A cold case of hemolytic disease of the fetus and newborn resolved by genomic sequencing and population studies to define a new antigen in the Rh system.

BACKGROUND: We report an obstetric case involving an RhD-positive woman who had developed a red blood cell (RBC) antibody that was not detected until after delivery of a newborn, who presented with a positive direct antiglobulin test result. Immunohematology studies suggested that the maternal antibody was directed against a low-prevalence antigen on the paternal and newborn RBCs. RESULTS: Comprehensive blood group profiling by targeted exome sequencing revealed a novel nonsynonymous single nucleotide variant (SNV) RHCE c.486C>G (GenBank MZ326705) on the RHCE*Ce allele, for both the father and newborn. A subsequent genomic-based study to profile blood groups in an Indigenous Australian population revealed the same SNV in 2 of 247 individuals. Serology testing showed that the maternal antibody reacted specifically with RBCs from these two individuals. DISCUSSION: The maternal antibody was directed against a novel antigen in the Rh blood group system arising from an RHCE c.486C>G variant on the RHCE*Ce allele linked to RHD*01. The variant predicts a p.Asn162Lys change on the RhCE protein and has been registered as the 56th antigen in the Rh system, ISBT RH 004063. CONCLUSION: This antibody was of clinical significance, resulting in a mild to moderate hemolytic disease of the fetus and newborn (HDFN). In the past, the cause of such HDFN cases may have remained unresolved. Genomic sequencing combined with population studies now assists in resolving such cases. Further population studies have potential to inform the need to design population-specific red cell antibody typing panels for antibody screening in the Australian population.

Genomic characterisation of clinically significant blood group variants in Aboriginal Australians.

BACKGROUND: Hematological disorders are often treated with blood transfusions. Many blood group antigens and variants are population-specific, and for patients with rare blood types, extensive donor screening is required to find suitable matches for transfusion. There is a scarcity of knowledge regarding blood group variants in Aboriginal Australian populations, despite a higher need for transfusion due to the higher prevalence of renal diseases and anaemia. MATERIALS AND METHODS: In this study, we applied next-generation sequencing and analysis to 245 samples obtained from Aboriginal Australians from South-East Queensland, to predict antigen phenotypes for 36 blood group systems. RESULTS: We report potential weak antigens in blood group systems RH, FY and JR that have potential clinical implications in transfusion and pregnancy settings. These include partial DIII type 4, weak D type 33, and Del RHD (IVS2-2delA). The rare Rh phenotypes D+ C+ E+ c- e+ and D+ C+ E+ c+ e- were also detected. DISCUSSION: The comprehensive analyses of blood group genetic variant profiles identified in this study will provide insight and an opportunity to improve Aboriginal health by aiding in the identification of appropriate blood products for population-specific transfusion needs.

Genetic characterization of HIV type 1 Nef-induced vesicle secretion.

The HIV-1 Nef protein is known to be secreted, and our group has shown that Nef is secreted from nef-transfected and HIV-1-infected cells in small exosome-like vesicles (d. 40-100 nm). The role of secreted Nef remains to be fully characterized. Thus, it is important to characterize the nature of and the mechanisms regulating Nef secretion. We hypothesized that specific structural domains on the Nef protein interact with components of the endosomal trafficking machinery, sorting Nef into multivesicular bodies (MVB) and packaging it in exosome-like vesicles. To identify those domains, a series of mutants spanning the entire nef sequence were made and cloned into the expression vector pQB1, which expresses the mutants as Nef-GFP fusion proteins. These constructs were used in transient transfection assays to identify sequences necessary for secretion of the Nef-GFP fusion protein. N-terminal domains were identified as critical for Nef-induced vesicle secretion: (1) a basic cluster of four arginine residues (aa 17, 19, 21, 22), (2) the phosphofurin acidic cluster sequence (PACS; Glu62-65), and (3) a previously uncharacterized domain spanning amino acid residues 66-70 (VGFPV), which we named the secretion modification region (SMR). Additional amino acids P25, 29GVG31, and T44 were identified in HIV-1 Nef as regulating its secretion. These residues have not been associated with other reported Nef functions. The myristoylation domain, ubiquitination lysine residues, and the C-terminal portion of Nef (aa 71-206) had no effect on secretion. A minimal HIV-1 Nef sequence, comprising the identified motifs, was sufficient for Nef-induced vesicle secretion.

HIV-1 Nef protein is secreted into vesicles that can fuse with target cells and virions.

HIV-1 Nef is a major determinant in HIV-1 pathogenicity. However, its properties have mainly been associated with its biochemical activities within the producer cell. Nef is also secreted from infected and transfected cells. Our primary objective was to determine the nature of secreted Nef protein and its effect on target cells. We determined that HIV-1 Nef is secreted in the form of exosome-like vesicles. Nef protein present in these vesicles is largely protected from protease digestion, which suggests that most of the protein is present on the lumenal side of the vesicles. We observed that HEK293 cells, transfected with a Nef-GFP expression vector, can secrete vesicles containing Nef-GFP fusion protein into the extracellular medium. When the conditioned medium was used to treat Jurkat cells, we found that the cells can take up the Nef fusion protein. The pattern of distribution of the Nef-GFP fusion protein within the target cells is mainly cytoplasmic and results in a punctate staining pattern. We also observed that Nef (-) virions treated with Nef-conditioned medium have their infectivity restored to near wild-type levels. This implies that the Nef contained within vesicles has the ability to fuse with HIV-1 virions and deliver functional Nef to these virions. It also demonstrates that Nef protein delivered in trans can restore infectivity even after virion maturation has occurred. These studies suggest that secreted Nef could play a role in HIV-1 pathogenesis by inducing effects in noninfected bystander cells.