Homozygous Hemoglobin Constant Spring Research Articles

Generation of human induced pluripotent stem cell line (MUi034-A) from an unusual case of hydrops fetalis associated with homozygous hemoglobin Constant Spring

Hemoglobin Constant Spring (HbCS) is unstable hemoglobin resulting from a nucleotide substitution at the termination codon of the HBA2 gene (c.427 T > C). The homozygous state for HbCS is non-transfusion dependent in adults. Nevertheless, severe anemia is often observed in fetuses. Here, human induced pluripotent stem cell line MUi034-A was generated from peripheral blood CD34+ hematopoietic stem/progenitor cells (HSPCs) derived from a 14-year-old female with homozygous HbCS who had a history of severe anemia and hydrops during fetal period. The MUi034-A cell line represented embryonic-like characteristics as they expressed specific pluripotency markers, differentiated into the three germ layers, and retained normal karyotyping.

Prenatal diagnosis and management of homozygous hemoglobin constant spring disease.

To describe fetal management of homozygous hemoglobin constant spring (Hb CS). Six fetuses with homozygous Hb CS disease undergoing intrauterine transfusion (IUT) were comprehensively reviewed. Additionally, when combined with 8 cases previously reported, a total of 14 cases were analyzed. The first clues of diagnosis were hydropic changes suggesting fetal anemia. Increased cardiothoracic diameter ratio (CTR) was the most sensitive sonographic marker but slowly changed after IUT, whereas MCA-PSV was the most sensitive in response to IUT. Pre-IUT Hb varied from 1.1% to 6.8%. Gestational age at diagnosis was 17-29 (22.8 ± 3.3) weeks. Rates of adverse obstetric outcomes were relatively high; preterm birth: 35.7%, low birthweight: 42.9%, and fetal growth restriction: 28.6%. All showed good response to IUT with disappearance of hydropic signs and all survived without short-term complications. Their anemia gradually improved in childhood and transfusion independent. Homozygous Hb CS can cause severe fetal anemia. Early diagnosis and IUT can improve neonatal outcomes, probably preventing adult diseases caused by fetal programming.

Clinical Course of Homozygous Hemoglobin Constant Spring in Pediatric Patients.

Hemoglobin (Hb) Constant Spring is an alpha-globin gene variant due to a mutation of the stop codon resulting in the elongation of the encoded polypeptide from 141 to 172 amino acid residues. Patients with homozygous Hb Constant Spring are usually mildly anemic. We retrospectively describe clinical manifestations, diagnosis, laboratory investigations, treatment, and associated findings in pediatric patients with homozygous Hb Constant Spring followed-up at Srinagarind Hospital. Sixteen pediatric cases (5 males and 11 females) were diagnosed in utero (N=6) or postnatal (n=10). Eleven cases were diagnosed with homozygous Hb Constant Spring, 4 with homozygous Hb Constant Spring with heterozygous Hb E, and 1 with homozygous Hb Constant Spring with homozygous Hb E. Three cases were delivered preterm. Six patients had low birth weights. Clinical manifestations included fetal anemia in 6 cases, hepatomegaly in 1 case, hepatosplenomegaly in 2 cases, splenomegaly in 1 case. Twelve cases exhibited early neonatal jaundice, 9 of which required phototherapy. Six cases received red cell transfusions; 1 (3), >1 (3). After the first few months of life, almost all patients had mild microcytic hypochromic anemia and an increased reticulocyte count with a wide red cell distribution (RDW), but no longer required red cell transfusion. At 1 to 2 years of age, some patients still had mild microcytic hypochromic anemia and some had normocytic hypochromic anemia with Hb around 10 g/dL, increased reticulocyte count and wide RDW. Associated findings included hypothyroidism (2), congenital heart diseases (4), genitourinary abnormalities (3), gastrointestinal abnormalities (2), and developmental delay (1). Pediatric patients with homozygous Hb Constant Spring developed severe anemia in utero and up to the age of 2 to 3 months postnatal, requiring blood transfusions. Subsequently, their anemia was mild with no evidence of hepatosplenomegaly. Their Hb level was above 9 g/dL with hypochromic microcytic blood pictures as well as wide RDW. Blood transfusions have not been necessary since then.

Derivation of the human induced pluripotent stem cell line MUi017-A from a patient with homozygous Hemoglobin Constant Spring

Hemoglobin Constant Spring (HbCS, HBA2: c.427T>C) is a common nondeletional α-thalassemia resulting from a nucleotide substitution at the termination codon of the HBA2 gene. Homozygosity for HbCS is characterized with mild anemia, jaundice, and splenomegaly. In this study, the human induced pluripotent stem cell line MUi017-A was successfully generated from peripheral blood CD34+ hematopoietic progenitors of a 52year old female with homozygous HbCS. The MUi017-A cell line exhibited embryonic stem cell characteristics with consistent expression of specific pluripotency markers and the capability of differentiating into the three germ layers. The cell line may be used for the disease modeling.

Iron deficiency anemia interfering the diagnosis of compound heterozygosity for Hb constant spring and Hb Paksé: The first case report

Diagnosis of thalassemia or hemoglobinopathy concomitant with iron deficiency anemia (IDA) is challenging. We report a case of 43-year-old female whose diagnosis of compound heterozygosity for hemoglobin Constant Spring (HbCS) and Hb Paksé became apparent after the treatment of IDA. Prior to treatment, Hb analysis using isoelectric focusing (IEF) showed HbA 95.6%, HbA2 2.7%, and HbCS 1.7% compatible with heterozygous HbCS. After 4months of oral iron therapy resulting in an improved Hb level, her HbCS level was substantially increased to 8.7% on IEF suggesting homozygous HbCS. Subsequent DNA analysis using multiplex amplification refractory mutation system analysis revealed compound heterozygosity for HbCS and Hb Paksé. This case demonstrated that IDA can significantly reduce HbCS/Hb Paksé levels and probably mask the diagnosis of homozygous HbCS, homozygous Hb Paksé or the compound heterozygosity for both hemoglobinopathies by hemoblogin analysis. The test should be repeated after resolution of IDA, or molecular testing should be performed to confirm the diagnosis.

Rapid Identification of Heterozygous and Homozygous Hemoglobin Constant Spring by SYTO9 with a High Resolution Melting Analysis

Gel-electrophoresis and ethidium bromide are not ideally suited to large scale analysis in clinical laboratories. Amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) specific for Hb CS was performed in 10 blood samples from normal individuals and 61 samples containing a peak of Hb CS when analyzed by capillary electrophoresis. Heterozygosity of Hb CS was identified using SYTO9 and high resolution melting (HRM) analysis method. Specific peak heights of amplified fragments of wild type and Hb CS alleles were observed in the heterozygote. Only one peak height of amplified fragments of the wild type allele was observed in the normal individual while only one peak height of amplified fragments of Hb CS allele was observed in the homozygote. HRM analysis interpretation results were completely consistent with the interpretation results from gel-electrophoresis. SYTO9 HRM analysis may be used as an alternative for rapid diagnosis of heterozygosity of Hb CS.

Anemia and Hydrops in a Fetus With Homozygous Hemoglobin Constant Spring

Hemoglobin Constant Spring (Hb CS) is an unstable hemoglobin (Hb) variant that results from a nucleotide substitution at the termination codon of the alpha2-globin gene. The compound heterozygosity of alpha-thalassemia and Hb CS (--/alphaCSalpha) results in a Hb H/CS disease which is clinically more severe than deletional Hb H disease. Homozygosity of Hb CS (alphaCSalpha/alphaCSalpha) is generally characterized with mild hemolytic anemia, jaundice, and splenomegaly. The authors report 1 case with Hb CS homozygosity who presented with fetal anemia and hydrops. Intrauterine transfusions were given which rendered a favorable outcome. This report demonstrates an unusual and serious in utero complication in a fetus with Hb CS/CS.

Hemoglobin H Hydrops Fetalis Associated with Homozygous Hemoglobin Constant Spring. Case Report.

While Barts hydrops is predicted when all 4 α globin genes are absent or non functional, hydrops has rarely been described with a hemoglobin H (Hb H) genotype. Hb H disease has a varied hematologic phenotype ranging from asymptomatic to transfusion dependant. The severest clinical expression is non immune hydrops and may result in fetal demise. The commonest form of Hb H disease is deletional-type. This occurs when there is deletion of α2 and α1 genes in cis and deletion of either α2 or α1 in trans. The less frequent type, but clinically more severe, is non-deletional. This is usually due to a mutation in either α2 or α1 in cis and a deletion of both α2 and α1 in trans. There are rare reports of non-deletional Hb H due to homozygous α2 mutations without any α1 deletions or mutations. Heretofore, there have been no reports of hydrops with homozygous hemoglobin Constant Spring (Hb CS). A 31-year-old Laotian woman presented at 22 weeks with polyhydramnios. Besides this, the pregnancy was uneventful for the mother. On ultrasound the fetus had a pericardial effusion and signs of congestive heart failure. The hematological and genotypic parameters of the parents and fetus are outlined in table 1. Intrauterine transfusions were performed at 22 and 29 weeks. A male infant was born vaginally at 36 weeks gestation weighing 2.3 kg. The hemoglobin was 15.9 g/dL and bilirubin was 8.4mg/dL. There was no evidence of maternofetal alloimmunization. G6PD screen was negative. By 4 weeks post-partum, the bilirubin had decreased to 0.9 mg/dL but the hemoglobin had progressively fallen to 6.9 g/dL, requiring transfusion. He had two additional transfusions at 6 and 8 weeks of life but has subsequently maintained a hemoglobin above 10 g/dL and has steadily gained weight. As in this patient, the genotypes of the other reported cases of Hb H hydrops are usually associated with a thalassemia intermedia phenotype. Thus the α globin genotypes alone do not explain why hydrops occurs. This suggests that there may be unknown genetic and/or acquired modifiers contributing to the severity of Hb H disease that results in hydrops as in this fetus with homozygous Hb CS.[Table 1]MotherFatherFetus at 22 weeksHemoglobin g/dL10.012.55.2MCV fL86.381.8143.8MCH pg26.525.6.846.4RDW %16.713.621.4Reticulocyte %9.9ND19.0NRBC/100WBC104756Hemoglobin Fractions by HPLCHb A73.3%97.8%3.4%Hb F0.8%0.0%77.7%Hb A20.9%2.1%0.0%Barts0.0%0.0%17.7%Hb Constant Spring2.8%0.1%1.2%HbE 22.2%0.0%0.0%αGlobin Gene Dosage Assay by Linear PCR444αGlobin Gene Deletions by PCR - 3.7, - 4.2, _SEA, - 20.5, _MED,_FIL,_THAINone detectedNone detectedNone detectedα2 Globin Gene Sequencingα-2CS/α-2CSα-2CS/WTα-2CS/α-2CSα1 Globin Gene SequencingWT/WTWT/WTWT/WTβGlobin Gene SequencingHeterozygous Hb EWT/WTWT/WT E

A correlation of erythrokinetics, ineffective erythropoiesis, and erythroid precursor apoptosis in Thai patients with thalassemia: Presented in part at the XXI Congress of the International Society of Hematology, Sydney, Australia, May 1985, and the Annual Meeting of the American Society of Hematology, San Diego, CA, December 1997.

AbstractThe variety of patients with thalassemia in Thailand offers an opportunity to fully characterize the kinetic causes of the anemia and to study apoptosis of marrow erythroid precursors as a possible factor contributing to its severity. Kinetic studies showed that in hemoglobin H (HbH) disease, the extent of hemolysis, as well as the minimally ineffective erythropoiesis, usually falls within the compensatory capacity of normal erythropoiesis; therefore, anemia in patients with HbH partly represents a failure to expand erythropoiesis adequately. Hemoglobin Constant Spring (HbCS), a common variant of α thalassemia in Bangkok, causes more severe hemolysis and a distinct increase in ineffective erythropoiesis. Ineffective erythropoiesis plays a much more prominent role in β thalassemia/hemoglobin E (β-thal/HbE) disease, in which the variability of the anemia is puzzling. We compared mild and severe cases and found that patients with severe disease had a maximal marrow erythropoietic response that failed to compensate for very short survival of red blood cells and a marked quantitative increase in ineffective erythropoiesis. Analysis of apoptosis of marrow erythroid precursors done both on shipped samples and in Bangkok showed a moderate increase in HbH disease, consistent with the small increase in ineffective erythropoiesis. In patients with homozygous HbCS, there was a further increase in apoptosis, consistent with the additional increase in ineffective erythropoiesis. Patients with β-thal/HbE disease had the most ineffective erythropoiesis and the most erythroid apoptosis. Thus, it appears that α-chain deposition in erythroid precursors, either αA or αcs, leads to accelerated apoptosis and ineffective erythropoiesis.

A correlation of erythrokinetics, ineffective erythropoiesis, and erythroid precursor apoptosis in Thai patients with thalassemia

The variety of patients with thalassemia in Thailand offers an opportunity to fully characterize the kinetic causes of the anemia and to study apoptosis of marrow erythroid precursors as a possible factor contributing to its severity. Kinetic studies showed that in hemoglobin H (HbH) disease, the extent of hemolysis, as well as the minimally ineffective erythropoiesis, usually falls within the compensatory capacity of normal erythropoiesis; therefore, anemia in patients with HbH partly represents a failure to expand erythropoiesis adequately. Hemoglobin Constant Spring (HbCS), a common variant of alpha thalassemia in Bangkok, causes more severe hemolysis and a distinct increase in ineffective erythropoiesis. Ineffective erythropoiesis plays a much more prominent role in beta thalassemia/hemoglobin E (beta-thal/HbE) disease, in which the variability of the anemia is puzzling. We compared mild and severe cases and found that patients with severe disease had a maximal marrow erythropoietic response that failed to compensate for very short survival of red blood cells and a marked quantitative increase in ineffective erythropoiesis. Analysis of apoptosis of marrow erythroid precursors done both on shipped samples and in Bangkok showed a moderate increase in HbH disease, consistent with the small increase in ineffective erythropoiesis. In patients with homozygous HbCS, there was a further increase in apoptosis, consistent with the additional increase in ineffective erythropoiesis. Patients with beta-thal/HbE disease had the most ineffective erythropoiesis and the most erythroid apoptosis. Thus, it appears that alpha-chain deposition in erythroid precursors, either alpha(A) or alpha(cs), leads to accelerated apoptosis and ineffective erythropoiesis.

Selective Enzymatic Amplification of α2-Globin DNA for Detection of the Hemoglobin Constant Spring Mutation

Hemoglobin Constant Spring is an elongation mutation of the alpha 2-globin locus that results in a thalassemic phenotype. It has a high prevalence in Asian populations. When inherited with other alpha-thalassemia determinants, the Constant Spring gene has the potential to cause severe forms of alpha-thalassemia. Accurate diagnosis of the condition with standard hemoglobin electrophoresis is unreliable due to the small to undetectable amounts of the mutant hemoglobin present. Because of the extensive sequence homology of the alpha 1 and alpha 2 loci, allele-specific hybridization to total genomic DNA containing the Constant Spring gene would not distinguish between heterozygous and homozygous hemoglobin Constant Spring. Selective enzymatic amplification of alpha 2-globin DNA sequences, however, allows unambiguous diagnoses to be made using allele-specific hybridization. This method is useful for providing accurate genetic counseling and prenatal diagnosis in populations and specific families in which precise diagnosis is important.

Selective enzymatic amplification of alpha 2-globin DNA for detection of the hemoglobin Constant Spring mutation

Hemoglobin Constant Spring is an elongation mutation of the alpha 2- globin locus that results in a thalassemic phenotype. It has a high prevalence in Asian populations. When inherited with other alpha- thalassemia determinants, the Constant Spring gene has the potential to cause severe forms of alpha-thalassemia. Accurate diagnosis of the condition with standard hemoglobin electrophoresis is unreliable due to the small to undetectable amounts of the mutant hemoglobin present. Because of the extensive sequence homology of the alpha 1 and alpha 2 loci, allele-specific hybridization to total genomic DNA containing the Constant Spring gene would not distinguish between heterozygous and homozygous hemoglobin Constant Spring. Selective enzymatic amplification of alpha 2-globin DNA sequences, however, allows unambiguous diagnoses to be made using allele-specific hybridization. This method is useful for providing accurate genetic counseling and prenatal diagnosis in populations and specific families in which precise diagnosis is important.