The paper by Girard et al. is the first of a number of papers that will soon appear searching for rare single nucleotide DNA sequence variants in patients with schizophrenia. It marches to the popular drumbeat that rare single nucleotide variants, like rare CNVs, represent part of the complex genetic architecture of the population of individuals with the diagnosis of schizophrenia. While this is likely to be true, de-novo variants in particular (i.e., those not inherited) cannot account for the “missing heritability” that has so far not been accounted for by common variant association studies, unless for some improbable reason the genetics of the machinery that leads to DNA replication errors is associated with risk for schizophrenia. The findings of Girard et al.—that there is an increased burden of coding, likely functional, de-novo mutations in individuals with schizophrenia—are based on a very small sample (n = 14 patients). The reference sequence data to which they compare their results are based on even smaller numbers of fully sequenced individuals.

I believe the jury is still out about the frequency of putatively functional single nucleotide mutations in the healthy population. Moreover, it is even more obscure how pathogenic a single nucleotide mutation actually is, even when it looks highly deleterious. In a recent, very important study of exon sequencing of 237 ion channel genes implicated in risk for inherited epilepsy syndromes in a much larger sample (391 subjects), consisting of individuals with epilepsy and normal controls (Klassen et al., 2011), the ratio of missense to synonymous coding mutations was 1:2, even higher than that found in the results of Girard et al. In addition, functional mutations that had previously been shown to be causative for inherited epilepsy were also found in healthy individuals. The attribution of causation or even high penetrance to rare, de-novo, and in most cases, private sequence mutations will be a very tough challenge.

References:

Klassen T, Davis C, Goldman A, Burgess D, Chen T, Wheeler D, McPherson J, Bourquin T, Lewis L, Villasana D, Morgan M, Muzny D, Gibbs R, Noebels J. Exome sequencing of ion channel genes reveals complex profiles confounding personal risk assessment in epilepsy. Cell . 2011 Jun 24 ; 145(7):1036-48. Abstract

View all comments by Daniel Weinberger

Girard et al. sequenced the exomes of 14 parent-child trios, each comprising an individual with sporadic schizophrenia and his or her unaffected parents. They found 15 de-novo mutations from eight probands, and suggest that individuals with schizophrenia are more likely to have de-novo and deleterious mutations specific to coding sequences. This is the first paper in this area, and the third in neuropsychiatric disorders following mental retardation (Vissers et al., 2010) and sporadic autism spectrum disorders (O'roak et al., 2011).

However, some major issues complicate interpretation of the results reported in Girard et al. and weaken the conclusions the authors wish to draw.

First, regarding sequencing coverage, mutation discovery from exome data is a challenging problem, and identifying de-novo mutations is particularly enriched by technical errors. The authors reported that the exome capture resulted in an average of 72 percent targeted regions covered with read depth >20x, which is inadequate for producing accurate genotype calls across a large fraction of the exome (Ajay et al., 2011). The authors removed false-positive de-novo calls by carrying out Sanger sequencing, but the false-negative rate of this experiment is not established.

Second, regarding control data, Girard et al. used genomewide de-novo mutation rates from genome sequencing studies in the literature. The authors are forced to assume that de-novo mutation rates across the entire genome are comparable in those with or without schizophrenia. Unfortunately, the literature studies did not use the same experimental conditions as Girard et al., and can be expected to have different false-positive and false-negative rates. Therefore, it is difficult to conclude that an increased exonic de-novo mutation rate is found in individuals with schizophrenia.

Third, in regard to case definition, it is important to remind readers that it is typical for individuals with schizophrenia to be family history-negative. The authors could have provided more detail about their efforts to exclude environmental causes. One wonders whether the study of sporadic cases truly identified what the authors intended to study.

Fourth, it is of interest that six probands did not have a detectable mutation. There could have been more discussion of this interesting finding.

Fifth, the mere presence of a de-novo exonic mutation is far from sufficient for causality. We all have multiple such mutations in our exomes, and many such single-copy mutations are functionally silent. The authors appear to assume that the mutations they identify have a dominant mode—that a single-copy mutation overrides the opposing wild-type allele. While this can occur, it is not the only outcome, and some functional data would have strengthened the authors' assertions.

Sixth, the algorithms that predict the functional consequences of a mutation are far from perfect. Even a high-confidence predicted deleterious mutation may have no major consequences (e.g., the prediction could be incorrect, or the exon containing the mutation might not be used in the CNS). Moreover, some predicted "silent" mutations can have major consequences (e.g., via the creation of a splice site or miRNA target site).

We can expect many of these studies in the next year. Many investigators are eagerly following this area, as this may be a part of the allelic spectrum that contains variation of particular use to biologists.

If there is any one lesson to be learned from the history of schizophrenia genetics, far larger samples will be required than reported here. The field needs integrated and standardized analyses of ~1,000 times more cases than studied here in order to elucidate the genetic basis of schizophrenia. Data generation is in progress, and multiple groups (including the Psychiatric GWAS Consortium) are preparing for the necessary meta-analyses.

References:

Vissers LE, de Ligt J, Gilissen C, Janssen I, Steehouwer M, de Vries P, van Lier B, Arts P, Wieskamp N, del Rosario M, van Bon BW, Hoischen A, de Vries BB, Brunner HG, Veltman JA. A de novo paradigm for mental retardation. Nat Genet. 2010 Dec;42(12):1109-12. Abstract

O'roak BJ, Deriziotis P, Lee C, Vives L, Schwartz JJ, Girirajan S, Karakoc E, Mackenzie AP, Ng SB, Baker C, Rieder MJ, Nickerson DA, Bernier R, Fisher SE, Shendure J, Eichler EE. Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations. Nat Genet. 2011 Jun;43(6):585-9. Abstract

Ajay SS, Parker SC, Ozel Abaan H, Fuentes Fajardo KV, Margulies EH. Accurate and comprehensive sequencing of personal genomes. Genome Res. 2011 Jul 19. Abstract

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