Identification of sixteen novel candidate genes for late onset Parkinson's disease

Abstract

Background: Parkinson's disease (PD) is a neurodegenerative movement disorder affecting 1-5% of the general population for which neither effective cure nor early diagnostic tools are available that could tackle the pathology in the early phase. Here we report a multi-stage procedure to identify candidate genes likely involved in the etiopathogenesis of PD.

Methods: The study includes a discovery stage based on the analysis of whole exome data from 26 dominant late onset PD families, a validation analysis performed on 1542 independent PD patients and 706 controls from different cohorts and the assessment of polygenic variants load in the Italian cohort (394 unrelated patients and 203 controls).

Results: Family-based approach identified 28 disrupting variants in 26 candidate genes for PD including PARK2, PINK1, DJ-1(PARK7), LRRK2, HTRA2, FBXO7, EIF4G1, DNAJC6, DNAJC13, SNCAIP, AIMP2, CHMP1A, GIPC1, HMOX2, HSPA8, IMMT, KIF21B, KIF24, MAN2C1, RHOT2, SLC25A39, SPTBN1, TMEM175, TOMM22, TVP23A and ZSCAN21. Sixteen of them have not been associated to PD before, were expressed in mesencephalon and were involved in pathways potentially deregulated in PD. Mutation analysis in independent cohorts disclosed a significant excess of highly deleterious variants in cases (p = 0.0001), supporting their role in PD. Moreover, we demonstrated that the co-inheritance of multiple rare variants (≥ 2) in the 26 genes may predict PD occurrence in about 20% of patients, both familial and sporadic cases, with high specificity (> 93%; p = 4.4 × 10^{- 5}). Moreover, our data highlight the fact that the genetic landmarks of late onset PD does not systematically differ between sporadic and familial forms, especially in the case of small nuclear families and underline the importance of rare variants in the genetics of sporadic PD. Furthermore, patients carrying multiple rare variants showed higher risk of manifesting dyskinesia induced by levodopa treatment.

Conclusions: Besides confirming the extreme genetic heterogeneity of PD, these data provide novel insights into the genetic of the disease and may be relevant for its prediction, diagnosis and treatment.

Keywords: Late onset Parkinson’s disease; Novel candidate genes for Parkinson’s disease; Rare variant burden analysis; Whole exome sequencing.

Fig. 1

Graphical representation of the 26 families used as discovery cohort. Pedigrees of the 26 Italian families with supposedly dominant PD forms. Affected individuals are indicated with dark symbols. Patients who underwent WES analysis are shown with dark arrows. Additional family members for which DNA was available are indicated with an asterisk. Only affected subjects were used for segregation analysis. For each family, the rare deleterious variants reported in Table 1 were carried by the affected family members

Fig. 2

Venn diagram and STRING analysis representing the logical relations between the 16 novel gene products and the network of PD-interacting genes products. a Venn diagram showing the 16 novel identified genes involved across different interconnected pathways: interactors of PD related genes, genes involved in mitochondrial metabolism and those involved in vesicular trafficking and autophagy. b Schematic representation of the network of interactions of the 16 novel PD gene products with known PD-associated genes. STRING database analysis identified 9 genes interacting with PD-associated genes

Fig. 3

Graphical representation of the protein domains and mapped mutations. For those genes whose protein structure was available (AIMP2, GIPC1, KIF24, KIF21B, RHOT2, SLC25A39, SPTBN1, TMEN175), we report the protein domains and mapped amino acid changes identified in the cohort of 1542 patients. Amino acid changes are referred to by their single letter code

Fig. 4

Expression analysis of the novel identified Parkinson’s disease genes. qPCR experiment showed that the 16 novel genes were expressed in mesencephalon of P45 adult mice. Pitx3 was used as an example of mdDA neurons restricted gene expression. Gapdh was used to normalize the results. Data were shown as absolute values with standard deviation calculated from three different experiments. Genes were grouped on the basis of their expression level in three different scales

Fig. 5

Expression analysis of TOMM22, GIPC1, ZSCAN21, SLC25A39 and HSPA8 in human and mouse DA neurons. a-g Immunohistochemistry experiments performed on human (a) and P45 mouse (b-g) adult brain sections showed that TOMM22, GIPC1, ZSCAN21, SLC25A39 and HSPA8 were co-expressed with TH in DA neurons of the human SN (a) and in mouse DA neurons of the SN (b-e) and VTA (f, g). Images in (c, e, g) correspond to magnification of the boxed area reported in (b, d, f). Scale bars correspond to 100 μm. Abbreviations: SN stands for substantia nigra and VTA for ventral tegmental area

Fig. 6

Distribution of variants load, ROC curve and analysis of PD endophenotypes. a, b Very rare (MAF ≤ 0.001), exonic variants (exclusion of synonymous changes) were annotated in 26 PD genes in 394 unrelated Italian cases and in 203 controls. a The histogram shows the percentage of samples (cases in blue and controls in green) carrying 0, 1, 2, 3, 4 variants in the selected genes; b ROC curve and analysis of sensitivity and specificity. The test shows that the distribution is high significant and the test may predict the disease in about 17% of at risk individuals in the general population, carrying at least 2 variants, with specificity > 93%. c Percentage of PD cases manifesting L-dopa-induced dyskinesia (LID), and earlier age at onset (AAO) of Parkinson’s disease. d Polygenic variant load, including GBA variants, was inversely associated with age at PD onset at the nominal significance level (p 0.044). For each comparison, we set statistical significance threshold at p < 0.05. Statistical significance was reported with asterisk