A 3-year-old girl was referred to our unit for evaluation of congenital transfusion-dependent anemia. She was born at 38 weeks gestational age, small for gestational age (SGA) for length (42 cm), but of normal weight (3,260 g). Before admission, her hemoglobin (Hb) values fluctuated between 5 and 10 g/dl, requiring approximately 1 unit of concentrated red blood cell every 3 to 4 months. Erythropoietin supplementation 500 U/kg/week and prednisone 1 mg/kg/day were not able to improve anemia. On clinical examination, she showed black curly hair, no visible hand or skeletal malformations, or abnormal facial features; splenomegaly was noted and later confirmed by ultrasonography (11 cm in longitudinal diameter, without signs of hepatic cirrhosis or portal hypertension). Upon laboratory evaluation, she showed borderline macrocytosis (mean corpuscular volume 95 fl, range of normal for age 74-94 fl), non-compensatory reticulocyte count (56,000/mul), elevated erythropoietin levels (3,316 IU/L, normal values 3.7-31.5 IU/L), increased Hb F (5.1%, normal values < 1%) and vitamin B12 (1,263 pg/ml, normal values 190-660 pg/ml), normal iron balance, no evidence of hemolysis, and a mildly positive Direct Antiglobulin Test (DAT). Polymerase chain reaction for Parvovirus B19 DNA was negative. Erythrocyte adenosine deaminase (eADA) was increased at 8.4 U/gHb (normal values < 1.2 U/gHb). Bone marrow biopsy revealed erythroid hypoplasia, as reflected by an increased granulocyte/erythroid precursor ratio (4/1), and some degree of dyserythropoiesis. In vitro erythroid precursor growth (methyl cellulose assay) was severely impaired compared to normal controls and was not rescued after lymphodepletion of the sample. Patient bone marrow plasma did not inhibit erythroid colony formation from the marrow of a healthy donor. These features were in keeping with the intrinsic erythropoietic failure associated with Diamond Blackfan Anemia (DBA), but both Sanger sequencing and the Multiple Ligand Probe Assay of the genes more frequently associated with DBA (RPS19, RPL5, RPL11, RPS17, RPS26, and RPL35a) did neither reveal any single-nucleotide variants or copy number variants. The dyepoxybutane chromosomal fragility test was normal, thus excluding Fanconi anemia and related genetic DNA repair defects. Telomere length, assayed by flow-fluorescence in situ hybridization (FISH), was normal in granulocytes and unmeasurable in lymphocytes due to the insufficient number of cells to perform the test. Ribosomal RNA (rRNA) analysis (i.e., quantification of long non-coding rRNA precursors, which accumulate in the case of ribosomal processing malfunction) was attempted on multiple occasions but failed due to the insufficient quantity of nucleic acid retrieved from the patient's lymphocytes.
At 7.5 years of age, the patient was hospitalized for septic shock due to Streptococcus viridans. At this point in time, she was mildly lymphopenic, with undetectable serum IgA and normal IgG and IgM values. Further immunologic testing revealed low CD4+ and CD8+ T lymphocytes, decreased mitogen-induced lymphocyte proliferation, moderately increased alpha-beta T lymphocyte CD4-CD8- ("double-negative" T lymphocytes, DNTs), and markedly reduced thymic output (i.e., low naive T cell and absent T excision circles) ( Table 1 ). FAS-mediated lymphocyte apoptosis was within the normal limit. A neck ultrasound revealed thymic hypoplasia. FISH analysis of chromosome 22 was normal, most likely excluding a diagnosis of Di George syndrome. A custom panel of 312 genes related to primary immune deficiencies and bone marrow failure syndromes was analyzed through a next-generation sequencing (NGS) tool in use in our center but no pathogenic variants were detected. This version of our NGS panel did not evaluate for the RMRP gene, which was included in later versions. Whole-exome sequencing (WES) was then performed, and it did not detect any significant mutation. Prophylaxis with trimethoprim-sulfamethoxazole was started to prevent opportunistic infections due to Pneumocystis jiroveci.
Since splenomegaly, increased DNTs, increased B12 serum level, reduced thymic output, and positive DAT were consistent with a diagnosis of the primary immune regulatory disorder (PIRD) autoimmune lymphoproliferative syndrome (ALPS), we started treating our patient with sirolimus (2 mg/mq/day): her Hb values improved, reaching a plateau at 12-13 g/dl, DAT became negative, spleen volume progressively reduced to normal (8 cm of longitudinal diameter after 6 months of therapy), and DNTs decreased below 1%. Trephine biopsy showed normal erythroid cellularity and only mild signs of dyserythropoiesis; in vitro erythroid precursor growth became normal.
Over the years, the patient developed progressive lymphopenia with severe B-cell lymphopenia, absence of memory B cells, symptomatic hypogammaglobulinemia with recurrent respiratory tract infections, and bronchiectasis, treated with subcutaneous immune globulins and azithromycin. Chronic inflammatory skin disease and recurrent non-infectious diarrhea also appeared. Progressive growth delay associated with growth hormone deficiency poorly responding to replacement therapy also developed, which became particularly evident in peri-pubertal years (final height at 15 years of age: 142.5 cm, corresponding to -2.9 standard deviation from average). A total body radiological skeletal survey then revealed metaphyseal dysplasia ( Figure 1 ). At age 15 years, she developed severe headache, neck stiffness, photophobia, wide-based gait, and microzoopsia. She was diagnosed with cryptococcal meningoencephalitis and successfully treated with daily liposomal amphotericin B for 4 weeks and flucytosine for 2 weeks, followed by 10 weeks of fluconazole at a therapeutic dosage, later continued at a prophylactic dosage.
Sirolimus was stopped and the patient rapidly became anemic (Hb nadir at 6 g/dl, 6 weeks after therapy interruption); marrow evaluation again showed features of erythropoietic failure, with severely impaired erythroid colony formation and increased DNTs up to 3%. Once infection resolved, we estimated that the benefit of sirolimus on erythropoiesis outbalanced the mild immunosuppressive effect, and thus, we restarted treatment that led to resolution of anemia within 3 weeks. The patient had no neurological sequelae or cryptococcosis relapses following this episode.
Whole-genome sequencing (WGS) identified two compound heterozygous mutations in the gene encoding the long non-coding RNA RMRP (ENST00000363046.1): a maternally inherited single-nucleotide rare variant, n.67G>A (rs1245480029), and a novel paternally inherited one-nucleotide insertion, n.64_65insA. Both variants map to a proximal highly conserved region of the gene, where several other variants associated with CHH-anauxetic dysplasia spectrum disorders are localized. For this reason, the two variants detected in the proband were considered pathogenic, and a diagnosis of CHH was made.
The progressively severe immune deficiency along with the cryptococcal meningoencephalitis:that in these patients, despite chronic antifungal treatment, has a high risk of lethal recurrence:strongly supported the indication to HSCT. In the absence of a matched family or unrelated donor, she received at 15 years of age a haploidentical alpha-beta-T-depleted HSCT from her heterozygous, clinically, and immunologically healthy father. The conditioning regimen included thiotepa, treosulfan, and fludarabine. No toxic events or recurrence of cryptococcal meningoencephalitis occurred during the early period after HSCT; sustained neutrophil and platelet engraftment occurred within 16 days post-infusion. At the time of writing, the patient is 2 months post-transplant and shows normal blood counts with no signs of graft-versus-host disease and full donor chimerism. She will receive fluconazole prophylaxis until immunological reconstitution is complete.