Exome sequencing identifies WDR35 variants involved in Sensenbrenner syndrome

Abstract

Sensenbrenner syndrome/cranioectodermal dysplasia (CED) is an autosomal-recessive disease that is characterized by craniosynostosis and ectodermal and skeletal abnormalities. We sequenced the exomes of two unrelated CED patients and identified compound heterozygous mutations in WDR35 as the cause of the disease in each of the two patients independently, showing that it is possible to find the causative gene by sequencing the exome of a single sporadic patient. With RT-PCR, we demonstrate that a splice-site mutation in exon 2 of WDR35 alters splicing of RNA on the affected allele, introducing a premature stop codon. WDR35 is homologous to TULP4 (from the Tubby superfamily) and has previously been characterized as an intraflagellar transport component, confirming that Sensenbrenner syndrome is a ciliary disorder.

Figure 1

Two Patients with Sensenbrenner Syndrome for Whom Exome Sequencing Was Performed (A) Patient 1: small thorax, pectus excavatum, rhizomelic shortening of limbs. (B) Shortening of proximal second phalanx. The proximal phalanx is indicated by “P”; the middle phalanx is indicated by “M.” (C) Syndactyly 2-3 right foot, 2-3-4 left foot. (D) Hypertelorism, unilateral ptosis of left eye, low-set ears, everted lower lip. (E) Patient 2: small thorax, pectus excavatum, rhizomelic shortening of limbs. (F) Short, broad hands. (G) Bilateral sandal gap between first and second toe. (H) Hypertelorism, low-set simple ears, thin hair.

Figure 2

Splice-Site Mutation in Patient 1 (A) Gene and protein structure of WDR35. WD domains are indicated by triangles; the box indicates a low-complexity region. (B) Sequencing reads showing the heterozygous splice-site mutation at the splice-acceptor site of exon 2 as well as a nearby exonic polymorphism (rs1060742). (C) Maternal inheritance of the splice-site mutation in patient 1 shown by Sanger sequencing. (D) Effect of the splice-site mutation on the RNA shows two different products of equal intensity in lane 1 (523 bp and 465 bp for the upper and lower band, respectively). Lane 2 shows the product of an unrelated control (465 bp). (E) Sequence of the two WDR35 RT-PCR products from Figure 2D, showing that the polymorphism (rs1060742) is predominantly present in the normal spliced product as a C (Figure 2D, lane 1, arrow a), whereas this is a T in the alternatively spliced product (Figure 2D, lane 1, arrow b). The sequence of the RT-PCR product of an unrelated individual with the same heterozygous SNP (rs1060742) is shown as a control (Figure 2D, lane 3, arrow c).