Supravalvular aortic stenosis (SVAS; Online Mendelian Inheritance in Man [OMIM] #185500) is a congenital heart disease with an incidence of approximately 1 in 25,000 live births (Ewart et al., 1993). Peripheral pulmonary stenosis (PPS) is known to occasionally coexist with SVAS, and induces increased resistance to blood flow and causes elevated ventricular pressure and hypertrophy resulting in heart failure (Collins et al., 2010).

SVAS is the main feature of elastin arteriopathy, and is related to haploinsufficiency of the elastin (ELN) gene located at 7q11.23 that encodes the elastin protein (Denie and Verheugt, 1958). The syndromic form, Williams-Beuren syndrome (WBS; OMIM #194050), accounts for approximately 30%–50% of SVAS patients. It is a complex genetic disorder caused by heterozygous microdeletions on chromosome 7 at 7q11.23, characterized by intellectual disability, hypercalcemia, impaired social interactions, facial dysmorphism, and SVAS (Collins, 2018; Duque Lasio and Kozel, 2018; Williams syndrome, 2021). Autosomal dominant non-syndromic “familial SVAS” accounts for 20% of SVAS patients. The ELN gene which encodes elastin is causatively involved in non-syndromic familial SVAS. Hemizygosity for elastin causes SVAS, and sometimes peripheral artery stenosis and hernias, but not the other features of WBS (Metcalfe et al., 2000; Hayano et al., 2019). SVAS can also occur sporadically with unknown etiology.

To date more than 100 pathogenic or suspected pathogenic mutations of the ELN gene have been described in the literature, the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar) and the human gene mutation database (http://www.hgmd.cf.ac.uk) (Duque Lasio and Kozel, 2018). ELN mutations mainly include missense mutations affecting methionine, nonsense mutations, and frameshift mutations resulting from insertions and deletions. Previous studies indicate that point mutations can lead to premature stop codons (PTCs), resulting in functional elastin haploinsufficiency through a nonsense-mediated mRNA decay mechanism (Micale et al., 2010; Hayano et al., 2019). It remains unclear whether ELN can explain the remaining cases of SVAS, or whether there are unidentified causative genes. The molecular mechanism of the disease requires further study.

The current study included 42 patients with SVAS from 11 families. ELN mutations were assessed via whole-exome sequencing (WES) to elucidate the genetic background of SVAS. Aortic tissues were collected from some patients after surgery to further define the molecular pathology of familial SVAS.

Mutation or microdeletion within the ELN gene can lead to SVAS (Duque Lasio and Kozel, 2018). In an attempt to elucidate the molecular pathology of SVAS we performed mutation screening of the ELN gene. Pathogenic mutations of the ELN gene were found in 11 autosomal dominant SVAS families, and nine of them were novel mutations that had not been reported and were not included in any database. Two different families had the same mutation. Among the 11 SVAS families, nine had nonsense mutations and 2 had missense mutations. Analysis of aortic tissue revealed reduced elastin expression. These results suggest that reduced elastin expression caused by heterozygous pathogenic mutations in the ELN gene is the major genetic cause in hitherto unexplained cases of familial SVAS in China.

Elastin is encoded by the ELN gene and is expressed in a variety of tissues and organs including large artery smooth muscle cells, and it contributes to tissue elasticity (Yeo et al., 2016). Although the molecular mechanism of SVAS has not been fully elucidated, it is mainly related to insufficient secretion of elastin by aortic smooth muscle cells caused by ELN gene mutation or deletion (Hayano et al., 2019; Min et al., 2020; Dave et al., 2022). Consistent with previous studies (Micale et al., 2010), in the current cohort of patients the disease was mainly caused by nonsense mutations in the ELN gene leading to PTCs. Analysis of aortic mRNA expression in the patients with PTC mutations showed that haploinsufficiency of ELN expression caused SVAS and nonsense mutation-mediated mRNA degradation. However, no differences in ELN expression levels in aortic tissue between patients with different mutations were found in this study. This may be related to the fact that the sample size of our study was not very large, which remains to be further explored in future studies.

The same mutation was found in two different families (c.1621C>T in families B and D), and this mutation has also been reported in ClinVar. Therefore, we hypothesized that this locus may be a hotspot for mutation of SVAS patients.

The main clinical phenotypes of patients with ELN mutations are arterial stenosis, especially SVAS and pulmonary artery stenosis (Keating, 1994; Park et al., 2006). These symptoms are similar to those in patients with WBS (Eronen et al., 2002; Delio et al., 2013) thus the two groups of patients are sometimes difficult to distinguish based on clinical findings alone. In the present study some patients were initially suspected of having WBS, but no abnormalities were found after Fluorescence in situ hybridization (Ramirez-Velazco et al., 2019), multiplex ligation-dependent probe amplification (Honjo et al., 2015), and chromosomal microarray analysis (Kuo et al., 2019) targeting probes containing the WBS chromosomal region. Therefore, in such patients the use of methods with higher sequencing depth is recommended, such as whole-exon sequencing and whole-genome sequencing, to increase the gene mutation detection rate.

In most individuals in the present study there was a consistent separation of symptoms and mutations, and most mutations resulted in PTCs. However, there were also individuals in the same family with ELN mutations who did not suffer from cardiovascular malformations or present with other types of defects. Moreover, no other related genes mutation was found according to the whole exome sequencing data that may explain this clinical manifestation variation. These cases are consistent with previous studies (Metcalfe et al., 2000; Jakob et al., 2011; Jelsig et al., 2017) in which SVAS families had highly variable cardiovascular phenotypes ranging from asymptomatic to multiarterial severe stenosis. Such differences may be related to epigenetics (Parrish et al., 2020; Procknow and Kozel, 2022), or some individuals may initially have undetected arterial stenosis then undergo self-healing, suggesting that pulmonary artery stenosis caused by elastin depletion has a high probability of undergoing gradual improvement as reported by Collins et al. (2010). Taken together, findings to date indicate that cardiac screening and genetic counseling should be administered to relatives of SVAS patients, as unaffected carriers may give birth to severely affected individuals.

Consistent with previous findings (Micale et al., 2010) PTC mutations were associated with ELN mRNA substrate deficiency, resulting in reduced elastin expression, which may be caused by nonsense-mediated attenuation of PTC mutations. Interestingly, we also identified two families with ELN missense mutations, which are not common in previous reports. We suspect that this may be related to normal splicing affecting ELN gene transcription, resulting in truncation of the protein (Zhang et al., 1999; Kozel et al., 2003; Ott et al., 2011).

The current study indicates that point mutations within the ELN gene are an important genetic cause of familial SVAS in China. The use of whole-exome sequencing or whole-genome sequencing gene testing technology is therefore recommended for genetic diagnosis in such patients, to improve the mutation detection rate. The study also illustrates the importance of screening for ELN gene mutations in patients with arterial stenosis, especially SVAS and pulmonary stenosis, in order to identify the genetic etiology involved. Notably, our findings revealed that insufficient elastin expression is the main cause of these vascular lesions. Further investigation is required to identify the specific molecular mechanism involved however, which may in turn contribute to the identification of potentially therapeutic drug targets.