Demirci F Y, White N J, Rigatti B W, Lewis K F, Gorin M B
Departments of Ophthalmology and Human Genetics, University of Pittsburgh, Pittsburgh, PA 15213, USA.
Mol Vis. 2001 Oct 5;7:234-9.
Our goal is to identify the gene responsible for X-linked cone-rod dystrophy (COD1) that has been localized to a limited region of Xp11.4.
A complete physical contig of the COD1 region was partially sequenced and subjected to BLAST searches to identify homologies with GenBank ESTs. ESTs were analyzed for overlapping or related cDNA sequences and retinal expression by PCR screening of multiple human retina cDNA libraries. RACE was performed to complete the missing 5' end of the transcripts. Transcripts were compared with genomic sequences to specify intron-exon boundaries. Genomic DNAs from COD1-affected males from 3 families were screened for mutations using direct PCR sequencing of the exons.
The vacuolar proton-ATPase membrane sector-associated protein M8-9 (APT6M8-9) gene was identified within our critical region. We confirmed its retinal expression and its genomic location in our physical contig. Eight exons (with flanking intronic sequences) were characterized from partial cDNA sequence and genomic sequence data. An additional 5' end exon was identified using RACE. No mutations were found in the COD1-affected males.
The combination of disease mapping and information from the Human Genome project has enabled us to identify candidate genes within the COD1 region, including APT6M8-9 gene. We found no evidence that this gene is responsible for COD1 in our families, but it may be an important candidate for other diseases that have been mapped to this region of the X chromosome.
我们的目标是鉴定导致X连锁性锥体-杆体营养不良（COD1）的基因，该基因已被定位到Xp11.4的一个有限区域。
对COD1区域的完整物理重叠群进行部分测序，并进行BLAST搜索以鉴定与GenBank EST的同源性。通过对多个人类视网膜cDNA文库进行PCR筛选，分析EST的重叠或相关cDNA序列以及视网膜表达情况。进行RACE以完成转录本缺失的5'端。将转录本与基因组序列进行比较以确定内含子-外显子边界。使用外显子的直接PCR测序对来自3个家族的受COD1影响的男性的基因组DNA进行突变筛选。
在我们的关键区域内鉴定出液泡质子-ATP酶膜扇区相关蛋白M8-9（APT6M8-9）基因。我们证实了其在视网膜中的表达及其在我们物理重叠群中的基因组位置。从部分cDNA序列和基因组序列数据中鉴定出8个外显子（带有侧翼内含子序列）。使用RACE鉴定出另外一个5'端外显子。在受COD1影响的男性中未发现突变。
疾病定位与人类基因组计划信息的结合使我们能够在COD1区域内鉴定候选基因，包括APT6M8-9基因。我们没有发现该基因在我们家族中导致COD1的证据，但它可能是其他已定位到X染色体该区域疾病的重要候选基因。
