PDF(Pubmed)
Abstract：
Human vertebral malformations (VMs) have an estimated incidence of 1/2000 and are associated with significant health problems including congenital scoliosis (CS) and recurrent organ system malformation syndromes such as VACTERL (vertebral anomalies; anal abnormalities; cardiac abnormalities; tracheo-esophageal fistula; renal anomalies; limb anomalies). The genetic cause for the vast majority of VMs are unknown. In a CS/VM patient cohort, three COL11A2 variants (R130W, R1407L and R1413H) were identified in two patients with cervical VM. A third patient with a T9 hemivertebra and the R130W variant was identified from a separate study. These substitutions are predicted to be damaging to protein function, and R130 and R1407 residues are conserved in zebrafish Col11a2. To determine the role for COL11A2 in vertebral development, CRISPR/Cas9 was used to create a nonsense mutation (col11a2L642*) as well as a full gene locus deletion (col11a2del) in zebrafish. Both col11a2L642*/L642* and col11a2del/del mutant zebrafish exhibit vertebral fusions in the caudal spine, which form due to mineralization across intervertebral segments. To determine the functional consequence of VM-associated variants, we assayed their ability to suppress col11a2del VM phenotypes following transgenic expression within the developing spine. While wildtype col11a2 expression suppresses fusions in col11a2del/+ and col11a2del/del backgrounds, patient missense variant-bearing col11a2 failed to rescue the loss-of-function phenotype in these animals. These results highlight an essential role for COL11A2 in vertebral development and support a pathogenic role for two missense variants in CS.
摘要：
人类脊椎畸形（VM）的发病率估计为1/2000，并与严重的健康问题有关，包括先天性脊柱侧凸（CS）和复发性器官系统畸形综合征，例如VACTERL。绝大多数VM的遗传原因尚不清楚。在CS/VM患者队列中，三种COL11A2变体(R130W,R1407L,在两名宫颈VM患者中鉴定出R1413H）。从另一项研究中鉴定出具有T9半椎骨和R130W变体的第三名患者。预计这些取代会损害蛋白质功能，并且R130和R1407残基在斑马鱼Col11a2中是保守的。为了确定COL11A2在椎体发育中的作用，CRISPR/Cas9用于在斑马鱼中产生无义突变(col11a2L642*)以及全基因基因座缺失(col11a2del)。col11a2L642*/L642*和col11a2del/del突变斑马鱼都在尾脊表现出椎骨融合，由于椎间段的矿化而形成。要确定VM相关变体的功能后果，我们测定了它们在发育中脊柱内转基因表达后抑制col11a2delVM表型的能力。虽然野生型col11a2表达抑制col11a2del/+和col11a2del/del背景中的融合，携带错义变异的col11a2未能挽救这些动物的LOF表型.这些结果突出了COL11A2在椎骨发育中的重要作用，并支持了CS中两个错义变体的致病作用。
