Nadia Soriani

Genetic hyperferritinaemia and reticuloendothelial iron overload associated with a three base pair deletion in the coding region of the ferroportin gene (SLC11A3).

Summary. Iron overload may predominantly involve parenchymal or reticuloendothelial cells, the prototype of parenchymal iron overload being HFE‐related genetic haemochromatosis. We studied a family with autosomal dominant hyperferritinaemia in whom the proband showed selective iron accumulation in the Kupffer cells on liver biopsy. Analysis of L and H ferritin genes excluded mutations responsible for hereditary hyperferritinaemia/cataract syndrome or similar translational disorders. Sequence analysis of the ferroportin gene (SLC11A3) in four individuals with hyperferritinaemia singled out a three base pair deletion in a region that contains four TTG repeats. This mutation removes a TTG unit from 780 to 791, and predicts the loss of one of three sequential valine residues 160–162. Denaturing high performance liquid chromatography can be used for its detection. SLC11A3 polymorphism analysis indicates that this probably represents a recurrent mutation due to slippage mispairing. Affected individuals may show marginally low serum iron and transferrin saturation, and young women may have marginally low haemoglobin concentration levels. Serum ferritin levels are directly related to age, but are 10–20 times higher than normal. Heterozygosity for the ferroportin Val 162 deletion represents the prototype of selective reticuloendothelial iron overload, and should be taken into account in the differential diagnosis of hereditary or congenital hyperferritinaemias.

Genetic and clinical heterogeneity of ferroportin disease

Ferroportin is encoded by the SLC40A1 gene and mediates iron export from cells by interacting with hepcidin. SLC40A1 gene mutations are associated with an autosomal type of genetic iron overload described as haemochromatosis type 4, or HFE4 (Online Mendelian Inheritance in Man number 606069), or ferroportin disease. We report three families with this condition caused by novel SLC40A1 mutations. Denaturing high‐performance liquid chromatography was employed to scan for the SLC40A1 gene. A D181V (A846T) mutation in exon 6 of the ferroportin gene was detected in the affected members of an Italian family and shown to have a de novo origin in a maternal germinal line. This mutation was associated with both parenchymal and reticuloendothelial iron overload in the liver, and with reduced urinary hepcidin excretion. A G80V (G543T) mutation in exon 3 was found in the affected members of an Italian family with autosomal hyperferritinaemia,. Finally, a G267D (G1104A) mutation was identified in exon 7 in a family of Chinese descent whose members presented with isolated hyperferritinaemia. Ferroportin disease represents a protean genetic condition in which the different SLC40A1 mutations appear to be responsible for phenotypic variability. This condition should be considered not only in families with autosomal iron overload or hyperferritinaemia, but also in cases of unexplained hyperferritinaemia.

Mutational scanning of the ABCR gene with double-gradient denaturing-gradient gel electrophoresis (DG-DGGE) in Italian Stargardt disease patients

Mutations in the retina-specific ABC transporter (ABCR) gene are responsible for autosomal recessive Stargardt disease (arSTGD). Mutation detection efficiency in ABCR in arSTGD patients ranges between 30% and 66% in previously published studies, because of high allelic heterogeneity and technical limitations of the employed methods. Conditions were developed to screen the ABCR gene by double-gradient denaturing-gradient gel electrophoresis. The efficacy of this method was evaluated by analysis of DNA samples with previously characterized ABCR mutations. This approach was applied to mutation detection in 44 Italian arSTGD patients corresponding to 36 independent genomes, in order to assess the nature and frequency of the ABCR mutations in this ethnic group. In 34 of 36 (94.4%) STGD patients, 37 sequence changes were identified, including 26 missense, six frameshift, three splicing, and two nonsense variations. Among these, 20 had not been previously described. Several polymorphisms were detected in affected individuals and in matched controls. Our findings extend the spectrum of mutations identified in STGD patients and suggest the existence of a subset of molecular defects specific to the Italian population. The identification of at least two disease-associated mutations in four healthy control individuals indicates a higher than expected carrier frequency of variant ABCR alleles in the general population. Genotype-phenotype analysis in our series showed a possible correlation between the nature and location of some mutations and specific ophthalmoscopic features of STGD disease.

Scanning mutations of the 5'UTR regulatory sequence of L-ferritin by denaturing high-performance liquid chromatography: identification of new mutations.

Summary. Hereditary hyperferritinaemia cataract syndrome is an autosomal dominant disorder caused by heterogeneous mutations of the iron regulatory element (IRE) in the ferritin l‐chain mRNA. The mutations are rare and fast DNA scanning would facilitate diagnosis. The aim of the study was to compare the analytical performances of two fast DNA scanning techniques: denaturing high‐performance liquid chromatography (DHPLC) and double‐gradient denaturing gradient gel electrophoresis (DG‐DGGE). We analysed the sequence encoding the 5′ untranslated flanking region of ferritin l‐chain mRNA, which includes an IRE stem loop structure. The two systems unambiguously identified all the 12 accessible mutations in a single run, including the difficult C–G transversions. DHPLC and DG‐DGGE identified seven abnormal patterns in DNA samples from 47 subjects with unexplained hyperferritinaemia; all had mutations in the IRE sequence, including two not reported before: C36G and A37G. The scanning of 250 DNA samples from subjects genotyped for HFE led to the identification of four new mutations, all outside the IRE structure: C10T, C16T, C90T and del‐T156. We conclude that DHPLC, similar to DG‐DGGE, detects all the mutations in the l‐ferritin 5‘UTR sequence in a single run, and that various mutations occur outside the IRE structure.

Seven novel additional small mutations and a new alternative splicing in the human dystrophin gene detected by heteroduplex analysis and restricted RT-PCR heteroduplex analysis of illegitimate transcripts.

Around 35% of Duchenne and Becker muscular dystrophy (DMD/BMD) patients cannot be identified by techniques which identify major DMD rearrangements in the dystrophin gene. In order to characterize the gene defect in these patients, we screened 40 exons of the dystrophin gene by heteroduplex analysis on genomic DNA in 50 affected Italian males. Using conventional heteroduplex analysis and a modified heteroduplex analysis on restricted RT-PCR products of illegitimate transcripts, restricted RT-PCR heteroduplex analysis, we were able to identify 7 novel small mutations and a new alternative splicing involving exon 25 of the dystrophin gene in peripheral blood lymphocytes and skeletal muscle transcripts.

Droplet digital polymerase chain reaction for DNMT3A and IDH1/2 mutations to improve early detection of acute myeloid leukemia relapse after allogeneic hematopoietic stem cell transplantation.

Over the last decades, allogeneic hematopoietic stem cell transplantation (allo-HSCT) has considerably improved the outcome of acute myeloid leukemia (AML). Unfortunately, disease relapse remains a frequent occurrence, and a major cause of post-transplant mortality.[1][1] Most salvage treatments do

HIF1A and MIF as potential predictive mRNA biomarkers of pre-eclampsia: a longitudinal prospective study in high risk population.

Abstract Background: Pre-eclampsia (PE) is a hypertensive multisystem disorder, causing significant fetal-maternal mortality and morbidity worldwide. This study aims to define possible longitudinal predictive mRNA markers involved in the main pathogenic pathways of PE: inflammation [macrophage migration inhibitory factor (MIF)], hypoxia and oxidative stress [hypoxia inducible factor 1-α subunit (HIF1A) and β-site APP-cleaving enzyme-2 (BACE2)] and endothelial dysfunction [endoglin (ENG), fms-related tyrosine kinase-1 (FLT1) and vascular endothelial growth factor (VEGF)]. Methods: Peripheral blood was collected from 33 singleton pregnancies characterized by a high cardiovascular profile risk sampled consecutively at 6–16; 17–23; 24–30; 31–34; ≥35 weeks followed by the Obstetrics and Gynecology Unit of the San Raffaele Hospital in Milan. A real-time quantitative PCR reaction was performed on plasma RNA. Results: Of the 33 women enrolled, nine developed PE. Until 23 weeks HIF1A was significantly higher in women who later developed PE compared to women who did not (p=0.049 and p=0.012 in the first and second blood collection). In the third time interval MIF (p=0.0005), FLT1 (p=0.024), ENG (p=0.0034) and BACE2 (p=0.044) appeared to be significantly increased while HIF1A was elevated even from 24 week onwards but not reaching the statistical significance. In the fourth time interval ENG mRNA still remained increased (p=0.037). Conclusions: HIF1A, marker of hypoxia and oxidative stress, and MIF, marker of inflammation, seemed to be the most promising RNA markers, suggesting that hypoxia, principally, and inflammation may play an important role in PE pathogenesis.

COLD-PCR and microarray: two independent highly sensitive approaches allowing the identification of fetal paternally inherited mutations in maternal plasma

Background Until now, non-invasive prenatal diagnosis of genetic diseases found only limited routine applications. In autosomal recessive diseases, it can be used to determine the carrier status of the fetus through the detection of a paternally inherited disease allele in cases where maternal and paternal mutated alleles differ. Methods Conditions for non-invasive identification of fetal paternally inherited mutations in maternal plasma were developed by two independent approaches: coamplification at lower denaturation temperature-PCR (COLD-PCR) and highly sensitive microarrays. Assays were designed for identifying 14 mutations, 7 causing β-thalassaemia and 7 cystic fibrosis. Results In total, 87 non-invasive prenatal diagnoses were performed by COLD-PCR in 75 couples at risk for β-thalassaemia and 12 for cystic fibrosis. First, to identify the more appropriate methodology for the analysis of minority mutated fetal alleles in maternal plasma, both fast and full COLD-PCR protocols were developed for the most common Italian β-thalassaemia Cd39 and IVSI.110 mutations. In 5 out of 31 samples, no enrichment was obtained with the fast protocol, while full COLD-PCR provided the correct fetal genotypes. Thus, full COLD-PCR protocols were developed for all the remaining mutations and all analyses confirmed the fetal genotypes obtained by invasive prenatal diagnosis. Microarray analysis was performed on 40 samples from 28 couples at risk for β-thalassaemia and 12 for cystic fibrosis. Results were in complete concordance with those obtained by both COLD-PCR and invasive procedures. Conclusions COLD-PCR and microarray approaches are not expensive, simple to handle, fast and can be easily set up in specialised clinical laboratories where prenatal diagnosis is routinely performed.

High resolution melting for the identification of mutations in the iron responsive element of the ferritin light chain gene

Abstract Background: Among the causes of hyperferritinemia, hereditary hyperferritinemia cataract syndrome (HHCS) is an autosomal dominant disease characterized by distinctive cataracts and high serum ferritin. It is caused by mutations in the iron responsive element (IRE) of the ferritin light chain gene (FTL). Methods: To speed up and simplify mutational scanning in this genomic region, we developed a protocol based on high-resolution melting (HRM) analysis. Results: Validation was carried out using 18 wild-type and 14 DNA samples carrying different mutations, each analyzed in replicates of 20. The method allowed for correct identification and genotyping of all mutant samples, and each variant generated a specific profile distinguishable from the wild type. A 5.5% proportion of false positive results were obtained. In addition, in two patients with HHCS, two new mutations were identified by HRM based on an altered melting profile. These mutations were subsequently characterized by direct sequencing (7C>G+40A>G and 49A>C). Conclusions: The high reliability of HRM in detecting known and new DNA variations indicate that this could be an effective and sensitive method for molecular scanning of mutations in the IRE of the FTL gene in patients presenting with either HHCS or unexplained hyperferritinemia. Clin Chem Lab Med 2010;48:1415–8.

A nonsense mutation (Gln-673-Term) in exon 17 of the human dystrophin gene detected by heteroduplex analysis

Heteroduplex analysis was used to search for small mutations in a sample of 40 Italian DMD/BMB patients in whom large rearrangements were not found. A novel nonsense mutation in exon 17 of the dystrophin gene, consisting of a C to T transition, is described.