Lack of phenotypic effect of triallelic variation in SPATA7 in a family with Leber congenital amaurosis resulting from CRB1 mutations

Abstract

Purpose: To identify the causative gene for autosomal recessive Leber congenital amaurosis (LCA) in a Chinese family.

Methods: One Chinese LCA family was identified and an ophthalmologic examination was performed. The genetic defects were analyzed simultaneously by a genome-wide linkage scan with 382 polymorphic microsatellite markers, as well as by comprehensive mutational screening of 15 genes known to associate with LCA on the genomic DNA of this family.

Results: Suggestive linkages were found in 13 chromosomal regions, of which only one harbored a known causative gene, crumbs homolog 1 (CRB1), on chromosome 1. Sanger sequencing of CRB1 identified two novel heterozygous mutations, c.3221T>C (p.L1074S) and c.2677-2A>C. In addition, a novel missense heterozygous mutation, c.938C>A (p.A313D), in spermatogenesis associated 7 (SPATA7), was detected in the proband after screening of the other 14 LCA causative genes. All three affected individuals of the family had compound heterozygous CRB1 mutations, and one of the three (the proband) had an additional mutation in SPATA7. The unaffected mother had the heterozygous c.3221T>C mutation in CRB1 and the heterozygous c.938C>A mutation in SPATA7. The unaffected father could not be tested, but presumably had the heterozygous c.2677-2A>C mutation in CRB1. The proband, with triallelic mutations in CRB1 and SPATA7, had a phenotype similar to other two affected brothers, suggesting the additional mutant allele in SPATA7 might not contribute to the disease. Similarly, the mother, with digenic mutations in CRB1 and SPATA7, had normal vision and fundi, suggesting the digenic mutations in these two genes might not cause disease.

Conclusions: Digenic and triallelic mutations of CRB1 and SPATA7 were detected in a family with LCA. Our results imply that CRB1 and SPATA7 may not interact with each other directly. This emphasizes that care should be taken in invoking a mutation-disease association for digenic and triallelic mutations.

Figure 1

Pedigree and haplotypes: sequence changes of family 83002. (The DNA sample for I:1 was not available.) A: Haplotypes of the CRB1 region of family 83002 showing the CRB1 c.3221T>C and c.2677–2A>C mutations and surrounding microsatellite markers included in Table 1. The risk haplotype is shown in black and green. B, C: Electropherograms demonstrate sequences in the regions with the CRB1 c.3221T>C and c.2677–2A>C mutations in individuals I:2, II:1, II:2, II:3, and a normal control. D: Electropherograms show the c.938C>A mutation in SPATA7 of individuals I:2 and II:1.

Figure 2

Protein sequence alignment. A: CRB1 L1047 amino acid(red) in ten species ranging from humans to zebrafish. L1047 is conserved in all species from Homo sapiens to Danio rerio as ascertained by a bioinformatic search of NCBI-Blast by means of human CRB1 DNA and protein and MegAlign (DNASTAR Lasergene, Madison, WI). And this may indicate functional or structural significance in L1047. B: SPATA7 A313 amino acid(red) in ten species ranging from humans to mice. A313 is relatively conserved among mammal.

Figure 3

Sagittal curvature of the cornea. Only the right eye of the four members of the family are shown, since the sagittal curvatures of both eyes in each individual were similar. The number of color scale presents the degree of the curvature of cornea. The corneal diameters and corneal curvatures of the three patients(II:1, II:2 and II:3) were similar with unaffected mother (I:2) and within normal range.

Figure 4

Fundus photos of the four members of the family. The mother (I:2), with digenic mutations, had a normal fundus appearance. All three patients (II:1, II:2, and II:3) from the family had similar fundus changes, including waxy, pale optic discs, artery attenuation, generalized carpetlike retinal degeneration, macular atrophy, nummular pigmentation at the posterior pole, and irregular pigmentation and white dots in the midperipheral region. The fundus changes in the proband (II:1) with triallelic mutations in CRB1 and SPATA7 were similar to the those of the other two patients (II:2 and II:3) with compound heterozygous CRB1 mutations.

Figure 5

Optical coherence tomography of three patients shows thinning of the retina and loss of photoreceptor layers compared with the mother (I:2) who has normal fundus. Patient II:1 had similar changes, compared to patients II:2 and II:3. It indicated the affected member II:1 who carried a third mutant allele in a second gene may not have more severe phenotypes than other affected members in the family.