The American Journal of Human Genetics. June.6.2013

Mutations in POFUT1, Encoding Protein O-fucosyltransferase 1, Cause Generalized Dowling-Degos Disease. (IF: 10.603)

Ming Li,1 Ruhong Cheng,1 Jianying Liang,1 Heng Yan,2 Hui Zhang,1 Lijia Yang,3 Chengrang Li,4 Qingqing Jiao,1 Zhiyong Lu,1 Jianhui He,5 Jin Ji,3 Zhu Shen,2 Chunqi Li,5 Fei Hao,2 Hong Yu,1,*and Zhirong Yao1,*     （该文章的芯片和测序实验全部在dafabet黄金手机版公司完成）

文章链接 http://dx.doi.org/10.1016/j.ajhg.2013.04.022

背景

1. 课题组在国际上首次确定了POFUT1基因为泛发性屈侧网状色素异常的致病基因，并证实该基因在黑素合成和转运方面发挥重要作用。

2. 课题组首次揭示了POFUT1基因以及Notch信号通路在人类黑素合成与转运的作用，为疾病的分子诊断、预防与治疗，以及探索人类色素性疾病的发病机制奠定了基础。

研究方法

1. 采集DDD家系血样并抽提DNA；

2.  利用Linkage SNP芯片进行家系的连锁分析，找出疾病的候选区域；

3. 运用全外显子测序手段，筛选得到两个候选基因；

4. 通过Sanger测序对两个家系样本进行验证，最终锁定POFUT1为致病基因；

5. 构建敲除POFUT1基因的斑马鱼模型，揭示pofut1基因对黑素合成与转运的重要作用。

结果分析

1．为了确定泛发性褶皱部网状色素异常（DDD）致病基因的候选基因，课题组对家系1中的14名成员进行芯片实验，通过家系连锁分析找出候选基因区域。

Fig3A. Haplotype analysis of family 1. Haplotype indicates heterozygosity between rs1293713 and rs244123 in affected family members. The recombination events defined the susceptibility region in 20p11.21–20q13.12 to a 14.79 cM interval. Red bars indicate the chromosomal region shared by affectedmembers of the pedigree.

2. 将高通量测序筛选到的候选位点进行Sanger测序验证，最终找到在两个家系中共有的致病基因POFUT1。

Fig3(C and D). POFUT1 mutations in the two families: c.430G>T (p.Glu144*) in family 1 (C) and c.482delA (p.Lys161Serfs*42) in family 2 (D). The black arrows indicate mutations.

3. 利用反义吗啡啉基因沉默技术，成功构建了疾病的斑马鱼模型，充分验证了pofut1基因对黑素合成与转运的重要作用。

Fig4(C)

 Fig4(D)

Fig4 (C and D).  Zebrafish phenotype observation at 48 and 72 hpf. The melanin contents in the zebrafish tail at 48 hpf (C) and in both the tail and body axis at 72 hpf (D) decreased significantly in the pofut1-MO group compared with controls, and the melanin distribution in the zebrafish tail was presented as dispersion in the pofut1-MO group (D). The black arrow indicates abnormal melanin distribution in the zebrafish fin.

参考文献

[1] Yan XJ, Xu J, Gu ZH,et al.Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic leukemia. Nature Genetics, 2011, 43:309-15.

[2] Lossifov, I. et al. De novo gene disruptions in children on the autistic spectrum. Neuron, 2012, 74:285–299.