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The two RH genes and their Rhesus boxes FF Wagner Priv.-Doz. Dr. Franz F Wagner Abt. Spenderlabor Institut Springe 31830 Springe DRK-Blutspendedienst NSTOB - Institut Springe RH: The most complex blood group Immunohematology 48 different antigens Antigen D split in >30 different epitopes Genetics >100 known haplotypes Similar phenotypes of different alleles Proteomics Membrane expression depends on RhAG Direct binding to Ankyrin DRK-Blutspendedienst NSTOB - Institut Springe 2 RH Blood Group Rh protein structure Structural model Differences of RhD and RhCE Genetics Structure of an RH gene Structure of RH locus and the RHD deletion Prediction of the phenotype Molecular basis of antigen D Molecular basis of other main antigens Testing strategies, pitfalls and limitations DRK-Blutspendedienst NSTOB - Institut Springe 3 RH Blood Group Rh protein structure Structural model Differences of RhD and RhCE Genetics Structure of an RH gene Structure of RH locus and the RHD deletion Prediction of the phenotype Molecular basis of antigen D Molecular basis of other main antigens Testing strategies, pitfalls and limitations DRK-Blutspendedienst NSTOB - Institut Springe 4 Rh protein in the membrane Complex with RhAG Direct interaction with ankyrin Nicolas JBC 2003 DRK-Blutspendedienst NSTOB - Institut Springe 5 Schematic structure of Rh protein 6 exofacial loops 32 53 11 72 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 307 370 333 389 417 2 12 transmembraneous helices DRK-Blutspendedienst NSTOB - Institut Springe 6 Added complexity: AmtB structure resolved AmtB: Ammonia transporter of E. coli Homology to the Rh proteins 3D structure resolved: 3 protein complex Khademi et al. Science 2004; 305: 1587 11 helices DRK-Blutspendedienst NSTOB - Institut Springe 7 AmtB extracellular aminoterminal end helix 1 near the axis Khademi et al. Science 2004; 305: 1587 DRK-Blutspendedienst NSTOB - Institut Springe 8 RhD structure modeled on AmtB Exofacial 32 35 80 49 107 158 52 131 135 167 230 186 231 282 201 263 290 266 321 Intracellular DRK-Blutspendedienst NSTOB - Institut Springe 347 358 324 391 417 9 RhD structure modeled on AmtB Mature protein starts at position 2 Exofacial 32 35 80 49 107 158 52 131 135 167 230 186 231 282 201 263 290 266 321 Intracellular DRK-Blutspendedienst NSTOB - Institut Springe 347 358 324 391 417 10 RhD structure modeled on AmtB Mature protein starts at position 2 Exofacial 32 35 80 49 107 158 52 131 135 167 230 186 231 282 201 263 290 266 321 347 358 324 391 N-terminal end intracellular Intracellular DRK-Blutspendedienst NSTOB - Institut Springe 417 11 RhD structure modeled on AmtB Mature protein starts at position 2 Exofacial 32 35 80 49 107 158 52 131 135 167 230 186 231 282 201 263 290 266 321 347 358 324 391 N-terminal end Trypsin site intracellular 42/43 Intracellular DRK-Blutspendedienst NSTOB - Institut Springe 417 12 RhD structure modeled on AmtB Mature protein starts at position 2 The AmtB model seems to fail at the aminoterminal end. 32 35 80 49 107 158 52 131 135 167 230 186 231 282 201 263 290 266 321 347 358 324 391 N-terminal end Trypsin site intracellular 42/43 Intracellular DRK-Blutspendedienst NSTOB - Institut Springe 417 13 RhD structure partially modeled on AmtB Exofacial 32 53 11 72 2 94 107 158 75 131 135 167 230 186 231 282 201 263 290 266 321 Intracellular DRK-Blutspendedienst NSTOB - Institut Springe 347 358 324 391 417 14 RhD structure and AmtB Amino acids lining Ammonia channel 103 Phe 107 Phe 148 Trp 219 Ser 168 His 318 His DRK-Blutspendedienst NSTOB - Institut Springe 15 RhD structure and AmtB Amino acids lining Ammonia channel 103 Phe 107 Phe conserved through RhAG, RhBG, RhCG 148 Trp 219 Ser 168 His conserved through RhAG, RhBG, RhCG 318 His conserved through RhAG, RhBG, RhCG DRK-Blutspendedienst NSTOB - Institut Springe 16 RhD structure and AmtB Amino acids lining Ammonia channel 103 Phe not conserved in RhCE and RhD 107 Phe not conserved in RhCE and RhD 148 Trp not conserved in RhCE and RhD 219 Ser not conserved in RhCE and RhD 168 His not conserved in RhCE and RhD 318 His not conserved in RhCE and RhD DRK-Blutspendedienst NSTOB - Institut Springe 17 RH Blood Group Rh protein structure Structural model Differences of RhD and RhCE Genetics Structure of an RH gene Structure of RH locus and the RHD deletion Prediction of the phenotype Molecular basis of antigen D Molecular basis of other main antigens Testing strategies, pitfalls and limitations DRK-Blutspendedienst NSTOB - Institut Springe 18 RhD vs RhCE 32 53 11 72 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 307 370 333 389 417 2 DRK-Blutspendedienst NSTOB - Institut Springe 19 RhD vs RhCE 32 53 11 72 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 307 370 333 389 417 2 DRK-Blutspendedienst NSTOB - Institut Springe 20 RhD vs RhCE (new model) Exofacial 32 53 11 72 2 94 107 158 75 131 135 167 230 186 231 282 201 263 290 266 321 Intracellular DRK-Blutspendedienst NSTOB - Institut Springe 347 358 324 391 417 21 RhD vs RhCE (new model) Exofacial 32 53 11 72 2 94 107 158 75 131 135 167 230 186 231 282 201 263 290 266 321 Intracellular DRK-Blutspendedienst NSTOB - Institut Springe 347 358 324 391 417 22 RhD vs RhCE (new model) Exofacial 32 53 11 72 2 94 107 158 75 131 135 167 230 186 231 282 201 263 290 266 321 Intracellular DRK-Blutspendedienst NSTOB - Institut Springe 347 358 324 391 417 23 RH Blood Group Rh protein structure Structural model Differences of RhD and RhCE Genetics Structure of an RH gene Structure of RH locus and the RHD deletion Prediction of the phenotype Molecular basis of antigen D Molecular basis of other main antigens Testing strategies, pitfalls and limitations DRK-Blutspendedienst NSTOB - Institut Springe 24 Genomic structure Exofacial Exofacial 32 53 11 72 2 94 110 154 75 131 169 134 226 188 238 207 283 257 286 352 264 Intrac ellular Insertion in intron 2 of C 307 32 370 333 53 389 11 72 94 107 158 75 131 135 167 230 186 231 282 201 263 290 266 321 347 358 324 391 417 2 Intrac ellular 417 Partial deletion of intron 4 in RHD DRK-Blutspendedienst NSTOB - Institut Springe 25 RH Blood Group Rh protein structure Structural model Differences of RhD and RhCE Genetics Structure of an RH gene Structure of RH locus and the RHD deletion Prediction of the phenotype Molecular basis of antigen D Molecular basis of other main antigens Testing strategies, pitfalls and limitations DRK-Blutspendedienst NSTOB - Institut Springe 26 RH locus P N SMP1 RH Ancestral (Mouse) P N SMP1 RHD Upstream RHCE RHD positive Downstrea m Rhesus box Man P N Hybrid SMP1 RHCE RHD negative 50,000 bp DRK-Blutspendedienst NSTOB - Institut Springe 27 Detection of RHD deletion by PCR 5’ RHD 5’ SMP1 RHCE SMP1 RHCE 23,130 bp 9,416 bp DRK-Blutspendedienst NSTOB - Institut Springe cDE/cDE CDe/cDE CDe/CDe cDE/c de c De/c de CDe/c de Cde/Cde c dE/c dE cde/c de 6,557 bp 28 Detection of RHD deletion by PCR PstI Sequence-specific Priming Hybrid Rhesus box Upstream Downstream Rhesus box Rhesus box 1,888 bp 800 bp 100 bp DRK-Blutspendedienst NSTOB - Institut Springe 744 bp 564 bp 397 bp 179 bp 29 Detection of RHD deletion by PCR PstI Sequence-specific Priming Hybrid Rhesus box Upstream Downstream Upstream Rhesus box Rhesus box Rhesus box (RHD psi, DAU-1, -3) Downstream Rhesus box (Weak D type 4.1) DRK-Blutspendedienst NSTOB - Institut Springe 30 RH locus A RHD SMP1 B RHCE D B B C E RHD-CE(4-7)-D RHCE RHD(1-9)-CE(10) DRK-Blutspendedienst NSTOB - Institut Springe RHCE(1)-D(2-10) 31 RH Blood Group Rh protein structure Structural model Differences of RhD and RhCE Genetics Structure of an RH gene Structure of RH locus and the RHD deletion Prediction of the phenotype Molecular basis of antigen D Molecular basis of other main antigens Testing strategies, pitfalls and limitations DRK-Blutspendedienst NSTOB - Institut Springe 32 Three types of D neg D+ 5’ SMP1 RHCE SMP1 RHCE RHD SMP1 RHCE RHD SMP1 RHCE RHD D - 5’ RHD deletion D - 5’ „large“ hybrid D - 5’ Stop codon, frameshift, splice site mutation ... DRK-Blutspendedienst NSTOB - Institut Springe 33 Molecular basis of antigen D 32 53 11 72 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 307 370 333 389 417 2 DRK-Blutspendedienst NSTOB - Institut Springe 34 RhD loops and D antigen FPTT DFR BARC D cat VI 6 DW 4 D 3 D cat V Rh32 DBT Evans D cat IV CE Rh33 DHAR DRK-Blutspendedienst NSTOB - Institut Springe 35 D negative RHD-CE-D hybrid alleles Exofacial Exofacial 32 53 11 72 2 94 110 154 75 131 169 134 226 188 238 207 283 257 286 352 264 Intracellular Insertion in intron 2 of C 307 32 370 333 53 389 11 72 94 107 158 75 131 135 167 230 186 231 282 201 263 290 266 321 347 358 324 391 417 2 Intracellular 417 Partial deletion of intron 4 in RHD DRK-Blutspendedienst NSTOB - Institut Springe 36 Three types of D neg D+ 5’ SMP1 RHCE SMP1 RHCE RHD SMP1 RHCE RHD SMP1 RHCE RHD D - 5’ RHD deletion D - 5’ „large“ hybrid D - 5’ Stop codon, frameshift, splice site mutation ... DRK-Blutspendedienst NSTOB - Institut Springe 37 Other D negative RHD alleles Nonsense mutations RHD(W16X), RHD(W90X), RHD(Y330X) „Small“ deletions/insertions causing frameshift RHD(488del4), RHD(del711),RHD(1253instT) Splice site mutations (may express some D) RHD(IVS3+1G>A), RHD(G212V), RHD(1227G>A) Complex changes RHDΨ Unusual hybrid pattern, „normal“ RHD DRK-Blutspendedienst NSTOB - Institut Springe 38 Importance of C-terminal end Exofacial 32 53 11 72 2 94 107 158 75 131 Intracellular 135 167 230 186 231 282 201 263 290 266 321 347 358 324 391 Ankyrininteracting residues 417 (Nicolas V et al. 2004) DRK-Blutspendedienst NSTOB - Institut Springe 39 Shortened proteins don‘t fit the membrane Exofacial 32 53 11 72 2 94 107 158 75 131 Intracellular 135 167 230 186 231 282 201 263 290 266 321 347 358 324 391 Ankyrininteracting residues 417 (Nicolas V et al. 2004) DRK-Blutspendedienst NSTOB - Institut Springe 40 D variants Partial D Qualitative change of antigen D Anti-D immunization possible Loss of epitopes defined by monoclonal antibodies Antigen density normal, decreased or enhanced Molecular causes: RHD-CE-D hybrid alleles Single amino acid substitution in or near the extracellular loops Multiple dispersed amino acid substitutions DRK-Blutspendedienst NSTOB - Institut Springe 41 RhD loops and D antigen FPTT DFR BARC D cat VI 6 DW 4 D 3 D cat V Rh32 DBT Evans D cat IV CE Rh33 DHAR DRK-Blutspendedienst NSTOB - Institut Springe 42 Single amino acid substitutions in partial D 32 53 11 72 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 307 370 333 389 417 2 DRK-Blutspendedienst NSTOB - Institut Springe 43 Single amino acid substitutions in partial D New RhD model 32 Exofacial 53 11 72 2 94 107 158 75 131 135 167 230 186 231 282 201 263 290 266 321 Intracellular DRK-Blutspendedienst NSTOB - Institut Springe 347 358 324 391 417 44 D variants Partial D Qualitative change of antigen D Anti-D immunization possible Loss of epitopes defined by monoclonal antibodies Antigen density normal, decreased or enhanced Molecular causes: RHD-CE-D hybrid alleles Single amino acid substitution in or near the extracellular loops Multiple dispersed amino acid substitutions DRK-Blutspendedienst NSTOB - Institut Springe 45 RH phylogeny cE Ce weak D type 1 DNB wea k D typ e 2 DCe ce Dc E D ca teg ory VI t ype II DNU D c ategory VI type I Dce Dc(W16C)e Cluster of Dc ategory IVa allele s Cluster of wea k D typ e 4 alleles Clust er of normal RHD allele DRK-Blutspendedienst NSTOB - Institut Springe Cluster of DAU a llele 46 D variants Weak D and Del Mainly quantitative reduction of antigen D Usually no Anti-D immunization observed Molecular causes: Single amino acid substitution in transmembraneous and intracellular parts of the Rh protein Multiple dispersed amino acid substitutions Splice site mutations (Del) DRK-Blutspendedienst NSTOB - Institut Springe 47 Single amino acid substitutions in weak D 32 53 11 72 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 307 370 333 389 417 2 DRK-Blutspendedienst NSTOB - Institut Springe 48 Single amino acid substitutions in weak D New RhD model 32 Exofacial 53 11 72 2 94 107 158 75 131 135 167 230 186 231 282 201 263 290 266 321 Intracellular DRK-Blutspendedienst NSTOB - Institut Springe 347 358 324 391 417 49 RH Blood Group Rh protein structure Structural model Differences of RhD and RhCE Genetics Structure of an RH gene Structure of RH locus and the RHD deletion Prediction of the phenotype Molecular basis of antigen D Molecular basis of other main antigens Testing strategies, pitfalls and limitations DRK-Blutspendedienst NSTOB - Institut Springe 50 Molecular basis of antigen Cw Arg at pos. 41 32 53 11 72 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 307 370 333 389 417 2 DRK-Blutspendedienst NSTOB - Institut Springe 51 Molecular basis of antigen E Pro at pos. 226 32 53 11 72 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 307 370 333 389 417 2 DRK-Blutspendedienst NSTOB - Institut Springe 52 Molecular basis of antigen E Pro at pos. 226 32 53 11 72 2 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 Additional mutations may cause „partial E“ DRK-Blutspendedienst NSTOB - Institut Springe 307 370 333 389 417 53 Molecular basis of antigen E RhcE(M167K): E var I, expresses Ew antigen 32 53 11 72 2 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 Additional mutations may cause „partial E“ DRK-Blutspendedienst NSTOB - Institut Springe 307 370 333 389 417 Strobel et al. 2004 54 Molecular basis of antigen c Pro at pos. 103 32 53 11 72 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 307 370 333 389 417 2 DRK-Blutspendedienst NSTOB - Institut Springe 55 Molecular basis of antigen G Ser at pos. 103 32 53 11 72 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 307 370 333 389 417 2 DRK-Blutspendedienst NSTOB - Institut Springe 56 Molecular basis of antigen C Ser at pos. 103 32 53 11 72 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 307 370 333 389 417 2 DRK-Blutspendedienst NSTOB - Institut Springe 57 Molecular basis of antigen C Ser at pos. 103 32 53 11 72 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 307 370 333 389 417 2 Overall structure must be RhCE DRK-Blutspendedienst NSTOB - Institut Springe 58 Molecular basis of antigen C Ser at pos. 103 32 53 11 72 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 307 370 333 389 Cys 16 must be present 417 2 Overall structure must be RhCE DRK-Blutspendedienst NSTOB - Institut Springe 59 Molecular basis of antigen e Ala at pos. 226 32 53 11 72 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 307 370 333 389 417 2 DRK-Blutspendedienst NSTOB - Institut Springe 60 Molecular basis of antigen e Ala at pos. 226 32 53 11 72 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 307 370 333 389 417 2 Overall structure must be RhCE DRK-Blutspendedienst NSTOB - Institut Springe 61 Molecular basis of antigen e Exon 5 may be RHD Ala at pos. 226 32 53 11 72 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 307 370 333 389 417 2 Overall structure must be RhCE DRK-Blutspendedienst NSTOB - Institut Springe 62 Molecular basis of antigen Ce (Rh7) Ser at pos. 103 and Ala at pos. 226 32 53 11 72 94 110 75 131 154 169 134 226 188 238 207 283 257 286 352 264 307 370 333 389 417 2 Overall structure must be RhCE DRK-Blutspendedienst NSTOB - Institut Springe 63 Further complexities „African“ RHCE-variants -D Rhnull Amorph type: defect of RH Regulator type: defect of RHAG DRK-Blutspendedienst NSTOB - Institut Springe 64 RH Blood Group Rh protein structure Structural model Differences of RhD and RhCE Genetics Structure of an RH gene Structure of RH locus and the RHD deletion Prediction of the phenotype Molecular basis of antigen D Molecular basis of other main antigens Testing strategies, pitfalls and limitations DRK-Blutspendedienst NSTOB - Institut Springe 65 Purpose of molecular testing Antigen prediction in the absence of suitable RBC Prenatal diagnoses Recently transfused patients Positive DAT and similar serologic problems Detection of weak antigens Donor screening Confirmation of serologic results Characterization of antigenic variants Discrimination weak D / partial D alleles Characterization of variants of other antigens DRK-Blutspendedienst NSTOB - Institut Springe 66 Testing strategies Direct check for antigenic polymorphisms Antigen Cw prediction Indirect check for antigenic polymorphisms Antigen C prediction based on intron 2 Antigen D prediction based on intron 4 Antigen D prediction based on non-exofacial positions Exclusion of additional polymorphisms DRK-Blutspendedienst NSTOB - Institut Springe 67 Strategies for the prediction of D antigen RHD PCR should focus on loops 3 to 6 Corresponds to RHD exon 4 to 7 Absence of RHD in these loops => D negative Presence of RHD in all loops => likely D positive Presence of RHD in some loops => likely partial D Many partial D and hybrid-type D negative will be typed correctly „Other“ changes must be detected individually RHDpsi, frequent stop/splice/frameshift mutations DRK-Blutspendedienst NSTOB - Institut Springe 68 A solution to antigen D prediction A c ontrol Intron 4 Exon 7 B D cat IV type III D cat VI type II s Cde RHDΨ RHD(W16X) RHD-CE(8-9)-D RHD negative standard RHD c ontrol Intron 7 RHD(W16X) RHDΨ DRK-Blutspendedienst NSTOB - Institut Springe 69 Exclusion of additional polymorphisms Which alleles must be detected? Alleles found as single case report? Alleles detected in a single region? Alleles detected by a single group? Alleles with very low frequency? D negative alleles ? Del alleles ? Partial D alleles ? Weak D alleles ? RHCE-alleles with unexpected D-expression (e.g. ceRT) DRK-Blutspendedienst NSTOB - Institut Springe 70 Strategies for the prediction of antigen C Cys at position 16 Shared by most c-alleles of cDe (Africans!) Absence predicts C negative phenotype RHD exon 2 Also present in RHD (e.g. cDE) Absence predicts C negative phenotype C-specific insertion in intron 2 Best predicting polymorphism in Europeans Fails in Ccdes DRK-Blutspendedienst NSTOB - Institut Springe 71 Strategies for the prediction of antigen e Usual approach: Ala 226 in RHCE exon 5 Fails in RHCE-D(5)-CE No information for RHD-CE(5)-D Minimal RHCE-context unknown DRK-Blutspendedienst NSTOB - Institut Springe 72 Prediction of antigen Ce (Rh7) Simultaneous presence of Ser 103 and Ala 226 in RHCE Ser 103 coded by exon 2 Ala 226 coded by exon 5 Two problems: Distance on DNA: ~ 17,000 bp Simultaneous testing of 3 determinants: Ser 103, Ala 226, RhCE DRK-Blutspendedienst NSTOB - Institut Springe 73 Additional pitfalls in Rh antigen prediction Regulator-type RhNull Normal Rh genes, no Rh antigens Sporadic mutations How many samples and regions must be tested? Unexplained non-expression or Rh proteins D negative type with anti-D despite „normal“ RHD DRK-Blutspendedienst NSTOB - Institut Springe 74 More facts --- more questions DRK-Blutspendedienst NSTOB - Institut Springe 75
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