Mahin Azimi

Newborn Screening for Hemoglobinopathies in California

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.

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.

Universal Newborn Screening for Hb H Disease in California

Newborn screening is an accepted public health measure to ensure that appropriate health care is provided in a timely manner to infants with hereditary/metabolic disorders. Alpha-thalassemia is a common hemoglobin (Hb) disorder, and causes Hb H (beta4) disease, and usually fatal homozygous alpha(0)-thalassemia, also known as Hb Barts (gamma4) hydrops fetalis syndrome. In 1996, the State of California began to investigate the feasibility of universal newborn screening for Hb H disease. Initial screening was done on blood samples obtained by heel pricks from newborns, and stored as dried blood spots on filter paper. Hb Barts levels were measured as fast-moving Hb by automated high-performance liquid chromatography (HPLC) identical to that currently used in newborn screening for sickle cell disease. Subsequent confirmation of Hb H disease was done by DNA-based diagnostics for alpha-globin genotyping. A criterion of 25% or more Hb Barts as determined by HPLC detects most, if not all cases of Hb H disease, and few cases of alpha-thalassemia trait. From January, 1998, through June, 2000, 89 newborns were found to have Hb H disease. The overall prevalence for Hb H disease among all newborns in California is approximately 1 per 15,000. Implementation of this program to existing newborn hemoglobinopathy screening in populations with significant proportions of southeast Asians is recommended. The correct diagnosis would allow affected infants to be properly cared for, and would also raise awareness for the prevention of homozygous alpha(0)-thalassemia or Hb Barts hydrops fetalis syndrome.

Application of an expanded multiplex genotyping assay for the simultaneous detection of Hemoglobin Constant Spring and common deletional α‐thalassemia mutations

Hemoglobin Constant Spring (HbCS) is the most common nondeletional α‐thalassemia variant causing HbH disease, making its detection crucial in populations at risk. Universal newborn screening for HbH is carried out in California. Identification of α‐thalassemia genotypes responsible for HbH and HbH‐CS requires rapid, accurate and cost‐effective genotyping methods suitable for population screening. We incorporated the HbCS mutation into our existing seven‐plex genotyping assay for common α‐thalassemia deletions. To assess the feasibility and diagnostic utility of this expanded multiplex gap‐PCR assay, we determined genotypic frequencies of HbCS in samples referred for α‐thalassemia testing between 1 January 2006 and 31 December 2008. During the 3‐year study period, 1436 samples were genotyped for α‐thalassemia. HbH‐CS accounted for 23 (13%) of the 176 cases of HbH disease identified. In a subset of 145 newborns referred by the California NBS program with an elevated Hb Bart’s level at birth, HbH disease was confirmed in 134 (93%) and HbH‐CS identified in 13 (10%) of these. This expanded genotyping assay has proven to be a rapid, reliable and clinically useful diagnostic tool for the detection of HbH‐CS disease.

Detection of Anti-Lepore Hb P-Nilotic by Multiplex Ligation-Dependent Probe Amplification

Anti-Lepore hemoglobins (Hbs) are rare βδ 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 β and δ gene segments consistent with an anti-Lepore βδ fusion gene. Resolution of the hybrid gene by DNA sequencing identified the variant as Hb P-Nilotic (β31-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 β-globin gene locus.

Three New α-Thalassemia Point Mutations Ascertained Through Newborn Screening

We report three new α-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 α2-globin gene, detected in a newborn of Hmong ethnicity who carried the Southeast Asian α0-thal deletion (αTα/– –SEA). The second mutation, a frameshift caused by a single nucleotide deletion in exon 2 of the α1-globin gene [codon 78 (−C)], was detected in a Black/Chinese newborn who also carried the Southeast Asian α0-thal deletion (ααT/– –SEA). The third mutation was a frameshift in exon 3 of the α2-globin gene, codons 113/114 (−C). This mutation was detected in a newborn who carried the 3.7 kb α+-thal deletion (αTα/–α3.7).

Application of Multiplex Ligation-Dependent Probe Amplification to Screen for β-Globin Cluster Deletions: Detection of Two Novel Deletions in a Multi Ethnic Population

Hereditary persistence of fetal hemoglobin (HPFH) and δβ-thalassemia (δβ-thal) are heterogeneous disorders caused by deletions within the β-globin gene cluster. When combined with other β-thal mutations or structural hemoglobin (Hb) variants, these deletions give rise to clinical phenotypes ranging from an asymptomatic condition to β-thal major (β-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 β-globin gene cluster deletions in 49 unresolved samples referred for a suspected β-thal anomaly. The 1.39 kb Black β0, 3.5 kb Thai β0, 118 kb Filipino β0, 11.8 kb Black (δβ)0, 13.4 kb Sicilian (δβ)0, 35.8 kb Black (Aγδβ)0, Hb Lepore-Boston-Washington (Hb LBW) and HPFH-2 deletions, and two novel deletions, a 61.7 kb Pakistani β0 deletion and an (Aγδβ)0 deletion, were identified in 15 cases. Detection of both known and unknown deletional Hb disorders provides for appropriate clinical management and genetic counseling.

Three New β-Globin Gene Promoter Mutations Identified Through Newborn Screening

We report three new β-globin gene promoter mutations identified in newborns with hemoglobin (Hb) profiles consistent with Hb S/β+-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.

Hemoglobinopathy testing: the significance of accuracy and pitfalls in HbA 2 determination

The WHO estimates that more than 270 million individuals are carriers of thalassemia. Each year, 500 000 births are registered with a thalassemia gene or a hemoglobin variant. Eighty percent of those are born in developing countries, 83% concern sickle-cell disease, and 17% severe thalassemias [1]. Identification of hemoglobinopathies is easier to perform by routine hematological or biochemical tests than by DNA analysis [2]. When routine test is abnormal, additional investigations might be needed for definitive diagnosis and DNA analysis required in cases of risk assessment [3]. Identification of carriers for thalassemias and other hemoglobinopathies associates determination of red cell indices with qualitative and quantitative analysis of Hb fractions. Microcytosis with mean cellular volume (MCV) < 80 fL, hypochromia with mean cellular hemoglobin (MCH) < 27 pg, and a relatively elevated RBC count suggest either a thalassemia mutation or a structural variant with thalassemic effects, such as Hb E (b26 Glu > Lys) or Hb Knossos (b27 Ala > Ser). Abnormal findings on the complete blood count (CBC) should prompt additional testing [4]. As the most common Hb variants (Hb S and Hb C) present usually with normocytic indices, an analysis of the Hb fractions should always be performed together with CBC specially in populations with high prevalence of these traits. During the second year of life, HbA2 is raised in most carriers of b-thalassemia to values between 4% and 8%. Low or borderline values (3.2–3.9%) in a microcytic patient may indicate iron depletion or ‘normal HbA2 bthalassemia’ conditions. Levels higher than 8% may indicate Hb Lepore or Hb E requiring further investigation. Several methods are available for HbA2 testing, but the two most frequently used are automated high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). While measurement of HbA2 is essential for routine identification of b-thalassemia carriers, DNA analysis is needed to characterize the mutation[4]. Often more information is required for interpretation of the hematological condition and should be considered: (i) iron studies to exclude iron-deficiency anemia [e.g., measurement of serum iron transferrin, ferritin, or zinc-protoporphyrin (ZnPP)], (ii) recent blood transfusion (3 months), (iii) ethnic origin, (iv) clinical history, (v) drug therapy (e.g., hydroxyurea, antiretroviral drugs).

Detection of a Novel βδ-Globin Fusion Gene, Anti-Lepore Hb CHORI (βthrough IVS-I-57/δfrom IVS-I-101), by Multiplex Ligation-Dependent Probe Amplification

Abstract Anti-Lepore hemoglobins (Hbs) are rare βδ 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 β 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) (βthrough IVS-I-57/δfrom IVS-I-101).