**1. Introduction**

Pregnancy is a critical period during which a mother's nutrition and lifestyle have a decisive influence on maternal and child outcomes. Multiple factors can affect pregnancy

**Citation:** Rodriguez-Carnero, G.; Lorenzo, P.M.; Canton-Blanco, A.; Mendizabal, L.; Arregi, M.; Zulueta, M.; Simon, L.; Macia-Cortiñas, M.; Casanueva, F.F.; Crujeiras, A.B. Genetic Variants in Folate and Cobalamin Metabolism-Related Genes in Pregnant Women of a Homogeneous Spanish Population: The Need for Revisiting the Current Vitamin Supplementation Strategies. *Nutrients* **2022**, *14*, 2702. https:// doi.org/10.3390/nu14132702

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Academic Editor: Marloes Dekker Nitert

Received: 16 May 2022 Accepted: 27 June 2022 Published: 29 June 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/).

health, including maternal sociodemographic characteristics, environmental exposures, maternal nutrition, age, obesity, lifestyle, and socioeconomic status, as well as genetic background and gene–environment interactions [1–3].

The maternal diet and nutritional stores provide nutrients for the developing embryo and foetus [4–8]. Among nutrients, folic acid (vitamin B9) and cobalamin (vitamin B12) stand out for their role in foetal growth and prevention of neural tube defects, through their role as essential co-factors in the one-carbon metabolism pathway [9–11]. Moreover, deficiencies in these vitamins were also associated with important impacts on the health of mothers such as preeclampsia [12], gestational diabetes [13], or maternal neurocognitive symptoms [14]. Considering this issue, supplementation with folic acid was particularly recommended by the World Health Organisation (WHO) in order to prevent pregnancy outcomes [15]. However, maternal vitamin levels depend on dietary and supplement intake but also are influenced by genetic polymorphisms in the gene coding for enzymes involved in vitamin metabolism and transport, which may lead to changes in their catalytic activity. Functional polymorphisms of genes encoding enzymes involved in one-carbon metabolism can cause disturbances in B9 and B12 vitamin status due to a reduction in enzyme activity [16–19]. Polymorphisms of genes involved in the metabolism and transport of these vitamins were associated with disturbances in the health of mother and child [20].

In this regard, determining genetic variants in folate and cobalamin metabolism-related genes in pregnan<sup>t</sup> women can lead to personalised treatment with higher amounts of folic acid and cobalamin for the sake of improving pregnancy and neonatal health outcomes. Therefore, the aim of the present study was to assess the prevalence of the common target genetic polymorphisms of folate and cobalamin metabolism-related genes in the literature such as methylenetetrahydrofolate reductase (*MTHFR*), methionine synthase (*MTR*), cubilin (*CUBN*), and *SLC19A1* (commonly known as reduced folate carrier (RFC1)) in pregnan<sup>t</sup> women of a homogeneous Spanish population according to conception, pregnancy, delivery, and newborn complications.

### **2. Patients and Methods**

### *2.1. Study Population and Design*

This study was conducted on 149 nulliparous women with singleton pregnancies. Pregnant women were recruited in the Endocrinology and Obstetrics departments of the "Complejo Hospitalario Universitario de Santiago de Compostela (CHUS)" in Santiago de Compostela, Galicia (northeastern Spain) from September 2018 to February 2020. Women were recruited between 24 and 28 weeks of pregnancy and then followed up throughout the pregnancy to delivery. The inclusion criteria were an age of 16 years old or older, singleton pregnancy, lack of any chronic disease or being under medical treatment, absence of any language barrier, and correct monitoring of pregnancy and delivery in our centre.

### *2.2. Sociodemographic and Obstetrics Variables*

At the time of recruitment, the following data were collected: age; ethnicity; maternal lifestyles (tobacco smoking, alcohol consumption, or drug abuse) during the pregnancy; singleton or multiple pregnancies; previous miscarriages, abortions, or ectopic pregnancies; use of assisted reproductive technologies and medical history (hypertension, diabetes); maternal height; and self-reported pre-pregnancy weight and weight at week 36 (or last weight in the case of preterm delivery) of pregnancy, which allowed for calculation of weight gain during pregnancy and body mass index pre-pregnancy.

#### *2.3. Vitamin B12 and Folic Acid Supplementation Use*

Information on vitamin supplementation was obtained by reviewing electronic medical records and asking pregnan<sup>t</sup> women from recruitment to delivery. Information on supplement intake included brand name, dosage per day, and the start and end dates of consumption. This information was used to determine supplemental vitamin B12 and folate doses per day for each woman.

### *2.4. Maternal and Neonatal Outcomes and Definitions*

Participants were followed prospectively from recruitment until delivery. Maternal and neonatal outcomes were collected from electronic medical records.

Maternal outcomes such as gestational diabetes mellitus (GDM), gestational hypertension (GHT), and preeclampsia were obtained. GDM was defined as diabetes diagnosed in the second or third trimester of pregnancy that was not clearly overt diabetes prior to gestation and was diagnosed according to our Hospital's protocol and the one-step approach [21]. GHT was defined as blood pressure ≥140/90 mmHg arising after 20-week gestation, without any other feature of the multisystem disorder that resolves within 3 months postpartum [22]. PE was defined as GHT with ≥1 proteinuria/abnormal renal or liver function tests or platelet count/symptoms and signs consistent with end-organ damage of preeclampsia [22].

Gestational age and anthropometric measurements (weight, height, head circumference, and chest circumference) at birth were obtained from electronic medical records. Weight index was calculated as the ratio of birth weight (grams) to height (cm3). Size for gestational age was estimated based on Carrascosa et al. (2004) [23], using neonatal gestational age at delivery, anthropometric measurements (AM: weight, height, and head circumference), and sex. Newborns were categorised into three groups: small for gestational age (SGA) (AM less than 10th percentile for gestational age); appropriate for gestational age (AGA) (AM 10th to 90th percentile for gestational age, which was the reference group); and large for gestational age (LGA) (AM greater than 90th percentile for gestational age). For birth weight, newborns were considered to be LGA if their birth weight was >2.0 standard deviation (SD) or over the 90th percentile for sex and gestational age, and SGA infants were considered to have a birth weight that was < −2.0 SD or under the 10th percentile for sex and gestational age. Normal birth weight was considered for values between −2.0 and +2.0 SD for sex and gestational age (between the 10th and 90th percentile). Low birth weight (LBW) was defined as birth weight less than 2500 g. Macrosomia was defined as neonates whose birth weight was equal to or greater than 4000 g.

Other obstetric complications such as miscarriage, stillbirth, and neonatal death were also collected. Delivery data were obtained, with special attention to the type of delivery (spontaneous onset of labour, induced labour, instrumental delivery, and route of delivery), preterm delivery, perinatal complications, admission to intensive care unit, and hospital stay. Spontaneous preterm birth (PTB) was spontaneous preterm labour or preterm premature rupture of the membranes, resulting in birth at <37-week gestation. Uncomplicated pregnancy was defined as a pregnancy in which no antenatal medical or obstetric complication had been diagnosed, resulting in the delivery of an appropriately grown, healthy baby at ≥37 weeks of gestation.

### *2.5. Sample Collection*

Participants were asked to refrain from eating, drinking, brushing teeth, and using mouthwash for at least 30 min prior to sample collection for which a buccal swab of the Puritan Medical Products PurFlock Ultra® (25-3606-U, Guilford, NC, USA) was used. Genomic DNA was extracted from the buccal swab using a MagMax DNA Multi-Sample Ultra Kit (Applied biosystems by Thermo Fisher Scientific, Waltham, MA, USA).
