Genotype-phenotype correlation in Italian families with Stargardt disease

Autosomal recessive Stargardt disease (STGD) has been associated with substantial genetic and phenotypic heterogeneity. By systematic clinical analyses of STGD patients with complete genetic data (i.e. identified mutations on both alleles of the ABCA4 gene), we set out to determine phenotypic subtypes and to correlate these with specific ABCA4 alleles. Twenty-eight patients from 18 families with STGD/fundus flavimaculatus were investigated. All patients were submitted to complete ophthalmologic examination, electrophysiology, fluorescein angiography and ABCA4 gene chip analysis. Two main clinical phenotypes were observed among the examined patients. The severe phenotype was characterized by the onset of the disease <20 years and reduced ERG response, whereas the mild phenotype presented with later onset of the disease and a normal ERG response. Genetic analysis of the ABCA4 gene revealed, in the severe group, more frequently deletions, stop codons and insertions as compared to the mild phenotype group (p=0.0113 by Fisher's exact test). Moreover, the compound heterozygous mutations G1961E/5018+2T-->C found in 7 patients from 3 unrelated STGD families were associated with a mild phenotype in all subjects, except 1. This study documented variability of the clinical expression of STGD in relation to the age of onset of the disease, fundus appearance and the ERG response and allowed to subdivide patients into a severe and a mild phenotype group. These findings suggest that an extensive and comprehensive genetic analysis of STGD patients combined with thorough clinical evaluation, including the careful recording of the age of onset of the disease, would allow a more precise prognostic evaluation.     

Genotype-phenotype correlation of a pyridoxine-responsive form of gyrate atrophy

Two clinical subtypes of gyrate atrophy (GA) have been defined based on in vivo or in vitro evidence of response to vitamin B6 (pyridoxine), which is the cofactor of the enzyme ornithine aminotransferase (OAT) shown to be defective in GA. We identified the E318K mutation in the OAT gene, heterozygously in three patients and homozygously in one patient, all of whom were vitamin B6-responsive by previous in vivo and in vitro studies. Dose-dependent effects of the E318K mutation were observed in the homo- and heterozygotes in the OAT activity, increase of OAT activity in the presence of pyridoxal phosphate, and apparent Km for pyridoxal phosphate. The highest residual level of OAT activity and mildness of clinical disease correlated directly with the dose of the mutant E318K allele present in the patient.     

Genotype-phenotype correlation in X-linked retinitis pigmentosa 2 (RP2)

In order to understand the underlying molecular genetic defect causing aniridia in India, eight probands from sporadic cases were screened for all 14 exons of the PAX6 gene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Direct sequencing of the SSCP variants revealed a nonsense mutation (R317X) in the eleventh exon leading to a premature termination of the PAX6 protein in the proline-serine-threonine (PST)-rich domain in two probands. Another proband exhibited an intronic polymorphism (IVS 9–12 C-T). The mutation resulted in loss of function of the PAX6 protein along with variable phenotypic manifestations in the probands. This is the first report describing a PAX6 gene mutation in aniridia cases from India and highlights the variable expressivity in phenotypes due to haploinsufficiency.     

Genotype-phenotype correlation in a family with Arg135Leu rhodopsin retinitis pigmentosa

AIM: To describe the clinical characteristics and disease course of a large family with retinitis pigmentosa (RP) from an Arg135Leu change in rhodopsin. METHODS: 29 patients in this family were evaluated. Goldmann visual fields were performed on 14 affected individuals, Ganzfeld electroretinography (ERG) on eight individuals (11-56 years), and blood samples collected on 10 individuals (11-58 years). Patient visual field data were compared with previously reported patients with different rhodopsin mutations using linear regression. RESULTS: An Arg135Leu mutation was identified in rhodopsin. Distinct stages of clinical evolution were identified for this family ranging from normal, white dots, classic bone spicules and, finally, ending with extensive retinal pigment epithelium (RPE) atrophy. 9/16 patients over the age of 20 years also demonstrated marked macular atrophy. All patients who underwent full field ERG testing demonstrated non-recordable ERGs. The overall regression model comparing solid angles of visual fields from patients with rhodopsin mutations (Pro23His, Pro347Ala, Arg135Leu) shows significant effects for age (p = 0.0005), mutation (p = 0.0014), and interaction between age and mutation (p = 0.018) with an R(2) of 0.407. CONCLUSIONS: An Arg135Leu change in rhodopsin results in a severe form of RP that evolves through various fundus appearances that include white dots early in life and classic appearing RP later. This transmembrane change in rhodopsin proves to be more severe than in a family with an intradiscal change and a family with a cytoplasmic change.     

Genotype-phenotype correlation in British families with X linked congenital stationary night blindness

AIM: To correlate the phenotype of X linked congenital stationary night blindness (CSNBX) with genotype. METHODS: 11 CSNB families were diagnosed with the X linked form of the disease by clinical evaluation and mutation detection in either the NYX or CACNA1F gene. Phenotype of the CSNBX patients was defined by clinical examination, psychophysical, and standardised electrophysiological testing. RESULTS: Comprehensive mutation screening identified NYX gene mutations in eight families and CACNA1F gene mutations in three families. Electrophysiological and psychophysical evidence of a functioning but impaired rod system was present in subjects from each genotype group, although the responses tended to be more severely affected in subjects with NYX gene mutations. Scotopic oscillatory potentials were absent in all subjects with NYX gene mutations while subnormal OFF responses were specific to subjects with CACNA1F gene mutations. CONCLUSIONS: NYX gene mutations were a more frequent cause of CSNBX than CACNA1F gene mutations in the 11 British families studied. As evidence of a functioning rod system was identified in the majority of subjects tested, the clinical phenotypes "complete" and "incomplete" do not correlate with genotype. Instead, electrophysiological indicators of inner retinal function, specifically the characteristics of scotopic oscillatory potentials, 30 Hz flicker and the OFF response, may prove more discriminatory.     

Genotype-phenotype correlation and longitudinal course in ten families with Best vitelliform macular dystrophy

AIM: Longitudinal course and genotype-phenotype correlation in patients and carriers with heterozygous mutations in hBEST1 (bestrophin). METHODS: Thirteen patients and seven possible carriers were characterised by mutation analysis with SSCPA and direct sequencing, clinical examination and fundus autofluorescence (AF). Electrophysiology (EOG and mfERG) and optical coherence tomography (OCT) were additionally performed whenever possible. RESULTS: We identified seven different heterozygous mutations in ten unrelated families with Best disease. I296del was the most frequent mutation. Five of nine individuals with I295del and two of three with N99K were asymptomatic carriers. One patient with I295del mutation had funduscopically unilateral Best disease. In three children (all with I295del), EOG initially showed a clearly present light peak that deteriorated during 5 years of follow-up in two of them. Increased AF corresponded well to funduscopically visible lesions. During 3-6 years of follow-up, the lesion area did not change significantly, but there were obvious changes in the inner structure of the lesion. CONCLUSION: In the present series I295del, the most frequent mutation in our study, and N99K showed reduced penetrance. EOG was normal in young patients even if prime signs were visible. The lesion area did not depend on the mutation and did not correlate with VA. Lower VA was associated with a more irregular AF pattern due to scarring or haemorrhage. Our results indicate a disease causing effect that is cumulative over time.     

Genotype-phenotype correlations in Axenfeld-Rieger malformation and glaucoma patients with FOXC1 and PITX2 mutations

PURPOSE: To improve the understanding of Axenfeld-Rieger Malformation (ARM)-associated glaucoma and to determine the best glaucoma treatment for patients with ARM who have known genetic defects in FOXC1 or PITX2. METHODS: Clinical data were collected from patients with diagnosed ARM, in whom we had previously identified disease-causing mutations in either the FOXC1 or PITX2 genes, by examination of patient records and use of clinical questionnaires. One hundred twenty-six patients with ARM, representing 20 different probands, with FOXC1 and PITX2 alterations were included in the study. RESULTS: ARM-associated glaucoma is a bilateral anterior segment dysgenesis disease that affects males and females equally. Seventy-five percent of the patients with ARM who participated in this study had glaucoma that had developed in adolescence or early adulthood. Of note, the patients with nonocular findings were more likely to have PITX2 defects than FOXC1 defects. Glaucoma in only 18% of patients with either PITX2 or FOXC1 genetic defects responded to medical or surgical treatment (used solely or in combination). CONCLUSIONS: Patients with FOXC1 mutations have the mildest prognosis for glaucoma development, whereas patients with PITX2 defects and patients with FOXC1 duplication have a more severe prognosis for glaucoma development than do patients with FOXC1 mutations. In the present study, current medical therapies do not successfully lower intraocular pressure or prevent progression of glaucoma in patients with ARM who have FOXC1 or PITX2 alterations. This clinical study also provides useful diagnostic criteria to identify the gene responsible for ARM.     

Peters异常PITX2及PAX6基因型表型分析

目的：分析中国人Peters异常(PA)患者的临床特征,研究Peters异常患者PITX2及PAX6基因变异情况。

方法：选取2016/2019年在常州市第二人民医院及第三人民医院眼科就诊的15例Peters异常患者,并收集详细的相关临床资料。征得患者及其家系成员的同意后抽血制备基因组DNA,用聚合酶链反应(PCR)对致病基因PITX2及PAX6的编码区及其临接内含子进行扩增后,直接测序筛查中国人群Peters异常患者PITX2及PAX6基因变异,异源双链-单链构象多态性分析(HA-SSCP)的方法对突变患者及其家系成员及80例正常对照进行验证; 分析比较国内已报道的Peters异常患者PITX2及PAX6基因突变并研究其相关表型。

结果：Peters异常患者15例PITX2基因突变筛查结果发现了1种新PITX2的突变c.296delG(P.R99fsx56),导致该基因的功能异常,分析突变患者临床特征,该患者右眼诊断为Axenfeld-Rieger综合征(ARS),左眼诊断为Peters异常。而家系成员中该患者父母及无亲缘关系的正常对照者均未发现相同突变,故此突变为新生突变。PAX6基因突变筛查未能发现突变。

结论：PA患者15例中检测到1个新PITX2基因突变,丰富了PITX2基因突变频谱,进一步明确了PA合并ARS眼病的临床特点,为该种少见眼病的临床诊断和发病原因提供了依据。     

Identification and genotype phenotype correlation of novel mutations in SIX6 gene in primary open angle glaucoma

Background: Recently SIX1 and SIX6 genes have been associated with primary open angle glaucoma (POAG). This study was planned to do mutation screening in SIX1 and SIX6 genes in North Indian POAG patients and correlate with clinical phenotypes.

Materials and Methods: SIX1 and SIX6 genes were amplified by PCR and sequenced in 115 POAG cases and 105 controls. Four pathogenecity prediction tools (MutationTaster, PolyPhen-2 HumDiv, PolyPhen-2 HumVar and SIFT) were used to predict the pathogenicity of the missense mutations. Protein modeling studies were done to predict the effect of the missense mutations on the protein structure and function.

Results: Two novel mutations p.R116G and p.R116E were observed in the SIX6 gene of patients with POAG. The mutations p.R116G and p.R116E were predicted to be pathogenic and replacement of R116 by G or E might lead to loss of interaction between DNA and R116 of wild type SIX6 protein. The patients with the mutation p.R116E had significantly more visual field damage (MD) and early age of onset of the disease. No sequence variations were observed in the SIX1 gene.

Conclusion: These results expand the mutation spectrum of SIX6 gene and suggest that SIX6 gene plays an important role in POAG pathogenesis.     

视网膜变性Pde6b基因突变小鼠治疗的研究进展

视网膜变性动物模型是研究人类遗传性视网膜变性疾病治疗的重要工具,目前常用于基础研究的视网膜变性动物模型包括系列视杆细胞变性小鼠(rd),如rd1小鼠、rd10小鼠和nmf137小鼠,这些模型鼠均为磷酸二酯酶β亚基(Pde6b,PDEβ)基因突变的小鼠模型,因其发病机制与人类具有某些同源性,因而已大量应用于视网膜变性疾病的基因诊断和治疗研究中.为了探索更为有效的治疗方法,就3种模型鼠的发病机制、药物治疗、神经营养治疗以及干细胞治疗方法、疗效等的研究进展进行综述.     

RB1 gene mutations in retinoblastoma and its clinical correlation

Purpose

To find correlation between the type of mutations observed and the severity of the disease using multiple techniques like polymerase chain reactions (PCR), quantitative multiplex PCR, sequencing and RNA analysis.

Methods

Prospective, observational study. Patients who had been screened for mutations in the RB1 gene were included in the study. Patient details including demographic data; age and sex, laterality, international classification of intraocular retinoblastoma (ICIOR) staging, modality of management, histopathology high risk factors if the eyes were enucleated and metastasis rate were assessed.

Results

Seventy four patients were studied. Fifty three patients had bilateral and 21 unilateral disease. Complete genetic data was analyzed for 74 patients and complete clinical correlation was established for all the 49 patients with mutations. Of the total mutations identified, 11/49 (22.4%) of patients had large deletions, 12/49 (24.5%) had small deletions or insertions, 14/49 (28.6%) had nonsense mutations, 7/49 (14.3%) had splice mutations and 5/49 (10.2%) of patients had missense mutations. Four cases were familial. Group E ICIOR stage at presentation was noted in 40% of patients with large deletions, 33% with small deletions whereas 38.5% with splice mutations and 44.4% of patients with missense mutations presented with Group B ICIOR. Twenty five percentages of eyes with large deletions had high risk features on histopathology and one patient among these developed metastasis.

Conclusion

Current laboratory testing of RB1 mutations may be feasible in determining the severity of the disease and patient counseling. The study provides a starting point for looking at correlations.     

Spatial correlation of mouse photoreceptor-RPE thickness between SD-OCT and histology

Spectral Domain Optical Coherence Tomography (SD-OCT) applied to the mouse retina has been limited due to inherent movement artifacts and lack of resolution. Recently, SD-OCT scans from a commercially available imaging system have yielded retinal thickness values comparable to histology. However, these measurements are based on single point analysis of images. Here we report that using the Spectralis HRA + OCT Spectral Domain OCT and Fluorescein Angiography system (Heidelberg Engineering, Heidelberg, Germany), retinal thickness of linear expanses from SD-OCT data can be accurately assessed. This is possible by the development of a Spectralis-compatible ImageJ plug-in that imports 8-bit SLO and 32-bit OCT B-scan images, retaining scale and segmentation data and enabling analysis and 3D reconstruction. Moreover, mouse retinal layer thickness values obtained with this plug-in exhibit a high correlation to thickness measurements from histology of the same retinas. Thus, use of this ImageJ plug-in results in reliable quantification of long retinal expanses from in vivo SD-OCT images.     

Ophthalmic manifestations of Heimler syndrome due to PEX6 mutations

Background/Aims: Pigmentary retinal dystrophy and macular dystrophy have been previously reported in Heimler syndrome due to mutations in PEX1. Here we reported the ocular manifestations in Heimler syndrome due to mutations in PEX6.

Materials and methods: Medical records were reviewed to identify patient demographics, ophthalmic and systemic findings, and results of diagnostic testing including whole genome sequencing.

Results: Patient 1 is 12-year-old boy with a novel mutation c.275T>G (p.Val92Gly) and known mutation c.1802G>A (p.Arg601Gln) in PEX6. Patient 2 is a 7-year-old girl with the same known c.1802G>A (p.Arg601Gln) mutation and another novel missense mutation c.296G>T (p.Arg99Leu). Both patients exhibited a pigmentary retinopathy. Visual acuity in patient 1 was 20/80 and 20/25 following treatment of intraretinal cystoid spaces with carbonic anhydrase inhibitors, while patient 2 had visual acuity of 20/20 in both eyes without intraretinal cysts. Fundus autofluorescence showed a multitude of hyperfluorescent deposits in the paramacular area of both eyes. OCTs revealed significant depletion of photoreceptors in both patients and macular intraretinal cystoid spaces in one patient. Full field electroretinograms showed normal or abnormal photopic but normal scotopic responses. Multifocal electroretinograms were abnormal.

Conclusions: Heimler syndrome due to biallelic PEX6 mutations demonstrates a macular dystrophy with characteristic fundus autofluorescence and may be complicated by intraretinal cystoid spaces.     

PAX6 mutations may be associated with high myopia

PAX6 is a key regulator of eye development and there are many well recognized ophthalmic sequelae of mutations at this locus. The 14 exon PAX6 gene is well conserved across species and phyla. Coding region mutations manifest in a variety of phenotypes. Predicted premature protein truncations are generally associated with classical aniridia. Missense mutations are often found in cases with variant phenotypes such as ectopia pupillae; isolated foveal hypoplasia; nystagmus and hyaloid vessel proliferation. The locus has also been implicated, through a genome-wide sib-pair scan, to be important in the normal variation of myopia. We investigated the association between identified PAX6 mutations and refractive error in Australian patients from four pedigrees. Two of eight subjects with a 1410delC PAX6 mutation had a mean spherical equivalence < -9D, whilst a mean spherical equivalence < or = -5D was recorded in two from four subjects with an Arg240Stop PAX6 mutation and one of two subjects with a Glu93Stop mutation. One individual identified with a Pro346Ala PAX6 mutation had a mean spherical equivalence of +2.8 D. Thus, our observations generally support other incidental findings, that PAX6 mutation, particularly predicted haploinsufficiency, may be associated with extreme refractive error, although the mechanism by which this occurs is not clear.