WO2014181962A1 - Method and apparatus for predicting metabolic syndrome using pomc gene - Google Patents

Abstract

The present invention provides a method and apparatus for predicting childhood metabolic syndrome including birth weight, body mass index (BMI), body fat mass (BFM), serum triglyceride (TG), glucose, and insulin level by detecting the methylation level of cord blood proopiomelanocortin (POMC).

Description

Method and apparatus for predicting metabolic syndrome using POMC gene

The present invention relates to a method and apparatus for predicting metabolic syndrome tendency after growth by detecting methylation levels of POMC genes in cord blood.

The prevalence of overweight and obesity in children and adults has increased worldwide in recent decades. Until recently, there was a relatively low prevalence in overweight and obesity in Korea, and the effects of obesity on disease status and mortality were ignored. However, the prevalence of overweight and obesity has recently increased dramatically in childhood. In Korea, childhood overweight and obesity rates nearly doubled from 1997 (12.3%) to 2005 (20.9%). Childhood obesity often persists to adults and is associated with the risk of chronic diseases such as type 2 diabetes, cardiovascular disease and hypertension.

On the other hand, eating patterns during pregnancy are important because pregnant women who have skipped meals and / or snacks for long periods of time without food are subject to physiological stress during pregnancy. Maternal nutritional imbalance is a pathological condition that increases the risk of complications for both mother and fetus. However, most young women are on a diet because they want to look better, and dietary restrictions on the mother during pregnancy cause the fetus to have a lower birth weight.

Recent animal experiments have shown that babies born due to artificial limitations in gestation are more obese than other rats. Other studies suggest that the prenatal period is an important time for metabolic programming induced by high-risk intrauterine fetal delay (IUGR), which leads to adult malnutrition, as well as premature overweight and obesity.

Physiological and genetic factors for fetal programming have recently been characterized. Epigenetic mechanisms, such as DNA methylation, are thought to be factors that affect the phenotype of an adult. The establishment and modification of DNA methylation has been studied in terms of various environmental and lifestyle exposures. In humans and animals, the ability of epigenetic markers to persist during development and potentially be delivered to offspring is necessary to produce a wide range of different phenotypes from the same genotype. In addition to natural diversity, changes in epigenetic markers are closely linked to the pathogenesis of offspring, such as childhood metabolic syndrome.

There is a need for a method for predicting metabolic syndrome in childhood through genes that cause such epigenetic changes, and to predict and prevent metabolic syndrome tendency after growth in the neonatal period.

It is an object of the present invention to provide a method and apparatus for predicting metabolic syndrome, which can more effectively predict the occurrence of metabolic syndrome in the growth process by a simple method such as blood tests of newborns.

In order to achieve the above object, the present invention, the step of detecting the methylation level of the POMC gene from the DNA of the newborn blood sample; And predicting metabolic syndrome tendency of the newborn according to the methylation level detected in the step.

The detecting of the methylation level may include detecting methylation of CpG at the exon 3 region of the POMC gene, and selecting from the group consisting of PCR, methylation specific PCR, quantitative PCR, pyro sequencing, and bisulfite sequencing. Characterized in that it is carried out.

The newborn blood sample is characterized in that it is taken from umbilical cord blood.

On the other hand, according to another embodiment of the present invention, a POMC gene comprising a PCR primer pair for amplifying a fragment containing a CpG of the POMC gene and a sequencing primer for pyro sequencing a PCR product amplified by the primer pair Provided is an apparatus for predicting metabolic syndrome.

The apparatus for predicting metabolic syndrome using the POMC gene may be a diagnostic kit or a nucleic acid chip for predicting metabolic syndrome tendency after growth of the newborn.

According to the present invention, by detecting the methylation of CpG of the POMC gene of umbilical cord blood of a newborn, there is an effect of providing a method for predicting the metabolic syndrome tendency after growth of the newborn.

By using the method for predicting metabolic syndrome tendency, diagnostic kit, and nucleic acid chip according to the present invention, it is possible to predict metabolic syndrome tendency simpler, more accurately, and faster than conventional methods, and in particular, it is possible to predict metabolic syndrome tendency beforehand in obesity. Prevent disease such as

1 is a schematic representation of a human POMC gene.

2 shows the relationship between POMC methylation of cord blood and peripheral blood of a child. Blue dots represent individual methylation values and red lines represent correlation values (r = 0.80, y = 0.8429x + 7.7534). Correlation coefficients and significance are derived from Pearson's correlation test.

3 shows changes in birth weight (KG) and childhood BMI values according to cord blood methylation status.

The following abbreviations are used herein to abbreviate the following words: ACTH is adrenocorticotrophic hormone; BMI, Body mass index; BFM, body fat mass; HOMA-IR, Homeostasis Model Assessment of Insulin Resistance; IUGRs include intrauterine growth restriction; MSH, melanocytestimulating hormones; POMC, proopiomelanocortin; TG, Triglyceride; WC is a wafer circumference.

According to an embodiment of the present invention, the cord blood proopiomelanocortin (POMC) methylation level is detected by detecting birth weight, body mass index (BMI), body fat mass (BFM), serum triglyceride (TG), Methods and devices for diagnosing childhood metabolic syndrome, including glucose, and insulin levels.

That is, the present invention uses, as a biomarker, CpG of the hypothalamic neuronal peptide POMC, which acts to suppress food intake and alter glucose processing and glucocorticoids, in order to predict the tendency of metabolic syndrome expression. The POMC gene has a nucleotide sequence of SEQ ID NO: 1, which is located on chromosome 2p23 and spans 7665 base pairs. POMC is expressed in immature neurons in the very early embryonic stage. In humans, the POMC gene has two CpG islands: one is the promoter region associated with exon1 and the other downstream is over exon3. If methylation is increased in obese children, methylation extends the CpG-hymethylated region to non-methylated CpG island regions (Intron 2 and Exon 3).

Next, an experimental example for detecting methylation level of POMC gene of newborn and predicting metabolic syndrome tendency in childhood or adulthood after growth will be described in detail.

Experimental Example

This experimental example is the result of a follow-up study from July to August 2011 of 249 newborns born between September 2001 and September 2003.

Anthropometric data including blood samples were collected. Information on birth weight and gestational age and maternal physical characteristics were used. In this experiment, 90 children with usable umbilical cord blood were included in the test subject. There is no difference in height, weight, or BMI among the children included or excluded. The characteristics of the subjects of the sex group are set forth in Table 1 below.

Table 1

Table 1 shows the basic characteristics of the test subject.

Body mass index (BMI) and body fat mass (BFM)

All anthropometric data was collected by trained experimenters. Each child's height, waist circumference (WC), and weight were measured using an automatic electronic measuring device (Model: DS-102, Dong-Sahn Jenix Co. Ltd, Seoul, Korea). Measure in kg. BMI is weight divided by height squared (kg / m ² ). BFM was measured using an automatic body composition analyzer (Model: Inbody 230, GE Healthcare, Madison, Wis., USA).

Blood Control and Biochemical Assessment

Childhood blood samples are obtained from the middle vein of the arm with a vacuum vessel tube containing EDTA or serum tubes after an overnight fast. All blood samples obtained from the subject are stored at -70 ° C. Biochemistry (glucose, triglyceride (TG), cholesterol, and HDL-cholesterol) concentrations in blood are measured with an automated analyzer (Model: 7180, Hitachi, Tokyo, Japan). Serum insulin resistance is determined by the commonly used Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) method, which is calculated by (plasma glucose [mmol / L] x insulin [μIU / mL]) / 22.5.

Numerical Analysis of DNA Methylation by Pyro Sequencing

Genomic DNA in umbilical cord blood is extracted from 250 μl of whole blood according to the product protocol using the DNeasy Blood & Tissue Kit Kit (Qiagen, Valencia, CA, USA). Purity and concentration of the isolated DNA is determined using a spectrophotometer (Model: ND-2000, Nanodrop Technologies, Wilmington, DE, USA).

Downstream exon 3 of POMC is amplified using a biotin reverse primer (SEQ ID NO: 3) and a forward primer (SEQ ID NO: 2) designed with PSQ Assay Design (Biotage AB, Uppsala, Sweden) (Table 2, Figure 1). .

TABLE 2

Table 2 shows the primers and PCR conditions of POMC for pyrosequencing reactions.

Each sample genomic DNA (20 ng) is treated with sodium hydrogen sulfite according to the manufacturer's instructions using the EZ DNA methylation kit (ZYMO Research, CA, USA). Each target region of the hydrogen sulfite-converted DNA is amplified by reacting 25 μl with a primer set and 5 units of Taq polymerase (Solgent Co., Daejeon, Korea). The amplification reaction is as follows. First, the DNA sample is heated to 94 ° C. for 10 minutes and then amplified for 45 cycles including 30 seconds at 94 ° C., 30 seconds at annealing temperature, and 30 seconds at 72 ° C. All reactions are then incubated at 72 ° C. for 10 minutes and cooled to 4 ° C. PCR products are visualized on 1.5% agarose gels by bromination staining for confirmation.

The pyrosequencing reaction consists of sequencing primers (Table 2, SEQ ID NO: 4) according to the manufacturer's instructions in the PSQ HS 96A system (Biotage AB). The methylation index (MtI) of each gene promoter and of each sample is calculated as the mean value of mC (mC + C), which is the ratio of methylated cytosine (mC) and cytosine (C) for all test CpGs in the target region.

Statistical analysis

Insulin and TG are log-transformed to meet steady-state statistical assumptions. Since the individual correlations at each site are quite high, the average POMC methylation value is used. POMC methylation levels in cord blood are classified into high, medium and low methylation groups according to their methylation status (above 90th percentile, 11-89th percentile, below 10th percentile). Multiple correlation analysis of the tested parameters is performed and given Pearson's correlation index. Metabolic components in the blood and anthropometric measurements are compared using ANOVA by POMC methylation status. The association between birth weight and cord blood POMC methylation is assessed using an analysis of controlled covariance (ANCOVA) for prepregnancy weight, sex of offspring and gestational age. To control potential disturbances, according to the variance of the description, a covariate factor with a meaning of <0.1 is chosen for ANCOVA and multiple linear regression models. The association size of body fat or metabolic index with POMC methylation is assessed using multiple linear regression analysis adjusted for gender, birth weight, age, and POMC methylation in children. Statistical analysis is performed using SAS version 9.3 (SAS Institute Inc., Cary, NC, USA). All analyzes were bilaterally validated and p values <0.05 are considered statistically significant.

Experiment result

The mean cord blood methylation of the POMC gene value is 49.53 and the methylation of each site is 52.48 (site 1), 50.30 (site 2), 47.57 (site 3), 47.78 (site 4). Childhood mean methylation at 49.51 is similar to cord blood (see Table 3).

TABLE 3

Table 3 is an overview of POMC DNA methylation at birth and childhood.

Umbilical cord blood pomc methylation is significantly associated with childhood POMC methylation values ( r = 0.80, p = 0.0001) (see Table 4 and FIG. 2).

Table 4

Table 4 shows the correlation between maternal POMC methylation in cord blood (birth) and POMC methylation in children.

Table 5

Table 5 shows a comparison of post-natal POMC methylation (%) according to birth weight, maternal characteristics during pregnancy, and cord blood pro-opiomelanocortin methylation status.

Dividing the study group into three groups according to the degree of POMC methylation (below 10th percentile, 11-9th percentile, above 90th percentile), and the birth weight of the highly methylated POMC group (above 90th percentile) (p = 0.04) ) Was significantly lower than the other groups (see Table 5, Figure 3). However, gestational characteristics, such as gestational age, prenatal weight and BMI, and increased weight during pregnancy, did not differ between the POMC methylated groups (see Table 5). The average POMC methylation values here are 35.57% (10 percentile or less), 49.77 (11-89 percentile), and 59.60 (above 90 percentile) (p = 0.001).

Table 6

	Umbilical Cord Blood POMC Methylation
	≤10 th (n = 10)	p value †	11 ~ 89 th (n = 70)	p-value †	≥90 th (n = 10)	p-value ‡
BMI (kg / m 2 )	15.95 (0.90)	0.57	16.49 (0.28)	0.19	17.76 (0.92)	0.40
BFM (kg)	6.13 (1.09)	0.80	6.42 (0.34)	0.16	8.09 (1.12)	0.37
WC (cm)	56.55 (2.30)	0.74	57.36 (0.71)	0.67	58.43 (2.34)	0.88
Log TG	4.06 (0.19)	0.45	4.21 (0.06)	0.01	4.74 (0.19)	0.04
HDL-c (mg / dL)	63.04 (4.15)	0.49	60.04 (1.29)	0.06	51.50 (4.24)	0.16
Cholesterol (mg / dL)	161.75 (8.95)	0.63	166.32 (2.79)	0.14	152.10 (9.14)	0.26
Schlin	1.96 (0.11)	0.53	2.03 (0.03)	0.02	2.32 (0.11)	0.07
Glucose (mmol / L)	4.44 (0.14)	0.57	4.36 (0.04)	0.65	4.29 (0.15)	0.80
HOMA indicator	1.39 (0.23)	0.46	1.57 (0.07)	0.06	2.03 (0.23)	0.17

In childhood, the BMI of the hypermethylated POMC group (above 90 percentile) is not significantly higher than the other groups (p = 0.40) (see Table 6, Figure 3). Instead of the POMC methylated group 3, the BFM values were 6.13 and 6.42 kg below the 10th and 11-89th percentile groups, while 8.09 kg for the high methylated group (above 90th percentile). However, waist circumference was similar in all groups (P = 0.88). In child blood biochemical components, stratified according to cord blood POMC methylation levels, TG (log converted, p = 0.04) is a significantly significantly higher value in the hyper methylation group. TG values are significantly higher in the hypermethylated group than in the middle group (4.74 vs 4.21, p = 0.01), such as dummy variables obtained by covariance analysis adjusted to gender, birth weight, age, and methylation values. Insulin (log converted, p = 0.07) is a significant increase in the difference of hypermethylated groups. In addition, insulin (log converted) is a significantly higher increase in hypermethylated groups from the ANCOVA model (2.32 vs 2.03, p = 0.02). HDL-cholesterol values vary greatly, but tend to be lower in the hypermethylated group (p = 0.06). Insulin resistance to HOMA index values is statistically significant (p = 0.06) between the two groups when the middle group (11-89 percentile) is 1.57 and the high methyl group (over 90 percentile) is 2.03. Plasma glucose values in all groups showed similar size results (p = 0.80).

Result analysis

From these experiments it can be seen that POMC methylation in cord blood is involved as an early marker of metabolic syndrome. Recent studies on nutrition during pregnancy have found that at relatively low nutritional levels, obesity, glucose tolerance and insulin sensitivity of the offspring are relatively worsened and the risk of obesity is increased. These changes in pubertal development include the growth of type 2 diabetes, cardiovascular disease, short growth and IUGR related diseases including polycystic ovary syndrome. IUGR can be symmetrical or asymmetrical depending on the timing of the insult during pregnancy. Adolescent obesity can be explained by a change in phenotype during exposure to adverse environmental conditions in the uterus.

The experiments show that the POMC methylation status and the childhood methylation status of cord blood are significantly higher. Maternal diet during pregnancy has a devastating effect on the DNA methylation patterns of genes involved in cardiovascular disease and blood pressure control. High expression of the POMC gene affects immature neurons in early embryonic stages. Recent research shows that IUGR is due to epigenetics. In particular, epigenetic changes can be active. From these experiments it can be seen that environmental factors act on the genotype during development regarding the epigenetic profile of the adult. Fetal hypothalamic appetite control network is a major target for epigenetic fetal programming and IUGR. Methylation of the POMC gene in the hypothalamic pathway is centrally regulated by the circulation of leptin and insulin. Therefore, it can be predicted that the POMC methylation level at the fetal developmental stage is determined.

In this experiment, the hypermethylated group has significantly lower birth weight and childhood metabolic syndrome tendency. In addition, hypermethylated groups show TG concentration indicators of childhood metabolic syndrome. This includes evaluating metabolic syndrome with log TG, log HDL ratio, and the occurrence of mutual reinforcement or proliferation of disturbing factors that affect factors such as eating and exercise status.

Regulation of food intake and energy balance appears to affect the loss of function in the melanocortin MC4R gene and in POMC. The hypothalamus also responds to appetite suppressing leptin by interacting with MC4R expressing neurons in the arch nucleus. Leptin intake during lactation continues to influence the expression of hypothalamic factors involved in food intake control and regulated at the median level by leptin, and in particular the POMC gene is exposed to high fat calories. This is an important role combined with appetite control and fat metabolism by POMC methylation.

In particular, this example shows an association between birth weight and childhood metabolic syndrome and among exon 3 regions of the POMC gene on epigenetic markers. Hypermethylation parameters targeting individual CpGs at the intro 2 (Alu sequence) and exon 3 boundaries of POMC by hydrogen sulfite salt sequencing are significantly associated with childhood obesity. A limitation in previous studies is the use of peripheral blood from newborns.

However, it is important to note that this example uses the cord blood review analysis of the first study as predicting a marker of the methylation level status of the neonatal POMC gene. In addition, downstream region exon 3 hypermethylation within the POMC site represents the DNA methylation variable first identified as being associated with risk for childhood metabolic syndrome.

POMC methylation and low birth weight increase the likelihood of developing metabolic syndrome in childhood and adulthood. Thus, the present invention provides that POMC methylation in cord blood is closely related to the obesity diagnostic label and that methylation of cord blood at birth can be an early predictor of metabolic syndrome in childhood.

When the subjects were divided into three groups according to the POMC methylation status, the birth weight of the hypermethylated POMC group (p = 0.04) was significantly lower than the other groups. In the examples, the POMC methylation values for each group are 37.57% (up to 10 percentile), 49.77% (11 to 89 percentile), and 59.60 (above 90 percentile) (p = 0.001). In this example, stratification of cord blood POMC methylation levels in the biochemical components of childhood blood results in high TG (log transformation, 4.74 vs. 4.21, p = 0.21) and insulin (log transformation, 2.32 vs 2.03, p = 0.02). Methylated groups (above 90th percentile) are significantly higher than intermediate groups (11-89th percentile). HDL-cholesterol values tend to be slightly lower in the high methylated group (p = 0.06). The HOMA index value of the middle group was 1.57 and the hypermethylated group was 2.03, but the difference between the two groups was slightly significant (p = 0.06).

POMC methylation and low birth weight increase the risk of metabolic syndrome in childhood and adulthood. The results of the present invention indicate that POMC methylation in cord blood is closely related to metabolic syndrome, and POMC methylation of cord blood at birth is an early predictive marker of childhood metabolic syndrome.

The present invention detects cord blood proopiomelanocortin (POMC) methylation levels to detect birth weight, body mass index (BMI), body fat mass (BFM), serum triglyceride (TG), glucose, and insulin levels. It provides a method and apparatus for predicting childhood metabolic syndrome comprising a.

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Claims (11)

1. Detecting the methylation level of the POMC gene from the DNA of the blood sample of the newborn; And

   Predicting metabolic syndrome tendency of the newborn according to the methylation value detected in the step metabolic syndrome prediction method using the POMC gene.
2. The method of claim 1,

   The detecting of the methylation level is metabolic syndrome prediction method using the POMC gene for detecting the methylation of CpG of the exon 3 region of the POMC gene.
3. The method of claim 2,

   Exon 3 site of the POMC gene metabolic syndrome prediction method using the POMC gene comprising the nucleotide sequence of SEQ ID NO: 1.
4. The method of claim 1,

   The step of detecting the methylation level is a metabolic syndrome prediction method using the POMC gene is performed by a method selected from the group consisting of PCR, methylation specific PCR, quantitative PCR, pyro sequencing and bisulfite sequencing.
5. The method of claim 1,

   The blood sample is a method for predicting metabolic syndrome using the POMC gene collected from the cord blood of the newborn.
6. An apparatus for predicting metabolic syndrome using a POMC gene comprising a PCR primer pair for amplifying a fragment including a CpG of a POMC gene and a sequencing primer for pyro sequencing a PCR product amplified by the primer pair.
7. The method of claim 6,

   The PCR primer pair is a metabolic syndrome prediction device using the POMC gene which is a nucleotide sequence represented by SEQ ID NO: 2 or 3.
8. The method of claim 6,

   The sequencing primer is a metabolic syndrome prediction device using the POMC gene, characterized in that the base sequence represented by SEQ ID NO: 4.
9. The method of claim 6,

   The CpG is a device for predicting metabolic syndrome using the POMC gene located in the exon 3 region of the POMC gene.
10. A pair of PCR primers for amplifying fragments containing CpG of the POMC gene of DNA of a blood sample of a newborn and sequencing primers for pyro sequencing the PCR product amplified by the primer pairs, and metabolizing after growth of the newborn Diagnostic kit for predicting syndrome tendency.
11. PCR primer pairs for amplifying fragments containing CpG of POMC gene of POMC gene of DNA of blood samples of newborns and sequencing primers for pyro sequencing PCR products amplified by the primer pairs. Diagnostic nucleic acid chip predicting metabolic syndrome tendency after growth.

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