A case of retinitis pigmentosa homozygous for a rare CNGA1 causal variant

Retinitis pigmentosa (RP) is a heterogenous hereditary disorder leading to blindness. Despite using next-generation sequencing technologies, causal variants in about 60% of RP cases remain unknown. The heterogeneous genetic inheritance pattern makes it difficult to pinpoint causal variants. Besides, rare penetrating variants are hardly observed in general case–control studies. Thus, a family-based analysis, specifically in a consanguineous family, is a clinically and genetically valuable approach for RP. We analyzed a Japanese consanguineous family with a member suffering from RP with a typical autosomal recessive pattern. We sequenced five direct descendants and spouse using Whole-exome sequencing (WES) and Whole-genome sequencing (WGS). We identified a homozygous pathogenic missense variant in CNGA1 (NM_000087.3, c.839G > A, p.Arg280His) in the proband, while we found no homozygous genotypes in the other family members. CNGA1 was previously reported to be associated with RP. We confirmed the genotypes by the Sanger sequencing. Additionally, we assessed the homozygous genotype in the proband for the possibility of a founder mutation using homozygosity analysis. Our results suggested the two copies of the variant derived from a founder mutation. In conclusion, we found the homozygotes for c.839G > A in CNGA1 as causal for RP.


Results
The family history showed a typical AR inheritance pattern (Fig. 1). We performed the whole genome sequence (WGS) for the proband (II-1) and the whole exome sequence (WES) for five unaffected members (II-2, III-1, III-2, III-3, III-4). The coverage of the targeted exon site was × 38 and × 106 ± 2.3(mean ± SD) for WGS and WES, respectively. After joint calling, we identified a total of 98,841 variants in at least one of the six subjects. Next, we filtered out variants with either DP < 20 or GQ < 20. As a result, 61,634 variants remained including 57,430 Single-Nucleotide Polymorphisms (SNPs), 1906 insertions, and 2301 deletions. Functional annotations revealed 13,331 (21.6%) missense, and 156 (0.25%) nonsense variants. Transition-transversion ratio (Ts/Tv) was 2.6. The Ts/Tv has been used as a QC metric in WES and is expected to above 2.0 within coding regions 10,11 . Supposing a rare pathogenic causal variant in this family, we further filtered variants with a maximum minor allele frequency < 0.01 in 1000 Genomes project 12 , NHLBI Exome Variant Server (https ://evs.gs.washi ngton .edu/EVS/), and gnomAD 13 (Fig. 2).   www.nature.com/scientificreports/ We then excluded variants for which proband's genotypes contained alternative alleles (i.e., not homozygous for a reference allele, 0/0). After the filtering, the 2725 variants remained. We further restricted candidates of a causal variant in 790 genes related to retinal diseases in the RetNet database (https ://sph.uth.edu/retne t/, updated on June 20, 2020), resulting in 43 variants. Since there was no loss of function variants, we focused on the 10 missense variants with the functional impact of "High" or "Moderate". We could not identify the genotype of the I-1, as we could not obtain the sample. However, we speculate I-1 may have this variant as a homozygous manner due to possible consanguineous marriage of his parents, as the regional characteristics of their living area. Thus, with the consideration of the AR inheritance pattern, we identified homozygous suspected-pathogenic variants in the two genes, namely, the CNGA1 (NM_000087.3, c.839G > A, p.Arg280His) and KCNV2 (NM_133497.4, c.1063T > C, p.Phe355Leu). CNGA1 is a well-known gene associated with RP. CNGA1 is a well-known gene associated with RP, and according to HDMG (http://www.hgmd.org), the variant was "disease causing" to RP (reported to be disease-causing in the corresponding literature) 9 . The variant in the KCNV2 was found in the Clinvar database with conflicting interpretations of pathogenicity for non-RP disorders (likely benign for dystrophy with supernormal rod response and uncertain significance for Retinal dystrophy). According to ACMG/ AMP guidelines 14 , the variant in the CNGA1 was classified as pathogenic (Supplemental Table S1), and the variant in the KCNV2 was classified as Uncertain Significant (Supplemental Table S1). From these annotation analyses, we considered the variant in the CNGA1 as a causal variant for arRP in this family. Then, to analyze whether the homozygote variants derived from a common ancestor, we performed ROH analysis. As a result, the homozygous regions in the affected proband (II-1) had a total of 31 (129 Mb) homozygous regions that contained the homozygous variants in the CNGA1. The unaffected four descendants had no homozygous regions, and the mother had 1 homozygous part on chromosome 3 (3.4 M). These findings confirmed strong consanguinity in the proband and indicated that the homozygous variants in the CNGA1 derived from a founder mutation.
We performed direct sequencing for the variant in the CNGA1 in all subjects using the Sanger Sequence. We confirmed that the affected proband was the only one who carried two copies of the variant (Fig. 1).

Discussion
In the present study, we identified a homozygous disease-causing variant c.839G > A in the CNGA1 in a consanguineous Japanese family with arRP using NGS sequencing and Sanger sequencing.
Our study showed two important novel points. First, this is the first time to classify the variant in the CNGA1 (NM_000087.3, c.839G > A, p.Arg280His) as pathogenic according to the ACMG/AMP guideline 14 (Supplementary Table S1). Although the variant has already been found to be associated with RP in a large sequencing study in a Japanese population 9 , to predict the pathogenicity, the previous study did not follow the ACMG/ AMP guideline 14 but depended on in-silico prediction algorithms and conservation scores. The evaluation of pathogenicity based on the ACMG/AMP guideline is clinically important since clinicians see their patients and diagnose genetic diseases in the clinical settings based on evaluations of variants by the ACMG/AMP guideline. Furthermore, it is essential to validate a variant's pathogenicity in independent samples. Otherwise, a single case with the variant is used to define its pathogenicity, and the definition will affect many potential patients. Second, in our study, we found that the variant in the family of the current analysis derived from a founder mutation.
CNGA1 is known to be a susceptibility gene to RP, especially arRP. The estimated prevalence of RP with variants in the CNGA1 is approximately 2-5% with a slight deviation to the Asian population [15][16][17] . Among the causative gene with arRP, the CNGA1 was estimated to account for a similar proportion 17 to the EYS gene (5-16%) 18,19 The first report of patients with arRP with variants in the CNGA1 was in 1995 20 . To date, 28 missense/nonsense, 10 small deletion mutations, and 1 splicing substitutions in the CNGA1 are found in the HDMG database (http:// www.hgmd.org). The variant c.839G > A in the CNGA1 is too rare (allele frequency: 0.0032% from gnomAD) to be found in the general population. Interestingly, this variant has also been identified homozygous in a Japanese RP patient 9 . However, there are no details about clinical and family information, and no functional analysis was conducted other than in silico analysis 9 .
Despite the genetic evidence that the homozygous genotype of this variant leads to RP, its detailed mechanism is still unknown. The CNGA1 encodes the α subunit of cyclic nucleotide-gated (CNG) channels, one of the cGMP-binding transmembrane channels of cone cells 20 . CNG channel is essential for maintaining the structure and function of photoreceptor cells [21][22][23] . In the UniProtKB (P29973), there are four known functional domains CNGA1 protein as follows: P-helix (residues 350-360), Selectivity filter (residues 361-369), C-linker (residues 402-484), Cyclic nucleotide-binding domain (residues 485-612) and C-terminal coiled-coil domain (residues 623-666). However, the region containing c.839G > A (p.Arg280His) does not overlap with the functional domains. Therefore, further molecular experiments are necessary to understand the molecular mechanism of this variant.
We found another rare homozygous candidate variant c.1063T > C in the KCNV2. The KCNV2 is known as causing Cone dystrophy retinal 3B (RCD3B) 24 . RCD3B is a rare disease with supernormal rod responses, which is distinct from RP with peripheral retina atrophy 25 . We found that KCNV2 was also in the homozygous region, which implicated this variant also derived from the same ancestor in spite of unknown functional significance.
In conclusion, we identified a homozygous rare pathogenic variant c.839G > A in CNGA1 in a consanguineous Japanese family with arRP using NGS sequencing and the Sanger Sequence. Additionally, this is the first study to classify the variant's pathogenicity according to the ACMG/AMP guideline. Our results also suggested the variant c.839G > A derived from a founder mutation. Furthermore, future functional studies are necessary to conclude the effects of the variant in this study as well as the other known pathogenic variants. www.nature.com/scientificreports/

Methods
Ethics. This study complied with the standards of the Declaration of Helsinki and the ethics committee approved this study of Shizuoka General Hospital, and we obtained written informed consent from all the participants.

Subject.
A total of six members participated in this study (Fig. 1). An ophthalmologist diagnosed the proband (II-1) with RP based on comprehensive ophthalmologic examinations, slit-lamp biomicroscopy, color fundus photography, fundus autofluorescence, and ISCEV Standard electroretinogram (The imaging findings of the proband was shown in Fig. 3). All the six participants have performed a fundus examination and electroretinogram. The interviews with the proband determined the affection of the other family members, including their information on daily visual matters. The proband (II-1) is 67 years old. He was diagnosed with RP at the age of 56 and has suffered from visual impairment since then.
DNA sequence using the Next-generation sequencer. According to the manufacturer's instruction, the genomic DNA of each member was extracted from peripheral blood using PAXgene Blood DNA Kit (Qiagen). The samples were sent to Macrogen Japan Corp. at the University of Kyoto to perform NGS using Novaseq 6000 (Illumina, San Diego, CA, United States) on a 150-bp paired-end read protocol. We conducted the wholeexome sequence (WES), using the SureSelect Human All Exon Kit v6 (Agilent, Santa Clara, CA, United States). We conducted the whole-genome sequence (WGS), using the TruSeq DNA PCR-Free Library Preparation Kit (Illumina, San Diego, CA, United States).

PCR.
To validate genotypes of a pathogenic variant in CNGA1, we conducted direct sequencing. A part of exon 11 of CNGA1 was amplified by PCR using primers of 3′ GGA CTG CTG GTA AAG GAA GAA CTT AAA CTC 5′ for sense primer and 3′ CAC CAA TGG TAG TCA AAG TCA GTG TAG ACC 5′ for antisense primer. The sequencing was carried out by using a sense primer. with a 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA).

Data availability
The list of variants analyzed in this study is available upon reasonable request to the corresponding author. Data not available due to ethical restrictions.