- Email: [email protected]
Introduction: genetic principles, approaches and technologies
Clinics in Dermatology (2005) 23, 2 – 5
Introduction: genetic principles, approaches and technologies Mary Beth Dinulos, MD* Department of Pediatrics, Section of Genetics and Child Development, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire 03756, USA
Introduction The Human Genome Project has catapulted the field of genetics into the forefront of 21st century medicine. There are well over 200 skin disorders with an identifiable genetic cause. Genetic factors may contribute to most, if not all, skin diseases. Thus, the field of genetics has become a fundamental part of clinical dermatology. The purpose of this chapter is to familiarize the clinician with basic genetic principles and technologies. The chapter progresses from chromosomal and Mendelian genetics to techniques used in gene mapping and identification. These core concepts serve as the language of clinical genetics and are essential to understanding the genetic basis of skin disease.
Glossary Allele: different forms of a gene in the population Allelic heterogeneity: mutations in the same gene can cause variable expression of a disease, or even 2 distinct diseases Aneuploidy: chromosome number is not a multiple of the haploid set (23) – ie trisomy, monosomy Functional cloning: an approach to gene cloning whereby the gene product and its function are used to identify the gene Gonadal (germ line) mosaicism: the presence of two or more different cell lines derived from post-zygotic mutation Linkage analysis: an approach to gene mapping that utilizes DNA markers and determines if their alleles co-segregate with the disease (ie, if markers and disease genes are dlinkedT) Locus: the position of a gene on a chromosome * Tel.: +603 653 6044; fax: +603 653 3585. E-mail address: [email protected]. 0738-081X/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.clindermatol.2004.09.004
Locus heterogeneity: mutations in different genes result in the same disease phenotype LOD score: the logarithm of the odds in favor of linkage Missense mutation: single nucleotide substitution that changes the coding triplet for another amino acid Nonsense mutation: single nucleotide substitution that changes the coding triplet to an termination codon Microdeletion: submicroscopic deletion detectable by fluorescence in situ hybridization (FISH) Penetrance: an all-or-none phenomenon in which a mutation does or does not result in the disease phenotype Phenotype: the outward expression of genes and environment Polymorphism: normal genetic variant in the general population Polyploidy: chromosome number is a multiple of the haploid set (23), but greater than the diploid number (46) – ie triploidy (69) Positional cloning: a traditional approach to gene cloning whereby the region surrounding a linked marker is scanned for the disease gene Silent mutation: single nucleotide substitution that does not change the encoded amino acid Splice site mutation: mutation that alters a conserved, intronic splice donor or acceptor site flanking the coding exons Variable expressivity: the same mutation may result in phenotypes of varying severity and features X-inactivation: inactivation of genes on one X chromosome in each cell of the developing female embryo
Chromosomal disorders The human genome consists of 6 to 7 billion base pairs of DNA organized into 23 pairs of chromosomes.1 The chromosomes are numbered 1 through 22 (referred to as autosomes), with the 23rd pair designated the bsex chromosomesQ ( XX = female, XY = male). Chromosomal abnormalities are classified into two major groups – numerical and structural. Numerical chromosomal abnormalities
Introduction: genetic principles, approaches and technologies are due to extra or missing chromosomes (aneuploidy), or multiples of the haploid set (polyploidy). Aneuploidy may involve either autosomes or sex chromosomes. Turner syndrome (45,X) is an example of aneuploidy involving the sex chromosomes (X chromosome monosomy). These girls typically have short stature, gonadal dysgenesis, cardiovascular and renal abnormalities as well as multiple nevi. Chromosome abnormalities involving chromosomal rearrangements, deletions or duplications are called structural chromosomal abnormalities.2 Deletions are a major component of this category and are typically associated with birth defects, growth retardation and mental retardation. Microdeletion syndromes are caused by submicroscopic deletions detectable by a molecular cytogenetic technique termed fluorescence in situ hybridization or FISH. The majority of males with X-linked ichthyosis have a complete deletion of the steroid sulfatase (STS) gene on chromosome Xp22.32 detectable by FISH.
3
other words, an individual who has inherited an NF1 gene mutation will show the phenotype, but it may be mild, moderate or severe. Penetrance is an all-or-none phenomenon. If a gene is fully penetrant, then an individual who inherits the mutant gene will show the phenotype. Darier’s disease is an example of age-related penetrance.3 Individuals who inherit a ATP2A2 gene mutation are typically normal at birth and do not develop the main features of the disease until early adulthood. Thus, the penetrance of the gene mutation is related to the age of the individual. This phenomenon often presents difficulties when assessing the recurrence risk for various individuals within a family, especially if the age of onset is in the later decades.
Allelic and locus heterogeneity
Each chromosome is composed of 100’s to 1000’s of genes. There is an estimated 30 000 to 40 000 genes in the entire human genome.1 A change or mutation in a gene may result in a recognizable genetic disorder. These disorders are referred to as singe gene or Mendelian disorders. Single gene disorders are typically inherited in an autosomal dominant, autosomal recessive or X-linked fashion.
The GJB6 gene (encoding connexin 30) provides an excellent example of allelic heterogeneity. Different mutations in the GJB6 gene can result in at least three different diseases - autosomal dominant Clouston syndrome (GJB6), autosomal dominant nonsyndromic deafness (DFNA3) or autosomal recessive nonsyndromic deafness (DFNB1).4 In contrast, locus heterogeneity implies that mutations in different genes may result in the same disease phenotype. The clinical phenotype of tuberous sclerosis ( TS) can be caused by mutations in genes on either chromosome 9 (TSC1) or chromosome 16 ( TSC2). Both of these genes produce proteins that may act as tumor suppressors.
Autosomal dominant disorders
De novo mutations and gonadal mosaicism
Autosomal dominant conditions are usually transmitted from an affected parent to child and require only one copy of a mutant gene to convey disease. Males and females are equally affected and can transmit the trait. The child of an affected individual has a 50% chance of inheriting the condition. Examples of autosomal dominant genodermatoses include neurofibromatosis type 1 (due to mutations in the NF1 gene), DarierTs disease (due to mutations in the ATP2A2 gene), and Clouston syndrome (due to mutations in GJB6).
In autosomal dominant conditions in particular, affected individuals may not have an affected parent. We surmise that the disease in these individuals arose from a bde novoQ mutation in the specific gene. Some conditions have a high mutation rate, such as achondroplasia (disproportionate short stature) whereas other genetic conditions have a low mutation rate and are almost always inherited (ie Huntington disease, a neurodegenerative disorder).1 In rare instances, a child with an autosomal dominant disorder (such as NF1) will have unaffected parents, but an affected sibling. This is usually due to gonadal, or germ line, mosaicism. In other words, the gene mutation is present in only some cells of the mother or father, presumably including the gonads. This is possible if a post-zygotic mutation occurred in one of the parents during early embryonic development.
Mendelian disorders
Variable expressivity and penetrance NF1 provides a nice example of variable expressivity.1 All individuals with NF1 will have the minimal clinical diagnostic features by definition, but the expression of the disease can be quite variable. Some individuals are severely affected, while others are quite mildly affected, even within the same family. It is thought that this phenomenon may be due to the effect of modifier genes as well as the environment. However, although NF1 has highly variable expression, the penetrance of this gene is close to 100%. In
Autosomal recessive disorders Autosomal recessive disorders are usually transmitted from unaffected carrier parents to a child and require two copies of a mutant gene to convey disease. Males and females are equally affected. Carrier parents have a 25% chance of
4
M.B. Dinulos
having an affected child with each pregnancy. Examples of autosomal recessive genodermatoses include ataxia-telangiectasia (ATM gene), Netherton syndrome (SPINK5 gene) and Chediak-Higashi syndrome (LYST gene).
eration of thousands of new polymorphic markers, has facilitated this process.
X-linked disorders
Locating a gene, or gene mapping, is usually the first step in cloning a gene. For most disorders, a linkage analysis approach is used to map the location of the disease gene. Linkage analysis works on the premise that two or more gene loci may occupy the same region of a chromosome, and may be transmitted together from one generation to the next. In linkage analysis, the disease gene is unknown and polymorphic loci or markers are used to track the disease through the pedigree. The goal is to find a marker that is inherited in a pattern identical to the disease gene. Such loci are presumed to be linked. In order to determine whether linkage has occurred, statistical analysis must be performed on the data collected. The standard statistical method used in linkage analysis is called the LOD score (bZQ). The LOD score is defined as the logarithm of the odds in favor of linkage. A LOD score of 3.0 or greater is accepted as linkage; a LOD score of 2.0 or less excludes linkage. The LOD score tests the linkage between two markers (one polymorphic locus and one disease locus) and is referred to as two-point analysis. In contrast, the utilization of more than two markers to determine linkage is called multipoint analysis.
X-linked disorders refer to those conditions in which the mutant gene resides on the X chromosome. In X-linked recessive disorders, affected individuals are usually male; however, female carriers may show a mild form of the disorder due to X-inactivation. There is no male-to-male transmission as the father passes the Y chromosome (not the X) to his sons. An affected male passes a mutant X chromosome on to all of his daughters, who will be carriers, and subsequently will have an increased risk of having an affected male child. Examples of X-linked recessive genodermatoses include X-linked hypohidrotic ectodermal dysplasia (EDA gene), Menkes syndrome (ATP7A gene) and Wiskott-Aldrich syndrome (WAS gene). In X-linked dominant disorders, both males and females can be affected; however, males are typically much more severely affected and often there is lethality in utero. CHILD syndrome ( NSDHL gene) and incontinentia pigmenti ( NEMO and IKGKG genes) are examples of X-linked dominant genodermatoses.
Mitochondrial inheritance There are several types of inheritance that do not follow the basic Mendelian patterns. One example is mitochondrial inheritance. Mitochondria are present within the various cells of the body and contain one or more copies of the mitochondrial genome (16 kb circular DNA molecule). Mitochondria are inherited only from the mother, and thus, in the majority of cases, maternal inheritance will be evident in the pedigree. However, one exception includes those mitochondrial disorders that are due to mutations in nuclear DNA (not mitochondrial DNA). In these cases, inheritance will follow the classical Mendelian pattern. An example of a genetic skin condition that is inherited in the classic maternal inheritance pattern of mitochondrial disorders is one type of palmarplantar keratoderma with sensorineural deafness that is caused by a mutation in the MTTS1 mitochondrial gene.
Gene identification: gene mapping and cloning Identification of the gene responsible for a Mendelian disorder may be beneficial in terms of diagnostic testing, treatment options and recurrence risk counseling. Gene identification, however, is an arduous task. Recently, information gained from the Human Genome Project, including the gen-
Gene mapping and linkage analysis
Gene cloning Once the disease gene has been localized to a specific chromosomal region, several approaches may be used to identify the gene. In the functional cloning approach, the gene product and its function are used to aid in the identification of the disease gene. However, this technique is only utilized when the gene product responsible for a genetic condition is known, which is uncommon. Traditionally, the vast majority of genes have been identified by an approach called positional cloning. Using this technique, the region surrounding a linked marker is meticulously scanned for the disease gene. This tedious approach to gene identification has been largely supplanted by the positional candidate approach to gene cloning. In this approach, a search of the disease locus interval for candidate genes is performed. A candidate gene is thought to be a likely candidate for the disease gene based upon it protein product. The information gleaned from the Human Genome Project has allowed great success with the positional candidate approach. The identification of the gene for Clouston syndrome is an example of the positional candidate approach. The gene was mapped to the pericentromeric region of chromosome 13q by linkage analysis. After refinement of the region, two candidate genes were proposed based upon their protein products – connexins. These genes were GJB2 and GJB6,
Introduction: genetic principles, approaches and technologies the latter found to harbor mutations in patients with Clouston syndrome.
Mutational identification Once a candidate gene is isolated, it is examined for the presence of disease-causing (pathogenic) variations in the nucleotide sequence, so-called mutations. Techniques used for mutation screening include single-stranded confirmation polymorphisms (SSCP), conformation-sensitive gel electrophoresis (CSGE), denaturing high-performance liquid electrophoresis (dHPLC) or direct DNA sequencing. Most pathogenic mutations occur in the coding sequence of a gene or at the conserved splice junctions. There are different classes of mutations. Small nucleotide deletions or insertions usually shift the reading frame and introduce premature termination codons. Single nucleotide substitutions may not alter the amino acid sequence (silent mutations), change one amino acid codon with another (missense mutations) or with a termination codon (nonsense mutations). Less common are large gene deletions or genomic rearrangements. Nonsense, frameshift and splice site mutations are often inherited in an autosomal recessive manner and interfere with the production of the encoded protein or impair its function (null alleles). Missense mutations are often autosomal dominantly transmitted since they exert their pathological effects due to a dominant interference with the gene product from the wildtype allele or other interacting proteins.
5
Polymorphisms Of note, most changes in the human genome do not convey disease on their own but represent polymorphisms, or variations among the general population, which may contribute to the great phenotypic variability of the human race. Combinations of polymorphisms may also convey susceptibility for developing complex disorders, such as atopic eczema or psoriasis.
References 1. Jorde LB, Carey JC, Bamshad MJ, et al. Medical genetics. 2nd ed. St. Louis (MO)7 Mosby; 1999. 2. Dinulos MB. Cytogenetics. In: Horwitz M, editor. Basic concepts in medical genetics: a student’s survival guide. 1st ed. New York (NY)7 McGraw-Hill; 2000. p. 83 - 105. 3. Sybert VP. Genetic skin disorders. New York (NY)7 Oxford University Press; 1997. 4. Richard G. Connexin gene pathology. Clin Exp Dermatol 2003;28: 397 - 409.
Online Resources 1. GeneTests: http://www.genetests.org. GeneTestsk provides reliable, easy-to-use and current genetic counseling and testing information for the benefit of families and their healthcare providers. 2. Online Mendelian Inheritance in Man: http://www.ncbi.nlm.nih.gov/ Omim. This database is a catalog of human genes and genetic disorders developed for the World Wide Web by the National Center for Biotechnology Information.
Introduction: genetic principles, approaches and technologies Mary Beth Dinulos, MD* Department of Pediatrics, Section of Genetics and Child Development, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire 03756, USA
Introduction The Human Genome Project has catapulted the field of genetics into the forefront of 21st century medicine. There are well over 200 skin disorders with an identifiable genetic cause. Genetic factors may contribute to most, if not all, skin diseases. Thus, the field of genetics has become a fundamental part of clinical dermatology. The purpose of this chapter is to familiarize the clinician with basic genetic principles and technologies. The chapter progresses from chromosomal and Mendelian genetics to techniques used in gene mapping and identification. These core concepts serve as the language of clinical genetics and are essential to understanding the genetic basis of skin disease.
Glossary Allele: different forms of a gene in the population Allelic heterogeneity: mutations in the same gene can cause variable expression of a disease, or even 2 distinct diseases Aneuploidy: chromosome number is not a multiple of the haploid set (23) – ie trisomy, monosomy Functional cloning: an approach to gene cloning whereby the gene product and its function are used to identify the gene Gonadal (germ line) mosaicism: the presence of two or more different cell lines derived from post-zygotic mutation Linkage analysis: an approach to gene mapping that utilizes DNA markers and determines if their alleles co-segregate with the disease (ie, if markers and disease genes are dlinkedT) Locus: the position of a gene on a chromosome * Tel.: +603 653 6044; fax: +603 653 3585. E-mail address: [email protected]. 0738-081X/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.clindermatol.2004.09.004
Locus heterogeneity: mutations in different genes result in the same disease phenotype LOD score: the logarithm of the odds in favor of linkage Missense mutation: single nucleotide substitution that changes the coding triplet for another amino acid Nonsense mutation: single nucleotide substitution that changes the coding triplet to an termination codon Microdeletion: submicroscopic deletion detectable by fluorescence in situ hybridization (FISH) Penetrance: an all-or-none phenomenon in which a mutation does or does not result in the disease phenotype Phenotype: the outward expression of genes and environment Polymorphism: normal genetic variant in the general population Polyploidy: chromosome number is a multiple of the haploid set (23), but greater than the diploid number (46) – ie triploidy (69) Positional cloning: a traditional approach to gene cloning whereby the region surrounding a linked marker is scanned for the disease gene Silent mutation: single nucleotide substitution that does not change the encoded amino acid Splice site mutation: mutation that alters a conserved, intronic splice donor or acceptor site flanking the coding exons Variable expressivity: the same mutation may result in phenotypes of varying severity and features X-inactivation: inactivation of genes on one X chromosome in each cell of the developing female embryo
Chromosomal disorders The human genome consists of 6 to 7 billion base pairs of DNA organized into 23 pairs of chromosomes.1 The chromosomes are numbered 1 through 22 (referred to as autosomes), with the 23rd pair designated the bsex chromosomesQ ( XX = female, XY = male). Chromosomal abnormalities are classified into two major groups – numerical and structural. Numerical chromosomal abnormalities
Introduction: genetic principles, approaches and technologies are due to extra or missing chromosomes (aneuploidy), or multiples of the haploid set (polyploidy). Aneuploidy may involve either autosomes or sex chromosomes. Turner syndrome (45,X) is an example of aneuploidy involving the sex chromosomes (X chromosome monosomy). These girls typically have short stature, gonadal dysgenesis, cardiovascular and renal abnormalities as well as multiple nevi. Chromosome abnormalities involving chromosomal rearrangements, deletions or duplications are called structural chromosomal abnormalities.2 Deletions are a major component of this category and are typically associated with birth defects, growth retardation and mental retardation. Microdeletion syndromes are caused by submicroscopic deletions detectable by a molecular cytogenetic technique termed fluorescence in situ hybridization or FISH. The majority of males with X-linked ichthyosis have a complete deletion of the steroid sulfatase (STS) gene on chromosome Xp22.32 detectable by FISH.
3
other words, an individual who has inherited an NF1 gene mutation will show the phenotype, but it may be mild, moderate or severe. Penetrance is an all-or-none phenomenon. If a gene is fully penetrant, then an individual who inherits the mutant gene will show the phenotype. Darier’s disease is an example of age-related penetrance.3 Individuals who inherit a ATP2A2 gene mutation are typically normal at birth and do not develop the main features of the disease until early adulthood. Thus, the penetrance of the gene mutation is related to the age of the individual. This phenomenon often presents difficulties when assessing the recurrence risk for various individuals within a family, especially if the age of onset is in the later decades.
Allelic and locus heterogeneity
Each chromosome is composed of 100’s to 1000’s of genes. There is an estimated 30 000 to 40 000 genes in the entire human genome.1 A change or mutation in a gene may result in a recognizable genetic disorder. These disorders are referred to as singe gene or Mendelian disorders. Single gene disorders are typically inherited in an autosomal dominant, autosomal recessive or X-linked fashion.
The GJB6 gene (encoding connexin 30) provides an excellent example of allelic heterogeneity. Different mutations in the GJB6 gene can result in at least three different diseases - autosomal dominant Clouston syndrome (GJB6), autosomal dominant nonsyndromic deafness (DFNA3) or autosomal recessive nonsyndromic deafness (DFNB1).4 In contrast, locus heterogeneity implies that mutations in different genes may result in the same disease phenotype. The clinical phenotype of tuberous sclerosis ( TS) can be caused by mutations in genes on either chromosome 9 (TSC1) or chromosome 16 ( TSC2). Both of these genes produce proteins that may act as tumor suppressors.
Autosomal dominant disorders
De novo mutations and gonadal mosaicism
Autosomal dominant conditions are usually transmitted from an affected parent to child and require only one copy of a mutant gene to convey disease. Males and females are equally affected and can transmit the trait. The child of an affected individual has a 50% chance of inheriting the condition. Examples of autosomal dominant genodermatoses include neurofibromatosis type 1 (due to mutations in the NF1 gene), DarierTs disease (due to mutations in the ATP2A2 gene), and Clouston syndrome (due to mutations in GJB6).
In autosomal dominant conditions in particular, affected individuals may not have an affected parent. We surmise that the disease in these individuals arose from a bde novoQ mutation in the specific gene. Some conditions have a high mutation rate, such as achondroplasia (disproportionate short stature) whereas other genetic conditions have a low mutation rate and are almost always inherited (ie Huntington disease, a neurodegenerative disorder).1 In rare instances, a child with an autosomal dominant disorder (such as NF1) will have unaffected parents, but an affected sibling. This is usually due to gonadal, or germ line, mosaicism. In other words, the gene mutation is present in only some cells of the mother or father, presumably including the gonads. This is possible if a post-zygotic mutation occurred in one of the parents during early embryonic development.
Mendelian disorders
Variable expressivity and penetrance NF1 provides a nice example of variable expressivity.1 All individuals with NF1 will have the minimal clinical diagnostic features by definition, but the expression of the disease can be quite variable. Some individuals are severely affected, while others are quite mildly affected, even within the same family. It is thought that this phenomenon may be due to the effect of modifier genes as well as the environment. However, although NF1 has highly variable expression, the penetrance of this gene is close to 100%. In
Autosomal recessive disorders Autosomal recessive disorders are usually transmitted from unaffected carrier parents to a child and require two copies of a mutant gene to convey disease. Males and females are equally affected. Carrier parents have a 25% chance of
4
M.B. Dinulos
having an affected child with each pregnancy. Examples of autosomal recessive genodermatoses include ataxia-telangiectasia (ATM gene), Netherton syndrome (SPINK5 gene) and Chediak-Higashi syndrome (LYST gene).
eration of thousands of new polymorphic markers, has facilitated this process.
X-linked disorders
Locating a gene, or gene mapping, is usually the first step in cloning a gene. For most disorders, a linkage analysis approach is used to map the location of the disease gene. Linkage analysis works on the premise that two or more gene loci may occupy the same region of a chromosome, and may be transmitted together from one generation to the next. In linkage analysis, the disease gene is unknown and polymorphic loci or markers are used to track the disease through the pedigree. The goal is to find a marker that is inherited in a pattern identical to the disease gene. Such loci are presumed to be linked. In order to determine whether linkage has occurred, statistical analysis must be performed on the data collected. The standard statistical method used in linkage analysis is called the LOD score (bZQ). The LOD score is defined as the logarithm of the odds in favor of linkage. A LOD score of 3.0 or greater is accepted as linkage; a LOD score of 2.0 or less excludes linkage. The LOD score tests the linkage between two markers (one polymorphic locus and one disease locus) and is referred to as two-point analysis. In contrast, the utilization of more than two markers to determine linkage is called multipoint analysis.
X-linked disorders refer to those conditions in which the mutant gene resides on the X chromosome. In X-linked recessive disorders, affected individuals are usually male; however, female carriers may show a mild form of the disorder due to X-inactivation. There is no male-to-male transmission as the father passes the Y chromosome (not the X) to his sons. An affected male passes a mutant X chromosome on to all of his daughters, who will be carriers, and subsequently will have an increased risk of having an affected male child. Examples of X-linked recessive genodermatoses include X-linked hypohidrotic ectodermal dysplasia (EDA gene), Menkes syndrome (ATP7A gene) and Wiskott-Aldrich syndrome (WAS gene). In X-linked dominant disorders, both males and females can be affected; however, males are typically much more severely affected and often there is lethality in utero. CHILD syndrome ( NSDHL gene) and incontinentia pigmenti ( NEMO and IKGKG genes) are examples of X-linked dominant genodermatoses.
Mitochondrial inheritance There are several types of inheritance that do not follow the basic Mendelian patterns. One example is mitochondrial inheritance. Mitochondria are present within the various cells of the body and contain one or more copies of the mitochondrial genome (16 kb circular DNA molecule). Mitochondria are inherited only from the mother, and thus, in the majority of cases, maternal inheritance will be evident in the pedigree. However, one exception includes those mitochondrial disorders that are due to mutations in nuclear DNA (not mitochondrial DNA). In these cases, inheritance will follow the classical Mendelian pattern. An example of a genetic skin condition that is inherited in the classic maternal inheritance pattern of mitochondrial disorders is one type of palmarplantar keratoderma with sensorineural deafness that is caused by a mutation in the MTTS1 mitochondrial gene.
Gene identification: gene mapping and cloning Identification of the gene responsible for a Mendelian disorder may be beneficial in terms of diagnostic testing, treatment options and recurrence risk counseling. Gene identification, however, is an arduous task. Recently, information gained from the Human Genome Project, including the gen-
Gene mapping and linkage analysis
Gene cloning Once the disease gene has been localized to a specific chromosomal region, several approaches may be used to identify the gene. In the functional cloning approach, the gene product and its function are used to aid in the identification of the disease gene. However, this technique is only utilized when the gene product responsible for a genetic condition is known, which is uncommon. Traditionally, the vast majority of genes have been identified by an approach called positional cloning. Using this technique, the region surrounding a linked marker is meticulously scanned for the disease gene. This tedious approach to gene identification has been largely supplanted by the positional candidate approach to gene cloning. In this approach, a search of the disease locus interval for candidate genes is performed. A candidate gene is thought to be a likely candidate for the disease gene based upon it protein product. The information gleaned from the Human Genome Project has allowed great success with the positional candidate approach. The identification of the gene for Clouston syndrome is an example of the positional candidate approach. The gene was mapped to the pericentromeric region of chromosome 13q by linkage analysis. After refinement of the region, two candidate genes were proposed based upon their protein products – connexins. These genes were GJB2 and GJB6,
Introduction: genetic principles, approaches and technologies the latter found to harbor mutations in patients with Clouston syndrome.
Mutational identification Once a candidate gene is isolated, it is examined for the presence of disease-causing (pathogenic) variations in the nucleotide sequence, so-called mutations. Techniques used for mutation screening include single-stranded confirmation polymorphisms (SSCP), conformation-sensitive gel electrophoresis (CSGE), denaturing high-performance liquid electrophoresis (dHPLC) or direct DNA sequencing. Most pathogenic mutations occur in the coding sequence of a gene or at the conserved splice junctions. There are different classes of mutations. Small nucleotide deletions or insertions usually shift the reading frame and introduce premature termination codons. Single nucleotide substitutions may not alter the amino acid sequence (silent mutations), change one amino acid codon with another (missense mutations) or with a termination codon (nonsense mutations). Less common are large gene deletions or genomic rearrangements. Nonsense, frameshift and splice site mutations are often inherited in an autosomal recessive manner and interfere with the production of the encoded protein or impair its function (null alleles). Missense mutations are often autosomal dominantly transmitted since they exert their pathological effects due to a dominant interference with the gene product from the wildtype allele or other interacting proteins.
5
Polymorphisms Of note, most changes in the human genome do not convey disease on their own but represent polymorphisms, or variations among the general population, which may contribute to the great phenotypic variability of the human race. Combinations of polymorphisms may also convey susceptibility for developing complex disorders, such as atopic eczema or psoriasis.
References 1. Jorde LB, Carey JC, Bamshad MJ, et al. Medical genetics. 2nd ed. St. Louis (MO)7 Mosby; 1999. 2. Dinulos MB. Cytogenetics. In: Horwitz M, editor. Basic concepts in medical genetics: a student’s survival guide. 1st ed. New York (NY)7 McGraw-Hill; 2000. p. 83 - 105. 3. Sybert VP. Genetic skin disorders. New York (NY)7 Oxford University Press; 1997. 4. Richard G. Connexin gene pathology. Clin Exp Dermatol 2003;28: 397 - 409.
Online Resources 1. GeneTests: http://www.genetests.org. GeneTestsk provides reliable, easy-to-use and current genetic counseling and testing information for the benefit of families and their healthcare providers. 2. Online Mendelian Inheritance in Man: http://www.ncbi.nlm.nih.gov/ Omim. This database is a catalog of human genes and genetic disorders developed for the World Wide Web by the National Center for Biotechnology Information.