Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy.
Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy.
复制标题
DOI：
10.1136/jmg.2010.082552
复制
发表时间：
2011-02
影响因子：
4
通讯作者：
GPN Study Group
中科院分区：
文献类型：
--
作者：
Otto EA;Ramaswami G;Janssen S;Chaki M;Allen SJ;Zhou W;Airik R;Hurd TW;Ghosh AK;Wolf MT;Hoppe B;Neuhaus TJ;Bockenhauer D;Milford DV;Soliman NA;Antignac C;Saunier S;Johnson CA;Hildebrandt F;GPN Study Group
肾单位结核相关性纤毛病变（NPHP-AC）包括一组常染色体隐性囊性肾病，包括肾单位结核（NPHP）、Senior-Loken综合征（SLS）、Joubert综合征（JBTS）和Meckel-Gruber综合征（MKS）。迄今为止，已经描述了18个不同基因的NPHP-AC中的致病突变，使得突变分析繁琐且昂贵。为了克服广泛的遗传位点异质性，我们设计了一种策略，DNA池连续大规模平行重测序（MPR）。在120例严重NPHP-AC表型患者中，我们制备了5个基因组DNA池，每个池24例患者，用作模板，以PCR扩增18个NPHP-AC基因的所有376个外显子（NPHP 1、INVS、NPHP 3、NPHP 4、IQCB 1、CEP 290、GLIS 2、RPGRIP1L、NEK 8、TMEM 67、INPP 5E、TMEM 216、AHI 1、ARL 13 B、CC2D2A、TTC 21 B、MKS 1和XPNPEP 3）。然后在Illumina基因组分析仪上对PCR产物进行MPR，随后通过基于CEL I内切核酸酶的异源双链体筛选将突变分配给它们各自的突变载体，并通过桑格测序确认。为了证明原理，我们使用了来自具有已知突变的患者的DNA，并证明了24种不同等位基因中的22种的检测（92%的灵敏度）。MPR导致30/120例患者（25%）的分子诊断，我们在7个不同的NPHP-AC基因中鉴定出54个致病性突变（27个新突变）。此外，在24例患者中，我们只发现了意义不明的单一杂合变异。结合DNA池，然后MPR的方法大大有利于突变分析广泛异质性单基因疾病。在我们的队列中，75%的患者缺乏突变，表明NPHP-AC存在更广泛的异质性。
Nephronophthisis-associated ciliopathies (NPHP-AC) comprise a group of autosomal recessive cystic kidney diseases that includes nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS), and Meckel-Gruber syndrome (MKS). To date, causative mutations in NPHP-AC have been described for 18 different genes, rendering mutation analysis tedious and expensive. To overcome the broad genetic locus heterogeneity we devised a strategy of DNA pooling with consecutive massively parallel resequencing (MPR). In 120 patients with severe NPHP-AC phenotypes we prepared 5 pools of genomic DNA with 24 patients each which were used as templates in order to PCR-amplify all 376 exons of 18 NPHP-AC genes (NPHP1, INVS, NPHP3, NPHP4, IQCB1, CEP290, GLIS2, RPGRIP1L, NEK8, TMEM67, INPP5E, TMEM216, AHI1, ARL13B, CC2D2A, TTC21B, MKS1, and XPNPEP3). PCR products were then subjected to MPR on a Illumina Genome-Analyzer and mutations were subsequently assigned to their respective mutation carrier via CEL I endonuclease-based heteroduplex screening and confirmed by Sanger sequencing. For proof of principle we used DNA from patients with known mutations and demonstrated the detection of 22 out of 24 different alleles (92% sensitivity). MPR led to the molecular diagnosis in 30/120 patients (25%) and we identified 54 pathogenic mutations (27 novel) in 7 different NPHP-AC genes. Additionally, in 24 patients we only found single heterozygous variants of unknown significance. The combined approach of DNA pooling followed by MPR strongly facilitates mutation analysis in broadly heterogeneous single-gene disorders. The lack of mutations in 75% of patients in our cohort indicates further extensive heterogeneity in NPHP-AC.
登录
查看更多内容
影响因子：
9.8
作者：
Gorden, Nicholas T.;Arts, Heleen H.;Doherty, Dan
通讯作者：
Doherty, Dan
影响因子：
30.8
作者：
Kyttälä, M;Tallila, J;Kestïla, M
通讯作者：
Kestïla, M
影响因子：
30.8
作者：
Otto, EA;Schermer, B;Hildebrandt, F
通讯作者：
Hildebrandt, F
影响因子：
3.9
作者：
Brancati, Francesco;Iannicelli, Miriam;Travaglini, Lorena;Mazzotta, Annalisa;Bertini, Enrico;Boltshauser, Eugen;D'Arrigo, Stefano;Emma, Francesco;Fazzi, Elisa;Gallizzi, Romina;Gentile, Mattia;Loncarevic, Damir;Mejaski-Bosnjak, Vlatka;Pantaleoni, Chiara;Rigoli, Luciana;Salpietro, Carmelo D.;Signorini, Sabrina;Stringini, Gilda Rita;Verloes, Alain;Zabloka, Dominika;Dallapiccola, Bruno;Gleeson, Joseph G.;Valente, Enza Maria
通讯作者：
Valente, Enza Maria
影响因子：
9.8
作者：
Baala, Lekbir;Romano, Stephane;Attie-Bitach, Tania
通讯作者：
Attie-Bitach, Tania