Resolution of SLC6A1 variable expressivity in a multi-generational family using deep clinical phenotyping and Drosophila models.

Purpose: Variants in SLC6A1 result in a rare neurodevelopmental disorder characterized by a variable clinical presentation of symptoms including developmental delay, epilepsy, motor dysfunction, and autism spectrum disorder. SLC6A1 haploinsufficiency has been confirmed as the predominant pathway of SLC6A1-related neurodevelopmental disorders (NDDs), however, the molecular mechanism underlying the variable clinical presentation remains unclear. Methods: Here, through work of the Undiagnosed Diseases Network, we identify an undiagnosed individual with an inherited p.(A334S) variant of uncertain significance. To resolve this case and better understand the variable expressivity with SLC6A1, we assess the phenotypes of the proband with a cohort of cases diagnosed with SLC6A1-related NDDs. We then create an allelic series in the Drosophila melanogaster to functionally characterize case variants. Results: We identify significant clinical overlap between the unsolved case and confirmed cases of SLC6A1-related NDDs and find a mild to severe clinical presentation associated with missense variants. We confirm phenotypes in flies expressing SLC6A1 variants consistent with a partial loss-of-function mechanism. Conclusion: We conclude that the p.(A334S) variant is a hypomorphic allele and begin to elucidate the underlying variability in SLC6A1-related NDDs. These insights will inform clinical diagnosis, prognosis, treatment and inform therapeutic design for those living with SLC6A1-related NDDs.

Genetic analysis of the X-linked Adrenoleukodystrophy ABCD1 gene in Drosophila uncovers a role in Peroxisomal dynamics.

X-linked adrenoleukodystrophy (X-ALD) is a progressive neurodegenerative disorder caused by a loss-of-function (LOF) mutation in the ATP-binding cassette subfamily D member 1 (ABCD1) gene, leading to the accumulation of very long-chain fatty acids (VLCFAs). This disorder exhibits striking heterogeneity; some male patients develop an early childhood neuroinflammatory demyelination disorder, while other patients, including adult males and most affected female carriers, experience a chronic progressive myelopathy. Adrenocortical failure is observed in almost all male patients, with age of onset varying sometimes being the first diagnostic finding. The gene underlying this spectrum of disease encodes an ATP-binding cassette (ABC) transporter that localizes to peroxisomes and facilitates VLCFA transport. X-ALD is considered a single peroxisomal component defect and does not play a direct role in peroxisome assembly. Drosophila models of other peroxisomal genes have provided mechanistic insight into some of the neurodegenerative mechanisms with reduced lifespan, retinal degeneration, and VLCFA accumulation. Here, we perform a genetic analysis of the fly ABCD1 ortholog Abcd1 (CG2316). Knockdown or deficiency of Abcd1 leads to VLCFA accumulation, salivary gland defects, locomotor impairment and retinal lipid abnormalities. Interestingly, there is also evidence of reduced peroxisomal numbers. Flies overexpressing the human cDNA for ABCD1 display a wing crumpling phenotype characteristic of the pex2 loss-of-function. Surprisingly, overexpression of human ABCD1 appears to inhibit or overwhelm peroxisomal biogenesis to levels similar to null mutations in fly pex2, pex16 and pex3. Drosophila Abcd1 is therefore implicated in peroxisomal number, and overexpression of the human ABCD1 gene acts a potent inhibitor of peroxisomal biogenesis in flies.

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

PURPOSE: Epigenetic dysregulation has been associated with many inherited disorders. RBBP5 (HGNC:9888) encodes a core member of the protein complex that methylates histone 3 lysine-4 and has not been implicated in human disease. METHODS: We identify 5 unrelated individuals with de novo heterozygous variants in RBBP5. Three nonsense/frameshift and 2 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. RESULTS: Both missense p.(T232I) and p.(E296D) variants affect evolutionarily conserved amino acids located at the interface between RBBP5 and the nucleosome. In Drosophila, overexpression analysis identifies partial loss-of-function mechanisms when the variants are expressed using the fly Rbbp5 or human RBBP5 cDNA. Loss of Rbbp5 leads to a reduction in brain size. The human reference or variant transgenes fail to rescue this loss and expression of either missense variant in an Rbbp5 null background results in a less severe microcephaly phenotype than the human reference, indicating both missense variants are partial loss-of-function alleles. CONCLUSION: Haploinsufficiency of RBBP5 observed through de novo null and hypomorphic loss-of-function variants is associated with a syndromic neurodevelopmental disorder.

Dicarboxylic acylcarnitine biomarkers in peroxisome biogenesis disorders.

The peroxisome is an essential eukaryotic organelle with diverse metabolic functions. Inherited peroxisomal disorders are associated with a wide spectrum of clinical outcomes and are broadly divided into two classes, those impacting peroxisome biogenesis (PBD) and those impacting specific peroxisomal factors. Prior studies have indicated a role for acylcarnitine testing in the diagnosis of some peroxisomal diseases through the detection of long chain dicarboxylic acylcarnitine abnormalities (C16-DC and C18-DC). However, there remains limited independent corroboration of these initial findings and acylcarnitine testing for peroxisomal diseases has not been widely adopted in clinical laboratories. To explore the utility of acylcarnitine testing in the diagnosis of peroxisomal disorders we applied a LC-MS/MS acylcarnitine method to study a heterogenous clinical sample set (n = 598) that included residual plasma specimens from nineteen patients with PBD caused by PEX1 or PEX6 deficiency, ranging in severity from lethal neonatal onset to mild late onset forms. Multiple dicarboxylic acylcarnitines were significantly elevated in PBD patients including medium to long chain (C8-DC to C18-DC) species as well as previously undescribed elevations of malonylcarnitine (C3-DC) and very long chain dicarboxylic acylcarnitines (C20-DC and C22-DC). The best performing plasma acylcarnitine biomarkers, C20-DC and C22-DC, were detected at elevated levels in 100% and 68% of PBD patients but were rarely elevated in patients that did not have a PBD. We extended our analysis to residual newborn screening blood spot cards and were able to detect dicarboxylic acylcarnitine abnormalities in a newborn with a PBD caused by PEX6 deficiency. Similar to prior studies, we failed to detect substantial dicarboxylic acylcarnitine abnormalities in blood spot cards from patients with x-linked adrenoleukodystrophy (x-ald) indicating that these biomarkers may have utility in quickly narrowing the differential diagnosis in patients with a positive newborn screen for x-ald. Overall, our study identifies widespread dicarboxylic acylcarnitine abnormalities in patients with PBD and highlights key acylcarnitine biomarkers for the detection of this class of inherited metabolic disease.

A de novo missense variant in EZH1 associated with developmental delay exhibits functional deficits in Drosophila melanogaster.

EZH1, a polycomb repressive complex-2 component, is involved in a myriad of cellular processes. EZH1 represses transcription of downstream target genes through histone 3 lysine27 (H3K27) trimethylation (H3K27me3). Genetic variants in histone modifiers have been associated with developmental disorders, while EZH1 has not yet been linked to any human disease. However, the paralog EZH2 is associated with Weaver syndrome. Here we report a previously undiagnosed individual with a novel neurodevelopmental phenotype identified to have a de novo missense variant in EZH1 through exome sequencing. The individual presented in infancy with neurodevelopmental delay and hypotonia and was later noted to have proximal muscle weakness. The variant, p.A678G, is in the SET domain, known for its methyltransferase activity, and an analogous somatic or germline mutation in EZH2 has been reported in patients with B-cell lymphoma or Weaver syndrome, respectively. Human EZH1/2 are homologous to fly Enhancer of zeste (E(z)), an essential gene in Drosophila, and the affected residue (p.A678 in humans, p.A691 in flies) is conserved. To further study this variant, we obtained null alleles and generated transgenic flies expressing wildtype [E(z)WT] and the variant [E(z)A691G]. When expressed ubiquitously the variant rescues null-lethality similar to the wildtype. Overexpression of E(z)WT induces homeotic patterning defects but notably the E(z)A691G variant leads to dramatically stronger morphological phenotypes. We also note a dramatic loss of H3K27me2 and a corresponding increase in H3K27me3 in flies expressing E(z)A691G, suggesting this acts as a gain-of-function allele. In conclusion, here we present a novel EZH1 de novo variant associated with a neurodevelopmental disorder. Furthermore, we found that this variant has a functional impact in Drosophila.

De novo variants in MRTFB have gain-of-function activity in Drosophila and are associated with a novel neurodevelopmental phenotype with dysmorphic features.

PURPOSE: Myocardin-related transcription factor B (MRTFB) is an important transcriptional regulator, which promotes the activity of an estimated 300 genes but is not known to underlie a Mendelian disorder. METHODS: Probands were identified through the efforts of the Undiagnosed Disease Network. Because the MRTFB protein is highly conserved between vertebrate and invertebrate model organisms, we generated a humanized Drosophila model expressing the human MRTFB protein in the same spatial and temporal pattern as the fly gene. Actin binding assays were used to validate the effect of the variants on MRTFB. RESULTS: Here, we report 2 pediatric probands with de novo variants in MRTFB (p.R104G and p.A91P) and mild dysmorphic features, intellectual disability, global developmental delays, speech apraxia, and impulse control issues. Expression of the variants within wing tissues of a fruit fly model resulted in changes in wing morphology. The MRTFBR104G and MRTFBA91P variants also display a decreased level of actin binding within critical RPEL domains, resulting in increased transcriptional activity and changes in the organization of the actin cytoskeleton. CONCLUSION: The MRTFBR104G and MRTFBA91P variants affect the regulation of the protein and underlie a novel neurodevelopmental disorder. Overall, our data suggest that these variants act as a gain of function.

A de novo missense variant in EZH1 associated with developmental delay exhibits functional deficits in Drosophila melanogaster.

EZH1 ( Enhancer of Zeste, homolog 1) , a Polycomb Repressive Complex-2 (PRC2) component, is involved in a myriad of cellular processes through modifying histone 3 lysine27 (H3K27) residues. EZH1 represses transcription of downstream target genes through H3K27 trimethylation (H3K27me3). Genetic mutations in histone modifiers have been associated with developmental disorders, while EZH1 has not yet been linked to any human disease. However, the paralog EZH2 is associated with Weaver syndrome. Here we report a previously undiagnosed individual with a novel neurodevelopmental phenotype identified to have a de novo variant in EZH1 , p.Ala678Gly, through exome sequencing. The individual presented in infancy with neurodevelopmental delay and hypotonia and was later noted to have proximal muscle weakness. The variant, p.A678G, is in the SET domain, known for its methyltransferase activity, and was the best candidate variant found in the exome. Human EZH1 / 2 are homologous to fly Enhancer of zeste E(z) , an essential gene in flies, and the residue (A678 in humans, A691 in Drosophila ) is conserved. To further study this variant, we obtained Drosophila null alleles and generated transgenic flies expressing wild-type (E(z) WT ) and the variant (E(z) A691G ) . The E(z) A691G variant led to hyper H3K27me3 while the E(z) WT did not, suggesting this is as a gain-of-function allele. When expressed under the tubulin promotor in vivo the variant rescued null-lethality similar to wild-type but the E(z) A691G flies exhibit bang sensitivity and shortened lifespan. In conclusion, here we present a novel EZH1 de novo variant associated with a neurodevelopmental disorder. Furthermore, we found that this variant has a functional impact in Drosophila . Biochemically this allele leads to increased H3K27me3 suggesting gain-of-function, but when expressed in adult flies the E(z) A691G has some characteristics of partial loss-of-function which may suggest it is a more complex allele in vivo .

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

Individuals with autism spectrum disorder (ASD) exhibit an increased burden of de novo mutations (DNMs) in a broadening range of genes. While these studies have implicated hundreds of genes in ASD pathogenesis, which DNMs cause functional consequences in vivo remains unclear. We functionally test the effects of ASD missense DNMs using Drosophila through "humanization" rescue and overexpression-based strategies. We examine 79 ASD variants in 74 genes identified in the Simons Simplex Collection and find 38% of them to cause functional alterations. Moreover, we identify GLRA2 as the cause of a spectrum of neurodevelopmental phenotypes beyond ASD in 13 previously undiagnosed subjects. Functional characterization of variants in ASD candidate genes points to conserved neurobiological mechanisms and facilitates gene discovery for rare neurodevelopmental diseases.

Rare deleterious de novo missense variants in Rnf2/Ring2 are associated with a neurodevelopmental disorder with unique clinical features.

The Polycomb group (PcG) gene RNF2 (RING2) encodes a catalytic subunit of the Polycomb repressive complex 1 (PRC1), an evolutionarily conserved machinery that post-translationally modifies chromatin to maintain epigenetic transcriptional repressive states of target genes including Hox genes. Here, we describe two individuals, each with rare de novo missense variants in RNF2. Their phenotypes include intrauterine growth retardation, severe intellectual disabilities, behavioral problems, seizures, feeding difficulties and dysmorphic features. Population genomics data suggest that RNF2 is highly constrained for loss-of-function (LoF) and missense variants, and both p.R70H and p.S82R variants have not been reported to date. Structural analyses of the two alleles indicate that these changes likely impact the interaction between RNF2 and BMI1, another PRC1 subunit or its substrate Histone H2A, respectively. Finally, we provide functional data in Drosophila that these two missense variants behave as LoF alleles in vivo. The evidence provide support for deleterious alleles in RNF2 being associated with a new and recognizable genetic disorder. This tentative gene-disease association in addition to the 12 previously identified disorders caused by PcG genes attests to the importance of these chromatin regulators in Mendelian disorders.

A Genetic Screen for Genes That Impact Peroxisomes in Drosophila Identifies Candidate Genes for Human Disease.

Peroxisomes are subcellular organelles that are essential for proper function of eukaryotic cells. In addition to being the sites of a variety of oxidative reactions, they are crucial regulators of lipid metabolism. Peroxisome loss or dysfunction leads to multi-system diseases in humans that strongly affect the nervous system. In order to identify previously unidentified genes and mechanisms that impact peroxisomes, we conducted a genetic screen on a collection of lethal mutations on the X chromosome in Drosophila Using the number, size and morphology of GFP tagged peroxisomes as a readout, we screened for mutations that altered peroxisomes based on clonal analysis and confocal microscopy. From this screen, we identified eighteen genes that cause increases in peroxisome number or altered morphology when mutated. We examined the human homologs of these genes and found that they are involved in a diverse array of cellular processes. Interestingly, the human homologs from the X-chromosome collection are under selective constraint in human populations and are good candidate genes particularly for dominant genetic disease. This in vivo screening approach for peroxisome defects allows identification of novel genes that impact peroxisomes in vivo in a multicellular organism and is a valuable platform to discover genes potentially involved in dominant disease that could affect peroxisomes.

Disruptive mutations in TANC2 define a neurodevelopmental syndrome associated with psychiatric disorders.

Postsynaptic density (PSD) proteins have been implicated in the pathophysiology of neurodevelopmental and psychiatric disorders. Here, we present detailed clinical and genetic data for 20 patients with likely gene-disrupting mutations in TANC2-whose protein product interacts with multiple PSD proteins. Pediatric patients with disruptive mutations present with autism, intellectual disability, and delayed language and motor development. In addition to a variable degree of epilepsy and facial dysmorphism, we observe a pattern of more complex psychiatric dysfunction or behavioral problems in adult probands or carrier parents. Although this observation requires replication to establish statistical significance, it also suggests that mutations in this gene are associated with a variety of neuropsychiatric disorders consistent with its postsynaptic function. We find that TANC2 is expressed broadly in the human developing brain, especially in excitatory neurons and glial cells, but shows a more restricted pattern in Drosophila glial cells where its disruption affects behavioral outcomes.

Biallelic Mutations in ATP5F1D, which Encodes a Subunit of ATP Synthase, Cause a Metabolic Disorder.

ATP synthase, H+ transporting, mitochondrial F1 complex, δ subunit (ATP5F1D; formerly ATP5D) is a subunit of mitochondrial ATP synthase and plays an important role in coupling proton translocation and ATP production. Here, we describe two individuals, each with homozygous missense variants in ATP5F1D, who presented with episodic lethargy, metabolic acidosis, 3-methylglutaconic aciduria, and hyperammonemia. Subject 1, homozygous for c.245C>T (p.Pro82Leu), presented with recurrent metabolic decompensation starting in the neonatal period, and subject 2, homozygous for c.317T>G (p.Val106Gly), presented with acute encephalopathy in childhood. Cultured skin fibroblasts from these individuals exhibited impaired assembly of F1FO ATP synthase and subsequent reduced complex V activity. Cells from subject 1 also exhibited a significant decrease in mitochondrial cristae. Knockdown of Drosophila ATPsynδ, the ATP5F1D homolog, in developing eyes and brains caused a near complete loss of the fly head, a phenotype that was fully rescued by wild-type human ATP5F1D. In contrast, expression of the ATP5F1D c.245C>T and c.317T>G variants rescued the head-size phenotype but recapitulated the eye and antennae defects seen in other genetic models of mitochondrial oxidative phosphorylation deficiency. Our data establish c.245C>T (p.Pro82Leu) and c.317T>G (p.Val106Gly) in ATP5F1D as pathogenic variants leading to a Mendelian mitochondrial disease featuring episodic metabolic decompensation.

A Pak-regulated cell intercalation event leading to a novel radial cell polarity is involved in positioning of the follicle stem cell niche in the Drosophila ovary.

In the germarium of the Drosophila ovary, germline cysts are encapsulated one at a time by a follicular epithelium derived from two follicle stem cells (FSCs). Ovaries in flies mutant for the serine/threonine kinase Pak exhibit a novel phenotype, in which two side-by-side cysts are encapsulated at a time, generating paired egg chambers. This striking phenotype originates in the pupal ovary, where the developing germarium is shaped by the basal stalk, a stack of cells formed by cell intercalation. The process of basal stalk formation is not well understood, and we provide evidence that the cell intercalation is driven by actomyosin contractility of DE-Cadherin-adhered cells, leading to a column of disk-shaped cells exhibiting a novel radial cell polarity. Cell intercalation fails in Pak mutant ovaries, leading to abnormally wide basal stalks and consequently wide germaria with side-by-side cysts. We present evidence that Pak mutant germaria have extra FSCs, and we propose that contact of a germline cyst with the basal stalk in the pupal ovary contributes to FSC niche formation. The wide basal stalk in Pak mutants enables the formation of extra FSC niches which are mispositioned and yet functional, indicating that the FSC niche can be established in diverse locations.