Distinct variants affecting differential splicing of TGFBR1 exon 5 cause either Loeys–Dietz syndrome or multiple self-healing squamous epithelioma

Abstract

Variants in TGFBR1 have been reported to induce two completely distinct diseases, namely Loeys-Dietz syndrome (LDS) and multiple self-healing squamous epithelioma (MSSE). However, detailed mechanisms underlying this effect remain unknown. We report a Japanese familial case of LDS with a novel splice donor site variant in TGFBR1 gene (c.973 + 1 G > A; NG_007461.1). The intronic variant was predicted to mediate in-frame exon 5 skipping within the serine/threonine kinase (STK) domain, which may also be mediated by a similar TGFBR1 variant of a splice acceptor site in intron 4 (c.806-2 A > C), identified in a British familial case of MSSE. Therefore, ex vivo splicing and functional assays were performed in mammalian cells to evaluate the effect of these sequence variants. The MSSE variant activated a cryptic acceptor site at 76 bp downstream of the 3' natural splice acceptor site, which produced an out-of-frame transcript (r.807_882del, p.Asn270Thrfs*8). In contrast, the LDS variant generated two types of in-frame transcription products, r.[806_973del, 965_973 del], and produced two functionally inactivated proteins, p.[Asp269_Gln324del, Thr323_Gly325del], as a result of exon 5 skipping and the activation of a cryptic donor splice site at 9 bp upstream of the 5' natural splice donor site, respectively. Our results support the previously proposed but not yet approved mechanism that dominant-negative and truncating variants in STK domain induce LDS and MSSE, respectively.

TGF-β Signaling-Related Genes and Thoracic Aortic Aneurysms and Dissections

“…The IVS4-1A > C variant generated an out-of-frame transcript due to cryptic acceptor splice-site activation. Our results strongly supported that missense and truncating variants in the STK domain induce LDS and MSSE, presumably through the DN and HI effect, respectively [ 91 ].…”

“…Goudie et al reported familial cases with primary multiple self-healing squamous epithelioma (MSSE) of the skin, caused by truncating mutations in the STK domain of the TGFBR1 gene without LDS-like phenotypes [ 89 , 90 ]. DN-type and HI-type mutations in the STK domain induce LDS and MSSE, respectively [ 90 ], and we have recently observed pathology in LDS patients that may support this proposed but not yet validated mechanism [ 91 ]. Fujiwara et al focused on a novel splice donor site variant in the TGFBR1 gene (IVS5 + 1G > A) causing a familial case of LDS without MSSE phenotypes, which was predicted to mediate in-frame exon 5 skipping within the STK domain.…”

“…Fujiwara et al focused on a novel splice donor site variant in the TGFBR1 gene (IVS5 + 1G > A) causing a familial case of LDS without MSSE phenotypes, which was predicted to mediate in-frame exon 5 skipping within the STK domain. A similar variant is also expected to cause the in-frame deletion of exon 5 (IVS4-2A > C) and reported to induce MSSE without LDS-like phenotypes [ 91 ]. Analysis of in vivo RNA transcription products and ex vivo functional splicing assays revealed that the IVS5 + 1G > A variant produced two in-frame transcripts as a result of exon skipping and cryptic donor splice-site activation.…”

“…( B ) Basal levels of TGF-β signaling, through the wild-type/wild-type receptors, are maintained, in heterozygous Tgfbr1 and Tgfbr2 knock-out (KO) mice. This scheme holds true in patients with MSSE carrying truncating pathogenic variants in the STK domain of TGFBR1, who do not develop aortic aneurysms [ 91 ]. ( C ) Postnatal homozygous TGF-β receptor knockout mice, such as Myh11-CreERT2.Tgfbr2 f/f [ 70 ], develop aortic aneurysms without activation of intracellular TGF-β signaling.…”

Unique Mechanism by Which <i>TGFBR1</i> Variants Cause 2 Distinct System Diseases ― Loeys-Dietz Syndrome and Multiple Self-Healing Squamous Epithelioma ―

“…6,7 Exon 5 encodes part of the STK domain, and LDS and MSSE family members do not present with overlap features; thus, we performed ex vivo minigene splicing assays of 2 variants (Figure 3B,C) to elucidate the mechanism by which these 2 apparently similar variants produce different system diseases. 7 On analysis of the resulting transcripts, the LDS variant was found to produce 2 types of in-frame products as a result of exon 5 skipping (r.806_973del, p.Asp269_Gln324del), Very recently, MacFarlane et al reported a partial solution to this problem (Figure 2). 16 The severely affected aortic root and ascending aorta are composed of 2 types of vascular smooth muscle cells (VSMCs): secondary heart field (SHF)-and cardiac neural crest (CNC)-derived VSMCs.…”