Hematopoiesis is an intricately regulated homeostatic process that maintains all of the differentiated blood cell lineages. N-ethyl-N-nitrosurea (ENU) is a powerful mutagen that induces point mutations randomly in the genome. ENU was used in a dominant forward genetic screen to identify novel mutations in regulators of hematopoiesis and to create new mouse models of hematopoietic disease. The objectives of this thesis were to characterize two mutants that originated from the dominant screen (7192 and 7238) and to develop a pharmacologically sensitized screen that would detect a unique set of mutations undetectable in the dominant screen. The 7192 mutant from the ENU dominant screen presented with elevated microcytic red blood cells (RBC) and increased polychromasia. The causative mutation was identified as a nonsense mutation in Ank1 (Q895X) that coded for a truncated ANK1 protein. Ank17192 is a novel mouse model of hereditary spherocytosis (HS), a human disease that results from increased RBC fragility. We have demonstrated that Ank17192/+ mice model a mild HS and Ank17192/7192 mice model severe HS. The 7238 mutant from the dominant ENU screen was macrothrombocytic and carried a missense mutation in Myh9 (Q1443L). The Myh97238/7238 mice are viable and have a more severe phenotype of macrothrombocytopenia. Myh97238 is the first mouse model for Myh9 related disorders that accurately models the genetic origins and the systemic manifestations of the disorder. A pharmacologically sensitized screen using chemotherapeutic drugs was designed to induce stress hematopoiesis to detect mutations that alter cell cycle of hematopoietic progenitors or stress hematopoiesis. Analysis of both peripheral blood and progenitor recovery kinetics, determined that 5-fluorouracil (5FU) and phenylhydrazine were good candidates for a pharmacologically sensitized screen. 5FU was successfully incorporated into an ENU dominant screen, and 13 platelet recovery outliers were detected. From these outliers, three mutant lines were successfully established.

Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The purpose of this study was to investigate the association of the selected MYH9 single nucleotide polymorphisms (SNPs) with chronic kidney disease (CKD) and its related co-morbidities in the South African mixed ancestry population residing in Bellville South, Cape Town. In 2008, two landmark studies identified SNPs in the MYH9 gene which explained most of the increased risk for non-diabetic CKD in African Americans. These polymorphisms were later found to be weakly associated with diabetic nephropathy. Three SNPs that exhibited independent evidence for association with CKD were selected (rs5756152, rs4821480 and rs12107). These were genotyped using a Taqman genotyping assay on a BioRad MiniOpticon and confirmed by sequencing in 724 subjects from Bellville South, Cape Town, South Africa. Prevalent CKD was defined based on the estimated glomerular filtration rate calculated using the modification of diet in renal disease (MDRD) formula. Chronic kidney disease was present in 214 subjects (29.6%), 96.3% were stage 3 and only 8 subjects were stage 4. In additive allelic models, adjusted for age and gender, rs5756152 demonstrated an association with kidney function whereby each G allele of rs5756152 increased eGFR by 3.67 ml/min/1.73, reduced serum creatinine by 4.5% and increased fasting plasma glucose by 0.51 mmol/L. When an interaction model was used, the effect of rs5756152 on serum creatinine, eGFR and blood glucose levels was retained, and enhanced, but only in diabetic subjects. In addition, rs4821480 T allele increased eGFR while rs12107 A allele decreased glucose levels in diabetic subjects. In contrast to reports that MYH9 SNPs are strongly associated with non-diabetic end stage renal disease, our study demonstrated that rs5756152 and rs4821480 are associated with early kidney function derangements in type 2 diabetes whilst rs12107 is associated with glucose metabolism. Our findings, along with previous reports, suggest that the MYH9 gene may have a broader genetic risk effect on different types of kidney diseases than previously thought. / AFRIKAANSE OPSOMMING: Hierdie studie het ondersoek ingestel na die verband tussen drie gekose MYH9-enkelnukleotied-polimorfismes (SNP’s) en chroniese niersiekte (hierna ‘niersiekte’), wat verwante ko-morbiditeite insluit, onder ’n Suid-Afrikaanse populasie van gemengde afkoms in Bellville-Suid, Kaapstad. Twee rigpuntstudies het in 2008 op SNP’s in die MYH9-geen afgekom wat verklaar het waarom Afro-Amerikaners ’n hoër risiko vir niediabetiese niersiekte toon. Later is bevind dat hierdie polimorfismes ook ’n swak verband met diabetiese nefropatie het. Drie SNP’s wat elk onafhanklik bewys gelewer het van ’n verband met niersiekte is vervolgens gekies (rs5756152, rs4821480 en rs12107). Die SNP’s is daarná met behulp van die Taqman-toets op ’n BioRad MiniOpticon aan genotipering onderwerp, en is toe deur middel van reeksbepaling by 724 proefpersone van Bellville-Suid, Kaapstad, Suid-Afrika, bevestig. Die voorkoms van niersiekte is bepaal op grond van die geraamde glomerulêre filtrasietempo (eGFR), wat aan die hand van die ‘niersiekte-dieetveranderings’- (MDRD-)formule bereken is. Daar is bevind dat 214 proefpersone (29,6%) aan chroniese niersiekte ly – 96,3% was in fase 3 en slegs agt proefpersone in fase 4. In toegevoegde alleliese modelle wat vir ouderdom en geslag aangepas is, het rs5756152 ’n verband met nierfunksie getoon: Elke G-allel van rs5756152 het eGFR met 3,67 ml/min/1,73 verhoog, serumkreatinien met 4,5% verlaag en vastende plasmaglukose met 0,51 mmol/L verhoog. Toe ’n interaksiemodel gebruik is, is die effek van rs5756152 op serumkreatinien, eGFR en bloedglukosevlakke behou en versterk, hoewel slegs by diabetiese proefpersone. Daarbenewens het die T-allel van rs4821480 eGFR verhoog, terwyl die A-allel van rs12107 ook glukosevlakke by diabetiese proefpersone verlaag het. In teenstelling met bewerings dat MYH9-SNP’s ’n sterk verband met niediabetiese eindstadiumniersiekte toon, het hierdie studie bewys dat rs5756152 en rs4821480 met vroeë nierfunksieversteurings by tipe 2-diabetes verband hou, terwyl rs12107 weer met glukosemetabolisme verbind word. Tesame met vorige studies, doen hierdie navorsingsbevindinge dus aan die hand dat die MYH9-geen dalk ’n groter genetiese risiko-effek op verskillende tipes niersiekte het as wat voorheen vermoed is. / Cape Peninsula University of Technology Research Fund / University of Stellenbosch Merit Bursary