Glucose-6-phosphate dehydrogenase (G6PD) deficiency, caused by pathogenic variants in the G6PD gene, is the most common X-linked enzymopathy in the world. The clinical manifestations range from drug-induced hemolytic anemia to neonatal hyperbilirubinemia and chronic hemolysis. The G6PD status needs to be accurately assessed in several clinical settings, including drug prescription, assisted reproductive technologies, and the diagnostic workflow of hemolytic anemia. In this study, the contribution of molecular analysis in the diagnostic workflow of patients with suspected G6PD deficiency was retrospectively evaluated over one year. A total of 340 samples were analyzed for the enzymatic activity of G6PD, including 28 organ donors, 62 pediatric patients, 157 hospitalized patients, and 93 referred from the Genetics, Internal Medicine, Gastroenterology, and Hematology units. Molecular analysis was performed for 52 cases (17 males and 35 females): 37 with reduced enzymatic activity, five with inconclusive results due to recent blood transfusions, and 10 with specific clinical indications. Through molecular testing, eight previously reported and five novel disease-causing variants (c.769 C > T, p.(Arg257Gly); c.488G > T, p.(Gly163Val); c.1124 A > G, p.(Asn375Gly); c.1152G > C p.(Gln384His); c.582 C > G, p.(Asp194Glu)) were identified in 36 patients, along with three common polymorphic variants in 11 other patients. No variants were identified in five patients, consistent with borderline or difficult-to-interpret activity results. Overall, lower G6PD activity was found in 66 of 340 samples (19.4

Porphyrias are a group of diseases that are clinically and genetically heterogeneous and originate mostly from inherited dysfunctions of specific enzymes involved in heme biosynthesis. Such dysfunctions result in the excessive production and excretion of the intermediates of the heme biosynthesis pathway in the blood, urine, or feces, and these intermediates are responsible for specific clinical presentations. Porphyrias continue to be underdiagnosed, although laboratory diagnosis based on the measurement of metabolites could be utilized to support clinical suspicion in all symptomatic patients. Moreover, the measurement of enzymatic activities along with a molecular analysis may confirm the diagnosis and are, therefore, crucial for identifying pre-symptomatic carriers. The present review provides an overview of the laboratory assays used most commonly for establishing the diagnosis of porphyria. This would assist the clinicians in prescribing appropriate diagnostic testing and interpreting the testing results.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive hemolytic anemia caused by mutations in G6PD gene. The distribution and frequency of genetic variants differ depending on ethnicity and geographical areas. Because of new migrations different variants are now present in Europe. This retrospective study aims to identify variants among the G6PD deficient subjects referred since 2004 to IRCCS Ca’ Granda Foundation Hospital in Milan. The subjects were divided into 3 groups: group 1 (2004–2008), group 2 (2009–2013), and group 3 (2014–2018). During 15 years a significant decrease of the Mediterranean and an important increase of the African, Asian, and uncommon variants (classified as Others) have been observed. Three new mutations were found: in group 2 heterozygosity for c.[1454G > A] (Gly485Asp) in an adult female with severe anemia, high bilirubin levels and G6PD activity of 0,69 (IU/gHb) and heterozygosity for c.[584A > G] (Gln195Arg) in an elderly woman of Italian origin showing only anemia and enzymatic activity of 1,54 (IU/gHb) were detected. In group 3 hemizygosity for c.[670A > T] (Ile224Phe) in an adult Chinese man without anemia but with total absence of G6PD activity was found. These data reflect the appearance of uncommon G6PD mutations in northern Italy, probably due to new migrations, as consequence G6PD characterization becomes a diagnostic issue.

To the Editor: Gilbert's syndrome (GS) is a condition characterized by intermittent unconjugated hyperbilirubinemia without structural liver damage, affecting about 10% of the caucasian population. It is an autosomal recessive disorder, mainly associated with variations in uridine50-diphosphate (UDP)-glucuronosyltransferase gene (UGT1A1). In Caucasians, the most common variation is the TATA box polymorphism, in which an insertion of an additional TA-repeat into the promoter region of the gene results in a A(TA) 7TAA sequence, that differs from the more prevalent A(TA) 6TAA. Gilbert's syndrome is a benign condition which does not lead to liver inflammation, cellular destruction, fibrosis, or cirrhosis but is merely clinically characterized by intermittent episodes of uncomplicated unconjugated hyperbilirubinemia. The effect of GS on liver complication and outcome of hematological malignancies has been scarcely investigated, with only a previous study in a series of children diagnosed with acute lymphoblastic leukemia, a series of adult patients with Hodgkin lymphoma and some anecdotal case reports in acute leukemia patients. Tyrosine-kinase inhibitors (TKIs) are currently used for the treatment of chronic myeloid leukemia (CML). Nilotinib inhibits bilirubin metabolism via UGT1A1, thereby increasing bilirubin levels; not surprisingly, GS has been associated with nilotinib-induced hyperbilirubinemia in patients affected by CML. Similar increases in bilirubin were also reported in patients with GS treated with imatinib or dasatinib. The aim of the present study is to assess if in CML-chronic phase (CML-CP) patients treated with either firstor second-generation TKIs, GS has an impact on clinical outcomes, assessed by cytogenetic/molecular response rates and progression-free survival (PFS), as well as on hematological or extra-hematological toxicities. We retrospectively collected data on CML-CP patients consecutively treated with TKIs at our institution between February 2002 and November 2018. All the following data were collected at baseline before TKI initiation: sociodemographic and hematological variables, disease risk scores (Sokal, Eutos, Hasford, and ELTS), and TKI starting dose. Monitoring and responses evaluation followed the current European LeukemiaNet recommendations. Progression-free survival was calculated from the start of first-line TKI to any of the following events: primary or secondary resistance, and/or discontinuation due to intolerance. Hematological and extra-hematological toxicities were graded according to the Common Toxicity Criteria Adverse Events (CTCAE) version 5.0. Gilbert's syndrome genotype was investigated by polymerase chain reaction (PCR) amplification of a region encompassing the TATA-box of the UGT1A1 gene (forward primer: 5’-GTC ACG TGA CAC AGT CAA AC-30: reverse primer: 50-TTT GCT CCT GCC AGA GGT-30; annealing temperature: 62°C. RefSeq UGT1A1 NG_033238.1). DNA fragment length analysiswas performed by 12%polyacrylamide gel electrophoresis (PAGE): a 98 bp fragment indicating the presence of the (TA)6TAA wildtype allele, while a 100 bp fragment accounts for the (TA)7TAA allele (c.-41_-40dupTA). The more rare (TA)8TAA allele (c.-43_-40dupTATA) could be as well evidenced by this technique as a 102 bp PCR product, but it was not detected in any of the patient studied. Clinical and sociodemographic characteristics were described using absolute and relative frequencies. We evaluated time to resistance (truncated at 10 years) according to GS genotype by calculating the Kaplan-Meier function and by fitting univariate and multivariable Cox models adjusted for gender, age, and line of therapy. P values were obtained from post-estimation global Wald tests. Statistical analysis was performed with Stata 15 (StataCorp. 2017). One hundred and five CML-CP patients consecutively treated with either firstor second-generation TKIs were evaluated. Clinicaldemographic data at diagnosis are showed in Table S1. In particular, concomitant drugs were reported in 49 patients (46.7%), with seven patients taking more than five medications. Gilbert's syndrome genotypes were distributed as follows: 17 (16.2%) patients were 7/7, 44 (41.9%) 6/7 and the remaining cases were wild-type. The majority of the patients were treated with imatinib (69.5%), following by nilotinib (17.1%) and dasatinib (13.3%). Complete cytogenetic response (CCyR) was obtained in 79 (75.2%) patients, of whom 55 (52.4%) were within 3 months of treatment. Among 7/7, 6/7 and 6/6 genotypes, CCyR was achieved by 12 (70.6%), 31 (70.4%) and 29 (65.9%) patients, respectively. Major molecular response (MMR) was obtained in 73 patients (69.5%); among them eight (47%) were 7/7, 23 (52.3%) 6/7 and 20 (45.4%) 6/6. Deep molecular response (DMR) was achieved in 51 (48.6%) patients, of whom 8 (47.1%), 23 (52.3%) and 20 (45.4%) showed a 7/7, 6/7, or 6/6 genotype, respectively. Interestingly, none of these differences was statistically significant. Forty-seven patients switched to second-line therapy, of whom 18 (38.3%) for primary resistance, 14 (29.8%) for secondary resistance and 15 (14.3%) for intolerance or unacceptable toxicity. Among patients who experienced primary resistance, two (22.2%) were 7/7, seven (36.8%) were 6/7 and nine (45%) wild-type. A similar distribution was also recorded among patients who switched for toxicity. Received: 4 July 2019 Revised: 23 July 2019 Accepted: 24 July 2019

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive hemolytic anemia caused by G6PD gene mutations which affect 400 million people worldwide. To date, more than 140 G6PD mutations have been reported. The distribution and frequency of genetic variants differ depending on ethnicity and geographical areas. Because of new migrations different variants are now present in Europe. Aims: This retrospective study aims to identify variants among the G6PD deficient subjects referred since 2004 to “Ca’ Granda” Foundation Hospital “in Milan. The subjects were divided into 3 groups: group 1 (2004–2008), group 2 (2009–2013) and group 3 (2014–2018).These subjects were studied because of known or suspected G6PD deficiency or for genetic counselling. Methods: The molecular analysis has been carried out by restriction fragments length polymorphisms (RLFP) and direct DNA sequencing. Results: Three hundred and thirty nine G6PD mutated subjects were identified, classified and divided as shown in table 1:Table 1 - G6PD variants identified in glucose 6-phosphate dehydrogenase-deficient subjects diagnosed since 2004 (number of subjects with G6PD mutations in italic, percentage % in brackets). During 12 years a significant decrease of the Mediterranean and an important increase of the African, Asian and uncommon variants (classified as Others) have been observed. In the first eight years Cassano and Chatham variants have an increase that does not change in the last four years. During each period new mutations were found: in group 1 the Gly485Asp (c.[1454G> A]) in homozygosity (female), in group 2 the Glu195Arg (c.[584A> G]) in heterozygosity and in group 3 the Ile224Phe (c.[670A>T]) in hemizygosity. Referring to the entire period of the study 2004–2018, in 3 subjects (1.1%) were found Portici variant, responsible for chronic non-spherocytic haemolytic anaemia, as well as Puerto Limon and Georgia (0,4%). Other variants identified in less than 1% of subjects studied were: Cairo, Mahidol, Ludhiana, Rignano, Santamaria, Sant’Antioco, Canton, Sibari, Cosenza, Abruzzo, Viangchan, Orissa, and Union. Two cases showed the 1365–13T>C polymorphism coupled with 1311C>T variation, generally associated with Mediterranean variant. Summary/Conclusion: These data reflect the appearance of uncommon G6PD mutations in Northern Italy, probably due to new migrations, as consequence G6PD characterization becomes a diagnostic issue. The evidence of polymorphisms in G6PD deficient subjects suggests that not only single variations in the exonic/intronic boundaries, but also specific haplotypes could be causative for G6PD deficiency.

American Journal of HematologyVolume 93, Issue 11 p. E365-E368 E-ONLY ARTICLEFree Access Circulating cell-free DNA and ineffective erythropoiesis in nontransfusion-dependent β-thalassemia Dario Tavazzi, Dario Tavazzi Università degli Studi di Milano, Dip. Scienze Cliniche e di Comunità, Milan, ItalySearch for more papers by this authorIrene Motta, Irene Motta orcid.org/0000-0001-5701-599X Fondazione IRCCS 'Cà Granda' Ospedale Maggiore Policlinico, UO Medicina Interna, Milan, ItalySearch for more papers by this authorGiovanna Graziadei, Giovanna Graziadei Fondazione IRCCS 'Cà Granda' Ospedale Maggiore Policlinico, UO Medicina Interna, Milan, ItalySearch for more papers by this authorMaurizio Sampietro, Maurizio Sampietro Università degli Studi di Milano, Dip. Fisiopatologia Medico-Chirurgica e dei Trapianti, Milan, ItalySearch for more papers by this authorLorena Duca, Lorena Duca Fondazione IRCCS 'Cà Granda' Ospedale Maggiore Policlinico, UO Medicina Interna, Milan, ItalySearch for more papers by this authorMaria Domenica Cappellini, Corresponding Author Maria Domenica Cappellini maria.cappellini@unimi.it orcid.org/0000-0001-8676-6864 Fondazione IRCCS 'Cà Granda' Ospedale Maggiore Policlinico, UO Medicina Interna, Milan, Italy Università degli Studi di Milano, Dip. Scienze Cliniche e di Comunità, Milan, Italy Correspondence Maria Domenica Cappellini, Dipartimento di Scienze Cliniche e di Comunità, Università degli Studi di Milano, Fond. IRCCS Cà Granda Policlinico, Milan, Italy. Email: maria.cappellini@unimi.itSearch for more papers by this author Dario Tavazzi, Dario Tavazzi Università degli Studi di Milano, Dip. Scienze Cliniche e di Comunità, Milan, ItalySearch for more papers by this authorIrene Motta, Irene Motta orcid.org/0000-0001-5701-599X Fondazione IRCCS 'Cà Granda' Ospedale Maggiore Policlinico, UO Medicina Interna, Milan, ItalySearch for more papers by this authorGiovanna Graziadei, Giovanna Graziadei Fondazione IRCCS 'Cà Granda' Ospedale Maggiore Policlinico, UO Medicina Interna, Milan, ItalySearch for more papers by this authorMaurizio Sampietro, Maurizio Sampietro Università degli Studi di Milano, Dip. Fisiopatologia Medico-Chirurgica e dei Trapianti, Milan, ItalySearch for more papers by this authorLorena Duca, Lorena Duca Fondazione IRCCS 'Cà Granda' Ospedale Maggiore Policlinico, UO Medicina Interna, Milan, ItalySearch for more papers by this authorMaria Domenica Cappellini, Corresponding Author Maria Domenica Cappellini maria.cappellini@unimi.it orcid.org/0000-0001-8676-6864 Fondazione IRCCS 'Cà Granda' Ospedale Maggiore Policlinico, UO Medicina Interna, Milan, Italy Università degli Studi di Milano, Dip. Scienze Cliniche e di Comunità, Milan, Italy Correspondence Maria Domenica Cappellini, Dipartimento di Scienze Cliniche e di Comunità, Università degli Studi di Milano, Fond. IRCCS Cà Granda Policlinico, Milan, Italy. Email: maria.cappellini@unimi.itSearch for more papers by this author First published: 13 August 2018 https://doi.org/10.1002/ajh.25248Citations: 1AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abbreviations SPX splenectomized cfDNA circulating cell-free DNA (ng/mL) EPO erythropoietin (mU/mL) sTfR soluble transferrin receptor (μg/mL) GDF15 growth differentiation factor 15 (pg/mL) Ca15.3 cancer antigen 15.3 (U/mL) Hb hemoglobin (g/dL) RBC red blood cells (x106/mm3) Retics retyculocytes (x106/mm3) EBL erythroblasts (x103/mm3) Fe serum iron (μg/dL) Tf transferrin (mg/dL) SF serum ferritin (ng/mL) TS transferrin saturation (%) ALT alanine transaminase (mU/mL) AST aspartate transaminase (mU/mL) LDH lactate dehydrogenase (mU/mL) BIL TOT/IND total and conjugated bilirubin (mg/dL). To the Editor: Circulating cell-free DNA (cfDNA) is a pool of single- or double-stranded DNA fragments that originate from apoptotic/necrotic tissue cells or from cells released and lysed into the bloodstream. Small amounts of cfDNA are detectable in plasma of healthy individuals, while increased levels are observed in pathologic conditions, including cancer, trauma, stroke, chronic inflammation and infection, myocardial ischemia, and autoimmune diseases, proportionally to clinical severity.1 Hematopoietic cells are likely to contribute to a certain amount of measurable cfDNA, being in close contact with the plasma moiety. Consistently, it has been reported that cfDNA after sex-mismatched bone-marrow transplantation is mostly of hematopoietic origin.2 Recently, it has been shown that either white blood cells or erythroid cells significantly contribute to circulating cfDNA and that the erythroid pool accounts for approximately 30% of it.3 Circulating cfDNA was described in sickle cell disease (SCD) as a valuable marker of increased cellular turnover and chronic hemolysis; it is considered a good candidate marker to confirm and monitor vaso-occlusive crises and the response to hydroxyurea therapy.4 Preliminary data obtained in anemias of different etiologies suggest also that circulating erythroid DNA could reflect the erythropoietic activity in the bone marrow3 and hence represent a noninvasive biomarker of erythropoiesis. However, no data are available in the literature about cfDNA and ineffective erythropoiesis (IE). IE is the hallmark of β-thalassemia syndrome. This is the most clinically relevant hemoglobinopathy in the Mediterranean and Middle Eastern regions, and its severity strongly relates with the extent of the imbalance between α- and β-chains of hemoglobin (Hb).5 The aim of this study was to evaluate cfDNA in nontransfusion-dependent β-thalassemia (β-NTDT) patients, and to investigate its possible role as biomarker of IE in clinical practice. Forty-nine adults with β-NTDT (22M/27F, aged between 16 and 65 years) were enrolled. The diagnosis of β-NTDT was based on clinical criteria. All subjects had not received any blood transfusion during the previous 3 months and had no evidence of tumor, trauma, inflammation, or autoimmune disease at the time of blood collection. According to the severity of mutation in the β-globin gene, patients were divided into mild (β+/β+) and moderate-severe (β0/β+) groups. Twenty-three (12M/11F) out of 49 patients were splenectomized. Eighteen healthy subjects acted as controls. The study was approved by the Ethics Committee of the Fondazione IRCCS Ca' Granda Policlinico in Milan; enrolled subjects gave their informed written consent. We evaluated (1) markers of erythropoiesis: erythropoietin (EPO), soluble transferrin receptor (sTfR) and growth differentiation factor 15 (GDF-15),6 (2) plasma cfDNA concentration, and (3) routine biochemical and hematological parameters, including iron status, hemolysis markers (ie, lactate dehydrogenase [LDH] and aspartate transaminase [AST]) and cancer antigen 15.3 (CA15.3), a known marker of IE.7 Statistical analysis was performed with nonparametric Mann-Whitney U test and Spearman's rank correlation test. We compared hematological, iron parameters and markers of IE between splenectomized and nonsplenectomized patients. As reported in previous works, splenectomized patients showed a more enhanced IE (Table 1). Table 1. Hematologic parameters, plasma cell-free DNA and indicators of erythropoiesis in non transfusion-dependent beta-thalassemia patients (descriptive statistics) Figure 1Open in figure viewerPowerPoint Correlations between markers of erythropoiesis and plasma cfDNA in nontransfusion-dependent β-thalassemic patients; data are reported for nonsplenectomized () and splenectomized () patients. A, sTfR vs cfDNA, total: y = .042x + 7.853; R = .436, P = .0022; splenectomized (SPX): y = .025x + 8.629; R = .475, P = .0253. B, growth differentiation factor 15 (GDF15) vs cfDNA, total: y = 340x + 4072; R = .554, P = .0001; SPX: y = 305x + 7442; R = .518, P = .0135. C, EPO vs cfDNA, total: y = .681x + 87.013; R = .071, P = .6249; SPX: y = −.188x + 117.101; R = 0.032, P = .9144. D, EBL vs cfDNA, total: y = .748x–10.425; R = .689, P < .0001; SPX: y = .862x–7.818; R = .754, P < .0001 Small amounts of cfDNA were observed in healthy subjects (median 10.39 [3.85-15.81] ng/mL) while cfDNA concentration was significantly higher in β-NTDT patients (21.84 [6.33-93.10] ng/mL, P < .0001). cfDNA was higher in splenectomized group (median 29.45 [10.41-93.10] ng/mL) than in nonsplenectomized (19.35 [6.33-59.88] ng/mL) patients (P = .0085). Cell-free DNA correlated with markers of IE, namely sTfR (R = .436, P = .0022), GDF-15 (R = .554, P < .0001) and CA15.3 (R = .454, P = .0010), while no significant correlation was observed with EPO concentrations (Figure 1). Similarly, considering hemolysis markers, LDH showed a positive correlation with cfDNA (R = .511, P = .0002); furthermore, AST and alanine transaminase were higher in splenectomized patients and positively correlated with plasma cfDNA (R = .557, P < .0001, and R = .326, P = .0223, respectively). Considering all the β-NTDT patients, erythroblasts (EBL) count showed positive correlation (R = .689, P < .0001) with cfDNA and this correlation remained significant in the splenectomized group (R = .754, P < .0001; Figure 1, lower panel). Taking into account genotypes of β-NTDT patients, in the moderate-severe group we observed higher EBL count (P < .0001), serum ferritin (P = .0058), AST (P = .0052), and LDH (P = .0356), suggesting a more pronounced IE. Consistently, in the moderate-severe group plasma cfDNA was significantly higher (median 29.44 [10.41-93.10] vs 20.56 [6.33-75.43] ng/mL, P = .0314) regardless of splenectomy. In β-thalassemia syndromes the impaired production of β-globin chains causes the accumulation of free α-globin chains in erythroid cells, generating hemichromes that cause oxidative stress damage and cell death, leading to IE and chronic hemolytic anemia. The diversity of β-globin gene mutations and the consequent variable degree of α/β-globin chain imbalance are the main determinants of phenotype severity. In the past, splenectomy was generally used to increase total Hb and to overcome blood transfusion therapy. Conversely, nowadays splenectomy is recommended only for selected cases with significant hypersplenism and mechanical issues due to massive organomegaly. As previously described, we demonstrate here that IE is greater in splenectomized than in nonsplenectomized patients. This might be related to either a more severe disease (requiring splenectomy), or a reduced "filter" by the spleen, resulting in more circulating erythroid precursors. Increased iron overload in splenectomized patients, in the presence of similar Hb levels, further support the concept of greater IE. Plasma cfDNA concentration was higher in β-NTDT patients compared to controls and, among β-NTDT patients, cfDNA was higher in the splenectomized group and in the group with more severe genotypes. Moreover, cfDNA concentration correlated with parameters of IE such as erythroblasts count and sTfR, as well as with the indirect markers GDF-15 and CA15.3. Therefore, plasma cfDNA concentration may be a suitable clinical marker of erythropoiesis in β-NTDT patients. Our results need to be confirmed in a larger cohort of patients. Of note, the method we used for measuring cfDNA consisted of DNA extraction from plasma and quantification by the PicoGreen fluorescent dye, which resulted a reproducible, inexpensive, and fast technique. Thus, cfDNA could represent a candidate marker of IE beyond those commonly used. Furthermore, to establish its potential use as marker of IE, cfDNA should be evaluated in patients under treatment with new experimental drugs that target IE in β-thalassemia.8 ACKNOWLEDGEMENTS This work was supported by grants from Ministero dell'Istruzione, Università e Ricerca (PRIN 2012 Nr. 20128PNX83) and by Fondazione IRCCS Cà Granda Ospedale Policlinico (Ricerca Corrente Nr. 099/02). CONFLICT OF INTEREST None of the authors declared any conflict of interest regarding matters pertinent to the current manuscript. AUTHOR CONTRIBUTIONS DT performed laboratory analysis, collected and analyzed data, wrote the paper. IM followed patients, wrote and revised the paper for intellectual content. GG followed patients and revised the paper for intellectual content. LD performed laboratory analysis, collected and analyzed data. MS revised the paper for intellectual content. MDC planned the study, followed patients and revised the paper for intellectual content. REFERENCES 1Gormally E, Caboux E, Vineis P, Hainaut P. Circulating free DNA in plasma or serum as biomarker of carcinogenesis: practical aspects and biological significance. Mutat Res. 2007; 635: 105- 117. 2Lui YYN, Chik KW, Chiu RW, Ho CY, Lam CW, Lo YMD. Predominant hematopoietic origin of cell-free DNA in plasma and serum after sex-mismatched bone marrow transplantation. Clin Chem. 2002; 48: 421- 427. 3Lam WKJ, Gai W, Sun K, et al. DNA of erythroid origin is present in human plasma and informs the types of anemia. Clin Chem. 2017; 63: 1614- 1623. 4Al-Hummood S, Zueriq R, Al-Faris L, Marouf R, Al-Mulla F. Circulatimg cell-free DNA in sickle cell disease: is it a potentially useful biomarker? Arch Pathol Lab Med. 2014; 138: 678- 683. 5Gupta R, Musallam KM, Taher AT, Rivella S. Ineffective erythropoiesis: anemia and iron overload. Hematol Oncol Clin N Am. 2018; 32: 213- 221. 6Musallam KM, Taher AT, Duca L, Cesaretti C, Halawi R, Cappellini MD. Levels of growth differentiation factor-15 are high and correlate with clinical severity in transfusion-independent patients with β thalassemia intermedia. Blood Cells Mol Dis. 2011; 47: 232- 234. 7Symeonidis A, Kouraklis-Symeonidis A, Constandinidou I, et al. Increased CA-15.3 levels in the serum of patients with homozygous β-thalassaemia and sickle cell/β-thalassaemia. Br J Haematol. 2006; 133: 690- 694. 8Cappellini MD, Motta I. New therapeutic targets in transfusion-dependent and -independent thalassemia. Hematology 2017 Am Soc Hematol Educ Program. 2017; 1: 278- 283. Citing Literature Volume93, Issue11November 2018Pages E365-E368 FiguresReferencesRelatedInformation

Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by molecular abnormalities in the HMBS gene. This gene is transcribed from two promoters to produce ubiquitous and erythroid specific isoforms of porphobilinogen deaminase (PBGD). In the classical form of AIP, both isoforms are deficient, but about 5% of families have the non-erythroid variant in which only the ubiquitous isoform is affected. Only one mutation sited in the housekeeping promoter has been previously reported as causative for this form of AIP. In this study, we identified one small deletion and six nucleotide substitutions within the 5′UTR and the housekeeping promoter of HMBS gene: c.1–440_-427del14bp; c.1–421G>A; c.1–331C>T; c.1–270G>A; c.1–122T>A; c.1–103C>T; c.1–28A>C. Using luciferase reporter assays and quantitative PCR experiments, we characterized the functional role of these seven novel genetic variants demonstrating that all mutations cause a significant loss of transcriptional activity. Our investigations suggest that these nucleotide substitutions may alter critical binding sites for transcriptional factors, which confirms that these regions represent an important molecular target for pathogenesis of non-erythroid form of acute intermittent porphyria.

Background and Aims: The orphan nuclear receptor hepatocyte nuclear factor 4a (HNF4a) is a transcriptional factor playing a key role in the liver development and hepatocyte identity and function. The HNF4-driven gene expression depends from both its activator and repressor activity. Our efforts are aimed to the comprehension of the molecular mechanisms allowing soluble niche factors to induce dynamic hepatocyte responses. Methods: Endogenous or over-expressed HNF4 recruitment on target genes promoters was determined by EMSA and ChIP assays in different hepatocyte cell lines, some of which treated with thhe cytokines Wnt3a and TGFbeta. EMT-master genes, mesenchymal and epithelial gene expression was assessed by qRTPCR. Migration and invasivity assays were performed on hepatoma cells overexpressing HNF4. Results: We showed that HNF4 drives the formation of activator and repressor complexes which dynamic recruitment on promoters of several genes is crucial for: – Post-differentiative patterning of hepatocyte controlling metabolic liver zonation; in particular we demonstrated as the switch from a peri-portal to peri-venular hepatocytes in response to Wnt cytokines depends from changes in recruitment/displacement of HNF4, in different combinations with co-activators or corepressors, on/from promoters of several target genes; (Colletti et al., 2009); – Maintenance of epithelial identity; we showed that, in differentiated hepatocyte, HNF4 simultaneously activates the epithelial program and inhibits of the mesenchymal/stemness one; in particular this transcriptional factor, in cooperation with its target HNF1a, directly inhibits transcription of Snail, Slug and HMGA2 (previously demonstrated to act as EMT masters and stemness related genes) and mesenchymal markers; the relevance of HNF4a in the active repression of mesenchymal program was confirmed in the in vivo model of Hnf4a KO mice (Santangelo et al., 2011); – Induction of mesenchymal to epithelial transition in hepatomas; we provide evidence on the molecular mechanisms by which HNF4a induces MET in undifferentiated, invasive hepatoma cells reestablishing a differentiated phenotype (Santangelo et al., 2011). Conclusions: In conclusion, our efforts, aimed to the comprehension of the molecular mechanisms allowing soluble niche factors to induce dynamic hepatocyte responses, highlighted as several pathways converge on HNF4a-mediated transcription activation and repression.

Porphyrias are a group of eight rare inherited metabolic disorders of heme biosynthesis pathway. Porphyrias are still underdiagnosed, although examinations of urine and plasma are first-line tests for detecting excess of porphyrins or heme precursors in suspected patients. Diagnosis, particularly for the acute forms, is essential to avoid precipitating factors and the use of triggering drugs. Mutation screening of family members is recommended to identify presymptomatic carriers and to prevent acute attacks. The therapeutic approach should be appropriate regarding specific forms of porphyria and treatment should be started promptly.

Variegate Porphyria (VP) is an autosomal dominant disorder found worldwide but is rare in Italy. In this study we provide an overview of clinical, biochemical and genetic background of 33 Italian VP patients diagnosed in the last fifteen years. About 70% of patients had experienced clinical symptoms: 43.4% had photosensivity, 8.7% acute attacks and 47.8% both. Among the 33 patients, 14 different mutations were identified. Of these only 6 defects have been previously described in other countries and 8 are unique having been identified for the first time in Italy. Two of these, the c.851G>T and the c.1013C>G, were found in two and four unrelated families respectively. No mutation has been found in homozygosis and no significant correlation has been observed between specific clinical and biochemical manifestations and the type of mutation. In contrast, normal faecal protoporphyrin excretion was high predictive of silent phenotype. Normal urinary excretion of PBG and ALA, predicted absence of neurovisceral symptoms. This paper represents the first compilation of data on genotype-phenotype relation in Italian patients with VP.

Objectives: Aim of this study was to evaluate prevalence and characteristics of cholelithiasis in a large population of patients with thalassemia major (TM). Methods: Data from 858 consecutive patients with transfusion-dependent thalassemia at five major Italian centers were analyzed. In these centers, a complete abdomen ultrasonography is performed yearly after the beginning of the transfusion regimen. The role of co-inheriting Gilbert's syndrome genotype was investigated studying the promoter region of the UGT1-A1 gene by automated sequencing. Results: Thirty percent of TM patients had gallstones. The Gilbert's genotype [homozygosity for (TA) 7 motif at UGT1A promoter gene], influenced both the prevalence of cholelithiasis and the age at which it developed. Conclusions: Cholelithiasis has a remarkable frequency and precocity in patients with TM and especially in those with (TA)(7)/(TA)(7) UGT1-A1 genotype. An early biliary ultrasonography is recommended from childhood and a closer follow-up in patients with thalassemia and associated Gilbert's syndrome may be indicated.