Genome-wide expression profiling of human blood reveals biomarkers for Huntington's disease

Abstract

Huntington's disease (HD) is an autosomal dominant disorder caused by an expansion of glutamine repeats in ubiquitously distributed huntingtin protein. Recent studies have shown that mutant huntingtin interferes with the function of widely expressed transcription factors, suggesting that gene expression may be altered in a variety of tissues in HD, including peripheral blood. Affymetrix and Amersham Biosciences oligonucleotide microarrays were used to analyze global gene expression in blood samples of HD patients and matched controls. We identified 322 mRNAs that showed significantly altered expression in HD blood samples, compared with controls (P < 0.0005), on two different microarray platforms. A subset of up-regulated mRNAs selected from this group was able to distinguish controls, presymptomatic individuals carrying the HD mutation, and symptomatic HD patients. In addition, early presymptomatic subjects showed gene expression profiles similar to those of controls, whereas late presymptomatic subjects showed altered expression that resembled that of symptomatic HD patients. These elevated mRNAs were significantly reduced in HD patients involved in a dose-finding study of the histone deacetylase inhibitor sodium phenylbutyrate. Furthermore, expression of the marker genes was significantly up-regulated in postmortem HD caudate, suggesting that alterations in blood mRNAs may reflect disease mechanisms observed in HD brain. In conclusion, we identified changes in blood mRNAs that clearly distinguish HD patients from controls. These alterations in mRNA expression correlate with disease progression and response to experimental treatment. Such markers may provide clues to the state of HD and may be of predictive value in clinical trials.

Figures

Fig. 1.

Altered gene expression in the blood of HD patients. Cluster analysis of the 322 most differentially expressed genes on Affymetrix (A) and Amersham Biosciences (B) microarrays is shown. The genes were selected from 17 HD-affected subjects and 14 healthy control subjects according to P value (P < 0.0005), fold change (>1.8 or <0.6), and expression maximum >100 (Affymetrix) or >1 (Amersham Biosciences). Each column represents a sample and each row a gene. The colorgram depicts high (red) and low (green) relative levels of gene expression. The samples were normalized (median-polished) for each platform. Hierarchical clustering using cosine correlation with complete linkage was performed on the pool of all samples from both platforms to determine sample and gene clustering. The two groups were then separated in the display to compare the gene profiles between the two platforms (healthy control subjects C1-C14, late presymptomatic carriers of the HD mutation P1-P5, and symptomatic HD patients S1-S12).

Fig. 2.

Differential expression of a subset of genes was confirmed with QRT-PCR. (A) The up-regulation of expression of the 12 selected genes in blood of HD subjects was confirmed on the training set of 11 HD patients and 5 controls and a test set of 30 new HD subjects and 25 controls by using QRT-PCR. Values represent average fold change = 2-(average ΔΔCt) in mRNAs in HD relative to healthy control subjects (in every case, P < 0.05). Error bars are presented as (average fold change) × (2SEM - 1). (B) Gene descriptions of the 12 genes with representative gene symbols, GenBank accession numbers, LocusLink ID numbers, and probe IDs for Affymetrix and Amersham Biosciences microarrays.

Fig. 3.

The marker gene set was differentially expressed in presymptomatic and symptomatic HD subjects. PCA in the training set confirmed the separation of the presymptomatic carriers of the HD mutation and HD patients from healthy control subjects (A). Using the first two principal components from the QRT-PCR training set, PCA for the 12 genes in the test set confirmed the separation of symptomatic HD patients from healthy controls (B). Early presymptomatic carriers of the HD mutation (average age, 22.5 ± 2.6 years) cluster predominantly with control samples (C), whereas late presymptomatics (average age, 39 ± 6.1 years) mostly group with symptomatic HD patients (D). PCA was performed by using the ΔCt values. For visualization in a 2D plot, the first two principal components for the training set data were chosen representing 81% of the variance in the training set. The subgroups are colored according to their clinical classification (healthy control subjects ♦, early presymptomatics ▪, late presymptomatics ▴, symptomatic HD patients •). Analysis of the individual genes revealed up-regulation of expression with progression from early presymptomatic to symptomatic stage of HD (E). All of the 12 genes were significantly up-regulated in symptomatic HD patients (▪) and late presymptomatic carriers of HD mutation (▪), compared with controls (P < 0.05). In the early presymptomatic group (▪), only annexin A1 (ANXA1), TAF7, and purinergic receptor P2Y (P2Y5) were significantly up-regulated (P < 0.05). Gene expression of the 12 selected genes in A-E was analyzed by QRT-PCR.

Fig. 4.

Gene expression of the marker gene set was decreased in HD patients treated with phenylbutyrate. RNA was isolated from blood samples taken from 12 HD patients (HD1-HD12) before treatment and after 4 weeks of treatment with phenylbutyrate. QRT-PCR analysis of gene expression of the 12 selected genes (A) showed significant decrease in expression after treatment in 10 of 12 patients (P < 0.05). In patients HD2 and HD12, no changes were observed. The values are presented as average fold change = 2-(average ΔΔCt) for gene set in treated relative to untreated samples. Error bars represent (average fold change) × (2SEM - 1). In B, QRT-PCR analysis of each individual gene from the marker set in patients treated with phenylbutyrate showed statistically significant decrease in expression of 8 of 12 genes (P < 0.05) after treatment. The values represent average fold change = 2-(average ΔΔCt) for the samples after treatment, compared with untreated samples. Error bars are presented as (average fold change) × (2SEM - 1).

Fig. 5.

Genes altered in HD blood are differentially expressed in HD brain. Expression of the 12 marker genes was analyzed in five postmortem HD caudate samples (Vonsattel grades 0-2) and four control samples by using QRT-PCR. Seven genes were significantly up-regulated in brain tissue samples from HD patients, compared with controls (P < 0.05). Values are presented as average fold change = 2-(average ΔΔCt) for HD brain samples, compared with controls. Error bars are presented as (average fold change) × (2SEM - 1).