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Genetic linkage analysis provides a powerful approach with which to elucidate the underlying genetic factors in inherited disorders. Linkage analysis serves to demonstrate the existence of a major locus to clarify the observed pattern of inheritance of a particular trait within a family (Alsobrook and Pauls, 1998). The objective is to establish the co-segregation of polymorphic genetic markers of known chromosomal location with the disease phenotype within a family unit or pedigree (i.e., the non-random sharing of marker alleles between affected members of each family).

After initial localisation of a putative genetic marker for a disease susceptibility locus by means of linkage analysis, it is possible to type additional polymorphic markers in order to generate a high resolution map of the relevant genomic region surrounding the disease-causing gene (Alsobrook and Pauls, 1998). Thereafter, the location of the causative genetic variant(s) may be inferred by means of, for example, fine mapping procedures.

Reference No. proband

families

No. control Mode of transmission most compatible families (i.e models that were not rejected) Nicolini et al. (1991) 24^a 0 Autosomal dominant or recessive model

107 (418 1^st degree

relatives; 1121 2 Autosomal dominant model, with possibility of polygenic inheritance Cavallini et al. (1999) nd

degree relatives)

0

All models except mixed model were rejected for whole sample;

Unfortunately, linkage analysis has had limited success in detecting genes involved in psychiatric disorders due to their complex, multifactorial nature. Nowadays, searching for linkage between candidate loci and complex psychiatric disorders involves conducting a whole genome screen (Risch and Merikangas, 1996; Collins et al., 1997). This strategy involves screening the entire genome using evenly spaced genetic markers, to allow identification of regions of potential linkage. The regions are recognised by calculating an appropriate linkage statistic at each position along the genome, and identifying those regions in which the statistic indicates a significant deviation from what would be expected under the rule of independent assortment (Visscher and Haley, 2001). A peak is produced where the test statistic exceeds a predetermined significance threshold caused by one or more loci that may influence the trait. A second stage of mapping may then be applied to fine-map the linked chromosomal region.

In genome-wide linkage studies, either sib-pairs or families can be used. This method has met with much success in identifying susceptibility loci for psychiatric disorders, including social phobia (Gelernter et al., 2004); simple phobia (Gelernter et al., 2003); schizophrenia (Stefansson et al., 2002); irritable bowel syndrome (Hugot et al., 2001; Rioux et al., 2001;

Stoll et al., 2004); bipolar disorder (Middleton et al., 2004), panic disorder (Knowles et al., 1998) and panic syndrome (Hamilton et al., 2003).

To date, one OCD genome-wide linkage study has been published (Hanna et al., 2002). This study was conducted using seven probands (who ranged in age from six to 17 years), whose age at onset was between three and 14 years. Fifty-six individuals from the seven families were initially genotyped with 349 microsatellite markers spaced at any average of 11.3 centiMorgan (cM). A region on the telomere of chromosome 9p met with the criterion for suggestive linkage (using the dominant model, the logarithm of odds score [LOD score] was equal to 2.25). The investigators also observed weak evidence for linkage between OCD and regions on chromosomes 2q, 6q, 16q, 17q and 19q.

Hanna et al. (2002) then followed up their initial findings on chromosomes 2q, 9p and 16q (i.e., those regions with dominant LOD scores of greater than 1) by genotyping 24 additional markers at an average spacing of 1.6 cM in the original 56 subjects, as well as in 10 additional family members from one of the families. They found the region that displayed the strongest evidence for linkage (with a LOD score of 1.97) was 9p24. Interestingly, there has been a

report of 9p monosomy in a patient with TS (Taylor et al., 1991) and a region on chromosome 6p has also been found to be associated with TS in an Afrikaner subject sample (Simonic et al, 1998), providing further support for the proposed genetic link between the two disorders.

Recently, the evidence for linkage on 9p24 has been replicated (Willour et al, 2004) using 50 pedigrees (consisting of 193 subjects) from the John Hopkins OCD family study (Nestadt et al., 2000[a]). Here, genomic DNA samples from all individuals were genotyped for 13 microsatellite markers spanning a distance of 19 cM across 9p24, with an average inter-marker spacing of 15 cM. The narrow phenotype model for OCD, combined with dominant parameters and penetrance of 0.5, produced the strongest findings in the study (LOD score = 2.26), with the strongest nonparametric findings also observed under a narrow phenotype model (nonparametric linkage signal [NPL] = 2.52; p = 0.006).

It is notable that the original LOD score for chromosome 9p24 (LOD = 2.25) obtained by Hanna et al. (2002) resembled the LOD score in the study by Willour et al. (2004) very closely. The NPL peak observed by Willour et al. (2004) was situated at D9S1813, which lies only 350kb away from that observed by Hanna et al. (2002) at D9S288, suggesting the location of a susceptibility locus in that region. The 9p24 chromosomal region spans approximately 75 megabases (Mb) and thus contains numerous potential candidate genes that could be investigated for the role they may play in the development of OCD. Veenstra-vanderWeele et al. (2001) conducted a mutation screen of the gene encoding the neuronal and epithelial glutamate transporter (situated approximately 350cM centromeric to the NPL peak identified by Willour et al. [2004]). They reported eight exonic synonymous single nucleotide polymorphisms (SNPs) that did not appear to alter the functioning of the gene. Using a family-based association study, the investigators observed no statistically significant association between two of the intronic polymorphisms and OCD.

Nonetheless, the results from both the genome scans are noteworthy, and a collaborative effort is underway to collect almost 300 sib-pair and multiplex families, in an attempt to improve the power of the previous studies, and replicate the results (Willour et al., 2004).

Limitations of using pedigree linkage analyses in complex psychiatric disorders

In OCD, as for all psychiatric disorders, the underlying mechanisms, degree of penetrance and mode of inheritance remain unknown (Crowe, 1993). The environmental contribution to the aetiology of the disorder, diagnostic misclassification, and the epistatic and additive interactions between genes conferring susceptibility to mental illness may also limit the success of identifying susceptibility genes utilising linkage strategy (Crowe, 1993; Nothen et al., 1993; Souery et al., 2001; Stoltenberg and Burmeister, 2000; Ghosh and Schork, 1996).

Moreover, identifying families with multiple affected individuals poses a problem – there still seems to be an amount of stigma attached to being diagnosed with OCD; consequently a large proportion of affected individuals prefer to keep their affliction hidden from family members.

The nature of the susceptibility allele further weakens the power of linkage analysis for complex disorders, since the susceptibility allele is neither necessary nor sufficient to produce the clinical phenotype (Greenberg, 1993; Hodge, 1993), but may simply increase the chances that the allele-carrier will develop the disorder. Therefore, a proportion of the patients will not possess the associated allele, in which case the “susceptibility” allele may not necessarily co-segregate with the disorder (Nothen et al., 1993; Propping et al., 1993).

Overall, it has been found that association designs have a greater power to detect disease alleles in disorders with mild risk factors (with a genotypic risk ratio [GRR] < 4) than in comparably sized linkage analyses (parametric and non-parametric). This is because for diseases with a GRR <2, unrealistic family sample sizes (in excess of 2500) are required to achieve the same power as for association studies (Risch and Merikangas, 1996; Risch and Botstein, 1996).