NAA10 polyadenylation signal variants cause syndromic microphthalmia

Abstract

Background: A single variant in NAA10 (c.471+2T>A), the gene encoding N-acetyltransferase 10, has been associated with Lenz microphthalmia syndrome. In this study, we aimed to identify causative variants in families with syndromic X-linked microphthalmia.

Methods: Three families, including 15 affected individuals with syndromic X-linked microphthalmia, underwent analyses including linkage analysis, exome sequencing and targeted gene sequencing. The consequences of two identified variants in NAA10 were evaluated using quantitative PCR and RNAseq.

Results: Genetic linkage analysis in family 1 supported a candidate region on Xq27-q28, which included NAA10. Exome sequencing identified a hemizygous NAA10 polyadenylation signal (PAS) variant, chrX:153,195,397T>C, c.*43A>G, which segregated with the disease. Targeted sequencing of affected males from families 2 and 3 identified distinct NAA10 PAS variants, chrX:g.153,195,401T>C, c.*39A>G and chrX:g.153,195,400T>C, c.*40A>G. All three variants were absent from gnomAD. Quantitative PCR and RNAseq showed reduced NAA10 mRNA levels and abnormal 3' UTRs in affected individuals. Targeted sequencing of NAA10 in 376 additional affected individuals failed to identify variants in the PAS.

Conclusion: These data show that PAS variants are the most common variant type in NAA10-associated syndromic microphthalmia, suggesting reduced RNA is the molecular mechanism by which these alterations cause microphthalmia/anophthalmia. We reviewed recognised variants in PAS associated with Mendelian disorders and identified only 23 others, indicating that NAA10 harbours more than 10% of all known PAS variants. We hypothesise that PAS in other genes harbour unrecognised pathogenic variants associated with Mendelian disorders. The systematic interrogation of PAS could improve genetic testing yields.

Keywords: Naa10; polyadenylation signal.

Figure 1

Pedigrees for families 1–3. Clinically affected individuals are depicted by filled symbols, black symbols depict individuals with eye findings and grey symbols depict individuals without eye findings, genotypes of tested individuals are noted.

Figure 2

Reverse transcription and quantitative PCR analysis of NAA10 expression in mRNA; data were normalised to ACTB mRNA levels. (A) relative NAA10 expression in whole blood from affected individuals (family 1, individuals III-2, III-5 and IV-3), carrier females (family 1, individuals II-2, II-6 and III-15) and male and female control individuals (C1–C4). All values are shown relative to control C1. (B) NAA10 expression levels in lymphoblasts from affected male IV-6 shown relative to the expression level in unaffected male V-9, family 2.

Figure 3

RNA-seq data comparing reads from four affected individuals, two carrier females and three control individuals, one female and two males, for NAA10. The variant positions in the NAA10 polyadenylation signal are marked by a filled red arrow. Transcription in affected individuals (family 1, individuals III-2, III-5 and IV-3 and family 2, individual IV-6) and carrier females (family 1, individuals II-2 and III-15) continues past the normal polyadenylation cleavage site and uses a cryptic signal approximately 600 bp downstream at an alternate polyadenylation cleavage site depicted by an open red arrow. NAA10 gene models are from GENCODE V.19 as included in the UCSC genome browser (GRCh37/hg19).

Figure 4

Comparison of the effects of AAUAAA mutations on cleavage and poly(A) addition. Variants identified in this study are noted with an asterisk. Figure is reproduced from Sheets et al with permission from Oxford University Press.