Heterogeneity of hemoglobin H disease in childhood.

BACKGROUND: Early diagnosis during newborn screening or infancy has enabled the observation of the natural history of hemoglobin H disease, a subtype of α-thalassemia. METHODS: We analyzed longitudinal clinical data for patients with hemoglobin H disease arising from the deletion of three of four α-globin genes (HbH) and from hemoglobin H Constant Spring (HCS), caused by the deletion of two α-globin genes and the Constant Spring mutation. RESULTS: We identified 86 patients with hemoglobin H disease (48 through newborn screening). Of these patients, 60 (70%) had HbH, 23 (27%) had HCS, and 3 (3%) had other, nondeletional forms of hemoglobin H disease. The parental ethnic background was Asian in 81% of patients, Hispanic in 5%, and African American in 3%, whereas mixed ancestry was observed in 10% of patients. Among the patients with deletional hemoglobin H disease, 15% had one or both parents with African-American ancestry. Growth was normal in patients with HbH during the first decade, but growth deficits began during infancy in those with HCS. Anemia was more severe in patients with HCS at all ages (P<0.001). Acute worsening of anemia with infections requiring urgent blood transfusion was observed in patients with HCS but not in those with HbH. The probability of receiving at least one transfusion by the age of 20 years was 3% for patients with HbH and 80% for those with HCS (P<0.001). Among patients with HCS, transfusions occurred in 13% of infants and 50% of children under the age of 6 years; splenectomy was associated with a significant improvement in hemoglobin levels (P=0.01) and a reduction in the number of transfusions. CONCLUSIONS: HCS should be recognized as a distinct thalassemia syndrome with a high risk of life-threatening anemia during febrile illnesses. HbH was not associated with an increased rate of severe anemia with infections and was managed without blood transfusions. Many patients with these disorders had mixed ethnic backgrounds, which highlights the need for extended newborn screening in populations that are traditionally considered to be at low risk for hemoglobin H disease.

Detection of a novel ßδ-globin fusion gene, anti-lepore Hb CHORI (ß(through IVS-I-57)/δ(from IVS-I-101)), by multiplex ligation-dependent probe amplification.

Anti-Lepore hemoglobins (Hbs) are rare ßδ fusion variants that arise from non homologous crossover during meiosis. Using multiplex ligation-dependent probe amplification (MLPA), we identified a novel anti-Lepore Hb in an individual with an ambiguous Hb variant detected on routine screening by electrophoresis and high performance liquid chromatography (HPLC). The results of MLPA revealed duplication of ß and δ gene segments. Resolution of the rearrangement by DNA sequencing confirmed a novel anti-Lepore Hb, molecularly distinct from Hb P-Nilotic, which we have named anti-Lepore Hb CHORI (Children's Hospital Oakland Research Institute) (ß(through IVS-I-57)/δ(from IVS-I-101)).

Application of multiplex ligation-dependent probe amplification to screen for ß-globin cluster deletions: detection of two novel deletions in a multi ethnic population.

Hereditary persistence of fetal hemoglobin (HPFH) and δß-thalassemia (δß-thal) are heterogeneous disorders caused by deletions within the ß-globin gene cluster. When combined with other ß-thal mutations or structural hemoglobin (Hb) variants, these deletions give rise to clinical phenotypes ranging from an asymptomatic condition to ß-thal major (ß-TM). Overlap in hematological parameters and variability in expression of Hbs A2 and F make molecular testing necessary to distinguish clinically relevant deletions. Multiplex ligation-dependent probe amplification (MLPA) was used to screen for ß-globin gene cluster deletions in 49 unresolved samples referred for a suspected ß-thal anomaly. The 1.39 kb Black ß(0), 3.5 kb Thai ß(0), 118 kb Filipino ß(0), 11.8 kb Black (δß)(0), 13.4 kb Sicilian (δß)(0), 35.8 kb Black ((A)γδß)0, Hb Lepore-Boston-Washington (Hb LBW) and HPFH-2 deletions, and two novel deletions, a 61.7 kb Pakistani ß(0) deletion and an ((A)γδß)(0) deletion, were identified in 15 cases. Detection of both known and unknown deletional Hb disorders provides for appropriate clinical management and genetic counseling.

Identification of three novel Hb F variants: Hb F-Hayward [Gγ1(NA1)Gly→Asp, GGT>GAT], Hb F-Chori-I [AγT16(A13)Gly→Asp, GGC>GAC] and Hb F-Chori-II [AγI29(B11)Gly→Glu, GGA>GAA].

Three new γ-globin chain mutations were identified in four newborn samples referred to the Hemoglobinopathy Reference Laboratory at the Children's Hospital & Research Center Oakland, Oakland, CA, USA, for diagnostic testing. The variants were characterized by sequencing of amplified γ-globin genes. These three novel variants have been named Hb F-Hayward [(G)γ1(NA1)Gly→Asp, GGT>GAT], Hb F-Chori-I [(A)γ(T)16(A13)Gly→Asp, GGC>GAC] and Hb F-Chori-II [(A)γ(I)29(B11)Gly→Glu, GGA>GAA], respectively. No functional studies could be performed.

Detection of anti-Lepore Hb P-Nilotic by multiplex ligation-dependent probe amplification.

Anti-Lepore hemoglobins (Hbs) are rare ßδ fusion variants that arise from non homologous crossover during meiosis. We describe the application of multiplex ligation-dependent probe amplification (MLPA) to test for a suspected anti-Lepore Hb in an individual with an ambiguous Hb variant detected on routine screening by electrophoresis and high performance liquid chromatography (HPLC). The results of MLPA revealed duplication of ß and δ gene segments consistent with an anti-Lepore ßδ fusion gene. Resolution of the hybrid gene by DNA sequencing identified the variant as Hb P-Nilotic (ß31-δ50) HBB/HBD hybrid; HBB through 22; HBD from 50 (NG_000007.3:g.63290_70702dup). Multiples ligation-dependent probe amplification allows for rapid detection of hybrid globin variants caused by duplications in the ß-globin gene locus.

Newborn screening for hemoglobinopathies in California.

BACKGROUND: Newborn screening (NBS) for hemoglobinopathies facilitates early identification of affected individuals to ensure the prompt institution of comprehensive medical care for affected newborns in California. When linked to extensive follow-up and education, NBS has been shown to significantly reduce mortality in children with sickle cell disease. Due to changing immigration patterns from Asia and Latin America, the State of California has witnessed an increased prevalence of clinically significant hemoglobin (Hb) disorders, including those resulting from novel genotypes. In 1999, newborn screening for Hb H disorders was incorporated in the statewide hemoglobinopathy screening program. PROCEDURE: Primary screening for hemoglobin variants was performed using high performance liquid chromatography. Confirmatory testing on hemoglobinopathy mutations was performed by electropheresis techniques and genotyping methods. RESULTS: Of 530,000 newborn samples screened annually in California, 2,118 samples were referred to the Hemoglobin Reference Laboratory (HRL) for confirmatory testing between January 1, 1998 and June 30, 2006 (0.05%). Sickle cell disease was most frequently observed (1 in 6,600 births) followed by alpha-thalassemia (1 in 9,000 births) and beta-thalassemia disease (1 in 55,000 births). The confirmatory analysis modified the initial screening in 5% of cases and revealed 25 rare or new genotypes. Diverse ethnicities were associated with hemoglobin mutations including Southeast Asian, Black, Indian/Asian, Middle Eastern, and Hispanic. CONCLUSIONS: The California hemoglobinopathy screening program provides accurate diagnosis of hemoglobinopathies. Increasing incidence of diverse mutations require new strategies of laboratory screening, counseling, and patient management.

Three new beta-globin gene promoter mutations identified through newborn screening.

We report three new beta-globin gene promoter mutations identified in newborns with hemoglobin (Hb) profiles consistent with Hb S/beta(+)-thalassemia (thal) (Hbs FSA). All three mutations are in close proximity to the conserved ATAA sequence located at positions -31 to -28 relative to the mRNA Cap site. Two cases involved single base substitutions at positions -25 (G-->C) and -32 (C-->T). The remaining case involved the deletion of two bases (-AA) at positions -27 and -26.

Three new alpha-thalassemia point mutations ascertained through newborn screening.

We report three new alpha-thalassemia (thal) point mutations detected during newborn screening for hemoglobinopathies. The first mutation is a single nucleotide deletion (-A) that abolishes the translation initiation codon of the alpha2-globin gene, detected in a newborn of Hmong ethnicity who carried the Southeast Asian alpha(0)-thal deletion (alpha(T)alpha/- -(SEA)). The second mutation, a frameshift caused by a single nucleotide deletion in exon 2 of the alpha1-globin gene [codon 78 (-C)], was detected in a Black/Chinese newborn who also carried the Southeast Asian alpha0-thal deletion (alphaalpha(T)/- -(SEA)). The third mutation was a frameshift in exon 3 of the alpha2-globin gene, codons 113/114 (-C). This mutation was detected in a newborn who carried the 3.7 kb alpha(+)-thal deletion (alpha(T)alpha/-alpha(3.7)).