Quantitative analyses of postmortem heat shock protein mRNA profiles in the occipital lobes of human cerebral cortices: implications in cause of death

Abstract

Quantitative RNA analyses of autopsy materials to diagnose the cause and mechanism of death are challenging tasks in the field of forensic molecular pathology. Alterations in mRNA profiles can be induced by cellular stress responses during supravital reactions as well as by lethal insults at the time of death. Here, we demonstrate that several gene transcripts encoding heat shock proteins (HSPs), a gene family primarily responsible for cellular stress responses, can be differentially expressed in the occipital region of postmortem human cerebral cortices with regard to the cause of death. HSPA2 mRNA levels were higher in subjects who died due to mechanical asphyxiation (ASP), compared with those who died by traumatic injury (TI). By contrast, HSPA7 and A13 gene transcripts were much higher in the TI group than in the ASP and sudden cardiac death (SCD) groups. More importantly, relative abundances between such HSP mRNA species exhibit a stronger correlation to, and thus provide more discriminative information on, the death process than does routine normalization to a housekeeping gene. Therefore, the present study proposes alterations in HSP mRNA composition in the occipital lobe as potential forensic biological markers, which may implicate the cause and process of death.

Fig. 1.

Relative mRNA levels of various HSP genes in postmortem human occipital lobes of cerebral cortices. mRNA levels of HSPs obtained from microarray analyses were normalized to those of GAPDH. Signal intensities from two independent replicates were averaged and expressed as % intensity of GAPDH in a log scale. Based on negative control probes, mRNA levels of HSPs higher than 5% of GAPDH were considered to be significantly detected.

Fig. 2.

HSP mRNA levels in the occipital lobes influenced by different causes of death. Occipital lobe-enriched HSP mRNA species were quantitatively analyzed by real-time qRT-PCR in subjects who died by traumatic injury (T), mechanical asphyxiation (A), or sudden cardiac death (S). Data were normalized to GAPDH mRNA levels and expressed the mean ± SE in arbitrary units (A.U.), where the mean values of the T group were set as 1. The group differences were statistically evaluated by one-way ANOVA followed by Newman-Keuls test (*p < 0.05 and **p < 0.01 vs. T; ^†p < 0.05 vs. A).

Fig. 3.

Correlation of individual HSP mRNA levels to GAPDH. The Ct values for HSPA2 and A8 were plotted with those of GAPDH, and linear regression was carried out for each group (TI, open; ASP, gray; SCD, closed circles). Slopes and linear correlation coefficients expressed as R² are summarized in Table 2.

Fig. 4.

Variations of selected HSP mRNA levels within the group. Relative mRNA levels of the differentially regulated HSPs were expressed by box-and-whisker diagrams in log scales. The median values are expressed as lines in the middle of the boxes.

Fig. 5.

Relative mRNA abundances between two differentially expressed HSP gene transcripts. Relative mRNA abundances between HSPA2 and A7 (left) or A13 (right) were examined by use of 2^−ΔCt indices of two mRNA transcripts (ΔCt = Ct_HSPA2 − Ct_HSPA7/A13) and then plotted using standard bar charts indicating the mean ± SE (A; *p < 0.05 vs. T by Newman-Keuls test as post-hoc comparison) or box-and-whisker diagrams (B).

Fig. 6.

Individual profiles of the TI and ASP subjects in relation to PMI and RNA integrity. (A) HSPA2 mRNA abundance relative to A7 (left), A13 (middle), or GAPDH (right) of each subject were plotted in relation to PMI (TI, open; ASP, closed symbols). (B) Subjects from the TI and ASP groups were categorized into two subgroups according to the 28S:18S rRNA ratio, and HSPA2 mRNA abundance relative to A7 (left), A13 (middle), or GAPDH (right) of a subject were plotted in relation to the 28S:18S rRNA ratio (TI, open; ASP, closed symbols).