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.

Hb A2-Wrens or alpha 2 delta 2 98(FG5) Val----Met, an unstable delta chain variant identified by sequence analysis of amplified DNA.

Data are reported for an 85-year-old black make who had an HPFH condition on one chromosome and a suspected 'delta-thalassemia' on the other. Sequence analysis of amplified DNA of an appropriate segment of the delta-globin gene identified a GTG to ATG mutation for codon 98 and thus a Val----Met replacement in the delta chain. This abnormality was confirmed by hybridization of amplified DNA with 32P-labeled synthetic probes and by the amino-acid composition of the isolated tryptic peptide delta T-11. Thus, the 'delta-thalassemia' is caused by the presence of an Hb A2 variant that is considered to be unstable to a similar extent as is Hb Köln, its beta chain counterpart.

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.

gamma-Globin gene triplication and quadruplication in Japanese newborns. Evidence for a decreased in vivo expression of the 3'-A gamma-globin gene.

Cord blood samples of 113 Japanese babies from Kurashiki, Japan, were analyzed for the presence of possible modifications in gamma-globin gene arrangements and for changes in the relative quantities of the gamma chains G gamma, A gamma, and its variant A gamma T. As many as 9 babies had the gamma-globin gene triplication, one a gamma-globin gene quadruplication, and two a gamma-globin gene deletion. The triplication and quadruplication involved cross-overs within the G gamma- and A gamma-globin genes resulting in a hybrid gene with the 5'-segment being derived from the A gamma and the 3'-segment from the G gamma-globin gene (-AG gamma-). The G gamma value of the 9 babies with the -G gamma-AG gamma-A gamma-globin gene arrangement averaged approximately 80% and that of the baby with -G gamma-AG gamma-AG gamma-A gamma- was 78%. Four of the 9 babies with the triplication and the baby with the quadruplication had an A gamma T mutation in trans; in these babies, the A gamma chain level (from the A gamma-globin gene in 3'-position in the tri- and quadruplication arrangements) was decreased to about one-third and one-sixth of the level observed in the simple A gamma T heterozygote. This observation supports the suggestion that transcription beginning at the 5'-gamma-globin gene interferes with that of the 3'-gamma-globin gene.

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.

Chinese in west Malaysia: the geography of beta thalassaemia mutations.

The overseas Chinese in West Malaysia are almost exclusively from the south-eastern provinces of China-Kwangtung, Fukien, and Kwangsi. To institute a comprehensive thalassaemia control programme for this region we have characterised the beta thalassaemia mutations in 16 Chinese patients from West Malaysia: 4 beta thalassaemia mutations were seen: a) an A----G substitution in the TATA box [-28 base pairs (bp)], an A----T substitution in codon 17 [17 A----T], c) a 4 base pairs - TCTT deletion in codon 41-42 [frameshift mutation (FSC 41-42)], and d) a C----T substitution at the second intervening sequence (IVS 11) position 654. Similar mutations have been described in patients from the south-eastern provinces of China. The delineation of the specific mutations present will enable effective prenatal diagnosis for beta thalassaemia of ethnic Chinese in West Malaysia to be instituted.

beta S-haplotypes and alpha-thalassemia along the coastal belt of Kenya.

In this paper, we present data on studies of beta S-haplotypes and alpha-thalassemia gene in subjects from the indigenous population of the Coast Province of Kenya Of the 7SS patients studied, four were homozygous for beta S-haplotype 20 characteristically associated with the severe form of sickle cell anemia found in the Central African Republic and Western Kenya. Two had haplotype 20 combined with haplotype 19 (Benin Type) and one had haplotype 20 combined with a new haplotype (20x). Alpha thalassaemia-2 gene (-3.7kb deletion) was detected in 45.6% of the 57AA subjects studied. An alpha globin gene triplication was detected in one subject whereas eight had gamma globin gene triplication.

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.

[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.

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.

Separation of normal and abnormal hemoglobin chains by reversed-phase high-performance liquid chromatography.

A review is presented of the elution patterns on reversed-phase columns of the normal and abnormal globin chains of different hemoglobin types, including 16 beta-chain variants, 7 alpha-chain variants, 9 gamma-chain variants, and 4 variants with fusion or hybrid chains. Separations appear to be based primarily on differences in hydrophobicity. The method is ideally suited for the detection of abnormal globin chains, their quantitation and their isolation. Semi-quantitative data based on the calculation of the delta/non-alpha ratios allow the detection of beta-thalassemic conditions in situations where the quantitation of hemoglobin A2 by other procedures is impossible or complicated.

Gamma chain abnormalities and gamma-globin gene rearrangements in newborn babies of various populations.

The present review provides a summary of quantitative hemoglobin data and lists the results of gene mapping and sequencing analyses for blood samples from newborn babies of different countries. Methodology suitable for such studies is reviewed, various abnormal fetal hemoglobins are discussed, the occurrence of Hb Bart's (gamma 4) and of the embryonic zeta chain is evaluated, and the various types of gamma-globin gene rearrangements (-A gamma.A gamma-; -G gamma.G gamma-; gamma-thalassemia; gamma-globin gene triplications, quadruplications, and quintuplications) are compared. The several tables list the frequencies of the common A gamma T variant and of the different gamma gene rearrangements in various populations, while the results of quantitative analyses suggest that most anomalies are not associated with disease.

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.

Haplotypes of beta S chromosomes among patients with sickle cell anemia from Georgia.

Fetal hemoglobin and G gamma levels have been correlated with the presence or absence of eight restriction sites within the beta globin gene cluster (haplotypes) for numerous sickle cell anemia patients from Georgia. The most common haplotypes were #19 (Benin) and #20 (CAR); all patients with haplotype combinations 19/19, 20/20, and 19/20 were severely affected with low Hb F and low G gamma levels. A modified #19 beta S chromosome with a -G gamma-G gamma- globin gene arrangement, instead of -G gamma-A gamma-, was present in SS and SC newborn babies with G gamma values above 80%. Haplotype #3 (Senegal) was present among 15% of the beta S chromosomes; the two adult patients with the 3/3 combination were mildly affected with high Hb F and G gamma values. The haplotype AT with the variant A gamma T chain was a rarity. A new haplotype was found in one 17-year-old SS patient and five of his Hb S heterozygous relatives. This haplotype is associated with an increased production of Hb F in heterozygous and homozygous Hb S individuals; this Hb F contained primarily A gamma chains. A comparison was made between the different haplotypes among SS patients and normal Black individuals, and a remarkable similarity was noted in the fetal hemoglobin data for subjects with these different chromosomes.

Hb F-Catalonia or alpha 2G gamma(2)15(A12)Trp----Arg.

Hb F-Catalonia, a G gamma chain variant with a Trp----Arg substitution at position gamma 15(A12), was observed in two Spanish newborn babies from Northeastern Spain. Analyses were performed with different reversed phase high performance liquid chromatographic procedures that allowed the identification of the amino acid replacement in only a minute quantity of the isolated G gamma X chain.

Hb F-Charlotte, an A gamma variant with a threonine residue in position gamma 75 and a glycine residue in position gamma 136.

Structural analyses of an abnormal gamma chain, present in a relative amount of approximately 10% in a cord blood sample of a Black newborn baby, identified two substitutions (gamma 75 Ile----Thr and gamma 136 Ala----Gly) in an apparent variant of the A gamma chain. Gene mapping analysis of genomic DNA and hybridization with specific probes confirmed the presence of these two mutations and provided the evidence to indicate that the abnormal gamma chain was indeed a variant of A gamma with the two listed mutations.

Hydrops fetalis due to homozygosity for alpha-thalassemia-1, -(alpha)-20.5 kb: the first observation in a Turkish family.

We report data on a fetus with hydrops fetalis due to a homozygosity for alpha-thalassemia-1, type -(alpha)-20.5 kb; this is the first reported case in a Turkish family. Characterization of the abnormality was based on data from family studies and from alpha-globin gene mapping of the DNA from the parents.

Fetal hemoglobin in normal adults and beta-thalassemia heterozygotes.

A recently developed high performance liquid chromatographic (HPLC) procedure using a weak cation exchanger (PolyCAT) in columns of different sizes was used to quantify fetal hemoglobin (HbF) in blood of normal adults and beta-thalassemia (beta-thal) heterozygotes with ten different types of mutations. Preparative PolyCAT-HPLC greatly facilitated the characterization of isolated HbF, i.e., the determination of the relative quantities of the G gamma and A gamma chains. The method is accurate and allows quantitation of Hb F at the 0.5% level; preparative PolyCAT-HPLC allows isolation of (nearly) pure Hb F from blood samples with low (less than 1%) Hb F. Adult Hb F levels were determined in 69 normal adults (including 24 diabetics); Hb F levels fell below 1% except for subjects with abnormal -- G gamma -- G gamma -- arrangement and a C----T mutation at position -158 relative to the Cap site of both G gamma genes. The effect of the same mutation in the normal -- G gamma -- A gamma-arrangement was variable. Certain beta-thal mutations (namely, those at positions -29; -88; IVS-I-1; IVS-II-1) were associated with high Hb F levels in heterozygotes, while those at nucleotide (nt) positions IVS-I-6; IVS-I-110; codon 24; codon 39; codons 41/42; IVS-II-745 were not. G gamma values varied and often fell into two groups (high G gamma and low G gamma); high G gamma values were not associated with high Hb F values. The chromatographic procedure is ideally suited for Hb A2 quantitation. Average values of Hb A2 in beta-thal heterozygotes with any one of nine of the ten mutations were twice that of normals; the one exception was the beta-thal heterozygote with the IVS-I-6 (T----C) mutation with an average low Hb A2 value of 3.6%.

Variation in the level of fetal hemoglobin in (delta beta) (0)-thalassemia heterozygotes with different numbers of alpha-globin genes.

The Sicilian type of (delta beta) (0)-thalassemia characterized by a approximately 13 kb deletion, was present in a Turkish boy who is a homozygote and in his heterozygous parents who are first cousins. The father with approximately 21% Hb F had five alpha-globin genes (alpha alpha/alpha alpha alpha) and the mother with approximately 10% Hb F had an alpha-thal-2 heterozygosity (alpha alpha/-alpha). The difference in Hb F level is explained by a decreased formation of alpha 2 gamma 2 tetramers in the mother with an alpha-chain deficiency while the extra alpha-globin gene in the father will promote Hb F production.