DNA to RNA to Protein

The central dogma of genetics has been that DNA is transcribed into RNA that is than translated into protein—the “business” end of the process. So, following its transcription from DNA in the nucleus, mRNA is transported out of the nucleus to the cytoplasm, and possibly to a specific subcellular location depending on the mRNA, where it can be deciphered by the cell. This takes place via interaction with a complex known as the ribosome, which binds the mRNA and converts its genetic information into protein via the process of translation. The ribosome initiates translation at a pre-encoded start site and converts the mRNA sequence into protein until a designated termination site is reached. Sequence information is read in three-nucleotide groups called codons, each of which specifies an individual amino acid. With the four distinct bases, there are mathematically 64 possible codons, but these have an element of redundancy and code for only 20 different amino acids and 3 termination signals (UAG, UGA, and UAA), also called stop codons. The start codon is ATG and codes for methionine. These amino acids are joined by the ribosome to synthesize a protein. This protein, which may undergo further modification, will ultimately carry out a programmed biological function in the cell. Regulation of this process is highly coordinated and important in learning, for example, where activity-dependent translation at the synapse underlies some aspects of synaptic plasticity, which may go awry in certain disorders such as fragile X syndrome and autism (Morrow et al., 2008).

Over the last decade, the discovery of several classes of functional non–protein coding RNAs has added additional complexity to our understanding of how the genetic code is manifest at the level of cellular function. Of these, microRNAs (miRNAs) are increasingly being recognized as vital players in gene regulation and neurological disease (Weinberg and Wood, 2009). Nascent miRNA molecules are processed to form short (approximately 22-nucleotide) RNA duplexes that target endogenous cellular machinery to specific coding RNAs and induce posttranscriptional gene silencing through a diverse repertoire including RNA cleavage, translational blocking, transport to inactive cell sites, or promotion of RNA decay (Filipowicz et al., 2008; Weinberg and Wood, 2009). Depending on the cell and the context, miRNA activity can result in specific gene inactivation, functional repression, or more subtle regulatory effects and may involve multiple RNAs in a given biological pathway (Flynt and Lai, 2008). Estimates suggest that miRNAs may regulate 30% of protein-coding genes, implicating these molecules as important targets for future research into the biology of neurological disease (Filipowicz et al., 2008; Weinberg and Wood, 2009).

For a specific disease-related gene, the DNA sequence present within an individual is referred to as their genotype, and the expression of that code often results in a feature (or features) that can be observed or measured, known as the phenotype. Genes are further organized into higher-order structures termed chromosomes, which together comprise the entire set of DNA, or genome, of the individual. The human genome is diploid, meaning we possess 23 pairs of chromosomes, 22 autosomes and 1 sex chromosome. Consequently, normal individuals possess two copies (or alleles) of every autosomal gene, one from the mother and one from the father. Because there are two distinct sex chromosomes, X and Y, genes on these chromosomes are expressed in a slightly different manner, discussed in more detail later for the sex-linked disorders.

It is important to emphasize that most genes are not simply “on” or “off.” In reality, cells maintain strict regulatory control over their genes. Some genes, such as those required for cell structure or maintenance, must be expressed constitutively, but genes with specific precise functions may only be needed in certain cells at certain times under certain conditions. Potential levels of regulation are depicted in Fig. 40.2 and include virtually every stage of gene expression. Initially, genes can be regulated at the level of transcription, ranging from the regulated binding of histone proteins, which leads to chromosome condensation, inactivating genes, to the coordinated activity of protein factors that activate or repress gene transcription in response to cell state, environmental conditions, or other factors. Once expressed, the RNA is subject to processing regulation, particularly through alternative splicing as already discussed. Transport of the mRNA and its translation provide additional steps for cellular regulation. Lastly, the final protein can be subject to control via posttranslational modifications or interactions with other proteins. To operate, all these levels of regulation require trans-acting factors, such as proteins, which stimulate or repress a particular step, as well as cis-acting elements, sequences recognized and bound by the regulatory factors.

These detailed levels of regulation provide a dynamic and expansive capability to precisely control cellular function, essential for growth, development, and survival in an unpredictable environment. However, this also provides many potential points at which disease can arise from disrupted regulation. Consequently, a defective gene could cause disease directly through its own action or indirectly by disrupting regulation of other cellular pathways. For example, the forkhead box P2 (FOXP2) transcription factor regulates the expression of genes thought to be important for the development of spoken language (Konopka et al., 2009). Mutations in this gene cause an autosomal dominant disorder characterized by impairment of speech articulation and language processing (Lai et al., 2001). However, other mutations in this gene are responsible for approximately 1% to 2% of sporadic developmental verbal dyspraxia (MacDermot et al., 2005), likely via downstream effects. Mutation of the methyl-CpG-binding protein 2 (MECP2), which regulates chromatin structure, causes the neurodevelopmental disorder, Rett syndrome, but other mutations in this gene can cause intellectual disability or autism (Gonzales and LaSalle, 2010). Similarly, the FOX1 protein (also called ataxin 2 binding protein 1, or A2BP1), a neuron-specific RNA splicing factor (Underwood et al., 2005) predicted to regulate a large network of genes important to neurodevelopment (Yeo et al., 2009; Zhang et al., 2008), causes autistic spectrum disorder when disrupted (Martin et al., 2007) but has also been implicated as a susceptibility gene associated with both primary biliary cirrhosis (Joshita et al., 2010) and hand osteoarthritis (Zhai et al., 2009), presumably due to downstream effects or specific effects in non-neural tissues. This concept of genes acting on other genes will be further explored later (see Common Neurological Disorders and Complex Disease Genetics).

In addition to the complexity of regulatory mutations that affect gene expression by altering RNA or protein levels or by disrupting RNA splicing, there are certain mutations that do not cause protein dysfunction, but instead have effects restricted to the RNA itself. For example, RNA inclusions are found in several forms of triplet repeat disorders (see Repeat Expansion Disorders) including myotonic dystrophy type 1 and the fragile X–associated tremor/ataxia syndrome (FXTAS) (Garcia-Arocena and Hagerman, 2010; Orr and Zoghbi, 2007). The latter is particularly interesting from a genetic standpoint, because a disorder of late-onset progressive ataxia, tremor, and cognitive impairment occurs in carriers of FMR1 alleles of intermediate sizes, which are not full fragile X–causing mutations (Garcia-Arocena and Hagerman, 2010). FXTAS is a dominant gain-of-function disease that occurs via an entirely different mechanism than the recessive loss-of-function disease, fragile X syndrome (Garcia-Arocena and Hagerman, 2010; Penagarikano et al., 2007). Primary disorders of RNA still represent relatively uncharted territory, and it is likely that more RNA-specific diseases will be identified. This is particularly exciting for many reasons, not the least of which is that certain classes of these disorders may be amendable to therapy (Nakamori and Thornton, 2010; Wheeler et al., 2009).

Rare versus Common Variation

As dictated by the principles of natural selection, most genetic variation is not deleterious, and the induced phenotypic variability can be beneficial as a source on which evolution may act. From a clinical standpoint, it is helpful to dichotomize genetic variation into common and rare variation, while accepting that genetic variation is likely a continuum, and the choice of cutoff could be considered arbitrary. Rare genetic variants are of low frequency in the population (<1% frequency), either because they are deleterious and selected against, or because they are new and most often benign. Common genetic variation (>1% to 5% population frequency), on the other hand, is either adaptive, neutral, or not deleterious enough to be subject to strong negative selection; such variants are referred to as polymorphisms. The preeminent genetic model has been that common disease susceptibility is related to common genetic variation, and more rare forms of disease are caused by rare genetic variants, so-called mutations, which act in a Mendelian fashion. In contrast, common variants or polymorphisms may increase susceptibility for disease, but alone are not sufficient to cause disease (see Common Neurological Disorders and Complex Disease Genetics).

Polymorphisms and Point Mutations

The most prevalent form of genetic polymorphism is the single nucleotide polymorphism (SNP), which occurs on average every 300 to 1000 base pairs in the human genome. Most of these SNPs are relatively benign on their own and do not directly cause disease, so for the purposes of this initial discussion, we will concern ourselves primarily with mutations: rare genetic variants sufficient to cause disease. Pathogenic mutations can occur in numerous ways and vary from single nucleotide changes to gross rearrangements of chromosomes (Fig. 40.4). Owing to the large volume of DNA in the human genome, heritable mutations can arise spontaneously in the germline over time through errors in DNA replication or from DNA damage by metabolic or environmental sources despite the constant surveillance of extensive cellular preventive proofreading and repair mechanisms. Thus, mutations can be inherited from the parent or occur de novo in the germline. An example of a common de novo variant is trisomy 21, which causes Down syndrome (discussed further in Chromosomal Analysis and Abnormalities). The smallest pathogenic alterations, termed point mutations, involve a change in a single nucleotide within a DNA sequence. A point mutation can result in one of three possible effects with respect to protein: (1) a change to a different amino acid, called a missense mutation, (2) a change to a termination codon, called a nonsense mutation, or (3) creation of a new sequence that is silent with regard to protein sequence but alters some aspect of gene regulation, such as RNA splicing or transcriptional expression levels. Nonsense mutations can cause premature truncation of a protein, whereas a missense mutation can affect a protein in different ways depending on the chemical properties of the new amino acid and whether the change is located in a region of functional importance.

Fig. 40.4 Genetic mutations. A, Categories of chromosomal aberrations. Paired homologous chromosomes are shown, with various anomalies indicated. An insertional translocation is depicted; other common types include reciprocal translocations and centric fusions (Robertsonian translocations). B, Types of point mutations. A generic DNA sequence is shown (boxed) along with its corresponding mRNA sequence. Codons are indicated, as are their translation into protein (designed by the standard three-letter code). Mutations are in purple, as are the corresponding alterations in the mRNA and protein if present. Note that silent point mutations do not alter the protein sequence. C, Repeat expansion disorders. An example mRNA is shown with a CAG-codon (polyglutamine) repeat region indicated. In the expanded form, an additional number of repeats are present which may perturb the function of the protein produced and/or lead to cell damage via the expanded polyglutamine region (see text for details).

It should be emphasized that not all point mutations are disease-causing variants, although until recently, many considered that a premature stop codon was a “smoking gun.” Whole genome sequencing demonstrates that more than 100 such nonsense mutations may exist per genome, and the vast majority are expected to be relatively benign (Lupski et al., 2010; see Whole Genome/Exome Sequencing in Disease Gene Discovery). So in many cases, the pathogenicity of rare variants is not immediately discernable, and without strong statistical or functional evidence, labeling such genetic variation a mutation is premature and may be misleading. It is likely that most of these, including some variants thought previously to cause rare Mendelian diseases, may simply be benign genetic variation. This is because even a complete knockout of one allele caused by a premature stop codon (haploinsufficiency) may have no discernable effect on gene function for a majority of genes in the human genome (Lupski et al., 2010; Ng et al., 2009; Yngvadottir et al., 2009).

Occasionally, silent coding mutations or point mutations in noncoding regions may be significant for disease if they damage sequences important for gene expression (e.g., transcriptional and/or RNA processing regulatory elements). It has been estimated that up to half of all disease-causing mutations impact RNA splicing, which can have dire consequences given the importance of splicing to regulated gene expression. Such is the case for frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), where in some populations, the most common mutations disrupt splicing, causing a pathogenic imbalance in tau isoforms (D’Souza and Schellenberg, 2005). As for noncoding mutations, given the large volume of such sequences in the human genome—perhaps up to 96%—and our still imprecise ability to predict sequences required for regulation or to interpret identified sequence changes without direct experimentation, the majority of these mutations likely go unrecognized. It is hoped that the next generation of sequencing and bioinformatic technologies will allow for a better understanding of the role of these types of mutation in human disease.

Structural Chromosomal Abnormalities and Copy Number Variation

Small deletions and insertions can occur through slippage and strand mispairing at regions of short, tandem DNA repeats during replication. If the deletion or insertion is not a multiple of three, a frameshift will result, which leads to the translation of an altered protein sequence from the site of the mutation. On a larger scale, errors of chromosomal replication or recombination can result in inversions, translocations, deletions, duplications, or insertions (Stankiewicz and Lupski, 2010). When the region of deletion or duplication is greater than 1 kb, this is referred to as a copy number variation (CNV). Copy number variation is far more common than previously suspected, and it is estimated that at least 4% of the human genome varies in copy number (Conrad et al., 2010; Redon et al., 2006), much of which is commonly observed in the population and benign (Conrad et al., 2010). However, some rare CNVs such as the recurrent chromosome 17p12 duplication underlying most cases of Charcot-Marie-Tooth type 1A (Shchelochkov et al., 2010) or the alpha-synuclein triplication that can cause Parkinson disease (PD)(Singleton et al., 2003) are pathogenic and act in a Mendelian fashion. Even though such changes may be extensive, they may not be pathogenic if they do not disrupt expression of any key genes. This is particularly true for balanced translocations where genetic material is rearranged between chromosomes, yet no significant portion is actually lost. Although an individual with such a condition may be normal, if the germline is affected, their offspring may receive unbalanced chromosomal material and consequently develop a clinical phenotype (Kovaleva and Shaffer, 2003). CNVs will be discussed in greater detail when we consider common and complex disease genetics (see Copy Number Variation and Comparative Genomic Hybridization).

Repeat Expansion Disorders

Most mutations thus far discussed pass from parent to offspring unaltered, and in large affected families, the identical mutation can potentially be traced back generations. In contrast, there is a specific class of mutation, the repeat expansion (Orr and Zoghbi, 2007) (Table 40.2), which is unstable and can present with earlier onset and increasing severity in successive generations, a process known as anticipation. There are several examples of diseases caused by expanded repeats in coding sequence (e.g., most spinocerebellar ataxias, HD), as well as examples in noncoding sequence (e.g., fragile X syndrome, myotonic dystrophy) and within an intron (e.g., Friedreich ataxia). Interestingly, virtually all these disorders show neurological symptoms that can include such features as ataxia, intellectual disability, dementia, myotonia, or epilepsy, depending on the disease. The most common repeated sequence seen in these diseases is the CAG triplet, which codes for glutamine and expansion of which is seen in a variety of the spinocerebellar ataxias (SCAs) including SCA types 1, 2, 3, 6, 7, 17, and dentatorubropallidoluysian atrophy (DRPLA). In addition to protein-specific effects, these disorders likely share a common pathogenesis due to the presence of the polyglutamine repeat regions. In some disorders, the phenotype can be quite different depending on the number of repeats, such as in the FMR1 gene, where more than 200 CCG repeats causes fragile X syndrome, but repeats in the premutation range of 60 to 200, from which fully expanded alleles arise, can result in FXTAS or premature ovarian failure (Oostra and Willemsen, 2009). Although in general, the underlying mutation is similar, each specific repeat expansion has distinct effects on its corresponding gene, and thus in addition to varying phenotypes, they may also show very different inheritance patterns as illustrated later (see Disorders of Mendelian Inheritance).

Autosomal Dominant Disorders

Diseases involving autosomal genes that require mutation of only one allele are defined as dominant. In most cases, the affected individual has two distinct alleles of a gene (in this case, one normal and one pathogenic) and is described as being heterozygous. Often these pathogenic mutations impart new functionality, referred to as a toxic gain of function, meaning that the phenotype is produced as a result of the expression of the mutated protein. Other disease mechanisms in dominantly inherited conditions include: (1) haploinsufficiency, where inactivation of a single allele is sufficient to produce disease despite the presence of another normal copy, and (2) dominant negative effects, where a mutated protein disrupts function of the normal protein transcribed from the other nonmutant allele.

Autosomal dominant inheritance is characterized by direct transmission of the disorder from parent to child (Fig. 40.6). Affected individuals are seen in all generations, and a vertical line can be drawn on the pedigree to illustrate the passage of the disorder. Since only one deleterious copy of the disease gene is necessary, risk of transmission from an affected parent is 50%. Since the disorder is autosomal, there is no sex preference, and both males and females can present with the disease. One caveat involves the concept of penetrance, or the percent likelihood that a trait will manifest in a person with a specific genotype. A dominant gene is considered to have complete penetrance if all individuals with a given mutation develop disease. In practice, however, many autosomal dominant genes show varying degrees of penetrance or expressivity, most likely due to the influence of other genes and environmental factors.

Fig. 40.6 Autosomal dominant inheritance. A pedigree diagram is shown, using standard nomenclature. Generations are numbered consecutively on the left, and individuals are numbered within each generation. Males are depicted as squares and females as circles. Affected persons are indicated by filled icons. Death is indicated by a diagonal line. A union producing offspring is indicated by horizontal lines. A diamond represents individuals (n) of unknown sex. A triangle represents a spontaneous abortion. Individuals V-2 and V-3 illustrate the diagramming of dizygotic twins. The proband of the pedigree is indicated by an arrow. An autosomal dominant pedigree demonstrates vertical transmission of disease without a sex preference. On average, 50% of offspring are affected. Individual III-4 represents a case of incomplete penetrance (dark circle) where the individual carries the mutation but does not manifest disease. Anticipation (see text) would be illustrated by increasing severity/onset in patients III-1, IV-2, and V-4.

There are over 400 examples of diseases with neurological phenotypes that show autosomal dominant inheritance (OMIM, 2010). These conditions include hyperkalemic periodic paralysis (voltage-gated sodium channel Na_V1.4 on chromosome 17, often caused by missense mutations), HD (Huntington on chromosome 4, caused by CAG repeat expansion), SCA type 3 (ataxin-3 on chromosome 14, caused by CAG repeat expansion), Charcot-Marie-Tooth type 1B (myelin protein zero on chromosome 1, often caused by missense mutations), early-onset familial Alzheimer disease (AD)(presenilin-1, often caused by missense mutations), frontotemporal dementia with parkinsonism (microtubule-associated protein tau on chromosome 17, often caused by missense or splicing mutations), tuberous sclerosis type 1 (hamartin on chromosome 9, often caused by nonsense mutations and frameshifts), neurofibromatosis type 1 (neurofibromin on chromosome 17, caused by point mutations, frameshifts, and splicing mutations), and familial amyotrophic lateral sclerosis (ALS) (superoxide dismutase-1 on chromosome 21, caused by missense mutations), to name a few. Even rare Mendelian forms of more common syndromes such as epilepsy or sleep disorders (e.g., familial advanced sleep-phase syndrome) have been identified. More detailed lists can be found using the recommended online resources (Table 40.3).

Table 40.3 Selected Online Clinical Neurogenetics Resources

Autosomal Recessive Disorders

Disease involving autosomal genes that require mutation of both alleles is defined as recessive. An unaffected individual who harbors one disease-causing allele is referred to as a carrier of that allele. For some disorders, a mild phenotype can be seen in these individuals, who are then described as symptomatic carriers. An individual with two identical alleles (in this case both pathogenic) is described as being homozygous. Alternatively, if they possess two different pathogenic alleles this is described as being compound heterozygous. In general, autosomal recessive mutations modify the function of the protein in a negative way, meaning that the phenotype is produced because of the absence of the mutated protein. This is referred to as a loss of function.

Autosomal recessive inheritance is characterized by lack of intergenerational transmission, in contrast to dominantly inherited disorders (Fig. 40.7