF, its clinical and genetic spectrum, and the pathophysiological mechanisms involved. METHODS: Clinical and genetic data were collected through GeneMatcher and clinical or genetic networks for 41 novel patients harboring various types ofSOX5 alterations. Functional consequences of selected substitutions were investigated. RESULTS: Microdeletions and truncating variants occurred throughout SOX5. In contrast, most missense variants clustered in the pivotal SOX-specific high-mobility-group domain. The latter variants prevented SOX5 from binding DNA and promoting transactivation in vitro, whereas missense variants located outside the high-mobility-group domain did not. Clinical manifestations and severity varied among patients. No clear genotype-phenotype correlations were found, except that missense variants outside the high-mobility-group domain were generally better tolerated. CONCLUSIONS: This study extends the clinical and genetic spectrum associated with LAMSHF and consolidates evidence that SOX5 haploinsufficiency leads to variable degrees of intellectual disability, language delay, and other clinical features.

A recurrent missense variant in HARS2 results in variable sensorineural hearing loss in three unrelated families.

HARS2 encodes mitochondrial histidyl-tRNA synthetase (HARS2), which links histidine to its cognate tRNA in the mitochondrial matrix. Biallelic variants in HARS2 are associated with Perrault syndrome, a rare recessive condition characterized by sensorineural hearing loss in both sexes and primary ovarian insufficiency in 46,XX females. Some individuals with Perrault syndrome have a broader phenotypic spectrum with neurological features, including ataxia and peripheral neuropathy. Here, we report a recurrent variant in HARS2 in association with sensorineural hearing loss. In affected individuals from three unrelated families, the variant HARS2 c.1439G>A p.(Arg480His) is present as a heterozygous variant in trans to a putative pathogenic variant. The low prevalence of the allele HARS2 c.1439G>A p.(Arg480His) in the general population and its presence in three families with hearing loss, confirm the pathogenicity of this variant and illustrate the presentation of Perrault syndrome as nonsyndromic hearing loss in males and prepubertal females.

Primrose syndrome: Characterization of the phenotype in 42 patients.

Primrose syndrome (PS; MIM# 259050) is characterized by intellectual disability (ID), macrocephaly, unusual facial features (frontal bossing, deeply set eyes, down-slanting palpebral fissures), calcified external ears, sparse body hair and distal muscle wasting. The syndrome is caused by de novo heterozygous missense variants in ZBTB20. Most of the 29 published patients are adults as characteristics appear more recognizable with age. We present 13 hitherto unpublished individuals and summarize the clinical and molecular findings in all 42 patients. Several signs and symptoms of PS develop during childhood, but the cardinal features, such as calcification of the external ears, cystic bone lesions, muscle wasting, and contractures typically develop between 10 and 16 years of age. Biochemically, anemia and increased alpha-fetoprotein levels are often present. Two adult males with PS developed a testicular tumor. Although PS should be regarded as a progressive entity, there are no indications that cognition becomes more impaired with age. No obvious genotype-phenotype correlation is present. A subgroup of patients with ZBTB20 variants may be associated with mild, nonspecific ID. Metabolic investigations suggest a disturbed mitochondrial fatty acid oxidation. We suggest a regular surveillance in all adult males with PS until it is clear whether or not there is a truly elevated risk of testicular cancer.

PLPHP deficiency: clinical, genetic, biochemical, and mechanistic insights.

Biallelic pathogenic variants in PLPBP (formerly called PROSC) have recently been shown to cause a novel form of vitamin B6-dependent epilepsy, the pathophysiological basis of which is poorly understood. When left untreated, the disease can progress to status epilepticus and death in infancy. Here we present 12 previously undescribed patients and six novel pathogenic variants in PLPBP. Suspected clinical diagnoses prior to identification of PLPBP variants included mitochondrial encephalopathy (two patients), folinic acid-responsive epilepsy (one patient) and a movement disorder compatible with AADC deficiency (one patient). The encoded protein, PLPHP is believed to be crucial for B6 homeostasis. We modelled the pathogenicity of the variants and developed a clinical severity scoring system. The most severe phenotypes were associated with variants leading to loss of function of PLPBP or significantly affecting protein stability/PLP-binding. To explore the pathophysiology of this disease further, we developed the first zebrafish model of PLPHP deficiency using CRISPR/Cas9. Our model recapitulates the disease, with plpbp-/- larvae showing behavioural, biochemical, and electrophysiological signs of seizure activity by 10 days post-fertilization and early death by 16 days post-fertilization. Treatment with pyridoxine significantly improved the epileptic phenotype and extended lifespan in plpbp-/- animals. Larvae had disruptions in amino acid metabolism as well as GABA and catecholamine biosynthesis, indicating impairment of PLP-dependent enzymatic activities. Using mass spectrometry, we observed significant B6 vitamer level changes in plpbp-/- zebrafish, patient fibroblasts and PLPHP-deficient HEK293 cells. Additional studies in human cells and yeast provide the first empirical evidence that PLPHP is localized in mitochondria and may play a role in mitochondrial metabolism. These models provide new insights into disease mechanisms and can serve as a platform for drug discovery.

Variants in nuclear factor I genes influence growth and development.

The nuclear factor one (NFI) site-specific DNA-binding proteins represent a family of transcription factors that are important for the development of multiple organ systems, including the brain. During brain development in mice, the expression patterns of Nfia, Nfib, and Nfix overlap, and knockout mice for each of these exhibit overlapping brain defects, including megalencephaly, dysgenesis of the corpus callosum, and enlarged ventricles, which implies a common but not redundant function in brain development. In line with these models, human phenotypes caused by haploinsufficiency of NFIA, NFIB, and NFIX display significant overlap, sharing neurodevelopmental deficits, macrocephaly, brain anomalies, and variable somatic overgrowth. Other anomalies may be present depending on the NFI gene involved. The possibility of variants in NFI genes should therefore be considered in individuals with intellectual disability and brain overgrowth, with individual NFI-related conditions being differentiated from one another by additional signs and symptoms. The exception is provided by specific NFIX variants that act in a dominant negative manner, as these cause a recognizable entity with more severe cognitive impairment and marked bone dysplasia, Marshall-Smith syndrome. NFIX duplications are associated with a phenotype opposite to that of haploinsufficiency, characterized by short stature, small head circumference, and delayed bone age. The spectrum of NFI-related disorders will likely be further expanded, as larger cohorts are assessed.

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

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

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

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