Congenital dyserythropoietic anemia types Ib, II, and III: novel variants in the CDIN1 gene and functional study of a novel variant in the KIF23 gene.

Congenital dyserythropoietic anemias (CDA) are disorders characterized by ineffective erythropoiesis and morphological anomalies in erythrocytes and erythroblasts. The purpose of this study is to identify the gene variants in patients diagnosed with CDA. We analyzed five unrelated patients and two siblings with a targeted panel of genes to CDA: CDAN1, CDIN1, SEC23B, KIF23, KLF1, and GATA1 genes. We found three novel variants in the CDIN1 gene (p.Leu136Val, p.Tyr247Cys, and p.Ile273Thr), four known variants in the SEC23B gene (p.Arg14Trp, p.Arg554Ter, p.Asp239Gly, and p.Ser436Leu), and one novel variant in the KIF23 gene (p.Leu945Trpfs*31). The in silico analysis of novel variants predict that they are pathogenic and, the in vitro study confirms the functional impact of the KIF23 variant on the protein location.

Unexpected Cause of Persistent Microcytosis and Neurological Symptoms in a Child: Niemann-Pick Disease Type C.

Atypical microcytic anemias are rare diseases of iron/heme metabolism that can be diagnostically challenging. We report the case of a 2-year-old twin boy with neurodevelopmental delay and persistent microcytosis in whom atypical microcytic anemias was initially suspected. He had low blood iron and transferrin saturation with normal/high ferritin despite iron therapy. Hemoglobinopathies were excluded by conventional/DNA studies. Hepcidin was high but iron-refractory-iron-deficiency anemia was ruled out by a genetic panel. Bone marrow aspiration revealed foamy cells and iron depletion. A genetic study confirmed the diagnosis of Niemann-Pick disease type C which was finally considered the origin of microcytosis through anemia of chronic disease.

Clinical and genetic features of congenital dyserythropoietic anemia (CDA).

INTRODUCTION: Congenital dyserythropoietic anemias (CDA) are characterized by hyporegenerative anemia with inadequate reticulocyte values, ineffective erythropoiesis, and hemolysis. Distinctive morphology of bone marrow erythroblasts and identification of causative genes allow classification into 4 types caused by variants in CDAN1, c15orf41, SEC23B, KIF23, and KLF1 genes. OBJECTIVE: Identify pathogenic variants in CDA patients. METHODS: Massive parallel sequencing with a targeted gene panel, Sanger sequencing, Comparative Genome Hybridization (CGH), and in silico predictive analysis of pathogenicity. RESULTS: Pathogenic variants were found in 21 of 53 patients studied from 44 unrelated families. Six variants were found in CDAN1: two reported, p.Arg714Trp and p.Arg725Trp and, four novel, p.Arg623Trp, p.Arg946Trp, p.Phe1125Ser and p.Ser1227Gly. Twelve variants were found in SEC23B: seven reported, p.Arg14Trp, p.Glu109Lys, p.Arg217Ter, c.835-2A>G, p.Arg535Ter, p.Arg550Ter and p.Arg718Ter and, five novel, p.Val164Leu, p.Arg190Gln, p.Gln521Ter, p.Arg546Trp, and p.Arg611Gln. The variant p.Glu325Lys in KLF1 was found in one patient and p.Tyr365Cys in ALAS2 in an other. Moreover, we identified genomic rearrangements by CGH in some SEC23B-monoallelic patients. CONCLUSIONS: New technologies for genetic studies will help to find variants in other genes, in addition to those known, that contribute to or modulate the CDA phenotype or support the correct diagnosis.

Juvenile Hemochromatosis due to a Homozygous Variant in the HJV Gene.

Hemochromatosis type 2 or juvenile hemochromatosis has an early onset of severe iron overload resulting in organ manifestation such as liver fibrosis, cirrhosis, cardiomyopathy, arthropathy, hypogonadism, diabetes, osteopathic medicine, and thyroid abnormality, before age of 30. Juvenile hemochromatosis type 2a and 2b is an autosomal recessive disease caused by pathogenic variants in HJV and HAMP genes, respectively. We report a child with hepatic iron overload and family history of hemochromatosis. We aim to raise awareness of juvenile hemochromatosis, especially in families with a positive family history, as early diagnosis and treatment may prevent organ involvement and end-stage disease. The purpose of this study was to identify the gene variant that causes the disease. The genetic study was performed with a targeted gene panel: HFE, HJV, HAMP, TFR2, SLC40A1, FTL, and FTH1. We identified the variant c.309C > G (p.Phe103Leu) in the HJV gene in the homozygous state in the patient.

Molecular Analysis of 55 Spanish Patients with Acute Intermittent Porphyria.

Acute intermittent porphyria (AIP) results from a decreased activity of hepatic hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway. AIP is an autosomal dominant disorder with incomplete penetrance, characterized by acute neurovisceral attacks precipitated by several factors that induce the hepatic 5-aminolevulinic acid synthase, the first enzyme in the heme biosynthesis. Thus, a deficiency in HMBS activity results in an overproduction of porphyrin precursors and the clinical manifestation of the disease. Early diagnosis and counselling are essential to prevent attacks, and mutation analysis is the most accurate method to identify asymptomatic carriers in AIP families. In the present study, we have investigated the molecular defects in 55 unrelated Spanish patients with AIP, identifying 32 HMBS gene mutations, of which six were novel and ten were found in more than one patient. The novel mutations included a missense, an insertion, two deletions, and two splice site variants. Prokaryotic expression studies demonstrated the detrimental effect for the missense mutation, whereas reverse transcription-PCR and sequencing showed aberrant splicing caused by each splice site mutation. These results will allow for an accurate diagnosis of carriers of the disease in these families. Furthermore, they increase the knowledge about the molecular heterogeneity of AIP in Spain.

Molecular analysis of 19 Spanish patients with mixed porphyrias.

Porphyrias are rare diseases caused by alterations in the heme biosynthetic pathway. Depending on the afected enzyme, porphyrin precursors or porphyrins are overproduced, causing acute neurovisceral attacks or dermal photosensitivity, respectively. Hereditary Coproporphyria (HCP) and Variegate Porphyria (VP) are mixed porphyrias since they can present acute and/or cutaneous symptoms. These diseases are caused by a deficiency of coproporphyrinogen oxidase (CPOX) in HCP, and protoporphyrinogen oxidase (PPOX) in VP. Herein, we studied nineteen unrelated Spanish patients with mixed porphyrias. The diagnosis of either, HCP or VP was made on the basis of clinical symptoms, biochemical findings and the identification of the mutation responsible in the CPOX or PPOX genes. Two patients presented both acute and cutaneous symptoms. In most patients, the biochemical data allowed the diagnosis. Among eleven patients with HCP, ten CPOX mutations were identified, including six novel ones: two frameshift (c.32delG and c.1102delC), two nonsense (p.Cys239Ter and p.Tyr365Ter), one missense (p.Trp275Arg) and one amino acid deletion (p.Gly336del). Moreover, seven previously described PPOX mutations were identified in eight patients with VP. The impacts of CPOX mutations p.Trp275Arg and p.Gly336del, were evaluated using prediction softwares and their functional consequences were studied in a prokaryotic expression system. Both alterations were predicted as deleterious by in silico analysis. Aditionally, when these alleles were expressed in E. coli, only p.Trp275Arg retained some residual activity. These results emphasize the usefulness of integrated the biochemical tests and molecular studies in the diagnosis. Furthermore, they extend knowledge on the molecular heterogeneity of mixed porphyrias in Spain.

A case of congenital dyserythropoietic anemia type IV.

Congenital dyserythropoietic anemias (CDAs) are displayed by ineffective erythropoiesis. The wide variety of phenotypes observed in CDA patients makes differential diagnosis difficult; identification of the genetic variants is crucial in clinical management. We report the fifth case of a patient with unclassified CDAs, after genetic study, with CDA type IV.

Sideroblastic anemia: functional study of two novel missense mutations in ALAS2.

BACKGROUND: X-linked sideroblastic anemia (XLSA) is a disorder characterized by decreased heme synthesis and mitochondrial iron overload with ringed sideroblasts in bone marrow. XLSA is caused by mutations in the erythroid-specific gene coding 5-aminolevulinate synthase (ALAS2). Anemia in XLSA is extremely variable, characteristically microcytic and hypochromic with poikilocytosis, and the red blood cell distribution width is increased and prominent dimorphism of the red cell population. Anemia in XLSA patients responds variably to supplementation with pyridoxine. METHODS AND RESULTS: We report four patients with XLSA and three mutations in ALAS2: c.611G>A (p.Arg204Gln), c.1218G>T (p.Leu406Phe) and c.1499A>G (p.Tyr500Cys). The in silico predictions of three ALAS2 mutations and the functional consequences of two ALAS2 mutations were assessed. We performed in silico analysis of these mutations using ten different softwares, and all of them predicted that the p.Tyr500Cys mutation was deleterious. The in vitro prokaryotic expression showed that the p.Leu406Phe and p.Tyr500Cys mutations reduced the ALAS2 specific activity (SA) to 14% and 7% of the control value, respectively. CONCLUSION: In view of the results obtained in this study, a clear relationship between genotype and phenotype cannot be established; clinical variability or severity of anemia may be influenced by allelic variants in other genes or transcription factors and environmental conditions.

Volatile anaesthetics induce biochemical alterations in the heme pathway in a B-lymphocyte cell line established from hepatoerythropoietic porphyria patients (LBHEP) and in mice inoculated with LBHEP cells.

Hepatoerythropoietic porphyria (HEP) is the homozygous form of Porphyria Cutanea Tarda (PCT), characterized by an accumulation of porphyrins due to uroporphyrinogen decarboxylase deficiency. Fluorinated volatile anaesthetics are often used to produce general anaesthesia. Anaesthesia has certainly been implicated in the triggering of acute porphyria crisis. The effects of volatile anaesthetics in a B-lymphocyte cell line established from HEP patients (LBHEP) on heme metabolism have been investigated.LBHEP cells were exposed to sodium phosphate buffer containing dissolved Enflurane, Isoflurane or Sevoflurane (10mM) during 20min. Aminolevulinate synthase (ALA-S) activity, the regulatory enzyme of heme synthesis, was 300% induced by the anaesthetics. A 25-30% diminution of porphobilinogenase (PBG-ase) activity was found when Isoflurane or Sevoflurane were added to the cells, while no significant changes were detected after Enflurane treatment. Although some oxidative stress has been induced by the anaesthetics, reflected by the 35% diminution of glutathione (GSH), no alteration in heme oxygenase (HO) activity, the enzyme involved in heme breakdown and frequently induced as a response to stress stimuli, was observed. Studies using animals inoculated with LBHEP cells were also performed. Findings here described mimic biochemical alterations in the heme pathway, which are characteristic of another hepatic porphyria, similar to those previously reported when these anaesthetics were administered to animals, and they also advertise about the possible unsafe use of these drugs in the case of hepatic non-acute porphyrias.

Erythrocyte aminolevulinate dehydratase activity as a lead marker in patients with chronic renal failure.

BACKGROUND: Overexposure to lead may result in an increased risk for developing chronic renal failure (CRF) and hypertension. Subclinical lead poisoning is difficult to identify. Because the heme biosynthetic pathway is highly sensitive to lead, we considered the study of enzymes involved in this pathway as a method to detect an excessive body lead burden. METHODS: Main concerns in assessing the heme pathway in patients with CRF were related to aminolevulinate dehydratase (ALAD) activity. We first selected a number of patients with CRF at a predialysis stage, subsequently dividing them into two groups after the EDTA mobilization test had determined whether lead pools were expanded. The study included 24 healthy controls, 12 patients with clinical plumbism and biochemical demonstration of lead poisoning (Pb-CONT), 18 patients with CRF with no evidence of high lead storage (CRF/-), and 8 patients with CRF with high urinary excretion of lead in contrast to normal blood lead levels (CRF/+). RESULTS: As expected, symptoms of plumbism (Pb-CONT) were accompanied by an increased erythrocyte zinc-protoporphyrin-free protoporphyrin ratio and high urine coproporphyrin excretion, whereas both these values were within the normal range in all patients with CRF. CRF/- patients showed minor abnormalities of erythrocyte heme metabolism, such as low ALAD activity, both baseline and in vitro restored. The ALAD-restored ALAD ratio correlated closely with urine lead excretion; it was normal in healthy controls and CRF/- patients and significantly reduced in Pb-CONT and CRF/+ patients. CONCLUSION: The erythrocyte ALAD-restored ALAD ratio may be a useful tool to show otherwise subclinical lead poisoning in patients with CRF.

Successful and safe treatment of hypertrichosis by high-intensity pulses of noncoherent light in a patient with hepatoerythropoietic porphyria.

Hypertrichosis is a common feature in cutaneous porphyrias, characterized by high accumulation of photoreactive porphyrins. Photothermolysis induced by noncoherent light (755-1200 nm) and energy fluence of 42 J/cm(2) was applied to a patient with hepatoerythropoietic porphyria. Hypertrichosis was almost completely removed after seven sessions without development of skin lesions.

Relationship between biomarker expression and allelic alteration in esophageal carcinoma.

BACKGROUND AND AIM: Expression of biomarkers and probable allelic alterations were studied in esophagus tissue samples from patients with esophageal carcinoma. METHODS: A total of 116 esophagus tissue samples were obtained from 25 patients with esophagus cancer. Histological studies revealed 23 samples were adenocarcinoma and 14 samples were epidermoid carcinoma while 79 samples were non-tumor. Expression of biomarkers was determined by enzyme immunoassay, and allelic alterations on chromosome 17p were performed by polymerase chain reaction (PCR) using primers D17S513 and D17S514. RESULTS: The adenocarcinoma group exhibited an increase of matrix metalloproteinase (MMP)-1 (P < 0.0001) and sialyl Le (a) (P < 0.001) mean levels when compared with the non-tumor group. Adenocarcinoma samples from patients with more than three positive lymph nodes had lower levels of tissue-inhibitor metalloproteinase (TIMP)-1 than those with negative nodes (P < 0.0005). Positive allelic alteration was associated with high levels of MMP-1 expression (P = 0.003). Epidermoid carcinoma samples showed higher expression of MMP-1 (P < 0.0001) and TIMP-1 (P < 0.02) than non-tumor samples. Both epidermal growth factor receptor and sialyl Le (a) levels were overexpressed in tumors of patients with more than three positive lymph nodes (P < 0.005). Carcinoembryonic antigen levels were higher in tumors associated with allelic wild type group (P = 0.0001) and patients with negative lymph nodes (P < 0.05). Furthermore, variability in expression of biomarkers was observed according to sample location, and allelic alterations were also found both in tumor and in some non-tumor samples. CONCLUSION: The data suggest that overexpression of tissue biomarkers associated with allelic alterations may have potential prognostic implications with different behavior in esophagus cancer.

Increased mitochondrial respiratory chain enzyme activities correlate with minor extent of liver damage in mice suffering from erythropoietic protoporphyria.

Mitochondrial dysfunction might play a role in the pathogenesis of liver damage in erythropoietic protoporphyria (EPP). Changes in mitochondrial respiratory chain activities were evaluated in the Fech(m1pas)/Fech(m1pas) mouse model for EPP. Mice from different strains congenic for the same ferrochelatase germline mutation manifest variable degrees of hepatobiliary injury. Protoporphyric animals bred into the C57BL/6J background showed a higher degree of hepatomegaly and liver damage as well as higher protoporphyrin (PP) accumulation than those bred into the SJL/J and BALB/cJ backgrounds. Whereas mitochondrial respiratory chain activities remained unchanged in the liver of protoporphyric mice C57BL/6J, they were increased in protoporphyric mice from both SJL/J and BALB/cJ backgrounds, when compared to wild-type animals. Mitochondrial respiratory chain activities were increased in Hep G2 cell line after accumulation of PP following addition of aminolevulinic acid. As a direct effect of these elevated mitochondrial activities, in both hepatic cells from mutant mouse strains and Hep G2 cells, adenosine 5'-triphosphate (ATP) levels significantly increased as the intracellular PP concentration was reduced. These results indicate that PP modifies intracellular ATP requirements as well as hepatic mitochondrial respiratory chain enzymatic activities and further suggest that an increase of these activities may provide a certain degree of protection against liver damage in protoporphyric mice.

Hemoglobin content is increased in the human erythroleukemia K562 cell line by a 56.2-kD peptide from uremic plasma.

BACKGROUND: Some abnormalities in porphyrin metabolism have been described in erythrocytes from patients with end-stage renal failure. A peptidic fraction of 56.2 kD isolated from plasma of these patients was previously identified as an aminolevulinate dehydratase inhibitor. The aim of this study was to examine the in vitro effect of this peptide on heme synthesis in the erythroleukemia K562 cells. METHODS: The 56.2-kD fraction was purified from uremic plasma by protein electroelution and, its action on the mitochondrial rate-limiting steps of heme synthesis, as well as the hemoglobin content during erythroid differentiation induced by sodium butyrate, was investigated in K562 cells. RESULTS: Two hours after addition of the 56.2-kD peptide, the activities of aminolevulinate acid synthase and aminolevulinate dehydratase were reduced while the activity of the ferrochelatase was enhanced, indicating that this peptide easily across the membranes. A 3-day incubation with this peptide enhanced approximately twofold the hemoglobin and porphyrin levels during erythroid differentiation of K562 cells without variation of cell growth. CONCLUSION: This study shows that the addition of the 56.2-kD uremic factor to K562 cells was clearly implicated in heme disturbances existing in chronic renal failure but it did not play a negative role in the pathogenesis of the uremic anemia.