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Homozygosity for the R1268Q Mutation in MRP6, the Pseudoxanthoma Elasticum Gene, Is Not Disease-Causing
Biochemical and Biophysical Research Communications 274, 297–301 (2000) doi:10.1006/bbrc.2000.3101, available online at http://www.idealibrary.com on
Homozygosity for the R1268Q Mutation in MRP6, the Pseudoxanthoma Elasticum Gene, Is Not Disease-Causing Dominique P. Germain, 1 Je´roˆme Perdu, Ve´ronique Remones, and Xavier Jeunemaitre De´partement de Ge´ne´tique, Hoˆpital Europe´en Georges Pompidou, Universite´ Paris VI, Paris, France
Received June 13, 2000
Pseudoxanthoma elasticum (PXE) is an inherited systemic disorder of connective tissue, characterized by progressive calcification of the elastic fibers in the eye, the skin, and the cardiovascular system, resulting in decreased vision, skin lesions, and life-threatening vascular disease, with highly variable phenotypic expression. The PXE locus has been mapped to chromosome 16p13.1, and was recently further refined to a 500 kb-region, containing two pseudogenes and four candidate genes. In a comprehensive mutational screening, we were able to exclude the responsibility of pM5, UNK, and MRP1 genes, candidate on the basis of their genetic localization. Conversely, we have found pathogenetic mutations in the MRP6 gene, in patients affected with PXE, indicating that human MRP6, which encodes a 1503 amino-acids membrane protein, member of the human ATP binding cassette (ABC) transporters superfamily, is the gene responsible for PXE. In one large PXE pedigree for which we had identified a nonsense mutation (R1141X), we came across a G to A transition at position 3803 of the MRP6 cDNA sequence (R1268Q). Astonishingly, this latter variant was found at the homozygous state in the proband’s unaffected husband. We investigated the R1268Q mutation, and found the Q1268 allele at a relatively high frequency (0.19) in a Caucasian control population (n ⴝ 62 subjects). Genotype frequencies were in HardyWeinberg equilibrium, and three healthy volunteers were homozygous for the Q1268 allele. These data indicate that the R1268Q variant in the MRP6 gene does not cause PXE per se. Further studies will elucidate if it may play a role when found in compound heterozygotes. © 2000 Academic Press Key Words: MRP6; multidrug resistance protein 6; ATP binding cassette transporters; pseudoxanthoma elasticum.
Pseudoxanthoma elasticum (PXE) is an inherited disorder of connective tissue in which the elastic fibers of the skin, eyes, and cardiovascular system slowly become calcified, causing a spectrum of disease involving these three organ systems (1). Most cases of PXE are sporadic, but autosomal recessive inheritance (OMIM 264800) and autosomal dominant segregation (OMIM 177850) have also been described (2). Previous efforts to link the disease, in limited numbers of families, to several potential candidate genes, such as elastin, fibrillins I and II, and lysyl oxidase, were negative or equivocal. Following a systematic genome search, two groups have independently reported linkage of both autosomal dominant and recessive forms of PXE to a 3- to 5-cM domain within the p13.1 locus of chromosome 16 (3, 4). The PXE locus was recently further refined within 16p13.1 to a region of 500 kb containing two pseudogenes and four candidate genes: MRP1, MRP6, pM5 and two copies of a previously unknown gene (UNK) (5, 6). In a comprehensive mutational analysis, we were able to exclude PM5, UNK and MRP1 as the genes responsible for PXE. Conversely, pathogenetic mutations in the MRP6 gene, have been found in patients affected with PXE, indicating this gene as responsible for the disease (7, Germain et al., in preparation). During our molecular analysis of the MRP6 gene in PXE patients, we came across a G to A transition at position 3803 of the cDNA sequence, altering the codon (CGG) for arginine to the codon (CAG) for glutamine (R1268Q) in a pedigree in which we had previously identified a nonsense mutation (R1141X). In the present study, the R1268Q mutation was investigated in relatives of our PXE patients and in a control population, to determine if this amino-acid change was a disease-causing mutation or a neutral polymorphism. PATIENTS
1
To whom correspondence should be addressed at Laboratoire de Ge´ne´tique. Hoˆpital Broussais, 96, rue Didot, 75014 Paris, France. Fax: ⫹ 33 1 45 41 02 34. E-mail: [email protected].
Two sisters presenting with PXE, eight unaffected members of their family, and 62 healthy volunteers
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Vol. 274, No. 2, 2000
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were evaluated in this study (Fig. 1). Both patients were examined by one of us (D.P.G), and diagnosis of PXE was consistent with previously reported consensus criteria, which include a positive von Kossa stain of a skin biopsy, indicating calcification of elastic fibers, in combination with specific cutaneous, and ocular manifestations (angioid streaks), and a family history of PXE. All participants in the study provided written informed consent, using a form that was approved by the Institutional Review Board of our academic institution. MATERIALS AND METHODS Amplification of genomic DNA fragments. Whole-blood samples were obtained with informed consent from both affected sisters, 8 family relatives, and 62 Caucasians controls. High molecular weight DNA was isolated from peripheral blood leukocytes according to standard procedures. We designed primers for amplification of the MRP6 gene from the published sequence of human chromosome 16 BAC clone A-962B4 (GenBank Accession No. U91318). Exon 24 and exon 27 of the MRP6 gene were PCR-amplified, using the following primers: 5⬘-AAGCAACTAGCCCAAGGTCA-3⬘ (E24forward), 5⬘-GCCCACGGTGAGAACTGATA-3⬘ (E24 reverse), 5⬘-AAAAGTAGCCACCACCATCT-3⬘ (E27-forward), and 5⬘-GAAGGAAGTCACGGAGTTGC-3⬘ (E27-reverse). For all patients and control subjects, the PCR reactions were performed on genomic DNA (100 ng) in 20 L final volume, using 5 pmol of each primer, 1.5 mM MgCl 2, 250 M dNTPs, 1 unit Taq DNA Polymerase (ATGC Biotechnologie) under the following conditions 95°C 5 min ⫻ 1; 95°C 1 min, 58°C 1 min, 72°C 90 s for 35 cycles; 72°C 10 min ⫻ 1, on a Robocycler gradient 96 (Stratagene). An aliquot of each amplicons was analyzed by ethidium bromide visualization on a 2% agarose gel to assess the size of the fragment(s). Sequencing. MRP6 PCR products of PXE patients were purified by using QIAquick Spin PCR Purification Kit (Qiagen) to remove unincorporated nucleotides, and subjected to automated nucleotide sequencing on an automated ABI 310 DNA sequencer (PE Biosystems), with the Big Dye Sequencing Kit (PE Biosystems) according to the manufacturer’s recommendations. DNA sequences were handled with Navigator 2.0 software (PE Biosystems). Restriction digests. Mutation R1268Q predicted the creation of a novel restriction site for BstX I. We performed restriction analysis using BstX I on PCR-amplified genomic DNA, to examine family members and 14 Caucasian controls for the presence or absence of the nucleotide change. Reactions were performed in 20 l final volume, following the manufacturer’s instructions (New England Biolabs). Mutation R1268Q also predicted the loss of a Msp I restriction site. We performed restriction digests using Msp I on PCR-amplified genomic DNA, to confirm data obtained for family relatives, and to test for the presence or absence of the nucleotide change in 48 additional healthy volunteers. Nomenclature. The designations for the mutations refer to the position of the amino acid substitution, where amino acid 1 is the amino terminus of the MRP6 protein. The cDNA base numbers refer to the nucleotide in the cDNA, where nucleotide 1 is the A of the first ATG (GenBank Accession No. AF076622) (8).
RESULTS The MRP6 gene (8), a candidate on the basis of its genetic localization at 16p13.1 (5, 6), was very recently
demonstrated as the gene responsible for pseudoxanthoma elasticum, an inherited disorder of connective tissue (7, Germain et al., unpublished data). In the present study, the molecular pathology of the MRP6 gene was investigated in 2 female patients from a newly ascertained PXE pedigree of French/Portuguese ancestry. During our mutational analysis, we found a heterozygous C to T transition at cDNA position 3421 of the MRP6 gene, predicting termination of translation at codon 1141 (R1141X) (Fig. 2). Another mutation, corresponding to a G to A transition was also evidenced by direct sequencing, at position 3803 in the cDNA sequence of the MRP6 gene, in both affected sisters. This nucleotide substitution alters the codon (CGG) for arginine to the codon (CAG) for glutamine (R1268Q). Restriction digests using BstX I, performed to examine family relatives, showed that both affected patients (II-3 and II-5) and their asymptomatic mother (I-2) were heteroallelic for R1268Q at the genomic level, the father (I-1) being a wild type homozygote. However, the proband’s asymptomatic husband (individual II-6 in Fig. 1) was found to be homozygote for R1268Q, ruling out a causative role for homozygosity for this mutation in the clinical phenotype observed in PXE patients. Mutation R1268Q also predicted the loss of a Msp I restriction site. Restriction digests, using Msp I, carried out in the aforementioned relatives, confirmed the previous data. This prompted us to test 62 healthy volunteers for the presence or absence of the c3803G ⬎ A nucleotidic change, using either BstX I or Msp I. The R1268Q mutation was identified in 24 of the 124 tested alleles, and its overall frequency was 0.19 (Fig. 3). Among controls, 18 individuals were heterozygotes and 3 were homozygotes for the glutamine residue at position 1268 of the protein sequence (Fig. 3). Direct sequencing of exon 27 of the MRP6 gene formally confirmed the homozygosity for the G3803 to A transition in a healthy volunteer, unaffected with PXE (Fig. 4). Our results indicate that the R1268Q mutation in the MRP6 gene is a neutral polymorphism, which does not cause pseudoxanthoma elasticum when present at the homozygous state. DISCUSSION We have recently identified mutations in the MRP6 gene as the genetic defect which underlies the phenotype observed in patients affected with pseudoxanthoma elasticum (PXE), an inherited disorder of connective tissue. The human MRP6 gene is located on chromosome 16p13.1, 9 kb centromeric to MRP1. The 4512 bp MRP6 transcript encodes a protein of 1503 amino acids, with a predicted molecular weight of M r 165,000 (8). The MRP6 protein is a polytopic integral
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FIG. 1. Pedigree of a family of French/Portuguese ancestry affected with pseudoxanthoma elasticum (PXE). Filled symbols, affected individuals; open symbols, unaffected. Arrow indicates the proband. Segregation of the allele at codon 1268 of the MRP6 protein is indicated. R, arginine; Q, glutamine.
membrane protein, which belongs to the multidrug resistance protein (MRP) subfamily of the ABC transporters superfamily (9). Hydrophobicity analysis of the MRP6 amino acid sequence indicates a conserved organization of putative transmembrane domains, resulting in a similar topology as suggested for MRP1, which spans the membrane 17 times, with the NH 2 terminus being extracellular (8). During our mutational scanning of the MRP6 gene, in two sisters affected with pseudoxanthoma elasticum, a G to A transition, was detected at position 3803
FIG. 2. Detection of the R1141X nonsense mutation by direct sequencing of the MRP6 gene in the proband. The arrow indicates the heterozygous C to T transition at position 3421 of the cDNA sequence, altering the codon for arginine (CGA) to a stop codon (TGA).
of the MRP6 cDNA sequence, predicting a R1268Q missense mutation at the protein level. This change is non conservative, converting a positively charged amino-acid in an uncharged residue, and could on this basis be predicted to be disease-causing. The amino acid at codon 1268 is predicted to belong to the cytoplasmic carboxyterminal domain of the MRP6 protein (10), and is evolutionarily conserved in the rat and the
FIG. 3. (A) BstX I digests of PCR-amplified exon 27 of the MRP6 gene in six healthy volunteers show homozygosity for the R1268 allele in three subjects (lanes 2, 3, and 4), heterozygosity for two other individuals (lanes 5 and 6), and homozygosity for the Q1268 allele in the last control (lane 7). Lanes 1 and 8, 100-bp molecular weight ladder. (B) Msp I restriction digests of the same amplicon in six other unaffected controls show homozygosity for Q1268 allele in one of them (lane 5). A 100-bp ladder is used as a size reference.
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FIG. 4. Direct sequencing of the MRP6 genomic sequence (exon 27) in a healthy volunteer shows homozygous G ⬎ A transition at position 3803, predicting the substitution of glutamine for arginine at codon 1268 of the MRP6 protein.
mouse mrp6 proteins (GenBank Accession Nos.: rat mrp6, RNU73038; mouse mrp6, AB028737), as well as in human MRP1, and human MRP3 (GenBank Accession Nos.: human MRP1, L05628; human MRP3, AF009670) (10 –12). Accuracy is essential when reporting variations in genome sequences, since they will subsequently be included in mutation databases, and used as a basis for genetic counselling in affected pedigrees (13). Incomplete scanning for mutations may lead to wrongly propose a harmless neutral polymorphism to be a cause of a genetic disease (14). Once a mutation is found which might cause disease, the search elsewhere in the gene is often stopped. Therefore, the investigator may miss a second, or disease-causing, mutation (13). Incriminating the variation as disease-causing is a critical step in medical genetics where disease-causing sequence variations are referred to as mutations, and neutral sequence variations as polymorphisms (15). The ultimate test whether a particular variation causes disease, is expression of the protein from the gene with the putative disease-causing base change and measuring its activity (13). However, to our knowledge, very few is known regarding the specific function of the MRP6 protein. It does not contribute to the multidrug-resistant phenotype, and its overexpression has been found to be invariably associated with amplification of the MRP1 gene, which is localized 9 kb telomeric, in opposite orientation, on human chromosome 16p13.1 (8). The recent demonstration that PXE fibroblasts have altered cell-cell and cell-matrix interactions, associated with modified proliferation capabilities (16), is consistent with a broad regulatory role on the cellular machinery for MRP6, but to our knowledge, the physiological role of MRP6 remains unknown, although its obvious similarity to the MRP family suggests an involvement in transport across the plasma membrane. When expression studies cannot be performed, a series of issues can be addressed to help in deciding if a particular base change is harmless or not (13). The nature and location of the base change may be considered. However, it is certainly an oversimplification to conclude on the severity of a given missense mutation, when only considering the aminoacids involved in the substitution. The nature and location of the change
must be interpreted in relation to the 3D structure, or potentially important domains of the protein (17). However, data available for the MRP6 protein are limited (8). Another argument comes from the frequency of the mutation in the affected and general population. We have therefore investigated the R1268Q substitution to determine its frequency, and have found its frequency to be high in a Caucasian control population. After we had completed our work, mutations in the MRP6 gene were formally demonstrated as the genetic defect which underlies PXE (7). It is interesting to note that the authors also detected the R1268Q mutation in their patients population, although always in the context of compound heterozygosity. Whether the R1268Q mutation may lead to the clinical phenotype observed in patients with PXE, when found in association with another, potentially more severe, mutation in the MRP6 gene remains to be elucidated. However, since PXE is a rare disease, with an estimated prevalence of 1 in 70,000 (1), the high frequency observed for the Q1268 allele in our control population is much against this latter hypothesis. In conclusion, while screening the MRP6 gene in patients affected with PXE, we have identified, beside disease-causing mutations, a 3803G ⬎ A transition (R1268Q) which was shown to be a harmless polymorphism when present at the homozygous state. Our results should prove invaluable for future DNA-based genetic counselling and prenatal diagnosis in families affected with PXE. ACKNOWLEDGMENTS The authors thank the PXE patients, their families and clinicians (Drs. M. Blayau, J. N. Fiessinger, E. Flori, B. Le Marec, L. Van Maldergem, and M. T. Zabot) for their help during this project. We gratefully acknowledge J. Jean and T. Joao for expert technical assistance.
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7. Ringpfeil, F., Lebwohl, M. G., Christiano, A. M., and Uitto, J. (2000) Proc. Natl. Acad. Sci. USA 97, 6001– 6006. 8. Kool, M., van der Linden, M., de Hass, M., Baas, F., and Borst, P. (1999) Cancer Res. 59, 175–182. 9. Hyde, S. C., Emsley, P., Hartshorn, M. J., Mimmack, M. M., Gileadi, U., Pearce, S. R., Gallagher, M. P., Gill, D. R., Hubbard, R. E., and Higgins, C. F. (1990) Nature 346, 362–365. 10. Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T., and Sugiyama, Y. (1998) Mol. Pharmacol. 53, 1068 –1075. 11. Grant, D. E., Kurz, E. U., Cole, S. P. C., and Deeley, R. G. (1997) Genomics 45, 368 –378. 12. Kool, M., van der Linden, M., de Haas, M., Scheffer, G. L., de
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Homozygosity for the R1268Q Mutation in MRP6, the Pseudoxanthoma Elasticum Gene, Is Not Disease-Causing Dominique P. Germain, 1 Je´roˆme Perdu, Ve´ronique Remones, and Xavier Jeunemaitre De´partement de Ge´ne´tique, Hoˆpital Europe´en Georges Pompidou, Universite´ Paris VI, Paris, France
Received June 13, 2000
Pseudoxanthoma elasticum (PXE) is an inherited systemic disorder of connective tissue, characterized by progressive calcification of the elastic fibers in the eye, the skin, and the cardiovascular system, resulting in decreased vision, skin lesions, and life-threatening vascular disease, with highly variable phenotypic expression. The PXE locus has been mapped to chromosome 16p13.1, and was recently further refined to a 500 kb-region, containing two pseudogenes and four candidate genes. In a comprehensive mutational screening, we were able to exclude the responsibility of pM5, UNK, and MRP1 genes, candidate on the basis of their genetic localization. Conversely, we have found pathogenetic mutations in the MRP6 gene, in patients affected with PXE, indicating that human MRP6, which encodes a 1503 amino-acids membrane protein, member of the human ATP binding cassette (ABC) transporters superfamily, is the gene responsible for PXE. In one large PXE pedigree for which we had identified a nonsense mutation (R1141X), we came across a G to A transition at position 3803 of the MRP6 cDNA sequence (R1268Q). Astonishingly, this latter variant was found at the homozygous state in the proband’s unaffected husband. We investigated the R1268Q mutation, and found the Q1268 allele at a relatively high frequency (0.19) in a Caucasian control population (n ⴝ 62 subjects). Genotype frequencies were in HardyWeinberg equilibrium, and three healthy volunteers were homozygous for the Q1268 allele. These data indicate that the R1268Q variant in the MRP6 gene does not cause PXE per se. Further studies will elucidate if it may play a role when found in compound heterozygotes. © 2000 Academic Press Key Words: MRP6; multidrug resistance protein 6; ATP binding cassette transporters; pseudoxanthoma elasticum.
Pseudoxanthoma elasticum (PXE) is an inherited disorder of connective tissue in which the elastic fibers of the skin, eyes, and cardiovascular system slowly become calcified, causing a spectrum of disease involving these three organ systems (1). Most cases of PXE are sporadic, but autosomal recessive inheritance (OMIM 264800) and autosomal dominant segregation (OMIM 177850) have also been described (2). Previous efforts to link the disease, in limited numbers of families, to several potential candidate genes, such as elastin, fibrillins I and II, and lysyl oxidase, were negative or equivocal. Following a systematic genome search, two groups have independently reported linkage of both autosomal dominant and recessive forms of PXE to a 3- to 5-cM domain within the p13.1 locus of chromosome 16 (3, 4). The PXE locus was recently further refined within 16p13.1 to a region of 500 kb containing two pseudogenes and four candidate genes: MRP1, MRP6, pM5 and two copies of a previously unknown gene (UNK) (5, 6). In a comprehensive mutational analysis, we were able to exclude PM5, UNK and MRP1 as the genes responsible for PXE. Conversely, pathogenetic mutations in the MRP6 gene, have been found in patients affected with PXE, indicating this gene as responsible for the disease (7, Germain et al., in preparation). During our molecular analysis of the MRP6 gene in PXE patients, we came across a G to A transition at position 3803 of the cDNA sequence, altering the codon (CGG) for arginine to the codon (CAG) for glutamine (R1268Q) in a pedigree in which we had previously identified a nonsense mutation (R1141X). In the present study, the R1268Q mutation was investigated in relatives of our PXE patients and in a control population, to determine if this amino-acid change was a disease-causing mutation or a neutral polymorphism. PATIENTS
1
To whom correspondence should be addressed at Laboratoire de Ge´ne´tique. Hoˆpital Broussais, 96, rue Didot, 75014 Paris, France. Fax: ⫹ 33 1 45 41 02 34. E-mail: [email protected].
Two sisters presenting with PXE, eight unaffected members of their family, and 62 healthy volunteers
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0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 274, No. 2, 2000
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were evaluated in this study (Fig. 1). Both patients were examined by one of us (D.P.G), and diagnosis of PXE was consistent with previously reported consensus criteria, which include a positive von Kossa stain of a skin biopsy, indicating calcification of elastic fibers, in combination with specific cutaneous, and ocular manifestations (angioid streaks), and a family history of PXE. All participants in the study provided written informed consent, using a form that was approved by the Institutional Review Board of our academic institution. MATERIALS AND METHODS Amplification of genomic DNA fragments. Whole-blood samples were obtained with informed consent from both affected sisters, 8 family relatives, and 62 Caucasians controls. High molecular weight DNA was isolated from peripheral blood leukocytes according to standard procedures. We designed primers for amplification of the MRP6 gene from the published sequence of human chromosome 16 BAC clone A-962B4 (GenBank Accession No. U91318). Exon 24 and exon 27 of the MRP6 gene were PCR-amplified, using the following primers: 5⬘-AAGCAACTAGCCCAAGGTCA-3⬘ (E24forward), 5⬘-GCCCACGGTGAGAACTGATA-3⬘ (E24 reverse), 5⬘-AAAAGTAGCCACCACCATCT-3⬘ (E27-forward), and 5⬘-GAAGGAAGTCACGGAGTTGC-3⬘ (E27-reverse). For all patients and control subjects, the PCR reactions were performed on genomic DNA (100 ng) in 20 L final volume, using 5 pmol of each primer, 1.5 mM MgCl 2, 250 M dNTPs, 1 unit Taq DNA Polymerase (ATGC Biotechnologie) under the following conditions 95°C 5 min ⫻ 1; 95°C 1 min, 58°C 1 min, 72°C 90 s for 35 cycles; 72°C 10 min ⫻ 1, on a Robocycler gradient 96 (Stratagene). An aliquot of each amplicons was analyzed by ethidium bromide visualization on a 2% agarose gel to assess the size of the fragment(s). Sequencing. MRP6 PCR products of PXE patients were purified by using QIAquick Spin PCR Purification Kit (Qiagen) to remove unincorporated nucleotides, and subjected to automated nucleotide sequencing on an automated ABI 310 DNA sequencer (PE Biosystems), with the Big Dye Sequencing Kit (PE Biosystems) according to the manufacturer’s recommendations. DNA sequences were handled with Navigator 2.0 software (PE Biosystems). Restriction digests. Mutation R1268Q predicted the creation of a novel restriction site for BstX I. We performed restriction analysis using BstX I on PCR-amplified genomic DNA, to examine family members and 14 Caucasian controls for the presence or absence of the nucleotide change. Reactions were performed in 20 l final volume, following the manufacturer’s instructions (New England Biolabs). Mutation R1268Q also predicted the loss of a Msp I restriction site. We performed restriction digests using Msp I on PCR-amplified genomic DNA, to confirm data obtained for family relatives, and to test for the presence or absence of the nucleotide change in 48 additional healthy volunteers. Nomenclature. The designations for the mutations refer to the position of the amino acid substitution, where amino acid 1 is the amino terminus of the MRP6 protein. The cDNA base numbers refer to the nucleotide in the cDNA, where nucleotide 1 is the A of the first ATG (GenBank Accession No. AF076622) (8).
RESULTS The MRP6 gene (8), a candidate on the basis of its genetic localization at 16p13.1 (5, 6), was very recently
demonstrated as the gene responsible for pseudoxanthoma elasticum, an inherited disorder of connective tissue (7, Germain et al., unpublished data). In the present study, the molecular pathology of the MRP6 gene was investigated in 2 female patients from a newly ascertained PXE pedigree of French/Portuguese ancestry. During our mutational analysis, we found a heterozygous C to T transition at cDNA position 3421 of the MRP6 gene, predicting termination of translation at codon 1141 (R1141X) (Fig. 2). Another mutation, corresponding to a G to A transition was also evidenced by direct sequencing, at position 3803 in the cDNA sequence of the MRP6 gene, in both affected sisters. This nucleotide substitution alters the codon (CGG) for arginine to the codon (CAG) for glutamine (R1268Q). Restriction digests using BstX I, performed to examine family relatives, showed that both affected patients (II-3 and II-5) and their asymptomatic mother (I-2) were heteroallelic for R1268Q at the genomic level, the father (I-1) being a wild type homozygote. However, the proband’s asymptomatic husband (individual II-6 in Fig. 1) was found to be homozygote for R1268Q, ruling out a causative role for homozygosity for this mutation in the clinical phenotype observed in PXE patients. Mutation R1268Q also predicted the loss of a Msp I restriction site. Restriction digests, using Msp I, carried out in the aforementioned relatives, confirmed the previous data. This prompted us to test 62 healthy volunteers for the presence or absence of the c3803G ⬎ A nucleotidic change, using either BstX I or Msp I. The R1268Q mutation was identified in 24 of the 124 tested alleles, and its overall frequency was 0.19 (Fig. 3). Among controls, 18 individuals were heterozygotes and 3 were homozygotes for the glutamine residue at position 1268 of the protein sequence (Fig. 3). Direct sequencing of exon 27 of the MRP6 gene formally confirmed the homozygosity for the G3803 to A transition in a healthy volunteer, unaffected with PXE (Fig. 4). Our results indicate that the R1268Q mutation in the MRP6 gene is a neutral polymorphism, which does not cause pseudoxanthoma elasticum when present at the homozygous state. DISCUSSION We have recently identified mutations in the MRP6 gene as the genetic defect which underlies the phenotype observed in patients affected with pseudoxanthoma elasticum (PXE), an inherited disorder of connective tissue. The human MRP6 gene is located on chromosome 16p13.1, 9 kb centromeric to MRP1. The 4512 bp MRP6 transcript encodes a protein of 1503 amino acids, with a predicted molecular weight of M r 165,000 (8). The MRP6 protein is a polytopic integral
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FIG. 1. Pedigree of a family of French/Portuguese ancestry affected with pseudoxanthoma elasticum (PXE). Filled symbols, affected individuals; open symbols, unaffected. Arrow indicates the proband. Segregation of the allele at codon 1268 of the MRP6 protein is indicated. R, arginine; Q, glutamine.
membrane protein, which belongs to the multidrug resistance protein (MRP) subfamily of the ABC transporters superfamily (9). Hydrophobicity analysis of the MRP6 amino acid sequence indicates a conserved organization of putative transmembrane domains, resulting in a similar topology as suggested for MRP1, which spans the membrane 17 times, with the NH 2 terminus being extracellular (8). During our mutational scanning of the MRP6 gene, in two sisters affected with pseudoxanthoma elasticum, a G to A transition, was detected at position 3803
FIG. 2. Detection of the R1141X nonsense mutation by direct sequencing of the MRP6 gene in the proband. The arrow indicates the heterozygous C to T transition at position 3421 of the cDNA sequence, altering the codon for arginine (CGA) to a stop codon (TGA).
of the MRP6 cDNA sequence, predicting a R1268Q missense mutation at the protein level. This change is non conservative, converting a positively charged amino-acid in an uncharged residue, and could on this basis be predicted to be disease-causing. The amino acid at codon 1268 is predicted to belong to the cytoplasmic carboxyterminal domain of the MRP6 protein (10), and is evolutionarily conserved in the rat and the
FIG. 3. (A) BstX I digests of PCR-amplified exon 27 of the MRP6 gene in six healthy volunteers show homozygosity for the R1268 allele in three subjects (lanes 2, 3, and 4), heterozygosity for two other individuals (lanes 5 and 6), and homozygosity for the Q1268 allele in the last control (lane 7). Lanes 1 and 8, 100-bp molecular weight ladder. (B) Msp I restriction digests of the same amplicon in six other unaffected controls show homozygosity for Q1268 allele in one of them (lane 5). A 100-bp ladder is used as a size reference.
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FIG. 4. Direct sequencing of the MRP6 genomic sequence (exon 27) in a healthy volunteer shows homozygous G ⬎ A transition at position 3803, predicting the substitution of glutamine for arginine at codon 1268 of the MRP6 protein.
mouse mrp6 proteins (GenBank Accession Nos.: rat mrp6, RNU73038; mouse mrp6, AB028737), as well as in human MRP1, and human MRP3 (GenBank Accession Nos.: human MRP1, L05628; human MRP3, AF009670) (10 –12). Accuracy is essential when reporting variations in genome sequences, since they will subsequently be included in mutation databases, and used as a basis for genetic counselling in affected pedigrees (13). Incomplete scanning for mutations may lead to wrongly propose a harmless neutral polymorphism to be a cause of a genetic disease (14). Once a mutation is found which might cause disease, the search elsewhere in the gene is often stopped. Therefore, the investigator may miss a second, or disease-causing, mutation (13). Incriminating the variation as disease-causing is a critical step in medical genetics where disease-causing sequence variations are referred to as mutations, and neutral sequence variations as polymorphisms (15). The ultimate test whether a particular variation causes disease, is expression of the protein from the gene with the putative disease-causing base change and measuring its activity (13). However, to our knowledge, very few is known regarding the specific function of the MRP6 protein. It does not contribute to the multidrug-resistant phenotype, and its overexpression has been found to be invariably associated with amplification of the MRP1 gene, which is localized 9 kb telomeric, in opposite orientation, on human chromosome 16p13.1 (8). The recent demonstration that PXE fibroblasts have altered cell-cell and cell-matrix interactions, associated with modified proliferation capabilities (16), is consistent with a broad regulatory role on the cellular machinery for MRP6, but to our knowledge, the physiological role of MRP6 remains unknown, although its obvious similarity to the MRP family suggests an involvement in transport across the plasma membrane. When expression studies cannot be performed, a series of issues can be addressed to help in deciding if a particular base change is harmless or not (13). The nature and location of the base change may be considered. However, it is certainly an oversimplification to conclude on the severity of a given missense mutation, when only considering the aminoacids involved in the substitution. The nature and location of the change
must be interpreted in relation to the 3D structure, or potentially important domains of the protein (17). However, data available for the MRP6 protein are limited (8). Another argument comes from the frequency of the mutation in the affected and general population. We have therefore investigated the R1268Q substitution to determine its frequency, and have found its frequency to be high in a Caucasian control population. After we had completed our work, mutations in the MRP6 gene were formally demonstrated as the genetic defect which underlies PXE (7). It is interesting to note that the authors also detected the R1268Q mutation in their patients population, although always in the context of compound heterozygosity. Whether the R1268Q mutation may lead to the clinical phenotype observed in patients with PXE, when found in association with another, potentially more severe, mutation in the MRP6 gene remains to be elucidated. However, since PXE is a rare disease, with an estimated prevalence of 1 in 70,000 (1), the high frequency observed for the Q1268 allele in our control population is much against this latter hypothesis. In conclusion, while screening the MRP6 gene in patients affected with PXE, we have identified, beside disease-causing mutations, a 3803G ⬎ A transition (R1268Q) which was shown to be a harmless polymorphism when present at the homozygous state. Our results should prove invaluable for future DNA-based genetic counselling and prenatal diagnosis in families affected with PXE. ACKNOWLEDGMENTS The authors thank the PXE patients, their families and clinicians (Drs. M. Blayau, J. N. Fiessinger, E. Flori, B. Le Marec, L. Van Maldergem, and M. T. Zabot) for their help during this project. We gratefully acknowledge J. Jean and T. Joao for expert technical assistance.
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