Two Novel NYX Gene Mutations in the Chinese Families with X-linked Congenital Stationary Night Blindness

Mutations in NYX and CACNA1F gene are responsible for the X-linked congenital stationary night blindness (CSNB). In this study, we described the clinical characters of the two Chinese families with X-linked CSNB and detected two novel mutations of c. 371_377delGCTACCT and c.214A>C in the NYX gene by direct sequencing. These two mutations would expand the mutation spectrum of NYX. Our study would be helpful for further studying molecular pathogenesis of CSNB.


Materials and Methods
Patient Ascertainment. Two families with X-linked CSNB (CSNB-01, and CSNB-02) were recruited for this study (Fig. 1). All participants underwent an ophthalmologic and orthoptic examination, including best corrected visual acuities, visual field, anterior segment of the eyes, vitreous and fundus, measurement of the deviation angle in the cardinal eye positions, examination of ocular movement and binocular vision, and Electroretinography (ERG). ERG were recorded according to the standards of the International Society for Clinical Electrophysiology of Vision (http://www.iscev.org). Cycloplegic refraction were performed for children under the age of 14. The spherical equivalent of the refractive error is given in diopters (D). After informed consent, 7 affected and 13 unaffected individuals from the two unrelated families were taken 3 ml blood samples from their blood vessels and DNA was extracted from blood lymphocytes according to the standard methods of protocol (Roche Biochemical, Inc). This study obtained IRB approval from the Tianjin Eye Hospital and conformed to the tenets of the Declaration of Helsinki.
Mutation Analysis. Because NYX and CACNA1F are two candidate genes for X-linked congenital stationary night blindness, both of these two genes were directly sequenced in the CSNB-01 and CSNB-02 families. The coding regions and exon-intron boundaries of these two genes were amplified by PCR reaction using the primer pairs designed by the online software of the Primer 3. The exon 3 of NYX gene was split into five overlap fragments. The primer sequences were listed in the supplement Table. PCR were carried 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 2min denaturation at 98 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 55 °C, 1 min at 72 °C, and a final 7 min extension step at 72 °C. The PCR products were bi-directionally sequenced using the BigDye Terminator Cycle Sequencing V3.1 kit on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) after purification with the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). Sequencing results were assembled and analyzed with the Seqman program of DNASTAR software (DNASTAR Inc, Madison, WI). The reference cDNA sequences of the NYX and CACNA1F were obtained from Genebank (NM_022567 for NYX mRNA, and NM_001256789 for CACNA1F mRNA) and + 1 corresponds to the A of the ATG translation initiation codon. Mutation naming followed the nomenclature recommended by the Human Genomic Variation Society (HGVS).

Results
Clinical features. In the family CSNB-01, all affected males complained about night blindness since their early childhood. Ophthalmologic examination showed that the patients had decreased visual acuity, variable degrees of myopia, latent nystagmus, and extropia combined with dissociated vertical deviation (DVD). Their detail clinical data were listed in the table 1. The proband (individual III:4, Fig. 1A) was a 24-year-old affected male and feel night blindness at the age of 4. Fundus examination revealed no obvious anomalies. Refraction examination showed a refractive error of − 6.25D in his right eye and of − 6.0D in his left eye. The best corrected visual acuity of his both eyes were 0.4. He had an extropia of about 15° at the primary eye position, and had fairly pronounced latent nystagmus combined with dissociated vertical deviation. The ERG showed absence of rod b-wave and oscillatory potentials under the scotopic condition, but a normal 30 HZ flicker response (Fig. 2). Thus, the clinical phenotype of the family CSNB-01 could be classified as the complete form of CSNB according to the ERG. The CSNB-02 family consisted of 6 affected males. The proband of the CSNB-02 family (individual IV:1, Fig. 1B) was a 7-year-old affected boy. He was diagnosed with CSNB by inquiring family history because his two uncles (individual III:1,III:4) were diagnosed with CSNB in other hospital. Refraction examination showed a refractive error of − 9.0D sph in his right eye and of − 10.0D sph in his left eye. The best corrected visual acuity of both eyes were 0.7. He had an intermittent extropia of 30 prism diopter (PD) at distance and 40 PD at near fixation. However, nystagmus and DVD were unremarkable. Fundus examination were high myopia changes but were otherwise normal. ERG examination was failed because he cannot cooperate very well. The CSNB form were also not able to established without ERG.

Identification of Mutations.
After sequence analysis to the NYX and CACNA1F genes, a 7-base pair deletion of c. 371_377delGCTACCT(p.Y125TfsX138) was detected in exon 3 of NYX gene in all affected males and female carriers in the family CSNB-01, while in the same exon, a missense mutation of c.214A> C( p.N72H) was identified in the family CSNB-02 (Fig. 3). Multiple sequence alignment of the NYX protein shows that N72 is conserved among Homo sapiens, Pongo abelii, Rattus norvegicus, Mus musculus, Bos taurus, Gallus gallus, Xenopus, and Danio rerio (Fig. 4 ). A PISC score of 1.0 and a SIFT score of 0.02 were yielded after using the POLYPHEN 2 (http://coot.embl.de/PolyPhen/) and SIFT (http://sift.bii.a-star.edu.sg/) program to predict the functional and structural changes of the amino acid substitution, which means that the substitution of Asparagine (Asn, N) at codon 72 by Histidine would be deleterious to the structure and function of the NYX protein. The above two mutations in NYX were absent in 100 normal controls after sequencing of the NYX.

Discussion
NYX gene is located on chromosome Xp11.3 and consists of 3 exons spanning approximately 28 kb. It is mainly expressed in the kidney and retina. Within the retina, NYX is expressed in the inner segment of photoreceptors, outer and inner nuclear layers and the ganglion cell layer 9 . Nyctalopin is the encoded protein of the NYX gene, and consists of 481 amino acids. Structurally, nyctalopin belongs to a member of the leucine-rich repeat (LRR) superfamilies, containing 11 consecutive LRRs flanked by N-and C-terminal cysteine-rich LRRs, as well as an N-terminal ER signal peptide and a C-terminal glycosylphosphatidylinositol (GPI) membrane anchor 10 . LRRs are short-sequence motifs presenting in a number of proteins and are believed to be involved in the protein-protein interactions, cell adhesion and axon guidance [11][12][13] . Up to date, more than 50 mutations in NYX have been reported to be associated with congenital stationary night blindness. These mutations include missense and nonsense, splicing site mutations, as well as insertions and deletions. However, the exact molecular pathogenesis of CSNB caused by NYX mutations remains to be elucidated. It was suggested that nyctalopin might play an important role in keeping localization of TRPM1 in the dendritic tips of ON bipolar cells 14,15 . As an accessory subunit of the transient receptor potential (TRP) channel, nyctalopin might interact directly with TRPM1, or together with LRIT3 (another LRR family member), to keep the signaling channel proteins in the right place. The dislocation of TRPM1 would lead to abnormal function of retinal depolarizing bipolar cells (DBCs) and be predicated to result in abnormal signal transmission from photoreceptors. Mutations in these three interacted proteins (Nyctalopin, LRIT3 and TRPM1) have been documented to be associated with the complete form CSNB. The mutation of c. 371_377delGCTACCT(p.Y125TfsX138) is located in exon 3 of the NYX gene. The 7 base pair of GCTACCT deletion would result in a frameshift change of NYX gene and produce a truncated proteins with initial 125 amino acids similar to the wild type NYX protein and additional 12 aberrant amino acids followed by a premature stop codon. Thus, the NMD surveillance mechanism could be activated by a PTC-containing mRNA. The abnormal mRNA with PTC would be degraded under the NMD surveillance mechanism or produce a defective truncated protein through escaping the NMD surveillance 16,17 .
Mutation of c.214A> C (p.N72H) in the family CSNB-02 is a novel mutation and not detected in 100 normal controls. N72 is located in the first repeat of leucine-rich repeat (LRR) in the nyctalopin, and is highly conserved among human and the lower animals. Substitution of Asparagine by Histidine would be probably damaging to the protein structure and/or the protein function, predicted by either POLYPHEN 2 or SIFT program. In addition, Asparagine 72 forms the inter-side chain hydrogen bonds with Cysteine 48, Leucine 53, Leucine 69 and Aspartate 70. Substitution of Asparagine by Histidine would break hydrogen bonds with Leu 53 and Leu 69, and forms a new hydrogen bond with Alanine 51 in the Swiss-model (Fig. 5), which could be expected to change the protein structure. However, changing Asparagine to Histidine were not able to disrupt the concave exterior surface of the secondary molecular structure in the constructed model as to be expected (Fig. 5). Previous study documented that different constructs containing NYX mutations could not produce any effect on the cellular localization in comparison to wild type constructs 18 , and the mutated variants in the LRR might cause CSNB through disrupting the protein-protein interactions 11,19 . The detailed molecular mechanism should be elucidated further by in vitro and in vivo experiments.
Although more than 50 mutations in NYX have been reported and could be responsible for 45% of the X-linked CSNB, only three NYX mutations, including p.Arg94Pro 20 , p.Leu184Arg 20 and p.Thr258Pro 21 , have been reported in the Chinese patients with CSNB. Mutations of p.Arg94Pro and p.Leu184Arg are located in the second and the third LRR motif respectively, whereas mutation of p.Thr258Pro located in the ninth LRR motif. To our knowledge, mutation of N72H in the first LRR motif have not been reported before. With exception of the CSNB, NYX mutations could be responsible for some Chinese high myopia patients without CSNB 22,23 .  The forms of CSNB1 and CSNB2 can be differentiated by ERG. However, the correlation of the genotype with the clinical phenotypes "complete" and "incomplete" cannot be established very well 24 . Previous study suggested that patients with CSNB1 mainly had rod-related problems, and patients with CSNB2 had both rod-and cone-related problems, so that the visual acuity on average was better in CSNB1 than in CSNB2. However, the expressivity may be variable within the CSNB1 patients due to the clinical and genetic heterogeneity. Some CSNB1 patients may have dramatically decreased visual acuity, whereas a few patients might have good visual acuity even better than 0.8, and experience only high refractive error and nyctalopia 25,26 . In our patients, the patients carrying NYX mutation of c.371_377del GCTACCT (p.Y125TfsX138) had reduced visual acuity ≤ 0.4, myopia of the spherical equivalent range from − 3.0D to − 10.0D, dissociated vertical deviation, extropia and nystagmus, whereas the patient carrying NYX mutation of c.214A> C (p.N72H) had only high myopia of − 10.0D and intermittent extropia. Although we cannot establish the correlation of the NYX mutations with the clinical phenotype because we don't have enough patients to be observed, we think that variable expressivity might be related to other genetic or environmental factors rather than to the different mutant types in the same gene.
In summary, we report the clinical characterization of two Chinese families with X-linked CSNB and identified two novel mutations of c. 371_377delGCTACCT and c.214A> C in the NYX gene. These two mutations would expand the mutation spectrum of NYX and help to study molecular pathogenesis of CSNB.