Editorial Article: Identification of Schizophrenia Risk Factors Using Advanced Genetic Analyses at Harvard Medical School

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Steven McCarroll, Associate Professor in the Department of Genetics at Harvard Medical School, USA and colleagues, recently published a study in Nature that reported an association of the major histocompatibility complex (MHC) locus with schizophrenia risk. Copy number variations (CNVs), other complex forms of structural variation and paralogous sequence variations that resulted in increased expression of complement component 4 (C4) were particularly associated with an increased schizophrenia risk.

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Schizophrenia is a multifactorial, neuropsychological disorder known to have a genetic component. More than 100 loci in the human genome contain genetic markers that associate with risk of developing schizophrenia, including the MHC locus, which spans several megabases of chromosome 6. This has made identification of functional candidate risk alleles more difficult. However, McCarroll and colleagues were able to identify a strong association of C4 alleles with schizophrenia risk by analysing genomes from large populations of patients and healthy controls using droplet digital PCR (ddPCR) to identify structural haplotypes of the C4 genes.

Schizophrenia risk from complex variation of complement component C4

Human C4 exists as two functionally distinct genes, which vary in structure and copy number. Using droplet digital PCR, the McCarroll group developed a method to identify four structural haplotypes of C4 that were defined by the presence or absence of a human endogenous retroviral insertion and by their copy number. Further assessment of gene expression levels in post-mortem brains from schizophrenia patients and healthy controls revealed that structural haplotypes that predisposed to elevated C4 expression were more strongly associated with increased schizophrenia incidence and risk. 

C4 is a critical component of the complement cascade, which has been implicated in the elimination or ‘pruning’ of synapses and immunohistochemistry showed C4 localized to synapses. These data suggested a role for aberrant synaptic pruning, mediated by altered complement expression, in the pathology of schizophrenia.

Application of ddPCR for CNV and gene expression analysis

The development of a quantitative assay that could reliably differentiate between the long and short variants and determine copy numbers of the C4 alleles was crucial to this study and achieved through the use of Bio-Rad’s QX100 Digital Droplet PCR system. Although based on similar principles as real-time quantitative PCR, ddPCR technology is far more precise and sensitive, as it does not rely on relative measurements. DNA samples are partitioned into approximately 20,000 droplets using a microfluidic droplet generator and amplification is performed within each droplet, using primers and fluorescent probes specific to the target sequence. Fluorescent droplets are then scored as positive for the target sequence, whilst droplets lacking fluorescence are scored as negative, providing an absolute quantification of target DNA within the sample. These data were then used to connect structural variations in C4 genomic structure with RNA expression levels and association with schizophrenia genetic risk.  

Future perspectives

These findings, together with other published data, further implicate excessive or inappropriate synaptic pruning as an underlying pathology of schizophrenia. Aberrant complement activity, which is involved in pruning mechanisms, may correspond to the reduced numbers of synapses found in the brains of affected individuals. This study is one of the few published that is able to connect genome-wide association study (GWAS) associations with functional alleles, and may point to a future strategy for effective investigation of the contribution of other GWAS identified risk factors to schizophrenia and other brain diseases.

This research was a collaboration between researchers at Harvard Medical School, Broad Institute of MIT and Harvard, Boston Children’s Hospital and Massachusetts General Hospital and the Schizophrenia Working Group of the Psychiatric Genomics Consortium, and represents a big step forward in understanding the pathogenic mechanisms underlying this complex disease. 

Visit the McCarroll lab website to find out more about their research and read the Nature research paper here.  

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