In recent years, our understanding of the genetic architecture of schizophrenia, a
phrase which denotes the numbers of risk variants, their frequencies and effect sizes,
has been transformed. This has come about through advances in technology that have
allowed almost the entire human genome to be simultaneously interrogated for the presence
of disease-associated genetic variation and allows this to be performed in sample
sizes powered for a realistic possibility of success. Another development has been
the emergence of international consortia willing to share raw data and their coalescence
into super-consortia to achieve sample sizes and bodies of clinical and analytic expertise
that was unimaginable a decade ago. These innovations have driven the emergence of
statistically robust and replicable genetic findings in schizophrenia, and a rapid
escalation in the number of those findings over the last 5 years.
The latest example comes from the Schizophrenia Working Group of the Psychiatric Genomics
Consortium (PGC-SCZ) which, at the time of publication, included contributions from
around 37 000 individuals with schizophrenia, 302 investigators, 35 countries, and
4 continents.
1
In their recent paper, published in Nature in July 2014, the PGC-SCZ group report
128 statistically independent genetic associations, implicating a minimum of 108 conservatively
defined schizophrenia-associated genetic loci.
1
Of the identified loci, 83 have not been previously robustly supported as playing
a role in schizophrenia, but it is also important to note the findings are consistent
with previous literature; 25 loci that had previously been reported as associated
with schizophrenia in large samples were again supported in this much larger analysis,
confirming that the use of large samples and stringent statistical cut-offs results
in reproducible findings. The availability of so many robustly supported findings
offers immense opportunities for investigating and advancing our understanding of
etiology.
Large Numbers of Alleles Across the Frequency Spectrum
While each individual variant themselves have a small effect size (less than 1.4),
it has been estimated that common variation as a whole accounts for around a third
to half of the genetic variance in schizophrenia
2,3
though this may turn out to be an underestimate when heterogeneity and interaction
effects are taken into account. Nevertheless, rare variants also play a role. This
has been well established for around 20 years with respect to small deletions and
duplications known as copy number variants (CNVs)
4,5
but systematic surveys for other forms of rare genetic variation have had to await
the development of high capacity next generation sequencing technology. These studies,
particularly the larger ones,
6,7
have been commented on in a recent review in the journal
8
and will not be considered in detail here. However, like the early genome-wide association
studies (GWASs),
2,9
rather than striking evidence for individual susceptibility genes, the recent sequencing
studies provided evidence that many loci contribute to risk and for enrichment of
rare mutations in certain gene sets. This strongly suggests that, as we have seen
for GWAS, better powered studies will implicate specific genes.
10,11
The evidence that risk variants for schizophrenia occur across the range of allele
frequencies is compelling. As noted above, the contribution to risk of schizophrenia
arising from the aggregate effect of common variants is not trivial, but even a single
common variant can have a similar effect on the variance in the population as rare
variants with larger effects simply because they occur much more frequently in the
population.
12
This suggests that in order to understand genetic risk mechanisms we will need to
explore all parts of the allele frequency spectrum. Moreover, it seems likely that
advantages may accrue from a combined approach to gene identification as the PGC-SCZ
study pointed to an overlap between genes in schizophrenia GWAS regions and those
with de novo mutations. This suggests that a combination of the superior power of
GWAS (via sample size) and the superior precision of sequencing with respect to gene
identity might on the one hand be usefully harnessed to identify likely causal genes
within GWAS-associated loci and on the other to enhance the power of sequencing by
providing targets with enhanced prior probability.
Genetic Associations to Biological Mechanisms
The identity of individual associations, and patterns of enrichment of various types
of associations and disease-linked mutations are beginning to shed light on areas
of biology that are likely relevant to schizophrenia, although we stress detailed
mechanistic conclusions will require other types of research, and that the associations
at each locus have not yet been firmly linked to specific genes. Nevertheless, it
is notable that within the schizophrenia-associated loci are multiple genes involved
in synaptic function and plasticity, particularly genes involved in glutamatergic
neurotransmission (GRM3, GRIN2A, GRIA1, and SLC38A7) and neuronal calcium signaling
(CACNA1C, CACNB2, CAMKK2, CACNA1I, NRGN, and RIMS1). DRD2, which a priori is possibly
the strongest of all conceivable candidate genes for schizophrenia based on function,
is also associated with the disorder.
Given that most of the common variant associations do not appear to result from DNA
changes that affect protein sequence, it is presumed, with some evidence,
13
that they exert their effects through influencing gene expression. Investigating this
hypothesis further, the PGC-SCZ group sought to determine if schizophrenia-associated
common variants are concentrated in regulatory elements marked as activating gene
expression in particular tissues or cell lines. Importantly, though largely as predicted,
associations were enriched in these regulatory elements in various brain tissues and
in genes showing high expression in neurons/interneurons. A much more novel and potentially
important finding was they were also enriched in these regulatory elements in the
immune system, particularly B-lymphocyte cell lineages. This finding is intriguing
as it provides some genetic, and therefore etiological, support for the general hypothesis
that immune dysregulation plays a role in the development of schizophrenia.
14
However, we need to move beyond general enrichment analyses to identify specific causal
variants in specific regulatory elements, understand which genes/proteins are affected
by those regulatory elements, and show how genetic variation directly affects immune
but not neuronal function, before the immune hypothesis of schizophrenia can be considered
to be genetically confirmed.
The limited network analyses conducted by the PGC-SCZ did not identify any generically
annotated biological pathways that were enriched for associations. This may reflect
the restricted analyses presented, and a more thorough evaluation of the data is underway.
But it may also reflect either high polygenicity and/or limitations in the quality
and availability of data upon which to base these bioinformatic-driven analyses. Not
only are the functions of many proteins unknown, but even less well documented are
the elements that regulate the expression and processing of protein isoforms in specific
cellular, developmental, and physiological contexts especially in the brain. Proteomic
studies in particular lack the comprehensive scale of transcriptomic studies and are
currently limited to targeted approaches. Interestingly, in PGC-SCZ, support was found
for one of the most consistently implicated gene sets in schizophrenia, a set comprised
of brain-expressed genes that interact with the Fragile X mental retardation (FMRP)
protein.
3,6,7,15
While the biological implications of this are not yet understood, it is likely that
improving knowledge of the protein interactome and of the constituent members of sets
of proteins involved in brain function will improve our ability to move from patterns
of association to pathogenesis. This is likely to require more extensive experimental
validation, and iterative refinement, of bioinformatics tools. For example, experimental
studies reveal little overlap between genes predicted to be targets of microRNA-137,
whose encoding gene lies within a schizophrenia-associated locus, and those whose
expression is affected by knockdown or overexpression of the microRNA
16
demonstrating that experimental validation of bioinformatics predictions is essential.
Targets of FMRP are not the only consistently implicated gene set in schizophrenia.
Both GWAS
1,3
and exome sequencing
7
points to the involvement of multiple calcium channels and multiple genes involved
in calcium signaling, a process also implicated in bipolar disorder.
17,18
Proteins affiliated with the N-methyl-d-aspartate receptor and the activity-regulated
cytoskeleton-associated protein have been implicated by CNVs analysis
19
and by exome sequencing,
6,7,20
while as noted above, GRIN2A and multiple proteins related to glutamatergic signaling
are associated with the disorder in the recent GWAS.
1
These gene sets plausibly converge at the functional level of synaptic plasticity
and remodeling,
6,21
although this hypothesis requires testing through mechanistic experimental studies.
The complexity and inaccessibility of human brain tissue has made it challenging to
understand basic disease mechanisms and to translate genetic findings into biology.
Discussed more fully in a recent review,
8
a synthesis of gene discovery with recent advancements in stem cell technology and
genome engineering mean that an exciting avenue of research has been opened for psychiatry.
This could be either through patient-derived induced pluripotent stem cells
22
or through manipulations of cell lines by emerging gene engineering technologies.
23,24
Further discussion of these developing areas is outside the scope of the current article
but have been discussed elsewhere.
25–33
Pleiotropy
Pleiotropy occurs when one gene or genetic variant contributes to multiple phenotypes,
a phenomenon fast becoming a characteristic of identified genetic risk factors for
neuropsychiatric disorders. There is already evidence for extensive sharing of common
genetic risk variants between schizophrenia, bipolar disorder major depressive disorder,
and attention deficit hyperactivity disorder (ADHD)
17,34,35
though the evidence for ADHD is somewhat less consistent than that for the other phenotypes.
The high genetic correlation is not explained by diagnostic misclassification,
36
and instead points to considerable genetic pleiotropy in terms of these categorically
defined diagnoses. The same is true for rare genetic risk factors for schizophrenia.
It has been clear for several years that the same CNVs that confer risk for schizophrenia
also do so for neurodevelopmental disorders including autism spectrum disorders (ASD),
intellectual disability, ADHD, and epilepsy.
4,5,37,38
Given most of these CNVs affect multiple genes, it could conceivably be argued that
different genes were involved in each phenotype, but a recent analysis of point mutations
supports the view that sharing of risk occurs at the level of genes and types of mutation.
6
Moreover, the PGC-SCZ study found overlap between the genes in schizophrenia GWAS
regions and those with de novo mutations in intellectual disability and ASD providing
further support for overlapping genetic risk, and presumably pathophysiology.
The extent of pleiotropy may be surprising, but it is consistent with other disciplines
which generally show high rates of comorbidity and lack of specificity in disease
associations. Pleiotropy can also facilitate understanding of disease mechanisms by
identifying novel intermediate phenotypes on the causal chain. For example, many risk
factors for type 2 diabetes are pleiotropic for body mass and are likely to mediate
their effects on the former through the latter. Of course in type 2 diabetes, there
was a strong hypothesized link with body mass, and exploiting pleiotropy is not going
to be quite so simple in psychiatry. Rather, finding pleiotropic links may require
deep, and largely speculative, mass phenotyping. Deep phenotyping refers to the collection
of large amounts of information on individuals beyond categorical diagnostic status,
eg, cognitive and neuroimaging data, or given recent findings (see above) immune function.
It is early days with respect to this type of work, but promising results have already
been demonstrated for the first GWAS-identified schizophrenia risk gene, ZNF804A,
9
which have been associated with cognition, clinical subdimensions, and brain phenotypes.
39–42
Small samples limit the robustness of the conclusions that have emerged so far from
this sort of research.
Exploiting pleiotropy is likely to require very large cohorts, although much smaller
samples ascertained for the same relatively high penetrance mutation, eg, a CNV, are
also likely to be highly informative. The considerable genetic overlap between disorders
also suggests it will be important that much of this work should be undertaken across
current diagnostic boundaries (and also include unaffected individuals given we all
carry large numbers of common risk alleles) in order to characterize the impact of
genetic variants.
From Genetics to Treatment
Antipsychotics are only partly effective for the positive symptoms of schizophrenia
and do little to alleviate the negative symptoms or cognitive deficits. No novel class
of drug for schizophrenia has emerged since the 1960s,
43
presumably due to the limited understanding and insight into the molecular underpinnings
of schizophrenia. It is therefore some cause for optimism that the recent PGC-SCZ
publication implicates genetic variants at the locus that contains DRD2, the gene
that encodes the D2 dopamine receptor, the target of all known effective antipsychotics.
The association of genetic variants at this gene can be seen as a “proof of principle”
finding in reverse, supporting the hypothesis that pharmacological manipulation of
proteins highlighted by common variants can have substantial therapeutic effects regardless
of the fact that the genetic effect size on risk is meagre.
44
It also suggests that other loci implicated by GWAS harbor the potential to guide
effective drug discovery for schizophrenia, although we need better knowledge of the
actual risk variants, and the proximal and distal functional consequences of those
variants in the pathway to disease.
Furthermore, each gene implicated in the development of schizophrenia does not work
independently. If the gene itself is an unsuitable drug target, there is the potential
for manipulation via interacting proteins or other members participating in the same
biological pathway. Therefore, it is important to fully characterize these risk genes
and the cellular process, pathways, and phenotypes which they regulate. Translating
just one associated locus into an effective treatment for schizophrenia would amply
justify the contribution made by tens of thousands of patients with the disorder that
have made the recent genetic advances possible, and the investments of effort and
resource by researchers and funding bodies, governmental and charitable.
Conclusions
The recent PGC-SCZ publication is a landmark in the process of identifying genetic
risk variants for schizophrenia, but it is not one that indicates the end of the journey.
As sample sizes and power increases, and with the additional detail provided by sequencing,
discovery is likely to accelerate. However, even now, the genetic findings provide
the basis for a wealth of research, from molecular and cellular investigations through
to defining novel clinical classifications. It is too early to know the implications
for new treatments, although some of the associations may immediately inspire potential
therapeutic targets. What would seem inevitable is that if as yet undiscovered treatments
for the disorder are possible, and it seems unlikely that the only effective treatment
has already been discovered, the opportunities for better understanding of pathogenesis
that flow from the genetic data must surely accelerate their discovery. For this to
happen, scientists of many disciplines must move away from the comfort of some of
the “old favourites,” for which the evidence is much less secure and devote their
energies and intellect in pursuit of the new findings that are well grounded in evidence.
Looking beyond schizophrenia, the findings definitively demonstrate the power of genetics
can be harnessed for psychiatric phenotypes despite their presumed high heterogeneity,
the absence of tests to validate diagnosis, and uncertain biological validity.
Funding
The work at Cardiff University was funded by Medical Research Council (MRC) Centre
(G0800509) and Program Grants (G0801418); European Community’s Seventh Framework Programme
(HEALTH-F2-2010-241909, Project EU-GEI).