Genetic Mechanisms of Retinal Disease

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Chapter 31 Genetic Mechanisms of Retinal Disease

Stephen P. Daiger, Lori S. Sullivan, Sara J. Bowne

Introduction

The purpose of this chapter is to provide an overview of concepts underlying our current understanding of the genetic basis of inherited retinal diseases (iRDs). iRDs are perhaps the best understood of human hereditary disorders. In part this is because diseases that affect vision are easily recognized and the retina is an accessible and well-characterized tissue. In many ways, though, we are still at an early stage of understanding the causes and consequences of these diseases. In fact, the causes of iRDs are highly varied: many different types of retinal disease are known, many different genes are involved, and there may be dozens of disease-causing mutations reported within a single gene. For example, currently at least 220 genes are known which can cause one or another form of retinal disease,1 and over 5000 mutations have been reported, in total, in these genes.2 In spite of the underlying complexity, it is now possible to identify the disease-causing gene and mutation, or mutations, in a substantial fraction of affected individuals and families.3,4

A useful concept in medical genetics is the distinction between single-gene diseases and multifactorial diseases. Inherited diseases such as retinitis pigmentosa (RP) are considered to be single-gene because there is a specific, underlying cause in each affected individual, that is, an inherited difference in DNA sequence that has a direct cause-and-effect relationship to the disease. There may be one DNA difference for dominant diseases, or two for recessive diseases, but only one gene is involved. These are also referred to as monogenic or Mendelian diseases. In contrast, for diseases such as age-related macular degeneration (AMD), genetic differences play a role in lifetime risk and/or clinical expression, but the differences are merely contributory and do not have a clear cause-and-effect relationship to the disease. These are “multifactorial” diseases because multiple factors, genetic, environmental, and stochastic, play a role in determining who is affected and who is not.

Therefore the cause of disease in an individual with an inherited condition such as RP is “simple,” in the sense that only one gene is affected (and usually affected in an obvious way), whereas there may be multiple contributory factors in an individual with AMD and the differences may be subtle. We already known exceptions to this rule – for example, there are digenic forms of RP with two affected genes5 – but the exceptions are rare.

This chapter focuses on genetic differences that are single-gene in nature and have a direct cause-and-effect relationship with disease, that is, inherited diseases of the retina. Genetic factors contributing to AMD are discussed in Chapter 64 (AMD: Etiology, genetics, and pathogenesis).

Basic concepts in human genetics

Inheritance

Figure 31.1 shows pedigrees illustrating autosomal dominant, autosomal recessive, and X-linked recessive inheritance (see Nussbaum et al.6 for details).

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Fig. 31.1 Pedigrees illustrating autosomal dominant, autosomal recessive, and X-linked recessive inheritance.

iRDs follow textbook patterns of Mendelian inheritance: autosomal dominant, autosomal recessive or X-linked. However, real families are often more complicated, especially for late-onset, progressive forms of retinal disease. This section reviews the conventional modes of inheritance and possible complexities.

Autosomal dominant inheritance

Autosomal dominant inheritance occurs when a single copy of a mutation on an autosomal chromosome is sufficient to cause disease. That is, an affected individual is heterozygous for the mutation. Diseases caused by dominant mutations pass from generation to generation, i.e., most families have affected individuals in multiple generations. Males are as likely to be affected as females and approximately 50% of children of an affected individual will be affected. Forms of retinal disease that are often autosomal dominant include maculopathies such as Best disease.

Two phenomena that can confuse the picture of autosomal dominant disease are variable expression and incomplete penetrance.

Variable clinical expression means that individuals with the same mutation may vary in onset, progression, or severity of disease or, in some cases, may have distinctly different clinical findings. Autosomal dominant RP is notoriously variable in expression. For example, mutations in one autosomal gene, PRPH2 (also known as RDS), can cause dominant RP, dominant macular degeneration, or dominant panretinal maculopathy, even among members of the same family.7–11

Variable expression is a problem in determining mode of inheritance because some individuals may not show symptoms until late in life, and individuals with different symptoms may be diagnosed with different diseases.

Incomplete penetrance, or nonpenetrance, means that some individuals with a disease-causing mutation will not be affected. For instance, 20% of individuals with a dominant-acting mutation in PRPF31 will have normal vision by age 60 even though relatives with the same mutation may have RP by age 20.12–15 One indicator of nonpenetrance in a multigenerational family is a “skipped generation,” that is, an unaffected individual with an affected parent and an affected child. This is often seen in families with PRPF31 mutations.

Although variable expression and incomplete penetrance are seen as distinct phenomena, they are actually part of a continuum, with nonpenetrance just the extreme. The difference between late onset and no onset may simply be the age of the patient when examined. Whatever the terminology, the underlying finding is that dominant retinal disease mutations may have highly variable consequences, confounding diagnosis.

Autosomal recessive inheritance

Autosomal recessive inheritance occurs when both copies of an autosomal gene must be affected to cause disease. An affected individual can be either homozygous for a single mutation or heterozygous for two distinct mutations. An individual with two distinct recessive mutations is also called a compound heterozygote. Note that a pair of recessive mutations must be on opposite chromosomes. If two variants are in the same gene on the same chromosome, they are in cis to each other; if they are on opposite chromosomes they are in trans. Recessive mutations must be in trans.

Examples of autosomal recessive retinopathies include Leber congenital amaurosis and Usher syndrome.

Unless one of the two mutations in a recessive case is a new mutation, the parents must be carriers of the mutation or mutations, that is, they must be heterozygous. Carriers are usually not affected. Approximately one-fourth of children of carrier parents are affected and one-half of children are carriers. Many recessive cases are isolated or simplex cases, i.e., one affected family member only. Families with multiple affected sibs are “multiplex.”

Finally, in consanguineous families with marriage between relatives, an identical recessive mutation may be passed to multiple family members. Affected individuals may occur in more than one generation and in more than one branch of these families. Two identical mutations that derive from a recent ancestor are identical by descent (IBD). Marriage between relatives is more common in some cultures than others, hence IBD inheritance of retinal diseases is more frequent in those societies.

Because carriers are not self-evident, the mode of inheritance is often hard to assign in recessive families.

X-linked or sex-linked inheritance

X-linked or sex-linked inheritance is a single mutation on the X chromosome which causes disease. Males, who are hemizygous for the X chromosome, are always affected, often severely affected. For many inherited diseases, female carriers of an X-linked mutation are not affected. Since females have two Xs, this implies that most X-linked mutations will be recessive in females. For a truly recessive X-linked mutation, one-half of the sons of a carrier female are affected, one-half of her daughters are unaffected carriers, and none of the sons of an affected male are affected. This produces a notable pattern of inheritance, with the salient feature that male-to-male transmission of an X-linked mutation is not possible.

The disease status of female carriers is more complex, though. Although females have two Xs, one of the Xs, selected at random in each cell, is inactivated in most tissues. This is X-inactivation or lyonization, named for Mary Lyon, who first described the phenomenon.16,17 Lyonization increases the likelihood that a female carrier will be affected since some cells will express only the mutant protein. In fact, many female carriers of X-linked RP mutations show clinical symptoms. Females are less severely affected than males with the same mutation, but female carriers of X-linked RP mutations may have significant loss of vision by midlife or earlier.18–22

One consequence of clinical disease in carrier females is that families with X-linked RP may appear to have autosomal dominant RP if several females are affected.23 This is an example of complexities that arise in determining the mode of inheritance of iRDs.

Isolated cases

Isolated cases deserve an entry of their own because the mode of inheritance is often obscure. A practical definition of an isolated case is an affected individual with no affected first-degree relatives (parents, sibs, and children) and no reports of more distant affected relatives. One immediate concern is that there may be other affected family members but the person describing the family is unaware or uninformed. Clinical examination of first-degree relatives is often revealing.

Assuming a case is genuinely isolated, there are several possibilities. The most likely prospect is that this is an autosomal recessive case and the parents are carriers. Or, perhaps one parent is a carrier but the other mutation is de novo (new). Alternatively, this may be a new dominant-acting or X-linked mutation. Another possibility is autosomal dominant or X-linked inheritance with nonpenetrance in prior generations. Ultimately, for most isolated cases the mode of inheritance will have to be determined at a molecular level by genetic testing.

Digenic and polygenic inheritance

Nearly all iRDs are monogenic, with only one gene affected per person. This is based on empirical observation, but it may be misleading since more complex forms of inheritance are hard to prove. Two counter examples are known for iRDs. First, one form of RP is caused by a combination of one mutation in the PRPH2 (RDS) gene and another mutation in the ROM1 gene.5,22 These two mutations are benign alone but pathogenic in combination. This is digenic inheritance. Secondly, Bardet–Biedl syndrome (BBS), a form of RP combined with congenital abnormalities, is in most instances a recessive disease with mutations in any one of at least 15 known BBS genes.1,24 Some cases of BBS, though, require a third mutation in a second BBS gene for disease expression.25,26 This is called trigenic or triallelic inheritance. Whether these examples of polygenic inheritance of iRDs are just rare anomalies or hint at greater complexity of retinal diseases is unclear.

Chromosomes

Chromosomes are dark-staining bodies seen in the nucleus of dividing eukaryotic cells. In diploid organisms, such as humans, the earliest diploid cell before division results from fusion of a haploid cell from the male parent and a haploid cell from the female parent. That is, the first human cell has a diploid count of 23 pairs of chromosomes (n), or 46 total chromosomes (2n), and derives from fusion of a haploid sperm and haploid ovum. This is the primary germline cell and contains the germline genetic information in the nucleus. All subsequent cells, known as somatic cells, contain a nearly perfect copy of the original chromosomes and genetic information. Exceptions in humans are sperm- and ovum-producing cells (also known as germline cells) which produce haploid cells, and certain blood cells that do not contain a nucleus.

Eukaryotic chromosomes have been referred to as “information-carrying organelles” because they are highly structured, highly compressed complexes of proteins, RNAs, DNA, and other factors, with the primary function of transmitting genetic information from one generation to the next, or from a parent cell to a daughter cell. However, at the heart of each chromosome is a single, double-stranded DNA molecule. DNA length is measured in basepairs (bp): each single strand of DNA is composed of nucleotide bases, and each base interacts (pairs) with an alternate base in double-stranded DNA, so bp are the natural units. DNA is also measured in kilobases (kb), megabases (Mb), and gigabases (Gb). The DNA molecule within a chromosome may be hundreds of Mb in length. This is, by far, the largest single biomolecule known. One reason for the chromosomal superstructure may simply be to keep this giant molecule intact. However, chromosomes also participate directly in DNA duplication and expression.

DNA, RNA, and proteins

Figure 31.2 shows the steps in DNA duplication, RNA translation, and protein synthesis.27

image

Fig. 31.2 Steps in DNA duplication, RNA translation, and protein synthesis.

(Reproduced from Nussbaum RL, McInness RR, Willard HF. Thompson and Thompson’s genetics in medicine, 7th ed. Philadelphia, PA: Saunders Elsevier; 2007, p. 31, with permission from Elsevier.)

DNA is deoxyribonucleic acid, a linear molecule composed of four monomers: adenine (A), thymine (T), guanine (G), and cytosine (C). Two antiparallel DNA strands pair through hydrogen bonds to form a double-stranded molecule which carries genetic information.

RNA is ribonucleic acid, a linear molecule, like DNA, composed of adenine, uracil (U), guanine, and cytosine. RNA is single-stranded in most circumstance but it can form complex folded shapes by pair bonding within the linear strand. Messenger RNA (mRNA) transfers genetic information within cells, but other RNA molecules play diverse roles in several biological processes.

Proteins, composed of various combinations of 20 amino acids, are linear molecules which can fold into many shapes, and which play essential and highly diverse roles in all biological processes.

DNA function is called the central dogma of DNA in recognition of the landmark explanation of DNA structure and function by Watson and Crick in 1953, and subsequent unraveling of the genetic code over the next decade.28,29 DNA is comprised of a phosphate backbone with nucleotide bases, A, T, G, or C, in linear array along the backbone. The backbone is conventionally drawn from the 5’ phosphate on one end to the 3’ phosphate on the other end. The opposite strand forms by pairing of cognate bases, A to T and G to C, on the parent strand. The opposite strand naturally aligns in a helical, antiparallel fashion, from 3’ to 5’ phosphates. This arrangement essentially explains inheritance in all living things.

In DNA duplication, the two antiparallel strands unwind, and a nearly exact antiparallel copy is synthesized on each single strand. The principal enzyme involved is DNA polymerase, but additional enzymes are involved in unwinding, patching, and repairing the DNA. DNA duplication occurs in the nucleus of cells only.

In DNA-RNA transcription, the DNA strands unwind, and a single-stranded RNA molecule is synthesized as a short antiparallel copy of one of the DNA strands, pairing each DNA nucleotide with the corresponding RNA nucleotide. The primary enzyme involved is RNA polymerase, and the first steps occur in the nucleus. Thereafter the RNA molecule is processed through many steps, and eventually exported from the nucleus to the protein-forming machinery. The final molecule is mRNA since it carries the DNA message to the cytoplasm.

In protein translation, mRNA is read by the protein-forming machinery and the corresponding protein is built by adding one amino acid to the next in succession, from the amino (NH2-) end of the protein to the carboxy terminus (-COOH). Each amino acid is coded for by three RNA bases, that is, a nucleotide triplet or codon. After synthesis, most proteins are further modified through posttranslational modification, then the linear protein folds into its active shape, often with the assistance of proteins known as chaperones.

Gene structure

Figure 31.3 shows gene structure based on the relationship between the protein sequence, mRNA intermediate, and original DNA gene sequence.27

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Fig. 31.3 (A, B) Gene structure based on the relationship between the protein sequence, mRNA intermediate, and original DNA gene sequence.

(B, reproduced from Nussbaum RL, McInness RR, Willard HF. Thompson and Thompson’s genetics in medicine, 7th ed. Saunders Elsevier; 2007, p. 29, with permission from Elsevier.)

The modern concept of a gene is clouded by arguments as to where a gene starts and stops, and whether segments of DNA that do not code for proteins but still influence traits are “genes.” This discussion is limited to defining a gene in terms of proteins, while acknowledging the broader complexities.

Gene expression is principally the steps from DNA transcription to protein translation. Gene expression starts with separation of double-stranded DNA, exposing a single-stranded sequence on which DNA-to-RNA transcription can occur. This is accompanied by binding of a complex set of proteins, “expression factors,” which facilitate binding and activity of RNA polymerase.

The primary RNA strand begins at the start of transcription and ends far beyond the length sufficient to code for a protein. The first RNA-processing steps add a methyl cap to the first RNA nucleotide, trim the 3’ end, and add a polyadenosine tail (poly-A tail). Next the RNA moves to a complex assembly of proteins and small, functional RNAs, known as a splicesome. The splicesome then removes anywhere from one to many internal segments of the RNA transcript and reassembles the remainder. This is, largely, the finished mRNA, which is then exported from the nucleus to the protein synthesis machinery in the cytoplasm.

RNA splicing has profound consequences for gene structure and protein variation. Splicing occurs in nearly all eukaryotes and almost all human genes are spliced. The spliced-out segments are called introns and the remaining, reassembled segments are exons. The splice sites are defined by short, canonical sequences, highly conserved across species, known respectively as splice donor and splice acceptor sites.

The evolutionary significance of splicing is still disputed, but its functional consequence is clear: it vastly increases the number of distinct proteins. This is because when splicing occurs, alternate combinations of introns may be removed. Alternate splicing is the norm in human genes, not the exception, and usually results in alternate mRNAs and alternate protein isoforms – all from a “single” gene. There are many examples of alternately spliced retinal genes producing multiple protein isoforms.30,31

Following splicing and export from the nucleus, mRNA is translated into protein by ribosomes in the endoplasmic reticulum. The start of translation is usually not at the beginning of the mRNA, and the end is not at the end. The segment upstream of the start of translation is the 5’ untranslated region (5’-UTR). Similarly, the segment downstream of the end of translation is the 3’ untranslated region (3’-UTR). The 5’ and 3’-UTRs may sit within the first and last exons, or may stretch across exons.

In addition to alternate splicing and alternate protein isoforms, there are alternate starts of transcription, alternate starts of translation, alternate ends of translation, and alternate poly-A sites.

Mitosis, meiosis, and linkage

Figure 31.4 shows the steps in meiosis.6

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Fig. 31.4 The steps in meiosis.

(Reproduced from Nussbaum RL, McInness RR, Willard HF. Thompson and Thompson’s genetics in medicine, 7th ed. Philadelphia, PA: Saunders Elsevier; 2007, p. 19, with permission from Elsevier.)

In normal cell division, in which a somatic cell divides to produce a daughter cell, the DNA in all 46 human chromosomes is copied and a complete copy is passed to each of the two resulting cells. This is mitosis, and it provides a nearly perfect copy of the DNA sequence to each somatic cell.

Chromosome distribution and DNA processing are substantially different in meiosis. Meiosis occurs in cells producing sperm and ova. In the first phase of meiosis chromosomal DNA is duplicated as in mitosis, but the duplicated DNA strands do not separate. Then, in subsequent phases, homologous chromosomes bind to each other and exchange DNA strands. Homologous chromosomes are pairs of similar chromosomes from each parent, e.g., the maternal and paternal chromosome 1. The result of homologous exchange or recombination is to produce novel DNA sequences that are mixed linear combinations of chromosomal segments from each parent. The resulting duplicated, recombined chromosomes are then separated into four haploid-containing cells, which eventually become sperm or ovum.

The result is, first, that each sperm or ovum contains only a haploid set of chromosomes. However, when a sperm fertilizes an ovum the resulting cell has the full diploid set of chromosomes. Second, each chromosome in the offspring is a recombined mix of the chromosomes of each parent. Thus the chromosomes in each individual are a unique, one-of-a-kind combination of previous generations (excepting identical twins).

As a result, genes on different chromosomes segregate independently, but genes which are close together on a chromosome segregate together. More generally, the further a pair of genes are apart on a chromosome, the more likely a recombination will have occurred between them during meiosis. Two loci which are close enough on a chromosome such that recombination is unlikely are “linked.” The phenomenon of genes that do not segregate independently is linkage.

To put this in context, the average chromosome is 100 Mb in length. Genes that are 50 Mb apart are unlinked, and genes within 10 Mb of each other show linkage. If genes are within 1 Mb of each other the chance of recombination is less than 1%. There are roughly 5–20 genes per Mb in the human genome, so hundreds of contiguous genes on a chromosome may show linkage to each other.32

Linkage has significant evolutionary and functional consequences, but its importance in medical genetics is as a tool to locate disease-causing genes. If a neutral genetic variant is found in association with an inherited disease in a family, then the variant, called a marker in this case, may be physically close to the disease gene, that is, they may be linked. If so, then knowing the location of the marker fixes the location of the disease gene.

Evolution

Theodosius Dobzhansky said “nothing in biology makes sense except in the light of evolution.”33 This observation serves to emphasize the fundamental organizing principal of biology. Without evolution, biology is largely a random collection of facts and principles; with evolution, biology (and medicine) is a coherent science. Evolution explains why the central dogma is true for all species, why all eukaryotic cells share a common architecture, why animal models are useful in understanding and treating human diseases, why human DNA variants are evaluated by comparison to other species. Evolution is so central to biology and medicine that we often forget its impact, but modern, 21st-century biomedicine is unimaginable without it.

The human genome

Overview

The human genome is the combined DNA sequence of the haploid set of all chromosomes. Humans have 23 pairs of chromosomes; these consist of 22 pairs shared by males and females (autosomes) and a pair of Xs in females or an X and Y in males (sex chromosomes). Autosomes are labeled from the largest, 1, to the smallest, 22. Thus the human genome is the sequence from the top of chromosome 1 through the bottom of chromosome 22, plus the X and Y chromosomes.

In one of the greatest scientific achievements in modern history, the Human Genome Project produced the first human genome sequences in 2001.34,35 Since then hundreds of complete human genomes have been sequenced, and the genomes of thousands of other species are known.36

There is a distinction between the generic human genome and the genome of a specific person. “The” human genome, or “the” genome of any species, is neither an average nor a consensus. It is simply a sampling of the first individuals sequenced. Therefore the reference human sequence and reference gene sequences are just examples, not definitive sequences. In contrast, the genome of a specific human would cover all 46 chromosomes and be unique to that person. Thus, in this sense, there are billions of human genomes and no two are alike.

Nonetheless, both the generic human genome sequence and the many specific sequences are extremely valuable contributions to medicine and biology.

The human haploid genome is 3.3 Gb in length; the complete genome of an individual is 6.6 Gb. There are 21 000–25 000 protein-producing genes in the generic human genome. This is far fewer than expected, but the discrepancy is partly explained by alternate splicing and multiple protein isoforms.

Most genes, including introns, exons, and regulatory elements, are 10–100 kb in length. Therefore the portion of the genome devoted to genes is less than 10% of the total. Considering exons only, the fraction of the genome actually coding for proteins is only 1.5% of the total.

Roughly 50% of the human genome consists of thousands or millions of short, repetitive DNAs. For example, there are more than one million copies of Alu sequences dispersed throughout the genome. Alu’s are short retrotransposon-related sequences which have the capacity to copy themselves into new locations in the genome. Some repetitive DNAs are functional in that they play a role in chromosome structure and protein activity. However, it is fair to say that this is principally “junk DNA,” at least in relation to actual genes.

The remaining 50% consists of single-copy DNA (that is, not repetitive), including coding sequences. Most of this single-copy DNA is transcribed into RNA but not translated into proteins. This noncoding RNA (ncRNA) must play a role in normal biological processes, and probably contributes to human diseases, but it is poorly understood at present.

Polymorphisms

Humans are 99.9% identical to each other at a DNA level. Since there are 6.6 Gb in the human diploid genome, this means that one person differs from another at millions of sites. Some of these differences contribute to diseases, both simple and complex, but the vast majority are neutral or only slightly advantageous or disadvantageous. This is the extremely varied “genetic background” that distinguishes one individual from another.

An identifiable site in a genome, usually a gene, is called a locus, and any variant at this site is an allele. If there is more than one allele at a locus in a group of people, and the second most common allele has a frequency of 1% or greater, then this is called a genetic polymorphism. If there are only two alleles at a locus then the less common allele is the minor allele. Note that locus and allele apply to an individual whereas polymorphism only makes sense in the context of a population in which more than one allele is found.

The 1% criterion is arbitrary. A specific allele may occur only once in one individual or may range up to 100% of all chromosomes in a population. One percent was chosen because this frequency can be measured in a realistic survey and it was presumed that any more common allele must be largely benign. (The latter is definitely not true; for instance, the sickle-cell and cystic fibrosis mutations are both polymorphic in their respective populations.) Polymorphism is a useful concept, though, despite the arbitrary definition.

A person who has identical alleles at a locus is homozygous at that site; a person with two different alleles is heterozygous. (This applies to autosomal chromosomes and the Xs in a female. Alleles on the male Y are hemizygous.) The frequency of heterozygotes in a population is predicted from Hardy–Weinberg equilibrium: if there are two alleles with frequencies p and q, then the frequency of the two homozygote types is p2 and q2, respectively, and the heterozygote type is 2 × p × q. The total fraction of heterozygotes at a locus in a population is heterozygosity, basically the sum of all heterozygote combinations.

Several broad classes of polymorphism are found in the human genome

Single-nucleotide polymorphisms

Single-nucleotide polymorphisms (SNPs) are sites at which more than one nucleotide is found in a population. There are usually only two alleles at a SNP locus, e.g., an A or a T. Millions of SNP sites have been identified in humans.37 Some are found in only one major population, such as Africans, but many are polymorphic in all major human groups. A SNP site at which the nucleotide substitution leads to an amino acid substitution, or otherwise affects a protein, is a coding SNP (cSNP). There are tens of thousands of cSNPs, at least one in almost every gene.

If a pair of human chromosomes are compared base by base, each of us is heterozygous for a nucleotide substitution roughly every 1000 bp. Not all of these are at polymorphic sites; some are private or rare variants (often called single-nucleotide variants). However, most are true SNPs.

Short tandem repeats

Another type of polymorphism is short tandem repeats (STRs). STRs are short stretches of DNA, typically 2–5 bp in length, which are repeated a variable number of times, say from 10 to 20 times. Alleles differ in the number of repeats. Because many different lengths are possible, there are usually several or many alleles at a locus. These are also called microsatellites.

Other polymorphisms

There are many other human DNA polymorphisms. There are large polymorphic elements called minisatellites or VNTRs (variable number tandem repeats); Alu insertions may be polymorphic; chromosome deletions and rearrangements are polymorphic in humans; gene structure and copy number may be polymorphic. (The latter are copy number variants, which are common.) The extent of polymorphic variation in humans is exceptional, reflecting our high mobility, heterogeneous habitats, and variable mating patterns.

Finally, in addition to polymorphic variants, there are many private or rare variants. Some, but not all, of these are deleterious, e.g., mutations causing retinal diseases. The extent of rare variants is not well known, and it is often difficult to determine which are benign and which are pathogenic. However, it is clear that each of us harbors millions of variants in our genomes that are rare or unique to us or our families, some of which affect our health and well-being.

Mutations

Figure 31.5 illustrates several of the types of mutation that cause iRDs.

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Fig. 31.5 Types of mutation that cause inherited retinal diseases.

Technically, a mutation is a change in DNA from one generation to the next. In practice, the word often refers to any damaging, deleterious DNA variant, one that acts in either a dominant or recessive fashion. This chapter uses the word largely in the second sense, that is, a mutation is “bad.” However, it is important to recognize that a rare, novel variant is not necessarily pathogenic and a polymorphic variant with an allele frequency of 1% or greater is not necessarily benign.

Most disease-causing mutations affect a protein, either directly or indirectly. However, this observation may simply reflect the fact that only proteins have been well studied.

A mutation may eliminate a protein, reduce or eliminate its function, alter or add to its function, or convert the protein into a toxic factor. One way of describing mutations is by their consequences: an absent protein, a functional null, a gain or loss of function, or a toxic protein. Dominant mutations may also be described as dominant negative: the mutant protein interferes with the wild-type protein. As a sweeping generalization, with many exceptions, autosomal recessive mutations often lead to an absence of a protein or a functional null, and autosomal dominant mutations often result in a gain of function, toxic protein, or dominant negative effect. X-linked mutations are less predictable because of the differential effect in males and females.

Another way to categorize mutations is to work from DNA to mRNA to proteins:

1. DNA deletions or rearrangements may result in the absence of a protein or a critical part of a protein. Surprisingly, humans harbor large, polymorphic deletions, some 100 kb in length or longer.38 In apparently healthy people, these are in a heterozygous state: one chromosome segment is deleted and the matching segment on the homologous chromosome is intact. Large, homozygous deletions are severely deleterious. Smaller deletions, usually the size of a gene or less, affecting one or a few proteins, may cause autosomal dominant, autosomal recessive, or X-linked retinal diseases.14,39,40

2. DNA changes 5’ to the start of transcription may block synthesis of mRNA or may affect timing or amounts of mRNA. These are promoter or expression mutations. Further, any changes to the canonical nucleotides that define the donor or acceptor slice sites of introns may profoundly affect mRNA splicing. These are splice site mutations. Splice site mutations typically result in a structurally abnormal protein or no protein at all.

3. Finally, mutations can alter proteins directly in a myriad of ways. Mutations in DNA that directly affect proteins are broadly classed into nucleotide substitutions in contrast to small insertions or deletions, typically 1–15 bp in length, called indels. A nucleotide substitution which causes replacement of one amino acid with another is a missense mutation. Missense mutations may alter protein function, or produce a toxic protein or a dominant negative protein. (A nucleotide substitution that changes a codon but does not alter an amino acid is a silent substitution. Most silent substitutions are benign.) An indel that alters the order of codons is a frameshift mutation or an inframe amino acid deletion. A nucleotide substitution that introduces a signal to stop translation of a message prior to the normal stop is a nonsense mutation or a premature-stop mutation. Indels and premature-stop mutations produce a severely abnormal protein or no protein.

It is likely that new ways in which mutations can cause disease will be revealed in the future as the complexity of the human genome and inherited diseases is better understood.

Genetic testing methods

The primary purpose of genetic testing for iRDs is to determine the underlying cause of disease in an affected individual and in his or her family. A broader goal is to use genetic testing for research purposes, e.g., to find novel mutations, to discover new disease-causing genes, to identify patients for clinical trials, or to study the natural history of disease. There is no clear demarcation between diagnostic testing and research, but there are practical distinctions. Diagnostic testing is often limited to testing affected individuals, screening known genes only, and is usually conducted in a certified laboratory. (Diagnostic laboratories in the USA have Clinical Laboratory Improvement Amendments (CLIA)41 and/or College of American Pathologists (CAP)42 certification.) Research testing may involve other family members, may screen novel genes, and is often conducted in facilities which are not certified.

However, for this section, no distinction is made between diagnostic testing and research testing, in part because the boundary between them is frequently shifting. In general, diagnostic testing and research are highly interdependent activities.

Current methods for genetic testing can be grouped into three categories: (1) screening known genes and mutations; (2) mapping to localize the disease-causing gene; and (3) high-throughput DNA sequencing. However, the essential first step is an informed clinical examination, which must precede testing.

Informed clinical examination

The clinical examination is critical in selecting the correct tests and in making sense of the results. An effective examination and good family history are necessary to establish the mode of inheritance, may identify subtle clinical findings suggesting possible causes, and may rule out certain genes and diseases. The exam may involve additional affected and unaffected family members which will facilitate follow-up studies. Finally, without good clinical and family information it may be difficult or impossible to interpret and explain the testing results.

Screening known genes and mutations

For each category of disease there is a set of known, possible disease-causing genes and a set of known mutations within each gene. Among the known genes and mutations, some are more common causes of disease than others. For example, mutations in 22 genes are known to cause autosomal dominant RP1 and more than 680 mutations have been reported in these genes.2 However, mutations in one gene, rhodopsin, account for at least 25% of cases of autosomal dominant RP in the USA.4 Also, more than 150 mutations have been reported in rhodopsin alone, but six of these mutations account for more than half of all cases.22

As a consequence, the natural next step is to test the most likely disease-causing genes in an affected individual, based on clinical findings and family history. Testing is usually done by polymerase chain reaction-based di-deoxy chain termination cycle sequencing, also called Sanger sequencing, the “gold standard” of DNA sequencing.43,44 Sequencing laboratories and services in the USA for iRDs include commercial facilities, university-based services, and federally supported programs.45 A current list of testing facilities in Europe and the USA is maintained by GeneTests.46

An alternative to DNA sequencing is to detect known mutations only. One approach is to use microarrays with short single-stranded DNA sequences that bind to the region containing the targeted mutation and detect the presence or absence of the mutation. An example of this technology is arrayed primer extension.47,48 The advantage of DNA sequencing is that it detects both known and unknown mutations in the genes tested. The advantage of testing known mutations only is that it is much less expensive than sequencing.

Additional possible tests include sequencing all the known disease-causing genes, even the rare causes of disease, and using methods other than sequencing to detect mutations. An example of the latter is the use of multiplex ligation-dependent probe amplification to detect large deletions.14,49,50

Linkage and homozygosity mapping

If sequencing known genes fails to detect a disease-causing mutation in an affected individual, then an alternative approach is to determine the chromosomal site of the disease locus using linkage mapping. This does not immediately identify the disease gene, but it can reduce the location harboring the gene from the entire genome to a region containing, at most, a few hundred genes. From this information, other methods can be used to find the specific gene.

Linkage testing is the principal approach used to map disease genes. This involves determining whether a neutral allele at a polymorphic site is tracking with disease in a family, taking into consideration the proposed mode of inheritance. A large number of polymorphic sites are assayed, and alleles at each locus are tested for association with the putative disease mutation or mutations in each family member. If a particular allele is segregating with disease in the family, then the polymorphic locus and the disease gene are linked, that is, close together on the same chromosome. The chromosomal location of each polymorphic site is known exactly (an outcome of the Human Genome Project), so the location of the disease gene is also fixed.

In practice, linkage testing is most useful in large families with autosomal dominant or X-linked disease since more affected family members are available for testing. Currently, linkage testing is often done using microarrays that assay up to a million SNPs, made by Affymetrix, among others.51,52 Several computer programs are available for analysis of linkage data.53–55 Once linkage is established, genes in the linkage region are sequenced, by a variety of methods, looking for mutations. Determining whether a rare variant in the linkage region is pathogenic can be challenging.51

In some cases, linkage can be useful in small recessive families, too. One effective approach is homozygosity mapping.56 If the mutations in a family with recessive disease are IBD, then not only are the mutations themselves identical, but DNA sequences within thousands of basepairs of the mutations are also identical, because of linkage. This manifests as long chromosomal segments surrounding the disease gene that contain no variant sites, for example, no heterozygous SNP sites. Long, homozygous DNA sequences are rare in humans, and often indicative of IBD. They can be detected using the same SNP-testing arrays used for conventional linkage testing. Once the region containing the disease gene is located by homozygosity mapping, strategies to identify mapped genes can be applied. This has been a fruitful approach to finding genes causing recessive iRDs.57,58

High-throughput DNA sequencing

In recent years, novel methods for very rapid DNA sequencing have become available. This is referred to as next-generation sequencing or deep sequencing.59,60 These approaches can be up to 10 000 times as fast as conventional di-deoxy cycle sequencing. The principle is to shear human DNA into short fragments and to sequence these fragments, up to 1 million simultaneously, in micron-sized wells or slots. The isolated sequences are then assembled computationally by comparison to the reference human genome. This is called “shotgun sequencing” because many short, random fragments are sequenced and later assembled into long, useful sequences. Next-generation sequencing is more error-prone than conventional sequencing, but it more than compensates by the extremely high sequencing rate.

With next-generation sequencing it is practical and inexpensive to sequence large tracts of the genome in individual patients. One application is to sequence the coding regions of all human genes. This is referred to as whole-exome sequencing – the “exome” is all known exons. Another application is whole-genome sequencing, that is, sequencing the entire genome of an individual. Since exons are only 1.5% of the genome, whole-exome sequencing is currently faster and cheaper than whole-genome sequencing, but whole-genome sequencing will be common within a few years. Already, whole-exome and whole-genome sequencing have shown promise in finding novel iRD genes and mutations.51,61–63

Future prospects

A safe prediction is that high-throughput sequencing, and even faster sequencing methods, will dominate genetic testing within several years. One problem now, and for the foreseeable future, is distinguishing pathogenic mutations from the exceptional number of rare variants in each of us – variants that help define our individuality. Also, the sheer amount of DNA sequence data per patient is potentially overwhelming. At the same time, though, robust computational methods are under development to address these problems. The divide between diagnostic testing and research is likely to fade further. Genetic testing will play an increasingly important role in the diagnosis and treatment of iRDs, with clinical ophthalmology serving as the indispensable link between patients, families, and testing facilities.

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