Clinical Neurogenetics

DNA to RNA to Protein

The central dogma of genetics has been that DNA is transcribed into RNA that is then 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 ( ). In other cases, such as repeat-associated non-ATG (RAN) translation, specific mutations causing disruption of this fundamental process can be an important mechanism underlying neurological disease ( ; see Repeat Expansion Disorders).

Over the past 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 ( ). Nascent miRNA molecules are processed to form short (approximately 22-nucleotide) RNA duplexes that target endogenous cellular machinery to specific coding RNAs and induce post-transcriptional gene silencing through a diverse repertoire including RNA cleavage, translational blocking, transport to inactive cell sites, or promotion of RNA decay ( ). 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 ( ). 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 ( ).

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 compose 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. 48.1 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. Last, the final protein can be subject to control via post-translational 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.

Epigenetics , or the study of heritable changes in gene expression that do not involve changes in the DNA sequence itself, is emerging as an important aspect of both gene regulation and neurological disease ( ). These changes can involve several mechanisms including methylation of the DNA, modification of histone proteins, chromatin remodeling, expression of noncoding RNAs, and RNA editing, all of which may occur in response to a variety of intracellular or environmental signals ( ). Disruption of epigenetic mechanisms can cause Mendelian neurological disease (see Imprinting), as can impairment of the function of factors that mediate these epigenetic mechanisms ( ). Epigenetics may also play a role in sporadic disease, as a recent study reported the H1 haplotype of the MAPT gene to be differentially methylated in a dose-dependent manner in patients with progressive supranuclear palsy ( ), suggesting an epigenetic mechanism for the disease risk associated with the presence of that haplotype. Further studies investigating the role of these pathways genome-wide in clinical populations will likely uncover more associations with disease and disease risk ( ).

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. This also provides many potential points at which disease can arise from disrupted regulation, however. 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 ( ). Mutations in this gene cause an autosomal-dominant disorder characterized by impairment of speech articulation and language processing ( ). However, other mutations in this gene are responsible for approximately 1%–2% of sporadic developmental verbal dyspraxia ( ), 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 (ID) or autism ( ). Similarly, the RBFOX1 protein (also called ataxin 2 binding protein 1, or A2BP1), a neuron-specific RNA splicing factor ( ) predicted to regulate a large network of genes important to neurodevelopment ( ), causes autistic spectrum disorder when disrupted ( ) but has also been implicated as a susceptibility gene associated with both primary biliary cirrhosis ( ) and hand osteoarthritis ( ), presumably due to downstream effects or specific effects in non-neural tissues. This concept of genes acting on other genes will be explored further 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; ). 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 ( ). FXTAS is a dominant gain-of-function disease that occurs via an entirely different mechanism than the recessive loss-of-function disease that is fragile X syndrome ( ). FXTAS pathogenicity appears related to repeat-associated non-AUG-initiated translation of a cryptic polyglycine protein ( ), an example of a rapidly emerging mechanism in several RNA-mediated neurodegenerative disorders, including DM1 myotonic dystrophy, C9orf72-mediated amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), and several others ( ). 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 amenable to therapy ( ).

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%–5% population frequency), on the other hand, is 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 each variant alone is not sufficient to cause disease. Instead, hundreds or thousands of variants necessarily act in combination to lead to disease risk, which is termed “ polygenic risk ” ( ; 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 300–1000 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. 48.3 ). 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, a potentially disastrous effect, often leading to production of a nonfunctional protein. Missense mutations, in contrast, 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.

It is often not possible to determine the outcome of specific missense changes, or even nonsense ones, without experimental evidence. The term “mutation” thus should be reserved only for those changes known to cause disease, with the typical description of such alterations being “variants.” For example, genome sequencing demonstrates that more than 100 nonsense variants may exist per genome, and the vast majority are expected to be relatively benign ( ; see Genome/Exome Sequencing in Clinical Practice, Disease Gene Discovery, and Gene Therapy). In many cases, the pathogenicity of rare missense variants is not immediately discernible, and without strong statistical or functional evidence, labeling such genetic variation a mutation is premature and may be misleading. As our knowledge of normal human genetic variation grows, it is likely that most of these, including even some variants thought previously to cause rare Mendelian diseases, will be reclassified as benign genetic variation. This is not surprising, as even a complete knockout of one allele caused by a premature stop codon (haploinsufficiency) may have no discernible effect on gene function for a majority of genes in the human genome ( ). In some cases, Mendelian diseases may even require combinations of variants in more than one gene ( ) before a phenotype is observed, further illustrating the challenge of predicting pathogenicity.

Occasionally, silent coding or noncoding variants may cause 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 FTD 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 ( ). 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. Advances in next-generation sequencing and bioinformatic technologies are beginning to examine larger populations of patients globally for both coding and noncoding variants and are expected to expand our 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 ( ). When the region of deletion or duplication is greater than 1 kb, this is referred to as a copy number variation (CNV). CNV is far more common than previously suspected, and it is estimated that at least 4% of the human genome varies in copy number ( ), much of which is commonly observed in the population and benign ( ). However, some rare CNVs such as the recurrent chromosome 17p12 duplication underlying most cases of Charcot-Marie-Tooth (CMT) type 1A ( ) or the alpha-synuclein triplication that can cause Parkinson disease (PD; ) 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 ( ), which may be quite severe. CNVs will be discussed in greater detail when we consider common and complex disease genetics (see Copy Number Variation and Chromosomal Microarray Analysis).

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 ( ; Table 48.2 ), 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., the common dominant spinocerebellar ataxias [SCAs], 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, ID, 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. Its expansion is seen in a variety of the 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 enlarged polyglutamine repeat regions. Furthermore, recent studies have shown that bidirectional transcription may occur from these repeat regions in many of the disorders, through a process termed RAN translation, producing polypeptide proteins which may disrupt metabolism, induce toxic cellular effects, and lead to disease ( ). 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 cause fragile X syndrome, but repeats in the premutation range of 60–200, from which fully expanded alleles arise, can result in FXTAS or premature ovarian failure ( ). 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).