【作者】 张霞；

【导师】 余永国；

【作者基本信息】 上海交通大学 ， 儿科学， 2019， 硕士

【摘要】 第一部分 中国苯丙酮尿症患者的基因型和表型相关性目的:分析中国苯丙酮尿症(PKU)患者的表型特点和PAH基因突变谱,研究中国PKU患者的基因型与表型相关性。方法:总结2010.01-2018.12期间就诊于上海市新华医院的PKU患者的基因型和表型,分析中国地区PAH基因突变谱特征,并运用APV(Allelic phenotype value)预测方法对中国PKU患者进行表型预测。结果:共纳入PKU患者1383例,其中91.6%的患者携带PAH基因复合杂合变异,与BIOPKU收录患者相比存在显著性差异(P<0.0001);共发现PAH变异239种,57.3%位于催化区域,20.5%位于调节区域,5.9%位于四聚化区域,变异位置与PAHvdb收录变异无显著性差异(P=0.6686);最常见的变异类型为错义突变,占58.6%,其次为缺失突变14.2%,剪接突变13.4%,无义突变10.0%,与PAHvdb收录变异无显著性差异(P=0.2411);中国患者中最常见变异为c.728G>A,其频率为16.7%,其次为c.611A>G和c.721C>T,频率均为7.9%;对982例患者进行临床分型,其中31%表现为经典型PKU(c PKU),33.6%表现为轻度PKU(m PKU),35.4%表现为轻度高苯丙氨酸血症(MHP),表型构成与BIOPKU收录患者差异性显著(P<0.0001)。APV表型预测结果与90.04%的c PKU患者,43.43%的m PKU患者和35.57%的MHP患者表型相吻合。结论:中国地区PKU患者的基因型表型与BIOPKU收录患者存在显著性差异,APV法对中国c PKU患者表型预测率较高,但是对m PKU和MHP的表型预测欠佳。第二部分苯丙氨酸羟化酶基因新变异体外功能测定目的:检测20种变异型PAH的蛋白表达量及残余酶活性,研究PAH酶活性与PKU患者表型的相关性。方法:构建野生型及20种变异型(p.L52F,p.R86P,p.L128P,p.L142P,p.D163N,p.C203G,p.E214G,p.F260L,p.M276T,p.L311R,p.P314A,p.L364F,p.Q375H,p.F382I,p.A395S,p.V412D,p.E108*,p.C203*,p.C284*和p.E353*)PAH的真核表达载体,瞬时转染COS-7细胞,提取蛋白后分别用Western Blotting和LC-ESIMS/MS的方法检测PAH蛋白表达量和残余酶活性,根据残余酶活性预测PKU患者的临床表型,并比较APV和残余酶活性预测表型的一致性。结果:与野生型相比,8种变异型PAH(R86P,L142P,D163N,M276T,L364F,Q375H,F382I和A395S)的蛋白表达量没有明显下降,其残余酶活性轻度受损(48%-92%);9种变异型PAH(p.L128P,p.C203G,p.E214G,p.L311R,p.V412D,p.E108*,p.C203*,p.C284*和p.E353*)的蛋白表达量严重下降,残余酶活性也受到严重影响(0-34%);另外3种变异(L52F,F260L和P314A)的蛋白表达量没有明显降低,但是残余酶活性显著下降(分别是5%,32%和29%)。我们计算了14种变异的APV值,利用PAH酶活性预测表型结果与APV法一致性达12/14(86%)。根据残余酶活性可正确预测17/22(77%)的患者表型,其中在MHP患者中的预测率为14/18(78%)。结论:20种变异不同程度影响了PAH酶活性,PAH残余酶活性是预测PKU患者临床表型的重要依据,在MHP患者中运用酶活性方法较APV法预测率更高。第三部分初探女性PHF6基因新变异的致病性目的:探索女性PHF6基因新变异的致病性,分析Borjeson-Forssman-Lehmann综合征(BFLS)患者的基因型与表型特点,评估生长激素治疗的效果。方法:收集患者临床资料,提取患者及其父母外周血基因组DNA,对患者进行全外显子检测,Sanger测序对患者及其父母进行验证,甲基化PCR的方法检测患者及母亲X-染色体失活情况,总结分析已报道BFLS患者的基因型表型特点和治疗效果。结果:患者除表现为生长发育迟缓,面容异常,手指及脚趾异常和色素沉着等经典BFLS症状外,还表现为马蹄肾和生长激素缺乏,给予生长激素治疗后患者出现双下肢水肿症状。全外显子检测发现患者携带PHF6基因杂合变异c.673C>T(p.R225X),Sanger测序发现此变异遗传自母亲,患者与母亲的X-染色体失活比例分别为98:2和95:5。结论:X-染色体失活不能作为判断PHF6基因杂合突变是否导致女性患者致病的依据,BFLS患者用生长激素治疗效果不理想。更多还原

【Abstract】 Part 1 Genotype-Phenotype Correlation in Chinese Phenylketonuria PatientsObjectives:To analyze the phenotypic characteristics and PAH gene spectrum of Chinese phenylketonuria(PKU)patients,explore the genotype-phenotype correlation of PKU patients.Methods:The genotypes and phenotypes of PKU patients in Shanghai Xinhua Hospital from January 2010 to December 2018 were summarized,and phenotypic prediction was performed based on APV scores.Results:A total of 1383 patients with PKU were enrolled from,91.6%of them carried two compound heterozygous variations,and there was significant differences compared with patients included in the BIOPKU database(P<0.0001);239 PAH mutations were found,20.5%in the regulatory region,57.3%in the catalytic region and 5.9%in the tetramerization region,there was no significant difference compared with those in the PAHvdb(P=0.6686);the most common is missence mutations(58.6%),followed by the deletion mutations(14.2%),the splicing mutations(13.4%),the nonsense mutations(10.0%),and there was no significant difference compared with those in the PAHvdb(P=0.2411);the most common variations in Chinese patients was c.728G>A(p.R243Q)(16.7%),followed by c.611A>G(Ex6-96A>G)(7.9%)and c.721C>T(p.R241C)(7.9%);among the 982 patients,31%were classic PKU(c PKU),33.6%were mild PKU(m PKU)and 35.4%were mild hyperphenylalaninemia(MHP),the phenotypes were significantly different from those in BIOPKU(P<0.0001).The APV scores correctly predicted the phenotype in 90.04%of classic PKU,43.43%of mild PKU patients,and 35.57%of MHP patients.Conclusion:The phenotypes and genotypes of Chinese PKU patients are significant difference with those included in the public database.APV score is an effective predictor for classic PKU,but not for mild PKU and MHP.Part 2 In Vitro Residual Activity in 20 Variants of Phenylalanine HydroxylaseObjectives: Detect the in vitro protein expression and residual activity of 20 PAH variants,analysis the correlation between PAH activity and phenotype in PKU patients.Methods: 20 variants(p.L52 F,p.R86 P,p.L128 P,p.L142 P,p.D163 N,p.C203 G,p.E214 G,p.F260 L,p.M276 T,p.L311 R,p.P314 A,p.L364 F,p.Q375 H,p.F382 I,p.A395 S,p.V412 D,p.E108*,p.C203*,p.C284* and p.E353*)of PAH were expressed in COS-7 cells.The residual activities and protein expression levels were detected by isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry(LC-ESI-MS/MS)and Western blotting,respectively.We compared the results of the phenotypic prediction based on APV and PAH activity respectively,and further explored the relationship between residual activity and phenotype in PKU patients.Results: Compared to the wild-type PAH,8 variants showed mild impaired residual activities(48-92%)and relatively normal protein expression levels.In contrast,12 variants showed severely impaired residual activities(0-34%)and reduced protein expression except for p.L52 F,p.F260 L and p.P314 A.We assigned APV scores for 12 variants,in which the results of the phenotypic prediction were consistent for 12/14(86%)variants based on APV and residual activity respectively.The residual activity correctly predicted 17/22(77%)of the patients,and 14/18(78%)of the MHP patients.Conclusion: Residual enzyme activities of 20 variant PAHs were affected.PAH activity is an important factor for phenotypic prediction and is better than APV in MHP patients.Part 3 Pathogenesis of PHF6 gene new Mutation in Female PatientObjectives:To explore the mechanism of heterozygous mutation of PHF6 gene in female patients,analyze the genotypes and phenotypes of Borjeson-ForssmanLehmann Sydrome(BFLS),and evaluate the efficacy of growth hormone therapy in patients with BFLS.Methods: Peripheral blood genomic DNA was extracted and detected by whole exome sequencing,Sanger sequencing was performed to verify her parents,and the methylation-specific PCR was used to detect the X-inactivation of the patient and her mother.Results: The patient presented with developmental disorder,characteristic face,anomalies of fingers and toes,hyperpigmentation,horseshoe kidney and growth hormone deficiency.The whole exon sequencing revealed that the patient carried a heterozygous mutation of PHF6 gene c.673C>T(p.R225X).Sanger sequencing found that the mutation was inherited from her mother,highly skewed X-inactivation was found in the patient(98:2)and in her mother(95:5).Conclusion: Our findings further widen the spectrum of mutations involving the PHF6 gene,and it suggested the X-inactivation pattern is not a reliable predictor of the BFLS phenotype.It may not be appropriate to treat BFLS patients with growth hormone.更多还原