This study used a combined approach of linkage to identify a schizophrenia susceptibility region and gene expression to identify candidate genes within the linkage region. The genome-wide scan with microsatellite markers and SNPs suggested linkage of schizophrenia to chromosome 4q33−35.1 in a high-density multiplex pedigree. The pedigree was followed up with microarray screening of gene expression. We were not able to demonstrate a significant linkage between the putative haplotype for disease and for gene expression. This may be due to a suggestive but nonsignificant LOD score generated for schizophrenia in this single pedigree. Thus, we emphasize that these results indicate the methods of using gene expression and linkage with a disease to attempt to pinpoint regulatory and possibly causative loci.
We found that ADH1B differential gene expression is validated by Q-PCR, and showed a significant LOD score to a regulatory locus. After considering all 1,327 nominally significant gene expression differences for linkage to chromosome 4, both ADH1B and GALNT7 showed significant LOD scores for linkage to chromosome 4 (LOD>3.0). Interestingly, both genes showed suggestive linkage of a smaller magnitude to the haplotype region (LOD>2.0). We focused our search on chromosome 4q haplotype, as an exhaustive analysis of all gene expression and SNP markers would require ~5×108 statistical tests. However, with additional subjects it would be worthwhile to pursue a genome-wide scan of gene expression regulatory loci.
Gene expression in peripheral white blood cells has been tested in schizophrenia, bipolar, and controls (Tsuang et al. 2005
; Middleton et al. 2005
). Linkage regions in schizophrenia and bipolar disorder have been further scanned for changes in gene expression of peripheral white blood cells (Middleton et al. 2005
). We have also found that three genes within the 4q33−35.1 region are differentially expressed in DLPFC (AGA
, and HMGB2
). Thus, another potential use for this type of investigation is for determining whether the lymphocyte expression differences serve as potential biomarkers, especially if the gene is also expressed in the brain.
Summary of genetic studies in schizophrenia reporting findings on chromosome 4q
We have used Q-PCR to validate the microarray data. However, we were unable to validate two candidate genes in the 4q33−35.1 region. In reanalysis of the data using unequal variance, the t test for both genes became nonsignificant. Thus, the Q-PCR failure was due to high variation in samples. However, we were able to validate 7 out of 14 genes tested by Q-PCR.
might be a candidate gene based upon gene expression differences in schizophrenia and linkage to regulatory loci within the schizophrenia haplotype and outside of the haplotype. ADH1B
metabolizes substrates in pathways involving serotonin, norepinephrine, dopamine, and alcohol (Consalvi et al. 1986
; Helander et al. 1994
; Svensson et al. 1999
; Matsuo and Yokoyama 1989
; Matsuo et al. 1989
) and thus is relevant to psychiatric disorders. Although we did not find a predisposing mutation in any exons of AGA
for schizophrenia, there are known mutations of AGA
that cause the lysosomal storage disease, Aspartylglucosaminuria (OMIM# 208400). Aspartylglucosaminuria is characterized by predominant cognitive deterioration, mental retardation, and emotional lability.
Potential regions for control of gene expression were mapped to cis
- and trans
-regulatory sites. The results of the study are consistent with prior reports demonstrating that gene expression traits in lymphocytes are heritable in multiplex pedigrees (Morley et al. 2004
; Monks et al. 2004
; Schadt et al. 2003a
). These results suggest that studying gene expression traits in combination with linkage studies helps identify regulatory regions for candidate genes. A high LOD score between a quantitative trait (gene expression) and a genetic marker suggests that possible regulatory elements within the linkage region may regulate the quantitative trait (gene expression) (Schadt et al. 2003a
). Schadt et al. (2003a
reported that 423 genes were linked to a chromosome 2 locus of interest, which was a quantitative trait of subcutaneous fat deposition. Notably, only four genes were within 2 cM of the peak where gene expression traits showed linkage. “Most of the genes linked to the chromosome 2 locus do not physically reside on chromosome 2, and so, are at least partially regulated by one or more loci in the chromosome 2 hotspot region” (Schadt et al. 2003a
). This reasoning can apply to the present results, as most genes with differential expression showed regulatory loci in a trans
- location, while some genes also showed evidence of being partially regulated by two distinct loci.
The differentially expressed gene, such as ADH1B
, located outside of the haplotype but linked to the haplotype may not confer risk to illness, but may modify gene expression. This modification in gene expression could confer a variation in symptoms, onset, or subtype in the schizophrenia syndrome. Consistent with the Schadt et al. (2003a
study, we find differentially expressed candidate genes on other chromosomes with suggestive linkage to the haplotype (). Both cis
- and trans
- acting factors regulate genes, perhaps in combination in a complex disorder, as suggested by our results and others (Morley et al. 2004
). Improvements in the analytical methods are needed to integrate differential gene expression as a phenotype and linkage to genomic markers (Schadt et al. 2003a
; Kraft et al. 2003
; Sham et al. 2002
; Horvath and Baur 2000
; Middleton et al. 2005
). However, taken together, linkage analysis of the inheritability of gene expression traits will be useful for mapping regulators of gene expression.