RNA sequencing of the human milk fat globule transcriptome at three stages of lactation

Transcription

1 RNA sequencing of the human milk fat globule transcriptome at three stages of lactation Danielle G Lemay, 1 Olivia A Ballard, 2 Nelson D Horseman, 3 Ardythe L Morrow, 2,3 and Laurie A Nommsen-Rivers 2,3 1 UC Davis Genome Center, 2 Cincinnati Children s Hospital Medical Center, and 3 University of Cincinnati College of Medicine Danielle G. Lemay, PhD Assistant Professional Researcher UC Davis Genome Center dglemay@ucdavis.edu October 10, 2012

2 Lactation problems in the U.S. Only 13% of moms exclusively breastfeed for 6 months. Over half of women who breastfed < 6 months cited not enough milk as an important factor in weaning. (Li, 2008) IBCLC Even with support, some moms cannot produce enough milk.

3 Biological contributors to lactation success Genetic selection in dairy cattle Milk production increased 100 lbs of milk per cow per year What we can expect in humans? Some contributors to lactation success may be heritable Large variation in milk synthesis rates between moms

4 Non-invasive assay of milk-producing cells What are the genetic, physiological, and environmental contributors to lactation success in humans? Need to know the molecular status of milk-producing cells Maningat PD, et al. Haymond Lab at Baylor College of Medicine

5 Milk as a source of mammary epithelial RNA Cytoplasmic CRESCENT Milk fat globule (a.k.a. lipid droplet) Mammary epithelial cell (a.k.a. MEC) Mather, I.H. and Keenan, T.W. (1998) J. Mammary Gland Biol. Neoplasia

6 RNA origin and quality Please see our posters Effects of milk collection and processing methods on origin and integrity of RNA in milk Danielle G Lemay, Katie Hinde, Russell C Hovey, Jennifer T Smilowitz, Stella R Hartono, Frank Ventimiglia, Kimberli A Schmidt, Joyce W.S. Lee, Ian Korf, Peter A Barry, J. Bruce German An automated method to quantify milk fat globules and sources of RNA in milk Stella Hartono, Frank Ventimiglia, Jennifer T Smilowitz, J. Bruce German, Ian Korf, and Danielle G Lemay

7 Methods 12 human milk samples: 6 Day 2, 6 mature Spin 15000g, 10 minutes, 4 C Milk Fat TRIzol method RNA Library prep (Illumina TruSeq) Sequencing on Illumina HiSeq Sequence Reads Transcriptome Results Bioinformatics analysis

8 Transcript abundance, Mature milk LALBA? CSN2?

9 Mature milk, RNA-Seq vs Microarray RNA-Seq Microarray Data Derived from Maningat et al 2009

10 Functional analysis, Mature Milk Highest expressing genes are milk protein genes What are the functions of other highly expressed genes? (top 10%) mrna translation (making milk proteins!) ATP synthesis (getting energy to make milk proteins!) Golgi vesicle transport (packaging milk proteins!) Endoplasmic reticulum (making milk proteins!)

11 Day 2 samples, Colostrum or Transitional? Day 2 samples Dendogram-Heatmap showing distances of samples based on variance-stabilized transformation of count data

12 Day 2 samples, Colostrum or Transitional? Colostrum: Na:K 2 Transitional: Na:K < 2 ID Stage Meth. Na:K 187 Col Hard Col Wash Col Wash Tra Hard Tra Wash Tra Wash Tra Soft Mat Hard Mat Wash Mat Wash Mat Soft Mat Hard Mat Wash Tra Hard Mat Hard Mat Wash 0.33

13 Transcript abundance throughout lactation LALBA CSN2 LALBA CSN2 LALBA CSN2 Colostrum Transitional Mature

14 Transcript abundance, Colostrum CTSD (cathepsin D) FTL (ferritin) FTH1 (ferritin) Lactoferrin (LTF) has received much attention, but the isoferritins (FTL and FTH1) are far more abundant in colostrum.

15 Functional analysis across lactation Enrichment of functional annotations for top 10% of expressing genes Colostrum Transitional Mature Make and deliver milk proteins Immune functions Inhibition of protein degradation, response to milk-producing hormones Make lipids and cofactors Hallmarks of each stage

16 Distinct gene expression by stage Colostrum Transitional Mature

17 Primary gene expression trajectories Insulin signaling pathway

18 Insulin signaling pathway Colostrum Transitional Transitional Mature

19 Lactose synthesis Key regulatory proteins: PGM1 UGP2 NME2 Colostrum Transitional Transitional Mature Fold-increase = , = , = , = , = Fold-decrease = , =

20 Take-home messages on Methods Got microarray data? Publish quickly. (RNA-Seq is the bomb) Got early milk samples? Classify them by Na:K, not day of lactation Milk fat globule RNA + sequencing = exquisite detail of milk-producing cells

21 Conclusions Primary function of milk-producing cells? Making and delivering milk proteins Hallmarks of lactation stages: Colostrum: Immune function Transition: Turn off protein degradation, respond to milk hormones Mature: Massive lipid synthesis Identified key genes for further investigation

22 Next steps Investigate new hypotheses Rate-limiting steps of human milk production Role of ferritins in colostrum Dysregulation of insulin signaling Apply this new assay Characterize phenotypes Evaluate interventions Understand mechanisms to design better interventions

23 Acknowledgments Moms who donated their milk Funding: Cincinnati Obesity and Diabetes Center and NIH HD1302 Milk Collection:CCHMC Jeanne Kleiman, Erin Wagner, Anne Minter; The Christ Hospital Charla Payne and Beth Von Luehrte Milk processing: Barbara Davidson, Diana Taft, Maria Hughes, Amy Lefevers, Myra Johnson Technical Advising: Susan Wert, Jeffrey Whitsett, Mike Burhans RNA-seq: CCHMC Gene Discovery Core Facility Leadership: Laurie Nommsen-Rivers, Ardythe Morrow Morrow and Nommsen-Rivers Labs (not pictured: Ardythe Morrow, Barbara Davidson, Diana Taft, Amy Lefevers)