Schizophrenia Genetics 1: Linkage Studies—What’s Under Your Peak?
In SRF's schizophrenia genetics overview, writer Pat McCaffrey surveys the range of experimentation and opinion in the field in a five-part series.
Editor's note: From the large-scale collaboration of the Psychiatric Genomewide Association Study Consortium, to the very public debate about priorities and funding, schizophrenia researchers had a lot to talk and argue about in 2009. To quote a popular metaphor in this field, some describe the genetics glass as half empty, a vessel of as-yet unfulfilled promise, while others see the cup running over with new discoveries and possibilities. Undaunted by the roiling waters, SRF writer Pat McCaffrey dove in and asked a handful of prominent researchers such questions as, What is going on in schizophrenia genetics? What have we learned about the genetic landscape and the genomic substrates of schizophrenia? What paths are available to researchers, and what is the best way to proceed?
What follows is the first in a series of five articles that will air a spectrum of views (see also the interview with Lars Bertram on the new meta-analysis results from SZGene). Our molecular genetics story starts in the 1980s, with the early linkage studies that produced the first flock of gene candidates. Then, it moves on to the controversial common disease/common variant hypothesis and the large genomewide association studies (GWAS) it inspired, followed by a look at the hunt for rare mutations in schizophrenia. Finally, we gaze ahead at the next steps and ask how the accumulated wisdom is contributing to the search for new treatments. As always, we hope these articles will stimulate comments or corrections (not to mention commendations for Pat!) from our readers.
12 March 2010. The hunt for the genetic basis of schizophrenia started well before the Human Genome Project or the International HapMap. Buoyed by the evidence of inherited risk and the hope of identifying single-gene Mendelian forms of the disease, in the late 1980s researchers began to deploy new molecular genetic techniques in linkage studies to identify large chunks of DNA that segregated with schizophrenia in affected families. The first crop of candidate genes from beneath the linkage peaks—dysbindin (DTNBP1) at chromosome 6p, neuregulin (NRG1) at 8p, and the D-amino acid oxidase activator (DAOA, formerly G72/G30) at 13q—were all identified in 2002 by linkage scans in relatively small family samples, followed by linkage disequilibrium mapping (Straub et al., 2002; Stefansson et al., 2002; Chumakov et al., 2002). Many subsequent studies bore out these linkages, and meta-analyses in the SchizophreniaGene (SZGene) database continued to support the role of these genes when this story went to press (see the most up-to-date results of SchizophreniaGene meta-analyses, revised 29 January 2010 to include criteria that assess the quality of the data; see interview with Lars Bertram on SZGene).
In addition to linkages, the candidate gene approach yielded some early successes by focusing studies on genes with a biologically plausible link to schizophrenia, such as those encoding dopamine system molecules. These early linkage and candidate gene finds are now among the best-supported candidate genes for schizophrenia, though they are not universally accepted.
A promising start
At the time, debates sometimes grew contentious about specific candidate genes, but the linkage studies proved that schizophrenia geneticists could collaborate for the greater good. "As a group, we talked very intensely at meetings, we collaborated well prior to publication, and we fed each other our best markers and regions," said Richard Straub of the National Institute of Mental Health, Bethesda, Maryland, one of the first researchers to do linkage studies in schizophrenia. This spirit of cooperation culminated in a meta-analysis of 20 different studies, which identified genomewide significant linkage at 2q, and an additional nine locations that the researchers agreed were strong candidate gene regions (Lewis et al., 2003).
In retrospect, linkage studies in pools of unrelated families should not have worked, if indeed schizophrenia springs from many genes of small effect size or from rare variants in many different genes (as is discussed in Part 2 and Part 3 of this series). Small effect sizes and genetic heterogeneity make it hard to separate linkage peaks from noise. The definition of schizophrenia presented another difficulty. A clinical syndrome with variable manifestations, schizophrenia has no biological diagnostic criteria. Is it one disease or many? A problematic phenotype just added to the uncertainty around the search for genes responsible for the disorder.
Nonetheless, linkage studies turned out to be surprisingly good at finding evidence of genes of small effect size, says Straub, probably because some peaks span multiple susceptibility genes that sum up to a stronger signal. That is the advantage of linkage over association studies, because a genomic region can “get credit” for the disease in different families, even if the actual genes involved are different. In addition, a signal can sum up the effects of different alleles of the same gene, said Straub. In linkage, common variation and rare changes all contribute to the linkage signal, so that despite the small size and low resolution of the early studies, gene candidates did emerge as researchers looked for biologically plausible genes under the peaks.
The early linkage studies were “shockingly successful,” said Straub, and really opened up the field. “Having one gene allowed nomination of other candidate genes,” and in that way neuregulin led to ErbB4, and dysbindin set researchers onto other potential genes. For instance, Straub and colleagues found that dysbindin is part of an eight-protein macromolecular complex that is involved in vesicle trafficking and dopamine receptor stability (the BLOC-1 complex; see SRF related news story). That finding led them to another candidate gene. “We did a few SNPs in the gene for MUTED, one of the three known dysbindin binding partners at the time," said Straub. "We got a beautiful signal in the 3’ UTR that may involve a microRNA binding site.”
Two studies from other labs on MUTED have not been positive (see SZGene entry for MUTED), but Straub stands by his analysis. He argues that in a genetically heterogenous disease like schizophrenia, replications in different samples should not be the gold standard and are, in fact, statistically unlikely. “It is so much harder for competent investigators to generate a false positive than a false negative in the detection of genes in complex disorders that negative studies should never be weighted as strongly as positive,” he said. In fact, he added, “I’ve maintained for many years that a negative really means nothing.” Instead, researchers need to look at the cumulative evidence for a gene’s involvement, be it genetic or biological.
Not everyone agrees on the strength of the evidence for genes implicated by linkage studies. In his recent talk at the 2009 World Congress on Psychiatric Genetics, Michael Owen of Cardiff University in the United Kingdom said that while some of the genes (NRG1, DTNBP1, and DAOA) had strong support, there were still some holes in the data. For one, there were no clear risk alleles identified for the genes. “These findings are better than we had previously, but they fall short of the strength and consistency one would hope to see and that have been seen in other diseases,” he said.
Biology: help or hindrance?
Owen also spoke critically of the "contamination” of genetics research by biology. “Some say that geneticists are obsessed by statistics, and what you need is biology. If you are looking at biologically plausible things, you don’t need huge samples, and you should focus on a biology-based interpretation of the data, they argue. That’s a view I happen to profoundly disagree with,” he said, because researchers do not know what biology underpins schizophrenia.
Straub thinks the weight of the evidence justifies a stronger conclusion. “The chances of me analyzing eight proteins related to dysbindin and then getting a beautiful signal out of MUTED, if dysbindin wasn’t a schizophrenia gene, is basically zero,“ he says. “It turns out that within two protein binding partners of dysbindin are five schizophrenia genes, and that immediately validates dysbindin."
Kenneth Kendler, of Virginia Commonwealth University in Richmond, was also involved in the early linkage studies, and he, like Owen, is reserving judgment on whether neuregulin and dysbindin are susceptibility genes. He is also skeptical about the candidate gene approach, which has nominated the dopamine receptor genes and other genes in the dopamine pathway. “The candidate gene approach is basically a Bayesian approach where researchers take prior knowledge about genes that are involved to up the chances that P values produced will be true and not false positives,” Kendler said.
With schizophrenia, however, that approach makes little sense to him, “because we don’t have a clue what causes the illness.” He noted, for example, that while drugs targeting the dopamine system produce a treatment response, the evidence that dopamine is involved in pathophysiology is not strong.
Though the debate continues on the results of linkage and candidate gene studies, the work done in this arena has left its imprint on the field. The prospect of causative genes drew neurobiologists into schizophrenia research when they could see, for the first time, a path to molecular and cellular studies. And because of the uncertainty in the genetic studies, geneticists needed biological support to complement their own work. “These discoveries ushered in a new era in schizophrenia research,” said Straub. “Because they were not definitive, they encouraged scientists to assemble other evidence—both genetic and biological—to support genetic evidence that on its own might not be compelling.” That situation led to an influx of physiologists and biologists into the field, pursuing studies aimed at building a biological rationale for a candidate gene, he said.
Wherever studies of the early candidate genes lead in the future, linkage studies kicked off the transformation of the field of psychiatric research from clinical studies and epidemiology to biology. “Genes are the first objective signs to point us to the cause of these illnesses,” said Daniel Weinberger, also of the National Institute of Mental Health. “These are the first clues to the biological mechanisms of causation, which we never had before. That’s the sea change we have seen.”—Pat McCaffrey.