Human Molecular Genomics Group, Max Planck Institute for Molecular Genetics, Berlin, Germany.
Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.
J Med Genet. 2019 Apr;56(4):246-251. doi: 10.1136/jmedgenet-2018-105793. Epub 2019 Feb 2.
Structural variants (SVs) affecting non-coding -regulatory elements are a common cause of congenital limb malformation. Yet, the functional interpretation of these non-coding variants remains challenging. The human Liebenberg syndrome is characterised by a partial transformation of the arms into legs and has been shown to be caused by SVs at the locus leading to its misregulation in the forelimb by its native enhancer element Pen. This study aims to elucidate the genetic cause of an unsolved family with a mild form of Liebenberg syndrome and investigate the role of promoters in long-range gene regulation.
Here, we identify SVs by whole genome sequencing (WGS) and use CRISPR-Cas9 genome editing in transgenic mice to assign pathogenicity to the SVs.
In this study, we used WGS in a family with three mildly affected individuals with Liebenberg syndrome and identified the smallest deletion described so far including the first non-coding exon of . To functionally characterise the variant, we re-engineered the 8.5 kb deletion using CRISPR-Cas9 technology in the mouse and showed that the promoter of the housekeeping gene insulates the Pen enhancer from in forelimbs; its loss leads to misexpression of by the pan-limb activity of the Pen enhancer causing Liebenberg syndrome.
Our data indicate that housekeeping promoters may titrate promiscuous enhancer activity to ensure normal morphogenesis. The deletion of the promoter as a cause of Liebenberg syndrome highlights this new mutational mechanism and its role in congenital disease.
影响非编码调控元件的结构变异（SVs）是先天性肢体畸形的常见原因。然而，这些非编码变异的功能解释仍然具有挑战性。人类 Liebenberg 综合征的特征是手臂部分转化为腿部，并已被证明是由 基因座上的 SV 引起的，导致其通过天然增强子元件 Pen 在前肢中失调。本研究旨在阐明一个未解决的具有轻度 Liebenberg 综合征家族的遗传原因，并研究启动子在长距离基因调控中的作用。
我们通过全基因组测序（WGS）鉴定 SVs，并使用 CRISPR-Cas9 基因组编辑在转基因小鼠中鉴定 SVs 的致病性。
在这项研究中，我们使用 WGS 对一个具有三个轻度 Liebenberg 综合征的家族进行了研究，并鉴定了迄今为止描述的最小缺失，包括 的第一个非编码外显子。为了对变体进行功能表征，我们使用 CRISPR-Cas9 技术在小鼠中重新设计了 8.5kb 的缺失，并表明管家基因 的启动子将 Pen 增强子与 隔离在前肢中；其缺失导致 Pen 增强子的泛肢活性错误表达 ，从而导致 Liebenberg 综合征。
我们的数据表明，管家基因启动子可能会抑制混杂增强子活性以确保正常形态发生。作为 Liebenberg 综合征原因的 启动子缺失突出了这种新的突变机制及其在先天性疾病中的作用。
