Research On The Genetics And Clinical Characteristics Of The Chinese Bietti Crystalline Corneoretinal Dystrophy

BackgroundBietti crystalline corneoretinal dystrophy（BCD） is a serious inherited retinal degeneration disease. Patients with BCD usually present in the 2nd or 3rd decade and they progress to legal blindness by the 5th or 6th decade of life. BCD inheritance pattern is autosomal recessive heredity, its pathogenic gene is CYP4V2 with 19 Kp and containing 11 exons, located in 4q35⁴qter of chromosome, and belonged to the family of CYP450, the encoding protein of the gene include 525 amino acid. There are 62 species change on CYP4V2 gene in 238 patients around the world by DNA sequencing analysis（as of January 2015）. Study found that there is a high morbidity of BCD in China, according to the forecast for 1/24000. So far, a few cases reported that gene mutations were found and described the incomplete clinical features. There are lack of researches for BCD in demographic characteristics, regional distribution, epidemic characteristics, clinical characteristics and genetic type on these case.The aim of this study is to know well the genetic characteristics, morbidity, regional distribution, hot distribution of patho-mutation, the correlation between genotype and phenotype, the curative effect of drug treatment, and the relationship between the genotypes and the curative effect, based on the characteristic analyzing of demography, genetics and clinical cases in the propositus of BCD family by large sample genetic testing in Chinese population, to lay the foundation for further study of pathogenesis and individualized treatment of BCD.Methods1. The normative registration of demography and genetic information was carried out among the 215 patients with BCD, including the information of patients, occupation, age, level of education, life skills, nutrition, birthplace and ancestral home, family history of the retinal degeneration disease, history of incest, and other diseases, etc.The mobidity statistics and characteristic analying of family and geographic distribution of patients with BCD were done according to the case number and sources of these patients.2. Mutational screening of CYP4V2 coding regions and flanking intron sequences were examined via directional Sanger sequencing, with allele separation confirmed by screening other family members in 92 probands. Subsequent in silico analysis of the mutational consequence on protein function was undertaken, with the impact of the novel mutation on pre-mRNA splicing examined via RT-PCR.Targeted region capture and next-generation sequencing: A sequence capture array was designed to capture the coded exons of the 55 HRD genes, including the CYP4V2 and 23 probands were detected with it. A family with five-generation RP patient were tested for pathogenic gene screening by NGS technology and Sanger validation with a target seguence capture chip.3. The linical features of BCD were investigated by checking Best Corrected Visual Acuity（BCVA）, Vision Field（VF）, Full field Flash Electroretinogram（FERG）, Multifocal Electroretinogram（mf FERG）, Fundus Photography, Autofluorescence（AF）, and Optical coherence tomograpy（OCT） in macular area for 292 eyes of 146 patients with BCD. Combined with the results of genetic test, the correlation between the pathogenicity gene and clinical features of BCD was also investigated for 115 cases among these patients and for family with many BCD patients.Results1.215 BCD patients, 110 men and 105 women, belong to 184 familys with the autosomal recessive heredity. All of them are the han nationality. The BCD mobidity due to consanguineous marriage is 3.2%. A pair of twins female patients accounts for 0.6% of the BCD families. The ratio between BCD phenotype and normal phenotype is 1:3.8. The ratio of falling in BCD is 1: 0: 0 among patients, adopted childs and adopter parents. About 25.1% of first-degree relatives in these family have systemic disease. BCD in RP is 22.70%. The prevalence rate of patients at our hospital was 4.6/ 100000. The mean age is 31.7±16.8（years） for the patients with initial symptoms of BCD, 41.8 ±23.1（years）for the patients with initial consult, there is statistical significance between these age（p=0.01）. The BCD mobidity among each age groups also has notable statistical significance（p=0.03）. Patients born in southwestern China account for 95%, and that ancestral home belonged to Hunan, Hubei, and Guangdong provience for 30.7%, 80% of patients come from the countryside. Aggravation of BCD for 20.1% female patients is thought to be related to pregnancy, childbirth, breastfeeding, and menopause.10% of the patients with BCD approval that the bad living and working environment aggravate disease.2.A total of 22 alleles were identified in 115 probands and nine were novel. The most common mutation are c.802₈10del17ins GC, with 48.7%（112/230） allele frequency. There were did not found these new pathogenic mutations in 100 controls and dbSNP prediction showed that these novel mutation are harmful.In silico analysis predicted that the missense variant, p.Tyr343 Asp, disrupted the CYP4V2 surface electrostatic potential distribution and spatial conformation.Two separata genes and gene mutation screening coexist in a family.3.100% crystal particles are observed in retina and vitreum in 146 patients with BCD, and shown best in infrared fundus photography. It is consistent that local RPE damaged caused crytal deposition and lesions of retinal morphology and function from AF typ e, OCT style,and mf ERG parting.Early patients with BCD are not symptoms of decreased vision and night blindness.Vision loss rate of patients with BCD per year is 6.44%, and the fundus changes is slow and the AF changes is very clear in following patients w ith BCD within three years. There are significant correlations（p < 0.01） between the AF type,VA, the sum of amplitude of 1 ring, 1⁴ring and 1⁶ ring of P1 wave with mf ERG, CSFT,ages and fundus stage.AF parting,OCT installment and mfERG type is highly correlated.The patients with type 1 with pepper salt of AF is characterized by OCT phase1²,normal or reduce model mf ERG, Patients with type 2 with map hypo-AF showed OCT phase2³, reduce and model extinguished mf ERG. Patients with type 3 with diffuse hypo-AF indicated OCT phase3⁵, extinguished mfERG. Patients with type 4 of vascular sclerosis type hype-AF corresponding OCT parting for 4⁵ period, extinguished mf ERG. FERG style shows an diversity of damage of retinal photoreceptor cells: the mfERG of early BCD patients with normal FERG is exception; the type of FERG is Cone-dominant for the BCD patients with starting symptoms of night blindness, and Rod-dominant for the BCD patients with starting symptoms of vision decrease.4. Among four types of mutations,homozygous mutations c. 802-8₈10del17ins GC and c. 1091-2 A > G, compound heterozygous mutations c. 802-8₈10del17ins GC + c. 992 a > c, p.H 331 p and c. 802-8₈10del17ins GC + c. 1091-2 A > G, account respectively for 28.1%, 5.3%, 14.0%, and 5.3% of all alleles in the CYP4V2 screening of 114 cases of BCD probands.There is no difference in the onset age and duration of disease between two of them（p > 0.05） and there is correlation between FERG and mfERG of the patients with homozygous mutations c. 802-8₈10del17ins GC.The patients of the same family are different both in the onset ages and in fundus stages of BCD.5.Two independent disease-causing genes, CYP4V2 and PRPF3, are found to coexist in same big family of BCD by gene screening, 12 patients of the family carry PRPF3 and show typical phenotype of RP with autosomal dominant inheritance, however,the proband was identified CYP4V2 mutations and show clinical phenotypes of BCD with autosomal recessive inheritance.Conclusions1. The onset age of BCD among Chinese is late, all of them show autosomal recessive inheritance, the sicken rate in the RP is as high as 1:10, the geographical and environmental factors may affect the occurrence and development process of BCD.2. The c.802₈10del17ins CG mutation was found to be the most common mutation in 115 BCD Chinese probands examined. Nine novel mutations were identified, contributing to the spectrum of CYP4V2 mutations associated with BCD.3. BCD patients with AF classification are more reasonable than traditional fundus stage and reflects the changes of outer shape changes and dysfunction in the post pole of retina and can be used as a BCD evaluation standard of clinical research.4. BCD genotype is not always associated with onset age, course of the disease or ERG changes, that is the patients with same BCD genotype may have very different clinical phenotype even in same family.5.The phenotype differences of complex big family due to gene mutation of more than two coexisting independent genes are associated with pathogenic genes and genetic model.