Bi-allelic loss-of-function variants in WBP4, encoding a spliceosome protein, result in a variable neurodevelopmental syndrome

Abstract

Over two dozen spliceosome proteins are involved in human diseases, also referred to as spliceosomopathies. WW domain-binding protein 4 (WBP4) is part of the early spliceosomal complex and has not been previously associated with human pathologies in the Online Mendelian Inheritance in Man (OMIM) database. Through GeneMatcher, we identified ten individuals from eight families with a severe neurodevelopmental syndrome featuring variable manifestations. Clinical manifestations included hypotonia, global developmental delay, severe intellectual disability, brain abnormalities, musculoskeletal, and gastrointestinal abnormalities. Genetic analysis revealed five different homozygous loss-of-function variants in WBP4. Immunoblotting on fibroblasts from two affected individuals with different genetic variants demonstrated a complete loss of protein, and RNA sequencing analysis uncovered shared abnormal splicing patterns, including in genes associated with abnormalities of the nervous system, potentially underlying the phenotypes of the probands. We conclude that bi-allelic variants in WBP4 cause a developmental disorder with variable presentations, adding to the growing list of human spliceosomopathies.

Keywords: WBP4; abnormal splicing; pre-mRNA splicing; recessive disorder; spliceosome; spliceosomopathy; syndromic neurodevelopmental disorder.

Graphical abstract

Figure 1

Clinical and genetic findings in eight families with WBP4-related syndrome (A) Pedigrees of eight families with WBP4-related syndrome. Symbols: filled black, affected individuals; diagonal line, deceased; double line, consanguinity; arrows indicate the probands. In each family the likely pathogenic variant in WBP4 is written beneath the pedigree using GenBank: NM_007187.5 transcript. Homozygotes for the likely pathogenic variant are noted as −/− and heterozygotes as +/−. F2-II:1 died of aspiration pneumonia at the age of 3 years. F3-II:3 was reported as phenotypically similar to her affected sister, but clinical details were not available. F4-III:1 has Wilson’s disease. F5-II:4 died of intestinal obstruction at the age of 55 days; the parents of the proband (I:1 and I:2) are from the same village. F8-II:1 died in infancy due to congenital heart defects. (B) Photos of individuals F2-II:1, F4-III:2, and F7-II:1. F2-II:1, profile and frontal photos at the age of 1 day, frontal photo at the age of 5 months, and a magnetic resonance image (MRI) at the age of 1.5 years showing agenesis of corpus callosum (arrow). F4-III:2, frontal and profile photos at ∼10 years of age and a midsagittal T1-weighted MRI at the age of 1.5 years showing hypoplastic corpus callosum (arrow). F7-II:1, frontal and profile pictures as a newborn and at the age of 4 years and 10 months. (C) Schematic diagram of WBP4 transcript GenBank: NM_007187.5 and the five LOF variants identified in our study. The gray rectangles represent exons 1–10 starting from left to right. The functional domains (zinc finger, WW1, WW2) of the protein are drawn below as blue and green rectangles. The purple rectangle represents the deletion found in family 2.

Figure 2

Immunoblotting analysis of WBP4 levels in families 1 and 2 (A) Western blot was performed to analyze the levels of WBP4 and actin proteins in fibroblast cell cultures from control fibroblasts (CCD-1092Sk), family 1 (F1-II:2 mother, F1-II:1 father, F1-III:1 proband), and family 2 (F2-I:2 mother, F2-I:1 father, F2-II:1 proband). (B) Quantification and relative amounts calculated from the band intensities.

Figure 3

Common abnormal splicing events in F1-III:1 −/− and F2-II:1 −/− (A) RNA was extracted from fibroblasts of F1-II:2 +/−, F1-II:1 +/−, F1-III:1 −/−, F2-I:2 +/−, F2-I:1 +/−, and F2-II:1 −/− and subjected to sequencing. Venn diagram representing the overlap in genes with splicing anomalies between the two affected individuals (F1-III:1 and F2-II:1); p value < 0.00001, Fisher's exact test. (B) Gene set enrichment analysis for 619 overlapping abnormally spliced genes. Gene set enrichment for human phenotypes, with enrichment represented as the number of genes matching each human phenotype and the enrichment significance (−(Log₁₀) p value). (C and D) Analysis of RNA-seq of probands (F1-III:1 and F2-II:1) relative to available RNA-seq data of age-matched healthy fibroblasts. Summary of significant abnormal splicing events (FDR < 0.05, IncLDiff < |0.1|) identified using rMATS. A5SS, alternative 5′ splice site; A3SS, alternative 3′ splice site, MXE, mutually exclusive exons (C). Gene set enrichment for human phenotypes, with enrichment represented as the number of genes matching each human phenotype and the enrichment significance (−(Log₁₀) p value) (D).