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.

A Distinctive Type of Mosaic Variegated Aneuploidy: Case Report and Review of the Literature.

Mosaic variegated aneuploidy (MVA) is an autosomal recessive disorder characterized by mosaic aneuploidies, predominantly trisomies, involving multiple different chromosomes and tissues. The proportion of aneuploid cells varies, and most patients present with intrauterine growth delay, microcephaly, and a broad spectrum of congenital abnormalities. We report a patient with a distinctive type of MVA discovered in bone marrow (BM) when she was 3-month-old due to neutropenia and hypocellular bone marrow. She was followed up for more than 20 years, and different trisomic cells were repeatedly discovered in different tissues, whereas her clinical picture has never been severe. The main sign remained intermittent neutropenia, not cyclic and often not too severe, occasionally with anemia and thrombocytopenia. Retromicrognathia was the only dysmorphic sign. Unlike other patients with MVA, the trisomies in all tissues involved almost invariably chromosomes 18 and 19. Therefore, the peculiarities of our patient were the clinical and the atypical cytogenetic pictures. Nevertheless, we looked for mutations in the seven causative genes of the known types of MVA, but the results were negative. Then, we analyzed the entire exome to find out other possible causing mutations, but also this attempt failed to discover a possible cause of this distinctive form of MVA.

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.

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.

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.

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.

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.

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.

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.

FAS-L, IL-10, and double-negative CD4- CD8- TCR alpha/beta+ T cells are reliable markers of autoimmune lymphoproliferative syndrome (ALPS) associated with FAS loss of function.

Autoimmune lymphoproliferative syndrome (ALPS) is characterized by splenomegaly, lymphadenopathy, hypergammaglobulinemia, accumulation of double-negative TCRalphabeta(+) CD4(-)CD8(-) T cells (DNT cells), and autoimmunity. Previously, DNT cell detection and a functional defect of T cells in a FAS-induced apoptosis test in vitro had been used for ALPS diagnosis. However, a functional defect can also be detected in mutation-positive relatives (MPRs) who remain free of any ALPS-related disease. In contrast, lymphocytes from patients carrying a somatic mutation of FAS exhibit normal sensitivity to FAS-induced apoptosis in vitro. We assessed the soluble FAS-L concentration in the plasma of ALPS patients carrying FAS mutations. Overall, we showed that determination of the FAS-L represents, together with the IL-10 concentration and the DNT cell percentage, a reliable tool for the diagnosis of ALPS.

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.

Presence of cytosolic peroxiredoxin 2 in the erythrocyte membrane of patients with hereditary spherocytosis.

We studied 82 Portuguese individuals, 57 with hereditary spherocytosis (HS) and 25 unaffected controls. We performed standardized diagnosis tests, including electrophoretic membrane protein analysis to identify and quantify protein deficiencies underlying HS. Membrane bound hemoglobin (MBH) and band 3 profiles were determined as oxidative stress and aging markers. A protein of about 22 kDa, present in 21 of 57 HS patients, but not in controls, was identified as peroxiredoxin 2 (Prx2), by mass-spectroscopy and by immunoblotting. Human erythrocyte Prx2 is a peroxiredoxin with thiol-specific antioxidant activity. The presence of Prx2 in erythrocyte membranes was linked to higher levels of oxidative stress, as reflected by significantly increased MBH in those HS patients. No relation with HS clinical severity was observed and Prx2 was detected in all types of membrane protein abnormalities. Prx2 membrane linkage is associated with a higher oxidative stress susceptibility of HS erythrocytes.

Audiologic abnormalities of Fanconi anaemia.

CONCLUSIONS: The most common audiologic manifestation in Fanconi anaemia (FA) was asymmetrical bilateral conductive hearing loss that was more severe at lower frequencies and in some cases had a progressive character. The routine screening of all patients diagnosed with FA allowed the recognition of mild hearing loss and the prevention of the deleterious effects of its progression with adequate rehabilitation measures. OBJECTIVES: FA is an autosomal recessive disease characterized by bone marrow failure, multiple congenital anomalies and increased susceptibility to malignancy. Otologic manifestations in FA include morphologic anomalies affecting the ear structures and hearing loss. This report is a retrospective review of the most important features, including audiologic features, in children with FA attending a paediatric hospital. SUBJECTS AND METHODS: The medical records of eight patients with FA were reviewed and patient demographics, physical abnormalities, haematological characteristics at diagnosis and otological and audiological features were analysed. RESULTS: Eight patients (five females, three males), aged between 3 and 13 years old, have been followed for at least 1 year in our hospital. In all, 50% (four of eight) of our population has hearing loss. It is an asymmetrical, bilateral, conductive hearing loss that is more severe at lower frequencies. Two patients (25%) have unilateral type I microtia and stenotic external ear canal.

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.