Holt–Oram syndrome (HOS) is a rare disorder characterized by cardiac and upper-limb defects. Pathogenic variants in TBX5—a gene encoding a transcription factor important for heart and skeletal development—are the only known cause of HOS. Here, we present the identification and functional analysis of two novel TBX5 pathogenic variants found in two individuals with HOS presenting distinct phenotypes. The individual with the c.905delA variant has a severe cardiac phenotype but mild skeletal defects, unlike the individual with the c.246_249delGATG variant who has no cardiac problems but severe upper limbs malformations, including phocomelia. Both frameshift variants, c.246_249delGATG and c.905delA, generate mRNAs harbouring premature stop codons which, if not degraded by nonsense mediated decay, will lead to the production of shorter TBX5 proteins, p.Gln302Argfs*92 and p.Met83Phefs*6, respectively. Immunocytochemistry results suggest that both mutated proteins are produced and furthermore, like the wild-type protein, p.Gln302Argfs*92 mutant appears to be mainly localized in the nucleus, in contrast with p.Met83Phefs*6 mutant that displays a higher level of cytoplasmic localization. In addition, luciferase activity analysis revealed that none of the TBX5 mutants are capable of transactivating the NPPA promoter. In conclusion, our results provide evidence that both pathogenic variants cause a severe TBX5 loss-of-function, dramatically reducing its biological activity. The absence of cardiac problems in the individual with the p.Met83Phefs*6 variant supports the existence of other mechanisms/genes underlying the pathogenesis of HOS and/or the existence of an age-related delay in the development of a more serious cardiac phenotype. Further studies are required to understand the differential effects observed in the phenotypes of both individuals.

Holt-Oram综合征（HOS）是一种罕见的疾病，其特征是心脏和上肢缺损。TBX5中的致病性变异体是一种编码HOS的唯一病因，TBX5是一种编码对心脏和骨骼发育至关重要的转录因子的基因。在这里，我们介绍两种新型TBX5的鉴定和功能分析在两个具有不同表型的HOS个体中发现的致病变体。具有c.905delA变体的个体具有严重的心脏表型，但轻度的骨骼缺陷，与具有c.246_249delGATG变体的个体没有心脏问题，但上肢严重畸形，包括phocomelia。两种移码变体c.246_249delGATG和c.905delA均产生带有过早终止密码子的mRNA，如果不被无意义的介导的衰变降解，将导致产生较短的TBX5蛋白，p.Gln302Argfs * 92和p.Met83Phefs * 6，分别。免疫细胞化学结果表明，两种突变蛋白均产生，此外，与野生型蛋白一样，p.Gln302Argfs * 92突变体似乎主要位于细胞核内，与p。Met83Phefs * 6突变体，具有较高的细胞质定位水平。此外，萤光素酶活性分析表明，没有TBX5突变体能够反式激活NPPA启动子。总之，我们的结果提供了证据，表明两种致病变体均导致严重的TBX5功能丧失，从而大大降低了其生物学活性。患有p.Met83Phefs * 6变体的个体中没有心脏问题，这支持存在其他机制/基因，这些机制/基因是HOS发病机理的基础，和/或存在与年龄相关的更严重的心脏表型发育中的延迟。需要进一步的研究以了解在两个人的表型中观察到的差异作用。