Molecular scanning of the ABCA4 gene in Spanish patients with retinitis pigmentosa and Stargardt disease: identification of novel mutations

PURPOSE: Among the 500 ABCA4 mutations identified so far in the retina-specific ABC transporter (ABCA4) gene, only 20 have been described in patients with autosomal recessive retinitis pigmentosa (arRP). In this study the gene was screened for mutations in a cohort of 25 patients of Spanish origin, to further assess ABCA4 involvement in retinal dystrophies. METHODS: The 50 exons of the ABCA4 gene have been analyzed through denaturing high performance liquid chromatography (DHPLC) and direct sequencing of samples displaying altered elution profiles. RESULTS: Four new and five known mutations were identified in our patients; except for one new deletion detected in a patient with Stargardt disease, all the remaining variations are single nucleotide substitutions resulting in missense mutations. CONCLUSIONS: The results further underline the genetic heterogeneity of retinal disorders, suggesting that more than one gene could differentially contribute to at least some forms of retinal degeneration. Finding a high proportion of novel mutations merits the use of scanning methodologies to analyze the whole coding region of the ABCA4 gene.     

Null missense ABCR (ABCA4) mutations in a family with stargardt disease and retinitis pigmentosa.

PURPOSE: To determine the type of ABCR mutations that segregate in a family that manifests both Stargardt disease (STGD) and retinitis pigmentosa (RP), and the functional consequences of the underlying mutations. METHODS: Direct sequencing of all 50 exons and flanking intronic regions of ABCR was performed for the STGD- and RP-affected relatives. RNA hybridization, Western blot analysis, and azido-adenosine triphosphate (ATP) labeling was used to determine the effect of disease-associated ABCR mutations in an in vitro assay system. RESULTS: Compound heterozygous missense mutations were identified in patients with STGD and RP. STGD-affected individual AR682-03 was compound heterozygous for the mutation 2588G-->C and a complex allele, [W1408R; R1640W]. RP-affected individuals AR682-04 and-05 were compound heterozygous for the complex allele [W1408R; R1640W] and the missense mutation V767D. Functional analysis of the mutation V767D by Western blot and ATP binding revealed a severe reduction in protein expression. In vitro analysis of ABCR protein with the mutations W1408R and R1640W showed a moderate effect of these individual mutations on expression and ATP-binding; the complex allele [W1408R; R1640W] caused a severe reduction in protein expression. CONCLUSIONS: These data reveal that missense ABCR mutations may be associated with RP. Functional analysis reveals that the RP-associated missense ABCR mutations are likely to be functionally null. These studies of the complex allele W1408R; R1640W suggest a synergistic effect of the individual mutations. These data are congruent with a model in which RP is associated with homozygous null mutations and with the notion that severity of retinal disease is inversely related to residual ABCR activity.     

Screening for mutations in the IMPDH1 gene in Japanese patients with autosomal dominant retinitis pigmentosa

PURPOSE: To determine the presence and frequency of mutations in the IMPDH1 gene in Japanese patients with autosomal dominant retinitis pigmentosa (ADRP), and to characterize the clinical characteristics of patients with the Lys238Arg mutation in the IMPDH1 gene. DESIGN: Case reports and results of DNA analysis. METHODS: All 14 coding exons of the IMPDH1 gene were directly sequenced in 96 unrelated patients with ADRP. The clinical features were determined by visual acuity, slit-lamp biomicroscopy, and kinetic visual field tests. RESULTS: Two novel mutations, a Leu227Pro and Lys238Arg, in the IMPDH1 gene were identified in two unrelated families with ADRP. The clinical features associated with the Lys238Arg mutation were an early-onset and severe retinal degeneration. CONCLUSIONS: The most commonly reported Asp226Asn mutation was not found in the Japanese population, instead two novel mutations were found. These findings suggest that mutations of the IMPDH1 gene cause ADRP in the Japanese population.     

Rhodopsin mutations in Chinese patients with retinitis pigmentosa

AIM: To determine the pattern of rhodopsin mutations in Chinese retinitis pigmentosa (RP) patients. METHODS: The rhodopsin gene was examined in 101 RP patients and 190 controls from Hong Kong. RESULTS: Three coding changes were identified: Pro347Leu, Ala299Ser, and 5211delC. Each protein sequence alteration was found in one patient. Ala299Ser also existed in two controls. CONCLUSION: The C-terminal nonsense mutation may cause mis-sorting of rhodopsin protein. The finding of controls with Ala299Ser suggests this is only the third missense alteration reported that does not cause RP. The expected frequency of rhodopsin mutations in RP is <7% (2/101=2.0%, 95% confidence interval: 0.2%-7.0%).     

Mutation spectrum and founder chromosomes for the ABCA4 gene in South African patients with Stargardt disease

PURPOSE: To assess the mutation spectrum of ABCA4 underlying Stargardt disease (STGD) in South Africa (SA) and to determine whether there is a single or a few founder chromosomes in SA STGD families. METHODS: Sixty-four probands exhibiting the STGD phenotype were screened for mutations in the 50 exons of ABCA4 by single-strand conformational polymorphism-heteroduplex analysis sequencing and restriction fragment length polymorphism analysis. Microsatellite marker haplotyping was used to determine the ancestry in 10 families. RESULTS: Fifty-seven ABCA4 disease-associated alleles were identified that comprised 16 different sequence variants, of which two were novel, in 40 individuals of the cohort of 64 subjects. The most common variants identified included the C1490Y, L2027F, R602W, V256splice, R152X, and 2588G-->C mutations. The C1490Y variant was the most common disease-associated variant identified (19/64 subjects) and was absent in 392 control chromosomes. At least 10 ABCA4 disease-associated haplotypes were identified. Two of these haplotypes, which carried the C1490Y mutation, were identified in three unrelated families. CONCLUSIONS: Results suggest that ABCA4 is the major gene underlying STGD in the cohort investigated. Five of the six common sequence variants identified were at a higher frequency in the SA cohort than reported in published data on individuals of similar ancestry. The mutation and haplotype data suggests that there are several ancestral haplotypes underlying STGD in SA. There seems to be at least two different origins for the common C1490Y mutation, as well as two for the R602W mutation, thereby suggesting several founder effects for STGD in SA.     

视网膜色素变性1基因在常染色体显性遗传视网膜色素变性家系中的突变分析

目的:研究常染色体显性遗传视网膜色素变性(autosomal dominant retinitis pigmentosa,ADRP)家系中视网膜色素变性1(retinitis pigmentosa-1,RP1)基因的突变特征及其在RP发病机制中的作用。方法:运用聚合酶链反应和直接测序方法,对6个ADRP家系的47例成员和50例对照者进行了RP1基因全编码区和邻近剪切位点的内含子区域序列突变的筛选与检测。运用单因素分析、多因素Logistic回归分析研究RP1基因点突变在RP发病中的作用。结果:ADRP家系成员和对照组RP1基因第4外显子上检测出2个变异位点。在1691和1725密码子存在杂合的两种类型的密码子(S1691P,Ser-Pro,TCT→CCT;Q1725Q,Gln-Gln,CAA→CAG)。ADRP家系成员中Ser-1691-Pro及Gln-1725-Gln位点突变率显著高于正常对照组(χ2=11.202,P<0.05)。结论:RP1基因Ser-1691-Pro及Gln-1725-Gln位点多态性可增高RP的危险性,具有潜在的致病性,考虑为ADRP家系的易感基因。     

CFH Y402H polymorphism in Italian patients with age-related macular degeneration,retinitis pigmentosa,and Stargardt disease

ABSTRACT

Background: The complement system has been implicated in the pathogenesis of age-related macular degeneration (AMD) and the CFH Y402H polymorphism has been suggested as a major risk factor for AMD. Recent evidences supported the role of inflammation in the pathogenesis of some retinal dystrophies. Aim of this study was to evaluate the prevalence of CFHY402H polymorphism in a group of Italian patients affected by atrophic AMD, Stargardt disease (STGD), or retinitis pigmentosa(RP).

Materials and Methods: Our case–control association study included 116 patients with atrophic AMD, 77 with RP, 86 with STGD, and 100 healthy controls. All the patients were evaluated by a standard ophthalmologic examination and OCT. ERG was performed on STGD and RP patients. All the subjects underwent a blood drawing for genetic testing and the CFHY402H polymorphism was genotyped with the TaqMan real-time polymerase chain reaction single nucleotide polymorphism assay.

Results: The prevalence of the risk genotype C/C was higher in the AMD group than in controls (p < 0.001). The risk allele C was more frequent in the AMD group than in controls (p < 0.001). The prevalence of the risk genotype was higher in the RP patients than in controls (p < 0.001) and similarly the risk allele C was more frequent in the RP group (p = 0.008). The CFHY402H genotype distribution was not different between patients with STGD and the controls, for the biallelic (p = 0.531) and for the monoallelic (p = 0.318) evaluation.

Conclusions: In our series of Italian patients, the CFHY402H genotype is associated with atrophic AMD and RP, but not with STGD. This result may support the hypothesis of a complement system dysregulation in the pathogenesis of AMD and RP     

目标区域捕获测序检测到一视网膜色素变性家系ABCA4基因新突变

目的研究我国一个常染色体隐性遗传的视网膜色素变性（RP）家系患者的临床表型及致病基因突变,并分析表型与基因型间的关系。方法实验研究。收集了宁夏人民医院眼科诊治的一个RP家系的临床资料,共有7名家庭成员参与研究,其中包括2名患者和5名正常成员。完善家系内所有参与成员的眼科检查,包括最佳矫正视力（BCVA）、视野、眼底照相、光学相干断层扫描（OCT）及全视野视网膜电流图（ERG）等。针对180个已知的遗传性视网膜疾病致病基因及9个高度可疑的候选基因设计目标区域捕获芯片,利用该捕获芯片对先证者（Ⅳ-4）进行目标区域内的高通量二代测序,借助优化的生物信息学分析对捕获的遗传变异进行筛查过滤,最终通过家系内共分离验证确认致病突变,进一步分析该突变与患者表型间的关系。结果临床检查结果表明该家系内的2名患者的临床表现均符合典型的RP改变,遗传学分析结果证实了ABCA4基因c.419G>A突变是该家系的致病突变。该突变导致了ABCA4基因所编码的蛋白第140号氨基酸由精氨酸变为谷氨酰胺（p.Arg140Gln）,保守性分析显示该突变位点在各物种中高度保守,PloyPhen-2软件预测结果表明该突变具有较高的致病性。结论本研究借助基于高通量二代测序平台的目标区域捕获测序,首次发现了ABCA4基因新突变p.Arg140Gln是一个常染色体隐性遗传RP家系的致病突变,进一步扩充了ABCA4基因的遗传突变谱及表型谱。     

Mutations in the ABCA4 gene in a family with Stargardt's disease and retinitis pigmentosa (STGD1/RP19)

BACKGROUND: Demonstrating the types of ABCA4 mutations in the STGD1 gene in a family manifesting both Stargardt's disease and retinitis pigmentosa (RP19). METHODS: Clinical ophthalmological examination included funduscopy, ERG, Arden Colour contrast test, fluorescein angiography in one patient, perimetry and SLO perimetry. The 50 exons of the ABCA4 gene were screened using a combination of denaturating gradient gel electrophoresis (DGGE), high performance electrophoresis (dHPLC) and SSCP analysis. RESULTS: Patient I/1 showed typical signs of Stargardt's disease, while her son, II-1 demonstrated functional signs and morphological features of retinitis pigmentosa. Mutational analysis of the ABCA4 gene revealed a missense mutation in exon 42 (G5882G > A) and a frameshift mutation in exon 43 (5917delG) of patient I-1. Patient II/1 demonstrated a homozygous 5917delG mutation in exon 43, resulting in a functional null-mutation. CONCLUSIONS: The combination of ABCA4 alleles with various functional consequences to protein activity can lead to different clinical phenotypes in one and the same family, resulting either in typical Stargardt's disease or in autosomal recessive retinitis pigmentosa (RP19).     

Disease course of patients with X-linked retinitis pigmentosa due to RPGR gene mutations

PURPOSE: To measure the rates of visual acuity, visual field, and ERG loss in patients with X-linked retinitis pigmentosa due to RPGR mutations and to determine whether these rates differ from those of patients with dominant retinitis pigmentosa due to RHO mutations. METHODS: Snellen visual acuities, Goldmann visual field areas (V4e white test light), and 30 Hz (cone) full-field ERG amplitudes were recorded for an average of 9.8 years in 113 patients with RPGR mutations. After censoring data to eliminate ceiling and floor effects, we used longitudinal regression to estimate mean rates of change and to compare these rates with those of a previously studied cohort of 134 patients with dominant retinitis pigmentosa due to RHO mutations, who were followed for an average of 8.9 years. Survival analysis was used to compare the age distribution of legal blindness in these two groups. To explain group differences in visual acuity, optical coherence tomograms were recorded in some patients to visualize central retinal structure. RESULTS: Mean annual exponential rates of decline for the patients with RPGR mutations were 4.0% for visual acuity, 4.7% for visual field area, and 7.1% for ERG amplitude. Each of these rates was significantly different from zero (P < 0.001). The rates of visual acuity and visual field loss were significantly faster than the corresponding rates in the RHO patients (1.6%, P < 0.001 and 2.9%, P = 0.002, respectively), whereas the rate of ERG amplitude loss was comparable to that in the RHO patients (7.7%, P = 0.39). The median age of legal blindness was 32 years younger in the RPGR patients than in the RHO patients, due primarily to loss of visual acuity rather than to loss of visual field. Loss of acuity in RPGR patients appeared to be associated with foveal thinning. CONCLUSIONS: Patients with X-linked retinitis pigmentosa due to RPGR mutations lose visual acuity and visual field more rapidly than do patients with dominant retinitis pigmentosa due to RHO mutations.     

常染色体显性遗传视网膜色素变性患者视紫红质基因突变的研究

目的探讨常染色体显性遗传型视网膜色素变性(autosomal dominant retinitis pigmentosa,ADRP)患者视紫红质(rhodopsin,RHO)基因是否存在突变。方法应用聚合酶链反应(polymerase chain reaction,PCR)扩增RHO第1、5外显子基因片段，以限制性核酸内切酶酶切消化技术检测38个ADRP家系的57名患者和60名正常对照者RHO第58、347密码子的突变。结果1个ADRP家系的4名患者第58密码子发生点突变，另2个ADRP家系的6名患者第347密码子也出现点突变，而对照者未发现上述两种突变。结论ADRP存在分子水平的遗传异质性，某些ADRP是由于RHO基因突变所致。(中华眼底病杂志,1998,14:108-110)     

Disease progression in patients with dominant retinitis pigmentosa and rhodopsin mutations

PURPOSE: To measure the rate of progression of retinal degeneration in patients with retinitis pigmentosa due to dominant rhodopsin mutations and to determine whether the rate of progression correlates with the location of the altered amino acid in the rhodopsin molecule. METHODS: Change in ocular function was observed for an average of 8.7 years in 140 patients. After censoring data to eliminate "ceiling" and "floor" effects, longitudinal rates of change were compared, after weighting by follow-up time and number of visits, with rates inferred from cross-sectional analyses of the data from baseline visits. Mean rates of change were compared among groups of patients with mutations affecting the globule, plug, or C-terminal region of the protein after adjusting for age, gender, and baseline function. RESULTS: Mean annual exponential rates of decline were 1.8% for visual acuity, 2.6% for visual field area, and 8.7% for ERG amplitude. The rates of visual acuity and ERG amplitude decline were significantly faster, and the rate of visual field area decline was significantly slower, than those inferred from baseline visits. Rates of acuity loss did not vary significantly with the region affected by the mutation. In contrast, the mean annual rate of field loss in the C terminus group (7.4%) was significantly faster than that in the globule (1.7%) or plug (1.1%) group. The mean annual rate of ERG decline was also significantly faster in the C terminus group (13.5%) than in the globule (8.5%) or plug (3.7%) groups and significantly faster in the globule group than in the plug group. CONCLUSIONS: Rates of decline in visual function for groups of patients with rhodopsin mutations cannot be accurately inferred from cross-sectional analyses of baseline visits. Average rates of decline of visual field area and ERG amplitude are fastest in patients with mutations affecting the C-terminal region.     

常染色体显性视网膜色素变性NRL基因的突变研究

目的 对中国人常染色体显性视网膜色素变性（RP）NRL基因的突变现状进行研究。方法 抽取120例RP先证者的静脉血提取DNA。行NRL基因的引物设计合成，PCR扩增，琼脂糖电泳鉴定，异源双连-SSCP电泳。结果 120例散发型RP患者未发现NRL基因突变。结论 中国人RP NRL基因的突变频率很低。     

视网膜色素变性和视锥-视杆细胞营养不良患者基因突变频谱分析

目的：分析中国宁夏地区常染色体隐性遗传视网膜色素变性(ARRP)及视锥-视杆细胞营养不良(CORD)的基因突变频谱。

方法：纳入2016-09/2020-02在宁夏人民医院眼科医院就诊的35例ARRP患者和18例常染色体隐性CORD患者,行详细的眼科检查。抽取外周静脉血,对先证者应用包含232个致病基因的遗传性视网膜疾病捕获芯片进行靶向捕获富集高通量测序。利用在线分析软件对可疑基因变异致病性进行预测,利用Sanger测序对家系成员进行共分离分析。

结果：ARRP患者35例中,检测到致病基因16个,以RP1基因突变率最高,占14%(5/35),其次为ABCA4、CRB1和EYS基因,均占11%(4/35); 18例常染色体隐性CORD患者中,检测到致病基因10个,以ABCA4基因突变率最高,占28%(5/18),其次为ALMS1、PROM1、RPE65、USH2A基因,均占11%(2/18); ARRP和CORD患者中,共同致病基因有ABCA4、CLN3、CRB1、PROM1、NRL共5个,占42%(22/53)。

结论：ARRP及CORD两种疾病在表型之间具有一定程度的相似性和交叉性,致病基因突变谱上存在一定重叠性。宁夏地区最常见的重叠基因为ABCA4。     

全外显子组测序检测到一视网膜色素变性家系ABCA4基因致病剪切新突变

目的探讨一个中国视网膜色素变性（RP）家系的致病基因及其位点。方法实验研究。对一个RP家系的成员进行病史采集、视力检查、眼底检查及多焦视网膜电图（mfERG）检查。绘制家系图,对家系成员采血,进行DNA提取、全外显子组测序、数据分析和Sanger测序验证,并在500例健康对照者中进行测序验证。结果共纳入该家系成员5例,含2例患者。患者表现为青少年期发病,夜盲,进行性周边视力受损,逐渐累及中央区。眼底检查显示视网膜色素沉着。mfERG显示a波、b波振幅明显下降甚至记录不到。家系特点为2例患者为姐妹,另一位姐妹正常,父母均正常,符合常染色体隐性遗传模式特征。经外显子组测序、数据分析和Sanger测序验证后,发现ABCA4变异性剪切位点c.1761-2A>G发生纯合突变。另外,在500例健康对照者中,Sanger测序没有发现该位点纯合突变。结论本研究发现了一个新的视网膜色素致病基因突变位点c.1761-2A>G,扩大了ABCA4致病基因位点谱,为临床的诊断和治疗提供了新靶点。     

Molecular biological study of the rhodopsin gene in Japanese patients with autosomal dominant retinitis pigmentosa]

The author analyzed codon 347 of the rhodopsin gene using PCR (polymerase chain reaction) amplification and restriction enzymes in 19 unrelated Japanese families including 28 patients with autosomal dominant retinitis pigmentosa (ADRP). An allele of codon 347 mutation was found in a family (father and daughter). Sequence analysis shows that the mutation is from CCG to CTG. This mutation appears to be the cause of one form of ADRP, since it was also found in Japanese cases of ADRP which have a different racial background from families reported by Dryja et al.