Many neuropsychiatric developmental disorders, including schizophrenia, autism spectrum disorders (ASD) and Tourette’s syndrome, may be the consequence of subtle alterations in the overall scheme of CNS development. This is likely to be caused by a combination of a number of gene variants as well as environmental factors, although in rare cases a single gene variant of strong effect may be sufficient to cause altered development and thus illness. Recent advances in genetics and developmental neurobiology using animal models have unveiled in astonishing depths the fundamental history of CNS development (see the article of JLR Rubenstein in this issue). The development of gene knockout and transgenesis in mouse, transplantation experiments in amphibians and avian embryos, single cell ablation and RNA interference in nematodes and fruit flies, and sophisticated cell culture techniques in a variety of species including human have revealed common laws guiding the morphogenesis and cellular differentiation of the vertebrate CNS, and particularly its most complex portion, the brain. These fundamental studies focus on the commonalities of neural development amongst different organisms, but are most likely insufficient to understand the developmental origin of psychiatric illnesses.
The characteristics that distinguish the recently evolved primate and human brain from other mammalian brains include the proportionally larger growth of the cerebral cortex, the diversification of cortical area maps and a much more extensive degree of connectivity (Rakic, 1995
). It can be argued that these differences in scale and complexity have driven an increase in size of neurons, a larger metabolic demand and an increased proportion of glial cells. Another important aspect of the human brain is its diversity from one individual to another. Not one human is identical to the other with regard to the location of sulci and gyri on the cortical surface, the pattern of cortical area activation in response to stimuli, and other characteristics of the neural network. The much greater variation in both morphology and connectivity amongst human brains is a great challenge for investigators that wish to draw statistical inferences; but it can be considered an important clue for understanding the basis of normal and less typical cognitive functions.
Mirroring the diversity and degree of variation in the physical characteristics of the human brain is the variation found in genomic sequence, when comparing individual human genomes (Kim et al., 2008
, McCarroll et al., 2008
). This genomic variation is to be found in their complement of SNPs (single nucleotide polymorphisms) affecting about 0.1% of the total genomic sequence. In addition to this it has become clear over just the last few years, with the advent of novel genomics technology and also the completion of additional human genome sequences that the degree of variation between two individual genomes is even larger than what can be accounted for by SNPs. The nature of this variation is very complex: in addition to SNPs each genome contains an abundance of copy-number-variation and structural variation (CNV and SV). CNVs consist of all variations leading to changes in amount of genomic material such as deletions and duplications, whereas the term SV indicates other types of structural changes, such as insertions, translocations and inversions. It is now clear that entire blocks of sequence in size from less than 1 kb to several millions bp have been deleted, duplicated, inserted, translocated or inverted in the human genome (Hurles et al., 2008
, Korbel et al., 2007
, Levy et al., 2007
) (). The methodology for efficiently identifying CNV/SV in DNA samples is still evolving and not yet as robust or inexpensive as SNP genotyping. The average number of such CNV/SV per individual is variously estimated between 700 and 1400 depending on the methods chosen for analysis and the ethnicity of the subject (Park et al., 2010
, Conrad et al., 2010
). In total, CNV/SVs may alter the coding potential of at least 5% of the known genes. A substantial fraction, but by no means all, of their impact on disease can be estimated by analysis of SNPs in linkage disequilibrium with the CNVs.
Extensive structural variation in the human genome
CNV/SVs are incompletely assessed by commercial SNP arrays and only recently have studies begun to explore them as a potential cause of complex traits, including neuropsychiatric disorders. These CNV/SV are scattered all over the genome, although “hot spots” have been identified, i.e., an 8-megabase (Mb) region in chromosome 22q11.2 and an 18-Mb region at 7q11 (Korbel et al., 2007
) (). These two hotspot regions, for example, harbor deletions in two developmental neuropsychiatric disorders of genomic etiology, velocardiofacial syndrome (VCFS) and William-Beuren Syndrome, respectively. Patients with VCFS have a high frequency of learning disorders, ASD and schizophrenia. As mapping studies continue the total extent and degree of complexity of human genomic variation is being unveiled. In parallel to this, an increasing number of studies link CNV/SV to phenotypic effects. Disease-associated CNV/SVs detected so far include both rare variants with large effect and common variants with more modest effect sizes, but carried by a large proportion of the population (Manolio et al., 2009
). Rare CNV/SVs include deletions/duplications at 16p11.2, associated with ASD and idiopathic mental retardation, and deletions at 1q21.1, 15.q13.3 and 22.q11.2, found in schizophrenia (Sebat et al., 2007
, Weiss et al., 2008
, Stefansson et al., 2008
). An increase in the occurrence of de novo
large deletions has been reported in individuals withASD (Christian et al., 2008
, Sebat et al., 2007
). Common CNVs, which tend to be smaller in size, are under-ascertained by the current methods. Thus, human genomic variation is widespread and varied and has to be taken into account when trying to understand complex phenotypes, both in terms of causative and modifying variation events, if indeed a complex condition is caused not by a single genetic event of strong effect but a combination of variants each with small effect. The large degree of interindividual variability, mostly rare, raises the crucial question of what may be the best approach for determining which of the multitude of genomic sequence variants carried by an individual is responsible for a given phenotype, especially if the functional consequences of such variants at the protein level are not known. Genome-wide association studies have tended to focus exclusively on statistical evidence, but the assessment of the significance of human genomic variants for disease is going to be difficult on statistical grounds alone, and we need to pay more attention to biology in deciding which genetic variants to pursue for diagnostic and treatment purposes.
Recent studies suggest that gene transcripts expressed in the developing human brain encompass a much larger set of mRNA variants and splice patterns, not found at corresponding stages of animal brain development (Johnson et al., 2009
). Unfortunately, how human genetic variation leads to morphological and thus functional variation, the extremes of which may represent mental disorders, is extremely difficult to investigate. It would require the ability to follow neural development in specific individuals at the cellular and systems level, and correlate the particulars of this development to the underlying structure of their genome. The experimental analysis of human neural development in its relation to differences in genetic activity is hampered by seemingly insurmountable challenges. These challenges have so far impeded a rigorous exploration of gene transcripts that are specifically expressed at specific stages of human brain development.
The derivation of iPSCs from skin or other differentiated somatic cells might allow the study of human neural development for individual genomes, albeit in vitro. There is a potentially large impact of this type of studies for psychiatry, neurology, and psychology, if, indeed, they will permit study on how individual, natural genetic variation affects neurodevelopment and how genetic variation is linked to individual differences in brain function and behavior. The success of this approach will depend to a large extent on our ability to recapitulate in vitro the biological steps that enable an embryonic stem cell to differentiate into neurons and glia from specific regions of the CNS. If, using patient-specific iPSCs, we can reproducibly recreate key milestones of neural differentiation in vitro, leading to a highly coordinated generation of specific repertoires of neuronal cell types, we could, in principle, understand the molecular programs of development that may underpin specific disease phenotypes in human ().
Integrated data analysis using patient-specific iPSCs
Another question that could be approached using the iPSC technology is that of how, given the substantial similarity in gene number between mammalian species, the substantial differences in brain development amongst mammals are encoded at the genomic level. It seems possible that differences in sequence of non-coding areas of the genome, and therefore in gene regulation, might be a crucial component of such differences. With the term “regulation” we refer here not only to the functional effects of specific transcription factors bound to their enhancers, and thus the regulation of the amount of mRNA expression in space/time, but also the incompletely understood process of alternative splicing of a single mRNA into different types of transcripts. Regulation at the level of control of the rate of translation of specific mRNAs is a comparably important aspect of regulation of protein production that has been much less extensively studied in neural tissues.
Another important component to consider is the regulation of the chromatin due to histone modifications and the methylation of cytosines in the DNA, i.e., epigenetics. The development of iPSCs would enable us to study regulatory processes that establish the dynamic gene networks driving the differentiation of a particular cell type at a particular time, whether determined by DNA sequence variants, mRNA variants, or altered states of chromatin (). Hence, iPSCs promise to offer a unique opportunity to begin to understand the direct biological consequences of human gene sequence variation as it applies to the structure and development of the CNS. Importantly, the environmental, hormonal and toxic effects on the differentiation dynamics and related gene expression trajectories can also be explored.