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Submitted URL:
http://bloodjournal.hematologylibrary.org/content/106/9/3043.full
Effective URL:
https://ashpublications.org/blood/article/106/9/3043/21926/Aminoglycoside-suppression-of-nonsense-mutations
Submission: On August 05 via api (August 5th 2023, 3:10:20 am UTC) from US — Scanned from DE

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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY| November 1, 2005

AMINOGLYCOSIDE SUPPRESSION OF NONSENSE MUTATIONS IN SEVERE HEMOPHILIA

Clinical Trials & Observations
Paula D. James,
Paula D. James
From the Departments of Medicine, Pharmacy, Pathology, and Molecular Medicine,
Queen's University, Kingston, ON, Canada; National Institute for Biological
Standards and Control, Potters Bar, United Kingdom; Department of Hematology,
Hôpital Sainte-Justine, Montreal, QC, Canada; Departments of Medicine,
Pediatrics, and Oncology, University of Calgary, Calgary, AB, Canada; and
Department of Medicine, McGill University, Montreal, QC, Canada.
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Sanj Raut,
Sanj Raut
From the Departments of Medicine, Pharmacy, Pathology, and Molecular Medicine,
Queen's University, Kingston, ON, Canada; National Institute for Biological
Standards and Control, Potters Bar, United Kingdom; Department of Hematology,
Hôpital Sainte-Justine, Montreal, QC, Canada; Departments of Medicine,
Pediatrics, and Oncology, University of Calgary, Calgary, AB, Canada; and
Department of Medicine, McGill University, Montreal, QC, Canada.
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Georges E. Rivard,
Georges E. Rivard
From the Departments of Medicine, Pharmacy, Pathology, and Molecular Medicine,
Queen's University, Kingston, ON, Canada; National Institute for Biological
Standards and Control, Potters Bar, United Kingdom; Department of Hematology,
Hôpital Sainte-Justine, Montreal, QC, Canada; Departments of Medicine,
Pediatrics, and Oncology, University of Calgary, Calgary, AB, Canada; and
Department of Medicine, McGill University, Montreal, QC, Canada.
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Man-Chiu Poon,
Man-Chiu Poon
From the Departments of Medicine, Pharmacy, Pathology, and Molecular Medicine,
Queen's University, Kingston, ON, Canada; National Institute for Biological
Standards and Control, Potters Bar, United Kingdom; Department of Hematology,
Hôpital Sainte-Justine, Montreal, QC, Canada; Departments of Medicine,
Pediatrics, and Oncology, University of Calgary, Calgary, AB, Canada; and
Department of Medicine, McGill University, Montreal, QC, Canada.
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Margaret Warner,
Margaret Warner
From the Departments of Medicine, Pharmacy, Pathology, and Molecular Medicine,
Queen's University, Kingston, ON, Canada; National Institute for Biological
Standards and Control, Potters Bar, United Kingdom; Department of Hematology,
Hôpital Sainte-Justine, Montreal, QC, Canada; Departments of Medicine,
Pediatrics, and Oncology, University of Calgary, Calgary, AB, Canada; and
Department of Medicine, McGill University, Montreal, QC, Canada.
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Susan McKenna,
Susan McKenna
From the Departments of Medicine, Pharmacy, Pathology, and Molecular Medicine,
Queen's University, Kingston, ON, Canada; National Institute for Biological
Standards and Control, Potters Bar, United Kingdom; Department of Hematology,
Hôpital Sainte-Justine, Montreal, QC, Canada; Departments of Medicine,
Pediatrics, and Oncology, University of Calgary, Calgary, AB, Canada; and
Department of Medicine, McGill University, Montreal, QC, Canada.
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Jayne Leggo,
Jayne Leggo
From the Departments of Medicine, Pharmacy, Pathology, and Molecular Medicine,
Queen's University, Kingston, ON, Canada; National Institute for Biological
Standards and Control, Potters Bar, United Kingdom; Department of Hematology,
Hôpital Sainte-Justine, Montreal, QC, Canada; Departments of Medicine,
Pediatrics, and Oncology, University of Calgary, Calgary, AB, Canada; and
Department of Medicine, McGill University, Montreal, QC, Canada.
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David Lillicrap
David Lillicrap
From the Departments of Medicine, Pharmacy, Pathology, and Molecular Medicine,
Queen's University, Kingston, ON, Canada; National Institute for Biological
Standards and Control, Potters Bar, United Kingdom; Department of Hematology,
Hôpital Sainte-Justine, Montreal, QC, Canada; Departments of Medicine,
Pediatrics, and Oncology, University of Calgary, Calgary, AB, Canada; and
Department of Medicine, McGill University, Montreal, QC, Canada.
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Blood (2005) 106 (9): 3043–3048.
https://doi.org/10.1182/blood-2005-03-1307
Article history
Submitted:
April 5, 2005
Accepted:
June 30, 2005

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Citation

Paula D. James, Sanj Raut, Georges E. Rivard, Man-Chiu Poon, Margaret Warner,
Susan McKenna, Jayne Leggo, David Lillicrap; Aminoglycoside suppression of
nonsense mutations in severe hemophilia. Blood 2005; 106 (9): 3043–3048. doi:
https://doi.org/10.1182/blood-2005-03-1307

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ABSTRACT

Aminoglycoside antibiotics exhibit their bactericidal effect by interfering with
normal ribosomal activity. In this pilot study, we have evaluated the effect of
the aminoglycoside antibiotic gentamicin on the factor VIII (FVIII) and IX
levels of severe hemophiliacs with known nonsense mutations. Five patients were
enrolled and each patient was given 3 consecutive days of gentamicin at a dose
of 7 mg/kg intravenously every 24 hours. Two patients (patient no. 1: hemophilia
A, Ser1395Stop; and patient no. 5: hemophilia B, Arg333Stop) showed a decrease
in their activated partial thromboplastin time (aPTT), an increase in their
FVIII (0.016 IU/mL, 1.6%) or FIX (0.02 IU/mL, 2%) levels, and an increase in
thrombin generation. The remaining 3 patients (patient no. 2: hemophilia B,
Arg252Stop; patient no. 3: hemophilia A, Arg2116Stop; and patient no. 4:
hemophilia A, Arg427Stop) showed no response in the aPTTs or factor levels, but
one (patient no. 2: hemophilia B, Arg252Stop) showed an increase in the factor
IX antigen level (2%-5.5%) that persisted throughout the period of the study and
was concordant with an increase in thrombin generation. Gentamicin is unlikely
to be an effective treatment for severe hemophilia due to its potential
toxicities and the minimal response documented in this report. This study,
however, does provide a proof of principle, suggesting that ribosomal
interference with a less toxic agent may be a potential therapeutic mechanism
for severe hemophilia patients with nonsense mutations.

Subjects:
Clinical Trials and Observations, Free Research Articles, Hemostasis,
Thrombosis, and Vascular Biology
Topics:
activated partial thromboplastin time measurement, aminoglycosides, gentamicin
sulfate (usp), gentamicins, hemophilia a, hemophilias, mutation, nonsense,
thrombin, hemophilia b, antigen assay

INTRODUCTION

Hemophilia A and B are inherited deficiencies of coagulation factor VIII (FVIII)
and FIX, respectively, the genes for which are both located on the human X
chromosome. Hemophilia A occurs at an incidence of 1 in 5000 live male births
compared with 1 in 30 000 for hemophilia B.1 Both conditions occur in mild,
moderate, and severe forms, corresponding to plasma clotting factor levels of 6%
to 30%, 1% to 5%, and less than 1%, respectively. Mild hemophiliacs usually
bleed only after trauma or surgery, however, those with severe hemophilia A or B
bleed spontaneously into joints and muscles. Current treatment for correction of
hemostasis involves the infusion of coagulation factor concentrates.

Advances in the understanding of the molecular genetics of hemophilia have
allowed many innovations in management, including the production of recombinant
clotting factor concentrates. Identification of the disease-causing hemophilic
mutations is, in many cases, now possible and allows carrier detection and
antenatal diagnosis as well as the tailoring of treatment in specific cases. In
the past few years, studies of somatic cell gene therapy have also begun, and
results from preclinical animal studies2-7 as well as initial phase 1/2 human
trials8-11 have been promising, although evidence of long-term hemostatic
benefit from this treatment strategy has still to be achieved. In addition to
“classical” substitutive hemophilia gene therapy, other specific molecular
therapies, such as the use of SMaRT (spliceosome-mediated RNA trans-splicing) to
repair mutant DNA,12 and chimeric RNA/DNA oligonucleotide therapies13 are also
being evaluated.

The molecular defects that cause hemophilia are highly variable. While about 45%
of severe hemophilia A cases are caused by an inversion within intron 2214 and
3% by an inversion in intron 1,15 the vast majority of molecular defects
causing hemophilia are a diverse array of either missense or nonsense mutations,
deletions/insertions, or splicing abnormalities. Hemophilia B is also caused by
a wide array of mutations, the majority of which are unique to each kindred. A
review of the factor VIII HAMSTeRS database16 shows that of 952 reported
hemophilia A mutations, 100 are nonsense mutations (∼9%). Similarly, review of
the hemophilia B mutation database17 shows that of 622 reported mutations, 70
are nonsense mutations (∼9%).

Aminoglycoside antibiotics are commonly used to treat Gram-negative infections.
They exert their antimicrobial effect by interfering with the normal function of
the ribosome, specifically with the process of “proofreading” that allows the
discrimination against mismatched amino acyl-transfer RNA from becoming
incorporated into the growing polypeptide chain.18 This mechanism has been
shown to suppress premature termination codons by causing the disregard of the
termination codon and the incorporation of another random amino acid into that
position, thus allowing translation of the polypeptide chain to continue.19-21
Clinical studies exploiting this mechanism for therapeutic benefit have been
performed in a number of conditions including cystic fibrosis,22 muscular
dystrophy,23 Hurler syndrome,24 ataxia-telangiectasia,25 and late infantile
neuronal ceroid lipofuscinosis.26 Furthermore, Srivastava et al27 treated 4
hemophilia B patients with gentamicin in an attempt to override their premature
termination codons but did not see any increase in FIX levels. In this pilot
study, we treated 5 patients (3 with severe hemophilia A and 2 with severe
hemophilia B; Table 1), all with known nonsense mutations, with 3 consecutive
days of gentamicin in an attempt to override their premature stop codons and
increase their levels of FVIII or FIX and to increase overall thrombin
generation.

Table 1.

Patients with severe hemophilia with known nonsense mutations treated with
gentamicin

--------------------------------------------------------------------------------

Patient no.

--------------------------------------------------------------------------------

Hemophilia

--------------------------------------------------------------------------------

Nucleotide

--------------------------------------------------------------------------------

Mutation

--------------------------------------------------------------------------------

Conserved amino acid

--------------------------------------------------------------------------------

. 1 A 4241C > A Ser1395Stop No 2 B 30875C > T Arg252Stop Yes
3 A 6403C > T Arg2116Stop Yes 4 A 1536C > T Arg427Stop Yes
5

--------------------------------------------------------------------------------

31118C > T

--------------------------------------------------------------------------------

Arg333Stop

--------------------------------------------------------------------------------

Yes

--------------------------------------------------------------------------------

Patient no.

--------------------------------------------------------------------------------

Hemophilia

--------------------------------------------------------------------------------

Nucleotide

--------------------------------------------------------------------------------

Mutation

--------------------------------------------------------------------------------

Conserved amino acid

--------------------------------------------------------------------------------

. 1 A 4241C > A Ser1395Stop No 2 B 30875C > T Arg252Stop Yes
3 A 6403C > T Arg2116Stop Yes 4 A 1536C > T Arg427Stop Yes
5

--------------------------------------------------------------------------------

31118C > T

--------------------------------------------------------------------------------

Arg333Stop

--------------------------------------------------------------------------------

Yes

--------------------------------------------------------------------------------

View Large

PATIENTS, MATERIALS, AND METHODS

PATIENTS AND STUDY PROTOCOL

Potential patients were identified through the Canadian National Hemophilia
Genotyping Program (Kingston, ON, Canada) and invited to participate in the
study by their local Hemophilia Comprehensive Care Clinic. Research Ethics Board
approval was obtained from Queen's University (Kingston, ON, Canada) and from
all treating centers. Investigational New Drug approval was obtained from Health
Canada for this off-label use of gentamicin (control no. 079440). Patients were
eligible if they were severe hemophiliacs older than 12 years of age with known
nonsense mutations. Exclusion criteria included preexisting renal impairment,
preexisting hearing impairment, presence of an inhibitor to FVIII or FIX,
significant hepatic or cardiac impairment, or a known allergy to gentamicin or
other aminoglycoside antibiotic. Patients were also excluded if they were taking
furosemide, amphotericin B, vancomycin, or acyclovir or if they had been
diagnosed with myasthenia gravis. Patients had to observe a 7-day washout for
coagulation factor concentrates prior to the first treatment day of the study
and were excluded from the study or the study was postponed if they had a bleed
that required treatment within the 7-day period.

Patients underwent a number of prestudy investigations including FVIII or FIX
levels and inhibitor studies, serum creatinine levels, electrolyte levels, liver
enzyme levels and liver function tests, and audiometry. All patients gave
informed consent. Each patient was treated with 3 consecutive days of gentamicin
at a dose of 7 mg/kg intravenously every 24 hours. Pretreatment and
posttreatment (1 hour and 6 hours) activated partial thromboplastin times
(aPTTs), FVIII or FIX levels, thrombin generation assays, gentamicin levels, and
serum creatinine levels were performed on each treatment day. Coagulation
testing and a serum creatinine level were repeated on days 4 and 10. All
patients had repeat audiometry within one month of completion of the study.
Patients were monitored for bleeds throughout the study period and the protocol
was terminated prematurely if a bleed developed that required treatment with a
coagulation factor concentrate.

COAGULATION TESTING

Blood for all coagulation testing was obtained by a 2-syringe technique and
anticoagulated with 0.105 M buffered trisodium citrate at a ratio of 1:9
(anticoagulant-blood). After centrifugation at 10 000g for 15 minutes, the
platelet-poor plasma was removed and stored at –70°C until testing was
performed. All testing was carried out in a central reference laboratory after
transfer of plasma samples on dry ice. Frozen plasma samples were thawed at 37°C
for 5 minutes prior to testing. Testing was performed in such a way that the
technologist was “blinded” to the identity of individual samples.

Functional FVIII and FIX studies were performed with one-stage assays employing
aPTTs using the MDA Platelin L reagent from bio Merieux (St Laurent, QC, Canada)
and factor-deficient plasmas from Precision Biologicals (Dartmouth, NS, Canada).
Tests were performed on an MDA 180 automated coagulometer, and the sensitivity
of the one-stage assays is 0.01 IU/mL (1%). A chromogenic FVIII assay was also
performed using the Chromogenix COAMATIC assay (Diapharma, West Chester, OH) and
is sensitive to a level of 0.005 IU/mL (0.5%). All assays were performed using a
normal plasma pool from Precision Biologicals that is referenced to the
appropriate World Health Organization (WHO) standards (FVIII WHO 97/586; FIX WHO
94/746). Factor VIII:Ag and factor IX:Ag assays were performed using polyclonal
antibodies from Affinity Biologicals (Hamilton, ON, Canada). The level of
sensitivity of the antigen assays is 0.01 IU/mL (1%). Factor VIII and IX
inhibitor assays were performed using the Nijmegen modification of the original
Bethesda method for FVIII28 and a standard Bethesda protocol for FIX.

FLUOROGENIC THROMBIN GENERATION TEST

The fluorogenic thrombin generation test (FTGT) is based on the use of a
fluorogenic thrombin substrate that allows thrombin generation to be determined
continuously in a more physiologic system than prior TGT assays, without the
need for subsampling or defibrination of plasma samples. This method is
sensitive to low levels of FVIII (< 0.001 IU/mL),29 significantly below the
level that is detectable by current coagulation factor assays (Figure 4).
Thrombin generation is initiated by the addition of one part nondefibrinated
plasma (40 μL) to 2 parts substrate mixture (80 μL; fluorogenic substrate, 0.238
mM; FIXa, 5 nM; phospholipids, 3 μg/mL; and Ca2+, 7 mM) in a black microtiter
plate (Greiner, Frickenhausen, Germany). The plate is read in a Spectramax
Gemini XS Fluorimeter (Molecular Devices, Sunnyvale, CA) at 30°C at 30-second
intervals for 1 hour with excitation at 390 nm and reading at 460 nm. The data
are exported into an Excel file and the amount of thrombin generated is
calculated according to the method of Hemker and Begiun.30 This method allows
determination of free thrombin in a continuous thrombin generation test with
chromogenic and fluorogenic substrates.27 For hemophilia B plasma samples, the
trigger is via contact activation. Factor IXa is omitted from the substrate
mixture and the reaction is first carried out in a glass tube, prior to transfer
to a microtiter plate. The data were analyzed and quantified for the area under
the curve (AUC) parameter.

RESULTS

Five patients were recruited and treated under the study protocol: 3 with
hemophilia A (Ser1395Stop, Arg2116Stop, and Arg427Stop) and 2 with hemophilia B
(Arg333Stop and Arg252Stop; Table 1). Patient no. 1 (hemophilia A: Ser1395Stop)
revealed that he had prophylactically treated himself with recombinant FVIII 7
days prior to the start of the trial. Based on half-life calculations, we
estimated that the exogenous factor would have disappeared from the plasma by
the start of the trial and we therefore proceeded. One patient (hemophilia A:
exon 9 Arg427Stop) did not continue past the day-2 6-hours posttreatment sample,
due to the development of hematuria that required treatment. None of the
patients showed any signs of renal or otoxicity or any other adverse events
during or after the treatment protocol.

Two patients showed a change in the aPTTs and factor levels during the
gentamicin treatment period. Patient no. 1 (hemophilia A: Ser1395Stop) showed a
25-second decrease in the aPTT at the day-2 6-hours posttreatment sample that
coincided with an increase in the FVIII level from a baseline of 0.006 IU/mL to
0.014 IU/mL (or 1.4%) by chromogenic FVIII assay. On day 3, the aPTT returned to
baseline however the FVIII level peaked at 0.016 IU/mL (1.6%; Figure 1). Patient
no. 5 (hemophilia B: Arg333Stop) showed a similar decrease in the aPTT of
approximately 20 seconds that started at the day-1 6-hours sample and continued
into day 2. These results coincided with an increase in the FIX level to 0.02
IU/mL (2%) by one-stage FIX assay. Both the aPTT and FIX level returned to the
baseline on day 3 (Figure 2). Significant changes in the factor levels or aPTTs
were not seen in patients no. 2 (hemophilia B: Arg252Stop), no. 3 (hemophilia A:
Arg2116Stop), or no. 4 (hemophilia A: Arg427Stop).

The FVIII enzyme-linked immunosorbent assay (ELISA) results for patient no. 1
(hemophilia A: Ser1395Stop) showed an increase in FVIII antigen above baseline
to a peak of 0.07 IU/mL (7%), which is higher than the 1.6% detected by
chromogenic assay, suggesting the production of a mixture of functional and
nonfunctional proteins (Figure 1). Likewise, the FIX ELISA results for patient
no. 2 (hemophilia B: Arg252Stop) showed a persistent increase in FIX antigen
during the study between 0.02 (2%) and 0.055 IU/mL (5.5%; Figure 3).
Interestingly, patient no. 2 showed no response in either the aPTT or the FIX
level, suggesting that in this case any new FIX protein synthesized in response
to the gentamicin possessed insufficient procoagulant function to register an
effect in the aPTT-based clotting assay.

Figure 1.
View largeDownload PPT

Patient no. 1 aPTT, chromogenic FVIII results, mean thrombin generation area
under the curve (AUC), and FVIII antigen level (ELISA). (Top) There is good
correlation between the drop in aPTT and rise in FVIII in the day-2 pretreatment
(pre), day-2 1-hour, and day-2 6-hour samples. There is a peak in FVIII activity
at the day-3 6-hour sample, and both the aPTT and FVIII return to baseline by
the day-10 sample. (Bottom) There is a peak in FVIII:Ag at the day-3 1-hour
sample to a level of 0.07 IU/mL (7%). The mean thrombin generation AUC is also
shown and begins with the greatest AUC in the baseline sample that most likely
reflects the exogenous recombinant FVIII that the patient self-administered 7
days before the start of the study. The AUC steadily declines from that time
point on and it is possible that this exogenous factor masked any contribution
to overall thrombin generation that the endogenous factor synthesized under the
influence of gentamicin could have made. Error bars indicate the standard error
of the mean (SEM).

Figure 1.
View largeDownload PPT

Close modal
Figure 2.
View largeDownload PPT

Patient no. 5 aPTT, one-stage FIX results and mean thrombin generation AUC.
(Top) There is good correlation between the drop in aPTT and the rise in FIX in
the day-1 6-hours and the day-2 pretreatment samples, however the changes are
not sustained, and both the aPTT and FIX essentially return to baseline by the
day-2 1-hour sample. The greatest mean thrombin generation (bottom) also
correlates with the peak FIX and drop in the aPTT seen in the day-2 pretreatment
sample. Error bars indicate SEM.

Figure 2.
View largeDownload PPT

Close modal

The thrombin generation assay (FTGT) for patient no. 1 (hemophilia A:
Ser1395Stop) showed a peak in the day-1 pretreatment sample, which steadily
declined from that point on (Figure 5A). As mentioned above, this patient had
given himself a prophylactic infusion of recombinant FVIII concentrate 7 days
prior to the start of the trial. The effects of this treatment were not
detectable by either the FVIII chromogenic assay or the aPTT in the day-1
pretreatment sample (Figure 1). FTGT for patient no. 5 (hemophilia B:
Arg333Stop) showed a peak of thrombin generation in the day-2 pretreatment
sample, followed by the day-4 sample, followed by the day-1 6-hours sample
(Figure 5B). The changes seen in the day-1 6-hours and day-2 pretreatment sample
correlated with the increase in the FIX level and the decrease in the aPTT, but
the thrombin generation seen in the day-4 sample did not. FTGT for patient no.
2, in whom FIX antigen values between 0.02 and 0.055 IU/mL were documented
throughout the 3-day study, showed a peak of thrombin generation in the day-2
6-hours posttreatment sample. Moreover, minor but definite increases in thrombin
generation were seen at every time point during the study period in patient no.
2 (Figure 5C). The results in this patient suggest that low levels of FIX
synthesis were stimulated by gentamicin administration sufficient to generate
low levels of thrombin but insufficient to influence aPTT-based coagulant
assays. Increased thrombin generation was not observed in patient no. 3
(hemophilia A: Arg2116Stop) or patient no. 4 (hemophilia A: Arg427Stop).

Figure 3.
View largeDownload PPT

Patient no. 2 FIX ELISA and mean thrombin generation AUC. There is a sustained
increase in the FIX:Ag throughout the study period that returns to baseline by
the day-10 sample. This correlates with a sustained increase in the mean
thrombin AUC that is seen throughout the study period as well. There was no
change seen in the aPTT or the FIX:C level in this patient (not shown). Error
bars indicate SEM.

Figure 3.
View largeDownload PPT

Close modal
Figure 4.
View largeDownload PPT

FTGT on normal plasma (NP). (A) The thrombin generation dose response for normal
plasma with differing FVIII concentrations. (B) The thrombin generation dose
response for normal plasma with differing FIX concentrations. (C) The calculated
area under the thrombin generation curve (AUC) for different dilutions of both
FVIII and FIX plasma.

Figure 4.
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Close modal

DISCUSSION

In this pilot study, we show a change in standard functional hemostatic
parameters (chromogenic and aPTT-based coagulant assays) in 2 patients with
severe hemophilia caused by nonsense mutations and changes in antigen level and
thrombin generation in one additional patient during treatment with the
aminoglycoside antibiotic gentamicin. Despite this interesting difference
between those who showed hemostatic changes and those who did not, the magnitude
of hemostatic response documented in this study was small. Based on our results,
the potential for gentamicin to be an effective treatment for severe
hemophiliacs with nonsense mutations appears limited, given the minimal
hemostatic benefit, the potential toxicities, and the intravenous route of
administration of this agent. Additionally, there are a number of factors to
consider in terms of the changes in hemostatic parameters that we saw, including
the lack of complete correlation between the aPTTs, clotting factor assay
results, and the FTGT results.

In patient no. 1 the nonsense mutation Ser1395Stop is located at the position of
a nonconserved amino acid. However, in the other 4 patients the mutation is at a
conserved position. It can be speculated that in patient no. 1, the insertion of
a random amino acid would be more likely to produce a functional protein, given
the potential lesser importance of that amino acid position. This hypothesis
cannot be extended to explain the positive hemostatic changes seen in patient
nos. 5 and 2, however. We assume that the amino acid that is inserted under the
influence of gentamicin is randomly selected, however, this is not certain, and
the true sequence and structure of the resultant protein is unknown.

In both patients who showed a response in their aPTTs and clotting factor
levels, there was a lack of complete correlation between the shortening of the
aPTT and the increase in factor levels, and both patients showed the changes
early in the treatment protocol. This change was not sustained through to the
end of the protocol in either patient no. 1 or patient no. 5. This may be
related to specific nonsustained interactions between gentamicin and the
ribosome in the hemophiliac system, and, certainly, fluctuations in responses to
aminoglycosides have been observed in other conditions.31,32 However, the lack
of correlation may simply reflect the relative sensitivity of the assays to
small changes in the determinants of coagulation. In this study, all of the more
routine tests of hemostasis are being conducted at their lower limits of
detection. Standard clotting assays such as the aPTT achieve their end point
when approximately 4% of the total thrombin has been generated33 and may not be
the most reflective measure of the true hemostatic response. Much work has been
done recently in order to more accurately characterize global hemostatic
responses including evaluating the contribution that a number of procoagulation
factors including FVII/VIIa, FIX, FX, FII, FV, FVIII, and the anticoagulants
tissue factor pathway inhibitor (TFPI) and antithrombin (AT) make to overall
thrombin generation in in vitro experiments and in computer-generated active
thrombin profiles.34,35 In the paper by Brummel-Ziedins et al,34 data are
presented on 4 severe hemophiliacs, showing significant variability in the
ability to generate thrombin in both the untreated and treated states. It may be
that small changes in determinants of coagulation other than FVIII and FIX in
our patients are responsible for the variable and discordant responses seen both
within and between individuals. In light of these additional complicating
factors, we performed the FTGT in an attempt to evaluate global hemostasis.

The thrombin generation data for patient no. 1 may reflect, at least in part,
the exogenous factor that was self-administered 7 days prior to the start of the
trial because of the clear peak in the day-1 pretreatment sample and the
subsequent, progressive decline. Although calculations of the conventionally
accepted half-life for FVIII would suggest that no residual coagulant activity
should be detectable after a 7-day period, there is growing evidence from
hemophilia prophylaxis studies to question this fact.36 It is possible that
this exogenous factor masked any contribution to overall thrombin generation
that the endogenous factor synthesized under the influence of gentamicin could
have made. The thrombin generation data for patient no. 5 correlated, although
not completely, with the results of the FIX assays and the aPTTs, and although
there was no correlation between the thrombin generation data in patient no. 2
and the clotting factor or aPTT results, there was good correlation between the
FTGT and the FIX ELISA results. This suggests that the FTGT may be capable of
detecting levels of FIX protein that possess partial functionality below the
level of detection of the FIX one-stage assay or the aPTT. Another difference
that merits comment is the discrepancy between the time-to-peak thrombin
generation as seen in the dose-response curves for normal plasma (Figure 5) and
the time to peak seen in our patients. In patient no. 5 and patient no. 2, the
increase in thrombin generation is likely reflecting the endogenous production
of FIX under the influence of gentamicin; however, it may be that differences
between this endogenously produced protein and normal protein are responsible
for the increased time to peak.

In addition to the potential differences in other determinants of the global
hemostatic response, there are other factors that may contribute to the
variability of response seen in this study. Both the specific stop codon and the
DNA sequence context of that stop codon have been shown to have an effect on the
efficiency of translation termination,37-39 however, these factors did not
appear important in our patients.

The identification of therapeutic methods for overriding nonsense mutations
would be beneficial for approximately 10% of hemophiliacs and for individuals
with other inherited conditions caused by nonsense mutations. In this
proof-of-principle study, we have documented small positive effects on the
clotting factor levels in 2 of 5 patients, with additional benefits seen in the
FIX antigen level and thrombin generation in another patient. However, our
results do not support the use of gentamicin as a clinical therapy to suppress
nonsense mutations in severe hemophiliacs. The changes documented in 3 of these
patients support the proof of principle that ribosomal interference with a less
toxic, more efficient, and potentially orally administered agent may present a
viable therapy for severe hemophiliacs with nonsense mutations in the future.

Figure 5.
View largeDownload PPT

FTGT results on patient nos. 1, 5, and 2. (A) The fluorogenic thrombin
generation curve for patient no. 1 before and throughout the study period. There
is a clear high peak in the baseline sample that most likely reflects the
exogenous recombinant FVIII that the patient self-administered 7 days before the
start of the study. The peak of thrombin generation steadily declines from that
time point on and it is possible that this exogenous factor masked any
contribution to overall thrombin generation that the endogenous factor
synthesized under the influence of gentamicin could have made. (B) The FTGT for
patient no. 5 before and throughout the study period. There is a peak of
thrombin generation in the day-2 pretreatment (PT) sample that correlates with
the drop in aPTT and rise in FIX level seen in Figure 2. (C) The FTGT for
patient no. 2 before and throughout the study period. There is an increase in
thrombin generation throughout the study period that correlates with the rise in
the FIX antigen level (Figure 3) but is not reflected in the aPTT or FIX
one-stage assay. RFU indicates relative fluorescence units.

Figure 5.
View largeDownload PPT

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Prepublished online as Blood First Edition Paper, July 28, 2005; DOI
10.1182/blood-2005-03-1307.

Supported by a Canadian Hemophilia Society Care Until Cure Research Grant and an
operating grant from the Canadian Institutes for Health Research (MOP-10912).
P.D.J. held the Aventis-Behring–Canadian Hemophilia Society—Association of
Hemophilia Clinic Directors of Canada (CHS-AHCDC) Fellowship in Hemophilia at
the time of this study. D.L. holds a Canada Research Chair in Molecular
Hemostasis and a Career Investigator Award from the Heart and Stroke Foundation
of Ontario.

D.L. and P.D.J. designed research, analyzed data, and wrote the paper; S.R.
performed research, contributed vital investigations, analyzed data, and
contributed to final manuscript; G.E.R. and M.-C.P. performed research, analyzed
data, and contributed to final manuscript; M.W. performed research; S.M.
designed research, and J.L. performed research.

The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 U.S.C. section 1734.

The authors acknowledge the invaluable contributions of Francine Derome, Morna
Brown, Colleen Notley, and Caroline Hensman. D.L. is a Career Investigator of
the Heart and Stroke Foundation of Ontario, and holds a Canada Research Chair in
Molecular Hemostasis.

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Copyright © 2005 by The American Society of Hematology
2005

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