Description of a Cohort with a New Truncating MYBPC3 Variant for Hypertrophic Cardiomyopathy in Northern Spain

Background: The pathogenicity of the different genetic variants causing hypertrophic cardiomyopathy (HCM) and the genotype/phenotype correlations are difficult to assess in clinical practice, as most mutations are unique or identified in non-informative families. Pathogenic variants in the sarcomeric gene MYBPC3 inherited with an autosomal dominant pattern, whereas incomplete and age-dependent penetrance are the most common causes of HCM. Methods: We describe the clinical characteristics of a new truncating MYBPC3 variant, p.Val931Glyfs*120, in 75 subjects from 18 different families from northern Spain with the p.Val931Glyfs*120 variant. Results: Our cohort allows us to estimate the penetrance and prognosis of this variant. The penetrance of the disease increases with age, whereas 50% of males in our sample developed HCM by the age of 36 years old, and 50% of women developed the disease by the time they reached 48 years of age (p = 0.104). Men have more documented arrhythmias with potential risk of sudden death (p = 0.018), requiring implantation of cardioverter defibrillators (p = 0.024). Semi-professional/competitive sport among males is related to earlier onset of HCM (p = 0.004). Conclusions: The p.Val931Glyfs*120 truncating variant in MYBPC3 is associated with a moderate phenotype of HCM, with a high penetrance, onset in middle age, and a worse outcome in males due to higher risk of sudden death due to arrhythmias.


Introduction
Hypertrophic cardiomyopathy (HCM) is a disease prevalent in our setting (1/500 individuals) that is genetic based (generally with autosomal dominant inheritance) and inherited through families [1,2]. There are more than 1000 known variants in the genes that code for cardiac sarcomeric proteins, which alter the functionality of the cardiac sarcomere, generating an increase in muscle mass and myofibrillar disarray [3]. This leads to an obstruction in the outflow tract of the left ventricle, coronary ischemia due to narrowing of the small blood vessels, and severe arrhythmias [4], and is the main cause of sudden death of cardiac origin in children and young people. The existing treatments (β-blockers, myomectomy, ICD, transplant, etc.) are palliative [5].

Materials and Methods
The study included 108 individuals from 18 unrelated Cantabrian families with HCM and carriers of the p.Val931Glyfs*120 MYBPC3 gene variant (NM_000256.3:c.2791_2792insG).
The recruited participants were being monitored by the Pediatric Cardiology and Family Heart Disease Units of the "Marqués de Valdecilla" University Hospital.
All 18 index cases (13 males, 5 females) were recruited from the Cardiology Units of the "Marqués de Valdecilla" University Hospital, where 13 patients (72.2%; 9 males, 4 females) attended for cardiac symptoms (dyspnea, chest pain, syncope and/or palpitations); one male (5.5%) was referred after a cardiorespiratory arrest (CRA) and 4 patients (22.2%; 3 males, 1 female) were diagnosed from an ECG suspicious for HCM performed in a routine health check-up. From these cases, a pedigree was designed for each case and a genetic study was offered to 90 first-to third-degree relatives [7]. The DNA was extracted using standard peripheral blood sample protocols. Probands were studied by Next generation sequencing (NGS) using an 18/118 gene panel at the Health in Code ® laboratory in A Coruña, and family members were studied by Sanger sequencing of the variants detected in the proband patient (Supplementary Material, Figure S1). They were all duly informed. The adults signed an informed consent form, whereas the minors gave their consent verbally, with their parents/guardians signing the written consent document. The study has the approval of the Cantabria Clinical Research Ethics Committee (CEIC-Idival, Code 2018.286; 15 February 2019).
All the affected subjects and the carrying relatives are currently being monitored by the Cardiology Units of our hospital. A medical history was produced for all the carriers, and they were all given a physical examination, ECG, and echocardiogram, according to the latest HCM guidelines published by the European Society of Cardiology [18]. According to the monitoring protocols and the outcome of each patient, magnetic resonance imaging (MRI) studies were carried out.
The diagnosis of HCM based on the aforementioned Guides [18] included the following criteria: maximum thickness of the left ventricle wall ≥15 mm in adult index patients, ≥13 mm in adult relatives, or >2 SD in children in the absence of any other disease that Genes 2023, 14, 840 3 of 16 could cause left ventricular hypertrophy. The patients underwent risk stratification and were managed in accordance with the latest recommendations for this disease [19].
For the statistical analysis, a database was created (Excel 2000, Microsoft) and the statistical program IBM SPSS for PC (version 28.0.1) was used. Parametric tests were performed (Student's t-test and ANOVA) for a comparison of independent sample means where the distribution permitted (Levene's test for homogeneity of variance). Non-parametric tests were used (Mann-Whitney U-test and Kruskal-Wallis test), where the distribution did not meet the above-mentioned criterion. Independence between the two qualitative variables was tested using the chi-square test. The log-rank test was used to compare the disease-free survival between the sexes. Statistically significant differences were considered where p < 0.05.

Results
The study included 108 subjects: relatives of 18 index cases from 18 non-related first grade families, carriers of the p.Val931Glyfs*120 MYBPC3 gene variant, and classified as pathogenic following ACMG criteria [20]. We identified 75 subjects carrying the variant and 33 non-carrier relatives ( Figure 1).
The diagnosis of HCM based on the aforementioned Guides [18] included the follow ing criteria: maximum thickness of the left ventricle wall ≥15 mm in adult index patient ≥13 mm in adult relatives, or >2 SD in children in the absence of any other disease tha could cause left ventricular hypertrophy. The patients underwent risk stratification an were managed in accordance with the latest recommendations for this disease [19].
For the statistical analysis, a database was created (Excel 2000, Microsoft) and th statistical program IBM SPSS for PC (version 28.0.1) was used. Parametric tests were per formed (Student's t-test and ANOVA) for a comparison of independent sample mean where the distribution permitted (Levene's test for homogeneity of variance). Non-para metric tests were used (Mann-Whitney U-test and Kruskal-Wallis test), where the distr bution did not meet the above-mentioned criterion. Independence between the two qua itative variables was tested using the chi-square test. The log-rank test was used to com pare the disease-free survival between the sexes. Statistically significant differences wer considered where p < 0.05.

Results
The study included 108 subjects: relatives of 18 index cases from 18 non-related firs grade families, carriers of the p.Val931Glyfs*120 MYBPC3 gene variant, and classified a pathogenic following ACMG criteria [20]. We identified 75 subjects carrying the varian and 33 non-carrier relatives ( Figure 1). In 67 cases (89.3%), the p.Val931Glyfs*120 variant in MYBPC3 was identified, wherea in 8 cases (10.6%), the genetic analyses revealed an association with a second varian In 67 cases (89.3%), the p.Val931Glyfs*120 variant in MYBPC3 was identified, whereas in 8 cases (10.6%), the genetic analyses revealed an association with a second variant related to the development of HCM, in the sarcomeric genes MYL2, MYH6, or TNNT2 or in the protein-coding gene RBM20 [21,22]. The variants in the MYL2, MYH6, and RBM20 (Appendix B) genes present in 6 subjects were classified as of uncertain/unknown significance, whereas the p.Arg286His variant in the TNNT2 (Appendix B) gene present in two individuals from the same family was classified as pathogenic [23]. No significant differences were found in the incidence of HCM among carriers of one or two variants (p = 0.853). Moreover, three relatives (3.3%) inherited only the variants of uncertain significance in RBM20 and MYL2, and their cardiac phenotypes were normal.
The clinical assessment confirmed the presence of HCM in 44 of the 75 subjects carrying the p.Val931Glyfs*120 variant (58.7%), while one patient (2.2%) developed dilated cardiomyopathy (DCM) and ischemic cardiomyopathy, and was therefore excluded from the statistical analysis. Five of the patients under study died at a relatively early age (60, 62, 64, 70, and 77 years old), three of them due to heart failure as an advanced stage of HCM ( Figure 2). related to the development of HCM, in the sarcomeric genes MYL2, MYH6, or TNNT2 or in the protein-coding gene RBM20 [21,22]. The variants in the MYL2, MYH6, and RBM20 (Appendix B) genes present in 6 subjects were classified as of uncertain/unknown significance, whereas the p.Arg286His variant in the TNNT2 (Appendix B) gene present in two individuals from the same family was classified as pathogenic [23]. No significant differences were found in the incidence of HCM among carriers of one or two variants (p = 0.853). Moreover, three relatives (3.3%) inherited only the variants of uncertain significance in RBM20 and MYL2, and their cardiac phenotypes were normal.
The clinical assessment confirmed the presence of HCM in 44 of the 75 subjects carrying the p.Val931Glyfs*120 variant (58.7%), while one patient (2.2%) developed dilated cardiomyopathy (DCM) and ischemic cardiomyopathy, and was therefore excluded from the statistical analysis. Five of the patients under study died at a relatively early age (60, 62, 64, 70, and 77 years old), three of them due to heart failure as an advanced stage of HCM ( Figure 2).  In the group of non-carrier relatives, an ECG was performed on all of them and an echocardiogram on 25 of the 33 (75.7%); both studies were normal in all cases.
An ECG and echocardiogram were performed on all the carriers. In this group, in 44 patients (16 women and 28 men), the echocardiograms were consistent with HCM according to the latest Guidelines [18]. The average age of the carriers with HCM was 55 ± 17 years, whereas in the carriers with a negative phenotype, that is to say clinically asymptomatic and with a normal echocardiography, the average age was 26 ± 15 years (p < 0.001). Additionally, 45.7% of female carriers had developed the disease at the time of the study (5 years) compared with 71.8% of the male carriers (p = 0.009). No significant differences were found in the current average age of the men and women with a positive phenotype ( Table 1, Appendix A). However, there is an almost significant difference (p = 0.063) in the current average age of males and females with a negative phenotype (20 ± 14 years and 30 ± 14 years, respectively), being higher in the women who have not yet developed the disease. In the group of non-carrier relatives, an ECG was performed on all of them and an echocardiogram on 25 of the 33 (75.7%); both studies were normal in all cases.
An ECG and echocardiogram were performed on all the carriers. In this group, in 44 patients (16 women and 28 men), the echocardiograms were consistent with HCM according to the latest Guidelines [18]. The average age of the carriers with HCM was 55 ± 17 years, whereas in the carriers with a negative phenotype, that is to say clinically asymptomatic and with a normal echocardiography, the average age was 26 ± 15 years (p < 0.001). Additionally, 45.7% of female carriers had developed the disease at the time of the study (5 years) compared with 71.8% of the male carriers (p = 0.009). No significant differences were found in the current average age of the men and women with a positive phenotype (Table 1, Appendix A). However, there is an almost significant difference (p = 0.063) in the current average age of males and females with a negative phenotype (20 ± 14 years and 30 ± 14 years, respectively), being higher in the women who have not yet developed the disease. The penetrance of the disease increases with age, meaning that 50% of the men in our sample developed HCM at the approximate age of 36 years old, while 50% of the women developed the disease at the age of 48 years old. These differences are not statistically significant (p = 0.104) (Figure 3). The average age at diagnosis was 40 ± 16 years old, with the youngest patient being diagnosed at 3 years of age and the oldest at 69 years of age. We did not find any significant differences between the sexes in this regard (Table 1, Appendix A).

PEER REVIEW 7 of 17
We did not find any significant differences between the sexes in this regard (  Regarding the reason for the cardiological examination of our patients, 45.5% was due to the presence of suspected symptoms of heart disease (breathlessness, chest pain, palpitations), followed by a family history of HCM (43.2%) and, finally, asymptomatic patients, where possible signs of HCM were revealed by an ECG (11.4%). Among the men, the predominant reason was an examination due to symptoms (50%), whereas the women were generally referred to the cardiologist because they were relatives of patients with HCM (56.3%), although the difference is not significant (p = 0.382) ( Table 1; Appendix A).
In this regard, it should be noted that in 50% of the families, there were one or more cases of SCD in persons not under study (Figure 1).
In our sample of patients with HCM, we found no significant differences between sexes respecting the average thickness of the interventricular septum (IVS) (p = 0.208), nor the average thickness of the left ventricle posterior wall (LVPW) (p = 0.759), although in both cases, women had slightly lower thicknesses than males. Regarding the type of hypertrophy, asymmetric forms with septum impairment were predominant in both sexes. We did not observe any significant differences in the average thickness of the IVS in carriers with a negative phenotype or in the average thickness of the LVPW (p = 0.802 and p = 0.632, respectively). However, as was to be expected, there was a clear difference in IVS thickness between carriers with a positive and negative phenotype (p < 0.001), and the same is also true of the LVPW values (p = 0.001).
Regarding the electrocardiogram findings, three carriers with a negative phenotype (10.0%) had irregularities in their ECG that were consistent with HCM (high voltages in left precordial leads, left axis, and negative T wave in III, and/or negative T waves in left Regarding the reason for the cardiological examination of our patients, 45.5% was due to the presence of suspected symptoms of heart disease (breathlessness, chest pain, palpitations), followed by a family history of HCM (43.2%) and, finally, asymptomatic patients, where possible signs of HCM were revealed by an ECG (11.4%). Among the men, the predominant reason was an examination due to symptoms (50%), whereas the women were generally referred to the cardiologist because they were relatives of patients with HCM (56.3%), although the difference is not significant (p = 0.382) ( Table 1; Appendix A).
In this regard, it should be noted that in 50% of the families, there were one or more cases of SCD in persons not under study (Figure 1).
In our sample of patients with HCM, we found no significant differences between sexes respecting the average thickness of the interventricular septum (IVS) (p = 0.208), nor the average thickness of the left ventricle posterior wall (LVPW) (p = 0.759), although in both cases, women had slightly lower thicknesses than males. Regarding the type of hypertrophy, asymmetric forms with septum impairment were predominant in both sexes. We did not observe any significant differences in the average thickness of the IVS in carriers with a negative phenotype or in the average thickness of the LVPW (p = 0.802 and p = 0.632, respectively). However, as was to be expected, there was a clear difference in IVS thickness between carriers with a positive and negative phenotype (p < 0.001), and the same is also true of the LVPW values (p = 0.001).
Regarding the electrocardiogram findings, three carriers with a negative phenotype (10.0%) had irregularities in their ECG that were consistent with HCM (high voltages in left precordial leads, left axis, and negative T wave in III, and/or negative T waves in left precordial leads). These irregularities were evident in 37 of the subjects with HCM (84.1%), and only three relatives with an echocardiographic diagnosis of HCM had a normal ECG (6.8%). Therefore, there are significant differences between carriers with a developed phenotype and those with a negative phenotype in the ECG (p < 0.001).
A total of 54.5% of the patients with a positive phenotype had symptoms, the most common of which was breathlessness. No significant differences were recorded between the sexes in this regard (p = 0.647). Most of the patients were in the NYHA class I-II (75%), without any significant differences between the sexes (p = 0.897).
A total of 25% of patients of both sexes developed hypertrophic obstructive cardiomyopathy (HOCM); 15.9% showed signs of systolic dysfunction, and 75% diastolic dysfunction in the echocardiogram. Both are more frequent in males, but the differences are not significant (Table 1, Appendix A).
If the left atrium is dilated when the diameter is ≥45 mm [24], we observed that 50% of patients had dilatation of the left atrium. Consequently, 28.6% of the sick males and 43.8% of the sick females (p = 0.307) had atrial fibrillation (AF).
Moreover, although more than 60% of patients of both sexes had arrhythmias, the episodes of non-sustained ventricular tachycardia (NSVT) detected in Holter monitoring were significantly more common in men (p = 0.018).
A cardiac MRI was performed on 63.6% of the patients diagnosed with HCM, all being consistent with the disease; 70.6% of the sick men showed signs of fibrosis, compared with 36.4% of the sick women (p = 0.074).
On the other hand, 53.6% of the sick men had an ICD compared with 18.8% of the sick women (p = 0.024). In most cases, an ICD was recommended for primary prevention and only two patients (11.1%) received appropriate shocks.
A total of 61.4% of patients were receiving drug treatment. Two patients required a heart transplant, three a Morrow myomectomy, and one an alcohol septal ablation.
Nine patients suffered events, with no significant differences detected between the sexes (p = 0.394). Three patients died from heart failure and another two from causes unrelated to this disease.
A total of 25% of patients played sport at semi-professional/competitive levels prior to diagnosis, compared with 10.5% of the carriers who still have a negative phenotype (p = 0.870). Regarding the men, 39.3% of the patients played sport at this level, compared with 45.5% of the carriers with a negative phenotype (p = 0.165). Moreover, the average onset age of the sick men who played high-level sport was 29 ± 8 years, compared with those who did not play sport, with an average onset age of 45 ± 11 years, resulting in a statistically significant difference (p < 0.001). None of the sick women previously played sport at this level, and only two who still have a negative phenotype play sport.

Discussion
As far as we are aware, this is the first study to describe the clinical phenotype of the p.Val931Glyfs*120 MYBPC3 gene variant, demonstrating co-segregation in 18 not-related families affected by HCM.
This variant was first referred to in Gómez et al. (2014) as the cause of hypertrophic cardiomyopathy [25]. However, this is the first study to describe the genotype-phenotype of a cohort for this variant.
The pathogenic mechanism of the p.Val931Glyfs* variant would be by truncation, given that the insertion of a guanine would produce a change in the translation of a valine by a glycine at position 931, with a change in the reading frame that would generate a premature stop codon 120 amino acids downstream. Aberrant translations could be degraded by the cellular machinery without being translated, or could do so, giving rise to non-functional truncated peptides [26]. The purpose of the MYBPC3 protein is to anchor the myosin to the sarcomere A-band, providing stability during muscle contraction; the disease would therefore occur through haploinsufficiency [27] (Figure 4).  As described in most series with truncating MYBPC3 variants, the penetrance for variant increases with age (p < 0.001) [26,28]. In our cohort, there are no significant diff ences between the sexes in terms of the disease onset age or the penetrance (Figure 3). T is in line with the results of other Spanish and international studies carried out in la populations of patients with HCM [29][30][31]. However, other published cohorts have fou significant differences in the onset age, with men being younger at diagnosis [10,31, We postulate a possible protective role of endocrine factors in women [10] or a lack attention to early signs of disease in women and the absence of any special recommen tion for screening programs [32]. In fact, we found an almost significant difference 0.063) in the current average age of males and women with a negative phenotype, w women who are yet to develop the disease being older. In our case, the lack of a big difference between the sexes could be since we carried out an active search for relat carrying the disease, both with symptoms and asymptomatic, irrespective of their sex The predominant clinical presentation in our population is moderate HCM with s tal predominance, limited obstruction, but a high percentage of arrhythmias. These ch acteristics are like those described for other variants in the MYBPC3 gene that lead to tr cation of the protein [12,17,28,33].
One of the carrying relatives developed a phenotype consistent with dilated card myopathy. It is possible that they may be in an advanced phase of hypertrophic card myopathy, where dilatation and systolic dysfunction are predominant, or that this is initial manifestation. These findings had already been previously described in other a cles as the final evolving forms of HCM [12,34].
Mention should be made of the fact that in the cardiac MRI of the men with HC more fibrosis was observed than in women. This would explain the higher number of isodes of NSVT in the Holter monitoring of the men. We consider this one of the m As described in most series with truncating MYBPC3 variants, the penetrance for this variant increases with age (p < 0.001) [26,28]. In our cohort, there are no significant differences between the sexes in terms of the disease onset age or the penetrance (Figure 3). This is in line with the results of other Spanish and international studies carried out in large populations of patients with HCM [29][30][31]. However, other published cohorts have found significant differences in the onset age, with men being younger at diagnosis [10,31,32]. We postulate a possible protective role of endocrine factors in women [10] or a lack of attention to early signs of disease in women and the absence of any special recommendation for screening programs [32]. In fact, we found an almost significant difference (p = 0.063) in the current average age of males and women with a negative phenotype, with women who are yet to develop the disease being older. In our case, the lack of a bigger difference between the sexes could be since we carried out an active search for relatives carrying the disease, both with symptoms and asymptomatic, irrespective of their sex.
The predominant clinical presentation in our population is moderate HCM with septal predominance, limited obstruction, but a high percentage of arrhythmias. These characteristics are like those described for other variants in the MYBPC3 gene that lead to truncation of the protein [12,17,28,33].
One of the carrying relatives developed a phenotype consistent with dilated cardiomyopathy. It is possible that they may be in an advanced phase of hypertrophic cardiomyopathy, where dilatation and systolic dysfunction are predominant, or that this is the initial manifestation. These findings had already been previously described in other articles as the final evolving forms of HCM [12,34].
Mention should be made of the fact that in the cardiac MRI of the men with HCM, more fibrosis was observed than in women. This would explain the higher number of episodes of NSVT in the Holter monitoring of the men. We consider this one of the main prognostic differences between the sexes. In fact, these findings would be in line with the fact that the percentage of male patients who have an ICD in our sample is much higher than that of women.
In our cohort, there are no significant differences regarding symptoms by sex. However, in the study by Sabater et al. [28], female patients had more breathlessness and chest pain. This could be because in our study, younger people have been diagnosed in earlier phases of the disease.
As regards the advice given to young male carriers on lifestyle and sport, we find it interesting to note that there is a significant difference between the age of onset of patients who practice any sport at a semi-professional/competitive level and those who do not, being the lowest age in the first group. The practice of intense exercise on a regular basis generates certain physiological changes in the heart in "healthy" subjects, including an increase in the mass and volume of the left ventricle [35]. Pathological studies of hearts with HCM show increased left ventricular muscle mass at the expense of hypertrophy of cardiac myocytes, with decreased ventricular volume, as well as myofibrillar disorganization and coronary small vessel disease [18]. It has been shown that intense exercise increases the gradient through the left ventricular outflow tract (the obstruction increases), and shortens diastolic filling time; it can cause cardiac ischemia due to a mismatch between demand and supply and small vessel disease, and increases the risk of atrial and ventricular arrhythmias due to sympathetic activation [36]. For this reason, it has traditionally been considered that competitive sport was a factor which could accelerate the advance of HCM in genetically predisposed subjects and even contribute to the development of malignant arrhythmias. The Guidelines, in the past, were very restrictive in respect of the practice of competitive sport [37,38].
More recent studies, in both transgenic mice and in humans, have not only questioned this but also observed a better outcome in patients who play sport, with less cardiac wall hypertrophy and less systolic dysfunction in the outcome [39][40][41].
Taking a detailed three-generation family history, we were able to estimate penetrance for this familial condition and estimate the impact of some aspects as physical activity. Although we did not observe a statistically significant difference, according to the long-rank graph, it is remarkable that at the age of 50 years, 25% of men remain with a negative phenotype, while at the same age, 46% of women remain with a normal echocardiography. The estimate of penetrance has some limitations. The data were generated assuming a simple copula model, and actual familial disease data may exhibit more complex patterns of residual disease correlation so they can be extrapolated to the general population.
Our cohort shared both variants and environment, thus some interactions between them can be assessed. According to our study, the p.Val931Glyfs*120 MYBPC3 variant is clearly pathogenic, with a strong founder effect in the northern Spanish region of Cantabria, where all the families are from. In addition, the surnames of several affected families are of very specific origin (Pasiego origin). The Pasiegos Valleys have traditionally been considered a "closed population with strong endogamy" [42]. This possible endogamy has been proven in numerous studies analyzing the polymorphic genetic markers of both the HLA system and the mitochondrial DNA or the Y chromosome. Moreover, Cardoso et al. [43] observed a low diversity of female lineages through an analysis of the mitochondrial DNA of a group of individuals of Pasiego origin with no maternal lineage. The founder effect would reduce the amount of existing genetic variability within our population, and as a result of this, certain phenotypes or particular genes might be highlighted in this population, such as the p.Val931Glyfs*120 MYBPC3 variant.

Limitations
As is the case in most studies of genotype-phenotype correlation in HCM, our study is also limited by the small size of the families and the scarcity of families with identical variants [15,44]. In addition, in our cohort, some families present some other mutation in sarcomeric genes or genes related to cardiomyopathy, which could produce a synergy effect with the variant under study. Furthermore, the penetrance data is only applicable to our sample, since the variant frequency has not been tested in a local population. On the other hand, our study is both retrospective and prospective, with a follow-up time from its beginning of 5 years. Future studies with larger cohorts will therefore be necessary to help to clarify the genotype-phenotype relations for this pathogenic variant.

Conclusions
According to the results of our cohort, the pathogenic variant Val931Glyfs*120 has a phenotype pattern like that described for other MYBPC3 gene variants with a more advanced onset age, a more benign clinical course, and a lower incidence of SCD in comparison with variants affecting the MYH7 gene. There is a clear difference between men and women in terms of the presence of NSVT, which means a higher theoretical risk of SCD. Moreover, an earlier onset in men who do exercise at the semi-professional/competitive level was observed. Our group believes that it is particularly important to give these young men appropriate advice and to maintain a high degree of vigilance of arrhythmic events by carrying out regular Holter monitoring.  Table 1 separated by gender a ences expressed with p value. Simple bar graphs represent percentage of patients.
Appendix B. Table of Additional Genetic Variants Mentioned   Table A1. Additional genetic variants identified in the studied cohort, within the tributed clinical significance and estimated frequency in general population acco the literature [23,45,46].  Figure A1. Graphic Representation of the Data Included in Table 1 separated by gender and differences expressed with p value. Simple bar graphs represent percentage of patients.