Cytogenetics and Gene Discovery in Psychiatric Disorders

Cytogenetics and gene discovery in psychiatric disorders

BS Pickard1 ABSTRACT 1 The disruption of genes by balanced translocations and other rare germline JK Millar chromosomal abnormalities has played an important part in the discovery of 1 DJ Porteous many common Mendelian disorder genes, somatic oncogenes and tumour WJ Muir2 supressors. A search of published literature has identified 15 genes whose DHR Blackwood2 genomic sequences are directly disrupted by translocation breakpoints in individuals with neuropsychiatric illness. In these cases, it is reasonable to 1Medical Genetics, School of Molecular and hypothesise that haploinsufficiency is a major factor contributing to illness. Clinical Medicine, Molecular Medicine Centre, These findings suggest that the predicted polygenic nature of psychiatric University of Edinburgh, Western General illness may not represent the complete picture; genes of large individual Hospital, Edinburgh, UK; 2Psychiatry, School of Molecular and Clinical Medicine, University of effect appear to exist. Cytogenetic events may provide important insights Edinburgh, Kennedy Tower, Royal Edinburgh into neurochemical pathways and cellular processes critical for the develop- Hospital, Morningside Park, Edinburgh, UK ment of complex psychiatric phenotypes in the population at large. The Pharmacogenomics Journal (2005) 5, 81–88. doi:10.1038/sj.tpj.6500293 Correspondence: Published online 25 January 2005 Dr BS Pickard Medical Genetics, School of Molecular and Clinical Medicine, Molecular Medicine Keywords: cytogenetics; chromosomal translocation; haploinsufficiency; psychia- Centre, University of Edinburgh, Western tric illness; candidate gene; complex genetic disorders General Hospital, Crewe Road, Edinburgh EH4 2XU, UK. Tel: þ 44 131 651 1056/1043 Fax: þ 44 131 651 1059 E-mail: [email protected] THE GENETIC CONTRIBUTION TO PSYCHIATRIC ILLNESS IS COMPLEX Twin and family studies have made it abundantly clear that powerful genetic risk factors act in concert with environmental factors to give rise to psychiatric illnesses such as schizophrenia, bipolar affective disorder and unipolar depression.1 However, the precise nature of this contribution is not well understood. The number of genes contributing to illness susceptibility has yet to be defined either at the individual or population levels and considerable debate remains as to whether ‘oligogenic’ or ‘polygenic’ is the most accurate term for these disorders. Moreover, at the level of a hypothetical causative gene, it is not certain whether a single mutation or multiple independent mutations (allelic heterogeneity) will be found in the case population.2 After a period of confusing and unreplicated results, the principal methodol- ogies used to explore this genetic contribution, namely family linkage studies and case–control association studies, have begun to yield promising findings. The rather vaguely defined chromosome loci identified as housing risk factors have now, with the advent of the Human Genome Project data, been translated into a number of candidate genes. These include neuregulin (NRG1), dysbindin (DTNBP1), G72, COMT and RGS4 (recently reviewed in O’Donovan et al3 and Harrison and Weinberger4). All these are potentially interesting functional Received 8 April 2004 candidates, but their involvement in psychiatric illness has yet to be confirmed. Revised 12 October 2004 Accepted 19 October 2004 Biased distributions of single-nucleotide polymorphisms (SNPs), or groups of Published online 25 January 2005 SNPs (haplotypes), have been observed for the genes in case–control studies. Cytogenetics and gene discovery in psychiatric disorders BS Pickard et al 82

However, these polymorphisms are unlikely to be the actual abnormalities may still be underestimated within a given disease-causing mutations but rather present in linkage condition, especially when those previously cryptic to disequilibrium with them. In only one instance, the standard microscopic methods are included. In a recent ZDHHC8 gene, has an associated SNP been shown to study of aniridia, 30 of 77 patients studied were found to have a defined effect on gene function.5 In addition, there have a variety of chromosomal abnormalities including are marked differences between studies in the details of deletions and translocations around the Wilms tumour the associated markers or haplotypes implying allelic locus on 11p.12 In addition to the germline chromosome heterogeneity at individual gene loci: particularly between rearrangements described above, somatic rearrangements in different geographical populations, as is the case in simple tumours and leukaemias have aided the identification of Mendelian disorders. many new oncogenes and tumour suppressors.13 In summary, the detection of pathological gene mutations Although a full description is outside the remit of this lags behind the identification of candidate genes and review, several models have been put forward to explain the chromosomal loci in psychiatric illnesses. mechanistic basis of translocation events. These suggest that either particular chromosomal motifs, for example, low copy repeats, or AT-rich palindromes or, alternatively, the CYTOGENETICS AS A TOOL FOR HUNTING DISEASE GENES physical properties of the DNA sequences increase the risk of In the last half-century human cytogenetics has emerged as architectural rearrangements.14–20 Different explanations a distinct discipline following the discovery of the number are likely to be required for random (unique) or conserved and form of the human chromosomal complement.6–8 (recurrent) rearrangements. Within the last decade, it has become possible to translate chromosome position into annotated genomic DNA sequence allowing cytogenetics to proceed from an observa- THE SCOPE OF THE REVIEW tional science to a diagnostic tool and, more recently, a This review focuses on psychiatric disorders and is limited to direct means to isolate disease genes. those cases where apparently simple events, translocations, The advantage of a cytogenetic approach to disease gene inversions or insertions, have been shown to disrupt a gene identification in the postgenomic era is that the chromo- between the first and last exons. Many other examples exist some abnormality is both the physical ‘flag’ by which the of abnormalities where the breakpoint lies outside the candidate gene can be speedily identified and the functional coding portion of the bona fide disease gene (by up to 1 Mb mutation itself. in some cases) but still perturbs function through a position The rarity of these cases is perhaps their biggest drawback effect or the separation of enhancer elements from the to the investigator. Discrete cytogenetic abnormalities, in promoter.21–23 However, these instances have required contrast to numerical chromosomal anomalies such as considerable confirmatory experimentation. For similar trisomies, are rarely found in association with complex reasons, we have not included the contribution of cytoge- disorders such as schizophrenia and the affective disorders. netics to important neurological associates such as mental The reason for this rarity becomes clear when the factors retardation (UK equivalent terminology is learning disabi- involved are considered: the prevalence and the number lity), dup15 (associated with joint laxity and panic disorder) of genes that can give rise to the disorder, the frequency of or genomic disorders that are associated with clear beha- chromosome abnormalities in the population, the size of vioural phenotypes such as Williams syndrome (overfriend- the human genome, the size of the gene loci themselves and liness and anxiety), Smith–Magenis syndrome (self-injury), the efficiency of clinical ascertainment. Assuming an Prader–Willi syndrome (hyperphagia) or chromosome 22 average gene size of 27 kb,9 a genome size of 3 billion bases, deletion syndrome (associated with psychosis).24–28 Neither two breakpoints per chromosome abnormality (eg a typical shall we discuss the important work being carried out on the reciprocal translocation/inversion) and a frequency of such phenomenon of subtelomeric deletions associated with abnormalities in the population of 0.29–0.56%10,11 then, idiopathic mental retardation29,30 as well as autism.31–34 providing the disruption is compatible with life, any given The purpose of the selection in this review is to collate and gene would be cytogenetically disrupted in 3–6 individuals describe the function of genes where one parental copy is in the UK population of B60 million. However, for any definitively damaged and most likely nonfunctional in a specific locus this is probably an underestimate because patient with a major mental disorder. breakpoints are unlikely to occur as a truly random The phenotypes covered in this review have been loosely phenomenon. grouped under the term ‘neuropsychiatric’ conditions or Naturally, such opportunities have been exploited by ‘functional’ disorders with particular emphasis on mental human geneticists. A combination of positional evidence health and cognitive function. A list of the 15 genes from linkage studies and the analysis of rare cytogenetic identified by this strategy together with their various lesions in patients has led to the discovery of the underlying functions and properties is presented in Table 1. Three of genetics of Duchenne muscular dystrophy, retinoblastoma, these genes have been identified in our laboratory in the neurofibromatosis type 1, Wilms tumour, tuberous sclerosis, course of a detailed molecular characterisation of over 12 polycystic kidney disease, aniridia, familial polyposis coli chromosomal rearrangements in individuals with psychia- and fragile-X syndrome among others. The rate of such tric illness (principally schizophrenia). The majority of cases

The Pharmacogenomics Journal Table 1 Neuropsychiatric genes identified through cytogenetic disruption

Disorder Gene Alternative name Domains and function Ref. seq. Chromosome Carriers Association Result Ref.

Asperger syndrome USP6 Ubiquitin-specific Protease component of protein degradation NM_004505 17p13.2 1 de novo No N/A 35 protease 6 pathway Autism NBEA Neurobeachin/ A kinase anchoring protein (AKAP) function, NM_015678 13q13.3 1 de novo No N/A 36,37 BCL8B PH-BEACH-WD40 domains—protein interaction/subcellular localisation Autism ST7 RAY1/FAM4A1 No domains, but mouse/Drosophila/nematode NM_013437 7q31.2 2 Yes, exonic SNPs No 38,39 orthologues identified Autism AUTS2 ARG1 No domains, but mouse orthologue and other NM_015570 7q11.22 2 Yes, exonic SNPs Sugg. 40 related sequences identified Autism SSBP1 Single-stranded Mitochondrial single-stranded DNA-binding NM_003143 7q34 3 Yes, small scale No 41 DNA-binding protein protein 1 Autism/multiple GRPR Gastrin-releasing Gastrin-releasing peptide G-protein-coupled NM_005314 Xp22.13 1 No N/A 42 exostoses/MR/ peptide receptor receptor epilepsy Gilles de la Tourette IMMP2L Mitochondrial inner Mitochondrial inner membrane peptidase NM_032549 7q31.1 1 No N/A 43 syndrome membrane processing polypeptides shuttled into peptidase subunit 2 mitochondrion like Gilles de la Tourette CNTNAP2 Contactin-associated Neurexin family, potassium channel interaction, NM_014141 7q35 3 No N/A 44,45 syndrome/OCD protein-like 2 membrane protein located near axonal nodes of Ranvier disorders psychiatric Pickard in BS discovery gene and Cytogenetics Developmental DYX1C1 Three tetratricopeptide domains—protein NM_130810 15q21 4 Yes, small Mutation 46 dyslexia interaction tal et Speech and FOXP2 Forkhead box P2 Transcription factor, winged helix type NM_014491 7q31.1 1 Mutation Mutation 47–49 language detection development Schizophrenia NPAS3 Neuronal PAS Transcription factor, basic–loop–helix and PAS NM_022123 14q13.1 2 Mutation No 50, 51,52 domain protein 3 domain type detection (Fahr’s) Schizophrenia DISC1/2 Disrupted in DISC1: globular N-terminal, helical C-terminal— NM_018662 1q42.2 29 Several Yes 53 (+affective disorders) schizophrenia 1/2 protein interaction (see text for more detail) Schizophrenia/MR MGAT5 Mannosyl (a-1,6-)- N-linked glycosylation pathway. (NB: unbalanced NM_002410 2q21.2– 1NoN/A54–56 glycoprotein b-1,6- rearrangement—gene dosage changes may 2q21.3 N-acetyl- contribute to phenotype). glucosaminyl- transferase BPAD/MR GRIA3 AMPA3, GLUR3 Ionotropic glutamate receptor, AMPA type NM_181894 Xq25 1 Linkage study No 57,58 BPAD DIBD1 Disrupted in bipolar N-linked glycosylation pathway NM_024740 11q23.1 11 Yes No 59 disorder 1

www.nature.com/tpj ‘Association’ column, indicates if a published association study exists; ‘Result’ column, association study result where carried out; Sugg., suggestive positive association; MR, mental retardation; OCD, obsessive– compulsive disorder, BPAD, bipolar affective disorder. We have collated information on 15 disrupted genes matching the criteria described above. These genes were found using PubMed60 literature searches using the following text queries singly or combined: ‘psychiatric illness’, ‘schizophrenia’, ‘bipolar affective disorder’, ‘autism’, ‘chromosome abnormality’, ‘translocation’, ‘breakpoint’ and ‘cytogenetics’. Overall, the described experimental strategies employed to progress from abnormality to gene principally involved fluorescence in situ hybridisation (FISH) coupled, in some instances, with Southern-based fine mapping of breakpoints. One gene, SSBP1, was promoted to the class of disrupted genes by the identification by the authors of this review, in recently annotated genome sequence releases, of 30 splice variants spanning the breakpoint. References in bold are the primary gene identification papers. 83 Cytogenetics and gene discovery in psychiatric disorders BS Pickard et al 84

studied possessed direct gene disruptions or breakpoints There are more cases of schizophrenia, autism and close to good candidate genes, which suggests that the asperger syndrome in Table 1 than bipolar disorder and chromosomal rearrangements are the underlying cause of unipolar depression. In a recently collated list of published the psychiatric conditions in these individuals. In this chromosome abnormalities associated with psychiatric ill- article, we aim to identify common themes linking the ness, few of which have been studied at the molecular level, function of individual genes whose disruptions, albeit 22 cases match our criteria for selection based on phenotype extremely rare events, may give clues about the cellular and rearrangement type.64 Of these, diagnoses of schizo- processes and neurochemical pathways critical to complex phrenia and related conditions occurred far more com- psychiatric phenotypes. monly than bipolar/unipolar disorders. Moreover, a network of Scandinavian cytogenetics laboratories has compiled data summarising 71 739 postnatal cytogenetic tests, of which CHANCE FINDINGS OR CAUSAL EVENTS? 216 showed balanced chromosome rearrangements.65 Of When considering the biological significance of these data, these, five were associated with psychotic mental illness and we must ask if the chromosomal abnormalities and disrupted three with autism. We have also observed a similar genes observed are chance findings or causal to the diagnostic bias in unpublished cytogenetic cases under phenotypes of the carriers. Support for a causal involvement study in our laboratory. The reasons for this observed bias in the disorder can be provided by establishing significant are not clear but there are two strong possibilities. There linkage between the chromosome abnormality and illness in could be an ascertainment bias due to the more frequent an extended family. Unfortunately, to date, access to referral of patients with schizophrenia or autism for necessarily large pedigrees has been restricted to the t(1;11) cytogenetic analysis and greater resources for research into family in which DISC1 is disrupted (see ‘carriers’ column in those disorders. The bias may also be due to the underlying Table 1). In this case, an LOD score of 7.1 confirmed linkage genetic aetiology of these conditions. We predict that most between the translocation and illness in this family.61 instances of direct disruption will result in a decrease in Alternatively multiple independent observations of a rear- transcription of the gene rather than the production of rangement affecting a particular gene as reported for SATB2 transcripts encoding aberrant forms of the protein because in cleft palate would be good evidence of its involvement.62 these would be liable to degradation by nonsense-mediated Owing to the large number of genes presumably involved and decay. We can postulate that schizophrenia and autism, in the small number of reported rearrangements, it is perhaps contrast to bipolar affective disorder or unipolar depression, not surprising that independent reports of disruptions to the are more frequently associated with gene haploinsufficiency. same gene have yet to be reported for neuropsychiatric By analogy, a simple view of cancer-causing gene mutations conditions. However, this also means that we have not yet is that they fall into two groups: gain-of-function in the case fully exploited cytogenetics as a gene-finding approach. of oncogenes and loss-of-function in the case of tumour It is important to investigate whether these disrupted suppressor genes. Indeed, a derived classification of suscept- genes are unique, case-specific, findings or whether they ibility genes into ‘psychogenes’ and ‘psychosis suppressor’ contribute more widely to illness in the general population genes has already been proposed for psychiatric condi- by alternative mutation mechanisms, as was found to be the tions.66 Therefore, cytogenetics may help to identify the case for Duchenne muscular dystrophy and retinoblastoma. ‘suppressor’ type of gene implicated in the aetiology of a A proportion of these disrupted genes may be unique subset of neuropsychiatric conditions. representing a set of genes with the potential when mutated Three cases in Table 1 are patients with mental retardation to cause disease but not represented by mutations at the comorbid with autism, schizophrenia or bipolar disorder. population level. Studies to detect linkage disequilibrium Individuals with mental retardation are more likely to have between disease- and gene-related polymorphisms in the psychiatric illness than the general population67 and to have general population have been performed in a number of the a contiguous gene syndrome, resulting from the hemizygous instances as detailed in Table 1, but statistically robust loss or gain of several genes simultaneously through association studies have not been carried out for all of these microdeletions and duplications. However, Table 1 has genes to assess their population attributable risk. included cases where the genetic lesion appears precisely limited to one gene and yet mental retardation still exists. PHENOTYPES OF TRANSLOCATION CARRIERS One explanation might be that the gene has pleiotropic In major psychiatric disorders, there is often variable action, causing a neuropsychiatric disorder and, indepen- penetrance and a spectrum of phenotypes within a family. dently, mental retardation. Alternatively, gene disruption The t(1;11) family segregates for both schizophrenia and may have a greater effect on gene expression, giving rise to affective psychosis, for example. Such pleiomorphic a more severe form of illness than a point mutation in a phenotypes are increasingly being described within mono- karyotypically normal patient. genic conditions and also for chromosomal deletions with relatively stable disruption boundaries such as the chromosome 22 deletion syndrome. Mouse studies have COMMON THEMES IN GENE FUNCTION replicated this variability, ascribed largely to genetic modifiers With the caveat that it is relatively easy to propose a model elsewhere in the genome.63 placing a candidate gene at the heart of the presumed

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pathophysiology of psychosis when independent knowl- enzymes might act as modifiers of other gene defects. edge of the underlying biology is limited, there are some Indeed, within the CDGs themselves, interactions of mutant general and specific features of several genes described in genes have been shown to modulate the severity and nature Table 1 to support their role in the psychoses. of disease. Recently, another glycosylation enzyme, B3GAT1, It is interesting that a glutamate receptor, GRIA3,is was identified near to, but not directly disrupted by, a present within the list. The glutamate hypothesis, primarily breakpoint in a family with schizophrenia-like psychosis applied to schizophrenia and related psychoses, was initially and a t(6;11)(q14.2;q25) translocation. The proposed asso- based on the observation that administration of phencycli- ciation has been complicated by the emergence of another dine and ketamine, antagonists of the NMDA subtype of gene transcript, FLJ37762, between this gene and the 11q25 glutamate receptor, can produce schizophrenia-like states.68 breakpoint.75 The hypothesis has been strengthened by recent functional and behavioural data from transgenic knockdown experi- ments on an NMDA receptor gene (Grin1) in mice.69 Loss-of- DISC1 AS A MODEL FOR FUNCTIONAL AND GENETIC function mutants of glutamate receptors by cytogenetic ANALYSES disruption might be expected to have similar phenotypes. DISC1 was identified as a likely genetic risk factor for Overall, with the exception of the two receptors, GRIA3 psychiatric illness from studies of a single large Scottish and GRPR, it is possible to group the candidates loosely into family multiply affected with severe psychiatric illness, a class of genes encoding proteins involved in processes with including schizophrenia and affective disorders. In this multiple inputs (substrates, interacting proteins, DNA-bind- family, a balanced (1;11)(q42;q14) translocation cosegre- ing sites) or outputs (products, interacting proteins, tran- gates with psychosis, generating a highly significant scriptionally regulated genes). NBEA, CNTNAP2, DYX1C1 maximum LOD score of 7.1.61,76 Thus, in this family, and DISC1 proteins may all mediate interactions with other translocation-induced gene disruption is causal, and a proteins, perhaps participating in the formation of localised genetic locus of major effect is responsible for the psychosis functional complexes. The three DNA-binding proteins, in family members. On chromosome 1, the translocation NPAS3, SSBP1 and FOXP2, are likely to have many targets. directly disrupts two overlapping genes, DISC1 and DISC2, The proteasome complex component, USP6, the mitochon- and it is therefore likely that altered expression of DISC1 drial peptidase, IMMP2L, and the two enzymes within the and/or DISC2 underlies disease pathology in translocation N-linked glycosylation pathway, discussed in more detail carriers.53,77 below, are also examples of proteins with many potential The DISC region has subsequently been highlighted as a substrates. It can be predicted that mutation of such likely susceptibility locus for psychiatric illness by a number focal points or ‘hubs’ of activity within a ‘scale-free’ of independent genetic studies, with the most significant biological network will be particularly harmful to cellular results generated by analysis of individuals diagnosed with activity and function.70,71 schizophrenia in Finland. A genome-wide linkage study and subsequent follow-up studies using dense marker sets identified a peak of linkage with a polymorphism that lies RARE DISRUPTIONS MAY SIGNPOST COMMON within DISC1 itself, with a maximum LOD score of 3.2.78 AETIOLOGIES Several associated haplotypes have been identified within The goal of identifying genes from rare breakpoints is to the same population.79 Other reports of linkage to this understand the biological processes and pathways under- region include studies of North American, Taiwanese, British lying the common forms of the disorder and to identify and Icelandic families diagnosed variously with schizophre- other constituent genes that show a measurable effect in the nia and affective disorders.80–82 Both of these clinical general population. phenotypes are expressed in the family segregating for the An example of breakpoints uncovering a common path- translocation. The DISC locus, although initially identified way comes with the identification of MGAT5 and DIBD1 as as a major risk factor only in a single family, is now emerging disrupted genes in schizophrenia comorbid with mental as a susceptibility locus for schizophrenia and affective retardation and bipolar affective disorder, respectively. disorders in several different populations. These two genes participate in N-linked glycosylation, a Little is known of DISC1 and DISC2 function. Most studies post-translational process involving the modification of have focused upon DISC1 and all information to date secreted and membrane proteins through the addition of indicates an important role for this protein in the adult and oligosaccharide moieties. Several other genes in this path- foetal central nervous system. In particular, DISC1 is way have been shown to be causative for a varied group of expressed in the hippocampus and limbic regions of the disorders collectively known as congenital disorders of adult brain83 and its possible involvement in neurodevelop- glycosylation or CDGs.72,73 These, predominantly recessive, mental processes has been highlighted by its interaction disorders are often characterised by central nervous system with other proteins including FEZ184 and Nudel.85–88 FEZ1 is phenotypes, suggesting that the brain is particularly suscep- thought to function in axon outgrowth and is associated tible to perturbations in protein glycosylation.74 With a with DISC1 at axon growth cones, suggesting a requirement large set of target proteins predicted to be affected by these for DISC1 in neurite extension, a process critical for synaptic disorders, it is not hard to see how N-linked glycosylation connectivity.84 Interaction between DISC1 and Nudel

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suggests an additional significant neurodevelopmental role breakpoint genes correspond to the former, then it is clear because Nudel complexes with LIS1, a molecule required for that much useful biological information is waiting to be neuronal migration and cortical lamination. LIS1 mutations gained from their study. are a major cause of lissencephaly, a neurodevelopmental condition with variable degrees of cognitive impairment. REFERENCES 1 Berrettini WH. Genetics of psychiatric disease. Annu Rev Med 2000; 51: 465–479. IMPLICATIONS FOR THE GENETICS OF NEUROPSYCHIA- 2 Neale BM, Sham PC. The future of association studies: gene-based analysis and replication. Am J Hum Genet 2004; 75: 353–362. TRIC ILLNESSES 3 O’Donovan MC, Williams NM, Owen MJ. Recent advances in the In light of the data presented here, the authors suggest that genetics of schizophrenia. Hum Mol Genet 2003; 12: 125–133. karyotype screening should be implemented as a potentially 4 Harrison PJ, Weinberger DR. 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