Albertine Donker

TSAT/Hepcidin Ratio discriminates TMPRSS6 -related Anemia from IDA 279 8 METHODS IRIDA patients We screened the Radboudumc Iron Registry Biobank, Nijmegen, the Netherlands for patients who were diagnosed with TMPRSS6 -related IRIDA (called ‘IRIDA’ hereafter) between 2010 and May 2019. 20 This registry is a facility to optimize the use and distribution of biomaterial and/or demographic and clinical patient data for scientific research of iron disorders, for which ethical permission has been obtained. IRIDA probands were defined as patients having both an ‘IRIDA phenotype’ and an ‘IRIDA genotype’, i.e. microcytic anemia, TSAT<10%, Hb not or partially responsive to oral iron (<2g/dL Hb increment after three weeks of oral iron therapy), 21 and mono- or bi-allelic pathogenic TMPRSS6 variants. Only patients living in the Netherlands for which the TSAT/hepcidin ratio was assessed in absence of moderate or severe inflammation (defined as CRP≤10 mg/L) were included. Genotyping of IRIDA patients was performed by PCR, DNA Sanger sequencing (until March 2014) and by Ion Torrent sequencing (after March 2014) of the exons of TMPRSS6 , thereby analyzing the coding part of the gene. Multiplex ligation-dependent probe amplification (MLPA) was conducted in patients with an IRIDA phenotype in whom only a mono-allelic pathogenic TMPRSS6 variant was found, in order to exclude large deletions and/or duplications in the ‘healthy’ allele. 22 The pathogenicity of genetic variants was assessed by review of the literature on previous reported cases and functional studies, association of the variant with the phenotype within a family and by bioinformatic tools (SIFT, Align GVGD, Polyphen, SpliceSiteFinder-like, MaxEntScan, NNSplice, GeneSplicer, and Human Splicing Find, all as part of Alamut software, Alamut), as described in detail before. 11,23,24 IDA controls Participants of the control group were consecutively enrolled between February 2018 and May 2019 while attending different departments of the Máxima MC (MMC) Veldhoven, the Netherlands, for work-up of IDA. For ethical reasons, we only included IDA controls aged ≥18 years. A total of 161 patients underwent laboratory testing for IDAandwere screened for potential study inclusion by checking laboratory results, medical history and medication use. IDA