Biology subjects

Rosenfeld, J. A.

Publications and source records attributed to Rosenfeld, J. A..

18 recordsLinked to original sources

bioRxiv2026

ARID5B mutations cause a neurodevelopmental syndrome with neuroinflammation episodes

Genetic disorders affecting the epigenetic machinery constitute a major group of neurodevelopmental conditions. Pathogenic variants in several ARID transcription factors--particularly ARID1A, ARID1B, and ARID2--cause Coffin-Siris syndromes, all characterized by intellectual disability (ID). These genes encode core subunits of the BRG1/BRM-associated factor (BAF) chromatin remodeling complex. In contrast, ARID family members that function in other regulatory complexes have remained largely unexplored in neurodevelopmental disease. Here, we identify 29 individuals carrying heterozygous ARID5B variants, of which 24 (83%) introduce premature termination codons in the exceptionally long final exon, one affects the exon 9 splice donor site, and four are missense variants in conserved domains within the N-terminal half of the protein. Using a CRISPR-Cas9 knock-in mouse model harboring the p.Q522Ter variant, together with in vitro assays, we investigated the functional consequences of C-terminal ARID5B truncations. All affected individuals presented with global developmental delay or ID--most commonly mild--and frequent speech and language impairment. Recurrent features included kidney malformations, behavioral difficulties, and recurrent infections of the respiratory and urinary tracts. Two individuals experienced central nervous system inflammation, and two infants presented with persistent pulmonary hypertension. Remarkably, 19 of 29 variants (66%) cluster within the first quarter of exon 10, are de novo, and escape nonsense-mediated mRNA decay (NMD), which we confirmed for two variants affecting seven individuals. Variants outside this region were inherited. Heterozygous mice exhibited developmental and behavioral abnormalities, while homozygous mutations was perinatally lethal. Truncations and a small deletion within a predicted nuclear localization signal (NLS) caused cytosolic mislocalization of ARID5B, whereas the isolated C-terminal half retained nuclear localization, suggesting an independent distal NLS. Collectively, these findings define ARID5B-related neurodevelopmental disorder as a distinct clinical entity and reveal how disruption of specific ARID5B domains impacts protein localization, mammalian development, immune and neurobehavioral function. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/698931v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@158e8d9org.highwire.dtl.DTLVardef@1f014dorg.highwire.dtl.DTLVardef@18e6e7forg.highwire.dtl.DTLVardef@1f886e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2024

Loss of function in RBBP5 results in a syndromic neurodevelopmental disorder associated with microcephaly

PurposeEpigenetic dysregulation has been associated with many inherited disorders. RBBP5 encodes a core member of the protein complex that methylates histone 3 lysine-4 (H3K4) and has not been implicated in human disease. MethodsWe identify five unrelated individuals with de novo heterozygous pathogenic variants in RBBP5. Three truncating and two missense variants were identified in probands with neurodevelopmental symptoms including global developmental delay, intellectual disability, microcephaly, and short stature. Here, we investigate the pathogenicity of the variants through protein structural analysis and transgenic Drosophila models. ResultsBoth missense p.T232I and p.E296D variants affect evolutionarily conserved amino acids and are expected to interfere with the interface between RBBP5 and the histones. In Drosophila, ubiquitous overexpression of human RBBP5 is lethal in the larval developmental stage. Loss of Rbbp5 leads to a reduction in brain size, and the human reference, p.T232I, or p.E296D variant transgenes fail to rescue loss of Rbbp5. Expression of either missense variant in an Rbbp5 null background results in a less severe microcephaly phenotype than the human reference, indicating both p.T232I and p.E296D variants are loss-of-function alleles. ConclusionDe novo heterozygous variants in RBBP5 are associated with a syndromic neurodevelopmental disorder. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/578086v1_ufig1.gif" ALT="Figure 1"> O_LINKSMALLFIG WIDTH=200 HEIGHT=24 SRC="FIGDIR/small/578086v1_ufig1a.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@11337daorg.highwire.dtl.DTLVardef@b7f4d8org.highwire.dtl.DTLVardef@12bc283org.highwire.dtl.DTLVardef@1ef4e09_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2024

Behavioral screening of conserved RNA-binding proteins reveals CEY-1/YBX RNA-binding protein dysfunction leads to impairments in memory and cognition

RNA-binding proteins (RBPs) regulate translation and plasticity which are required for memory. RBP dysfunction has been linked to a range of neurological disorders where cognitive impairments are a key symptom. However, of the 2,000 RBPs in the human genome, many are uncharacterized with regards to neurological phenotypes. To address this, we used the model organism C. elegans to assess the role of 20 conserved RBPs in memory. We identified eight previously uncharacterized memory regulators, three of which are in the C. elegans Y-Box (CEY) RBP family. Of these, we determined that cey-1 is the closest ortholog to the mammalian Y-Box (YBX) RBPs. We found that CEY-1 is both necessary in the nervous system for memory ability and sufficient to increase memory. Leveraging human datasets, we found both copy number variation losses and single nucleotide variants in YBX1 and YBX3 in individuals with neurological symptoms. We identified one predicted deleterious YBX3 variant of unknown significance, p.Asn127Tyr, in two individuals with neurological symptoms. Introducing this variant into endogenous cey-1 locus caused memory deficits in the worm. We further generated two humanized worm lines expressing human YBX3 or YBX1 at the cey-1 locus to test evolutionary conservation of YBXs in memory and the potential functional significance of the p.Asn127Tyr variant. Both YBX1/3 can functionally replace cey-1, and introduction of p.Asn127Tyr into the humanized YBX3 locus caused memory deficits. Our study highlights the worm as a model to reveal memory regulators and identifies YBX dysfunction as a potential new source of rare neurological disease. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=66 SRC="FIGDIR/small/574402v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@c1e04forg.highwire.dtl.DTLVardef@6d82b9org.highwire.dtl.DTLVardef@1a15571org.highwire.dtl.DTLVardef@f0ad20_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Differential effects of MATR3 variants on its cryptic splicing repression function

MATR3 is an RNA-binding protein implicated in neurodegenerative and neurodevelopmental diseases. However, little is known regarding the role of MATR3 in cryptic splicing within the context of functional genes and how disease-associated variants impact this function. We show that loss of MATR3 leads to cryptic exon inclusion in many transcripts. We reveal that ALS-linked S85C pathogenic variant reduces MATR3 solubility but does not impair RNA binding. In parallel, we report a novel neurodevelopmental disease-associated M548T variant, located in the RRM2 domain, which reduces protein solubility and impairs RNA binding and cryptic splicing repression functions of MATR3. Altogether, our research identifies cryptic events within functional genes and demonstrates how disease-associated variants impact MATR3 cryptic splicing repression function.

molecular biology↗

bioRxiv2022

Functional analysis of a novel de novo variant in PPP5C associated with microcephaly, seizures, and developmental delay

We describe a proband evaluated through the Undiagnosed Diseases Network (UDN) who presented with microcephaly, developmental delay, and refractory epilepsy with a de novo p.Ala47Thr missense variant in the protein phosphatase gene, PPP5C. This gene has not previously been associated with a Mendelian disease, and based on the population database, gnomAD, the gene has a low tolerance for loss-of-function variants (pLI=1, o/e=0.07). We functionally evaluated the PPP5C variant in C. elegans by knocking the variant into the orthologous gene, pph-5, at the corresponding residue, Ala48Thr. We employed assays in three different biological processes where pph-5 was known to function through opposing the activity of genes, mec-15 and sep-1. We demonstrated that, in contrast to control animals, the pph-5 Ala48Thr variant suppresses the neurite growth phenotype and the GABA signaling defects of mec-15 mutants, and the embryonic lethality of sep-1 mutants. The Ala48Thr variant did not display dominance and behaved similarly to the reference pph-5 null, indicating that the variant is likely a strong hypomorph or complete loss-of-function. We conclude that pph-5 Ala48Thr is damaging in C. elegans. By extension in the proband, PPP5C p.Ala47Thr is likely damaging, the de novo dominant presentation is consistent with haplo-insufficiency, and the PPP5C variant is likely responsible for one or more of the probands phenotypes.

genetics↗

bioRxiv2021

Drosophila functional screening of de novo variants in autism uncovers deleterious variants and facilitates discovery of rare neurodevelopmental diseases

Individuals with autism spectrum disorders (ASD) exhibit an increased burden of de novo variants in a broadening range of genes. We functionally tested the effects of ASD missense variants using Drosophila through humanization rescue and overexpression-based strategies. We studied 79 ASD variants in 74 genes identified in the Simons Simplex Collection and found 38% of them caused functional alterations. Moreover, we identified GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in eight previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes point to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.

genetics↗

bioRxiv2020

COPB2 haploinsufficiency causes a coatopathy with osteoporosis and developmental delay

Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures and developmental delay of variable severity. Because the role of COPB2 in bone has not been characterized, we studied the effect of COPB2 deficiency on skeletal development in mice and zebrafish. Copb2+/- mice showed low bone mass and decreased bone strength. In zebrafish, larvae carrying a copb2 heterozygous frameshift variant showed delayed mineralization. copb2-null embryos showed endoplasmic reticulum (ER) and Golgi disorganization, and embryonic lethality. COPB2 siRNA-treated fibroblasts showed delayed collagen trafficking with retention of type I collagen in the ER and Golgi, and altered distribution of Golgi markers. Our data suggest that COPB2 haploinsufficiency leads to disruption of intracellular collagen trafficking and osteoporosis, which may improve with ascorbic acid supplementation. This work highlights the role of COPI complex as a critical regulator of bone mass and identifies a new form of coatopathy due to COPB2 deficiency.

genetics↗

bioRxiv2020

Benchmarking challenging small variants with linked and long reads

Genome in a Bottle (GIAB) benchmarks have been widely used to help validate clinical sequencing pipelines and develop new variant calling and sequencing methods. Here, we use accurate linked reads and long reads to expand the prior benchmarks in 7 samples to include difficult-to-map regions and segmental duplications that are not readily accessible to short reads. Our new benchmark adds more than 300,000 SNVs, 50,000 indels, and 16 % new exonic variants, many in challenging, clinically relevant genes not previously covered (e.g., PMS2). For HG002, we include 92% of the autosomal GRCh38 assembly, while excluding problematic regions for benchmarking small variants (e.g., copy number variants and reference errors) that should not have been in the previous version, which included 85% of GRCh38. By including difficult-to-map regions, this benchmark identifies eight times more false negatives in a short read variant call set relative to our previous benchmark.We have demonstrated the utility of this benchmark to reliably identify false positives and false negatives across technologies in more challenging regions, which enables continued technology and bioinformatics development.

genomics↗

bioRxiv2020

Cell-based analysis of CAD variants identifies individuals likely to benefit from uridine therapy

PurposePathogenic autosomal recessive variants in CAD, encoding the multienzymatic protein initiating pyrimidine de novo biosynthesis, cause a severe inborn metabolic disorder treatable with a dietary supplement of uridine. This condition is difficult to diagnose given the large size of CAD with over 1000 missense variants and the non-specific clinical presentation. We aimed to develop a reliable and discerning assay to assess the pathogenicity of CAD variants and to select affected individuals that might benefit from uridine therapy. MethodsUsing CRISPR/Cas9, we generated a human CAD-knockout cell line that requires uridine supplements for survival. Transient transfection of the knockout cells with recombinant CAD restores growth in absence of uridine. This system determines missense variants that inactivate CAD and do not rescue the growth phenotype. ResultsWe identified 25 individuals with biallelic variants in CAD and a phenotype consistent with a CAD deficit. We used the CAD-knockout complementation assay to test a total of 34 variants, identifying 16 as deleterious for CAD activity. Combination of these pathogenic variants confirmed 11 subjects with a CAD deficit, for whom we describe the clinical phenotype. ConclusionsWe designed a cell-based assay to test the pathogenicity of CAD variants, identifying 11 CAD deficient individuals, who could benefit from uridine therapy.

genetics↗

bioRxiv2019

Whole Genome Sequencing and Assembly of the Asian Honey Bee Apis dorsata

The Asian honey bee (Apis dorsata) is distinct from its more widely distributed cousin A. mellifera by a few key characteristics. Most prominently, A. dorsata, nest in the open by forming a colony clustered around the honeycomb, while A. mellifera nest in concealed cavities. Additionally, the worker and reproductive castes are all of the same size in A. dorsata. In order to investigate these differences, we performed whole genome sequencing of A. dorsata using a hybrid Oxford Nanopore and Illumina approach. The 223MB genome has an N50 of 35kb with the largest scaffold of 302kb. We have found that there are many genes in the dorsata genome that are distinct from other hymenoptera and also large amounts of transposable elements, and we suggest some candidate genes for A. dorsatas exceptional level of defensive aggression.

genomics↗

bioRxiv2019

De novo EIF2AK1 and EIF2AK2 variants are associated with developmental delay, leukoencephalopathy, and neurologic decompensation

EIF2AK1 and EIF2AK2 encode members of the Eukaryotic Translation Initiation Factor 2 Alpha Kinase (EIF2AK) family that inhibits protein synthesis in response to physiologic stress conditions. EIF2AK2 is also involved in innate immune response and the regulation of signal transduction, apoptosis, cell proliferation, and differentiation. Despite these findings, human disorders associated with deleterious variants in EIF2AK1 and EIF2AK2 have not been reported. Here, we describe the identification of eight unrelated individuals with heterozygous de novo missense variants in EIF2AK1 (1/8) or EIF2AK2 (7/8). Features seen in these eight individuals include white matter alterations (8/8), developmental delay (8/8), impaired language (8/8), cognitive impairment (7/8), ataxia (6/8), dysarthria in probands with verbal ability (6/6), hypotonia (6/8), hypertonia (5/8), and involuntary movements (3/8). Individuals with EIF2AK2 variants also exhibit neurological regression in the setting of febrile illness or infection. We use mammalian cell lines and patient-derived fibroblasts to further confirm the pathogenicity of variants in these genes and found reduced kinase activity. EIF2AKs phosphorylate Eukaryotic Translation Initiation Factor 2 Subunit 1, (EIF2S1, also known as EIF2), which then inhibits EIF2B activity. Deleterious variants in genes encoding EIF2B proteins cause childhood ataxia with central nervous system hypomyelination/vanishing white matter disease (CACH/VWM), a leukoencephalopathy characterized by neurologic regression in the setting of febrile illness and other stressors. Our findings indicate that EIF2AK2 missense variants cause a neurodevelopmental syndrome that may share phenotypic and pathogenic mechanisms with CACH/VWM.

genetics↗

bioRxiv2019

Single-Cell, Human Sperm Transcriptomes and Variants from Fathers of Autistic and Healthy Children

The human sperm is one of the smallest cells in the body, but also one of the most important, as it serves as the entire paternal genetic contribution to a child. This is especially relevant for diseases such as Autism Spectrum Disorders (ASD), which have been correlated with advance paternal age. Historically, most studies of sperm have focused on the assessment of a bulk sperm, wherein millions of individual sperm are present and only high-frequency variants can be detected. Using 10X Chromium single cell sequencing technology, we have assessed the RNA from >65,000 single sperm cells across 6 donors (scsperm-RNA-seq), including two of whom have autistic children and four that do not. Using multiple RNA-seq methods for differential expression and variant analysis, we found clusters of sperm mutations in each donor that are indicative of the sperm being produced by different stem cell pools. Moreover, by comparing the two groups, we have found expression changes that can separate out the two sets of donors. Finally, through our novel variant calling from single-cell RNA-seq methods, we have shown that we can detect mutation rates in sperm from ASD donors that is distinct from the controls, highlighting this method as a new means to characterize ASD risk.

genomics↗

bioRxiv2019

DYRK1A-related intellectual disability: a syndrome associated with congenital anomalies of the kidney and urinary tract

PurposeHaploinsufficiency of DYRK1A causes a recognizable clinical syndrome. The goal of this paper is to investigate congenital anomalies of the kidney and urinary tract (CAKUT) and genital defects (GD) in patients with DYRK1A mutations.\n\nMethodsA large database of clinical exome sequencing (ES) was queried for de novo DYRK1A mutations and CAKUT/GD phenotypes were characterized. Xenopus laevis (frog) was chosen as a model organism to assess Dyrk1as role in renal development.\n\nResultsPhenotypic details and mutations of 19 patients were compiled after an initial observation that one patient with a de novo pathogenic mutation in DYRK1A had GD. CAKUT/GD data were available from 15 patients, 11 of whom present with CAKUT/GD. Studies in Xenopus embryos demonstrate that knockdown of Dyrk1a disrupts the development of segments of developing embryonic nephrons, which ultimately give rise to the entire genitourinary (GU) tract. These defects could be rescued by co-injecting wildtype human DYRK1A RNA, but not with truncated DYRK1AR205* RNA.\n\nConclusionEvidence supports routine GU screening of all individuals with de novo DYRK1A pathogenic variants to ensure optimized clinical management. Collectively, the reported clinical data and loss of function studies in Xenopus substantiate a novel role for DYRK1A in GU development.

genetics↗

bioRxiv2018

Evidence for secondary-variant genetic burden and non-random distribution across biological modules in a recessive ciliopathy

The influence of genetic background on driver mutations is well established; however, the mechanisms by which the background interacts with Mendelian loci remains unclear. We performed a systematic secondary-variant burden analysis of two independent Bardet-Biedl syndrome (BBS) cohorts with known recessive biallelic pathogenic mutations in one of 17 BBS genes for each individual. We observed a significant enrichment of trans-acting rare nonsynonymous secondary variants compared to either population controls or to a cohort of individuals with a non-BBS diagnosis and recessive variants in the same gene set. Strikingly, we found a significant over-representation of secondary alleles in chaperonin-encoding genes, a finding corroborated by the observation of epistatic interactions involving this complex in vivo. These data indicate a complex genetic architecture for BBS that informs the biological properties of disease modules and presents a model paradigm for secondary-variant burden analysis in recessive disorders.

genetics↗

bioRxiv2018

Loss-of-function in IRF2BPL is associated with neurological phenotypes

The Interferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) gene encodes a member of the IRF2BP family of transcriptional regulators. Currently the biological function of this gene is obscure, and the gene has not been associated with a Mendelian disease. Here we describe seven individuals affected with neurological symptoms who carry damaging heterozygous variants in IRF2BPL. Five cases carrying nonsense variants in IRF2BPL resulting in a premature stop codon display severe neurodevelopmental regression, hypotonia, progressive ataxia, seizures, and a lack of coordination. Two additional individuals, both with missense variants, display global developmental delay and seizures and a relatively milder phenotype than those with nonsense alleles. The bioinformatics signature for IRF2BPL based on population genomics is consistent with a gene that is intolerant to variation. We show that the IRF2BPL ortholog in the fruit fly, called pits (protein interacting with Ttk69 and Sin3A), is broadly expressed including the nervous system. Complete loss of pits is lethal early in development, whereas partial knock-down with RNA interference in neurons leads to neurodegeneration, revealing requirement for this gene in proper neuronal function and maintenance. The nonsense variants in IRF2BPL identified in patients behave as severe loss-of-function alleles in this model organism, while ectopic expression of the missense variants leads to a range of phenotypes. Taken together, IRF2BPL and pits are required in the nervous system in humans and flies, and their loss leads to a range of neurological phenotypes in both species.

genetics↗

bioRxiv2018

Microdeletions excluding YWHAE and PAFAH1B1 cause a unique leukoencephalopathy and hypermobility syndrome: Further delineation of the 17p13.3 microdeletion spectrum

PurposeBrain malformations are caused by 17p13.3 deletions: lissencephaly with deletions of the larger Miller-Dieker syndrome region or smaller deletions of only PAFAH1B1, as well as various abnormalities, including white matter changes, in the distinct syndrome due to deletions including YWHAE and CRK but sparing PAFAH1B1. We sought to understand the significance of 17p13.3 deletions between the YWHAE/CRK and PAFAH1B1 loci.\n\nMethodsWe analyzed the clinical features of five individuals from four families with 17p13.3 deletions between and not including YWHAE/CRK and PAFAH1B1 identified among individuals undergoing clinical chromosomal microarray testing.\n\nResultsFour individuals from three families have multi-focal white matter lesions and hypermobile joints, while a fifth had a normal MRI. A combination of our patients and a review of those in the literature with white matter changes and deletions in this chromosomal region narrows the overlapping region for this brain phenotype to [~]345 kb, including 11 RefSeq genes, with RTN4RL1 haploinsufficiency as the best candidate for causing this.\n\nConclusionWhile previous literature has hypothesized dysmorphic features and white matter changes related to YWHAE, our cohort contributes evidence to the presence of additional genes within 17p13.3 required for proper brain development.

genetics↗

bioRxiv2018

Phenotypic expansion in DDX3X -- a common cause of intellectual disability in females

De novo variants in DDX3X account for 1-3% of unexplained intellectual disability (ID), one of the most common causes of ID, in females. Forty-seven patients (44 females, 3 males) have been described. We identified 29 additional individuals carrying 27 unique DDX3X variants in the setting of complex clinical presentations including developmental delay or ID. In addition to previously reported manifestations, rare or novel phenotypes were identified including respiratory problems, congenital heart disease, skeletal muscle mitochondrial DNA depletion, and late-onset neurologic decline. Our findings expand the spectrum of DNA variants and phenotypes associated with DDX3X disorders.

genetics↗

bioRxiv2018

Rare variants in the genetic background modulate the expressivity of neurodevelopmental disorders

PurposeTo assess the contribution of rare variants in the genetic background towards variability of neurodevelopmental phenotypes in individuals with rare copy-number variants (CNVs) and gene-disruptive mutations.\n\nMethodsWe analyzed quantitative clinical information, exome-sequencing, and microarray data from 757 probands and 233 parents and siblings who carry disease-associated mutations.\n\nResultsThe number of rare secondary mutations in functionally intolerant genes (second-hits) correlated with the expressivity of neurodevelopmental phenotypes in probands with 16p12.1 deletion (n=23, p=0.004) and in probands with autism carrying gene-disruptive mutations (n=184, p=0.03) compared to their carrier family members. Probands with 16p12.1 deletion and a strong family history presented more severe clinical features (p=0.04) and higher burden of second-hits compared to those with mild/no family history (p=0.001). The number of secondary variants also correlated with the severity of cognitive impairment in probands carrying pathogenic rare CNVs (n=53) or de novo mutations in disease genes (n=290), and negatively correlated with head size among 80 probands with 16p11.2 deletion. These second-hits involved known disease-associated genes such as SETD5, AUTS2, and NRXN1, and were enriched for genes affecting cellular and developmental processes.\n\nConclusionAccurate genetic diagnosis of complex disorders will require complete evaluation of the genetic background even after a candidate gene mutation is identified.

genomics↗