Hb Q-India: an uncommon variant diagnosed in three Punjabi patients with diabetes is identified by a novel DNA analysis test

AIMS: An abnormality in the glycated haemoglobin peak (Hb A1c) on Diastat (Bio-Rad) cation exchange low pressure liquid chromatography (LPLC) was found in three Punjabi patients with diabetes. The aims of this study were to identify the variant by chromatography and electrophoresis and to determine whether a DNA analysis test could be designed for confirmation that could be generally applied for the identification of any unusual abnormal haemoglobin. METHODS: The presence of an Hb variant was confirmed by cellulose acetate electrophoresis at pH 8.6. The variant was characterised further by high performance liquid chromatography (HPLC; Bio-Rad Variant) and isolelectric focusing (IEF) electrophoresis. A novel DNA analysis test based on the amplification refractory mutation system (ARMS) and the polymerase chain reaction (PCR) was developed to confirm the presence of the mutation for the uncommon variant. RESULTS: Comparison of the HPLC retention time and IEF band position determined the presence of the variant Hb Q-India in all three cases. Hb Q-India is caused by the mutation GAC --> CAC at codon 64 of the alpha-1 globin gene and is clinically silent. ARMS-PCR specific primers were designed and used successfully to confirm the presence of the mutation for Hb Q-India. CONCLUSIONS: The results show that the ARMS-PCR technique, developed previously for the diagnosis of beta thalassaemia mutations, can also be adapted to provide a simple, rapid, and inexpensive approach for the identification of abnormal haemoglobins.     

Thalassemic hemoglobinopathies.

Hemoglobinopathies are due to changes in the normal amino acid sequence of globin. Thalassemias result from imbalance in the normal coordinated synthesis of the globin subunits that make up the hemoglobin tetramer. It is now apparent that a single globin gene can have coding region mutations which simultaneously produce a structural defect (hemoglobinopathy) and a biosynthetic defect (thalassemia). It is likely that two distinct mutations within the same gene can occur and produce a hemoglobinopathy with features of thalassemia. In this review the authors discuss such disorders and include the Hb Lepore and Constant Spring variants, hyper-unstable globins, mutations which create alternative sites for mRNA splicing, and amino acid substitutions likely to be associated with an additional thalassemia lesion within the same gene.     

Phenotype-genotype correlation in haemoglobin H disease in childhood.

In this study we used restriction endonuclease mapping to characterise the molecular defect responsible for haemoglobin H disease in 14 Sardinian children. The resulting genotypes were then correlated with the respective clinical and haematological phenotypes. We found that patients with the combination of non-deletion alpha(+)-thalassaemia [(alpha alpha)th] and deletion alpha(0)-thalassaemia (-Med) have a more severe phenotype than that resulting from the interaction of deletion alpha(0)-thalassaemia (-Med) and alpha(+)-thalassaemia (-alpha) determinants. Clinically, presentation was earlier and with moderate anaemia or haemolytic crisis, enlargement of the liver and spleen, and thalassaemic bone changes. Haematologically, the anaemia was more severe and there were higher bilirubin levels, reticulocyte counts, Hb H levels, and percentage of red blood cells with inclusion bodies. These results suggest that in those Hb H disease patients with the non-deletion [(alpha alpha)th] determinant, two alpha globin genes produce fewer alpha globin chains than a single alpha globin locus.     

Should we screen for globin gene mutations in blood samples with mean corpuscular volume (MCV) greater than 80 fL in areas with a high prevalence of thalassaemia?

AIMS: To investigate whether it is worthwhile, in areas where thalassaemia is common, to screen for globin gene mutations in subjects with a mean corpuscular volume (MCV) above 80 fL, especially in partners of known thalassaemia carriers. METHODS: Blood samples from 95 subjects with MCV between 80 and 85 fL were screened for the presence of alpha globin gene mutations and the haemoglobin (Hb) E mutation. RESULTS: Thirty four subjects harboured globin gene mutations. Of these, 31 had deletions of one alpha globin gene, one had Hb Constant Spring, and three had Hb E mutations. CONCLUSION: Based on the above figures and known prevalence rates of thalassaemia carriers, it would seem worthwhile to screen for globin gene mutations in partners of known thalassaemia carriers, regardless of MCV, to identify pregnancies at risk of Hb H disease or Hb E/beta thalassaemia.     

Co-inheritance of compound heterozygous Hb Constant Spring and a single -alpha(3.7) gene deletion with heterozygous deltabeta thalassaemia: a diagnostic challenge

Haemoglobin Constant Spring (Hb CS) mutation and single gene deletions are common underlying genetic abnormalities for alpha thalassaemias. Co-inheritance of deletional and non-deletional alpha (alpha) thalassaemias may result in various thalassaemia syndromes. Concomitant co-inheritance with beta (beta) and delta (delta) gene abnormalities would result in improved clinical phenotype. We report here a 33-year-old male patient who was admitted with dengue haemorrhagic fever, with a background history of Grave's disease, incidentally noted to have mild hypochromic microcytic red cell indices. Physical examination revealed no thalassaemic features or hepatosplenomegaly. His full blood picture showed hypochromic microcytic red cells with normal haemoglobin (Hb) level. Quantitation of Hb using high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) revealed raised Hb F, normal Hb A2 and Hb A levels. There was also small peak of Hb CS noted in CE. H inclusions was negative. Kleihauer test was positive with heterocellular distribution of Hb F among the red cells. DNA analysis for alpha globin gene mutations showed a single -alpha(-3.7) deletion and Hb CS mutation. These findings were suggestive of compound heterozygosity of Hb CS and a single -alpha(-3.7) deletion with a concomitant heterozygous deltabeta thalassaemia. Co-inheritance of Hb CS and a single -alpha(-3.7) deletion is expected to result at the very least in a clinical phenotype similar to that of two alpha genes deletion. However we demonstrate here a phenotypic modification of alpha thalassemia presumptively as a result of co-inheritance with deltabeta chain abnormality as suggested by the high Hb F level.     

Pitfalls in genetic counselling for β-thalassemia: an individual with 4 different thalassemia mutations

This paper describes a complex combination of four thalassemia genes (delta(+), beta(0), nondeletion and deletion alpha-thalassemia) in the spouse of a typical high Hb A2 beta-thalassemia carrier presenting for genetic counselling. This complex gene combination resulted in a hematological phenotype, characterized by thalassemia-like red cell indices, normal Hb A2 and Hb F levels and slightly reduced alpha/beta globin chain synthesis ratio, and therefore not indicative for the presence of beta-thalassemia trait. Family studies in combination with alpha-globin gene mapping, haplotype analysis at the beta-globin gene cluster and definition of the beta-thalassemia mutation by oligonucleotide hybridization led us to identify a beta-thalassemia mutation, to define the molecular basis for this phenotype and give the appropriate genetic counselling.     

PCR-LIS-SSCP快速分析非缺失型α-地中海贫血点突变

目的建立一种简便快速的筛查非缺失型α－地中海贫血点突变的ＳＳＣＰ分析方法。方法在选择性扩增α２珠蛋白基因的基础上,巢式ＰＣＲ扩增突变热点区域,低离子强度（ＬＩＳ）条件下将ＰＣＲ产物热变性,用非变性胶电泳分析其ＳＳＣＰ。结果ＬＩＳ条件下处理样品,ＰＣＲ产物变性效率明显提高,电泳结果可检出区别于野生型对照的３种含基因突变的电泳带型,且电泳时间仅需３小时。结论ＰＣＲ－ＬＩＳ－ＳＳ－ＣＰ可用于快速筛查非缺失型α－地中海贫血点突变。     

Bone marrow and peripheral blood globin chain synthesis in sickle cell beta zero thalassaemia.

A similar imbalance of globin chain synthesis, with low non-alpha/alpha ratios, was shown in peripheral blood and in bone marrow of compound heterozygotes for both the Hb S and beta zero thalassaemia genes (S/beta zero thalassaemia). Previous purification of whole cell globin obtained from the bone marrow did not change the non-alpha/alpha ratio. The mean non-alpha/alpha ratios were 0.57 +/- 0.13 (means +/- SD) for the peripheral blood of 12 patients, 0.52 +/- 0.10 for five patients using bone marrow globin purified on Sephadex G100, and 0.55 +/- 0.16 for the unfiltered bone marrow globin of five patients. The data show that patients with S/beta zero thalassaemia have a similar beta chain deficiency in reticulocytes and in bone marrow cells, provided whole cell globin is used which avoids the removal of the free alpha chains. The non-alpha/alpha ratios in the peripheral blood of an S/beta zero thalassaemia patient and a beta thalassaemia heterozygote from the same family were compared in seven families and no significant difference was found.     

Occurrence of the alpha thalassaemia-mental retardation syndrome (non-deletional type) in an Australian male.

The rare association of alpha thalassaemia and mental retardation has been described previously. Molecular studies of the alpha globin cluster in these cases have been heterogeneous, with some patients having large deletions while in others the alpha globin complex appears to be intact (non-deletional). The non-deletional cases form a distinct group whose features include severe mental retardation, haematological changes of haemoglobin H (Hb H) disease, developmental defects, and unusual patterns of inheritance. To date, five cases have been described with non-deletional alpha thalassaemia-mental retardation. We present here a further example of a young male of Northern European origin who appears to have the non-deletional form of the disease. Clinical features included severe mental retardation, Hb H disease, and developmental defects similar to those reported previously. DNA mapping, including pulsed field electrophoresis, showed no evidence of deletions within the alpha globin cluster. Karyotypic analysis indicated an increase in random breakage, which has been observed previously in one case of deletional alpha thalassaemia-mental retardation. Profuse Hb H bodies and Hb H on electrophoresis were consistent with Hb H disease. However, the latter was present at a relatively low level (1.6%) and, as well, the mean corpuscular volume (82.8 fl) and mean corpuscular haemoglobin (26.4 pg) were surprisingly high. Our findings are compared to other cases described with the non-deletional Hb H-mental retardation syndrome.     

Phenotyping and genotyping studies in a family with the compound heterozygosity deltabeta Thalassemia/beta(IVSII-849) Thalassemia

The propositus is a two year old child with a severe hemolytic anemia and increased level of Hb F. The Hbs A, A2 and F were eluted and quantitated by cation exchange high-performance liquid chromatography (HPLC-CE). DNA was isolated from peripheral blood leukocytes by a salting-out extraction procedure. The beta globin gone was amplified and the presence of the beta thalassemia mutation was determined by PCR followed of Reverse Dot Blot. Her hematological parameters were as follows: Hb: 7.0 g/dL, Hct: 24.8%, VCM: 87.4 fl, CHCM: 27.8 fl. The haemoglobin study showed an 97% increase of Hb F and Hb A2 normal. The molecular study suggested the presence of beta(IVSII-829) mutation in trans to deltabeta Thalassemia. The propositus inherited her mother's deltabeta-thalassemia gene mutation and her father's beta(IVSH-829) mutation. This is the first time the diagnosis has been performed in a Venezuelan family at-risk of compound heterozygotes for beta-thalassemia and delta beta-thalassemia.     

A genetic combination of silent beta-thalassaemia, high Hb A2 beta-thalassaemia, and single alpha globin gene deletion causing mild thalassaemia intermedia.

This paper reports a Sardinian patient, who was a compound heterozygote for silent beta-thalassaemia and high Hb A2 beta o-thalassaemia with the clinical phenotype of mild thalassaemia intermedia; alpha globin gene mapping showed a single alpha globin gene deletion. The reduced alpha globin chain output resulted in more balanced globin chain synthesis, which in turn accounted for the mild clinical phenotype.     

广西地区α2珠蛋白基因密码子30缺失导致非缺失型血红蛋白H病

目的　分析广西地区血红蛋白 (hemoglobin,Hb) H病的基因型 ,了解其表型与基因型的相互关系。方法　对 2 98个病例进行血液学分析、血红蛋白分析 ,及用聚合酶链反应方法和 DNA测序确诊基因突变。结果　在 2 98例 Hb H病患者中发现 1例罕见的 Hb H病。患者为男性 ,2 5岁。自幼有黄疸、脾肿大 ,从未输过血。血液学分析示血红蛋白 10 7g/ L,RBC4 .9× 10 1 2 / L,MCV76 .2 fl,血红蛋白分析 Hb H Hb Bart's34.4 1%。基因分析证实其为 α2珠蛋白基因密码子 30缺失复合东南亚缺失型 α地中海贫血 - 1。结论　我国曾报道的非缺失型 Hb H病主要有 Hb CS- H,Hb QS- H及 α2珠蛋白基因密码子 31基因突变复合东南亚缺失型 α地中海贫血 - 1,临床上表现为中至重度贫血的非缺失型 Hb H病。与之不同的是 ,该患者无贫血 ,但 Hb H Hb Bart's水平高于上述疾病。此基因突变在 α地中海贫血高发区之一的广西地区的认识和发现 ,对该地区的遗传咨询和产前诊断具有重要意义。     

Iron deficiency in sickle cell anaemia.

Thirty-seven patients with SCD were studied: 24 were diagnosed as homozygous Hb S on the basis of their haematological findings, and alpha:non-alpha globin chain ratios were found to be balanced in all. Thirteen patients were thought to have alpha or beta thalassaemia interaction with Hb S on the basis of low MCV and MCH, family history and/or presence of Hb A on electrophoresis. Six of them had abnormal alpha:non-alpha ratio (one had a ratio of 0.72 suggestive of alpha thalassaemia, and five had ratios between 1.4 and 1.9, compatible with beta thalassaemia interaction). The remaining seven patients with microcytosis had balanced globin chain synthesis and five were found to be iron deficient. Five additional patients (3 with Hb SS and 2 with Hb S/beta thalassaemia) had lower than normal serum ferritin concentration. The analysis of case histories disclosed that peptic ulceration, recurrent epistaxis and multiple pregnancies could account for iron loss in seven patients. These findings indicate that iron deficiency may be common in SCD and should be excluded as a cause of microcytosis. Microcytosis, in the absence of conclusive family studies and/or presence of Hb A on electrophoresis, is an unreliable indicator of alpha or beta thalassaemia interaction with Hb S.     

G gamma beta + type of hereditary persistence of fetal haemoglobin in association with Hb C.

This report describes a Negro family with the G gamma beta + type of hereditary persistence of fetal haemoglobin. Family members with levels of haemoglobin F of 17 to 23% had normal red cell indices, balanced globin chain synthesis, and a pancellular distribution of the fetal haemoglobin, showing that these subjects have a form of HPFH. The production of Hb A and C in addition to the large amount of Hb F in one family member showed that there was an active beta A gene in cis to the HPFH determinant, while structural analysis of the Hb F revealed the presence of only G gamma chains. The criteria for the diagnosis of G gamma beta + HPFH, and the relevance of such conditions to the control of globin gene expression, are discussed.     

The clinical features of spondyloepiphyseal dysplasia congenita resulting from the substitution of glycine 997 by serine in the alpha 1(II) chain of type II collagen.

The features of a child with spondyloepiphyseal dysplasia congenita resulting from a mutation in one COL2A1 allele were studied. The child was heterozygous for a G to A transition in exon 48 that resulted in the substitution of glycine 997 by serine in the triple helical domain of alpha 1(II) chains of type II collagen. Her longitudinal growth was close to the mean growth curve for children with this chondrodysplasia. Expression of the mutation by chondrocytes would account for the abnormal growth and development of the bones of the limbs and spine. Early expression of the mutation by epithelial cells and later expression by chondrocytes of the developing craniofacial structures would also account for her complex pattern of craniofacial anomalies. The findings in this study confirm that mutations of exon 48 of the COL2A1 gene, that alter the normal Gly-X-Y triplet structure of the corresponding region of alpha 1(II) chains of type II collagen, produce the spondyloepiphyseal dysplasia congenita phenotype.     

抗人宫颈癌单链抗体的基因构建、空间结构和进化分析

目的:构建抗人宫颈癌单链抗体(scFv)基因CSAs-1,并进行二级结构和三级结构预测、理化性质分析及进化树构建。方法:采用重叠延伸PCR方法,以能特异性分泌抗人宫颈癌mAb的杂交瘤细胞株CSA125为原料,构建抗人宫颈癌scFv基因CSAs-1,进行测序;并通过Internet对其进行结构预测和进化树构建。结果:抗人宫颈癌scFv基因CSAs-1有834bp,对应278个氨基酸的多肽,等电点预测值7.215,为碱性蛋白质;PHDsec二级结构显示CSAs-1属于α β蛋白,VH和VL区均有多个蛋白激酶C磷酸化位点、酪蛋白激酶II磷酸化位点;CSAs-1三级结构建模显示VL和VH形成一个疏水的“口袋”,Linker游离于此结构之外。以上结果表明:CSAs-1符合scFv的结构特点,明显具有抗原结合位点的空间构象,理论上应该具有良好的抗原结合活性。进化分析发现脊椎动物V基因形成三个进化群,CSAs-1V区分布于进化树的A群中。结论:构建一个鼠源性抗人宫颈癌scFv基因CSAs-1,为该基因的进一步原核和真核表达打下了基础;应用信息学技术所获得的预测和分析结果为CSAs-1表达、纯化和活性研究提供了大量的信息;为进一步分析抗体VH、VL在抗体功能中的作用、改造抗体、实现抗体的人源化、提高抗体的活性等方面提供了一定的依据。     

Filipino beta zero thalassaemia: a high Hb A2 beta zero thalassaemia resulting from a large deletion of the 5'' beta globin gene region.

A large novel deletional beta zero thalassaemia mutation associated with unusually high levels of haemoglobin (Hb) A2 in heterozygotes is described in two unrelated subjects of Filipino background. The deletion was characterised by DNA mapping including pulsed field gel electrophoresis. Filipino beta zero thalassaemia extends for approximately 45 kb beginning approximately 1.5 kb 3' to the delta globin gene. It is the largest deletion to date which gives rise to the beta zero thalassaemia phenotype. This mutation, similar to previously described deletional beta zero thalassaemias associated with high Hb A2, removes sequences 5' to the beta globin gene promoter and emphasises the functional importance of the 5' beta globin region in eliciting the unusually high level of Hb A2. This example also suggests that it is the 3' sequences which are transposed rather than the actual deletion size which are significant in the raised fetal haemoglobin (Hb F) found with some of the thalassaemias.     

Alpha thalassemia British type (alpha alpha/--Brit) in an Australian family

Alpha thalassemia is rarely diagnosed in Australian families of British or Northern European ancestry. In 1972, a third generation Australian was shown to have alpha thalassemia. In the absence of known Mediterranean or South East Asian ancestry it was reported as being the first example of alpha thalassemia in an Australian family. Further study of the proposita in 1985 using DNA mapping of the alpha globin gene complex, shows a distinctive molecular defect identical to the British type of alpha thalassemia. The latter is clearly different from the commonly encountered Mediterranean and South East Asian alpha zero haplotypes. Recognition that alpha zero thalassemia occurs in Australians is important since it may produce a microcytic hypochromic anemia. Its inheritance together with other forms of alpha thalassemia may lead to severe Hb H disease or Hb Bart's hydrops fetalis.     

应用温度梯度凝胶电泳分析非缺失型α-地中海贫血突变

目的 建立用于非缺失型α-地中海贫血（α-地贫）突变筛查的温度梯度凝胶电泳（temperature gradient gel electrophoresis,TGGE)技术。方法 以不同基因型人基因组DNA为模板,选择性扩增全长α2－珠蛋白基因,继以巢式PCR扩增突变热点区域,然后建立并优化TGGE参数,在此基础上,筛查表型阳性的15例非缺失型α－地贫疑诊诊样品,阳性样品经DNA序列测定确认突变,结果 建立了分析543 bp PCR片段和α2珠蛋白基因全长1085bp片段的TGGE技术,可检测出中国人群中常见的两种α-地贫点突变：Hb Constant Spring (Hb CS)和Hb Quong Sze(Hb QS)及Hb Westmead a2 CD122CAC→CAG（His-Gln),15例样品中,10例为Hb CS,2例为Hb QS,2例为Hb Westmead,1例为新的Hb H病相关的突变a2 CD31 AGG－AAG（Arg-Lys),结论 所建立的PCR－TGGE技术可用于非缺失型a-地贫点突变和a-珠蛋白基因多态性的分子筛查。