Thalidomide as an Effective Treatment in Sideroblastic Anemia, Immunodeficiency, Periodic Fevers, and Developmental Delay (SIFD)

Written informed consent was obtained from the parents of both participants. This study was conducted in accordance with the tenets of the Declaration of Helsinki 1964 and its later amendments. The study protocol was approved by the ethics committee of Beijing Children’s Hospital (Beijing, China).

Two children admitted to our department between 1 April 2021 and 1 November 2022 were diagnosed with SIFD. The medical history of the children was complete. The duration of follow-up after initial treatment was > 12 months. The clinical manifestations, laboratory data, treatments, and prognoses of both patients were collected and analyzed. The patients were diagnosed with SIFD according to their medical history, diagnostic test result, and genetic data.

Peripheral blood was collected from members of the patients’ families after obtaining written informed consent. Samples of whole blood were sent to MyGenostics (Beijing, China) and subjected to whole-exome sequencing (WES). The pathogenicity of the mutations was assessed following the American College of Medical Genetics and Genomics guidelines. Mutations in TRNT1 were confirmed using Sanger sequencing. Briefly, genomic DNA was extracted from peripheral blood mononuclear cells using a QIAamp DNA Mini Kit (Qiagen, Shanghai, China). Polymerase chain reaction (PCR) was performed to amplify the genomic TRNT1 gene. The PCR products were sent for Sanger sequencing.

All protein models were visualized with the molecular graphics program PyMol. The homology modeling of TRNT1 structure was performed based on the crystal structure of human CCA-adding enzyme (PDB ID: 1OU5). The mutant structure of TRNT1 was generated using the PyMol mutagenesis tool. Hydrophobic surfaces were colored based on a normalized hydrophobicity scale by running the python script “Color_h” (https://​pymolwiki.​org/​index.​php/​ Color_h). The electrostatic potentials were performed in PyMol using the vacuum electrostatics function.

A literature review of SIFD was undertaken using databases (PubMed, Web of Science, and EMBASE). The search terms were “Sideroblastic anemia, immunodeficiency, periodic fevers and developmental delay,” “SIFD,” and “TRNT1.” All studies (articles, reviews, letters, or case reports) published online before 1 December 2022 were eligible for review.

Patient 1 was born to non-consanguineous, healthy parents of Chinese descent following an uneventful pregnancy. He had developmental delay and intermittent eczema after birth. He developed recurrent fever at 1.5 months of age, occurred once a week for 3 days in each instance. Antibiotics were not efficacious. Vomiting and diarrhea occurred simultaneously during episodes of fever. Laboratory tests revealed a normal leukocyte count, increased levels of C-reactive protein (CRP) and ferritin, and an increased erythrocyte sedimentation rate (ESR). The symptoms disappeared and levels of inflammatory markers decreased during afebrile intervals. Microcytic hypochromic anemia was detected with normal reticulocytes at an age of 2.5 months, but sideroblastic anemia was not detected in peripheral blood or bone marrow during the same period. Examination of immune function during fever at 3 months of age is listed in Table 1. Humoral immune function revealed a reduced level of immunoglobulin (Ig) A levels and normal levels of IgG and IgM. The test results of lymphocyte subtypes showed a normal number of T lymphocytes, reduced number of B cells, and an increased number of natural killer (NK) cells. The interleukin (IL)-6 level was increased. The patient experienced severe infections, including pneumonia (adenovirus and extended-spectrum-β-lactamase-producing Klebsiella pneumoniae in sputum), enteritis (Clostridium difficile in stools), and urinary-tract infection (extended-spectrum-β-lactamase-producing K. pneumoniae in urine). High-resolution computed tomography of the lungs revealed bilateral inflammatory changes. Magnetic resonance imaging (MRI) of the brain disclosed widening of bilateral ventricles, cisterna magna, and left extracerebellar space. The patient’s oldest sister had identical clinical manifestations. She died of severe pneumonia at 2 years of age without undergoing genetic testing.

Patient 2 was born to unrelated Chinese parents after a normal pregnancy. She developed periodic high fever 17 h after birth accompanied by vomiting and diarrhea that did not respond to antibiotics. Fever persisted for 5 days, followed by 7 days of normal body temperature. Her inflammatory indices (CRP level, ESR, and ferritin level) were increased during fever and decreased in the afebrile interval. Non-infectious erythematous rash with partial ulceration appeared during periods of fever (Fig. 1a). The pathology of the erythema included degeneration and necrosis in the epidermis and subcutaneous tissue. At the age of 1 month, moderate-to-severe microcytic hypochromic anemia and thrombocytopenia were observed, and examination of bone marrow revealed occasional nucleated red cells with possible sideroblastic granules (but no ringed sideroblasts) and normal megakaryocytes. Conduction in the auditory nerve was aberrant. MRI of the brain revealed mild dilatation of bilateral lateral ventricles. The patient coughed intermittently and experienced pneumonia 2–3 times a year, which was cured by antibiotics. Sputum culture disclosed K. pneumoniae. Her gross motor and language skills were delayed. She had mild facial dysmorphism (Fig. 1b) and a failure to thrive. She was admitted to our department when she was 5 years of age. Testing of immune function was done during hospitalization, and the results indicated hypogammaglobulinemia, B cell lymphopenia, an increased number of T lymphocytes and NK cells, and a significantly increased number of γδT cells. In addition, levels of IL-6, IL-8, and interferon (IFN)-γ were increased, but levels of IFN-α and IFN-γ-induced protein 10 (IP-10) were normal (Table 1).

Based on clinical and laboratory information, WES was performed for the two patients and their families. A homozygous missense mutation in exon 6 (c.706G > A and p.Glu236Lys) of the TRNT1 gene was identified in patient 1. Both his parents and 3-year-old brother were heterozygous mutation carriers of this mutation (Fig. 2a). In addition, the mutation was not found in the gnomAD (v2.1.1), ClinVar, or Human Gene Mutation Database. Compound heterozygous mutations of TRNT1 (c.907C > G, p.Gln303Glu and c.88A > G, p.Met30Val) were found in patient 2, and these mutations were inherited from her mother and father, respectively (Fig. 2b and c). Of these, p.Met30Val was known as a pathogenic mutation, whereas p.Gln303Glu has not been reported previously. Further pathogenicity analysis by MutationTaster and GERP + found that the p.Gln303Glu mutation was predicted to be pathogenic.

In silico studies were performed to evaluate the impacts of these mutations on TRNT1 structure. The amino acid at position 236 of TRNT1 was located in the PcnB domain and near a highly conserved position, and the mutation resulted in a substitution of glutamic acid (Glu) with lysine (Lys). The mutation did not change the number of hydrogen bond or the distance between Glu236 and Val240 (Fig. 2d). However, it introduced an opposite charge and changed from negative (Glu) to positive (Lys), which may disrupted interaction with other molecules (Fig. 2e). Besides, the mutation also decreased local hydrophobic interactions, which could alter the physicochemical properties of the enzyme (Fig. 2f). The mutant residue Gln303 was located on the surface of PcnB domain in the TRNT1 protein. The hydrogen bond (yellow dotted line) distance between Gln303 and Asn329 was 2.6 Å, whereas the Glu303 mutation did not form a hydrogen bond with Asn329 (Fig. 2d). Beyond that, according to the electrostatic potential calculation, the mutation, charge changed from neutral (Gln) to negative (Glu), may cause a clash and repulsion with neighboring residues (Fig. 2g). The change in surface hydrophobicity potential was not significant (Fig. 2h). Collectively, these results strongly indicated that these mutations might alter the structure and function of TRNT1.

Patient 1 was diagnosed with SIFD and treated with IVIG replacement once a month. He died of severe infection at 1 year of age.

Patient 2 received blood transfusions, IVIG replacement, empiric antibiotics, and systemic corticosteroids, but the efficacy of those treatments was poor. At the age of 5 years, she was treated with adalimumab combined with IVIG replacement (400 mg/kg/m) in our hospital. At the beginning of treatment, her temperature was normal. Adalimumab was discontinued 5 months later because of the reappearance of periodic fever after 1 month of treatment. Thalidomide was administered together with IVIG supplementation therapy. After 11 months of observation, the patient no longer developed periodic fever and infection, and the inflammatory indicators were normal (Fig. 3). Changes in immune function and cytokines releases were monitored before and after thalidomide administration (Table 2). We documented normalization of the number of T lymphocytes, γδT cells, NK cells, and IgG levels, an increased number of B cells, and reduced levels of IL-6 and IFN-γ. Initially, she was receiving IVIG (400 mg/kg, i.v.) supplementation every month. For nearly 6 months, IVIG was given (i.v.) once every 50–60 days, but the trough concentration of IgG before each infusion was maintained at a normal level. The hemoglobin level increased to 100 g/L after 3 months of thalidomide treatment, but decreased again upon recent retesting. The influence of thalidomide on microcytic anemia, growth, and development requires further long-term observation, but its curative effect on SIFD was significant in this patient.

To date, 71 cases of SIFD have been reported based on 33 articles (Supplementary Table 1) [3‐36]. In those articles, 23 patients had a family history of the disease. Among the 65 patients whose sex was reported, the male: female ratio was 1: 1. Patients developed SIFD as early as the fetal period [13], and the older age of presentation was 40 years [35]. Only one patient had adult-onset SIFD [35]. The mean age of onset was 3.0 (Q1–Q3 = 1.0–7.2) months.