Rapid Detection of Known Mutations and Its Application to Carrie Testing

本研究开展了以下工作：1.全羧酶合成酶(HCS)基因的克隆HCS的缺陷会导致生物素应答的多个羧基酶。我们克隆了人HCScDNA，它与BIRA同源，并定位在染色体21q22.1.2上。各种遗传性疾病的突变研究：1)对6例日本患者进行突变分析，发现T997C、G1935A错义突变和一个碱基缺失(G1067)。这些突变加在一起约占日本人HCS等位基因的83%。2)GSD患者的突变：R83H、P257L、R170X、g727t和IVS 1NT-a共占日本人GSD突变等位基因的98%，其中g727t剪接突变是日本人最常见的突变。3)PKU患者的突变：R413P、IVS-4NT-1、R111X、Y204C、R243Q、R252W、R241C、R278I和IVS-9，占日本PKU突变等位基因的67%。4)PKU患者的突变：R243Q、R413P、IVS-4、R365X、R111X、R261Q、Y204C，占中国人PKU突变等位基因的61%。5)日本甲基丙二酸血症突变；G425T和769 DELTA突变；6)日本CPT II缺陷患者突变；7)日本家族性ALS突变；H46R.3。多重等位基因特异性聚合酶链式反应的建立我们已将ASPCR方法应用于PAH基因突变的检测。对在日本和中国发现的12个突变进行了单次ASPCR法检测。3个或4个突变的ASPCR反应已被多重进行。这两项多重检测将分别检测R111X、R261Q和Y204C突变以及R243Q、R413P、IVS 4NT-1和Y356X突变。

Present study carried out the following projects ;1. Cloning of holocarboxylase synthetase (HCS) geneDeficiency of HCS causes biotin-responsive multiple carboxylase. We have choned the human HCScDNA,which shows homology to BirA and maps to chromosome 21q22.1.2. Mutation studies from various genetic disorders :1) Mutation analysis in 6 Japanese patients revealed the misssense mutation of T997C,G1935A and one base deletion (G1067). These mutations together account for about 83% of Japanese HCS alleles. 2) Mutations of GSD patients ; R83H,P257L,R170X,g727t and IVS 1nt-a, together account for 98% of Japanese GSD mutant alleles.The g727t splicing mutation was the most common mutation among the Japanese patients. 3) Mutaions of PKU patients ; R413P,IVS-4nt-1, R111X,Y204C,R243Q,R252W,R241C,R278I and IVS-9, account for 67% of Japanese PKU mutant alleles. 4) Mutaions of PKU patients ; R243Q,R413P,IVS-4, R365X,R111X,R261Q,Y204C,account for 61% of Chinese PKU mutant alleles. 5) Mutations of Japanese methylmalonic acidemia ; G425T and 769DELTACA.6) Mutations of Japanese CPT II deficient Patients ; F352C,V368I and M647V.7) Mutation of Japanese familial ALS ; H46R.3. Development of multiplex allele specific PCR ; We have applied ASPCR methodology to the detection of mutations in PAH gene. Single ASPCR tests have been developed for 12 mutations found in Japan and China. ASPCR reactions for 3 or 4 mutaions have been multiplexed. The two multiplex tests will detect the presence of the R111X,R261Q and Y204C mutations and the R243Q,R413P,IVS 4nt-1 and Y356X mutations, respectively.