De novo variants in POLR3B cause ataxia, spasticity, and demyelinating neuropathy.

POLR3B encodes the second-largest catalytic subunit of RNA polymerase III, an enzyme involved in transcription. Bi-allelic pathogenic variants in POLR3B are a well-established cause of hypomyelinating leukodystrophy. We describe six unrelated individuals with de novo missense variants in POLR3B and a clinical presentation substantially different from POLR3-related leukodystrophy. These individuals had afferent ataxia, spasticity, variable intellectual disability and epilepsy, and predominantly demyelinating sensory motor peripheral neuropathy. Protein modeling and proteomic analysis revealed a distinct mechanism of pathogenicity; the de novo POLR3B variants caused aberrant association of individual enzyme subunits rather than affecting overall enzyme assembly or stability. We expand the spectrum of disorders associated with pathogenic variants in POLR3B to include a de novo heterozygous POLR3B-related disorder.

SLC6A1 variant pathogenicity, molecular function and phenotype: a genetic and clinical analysis.

Genetic variants in the SLC6A1 gene can cause a broad phenotypic disease spectrum by altering the protein function. Thus, systematically curated clinically relevant genotype-phenotype associations are needed to understand the disease mechanism and improve therapeutic decision-making. We aggregated genetic and clinical data from 172 individuals with likely pathogenic/pathogenic (lp/p) SLC6A1 variants and functional data for 184 variants (14.1% lp/p). Clinical and functional data were available for a subset of 126 individuals. We explored the potential associations of variant positions on the GAT1 3D structure with variant pathogenicity, altered molecular function and phenotype severity using bioinformatic approaches. The GAT1 transmembrane domains 1, 6 and extracellular loop 4 (EL4) were enriched for patient over population variants. Across functionally tested missense variants (n = 156), the spatial proximity from the ligand was associated with loss-of-function in the GAT1 transporter activity. For variants with complete loss of in vitro GABA uptake, we found a 4.6-fold enrichment in patients having severe disease versus non-severe disease (P = 2.9 × 10-3, 95% confidence interval: 1.5-15.3). In summary, we delineated associations between the 3D structure and variant pathogenicity, variant function and phenotype in SLC6A1-related disorders. This knowledge supports biology-informed variant interpretation and research on GAT1 function. All our data can be interactively explored in the SLC6A1 portal (https://slc6a1-portal.broadinstitute.org/).

The clinical and molecular spectrum of QRICH1 associated neurodevelopmental disorder.

De novo variants in QRICH1 (Glutamine-rich protein 1) has recently been reported in 11 individuals with intellectual disability (ID). The function of QRICH1 is largely unknown but it is likely to play a key role in the unfolded response of endoplasmic reticulum stress through transcriptional control of proteostasis. In this study, we present 27 additional individuals and delineate the clinical and molecular spectrum of the individuals (n = 38) with QRICH1 variants. The main clinical features were mild to moderate developmental delay/ID (71%), nonspecific facial dysmorphism (92%) and hypotonia (39%). Additional findings included poor weight gain (29%), short stature (29%), autism spectrum disorder (29%), seizures (24%) and scoliosis (18%). Minor structural brain abnormalities were reported in 52% of the individuals with brain imaging. Truncating or splice variants were found in 28 individuals and 10 had missense variants. Four variants were inherited from mildly affected parents. This study confirms that heterozygous QRICH1 variants cause a neurodevelopmental disorder including short stature and expands the phenotypic spectrum to include poor weight gain, scoliosis, hypotonia, minor structural brain anomalies, and seizures. Inherited variants from mildly affected parents are reported for the first time, suggesting variable expressivity.

Characterization of SETD1A haploinsufficiency in humans and Drosophila defines a novel neurodevelopmental syndrome.

Defects in histone methyltransferases (HMTs) are major contributing factors in neurodevelopmental disorders (NDDs). Heterozygous variants of SETD1A involved in histone H3 lysine 4 (H3K4) methylation were previously identified in individuals with schizophrenia. Here, we define the clinical features of the Mendelian syndrome associated with haploinsufficiency of SETD1A by investigating 15 predominantly pediatric individuals who all have de novo SETD1A variants. These individuals present with a core set of symptoms comprising global developmental delay and/or intellectual disability, subtle facial dysmorphisms, behavioral and psychiatric problems. We examined cellular phenotypes in three patient-derived lymphoblastoid cell lines with three variants: p.Gly535Alafs*12, c.4582-2_4582delAG, and p.Tyr1499Asp. These patient cell lines displayed DNA damage repair defects that were comparable to previously observed RNAi-mediated depletion of SETD1A. This suggested that these variants, including the p.Tyr1499Asp in the catalytic SET domain, behave as loss-of-function (LoF) alleles. Previous studies demonstrated a role for SETD1A in cell cycle control and differentiation. However, individuals with SETD1A variants do not show major structural brain defects or severe microcephaly, suggesting that defective proliferation and differentiation of neural progenitors is unlikely the single underlying cause of the disorder. We show here that the Drosophila melanogaster SETD1A orthologue is required in postmitotic neurons of the fly brain for normal memory, suggesting a role in post development neuronal function. Together, this study defines a neurodevelopmental disorder caused by dominant de novo LoF variants in SETD1A and further supports a role for H3K4 methyltransferases in the regulation of neuronal processes underlying normal cognitive functioning.

Heterozygous variants in PRPF8 are associated with neurodevelopmental disorders.

The pre-mRNA-processing factor 8, encoded by PRPF8, is a scaffolding component of a spliceosome complex involved in the removal of introns from mRNA precursors. Previously, heterozygous pathogenic variants in PRPF8 have been associated with autosomal dominant retinitis pigmentosa. More recently, PRPF8 was suggested as a candidate gene for autism spectrum disorder due to the enrichment of sequence variants in this gene in individuals with neurodevelopmental disorders. We report 14 individuals with various forms of neurodevelopmental conditions, found to have heterozygous, predominantly de novo, missense, and loss-of-function variants in PRPF8. These individuals have clinical features that may represent a new neurodevelopmental syndrome.

Variants in GNAI1 cause a syndrome associated with variable features including developmental delay, seizures, and hypotonia.

PURPOSE: Neurodevelopmental disorders (NDDs) encompass a spectrum of genetically heterogeneous disorders with features that commonly include developmental delay, intellectual disability, and autism spectrum disorders. We sought to delineate the molecular and phenotypic spectrum of a novel neurodevelopmental disorder caused by variants in the GNAI1 gene. METHODS: Through large cohort trio-based exome sequencing and international data-sharing, we identified 24 unrelated individuals with NDD phenotypes and a variant in GNAI1, which encodes the inhibitory Gαi1 subunit of heterotrimeric G-proteins. We collected detailed genotype and phenotype information for each affected individual. RESULTS: We identified 16 unique variants in GNAI1 in 24 affected individuals; 23 occurred de novo and 1 was inherited from a mosaic parent. Most affected individuals have a severe neurodevelopmental disorder. Core features include global developmental delay, intellectual disability, hypotonia, and epilepsy. CONCLUSION: This collaboration establishes GNAI1 variants as a cause of NDDs. GNAI1-related NDD is most often characterized by severe to profound delays, hypotonia, epilepsy that ranges from self-limiting to intractable, behavior problems, and variable mild dysmorphic features.

Challenging the Norm: Is Routine Use of Cranial CT in Evaluation of Craniosynostosis Necessary?

BACKGROUND: Cranial CT is routinely taught to be the gold standard for diagnosis of craniosynostosis and used by craniofacial teams for suspected nonsyndromic single suture craniosynostosis. Given the risks associated with infant CTs, do these scans provide significantly enhanced diagnostic accuracy compared to the physical exam when performed by an experienced clinical provider? METHOD: A retrospective chart review was performed for children who underwent corrective surgery for nonsyndromic, single-suture craniosynostosis over an 11 year period by a single craniofacial team. Ages at presentation and surgery, preoperative clinical diagnosis and imaging, co-existing radiographic findings, and correlation with the intraoperative diagnosis were analyzed. RESULTS: A total of 138 patients were included in this study. The mean age was 4.2 months at initial craniofacial evaluation, and 8.0 months at time of surgery. Twenty-seven patients received imaging prior to our clinic. Of those, 21 had plain radiography and 6 had CT scans. Of the remaining 111 patients referred without imaging, craniosynostosis was clinically diagnosed in 102 (92%), whereas 9 (8%) had an unclear clinical diagnosis. Of these 9, 1 (1%) was diagnosed clinically at follow-up exam, and the remaining 8 (7%) were diagnosed using radiography (3 CT scans, 5 plain radiographs). In all patients, the preoperative diagnosis was confirmed during intraoperative assessment. CONCLUSIONS: Cranial CT was not needed by experienced craniofacial providers in 93% of nonsyndromic, single-suture craniosynostosis. Imaging obtained before craniofacial clinic referral may have been unnecessary. These findings question the classic teaching that preoperative cranial CT is the gold standard for diagnosis in infants with nonsyndromic, single-suture craniosynostosis.

Rabconnectin-3a regulates vesicle endocytosis and canonical Wnt signaling in zebrafish neural crest migration.

Cell migration requires dynamic regulation of cell-cell signaling and cell adhesion. Both of these processes involve endocytosis, lysosomal degradation, and recycling of ligand-receptor complexes and cell adhesion molecules from the plasma membrane. Neural crest (NC) cells in vertebrates are highly migratory cells, which undergo an epithelial-mesenchymal transition (EMT) to leave the neural epithelium and migrate throughout the body to give rise to many different derivatives. Here we show that the v-ATPase interacting protein, Rabconnectin-3a (Rbc3a), controls intracellular trafficking events and Wnt signaling during NC migration. In zebrafish embryos deficient in Rbc3a, or its associated v-ATPase subunit Atp6v0a1, many NC cells fail to migrate and misregulate expression of cadherins. Surprisingly, endosomes in Rbc3a- and Atp6v0a1-deficient NC cells remain immature but still acidify. Rbc3a loss-of-function initially downregulates several canonical Wnt targets involved in EMT, but later Frizzled-7 accumulates at NC cell membranes, and nuclear B-catenin levels increase. Presumably due to this later Wnt signaling increase, Rbc3a-deficient NC cells that fail to migrate become pigment progenitors. We propose that Rbc3a and Atp6v0a1 promote endosomal maturation to coordinate Wnt signaling and intracellular trafficking of Wnt receptors and cadherins required for NC migration and cell fate determination. Our results suggest that different v-ATPases and associated proteins may play cell-type-specific functions in intracellular trafficking in many contexts.

Robust regeneration of adult zebrafish lateral line hair cells reflects continued precursor pool maintenance.

We have examined lateral line hair cell and support cell maintenance in adult zebrafish when growth is largely complete. We demonstrate that adult zebrafish not only replenish hair cells after a single instance of hair cell damage, but also maintain hair cells and support cells after multiple rounds of damage and regeneration. We find that hair cells undergo continuous turnover in adult zebrafish in the absence of damage. We identify mitotically-distinct support cell populations and show that hair cells regenerate from underlying support cells in a region-specific manner. Our results demonstrate that there are two distinct support cell populations in the lateral line, which may help explain why zebrafish hair cell regeneration is extremely robust, retained throughout life, and potentially unlimited in regenerative capacity.

Loss of symmetric cell division of apical neural progenitors drives DENND5A-related developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEEs) feature altered brain development, developmental delay and seizures, with seizures exacerbating developmental delay. Here we identify a cohort with biallelic variants in DENND5A, encoding a membrane trafficking protein, and develop animal models with phenotypes like the human syndrome. We demonstrate that DENND5A interacts with Pals1/MUPP1, components of the Crumbs apical polarity complex required for symmetrical division of neural progenitor cells. Human induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division with an inherent propensity to differentiate into neurons. These phenotypes result from misalignment of the mitotic spindle in apical neural progenitors. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles, biasing daughter cells towards a more fate-committed state, ultimately shortening the period of neurogenesis. This study provides a mechanism for DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families.

Loss of symmetric cell division of apical neural progenitors drives DENND5A-related developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEEs) are a heterogenous group of epilepsies in which altered brain development leads to developmental delay and seizures, with the epileptic activity further negatively impacting neurodevelopment. Identifying the underlying cause of DEEs is essential for progress toward precision therapies. Here we describe a group of individuals with biallelic variants in DENND5A and determine that variant type is correlated with disease severity. We demonstrate that DENND5A interacts with MUPP1 and PALS1, components of the Crumbs apical polarity complex, which is required for both neural progenitor cell identity and the ability of these stem cells to divide symmetrically. Induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division during neural induction and have an inherent propensity to differentiate into neurons, and transgenic DENND5A mice, with phenotypes like the human syndrome, have an increased number of neurons in the adult subventricular zone. Disruption of symmetric cell division following loss of DENND5A results from misalignment of the mitotic spindle in apical neural progenitors. A subset of DENND5A is localized to centrosomes, which define the spindle poles during mitosis. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles, biasing daughter cells towards a more fate-committed state and ultimately shortening the period of neurogenesis. This study provides a mechanism behind DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families.

Next-generation universal hereditary cancer screening: implementation of an automated hereditary cancer screening program for patients with advanced cancer undergoing tumor sequencing in a large HMO.

Variants in hereditary cancer risk genes are frequently identified following tumor-based DNA sequencing and represent an opportunity to diagnose hereditary cancer. We implemented an automated hereditary cancer screening program in a large HMO for all patients who underwent tumor-based DNA sequencing to identify patients with hereditary cancer and determine if this approach augmented existing genetic counseling approaches driven by personal/family history criteria. Regular automated searches of a centralized tumor DNA variant database were performed for ATM, BRCA1, BRCA2, MLH1, MSH2, MSH6, PALB2, and/or PMS2 variants, and germline hereditary cancer gene panel testing was offered to patients with tumor variants who had never undergone germline testing. Patients completing germline testing due to their tumor DNA test results were considered part of the tumor DNA safety net. Patients previously completing germline testing via traditional genetic counseling and tumor DNA safety net were compared for demographics, tumor type, presence of germline pathogenic/likely pathogenic (P/LP) variant, and whether NCCN criteria were met for hereditary cancer genetic testing. Germline P/LP variants were common in both groups. Patients who received germline testing through traditional genetic counseling were more likely to have cardinal hereditary tumors than the tumor DNA safety net group. Patients identified with hereditary cancer through traditional genetic counseling were more likely to meet NCCN personal/family history criteria for germline testing than the tumor DNA safety net group (99% versus 34%). A universal tumor DNA safety net screen is an important diagnostic strategy which augments traditional genetic counseling approaches based on personal/family history.

Brain magnetic resonance imaging findings in 49,XXXXY syndrome.

Klinefelter syndrome is a chromosomal disorder characterized by one or more supernumerary X chromosomes, in addition to the normal 46,XY male karyotype. Whereas classic Klinefelter syndrome (47,XXY) occurs in 1:400 births, the most severe Klinefelter variant (49,XXXXY) occurs in only 1:85,000 births. The degree of cognitive impairment, specific skeletal changes, and genital abnormalities in Klinefelter syndrome variants is thought to correlate with the number of additional X-chromosomes present. Magnetic resonance imaging studies in individuals with classic Klinefelter syndrome show smaller brain volumes, but magnetic resonance imaging data are lacking for individuals with rarer and more severe Klinefelter variants. We present case reports and magnetic resonance imaging studies on 3 individuals with 49,XXXXY. All 3 patients exhibited varying degrees of volume loss and abnormalities in white matter. Changes in white matter may represent a specific finding in patients with severe Klinefelter variants such as 49,XXXXY, and karyotype analysis should be considered in patients with unexplained white-matter disease, especially when developmental delay or genital abnormalities are present.

Genetics of craniosynostosis.

Craniosynostosis is a defect of the skull caused by early fusion of one or more of the cranial sutures and affects 3 to 5 individuals per 10,000 live births. Craniosynostosis can be divided into two main groups: syndromic and nonsyndromic. Nonsyndromic craniosynostosis is typically an isolated finding that is classified according to the suture(s) involved. Syndromic craniosynostosis is associated with various dysmorphisms involving the face, skeleton, nervous system, and other anomalies and is usually accompanied by developmental delay. More than 180 syndromes exist that contain craniosynostosis. Secondary effects of craniosynostosis may include vision problems and increased intracranial pressure, among others. The molecular basis of many types of syndromic craniosynostosis is known, and diagnostic testing strategies will often lead to a specific diagnosis.

Tfap2 transcription factors in zebrafish neural crest development and ectodermal evolution.

Transcription factor AP2 (Tfap2) genes play essential roles in development of the epidermis and migratory cells of the neural crest (NC) in vertebrate embryos. These transcriptional activators are among the earliest genes expressed in the ectoderm and specify fates within the epidermis/crest through both direct and indirect mechanisms. The Tfap2 family arose from a single ancestral gene in a chordate ancestor that underwent gene duplication to give up to five family members in living vertebrates. This coincided with the acquisition of important roles in NC development by Tfap2 genes suggesting that this gene family was important in ectodermal evolution and possibly in the origin of NC. Here, we show that a zebrafish tfap2c is expressed in the nonneural ectoderm during early development and functions redundantly with tfap2a in NC specification. In zebrafish embryos depleted of both tfap2a and tfap2c, NC cells are virtually eliminated. Cell transplantation experiments indicate that tfap2c functions cell-autonomously in NC specification. Cells of the enveloping layer, which forms a temporary skin layer surrounding the ectoderm, also fail to differentiate or to express appropriate keratins in tfap2c deficient embryos. The role of Tfap2 genes in epidermal and NC development is considered here in the broader context of ectodermal evolution. Distinct, tissue-specific functions for Tfap2 genes in different vertebrates may reflect subfunctionalisation of an ancestral gene that consequently led to the gain of novel roles for different subfamily members in patterning the epidermis and NC.

Inca: a novel p21-activated kinase-associated protein required for cranial neural crest development.

Inca (induced in neural crest by AP2) is a novel protein discovered in a microarray screen for genes that are upregulated in Xenopus embryos by the transcriptional activator protein Tfap2a. It has no significant similarity to any known protein, but is conserved among vertebrates. In Xenopus, zebrafish and mouse embryos, Inca is expressed predominantly in the premigratory and migrating neural crest (NC). Knockdown experiments in frog and fish using antisense morpholinos reveal essential functions for Inca in a subset of NC cells that form craniofacial cartilage. Cells lacking Inca migrate successfully but fail to condense into skeletal primordia. Overexpression of Inca disrupts cortical actin and prevents formation of actin "purse strings", which are required for wound healing in Xenopus embryos. We show that Inca physically interacts with p21-activated kinase 5 (PAK5), a known regulator of the actin cytoskeleton that is co-expressed with Inca in embryonic ectoderm, including in the NC. These results suggest that Inca and PAK5 cooperate in restructuring cytoskeletal organization and in the regulation of cell adhesion in the early embryo and in NC cells during craniofacial development.

Biotinidase deficiency: the importance of adequate follow-up for an inconclusive newborn screening result.

UNLABELLED: Biotinidase deficiency is an inherited metabolic disorder characterized by inability to recycle protein-bound biotin. It usually presents with ataxia and seizures, though atypical presentations have also been described. We report a 15-month-old boy with profound biotinidase deficiency who presented with laryngeal stridor and subsequently developed severe ataxia and lactic acidosis. Subsequently, it was discovered that the patient's newborn screening test for biotinidase activity had been inconclusive, but confirmatory testing had not been done. Brain magnetic resonance imaging showed multiple white matter non-enhancing T2 hyperintensities, which largely resolved following 6 months of biotin therapy; however, there was residual deafness and mental retardation. CONCLUSION: An argument is made for universal newborn screening in biotinidase deficiency and improved mechanisms for follow-up of positive screens, because delay in diagnosis results in irreversible morbidity, newborn screening is cost effective, and early therapy prevents the neurologic sequelae.