Albertine Donker

Chapter 4 152 were decreased). Only the mother of patient 17, who had the same heterozygous genotype as her daughter, had an IRIDA phenotype. She had a history of unexplained iron deficiency ( Supplemental Table 4 ). Molecular characterization We observed 14 different defects in 21 patients ( Table 1, Supplemental Table 5 ). Five out of 14 are previously described TMPRSS6 defects: three missense variants, 3,15,27 and two frameshift variants. 3,15 Of these variants, the frameshift alteration c.497delT (p.Leu166Argfs*37) was the most prevalent defect in our population and was found in eight out of 14 bi-allelic patients and in two out of eight mono- allelic patients (12 out of 36 affected alleles = 33%) ( Table 1 ). All patients with the c.497delT (p.Leu166Argfs*37) were of Dutch origin, whereas all three patients with the c.1904_1905dup (p.Lys636Alafs*17) were of Turkish origin ( Supplemental Table 6 ). For the non-synonymous missense variant c.2383G>A (p.Val795Ile) a functional test had previously been performed. This showed physiological inhibition of HAMP1 expression ( in vitro studies by site directed mutagenesis into murine TMPRSS6 , 15 despite the IRIDA phenotype of the patient who harbored this variant. However, although considered as the most reliable method to predict the pathogenicity of an allele variant, the results of cell based functional testing by site directed mutagenesis should be interpreted with caution. The technique has been described as error prone and is likely to be limited in its extrapolation because the test environment may not fully reflect the in vivo situation. 15,28 Allele frequency of the c.2383G>A (p.Val795Ile) missense variant is < 1% ( Supplemental Table 5 ), which argues against a neutral or beneficial effect of this nucleotides change. Because of the not fully reliable functional tests, the low allele frequency and the in-silico analysis that point towards pathogenicity, we considered this missense variant as pathogenic. 29 Nine novel pathogenic TMPRSS6 defects were demonstrated in our case series; two large deletions, four missense mutations, one nonsense mutation and two splice defects ( Table 2 ). Pathogenicity of the new mutations was predicted with in-silico software. In all seven heterozygous IRIDA patients, no deletions and/or duplications in the TMPRSS6 gene of the ‘healthy’ allele were found with MLPA.