Genetics for the ophthalmologist
Karthikeyan A. Sadagopan, Jenina Capasso, and Alex V. Levin
Abstract
The eye has played a major role in human genomics including gene therapy. It is the fourth most common organ system after integument (skin, hair and nails), nervous system, and musculoskeletal system to be involved in genetic disorders. The eye is involved in single gene disorders and those caused by multifactorial etiology. Retinoblastoma was the first human cancer gene to be cloned. Leber hereditary optic neuropathy was the first mitochondrial disorder described. X-Linked red-green color deficiency was the first X-linked disorder described. The eye, unlike any other body organ, allows directly visualization of genetic phenomena such as skewed X-inactivation in the fundus of a female carrier of ocular albinism. Basic concepts of genetics and their application to clinical ophthalmological practice are important not only in making a precise diagnosis and appropriate referral, but also in management and genetic counseling.
Introduction
The field of genetics is evolving at a rapid pace. Almost 50% of pediatric blindness is due to a genetic etiology.[1,2] The eye is second only to the brain as an individual organ in its frequency of involvement in genetic disorders.[3] The ophthalmologist plays important roles not only in clinical diagnosis and management but may also be asked by patients and their families to provide genetic counseling and referrals to appropriate subspecialty services. An understanding of clinical genetic concepts and accurate diagnosis are prerequisites for effective genetic counseling. Examination of other family members might be required.
This article offers the ophthalmologist an understanding of basic genetics, including patterns of inheritance, genetic concepts and terminology.
Chromosomes
Each nucleated cell in humans has twenty three pairs of chromosomes except sperm and ovum which each have only twenty three chromosomes. The first twenty two pairs, numbered 1 through twenty two, are referred to as autosomes. The remaining pair is the sex chromosomes: XY in males, XX in females. Each chromosome has two arms which join at a central constriction (centromere). The terminal part of a chromosome is the telomere. The short arm is called “p”, and “q” is the long arm. Each chromosome is further subdivided into regions based on banding patterns that appear after staining with various chemicals. Numbering of subsections on each arm starts from the centromere and proceeds towards the telomere.[4] For example; 13q23.1 indicates chromosome number 13, long arm, region 2, subsection 3, and sub-subsection 1.
Each chromosome is made up of double stranded DNA in a double helix configuration. Each strand is a sequence of nucleotides: Adenine, Guanine, Thymine and Cytosine. Uracil substitutes for Thymine in RNA. DNA is transcribed into messenger RNA (m-RNA) which is then translated into protein.
Genes
There are approximate thirty five thousand genes in each human cell.[5] The use of different genes differs in each cell depending on that cell's function: Eye genes (e.g. rhodopsin) are used in the eye but not used in liver. Some genes (e.g. collagen) are used both in the eye and body areas. If such a gene is mutated, then one sees eye abnormalities and abnormalities in other parts of the body. Genes may be responsible for making a structural component (e.g. fibrillin) or enzyme. Other genes manufacture proteins, called transcription factors that influence and direct function of other genes. These genes are also often used (expressed) in multiple tissues.
Structure of a gene
Each gene has a promoter which is responsible for turning the gene on, sequences that code for protein (exons) and non-coding sequences (introns). Introns are segments between exons that are required for appropriate linking of the protein parts generated by transcribed exons to form the complete protein product of the gene. Exons represent only 3% of human DNA with the remaining 97% being introns.[6] Within each exon, triplet nucleotide sequences form codons. Each codon codes for an amino acid. Codons may also cause a stop of normal transcription and hence determine that the gene has completely transcribed.
Mutation and polymorphism
A change in the sequence of the nucleotides with in a gene can occur in an exon or intron. Changes in a codon sequence may result in a different amino acid product. When a sequence change results in an abnormal phenotype it is referred as a mutation.[7] More commonly, sequence changes do not result in abnormal phenotypes, but result in subtle protein variability that is not disease causing, yet results in human variation. These sequence changes are benign polymorphisms.[7] Unlike true mutations, polymorphisms do not segregate with the disease in a family: Patients with the polymorphism may or may not have the disease. With mutations, only clinically affected individuals in a pedigree will have the mutation. Polymorphisms are seen in ethnically matched controls, do not have a biologically significant predicted effect on the protein product of the gene, and in the laboratory don’t show a disruption of cellular function or an animal phenotype.
Type of mutations
A point mutation involves a single nucleotide change. Missense mutations involve substitution of one nucleotide by another, resulting in coding by the codon for a different amino acid. Almost half of all genetic disorders in humans are due to missense mutations.[8] A nonsense mutation occurs when nucleotide substitution results in a premature stop codon resulting in a termination of protein synthesis and a truncated protein.[9] A frameshift mutation occurs when nucleotides are skipped or added. This causes a shift in the reading of the code resulting in an abnormal functionless protein. Intronic mutations may also disrupt proper splicing of exon products.
Mutations can be inherited from one or both parents or occur as a new event. New and spontaneous changes are referred to as de novo or sporadic mutations. A novel (sometimes referred to as “private”) mutation is one that has not been reported previously in the literature.[10] Previously reported mutations are often stored in web-based disease or gene specific universal genome databases accessible globally.
Pedigree
A critical part of a genetic evaluation is the pedigree. Typically, at least three generations are constructed. The common symbols used in a pedigree construction are shown in Figure 1. Generations are represented in Roman numerals with the oldest generation designated as I. In each generation, individual members are annotated with Arabic numerals starting with 1 to the left and proceeding sequentially to the right. The ethnicity on both paternal and maternal sides is documented. Important clinical information obtained and advantages of pedigrees are listed in Table 1.
Inheritance patterns
Mendelian disorders are caused by mutations in single genes and follow the principles of inheritance initially described by Gregor Mendel in 1866. Pedigree examples are shown in Figure 2.
Autosomal dominant [Figure 2a]
Autosomal dominant disorders manifest when mutation involves only one autosomal allele (heterozygous). Both genders are equally affected. Transmission occurs regardless of gender. Offspring affected individuals have a 50% chance of being affected with each conception. Within a family, patients inheriting the mutation may manifest different levels of severity (variable expressivity).[11] This is due to phenotype modification by other genes or environmental factors. For example, in Marfan syndrome, some family members have tall stature and cardiac lesions but no lens subluxation whereas other family members might have significant lens subluxation but lesser cardiac involvement. When an individual has an autosomal dominant mutation but shows no clinical signs of the disease, the individual is non-penetrant. Other examples of autosomal dominant disorders include Stickler syndrome, Best vitelliform macular dystrophy, some pediatric cataracts and anterior segment dysgenesis syndromes and aniridia.
Autosomal recessive [Figure 2b]
Autosomal recessive disorders occur only when both copies of a gene are mutated. When mutations in both copies of the gene are identical the individual is homozygous. If the mutation on each allele is different, the individual is a compound heterozygote.[12] Autosomal recessive disorders are more common in consanguineous matings and constricted gene pools. Therefore it is important to know ethnicity and demographic details of parents of an affected patient. Both genders are affected equally and transmission is independent of gender. For offspring of two carriers (heterozygotes), there is a risk with each pregnancy of 50% that the offspring will be a carrier and 25% that the offspring is affected. This risk increases to 50% for affectation, when one parent is a carrier and the other is affected. This can mimic an autosomal dominant disorder and is referred to as a pseudodominant pattern.[13] Most forms of Leber congenital amaurosis, oculo- cutaneous albinism, and many inherited metabolic disorders are examples of autosomal recessive disorders.
X-Linked recessive [Figure 2c]
In an X-linked recessive disorder, males who have the mutation on their single X chromosome manifest the phenotype. An affected male is hemizygous. Females are carriers but do not manifest the disease as their second X chromosome has a normal copy of the gene (i.e. heterozygous). For offspring of female carriers, the risk of a son being affected is 50% and for daughters, there is a 50% chance of being a carrier, with each conception. There is no male to male transmission as an affected male must use his Y chromosome to create a son. In a pedigree, there is a predominant affliction of males.
All normal females have one X chromosome inactivated in every cell, a process called Lyonization. In normal individuals this is a random process which results, on average, in half of the cells of any organ using one X and half using the other X. A female might be affected with an X-linked recessive disorder either due to skewed X inactivation, if she has a mutation in both copies of the X chromosome gene, or if she has Turner syndrome (XO). In the latter case, like an affected male, she does not have a protective second normal copy of the gene. If the gene mutation frequency is high in a population (e.g. red-green color deficiency), then it becomes not unlikely that affected males could mate with female carriers resulting in daughters who inherit the mutation from each parent thus becoming affected compound heterozygotes or homozygotes ocular albinism, juvenile retinoschisis, some forms of congenital stationary night blindness, and Lowe syndrome are other examples of X-linked recessive disorders.
X-linked dominant [Figure 2d]
In X-linked dominant disorders, only one copy of the X chromosome gene needs to be abnormal for disease to occur. Females who have heterozygous mutations are therefore affected. Males who are hemizygous for the mutation usually have very severe disease or die, as they do not have another copy of the gene. Pedigrees are often characterized by multiple miscarriages of male fetuses and exclusively female affliction. There is no male to male transmission. Affected females have a 50% risk of having an affected son/daughter with each conception. Aicardi syndrome and Incontinentia pigmenti are examples.
Non mendelian patterns of inheritance
Mitochondrial inheritance [Figure 3]
Mitochondria have their own DNA which is distinct from nuclear DNA. All mitochondria are inherited from the mother. The sperm loses its mitochondria soon after entry into the ovum. Hence ovum contributes all mitochondria of the zygote. All children born to affected females have the mutated mitochondrial DNA. Severity of disease in each child depends upon the proportion of abnormal mitochondria in affected tissues (heteroplasmy). Affected male do not transmit the disease to their children. Examples include Kearn-Sayre syndrome, mitochondrial encephalopathy with lactic acidosis and seizure-like episodes (MELAS), and Leber hereditary optic neuropathy (LHON). Females and males are usually affected equally. The one exception is LHON for which there is preferential affectation of males.
Genetic disorders caused by expansion of repeat nucleotides
Repeat nucleotide sequences occur in some human genes; for example, a strand of repeated CAG tri-nucleotides. Within a limit, there is no disease phenotype. An excess number of repeats disrupt the gene function leading to disease. The number of repeat sequences increases in subsequent generations resulting in earlier and more severe manifestations (anticipation). Myotonic dystrophy and Fragile X syndrome are examples.
Digenic and triallelic inheritance
When involvement of two different genes is required to manifest a phenotype the pattern is digenic. Digenic retinitis pigmentosa is caused by a co-existing mutation involving ROM1 and peripherin/ RDS (PRPH) genes.[14] With only the mutation in ROM1 or PRPH, there is no disease. In Bardet-Beidl syndrome, triallelic disease has been observed: For example, co-existing mutations in two copies of one gene and one mutation in a second gene.[15] Polygenic disease involves more than two genes and differs from multifactorial disease in which environmental exposures also contribute to the phenotype.
Chromosomal aberrations
Whole chromosomes, or chromosomes pieces, may be deleted or duplicated. Most chromosomal aberrations are lethal. There's usually no affected parent. As deletions or duplications almost always involve more than one gene, patients usually have multisystem involvement, often including developmental delay. A karyotype examines chromosomes microscopically for deletions/ duplications. Microarray technology allows for detection of submicroscopic variations in DNA copy number.
Deletion/duplication results in a contiguous gene syndrome. WAGR syndrome (Wilms tumor, Aniridia, Genital abnormalities, Retardation) is due to deletion at 11p13 involves several genes including PAX 6 and WT1.[16,17] Deletion of a portion of 13q causes dysmorphic facial features and retinoblastoma.[18] Contiguous gene syndromes should be suspected when patients present with features not usually seen together with a single gene disorder.
Translocation refers to transfer of genetic material from one chromosome to another. Reciprocal translocation involves an exchange of segments between two chromosomes. In balanced translocations there is no net loss of genetic material and the individual is usually normal unless the break causes a disruption of a single gene. Offspring may have an unbalanced translocation with either excess or deficient chromosomal material leading to a phenotype, usually with more than one organ system involved. Karyotype is essential for diagnosis as translocation.
Mosaicism
After fertilization of the egg, the zygote begins to divide. Mutation or chromosomal aberration that develops in one cell thereafter will affect all progeny of that cell. The patient will then have a separate population of cells throughout the body or localized to a region if the mutation occurs late in development. This is mosaicism. Mosaicism can affect somatic cells (somatic mosaicism) or sperm or unfertilized eggs (germ line mosaicism). Mosaicism is not heritable unless germ cells are affected. Clinical phenotypes vary depending on size and distribution of the affected population of cells. Down syndrome may be caused by mosaic trisomy 21.[19] Peripheral blood testing might fail to detect the mutation/ aberration confined to another tissue. For example, chromosomal aberrations may cause hyper pigmentation along the Lines of Blaschko (hypomelanosis of Ito), the developmental streams of ectodermal cells during development that form our integumental system. By performing a biopsy of the hyper pigmented skin, one may detect the mosaic chromosomal aberration that will not be found in the neighboring normal skin.
Loss of heterozygosity
Retinoblastoma was the first disease to be described as secondary to a “two hit process” known as loss of heterozygosity. The first mutation usually occurs in the germ cells such that it is harbored by every cell in the body. Tumors only develop when a “second hit” occurs in a somatic cell, thus causing that cell to contain a mutation in both copies of the gene. Such disorders are inherited as autosomal dominant but behaves as recessive disorder at the molecular level.[20]
Genetic testing
Genetic testing provides opportunity to gain information regarding diagnosis, prognosis, the need to screen other organ systems, surveillance, therapy, counseling and research.[21] Testing may involve karyotype, microarray, fluorescent in situ hybridization (FISH), gene sequencing or other tests. Gene sequencing may be done on a clinical or research basis.[22] Cost, turn around time, reporting and interpretation of results are complex issues. Ethical concerns add further complexity. For example, genetic testing might reveal non paternity or unexpected findings suggestive of another serious underlying genetic disorder for which the patient is asymptomatic. Genetic testing of children,[23–25] adds additional layers of concern, especially in asymptomatic patients (i.e. predictive testing). Other ethical issues may include insurance concerns, prenatal diagnosis,[26] family dynamics, duty to notify patients of future developments, involvement in research, and DNA storage, Counseling is necessary prior to and after genetic testing especially.
Karyotype is mainly useful to detect structural and numerical abnormalities of chromosomes. It is most useful when the patient has multiple congenital anomalies, suggesting involvement of multiple genes, or when a parent has had multiple miscarriages. Disorders may also result from copy number variations smaller than that detectable by karyotype. Microarray or FISH technology may then be useful. FISH uses fluorescent probes that bind to specific chromosome loci. One must have a specific syndromic suspicion to select the desired probe.[27] Microarray screens the entire genome for copy number variations. Microarray cannot detect balanced translocations as there is no net change in DNA content. It also cannot detect single gene mutations. In the future, whole exon sequencing, wherein the coding regions of all genes are sequenced simultaneously, will likely attain utility. The major challenge of this technique and microarray is sorting out physiologic non-disease causing variants from pathogenic changes.
Once a mutation or chromosomal aberration has been identified in the proband, targeted analysis can be performed for at risk family members. Prenatal and pre-implantation genetic diagnosis (PGD),[28] may also be available. Interpretation of genetic test results is often challenging. Nucleotide sequence changes may be pathogenic (mutations), non-pathogenic (polymorphisms) or of unknown significance. Software programs which study possible effects of sequence change on protein structure and behavior and bio-informatics are utilized in predicting the possible role of a particular sequence change. The failure to detect a mutation may be due to incomplete sequencing of a gene, mutation in the gene's promoter region, deletion of the entire gene or mutation in another gene.
Important Resources
Excellent internet resources provide valuable information about genetic eye disorders [Table 2]. Online Mendelian Inheritance in Man (OMIM) is an extensive and searchable compendium of all syndromes and genetic disorders as well as gene mutations chromosomal mapping.
Conclusion
Ophthalmic genetics is a rapidly advancing clinical specialty. Great technological advances in the field of molecular diagnostics have paved the way for better understanding of genotypic- phenotypic correlations. It has also the opened the doors for gene therapy. Ophthalmologists should understand the basics of ophthalmic genetics and its implications in clinical practice, so as to make appropriate referrals to ensure that patients get complete care.
Acknowledgement
Funded in part by The Foerderer Fund (AVL) and Alcon Ocular Genetics Fellowship (ASK).
Footnotes
Source of Support: Funded in part by The Foerderer Fund (AVL) and Alcon Ocular Genetics Fellowship (ASK)
Conflict of Interest: No.
