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Two novel mutations in the tmprss6 gene associated with iron‐refractory iron‐deficiency anaemia (irida) and partial expression in the heterozygous form

2012; Wiley; Volume: 158; Issue: 5 Linguagem: Inglês

10.1111/j.1365-2141.2012.09198.x

1365-2141

Rosa Maria Pellegrino, Maria Francisca Coutinho, Domenico Giuseppe D’Ascola, Ana M. Lopes, A Palmieri, Francesca Carnuccio, Mónica Costa, Gabriella Zecchina, Giuseppe Saglio, Emília Costa, José Barbot, Graça Porto, Jorge P. Pinto, Antonella Roetto,

Erythropoietin and Anemia Treatment

Iron-refractory iron-deficiency anaemia (IRIDA, Online Mendelian Inheritance in Man number 206200) is an autosomal recessive genetic disorder characterized by iron deficiency anaemia unresponsive to oral iron treatment but partially responsive to parenteral iron therapy (Finberg, 2009). IRIDA is due to mutations in the TMPRSS6 gene, which encodes the serine-protease matriptase 2 (Finberg et al, 2008), an inhibitor of the iron-related hormone, hepcidin. Several mutations in the TMPRSS6 gene have been characterized in IRIDA families of different ethnic origins. Recent findings on Tmprss6-haploinsufficient mice support the hypothesis that susceptibility to iron deficiency may be increased by the presence of TMPRSS6 mutations even in the heterozygous state (Nai et al, 2010). Here we present two new TMPRSS6 variants that were associated, in the heterozygous form, with manifest IRIDA in two un-related families. In the Italian Family 1, the proband was a 9-year-old girl with microcytic anaemia, low serum iron and normal ferritin (Fig 1A, C). She had no response to iron oral therapy but she responded to i.v. iron therapy, reaching haemoglobin values stably higher than 110 g/l with ferritin values around 440 pmol/l. The molecular study of family members revealed the absence of HBA1/HBA2 and HBB gene defects. The proband's mother also presented blood parameters consistent with iron-deficient anaemia. She referred mild hyper-menorrhea but was negative for the most common malabsorption causes. She was treated with oral iron, with poor results (haemoglobin 75 g/l vs. 91 g/l after 5 months oral iron therapy). In the Portuguese Family 2, a 9-year-old girl was diagnosed with microcytic anaemia, low serum iron and normal ferritin (Fig 1B, C). After no response to oral iron therapy, she started with i.v. iron, with no response (haemoglobin increase 5 g/l, with no reticulocytosis). Ferritin increased significantly (360 pmol/l) but iron remained low. Specific tests excluded other causes of iron-deficiency anaemia, such as malabsorption and bleeding, and the common alpha/beta thalassemia mutations. Her mother had a non-symptomatic mycrocitic and hypochromic anaemia (Fig 1C). Again, specific tests excluded secondary causes of iron deficient anaemia or thalassemia trait (including common alpha/beta thalassemia mutations). The father had hyperferritinaemia, probably due to heavy alcohol consumption. A microcytic anaemia refractory to oral iron therapy was also documented in a 6-year-old maternal cousin. Serum hepcidin-25 values and serum transferrin saturation/hepcidin-25 ratio, a marker for IRIDA, showed values below the reference for both the proband and her mother (Fig 1C). After all family members provided informed consent, DNA was extracted from peripheral blood. TMPRSS6 exons and exon-intron boundaries were amplified and automatically sequenced. HBA1/HBA2 and HBB molecular analysis was performed with a reverse dot blot kit (Nuclear Laser Medicine Strip Assay, Italy). The effect of mutations on TMPRSS6 mRNA maturation was predicted utilizing the alamut (Interactive Biosoftware, Rouen, France) and the Human Splicing Finder (Desmet et al, 2009) software. For expression analysis of mutated transcripts, total RNA was extracted from peripheral blood mononuclear cells, reverse-transcribed and amplified by qualitative or quantitative real time polymerase chain reaction (PCR). A previously reported mutation in exon 8 (1025 C>T, S304L) (De Falco et al, 2010) was found in heterozygosity in Family 1 proband (Fig S1), which was also present in her father and brother (Fig 1A). On the other patient's allele, a 9 bp deletion was identified in heterozygous form at the end of intron 8 (IVS8-1433 Δ9) (Fig S1), which was inherited from her mother (Fig 1A). In silico analysis predicted that the deletion abolishes the constitutive splice site and favours the use of a cryptic site 17 bp upstream (Fig 2A). As a consequence of this, 8 bp are added to the coding sequence, causing a frameshift and the creation of a premature stop codon (Fig 2B). Real time PCR analysis revealed the presence of two mRNA forms in the proband, one of which was found at lower amounts compared to the other (Fig S2). This is probably due to the faster degradation of the mutated mRNA in comparison to the wild-type. A heterozygous mutation at the end of intron 15 (c. 1869-21 C>G) was identified in the proband from Family 2 (Fig S1), which was inherited from her mother (Fig 1B). Two synonymous polymorphisms (rs11704654 and rs2235321, corresponding to Pro32 and Tyr738 respectively), previously associated with IRIDA (Delbini et al, 2010) were also found, both of which were also present in the proband's father. In silico analysis of the effect of the c. 1869-21 C>G mutation predicted the creation of a new splicing site, approximately 50% stronger than the native site, localized 21 bp downstream (Fig 2A). The mutated mRNA would contain an extra 20 bp, causing a frameshift and the creation of a premature stop codon. As a consequence, the new protein would lack 96% of the serine protease domain (Fig 2C). Analysis of mRNA expression using primers amplifying the mutated locus showed the presence of two mRNA forms differing by approximately 20 bp, while amplification with primers specific for the mutation was observed only for the proband and her mother (Fig S2). The characterization of new mutations in the TMPRSS6 gene in two young patients with IRIDA further confirms the allelic heterogeneity of this disorder. In Family 1, the presence of the IVS8-1433Δ9 variation at the heterozygous state in the mother was associated with a consistent anaemia that was resistant to oral iron therapy and not completely explainable by secondary causes. In Family 2, the new 1869-21 C>G mutation is associated with two iron deficiency-related polymorphisms both in the proband and her symptomatic cousin. The two polymorphisms alone cannot be accountable for the disease, as these are both present in the proband's asymptomatic father. Thus, it is apparent that the development of IRIDA symptoms in Family 2 needs the presence of the 1869-21 C>G mutation. Curiously, the proband's mother, who carries the 1869-21 C>G mutation, but not the two polymorphisms, although asymptomatic, has a picture of mild iron-deficient anaemia. In conclusion, the data suggest that although heterozygous TMPRSS6 mutations may not be able to induce a clear IRIDA phenotype, some of them may increase the susceptibility to iron deficiency. This work was supported by grants from FCT (PTDC/SAU-MET/113011/2009), the American Portuguese Biomedical Research Fund (APBRF), and the INNOVA foundation. RMP and MC collected clinical data and performed the molecular analysis, DDA, GZ; E Costa and JB referred patients for molecular diagnosis; AML, AP, MC and FC performed DNA and RNA extraction and sequencing, GP, JPP and AR coordinated experiments and wrote the paper, GS read the manuscript and added critical suggestions. All the authors declare no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.