Identification and Functional Analysis of Two Novel Mutations in the Multidrug Resistance Protein 2 Gene in Israeli Patients with Dubin-Johnson Syndrome

Dubin-Johnson syndrome (DJS) is an inherited disorder characterized by conjugated hyperbilirubinemia and is caused by a deficiency of the multidrug resistance protein 2 (MRP2) located in the apical membrane of hepatocytes. The aim of this study was to identify the mutations in two previously characterized clusters of patients with Dubin-Johnson syndrome among Iranian and Moroccan Jews and determine the consequence of the mutations on MRP2 expression and function by expression studies. All 32 exons and adjacent regions of the MRP2 gene were screened by polymerase chain reaction and DNA sequencing. Two novel mutations were identified in exon 25. One mutation, 3517A→T, predicting a I1173F substitution, was found in 22 homozygous Iranian Jewish DJS patients from 13 unrelated families and a second mutation, 3449G→A, predicting a R1150H substitution, was found in 5 homozygous Moroccan Jewish DJS patients from 4 unrelated families. Use of four intragenic dimorphisms and haplotype analyses disclosed a specific founder effect for each mutation. The mutations were introduced into an MRP2 expression vector by site-directed mutagenesis, transfected into HEK-293 cells, and analyzed by a fluorescence transport assay, immunoblot, and immunocytochemistry. Continuous measurement of probenecid-sensitive carboxyfluorescein efflux revealed that both mutations impaired the transport activity of MRP2. Immunoblot analysis and immunocytochemistry showed that MRP2 (R1150H) matured properly and localized at the plasma membrane of transfected cells. In contrast, expression of MRP2 (I1173F) was low and mislocated to the endoplasmic reticulum of the transfected cells. These findings provide an explanation for the DJS phenotype in these two patient groups. Furthermore, the close localization of the two mutations identify this region of MRP2 as important for both activity and processing of the protein. Dubin-Johnson syndrome (DJS) is an inherited disorder characterized by conjugated hyperbilirubinemia and is caused by a deficiency of the multidrug resistance protein 2 (MRP2) located in the apical membrane of hepatocytes. The aim of this study was to identify the mutations in two previously characterized clusters of patients with Dubin-Johnson syndrome among Iranian and Moroccan Jews and determine the consequence of the mutations on MRP2 expression and function by expression studies. All 32 exons and adjacent regions of the MRP2 gene were screened by polymerase chain reaction and DNA sequencing. Two novel mutations were identified in exon 25. One mutation, 3517A→T, predicting a I1173F substitution, was found in 22 homozygous Iranian Jewish DJS patients from 13 unrelated families and a second mutation, 3449G→A, predicting a R1150H substitution, was found in 5 homozygous Moroccan Jewish DJS patients from 4 unrelated families. Use of four intragenic dimorphisms and haplotype analyses disclosed a specific founder effect for each mutation. The mutations were introduced into an MRP2 expression vector by site-directed mutagenesis, transfected into HEK-293 cells, and analyzed by a fluorescence transport assay, immunoblot, and immunocytochemistry. Continuous measurement of probenecid-sensitive carboxyfluorescein efflux revealed that both mutations impaired the transport activity of MRP2. Immunoblot analysis and immunocytochemistry showed that MRP2 (R1150H) matured properly and localized at the plasma membrane of transfected cells. In contrast, expression of MRP2 (I1173F) was low and mislocated to the endoplasmic reticulum of the transfected cells. These findings provide an explanation for the DJS phenotype in these two patient groups. Furthermore, the close localization of the two mutations identify this region of MRP2 as important for both activity and processing of the protein. Dubin-Johnson syndrome multidrug resistance protein 5-carboxyfluorescein polymerase chain reaction peptide N-glycosidase F green fluorescent protein wild type phosphate-buffered saline Dubin-Johnson syndrome (DJS)1 is an autosomal recessive disorder manifested by chronic conjugated hyperbilirubinemia and accumulation of a dark pigment in liver parenchymal cells (1Dubin I.N. Johnson F.B. Medicine. 1954; 33: 155-172Crossref PubMed Scopus (261) Google Scholar, 2Sprinz H. Nelson R.S. Ann. Intern. Med. 1954; 41: 952-962Crossref PubMed Scopus (89) Google Scholar). The disorder has recently been associated with several mutations in the multidrug resistance protein 2 (MRP2) gene (3Paulusma C.C. Kool M. Bosma P.J. Scheffer G.L. ter Borg F. Scheper R.J. Tytgat G.N. Borst P. Baas F. Oude Elferink R.P. Hepatology. 1997; 25: 1539-1542Crossref PubMed Scopus (500) Google Scholar, 4Wada M. Toh S. Taniguchi K. Nakamura T. Uchiumi T. Kohono K. Yoshida I. Kimura A. Sakisaka S. Adachi Y. Kuwano M. Hum. 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Courtois A. Campion J.P. Guillouzo A. Fardel O. Biochem. Pharmacol. 2000; 60: 1967-1975Crossref PubMed Scopus (67) Google Scholar). The results obtained by these measurements have a relatively poor temporal resolution and require transfection with high efficiency or development of stable cell lines. In this study we identified two novel mutations causing DJS in the Iranian and Moroccan Jewish patients, respectively, and obtained evidence for specific founder effects which account for the observed clusters. Both mutations were functionally analyzed by expressing the mutated MRP2 proteins in HEK-293 cells, testing their transport activity by a CF transport assay in single cells with high temporal resolution, and determining their cellular localization. Most patients with DJS were ascertained previously (23Shani M. Seligsohn U. Gilon E. Sheba C. Adam A. Q. J. Med. 1970; 39: 549-567PubMed Google Scholar) and have been followed for more than 3 decades. The diagnosis was based on the finding of chronic or intermittent conjugated hyperbilirubinemia, and on either finding in liver biopsy material the typical pigment in hepatocytes or a predominance of coproporphyrin I urinary excretion (37Ben-Ezzer J. Rimington C. Shani M. Seligsohn U. Sheba C. Szeinberg A. Clin. Sci. 1971; 40: 17-30Crossref PubMed Scopus (50) Google Scholar). A total of 35 patients of 24 unrelated families were included in the study. Twenty-two were of Iranian Jewish origin (13 families), 5 were of Moroccan Jewish origin (4 families), 2 were offspring of a Moroccan Jewish mother and an Iranian Jewish father, 3 were of Ashkenazi Jewish origin (3 families) and 3 were of Turkish, Kurdish, and Afghani Jewish origins, respectively. Control subjects were patients consecutively admitted to the Sheba Medical Center or healthy individuals who were Sheba Medical Center personnel. Definition of the ethnic origin of each subject was based on the country of birth of the individual's 4 grandparents. The human subject ethics committee of Sheba Medical Center approved the performance of the study. LipofectAMINE and Geneticin (G418) were obtained from Life Technologies, Inc. 5-(and-6)-Carboxyfluorescein diacetate was obtained from Molecular Probes, Inc. (Eugene, OR). Probenecid was from Sigma and cyclosporine A was from Sandoz Research Institute (Hanover, NJ). The monoclonal antibody M2III-6 (38Scheffer G.L. Kool M. Heijn M. de Haas M. Pijnenborg A.C. Wijnholds J. van Helvoort A. de Jong M.C. Hooijberg J.H. Mol C.A. van der Linden M. de Vree J.M. van der Valk P. Elferink R.P. Borst P. Scheper R.J. Cancer Res. 2000; 60: 5269-5277PubMed Google Scholar) was obtained from Kamiya Biomedical Co. (Seattle, WA). PNGase F was obtained from New England Biolabs (Beverly, MA). The human MRP2 expression vector, pcDNA3.1/MRP2, was a gift from Professor D. Keppler and has been described previously (27Cui Y. König J. Buchholz U. Spring H. Leier I. Keppler D. Mol. Pharmacol. 1999; 55: 929-937PubMed Google Scholar). Green fluorescent protein (GFP)-expressing plasmid was purchased from Life Technologies, Inc. Genomic DNA was extracted from peripheral blood leukocytes by the desalting procedure (39Miller S.A. Dykes D.D. Polesky H.F. Nuc. Acids. Res. 1988; 16: 1215-1219Crossref PubMed Scopus (17908) Google Scholar). Polymerase chain reaction (PCR) was used to amplify each of the 32 exons of the MRP2 gene and their intronic-exonic boundaries. Primers were designed according to intronic sequences flanking the exons (6Toh S. Wada M. Uchiumi T. Inokuch I.A. Makino Y. Horie Y. Adachi Y. Sakisaka S. Kuwano M. Am. J. Hum. Genet. 1999; 64: 739-746Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 7Tsujii H. König J. Rost D. Stöckel B. Leuschner U. Keppler D. Gastroenterology. 1999; 117: 653-660Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) and are presented in Table I. PCR was performed in a 20-μl PCR buffer containing 1.5 mm MgCl2, 100 to 200 ng of genomic DNA, 500 nm each primer, 200 μm each dNTP, and 0.125 unit of Taq polymerase (Super Nova; Laboratory Products, Kent, United Kingdom). The reactions were subjected to 35 cycles of 45 s of denaturation at 94 °C, 45 s of annealing at 55 °C, and 1 min of extension at 72 °C.Table IPrimers used for amplification of the 32 exons of the MRP2 geneExonFragment sizeForward primerReverse primerNameSequenceNameSequencebase pairs1404 1F5′-TTGTTGGCCAGCTCTGTTG-3′ 1R*5′-ACTACCACTTGTTCTGAGTC-3′2310 2F*5′-TGAAAGCAGTGGGATGTGC-3′ 2R*5′-CTCTACTGTGCAGCCAAGG-3′3295 3F*5′-ATCTGAATCACTGCATACCG-3′ 3R*5′-TCACCTAGATGCCTATGGG-3′4251 4F*5′-CTCAGTCCTCGGTTAGTGG-3′ 4R*5′-CTATGAGTTAGAGGTTGCCC-3′5266 5F*5′-GCCATGTAGACTTCCTTTGG-3′ 5R*5′-ACCTTATTCTGGGCTTGTGG-3′6185 6F*5′-TTAGAGTCCCATGAAGTTCC-3′ 6R*5′-AGTAAGGATACAGCCAATCC-3′7406 7F*5′-TGGAGATAGCCTCTGACCC-3′ 7R5′-TGCACTGAGAAGTATGAAGTGC-3′8428 8F5′-CCTGTACAGAGAAGGCCACG-3′ 8R5′-CGGTCTTCATGACACAATGC-3′9515 9F*5′-GATAGTGTAGTCTAGCTGGC-3′ 9R*5′-TGAGCACCAGAACAGCTTGC-3′1043510F*5′-ACTCCCTAGTATCCTTGGC-3′10R*5′-GATGGTAGAAAGTCTTCCACCAGC-3′1134811F5′-ACAGTCAGGCAAGGGCTATG-3′11R5′-TCCTTACCCACAGAGAGCC-3′1238912F*5′-GGATCAGATACACCTGGTGC-3′12R*5′-ACGAAGGTGAAACTAGAGC-3′1351213F*5′-AAGGATTGGCTTAGGAGGC-3′13R*5′-AGTCATTCTGGACTCCAAGG-3′1425614F*5′-TTAGGAGATGCCAGCTGTGG-3′14R*5′-ATTCTGGCACCAGTACTGCG-3′1528615F*5′-GCACTTAGCAGAAACAATCC-3′15R*5′-ACCGAAGACATGCACATAGC-3′1634316F*5′-CCTGATACCAGACTTCATGG-3′16R*5′-GTCGGATGTCTCCAAGACC-3′1728917F*5′-CTTCAACCCTGCGTTTCTGG-3′17R*5′-CTCTTCAATATGCCTTCACCC-3′18 + 1979218F5′-TCACAGGGTGACAAGCAAC-3′19R5′-TTTACCATTCCACCCATGGC-3′2035220F5′-GTGTCTCCCTAGTCCATGATGG-3′20R5′-TCACTCAGCTGGCATCAAAG-3′2133021F*5′-ATGCGCTTTGATGCCAGCTG-3′21R*5′-ATTGCTCCTGTAAGTATGCG-3′2241722F*5′-TTGGCATTCTAGGTGATTCC-3′22R*5′-CACCATGCACAGGAATCCC-3′2335223F*5′-CACAAGTCTTCAGGGATTCC-3′23R*5′-GGTACTCAAGAAACACTTGC-3′2430724F*5′-TTACATGAAGGAGTACTGGG-3′24R*5′-GGAAGGATGACTTAGCAATTTCC-3′2546025F5′-GGAGCCTCTCATCATTCTGC-3′25R5′-TTTCACACCACTAGCCATGC-3′2640226F5′-GAGGCATTGCCTAAGAGTGC-3′26R5′-AAAGATGGAGCCAGGGTTTG-3′2721427F5′-TTGGTTTCTGTGCCTATGATG-3′27R*5′-GCACTCTCGAAGGAGTTGC-3′2832328F*5′-TCTATGTCTCGAGTCCTGGG-3′28R*5′-CAAATGATGAAGGCTTAGGG-3′2928529F*5′-ATGGAGTAGCCAGTCACTGC-3′29R*5′-CCCGAGTAAGTTCTAGAGC-3′3032130F*5′-CAGGAATCCATCTCAGGCC-3′30R*5′-CACATCCTCTCATTGCCTGC-3′3128231F*5′-CTTTAGGAGCTAACACATGG-3′31R*5′-GAGCAAGGGTTAAGCCATCC-3′3219232F*5′-AATGCCTAGACTTGAGATGC-3′32R5′-CTGCTAGAATTTTGTGCTGTTCACATTC-3′Asterisks indicate primers designed by the authors. The rest of the primers have been published previously (3,6). Open table in a new tab Asterisks indicate primers designed by the authors. The rest of the primers have been published previously (3,6). PCR fragments were sequenced either directly or after subcloning into pGEM-T vector using pGEM-T vector System (Promega, Madison, WI) and transformation into JM109-competent cells (Promega). Plasmid DNA was isolated using Wizard™ Plus Minipreps DNA purification system (Promega). Sequencing was carried out in an automatic sequencer (ABI Prism 377, PerkinElmer Life Sciences). Following identification of mutations and intragenic polymorphisms by sequence alterations, methods for their easy detection were designed using amplified DNA segments and restriction analyses. Amplified DNA segments were digested by 5U of the respective enzymes (EcoRV, BsaHI,BbsI, BsaBI, Psp1406I, andNlaIII) and the products were separated on 3–4% agarose or metaphor gels (FMC-Bioproducts, Rockland, ME). Allele frequencies for identified mutations and polymorphisms were determined in patients with DJS and in control populations. Haplotypes of informative alleles from patients with DJS and controls were determined. Assessments of founder haplotypes were based on χ2 analysis. Mutations were introduced into the pcDNA3.1/MRP2 vector using the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA). Incorporation of the mutations was verified by DNA sequencing. Two sets of primers were used to introduce the I1173F mutation:1) the forward primer (5′-CAGGTTTGCCAGTTTTCCGTGCCTTTGAGC-3′) and the reverse primer (5′-GCTCAAAGGCACGGAAAACTGGCAAACCTG-3′); 2) the forward primer (5′-CCGTATCAGGTTTGCCAGTTTTCCGTGCCTTTGAGC-3′) and the reverse primer (5′-GCTCAAAGGCACGGAAAACTGGCAAACCTGATACGG-3′). This was done to ensure that low expression of this construct was not due to unexpected interference in any region of the vector except for the desired mutation. The primers used to introduce the R1150H mutation are the forward primer (5′-GCCAGCTGAGGCATCTGGACTCTGTCACCAG-3′) and the reverse primer (5′-CTGGTGACAGAGTCCAGATGCCTCAGCTGGC-3′). All procedures were as suggested by the manufacturer. Positive clones were selected on ampicillin agar plates and all clones were verified by sequencing. At least three clones of each mutant were analyzed for activity, and all six clones of the I1173F mutant were analyzed for expression by Western blot. HEK-293 cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal bovine serum, 100 μg/ml penicillin, and 100 μg/ml streptomycin. The expression vectors containing WT or the mutated MRP2 were transfected into HEK-293 cells using LipofectAMINE reagent according to instructions provided by the manufacturer. Cells were used 48–72 h after transfection. For stable expression, cells were selected with 1000 μg/ml G418. HEK-293 cells were plated on a sterile 22 × 22-mm coverslips. On the following day, WT or mutant MRP2 plasmids and GFP-expressing plasmid were co-transfected into the cells. 48–72 h after transfection, coverslips with cells attached to them were washed once with a Hepes-buffered solution and assembled to form the bottom of a perfusion chamber. The Hepes-buffered solution contained (in mm) 140 NaCl, 5 KCL, 1 MgCl2, 1 CaCl2, 10 glucose, 10 Hepes (pH 7.4 with NaOH, osmolarity 310 with NaCl). GFP-expressing cells were identified by viewing GFP fluorescence and one cell was selected. GFP fluorescence intensity was recorded at an excitation wavelength of 490 nm and used to normalize MRP2 expression in different experiments. Subsequently, cells were loaded with carboxyfluorescein (CF) by a 10-min incubation at room temperature in Hepes-buffered solution containing 100 μm carboxyfluorescein diacetate and 1.5 mm probenecid. CF fluorescence was continuously monitored, and after it exceeded GFP fluorescence by at least 10-fold, the cells were perfused with Hepes-buffered solution containing 1.5 mm probenecid to establish a base line. Since inhibition by probenecid is completely reversible, transport of CF by MRP2 was initiated by removing probenecid from the perfusate. CF fluorescence was measured from single cells at excitation wavelengths of 490 and 440 nm using the recording setup from PTI (PTI Delta Ram, Brunswick, NJ). Since the fluorescence at the excitation wavelength of 440 nm is the isosbestic point for pH sensitivity of CF, fluorescence recorded at 440 nm was used to evaluate CF efflux. The fluorescence recorded at 490 nm was used to follow intracellular pH during the experiment to verify that the observed effect was not secondary to changes in pHi. HEK-293 cells, transiently or stably transfected with WT or mutant MRP2, were disrupted in RIPA buffer (10 mm Tris-HCl, pH 7.4, 1 mm EDTA, 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 0.2 mm phenylmethylsulfonyl fluoride, 10 μm leupeptin, 10 μm pepstatin, 10 μm aprotinin) and centrifuged at 15,000 rpm for 15 min. The clarified supernatant was recovered, and protein concentrations were determined with a Bio-Rad protein assay. Between 13 and 55 μg of protein was separated on a 7.5% SDS-polyacrylamide gel and electrophoretically transferred to nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% skim milk in 20 mmTris/HCl, pH 7.4, 150 mm NaCl, 4 mm Trizma base, 0.1% Tween 20 and probed with a 1:100 dilution of M2III-6 antibody in 5% skim milk in 20 mmTris/HCl, pH 7.4, 150 mm NaCl, 4 mm Trizma base, 0.1% Tween 20. Horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad) at a 1:1000 dilution was used as a secondary antibody, and the signal was detected using ECL detection system (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). For PNGase F digestion assay, samples (25 μl) were incubated with 1000 units of PNGase F for 2 h at 37 °C before separation and probing as above. Stable or transiently transfected HEK-293 cells cultured on 12-mm glass coverslips were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.05% Triton X-100 in PBS. After washing with PBS, cells were incubated for 1 h in blocking serum (1% bovine serum albumin, 0.1% gelatinin, 0.01% sodium azide, and 5% normal goat serum in PBS). The cells were then incubated for 2 h at room temperature with the M2III-6 antibody diluted 1:20 in the blocking serum, washed with PBS, and reincubated for 1 h at room temperature with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories, Inc., West Grove, PA; dilution 1:100 in the blocking serum). The cells were than mounted on glass slides and examined with a confocal laser-scanning microscope (MRC 1024, Bio-Rad). Screening of all 32 exons of the MRP2 gene in an Iranian Jewish patient disclosed a 3517A→T transition in exon 25, predicting an I1173F substitution. The mutation was detectable by restriction analysis of nested PCR performed as follows. First, exon 25 was amplified by the forward and reverse primers depicted in TableI, and then nested PCR was carried out using a mutated forward primer (5′-ACCGTATCAGGTTTGCCAGAT-3′) that created with the reverse primer an EcoRV restriction site in the normal sequence of exon 25. The amplified fragment was digested with EcoRV and its products analyzed by 4% metaphor gel electrophoresis (Fig.1A). All 22 Iranian Jewish patients were homozygous for the 3517A→T mutation, 2 affected siblings of mixed Iranian and Moroccan Jewish origin were heterozygous, and 11 non-Iranian Jewish patients did not bear this mutation. In the general Iranian Jewish population, heterozygosity for this mutation was observed in 14/243 subjects examined (5.8%), whereas none of 164 Moroccan Jews and 108 Ashkenazi Jews carried this mutation. The estimated allele frequency of the mutation in the general Iranian Jewish population was 2.9% (95% confidence interval of 1.6–4.8%) (Table II).Table IIAllele frequencies of the 3517A →T and the 3449G→A mutations and of the 4 polymorphisms in the MRP2 gene in Iranian, Moroccan, and Ashkenazi JewsEthnic groupMutationsPolymorphismsExon 25 3517A→TExon 25 3449G→A5′-Untranslated region −24C→TExon 7 842G→AExon 10 1249G→AIntron 29 IVS −35G→AAlleleFrequencyAlleleFrequencyAlleleFrequencyAlleleFrequencyAlleleFrequencyAlleleFrequency%%%%%%Iranian JewsT2.9A0T12.3A0.6A31.2A5.4A97.1G100C87.7G99.4G68.8G94.6(n = 486)2-an, number of alleles examined.(n = 312)(n = 228)(n = 336)(n = 202)(n = 280)Moroccan JewsT0A0.9T26.1A5.6A18.4A5.9A100G99.1C73.9G94.4G81.6G94.1(n = 328)(n = 452)(n = 284)(n = 284)(n = 222)(n = 238)Ashkenazi JewsT0A0T21.9A0.9A18.3A3.5A100G100C78.1G99.1G81.7G96.5(n = 216)(n = 312)(n = 224)(n = 222)(n = 180)(n = 286)2-a n, number of alleles examined. Open table in a new tab A different mutation in the MRP2 gene, a 3449G→A transition in exon 25, was identified in a Moroccan Jewish patient. The mutation predicts an R1150H substitution and results in loss of a BsaHI restriction site. For detection of this mutation, exon 25 was amplified by the primers depicted in Table I, then digested with BsaHI (New England Biolabs) and the products analyzed by 3% agarose gel electrophoresis (Fig. 1B). All 5 Moroccan Jewish patients were homozygous for the 3449G→A mutation, whereas none of the 28 non-Moroccan patients carried it. The 2 affected siblings of the mixed Moroccan and Iranian Jewish origin were heterozygous for the mutation, and were thus compound heterozygotes for the 3449G→A and the 3517A→T mutations (Fig. 1C). The mother of these siblings of Moroccan Jewish origin was heterozygous for the 3449G→A mutation, and the father, of Iranian Jewish origin, was heterozygous for the 3517A→T mutation. In the general Moroccan Jewish population, heterozygosity for the 3449G→A mutation was observed in 4/226 subjects examined (1.8%), whereas none of 156 Iranian Jews and 156 Ashkenazi Jews carried this mutation. The estimated allele frequency of the mutation in the general Moroccan Jewish population was 0.9% (95% confidence interval, 0.24–2.27%) (Table II). Four dimorphisms in the MRP2 gene were identified: 1) −24C→T in the 5′-untranslated region, 2) 842G→A in exon 7 predicting a S281N substitution, 3) 1249G→A in exon 10 predicting a V417I substitution, and 4) IVS29–35G→A. 842G→A and IVS29–35G → A are novel polymorphisms, whereas −24C→T and 1249G→A were recently published (40Ito S. Ieiri I. Tanabe M. Suzuki A. Higuchi S. Otsubo K. Pharmacogenetics. 2001; 11: 175-184Crossref PubMed Scopus (234) Google Scholar). Methods were devised for simple detection of these four polymorphisms by PCR and restriction analysis (Fig.2). Table II shows the allele frequencies of the four dimorphisms in Iranian, Moroccan, and Ashkenazi Jews. A pronounced difference was observed for the rare 842G→A dimorphism; in Moroccan Jews, its frequency was 6-fold higher than in Ashkenazi Jews and 9-fold higher than in Iranian Jews. Haplotype analysis disclosed that all 26 alleles of 13 unrelated Iranian Jewish patients carried the same haplotype (−24C, 842G, 1249A, IVS29–35A), whereas none of 144 informative alleles (out of 168 examined) of control Iranian Jews carried the same haplotype (Table III). The common haplotype among Iranian Jewish controls was −24C, 842G, 1249G, IVS29–35G; it accounted for 63% of the alleles. Statistical analysis yielded highly significant differences in the haplotype distribution between patients and controls (χ2 = 271, p < 0.0001).Table IIIFrequency of haplotypes in Iranian Jewish and Moroccan Jewish controls and DJS patientsHaplotype5′-Untranslated region −24C→TExon 7 842G→AExon 10 1249G→AIVS29–35 G→AFrequency in Iranian Jews3-an indicates number of informative alleles of unrelated individ