key: cord-0860854-ixde41wc
authors: Yu, Tingting; Yu, Ying; Li, Xiaoqing; Xue, Peng; Yu, Xiaodan; Chen, Yao; Kong, Huijun; Lin, Cuilan; Wang, Xiumin; Mei, Hao; Wang, Dan; Liu, Shijian
title: Effects of childhood obesity and related genetic factors on precocious puberty: protocol for a multi-center prospective cohort study
date: 2022-05-27
journal: BMC Pediatr
DOI: 10.1186/s12887-022-03350-x
sha: f496e1f5ba8393c2672daa8f63b8fb2a28c62c38
doc_id: 860854
cord_uid: ixde41wc

BACKGROUND: Childhood obesity has important effects on the onset and development of puberty. Although a number of studies have confirmed the relationship between obesity and precocious puberty, little is known about the pleiotropic genes of obesity and precocious puberty and the interaction between genes and environment. There are four objectives: (1) to analyze the incidence of precocious puberty in the general population in China; (2) to verify the direct effect of obesity on children’s precocious puberty using a variety of methods; (3) to verify the effect of obesity and its risk gene polymorphism on precocious puberty in a prospective cohort study; and (4) to analyze the interaction effect of genes and environment on pubertal development. METHODS: We will conduct a multi-center prospective cohort study in three cities, which are selected in southern, central, and northern China, respectively. Primary schools in these cities will be selected by a stratified cluster random sampling method. Primary school students from grade 1 to grade 3 (6 to 10 years old) will be selected for the cohort with extensive baseline data collection, including assessment of pubertal development, family demographic information, early development, sleep pattern, dietary pattern, and physical activity. Participants will be followed up for at least three years, and long-term follow-up will depend on future funding. DISCUSSION: The findings of this multicenter prospective population-based cohort study may expand previous related puberty development research as well as provide important information on the mechanism of early puberty. Targeted interventions can also be developed to improve adolescent health problems related to puberty development based on the available evidence. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04113070, prospectively registered on October 2, 2019.

The high prevalence of childhood obesity has become a concerning public health problem. In 2016, over 340 million children and adolescents aged 5-19 globally were overweight or obese [1] . Moreover, an increasing number of countries are seeing earlier onset of puberty in children [2] [3] [4] . Early puberty has been reported to have a positive association with psychological symptoms, conduct problems, and reproductive diseases Open Access † Tingting Yu and Ying Yu contributed equally to this work. [5, 6] . Puberty is a critical but complex process, since many factors, including genetic [7] [8] [9] , nutritional [10] , and environmental factors [11] , may influence its development.

Recently, growing evidence from cross-sectional [12] [13] [14] , and longitudinal studies [15] has indicated that obesity or a higher body mass index (BMI) is associated with earlier timing of puberty in girls. However, the relationship between obesity and timing of puberty onset in boys is inconsistent. Although several studies reported that boys with higher BMI or obesity are more likely to experience early puberty [12, 15, 16] , some studies have suggested that delayed puberty is associated with obesity [17, 18] .

The occurrence of precocious puberty is closely related to obesity, and there exists some common pathogenesis. A previous study found a positive correlation between obesity and the level of leptin, a protein secreted by peripheral mature adipocytes; moreover, leptin and its receptor play key roles in the initiation of puberty onset and are important biomarkers involved in the regulation of both obesity and precocious puberty [19] . Genetically speaking, over 600 genetic locus are associated with obesity, and many studies have focused on the LEPR, MC4R, and FTO genes [20, 21] . In addition, precocious puberty is related to the KISS1, MC4R, and LEPR genes [7] [8] [9] . Consequently, pleiotropic genes like LEPR and MC4R could influence the phenotype of both obesity and precocious puberty; however, their effect on precocity puberty and its magnitude are still unknown.

Previous studies on the relationship between obesity and precocious puberty have mostly been crosssectional studies, with few studies on pleiotropic genes of obesity and puberty. To explore the relationship between obesity and puberty development, this prospective cohort study will investigate the genetic, parental, social and family environment, lifestyle, and other determinants of puberty development. This protocol describes an overview of the design and methods of this cohort study, which is funded by the National Natural Science Foundation of China (grant number 81872637). The study objectives are as follows: I) analyzing the incidence of precocious puberty in the general population in China, examining the direct effect of childhood obesity on precocious puberty; II) exploring the relationship between prepuberty obesity and its risk genes and precocious puberty; III) determining the relationship between prepuberty obesity and its risk genes and hormone levels among children with precocious puberty; and IV) analyzing the interaction effect between genes and environmental factors on precocious puberty.

This study is a prospective multi-center cohort study sponsored by the Shanghai Children's Medical Center affiliated to the School of Medicine, Shanghai Jiao Tong University and performed in three cities in southern, central, and northern China, namely, Zhongshan, Guangdong Province, Qufu, Shandong Province, and Dalian, Liaoning Province. According to socioeconomic level and geographical distribution, the districts of Zhongshan, Qufu, and Dalian are stratified into urban districts in the central area and suburban districts in suburban areas. Using a stratified cluster random sampling method, districts and schools will be selected according to geographical location and school size by a random number generator. Grade 1, 2, and 3 children (6 to 10 years old) from primary schools will be included in this study. However, participants with overweight or obesity with a history of hormone treatment in the past 6 months as reported by parents in the questionnaire will be excluded.

We will carry out a nested case-control study, which will be nested in this cohort study. Participants in casecontrol study will be enrolled in four groups: children with normal weight and normal puberty, with normal weight and precocious puberty, with overweight/obesity and normal puberty, and with overweight/obesity and precocious puberty. And they will be measured body composition, collected venous blood, and stool at baseline, and collected urine samples at baseline and followup periods.

Based on the formula for calculating the sample size of a cohort study at one-side significance level: [22] .

According to the previous studies, the prevalence of precocious puberty was 4.85% (P 2 ) and 8.43% (P 1 ) of children aged 7-10 years with normal weight and overweight, respectively [12, 23] . A type I error (α) of 0.05, u 0.05 = 1.96;, β = 0.10, u 0.10 = 1.28; and a loss of follow up rate of 15%, a total of 4152 children will be required in each center. Finally, inverse-variance weighting (IVW) methods will be used to adjust the sample size.

Based on the SNP gene differences between different groups reported in the literature, Quanto 1.2.4 software (John Morrison, W. James Gauderman 2009, University of Southern California) was used to estimate sample sizes for each group. According to the previous

pre-experimental results of obesity study showed that the allele frequency was 0.30 and the minimum allele frequency (MAF) was 0.10 using Additive Genetic Model (ADD), the type I error (α) of 0.05, the actual Power is 0.80, we need average of 800 participants per group and 3200 cases for four groups. Cases among four groups will be controlled by sex and age.

Participants will accept anthropometric measuring by clinicians from the three collaborating hospitals, including Bo Ai Hospital of Zhongshan, People's Hospital of Qu Fu, and Da Lian Children's Hospital. The cohort study was scheduled to begin in October, 2019, and the first and secondary follow-up visits will be conducted one and two years later, respectively. A flowchart of the study design was shown in Fig. 1 , and the investigation contents required at baseline, first visit, and second visit were shown in Table 1 .

Due to differences in gene polymorphism distribution in different ethnic groups, we screened ten candidate genes and selected one or two representative single-nucleotide polymorphisms (SNPs) each gene on the basis of the Hans according to GWAS database (www. genome. gov/ gwast udies) and the related literatures, the candidate genes and SNPs were shown in Table 2 .

Anthropometric data, including weight (kg), height (cm), and waist circumference (cm), will be measured for each child standing without shoes and lightly clothed using standard techniques by one observer at baseline and the first and second visit. Waist circumference will be measured midway between the lower edge of the rib and the upper edge of the ilium. All of the measurement indexes will be measured twice and averaged. Overweight and obesity are the main predictors of interest and will be defined by BMI value, which is calculated as weight (kg) divided by height squared (m 2 ). Participants will be defined as normal weight, overweight, or obese if their BMI index is between 18.5 and 25 kg/m 2 , greater than [24, 25] . Children with overweight or obesity and some participants with normal weight (matched for sex and age with children with obesity and overweight) will be recruited to analyze their body composition, including height, weight, fat-free mass, body fat mass, body fat rate, body water, muscle mass, basal metabolic rate, total energy consumption, protein, inorganic salt, and impedance.

The primary outcome is the difference in incidence of precocious puberty between normal-weight and obese children. Secondary sexual characteristics assessment for boys and girls will be conducted by male and female professional pediatric endocrinologists or pediatric care physicians separately in different rooms to protect the children's privacy. Breast development will be evaluated by visual inspection and palpation. Girls with overweight or obesity will be subjected to breast ultrasound to evaluate their nodules. In boys, testicular volume (TV) will be determined by palpation and an orchidometer. According to Marshall and Tanner [26, 27] , breast and pubic hair development are divided into 5 stages (stages I to V). Precocious puberty will be diagnosed according to the Tanner stage: onset of breast development stage II (B 2 ) or pubic hair development stage II (PH 2 ) or above before 8 years or menarche before 10 years of age in girls, and PH 2 or testicular enlargement (TV ≥ 4 ml) before 9 years of age in boys [28] [29] [30] .

The presence of early puberty will be defined as children reaching a certain stage of puberty earlier than the median age of that stage [31] . According to previous studies, the median age of puberty onset (girls: breast or pubic hair development; boys: pubic hair or testicular volume development) of Chinese adolescents will be referenced [32, 33] , and the onset of menarche before 12 years of age in girls and of voice breaking before 13 years of age in boys will also be defined as early puberty [34] .

We separately evaluated the tanner stage for each breast among obese girls by using portable ultrasound (US), and chose the larger one as evaluation result. Tanner II by US is characterized as the presence of a hyperechoic retro-areolar nodule with a central star-shaped or linear hypoechoic area, this stage is assumed to mark the start of breast development [35] .

Children with overweight or obesity and some of the participants with normal weight (matched for sex and age) will be recruited to collect venous blood at baseline; urine samples will be collected at baseline and 1-year follow-up. A total of 2 ml and 1 ml venous blood will be collected in coagulation and anticoagulation collection tubes, respectively. These participants will receive a vacuum stool collection tube and 15-ml urine tube 1 day before physical inspection, and they will be asked to collect stool and urine samples in the morning and take them to the researchers at school on the physical inspection day. All of the biological sample will be transferred with an ice bag to a local collaborating hospital within 2 hours after collection. Blood samples in coagulation collection tubes will be centrifuged to extract serum. Then, all biospecimens will be transferred with dry ice to the Biobank of Shanghai Children's Medical Center and stored at − 80 °C.

Part of the serum sample will be used to test folliclestimulating hormone (FSH), luteinizing hormone (LH), and estrogens (E2) in girls and testosterone (T) in boys; the residual serum will be used to test leptin, kisspeptin, and ghrelin levels. A whole-blood sample will be used to analyze the pleiotropic genes of precocious puberty and obesity.

Simultaneous quantification of 64 steroids, including EED in pediatric plasma and urine by LC-MS/MS. Using the SNaPshot technology of ultra-high throughput SNP typing to detect genetic markers, thousands of SNP loci can be detected in each reaction. ABI3730XL sequencer was used for simultaneous detection of several samples.

i. Basic information of children, including age, sex, birth status, siblings, adoption, and physical and sexual development.

Environment, including parents' educational level, , is used to document the sleep/wake patterns of children in the past week. iv. The dietary habit questionnaire, which consists of food consumption frequency, frequency of consuming plastic or tin packaged products, and frequency of consuming take-out food during the past week [36, 37] . v. Physical activity habits are assessed using the Physical Activity Questionnaire to record the days and times of high-, moderate-, and low-intensity activities of children during the past week [38] . vi. Items from the Pubertal Development Scale (PDS) are also adapted to assess puberty development, which is indicated by 4 items (growth spurt, pubic hair, skin changes, and breast development) in girls and by 5 items (growth spurt, pubic hair, skin changes, voice breaking, and facial hair) in boys. For each, the parent rates his or her child's development as "has not started yet, " "has barely started, " "has definitely started, " "seems complete, " or "unclear. " For each adolescent, the PDS score is the mean of the sum of items [39] .

Categorical variables will be reported as the number and percentage and analyzed by a chi-square test to compare the distribution between groups. The t-test, analysis of variance, and Wilcoxon rank-sum test will be used to compare the differences in distribution of continuous variables, which will be described by mean ± standard deviation (SD) for normal data or median with interquartile range for non-normal data. Based on the discovered pleiotropic genes and obesityrelated risk loci (SNPS), we will examine the distribution of gene polymorphisms (SNPS). For obesity-related SNPs of these genes, Effect size Weighting was carried out according to the method reported by Speliotes EK et al. [40] , and then genetic risk scores (GRS) will been calculated using the method established in our previous study [41] .That is, the number of effective Allele genes (Allele) was calculated, then the weighted value was multiplied, and finally the combined GRS of SNPs of all candidate genes in this study was accumulated.

The effects of exposure factors on outcomes will be evaluated after adjusting confounding factors using logistic regression or multiple linear regression. To analyze whether there is a linear trend of GRS among four groups: children with normal weight and normal puberty, with normal weight and precocious puberty, with overweight/ obesity and normal puberty, and with overweight/obesity and precocious puberty, the Cochran-Mantel-Haenszel Trend Test will be used. After 3 years' follow-up, a causal relationship model between exposure factors and outcomes will be established by using Mendelian randomization (MR) and Egger's regression models and assessed by sensitivity analysis. Statistical analyses will be performed by using IBM SPSS, STATA and R statistical packages. and the level of significance will be set at p < 0.05.

In this protocol, we described the study design, data collection methods, and analysis of a prospective cohort study in a population-based primary school children. According to the geographical distribution, we chose three cities in northern, central, and southern China and did our best to carry out this study to ensure the representativeness of the population. Students in grades 1 to 3 in randomly selected primary schools are expected to represent local school-age children.

By collecting information on factors such as children's family environment, lifestyle, and genes, this study will analyze the effect of these factors on children's physical development from the perspectives of heredity and environment. As a longitudinal study, this study will start earlier in development than previous studies to capture the impact of early childhood experiences on puberty development. Puberty is defined through visual and palpation inspection by a clinical doctor combined with ultrasonic diagnostics; previous studies have depended on selfreports or clinical palpation by non-professional volunteers, which are less sensitive and precise measurements, especially for breast development assessment among obese girls, which may lead to overestimating the precocious puberty detection rate among obese girls, since it is difficult to distinguish adipose tissue and breast tissue, so we will use ultrasonic examination in this study. Although the relationship between obesity and the sexual development of children has been confirmed in both genders by many earlier studies, due to the racial differences in the influence of obesity on sexual precocity, the relationship between obesity and early development is controversial among boys. Through this prospective cohort study, we will further investigate the relationship between obesity and development among Chinese mainland children.

To expand the previous research on puberty development, this study starts from the gene pleiotropy of obesity and takes the lead in studying the influence of the genetic polymorphism of obesity on precocious puberty. Although studies on genetic polymorphism of obesity or precocious puberty in girls have been performed previously, there has been no report on the combination of the two phenotypes, which is expected to reveal the genetic mechanism of the association between childhood obesity and precocious puberty.

At the same time, this study adopts several new analysis techniques, such as Mendelian randomization and Egger's test, to verify potential pleiotropic genes and further identify the mediating role of obesity in the relationship between pleiotropic genes and precocity. There is a lack of understanding of the direction and extent of the effect of life behaviors and habits on the internal rhythm and timing of puberty and how external factors interact with genetics to influence the indicators of adolescent development, such as secondary sexual characteristics development and sex hormone levels. Through these analysis methods, we can understand the direct and indirect effects of these influence factors on child growth and development.

This study has several limitations that should be considered. First, information on the diets and physical activity of children will be collected through parent reports, which may lead to bias due to a lack of information on children' behaviors at school. Second, detection of the number of genes and SNP is limited by research funding, and there can be differences in gene polymorphism distribution in different ethnic groups; therefore, we are basing our analysis on Han people according to the GWAS directory database (www. genome. gov/ gwast udies) and literature reports on potential candidate genes to screen pleiotropic genes related to obesity and precocious puberty. However, only 2 SNPs per candidate gene will be tested in this study. Despite these limitations, China is a country of great ethnic and genetic diversity. Up to now, there have been few prospective cohort studies on obesity and puberty development in China. Through this multicenter prospective population-based cohort study, we expect to find that obesity and the external environment indeed have an impact on puberty development in Chinese children. It is possible to develop directional intervention measures on the basis of the established evidence for youth at risk in families, communities, or schools to improve the adolescent health.

Enrollment of prospective longitudinal study participants had begun in October 2019. However, due to the COVID-19 epidemic, our investigation in different city cannot carried out simultaneously. Currently (as of April 2022), data collection of baseline survey in Zhongshan, Guangdong province and Qufu, Shandong province were done in December 2020, and the first year follow-up in two of above cities were finished in January 2022. Data collection and the analysis of the results is ongoing. The baseline survey in Dalian, Liaoning province is scheduled to begin in December 2022. The baseline characters of this survey in Zhongshan, Guangdong province and Qufu, Shandong province were summarized in Table 3 . 

Age of onset of puberty in Chilean boys according to testicular volume and Tanner stage

Timing of puberty in boys and girls: a population-based study

Onset of breast development in a longitudinal cohort

Age at menarche, depression, and antisocial behavior in adulthood

Public health implications of altered puberty timing

Mutations of the KISS1 gene in disorders of puberty

Analysis of the role of the Mc4r system in development, growth, and puberty of Medaka

Single-nucleotide polymorphisms in the LEPR gene are associated with divergent phenotypes for age at onset of puberty in Davisdale ewes

Nutritional determinants of the timing of puberty

A prospective study of childhood social hardships and age at menarche

Investigating the relationship between precocious puberty and obesity: a cross-sectional study in Shanghai

Sexual dimorphisms in the associations of BMI and body fat with indices of pubertal development in girls and boys

Age at menarche and its relationship to body mass index among adolescent girls in Kuwait

Early life growth predicts pubertal development in south African adolescents

Influence of the body weight on the onset and progression of puberty in boys

Timing of puberty in overweight versus obese boys

Obesity in children and adolescents: relationship to growth, pubarche, menarche, and voice break

Normative data for adiponectin, resistin, interleukin 6, and leptin/receptor ratio in a healthy Spanish pediatric population: relationship with sex steroids

Common variants of IL6, LEPR, and PBEF1 are associated with obesity in Indian children

Association between LEPR, FTO, MC4R, and PPARG-2 polymorphisms with obesity traits and metabolic phenotypes in school-aged children

Sample size for a Cohort study

Prevalence of grade 1, 2 and 3 thinness is associated with lower socio-economic status in children in Shanghai

Establishing a standard definition for child overweight and obesity worldwide: international survey

Body mass index cut offs to define thinness in children and adolescents: international survey

Variations in the pattern of pubertal changes in boys

Variations in pattern of pubertal changes in girls

Sexual precocity: sex incidence and aetiology

Management of central isosexual precocity: diagnosis, treatment, outcome

Update on precocious puberty: who should be treated?

Is obesity associated with early sexual maturation? A comparison of the Association in American Boys Versus Girls

National estimates of the pubertal milestones among urban and rural Chinese girls

National estimates of pubertal milestones among urban and rural Chinese boys

Timing of voice breaking in males associated with growth and weight gain across the life course

Breast US in children and adolescents

Urine bisphenol-a level and habits of life in relation to precocious puberty in girls

Association between television viewing and dietary patterns among preschool children

Physical activity and obesity mediate the association between childhood motor function and adolescents' academic achievement

Measurement of pubertal status with a Chinese self-report pubertal development scale

convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year submit your research ?

Association analyses of 249,796 individuals reveal 18 new loci associated with body mass index

Multi-variant study of obesity risk genes in African Americans: the Jackson heart study

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations

We would like to thank the children, parents, and teachers who participated in this research for their help and cooperation.Authors' contributions X.Y., Y.C., X.W., H.M., and S.L. conceived and designed the study. T.Y., X.L., P.X., H.K., C.L., and S.L. will be responsible for performing the research, including participants enrollment, data collection, and data extraction. H.M. and S.L. will be responsible for data analysis. T.Y., Y.Y., D.W. and S.L. drafted and revised the final manuscript. All of the authors have read and approved the manuscript.

According to the policy of the International Committee of Medical Journal Editors, the study has been registered in the ClinicalTrials.gov Protocol Registration and Results System (ClinicalTrials.gov, NCT04113070). Prior to the study, all of the personnel will receive unified training. Before data collection, personnel will explain the purpose of this study to the participants, and written consent will be obtained from the participants and their guardians.

Not applicable.

The authors declare that they have no competing interests. 1 Sanya Women and Children's Hospital, managed by Shanghai Children's