Aspartic acid at position 57 of the HLA-DQ p chain protects against

Transcription

1 Proc. Nati. Acad. Sci. USA Vol. 85, pp , November 1988 Genetics Aspartic acid at position 57 of the HLA-DQ p chain protects against type I diabetes: A family study (histocompatibility antigens/oligonucleotide/polymerase chain reaction/multiplex families) PENELOPE A. MOREL*, JANICE S. DORMANt, JOHN A. TODD4, HUGH 0. MCDEVITTf, AND MASSIMO TRUCCO*t *Department of Pediatrics, Division of Endocrinology, University of Pittsburgh School of Medicine, Children's Hospital, and tdepartment of Epidemiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15213; tdepartments of Microbiology and Immunology and of Medicine, Stanford University School of Medicine, Stanford, CA 94305; and tnuffield Departments of Clinical Medicine and Surgery, John Radcliffe Hospital, Headington, Oxford, 0X3 9DU United Kingdom Contributed by Hugh 0. McDevitt, July 5, 1988 ABSTRACT One hundred seventy-two members from 27 randomly selected multiple case Caucasian families of patients with insulin-dependent diabetes mellitus (IDDM) were studied at the DNA level to ascertain the reliability of codon 57 of the HLA-DQ a-chain gene as a disease protection/susceptibility marker. The analysis was carried out by polymerase chain reaction amplification of DNA encoding the first domain of the DQ f chain and by dot blot analysis of the amplified material with allele-specific oligonucleotide probes. One hundred twenty-three randomly selected healthy Caucasian donors were also tested. The results demonstrated that haplotypes carrying an aspartic acid in position 57 (Asp-57) of their DQ.8 chain were significantly increased in frequency among nondiabetic haplotypes (23/38), while non-asp-57 haplotypes were significantly increased in frequency among diabetic haplotypes (65/69). Ninety-six percent of the diabetic probands in our study were homozygous non-asp/non-asp as compared to 19.5% of healthy unrelated controls. This conferred a relative risk of 107 (x2 = 54.97; P ) for non-asp-57 homozygous individuals. Even though the inheritance and genetic features of IDDM. are complex and are not necessarily fully explained by DQ p chain polymnorphism, this approach is much more sensitive than HLA serolog in assessing risk for IDDM. A striking feature of many autoimmune diseases is their association with the expression of certain cell-surface antigens of the major histocompatibility complex (1). Major histocompatibility complex class II antigens are cell-surface glycoproteins encoded by the human leukocyte antigen (HLA) D region on human chromosome 6. The HLA-D region has three different subregions, DR, DQ, and DP, each encoding at least one heterodimer of a and f chains. Insulin-dependent diabetes mellitus (IDDM) has been associated with the serologically defined antigens HLA-DR3 and HLA- DR4: 95% of Caucasian IDDM patients possess DR3 and/or DR4 (2-7). Restriction fragment length polymorphism studies have demonstrated that the association is stronger with molecules encoded by the HLA-DQ locus rather than by the HLA-DR locus (8-12). HLA-DQ is in strong linkage disequilibrium with HLA-DR (13). Recently, it has been reported that the predicted amino acid sequence of the DQ 3 chain is strongly correlated with IDDM susceptibility (14). By DNA sequence and oligonucleotide dot blot analyses of the DQ p-chain genes, it was shown that amino acid 57 of the DQ /3-chain gene correlates with susceptibility and resistance to IDDM. The presence of an aspartic acid (Asp) in this position seems to provide protection against the development of IDDM. Conversely, the presence of a gene that encodes a The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. non-charged amino acid (non-asp), on both DQ B chain alleles of an individual, appears to predispose to IDDM. The mechanism by which this protection and predisposition are conferred is at present unknown. To determine whether molecular typing of DQ 8-chain codon 57 provides a more reliable marker for IDDM than serological markers, we have conducted studies on 27 multiple case IDDM families collected in the Pittsburgh area. Healthy unrelated individuals from the same population were used as controls. The genomic DNA segment encoding the first domain of both DQ,3 chains from each donor was amplified by polymerase chain reaction (15) and the amplified material was subjected to oligonucleotide dot blot analysis with allelespecific oligonucleotide (ASO) probes. From these studies, we have found that 96% of diabetic probands are homozygous for non-asp at position 57 of their DQ,8 chain as compared with 19.5% of randomly selected controls. Four percent of the diabetics (vs. 46% of controls) were heterozygous Asp/non- Asp at position 57, and none of the diabetics was Asp-57 homozygous (vs. 34.1% of controls). Thus, aspartic acid at position 57 of one of the two DQ /3 chains of an individual appears to protect against the development of IDDM. These data permitted estimation of the relative risk for the disease for each molecular marker present in this population. The significantly high values obtained have important implications for IDDM genetic counseling. MATERIALS AND METHODS Study Population. The families selected for this investigation were identified from the Allegheny County IDDM registry for and the Children's Hospital of Pittsburgh IDDM registry for the years All probands were on insulin therapy at hospital discharge and were <17 years old at IDDM onset. After reviewing family history data collected from >75% of 2700 probands, >300 families with at least one other diabetic sibling or parent, who were diagnosed before age 40 and on insulin therapy, were identified. To date, 80 complete families have agreed to participate in our genetic studies of IDDM by donating a blood sample for HLA typing, lymphocyte storage, and the development of lymphoblastoid cell lines. Twenty-seven of these 80 families were randomly selected for the current investigation and represent those for whom the serological typing was completely informative and in whom cell transformations were complete. Included in these 27 families were five parent/offspring families. Family 5752 was originally thought to be a parent/offspring family. Abbreviations: IDDM, insulin-dependent diabetes mellitus; ASO, allele-specific oligonucleotide. $To whom reprint requests should be addressed at: Pittsburgh Cancer Institute, 3343 Forbes Avenue, Room 113, Pittsburgh, PA

2 8112 Genetics: Morel et al. In this analysis, however, the father was considered nondiabetic because he was diagnosed after the age of 40. Lymphoblastoid cell lines were derived from peripheral blood lymphocytes as described (16). The 123 randomly selected healthy caucasoid controls were blood donors and employees of the Pittsburgh Blood Bank. All cells used in this study were typed for HLA-A, -B, -C, HLA-DR, and HLA- DQ by standard National Institutes of Health microlymphocytotoxicity techniques and the sera of the 9th International Histocompatibility Workshop. DNA Extraction and Polymerase Chain Reaction. DNA was extracted from each of the cell lines by standard techniques (17). The genomic DNA was subjected to polymerase chain reaction to amplify the first domain of the DQ P-chain gene as described (14, 15, 18). The primers used were P2 (5'- GATTTCGTGTACCAGTTTAAGG-3') and P4 (5'- CCACCTCGTAGTTGTGTCTGC-3'), and these yield a product of 241 base pairs (Fig. 1). The procedure was carried out using the thermostable Taq polymerase (Perkin-Elmer- Cetus, Norwalk, CT). One to two micrograms of DNA was subjected to 30 cycles of amplification. Dot Blot Analysis with ASO Probes. Dot blots were done as described (14). The DNA was crosslinked to the membrane by UV irradiation and prehybridized at 420C. The dot blots were probed at 420C overnight with ASO probes. The filters were then washed in 6 x SSC (1 x SSC = 0.15 M NaCl/0.015 M sodium citrate)/0.1% sodium dodecyl sulfate at a temperature based on Td [Td = (number of G C base pairs) x 4 + (number of A T base pairs) x 2]. This removed any probe that had one mismatch or more with the target sequence. The oligonucleotide probes used to identify specific alleles are shown in Fig. 1. This procedure proved to be rapid, efficient, and unambiguous. We were able in all cases but one ("?" haplotype in family 8658) to assign both alleles in each member of each family. In the case of DQw3.1, -3.2, and -3.3, the use of the probe specific for DQw (Fig. 1) was necessary to distinguish between these three closely related alleles. Statistical Analyses. Four parental haplotypes were identified for each family. Those present among IDDM siblings or the parents with IDDM were defined as "diabetic" haplotypes. Therefore, for each family a minimum of two DQWl. I DQwI.2 DQw1. 9 DQwI.AZH DQw3.1 DQwI. I DQwi.2 DQw. 9 DQwl.AZH DQw3.1 DQwi. I DQwI.2 DQwl. 9 DQVii.AZH DQw3. 1 Proc. Natl. Acad. Sci. USA 85 (1988) diabetic haplotypes (if affected siblings were HLA identical) and a maximum of four diabetic haplotypes (if affected individuals were HLA non-identical) were identified. The remaining one or two parental haplotypes present only anibng nonaffected individuals in the family were defined as "nondiabetic" haplotypes (19). The haplotype frequencies were obtained by counting the number of specific DR (serologically defined) and DQ (defined by ASO probe analysis) genes among the diabetic and nondiabetic haplotypes. Therefore, each allele was counted only once and the genotype information obtained from each family was equally weighted in the analyses. x2 values were calculated by using the Yates correction. When the expected frequency in one of the four cells of the 2 x 2 tables was <5, the Fisher exact test was used instead. All the P values were corrected for multiple comparisons. Because of the reported association between IDDM and both the DR and DQ loci, stratified analyses were conducted to determine whether genetic susceptibility was primarily related to DQ P-chain non-asp-57 haplotypes, as opposed to the HLA-DR locus (20). Sibling pair analysis was conducted by comparing the HLA haplotype identity of the proband, as defined by the DQ-ASO molecular probes, with that of either one diabetic or one healthy sibling within each family. In each case, the eldest sibling was selected. Sibling pairs were classified as sharing 2, 1, or 0 DQ alleles with the diabetic proband, defined as identical by state (21). Finally, phenotype frequencies for the 27 probands and the 123 healthy unrelated controls were used to estimate the relative risk (odds ratio) associated with specific molecular markers. RESULTS Demographic Characteristics. The average age at onset of IDDM for the probands in the 27 families studied was 7.9 years and 13.7 years for their 28 diabetic siblings. The current average ages of probands, diabetic, and nondiabetic siblings are 33.5, 33.2, and 32.7 years, respectively. The family trees of the 27 families studied are shown in Fig. 2 with the AGA GAC TCT CCC GAG [GAT TTC GTG TAC CAG TTT MG G3C CTG TGC TAC TTC ACC MAC GGG ACG GAG CGC GTG COG GIT GTG ACC AGA CAC A A T CT- -G- AG C- A C -- TA- T A C T CT T A C T CT T T A C T ATC TAT AAC CGA GAG GAG TAC GTG CGC TTC GAC AGC GAC GTG GGG GTG TAC CGG GCA GTG ACG CCG CAG E G COG CCT GTT GCC GAG TAC C C --G T G AC G G _----AGC A --- AT A- -T G T- -CC +_ CA GA T I-C-- -AC CA T G T- -_C CA T G T C- ---AC C T G T s 1_ -T -A TGG AAC AGC CAG AAG GAA GTC CTG GAG GGG GCC CGG GCG TCG GTG GAC AGG GTG SiC GA dcac MC TAC GAG GTGGiG TAC CGC GGG ATC A GA- T C T A A C A-- A-- AAA G C-- T-- -A- CT- ---AC- -C A-- A GA- T C-- T-- -A- CT- --- AC- -C A-- A GA- T C-- T-- -A- CT- ---AC- -C A-- A GA- T C-- T-- -A- CT- --- AC- -C- --T C A A-- -A CC --A C-- T-- -A- CT- --- AC- -C- FIG. 1. Coding sequences for the first domain of allelic DQ A chains. The stretch of DNA recognized by the oligonucleotides used for the amplification (-- -) as well as the stretches used as probes (-) to distinguish the various alleles are boxed. 60

3 Genetics: Morel et al. HLA-DR typing and non-asp or Asp DQ a-chain status of each family member. Haplotype Frequencies and Statistical Significance. The haplotype frequencies for all the known DR antigens identified in this population are shown in Table 1. The frequencies of DR3 and DR4 among diabetic haplotypes were 29o and 48%, respectively, as compared to 2.5% and 7.7% among the nondiabetic haplotypes, confirming that these are the most common diabetogenic haplotypes. These increases were significant with X2 of 9.39 (P = 0.02) for DR3 and (P = ) for DR4. The frequencies of DR2 and DR7 were significantly decreased among diabetic haplotypes (P = 0.03 and P = 0.012, respectively). All the other DR alleles showed a nonsignificant P value. The three DQw genes having Asp at position 57 (DQwJ.2, DQw3.1, and ) showed a statistically significant negative association with the disease. Of the three genes having non-asp at position 57 (DQwl.J,, and ), and were both found to be increased in frequency among diabetic haplotypes, but this was significant (x2 = 15.16; P = ) only in the case of. It is important to note that all but two of the DR4- associated DQw3 genes in this series were of the type (94%) (Table 1) as compared to 75% of DR4 usually found in the general population (14). DR7 can be associated with or and Table 1 indicates the distribution of the DR7 haplotypes that were observed in this population. The increase in frequency among diabetics was not /4 3/ Proc. Natl. Acad. Sci. USA 85 (1988) 8113 significant, presumably because of the number of DR7- associated genes among the nondiabetic haplotypes. DR6 has been reported to be associated with the DQw p-chain subtypes DQwL.19, DQw1.18, DQwL.9, and DQw3.1 (22). The distribution of the different DR6 haplotypes is also shown in Table 1. The only DR2-positive diabetic haplotype found in family 0200 was of the DQwl.AZH type, which has a serine at position 57. DQwL.I-containing genes were slightly increased in frequency among diabetics, but again this was not significant. However, when the data were pooled and expressed as Asp- or non-asp-containing DQw genes, it was found that 94% (65/69) of the diabetic haplotypes were non-asp containing, and this was highly significant (2 = 35.72; P = ). Furthermore, stratified analysis (Table 2) demonstrated that the choice of amino acid at position 57 of the DQ,8 chain is critical in determining susceptibility to IDDM when the DR haplotypes are DR3 and/or DR4 negative (X2 = 10.9; P = 0.001). Sibling pair analysis was also conducted and revealed that 87.5% (21/24) of affected sibling pairs shared both DQ alleles with the proband, 4.2% (1/24) shared one, and 8.3% (2/24) shared zero. Among the nonaffected siblings, the distribution of DQ allele sharing with the proband was as follows: 2, 6/22 (27.3%); 1, 10/22 (45.4%); 0, 6/22 (27.3%). To estimate the relative risks associated with serologic and ASO-defined alleles, the phenotype frequencies for the diabetic probands, one from each of the 27 families, were calculated together with the corresponding frequencies from *5/6* 3/4 3/4F 3.22/ /3.1 *S/6* 3/4 3/ /4 *2/ / /2 3/4 *2/4 3/ *2/4* 1/4 4/4* /2* 1*2/ / /3.1 3/ / 2 / 1/.2 *2/4*44*4/ 4/ 14 *P / /4 0 5N 1/4 73/43 3/4 3/4 3/4 4/50 N.T. *7/7 3/4 3/4 3/ /10 3/4 3 n4/4 *23 3/4/7 4P 3.2 / / / 3. / / 423.2/4? 3.2 / / /3 32/.2 4/4 3/10 4/10 4/4 Vn 4/7 3/4 Vn 3/4 3/4 02/4 *2/4 *5f7 4/4 4/4 V7 3 3n3/3 3/3 V 3/4 4/4 4/4 3/60 4/6* 4/ /6* 3/0 3.2/3.1 2/1.1 4/6* 1/3 3.2 / /2 1/6* 4*2/4 1.1 / /3.2 4/6 3/4 3/4 3/4 4/6 4/6 3/4 3/4 3/6* 1/4 3/4 3/4 3/6* 4/60 4/6* 1/4 1/4 V6O 1/4 *2^* N.T. 4/9* 4/4 4/4 4/ 4n N.T / / IAZHI /6 46 1/ /6 1 /6 4/6* N.T. 1/3 1/ 1/7$ In 2/3 2/3 Ur 2n* 2/* 2n V2/3 *2f/71/? N.T. 3/? *2/3 *7/? 3/? *7/? 3? *2n/ 3n FIG. 2. Family trees of the individuals used in this study. Patients with IDDM, as defined by the criteria stated in Materials and Methods, are shown as solid symbols. The HLA-DR typing of each individual is also indicated. The DQ 3-chain alleles are indicated for each parent haplotype. * indicates an Asp-57-containing DQ (-chain gene. N.T. indicates that these individuals were not available for study. A diagonal line through a symbol indicates the death of this individual. The? in family 5019 means that one allele could not be typed for DR with the available sera, but it was possible to type it by using DQ P-chain ASO probes. The? in family 8658 indicates that one allele could not be typed for either DR or DQ.

4 8114 Genetics: Morel et al. Proc. Natl. Acad. Sci. USA 85 (1988) Table 1. Genotype frequencies of diabetic and nondiabetic haplotypes from 27 families Diabetics Nondiabetic (n = 69) (n = 39) Haplotype n Frequency n Frequency DRJ/DQwJ.J (NA) DR2/DQwJ.2 (A) DR2/DQwl.AZH (NA) DR3/ (NA) DR4/DQw3.1 (A) DR4/ (NA) DR5/DQw3.1 (A) DR6/DQwl.l (NA) DR6/DQwJ.2 (A) DR6/DQw3.1 (A) DR7/ (NA) DR7/ (A) DR8/ (NA) DR9/ (A) DR10/DQwJ.J (NA) DRbJ/DQwl.l (NA) DRbJ/DQw? NA A All the haplotypes observed in the 27 families are shown with their respective frequencies. The DR6 haplotype can be associated with 3 DQwJ alleles: DQwl.19, DQwJ.18, and DQwl.9. DQwl.19 is identical to DQwl.l between amino acids 40 and 70 and therefore was detected by the DQw1.1 probe. DQw1.18 is identical to DQwI.2 in the same region and thus was detected by the DQwJ.2 probe. We have named them in the table according to the probe used. There were no DR6 DQwl.9 haplotypes in this series. The DR6-associated DQw3.1 allele is identical to that found associated with DR4 and DR5. DRbl, DR blank. DQw? indicates that we were unable to detect this allele with our panel of ASO probes. There are only 38 nondiabetic haplotypes here because we were unable to assign a non-asp (NA) or Asp (A) status to the DQw? allele. the 123 healthy controls (Table 3). Of the 27 diabetic probands, 33% typed DR3,-4, 7% were DR3 homozygous, and 26% were DR4 homozygous, as compared with 5.6%, 0.8%, and 0% of the controls, respectively. This distribution reflects the expected increased percentage of DR3 and DR4 haplotypes in the diabetic individuals. In fact, 96% of the probands in our population were DR3 and/or DR4 positive, in good agreement with results obtained in other study populations (2-7). The relative risk calculated for DR3/DR4 heterozygotes was equal to 8.29 (P = 0.002). At the DNA level, 26 (96%) of these probands were homozygous for Table 2. Prevalence of non-asp and Asp alleles among non-dr3 non-dr4 haplotypes Haplotype Non-Asp-57 Asp-57 Total Diabetic Nondiabetic Total %2 = 10.90; P = non-asp at position 57, and the one (4%) who was not was heterozygous non-asp/asp, and the Asp-containing gene was DQw3.1. In the controls, the frequencies were 19.5% (24/123) for the non-asp-57 homozygous, 46.3% (57/123) for the non-asp-57/asp-57 heterozygous, and 34.1% (42/123) for the Asp-57/Asp-57 homozygous individuals. These figures indicated that the non-asp-57/non-asp-57 homozygous phenotype was significantly (X2 = 54.97; P = ) associated with IDDM, and with an estimated relative risk of Non-Asp-57/Asp-57 heterozygotes were significantly (X2 = 15.12; P = ) negatively associated with the disease (relative risk, 0.04). The negative association of Asp-57/Asp-57 homozygotes was also found to be highly significant (X2 = 11.09; P = ). DISCUSSION In this paper, we have presented data that confirm the importance of the HLA-DQ B amino acid 57 in determining susceptibility to IDDM. Of the 27 diabetic probands studied to date at the DNA level, 26 were homozygous for non-asp at position 57 and this conferred a relative risk of 107. This figure may be a slight overestimate because these probands were all diagnosed before the age of 17 and were from multiplex families. The presence ofone Asp-containing DQ gene appeared to significantly reduce susceptibility to IDDM (relative risk, 0.04). DQw1.2 and DQw3. -regardless of whether they were associated with DR2, DR6 (DQwJ.18), DR6 (DQw3.1), or DRS-appeared to protect against the development of IDDM. The fact that only 4% of diabetic individuals are heterozygous non-asp/asp suggests that the protection observed for Asp behaves in a dominant-like manner. This implies a specific immune mechanism (23, 24). It is noteworthy that of all the DR7 haplotypes found in 13 families, five (38%) of them carried the gene (these were all nondiabetic haplotypes) and nine (69%) carried. Of these nine, three were associated with diabetes and six were not. Even though DR7 is commonly associated with an identical non-asp-57 DQ chain () to that found on Caucasian DR3 haplotypes (25), it is not found Table 3. HLA DR and DQ phenotype frequencies in the probands of 27 Pittsburgh IDDM families compared with the corresponding frequencies found in random selected healthy controls from the same population Diabetics Nondiabetics (n = 27) (n = 123) Corrected Phenotype n Frequency n Frequency X2 P value RR DR (serology) DR3/DR DR3/DR NS 9.76 DR3/DR* NS 0.05 DR4/DR DR4/DR* NS 1.47 DR*/DR* DQ (ASO probes) NA/NA NA/A A/A NA, non-asp; A, Asp; RR, relative risk (odds ratios);, invalid; NS, not significant, P > *Non-DR 3 non-dr 4.

5 Genetics: Morel et al. increased in frequency in IDDM patients in this as well as in other studies (2-7). It is possible that the higher than expected frequency for the DR7-linked allele could account for the observed reduction of DR7 frequency among diabetic haplotypes in this study. Another explanation for this is that since the DR7DQ a chain has a unique structure (26), it limits susceptibility provided by the 8 chain. This implies that the DQ a-chain gene is an important predisposing factor on DR3 haplotypes as previously suggested (23, 27). It is also possible that DQ a-chain gene polymorphism (28) accounts for the observed differential IDDM susceptibility associated with different DR3 haplotypes (29, 30). Polymorphisms lying outside the DQ structural genes may also be responsible. The Dw4 and DwJO subtypes of DR4, as defined by DR,8 1 sequence (31), appear to be increased in frequency in IDDM (32, 33). Dw4 can be associated with either DQw3.1 or DQw.:.2, whereas DwJO is associated with only. However, the Dw14 subtype, which is associated with the allele, is not increased in IDDM, implying that, at least in the case of DR4, DR,3 1 alleles may influence IDDM predisposition. The reduction in susceptibility associated with DQw3.1 is observed, however, regardless of which allelic subtype of DR4 is present. In the diabetics that are non-dr3/non-dr4, the role of the amino acid in position 57 is much clearer. The non-asp or Asp status of the DQ,8-chain gene is significantly involved in determining susceptibility to IDDM: the presence of Asp at this position is associated with protection. The high rate ofdq allele sharing between affected siblings suggests that this gene is very significantly involved in IDDM susceptibility. These data suggest that DQ may be "the" HLA-associated gene that is responsible for IDDM. The fact that not all HLA identical siblings, as well as identical twins (34), develop IDDM may indicate the effect of non-hlalinked genes, low penetrance of HLA-linked genes, and/or differences in environmental exposure. IDDM is the result of a selective autoimmune destruction of insulin-producing 3 cells that is T-cell dependent (35-37). The causative agent for IDDM is unknown but there is accumulating evidence that exposure to certain viruses may be important (38, 39). T cells generally recognize peptide fragments from foreign and self antigens directly bound to class II molecules (40-42). T-cell recognition [which involves the formation of a ternary complex between T-cell antigen receptor, antigenic peptide, and class II molecules (43)] of antigens implicated in IDDM is likely to be influenced by class II allelic variation. In a model of class II structure (44), the amino acid at residue 57 is placed within the putative antigen binding cleft and thus may affect T-cell recognition and/or antigen binding. These data demonstrate that the use of DQ p ASO typing is of great value in determining whether the siblings of a diabetic will develop IDDM. It should now be possible to identify siblings who would be at greater risk and monitor them closely for early signs of glucose intolerance and the presence of islet cell autoantibodies. We would like to thank Glenys Thomson, Richard Spielman, and Henry Erlich for their useful criticisms and suggestions; Ronald LaPorte for his continual organizational support; Rene Duquesnoy, Marilyn Marrari, and Bruce Rabin for the HLA typing of family members. The expert technical assistance of Craig Robinson and Read Fritsch is greatly appreciated. P.A.M. is supported by a postdoctoral fellowship from the Arthritis Foundation. 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