Novel homozygous large deletion including the 5' part of the SPATA7 gene in a consanguineous Israeli Muslim Arab family

Abstract

Purpose: To identify the genetic defect in a consanguineous Israeli Muslim Arab family with juvenile retinitis pigmentosa (RP).

Methods: DNA samples were collected from the index patient, her parents, her affected sister, and two non-affected siblings. Genome-wide linkage analysis with 250 K single nucleotide polymorphism (SNP) arrays was performed using DNA from the two affected patients. Owing to consanguinity in the family, we applied homozygosity mapping to identify the disease-causing gene. The candidate gene SPATA7 was screened for mutations with PCR amplifications and direct Sanger sequencing.

Results: Following high-density SNP arrays, we identified several homozygous genomic regions one of which included the SPATA7 gene. Several mutations in SPATA7 have been reported for various forms of retinal dystrophy, including Leber congenital amaurosis (LCA) and juvenile RP. PCR-based sequence content mapping, long-distance PCR amplifications, and subsequent sequencing analysis revealed a homozygous 63.4 kb large deletion that encompasses the 5' part of the SPATA7 gene including exons 1-5. The mutation showed concordant segregation with the phenotype in the family as expected for autosomal recessive mode of inheritance and is consistent with a diagnosis of juvenile RP.

Conclusions: We report a novel homozygous large deletion in SPATA7 associated with juvenile RP in a consanguineous Israeli Muslim Arab family. This is the first larger deletion mutation reported for SPATA7.

Figure 1

Pedigree of family TR 11. The parents were first-degree cousins. Affected members are indicated with filled symbols, and unaffected members are represented by open symbols. The arrow indicates the index patient.

Figure 2

Retinal imaging findings. A: Funduscopy revealed the salt-and-pepper appearance of the retina, characteristic of juvenile retinitis pigmentosa (RP). B: Optical coherence tomography (OCT) image of the right eye. C: OCT image of the left eye. OCT imaging showed an overall decrease in retinal thickness with shortening of the photoreceptor outer segments, reduction in the outer nuclear layer, and RPE atrophy. The foveal photoreceptor layer was more preserved.

Figure 3

Breakpoint sequence of the homozygous deletion at the SPATA7 locus. Electropherogram of the breakpoint sequence was obtained from PCR amplification covering the deletion. The deletion allele sequence is given in red, and the left and right junction sequences are indicated by black and gray letters. While the centromeric junction of the deletion is located within the intergenic sequence between SPATA7 and its upstream gene KCNK10, the telomeric junction is located within the intronic sequence between exons 5 and 6 of SPATA7. Sequencing of the deletion junction showed that the deletion is accompanied by a small insertion of three extra nucleotides (TGG) at the deletion site.

Figure 4

Segregation analysis of the SPATA7 mutation in family TR 11. Affected members are indicated with filled symbols, and unaffected members are represented by open symbols. The arrow indicates the index patient. Segregation analysis was performed with all family members as denoted by the genotype data (underneath). Two distinct PCR amplifications were used to analyze deletion and wild-type alleles, respectively: (1) one PCR with primers directly flanking the deletion breakpoints (top panel, Del) and (2) one PCR with primers amplifying exon 2 of SPATA7 located within the deletion (bottom panel, Ex. 2). The latter indicates the presence of the wild-type allele. All PCR products were analyzed on a 2% agarose gel. According to autosomal recessive inheritance, both parents and both unaffected siblings are heterozygous, and the two affected sisters are homozygous mutation carriers. Del, deletion allele; +, wild-type allele; Ex. 2, exon 2 (in place of wild-type allele); M, size marker (KEB, pcDNA 3.1zeo/TaqI-digested, fragment sizes given in bp); Co, control (DNA of an unaffected, not related person); NC, negative control (water control).