Mutational analysis of the mitochondrial tRNA genes and flanking regions in umbilical cord tissue from uninfected infants receiving AZT-based therapies for prophylaxis of HIV-1

Abstract

A sensitive vertical denaturing gradient gel electrophoresis (DGGE) method, using 13 unipolar psoralen-clamped PCR primer pairs, was developed for detecting sequence variants in the 22 tRNA genes and flanking regions (together spanning approximately 21%) of the human mitochondrial genome. A study was conducted to determine (i) if mitochondrial DNA (mtDNA) polymorphisms and/or mutations were detectable in healthy newborns and (ii) if prepartum 3'-azido-2',3'-dideoxythymidine (AZT) based HIV-1 prophylaxis was associated with significant increases in mtDNA mutations and changes in the degree of heteroplasmy of sequence variants in uninfected infants born to HIV-1-infected mothers. DGGE analysis of umbilical cord tissue (where vascular endothelium and smooth muscle cells are the major source of mtDNA) showed that mtDNA sequence variants were significantly elevated by threefold in AZT-treated infants compared with unexposed controls (P < 0.001), with 24 changes observed in 19/52 (37%) treated newborns (averaging 0.46 changes/subject) versus only eight changes found in 7/55 (13%) unexposed newborns (averaging 0.15 changes/subject). Six distinct sequence variants occurring in unexposed controls were predominately synonymous and homoplasmic, representing previously reported polymorphisms. Uninfected infants exposed to a combination of AZT and 2',3'-dideoxy-3'-thiacytidine and "maternal HIV-1" had a significant shift in the spectrum of mutations (P = 0.04) driven by increases in nonsynonymous heteroplasmic sequence variants at polymorphic sites (10 distinct variants) and novel sites (four distinct variants). While the weight of evidence suggests that prepartum AZT-based prophylaxis produces mtDNA mutations, additional research is needed to determine the degree to which fetal responses to maternal HIV-1 infection, in the absence of antiretroviral treatment, contribute to prenatal mtDNA mutagenesis.

Fig. 1

Map positions of human mtDNA segments amplified by PCR for sequence variations in the tRNA genes. Thirteen segments of human mtDNA (Table I) were selected to screen all tRNA-coding regions using the Primo Melt 3.4 computer program.

Fig. 2

Schematic representations of cloverleaf structures for mitochondrial tRNA^Arg (A) and tRNA^Thr (B) showing the location of each mutation (shown in bold) in umbilical cord tissue from two infants receiving prepartum AZT-3TC for prophylaxis of HIV-1 (PID #s 371 and 606) and one unexposed infant (PID # 310). D-loop, dihydrouridine loop; T-loop, TCC loop. Criteria used for assessing pathogenicity are those described by McFarland et al. [2004]: (1) the mutation occurs in a stem structure, (2) the mutation occurs in an acceptor or anticodon stem structure, (3) the mutation disrupts Watson-Crick base pairing, (4) the mutation disrupts GC base pairing, (5) the mutation, if in a loop, is in a loop of unusual size or affects tertiary folding. The A15924G transition meets criteria 1–3, while the T10454C transition and single-base deletion at base 15940 occur in small T-loops and fail to meet criterion 5.