Next-generation sequencing of iron-metabolism related genes in Portuguese patients with iron overload: novel pathogenic genetic variants

Objective: In Southern European countries up to one-third of the patients with hereditary hemochromatosis (HH) do not present the common HFE risk genotype. In order to investigate the molecular basis of these cases we have designed a gene panel for rapid and simultaneous analysis of 6 HH-related genes (HFE, TFR2, HJV, HAMP, SLC40A1 and FTL) by next-generation sequencing (NGS). Materials and Methods: Eighty-eight iron overload Portuguese patients, negative for the common HFE mutations, were analysed. A TruSeq Custom Amplicon kit (TSCA, by Illumina) was designed in order to generate 97 amplicons covering exons, intron/exon junctions and UTRs of the mentioned genes with a cumulative target sequence of 12115bp. Amplicons were sequenced in the MiSeq instrument (IIlumina) using 250bp paired-end reads. Sequences were aligned against human genome reference hg19 using alignment and variant caller algorithms in the MiSeq reporter software. Novel variants were validated by Sanger sequencing and their pathogenic significance were assessed by in silico studies. Results: We found a total of 55 different genetic variants. These include novel pathogenic missense and splicing variants (in HFE and TFR2), a very rare variant in IRE of FTL, a variant that originates a novel translation initiation codon in the HAMP gene, among others. Conclusion: The merging of TSCA methodology and NGS technology appears to be an appropriate tool for simultaneous and fast analysis of HH-related genes in a large number of samples. However, establishing the clinical relevance of NGS-detected variants for HH development remains a hard-working task, requiring further functional studies.

Allocation of uranium enrichment services to fuel foreign and domestic nuclear reactors, Energy Research and Development Administration, Department of State : report to the Committee on Foreign Affairs, House of Representatives /

Publication date stamped on cover.

Unclassified U.S. Atomic Energy Commission reports on civilian power reactors and test reactors : a literature search /

"November 1956."

Pressurized water reactors : a literature search /

"June 1959."

Industrial process and space heat from nuclear reactors : a literature search /

"October 1959."

Selected reactors of the Power Reactor Demonstration Program : a literature search /

U.S. Atomic Energy Commission Report No. TID-3556.

Selected reactors of the Power Reactor Demonstration Program : a literature search /

AEC Report No. TID-3556(REV. 1).

Fast neutron pulse reactors and fast neutron critical assemblies : a literature search /

"Reactor Technology."

The LRL advanced reactors studies program a review /

"Issued April 3, 1962"

Selected reactors of the Power Reactor Demonstration Program : a literature search /

"Listing of reports dealing with eleven reactors of the Power Reactor Demonstration Program."

Nuclear reactors and earthquakes /

Issuance date: August 1963.

Fuel-efficiency tests on batch oil stills /

Includes index.

Experimental reactors : power and test : a technical and pictorial summary /

"November 1956"--Page [i] of cover.

Applicability of two mathematical models to the batch settling of activated sludge.

"Supported by Federal Water Pollution Control Administration, U.S. Department of the Interior. Research project WP-01011."

An experiment on CAI sequencing constructs /

Includes bibliographical references.