Abstract Previous studies have shown that Japanese people exhibit a higher abundance of Bifidobacterium compared to people from other countries. Among the possible factors affecting the gut microbiota composition, an association of functional lactase gene variants with a higher abundance of Bifidobacterium in the gut has been proposed in some reports. However, no Japanese subjects were included in these studies. In this study, we investigated the possible contribution of functional lactase loci to the high abundance of Bifidobacterium in Japanese populations. Based on a data analysis assessing 1,068 healthy Japanese adults, a number of subjects is at least seven times greater than that reported in available online data. all subjects possessed CC genotype at rs4988235 and the GG at rs182549, which are associated with low lactase activity. We observed a positive correlation between dairy product intake and Bifidobacterium abundance in the gut. Considering previous reports, which revealed that four additional functional lactase loci, rs145946881, rs41380347, rs41525747 and rs869051967 (ss820486563), are also associated with low lactase activity in Japanese people, our findings imply the possible contribution of host genetic variation-associated low lactase activity to the high abundance of Bifidobacterium in the Japanese population.

Citation: Kato K, Ishida S, Tanaka M, Mitsuyama E, Xiao J-z, Odamaki T (2018) Association between functional lactase variants and a high abundance of Bifidobacterium in the gut of healthy Japanese people. PLoS ONE 13(10): e0206189. https://doi.org/10.1371/journal.pone.0206189 Editor: Brenda A. Wilson, University of Illinois at Urbana-Champaign, UNITED STATES Received: July 10, 2018; Accepted: October 7, 2018; Published: October 19, 2018 Copyright: © 2018 Kato et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: Our work was funded by Morinaga Milk Industry Co., LTD., DeNA Life Science, Inc., and the Kanagawa Prefecture’s model project related to the creation of ME-BYO industry in FY2016. Employees of Morinaga Milk Industry Co., LTD., KK, EM, JX and TO, and those of DeNA Life Science, Inc., SI and MT, were received salary from each company. The specific roles of these authors are articulated in the ‘author contributions’ section. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: I have read the journal's policy, and the authors of this manuscript have the following competing interests: Kumiko Kato, Eri Mitsuyama, Jin-zhong Xiao and Toshitaka Odamaki were employed by Morinaga Milk Industry Co., Ltd., and Sachiko Ishida and Masami Tanaka were employed by DeNA Life Science, Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Introduction The life expectancy of Japanese population is one of the longest life expectancies of any country in the world[1]. The underlying causes of long life in Japan have been debated, and the possibilities include good hygiene, a high level of health consciousness in Japan, and the Japanese diet[2]. In previous reports, Japanese healthy adults exhibited a higher abundance of Bifidobacterium compared to people from other countries[3–5]. Considering that Bifidobacterium naturally inhabits the human gastrointestinal tract (GIT) and is thought to play pivotal roles in maintaining human health[6], the higher abundance of this genus seems to be another potential reason for long life in Japan. The proportion of Bifidobacterium in the gut microbiota is affected by many factors, such as host age[4], stress[7] and diet[8]. Host genetic variations related to fucosyltransferase 2[9] and lactase[10–13] have been reported to contribute to Bifidobacterium abundance. In particular, the functional lactase gene (LCT) variant rs4988235 has been associated with Bifidobacterium abundance in the gut in multiple reports[14–16]. The presence of the CC genotype at rs4988235 is related to low lactase activity and milk indigestibility in adulthood. Because Bifidobacterium assimilates lactose as a preferred carbon source for growth, it is reasonable that subjects with the CC genotype at this locus have a higher Bifidobacterium abundance in their gut. The locus rs4988235 is mainly reported as a characteristic locus related to lactase activity in Europeans. However, this single locus is insufficient to explain the frequency of the lactase phenotype present in various populations worldwide[11]. Another variant, the GG genotype at rs182549, has been reported to contribute to lactase persistence in Japanese-Brazilian and Chinese populations, whose genetic backgrounds are related more closely to those of the Japanese population than to those of the European population[11]. Therefore, the rs182549 locus might also be an important contributor to the lactase activity in the Japanese population. Here, we investigated the associations between these two single nucleotide polymorphisms (SNPs), which have been reported as functional lactase variants, and the proportion of Bifidobacterium in the gut microbiota of 1,068 healthy Japanese adults. Furthermore, information for an additional four functional lactase variants that were reported previously[12,13] but were missing from our data set was also included using Japanese genome data obtained from two previous studies[13,17].

Material and method Study subjects and sample collection A total of 1,250 healthy Japanese adults were enrolled in this study as part of MYCODE Research, a research platform based on customers of MYCODE (DeNA Life Science Inc., Tokyo, Japan), a personal genome service in Japan. A total of 182 subjects were removed from the data analysis based on the following criteria (see also S1 Fig): 64 subjects declined participation, seven subjects were pregnant or lactating women, 96 subjects had taken medication within the last two weeks, and 15 subjects did not meet the criteria for quality control of the genotypic data as described in detail below. Finally, the 1,068 participants consisted of 541 women and 527 men with a median age of 41 years (range 20–64 years) and median body mass index of 21.4 kg/m2 (inter-quartile range 19.7–23.7 kg/m2). The entire study was approved by both the ethics committee of DeNA Life Science Inc. (protocol #20160727_1) and the Institute of Medical Science, University of Tokyo (protocol #28-29-1125) (Tokyo, Japan). Written informed consent was initially obtained for MYCODE Research covering diverse genomic research comprehensively. Then, additional informed consent for this specific study was obtained from all subjects on the MYCODE website. Saliva samples were collected for MYCODE genetic testing, and these genetic data were used for this study. All participants submitted their own stool samples for gut microbiota analyses. A stool sample aliquot was mixed with 1 ml of guanidine thiocyanate (GuSCN) solution (TechnoSuruga Laboratory Co., Ltd, Shizuoka, Japan)[24] and was transported to the laboratory by postal mail at room temperature. Immediately upon receipt, the faecal samples were stored at −80°C until the day of analysis. Genotyping and quality control Genotyping of SNPs, was performed using either an Infinium OmniExpress-24+ BeadChip or a Human OmniExpress-24+ BeadChip (Illumina Inc., San Diego, CA, United States). Based on the genotypic data, a total of 15 subjects were removed in the process of quality controls using PLINK version 1.9[25] as follows: two subjects with call rates under 95% which indicated low reliability of their genotyping results; nine subjects who were one of a pair with a proportion of identical by descent (IBD) >0.185; which indicates kinship; one subject showing a discordance between self-reported sex and genotyped sex; and three subjects determined to have non-Japanese ancestry by principal component analysis. DNA extraction from faecal samples DNA extraction from human faecal samples was performed using the bead-beating method as previously described[4] with some modifications. Briefly, 500 μl of faecal sample in GuSCN solution was vigorously vortexed with glass beads (300 mg; 0.1 mm in diameter) and 500 μl of buffer-saturated phenol using a Multi-Beads Shocker (Yasui Kikai Co., Osaka, Japan) at a speed of 2,700 rpm for 180 s. After centrifugation at 10,000×g for 10 min, 400 μl of the supernatant was extracted with phenol-chloroform, and 250 μl of the supernatant was precipitated with isopropanol. The purified DNA was suspended in 1,000 μl of Tris-EDTA buffer (pH 8.0). Sequencing and data processing of bacterial 16S rRNA sequences 16S rRNA gene sequencing was performed as previously described with minor modifications[4]. Briefly, the V3-V4 region of the bacterial 16S rRNA gene was amplified by PCR in triplicate using the TaKaRa Ex Taq HS Kit (TaKaRa Bio, Shiga, Japan) and the primer sets Tru357F (5′-CGCTCTTCCGATCTCTGTACGGRAGGCAGCA G-3′) and Tru806R (5′-CGCTCTTCCGATCTGACG- GACTACHVGGGTWTCTAAT-3′) with the following program: preheating at 94°C for 3 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s and extension at 72°C for 30 s; and terminal extension at 72°C for 5 min. A 1-μl sample of the combined PCR products was amplified with barcoded primers adapted for Illumina MiSeq sequencing: Fwd 5′-AATGATACGGCGACCACCGAGATCTACACXXXXXXXXACACTCTTTCCCTACACGACGCTCTTCCGATCTCTG-3’ and Rev 5′-CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC-3′, where X represents a barcode base. Amplification was performed according to the program described above except only eight cycles were performed. The products were purified and quantified by a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, United States) and Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s protocols. Equal amounts of amplicons were pooled and purified with the GeneRead Size Selection Kit (Qiagen) according to the manufacturer’s protocol. The pooled libraries were sequenced with an Illumina MiSeq instrument and the MiSeq v3 Reagent Kit (Illumina Inc., San Diego, CA, United States). After acquiring the Illumina paired-end reads, the Bowtie-2 program[26] (ver. 2–2.2.4) was used to remove reads mapped to the PhiX 174 sequence and the Genome Reference Consortium human build 38 (GRCh38). Thereafter, the 3’ region of each read with a PHRED quality score of less than 17 was trimmed. Trimmed reads less than 150 bp in length with an average quality score of less than 25 or those lacking paired reads were also removed. The trimmed paired-end reads were combined by the fastq-join script in EA-Utils[27] (ver. 1.1.2–537). Potential chimeric sequences were removed by reference-based chimaera checking in USEARCH[28] (ver. 5.2.32) and the Genomes OnLine Database (GOLD) (http://drive5.com/otupipe/gold.tz). Non-chimeric sequences were analysed via the QIIME software package version 1.8.0[29,30]. For genus-level analysis, the sequences were assigned to operational taxonomic units (OTUs) by open-reference OTU picking[31] with a 97% pairwise identity threshold and the Greengenes reference database[32]. The frequencies of LCT variants in different populations around the world The frequencies of LCT variants in Japanese, African, American, East Asian, European, and South Asian populations were obtained from the Phase 3 1000 Genomes Project data (http://phase3browser.1000genomes.org/index.html)[17]. Assessment of dairy product intake with a BDHQ A BDHQ[33] was used to assess food intake habits during the month before sample collection. To estimate the amount of lactose contained in an individual’s diet, we used combined data with the categories “normal-fat milk” and “low-fat milk” (designated here as dairy products), after energy adjustment using the density method. Statistical analysis All analyses were performed using the IBM SPSS Statistics, version 22.0, statistical software package (IBM Corp., Armonk, NY, USA). Intergroup differences in the composition of Bifidobacterium were analysed using the Kruskal-Wallis test. Spearman’s correlation coefficient was used to determine the relationship of Bifidobacterium abundance with the amount of dairy product intake. For all tests, p<0.05 was considered statistically significant.

Acknowledgments We would like to thank Dr. Chyn Boon Wong for her critical review of this manuscript.