Xlid Syndrome

[Edie Staniszewski]

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RNA-binding proteins play important roles in X-linked intellectual disability (XLID). In this study, we investigate the contribution of the XLID-associated RBMX in neuronal differentiation. We show that RBMX-depleted cells exhibit aberrant activation of the p53 pathway. Moreover, we identify that the RBMX RGG/RG motif is methylated by protein arginine methyltransferase 5 (PRMT5), and this regulates assembly with the SRSF1 splicing factor into higher-order complexes. Depletion of RBMX or disruption of the RBMX/SRSF1 complex in PRMT5-depleted cells reduces SRSF1 binding to the MDM4 precursor (pre-)mRNA, leading to exon 6 exclusion and lower MDM4 protein levels. Transcriptomic analysis of isogenic Shashi-XLID human-induced pluripotent stem cells (hiPSCs) generated using CRISPR-Cas9 reveals a dysregulation of MDM4 splicing and aberrant p53 upregulation. Shashi-XLID neural progenitor cells (NPCs) display differentiation and morphological abnormalities accompanied with excessive apoptosis. Our findings identify RBMX as a regulator of SRSF1 and the p53 pathway, suggesting that the loss of function of the RBMX RGG/RG motif is the cause of Shashi-XLID syndrome.

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The clinical features of the family K9677 male proband (II-1) overlapped with affected males in K8100 (Fig. 1e and Table 1; Supplementary Note 1); he exhibited global developmental delay, dysmorphic facial features and exotropia. Trio-based WES and bioinformatic filtering identified a rare de novo variant in FAM50A (c.763G>A, p.Asp255Asn; Fig. 1e; Supplementary Fig. 1a, b), that was predicted to be likely pathogenic (Supplementary Table 1). Protein modeling suggests that this change introduces a polar residue, Asn, and thus does not form an H-bond with residue Arg180 as does the WT amino acid (Supplementary Fig. 2). Protein stability predictions are inconsistent (Supplementary Table 2). However, the variant alters the charge at residue 255, potentially affecting FAM50A function.

Together, we identified a cohort of nine males from five unrelated families who carry rare FAM50A variants. Affected individuals share syndromic ID and comorbid phenotypes impacting growth, facial gestalt, and ocular development (Fig. 1 and Table 1). These nonsynonymous changes segregate with disease status in pedigrees, are absent from gnomAD, and reside within highly conserved regions of the XAP domain in the C-terminal portion of FAM50A (Fig. 1; Supplementary Fig. 1a, b and Supplementary Table 1).

To show direct interaction of FAM50A with bona fide spliceosome effectors, we performed co-immunoprecipitation (co-IP) assays. We transiently transfected U-87 glioblastoma cells with V5-tagged EFTUD2 and DDX41 plasmids, which are part of the spliceosome U5 and C-complex, respectively7 (Fig. 6a, b). Immunoblotting using anti-V5 and anti-GAPDH antibodies detected the overexpressed tagged proteins in input lysates (Fig. 6c, d). Next, we immunoprecipitated proteins with anti-FAM50A antibody in transfected and negative control samples. Western blot against FAM50A in co-IP lysates discovered the pulled down FAM50A protein in all samples. Immunoblot with anti-V5 in IP lysates detected both Co-IP partners in their respective transfected samples but not in negative controls, indicating a specific physical interaction of FAM50A with spliceosome binding partners that are active during the two-step splicing reaction (Fig. 6c, d).

We report partial loss-of-function missense variants in FAM50A as the genetic basis of Armfield XLID syndrome. Our work epitomizes the challenges of understanding rare disease pathogenesis. Using candidate gene sequencing, we identified the causal FAM50A variant in the original Armfield XLID syndrome family in 2001. Next-generation sequencing technology and data sharing platforms were required to identify four additional cases with FAM50A variants several years later. Even with bolstered support for genetic causality, this work required a vertebrate model to gain insight into variant pathogenicity and cellular mechanism. Our experience is not unique. Of the estimated 9000 Mendelian phenotypes that have been described32, a substantial proportion of gene-phenotype pairs identified in the last 5 years required partnering of a rare human finding with a model organism or relevant in vitro functional assay33.

In all genetic studies, candidate causal variants were considered if they: (1) altered coding sequence or splice junctions; (2) were absent from healthy control males; and (3) segregated with disease in pedigrees. FAM50A variants were confirmed using Sanger sequencing of an independent genomic DNA sample from the proband (Supplementary Table 3). Segregation analysis was conducted on DNA samples from all available family members.

Due to lack of an experimentally-determined structure of FAM50A protein, we used in silico modeling. Homology modeling is not suitable for predicting FAM50A 3D protein structure, since no template can be found that covers the entire sequence of FAM50A. Thus, we used I-TASSER, which is an iterative threading method47. We uploaded the full sequence of FAM50A (NP_004690.1) and specified one template structure (PDB: 3AG7), which has 27% sequence identity and covers residues from 173 to 248. In addition, we predicted the secondary structure elements (SSEs) of FAM50 using YASPIN48.

In the original version of this article, the given and family names of Won Do Heo were incorrectly structured. The name was displayed correctly in all versions at the time of publication. The original article has been corrected.

Peer review information Nature Communications thanks Maurice Swanson, Corinne Houart, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer review reports are available.

New findings from Northwestern Medicine scientists have revealed previously unknown information about the genetic basis for Armfield XLID syndrome, a rare intellectual disability linked to genetic defects in the X chromosome.

Published in Nature Communications, these findings point to defects in mRNA splicing as a major cause for Armfield XLID. This, along with other recent studies, shows a rising class of diseases called spliceosomopathies that interfere with neurodevelopment, according to Erica Davis, PhD, associate professor of Pediatrics and co-senior author of the study.

Intellectual disability (ID) affects one to three percent of people, but is 20 to 30 percent more common in males due to a concentration of genes on the X chromosome that are required for neurodevelopment.

First reported in 1999, Armfield XLID syndrome is characterized by impaired growth and causes dysmorphic facial features and seizures. While the causal region of the X chromosome has been known, the specific gene and underlying cellular process impacted by mutations has eluded investigators for over two decades, according to the authors.

In the current study, investigators used GeneMatcher, an online platform that enables the identification of affected individuals with overlapping phenotypes and gene mutations in order to accelerate disease gene discovery. GeneMatcher revealed four unrelated males who all displayed symptoms similar to Armfield XLID, and each one had different rare mutations in FAM50A.

Using zebrafish models, the investigators knocked out FAM50A to study its effects on early development, finding that the animals exhibited similar physical abnormalities to humans with Armfield XLID syndrome. They also studied the specific mechanisms of the FAM50A variants seen in human patients, and discovered that the mutation caused aberrant mRNA splicing and depletion of genetic material that is vital for neurodevelopment.

These findings, joined with previous studies of other XLIDs, show that defective splicing is an emergent contributor to this class of syndromes. According to the authors, further investigation is warranted in order to better understand the cause of these conditions and to help find treatments.

GGC has a long and successful history of research on X-linked intellectual disability (XLID). The JC Self Research Institute, a division of GGC, has been renowned as an international resource for scientists and clinicians seeking greater understanding of the diagnosis and causes of XLID, as well as prevention and treatment strategies. Many avenues of XLID research have been explored at the Institute, from studying the effects of a single gene mutation, to mapping genes along the X chromosome to developing strategies that help families in prevention of recurrences.

An intellectual disability is identified as a condition causing significant limitations in social and behavioral skills and cognitive abilities. It is estimated that up to 3% of the population lives with an intellectual disability (ID). Intellectual disability can be caused by environmental influences, but genetic causes, such as chromosomal anomalies and single-gene disorders account for more than half of moderate to severe cases.

XLID comprises a group of genetic conditions in which the causative gene mutation is located on the X chromosome. Because males have a single X chromosome, XLID predominantly affects males and is often passed through an unaffected carrier mother. Females with a single X-linked mutation have a 50 percent chance of passing on the mutation to their offspring.

Newly identified X-linked intellectual disability syndromes and localizations, new gene identifications, the status of nonsyndromal XLID (IDX), and the status of duplications of genes associated with XLID are presented in abbreviated form with appropriate references.

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