Albertine Donker

Chapter 9 318 a family will support but not prove pathogenicity since the true cause of the disease might be located elsewhere in the gene, in the cis position. If SNP databases and co- segregation data within a family do not answer the question whether a new variant is pathogenic or not, in silico predictions can be helpful. 78 However, performing functional studies is still the gold standard for determining the (non)pathogenicity of variants of unknown significance (VUS). For this reason, we suggest to perform functional studies of VUS that will be found by WES sequencing of candidate genes in mono-allelic IRIDA patients by the above-mentioned approach. Functional studies might also be helpful in determining the pathogenicity of VUS in TMPRSS6 variants that are often found in suspected IRIDA cases. However, the development of functional in vitro assays that represent the in vivo situation is challenging. Moreover, the exact function of matriptase 2 is still not completely certain. As reviewed earlier in this thesis, in vitro experiments have shown that matriptase 2 (MT2) acts by cleaving hemojuvelin, the BMP co-receptor that upregulates HAMP transcription. 79 IRIDA-causing TMPRSS6 variants result in aberrant MT2 with impaired HJV-cleaving capacity, resulting in inappropriately increased hepcidin levels. 79 These data and other observations 80 suggest that HJV is the substrate of MT2. However, recent in vivo studies in Hjv knock out mice overexpressing TMPRSS6 s uggest that also BMP receptors of the BMP/SMAD pathway are cleaved by MT2. 81 Whether BMP receptors are substrates of MT2, next to HJV, needs further exploration in functional studies. Performing these studies might identify novel working mechanism for matriptase 2, form the basis for novel functional assays for TMPRSS6 variants, and might help to further elucidate the pathophysiology of IRIDA, with the ultimate goal of improving the diagnosis and treatment for the individual patient. The lifetime course of IRIDA is intriguing and raises questions concerning the effects of TMPRSS6 defects during the journey from fetus to adult The lifetime course of IRIDA in human life is intriguing. According to our observations and those of others, 82 suspicion of IRIDA because of unexplained and therapy- resistant IDA predominantly occurs during childhood, but not at birth or during the first postnatal months. This raises the following questions: i) Is hepcidin suppressed by other, MT2 independent pathways in the fetus and neonate? Noteworthy, in TMPRSS6 -/- fetal mice mRNA expression of liver HAMP is significantly higher than in control mice, arguing against this suggestion. 83,84 ii) Are the fetus and neonate protected for overexpression of hepcidin, and if so, by which mechanism(s)? One