The province of Ontario has a total population of approximately 10 million people, with approxima... more The province of Ontario has a total population of approximately 10 million people, with approximately 20% being of African, Southeast Asian, East Indian, Mediterranean, or Middle Eastern ancestry in whom the gene frequency for hemoglobinopathies is relatively high. In 1989, the Ontario Ministry of Health funded the establishment of the Provincial Hemoglobinopathy DNA Diagnostic Laboratory located at the McMaster University Medical Centre in Hamilton, Ontario. The Laboratory provides DNA analysis to identify the globin gene mutations in carriers and affected individuals, and performs prenatal diagnosis for severe hemoglobinopathies. Annually, more than 400 patient samples are referred to the Laboratory for investigation, of which 25-35 are fetal samples from pregnancies at risk for either homozygous alpha-thalassemia, beta-thalassemia major, or sickling disorders. We have detected more than 70 different globin gene mutations, including several mutations not previously reported in the literature. Here we present examples of the approaches used to detect globin gene mutations in a heterogeneous "at risk" population such as in Ontario, and discuss the impact of this service on patient care, genetic counselling, and the incidence of severe hemoglobinopathies in Ontario.
Traditional diagnosis of hemoglobinopathies rely on separation and accurate quantification of hem... more Traditional diagnosis of hemoglobinopathies rely on separation and accurate quantification of hemoglobin (Hb) fractions using alkaline and acid electrophoresis, isoelectric focusing (IEF) and/or High Performance Liquid Chromatography (HPLC). Recent reports have suggested capillary electrophoresis (CE) as an alternate method for screening. We evaluated a new CE system the Sebia CapillaryS 2 (Evry Cedex, France) and compared it to IEF and HPLC. For this evaluation samples referred to our laboratory for routine hemoglobinopathy screening or as cord bloods sent for confirmation of a variant detected as a result of newborn screening were analyzed using all three techniques. IEF was performed with the Resolve IEF kit (Perkin Elmer,Wallac Oy, Finland). HPLC was done on the BioRad Variant II (Munich, Germany) using the Hb A2/Hb A1c Dual program. The CE was performed using the “Capillarys Hemoglobin(E) kit”. Suspected rare variants were further investigated with DNA investigations, including PCR and direct nucleotide sequencing. Variant hemoglobins were detected in 156 of 764 samples (Table 1). The correlation between IEF, HPLC and CE was excellent. One variant (Hb Toulon) was not detected by the CE but ran with Hb F. The CE system did not report the Hb F value on samples with very low (< 1.0%) Hb F by HPLC. Two cases of Hb Constant Spring were identified by CE but not by either IEF or HPLC. The correlation of both Hb F and Hb A2 reported between the CE and HPLC systems were excellent (R…
A 10-year-old Danish girl with congenital anemia is described. At birth, she had severe anemia an... more A 10-year-old Danish girl with congenital anemia is described. At birth, she had severe anemia and erythroblastosis and was transfused a number of times during the first year. The need for transfusions has since declined steadily. Her reticulocyte counts varied between 2% and 15%. and her bone marrow aspirate showed some dyserythropoietic features. Her hemoglobin F level was consistently elevated, EMOGLOBINS containing either embryonic [-globin H or e-globin chains are the predominant hemoglobins in fetuses during the first 7 weeks ofgestation.' Subsequently, {-globin chains are present in circulating erythrocytes at a very low but detectable level until about 3 months after birth.2 Embryonic t-globin chains also are present at a very low level until about 20 weeks of ge~tation.~ In normal adults, embryonic [-and e-globin chains are not detected in circulating erythrocytes even by very sensitive immunoassays.2-6 [Globin chains are present in minute amounts in the erythrocytes of adult carriers of (-SEA/), (-MED/), and (-SPAN/) types of deletional a-thala~semias.~-' In addition, both {-and €-globin chains are present in erythroblasts derived from erythroid burst-forming units (BFU-E) cultured in vitro from patients with juvenile chronic myelocytic We describe here a 10-year-old girl with congenital anemia. Both embryonic {-globin and €-globin chains were found to be present in some of her circulating erythrocytes. MATERIALS AND METHODS Hematologic studies. Peripheral blood counts, red blood cell indices, and reticulocyte counts were determined by standard laboratory procedures, including the use of electronic cell counters. Hemoglobin (Hb) studies included cellulose acetate electrophoresis at pH 8.4, as well as determinations of HbA2 either by microcolumn chromatography or by high-performance liquid chromatography (HPLC) and of HbF by either isoelectric focusing, a modification of the Betke method, or HPLC.10'12 Triton X-100/acid urea/polyacrylamide gel electrophoresis of globin chains was carried out as previously described? Genomic DNA was isolated from peripheral blood, digested with restriction endonucleases, separated by
reproducibility in practice among interpretation from IHC and FISH, especially for cases with equ... more reproducibility in practice among interpretation from IHC and FISH, especially for cases with equivocal HER2 results. Alternative HER2 assessment using molecular inversion probe array (MIP) microarray has recently been reported. In this study, we report on a 64-yearold patient with 2 cm breast cancer equivocal for HER2 amplification by IHC and FISH. Material and methods: Representative section was tested for HER2 overexpression and amplification by IHC and FISH, respectively. Due to equivocal result, additional tumor block was tested by FISH and both tumor blocks were subjected to MIP microarray using OncoScan FFPE Assay kit (Affymetrix). Copy number analysis was performed by OncoScan Console and data were reviewed by OncoScan™ Nexus Express (BioDiscovery). Results: Initial tumor block was equivocal for HER2 overexpression (2+) and negative for HER2 amplification (average HER2 copies/ cell 3.5, HER2/CEP17 ratio 1.70). FISH performed on additional tumor block was equivocal for HER2 amplification (average HER2 copies/cell 4.3, HER2/CEP17 ratio 1.6). MIP microarray showed 1q, 16p, and 17q gain (3×) and 16q and 17p loss for both sites. Using previously established threshold of 4, the case is best interpreted as negative for HER2 amplification. Conclusion: MIP microarray might be an alternative test for breast cancer patient with equivocal HER2 status.
Molecular variants including single nucleotide variants (SNVs), copy number variants (CNVs) and f... more Molecular variants including single nucleotide variants (SNVs), copy number variants (CNVs) and fusions can be detected in the clinical setting using deep targeted sequencing. These assays support low limits of detection using little genomic input material. They are gaining in popularity in clinical laboratories, where sample volumes are limited, and low variant allele fractions may be present. However, data on reproducibility between laboratories is limited. Using a ring study, we evaluated the performance of 7 Ontario laboratories using targeted sequencing panels. All laboratories analysed a series of control and clinical samples for SNVs/CNVs and gene fusions. High concordance was observed across laboratories for measured CNVs and SNVs. Over 97% of SNV calls in clinical samples were detected by all laboratories. Whilst only a single CNV was detected in the clinical samples tested, all laboratories were able to reproducibly report both the variant and copy number. Concordance for ...
We identify and characterize a novel Po-thalassemia mutation that is associated with an unusually... more We identify and characterize a novel Po-thalassemia mutation that is associated with an unusually high level of hemoglobin (Hb) A, in the heterozygote. This newly discovered mutation is caused by a 532-basepair deletion that extends from positions-454 to +78 relative to the mRNA cap site of the ETA-THALASSEMIA is an autosomal recessive dis-B ease characterized by a deficiency (P'-thalassemia) or absence (Po-thalassemia) of p-globin chain synthesis. In general, the Poand P'-thalassemia mutations are caused by base substitutions and small insertions or deletions in the P-globin gene. Almost 100 such mutations have now been reported.'.' In addition, there are larger deletions that result in P"-thalassemia. In this report, we identify and characterize a Pothalassemia mutation caused by a deletion of the 5' coding region of the P-globin gene and several hundred basepairs (bp) of upstream sequences. In the heterozygote, this deletion is associated with an unusually high level of hemoglobin (Hb) A, and typical P-thalassemia trait. The endpoints of this new P-globin gene deletion reaffirm the notion that removal of the 5' region of the P-globin gene and adjacent upstream sequences is associated with elevated Hb A, levels, and further localize the essential sequences that, when deleted, can lead to this phenotypic anomaly.
... 1 , 2 , Barry Eng 2 , Margaret Patterson 2 , Manuel D. Carcao 3 , Lebe Chang 3 , Nancy F. Oli... more ... 1 , 2 , Barry Eng 2 , Margaret Patterson 2 , Manuel D. Carcao 3 , Lebe Chang 3 , Nancy F. Olivieri 3 and David HK Chui 2 ... Higgs DR, Vickers MA, Wilkie AOM, Pretorius I.-M., Jarman AP, Weatherall D. J. A Review of the Molecular Genetics of the Human α-Globin Gene Cluster. ...
We describe a case of Hb S/β-thalassemia (thal) involving a 468 bp deletion that removes the β-gl... more We describe a case of Hb S/β-thalassemia (thal) involving a 468 bp deletion that removes the β-globin gene promoter but leaves the coding regions intact. This is the second report of this deletion, and our family study establishes that this deletion causes β0-thal with unusually high levels of Hb A2 and Hb F. As with other genotypes involving deletions of the 5′ region of the β-globin gene, our patient had a mild form of Hb S/β-thal.
We report three new b-globin gene promoter mutations identified in newborns with hemoglobin (Hb) ... more We report three new b-globin gene promoter mutations identified in newborns with hemoglobin (Hb) profiles consistent with Hb S/b +-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.
Two novel pthalassemia mutations are described. The first mutation, found in an Italian family, i... more Two novel pthalassemia mutations are described. The first mutation, found in an Italian family, is a G+A substitution in nucleotide (nt) +22 relative to the B-globin gene Cap site. This mutation creates a cryptic ATG initiation codon, the utilization of which for translation would result in premature termination 36 bp 3' downstream. The second mutation, HE P-THALASSEMIAS are hereditary disorders due T to mutations in the P-globin gene on chromosome 11, leading to either decreased or absent P-globin chain synthesis. The most common genetic defects in P-thalassemias are due t o point mutations or mutations involving small deletions or insertions in the @-globin gene. More than 100 such naturally occurring mutations are known.' The characterization of these mutations is essential for performing the prenatal diagnosis of fetuses at risk. Furthermore, they provide additional insight into the identification or confirmation of nucleotide sequences that are important in the regulation and expression of the p-globin gene? In this investigation, we have identified two new P-thalassemia mutations in the 5' and 3' noncoding regions of the P-globin gene. The proposed mechanisms whereby these two mutations cause a decrease in normal P-globin chain synthesis are novel. MATERIALS AND METHODS Hematologic studies. Peripheral blood counts and erythrocyte indices were determined using an electronic cell counter. Hemoglobin (Hb) electrophoresis was performed on cellulose acetate membranes at pH 8.6. Hb F was measured with a modification of the alkali-denaturation method of Chui et a1 as has been previously described.' Genomic DNA was isolated from peripheral blood, digested with restriction endonucleases, separated by electrophoresis through 0.8% agarose slab gel, and transferred to nylon membranes using standard procedures. Probes specific for Gene mappingstudies.
We report two novel β-thalassemia (β-thal) deletions involving the 5&... more We report two novel β-thalassemia (β-thal) deletions involving the 5' region of the β-globin gene (HBB). The first deletion spans 538 bp and removes the β-globin promoter, 5' untranslated region (5'UTR) and most of exon 1. This deletion was identified in a 3-year-old Vietnamese boy with non transfusion dependent Hb E (HBB: c.79G>A)/β0-thal. The second deletion spans 1517 bp and removes the β-globin gene promoter, 5'UTR, and exons 1 and 2. This deletion was identified in two unrelated adults of European descent who had β-thal trait with unusually high Hb A2 levels. Deletions such as these are generally associated with higher levels of Hb A2 and Hb F than typical β-thal alleles, which may ameliorate the severity of the disease.
ABSTRACT A 73-year-old female of Dutch descent was referred for investigation of a high oxygen af... more ABSTRACT A 73-year-old female of Dutch descent was referred for investigation of a high oxygen affinity hemoglobin variant. The β-globin gene was amplified using the polymerase chain reaction. Direct nucleotide sequencing of the polymerase chain reaction amplified DNA revealed that she is heterozygous for a novel p-globin gene mutation at codon 139, AAT→TAT. The resulting hemoglobin variant has been designated Hb Aurora [β139 (H17) Asn→Tyr].
@Globin chain expression in carriers of a number of deletional a-thalassemias is investigated by ... more @Globin chain expression in carriers of a number of deletional a-thalassemias is investigated by radioimmunoassay. In a few cases, &-globin mRNAs are also studied. {-Globin chains are detected in (-SEA/), (-MED/), and (-SPAN/) deletions, but not in six other deletional mutations. These results suggest that the DNA element capable of suppressing @globin expression in adult erythroid cells is present within the (-SPAN/) deletion, while the DNA fragment between the 5' breakpoints of the (-SA/) and the (-SEA/) deletions may contain sequences necessary for HE HUMAN EMBRYONIC (-globin chains are a-glo-Reticulocyte RNA was extracted from peripheral reticulocyte polysomes as previously reported.I2 Each RT/PCR reaction contained two sets of specific primers so that two specific messenger RNAs (mRNAs) (a and 1; or (and 0) were coamplified. The
Sickle cell disease (SCD) is most often due to homozygosity for the hemoglobin sickle (Hb S) miss... more Sickle cell disease (SCD) is most often due to homozygosity for the hemoglobin sickle (Hb S) missense mutation of the β-globin gene (HBB:c.20A>T). SCD can also result from compound heterozygosity for Hb S and other β-chain variants or β-thalassemia (β-thal). Loss-of-function point mutations of the β-globin gene that abolish (β0) or reduce (β+) production of normal β-chains are the most common cause of β-thal, with a minority of alleles being larger deletions. Patients with Hb S/β0-thal typically have severe SCD, whereas residual β-chain synthesis in Hb S/β+-thal is associated with lower hemoglobin S concentrations and less severe disease. It has long been recognized that the high-level production of β-like chains throughout development is controlled by a cis regulatory element, the β-globin locus control region (βLCR). The βLCR is located 5.7 kb to 21.2 kb upstream of the ε-globin gene, and consists of five DNase I hypersensitivity sites designated HS1 through HS5. Twelve naturally occurring βLCR deletions have been reported, most resulting in complete loss of expression of the β-like genes and a carrier phenotype that resembles (εγδβ)0-thal. In these patients, neonatal hemolytic anemia is common due to impaired γ-chain synthesis required for Hb F. Once the γ→β switch has occurred during the first six months of infancy, the phenotype resolves to one of thalassemia trait with normal Hb A2. While the phenotype is well established for carriers of large deletions that remove all or most of the HS regions, the contribution of individual HS regions to β-globin gene expression in human has yet to be elucidated. To this end, it is important to identify and characterize naturally occurring deletions that involve individual HS regions or combinations thereof. Here, we report a case of SCD due to a novel βLCR deletion involving only HS3 and HS4. The proband is a 6-year old boy born to healthy non-sanguineous parents of Caribbean decent. Newborn screening was negative for SCD, with the Hb profile being consistent with Hb S trait (Hb F 79.1%, Hb A 6.0%, Hb S 4.0%, Hb Bart’s 9.1%). Postnatally there was no significant jaundice or clinically diagnosed anemia. The proband had no clinical complaints and growth and development were normal until age 5 years when he was diagnosed with SCD during an admission for unexplained abdominal pain and an enlarged spleen. He was noted to have microcytic anemia (Hb 87 g/L, MCV 68.2 fL), and the peripheral blood smear showed sickle cells, Howell-Jolly bodies and target cells. The Hb profile was suggestive of Hb S/β+-thal with 19.4% Hb A, 72.7% Hb S, and Hb A2 within normal range. He has had one vasoocclusive event since the diagnosis and had tonsillectomy for obstructive sleep apnea. Nucleotide sequence analysis demonstrated that the proband is heterozygous for the Hb S mutation (HBB:c.20A>T) with no other mutations of the β-globin gene. He was also shown to be heterozygous for the 3.7 kb α-globin gene deletion (-α3.7/αα). As this genotype does not explain the reduced expression of Hb A and SCD phenotype,…
We report two Italian-Canadian families with α(+)-thalassemia (α(+)-thal) trait caused by a novel... more We report two Italian-Canadian families with α(+)-thalassemia (α(+)-thal) trait caused by a novel mutation of the translation initiation codon of the α1-globin gene (ATG>AAG or HBA1:c.2T>A). This is the tenth reported α-thal mutation involving the translation initiation codon or the conserved Kozak consensus sequences of the HBA2 or HBA1 genes.
We report two Canadian families in which there are four carriers of a novel (G)gamma((A)gammadelt... more We report two Canadian families in which there are four carriers of a novel (G)gamma((A)gammadeltabeta)(0)-thalassemia deletion. The patients all have mild microcytosis and hypochromia, and elevated levels of Hb F ranging from 9.7 to 17.3%. The precise endpoints of the deletion have been identified and are unique relative to other forms of (G)gamma((A)gammadeltabeta)(0)-thal reported in the literature. The deletion encompasses approximately 55.1 kb, beginning approximately 1.6 kb downstream of the (G)gamma-globin gene and extending approximately 29.0 kb downstream of the beta-globin gene.
We report a case of δβ-thalassemia (δβ-thal) trait in an adult male originally from Sudan. Multip... more We report a case of δβ-thalassemia (δβ-thal) trait in an adult male originally from Sudan. Multiplex ligation-dependent probe amplification (MLPA) was used to localize the approximate boundaries of the deletion, followed by polymerase chain reaction (PCR) amplification and sequence analysis of the junction fragment to determine the precise deletion endpoints. The deletion spans 9594 bp, with the 5' deletion endpoint located 1560 bp upstream of the δ-globin gene and the 3' endpoint within the second intervening sequence (IVS-II) of the β-globin gene.
The province of Ontario has a total population of approximately 10 million people, with approxima... more The province of Ontario has a total population of approximately 10 million people, with approximately 20% being of African, Southeast Asian, East Indian, Mediterranean, or Middle Eastern ancestry in whom the gene frequency for hemoglobinopathies is relatively high. In 1989, the Ontario Ministry of Health funded the establishment of the Provincial Hemoglobinopathy DNA Diagnostic Laboratory located at the McMaster University Medical Centre in Hamilton, Ontario. The Laboratory provides DNA analysis to identify the globin gene mutations in carriers and affected individuals, and performs prenatal diagnosis for severe hemoglobinopathies. Annually, more than 400 patient samples are referred to the Laboratory for investigation, of which 25-35 are fetal samples from pregnancies at risk for either homozygous alpha-thalassemia, beta-thalassemia major, or sickling disorders. We have detected more than 70 different globin gene mutations, including several mutations not previously reported in the literature. Here we present examples of the approaches used to detect globin gene mutations in a heterogeneous "at risk" population such as in Ontario, and discuss the impact of this service on patient care, genetic counselling, and the incidence of severe hemoglobinopathies in Ontario.
Traditional diagnosis of hemoglobinopathies rely on separation and accurate quantification of hem... more Traditional diagnosis of hemoglobinopathies rely on separation and accurate quantification of hemoglobin (Hb) fractions using alkaline and acid electrophoresis, isoelectric focusing (IEF) and/or High Performance Liquid Chromatography (HPLC). Recent reports have suggested capillary electrophoresis (CE) as an alternate method for screening. We evaluated a new CE system the Sebia CapillaryS 2 (Evry Cedex, France) and compared it to IEF and HPLC. For this evaluation samples referred to our laboratory for routine hemoglobinopathy screening or as cord bloods sent for confirmation of a variant detected as a result of newborn screening were analyzed using all three techniques. IEF was performed with the Resolve IEF kit (Perkin Elmer,Wallac Oy, Finland). HPLC was done on the BioRad Variant II (Munich, Germany) using the Hb A2/Hb A1c Dual program. The CE was performed using the “Capillarys Hemoglobin(E) kit”. Suspected rare variants were further investigated with DNA investigations, including PCR and direct nucleotide sequencing. Variant hemoglobins were detected in 156 of 764 samples (Table 1). The correlation between IEF, HPLC and CE was excellent. One variant (Hb Toulon) was not detected by the CE but ran with Hb F. The CE system did not report the Hb F value on samples with very low (< 1.0%) Hb F by HPLC. Two cases of Hb Constant Spring were identified by CE but not by either IEF or HPLC. The correlation of both Hb F and Hb A2 reported between the CE and HPLC systems were excellent (R…
A 10-year-old Danish girl with congenital anemia is described. At birth, she had severe anemia an... more A 10-year-old Danish girl with congenital anemia is described. At birth, she had severe anemia and erythroblastosis and was transfused a number of times during the first year. The need for transfusions has since declined steadily. Her reticulocyte counts varied between 2% and 15%. and her bone marrow aspirate showed some dyserythropoietic features. Her hemoglobin F level was consistently elevated, EMOGLOBINS containing either embryonic [-globin H or e-globin chains are the predominant hemoglobins in fetuses during the first 7 weeks ofgestation.' Subsequently, {-globin chains are present in circulating erythrocytes at a very low but detectable level until about 3 months after birth.2 Embryonic t-globin chains also are present at a very low level until about 20 weeks of ge~tation.~ In normal adults, embryonic [-and e-globin chains are not detected in circulating erythrocytes even by very sensitive immunoassays.2-6 [Globin chains are present in minute amounts in the erythrocytes of adult carriers of (-SEA/), (-MED/), and (-SPAN/) types of deletional a-thala~semias.~-' In addition, both {-and €-globin chains are present in erythroblasts derived from erythroid burst-forming units (BFU-E) cultured in vitro from patients with juvenile chronic myelocytic We describe here a 10-year-old girl with congenital anemia. Both embryonic {-globin and €-globin chains were found to be present in some of her circulating erythrocytes. MATERIALS AND METHODS Hematologic studies. Peripheral blood counts, red blood cell indices, and reticulocyte counts were determined by standard laboratory procedures, including the use of electronic cell counters. Hemoglobin (Hb) studies included cellulose acetate electrophoresis at pH 8.4, as well as determinations of HbA2 either by microcolumn chromatography or by high-performance liquid chromatography (HPLC) and of HbF by either isoelectric focusing, a modification of the Betke method, or HPLC.10'12 Triton X-100/acid urea/polyacrylamide gel electrophoresis of globin chains was carried out as previously described? Genomic DNA was isolated from peripheral blood, digested with restriction endonucleases, separated by
reproducibility in practice among interpretation from IHC and FISH, especially for cases with equ... more reproducibility in practice among interpretation from IHC and FISH, especially for cases with equivocal HER2 results. Alternative HER2 assessment using molecular inversion probe array (MIP) microarray has recently been reported. In this study, we report on a 64-yearold patient with 2 cm breast cancer equivocal for HER2 amplification by IHC and FISH. Material and methods: Representative section was tested for HER2 overexpression and amplification by IHC and FISH, respectively. Due to equivocal result, additional tumor block was tested by FISH and both tumor blocks were subjected to MIP microarray using OncoScan FFPE Assay kit (Affymetrix). Copy number analysis was performed by OncoScan Console and data were reviewed by OncoScan™ Nexus Express (BioDiscovery). Results: Initial tumor block was equivocal for HER2 overexpression (2+) and negative for HER2 amplification (average HER2 copies/ cell 3.5, HER2/CEP17 ratio 1.70). FISH performed on additional tumor block was equivocal for HER2 amplification (average HER2 copies/cell 4.3, HER2/CEP17 ratio 1.6). MIP microarray showed 1q, 16p, and 17q gain (3×) and 16q and 17p loss for both sites. Using previously established threshold of 4, the case is best interpreted as negative for HER2 amplification. Conclusion: MIP microarray might be an alternative test for breast cancer patient with equivocal HER2 status.
Molecular variants including single nucleotide variants (SNVs), copy number variants (CNVs) and f... more Molecular variants including single nucleotide variants (SNVs), copy number variants (CNVs) and fusions can be detected in the clinical setting using deep targeted sequencing. These assays support low limits of detection using little genomic input material. They are gaining in popularity in clinical laboratories, where sample volumes are limited, and low variant allele fractions may be present. However, data on reproducibility between laboratories is limited. Using a ring study, we evaluated the performance of 7 Ontario laboratories using targeted sequencing panels. All laboratories analysed a series of control and clinical samples for SNVs/CNVs and gene fusions. High concordance was observed across laboratories for measured CNVs and SNVs. Over 97% of SNV calls in clinical samples were detected by all laboratories. Whilst only a single CNV was detected in the clinical samples tested, all laboratories were able to reproducibly report both the variant and copy number. Concordance for ...
We identify and characterize a novel Po-thalassemia mutation that is associated with an unusually... more We identify and characterize a novel Po-thalassemia mutation that is associated with an unusually high level of hemoglobin (Hb) A, in the heterozygote. This newly discovered mutation is caused by a 532-basepair deletion that extends from positions-454 to +78 relative to the mRNA cap site of the ETA-THALASSEMIA is an autosomal recessive dis-B ease characterized by a deficiency (P'-thalassemia) or absence (Po-thalassemia) of p-globin chain synthesis. In general, the Poand P'-thalassemia mutations are caused by base substitutions and small insertions or deletions in the P-globin gene. Almost 100 such mutations have now been reported.'.' In addition, there are larger deletions that result in P"-thalassemia. In this report, we identify and characterize a Pothalassemia mutation caused by a deletion of the 5' coding region of the P-globin gene and several hundred basepairs (bp) of upstream sequences. In the heterozygote, this deletion is associated with an unusually high level of hemoglobin (Hb) A, and typical P-thalassemia trait. The endpoints of this new P-globin gene deletion reaffirm the notion that removal of the 5' region of the P-globin gene and adjacent upstream sequences is associated with elevated Hb A, levels, and further localize the essential sequences that, when deleted, can lead to this phenotypic anomaly.
... 1 , 2 , Barry Eng 2 , Margaret Patterson 2 , Manuel D. Carcao 3 , Lebe Chang 3 , Nancy F. Oli... more ... 1 , 2 , Barry Eng 2 , Margaret Patterson 2 , Manuel D. Carcao 3 , Lebe Chang 3 , Nancy F. Olivieri 3 and David HK Chui 2 ... Higgs DR, Vickers MA, Wilkie AOM, Pretorius I.-M., Jarman AP, Weatherall D. J. A Review of the Molecular Genetics of the Human α-Globin Gene Cluster. ...
We describe a case of Hb S/β-thalassemia (thal) involving a 468 bp deletion that removes the β-gl... more We describe a case of Hb S/β-thalassemia (thal) involving a 468 bp deletion that removes the β-globin gene promoter but leaves the coding regions intact. This is the second report of this deletion, and our family study establishes that this deletion causes β0-thal with unusually high levels of Hb A2 and Hb F. As with other genotypes involving deletions of the 5′ region of the β-globin gene, our patient had a mild form of Hb S/β-thal.
We report three new b-globin gene promoter mutations identified in newborns with hemoglobin (Hb) ... more We report three new b-globin gene promoter mutations identified in newborns with hemoglobin (Hb) profiles consistent with Hb S/b +-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.
Two novel pthalassemia mutations are described. The first mutation, found in an Italian family, i... more Two novel pthalassemia mutations are described. The first mutation, found in an Italian family, is a G+A substitution in nucleotide (nt) +22 relative to the B-globin gene Cap site. This mutation creates a cryptic ATG initiation codon, the utilization of which for translation would result in premature termination 36 bp 3' downstream. The second mutation, HE P-THALASSEMIAS are hereditary disorders due T to mutations in the P-globin gene on chromosome 11, leading to either decreased or absent P-globin chain synthesis. The most common genetic defects in P-thalassemias are due t o point mutations or mutations involving small deletions or insertions in the @-globin gene. More than 100 such naturally occurring mutations are known.' The characterization of these mutations is essential for performing the prenatal diagnosis of fetuses at risk. Furthermore, they provide additional insight into the identification or confirmation of nucleotide sequences that are important in the regulation and expression of the p-globin gene? In this investigation, we have identified two new P-thalassemia mutations in the 5' and 3' noncoding regions of the P-globin gene. The proposed mechanisms whereby these two mutations cause a decrease in normal P-globin chain synthesis are novel. MATERIALS AND METHODS Hematologic studies. Peripheral blood counts and erythrocyte indices were determined using an electronic cell counter. Hemoglobin (Hb) electrophoresis was performed on cellulose acetate membranes at pH 8.6. Hb F was measured with a modification of the alkali-denaturation method of Chui et a1 as has been previously described.' Genomic DNA was isolated from peripheral blood, digested with restriction endonucleases, separated by electrophoresis through 0.8% agarose slab gel, and transferred to nylon membranes using standard procedures. Probes specific for Gene mappingstudies.
We report two novel β-thalassemia (β-thal) deletions involving the 5&... more We report two novel β-thalassemia (β-thal) deletions involving the 5' region of the β-globin gene (HBB). The first deletion spans 538 bp and removes the β-globin promoter, 5' untranslated region (5'UTR) and most of exon 1. This deletion was identified in a 3-year-old Vietnamese boy with non transfusion dependent Hb E (HBB: c.79G>A)/β0-thal. The second deletion spans 1517 bp and removes the β-globin gene promoter, 5'UTR, and exons 1 and 2. This deletion was identified in two unrelated adults of European descent who had β-thal trait with unusually high Hb A2 levels. Deletions such as these are generally associated with higher levels of Hb A2 and Hb F than typical β-thal alleles, which may ameliorate the severity of the disease.
ABSTRACT A 73-year-old female of Dutch descent was referred for investigation of a high oxygen af... more ABSTRACT A 73-year-old female of Dutch descent was referred for investigation of a high oxygen affinity hemoglobin variant. The β-globin gene was amplified using the polymerase chain reaction. Direct nucleotide sequencing of the polymerase chain reaction amplified DNA revealed that she is heterozygous for a novel p-globin gene mutation at codon 139, AAT→TAT. The resulting hemoglobin variant has been designated Hb Aurora [β139 (H17) Asn→Tyr].
@Globin chain expression in carriers of a number of deletional a-thalassemias is investigated by ... more @Globin chain expression in carriers of a number of deletional a-thalassemias is investigated by radioimmunoassay. In a few cases, &-globin mRNAs are also studied. {-Globin chains are detected in (-SEA/), (-MED/), and (-SPAN/) deletions, but not in six other deletional mutations. These results suggest that the DNA element capable of suppressing @globin expression in adult erythroid cells is present within the (-SPAN/) deletion, while the DNA fragment between the 5' breakpoints of the (-SA/) and the (-SEA/) deletions may contain sequences necessary for HE HUMAN EMBRYONIC (-globin chains are a-glo-Reticulocyte RNA was extracted from peripheral reticulocyte polysomes as previously reported.I2 Each RT/PCR reaction contained two sets of specific primers so that two specific messenger RNAs (mRNAs) (a and 1; or (and 0) were coamplified. The
Sickle cell disease (SCD) is most often due to homozygosity for the hemoglobin sickle (Hb S) miss... more Sickle cell disease (SCD) is most often due to homozygosity for the hemoglobin sickle (Hb S) missense mutation of the β-globin gene (HBB:c.20A>T). SCD can also result from compound heterozygosity for Hb S and other β-chain variants or β-thalassemia (β-thal). Loss-of-function point mutations of the β-globin gene that abolish (β0) or reduce (β+) production of normal β-chains are the most common cause of β-thal, with a minority of alleles being larger deletions. Patients with Hb S/β0-thal typically have severe SCD, whereas residual β-chain synthesis in Hb S/β+-thal is associated with lower hemoglobin S concentrations and less severe disease. It has long been recognized that the high-level production of β-like chains throughout development is controlled by a cis regulatory element, the β-globin locus control region (βLCR). The βLCR is located 5.7 kb to 21.2 kb upstream of the ε-globin gene, and consists of five DNase I hypersensitivity sites designated HS1 through HS5. Twelve naturally occurring βLCR deletions have been reported, most resulting in complete loss of expression of the β-like genes and a carrier phenotype that resembles (εγδβ)0-thal. In these patients, neonatal hemolytic anemia is common due to impaired γ-chain synthesis required for Hb F. Once the γ→β switch has occurred during the first six months of infancy, the phenotype resolves to one of thalassemia trait with normal Hb A2. While the phenotype is well established for carriers of large deletions that remove all or most of the HS regions, the contribution of individual HS regions to β-globin gene expression in human has yet to be elucidated. To this end, it is important to identify and characterize naturally occurring deletions that involve individual HS regions or combinations thereof. Here, we report a case of SCD due to a novel βLCR deletion involving only HS3 and HS4. The proband is a 6-year old boy born to healthy non-sanguineous parents of Caribbean decent. Newborn screening was negative for SCD, with the Hb profile being consistent with Hb S trait (Hb F 79.1%, Hb A 6.0%, Hb S 4.0%, Hb Bart’s 9.1%). Postnatally there was no significant jaundice or clinically diagnosed anemia. The proband had no clinical complaints and growth and development were normal until age 5 years when he was diagnosed with SCD during an admission for unexplained abdominal pain and an enlarged spleen. He was noted to have microcytic anemia (Hb 87 g/L, MCV 68.2 fL), and the peripheral blood smear showed sickle cells, Howell-Jolly bodies and target cells. The Hb profile was suggestive of Hb S/β+-thal with 19.4% Hb A, 72.7% Hb S, and Hb A2 within normal range. He has had one vasoocclusive event since the diagnosis and had tonsillectomy for obstructive sleep apnea. Nucleotide sequence analysis demonstrated that the proband is heterozygous for the Hb S mutation (HBB:c.20A>T) with no other mutations of the β-globin gene. He was also shown to be heterozygous for the 3.7 kb α-globin gene deletion (-α3.7/αα). As this genotype does not explain the reduced expression of Hb A and SCD phenotype,…
We report two Italian-Canadian families with α(+)-thalassemia (α(+)-thal) trait caused by a novel... more We report two Italian-Canadian families with α(+)-thalassemia (α(+)-thal) trait caused by a novel mutation of the translation initiation codon of the α1-globin gene (ATG>AAG or HBA1:c.2T>A). This is the tenth reported α-thal mutation involving the translation initiation codon or the conserved Kozak consensus sequences of the HBA2 or HBA1 genes.
We report two Canadian families in which there are four carriers of a novel (G)gamma((A)gammadelt... more We report two Canadian families in which there are four carriers of a novel (G)gamma((A)gammadeltabeta)(0)-thalassemia deletion. The patients all have mild microcytosis and hypochromia, and elevated levels of Hb F ranging from 9.7 to 17.3%. The precise endpoints of the deletion have been identified and are unique relative to other forms of (G)gamma((A)gammadeltabeta)(0)-thal reported in the literature. The deletion encompasses approximately 55.1 kb, beginning approximately 1.6 kb downstream of the (G)gamma-globin gene and extending approximately 29.0 kb downstream of the beta-globin gene.
We report a case of δβ-thalassemia (δβ-thal) trait in an adult male originally from Sudan. Multip... more We report a case of δβ-thalassemia (δβ-thal) trait in an adult male originally from Sudan. Multiplex ligation-dependent probe amplification (MLPA) was used to localize the approximate boundaries of the deletion, followed by polymerase chain reaction (PCR) amplification and sequence analysis of the junction fragment to determine the precise deletion endpoints. The deletion spans 9594 bp, with the 5' deletion endpoint located 1560 bp upstream of the δ-globin gene and the 3' endpoint within the second intervening sequence (IVS-II) of the β-globin gene.
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Papers by Barry Eng