We screened the DNA of 135 patients with RP, 25 patients with CRD, and 30 with LCA using SSCP and direct DNA sequencing for mutations in the SEMA4A gene. The results are summarised in table 2. During SSCP analysis for exon 10, two types of variant bands were seen in 20 different samples. The variant bands were further subjected to genomic DNA sequencing. Sequencing analysis revealed two heterozygous mutations in codon 345 and 350. A heterozygous G→C substitution (c.345GAC→CAC; aspartic acid→histidine) in codon 345 results in a p.D345H mutation that is conservative in nature. The second T→G substitution in codon 350 (c.350TTT→TGT; phenylalanine→cysteine) results in a non‐conservative p.F350C mutation (fig 1A). Both the p.D345H and p.F350C mutations were identified in four patients (RODS002, 006, 067, and 119). Of these, two were diagnosed with RP and two with CRD. It is noteworthy that all the patients had both mutations and none had only one. Subsequently the sequencing analysis of the parents of one of the CRD patients (RODS006) revealed that he inherited the p.D345H mutation from his father while the p.F350C mutation came from his mother (fig 2A). Upon clinical examination, both parents appeared to be normal. None of the normal controls had either of these mutations. It can therefore be inferred that compound heterozygous mutations cause this disease phenotype.
Table 2Summary of Sema4A gene sequencing results
Figure 1Selected electropherograms (forward and reverse) of the patients identified with SEMA4A gene mutations. (A) Heterozygous G→C and T→G substitutions in exon 10. (B) Heterozygous G→A substitution in exon 15. Arrows (more ...)
Figure 2Pedigrees of families of (A) a CRD patient with compound heterozygosity for the D345H, & F350C (G→C and T→G) mutations and (B) of an autosomal dominant RP patient in which sequencing analysis showed the segregation (more ...)
The remaining 16 patients (of the 20 who showed a mobility shift in SSCP analysis) had a 2 bp deletion in intron 10, 26 bp downstream of exon 10. In addition, a large number of samples from the normal controls were also identified as having the aforementioned 2 bp deletion. This polymorphic deletion was heterozygous in all the samples that were examined. It was considered nonpathogenic because it was found in both patients and controls. In addition, three isocoding substitutions (C→A, T→C, and C→T) were also identified in exon 2, 8, and 15, respectively.
In exon 15, a heterozygous G→A transition mutation was identified. This mutation causes a change in codon 713, whereby arginine (CGG) is replaced by glutamine (CAG) (fig 1B). This R713Q mutation was found in four patients. Of these, one patient had congenital blindness while the remaining three had RP. Sequence analysis of the family members of an RP patient (RODS52) confirmed that the R713Q mutation was segregating with the disease phenotype (fig 2B), with an autosomal dominant mode of inheritance. This mutation was not present in the 100 ethnically matched control subjects.
Of the 190 patients analysed, three novel point mutations were found in SEMA4A
. These mutations could be considered pathogenic for two reasons. Firstly, they were not observed in any of the normal members or the 100 control subjects. Secondly, the p.D345H and p.F350C mutations identified in this human study occur in the conserved semaphorin domain. In the mouse model, it has been shown that disruption in this domain causes severe retinal degeneration, including attenuated retinal blood vessels and depigmentation.11
However, the R713Q mutation, found in the RP and congenitally blind patients, occurs in the cytoplasmic tail. This mutation probably disrupts the signal that activates the biochemical pathways required for the normal function of the cell. Multiple sequence alignments using Clustal analysis showed that R713Q is a conserved substitution and D345H is a semi‐conserved substitution in which an acidic amino acid is changed into a basic amino acid (http://www.ebi.ac.uk/clustalw/index.html
The novel identification of these mutations in patients as a cause of various retinal degenerations could be helpful to further understand the function of SEMA4A in the visual system and the role that it plays in the signalling mechanism to control the development of the outer retina.