ObjectiveTo observe the effects of penetrance, different time of onset and mutation sites on retinal nerve fiber layer (RNFL) and macular thickness in patients with Leber's hereditary optic neuropathy (LHON).MethodsThis was a cross-sectional observational study. A total of 88 patients with LHON and 1492 relatives of the maternal relatives (gene carriers) who received treatment in People’s Liberation Army General Hospital from 2015 to 2017 were included in the study. Among the 1492 family members, there were 694 males and 798 females. Peripheral venous blood was extracted from all subjects for mitochondrial DNA testing, and penetrance was calculated. A total of 117 patients underwent BCVA and SD-OCT examinations, including 82 patients and 35 gene carriers. The BCVA examination was performed using the Snellen visual acuity chart, which was converted into logMAR visual acuity. The thickness of RNFL, ganglion cell complex (GCC) and inner limiting membrane (ILM)-RPE were measured with OCT instrument. The mean follow-up was 50.02±86.27 months. The disease course was divided into 6 stages including ≤3 months, 4-6 months, 7-12 months and >12 months. The thickness of RNFL, GCC and ILM-RPE in patients with different time of onset and mutation sites were comparatively analyzed by covariance analysis. Categorical variables were expressed as a percentage, and the χ2 test was used for comparison among multiple groups.ResultsAmong the 1492 family members, 285 were diagnosed with LHON and highly suspected clinical manifestations (19.10%), including 190 males (21.98%) and 95 females (11.90%). The total penetrance rates of 11778, 14484 and rare mutation sites were 19.84% (228/1149), 20.50% (33/161), and 13.19% (24/182) respectively; male penetrance rates were 28.87% (153/530), 27.28% (20/72), and 18.48% (17/92) and female penetrance rates were 12.12% (75/619),14.61% (13/89) and 7.78% (7/90). There was no significant difference in total (χ2=4.732), male (χ2=4.263) and female (χ2=4.263) penetrance between different mutation sites (P=0.094, 0.110, 0.349). Compared with non-pathogenic carriers, the thickness of the RNFL, GCC and ILM-RPE were all different in the four stages ( ≤3months, 4-6 months, 7-12 months and >12 months). The thickness of RNFL, GCC and ILM-RPE decreased with the time of onset (P=0.000). There were significant differences in the thickness of each of the GCC and ILM-RPE layers in the macular area of LHON patients with different mutation sites (P＜0.05). Among them, the site 11778 and 3460 had the most severe damage in all quadrants of macular GCC and ILM-RPE layer, followed by 14484 site, and the rare site had the least damage in all quadrants.ConclusionsThe penetrance of LHON patients is 19.10%. With the extension of the onset time (within 1 year), the RNFL layer of the optic disc and all quadrants of the macular GCC and ILM-RPE layer gradually thinned. Compared with 11778 and rare site, 14484 site, and the rare site had the lighter damage on the thickness of RNFL, GCC and ILM-RPE.
ObjectiveTo observe the disease-causing genes and the inheritance in sporadic retinitis pigmentosa (sRP) in Ningxia region. Methods49 sRP patients and 128 family members were recruited for this study. All the patients and family members received complete ophthalmic examinations including best corrected visual acuity, slit-lamp microscope, indirect ophthalmoscopy, fundus color photography, visual field, optic coherence tomography, full view electroretinogram. DNA was extracted from patients and family members. Using exon combined target region capture sequencing chip to screen the 230 candidate disease-causing gene mutations, polymerase chain reaction and direct sequencing were used to confirm the disease-causing mutations. Results24/49 patients (49.0%) had identified disease-causing genes, totally 16 genes were involved. There were 41 mutation sites were found, including 32 new mutations (78.0%). The disease-causing genes include USH2A, C2orf71, GNGA1, RPGR1, IFT140, TULP1, CLRN1, RPE65, ABCA4, GUCA1, EYS, CYP4V2, GPR98 and ATXN7. Based on pedigree analysis, 20 patients were autosomal recessive retinitis pigmentosa, 3 patients were autosomal dominant retinitis pigmentosa and 1 patient was X linked retinitis pigmentosa. 3/7 patients with USH2A mutations were identified as Usher syndrome. ConclusionsUSHZA is the main disease-causing of sRP patients in Ningxia region. 83.3% of sRP in this cohort are autosomal recessive retinitis pigmentosa.
ObjectiveThe clinical phenotypes and pathogenicity of isolated cone-rod dystrophy (CORD) caused by two novel complex heterozygous variants of the CEP290 gene were analyzed using high-resolution multi-mode imaging and gene detection techniques. MethodsA retrospective study. Two patients and two family members from a CORD family who were diagnosed by genetic testing at Henan Provincial People's Hospital in December 2021 were included in the study. All subjects underwent best-corrected visual acuity (BCVA), color fundus photography, autofluorescence, swept-source optical coherence tomography (SS-OCT), adaptive optics fundus imaging, static threshold field, full field and multiple electroretinogram (ERG) examination, as well as other systemic examinations throughout the body. The peripheral venous blood of the subjects was collected, and the whole genome DNA was extracted. DNA sequencing was performed using the Inherited Retinal Disease Kit PS400, and Sanger verification and pedigree co-segregation analysis were performed on the suspected pathogenic mutation sites. Validation was performed by Sanger sequencing, pathogenicity analysis was performed in accordance with the American College of Medical Genetics and Genomics (ACMG) guidelines. Conservation of variation among different species was analyzed by GERP++, Clustal Omega and Weblogo. ResultsBoth patients were male, and their ages were 21 and 29 years old, respectively. The right eye and left eye about BCVAs were 0.7, 0.4 and 0.3, 0.4, respectively. The full field and multiple electroretinogram ERG showed a decreased function of cones and rods, especially cones. SS-OCT showed thinning of the outer nuclear layer of macular, and attenuation of ellipsoid zone reflectivity in B-scan. Adaptive optics fundus imaging examination showed that the arrangement of cone cells in the fovea of the fovea was disordered and the density decreased, and the retinal pigment epithelial cells were seen through the atrophy of cone cells in some areas at 10°visual angle. No obvious abnormality was found in other systemic examinations of the whole body. Genetic testing showed that 2 novel compound heterozygous variants c.950T >A (p.Leu317*) (M1) and c.4144_4149del (p.Tyr1382_Glu1383del) (M2) in CEP290 were found in two patients. The first variant was predicted to be harmful in MutationTaster and CADD. GERP++ showed highly conserved among different species. The pathogenicity of the variant was suspected to be likely pathogenic according to ACMG guidelines. The pathogenicity of the second variant was uncertain significance. The parents of the proband had no similar ocular abnormalities. Verified by Sanger sequencing, it was consistent with co-separation in the family. ConclusionsPatients with pure CORD caused by CEP290 gene mutation still retain better vision when the cone structure is abnormal, the density is decreased, and the function of cone and rod cells is decreased. CEP290 M1 and M2 are newly discovered nonsense mutations and newly discovered deletion mutations, which expanded the causative gene spectrum of pure CORD.
ObjectiveTo identify the pathogenic genes and mutations in a family with Usher syndrome type 2.MethodsA three-generation family including 7 individuals was enrolled in this study. There were 2 male patients and 5 unaffected individuals. All participants was underwent related ophthalmologic examination, including best corrected visual acuity, slit-lamp, indirect ophthalmoscopy, electroretinogram (ERG), optical coherence tomography and visual field test. DNA was extracted from 3 ml peripheral venous blood of all participants. A total of 136 hereditary retinal disease target genes were screened and the DNA sequence was performed by Next-generation sequence analysis. Then the suspected mutations compared with databases to identify the suspected mutations, which should be verified with non-affected family members and 100 normal subjects by PCR and Sanger sequence.ResultsThe sequence result showed that 2 patients, the proband and his brother, carried complex heterozygous mutations in the USH2A gene: c.5459T＞C (p.M1820T) in exon 27, c.802G＞A (p.G268R) in exon 5 and c.1190T＞A (p.I397K) in exon 7. The c.5459T＞C and c.1190T＞A mutations in USH2A have not been reported in the literature and database. Although their mother carried c.5459T＞C (p.M1820T) and c.802G＞A (p.G268R), and their father carried c.1190T＞A (p.I397K) heterozygous mutations, the parents did not present phenotype. These mutations were not detected in other normal family members. The result was supported by co-segregation analysis.ConclusionThe heterozygous mutations c.5459T＞C (p.M1820T), c.1190T＞A (p.I397K) and c.802G＞A (p.G268R) in USH2A gene cause Usher syndrome in this family.
Objective To find the new mutations of Leber's hereditary optic neuropathy (LHON). Methods Two LHON families were enrolled in this study. The probands and all maternal members in this two families were underwent ophthalmologic examinations. The ages of probands were seven and 14 years old respectively. A total of 358 healthy adults were enrolled in this study as control group. The genomic DNA from whole blood of participants were extracted. The entire mitochondrial genome of probands were PCR amplified and sequenced in 24 overlapping fragments using primers as designed. At the same time, the mtDNA of maternal relatives and 358 controls were also detected. Fourteen primate species were selected from GenBank to analyzed the phylogenetics of mitochondrial sequence. Results There was no ND4 G11778A, ND1 G3460A, ND6 T14484C mutational site in all maternal members. Molecular analysis of mtDNA in this two families identified the homoplasmic tRNAGluA14683G mutation and distinct set of variants belonging to the Asian haplogroup F1a1 and G2. The site was at theTpsi;C stem oftRNAGlu and extremely conserved among 14 primate species. It was anticipated that the A14683G increased the highly conserved C-G basepairing. Furthermore, the A14683G was absence in control group. Conclusion The tRNAGluA14683G mutation is likely a new mutation associated with LHON.
Objective To screen and analyze NR2E3 gene mutations in rentinitis pigmentosa (RP) patients from Ningxia area of China. Method 120 RP patients were enrolled in this study. The patients include 33 autosomal dominant RP (ADRP) patients from 18 families, 20 autosomal recessive RP (ARRP) patients from 15 families, and 67 simplex RP (SRP) patients.100 healthy people were collected as the control group. PCR and direct DNA sequencing were used to screen the entire coding region and splice sites of NR2E3 gene. Multiple analysis was used to study the effects of NR2E3 gene on RP. ResultsA total of 12 different sequence variants in the NR2E3 gene were identified, including 6 novel sequence variants. 5 variants were detected in non-coding regions; 7 variants were detected on the 4th, 6th, 7th exon which including 3 synonymous mutations and 4 missense mutations. All of them were NR2E3 gene polymorphisms and showed no positive correlation with the RP confirmed by the multivariate logistic regression analysis. The missense mutation of p.Glu121Lys was first found in 1 ADRP proband, 2 SRP patients and 2 control subjects. Among other 8 affected individuals in this ADRP family, 5 patients also had the p.Glu121Lys variant. Notably, the 6 affected individuals with p.Glu121Lys showed more serious ophthalmic findings (early onset and early central visual impairment) than other 3 affected individuals without p.Glu121Lys.Conclusion The mutation frequency of NR2E3 and p.Glu121Lys variant in NR2E3 gene in Ningxia RP patients were lower than previous reports in other populations.
ObjectiveTo analyze the pathogenic gene types and phenotypic characteristics of 6 albinism families. Methods A retrospective series of case studies. Six probands of albinism and 20 family members were recruited for this study, 5 probands with clinical manifestations of oculocutaneous albinism (OCA) and 1 proband of ocular albinism (OA). Genomic DNA was extracted from peripheral venous blood which was collected from 6 probands and 20 family members. Genetic variations were screened by whole-exome sequencing or Sanger sequencing and then analyzed the relationship between genotypes and phenotypes. Results Genetic sequencing identified 6 potential pathogenic variants in 4 probands, including 2 compound heterozygous mutations in the 2 genes [TYR (c.1037-7T＞A, c.925_c.926insC), OCA2 (c.2359G＞A, c.587T＞C)] associated with OCA1 and OCA2, and 2 hemizygous mutations in the GPR143[GPR143 (c.11C＞G), GPR143 (c.333G＞A)] associated with OA1, respectively. In which, 5 were novel mutations and confirmed by Sanger sequencing. One case was accorded with OCA in clinical phenotype, but genetic diagnosis was OA1, the others were agreement between clinical diagnosis and genetic diagnosis. Conclusion There are 4 families with mutations in 6 families, representative of 3 type of albinism (OCA1, OCA2, OA1).
Objective To screen mitochondrial DNA mutations in 3 Chinese pedigrees with Leberprime;s hereditary optic neuropathy (LHON) carrying the ND1 G3635A mutation. Methods 88 members（53 maternal relatives and 35 paternal relatives）in 3 pedigrees were enrolled. The ophthalmologic examinations were performed for all members, including visual acuity (standard logarithmic visual acuity charts), fundus photography (Canon fundus camera),visual field (Humphrey Visual Field Analyzer), color vision (Yu zhiping color vision plate), and visual evoked potentials (Roland Consult RETI port gamma, flash VEP). 16 members had LHON, 72 members did not have LHON. 135 healthy people from Wenzhou were included as the control group. Genomic DNA was extracted from peripheral blood leukocytes of all subjects. G3635A mutation was screened by PCR mplification of mitochondrial DNA for all subjects. Mitochondrial haplotypes and other mutations in the entire mitochondrial genome were also determined by PCR using 24 pairs of primers for the probands. Results Analysis of mitochondrial DNA (mtDNA) in 3 pedigrees revealed the presence of ND1 G3635A mutation in 3 probands and all maternal relatives, but not in paternal relatives and healthy controls. Probandprime;s haplogroup belong to East Asia group N9a3, D4, and R11a. In addition to the G3635A mutation, probands also had other variants including 12 variants in D-loop region, 6 variants in RNA gene, and 36 variants in protein-encoding gene. Conclusions G3635A mutation was identified in probands and maternal relatives of 3 pedigrees of LHON. It showed that G3635A mutation was the pathogenic molecular basis for those patients.
Objective To investigate the correlation between mutation genotypes and phenotypes of X-linked retinoschisis (XLRS) patients. Methods 33 male XLRS patients, 26 female carriers and 100 normal subjects were enrolled in this study. All 33 XLRS patients were bilateral, which included 18 patients from 8 families and 15 sporadic patients. Among 66 XLRS eyes, there are microcystis-like foveal splitting in 49 eyes (74.2%), lamellar macular splitting in 43 eyes (65.2%), peripheral splitting in 32 eyes (48.5%), retinal detachment in 17 eyes (25.8%), and vitreous hemorrhage in 8 eyes (13.6%). Electroretinogram was performed on 42 eyes which showed decreased amplitude of b-wave. The 6 exons of RS1 gene were amplified by polymerase chain reaction and then directly sequenced.The correlation analysis was performed between mutation genotypes and phenotypes. Results There were 19 RS1 gene mutations including 6 novel mutations (p.Gly70Cys, p.Trp112Arg, p.Arg156Trp, p.His207ProfsX56, p.Arg209AlafsX28, p.Cys223Tyr). There was no correlation between mutation genotypes and phenotypes (chi;2=0.731, 3.438, 0.820, 3.208, 1.992; P＞0.05 ).Conclusions RS1 gene mutation is a major cause of XLRS. The RS1 mutation genotype is not correlated with phenotype, so that the prognosis cannot be predicted by the genotypes.
Objective To investigate the clinical manifestations and gene mutation of a pedigree with retinal lattice degeneration and granular corneal dystrophy (GCD) type 2. Methods Ten members in 3 generations of a pedigree with retinal lattice degeneration and GCD2 were included in the study, including 6 patients (3 males and 3 females) and 4 healthy family members. All members underwent visual acuity, slit lamp microscope, three-mirror lens, fundus color photography, optical coherence tomography, and corneal endothelial cells counting. Genomic DNA was extracted from peripheral venous blood (2 ml) from all the subjects and their spouses, who had no related inherited diseases. The next generation sequencing method was used to detect the mutation sites of transforming growth factor β (TGFBI), and all results underwent Sanger verification. Results Among the 12 eyes of 6 patients, the visual acuity was FC/20 cm-1.0. In the superficial central corneal stroma, snowflake-like deposits were observed in three cases (6 eyes), and a small amount of granular deposits were observed in three cases (6 eyes). Corneal endothelial cell counts were normal. Retinal lattice degeneration were observed in 3 cases, 6 eyes (including 3 cases of rhegmatogenous retinal detachment in 4 eyes); retinal thinning without obvious lattice degeneration in 4 eyes of 2 patients. Nystagmus in 1 patient and fundus examination showed no significant abnormalities. DNA sequencing results showed that the proband and 4 patients had missense mutation of TGFBI gene in exon 4 c.371G＞ A, the mutation site corresponding to the amino acid change encoded by TGFBI gene No. 124 Amino acids, from arginine to histidine (p.R124H). Patients with this mutation have varying degrees of clinical phenotype. Conclusions The mutation of c.701G＞ A (p.R124H) in TGFBI gene is the causative gene of GCD in this pedigree. The patients with this mutation have different clinical phenotypes.