*Article* **Association and Interaction Effect of** *BHMT* **Gene Polymorphisms and Maternal Dietary Habits with Ventricular Septal Defect in Offspring**

**Manjun Luo 1, Tingting Wang 2,\*, Peng Huang 3, Senmao Zhang 1, Xinli Song 1, Mengting Sun 1, Yiping Liu 1, Jianhui Wei 1, Jing Shu 1, Taowei Zhong 1, Qian Chen 1, Ping Zhu 4,\* and Jiabi Qin 1,2,4,5,6,\***


**Abstract:** This study attempted to learn the association between maternal betaine-homocysteine methyltransferase (*BHMT*) gene polymorphisms, maternal dietary habits, and their interactions with the risk of ventricular septal defects (VSD) in offspring. A total of 426 mothers of VSD children and 740 control mothers were included in the study. Logistic regression was used to evaluate the level of associations and interaction effects. Our study suggested that mothers reporting excessive intake of smoked foods (aOR = 2.44, 95%CI: 1.89–3.13), barbecued foods (aOR = 1.86, 95%CI: 1.39–2.48), fried foods (aOR = 1.93, 95%CI: 1.51–2.46), and pickled vegetables (aOR = 2.50, 95%CI: 1.92–3.25) were at a significantly higher risk of VSD in offspring, instead, mothers reporting regular intake of fresh fruits (aOR = 0.47, 95%CI: 0.36–0.62), fish and shrimp (aOR = 0.35, 95%CI: 0.28–0.44), fresh eggs, (aOR = 0.56, 95%CI: 0.45–0.71), beans (aOR = 0.68, 95%CI: 0.56–0.83), and milk products (aOR = 0.67, 95%CI: 0.56–0.80) were at a lower risk of VSD in offspring. In addition, maternal *BHMT* gene polymorphisms at rs1316753 (CG vs. CC: aOR = 2.01, 95%CI: 1.43–2.83) and rs1915706 (CT vs. TT: (aOR = 1.81, 95%CI: 1.33–2.46) were significantly associated with increased risk of VSD in offspring. Furthermore, a significant interaction between *BHMT* polymorphisms and maternal bean intake was identified in the study. In conclusion, Maternal *BHMT* polymorphisms at rs1316753 and rs1915706, dietary habits as well as their interaction were observed to be significantly associated with the risk of VSD in offspring.

**Keywords:** ventricular septal defects; *BHMT* gene plolymorphisms; dietary habits; interaction effects

#### **1. Introduction**

Congenital heart disease (CHD) is typically defined as a gross structural abnormality of the heart and/or great vessels that is present at birth [1,2]. It has been reported that the birth prevalence of CHD has increased significantly since the 1930s and reached a maximum of over 9 per 1000 live births since 1995 [1,3]. Ventricular septal defect (VSD) has been recognized as the most common congenital cardiac malformation and accounts for roughly 30–40% of all cardiac anomalies [1,4]. Over the past decades, considerable inherited

**Citation:** Luo, M.; Wang, T.; Huang, P.; Zhang, S.; Song, X.; Sun, M.; Liu, Y.; Wei, J.; Shu, J.; Zhong, T.; et al. Association and Interaction Effect of *BHMT* Gene Polymorphisms and Maternal Dietary Habits with Ventricular Septal Defect in Offspring. *Nutrients* **2022**, *14*, 3094. https:// doi.org/10.3390/nu14153094

Academic Editors: Louise Brough, Gail Rees and Jamileh Movassat

Received: 20 May 2022 Accepted: 25 July 2022 Published: 28 July 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

causes and noninherited modifiable factors have been implicated in the development of CHD and its subgroups [5–8]. Recently, there has been a consensus that genetic factors and environmental factors interact in the etiology of most nonsyndromal forms of CHD [9,10], naturally including VSD.

A recent review showed strong evidence that oral prenatal fortification and supplementation dosing of folic acid (FA) can prevent the incidence of VSD and atrial septal defect (ASD) [11]. Women with a diverse diet during pregnancy (dietary diversity score, DDS ≥5) had lower risks of having fetuses with total CHD and VSD [12]. Furthermore, the dietary intake of vitamins and minerals was found to be associated with a reduced risk of CHD in offspring, including B-vitamin, vitamin D, zinc, and selenium [13–15]. Since different nutrients interact with one another in many metabolic pathways, it seems that the association would not remain constant when various nutrients coexist in the same food. In addition, the dietary pattern differs a lot owing to the discrepancy in economics, geographical environment, social culture, race, and so on. Therefore, the first concern we would care to discuss is the association between maternal dietary habits and VSD in offspring.

The human betaine-homocysteine methyltransferase (BHMT) gene maps to 5q13.1–q15, spans about 20 kilobases of DNA and contains eight exons and seven introns [16,17]. The enzyme it encodes, betaine-homocysteine methyltransferase, catalyzes the transfer of a methyl group from betaine to homocysteine (Hcy), forming dimethylglycine and methionine. Generally, the homeostasis of plasma homocysteine benefits from the transulfuration pathway involving cystathionine β synthase (CBS) and the remethylation pathway involving BHMT, BHMT2, and methionine synthase (MS) (Figure 1) [18]. In the latter pathway, the catalytic activity of BHMT2 is absolutely diet-dependent since its substrate, S-methylmethionine, can only be biosynthesized by various plants mainly belonging to the Brassicaceae family rather than mammals [19,20]. Experimental research conducted in mice suggested that BHMT is a predominant enzyme for the elimination of Hcy while the MS has little excess capacity to methylate the Hcy [18]. Therefore, the remethylation reaction catalyzed by BHMT seems to play a vital role in preventing the toxic accumulation of Hcy. In fact, BHMT catalyzes up to 50% of homocysteine metabolism in the human liver, where the enzyme is highly expressed [21,22]. The latest literature revealed that elevated Hcy concentrations acted as a risk factor for multiple congenital anomalies in human production, mainly comprising neural tube defects (NTD), orofacial clefts, and CHD [23–25]. The discovery has been generally accepted that the 677 C→T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene contributed to elevated tHcy and is a genetic risk factor for diseases associated with hyperhomocysteinaemia [26]. Moreover, this mutation has been applied to antenatal screening for pregnant women in China. The thought naturally emerged that polymorphisms of the BHMT gene exist that reduce BHMT activity and increase plasma Hcy levels and thus increase malformation risk. In fact, research has been dedicated to exploring the association between BHMT gene polymorphisms and CHD, but with fixed results and little involving subgroups of CHD [27–30]. In this study, we focused on the largest subcategory of CHD, namely, VSD, to detect its association with polymorphisms of the maternal BHMT gene.

In addition, betaine, the substrate of BHMT, can be either obtained from food resources or produced from choline endogenously [31]. Likewise, choline can also be produced endogenously via the hepatic phosphatidylethanolamine N-methyltransferase (PEMT) pathway. However, most people must consume this nutrient exogenously to prevent deficiency [32]. Therefore, the BHMT activity, to a certain degree, is diet dependent. Animal studies did observe that pane of nutrition or the supply of some nutrients, including choline and methionine, can alter BHMT activity [33–35]. In addition, it has been reported that women with a high intake of one-carbon cofactors had a lower risk of congenital anomalies in offspring, such as the neural tube defect (NTD) and perimembranous ventricular septal defect (VSDpm) [36,37]. Overall, these valuable clues were collected to put forward a

reasonable hypothesis that BHMT gene polymorphisms may interact with maternal dietary habits on congenital anomalies.

**Figure 1.** Pathways of homocysteine metabolism. Abbreviation: BHMT Betaine-homocysteine S-methyltransferase; MS methionine synthase; CBS cystathionine β-synthase; CSE cystathionineγ-lyase; THF tetrahydrofolate; DMG dimethylglycine; SAM S-adenosylmethionine; SAH Sadenosylhomocysteine.

In this study, we determined VSD, the most common subgroup in CHD, as the interested outcome variable, which is relatively more sensitive to maternal nutrient intake. A hospital-based case-control study was carried out in an attempt to learn the following questions: a. the association of maternal dietary habits with risk of VSD in offspring; b. the association of polymorphisms of maternal BHMT gene with risk of VSD in offspring; c. the interaction between BHMT genetic variants and maternal dietary habits on VSD.

#### **2. Materials and Methods**

#### *2.1. Design and Participants*

This is a hospital-based case-control study that started in February 2018 and was over in March 2020. The cases and controls came from different departments in the same hospital, Hunan children's hospital, which is famous partly for its sophisticated diagnosis and treatment techniques for CHD within the province. Considering the characteristics of the relatively low incidence of VSD compared with other chronic diseases, a convenient sampling method was used in the recruitment of the cases. VSD children, verified by both doppler echocardiography and surgery, were consecutively recruited from the Department of Cardiothoracic Surgery. Children in the control, free of any congenital malformations, were randomly selected from the Department of Child Healthcare. It is worth noting that cases only included VSD children that may or may not be diagnosed with other congenital heart diseases; those coexisting with any other extra-cardiac malformations were excluded from the study. Additionally, informed consent was obtained from all of the participants, and the possible consequences of the study were explained. The exclusion criterions mainly included: minority mothers, mothers conceiving children through in vitro fertilization or other conception methods, adoptive mothers or stepmothers, and mothers suffering from mental disorders or any other physical diseases so that this did not hinder the provision of accurate exposure information and blood samples. Finally, a total of 426 mothers of VSD children and 740 control mothers were included in the study.

The protocol of this study was in accordance with the guidelines of the 1964 Helsinki Declaration, and the Ethics Committee of Xiangya School of Public Health, Central South University, officially approved this study in January 2018. (no. XYGW-2018-36).

#### *2.2. Information Collection*

The outcome we focused on in the study was VSD in offspring, which was diagnosed by professional physicians via both doppler echocardiography and surgery. The interested exposures were maternal dietary habits in early pregnancy, which were collected from a self-designed food frequency questionnaire. We consulted The Dietary Guidelines for Chinese Residents and went deep into the local food culture to develop the questionnaire. Eleven main categories were determined, involving smoked foods, barbecued foods, fried foods, pickled vegetables, fresh vegetables, fresh fruits, fresh meat, fish and shrimp, fresh eggs, beans, and milk products. Each category was provided with three choices: a. hardly (less than or equal to two times per week); b. sometimes (three to five times per week); c. often (more than or equal to six times per week). The questionnaire was pre-investigated using eligible mothers (test–retest reliability: r = 0.826; internal consistency: α = 0.769).

In addition, we also collected various pieces of maternal information that might influence the outcomes of their offspring, mainly including the child-bearing age (<35 years or ≥35 years), pre-pregnancy BMI (calculated with their pre-pregnancy height and weight, <18.5, 18.5–23.9, 24–26.9, or ≥27), education level (less than primary or primary, junior high school, high school or technical secondary school, college or above), consanguineous marriages (yes or no), gestational diabetes mellitus (yes or no), gestational hypertension (yes or no), abnormal pregnancy history before this pregnancy (yes or no), congenital malformations in family members (yes or no), exposure of environmental pollutants (yes or no), antibiotic use in early pregnancy (yes or no), tobacco exposure in early pregnancy (yes or no), alcohol exposure in early pregnancy (yes or no), and periconceptional folate use (yes or no).

An epidemiological survey was conducted by well-trained investigators when participants were waiting for their operation arrangements in the wards or medical check-ups in the Department of Child Health. In China, every expectant mother has a personal Maternal and Child Health Manual, which provides their sociodemographic information, the results of regular medical check-ups, and necessary exposure information. So, in the course of the investigation, we consulted the participants' manual to further confirm the abovementioned information obtained from face-to-face interviews, which enabled us to reduce recall bias to a certain extent.

#### *2.3. Sample Collection and Genotyping*

Five milliliters of peripheral venous blood were collected from every single participant after the face-to-face interview. All of the obtained blood samples would be brought back to the laboratory at low temperatures (≤4 ◦C) within twelve hours and then divided into two layers using a high-speed centrifuge: the blood cell layer and the plasma layer. Both were stored in an ultra-low-temperature freezer until genotyping. The DNA was extracted from the blood cell samples with the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA). Genotyping was performed by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry MassARRAY system (Agena iPLEX assay, San Diego, CA, USA). The laboratory technicians who performed SNP detection and recorded the genotype data were blind to whether each sample was from the cases or controls, thereby reducing selection bias to some extent.

Before genotyping, we consulted the NCBI and HapMap databases to determine the major SNP sites of the *BHMT* gene and simultaneously excluded the SNPs whose minor allele frequencies (MAF) were less than 10%. Furthermore, we imposed a minimum SNP genotyping call rate at the level of 50%, which was applied to ensure the data integrity of the individual's genotypes. Moreover, the success rates for the SNPlex assays were >94% for 2 SNPs, from 90 to 94% for 2 SNPs. Finally, these genetic loci (rs3733890, rs1316753, rs567754, and rs1915706) were selected as candidate loci for this study.

#### *2.4. Statistical Analysis*

The data for the qualitative variables were expressed as absolute numbers (percentages). The chi-square test was used to assess the differences in qualitative variables across groups. The Hardy–Weinberg equilibrium (HWE) test was used to compare the differences in genotype distribution frequency in the control group (significance level at *p* < 0.01). We utilized a logistic regression model to detect whether the association between maternal dietary habits in early pregnancy, *BHMT* gene polymorphisms, and VSD in offspring existed and the level of the association. Both univariate and multivariate logistic regressions were adopted; the crude odds ratio (cOR) and its 95% confidential intervals (CI) were calculated by the former one without any adjustment; the adjusted odds ratio (aOR) and its 95% confidential intervals (CI) were calculated by the latter one, which adjusted for the significant confounders found using the chi-square test. For the significant SNPs and maternal dietary habits, these originally ternary variables were converted into binary variables. We then introduced all of the potential confounders, genetic factors, environmental factors, and their multiplicative interaction term into the same logistic regression model to determine the presence or absence of gene–environment interaction and assess its significance. When it comes to multiple hypothesis testing, the false discovery rate (FDR) based on the Benjamini–Hochberg method was used to correct for bias. A false discovery rate P value (FDR\_P) of <0.1 was considered to be statistically significant. The calculation of FDR\_P was completed using R software (version 4.1.3, stats package). Basic analyses were performed using SPSS 26.0 software (SPSS Inc., Chicago, IL, USA). The statistically significant results were those with the two-sided *p*-value < 0.05, except where otherwise specified.

#### **3. Results**

#### *3.1. Comparison of Maternal Baseline Characteristics*

In the study, we recruited a total of 426 mothers of VSD children for cases and 740 mothers of non-congenital malformation children for controls. The selection of participants conformed strictly to the pre-made inclusion and exclusion criteria. The median (interquartile range) age of the children the in cases and controls was 8.4 (5.7) months and 7.8 (4.3) months, respectively. The comparisons of the maternal baseline characteristics between cases and controls are summarized in Table 1. There were statistically significant differences between the two groups in the following factors: pre-pregnancy BMI, education level, consanguineous marriages, gestational diabetes mellitus, gestational hypertension, abnormal pregnancy history before this pregnancy, congenital malformations in family members, exposure to environmental pollutants, antibiotic use in early pregnancy, tobacco exposure in early pregnancy, alcohol exposure in early pregnancy, and periconceptional folate use (all *p* values < 0.05). These abovementioned factors would be adjusted as confounders when evaluating the association of maternal dietary habits, SNPs of the *BHMT* gene, and their interactions with VSD in offspring.

#### *3.2. Maternal Dietary Habits and the Risk of VSD in Offspring*

The association of maternal dietary intake in early pregnancy with the risk of VSD in offspring is shown in Table 2. Both univariate and multivariate logistic regression indicated that smoked foods, barbecued foods, fried foods, pickled vegetables, fresh fruits, fish and shrimp, fresh eggs, beans, and milk products were significantly associated with the risk of VSD in offspring. Specifically, children were predisposed to VSD when their mothers reported excessive intake of smoked foods (aOR = 2.44, 95%CI: 1.89–3.13), barbecued foods (aOR = 1.86, 95%CI: 1.39–2.48), fried foods (aOR = 1.93, 95%CI: 1.51–2.46), and pickled vegetables (aOR = 2.50, 95%CI: 1.92–3.25). Instead, a significantly decreased risk of VSD was observed in children whose mothers reported regular intake of fresh fruits (aOR = 0.47, 95%CI: 0.36–0.62), fish and shrimp (aOR = 0.35, 95%CI: 0.28–0.44), fresh eggs, (aOR = 0.56, 95%CI: 0.45–0.71), beans (aOR = 0.68, 95%CI: 0.56–0.83), and milk products (aOR = 0.67, 95%CI: 0.56–0.80).


**Table 1.** Comparison of maternal baseline characteristics in cases and controls.

Abbreviations: BMI body mass index. <sup>a</sup> Classification according to Chinese standard for obesity BMI.

#### *3.3. Maternal BHMT Gene Polymorphisms and the Risk of VSD in Offspring*

Table 3 displays the genotypic distribution of four SNPs between two groups and the results of the HWE test in the controls. All of the SNPs were in accordance with the Hardy–Weinberg equilibrium (all of the *p* values were <0.05), indicating that the sample was qualified for good group representativeness.


**Table 2.** Maternal dietary habits and the risk of VSD in offspring.

Abbreviations: VSD ventricular septal defect, cOR crude odds ratio, aOR adjusted odds ratio, CI confidence interval. <sup>a</sup> Adjusted for pre-pregnancy BMI, education level, consanguineous marriages, gestational diabetes mellitus, gestational hypertension, abnormal pregnancy history before this pregnancy, congenital malformations in family members, exposure to environmental pollutants, antibiotic use in early pregnancy, tobacco exposure in early pregnancy, alcohol exposure in early pregnancy, periconceptional folate use. <sup>b</sup> Hardly was defined as less than or equal to two times per week. <sup>c</sup> Sometimes was defined as three to five times per week. <sup>d</sup> Often was defined as more than or equal to six times per week.


**Table 3.** Genotypic frequencies of maternal *BHMT* polymorphisms and *P* values of HWE test.

Abbreviations: *BHMT* betaine-homocysteine methyltransferase, HWE Hardy–Weinberg equilibrium, SNP single nucleotide polymorphism, MAF minimum allele frequency. <sup>a</sup> AA = homozygous wild—type; AB = heterozygous variant type; BB = homozygous variant type.

The association of maternal *BHMT* gene polymorphisms with the risk of VSD in offspring based on logistic regression analysis was summarized in Table 4. After adjusting for potential confounders, statistically significant associations were found between the polymorphisms of the *BHMT* gene at rs1316753, rs1915706, and VSD in offspring. For rs1316753, mothers carrying the CG genotype (aOR = 2.01, 95%CI: 1.43–2.83) were at a significantly higher risk of VSD in offspring compared with those who had the CC genotype. In addition, the dominant model (aOR = 1.88, 95%CI: 1.36–2.61) and the additive model (aOR = 1.30, 95%CI: 1.06–1.60) of rs1316753 were also observed to be significantly associated with increased risk of VSD in offspring. For rs1915706, compared to the TT genotype, mothers with the CT genotype (aOR = 1.81, 95%CI: 1.33–2.46) were more likely to have VSD children. Additionally, the dominant model (aOR = 1.84, 95%CI: 1.37–2.48) and the additive model (aOR = 1.61, 95%CI: 127–2.05) of rs1915706 were significantly associated with an increased risk of VSD in offspring.

**Table 4.** Polymorphisms of maternal *BHMT* gene associated with risk of VSD in offspring based on logistic regression analysis.



**Table 4.** *Cont.*

Abbreviations: *BHMT* betaine-homocysteine methyltransferase, VSD ventricular septal defect, SNP single nucleotide polymorphism, cOR crude odds ratio, aOR adjusted odds ratio, CI confidence interval, FDR\_P, false discovery rate P value. <sup>a</sup> Adjusted for pre-pregnancy BMI, education level, consanguineous marriages, gestational diabetes mellitus, gestational hypertension, abnormal pregnancy history before this pregnancy, congenital malformations in family members, exposure of environmental pollutants, antibiotic use in early pregnancy, tobacco exposure in early pregnancy, alcohol exposure in early pregnancy, periconceptional folate use. <sup>b</sup> The dominant model means heterozygote and mutant type homozygote vs. wild type homozygote. <sup>c</sup> The recessive model means mutant type homozygote vs. heterozygote and wild type homozygote. <sup>d</sup> The additive model means mutant type homozygote vs. heterozygote vs. mutant type homozygote.

#### *3.4. Interaction of the Polymorphisms of BHMT Gene and Maternal Dietary Habits on the Risk of VSD in Offspring*

Figure 2 shows the level of association between genetic variants of the *BHMT* gene, maternal dietary intake, and VSD in offspring. The interaction of *BHMT* gene polymorphisms and maternal dietary habits in early pregnancy based on multivariate logistic regression analysis is displayed in Table 5. For rs1316753, statistically significant interactions were observed between the variant genotypes (CG + GG) and excessive intake of pickled vegetables (aOR = 0.48, 95%CI: 0.24–0.95) and beans (aOR = 0.40, 95%CI: 0.17–0.95). Nevertheless, this significance vanished from the multiple test corrections based on the Benjamini–Hochberg method (both FDR-*p* values > 0.1). As for rs1915706, there were significant interactions between the variant genotypes (CT + CC) and a regular intake of beans (aOR = 0.33, 95%CI: 0.15–0.73, FDR-*p* = 0.035).

The crossover analysis was conducted to further elucidate the interaction between *BHMT* gene polymorphisms at rs1915706 and maternal bean intake on the risk of VSD in offspring (Table 6 and Figure 3). Mothers who had the wild genotype (TT) and meanwhile reported regular intake of beans in early pregnancy were seen as the reference group. After adjustment for potential confounders detected in Table 1, mothers who had the variant genotypes (CT + CC) and meanwhile reported regular intake of beans (aOR = 1.52, 95%CI: 1.09–2.11) and a small intake of beans (aOR = 4.00, 95%CI: 2.17–7.40) were at a significantly higher risk of VSD in offspring compared with those who were in the reference group.

**Figure 2.** The level of association between genetic variants of *BHMT* gene, maternal dietary intake and VSD in offspring.

**Table 5.** Interactions of polymorphisms of *BHMT* gene and maternal dietary habits based on multivariate logistic regression.


Abbreviations: *BHMT* betaine-homocysteine methyltransferase, aOR adjusted odds ratio, CI confidence interval, FDR\_P, false discovery rate *p*-value. <sup>a</sup> Maternal dietary habits were classified as hardly and sometimes/often. <sup>b</sup> Single nucleotide polymorphisms were classified as wild-type and variant genotypes. <sup>c</sup> Adjusted for pre-pregnancy BMI, education level, consanguineous marriages, gestational diabetes mellitus, gestational hypertension, abnormal pregnancy history before this pregnancy, congenital malformations in family members, exposure to environmental pollutants, antibiotic use in early pregnancy, tobacco exposure in early pregnancy, alcohol exposure in early pregnancy, periconceptional folate use.

**Table 6.** Interaction of rs1915706 and maternal beans intake based on crossover analysis.


Abbreviations: cOR crude odds ratio, aOR adjusted odds ratio, CI confidence interval. <sup>a</sup> For rs1915706, '-' means wild type, '+' means variant genotype. <sup>b</sup> For maternal beans intake, '–' means regular intake (namely, sometimes/often), '+' means little intake (namely, hardly). <sup>c</sup> Adjusted for pre-pregnancy BMI, education level, consanguineous marriages, gestational diabetes mellitus, gestational hypertension, abnormal pregnancy history before this pregnancy, congenital malformations in family members, exposure to environmental pollutants, antibiotic use in early pregnancy, tobacco exposure in early pregnancy, alcohol exposure in early pregnancy, and periconceptional folate use.

**Figure 3.** Interaction of rs1915706 and maternal beans intake on the risk of VSD in offspring.

#### **4. Discussion**

Research on the causes of CHD has made great strides, and more than 400 genes and important environmental factors have been determined to have substantial evidence in relation to the risk of developing CHD and its subgroups [5–8]. However, the conclusion can be easily drawn from the other hand that a single genetic or environmental factor may impose minimal effects on CHD. Moreover, the interaction of the two factors cannot be overlooked in the occurrence and development of CHD and its subgroups. In this study, we attempted to achieve an insight into the etiology of VSD, the most common subtype of CHD. The main purpose was to decide whether the association and interaction effect of BHMT gene polymorphisms and maternal dietary habits with VSD exists, which is conducive to the achievement of molecular genetic diagnostics and provide diet instruction to expectant mothers in early pregnancy.

Firstly, we explored the association between maternal dietary intake during early pregnancy and the risk of VSD in offspring. The results sent two messages. On the one hand, mothers who reported excessive intake of smoked foods (aOR = 2.44), barbecued foods (aOR = 1.86), fried foods (aOR = 1.93), and pickled vegetables (aOR = 2.50) were more likely to have a VSD-affected child. On the other hand, mothers with an excessive intake of fresh fruits (aOR = 0.47), fish and shrimp (aOR = 0.35), fresh eggs (aOR = 0.56), beans (aOR = 0.68), and milk products (aOR = 0.67) were less likely to have a VSD-affected child. Generally, various harmful chemicals can be generated from improper food processing, and most of them are teratogens and carcinogens, such as nitrites, acrylamide (ACR), polycyclic aromatic hydrocarbons (PAH), and so on. Pickled vegetables have a wide range of nitrite and nitrate contents. Gravidas, who have an excessive intake of pickles, may suffer hypoxemia because increased nitrite can react with hemoglobin, rendering it incapable of carrying oxygen [38]. A recent experimental study established a rodent model and reported that hypoxia was able to cause numerous abnormalities in cardiomyocyte gene expression, the electrophysiologic substrate of the heart, and contractile function, thus delaying cardiac maturation [39]. ACR, identified in food in 2002, is mainly formed via the Maillard reaction, whereby a carbonyl compound reacts with the amino group of asparagine processed at high temperatures (>120 ◦C, such as barbecuing, frying, and baking) [40,41]. Although no direct evidence manifested its relation to heart defects, a number of animal studies have shown strong neurotoxic, genotoxic, carcinogenic, mutagenic, and teratogenic effects [42]. Food is readily contaminated by PAH during the smoking process involving the combustion of fuel. A recent study reported that greater maternal levels of PAH exposure during

pregnancy might be associated with an elevated prevalence of fetal CHD [43]. Moreover, prior experimental research provided strong evidence that PAH exposure can result in abnormal heart looping, an enlarged ventricle with a thinner ventricular wall, and even developmental cardiac defects [44,45]. The protective foods detected in the study, such as fruits, fish and shrimp, eggs, beans, and milk products, are common foods on tables. Furthermore, they are packed with numerous nutrients, such as high-quality protein, vitamins, minerals, and so on, which have been extensively accepted to play a vital role in maintaining the health of gravidas and fetuses.

Moreover, we determined the association between maternal BHMT gene polymorphisms and the risk of VSD in offspring. Four SNPs (rs3733890, rs1316753, rs567754, and rs1915706) were considered in this study, and two SNPs (rs1316753 and rs1915706) were for the first time found to have a statistically significant association with VSD. To date, the BHMT gene remained relatively little studied compared with other folate- and homocysteine-metabolizing genes. The results in our study were only partly in accordance with prior studies. The rs3733890 polymorphism is a well-studied exon of the BHMT gene and undergoes a G-to-A change at nucleotide 716, leading to an arginine-to-glutamine substitution at amino acid 239. Its association with congenital defects has been widely explored but with contradictory results [27,46–48]. In the present study, we did not detect its significance in the occurrence of VSD. Rs567754 is an intronic variant of the BHMT gene, and neither previous data nor our data revealed an association with CHD or VSD in offspring [27]. The interesting thing is that two other SNPs, rs1316753 and rs1915706, were observed to be statistically associated with a remarkably-increased risk of VSD in offspring. To the best of our knowledge, experimental or epidemiological research involving these two polymorphisms remains a nearly unworked area, meaning that their potential functional effects on BHMT are largely unknown. Qiping Feng et al. performed a genotype–phenotype correlation analysis on betaine-homocysteine methyltransferase and found that three introns (rs41272270, rs6875201, and rs7700790) and an intergenic variant (rs16876512) were significantly correlated with both BHMT activity and protein levels [22]. Although this convincing research did not cover the two significant SNPs in our study, the analogy seems to be reasonable that the two intergenic variants, rs1316753 and rs1915706, are also capable of producing potential effects on the expression of the BHMT gene and subsequently influencing plasma hcy concentrations. The correlation between maternal hyperhomocysteinemia and CHD has been extensively studied and reviewed [25,49,50]. Meanwhile, hcy-induced CHDs, such as the transposition of the great arteries (TGA), single ventricle defects (SVD), and VSD, have been found in embryos of different species (mice and chicken) [51]. Therefore, the statistical association between maternal BHMT polymorphisms and VSD in offspring might be explained by the pathway from BHMT activity to elevated hcy levels to multiple congenital anomalies. Nonetheless, more related research is encouraged to provide clearer evidence, thus elucidating the potential mechanism.

Lastly, we also analyzed the gene–environment interaction and observed a significant interaction between genetic variants of the BHMT gene at rs1915706 and maternal bean intake on the risk of VSD in offspring. The expectant mothers who had the variant genotypes (CT + CC) and meanwhile reported a small intake of beans were at a significantly higher risk of VSD in offspring (aOR = 4.00) compared with those with the wild genotype (TT) and reported having a regular intake of beans in early pregnancy. Beans are an excellent source of zinc, choline, and multiple B vitamins (such as folate, thiamin, niacin, riboflavin, and pyridoxine) [52,53]. Notably, BHMT is a zinc-dependent cytosolic enzyme, and its substrate, betaine, is partly derived from dietary choline [31,54]. In addition, a stronger risk reduction in CHD has been found in the maternal folate + B-vitamin supplementation group compared with the maternal folate supplementation group, both setting the non-users as the reference group [14]. Concerning whether a single nutrient can exist in various foods, we speculated that the deficiency of diverse nutrients coexisting in beans coincided with a genetic variant that contributes to the occurrence of VSD. This speculation seemed plausible since similar research had been conducted not long ago. Hartmut Cuny et al. demonstrated

that when dietary undersupply during pregnancy was combined with a maternal heterozygous variant in Haao, a gene of the nicotinamide adenine dinucleotide (NAD) synthesis pathway, the incidence of embryo loss and malformations was significantly higher [55]. This is a classical experimental study forcibly indicating a gene-diet interaction. As Gibson G and Berger K commented, the discovery of such interaction suggests that the close monitoring of nutrition in at-risk carrier mothers would be the type of personalized and predictive intervention that advocates of genomic health call for [56]. Regardless, what we found in our study necessitates more convincing experimental research and crowd investigation to confirm it repeatedly.

Furthermore, several limitations in our study should be acknowledged. Firstly, although we perfected the study design and executed it strictly during the whole process as far as possible, the association found in this study, an observational case-control study, was relatively limited compared to a cohort study or an experimental study. So, in other words, well-designed prospective cohorts or reasonable experimental research are needed to validate our findings further. Secondly, the information on food frequency and related exposure in the questionnaire were obtained through retrospective investigation; we cannot rule out the possible limitation of recall bias. Thirdly, this is a hospital-based case-control study, and all of the cases came from the same department in a hospital; though its representativeness in the province for sophisticated diagnosis and treatment techniques, the selection bias still cannot be ignored. Fourthly, several potential confounders were determined and adjusted in the study, but there undoubtedly are other confounding covariates that might also influence the outcomes, such as common genetic polymorphisms and some nutritional biomarkers. The findings would be more convincing if taking these factors into consideration. Last but not least, maternal hcy concentration was not available in our research, which excludes the possibility of verifying the potential explanation that genetic variants of the BHMT gene may cause VSD by elevating maternal hcy levels.

#### **5. Conclusions**

In this hospital-based case-control study, statistically significant associations were found between the polymorphisms of the *BHMT* gene at rs1316753, rs1915706, and VSD in offspring. Maternal dietary habits were also observed to have a significant impact on the occurrence and development of VSD. A significant interaction between *BHMT* polymorphisms and maternal bean intake was identified in the study. Concerning the limitations of our study, more convincing crowd investigation and experimental research are necessary to repeatedly verify the findings and further elucidate the potential mechanism.

**Author Contributions:** Conceptualization, T.W., P.Z. and J.Q.; Data curation, M.L.; Formal analysis, M.L.; Funding acquisition, J.Q.; Investigation, X.S., M.S., Y.L., J.W., T.Z. and Q.C.; Methodology, M.L.; Project administration, J.Q.; Resources, P.H.; Software, S.Z. and J.S.; Supervision, T.W.; Visualization, M.L.; Writing—original draft, M.L.; Writing—review & editing, M.L. and T.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Project Funded by National Key Research and Development Project (2018YFE0114500), National Natural Science Foundation Program of China (82073653 and 81803313), China Postdoctoral Science Foundation (2020M682644), Hunan Provincial Science and Technology Talent Support Project (2020TJ-N07), Hunan Provincial Key Research and Development Program (2018SK2063 and 2018SK2064), Natural Science Foundation of Hunan Province (2018JJ2551), Open Project from NHC Key Laboratory of Birth Defect for Research and Prevention (KF2020006), and Science and Technology Planning Project of Guangdong Province (2020A1414010152), Hunan Outstanding Youth Fund Project (2022JJ10087), Natural Science Foundation of Hunan Province of China (2022JJ40207); Changsha Municipal Natural Science Foundation (kq2202470), Postgraduate Scientific Research Innovation Project of Central South University(1053320215987).

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee for Clinical Research of Xiangya School of Public Health of Central South University (no. XYGW-2018-36).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The department of cardiothoracic surgery in Hunan Children's Hospital lent full support to the research. The staff in the department helped us collect data and blood samples of cases and their busy work provided support for our epidemiological investigation. We want to express our sincere appreciation to all of them.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Dietary Quality during Pregnancy and Congenital Heart Defects**

**Jiaomei Yang 1,\*, Qianqian Chang 1, Shaonong Dang 1, Xin Liu 1, Lingxia Zeng <sup>1</sup> and Hong Yan 1,2,3**

	- Ministry of Education, Xi'an 710061, China

**Abstract:** Limited studies on maternal dietary quality indices and congenital heart defects (CHD) are available. This study aimed to explore the relationship between dietary quality in pregnancy and CHD among the Chinese population. A case-control study was performed in Northwest China, and 474 cases and 948 controls were included. Eligible women waiting for delivery were interviewed to recall diets and other information during pregnancy. Dietary quality was assessed by the Global Diet Quality Score (GDQS) and Mediterranean Diet Score (MDS). Logistic regression models were adopted to evaluate the associations of dietary quality scores with CHD. Pregnant women with higher scores of GDQS and MDS were at a lower risk of fetal CHD, and the adjusted ORs comparing the extreme quartiles were 0.26 (95%CI: 0.16–0.42; *P*trend < 0.001) and 0.53 (95%CI: 0.34–0.83; *P*trend = 0.007), respectively. The inverse associations of GDQS and MDS with CHD appeared to be stronger among women with lower education levels or in rural areas. Maternal GDQS and MDS had good predictive values for fetal CHD, with the areas under the receiver operating characteristic curves close to 0.8. Efforts to improve maternal dietary quality need to be strengthened to decrease the prevalence of CHD among the Chinese population.

**Keywords:** dietary quality; congenital heart defects; pregnancy; Global Diet Quality Score; Mediterranean Diet Score

#### **1. Introduction**

Congenital heart defects (CHD) refer to the structural abnormalities of the heart and/or vessels at birth. As the most common congenital anomaly worldwide, the CHD birth prevalence is estimated to be 9.41‰, with millions of newborns diagnosed with CHD each year [1]. In China, the estimated CHD prevalence at live birth is about 9.00‰, with more than 0.15 million incident cases yearly [2]. CHD accounted for over 0.26 million deaths worldwide in 2017, and remained the leading cause of infant morbidity and mortality from congenital abnormalities [3]. Nowadays nearly 20 million people live with CHD globally, causing great burdens on the family and society [3]. Although some environmental and genetic factors have been generally accepted as the risk factors for CHDs, the etiology of CHD remains to be largely unclear [4,5].

Maternal diet in pregnancy, as an important modifiable factor, has been the focus of interventions to improve birth outcomes because of the low cost and low risk. Existing evidence suggests that maternal low intakes of some nutrients including iron, zinc, selenium, folate, and niacin increase the risk of CHD [6–10], while maternal high intake of vitamin E increase the risk of CHD [11]. Previous researches also suggest that mothers with excessive intake of barbecued foods, smoked foods, fried foods, and pickled vegetables are at higher risks of fetal CHD and ventricular septal defects (VSD), while mothers with regular intake of fresh fruits, dairy, and fish and shrimp are at lower risks of fetal CHD

**Citation:** Yang, J.; Chang, Q.; Dang, S.; Liu, X.; Zeng, L.; Yan, H. Dietary Quality during Pregnancy and Congenital Heart Defects. *Nutrients* **2022**, *14*, 3654. https://doi.org/ 10.3390/nu14173654

Academic Editor: Leanne M. Redman

Received: 22 July 2022 Accepted: 2 September 2022 Published: 4 September 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and VSD [12–14]. However, most studies on the association between nutrition and CHD to date focus on individual nutrients or foods, which does not fully capture the complex interactions among nutrients and foods. Despite the emphasis of recent dietary recommendations on healthy dietary patterns, limited studies have been published on optimal dietary patterns in pregnancy to reduce the risk of CHD [15–18], especially for the use of dietary quality indices [18]. Dietary quality indices have great potential for use among people because of their easier collection and interpretation, especially in low- and middle-income countries [19,20]. However, the associations between maternal dietary quality indices and CHD have not been investigated in the population except one in America [18].

The Global Diet Quality Score (GDQS) is a novel food group-based dietary score according to the data from 14 countries [21]. The GDQS has the ability to capture nutrient adequacy and diet-related non-communicable disease, and is a promising candidate for global monitoring platforms [21]. The Mediterranean Diet Score (MDS) is developed on the basis of the Mediterranean diet, which is high in fruits, vegetables, whole grains, legumes, fish, and nuts, high in olive oil but low in saturated lipids, low to moderate in dairy, and limited in red meat and wine [22]. Previous studies have shown that the Mediterranean diet in pregnancy benefits maternal and offspring health [19]. To our knowledge, the GDQS has not been assessed for the associations with pregnancy outcomes including CHD. For the MDS, only one study in America has used it to examine the association with CHD [18]. It remains unknown whether maternal MDS during pregnancy could be used to evaluate the association with CHD in Asian countries, where the dietary habits are distinct from those in western countries. The prevalence of CHD (the mild lesions in particular) is higher in Asia than that in other regions, and the increase rate of CHD prevalence (atrial septal defects (ASD) in particular) in Asia is also greater than in other regions [1], which may partly come from the difference in dietary habits. Some studies have described maternal predictors for CHD [23–26], providing a reference for the early prediction of CHD. However, the prediction values for dietary quality indices on CHD have not been explored.

The current study in Northwest China aimed to explore the associations of maternal dietary quality during pregnancy assessed by GDQS and MDS with the risk of CHD, and evaluate the prediction values for dietary quality indices on CHD.

#### **2. Materials and Methods**

#### *2.1. Study Design and Participants*

We performed a case-control study in six comprehensive hospitals from August 2014 to August 2016 in Xi'an City, Northwest China. These six hospitals were selected according to their qualification to perform the diagnoses of birth defects and their willingness to cooperate. The detailed fetal echocardiography during the 20th–24th month of gestation was in the routine prenatal screening program in the six hospitals, and used as the prenatal diagnosis of CHD. The study design has been published in detail previously [8,15]. Briefly, we recruited participants among the pregnant women waiting for delivery in the cooperated hospitals. Pregnant women whose fetuses had isolated CHD and no genetic abnormalities were included in the case group, and pregnant women whose fetuses had no diagnosed congenital anomalies were included in the control group. Mothers with multiple gestations or diabetes were excluded because of potentially different etiologies. Qualified specialists in the ultrasound, obstetrics, and pediatrics departments strictly enforced the diagnostic standard criteria to finish the diagnoses in cases and controls. We further conducted a telephone follow-up within one year after birth to confirm the diagnosis. All the CHD diagnoses were ascertained by echocardiography and/or cardiac catheterization and/or surgery. We randomly selected controls each month in each hospital, and the ratio of the number of controls to cases included in the same month in the same hospital was 2:1. To detect a significant (*p* < 0.05) OR of 0.75 between groups of good and poor dietary quality with a statistical power of 80%, 443 cases, and 886 controls would be required. Finally, 474 cases and 948 controls with completed questionnaires were included, meeting the requirements of the sample size.

All participants gave written informed consent. The ethics committee of Xi'an Jiaotong University Health Science Center approved this study (No. 2012008).

#### *2.2. Dietary Quality Evaluation*

We used a 111-item semi-quantitative food frequency questionnaire (FFQ) to collect maternal diets throughout the entire pregnancy when women were waiting for delivery in the hospital. The median time from the end of the interview to delivery was two days among the cases and controls. Maternal dietary habits tend to be stable across pregnancy [27]; thus, maternal diets during the entire pregnancy are comparable with those in the 3rd–8th week of gestation, the critical period of cardiac development [6,8,15,28]. The FFQ was established according to a validated FFQ designed for pregnant women in Northwest China [29]. Women recalled consumption frequency based on eight predefined categories ranging from never to two or more times per day and reported portion sizes with the help of food portion images [30,31]. The nutrient contents of foods were derived from the Chinese Food Composition Tables [32,33].

The GDQS and MDS were used to assess dietary quality because these priori-defined indices were previously validated and reflected common recommended dietary guidelines. The GDQS consists of 16 healthy food groups (citrus fruits, deep orange fruits, other fruits, dark green leafy vegetables, cruciferous vegetables, deep orange vegetables, other vegetables, legumes, deep orange tubers, nuts and seeds, whole grains, liquid oils, fish and shellfish, poultry and game meat, low-fat dairy, and eggs), 7 unhealthy food groups (processed meat, refined grains, and baked goods, sweets and ice cream, sugar-sweetened beverages, juice, white roots and tubers, and purchased deep fried foods), and 2 food groups regarded as unhealthy in excessive amounts (high-fat dairy, and red meat) [21]. The daily intake of each food group was classified into 3 or 4 categories. For 16 healthy food groups, points from 0 to 4 were given to each intake category, with higher intake receiving more points. For the other 9 food groups, points from 0 to 2 were given according to the intake categories. The GDQS was a sum of all 25 food group scores, with a range of 0 to 49 [21]. The higher the GDQS was, the better the diet quality was. A previously modified version of MDS for pregnant women was used in this study [34], in which 8 components were positively scored (fruits, vegetables, legumes, whole grains, fish, dairy, nuts, monounsaturated-to-saturated fat ratio), and 1 component was negatively scored (red and processed meat). Zero or one points were assigned according to the median intake for each component. The MDS was summed for each component score, and the range was 0 to 9, with higher scores showing better adherence to the Mediterranean diet. Alcohol consumption was excluded from the original MDS because alcohol intake was not recommended during pregnancy and our participants rarely drank alcohol during pregnancy.

#### *2.3. Covariates*

We collected the general information about pregnancy face-to-face by a standard questionnaire. The study covariates included (1) socio-demographic characteristics: maternal age (<30 years/≥30 years), work (in employment/without employment), education (junior high school or below/senior high school or above), residence (urban/rural), and parity (0/≥1); (2) maternal health-related factors in early pregnancy: passive smoking (no/yes), anemia (no/yes), medication use (no/yes), and folate/iron supplements use (yes/no). Women with no paid employment outside their homes were regarded as without employment. People exposed to others' tobacco smoke for ≥15 min/d was defined as exposure to passive smoking. Women with hemoglobin concentration <110 g/L in early pregnancy were diagnosed with anemia.

#### *2.4. Statistical Analysis*

Univariate comparisons between groups were tested by the χ<sup>2</sup> test for categorical variables, and by the Mann-Whitney U test for continuous variables because of the nonnormal distributions observed by the Shapiro–Wilk test. Considering the clustering in

the design through hospitals, we used mixed logistic regression models to estimate ORs (95%CIs) for total CHDs and CHDs subtypes associated with GDQS and MDS. The GDQS and MDS were divided into four groups according to quartiles of the control distribution. The confounding variables were adjusted in the models if they were reported to be risk factors for CHD [4,5] and changed the estimated by more than 10% [35], which finally included maternal age, work, education, residence, parity, and maternal passive smoking, anemia, medication use, and folate/iron supplement use in early pregnancy. Total energy intake during pregnancy was adjusted in the models to indirectly reflect maternal BMI status that was not collected in the survey. *p* for trend was calculated by including quartilespecific median value in the model. The risk of CHD associated with per 1 higher score of GDQS and MDS was assessed by mixed logistic regression models. Subgroup analyses were performed by baseline characteristics including maternal age, work, education, residence, parity, and maternal passive smoking, anemia, medication use, and folate/iron supplement use in early pregnancy. The interaction between GDQS or MDS and each of the subgroup factors was tested by the likelihood ratio test comparing regression models with and without an interaction term. The receiver operating characteristic curves (ROC) were constructed to determine the optimal cut-off values of GDQS and MDS in pregnancy for CHD with the maximum Youden index (sensitivity + specificity − 1). The areas under the ROC (AUCs) showed the accuracy of GDQS and MDS as predictive markers for CHD. When the AUC > 0.5, the closer the AUC was to 1, the better the predictive power of the model was, as follows: AUC > 0.9, very good; AUC > 0.8, good; and AUC > 0.7, useful [36].

We used the Stata software (version 15.0; StataCorp, College Station, TX, USA) to conduct the statistical analyses. We set the statistical significance at 0.05 with two-sided.

#### **3. Results**

#### *3.1. Baseline Characteristics of the Study Population*

Among the 474 CHD babies, 46.8% were diagnosed with VSD, and 46.0% with ASD, followed by atrioventricular septal defects, patent ductus arteriosus, and tetralogy of fallot (Table S1). Women in the cases were less likely to be in employment, have higher education levels, live in an urban area, and be nulliparity when compared with those in the controls (Table 1). Passive smoking, anemia, and medication use in early pregnancy were more common in the cases than in the controls, while folate/iron supplements use in early pregnancy was less common in the cases than in the controls. The proportion of babies with birth weight lower than 2500 g was higher in the cases than in the controls, while no difference in gestational age was found between the two groups. Pregnant women in the cases had lower intakes of energy and most nutrients except carbohydrate in comparison with those in the controls. Women in the cases had significantly lower GDQS and MDS in pregnancy than the controls (both *p* < 0.001).

#### *3.2. The Distribution of Food Components in GDQS and MDS among Cases and Controls*

The GDQS scores for all 16 healthy food groups except dark green leafy vegetables, whole grains, and low-fat dairy were significantly lower in the cases than in the controls (all *p* < 0.05) (Table 2). The GDQS scores for juice, purchased deep-fried foods, high-fat dairy, and red meat were also significantly lower in the cases than the controls (all *p* < 0.05), while the GDQS scores for the other five unhealthy food groups were not significantly different between groups (Table 2). The proportions of women consuming fruits, vegetables, legumes, fish, dairy, and nuts above the median intake levels during pregnancy were significantly lower in the cases than in the controls, while the proportion of women consuming red and processed meat above the median intake level during pregnancy was significantly higher in the cases than the controls (Figure 1).


**Table 1.** Baseline characteristics of the study population.

GDQS, Global Diet Quality Score; MDS, Mediterranean Diet Score; <sup>1</sup> Categorical variables are compared between groups by the χ<sup>2</sup> test, and continuous variables are compared between groups by the Mann–Whitney U test.


GDQS, Global Diet Quality Score. 1 Scoring ranges: the 3 categories here are low, middle, and high separated by a solidus; for high-fat dairy, 4 categories were classified: low, lower middle, high middle, and high (from left to right). 2 Values are present as median (25th percentile, 75th percentile), and compared between groups by Mann–Whitney U test.

#### *Nutrients* **2022**, *14*, 3654

**Table 2.** The distribution

 of food components

 in the Global Diet Quality Score among cases and controls.

**Figure 1.** The proportion of women consuming food components in the Mediterranean Diet Score during pregnancy by the median intake levels among cases and controls. MUFA/SFA, monounsaturatedto-saturated fat ratio. Dark shaded bars indicate maternal consumption above the median levels, and light shaded bars indicate maternal consumption equal to or below the median levels. Statistically significant differences were found for the groups of fruits, vegetables, legumes, fish, dairy, nuts, and red and processed meat between cases and controls by the χ<sup>2</sup> test (all *p* < 0.05).

Daily nutrient intakes during pregnancy by GDQS and MDS categories among cases and controls are shown in Tables S2 and S3, respectively. In both cases and controls, daily intakes of energy, macronutrients, and micronutrients were increased with higher scores of GDQS and MDS (all *p* < 0.001).

#### *3.3. Associations of Maternal GDQS and MDS during Pregnancy with CHD*

The risk of total CHD was reduced with the increasing quartiles of GDQS and MDS, and the tests for trend were statistically significant (both *p* for trend <0.05) (Table 3). The fully adjusted ORs comparing the highest with the lowest quartiles of the GDQS and MDS were 0.26 (95%CI: 0.16–0.42) and 0.53 (95%CI: 0.34–0.83), respectively. The risk of total CHD was reduced by 12% (OR: 0.88, 95%CI: 0.85–0.91) and 12% (OR: 0.88, 95%CI: 0.80–0.95) for per 1 higher score of GDQS and MDS, respectively. The fully adjusted models also showed inverse associations of GDQS and MDS with the risks of VSD and ASD (all *p* for trend <0.05) (Table 3). Per 1 higher score of GDQS and MDS was associated with 12% (OR: 0.88, 95%CI: 0.84–0.92) and 11% (OR: 0.89, 95%CI: 0.81–0.98) lower risk of VSD, respectively. Per 1 higher score of GDQS and MDS was associated with 13% (OR: 0.87, 95%CI: 0.84–0.91) and 10% (OR: 0.90, 95%CI: 0.83–0.98) lower risk of ASD, respectively.

Subgroup analyses showed that the associations of maternal GDQS and MDS during pregnancy with total CHD did not change by maternal age, work, parity, or maternal health-related factors in early pregnancy (passive smoking, anemia, medication use, or folate/iron supplements use) (Figures S1 and S2). However, the inverse association between maternal GDQS and the risk of total CHD appeared to be stronger among women with lower education levels and in rural areas, and the tests for interaction were significant (both *p* < 0.05) (Figure S1). The inverse association between maternal MDS and the risk of total CHD appeared to be stronger among women in rural areas, and the test for interaction was significant (*p* = 0.004) (Figure S2).


(maternal age, work, education, residence, and parity), and maternal health-related factors in early pregnancy (passive smoking, anemia, medication folate/ironsupplementsuse).

use,

**Table 3.**

Associations

 of maternal GDQS and MDS during pregnancy with congenital heart defects.

#### *3.4. The Prediction Values for Maternal GDQS and MDS during Pregnancy on CHD*

The ROC suggested that the performances of maternal GDQS during pregnancy were useful in predicting total CHD, VSD, and ASD, with the AUCs being 0.80 (95%CI: 0.78–0.83), 0.80 (95%CI: 0.76–0.83), and 0.78 (95%CI: 0.74–0.81), respectively (Figure 2). The optimal GDQS cut-off scores were 30 for total CHD (sensitivity: 69.6%; specificity: 77.7%), 28 for VSD (sensitivity: 63.5%; specificity: 82.7%), and 30 for ASD (sensitivity: 60.6%; specificity: 84.8%), respectively. The ROC also suggested that the performances of maternal MDS during pregnancy were useful in predicting total CHD, VSD, and ASD, with the AUCs being 0.79 (95%CI: 0.76–0.81), 0.79 (95%CI: 0.76–0.82), and 0.77 (95%CI: 0.74–0.80), respectively (Figure 3). The optimal MDS cut-off scores were 8 for total CHD (sensitivity: 65.2%; specificity: 79.5%), 7 for VSD (sensitivity: 70.7%; specificity: 73.7%), and 7 for ASD (sensitivity: 69.3%; specificity: 73.5%), respectively.

**Figure 2.** The ROC for the Global Diet Quality Score during pregnancy in the prediction of (**A**) total congenital heart defects, (**B**) ventricular septal defects, and (**C**) atrial septal defects. ASD, atrial septal defects; CHD, congenital heart defects; ROC, receiver operating characteristic curves; VSD, ventricular septal defects. The dotted line refers to the reference line, resulting from random selection.

**Figure 3.** The ROC for the Mediterranean Diet Score during pregnancy in the prediction of (**A**) total congenital heart defects, (**B**) ventricular septal defects, and (**C**) atrial septal defects. ASD, atrial septal defects; CHD, congenital heart defects; ROC, receiver operating characteristic curves; VSD, ventricular septal defects. The dotted line refers to the reference line, resulting from random selection.

#### **4. Discussion**

This case-control study suggested that pregnant women with better dietary quality, defined by higher scores of GDQS and MDS, had a reduced risk of having fetuses with total CHD and subtypes. The inverse associations of maternal GDQS and MDS during pregnancy with CHD appeared to be stronger among women with lower education levels or in rural areas. This case-control study also suggested that maternal GDQS and MDS during pregnancy had good predictive values for total CHD and the subtypes in fetuses, with the AUCs close to 0.8.

To date, there was only one study investigating the relationship between maternal dietary quality and CHD [18]. This previous study conducted in America found that better dietary quality, assessed by MDS and Dietary Quality Index for Pregnancy, reduced the risk of CHD, which was consistent with the results in the current study. Some studies evaluated priori dietary quality indices in association with birth defects [37–39], but not CHD. These studies reported significant inverse associations of maternal MDS with the risks of some birth defects (orofacial clefts, neural tube defects, and gastroschisis) [37,38], but not hypospadias [40]. There were three studies examining the risk of CHD in relation to dietary patterns identified by posterior statistical analyses [15–17], rather than the priori indices. A study from America reported that a prudent dietary pattern high in reduced-fat milk, yogurt, fortified cereal, whole-wheat bread, and fish, and low in vegetables and fruits reduced CHD risk [16]. The study in the Netherlands observed that the one-carbon-rich dietary pattern, which was high in fish and seafood, lowered the CHD risk [17]. The study in Shaanxi China found that the prudent dietary pattern, which was high in white meats, red meats, vegetables, legumes, dairy, and snacks, and the dairy and egg pattern, which was high in dairy, nuts, and eggs, and low in beverages, decreased the CHD risk [16]. Although these three studies have identified some posterior dietary patterns associated with CHD, it could hardly be applied in other populations because of the subjective. Our study focused on the priori dietary quality indices, to be more easily reproducible and comparable to prior related studies. The magnitude of the risk for CHD associated with maternal MDS in our study was approximately similar to that observed for CHD in the previous study, with the lowest OR comparing the extreme quartiles being 0.53 (95%CI: 0.34–0.83) and 0.43 (95%CI: 0.25–0.75), respectively [18]. The strength of the inverse association between dietary quality and CHD risk seemed to be stronger for GDQS than MDS in the current study, similar to the stronger association for Dietary Quality Index for Pregnancy than MDS with CHD and other birth defects in the previous studies [18,37]. Since there has been no study investigating health outcomes in relation to maternal GDQS in pregnancy, it is hard for us to compare the results about GDQS in our study with other studies. More studies are needed to explore the relationships between maternal GDQS and health outcomes including CHD and to validate the prediction of maternal GDQS on health outcomes.

Maternal diet is critical to fetal growth and development. Suboptimal diets in pregnancy can cause adverse pregnancy outcomes including congenital abnormalities [28]. Maternal low intakes of zinc, selenium, iron, niacin, and folate have been reported to reduce the risk of CHD [6–8,11]. Maternal diets rich in these nutrients may benefit the development of the cardiovascular system in fetuses. Women with higher scores of GDQS and MDS during pregnancy had higher intakes of these micronutrients in the current study, which may partly explain the protective effect of better dietary quality on CHD. Pregnant women with better dietary quality may have more opportunities to obtain nutrition knowledge and pay more attention to healthy diets, and thus have better nutritional status [15]. In addition, maternal adherence to better dietary quality was reported to decrease maternal oxidative DNA damage and lipid oxidation [39], which may further benefit the normal development of the fetal cardiovascular system [41]. Maternal dietary quality is also likely to be a mediator of metabolic diseases including gestational diabetes mellitus and hypertension in pregnancy, which further influences the fetal cardiovascular system [42]. However, we could not further conduct the mediation analyses because women with diabetes have been excluded and there were only two women with gestational hypertension.

The current study provides valuable evidence for the relationship between maternal dietary quality in pregnancy and the risk of CHD. However, we should acknowledge some limitations. First, due to the limited sample size, the associations of dietary quality with other CHD subtypes could not be separately investigated. Further studies with large sample sizes are needed to explore the association with other CHD subtypes. Second, selection bias cannot be excluded because pregnant women having CHD fetuses tend to choose comprehensive hospitals for delivery. Selection bias may also come from the fact that this study did not recruit CHD fetuses that did not survive. Third, recall bias cannot be excluded because maternal information during pregnancy was retrospectively reported by mothers waiting for delivery. However, previous studies have indicated that diets and events in pregnancy can be recalled well after years [43,44]. To reduce this bias, standard questionnaires and supporting materials such as food portion images and calendars were

used to collect information to help participants recall accurately in the survey. Fourth, exposure misclassification may cause because we collected dietary information during the entire pregnancy, rather than in the critical period of heart development in the 3rd–8th week of gestation. However, previous studies have shown that maternal dietary habits tend to be stable throughout pregnancy [27]. Finally, the possibility of residual confounding from unobserved and unknown factors cannot be excluded. For example, we did not collect information on gestational weight gain or BMI, which was reported to be associated with fetal CHD [45]. In fact, the real causal relationship between dietary quality during pregnancy and CHD cannot be revealed in the case-control study. Further intervention studies are needed to examine the influence of maternal dietary quality on fetal CHD.

#### **5. Conclusions**

In summary, this case-control study suggested that maternal GDQS and MDS, indicators of dietary quality, were negatively associated with the CHD risk. Moreover, maternal GDQS and MDS during pregnancy had good predictive values for CHD in offspring. These results implied the importance of improving dietary quality in pregnancy to decrease the prevalence of CHD in Northwest China. Further research is warranted to assess the validity of these scoring systems as predictive tools for CHD in other populations.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/nu14173654/s1, Table S1: The constitution of congenital heart defects subtypes in the cases; Table S2: Daily nutrients intakes during pregnancy by Global Diet Quality Score category among cases and controls; Table S3: Daily nutrients intakes during pregnancy by Mediterranean Diet Score category among cases and controls; Figure S1: Subgroup analyses for the risk of total congenital heart defects associated with per 1 higher score of the Global Diet Quality Score; Figure S2: Subgroup analyses for the risk of congenital heart defects associated with per 1 higher score of the Mediterranean Diet Score.

**Author Contributions:** Conceptualization, J.Y.; methodology, J.Y., Q.C., X.L., S.D., L.Z. and H.Y.; formal analysis, J.Y., Q.C. and X.L.; investigation, J.Y., Q.C. and X.L.; data curation, L.Z., S.D. and H.Y.; writing—original draft preparation, J.Y. and Q.C.; writing—review and editing, J.Y.; supervision, J.Y., S.D. and H.Y.; project administration, S.D. and L.Z.; funding acquisition, J.Y., S.D. and H.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (82103852, 81230016), China Postdoctoral Science Foundation (2019M663751), Shaanxi Health and Family Planning Commission (Sxwsjswzfcght2016-013), and National Key R&D Program of China (2017YFC0907200, 2017YFC0907201).

**Institutional Review Board Statement:** The study was in accordance with the guidelines of the Declaration of Helsinki, and approved by the ethics committee of Xi'an Jiaotong University Health Science Center (No.2012008).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data present in this study are available on request from the corresponding authors.

**Acknowledgments:** The authors are grateful to all medical staff involved in the study for recruiting the participants. The authors are also grateful to all mothers and infants who participated in the study and all investigators who contributed to data collection.

**Conflicts of Interest:** The authors declare no conflict of interest.

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