David H. K. Chui

Filipino β-thalassemia due to a large deletion: identification of the deletion end points and polymerase chain reaction (PCR)-based diagnosis

The gene frequency of β-thalassemia among Filipinos is estimated to be 0.02, although little is known about the mutations involved. Recently, an extensive β-thalassemia deletion was reported in several unrelated individuals of Filipino descent. The deletion begins approximately 4 kb upstream of the β-globin gene, and extends 3′ beyond the β-globin gene. In this report, we identify the 5′ and 3′ deletion endpoints and present a polymerase chain reaction (PCR) strategy for rapid DNA diagnosis of the Filipino β-thalassemia deletion.

A rapid and simple electrophoretic method for the detection of mutations involving small insertion or deletion: application to β-thalassemia

SummaryThe 1.8-kb β-globin gene fragments of DNAs from individuals heterozygous for nine different β-thalassemia mutations involving 1, 2, 3, 4, or 25 basepair (bp) insertions or deletions were amplified by the polymerase chain reaction (PCR). The PCR products were subjected to electrophoresis on aqueous 8% polyacrylamide gel. In each heterozygote with either a 2 to 25 bp deletion, but not with a 1 bp insertion, two slower migrating bands representing heteroduplexes in addition to the 1.8-kb homoduplex band were seen. The electrophoretic positions of these slower migrating bands were characteristic of each mutation studied. By co-amplification with known normal DNA, it was also possible to distinguish DNAs from normal individuals and from individuals who are homozygous for the small insertion/deletion mutations. These studies demonstrate that the heteroduplex formation generated in PCR can be applied as a simple method in the diagnosis of insertion/deletion mutations involving 2 to 25 bp in β-thalassemias as well as in other genetic disorders.

Spectrum of beta-thalassemia mutations in Egypt.

Carrier screening and prenatal diagnosis programs have successfully reduced the incidence of β-thalassemia (thal) in countries where the carrier rates are high (1-3). The incidence of β-thal is particularly high in Egypt (4) due to the combined effects of high carrier rates (5) and consanguineous marriages (6). As a prerequisite to establishing effective programs for prenatal diagnosis, it is important to identify the spectrum of mutations within the population. To this end, we have surveyed the β-thal mutations for a cohort of 55 transfusion-dependent β-thal patients from the Alexandria region of Egypt and the adjacent rural governorates. Affected siblings were not included in the study. The patients were either Moslem (46/55, 84%) or Christian (9/55, 16%). Almost three-quarters (40/55, 73%) of the patients had consanguineous parents.

Identification of a New β-Thalassemia Nonsense Mutation [Codon 59 (AAG→TAG)]

We report the case of an Italian-American girl with transfusion-dependent b-thalassemia (thal). Blood samples were obtained from the proband and the b-globin gene was screened for the most common b-thal mutations using the polymerase chain reaction (PCR) and the allele-specific amplification refractory mutation system (ARMS) as described by Old et al. (1). This test demonstrated that the proband is positive for the common IVS-I-110 (G!A) bþ-thal mutation (HBB g.202G!A). Direct nucleotide sequence analysis of the entire b-globin gene was used to identify the other mutant allele. The b-globin gene was amplified by PCR and sequenced on the ABI PRISM 310 Genetic Analyzer using internal primers and the BigDye TM Terminator Cycle Sequencing Kit (PE Applied BioSystems, Foster City, CA, USA). The 50 region of the b-globin gene was amplified using primers P1 (50-TCCTAAGCCAGTGCCAGAAG-30, positions 181 to 162 relative to mRNA Cap site) and P2 (50-TCATTCGTCTGTTTCCCATT-30, positions þ610 to þ591), and sequenced using internal primers P3 (50-AATAGACCAA

Dominant beta-thalassemia due to a newly identified frameshift mutation in exon 3 (codon 113, GTG-->Tg).

Provincial Hemoglobinopathy DNA Diagnostic Laboratory, Hamilton Regional Laboratory Medicine Program, Hamilton, Ontario L8N 3Z5, Canada Department of Pathology and Molecular Medicine, McMaster University Medical Centre, Room 3N17, McMaster University Faculty of Health Sciences, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada St. Joseph’s Hospital, Hamilton, Ontario L8N 4A6, Canada Division of Medical Genetics, Department of Pediatrics, University of Saskatchewan and Royal University Hospital, Saskatoon Saskatchewan S7N 0W8, Canada

IDENTIFICATION OF TWO NEW β-THALASSEMIA SPLICE MUTATIONS: IVS-I-1 (G → C) AND IVS-I (−2) (A → C)

b-Thalassemia (thal) is a common hereditary anemia caused by mutations that reduce or abolish expression from the b-globin genes. More than 180 different b-thal mutations have been reported, the vast majority of which are point mutations or small insertions=deletions within the b-gene (1). b-Thal is most common among populations originating from regions of the world where malaria is or has been endemic, including the Mediterranean Basin, the Middle East, Africa, the Indian subcontinent, and Southeast Asia. Relatively small numbers of common b-thal mutations can be found for each high-risk population, thereby facilitating efficient strategies for mutation detection. In heterogeneous multicultural populations such as Canada, mutation screening must accommodate a broader spectrum of mutations. Here we report two cases of high Hb A2 b-thal trait, due to previously unreported mutations involving the IVS-I donor splice sequences. Both patients are 20-year-old Canadian women whose ethnic backgrounds are uncertain. Patient #1 had a history of thalassemia trait on her mother’s side of the family, which was of Italian descent. She was pregnant at the time of examination and her hematology results were as follows: Hb 8.6 g=dL, MCV 72.7 fL, MCH 22.5 pg, MCHC HEMOGLOBIN, 26(1), 87–89 (2002)

Identification of a new hemoglobin variant: Hb St. Joseph's [β77(EF1)His→Leu]

We report the case of a 30-year-old Canadian man who was originally referred for Hb A1c quantification using cation exchange high performance liquid chromatography (HPLC) (VARIANT II; Bio-Rad Laboratories, Hercules, CA, USA). His hematology results are as follows: Hb 14.8 g dL , MCV 91.0 fL, Hb A2 3.3%, Hb F< 1.0%, Hb X 43.0%. The variant migrated ahead of Hb A on the b-Thalassemia Short Program, to the position of Hb J using isoelectrofocusing (IEF), and between Hb A and Hb F using acid gel electrophoresis. The available sample was insufficient for studies of stability or oxygen affinity. The b-globin gene was amplified by polymerase chain reaction (PCR) and sequenced on the ABI PRISM 1 310 Genetic Analyzer using internal primers and the BigDye Terminator Cycle Sequencing Kit (PE Applied BioSystems, Foster City, CA, USA). The 50 region of the b-globin gene was amplified using primers P1 (50-TCCTAAGCCAGTGC CAGAAG-30, positions 181 to 162 relative to the mRNA Cap site) and P2 (50TCATTCGTCTGTTTCCCATT-30, positions þ610 to þ591), and sequenced using internal primers P3 (50-AATAGACCAATAGGCAGAG-30, positions þ 261 to þ243) and P4 (50GCAGGTTGGTATCAAGGTT-30, positions þ140 to þ158). The 30 region of the b-globin gene was amplified by PCR using primers P5 (50-GTGTACACATATTGACCAAA-30,

DEVELOPMENTAL HEMOGLOBINS IN THE MOUSE

Publisher Summary Erythropoiesis in the normal fetal mouse occurs first in the yolk sac blood islands beginning at around day 8 of gestation. These erythroid cells are subsequently released into the circulation where they proliferate and mature in a relatively synchronized manner. These circulating primitive nucleated erythrocytes synthesize three embryonic hemoglobins, EI (X2Y2), EII (α2Y2), and EIII (α2Z2). The second site of erythropoiesis occurs in the fetal liver shortly after day 10 of gestation. The fetal hepatic erythroblasts proliferate and differentiate within the hepatic parenchyma and are released into the circulation as non-nucleated reticulocytes beginning at around day 12 of gestation. These definitive erythrocytes contain adult hemoglobin, α2β2. Some of the experiments indicate that the primitive erythroid cell line of yolk sac blood island origin produce embryonic hemoglobins early in gestation but later in gestation, these erythroid cells are also capable of producing adult hemoglobins in addition to embryonic hemoglobins. The in vitro culture experiments indicate that there are hemopoietic stem cells in circulation at a time when the fetal liver is not yet formed.