Transfer RNA and syndromic sideroblastic anemia

In this issue of Blood, Chakraborty et al¹  reported loss-of-function of TRNT1 gene causes a syndromic form of congenital sideroblastic anemia (SA) associated with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD). This new syndrome, inherited with a recessive pattern, was described in this Journal 1 year ago studying 12 subjects from 10 different families.²  It is a severe condition with neonatal or infancy onset and systemic tissue involvement that can partially benefit from bone marrow transplantation.

Identification of TRNT1 mutations as SIFD-causing genetic alterations was achieved by using 2 independent technical approaches: genome-wide next-generation sequencing and descendent mapping linkage analysis. Both methods identified germline point mutations of the TRNT1 gene (EC 2.7.7.25) that encodes an essential enzyme that catalyzes the addition of the CCA terminus to the 3′ end of transfer RNA (tRNA) precursors. This reaction is a fundamental prerequisite for mature cytosolic and mitochondrial tRNA aminoacylation and quality control as well as for stress response.

Inherited SAs comprise heterogeneous phenotypes depending on the original function(s) of the mutated genes but all characterized by the presence of ringed sideroblasts in the bone marrow aspirate. The latter are erythroblasts with pathological coarse granules of iron deposition in mitochondria, as demonstrated by electron microscopy. Such iron-encrusted mitochondria surround the nucleus of the erythroid cell forming a characteristic “ring,” when stained with Perls’ Prussian blue.

Inherited SA is rarer than the acquired form and could be syndromic or not syndromic, with heterogeneous patterns of inheritance. To date, several genes responsible for inherited SA have been identified and they all play important roles in heme biosynthesis, Fe-S cluster biogenesis, or biology of mitochondria³  (see figure).

The most frequent form is X-linked SA (XLSA), caused by mutations in the erythroid-specific d-aminolevulinate synthase gene (ALAS2). Bergmann et al⁴  systematically examined gene mutations in 60 inherited SA probands, and identified mutations of ALAS2, SLC25A38, mitochondrial DNA, and PUS1 in 37% 15%, 2.5%, and 2.5% of cases, respectively. Disease-causing mutations were not found in the remaining 43% of cases, suggesting that there are as-yet-unidentified gene mutations that can cause inherited SA.

The observations of Chakraborty et al¹  reduce the number of syndromic forms lacking a causative gene, in particular those with SIFD. Because of the large heterogeneity of SIFD expressivity, it is difficult to calculate the exact percentage of patients carrying TRNT1 mutations because many cases might be misdiagnosed for their mild phenotype. Such cases could now be identified by TRNT1 gene sequencing. The loss of function effect of SIFD-associated TRNT1 mutations was clearly shown in both human fibroblasts and in yeast, although the specific mechanism by which these mutated proteins cause specifically anemia, immunodeficiency, fever, and developmental delay is still largely unknown. This is another example of an inherited disease in which insufficiency of a single protein, TRNT1, simultaneously promotes the deficiency of many other proteins via the perturbation of protein biosynthesis pathway. Noteworthy is that TRNT1 gene function appears crucial and not redundant for cellular homeostasis and fitness, as demonstrated by the observations that almost all patients retain some enzyme activity and most mutations are hypomorphic. In addition, silencing of the TRNT1 gene is accompanied by cell death in vitro.

However, as in Diamond–Blackfan anemia for the ribosome synthesis⁵  and in congenital dyserythropoietic anemia type II for COPII assembly, even if the mutated gene is expressed in a variety of tissues, the clinical symptoms appeared limited to a single or few organs and tissues.⁶  How can such an oddness be explained? One possibility could be that, in SIFD, tissues devoted to producing a large amount of a limited set of proteins or even a single one (as hemoglobin for erythroid precursors or immunoglobulin for B lymphocytes) could suffer more from inefficient protein synthesis.

In both the syndromic and not-syndromic forms of SA, the final effect is deregulation of iron metabolism that causes an iron overload in the erythroblast mitochondria associated with increased mitochondrial ferritin, which is, indeed, the hallmark of these anemias. Thus, such iron excess in these organelles needs to be sheltered by mitochondrial ferritin to avoid Fenton-type reactions and iron-induced oxidative damage.⁷ 

In several nonsyndromic cases (such as those caused by mutations of XLSA, SLC25A38, GLRX5, and FECH), the iron overload appears as a direct consequence of the involvement of a gene whose product is directly related to iron or heme metabolism. In these cases, the symptoms are mainly confined to anemia. On the other hand, syndromic forms appear to be associated with a defect in mitochondrial/cytosolic protein translation (ABCB7, SIFD, Pearson syndrome, myopathy lactic acidosis and SA, thiamine-responsive megaloblastic anemia) and in particular with abnormal tRNA function caused by defects in tRNA maturation/modification or even deletion of mitochondrial tRNA genes. In accordance, TRNT1 is essential for the tRNA maturation, and its reduced activity, as in other syndromic SA, is predicted to have profound effects on cell viability by affecting overall protein biosynthesis and causing a plethora of dysfunctions in patients.

In conclusion, the article by Chakraborty et al opens new frontiers for molecular diagnosis in the pre- and postnatal periods and genetic treatment of patients with SIFD, although many efforts still need to be undertaken to understand the molecular mechanism underlying this complex disease phenotype.