Altered mRNA Splicing, Chondrocyte Gene Expression and Abnormal Skeletal Development due to <i>SF3B4</i> Mutations in Rodriguez Acrofacial Dysostosis

<div><p>The acrofacial dysostoses (AFD) are a genetically heterogeneous group of inherited disorders with craniofacial and limb abnormalities. Rodriguez syndrome is a severe, usually perinatal lethal AFD, characterized by severe retrognathia, oligodactyly and lower limb abnormalities. Rodriguez syndrome has been proposed to be a severe form of Nager syndrome, a non-lethal AFD that results from mutations in <i>SF3B4</i>, a component of the U2 small nuclear ribonucleoprotein particle (U2 snRNP). Furthermore, a case with a phenotype intermediate between Rodriguez and Nager syndromes has been shown to have an <i>SF3B4</i> mutation. We identified heterozygosity for <i>SF3B4</i> mutations in Rodriguez syndrome, confirming that the phenotype is a dominant disorder that is allelic with Nager syndrome. The mutations led to reduced SF3B4 synthesis and defects in mRNA splicing, primarily exon skipping. The mutations also led to reduced expression in growth plate chondrocytes of target genes, including the <i>DLX5</i>, <i>DLX6</i>, <i>SOX9</i>, and <i>SOX6</i> transcription factor genes, which are known to be important for skeletal development. These data provide mechanistic insight toward understanding how <i>SF3B4</i> mutations lead to the skeletal abnormalities observed in the acrofacial dysostoses.</p></div

SF3B4 and DLX5 are co-expressed in the human growth plate.

<p>Immunohistochemical staining of the distal femur growth plate from a control fetus with (A) anti-SF3B4 and (B) anti-DLX5 antibodies. The hypertrophic zone is marked with double arrows. The periosteum is identified by white arrows. Images were obtained at 20X (SF3B4) and 10X (DLX5) magnification. Scale bars are 100 μM.</p

Summary of the ultrasound, clinical and radiographic findings.

<p>Summary of the ultrasound, clinical and radiographic findings.</p

SF3B4 mutation leads to altered splicing.

<p>(A) Bar graph representation of altered splicing events in case R08-269A. The number of events in each category is indicated, with exon exclusion to the left (blue bars) and exon inclusion to the right (red bars). (B) GO enrichment analysis of alternative splicing events. The number of genes in each GO term is shown. SE, skipped exon; MXE, mutually exclusive exon; A5SS, alternative 5’ splice site; A3SS, alternative 3’ splice site; RI, retained intron.</p

SF3B4 mutation leads to altered gene expression.

<p>(A) GO analysis of down-regulated genes in growth plate chondrocytes from case R08-269A. The number of genes in each GO term is indicated. (B-E) Expression changes of (B) <i>DLX5</i>, (C) <i>SOX6</i>, (D) <i>SOX9</i> and (E) <i>SF3B4</i>. The gene expression is represented as FPKM values. (F) Real-time quantitative RT-PCR validation of the RNA-seq data. mRNA expression was normalized to <i>GAPDH</i>. **p<0.01. (G) From top to bottom, enriched <i>DLX5</i>, <i>DLX6</i>, <i>SOX5</i> and <i>SOX9</i> binding motifs among the promoter regions of the down-regulated genes. The p values for motif enrichment are shown in parentheses below each motif.</p

Pronounced disorganization of hypertrophic chondrocytes caused by <i>SF3B4</i> mutations.

<p>Toluidine blue staining of the distal femur growth plate from (A) a control fetus, (B) R14-123A, (C) R08-269A and (D) R08-269B. The hypertrophic zone is marked with double arrows. Images were obtained at 20X magnification. Scale bars are 100 μM.</p

Skeletal developmental genes with reduced expression in R08-269A.

<p>Skeletal developmental genes with reduced expression in R08-269A.</p

Radiographic phenotypes of cases R14-123A and R08-269A & B.

<p>(A) A/P radiograph of the chest of R14-123^a showing small scapulae, 11 ribs, and abnormally formed hypoplastic humeri with radioulnar synostosis. (B) Hand radiograph showing oligodactyly, hypoplastic carpal bones and preaxial polydactyly. (C) Bilateral lower extremities showing hypoplastic or absent fibulae with small stippled calcanei. (D) A/P radiograph of R08-269A showing hypoplastic radii, oligodactyly, absent thumbs, thin fibulae, and club foot. (E) A/P radiograph of R08-269B showing 11 ribs, absent radii and ulnae.</p

Mutations in <i>SF3B4</i>.

<p>(A,B) Electropherogram representation of genomic DNA fragments from controls (top), (A) case R14-123A (bottom), (B) case R08-269B (bottom). (C) The insertion in the <i>SF3B4</i> cDNA of R14-123A (bottom) as compared with control (top). The positions of the insertion mutations are indicated by arrows. (D) Schematic diagram of predicted protein alterations caused by <i>SF3B4</i> frameshift mutations. The blue bars correspond to the reference amino acid sequence and the red bars indicate altered amino acid sequences that begin at the mutation site. RNA recognition motifs (RRM) are shown as purple ovals.</p

MOESM1 of Mutations in IFT-A satellite core component genes IFT43 and IFT121 produce short rib polydactyly syndrome with distinctive campomelia

Additional file 1: Figure S1. Clinical findings in R06-303A. Major findings in R06-303A were small thorax, poor ossification of the phalanges, polydactyly in both hands and feet (a, b), kidneys showing thin cortex and areas of fibrosis (c, e, f, g) and pancreas (d) showing cystic changes in the tail