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

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

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

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

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

Use of Targeted Exome Sequencing for Molecular Diagnosis of Skeletal Disorders

<div><p>Genetic disorders of the skeleton comprise a large group of more than 450 clinically distinct and genetically heterogeneous diseases associated with mutations in more than 300 genes. Achieving a definitive diagnosis is complicated due to the genetic heterogeneity of these disorders, their individual rarity and their diverse radiographic presentations. We used targeted exome sequencing and designed a 1.4Mb panel for simultaneous testing of more than 4,800 exons in 309 genes involved in skeletal disorders. DNA from 69 individuals from 66 families with a known or suspected clinical diagnosis of a skeletal disorder was analyzed. Of 36 cases with a specific clinical hypothesis with a known genetic basis, mutations were identified for eight cases (22%). Of 20 cases with a suspected skeletal disorder but without a specific diagnosis, four causative mutations were identified. Also included were 11 cases with a specific skeletal disorder but for which there was at the time no known associated gene. For these cases, one mutation was identified in a known skeletal disease genes, and re-evaluation of the clinical phenotype in this case changed the diagnoses from osteodysplasia syndrome to Apert syndrome. These results suggest that the NGS panel provides a fast, accurate and cost-effective molecular diagnostic tool for identifying mutations in a highly genetically heterogeneous set of disorders such as genetic skeletal disorders. The data also stress the importance of a thorough clinical evaluation before DNA sequencing. The strategy should be applicable to other groups of disorders in which the molecular basis is largely known.</p></div