We investigated 32 individuals (27 families) from 17 countries. Consanguinity was reported in 13 families. At the time of referral for genetic testing, the age of the individuals in the cohort ranged from 3 months to 28 years (median age 4 years [IQR 1–9]). We describe 20 previously unreported individuals with TRMA syndrome and present follow-up data for 12 individuals reported previously [16, 26].
Genotype
Twenty-four different SLC19A2 mutations were identified in the 32 individuals from 27 families, confirming a genetic diagnosis of TRMA syndrome. Four individuals from three families were compound heterozygotes for SLC19A2 mutations and the remaining participants had a homozygous mutation (Table 1, Fig. 1 and electronic supplementary material [ESM] Table 1).
Table 1 Mutation description and phenotype of study participants
Nine of the mutations identified were novel (three missense, three nonsense, two frameshift and one splice site mutation). The three novel missense mutations (p.Leu64Pro, p.Ala91Pro and p.Gly334Asp) affect residues highly conserved across species and in silico investigations predict these mutations to be pathogenic. Only p.Gly334Asp was listed in the Exome Aggregation Consortium (ExAC) database (minor allele frequency (MAF) 1.499 × 10−05; http://exac.broadinstitute.org/, accessed November 2016) in a single heterozygous individual of European ancestry. The two frameshift (c.993_996dup and c.1201_1202del) and three nonsense (p.Trp30*, p.Trp302* and p.Trp387*) mutations are predicted to cause the insertion of a premature stop codon, which probably results in mRNA degradation via nonsense mediated decay. The splicing mutation (c.204+2T>G) is predicted to abolish the splicing donor site in intron 1 and cause aberrant splicing.
Phenotype
The clinical characteristics and demographics of each individual are summarised in Table 1, Figs 2, 3 and ESM Table 1. The exact age at presentation was not available for three individuals (2.1, 4.1 and 13.1). In the remaining 29 participants, the age at diagnosis of the first symptom ranged from birth to 4 years (median 26 weeks [IQR 12–104]). Variable age at onset was observed between individuals with the same mutation, including siblings, indicating intra-familial variability.
Three individuals (5.1, 7.1 and 15.1) presented with the classical triad of TRMA syndrome, whilst ten were initially diagnosed with diabetes and anaemia with deafness subsequently confirmed on hearing test. The remaining individuals developed these clinical features in variable order.
All 32 individuals had diabetes, with age at onset ranging from 6 weeks to 8 years (median 43 weeks [IQR 21–116]). Diabetes was diagnosed in the first year of life in 15 individuals, with nine of them being diagnosed before the age of 6 months, fulfilling diagnostic criteria for neonatal diabetes [24]. In three individuals (1.1, 19.1 and 21.1) diabetes was diagnosed before appearance of the other clinical features, whilst in 11 individuals hyperglycaemia was the presenting symptom along with anaemia and/or deafness. Three children presented with diabetic ketoacidosis (DKA), 17 with symptomatic hyperglycaemia and in the remaining 12 individuals hyperglycaemia was an incidental finding. Islet cell autoantibodies were measured in three participants and the tests were negative. C-peptide was measured in four individuals, at least 1 year after starting insulin, and the level was <0.165 nmol/l for all of them. After a diagnosis of diabetes, all patients started insulin therapy and all except four are still on insulin with variable glycaemic control. Recurrent DKA episodes were reported for two individuals and one child died of DKA in a remote hospital during a family holiday.
Anaemia was documented in all except two individuals: individual 19.1 who still has normal haemoglobin (Hb) and mean corpuscular volume (MCV) at the age of 3.9 years; and individual 6.2 who was reported to have macrocytosis in the presence of normal Hb [21]. In the other participants in the cohort anaemia presented between birth and 7.5 years (median 47 weeks [IQR 14–155]) and initial Hb levels ranged from 43–75 mg/l (normal range 115–150 mg/l). In 18 individuals anaemia was the first presenting symptom, either in isolation (individuals 3.1, 9.1, 11.1, 12.1, 14.1, 24.1 and 27.1) or in combination with other features. For the rest of the cohort, anaemia developed after the onset of diabetes. All individuals with anaemia needed multiple blood transfusions prior to starting thiamine therapy.
All except two individuals (3.1 and 14.1) were reported to have sensorineural deafness. This was diagnosed between birth and 4.5 years of age (median 60 weeks [IQR 26–156]). Deafness was the presenting feature in seven individuals. In individuals 17.1, 17.2 and 18.1 it was detected soon after birth and in individuals 1.2, 1.3, 6.2 and 10.1 it was diagnosed up to 4.5 years before the appearance of the other features. In the remaining individuals deafness was detected by hearing tests following the appearance of other clinical features. Two individuals (8.1 and 27.1) had cochlear implantation, while the rest have hearing aids. Regarding the two individuals with no reported deafness, individual 14.1 died at the age of 15 months and individual 3.1 has severe neurodevelopmental delay with no follow-up audiogram since initial presentation.
Additional clinical features, other than anaemia, diabetes and deafness, were reported in 22 individuals (Table 1 and Fig. 2). These included neurological deficit/developmental delay (37.5%), visual defects (25.0%), cardiac anomalies (15.6%), short stature (9.4%) and thrombocytopenia (12.5%).
Follow-up and response to thiamine therapy
Follow-up data was available for 16 individuals (Table 2). This includes individual 14.1 who died at the age of 15 months following a viral infection before starting thiamine therapy. For the other 15 participants, thiamine therapy was commenced following the genetic diagnosis. The age at starting thiamine treatment ranged from 6 months to 14 years (median 24 years [IQR 12.5–76]) and the maximum dose ranged between 25 and 300 mg/day. However, none of the clinicians observed any additional benefits of thiamine doses >150 mg/day. The duration of thiamine therapy was between 2 and 17.5 years (median 4.7 years) giving a total period of 103.5 treatment years.
Table 2 Long-term follow-up data and response to thiamine in 16 individuals
A clear benefit of thiamine therapy on diabetes control was observed in 11 out of 15 patients. Seven individuals achieved improved HbA1c levels, at least for the following 6 months (Fig. 4), with reduced insulin requirement, and four managed to become insulin-independent. Participants 16.1, 19.1 and 27.1 have been off insulin since starting thiamine at the ages of 8 months, 13 months and 9 months, respectively. Individual 20.1 (currently aged 11 years) has recently started to require insulin again following a period of 5 years of insulin independence on thiamine treatment alone. The 11 patients who showed a glycaemic benefit from thiamine therapy (responders) had a significantly younger age at diagnosis of diabetes (p = 0.042), were referred for genetic testing earlier (p = 0.01) and had started thiamine treatment earlier (p = 0.007) compared with the non-responder group (Table 3). Our study did not detect a significant difference in sex, age at onset of the first TRMA symptom, thiamine dose or duration of therapy between the responder and non-responder groups (Table 3).
Table 3 Analysis of demographic and clinical factors associated with response to thiamine
In all individuals with anaemia, Hb rose to normal levels within 6–8 weeks of starting thiamine therapy and all living participants are currently transfusion-independent. Similarly, participants with thrombocytopenia have maintained normal platelet counts while on thiamine. No impact of thiamine therapy was reported on hearing function or other extra-pancreatic features.
Five of the 15 participants for whom follow-up data on thiamine treatment was collected are currently aged 13 years or older. All of them have achieved normal puberty and are still transfusion-independent. These five patients are insulin-dependent and three had no improvement in their initial poor diabetes response to thiamine.
There were no reported side effects of thiamine therapy. One individual (9.1) had a stroke at the age of 2 years while on combined insulin and thiamine therapy. Individual 10.1 died at the age of 7 years. He had diabetes diagnosed at the age of 2 years and was initially treated with insulin. Subsequently thiamine was added and his glycaemic control improved during the following 4 years. However, he died of DKA following a viral illness during a family holiday to a remote area. Individual 27.1 developed hyperglycaemia during intercurrent illnesses while she was off thiamine therapy.