High Prevalence of Rare Monogenic Forms of Obesity in Obese Guadeloupean Afro-Caribbean Children

High Prevalence of Rare Monogenic Forms of Obesity in Obese Guadeloupean Afro-Caribbean Children Abstract Context The population of Guadeloupe Island exhibits a high prevalence of obesity. Objective We aimed to investigate whether rare genetic mutations in genes involved in monogenic obesity (or diabetes) might be causal in this population of Afro-Caribbean ancestry. Design and Setting This was a secondary analysis of a study on obesity conducted in schoolchildren from Guadeloupe in 2013 that aimed to assess changes in children’s profiles after a lifestyle intervention program. Through next-generation sequencing, we sequenced coding regions of 59 genes involved in monogenic obesity or diabetes in participants from this study. Participants and Interventions A total of 25 obese schoolchildren from Guadeloupe were screened for rare mutations (nonsynonymous, splice-site, or insertion/deletion) in 59 genes. Main Outcome Measures Correlation between phenotypes and mutations of interest. Results We detected five rare heterozygous mutations in five different children with obesity: MC4R p.Ile301Thr and SIM1 p.Val326Thrfs*43 mutations that were pathogenic; SIM1 p.Ser343Pro and SH2B1 p.Pro90His mutations that were likely pathogenic; and NTRK2 p.Leu140Phe that was of uncertain significance. In parallel, we identified seven carriers of mutations in ABCC8 (p.Lys1521Asn and p.Ala625Val) or KCNJ11 (p.Val13Met and p.Val151Met) that were of uncertain significance. Conclusions We were able to detect pathogenic or likely pathogenic mutations linked to severe obesity in >15% of this population, which is much higher than what we observed in Europeans (∼5%). Obesity, a major public health problem, is a highly complex disorder for which underlying genetic variations are not completely known and understood. Obesity heritability ranges from 40% to 70% according to family, twin, and population studies (1, 2), although monogenic forms of obesity have also been described in <5% of obese patients of European origin (3). These monogenic obesity disorders have been shown to be caused by mutations in leptin (LEP), leptin receptor (LEPR), proopiomelanocortin (POMC), or melanocortin 4-receptor (MC4R), highlighting the importance of the hypothalamic melanocortin pathway that mediates leptin’s effects on controlling energy homeostasis and food intake (4). Leptin, an adipocyte-derived hormone, modulates insulin secretion and action via the leptin receptor that is expressed in pancreatic β cells, adipose tissue, and muscle (5). Pathogenic mutations in both LEP and LEPR genes are very rare and have been described in obese individuals worldwide, mainly from consanguineous families (6). Among the melanocortin receptors and their ligands involved in the central melanocortin system, MC4R was shown to be distributed in several brain regions, including the appetite-regulating nuclei of the hypothalamus (7, 8). Rare mutations in MC4R were found in 5% to 6% of patients with severe early-onset obesity (9). Guadeloupe Island is a French “departement,” part of the Lesser Antilles in the Caribbean, with a high prevalence of obesity. To our knowledge, no study has investigated the contribution of gene mutations to childhood obesity in this population with African ancestry. In the current study, we took advantage of our next-generation sequencing panel, which targets all genes involved in monogenic obesity or monogenic diabetes with high sensitivity (10), to screen 25 Afro-Caribbean children with obesity from Guadeloupe Island. Materials/Subjects and Methods Study population The present investigation included a sample of 25 unrelated children with obesity [standard deviation score (z score) of body mass index (BMI): 2.0 to 2.8] from a previous study on overweight conducted in Guadeloupe Island (11). This study was performed in 2013 to evaluate the basal data of middle school children between 11 and 15 years of age and to evaluate the changes in the children’s profiles after 1 year of a lifestyle intervention program. The study was approved by the ethic committee (South West, Overseas III, Bordeaux, France) in October 2012 and the French National Agency for Medicines and Health Products Safety. Written informed consent for participation in this study was obtained from all children and their parents. The children and accompanying parents were interviewed. Anthropometric measurements were obtained by trained nurses. Height in centimeters (cm) and weight in kilograms (kg) were measured with participants standing without shoes and lightly clothed. BMI was calculated as weight/height2 (kg/m2). The BMI z score was calculated as [(measured value − average value in the reference population)/standard deviation in the reference population] using a pediatric z score calculator. Pubertal stage was assessed according to Tanner (12). Tanner stages 3, 4, and 5 defined pubertal/postpubertal development. Family history of obesity or diabetes was available only for the accompanying parent (mother or father). Laboratory measurements Blood samples were collected by venipuncture for adipokine and hormone estimations and for DNA extractions after an overnight fast. Blood lipid and blood glucose (mmol/L), serum insulin (µU/mL), leptin (ng/mL), and total adiponectin (µg/mL) levels were measured as previously described (11). Blood lipid abnormalities were defined as high-density lipoprotein cholesterol <1.03 mmol/L or triglycerides ≥1.7 mmol/L (13). The homeostasis model assessment to determine insulin resistance (HOMA-IR) was calculated using the following formula: [fasting insulin (μIU/mL) × fasting glucose (mmol/L)/22.5] (14). Insulin resistance was defined by a HOMA-IR >3.16 (15). Genomic DNA was extracted from whole blood using the Gentra Puregene Blood Kit (Qiagen, Crawley, UK) per manufacturer’s instructions. Next-generation sequencing The coding regions (including at least 40 base pairs of the flanking intron of each exon) of 59 genes involved in monogenic forms of obesity or diabetes (Table 1) were sequenced in the 25 children. For this purpose, we used targeted polymerase chain reaction‒based enrichment in lipid microdroplets (RainDance Technologies, Lexington, MA) in combination with Illumina sequencing (San Diego, CA) as previously described by us (10, 16). The reads were mapped to the hg19 human genome assembly, and genetic variants (punctual mutations and insertions/deletions) were detected as previously described (10). The mean depth of coverage was between 240× and 428× [median (first quartile – third quartile): 318 (286–352)], and 99% of the target on average was covered with more than eight reads. The rare variants of potential interest described later had a high quality (Phred) score of at least 50 (mean quality score of 170). We used standards and guidelines of the American College of Medical Genetics and Genomics for the interpretation of sequence variants (17). Of note, we were unfortunately not able to sequence DNA samples from the relatives of the children carrying loss-of-function mutations. Table 1. List of Genes Involved in Monogenic Obesity or Diabetes Gene  Name  Location  Disease  Inheritance  GNAS  GNAS complex locus  20q13.32  AHO  AD (from the mother)  ALMS1  ALMS1, centrosome and basal body associated protein  2p13.1  ALMS  AR  BBS1  Bardet-Biedl syndrome 1  11q13.2  BBS1  AR  BBS2  Bardet-Biedl syndrome 2  16q13  BBS2  AR  ARL6  ADP ribosylation factor like GTPase 6  3q11.2  BBS3  AR  BBS4  Bardet-Biedl syndrome 4  15q24.1  BBS4  AR  BBS5  Bardet-Biedl syndrome 5  2q31.1  BBS5  AR  MKKS  McKusick-Kaufman syndrome  20p12.2  BBS6  AR  BBS7  Bardet-Biedl syndrome 7  4q27  BBS7  AR  TTC8  Tetratricopeptide repeat domain 8  14q31.3  BBS8  AR  BBS9  Bardet-Biedl syndrome 9  7p14.3  BBS9  AR  BBS10  Bardet-Biedl syndrome 10  12q21.2  BBS10  AR  TRIM32  Tripartite motif containing 32  9q33.1  BBS11  AR  BBS12  Bardet-Biedl syndrome 12  4q27  BBS12  AR  MKS1  Meckel syndrome, type 1  17q22  BBS13  AR  CEP290  Centrosomal protein 290  12q21.32  BBS14  AR  WDPCP  WD repeat containing planar cell polarity effector  2p15  BBS15  AR  SDCCAG8  Serologically defined colon cancer antigen 8  1q43-q44  BBS16  AR  FOXP3  Forkhead box P3  Xp11.23  IPEXS  XR  PCBD1  Pterin-4 alpha-carbinolamine dehydratase 1  10q22.1  MODY  AR  HNF1A  HNF1 homeobox A  12q24.31  MODY  AD  KLF11  Kruppel like factor 11  2p25.1  MODY  AD  HNF4A  Hepatocyte nuclear factor 4α  20q13.12  MODY  AD  TRMT10A  tRNA methyltransferase 10A  4q23  MODY  AR  PAX4  Paired box 4  7q32.1  MODY  AD  CEL  Carboxyl ester lipase  9q34.13  MODY  AD  SLC19A2  Solute carrier family 19 member 2  1q24.2  NDM  AR  NEUROG3  Neurogenin 3  10q22.1  NDM  AR  PTF1A  Pancreas specific transcription factor, 1a  10p12.2  NDM  AR  IER3IP1  Immediate early response 3 interacting protein 1  18q21.1  NDM  AR  NKX2-2  NK2 homeobox 2  20p11.22  NDM  AR  SLC2A2  Solute carrier family 2 member 2  3q26.2  NDM  AR  RFX6  Regulatory factor X6  6q22.1  NDM  AR  MNX1  Motor neuron and pancreas homeobox 1  7q36.3  NDM  AR  GLIS3  GLIS family zinc finger 3  9p24.2  NDM  AR  ABCC8  ATP binding cassette subfamily C member 8  11p15.1  NDM/MODY  S or AD  INS  Insulin  11p15.5  NDM/MODY  (S, AD, or AR)/AD  KCNJ11  Potassium voltage-gated channel subfamily J member 11  11p15.1  NDM/MODY  (S or AD)/AD  PDX1  Pancreatic and duodenal homeobox 1  13q12.2  NDM/MODY  AR/AD  HNF1B  HNF1 homeobox B  17q12  NDM/MODY  (S or AD)/AD  GATA6  GATA binding protein 6  18q11.2  NDM/MODY  S or AD  NEUROD1  Neuronal differentiation 1  2q31.3  NDM/MODY  AR/AD  GCK  Glucokinase  7p13  NDM/MODY  AR/AD  GATA4  GATA binding protein 4  8p23.1  NDM/MODY  S or AD  LEPR  Leptin receptor  1p31.3  SEO  AR  BDNF  Brain-derived neurotrophic factor  11p14.1  SEO  S  TUB  Tubby bipartite transcription factor  11p15.4  SEO  AR  SH2B1  SH2B adaptor protein 1  16p11.2  SEO  AD  MC4R  Melanocortin 4 receptor  18q21.32  SEO  AD or AR  DYRK1B  Dual specificity tyrosine phosphorylation regulated kinase 1B  19q13.2  SEO  AD  POMC  Proopiomelanocortin  2p23.3  SEO  AR  CEP19  Centrosomal protein 19  3q29  SEO  AR  PCSK1  Proprotein convertase subtilisin/kexin type 1  5q15  SEO  AD or AR  MRAP2  Melanocortin 2 receptor accessory protein 2  6q14.2  SEO  AD  LEP  Leptin  7q32.1  SEO  AR  NTRK2  Neurotrophic receptor tyrosine kinase 2  9q21.33  SEO  S  SIM1  Single-minded family bhlh transcription factor 1  6q16.3  SEO/PWL  AD  EIF2AK3  Eukaryotic translation initiation factor 2α kinase 3  2p11.2  WRS  AR  WFS1  Wolframin ER transmembrane glycoprotein  4p16.1  WS  AR  Gene  Name  Location  Disease  Inheritance  GNAS  GNAS complex locus  20q13.32  AHO  AD (from the mother)  ALMS1  ALMS1, centrosome and basal body associated protein  2p13.1  ALMS  AR  BBS1  Bardet-Biedl syndrome 1  11q13.2  BBS1  AR  BBS2  Bardet-Biedl syndrome 2  16q13  BBS2  AR  ARL6  ADP ribosylation factor like GTPase 6  3q11.2  BBS3  AR  BBS4  Bardet-Biedl syndrome 4  15q24.1  BBS4  AR  BBS5  Bardet-Biedl syndrome 5  2q31.1  BBS5  AR  MKKS  McKusick-Kaufman syndrome  20p12.2  BBS6  AR  BBS7  Bardet-Biedl syndrome 7  4q27  BBS7  AR  TTC8  Tetratricopeptide repeat domain 8  14q31.3  BBS8  AR  BBS9  Bardet-Biedl syndrome 9  7p14.3  BBS9  AR  BBS10  Bardet-Biedl syndrome 10  12q21.2  BBS10  AR  TRIM32  Tripartite motif containing 32  9q33.1  BBS11  AR  BBS12  Bardet-Biedl syndrome 12  4q27  BBS12  AR  MKS1  Meckel syndrome, type 1  17q22  BBS13  AR  CEP290  Centrosomal protein 290  12q21.32  BBS14  AR  WDPCP  WD repeat containing planar cell polarity effector  2p15  BBS15  AR  SDCCAG8  Serologically defined colon cancer antigen 8  1q43-q44  BBS16  AR  FOXP3  Forkhead box P3  Xp11.23  IPEXS  XR  PCBD1  Pterin-4 alpha-carbinolamine dehydratase 1  10q22.1  MODY  AR  HNF1A  HNF1 homeobox A  12q24.31  MODY  AD  KLF11  Kruppel like factor 11  2p25.1  MODY  AD  HNF4A  Hepatocyte nuclear factor 4α  20q13.12  MODY  AD  TRMT10A  tRNA methyltransferase 10A  4q23  MODY  AR  PAX4  Paired box 4  7q32.1  MODY  AD  CEL  Carboxyl ester lipase  9q34.13  MODY  AD  SLC19A2  Solute carrier family 19 member 2  1q24.2  NDM  AR  NEUROG3  Neurogenin 3  10q22.1  NDM  AR  PTF1A  Pancreas specific transcription factor, 1a  10p12.2  NDM  AR  IER3IP1  Immediate early response 3 interacting protein 1  18q21.1  NDM  AR  NKX2-2  NK2 homeobox 2  20p11.22  NDM  AR  SLC2A2  Solute carrier family 2 member 2  3q26.2  NDM  AR  RFX6  Regulatory factor X6  6q22.1  NDM  AR  MNX1  Motor neuron and pancreas homeobox 1  7q36.3  NDM  AR  GLIS3  GLIS family zinc finger 3  9p24.2  NDM  AR  ABCC8  ATP binding cassette subfamily C member 8  11p15.1  NDM/MODY  S or AD  INS  Insulin  11p15.5  NDM/MODY  (S, AD, or AR)/AD  KCNJ11  Potassium voltage-gated channel subfamily J member 11  11p15.1  NDM/MODY  (S or AD)/AD  PDX1  Pancreatic and duodenal homeobox 1  13q12.2  NDM/MODY  AR/AD  HNF1B  HNF1 homeobox B  17q12  NDM/MODY  (S or AD)/AD  GATA6  GATA binding protein 6  18q11.2  NDM/MODY  S or AD  NEUROD1  Neuronal differentiation 1  2q31.3  NDM/MODY  AR/AD  GCK  Glucokinase  7p13  NDM/MODY  AR/AD  GATA4  GATA binding protein 4  8p23.1  NDM/MODY  S or AD  LEPR  Leptin receptor  1p31.3  SEO  AR  BDNF  Brain-derived neurotrophic factor  11p14.1  SEO  S  TUB  Tubby bipartite transcription factor  11p15.4  SEO  AR  SH2B1  SH2B adaptor protein 1  16p11.2  SEO  AD  MC4R  Melanocortin 4 receptor  18q21.32  SEO  AD or AR  DYRK1B  Dual specificity tyrosine phosphorylation regulated kinase 1B  19q13.2  SEO  AD  POMC  Proopiomelanocortin  2p23.3  SEO  AR  CEP19  Centrosomal protein 19  3q29  SEO  AR  PCSK1  Proprotein convertase subtilisin/kexin type 1  5q15  SEO  AD or AR  MRAP2  Melanocortin 2 receptor accessory protein 2  6q14.2  SEO  AD  LEP  Leptin  7q32.1  SEO  AR  NTRK2  Neurotrophic receptor tyrosine kinase 2  9q21.33  SEO  S  SIM1  Single-minded family bhlh transcription factor 1  6q16.3  SEO/PWL  AD  EIF2AK3  Eukaryotic translation initiation factor 2α kinase 3  2p11.2  WRS  AR  WFS1  Wolframin ER transmembrane glycoprotein  4p16.1  WS  AR  Abbreviations: AD, autosomal dominant; AHO, Albright hereditary osteodystrophy; ALMS, Alström syndrome; AR, autosomal recessive; BBS, Bardet-Biedl syndrome; IPEXS, immune dysregulation polyendocrinopathy enteropathy X-linked syndrome; MODY, maturity-onset diabetes of the young; NDM, neonatal diabetes mellitus; PWL, Prader-Willi‒like syndrome; S, spontaneous; SEO, severe early-onset obesity; WRS, Wolcott-Rallison syndrome; WS, Wolfram syndrome; XR, X-linked recessive. View Large Statistical analysis A descriptive analysis of children carrying gene mutations was performed. Data are presented as mean ± standard deviation for quantitative parameters and as percentage for categorical parameters. Results The 25 obese children from Guadeloupe Island were 12 boys and 13 girls. The mean age of the study population was 12.4 ± 1.1 years (12.3 ± 1.2 years in boys and 12.2 ± 1.1 years in girls). All the children were at a pubertal or postpubertal stage (Tanner stage ≥3). Tanner stage was distributed as follows: in boys, five at stage 3, five at stage 4, and two at stage 5; in girls, nine at stage 3 and four at stage 4. BMI z scores ranged from 2.00 to 2.80. Seventeen children (68%) had insulin-resistance (HOMA-IR >3.16). A family history of obesity (mother or father) was observed in eight children, and three of the accompanying parents presented with type 2 diabetes. All the children had abnormal eating behavior, favoring foods that were too sweet, rich in fat, and high in calories and sometimes secretly taken, combined with an insufficient intake of fruits and vegetables. However, none of the children was a binge eater. Furthermore, none of the children had a gonadotrophic abnormality or mental retardation. Among the 25 obese schoolchildren, we identified five carriers of rare heterozygous mutations [with a frequency below 0.5% in the Genome Aggregation Database (gnomAD) that provided 123,136 exome sequences and 15,496 whole-genome sequences from unrelated individuals in August 2017] that were either nonsynonymous or frameshift within genes involved in monogenic obesity (MC4R, NTRK2, SH2B1, SIM1): An obese boy (ID #1; BMI, 30.5 kg/m2; BMI z score, 2.39; age, 11.8 years) who presented with insulin resistance and blood lipid abnormalities and carried a nonsynonymous mutation (p.Ile301Thr) in MC4R (Tables 2 and 3). This mutation was not present in the gnomAD browser and was predicted to be damaging according to every in silico prediction tool used in the current study (i.e., SIFT, MutationTaster, Align GVGD, and PolyPhen-2). Of note, we also found that the boy (ID #1) carried a rare nonsynonymous mutation (p.Thr1068Met) in ABCC8. This mutation was not novel (rs139524121; with a frequency of 0.32% in Africans according to the gnomAD browser) and was damaging according to one in silico prediction tool. Of note, the mother of this boy was obese and had type 2 diabetes, but we were unable to sequence her DNA. A severely obese girl (ID #2; BMI, 36.6 kg/m2; BMI z score, 2.43; age, 13.5 years) who presented with severe insulin resistance and impaired fasting plasma glucose level (>5.6 mmol/L) according to the 2017 American Diabetes Association guidelines (19) and carried a nonsynonymous mutation (p.Leu140Phe) in NTRK2 (Tables 2 and 3 ). This mutation was not novel (rs150692457; with a frequency of 0.43% in Africans according to the gnomAD browser), and it was damaging according to two in silico prediction tools. An obese boy (ID #3; BMI, 30.4 kg/m2; BMI z score, 2.22; age, 12.7 years) who presented with insulin resistance and carried a nonsynonymous mutation (p.Pro90His) in SH2B1 (Tables 2 and 3 ). This mutation was not novel (rs149091795; with a frequency of 0.47% in Africans according to the gnomAD browser) and was not damaging according to the four in silico prediction tools. An obese girl (ID #4; BMI, 29.7 kg/m2; BMI z score, 2.14; age, 11.9 years) who presented with insulin resistance and carried a nonsynonymous mutation (p.Ser343Pro) in SIM1 (Tables 2 and 3 ). This mutation was not present in the gnomAD browser and was damaging according to three in silico prediction tools. A morbidly obese girl (ID #5; BMI, 51.2 kg/m2; BMI z score, 2.80; age, 14.7 years) who presented with insulin resistance and carried a frameshift mutation (p.Val326Thrfs*43) in SIM1 (Tables 2 and 3 ). This mutation was not present in the gnomAD browser and was probably deleterious because it led to a premature STOP codon in the reading frame. Of note, the girl (ID #5) also presented with an impaired fasting glucose level (Table 2) and carried a rare nonsynonymous mutation (p.Ala625Val) in ABCC8. This mutation was not novel (rs148709148; with a frequency of 0.21% in Africans according to the gnomAD browser), and it was damaging according to one in silico prediction tool. Table 2. Characteristics of Obese Children With Rare Mutations of Potential Interest in Genes Involved in Monogenic Obesity and Diabetes ID  Mutation in Gene Causing:   Sex  Birth Weight (kg)  Age (y)  BMI (kg/m2)  BMI z Score  Fasting Glucosea (mmol/L)  Low HDL-C  High TG  Fasting Insulina (µIU/mL)  HOMA-IR  IR  Leptina (ng/mL)  Adiponectina (µg/mL)  Monogenic Obesity  Monogenic Diabetes  1  MC4R  ABCC8  M  3.4  11.8  30.5  2.39  4.6  Yes  No  14.1  4.59  Yes  50  3.0  2  NTRK2  0  F  3.23  13.5  36.6  2.43  6.1  No  Yes  47.4  12.87  Yes  96  5.0  3  SH2B1  0  M  3.1  12.7  30.4  2.22  5.4  No  No  37.2  8.87  Yes  20  3.4  4  SIM1  0  F  3.0  11.9  29.7  2.14  4.2  No  No  20.7  5.56  Yes  35  4.8  5  SIM1  ABCC8  F  3.5  14.7  51.2  2.80  5.6  Yes  No  24.7  6.16  Yes  114  2.6  6  0  ABCC8  F  3.8  12.4  34.8  2.44  5.2  No  No  20.6  4.78  Yes  32  2.6  7  0  ABCC8  F  —  12.4  29.8  2.09  5.6  Yes  No  23.8  5.93  Yes  29  1.7  8  0  ABCC8  F  —  13.2  29.8  2.00  4.8  Yes  No  28.6  6.14  Yes  53  2.0  9  0  KCNJ11  M  —  14.2  35.8  2.49  5.0  No  No  20.4  5.09  Yes  31  3.5  10  0  KCNJ11  M  4.3  11.0  31.1  2.38  4.9  Yes  No  9.1  1.95  No  27  3.6  ID  Mutation in Gene Causing:   Sex  Birth Weight (kg)  Age (y)  BMI (kg/m2)  BMI z Score  Fasting Glucosea (mmol/L)  Low HDL-C  High TG  Fasting Insulina (µIU/mL)  HOMA-IR  IR  Leptina (ng/mL)  Adiponectina (µg/mL)  Monogenic Obesity  Monogenic Diabetes  1  MC4R  ABCC8  M  3.4  11.8  30.5  2.39  4.6  Yes  No  14.1  4.59  Yes  50  3.0  2  NTRK2  0  F  3.23  13.5  36.6  2.43  6.1  No  Yes  47.4  12.87  Yes  96  5.0  3  SH2B1  0  M  3.1  12.7  30.4  2.22  5.4  No  No  37.2  8.87  Yes  20  3.4  4  SIM1  0  F  3.0  11.9  29.7  2.14  4.2  No  No  20.7  5.56  Yes  35  4.8  5  SIM1  ABCC8  F  3.5  14.7  51.2  2.80  5.6  Yes  No  24.7  6.16  Yes  114  2.6  6  0  ABCC8  F  3.8  12.4  34.8  2.44  5.2  No  No  20.6  4.78  Yes  32  2.6  7  0  ABCC8  F  —  12.4  29.8  2.09  5.6  Yes  No  23.8  5.93  Yes  29  1.7  8  0  ABCC8  F  —  13.2  29.8  2.00  4.8  Yes  No  28.6  6.14  Yes  53  2.0  9  0  KCNJ11  M  —  14.2  35.8  2.49  5.0  No  No  20.4  5.09  Yes  31  3.5  10  0  KCNJ11  M  4.3  11.0  31.1  2.38  4.9  Yes  No  9.1  1.95  No  27  3.6  Abbreviations: F, female; HDL-C, high-density lipoprotein cholesterol; IR, insulin resistance; M, male; TG, triglycerides. a The values previously reported in nonobese adolescents were (mean ± standard deviation): fasting glucose, 4.9 ± 0.1 mmol/L; fasting insulin, 11 ± 1.4 µIU/mL; leptin, 5 ± 1 ng/mL; and adiponectin, 14.0 ± 1.6 µg/mL (18). View Large Table 3. Rare Heterozygous Mutations of Potential Interest Identified in Obese Children ID  Genea  rs ID  Mutation (cDNA)  Mutation (Protein)  Frequency in gnomAD (T/A) (%)b  SIFT  Mutation-Taster  Align GVGD  PPh2 HumDiv  Ref  1  MC4R  Novel  NM_005912.2:c.902T>C  p.Ile301Thr  Not reported  D  DC  C65  D  Yes  ABCC8  rs139524121  NM_000352.4:c.3203C>T  p.Thr1068Met  0.031/0.32  T  DC  C0  B  No  2  NTRK2  rs150692457  NM_006180.4:c.420G>C  p.Leu140Phe  0.041/0.43  T  DC  C0  D  Yes  3  SH2B1  rs149091795  NM_001145795.1:c.269C>A  p.Pro90His  0.043/0.47  T  P  C0  B  Yes  4  SIM1  Novel  NM_005068.2:c.1027T>C  p.Ser343Pro  Not reported  D  DC  C0  D  No  5  ABCC8  rs148709148  NM_000352.4:c.1874C>T  p.Ala625Val  0.021/0.21  T  DC  C0  B  No  SIM1  Novel  NM_005068.2:c.975_976 insACTCCTG  p.Val326Thrfs*43  Not reported  NA  NA  NA  NA  No  6  ABCC8  rs142272833  NM_000352.4:c.4563G>T  p.Lys1521Asn  0.039/0.43  T  DC  C0  B  Yes  7  ABCC8  rs142272833  NM_000352.4:c.4563G>T  p.Lys1521Asn  0.039/0.43  T  DC  C0  B  Yes  8  ABCC8  rs148709148  NM_000352.4:c.1874C>T  p.Ala625Val  0.021/0.21  T  DC  C0  B  No  9  KCNJ11  rs139079635  NM_000525.3:c.37G>A  p.Val13Met  0.035/0.071  D  P  C0  D  No  10  KCNJ11  rs529884745  NM_000525.3:c.451G>A  p.Val151Met  0.0036/0.021  D  DC  C0  PD  No  ID  Genea  rs ID  Mutation (cDNA)  Mutation (Protein)  Frequency in gnomAD (T/A) (%)b  SIFT  Mutation-Taster  Align GVGD  PPh2 HumDiv  Ref  1  MC4R  Novel  NM_005912.2:c.902T>C  p.Ile301Thr  Not reported  D  DC  C65  D  Yes  ABCC8  rs139524121  NM_000352.4:c.3203C>T  p.Thr1068Met  0.031/0.32  T  DC  C0  B  No  2  NTRK2  rs150692457  NM_006180.4:c.420G>C  p.Leu140Phe  0.041/0.43  T  DC  C0  D  Yes  3  SH2B1  rs149091795  NM_001145795.1:c.269C>A  p.Pro90His  0.043/0.47  T  P  C0  B  Yes  4  SIM1  Novel  NM_005068.2:c.1027T>C  p.Ser343Pro  Not reported  D  DC  C0  D  No  5  ABCC8  rs148709148  NM_000352.4:c.1874C>T  p.Ala625Val  0.021/0.21  T  DC  C0  B  No  SIM1  Novel  NM_005068.2:c.975_976 insACTCCTG  p.Val326Thrfs*43  Not reported  NA  NA  NA  NA  No  6  ABCC8  rs142272833  NM_000352.4:c.4563G>T  p.Lys1521Asn  0.039/0.43  T  DC  C0  B  Yes  7  ABCC8  rs142272833  NM_000352.4:c.4563G>T  p.Lys1521Asn  0.039/0.43  T  DC  C0  B  Yes  8  ABCC8  rs148709148  NM_000352.4:c.1874C>T  p.Ala625Val  0.021/0.21  T  DC  C0  B  No  9  KCNJ11  rs139079635  NM_000525.3:c.37G>A  p.Val13Met  0.035/0.071  D  P  C0  D  No  10  KCNJ11  rs529884745  NM_000525.3:c.451G>A  p.Val151Met  0.0036/0.021  D  DC  C0  PD  No  Abbreviations: B, benign; cDNA, complementary DNA; D, deleterious; DC, disease causing; NA, not applicable; P, polymorphism; PD, possibly damaging; PPh2 HumDiv, PolyPhen-2 for monogenic disease; rs ID, reference single nucleotide polymorphism ID number; T, tolerated. a Monogenic obesity genes are in bold. b Frequency in gnomAD in (T) the total population and (A) Africans. View Large In parallel, we found five obese girls and boys (ID #6 through ID #10) carrying a rare heterozygous missense mutation in ABCC8 (p.Ala625Val, p.Lys1521Asn) or KCNJ11 (p.Val13Met, p.Val151Met) (Tables 2 and 3 ). The p.Lys1521Asn mutation in ABCC8 was identified in two obese girls (ID #6 and ID #7), including one with an impaired fasting plasma glucose level (Table 2). This mutation was not novel (rs142272833; with a frequency of 0.43% in Africans according to the gnomAD browser), and it was damaging according to one in silico prediction tool. Of note, the mothers of these two girls had type 2 diabetes, but we were unable to sequence their DNA. The ABCC8 p.Ala625Val mutation (carried by ID #8) was described previously because it was also carried by the girl with ID #5. Finally, we found two mutations (p.Val13Met, p.Val151Met) in KCNJ11 in two obese boys (ID #9 and ID #10) with normal glucose levels (Tables 2 and 3 ). These two mutations were very rare (<0.1% in Africans according to the gnomAD browser), and they were both damaging according to several in silico prediction tools. Discussion In the multiethnic population of Guadeloupe Island that includes 472,124 inhabitants, 80% of subjects are of African descent (known as African-Caribbeans). On this island, overweight and obesity were recently estimated at 23% and 9%, respectively, among children aged 5 to 14 years. Diabetes is also highly prevalent (>8%) in the adult population. In the current study, which included 25 obese children of African-Caribbean ancestry, we found five mutations of potential interest in four genes involved in monogenic obesity. The MC4R p.Ile301Thr mutation that we identified in an obese boy (ID #1) was previously reported in a morbidly obese adult (20). We showed that this mutation leads to a reduction in MC4R activation (20). Furthermore, the specific binding of the [125I]NDP-αMSH MC4R agonist to the p.Ile301Thr mutant was reduced by more than 80% compared with the wild-type receptor (20). Therefore, the MC4R p.Ile301Thr mutation was pathogenic and probably caused obesity in the boy. However, the ABCC8 p.Thr1068Met mutation carried by the same boy was of uncertain significance. The NTRK2 p.Leu140Phe mutation carried by the severely obese girl (ID #2) was previously associated with smoking status in Africans (21). However, according to our knowledge, no NTRK2 mutation causing monogenic obesity has been identified since the primary study by Yeo et al. (22). The girl did not have an intellectual disability, in contrast to the previously reported subject with a mutation for NTRK2 (22). With regard to the frequency of the present mutation and its effect prediction, the NTRK2 p.Leu140Phe mutation was of uncertain significance. Further conclusive studies are needed on this case. The SH2B1 p.Pro90His mutation carried by an obese boy (ID #3) did not appear to be pathogenic with regard to its frequency and prediction effect. However, Doche et al. (23) considered it pathogenic as they demonstrated that this rare nonsynonymous mutation significantly impaired the ability of SH2B1β to enhance neuronal differentiation compared with the wild-type protein. Furthermore, in contrast to the wild-type protein, which was stimulated with both basal and growth hormone‒induced motilities, the p.Pro90His mutation inhibited growth hormone‒induced motility (23). In line with the phenotype of the boy analyzed in the current study, the two carriers reported in Doche et al. (23) presented with hyperinsulinemia. Of note, they also presented with social isolation and aggression, which is not the case with the boy from the current study. The SH2B1 p.Pro90His mutation possibly caused obesity in the boy, although further investigation is needed to confirm this result. Neither the SIM1 p.Ser343Pro nor the p.Val326Thrfs*43 mutation carried by two obese girls (ID #4 and ID #5) has been previously described, according to our knowledge. We and others previously demonstrated that rare loss-of-function mutations in SIM1 caused obesity potentially associated with Prader-Willi‒like clinical features (24–26). In the current study, we did not notice any intellectual disability, compulsive eating, neonatal hypotonia, or other features related to Prader-Willi‒like syndrome. However, the two rare mutations in SIM1 probably caused obesity in the two girls. The ABCC8 p.Ala625Val mutation carried by the morbidly obese girl (ID #5) was of uncertain significance. In parallel, we found rare heterozygous missense mutations in ABCC8 and KCNJ11 in five obese girls and boys (ID #6 through ID #10). The ABCC8 p.Lys1521Asn mutation was previously associated with adult-onset type 2 diabetes, but investigations failed to show a functional effect of this mutation on protein activity (27). Therefore, this mutation was of uncertain significance. The other ABCC8 mutation (p.Ala625Val) was of uncertain significance, as well as the two mutations (p.Val13Met, p.Val151Met) in KCNJ11 carried by two obese boys (ID #9 and ID #10). In summary, among 25 obese children, we identified three carriers of pathogenic or likely pathogenic mutations in SIM1, MC4R, and SH2B1, which probably caused the obesity. Therefore, we were able to detect mutations linked to severe obesity in more than 15% of this population, which is higher than what we found in Europeans (∼5%) (3). Limitations of our study include its small sample size; however, its strength is related to the fact that it concerns a homogeneous sample of Afro-Caribbean children with obesity. Larger family and longitudinal studies in this population appear relevant for the screening of monogenic obesity/diabetes genes. Abbreviations: BMI body mass index gnomAD Genome Aggregation Database HOMA-IR homeostasis model assessment to determine insulin resistance MC4R melanocortin 4-receptor. Acknowledgments The authors thank the children and their families for their cooperation, the nurses and physicians of the AGREXAM Health Centre, and Mrs. L. Nesty, the headmaster of Collège Saint John Perse middle school in Guadeloupe. Financial Support: This work was supported by grants from the University Hospital of Guadeloupe (to L.F.), the French National Research Agency [ANR-10-LABX-46 (European Genomics Institute for Diabetes) and ANR-10-EQPX-07-01 (LIGAN-PM)] (to P.F.), the European Research Council (ERC GEPIDIAB–294785) (to P.F.), and the Fonds européen de développement régional (FEDER; to P.F.). 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Endocrine Society
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Copyright © 2018 Endocrine Society
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0021-972X
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1945-7197
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10.1210/jc.2017-01956
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Abstract

Abstract Context The population of Guadeloupe Island exhibits a high prevalence of obesity. Objective We aimed to investigate whether rare genetic mutations in genes involved in monogenic obesity (or diabetes) might be causal in this population of Afro-Caribbean ancestry. Design and Setting This was a secondary analysis of a study on obesity conducted in schoolchildren from Guadeloupe in 2013 that aimed to assess changes in children’s profiles after a lifestyle intervention program. Through next-generation sequencing, we sequenced coding regions of 59 genes involved in monogenic obesity or diabetes in participants from this study. Participants and Interventions A total of 25 obese schoolchildren from Guadeloupe were screened for rare mutations (nonsynonymous, splice-site, or insertion/deletion) in 59 genes. Main Outcome Measures Correlation between phenotypes and mutations of interest. Results We detected five rare heterozygous mutations in five different children with obesity: MC4R p.Ile301Thr and SIM1 p.Val326Thrfs*43 mutations that were pathogenic; SIM1 p.Ser343Pro and SH2B1 p.Pro90His mutations that were likely pathogenic; and NTRK2 p.Leu140Phe that was of uncertain significance. In parallel, we identified seven carriers of mutations in ABCC8 (p.Lys1521Asn and p.Ala625Val) or KCNJ11 (p.Val13Met and p.Val151Met) that were of uncertain significance. Conclusions We were able to detect pathogenic or likely pathogenic mutations linked to severe obesity in >15% of this population, which is much higher than what we observed in Europeans (∼5%). Obesity, a major public health problem, is a highly complex disorder for which underlying genetic variations are not completely known and understood. Obesity heritability ranges from 40% to 70% according to family, twin, and population studies (1, 2), although monogenic forms of obesity have also been described in <5% of obese patients of European origin (3). These monogenic obesity disorders have been shown to be caused by mutations in leptin (LEP), leptin receptor (LEPR), proopiomelanocortin (POMC), or melanocortin 4-receptor (MC4R), highlighting the importance of the hypothalamic melanocortin pathway that mediates leptin’s effects on controlling energy homeostasis and food intake (4). Leptin, an adipocyte-derived hormone, modulates insulin secretion and action via the leptin receptor that is expressed in pancreatic β cells, adipose tissue, and muscle (5). Pathogenic mutations in both LEP and LEPR genes are very rare and have been described in obese individuals worldwide, mainly from consanguineous families (6). Among the melanocortin receptors and their ligands involved in the central melanocortin system, MC4R was shown to be distributed in several brain regions, including the appetite-regulating nuclei of the hypothalamus (7, 8). Rare mutations in MC4R were found in 5% to 6% of patients with severe early-onset obesity (9). Guadeloupe Island is a French “departement,” part of the Lesser Antilles in the Caribbean, with a high prevalence of obesity. To our knowledge, no study has investigated the contribution of gene mutations to childhood obesity in this population with African ancestry. In the current study, we took advantage of our next-generation sequencing panel, which targets all genes involved in monogenic obesity or monogenic diabetes with high sensitivity (10), to screen 25 Afro-Caribbean children with obesity from Guadeloupe Island. Materials/Subjects and Methods Study population The present investigation included a sample of 25 unrelated children with obesity [standard deviation score (z score) of body mass index (BMI): 2.0 to 2.8] from a previous study on overweight conducted in Guadeloupe Island (11). This study was performed in 2013 to evaluate the basal data of middle school children between 11 and 15 years of age and to evaluate the changes in the children’s profiles after 1 year of a lifestyle intervention program. The study was approved by the ethic committee (South West, Overseas III, Bordeaux, France) in October 2012 and the French National Agency for Medicines and Health Products Safety. Written informed consent for participation in this study was obtained from all children and their parents. The children and accompanying parents were interviewed. Anthropometric measurements were obtained by trained nurses. Height in centimeters (cm) and weight in kilograms (kg) were measured with participants standing without shoes and lightly clothed. BMI was calculated as weight/height2 (kg/m2). The BMI z score was calculated as [(measured value − average value in the reference population)/standard deviation in the reference population] using a pediatric z score calculator. Pubertal stage was assessed according to Tanner (12). Tanner stages 3, 4, and 5 defined pubertal/postpubertal development. Family history of obesity or diabetes was available only for the accompanying parent (mother or father). Laboratory measurements Blood samples were collected by venipuncture for adipokine and hormone estimations and for DNA extractions after an overnight fast. Blood lipid and blood glucose (mmol/L), serum insulin (µU/mL), leptin (ng/mL), and total adiponectin (µg/mL) levels were measured as previously described (11). Blood lipid abnormalities were defined as high-density lipoprotein cholesterol <1.03 mmol/L or triglycerides ≥1.7 mmol/L (13). The homeostasis model assessment to determine insulin resistance (HOMA-IR) was calculated using the following formula: [fasting insulin (μIU/mL) × fasting glucose (mmol/L)/22.5] (14). Insulin resistance was defined by a HOMA-IR >3.16 (15). Genomic DNA was extracted from whole blood using the Gentra Puregene Blood Kit (Qiagen, Crawley, UK) per manufacturer’s instructions. Next-generation sequencing The coding regions (including at least 40 base pairs of the flanking intron of each exon) of 59 genes involved in monogenic forms of obesity or diabetes (Table 1) were sequenced in the 25 children. For this purpose, we used targeted polymerase chain reaction‒based enrichment in lipid microdroplets (RainDance Technologies, Lexington, MA) in combination with Illumina sequencing (San Diego, CA) as previously described by us (10, 16). The reads were mapped to the hg19 human genome assembly, and genetic variants (punctual mutations and insertions/deletions) were detected as previously described (10). The mean depth of coverage was between 240× and 428× [median (first quartile – third quartile): 318 (286–352)], and 99% of the target on average was covered with more than eight reads. The rare variants of potential interest described later had a high quality (Phred) score of at least 50 (mean quality score of 170). We used standards and guidelines of the American College of Medical Genetics and Genomics for the interpretation of sequence variants (17). Of note, we were unfortunately not able to sequence DNA samples from the relatives of the children carrying loss-of-function mutations. Table 1. List of Genes Involved in Monogenic Obesity or Diabetes Gene  Name  Location  Disease  Inheritance  GNAS  GNAS complex locus  20q13.32  AHO  AD (from the mother)  ALMS1  ALMS1, centrosome and basal body associated protein  2p13.1  ALMS  AR  BBS1  Bardet-Biedl syndrome 1  11q13.2  BBS1  AR  BBS2  Bardet-Biedl syndrome 2  16q13  BBS2  AR  ARL6  ADP ribosylation factor like GTPase 6  3q11.2  BBS3  AR  BBS4  Bardet-Biedl syndrome 4  15q24.1  BBS4  AR  BBS5  Bardet-Biedl syndrome 5  2q31.1  BBS5  AR  MKKS  McKusick-Kaufman syndrome  20p12.2  BBS6  AR  BBS7  Bardet-Biedl syndrome 7  4q27  BBS7  AR  TTC8  Tetratricopeptide repeat domain 8  14q31.3  BBS8  AR  BBS9  Bardet-Biedl syndrome 9  7p14.3  BBS9  AR  BBS10  Bardet-Biedl syndrome 10  12q21.2  BBS10  AR  TRIM32  Tripartite motif containing 32  9q33.1  BBS11  AR  BBS12  Bardet-Biedl syndrome 12  4q27  BBS12  AR  MKS1  Meckel syndrome, type 1  17q22  BBS13  AR  CEP290  Centrosomal protein 290  12q21.32  BBS14  AR  WDPCP  WD repeat containing planar cell polarity effector  2p15  BBS15  AR  SDCCAG8  Serologically defined colon cancer antigen 8  1q43-q44  BBS16  AR  FOXP3  Forkhead box P3  Xp11.23  IPEXS  XR  PCBD1  Pterin-4 alpha-carbinolamine dehydratase 1  10q22.1  MODY  AR  HNF1A  HNF1 homeobox A  12q24.31  MODY  AD  KLF11  Kruppel like factor 11  2p25.1  MODY  AD  HNF4A  Hepatocyte nuclear factor 4α  20q13.12  MODY  AD  TRMT10A  tRNA methyltransferase 10A  4q23  MODY  AR  PAX4  Paired box 4  7q32.1  MODY  AD  CEL  Carboxyl ester lipase  9q34.13  MODY  AD  SLC19A2  Solute carrier family 19 member 2  1q24.2  NDM  AR  NEUROG3  Neurogenin 3  10q22.1  NDM  AR  PTF1A  Pancreas specific transcription factor, 1a  10p12.2  NDM  AR  IER3IP1  Immediate early response 3 interacting protein 1  18q21.1  NDM  AR  NKX2-2  NK2 homeobox 2  20p11.22  NDM  AR  SLC2A2  Solute carrier family 2 member 2  3q26.2  NDM  AR  RFX6  Regulatory factor X6  6q22.1  NDM  AR  MNX1  Motor neuron and pancreas homeobox 1  7q36.3  NDM  AR  GLIS3  GLIS family zinc finger 3  9p24.2  NDM  AR  ABCC8  ATP binding cassette subfamily C member 8  11p15.1  NDM/MODY  S or AD  INS  Insulin  11p15.5  NDM/MODY  (S, AD, or AR)/AD  KCNJ11  Potassium voltage-gated channel subfamily J member 11  11p15.1  NDM/MODY  (S or AD)/AD  PDX1  Pancreatic and duodenal homeobox 1  13q12.2  NDM/MODY  AR/AD  HNF1B  HNF1 homeobox B  17q12  NDM/MODY  (S or AD)/AD  GATA6  GATA binding protein 6  18q11.2  NDM/MODY  S or AD  NEUROD1  Neuronal differentiation 1  2q31.3  NDM/MODY  AR/AD  GCK  Glucokinase  7p13  NDM/MODY  AR/AD  GATA4  GATA binding protein 4  8p23.1  NDM/MODY  S or AD  LEPR  Leptin receptor  1p31.3  SEO  AR  BDNF  Brain-derived neurotrophic factor  11p14.1  SEO  S  TUB  Tubby bipartite transcription factor  11p15.4  SEO  AR  SH2B1  SH2B adaptor protein 1  16p11.2  SEO  AD  MC4R  Melanocortin 4 receptor  18q21.32  SEO  AD or AR  DYRK1B  Dual specificity tyrosine phosphorylation regulated kinase 1B  19q13.2  SEO  AD  POMC  Proopiomelanocortin  2p23.3  SEO  AR  CEP19  Centrosomal protein 19  3q29  SEO  AR  PCSK1  Proprotein convertase subtilisin/kexin type 1  5q15  SEO  AD or AR  MRAP2  Melanocortin 2 receptor accessory protein 2  6q14.2  SEO  AD  LEP  Leptin  7q32.1  SEO  AR  NTRK2  Neurotrophic receptor tyrosine kinase 2  9q21.33  SEO  S  SIM1  Single-minded family bhlh transcription factor 1  6q16.3  SEO/PWL  AD  EIF2AK3  Eukaryotic translation initiation factor 2α kinase 3  2p11.2  WRS  AR  WFS1  Wolframin ER transmembrane glycoprotein  4p16.1  WS  AR  Gene  Name  Location  Disease  Inheritance  GNAS  GNAS complex locus  20q13.32  AHO  AD (from the mother)  ALMS1  ALMS1, centrosome and basal body associated protein  2p13.1  ALMS  AR  BBS1  Bardet-Biedl syndrome 1  11q13.2  BBS1  AR  BBS2  Bardet-Biedl syndrome 2  16q13  BBS2  AR  ARL6  ADP ribosylation factor like GTPase 6  3q11.2  BBS3  AR  BBS4  Bardet-Biedl syndrome 4  15q24.1  BBS4  AR  BBS5  Bardet-Biedl syndrome 5  2q31.1  BBS5  AR  MKKS  McKusick-Kaufman syndrome  20p12.2  BBS6  AR  BBS7  Bardet-Biedl syndrome 7  4q27  BBS7  AR  TTC8  Tetratricopeptide repeat domain 8  14q31.3  BBS8  AR  BBS9  Bardet-Biedl syndrome 9  7p14.3  BBS9  AR  BBS10  Bardet-Biedl syndrome 10  12q21.2  BBS10  AR  TRIM32  Tripartite motif containing 32  9q33.1  BBS11  AR  BBS12  Bardet-Biedl syndrome 12  4q27  BBS12  AR  MKS1  Meckel syndrome, type 1  17q22  BBS13  AR  CEP290  Centrosomal protein 290  12q21.32  BBS14  AR  WDPCP  WD repeat containing planar cell polarity effector  2p15  BBS15  AR  SDCCAG8  Serologically defined colon cancer antigen 8  1q43-q44  BBS16  AR  FOXP3  Forkhead box P3  Xp11.23  IPEXS  XR  PCBD1  Pterin-4 alpha-carbinolamine dehydratase 1  10q22.1  MODY  AR  HNF1A  HNF1 homeobox A  12q24.31  MODY  AD  KLF11  Kruppel like factor 11  2p25.1  MODY  AD  HNF4A  Hepatocyte nuclear factor 4α  20q13.12  MODY  AD  TRMT10A  tRNA methyltransferase 10A  4q23  MODY  AR  PAX4  Paired box 4  7q32.1  MODY  AD  CEL  Carboxyl ester lipase  9q34.13  MODY  AD  SLC19A2  Solute carrier family 19 member 2  1q24.2  NDM  AR  NEUROG3  Neurogenin 3  10q22.1  NDM  AR  PTF1A  Pancreas specific transcription factor, 1a  10p12.2  NDM  AR  IER3IP1  Immediate early response 3 interacting protein 1  18q21.1  NDM  AR  NKX2-2  NK2 homeobox 2  20p11.22  NDM  AR  SLC2A2  Solute carrier family 2 member 2  3q26.2  NDM  AR  RFX6  Regulatory factor X6  6q22.1  NDM  AR  MNX1  Motor neuron and pancreas homeobox 1  7q36.3  NDM  AR  GLIS3  GLIS family zinc finger 3  9p24.2  NDM  AR  ABCC8  ATP binding cassette subfamily C member 8  11p15.1  NDM/MODY  S or AD  INS  Insulin  11p15.5  NDM/MODY  (S, AD, or AR)/AD  KCNJ11  Potassium voltage-gated channel subfamily J member 11  11p15.1  NDM/MODY  (S or AD)/AD  PDX1  Pancreatic and duodenal homeobox 1  13q12.2  NDM/MODY  AR/AD  HNF1B  HNF1 homeobox B  17q12  NDM/MODY  (S or AD)/AD  GATA6  GATA binding protein 6  18q11.2  NDM/MODY  S or AD  NEUROD1  Neuronal differentiation 1  2q31.3  NDM/MODY  AR/AD  GCK  Glucokinase  7p13  NDM/MODY  AR/AD  GATA4  GATA binding protein 4  8p23.1  NDM/MODY  S or AD  LEPR  Leptin receptor  1p31.3  SEO  AR  BDNF  Brain-derived neurotrophic factor  11p14.1  SEO  S  TUB  Tubby bipartite transcription factor  11p15.4  SEO  AR  SH2B1  SH2B adaptor protein 1  16p11.2  SEO  AD  MC4R  Melanocortin 4 receptor  18q21.32  SEO  AD or AR  DYRK1B  Dual specificity tyrosine phosphorylation regulated kinase 1B  19q13.2  SEO  AD  POMC  Proopiomelanocortin  2p23.3  SEO  AR  CEP19  Centrosomal protein 19  3q29  SEO  AR  PCSK1  Proprotein convertase subtilisin/kexin type 1  5q15  SEO  AD or AR  MRAP2  Melanocortin 2 receptor accessory protein 2  6q14.2  SEO  AD  LEP  Leptin  7q32.1  SEO  AR  NTRK2  Neurotrophic receptor tyrosine kinase 2  9q21.33  SEO  S  SIM1  Single-minded family bhlh transcription factor 1  6q16.3  SEO/PWL  AD  EIF2AK3  Eukaryotic translation initiation factor 2α kinase 3  2p11.2  WRS  AR  WFS1  Wolframin ER transmembrane glycoprotein  4p16.1  WS  AR  Abbreviations: AD, autosomal dominant; AHO, Albright hereditary osteodystrophy; ALMS, Alström syndrome; AR, autosomal recessive; BBS, Bardet-Biedl syndrome; IPEXS, immune dysregulation polyendocrinopathy enteropathy X-linked syndrome; MODY, maturity-onset diabetes of the young; NDM, neonatal diabetes mellitus; PWL, Prader-Willi‒like syndrome; S, spontaneous; SEO, severe early-onset obesity; WRS, Wolcott-Rallison syndrome; WS, Wolfram syndrome; XR, X-linked recessive. View Large Statistical analysis A descriptive analysis of children carrying gene mutations was performed. Data are presented as mean ± standard deviation for quantitative parameters and as percentage for categorical parameters. Results The 25 obese children from Guadeloupe Island were 12 boys and 13 girls. The mean age of the study population was 12.4 ± 1.1 years (12.3 ± 1.2 years in boys and 12.2 ± 1.1 years in girls). All the children were at a pubertal or postpubertal stage (Tanner stage ≥3). Tanner stage was distributed as follows: in boys, five at stage 3, five at stage 4, and two at stage 5; in girls, nine at stage 3 and four at stage 4. BMI z scores ranged from 2.00 to 2.80. Seventeen children (68%) had insulin-resistance (HOMA-IR >3.16). A family history of obesity (mother or father) was observed in eight children, and three of the accompanying parents presented with type 2 diabetes. All the children had abnormal eating behavior, favoring foods that were too sweet, rich in fat, and high in calories and sometimes secretly taken, combined with an insufficient intake of fruits and vegetables. However, none of the children was a binge eater. Furthermore, none of the children had a gonadotrophic abnormality or mental retardation. Among the 25 obese schoolchildren, we identified five carriers of rare heterozygous mutations [with a frequency below 0.5% in the Genome Aggregation Database (gnomAD) that provided 123,136 exome sequences and 15,496 whole-genome sequences from unrelated individuals in August 2017] that were either nonsynonymous or frameshift within genes involved in monogenic obesity (MC4R, NTRK2, SH2B1, SIM1): An obese boy (ID #1; BMI, 30.5 kg/m2; BMI z score, 2.39; age, 11.8 years) who presented with insulin resistance and blood lipid abnormalities and carried a nonsynonymous mutation (p.Ile301Thr) in MC4R (Tables 2 and 3). This mutation was not present in the gnomAD browser and was predicted to be damaging according to every in silico prediction tool used in the current study (i.e., SIFT, MutationTaster, Align GVGD, and PolyPhen-2). Of note, we also found that the boy (ID #1) carried a rare nonsynonymous mutation (p.Thr1068Met) in ABCC8. This mutation was not novel (rs139524121; with a frequency of 0.32% in Africans according to the gnomAD browser) and was damaging according to one in silico prediction tool. Of note, the mother of this boy was obese and had type 2 diabetes, but we were unable to sequence her DNA. A severely obese girl (ID #2; BMI, 36.6 kg/m2; BMI z score, 2.43; age, 13.5 years) who presented with severe insulin resistance and impaired fasting plasma glucose level (>5.6 mmol/L) according to the 2017 American Diabetes Association guidelines (19) and carried a nonsynonymous mutation (p.Leu140Phe) in NTRK2 (Tables 2 and 3 ). This mutation was not novel (rs150692457; with a frequency of 0.43% in Africans according to the gnomAD browser), and it was damaging according to two in silico prediction tools. An obese boy (ID #3; BMI, 30.4 kg/m2; BMI z score, 2.22; age, 12.7 years) who presented with insulin resistance and carried a nonsynonymous mutation (p.Pro90His) in SH2B1 (Tables 2 and 3 ). This mutation was not novel (rs149091795; with a frequency of 0.47% in Africans according to the gnomAD browser) and was not damaging according to the four in silico prediction tools. An obese girl (ID #4; BMI, 29.7 kg/m2; BMI z score, 2.14; age, 11.9 years) who presented with insulin resistance and carried a nonsynonymous mutation (p.Ser343Pro) in SIM1 (Tables 2 and 3 ). This mutation was not present in the gnomAD browser and was damaging according to three in silico prediction tools. A morbidly obese girl (ID #5; BMI, 51.2 kg/m2; BMI z score, 2.80; age, 14.7 years) who presented with insulin resistance and carried a frameshift mutation (p.Val326Thrfs*43) in SIM1 (Tables 2 and 3 ). This mutation was not present in the gnomAD browser and was probably deleterious because it led to a premature STOP codon in the reading frame. Of note, the girl (ID #5) also presented with an impaired fasting glucose level (Table 2) and carried a rare nonsynonymous mutation (p.Ala625Val) in ABCC8. This mutation was not novel (rs148709148; with a frequency of 0.21% in Africans according to the gnomAD browser), and it was damaging according to one in silico prediction tool. Table 2. Characteristics of Obese Children With Rare Mutations of Potential Interest in Genes Involved in Monogenic Obesity and Diabetes ID  Mutation in Gene Causing:   Sex  Birth Weight (kg)  Age (y)  BMI (kg/m2)  BMI z Score  Fasting Glucosea (mmol/L)  Low HDL-C  High TG  Fasting Insulina (µIU/mL)  HOMA-IR  IR  Leptina (ng/mL)  Adiponectina (µg/mL)  Monogenic Obesity  Monogenic Diabetes  1  MC4R  ABCC8  M  3.4  11.8  30.5  2.39  4.6  Yes  No  14.1  4.59  Yes  50  3.0  2  NTRK2  0  F  3.23  13.5  36.6  2.43  6.1  No  Yes  47.4  12.87  Yes  96  5.0  3  SH2B1  0  M  3.1  12.7  30.4  2.22  5.4  No  No  37.2  8.87  Yes  20  3.4  4  SIM1  0  F  3.0  11.9  29.7  2.14  4.2  No  No  20.7  5.56  Yes  35  4.8  5  SIM1  ABCC8  F  3.5  14.7  51.2  2.80  5.6  Yes  No  24.7  6.16  Yes  114  2.6  6  0  ABCC8  F  3.8  12.4  34.8  2.44  5.2  No  No  20.6  4.78  Yes  32  2.6  7  0  ABCC8  F  —  12.4  29.8  2.09  5.6  Yes  No  23.8  5.93  Yes  29  1.7  8  0  ABCC8  F  —  13.2  29.8  2.00  4.8  Yes  No  28.6  6.14  Yes  53  2.0  9  0  KCNJ11  M  —  14.2  35.8  2.49  5.0  No  No  20.4  5.09  Yes  31  3.5  10  0  KCNJ11  M  4.3  11.0  31.1  2.38  4.9  Yes  No  9.1  1.95  No  27  3.6  ID  Mutation in Gene Causing:   Sex  Birth Weight (kg)  Age (y)  BMI (kg/m2)  BMI z Score  Fasting Glucosea (mmol/L)  Low HDL-C  High TG  Fasting Insulina (µIU/mL)  HOMA-IR  IR  Leptina (ng/mL)  Adiponectina (µg/mL)  Monogenic Obesity  Monogenic Diabetes  1  MC4R  ABCC8  M  3.4  11.8  30.5  2.39  4.6  Yes  No  14.1  4.59  Yes  50  3.0  2  NTRK2  0  F  3.23  13.5  36.6  2.43  6.1  No  Yes  47.4  12.87  Yes  96  5.0  3  SH2B1  0  M  3.1  12.7  30.4  2.22  5.4  No  No  37.2  8.87  Yes  20  3.4  4  SIM1  0  F  3.0  11.9  29.7  2.14  4.2  No  No  20.7  5.56  Yes  35  4.8  5  SIM1  ABCC8  F  3.5  14.7  51.2  2.80  5.6  Yes  No  24.7  6.16  Yes  114  2.6  6  0  ABCC8  F  3.8  12.4  34.8  2.44  5.2  No  No  20.6  4.78  Yes  32  2.6  7  0  ABCC8  F  —  12.4  29.8  2.09  5.6  Yes  No  23.8  5.93  Yes  29  1.7  8  0  ABCC8  F  —  13.2  29.8  2.00  4.8  Yes  No  28.6  6.14  Yes  53  2.0  9  0  KCNJ11  M  —  14.2  35.8  2.49  5.0  No  No  20.4  5.09  Yes  31  3.5  10  0  KCNJ11  M  4.3  11.0  31.1  2.38  4.9  Yes  No  9.1  1.95  No  27  3.6  Abbreviations: F, female; HDL-C, high-density lipoprotein cholesterol; IR, insulin resistance; M, male; TG, triglycerides. a The values previously reported in nonobese adolescents were (mean ± standard deviation): fasting glucose, 4.9 ± 0.1 mmol/L; fasting insulin, 11 ± 1.4 µIU/mL; leptin, 5 ± 1 ng/mL; and adiponectin, 14.0 ± 1.6 µg/mL (18). View Large Table 3. Rare Heterozygous Mutations of Potential Interest Identified in Obese Children ID  Genea  rs ID  Mutation (cDNA)  Mutation (Protein)  Frequency in gnomAD (T/A) (%)b  SIFT  Mutation-Taster  Align GVGD  PPh2 HumDiv  Ref  1  MC4R  Novel  NM_005912.2:c.902T>C  p.Ile301Thr  Not reported  D  DC  C65  D  Yes  ABCC8  rs139524121  NM_000352.4:c.3203C>T  p.Thr1068Met  0.031/0.32  T  DC  C0  B  No  2  NTRK2  rs150692457  NM_006180.4:c.420G>C  p.Leu140Phe  0.041/0.43  T  DC  C0  D  Yes  3  SH2B1  rs149091795  NM_001145795.1:c.269C>A  p.Pro90His  0.043/0.47  T  P  C0  B  Yes  4  SIM1  Novel  NM_005068.2:c.1027T>C  p.Ser343Pro  Not reported  D  DC  C0  D  No  5  ABCC8  rs148709148  NM_000352.4:c.1874C>T  p.Ala625Val  0.021/0.21  T  DC  C0  B  No  SIM1  Novel  NM_005068.2:c.975_976 insACTCCTG  p.Val326Thrfs*43  Not reported  NA  NA  NA  NA  No  6  ABCC8  rs142272833  NM_000352.4:c.4563G>T  p.Lys1521Asn  0.039/0.43  T  DC  C0  B  Yes  7  ABCC8  rs142272833  NM_000352.4:c.4563G>T  p.Lys1521Asn  0.039/0.43  T  DC  C0  B  Yes  8  ABCC8  rs148709148  NM_000352.4:c.1874C>T  p.Ala625Val  0.021/0.21  T  DC  C0  B  No  9  KCNJ11  rs139079635  NM_000525.3:c.37G>A  p.Val13Met  0.035/0.071  D  P  C0  D  No  10  KCNJ11  rs529884745  NM_000525.3:c.451G>A  p.Val151Met  0.0036/0.021  D  DC  C0  PD  No  ID  Genea  rs ID  Mutation (cDNA)  Mutation (Protein)  Frequency in gnomAD (T/A) (%)b  SIFT  Mutation-Taster  Align GVGD  PPh2 HumDiv  Ref  1  MC4R  Novel  NM_005912.2:c.902T>C  p.Ile301Thr  Not reported  D  DC  C65  D  Yes  ABCC8  rs139524121  NM_000352.4:c.3203C>T  p.Thr1068Met  0.031/0.32  T  DC  C0  B  No  2  NTRK2  rs150692457  NM_006180.4:c.420G>C  p.Leu140Phe  0.041/0.43  T  DC  C0  D  Yes  3  SH2B1  rs149091795  NM_001145795.1:c.269C>A  p.Pro90His  0.043/0.47  T  P  C0  B  Yes  4  SIM1  Novel  NM_005068.2:c.1027T>C  p.Ser343Pro  Not reported  D  DC  C0  D  No  5  ABCC8  rs148709148  NM_000352.4:c.1874C>T  p.Ala625Val  0.021/0.21  T  DC  C0  B  No  SIM1  Novel  NM_005068.2:c.975_976 insACTCCTG  p.Val326Thrfs*43  Not reported  NA  NA  NA  NA  No  6  ABCC8  rs142272833  NM_000352.4:c.4563G>T  p.Lys1521Asn  0.039/0.43  T  DC  C0  B  Yes  7  ABCC8  rs142272833  NM_000352.4:c.4563G>T  p.Lys1521Asn  0.039/0.43  T  DC  C0  B  Yes  8  ABCC8  rs148709148  NM_000352.4:c.1874C>T  p.Ala625Val  0.021/0.21  T  DC  C0  B  No  9  KCNJ11  rs139079635  NM_000525.3:c.37G>A  p.Val13Met  0.035/0.071  D  P  C0  D  No  10  KCNJ11  rs529884745  NM_000525.3:c.451G>A  p.Val151Met  0.0036/0.021  D  DC  C0  PD  No  Abbreviations: B, benign; cDNA, complementary DNA; D, deleterious; DC, disease causing; NA, not applicable; P, polymorphism; PD, possibly damaging; PPh2 HumDiv, PolyPhen-2 for monogenic disease; rs ID, reference single nucleotide polymorphism ID number; T, tolerated. a Monogenic obesity genes are in bold. b Frequency in gnomAD in (T) the total population and (A) Africans. View Large In parallel, we found five obese girls and boys (ID #6 through ID #10) carrying a rare heterozygous missense mutation in ABCC8 (p.Ala625Val, p.Lys1521Asn) or KCNJ11 (p.Val13Met, p.Val151Met) (Tables 2 and 3 ). The p.Lys1521Asn mutation in ABCC8 was identified in two obese girls (ID #6 and ID #7), including one with an impaired fasting plasma glucose level (Table 2). This mutation was not novel (rs142272833; with a frequency of 0.43% in Africans according to the gnomAD browser), and it was damaging according to one in silico prediction tool. Of note, the mothers of these two girls had type 2 diabetes, but we were unable to sequence their DNA. The ABCC8 p.Ala625Val mutation (carried by ID #8) was described previously because it was also carried by the girl with ID #5. Finally, we found two mutations (p.Val13Met, p.Val151Met) in KCNJ11 in two obese boys (ID #9 and ID #10) with normal glucose levels (Tables 2 and 3 ). These two mutations were very rare (<0.1% in Africans according to the gnomAD browser), and they were both damaging according to several in silico prediction tools. Discussion In the multiethnic population of Guadeloupe Island that includes 472,124 inhabitants, 80% of subjects are of African descent (known as African-Caribbeans). On this island, overweight and obesity were recently estimated at 23% and 9%, respectively, among children aged 5 to 14 years. Diabetes is also highly prevalent (>8%) in the adult population. In the current study, which included 25 obese children of African-Caribbean ancestry, we found five mutations of potential interest in four genes involved in monogenic obesity. The MC4R p.Ile301Thr mutation that we identified in an obese boy (ID #1) was previously reported in a morbidly obese adult (20). We showed that this mutation leads to a reduction in MC4R activation (20). Furthermore, the specific binding of the [125I]NDP-αMSH MC4R agonist to the p.Ile301Thr mutant was reduced by more than 80% compared with the wild-type receptor (20). Therefore, the MC4R p.Ile301Thr mutation was pathogenic and probably caused obesity in the boy. However, the ABCC8 p.Thr1068Met mutation carried by the same boy was of uncertain significance. The NTRK2 p.Leu140Phe mutation carried by the severely obese girl (ID #2) was previously associated with smoking status in Africans (21). However, according to our knowledge, no NTRK2 mutation causing monogenic obesity has been identified since the primary study by Yeo et al. (22). The girl did not have an intellectual disability, in contrast to the previously reported subject with a mutation for NTRK2 (22). With regard to the frequency of the present mutation and its effect prediction, the NTRK2 p.Leu140Phe mutation was of uncertain significance. Further conclusive studies are needed on this case. The SH2B1 p.Pro90His mutation carried by an obese boy (ID #3) did not appear to be pathogenic with regard to its frequency and prediction effect. However, Doche et al. (23) considered it pathogenic as they demonstrated that this rare nonsynonymous mutation significantly impaired the ability of SH2B1β to enhance neuronal differentiation compared with the wild-type protein. Furthermore, in contrast to the wild-type protein, which was stimulated with both basal and growth hormone‒induced motilities, the p.Pro90His mutation inhibited growth hormone‒induced motility (23). In line with the phenotype of the boy analyzed in the current study, the two carriers reported in Doche et al. (23) presented with hyperinsulinemia. Of note, they also presented with social isolation and aggression, which is not the case with the boy from the current study. The SH2B1 p.Pro90His mutation possibly caused obesity in the boy, although further investigation is needed to confirm this result. Neither the SIM1 p.Ser343Pro nor the p.Val326Thrfs*43 mutation carried by two obese girls (ID #4 and ID #5) has been previously described, according to our knowledge. We and others previously demonstrated that rare loss-of-function mutations in SIM1 caused obesity potentially associated with Prader-Willi‒like clinical features (24–26). In the current study, we did not notice any intellectual disability, compulsive eating, neonatal hypotonia, or other features related to Prader-Willi‒like syndrome. However, the two rare mutations in SIM1 probably caused obesity in the two girls. The ABCC8 p.Ala625Val mutation carried by the morbidly obese girl (ID #5) was of uncertain significance. In parallel, we found rare heterozygous missense mutations in ABCC8 and KCNJ11 in five obese girls and boys (ID #6 through ID #10). The ABCC8 p.Lys1521Asn mutation was previously associated with adult-onset type 2 diabetes, but investigations failed to show a functional effect of this mutation on protein activity (27). Therefore, this mutation was of uncertain significance. The other ABCC8 mutation (p.Ala625Val) was of uncertain significance, as well as the two mutations (p.Val13Met, p.Val151Met) in KCNJ11 carried by two obese boys (ID #9 and ID #10). In summary, among 25 obese children, we identified three carriers of pathogenic or likely pathogenic mutations in SIM1, MC4R, and SH2B1, which probably caused the obesity. Therefore, we were able to detect mutations linked to severe obesity in more than 15% of this population, which is higher than what we found in Europeans (∼5%) (3). Limitations of our study include its small sample size; however, its strength is related to the fact that it concerns a homogeneous sample of Afro-Caribbean children with obesity. Larger family and longitudinal studies in this population appear relevant for the screening of monogenic obesity/diabetes genes. Abbreviations: BMI body mass index gnomAD Genome Aggregation Database HOMA-IR homeostasis model assessment to determine insulin resistance MC4R melanocortin 4-receptor. Acknowledgments The authors thank the children and their families for their cooperation, the nurses and physicians of the AGREXAM Health Centre, and Mrs. L. Nesty, the headmaster of Collège Saint John Perse middle school in Guadeloupe. Financial Support: This work was supported by grants from the University Hospital of Guadeloupe (to L.F.), the French National Research Agency [ANR-10-LABX-46 (European Genomics Institute for Diabetes) and ANR-10-EQPX-07-01 (LIGAN-PM)] (to P.F.), the European Research Council (ERC GEPIDIAB–294785) (to P.F.), and the Fonds européen de développement régional (FEDER; to P.F.). 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Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Feb 1, 2018

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