Biased Homozygous Haplotypes Across the Human Caveolin 1 Upstream Purine Complex in Parkinson’s Disease

One hundred and forty-one unrelated subjects receiving the diagnosis of PD were included in the study. Those cases included late-onset (n = 112) and early-onset PD (n = 29). The average age of onset in the late-onset group was 55 years and in the early-onset group at 30 years. The diagnostic criteria for PD were based upon the UK Parkinson’s disease Society Brain Bank Clinical Diagnostic Criteria. The control samples included 150 newly collected individuals and 610 individuals from the previous studies (Heidari et al. 2011). The term “control pool” is herein used when the summation of the controls studied to date is considered. Cases and controls were matched based on age, gender, and ethnicity. All participants provided informed consent. Different haplotypes of the CAV1 purine complex were PCR amplified and cloned into basic pGL3 vector (Promega UK) as previously described (Heidari et al. 2011; Heshmati et al. 2009). Human neuronal cell line LAN-5 was cultured for expression analysis and transfected with FuGENE HD (Roche Biochemicals, Germany), as previously described (Heidari et al. 2012b),

Overall, 20 haplotypes were homozygous in patients with neurodegenerative disorders, which contained motif lengths nonexistent in the control pool (Fisher exact p < 1 × 10⁻⁶) (Table 1). We compared the expression activity of the disease vs. control haplotypes in human LAN-5 neuronal cell line (Fig. 1). Significant difference in gene expression was observed in the PD homozygous haplotypes vs. control homozygous haplotype (Table 3). The disease haplotypes significantly increased promoter activity comparing with the control haplotype (p < 9 × 10⁻⁶). The amount of gene expression did not correlate with the length of the haplotypes.

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Table 3

Luciferase analysis of four haplotypes across the human CAV1 upstream purine complex in human neuronal cell line, LAN-5

Haplotypes	Firefly	Renilla	RLU	p valuea
PGL3	23,451	43,451	0.57 ± 0.02
5-11-7	9,621,451	87,077	110.49 ± 3.05	1 × 10−7
5-12-7	1,781,561	56,326	31.63 ± 2.37
5-11-9	2,480,095	43,792	56.63 ± 2.07	1 × 10−7
5-13-9	5,656,841	97,613	57.55 ± 0.06	9 × 10−6

RLU relative luminescence unit

^a t test was used to compare each disease haplotypes (106-, 114-, and 122-bp) with the control haplotype (110 bp)

The alpha-synuclein–CAV1 axis has been suggested to be of role in the pathogenesis of PD in cell line models (Madeira et al. 2011). The purine complex at the 1.5 kb upstream region of the human CAV1 gene is conserved across several species, including macaque, mouse, and guinea pig (Heidari et al. 2011). Over 150 haplotypes encompassing the purine complex, most of them with very low frequencies, have been detected to date. Correlating the haplotypes to disease in the heterozygous status necessitates studying very large numbers of cases and controls. The homozygous haplotype compartment, on the other hand, is more effective in studying the possible implication of each haplotype in disease pathogenesis. For example, the long extreme homozygous haplotypes of 146-, 142-, and 138-bp length were not detected in the control pool in a homozygous status (Table 1). We observed novel homozygous haplotypes across the region that were unique to PD (i.e., they were not detected in other neurodegenerative disorders including AD and MS). Remarkably, in contrast with the late-onset PD homozygous haplotypes that contained one motif length absent from the control homozygous pool, haplotype 5-11-9 contains two motif lengths that are nonexistent in the control homozygous pool and co-occurs with early-onset PD.

We also observed haplotypes that overlap in AD and MS. Pooling of the neurodegenerative (i.e., PD, MS, and AD cases) (Table 1) vs. non-neurodegenerative disorders and disease-free controls revealed significant bias in the homozygous compartment in neurodegenerative disorders. The range and variety of the homozygous haplotypes were significantly more restricted in the controls. Twenty haplotypes were specifically homozygous in the neurodegenerative disorders, which contained motif lengths nonexistent in the control pool (Table 1). Only six haplotypes in the middle of the haplotype length (i.e., 106 to 122 bp) were found in the control homozygous compartment. Homozygosity for the disease haplotypes supports additive role for them in the pathogenesis of neurodegeneration. In support of this notion, Lemos and coworkers provide evidence of dominance and the evolutionary accumulation of cis- and trans-effects on gene expression (Lemos et al. 2008). They used chromosome substitution lines of Drosophila melanogaster to show that the majority of the genes that are differentially expressed between two homozygous lines are masked in the heterozygous status.

Homozygosity for exceedingly rare haplotypes in neurodegenerative disorders, which contain motif lengths nonexistent in the control homozygous compartment, provides genetic support for those haplotypes in disease. Functionality of those haplotypes to increase gene expression further strengthens the role of this region in the pathophysiology of PD. An increase in gene expression activity has also been observed in two additional homozygous haplotypes in neurodegenerative patients in human and mouse neuronal cell lines (Heidari et al. 2012a).

In line with our findings, one of the hallmarks of the three neurodegenerative disorders presented in this paper is aberrant increase in CAV1 gene expression (Hashimoto et al. 2003; Kim et al. 2006; Gaudreault et al. 2004; Kang et al. 2006). Mechanisms underlying inflammation in neurodegeneration (Glass et al. 2010) may explain the involvement of CAV1 purine complex in major neurodegenerative disorders, as this complex contains binding sites for numerous inflammatory transcription factors including the IRF and STAT family members.

We have recently identified exceptional purine island human promoter compositions (albeit in the proximal core promoter region) with sequence bias between human and mouse (Darvish et al. 2011), and shown that haplotypes across those purine complexes can significantly alter gene expression (Heidari et al. 2012b).

Our data are preliminary at this stage and warrant confirmation in independent cohorts of patients. It is also warranted that the region is screened in other neurodegenerative disorders.