Targeted sequencing and in vitro splice assays shed light on ABCA4-associated retinopathies missing heritability.
Targeted sequencing and in vitro splice assays shed light on ABCA4-associated retinopathies missing heritability.
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DOI：
10.1016/j.xhgg.2023.100237
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发表时间：
2023-10-12
期刊：
影响因子：
--
通讯作者：
Cremers, Frans P. M.
中科院分区：
文献类型：
--
作者：
Corradi, Zelia;Khan, Mubeen;Hitti-Malin, Rebekkah;Mishra, Ketan;Whelan, Laura;Cornelis, Stephanie S.;Hoyng, Carel B.;Kampjarvi, Kati;Klaver, Caroline C. W.;Liskova, Petra;Stoehr, Heidi;Weber, Bernhard H. F.;Banfi, Sandro;Farrar, G. Jane;Sharon, Dror;Zernant, Jana;Allikmets, Rando;Dhaenens, Claire-Marie;Cremers, Frans P. M.
ABCA 4基因是最常突变的孟德尔视网膜病相关基因。双等位基因变体导致多种表型，然而，对于数千例病例，潜在的变体仍然未知。在这里，我们的目的是通过分析一个大型的黄斑营养不良先证者队列，进一步阐明ABCA 4相关视网膜病变的缺失遗传性。从26个中心收集了858名先证者，其中722人不携带或携带一种致病性ABCA 4变异体，而136例携带两种ABCA 4等位基因，其中一种是常见的轻度变异体，这表明可能遗漏了深度内含子变异体（DIV）或其他顺式修饰物。在对完整的128-kb ABCA 4基因座进行基于单分子分子倒置探针（smMIP）的测序后，通过HEK 293 T细胞中的midigene剪接测定在体外评估推定剪接变体的影响。通过连接PCR和桑格测序确定拷贝数变异（CNVs）的断点。ABCA 4序列分析解决了520例初治或未解决病例中的207例（39.8%）和202例单等位基因病例中的70例（34.7%），而在136例携带两种变体的先证者中的54例（39.7%）中鉴定出了其他致病变体。在总共35个等位基因中检测到7个新的DIV和6个新的非典型剪接位点变体，并进行了表征，包括导致复杂剪接缺陷的c.6283- 321 C>G变体。此外，在五个等位基因中鉴定和表征了四个新的CNV。这些结果证实，完整ABCA 4基因的基于smMIP的测序提供了一种具有成本效益的方法来遗传解决视网膜病变病例，并且在Stargardt病病例中仍未发现几种罕见的结构和剪接改变缺陷。在ABCA 4相关视网膜病变先证者的异质性队列中进行的ABCA 4序列分析解决了722例病例中的277例（38.4%），而在已知已经携带两种变体的136例个体中的54例（39.7%）中鉴定出了其他变体。此外，完整的ABCA 4基因座测序导致13个新的剪接变体和4个新的拷贝数变体的鉴定。
The ABCA4 gene is the most frequently mutated Mendelian retinopathy-associated gene. Biallelic variants lead to a variety of phenotypes, however, for thousands of cases the underlying variants remain unknown. Here, we aim to shed further light on the missing heritability of ABCA4-associated retinopathy by analyzing a large cohort of macular dystrophy probands. A total of 858 probands were collected from 26 centers, of whom 722 carried no or one pathogenic ABCA4 variant, while 136 cases carried two ABCA4 alleles, one of which was a frequent mild variant, suggesting that deep-intronic variants (DIVs) or other cis-modifiers might have been missed. After single molecule molecular inversion probes (smMIPs)-based sequencing of the complete 128-kb ABCA4 locus, the effect of putative splice variants was assessed in vitro by midigene splice assays in HEK293T cells. The breakpoints of copy number variants (CNVs) were determined by junction PCR and Sanger sequencing. ABCA4 sequence analysis solved 207 of 520 (39.8%) naive or unsolved cases and 70 of 202 (34.7%) monoallelic cases, while additional causal variants were identified in 54 of 136 (39.7%) probands carrying two variants. Seven novel DIVs and six novel non-canonical splice site variants were detected in a total of 35 alleles and characterized, including the c.6283-321C>G variant leading to a complex splicing defect. Additionally, four novel CNVs were identified and characterized in five alleles. These results confirm that smMIPs-based sequencing of the complete ABCA4 gene provides a cost-effective method to genetically solve retinopathy cases and that several rare structural and splice altering defects remain undiscovered in Stargardt disease cases. ABCA4 sequence analysis in a heterogeneous cohort of ABCA4-associated retinopathy probands solved 277 of 722 (38.4%) cases, while additional variants were identified in 54 of 136 (39.7%) individuals known to already carry two variants. Additionally, complete ABCA4 locus sequencing lead to the identification of 13 novel splicing variants and four novel copy number variants.
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影响因子：
3.5
作者：
Braun TA;Mullins RF;Wagner AH;Andorf JL;Johnston RM;Bakall BB;Deluca AP;Fishman GA;Lam BL;Weleber RG;Cideciyan AV;Jacobson SG;Sheffield VC;Tucker BA;Stone EM
通讯作者：
Stone EM
影响因子：
14.8
作者：
Kumar, Prateek;Henikoff, Steven;Ng, Pauline C.
通讯作者：
Ng, Pauline C.
影响因子：
14.9
作者：
Leman R;Gaildrat P;Le Gac G;Ka C;Fichou Y;Audrezet MP;Caux-Moncoutier V;Caputo SM;Boutry-Kryza N;Léone M;Mazoyer S;Bonnet-Dorion F;Sevenet N;Guillaud-Bataille M;Rouleau E;Bressac-de Paillerets B;Wappenschmidt B;Rossing M;Muller D;Bourdon V;Revillon F;Parsons MT;Rousselin A;Davy G;Castelain G;Castéra L;Sokolowska J;Coulet F;Delnatte C;Férec C;Spurdle AB;Martins A;Krieger S;Houdayer C
通讯作者：
Houdayer C
影响因子：
3.9
作者：
Khan, Mubeen;Cornelis, Stephanie S.;Cremers, Frans P. M.
通讯作者：
Cremers, Frans P. M.
感光体ABC转运蛋白ABCA4在脂质转运和Stargardt黄斑变性中的作用。
DOI：
10.1016/j.bbalip.2009.02.004
发表时间：
2009-07
影响因子：
4.8
作者：
Molday, Robert S.;Zhong, Ming;Quazi, Faraz
通讯作者：
Quazi, Faraz