Claudio Sandoval

Identification of an Intestinal Folate Transporter and the Molecular Basis for Hereditary Folate Malabsorption

Folates are essential nutrients that are required for one-carbon biosynthetic and epigenetic processes. While folates are absorbed in the acidic milieu of the upper small intestine, the underlying absorption mechanism has not been defined. We now report the identification of a human proton-coupled, high-affinity folate transporter that recapitulates properties of folate transport and absorption in intestine and in various cell types at low pH. We demonstrate that a loss-of-function mutation in this gene is the molecular basis for hereditary folate malabsorption in a family with this disease. This transporter was previously reported to be a lower-affinity, pH-independent heme carrier protein, HCP1. However, the current study establishes that a major function of this gene product is proton-coupled folate transport required for folate homeostasis in man, and we have thus amended the name to PCFT/HCP1.

Clinical and hematologic effects of hydroxyurea in children with sickle cell anemia

PURPOSE This open-label pilot study was designed (1) to determine the effect of hydroxyurea on the hemoglobin level in children with sickle cell anemia, (2) to evaluate the toxicity of hydroxyurea, and (3) to assess any impact of hydroxyurea on the frequency of vaso-occlusive crises (VOCs). PATIENTS AND METHODS Ten children (group 1) with three or more VOCs of the extremities or two or more VOCs of the lungs (acute chest syndrome) in the preceding 12 months, and five children (group 2) with hemoglobin levels less than 70 gm/L were treated with hydroxyurea in doses of 20 to 35 mg/kg per day. The frequency of VOCs before hydroxyurea therapy was compared with the frequency during therapy, and the peak hemoglobin levels during hydroxyurea therapy were compared with the pretreatment values. RESULTS One patient in group 1 was removed from the study within 1 month because of nausea. Seven of the remaining nine patients in group 1 had a decrease in the frequency of VOCs. The number of VOCs per patient-year for all 14 patients decreased from 2.5 before hydroxyurea therapy to 0.87 during hydroxyurea therapy, a decrease of 65% (p < 0.00001). Two of five patients in group 2 had an increase in hemoglobin of 27 gm/L and 34 gm/L over the baseline. The median rise in hemoglobin was 19 gm/L (range, 7 to 37) for all 14 patients. Nine patients are still receiving hydroxyurea for a median period of 23 months (range, 18 to 59). CONCLUSIONS Hydroxyurea decreases the severity of anemia in some patients, and it may decrease the frequency of VOC. Its short-term hematologic toxicity is minimal.

Treatment of lymphoid malignancies in patients with ataxia-telangiectasia.

BACKGROUND Patients with ataxia-telangiectasia (A-T) are at an increased risk for developing lymphoid malignancies, yet the appropriate therapy remains unknown. Radiation therapy at conventional doses results in destruction of normal tissue, which has suggested that full-dose chemotherapy might result in unacceptable toxicity in A-T patients with cancer. PROCEDURE The medical records of 412 A-T patients were reviewed to identify those patients who developed lymphoid malignancies and to analyze the type and duration of therapy, events during therapy, and off-therapy follow-up. RESULTS Of 74 A-T patients with lymphoid malignancies, 32 patients received chemotherapy. The 21 patients treated with standard chemotherapy had a significantly better median survival (9 months, range, 1-162+ months vs. 5 months, range, 0.5-28 months) (P = 0.03) and complete remission rate (76% vs. 9%) (P = 0.001) than the 11 treated with reduced dose chemotherapy. Three of the 21 full-dose chemotherapy patients required dose reductions because of neutropenia. Seven of the 14 patients exposed to 1,200 mg/m2 or greater of cyclophosphamide developed hemorrhagic cystitis. All three patients exposed to bleomycin developed pulmonary disease which was fatal in two. Of the 16 standard-dose chemotherapy patients who achieved a complete remission, two remain disease-free, five have died of recurrent disease, and five died of pulmonary disorders and four of other causes while in remission. CONCLUSIONS Standard-dose chemotherapy should be given to each A-T patient with a lymphoid malignancy unless additional physical or emotional problems make it unlikely that the patient will benefit. Morbidity and mortality may be reduced by prophylaxis against hemorrhagic cystitis and early detection and treatment of pulmonary disorders.

Telomerase activity and telomere length in pediatric patients with malignancies undergoing chemotherapy

Telomerase activity and telomere length in mononuclear cells (MNCs) and granulocytes from peripheral blood (PB) and bone marrow (BM) specimens were studied in pediatric acute leukemia (ALL, n = 15; AML, n = 1) and pediatric solid tumor (ST) patients (n = 9) at diagnosis, during and after chemotherapy. In four ST patients, tumor tissue was also available. For comparative analysis, MNCs from healthy donors (n = 53) were analyzed. Telomerase was evaluated using a modified telomeric repeat amplification protocol (TRAP) assay, and telomere length by terminal restriction fragment (TRF) analysis. At diagnosis, high telomerase activity was detected in MNCs from all leukemia patients, which was similar to the activity from ST biopsy specimens. This exceeded by 10- to 20-fold the activity in PB MNCs from ST patients and healthy donors (P < 0.05). granulocyte fractions lacked telomerase activity in all groups. bm mncs in leukemia patients revealed a four-fold higher telomerase activity than pb (P = 0.005). After induction chemotherapy and response to treatment, telomerase activity decreased to borderline or undetectable levels in PB MNCs in leukemia (P < 0.01). average telomeres in pb mncs from pediatric patients were significantly longer (n = 25; 10.9 kbp) than telomeres in PB and BM MNCs from adult healthy donors (7.45 kbp) (P < 0.0001). at diagnosis, telomeres were shorter from bm compared to pb specimens in leukemia (P < 0.05), and two peak trfs were observed corresponding to the malignant and normal cell clones. with the attainment of remission, the lower trf peak, reflecting the leukemic population, was lost. in leukemia patients, mean trfs increased on average 2.2 kbp after induction chemotherapy, but decreased thereafter on consolidation and maintenance chemotherapy (1 kbp). this was comparable to an average telomere loss of 1.2 kbp in pb specimens from st patients after chemotherapy. in all patients, telomere loss in granulocytes as compared to mncs was more pronounced with 1.8 vs 1 kbp, respectively (P = 0.014). Our results demonstrate that at diagnosis, telomerase was consistently and highly upregulated in BM and PB specimens in leukemia, decreased after induction therapy, and correlated with remission. BM specimens in leukemia had higher telomerase activity, probably due to the greater leukemic burden than in PB. Telomeres were significantly longer in children than in adults, but shortened as a consequence of chemotherapy with repeated cycles of hematopoietic regeneration. In acute leukemia, with the loss of the leukemic burden after induction chemotherapy, longer mean TRFs were found, a reflection of the repopulation with normal cells. Our findings suggest that telomerase activity may be useful in the management of childhood malignancies. The significance of telomere length shortening in pediatric patients undergoing chemotherapy and possible telomere regeneration after myelosuppressive treatment remain to be determined.

Clinical and laboratory features of 178 children with recurrent epistaxis.

Purpose To determine the clinical and laboratory features of 178 children referred for the evaluation of recurrent epistaxis to an outpatient hematology clinic in a university medical center. Patients and Methods Medical records of 3681 outpatient pediatric hematology referrals were retrospectively review, and 178 children with recurrent epistaxis from 1985 to 1999 were identified. Historic (other bleeding symptoms: gingival bleeding, easy bruising, menorrhagia, and gross blood in the urine or stool; duration and severity of the epistaxis episodes; and family history of bleeding) and laboratory (complete blood count and coagulation tests) data were analyzed. Results There were 103 boys and 75 girls with a median age of 84 months (range 15–219 months). Sixty-seven percent (n = 119) did not have a coagulopathy diagnosed and 33% (n = 59) did. The diagnoses included von Willebrand disease in 33, platelet aggregation disorders in 10, thrombocytopenia in seven, mild factor VIII deficiency in three, Bernard–Soulier syndrome in two, factor VII deficiency in one, factor IX deficiency in one, and factor XI deficiency in one, and coagulation inhibitor in one. Of the historic data, only a family history of bleeding was predictive of diagnosing a coagulopathy (P = 0.023). The duration and severity of the epistaxis and the presence of other bleeding symptoms had no predictive value. Children with a coagulopathy diagnosed had a longer median partial thromboplastin time (PTT) (33.1 vs. 30.5 seconds;P = 0.012). Conclusions One-third of children presenting with recurrent epistaxis have a diagnosable coagulopathy. A positive family history and a prolonged PTT are useful predictive data.

Intravenous anesthesia with propofol for painful procedures in children with cancer

Objective To study the safety and efficacy of propofol-based intravenous anesthesia in children with cancer undergoing painful procedures. Methods This study is a retrospective analysis of data collected from 52 consecutive children who underwent 335 procedures using propofol anesthesia. These data were routinely collected in all patients: time to induction, duration of the procedure, time to recover, and the doses of the drugs used. Monitoring with electrocardiography and pulse oximetry was continuous during the procedure; blood pressures were recorded before and after the procedure and every 5 to 10 minutes during the procedure. The patients received one of these four propofol-based intravenous regimens according to the anesthesiologists preference: propofol only; propofol plus fentanyl; propofol plus midazolam; or propofol, fentanyl, and midazolam. The efficacy of sedation was rated by this scoring system: 3 = no movement during procedure; 2 = minimal movement that did not interfere with the procedure; 1 = moderate movement requiring physical restraint to complete the procedure. Results There were six episodes of mild hypoxia (oxygen saturation 85%–94%) and one episode of laryngospasm. None required intubation. Two patients had agitation and one patient had emesis during the postrecovery phase. There was no difference in the efficacy of sedation between the four regimens. Patients receiving the combination of propofol, fentanyl, and midazolam received the least amount of propofol and required the least time to recover. There were no life-threatening complications. Conclusions Propofol-based anesthesia, when administered by an anesthesiologist in a controlled setting, is safe and effective for performing painful procedures in children with cancer.

Diamond–Blackfan anemia with mandibulofacial dystostosis is heterogeneous, including the novel DBA genes TSR2 and RPS28

Patients with physical findings suggestive of Treacher Collins syndrome (TCS) or mandibulofacial dysostosis (MFD) and macrocytic anemia diagnostic of Diamond–Blackfan anemia (DBA) have been reported. Disease‐causing genes have been identified for TCS and other MFDs. Mutations in several ribosomal protein genes and the transcription factor GATA1 result in DBA. However, no disease‐causing mutation had been identified in the reported patients with the combination of TCS/MFD and DBA phenotype, and we hypothesized that pathogenic mutations in a single gene could be identified using whole exome analysis. We studied probands from six unrelated families. Combining exome analysis and Sanger sequencing, we identified likely pathogenic mutations in 5/6 families. Two mutations in unrelated families were seen in RPS26, the known DBA10 gene. One variant was predicted to affect mRNA splicing, and the other to lead to protein truncation. In another family a likely pathogenic X‐linked mutation affecting a highly conserved residue was found in TSR2, which encodes a direct binding partner of RPS26. De novo mutations affecting the RPS28 start codon were found in two unrelated probands, identifying RPS28 as a novel disease gene. We conclude that the phenotype combining features of TCS with DBA is genetically heterogeneous. Each of the pathogenic variants identified is predicted to impede ribosome biogenesis, which in turn could result in altered cell growth and proliferation, causing abnormal embryologic development, defective erythropoiesis and reduced growth. The phenotype combining TCS/MFD and DBA is highly variable, overlaps with DBA and lies within the phenotypic spectrum of ribosomopathies.

Infections Due to Nontuberculous Mycobacteria in Children with Leukemia

We reviewed the spectrum of infections due to nontuberculous mycobacteria (NTM) in children with leukemia. Three children acquired such infections. One patient developed pneumonia after the cessation of chemotherapy when Mycobacterium xenopi was identified in his lung biopsy specimen. He required 2 years of treatment with antituberculous agents and clarithromycin. Cultures of central and peripheral blood specimens from two patients yielded Mycobacterium fortuitum and Mycobacterium chelonae, respectively. Broviac catheters were likely the source of infection. Removal of the catheters and antibiotic treatment resulted in cure. Central venous catheters in leukemic children are potential sources of infection. For febrile neutropenic children with leukemia who do not respond to antibiotic therapy, cultures positive for diphtheroids or negative routine bacterial and fungal cultures should raise a suspicion for infections due to NTM. Systemic infections may require up to 2 years of therapy. Removal of the infected catheters during persistent or recurrent infections in necessary for control of the infection.

Lymphocyte-predominant Hodgkin disease in children.

Purpose To describe the clinicobiological features, treatment, treatment outcome, and sequelae of children with lymphocyte-predominant Hodgkin disease. Patients and Methods The authors performed a retrospective chart review of 754 patients with Hodgkin disease diagnoses at New York Medical College and St. Jude Childrens Research Hospital from 1962 to 2000 to identify those with lymphocyte-predominant histology. Hematopathologists at the treating institutions reviewed stored tissue specimens and reconfirmed the histopathology of each case. Results Fifty-one children (44 boys, 7 girls) were identified. The median age was 10.5 years (range 3.2–18.5); five children were younger than age 60 months. The median duration of lymphadenopathy before diagnosis was 4 months (range 0.5–30). Thirty-six children had stage 1 disease, eight had stage 2 disease, four had stage 3 disease, and three had stage 4 disease. Fifteen children underwent staging laparotomy, and four of these were upstaged. Treatment comprised combined modality therapy (n = 27), radiation therapy alone (n = 17), and chemotherapy alone (n = 7). Four children had a Hodgkin disease recurrence. Forty-eight (94%) patients were alive and disease-free at a median follow-up of 8 years (range 0.4–32.6). Eleven patients had long-term, therapy-related adverse effects (cardiac, infertility, pulmonary, and second malignant neoplasms). Three patients died. Two died of complications of second malignant neoplasms and one died of infectious complications after Hodgkin disease recurrence. Conclusions Children with lymphocyte-predominant Hodgkin disease respond favorably to a variety of treatment modalities and are ideal candidates for less toxic therapy.

Long-term outcome of chronic idiopathic thrombocytopenic purpura in children.

Objectives: Children with chronic idiopathic thrombocytopenic purpura (ITP) generally have a favorable outcome, but it is not known whether there are any prognostic factors to predict outcome. The objectives of this study were to assess the spontaneous remission rate and the prognostic significance of age, gender, initial platelet count, initial treatment, and response to treatment. Methods: In this retrospective review of 62 consecutive children with chronic ITP, 37 were girls and 27 were 10 years of age or older (median age 9 years; range, 0.75-19). Results: Thirty-five patients (56%) achieved spontaneous remission (remission without splenectomy), 30 of them (48%) within 4 years from diagnosis. Twenty-eight (45%) were complete remissions (platelet counts of ≥100,000) and 7 (11%) were partial remissions (50,000-99,000). There was no significant difference in the spontaneous remission rate between the younger (<10 years) and older children (55.8% vs. 57.1%, P = 0.95) or between boys and girls (56% vs. 56.7%, P = 0.98). Similarly, platelet count at initial diagnosis, initial therapy, or response to initial therapy did not have any prognostic significance. All 14 patients who underwent splenectomy achieved complete remission. Conclusions: More than 50% of children with chronic ITP achieve spontaneous remission. Age, gender, platelet count at initial diagnosis, initial treatment, and response to initial treatment do not have any prognostic significance toward the outcome of chronic ITP.