200 Years After Darwin

On February 12, 2009, we celebrate the 200th anniversary of the birth of Charles Darwin. He has earned the scientific reputation as being the most influential naturalist-biologist of his generation and perhaps of all time. Achievement of his esteemed status resulted initially from studying the geographical distribution of wildlife and fossils collected on his 5-year voyage on the Beagle and concluding that the “transmutation of species” was due to natural selection.¹ In 1858, he was in the process of documenting his research when he received an essay from Alfred Russel Wallace putting forth the same concept of natural selection as the mechanism for generation of new species, resulting in a joint publication that same year.² On the Origin of Species³ was published by Darwin in 1859, making it now the 150th anniversary of its publication. This book proposed evolution as the scientific basis of diversity and species generation produced through the effects of natural selection. Evolution has been proven to be the mechanism for species generation and the engine for change is natural selection, providing the evolved species, beginning from a common organism, adaptation and fitness to maximize reproduction and survival. Dobzhansky put it succinctly by saying that “nothing in biology makes sense except in the light of evolution.”⁴ Darwin expressed it eloquently in 1859:

There is grandeur in this view of life, with its several powers, having been originally breathed into a few forms or into one; and that, whilst this planet has gone cycling on according to the fixed law of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been, and are being evolved.³

Two hundred years after Darwin's birth, molecular genomic analyses of the human genome have been implemented, seeking the genetic basis for natural selection providing biological fitness and also risk of developing disease. Genome-wide association studies (GWASs) seeking gene variations, or single-nucleotide polymorphisms (SNPs), causal of several human diseases have been conducted in recent years. These diseases include autism, schizophrenia, obesity, diabetes, and heart disease.^{5 - 6}

Several GWASs for risk association with neurological diseases, or neuromic studies, have been reported. Increased risks for amyotrophic lateral sclerosis, Alzheimer disease (AD), restless legs syndrome, and multiple sclerosis have been associated with polymorphisms in specific genes. It was hoped that these observations would advance an understanding of the causation of inherited, complex polygenetic, multifactorial neurological diseases. They have been made possible by the publication of the human genome and haplotype studies (HapMap analyses).

A whole-genome analysis of sporadic amyotrophic lateral sclerosis was published by Dunckley et al.⁷ They identified 10 genetic loci that were associated with sporadic amyotrophic lateral sclerosis in 3 independent series of patients and control subjects. The most significant association with disease was found for a SNP near an uncharacterized gene known as FLJ10986 on chromosome 1. Thus, they described specific SNPs that are associated with onset of sporadic amyotrophic lateral sclerosis.

Reiman et al⁸ conducted a genome-wide survey of 502 627 SNPs to characterize and confirm late-onset AD susceptibility genes. They found that late-onset AD was associated with 6 SNPs from the GRB-associated binding protein 2 (GAB2) gene and a common haplotype encompassing the entire GAB2 gene. The GAB2 gene was overexpressed in pathologically vulnerable neurons, and interference with GAB2 gene expression increased tau phosphorylation. They concluded that GAB2 modifies late-onset AD risk in apolipoprotein E (APOE) ε4 allele carriers.

Coon et al⁹ found a single SNP in linkage disequilibrium to the APOE ε4 allele in a neurome-wide association study. Grupe et al¹⁰ described 3 additional SNPs on chromosome 19 in linkage disequilibrium to the APOE ε4 locus also in a neurome-wide association study. Liu et al¹¹ described significant linkage to 3q23 markers associated with AD in a genetically isolated Dutch population.

In 2008, Li et al¹² described SNPs associated with risk and age at onset of AD in a GWAS of 469 438 SNPs. They identified the APOE linkage disequilibrium region as the strongest genetic risk factor for AD. They concluded that this event could be a consequence of the coevolution of more than 1 susceptibility allele, including APOC1, in the same region. Additional potential at-risk genes for AD were of interest and require further studies.

Waring and Rosenberg¹³ outlined their approach for a new GWAS from the Texas Alzheimer Disease Consortium comprising 4 Alzheimer Disease Centers at universities in Texas. They considered recent reports that have applied genome-wide association methods to investigations by taking advantage of the currently available high-throughput arrays, bioinformatics, and software advances. The inherent strengths, limitations, and challenges associated with study design issues in the context of AD were presented.

A positive neuromic finding of considerable importance was reported by Rogaeva et al¹⁴ in a study of multiple cohorts with late-onset AD. They found genetic variants of sorLA associated with late-onset AD. They genotyped VPS35, VPS26, and the family of VPS10-containing molecules. These results are considered in the context of the retromer sorting pathway and provide positive data that VPS35 binds sorLA, and reducing VPS26 in cell culture resulted in increased β-amyloid (Aβ) production.

Winkelmann et al¹⁵ have reported evidence from a genome-wide case-control study that showed an association with restless legs syndrome and 3 genetic loci: 1 within MEISI, 1 within BTBD9, and 1 between MAP2K5 and LBXCOR1. Stefansson et al¹⁶ presented a GWAS of restless legs syndrome and periodic limb movements, stereotypical repetitive limb or leg twitches that are associated with disturbances of sleep onset. They found an association with 1 of these genes, BTBD9.

Hafler et al¹⁷ and Gregory et al¹⁸ as collaborating groups simultaneously published GWASs to identify alleles associated with the risk of multiple sclerosis. A test of 334 923 SNPs in 931 family trios showed 49 SNPs having an association with multiple sclerosis. Thirty-eight SNPs from this original number were selected for a second-stage analysis. A comparison between the 931 case subjects from the family trios and 2431 control subjects identified an additional nonoverlapping 32 SNPs. An additional 40 SNPs that had less stringent P values were also selected. A total of 110 SNPs were identified in the second-stage analysis. From these SNPs, 2 within the interleukin-2 receptor α gene (IL2RA) were strongly associated with multiple sclerosis. A nonsynonymous SNP in the interleukin-7 receptor α gene (IL7RA) and multiple SNPs in the HLA-DRA locus were also strongly associated with multiple sclerosis. They indicate that the likely causal SNP, rs6897932 located within the alternatively spliced exon 6 of IL7RA, has a functional effect on gene expression.^{17 - 18}

Byun et al¹⁹ reported a neurome-wide pharmaconeuromic analysis of the response to interferon beta in patients with multiple sclerosis. They found that responders to the drug compared with nonresponders had significantly different genotype frequencies for SNPs located in many genes, including glypican 5, collagen type XXV α1, hyaluronan proteoglycan link protein, calpastatin, and neuronal PAS domain protein 3. Many of the detected differences they found between responders and nonresponders were genes associated with ion channels and signal transduction pathways. They believed that genetic variants in heparin sulfate proteoglycan genes may be of clinical interest in multiple sclerosis as predictors of the response to therapy.

The hope with neurome-wide association studies is that the complete complement of variant genes causal of the major neurodegenerative diseases will be identified. Then, pharmaconeuromic therapy could not be far behind. However, it may not be turning out that way. The GWASs promised to provide the major genes responsible for major human traits and common diseases. The GWASs would provide insights into gene variations in low-penetrant genes causal for polygenetic, multifactorial neurological disease such as AD. Overall, about 400 genetic variants that contribute to human traits and diseases, including neurological diseases, have been identified. However, the cumulative effects are disappointingly small and have not provided clear insights into disease causation and prior estimates of heritability.²⁰ Francis Collins, MD, PhD, former head of the National Human Genome Research Institute in Bethesda, Maryland, has expressed his own dismay at the low level of convincing genetic variability, or missing heritability, found in GWASs to explain the genetics of common human disease.²⁰

Sequencing candidate genes for disease including their surrounding regions in thousands of people will be needed to discover more associations with disease. The SNPs are turning out not to be a stringent-enough level of analysis in seeking genetic risks for disease. The change in mind-set is going from seeking analyses of common, low-penetrance variants causal of common diseases to seeking rare low- or moderate-penetrance variants that have been missed by GWASs.²⁰ It may be necessary to move beyond sequencing candidate genes and surrounding regions for disease association and begin sequencing whole genomes to find the missing heritability. Collins has suggested that the National Human Genome Research Institute's 1000 genomes project, designed to sequence the genomes of at least 1000 people from all over the world, would provide a powerful approach to finding the hidden heritability.²⁰

The genetic explanations that would be of primary interest to find the missing heritability for genetic neurological disease missed by GWASs include copy-number variation, epistatic effects, and epigenetics. Copy-number variation refers to regions of DNA that are up to hundreds of base pairs long and are deleted or duplicated between individuals.¹⁹ There are strong copy-number variation associations between schizophrenic individuals compared with healthy individuals, and they may arise de novo in persons without a family history of the mutation.¹⁹ Epistasis, where 1 or more modifying genes reduce or enhance the effect of another gene, may be an important genetic mechanism at work to explain heritability not found by GWASs. Epigenetics is another vital area to be explored. It refers to changes in gene expression that are inherited but not caused by alteration in the sequence of the gene. Gene expression is altered by methylation or acetylation and also by inhibition of messenger RNA expression by interfering RNA or microRNA binding.^{20 - 22}

The 24 000 protein-coding genes in the human genome make up less than 1.2% of the human genome. Analysis of the remaining 98.8% of the human genome and its role in the cause of human neurological diseases, both inherited and acquired, is a formidable challenge yet unexplored to any degree. The RNA transcripts and their effects on regulation and levels of gene expression are the next frontier for neuromics.

Then there is the issue that natural selection functions only before or during the reproductive years and not afterward, when AD and Parkinson disease occur. The major biological function of natural selection is to select for fitness, allowing for reproduction and maintenance of a lineage or species. Aging and neurodegenerative diseases seem to have escaped the forces of natural selection by occurring after the reproductive years. On the other hand, perhaps evolution has actually selected for aging and neurodegenerative diseases as a means to maintain the limits of a finite lifespan. Clearly, neuromics must address the molecular basis of brain aging and why the aging process provides a permissive environment to allow the opportunistic neuromic program causal of late-onset neurodegenerative diseases to be expressed.

Alzheimer disease is due to both genetic polymorphisms and environmental stimuli. In this view, environmental stimuli that have yet to be determined influence the production of an abnormal pattern of gene expression causal of AD. So, we will have to understand the process of natural selection in the context of the selection pressures from the environments that we inhabit. Darwin emphasized adaptation to a changing environment as the principal selective influence for evolution. This principle is valid, studying the interaction of environmental stimuli and the genetic factors causal of neurodegenerative diseases. Future research directions become highly variable and complex. Deriving induced pluripotential stem cells from patients with late-onset AD and differentiating them into neuroblasts would be a way to screen compounds to determine whether an abnormal pattern of gene expression is produced compared with derived neuroblasts from healthy control subjects. Here would be a method to link environment to the genetic program causal of AD. It would also be a means to screen potential therapeutic agents that correct an abnormal pattern of gene expression in patients with AD as a prelude to a clinical trial.

The 200 years since Darwin's birth and the 150 years since the publication of On the Origin of Species are a brief time in human experience. We believe Darwin would be delighted to see how far natural selection has come to explain the evolution of the human brain. He would encourage us to pursue genome sequencing, copy-number variation, epistasis, epigenetics, and RNA regulation of gene expression to decipher how natural selection has served to promote fitness and survival and how mutations in these genetic functions are causal of neurological diseases.

Charles Darwin would challenge the neurological community to be at the forefront of developing ethical and humane applications for the discoveries being made in neurogenetics. How will genetic testing affect medical policy making? How do we protect patients and their families from inappropriate distribution and use of their genetic code? How do we educate the public on medical genetics when it is becoming increasingly difficult even for clinician-scientists to grasp the complexity of advances in the field?

The Archives of Neurology has a long-standing history of publishing articles that explain the genetics of neurological diseases. We will continue to accompany the academic neurological community on this exciting quest.