Massively parallel sequencing of exons on the X chromosome identifies RBM10 as the gene that causes a syndromic form of cleft palate

Abstract

Micrognathia, glossoptosis, and cleft palate comprise one of the most common malformation sequences, Robin sequence. It is a component of the TARP syndrome, talipes equinovarus, atrial septal defect, Robin sequence, and persistent left superior vena cava. This disorder is X-linked and severe, with apparently 100% pre- or postnatal lethality in affected males. Here we characterize a second family with TARP syndrome, confirm linkage to Xp11.23-q13.3, perform massively parallel sequencing of X chromosome exons, filter the results via a number of criteria including the linkage region, use a unique algorithm to characterize sequence changes, and show that TARP syndrome is caused by mutations in the RBM10 gene, which encodes RNA binding motif 10. We further show that this previously uncharacterized gene is expressed in midgestation mouse embryos in the branchial arches and limbs, consistent with the human phenotype. We conclude that massively parallel sequencing is useful to characterize large candidate linkage intervals and that it can be used successfully to allow identification of disease-causing gene mutations.

Figure 1

Pedigree of TARP Syndrome Family 2 The three affected male individuals are shown with darkened symbols. Obligate female carriers have a dot within their symbol. Nine family members were genotyped for the c.1235G>A mutation; their status is indicated by the + (wild-type) or m (mutant) designations below each pedigree symbol. Also shown are Sanger electropherograms of the two mutations; on the left is the nonsense mutation c.1235G>A found in family 2 below the control sequence, and on the right is the insertion mutation c.1893_1894insA found in family 1, again below the control sequence.

Figure 2

Cartoon of the RBM10 Gene Structure, Alternative Splicing, Conserved Domains, and Mutations Found in Two Families with TARP Syndrome For clarity, the width of the rectangles is the same for all exons and is therefore not proportional to the actual length of the exons. The 5′ and 3′ UTRs are shown as rectangles with reduced vertical height. The gene has two mRNA isoforms: variant 1, which includes exon 4, and variant 2, which does not. Note that all other exons are believed to be constitutively spliced into both isoforms, so splicing lines are not shown for those exons. The portions of the gene that encode for the four recognized conserved domains are shown (two RRM, RNA recognition motif znfRBP, a zinc finger Ran binding protein, and a G-patch domain). Finally, the two independent mutations found in families 1 and 2 are shown.

Figure 3

Expression of the Murine Ortholog Rbm10 in Midgestation Embryos (A) In situ hybridization of a probe to murine Rbm10 in a wild-type E10.5 mouse embryo. There is expression of the transcript primarily in branchial arches 1 and 2. There is some expression in the limb (L), in a region that partially overlaps the apical ectodermal ridge. (B) Expression of Rbm10 in an E9.5 mouse embryo. At this stage, the expression is slightly less strong in the second branchial arch and limb but is strong in the first branchial arch. Expression was also noted in the tail (T) at both stages.