Serologic and molecular characterization of weak D type 29

Additional Supporting Information may be found in the online version of this article at the publisher's website. Appendix S1. Supplemental material. Table S1. D antigen density (D antigens per red blood cell). Table S2. Serologic reactivity with panels of anti-D. Fig. S1. Nucleotide sequencing strategy for the RHD and RHCE genes. The nucleotides sequenced are indicated for the 5'-UTR, 10 exons, 9 introns and 3'-UTR of the RHD gene and the inversely oriented RHCE gene. The total number of nucleotides sequences is 5,483 for RHD and 5,851 for RHCE. Fig. S2. RH haplotypes in the family. The inheritance of the RHD*weak D type 29 – RHCE*ce haplotype is shown (KY617089). The trio allowed determining 3 more RH haplotypes (KY617090 to KY617092). The RHD allele RHD*weak D type 29 is identical to the previously published nucleotide sequence RHD(178A>C, 201G>A, 203G>A, 594A>T, 667T>G, 744C>T, 957G>A, 1025T>C),7,10 encoding the 5 amino acid substitutions I60L, S68N, K198N, F223V, and I324T. Fig. S3. Comparison of Rhesus boxes. Nucleotide positions are numbered according to the standard upstream Rhesus box. Position 1 represents the start of the homology between upstream and downstream Rhesus boxes and corresponds to nucleotide number 33 in GenBank accession number AJ252311. Nucleotides typical for the upstream Rhesus box are shown in orange, and those typical for the downstream Rhesus box in green. Yellow denotes nucleotides not reported for the standard upstream or downstream Rhesus box, gray denotes nucleotides common to upstream and downstream Rhesus box; –, indicates a one-nucleotide deletion; the numbers at nucleotide position 6594 indicate approximate lengths of the poly(T) tracts; further details as described previously.9 Fig. S4. Phylogenetic position of weak D type 29 among alleles in the 4 RHD clusters. The phylogenetic tree is based on a previous tree for RHD1 but limited to typical alleles of each cluster. Four main clusters of Rhesus haplotypes may be discerned. The RHD alleles of the DIVa cluster share three ancestral amino acids (62F, 137V, 152T), those of the weak D type 4 cluster a F223V substitution and of those of the DAU cluster a T379M substitution, two alleles are shown as “orphan alleles”, because their ce-association and observation in samples of African ancestry suggest an early divergence from the “Eurasian” cluster mainly comprised of alleles associated with cE, Ce or CE. Because current data are insufficient to resolve the order of recombination and substitution events, only one possible tree is shown, as it is for the 744CA>T and 957G>A substitution in weak D type 4.2.2. Fig. S5. Model of the RhD protein in weak D type 29. A homology model of the RhD protein is depicted as it is situated in the RBC membrane. The 2 side views are rotated 90° relative to each other (center panels); their top faces the RBC surface and bottom the RBC inside. The views to the RhD protein from the outside (top panel) and from the inside of an RBC (bottom panel) are depicted, rotated 90° relative to the side view (center left). 4 amino acid substitutions (I60L – red, S68N – green, F223V – orange, and I342T – yellow) occur in the transmembraneous helices (gray ribbons) and 1 substitution in an intracellular loop (K198N – pink). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.