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Human breast milk miRNA, maternal probiotic
supplementation and atopic dermatitis Melanie Rae Simpson1*, Gaute Brede2, Jostein Johansen2, Roar Johnsen1, Ola Storrø1, Pål Sætrom2,3, Torbjørn Øien1
*Presenting author: [email protected]
Background
Perinatal probiotic ingestion has been shown to prevent atopic dermatitis (AD) in
infancy.[1,2] The clinical trials underpinning this conclusion used a variety probiotics
strains and supplementation regimes, and the strongest evidence comes from studies
which include both pre- and post-natal supplementation. Results from our own study,
Probiotics in Prevention of Allergy among Children in Trondheim (ProPACT), suggest
that maternal supplementation alone is sufficient to prevent AD in offspring.[3] The
mechanisms behind this preventative effect are incompletely understood and may be
partially mediated by changes in breast milk composition. Micro-RNAs (miRNA) are
short non-coding RNA molecules which modify gene expression at the post-
transciptional level. They are abundant in mammalian milk and may influence the
developing gastrointestinal and immune systems of newborn infants.
Objectives
1. Describe the miRNA profile of human breast milk,
2. investigate the association between miRNAs and maternal probiotic supplementation, and
3. Determine if changes in breastmilk miRNAs are mediators of the beneficial effect of maternal
probiotic supplementation on the development of atopic dermatitis in offspring.
Methods
Conclusions
There appears to be a stable group of core breast milk miRNAs, which are at least partially conserved across a
number of mammalian species. Functional analysis of the potential gene targets of highly expressed breast milk
miRNAs revealed enrichment in a broad range of biological processes and molecular functions. Finally, individual
miRNAs in breast milk 3 months postpartum are unlikely to play a major role in the prevention of atopic dermatitis in
infancy by probiotics ingested in the perinatal period.
Results
Future plans
Current research in the ProPACT research group uses the various biological samples collected during the trial to
investigate the biological mechanisms by which maternal probiotic supplementation results in a reduced risk of
atopic dermatitis in offspring. We also plan to further investigate the general physiological role of breast milk
miRNAs and other non-coding RNAs through in vitro experiments and computational modelling. Particularly, with
respect to the other non-coding RNA (the tRNA and rRNA fragments) we will be conducting quality control
experiments using paired fresh and frozen samples to establish if these are observed because of storage.
Impact for the dairy industry
The ProPACT study provides scientific support for health
benefits of probiotic containing milk products. Indeed the
milk used in this study is a commercially available product
in Norway. More generally, the investigation of breast milk
constituents, such as miRNA, may prove to be of more
interest to the infant formula industry.
125 miRNAs
98.5% of reads
6568 targets
Top 20 miRNAs
76.0% of reads
3498 targets
Top 10 miRNAs
63.0% of reads
1759 targets
Top 5 miRNAs
53.8% 1371 predicted targets
Figure 3: Proportion of reads accounted for by the top
5, 10, 20 and 125 miRNAs and the number of
predicted unique target genes.
0
10
20
30
40
50
Pe
rce
nta
ge o
f re
ad
s
miRNA tRNA rRNA
Figure 1: Average percentage of small RNA sequences
aligned to different RNA species, with 95% CIs.
0
50,000
100000
150000
200000
250000
CP
M m
atu
re m
iRN
As
miR
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miR
-22
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miR
-30
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let-
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miR
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let-
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let-
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5p
miR
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6b
-5p
miR
-24
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miR
-21
-5p
miR
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miR
-29
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miR
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miR
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miR
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miR
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miR
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Figure 2: Box plot of counts per million (CPM) mature
miRNA of the 20 most abundant miRNAs
Literature 1. Pelucchi C, Chatenoud L, Turati F, Galeone C, Moja L, Bach JF, et al. Probiotics
supplementation during pregnancy or infancy for the prevention of atopic
dermatitis: a meta-analysis. Epidemiology. 2012;23(3)402-414
2. Panduru M, Panduru NM, Salavastru CM, Tiplica GS. Probiotics and primary
prevention of atopic dermatitis: a meta-analysis of randomized controlled
studies. J Eur Acad Dermatol Venereol. 2015;29(2):232-42
3. Dotterud CK, Storrø O, Johnsen R, Øien T. Probiotics in pregnant women to
prevent allergic disease: a randomized, double-blind trial. Br J Dermatol. 2010;
163(3):616-23
1Department of Public Health and General Practice 2Department of Cancer Research and Molecular Medicine 3Department of Computer and Information Science
Participants
Fifty-four (54) mother-infant pairs were included in this analysis, 32 from the probiotics
group and 22 from the placebo group. Among the children, 11 and 18 had developed
atopic dermatitis by 2 years of age in the respective groups.
Small RNA profile of human breast milk
The RNA isolates obtained from the human milk samples contained between 12.4
and 247.5ng/µL of RNA, and were found to contain RNA fragments up to 1000nt.
Sequencing analysis of RNA molecules 11-28nt in length aligned primarily to rRNA,
tRNA and miRNA (Fig. 1).
miRNA profile
The miRNA profile revealed a relatively stable core group of highly expressed
miRNAs, including miR-148a-3p, miR-22-3p, miR-30d-5p, let-7b-5p and miR-200a-
3p (Fig. 2). These five miRNAs were responsible for over half of all miRNA found in
breast milk (Fig 3).
Functional predictions
The 20 most highly expressed miRNAs have 3498 predicted mRNA targets (Fig
3). Functional annotation clusters of these target genes included a) the positive
and negative regulation of metabolic processes involving nitrogen compounds,
RNA, DNA and macromolecules and the positive regulation of transcription and
gene expression, b) embryonic development, c) angiogenesis, d) catabolic
processes and e) cell migration and localisation. The first mentioned group of
clusters is large interwoven network of genes (Fig 4). There was also significant
overlap with genes that are upregulated in brain and epithelial tissues. Epithelial
cells in the gastrointestinal tract may be the first “recipients” of breast milk
miRNAs.
Differential expression associated with
maternal probiotic supplementation and
atopic dermatitis
Although several miRNAs were found to be
differentially expressed on comparison of the
probiotic vs placebo and AD vs non-AD groups,
none had an acceptable false discovery rate
(FDR) and their biological significance in the
development of AD is not immediately apparent
from their predicted functional consequences.
ProPACT Study
The Probiotics in the Prevention of Allergy among Children in Trondheim (ProPACT) trial is a
double blinded, placebo controlled, randomised trial investigating the effect of maternal probiotic
supplementation on the development of allergy related diseases in early childhood.[3]
2 yrs 1 yr Birth -4 wks 6 wks 3 mths
Intervention period
Biological samples collected from mother: • Stool samples (recuitment and 3mths)
• Vaginal swabs (recruitment and birth)
• PBMC and plasma (4 week before due date)
• Breast milk (10 days, 3 mths)
• Mouth swabs (10 days, 3mths)
Biological samples collected from child: • Cord blood (birth)
• Stool samples (10 days, 3mths, 1yr, 2yrs)
• PBMC and plasma (10 days, 3mths, 1yr, 2yrs)
• Mouth swabs (10 days, 3mths)
Lactobacillus rhamnosus GG (LGG; 5 x 1010 CFU)
Bifidobacterium animalis subsp. lactis Bb-12 (Bb-12; 5 x 1010 CFU)
L. acidophilus La-5 (La-5; 5 x 109 CFU).
Probiotic milk (n=211)
Placebo milk (n=204) Cultured milk, sterilised after culture process
PBMC: peripheral blood mononuclear cell
n=287 n=415
UCAGUGCACUACAGAACUUUGU
AAGCUGCCAGUUGAAGAACUGU
hsa-miR-148a-3p
hsa-miR-22-3p
Target genes and
their functions
Fifty-four breast milk samples from 3 months postpartum collected
during the ProPACT trial were used. Samples had been stored at -
80ºC for 7 to 9 years at time of analysis.
Extracellular vesicle and RNA isolation: breast milk samples
were enriched for extracellular vesicles using ExoQuick™, and total
RNA was isolated using Quigen miRNeasy kit.
Sequencing and bioinformatics pipeline: Small RNA sequencing
was conducted using 50-bp single-end reads on Illumina’s HiSeq
2000 sequencing system. Raw reads were processed, filtered and
mapped to the human genome and to miRNAs in miRBase version
20.0. Differential expression of miRNAs was assessed for the
probiotic vs placebo and the AD vs non-AD groups using the voom
and limma R-packages.
Functional predictions: Target genes were predicted for the 20
most highly expressed miRNAs and for the differentially expressed
miRNA lists using TargetScan (v7.0). Insight into the potential
functional consequences of these miRNA was gained by uploaded
gene target lists to the Database for Annotation, Visualization and
Integrated Discovery (DAVID) v6.7.
n=54
Trial registration and ethics approval: The clinical trial and this sequencing study were approved by the Regional
Committee for Medical Research Ethics for Central Norway (Ref. 097-03) and written consent was obtained from the
participating families. The original trial protocol is registered in ClinicalTrials.gov (identifier NCT00159523).
Figure 4: Enrichment map of the 50 highest ranked GO terms. The large network to the
left represents an interwoven network of terms associated with transcription, gene
expression, metabolic and biosynthetic processes. The red nodes represent negative
regulation of these processes, the green nodes positive regulation and the orange
nodes general regulation. Node size inversely weighted according to FDR. Edge colour
and width is weighted by similarity coefficient with wider and darker edges representing
a high coefficient.
A collaborative project between: