Transcript Fold - Change ( HAI vs. C)

MicroRNA-1246 is a Potential Regulator
of Factor 8 Gene*
C.D. Atreya, Ph.D.
Associate Director for Research
Office of Blood Research and Review
Center for Biologics Evaluation and Research
US Food and Drug Administration
US Department of Health and Human Services
5th World Hematologists Congress, Aug 18-19, 2016
*Sarachana T, Dahiya N, Simhadri VL, Pandey GS, Saini S, Guelcher C, Guerrera MF, KimchiSarfaty C, Sauna ZE, Atreya CD. PLoS One. 2015 Jul 15;10(7):e0132433.
>>This presentation reflects the views of Dr. C.D. Atreya and should not be construed
to represent FDA’s views or policies<<
Introduction
• Hemophilia A disease
– Symptoms
– Severity
• microRNAs (mi-RNAs), the gene regulators
– Biogenesis
– Mechanism of action
• New insights into FVIII regulation?
Hemophilia A (HA) Disease
 An X chromosome-linked bleeding disorder, caused by
mutations in the F8 gene that results in a dysfunctional
clotting Factor VIII (FVIII)
 Symptom Severity (percentage breakdown of overall
hemophilia population by severity)
 Severe (factor levels < 1% in blood) represent ~ 60% of cases
 Moderate (factor levels of 1-5%in blood) represent ~ 15% of cases
 Mild (factor levels of 6%-40% in blood) represent ~ 25% of cases
 In severe and moderate cases, repeat infusions of FVIII
are required, which leads the development of FVIII
inhibitors (antibodies)
 Inhibitors interfere with the FVIII therapy
Frequencies of F8 mutation types and HA disease manifestation
F8
Severe Moderate
Mutation Enrolled Enrolled
661
139
None
Yes*
15
646
7
132
Mild
Total
Enrolled
187
Enrolled
987
4
183
26
961
% of Total
enrolled
2.6**
97.4
*Intron 22 inversion, Frameshift, Nonsense, Splice site, Large deletion, Intron 1 inversion,
Small deletion and Duplication
**In addition to the 2.6% with no mutations in F8, there are a few HA patients
(~1.3%) with synonymous mutations in F8 where codon changes do not change
the primary amino acid sequence of the translated FVIII protein (Total ~3.99%)
Source: CDC HA Mutation Project, aka CHAMP database
Hemophilia A
Observation
 Over 3.9% of patients had either no mutations in F8 gene or had
synonymous mutations, but exhibit the complete range of HA phenotypes mild to severe, with over 50% of the patients being severe (CDC HA
Mutation Project, aka CHAMP) and develop FVIII inhibitors
 This suggests that mechanisms other than mutations in the F8 coding region
per se may play either a primary or secondary role in the manifestation of
HA
Possible mechanism
 Posttranscriptional regulation of F8 mRNA (i.e. translation control)?
Hypothesis
 Since microRNAs (miRNAs) are potent post-transcriptional negative
regulators of mRNAs, they must be affecting FVIII expression, especially
in patients with no mutation in F8 or with synonymous mutations
Gene Regulation
MicroRNAs
MicroRNAs (miRNAs)
Short single-stranded
noncoding regulatory RNAs
(17-22 nts in size)
Biogenesis and
mechanism of action
Drsha
Nuclear RNAse III
Dicer
Cytoplasmic RNAse III
DGCR8 protein
Digeorge Syndrome Critical Region 8
RISC
RNA interfering silence complex
http://www.sigmaaldrich.com/
Study Methods
• 15 confirmed HA patients (9 with and, 6 without
inhibitors)
• 5 healthy controls
• Total RNA was isolated from all 20 samples
• Affimetrix GeneChip miRNA 3.0 microarrays were
used (19,724 probe set covering over 5,600
human miRNAs, pre-miRNAs, snoRNAs, and
scaRNAs
• Appropriate bioinformatics and statistical analyses
software were used to analyze and validate the
data
Significantly differentially regulated ncRNAs (6)
identified by SAM* analysis
(*Significance Analysis of Microarrays)
Transcript ID
Fold-Change
(all HA vs. C)
Fold-Change
(HAI vs. C)
Fold-Change
(HAWI vs. C)
Fold-Change
(HAI vs. HAWI)
miR-1246
5.001
1.949
12.834
-6.585
HBII-13
2.538
3.646
1.766
2.065
miR-4521
2.288
2.885
1.814
1.591
miR-181d
1.951
2.234
1.703
1.312
SNORD121B
1.498
1.701
1.318
1.290
A 3-class SAM analysis of ncRNA microarray data revealed 7 ncRNA probes that were
differentially expressed between hemophilia A (HA) patients with inhibitor development
(HAI), hemophilia A patients without inhibitor development (HAWI), and controls (C) with
the false discovery rate less than 5%. (Sarachana et al, PLOS ONE 2015. 10 (7):e0132433)
Validation of microarray results by qRT-PCR
ncRNA
miR-1246
miR-4521
HBII-13
Fold-Change
(all HA vs. C)
qRT-PCR
Microarray
(p- value)
30.056
5.001
(0.382)
2.328
2.288
(0.014)
3.357
2.538
(0.006)
Fold-Change
Fold-Change
( HAI vs. C)
(HAWI vs. C)
qRT-PCR
qRT-PCR
Microarray
Microarray
(p- value)
(p- value)
3.107
43.534
1.949
12.834
(0.146)
(0.297)
2.173
2.656
2.885
1.814
(0.013)
(0.027)
3.127
3.110
3.646
1.766
(0.047)
(0.066)
miR-1246 and F8 mRNA levels in HA patients
compared to normal donors
(A) The fold change in miR-1246 was determined by RT-PCR. Total RNA was isolated from the blood of 5
control (CON) donors and 15 HA patients (HA). There is a significant increase in the miR-1246 levels in
samples from HA patients compared to controls. (B) The fold change in F8 mRNA was determined by RTPCR. Total RNA was isolated from the blood of 5 control (CON) donors and 15 HA patients (HA). There is a
significant decrease in the F8 mRNA levels in samples from HA patients compared to controls
So far….
 6-fold up-regulation of miR-1246 was observed in HAWI
patients relative to HAI patients
 miR-1246 target site is present in the F8 mRNA 3’UTR
Testable hypothesis:
miR-1246 down regulates F8 mRNA expression. If this is
true, it could explain why 3.9% of the CHAMP database
manifested HA phenotype without a mutation in F8 gene.
Suppression of F8 mRNA expression by miR-1246 in a
stable lymphoblastoid cell line expressing FVIII
P=0.0005
P<0.0001
miR 1246 Precursor
-
+
-
+
Fold Change in miR 1246 Fold Change in F8 mRNA
Mir-1246
Fold-Change in miR-1246 (with
precursor
respect to non-targeted miR)
0 nM
1
P = 0.0005
10 nM
1.5 ± 0.05
Fold-Change in F8 mRNA (with
respect to non-targeted miR)
1.00
P < 0.0001
0.74 ± 0.02
Conclusions so far….
 miR-1246 has the potential to downregulate FVIII
levels in normal F8, which could lead to HA phenotype
 miR-1246 is a potential biomarker for
o ~3.9% CHAMP subset (no mutations/synonymous
mutation in F8)
o HAWI subset
Next steps….
 More HA patients will be recruited to validate this
study
 F8 gene from miR-1246 high expresser (HAWI) patients
will be sequenced to confirm their F8 gene status
Future work….
Testable hypothesis..
Since high levels of serum FVIII (>150 IU/L) is predictive
of deep-vein thrombosis (DVT), miR-1246 might be
present at low levels in such patient population and
hence, low levels of miR-1246 might serve as a
predictive biomarker for DVT!
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