Gargiulo Annagiusi, Auricchio Renata, Barone Maria Vittoria, Cotugno Gabriella, Reardon William, Milla Peter J, Ballabio Andrea, Ciccodicola Alfredo, Auricchio Alberto
Telethon Institute of Genetics and Medicine, Naples, Italy.
Am J Hum Genet. 2007 Apr;80(4):751-8. doi: 10.1086/513321. Epub 2007 Feb 26.
We have previously reported that an X-linked recessive form of chronic idiopathic intestinal pseudo-obstruction (CIIPX) maps to Xq28. To select candidate genes for the disease, we analyzed the expression in murine fetal brain and intestine of 56 genes from the critical region. We selected and sequenced seven genes and found that one affected male from a large CIIPX-affected kindred bears a 2-bp deletion in exon 2 of the FLNA gene that is present at the heterozygous state in the carrier females of the family. The frameshift mutation is located between two close methionines at the filamin N terminus and is predicted to produce a protein truncated shortly after the first predicted methionine. Loss-of-function FLNA mutations have been associated with X-linked dominant nodular ventricular heterotopia (PVNH), a central nervous system (CNS) migration defect that presents with seizures in females and lethality in males. Notably, the affected male bearing the FLNA deletion had signs of CNS involvement and potentially has PVNH. To understand how the severe frameshift mutation we found can explain the CIIPX phenotype and its X-linked recessive inheritance, we transiently expressed both the wild- type and mutant filamin in cell culture and found that filamin translation can start from either of the two initial methionines in these conditions. Therefore, translation of a normal shorter filamin can occur in vitro from the second methionine downstream of the 2-bp insertion we found. We confirmed this, demonstrating that the filamin protein is present in the patient's lymphoblastoid cell line that shows abnormal cytoskeletal actin organization compared with normal lymphoblasts. We conclude that the filamin N terminal region between the initial two methionines is crucial for proper enteric neuron development.
我们之前报道过，一种X连锁隐性形式的慢性特发性肠道假性梗阻（CIIPX）定位于Xq28。为了筛选该疾病的候选基因，我们分析了关键区域56个基因在小鼠胎脑和肠中的表达情况。我们挑选并测序了7个基因，发现来自一个受CIIPX影响的大家系中的一名患病男性，其FLNA基因外显子2中有一个2碱基对的缺失，该缺失在家族的携带者女性中呈杂合状态。该移码突变位于细丝蛋白N端的两个相邻甲硫氨酸之间，预计会产生一种在第一个预测的甲硫氨酸之后不久就被截断的蛋白质。功能丧失型FLNA突变与X连锁显性结节性室管膜下异位（PVNH）相关，PVNH是一种中枢神经系统（CNS）迁移缺陷，女性表现为癫痫发作，男性则具有致死性。值得注意的是，携带FLNA缺失的患病男性有中枢神经系统受累的迹象，可能患有PVNH。为了理解我们发现的严重移码突变如何解释CIIPX表型及其X连锁隐性遗传，我们在细胞培养中瞬时表达了野生型和突变型细丝蛋白，发现在这些条件下细丝蛋白的翻译可以从两个起始甲硫氨酸中的任何一个开始。因此，在体外可以从我们发现的2碱基对插入下游的第二个甲硫氨酸开始翻译出正常的较短细丝蛋白。我们证实了这一点，表明细丝蛋白存在于患者的淋巴母细胞系中，与正常淋巴细胞相比，该细胞系显示出异常的细胞骨架肌动蛋白组织。我们得出结论，最初两个甲硫氨酸之间的细丝蛋白N端区域对于正常的肠神经元发育至关重要。
