Compound inheritance of a low-frequency regulatory SNP and a rare null mutation in exon-junction complex subunit RBM8A causes TAR syndrome

Abstract

The exon-junction complex (EJC) performs essential RNA processing tasks. Here, we describe the first human disorder, thrombocytopenia with absent radii (TAR), caused by deficiency in one of the four EJC subunits. Compound inheritance of a rare null allele and one of two low-frequency SNPs in the regulatory regions of RBM8A, encoding the Y14 subunit of EJC, causes TAR. We found that this inheritance mechanism explained 53 of 55 cases (P < 5 × 10(-228)) of the rare congenital malformation syndrome. Of the 53 cases with this inheritance pattern, 51 carried a submicroscopic deletion of 1q21.1 that has previously been associated with TAR, and two carried a truncation or frameshift null mutation in RBM8A. We show that the two regulatory SNPs result in diminished RBM8A transcription in vitro and that Y14 expression is reduced in platelets from individuals with TAR. Our data implicate Y14 insufficiency and, presumably, an EJC defect as the cause of TAR syndrome.

Figure 1. Most TAR syndrome cases have a low-frequency regulatory variant and a rare null allele in the RBM8A locus

a) Fifty-three out of 55 TAR cases are heterozygous carriers of a rare 1q21.1 deletion of varying size. The red bar indicates the region that is deleted in all 53 cases with a deletion. b) The RBM8A transcript is shown in genomic coordinates with the RNA binding domain (RRM) indicated by the orange bar above the transcript. c) We identified two low-frequency regulatory SNPs in 53 of a total of 55 TAR cases studied. The first, 145507646 G/A, is located in the 5′UTR of RBM8A and has a population minor allele frequency of 3.05% (shown in blue). The second, 145507765 G/C is located in the first intron of RBM8A and has a population minor allele frequency of 0.41% (shown in green). Thirty-nine TAR cases carried the minor allele of the 5′UTR SNP on one chromosome and the 1q21.1 deletion on the other; 12 TAR cases carried the minor allele of the intronic SNP on one chromosome and the 1q21.1 deletion on the other. The compound inheritance of the 1q21.1 deletion and one of the two regulatory SNPs is strongly associated with TAR with a p-value <5 × 10^×228. Two further TAR cases were found to have the 5′UTR minor allele in combination with either a frameshift insertion (shown in purple) and a nonsense mutation in RBM8A (shown in light blue) instead of the 1q21.1 deletion, implicating RBM8A as the causative gene for TAR syndrome. d) Sequencing of RNA from cord-blood-derived megakaryocytes shows that RBM8A is expressed in megakaryocytes. e) Histone modifications from the ENCODE Project in 7 cell lines (GM12878, H1-hESC, HSMM, HUVEC, K562, NHEK, and NHLF) indicate the presence of regulatory elements at the promoter and first intron of RBM8A. H3K4Me1 is often found near regulatory elements, H3K4Me3 is often found near promoters and H3K27Ac is often found near active regulatory elements (UCSC Genome Browser). f) FAIRE marking regions of open chromatin shows that the 5′UTR SNP and first intron SNP are accessible in megakaryocytes. g) Computational modeling predicts that the 5′UTR SNP minor allele creates a binding site for the transcription factor Evi1, whilst the intronic SNP minor allele is predicted to disrupt binding of Mzf1 and Rbpj.

Figure 2. Effect of the regulatory SNPs on transcription factor binding, RBM8A promoter activity and protein expression in platelets

a) Electrophoretic mobility shift assays (EMSA) in nuclear protein extracts from the megakaryocytic cell line CHRF-288-11 showed higher protein affinity of the probe containing the A-allele (lane 7) than the G-allele (lane 2) of the 5′UTR non-coding SNP. Protein binding of A-allele-probes was competed by specific unlabeled probes (lane 8), but not by unspecific G-allele-probes (lane 9). We observed a supershift with Evi1 antibodies for DNA-protein complexes containing the A-allele-probe (lane 10), indicating that the 5′UTR SNP minor allele increases binding affinity for the transcription factor EVI1 in vitro. b) Luciferase reporter assays in cell lines representative of megakaryocytes (CHRF, DAMI) and osteoblasts (mouse MC3T3) showed significantly decreased RBM8A promoter activity for the minor alleles of both the 5′UTR and intronic non-coding SNPs (indicated by asterisks,). No effect of the 5′UTR SNP was observed in human endothelial EAHY926 and HEK293 cells. Error bars represent s.d. Statistical analysis by Tukey–Kramer multiple comparisons test. c) Densitometry analysis of immunoblot staining for Y14, the protein encoded by the RBM8A gene, in platelet lysates from seven TAR cases, six parents (three with the 1q21.1 deletion, one heterozygote for the 5′UTR SNP, one homozygote for the 5′UTR SNP and one compound heterozygote for the 5′UTR and intronic SNPs) and six controls. Results show a significantly reduced Y14 protein level in TAR cases when compared to parental and control samples. Immunoblots are presented in Supplementary Fig. 3. Error bars represent s.d. Statistical analysis by heteroscedastic t-test. Only genotype configurations indicated by lines were compared. n.s., not significant.