The Complete Genomic Sequence of the Machado-Joseph Disease Gene

Machado-Joseph病（MJD）是一种进行性神经退行性疾病，临床表现为小脑共济失调和各种相关症状。该疾病是遗传性脊髓小脑共济失调中最常见的类型，以常染色体显性遗传方式遗传。致病的遗传异常是定位于染色体14q32.1的MJD基因中CAG重复序列的不稳定扩增。该基因的基因组结构、表达和生理功能尚不清楚。在本项目中，我们确定了MJD的全基因组序列，以阐明其基因组结构。通过筛选4个人类cDNA文库获得27个cDNA克隆，通过筛选14号染色体特异性文库获得13个cosmid克隆，通过筛选RPCI11人类BAC文库获得8个BAC克隆。用FISH法构建了一个包含MJD的约300kb的contig。测定了BAC克隆B445M7的完整序列（175,330bp）。基因组、cDNA和EST序列比较表明，MJD基因全长48240bp，由11个外显子组成，通过选择性剪接和聚adenylation转录出至少5个信使（约1.4、1.9、2.0、4.8和7.0kb）。Northern blot分析证实了这些结果，显示了该基因的普遍表达。我们在编码区发现了3个单核苷酸多态性位点；GT^<527>T/GTC ^<669>ATG/GTG和TA^<1118>A/TAC。单倍型分析表明，日本人群中只有2个单倍型，CAG扩增与527T-669A-987C-1118A单倍型之间存在较强的连锁不平衡。

Machado-Joseph disease (MJD) is a progressive neurodegenerative disease that is characterized clinically by cerebellar ataxia and various associated symptoms. The disorder is the most common type among the hereditary spinocerebellar ataxias and is inherited with autosomal dominant manner. The causative genetic abnormality is an unstable expansion of the CAG repeat in the MJD gene that maps to chromosome 14q32.1. The genomic structure, expression and physiological function of the gene have been obscure. In this project we determined the complete genomic sequence of MJD to elucidate its genomic structure. We obtained totally 27 cDNA clones by screening four human cDNA libraries, 13 cosmid clones by screening the chromosome 14 specific library and 8 BAC clones by screening RPCI11 human BAC library. A contig of the size of approximately 300kb, including MJD, was constructed by the fiber FISH method. The complete sequence of a BAC clone, B445M7, was determined (175,330bp). The comparison of the genomic, cDNA and EST sequences indicated that the MJD gene spanned 48,240bp, was composed of 11 exons and transcribed at least 5 messengers (approximately 1.4, 1.9, 2.0, 4.8 and 7.0kb) by alternative splicing and polyadenylation. Northern blot analysis confirmed these results and showed the ubiquitous expression of the gene. We found three single nucleotide polymophism (SNP) sites in the coding region ; GT^<527>T/GTC, ^<669>ATG/GTG and TA^<1118>A/TAC.Haplotyping analysis showed that there were only two haplotypes in the Japanese population and the strong linkage disequilibrium was found between the CAG expansion and 527T-669A-987C-1118A haplotype.