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How Genetic Defects Are Identified
How Genetic Defects Are Identified SHIGERU SASSA, MD, PhD KAZUMICHI FURUYAMA, MD, PhD
A
bout one hundred years ago, Archibald Garrod advanced the concept of inborn errors of metabolism. Until then, alkaptonuria was thought to be due to the action of microorganisms in the human intestine, an idea that Garrod seriously questioned. He thought that alkaptonuria was due to a chemical aberration, and coined the term an inborn error of metabolism. Later, he recognized additional inborn errors of metabolism, in which he included porphyrias.1 Since then, medical research has made enormous progress, particularly through the biochemical and genetic research on inherited disorders. In recent years, the main stream of medicine has also developed a molecular aspect, through the application of molecular biological approaches to these questions. The research involving the porphyrias is a good example of the developmental history of genetic studies of inherited metabolic disorders. Garrod’s concept of inborn errors of metabolism was subsequently supported by the one gene-one enzyme hypothesis,2 which was originally found in yeast but extended to all cells to cover general proteins, including nonenzymatic proteins and complex proteins composed of nonidentical polypeptide subunits. A cistron, a term that refers to the functional unit of DNA that controls the structure of a single polypeptide, is now considered as the functional unit of inheritance, thus the one gene-one enzyme hypothesis has been redefined as the one cistron-one polypeptide concept. Analysis of cistronic mutations and their abnormal consequences are the target of genetic studies.
Diagnosis of Inherited Porphyrias Porphyrias may be first suspected by the characteristic clinical history and biochemical features of the patient. Establishment of the diagnosis of porphyrias, however, depends on characteristic findings for these disorders, which are obtained by several different methods. First, diagnosis of clinically manifest porphyrias can be made by the determination of porphyrins and their precursors that are excreted in excess into urine or feces or are From the Laboratory of Biochemical Hematology, The Rockefeller University, New York, New York. Address correspondence to Dr. Shigeru Sassa, Laboratory of Biochemical Hematology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399. © 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
elevated in erythrocytes. This is still the key for the practical diagnosis of an acute porphyria attack. It is also important in the evaluation of the efficacy of treatment. Secondly, more specific enzymatic assays should be used to distinguish among similar porphyrias, or to differentiate a porphyria from other porphyria-like syndromes. These assays will recognize not only patients with overt porphyrias, but also latent gene carriers. There are two types of latent gene carriers, one that is entirely latent and the other that is clinically latent but biochemically active. It is unclear whether the biochemically active carriers will develop an acute porphyria attack, but they should be considered at high risk for such a condition, and they should be monitored carefully. These enzymatic assays, combined with metabolite determinations, offer essential information for the genetic counseling of family members of the patient. Unfortunately, enzyme assays are not available for all porphyrias. Commercial assays are available only for ALA dehydratase (ALAD) and PBG deaminase (PBGD) activities using blood samples. These cytosolic enzymes appear to be stable in blood. In contrast, mitochondrial enzyme assays are difficult to perform using whole blood samples, because there are few mitochondria in erythrocytes. While there are commercial assays for mitochondrial enzymes such as coproporphyrinogen oxidase activity in blood, their significance is therefore questionable. Thirdly, using recombinant DNA techniques, gene mutations can be analyzed directly, and the exact disease-responsible mutations can be defined. This is obviously the ultimate goal of genetic diagnosis. Defined gene mutations, such as point mutations or gene deletions, are very useful for screening for the gene defect in the patient’s family. Specific diagnosis such as for a founder mutation can also be made by this technique. It would also be very useful for prenatal diagnosis. On the other hand, molecular genetics does not predict which gene carriers may develop clinical porphyrias. Thus in the practical management of porphyrias, clinical and biochemical assays remain crucial.3
Molecular Defects in the Porphyrias Porphyrias are due to inherited defects of one of the enzymes in the heme biosynthetic pathway, with the 0738-081X/98/$19.00 PII S0738-081X(97)00203-4
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Figure 1. A scheme for DNA diagnosis of porphyrias.
exception of the first enzyme, ALA synthase (ALAS). These enzyme deficiencies can be either partial or nearly complete. As a result, abnormally elevated levels of porphyrins, and/or their precursors accumulate in tissues, leading to neurological disturbances and/or photosensitive reactions; and they are also excreted in excess into urine and stool.3 Complementary DNA (cDNA) clones have been isolated for all eight enzymes in the heme biosynthetic pathway. Genomic sequences have also been reported for these enzymes, except uroporphyrinogen III synthase (UROIIIS). Some enzymes have been shown to occur as tissue-specific isozymes, for example, ALAS and PBGD,4,5 or as tissue-specific mRNAs such as ALAD.6 The isozyme-specificity occurs principally between the erythroid tissues and the nonerythroid tissues such as the liver, suggesting that there may be significant differences in the regulation of gene expression of these enzymes between these tissues.7 cDNA clones from many patients with various forms of the porphyrias have been obtained, their sequences determined, and the properties of some of their phenotypes studied using heterologous expression systems.
Cloning Gene Defects in the Porphyrias To define a mutation, it is necessary to determine the structure of its normal gene, and to elucidate its regulation and its phenotype in normal cells. For such a purpose, (1) the sequence of cDNA and structure of genomic DNA, (2) the regulatory mechanism of gene expression in normal cells must be elucidated, and (3)
the phenotypic consequences of gene expression should be examined. Since there are a number of excellent technical protocols for genetic analysis,8,9 only the general aspects of this approach are summarized below, and an outline of common approaches is shown in Fig 1. There are also excellent reviews specifically dealing with mutation detection.10 –12
Cloning of cDNA and Genome DNA cDNA Cloning Cloning refers to the process by which a DNA molecule is joined to another DNA molecule (termed a cloning vector) that can replicate when introduced into an appropriate host, usually a bacterium or yeast. A cDNA is a reverse copy of mRNA, which is manufactured in vitro from mRNA by reverse transcriptase, cDNA cloning is a useful strategy to obtain specific information about the desired mRNA. Because mRNA is a nuclear RNA transcript from which introns have been spliced out, cDNA represents principally the protein-coding sequence, plus a short stretch of an upstream (59 untranslated) as well as a downstream (39 untranslated) sequence of mRNA that includes a poly A tail(s). The most common approach for cDNA cloning utilizes nucleic-acid hybridization; however, it requires knowledge of the sequences being sought. Replicas of plasmid colonies or phage plaques are lysed on nitrocellulose membranes, denatured, immobilized, and then detected using radiolabeled single-stranded DNA fragments specific for a gene sequence of interest. In some cases, part of the gene may already have been
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cloned, and this information can be used to search clones that contain additional sequences flanking the original clone. In other cases, a closely related gene (a homologous gene) may already have been cloned and can be used in screening for the gene that has sufficient homology with the starting clone under an appropriate condition. It is possible to perform sequence analysis of a purified protein by an automated protein sequencer. In this manner, amino acid sequences can be determined from a very small amount of protein, for example, 50 pmol; and sequence information can be used to generate a corresponding RNA sequence of its mRNA by using the known triplet codon for each amino acid. Because there is redundancy in the triplet codon where one amino acid can be encoded by multiple codons, there are multiple DNA sequences encoding a short stretch of a polypeptide that are slightly different from each other. This problem can be corrected by using degenerate pools of oligonucleotides that take this redundancy, or the ‘wobbling’, of triplets into account. These oligonucleotides can be used as probes to screen the cDNA library. When a candidate cDNA is cloned, its nucleotide sequences should be determined. With the help of DNA sequence-analysis computer programs, sequence data can be used (1) to determine all known restriction enzyme sites within the gene, and (2) to predict the protein product encoded by the gene. This method will help to determine whether the cloned cDNA is a bona fide clone of interest. cDNA clones can be used as a probe to search gene expression in various organs, and to define tissue- and/or cell-specific expression by Northern blot analysis, or in situ hybridization. Additionally, it is also useful in screening cDNA and genomic libraries, and patient’s cell cDNA libraries. Genomic DNA Cloning Generally, cDNA is used as a probe to isolate its genomic DNA. The genomic DNA is always larger than its cDNA, because it contains introns. Thus, while the overall technique of isolating genomic DNA is similar to that used in cDNA cloning, many DNA fragments are usually needed for genomic DNA cloning. When a genomic DNA is cloned, its nucleotide sequences should be determined. The nucleotide sequences thus obtained provide invaluable information on introns in genomic DNA. Nucleotide sequence analysis of a genomic DNA permits the analysis of control regions such as promoter and/or regulatory regions in DNA. Such information is essential for elucidating the mechanism of gene expression, because these regions contain DNA sequences that may bind specific nuclear proteins that regulate transcription (transcription factors), and confer upon the gene temporal or tissue-specific control.
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Tissue-Specific Gene Regulation Of the estimated approximately 100,000 human genes, many genes are transcribed ubiquitously in all tissues, and they are called “house-keeping genes”. The housekeeping genes are important in regulating essential function of cellular proteins, although their specific roles in cellular function remains unclear for most of them. In contrast, there are certain genes whose transcription is restricted to a specific tissue lineage, or to a specific time in organ development, or they are induced in response to a specific humoral factor(s), such as hormones. In these genes, tissue-, time-, or inductionspecific transcription is not due to a difference in the genotype, but rather to a difference in the mechanism(s) that control gene expression. Tissue-specific gene expression can be controlled at variable stages, for example, directly at the level of transcription (transcriptional control), at the level of splicing (posttranscriptional control), or at the level of translation (translational control). While all these mechanisms may contribute, by far the most common mechanism of control is at the level of transcription. It has also been shown that gene expression is critically regulated by a hierarchical control of transcription at the level of the structure of the chromosome (chromatin structure) and by combinational interaction between a limited number of cell-specific transcriptional regulatory proteins and RNA polymerase. The initial step of RNA synthesis is an association of RNA polymerase and general transcription factors with the promoter of the gene to form a basal transcription complex that is to be transcribed. The basal transcription complex acts as a target for transcriptional activation, or repression by tissue-specific transcriptional regulatory proteins. These tissue-specific transcription factors also bind specifically to other sequences in the control region of the genes, which then results in regulated tissue-specific gene expression. The ability of transcription factors, either general or tissue-specific, to interact directly with DNA is fundamental to such control of gene transcription.
DNA Diagnosis Nature of the Genetic Materials A tissue sample for genetic analysis should be obtained from both the patient and the family members. If an abnormal enzyme is expressed in a tissue-specific manner, it is essential to analyze mRNA in that tissue. Because it is often difficult to obtain a tissue sample by biopsy and nucleated cells from blood can be used instead, blood is the most common source of genetic materials. For example, mononuclear cells can be separated by centrifugation, and they can be used to prepare genomic DNA. Lymphocytes can be separated by Ficoll-Hypaque gradient centrifugation; and they can be used by stimulation with phytohemagglutinin and
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pokeweed mitogen or by transformation by EpsteinBarr (EB) virus, which permits establishment of an immortal cell line. The use of cell cultures for the diagnosis of genetic disorders, including porphyrias, is now very popular.13–15 Skin fibroblast cultures and EB virus transformed lymphoblastoid cells are the standard in this technique, though cells from other tissues have also been used. The advantage of cultured cells are: (1) they are readily established from patient’s tissues; (2) they can be propagated, or stored for an indefinite period, thus avoiding repeated sampling from patients; (3) they are often useful for complex biochemical analysis, including enzyme assays; (4) they are suitable for diagnosis of all mutations at a DNA level, and many at a RNA level; and (5) they are the only material that can be used for radioisotopic studies, such as protein turnover.
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by autoradiography and a color reaction, respectively. This method reveals details of sequence organization. Thus, in many diseases that are due to a large deletion of the gene, or rearrangement between several genes, Southern blot analysis is a very useful technique to detect such changes. NORTHERN BLOT HYBRIDIZATION This technique is analogous to Southern blotting of DNA, except RNA is transferred to a nitrocellulose filter (Northern transfer) on which it can be hybridized to complementary (radioactive or biotinylated) single-stranded DNA or RNA probes. If a large amount of mRNA (in mg quantities) can be prepared, Northern blot analysis is the method of choice to identify a major alteration in mRNA, such as a large deletion or an insertion, and to determine the extent of expression of an abnormal mRNA.
cDNA CLONING FROM THE PATIENT’S mRNA cDNA is made from the patient’s mRNA by using reverse transcriptase. This procedure involves generation of a single strand of cDNA, very often begun (primed) by large tracts of deoxythymidine [poly(dT)], which physically associates with, or anneals to the poly (A) tract at the 39 end of mRNA (the site of polyadenylation) by its DNA complementarity. Alternatively, priming can be carried out by a large pool of random oligonucleotides (random priming), which will anneal all along the RNA molecule. A single-stranded cDNA is then used as a template to generate double-stranded cDNA by the use of DNA polymerase. These double-stranded cDNAs are then inserted into a l phage, or a plasmid DNA. Because as only one recombinant l phage and a plasmid can enter each bacteria, each individual cDNA is replicated in a single bacteria; and this permits clonal propagation of a single cDNA in a clone of bacteria (cDNA clone). These bacteria carry cDNAs representing every expressed RNA from a cell, or a tissue of interest (cDNA library). Alternatively, a cDNA of interest can be directly cloned into a plasmid if its sequence information is available from a DNA database. When cDNA is made from the mRNA of a patient, information on a mutant cDNA can be obtained by screening its library with normal cDNA followed by polymerase chain reaction (PCR). Cloning of the patient’s cDNA is a very useful technique for isolating the mRNA of interest.
POLYMERASE CHAIN REACTION The polymerase chain reaction (PCR) is a technique used for enzymatic in vitro amplification (PCR amplification) of specific DNA sequences without utilizing conventional procedures, that is, molecular cloning. It is an extremely powerful technique for selective and rapid amplification of a target DNA, or an RNA sequence. It permits the amplification of a DNA (genome or cDNA) fragment situated between two convergent primers, and it utilizes oligonucleotide primers that hybridize to opposite strands. Primer extension proceeds inward across the region between the two primers. The product of DNA synthesis of one primer serves as a template for the other primer. A series of reactions starting with denaturation, annealing and DNA synthesis is cycled for many times, resulting in an exponential increase in the number of copies of the region bounded by the primers. The sequence of the double-stranded DNA fragment can then be identified by hybridization to allele-specific oligonucleotide probes representing various alleles studied or whose sequence can be determined. The advantages of this method are: (1) only a very small amount of DNA is needed; and (2) results are achieved quickly, typically in less than one day. On the other hand, it is essential to have sequence information for the synthesis of specific oligonucleotide primers for PCR. When PCR is performed following a reverse transcription (RT) reaction, the combination is called “RT-PCR”; and this method is extremely useful in the amplification and analysis of a cDNA of interest.
SOUTHERN BLOT HYBRIDIZATION The Southern blot analysis is a common approach to DNA-based diagnosis. It involves transfer of single-stranded, restricted DNA fragments, separated in an agarose gel, to a binding matrix, for example, nitrocellulose membrane (Southern transfer). DNA fragments are then analyzed by hybridization to radioactive or biotinylated singlestrand DNA, or RNA probes; and hybrids are detected
DNA SEQUENCING The most precise method for the characterization of a segment of DNA is to obtain its exact nucleotide sequence. The procedure consists of (1) selection of DNA clones, (2) treatment of these clones individually by base-specific reactions, (3) separation of the reaction products by size on sequencing gels, and (4) computer-assisted reading of separated bands as a sequence of A, C, G, and T. There are two major meth-
Techniques for DNA Analysis
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ods for nucleotide sequencing, one of which utilizes base-specific chemical cleavage, while the other uses termination of in vitro DNA synthesis.16,17 The former requires at least partial restriction mapping and is more labor-intensive, while the latter is less difficult and is now commonly used for most applications. Abnormal variations in DNA sequences can be identified in this manner and then used to detect mutations in family members, and other individuals. DNA sequence analysis can be performed with products of a PCR reaction (direct sequencing), which permits rapid determination of nucleotide sequences of PCR products. ELECTROPHORESIS OF SINGLE-STRANDED OR DENATURED DNA Structural variation in single-stranded DNA due to sequence-dependent intrastrand secondary structure is called single-stranded conformational polymorphism (SSCP). It is detectable by electrophoresis in nondenaturing gels, and it is useful for point mutation scanning.18 Point mutation scanning is also possible by a method called denaturing gradient gel electrophoresis (DGGE), which utilizes electrophoresis in a gel containing a gradient of denaturants (urea and formamide), allowing detection of sequence-dependent differences under denaturation conditions.19 Both SSCP and DGGE permit quick identification of differences in DNA sequences. Because these methods also detect normal DNA variation or polymorphism, as well as DNA sequence changes that are related to other diseases, nucleotide sequences of the abnormal fragment must be determined to ultimately confirm that the detected change is a mutation responsible for the disease. ALLELE-SPECIFIC OLIGONUCLEOTIDE (ASO) HYBRIDIZATION ANALYSIS This technique uses two synthetic oligonucleotide probes, one specific for a normal gene and the other specific for a mutation identified in the patient. They are used as hybridization probes to determine whether a patient and his or her family members have the normal gene or the abnormal gene. This analysis is very helpful for the diagnosis of a genetic disease that typically arises from a single predominant mutation, such as in the porphyrias. When this technique is used with a DNA segment that has been selectively amplified by PCR, it provides a rapid and an accurate DNAbased diagnosis. On the other hand, application of ASO hybridization analysis is limited in many cases to a specific known mutation occurring in each pedigree. RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLP) There are variations in the length of restriction fragments, termed restriction fragment length polymorphisms (RFLP) that are generated after DNA is digested with different restriction enzymes, and analyzed with a variety of cloned DNA probes that detect specific homologous DNA fragments. RFLPs are due to either introduction or removal of restriction sites, or sequence
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deletions, additions, and rearrangements that affect the length of DNA between restriction sites. They represent codominant, typically neutral, genetic markers; and they can be used to construct detailed genetic linkage maps (RFLP linkage maps), to follow the mode of inheritance of genetic diseases and traits, and to examine variation between and within populations. Because RFLPs are not rare events, occurring every 200 –300 bp in the human genome, and they are inherited in simple Mendelian fashion, they provide a very rapid and efficient method for studying the inheritance of a relevant allele over the generations by following the inheritance of detectable RFLPs linked to it. Similar limitations, however, apply as in the case of ASO hybridization analysis of a mutation. A TYPICAL DNA DIAGNOSIS Generally, DNA diagnosis is performed by using a combination of several methods described above. A typical example of DNA diagnosis is shown here for d-aminolevulinate dehydratase (ALAD) porphyria.20 1. Lymphocytes of a patient with ALAD porphyria were isolated and transformed by EB virus to create an immortalized cell line. Then, mRNA was prepared from these cells, and cDNA was synthesized with reverse transcriptase using an oligo(dT) primer. 2. cDNA was amplified by PCR using the primers that correspond to the 59-untranslated and the 39-untranslated regions of ALAD cDNA. The amplified fragment was cloned into a plasmid vector and nucleotide sequence analysis was performed. To confirm the mutation, direct sequencing analysis was performed using the patient’s mRNA. 3. Following the determination of the mutations in the proband, ALAD phenotype analysis in the proband’s family were carried out using RT-PCR combined with ASO hybridization analysis. 4. As a result, a silent mutation (without an accompanying amino acid change) and a mutation with an amino acid substitution were found on one allele. In addition, three independent silent mutations and another mutation were found on the other allele. cDNA cloning as shown in this example is useful if several mutations are found in both alleles and when the coding region of the gene is not huge. Types of Mutations A number of mutations have been described in the porphyrias. While gross mutations that encompass the entire genome, or deletion of a very large segment of a chromosome have not been reported, essentially all other types of mutations have been described in the porphyrias (Table 1). MISSENSE MUTATION If codons are affected by a single or a few nucleotide substitution, such a mutation is
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Table 1.
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Types of Mutations and Prenatal Diagnosis Reported in the Porphyrias
Missense mutation Translational mutation* RNA splicing mutation Prenatal diagnosis
ADP
AIP
CEP
PCT
HEP
HCP
VP
EPP
1 2 2 2
1 1 1 1
1 1 2 1
1 1 1 2
1 1 2 2
1 1 1 2
1 1 2 2
1 1 1 2
* Including nonsense, frame-shift (insertion, deletion) mutations and gene deletion.
called a missense mutation. Missense mutations have been described in all the porphyrias, and they are by far more common than other kinds of mutations. A point mutation was identified in most cases of missense mutation. It is interesting to note that, in a patient with hepatoerythropoietic porphyria (HEP) and a patient with familial porphyria cutanea tarda (PCT) from different pedigrees, a very rare but identical three-sequential-base substitution was detected; however, it resulted in only one amino-acid change.21,22 TRANSLATIONAL
(NONSENSE MUTATION AND Mutations affecting translation have also frequently been described in the porphyrias. Nonsense mutation affects normal translation by a nucleotide substitution, which changes a triplet codon for an amino acid to a stop codon. It results in a truncated protein that is often unstable and readily degraded. A frameshift mutation is either deletion or insertion of one or a few nucleotides in the coding region of a gene. These mutations change the coding message for all subsequent codons, thus completely altering the aminoacid sequence from the mutation site. In many instances, a frameshift mutation results in premature termination of the protein, because it often creates a stop codon.
2.
3.
MUTATION
FRAMESHIFT MUTATION)
RNA SPLICING MUTATION RNA splicing is critical to normal gene expression, because introns must be precisely spliced to produce the appropriate mRNA for translation into protein. RNA splicing may be affected by mutation, either by altering a normal splice site at the intron and exon junction, or by creating a new splice site within introns or exons. The 59 end of the intron always begins with nucleotides GT, and the 39 end of the intron always has the nucleotides AG. Mutations that alter the AG or GT at the splice junction site lead to complete absence of normal splicing, which results in an unstable mRNA that is rapidly degraded. Thus, the protein product of this mutation is usually undetectable.
4.
5.
6.
7.
IMPORTANT ASPECTS A number of important aspects have been elucidated by genetic studies of porphyrias. 1. In every porphyria, the molecular defect of the enzyme is heterogeneous. In other words, there is more than one mutation resulting clinically in a single porphyria disease. For example, there are more than 100 mutations found in the PBGD gene in patients
8.
with acute intermittent porphyria (AIP). There are few founder effect mutations, while a pedigree-specific mutation of the enzyme gene is very common. Many different kinds of mutations have been found in the porphyrias, for example, promoter mutations, splicing mutations, consensus sequence mutations, mutations producing nonfunctional mRNAs, mutations resulting in unstable proteins, gene deletions, and so forth (Table 1). Even among the dominant form of porphyrias, a homozygous form of the disease can be found in a few patients. The homozygous form appears to be due to mutations with lesser pathophysiological effects than that found in the heterozygous form of the disease. A subset of homozygous diseases among commonly autosomal dominant diseases has been observed in AIP, hereditary coproporphyria (HCP), and variegate porphyria (VP). Clinically homozygous mutations can be due either to a homoallelic or a heteroallelic mutation; however, heteroallelic mutation, that is, compound heterozygosity for two separate mutations, is far more common than homoallelic mutation. Tissue-specific regulation of heme pathway enzymes may likely be the basis for the tissue-specific expression of different porphyrias. It probably explains why some porphyrias occur principally in the liver, while others occur in erythroid tissues. In the case of PBGD, a mutation affecting the splicing of the first intron results in an abnormal enzyme in nonerythroid cells, while the same mutation has no effect on the erythroid-specific PBGD, because the transcription of the erythroid PBGD mRNA utilizes a second AUG which is located approximately 3 kb downstream of the mutated site.5 Thus, even in a single form of acute hepatic porphyria, there may be distinct tissue-specific expression of the disease, depending on the type of mutation. Most of the acute hepatic porphyrias, such as AIP, HCP, and VP, require an additional factor(s) for their clinical expression.3 Such a factor can be either genetic or environmental in nature. Thus, porphyrias are not only inborn errors of metabolism, but also diseases in which environmental factors have an immense impact on their gene expression. Some hepatic porphyria-like symptoms can be elicited by environmental chemicals that strongly inhibit
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heme pathway enzymes. For example, porphyria cutanea tarda (PCT) may be caused by hexachlorobenzene in normal individuals. Genetic analysis has elucidated the complex nature of molecular defects in the porphyrias and the possible consequences of their gene defects on cellular function. It also offers an explanation as to why hepatic porphyrias do not accompany anemia, or conversely why erythropoietic porphyrias do not exhibit the defective drug metabolism that largely reflects the function of hepatic cytochrome P450.
Gene-Environment Interaction While inherited porphyrias are due to a gene defect of the enzymes in the heme biosynthetic pathway, their clinical phenotype often remains silent; and their clinical expression requires another insult, either genetic or environmental. Porphyrias represent a group of disorders in which there is significant gene-environment interaction, and this aspect must also be evaluated in the diagnosis of the disease. Some 90% of subjects with deficiencies of PBGD activity remain asymptomatic throughout their lifetimes. Such asymptomatic heterozygotes may be AIP carriers who display no abnormalities in concentrations of heme pathway intermediates, or who may have latent AIP characterized by increased production and excretion of heme pathway intermediates, such as ALA or PBG, but without any other clinical manifestation of the disease. Persons with both latent and clinically expressed AIP have similar levels of PBG deaminase activity, which is typically 50% of the value of that occurring in normal subjects in their pedigree,3 and its value is unaffected in relapse as compared with the level in remission.23 Both latent gene carriers and patients with clinically overt AIP can be precipitated into an acute AIP attack by either endogenous or exogenous environmental factors. Most, but not all, such precipitating factors can be related to an associated increase in the gene expression of ALAS-N, the nonspecific isoform of ALA synthase in liver.3 Endocrine factors play a major role in the induction of AIP. For example, clinically expressed AIP is exceedingly rare before puberty, but relatively common in women, especially at the time of menses; and a subset of female patients experiences regular cyclical premenstrual exacerbation of this disease. The incidence and the severity of AIP decline markedly after menopause.3 Inadequate nutrition is another important precipitating factor. Conversely, additional carbohydrate-derived calories added to a preexisting adequate diet often decrease PBG excretion; and this constitutes one of the important modes of treatment.3 The third major category of inducers of acute attacks
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of AIP comprises drugs and other foreign chemicals. Many chemicals that exacerbate porphyria are thought to have the potential to induce cytochrome P450 in the liver, and the resultant enhanced demand for de novo heme synthesis leads to induction of ALAS-N.3
Prenatal Diagnosis and Gene Therapy Prenatal Diagnosis Theoretically, prenatal diagnosis is possible for all porphyrias, particularly if a mutation of the patient has been elucidated. Even before the advent of recombinant technology, measurement of enzyme activities of the heme biosynthetic pathway had been carried out on amniotic cells, which led to the diagnosis of fetuses with porphyric gene defects (Table 1). Additionally, in the case of congenital erythropoietic porphyria (CEP), a remarkable finding was reported that amniotic fluid obtained at 16 weeks of gestation from a mother of a patient with CEP displayed a pink-brown discoloration and contained high concentrations of porphyrins (predominantly uroporphyrin I, the biochemical hallmark of this disorder). The presence of uroporphyrin I in the fetus was also confirmed in the aborted tissue. Thus the marked derangement of uroporphyrin I formation and its excretion into amniotic fluid appears to be a useful index for the prenatal diagnosis of this rare disorder.24 Subsequently, this finding was confirmed during the 15th week of pregnancy in another fetus that also showed brown amniotic fluid.25 A marked deficiency of PBGD activity has also been demonstrated at 17 weeks of gestation in amniotic cells isolated from a woman with AIP.13 The prenatal diagnosis of the AIP trait in this fetus (Table 1) was confirmed postnatally by the demonstration in the child of a low level of erythrocyte PBGD activity, which was comparable to those found in her mother and a sibling, both of whom had AIP; but it was approximately onehalf the level found in her normal father and other normal siblings.13 Only one case of prenatal diagnosis of porphyria with direct detection of the gene mutation has been reported for CEP (Table 1).26 A French couple, whose first child was diagnosed with CEP, requested prenatal diagnosis at 16 weeks of gestation. Uroporphyrin I was drastically increased in amniotic fluid as in the other fetal cases of CEP, and the fetus was shown to be homozygous for a C73R mutation, the most common mutation in this disease. Based on these findings, the pregnancy was terminated. Despite the theoretical or practical ability to perform prenatal diagnosis of porphyrias, care must be taken with its application. First, as discussed earlier, because fewer than 10% of individuals with PBGD deficiency develop AIP, the diagnosis of a gene-carrier state per se does not predict the development of an actual porphy-
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ria disease.3 In addition, low rates of clinical expression are also suspected in other forms of acute hepatic porphyrias, such as HCP and VP. Most parents and physicians faced with these odds following a diagnostic amniocentesis elect for continuation of the pregnancy. Therefore, other than for recessive porphyrias such as CEP, the significance of prenatal diagnosis of porphyrias seems questionable.
Gene Therapy of Porphyrias Animal models of porphyrias, either naturally occurring or created by gene knockout, may be useful for the analysis of the pathogenesis and treatment of porphyrias.27–29 For example, PBGD-knockout mice display neurological disturbances similar to those that are observed in patients with AIP.29 The enzyme defect of CEP in cultured murine fibroblasts expressing the human UROIIIS deficiency, or bone marrow cells from patients with CEP can be corrected in vitro in cell culture by gene transfer.30,31 These findings suggest the potential usefulness of somatic gene therapy for the treatment of CEP. Gene therapy would also be valuable for other autosomal recessive porphyrias, such as ALAD porphyria (ADP) and HEP; however, its value in other porphyrias remains questionable, for the same reasons as discussed above for the validity of the prenatal diagnosis of porphyrias. Acknowledgments We are grateful to Drs. George Drummond, James Krueger, and Hiroyoshi Fujita for their critical reading of our manuscript. This work is supported in part by USPHS grant DK-32890, and the Yamanouchi Molecular Medicine Research Fund.
References 1. Garrod AE. Inborn Errors of Metabolism. London: Hodder & Stoughton, 1923;1–216. 2. Beadle GW, Tatum EL. Genetic control of biochemical reactions in Neurospora. Proc Natl Acad Sci U S A 1941; 27:499 –506. 3. Kappas A, Sassa S, Galbraith RA, et al. The porphyrias. In: Scriver CR, Beaudet AL, Sly WS, editors. The Metabolic and molecular basis of inherited disease, New York: McGraw-Hill, 1995;2103–59. 4. Bishop DF. Two different genes encode d-aminolevulinate synthase in humans: Nucleotide sequences of cDNAs for the housekeeping and erythroid genes. Nucleic Acids Res 1990;18:7187– 8. 5. Chretien S, Dubart A, Beaupain D, et al. Alternative transcription and splicing of the human porphobilinogen deaminase gene result either in tissue-specific or in housekeeping expression. Proc Natl Acad Sci U S A 1988;85: 6 –10. 6. Bishop TR, Miller MW, Beall J, et al. Genetic regulation of delta-aminolevulinate dehydratase during erythropoiesis. Nucleic Acids Res 1996;24:2511– 8.
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7. Sassa S, Nagai T. The role of heme in gene expression. Int J Hematol 1996;63:167–78. 8. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989. 9. Ausubel FM, Brent R, Kingston RE, et al. Current Protocols in Molecular Biology. New York: John Wiley & Sons, 1990. 10. Cotton RG. Current methods of mutation detection. Mutat Res 1993;285:125– 44. 11. Grompe M. The rapid detection of unknown mutations in nucleic acids. Nat Genet 1993;5:111–7. 12. Cotton RG. Slowly but surely towards better scanning for mutations. Trends Genet 1997;13:43– 6. 13. Sassa S, Solish G, Levere RD, et al. Studies in porphyria. IV. Expression of the gene defect of acute intermittent porphyria in cultured human skin fibroblasts and amniotic cells: Prenatal diagnosis of the porphyric trait. J Exp Med 1975;142:722–31. 14. Sassa S, Zalar GL, Kappas A. Studies in porphyria. VII. Induction of uroporphyrinogen-I synthase and expression of the gene defect of acute intermittent porphyria in mitogen-stimulated human lymphocytes. J Clin Invest 1978; 61:499 –508. 15. Sassa S, Zalar GL, Poh-Fitzpatrick MB, et al. Studies in porphyria: Functional evidence for a partial deficiency of ferrochelatase activity in mitogen-stimulated lymphocytes from patients with erythropoietic protoporphyria. J Clin Invest 1982;69:809 –15. 16. Maxam AM, Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A 1977;74:560 – 4. 17. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 1977;74:5463–7. 18. Orita M, Iwahana H, Kanazawa H, et al. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci U S A 1989;86:2766 –70. 19. Fischer SG, Lerman LS. Separation of random fragments of DNA according to properties of their sequences. Proc Natl Acad Sci U S A 1980;77:4420 – 4. 20. Ishida N, Fujita H, Fukuda Y, et al. Cloning and expression of the defective genes from a patient with d-aminolevulinate dehydratase porphyria. J Clin Invest 1992;89: 1431–7. 21. Meguro K, Fujita H, Ishida N, et al. Molecular defects of uroporphyrinogen decarboxylase in a patient with mild hepatoerythropoietic porphyria. J Invest Dermatol 1994; 102:681–5. 22. McManus JF, Begley CG, Sassa S, et al. Five new mutations in the uroporphyrinogen decarboxylase gene identified in families with cutaneous porphyria. Blood 1996; 88:3589 – 600. 23. Sassa S, Kappas A. Lack of effect of pregnancy or hematin therapy on erythrocyte porphobilinogen deaminase activity in acute intermittent porphyria. N Engl J Med 1989; 321:192–3. 24. Nitowsky HM, Sassa S, Nakagawa M, et al. Prenatal diagnosis of congenital erythropoietic porphyria. Pediatr Res 1978;12:455 A.
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25. Kaiser IH. Brown amniotic fluid in congenital erythropoietic porphyria. Obstet Gynecol 1980;56:383. 26. Ged C, Moreau-Gaudry F, Taine L, et al. Prenatal diagnosis in congenital erythropoietic porphyria by metabolic measurement and DNA mutation analysis. Prenat Diagn 1996;16:83– 6. 27. Ruth GR, Schwartz S, Stephenson B. Bovine protoporphyria: The first nonhuman model of this hereditary photosensitizing disease. Science 1977;198:199 –201. 28. Boulechfar S, Lamoril J, Montagutelli X, et al. Ferrochelatase structural mutant (Fechm1Pas) in the house mouse. Genomics 1993;16:645– 8.
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29. Lindberg RLP, Porcher C, Grandchamp B, et al. Porphobilinogen deaminase deficiency in mice causes a neuropathy resembling that of human hepatic porphyria. Nat Genet 1996;12:195–9. 30. Moreau-Gaudry F, Ged C, Barbot C, et al. Correction of the enzyme defect in cultured congenital erythropoietic porphyria disease cells by retrovirus-mediated gene transfer. Hum Gene Ther 1995;6:13–20. 31. Moreau-Gaudry F, Barbot C, Mazurier F, et al. Correction of the enzyme deficit of bone marrow cells in congenital erythropoietic porphyria by retroviral gene transfer. Hematol Cell Ther 1996;38:217–20.
A
bout one hundred years ago, Archibald Garrod advanced the concept of inborn errors of metabolism. Until then, alkaptonuria was thought to be due to the action of microorganisms in the human intestine, an idea that Garrod seriously questioned. He thought that alkaptonuria was due to a chemical aberration, and coined the term an inborn error of metabolism. Later, he recognized additional inborn errors of metabolism, in which he included porphyrias.1 Since then, medical research has made enormous progress, particularly through the biochemical and genetic research on inherited disorders. In recent years, the main stream of medicine has also developed a molecular aspect, through the application of molecular biological approaches to these questions. The research involving the porphyrias is a good example of the developmental history of genetic studies of inherited metabolic disorders. Garrod’s concept of inborn errors of metabolism was subsequently supported by the one gene-one enzyme hypothesis,2 which was originally found in yeast but extended to all cells to cover general proteins, including nonenzymatic proteins and complex proteins composed of nonidentical polypeptide subunits. A cistron, a term that refers to the functional unit of DNA that controls the structure of a single polypeptide, is now considered as the functional unit of inheritance, thus the one gene-one enzyme hypothesis has been redefined as the one cistron-one polypeptide concept. Analysis of cistronic mutations and their abnormal consequences are the target of genetic studies.
Diagnosis of Inherited Porphyrias Porphyrias may be first suspected by the characteristic clinical history and biochemical features of the patient. Establishment of the diagnosis of porphyrias, however, depends on characteristic findings for these disorders, which are obtained by several different methods. First, diagnosis of clinically manifest porphyrias can be made by the determination of porphyrins and their precursors that are excreted in excess into urine or feces or are From the Laboratory of Biochemical Hematology, The Rockefeller University, New York, New York. Address correspondence to Dr. Shigeru Sassa, Laboratory of Biochemical Hematology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399. © 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
elevated in erythrocytes. This is still the key for the practical diagnosis of an acute porphyria attack. It is also important in the evaluation of the efficacy of treatment. Secondly, more specific enzymatic assays should be used to distinguish among similar porphyrias, or to differentiate a porphyria from other porphyria-like syndromes. These assays will recognize not only patients with overt porphyrias, but also latent gene carriers. There are two types of latent gene carriers, one that is entirely latent and the other that is clinically latent but biochemically active. It is unclear whether the biochemically active carriers will develop an acute porphyria attack, but they should be considered at high risk for such a condition, and they should be monitored carefully. These enzymatic assays, combined with metabolite determinations, offer essential information for the genetic counseling of family members of the patient. Unfortunately, enzyme assays are not available for all porphyrias. Commercial assays are available only for ALA dehydratase (ALAD) and PBG deaminase (PBGD) activities using blood samples. These cytosolic enzymes appear to be stable in blood. In contrast, mitochondrial enzyme assays are difficult to perform using whole blood samples, because there are few mitochondria in erythrocytes. While there are commercial assays for mitochondrial enzymes such as coproporphyrinogen oxidase activity in blood, their significance is therefore questionable. Thirdly, using recombinant DNA techniques, gene mutations can be analyzed directly, and the exact disease-responsible mutations can be defined. This is obviously the ultimate goal of genetic diagnosis. Defined gene mutations, such as point mutations or gene deletions, are very useful for screening for the gene defect in the patient’s family. Specific diagnosis such as for a founder mutation can also be made by this technique. It would also be very useful for prenatal diagnosis. On the other hand, molecular genetics does not predict which gene carriers may develop clinical porphyrias. Thus in the practical management of porphyrias, clinical and biochemical assays remain crucial.3
Molecular Defects in the Porphyrias Porphyrias are due to inherited defects of one of the enzymes in the heme biosynthetic pathway, with the 0738-081X/98/$19.00 PII S0738-081X(97)00203-4
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Figure 1. A scheme for DNA diagnosis of porphyrias.
exception of the first enzyme, ALA synthase (ALAS). These enzyme deficiencies can be either partial or nearly complete. As a result, abnormally elevated levels of porphyrins, and/or their precursors accumulate in tissues, leading to neurological disturbances and/or photosensitive reactions; and they are also excreted in excess into urine and stool.3 Complementary DNA (cDNA) clones have been isolated for all eight enzymes in the heme biosynthetic pathway. Genomic sequences have also been reported for these enzymes, except uroporphyrinogen III synthase (UROIIIS). Some enzymes have been shown to occur as tissue-specific isozymes, for example, ALAS and PBGD,4,5 or as tissue-specific mRNAs such as ALAD.6 The isozyme-specificity occurs principally between the erythroid tissues and the nonerythroid tissues such as the liver, suggesting that there may be significant differences in the regulation of gene expression of these enzymes between these tissues.7 cDNA clones from many patients with various forms of the porphyrias have been obtained, their sequences determined, and the properties of some of their phenotypes studied using heterologous expression systems.
Cloning Gene Defects in the Porphyrias To define a mutation, it is necessary to determine the structure of its normal gene, and to elucidate its regulation and its phenotype in normal cells. For such a purpose, (1) the sequence of cDNA and structure of genomic DNA, (2) the regulatory mechanism of gene expression in normal cells must be elucidated, and (3)
the phenotypic consequences of gene expression should be examined. Since there are a number of excellent technical protocols for genetic analysis,8,9 only the general aspects of this approach are summarized below, and an outline of common approaches is shown in Fig 1. There are also excellent reviews specifically dealing with mutation detection.10 –12
Cloning of cDNA and Genome DNA cDNA Cloning Cloning refers to the process by which a DNA molecule is joined to another DNA molecule (termed a cloning vector) that can replicate when introduced into an appropriate host, usually a bacterium or yeast. A cDNA is a reverse copy of mRNA, which is manufactured in vitro from mRNA by reverse transcriptase, cDNA cloning is a useful strategy to obtain specific information about the desired mRNA. Because mRNA is a nuclear RNA transcript from which introns have been spliced out, cDNA represents principally the protein-coding sequence, plus a short stretch of an upstream (59 untranslated) as well as a downstream (39 untranslated) sequence of mRNA that includes a poly A tail(s). The most common approach for cDNA cloning utilizes nucleic-acid hybridization; however, it requires knowledge of the sequences being sought. Replicas of plasmid colonies or phage plaques are lysed on nitrocellulose membranes, denatured, immobilized, and then detected using radiolabeled single-stranded DNA fragments specific for a gene sequence of interest. In some cases, part of the gene may already have been
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cloned, and this information can be used to search clones that contain additional sequences flanking the original clone. In other cases, a closely related gene (a homologous gene) may already have been cloned and can be used in screening for the gene that has sufficient homology with the starting clone under an appropriate condition. It is possible to perform sequence analysis of a purified protein by an automated protein sequencer. In this manner, amino acid sequences can be determined from a very small amount of protein, for example, 50 pmol; and sequence information can be used to generate a corresponding RNA sequence of its mRNA by using the known triplet codon for each amino acid. Because there is redundancy in the triplet codon where one amino acid can be encoded by multiple codons, there are multiple DNA sequences encoding a short stretch of a polypeptide that are slightly different from each other. This problem can be corrected by using degenerate pools of oligonucleotides that take this redundancy, or the ‘wobbling’, of triplets into account. These oligonucleotides can be used as probes to screen the cDNA library. When a candidate cDNA is cloned, its nucleotide sequences should be determined. With the help of DNA sequence-analysis computer programs, sequence data can be used (1) to determine all known restriction enzyme sites within the gene, and (2) to predict the protein product encoded by the gene. This method will help to determine whether the cloned cDNA is a bona fide clone of interest. cDNA clones can be used as a probe to search gene expression in various organs, and to define tissue- and/or cell-specific expression by Northern blot analysis, or in situ hybridization. Additionally, it is also useful in screening cDNA and genomic libraries, and patient’s cell cDNA libraries. Genomic DNA Cloning Generally, cDNA is used as a probe to isolate its genomic DNA. The genomic DNA is always larger than its cDNA, because it contains introns. Thus, while the overall technique of isolating genomic DNA is similar to that used in cDNA cloning, many DNA fragments are usually needed for genomic DNA cloning. When a genomic DNA is cloned, its nucleotide sequences should be determined. The nucleotide sequences thus obtained provide invaluable information on introns in genomic DNA. Nucleotide sequence analysis of a genomic DNA permits the analysis of control regions such as promoter and/or regulatory regions in DNA. Such information is essential for elucidating the mechanism of gene expression, because these regions contain DNA sequences that may bind specific nuclear proteins that regulate transcription (transcription factors), and confer upon the gene temporal or tissue-specific control.
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Tissue-Specific Gene Regulation Of the estimated approximately 100,000 human genes, many genes are transcribed ubiquitously in all tissues, and they are called “house-keeping genes”. The housekeeping genes are important in regulating essential function of cellular proteins, although their specific roles in cellular function remains unclear for most of them. In contrast, there are certain genes whose transcription is restricted to a specific tissue lineage, or to a specific time in organ development, or they are induced in response to a specific humoral factor(s), such as hormones. In these genes, tissue-, time-, or inductionspecific transcription is not due to a difference in the genotype, but rather to a difference in the mechanism(s) that control gene expression. Tissue-specific gene expression can be controlled at variable stages, for example, directly at the level of transcription (transcriptional control), at the level of splicing (posttranscriptional control), or at the level of translation (translational control). While all these mechanisms may contribute, by far the most common mechanism of control is at the level of transcription. It has also been shown that gene expression is critically regulated by a hierarchical control of transcription at the level of the structure of the chromosome (chromatin structure) and by combinational interaction between a limited number of cell-specific transcriptional regulatory proteins and RNA polymerase. The initial step of RNA synthesis is an association of RNA polymerase and general transcription factors with the promoter of the gene to form a basal transcription complex that is to be transcribed. The basal transcription complex acts as a target for transcriptional activation, or repression by tissue-specific transcriptional regulatory proteins. These tissue-specific transcription factors also bind specifically to other sequences in the control region of the genes, which then results in regulated tissue-specific gene expression. The ability of transcription factors, either general or tissue-specific, to interact directly with DNA is fundamental to such control of gene transcription.
DNA Diagnosis Nature of the Genetic Materials A tissue sample for genetic analysis should be obtained from both the patient and the family members. If an abnormal enzyme is expressed in a tissue-specific manner, it is essential to analyze mRNA in that tissue. Because it is often difficult to obtain a tissue sample by biopsy and nucleated cells from blood can be used instead, blood is the most common source of genetic materials. For example, mononuclear cells can be separated by centrifugation, and they can be used to prepare genomic DNA. Lymphocytes can be separated by Ficoll-Hypaque gradient centrifugation; and they can be used by stimulation with phytohemagglutinin and
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pokeweed mitogen or by transformation by EpsteinBarr (EB) virus, which permits establishment of an immortal cell line. The use of cell cultures for the diagnosis of genetic disorders, including porphyrias, is now very popular.13–15 Skin fibroblast cultures and EB virus transformed lymphoblastoid cells are the standard in this technique, though cells from other tissues have also been used. The advantage of cultured cells are: (1) they are readily established from patient’s tissues; (2) they can be propagated, or stored for an indefinite period, thus avoiding repeated sampling from patients; (3) they are often useful for complex biochemical analysis, including enzyme assays; (4) they are suitable for diagnosis of all mutations at a DNA level, and many at a RNA level; and (5) they are the only material that can be used for radioisotopic studies, such as protein turnover.
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by autoradiography and a color reaction, respectively. This method reveals details of sequence organization. Thus, in many diseases that are due to a large deletion of the gene, or rearrangement between several genes, Southern blot analysis is a very useful technique to detect such changes. NORTHERN BLOT HYBRIDIZATION This technique is analogous to Southern blotting of DNA, except RNA is transferred to a nitrocellulose filter (Northern transfer) on which it can be hybridized to complementary (radioactive or biotinylated) single-stranded DNA or RNA probes. If a large amount of mRNA (in mg quantities) can be prepared, Northern blot analysis is the method of choice to identify a major alteration in mRNA, such as a large deletion or an insertion, and to determine the extent of expression of an abnormal mRNA.
cDNA CLONING FROM THE PATIENT’S mRNA cDNA is made from the patient’s mRNA by using reverse transcriptase. This procedure involves generation of a single strand of cDNA, very often begun (primed) by large tracts of deoxythymidine [poly(dT)], which physically associates with, or anneals to the poly (A) tract at the 39 end of mRNA (the site of polyadenylation) by its DNA complementarity. Alternatively, priming can be carried out by a large pool of random oligonucleotides (random priming), which will anneal all along the RNA molecule. A single-stranded cDNA is then used as a template to generate double-stranded cDNA by the use of DNA polymerase. These double-stranded cDNAs are then inserted into a l phage, or a plasmid DNA. Because as only one recombinant l phage and a plasmid can enter each bacteria, each individual cDNA is replicated in a single bacteria; and this permits clonal propagation of a single cDNA in a clone of bacteria (cDNA clone). These bacteria carry cDNAs representing every expressed RNA from a cell, or a tissue of interest (cDNA library). Alternatively, a cDNA of interest can be directly cloned into a plasmid if its sequence information is available from a DNA database. When cDNA is made from the mRNA of a patient, information on a mutant cDNA can be obtained by screening its library with normal cDNA followed by polymerase chain reaction (PCR). Cloning of the patient’s cDNA is a very useful technique for isolating the mRNA of interest.
POLYMERASE CHAIN REACTION The polymerase chain reaction (PCR) is a technique used for enzymatic in vitro amplification (PCR amplification) of specific DNA sequences without utilizing conventional procedures, that is, molecular cloning. It is an extremely powerful technique for selective and rapid amplification of a target DNA, or an RNA sequence. It permits the amplification of a DNA (genome or cDNA) fragment situated between two convergent primers, and it utilizes oligonucleotide primers that hybridize to opposite strands. Primer extension proceeds inward across the region between the two primers. The product of DNA synthesis of one primer serves as a template for the other primer. A series of reactions starting with denaturation, annealing and DNA synthesis is cycled for many times, resulting in an exponential increase in the number of copies of the region bounded by the primers. The sequence of the double-stranded DNA fragment can then be identified by hybridization to allele-specific oligonucleotide probes representing various alleles studied or whose sequence can be determined. The advantages of this method are: (1) only a very small amount of DNA is needed; and (2) results are achieved quickly, typically in less than one day. On the other hand, it is essential to have sequence information for the synthesis of specific oligonucleotide primers for PCR. When PCR is performed following a reverse transcription (RT) reaction, the combination is called “RT-PCR”; and this method is extremely useful in the amplification and analysis of a cDNA of interest.
SOUTHERN BLOT HYBRIDIZATION The Southern blot analysis is a common approach to DNA-based diagnosis. It involves transfer of single-stranded, restricted DNA fragments, separated in an agarose gel, to a binding matrix, for example, nitrocellulose membrane (Southern transfer). DNA fragments are then analyzed by hybridization to radioactive or biotinylated singlestrand DNA, or RNA probes; and hybrids are detected
DNA SEQUENCING The most precise method for the characterization of a segment of DNA is to obtain its exact nucleotide sequence. The procedure consists of (1) selection of DNA clones, (2) treatment of these clones individually by base-specific reactions, (3) separation of the reaction products by size on sequencing gels, and (4) computer-assisted reading of separated bands as a sequence of A, C, G, and T. There are two major meth-
Techniques for DNA Analysis
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ods for nucleotide sequencing, one of which utilizes base-specific chemical cleavage, while the other uses termination of in vitro DNA synthesis.16,17 The former requires at least partial restriction mapping and is more labor-intensive, while the latter is less difficult and is now commonly used for most applications. Abnormal variations in DNA sequences can be identified in this manner and then used to detect mutations in family members, and other individuals. DNA sequence analysis can be performed with products of a PCR reaction (direct sequencing), which permits rapid determination of nucleotide sequences of PCR products. ELECTROPHORESIS OF SINGLE-STRANDED OR DENATURED DNA Structural variation in single-stranded DNA due to sequence-dependent intrastrand secondary structure is called single-stranded conformational polymorphism (SSCP). It is detectable by electrophoresis in nondenaturing gels, and it is useful for point mutation scanning.18 Point mutation scanning is also possible by a method called denaturing gradient gel electrophoresis (DGGE), which utilizes electrophoresis in a gel containing a gradient of denaturants (urea and formamide), allowing detection of sequence-dependent differences under denaturation conditions.19 Both SSCP and DGGE permit quick identification of differences in DNA sequences. Because these methods also detect normal DNA variation or polymorphism, as well as DNA sequence changes that are related to other diseases, nucleotide sequences of the abnormal fragment must be determined to ultimately confirm that the detected change is a mutation responsible for the disease. ALLELE-SPECIFIC OLIGONUCLEOTIDE (ASO) HYBRIDIZATION ANALYSIS This technique uses two synthetic oligonucleotide probes, one specific for a normal gene and the other specific for a mutation identified in the patient. They are used as hybridization probes to determine whether a patient and his or her family members have the normal gene or the abnormal gene. This analysis is very helpful for the diagnosis of a genetic disease that typically arises from a single predominant mutation, such as in the porphyrias. When this technique is used with a DNA segment that has been selectively amplified by PCR, it provides a rapid and an accurate DNAbased diagnosis. On the other hand, application of ASO hybridization analysis is limited in many cases to a specific known mutation occurring in each pedigree. RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLP) There are variations in the length of restriction fragments, termed restriction fragment length polymorphisms (RFLP) that are generated after DNA is digested with different restriction enzymes, and analyzed with a variety of cloned DNA probes that detect specific homologous DNA fragments. RFLPs are due to either introduction or removal of restriction sites, or sequence
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deletions, additions, and rearrangements that affect the length of DNA between restriction sites. They represent codominant, typically neutral, genetic markers; and they can be used to construct detailed genetic linkage maps (RFLP linkage maps), to follow the mode of inheritance of genetic diseases and traits, and to examine variation between and within populations. Because RFLPs are not rare events, occurring every 200 –300 bp in the human genome, and they are inherited in simple Mendelian fashion, they provide a very rapid and efficient method for studying the inheritance of a relevant allele over the generations by following the inheritance of detectable RFLPs linked to it. Similar limitations, however, apply as in the case of ASO hybridization analysis of a mutation. A TYPICAL DNA DIAGNOSIS Generally, DNA diagnosis is performed by using a combination of several methods described above. A typical example of DNA diagnosis is shown here for d-aminolevulinate dehydratase (ALAD) porphyria.20 1. Lymphocytes of a patient with ALAD porphyria were isolated and transformed by EB virus to create an immortalized cell line. Then, mRNA was prepared from these cells, and cDNA was synthesized with reverse transcriptase using an oligo(dT) primer. 2. cDNA was amplified by PCR using the primers that correspond to the 59-untranslated and the 39-untranslated regions of ALAD cDNA. The amplified fragment was cloned into a plasmid vector and nucleotide sequence analysis was performed. To confirm the mutation, direct sequencing analysis was performed using the patient’s mRNA. 3. Following the determination of the mutations in the proband, ALAD phenotype analysis in the proband’s family were carried out using RT-PCR combined with ASO hybridization analysis. 4. As a result, a silent mutation (without an accompanying amino acid change) and a mutation with an amino acid substitution were found on one allele. In addition, three independent silent mutations and another mutation were found on the other allele. cDNA cloning as shown in this example is useful if several mutations are found in both alleles and when the coding region of the gene is not huge. Types of Mutations A number of mutations have been described in the porphyrias. While gross mutations that encompass the entire genome, or deletion of a very large segment of a chromosome have not been reported, essentially all other types of mutations have been described in the porphyrias (Table 1). MISSENSE MUTATION If codons are affected by a single or a few nucleotide substitution, such a mutation is
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Table 1.
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Types of Mutations and Prenatal Diagnosis Reported in the Porphyrias
Missense mutation Translational mutation* RNA splicing mutation Prenatal diagnosis
ADP
AIP
CEP
PCT
HEP
HCP
VP
EPP
1 2 2 2
1 1 1 1
1 1 2 1
1 1 1 2
1 1 2 2
1 1 1 2
1 1 2 2
1 1 1 2
* Including nonsense, frame-shift (insertion, deletion) mutations and gene deletion.
called a missense mutation. Missense mutations have been described in all the porphyrias, and they are by far more common than other kinds of mutations. A point mutation was identified in most cases of missense mutation. It is interesting to note that, in a patient with hepatoerythropoietic porphyria (HEP) and a patient with familial porphyria cutanea tarda (PCT) from different pedigrees, a very rare but identical three-sequential-base substitution was detected; however, it resulted in only one amino-acid change.21,22 TRANSLATIONAL
(NONSENSE MUTATION AND Mutations affecting translation have also frequently been described in the porphyrias. Nonsense mutation affects normal translation by a nucleotide substitution, which changes a triplet codon for an amino acid to a stop codon. It results in a truncated protein that is often unstable and readily degraded. A frameshift mutation is either deletion or insertion of one or a few nucleotides in the coding region of a gene. These mutations change the coding message for all subsequent codons, thus completely altering the aminoacid sequence from the mutation site. In many instances, a frameshift mutation results in premature termination of the protein, because it often creates a stop codon.
2.
3.
MUTATION
FRAMESHIFT MUTATION)
RNA SPLICING MUTATION RNA splicing is critical to normal gene expression, because introns must be precisely spliced to produce the appropriate mRNA for translation into protein. RNA splicing may be affected by mutation, either by altering a normal splice site at the intron and exon junction, or by creating a new splice site within introns or exons. The 59 end of the intron always begins with nucleotides GT, and the 39 end of the intron always has the nucleotides AG. Mutations that alter the AG or GT at the splice junction site lead to complete absence of normal splicing, which results in an unstable mRNA that is rapidly degraded. Thus, the protein product of this mutation is usually undetectable.
4.
5.
6.
7.
IMPORTANT ASPECTS A number of important aspects have been elucidated by genetic studies of porphyrias. 1. In every porphyria, the molecular defect of the enzyme is heterogeneous. In other words, there is more than one mutation resulting clinically in a single porphyria disease. For example, there are more than 100 mutations found in the PBGD gene in patients
8.
with acute intermittent porphyria (AIP). There are few founder effect mutations, while a pedigree-specific mutation of the enzyme gene is very common. Many different kinds of mutations have been found in the porphyrias, for example, promoter mutations, splicing mutations, consensus sequence mutations, mutations producing nonfunctional mRNAs, mutations resulting in unstable proteins, gene deletions, and so forth (Table 1). Even among the dominant form of porphyrias, a homozygous form of the disease can be found in a few patients. The homozygous form appears to be due to mutations with lesser pathophysiological effects than that found in the heterozygous form of the disease. A subset of homozygous diseases among commonly autosomal dominant diseases has been observed in AIP, hereditary coproporphyria (HCP), and variegate porphyria (VP). Clinically homozygous mutations can be due either to a homoallelic or a heteroallelic mutation; however, heteroallelic mutation, that is, compound heterozygosity for two separate mutations, is far more common than homoallelic mutation. Tissue-specific regulation of heme pathway enzymes may likely be the basis for the tissue-specific expression of different porphyrias. It probably explains why some porphyrias occur principally in the liver, while others occur in erythroid tissues. In the case of PBGD, a mutation affecting the splicing of the first intron results in an abnormal enzyme in nonerythroid cells, while the same mutation has no effect on the erythroid-specific PBGD, because the transcription of the erythroid PBGD mRNA utilizes a second AUG which is located approximately 3 kb downstream of the mutated site.5 Thus, even in a single form of acute hepatic porphyria, there may be distinct tissue-specific expression of the disease, depending on the type of mutation. Most of the acute hepatic porphyrias, such as AIP, HCP, and VP, require an additional factor(s) for their clinical expression.3 Such a factor can be either genetic or environmental in nature. Thus, porphyrias are not only inborn errors of metabolism, but also diseases in which environmental factors have an immense impact on their gene expression. Some hepatic porphyria-like symptoms can be elicited by environmental chemicals that strongly inhibit
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heme pathway enzymes. For example, porphyria cutanea tarda (PCT) may be caused by hexachlorobenzene in normal individuals. Genetic analysis has elucidated the complex nature of molecular defects in the porphyrias and the possible consequences of their gene defects on cellular function. It also offers an explanation as to why hepatic porphyrias do not accompany anemia, or conversely why erythropoietic porphyrias do not exhibit the defective drug metabolism that largely reflects the function of hepatic cytochrome P450.
Gene-Environment Interaction While inherited porphyrias are due to a gene defect of the enzymes in the heme biosynthetic pathway, their clinical phenotype often remains silent; and their clinical expression requires another insult, either genetic or environmental. Porphyrias represent a group of disorders in which there is significant gene-environment interaction, and this aspect must also be evaluated in the diagnosis of the disease. Some 90% of subjects with deficiencies of PBGD activity remain asymptomatic throughout their lifetimes. Such asymptomatic heterozygotes may be AIP carriers who display no abnormalities in concentrations of heme pathway intermediates, or who may have latent AIP characterized by increased production and excretion of heme pathway intermediates, such as ALA or PBG, but without any other clinical manifestation of the disease. Persons with both latent and clinically expressed AIP have similar levels of PBG deaminase activity, which is typically 50% of the value of that occurring in normal subjects in their pedigree,3 and its value is unaffected in relapse as compared with the level in remission.23 Both latent gene carriers and patients with clinically overt AIP can be precipitated into an acute AIP attack by either endogenous or exogenous environmental factors. Most, but not all, such precipitating factors can be related to an associated increase in the gene expression of ALAS-N, the nonspecific isoform of ALA synthase in liver.3 Endocrine factors play a major role in the induction of AIP. For example, clinically expressed AIP is exceedingly rare before puberty, but relatively common in women, especially at the time of menses; and a subset of female patients experiences regular cyclical premenstrual exacerbation of this disease. The incidence and the severity of AIP decline markedly after menopause.3 Inadequate nutrition is another important precipitating factor. Conversely, additional carbohydrate-derived calories added to a preexisting adequate diet often decrease PBG excretion; and this constitutes one of the important modes of treatment.3 The third major category of inducers of acute attacks
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of AIP comprises drugs and other foreign chemicals. Many chemicals that exacerbate porphyria are thought to have the potential to induce cytochrome P450 in the liver, and the resultant enhanced demand for de novo heme synthesis leads to induction of ALAS-N.3
Prenatal Diagnosis and Gene Therapy Prenatal Diagnosis Theoretically, prenatal diagnosis is possible for all porphyrias, particularly if a mutation of the patient has been elucidated. Even before the advent of recombinant technology, measurement of enzyme activities of the heme biosynthetic pathway had been carried out on amniotic cells, which led to the diagnosis of fetuses with porphyric gene defects (Table 1). Additionally, in the case of congenital erythropoietic porphyria (CEP), a remarkable finding was reported that amniotic fluid obtained at 16 weeks of gestation from a mother of a patient with CEP displayed a pink-brown discoloration and contained high concentrations of porphyrins (predominantly uroporphyrin I, the biochemical hallmark of this disorder). The presence of uroporphyrin I in the fetus was also confirmed in the aborted tissue. Thus the marked derangement of uroporphyrin I formation and its excretion into amniotic fluid appears to be a useful index for the prenatal diagnosis of this rare disorder.24 Subsequently, this finding was confirmed during the 15th week of pregnancy in another fetus that also showed brown amniotic fluid.25 A marked deficiency of PBGD activity has also been demonstrated at 17 weeks of gestation in amniotic cells isolated from a woman with AIP.13 The prenatal diagnosis of the AIP trait in this fetus (Table 1) was confirmed postnatally by the demonstration in the child of a low level of erythrocyte PBGD activity, which was comparable to those found in her mother and a sibling, both of whom had AIP; but it was approximately onehalf the level found in her normal father and other normal siblings.13 Only one case of prenatal diagnosis of porphyria with direct detection of the gene mutation has been reported for CEP (Table 1).26 A French couple, whose first child was diagnosed with CEP, requested prenatal diagnosis at 16 weeks of gestation. Uroporphyrin I was drastically increased in amniotic fluid as in the other fetal cases of CEP, and the fetus was shown to be homozygous for a C73R mutation, the most common mutation in this disease. Based on these findings, the pregnancy was terminated. Despite the theoretical or practical ability to perform prenatal diagnosis of porphyrias, care must be taken with its application. First, as discussed earlier, because fewer than 10% of individuals with PBGD deficiency develop AIP, the diagnosis of a gene-carrier state per se does not predict the development of an actual porphy-
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ria disease.3 In addition, low rates of clinical expression are also suspected in other forms of acute hepatic porphyrias, such as HCP and VP. Most parents and physicians faced with these odds following a diagnostic amniocentesis elect for continuation of the pregnancy. Therefore, other than for recessive porphyrias such as CEP, the significance of prenatal diagnosis of porphyrias seems questionable.
Gene Therapy of Porphyrias Animal models of porphyrias, either naturally occurring or created by gene knockout, may be useful for the analysis of the pathogenesis and treatment of porphyrias.27–29 For example, PBGD-knockout mice display neurological disturbances similar to those that are observed in patients with AIP.29 The enzyme defect of CEP in cultured murine fibroblasts expressing the human UROIIIS deficiency, or bone marrow cells from patients with CEP can be corrected in vitro in cell culture by gene transfer.30,31 These findings suggest the potential usefulness of somatic gene therapy for the treatment of CEP. Gene therapy would also be valuable for other autosomal recessive porphyrias, such as ALAD porphyria (ADP) and HEP; however, its value in other porphyrias remains questionable, for the same reasons as discussed above for the validity of the prenatal diagnosis of porphyrias. Acknowledgments We are grateful to Drs. George Drummond, James Krueger, and Hiroyoshi Fujita for their critical reading of our manuscript. This work is supported in part by USPHS grant DK-32890, and the Yamanouchi Molecular Medicine Research Fund.
References 1. Garrod AE. Inborn Errors of Metabolism. London: Hodder & Stoughton, 1923;1–216. 2. Beadle GW, Tatum EL. Genetic control of biochemical reactions in Neurospora. Proc Natl Acad Sci U S A 1941; 27:499 –506. 3. Kappas A, Sassa S, Galbraith RA, et al. The porphyrias. In: Scriver CR, Beaudet AL, Sly WS, editors. The Metabolic and molecular basis of inherited disease, New York: McGraw-Hill, 1995;2103–59. 4. Bishop DF. Two different genes encode d-aminolevulinate synthase in humans: Nucleotide sequences of cDNAs for the housekeeping and erythroid genes. Nucleic Acids Res 1990;18:7187– 8. 5. Chretien S, Dubart A, Beaupain D, et al. Alternative transcription and splicing of the human porphobilinogen deaminase gene result either in tissue-specific or in housekeeping expression. Proc Natl Acad Sci U S A 1988;85: 6 –10. 6. Bishop TR, Miller MW, Beall J, et al. Genetic regulation of delta-aminolevulinate dehydratase during erythropoiesis. Nucleic Acids Res 1996;24:2511– 8.
Clinics in Dermatology
Y
1998;16:235–243
7. Sassa S, Nagai T. The role of heme in gene expression. Int J Hematol 1996;63:167–78. 8. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989. 9. Ausubel FM, Brent R, Kingston RE, et al. Current Protocols in Molecular Biology. New York: John Wiley & Sons, 1990. 10. Cotton RG. Current methods of mutation detection. Mutat Res 1993;285:125– 44. 11. Grompe M. The rapid detection of unknown mutations in nucleic acids. Nat Genet 1993;5:111–7. 12. Cotton RG. Slowly but surely towards better scanning for mutations. Trends Genet 1997;13:43– 6. 13. Sassa S, Solish G, Levere RD, et al. Studies in porphyria. IV. Expression of the gene defect of acute intermittent porphyria in cultured human skin fibroblasts and amniotic cells: Prenatal diagnosis of the porphyric trait. J Exp Med 1975;142:722–31. 14. Sassa S, Zalar GL, Kappas A. Studies in porphyria. VII. Induction of uroporphyrinogen-I synthase and expression of the gene defect of acute intermittent porphyria in mitogen-stimulated human lymphocytes. J Clin Invest 1978; 61:499 –508. 15. Sassa S, Zalar GL, Poh-Fitzpatrick MB, et al. Studies in porphyria: Functional evidence for a partial deficiency of ferrochelatase activity in mitogen-stimulated lymphocytes from patients with erythropoietic protoporphyria. J Clin Invest 1982;69:809 –15. 16. Maxam AM, Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A 1977;74:560 – 4. 17. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 1977;74:5463–7. 18. Orita M, Iwahana H, Kanazawa H, et al. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci U S A 1989;86:2766 –70. 19. Fischer SG, Lerman LS. Separation of random fragments of DNA according to properties of their sequences. Proc Natl Acad Sci U S A 1980;77:4420 – 4. 20. Ishida N, Fujita H, Fukuda Y, et al. Cloning and expression of the defective genes from a patient with d-aminolevulinate dehydratase porphyria. J Clin Invest 1992;89: 1431–7. 21. Meguro K, Fujita H, Ishida N, et al. Molecular defects of uroporphyrinogen decarboxylase in a patient with mild hepatoerythropoietic porphyria. J Invest Dermatol 1994; 102:681–5. 22. McManus JF, Begley CG, Sassa S, et al. Five new mutations in the uroporphyrinogen decarboxylase gene identified in families with cutaneous porphyria. Blood 1996; 88:3589 – 600. 23. Sassa S, Kappas A. Lack of effect of pregnancy or hematin therapy on erythrocyte porphobilinogen deaminase activity in acute intermittent porphyria. N Engl J Med 1989; 321:192–3. 24. Nitowsky HM, Sassa S, Nakagawa M, et al. Prenatal diagnosis of congenital erythropoietic porphyria. Pediatr Res 1978;12:455 A.
Clinics in Dermatology
Y
1998;16:235–243
25. Kaiser IH. Brown amniotic fluid in congenital erythropoietic porphyria. Obstet Gynecol 1980;56:383. 26. Ged C, Moreau-Gaudry F, Taine L, et al. Prenatal diagnosis in congenital erythropoietic porphyria by metabolic measurement and DNA mutation analysis. Prenat Diagn 1996;16:83– 6. 27. Ruth GR, Schwartz S, Stephenson B. Bovine protoporphyria: The first nonhuman model of this hereditary photosensitizing disease. Science 1977;198:199 –201. 28. Boulechfar S, Lamoril J, Montagutelli X, et al. Ferrochelatase structural mutant (Fechm1Pas) in the house mouse. Genomics 1993;16:645– 8.
HOW GENETIC DEFECTS ARE IDENTIFIED
243
29. Lindberg RLP, Porcher C, Grandchamp B, et al. Porphobilinogen deaminase deficiency in mice causes a neuropathy resembling that of human hepatic porphyria. Nat Genet 1996;12:195–9. 30. Moreau-Gaudry F, Ged C, Barbot C, et al. Correction of the enzyme defect in cultured congenital erythropoietic porphyria disease cells by retrovirus-mediated gene transfer. Hum Gene Ther 1995;6:13–20. 31. Moreau-Gaudry F, Barbot C, Mazurier F, et al. Correction of the enzyme deficit of bone marrow cells in congenital erythropoietic porphyria by retroviral gene transfer. Hematol Cell Ther 1996;38:217–20.