J Pediatr Endocrinol Metab 2016; 29(1): 55–61
Emil Poláka,*, Eva Vitáriušováa, Peter Celec, Zuzana Pribilincová, Ľudmila Košťálová, Anna Hlavatá, László Kovács and Ľudevít Kádaši
The prevalence of melanocortin-4 receptor gene mutations in Slovak obese children and adolescents DOI 10.1515/jpem-2015-0015 Received January 12, 2015; accepted April 24, 2015; previously published online August 5, 2015
Abstract: Melanocortin-4 receptor (MC4R) deficiency is the most frequent monogenic form of obesity. The contribution of MC4R mutations to the Slovak population has not been investigated as yet. We screened the coding sequence of the MC4R gene in a cohort of 210 Slovak obese children and adolescents. We identified four different mutations in four patients, giving a mutation detection rate of 0.95%. Of these, three were missense mutations previously identified and characterized by other research groups (p.R7C, p.S127L and p. R305W, respectively). One was a novel nonsense mutation p.W174* detected in a severely obese 7-year-old boy. This mutation was further analyzed in family segregation analysis and exhibited variable penetrance. Two known amino acid polymorphisms (p.V103I and p.I251L) were also identified in seven subjects of our cohort group. We also performed multifactorial statistical analysis to determine the influence of genotypes on
a Emil Polák and Eva Vitáriušová: These authors contributed to this work equally. *Corresponding author: Emil Polák, Faculty of Natural Sciences, Department of Molecular Biology, Comenius University, Mlynska dolina B2-210, 842 15 Bratislava, Slovak Republic, Phone: +421260296653, Fax: +421260296508, E-mail: [email protected]; and Institute of Molecular Physiology and Genetics, Slovak Academy of Science, Bratislava, Slovakia Eva Vitáriušová, Zuzana Pribilincová, Ľudmila Košťálová, Anna Hlavatá and László Kovács: Faculty of Medicine, 2nd Department of Paediatrics, Comenius University, University Children’s Hospital, Bratislava, Slovakia Peter Celec: Faculty of Natural Sciences, Department of Molecular Biology, Comenius University, Bratislava, Slovakia; Institute of Molecular Biomedicine, Comenius University, Bratislava, Slovakia; and Center for Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia Ľudevít Kádaši: Faculty of Natural Sciences, Department of Molecular Biology, Comenius University, Bratislava, Slovakia; and Center for Molecular Medicine, Slovak Academy of Science, Bratislava, Slovakia
standard biochemical blood markers. No significant influence was observed in carriers of DNA variants on tested parameters. We conclude that rare heterozygous MC4R mutations contribute to the onset of obesity only in a few cases in the Slovak population. Keywords: MC4R; melanocortin-4 receptor; mutation analysis; obesity; polymorphisms.
Introduction Obesity is the population disease of the 21st century. With its rapidly increasing incidence in children, it has become one of the main health concerns of the modern world (1), although its incidence has already stabilized or has a decreasing tendency in some pediatric populations (2, 3). Obesity is a multifactorial disease resulting from the interplay of several genetic loci and the environment (4). To date, eight genes are known to be related to the monogenic forms of obesity (5–11). Of these, the melanocortin-4 receptor (MC4R) deficiency is arguably the most frequent monogenic form encountered and the best understood (4). MC4R is a single exon gene mapped to chromosome 18q22 encoding a 332-residue-long protein, and the melanocortin-4 receptor is a G-coupled seven-transmembrane receptor predominantly expressed in the hypothalamus (12). Receptor activation is mediated via its natural agonist alpha-melano-stimulating hormone (α-MSH). This is a cleaveage product of pro-opiomelanocortin (POMC) expressed following adipocyte leptine secretion. Activation of MC4R leads to an anorexigenic state, where energy intake is decreased and expenditure increased (13). Thus, MC4R is the key element in the melanocortinergic pathway, and consequently in human energy homeostasis. The influence of MC4R on body weight regulation was initially demonstrated on rodents, where homozygous MC4R-deficient mice exhibited obesity, hyperphagia, hyperinsulinemia and increased linear growth. Mice with only one copy of the nonfunctional receptor had
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56 Polák et al.: The prevalence of melanocortin-4 receptor gene mutations in Slovak obese children an intermediate average body-weight increase, indicating haplo-insufficiency and co-dominant inheritance (14). However, a more complex phenotype is observed in humans. Several studies report that mutations altering the function, ligand-binding or cell-surface expression of MC4R in vitro reveal variable penetrance and expressivity in family segregation studies (15–18). Furthermore, the current obesogenic environment contributes more significantly to obesity onset compared to conditions that existed for previous generations (16), and the functional mutation impact here also influences disease penetrance (19). Interestingly, two MC4R variants (p.V103I and p.I251L) have been negatively correlated with the risk of obesity (20, 21). These non-synonymous amino acid polymorphisms are considered obesity-protective. Van der Berg and colleagues (2011) generated a list of MC4R variants, which is available online at http:// www.LOVD.nl/MC4R (18). To date, more than 200 different MC4R variants have been identified and listed in this online database. The primary aims of our study are to screen the MC4R coding region in Slovak obese children and adolescents, to asses the frequencies of identified variants and to compare obtained results with other European studies. Furthermore, we performed multifactorial statistical analysis to measure the significance of differences between selected biochemical blood markers together with standard deviation score of body mass index (SDS BMI) and tested variables such as age, sex and the genotypes. Herein, we present our findings in a cohort of 210 subjects.
Materials and methods Subjects for this study were recruited with the cooperation of the Second Department of Paediatrics in the Children’s Faculty Hospital in Bratislava and from local Slovak endocrinological outpatient departments. Following written informed consent from their legal representatives consistent with the Helsinki declaration, 210 unrelated Slovak obese patients (113 boys and 97 girls) each provided a blood sample for research purposes. Body weight and height were recorded for this group of children and adolescents, SDS BMI using Slovak national anthropometric references (22) was counted, and subsequent biochemical protocols assessed their glucose, triglycerides, total cholesterol and high density lipoprotein (HDL) cholesterol blood levels. The glucose blood level was quantified using the enzymatic hexokinase method, total and HDL cholesterol using the enzymatic colorimetric method and the levels of triglycerides were quantified using the glycerol phosphate oxidase and 4-aminophenazone enzymatic method. Reference ranges of glucose, triglycerides and HDL cholesterol levels were estimated according to Zimmet et al. (23). Total cholesterol
levels > 5.0 mmol/L were considered elevated (24). All blood tests were performed after 12-h fasting. Obesity onset in all subjects was before the 11th year of life; with organic and syndromic obesity causes excluded. The cohort group characteristics are summarized in Table 1. A control group of 110 adult Caucasian donors was also analyzed because a control group of healthy children and adolescents was not at our disposal. The control DNA samples were used for comparison, and they served to a certain extent as a defined control group. Furthermore, three family members of one obese patient were also analyzed in a family segregation analysis. The study was approved by the local Ethics Committee at the Children’s Faculty Hospital in Bratislava, Slovakia.
DNA isolation and MC4R screening Three-mL samples of peripheral blood were drawn for molecular analysis and diluted in ethylenediaminetetraacetic acid (EDTA). Genomic DNA was extracted using the Gentra Puregene Blood Kit (QIAGEN, Düsseldorf, Germany) under the manufacturer’s instructions. The entire coding region of the MC4R gene was polymerase chain reaction (PCR) amplified by MC4R-F (5′-ATCAATTCAGGGGGACACTG-3′) and MC4R-R (5′-TGCATGTTCCTATATTGCGTG-3′) primers from a
Table 1: Phenotype characteristics of Slovak children and adolescents cohort group. Parameters
Obese subjects
N Gender
210 Male 54% Female 46% Mean 11.45±4.16 Min 1 Max 19 Mean 1.53±0.21 Max 1.92 Min 0.76 Mean 79.19±30.43 Min 13.6 Max 190 Mean 4.86±1.7 Min 1.98 Max 16.54 Mean 4.55±0.6 Min 2.9 Max 6.67 Mean 4.26±0.81 Min 1.7 Max 8.87 Mean 1.09±0.23 Min 0.6 Max 1.74 Mean 1.48±0.73 Min 0.31 Max 4.12
Age, years
Height, m
Weight, kg
SDS BMI
Fasting blood glucose, mmol/L
Total cholesterol, mmol/L
HDL cholesterol, mmol/L
Triglycerides, mmol/L
Min, minimum; Max, maximum; ± s.d., standard deviation; HDL, high-density lipoprotein.
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Polák et al.: The prevalence of melanocortin-4 receptor gene mutations in Slovak obese children 57
previous study (25), using Eppendorf Mastercycler® pro S (Eppendorf, Hamburg, Germany) in 20 μl volumes. Each PCR product was subsequently purified under the standard protocol and sequenced using PCR primers MC4R-F, MC4R-R and MC4R-CF (5′-TGTAGCTCCTTGCTTGCATC-3′) and MC4R-CR (5′-GGCCATCAGGAACATGTGGA-3′) internal primers as previously described (26). Sequencing was performed using the Big Dye 3.1 terminator sequencing kit (Applied Biosystems/ Life Technologies, Foster City, USA) and the ABI 3100 genetic analyzer (Applied Biosystems/Life Technologies, Foster City, USA) according to the manufacturer’s instructions. Sequencing data was analyzed with Sequencing Analysis, version 5.2 (Applied Biosystems/Life Technologies, USA) and Chromas Pro software, version 1.7.1 (Technelysium, Brisbane, Australia). The obtained sequences were compared with the wild-type sequence of the MC4R gene (ENSG00000166603; www.ensembl.org).
Multifactorial statistical analysis Multifactorial analysis was conducted using multivariate analysis of variance (MANOVA) in Statistical Package for the Social Sciences (SPSS) (IBM, New York, USA). Dependent variables included SDS BMI, fasting blood glucose, total cholesterol, HDL cholesterol and triglycerides levels. Tested independent variables included age, sex and the genotypes, and p-values T c.380C > T c.522G > A c.913C > T c.751A > C c.307G > A
0.23% 0.23% 0.23% 0.23% 0.23% 1.66%
Missense Missense Nonsense Missense Missense Missense
Functional characterization
References
Decreased activity Decreased activity ND Decreased activity Benign/protective Benign/protective
(17) (17) This study (27) (20) (20)
ND, not determined.
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58 Polák et al.: The prevalence of melanocortin-4 receptor gene mutations in Slovak obese children Table 3: Genotypes and phenotype characteristics of variants’ carriers. Patient 1 2 3 4 5 6 7 8 9 10 11
Genotype p.R7C/− p.S127L/p.V103Ia p.R305W/− p.W174*/− p.V103I/− p.V103I/− p.V103I/− p.V103I/− p.V103I/− p.V103I/− p.I251L/−
Gender F F F M M M F F F F F
Age
SDS BMI
Fasting blood glucose, mmol/L
Total cholesterol, mmol/L
HDL cholesterol, mmol/L
Triglycerides, mmol/L
15.8 9.5 2.5 4 3.8 6.5 5.9 6.5 10 12 14
6.89 3.45 4.48 4.49 5.19 2.16 4.07 3.37 3.79 6.91 10.84
5.6 4.7 4.1 4.37 4.1 4.3 5.1 4.6 4.3 3 6
4.43 3.21 3.67 4.66 3.63 4.64 3.84 5.94 4 3.63 4.31
1.01 1.08 1.54 1.14 1.3 1.63 1.72 1.25 1.13 1.09 0.8
1.38 0.8 0.57 1.3 0.66 0.89 1.06 1.7 1.4 1.08 3.07
All parameters refer to the time of first consultation; HDL, high-density lipoprotein; F, female; M, male. Physiological reference values of tested biochemical parameters according to Zimmet et al. (23) and Gahagan et al. (24). Fasting blood glucose ≤ 5.6 mmol/L; total cholesterol 1.03 mmol/L; triglycerides ≤ 1.7 mmol/L. aTrans conformation of p.V103I was not confirmed by molecular analysis in parents.
ND
p.W174*
ND
p.W174*
p.V103I
p.W174*
Figure 1: Segregation of novel nonsense mutation p.W174* in family segregation analysis. Circles represent females; squares represent males. Black symbols represent obese individuals; gray symbols represent overweight individuals; and white symbols represent normal weight individuals. ND, genotype is not determined. Phenotype of relatives was based on verbal reports by the parents of the patient.
Discussion This is the first study in Slovak obese children and adolescents where the coding region of the MC4R gene has been analyzed. The aim of this work was to estimate the prevalence of MC4R variants in 210 Slovak obese patients and evaluate their phenotypes. We identified four different mutations and two non-synonymous obesity-protective variants in our cohort, giving a mutation detection rate of 0.95%. This is in concordance with the results of some other studies (28–30). However, the mutation prevalences in our cohort group have a significantly lower figure than
the estimated MC4R mutation frequencies in our neighboring Czech population and also in the European populations (15, 16). Obesity is generally considered a polygenic trait resulting from the interaction of several genetic loci with concurrent environmental factors (4), therefore many other genetic influences other than MC4R mutations and accompanying non-genetic factors may have contributed to the obesity manifestation in our cohort group. However, it is also possible that some of the analyzed patients are carriers of undetected MC4R promoter mutations as described by two previous authors groups (18, 31). Further genetic background may also be determined in the 3′ end of the MC4R gene, as has been shown in several genome wide association studies (GWAS), where two single nucleotide polymorphisms (SNPs) downstream of the MC4R gene were remarkably associated with obesity (32, 33). Recently, a high prevalence of homozygosity and heterozygosity for rs17782313 variant has been observed in the Greek population (34). In addition to the considerations mentioned above, a larger cohort sample could reveal some additional MC4R mutations. Stricter health criteria of cohort evaluation with regard to earlier obesity onset may be warranted. However, present concurrent hyperphagia and hyperinsulinemia may be irrelevant because these characteristics are not always present in all mutation carriers and they are dependent on mutation functionality (15, 18, 27). Thus, it is difficult to assess a reliable clinical setting for MC4R haplo-insufficient patients in the current obesogenic environment. Mutation p.R7C is located in the N-terminal extracellular domain of the MC4R, and this impairs the receptor α-MSH activation and decreases its constitutive activity
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Polák et al.: The prevalence of melanocortin-4 receptor gene mutations in Slovak obese children 59
2
4.5
1.8
4
1.6
3.5
1.4
3
TAG
HDL
1.2 1 0.8
2 1.5
0.6
1
0.4
0.5
0.2 0
2.5
0
5
10 Age
15
20
0
0
5
10 Age
15
20
Figure 2: Influence of age on triglycerides and HDL cholesterol blood levels in Slovak children and adolescents. HDL, high-density lipoprotein; TAG, triglycerides.
while cell surface expression remains unchanged (17, 27). The phenotypic outcome here should lead to excessive food intake. We identified this missense mutation in one morbidly obese 16-year-old girl with the onset of phenotypic manifestation from the age of 3 years (phenotype characteristics of all mutation carriers are summarized in Table 3). Unfortunately, we were unable to perform family segregation analysis for this allele. However, in the Czech cohort p.R7C cosegregated with obesity or overweight in all except one family member (15). Therefore, we estimate, that this mutation is most likely to be responsible for this patient’s clinical outcome, albeit with possible variable penetrance. Mutation p.S127L affects the transmembrane domain 3 and also shows in vitro alternations in α-MSH receptor activation (17, 27, 35). However, it exhibited a wide range of phenotypic manifestations in population analysis and obese subjects analyses (17), with its presence. It was detected in lean subjects as well as in severely obese patients (26, 29). This is likely due to variable penetrance and mutation expressivity. The carrier of this allele here was a 10-year-old obese girl demonstrating obesity onset from the age of 8 years. She was also a carrier of the heterozygous obesity-protective p.V103I variant (20, 36), which slightly improves receptor function by increasing agonist potency and decreasing inverse agonist potency (37). Interestingly, Hainerova et al. (15) reported that a patient who was also a carrier of p.S127L mutation and p.V103I underwent a 3-week weight reduction regimen and succeeded to lose more body weight compared to 21 non-carriers who underwent the same 3-week weight intervention program (15). It remains a matter of speculation whether the p.V103I variant could have influenced this finding or if it has a moderating effect on our patients’ phenotype.
Substitution of p.R305W in the C-terminal MC4R domain causes decreased cell surface expression and ligand-binding and also decreases basal metabolic activity, as reported and functionally characterized in one severely obese patient in a study by Lubrano-Berthelier and colleagues (27). The carrier of this mutation in our study was a 2.5-year-old girl who had obesity onset at 1 year of age. This patient’s phenotype supports findings that the majority of mutations altering cell surface expression lead to very early onset of this disease (38). We identified one novel nonsense mutation leading to a premature stop codon at position W174 in a morbidly obese 7-year-old boy with obesity onset at 4 years. This patient did not exhibit binge eating habits and did not have impaired glucose tolerance. Segregation analysis was performed in two generations to further examine this variant (Figure 1). Although nonsense MC4R mutations lead to truncated protein forms with lack of activity and cell surface expression (39), severe obesity due to MC4R haplo-insufficiency can be expected. Interestingly, our proband’s mother and grandmother who carried this mutation had a history of childhood obesity, but although slightly overweight they were not currently obese. A similar finding was reported in a case study, although regarding a different mutation (p.R165W) (40). These observations support a possible age-decrement in obesity (19, 41) and also that the current obesogenic environment could encourage an individual effect. We assessed standard biochemical markers in each subject of our cohort group commonly appendant in obesity to observe any differences between these parameters in mutation carriers compared to non-carriers and in carriers of obesity-protective variants compared to non-carriers. We did not observe any statistically
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60 Polák et al.: The prevalence of melanocortin-4 receptor gene mutations in Slovak obese children significant influence on tested parameters in variant carriers. However, it is noteworthy that p.I251L and p.V103I variant carriers had significantly higher HDL cholesterol levels compared to non-carriers in a recent German study (30). Nevertheless, we observed significant correlation between age and triacylglycerol levels and age and HDL cholesterol levels (Figure 2). In summary, MC4R mutations were detected as a cause of obesity in 1.9% of Slovak obese children and adolescents. We identified three missense mutations and one novel nonsense mutation, which was further studied in family segregation analysis. Our findings support variable penetrance of the MC4R gene variations and also agree with an age-dependent manifestation manner. As in similar studies, not all of the mutation carriers fulfilled the typical symptoms of MC4R deficiency regarding early onset of obesity, hyperphagia, increased linear growth and hyperinsulinemia. Acknowledgments: We would like to thank all the children and their families for their participation in this study. We are also grateful to pediatric endocrinologists Mária Kúseková MD†, Ľubica Tichá MD, Mária Ševecová MD, Adriana Dankovčíková MD, Marianna Debreová MD and Vilja Šandriková MD for their assistance in this study. This contribution is the result of projects implemented in the grants “Diagnostics of Socially Important Disorders in Slovakia, Based on Modern Biotechnologies” ITMS 26240220058 and “Creating Competitive Centre for research and development in the field of molecular medicine” ITMS 26240220071, supported by the Research and Developmental Operational Program funded by the ERDF. This work was also supported by VEGA 1/0497/08. Conflict of interest: The authors declare no conflict of interest.
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Emil Poláka,*, Eva Vitáriušováa, Peter Celec, Zuzana Pribilincová, Ľudmila Košťálová, Anna Hlavatá, László Kovács and Ľudevít Kádaši
The prevalence of melanocortin-4 receptor gene mutations in Slovak obese children and adolescents DOI 10.1515/jpem-2015-0015 Received January 12, 2015; accepted April 24, 2015; previously published online August 5, 2015
Abstract: Melanocortin-4 receptor (MC4R) deficiency is the most frequent monogenic form of obesity. The contribution of MC4R mutations to the Slovak population has not been investigated as yet. We screened the coding sequence of the MC4R gene in a cohort of 210 Slovak obese children and adolescents. We identified four different mutations in four patients, giving a mutation detection rate of 0.95%. Of these, three were missense mutations previously identified and characterized by other research groups (p.R7C, p.S127L and p. R305W, respectively). One was a novel nonsense mutation p.W174* detected in a severely obese 7-year-old boy. This mutation was further analyzed in family segregation analysis and exhibited variable penetrance. Two known amino acid polymorphisms (p.V103I and p.I251L) were also identified in seven subjects of our cohort group. We also performed multifactorial statistical analysis to determine the influence of genotypes on
a Emil Polák and Eva Vitáriušová: These authors contributed to this work equally. *Corresponding author: Emil Polák, Faculty of Natural Sciences, Department of Molecular Biology, Comenius University, Mlynska dolina B2-210, 842 15 Bratislava, Slovak Republic, Phone: +421260296653, Fax: +421260296508, E-mail: [email protected]; and Institute of Molecular Physiology and Genetics, Slovak Academy of Science, Bratislava, Slovakia Eva Vitáriušová, Zuzana Pribilincová, Ľudmila Košťálová, Anna Hlavatá and László Kovács: Faculty of Medicine, 2nd Department of Paediatrics, Comenius University, University Children’s Hospital, Bratislava, Slovakia Peter Celec: Faculty of Natural Sciences, Department of Molecular Biology, Comenius University, Bratislava, Slovakia; Institute of Molecular Biomedicine, Comenius University, Bratislava, Slovakia; and Center for Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia Ľudevít Kádaši: Faculty of Natural Sciences, Department of Molecular Biology, Comenius University, Bratislava, Slovakia; and Center for Molecular Medicine, Slovak Academy of Science, Bratislava, Slovakia
standard biochemical blood markers. No significant influence was observed in carriers of DNA variants on tested parameters. We conclude that rare heterozygous MC4R mutations contribute to the onset of obesity only in a few cases in the Slovak population. Keywords: MC4R; melanocortin-4 receptor; mutation analysis; obesity; polymorphisms.
Introduction Obesity is the population disease of the 21st century. With its rapidly increasing incidence in children, it has become one of the main health concerns of the modern world (1), although its incidence has already stabilized or has a decreasing tendency in some pediatric populations (2, 3). Obesity is a multifactorial disease resulting from the interplay of several genetic loci and the environment (4). To date, eight genes are known to be related to the monogenic forms of obesity (5–11). Of these, the melanocortin-4 receptor (MC4R) deficiency is arguably the most frequent monogenic form encountered and the best understood (4). MC4R is a single exon gene mapped to chromosome 18q22 encoding a 332-residue-long protein, and the melanocortin-4 receptor is a G-coupled seven-transmembrane receptor predominantly expressed in the hypothalamus (12). Receptor activation is mediated via its natural agonist alpha-melano-stimulating hormone (α-MSH). This is a cleaveage product of pro-opiomelanocortin (POMC) expressed following adipocyte leptine secretion. Activation of MC4R leads to an anorexigenic state, where energy intake is decreased and expenditure increased (13). Thus, MC4R is the key element in the melanocortinergic pathway, and consequently in human energy homeostasis. The influence of MC4R on body weight regulation was initially demonstrated on rodents, where homozygous MC4R-deficient mice exhibited obesity, hyperphagia, hyperinsulinemia and increased linear growth. Mice with only one copy of the nonfunctional receptor had
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56 Polák et al.: The prevalence of melanocortin-4 receptor gene mutations in Slovak obese children an intermediate average body-weight increase, indicating haplo-insufficiency and co-dominant inheritance (14). However, a more complex phenotype is observed in humans. Several studies report that mutations altering the function, ligand-binding or cell-surface expression of MC4R in vitro reveal variable penetrance and expressivity in family segregation studies (15–18). Furthermore, the current obesogenic environment contributes more significantly to obesity onset compared to conditions that existed for previous generations (16), and the functional mutation impact here also influences disease penetrance (19). Interestingly, two MC4R variants (p.V103I and p.I251L) have been negatively correlated with the risk of obesity (20, 21). These non-synonymous amino acid polymorphisms are considered obesity-protective. Van der Berg and colleagues (2011) generated a list of MC4R variants, which is available online at http:// www.LOVD.nl/MC4R (18). To date, more than 200 different MC4R variants have been identified and listed in this online database. The primary aims of our study are to screen the MC4R coding region in Slovak obese children and adolescents, to asses the frequencies of identified variants and to compare obtained results with other European studies. Furthermore, we performed multifactorial statistical analysis to measure the significance of differences between selected biochemical blood markers together with standard deviation score of body mass index (SDS BMI) and tested variables such as age, sex and the genotypes. Herein, we present our findings in a cohort of 210 subjects.
Materials and methods Subjects for this study were recruited with the cooperation of the Second Department of Paediatrics in the Children’s Faculty Hospital in Bratislava and from local Slovak endocrinological outpatient departments. Following written informed consent from their legal representatives consistent with the Helsinki declaration, 210 unrelated Slovak obese patients (113 boys and 97 girls) each provided a blood sample for research purposes. Body weight and height were recorded for this group of children and adolescents, SDS BMI using Slovak national anthropometric references (22) was counted, and subsequent biochemical protocols assessed their glucose, triglycerides, total cholesterol and high density lipoprotein (HDL) cholesterol blood levels. The glucose blood level was quantified using the enzymatic hexokinase method, total and HDL cholesterol using the enzymatic colorimetric method and the levels of triglycerides were quantified using the glycerol phosphate oxidase and 4-aminophenazone enzymatic method. Reference ranges of glucose, triglycerides and HDL cholesterol levels were estimated according to Zimmet et al. (23). Total cholesterol
levels > 5.0 mmol/L were considered elevated (24). All blood tests were performed after 12-h fasting. Obesity onset in all subjects was before the 11th year of life; with organic and syndromic obesity causes excluded. The cohort group characteristics are summarized in Table 1. A control group of 110 adult Caucasian donors was also analyzed because a control group of healthy children and adolescents was not at our disposal. The control DNA samples were used for comparison, and they served to a certain extent as a defined control group. Furthermore, three family members of one obese patient were also analyzed in a family segregation analysis. The study was approved by the local Ethics Committee at the Children’s Faculty Hospital in Bratislava, Slovakia.
DNA isolation and MC4R screening Three-mL samples of peripheral blood were drawn for molecular analysis and diluted in ethylenediaminetetraacetic acid (EDTA). Genomic DNA was extracted using the Gentra Puregene Blood Kit (QIAGEN, Düsseldorf, Germany) under the manufacturer’s instructions. The entire coding region of the MC4R gene was polymerase chain reaction (PCR) amplified by MC4R-F (5′-ATCAATTCAGGGGGACACTG-3′) and MC4R-R (5′-TGCATGTTCCTATATTGCGTG-3′) primers from a
Table 1: Phenotype characteristics of Slovak children and adolescents cohort group. Parameters
Obese subjects
N Gender
210 Male 54% Female 46% Mean 11.45±4.16 Min 1 Max 19 Mean 1.53±0.21 Max 1.92 Min 0.76 Mean 79.19±30.43 Min 13.6 Max 190 Mean 4.86±1.7 Min 1.98 Max 16.54 Mean 4.55±0.6 Min 2.9 Max 6.67 Mean 4.26±0.81 Min 1.7 Max 8.87 Mean 1.09±0.23 Min 0.6 Max 1.74 Mean 1.48±0.73 Min 0.31 Max 4.12
Age, years
Height, m
Weight, kg
SDS BMI
Fasting blood glucose, mmol/L
Total cholesterol, mmol/L
HDL cholesterol, mmol/L
Triglycerides, mmol/L
Min, minimum; Max, maximum; ± s.d., standard deviation; HDL, high-density lipoprotein.
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Polák et al.: The prevalence of melanocortin-4 receptor gene mutations in Slovak obese children 57
previous study (25), using Eppendorf Mastercycler® pro S (Eppendorf, Hamburg, Germany) in 20 μl volumes. Each PCR product was subsequently purified under the standard protocol and sequenced using PCR primers MC4R-F, MC4R-R and MC4R-CF (5′-TGTAGCTCCTTGCTTGCATC-3′) and MC4R-CR (5′-GGCCATCAGGAACATGTGGA-3′) internal primers as previously described (26). Sequencing was performed using the Big Dye 3.1 terminator sequencing kit (Applied Biosystems/ Life Technologies, Foster City, USA) and the ABI 3100 genetic analyzer (Applied Biosystems/Life Technologies, Foster City, USA) according to the manufacturer’s instructions. Sequencing data was analyzed with Sequencing Analysis, version 5.2 (Applied Biosystems/Life Technologies, USA) and Chromas Pro software, version 1.7.1 (Technelysium, Brisbane, Australia). The obtained sequences were compared with the wild-type sequence of the MC4R gene (ENSG00000166603; www.ensembl.org).
Multifactorial statistical analysis Multifactorial analysis was conducted using multivariate analysis of variance (MANOVA) in Statistical Package for the Social Sciences (SPSS) (IBM, New York, USA). Dependent variables included SDS BMI, fasting blood glucose, total cholesterol, HDL cholesterol and triglycerides levels. Tested independent variables included age, sex and the genotypes, and p-values T c.380C > T c.522G > A c.913C > T c.751A > C c.307G > A
0.23% 0.23% 0.23% 0.23% 0.23% 1.66%
Missense Missense Nonsense Missense Missense Missense
Functional characterization
References
Decreased activity Decreased activity ND Decreased activity Benign/protective Benign/protective
(17) (17) This study (27) (20) (20)
ND, not determined.
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58 Polák et al.: The prevalence of melanocortin-4 receptor gene mutations in Slovak obese children Table 3: Genotypes and phenotype characteristics of variants’ carriers. Patient 1 2 3 4 5 6 7 8 9 10 11
Genotype p.R7C/− p.S127L/p.V103Ia p.R305W/− p.W174*/− p.V103I/− p.V103I/− p.V103I/− p.V103I/− p.V103I/− p.V103I/− p.I251L/−
Gender F F F M M M F F F F F
Age
SDS BMI
Fasting blood glucose, mmol/L
Total cholesterol, mmol/L
HDL cholesterol, mmol/L
Triglycerides, mmol/L
15.8 9.5 2.5 4 3.8 6.5 5.9 6.5 10 12 14
6.89 3.45 4.48 4.49 5.19 2.16 4.07 3.37 3.79 6.91 10.84
5.6 4.7 4.1 4.37 4.1 4.3 5.1 4.6 4.3 3 6
4.43 3.21 3.67 4.66 3.63 4.64 3.84 5.94 4 3.63 4.31
1.01 1.08 1.54 1.14 1.3 1.63 1.72 1.25 1.13 1.09 0.8
1.38 0.8 0.57 1.3 0.66 0.89 1.06 1.7 1.4 1.08 3.07
All parameters refer to the time of first consultation; HDL, high-density lipoprotein; F, female; M, male. Physiological reference values of tested biochemical parameters according to Zimmet et al. (23) and Gahagan et al. (24). Fasting blood glucose ≤ 5.6 mmol/L; total cholesterol 1.03 mmol/L; triglycerides ≤ 1.7 mmol/L. aTrans conformation of p.V103I was not confirmed by molecular analysis in parents.
ND
p.W174*
ND
p.W174*
p.V103I
p.W174*
Figure 1: Segregation of novel nonsense mutation p.W174* in family segregation analysis. Circles represent females; squares represent males. Black symbols represent obese individuals; gray symbols represent overweight individuals; and white symbols represent normal weight individuals. ND, genotype is not determined. Phenotype of relatives was based on verbal reports by the parents of the patient.
Discussion This is the first study in Slovak obese children and adolescents where the coding region of the MC4R gene has been analyzed. The aim of this work was to estimate the prevalence of MC4R variants in 210 Slovak obese patients and evaluate their phenotypes. We identified four different mutations and two non-synonymous obesity-protective variants in our cohort, giving a mutation detection rate of 0.95%. This is in concordance with the results of some other studies (28–30). However, the mutation prevalences in our cohort group have a significantly lower figure than
the estimated MC4R mutation frequencies in our neighboring Czech population and also in the European populations (15, 16). Obesity is generally considered a polygenic trait resulting from the interaction of several genetic loci with concurrent environmental factors (4), therefore many other genetic influences other than MC4R mutations and accompanying non-genetic factors may have contributed to the obesity manifestation in our cohort group. However, it is also possible that some of the analyzed patients are carriers of undetected MC4R promoter mutations as described by two previous authors groups (18, 31). Further genetic background may also be determined in the 3′ end of the MC4R gene, as has been shown in several genome wide association studies (GWAS), where two single nucleotide polymorphisms (SNPs) downstream of the MC4R gene were remarkably associated with obesity (32, 33). Recently, a high prevalence of homozygosity and heterozygosity for rs17782313 variant has been observed in the Greek population (34). In addition to the considerations mentioned above, a larger cohort sample could reveal some additional MC4R mutations. Stricter health criteria of cohort evaluation with regard to earlier obesity onset may be warranted. However, present concurrent hyperphagia and hyperinsulinemia may be irrelevant because these characteristics are not always present in all mutation carriers and they are dependent on mutation functionality (15, 18, 27). Thus, it is difficult to assess a reliable clinical setting for MC4R haplo-insufficient patients in the current obesogenic environment. Mutation p.R7C is located in the N-terminal extracellular domain of the MC4R, and this impairs the receptor α-MSH activation and decreases its constitutive activity
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Polák et al.: The prevalence of melanocortin-4 receptor gene mutations in Slovak obese children 59
2
4.5
1.8
4
1.6
3.5
1.4
3
TAG
HDL
1.2 1 0.8
2 1.5
0.6
1
0.4
0.5
0.2 0
2.5
0
5
10 Age
15
20
0
0
5
10 Age
15
20
Figure 2: Influence of age on triglycerides and HDL cholesterol blood levels in Slovak children and adolescents. HDL, high-density lipoprotein; TAG, triglycerides.
while cell surface expression remains unchanged (17, 27). The phenotypic outcome here should lead to excessive food intake. We identified this missense mutation in one morbidly obese 16-year-old girl with the onset of phenotypic manifestation from the age of 3 years (phenotype characteristics of all mutation carriers are summarized in Table 3). Unfortunately, we were unable to perform family segregation analysis for this allele. However, in the Czech cohort p.R7C cosegregated with obesity or overweight in all except one family member (15). Therefore, we estimate, that this mutation is most likely to be responsible for this patient’s clinical outcome, albeit with possible variable penetrance. Mutation p.S127L affects the transmembrane domain 3 and also shows in vitro alternations in α-MSH receptor activation (17, 27, 35). However, it exhibited a wide range of phenotypic manifestations in population analysis and obese subjects analyses (17), with its presence. It was detected in lean subjects as well as in severely obese patients (26, 29). This is likely due to variable penetrance and mutation expressivity. The carrier of this allele here was a 10-year-old obese girl demonstrating obesity onset from the age of 8 years. She was also a carrier of the heterozygous obesity-protective p.V103I variant (20, 36), which slightly improves receptor function by increasing agonist potency and decreasing inverse agonist potency (37). Interestingly, Hainerova et al. (15) reported that a patient who was also a carrier of p.S127L mutation and p.V103I underwent a 3-week weight reduction regimen and succeeded to lose more body weight compared to 21 non-carriers who underwent the same 3-week weight intervention program (15). It remains a matter of speculation whether the p.V103I variant could have influenced this finding or if it has a moderating effect on our patients’ phenotype.
Substitution of p.R305W in the C-terminal MC4R domain causes decreased cell surface expression and ligand-binding and also decreases basal metabolic activity, as reported and functionally characterized in one severely obese patient in a study by Lubrano-Berthelier and colleagues (27). The carrier of this mutation in our study was a 2.5-year-old girl who had obesity onset at 1 year of age. This patient’s phenotype supports findings that the majority of mutations altering cell surface expression lead to very early onset of this disease (38). We identified one novel nonsense mutation leading to a premature stop codon at position W174 in a morbidly obese 7-year-old boy with obesity onset at 4 years. This patient did not exhibit binge eating habits and did not have impaired glucose tolerance. Segregation analysis was performed in two generations to further examine this variant (Figure 1). Although nonsense MC4R mutations lead to truncated protein forms with lack of activity and cell surface expression (39), severe obesity due to MC4R haplo-insufficiency can be expected. Interestingly, our proband’s mother and grandmother who carried this mutation had a history of childhood obesity, but although slightly overweight they were not currently obese. A similar finding was reported in a case study, although regarding a different mutation (p.R165W) (40). These observations support a possible age-decrement in obesity (19, 41) and also that the current obesogenic environment could encourage an individual effect. We assessed standard biochemical markers in each subject of our cohort group commonly appendant in obesity to observe any differences between these parameters in mutation carriers compared to non-carriers and in carriers of obesity-protective variants compared to non-carriers. We did not observe any statistically
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60 Polák et al.: The prevalence of melanocortin-4 receptor gene mutations in Slovak obese children significant influence on tested parameters in variant carriers. However, it is noteworthy that p.I251L and p.V103I variant carriers had significantly higher HDL cholesterol levels compared to non-carriers in a recent German study (30). Nevertheless, we observed significant correlation between age and triacylglycerol levels and age and HDL cholesterol levels (Figure 2). In summary, MC4R mutations were detected as a cause of obesity in 1.9% of Slovak obese children and adolescents. We identified three missense mutations and one novel nonsense mutation, which was further studied in family segregation analysis. Our findings support variable penetrance of the MC4R gene variations and also agree with an age-dependent manifestation manner. As in similar studies, not all of the mutation carriers fulfilled the typical symptoms of MC4R deficiency regarding early onset of obesity, hyperphagia, increased linear growth and hyperinsulinemia. Acknowledgments: We would like to thank all the children and their families for their participation in this study. We are also grateful to pediatric endocrinologists Mária Kúseková MD†, Ľubica Tichá MD, Mária Ševecová MD, Adriana Dankovčíková MD, Marianna Debreová MD and Vilja Šandriková MD for their assistance in this study. This contribution is the result of projects implemented in the grants “Diagnostics of Socially Important Disorders in Slovakia, Based on Modern Biotechnologies” ITMS 26240220058 and “Creating Competitive Centre for research and development in the field of molecular medicine” ITMS 26240220071, supported by the Research and Developmental Operational Program funded by the ERDF. This work was also supported by VEGA 1/0497/08. Conflict of interest: The authors declare no conflict of interest.
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