1. IN UTERO NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITOR EXPOSURE AND SIGNS OF POSSIBLE MITOCHONDRIAL DYSFUNCTION IN HIV-UNINFECTED CHILDREN IN PEDIATRIC AIDS CLINICAL TRIALS GROUP PROTOCOLS 219 & 219C
Susan Brogly, Nathalie Ylitalo, Lynne Mofenson, James Oleske, Russell Van Dyke, Marilyn Crain, Mark Abzug, Michael Brady, Patrick Jean-Philippe, Michael Hughes, George Seage III

2. NRTIs AND MITOCHONDRIA
NRTIs inhibit HIV DNA replication, but also have other unintended actions
NRTIs can impair mitochondrial function through inhibition of DNA polymerase-, and evidence of other mechanisms of impairment is emerging 1, 2
Clinical manifestations of NRTI-induced mitochondrial dysfunction include neuropathy, (cardio)myopathy, hepatic steatosis
NRTI are incorporated into the new DNA strand being synthesized by HIV RT; the lack of a 3’ hydroxyl group results in chain termination
Also can impair mitochondrial function
Dysfunction in one cell line may not be present in others
Does not explain the entire array of metabolic associated NRTI deficiencies
Neurological problems such as cognitive delay, behavioural issues, motor abnormalities, and convulsions have been described in children
1. Lewis W. Antiviral Therapy Côté H. Antiviral Therapy 2005.

3. IN UTERO NRTI EXPOSURE AND MITOCHONDRIAL DYSFUNCTION
In 1999 the Enquête Périnatale Française (EPF) described signs of mitochondrial dysfunction in 8 of 1,754 HIV-uninfected children exposed to NRTIs, 2 of whom died 1
None of the 30 deaths in HIV-uninfected children in 5 US cohorts were attributed to mitochondrial dysfunction 2
Case reports of mitochondrial dysfunction in HIV-uninfected infants exposed to ARVs in utero in the US, the Netherlands, and Italy 3-5
As well, there is some concern that NRTI taken by a woman during pregnancy may cause mitochondrial impairment, presumably through similar mechanisms of action, in their infants who do not contract HIV
The EPF subsequently documented sig higher rates of mitochondrial dysfunction HIV-uninfected children exposed vs. unexposed to NRTIs, respectively 0.3% (7/2644) vs. 0% (0/1,748)
Review of deaths - about half of whom were exposed to ARVs in utero (primarily ZDV alone)
1. Blanche S et al. Lancet Perinatal Surveillance Review Working Group. JAIDS Cooper E et al. Int AIDS Conference Tovo P et al. Int Medical Press Godfried M et al. Eur J of Pediatrics 2005

4. OBJECTIVE
To estimate the association between in utero NRTI exposure and possible mitochondrial dysfunction in HIV-uninfected children

5. STUDY METHODS
Source Population: children enrolled in PACTG protocols 219 & 219C, a multi-site US cohort study that began in 1993
Clinical, neurological, and laboratory data collected at enrollment and every 3-6 months before 2 years of age and annually thereafter
Study Population: 1,037 HIV-uninfected children with at least one year of follow-up before three years of age as of August 2003
Clinicians blinded to in utero ARV exposure reviewed the medical histories to identify possible cases according to the EPF definition
To identify children who developed early signs of mitochondrial dysfunction to be comparable to EPF and because many children had not yet been followed beyond three years of age
As expected, children in the study population were younger at enrollment than the 512 children not included (median age 6.8 vs months), enrolled earlier (median year of enrollment 1998 vs. 2001), were followed longer before three years of age (median follow-up 2.0 years vs. 5.8 months), and were more likely to have participated in protocol 219.
No significant difference in sex, race/ethnicity, or the proportion of children known to have been exposed to ARVs in utero in the two groups. However, children in the study population were significantly less likely to have been exposed to PIs (27.3% vs. 33.2%, p<0.001).

6. EPF DEFINITION
1 major sign at any visit, or 2 different minor signs at 2 different visits
Signs based on description of inherited mitochondrial disorders in children, and manifestations of NRTI-induced mitochondrial dysfunction in HIV-infected patients 1
1. Barret B et al. AIDS 2003.

7. STUDY METHODS CONT’D
Exact logistic regression used to estimate the association between:
1) overall in utero NRTI exposure
2) trimester of first in utero NRTI exposure
and possible mitochondrial dysfunction
while adjusting for potential confounders
The exact conditional maximum likelihood ratio test was used to assess whether the estimates for each trimester of first exposure were significantly different from zero and from one another
Is based on the hypergeometric distribution and doesn’t compare two binomial distributions

8. RESULTS

9. SIGNS OF POSSIBLE MITOCHONDRIAL DYSFUNCTION (N=20 CASES)

10. CHARACTERISTICS Characteristic Cases (N=20) Non-cases (N=1,017)
p-value
Birth weight (grams)
<2,500
2,500
Unknown
4 (20.0)
16 (80.0)
0 (0.0)
126 (12.4)
875 (86.0)
16 (1.6)
0.31
Premature birth
Yes No
13 (65.0)
3 (15.0)
139 (13.7)
654 (64.3)
224 (22.0)
0.52
Received ARV prophylaxis as a neonate
15 (75.0)
5 (25.0)
843 (82.9)
171 (16.8)
3 (0.3)
0.36
Median peak log10 maternal HIV RNA (range)
3.2 (2.1, 5.2)
3.4 (1.2, 5.6)
0.42
In utero illicit drug exposure
Exposed
Unexposed
162 (15.9)
607 (59.7)
248 (24.4)
0.24
Neonatal ARV prophylaxis [included neonatal ZDV monotherapy (75.0% vs. 82.4%) or ZDV/3TC use (0.0% vs. 4.3%)]
RNA data available for 16 cases and 603 non-cases.
Drug exposure includes intravenous drugs, cocaine, marijuana, methamphetamines, and barbiturates.

11. CHARACTERISTICS CONT’D
Cases
(N=20)
Non-cases
(N=1,017)
p-value
Sex
Male
Female
15 (75.0)
5 (25.0)
476 (46.8)
541 (53.2)
0.013
Year of birth
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
0 (0.0)
4 (20.0)
3 (15.0)
2 (0.2)
57 (5.6)
87 (8.6)
64 (6.3)
84 (8.3)
90 (8.9)
66 (6.5)
118 (11.6)
113 (11.1)
208 (20.5)
122 (12.0)
6 (0.6)
0.019
Of the potential confounders considered, only year of birth affected the estimated odds ratios (ORs) of in utero NRTI exposure and possible mitochondrial dysfunction; sex was significantly associated with possible mitochondrial dysfunction but not with in utero NRTI exposure.
Therefore, the final models included indicators for in utero NRTI exposure and year of birth categorized in one-year intervals.

12. OVERALL IN UTERO NRTI EXPOSURE AND MITOCHONDRIAL DYSFUNCTION
Cases
(N=20)
Non-cases
(N=1,017)
Adjusted OR
(95% CI)
Any NRTI
Unexposed
3 (15.0)
101 (9.9)
1.0
Exposed
17 (85.0)
916 (90.1)
1.16 ( )
3TC
10 (50.0)
509 (50.1)
508 (50.0)
4.26 ( )
ZDV
130 (12.8)
887 (87.2)
1.29 ( )
ZDV/3TC
538 (52.9)
479 (47.1)
4.85 ( )
Three of 20 cases were unexposed to any ARVs in utero.
There was no significant association between overall in utero NRTI exposurefor all NRTIs considered together or specific NRTIsand possible mitochondrial dysfunction in multivariate models adjusted for year of birth.
Among cases and non-cases exposed to ARVs in utero, there was no significant difference in the median duration of in utero NRTI (22.9 weeks vs weeks), ZDV (22.6 weeks vs weeks), or 3TC (9.3 weeks vs weeks) exposure.
In addition to the NRTIs considered above, two cases also were exposed to D4T and abacavir in utero

13. TRIMESTER OF FIRST IN UTERO NRTI EXPOSURE AND MITOCHONDRIAL DYSFUNCTION
Cases
(N=20)
Non-cases
(N=1,017)
Adjusted OR
(95% CI)
Any NRTI
Unexposed
3 (15.0)
101 (9.9)
1.0
1st trimester
5 (25.0)
338 (33.2)
0.77 ( )
2nd trimester
7 (35.0)
433 (42.6)
0.82 ( )
3rd trimester
145 (14.3)
1.73 ( )
ZDV
130 (12.8)
4 (20.0)
276 (27.1)
0.87 ( )
8 (40.0)
441 (43.4)
1.07 ( )
170 (16.7)
1.71 ( )
Adjusted model included all three trimesters and year of birth categorized in one-year intervals.

14. TRIMESTER OF FIRST IN UTERO NRTI EXPOSURE AND MITOCHONDRIAL DYSFUNCTION CONT’D
Cases
(N=20)
Non-cases
(N=1,017)
Adjusted OR
(95% CI)
3TC
Unexposed
10 (50.0)
509 (50.1)
1.0
1st trimester
3 (15.0)
206 (20.3)
2.29 ( )
2nd trimester
1 (5.0)
213 (20.9)
0.87 ( )
3rd trimester
6 (30.0)
89 (8.8)
10.06 ( )
ZDV/3TC
538 (52.9)
2 (10.0)
158 (15.5)
2.37 ( )
216 (21.2)
1.94 ( )
105 (10.3)
9.46 ( )
Same six cases in 3TC and ZDV/3TC group

15. CONCLUDING REMARKS
No significant association between overall in utero NRTI exposure and possible mitochondrial dysfunction
Higher odds of first in utero 3TC and ZDV/3TC exposure in the 3rd trimester among cases vs. non-cases
Small number of cases and limited use of individual NRTIs
We were unable to distinguish whether the increased odds was due to 3TC or to ZDV/3TC
We cannot rule out an etiologic association of in utero exposure to other NRTIs or NRTI combinations and possible mitochondrial dysfunction

16. CONCLUDING REMARKS CONT’D
Three children unexposed to NRTIs had clinical manifestations of possible mitochondrial dysfunction
Case definition based on clinical signs, which may have low positive predictive value
Possible that late initiation of ARVs is confounded by other determinants of fetal injury
Given the morbidity of HIV infection, the benefits of ARV prophylaxis, as recommended by the US Public Health Service, greatly outweigh the potential harms of perinatal ARV exposure
Rate of possible mitochondrial dysfunction among unexposed children 2.9% vs. 0% in the EPF; 1.8% (17/933) in utero NRTI exposed children with possible mitochondrial dysfunction vs. 0.3% in EPF
Unlike the French investigators, we did not have mitochondrial histological, enzymological, or magnetic resonance imaging results necessary for more definitive case identification, and some misclassification of cases may have occurred.
In utero cocaine exposure has been associated with cognitive impairments [17] and cardiovascular abnormalities [18], conditions included in our case definition.
There is evidence that HIV-uninfected infants born of HIV-infected women have depletions in mitochondrial DNA in the absence of in utero ARV exposure [22].

17. ACKNOWLEDGEMENTS
We would like to thank the children and families for their participation in PACTG protocols 219 & 219C
We would like to recognize the efforts of the 219 & 219C team and the institutions involved in this protocol
Funded by the National Institute of Allergy and Infectious Diseases, and the National Institute of Child Health and Development

18. EPF CASE DEFINITION
To be included as a possible case of early mitochondrial dysfunction, children must have fulfilled the definition of mitochondrial dysfunction proposed by the EPF investigators (Table 1) before three years of age [3]; additional signs, if present, that occurred after three years of age were included as supporting evidence for cases where cognitive delay was the only sign of mitochondrial dysfunction prior to three years of age.

19. 1,110 children without signs of possible
mitochondrial dysfunction
62 deemed not to have signs consistent with possible mitochondrial dysfunction before 3 years of age, to have an alternate etiology of the signs, or to have resolution of the signs
NON-CASES
1,017 children without signs of possible
before
3 years of age
2 unable to evaluate
1,220 children in the study population
110 children with signs of
possible mitochondrial dysfunction
63 children deemed not to have signs consistent with possible mitochondrial dysfunction before 3 years of age, to have an alternate etiology of the signs, or to have resolution of the signs
3 children with incomplete in utero ARV exposure information
5 children with unknown and 88 children with incomplete in utero ARV exposure information
45 children with signs consistent with possible mitochondrial dysfunction before 3 years of age
1 child with incomplete in utero ARV exposure information
22 children whose only sign of possible mitochondrial dysfunction
was cognitive developmental delay
CASES
20 children with signs consistent with possible mitochondrial dysfunction
To exclude children whose presentation could be explained by an etiology other than mitochondrial dysfunction (e.g. congenital abnormality, severe prematurity etc.), or who did not meet the EPF case definition of possible mitochondrial dysfunction on further review (e.g. lack of persistent abnormal findings), a thorough retrospective review of the medical histories recorded on the case report forms was performed by clinicians blinded to in utero ARV exposure.
Because another etiology likely explained the signs of possible mitochondrial dysfunction, the condition(s) subsequently resolved, or the child was deemed not to fulfill the case definition by three years of age