Spectrum of LYST mutations in Chediak-Higashi syndrome: a report of novel variants and a comprehensive review of the literature.

INTRODUCTION: Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disorder characterised by partial oculocutaneous albinism, a bleeding diathesis, immunological dysfunction and neurological impairment. Bi-allelic loss-of-function variants in LYST cause CHS. LYST encodes the lysosomal trafficking regulator, a highly conserved 429 kDa cytoplasmic protein with an unknown function. METHODS: To further our understanding of the pathogenesis of CHS, we conducted clinical evaluations on individuals with CHS enrolled in our natural history study. Using genomic DNA Sanger sequencing, we identified novel pathogenic LYST variants. Additionally, we performed an extensive literature review to curate reported LYST variants and classified these novel and reported variants according to the American College of Medical Genetics/Association for Molecular Pathology variant interpretation guidelines. RESULTS: Our investigation unveiled 11 novel pathogenic LYST variants in eight patients with a clinical diagnosis of CHS, substantiated by the presence of pathognomonic giant intracellular granules. From these novel variants, together with a comprehensive review of the literature, we compiled a total of 147 variants in LYST, including 61 frameshift variants (41%), 44 nonsense variants (30%), 23 missense variants (16%), 13 splice site variants or small genomic deletions for which the coding effect is unknown (9%), 5 in-frame variants (3%) and 1 start-loss variant (1%). Notably, a genotype-phenotype correlation emerged, whereby individuals harbouring at least one missense or in-frame variant generally resulted in milder disease, while those with two nonsense or frameshift variants generally had more severe disease. CONCLUSION: The identification of novel pathogenic LYST variants and improvements in variant classification will provide earlier diagnoses and improved care to individuals with CHS.

Increased red cell distribution width in Fanconi anemia: a novel marker of stress erythropoiesis.

BACKGROUND: Red cell distribution width (RDW), a classical parameter used in the differential diagnosis of anemia, has recently been recognized as a marker of chronic inflammation and high levels of oxidative stress (OS). Fanconi anemia (FA) is a genetic disorder associated to redox imbalance and dysfunctional response to OS. Clinically, it is characterized by progressive bone marrow failure, which remains the primary cause of morbidity and mortality. Macrocytosis and increased fetal hemoglobin, two indicators of bone marrow stress erythropoiesis, are generally the first hematological manifestations to appear in FA. However, the significance of RDW and its possible relation to stress erythropoiesis have never been explored in FA. In the present study we analyzed routine complete blood counts from 34 FA patients and evaluated RDW, correlating with the hematological parameters most consistently associated with the FA phenotype. RESULTS: We showed, for the first time, that RDW is significantly increased in FA. We also showed that increased RDW is correlated with thrombocytopenia, neutropenia and, most importantly, highly correlated with anemia. Analyzing sequential hemograms from 3 FA patients with different clinical outcomes, during 10 years follow-up, we confirmed a consistent association between increased RDW and decreased hemoglobin, which supports the postulated importance of RDW in the evaluation of hematological disease progression. CONCLUSIONS: This study shows, for the first time, that RDW is significantly increased in FA, and this increment is correlated with neutropenia, thrombocytopenia, and highly correlated with anemia. According to the present results, it is suggested that increased RDW can be a novel marker of stress erythropoiesis in FA.

Epigenetic Alterations in Fanconi Anaemia: Role in Pathophysiology and Therapeutic Potential.

Fanconi anaemia (FA) is an inherited disorder characterized by chromosomal instability. The phenotype is variable, which raises the possibility that it may be affected by other factors, such as epigenetic modifications. These play an important role in oncogenesis and may be pharmacologically manipulated. Our aim was to explore whether the epigenetic profiles in FA differ from non-FA individuals and whether these could be manipulated to alter the disease phenotype. We compared expression of epigenetic genes and DNA methylation profile of tumour suppressor genes between FA and normal samples. FA samples exhibited decreased expression levels of genes involved in epigenetic regulation and hypomethylation in the promoter regions of tumour suppressor genes. Treatment of FA cells with histone deacetylase inhibitor Vorinostat increased the expression of DNM3Tß and reduced the levels of CIITA and HDAC9, PAK1, USP16, all involved in different aspects of epigenetic and immune regulation. Given the ability of Vorinostat to modulate epigenetic genes in FA patients, we investigated its functional effects on the FA phenotype. This was assessed by incubating FA cells with Vorinostat and quantifying chromosomal breaks induced by DNA cross-linking agents. Treatment of FA cells with Vorinostat resulted in a significant reduction of aberrant cells (81% on average). Our results suggest that epigenetic mechanisms may play a role in oncogenesis in FA. Epigenetic agents may be helpful in improving the phenotype of FA patients, potentially reducing tumour incidence in this population.

Refractory iron-deficiency anemia and autoimmune atrophic gastritis in pediatric age group: analysis of 8 clinical cases.

INTRODUCTION: Refractory iron-deficiency anemia with no obvious etiology in pediatric age can be a puzzling problem. Screening of iron malabsorption conditions, including autoimmune atrophic gastritis (AAG), is emerging as a priority in the investigational procedure. MATERIALS AND METHODS: Retrospective analysis of clinical process of children/adolescents with the diagnosis of AAG. RESULTS: Eight patients (aged between 4.7 and 18 years old) were identified. The diagnosis was triggered on the basis of high serum gastrin levels and strong positivity of antiparietal cell antibodies. Upper endoscopy and biopsy revealed atrophic gastritis in all patients, with 2 of them with intestinal metaplasia. Four patients presented with Helicobacter pylori infection object of eradication therapy. After a medium follow-up of 36.6 months, antiparietal cell antibodies and hypergastrinemia did not show evidence of regression. Of the 3 patients who underwent endoscopic reevaluation, a similar anatomo-pathologic pattern was observed in 2 and intestinal metaplasia in 1 patient. Normalization of hematological parameters was achieved, using alternative iron formulas. CONCLUSIONS: AAG must be recognized as a pathology affecting pediatric patients. Gastric autoimmune lesion is a chronic process with potential evolution to malignancy. Management guidelines in childhood are not available. Their elaboration is important considering an important risk factor in these age group: a long life expectancy.

Fosfomycin increases chromosome instability in lymphocytes from Fanconi Anemia patients.

Fanconi Anemia (FA) is a chromosome instability (CI) syndrome, clinically characterized by progressive bone marrow failure and increased cancer predisposition. Lymphocytes from FA patients have hypersensitivity to alkylating agents, particularly to diepoxybutane (DEB). The antibiotic fosfomycin (FOM) is an alkylating agent. FOM is used as a large spectrum antibiotic and also as a prophylactic pre-surgery agent. FOM has been considered non-genotoxic. However, no specific genotoxic evaluation directed to patients with hypersensitivity to alkylating agents was performed. As FA patients are very susceptible to infections and may be submitted to several surgeries, FOM can eventually be prescribed to them during their lifetime. In the present study we evaluated the putative genotoxic effect of FOM in cultured lymphocytes from FA patients, compared to cultured lymphocytes from healthy donors (HD). Cultures from FA patients and HD were treated with 0.5mM FOM or with 0.6mM DEB and CI was evaluated. Results showed that FOM significantly increases CI in cultured lymphocytes from FA patients, compared to lymphocytes from HD, in which no effect was found. Additionally, a direct correlation between DEB and FOM toxicity was observed in lymphocytes from FA patients, indicating similar susceptibility to both agents.

Iron deficiency anemia due to lymphocytic gastritis with Helicobacter pylori infection in childhood: case report.

Lymphocytic gastritis (LG) is a chronic inflammatory process of poorly understood pathogenesis. We report the case of a 12-year-old premenstrual girl with refractory iron deficiency anemia in which the oral iron absorption challenge suggested iron malabsorption. Laboratory studies ruled out celiac disease and autoimmune gastritis, and carbon-13 urea breath test for Helicobacter pylori was also negative. Upper endoscopy with gastric body and antral mucosa biopsies revealed a LG with focal intestinal metaplasia and H. pylori infection. H. pylori eradication was undertaken with success and 3 months later her hematologic parameters normalized. Histologic reevaluation showed disappearance of LG. This case shows that investigation of malabsorption disease in the presence of refractory iron deficiency anemia can lead to the diagnosis of important gastric diseases, even in the absence of gastrointestinal symptoms. This nonceliac child was diagnosed with a severe histopathologic pattern of LG, with potential risk of malignant transformation, which was completely reverted with adequate H. pylori eradication treatment.

Infantile pyknocytosis: an under-recognized form of neonatal hemolytic anemia?

Infantile pyknocytosis (IP) is an under-recognized hematological entity of newborns that can cause a severe neonatal hemolytic anemia. A careful, prompt, and accurate peripheral blood smear examination is essential to establish the diagnosis. Here we describe the clinical features and histological parameters of 1 case of IP. Spontaneous resolution usually occurs by 4 to 6 months, but red blood cell transfusion may be needed if the anemia is severe.

Hb Iberia [α104(G11)Cys → Arg,TGC>CGC (α2) (HBA2:c.313T>C)], a new α-thalassemic hemoglobin variant found in the Iberian Peninsula: report of six cases.

We report a new structural defect of the α2-globin chain presenting with moderate microcytic hypochromic anemia, in six individuals from three unrelated families, living in Portugal and Spain. α-Globin gene deletions were ruled out by gap-polymerase chain reaction (gap-PCR) and multiplex ligation-dependent probe amplification (MLPA). Direct sequencing of the α2-globin gene revealed a substitution of codon 104 [α104(G11)Cys→Arg, TGC>CGC (α2) (HBA2:c.313T>C)]. This new variant, not detectable by high performance liquid chromatography (HPLC) or electrophoresis, was called Hb Iberia, as it was observed for the first time in families from the Iberian Peninsula. Although the mutant allele is transcribed, as indicated by the balanced mRNA α/ß ratio, the abnormal α2 chain could not form a stable tetramer as the cysteine and arginine residues, located at the α1ß1 contact, differ in size, charge and hydrophobicity. Hb Iberia is the third mutation described at codon 104 on the α-globin genes, namely, Hb Sallanches (α2, TGC>TAC) and Hb Oegstgeest (α1, TGC>AGC), also characterized as unstable hemoglobins (Hbs), present on an α-thalassemic phenotype.

Improvement of genetic stability in lymphocytes from Fanconi anemia patients through the combined effect of α-lipoic acid and N-acetylcysteine.

Fanconi Anemia (FA) is a rare genetic disorder, characterized by progressive bone marrow failure and increased predisposition to cancer. Despite being highly heterogeneous, all FA patients are hypersensitive to alkylating agents, in particular to 1,2:3,4-diepoxybutane (DEB), and to oxidative damage. Recent studies point to defective mitochondria in FA cells, which is closely related with increased production of reactive oxygen species (ROS) and concomitant depletion of antioxidant defenses, of which glutathione is a well-known biomarker.The objective of the present work is to evaluate the putative protective effect of α-lipoic acid (α-LA), a mitochondrial protective agent, and N-acetylcysteine (NAC), a direct antioxidant and a known precursor for glutathione synthesis, in spontaneous and DEB-induced chromosome instability (CI) in lymphocyte cultures from FA patients.For that purpose, lymphocyte cultures from 15 FA patients and 24 healthy controls were pre-treated with 20 µM α-LA, 500 µM NAC and α-LA plus NAC at the same concentrations, and some of them were exposed to DEB (0.05 µg/ml). A hundred metaphases per treatment were scored to estimate the relative frequency of spontaneous and DEB-induced chromosome breakage.The obtained results revealed that a cocktail of α-LA and NAC can drastically improve the genetic stability in FA lymphocytes in vitro, decreasing CI by 60% and 80% in cultures from FA patients and FA mosaic/chimera patients, respectively. These results suggest that the studied cocktail can be used as a prophylactic approach to delay progressive clinical symptoms in FA patients caused by CI, which can culminate in the delay of the progressive bone marrow failure and early cancer development.

Adams-Oliver syndrome and portal hypertension: fortuitous association or common mechanism?

Adams-Oliver syndrome (AOS) is a rare condition defined by combination of cutis aplasia and transverse limb abnormalities. Some authors have described a possible association between this syndrome and portal hypertension (PH) due to hepatoportal sclerosis (HPS). We present a boy with AOS who developed a progressive splenomegaly and hypersplenism at the age of 2 months, and was admitted for acute gastrointestinal bleeding (GI) at the age of 9 months. Subsequently, we documented an extrahepatic portal vein obstruction and esophageal varices. After several episodes of cataclysmic upper GI bleeding a mesentero-portal shunt (MPS) was performed at 10 months. The shunt thrombosed, and after three failed attempts of thrombectomy, it was removed. One month later a splenorenal shunt was performed, and this closed spontaneously by 3 years. The patient suffered from ischemic stroke after placing the first shunt, and has spastic diplegia, left frontal lobe epilepsy, hyperactivity and attention deficit disorder, and severe psychomotor delay. At 11 years and he presented with chronic liver failure and hyperammonemia and coagulopathy. We hypothesize that there may be an early embryonic vascular abnormality (vascular disruption) that may explain these vascular phenomena.

[Fanconi anemia: cytogenetic diagnosis of 40 cases]. / Anemia de Fanconi: diagnóstico citogenético de 40 casos.

Fanconi Anemia (FA) is a rare recessive disorder clinically characterized by progressive bone marrow failure, diverse congenital malformations and increased predisposition to cancer. Given the late onset of anemia, relatively to other cytopenias, and the high variability in the phenotype, a correct clinical diagnosis is difficult, and may be delayed or even missed. This fact may be prejudicial to patients, due to the need of avoiding exposure to toxic agents, programming the transplantation of hematopoietic progenitor cells and screening of neoplasia associated with the disease. Given the high genetic variability (thirteen complementation groups have been identified, each with genes presenting several different mutations), a rapid molecular diagnosis is not possible. However, there is an urgent need for a timely and correct diagnosis, due to the early evolution of the disease towards malignancy and to the early need of finding compatible donors for future hematopoietic stem cell transplantation. Fortunately, the hypersensitivity of FA cells to the clastogenic (chromosome breaking) effect of DNA cross-linking agents, in particular to diepoxybutane (DEB), provides a unique marker for the diagnosis. At present, cytogenetic analysis for detection of DEB-induced chromosome instability is the gold-standard test for the diagnosis of FA. In the present work we present the results from the DEB induced chromosome instability studies performed in the Laboratory of Cytogenetics of ICBAS between 1992 and 2009. Blood samples from 222 patients were obtained from different hospitals mainly from the north and centre of Portugal. This population includes not only patients with clinical suspicion of FA, but also patients presented with thrombocytopenia, pancitopenia or aplastic anemia, for confirmation/exclusion of FA. Two samples of amniotic fluid were also obtained for pre-natal diagnosis. A total of 34 FA patients were diagnosed. Cytogenetic studies were also performed in blood samples from AF relatives, which allowed the diagnosis of 6 new cases, 5 of them corresponding to asymptomatic individuals. In the total population of FA patients studied, 25% belong to the gypsy ethnic group. Periodic cytogenetic studies were also performed in blood samples from AF patients post transplantation, which confirmed the elimination of the original hematopoietic DEB sensitive cells.

Erythropoiesis versus inflammation in Hereditary Spherocytosis clinical outcome.

OBJECTIVES: This study aimed to evaluate the relationship between erythropoiesis and inflammation, in Hereditary Spherocytosis (HS) clinical outcome. DESIGN AND METHODS: We studied 26 controls and 82 HS patients presenting mild (n = 49) and severer (n = 33) HS forms. We evaluated plasma levels of EPO, sTfR, ferritin, iron, folic acid, vitamin B12, TNF-α, IFN-γ, elastase and lactoferrin; leukocyte and reticulocyte counts and RPI were determined. RESULTS: All HS patients showed significantly higher EPO, sTfR, reticulocytes and RPI but only mild HS presented normal hemoglobin levels; the positive significant correlations between EPO and sTfR, reticulocytes and RPI observed in mild HS were not observed in severer HS patients. HS patients presented with higher levels of neutrophils, TNF-α, IFN-γ, elastase, lactoferrin and ferritin. CONCLUSIONS: Our data show HS as a disease linked to enhanced erythropoiesis that is disturbed in the more severe forms, to which inflammation may contribute, at least in part.

Origin, functional role, and clinical impact of Fanconi anemia FANCA mutations.

Fanconi anemia is characterized by congenital abnormalities, bone marrow failure, and cancer predisposition. To investigate the origin, functional role, and clinical impact of FANCA mutations, we determined a FANCA mutational spectrum with 130 pathogenic alleles. Some of these mutations were further characterized for their distribution in populations, mode of emergence, or functional consequences at cellular and clinical level. The world most frequent FANCA mutation is not the result of a mutational "hot-spot" but results from worldwide dissemination of an ancestral Indo-European mutation. We provide molecular evidence that total absence of FANCA in humans does not reduce embryonic viability, as the observed frequency of mutation carriers in the Gypsy population equals the expected by Hardy-Weinberg equilibrium. We also prove that long distance Alu-Alu recombination can cause Fanconi anemia by originating large interstitial deletions involving FANCA and 2 adjacent genes. Finally, we show that all missense mutations studied lead to an altered FANCA protein that is unable to relocate to the nucleus and activate the FA/BRCA pathway. This may explain the observed lack of correlation between type of FANCA mutation and cellular phenotype or clinical severity in terms of age of onset of hematologic disease or number of malformations.

Complementary markers for the clinical severity classification of hereditary spherocytosis in unsplenectomized patients.

Hereditary spherocytosis (HS) is usually classified as mild, moderate or severe using conventional features, namely, hemoglobin (Hb) concentration, reticulocyte count and bilirubin levels, which do not always contribute to an adequate clinical classification. The aim of our study was to establish the importance of some laboratory routine parameters, as markers of HS clinical outcome, by studying a control group (n=26) and unsplenectomized HS patients (n=82) presenting mild, moderate or severe HS. We performed a basic hematologic study and evaluated the reticulocyte count, bilirubin, erythropoietin (EPO) and soluble transferrin receptor (sTfR) levels; the osmotic fragility (OFT) and criohemolysis tests (CHT); the ratios Hb/MCHC (mean cell hemoglobin concentration), Hb/RDW (red cell distribution width) and MCHC/RDW, were calculated. Hb changed significantly in accordance with HS severity, but not reticulocytes or bilirubin. We found that MCHC, RDW, EPO, sTfR, OFT, CHT and the calculated ratios were significantly changed in patients, and, therefore, were valuable as complementary diagnostic tools for HS. Moreover, RDW, Hb/MCHC, Hb/RDW and MCHC/RDW changed significantly with worsening of HS; thus, they are also good markers for the clinical outcome of HS. In conclusion, we propose the use of these routine parameters as useful to complement the analysis of HS severity.

Erythrocyte membrane protein destabilization versus clinical outcome in 160 Portuguese Hereditary Spherocytosis patients.

Hereditary Spherocytosis (HS) is a haemolytic anaemia caused by erythrocyte protein membrane defects - spectrin, ankyrin, band 3 or protein 4.2 - that lead to membrane destabilization. This study aimed to evaluate the prevalence of protein deficiencies and the role of membrane proteins or membrane-linked proteins in membrane disturbance and in HS clinical outcome. A total of 215 Portuguese individuals were studied - 203 from 71 families plus 12 individual unrelated subjects; 160 of them were diagnosed with HS. They were classified as presenting mild, moderate or severe forms of HS according to the degree of haemolytic anaemia. Standardized electrophoretic erythrocyte membrane protein analysis was used to identify and quantify protein deficiencies. Band 3 and ankyrin were found to account for the majority of the erythrocyte protein defects underlying HS. Increasing isolated protein deficiency or increasing imbalance between combined protein deficiencies seemed to underlie HS severity, by increasing membrane destabilization. There was an increased membrane linkage of the cytosolic proteins, glyceraldehyde-3-phosphate dehydrogenase and peroxiredoxin 2, and of denatured haemoglobin, suggesting that this linkage could interfere with membrane structure. Our data suggest that the quantification and the analysis of RBC membrane proteins may be helpful in predicting the clinical outcome of HS.