Chromatin assembly factor subunit CHAF1A as a monogenic cause for oculo-auriculo-vertebral spectrum.

Oculo-auriculo-vertebral spectrum (OAVS) is characterized by abnormal development of the 1st and 2nd branchial arches. Despite arguments against a monogenic condition, a few genes have been involved in a minority of cases. We now report heterozygous, presumably loss-of function variants in the CHAF1A gene in 8 individuals, including 3 members of the same family. Four cases fulfill stringent diagnostic criteria for OAVS, including asymmetric ear dysplasia, preauricular tags, mandibular asymmetry +/- vertebral malformations. Two patients also presented with kidney malformations. CHAF1A encodes a subunit of CAF-1 (chromatin assembly factor-1), a heterotrimeric protein complex responsible for the deposition of newly synthesized histones H3-H4 onto the newly synthetized DNA strand during replication. The identification of loss-of-unction variants in CHAF1A is consistent with the hypothesis of early developmental genes dysregulation driving OAVS and other associations recently lumped under the acronym Recurrent Constellations of Embryonic Malformations (RCEM).

Novel variants in the SOX11 gene: clinical description of seven new patients.

Pathogenic SOX11 variants have been associated with intellectual developmental disorder with microcephaly, and with or without ocular malformations or hypogonadotropic hypogonadism (HH) (IDDMOH, OMIM # 615866). In this article, we report seven new patients with de novo SOX11 variants. Five of the variants are missense, one nonsense, and one whole-gene deletion, most of them are novel variants. The main clinical features included neurodevelopmental delay (7/7) and intellectual disability (5/7), autism/attention deficit hyperactivity disorder (5/7), microcephaly (4/7), short stature (4/7), hypotonia (4/7), and clinodactyly of the 5th fingers (5/7). HH was confirmed in two female patients with primary amenorrhea, nonvisualized/prepubertal size of the uterus, and nonvisualized ovaries. Two of the male patients presented with micropenis, two had cryptorchidism, and one had decreased testicular size, which are suggestive findings of HH. This article contributes to the clinical characterization of patients with SOX11 variants and supports the role of this gene in HH.

Variant-specific pathophysiological mechanisms of AFF3 differently influence transcriptome profiles.

BACKGROUND: We previously described the KINSSHIP syndrome, an autosomal dominant disorder associated with intellectual disability (ID), mesomelic dysplasia and horseshoe kidney, caused by de novo variants in the degron of AFF3. Mouse knock-ins and overexpression in zebrafish provided evidence for a dominant-negative mode of action, wherein an increased level of AFF3 resulted in pathological effects. METHODS: Evolutionary constraints suggest that other modes-of-inheritance could be at play. We challenged this hypothesis by screening ID cohorts for individuals with predicted-to-be damaging variants in AFF3. We used both animal and cellular models to assess the deleteriousness of the identified variants. RESULTS: We identified an individual with a KINSSHIP-like phenotype carrying a de novo partial duplication of AFF3 further strengthening the hypothesis that an increased level of AFF3 is pathological. We also detected seventeen individuals displaying a milder syndrome with either heterozygous Loss-of-Function (LoF) or biallelic missense variants in AFF3. Consistent with semi-dominance, we discovered three patients with homozygous LoF and one compound heterozygote for a LoF and a missense variant, who presented more severe phenotypes than their heterozygous parents. Matching zebrafish knockdowns exhibit neurological defects that could be rescued by expressing human AFF3 mRNA, confirming their association with the ablation of aff3. Conversely, some of the human AFF3 mRNAs carrying missense variants identified in affected individuals did not rescue these phenotypes. Overexpression of mutated AFF3 mRNAs in zebrafish embryos produced a significant increase of abnormal larvae compared to wild-type overexpression further demonstrating deleteriousness. To further assess the effect of AFF3 variation, we profiled the transcriptome of fibroblasts from affected individuals and engineered isogenic cells harboring + / + , KINSSHIP/KINSSHIP, LoF/ + , LoF/LoF or KINSSHIP/LoF AFF3 genotypes. The expression of more than a third of the AFF3 bound loci is modified in either the KINSSHIP/KINSSHIP or the LoF/LoF lines. While the same pathways are affected, only about one third of the differentially expressed genes are common to the homozygote datasets, indicating that AFF3 LoF and KINSSHIP variants largely modulate transcriptomes differently, e.g. the DNA repair pathway displayed opposite modulation. CONCLUSIONS: Our results and the high pleiotropy shown by variation at this locus suggest that minute changes in AFF3 function are deleterious.

Rare predicted deleterious FEZF2 variants are associated with a neurodevelopmental phenotype.

FEZF2 encodes a transcription factor critical to neurodevelopment that regulates other neurodevelopment genes. Rare variants in FEZF2 have previously been suggested to play a role in autism, and cases of 3p14 microdeletions that include FEZF2 share a neurodevelopmental phenotype including mild dysmorphic features and intellectual disability. We identified seven heterozygous predicted deleterious variants in FEZF2 (three frameshifts, one recurrent missense in two independent cases, one nonsense, and one complete gene deletion) in unrelated individuals with neurodevelopmental disorders including developmental delay/intellectual disability, autism, and/or attention-deficit/hyperactivity. Variants were confirmed to be de novo in five of seven cases and paternally inherited from an affected father in one. Predicted deleterious variants in FEZF2 may affect the expression of genes that are involved in fate choice pathways in developing neurons, and thus contribute to the neurodevelopmental phenotype. Future studies are needed to clarify the mechanism by which FEZF2 leads to this neurodevelopmental disorder.

Variant-specific pathophysiological mechanisms of AFF3 differently influence transcriptome profiles.

Background: We previously described the KINSSHIP syndrome, an autosomal dominant disorder associated with intellectual disability (ID), mesomelic dysplasia and horseshoe kidney,caused by de novo variants in the degron of AFF3. Mouse knock-ins and overexpression in zebrafish provided evidence for a dominant-negative (DN) mode-of-action, wherein an increased level of AFF3 resulted in pathological effects. Methods: Evolutionary constraints suggest that other mode-of-inheritance could be at play. We challenged this hypothesis by screening ID cohorts for individuals with predicted-to-be deleterious variants in AFF3. We used both animal and cellular models to assess the deleteriousness of the identified variants. Results: We identified an individual with a KINSSHIP-like phenotype carrying a de novo partial duplication of AFF3 further strengthening the hypothesis that an increased level of AFF3 is pathological. We also detected seventeen individuals displaying a milder syndrome with either heterozygous LoF or biallelic missense variants in AFF3. Consistent with semi-dominance, we discovered three patients with homozygous LoF and one compound heterozygote for a LoF and a missense variant, who presented more severe phenotypes than their heterozygous parents. Matching zebrafish knockdowns exhibit neurological defects that could be rescued by expressing human AFF3 mRNA, confirming their association with the ablation of aff3. Conversely, some of the human AFF3 mRNAs carrying missense variants identified in affected individuals did not complement. Overexpression of mutated AFF3 mRNAs in zebrafish embryos produced a significant increase of abnormal larvae compared to wild-type overexpression further demonstrating deleteriousness. To further assess the effect of AFF3 variation, we profiled the transcriptome of fibroblasts from affected individuals and engineered isogenic cells harboring +/+, DN/DN, LoF/+, LoF/LoF or DN/LoF AFF3 genotypes. The expression of more than a third of the AFF3 bound loci is modified in either the DN/DN or the LoF/LoF lines. While the same pathways are affected, only about one-third of the differentially expressed genes are common to these homozygote datasets, indicating that AFF3 LoF and DN variants largely modulate transcriptomes differently, e.g. the DNA repair pathway displayed opposite modulation. Conclusions: Our results and the high pleiotropy shown by variation at this locus suggest that minute changes in AFF3 function are deleterious.

Neurodevelopmental and other phenotypes recurrently associated with heterozygous BAZ2B loss-of-function variants.

The bromodomain adjacent to zinc finger 2B (BAZ2B) gene encodes a chromatin remodeling protein that has been shown to perform a variety of regulatory functions. It has been proposed that loss of BAZ2B function is associated with neurodevelopmental phenotypes, and some recurrent structural birth defects and dysmorphic features have been documented among individuals carrying heterozygous loss-of-function BAZ2B variants. However, additional evidence is needed to confirm that these phenotypes are attributable to BAZ2B deficiency. Here, we report 10 unrelated individuals with heterozygous deletions, stop-gain, frameshift, missense, splice junction, indel, and start-loss variants affecting BAZ2B. These included a paternal intragenic deletion and a maternal frameshift variant that were inherited from mildly affected or asymptomatic parents. The analysis of molecular and clinical data from this cohort, and that of individuals previously reported, suggests that BAZ2B haploinsufficiency causes an autosomal dominant neurodevelopmental syndrome that is incompletely penetrant. The phenotypes most commonly seen in association with loss of BAZ2B function include developmental delay, intellectual disability, autism spectrum disorder, speech delay-with some affected individuals being non-verbal-behavioral abnormalities, seizures, vision-related issues, congenital heart defects, poor fetal growth, and an indistinct pattern of dysmorphic features in which epicanthal folds and small ears are particularly common.

Gain and loss of function variants in EZH1 disrupt neurogenesis and cause dominant and recessive neurodevelopmental disorders.

Genetic variants in chromatin regulators are frequently found in neurodevelopmental disorders, but their effect in disease etiology is rarely determined. Here, we uncover and functionally define pathogenic variants in the chromatin modifier EZH1 as the cause of dominant and recessive neurodevelopmental disorders in 19 individuals. EZH1 encodes one of the two alternative histone H3 lysine 27 methyltransferases of the PRC2 complex. Unlike the other PRC2 subunits, which are involved in cancers and developmental syndromes, the implication of EZH1 in human development and disease is largely unknown. Using cellular and biochemical studies, we demonstrate that recessive variants impair EZH1 expression causing loss of function effects, while dominant variants are missense mutations that affect evolutionarily conserved aminoacids, likely impacting EZH1 structure or function. Accordingly, we found increased methyltransferase activity leading to gain of function of two EZH1 missense variants. Furthermore, we show that EZH1 is necessary and sufficient for differentiation of neural progenitor cells in the developing chick embryo neural tube. Finally, using human pluripotent stem cell-derived neural cultures and forebrain organoids, we demonstrate that EZH1 variants perturb cortical neuron differentiation. Overall, our work reveals a critical role of EZH1 in neurogenesis regulation and provides molecular diagnosis for previously undefined neurodevelopmental disorders.

Deleterious, protein-altering variants in the transcriptional coregulator ZMYM3 in 27 individuals with a neurodevelopmental delay phenotype.

Neurodevelopmental disorders (NDDs) result from highly penetrant variation in hundreds of different genes, some of which have not yet been identified. Using the MatchMaker Exchange, we assembled a cohort of 27 individuals with rare, protein-altering variation in the transcriptional coregulator ZMYM3, located on the X chromosome. Most (n = 24) individuals were males, 17 of which have a maternally inherited variant; six individuals (4 male, 2 female) harbor de novo variants. Overlapping features included developmental delay, intellectual disability, behavioral abnormalities, and a specific facial gestalt in a subset of males. Variants in almost all individuals (n = 26) are missense, including six that recurrently affect two residues. Four unrelated probands were identified with inherited variation affecting Arg441, a site at which variation has been previously seen in NDD-affected siblings, and two individuals have de novo variation resulting in p.Arg1294Cys (c.3880C>T). All variants affect evolutionarily conserved sites, and most are predicted to damage protein structure or function. ZMYM3 is relatively intolerant to variation in the general population, is widely expressed across human tissues, and encodes a component of the KDM1A-RCOR1 chromatin-modifying complex. ChIP-seq experiments on one variant, p.Arg1274Trp, indicate dramatically reduced genomic occupancy, supporting a hypomorphic effect. While we are unable to perform statistical evaluations to definitively support a causative role for variation in ZMYM3, the totality of the evidence, including 27 affected individuals, recurrent variation at two codons, overlapping phenotypic features, protein-modeling data, evolutionary constraint, and experimentally confirmed functional effects strongly support ZMYM3 as an NDD-associated gene.

Biallelic variants in OGDH encoding oxoglutarate dehydrogenase lead to a neurodevelopmental disorder characterized by global developmental delay, movement disorder, and metabolic abnormalities.

PURPOSE: This study aimed to establish the genetic cause of a novel autosomal recessive neurodevelopmental disorder characterized by global developmental delay, movement disorder, and metabolic abnormalities. METHODS: We performed a detailed clinical characterization of 4 unrelated individuals from consanguineous families with a neurodevelopmental disorder. We used exome sequencing or targeted-exome sequencing, cosegregation, in silico protein modeling, and functional analyses of variants in HEK293 cells and Drosophila melanogaster, as well as in proband-derived fibroblast cells. RESULTS: In the 4 individuals, we identified 3 novel homozygous variants in oxoglutarate dehydrogenase (OGDH) (NM_002541.3), which encodes a subunit of the tricarboxylic acid cycle enzyme α-ketoglutarate dehydrogenase. In silico homology modeling predicts that c.566C>T:p.(Pro189Leu) and c.890C>A:p.(Ser297Tyr) variants interfere with the structure and function of OGDH. Fibroblasts from individual 1 showed that the p.(Ser297Tyr) variant led to a higher degradation rate of the OGDH protein. OGDH protein with p.(Pro189Leu) or p.(Ser297Tyr) variants in HEK293 cells showed significantly lower levels than the wild-type protein. Furthermore, we showed that expression of Drosophila Ogdh (dOgdh) carrying variants homologous to p.(Pro189Leu) or p.(Ser297Tyr), failed to rescue developmental lethality caused by loss of dOgdh. SpliceAI, a variant splice predictor, predicted that the c.935G>A:p.(Arg312Lys)/p.(Phe264_Arg312del) variant impacts splicing, which was confirmed through a mini-gene assay in HEK293 cells. CONCLUSION: We established that biallelic variants in OGDH cause a neurodevelopmental disorder with metabolic and movement abnormalities.

Phenotypic continuum of NFU1-related disorders.

Bi-allelic variants in Iron-Sulfur Cluster Scaffold (NFU1) have previously been associated with multiple mitochondrial dysfunctions syndrome 1 (MMDS1) characterized by early-onset rapidly fatal leukoencephalopathy. We report 19 affected individuals from 10 independent families with ultra-rare bi-allelic NFU1 missense variants associated with a spectrum of early-onset pure to complex hereditary spastic paraplegia (HSP) phenotype with a longer survival (16/19) on one end and neurodevelopmental delay with severe hypotonia (3/19) on the other. Reversible or irreversible neurological decompensation after a febrile illness was common in the cohort, and there were invariable white matter abnormalities on neuroimaging. The study suggests that MMDS1 and HSP could be the two ends of the NFU1-related phenotypic continuum.

Missense variants in ANKRD11 cause KBG syndrome by impairment of stability or transcriptional activity of the encoded protein.

PURPOSE: Although haploinsufficiency of ANKRD11 is among the most common genetic causes of neurodevelopmental disorders, the role of rare ANKRD11 missense variation remains unclear. We characterized clinical, molecular, and functional spectra of ANKRD11 missense variants. METHODS: We collected clinical information of individuals with ANKRD11 missense variants and evaluated phenotypic fit to KBG syndrome. We assessed pathogenicity of variants through in silico analyses and cell-based experiments. RESULTS: We identified 20 unique, mostly de novo, ANKRD11 missense variants in 29 individuals, presenting with syndromic neurodevelopmental disorders similar to KBG syndrome caused by ANKRD11 protein truncating variants or 16q24.3 microdeletions. Missense variants significantly clustered in repression domain 2 at the ANKRD11 C-terminus. Of the 10 functionally studied missense variants, 6 reduced ANKRD11 stability. One variant caused decreased proteasome degradation and loss of ANKRD11 transcriptional activity. CONCLUSION: Our study indicates that pathogenic heterozygous ANKRD11 missense variants cause the clinically recognizable KBG syndrome. Disrupted transrepression capacity and reduced protein stability each independently lead to ANKRD11 loss-of-function, consistent with haploinsufficiency. This highlights the diagnostic relevance of ANKRD11 missense variants, but also poses diagnostic challenges because the KBG-associated phenotype may be mild and inherited pathogenic ANKRD11 (missense) variants are increasingly observed, warranting stringent variant classification and careful phenotyping.

Semaphorin-Plexin Signaling: From Axonal Guidance to a New X-Linked Intellectual Disability Syndrome.

BACKGROUND: Semaphorins and plexins are ligands and cell surface receptors that regulate multiple neurodevelopmental processes such as axonal growth and guidance. PLXNA3 is a plexin gene located on the X chromosome that encodes the most widely expressed plexin receptor in fetal brain, plexin-A3. Plexin-A3 knockout mice demonstrate its role in semaphorin signaling in vivo. The clinical manifestations of semaphorin/plexin neurodevelopmental disorders have been less widely explored. This study describes the neurological and neurodevelopmental phenotypes of boys with maternally inherited hemizygous PLXNA3 variants. METHODS: Data-sharing through GeneDx and GeneMatcher allowed identification of individuals with autism or intellectual disabilities (autism/ID) and hemizygous PLXNA3 variants in collaboration with their physicians and genetic counselors, who completed questionnaires about their patients. In silico analyses predicted pathogenicity for each PLXNA3 variant. RESULTS: We assessed 14 boys (mean age, 10.7 [range 2 to 25] years) with maternally inherited hemizygous PLXNA3 variants and autism/ID ranging from mild to severe. Other findings included fine motor dyspraxia (92%), attention-deficit/hyperactivity traits, and aggressive behaviors (63%). Six patients (43%) had seizures. Thirteen boys (93%) with PLXNA3 variants showed novel or very low allele frequencies and probable damaging/disease-causing pathogenicity in one or more predictors. We found a genotype-phenotype correlation between PLXNA3 cytoplasmic domain variants (exons 22 to 32) and more severe neurodevelopmental disorder phenotypes (P < 0.05). CONCLUSIONS: We report 14 boys with maternally inherited, hemizygous PLXNA3 variants and a range of neurodevelopmental disorders suggesting a novel X-linked intellectual disability syndrome. Greater understanding of PLXNA3 variant pathogenicity in humans will require additional clinical, computational, and experimental validation.

Diagnostic assays for chronic granulomatous disease and other neutrophil disorders.

Inasmuch as neutrophils are the primary cellular defense against bacterial and fungal infections, disorders that affect these white cells typically predispose individuals to severe and recurrent infections. Therefore, diagnosis of such disorders is an important first step in directing long-term treatment/care for the patient. Herein, we describe methods to identify chronic granulomatous disease, leukocyte adhesion deficiency, and neutropenia. The assays are relatively simple to perform and cost effective and can be performed with equipment available in most laboratories.

Complex regulation of human cathelicidin gene expression: novel splice variants and 5'UTR negative regulatory element.

Cationic antimicrobial peptides play important roles in host defense, linking innate and adaptive immunity. hCAP18, the only human antimicrobial cathelicidin, consists of a conserved N-terminal cathelin-like domain and a C-terminal peptide, LL-37. Expression is regulated during myeloid differentiation, and tightly controlled during infection and inflammation, suggesting active regulation. Using 5' RACE (rapid amplification of cDNA ends), multiple transcription initiation sites were identified, as well as new splice variants leading to novel augmentations of hCAP18 amino acid composition in bone marrow but not peripheral blood neutrophils. Having expressed hCAP18 promoter constructs in cell lines, we found that full-length (-1739) and truncated (-978) promoter constructs had lower luciferase activities than 5'UTR deletion constructs. Transient transfection of progressively deleted constructs in the non-permissive K562 cell line led us to identify a negative regulatory element within the 53 bp immediately upstream of the ATG of hCAP18. Additionally, transient transfection of 5' deletion constructs identified a positive regulatory element within the 101 bases 5' of promoter sequence containing two GT-boxes. Negative and positive regulatory elements within the hCAP18 gene promoter provide new insights into the possible molecular basis of myeloid gene expression.

Diagnostic assays for chronic granulomatous disease and other neutrophil disorders.

Inasmuch as neutrophils are the primary cellular defense against bacterial and fungal infections, disorders that affect these white cells typically predispose individuals to severe and recurrent infections. Therefore, diagnosis of such disorders is an important first step in directing long-term treatment/care for the patient. Herein, we describe methods to identify chronic granulomatous disease (CGD), leukocyte adhesion deficiency (LAD), and neutropenia. The assays are relatively simple to perform, cost-effective, and can be performed with equipment available in most laboratories.