Monoallelic variants resulting in substitutions of MAB21L1 Arg51 Cause Aniridia and microphthalmia

Abstract

Classical aniridia is a congenital and progressive panocular disorder almost exclusively caused by heterozygous loss-of-function variants at the PAX6 locus. We report nine individuals from five families with severe aniridia and/or microphthalmia (with no detectable PAX6 mutation) with ultrarare monoallelic missense variants altering the Arg51 codon of MAB21L1. These mutations occurred de novo in 3/5 families, with the remaining families being compatible with autosomal dominant inheritance. Mice engineered to carry the p.Arg51Leu change showed a highly-penetrant optic disc anomaly in heterozygous animals with severe microphthalmia in homozygotes. Substitutions of the same codon (Arg51) in MAB21L2, a close homolog of MAB21L1, cause severe ocular and skeletal malformations in humans and mice. The predicted nucleotidyltransferase function of MAB21L1 could not be demonstrated using purified protein with a variety of nucleotide substrates and oligonucleotide activators. Induced expression of GFP-tagged wildtype and mutant MAB21L1 in human cells caused only modest transcriptional changes. Mass spectrometry of immunoprecipitated protein revealed that both mutant and wildtype MAB21L1 associate with transcription factors that are known regulators of PAX6 (MEIS1, MEIS2 and PBX1) and with poly(A) RNA binding proteins. Arg51 substitutions reduce the association of wild-type MAB21L1 with TBL1XR1, a component of the NCoR complex. We found limited evidence for mutation-specific interactions with MSI2/Musashi-2, an RNA-binding proteins with effects on many different developmental pathways. Given that biallelic loss-of-function variants in MAB21L1 result in a milder eye phenotype we suggest that Arg51-altering monoallelic variants most plausibly perturb eye development via a gain-of-function mechanism.

Fig 1. MAB21L1 Arg51 and Phe52 substitution causes microphthalmia and aniridia.

A. Pedigrees are shown for the six families with MAB21L1 variants and bilateral microphthalmia and/or aniridia. The pedigrees are ordered by variant: c.152G>A p.(Arg51Gln) (orange shaded box), c.152G>T p.(Arg51Leu) (green shaded box), c.152G>C p.(Arg51Pro) (yellow shaded box) and c.155T>G p.(Arg52Cys) (pink shaded box). A key to the pedigree symbols is shown to the left (grey shaded box). B. A schematic of MAB21L1 represented as a linear bar and with the first and last amino acid residue numbered. The linear positions of all pathogenic variants are shown: The monoallelic variants in this study are detailed above (red text) and the published biallelic variants are detailed below (bracketed black text). C. Clinical images of individuals with MAB21L1 Arg51-related eye malformations. R, right eye; L, left eye. Family 511 all have profound aniridia, microcornea, choroidal coloboma (just visible in II:1’s L fundus photo) and optic disc anomalies. The progression of disease in II:1 over one decade is shown between with the upper and lower photos, with worsening of phthisis in the right and pannus in the left. An enlarged retroilluminated image of III:1’s L eye is shown highlighting near-total aniridia. Individual 1434 II:1, showing bilateral partial aniridia and microphthalmia, worse on the L. Abbreviations: dn, de novo. Nucleotide and amino acid numbering are based on GenBank NM_005584.5 and GenPept NP_005575.1, respectively.

Fig 2

A. Nucleotidyltransferase activity: Graph showing the absence of nucleotidyltransferase activity in MAB21L1 and its mutant form Arg51Leu purified protein. OAS1 protein purified in the same way is a positive control and when incubated with ATP and double-stranded RNA (dsRNA), significant pyrophosphate release is detected indicating nucleotidyl transferase activity. MAB21L1 and Arg51Leu showed no activity with either ATP, CTP, GTP, UTP used as substrate separately or as an equal mixture of NTPs using DNA or RNA as an activator. The error bars represent standard errors.B: Cellular fractionation: Western blot analysis of cytoplasmic (C) and nuclear(N) extracts from HEK293-Flp-In cells with Tetracyclin (TET) inducible expression of GFP-tagged wild-type and mutant MAB21L1(Arg51Leu and Arg51Gln).Wild type and mutant proteins were present in cytoplasm(C) as well as nuclear fraction(N) as detected by anti-GFP antibody. Representative Coomassie stain gel image is shown.C: Differential Gene Expression: Gene expression analysis by RNA Sequencing performed on GFP-tagged wild-type and mutant MAB21L1 (Arg51Leu and Arg51Gln) cells. Heatmap showing top 20 differentially expressed genes in the datasets (padj < .05 and Log2F>1). The RNA sequencing data is available under the GSE166078 series at the NCBI Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/). D: Effect of PAX6 overexpression on SPARC transcripts levels: SPARC transcripts levels were quantified using quantitative RT-PCR using cells expressing GFP tagged Wild type and mutant MAB21L1 with or without overexpressing PAX6. GAPDH transcripts levels were used as normalization control. The levels of SPARC transcripts were significantly reduced in GFP tagged Wild type MAB21L1 cells in presence of overexpressed PAX6. There was no significant difference in the mutant cells in presence or absence of overexpressed PAX6.

Fig 3. The Effect of disease associated missense variants on protein structure and interactions.

A. A heatmap of the log-transformed quantitative mass spectroscopy (MS) results of biological triplicates of anti-GFP immunoprecipitates (IP) of control (GFP), tagged wild-type (WT) and mutant MAB21L1 (R51L and R51Q) from HEK293 cells. B. Representation of the structure of MAB21L1 with the position of the amino acid substitutions seen in MAB21L1 (L1) and MAB21L2 (L2) annotated. C-E Graphs showing the levels of the following classes of proteins in the GFP IP-MS from biological triplicates: C. MAB21L1. D. The five most abundant proteins interacting with WT, R51L and R51Q forms of MAB21L1 (MEIS1, PBX1, SUGT1, PBX3 & MEIS2), the dotted line box indicates the position of this class of protein on the heatmap. E. Wild-type specific interactor (TBL1XR1) and other components of the NCor complex (NCOR & HDAC3), the position in the heatmap is indicated by the closed arrowhead. F. Western blot analysis of the anti-GFP IP using the anti-TBL1XR1 antibody showing differential but not exclusive binding of the wild-type MAB21L1 compared to the mutant forms G. Mutant specific interactor MSI2/Musashi-2), the position in the heatmap is indicated by the open arrowhead. H. Western blot analysis of the anti-GFP IP using the anti-MSI2 antibody was unable to detect interaction with wild-type or mutant forms of MAB21L1 I-J. anti-MSI2 IP-MS analysis I. MSI2-derived peptides were present in all replicates and cell-lines in the anti-MSI2 IP J. MAB21L1-derived peptides were detectable in all replicates of the mutant forms of MAB21L1 but in only one replicate for the wild-type. Surprisingly peptides derived from endogenous MAB21L1 were detectable in two of the three GFP-only biological replicates.

Fig 4. Mab21l1^R51L mouse phenotype.

Left hand column shows wild type (WT) mice, middle column Mab21l1^R51L/+ heterozygous mice and right hand column homozygous Mab21l1^R51L/R51L mice. All mice were examined as young adults, at 2–3 months of age. (A) External photographs, showing normal external appearance of heterozygous and severe microphthalmia in homozygous mice. (B) Representative retinal photographs showing the anomalous, excavated optic disc phenotype (arrow) present in all the 13 heterozygous mice examined, and none of the WT (further images in S6 Fig). No retinal view (or OCT) was possible in homozygous animals as all eyes were microphthalmic, often with uveal tissue obscuring the cornea. (C) H&E-stained wax sections of the mouse eyes, with the abnormal features for each genotype labelled. The excavated optic nerve anomaly of the heterozygous mice is clearly seen. Note these mice had recently-administered dilating drops for fundal examination, but the iris appeared normal on slit lamp examination. Homozygous mice had a severe panocular eye malformation including microphthalmia, severely disorganised retina and uveal tissue, along with hypoplasia or aplasia of both the optic nerve and lens. Eyes from two different age-matched homozygous mice are shown to illustrate the spectrum of microphthalmia. Scale bars = 1 mm. (D) Optical coherence tomography (OCT) of WT and heterozygous mice, showing the enlarged, excavated optic nerve, with some persistent fetal vasculature seen above the optic nerve head.