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Elevation in plasma tRNA fragments precede seizures in humanepilepsy
Marion C. Hogg, … , David C. Henshall, Jochen H.M. Prehn
J Clin Invest. 2019. https://doi.org/10.1172/JCI126346.
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Transfer RNAs (tRNAs) are a major class of noncoding RNA. Stress-induced cleavage of tRNA is highly conserved andresults in tRNA fragments. Here we find specific tRNA fragments in plasma are associated with epilepsy. Small RNAsequencing of plasma samples collected during video-EEG monitoring of focal epilepsy patients identified significantdifferences in three tRNA fragments (5′GlyGCC, 5′AlaTGC, and 5′GluCTC) from controls. Levels of these tRNAfragments were higher in pre-seizure than post-seizure samples, suggesting they may serve as biomarkers of seizure riskin epilepsy patients. In vitro studies confirmed that production and extracellular release of tRNA fragments was lower afterepileptiform-like activity in hippocampal neurons. We designed PCR-based assays to quantify tRNA fragments in a cohortof pre- and post-seizure plasma samples from focal epilepsy patients and healthy controls. Receiver operatingcharacteristic analysis indicated that tRNA fragments potently distinguished pre- from post-seizure patients. ElevatedtRNA fragments levels were not detected in patients with psychogenic non-epileptic seizures, and did not result frommedication tapering. This study identifies a novel class of epilepsy biomarker and reveals the potential existence ofprodromal molecular patterns in blood that could be used to predict seizure risk.
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1
Elevation in plasma tRNA fragments precede seizures in human epilepsy 1
Marion C. Hogg1,2, Rana Raoof1,3, Hany El Naggar4, Naser Monsefi1, Norman Delanty2,4, Donncha 2
F. O’Brien5, Sebastian Bauer6,7,8, Felix Rosenow6,7,8, David C. Henshall1,2 & Jochen H.M. Prehn1,2* 3
1. Department of Physiology and Medical Physics, Royal College of Surgeons In Ireland, St. 4
Stephen’s Green, Dublin D02 YN77, Ireland. 5
2. FutureNeuro Research Centre, Royal College of Surgeons In Ireland, St. Stephen’s Green, 6
Dublin D02 YN77, Ireland. 7
3. Department of Anatomy, Mosul Medical College, University of Mosul, Mosul, Iraq. 8
4. Department of Neurology, Beaumont Hospital, Beaumont, Dublin, Ireland. 9
5. Department of Neurosurgery, Beaumont Hospital, Dublin, Ireland. 10
6. Epilepsy Center Hessen, Department of Neurology, Baldingerstr, 35043, Marburg, Germany. 11
7. Epilepsy Center Frankfurt Rhine-Main, Neurocenter, Goethe-University, Schleusenweg 2-16, 12
Haus 95, 60528, Frankfurt, Germany 13
8. LOEWE Center for Personalized Translational Epilepsy Research (CePTER), Frankfurt, Germany. 14
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*Corresponding author: Jochen H. M. Prehn, Department of Physiology and Medical Physics, 16
Royal College of Surgeons in Ireland, 123 St. Stephen’s Green, Dublin 2, Ireland. 17
Email: prehn@rcsi.ie 18
Tel: + 353 1 402 2255 19
Fax: + 353 1 402 2447 20
21
Word count: 4,000 22
2
Abstract: 191 23
Figures: 4 figures 24
Supplementary data: 1 document 25
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The authors have declared that no conflict of interest exists. 27
28
Keywords 29
Epilepsy, seizure, tRNA fragment, biomarker 30
3
Abstract 31
Transfer RNAs (tRNAs) are a major class of noncoding RNA. Stress-induced cleavage of tRNA is 32
highly conserved and results in tRNA fragments. Here we find specific tRNA fragments in plasma 33
are associated with epilepsy. Small RNA sequencing of plasma samples collected during video-34
EEG monitoring of focal epilepsy patients identified significant differences in three tRNA 35
fragments (5’GlyGCC, 5’AlaTGC, and 5’GluCTC) from controls. Levels of these tRNA fragments 36
were higher in pre-seizure than post-seizure samples, suggesting they may serve as biomarkers 37
of seizure risk in epilepsy patients. In vitro studies confirmed that production and extracellular 38
release of tRNA fragments was lower after epileptiform-like activity in hippocampal neurons. We 39
designed PCR-based assays to quantify tRNA fragments in a cohort of pre- and post-seizure 40
plasma samples from focal epilepsy patients and healthy controls. Receiver operating 41
characteristic analysis indicated that tRNA fragments potently distinguished pre- from post-42
seizure patients. Elevated tRNA fragments levels were not detected in patients with psychogenic 43
non-epileptic seizures, and did not result from medication tapering. This study identifies a new 44
class of epilepsy biomarker and reveals the potential existence of prodromal molecular patterns 45
in blood that could be used to predict seizure risk. 46
4
Introduction 47
Temporal lobe epilepsy (TLE) is the most prevalent form of focal epilepsy in adults, with around 48
0.1% of the world’s population affected. Diagnosis of epilepsy is complex and challenging. 49
Seizures are rarely witnessed by epileptologists and diagnosis is primarily based on clinical 50
history, supported by electroencephalography (EEG) and imaging (MRI) data. Accordingly, there 51
is an unmet need for biomarkers of epilepsy. To date, few studies have focussed on biomarkers 52
of seizure onset, and seizure prediction studies mainly rely on EEG recordings to provide a 53
patient-specific profile of seizure activity, which has limited predictive power in a subset of 54
patients (1). From a patient’s perspective, the ability to forecast seizures would allow greater 55
control in daily life and improve patient safety. 56
Circulating blood-based molecules represent an attractive source of epilepsy biomarker due to 57
the potential for fast analysis of easy-to-collect samples. Serum levels of High-Mobility Group Box 58
1 (HMGB1) protein have been shown to indicate epileptogenesis in animals and drug-refractory 59
epilepsy in patients (2). Small noncoding RNAs have recently emerged as potential biomarkers, 60
with several studies reporting differences in serum or plasma levels of miRNAs in patients with 61
epilepsy (3). Research into predictive biomarkers for seizure onset or imminence has been 62
limited. Interestingly, a recent study sampling blood from epilepsy patients in a video-EEG 63
monitoring unit identified four miRNAs that were significantly elevated immediately after onset 64
of generalised tonic-clonic seizures (GTCS) (4). 65
Transfer RNAs (tRNA) are ubiquitous noncoding RNAs that deliver amino acids to the ribosome 66
during protein synthesis. Cleavage of tRNAs occurs as part of a highly conserved stress response 67
5
present in single-celled organisms (5). In humans, tRNA fragments are generated by 68
ribonucleases including Dicer and Angiogenin (6). Numerous functions have been attributed to 69
tRNA fragments, including inhibition of protein translation (7), initiation of stress granule 70
formation (8), and regulation of gene expression (9, 10). tRNA fragments have also been 71
identified circulating in blood (11), indicating they are protected from degradation, which makes 72
them ideal candidates for investigation as biomarkers. 73
Here we identified plasma tRNA fragments in RNA sequencing (RNA-seq) data from focal 74
epilepsy patients (12). We found three specific tRNA fragments that were significantly elevated 75
in pre-seizure samples compared to post seizure samples and healthy controls. We performed 76
mechanistic and clinical validation studies to explore their potential as novel biomarkers of 77
seizure risk. Together, our study suggests that specific tRNA fragments may be a novel class of 78
epilepsy biomarker that could support prediction of seizure risk in patients diagnosed with 79
epilepsy. 80
81
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Results and Discussion 82
Blood samples were collected from 16 refractory focal epilepsy patients (majority with TLE) 83
upon arrival for video-EEG monitoring at the Epilepsy Monitoring Unit (EMU) at the Epilepsy 84
Centre in Hessen, Department of Neurology, Marburg. All patients had drug-refractory focal 85
epilepsy and were on polytherapy. Demographics and medications are shown in Supplementary 86
Table 1. Continuous video-EEG monitoring was performed for each patient using the 10-20 87
standard international electrode placement system. Recordings were manually reviewed by a 88
neurologist with special training in epilepsy. A second blood sample was collected 24 hours after 89
a confirmed electro-clinical seizure. Plasma samples were obtained at the same site from age and 90
gender-matched healthy volunteers. 91
Small RNA-seq (< 50 nt) was performed on pooled samples from 16 controls and pre-seizure or 92
post-seizure samples from 16 focal epilepsy patients. A custom tRNA library was used to quantify 93
reads aligning to tRNAs, summarised in supplementary Table 2. Reads were pooled for tRNAs of 94
the same isoacceptor type, and tRNAs were ranked by fold-change between pre-seizure and 95
control samples (Table 1). Three tRNA fragments were chosen for further investigation based on 96
abundance (counts per million, CPM) and fold-change: Glycine GCC, Alanine TGC, and Glutamic 97
acid CTC (Table 1). Read coverage plots revealed reads aligned to the 5’ end of the tRNA (Figure 98
1A, D, & G). The cleavage site was mapped onto the predicted secondary structure of each of the 99
tRNAs (Figure 1B, E, & H). Predicted secondary structures of the tRNA fragments indicated they 100
form short hairpin structures (Figure 1C, F, & I). Of note, tRNA fragments levels were highest in 101
pre-seizure samples and closer to control levels in post-seizure samples (Figure 1A, D, & G). 102
7
As these tRNA fragments are novel, no existing assays were available; therefore we designed 103
custom Taqman assays. These assays utilise standard qPCR techniques and allow fast, specific 104
quantification of short RNA fragments. As tRNAs are highly conserved, the assays can be used 105
on both mouse and human samples (see supplementary Figure 1). Next, we aimed to 106
experimentally model the observation that tRNA fragments levels decreased following epileptic 107
seizure. Seizure-like activity was induced in cultured mouse hippocampal neurons by withdrawal 108
of magnesium and assessed by measuring intracellular calcium levels with FLUO-4 (Figure 2A) 109
(13). Representative FLUO-4 traces in the presence (Mg+: 1 mM MgCl2) and absence (Mg-: 0 mM 110
MgCl2) of magnesium indicated spikes of increased intracellular calcium levels corresponding to 111
increased neuronal activity (Figure 2B). Intracellular 5’GluCTC levels were significantly decreased 112
following epileptiform activity (Figure 2C). As tRNA fragments are secreted from neurons (14), 113
we next quantified tRNA fragments in the extracellular medium, and found that both 5’GlyGCC 114
and 5’GluCTC were significantly reduced following increased neuronal activity (Figure 2D). This 115
confirmed tRNA fragments are generated in neurons, secreted from neurons, and that secretion 116
of tRNA fragments is reduced following neuronal hyperexcitation (mimicking epileptiform 117
activity). tRNA fragment levels were normalised to U6 RNA, which remained unchanged 118
throughout experiments (supplementary Figure 2). 119
120
We next used the custom Taqman assays to validate the RNA-seq data in the 16 focal epilepsy 121
patients and 16 healthy controls recruited in Marburg, with an additional 16 focal epilepsy 122
patients recruited at the EMU in Beaumont Hospital, Dublin (Supplementary Table 1), and 16 123
healthy controls. We found that levels of all three tRNA fragments were significantly elevated in 124
8
pre-seizure samples (Figure 3A-C). Wilcoxon signed-rank test indicated there was a highly 125
significant change in tRNA fragment levels between pre and post seizure samples, 5’GlyGCC: 126
3.17-fold change (p=0.0002), 5’AlaTGC: 1.93-fold change (p<0.0001), and 5’GluCTC: 2.89-fold 127
change (p=0.0003) (Figure 3D-F), with the majority of samples showing a decrease in tRNA 128
fragment levels post-seizure. Receiver Operating Characteristic (ROC) analysis indicated tRNA 129
fragments could distinguish pre- and post- seizure samples (Figure 3G-I). 5’GlyGCC had an area 130
under the curve (AUC) of 0.816 (p = 0.000027, Figure 3G), 5’AlaTGC AUC = 0.916 and was highly 131
significant (p = 1.86x10-8, Figure 3H), and 5’GluCTC AUC = 0.802 (p = 0.000069, Figure 3I). 132
Youden’s J statistic was used to determine the optimal cut-off for distinguishing pre and post 133
seizure samples and this indicated a value of 1.33 was most discriminatory for 5’AlaTGC with a 134
sensitivity of 96.8% and specificity of 87.1% (Figure 3H), for 5’GlyGCC 1.36 was optimal (Figure 135
3G) and for 5’GluCTC a 2.13 cut-off performed best (Figure 3I), ROC analyses are summarised in 136
supplementary Table 3. Analysing males and females separately revealed 5’AlaTGC was 137
significantly elevated in pre-seizure samples from both groups (supplementary Figure 3). 138
Importantly, levels of two other tRNA fragments detected in the plasma RNA-seq data (5’ValAAC 139
& 5’ProAGG) were validated by custom Taqman assay and were not elevated in pre-seizure 140
samples (supplementary Figure 4). Plasma collected at additional time points were available for 141
24/32 patients (T1: upon arrival to the EMU and T3: 1 hour post-seizure). Analysis of extra time 142
points indicates 5’AlaTGC levels spike pre-seizure and fall 1 hour post-seizure before rising again 143
by 24 hours post-seizure (supplementary Figure 5). Analysis of correlation between time interval 144
(pre-seizure blood collection to seizure onset) and tRNA fragment level indicates 5’GluCTC is 145
significantly elevated prior to seizure onset (Spearman r=-0.59, p=0.0035) when analysis is limited 146
9
to under 50 hours (supplementary Figure 6). 5’GlyGCC and 5’AlaTGC showed weaker correlation 147
that did not reach statistical significance. These analyses indicate specific tRNA fragments can 148
discriminate between pre- and post-seizure samples and may be of use as epilepsy biomarkers 149
in advance of seizure. 150
151
Having demonstrated tRNA fragments are elevated in focal epilepsy patient plasma in advance 152
of seizures occurred we sought to determine whether tRNA fragments were detectable in brain 153
tissues. Five patients from the Dublin cohort had undergone surgical resection in an attempt to 154
alleviate their seizures. Fresh frozen hippocampal and cortical tissue was cryosectioned, with 155
neighbouring sections mounted for histological analysis and collected for RNA analysis. All three 156
tRNA fragments were detectable in both hippocampus and cortex tissue (Supplementary Figure 157
7A-C); no significant difference in tRNA levels between tissues was detected. Histological analysis 158
indicated tissue samples were intact although components of the hippocampal formation were 159
only visible in two samples (Supplementary Figure 7D). Analysis of MRI data revealed 9 focal 160
epilepsy patients had no structural lesions detected (20 positive for lesions, 3 indeterminate), 161
and interestingly tRNA fragments were higher in MRI negative patients (supplementary Figure 8) 162
indicating tRNA fragments are not released as a result of structural damage. 163
164
One caveat to interpreting this data is the patients’ medication is withdrawn upon arrival at the 165
EMU and may therefore be higher in the pre-seizure than the post-seizure samples; however, we 166
do not believe that medication levels influence tRNA fragment levels for several reasons. Firstly, 167
10
we noted that four patients did not have their medication reduced and went on to experience 168
seizures, however we found no significant difference between pre-seizure tRNA fragment levels 169
in patients with and without AED reduction (supplementary Figure 9). Secondly, we have 170
analysed tRNA fragment levels in a cohort of patients where AEDs were reduced but patients did 171
not experience seizures and here we find no change in tRNA fragment levels (supplementary 172
Figure 10). Finally, we analysed plasma tRNA fragment levels in six patients subsequently 173
diagnosed with psychogenic non-epileptic seizures (PNES); here we found no change in tRNA 174
fragment levels between pre and post “seizure-like” event (supplementary Figure 11), indicating 175
elevated tRNA fragments are linked to electro-clinical seizures. 176
177
To investigate the mechanism leading to increased pre-seizure tRNA fragment levels we 178
examined whether abnormal inter-ictal neuronal activity may be occurring, which is not sufficient 179
to trigger an epileptic seizure but may instigate tRNA cleavage and secretion. One study using 180
continuous intra-cranial EEG-monitoring of a small group of patients identified long-term energy 181
bursts up to 7 hours before seizure onset, which increased in frequency in the hours immediately 182
preceding seizure onset (15). To examine this a Neurologist reviewed video-EEG recordings from 183
a period of 18-24 hours upon arrival to the EMU and patients were classified into three groups 184
based on v-EEG activity: rare, occasional, and frequent. There was no significant difference in 185
pre-seizure tRNA fragment levels between groups (supplementary Figure 12). Eight patients 186
developed GTCS; however, no difference in pre-seizure tRNA fragment levels were detected in 187
these patients compared to those who did not develop GTCS (supplementary Figure 13). 188
11
189
There are currently no reliable biomarkers of seizure onset and patients with drug refractory 190
epilepsy consistently report the unpredictable nature of seizures to be a challenging facet of their 191
disease (16). The ability to forecast seizure activity would allow patients to regain control over 192
their condition. Here we investigated RNA-seq data from patients with focal epilepsy, and 193
identified three tRNA fragments that were elevated in pre-seizure plasma samples. We showed 194
they are expressed and secreted from neurons, and that tRNA fragment levels are regulated in 195
response to neuronal activity. Finally, we validated the RNA-seq data results showing tRNA 196
fragments are elevated in pre-seizure plasma samples from a cohort of focal epilepsy patients 197
compared to healthy controls. We present an exciting proof-of-concept study, which indicates 198
plasma tRNA fragments warrant further investigation as prodromal biomarkers that could be 199
used to predict seizure risk. 200
201
The underlying stress that precipitates tRNA cleavage in our study is unknown. Seizures are 202
generally defined as excessive electrical activity in the brain, which can lead to involuntary 203
movement, changes in behaviour, and loss of consciousness. Many underlying causes can lead to 204
seizures such as infections, tumours, brain trauma, oxygen deprivation and exposure to drugs or 205
toxins. However, in most patients diagnosed with epilepsy the underlying cause of seizures is 206
unknown. At the cellular level, numerous changes during epileptogenesis have been identified 207
including neurodegeneration, gliosis, inflammation, neuronal growth and angiogenesis 208
(reviewed in (17)). tRNA cleavage occurs in response to stress in organisms from yeast to humans 209
12
indicating it is a highly conserved process (5, 6). tRNA cleavage can occur at several sites on the 210
tRNA molecule; Dicer cleaves tRNAs between the D-loop and the anticodon loop, whereas 211
Angiogenin primarily cleaves tRNAs within the anticodon loop (6). Interestingly Dicer levels are 212
significantly lower in TLE patients with hippocampal sclerosis (HS) than those without (18). tRNA 213
cleavage has been identified in response to infection (19) and ischaemia (20), indicating these 214
epileptogenic processes could contribute to the observed tRNA cleavage. A recent study has 215
shown that Kainic Acid (KA) treatment of synaptosome fractions induced spontaneous release of 216
specific miRNAs, which was inhibited by depolarisation, indicating dynamic regulation of non-217
coding RNA secretion by neuronal activity (21). An important limitation of the present study is 218
that despite detecting the tRNA fragments in resected brain tissue and cultured hippocampal 219
neurons, we cannot exclude that the tRNA fragments we detected in patient plasma may have 220
originated in another tissue, as both acute seizures and chronic epilepsy are system disorders, 221
with physical and biochemical effects on organs and tissues outside the CNS (22). 222
223
tRNA fragments share many features with miRNAs, which make them both ideal molecules for 224
use as biomarkers. In order to be of use to patients a biomarker of seizure imminence would 225
require a portable quantification device capable of assessing tRNA fragment levels rapidly in 226
whole blood. Recent advances in this area include development of the TORNADO device (23) 227
which is capable of accurately quantifying miRNA-134 in plasma from focal epilepsy patients 228
using an electrochemical quantification approach (12). This might complement other new 229
wearable technologies, such as seizure detection watches (24). Our data indicate that tRNA 230
fragments are quantifiable using standard laboratory-based PCR techniques and may provide 231
13
increased specificity over miRNAs as epilepsy biomarkers, which are the current focus of many 232
studies. 233
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Methods 234
Study Approval 235
Samples were collected at the Philipps University of Marburg, Germany (MAR) and the 236
Department of Neurology, Beaumont Hospital, Dublin, Ireland (DUB). Ethical approval was 237
obtained from the local medical ethics committees (MAR, 17/14; DUB 13/75), and written 238
consent was obtained from all participants according to the Declaration of Helsinki. Animal work 239
was approved by the Research Ethics Committee of the Royal College of Surgeons in Ireland (REC 240
1122). 241
242
Additional methods are provided in Supplementary data. 243
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Author Contributions 244
Study design: M.C.H., D.C.H., & J.H.M.P. 245
Sample collection and clinical evaluation: R.R., H.E.N., N.D., D.O’B., S.B., & F.R. 246
Performing and analysing experiments: M.C.H, R.R., & N.M. 247
Writing manuscript: M.C.H., D.C.H, & J.H.M.P 248
249
Acknowledgements 250
The authors wish to thank the patients that participated in this study, the neuropathologists who 251
assessed surgically resected patient tissue (Drs Jane Cryan, Francesca M. Brett, Alan Beausang, 252
and Michael A. Farrell), and Dr Heiko Duessman (RCSI) for help with imaging. This publication has 253
emanated from research supported in part by a research grant from Science Foundation Ireland 254
(SFI) under Grant Number 16/RC/3948 and co-funded under the European Regional 255
Development Fund and by FutureNeuro industry partners. This study was supported by SFI grants 256
SFI/13/IA/1891, financial support of the European Union’s ‘Seventh Framework’ Programme 257
(FP7) under Grant Agreement No. 602130 (EpimiRNA), and a fellowship from the Iraqi Ministry 258
of Higher Education and Scientific Research. 259
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References 261
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Figures 319
320
Figure 1: 5’tRNA fragments are differentially expressed in plasma from epilepsy patients. Small 321
RNA-seq on pooled samples from 16 focal epilepsy patients pre and post-seizure and 16 healthy 322
controls. Coverage plots showing reads aligned to A) GlyGCC, D) AlaTCG, & G) GluCTC tRNAs, in 323
counts per million (CPM, y-axis) and tRNA sequences (x-axis). B, E, & H the cleavage sites are 324
indicated (red triangle) on the mature tRNA structures (downloaded from GtRNAdb 2.0 (25)). C, 325
F, & I Predicted secondary structures of the tRNA fragments. 326
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327
Figure 2: tRNA fragments are regulated by epileptiform activity in hippocampal neurons. A) 328
Primary mouse hippocampal neurons were incubated with 5 M FLUO4 for 45 mins at 37oC and 329
imaged at 5 Hz. Scale bar = 40 m. B) Representative FLUO4 traces are shown for neurons in 1 330
mM Mg (Mg+, upper panel) or 0 mM Mg (Mg-, lower panel). Following 2 hours in Mg+ or Mg- 331
media, media was collected and total RNA extracted. Both intracellular (C) and extracellular (D) 332
tRNA fragment levels were lower following culture in Mg-free media, where extracellular 333
5’GlyGCC and 5’GluCTC levels were significantly lower (p < 0.05, Students t-test). In C & D 334
individual data points are plotted with mean indicated in red and error bars +/- SEM.N= 15-16 335
replicates from 5 independent preps. 336
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337
Figure 3: tRNA fragments are elevated in plasma from epilepsy patients pre-seizure. 32 epilepsy 338
patients had blood sampled pre and post-seizure and 32 healthy controls were analysed. A) 339
5’GlyGCC, B) 5’AlaTGC and C) 5’GluCTC tRNA fragments were significantly elevated in pre-seizure 340
samples, Kruskal-Wallis test indicated p = 0.0008, p < 0.0001, and p = 0.0001 respectively. No 341
significant difference was detected between post-seizure samples and controls. Mean +/- SEM 342
indicated. Most tRNA fragments decreased significantly post-seizure, Wilcoxon signed-rank test 343
results shown in D) 5’GlyGCC (p=0.0002), E) 5’AlaTGC (p<0.0001), and F) 5’GluCTC (p=0.0003). 344
ROC analysis indicated G) 5’GlyGCC had an area under the curve (AUC) of 0.816 (p = 0.000027), 345
H) 5’AlaTGC AUC = 0.916 (p = 1.86x10-8), and I) 5’GluCTC AUC = 0.802 (p = 0.000069). 346