A novel splicing mutation in the PRPH2 gene causes autosomal dominant retinitis pigmentosa in a Chinese pedigree

Retinitis pigmentosa (RP) (OMIM: 268000) is a rare, heterogeneous group of inherited ocular disorders that results in a progressive retinal degeneration.1, 2 The PRPH2 gene (NM_000322.4) (OMIM: 179605), also known as RDS, AOFMD, AVMD, CACD2, DS, MDBS1, PRPH, rd2, RP7 and TSPAN22, is located on chromosome 6p21.1 with three exons spanning 26 395 bp length in human genome (GRCh38/hg38) that encodes a putative protein with 346 amino acids.3 The PRPH2 protein ({"type":"entrez-protein","attrs":{"text":"NP_000313.2","term_id":"118572596"}}NP_000313.2) is a member of the transmembrane 4 superfamily, also known as the tetraspanin family. The majority of the members are cell‐surface proteins which were identified by the presence of four hydrophobic domains. The PRPH2 protein is a membrane‐associated glycoprotein, which is restricted to the area of photoreceptor outer segment discs.4 PRPH2 functions as an adhesion molecule by stabilization and compaction of outer segment discs. PRPH2 and ROM1 (OMIM: 180721) can be assembled into noncovalent tetramers (heterodimer) in vivo using disulphide bonds and higher order disulphide‐linked oligomers, thereby involving in photoreceptor disc morphogenesis.5 
 
Mutations in the PRPH2 gene are involved with assorted blinding diseases of the retina, inducing degenerations in both central retinal and peripheral retinal.6, 7, 8 The relationships between the mutations in the PRPH2 gene and the resultant diseases in the patients are variable; making genotype/phenotype correlations different. PRPH2 mutation in RP patients and genotype/phenotype relationship have not been well described in the Chinese population.


| INTRODUC TI ON
Retinitis pigmentosa (RP) (OMIM: 268000) is a rare, heterogeneous group of inherited ocular disorders that results in a progressive retinal degeneration. 1,2 The PRPH2 gene (NM_000322.4) (OMIM: 179605), also known as RDS, AOFMD, AVMD, CACD2, DS, MDBS1, PRPH, rd2, RP7 and TSPAN22, is located on chromosome 6p21.1 with three exons spanning 26 395 bp length in human genome (GRCh38/hg38) that encodes a putative protein with 346 amino acids. 3 The PRPH2 protein (NP_000313.2) is a member of the transmembrane 4 superfamily, also known as the tetraspanin family. The majority of the members are cell-surface proteins which were identified by the presence of four hydrophobic domains. The PRPH2 protein is a membrane-associated glycoprotein, which is restricted to the area of photoreceptor outer segment discs. 4 PRPH2 functions as an adhesion molecule by stabilization and compaction of outer segment discs. PRPH2 and ROM1 (OMIM: 180721) can be assembled into noncovalent tetramers (heterodimer) in vivo using disulphide bonds and higher order disulphide-linked oligomers, thereby involving in photoreceptor disc morphogenesis. 5 Mutations in the PRPH2 gene are involved with assorted blinding diseases of the retina, inducing degenerations in both central retinal and peripheral retinal. [6][7][8] The relationships between the mutations in the PRPH2 gene and the resultant diseases in the patients are variable; making genotype/phenotype correlations different. PRPH2 mutation in RP patients and genotype/phenotype relationship have not been well described in the Chinese population. proband ( Figure 1A, pedigree II: 1, arrow). For detailed clinical assessments, a clinical history and ophthalmic examination were previously described. 9,10 Genomic DNA (gDNA) was extracted using a reported phenol/chloroform method. 11

| Target sequencing and data analysis
Targeted next-generation sequencing (TGS) analyses were performed on the gDNA sample of the proband from family M074. 12,13 The capture Agilent probes were used as in previously published studies 2,12-14 with a retinal disease capture panel with 195 retinal disease-causing genes. Library construction and sequencing were used according to the manufacturer's protocols. 15 Data analysis and sequenced variants identification were described in detail previously. 2,10

| Sanger sequencing and co-segregation analysis
The amplified PCR products were then Sanger sequenced using a primer M074-PRPH2-868L. Co-segregation analysis in the pedigree was finished by using Sanger sequencing results.

| RNA extraction and reverse transcription-PCR
RNA was extracted from mice according to our previously reported standard protocol. 2 Semi-quantitative RT-PCR was was used as an internal control, which was described previously. 2

| Proband and clinical characteristics
The proband ( Figure 1A, II: 1) was a 60-year-old Chinese male, and

| TGS results and putative pathogenic mutation screening
By TGS with the proband gDNA ( Figure 1A

| Variant verification and segregation analysis
Confirmation of the variant and co-segregation analysis was done by Sanger sequencing (Figure 1). The c.582-2A>T mutation of the PRPH2 gene was verified to be heterozygous in the proband (pedigree II: 1; Figure 1D), while we revealed the wild-type gene in the proband's daughter without RP symptoms till test at the ages of 30 (pedigree III: 1; Figure 1E), and the proband's wife had two copies of the wild-type allele and a normal phenotype (pedigree II: 2; Figure 1F). Therefore, we validated that the c.582-2A>T mutation in the PRPH2 gene is co-segregated with the RP disease phenotype in these pedigree members. Furthermore, the c.582-2A>T variant was absent in 100 normal, ethnically matched controls by Sanger sequencing. Comprehensively, this finding shows co-segregation of the variant in this RP pedigree and pinpoints c.582-2A>T variant role in pathogenesis of this RP disease. Unfortunately, no DNA samples were available due to the death of proband's parents. The c.582-2A>T variant of the PRPH2 gene might be de vivo as no any RP phenotypes were claimed in the proband's parents.

| Expression profiles of Prph2 mRNA level
The Prph2 expression in the indicated tissues and developmental retinal stages was studied in mice ( Figure 2D and E). We found that Prph2 transcript is highly expressed in the retina, lens, sclera and cornea of the eye; is weakly expressed in the testis; have no detectable expression in the uterus, ovary, breast, spleen, kidney, liver, brain, intestine, skeletal muscle and blood ( Figure 2D

| D ISCUSS I ON
Earlier diagnosis and managements result in a better prognosis. In conclusion, our research is the first to identify the novel heterozygous mutation c.582-2A>T of PRPH2, which might causes RP disease in our Chinese family, thereby extending mutation spectrums. Our findings can also help in further understanding of adRP molecular pathogenesis, and assist the diagnosis and genetic counselling of the RP disease.