X-连锁无丙种球蛋白血症家系BTK基因突变分析及产前诊断研究

Mutation analysis and prenatal diagnosis in families of X-linked agammaglobulinemia caused by BTK gene mutation

目的对X-连锁无丙种球蛋白血症（XLA）家系进行致病基因突变分析及产前诊断。方法收集2011年1月至2012年6月在郑州大学第一附属医院就诊的3个XLA家系成员的外周血标本，应用PCR扩增和直接测序方法对3例xLA患者进行Bruton酪氨酸激酶（BTK）基因测序，分析BTK基因外显子区和剪切区DNA序列改变情况，采用PROVEN、PolyPhen-2和Clusta10软件对新发现的错义突变进行功能预测。在确定每个家系基因型后，对家系2和家系3中的2个高危胎儿抽取绒毛进行产前诊断。结果家系1中，先证者及其母亲携带BTK基因c．1117C〉A（p．L3731）错义突变，采用功能预测软件分析为致病突变的可能性大；家系2中，先证者及其母亲携带BTK基因c．126T〉G（P-Y42X）无义突变；家系3中，先证者及其母亲携带c．1679delC（p．P560fsX10）缺失突变。3个XLA家系发现的3种BTK基因突变为新突变，100名健康个体BTK基因相应区域测序，未发现有上述同样序列改变。对明确致病突变的家系2和家系3胎儿行孕早期产前诊断，家系2胎儿为p．Y42X女性杂合突变携带者，家系3胎儿为男性未携带突变基因者，2对夫妇均选择继续妊娠，胎儿娩出后随访结果与产前诊断结果一致。结论发现3个BTK基因新变异，BTK基因p．Y42X和p．P560fsX10突变是家系2和家系3患者的致病原因，BTK基因p．L373I突变可能是家系1患者致病的主要原因，但需要功能验证。对于有XLA生育史的夫妇再次生育时，应用基因测序技术行产前BTK基因突变分析可以有效地预防患儿出生。

Objective To evaluate the genetic diagnostic feasibility of Bruton＇s tyrosine kinase （BTK） gene in three families with X-linked agammagobulinemia （XLA） birth history, mutation analysis and prenatal genetic diagnosis of BTK gene for two families with XLA. Methods Polymerase chain reaction （PCR） was applied to amplify the regions of exon and exon-intron boundaries of BTK gene in 3 unrelated patients of XLA and their mothers from January 2011 to June 2012. The PCR products were further analyzed by direct sequencing. Prenatal genetic diagnosis was performed by chorionic villus sampling after genotyping of mothers of probands. Results Three novel mutations of BTK gene were identified in 3 pedigrees of XLA. A missense mutation c. 1117C 〉 A（ p. L373I）were detected in pedigree 1. The mutation was possible damage by predicting in sillico. A nonsense mutation c. 126T 〉 G（ p. Y42X）was found in pedigree 2. A single base deletion mutation c. 1679delC（p. P560fsX10）was found in pedigree 3. The three mutations, p. L373I, p. Y42X and p. P560fsX10 were novel. The three novel mutations were absent in the 100 normal controls. The male fetus in pedigree 3 was free of mutations identical to the proband and the female fetus in pedigree 2 was a carrier. The two families continued the pregnancies and the infants showed no symptom of XLA after one year old. Conclusions Three novel mutations were identified. The mutations of p. Y42X and p. P560fsX10 in BTK gene may be the major causes of pedigrees 2 and 3 with XLA. The mutation p. L373I of BTK gene is possibly the cause of pedigree 1 with XLA, but functional verification is needed. For pedigree of XLA, direct sequencing of BTK gene is available for providing genetic： counseling, prenatal diagnosis.