Normal HbA Research Articles

Unexpected Hemoglobin A1c Results

A 52-year-old woman with a medical history of hepatitis B, hyperlipidemia, hypertension, anemia, and depression presented to the internal medicine clinic for a routine visit. Laboratory tests 3 months previously had revealed an impaired fasting glucose concentration of 5.9 mmol/L (106 mg/dL) [reference interval, 3.9–5.6 mmol/L (70–100 mg/dL)]. Therefore, a hemoglobin (Hb)2 A1c analysis was performed. The initial Hb A1c evaluation by cation-exchange HPLC (CE-HPLC) (Hb A1c Program on the VARIANT II TURBO Link System; Bio-Rad Laboratories) showed an Hb A1c value of 115.8% (reference interval, 4.0%–6.0%) (Fig. 1). Fig. 1. CE-HPLC chromatogram for Hb A1c analysis. Hb S and an aberrant Hb A1c value of 115.8% represented the predominant Hb peaks in the chromatogram. In an effort to determine if the unusual Hb A1c result was due to potential hemoglobinopathies, we performed an Hb variant analysis with the Bio-Rad VARIANT CE-HPLC β-Thalassemia Short Program. The analysis revealed the absence of Hb A and the presence of sickle cell Hb (Hb S) (37.4%), along with normal Hb A2 (3.2%) and Hb F (<1.0%) (Fig. 2). Also evident was another large peak (53.0%) that eluted earlier than Hb A, which we called P2. This study suggested the presence of an Hb variant with a chromatographic retention time virtually identical to that of Hb A1c, in addition to Hb S (Figs. 1 and 2). A subsequent Hb electrophoretic analysis at pH 6.0 (QuickGel Acid; Helena Laboratories) identified Hb S and another abnormal band with a mobility similar to Hb F (not shown). Fig. 2. Chromatogram of Hb variants analysis with the CE-HPLC β-Thalassemia Short Program. Hb S (37.4%), wild-type Hb A2 (3.2%), and Hb F (<1.0%) were identified, but Hb A was not detected. A large peak, which we designated P2, was detected at 53.0%. ### PATIENT FOLLOW-UP To identify the Hb variants, we investigated DNA sequences corresponding to the patient's β-globin genes. This …

Role of iron deficiency anemia in the propagation of beta thalssemia gene

BackgroundThe diagnostic criterion for beta thalassemia trait (BTT) is elevated Hb-A2 levels. Iron deficiency anemia (IDA) reduces the synthesis of Hb-A2, resulting in reduced Hb-A2 levels, so patients with co-pathological conditions BTT with IDA, may have a normal level of Hb-A2. Many socio-economic factors like unawareness, poor diagnostic facilities, and cost of molecular diagnosis (for screening purposes) result in interpretation of these subjects as normal.MethodsVenous blood samples from 200 unmarried females having a family history of thalassemia were collected, and basic hematological parameters, hemoglobin electrophoresis, and molecular analysis for beta thalassemia were done. Patients with IDA and patients with co-pathological conditions BTT and IDA were treated with oral iron. These subjects were then followed for a period of 20 weeks.ResultsOf the 200 females, 34 were found to be anemic. Hemoglobin electrophoresis identified 16 of these patients as BTT. Molecular analysis of all patients confirmed this diagnosis, but identified 8 additional patients with BTT. Eight patients that were not detected with hemoglobin electrophoresis were found to have co-pathology of BTT with IDA.ConclusionPatients with the co-pathological condition BTT with IDA may be interpreted as being normal, as they have normal Hb-A2 levels. These misdiagnosed subjects when marry with BTT have the potential to produce beta thalassemia major in offspring. This is one of the factors playing a major role in the propagation of beta thalassemia gene in Pakistani population, and become a serious hindrance for the thalassemia prevention program in Pakistan.

Look for the silent when other tests cannot speak (a case of unexpected rare thalassaemia)

Microcytic anaemia is a common consultation in haematology practice. The common causes are iron deficiency, thalassaemia or anaemia of chronic disease. The diagnosis can be directly obtained through appropriate laboratory tests. However, in the absence of a positive history or a laboratory test, the diagnosis may be obscure and more sophisticated laboratory tests may be required. A 27-year-old Australian woman from a Scottish-Welsh ancestry was referred with long standing microcytic anaemia. The laboratory tests including iron studies, haemoglobin HPLC and electro-phoresis were normal and repeated twice to exclude pre-analytical and analytical variability. Other causes of microcytic anaemia including lead toxicity, copper or zinc deficiency, chronic inflammatory disease or sideroblastic anaemia were excluded. The patient’s blood sample was sent to the Royal Prince Alfred hospital for molecular testing for possible underlying thalassaemia. A heterozygous large deletion in the beta globin region with an intact alpha globin using DNA sequencing and multiplex ligation-dependent probe amplification (MLPA) was detected. The finding supports the diagnosis of epsilon-gamma-delta-beta (eγδβ)o thalassaemia. This type of normal HbA2 beta thalassaemia or silent beta thalassaemia has been previously reported in a family with Scottish-Irish ethnic background in NSW. 1

Low Molecular Weight Heparin Reduces sVCAM-1 and Lung Congestion In a Murine Model of Sickle Cell Disease

AbstractAbstract 1635There is evidence for increased levels of procoagulant proteins, thrombin generation and tissue factor activity in individuals with sickle cell disease. Both thrombin and tissue factor also have potent proinflammatory effects that likely further contribute to the vascular dysfunction and organ injury observed in sickle cell disease. In this study, we examine the effect of the LMW heparin, enoxaparin, on markers of vascular injury and inflammation in the Berkeley mouse model of severe sickle cell disease (HbSS mice). HbSS mice or control HbAA mice (that exclusively express normal human HbA) were treated with continuous subcutaneous infusion of enoxaparin (10 mg/kg/day) or saline for 14 days using a surgically implanted Alzet pump (n=12-14 mice per group). Blood was collected at baseline and following 14 days enoxaparin therapy. Anesthetized animals were perfused with PBS at constant physiologic pressure (80 mmg Hg) and lung and liver collected for pathologic analyses. Enoxaparin-treated mice had steady state LMW heparin levels of 0.45 ± 0.17 anti-Xa U/mL compared to <0.1 anti-Xa U/mL in saline treated animals. Enoxaparin reduced d-dimer levels by approximately 35% in both HbSS and HbAA mice compared to saline-treated mice (p=0.03 and p=0.02, respectively), showing a functional reduction in coagulation and fibrinolytic activity. The hemoglobin level did not change and no obvious hemorrhage was noted in enoxaparin-treated mice suggesting no major bleeding complications. sVCAM-1 is a measure of endothelial injury and is increased at baseline in HbSS mice compared to HbAA mice (p<0.001). sVCAM-1 levels were reduced in HbSS mice after 14 days of enoxaparin therapy (1424 ± 83 ng/mL baseline, 1314 ± 85 ng/mL post enoxaparin, p=0.02), but not in saline treated HbSS mice (1462 ± 69 ng/mL baseline, 1392 ± 85 ng/mL post saline, p=0.3). Furthermore, the sVCAM-1 level negatively correlated with the steady state heparin level (r=-0.48, p=0.02) suggesting a modest dose-dependent effect of heparin on sVCAM-1 level. There was no effect of enoxaparin or saline therapy on sVCAM-1 levels in control HbAA mice. Preliminary histological analysis revealed a reduced number of congested pulmonary arteries in enoxaparin-treated HbSS mice compared to saline treated HbSS mice (p=0.01). These data suggest that enoxaparin therapy may improve endothelial injury as measured by sVCAM-1 and pulmonary vascular congestion in a mouse model of severe sickle cell disease. The data also suggest that heparin-based anticoagulant therapy may be a novel therapeutic approach to improve vascular health in individuals who suffer from sickle cell disease. Disclosures:Field:Novartis: Honoraria.

Association of Non-Healing Wounds, Pain and Neurochemical Alterations In Sickle Cell Disease

AbstractAbstract 842The mechanisms underlying non-healing leg ulcers in sickle cell disease (SCD) remain unknown and their treatment is difficult. We hypothesized that leg ulcers in SCD cause injury to nerve fibers leading to neurogenic inflammation, and peripheral and central sensitization resulting in chronic pain and impairment of wound healing. We examined our hypotheses by creating 4 mm punch biopsies on the lower thigh of the left leg (LL) of 12 wk old BERK and hemizygous BERK (hBERK) mice expressing sickle hemoglobin (HbS). Since BERK did not survive wounding, hBERK and control HbA-BERK, expressing normal human HbA and wild type C57/BL6, were used. hBERK appeared to be a suitable model for this study because we found that >5 mo old BERK and hBERK show deep tissue and cutaneous hyperalgesia similar to pain in human SCD (Blood 116:456-65, 2010). Wounds were treated with topical morphine (3 mg/g Eucerin base cream) or with Eucerin base cream twice a day. Behavioral measures of hyperalgesia were assessed by paw withdrawal frequency (PWF) per 10 applications of a 1.0 g von Frey monofilament and paw withdrawal latency (PWL) in response to a radiant heat stimulus, applied to the plantar surface of the hind paw of wounded left leg and unwounded right leg. No hyperalgesia was observed in 12 wk old hBERK (baseline), but wounding led to a sharp increase in PWF and decrease in PWL, in LL for 24h, suggestive of wound-induced pain. Topical application of morphine significantly decreased mechanical and thermal hyperalgesia after 1h and 24h as compared to the base cream treated wound (p<0.05) in LL. Pain measures returned to baseline 2 wks post-wounding in morphine- but base cream-treated hBERK mice exhibited hyperalgesia for 4 wks (last measurement). The right leg exhibited increased mechanical but not thermal hyperalgesia following the wounding of left leg in hBERK. HbA-BERK and C57/BL6 wounds treated with base cream showed an increase in pain which was significantly less than hBERK and reached baseline 2 wks post-wounding, suggesting that wounding results in chronic pain in hBERK mice. Increased pain was accompanied by decreased wound closure in hBERK mice. Morphine-treated mice showed 100% wound closure at 4d Vs 8d in base cream treated hBERK, indicative of morphine-induced augmentation of wound healing. Laser scanning confocal microscopy (LSCM) of whole dorsal root ganglion (DRG) showed that, activating transcription factor 3 (ATF3), a marker of neuronal injury, was highly expressed in left lumbar DRG of base cream Vs morphine treated wounded hBERK, 30d post-wounding (p<0.05), suggestive of ongoing wound-induced neuronal injury which is abrogated by topical morphine. LSCM of 100 micron thick sections of healed wounds showed that substance P (SP), a neuropeptide expressed in nociceptive primary afferents, was significantly increased in base cream treated Vs morphine treated healed wounds 30d post wounding (p<0.01). Increased SP was accompanied by reduced and disorganized protein gene product (PGP 9.5)-ir nerve fibers and vasculature in base cream treated wounds. In contrast, morphine treated wounds showed normally organized dense PGP 9.5-ir nerve fibers and blood vessels replete with nerve and vascular plexus similar to their presentation in normal skin. Together, these data show that wounds stimulate chronic pain accompanied by nerve injury, central sensitization and neurogenic inflammation; and that morphine ameliorates this noxious insult and promotes wound healing. In addition, morphine may directly influence wound healing by promoting angiogenesis. It is possible that chronic pain may contribute to impaired wound healing and that non-healing wounds contribute to chronic pain in SCD. We propose that wound-induced nerve signal conduction (ATF3 in DRG) and neurogenic inflammation/sensitization of primary afferents (SP in skin) may contribute to chronic pain and in turn interfere with wound healing. Our observations suggest that neurochemical and behavioral alterations orchestrated by wounding may contribute to the impairment of healing of leg ulcers in SCD. We speculate that topical application of morphine may ameliorate pain and promote healing of painful leg ulcers in SCD. Disclosures:No relevant conflicts of interest to declare.

Variability In Hb A2 levels among Individuals with Beta-Thalassemia Trait: Is Iron Deficiency Associated with Abnormally Low Hb A2?

AbstractAbstract 4281 Introduction:Quantification of HbA2 is a well established screening test for beta-thalassemia trait (BTT). However, there is a small subset of individuals heterozygous for beta-thalassemia mutations who have HbA2 levels less than or equal to 3.5%. Some reports have suggested that iron deficiency in BTT patients causes HbA2 to be lower than expected, while others have found no significant relationship between iron deficiency and the level of HbA2. These conflicting reports have led to confusion amongst clinicians as to the reliability of HbA2 measurement when screening for BTT in iron deficient individuals. Methods:From a database of 444 individuals with heterozygous beta-thalassemia, confirmed by molecular testing, we assessed the variability in HbA2. Individuals were classified as “iron deficient” or “non-iron deficient” based on their serum ferritin, using two definitions of iron deficiency (serum ferritin <15 ug/L or <30 ug/L), and data was analyzed independently. The association of HbA2 levels with gender, iron deficiency or beta-thalassemia mutation type was evaluated using a two-sample T-test. The relationship of HbA2 with gender, hemoglobin level, Hb F percentage, reticulocyte percentage and/or the natural logarithm of serum ferritin was evaluated using single and multiple linear regression analysis. Results:HbA2 ranged from 2.0 to 8.1%, with a mean of 5.5%. Mean HbA2 in females (5.4%, SD 0.6%) was lower than that in males (5.6%, SD 0.6%) (p=0.003). Seven individuals had a HbA2 level below 3.5% (range 2.0–3.1%), all of whom had serum ferritin ≥15 ug/L. Of these people, five had the Chinese (Aγδβ)0-thalassemia deletion, and would thus be expected to have a normal HbA2 level; one woman had a HbA2 of 3.1% and was found to be heterozygous for a novel point mutation at codon 29 of the delta-globin gene (GGC>GAC or Gly29Asp); and another woman with HbA2 of 2.0% had unexplained elevation of Hb F (8.0%). The 30 patients with serum ferritin <15 ug/L had evidence of iron-deficient hematopoiesis (significantly lower hemoglobin concentration and MCV than patients with ferritin ≥ 15), and none had HbA2 <3.5%. However, HbA2 was lower (5.3%) versus HbA2 in non-iron deficient individuals (5.5%; p=0.04). Individuals with the Chinese (Aγδβ)0-thalassemia deletion had a mean Hb A2 of 2.8%. Of the remaining beta-thalassemia genotypes, IVSII-645 and Nt -29 beta+ mutations were associated with HbA2 values significantly below the group mean, and -28 beta+ and the Codon 17, Codons 41/42 and Codon 43 beta0 mutations were associated with significantly higher HbA2 values. Multiple linear-regression analysis demonstrated a significant association of low HbA2 with low ferritin (p=0.04) and beta+-thalassemia mutation type (p<0.001). There was no significant association with gender (p=0.1), hemoglobin (p=0.2), reticulocyte count (p=0.08) or hemoglobin F level (p=1.0). Conclusions:From our population of patients with BTT, we have shown that individuals with overt iron deficiency all had HbA2 within the range expected for an individual with BTT (Hb A2 >3.5%). Nonetheless, serum ferritin < 15 ug/L was associated with a small but significant decrease in HbA2 (mean HbA2 of 5.3%, versus 5.5% in non-iron deficient individuals; p=0.04). Although a multivariate analysis confirmed that ferritin under 15 ug/L was associated with lower HbA2 levels, beta+-thalassemia mutation type explained more of the variability than did iron status in individuals with lower HbA2 values. Six out of seven patients who had HbA2 <3.5% were found to have heterozygous deletion or mutation of the delta-globin gene. Based on the results of our study, HbA2 remains a reliable test for BTT screening, even in the presence of iron deficiency. In patients known or suspected to have BTT, but in whom HbA2 is found to be <3.5%, testing for delta-globin abnormalities should be pursued. Disclosures:No relevant conflicts of interest to declare.

Spectrum of α-Globin Gene Mutations in the Kerman Province of Iran

Mutation of the α-globin gene may result in α-thalassemia (α-thal) which is characterized by a reduction or complete absence of the gene expression. In this study, 607 individuals with low levels of blood cell indices and normal Hb A2 were referred to our laboratory for investigation of any α-thal mutations. We used the gap-polymerase chain reaction (gap-PCR) method and an α-globin strip assay kit to detect the mutation. Our results showed that −α3.7 was the most common mutation (83.8%) in the overall mutated alleles of the α-globin gene. The second and third most frequent α-globin gene defects were codon 19 (α2) and IVS-I, −5 nt/αα (α2), 5.7 and 4.2%, respectively. We found that the spectrum of α-globin gene mutation in Kerman Province was in accordance with what was previously reported in other Iranian provinces where malaria has selected these protective traits.

Genotype-Phenotype Relationship of the δ-Thalassemia and Hb A2 Variants: Observation of 52 Genotypes

The increase of Hb A2 (α2δ2) beyond the upper limit [2.0–2.2/3.3–3.4% of the total hemoglobin (Hb)] is an invaluable tool in the hematological screening of β-thalassemia (β-thal) carriers. Factors decreasing Hb A2 percentages can hinder correct diagnosis. In order to analyze the genotype-phenotype relationship, we characterized δ-, β- and α-globin genotypes in 190 families where the probands had Hb A2 values of ≤2.0% or were β-thal heterozygotes with normal Hb A2 levels. Hb A2 was measured with cation exchange high performance liquid chromatography (HPLC). Mutations were detected with allele-specific methods or DNA sequencing; two multiplex-ARMS (amplification refractory mutation system) assays were set up. The molecular basis underlying the decrease in Hb A2 was extremely heterogeneous. Nineteen δ-globin alleles (Hb A2-S.N. Garganico was new) were detected; their interaction with α- or β-globin alleles (10 and eight, respectively) led us to observe 52 genotypes in 261 carriers. The type of δ-globin mutations, the relative genotypes, the interaction with α0-thal traits, are the most important factors in decreasing the Hb A2 percentage. These results are extremely useful in addressing the molecular diagnosis of hemoglobinopathies and thalassemias.

New and Known β-Thalassemia Determinants Masked by Known and New δ Gene Defects [Hb A2-Ramallah or δ6(A3)Glu→Gln, GAG>>CAG

We report a novel thalassemia determinant found in a Nigerian woman living in the Netherlands, resulting from a 2 bp insertion at codons 9/10 of the β-globin gene (HBBc.28_29insTA p.Ser10LeufsX11). The novel defect causes a frameshift with a consequent premature TGA stop codon, located at 11 positions downstream from the mutated codon. The phenotype was typical of a β-thalassemia (β-thal), trait with high RBC counts and compensated mild microcytic anemia. However, the Hb A2 level was reported to be normal due to the presence of the common Hb A2' or Hb B2 [δ16(A13)Gly→Arg, GGC>CGC] variant that was not taken into account. We also present the opposite but comparable situation found in an a Palestinian man living in the USA. He was a carrier of a common β-globin gene defect [codon 6 (–A), HBB:c.20delA] in combination with a novel δ-globin gene defect at codon 6 [HBD. c.19G>C, Glu6Gln] that we have named Hb A2-Ramallah. In both cases, the provisional diagnosis could have been compromised when based on the measurement of the normal Hb A2 fraction only.

A REPORT OF 8 CASES WITH HEMOGLOBIN H DISEASE IN AN IRANIAN FAMILY

α-Thalassemia is a common genetic disorder in Iran. However, no comprehensive data on epidemiology of severe forms of α-thalassemia, including hemoglobin H (HbH) or hydrops fetalis, is available in this population. This is a first case report of an Iranian family with large number of HbH individuals. The proband is a 48-year-old woman, referred to our center with anemia and no history of previous blood transfusions. Similar clinical phenotype has been observed in all of her 5 siblings, 2 of her 4 children, and her granddaughter, whose parents are first cousins. A reverse hybridization assay covering 21 α globin mutations was performed to determine the genotype in 11 members of this family and a fetus. HbH genotype was identified in 9 individuals, representing 3 generations, including a fetus. The high prevalence of α-thalassemia carriers together with the high rate of consanguineous marriages could lead to a large number of individuals with HbH or even hydrops fetalis in Iranian families. Therefore, to avoid the risk of having affected offspring, carrier detection, genetic counseling, and prenatal diagnosis would be of vital importance for individuals with low red blood cell (RBC) indices, normal iron status, and normal HbA2 level, who are suspected to be α-thalassemia carriers.

Development of a quantitative real-time PCR assay for detection of unknown α-globin gene deletions

Background Alpha-Thalassemia is the most common inherited disorder of hemoglobin (Hb) synthesis in the world. Unlike beta-thalassemia, in which non-deletional mutations predominate, most of recognized α-thalassemia mutations include deletion of one or both α-globin genes. The importance of α-thalassemia detection is mainly due to its shared blood parameters with beta-thalassemia and its impact on discrimination between unknown α-thalassemia and normal HbA2 beta-thalassemia during thalassemia prevention program. Materials and methods Cases with hematologic profile of low MCV, MCH, and normal HbA2 were enrolled in this study. Common α-globin deletional mutations including α 3.7 kb, α 4.2 kb, α 20.5 kb, and α MED and point mutation including 5nt, Constant Spring (CS), and C19 were checked using either GAP-PCR or ARMS-PCR. Cases with unknown molecular defects were investigated further by direct gene sequencing. Finally, further study was done for probable unknown deletions by gene dosage analysis using real-time PCR. For this, five pairs of primers were used spanning from θ-globin gene up to the 3′ upstream of α 2 gene. Results After validation of primers specificity and performing serial dilution analysis in order to calculate PCR efficiency, the assay was performed on normal samples and cases with known alpha-globin gene deletions as positive and negative controls, respectively. The assay was able to diagnose the control groups successfully. In 21 out of 29 unknown cases (72.4%), the assay showed various patterns of deletions in at 2 to 5 screened regions (θ gene up to the upstream of alpha2 gene). In 8 (27.6%) cases, deletions were seen in all regions. Conclusion Gene dosage study by quantitative real-time PCR can be suggested as a rapid and reliable assay to screen probable carrier of α-thalassemia for unknown alpha-globin gene deletions.

Novel permeability characteristics of red blood cells from sickle cell patients heterozygous for HbS and HbC (HbSC genotype)

Individuals heterozygous for HbS and HbC (HbSC) represent about 1/3 rd of sickle cell disease (SCD) patients. Whilst HbSC disease is generally milder, there is considerable overlap in symptoms with HbSS disease. HbSC patients, as well as HbSS ones, present with the chronic anaemia and panoply of acute vaso-occlusive complications that characterize SCD. However, there are important clinical and haematological differences. Certain complications occur with greater frequency in HbSC patients (like proliferative retinopathy and osteonecrosis) whilst intravascular haemolysis is reduced. Patients with HbSC disease can be considered as a discrete subset of SCD cases. Although much work has been carried out on understanding the pathogenesis of SCD in HbSS homozygotes, including the contribution of altered red blood cell permeability, relatively little pertains directly to HbSC individuals. Results reported in the literature suggest that HbSC cells, and particularly certain subpopulations, present with similar permeability to HbSS cells but there are also important differences - these have not been well characterized. We hypothesise that their unique cell transport properties accounts for the different pattern of disease in HbSC patients and represents a potential chemotherapeutic target not shared in red blood cells from HbSS patients. The distinct pattern of clinical haematology in HbSC disease is emphasised here. We analyse some of the electrophysiological properties of single red blood cells from HbSC patients, comparing them with those from HbSS patients and normal HbAA individuals. We also use the isosmotic haemolysis technique to investigate the behaviour of total red blood cell populations. Whilst both HbSS and HbSC cells show increased monovalent and divalent (Ca 2+) cation conductance further elevated upon deoxygenation, the distribution of current magnitudes differs, and outward rectification is greatest for HbSC cells. In addition, although Gd 3+ largely abolishes the cation conductance of both HbSS and HbSC cells, only in HbSS ones are currents inhibited by the aminoglycosides like streptomycin. This distinction is retained in isosmotic lysis experiments where both HbSS and HbSC cells undergo haemolysis in sucrose solutions but streptomycin significantly inhibits lysis only in HbSS cells. These findings emphasise similarities but also differences in the permeability properties of HbSS and HbSC cells, which may be important in pathogenesis.

Significant haemoglobinopathies: guidelines for screening and diagnosis

Antenatal screening/testing of pregnant women should be carried out according to the guidelines of the NHS Sickle Cell and Thalassaemia Screening programme. Newborn screening and, when necessary, follow up testing and referral, should be carried out according to the guidelines of the NHS Sickle Cell and Thalassaemia Screening programme. All babies under 1 year of age arriving in the UK should be offered screening for sickle cell disease. Preoperative screening for sickle cell disease should be carried out in patients from ethnic groups in which there is a significant prevalence of the condition. Emergency screening with sickle solubility tests must always be followed by definitive analysis. Laboratories performing antenatal screening should utilize methods capable of detecting significant variants and be capable of quantitating haemoglobins A 2 and F at the cut-off points required by the national antenatal screening programme. The laboratory must ensure a provisional report is available within three working days from sample receipt.

Measurement of Urine Pyridinoline and Deoxypyridinoline as Markers of Increased Bone Degradation in Sickle Cell Disease.

Abstract 2572Poster Board II-549 Introduction:Sickle cell disease (SCD) is commonly manifested through skeletal involvement. Besides the characteristic acute musculoskeletal pain, SCD is also associated with chronic skeletal complications such as osteopenia and osteoporosis. During bone resorption, the collagen cross-links pyridinoline (PYD) and deoxypyridinoline (DPD) are released into circulation with subsequent urinary excretion. Measurements of urinary PYD and DPD could serve as valuable tools in detecting osteoporosis in the follow-up of SCD patients but perhaps also in determining the severity of bone infarction during painful crises. Therefore we compared urinary concentrations of PYD and DPD of SCD patients during asymptomatic state and painful crisis with those of race- and age-matched healthy controls. Methods:Urinary concentrations of PYD and DPD, adjusted for urine creatinine, were measured in SCD patients both during asymptomatic state (n=38) and painful crisis (n=27) and healthy controls with normal HbA hemoglobin (n=25) using high performance liquid chromatography (HPLC). Results:PYD and DPD concentrations were higher in asymptomatic SCD patients compared to controls ((54.8 (41.5–68.6) vs. 44.1 (37.7–49.9),P=0.005 and 11.6 (9.3–15.2) vs. 8.5 (6.8–10.4),P=0.004 respectively), with further increments during painful crisis (63.3 (51.8–76.0),P=0.041 and 15.3(13.0–21.5),P=0.003 respectively). In the asymptomatic patients levels of PYD and DPD were significantly correlated to the degree of hemolysis. Conclusion:In sickle cell patients bone resorption is increased and significantly correlated to the degree of hemolysis, compatible with their susceptibility to osteopenia and osteoporosis. Measurement of pyridinoline and deoxypyridinoline could have additional value as biomarkers of osteoporosis in SCD. During painful crises a further increment in bone degradation was observed. Disclosures:No relevant conflicts of interest to declare.

Two new -thalassemia deletions compromising prenatal diagnosis in an Italian and a Turkish couple seeking prevention

When the molecular background of couples requesting prevention is unclear, family analysis and tools to define rare mutations are essential. We report two novel deletion defects observed in an Italian and in a Turkish couple. The first proband presented with microcytic hypochromic parameters without iron deficiency, a normal HbA(2) and an elevated HbF (10.6%). His father presented with a similar phenotype and his wife was heterozygous for the common Mediterranean codon 39 (HBB:c.118C>T) mutation. Having excluded point mutations and common deletions, Multiplex Ligation-dependent Probe Amplification was performed revealing an unknown Ggamma(Agammadeltabeta)(0)-thalassemia defect spanning from the Agamma gene to downstream of the beta-globin gene provisionally named Leiden 69.5 kb deletion. In the second case, the wife presented with a mild thalassemic picture, normal HbA(2), elevated HbF (18.5%) and a beta/alpha globin chain synthesis ratio of 0.62, without iron deficiency or any known beta-thalassemia defect, while the husband was a simple carrier of the common Mediterranean IVS-I-110 (HBB:c.93-21 G>A) mutation. A new large deletion involving the beta-gene and part of the delta-gene was identified by Multiplex Ligation-dependent Probe Amplification provisionally named "Leiden 7.4 kb".

HbA2-Partinico or δ(A2)Pro→Thr, a new genetic variation in the δ-globin gene in cis to the β+ thal IVS-I-110 G&gt;A, and the heterogeneity of δ-globin alleles in double heterozygotes for β- and δ-globin gene defects

The study of the alleles of the delta-globin gene is relevant to the prevention of beta-thalassemia homozygosis; in fact, the increase of the HbA2 is an invaluable hematological marker of the beta-thalassemia heterozygosis and the double heterozygosis for alleles of delta- and beta-globin genes can cause the decrease of the HbA2 up to normal or borderline values. We carried out the characterization of alleles of the delta- and beta-globin genes, restriction fragment length polymorphism (RFLP) haplotype background, and hematologic phenotype in 23 double heterozygotes belonging to 18 unrelated families. A wide heterogeneity of the delta-globin alleles was detected; seven known alleles in trans to the beta-globin gene defects were revealed in 17 out of 18 families, while a new allele in cis to a beta-thalassemia allele was detected in one family. Moreover, the relative frequency of the delta-mutants was quite different from that found among heterozygotes. The new allele delta-cod 5 CCT>ACT, in cis to the allele beta(+) thal IVS-I-110 G>A, was found in five carriers of a Sicilian family. The new variant delta5(A2)Pro-->Thr, named HbA2-Partinico upon the origin of the family, was detected with high-performance liquid chromatography; it overlapped the HbA2 peak which was partially split. The double in cis heterozygotes had increased percentage of normal and variant HbA2 of comparable size. The variant originated most likely from a new mutational event because it was associated with RFLP haplotype I, commonly found with the beta(+) thal IVS-I-110 G>A, even if crossing over or gene conversion cannot be excluded.

Diagnostic value of zinc protoporphyrin in a screening strategy for α‐thalassemia

The definitive diagnosis of alpha-thalassemia involves detection of a deletion of one or more alpha-globin that encode the alpha-chains of Hb (hemoglobin). To determine whether DNA analysis is indicated, screening tests such as mean corpuscular volume (MCV) and Hb typing are employed. alpha-Thalassemia often correlates with normal or low HbA2 values. Zinc protoporphyrin (ZPP) is usually high in ferropenic anemia or lead-poisoning and is normal or slightly raised in beta-thalassemia. Therefore, ZPP is currently used as a marker to discriminate between ferropenic anemia and beta-thalassemia. We investigated the diagnostic potential of ZPP < 150 micromol/mol heme in a screening strategy for alpha-thalassemia. We measured ZPP and performed DNA analysis for detecting the seven most prevalent alpha-thalassemia deletions, namely, alpha3.7, SEA, alpha20.5, alpha4.2, MED, FIL, and THAI, in the blood samples of 200 patients with MCV < 70 fL and HbA2 < or = 3.5%. Deletions were detected in 9% subjects in the ZPP > or = 150 group (n = 175) and 56% subjects in the ZPP < 150 group (n = 29); this difference was statistically significant (chi-square test, P < 0.001). We conclude that ZPP < 150 micromol/mol heme can be used in a new screening strategy for alpha-thalassemia.

Sickle Hemoglobin Fiber Growth Rates Deduced Using Optical Channels

Sickle hemoglobin (HbS) is a point mutant of normal HbA, and will polymerize at concentrations above a well defined solubility. Polymerization occurs by a double nucleation mechanism in which homogeneous nuclei form in solution following a stochastic delay, and heterogeneous nuclei form on other polymers nucleated by either pathway. A fundamental element is the growth of individual fibers, whose diameter (20 nm) precludes direct optical visualization. Fiber growth and depolymerization have been measured by DIC microscopy, but the heterogeneous pathway makes it possible that bundles rather than single fibers have been observed, in addition to certain technical problems that make interpretation of the results less than simple. We have devised a method in which optical patterns are projected on a COHbS solution by laser photolyis, which creates deoxyHbS that can polymerize only in the illuminated area, easily allowing complex polymer structures to be created optically. In our experiment, polymers first form in an incubation circle. From the circle, a line of deoxyHbS is optically generated along which fibers can grow. Finally, a detection circle is illuminated and the connecting line is extinguished. If a polymer has entered the detection circle, thanks to heterogeneous nucleation it will fill the circle with easily observed polymers, otherwise the detection circle remains monomeric until, at some much later time, homogeneous nucleation occurs. Thus we can measure the elongation of a fiber too small to detect optically. Our preliminary results find growth rates greater than previously observed by DIC microscopy. We can also measure the growth rate of bundles by observing a mass of fibers that subsequently grow along the channel, and these grow at rates comparable to those determined by DIC microscopy. Implications of the measurements and the method will be discussed.

First Identification of a Point Mutation at Position −83 (G>A) of the β-Globin Gene Promoter

We report the first identification of a point mutation located within the promoter region of the β‐globin gene at position −83 (G>A) and associated with the common heterozygous deletional α‐thalassemia (α-thal) (−α3.7/αα). The patient was an adult male from Gabon belonging to the Obamba sub ethnic group, who was referred to our clinics for a mild microcytic anemia with a Hb A2 level at the upper limit of the normal value (3.5%). This observation is a new example of α- and β-thal co-inheritance with a normal Hb A2 level, and illustrates a potential source of pitfall in screening for α- and β-thal carriers.

Identification of a Point Mutation in Position – 83 (G>A) of the β Globin Gene Promoter and the Influence of Globin Genes Mutation on the HbA2 Level

Carriers of typical β thalassemia alleles have microcytic hypochromic red blood cells with elevated Hb A2 values and a normal or slightly elevated level of Hb F. The increased level of Hb A2 is a reliable marker for heterozygous beta-thalassemia while Hb A2 levels within normal ranges do not exclude heterozygous beta-thalassemia. A male patient of Gabonese ancestry with both parents belonging to the Obamba sub-population was referred to us because of persistent microcytosis with anisopoikylocytosis and spherocytosis in the absence of clinical signs. The Hb separation pattern on alkaline electrophoresis and on ion exchange HPLC was normal. The Hb A2 level was 3.5% and the HbF level was 0.8%. Direct sequencing of the β globin genes revealed a G>A transition in position − 83 upstream of the cap site. The − 83 G>A substitution is located in a region between the CACCC motif (from − 90 to − 86) and the CCAAT motif (from − 76 to − 72) and has not been reported before. An alpha thalassemia associated with the β mutation was considered as well as a θ gene defect. Both hypotheses were based on the Hb A2 level.The Gap-PCR revealed the presence of − α3.7 (rightward) deletion in the heterozygous state in the proband while the θ genes did not show any mutation when compared to the normal sequence. In some instances, carriers for β thalassemia mutation may have normal Hb A2 level and can be mistaken for α-thal and/or θ-thal or iron deficiency. We have revisited the Hb A2 level in different genotype conditions by using the VARIANT TM HPLC system with the β-Thalassemia short program (Bio-Rad Laboratories, Hercules, CA, USA). We have established thal carriers of typical nondeletional β-Thal alleles with no alpha thalassemia and in the absence of iron deficiency have a remarkable uniform level of Hb A2, rarely more than 6% and with an average value of 4.82 % ± 0.67( n=214). Point mutations located within the β globin gene promoter gives rise to an average of Hb A2 level of 5.42% ± 0.76 (n= 27). When a premature termination codon is present the Hb A2 percentage reaches 5.90 % ± 0.32 (n=12). The Hb A2 level drops to an average of 2.54 % ± 0.40 (n=108) in individuals carrying the − α3.7/αα genotype only. The association of the − α3.7/αα genotype with a non deletional β thalassemic allele shows an average of Hb A2 at 3.30 % ± 0.32 (n=134). Thus we explain the normal Hb A2 level in our patient by the co-inheritance of the β mutation together with the common heterozygous deletional α thalassemia (− α3.7/αα). Also, it is very unlikely that the − α3.7/αα genotype by its own may explain the red cell indices found in our patient (MCV=73 ; Hb = 12g/dl ; MCH = 23 pg) Co-inheritance of α and β thalassemia should always be considered when non iron deficient hypochromic microcytic anemia is diagnosed together with normal Hb A2 level, especially in regions with a high prevalence of both types of thalassemia. DNA analyses are needed for accurate diagnosis as this combination of genotypes can have implications for genetic counselling and prenatal diagnosis.