Hb M Dothan [beta 25/26 (B7/B8)/(GGT/GAG-->GAG//Gly/Glu-->Glu]; a new mechanism of unstable methemoglobin variant and molecular characteristics.

A new unstable beta globin chain variant associated with methemoglobin (Met-Hb) phenotype was found in a Caucasian infant. Molecular analysis of the beta globin gene using polymerase chain reaction (PCR) amplification and sequencing led to the detection of a new in frame deletion in exon-1. Direct sequencing of the PCR product revealed a 3 bp deletion (-GTG) between codons 25/26, which resulted in the loss of a single amino acid (-Gly). We propose that this newly discovered unstable M-hemoglobin (M-Hb) variant, named Hb Dothan [GGT/GAG-->GAG//Gly/Glu-->Glu], is caused by a shift in the amino acid sequence and altered packing of the B and E helices during beta globin synthesis, and also changes the orientation of the critical proximal and distal histidine in the F and E helices respectively. Phenotype/Genotype features and molecular characteristics of this new beta chain are presented in this communication.

The influence of uridine diphosphate glucuronosyl transferase 1A promoter polymorphisms, beta-globin gene haplotype, co-inherited alpha-thalassemia trait and Hb F on steady-state serum bilirubin levels in sickle cell anemia.

PURPOSE: Homozygosity for the (AT)7 allele of uridine diphosphate glucuronosyl transferase 1A (UGT1A1) gene polymorphism is associated with increased bilirubin levels in sickle cell anemia (SCA). In the present study, in addition to UGT1A1 promoter genotype, serum bilirubin level was related to other genetic modifiers -beta(S)-globin gene haplotype, Hb F, co-inherited alpha-thal trait, age and gender. METHODS: The patients were randomly selected from the sickle cell clinic, Medical College of Georgia. UGT1A1 promoter polymorphisms were determined using automated sequencing. Other investigations were with standard techniques. RESULTS: There were 67 SCA patients (41 males and 26 females), aged 2-44 yr (mean of 20.6 +/- 10.7). Ten (14.9%) patients were homozygous for the (AT)6 UGT1A1 allele, 35 (52.2%) were heterozygous for (AT)6 and (AT)7 alleles while 22 (32.8%) were homozygous for (AT)7. Serum bilirubin was significantly higher in the homozygous (AT)7 group (3.7 +/- 1.5, 3.8 +/- 2.3 and 5.6 +/- 2.4 mg/dL, respectively). It was also significantly higher in males than females and in patients aged >10 yr. There was a significant negative linear correlation (r = -0.304, P = 0.016) of serum bilirubin with Hb F. The beta-globin haplotype and co-existing alpha-thal trait did not have any significant influence on serum bilirubin levels. Patients on hydroxyurea were older, had lower Hb F, but higher mean serum bilirubin. The latter also was signifcantly higher among those with UGT1A1 (AT)7 homozygosity. CONCLUSIONS: Apart from UGT1A1 (AT)7 homozygosity, Hb F, age and gender are the other factors that significantly influence serum bilirubin level in SCA.

Postmortem molecular diagnosis of sickle beta thalassaemia.

This report describes a case in which the diagnosis of sickle cell disease (SCD) was established after death. The diagnosis of sickle cell syndrome was made in a 68 year old black patient who was found to have sickled red blood cells in many organs at necropsy although the disease had not been diagnosed during her lifetime. DNA was isolated from a peripheral blood smear obtained on the day of the patient's death. The beta globin gene was polymerase chain reaction amplified and sequenced, revealing that the patient had S-beta(+) thalassaemia. This study shows that blood smears are a suitable source for retrospective DNA analysis studies. This case illustrates that relatively "mild" forms of SCD can be overlooked, despite symptomatology suggestive of a sickle syndrome, and demonstrates the feasibility of the postmortem molecular diagnosis of haemoglobinopathies in such cases.

The methylene tetrahydrofolate reductase (C677T) mutation as a potential risk factor for avascular necrosis in sickle cell disease.

Avascular necrosis (AVN) of the humeral and femoral heads is a frequent and debilitating complication of sickle cell disease. Some of the risk factors for AVN are alpha-thalassemia and age. Recently, newly discovered thrombophilia mutations have been associated with AVN in patients without sickle cell disease. We studied the frequency of the thermolabile methylene tetrahydrofolate reductase (MTHFR) variant (C677T) in adult sickle cell patients with and without AVN. The frequency of the MTHFR mutation was 35.6% in patients with AVN and 12.9% in those without AVN (p = 0.006). These data suggest that the thermolabile MTHFR variant may be a contributing risk factor for AVN in some populations with sickle cell disease.

Frequency of the 677 C-->T mutation of the methylenetetrahydrofolate reductase gene among Kuwaiti sickle cell disease patients.

Sickle cell disease (SCD) is relatively mild among Kuwaiti Arabs. However, an atypical subset of patients exists with frequent, severe vaso-occlusive crisis and osteonecrosis. The thermolabile variant of MTHFR, resulting from a C-->T mutation at nucleotide 677, has been shown to be associated with hyperhomocysteinemia, which is an important risk factor for premature vascular disease. We have screened an unselected group of 41 Kuwaiti SCD patients (33 SS and 8 Sbeta(0)-thal) attending the Hematology Clinic of Kuwait University Teaching Hospital for the MTHFR mutation, using a PCR-RFLP method. The patients were aged 2-41 years (mean of 12.8 +/- 8.6). One (2.4%) individual was homozygous for the mutation while 15 (36.6%) were heterozygous, giving an allele frequency of 20.7%. Twenty-one patients (14 SS and 7 Sbeta(0)-thal) were screened for osteonecrosis using MRI of the hip (spin-echo T1- and T2-weighted images). Seven (33.3%) had varying degrees of osteonecrosis, among whom the frequency of the 677 C-->T allele was 21.4%. The frequency was identical among those without osteonecrosis. Although the allele frequency is higher among our patients compared to American SS patients, our results do not suggest an association with osteonecrosis.

[Two Turkish manuscripts on gems (jauhar) and the treatment by means of gems]. / Iki Türkçe cevâhir-nâmeye ve cevherlerin etkilerine dair.

Literary works of the genre called cevâhir-nâme or cevher-nâme are written in order to give information on how gems (cevher-jauhar) are formed, where they are found, their estimated values, how to distinguish them from the imitations, what their particular colours are and on their uses and harmful effects. These literary works are sometimes titled in accordance with the genre they belong to and sometimes they are given different titles. The literary works dealing with the subject of cevher are mostly written in prose and only a few of them are in verse. The Turkish versions of cevâhir-nâme texts started to be written in the 15th century. These texts, covering the earlier knowledge of medicine, give information on gems and perfumes; how they effect human health; and the way they should be used in medical treatment. Therefore, these texts should be discussed in relation with their place in the history of Turkish science. Two cevâhir-nâmes, which were translated into Turkish by Mustafâ bin Seydî and Za'îfî in the 15th and 16th centuries are still available. The translations by Mustafâ bin Seydî and Za'îfî, did not only convey the classical and medieval knowledge on cevhers to the contemporary readers, but they also confirm the fact that this information was a part of the Ottoman social life.

Identification of the Chinese IVS-II-654 (C-->T) beta-thalassemia mutation in an immigrant Turkish family: recurrence or migration?

In this study we describe the Chinese IVS-II-654 (C-->T) beta-thalassemia mutation for the first time in an immigrant Turkish family living in Istanbul and originating from Xanthe, Greece. Four members of the family, representing 3 generations, are heterozygous for this mutation. A detailed family history demonstrated a Greek origin for members of 5 generations with no records of migration or consanguineous marriages. Analysis of polymorphic nucleotides located at the 5' end of the beta-globin chromosomes bearing the IVS-II-654 mutation in the family described carried the (AT)9(T)5 type of microsatellite sequence and the ACATCCCCA haplotype. These 2 haplotype components favor a non-Eastern Asian origin for this chromosome, hence suggesting an independent origin for the IVS-II-654 mutation described in this family.

Hb Hammersmith [beta 42(CD1) Phe-->Ser]: occurrence as a de novo mutation in black monozygotic twins with multiple congenital anomalies.

PURPOSE: To present the occurrence of Hb Hammersmith as a de novo mutation in African-American twins with multiple congenital anomalies. METHODS: Standard hematologic methods were used. The presence of an unstable Hb variant was confirmed by brilliant cresyl blue staining and an isopropanol stability test. Hb Hammersmith was confirmed by the sequencing of polymerase chain reaction-amplified beta-globin gene. RESULTS: The presence of Hb Hammersmith was confirmed in female monozygotic twins of African-American origin with congenital Heinz body hemolytic anemia and multiple congenital anomalies. The variant occurred as a de novo mutation in the twins. CONCLUSION: This report describes the occurrence of Hb Hammersmith [B42(CD1)Phe-->Ser] in African-American twins. As with the other reported cases, both twins were female. In addition to Heinz body hemolytic anemia, a low arterial O2 saturation in the proposita was shown by pulse oximetry. Multiple congenital anomalies involving various systems were also found in both twins.

A long terminal repeat of the human endogenous retrovirus ERV-9 is located in the 5' boundary area of the human beta-globin locus control region.

Transcription of the human beta-like globin genes in erythroid cells is regulated by the far-upstream locus control region (LCR). In an attempt to define the 5' border of the LCR, we have cloned and sequenced 5 kb of new upstream DNA. We found an LTR retrotransposon belonging to the ERV-9 family of human endogenous retroviruses in the apparent 5' boundary area of the LCR. This ERV-9 LTR contains an unusual U3 enhancer region composed of 14 tandem repeats with recurrent GATA, CACCC, and CCAAT motifs. This LTR is conserved in human and gorilla, indicating its evolutionary stability in the genomes of the higher primates. In both recombinant constructs and the endogenous human genome, the LTR enhancer and promoter activate the transcription of cis-linked DNA preferentially in erythroid cells. Our findings suggest the possibility that this LTR retrotransposon may serve a relevant host function in regulating the transcription of the beta-globin LCR.

Hb Adana or alpha 2(59)(E8)Gly-->Asp beta 2, a severely unstable alpha 1-globin variant, observed in combination with the -(alpha)20.5 Kb alpha-thal-1 deletion in two Turkish patients.

We have identified a severely unstable hemoglobin variant through sequencing of amplified DNA involving the alpha 1-globin gene; the mutation is located in codon 59 (CCG CAG) and results in a Gly-->Asp replacement. This amino acid substitution concerns a glycine residue at an internal position in the E helix, which is in close contact with a glycine residue of the B helix; introduction of the larger and charged aspartic acid residue greatly affects the stability of the molecule. This variant was present in association with a common alpha-thalassemia-1 deletion [-(alpha)20.5 kb] in two adults and caused a severe type of Hb H disease with anemia, low levels of Hb A2, increased zeta chain, and Hb Bart's. In vitro chain synthesis in reticulocytes showed a high specific activity of the variant alpha chain. Only a minute quantity of Hb H was present but instead about 10% of Hb Bart's was observed. The increased synthesis of gamma chains was likely due to specific characteristics of a chromosome with haplotype #3, which was present in both patients. The same family was studied 18 years ago; the improved methodology presently available has led to a corrected diagnosis for these patients.

New ultra-micro high-performance liquid chromatographic method for determining the gamma chain composition of hemoglobin F in normal adults.

The difficulty in isolating the minute quantity of Hb F (< 1%) present in the red blood cells of normal adults greatly complicates the determination of its gamma chain composition. We have developed a rapid ultra-micro high-performance liquid chromatographic (HPLC) method and used it to analyze the gamma chain composition of Hb F in 47 adults with Hb F levels between 0.1-3.4%. The method involves the isolation of Hb from as little as 50 microliters of whole blood on an analytical size cation-exchange HPLC column, followed by concentration in a Centricon micro concentrator unit and by reversed-phase HPLC analysis. The entire procedure can be completed in one day and 3-4 analyses can be made simultaneously. We reanalyzed the blood samples from 22 subjects with known beta-globin gene cluster haplotypes, and confirmed the association of high, low, and very low G gamma levels with haplotypes A, B, and C, respectively. Also included are the results of DNA sequence analyses of the G gamma and beta promoters, and of the locus-control-region hypersensitive site-2 (LCR-HS-2) of the beta-globin gene cluster in five subjects homozygous for haplotypes A, B or C; the data obtained failed to provide a satisfactory explanation for all the variations in the G gamma levels that have been observed.

Spleen in sickle cell anemia: comparative studies of Nigerian and U.S. patients.

Anecdotal reports have attributed persistent splenomegaly in African sickle cell anemia (SS) patients to the effects of malaria. However, no comparative studies of patients in malarial and nonmalarial regions have been conducted, and few studies of malaria antibody titers have been reported. In the present study, age- and sex-matched Nigerian patients (n = 310), while it was found only in 8% of U.S. patients (n = 100) from Georgia. There was significant linear correlation between spleen size and Hb levels and with serum immunoglobulins in the Nigerian group. However, serum complement levels (C3 and C4) were not affected by spleen size. In both groups, patients with splenomegaly had fewer circulating pitted red cells than their counterparts without splenomegaly. The mean +/- SE of IgG-specific malaria antibody titer among the Nigerian patients without palpable spleens was 9,386 +/- 2,036; 9,334 +/- 2,980 in those with spleens between 1 and 5 cm, 16,201 +/- 4,502 in those with spleens between 6 and 10 cm, and 22,445 +/- 8,456 in those with spleens above 10 cm. Coexistent alpha-thalassemia did not influence the prevalence of splenomegaly among the Nigerian SS patients. This study provides additional evidence that malaria plays a significant role in the persistence of splenomegaly in African patients.

Beta-thalassemia mutations in the Portuguese; high frequencies of two alleles in restricted populations.

We report the characterization of seven different beta-thalassemia mutations in 131 newly diagnosed Portuguese beta-thalassemia heterozygotes. Methodology included the detection of abnormal fragments by agar gel electrophoresis of PCR-amplified DNA fragments after digestion with specific restriction endonucleases, as well as hybridization with synthetic nucleotide probes and sequencing of amplified DNA. Four mutations, including the newly discovered TGG-->TGA change at codon 15, occurred in excess of 10% and accounted for some 90% of the beta-thalassemia alleles in this population. The geographical distribution is uneven; the TGG-->TGA mutation at codon 15 was primarily observed in the coastal region north of Lisbon, while the IVS-I-6 (T-->C) mutation was confined to the central part of the country.

Usefulness of HPLC methodology for the characterization of combinations of the common beta chain variants Hbs S, C, and O-Arab, and the alpha chain variant Hb G-Philadelphia.

We have analyzed the blood samples from seven members of two families that contained mixtures of different hemoglobin types due to the simultaneous presence of different beta chain variants and the alpha chain variant Hb G-Philadelphia. The methods used were isoelectrofocusing and cation exchange high performance liquid chromatography for the separation of the hemoglobin components, and reversed phase high performance liquid chromatography for that of the various alpha and beta chains; the chromatographic procedures also provided quantitative data. Application of these fast and simple high performance liquid chromatographic methods greatly aided in defining the different hemoglobinopathies that were present in these persons, including an additional alpha-thalassemia-2 heterozygosity or homozygosity.

Black beta-thalassemia homozygotes with specific sequence variations in the 5' hypersensitive site-2 of the locus control region have high levels of fetal hemoglobin.

We have sequenced the 5' hypersensitive-2 (5'HS-2) site of the locus control region (LCR) and the promoters of the two gamma-globin genes located on chromosome 11 of a black patient with mild beta-thalassemia (beta-thal) major due to a homozygosity for the C----T mutation at position -88 of the beta promoter and with a high Hb F level. Sequence variations in the 5'HS-2 were the same as observed for the beta s chromosome with haplotype number 3, while most of the G gamma promoter and the A gamma promoter had sequences similar to that of the beta S chromosome with haplotype number 19. This atypical haplotype (number 19A) is apparently associated with an increased gamma chain production which is particularly evident during periods of severe hematopoietic stress. Additional studies on relatives of the proband and on 10 unrelated black beta-thal homozygotes with either the C----T mutation at -88 or the A----G mutation at -29, confirm the possible importance of the sequence differences in the 5'HS-2, and also suggest that at least two additional factors, namely a C----T mutation at position -158 of the G gamma promoter and a relative deficiency in alpha chain synthesis play a (perhaps less important) role in the increased Hb F synthesis in these patients.

Molecular characterization of beta-thalassemia in Czechoslovakia.

We have identified different beta-thalassemia mutations in 93 members of 34 families of Czech or Slovakian descent using gene amplification, hybridization with specific 32P-labeled oligonucleotide probes, sequencing of amplified DNA, and gene mapping. The G----A mutation at IVS-I-1 was found in 18 families; other Mediterranean mutations were IVS-II-1 (G----A), IVS-II-745 (C----G), IVS-I-110 (G----A), and codon 39 (C----T); these were present in 9 additional families. The G----T mutation at codon 121, known to cause Heinz-body beta-thalassemia, was present in 3 families, and the frameshift at codons 82/83 (-G), first described in the Azerbaijanian population, in 2 families. A newly discovered allele was a frameshift at codons 38/39 (-C). One beta-thalassemia allele was incompletely characterized. We observed in 2 families a T----C mutation at position +96 UTR (untranslated region) relative to the termination codon; this mutation likely is a rare polymorphism. alpha-Thalassemia was rare; only one person carried the -alpha 3.7 heterozygosity, and one other had a yet to be identified alpha-thalassemia-1, while seven had the alpha alpha alpha anti 3.7 triplication.

Heterogeneity of gamma-globin chain synthesis in Saudi newborns.

Cord blood samples from 655 unselected neonates born to Saudi mothers at King Fahad National Guard Hospital, Riyadh, Saudi Arabia were analysed to determine the levels of gamma-globin chains in Saudis. The percentage of three types of gamma-chains of human fetal hemoglobin (A gamma T, G gamma and A gamma I) was obtained by high-performance liquid chromatographic (HPLC) method. Although the majority of babies (631/655) had normal G gamma values in the range of 58-74%, there were only 69% with normal G gamma/A gamma ratio. The A gamma T chain or HbF Sardinia was present in 28% of the total neonates with a gene frequency of 0.160. The A gamma T values in this group ranged between 11-42%. Eight babies (1.2%) had G gamma levels 45% or less (mean 41 +/- 3%) and in 16 neonates (2.4%), G gamma values were highly elevated (mean 81.4 +/- 2.8%). The frequency of two G gamma-globin genes was 0.0061 and 0.0122, respectively, which is comparable with other ethnic or racial groups. The differences in G gamma to A gamma ratio in some Saudi babies could be due to an abnormal arrangement of gamma-globin genes of beta-globin gene cluster which is now being investigated.