Abstract
The craniofacial developmental disorder Burn-McKeown Syndrome (BMKS) is caused by biallelic variants in the pre-messenger RNA splicing factor gene TXNL4A/DIB1. The majority of affected individuals with BMKS have a 34 base pair deletion in the promoter region of one allele of TXNL4A combined with a loss-of-function variant on the other allele, resulting in reduced TXNL4A expression. However, it is unclear how reduced expression of this ubiquitously expressed spliceosome protein results in craniofacial defects during development. Here we reprogrammed peripheral mononuclear blood cells from a BMKS patient and her unaffected mother into induced pluripotent stem cells (iPSCs) and differentiated the iPSCs into induced neural crest cells (iNCCs), the key cell type required for correct craniofacial development. BMKS patient-derived iPSCs proliferated more slowly than both mother- and unrelated control-derived iPSCs, and RNA-Seq analysis revealed significant differences in gene expression and alternative splicing. Patient iPSCs displayed defective differentiation into iNCCs compared to maternal and unrelated control iPSCs, in particular a delay in undergoing an epithelial-to-mesenchymal transition (EMT). RNA-Seq analysis of differentiated iNCCs revealed widespread gene expression changes and mis-splicing in genes relevant to craniofacial and embryonic development that highlight a dampened response to WNT signalling, the key pathway activated during iNCC differentiation. Furthermore, we identified the mis-splicing of TCF7L2 exon 4, a key gene in the WNT pathway, as a potential cause of the downregulated WNT response in patient cells. Additionally, mis-spliced genes shared common sequence properties such as length, branch point to 3' splice site (BPS-3'SS) distance and splice site strengths, suggesting that splicing of particular subsets of genes is particularly sensitive to changes in TXNL4A expression. Together, these data provide the first insight into how reduced TXNL4A expression in BMKS patients might compromise splicing and NCC function, resulting in defective craniofacial development in the embryo.
Highlights
Burn-McKeown syndrome (BMKS; OMIM 608572) is a rare craniofacial developmental disorder
Two established induced pluripotent stem cells (iPSCs) clonal lines were generated for the patient and her mother, but attempts to reprogram Peripheral Blood Mononuclear Cells (PBMCs) from the patient’s father were unsuccessful
All iPSC lines displayed pluripotent stem cell-like morphology, had high mRNA expression levels of the pluripotency marker OCT3/4, NANOG and SOX2 transcripts, stained positively for a number of pluripotency markers and negatively for SSEA-1, a marker of early differentiation, and all iPSC lines had a normal karyotype (S1A-S1C Fig in S2 File)
Summary
Burn-McKeown syndrome (BMKS; OMIM 608572) is a rare craniofacial developmental disorder. The primary phenotype associated with BMKS is choanal atresia, observed in all patients to date. Intellectual development is usually unimpaired, at least one reported BMKS patient suffers severe intellectual disability and developmental delay [7]. In 2014, Wieczorek et al reported genetic variants in TXNL4A as causative in BMKS [4]. Most BMKS patients identified far have a 34bp deletion (chr: g.77,748,581_77,748,614del (GRCh37, hg19), type 1 Δ34bp) within the promoter region of TXNL4A on one allele combined with a loss-of-function variant on the other allele. Some individuals with BMKS do not have a compound heterozygous genotype, but are homozygous for a slightly different TXNL4A 34bp promoter deletion (chr: g.77,748,604_77,748,637 (GRCh37, hg19), type 2 Δ34bp) [4,5,8]. It is postulated that the BMKS phenotype is the result of a specific dosage of TXNL4A, where biallelic null variants in TXNL4A are likely to be incompatible with life
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