Heterogeneity of the epsilon gamma delta beta-thalassaemias: characterization of three novel English deletions

We have characterized three novel epsilon gamma delta beta-thalassaemia deletions in three English families. Two of the deletions, 114 and 439 kb, removed the entire beta-globin gene complex, including a variable number of flanking olfactory receptor (HOR) genes. The 98-kb deletion extended 90-kb upstream of the epsilon gene to 8 kb upstream of the G gamma-gene, leaving the gamma,delta and beta-genes intact. The 439 kb deletion is the largest deletion reported so far to cause epsilon gamma delta beta-thalassaemia; heterozygotes for this deletion were variably affected by neonatal haemolytic anaemia. Two of the deletions were de novo. Breakpoints of all three deletions occurred within regions of L1 or Alu repeats and contained short regions of direct homology between the flanking sequences, a feature that is likely to have contributed to the illegitimate recombinations.     

The entire beta-globin gene cluster is deleted in a form of gamma delta beta-thalassemia

We have used restriction endonuclease mapping to study a deletion involving the beta-globin gene cluster in a Mexican-American family with gamma delta beta-thalassemia. Analysis of DNA polymorphisms demonstrated deletion of the beta-globin gene from the affected chromosome. Using a DNA fragment that maps greater than 40 kilobases (kb) 5' to the epsilon-gene as a probe, reduced amounts of normal fragments were found in the DNA of affected family members. Similar analysis using radiolabeled DNA fragments located 3' to the beta-globin cluster has shown that the deletion extends more than 17 kb 3' to the beta-gene, but terminates before the 3' endpoint of the Ghanian HPFH deletion. Hence, this gamma delta beta-thalassemia deletion eliminates over 105 kb of DNA and is the first report of a deletion of the entire beta-globin gene cluster.     

A novel deletion causing (epsilon gamma delta beta) degrees thalassaemia in a Chilean family

We describe a novel deletion causing (epsilongammadeltabeta) degrees thalassaemia segregating in three generations of a Chilean family of Spanish descent. Heterozygotes for the deletion were all affected by neonatal haemolytic anaemia. The deletion of 152,569 bp extends from 77 kb upstream of the epsilon gene to 31 kb downstream of the beta gene, and includes the entire beta-globin gene cluster and two upstream olfactory receptor genes. Comparison of the sequences of the deletion junction with those of the flanking normal DNA suggests that the deletion results from a non-homologous recombination event. The insertion of 16 'orphan' nucleotides in the deletion junction creates a perfect inverted repeat of 12 nucleotides, forming a 12-bp stem with a four-nucleotide loop that could have contributed to the illegitimate recombination. The 3' breakpoint is located within an L1 family repeat that contains a perfect 160-bp palindrome, and is in close proximity to the 3' breakpoints of five other deletions in the beta cluster - Indian (HPFH-3), Italian (HPFH-4) and Vietnamese GgammaAgamma (deltabeta) degrees HPFH, German and Belgian Ggamma (Alphagammadeltabeta) degrees thalassaemia.     

A novel epsilon gamma delta beta thalassemia of 1.4 Mb deletion found in a Japanese patient

A novel large deletion, causing epsilon gamma delta beta thalassemia (here called, epsilon gamma delta beta thalassemia Jpn-I) was discovered in a 6-year-old Japanese boy. He was born uneventfully, but revealed thalassemia minor after birth. The mutation was inherited from his mother. The deletion, caused by an illegitimate recombination extended from 750 kb upstream to 660 kb downstream of e-globin gene, and removed about 1.4 Mb of DNA, the largest in epsilon gamma delta beta thalassemias. A 19-nucleotide orphan sequence and direct repeats were present at the junction. The deletion lost several functional genes, but no relevant symptoms manifested. The breakpoints were determined by relatively simple methods.     

Molecular and hematologic characterization of Scottish-Irish type (epsilon gamma delta beta)zero thalassemia

The DNA deletion associated with an example of (epsilon gamma delta beta)zero thalassemia (Scottish-Irish type) was characterized. The deletion is approximately 205 kb in length and involves the epsilon, G gamma, A gamma, delta, and beta globin genes. The breakpoint is located 263 bp 3' to exon 3 of the beta globin gene. An LI (KpnI) repeat element approximately 320 bp in size is found at the 3' end of the novel DNA sequence. Different clinical phenotypes for three heterozygous neonates suggest that the deletion alone does not predict severity of (epsilon gamma delta beta)zero thalassemia at this age.     

Deficiency of CD3gamma, delta, epsilon, and zeta expression in T cells from AML patients

In order to elucidate the feature of T-cell receptor (TCR) signal transduction in T-cells from acute myeloid leukemia (AML), the expression levels of CD3gamma, delta, epsilon and zeta chain genes in CD3+ T cells were analyzed using real-time PCR. CD3+ T cells sorted from peripheral blood of 10 AML patients and 10 healthy donors were used in the study. Significantly lower expression levels of all four CD3gamma, delta, epsilon, and zeta chain genes were found in the AML samples. The expression pattern of the four CD3 chains was epsilon>gamma>delta>zeta in CD3+ T cells from AML samples, which was different from the healthy control group. In conclusion, the results provide a global gene expression profile of CD3gamma, delta, epsilon, and zeta chains in AML patients. Deficiency of all four CD3 gene expression levels might represent the feature related to T-cell immunodeficiency.     

Hb Lepore-Leiden: a new delta/beta rearrangement associated with a beta-thalassemia minor phenotype

The Lepore hemoglobins (Hbs) are a group of structural defects resulting from different recombination events between the delta- and beta-globin genes. They may come with different beta-thalassemia (beta-thal) minor-like phenotypes in the carrier and with variably severe phenotypes in the rare homozygote, and in the common compound heterozygote with beta-thal. The most seriously affected patients are those of Yugoslavian origin presenting with severe transfusion-dependent hemolytic anemia, dyserythropoiesis, hepatosplenomegaly and skeletal malformations. Because of genetic risk, couples where both partners are carriers of these combinations may require prognosis and prenatal diagnosis. In these cases, recognition of the defect must be done with particular care. We report a case of Hb Lepore induced by a yet unknown crossover event found in a 24-year-old Turkish male and compare the novel mutation with those previously reported.     

A novel basis for delta beta-thalassemia in a Chinese family

We have studied a Chinese family in which beta-thalassemia and delta beta-thalassemia were found in simple and compound heterozygous states. The delta beta-thalassemia heterozygote (the mother) had 22.3% hemoglobin F, of which 40% was G gamma and 60% A gamma; globin chain studies showed an alpha/beta + gamma ratio of 1.36. The compound heterozygote for delta beta-thalassemia and beta-thalassemia (the child) had the clinical picture of thalassemia intermedia and an alpha/beta + gamma ratio of 4.44. Gene mapping studies were performed using DNA from the affected child. Seventy kilobases of DNA in the beta- globin gene cluster starting upstream from the epsilon-globin gene and ending downstream from the beta-globin gene were mapped, and no detectable deletions or rearrangements were detected. In addition, heterozygosity was detected at multiple polymorphic restriction sites in and 3' to the beta-globin gene, which excludes the possibility of a deletion of the entire beta-globin gene cluster. This is the first example of a nondeletion delta beta-thalassemia associated with increased expression of both G gamma and A gamma genes.     

Normal delta-globin gene sequences in Sardinian nondeletional delta beta-thalassemia.

In order to clarify the reasons for the reduced Hb A2 levels in Sardinian delta beta-thalassemia, we characterized, both by cloning and sequence analysis and by direct sequencing of amplified DNA, the delta-globin gene from an individual of Sardinian descent who is a compound heterozygote for the beta zero-thalassemia codon 39 (C-->T) nonsense mutation and the Sardinian delta beta-thalassemia [codon 39(C-->T)/-196(C-->T)A gamma]. The analysis of the delta-globin gene from the delta beta-thalassemia chromosome revealed an entirely normal sequence. The defective function of the delta-globin gene in this determinant is thus likely related to a suppressive effect of the in cis nondeletional high persistence of fetal hemoglobin mutation of the A gamma gene, probably resulting from an increased capability of the relative promoter to interact with the locus control region.     

Homozygosity for a new type of G gamma (A gamma delta beta)zero-thalassemia in a Malaysian male

Hematological and clinical data are presented for a young Malay patient with a homozygous (delta beta)zero-thalassemic condition. His red blood cells contained 100% fetal hemoglobin with alpha and G gamma chains only. Detailed gene mapping defined a large deletion with a 5' end between the Aha III and Apa I sites, some 200-400 bp 5' to the A gamma globin gene and a 3' end beyond sequences 17-18 kb 3' to the beta globin gene. This G gamma (A gamma delta beta)zero-type of thalassemia is different from all the other six types described before. Comparison of the hematological data of this patient with those of homozygotes for either the Sicilian or Spanish types of G gamma A gamma (delta beta)zero-thalassemia showed no differences; all homozygotes have a moderate anemia which is accentuated by the relatively high oxygen affinity of the Hb F containing erythrocytes.     

Two new large deletions in the low density lipoprotein receptor (LDLR) gene not revealed by PCR-based molecular diagnosis of familial hypercholesterolemia

In the French Canadian population six mutations appear to be responsible for about 85% of FH cases. Two of these mutations are large deletions. The most prevalent deletion is a >15 kb deletion of the promoter and first exon; the second, a 5 kb deletion that removes exons 2 and 3. The high frequency of these deletions in the French Canadian population has been attributed to a founder effect. Other mutations are present in the population but at a much lower prevalence. We recently identified two new large deletions in FH patients of French Canadian descent. Carriers of the new deletions were identified because of an unusual pattern of band migration on Southern blots. We have identified and sequenced the deletions' boundaries. The first deletion covers 3813 bp and removes exons 7 and 8. The second deletion covers 5994 bp and removes exons 3-6. These deletions have not been previously reported. They would have been missed if a PCR-based method had been used instead of Southern blot analysis.     

Hb A2-Zagreb or alpha 2 delta 2(125)(H3)Gln replaced by Glu, a new delta chain variant in association with delta beta-thalassemia

The structural identification of a new delta chain variant is described. The abnormal Hb A2 was found in two members of a family from Zagreb, Yugoslavia. The propositus also had a delta beta-thalassemia heterozygosity.     

The delta and epsilon errors in the assessment of cancer clinical trials

The error probabilities alpha and beta are widely used to compute sample sizes and to analyze results of clinical trials. These errors are, however, not the only probabilities to consider when assessing results of clinical studies. The rate of false positive (delta) and false negative (epsilon) results allows one to determine if an experimental finding is likely to reflect the true situation in the population of interest. The delta error is generally high in randomized phase III and in early phase II clinical trials in cancer patients, whereas the epsilon error is relatively low in these settings. This is essentially due to the small probability of detecting a more effective treatment or a new chemotherapeutic agent active in cancer. The delta error could be considerably reduced by increasing the sample sizes and by restricting the allowance made for the alpha error, which should be set at a 1% level as a minimum requirement.     

Identification of a new delta chain hemoglobin variant in a beta-thalassemia carrier: Hb A2-mumc [delta13(a10)Ala-->Asp

We describe a case of beta-thalassemia (thal) trait in which the patient also carries a novel delta chain variant due to a missense mutation at amino acid codon 13 (GCC-->GAC, Ala-->Asp). The level of Hb A2 was not elevated, raising the potential for misdiagnosis.     

Different 3'' end points of deletions causing delta beta-thalassemia and hereditary persistence of fetal hemoglobin: implications for the control of gamma-globin gene expression in man.

DNA at the end point of the gene deletion associated with one form of hereditary persistence of fetal hemoglobin (HPFH) was cloned and used as a probe in gene mapping experiments to analyze the extent and approximate 3' end points of various deletions associated with HPFH and delta beta-thalassemia. The deletions in the two known forms of deletion-type HPFH were shown to be considerably more extensive than in the two cases of delta beta-thalassemia studied. The overall extents of the deletions in the two types of HPFH were quite similar in both cases and the 3' end points were located at a minimum distance of approximately equal to 52 and 57 kilobases from the 3' extremity of the beta-globin gene. In contrast, the 3' end points of the deletions in the two forms of delta beta-thalassemia were located approximately equal to 5 and 10 kilobases to the 3' side of the beta-globin gene. The extent of these deletions and the nature of the DNA brought into the vicinity of the gamma-globin genes by the deletions may therefore be a more important influence on the phenotype of the deletions than the specific nature of the DNA sequences that are deleted within the non-alpha-globin gene cluster as a result of the mutations.     

A new insertion mutation in the beta-globin gene [codons 45/46 (+A)] resulting in a beta-thalassemia minor phenotype

The beta-globin gene of 306 newly diagnosed beta-thalassemia (thal) minor patients were sequenced. Analysis revealed that only one amongst all the identified mutations had not been previously reported. This new mutation, causing a beta(+)-thal minor phenotype, was found in a patient of Arabic origin. The insertion frameshift mutation (+A) between codons 45 and 46 [codons 45/46 (+A)] results in a premature termination signal at codon 52. No truncated beta-globin or abnormal hemoglobin (Hb) was identified.     

The expression of TCR-gamma delta/CD3 complex in neoplastic gamma delta T-cell

We describe that the neoplastic gammadelta T-cells from patients with hepatosplenic gammadelta T-cell lymphoma have a lower expression of TCR-gammadelta/CD3 complex compared to their normal or reactive counterparts. Interestingly, with the use of an appropriate antibody association, this feature is easily and rapidly observed on flow cytometry graphics.