Diagnostic Challenge Presented by “Hb F only” Pattern In Newborn Screening for Hemoglobinopathies: Approach to Accurate Diagnosis

Abstract 5173

Newborn screening for hemoglobinopathies has been proven to be an effective means of early diagnosis of clinically significant hemoglobinopathies such as sickle cell syndromes with early recruitment to clinical care and resultant reduction in morbidity and mortality. Neonatal screening for hemoglobinopathies is currently performed in 50 states utilizing screening methods such as hemoglobin electrophoresis or isoelectric focusing (IEF) complemented by High Performance Liquid Chromatography (HPLC) in a large number of states. These two methods yield accurate diagnosis of hemoglobinopathies especially of sickle cell disease in the vast majority of cases, although occasionally additional diagnostic approaches such as DNA based tests may be required for precise genetic diagnosis. One of the diagnostic challenges in newborn screening for hemoglobinopathies is posed by the observation of “Hb F only” pattern in electrophoresis/IEF and HPLC. This pattern can result from a spectrum of inherited globin gene abnormalities including homozygous β⁰ thalassemia, homozygous δβ-thalassemia, homozygous deletional HPFH (Hereditary Persistence of Fetal Hemoglobin), or a compound heterozygosity for any two of the above abnormalities. Since the clinical phenotype is dependent upon the underlying molecular defect, accurate diagnosis of the genotype in “Hb F only” cases is crucial in predicting the clinical course and planning therapy. The clinical spectrum of “Hb F only” (100% Hb F) cases in newborn screening ranges from severe transfusion dependent homozygous β⁰ thalassemia to completely asymptomatic homozygous deletional HPFH with intermediate phenotypes due to compound heterozygosity for any two of the above mentioned abnormalities. The prediction of the clinical phenotype based on the genotype is therefore important in ascertaining the significance of “Hb F only” cases. We present two cases of “F-only” phenotype detected during newborn screening, which were found to result from two distinct genotypes. Case 1 was an 8 mo old African-American male born in Sinai Hospital of Baltimore, MD at 39 weeks of gestation who was found to have 100% Hb F during newborn screening. He was referred to Pediatric Hematology at Sinai Hospital at two months of age, at which time a Hb electrophoresis confirmed the presence of 100% Hb F. His CBC showed a Hb of 13 g/dl, Hct 39%, RBC 4.68, MCV 84 fl, retic count 2.9%. At 8 months, he continued to be asymptomatic. She had a follow-up visit at 20 months of age and had Hbg 11.7, MCV 62; no repeat electrophoresis at that time. Work-up at MCG Sickle Cell Center Laboratory confirmed 100% Hb F with normal Hb and Hct. A gap PCR with primers specific for HPFH-1 and HPFH-2 deletions showed that the patient was a compound heterozygote for these two deletions. These findings were confirmed with family studies, which showed that the father was heterozygous for the HPFH-1 deletion, and the mother was heterozygous for HPFH-2. Case 2 was an African-American female born at UNC Hospital in Chapel Hill, NC. Newborn screening showed 100% Hb F. At age 2, she had a Hb of 11 g/dl, Hct 31.3%, MCV 80 fl. Hb analyses by HPLC revealed 96.2% Hb F, 2.9% Hb A, 0.9% Hb A2. Molecular analyses showed that the patient was a compound heterozygote for the HPFH-1 deletion and the β-globin promoter mutation, -90 C→T. These results were confirmed with family studies, which revealed that the father carried the HPFH-1 deletion while the mother was heterozygous for the rare -90 C→T mutation in the β-globin promoter. These two cases serve to illustrate the importance of detailed molecular diagnostic work up for the definite diagnosis of the genotype in “Hb F only” or 100% Hb F cases identified during neonatal screening for hemoglobinopathies. Since the phenotype of “100% Hb F” in a newborn can result from a variety of genotypes with a wide ranging spectrum of phenotypic expression, accurate genetic diagnosis is crucial in predicting the clinical course and determining the appropriate course of action. A stepwise approach to the work-up and the use of a combination of sensitive methods such as IEF, HPLC, and gene analysis will be helpful in establishing the correct diagnosis.