Albertine Donker

Chapter 4 144 Gene analysis Genotyping was performed by PCR, DNA Sanger sequencing (until March 2014) and Ion Torrent sequencing (after March 2014) of the coding part of TMPRSS6 ( Supplemental Table 1 ). Comparative Genomic Hybridization as performed in patient 1 and her relatives, for whom PCR products of the TMPRSS6 gene could not be obtained, using the Affymetrix CytoScan HD array platform according to the manufacturer’s protocol (Affymetrix, Santa Clara, CA, USA). Multiplex Ligation- dependent Probe Amplification (MLPA) analysis was performed in patients who had an IRIDA phenotype and a mono-allelic pathogenic TMPRSS6 variant to exclude large deletions and/or duplications in the ‘healthy’ allele, and also in patient 1 ( Supplemental Table 2 , The Netherlands, www.mlpa.com) . 24 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 bio-informatic tools (SIFT, Align GVGD, Polyphen, SpliceSiteFinder-like, MaxEntScan, NNSplice, GeneSplicer and Human Splicing Find, all as part of Alamut software, Alamut). 25 Alamut was used to assess pathogenicity in case of not previously reported TMPRSS6 variants. Haplotype analysis was performed in a search for a founder effect in families with identical pathogenic TMPRSS6 defects by using 10 different intra-gene exon High Frequency Variants (HFV’s) detected by Sanger DNA sequencing and 3 Short Tandem Repeats surrounding TMPRSS6 . We also performed parental haplotype analysis in two sibling pairs with identical heterozygous TMPRSS6 variants but different phenotypes, using 7 different intra- gene HFV’s also detected by Sanger DNA sequencing.