A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg in partial fulfillment of the requirements for the degree of Master of Medicine in the Branch of Medical Genetic. Johannesburg, South Africa, 2012 / Fanconi anaemia (FA) is a genotypically and phenotypically heterogeneous genetic condition , characterized microscopically by chromosomal breakage and instability and usually inherited in an autosomal recessive manner. Affected individuals often present with a diverse variety of physical congenital abnormalities and most progress to haematological disease including bone marrow aplasia and myelodysplasia in early childhood. In South Africa, Black individuals with FA share a common causative founder deletion mutation in the Fanconi G gene (FANCG del) in 82% cases. They are thus an ideal patient cohort for a genotype-phenotype correlation study. Thirty Black patients, homozygous for FANCG del, were ascertained from haematology/oncology clinics in Johannesburg and Bloemfontein. They were subjected to a comprehensive clinical examination to a document their physical features. A concurrent review of each participant's hospital file allowed data to be collected regarding disease presentation and haematological progression . Significant growth abnormalities and a high frequency of skin of skin pigmentary anomalies were found in the research cohort. Although subtle, anomalies of the eye, ears, and hands were noted in a high frequency. The overall physical phenotype does not appear to be appreciably different from that described in other Fanconi anaemia cohorts; however, affected Black individuals may present with more severe haematological indices and have poorer outcome that FA individuals of heterogeneous genotype. Further, it would appear that haematological disease progression cannot be predicted by the presence of abnormalities.

La tumeur de Wilms (WT) est la tumeur du rein la plus fréquente chez l'enfant âgé de moins de 5 ans. Bien que la majorité des enfants avec WT soit aujourd'hui soignée, 10 à 15% rechutent. L'identification de nouveaux marqueurs pronostic au diagnostic est donc nécessaire. Nous avons centralisé le matériel biologique de toutes les WTs françaises inclus dans le protocole SIOP-2001. Le blastème, la composante histologique la plus agressive de ces tumeurs, a été caractérisé par une analyse qualitative (architecture et aspect cytologique, index mitotique et prolifératif) et quantitative (volume et pourcentage). En collaboration avec les équipes européennes du Renal Tumor Study Group nous avons caractérisé les anomalies structurales de la WT par Multiple Ligation Probe Amplification (MLPA). De plus, nous nous sommes focalisés sur la région 11p15, où le gène IGF2 est localisé, en analysant les anomalies structurales et de méthylation au niveau de différents loci (IGF2-DMR0, ICR1, ICR2, H19), par MLPA et par ASMM RTQ- PCR (TaqMan allele-specific methylated multiplex real-time quantitative PCR). Nous avons de plus étudié l'expression d'IGF2 par RT-QPCR. Une dernière partie de notre étude a porté sur la caractérisation de la réponse immune intratumorale par immunohistochimie (CD3, CD4, CD8, PD1, PDL1). En conclusion, nous avons identifié des potentiels marqueurs pronostiques concernant la prise en charge des WTs: volume et pourcentage de blastème, index mitotique et de prolifération, perte de méthylation d'IGF2-DMR0 et rapport CD4/CD8. Ce travail pourrait contribuer à la détermination d'une nouvelle classification bio-pathologique de la WT. / Wilms tumor (WT) is the most common tumor of the kidney in children aged under 5 years. Although the majority of children with WT survives, 10-15% relapse. The identification of new prognosis markers at diagnosis is necessary. We centralized the biological material of all French WTs included in the SIOP-2001 protocol. The blastema, the most aggressive histological component of these tumors, was characterized by qualitative (architectural and cytological aspect, proliferative and mitotic index) and quantitative analysis (amount and percentage). In collaboration with European teams Renal Tumor Study Group we characterized the structural abnormalities of the WT by Multiple Ligation Probe Amplification (MLPA). In addition, we focused on the 11p15 region, where the IGF2 gene is located, analyzing the structural abnormalities and methylation at different loci (IGF2-DMR0, ICR1, ICR2, H19) by MLPA and ASMM RTQ - PCR (TaqMan allele-specific methylated multiplex quantitative real-time PCR). We also studied the expression of IGF2 by RT-QPCR. A final part of our study focused on the characterization of intratumoral immune response by immunohistochemistry (CD3, CD4, CD8, PD1, PDL1). In conclusion, we identified potential prognostic markers for the management of WTs: volume and percentage of blastema, mitotic and proliferation index, loss of IGF2 methylation-DMR0 and CD4 / CD8 ratio. This could contribute to the determination of a new bio-pathological classification of WT.

The imprinted expression of the IGF2 and H19 genes is controlled by the imprinting control region 1 (ICR1) located at chromosome 11p15.5. This methylation-sensitive chromatin insulator works by binding the zinc-finger protein CTCF in a parent-specific manner. CTCF binds the unmethylated maternal allele and is required for preventing de novo methylation at ICR1. DNA methylation defects involving the ICR1 IGF2/H19 domain result in two growth disorders with opposite phenotypes: an overgrowth disorder, the Beckwith-Wiedemann syndrome (ICR1 gain of methylation in 10% of BWS cases) and a growth retardation disorder, the Silver-Russell syndrome (ICR1 loss of methylation in 60% of SRS cases). Little information is available regarding the mechanism of ICR1 DNA methylation defects. Several deletions removing part of ICR1 (1.4 to 2.2 kb) have been described in a few familial BWS cases with dominant maternal transmission. In order to evaluate precisely the incidence of ICR1 mutations, we investigated, by long range PCR and sequencing, 21 BWS patients (including two brothers) with ICR1 gain of methylation and 16 SRS patients with ICR1 loss of methylation. No mutation of the seven CTCF binding sites was detected in the familial BWS cases. Two additional cases of constitutional genetic lesions were identified in BWS patients with apparently-sporadic forms. One patient was identified with a 8 bp deletion within the B3 repeat, 116 bases 3' of the CTCF binding site 4. Another patient was identified with a 1.8 kb deletion which eliminates CTCF binding sites 2 and 3. A single-nucleotide variation was identified in a SRS patient. Our data showed that ICR1 deletions, including new small deletions, account for apparently sporadic forms of BWS with ICR1 gain of methylation. ICR1 deletions are associated with a high incidence of Wilms' tumour, making their molecular diagnosis particularly important for genetic counseling and tumor surveillance.