Ellis J. Neufeld, MD, PhD, Division of Hematology and Oncology, Children's Hospital, Enders 720, 300 Longwood Avenue, Boston MA 02115, USA. E-mail: ellis.neufeld@tch. harvard.edu
Thiamine-responsive megaloblastic anaemia (TRMA) syndrome with diabetes and deafness was found in two patients from a Tunisian kindred. The proband was homozygous for a novel mutation, 287delG, in the high-affinity thiamine transporter gene, SLC19A2. We demonstrated that fibroblasts from this patient exhibited defective thiamine transport. These data confirm that the SLC19A2 gene is the high-affinity thiamine carrier and that this novel mutation is responsible for TRMA syndrome.
Treatment with pharmacological doses of thiamine (25–100 mg/d) usually corrects haemoglobin levels, although macrocytosis persists. A few patients, however, were refractory to thiamine (Raz et al, 1998). Diabetes mellitus may respond to thiamine therapy, as evidenced by a decreased insulin therapy in some patients, but long-term follow-up of two patients documented a slow progression of the pancreatic β-cell insufficiency with ultimate insulin dependence (reviewed in Valerio et al, 1998). All reported patients have had profound sensorineural deafness in early childhood. It is not clear if early thiamine therapy could arrest this process.
The disease gene was mapped to chromosome 1q23.2–23.3 by homozygosity mapping (Neufeld et al, 1997). Studies of additional families from different ethnic origins confirmed linkage to this locus and shortened the critical interval for the TRMA gene (Raz et al, 1998; Banikazemi et al, 1999).
Defects in thiamine transport were first demonstrated in red blood cells of TRMA patients (Poggi et al, 1984, 1989; Rindi et al, 1992, 1994). Nanomolar affinity thiamine transport is defective in fibroblasts from affected patients. Thiamine deprivation leads to apoptotic cell death in fibroblasts from patients but not from controls (Stagg et al, 1999). However, transfection of TRMA fibroblasts with the yeast thiamine transporter gene THI10 allowed them to grow in a milieu depleted of thiamine (Stagg et al, 1999). The TRMA gene, SLC19A2, was identified using candidate-gene methods by Fleming et al (1999), who demonstrated that it encodes a functional thiamine transporter and found mutations in two affected kindreds. Simultaneously, two additional groups used positional cloning to demonstrate TRMA defects in the SLC19A2 gene (Diaz et al, 1999; Labay et al, 1999). Substrate-specific assays indicated that the SLC19A2 protein is specific to thiamine (Dutta et al, 1999).
In this study, we describe a novel point mutation in the SLC19A2 gene that co-segregated with the disease phenotype in two TRMA patients, and show that the nucleotide change is the disease-causing mutation through functional studies.
Patients and methods
Patients A consanguineous Tunisian family with two affected individuals was studied (pedigree, Fig 1). Medical histories and physical examination were obtained, and skin biopsies and blood samples were drawn from these individuals after informed consent in accordance with the guidelines of local institutions.
Genotyping and mutation screening Genomic DNA was prepared from pelleted leucocytes using standard procedures (Sambrook et al, 1989). Genotyping was performed using six polymorphic markers spanning 16 cM of the TRMA critical region as follows: Cent-D1S2844-D1S2762-D1S196-D1S2658-D1S2851-D1S2786-Tel (Neufeld et al, 1997). Genomic DNA from all family members was used to construct haplotypes. The six exons and exon/intron boundaries were amplified by polymerase chain reaction (PCR) using genomic DNA from a TRMA homozygous patient, an obligate carrier and a wild-type control. Amplification was made using primer sets, as designed previously (Fleming et al, 1999). PCR fragments were purified with Qiaquick PCR purification kit (Qiagen) and sequenced on both strands using the PCR primers and the standard automated sequencing method.
Cell culture and thiamine uptake assays Fibroblasts were grown from skin samples in thiamine-replete α-minimal essential medium (α-MEM; 1 mg/l thiamine; Gibco BRL, Grand Island, NY, USA) with 15% fetal calf serum (FCS), 50 U/ml penicillin and 300 μg/ml streptomycin sulphate (Gibco BRL) in 5% CO2 at 37°C. α-MEM minus thiamine (thi-medium) with 15% dialysed FCS (Gibco BRL) was used for thiamine starvation. Experiments shown were performed on TRMA homozygous, heterozygous and normal fibroblasts.
Assays were carried out as previously described (Stagg et al, 1999). Cells were washed three times in thi-medium. One day after plating, thiamine uptake was determined by incubating the cells for 30 min at 37°C in thi-medium containing 23 nmol/l [3H]-thiamine hydrochloride (specific activity: 555 GBq/mmol, American Radiolabeled Chemicals, St. Louis, MO, USA). In unlabelled thiamine competition assays, thiamine hydrochloride was used at a final concentration of 3 μmol/l. After cellular labelling, cells were washed three times with phosphate-buffered saline (PBS) and harvested with 0·05% trypsin-EDTA (Gibco BRL). The amount of radioactive incorporation was determined by liquid scintillation counting in 5 ml of Ecolume scintillation fluid (ICN Radiochemicals, Costa Mesa, CA, USA) using a Beckman LS3801 instrument (Beckman Instrument, Palo Alto, CA, USA). Experiments were carried out in duplicate for all three patients and experimental results were confirmed by repeated assays.
Proband (case II-4 in Fig 1): a native Tunisian girl, the daughter of first-cousin parents, presented with diabetes mellitus aged 5 years. She was known to have bilateral sensorineural deafness since the age of 2 years, and had been found to be severely pancytopenic at age 8 months. Anaemia was macrocytic, with haemoglobin levels ranging from 2–6 g/dl and a mean cell volume (MCV) of 93–103 fl. She was treated with repeated transfusions. Thrombocytopenia (15–30 × 109 platelets/l) was present, with bruising and bleeding, which had also required platelet transfusions. She was occasionally neutropenic. Bone marrow examination revealed hypercellularity, with megaloblastic and some dysplastic changes. Full differentiation was observed in myeloid and megakaryocyte lineages. Other clinical findings included secundum atrial septal defect (ASD) with atrial dysrhythmia and, on some electrocardiograms, absent P-waves or first degree atrioventricular (AV) block. She had pronounced dilated cardiomyopathy predominant in the right cavities. Her ophthalmic examination was normal. By the age of 6 years, the patient had congestive heart failure, to which she succumbed. She was diagnosed with TRMA only few days before her death and brief treatment with thiamine at 25 mg/d did not improve the clinical status.
Case II-5: the proband's brother presented aged 1 month with severe pancytopenia and sensorineural deafness. Diabetes mellitus was observed at 2 years of age. When the diagnosis of TRMA was entertained, he was treated with thiamine 25 mg/d. Pancytopenia improved after 1 week, but macrocytosis persisted. Diabetic control improved, with HbA1C 21% before therapy, and 8% on a recent check. Like his sister, he was found to have secundum ASD with atrial dysrhythmia, and absent P waves, but had right bundle branch (RBB) block. ASD was surgically repaired. Bone marrow aspirate (Fig 2) taken 22 months after therapy at the age of 5 years showed normal cellularity with haemosiderin. Ringed sideroblasts were present and eosinophils were increased. His ophthalmic examination was normal. Hepatomegaly was noted at the age of 5 years.
Assessment of the family revealed mild normocytic anaemia and normal hearing in the mother. The father had mild bilateral high-frequency sensorineural hearing loss and normal blood counts. Three other siblings were healthy. Neither diabetes nor optic atrophy could be found in any of the family members.
Genotyping and mutation analyses
Genomic DNA was prepared from each family member shown in Fig 1 and genotyping was performed with six polymorphic markers spanning the TRMA critical region, as previously described (Neufeld et al, 1997). Haplotype analyses revealed that the proband and her brother were homozygous by descent for the telomeric five markers, while D1S2844 at the centromeric end revealed a cross-over in the paternal haplotype for individual II-5 (Fig 1). Neither the parents nor unaffected siblings were homozygous in this region, consistent with linkage of the disease phenotype to this region in the Tunisian family, as has been noted in every TRMA kindred to date.
Sequence analysis of the SLC19A2-coding region revealed a novel point mutation in exon 2, a single nucleotide deletion (guanine) at nucleotide 287 of the cDNA, relative to the translation start site, resulting in a frameshift and a premature stop codon at nucleotide 380 of the cDNA. Both patients were homozygous for the mutation. The father was heterozygous for the same mutation.
Association of secundum ASD together with conduction defects, AV block and RBB block in our patients also led us to consider possible co-segregation of the mutation in the NKX2–5 gene (Schott et al, 1998). Analysis of this gene revealed no detectable mutation (data not shown).
We assessed thiamine uptake by diploid fibroblasts from the proband (–/–, II-4), an obligate carrier (+/–, I-1) and a normal control (+/+) at nanomolar thiamine concentrations, as previously described (Stagg et al, 1999). Specific uptake in the mutant cells was only 3% of control values (0.007 pmol/30 min/106 cells versus 0.25 pmol/30 min/106 cells) at 23 nmol/l thiamine. The obligate heterozygous carrier (father) had an uptake pattern that was between that of the mutant and the normal individual. This is consistent with a severe defect of the specific high-affinity transport in the mutant.
We have identified a novel point mutation, 287delG in exon 2 of the SLC19A2 gene, leading to TRMA syndrome. This predicts a null phenotype, with frameshift and early termination. To date, every TRMA kindred tested has had a homozygous mutation in this gene (Diaz et al, 1999; Fleming et al, 1999; Labay et al, 1999; Raz et al, 2000; Scharfe et al, 2000). SLC19A2 encodes a transmembrane protein that confers high-affinity thiamine transport to TRMA mutant cells, or embryonic kidney cells when introduced by transfection (Fleming et al, 1999). Fibroblasts homozygous for 287delG were defective in the high-affinity thiamine transport compared with heterozygous and normal cells. To our knowledge, this is only the second report of defective nanomolar-affinity thiamine transport in TRMA confirming earlier hypotheses (Poggi et al, 1984, 1989; Rindi et al, 1992, 1994; Stagg et al, 1999).
Our patients exhibited severe pancytopenia early after birth, with bone marrow dysplastic changes, findings that were also reported by others (Rogers et al, 1969; Borgna-Pignatti et al, 1989; Poggi et al, 1989; Grill et al, 1991; Morimoto et al, 1992; Bazarbachi et al, 1998). The bone marrow picture in TRMA is unique. The combination of megaloblastic findings and ringed sideroblasts is suggestive of a ‘myelodysplastic’ state and has led others to term this condition ‘thiamine-responsive myelodysplasia’ (Bazarbachi et al, 1998). The two names appear certain to refer to the same genetic disorder, however. In patient II-5, ringed sideroblasts were still present ≈2 years after thiamine therapy (Fig 2A). The basis of mitochondrial iron deposition in this syndrome is not clear. One possible explanation is that low intracellular thiamine may produce defective α-ketoglutarate dehydrogenase which, in turn, is required for succinyl CoA formation, the first substrate for haem synthesis, leading to ineffective erythropoiesis of the sideroblastic type (Abboud et al, 1985). The basis of megaloblastosis in TRMA has not been satisfactorally explained (for example, see Haworth et al, 1982) and remains an area of active investigation.
Our patients eventually became insulin-dependent as did all reported TRMA patients (reviewed in Valerio et al, 1998). This diabetes was thought to be a non-type I in nature (Borgna-Pignatti et al, 1989; Mandel et al, 1993), however, one TRMA patient was found to have HLA-DR4 antigen and islet β-cell auto-antibodies (Akinci et al, 1993). It is noteworthy that SLC19A2 maps to chromosome 1q23.2–23·3, a locus known to be a candidate for type I and type II diabetes (Concannon et al, 1998; Hanson et al, 1998). SLC19A2 gene polymorphism analyses in type I and type II diabetic patients might shed light on the pathogenesis of these polygenic diseases.
In our patients, SLC19A2 mutation led to profound and irreversible deafness. The role of thiamine in hearing is unknown. However, an ion channel-regulatory role has recently been proposed for thiamine in excitable cells (Bettendorff, 1994). Therefore, thiamine starvation as a result of an SLC19A2 mutation might impair action potentials in inner ear cells by modifying endolymph electrolyte composition, leading to deafness.
It is interesting to speculate on the potential relationship of TRMA to cardiac defects. The rate of observed cardiac problems in TRMA patients is much greater than that in the population at large. Only a few cases of overt congestive cardiomyopathy have been noted (Mandel et al, 1984; Vossough et al, 1995), including our patients. Improvement in our younger patient on thiamine therapy suggests that SLC19A2 mutation has a role in the pathogenesis of cardiomyopathy. Our patients also had large secundum ASDs with conduction defects, which have been reported in other patients (Abboud et al, 1985). Although thiamine deficiency is not known to be teratogenic in humans, we cannot exclude SLC19A2-induced thiamine starvation as a cause of those malformations. Atrial dysrhythmia was described in several TRMA patients and was well controlled by thiamine therapy (reviewed in Rindi et al, 1994). This was also the case in our younger patient.
The father of our patients, proven heterozygous for SLC19A2, and many relatives of TRMA patients in the literature, have had a propensity for diabetes or deafness. We and colleagues have speculated that heterozygotes might have late onset or a milder form of TRMA that suggests an effect of heterozygosity for SLC19A2. In fact, most obligate heterozygotes are normal and every patient studied to date with the cardinal triad of anaemia, diabetes and deafness has been homozygous.
Overall, homozygous 287delG mutation led to severe bone marrow disease, insulin-dependent diabetes, profound deafness and dilated cardiomyopathy in our patients, probably by lack of the high-affinity transport. The multisystem involvement of the disease indicates that the SLC19A2 gene is important for normal function of haematopoietic cells, glucose homeostasis, normal hearing and cardiac function. With the exception of the pluripotent haematopoietic stem cells, which are capable of self-renewal and are not sensitive to cell death, thiamine starvation induced by SLC19A2 mutation might lead to apoptotic cell death of pancreatic β-cells, acoustic and cardiac cells, as was found in TRMA fibroblasts (Stagg et al, 1999).
We are indebted to the family members who participated in the study. We thank Dr Gianguido Rindi and Dr Cesare Patrini for BOM-thiamine administrated to the patients, Dr Anders Linde for his help with the manuscript and Dr Jonathan G. Seidman for assistance with NKX2–5 analysis. This work was supported by NIH grant HL04184 and March of Dimes Research grant to E.J.N., NIH training grant T32 HL07574 to E.T. and J.C.F.