The authors have declared that no competing interests exist.
Recruited the patients and controls: MdCC MdMG MGM EM IF ER. Conceived and designed the experiments: PD F. Vidal MB. Performed the experiments: MdCC MdMG MGM EM IF ER. Analyzed the data: PD F. Vidal MB FT. Contributed reagents/materials/analysis tools: JS MB JCD F. Villarroya. Wrote the paper: PD F. Villarroya F. Vidal FT MB.
Low expression thymidylate synthase (TS) polymorphism has been associated with increased stavudine triphosphate intracellular (d4T-TP) levels and the lipodystrophy syndrome. The use of d4T has been associated with acute pancreatitis and peripheral neuropathy. However, no relationship has ever been proved between TS polymorphisms and pancreatitis and/or peripheral neuropathy.
We performed a case-control study to assess the relationship of TS and methylene-tetrahydrofolate reductase (MTHFR) gene polymorphisms with acute pancreatitis and/or peripheral neuropathy in patients exposed to d4T. Student’s t test, Pearson’s correlations, one-way ANOVA with Bonferroni correction and stepwise logistic regression analyses were done.
Forty-three cases and 129 controls were studied. Eight patients (18.6%) had acute pancreatitis, and 35 (81.4%) had peripheral neuropathy. Prior AIDS was more frequent in cases than in controls (OR = 2.36; 95%CI 1.10–5.07, P = 0.0247). L7ow expression TS and MTHFR genotype associated with increased activity were more frequent in patients with acute pancreatitis and/or peripheral neuropathy than in controls (72.1% vs. 46.5%, OR = 2.97; 95%CI: 1.33–6.90, P = 0.0062, and 79.1% vs. 56.6%, OR = 2.90, 95%CI: 1.23–7.41, P = 0.0142, respectively). Independent positive or negative predictors for the development of d4T-associated pancreatitis and/or peripheral neuropathy were: combined TS and MTHFR genotypes (reference: A+A; P = 0.002; ORA+B = 0.34 [95%CI: 0.08 to 1.44], ORB+A = 3.38 [95%CI: 1.33 to 8.57], ORB+B = 1.13 [95%CI: 0.34 to 3.71]), nadir CD4 cell count >200 cells/mm3 (OR = 0.38; 95%CI: 0.17–0.86, P = 0.021), and HALS (OR = 0.39 95%CI: 0.18–0.85, P = 0.018).
Low expression TS plus a MTHFR genotype associated with increased activity is associated with the development of peripheral neuropathy in d4T-exposed patients.
The doubtless efficacy of highly active antiretroviral therapy (HAART) is still shadowed by drug toxicity, specially that appearing in the long-term
The ability of thymidine analogues to inhibit mitochondrial gamma polymerase will depend on the intracellular concentrations of their triphosphate metabolites
Our working hypothesis was that TS and MTHFR polymorphisms could be associated with other manifestations of mitochondrial toxicity, such as d4T-associated pancreatitis and d4T-associated peripheral neuropathy. To test this hypothesis we performed a case-control study in d4T-exposed patients who developed pancreatitis or peripheral neuropathy while on d4T-based therapy.
All patients and controls were recruited at the same HIV-1 infection clinic at the
A case of acute pancreatitis was defined as a clinical history consistent with pancreatitis (i.e. abdominal pain with or without findings of shock and hypotension where other causes of abdominal pain have been excluded) with supporting biochemical evidence of pancreatitis: elevated lipase (3 times the upper normal limit) or amylase (3 times the upper normal limit) and evidence of pancreatitis from radiological investigation or hemorrhagic pancreatitis at laparotomy or post-mortem exam
The diagnosis of neuropathy (sensory or mixed) required the presence of numbness, paresthesias, or dysesthesias in the patient’s lower or lower and upper extremities with onset after starting a d4T-based antiretroviral regime
The presence or absence of lipoatrophy, lipohypertrophy, and mixed syndrome was determined as previously described
All laboratory investigations were performed after a 12 h overnight fast and at least 15 minutes after the placement of a peripheral intravenous catheter, as previously described
The genomic DNA was extracted from the peripheral leucocytes by the salting-out procedure
The MTHFR gene polymorphisms (677C→T [rs1801133] and 1298 A→C [rs1801131) were determined. These two polymorphisms were analyzed using Fluidigm’s Biomark system. This technology is designed for the allelic discrimination 5′ nuclease assay. The samples and the TaqMan Gene expression assays (Applied Biosystems, Foster City, CA, USA) were prepared following manufacturer’s instructions. The 48.48 dynamic arrays used were automatically loaded using an IFC Controller (Fluidigm Corporation), and real-time reactions were performed and analyzed using BioMark Real-Time PCR System and Analysis software (Fluidigm Corporation), respectively. As a quality control, normal, heterozygote and homozygote sequenced samples were included on every array for each genotype. MTHFR genotypes were classified also into two groups: those associated with a decreased enzymatic activity (homozygous 677T, homozygous 1298C and compound heterozygous patients), and genotypes associated with an increased enzymatic acitivity (heterozygous and wild-type patients)
Data are expressed as median with interquartile range (IQR) or as otherwise specified. Continuous variables were assessed with the nonparametric Mann-Whitney test and categorical data such as genotype and allele frequencies were compared by use of the Fisher’s exact test. The level of significance was established at the 0.05 level and all reported P values are two-sided. A logistic regression analysis was used to examine the association of peripheral neuropathy and/or pancreatitis with TS and MTHFR polymorphisms and other parameters; variables associated with a P<0.1 in the univariate analyses were included in the multivariate stepwise analysis. All analyses were performed with the SAS version 9.1.3 software (SAS Institute Inc., Cary, NC).
From January 1994 to December 2010, there were 102 cases of pancreatitis and/or peripheral neuropathy in our cohort
Cases (N = 43) | Controls (N = 129) | P value | |
|
48.0 (43.0–57.7) | 50.0 (42.0–58.2) | 0.9042 |
|
32 (74.4) | 95 (73.6) | 0.9999 |
|
|||
|
17 (39.5) | 45 (34.9) | 0.3366 |
|
14 (32.5) | 55 (42.6) | |
|
11 (23.2) | 30 (21.7) | |
|
2 (4.6) | 1 0.8) | |
|
13.0 (9.0–15.7) | 13.0 (10.0–17.0) | 0.4065 |
|
24 (55.8) | 45 (34.9) | 0.0195 |
|
17 (39.5) | 70 (54.3) | 0.1138 |
|
1 (2.3) | 10 (7.7) | 0.2952 |
|
6 (13.9) | 11 (8.5) | 0.3751 |
|
13 (30.2) | 40 (31.0) | 0.9999 |
|
515 (346–730) | 543 (388–792) | 0.5430 |
|
386 (194–574) | 339 (207–553) | 0.6044 |
|
834 (582–1181) | 874 (617–1146) | 0.6521 |
|
555 (144–799) | 370 (122–594) | 0.1180 |
|
83 (23–206) | 182 (54–314) | 0.0091 |
|
24 (55.8) | 45 (35.1) | 0.0201 |
|
32 (74.4) | 71 (55.5) | 0.0314 |
|
1.28 (1.28–1.28) | 1.28 (1.28–1.55) | 0.8307 |
|
33 (76.7) | 91 (70.5) | 0.5564 |
|
5.46 (4.70–5.79) | 5.19 (4.29–5.58) | 0.1479 |
|
31 (68.9) | 75 (55.6) | 0.0643 |
|
3.78 (2.84–4.28) | 3.58 (2.53–4.16) | 0.3725 |
Values are expressed as median and interquartile range, unless indicated.
includes 2 patients with post-transfusion HIV and 1 with unknown risk, MsM = men who have sex with men, HTSX = heterosexuals, IDU = intravenous drug users, AIDS = acquired immune deficiency syndrome, HCV = hepatitis C virus, HBV = hepatitis B virus, ml = milliliters.
Most of the patients (124, 72.9%) had undetectable viral load at the time of the study. The median viral load for those who had it detectable was 2.0 (IQR: 1.68–3.06) log10 copies/ml. The mean CD4 count was 589±291 cells/mm3 (median: 539 [IQR: 383–784]). Nadir CD4 cell count was <200 cells/mm3 in 109 patients (60.5%) and <100 cells/mm3 in 75 (41.7%). A CD4 cell count nadir <200 cells/mm3 was significantly more frequent in cases (OR = 2.34; 95%CI: 1.03–5.58, P = 0.0437) (
Parameter | Cases (n = 43) | Controls (n = 129) | P value |
ART composition at event | 0.0154 | ||
|
33 (76.7) | 69 (53.5) | |
|
10 (23.2) | 51 (39.5) | |
|
0 (0.0) | 9 (7.0) | |
NRTI backbone at event | |||
|
15 (34.9) | 69 (53.5) | 0.3335 |
|
23 (53.5) | 44 (34.1) | |
|
2 (4.6) | 6 (4.6) | |
|
2 (4.6) | 6 (4.6) | |
|
1 (2.3) | 3 (2.3) | |
|
0 (0.0) | 1 (0.8) | |
Current d4T use, n (%) | 0(0) | 29 (22.5) | 0.0015 |
Current AZT use, n (%) | 13 (30.2) | 4 (3.1) | <0.0001 |
ART duration (m) | 110.0 (93.0–138.7) | 115.0 (94.7–143.2) | 0.4783 |
Individual drug exposure | |||
|
43.0 (2.0–70.7) | 11.0 (0.0–41.0) | 0.0013 |
|
559.6 (21.2–998.2) | 158.8 (0.0–583.8) | 0.0026 |
|
26.0 (9.0–47.7) | 57.0 (38.7–76.0) | <0.0001 |
|
62.9 (21.8–109.8) | 135.5 (86.7–177.2) | <0.0001 |
|
1.15 (1.04–1.27) | 1.06 (0.92–1.18) | 0.0126 |
|
68.0 (38.0–91.7) | 70.0 (36.0–103.2) | 0.6790 |
|
11.0 (3.2–33.0) | 17.0 (0.0–60.2) | 0.2688 |
|
0.0 (0.0–0.0) | 0.0 (0.0–0.0) | 0.7167 |
|
2.0 (0.0–46.2) | 0.0 (0.0–26.7) | 0.0406 |
|
0.0 (0.0–45.0) | 14.0 (0.0–43.0) | 0.3340 |
|
0.0 (0.0–0.0) | 2.0 (0.0–63.0) | 0.0947 |
|
6.0 (0.0–46.0) | 2.0 (0.0–50.2) | 0.6660 |
|
0.0 (0.0–0.0) | 0.0 (0.0–0.0) | 0.3068 |
|
109.0 (28.2–168.7) | 46.0 (22.7–97.5) | 0.0123 |
|
196.0 (157.2–247.5) | 224.0 (172.0–267.2) | 0.0819 |
All parameters expressed as median and (interquartile range) unless indicated. HALS = HIV-1/HAART-associated lipodystrophy syndrome, ART = antiretroviral therapy, PI = protease inhibitor, NNRTI = non-nucleoside reverse transcriptase inhibitor, NRTI = nucleoside reverse transcriptase inhibitor, d4T = stavudine, 3TC = lamivudine, FTC = emtricitabine, TDF = tenofovir, ddI = didanosine, AZT = zidovudine, ddC = zalcitabine, ABC = abacavir, EFV = efavirenz, NVP = nevirapine, ETV = etravirine, m = months, g = grams, kg = kilograms.
Metabolic and fat data are shown in
Cases (n = 43) | Controls (n = 129) | P value | |
|
64.7 (60.0–73.7) | 65.5 (58.0–73.0) |
|
|
23.5 (20.5–25.8) | 23.8 (21.3–25.7) |
|
|
89.0 (81.2–93.7) | 89.0 (82.7–95.0) |
|
|
0.96 (0.90–1.02) | 0.95 (0.90–1.02) |
|
|
4.5 (2.0–10.0) | 7.0 (3.4–11.1) |
|
|
1.0 (0.0–2.0) | 2.0 (0.7–2.0) |
|
|
120 (120–138) | 120 (110–130) |
|
|
75 (70–80) | 75 (70–80) |
|
|
21 (46.7) | 48 (35.5) |
|
|
5.07 (4.18–6.03) | 5.08 (4.44–5.92) |
|
|
1.90 (1.17–3.22) | 1.63 (1.12–2.54) |
|
|
1.11 (0.95–1.32) | 1.23 (0.97–1.53) |
|
|
2.89 (2.28–3.65) | 2.89 (2.46–3.50) |
|
|
0.88 (0.54–1.19) | 0.77 (0.52–1.11) |
|
|
5.30 (5.02–5.87) | 5.30 (4.90–6.00) |
|
|
18 (41.9) | 75 (58.1) |
|
All values expressed as median and (interquartile range) unless specified. HALS = HIV-1/HAART-associated lipodystrophy syndrome, BMI = body mass index, WHR = waist-hip ratio, LSGS = lipodystrophy severity grade score, mmol/l = milimoles per liter, HDL = high density lipoprotein, LDL = low density lipoprotein, VLDL = very low density lipoprotein, pmol/l = picomoles per liter, g = grams.
The distribution of the different genotypes is shown in
Cases (n = 43) | Controls (n = 129) | P value | |
|
|||
|
9 (20.9) | 19 (13.9) |
|
|
15 (34.9) | 30 (23.2) | |
|
7 (16.3) | 35 (27.1) | |
|
7 (16.3) | 11 (8.5) | |
|
5 (11.6) | 25 (19.4) | |
|
0 (0.0) | 10 (7.7) | |
|
31 (72.1) | 60 (46.5) |
|
|
12 (27.9) | 69 (53.5) | |
|
|||
|
21 (48.8) | 42 (32.5) |
|
|
18 (41.9) | 63 (48.8) | |
|
4 (9.3) | 24 (18.6) | |
|
|||
|
22 (51.2) | 64 (49.6) |
|
|
14 (32.5) | 58 (44.9) | |
|
7 (16.3) | 7 (5.4) | |
|
|||
|
34 (79.1) | 73 (56.6) |
|
|
9 (20.9) | 56 (20.9) | |
|
|||
|
25 (58.1) | 34 (26.3) |
|
|
6 (13.9) | 26 (20.1) | |
|
9 (20.9) | 39 (30.2) | |
|
3 (6.9) | 30 (23.2) |
TS = thymidylate synthase, MTHFR = methylene-tetrahydrofolate reductase,
Heterozygous and wild-type patients, †Homozygous 677T, homozygous 1298C and compound heterozygous patients.
Combined TS and MTHFR genotypes with respect to peripheral neuropathy or pancreatitis are shown in
A multivariable analysis was performed taking as the dependent variable the development of pancreatitis and/or peripheral neuropathy and as independent variables, age, sex, AIDS, CD4 count nadir <200/mm3, CD4 nadir <100/mm3, d4T exposure (m), d4T exposure (g), d4T exposure (mg/kg), AZT exposure (m), AZT exposure (g), ABC exposure (m), d4T plus ddI at event, EFV exposure (m), PI exposure (m), NRTI exposure (m), HALS, TS genotype (low vs. high expression),MTHFR genotype (increased vs. decreased enzymatic activity), and combined TS and MTHFR genotypes, all variables associated with a P value <0.1 in the univariate analysis. Independent positive or negative predictors for the development of d4T-associated pancreatitis and/or peripheral neuropathy were: combined TS and MTHFR genotypes (reference: A+A; P = 0.002; ORA+B = 0.34 [95%CI: 0.08 to 1.44], ORB+A = 3.38 [95%CI: 1.33 to 8.57], ORB+B = 1.13 [95%CI: 0.34 to 3.71]), nadir CD4 cell count >200 cells/mm3 (OR = 0.38; 95%CI: 0.17–0.86, P = 0.021), and HALS (OR = 0.39 95%CI: 0.18–0.85, P = 0.018).
Our study suggests an association between TS and MTHFR polymorphisms and the appearance of d4T-related toxicity in the form of acute pancreatitis or peripheral neuropathy. However, our work has inherent limitations. First, this is a case-control study and therefore no causal relationships should or must be drawn. Second, in case-control studies endpoint verification is of paramount importance. It is known that peripheral neuropathy or acute pancreatitis in the setting of HIV-1 infection treated with HAART may be caused not only by antiretroviral drugs but also by other drugs, alcohol abuse, or biliary stones and hypertriglyceridemia in the case of pancreatitis
Acute pancreatitis in the setting of HIV infection and antiretroviral therapy has wide incidence rates ranging from 1.27 to 22.6events/1000 PY
Acute pancreatitis has been associated with mutations in CTR and SPINK-1 genes both in the general population and in HIV-infected patients
Among the factors independently associated with the development of neuropathy or pancreatitis, we found a low CD4 cell count nadir, a common marker for most toxicities and co-morbidities
MTHFR gene is also polymorphic. The best known polymorphism consists of a 677 C
Stavudine-associated side effects may seem of only limited relevance today, because its use has greatly decreased in developed countries mainly due to its association with fat distribution abnormalities
In summary, our study suggests that d4T-associated acute pancreatitis and/or peripheral neuropathy are associated with low-degree TS expression and MTHFR genotype associated with an increased enzymatic activity. There is a plausible pathogenic mechanism for such an association since it is well-known that, in d4T-treated patients, the presence of low-degree TS expression genotype is associated with increased d4T-TP intracellular concentrations and MTHFR genotype associated with an increased enzymatic activity contributes to a decreased TS functionality. This may be of value in tailoring d4T therapy when needed.