| V OBJECTIVE:This study analyzed the clinical data of 43 patients with Alport syndrome,and preliminarily discussed the clinical characteristics of AS.Targeted sequence capture combined with high-throughput sequencing technology was used for gene detection of patients,which enriched the gene mutation spectrum of AS.The individualized diagnosis of AS was improved by gene detection,combined with clinical feature and pathological examination.The gene mutation types of AS were defined to analyze the relationship between genotype and clinical phenotype.Functional analysis was performed on three COL4A5 gene mutations using Mini gene experiment in vitro,in order to promote the understanding of the pathogenesis of AS.METHODS:1.43 patients with clinically confirmed or suspected AS were selected,who visited the pediatric or nephrology department of the eastern theater general hospital from January 2015 to December 2018,all patients were confirmed the diagnosis of AS by genetic testing.The clinical data of all patients were analyzed,including gender,age of onset,first symptoms,causes of first symptoms,presence or absence of hematuria proteinuria,eye and ear abnormalities,family history,renal function,renal and skin histopathological examination results,to preliminarily explore the clinical characteristics of AS patients.Using SPSS software for statistical analysis,measurement data was represented by mean ± standard deviation(？),and analyzed by the t test;measurement data was represented by number and percentage of case;Kaplan-meier method was used to analyze the time of progression to ESRD in AS patients of different genders.α=0.05 was used as the test level,P<0.05 represented the difference had statistically meaning,and P<0.01 represented significant difference.2.After the approval of the ethics committee of the eastern theater general hospital,all patients and family members signed the informed consents and were extracted the peripheral blood samples.Using the targeted sequence capture and high-throughput sequencing technology to conduct genetic testing on all probands,suspectable mutations were verified in family members.Combined with the clinical phenotype and pathological examination results,the individualized diagnosis of AS was improved.According to the genotypes of patients,XLAS families were divided into two groups: nonsense mutations,frame-shift mutations,and splicing mutations as the first group,missense mutations as the second group,to compare the relationship between genotypes and clinical phenotypes of the two groups.3.Bioinformatics softwares were used to predict whether the mutations might affect the splicing process of pre-mRNA.In vitro minigene experiment was used to carry out functional studies on one missense mutation of COL4A5 gene(c.2767G>T,p.G923C)and two splicing mutations occurring at the same site(c.2917+1 G> c and c.2917+1 G>A),in order to analyze the effects of different mutations on the splicing process of pre-mRNA.RESULTS:1.There were 43 AS patients,the ratio of male to female was 1:1.39.The age of onset of the patients ranged from 3 months to 22 years old,and the age of onset between men and women was no statistical difference(P>0.05).The onset age of AS was early,39.5% of the patients presented symptoms before the age of 3 years,and most of the patients(81.4%)presented symptoms before the age of 10 years.The first symptoms of AS patients were varied,mainly including macroscopic hematuria,microscopic hematuria and proteinuria(76.7%),in addition to frequent urination urgency,foam urine,edema,rash,hearing loss and other manifestations.Most of the patients(60.5%)had no self-conscious symptoms at the time of onset,and they often found abnormal conditions due to respiratory tract infection or other diseases.The family investigation found that 27 family members progressed to ESRD,including 16 males and 11 female patients,the mean age of progression to ESRD in males was 29.2±1.6 years and in females was 57.2±2.4 years,the difference was statistically significant(P<0.01).Hematuria was the most common clinical manifestation of AS,but extrarenal manifestations were rare.The diagnosis rate of electron microscopy examination,renal tissue collagen type Ⅳ alpha chain dyeing and skin tissue collagen type Ⅳ alpha chain dyeing were 77.8%,72.2% and 62.5%,respectively.2.After genetic testing,the diagnosis of AS was confirmed in 43 probands,including 36 cases of XLAS(83.7%),5 cases of ARAS(11.6%),2 cases of ADAS(4.7%).A total of 48 COL4A3,COL4A4,COL4A5 and COL4A6 gene mutations were detected,after searched HMGD,LOVD and Clinver databases and literature,29 gene mutations were novel.Pedigree validation showed that there were 36 cases of XLAS probands,24 of whom were inherited from their mothers,4 from their fathers,6 were de novo mutations,and 2 from unknown sources,while 5 cases of ARAS proband were detected complex heterozygous mutations,namely one inherited from their father and another one inherited from their mother,and two ADAS proband,one was inherited from mother,and one was of unknown origin.The XLAS families were divided into two groups according to genotypes: nonsense mutations,frame-shift mutations,and splicing mutations as the first group,missense mutations as the second group.There was no significant difference in the age of onset between the two groups(P>0.05).The average proteinuria level in the first group was 1.16±0.89 g/24 h,and in the second group was 0.58±0.54 g/24 h,with statistically significant difference(P<0.05).The mean age of ESRD of male patients in the first group was 22.89±3.02 years,and in the second group was 28.20±5.59 years old,the difference was statistically significant(P<0.05).3.36 COL4A5 gene mutations were predicted by bioinformatics softwares,results showed that a total of 11 mutations may affect the splicing process of pre-mRNA,including 4 classical splicing-site mutations(c.2918-1G>C,c.2917+1G>C,c.2917+1G>A,c.834+2T>G),3 non-classical splicing-site mutations(c.780+5G>A,c.2395+3A>G,c.1032+5delG),3 missense mutations(c.4706G>T,c.2767G>T,c.2509G>T)and 1 nonsense mutation(c.4705C>T).In vitro minigene experiment confirmed that the mutation c.2767G>T was actually splicing mutation,resulting in the deletion of exon 32 of COL4A5 gene.The splicing mutations c.2917+1 G>A and c.2917+1 G>C produced three transcription products:(1)partial deletion of exon 33(96bp);(2)complete deletion of exon 33;(3)both exon 33 and 34 were completely deleted,while the splicing effects of the two mutations were different: the primary produce of c.2917+1 G>A mutation was partial deletion of exon 33;while the primary produce of c.2917+1 G>C mutation was complete deletion of exon 33.CONCLUSIONS:1.The onset age of AS was early,with concealed onset.The clinical symptoms were diverse,mainly including gross hematuria,microscopic hematuria and proteinuria.Some patients were missed or delayed diagnosed.Male patients progressed to ESRD earlier than female patients.The cilincal phenotypes of truncated mutations and splicing mutations were more serious than that of missense mutations.2.Targeted sequence capture combined with high-throughput sequencing technology was an efficient and rapid gene detection method,which enriched the gene mutation spectrum of AS,improved the individualized diagnosis of AS,and contributed to the prognosis estimation of AS patients.3.The minigene experiment was a simple and effective splicing mutation analysis method.The analysis of both DNA and RNA levels of mutations was helpful to understand the molecular pathogenesis of diseases.Experiment confirmed that the COL4A5 gene mutation c.2767G>T was actually splicing mutation,furthermore,the two mutations located at the same site c.2917+1 G>A and c.2917+1 G>C had different splicing effects. |
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