PrEP_Study_Microarray_Analysis.md

PreP Study Microarray Analysis

Claire Levy

Experimental set up

We isolated RNA from four different sample types from 8 donors pre- and post- initiation of PreP. The pre-initation was called "Enrollment" and the post-initiation visit was called "Visit2"

Sample Types

• Duodenal biopsy
• Rectal biopsy
• PAXgene (whole blood collected into RNA preservative)
• PBMC (PBMC isolated from whole blood)

NOTE: PTID BG2305 may not have been adherent, left them in for now. May have had problems refilling prescription on time.

Plots of background-corrected but non-normalized data

These normalized samples all look good.

Non-specific filtering

There are 8 donors x 1 tissue type x 2 timepoints = 16 samples in this set. I will filter out any probes that are not expressed above the 0.05 p-value cut-off in at least 7 samples. I chose 7 because a probe may not be expressed at all in the 8 Enrollment samples (so 0/16 total), but may show up at Visit2 (8 possibilities). It would still be biologically interesting if a probe was expressed at Visit2 (and not at enrollment) even if it wasn't in all donors, so I'll require it to show up in at least 7 of them.

Number of probes removed from the data sets after filtering for expression. All started with 47323 probes.

Duod	Rectal	PBMC	PAXgene
26818	26787	28427	30127

Number of DE probes for each contrast

The duodenal samples were the only ones with any differentially expressed probes. For Visit2 relative to enrollment, there were 152 down-regulated probes and 120 up-regulated probes.

top 10 changes in the Duodenum pre vs post-PreP: p-value cut-off = 0.05

TargetID	logFC	DEFINITION	adj.P.Val
GK	-0.7041	Homo sapiens glycerol kinase (GK), transcript variant 2, mRNA.	0.007008
LCT	-1.182	Homo sapiens lactase (LCT), mRNA.	0.007008
GK	-0.6262	Homo sapiens glycerol kinase (GK), transcript variant 1, mRNA.	0.007008
DFNA5	-0.8681	Homo sapiens deafness, autosomal dominant 5 (DFNA5), transcript variant 1, mRNA.	0.007008
LAMA3	-0.6491	Homo sapiens laminin, alpha 3 (LAMA3), transcript variant 1, mRNA.	0.007008
BTNL3	-0.4034	Homo sapiens butyrophilin-like 3 (BTNL3), mRNA.	0.007008
MGC13057	-0.6181	Homo sapiens hypothetical protein MGC13057 (MGC13057), mRNA.	0.007008
AQP10	-1.664	Homo sapiens aquaporin 10 (AQP10), mRNA.	0.0131
KIAA1671	-0.5056	PREDICTED: Homo sapiens KIAA1671 protein (KIAA1671), mRNA.	0.01454
SLC36A1	-0.5032	Homo sapiens solute carrier family 36 (proton/amino acid symporter), member 1 (SLC36A1), mRNA.	0.01454

CAMERA testing

From the CAMERA documentation:

"camera performs a competitive test in the sense defined by Goeman and Buhlmann (2007). It tests whether the genes in the set are highly ranked in terms of differential expression relative to genes not in the set."

Hallmark gene sets: top 10 results

Duodenal sample results

Gene_Set	Direction	FDR	NGenes
interferon alpha response	Up	1.432e-10	118
e2f targets	Up	1.495e-09	269
g2m checkpoint	Up	2.482e-07	251
bile acid metabolism	Down	0.0003259	125
myc targets v1	Up	0.0007642	287
myc targets v2	Up	0.002901	79
interferon gamma response	Up	0.003457	241
xenobiotic metabolism	Down	0.006079	219
unfolded protein response	Up	0.006459	143
hypoxia	Down	0.02173	235

Rectal sample results

Gene_Set	Direction	FDR	NGenes
myc targets v2	Up	1.887e-07	79
oxidative phosphorylation	Up	8.293e-05	266
allograft rejection	Down	0.00145	232
cholesterol homeostasis	Up	0.002958	92
myc targets v1	Up	0.004697	288
estrogen response late	Up	0.0103	234
uv response up	Up	0.01245	175
pancreas beta cells	Up	0.01245	29
e2f targets	Up	0.02967	262
unfolded protein response	Up	0.02967	144

PBMC sample results

Gene_Set	Direction	FDR	NGenes
tnfa signaling via nfkb	Down	8.921e-09	218
interferon gamma response	Down	0.006626	250
apoptosis	Down	0.0113	204
complement	Down	0.0136	211
mtorc1 signaling	Down	0.02192	260
hypoxia	Down	0.02603	201
estrogen response late	Down	0.02715	168
myc targets v2	Up	0.03343	76
inflammatory response	Down	0.04017	198
cholesterol homeostasis	Down	0.0726	78

PAXgene sample results

Gene_Set	Direction	FDR	NGenes
interferon alpha response	Down	0.0002159	118
interferon gamma response	Down	0.01146	237
tgf beta signaling	Down	0.2055	53
myc targets v2	Up	0.2055	73
mitotic spindle	Up	0.4815	179
apical surface	Up	0.4815	35
hedgehog signaling	Up	0.5361	21
bile acid metabolism	Up	0.5361	85
dna repair	Up	0.5361	175
apical junction	Up	0.5525	155

GO biological process gene sets: top 10 results

Sean wrote some code (https://github.com/seaaan/Bioinformatics/tree/master/GOTermMappingsForCamera) to extract just the Biological Process gene sets out of all the GO gene sets.

Duodenal sample results

Gene_Set	Direction	FDR	NGenes
go flavonoid metabolic process	Down	6.613e-07	30
go neutral lipid biosynthetic process	Down	1.934e-06	34
go acylglycerol biosynthetic process	Down	1.934e-06	34
go dna replication	Up	3.922e-05	237
go glucuronate metabolic process	Down	3.922e-05	26
go uronic acid metabolic process	Down	3.922e-05	26
go response to type i interferon	Up	3.922e-05	78
go response to interferon alpha	Up	6.198e-05	28
go dna replication initiation	Up	7.884e-05	29
go response to xenobiotic stimulus	Down	9.868e-05	102

Rectal sample results

Gene_Set	Direction	FDR	NGenes
go hemidesmosome assembly	Up	0.002715	16
go oxidative phosphorylation	Up	0.03887	89
go ire1 mediated unfolded protein response	Up	0.04367	77
go electron transport chain	Up	0.04367	102
go regulation of cell projection size	Up	0.04367	13
go positive regulation of protein localization to cell periphery	Up	0.04367	52
go positive regulation of protein localization to plasma membrane	Up	0.04367	52
go cellular respiration	Up	0.04367	156
go energy homeostasis	Down	0.04367	15
go negative regulation of endoplasmic reticulum stress induced intrinsic apoptotic signaling pathway	Up	0.04367	22

PBMC sample results

Gene_Set	Direction	FDR	NGenes
go regulation of secondary metabolic process	Down	0.01013	9
go granulocyte migration	Down	0.01013	51
go positive regulation of transcription from rna polymerase ii promoter in response to stress	Down	0.01013	28
go lymph node development	Up	0.01013	15
go monocyte chemotaxis	Down	0.02234	23
go response to interferon gamma	Down	0.04605	139
go cellular response to interferon gamma	Down	0.04605	117
go positive regulation of vascular endothelial growth factor production	Down	0.04605	28
go negative regulation of viral genome replication	Down	0.05122	60
go epithelial cell maturation	Down	0.05122	14

PAXgene sample results

Gene_Set	Direction	FDR	NGenes
go regulation of secondary metabolic process	Down	0.01588	9
go negative regulation of viral genome replication	Down	0.1076	61
go response to type i interferon	Down	0.1213	78
go cellular defense response	Down	0.1994	53
go interferon gamma mediated signaling pathway	Down	0.222	82
go cellular response to interferon gamma	Down	0.222	112
go ganglion development	Down	0.3379	3
go respiratory burst	Down	0.3575	15
go response to interferon gamma	Down	0.3575	132
go cellular response to vitamin d	Down	0.5703	12

MTN-007 gene sets

Here I am comparing the probes in this experiment to significantly differentially expressed probes from MTN-007 9cm biopsies at 7 days.

Gene_Sets	Tissue	Direction	FDR	NGenes
MTN_007 Down	Duodenal	Down	1.333e-05	765
MTN_007 Up	Duodenal	Up	0.008987	181
MTN_007 Down	Rectal	Down	1.424e-05	787
MTN_007 Up	Rectal	Down	0.004617	191
MTN_007 Down	PBMC	Down	0.369	735
MTN_007 Up	PBMC	Down	0.369	152
MTN_007 Down	PAXgene	Down	0.08041	675
MTN_007 Up	PAXgene	Up	0.7308	144

Which DE probes from the PreP study overlap with those from MTN-007?

TargetID	DEFINITION	logFC in PreP Study
MUPCDH	Homo sapiens mucin-like protocadherin (MUPCDH), transcript variant 1, mRNA.	-0.5277
EDN3	Homo sapiens endothelin 3 (EDN3), transcript variant 3, mRNA.	-0.6583
GOLPH4	Homo sapiens golgi phosphoprotein 4 (GOLPH4), mRNA.	-0.5256
FAM23A	Homo sapiens family with sequence similarity 23, member A (FAM23A), mRNA.	-0.276
PTGDS	Homo sapiens prostaglandin D2 synthase 21kDa (brain) (PTGDS), mRNA.	0.4569
NUSAP1	Homo sapiens nucleolar and spindle associated protein 1 (NUSAP1), transcript variant 2, mRNA.	0.4414
TOP2A	Homo sapiens topoisomerase (DNA) II alpha 170kDa (TOP2A), mRNA.	0.4312
EDN3	Homo sapiens endothelin 3 (EDN3), transcript variant 2, mRNA.	-0.5274

CAMERA test of Hallmark interferon alpha geneset founder sets

Duodenal sample results

Gene_Set	Direction	FDR	NGenes
moserle ifna response	Up	4.674e-12	40
bennett systemic lupus erythematosus	Up	4.674e-12	36
einav interferon signature in cancer	Up	2.082e-10	42
zhang interferon response	Up	6.545e-10	31
dauer stat3 targets dn	Up	1.478e-09	64
hecker ifnb1 targets	Up	2.447e-09	104
browne interferon responsive genes	Up	3.261e-09	99
urosevic response to imiquimod	Up	2.097e-08	33
radaeva response to ifna1 up	Up	4.137e-08	69
stambolsky targets of mutated tp53 dn	Up	1.124e-07	56

Rectal sample results

Gene_Set	Direction	FDR	NGenes
honma docetaxel resistance	Up	0.005468	43
module 119	Down	0.005468	172
module 171	Down	0.01158	151
module 436	Down	0.01158	141
module 292	Down	0.01158	144
module 345	Down	0.01158	134
xu hgf targets induced by akt1 6hr	Up	0.01158	24
module 208	Down	0.01358	132
reactome interferon gamma signaling	Down	0.01358	68
becker tamoxifen resistance up	Up	0.02004	56

PBMC sample results

Gene_Set	Direction	FDR	NGenes
liang silenced by methylation 2	Down	7.635e-05	47
hecker ifnb1 targets	Down	7.635e-05	100
seitz neoplastic transformation by 8p deletion up	Down	0.0002599	66
moserle ifna response	Down	0.001732	39
jackson dnmt1 targets up	Down	0.001922	77
jison sickle cell disease up	Down	0.003179	208
roeth tert targets up	Down	0.003262	15
takeda targets of nup98 hoxa9 fusion 8d up	Down	0.003262	121
mel18 dn.v1 dn	Down	0.004138	93
dauer stat3 targets dn	Down	0.005241	65

PAXgene sample results

Gene_Set	Direction	FDR	NGenes
moserle ifna response	Down	0.0002304	40
einav interferon signature in cancer	Down	0.0002304	43
browne interferon responsive genes	Down	0.0002304	92
bowie response to tamoxifen	Down	0.0002304	29
hecker ifnb1 targets	Down	0.0002304	107
zhang interferon response	Down	0.0002304	32
bennett systemic lupus erythematosus	Down	0.0002881	40
dauer stat3 targets dn	Down	0.0003593	62
sana response to ifng up	Down	0.0003593	81
seitz neoplastic transformation by 8p deletion up	Down	0.000514	62