DISCUSSION:
We report IRIDA in fraternal twins who presented with severe microcytic anaemia, hypoferremia, hypoferritinemia and low transferrin saturation states. Two missense mutations (p. G442R; p.E522K) in the TMPRSS6gene presenting in a compound heterozygous form were detected using massively parallel gene sequencing. The mutations sequenced in our Australian twins have been previously identified in several other IRIDA patients [4,7,12]. To date, only one other case report of a French-Canadian kindred has identified the precise compound heterozygous missense mutations of TMPRSS6 seen in our patients [7]. In parallel with the findings reported by Khuong-Quang et al. (2013) [7], our patients presented with severe hypochromic microcytic anaemia, hypoferremia and negligible transferrin saturation. Similar blood count and serum iron abnormalities have been seen in other IRIDA patients with either the p.G442R or the p.E522K missense mutation [4,12]. However, unlike the French-Canadian siblings [7], our twins showed borderline normal to low serum ferritin levels and failed to respond to simple oral iron replacement therapies. It was only after the administration of parenteral iron infusions that profound and sustained improvements in the haemoglobin and serum ferritin of our patients were observed.
Reasons for these clinical differences despite a similar genotype has been a contentious issue within the IRIDA arena. Genotype-phenotype studies of a large European cohort of IRIDA patients across multiple unrelated families have proposed that missense mutations affecting theTMPRSS6 mutation through a compound heterozygous phenomenon typically present with less severe anaemia and milder hypoferremia [6]. In addition, they usually respond to oral iron therapy in comparison to patients with single homozygous mutations or other lesion types (e.g. frameshift, nonsense). However, this appears to be at odds with our patients. The clinical heterogeneity of IRIDA been attributed to diverse TMPRSS6 mutations affecting variable domains within the encoded MT-2 product, leading to different degrees of dysfunction [6,12]. The p.G442R mutation of the TMPRSS6 gene is known to target and alter the activity of the second C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1 (CUB) domain of MT-2, which is involved in its autoactivation through autocatalytic cleavage [4,6,12]. In contrast, the p.E522K mutation disrupts the second class A low-density lipoprotein receptor (LDLRA) domain further downstream [6,12]. This domain has been proposed to assist with the conformational folding of MT-2 to achieve autocatalytic cleavage, whilst also facilitating interactions between hemojuvelin and MT-2. Silvestri and co-workers (2009) [12] showed through a series of separately transfected HeLa cell lines with multiple missense mutations targeting the TMPRSS6 gene, including p.G442R and p.E522K, that the latter was more mutagenic. LDLRA domain alterations propagated through the p.E522K lesion showed reduced expression of MT-2 on the cell membrane of transfected cells and was unable to interfere with hemojuvelin [12]. In contrast, the p.G442R mutation and subsequent aberrations in the CUB domain did not alter the expression of the MT-2 protein on the cell membrane [12]. Moreover, CUB mutations still retained partial, albeit lowered hemojuvelin cleavage capabilities [12]. A compound heterozygous inheritance mechanism for both missense mutations may therefore cause a severe IRIDA phenotype that does not respond to oral iron therapy through complete dysfunction of the mutated MT-2 encoded product.
Of interest, we observed that despite a reasonable trial of oral iron replacement therapy, neither child showed any clinical improvement in red cell indices or serum iron levels. Previously published reports of IRIDA patients with missense mutations affecting the CUB and LDLRA domains, including the French-Canadian kindred, have successfully treated their patients with oral iron supplements [6,7]. Interestingly, the French-Canadian siblings only showed clinical improvements after one year of oral iron therapy in combination with ascorbic acid [7]. The shorter time course and exclusive use of oral iron supplementation without ascorbic acid may account for the lack of response seen in our patients. Nevertheless, parenteral iron infusions are reported to yield clinical benefits. It has been previously shown that recovery of red cell indices and ferritin is often slow and prolonged with an ongoing need for parenteral iron administration [2,6]. Surprisingly, we observed marked and sustained improvements in red cells indices and ferritin levels in our patients after a single iron infusion.
In summary, we have reported IRIDA in paediatric, fraternal twins with compound heterozygous missense mutations in the TMPRSS6 gene. We show that despite knowledge of these TMPRSS6 mutations [4,6,7,12], they confer extensive clinical heterogeneity. Therapy should therefore be directed on a case-by-case basis. Currently, the frequency of parenteral iron administration in IRIDA cases refractory to oral iron replacement therapy is not established. Future prospective cohort studies will hopefully elicit appropriate therapeutic guidelines for IRIDA.