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Myotonic dystrophy type 1 (DM1) is the most frequently inherited neuromuscular disease in adults. It is a multisystemic disorder with major cardiac involvement most commonly represented by first-degree atrioventricular heart block (AVB), followed by different degrees of bundle-branch and intraventricular blocks. In search for candidate genes, modifiers of the AVB phenotype in DM1, the expression of the small-conductance calcium activated potassium channel (SK3) gene was analysed in muscle biopsies from DM1 patients. The association between SK3 polymorphisms and the AVB phenotype was then studied analyzing 40 DM1 patients with AVB and 40 age-matched DM1 affected individuals with no ECG abnormalities. [CTG]n repeat length and cardiac clinical picture were also assessed for correlation. QRT-PCR experiments showed an over-expression of the SK3 transcript in DM1 muscle biopsies compared to healthy controls. However, no statistical association between the AVB phenotype and either the [CTG]n expansion length or the presence of specific SNPs in the SK3 gene were detected. These findings suggest that modifier genes, other than SK3, should be identified in order to explain the cardiac phenotypic variability among DM1 patients.
Myotonic dystrophy type 1 (DM1; MIM#160900), or Steinert’s disease, is the most prevalent form of muscular dystrophy in adults with a frequency of 1 in 8000 individuals (1). DM1 is an autosomal dominant disorder with incomplete penetrance and variable phenotypic expression caused by a [CTG]n expansion in the 3’-UTR of the myotonic dystrophy protein kinase gene (DMPK; MIM#605377), on chromosome 19q13.3 (2). Healthy individuals may have 5 to 37 [CTG]n repeats while in affected people this number may reach 50-8000 (2). DM1 patients variably present multisystem clinical features including myotonia, progressive muscle weakness, cardiac abnormalities, cataract and cognitive impairment (3, 4). Cardiac involvement manifests as a selective and extensive impairment of the conduction system, usually not associated with any apparent structural heart disease. Such degeneration of the conduction system has been correlated with the significant incidence of sudden death (SD) observed in DM1 patients, ranging from 2% to 30% according to data in the literature (5). In general, cardiac SD has been related to the development of conduction blocks, and, in fact, the implantation of a pacemaker is often (3-22% of cases) required in DM1 patients (6–13). It has been recently shown that severe electrocardiogram (ECG) abnormalities based on the rhythm disturbances, length of PR interval, QRS duration, and presence of atrioventricular block (AVB) predict sudden death in DM1 patients (14).
The observation of familial clustering of specific cardiac features (15–17) and the phenotypic variability among patients with the same class of [CTG]n expansion, strongly suggest the contribution of modifier genes other than the DMPK in the development of the AVB phenotype. Among many, SK3 (MIM #602983), a member of the SK channels, proved to be an intriguing candidate gene. SK channels are, in fact, the small conductance subset of the calcium-activated channel family (18). These channels are voltage independent and found to underlie the long-lasting after-hyperpolarization (AHP) following the action potential and its accompanying elevation in cytosolic calcium (19–22). At least three types of SK channels exist, namely SK1, SK2, and SK3 encoded by three different genes (KCNN1, KCNN2, and KCNN3, respectively) sharing up to 70% sequence homology (23). SK channels are expressed in myofibres of developing and denervated muscles, differentially regulated in atrial and ventricular myocytes, and down-regulated in adult skeletal muscle (24). Denervated muscles are hyperexcitable as they display trains of spontaneous action potential known as fibrillation (25, 26). Electric hyperactivity is also the cause of muscle stiffness in DM1 where, not surprisingly, SK3 is expressed at high levels in muscle cells (27, 28). It has also been shown that increased expression of SK3 has a role in causing the symptoms of myotonia, because muscle injection of the highly specific SK inhibitor –apamin- reduced the electrical activity associated with myotonia (18, 29, 30).
In the present study, in order to define the possible role of the SK3 gene in the development of conduction defects, the muscle-specific expression of this gene was investigated in DM1 patients. Furthermore, attention was focused on SK3 genetic variants possibly associated with the development of ABV in two cohorts of DM1 patients grouped to the cardiac phenotype (presence/absence of atrioventricular blocks).
A total of 80 DM1 patients, from unrelated pedigrees, were selected for this study among those regularly followed at the Cardiomyology and Medical Genetics Service of the Second University of Naples and the Neurology Service of the Catholic University of Rome. Exclusion criteria were: age < 20 years or > 65 years, presence of heart failure NYHA(New York Heart Association) class >/= 3), long-standing hypertension, chronic coronary artery disease, myocardial infarction, sarcoidosis, amyloidosis and primary cardiomyopathies in order to exclude causes of AVB other than DM1. The diagnosis of DM1, first established on the basis of the family history and clinical findings, was then confirmed by molecular testing in all patients. All patients underwent periodical cardiological investigations, such as standard and dynamic (24h Holter monitoring) ECG and echo-colour-doppler-cardiography. The cardiologic features of DM1 patients are listed in Table Table1.1. Patients were grouped, according to the presence/absence of AVB, into two age-matched groups. Presence of familial clustering for heart defects in the control group was excluded. Informed consent was obtained from all participants in the study.
Genomic DNA was extracted from blood samples by standard procedures (31) or according to the standard operator procedures (SOPs) published in the EBB network website (32) while [CTG]n repeat number in the DMPK gene was determined with a long-PCR (polymerase chain reaction) assay (33).
Needle or overt muscle biopsies were obtained from vastus lateralis or brachial biceps of patients of Caucasian origin, heterozygous for the DM1 (n = 7) mutation as well as from healthy controls (n = 2) followed by the Clinical Services involved in this study. Muscle specimens were snap frozen in liquid nitrogen and stored at -80°C until processing. Pathological assessment of the specimen was performed by an experienced pathologist.
Total RNA was extracted from muscle samples using the RNeasy mini kit (Qiagen Co., Valencia, CA, USA). Total RNA (3 μg) was reverse transcribed according to the cDNA protocol of the High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA). The expression of the potassium intermediate/small conductance calcium activated channel, subfamily N, member 3, (SK3: GenBank accession# NM_002249.4) was measured using the Hs00158463_m1 Assay-on-demand™ gene expression products. The β2-microglobulin gene (B2M: GenBank accession #NM_004048) was selected as housekeeping internal control gene. The expression level of SK3 gene and of the internal reference was measured by multiplex PCR using Assay-on-demand gene expression products labelled with FAM and VIC dye for SK3 and B2M transcripts, respectively (Applied Biosystems, Foster City, CA, USA). The simultaneous measurement of SK3-FAM over B2M-VIC transcripts expression allowed normalization of the amount of cDNA added per sample. Each PCR reaction was performed in triplicate using the Taqman Universal PCR Master Mix and the ABI PRISM 7000 Sequence Detection System. A comparative threshold cycle (Ct) was used to determine gene expression compared to a calibrator (median value of control subjects). Hence, steady-state mRNA levels were expressed as a n-fold difference relative to the calibrator. For each sample, Ct value of products was normalized using the formula ΔCt = Ctgenes/CtB2M. To determine relative expression levels, the following formula was used: ΔΔCt = ΔCt sample − ΔCt calibrator. The value adopted to plot relative gene expression was calculated using the expression 2−ΔΔCt.
The hypothesis of an association between the SK3 gene and the development of AVB in DM1 was tested using a case-control genetic study. Two single nucleotide polymorphisms (SNPs) (Genbank refSNP IDs rs6656494 and rs10128027) located at intron 1 and 5 of the SK3 gene (GenBank accession # NC_000001) were genotyped in both the case and the control populations (Fig. (Fig.2A).2A). Genotyping was carried out using standard PCR protocols, followed by restriction enzyme digestions. The primer pairs used were: rs6656494, F 5’-tctgacaggtctgcccca-3’ and R 5’-gaaaactgatgaaggcccaa-3’; rs10128027, F 5’-aaattccaggggtcccatta-3’ and R 5’-atcccatttcacagatgc-3’. PCR was performed with an initial denaturation of 2’ at 95°C followed by 30 cycles of 30’’ at 95°C, 30’’ at 60° (rs6656494) or 58°C (rs10128027) and 45’’ at 72°C, with a final extension of 5’ at 72°C. 20μl of the PCR were subjected to restriction enzyme digestion for 4 hours. The rs6656494 polymorphism was analyzed following digestion with BstNI and the rs10128027 with MboII restriction enzymes. 20μl of the digested products were resolved by gel electrophoresis (2.5% agarose gel) (Fig. (Fig.22 B, C). Reproducibility of genotyping was confirmed by bidirectional sequencing in 50 randomly selected samples, and the reproducibility was 100%.
The DMPK [CTG]n expansion was analysed for association with presence and severity of AVB by linear regression. The distribution of allelic and genotypic frequencies in the two DM1 groups was analysed by using the Chi square test and tested for multiple association by Bonferroni’s correction. All analyses were considered at 95% confidence interval (95% CI). and performed by SPSS 11.0 (http.//www.spss.com).
Among the genes possibly involved in the onset of AVB, in DM1 patients, attention was focused on SK3, the protein product of which regulates the electrical activity of the muscle (29). First, the SK3 mRNA expression was investigated in seven muscle biopsies from DM1 patients with a [CTG]n mutation ranging from 300 to 500 repetitions and in two muscle biopsies from healthy subjects. Biopsies of affected individuals were revised by an experienced pathologist thus allowing the homogeneous identification of a common hallmark in DM1 skeletal muscle, including atrophic fibres with increased fibre size variation, pyknotic nuclear clamps, and marked proliferation.
Expression levels of the SK3 transcript were assessed by qRT-PCR on total RNA extracted from muscle biopsies. The β2-microglobulin (B2M) housekeeping gene was used as an internal control for normalization and each experiment was conducted in triplicate. The average result of normal controls was given a value of 1. Consistently, over-expression of the SK3 transcript was found in all samples from DM1 patients, with a mean value of 3.28-fold changes. (range 1.85- 6.33-fold changes) (Fig. (Fig.1).1). A case-control study was then performed on the hypothesis of an association between genetic variants in the SK3 gene and the development of AVB in DM1 patients. Overall, 80 DM1 patients, age range 30 - 60 years were divided into two different cohorts recruited according to the study criteria (AVB-DM1 Patients and no AVB-DM1 Patients). The two groups were age and sex matched (Table (Table1).1). Two SK3 intragenic SNPs (rs6656494 and rs10128027) were selected for the genetic analysis in the different groups of DM1 patients discordant for the cardiac phenotype. These polymorphisms represent the distribution of the gene variants of the SK3 gene region and have been chosen on account of their highly polymorphic nature. The rs6656494 SNP is an A to G transition with an estimated heterozygosity rate of 0.495. The 403-bp PCR products corresponding to the rs6656494 SNP region were digested with BstNI restriction enzyme: four major DNA fragments of 102, 70, 65 and 46 bp were yielded for the G allele on 3% agarose gel and only 3 major bands of 172, 65 and 42 bp for the A allele (Fig. (Fig.2B).2B). The rs10128027 SNP is a C to T transition (heterozygosity rate of 0.464), localised in the SK3 intron 5, which abolishes a MboII enzyme restriction site. The PCR products containing the SNP region were, therefore, digested with the MboII enzyme: two DNA fragments of 226 and 177 bp were yielded for the C allele and only one band for the T allele (Fig. (Fig.2C).2C). The allelic and genotypic frequencies for the rs6656494 and rs10128027 SNPs observed in the AVB-DM1 and no AVB-DM1 patients are reported in Table Table2.2. The genotype distribution for both SNPs, in our sample, is in agreement with the Hardy-Weinberg equilibrium. Chi-square analysis of the allelic distribution between the two groups (AVB-DM1 and no AVB-DM1) revealed the lack of association with the AVB phenotype for both rs6656494 and rs10128027 SNPs (Χ2 = 1.61, p < 1 and Χ2 = 0.14, p < 1, respectively).
We therefore investigated the possibility of an association between the number of [CTG]n repetitions in the DMPK gene and the presence of the AVB phenotype in the present cohort of DM1 patients. Patients were stratified according to the three classes of expansion (E1:50-150 [CTG]n; E2: 150-1000 [CTG]n; E3: up to 1000 [CTG]n) currently applied for DM1 (1). Both groups showed a homogeneous distribution between the three classes (r = 0.918; Χ2 = 0.359, p = 8.36), thus excluding a direct correlation between the occurrence of AVB and the DMPK [CTG]n expansion. This result is in accordance with other previous studies (34, 35).
Over-expression of the SK3 gene, both at RNA and protein level in DM1, has been previously described (28, 36). This finding is confirmed by the present qRT-PCR experiments on muscle biopsies from DM1 patients. Despite up-regulation upon denervation and hyperexcitability, the absence of SK3 protein in a myotonic mouse (ADR) suggests that its over-expression in DM1 might be related to a differentiation defect (36). SK3 might, therefore, play a key role in DM1 pathogenesis, more than being a mere downstream target of disordered myocytes. Among other functions, SK channels have been found to play a prominent role in cardiac myocytes (37). In the mouse heart, SK3 showed homogeneous levels of expression both in the atria and ventricules and an intermediate sensitivity to apamin (37). On the other hand, SK1 and SK2 showed differential regulation in atria and ventricules, suggesting that these three genes may act together in order to regulate Ca2+-activated K+ channels in cardiac myocytes, particularly in atria (37, 38). Furthermore, tissue-specific truncated SK3 transcripts, SK3–1B and SK3–1C, have been identified leading to “dominant negative” suppression of K+ currents produced by SK1, SK2, and SK3 channels and, to a lesser extent, the intermediate-conductance Ca2+-activated K+ channels (39, 40). Based on these findings we hypothesized that the abnormal regulation of SK3 in DM1 could interfere with the cardiac conduction system and with the physiological repolarization of cardiomyocyte membranes. This study was performed in order to establish the possible role of SK3 variants in the modulation of cardiac feature of DM1 patients. Data obtained showed the lack of any significant association between SK3 variants and AVB in DM1 patients.
Until recently, genotype-phenotype studies aimed to analyse a specific correlation of disorders in heart conduction systems with the number of [CTG]n repeat amplification in DM1 patients, failed to demonstrate clear-cut results. In our dataset, the [CTG]n expansion class was not associated with AVB. However, a recent large multicentric prospective study demonstrated an association between the number of triplet repeats and the presence of severe abnormalities upon ECG, but no association with sudden death at the univariate analysis (14). Albeit the DM1 mutation alone cannot account for all the variability in phenotype in heart involvement in patients, thus indicating the need for more extensive efforts in order to identify not only genetic variants but also molecular mechanisms possibly affecting this variability. It is worthwhile pointing out, however, that our study has attempted to correlate the AVB phenotype and the length of [CTG]n expansion measured in DNA from lymphocytes. It is tempting to suggest that a significant correlation would be found if the mutation size were measured directly in the tissue affected by the pathological cardiac process. This approach to cardiac testing would be difficult to justify ethically, particularly at the level of the conduction system. Long-term follow-up of our patients will indicate more precisely the value of the measurement in the lymphocytes as a predictor of conduction disturbances, bearing in mind, the severity of peripheral muscle involvement or length of [CTG]n triplet repeats and ECG abnormalities. Interestingly, a recent study demonstrated the role of NKX2-5 over-expression as a genetic modifier of the DM1-associated RNA toxicity in the heart (41, 42). Moreover, for SK3, molecular and electrophysiological investigations are mandatory in order to identify mechanisms other than genetic variants associated with heart conduction defects in DM1 patients.
In conclusion, the present findings confirmed that SK3 over-expression is a hallmark in DM1 muscle tissues. However, genetic variants of the SK3 gene did not represent any additional risk of developing AVB, thus suggesting that different mechanisms underlie the SK3 misregulation and its possible involvement in the cardiac phenotype of DM1 patients.
This work was supported by Telethon grant #GGP07250 to GN, by MIUR grant # 2005064759 to GN, LP and GS (2005), and by AFM grant #13360 to GN (2008). Authors acknowledge the SUN-Naples Human Mutation Gene Bank (Cardiomyology and Medical Genetics), which is a partner of the Eurobiobank network, for providing muscle and DNA samples.