Christopher A. Fisher

Mutations in the chromatin-associated protein ATRX†

ATRX belongs to the SNF2 family of proteins, many of which have been demonstrated to have chromatin remodeling activity. Constitution mutations in the X‐encoded gene give rise to alpha thalassemia mental retardation (ATR‐X) syndrome and a variety of related conditions that are often associated with profound developmental delay, facial dysmorphism, genital abnormalities, and alpha thalassemia. Acquired mutations in ATRX are observed in the preleukemic condition alpha thalassemia myelodysplastic syndrome (ATMDS). Mutations in ATRX have been shown to perturb gene expression and DNA methylation. This is a comprehensive report of 127 mutations including 32 reported here for the first time. Missense mutations are shown to cluster in the two main functional domains. The truncating mutations appear to be “rescued” to some degree and so it appears likely that most if not all constitutional ATRX mutations are hypomorphs. Hum Mutat 29(6), 796–802, 2008.

A MULTI-CENTER STUDY IN ORDER TO FURTHER DEFINE THE MOLECULAR BASIS OF β-THALASSEMIA IN THAILAND, PAKISTAN, SRI LANKA, MAURITIUS, SYRIA, AND INDIA, AND TO DEVELOP A SIMPLE MOLECULAR DIAGNOSTIC STRATEGY BY AMPLIFICATION REFRACTORY MUTATION SYSTEM-POLYMERASE CHAIN REACTION

The spectrum of the β-thalassemia mutations of Thailand, Pakistan, India, Sri Lanka, Mauritius and Syria has been further characterized by a multi-center study of 1,235 transfusion-dependent patients, and the mutations discovered used to assess the fidelity of a simple diagnostic strategy. A total of 44 β-thalassemia mutations were identified either by allele-specific oligonucleotide hybridization, amplification with allele-specific primers, or DNA sequencing of amplified product. The results confirm and extend earlier findings for Thailand, Pakistan, India, Mauritius and Syria. This is the first detailed report of the spectrum of mutations for Sri Lanka. Two novel mutations were identified, codon 55 (−A) and IVS-I-129 (A → C), both found in Sri Lankan patients. Two β-thalassemia mutations were found to coexist in one β-globin gene: Sri Lankan patients homozygous for the β0 codon 16 (−C) frameshift were also homozygous for the β+ codon 10 (C → A) mutation. Studies of Sri Lankan, Pakistani, and Indian carriers suggest the codon 10 (C → A) mutation is just a rare polymorphism on an ancestral allele, on which the β0 codon 16 (−C) mutation has arisen. Each country was found to have only a few common mutations accounting for 70% or more of the β-thalassemia alleles. A panel of primers to diagnose the majority of the mutations by the amplification refractory mutation system was developed, enabling a simple molecular diagnostic strategy to be introduced for each country participating in the multi-center study.

The molecular basis for the thalassaemias in Sri Lanka

Summary. The β‐globin gene mutations and the α‐globin genes of 620 patients with the phenotype of severe to moderate thalassaemia from seven centres in Sri Lanka were analysed. Twenty‐four β‐globin gene mutations were identified, three accounting for 84·5% of the 1240 alleles studied: IVSI‐5 (G→C) 56·2%; IVSI‐1 (G→A) 15·2%; and haemoglobin E (codon (CD)26 GAG→GAA) 13·1%. Three new mutations were found; a 13‐bp deletion removing the last nucleotide in CD6 to CD10 inclusively, IVSI‐129 (A→C) in the consensus splice site, and a frame shift, CD55 (–A). The allele frequency of α+ thalassaemia was 6·5% and 1·1% for ‐α3·7 and ‐α4·2 deletions respectively. Non‐deletion α‐thalassaemia was not observed. Triplicate or quadruplicate α‐globin genes were unusually common. In 1·5% of cases it was impossible to identify β‐thalassaemia alleles, but in Kurunegala detailed family studies led to an explanation for the severe thalassaemia phenotype in every case, including a previously unreported instance of homozygosity for a quadruplicated α‐globin gene together with β‐thalassaemia trait. These findings have implications for the control of thalassaemia in high‐frequency populations with complex ethnic histories.

β-Thalassaemia intermedia in Lebanon

Abstract: Approximately one third of thalassaemia patients onrecord in Lebanon have thalassaemia intermedia. We haveanalysed three factors in a panel of 73 patients with this less severeform of the disease in our population: mild β‐globin gene mutations, deletions in the α‐globin gene and the presence of a polymorphismfor the enzyme XmnI in the Gγ‐promoter region. The results show that the most important contributing factor is the β‐genotype:68% of patients have a mild β+ mutation (IVSI‐6, cd29,−88 or −87), while 26% of patients are positive for the XmnI polymorphism associated with increased production ofHbF, which showed strong linkage to particular mutations(IVSII‐1, cd8 and cd30). However, the genotype–phenotypecorrelation is difficult, because many patients were initially misdiagnosed as thalassaemia major and were started early on regular blood transfusions, which was stopped later on. This illustrates well the importance of an early accurate diagnosis of thalassaemia intermedia for appropriate clinical management.

Adaptation to anemia in hemoglobin E-β thalassemia

Hemoglobin E β thalassemia is the commonest form of severe thalassemia in many Asian countries. Its remarkably variable clinical phenotype presents a major challenge to determining its most appropriate management. In particular, it is not clear why some patients with this condition can develop and function well at very low hemoglobin levels. Here, we demonstrate that patients with hemoglobin Eβ thalassemia have a significant decrease in the oxygen affinity of their hemoglobin, that is an increased P(50) value, in response to anemia. This may in part reflect the lower level of hemoglobin F in this condition compared with other forms of β thalassemia intermedia. The ability to right-shift the oxygen dissociation curve was retained across the spectrum of mild and severe phenotypes, despite the significantly higher levels of hemoglobin F in the former, suggesting that efforts directed at producing a modest increase in the level of hemoglobin F in symptomatic patients with this disease should be of therapeutic value.

Hemoglobin E-β-thalassemia : Progress report from the international study group

Abstract: A long‐term observational study of Hb E‐β‐thalassemia in Sri Lanka is beginning to define some of the genetic and environmental factors that are responsible for its remarkable phenotypic variability. In this population there is a very small difference between the steady‐state hemoglobin levels between the mild and severe phenotypes, and it has been possible to stop transfusion in many of those who have been on long‐term treatment of this kind. These preliminary observations, made over the last 7 years, provide directions for future research into this increasingly important disease.

Methemoglobinemia and ascorbate deficiency in hemoglobin E β thalassemia: metabolic and clinical implications

During investigations of the phenotypic diversity of hemoglobin (Hb) E β thalassemia, a patient was encountered with persistently high levels of methemoglobin associated with a left-shift in the oxygen dissociation curve, profound ascorbate deficiency, and clinical features of scurvy; these abnormalities were corrected by treatment with vitamin C. Studies of erythropoietin production before and after treatment suggested that, as in an ascorbate-deficient murine model, the human hypoxia induction factor pathway is not totally dependent on ascorbate levels. A follow-up study of 45 patients with HbE β thalassemia showed that methemoglobin levels were significantly increased and that there was also a significant reduction in plasma ascorbate levels. Haptoglobin levels were significantly reduced, and the high frequency of the 2.2 haptoglobin genotype may place an additional pressure on ascorbate as a free-radical scavenger in this population. There was, in addition, a highly significant correlation between methemoglobin levels, splenectomy, and factors that modify the degree of globin-chain imbalance. Because methemoglobin levels are modified by several mechanisms and may play a role in both adaptation to anemia and vascular damage, there is a strong case for its further study in other forms of thalassemia and sickle-cell anemia, particularly when splenic function is defective.

Alpha Thalassaemia and extended alpha globin genes in Sri Lanka

The α-globin genes were studied in nine families with unexplained hypochromic anaemia and in 167 patients with HbE β thalassaemia in Sri Lanka. As well as the common deletion forms of α(+) thalassaemia three families from an ethnic minority were found to carry a novel form of α(0) thalassaemia, one family carried a previously reported form of α(0) thalassaemia, --(THAI), and five families had different forms of non-deletional thalassaemia. The patients with HbE β thalassaemia who had co-inherited α thalassaemia all showed an extremely mild phenotype and reduced levels of HbF and there was a highly significant paucity of α(+) thalassaemia in these patients compared with the normal population. Extended α gene arrangements, including ααα, αααα and ααααα, occurred at a low frequency and were commoner in the more severe phenotypes of HbE β thalassaemia. As well as emphasising the ameliorating effect of α thalassaemia on HbE β thalassaemia the finding of a novel form of α(0) thalassaemia in an ethnic minority, together with an unexpected diversity of forms of non-deletion α thalassaemia in Sri Lanka, further emphasises the critical importance of micro-mapping populations for determining the frequency of clinically important forms of the disease.

Emerging insights in the management of hemoglobin E beta thalassemia.

Globally, hemoglobin (Hb) E β thalassemia accounts for approximately half the severe forms of β thalassemia. Because of its wide clinical diversity and the ability of patients with this condition to adapt unusually well to low hemoglobin levels, the management of Hb E β thalassemia, particularly the decision to instigate regular blood transfusion, is particularly difficult. Here, we present a summary of our work in patients with this condition, which attempts to define clinical, adaptive, and genetic factors of possible value in determining the early management of this condition.

A novel mutation in the last exon of ATRX in a patient with alpha-thalassemia myelodysplastic syndrome.

Abstract:u2002 We describe a patient with acquired alpha‐thalassemia myelodysplastic syndrome (ATMDS). A previously healthy 66‐year‐old man presented with hemoglobin of 9.3u2003g/dL, mean corpuscular volume 59u2003fL, and a bone marrow aspirate with increased erythroid precursors and hypolobulated megakaryocytes. Hemoglobin H inclusions were seen in most red cells after 1% brilliant cresyl blue supravital stain of the peripheral blood. At the molecular level, we identified of a novel mutation in the most 3′ exon of the ATRX gene (CGA→TGA substitution in codon 2407) resulting in a premature termination codon (p.R2407X). This case provides further evidence for a link between ATRX mutations and ATMDS, and suggests a possible role for the conserved Q‐box element in ATRX function.