Parkinson Disease: a common neurodegenerative disorder
Parkinson diseases (PD) is the second most common neurodegenerative disorder that usually has a late onset; it affects 1% of the population over age 60 and a total 4million people in the globe. The people with PD show either motor (muscle rigidity, slowness of movement, limb stiffness, and problems with balancing) or nonmotor (loss of mental sharpness, anxiety, delusion, or depression) symptoms. The disease is mostly sporadic (affected by both genetic and environmental risk factors) and there is only 5 to 10% familial (affected by Mendelian mode of monogenic inheritance of a few known genes). Because it is a complex disease, even today researchers are still looking for genes that contribute to the disease. The good news is that more and more genes are found using modern genomic tools and their functionality will be further studied with a hope for generating future therapies.
Genetic tools used for new gene discovery
The molecular genetics tools such as genome-wide linkage analysis are used to discover the familial genes that are involved in pathogenesis based on well-documented family studies and map them at particular chromosome loci. However, these gene variants are not found in many PD patients, thus a great effort has been focusing on the genes that are risk factors causing sporadic PD. There are two hypotheses to follow about discovering new gene affecting PD. One is the common disease common variants hypothesis and Genome-Wide Associated Studies (GWAS) are widely used. The other is the common disease rare variants hypothesis and whole exome sequencing (WES) has gained attraction for finding new genes.
According to the National Human Genome Research Institute, “a genome-wide association study (GWAS) is an approach used in genetics research to associate specific genetic variations with particular diseases. The method involves scanning the genomes from many different people and looking for genetic markers that can be used to predict the presence of a disease.” The genotyping technology of various platforms containing thousands of human SNPs (single nucleotide polymorphism) is easily available and affordable to discover common PD risk variants. In combination with functional genomics using large scale transcriptomic datasets, more genes whose expression or splicing levels are affected by PD can be identified. Although many susceptibility genes are discovered by GWAS especially for sporadic cases, pathogenic mutations in the majority of these genes, which often have large effect size, have not been found in both sporadic and familial PD.
With more than 20,000 SNPs analyzed for one sample, WES becomes affordable and invaluable tool to search for rare PD risk variants with large effect size. The WES method is especially useful for the identification of monogenic genes involved in Mendelian inheritance but can be used for both sporadic and familial PD gene discovery. The technique is more suitable for young-onset PD compared to late-onset PD. Through filtering of the minor allele frequency (MAF) less than 1%, for instance, plus other filtering criteria, novel genes associated with PD are identified. Different studies have used WES and various imperfect bioinformatics filtering criteria to narrow down the gene searches for potentially pathogenic genes.
The role of Sanger sequencing in discovery of new genes
Sanger sequencing is commonly used together with WES to confirm the variant sequence either after filtering the variants in sporadic PD or in the downstream cosegregation analysis of familial gene variants with Menlelian inheritance. Since the mutants filtered after WES could be important for downstream function studies in pathogenesis, Sanger sequencing makes sure all the filtered mutants truly exist and can be independently amplified and confirmed. Although some articles claim that Sanger sequencing is not necessary anymore for mutation confirmation based on their high concordance between NGS and Sanger sequencing, today it is still a common practice to use Sanger sequencing to confirm mutations filtered from WES screenings.