GPR143 Gene Mutations in Five Chinese Families with X-linked Congenital Nystagmus

The ocular albinism type I (OA1) is clinically characterized by impaired visual acuity, nystagmus, iris hypopigmentation with translucency, albinotic fundus, and macular hypoplasia together with normally pigmented skin and hair. However, it is easily misdiagnosed as congenital idiopathic nystagmus in some Chinese patients with OA1 caused by the G-protein coupled receptor 143 (GPR143) gene mutations. Mutations in the FERM domain–containing 7 (FRMD7) gene are responsible for the X-linked congenital idiopathic nystagmus. In this study, five Chinese families initially diagnosed as X-linked congenital nystagmus were recruited and patients underwent ophthalmological examinations. After direct sequencing of the FRMD7 and GPR143 genes, five mutations in GPR143 gene were detected in each of the five families, including a novel nonsense mutation of c.333G>A (p.W111X), two novel splicing mutations of c.360+1G>C and c.659-1G>A, a novel small deletion mutation of c.43_50dupGACGCAGC (p.L20PfsX25), and a previously reported missense mutation of c.703G>A (p.E235K). Optical coherence tomography (OCT) examination showed foveal hypoplasia in all the affected patients with nystagmus. Our study further expands the GPR143 mutation spectrum and contributes to the study of GPR143 molecular pathogenesis. Molecular diagnosis and optical coherence tomography (OCT) are two useful tools for differential diagnosis.

have been documented to be the cause of congenital idiopathic nystagmus 3 , while GPR143 gene mutations are believed to be responsible for ocular albinism type I (OA1) 4 .
The typical clinical features of the OA1 include impaired visual acuity, nystagmus, iris hypopigmentation with translucency, albinotic fundus, and macular hypoplasia together with normally pigmented skin and hair. As nystagmus can be the prevalent feature of OA1, it is easily misdiagnosed as congenital idiopathic nystagmus, especially for those patients without typical iris hypopigmentation and albinotic fundus changes. Molecular diagnosis and optical coherence tomography (OCT) are two useful tools for differential diagnosis. However, the treatment principle for OA1 is the same as that for congenital idiopathic nystagmus.
Here we describe five Chinese families with X-linked congenital nystagmus (CN). Molecular genetic analysis of the two candidate genes (FRMD7 and GPR143) indicated that five mutations in GPR143 were detected in these families. Macular hypoplasia was found by OCT in the patients with GPR143 mutations.

Patient Ascertainment
Five families with X-linked congenital nystagmus (NYS-17, NYS-18, NYS-19, NYS-20, and NYS-21) were recruited for this study (Fig. 1). All participants underwent an ophthalmologic examination, including best corrected visual acuities, anterior segment of the eyes, vitreous and fundus, OCT, Visual Evoked Potential (VEP) and electroretinography (ERG). After informed consent, 25 affected and 34 unaffected individuals from these five unrelated families had 3 ml blood samples taken and DNA was extracted from blood lymphocytes according to standard protocols (Roche Biochemical, Inc). This study obtained the institutional review board (IRB) approval from the Tianjin Eye Hospital and conformed to the tenets of the Declaration of Helsinki.
Mutation Analysis. The sequence analysis of FRMD7 and GPR143 was carried out by direct genomic DNA sequencing after PCR amplification of whole coding regions and exon-intron boundaries of these two genes using the previously published primers 5 . PCR was carried out in 20 μ L of standard PCR buffer containing 1.5 mM MgCl 2 , 0.2 mM of each dNTP, 0.5 μ M of each primer, 1 U of Taq polymerase (Sangon, Shanghai, China), and 50 ng of DNA. The amplification program was an initial 2 min denaturation at Reverse transcription PCR and real-time quantitative PCR. As we only obtained RNA from the NYS-17 family, real-time quantitative PCR was performed to analyze the GPR143 gene dosage change in this family. Total RNA was prepared from venous blood (0.2 ml) from the affected male individuals, the female carriers and the normal controls, using the RNA isolation kit for mammalian blood (Sangon, Shanghai, China). One microgram of total RNA from each sample was reverse transcribed into cDNA. The primer sequences for the GPR143 gene were CCGTGTGGTTAGGATTCCC (forward) and CCCACGCCATGATGTGATAC (reverse). As an endogenous control, the GAPDH gene with the primer sequences CCAAAATCAAGTGGGGCGAT (forward) and TGATCTTGAGGCTGTTGTCA (reverse) was used. For amplification of GPR143 and GAPDH, PCR reactions conditions were 95 °C, 3 min; 95 °C, 30 s; 58 °C 30 s and 72 °C, 1 min for 35 cycles with a final elongation step at 72 °C for 5 min. Real-time quantitative PCR was performed using the SYBR Premix ExTaq kit (Takara, Dalian, China) in accordance with the manufacturer's instructions. The PCR cycling conditions were as follows: 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 40 s. Real-time quantitative PCR was performed using Eppendorf Mastercycler ep realplex real-time PCR system. The cycle threshold (C T ) values were obtained from each PCR reaction. Data analyses were performed using the relative quantitative method (2 −ΔΔCT ) for calculating relative gene expression. Δ Δ C T = Δ C T sample -Δ C T control.

Results
Clinical features. All the families (NYS-17, NYS-18, NYS-19, NYS-20, and NYS-21) were referred to Tianjin Eye Hospital for congenital nystagmus. We evaluated the clinical features for 9 patients and 5 female carriers in these five families ( Table 1). The most common clinical features among the patients were nystagmus, impaired visual acuity, variable hypopigmentation in the fundus and macular hypoplasia detected by OCT. However, iris hypopigmentation with translucency was not evident. With the exception of the proband in the NYS-18 family, having a chin-down head position to compensate his vertical nystagmus, other probands in the NYS-17, NYS-19, NYS-20, and NYS-21 families showed a horizontal pendular nystagmus in the primary eye position without a compensative head position. In addition, exotropia was one of the clinical features in the NYS-17 family. The NYS-17 family was a large highly consanguineous family including 11 patients and 6 female carriers (Fig. 1). The individual III:1 was a 65-year-old male diagnosed with congenital nystagmus combined with exotropia. He married his cousin (individual III:2), a female carrier without nystagmus and exotropia. In his family, two of his   (Fig. 4). Real-Time quantitative PCR revealed that the level of GPR143 transcripts of the female carriers were about one-half the control level (Fig. 5). Two potential splicing mutations, c.360+ 1G> C and c.659-1G> A in GPR143, were detected in the families of NYS-18 and NYS-19 respectively. The Human Splicing Founder Server program predicted that these two splicing mutations would produce a consensus value (CV) variation score of -29.06 for c.360+ 1G> C and -32.5 for c.659-1G> A respectively, and lead to skipping of the wild type splice site.
In family NYS-20, an 8 bp duplication c.43_50dupGACGCAGC (p.L20PfsX25) in exon 1 of GPR143 was identified in the affected male and his mother. A previously reported missense mutation c.703G> A ( p.E235K) 7 was detected in exon 6 of GPR143 in NYS-21 family. The multiple sequence alignment of the GPR143 protein shows that E235 was conserved among Homo sapiens, Pongo abelii, Rattus norvegicus, Mus musculus, Bos taurus, Gallus gallus, Xenopus laevis, and Danio rerio (Fig. 6). A position-specific independent count (PISC) score of 1.0 was obtained after using the POLYPHEN program (http://coot. embl.de/PolyPhen/) to predict the functional and structural changes of the amino acid substitution. All the mutations described above were absent in 100 normal controls. Sequence analysis of the FRMD7 gene showed that no mutations were detected in these five families.

Discussion
In this study, we describe the clinical characteristics of five Chinese families with X-linked nystagmus, and have identified four novel mutations in these families, c.333G> A (p.W111X), c.360+ 1G> C, c.659-1G> A and c.43_50dupGACGCAGC (p.L20PfsX25), and a known mutation c.703G> A (p.E235K) in these families.
The GPR143 gene consists of 9 exons spanning approximately 40 kb on chromosome Xp22.3, and encodes a protein of 404 amino acids. It is highly expressed in the retinal pigment epithelium (RPE) and melanocytes. The GPR143 protein contains seven putative transmembrane domains, and is localized to RPE melanosomal membranes. It functions as a receptor of tyrosine, L-DOPA and dopamine, playing an important role in melanosome biogenesis, organization and signal transduction 8,9 . To date, more than one hundred mutations in GPR143 have been reported to be responsible for ocular albinism type I (Human Gene Mutations Database http://www.hgmd.cf.ac.uk). However, the exact molecular mechanism of how the mutated GPR143 causes ocular abnormalities, such as nystagmus and macular hypoplasia, is still unknown. It has been proposed that GPR143 could be involved in melanosomal biogenesis as a ligand of L-DOPA, a precursor in melanin synthesis. Mutations in GPR143 could affect melanin synthesis in the retinal pigment epithelium (RPE), and result in abnormal development of the retina and visual pathways, which could be the cause of the misrouting of the optic fibers, nystagmus, foveal hypoplasia, and reduced visual acuity [10][11][12][13][14] .
The nonsesnse mutation of c.333G> A (p.W111X) in exon 2 occurred in the transmembrane domain of the GPR143 protein and alters the length of the open reading frame, creating an aberrant truncated protein because of a wild type amino acid of tryptophan (Try, W) at codon 111 substituted by a premature termination codon (PTC). The nonsense-mediated mRNA decay (NMD) mechanism could be activated to degrade mRNA with a PTC in order to prevent the truncated harmful protein interfering with normal cellular processes 15 . We have demonstrated in this study that the GPR143 mRNA level was about 50% lower in female carriers than in normal controls in the family NYS-17.
The splicing mutation c.360+ 1G> C in GPR143 occurred in the 5′ consensus donor region for the splicing of intron 2-3, while the splicing mutation c.659-1G> A occurred in the 3′ consensus acceptor region for the splicing of intron 5-6. Both these splicing mutations would be predicted to result in loss of the original splicing site, exon skipping, activation of a cryptic splice site and introduction of a premature termination codon (PTC) 6 . Using the online tools of HSF program and ORF finder (https://www. ncbi.nlm.nih.gov), we found that the splicing mutation of c.360+ 1G> C would be predicted to produce a truncated protein with the initial 120 amino acids the same as wild type GPR143 and an additional 27 aberrant amino acids followed by a premature termination codon (PTC). While the splicing mutation c.659-1G> A would be predicted to produce a truncated protein with the initial 219 amino acids identical to the wild type GPR143 protein and an additional 3 aberrant amino acids followed by a premature termination codon (PTC). Similarly, the duplication mutation of c.43_50dupGACGCAGC (p.L20PfsX25) in exon 1 would be predicted to result in a frame shift and produce a truncated protein with the initial 19 amino acids the same as the wild type GPR143 protein and an additional 5 aberrant amino acids followed by a premature termination codon (PTC). Thus, the NMD surveillance mechanism could be activated by a PTC-containing mRNA. The abnormal mRNA with a PTC would be degraded under the NMD surveillance mechanism or produce a defective truncated protein through escaping the NMD surveillance 16,17 .
The missense mutation of c.703G> A (p.E235K) has been reported previously in North American patients with X-linked ocular albinism 7 .However, the clinical phenotypes caused by E235K mutation could be more complicated in North American Patients than in the Chinese patients, because Schnur and colleagues reported that the patient with E235K mutation had periodic alternating nystagmus, photophobia, high myopia, macular hypoplasia, albinotic fundus, iris transillumination, cleft lipand palate 7 , while the Chinese patients carrying with E235K mutation only showed pendular nystagmus, albinotic fundus and macular hypoplasia.
In addition, the patients carrying GPR143 mutations had macular hypoplasia obtained by OCT, which further supported clinically that congenital nystagmus in these patients sensory defect type and caused by the maldevelopment of the macular or visual pathways. Mutations in the GPR143 gene have been reported in Chinese families with nystagmus 5,18-23 . However, the previous reports that some patients with isolated X-linked congenital nystagmus 19,22,23 were caused by the GPR143 mutations should be considered carefully, because there were not sufficient clinical data in these reports to support the hypothesis of isolated nystagmus from GPR143 variants, suggested by Preising et al. 24 . Molecular diagnosis and OCT examination would be helpful in distinguishing between OA1 and isolated X-linked congenital nystagmus.