乾癬致病基因之鑑定與分析

Abstract

乾癬是一種慢性、非傳染性的皮膚病，主要特徵是角質細胞過度增生並且分化不完全，以及皮膚內有許多活化的免疫細胞堆積。乾癬人口在白種人中約佔2-3%，而在亞洲蒙古人種中約只佔0.3%。我們找到一個五代遺傳的乾癬家族並且著手研究，患者間的乾癬病灶輕重程度不同，但呈現典型自體顯性遺傳。為了找出此家族的乾癬易感致病基因位置，我們利用382個 microsatellite markers 進行全基因體掃描定位。第17號染色體q25區域有很顯著與乾癬連鎖的證據，最高的雙點LOD值是在D17S928標記處，LOD值是7.164。我們接著在附近另外挑選六個標記進一步做多點聯鎖分析，也證實了在D17S928處與乾癬有連鎖 (LOD值是4.58)。接著在17q25區域我們以202個SNP markers進一步定位分析，證實在兩處有很強的連鎖，分別在D17S784附近 (以高解析度雙點與多點分析分別LOD值為 4.45與 7.67) 以及 17號染色體末端附近。因此我們的研究將乾癬基因定位到一段新的染色體位置，約3.8 Mb長的區域，與之前報導17q25上可能的乾癬基因位置不同而比較偏17號染色體末端。 為了找出此乾癬家族的致病突變，我們在這個區域針對了78個基因進行定序分析，並且找到四個在家族中會與疾病一起傳遞的基因變異，分別是CD7中的p.Ala201Val、 ZNF750中的c.-625A>C、 C17orf56中的p.Asp189Asn 以及 AATK中的 p.Ala568Thr。後兩者基因變異在其他乾癬家族中並不會隨著疾病一起傳遞，並且在正常人對照組中也有出現，因此沒有繼續對此兩個基因繼續研究。對CD7功能進行研究的結果並不支持CD7就是致病基因。但相反的，ZNF750基因的c.-625A>C突變會造成promoter功能下降42%，EMSA實驗結果顯示突變C allele上有核蛋白結合。此c.-625A>C突變也在另一位偶發型 (sporadic) 乾癬患者身上找到，但188位正常對照組中卻沒有人帶有，此突變總共出現佔中國人種乾癬患者的1.7% (信賴區間為0.2-5.84%)。本研究推測ZNF750基因突變可能會造成乾癬誘發。

Psoriasis is a chronic, noncontagious skin disorder characterized by overgrowth and incomplete differentiation of keratinocytes, as well as infiltration of the skin by activated immune cells. Psoriasis affects 2-3% of the Caucasian population, but only 0.3% in the Mongoloid population. We have investigated a five-generation extended family with variable severity of psoriatic lesions followed a classical autosomal dominant inheritance pattern. To identify the psoriasis susceptibility locus in this family, a genome-wide scan with 382 microsatellite markers was performed. The 17q25 region showed a highly significant linkage, with a maximum two-point LOD score of 7.164 at D17S928 (theta=0.01). Multipoint linkage analysis with six additional flanking markers confirmed the linkage at D17S928 (LOD score of 4.58). Further mapping with 202 SNP markers in the 17q25 region confirmed the tight linkage to markers both around D17S784 (LOD score 4.45 and 7.67 by high-resolution two-point and multipoint analysis, respectively) and around 17q terminus (LOD score 4.472 and 3.404). Our study thus maps a new locus for psoriasis, a 3.8 Mb region, which is distal to and distinct from the recent report of a putative susceptibility gene located in 17q25. To identify the mutations responsible for the psoriasis in this family, we sequenced 78 genes within the region and found four genes variants, p.Ala201Val in CD7, c.-625A>C in ZNF750, p.Asp189Asn in C17orf56 and p.Ala568Thr in AATK co-segregated with the disease. The latter two variants were not studied further for absence of disease segregation in other familial psoriasis and presence of variants in normal subjects. Functional analyses of CD7 did not support CD7 as a disease-causing gene. In contrast, the c.-625A>C mutation in the ZNF750 resulted in a 42% reduction of the promoter activity, and the electrophoretic mobility shift assay demonstrated binding of nuclear protein(s) to the mutant C allele. The c.-625A>C mutation was found in another sporadic psoriasis patient but absent in 188 normal controls, together the mutation accounts for 1.7% (CI: 0.2-5.84%) of the Chinese psoriasis. This study suggests that ZNF750 mutations could contribute to psoriasis susceptibility.