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Background: The sodium iodide symporter is responsible for the transfer of iodine into breast milk and is encoded for by the SLC5A5 gene. The role of genetic variants in the SLC5A5 gene locus in relation to the transfer of iodine from plasma into breast milk in healthy lactating individuals has, to our knowledge, not been explored. Objective: To identify and characterize possible genetic variants of the SLC5A5 gene in women of African descent living in urban South Africa, and to study associations with breast milk iodine concentrations (BMIC) in lactating women. Methods: This study is affiliated to the Nutrition during Pregnancy and Early Development (NuPED) cohort study (n = 250 enrolled pregnant women). In a randomly selected sub-sample of 32 women, the SLC5A5 gene was sequenced to identify known and novel variants. Of the identified variants, genotyping of selected variants was performed in all pregnant women who gave consent for genetic analyses (n = 246), to determine the frequency of the variants in the study sample. Urinary iodine concentration (UIC) in spot urine samples and BMIC were measured to determine iodine status. Associations of SLC5A5 genetic variants with BMIC were studied in lactating women (n = 55). Results: We identified 27 variants from sequencing of gene exomes and 10 variants were selected for further study. There was a significant difference in BMIC between the genotypes of the rs775249401 variant (P = 0.042), with the homozygous GG group having lower BMIC [86.8 (54.9–167.9) μg/L] compared to the (A) allele carriers rs775249401(AG+AA) [143.9 (122.4–169.3) μg/L] (P = 0.042). Of the rs775249401(GG), 49% had UIC <100 μg/L and 61% had BMIC <100 μg/L. On the other hand, 60% of the rs775249401(AG+AA) carriers had UIC <100 μg/L, and none had a BMIC <100 μg/L. Conclusion: Our results suggest that A-allele carriers of rs775249401(AG+AA) are likely to have higher iodine transfer into breast milk compared to the homozygous GG counterparts. Thus, genetic variations in the SLC5A5 gene may play an important role in the transfer of iodine from plasma into breast milk and may partially explain inter-individual variability in BMIC.
The NuPED study was a prospective cohort study conducted in Johannesburg, South Africa from March 2016 to July 2018. The study protocol has be previously published (14). In brief, pregnant women (n = 250) were enrolled if they were between 18 and 39 years of age, <18 weeks gestational age, born in South Africa or a neighboring country, have lived in Johannesburg for at least 12 months, were able to communicate effectively in one of the local languages, non-smoking, and expecting a singleton. Pregnant women were excluded from participation if they reported use of illicit drugs, had a known non-communicable disease such as diabetes, renal disease, history of high blood cholesterol and hypertension, and had a known infectious disease such as tuberculosis or hepatitis, or known serious illness such as cancer, lupus or psychosis. HIV positive women were included in the study. Pregnant women were assessed at 10 and quality score of >500 frets were met. The sequence files were aligned against Genome Reference Consortium Human Build 37 (hg19), followed by coverage analysis and variant calling using the coverage analysis and variantCaller plugins from the Torrent Suite, respectively. Secondary data analyses of the variant caller files were annotated, filtered and mined following an in-house pipeline (15). In the subset of 32 sequenced samples, variants that passed the quality control assessment were considered for validation in the entire sample set using the iPLEX® MassARRAY system from Agena Bioscience™. IPLEX assays were designed and analyses were performed by the service provider Inqaba Biotech (Inqaba Biotechnology Pretoria South Africa). Assays were designed using the Assay Design Suite (ADS) software and dbSNP for metadata. gDNA was amplified in 96 microtiter plates using iPLEX reagent kits and a nano dispenser RS1000 was used to transfer samples from microtiter plates to a SpectroCHIP® array. Data were obtained from the SpectroCHIP® array using the MassARRAY® analyser. Reports were automatically generated by Typer (Company). Genotype calls were made in real-time during MALDI-TOF analysis and data was automatically saved to the MassARRAY database. Variants were assessed for quality, and tested for adherence to Hardy-Weinberg equilibrium (HWE) (16) by using Haploview and modified Pearson chi-square (χ2) test. Adherence to HWE was set at P < 0.001. From the 98 women participating in the 6-month follow-up, we collected a midstream spot urine sample (10–40 ml) into clean polystyrene cups between 07:00 and 12:00 noon, and approximately 5 ml was decanted into iodine-free screw-capped cups. The research team ensured that the urine samples were not used for any routine assessments using dipsticks (potential contamination with iodine). Samples were aliquoted and stored on-site at −20°C for a maximum of 7 days. Thereafter, samples were transported on dry ice to the CEN laboratories, for storage at −80°C until analysis. Urinary iodine concentration (UIC) in spot urine samples was measured in duplicate using the Pino modification of the Sandell-Kolthoff reaction with spectrophotometric detection at CEN laboratories (17, 18). All analyses were done using nanopure grade water and all laboratory glassware and plasticware were acid washed before use. Internal and external controls were used to ensure the quality of the analysis. Iodine concentrations in spot urine samples are expressed as median concentrations (μg/L). A UIC cut-off of <100 μg/L was used to indicate insufficient iodine intake in lactating women (19). From 58 lactating women who participated in the 6-month follow-up, a breast milk (foremilk) sample (≈5 ml) was collected by manual expression into an iodine-free screw-capped cup before feeding the infant. Iodine concentrations in breast milk (in μg/L) were measured using a multi-collector inductively coupled plasma mass spectrometer [MC-ICP-MS (Finnigan NEPTUNE, Thermo Scientific™ Waltham, MA, USA)] as described by Dold et al. (10). A BMIC cut-off of 500 μg/L) were considered outliers. These UIC outliers (n = 2) were excluded from the analysis because high intakes of iodine have previously been reported to lead to improved BMIC in an individual that harbored a SLC5A5 variant associated with the lower transfer of iodine into breast milk (23). Normally and non-normally distributed data are expressed as means ± standard deviation (SD) and medians (25th percentile, 75th percentile), respectively. Categorical data are expressed as frequencies and percentages. Participants were stratified according to UIC categories (UIC <100 μg/L and UIC ≥100 μg/L) or BMIC categories (BMIC <100 μg/L and BMIC ≥100 μg/L). The between-group analyses were performed using the Mann Whitney U test. Overlaid scatterplots were used to depict the relationship between total daily iodine excretion, fractional iodine excretion in breast milk and fractional iodine excretion in urine. Unadjusted general linear models were performed to compare UIC and BMIC between genetic variants with the recessive genetic model (GG vs. GA + AA) as categorical variables. For the significant models, effect sizes were calculated using Cohens' d and partial eta squared. Significance was set at p < 0.05.
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