Cloning and Expression of the Defective Genes from a Patient with Delta-Aminolevulinate Dehydratase Porphyria
Total Page：16
File Type：pdf, Size：1020Kb
Cloning and expression of the defective genes from a patient with delta-aminolevulinate dehydratase porphyria. N Ishida, … , A Kappas, S Sassa J Clin Invest. 1992;89(5):1431-1437. https://doi.org/10.1172/JCI115732. Research Article Cloning and expression of the defective genes for delta-aminolevulinate dehydratase (ALAD) from a patient with inherited ALAD deficiency porphyria (ADP) were carried out. Cloning of cDNAs for the defective ALAD were performed from EBV- transformed lymphoblastoid cells of the proband, and nucleotide sequences were determined. Two separate point mutations resulting in a single amino acid change in each ALAD allele were identified. One, C718----T, termed 'G1', occurred in the allele within the substrate-binding site, producing an Arg240----Trp substitution; the other, G820----A, termed 'G2', occurred downstream of this site in the other allele, resulting in an Ala274----Thr substitution. Using the reverse transcription-polymerase chain reaction, the mother, the brother, and the sister were shown to have the G1 defect. Expression of the G1 cDNA in Chinese hamster ovary cells produced ALAD protein with little activity; the G2 cDNA produced the enzyme with approximately 50% normal activity. Pulse-labeling studies demonstrated that the G1 enzyme had a normal half life, while the G2 enzyme had a markedly decreased half life. These data thus define the separate point mutations in each ALAD allele, as well as the altered properties of the two enzymic proteins encoded by the mutant genes in a patient with ADP. Find the latest version: https://jci.me/115732/pdf Cloning and Expression of the Defective Genes from a Patient with 6-Aminolevulinate Dehydratase Porphyria Nobuhiro Ishida,* Hiroyoshi Fujita,* Yoshiaki Fukuda,* Teruhisa Noguchi,t Manfred Doss,$ Attallah Kappas,* and Shigeru Sassa* *The Rockefeller University, New York 10021; tThe Suntory Institutefor Biomedical Research, Osaka, Japan; and OPhilipps University, Marburg, Federal Republic ofGermany Abstract chemicals, e.g., lead (5), succinylacetone (6, 7), as well as by an inherited ALAD deficiency (8, 9), which may result in the devel- Cloning and expression of the defective genes for b-aminolevu- opment of a clinically severe hepatic porphyria (3, 4). Four linate dehydratase (ALAD) from a patient with inherited unrelated cases of inherited ALAD porphyria (ADP) have been ALAD deficiency porphyria (ADP) were carried out. Cloning reported to date (9). In our previous study (10), we described a of cDNAs for the defective ALAD were performed from EBV- molecular defect in an ALAD allele, termed 'G2', in an adult- transformed lymphoblastoid cells of the proband, and nucleo- onset proband with ADP, originally reported from Germany tide sequences were determined. Two separate point mutations (8). This finding represented the first demonstration of a muta- resulting in a single amino acid change in each ALAD allele tion in the human ALAD gene, and suggested that the proband were identified. One, C718 -* T, termed 'Gl', occurred in the had two separate point mutations in the ALAD gene (10). In allele within the substrate-binding site, producing an Arge4 -- the present study, we describe the molecular analysis of the Trp substitution; the other, G'0' -- A, termed 'G2', occurred enzymatic defect encoded by the other ALAD allele in the pro- downstream of this site in the other allele, resulting in an Ala'4 band, termed 'GI', and ALAD phenotype studies in the pro- Thr substitution. Using the reverse transcription-polymer- band's family using allele-specific oligonucleotides (ASOs) for ase chain reaction, the mother, the brother, and the sister were the Gl and the G2 mutations. In addition, we report results on shown to have the Gi defect. Expression of the G1 cDNA in the activity and the turnover rates of the mutant enzymes ex- Chinese hamster ovary cells produced ALAD protein with little pressed by the GI and the G2 cDNAs in Chinese hamster ovary activity; the G2 cDNA produced the enzyme with 50% nor- (CHO) cells. This study thus represents the first complete mo- mal activity. Pulse-labeling studies demonstrated that the G1 lecular analysis ofthe aberrant enzymes expressed by both mu- enzyme had a normal half life, while the G2 enzyme had a tant ALAD alleles in a patient with ADP. markedly decreased half life. These data thus define the sepa- rate point mutations in each ALAD allele, as well as the altered Methods properties of the two enzymic proteins encoded by the mutant genes in a patient with ADP. (J. Clin. Invest. 1992. 89:1431- Patient andfamily members studied. The patient developed signs and 1437.) Key words: Point mutation * PCR * transfection * expres- symptoms of ADP at the age of 15 (8). His mother, a brother, and a sion * compound heterozygosity sister studied were all from family B, described previously (8). Cell cultures. Isolation oflymphocytes, transformation of cells with EBV, and cultivation of lymphoblastoid cells were carried out, as de- Introduction scribed previously (9). b-Aminolevulinate is a cy- Synthesis ofALAD cDNA. Poly(A)+ RNA was prepared from EBV- dehydratase (E.C.4.2. 1.24; ALAD)' transformed lymphoblastoid cells, as described previously (1 1, 12). The tosolic enzyme in the heme biosynthetic pathway that catalyzes first strand ofcDNA was synthesized using Moloney Murine Leukemia the condensation of two molecules of 5-aminolevulinic acid to Virus (MMLV) reverse transcriptase (Pharmacia Fine Chemicals) (Su- form a monopyrrole, porphobilinogen (1). The enzyme activ- perScript RNaseH- reverse transcriptase; Gibco-BRL, Gaithersburg, ity is present in great excess in normal cells, thus a partial defi- MD) primed with oligo(dT)1218(Pharmacia Fine Chemicals, Uppsala, ciency of this enzyme activity is not accompanied by any clini- Sweden). cal consequences (2-4). However, a marked enzyme deficiency Polymerase chain reaction (PCR). Two oligomers for amplifica- may occur through inhibition ofthe enzyme activity by certain tion, i.e., ALADI: 5'-CCGGAATTCCAACCAACTGATGCCC, and ALAD2: 5'-GTTCTAGAGCCTGGCACTGTCCTCC, were designed with the EcoRI site and the Xbal site at the 5'-terminus, respectively Address correspondence and reprint requests to Dr. Shigeru Sassa, The (10). These oligomers correspond to the 5'-untranslated and the 3'-un- Rockefeller University, New York, NY 10021. translated regions of ALAD cDNA (13), respectively. Amplifications Receivedfor publication 27 August 1991 and in revisedform 3 De- were performed three times in separate experiments, and further steps cember 1991. were processed separately as well. PCR was carried out using a DNA Thermal Cycler (Perkin-Elmer-Cetus Corp. Norwalk, CT), employing 1. Abbreviations used in this paper: ADP, ALAD deficiency porphyria; a thermal cycle program, as described previously (10). ALAD, delta-aminolevulinate dehydratase; A/M, activity/mass; ASOs, Cloning and sequencing of cDNAs. PCR products were digested allele-specific oligonucleotides; CHO, Chinese hamster ovary; CRIM, with EcoRI and Xbal, and cloned into pGEM4z vector (Promega Bio- cross-reactive immunological material; PCR, polymerase chain reac- tec, Madison, WI). Appropriate restriction fragments were subcloned tion; RT-PCR, reverse transcription PCR. into Ml 3mp18 or Ml 3mp 19. DNA sequencing was carried out by the dideoxy chain-termination method using a genetically engineered T7 J. Clin. Invest. DNA polymerase (Sequenase version 2.0; United States Biochemical © The American Society for Clinical Investigation, Inc. Corp., Cleveland, OH) (14-16). Sequence data were analyzed using 0021-9738/92/05/1431/07 $2.00 DNASIS software (Hitachi, San Bruno, CA). Volume 89, May 1992, 143 1-1437 Phenotype analysis. ALAD phenotype analyses in the proband's Molecular Defects ofDelta-Aminolevulinate Dehydratase Porphyria 1431 family were carried out using the reverse transcription-polymerase as polyadenylation sites derived from the rabbit ,B-globin gene, and the chain reaction (RT-PCR) (10). Purification of total RNA, synthesis of SV40 early gene. The construction of pdKCR-Neo was performed by a cDNA, amplification of ALAD cDNA, and hybridization with ASOs serial modification (19-21) of the plasmid, and by replacing the dhfr were performed, as described previously (10). Briefly, total RNA (1-5 gene with the neomycin resistant gene (S. Oikawa, personal communi- ,ug) was reverse transcribed with SuperScript RNaseH- reverse tran- cation). The resulting plasmid contained a unique EcoRI site for inser- scriptase, using random hexamers (Pharmacia Fine Chemicals) as a tion of foreign sequence (19), into which the EcoRI fragments of primer. ALAD cDNAs were then amplified by PCR, using ALAD1 and cDNAs were inserted. Recombinant plasmids, which encode the nor- ALAD2 as the primers. After 45 cycles of amplification, the aliquots of mal ALAD, a mutant ALAD with the GI defect, and a mutant ALAD RT-PCR products were subjected to hybridization with ASOs, or to with the G2 defect, were introduced into CHO cells by coprecipitation direct sequencing. with calcium phosphate (CellPhect Transfection kit; Pharmacia Fine Direct sequencing of RT-PCR products of ALAD mRNA. Single- Chemicals). Selection with G418 (Geneticin; Gibco-BRL) (1,600 ,g/ stranded DNAs were synthesized from aliquots of RT-PCR products, ml) was carried out, starting 48 h after transfection. using asymmetric PCR according to Gyllensten et al. (17). The reaction Western blot analysis. Human ALAD expressed in CHO cells was mixture, which included ALAD1 and ALAD2 as the specific primers at specifically detected by Western blot analysis, using 0.5-1.0 x 106 the molar ratio of either 100 pmol/5 pmol or 5 pmol/100 pmol, was CHO cells transfected with the normal or the mutant ALAD cDNAs. subjected to the following amplification cycles, using a Biocycler Oven Antibody used in this study was a rabbit IgG purified from a monospe- (BIOS Corp., New Haven, CT): 3 cycles of 1 min at 94°C, 30 s at 57°C, cific polyclonal antibody against human ALAD (22, 23). Detection of and 45 s at 72°C, followed by 27 cycles of 30 s at 940C, 30 s at 570C, the specific immune complex was carried out using an enhanced chemi- and 45 s at 720C.