The Rh blood group system is described as the most polymorphic of all blood group systems in humans. Since the first documented case of blood group incompatibility due to the RhD antigen, numerous reported case studies have contributed to our knowledge about the polymorphic nature of this blood group system, and altered expression of the Rh blood group genes. Abnormalities in the RH gene can range from being so minor that no clinical significance is associated with the mutation, to gross deletions that result in conformational protein changes of the antigens within the red cell membrane resulting in clinical manifestation of disease.
The aim of this thesis was to investigate the molecular basis for two forms of aberrant gene expression in the Rh blood group system: firstly, the identification of a silent mutation resulting in the Rhnull phenotype of an Australian blood donor and the inheritance of this aberrant gene through family studies, and secondly, to identify if the weakened expression of the RhD antigen in the red cell membrane of heterozygous RHD individuals phenotypically classified as weak D is the result of either a heterozygous mutation in the RHAG gene, or single point mutations in the RHD gene.
Analysis of the RHAG gene in Y.T., a well-documented Rhnull individual, was performed to identify the cause of a heterozygous trait previously identified. Earlier studies into the genetic cause for the Rhnull syndrome in this individual detected one abnormal RHAG gene (G836A) present in a single copy, indicating that a second silent gene with a separate unknown mutation was present. In this thesis, DNA sequencing identified mixed sequence at nt 155: the presence of both a G (wildtype) and A (mutant) base. This single point mutation, occurring at the exon 1 - intron 1 donor splice site region, prevents normal intron splicing from occurring, and results in a non-functional gene. Inheritance studies using PCR and RFLP analysis identified that the silent gene was inherited paternally, and has been passed on to two of Y.T.'s children. The two separate mutations identified in Y.T. result in the non-functional RHAG genes, leading to her extremely rare Rhnull classification as a composite heterozygote of the regulator type.
Mutation analysis of individuals classified as having a weakened expression of the RhD antigen was first performed using known mutations in the RHAG gene resulting in the Rh-deficiency syndrome in other reported RHnull individuals. None of the 6 known mutations in the RHAG gene were identified in 100 blood donors classified as weak D. The lack of RHAG involvement as a cause of reduced RhD antigen expression indicated that further study into the weak D samples was required.
Additional RFLP analysis on the weak D samples confirmed the presence of single point mutations in the RHD gene sequence. Recently reported findings of single point mutations in the RHD gene resulting in weakened expression of RhD were investigated in weak D blood donor samples. Three single point mutations: Weak D Type 1 (r809G), Weak D Type 2 (G1154C) and Weak D Type 3 (C8G) were identified in 88% of the samples tested and classified as having reduced RhD antigen expression. Of die DccEe (weak D) samples tested, 76% possessed the T809G mutation and 6% carried the C8G mutation. 95% of the DccEe (weak D) samples carried the G1154C mutation. Only one sample phenotyped as DccEe (weak D) had the G1154C mutation. In the 12 samples (12%) with no observed weak D types 1, 2 or 3 mutations, further examination showed 7 were Dvi variants, 1 was misclassified in initial serology as weak D, and 4 samples could not be classified further.
Out of 48 random samples tested with normal RhD antigen expression, one sample (DccEE) possessed the G1154C Weak D Type 2 mutation. Further testing determined this sample was homozygous for the RHD gene, with one mutant RHD gene, and one normal RHD gene.
This thesis has identified that mutations present in both the RHAG and the RHD gene can result in absent or reduced expression of the Rh antigens in the red cell membrane. Clinical manifestation of disease or reduced expression can be identified in those individuals with genetic alterations in conservative regions of the gene, emphasising the complex interaction of these proteins within the red cell membrane. The importance of correctly identifying these mutations and understanding their importance is vital in diagnostic treatment of individuals at risk of Rh associated disease, such as haemolytic disease of the newborn, Rh isoimmunisation or transfusion reactions.