A negative screen for mutations in calstabin 1 and 2 genes in patients with dilated cardiomyopathy
© Biagi et al; licensee BioMed Central Ltd. 2012
Received: 1 May 2011
Accepted: 11 January 2012
Published: 11 January 2012
Calstabins 1 and 2 bind to Ryanodine receptors regulating muscle excitation-contraction coupling. Mutations in Ryanodine receptors affecting their interaction with calstabins lead to different cardiac pathologies. Animal studies suggest the involvement of calstabins with dilated cardiomyopathy.
We tested the hypothesis that calstabins mutations may cause dilated cardiomyopathy in humans screening 186 patients with idiopathic dilated cardiomyopathy for genetic alterations in calstabins 1 and 2 genes (FKBP12 and FKBP12.6). No missense variant was found. Five no-coding variations were found but not related to the disease.
These data corroborate other studies suggesting that mutations in FKBP12 and FKBP12.6 genes are not commonly related to cardiac diseases.
KeywordsGenetic screening Calstabin FKBP12 FKBP12.6
Ryanodine receptors (RyRs) are the main proteins in the sarcoplasmic reticulum (SR) calcium-release channel and are modulated by other proteins in the regulation of muscle excitation-contraction coupling. Calstabins 1 and 2 (FKBP12 and FKBP12.6 genes, respectively) bind both RyR1 (skeletal form) and RyR2 (cardiac form) although calstabin1 has higher affinity to RyR1, and calstabin2 to RyR2 [2–4]. The RyR-calstabin interaction occurs to stabilize the RyR channel in the closed state, but its specific amino acids binding site is still controversial [5–7]. Although, calstabin1 deficient mice display normal skeletal muscle phenotype, they have severe Dilated Cardiomyopathy (DCM) and ventricular septal defects . Mice with a disruption in calstabin2 display hypertrophic cardiomyopathy only in males. However, both males and females present a similar dysregulation of Ca2+ release .
Disease-causing mutations in RyRs 1 and 2 affecting the interaction with calstabins were described as leading to different phenotypes . Wehrens and colleagues reported RyR2 mutations which decrease the affinity of RyR2 with calstabin2 in patients with Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) . However, no mutation in calstabin2 seemed to be involved with the disease . The association of calstabins with heart disease was also tested in a familial Hypertrophic Cardiomyopathy cohort, and no mutation was found in all 252 patients . Recently, Oyama and colleagues have reported an increased expression of FKBP12.6 gene in Great Dane dogs with DCM . These results, taken together with the dilated cardiomyopathy phenotype of calstabin1 knockout mice, suggest that calstabins may be associated with DCM development. Thus, the aim of the present study is to test the hypothesis that mutations in calstabins genes (FKBP12 and FKBP12.6) occur, and can cause human DCM phenotype.
Considering the 186 Idiopathic Dilated Cardiomyopathy (IDC) patients screened, 75% were male and 25% female. The mean age was 44.8 ± 12.4. The population had the following ethnic distribution: 14.5% blacks, 15.5% mullatos and 69.8% whites.
New allelic variants found in FKBP12 and FKBP12.6 gene.
(n = 186)
(n = 288)
Risk by bioinformatics analyses
Exon 1 - 5' UTR
Exon 1 - 5' UTR
Exon 4 - 3'UTR
SNPs found in FKBP12 and FKBP12.6 gene in cases.
(n = 186)
(YRI - CEU)
0.008 - 0
Exon 1 - 5' UTR
0.25 - 0
Exon 1 - 5' UTR
0 - 0.18
0.32 - 0
Exon 4 - 3'UTR
0.4 - 0.008
Exon 4 - 3'UTR
0 - 0.005
Data from animal models point out FKBPs as important candidate genes for cardiac diseases. In fact, several mutations in RyR1 and RyR2 proteins cause decreased affinity between RyRs and calstabins, leading to abnormal calcium cycling, a major hallmark of several cardiac phenotypes, including heart failure. Mutations affecting RyR-calstabin interactions are well described for some diseases such as: Left Ventricule Noncompaction (LVNC), Central Core Disease (CCD) and Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). However, little is known if mutations in calstabins could lead to disease by themselves. Genetic analyses of FKBP12 were done only in two cohorts of diseases (LVNC (48 patients)  and CCD (27 patients) ) and in one family with LVNC. No mutation was found in any of this studies. Recently, genetic analyses of FKBP12.6 have been done in 16 patients with CPVT and in 232 patients with hypertrophic cardiomyopathy and, also, no mutations were found.
To our knowledge, this study has been the first report of calstabins 1 and 2 genetic screening in idiopathic DCM patients. We found five new allelic variations and none of them caused amino acid change. The alterations c.*26G>A in FKBP12.6 gene and -40C>A in FKBP12 gene were also found in the control population. For the three other alterations we measured the risk of being disease-causing by the analyses of three different bioinformatics software, concerning their possible involvement in splicing, microRNA and transcription factor binding sites [15–17]. The -7G>A alteration in the promoter region of FKBP12 was predicted as low risk, although its create a new biding site for the NRF2 transcription factor (TF). NRF2 enhances gene expression in response to oxidative stress, which is involved in the development and progression of heart failure [18, 19]. This suggests that the -7G>A alteration could result in overexpression of FKBP12 gene. Although overexpression of FKBP12 gene has been described modulating RyR2 in rabbit myocytes , we believe that this mutation alone could not be responsible for disease development, once the concentration of calstabin1 in the heart is naturally much higher than calstabin2 and does not hinder calstabin2 interaction with RyR2 . Further studies are needed to assess the impact of the overexpression of calstabins1 in human cardiomyocytes. The c.-72C>G and c.-65C>G alterations in the exon 1 5'UTR region were predicted with none risk to be related with the disease. Overall, we did not find any alterations possibly related with the disease.
We found 7 silent SNPs with MAFs ranging between MAFs of European and African populations, which are the main founders of Brazilian population. Among the SNPs presented in the sequenced regions with allelic frequency described on dbSNP, only one was not found in our population (data not shown- rs77743612). This may be explained by its low MAF in the Yoruba population (0.014) and absence in the CEU population. Since these silent SNPs were not overrepresented in our population, it is quite improbable that they might be related with the disease.
Today, mutations in about 35 genes are described in idiopathic DCM. Together, they do not explain more than 10% of cases, indicating that more genes might be involved with the disease pathogenesis. Animal models suggested that calstabin proteins might be associated with DCM. Our result, taken together with other calstabin genetic screening efforts, leads us to believe that mutations in FKBP12 and FKBP12.6 genes are not commonly related to cardiac diseases.
Calstabin 1 and 2 are not involved in idiopathic DCM development in Brazilian patients.
Clinical and functional characteristics of IDC Cohort
12.7 ± 1.97
Total Cholesterol (mg/dL)
185 ± 55
106 ± 56
45.7 ± 17.3
116 ± 44
1.2 ± 0.3
25.08 ± 4.65
Heart Rate (pm)
76.35 ± 13.57
Diastolic Blood Pressure (mmHg)
67.41 ± 14.56
Systolic Blood Pressure (mmHg)
104.44 ± 18.9
Left Atrium Diameter, mm
47.52 ± 8.48
Aortic Diameter, mm
32.57 ± 4.26
Right Ventricle Diameter, mm
27.19 ± 6.14
Intraventricular Septum, mm
8.39 ± 1.48
Posterior Wall Thickness, mm
8.31 ± 1.21
Diastolic Diameter, mm
73.55 ± 10.06
Systolic Diameter, mm
64.01 ± 9.58
Ejection Fraction, (%)
35.08 ± 8.9
268.74 ± 77
Molecular Genetic Analyses
Primers for FKBP12 and FKBP12.6 amplification of coding exons and flanking region sequences.
5'- TCTCGGGGCTTCTGGGCTTC -3'
5'- GCGCCACTACTCACCGTCTC -3'
5'- CATGCCCGTCTCTGTCTCCTCA -3'
5'- AAGCCCCAGGCTGCCCCTGA -3'
5'- TGCTGCTGGTTTCTGACTTGT -3'
5'- TGGCATGAGGGTGAGACTTTTC -3'
5'- TTTCTTACAGAGACAGTTGGGG -3'
5'- GCCCTTCAGTATTCCATTTCCT -3'
5'- GAATGGATGGATGGAGGATG -3'
5'- TCCTAGACCCCCAAGAATCC -3'
The importance of variants out of coding regions that were not present in the control sample was analyzed in silico by FastSNP, MutationTaster  and miTarget ; concerning the transcription factor binding site, splicing and miRNA binding sites, respectively.
Data are presented as mean ± standard deviation (SD) for continuous variables and as frequencies for categorical variables. Statistical analyses were performed with SPSS software 13.0. Hardy-Weinberg equilibrium was evaluated by a Chi-square test. Allelic frequencies comparison was evaluated Fisher's exact test.
The investigation conforms to the principles outlined in the Declaration of Helsinki and the study protocol was approved by the Ethics Committee for Medical Research on Human Beings of the Hospital das Clínicas from University of São Paulo Medical School. Signed informed consent was obtained from all participants from both samples.
List of abbreviations
Central Core Disease
Catecholaminergic Polymorphic Ventricular Tachycardia
Idiopathic Dilated Cardiomyopathy
Left Ventricule Noncompaction
Minor Allelic Frequency
Single Nucleotide Polymorphism
We thank FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo for the financial support. (Grant Number: 2008/51652-0). We thank André Vaquero, Arnaldo Menezes, Camila Bonin, Débora Leite, Fernanda Sodré and Samantha Kuwada for English revision.
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