TGFBI and CHST6 gene analysis in Chinese stromal corneal dystrophies

AIM: To investigate whether mutations in TGFBI gene or CHST6 gene correlated with stromal corneal dystrophies (CD) in 8 Chinese probands. METHODS: Eight unrelated patients with stromal corneal dystrophies were recruited in this study; all affected members were assessed by completely ophthalmologic examinations. Genomic DNA was extracted from peripheral leukocytes, 17 exons of TGFBI gene and the exon of CHST6 gene were amplified by polymerase chain reaction (PCR), sequenced directly and compared with the reference database. RESULTS: Three heterozygous mutations in TGFBI gene were identified in six patients: c. 370C>T (p.Arg124Cys) was found in exon 4 of TGFBI gene in three members, c. 371G>A (p.Arg124His) was found in one patient; c. 1663C>T (p.Arg555Trp) was found in exon 12 in other two members. In addition,four polymorphisms with the nucleotide changes rs1442, rs1054124, rs4669, and rs35151677 were found in TGFBI gene. Mutations were not identified in the rest of 2 affected individuals in TGFBI gene or CHST6 gene. CONCLUSION: Within these patients, R124C, R124H and R555W mutations were co-segregated with the disease phenotypes and were specific mutations for lattice corneal dystrophy type I (LCD I), Avellino corneal dystrophy (ACD, GCDⅡ), granular corneal dystrophy type I (GCD I), respectively. Our study highlights the prevalence of codon 124 and codon 555 mutations in the TGFBI gene among the Chinese stromal corneal dystrophies patients.     

Diverse clinical phenotypes associated with a nonsense mutation in FAM161A

Purpose:

Mutations in the FAM161A gene have been reported in association with autosomal recessive retinitis pigmentosa (arRP) in several ethnic populations. This study aimed to assess the prevalence of FAM161A-related retinopathy in a British cohort and to characterise the phenotype associated with mutations in this gene.

Methods:

The FAM161A coding region and intron–exon boundaries were screened by Sanger sequencing in 120 retinitis pigmentosa (RP) patients (with likely autosomal recessive inheritance) in whom mutations in other known major RP genes have been ruled out by commercially available testing. Homozygosity mapping was performed in one consanguineous family, and high-throughput sequencing of candidate genes was performed to identify disease-associated changes. Clinical assessment of affected individuals included perimetry testing, fundus autofluorescence imaging, and optical coherence tomography.

Results:

Two patients of British origin with a homozygous mutation in FAM161A (c.1309A>T, p.Arg437*) were identified by Sanger sequencing. Homozygosity mapping and subsequent high-throughput sequencing analysis identified a further family of Pakistani origin with the same genotype. Clinical examination of affected members of these families revealed that this mutation was associated with a diverse clinical phenotype, ranging from mild disease with preservation of central acuity to severe visual impairment.

Conclusions:

Homozygosity for the c.1309A>T, p.Arg437* variant in FAM161A is a relatively common cause of arRP. The mutation occurs in diverse ethnic populations, associated with typical retinitis pigmentosa with disease onset usually in the second or third decade of life.     

Mutation c. 1142 del G in the <Emphasis Type="Italic">PRPF31</Emphasis> Gene in a Family with Autosomal Dominant Retinitis Pigmentosa (RP11) and Its Implications

Purpose

To identify a mutation in the PRPF31 gene in a family (Family K) with autosomal dominant retinitis pigmentosa (adRP) linked to 19q13.4 (RP11) and to find the frequency of mutations in the PRPF31 gene among Japanese families with adRP.

Methods

Genomic DNA specimens were prepared from five symptomatic and two asymptomatic members of Family K and an additional 39 patients of 39 unrelated families with adRP. Coding regions of the PRPF31 gene were amplified by polymerase chain reaction. The amplicons were analyzed by a direct sequencing method.

Results

All seven family members had a heterozygous c.1142delG mutation in the PRPF31 gene, which was identical to the mutation previously reported in a different Japanese family. No other mutation was found in the PRPF31 gene among the 39 additional patients with adRP.

Conclusion

Although the frequency of mutations in the PRPF31 gene is about 2.5% in Japanese families with adRP, it is possible that c.1142delG is a common mutation among Japanese patients with adRP associated with mutations in the PRPF31 gene.?Jpn J Ophthalmol 2007;51:45–48 © Japanese Ophthalmological Society 2007

     

Phenotype/genotype correlation in a case series of Stargardt's patients identifies novel mutations in the ABCA4 gene

Purpose

To investigate phenotypic variability in terms of best-corrected visual acuity (BCVA) in patients with Stargardt disease (STGD) and confirmed ABCA4 mutations.

Methods

Entire coding region analysis of the ABCA4 gene by direct sequencing of seven patients with clinical findings of STGD seen in the Retina Clinics of Southampton Eye Unit between 2002 and 2011.Phenotypic variables recorded were BCVA, fluorescein angiographic appearance, electrophysiology, and visual fields.

Results

All patients had heterozygous amino acid-changing variants (missense mutations) in the ABCA4 gene.A splice sequence change was found in a 30-year-old patient with severly affected vision. Two novel sequence changes were identified: a missense mutation in a mildly affected 44-year-old patient and a frameshift mutation in a severly affected 34-year-old patient.

Conclusion

The identified ABCA4 mutations were compatible with the resulting phenotypes in terms of BCVA.Higher BCVAs were recorded in patients with missense mutations. Sequence changes, predicted to have more deleterious effect on protein function, resulted in a more severe phenotype.This case series of STGD patients demonstrates novel genotype/phenotype correlations, which may be useful to counselling of patients. This information may prove useful in selection of candidates for clinical trials in ABCA4 disease.     

Correlation of structure and function of the macula in patients with retinitis pigmentosa

Purpose

To correlate the structure of the macula, as measured by spectral-domain optical coherence tomography (SD-OCT) and function, as measured by microperimetry (MAIA) in patients with retinitis pigmentosa (RP) and relatively good visual acuity.

Design

Prospective, cross-sectional, non-intervention study.

Subjects

Patients with RP.

Methods

Thirty patients with RP and good central visual acuity were identified. Each patient underwent SD-OCT of the macula and microperimetry. The images were overlaid using the custom-designed software. The retinal sensitivity by microperimetry was correlated with corresponding retinal thickness, as measured by the SD-OCT. ELM, COST, and IS/OS junction were scored as intact, disrupted, or absent.

Main outcome measures

Comparing the retinal sensitivity on the MAIA with various measurements on the SD-OCT.

Results

The retinal sensitivity on the MAIA showed a significant correlation with total retinal thickness and outer retinal thickness on the SD-OCT. There was no association with either the inner retinal thickness or the choroidal thickness. ORT showed a statistically significant correlation with the anatomical classification of ELM (r=−0.76, P<0.001), IS/OS (r=−0.800, P<0.001), and COST (r=−0.733, P<0.001).

Conclusion

This study determined that there was a high correlation of the structure and function of the central macula in patients with RP. These studies are important to establish surrogate markers that can be used as end points for various tests in future therapeutic clinical trials.     

Mutations in the CYP1B1 gene may contribute to juvenile-onset open-angle glaucoma

Purpose

Glaucoma is one of the leading causes of blindness in the world. Juvenile-onset open-angle is a subtype of glaucoma. In this context, we investigate the possible mutations in the promoter and coding regions of the CYP1B1 gene among patients suffering juvenile-onset open-angle glaucoma (JOAG).

Methods

The CYP1B1 gene was analysed for mutations in 61 unrelated Taiwanese probands with JOAG and in 100 healthy control subjects. Genomic DNA was extracted from peripheral blood leukocytes and then subjected to PCR. The amplified products were screened for base mutations by autosequence. Next, data from the two groups were compared using the χ² test. Additionally, three-dimensional (3D) modelling of the human wild-type and p.R390H mutation was performed using SWISS-MODEL, an automated homology modelling program. Finally, the figure was prepared for the modelled structures by using the Accelrys ViewerLite 5.0 program.

Results

Analysis results indicated two CYP1B1 mutations and five polymorphisms. The prevalence of CYP1B1 gene mutations in this study was 4.92% (3/61). The mutations included a missense mutation (p.Arg390His; 2/3) and a mutation in the 5′-untranslated region (c.1–313A>C; 1/3). Moreover, computer-assisted modelling revealed that this p.R390H mutation affects the intra-molecular interaction in the hydrogen-bonding interaction with Glu387 and Asn428, thus altering significantly the efficiency of the haem-binding and proper folding of the molecule.

Conclusions

As a result, the p.Arg390His mutation might affect the protein structure and, ultimately, the normal function of CYP1B1. Therefore, we suggest that the c.1169G>A (p.Arg390His) mutation of CYP1B1 may be a risk factor for the development of JOAG.     

Variability in clinical phenotypes of PRPF8-linked autosomal dominant retinitis pigmentosa correlates with differential PRPF8/SNRNP200 interactions

Purpose: To expand the genotype/phenotype correlations in patients with autosomal dominant retinitis pigmentosa (adRP) harboring PRPF8 variants.

Materials and Methods: Two patients, a father and his daughter, harboring a novel p.PRPF8-Glu2331* variant, underwent ophthalmic examination at 3-year-interval, including fundus photography, fundus autofluorescence, optical coherence tomography, and ISCEV standard full field ERGs. All reported disease-causing PRPF8 variants were collected and localized in the PRPF8 and PRPF8/SNRNP200 protein structures.

Results: The p.PRPF8-Glu2331* variant results in a truncated PRPF8 protein lacking the last five C-terminal amino acids and caused in the two patients a severe clinical phenotype, with the macula being affected from the second decade on. All but two adRP-linked variants are located in the last exon 43 encoding the C-terminal tail of the C-terminal PRPF8 Jab1 domain. The p.PRPF8-Ser2118Phe and -Asn2280Lys variants encoded by exons 39 and 42, respectively, are located at the basis of the C-terminal tail.

Conclusions: Frame-shift mutations and nonconservative amino acid changes in PRPF8 typically cause severe clinical phenotypes. The conservative missense variant p.PRPF8-Arg2310Lys that is not altering the global charge of the C-terminal tail, and variants located at the basis of the C-terminal tail show milder clinical phenotypes, in accordance with functional data on PRPF8/SNRNP200 interactions in yeast.     

Rapid and cost-effective molecular diagnosis using exome sequencing of one proband with autosomal dominant congenital cataract

Purpose

Due to high genetic heterogeneity, to exclude known mutations and map novel mutations in autosomal dominant congenital cataract (ADCC) using conventional candidate gene screening requires laborious laboratory work. We attempted to use a cost-effective exome sequencing strategy to identify disease-causing mutations in an ADCC pedigree.

Methods

An ADCC pedigree affected by nuclear cataract and 200 unrelated senile cataract controls were recruited and given comprehensive ophthalmic examination. Whole exome of the proband of the family was captured by the Illumina TruSeq Exome Enrichment Kit, followed by sequencing using Illumina HiSeq 2000 sequencer. Validation was performed by direct sequencing.

Results

The whole exome, including all exons of known ADCC disease-causing genes, was screened for possible disease-causing mutations. A recurrent missense mutation c.773C>T (p.S258F) in exon 2 of the gap junction protein alpha 8 gene (GJA8) was identified in the proband with nuclear cataract. The result was confirmed by direct sequencing. The mutation showed complete co-segregation with the disease phenotype in the family but was not observed in unrelated unaffected controls.

Conclusion

By successfully sequencing whole exome of only one proband and identifying a GJA8 mutation in one ADCC pedigree, the current study demonstrated that exome sequencing could serve as a rapid, robust, and cost-effective approach in clinical diagnosis and disease-causing gene discovery for ADCC.     

Assessment of Functional Vision Score and Vision-Specific Quality of Life in Individuals With Retinitis Pigmentosa

Purpose

To determine the relationship between the American Medical Association''s (AMA) functional vision score (FVS) and vision-specific quality of life in retinitis pigmentosa (RP) patients using the National Eye Institute''s Visual Functioning Questionnaire (NEI-VFQ 25).

Methods

One hundred eight patients with RP participated in the study. We measured best-corrected visual acuity, conducted Goldmann perimetry, and collected the self-reported NEI-VFQ 25. The FVS was calculated using the functional field score (FFS) and the functional acuity score (FAS). The correlations of the VFQ composite scores to the FVS, FFS, and FAS were determined using correlation and regression analyses.

Results

FVS was highly correlated to the BCVA (r=0.69, p<0.001), the FFS (r=0.86, p<0.001) and the FAS (r=0.73, p<0.001). Significant correlations of the VFQ composite score to the BCVA (r=0.60, p<0.001), FFS (r=0.44, p<0.001), FAS (r=0.60, p<0.001), FVS (r=0.58, p<0.001) were also found. However, the correlation strengths of BCVA, FVS, FAS, and FFS to NEI-FVQ were not different.

Conclusions

In RP patients, the vision-specific quality of life was correlated with the AMA guidelines'' FVS, FFS, and FAS. Their correlation degrees to NEI-FVQ were not different. This result suggests that vision-specific quality of life can be explained by both visual acuity and visual field in RP patients.     

Two novel mutations of the PAX6 gene causing different phenotype in a cohort of Chinese patients

Purpose

Aniridia (AN) is a rare congenital panocular disorder caused by the mutations of the paired box homeotic gene 6(PAX6) gene. The PAX6gene is also involved in other anterior segment malformations including Peters anomaly. We studied the PAX6gene mutations in a cohort of affected individuals with different clinical phenotype including AN, coloboma of iris and choroid, or anterior segment malformations.

Patients and methods

Six unrelated families and 10 sporadic patients were examined clinically. After informed consent was obtained, genomic DNA was extracted from the venous blood of all participants. Mutation screening of all exons of the PAX6gene was performed by direct sequencing of PCR-amplified DNA fragments. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect large deletions.

Results

By clinical examination, the patients and the pedigrees were divided into the following three groups: AN, coloboma of iris and choroids, and the anterior segment malformations including peters anomaly. Sequencing of the PAX6gene, three intragenic mutations including a novel heterozygous splicing-site mutations c.357-3C>G (p.Ser119fsX) were identified in the patients of the AN group. A novel missense mutation c.643T>C (p.S216P) was detected in the anterior segment malformation group. The mutation p.S216P located in the homeodomain region of the PAX6 caused the phenotype of Peters anomaly in family A6 with different expressing. Through MLPA analysis, a large deletion including the whole PAX6gene and DKFZ p686k1684gene was detected in one sporadic patient from the AN group. Neither intragenic mutation nor large deletion was identified in the group with coloboma of iris and choroid.

Conclusion

Our findings further confirmed that different kind of mutations might cause different ocular phenotype, and clearly clinical phenotype classification might increase the mutation detection rate of the PAX6gene.     

The ocular phenotype of stiff-skin syndrome

Purpose

Stiff skin syndrome (SSS; MIM#184900) is a rare autosomal dominantly inherited Mendelian disorder characterised by thickened and stone-hard indurations of the skin, mild hypertrichosis, and limitation of joint mobility with flexion contractures. It is autosomal dominant with high penetrance and results from mutations in the fibrillin 1 (FBN1; MIM*134797) gene. Here we present the associated ocular phenotype in a two generation nonconsanguineous Northern Irish family.

Methods

The affected patients underwent complete ophthalmic and orthoptic assessment and genetic testing.

Results

All three patients had ophthalmoplegia of varying degrees. Direct sequencing of the FBN1 gene detected a heterozygous pathogenic mutation (c.4710G>C; p.Trp1570Cys) in all affected patients.

Conclusions

This is the first report of ophthalmoplegia in association with SSS.     

A novel deletion mutation in RS1 gene caused X-linked juvenile retinoschisis in a Chinese family

Purpose

X-linked juvenile retinoschisis (XLRS), a leading cause of juvenile macular degeneration, is characterized by a spoke-wheel pattern in the macular region of the retina and splitting of the neurosensory retina. This study aimed to identify the underlying genetic defect in a Chinese family with XLRS.

Methods

The proband underwent complete ophthalmic examinations, including fundus examination, fundus autofluorescence, and optical coherence tomography. DNA extracted from proband and his younger brother was screened for mutations in RS1 gene. The detected RS1 mutation was tested in all available family members and 200 healthy controls.

Results

Reduced visual acuity, spoke-wheel pattern at the fovea, and split retina were observed in the proband. A novel frameshift mutation c.206-207delTG in the RS1 gene, leading to a truncated protein (p.L69fs16X), was identified in the proband and his younger brother. This mutation was not found in any unaffected member or in the healthy controls. The mother of the proband was hemizygous for this mutant allele.

Conclusions

We identified a novel causative mutation of RS1 in a Chinese family with XLRS. This finding expands the mutation spectrum of RS1 and provides evidence for a phenotype–genotype study in XLRS.     

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.     

Macular corneal dystrophy and associated corneal thinning

Purpose

To identify the molecular genetic cause of macular corneal dystrophy (MCD) in four probands, and characterize phenotypic similarities between MCD and keratoconus.

Methods

We performed ophthalmological examination, Scheimpflug imaging (Pentacam, Oculus Inc.), histopathological examination of excised corneal buttons, and direct sequencing of the CHST6 coding region.

Results

Pentacam measurements were taken in six eyes of three probands. All showed diffuse corneal thinning with paracentral steepening of the anterior corneal surface that was graded as keratoconus by the integrated software, but without associated ectasia of the posterior corneal surface or regional thinning. Homozygous or compound heterozygous CHST6 mutations were identified in all cases, including two novel mutations, c.13C>T; p.(Arg5Cys) and c.289C>T; p.(Arg97Cys).

Discussion

Localized elevation of the anterior corneal curvature can occur in MCD in the absence of other features of keratoconus. The identification of a further two Czech probands with the compound allele c.[484C>G; 599T>G] supports the enrichment of this allele in the study population.     

Chinese family with atypical granular corneal dystrophy type I caused by the typical R555W mutation in TGFBI

AIM: To investigate the clinical features and genetic defects in four generations of a Chinese family affected with atypical granular corneal dystrophy type I (GCD type I).METHODS: Family history and clinical data were recorded. Genomic DNA samples were obtained from peripheral blood leukocytes of all participated. Exons of the transforming growth factor-β-induced(TGFBI) gene were directly sequenced after being amplified by polymerase chain reaction (PCR), and multi-point linkage analysis using microsatellite makers flanking the gene was applied to identify the disease-causing mutation.RESULTS: Clinical features were quite variable in patients, some patients only had opacities in the epithelium, and others revealed multiple bilateral circular, discrete, crumb-like opacities mainly in the epithelium, with several in different depths of corneal stroma, and the performance was different bilaterally, even in the same patient. Directly nucleotide sequencing revealed a heterozygous p.R555W mutation in the coding sequence of the TGFBI gene in all affected individuals of the family, but was not found in all unaffected. The maximum logarithm of odds (LOD) score obtained by multi-point analysis was detected at marker locus D5S393 (LOD=2.740; α=1.000).CONCLUSION: Our case presented with clinical futures and the pathogenic mutations in TGFBI gene, the phenotype of the pedigree was quite different from typical GCD type I, so we suggested that this phenotype was a variant of GCD type I. These findings expand the knowledge about GCD type I, and demonstrate that molecular genetic analysis is important to make an accurate diagnosis of patients with variable corneal dystrophies in clinic.     

Best's macular dystrophy in Australia: phenotypic profile and identification of novel BEST1 mutations

Purpose

(1) To evaluate the spectrum of BEST1mutations within Australian Best Disease or vitelliform macular dystrophy (VMD) pedigrees, including any novel mutations; (2) to analyse the range of clinical presentations of this cohort; (3) to determine any possible genotype–phenotype correlations and (4) to compare clinical data of patients with phenotypic VMD, both with and without a BEST1mutation.

Patients and methods

Patients with suspected VMD were referred to clinical centres for ophthalmological assessment and genetic screening. When a mutation was identified in a proband, further family members were invited for clinical and genetic screening.

Results

We identified 42 patients with one of 13 BEST1mutations. Seven mutations were novel. There were a further 14 probands in whom a BEST1mutation was not identified. Median visual acuity in both VMD (mutation positive) and clinical VMD (no BEST1mutation identified) groups reached driving standards (6/12 or better).

Conclusion

We did not identify any firm genotype–phenotype correlations in our Australian VMD pedigrees, in which there was a spectrum of BEST1mutations and marked variation in clinical presentation. Genetic screening remains the gold standard for VMD diagnosis. Patients should be counselled that visual acuity might remain at or above driving standards in at least one eye even in the presence of a BEST1mutation.     

Ophthalmic and molecular genetic findings in Kniest dysplasia

Purpose

To study the variability of the ophthalmic phenotype in Kniest dysplasia. Kniest dysplasia is an inherited disorder associated with defects in type II collagen and characterised by short-trunked dwarfism, kyphoscoliosis, and enlarged joints with restricted mobility. Other features include marked hand arthropathy, cleft palate, hearing loss, and ocular abnormalities (myopia, abnormal vitreous, and high risk of developing retinal detachment).

Methods

Data from eight unrelated individuals with a clinical and molecular diagnosis of Kniest dysplasia are reported. Clinical assessment included an audiogram and ophthalmological examination in all but one patient who died in the immediate postnatal period. Sanger sequencing of the COL2A1 gene was performed.

Results

Six of the seven patients tested were high myopes with one patient being an emmetrope. Bilateral quandratic cataracts and subluxed lenses were noted in one subject. Variable but abnormal vitreous architecture was observed in all seven individuals tested. Six of the seven patients had significant hearing impairment and five of the seven patients exhibited clefting abnormalities. One patient had bilateral retinal detachments in his twenties. Six dominant disease-causing COL2A1 variants were detected. In three cases, testing of parental samples revealed that the disease-causing variant was not present in either parent.

Conclusion

The ophthalmic features in Kniest dysplasia are very similar to those in other disorders of type II collagen such as Stickler syndrome. It is likely that different type II collagenopathies have a similar level of ocular morbidity and regular ophthalmologic examination is recommended. Kniest dysplasia is associated with heterozygous COL2A1 mutations that are frequently de novo.     

A novel mutation of sgk-1 gene in central serous chorioretinopathy

AIM

To investigate the association of serum glucocorticoid kinase gene-1 (SGK-1) DNA variants with chronic central serous chorioretinopathy (CSC).

METHODS

We enrolled 32 eyes of 32 patients who were diagnosed with chronic CSC and composed 32 normal eyes as a control group. Peripheral blood was used for DNA extraction and polymerase chain reaction (PCR) amplification. SGK1 gene was sequenced by using BigDye^® Terminator v3.1 cycle sequencing KIT (Applied Biosystems, Foster City, CA, USA). The SGK1 gene and its variants were investigated in CSC patient group and control group.

RESULTS

We identified a new polymorphism M32V in two person in the patient group (Minor allele frequency (MAF)=0.009) on the region of 1-60 amino acids. The rs1057293 was located in the encoder region of the SGK 1 gene but not associated with CSC (P=0.68). An intrinsic rs1743966 is also not associated (P=0.28).

CONCLUSIONS

The new polymorphism M32V is located on the region of 1-60 amino acids which is necessary for localization to the mitochondria in CSC patient. This mutation is probably important for the energy metabolism and plays an important role in the cellular response to hyperosmotic stress and other stress stimuli. Both rs1057293 and rs1743966 are not associated with CSC.