Leber先天性黑矇患者不同基因新突变与临床表型分析

Phenotypic analysis of patients with Leber congenital amaurosis caused by new gene mutations

目的明确3个Leber先天性黑矇(LCA)家系的致病基因突变,初步分析其基因型和临床表型的关系。方法回顾性研究。2021年1~12月于宁夏回族自治区人民医院眼科检查确诊的4例LCA患者和7名家系成员纳入研究。4例患者来自3个无血缘关系家系。详细收集患者及家系成员临床资料。采集患者及家系成员外周静脉血,提取全基因组DNA。应用全基因组外显子测序技术进行致病基因突变检测,对可疑致病突变位点通过Sanger测序进行验证,通过蛋白质预测软件和保守性分析确定致病基因的突变位点,结合家系图与先证者进行共分离分析。结果4例患者中,男性1例,女性3例;年龄4~18岁。眼球震颤3例;指压眼征伴夜视力差1例;全视野视网膜电图重度下降4例。所有患眼黄斑中心凹反光可见,周边视网膜未见明显异常。黄斑区椭圆体带隆起强反射信号1只眼;视网膜层间隐约可见弱反射信号2只眼;血管分支增多、周边视网膜无灌注区伴毛细血管渗漏1只眼;黄斑区环状强自身荧光4只眼。家系成员表型未见明显异常。基因检测结果显示,家系1先证者(Ⅱ-1)携带PRPH2基因c.640T>A(p.C214S)(M1)纯合突变;家系2先证者(Ⅱ-2)携带TULP1基因c.1256G>A(p.R419Q)(M2)、c.1A>C(p.M1L)(M3)复合杂合突变;家系3先证者(Ⅱ-1)及其妹妹(Ⅱ-2)携带GUCY2D基因c.1943T>C(p.L648P)(M4)、c.380C>T(p.P127L)(M5)复合杂合突变。家系2先证者父母、姐姐(Ⅱ-1)及家系3先证者父母均为相应杂合变异携带者。其中,M1、M3、M4、M5为新发现突变。家系内基因型和疾病表型呈共分离。根据系谱及基因检测结果分析,3个家系均为常染色体隐性遗传。氨基酸保守性分析发现,M1、M2、M3、M4、M5在物种间具有高度保守性。生物信息学分析结果均为致病性变异。结论发现并证实PRPH2基因M1、TULP1基因M3和GUCY2D基因M4、M5为LCA的新发现突变位点,扩大了LCA的突变位点和基因突变频谱;LCA具有发病年龄小、眼底表现各异及视力损害严重等特点。

Objective To identify 3 the disease-causing genes and mutations of Leber congenital amaurosis(LCA),and to study the correlation of phenotype and genotype.Methods A retrospective study.Four LCA patients and seven family members who were diagnosed by eye examination in Ning Xia Eye Hospital of People's Hospital of Ningxia Hui Autonomous Region from January to December 2021 were included in the study.Four patients were from 3 unrelated families.Detailed collection of medical history and family history were received.Related ophthalmologic examination were collected and genomic DNA was extracted from peripheral blood.Whole-exome sequencing method was used for genetic diagnosis.The identified variant was confirmed with Sanger sequencing.Potential pathogenic mutation was analyzed using software and conserved domain analysis and performed co-separated analysis between the family member and the proband.Results Of the 4 patients,1 patient was males and 3 patients were females;the age was from 4 to 18 years.Nystagmus were seen in 3 cases,finger pressing eyes and night blindness was seen in 1 cases;electroretinogram showed 4 cases of extinction or near extinction.The foveal reflection was visible in all eyes,and there was no obvious abnormality in the peripheral retina.One eye had strong reflection signal with raised ellipsoid in macular area;two eyes had weak reflection signal faintly visible between retinal layers;1 eye had increased blood vessel branches,peripheral retinal non-perfusion area with capillary leakage;annular strong autofluorescence in macular area 4 eyes.No obvious abnormality was found in the phenotypes of family members.Genetic testing showed that the proband of pedigree 1(Ⅱ-1)was found a homozygous missense mutation in c.640A>T(p.C214S)(M1)of PRPH2 gene.The proband of pedigree 2(Ⅱ-2)was found compound heterozygous mutation in c.1256G>A(p.R419Q)(M2)and c.1A>C(p.M1L)(M3)of TULP1 gene.The proband 3(Ⅱ-1)and her sister(Ⅱ-2)were both found compound heterozygous mutation in c.1943T>C(p.L648P)(M4)and c.380C>T(p.P127L)(M5)of GUCY2D gene.The parents and sister(Ⅱ-1)of the proband in family 2 and the parents of the proband in family 3 were all carriers of the corresponding heterozygous variant.M1,M3,M4,M5 were novel mutations and unreported.The genotype and disease phenotype were co-segregated within the family.According to the analysis of pedigree and genetic testing results,all 3 families were autosomal recessive inheritance.The amino acid conservation analysis found that M1,M2,M3,M4,and M5 were highly conserved among species.The results of bioinformatics analysis were all pathogenic variants.Conclusions PRPH2 gene M1,TULP1 gene M3,and GUCY2D gene M4,M5 were novel mutations and not been reported in the literature and database.This research expanded the gene mutation spectrum of LCA.The patients with LCA have available characterristics,including onset age,varying ocular fundus and severe visual impairment.