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ICOSR 2007—Researchers Report from "Beyond Candidate Genes"

Editor's Note: On Thursday, 29 March 2007, the first full day of the International Congress on Schizophrenia Research in Colorado Springs, Anil Malhotra of Zucker Hillside Hospital in Glen Oaks, New York, chaired a session entitled, "Beyond candidate genes: new approaches to the identification of schizophrenia susceptibility loci." We are grateful to Eric Epping of the University of Iowa, one of the Young Investigator travel awardees to the meeting, who filed the following report.

11 April 2007. Joel Kleinman from the National Institute of Mental Health in Bethesda, Maryland, kicked off the symposium, presenting data on the identification and characterization of a novel schizophrenia susceptibility gene, KCNH2, a voltage-gated potassium channel. This gene candidate was identified from microarray expression studies, with follow-up work from a family-based association screen (NIMH Sibling Study), and further confirmation in additional datasets. Located on chromosome 7q, the gene encodes a protein involved in slow repolarization of the action potential in heart muscle and is involved in regulating the QT interval. Risk alleles were found to be associated with processing speed, IQ, visual memory, and reduced hippocampal volume in healthy subjects. In a postmortem brain collection, full length KCNH2 mRNA levels were reduced in subjects with schizophrenia compared to controls, and expression of a novel shorter isoform was increased. Functional properties of the isoforms were characterized in cell culture, indicating differences in channel activity. Antipsychotics also bind to the channel and inhibit its activity, although no frontal cortex changes in a rat model exposed to antipsychotics were found.

In his talk entitled “Transgenic manipulations of putative susceptibility genes,” Tyrone Cannon from the University of California, Los Angeles, described a novel mouse model of DISC1 that his group has used to elucidate functional effects of mutations in this gene. Using an inducible transgene expressing a variant of DISC1 that disrupts normal DISC1 binding to NUDEL and LIS1, the researchers found decreased performance in working memory in adult animals that had the transgene expressed at postnatal day (PND) 7. This effect on working memory was not seen in transgenic animals in whom the DISC1 variant was expressed in adulthood. PND7-induced animals also had worse performance on the forced swim test and reduced sociability. At the cellular level, the PND7-induced animals showed reduced dendritic arborization and reduced synaptic transmission. This model allows the researchers to test hypotheses about the effects of DISC1 variants at the molecular and whole animal level, and has shown that the effects are time-specific in development.

Using DNA chips capable of genotyping nearly 500,000 (500K) single nucleotide polymorphisms (SNPs), Todd Lencz of Zucker Hillside Hospital presented data on structural genomic variation in schizophrenia. In a search for regions of autozygosity, or segments of the genome with extended shared haplotypes (i.e., clusters of related alleles with the same sequence on both chromosomes), several runs of homozygosity (ROH) were found to be more common in patients with schizophrenia on multiple chromosomes. One gene within a ROH more frequent in schizophrenia includes the CAPON gene, which competes with PSD95 (a post-synaptic protein in NMDA neurons) for binding to neuronal nitric oxide synthase. These regions may indicate areas of low recombination or evolutionary selection. This technique also identifies regions of chromosomal copy number variation, which can be identified by measuring signal intensities on the SNP chips. Their initial analyses, according to Lencz, indicate that the ROH results are not primarily driven by copy number variation.

Anil Malhotra also presented results from a whole genome analysis of the SNP array data, which found an association with variants in a region containing the genes for two cytokine receptors on a pseudoautosomal region of the X and Y chromosomes (see SRF related news story).—Eric Epping.

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Related News: Genetic Homozygosity Runs in Schizophrenia Families

Comment by:  Ben Pickard
Submitted 7 December 2007
Posted 7 December 2007

Schizophrenia as genetic pelmanism
If you take a brand new pack of cards and start shuffling, it is not hard to appreciate that the longer you continue, the less likely it will be that you will find a series of cards in the same order as in the beginning. The European and Asian genomes are like a pack of cards that effectively started shuffling as humans first walked “Out of Africa” some 100,000 years ago. Meiotic recombination is the shuffling process and the result is a decreasing ability to predict at the gross level what combinations of marker alleles will be found together on a chromosome. African populations, with a longer “shuffling” time and without population bottlenecks (which effectively reorder the cards) show the least predictability (“linkage disequilibrium,” LD) across their genomes.

There are two counteracting forces to halt or even reverse this entropic breakdown. Firstly, if a particular region becomes strongly selected for, then its frequency increase in the population will, in the medium-term, outrun the shuffling effect such that the region flanking the selected genetic variant will maintain its order (Gibson et al., 2006; Li et al., 2006). This is known as a “selective sweep,” and numerous post-HapMap studies have successfully fished out regions of our genomes under this selective pressure (e.g., the lactose tolerance variant in populations where milk became a part of the staple prehistoric diet (Tishkoff et al., 2007). Secondly, and rather more obscurely, there can be physical restraints to recombination shuffling. These usually involve the physical reordering of sequence on our chromosomes, for example, in the case of paracentric inversions. The physical alignment of normal and inverted DNA sequences during meiosis is thus prevented and so recombination is suppressed, leading to greater LD.

Now imagine the situation where reasonably common stretches of less-shuffled chromosomes exist in the population. These are more likely to be found as matching pairs in any given individual compared to other parts of the genome. This appears to the researcher as a long stretch or tract of homozygous DNA. Such tracts have been studied elsewhere, particularly in the context of mapping and identifying recessive disease genes in remote, consanguineous (inbred) populations where the recessive mutations in genomic DNA of reduced allelic complexity are not only more likely to be exposed but occur within prominent tracts which co-segregate with the diagnosis. A newly published paper by Lencz et al. takes all of these ideas and combines them into a single strategy to hunt for schizophrenia-causing genes. They took raw data from their recently published genomewide association study of schizophrenia (178 cases of schizophrenia and 144 healthy controls: Lencz et al., 2007) and reassessed it for the presence of long “runs of homozygosity” (ROH) restricted to the case group. Their hypothesis was that if these regions existed, they would contain recessive mutations contributing to the disease.

Three hundred thirty-nine common ROHs were identified in the study, making up 12-13 percent of the total genome. The largest of these were predominantly found spanning the chromosome centromeres. This is perhaps not surprising since recombination rates have long been known to be reduced (through repression rather than selective sweep) at centromeres (see Kong et al., 2002). Nine of the commonest ROHs neatly overlap with previously described regions from selective sweep studies, as would be predicted. The key finding, however, was that when ROHs were compared between cases and controls, nine were found significantly more frequently in schizophrenia. Within these tracts, numerous genes were identified and, of these, there is pre-existing evidence in support of a few of them as potential candidates including NOS1AP, ATF2, NSF, MAPT, PIK3C3, and SNTG1.

One caveat to these findings is that a region of homozygosity, a loss of heterozygosity, copy number variation (CNV), and a deletion can, in some instances, all refer to the same genomic lesion and are not simple to distinguish by chip-based genotyping. The authors are careful to spell out technical and biological reasons for believing that their findings are a reflection of true homozygosity, but further independent verification would be reassuring, particularly in the context of how CNVs/genomic rearrangements might complicate recombination rates.

The significance of these findings is that we now have the potential to explore a brand new mutation class in a complex genetic disorder. Until now, the major research techniques such as linkage, association, and cytogenetics have only identified (and perhaps can only identify) dominantly behaving variants, albeit mostly with reduced penetrance. These are presumed to act through gain-of-function or, more likely, loss-of-function/haploinsufficiency mechanisms. The ROH regions described here are predicted to house reasonably common recessive risk variants: such properties meaning that they are not likely to be present in ascertained families with high densities of affected individuals but rather sporadic cases of illness where these alleles have, by chance, been inherited from both parents. It is not entirely clear why some of the more common ROHs didn’t feature in the original association study based on this data, particularly in genotype frequency rather than allele frequency analyses.

Nevertheless, the authors also make an additional, intriguing claim that these ROHs are not only overrepresented in the schizophrenia cohort because they are causative but because they have also been subject to positive selection. They cite the discovery of these ROHs in previous selective sweep scans, their more recent derivation from ancestral haplotypes, the presence of genes within which show selection pressure through alternative analyses, and their restriction to Caucasian populations as good evidence for such a claim. This effect may be due to some form of “heterozygote advantage” (also known as “overdominance”) which maintains or promotes the deleterious allele in the population. Examples where this phenomenon has been observed include recessive mutations giving rise to sickle-cell anemia, cystic fibrosis, and triose phosphate isomerase deficiency. Others have previously hypothesized that selection for the greater cognitive abilities in Homo sapiens compared to earlier hominins might have been at the cost of the emergence of schizophrenia, although the timescales of this kind of selection and the kind resulting in selective sweep are likely to be vastly different. An alternative explanation discussed in the paper is that rare recessive mutations could have “hitchhiked” their way to prominence within the selective sweep driven by a favorable variant in a closely linked gene. This latter idea seems more reasonable, given the difficulty in trying to imagine what cognitive or neurodevelopmental features would have been exclusively beneficial for the Caucasian population. It might also tally with some of the phenotypic epiphenomena that may coexist with schizophrenia (e.g., altered risk of rheumatoid arthritis, etc).

Finally, as an aside, this represents the third method of analysis, after the principal case-control studies and prediction of copy number variants, which can be applied to the large genomewide genotyping datasets being produced in numerous labs. Are there other aces waiting to be found in the hand?

References:

Gibson J, Morton NE, Collins A. Extended tracts of homozygosity in outbred human populations. Hum Mol Genet. 2006 Mar 1;15(5):789-95. Abstract

Kong A, Gudbjartsson DF, Sainz J, Jonsdottir GM, Gudjonsson SA, Richardsson B, Sigurdardottir S, Barnard J, Hallbeck B, Masson G, Shlien A, Palsson ST, Frigge ML, Thorgeirsson TE, Gulcher JR, Stefansson K. A high-resolution recombination map of the human genome. Nat Genet. 2002 Jul 1;31(3):241-7. Abstract

Lencz T, Morgan TV, Athanasiou M, Dain B, Reed CR, Kane JM, Kucherlapati R, Malhotra AK. Converging evidence for a pseudoautosomal cytokine receptor gene locus in schizophrenia. Mol Psychiatry. 2007 Jun 1;12(6):572-80. Abstract

Li LH, Ho SF, Chen CH, Wei CY, Wong WC, Li LY, Hung SI, Chung WH, Pan WH, Lee MT, Tsai FJ, Chang CF, Wu JY, Chen YT. Long contiguous stretches of homozygosity in the human genome. Hum Mutat. 2006 Nov 1;27(11):1115-21. Abstract

Tishkoff SA, Reed FA, Ranciaro A, Voight BF, Babbitt CC, Silverman JS, Powell K, Mortensen HM, Hirbo JB, Osman M, Ibrahim M, Omar SA, Lema G, Nyambo TB, Ghori J, Bumpstead S, Pritchard JK, Wray GA, Deloukas P. Convergent adaptation of human lactase persistence in Africa and Europe. Nat Genet. 2007 Jan 1;39(1):31-40. Abstract

Lencz T, Lambert C, DeRosse P, Burdick KE, Morgan TV, Kane JM, Kucherlapati R, Malhotra AK. (2007) Runs of homozygosity reveal highly penetrant recessive loci in schizophrenia. PNAS.

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Related News: Genetic Homozygosity Runs in Schizophrenia Families

Comment by:  Chris Carter
Submitted 20 December 2007
Posted 21 December 2007

This is a remarkable paper, not only for the genes described but also for its original and inventive design. As already stated by the authors, two genes identified in these regions (PIK3C3 and NOS1AP) have already been implicated in schizophrenia. A number of others are convincing candidates and can be related to genes and processes relevant to the disease. For example, Chimaerin 1 (CHN1) (found in roh52) binds to the NMDA receptor subunit GRIN2A and regulates the morphology and density of dendritic spines (Van de Ven et al., 2005; Buttery et al., 2006). Dendritic spine density is reduced in the frontal cortex in schizophrenia (Glantz and Lewis, 2000). ATF6 (found in roh15) is a key player in the endoplasmic reticulum stress pathway and regulates the expression of another gene implicated in schizophrenia, XBP1 (Hirota et al., 2006).

Perhaps even more interesting is EIF2S1 (found in roh291). This is an eif2α subunit phosphorylated by four stress-responsive eif2α kinases that are themselves activated by viruses (pkr/EIF2AK2), starvation (gcn2/EIF2AK4), oxidative stress (hri/EIF2AK1), and endoplasmic reticulum stress (perk/EIF2AK3) (cf ATF6 and XBP1). Phosphorylated eif2α turns off protein synthesis by inhibiting the actions of the translation initiation factor eif2b, and also activates the transcription factor ATF4, that turns on a series of programs designed to counter the effects of these stressors, including genes controlling glutathione homoeostasis (Carter, 2007). ATF4 is a binding partner of DISC1 (Morris et al., 2003), while mutations in eif2b are responsible for a disease that selectively attacks oligodendrocytes, vanishing white matter disease (van der Knaap et al., 2006). Famine (Susser et al., 1996) and viral infections, for example, prenatal influenza (Sham et al., 1992), are risk factors for schizophrenia, and oxidative stress (Gysin et al., 2007) and endoplasmic reticulum stress (XBP1, ATF6) also play a role in its pathology. Oligodendrocyte cell loss is also prevalent in schizophrenia (Uranova et al., 2007).

EIF2S1 is thus at the hub of a network activated by environmental risk factors implicated in schizophrenia. The outputs of this network (eif2b and ATF4) regulate oligodendrocyte function and glutathione homoeostasis (inter alia). As a recent clinical trial has reported some benefit with the glutathione precursor N-acetyl cysteine, in schizophrenic patients (Lavoie et al., 2007), this network and the genes therein may be extremely pertinent.

The genes and risk factors implicated in schizophrenia are annotated at Polygenic Pathways. This site is fairly regularly updated and now contains links to GeneCards from the Weizman Institute of Science and a selected set of Kegg pathways from the Kanehisa Laboratories (see also SchizophreniaGene).

References:

Van de Ven TJ, VanDongen HM, VanDongen AM. The nonkinase phorbol ester receptor alpha 1-chimerin binds the NMDA receptor NR2A subunit and regulates dendritic spine density. J Neurosci. 2005 Oct 12;25(41):9488-96. Abstract

Buttery P, Beg AA, Chih B, Broder A, Mason CA, Scheiffele P. The diacylglycerol-binding protein alpha1-chimaerin regulates dendritic morphology. Proc Natl Acad Sci U S A. 2006 Feb 7;103(6):1924-9. Abstract

Glantz LA, Lewis DA. Decreased dendritic spine density on prefrontal cortical pyramidal neurons in schizophrenia. Arch Gen Psychiatry. 2000 Jan;57(1):65-73. Abstract

Hirota M, Kitagaki M, Itagaki H, Aiba S. Quantitative measurement of spliced XBP1 mRNA as an indicator of endoplasmic reticulum stress. J Toxicol Sci. 2006 May;31(2):149-56. Abstract

Carter CJ. eIF2B and oligodendrocyte survival: where nature and nurture meet in bipolar disorder and schizophrenia? Schizophr Bull. 2007 Nov;33(6):1343-53. Epub 2007 Feb 27. Abstract

Morris JA, Kandpal G, Ma L, Austin CP. DISC1 (Disrupted-In-Schizophrenia 1) is a centrosome-associated protein that interacts with MAP1A, MIPT3, ATF4/5 and NUDEL: regulation and loss of interaction with mutation. Hum Mol Genet. 2003 Jul 1;12(13):1591-608. Abstract

van der Knaap MS, Pronk JC, Scheper GC. Vanishing white matter disease. Lancet Neurol. 2006 May 1;5(5):413-23. Abstract

Susser E, Neugebauer R, Hoek HW, Brown AS, Lin S, Labovitz D, Gorman JM. Schizophrenia after prenatal famine. Further evidence. Arch Gen Psychiatry. 1996 Jan 1;53(1):25-31. Abstract

Sham PC, O'Callaghan E, Takei N, Murray GK, Hare EH, Murray RM. Schizophrenia following pre-natal exposure to influenza epidemics between 1939 and 1960. Br J Psychiatry. 1992 Apr 1;160():461-6. Abstract

Gysin R, Kraftsik R, Sandell J, Bovet P, Chappuis C, Conus P, Deppen P, Preisig M, Ruiz V, Steullet P, Tosic M, Werge T, Cuénod M, Do KQ. Impaired glutathione synthesis in schizophrenia: convergent genetic and functional evidence. Proc Natl Acad Sci U S A. 2007 Oct 16;104(42):16621-6. Abstract

Uranova NA, Vostrikov VM, Vikhreva OV, Zimina IS, Kolomeets NS, Orlovskaya DD. The role of oligodendrocyte pathology in schizophrenia. Int J Neuropsychopharmacol. 2007 Aug;10(4):537-45. Epub 2007 Feb 21. Abstract

Lavoie S, Murray MM, Deppen P, Knyazeva MG, Berk M, Boulat O, Bovet P, Bush AI, Conus P, Copolov D, Fornari E, Meuli R, Solida A, Vianin P, Cuénod M, Buclin T, Do KQ. Glutathione Precursor, N-Acetyl-Cysteine, Improves Mismatch Negativity in Schizophrenia Patients. Neuropsychopharmacology. 2007 Nov 14; [Epub ahead of print] Abstract

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