A splice-site variant in the lncRNA gene RP1-140A9.1 cosegregates in the large Volkmann cataract family

Abstract

Purpose: To identify the mutation for Volkmann cataract (CTRCT8) at 1p36.33.

Methods: The genes in the candidate region 1p36.33 were Sanger and parallel deep sequenced, and informative single nucleotide polymorphisms (SNPs) were identified for linkage analysis. Expression analysis with reverse transcription polymerase chain reaction (RT-PCR) of the candidate gene was performed using RNA from different human tissues. Quantitative transcription polymerase chain reaction (qRT-PCR) analysis of the GNB1 gene was performed in affected and healthy individuals. Bioinformatic analysis of the linkage regions including the candidate gene was performed.

Results: Linkage analysis of the 1p36.33 CCV locus applying new marker systems obtained with Sanger and deep sequencing reduced the candidate locus from 2.1 Mb to 0.389 Mb flanked by the markers STS-22AC and rs549772338 and resulted in an logarithm of the odds (LOD) score of Z = 21.67. The identified mutation, rs763295804, affects the donor splice site in the long non-coding RNA gene RP1-140A9.1 (ENSG00000231050). The gene including splice-site junctions is conserved in primates but not in other mammalian genomes, and two alternative transcripts were shown with RT-PCR. One of these transcripts represented a lens cell-specific transcript. Meta-analysis of the Cross-Linking-Immuno-Precipitation sequencing (CLIP-Seq) data suggested the RNA binding protein (RBP) eIF4AIII is an active counterpart for RP1-140A9.1, and several miRNA and transcription factors binding sites were predicted in the proximity of the mutation. ENCODE DNase I hypersensitivity and histone methylation and acetylation data suggest the genomic region may have regulatory functions.

Conclusions: The mutation in RP1-140A9.1 suggests the long non-coding RNA as the candidate cataract gene associated with the autosomal dominant inherited congenital cataract from CCV. The mutation has the potential to destroy exon/intron splicing of both transcripts of RP1-140A9.1. Sanger and massive deep resequencing of the linkage region failed to identify alternative candidates suggesting the mutation in RP1-140A9.1 is causative for the CCV phenotype.

Figure 1

Figure shows the methods used to narrow down the location of the linkage region. A: Segregation of the CCV phenotype is shown for a small branch of the large multigenerational Volkmann family [10,11]. The fine-mapped CCV linkage region includes eight loci at 1pter, and the disease haplotype is shown boxed and marked in red. The haplotypes for individuals I:1 and I:2 are inferred, and the recombination events in individuals II:3, II:4, II:5, and IV:8 are shown with an arrow. Symbols: square, male; circle, female; filled symbol, affected individual; dot in a symbol, healthy carrier. B: The chromosomal map of 1p36.33–36.32 shows the Volkmann cataract locus found by the initial genome-wide linkage analysis [11] together with the regions covered by direct Sanger sequencing of protein coding genes and with targeted next-generation sequencing (NGS) and whole genome sequencing (WGS). The key sequence-tagged sites (STSs) and single nucleotides (SNPs) are shown for the 2 Mb telomeric chromosome 1 regions together with the Sanger sequenced genes in the linkage region flanked by STS-22AC/ rs761317190 and rs549772338. C: The exon/intron gene structure for the lncRNA gene RP1–140A9.1 (1) includes the position of the CCV splice site mutation, the PCR primer pair (F1 and R1) positions, and the length and structure of the corresponding PCR amplicons (2–4). The CCV mutation is localized distal in exon 1. The cDNA (218 bp) PCR detected (2) from transformed B-leucocytes. The cDNA 324 bp PCR product detected (3) from two different mixed carcinoma cell preparations and from a lens cDNA library. A 500 bp band (4) from gDNA. The alternative splice sites shown in (2,3) are proposed by the NetGene2 predictor. The enhancer-promoter-associated histone mark H3K27ac (5) is active in the lncRNA gene RP1–140A9.1. The positions are shown for the four CAGCTG repeats (6) found in the region. The CAGCTG palindrome is a putative binding site for hsa-miR-1207-3p (seed sequence CAGCUG) and a binding site for the transcription factor AP-4 [35] proposed by TFSEARCH. The palindrome sequence at the exon/intron border is present only in the mutant sequence.

Figure 2

RT–PCR amplification products from different tissues using the primer set CCV-mut-F/-R. Lane 1: Epstein Barr virus–transformed B-lymphocytes cell cDNA preparations showing the 218 bp transcripts. Donor splice site: 5′-AGA CCT CCA G^G TGA GGA AGG and the acceptor splice site: TTT CCT CCA G^G CTG CAC CA-3′. Lane 2: A 500 bp genomic product. Lane 3: Universal Human reference RNA (mixed carcinoma) shows the 324 bp product. Donor splice site: 5′-AGA CCT CCA G^G TGA GGA AGG and acceptor splice site: CTC TCC CCA G^C CAG CGC CCT-3′. Lane 4: cDNA from a human fetal eye from DNase-treated RNA. A 324 bp product (arrow) is seen. Lane 5: cDNA from human lens epithelial cells (HLEpiC cat. No. 6554 lot no. 2531, ScienCell) not DNase treated. A genomic DNA (500 bp) and a 324 bp PCR product (arrow) are seen from the spliced RP1–140A9.1. Lane 6: 100 bp ladder.