A Novel Missense SNRNP200 Mutation Associated with Autosomal Dominant Retinitis Pigmentosa in a Chinese Family

The SNRNP200 gene encodes hBrr2, a helicase essential for pre-mRNA splicing. Six mutations in SNRNP200 have recently been discovered to be associated with autosomal dominant retinitis pigmentosa (adRP). In this work, we analyzed a Chinese family with adRP and identified a novel missense mutation in SNRNP200. To identify the genetic defect in this family, exome of the proband was captured and sequencing analysis was performed to exclude known genetic defects and find possible pathogenic mutations. Subsequently, candidate mutations were validated in affected family members using Sanger sequencing. A novel missense mutation, c.2653C>G transition (p.Q885E), in exon 20 of SNRNP200 was identified. The mutation co-segregated with the disease phenotype over four generations and was absent in 100 normal unaffected individuals. This mutation occurs at highly conserved position in hBrr2 and is predicted to have a functional impact, suggesting that hBrr2-dependent small nuclear riboproteins (snRNPs) unwinding and spliceosome activation is important in the pathogenesis of some variants of RP.     

视网膜色素变性的分子遗传学研究进展

视网膜色素变性（RP）是常见的遗传性眼病,具有高度的遗传异质性,患者常有进行性夜盲和视野缺损。对于常染色体显性遗传RP已经发现有12个基因座与之有关,其中6个致病基因已被克隆,对于这些致病基因的结构、突变及其功能目前已有了新的研究进展。 Abstract:Retinitis pigmentosa (RP) describes a genetically and clinically heterogeneous group of disorders that are characterized by gradual degeneration of photoreceptor cells.Common clinical features include a progressive loss of night vision,leading to night blindness and peripheral-visual-field loss.At least 12 loci have been mapped to chromosomes,and mutations in an ever increasing number of genes have been found to cause autosomal dominant retinitis pigmentosa (ADRP).Six of the 12 genes known to cause ADRP have been cloned.New progress has been made on the studies of structure,mutation and function of these genes.     

De Novo Occurrence of a Variant in ARL3 and Apparent Autosomal Dominant Transmission of Retinitis Pigmentosa

Background

Retinitis pigmentosa is a phenotype with diverse genetic causes. Due to this genetic heterogeneity, genome-wide identification and analysis of protein-altering DNA variants by exome sequencing is a powerful tool for novel variant and disease gene discovery. In this study, exome sequencing analysis was used to search for potentially causal DNA variants in a two-generation pedigree with apparent dominant retinitis pigmentosa.

Methods

Variant identification and analysis of three affected members (mother and two affected offspring) was performed via exome sequencing. Parental samples of the index case were used to establish inheritance. Follow-up testing of 94 additional retinitis pigmentosa pedigrees was performed via retrospective analysis or Sanger sequencing.

Results and Conclusions

A total of 136 high quality coding variants in 123 genes were identified which are consistent with autosomal dominant disease. Of these, one of the strongest genetic and functional candidates is a c.269A>G (p.Tyr90Cys) variant in ARL3. Follow-up testing established that this variant occurred de novo in the index case. No additional putative causal variants in ARL3 were identified in the follow-up cohort, suggesting that if ARL3 variants can cause adRP it is an extremely rare phenomenon.     

Identification and Functional Analysis of GJA8 Mutation in a Chinese Family with Autosomal Dominant Perinuclear Cataracts

Congenital cataract is a clinically and genetically heterogeneous group of eye disorders that causes visual impairment and childhood blindness. The purpose of this study was to identify the genetic defect associated with autosomal dominant congenital perinuclear cataract in a Chinese family. A detailed family history and clinical data of the family were recorded, and candidate gene sequencing was performed to screen for mutation-causing disease in our study. Direct sequencing revealed a c.601G>A (p.E201K) transversion in exon 2 of GJA8. This mutation co-segregated with all affected individuals in the family and was not found in unaffected family members or 100 unrelated controls. The function and mechanism of novel GJA8 point mutation E201K in Chinese patients were then investigated in this study. We found E201K aberrantly located in cytoplasm and prevented its location in the plasma membrane. Induction of E201K expression led to a decrease in cell growth and viability by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Our study provides important evidence that GJA8 is a disease-causing gene for congenital cataract and that mutation of GJA8 has a potential causative effect.     

Identification of a Novel MYO15A Mutation in a Chinese Family with Autosomal Recessive Nonsyndromic Hearing Loss

Autosomal recessive nonsyndromic hearing loss (ARNSHL) is a genetically heterogeneous sensorineural disorder, generally manifested with prelingual hearing loss and absence of other clinical manifestations. The aim of this study is to identify the pathogenic gene in a four-generation consanguineous Chinese family with ARNSHL. A novel homozygous variant, c.9316dupC (p.H3106Pfs*2), in the myoxin XVa gene (MYO15A) was identified by exome sequencing and Sanger sequencing. The homozygous MYO15A c.9316dupC variant co-segregated with the phenotypes in the ARNSHL family and was absent in two hundred normal controls. The variant was predicted to interfere with the formation of the Myosin XVa-whirlin-Eps8 complex at the tip of stereocilia, which is indispensable for stereocilia elongation. Our data suggest that the homozygous MYO15A c.9316dupC variant might be the pathogenic mutation, and exome sequencing is a powerful molecular diagnostic strategy for ARNSHL, an extremely heterogeneous disorder. Our findings extend the mutation spectrum of the MYO15A gene and have important implications for genetic counseling for the family.     

Structure-Function Modeling of Optical Coherence Tomography and Standard Automated Perimetry in the Retina of Patients with Autosomal Dominant Retinitis Pigmentosa

Purpose

To assess relationships between structural and functional biomarkers, including new topographic measures of visual field sensitivity, in patients with autosomal dominant retinitis pigmentosa.

Methods

Spectral domain optical coherence tomography line scans and hill of vision (HOV) sensitivity surfaces from full-field standard automated perimetry were semi-automatically aligned for 60 eyes of 35 patients. Structural biomarkers were extracted from outer retina b-scans along horizontal and vertical midlines. Functional biomarkers were extracted from local sensitivity profiles along the b-scans and from the full visual field. These included topographic measures of functional transition such as the contour of most rapid sensitivity decline around the HOV, herein called HOV slope for convenience. Biomarker relationships were assessed pairwise by coefficients of determination (R²) from mixed-effects analysis with automatic model selection.

Results

Structure-function relationships were accurately modeled (conditional R²>0.8 in most cases). The best-fit relationship models and correlation patterns for horizontally oriented biomarkers were different than vertically oriented ones. The structural biomarker with the largest number of significant functional correlates was the ellipsoid zone (EZ) width, followed by the total photoreceptor layer thickness. The strongest correlation observed was between EZ width and HOV slope distance (marginal R² = 0.85, p<10⁻¹⁰). The mean sensitivity defect at the EZ edge was 7.6 dB. Among all functional biomarkers, the HOV slope mean value, HOV slope mean distance, and maximum sensitivity along the b-scan had the largest number of significant structural correlates.

Conclusions

Topographic slope metrics show promise as functional biomarkers relevant to the transition zone. EZ width is strongly associated with the location of most rapid HOV decline.     

A Novel DFNA36 Mutation in TMC1 Orthologous to the Beethoven (Bth) Mouse Associated with Autosomal Dominant Hearing Loss in a Chinese Family

Mutations in the transmembrane channel-like gene 1 (TMC1) can cause both DFNA36 and DFNB7/11 hearing loss. More than thirty DFNB7/11 mutations have been reported, but only three DFNA36 mutations were reported previously. In this study, we found a large Chinese family with 222 family members showing post-lingual, progressive sensorineural hearing loss which were consistent with DFNA36 hearing loss. Auditory brainstem response (ABR) test of the youngest patient showed a special result with nearly normal threshold but prolonged latency, decreased amplitude, and the abnormal waveform morphology. Exome sequencing of the proband found four candidate variants in known hearing loss genes. Sanger sequencing in all family members found a novel variant c.1253T>A (p.M418K) in TMC1 at DFNA36 that co-segregated with the phenotype. This mutation in TMC1 is orthologous to the mutation found in the hearing loss mouse model named Bth ten years ago. In another 51 Chinese autosomal dominant hearing loss families, we screened the segments containing the dominant mutations of TMC1 and no functional variants were found. TMC1 is expressed in the hair cells in inner ear. Given the already known roles of TMC1 in the mechanotransduction in the cochlea and its expression in inner ear, our results may provide an interesting perspective into its function in inner ear.     

Dominant Retinitis Pigmentosa,p.Gly56Arg Mutation in NR2E3: Phenotype in a Large Cohort of 24 Cases

Importance

This research is the single largest NR2E3 genotype-phenotype correlation study performed to date in autosomal dominant Retinitis Pigmentosa.

Objective

The aim of this study is to analyse the frequency of the p.Gly56Arg mutation in NR2E3 for the largest cohort of autosomal dominant Retinitis Pigmentosa patients to date and its associated phenotype.

Patients and Methods

A cohort of 201 unrelated Spanish families affected by autosomal dominant Retinitis Pigmentosa. The p.Gly56Arg mutation in the NR2E3 (NM_014249.2) gene was analysed in 201 families. In the 24 cases where the mutation had been detected, a haplotype analysis linked to the p.Gly56Arg families was performed, using four extragenic polymorphic markers D15S967, D15S1050, D15S204 and D15S188. Phenotype study included presence and age of onset of night blindness, visual field loss and cataracts; and an ophthalmoscopic examination after pupillary dilation and electroretinogram for the 24 cases.

Results

Seven of the 201 analyzed families were positive for the p.Gly56Arg, leading to a prevalence of 3.5%. Clinical data were available for 24 subjects. Night blindness was the first noticeable symptom (mean 15.9 years). Visual field loss onset was variable (23.3 ± 11.9 years). Loss of visual acuity appeared late in the disease´s evolution. Most of the patients with cataracts (50%) presented it from the third decade of life. Fundus changes showed inter and intrafamiliar variability, but most of the patients showed typical RP changes and it was common to find macular affectation (47.4%). Electroretinogram was impaired from the beginning of the disease. Two families shared a common haplotype. Additionally, all patients shared a 104Kb region between D15S1050 and the NR2E3 gene.

Conclusions

This study highlights the importance of p.Gly56Arg in the NR2E3 gene as a common mutation associated with adRP, and provides new clues to its phenotype, which can allow for a better clinical management and genetic counselling of patients and their families.     

The Severe Autosomal Dominant Retinitis Pigmentosa Rhodopsin Mutant Ter349Glu Mislocalizes and Induces Rapid Rod Cell Death

Mutations in the rhodopsin gene cause approximately one-tenth of retinitis pigmentosa cases worldwide, and most result in endoplasmic reticulum retention and apoptosis. Other rhodopsin mutations cause receptor mislocalization, diminished/constitutive activity, or faulty protein-protein interactions. The purpose of this study was to test for mechanisms by which the autosomal dominant rhodopsin mutation Ter349Glu causes an early, rapid retinal degeneration in patients. The mutation adds an additional 51 amino acids to the C terminus of the protein. Folding and ligand interaction of Ter349Glu rhodopsin were tested by ultraviolet-visible (UV-visible) spectrophotometry. The ability of the mutant to initiate phototransduction was tested using a radioactive filter binding assay. Photoreceptor localization was assessed both in vitro and in vivo utilizing fluorescent immunochemistry on transfected cells, transgenic Xenopus laevis, and knock-in mice. Photoreceptor ultrastructure was observed by transmission electron microscopy. Spectrally, Ter349Glu rhodopsin behaves similarly to wild-type rhodopsin, absorbing maximally at 500 nm. The mutant protein also displays in vitro G protein activation similar to that of WT. In cultured cells, mislocalization was observed at high expression levels whereas ciliary localization occurred at low expression levels. Similarly, transgenic X. laevis expressing Ter349Glu rhodopsin exhibited partial mislocalization. Analysis of the Ter349Glu rhodopsin knock-in mouse showed a rapid, early onset degeneration in homozygotes with a loss of proper rod outer segment development and improper disc formation. Together, the data show that both mislocalization and rod outer segment morphogenesis are likely associated with the human phenotype.     

Novel Mutations of RPGR in Chinese Retinitis Pigmentosa Patients and the Genotype-Phenotype Correlation

X-linked Retinitis Pigmentosa (XLRP) accounts for 10–20% of all RP cases, and represents the most severe subtype of this disease. Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) gene are the most common causes of XLRP, accounting for over 70–75% of all XLRP cases. In this work, we analyzed all the exons of RPGR gene with Sanger sequencing in seven Chinese XLRP families, two of these with a provisional diagnosis of adRP but without male-to-male transmission. Three novel deletions (c.2233_34delAG; c.2236_37delGA and c.2403_04delAG) and two known nonsense mutations (c.851C→G and c.2260G→T) were identified in five families. Two novel deletions (c.2233_34delAG and c.2236_37delGA) resulted in the same frame shift (p.E746RfsX22), created similar phenotype in Family 3 and 4. The novel deletion (c.2403_04delAG; p.E802GfsX31) resulted in both XLRP and x-linked cone-rod dystrophy within the male patients of family 5, which suggested the presence of either genetic or environmental modifiers, or both, play a substantial role in disease expression. Genotype-phenotype correlation analysis suggested that (1) both patients and female carriers with mutation in Exon 8 (Family 1) manifest more severe disease than did those with ORF15 mutations (Family 2&3&4); (2) mutation close to downstream of ORF15 (Family 5) demonstrate the early preferential loss of cone function with moderate loss of rod function.     

一个常染色体显性遗传白癜风家系

本文报道了一白癜风家系,对其发病原因进行了探讨。作者认为该家系的白癜风为常染色体显性遗传。 Abstract:A family history of vitiligo was reported in this paper,and the reason causing disease was discussed.We think that the vitiligo in the family history is caused by autosomal dominant inheritance.     

A Novel Cysteine-Sparing NOTCH3 Mutation in a Chinese Family with CADASIL

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult onset cerebral small vessel disorder caused by the mutations of the neurogenic locus notch homolog protein 3 (NOTCH3) gene. The extracellular part of NOTCH3 is composed of 34 epidermal growth factor-like (EGF-like) repeat domains. Each EGF-like domain is rich of cysteine and glycine to produce three loops that are essential for high-affinity binding to its ligand. Nearly all reported CADASIL-associated mutations result in gain or loss of a cysteine residue within the EGF-like domains. Only a few cysteine-sparing NOTCH3 mutations have been documented in the patients with CADASIL to date. Here, we reported a Chinese CADASIL family with a cysteine-sparing NOTCH3 mutation. In this family, affected patients had dizziness, memory loss, gait instability, or hemiplegia. Brain magnetic resonance imaging (MRI) showed diffuse leukoencephalopathy with confluent signal abnormalities in the periventricular white matter, basal ganglia, and centrum semiovale bilaterally. By screening the entire coding region of NOTCH3, a novel missense mutation p.G149V (c.446G>T) was found. This mutation was not detected in 400 normal controls. Considering the critical position of glycine within the C-loop of EGF-like domain and its high conservation through evolution, p.G149V mutation could be a potential pathogenic cause for CADASIL.     

Whole Exome Analysis Identifies Frequent CNGA1 Mutations in Japanese Population with Autosomal Recessive Retinitis Pigmentosa

Objective

The purpose of this study was to investigate frequent disease-causing gene mutations in autosomal recessive retinitis pigmentosa (arRP) in the Japanese population.

Methods

In total, 99 Japanese patients with non-syndromic and unrelated arRP or sporadic RP (spRP) were recruited in this study and ophthalmic examinations were conducted for the diagnosis of RP. Among these patients, whole exome sequencing analysis of 30 RP patients and direct sequencing screening of all CNGA1 exons of the other 69 RP patients were performed.

Results

Whole exome sequencing of 30 arRP/spRP patients identified disease-causing gene mutations of CNGA1 (four patients), EYS (three patients) and SAG (one patient) in eight patients and potential disease-causing gene variants of USH2A (two patients), EYS (one patient), TULP1 (one patient) and C2orf71 (one patient) in five patients. Screening of an additional 69 arRP/spRP patients for the CNGA1 gene mutation revealed one patient with a homozygous mutation.

Conclusions

This is the first identification of CNGA1 mutations in arRP Japanese patients. The frequency of CNGA1 gene mutation was 5.1% (5/99 patients). CNGA1 mutations are one of the most frequent arRP-causing mutations in Japanese patients.     

Macular Cone Abnormalities in Retinitis Pigmentosa with Preserved Central Vision Using Adaptive Optics Scanning Laser Ophthalmoscopy

Purpose

To assess macular photoreceptor abnormalities in eyes with retinitis pigmentosa (RP) with preserved central vision using adaptive optics scanning laser ophthalmoscopy (AO-SLO).

Methods

Fourteen eyes of 14 patients with RP (best-corrected visual acuity 20/20 or better) and 12 eyes of 12 volunteers underwent a full ophthalmologic examination, fundus autofluorescence, spectral-domain optical coherence tomography (SD-OCT), and imaging with a prototype AO-SLO system. Cone density and spatial organization of the cone mosaic were assessed using AO-SLO images.

Results

In 3 eyes with RP and preserved central vision, cones formed a mostly regular mosaic pattern with small patchy dark areas, and in 10 eyes, the cone mosaic patterns were less regular, and large dark regions with missing cones were apparent. Only one eye with RP demonstrated a normal, regular cone mosaic pattern. In eyes with RP, cone density was significantly lower at 0.5 mm and 1.0 mm from the center of the fovea compared to normal eyes (P<0.001 and 0.021, respectively). At 0.5 mm and 1.0 mm from the center of the fovea, a decreased number of cones had 6 neighbors in eyes with RP (P = 0.002 for both). Greater decrease in cone density was related to disruption of the photoreceptor inner segment (IS) ellipsoid band on SD-OCT images (P = 0.044); however, dark regions were seen on AO-SLO even in areas of continuous IS ellipsoid on SD-OCT. Decreased cone density correlated thinner outer nuclear layer (P = 0.029) and thinner inner segment and outer segment thickness (P = 0.011) on SD-OCT.

Conclusions

Cone density is decreased and the regularity of the cone mosaic spatial arrangement is disrupted in eyes with RP, even when visual acuity and foveal sensitivity are good. AO-SLO imaging is a sensitive quantitative tool for detecting photoreceptor abnormalities in eyes with RP.     

A Novel GJA8 Mutation (p.V44A) Causing Autosomal Dominant Congenital Cataract

Purpose

To examine the mechanism by which a novel connexin 50 (Cx50) mutation, Cx50 V44A, in a Chinese family causes suture-sparing autosomal dominant congenital nuclear cataracts.

Methods

Family history and clinical data were recorded and direct gene sequencing was used to identify the disease-causing mutation. The Cx50 gene was cloned from a human lens cDNA library. Connexin protein distributions were assessed by fluorescence microscopy. Hemichannel functions were analyzed by dye uptake assay. Formation of functional channels was assessed by dye transfer experiments.

Results

Direct sequencing of the candidate GJA8 gene revealed a novel c.131T>C transition in exon 2, which cosegregated with the disease in the family and resulted in the substitution of a valine residue with alanine at codon 44 (p. V44A) in the extracellular loop 1 of the Cx50 protein. Both Cx50 and Cx50V44A formed functional gap junctions, as shown by the neurobiotin transfer assay. However, unlike wild-type Cx50, Cx50V44A was unable to form open hemichannels in dye uptake experiments.

Conclusion

This work identified a unique congenital cataract in the Chinese population, caused by the novel mutation Cx50V44A, and it showed that the V44A mutation specifically impairs the gating of the hemichannels but not the gap junction channels. The dysfunctional hemichannels resulted in the development of human congenital cataracts.