1. 3rd HIV Exposed Uninfected (HEU) Child Workshop July 23, 2017
Mitochondrial DNA and neurodevelopmental disorders in HEU children – observations from the CARMA cohort
Hélène Côté, PhD
Pathology & Laboratory Medicine
The University of British Columbia
Canada
3rd HIV Exposed Uninfected (HEU) Child Workshop
July 23, 2017

2. HIV Epidemiology 72,000 17,000 200 11,000 ~25 1,850 All 34 M 17 M

3. CARMA cohort Children and Women: AntiRetrovirals and Markers of Aging
Initiated in 2008, ongoing
Enroll adults (primarily women), children, pregnant women & infants
Enrolled HIV+ and HEU children (0-19y) in four cities in Canada
Collect demographic and clinical data + bio-specimens
~4000 HEU children born in Canada since 1990
CARMA counts: ~150 HIV+ children (1-19y) ~300 HEU children (1-19y) ~230 HEU infants (0-3m) ~75 HUU infants (0-3m)
CARMA cohort Children and Women: AntiRetrovirals and Markers of Aging
Vancouver
Ottawa
Toronto
Montreal
CARMA

4. Autism spectrum disorder (ASD)
Neurodevelopmental disorder
Diagnosed by 2-3 years of age
Heterogeneous symptoms
Multi-hit (genetic/environment)
Prevalence in general population ~1.5%
Variable severity
Autism spectrum disorder (ASD)
Childhood
disintegrative
disorder
Asperger’s
Syndrome
Classic
autism

5. Factors associated with ASD
Substance use during pregnancy
Maternal infections
↑ age
Genetic susceptibility
Sex
Gastrointestinal abnormalities
Hypotonia
Intellectual disability
Seizures/epilepsy
Mitochondrial dysfunction

6. mtDNA as a marker of mitochondrial dysfunction
High prevalence of ASD among HEUs at one clinic (9/158 = 5.7%) Brophy et al., CAHR (2013)
Elevated blood mtDNA content in HEU infants compared to HIV- unexposed uninfected (HUU ) Cote et al., J Infect Dis (2008)
mtDNA as a marker of mitochondrial dysfunction
mitochondrion
mtDNA
Mitochondria contain circular DNA (mtDNA) – 16.5kb
Independent replication – polymerase 
Changes in mtDNA content – marker of mitochondrial function/health

7. mtDNA damage and mutations
ARVs and mtDNA
mtDNA content
Clonal expansion of existing mutations PI
Adaptive mitochondrial biogenesis
Oxidative stress
(Reactive oxygen species (ROS))
mtDNA damage and mutations
mitochondrial
dysfunction
Telomerase
mitophagy
NRTI
(mitochondria elimination)
NRTI
mitochondrial
polymerase γ
mtDNA content
Telomere shortening

8. Anonymous pediatric HUU
Hypothesis:
Blood mtDNA content will be different in HEU children diagnosed with ASD compared to 1:3 matched HEU children without ASD, HUU children with ASD, and HUU controls
ASD in HEU Study design
CARMA cohort
HUU
controls
n=51
Anonymous pediatric HUU
n=500
HUU non-ASD siblings
n=9
HEU
with ASD
n=14
Pediatric HEU
n=299
without ASD
n=42
Matched 1:3 for age, sex, and
ethnicity as best possible
HUU
with ASD
n=42
(BC Autism Spectrum Interdisciplinary Research Program): ASPIRE
ASPIRE DNA biobank

9. Study group characteristics
HEU
with ASD
n=14
without ASD
n=42
HUU
n=51
Male sex
10 (71)
30 (71)
36 (71)
Age at sample collection (years)
6 [4 – 8]
(2 – 16)
6 [4 – 9]
Self-reported ethnicity
Black/African Canadian
11 (79)
33 (79)
4 (10)
White/Caucasian
3 (21)
9 (21)
20 (48)
5(10)
Asian/South Asian
6 (14)
4 (8)
Other/Mixed Ethnicity
12 (29)
Unknown (anonymous)
42 (82)
Maternal cART regimen taken in pregnancy
None
2 (5) -
NRTIs only
1 (7)
1 (2)
Dual NRTI + PI
7 (50)
26 (62)
Dual NRTI + NNRTI
Other/unknown
2 (14)
10 (24)
Length of maternal cART exposure (weeks)
11 [6 – 35]
(0 – 38)
33 [20 – 39]
(0 – 41)
Received AZT prophylaxis
13 (93)
42 (100)
Study group characteristics

10. Comparing blood mtDNA content between groups
*mtDNA data not obtained for 2 participants *
Isolated blood mtDNA
Measured mtDNA content by qPCR
p = 0.2
Comparing blood mtDNA content between groups
p <
p = 0.004
p =
p = 0.02
p = 0.03
Possible cumulative effect of HEU status and ASD on mtDNA?
t-test
Mann Whitney U test

11. ASD may show a higher prevalence among HEU in our cohort – but could be subject to recruitment bias
MtDNA effects are associated with both HEU status and ASD diagnosis
Did not detect any relationship with perinatal ARV exposure but our study sample is small
HEU may get a “multi-hit”, perhaps increasing susceptibility
There may be confounder(s) we were not able to consider
Summary

12. Neuro developmental outcomes in BC’s HEU
Possible causal pathways
Neuro developmental outcomes in BC’s HEU
ARV exposure ?
HEU status
Preterm birth
Neurodevelopmental disorders
Maternal factors
A pilot study (n=103) conducted in BC suggested that a high proportion of HEU children required special aid in school
Substance use
Socioeconomic status, environment
HIV disease (CD4 cell count, viral load)
Infections in pregnancy

13. Study design Study objectives
To compare the frequency of neurodevelopmental disorders (ND) among HEU children compared to matched HUU
To examine possible associations with exposure to maternal ARVs
Study design: retrospective controlled cohort study
HEU (n = 446)
Born in BC
PH number on record
HUU (n = 1323)
Matched (98%) on:
Age, Sex, Geocode
matched 1:3
Dataset collected by
Data sources: BC ministry of Health (ICD-9 codes), Perinatal Service BC, Oak Tree Clinic, BC Vital Statistics

14. Population Data BC statement
“All inferences, opinions, and conclusions drawn in this presentation are those of the authors, and do not reflect the opinions or policies of the Data Steward(s).”

15. Frequency of ND among HEU and matched HUU 1990-2012 mean age 8.8 years
Neurodevelopmental disorder (ND)
% HEU/
% HUU
Odds Ratio
(95% CI)
P value
≥ 1 neurodevelopmental disorder
30 / 13
2.8 (2.2 – 3.6)
<0.0001
Infantile autism (aka ASD)
2 / 0.7
3.0 (1.2 – 7.6)
0.02
Disturbance of emotions
9 / 3
2.9 (1.9 – 4.6)
Hyperkinetic Syndrome (aka ADHD)
14 / 5
3.3 (2.3 – 4.7)
Developmental Delay
18 / 6
3.5 (2.5 – 4.9)
Intellectual Disability
Undisclosed (n < 6)
1.0 (0.2 – 4.9)
0.99
Epilepsy
5 / 3
1.6 (0.9 – 2.6)
0.08
Frequency of ND among HEU and matched HUU mean age 8.8 years
Other conditions
Neoplams (any)
6 / 9
0.68 (0.4 – 1.0)
0.06
Asthma
22 / 21
1.0 (0.8 – 1.3)
0.90

16. Maternal and child characteristics
Entire sample( )
Maternal and child characteristics
Characteristics
(missing variables removed in HEU + HUU pairs)
HEU
n = 411
HUU
n = 1224
P value
Maternal age (years)
29.9 ± 5.6
30.5 ± 5.6
0.02
Age of child at follow-up (years)
8.4 ± 5.6
0.9
Male sex
190 (46.2%)
573 (46.8%)
0.8
Gestational age at birth (weeks)
37.7 ± 3.1
38.9 ± 1.8
<0.0001
Preterm birth (<37 weeks)
83 (21%)
85 (7%)
Birth weight (kg)
2.98 ± 0.62
3.38 ± 0.57
APGAR 1 min
7.9 ± 1.8
8.1 ± 1.5
0.4
Data shown as mean ± SD or n (%)
P values calculated from Mann Whitney U test or Chi square test

17. Multivariable logistic regression for any ND 1990-2012
Model 1 ( )
Multivariable logistic regression for any ND
HEU (vs. HUU)
p <
Preterm birth (vs. term)
p <
Age at follow-up >11 years (vs. ≤11)
p <
Male sex (vs. female)
p <
Odds ratio (95% CI) of having at least one ND
Influence of maternal smoking/substance use?

18. Maternal substance use *2000-2012 mean age at follow-up now 6 years
Model 2 ( ; shorter follow-up)
Maternal substance use * mean age at follow-up now 6 years
Characteristics
(missing variables removed in HEU + HUU pairs)
HEU
n = 309
HUU
n = 917
P value
Maternal age (years)
30.2 ± 5.4
30.7 ± 5.6
0.08
Gestational age at birth (weeks)
37.7 ± 2.4
38.8 ± 1.8
<0.0001
Preterm birth (<37 weeks)
61 (20%)
63 (7%)
Birth weight (kg)
2.99 ± 0.59
3.38 ± 0.58
APGAR 1 min
7.9 ± 1.9
8.2 ± 1.5
0.03
Smoking during pregnancy (any)
107 (35%)
78 (9%)
0.0001
Alcohol use (identified as a risk)
26 (8%)
12 (1%)
Substance (illicit drug) use (any)
111 (36%)
24 (3%)
Data shown as mean ± SD or n (%)
P values calculated from Mann Whitney U test or Chi square test

19. Multivariable logistic regression for any ND 2000-2012
Model 2.0 ( )
Multivariable logistic regression for any ND
HEU (vs. HUU)
p = 0.011
Age at follow-up >6 years (vs. ≤6)
p <
Male sex (vs. female)
p <
Maternal smoking & substance use
p <
either or both (vs. never)
Odds ratio (95% CI) of having at least one ND
Influence of maternal smoking/substance use? Important

20. Model 2.1 ( )
Multivariable logistic regression for any ND (all variables included)
HEU (vs. HUU)
p = 0.055
Age at follow-up >6 years (vs. ≤6)
p <
Male sex (vs. female)
p <
Maternal smoking & substance use
p <
Either or both (vs. never)
Preterm birth (vs. term)
p <
Odds ratio (95% CI) of having at least one ND
If we also include preterm birth in the model, OR still borderline significant in HEU

21. Maternal ARV HEU births, and diagnoses of any ND in BC
Shorter follow-up
Maternal ARV HEU births, and diagnoses of any ND in BC
Percentage (%) n

22. Frequency of ND and preterm birth relative to duration of ARV exposure
Percentage (%) – 95% CI
No ARV
n = 76
n = 56
1-13 weeks
n = 95
n = 89
14-26 weeks
n = 162
n = 156
≥27 weeks
n = 107
n = 104
Any ND
Preterm Birth
Children with longer in utero exposure to ARV may have a lower risk of ND and preterm birth

23. Type of in utero ARV exposure (univariate association)
Preterm birth
Any ND
Type of in utero ARV exposure (univariate association)
No ARV
1-3 NRTIs
NRTIs + NNRTI
NRTIs + PI (unboosted)
NRTIs + PI (boosted)
Odds ratio (95% CI) of having at least one ND
Children whose mother was treated with combination ART (cART) may have a lower risk of ND
No evidence of higher risk of ND with boosted PI but maybe higher risk of preterm birth

24. Summary of preliminary analyses
Model 1 ( )
HEU  odds than HUU for 4 or the 6 ND studied (~3 times)
But…they do not have higher odds of being diagnosed with asthma
After adjusting for sex and age at follow-up, the OR of any ND in HEU is ~3
HEU are also more likely to:
be born preterm (~2.8 times)
have a mother who used substances during her pregnancy (~4-12 times), although this is likely underreported among HUU mothers
No evidence in this dataset that ARV exposure increases the risk of ND
Model 2 ( ; shorter follow-up)
After further adjusting for smoking/substance use, the odds ratio for:
having any ND  by 35% (from 2.3 to 1.5) = largest confounder
being born preterm decrease OR by 20% (from 3.3 to 2.7) but remain high
Higher risk of ND persist after all adjustments
Summary of preliminary analyses

25. Possible pathways to ND
ARV exposure ? ?
HEU status
Preterm birth
Neurodevelopmental disorders
Maternal factors
A pilot study (n=103) conducted in BC suggested that a high proportion of HEU children required special aid in school
Did not consider possible confounders such as maternal HIV disease state (CD4, viral load) as these data were not available

26. Acknowledgements CARMA participants CARMA Funding Cote Lab
Matthew Budd
Micah Piske
Patricia Janssen
Joel Singer
Jason Brophy
Lindy Samson
Acknowledgements
Hugo Soudeyns
Fatima Kakkar
Valerie Lamarre
Normand Lapointe
CARMA
CARMA participants
Ari Bitnun
Stanley Read
Ariane Alimenti
John Forbes
Laura Sauve
Deborah Money
Evelyn Maan
Funding