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Supravalvular aortic stenosis (SVAS) is a congenital narrowing of the ascending aorta, which can occur sporadically as an autosomal dominant condition or as one component of the Williams–Beuren syndrome, a complex developmental genomic disorder associated with cardiovascular, neurobehavioral, craniofacial, and metabolic abnormalities, caused by a microdeletion at 7q11.23. We report the identification of seven novel mutations within the elastin gene in 31 familial and sporadic cases of nonsyndromic SVAS. Five are frameshift mutations within the coding region of the ELN gene that result in premature stop codons (PTCs); the other two mutations abolish the donor splice site of introns 3 and 28, respectively, and are predicted to alter splicing efficiency resulting in the generation of a PTC within the same introns of the gene. In vitro analysis using minigenes and cycloheximide showed that some selected frameshift mutant alleles are substrates of nonsense-mediated mRNA decay (NMD), confirming that the functional haploinsufficiency of the ELN gene is the main pathomechanism underlying SVAS. Interestingly, molecular analysis on patient fibroblasts showed that the c.2044+5G>C mutant allele encodes for an aberrant shorter form of the elastin polypeptide that may hamper the normal assembly of elastin fibers in a dominant-negative manner.
Supravalvular aortic stenosis (SVAS) has an incidence of 1 in 20000 live births and was the first disorder to be associated with the ELN gene.1 SVAS may occur as a sporadic disease or it may be inherited in an autosomal dominant manner. It is also classically associated with the Williams–Beuren syndrome (WBS) (OMIM 194050), a complex developmental disorder caused by a microdeletion of ~1.5Mb of chromosome 7q11.23, which encompasses at least 25 genes, including the ELN gene.2, 3 The clinical and structural characteristics of SVAS are identical in both syndromic and nonsyndromic cases. In WBS patients, SVAS is caused by the deletion of one complete copy of the ELN gene. In nonsyndromic cases of SVAS, >60 mutations, including substitution, splicing, regulatory, deletion, insertion, and rearrangement mutations, have been identified so far (The Human Gene Mutation Database, www.hgmd.org). Among the point mutations described, there is a prevalence of premature termination mutations that result in null alleles through the nonsense-mediated mRNA decay (NMD) mechanism leading to functional elastin haploinsufficiency.1, 4
Here, by using DHPLC and directed sequencing of genomic DNA, we identified seven novel ELN mutations in a screening of a total of 31 patients mainly affected with familial (13) and sporadic (18) nonsyndromic SVAS, associated in a few cases with other clinical features (Table 1). Functional assay and expression analysis using minigenes and real-time quantitative PCR (QPCR) showed that two selected frameshift mutant alleles are substrates of NMD, confirming that the haploinsufficiency of the ELN gene is the main pathomechanism underlying nonsyndromic SVAS. Moreover, of the two ELN mutations affecting the splicing process, we tested and showed that the c.2044+5G>C mutant allele results in an aberrant shorter form of the elastin polypeptide, which, by a dominant-negative mechanism, might cause the SVAS phenotype in that family.
Patients were enrolled in this study after obtaining appropriate informed consent by the physician in charge and approval of local ethics committees. Patients were recruited from different Italian and Spanish Hospitals (see the authors' affiliations for recruitment origin of samples). Clinically, all probands in each family presented with SVAS or other vascular abnormalities, such as peripheral pulmonary stenosis, aortic coarctation, and interatrial defects (Table 1). In some cases, there was a family history of SVAS (Table 1). Genomic DNAs were extracted from fresh and frozen blood samples using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany).
Screening for large ELN deletions was carried out by fluorescence in situ hybridization (FISH) on metaphase cells following standard technique using LSI ELN, D7S486, and D7S522 probes (Vysis, Abbott Laboratories, Abbott Park, IL, USA). At least 30 metaphases for each available sample were analyzed. Putative ELN whole-exon deletions were investigated by QPCR (see below).
The entire ELN coding sequence was PCR amplified using the set of primers listed in Supplementary Table 1 in 32 amplicons, including complete exons and acceptor and donor splice-site sequences. The nucleotide numbering used in this study follows the coding region of ELN, as previously described.4
For mutational analysis, we first performed DHPLC analysis using Wave 3500 HT (Transgenomic, Omaha, NE, USA); afterward, when an altered migration profile was detected, purified PCR amplification products were directly sequenced on an ABI3100 automated sequencer (Applied Biosystems, Foster City, CA, USA). All the novel nucleotide variants were further analyzed by segregation analysis in the family patients and by screening 100 healthy unrelated individuals who were used as control samples.
From RNA of HeLa cells, we generated a RT-PCR fragment spanning from exon 11 to exon 24 of the elastin gene. We then used DNA of HeLa cells to amplify a genomic fragment of ELN, including exon 25, its upstream and downstream intronic sequence, and exon 26. PCR fragments were amplified using the primer listed in Supplementary Table 1. We then ligated the two fragments and cloned them into the pcDNA3.1 vector. This wild-type (wt) construct was used as a template to introduce c.1195delG and c.1161delC mutations by site-directed mutagenesis using the QuickChange II kit (Stratagene, La Jolla, CA, USA). Using similar strategies, we also generated the constructs for c.838_839insG and c.2044+5G>C mutations. Minigenes carrying wt or variant alleles were all verified by sequencing.
HEK 293 cells were maintained at 37°C in DMEM supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Invitrogen). Primary skin fibroblasts were obtained from a patient with SVAS and from control subject, and cultured, as previously described.5
Nonsense-mediated mRNA decay assays were carried out by transfecting each ELN minigene (wt and mutant) into HEK 293 cells (2 × 105 cells per plate) with Fugene HD (Roche Diagnostics, Monza, Italy), according to the manufacturer's instructions. GFP plasmid was used as a reference for transfection efficiency in each experiment. Twenty-four hours after transfection, 300μg/ml of cycloheximide (CHX) was added. After 4h, total RNA was extracted using the RNeasy Mini Kit (Qiagen) and reverse transcribed using the Quantitect Transcription kit (Qiagen), according to the manufacturer's instructions.
The level of mRNA transcribed from different constructs was normalized to the mRNA level of GFP. Then, the ratio between normalized mRNA levels transcribed from the mutant and wt construct after CHX treatment was calculated and compared with this ratio in untreated cells. The experiments were repeated at least thrice.
Oligos for QPCR were designed using the Primer3 program6 with default parameters (primer's sequence available on request). Amplicons and primer pairs were checked both by Blast and Blat against the human genome to ensure specificity. The reactions were run in triplicate in 10μl of final volume with 10ng of sample cDNA, 0.3μ of each primer, and 1 × Power SYBR Green PCR Master Mix (Applied Biosystems). Reactions were set up in a 384-well plate format with a Biomeck 2000 (Beckmann Coulter, Milan, Italy) and run in an ABI Prism7900HT (Applied Biosystems) with default amplification conditions. Raw Ct values were obtained using SDS 2.3 (Applied Biosystems). Calculations were carried out by the comparative Ct method.7 For ELN genomic copy number determination, four normalization assays mapping to HSA21 and four normalization DNAs were systematically included in each run as described.8
All probands have been diagnosed with vascular anomalies, mainly SVAS (Table 1). FISH and genomic QPCR excluded the presence of ELN deletion and/or the partial WBS-deleted region, respectively (data not shown). Mutation analysis of the ELN gene in our group of 31 patients enabled us to detect seven novel mutations (Figure 1 and Supplementary Figure 1). Both c.40delC and c.304delG mutations, detected in two unrelated families, result in the same L121X premature termination codon in exon 7. In the SVAS-7 family, we detected a segregating frameshift mutation c.1161delC in exon 20 of the elastin gene. The mutant allele is predicted to encode a 75-amino-acid-long peptide sequence with a premature stop codon (PTC) in the domain encoded by exon 22. Intriguingly, previous studies have shown a clustering of ELN mutation in exon 20.1, 5, 9 The mutation identified here expands the number of nucleotide variations identified in this putative hot spot mutation region. c.838_839insG was present in two siblings diagnosed with isolated SVAS. Interestingly, the patient's mother carried the same mutation, but she was asymptomatic for SVAS, indicating an incomplete penetrance of the mutation in this family. Finally, the c.1939_1940insTG mutation was detected in a 6-month-old boy and in his father, who also presented with isolated SVAS.
The other two identified base substitutions lie in splice sites of the ELN gene.
The c.2044+5G>C transversion was identified five bases downstream from the start of intron 28–29 and it was present in all SVAS-13 family members in whom SVAS had been diagnosed, as well as in one asymptomatic relative. The c.163+2T>C transition was located in the +2 donor splice site of intron 3–4. Both mutations were not seen in 100 healthy unrelated control samples, confirming their absence in the general population.
It has been clearly shown that a number of PTC mutations in ELN are substrates of the NMD pathway.5, 10 Therefore, we analyzed the expression level of ELN mutant alleles found in SVAS patients and investigated the involvement of the NMD mechanism in the modulation of ELN transcript expression. Owing to the impossibility of obtaining primary skin fibroblasts, we devised a strategy on the basis of the use of minigene constructs and transfection assays. We measured by QPCR the ELN mRNA levels transcribed by c.1161delC, c.838_839insG, and wt constructs after transfection into HEK 293 cells in the absence and presence of CHX, a widely used inhibitor of NMD. To substantiate the assay, we used a minigene containing the c.1195delG mutation, previously reported as NMD target.5 The QPCR analysis showed a significant increase in ELN mRNA expression for all the analyzed constructs after CHX treatment compared with untreated samples (Figure 2). These results confirm that functional haploinsufficiency of ELN through NMD-mediated degradation of aberrant mRNA is one of the primary pathomechanisms leading to SVAS.
The c.2044+5G>C and c.163+2T>C mutations abolish the normal donor splice site of introns 28–29 and 3–4, respectively, as predicted by algorithm analysis.
Hence, we investigated by RT-PCR the consequences of c.2044+5G>C on mRNA splicing using, initially, the minigene-based strategy and then in skin fibroblasts from an SVAS-affected patient and a family relative used as control.
As reported in Figure 3, when we transfected the c.2044+5G>C construct, we detected two distinct mRNA species compared with one transcript of the wt construct (compare lane 4 with lane 3 in Figure 3c). The upper band of about 300bp corresponds to the aberrant transcript (comprising 30% of normal transcripts), whereas the 200-bp band corresponds to the normally spliced wt mRNA product. Consistently, the same PCR pattern was observed in the skin fibroblasts of the affected patient compared with that of a normal individual (compare lane 4 with lane 2 in Figure 3d). Sequencing analysis of the RT-PCR fragments confirmed that the longer transcript resulted from a splicing failure and inclusion of intron 28–29 in the mRNA, predicting a shorter elastin protein with a PTC within the same intron. Importantly, by semiquantitative RT-PCR, we assessed the ELN mRNA expression level after incubating the fibroblast culture of the patient with NMD inhibitor CHX, but we did not find any significant increase in the ELN mRNA aberrant transcript, showing that this mutation is conceivably not a substrate of NMD (data not shown).
For the c.163+2T>C mutation, splice sites software predicted a complete exon 3 skipping in the processed transcript, resulting in a frameshift with a stop codon in introns 3–4. Unfortunately, because of the infeasibility of obtaining skin fibroblasts from the patient, the molecular consequence of this mutation was not further investigated.
A large spectrum of mutations within the ELN locus has been identified as being responsible for nonsyndromic SVAS.1, 4, 5, 9, 11, 12, 13 Reduction of ELN expression has been reported in the skin fibroblast and aortic smooth muscle cells of SVAS patients, with premature truncation mutations in ELN resulting in haploinsufficiency of elastin.5, 10 In an attempt to molecularly characterize our SVAS patient group, we performed a mutation screening of the ELN gene. We detected seven novel mutations, of which five result in PTC and two affect the natural splicing of the ELN gene.
Consistent with previous studies, our results of mRNA expression analysis of two identified PTC mutations showed that the functional haploinsufficiency of ELN, through nonsense-mediated degradation of mRNA, is one of the primary pathogenic mechanisms leading to SVAS. Even if not formally proven in this study, the other three PTC mutations are also probably targeted by NMD.
Interestingly, in one of the familial cases, the same change was also detected in one of the asymptomatic parents. This was not surprising because it has been reported that disease severity within SVAS families varies from asymptomatic carriers of the mutation to individuals who die in infancy from severe cardiac disease.1 Variable expressivity and reduced penetrance of elastin arteriopathy are observed in both WBS and nonsyndromic SVAS.14 This variability is typical of diseases associated with haploinsufciency, in which the genetic background is expected to have a major modifying effect.4
To date, only few ELN gene alterations reported in SVAS patients are missense mutations.1, 5, 12, 15 In our group of SVAS patients, we found two point mutations; they induce aberrant and/or additional splicing and are predicted to lead to truncated proteins as well.
We analyzed the effect of the c.2044+5G>C mutation in fibroblast culture and found an aberrant transcript that is predicted to produce a significant amount of abnormal tropoelastin lacking the domains encoded by the last six exons of the gene. The missing part contains several essential hydrophilic cross-linking domains and the well-conserved C-terminus region characterized by two cysteine residues forming a disulfide bond and the positively charged RKRK sequence. It has been shown that the disruption of the disulfide bond dramatically reduces the ability of tropoelastin to assemble and be included in developing fibers, as it does the removal of the C-terminal region.16 Therefore, we can speculate that the mutant tropoelastin, if secreted from the cell, would likely impair the tropoelastin from assembling in a dominant-negative manner, resulting in a deleterious effect on the normal elastogenesis in those patients. A definitive resolution of this issue would require arterial smooth muscle in order to investigate whether these mutations yield abnormal elastin and/or normal elastogenesis, but this kind of tissue is not available.
Finally, we did not find any ELN gene abnormality with our approach in the remaining sporadic and familial patients with clinically diagnosed SVAS included in the study. Familial cases were not investigated for linkage analysis because of the small number of family members available for analysis. DHPLC detection mutation rate and mutations in the regulatory regions of the gene might be the possible causes for the lack of ELN mutations in those patients. Alternatively, mutations in another gene could also cause SVAS. In this regard, further studies are required.
In summary, this study illustrates the importance of conducting mutation screening of the ELN gene among patients with vascular abnormalities, particularly SVAS and pulmonary stenosis, so that new mutations can be identified and characterized. Our findings also reinforce the idea that haploinsufficiency at elastin is the main cause of these vasculopathies, hence, therapeutic strategies should be directed toward the compensation of this pathogenic mechanism.
The authors declare no conflict of interest.
We are very grateful to the SVAS families, whose cooperation made this study possible. We thank Youngho Kim for ELN primer sequences. This study was conducted with the support of the Italian Ministry of Health (Ricerca Corrente 2006-2009), the Fondazione Banca del Monte di Foggia ‘Domenico Siniscalco Ceci' and the Jérôme Lejeune Foundation to GM, and the VI Framework Program of the European Commission (LSHG-CT-2006-037627) to LAPJ.
Supplementary Information accompanies the paper on European Journal of Human Genetics website (http://www.nature.com/ejhg)