Conclusions: Hfe+/− mice fed a HFD show evidence of partial phenotypic expression of the genetic defect. Despite the increase in HIC, Hfe+/− animals do not show increased susceptibility to HFD. Unlike homozygous deletion, heterozygosity for Hfe in this mouse model does not influence the severity of NAFLD. CJ McDONALD,1 DF WALLACE,1 DH CRAWFORD,2

VN SUBRAMANIAM1&2 1Queensland Institute of Medical Research, Brisbane, Australia. 2School of Medicine, University of R788 mouse Queensland, Brisbane, Australia Background: Most disorders of primary iron overload and iron-refractory anaemia have a genetic origin. Mutations in the HFE gene cause hereditary haemochromatosis (HH) and account for about 90% of HH in European populations, however mutations in other genes (termed non-HFE HH) are being increasingly identified in non-European populations. A combination of low awareness, high cost and non-standardized methodology for definitive diagnosis is likely leading to under-recognition of HH in these populations. As many Asia-Pacific

countries achieve improved nutrition and access to healthcare, it is possible that hitherto unrecognized hereditary iron overload conditions will be unmasked. We have developed Next Generation AZD0530 Sequencing technology to diagnose genetic iron overload and deficiency disorders in a systematic fashion. Methods: We have selected a panel of 41 genes either currently associated with genetic iron overload or anaemia, or for which evidence from MCE公司 animal models or in vitro experiments demonstrates a contribution to iron homeostasis. The whole transcripts of these genes, and promoter regions from 11 of them, were then used to generate a custom AmpliSeq sequencing panel. These target sequences were then simultaneously amplified from patient genomic DNA using AmpliSeq megaplex PCR, followed by high coverage sequencing on an Ion Torrent PGM. Patient sequences were mapped to the Human Genome

(HG19), and sequence variants annotated and analysed using IonReporter and Ingenuity Variant Analysis. Single Nucleotide Polymorphisms (SNP) were denoted as novel if they did not appear in the “1000 Genome” or “Exome Sequencing Project” databases. Non-synonymous amino acid changes were denoted as uncharacterised if there was no clinical or functional data associated with the SNP. The potential functional impact of identified SNPs was assessed using several algorithms, and finally potentially causative mutations confirmed by Sanger sequencing. Results: We have applied this system to identify the genetic basis of atypical iron overload in 18 cases to date. These cases represent a variety of ethnic backgrounds including European and Asian heritage. A significant amount of variation is seen within coding, UTR, and promoter regions of the sequenced genes with an average of 140 SNPs detected per case. Of these, an average of 12 cause non-synonymous amino acid changes.