ATR-X syndrome: genetics, clinical spectrum, and management.

ATR-X, an acronym for alpha thalassemia and mental retardation X-linked, syndrome is a congenital condition predominantly affecting males, characterized by mild to severe intellectual disability, facial, skeletal, urogenital, and hematopoietic anomalies. Less common are heart defects, eye anomalies, renal abnormalities, and gastrointestinal dysfunction. ATR-X syndrome is caused by germline variants in the ATRX gene. Until recently, the diagnosis of the ATR-X syndrome had been guided by the classical clinical manifestations and confirmed by molecular techniques. However, our new systematic analysis shows that the only clinical sign shared by all affected individuals is intellectual disability, with the other manifestations varying even within the same family. More than 190 different germline ATRX mutations in some 200 patients have been analyzed. With improved and more frequent analysis by molecular technologies, more subtle deletions and insertions have been detected recently. Moreover, emerging technologies reveal non-classic phenotypes of ATR-X syndrome as well as the description of a new clinical feature, the development of osteosarcoma which suggests an increased cancer risk in ATR-X syndrome. This review will focus on the different types of inherited ATRX mutations and their relation to clinical features in the ATR-X syndrome. We will provide an update of the frequency of clinical manifestations, the affected organs, and the genotype-phenotype correlations. Finally, we propose a shift in the diagnosis of ATR-X patients, from a clinical diagnosis to a molecular-based approach. This may assist clinicians in patient management, risk assessment and genetic counseling.

Mutant ATRX: uncovering a new therapeutic target for glioma.

INTRODUCTION: ATRX is a chromatin remodeling protein whose main function is the deposition of the histone variant H3.3. ATRX mutations are widely distributed in glioma, and correlate with alternative lengthening of telomeres (ALT) development, but they also affect other cellular functions related to epigenetic regulation. Areas covered: We discuss the main molecular characteristics of ATRX, from its various functions in normal development to the effects of its loss in ATRX syndrome patients and animal models. We focus on the salient consequences of ATRX mutations in cancer, from a clinical to a molecular point of view, focusing on both adult and pediatric glioma. Finally, we will discuss the therapeutic opportunities future research perspectives. Expert opinion: ATRX is a major component of various essential cellular pathways, exceeding its functions as a histone chaperone (e.g. DNA replication and repair, chromatin higher-order structure regulation, gene transcriptional regulation, etc.). However, it is unclear how the loss of these functions in ATRX-null cancer cells affects cancer development and progression. We anticipate new treatments and clinical approaches will emerge for glioma and other cancer types as mechanistic and molecular studies on ATRX are only just beginning to reveal the many critical functions of this protein in cancer.

Inherited germline ATRX mutation in two brothers with ATR-X syndrome and osteosarcoma.

We report a family in which two brothers had an undiagnosed genetic disorder comprised of dysmorphic features, microcephaly, severe intellectual disability (non-verbal), mild anemia, and cryptorchidism. Both developed osteosarcoma. Trio exome sequencing (using blood samples from the younger brother and both parents) was performed and a nonsense NM_000489.4:c.7156C>T (p.Arg2386*) mutation in the ATRX gene was identified in the proband (hemizygous) and in the mother's peripheral blood DNA (heterozygous). The mother is healthy, does not exhibit any clinical manifestations of ATR-X syndrome and there was no family history of cancer. The same hemizygous pathogenic variant was confirmed in the affected older brother's skin tissue by subsequent Sanger sequencing. Chromosomal microarray studies of both brothers' osteosarcomas revealed complex copy number alterations consistent with the clinical diagnosis of osteosarcoma. Recently, somatic mutations in the ATRX gene have been observed as recurrent alterations in both osteosarcoma and brain tumors. However, it is unclear if there is any association between osteosarcoma and germline ATRX mutations, specifically in patients with constitutional ATR-X syndrome. This is the first report of osteosarcoma diagnosed in two males with ATR-X syndrome, suggesting a potential increased risk for cancer in patients with this disorder.

Compromised genomic integrity impedes muscle growth after Atrx inactivation.

ATR-X syndrome is a severe intellectual disability disorder caused by mutations in the ATRX gene. Many ancillary clinical features are attributed to CNS deficiencies, yet most patients have muscle hypotonia, delayed ambulation, or kyphosis, pointing to an underlying skeletal muscle defect. Here, we identified a cell-intrinsic requirement for Atrx in postnatal muscle growth and regeneration in mice. Mice with skeletal muscle-specific Atrx conditional knockout (Atrx cKO mice) were viable, but by 3 weeks of age presented hallmarks of underdeveloped musculature, including kyphosis, 20% reduction in body mass, and 34% reduction in muscle fiber caliber. Atrx cKO mice also demonstrated a marked regeneration deficit that was not due to fewer resident satellite cells or their inability to terminally differentiate. However, activation of Atrx-null satellite cells from isolated muscle fibers resulted in a 9-fold reduction in myoblast expansion, caused by delayed progression through mid to late S phase. While in S phase, Atrx colocalized specifically to late-replicating chromatin, and its loss resulted in rampant signs of genomic instability. These observations support a model in which Atrx maintains chromatin integrity during the rapid developmental growth of a tissue.

Role of ATRX in chromatin structure and function: implications for chromosome instability and human disease.

Functional differentiation of chromatin structure is essential for the control of gene expression, nuclear architecture, and chromosome stability. Compelling evidence indicates that alterations in chromatin remodeling proteins play an important role in the pathogenesis of human disease. Among these, α-thalassemia mental retardation X-linked protein (ATRX) has recently emerged as a critical factor involved in heterochromatin formation at mammalian centromeres and telomeres as well as facultative heterochromatin on the murine inactive X chromosome. Mutations in human ATRX result in an X-linked neurodevelopmental condition with various degrees of gonadal dysgenesis (ATRX syndrome). Patients with ATRX syndrome may exhibit skewed X chromosome inactivation (XCI) patterns, and ATRX-deficient mice exhibit abnormal imprinted XCI in the trophoblast cell line. Non-random or skewed XCI can potentially affect both the onset and severity of X-linked disease. Notably, failure to establish epigenetic modifications associated with the inactive X chromosome (Xi) results in several conditions that exhibit genomic and chromosome instability such as fragile X syndrome as well as cancer development. Insight into the molecular mechanisms of ATRX function and its interacting partners in different tissues will no doubt contribute to our understanding of the pathogenesis of ATRX syndrome as well as the epigenetic origins of aneuploidy. In turn, this knowledge will be essential for the identification of novel drug targets and diagnostic tools for cancer progression as well as the therapeutic management of global epigenetic changes commonly associated with malignant neoplastic transformation.

Skeletal muscle structural and energetic characteristics in subjects with sickle cell trait, alpha-thalassemia, or dual hemoglobinopathy.

Previous studies have shown that subjects with sickle cell trait (SCT), alpha-thalassemia (alpha-t), and the dual hemoglobinopathy (SCT/alpha-t) manifest subtle, albeit significant, differences during exercise. To better understand such differences, we assessed skeletal muscle histomorphological and energetic characteristics in 10 control HbAA subjects (C), 5 subjects with alpha-t (alpha-t), 6 SCT carriers (SCT) and 9 SCT carriers with alpha-t (SCT/alpha-t). Subjects underwent a muscle biopsy and also performed an incremental maximal exercise and a time to exhaustion test. There were no observable differences in daily energy expenditure, maximal power output (Pmax), or time to exhaustion at 110% Pmax (Tex) among the groups. Blood lactate concentrations measured at the end of the Tex, muscle fiber type distribution, and mean phosphofructokinase (PFK), lactate dehydrogenase (LDH), beta-hydroxyacyl-CoA-dehydrogenase (HAD), and citrate synthase (CS) activities were all similar among the four groups. However, SCT was associated with a lower cytochrome-c oxidase (COx) activity in type IIa fibers (P<0.05), and similar trends were observed in fiber types I and IIx. Trends toward lower creatine kinase (CK) activity (P=0.0702) and higher surface area of type IIx fibers were observed in SCT (P=0.0925). In summary, these findings support most of the previous observations in SCT, such as 1) similar maximal power output and associated maximal oxygen consumption (VO2max) values and 2) lower exercise performances during prolonged submaximal exercise. Furthermore, performances during short supramaximal exercise were not different in SCT. Finally, the dual hemoglobinopathy condition does not seem to affect muscle characteristics.

Remodeling of skeletal muscle microvasculature in sickle cell trait and alpha-thalassemia.

The influence of sickle cell trait and/or alpha-thalassemia on skeletal muscle microvascular network characteristics was assessed and compared with control subjects [hemoglobin (Hb) AA] in 30 Cameroonian residents [10 HbAA, 5 HbAA alpha-thalassemia (alpha-t), 6 HbAS, and 9 HbASalpha-t] matched for maximal work capacity and daily energy expenditure. Subjects performed an incremental exercise to exhaustion and underwent a muscle biopsy. Muscle fiber type and surface area were not different among groups. However, sickle cell trait (SCT) was associated with lower capillary density (P < 0.05), lower capillary tortuosity (P < 0.001), and enlarged microvessels (P < 0.01). SCT carriers had reduced counts of microvessels <5-microm diameter, but a higher percentage of broader microvessels, i.e., diameter >10 microm (P < 0.05). alpha-Thalassemia seemed to be characterized by a higher capillary tortuosity and unchanged capillary density and diameter. Thus, while SCT is a priori clinically benign, we demonstrate for the first time that significant remodeling of the microvasculature occurs in SCT carriers. These modifications may possibly reflect protective adaptations against hemorheological and microcirculatory dysfunction induced by the presence of HbS. The remodeling of the microvascular network occurs to a lesser extent in alpha-thalassemia. In alpha-thalassemic subjects, increased capillary tortuosity would promote oxygen supply to muscle tissues and might compensate for the lower Hb content often reported in those subjects.

Alpha and beta thalassemia.

The thalassemias are a group of inherited hematologic disorders caused by defects in the synthesis of one or more of the hemoglobin chains. Alpha thalassemia is caused by reduced or absent synthesis of alpha globin chains, and beta thalassemia is caused by reduced or absent synthesis of beta globin chains. Imbalances of globin chains cause hemolysis and impair erythropoiesis. Silent carriers of alpha thalassemia and persons with alpha or beta thalassemia trait are asymptomatic and require no treatment. Alpha thalassemia intermedia, or hemoglobin H disease, causes hemolytic anemia. Alpha thalassemia major with hemoglobin Bart's usually results in fatal hydrops fetalis. Beta thalassemia major causes hemolytic anemia, poor growth, and skeletal abnormalities during infancy. Affected children will require regular lifelong blood transfusions. Beta thalassemia intermedia is less severe than beta thalassemia major and may require episodic blood transfusions. Transfusion-dependent patients will develop iron overload and require chelation therapy to remove the excess iron. Bone marrow transplants can be curative for some children with beta thalassemia major. Persons with thalassemia should be referred for preconception genetic counseling, and persons with alpha thalassemia trait should consider chorionic villus sampling to diagnose infants with hemoglobin Bart's, which increases the risk of toxemia and postpartum bleeding. Persons with the thalassemia trait have a normal life expectancy. Persons with beta thalassemia major often die from cardiac complications of iron overload by 30 years of age.

Plasma levels of adhesion molecules ICAM-1 and VCAM-1 in athletes with sickle cell trait with or without alpha-thalassemia during endurance exercise and recovery.

The aim of the study was to examine the effects of endurance exercise on circulating vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1) in sickle cell trait (SCT) athletes with or without alpha-thalassemia. Five athletes with SCT, 7 athletes with both SCT and alpha-thalassemia (SCTAT) and 8 control athletes (CONT) performed an incremental test on cycloergometer followed 72 hours later by a 60-min endurance exercise with a workload set at 70% P(peak) (peak power). We assessed levels of sICAM-1, sVCAM-1 and TNF-alpha at rest, immediately after endurance exercise and 1, 2, and 24 hours of recovery. Although, CONT and SCTAT groups exhibited similar basal plasma levels of adhesion molecules and TNF-alpha, SCT group had higher sVCAM-1 basal concentrations. No significant variation in sVCAM-1, sICAM-1 and TNF-alpha was measured following endurance exercise. Consequently, sVCAM-1 remained elevated in the SCT group after exercise and during the recovery period. In conclusion, our findings support the concept that SCT athletes might be at risk for microcirculatory disturbances, but these adhesive processes were not further impaired in response to endurance exercise. In addition, alpha-thalassemia co existing trait may be protective both at rest and after endurance exercise in SCT subjects.

Effects of progressive and maximal exercise on plasma levels of adhesion molecules in athletes with sickle cell trait with or without alpha-thalassemia.

The aim of the study was to examine the effects of exercise on soluble vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1) in sickle cell trait (SCT) athletes with or without alpha-thalassemia. Six athletes with SCT, seven athletes with both SCT and alpha-thalassemia (SCTAT), and seven control athletes (Cont) performed an incremental and maximal test on cycloergometer. Levels of sICAM-1 and sVCAM-1 were assessed at rest, immediately after the end of exercise, and 1, 2, and 24 h after exercise. Although Cont and SCTAT groups exhibited similar basal plasma levels of inflammatory and adhesion molecules, the SCT group had higher sVCAM-1 basal concentrations. Incremental exercise resulted in a significant increase of sVCAM-1 in all subjects, which remained elevated only in the SCT group during the recovery period. In conclusion, as sVCAM-1 increased with exercise and during the recovery period, our findings support the concept that SCT athletes might be at risk for microcirculatory disturbances and adhesive phenomena developing at rest and several hours after exercise. alpha-Thalassemia might be considered protective among exercising SCT subjects.

Use of the oral chelator deferiprone in the treatment of iron overload in patients with Hb H disease.

Seventeen non-transfusion-dependent Chinese haemoglobin H (Hb H) disease patients (age 29-76 years) with serum ferritin >900 microg/l were treated with deferiprone for up to 18 months. One patient withdrew and data from 16 patients were analysed. Sixteen other Hb H patients with ferritin <900 microg/l, matched for age and genotype, acted as controls. Treatment was well tolerated except for mild arthralgia. Serum ferritin fell with treatment, reaching significance at 6 and 18 months (from 1492.3 +/- 901.4 to 519.4 +/- 405.4 microg/l at 18 months, P = 0.0008). Nine of 16 patients had levels below 397 microg/l before 18 months. Serum ferritin remained stable 6 months after stopping treatment. In contrast, there was no change in ferritin levels in the control group. Magnetic resonance imaging was used for measurement of liver iron content. Spin echo T(1)-signal intensity ratio (T(1)-SIR) and gradient echo T(2)-signal intensity ratio (T(2)-SIR) increased with treatment. T(2)-SIR rose from 0.17 +/- 0.08 pretreatment to 0.58 +/- 0.50 at 2 years (P = 0.0055). Improvement occurred in 12 of 16 patients, reaching normal in three patients. Using echocardiography, peak early diastolic : late diastolic blood flow (E/A) remained unchanged with treatment, but isovolumic relaxation time (IVRT) was prolonged at 2 years indicating mild impairment of diastolic function. All systolic function parameters were normal. A longer treatment period is desirable to demonstrate improvement in cardiac function.

Thalassemia and related hemoglobinopathies.

Hemoglobinopathies are the most common single gene disorders in man. There are several hundred of these disorders though the thalassemias -- alpha and beta and the sickling disorders make up the vast majority. Recent advances in the understanding of the hemoglobin structure and the genetics of its synthesis has contributed significantly to the understanding of these diseases. Disorders include those with reduced globin synthesis, abnormal globin chains and failure to switch globin chain synthesis at the appropriate age. This review focuses on the clinical features, diagnosis and management strategies of the alpha and beta thalassemias, the sickling disorders and touches on a few rarer hemoglobinopathies. It also emphasizes prevention strategies and chronic transfusion safety in countries like India where there are limited resources.

Acquired somatic ATRX mutations in myelodysplastic syndrome associated with alpha thalassemia (ATMDS) convey a more severe hematologic phenotype than germline ATRX mutations.

Acquired somatic mutations in ATRX, an X-linked gene encoding a chromatin-associated protein, were recently identified in 4 patients with the rare subtype of myelodysplastic syndrome (MDS) associated with thalassemia (ATMDS). Here we describe a series of novel point mutations in ATRX detected in archival DNA samples from marrow and/or blood of patients with ATMDS by use of denaturing high-performance liquid chromatography (DHPLC), a technique sensitive to low-level mosaicism. Two of the new mutations result in changes in amino acids altered in previously described pedigrees with germ line ATRX mutations (ATR-X syndrome), but the hematologic abnormalities were much more severe in the patients with ATMDS than in the corresponding constitutional cases. In one ATMDS case where DNA samples from several time points were available, the proportion of ATRX-mutant subclones correlated with changes in the amount of hemoglobin H. This study strengthens the link between acquired, somatic ATRX mutations and ATMDS, illustrates how molecular defects associated with MDS and other hematologic malignancies masked by somatic mosaicism may be detected by DHPLC, and shows that additional factors increase the severity of the hematologic phenotype of ATRX mutations in ATMDS.

A normal beta-globin allele as a modifier gene ameliorating the severity of alpha-thalassemia in mice.

Thalassemia is a heritable human anemia caused by a variety of mutations that affect expression of the alpha- or the beta-chain of hemoglobin. The expressivity of the phenotype is likely to be influenced by unlinked modifying genes. Indeed, by using a mouse model of alpha-thalassemia, we find that its phenotype is strongly influenced by the genetic background in which the alpha-thalassemia mutation resides [129(sv/ev)/129(sv/ev) (severe) or 129(sv/ev)/C57BL/6 (mild)]. Linkage mapping indicates that the modifying gene is very tightly linked to the beta-globin locus (Lod score = 13.3). Furthermore, the severity of the phenotype correlates with the size of beta-chain-containing inclusion bodies that accumulate in red blood cells and likely accelerate their destruction. The beta-major globin chains encoded by the two strains differ by three amino acids, one of which is a glycine-to-cysteine substitution at position 13. The Cys-13 should be available for interchain disulfide bridging and consequent aggregation between excess beta-chains. This normal polymorphic variation between murine beta-globin chains could account for the modifying action of the unlinked beta-globin locus. Here, the variation in severity of the phenotype would not depend on a change in the ratio between alpha- and beta-chains but on the chemical nature of the normal beta-chain, which is in excess. This work also indicates that modifying genes can be normal variants that-absent an apparent physiologic rationale-may be difficult to identify on the basis of structure alone.

Cardiac blood flow studies in fetuses with homozygous alpha-thalassemia-1 at 12-13 weeks of gestation.

OBJECTIVE: Fetuses affected by homozygous alpha-thalassemia-1 develop anemia as early as the first trimester. Our objective was to study hemodynamic indices in affected fetuses at 12-13 weeks of gestation to determine whether these would be useful in the prediction of anemia. DESIGN: Prospective observational study. SUBJECTS: Women referred before 14 weeks of gestation for the prenatal diagnosis of homozygous alpha-thalassemia-1. METHODS: Transabdominal and/or transvaginal Doppler sonography was performed to measure the flow velocities in the fetal ascending aorta and pulmonary artery at 12-13 weeks. The Doppler indices were compared between those that were subsequently confirmed to be affected by homozygous alpha-thalassemia-1 and those that were unaffected. RESULTS: Between June 1997 and April 1998, 60 eligible women were recruited. Doppler examination was successful in 58 fetuses. Of these, 22 were subsequently confirmed to be affected by homozygous alpha-thalassemia-1. The diagnosis was made by chorionic villus sampling and DNA analysis in two affected fetuses and by cordocentesis and hemoglobin evaluation in 20 affected fetuses. Hemoglobin concentrations could be measured in ten fetuses and these ranged from 4 to 8 g/dl. The affected fetuses had significantly higher peak velocities at the pulmonary valve and ascending aorta and a larger inner diameter of the pulmonary valve than that in unaffected fetuses. The total cardiac output was increased by one-third in affected fetuses and was mainly due to an increase of the right-side cardiac output. CONCLUSION: In the early stage of anemia, the fetus responds mainly by increasing its right-side cardiac output. However, there is extensive overlap of the values of cardiac output between the affected and the unaffected fetuses, precluding its use in the prediction of anemia.

Endometrial extramedullary haemopoiesis.

Four cases of endometrial extramedullary haemopoiesis are reported, all with associated haematological disease. The diagnoses of a myeloproliferative disorder and thalassaemia trait were made as a consequence of the histological observations and subsequent haematological investigations in two cases. The third case occurred in a patient with an established diagnosis of chronic myeloid leukaemia. The diagnosis of extramedullary haemopoiesis in the final case was made on autopsy material from a patient with multiple myeloma. The endometrium from five other women with known myelofibrosis was examined but extramedullary haemopoiesis was not found. Endometrium from 32 fetuses did not contain haemopoietic elements, excluding the likelihood of the endometrium being a common site for extramedullary haemopoiesis in development. Endometrial extramedullary haemopoiesis is an uncommon finding, but it is worthy of note, as it may herald the presence of an underlying haematological abnormality.

Intrathoracic extramedullary hematopoietic tumor in hemoglobin H disease.

We report a Chinese patient with hemoglobin H (Hb H) disease who developed intrathoracic extramedullary hematopoiesis (EMH) 17 years following splenectomy for a blunt abdominal injury. The patient initially presented with extreme hyperbilirubinemia and multiple intrathoracic tumors. Hb H disease was diagnosed after investigation, and the marked jaundice, which declined gradually after supportive treatment, was attributed to his chronic hemolysis superimposed on an acute hepatitis C virus infection. A biopsy of the intrathoracic tumors revealed an EMH. Intrathoracic EMH, which is usually encountered in patients with beta-thalassemia and hereditary spherocytosis, has never been reported in Hb H disease. In areas where thalassemia is prevalent, EMH should be considered in the differential diagnosis of patients who have chronic anemia with asymptomatic intrathoracic tumor to avoid unnecessary surgical interventions.