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

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

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

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

Supplemental materials and methods.

(DOCX)</p

Significantly enriched terms relating to MAB21L1-interacting proteins using DAVID Functional Annotation Chart.

Significantly enriched terms relating to MAB21L1-interacting proteins using DAVID Functional Annotation Chart.</p

S6 File -

(DOCX)</p

Supplementary raw data table: Mass spectrometry.

(XLSX)</p

Dominant and recessive variants of MAB21L1 and MAB21L2.

Schematic representations of the linear form of MAB21L1 (blue filled bar) and MAB21L2 (purple filled bar) are shown, with the first and final amino acids numbered for each protein. For both MAB21L1 and MAB21L2 the linear positions of all published pathogenic variants are detailed on each cognate protein schematic, with the dominant heterozygous variants shown above and the recessive biallelic variants shown below. The MAB21L1 variants identified in this study are all dominantly inherited and are shown in red text. Abbreviations: dn, de novo. Nucleotide and amino acid numbering are based on GenBank NM_005584.5 and GenPept NP_005575.1, respectively. (DOCX)</p

Clinical and molecular features of individuals with <i>MAB21L1</i> heterozygous variants.

Clinical and molecular features of individuals with MAB21L1 heterozygous variants.</p

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.</p

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 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.</p