Jamaican Sbetañthalassaemia: mutations and haematology

The sickling disorders are a common cause of morbidity and mortality in Jamaica. Sickle cell betañthalassaemia is the fourth commonest form, occuring in one in every 3000 births. This is a heterogeneous condition, producing HbS, HbF and HbA2 with variable amounts of HbA, depending on the mutation and, within a defined population, only a few beta-thalassaemia mutations occur at high frequency. This study establishes the frequency of beta-thalassaemia mutations in Sbetañthalassaemia patients in Jamaica. In addition, comparison of the haematological phenotypes is possible by looking at the "average steady-state haematology" of the different mutational groups. Blood samples from 132 unrelated Sbetañthalassaemia patients attending the MRC Sickle Cell Unit at the University of the West Indies were analysed by amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) or sequencing to determine the nature and frequencies of the underlying beta-thalassaemia mutations. Ten mutations were identified, four of which accounted for 93 percent of the patients studied. These were 29 (A --> G) in 71 (54 percent), -88 (C --> T) in 27 (20 percent), polyA (T --> C) in 17 (13 percent) and IVS1-5 (G --> C) in nine (7 percent). The six remaining mutations found at lower frequency were C24 (T --> A) in two patients and one each of IVS2-848 (C --> A), -90 (C --> T), IVS1-5 (G --> T),IVS1-6 (T --> C). In one individual, no mutation was found. The three commonest mutations were all associated with levels of greater than 10 g/dl, whereas IVS1-5 (G --> C) had a more severe haematological phenotype. The predominance of -29 (A --> G) and -88 (C --> T) is in keeping with other studies on populations of African origin. IVS1-5 (G --> C) is found chiefly in Indian populations, and all affected families acknowledged Indian ancestry, reflecting the prominent Indian community in Jamaica. (AU)

Adeno-associated virus and development of cervical neoplasia

Evidence from several sources has suggested that adeno-associated virus (AAV) infection might protect against cervical cancer, in part, by interfering with human papillomavirus (HPV)-induced tumorigenesis. Detection of AAV type 2 (AAV-2) DNA in cervical tissues has been reported. However, there have been few in vivo studies of women with cervical HPV infection or neoplasia, and these have reported inconsistent results. Therefore, we used polymerase chain reaction (PCR) assays targeted to the AAV-2 rep and cap genes to test tissue specimens from women in an epidemiological study of cervical neoplasia in Jamaica. We tested 105 women with low-grade cervical intraepithelial neoplasia (CIN-1), 92 women with CIN-3/carcinoma in situ or invasive cancer (CIN-3/CA), and 94 normal subjects. PCR amplification of human beta-globin DNA was found in almost all cervical specimens, indicating that these materials were adequate for PCR testing. The prevalence of HPV DNA, determined by HPV L1 consensus primer PCR was, as expected, strongly associated with presence and grade of neoplasia. Each of the AAV PCR assays detected as few as 10 copies of the virus genome. However, none of the 291 cervical specimens from Jamaican subjects tested positive for AAV DNA. Negative AAV PCR results were also obtained in tests of cervical samples from 79 university students in the United States. Exposure to AAV was assessed further by serology. Using a whole virus AAV-2 sandwich enzyme-linked immunosorbent assay, we found no relationship between AAV antibodies and presence or grade of neoplasia in either the Jamaican study subjects or women enrolled in a U.S. cervical cancer case (n = 74) - control (n = 77) study. Overall, the data provide no evidence that AAV infection plays a role in cervical tumorigenesis or that AAV commonly infects cervical epithelial cells.(Au)

Benign clinical course in homozygous sickle cell disease: a search for predictors

AIMS: (1) To estimate the proportion of subjects with homozygous sickle cell disease who have a benign clinical course, and (2) to assess factors that may be predictive of benign disease. MATERIAL: Subjects (n = 280) were participants in a longitudinal cohort study of sickle cell disease. They were classified as benign or control based on clinical history from birth to age 13 years old. Associations with growth, hematology, and an index of social status were investigated. RESULTS: Benign disease occurred in 43 (15 percent) patients. Neither growth nor social status were related to benign disease. There were only two statistically independent association: alpha thalassemia status and average steady state fetal hemoglobin (HbF). Patients with a normal complement of alpha globin genes were 2.2 (1.0, 4.9) times more likely to have frequent painful crises, dactylitis, and bone necrosis. The odds of having benign disease were 1.09 (1.02, 1.17) times higher for each unit increase in HbF, and 44 percent of subjects with HbF in the top decile (HbF > 13.8 percent) of the distribution had benign disease. There was no evidence for a threshold effect of high HbF on benign disease. CONCLUSION: A benign clinical course of sickle cell disease may occur in Jamaica and is associated with a normal alpha globin gene complement, and high levels of HbF. Ability to predict benign disease at birth is limited.(AU)

The clinical features of sickle cell disease

Evidence from structural studies of DNA suggest that the sickle cell mutation has arisen on at least three separate occasions in Africa and as a fourth independent mutation in the Eastern Province of Saudi Arabia or India. The pathophysiology of sickle cell disease is essentially similar in these different areas although the frequency and severity of complications may vary between areas. Generally, the chronic haemolysis and resulting anaemia is well tolerated, although serious morbidity and occasionally mortality may be associated with the aplastic crisis or cholelithiasis. Exacerbation of anaemia below steady state levels occurs with chronic glomerular damage and renal failure, especially in older patients. Most of the morbidity of the disease arises from bone marrow necrosis in the painful crisis or from vaso-occlusive manifestation. Changes in the splenic circulation results in life-threatening episodes of acute splenic sequestration, the chronic morbidity of hypersplenism, and splenic dysfunction renders children prone to pneumococcal septicaemia. Chronic organ damage contributes to chronic leg ulceration in adolescence and progressive renal,pulmonary,and occasionally cardiovascular impairment in later life. The clinical spectrum of homozygous sickle cell disease varies widely between patients. Factors contributing to this variability include alpha-thalassaemia,persistence of high HbF levels, haematology, social circumstances, and geographical and climatic variation. Many of the causes of mortality may be prevented or more effectively treated,leading to increased survival and an increased quality of life in affected subjects (AU)

The red cell distribution width in sickle cell disease: is it of clinical value?

The red cell distribution width (RDW) has been studied during the clinical steady state in 1121 patients with homozygous sickle cell (SS) disease, 344 with sickle cell-haemoglobin C (SC) disease, 68 with sickle cell-beta+ thalassaemia, 49 with cell beta§ thalassaemia and in 130 control subjects with a normal (AA) genotype. The mean RDW was moderately increased in Sbeta+ thalassaemia and SC disease and markedly increased in Sbeta§ thalassaemia and SS disease. In SS, SC and Sbeta§ thalassaemia genotypes, lower RDW values occurred in females and with alpha thalassaemia. The RDW correlated negatively with total haemoglobin, mean cell haemoglobin concentration, mean cell volume and fetal haemoglobin (HbF) and positively with reticulocyte count in SS disease. A low RDW was associated with higher weight and less frequent dactylitis, painful crisis, acute chest syndrome, acute splenic sequestration and hospital admissions. A low RDW in SS disease is consistent with a high total haemoglobin, high HbF, low reticulocyte count, alpha thalassaemia and a more mild clinical course. (AU)

Clinical and laboratory features of sickle cell disease in the Netherlands

Out of about 200 patients with sickle cell disease (SCD) in the Netherlands, 6 percent are non-negroid patients from Turkey. 83 were assessed clinico-haematologically regarding the type of SCD, ethnic origin, concurrent O-thalassaemia (O-thal), and type of sickle cell gene (á-chromosome). 54 patients had homozygous sickle cell (SS),1 sickle cell haemoglobin D (SD) Punjab, 5 sickle cell á§-thalassaemia (Sá§-thal), 5 sickle cell á+-thalassaemia (Sá+-thal) and 18 sickle cell haemoglobin C (SC) disease. 14 percent of the 83 patients were from Turkey, the others were of West Indian and African origin, most (73 percent) of whom were from Surinam. The Netherlands may be the only country in the world where non-negroid SCD patients are present in such a proportion to negroid SCD patients. O-thal was detected in 16 patients and in 14 of their relative with sickle cell trait. Four main types of ás-chromosomes were identified: Benin, Central African Republic, Senegal and Saudi Arabia types. SS and Sá§-thal disease ran a more severe course than Sá+ +-thal and SC disease. No clinical difference was ascribable to ethnic origin, O-thal or HbF-level but in each ethnic group there were some patients with a remarkably mild course of SS disease, which was related to the type of ás-chromosome. These were the Senegal and Saudi Arabia ás-chromosomes. Proper differentiation between genotypes is of prognostic and therapeutic relevance,especially in SC disease as it is sometimes discovered too late. A proper screening program is encouraged not only for all negroid inhabitants or immigrants, but also for non-negroid immigrants especially from Turkey.(AU)

Fetal hemoglobin levels and beta s globin haplotypes in an Indian population with sickle cell disease

To further explore the cause for variation in hemoglobin F (HbF) levels in sickle cell disease, the á globin restriction-fragment length polymorphism haplotypes were determined in a total of 303 (126SS, 141AS, 17Sá§, 7Aá§, and 12AA) Indians from the state of Orissa. The ás globin gene was found to be linked almost exclusively to a ás haplotype (+++-++-), which is also common in Saudi Arabian patients from the Eastern province (referred to as the Asian ás haplotype). By contrast, the majority of áA and ᧠thalassemia globin genes are linked to hoplotypes common in all European and Asian populations (+-----[+/-];--++-++). Family studies showed that there is a genetic factor elevating HbF levels dominantly in homozygotes (SS). This factor appears to be related to the Asian ás globin haplotype, and a mechanism for its action is discussed. There is also a high prevalence of an independent Swiss type hereditary presistance of fetal hemoglobin (HPFH) determinant active in both the sickle cell trait and in sickle cell disease.(AU)

Interaction of the OOO globin gene haplotype and sickle haemoglobin

We have studied the interaction of the OOO/OO gene arrangements with various á globin genotypes (AA, AS, AC, SS and SC). Whereas this interaction has no detectable clinical or haematological effects in subjects with AA, SS or SC genotypes it is associated with a significantly increased level of Hb S or Hb C in heterozygotes for these variants. These findings indicate that the additional O globin gene in the OOO gene arrangement is functional (Summary)

G gamma beta+ hereditary persistence of fetal hemoglobin: cosmid cloning and identification of a specific mutation 5' to the G gamma gene

Hereditary persistence of fetal hemoglobin (HPFH) is a benign condition in which the normal shutoff of fetal hemoglobin (Hb F) production fails to occur. In the G gamma beta+ type of HPFH, erythrocytes of adult heterozygotes contain approximately equal to 20 percent Hb F, which is almost exclusively of the G gamma-globin variety, without increased levels of gamma-globin chains from the nearby A gamma-globin gene. Unlike some forms of HPFH, no major deletions in the globin gene cluster have been found by genomic blotting in the G gamma beta+ variety. We report here a family with this condition, from which cosmid clones of the beta-globin gene cluster from the G gamma beta+ HPFH allele have been obtained. Sequencing around the fetal genes has identified a point mutation 202 base pairs 5' to the G gamma-globin gene that is present in genomic DNA of 3/3 unrelated individuals with G gamma beta+ HPFH but in none of more than 100 non-HPFH individuals. Although the mutation could represent a tightly linked polymorphism, its location in a region suggested by recent data to be important in tissue-specific control of gene expression suggests the possibility that the -202 mutation accounts for the phenotype. The sequence created resembles elements of other eukaryotic promoters known to be important for efficient transcription. (AU)

Origin of the beta S-globin gene in blacks: the contribution of recurrent mutation or gene conversion or both

In order to investigate the origin(s) of the mutation(s) leading to the beta S-globin gene in North American populations of African ancestory, we analysed DNA polymorphisms in the beta-globin gene cluster in a large number of both beta A- and beta S-globin gene-bearing chromosomes in U.S. and Jamaican Blacks. We found 16 different haplotypes of polymorphic sites associated with 170 beta S-globin gene-bearing chromosomes. The three most common beta S haplotypes, which account for 151/170 of the beta S-globin gene-bearing chromosomes, are only rarely seen in the chromosomes bearing the beta A-globin gene in these populations (6/47). Two observations suggest multiple origins or interallelic gene conversion, or both, of the beta S mutation. First, the mutation is present in all three beta-globin gene frameworks. Second, the beta S haplotypes can be divided into four groups, each of which cannot be derived from any other by less than two crossing-over events. In summary, our observation of the beta S mutation on 16 different halotypes in African populations can be best explained by (i) a number of simple recombination events 5' to the beta-globin gene and (ii) up to four independent mutations and/or interallelic gene conversions. (AU)

The interaction of alpha-thalassemia and homozygous sickle-cell disease

Patients with homozygous sickle-cell disease may be homozygous for alpha-thalassemia 2 (O-/O-), may be heterozygous for alpha-thalessemia 2 (O-/OO), or may have a normal alpha-globin-gene complement (OO/OO). We compared the clinical and hematologic features of 44 patients who had sickle-cell disease and homozygous alpha-thalassemia 2 with those of controls with the two hematologic conditions. The patients with homozygous alpha-thalassemia 2 had significantly higher red-cell counts and levels of hemoglobin and hemoglobin Aý, as well as significantly lower hemoglobin F, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, reticulocyte counts, irreversibly-sickled-cell counts, and serum total billirubin levels, than those with a normal alpha-globin-gene complement. Heterozygotes (O-/OO) had intermediate values. In the group with homozygous alpha-thalassemia 2, fewer patients had episodes of acute chest syndrome and chronic leg ulceration and more patients had splenomegaly, as compared with patients in the other two subgroups. These data confirmed previous suggestions that alpha-thalassemia inhibits in vivo sickling in homozygous sickle-cell disease and may be an important genetic determinant of its hematologic severity.

Mapping of antigenic sites on human haemoglobin by means of monoclonal antibodies and haemoglobin variants

Monoclonal antibodies specific for human globin chains have been prepared and the following strategy has been applied in delimiting the antigenic sites involved in antibody binding. The structural sites of the human globin subunit that might be recognised by the monoclonal antibody were deduced from comparisons of the primary structures of mamalian globin chains that did or did not react with the antibody. The involvement of individual residues at these specific sites was subsequently tested by reacting the antibody with abnormal human haemoglobins in which there was either a substitution or a structural site recognised by monoclonal antibody HuHb á 3-2 (an antibody that reacts with the adult haemoglobins from man and macaque monkey, but not with those from baboon and mouse) includes the aspartic acid residue at position 52 of the á-globin subunit.(AU)

Hb F synthesis in sickle cell anaemia: a comparison of Saudi Arab cases with those of African origin

Fetal haemoglobin (Hb F) synthesis has been studied in 22 cases of sickle cell anaemia (SS) from Saudi Arabia and compared with an equal number of cases of African origin. Among the Saudi Arabs y chain synthesis ranged from 4.0 percent to 19.9 percent of the total non-x chain synthesis (mean 8.1 percent) while the corresponding range for the Negro cases was <0.3 percent to 4.6 percent (mean 1.7 percent). In both groups the peripheral blood HB F level was on average 3-4 times higher than the proportion synthesized, indicating that the selective survival of Hb F containing cells (F cells) was an important factor in determining the final Hb F levels. Among the Saudi Arab cases there was a significant negative correlation between the degree of F cell enrichment and either the Hb F level or the percentage y chain synthesis. No such correlation was observed among the Negro cases. A high proportion of the cases in both groups were carriers of x thalassaemia in addition to SS, but no effect of x talassaemia on Hb F production was observed (AU)

Detection of alpha thalassaemia in Negro infants

A prospective study of 2191 Negro infants in Jamaica showed that approximately 7 percent of them had detectable levels of Hb Bart's (Y4) in the neonatal period. The red cell indices, globin chain biosynthesis and restriction endonuclease mapping of DNA from these infants were used to determine the significance of Hb Bart's at birth. The results indicate that the genotypes OO/OO, - O/OO and - O/-O are associated with 0 percent, 0.1-2 percent, and greater than 2 percent Hb Bart's respectively. Although trace amounts of Hb Bart's may be associated with the genotype - O/OO this is not always the case and therefore haemoglobin analysis in the neonatal period cannot be used to diagnose this genotype with any certainty (AU)

Variations in globin chain synthesis in hereditary persistence of fetal haemoglobin

Globin synthesis was studied in four Negro families including 10 members with Hb A-HPFH and four with Hb S-HPFH. The á/O specific activity ratios in 10 of these patients with Hb A-HPFH heterozygotes were similar to those of the control group. In two patients with Hb A-HPFH, the á/O ratio was slightly decreased in one (0.84) and clearly decreased in another (0.78). In two of the patients with Hb S-HPFH the ratios were clearly decreased (0.71 and 0.75). The extended range of á/O ratios in these 14 patients is similar to that of Negro patients with á-thalassaemia trait. These studies indicate that a decreased á/O ratio may be found in HPFH, as well as in á-thalassaemia. Bone marrow globin synthesis was measured in two patients with Hb S-HPFH and decreased peripheral blood á/O ratios, and in one with Hb A-HPFH and a normal peripheral blood á/O ratio. In each patient the (á+y)/O ratio of radioactivities as well as the á/O specific activity ratio was close to I and therefore balanced, indicating more rapid decay of á-chain synthesis relative to O-chain during red cell maturation or extremely rapid destruction of newly synthesized excess O-chains in the bone marrow.(Summary)