Autosomal recessive retinitis pigmentosa locus maps on chromosome 1q in a large consanguineous family from Pakistan

A large Pakistani family with several consanguineous marriages is described, in which autosomal recessive retinitis pigmentosa is segregating. Linkage studies revealed close linkage between the disease locus and six loci on chromosome 1q (D1S158, F13B, D1S422, D1S412, D1S413, and D1S53) with maximum lod scores ranging from 0.988-4.657 at Θ=0.065-0.235. However, the analysis of individual nuclear families showed very close linkage without recombination in three branches and several recombinants and negative lod scores throughout in the fourth branch. These results strongly suggest that mutations of two different genes are responsible for the disease in the 'linked' and 'unlinked' branches. Parallel to the linkage heterogeneity, clear phenotypic differences have been observed among the 'linked' and 'unlinked' parts. Our findings demonstrate that in case of recessive disorders the possibility of non-allelic genetic heterogeneity should always be considered, even within the same kindred and in genetic isolates if a largely extended pedigree is analysed.     

视网膜色素变性患者中视网膜色素变性1基因点突变的研究

目的 研究中国视网膜色素变性（retinitis pigmentosa，RP)患者RP1基因的突变频率、特征及其在RP发病机理中所起的作用。方法 运用构象敏感凝胶电泳（conformation sensitive gel electrophoresis,CSGE)和DNA直接测序方法对101例香港地区RP患者的RP1基因全编码区进行突变的筛选与检测。结果 101例RP患者中检出1例患者携带常见的RP1致病突变－R677X，另外在3名正常个体及1例Stargardt患者中检出非致病的无义突变－R1933X。RP1基因在所有RP患者中的突变检出率为1／101。突变最终导致RP1蛋白严重截短。此外，在本研究人群中还发现10个错义突变，除M479I的病理意义未确定之外，其余均系RP1基因的多态现象。结论 R1933X无致病意义，提示羧基端224个氨基酸的区域可能为RP1蛋白非功能区，结合最近发现的RP1羧基端的移码突变－Y1053（1bp del)的病理意义，推测RP1蛋白中相应片段（密码子1052－1933）的缺失会导致RP的发生。为证实这种推测，大范围的RP1基因分型工作是有必要的，并且可同时发现更多的RP致病突变以及不同于其他种族人群的RP1基因多态变化。     

Novel high-throughput SNP genotyping cosegregation analysis for genetic diagnosis of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis

Retinitis pigmentosa (RP), the major cause of blindness in adults, is an extremely heterogeneous monogenic disorder. More than 32 causative genes have been identified, 18 of which are involved in autosomal recessive RP (arRP); however, more than 50% of the cases remain unassigned. There are no major causative genes identified for arRP nor any prevalent mutations, which make mutational screening of the already reported RP genes extremely time consuming and costly. Nonetheless, this step is unavoidable for genetic diagnosis of patients and potential carriers, and it is a prerequisite before approaching the identification of new RP genes and loci. We have designed an innovative high-throughput time- and cost-effective strategy for cosegregation analysis of 22 genes of arRP and Leber congenital amaurosis (LCA; an autosomal recessive retinal dystrophy that shares some of the RP genes and traits) by SNP genotyping. This novel indirect method has been validated in a panel of 54 consanguineous and nonconsanguineous arRP families. In a single and fast genotyping step: 1) we discarded all the 22 candidate genes in 13% of the pedigrees, highlighting the families of choice to search for novel arRP genes/loci; 2) we excluded an average of 18-19 genes per family, thus diminishing the number of genes to screen for pathogenic mutations; and 3) we identified CERKL as the causative RP gene in a family in which this candidate had been previously discarded by microsatellite cosegregation analysis. This type of approach can also be applied to other nonretinal diseases with high genetic heterogeneity, such as hereditary deafness or Parkinson disease.     

显性视网膜色素变性家系已知连锁位点的筛查

目的 用连锁分析法对1个中国人显性视网膜色素变性家系进行已知位点的筛查,寻找其致病基因.方法 随机选取已知致病基因上下约5cM(JB)范围内的27对微卫星标记,确立单倍型,用两点法计箅最大优势对数(Lod score)值.结果 所选微卫星标记与该家系表型间最大Lod值小于1.结论 基本排除由已知常染色体显性遗传视网膜色素变性的候选基因导致该家系的病变.     

Homozygosity mapping identifies the Crumbs homologue 1 (Crb1) gene as responsible for a recessive syndrome of retinitis pigmentosa and nanophthalmos

The association of retinitis pigmentosa (RP) and microphthalmia has been reported in a number of familial and isolated cases. Here, the results of genetic analysis in a familial case of early RP associated with nanophthalmos are described. Two affected sibs were ascertained from an endogamous population in Mexico. A genome-wide linkage analysis was performed by means of an Affymetrix 250K microarray. Five large regions of homozygosity were demonstrated. The largest interval comprised 15.08 Mb at chromosome 1q31-32.1 and contained the Crumbs homologue-1, CRB1, a gene responsible for a number of recessive retinal dystrophies. Nucleotide sequence analysis demonstrated a c.1125C>G transversion in CRB1 exon 5, predicting a novel p.Tyr375X variant. To our knowledge this is the first instance in which a CRB1 mutation has been associated with early RP and nanophthalmos. Our results suggest a role for CRB1 in promoting axial growth of the eye. Clinical analysis of additional subjects with retinal dystrophies due to CRB1 mutations will help to identify if the high hyperopia, a frequently observed trait in these subjects, could be related to decreased eye axial length (nanophthalmos).     

一个视网膜色素变性家系的视紫红质基因突变分析

目的　确定常染色体显性遗传视网膜色素变性家系的致病基因及其突变位点,并研究其临床表型。方法　对一个常染色体显性遗传视网膜色素变性(autosomal dominat retinitis pigmentosa,ADRP)家系成员进行了视力、视野及眼底镜检查,并对该家系中先证者进行了视网膜电流图分析。应用聚合酶链反应和直接测序技术,对该家系的所有现存人员的视紫红质基因的外显子进行测序分析。结果　该家系的2 5名成员中12例患者有视紫红质基因(rhodopsin,RH O)的5 12 C>T(P171L)突变,均呈杂合子,该错义突变使密码子171由CCA变成CTA。而未受累者的视紫红质基因表现为野生型。该家系患者的临床表现为5～6岁时出现夜盲,在2 0～30岁逐渐出现视力和视野损害,并先后在4 0～5 0岁前后失明,其中2例患者并发青光眼,先证者的闪烁视网膜电图呈熄灭型。结论　视紫红质基因RH O的一种已知突变5 12 C>T(P171L)是该家系的病因。与国外相同的基因突变类型相比较,该家系发病早、病情进展快、视功能损害较重。     

常染色体显性遗传视网膜色素变性家系的基因筛查

目的：确定一个常染色体显性遗传视网膜色素变性(autosomal dominant retinitis pigmentosa,ADRP）家系的致病基因及其突变位点和类型。方法：应用聚合酶链反应－单链构象多态性结合DNA测序技术，对来自同一家系的4例RP患者及4名正常人外周血DNA进行分子遗传学分析，筛查3个候选基因共8个外显子。结果：来自同一家系的4例RP患者均发现有视紫红质基因（rhodopsin,RHO)第1外显子第52密码子存在TTC→TAC的点突变（Phe52Tyr），而4名正常人未发现这种突变。结论：在这个中国ADRP大家系中，发现RHO基因的致病突变，表明ADRP存在明显遗传异质性。     

WDR19: An ancient,retrograde, intraflagellar ciliary protein is mutated in autosomal recessive retinitis pigmentosa and in Senior‐Loken syndrome

Autosomal recessive retinitis pigmentosa (arRP) is a clinically and genetically heterogeneous retinal disease that causes blindness. Our purpose was to identify the causal gene, describe the phenotype and delineate the mutation spectrum in a consanguineous Quebec arRP family. We performed Arrayed Primer Extension (APEX) technology to exclude ∼500 arRP mutations in ∼20 genes. Homozygosity mapping [single nucleotide polymorphism (SNP) genotyping] identified 10 novel significant homozygous regions. We performed next generation sequencing and whole exome capture. Sanger sequencing provided cosegregation. We screened another 150 retinitis pigmentosa (RP) and 200 patients with Senior‐Løken Syndrome (SLS). We identified a novel missense mutation in WDR19, c.2129T>C which lead to a p.Leu710Ser. We found the same mutation in a second Quebec arRP family. Interestingly, two of seven affected members of the original family developed ‘sub‐clinical’ renal cysts. We hypothesized that more severe WDR19 mutations may lead to severe ciliopathies and found seven WDR19 mutations in five SLS families. We identified a new gene for both arRP and SLS. WDR19 is a ciliary protein associated with the intraflagellar transport machinery. We are currently investigating the full extent of the mutation spectrum. Our findings are crucial in expanding the understanding of childhood blindness and identifying new genes.     

从rds小鼠视网膜克隆视网膜色素变性相关差异片段

目的 从遗传性视网膜色素变性动物模型rds小鼠中克隆视网膜色素变性发病过程中特异表达的基因。方法 应用差异显示技术,分析rds小鼠发病过程中视网膜的mRNA。对特异性表达的mRNA片段进行克隆测序。结果 在视网膜色素变性发病过程中,rds小鼠的视网膜存在着相当明显的基因表达差异。在所测序分析的5个差异显示片段中其中一个与GenBank刚登录的功能未知的、从成年男性睾丸组织中克隆出来的cDNA序列较高同源（同一率为86％）另外4个为低同源序列。25天rds小鼠高表达的一个差异片段,与37天正常鼠表达而rds小鼠不表达的另一个片段,长度相同,177个碱基只相差两个。结论 视网膜色素变性这类慢性病变,存在着多个基因的表达及调控。     

FLVCR1-related disease as a rare cause of retinitis pigmentosa and hereditary sensory autonomic neuropathy

FLVCR1 encodes for a transmembrane heme exporter protein and it is known to cause a rare form of syndromic retinitis pigmentosa: posterior column ataxia with retinitis pigmentosa. Recently, the FLVCR1-associated phenotype has been expanded with sporadic reports of hereditary sensory-autonomic neuropathy or non-syndromic retinitis pigmentosa. Here, we report a 23-year- old female with early onset hypomyelinating sensory-autonomic neuropathy and retinitis pigmentosa. Both features were present since childhood. The patient developed signs of advanced retinitis pigmentosa by the age of 10 years leading to legal blindness after the age of 18. Following candidate gene panel testing, which was negative, whole exome sequencing revealed compound heterozygous pathogenic FLVCR1 variants: NM_014053.3: c.3G > T; p.(Met1?) and NM_014053.3: c.730G > A; p.(Gly244Ser), the latter variant is novel. In this report we highlight the association of retinitis pigmentosa with hypomyelinating sensory-autonomic neuropathy, which could be underdiagnosed due to variable severity. To summarize, the phenotypic heterogeneity of FLVCR1 variants is broad and should include retinitis pigmentosa along with range of neurological features.     

Point mutations of rhodopsin gene found in Japanese families with autosomal dominant retinitis pigmentosa (ADRP)

Summary The mutations of codon 17, 23, 58, and 347 of rhodopsin gene were investigated in 24 unrelated Japanese families including 33 patients with autosomal dominant retinitis pigmentosa (ADRP). A patient with codon 17 mutation (Thr-17-Met, ACGATG) and a family including 4 patients with codon 347 mutation (Pro-347-Leu, CCGCTG) were detected among them. Their clinical findings were extremely different between the two mutations. The former showed type 2 and the latter showed type 1 ADRP. No mutation of codon 23 and 58 was detected in any families so far analyzed in the present study. Clinical findings associated with the mutation in codon 17 and 347 of the rhodopsin gene show an existence of allelic heterogeneity.     

Analysis of RPGR in a South African family with X-linked retinitis pigmentosa: research and diagnostic implications

Analysis of exon ORF15 of the RPGR gene has revealed a novel mutation in a South African family with X-linked retinitis pigmentosa (XLRP), which has implications for the rest of the family in terms of pre-symptomatic testing. The ability to test for this mutation will be beneficial for the accurate determination of carrier status in female relatives who may have been unaware of their risk before this study was performed. This work also highlights the need to be aware of the ramifications of mutation testing in what may appear to be small families. This is the first report of an RPGR ORF15 mutation in a South African family of mixed ancestry.     

Missense mutation of rhodopsin gene codon 15 found in Japanese autosomal dominant retinitis pigmentosa

Summary Heterozygous missense mutation in codon 15 of the rhodopsin gene was detected in a patient with autosomal dominant retinitis pigmentosa (ADRP), where a transition of adenine to guanine at the second nucleotide in codon 15 (AATAGT), corresponding to a substitution of serine residue for asparagine residue (Asn-15-Ser) was detected. None of the remaining unrelated 42 ADRP, 24 autosomal recessive RP (ARRP) and 34 normal individuals had this alteration. Her funduscopic findings were sectorial in type similar to that of the patients with the same mutation found in an Australian pedigree (Sullivanet al., 1993). This study shows phenotypic similarities in patients with the same mutation of a different ancestry.     

RP1 in Chinese: Eight novel variants and evidence that truncation of the extreme C-terminal does not cause retinitis pigmentosa

Heterozygous truncating mutations in the RP1 gene cause approximately 7% of autosomal dominant retinitis pigmentosa (RP) cases. To examine the role of RP1 mutations in RP, we screened 101 unrelated Chinese RP patients (unselected for mode of inheritance) and 190 elderly normal control subjects for sequence changes in the coding exons for the 2156 amino acid RP1 protein. One patient had a mutation, thus RP1 mutations cause about 0.0% to 5.4% (95% confidence interval) of all RP among Chinese. The mutation was R677X, the most common found in Americans. Five other known sequence changes were found. In addition, nine novel sequence alterations were identified: 746G>A (R249H), 1437G>T (M479I), 2116G>C (G706R), 3024G>A (Q1008Q), 3188G>A (Q1063R), 5797C>T (R1933X), 6423A>G (I2141M), and the variants 6542C>T and 6676T>A, both in the 3' untranslated region. One control subject and three members of a non-RP family were heterozygous for R1933X, which is therefore likely to be a non-disease-causing variant. The most C-terminal truncation previously reported was due to Tyr1053 (1-bp del) and occurred in RP patients. Thus the presence of a normal level of at least part of RP1 between amino acids 1052 and 1933 appears necessary to prevent RP. Hum Mutat 17:436, 2001.     

Autosomal recessive retinitis pigmentosa in Spain: evaluation of four genes and two loci involved in the disease

Autosomal recessive retinitis pigmentosa (ARRP) is a genetically heterogeneous form of retinal degeneration. The genes for the β-subunit of rod phosphodiesterase (PDEB), rhodopsin (RHO), peripherin/RDS (RDS) and the rod outer segment membrane protein 1 (ROM1), as well as loci at 6p and 1q, have previously been reported as the cause of ARRP. In order to determine whether they are responsible for the disease in Spanish pedigrees, linkage and homozygosity studies using markers at these loci were carried out on 47 Spanish ARRP families. SSCP analysis was performed to search for mutations in the genes cosegregating with the disease in particular pedigrees. Three homozygous mutations in the PDEB gene were found, thus accounting for 6% of the cases. No other disease-causing mutation was observed in the other genes analysed, nor was significant evidence found for the involvement of the loci at 6p or 1q. On the basis of these data, it is unlikely that these genes and loci account for a considerable proportion of ARRP cases.     

Homozygous tandem duplication within the gene encoding the β-subunit of rod phosphodiesterase as a cause for autosomal recessive retinitis pigmentosa

Autosomal recessive retinitis pigmentosa (ARRP) is a degenerative disease of photoreceptors in which defects in the rhodopsin and phosphodiesterase β-subunit (PDEB) loci have been reported. To assess the involvement of PDEB in ARRP families from Spain, we screened a panel of 19 families for linkage to markers within or close to the PDEB gene. Homozygosity was also tested in cases of consanguinity. This combined approach ruled out PDEB as the cause of the disease in all but one of the families. Molecular characterization of the gene in that family (a consanguineous pedigree) revealed a homozygous 71′bp tandem duplication in exon 1 of the affected member, the parents being heterozygous. This defect causes a frameshift mutation which leads to a premature stop codon, suggesting that this mutant allele is the underlying cause of ARRP in this patient. According to the data presented here, the PDEB gene is not the main gene responsible for ARRP, but accounts for about 5% of the cases.© 1995 wiley-Liss, Inc.     

一例视网膜色素变性患者 C2ORF71基因的变异分析

目的:分析1例视网膜色素变性(retinitis pigmentosa, RP)患者的基因变异,明确其可能的遗传学病因。方法:应用全外显子测序技术对先证者进行致病基因筛查,结合临床表型确定可疑变异,应用Sanger测序法验证检出的变异,分析双亲携带变异位点的情况。采用多种软件对所检出的变异进行致病性分析。结果:全外显子...     

Dominant Leber congenital amaurosis,cone‐rod degeneration,and retinitis pigmentosa caused by mutant versions of the transcription factor CRX

We summarize 18 mutations in the human CRX gene that have been associated with Leber congenital amaurosis (congenital retinal blindness), cone‐rod degeneration, or retinitis pigmentosa. Except for one obviously null allele not definitely associated with a phenotype (a frameshift in codon 9), all CRX mutations appear to be completely penetrant and cause disease in heterozygotes. These dominant alleles fall into two categories. In one group are missense mutations and short, in‐frame deletions; in the second group are frameshift mutations, all of which are in the last exon. All of these dominant mutations are likely to produce stable mRNA that is translated. Mutations in the missense group preferentially affect the conserved homeobox (codons 39–98), and all frameshift mutations leave the homeodomain intact but alter the OTX motif encoded by codons 284–295 at the carboxy terminus. We could not uncover any correlation between type of disease (congenital amaurosis vs. cone‐rod degeneration or retinitis pigmentosa) and the type of mutation (missense vs. frameshift). Four of the 18 mutations (～20%) were de novo mutations, and all of these were found in isolate cases of Leber congenital amaurosis. Dominant CRX mutations have not been associated with mental retardation or developmental delay that has sometimes been found in Leber congenital amaurosis caused by other genes. Implications regarding potential future therapies are discussed. Hum Mutat 18:488–498, 2001. © 2001 Wiley‐Liss, Inc.     

C1q,a subunit of the first component of complement,enhances antibody-mediated apoptosis of cultured rat glomerular mesangial cells

We have shown previously that IgG2a anti-Thy-1 MoAb (ER4G) induces apoptosis of rat mesangial cells (GMC) in vitro. Since the classical complement pathway plays an essential role in Thy-1 nephritis, we analysed whether C1q, a subunit of the first component of complement, enhances the ER4G-mediated apoptosis of rat GMC. Two different subclasses of anti-Thy-1 MoAb, ER4G (IgG2a) and ER14 (IgG1), were used. It was established that ER4G binds C1q efficiently, while ER14 reacts poorly with C1q. For the experiments of apoptosis, quiescent rat GMC were exposed for 1 h at 37°C to a fixed concentration of anti-Thy-1 MoAb and incubated further for 16 h at 37°C in the presence or absence of C1q. GMC exposed to medium (M-GMC) followed by incubation of the cells with medium alone was used as controls. Apoptosis was assessed by morphological studies and quantitative analysis on FACS using FITC-annexin V (the annexin V methods) or bicolour FACS analysis using FITC-annexin V and propidium iodide (the annexin V/PI method). With the annexin V method, M-GMC revealed 9.4 ± 1.4% apoptosis. C1q had only marginal effects on apoptosis of M-GMC. GMC exposed to ER4G (ER4G-GMC) and further incubated with medium in the absence of C1q resulted in 25.7 ± 5.7% apoptosis (P < 0.01 relative to control). Incubation of ER4G-GMC together with 100 μg/ml of C1q significantly increased GMC-apoptosis up to 39.4 ± 4.9% (P < 0.01 relative to ER4G-GMC incubated in the absence of C1q). This enhancing effect of C1q on apoptosis of ER4G-GMC was time- and dose-dependent. In contrast, C1q did not significantly alter the apoptosis of either GMC exposed to ER14 (ER14-GMC) or to F(ab′)₂-ER4G (F(ab′)₂-ER4G-GMC), while ER14-GMC or F(ab′)₂-ER4G-GMC incubated with medium resulted in significant apoptosis compared with control. These results were supported by morphological studies and bicolour FACS analyses in time course experiments using the annexin V/PI method. The effect of C1q is dependent on the presence of intact C1q-containing globular heads and does not occur with collagen-like fragments of C1q. Furthermore, incubation of ER4G-GMC with anti-mouse κ-chain antibodies also increased ER4G-mediated GMC-apoptosis. These results indicate for the first time that C1q enhances antibody-mediated apoptosis of rat GMC in vitro, presumably by its binding to ER4G and probably by additional cross-linking of Thy-1 on the surface of GMC.