The results of different studies [2, 3, 11], suggested a role for Melusin in cardiomyopathies as modifier gene, whose inactivation worsens the pathology and/or increases susceptibility to heart failure in response to different pathological conditions.
Analysis of the available data in the human genome database http://www.ncbi.nlm.nih.gov/Genbank/ indicated that Melusin ITGB1BP2 gene is highly conserved and only a limited number of single nucleotide polymorphisms have been described. In particular 35 SNPs are currently in the NCBI SNPs database http://www.ncbi.nlm.nih.gov/SNP/: 4 non-synonymous, 1 synonymous, 11 intronic, 19 in gene region. None of these SNPs, however, has been correlated with human pathologies and their frequency in the population has not been reported.
In this study we evidenced an extremely low number of variations in the ITGB1BP2 gene in nearly 1000 hypertensive/cardiopathic and healthy individuals, thus suggesting a high degree of conservation of the melusin gene, within the populations analyzed. In fact, only three genetic variants were detected in a total of 656 patients and 272 healthy controls screened by direct sequencing analysis; moreover the three variations have been found in three different patient and not in any control.
The 843C>T transition was found in a patient (#1) from HDCOC group of hypertensive patients that developed eccentric remodeling of the left ventricle. When analyzing the patient's family, three male subjects, all characterized by eccentric remodeling of the left ventricle, were hemizygous for the 843C>T SNP while the remaining three individuals with standard echocardiography profile showed the wild type DNA sequence. This SNP falls in the exon 11 coding region but is a silent substitution not affecting the coded amino acid. The sequence surrounding the 843C>T SNP, however, falls 3' to a consensus motif for an exonic splicing enhancer sequence (ESE), short nucleotide sequences that can stimulate splicing and regulate alternative splicing [12]. ESE sequences are present in most, if not all, exons, including constitutive ones and are thought to serve as binding sites for specific serine/arginine-rich (SR) proteins that promote exon definition by recruiting the splicing machinery. Nonsense, missense and even translationally silent mutations occurring in such ESE sequences can inactivate genes by inducing the splicing machinery to skip the mutant exons. Similarly, coding-region single-nucleotide polymorphisms might cause phenotypic variability by influencing splicing accuracy or efficiency [12, 13]. This hypothesis has been verified by a functional assay: we performed an analysis of the 843C>T SNP, by using a minigene system in order to test if the 843C>T SNP may affects Melusin gene splicing; our results indicate that the nucleotide substitution seems not to affect the splicing, similarly to what suggested by an in silico analysis we performed. The length and sequence of the mutated transcript were the same of the wild-type ones. However, these results should be cautiously considered, since they are just an in vitro model that may not completely reflect what happens in vivo in the patients, and the Hela cell line model (available to us) is quite different from the cardiomyocytes. Taking in account these limitations, we can not exclude the possibility that, in the in vivo context of muscle cell, the mutation detected can alter the splicing introducing stop codon(s) which can ultimately lead to nonsense-mediated decay of the aberrant transcript, as already reported for mutations in other genes [14, 15]. Testing this hypothesis requires analysis of Melusin transcripts in samples of skeletal muscle biopsies from patients, which, unfortunately, were not available for our study.
The second mutation detected in a hypertensive patient (#2) of the HDCOC group was the duplication IVS6+12_18dupTTTTGAG, occurring near the 5'donor splice site of intron 6. Such duplication was found in heterozygosis in a female patient presenting a modest cardiac remodeling. However, it was also found in an unaffected male as well as in an unaffected female, so this mutation reasonably does not correlate with the eccentric remodeling of the left ventricle and it is likely to represent an additional polymorphism of the Melusin gene whose association with the cardiac pathology is unlikely.
Similarly to what done for the 843C>T, also for the IVS6+12_18dupTTTTGAG we performed an in silico and a minigene splicing assay, without evidencing any difference in the transcript between the wild-type and the mutated sequence. However, the same arguments discussed for the 843C>T SNP, can be applied also to this mutation.
The third mutation detected is a C>T transition at position 37 in the exon 1 of the ITGB1BP2 gene and represents a missense mutation causing an amino acid change from His-13 to Tyr in the protein primary sequence. Such mutation falls in a highly conserved region of the Melusin molecule corresponding to the first CHORD domain characterized by a unique Cys and His signature [2] (Figure 2). The tertiary structure of Melusin CHORD domains has not been solved yet, and this fact restrains the possibility to predict the effect of the mutation. However, the substitution of an His residue, which is highly conserved and is part of the signature of the mammalian melusin CHORD 1 domain (Figure 2), strongly suggest that such mutation can modify the structure/function of the protein. This hypothesis, moreover, is also supported by preliminary bioinformatic analysis. The His/Tyr mutation in exon 1 was detected in heterozygosity in female patients II.2 and III.1 and in hemizygosity in male patients II.6 and III.3. No biological samples other than DNA were available from the subjects, thus impeding the possibility to perform functional study on the protein. The effect of this mutation remains to be disclosed, and it should also be noticed that while three of the patients (II.2, II.6, III.3) that presented the mutation are affected by HCM, one of the two heterozygote females (III.1) is not. This family also carries (II.2, III.1, III.3) an additional mutation in the 3' UTR of troponin T gene, whose molecular impact on the HCM status is unknown as well.
