Iron refractory iron deficiency anaemia (IRIDA) is caused by mutations in the TMPRSS6 gene, which encodes transmembrane protease, serine 6 (TMPRSS6, also known as matriptase-2) expressed by the liver. TMPRSS6 belongs to the family of type II serine proteases (IISPs) that are involved in the crucial processes of remodelling extracellular matrix components and regulating cell-surface receptors, adhesion molecules and proteolytic processes (Finberg et al, 2008; Ramsay et al, 2009).
It has been recently demonstrated that TMPRSS6 is an essential regulator of iron homeostasis, as it is a physiological suppressor of hepcidin. TMPRSS6 mutations cause IRIDA, as increased hepcidin concentrations degrade intestinal ferroportin and do not allow normal iron absorption (Finberg et al, 2008; Ramsay et al, 2009). Familial and sporadic cases of IRIDA have been described with different type of TMPRRS mutations and different clinical presentations at various ages. These may range from severe anaemia with parenteral iron requirement in infancy up to isolated microcytosis and low transferrin saturation with unremarkable anaemia in adult age (Tchou et al, 2009; Pellegrino et al, 2012).
Polyendocrine autoimmune syndromes (PAS) are acquired diseases with highly variable clinical phenotype. Patients develop several autoantibodies, eventually leading to the immune-mediated destruction of endocrine organs (Eisenbarth & Gottlieb, 2004). The main recognized categories are the rare monogenic Type I, which starts in childhood with a highly variable clinical phenotype encompassing hypoparathyroidism and adrenocortical failure, and the most frequent polygenic type II, which has variable onset from infancy to adulthood, includes autoimmune Addison disease plus type 1A diabetes or thyroid autoimmunity (TA) and is more varied in its manifestations. The coexistence of more than one endocrinopathy presumably results from shared genetic susceptibility leading to loss of tolerance towards multiple tissues.
Therefore, according to the ‘splitters’ who consider each of the combination of disorders as a separate syndrome, there are also PAS type III, referring to thyroid autoimmunity plus another autoimmunity (except Addison and type 1A diabetes) and PAS type IV, referring to two or more other organ-specific autoimmune diseases (Betterle & Zanchetta, 2003; Eisenbarth & Gottlieb, 2004). In more detail, PAS type III encompasses type IIIa, the combination of TA plus type 1A diabetes; type IIIb, the combination of TA plus anti-gastric parietal-cell (anti-GPC), also reported as ‘thyreogastric syndrome’; and type IIIc, the combination of TA plus coeliac disease (Sterzl et al, 2008; Checchi et al,2010).
A clear association between iron deficiency, autoimmune gastritis and Helicobacter pylori (HP) infection has been demonstrated (Hershko & Skikne, 2009; Tozzoli et al, 2010). However, to the best of our knowledge, the association of TMPRSS6 mutations and PAS had never been reported. Following the observation of TMPRSS6 mutation in one of our patients diagnosed with IgA glomerulonephritis in the setting of iron deficiency anaemia (IDA) and PAS type IIIb, we looked for TMPRSS6 mutation in other patients with IDA and PAS type III.
Patients were selected on the basis of the following criteria: (i) presence of unexplained iron deficiency leading to diagnosis of IRIDA, documented by personal history and failure of iron oral supplement the improve the status of IDA, (ii) diagnosis of PAS type III by the occurrence of TA plus type IA diabetes (Type IIIa), anti-GPC (type IIIb), or coeliac disease (type IIIc) and (iii) absence of other autoimmune diseases. All patients were enrolled after giving written informed consent and management of personal data was approved by the Local Ethics Committee as requested by the Italian Privacy Safeguard Act. Genomic DNA was isolated from peripheral blood lymphocytes and sequence variations in TMPRSS6 were evaluated by polymerase chain reaction (PCR) and direct sequencing. Direct sequencing was performed using a fluorescence-tagged dideoxy chain terminator method in an ABI BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions.
Eight patients fulfilled the inclusion criteria (Table 1). They were all Caucasian Italian women, seven with PAS type IIIb and one with PAS type IIIc; five of them had clinically evident autoimmune thyroid disease needing drug therapy. Gastritis was diagnosed in five patients, with previous HP infection in three of them.
at genetic diagnosis
|Class of PAS||IIIB||IIIB||IIIC||IIIB||IIIB||IIIB||IIIB||IIIB|
(normal range <60 IU/l)
|Anti-peroxidase titre (normal range <60 IU/ml)||123||492·8||59||82||>1300||430||65·2||282|
Anti-gastric mucosal antibodies titre
(normal range <10 IU/l)
|Mean cell volume (fl)||77||59·6||77·8||88·9||85·0||72·3||72·4||64·9|
|Serum iron (μmol/l)||4·8||2·1||3·75||1·8||7·1||2·3||6·8||1·9|
|Transferrin saturation (%)||8||2||6||18||9||4||10||2|
|TMPRSS6 mutation|| |
T>C (variant V736A)Exon 17
T>C (variant V736A) Exon 17
T>C (variant V736A) Exon 17
T>C (variant V736A) Exon 17
Deletion 1813G (variant A605fs) Exon 15
The patients had variable degrees of anaemia with microcytosis (mean cell volume, 59·6–88·9 fl), transferrin saturation 2–18% and serum ferritin 2–14 μg/l and all needed intravenous iron infusion. In six of the eight patients, TMPRSS6 variations were found: The single nucleotide polymorphism rs855791 in exon 17, resulting in non-synonymous (V736A) change in the serineprotease domain of PMPRSS6, was found in five patients (four homozygous and one heterozygous), while one patient showed a heterozygous G deletion (1813G) in exon 15 (A605fs).
Our data first demonstrate that TMPRSS6 variations are very frequently associated with IDA in patients suffering from PAS type III. Both TMPRSS6 mutations reported here have been previously associated with IRIDA (Tchou et al, 2009), but never in the setting of PAS.
All our patients fulfilled the criteria for IRIDA and PAS type III. A complex genetic influence in the pathogenesis of autoimmune diseases is well known, as demonstrated in coeliac disease or, for instance, for specific genetic polymorphisms of the insulin gene (INS) in type 1A diabetes (Eisenbarth & Gottlieb, 2004).
Based on the results of our study we speculate that mutations or polymorphisms modulating TMPRSS6 expression, which consequently alter iron absorption at the gastrointestinal mucosal level, might be linked to acquired changes in gastrointestinal molecules/peptides. These could make them more likely to be targets when the immune system fails to maintain self-tolerance, as in PAS (Fig 1).
The main limits of these preliminary observations are the lack of a control group of healthy people, which could help establish the usual incidence of TMPRSS6 mutations within the normal population, and the lack of a control group of patients with PAS but without IDA, in order to confirm and assess the strength of the reported association between TMPRSS6 and PAS.
We are currently working on both of these aspects, systematically analysing PAS patients for IRIDA. Despite all these limits, our findings could provide new insight into the genetic factors influencing anaemia, PAS and gastrointestinal-related disorders.