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

Clinical Manifestations of Patients

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.

Table 1 Laboratory examination of two cases of SIFD

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).

Fig. 1figure 1

Clinical manifestation of patient 1 we reported. (a) Erythematous rash with partial ulceration of patient 2. (b) Facial dysmorphism of patient 2 presenting with mild facial dysmorphism and protruding backed nose

Identification of TRNT1 Mutations in the Patients

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.

Fig. 2figure 2

TRNT1 mutations in our two patients. (a and b) Pedigree of family 1 (a) and family 2 (b). Circles, females; squares, males; filled symbol, affected subject; half-filled symbols, the heterozygous carriers; slash lines, dead individuals. (c) Sanger sequencing results of TRNT1 in patient 2 and his parents. (d) Crystal structure of the TRNT1 was shown in cartoon representation and colored by the secondary structure. Magnified pictures displayed the sticks of Glu236 and Val240 residues (top-left), Lys236 and Val240 residues (top-right), Gln303 and Asn329 residues (bottom-left), and Glu303 and Asn329 residues (bottom-right). (e and g) The calculated electrostatic potentials of the TRNT1 WT and mutant were mapped on the surfaces and colored in a gradient from red (negative) to blue (positive). Yellow circles represent the corresponding amino acid. (f and h) The predicted hydrophobicity of TRNT1 WT and mutant gradient colors from red to white indicate a hydrophobicity scale from high to low. Yellow circles represent the corresponding amino acid

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.

Treatment and Outcome of the Patients

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.

Fig. 3figure 3

Schematic diagram of clinical features and lab data of patient 2 following treatment

Table 2 The changes of immune function and cytokines of patient 2 before and after thalidomide treatmentReview of the Literature on SIFD

To date, 71 cases of SIFD have been reported based on 33 articles (Supplementary Table 1) [3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36]. 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.

Clinical Manifestation

Clinical information was available for 69 of 71 patients. The clinical manifestations and laboratory features of the 69 patients were analyzed (Table 3) [3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36]. The clinical features of SIFD were diverse. Microcytic anemia (82.6%), hypogammaglobulinemia/B cell lymphopenia (75.4%), recurrent fever (66.7%), and developmental delay (60.9%) were the most common manifestations of SIFD. Also, 36.2% (25/69) of patients had concurrent tetralogy. Three patients had none of the typical four manifestations of SIFD stated above [19], and they all had ocular symptoms accompanied by microcytosis and anisocytosis and were finally diagnosed as SIFD by gene sequencing. Microcytic anemia was present in 57 patients, of which 30 had sideroblastic anemia. Microcytosis was observed in four patients [19, 32]. Thrombocytopenia was documented in three patients, and one patient also had leukopenia [12, 36]. Three-quarters of patients with SIFD had hypogammaglobulinemia, B cell lymphopenia, and reduced production of protective antibodies [35]. They were prone to recurrent infections by multiple pathogens in different systems. Infections were present in 43.5% (30/69) of patients. In 2020, Zhou et al. reported a patient with severe coronavirus disease-2019 (COVID-19) who had been healthy in the past. Due to recurrence of severe infection by acute respiratory syndrome-coronavirus-2, genetic testing was conducted and a mutation in TRNT1 was found [35]. Specific autoantibodies of SIFD patients were all negative. Most of children had fever every 1–4 weeks, with each episode lasting 3–7 days. Developmental delay (another main feature of SIFD) can manifest as intellectual disability as well as disorders in motor development and language development.

Table 3 Clinical manifestations and laboratory features of TRNT1 deficiency

Vomiting and diarrhea during fever were the most common forms of gastrointestinal involvement in 30.4% (21/69) of patients. In other cases, patients had Crohn’s disease, necrotizing enterocolitis, or protein-losing bowel disease that manifested as food intolerance, gastrointestinal bleeding, chronic constipation, or diarrhea. Mucocutaneous lesions were observed in 40.6% (28/69) of patients, and erythema nodosum was the most common manifestation. Skin biopsies were carried out in four patients with erythema nodosum, which revealed panniculitis [7, 8, 23, 28]. Cutaneous vasculitis [16] and lichen sclerosus etatrophicus [18] were rare skin changes of SIFD, each occurring in one patient, respectively. Cardiomyopathy (one of the causes of death) was detected in 8.7% (6/69) of patients, four of whom were neonatal [3,

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