Fig 1.
Radiographic phenotypes of cases R14-123A and R08-269A & B.
(A) A/P radiograph of the chest of R14-123a 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.
Table 1.
Summary of the ultrasound, clinical and radiographic findings.
Fig 2.
(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 SF3B4 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 SF3B4 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.
Fig 3.
Decreased expression of SF3B4 in Rodriguez syndrome.
(A,B) Detection of SF3B4 mRNA by quantitative RT-PCR in (A) R14-123A fibroblasts and (B) R08-269A chondrocytes. (C) Western blots (left panel) and signal quantification (right panel) of SF3B4 in R14-123A fibroblasts. Data in the bar graphs were collected from at least three replicates and were represented as mean ± standard deviation. *p<0.05; **p<0.01. (D) Detection of SF3B4 protein in R14-123A fibroblasts by Western blotting with the antibodies against the N- and C-termini of the SF3B4 protein, respectively. The asterisk identifies the elongated form of SF3B4 that was presumed to be derived from the mutant allele. SF3B1 protein was used as a loading control. (E) Immunostaining of human fibroblasts with anti-SF3B4 (green). Cell nuclei were stained with DAPI (blue).
Fig 4.
Pronounced disorganization of hypertrophic chondrocytes caused by SF3B4 mutations.
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.
Fig 5.
SF3B4 mutation leads to altered gene expression.
(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) DLX5, (C) SOX6, (D) SOX9 and (E) SF3B4. 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 GAPDH. **p<0.01. (G) From top to bottom, enriched DLX5, DLX6, SOX5 and SOX9 binding motifs among the promoter regions of the down-regulated genes. The p values for motif enrichment are shown in parentheses below each motif.
Table 2.
Skeletal developmental genes with reduced expression in R08-269A.
Fig 6.
SF3B4 and DLX5 are co-expressed in the human growth plate.
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.
Fig 7.
SF3B4 mutation leads to altered splicing.
(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.