Two Novel Mutations in the EYS Gene Are Possible Major Causes of Autosomal Recessive Retinitis Pigmentosa in the Japanese Population

Retinitis pigmentosa (RP) is a highly heterogeneous genetic disease including autosomal recessive (ar), autosomal dominant (ad), and X-linked inheritance. Recently, arRP has been associated with mutations in EYS (Eyes shut homolog), which is a major causative gene for this disease. This study was conducted to determine the spectrum and frequency of EYS mutations in 100 Japanese arRP patients. To determine the prevalence of EYS mutations, all EYS exons were screened for mutations by polymerase chain reaction amplification, and sequence analysis was performed. We detected 67 sequence alterations in EYS, of which 21 were novel. Of these, 7 were very likely pathogenic mutations, 6 were possible pathogenic mutations, and 54 were predicted non-pathogenic sequence alterations. The minimum observed prevalence of distinct EYS mutations in our study was 18% (18/100, comprising 9 patients with 2 very likely pathogenic mutations and the remaining 9 with only one such mutation). Among these mutations, 2 novel truncating mutations, c.4957_4958insA (p.S1653KfsX2) and c.8868C>A (p.Y2956X), were identified in 16 patients and accounted for 57.1% (20/35 alleles) of the mutated alleles. Although these 2 truncating mutations were not detected in Japanese patients with adRP or Leber's congenital amaurosis, we detected them in Korean arRP patients. Similar to Japanese arRP results, the c.4957_4958insA mutation was more frequently detected than the c.8868C>A mutation. The 18% estimated prevalence of very likely pathogenic mutations in our study suggests a major involvement of EYS in the pathogenesis of arRP in the Japanese population. Mutation spectrum of EYS in 100 Japanese patients, including 13 distinct very likely and possible pathogenic mutations, was largely different from the previously reported spectrum in patients from non-Asian populations. Screening for c.4957_4958insA and c.8868C>A mutations in the EYS gene may therefore be very effective for the genetic testing and counseling of RP patients in Japan

Allelic copy number variation in FSCN2 detected using allele-specific genotyping and multiplex real-time PCRs

PURPOSE. Allelic copy number variation (CNV) may alter the functional effects of a heterozygous mutation. The underlying mechanisms and their roles in hereditary diseases, however, are largely unknown. In the present study an FSCN2 mutation was examined that has been reported, not only in patients with retinitis pigmentosa (RP), but also in the normal population. METHODS. Experiments were performed to investigate the gene and allele copy numbers of FSCN2 in patients with RP who have the c.72delG mutation as well as healthy subjects with or without the mutation. A real-time PCR-based genotyping approach was established that used a real-time PCR assay to qualify the copy numbers of both the wild-type and mutant alleles of the FSCN2 gene. RESULTS. Three patients with RP and three normal subjects had an equal ratio of the alleles. Of interest, another patient had an asymmetric allele ratio (4:1) of the copy number of the wildtype allele, compared with that of the mutant allele. These findings were further verified using quantitative assays. An allele-specific methylation assay demonstrated a random methylation pattern in the FSCN2 gene. CONCLUSIONS. The copy numbers of the FSNC2 gene and of each allele in the mutant samples were quantified. The findings excluded the possibility that allelic CNV was associated with RP, suggesting that the c.72delG variant is not the primary cause of RP. It is not likely that the FSCN2 gene is imprinted differentially. The real-time PCR-based genotyping method developed in this study is useful for investigations of allelic asymmetries within genomic regions with CNVs. (Invest Ophthalmol Vis Sci. 2008;49:3799 -3805

Retinitis Pigmentosa with EYS Mutations Is the Most Prevalent Inherited Retinal Dystrophy in Japanese Populations

The aim of this study was to gain information about disease prevalence and to identify the responsible genes for inherited retinal dystrophies (IRD) in Japanese populations. Clinical and molecular evaluations were performed on 349 patients with IRD. For segregation analyses, 63 of their family members were employed. Bioinformatics data from 1,208 Japanese individuals were used as controls. Molecular diagnosis was obtained by direct sequencing in a stepwise fashion utilizing one or two panels of 15 and 27 genes for retinitis pigmentosa patients. If a specific clinical diagnosis was suspected, direct sequencing of disease-specific genes, that is, ABCA4 for Stargardt disease, was conducted. Limited availability of intrafamily information and decreasing family size hampered identifying inherited patterns. Differential disease profiles with lower prevalence of Stargardt disease from European and North American populations were obtained. We found 205 sequence variants in 159 of 349 probands with an identification rate of 45.6%. This study found 43 novel sequence variants. In silico analysis suggests that 20 of 25 novel missense variants are pathogenic. EYS mutations had the highest prevalence at 23.5%. c.4957_4958insA and c.8868C>A were the two major EYS mutations identified in this cohort. EYS mutations are the most prevalent among Japanese patients with IRD

Predicted domain structure and distribution of identified <i>EYS</i> mutations.

<p>SMART (<a href="http://smart.embl-heidelberg.de/" target="_blank">http://smart.embl-heidelberg.de/</a>) and Pfam (<a href="http://pfam.sanger.ac.uk/" target="_blank">http://pfam.sanger.ac.uk/</a>) were used to search protein functional domains. A coiled-coil domain identified by Barragán et al. (2010) between the EGF-like domain and laminin G domain was also indicated. Novel very likely pathogenic mutations, novel possible pathogenic mutations, and a previously described mutation are shown in bold, normal, and italic type, respectively. Six out of 9 missense mutations were found in the EGF or laminin G domains. Furthermore, 7 were located in the latter half of the protein between the putative coiled-coil region and C-terminus.</p

Electropherograms of the 6 likely pathogenic <i>EYS</i> mutations.

<p>Partial sequence of the <i>EYS</i> gene showing the normal control sequences (A-1 through F-1), heterozygous mutation sequences (A-2 through F-2), and homozygous mutation sequences (A-3 and C-3). Deduced amino acids are indicated under the sequence trace. The mutation location is indicated either by an arrow (for a nucleotide change) or a horizontal line (to show 2 nucleotides between which the insertion occurred). (A) c.4957_4958insA; p.S1653KfsX2 (Exon 26), (B) c.6557G>A; p.G2186E (Exon 32), (C) c.8868C>A; p.Y2956X (Exon 44), (D) c.8351T>G; p.L2784R (Exon 44), (E) c.7793G>A; p.G2598D (Exon 40), (F) c.2522_2523insA; p.Y841X (Exon 16).</p

Pedigrees of the families that was available for mutation analysis.

<p>Below the individuals, genotypes are presented for either p.S1653KfsX2 (M1), p.L2784R (M2), p.Y2956X (M3), p.Y841X (M4), or p.G2186E (M5) detected to segregate with RP. M1/M1 represents homozygous mutation. M1/+ indicates heterozygous carriers, +/+ indicates individuals carrying 2 wild-type alleles, whereas M1/M2 represents individuals presenting both mutations as compound heterozygous. Square boxes indicate men, circles denote women, and affected individuals are pointed out by a black symbol. Slashed symbols indicate deceased individuals. The probands are indicated with an arrow. NA denotes unavailable DNA samples.</p

Summary of the very likely and possible pathogenic mutations identified in 100 Japanese arRP patients.

a<p>EYS contains a signal peptide, a putative coiled-coil, 29 EGF, and 5 laminin G domains. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031036#pone-0031036-g003" target="_blank">Fig. 3</a>.</p>b<p>hu/o/m/ho/d/op/p/c/z/dr denotes Human/Orangutan/Marmoset/Horse/Dog/Opossum/Platypus/Chicken/Zebrafish/Drosophila EYS orthologs, respectively. The hyphen (-) indicates that genomic sequence of corresponding region in the species was reported to be unknown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031036#pone.0031036-Barragn1" target="_blank">[5]</a>.</p>c<p>SIFT, PolyPhen2 (only the HumDiv data are shown), PMut, and SNAP were used as reference data to evaluate the pathogenicity of the missense mutations. c.77G>A, c.2923T>C, c.7793G>A, c.8351T>G, and c.9272T>C were predicted to be pathogenic by a number of different computational prediction programs. In addition, the c.6557G>A mutation, which had been previously reported as disease causing, was classified as pathogenic by the PolyPhen2, PMut, and SNAP programs.</p>d<p>Homozygous exon 32 deletion mutation was not detected in 200 controls.</p

Summary of the possible non-pathogenic sequence alterations in the <i>EYS</i> gene identified in this study.

<p>Fifty-four sequence alterations were identified in 100 patients. These alterations were predicted to be non-pathogenic for various reasons. Some have been reported as polymorphisms in previous reports. Newly identified alterations within the exons, except for c.334G>A and c.8923T>C, were also found in the control chromosome. The hyphen (-) indicates that genomic sequence of corresponding region in the species was reported to be unknown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031036#pone.0031036-Barragn1" target="_blank">[5]</a>.</p>a<p>hu/o/m/ho/d/op/p/c/z/dr denotes Human/Orangutan/Marmoset/Horse/Dog/Opossum/Platypus/Chicken/Zebrafish/Drosophila EYS orthologs, respectively.</p