Iron Refractory Iron Deficiency Anemia: Presentation With Hyperferritinemia and Response to Oral Iron Therapy

Abstract

Iron-refractory iron-deficiency anemia (IRIDA) is an autosomal recessive disorder caused by mutations in TMPRSS6. Patients have hypochromic microcytic anemia refractory to oral iron and are only partially responsive to parenteral iron administration. We report a French-Canadian kindred in which 2 siblings presented in early childhood with severe microcytic anemia, hypoferremia, and hyperferritinemia. Both children have been successfully treated solely with low-dose oral iron since diagnosis. Clinical and biological presentation did not fit any previously described genetic iron-deficiency anemia. Whole exome sequencing identified in both patients compound heterozygous mutations of TMPRSS6 leading to p.G442R and p.E522K, 2 mutations previously reported to cause classic IRIDA, and no additional mutations in known iron-regulatory genes. Thus, the phenotype associated with the unique combination of mutations uncovered in both patients expands the spectrum of disease associated with TMPRSS6 mutations to include iron deficiency anemia that is accompanied by hyperferritinemia at initial presentation and is responsive to continued oral iron therapy. Our results have implications for genetic testing in early childhood iron deficiency anemia. Importantly, they emphasize that whole exome sequencing can be used as a diagnostic tool and greatly facilitate the elucidation of the genetic basis of unusual clinical presentations, including hypomorphic mutations or compound heterozygosity leading to different phenotypes in known Mendelian diseases.

The main causes of iron-deficiency anemia are blood loss or an inadequate dietary iron intake. However, several genetic defects can lead to ineffective iron intestinal absorption or impaired iron recycling by macrophages, the 2 main processes that fulfill iron needs in mammals. Iron-refractory iron deficiency anemia (IRIDA; Online Mendelian Inheritance in Man database #206200) is an autosomal recessive disorder caused by loss-of-function mutations in TMPRSS6,¹ first described in 1981. Patients exhibit severe, congenital hypochromic, microcytic anemia with low serum iron and transferrin saturation that occurs in infancy and is refractory to oral iron treatment and partially recovered after parenteral iron administration.² They also show an inappropriate elevation of hepcidin, a circulating hormone that inhibits iron duodenal absorption and macrophage iron recycling. To date, >30 TMPRSS6 mutations have been identified in patients without any common ethnic or geographic distribution,^1,3–14 suggesting that TMPRSS6 mutations might be underestimated in patients with iron-deficiency anemia. Here, we report a family in which whole exome sequencing (WES) identified compound heterozygous TMPRSS6 mutations in 2 siblings with iron deficiency anemia that differed clinically and biologically from classic IRIDA.

Patient Presentation

Written informed consent and assents were obtained from the patients and their guardians for whole exome sequencing and publication of this case report.

In 1999, a 3-year-old boy was referred to our clinic at the Montreal Children’s Hospital for investigation of a microcytic hypochromic anemia diagnosed after symptoms of fatigue and abdominal pain. His growth and development were normal, and no anterior hemoglobin (Hb) measurement was available. He was of French Canadian descent with no known parental consanguinity. His parents and older sister had normal complete blood counts, reticulocyte counts, and ferritin levels. Laboratory values, described in Table 1, showed a severe microcytic, hypochromic anemia (Hb of 75 g/L, mean corpuscular volume [MCV] of 64 fL), low serum iron, low fractional transferrin saturation, and normal transferrin levels. Unexpectedly, the serum ferritin level was elevated at 348 µg/L, well above the normal range of 6 to 110 µg/L. Hb electrophoresis did not detect a hemoglobinopathy, there was no clinical or biological evidence of chronic inflammatory state, and gastroenterological investigations provided no evidence for occult blood loss or malabsorption. His diet was well diversified. During a course of oral iron supplementation (6–10 mg/kg/day of elemental iron) for 1 year, the proband’s symptoms disappeared, and he experienced a slow rise of Hb up to 119 g/L with normalization of the MCV. The transferrin saturation remained low (0.07), as did the circulating iron level at 4 µmol/L. However, ferritin rose up to 654 µg/L. The time course of hematologic and iron parameters under treatment is summarized in Table 2. After 9 years of follow-up, the patient has had a normal growth curve, normal physical activities, and improved Hb levels. On the basis of the increase in ferritin levels after oral iron therapy, oral iron supplementation has been continued with the dose tapered to 3.5 mg/kg per week, which has maintained ferritin levels similar to those seen at diagnosis and the Hb >110 g/L. The patient never required erythrocyte transfusion or intravenous iron administration.

TABLE 1.

TMPRSS6 Genotype and Hematologic and Biochemical Parameters in Patients, Their Parents, and Their Unaffected Sister

TMPRSS6 genotype	Pediatric reference range for patients 1 and 2	Proband, 3 y, p.G442R/p.E522K	Affected sister, 2 y, p.G442R/p.E522K	Father, 46 y,a WT/p.E522K	Mother, 45 y,a WT/p.G442R	Unaffected sister, 17 y,a WT/p.E522K
WBC	5.5–15.5 19^9/L	8.6	10.2	6.39	6.05	7.3
RBC	3.90–5.30 10^12/L	4.31	5	4.81	3.91	4.43
Hb	115–135 g/L	75	76	145	121	131
Hct	0.340–0.400	0.276	0.249	0.413	0.352	0.386
MCV	75.0–87.0 fL	64.1	49.7	85.9	90	87
MCH	24.0–30.0 pg/cell	17.4	15.1	30.2	30.9	29.6
MCHC	310–370 g/L	271	304	351	343	341
RDW	11.4% to 14.6%	16.9	22.4	13.2	13.4	14.1
Platelet	140–450 10^9/L	292	616	223	250	271
Reticulocytes	10^9/L	0.024	0.045	0.046	0.025	0.046
Sedimentation rate	3–13 mm/h	8	8	NA	NA	NA
Iron	4–25 µmol/L	<1	1.6	12.3	15.5	19.6
Transferrin	2.00–3.70 g/L	2	2.14	2.34	2.93	3.17
Fractional Transferrin saturation	0.20–0.55	0.02	0.07	0.21	0.21	0.24
Ferritin	6–110 µg/L	348	195	112	36	15
	Reference range	Proband, 15 y	Affected sister, 12 y	Father, 47 y	Mother, 46 y
Hepcidin	17–260 ng/mL	142.4	198.6	116.1	56.4
Hb		115	94
Iron		2.6	1.6

Parameters at diagnosis are shown in the upper panel. Mutations are described at the protein level. Plasma hepcidin was measured in 2011 while patients were receiving oral iron supplementation (3.5 mg/kg/wk). Results are shown in the lower panel along with Hb and serum iron. Hct, hematocrit; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; NA, not available; RBC, red blood count; RDW, red cell distribution width; WBC, white blood count; WT, wild type.

^aResults for both parents and their healthy sister were within the normal range.

TABLE 2.

Time Course of Hematologic and Biochemical Parameters in Probands Under Oral Iron Supplementation

Date	Tx duration, mo	Patient age, y	OEI suppl	Hb, g/L	MCV, fL	RDW, %	Retic, 10^9/L	Iron, µmol/L	Ferritin, µg/L	Transf, g/L	Transf saturation
9/16/1999	—	3	6 mg/kg/d	75	64.1	16.9	0.024	<1	348	2.00	0.02
1/19/2000	4	3	↑ up to 10mg/kg/d	100	64	16.4	0.050	3,0	217	2.24	0.05
10/14/2000	12	4	Stop Tx	112	71.9	14.9	ND	ND	ND	ND	ND
11/22/2000	—	4	Restart at 12 mg/kg/d	104	72.8	14.6	ND	ND	325	ND	ND
9/12/2001	9	5	Stop Tx	112	80.3	12.9	ND	ND	ND	ND	ND
5/3/2002	—	5	Restart at 10 mg/kg/d	101	73.4	13.6	ND	2.0	291	1.73	0.05
6/12/2002	1	6	↓ down to 6 mg/kg/d	107	73.3	15.1	0.068	3.0	379	2.16	0.05
9/6/2002	4	6	Stop Tx	113	75.5	13.8	ND	ND	ND	ND	ND
3/28/2003	—	6	Restart at 9 mg/kg/d	102	69.5	16.4	ND	4.0	376	ND	ND
11/3/2003	7	7	Stop Tx	114	75.7	14.6	0.066	4.0	654	2.12	0.07
5/11/2004	—	7	No Tx	109	81.6	14.5	ND	2.0	467	2.14	ND
10/18/2004	—	8	Restart at 4 mg/kg/d	106	73.6	15.3	0.057	2.0	391	2.06	0.04
3/17/2006	16	9	Stop Tx	119	78	14.3	ND	ND	ND	ND	ND
8/18/2006	—	10	Restart at 6 mg/kg/d	103	74.9	15.5	ND	3.1	439	2.07	0.06
10/26/2007	14	11	↓ down to 1.2 mg/kg/d	119	76.1	14.6	ND	5.0	598	ND	ND
5/23/2008	21	12	↓ down to 1.1 mg/kg/d	113	73.1	15.6	0.057	4.1	390	ND	ND
11/20/2009	39	13	↓ down to 0.7 mg/kg/d	119	72.2	17.2	ND	ND	512	ND	ND
12/17/2010	51	14	↓ down to 0.5 mg/kg/d	109	67.2	18.9	0.038	3.2	168	2.24	0.06
12/20/2011	64	15	0.5 mg/kg/d	115	66.8	19.1	ND	2.6	ND	ND	ND

ND, not determined; OEI suppl, oral elemental iron supplementation; RDW, red cell distribution width; Retic, reticulocytes; Transf, transferrin; Tx, treatment; ↑, increase; ↓, decrease.

The proband’s youngest sister presented with similar symptoms at age 2 years and also had microcytic anemia, hypoferremia, and high ferritin levels (Table 1). She was initially treated with 6 mg/kg per week of oral iron with elimination of symptoms; the oral iron dose was tapered to 3.5 mg/kg per week, also in response to a rising ferritin level during iron therapy. She is now aged 12 years and, like her brother, has a normal growth curve; she has a normal Hb level when she is compliant with her iron therapy. Notably, several trials to stop oral iron therapy were attempted in both siblings during periods ranging from 2 to 12 months. All resulted in increased fatigue hampering normal daily activities and significant drops in the Hb levels, leading us to resume oral iron therapy.

The pedigree suggested autosomal recessive transmission, and the clinical presentation of microcytic anemia, hypoferremia, and hyperferritinemia, which responded to oral iron supplementation with a concomitant increase in serum ferritin did not suggest any previously described inherited form of iron-deficiency anemia. In congenital hypotransferrinemia, serum transferrin is low.¹⁵ Patients with mutated SLC11A2 or glutaredoxin 5 deficiency exhibit hyperferremia.^16,17 Patients with IRIDA show no hematologic correction after oral iron. We thus performed WES on genomic DNA from both siblings, a strategy proven by us and others to identify novel genes associated with rare disorders.¹⁸ We identified compound heterozygous mutations in TMPRSS6 in both patients, causing p.G442R and p.E522K. To see if the unusual clinical presentation could be associated with a coding polymorphism, we searched the exome data for rare variants (allele frequency <1%) in genes known to be involved in iron metabolism (Supplemental Table 4). However, we found no uncommon polymorphisms in the probands for any of these genes. Other family members each carried 1 mutation.

Discussion

We describe here a family in which WES identified compound heterozygous TMPRSS6 mutations in the 2 siblings affected with iron deficiency anemia. The clinical presentation differed from classic IRIDA in that ferritin levels were high at diagnosis and the anemia in both children has been responsive to low-dose oral iron with >9 years of follow-up.

These 2 missense mutations have been previously reported in patients with the IRIDA phenotype who required parenteral iron.^1,6 To our knowledge, this specific compound heterozygosity is described here for the first time, in association with this atypical clinical and biological presentation of iron-deficiency anemia (Tables 1 and 2). TMPRSS6 encodes the hepatic transmembrane serine protease matriptase-2. p.G442R affects the matriptase-2 CUB domain, and p.E522K affects the second low-density lipoprotein receptor class A domain. Matriptase-2 inhibits hepcidin expression by cleaving hemojuvelin, a membrane-associated protein that promotes bone morphogenetic protein signaling in hepatocytes. In vitro, matriptase-2 p.G442R mutants show a partial defect in hemojuvelin cleavage, whereas p.E522K mutants are fully defective.⁶

Reported IRIDA cases show low or normal ferritin levels at diagnosis that increase after parenteral iron (Table 3). In our patients, however, ferritin was high at diagnosis and increased with oral iron treatment. Moreover, whereas typical IRIDA patients are refractory to oral iron, both patients were effectively treated exclusively by oral supplementation. Review of the literature indicates that the clinical and biological presentations of the siblings we report herein are unique (Table 3). Cau et al¹² report a 5-month-old Sardinian female with IRIDA and homozygous TMPRSS6 mutation who had normal ferritin at diagnosis. Unlike the patients in our study, this patient failed to respond to oral iron. Interestingly, after showing a partial response to intravenous iron, she responded to a combination of oral iron and ascorbic acid. Notably, other family members carrying the same homozygous TMPRSS6 mutation did not respond to oral iron. Beutler et al⁹ described a Belgian family in which the proband with compound TMPRSS6 heterozygous mutations was diagnosed at age 8 years when the Hb was 90 g/L and the ferritin was 20 ng/mL. The proband and his affected sibling both showed a partial response to oral iron. However, in contrast to the patients in our study, neither developed hyperferritinemia with long-term oral iron therapy.

TABLE 3.

Literature Review of Previously Reported Cases of IRIDA

Study	Year	Mutation 1	Mutation 2	Ethnic origin	Age,a y	Route of iron admin and additional therapies	Hb,a g/L	Ferritin,a µg/L	Ferritin under Tx, µg/Lb	Follow-up, y/age at Dx, y
Our study	2012	p.G442R	p.E522K	French Canadian	3	PO	75	348	654	FUP 13
		p.G442R	p.E522K	French Canadian	2	PO	76	195	309	FUP 11
Finberg et al1	2008	p.K636fs	p.K636fs	Turkish	6	IV	88	NAc	NA	NA
		p.A605fs	p.E527fs	Northern European	1.1	IV	92	NAc	NA	NA
		p.G713fs	unknown	Nigerian	1.4	IV	70	NAc	NA	NA
		p.G442R	p.D521N	Northern European	11	IV	82	NAc	NA	NA
		p.R774C	unknown	African American	7	IV	75	NAc	NA	NA
		IVS15-1 G>C	p.D622fs	Nigerian	3	IV	97	NAc	NA	NA
		p.Y355X	p.E461fs	African American	1.3	IV	79	NAc	NA	NA
Guillem et al3	2008	p.Y393X	p.R599X	English	1.5	IV	60	11	109	FUP 12
Melis et al4	2008	IVS6+1 G>C	IVS6+1 G>C	Sardinian	0.7–1	IV	100d	NA	53	NA
		IVS6+1 G>C	IVS6+1 G>C	Sardinian	14	IV	91d	116d	413	Dx between 0.7 and 1
		IVS6+1 G>C	IVS6+1 G>C	Sardinian	NA	IV	100d	NA	129	NA
		IVS6+1 G>C	IVS6+1 G>C	Sardinian	NA	IV	128d	NA	184	NA
		IVS6+1 G>C	IVS6+1 G>C	Sardinian	0.7–1	IV	139d	NA	466
Ramsay et al5	2009	p.A118D	p.P686fs	Spanish	15	IV + EPO	98d	123d	365	Dx at 1
		p.A118D	p.P686fs	Spanish	Twin sister	IV + EPO	1219d	388d	586	Dx at 1
Silvestri et al6	2009	p.D521N	p.E522K	French	0.8	PO (failure) then IV	10	4	180	FUP 6
Edison et al7	2009	IVS17(-1)G>C	IVS17(-1)G>C	Indian	35	IV	66d	20d	NA	Dx at 20
Techou et al8	2009	p.S304L	p.K478_K508del	Swiss	3	IV + fresh-frozen plasma	77d	46d	310	Dx at 0.3
		IVS15-1 G>C	IVS15-1 G>C	Italian	53	PO (failure)	114d	68d	NA	Dx at birth
Beutler et al9	2010	p.L674F	IVS13+1 G>A	Belgian	8	PO	90	20	70	FUP 8
		p.L674F	IVS13+1 G>A	Belgian	0.8	PO	82d	41d	33	FUP 15
		p.L166X+36	p.L166X+36	Dutch	6	IM	62.8	16	323	FUP 11
Altamura et al10	2010	p.Y141C	p.Y141C	Lebanese	10	PO (failure)	79d	86d	NA	Dx at 2
De Falco et al11	2010	p.Y141C	p.Y141C	Indian	8	IV	91	26d	25	Dx at 1.3 y
		p.I212T	p.R271Q	Italian	3	IV	80	25d	NA	NA
		p.S304L	p.S304L	Arabian	6	IV	80	112d	NA	NA
		p.S304L	p.S304L	Arabian	4	IV	85	32d	133	NA
		p.S304L	p.S304L	Arabian	2	IV	80	50d	113	NA
		p.L166fs	p.Q229fs	Austrian	3	IV	71	10	74	NA
		p.W247fs	p.W247fs	Greek	2.5	IV	58	8d	NA	NA
		p.W247fs	p.W247fs	Greek	2	IV	54	19d	NA	NA
		p.S561X	p.S561X	Arabian	7	IV	80	86	NA	NA
		p.S561X	p.S561X	Arabian	5	IV	88	101d	NA	Dx at 8
		p.S561X	p.S561X	Arabian	2	IV	79	38	NA	NA
		p.C510S	S570fs	Algerian	9	IV	104	228d	NA	Dx at 1.8
Cau et al12	2011	IVS6+1 G>C	IVS6+1 G>C	Sardinian	0.4	PO (failure), then IV, then PO with ascorbic acid	75	102	450 (IV)	FUP 2
Sato et al13	2011	p.K253E	p.K253E	Japanese	27	IV	108	4.3	35.1	NA
Choi et al14	2011	p.G603R	IVS6+1G>T	Korean	2	PO (failure) then IV	70	42d	106 (IV)	NA

Mutations are described at the protein level, except for the ones affecting splicing sites. Dx, diagnosis; EPO, erythropoietin; FUP, follow-up; IM, intramuscular; IV, intravenous; NA, not available; PO, per os; Tx, treatment.

^aAt evaluation.

^bHighest level reported.

^cIn the kindreds reported by Finberg et al¹, ferritin levels were reported to be low-normal, although specific ferritin values were not included.

^dHb and/or ferritin levels were measured under iron therapy. Age of patients at the time of diagnosis is included in the comments.

In these specific cases, iron absorption may be less impaired than reported IRIDA patients, perhaps because of the combined residual function of these particular mutant alleles. These phenotypic differences might also reflect modifier genes that promote iron uptake by enterocytes or reduce hepcidin release by hepatocytes.

Plasma hepcidin was assessed during iron therapy when Hb was improved but hypoferremia persisted. Thus, even if seemingly within normal range for the patients, hepcidin appeared inappropriately elevated relative to serum iron level. Comparison of hepcidin deregulation in our patients to reported IRIDA cases is, however, complicated by likely differences in erythropoiesis and hepatic iron stores, stimuli known to modulate hepcidin transcription. Because macrophages are a major source of serum ferritin,¹⁹ our patients’ presenting hyperferritinemia could reflect greater hepcidin sensitivity in macrophages versus enterocytes, in keeping with previous studies.²⁰

Recently, a single nucleotide polymorphism encoding V736A in the TMPRSS6 catalytic domain was found in several genome-wide association studies^21,22 to correlate with decreased Hb and serum iron in healthy populations. Our study suggests that TMPRSS6 sequence variants lead to a spectrum of matriptase-2 dysfunction, including severe loss-of-function mutations causing classic IRIDA, hypomorphic mutations as seen in our patients and potentially similar atypical ones, and the mild reduction in matriptase-2 activity associated with the common V736A SNP.²³ Accordingly, our results suggest that genetic testing for TMRSS6 mutation have clinical utility in cases of hypochromic, microcytic anemia with hypoferremia that do not exhibit the classic IRIDA phenotype. We suggest that TMPRSS6 sequencing should be considered in a subset of patients presenting with iron deficiency anemia of unknown cause in which blood loss, inadequate dietary intake, and chronic inflammatory conditions have been ruled out (see online Supplemental Fig 1 for a diagnostic algorithm). TMPRSS6 sequencing is available in several CLIA (clinically accredited) certified laboratories. Although hepcidin measurement is not yet widely available as clinical test, we note that the finding of a markedly reduced hepcidin level in the setting of iron deficiency anemia would indicate the anemia is unlikely to be attributed to TMPRSS6 mutation.

Our results further emphasize that next generation sequencing technologies, particularly WES, greatly facilitate the elucidation of the genetic basis of unusual clinical presentations exhibiting Mendelian inheritance, including hypomorphic mutations leading to different phenotypes. Clinical application of WES in undiagnosed clinical conditions has already been shown to be feasible, yielding an encouraging 50% rate of success in uncovering an underlying genetic defect in select clinical cases in which the probability of a genetic origin is high.²⁴ There is growing interest in its introduction into the clinic to aid in the diagnosis of conditions for which no genetic cause can be found with targeted testing. Although the mutations identified currently require validation in a Clinical Laboratory Improvement Amendments–certified laboratory, this technology is becoming accessible to clinicians through academic consortia (Finding of Rare Disease Genes in Canada[FORGE Canada], Broad Institute, etc.) or private companies that offer the possibility of sequencing the exome and performing the analysis. The broader use of WES will expand the range of clinical phenotypes associated with mutations in known disease genes.

Glossary

Hb

hemoglobin

IRIDA

iron refractory iron deficiency anemia

MCV

mean corpuscular volume

WES

whole exome sequencing