ANKRD26-related thrombocytopenia (ANKRD26-RT) is characterized by lifelong mild to moderate thrombocytopenia. Patients suffer from an increased susceptibility to acute or chronic myeloid leukemia, myelodysplastic syndrome, or chronic lymphocytic leukemia. We described here a patient with inherited thrombocytopenia initially misdiagnosed as immune thrombocytopenic purpura. A chromosomal deletion involving the ANKRD26 gene was identified. Gene and protein expression analyses suggest an alternative pathogenic mechanism of altered megakaryopoiesis: the synthesis of a chimeric protein with aberrant expression due to the unregulated action of a promoter from a gene located upstream of ANKRD26. This study highlights the importance of advanced genetic testing and functional analysis of patients’ primary cells in the case of the detection of previously unrecognized structural variants in order to understand pathogenic mechanisms. These investigations provided a definitive diagnosis for the patient and facilitated the development of a tailored clinical management strategy, especially concerning the potential for myeloid transformation.

Thalassemias and hemoglobinopathies are among the most common genetic diseases worldwide and have a significant impact on public health. The decreasing cost of next-generation sequencing (NGS) has quickly enabled the development of new assays that allow for the simultaneous analysis of small nucleotide variants (SNVs) and copy number variants (CNVs) as deletions/duplications of α- and β-globin genes. Background/Objectives: This study highlighted the efficacy and rapid identification of all types of mutations in the α- and β-globin genes, including silent variants, using the Devyser Thalassemia NGS kit. Furthermore, we report the frequency of mutations identified in a total population of 2649 individuals recruited from four Italian Medical Genetics Laboratories. Methods: All samples were first hematologically characterized, and sequence analysis was conducted by using the Devyser Thalassemia NGS kit. All variants were also validated in an independent sample by a conventional molecular test. Results: A total of 1789 subjects were identified with genetic variants in the globin genes, of which 966 (53.9%) had variations in the β-gene, 480 (26.8%) had variations in the α-gene; and 307 (17.1%) had variations in both α- and β-genes. Variant analysis evidenced a heterogeneous mutation spectrum enriched with variants not usually observed in the Italian population. Conclusions: This study showed the high effectiveness and the rapid identification of all mutation types in both α- and β-globin genes, including silent variants. It should be emphasized that the NGS approach greatly shortens turnaround reporting times, overcoming the classic diagnostic flowchart which envisages multistep, subsequent, diagnostic approaches, often requiring long resolution times.

Workflow of the study with some examples of the achieved results.

Topic: 28. Enzymopathies, membranopathies and other anemias Background: Approximately 1-5% of the global population is a carrier of a thalassemia mutation. Carriers of β-thalassemia, also defined as beta thalassemia trait (BT), are generally asymptomatic with mild, microcytic hypochromic anemia. We herein described a cohort of BT carriers presenting with anemia of variable degree, splenomegaly, alteration in hemolysis indices, and liver hemosiderosis (referred as symptomatic BT) analyzed for second- and third-line investigation in the suspicion of others erythrocyte defects. Aims: The aim of the study was to evaluate if the combination of the BT and other inherited RBC disorders could account for phenotypic variability. Methods: A cohort of 20 symptomatic BT carriers from 16 unrelated families followed in the specialized center for congenital anemias of Genoa (Italy) were analyzed for the diagnosis of RBC defects at the Medical Genetic Unit of AOU Federico II/CEINGE, Naples from November 2019 to December 2022 and they were compared to a cohort of BT carriers (n = 53) without anemia, alteration of hemolysis indices and iron parameters identified during a carrier screening for hemoglobinopathies. All the symptomatic BT patients were investigated by family history, blood count, peripheral blood smear, ektacytometry curve, molecular testing for HBB/HBA, next-generation sequencing for Hereditary Anemia (HA). Results: Among 20 BT symptomatic patients, we found causative variants of HA in 15/20 (75%). Overall, 66.7% (10/15) showed monogenic HA in addition to BT, while 33.3% (5/15) showed multi-locus HA. Of note, 14 out 15 symptomatic BT carriers showed mutations in PIEZO1 gene. The variants found in BT patients were classified by automated ACMG rule and then outputs were adjusted using additional available evidence for each patient, as well as data from literature studies and from in-house cohort of DHS patients. Additional analysis as PB smear evaluation revealed the presence of abnormal morphology. Similarly, the ektacytometry curves revealed alterations in all the 15 symptomatic BT patients compared to both healthy controls and asymptomatic BT. Overall, the clinical features of BT carriers with associated HA demonstrated the presence of splenomegaly in 87% (13/15), alteration in hemolysis indices in 73% (11/15), anemia in 100% (15/15). Analysis of the complete blood count showed decreased levels of both RBC count and Hb levels in symptomatic BT carriers with HA compared to asymptomatic subjects. On the contrary, the reticulocytes count resulted slightly increased in symptomatic BT carriers compared to the symptomatic ones. Of note, symptomatic BT carriers showed markedly increased hemolysis signs compared to asymptomatic BT subjects. Interestingly, no difference of the mean corpuscular volume (MCV) between the two groups was observed. Summary/Conclusion: The analysis of the present cohort of patients demonstrated that the clinical phenotype was more severe for BT carriers with dehydrated hereditary stomatocytosis, or multi-locus inheritance compared to pure BT carriers in terms of Hb levels, splenomegaly, and hemolytic indices. Our study highlighted the importance to investigate those cases of BT carriers in which the heterozygosity for a HBB variant is not enough to explain the entire clinical phenotype, even if the association of BT trait with RBC defects is a rare condition. This allows to underline that coinheritance of BT and RBC defects should be considered as part of thalassemic syndromes because these patients should be treated as NTDT or TDT thalassemic patients in terms of management and therapeutic treatment options Keywords: Red blood cell, Hemoglobinopathy, beta thalassemia, Genetic modifiers

Topic: 12. Bone marrow failure syndromes incl. PNH - Clinical Background: TNFRSF13B/TACI is the most common molecular defect related to Common Variable Immunodeficiency Disorders (CVID) which represents a heterogeneous group of diseases characterized by antibody deficiency and very low isotype switched memory B cell in the peripheral blood and variable clinical phenotype (ierecurrentinfections, lymphoprolipheration and autoimmune cytopenia) While this disorder is surely well recognized by the immunologists, it could probably a bit underestimated within the hematological setting. Aims: To describe the frequency of TNFRSF13B/TACI in a hematological population seen at a single center mainly for autoimmune cytopenia, ALPS/ALPS like disorders (NIH 2009 criteria) or in hypogammaglobulinemia with autoimmunity. Methods: All patients with the above diagnosis, studied with an Next Generation Sequencing (NGS) panel of 162 genes, including TNFRSF13B/TACI variant were considered eligible for the present study. Clinical, biochemical, immunological and genetic data were retrieved from our Cytopenia/ALPS Registry Results: From 2015 to 2022 at Unit of Hematology of Giannina Gaslini Children Hospital, 305 patients were screened with the estended NGS panel. A total of 28 subjects (43% females) affected with predominant feature of cytopenia 14(57%), ALPS/ALPSlike syndrome 9 (28%) or with a highly suspicion of CVID phenotype 5(18%) with median age at at our first observation of 10 years (0.1-48 yrs) were carriers of TACI variants (Table 1). As for the genetic definition according to Clin Var, 6/28 (21.5%) were P/LP, 6/28 (21.5%) were VUS and the remaining 16/28 (57%) were TACI variants with Conflicting Interpretation Results. Overall in 9/25 (36%) subjects, immunology was completely normal: neither depletion of immunoglobulins or lymphocyte count reduction were observed. This silent immunulogical aspect was observed in 50% of the cytopenic subjects and in 43% of ALPS/ALPS like affected subjects. Conversely, the reduction of at least of one class of was documented in all (100%) patients with a clear CVID phenotype as expected, while was shown in 50% and in 17% of patients respectevely affected with ALPS/ALPS like and citopenia (p=0.003). Lymphocytopenia at last FUP was observed in 46% of cytopenia subgroup, in 17% of ALPS/ALPS like group and in 40% of CVID affected subjects (p=ns). The impact of the type of TACI variants on hypogammaglobulinemia and/or lympocytopenia was not evident. Summary/Conclusion: In the present study, TACI variants studied in a cohort of patients affected with cytopenia and ALPS/ALPS like disorders are found in around 10 % of the subjects. The immune-dysregulation expressed by the described phenotype found in TACI the probable explanation. Unexpectedly in a high proportion of the subjects the typical immunological features of CVID including immunoglobulin and lymphocyte count depletions are not found. This observation encourages the application of diagnostic panels including TACI variants considering in ALPS/ ALPs like syndrome or of chronic/refractory cytopenias. These findings would surely deserve confirmation in prospective studies composed by larger numbers of patients.Keywords: Bone marrow failure

American Journal of HematologyVolume 97, Issue 9 p. E328-E331 CORRESPONDENCEFree Access TACI variants as underlying condition in autoimmune neutropenia: Description of four cases Francesca Fioredda, Corresponding Author Francesca Fioredda [email protected] orcid.org/0000-0002-7096-4580 Hematology Unit–IRCCS Istituto Giannina Gaslini, Genoa, Italy Correspondence Francesca Fioredda, Hematology Unit, IRCCS Istituto Giannina Gaslini, Largo G Gaslini 5, 16147 Genova, Italy. Email: [email protected]Search for more papers by this authorAndrea Beccaria, Andrea Beccaria Hematology Unit–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorElena Turrini, Elena Turrini Hematology Unit–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorPietro Casartelli, Pietro Casartelli Hematology Unit–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorDomenico Coviello, Domenico Coviello UOSD Genetics and Genomics of Rare Diseases, IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorMassimo Maffei, Massimo Maffei UOSD Genetics and Genomics of Rare Diseases, IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorMarina Lanciotti, Marina Lanciotti Hematology Unit–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorMichela Lupia, Michela Lupia Hematology Unit–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorPaola Terranova, Paola Terranova Hematology Unit–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorAlice Grossi, Alice Grossi Laboratory of Human Genetics–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorIsabella Ceccherini, Isabella Ceccherini Laboratory of Human Genetics–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorMaurizio Miano, Maurizio Miano orcid.org/0000-0002-9816-1704 Hematology Unit–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorCarlo Dufour, Carlo Dufour Hematology Unit–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this author Francesca Fioredda, Corresponding Author Francesca Fioredda [email protected] orcid.org/0000-0002-7096-4580 Hematology Unit–IRCCS Istituto Giannina Gaslini, Genoa, Italy Correspondence Francesca Fioredda, Hematology Unit, IRCCS Istituto Giannina Gaslini, Largo G Gaslini 5, 16147 Genova, Italy. Email: [email protected]Search for more papers by this authorAndrea Beccaria, Andrea Beccaria Hematology Unit–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorElena Turrini, Elena Turrini Hematology Unit–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorPietro Casartelli, Pietro Casartelli Hematology Unit–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorDomenico Coviello, Domenico Coviello UOSD Genetics and Genomics of Rare Diseases, IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorMassimo Maffei, Massimo Maffei UOSD Genetics and Genomics of Rare Diseases, IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorMarina Lanciotti, Marina Lanciotti Hematology Unit–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorMichela Lupia, Michela Lupia Hematology Unit–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorPaola Terranova, Paola Terranova Hematology Unit–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorAlice Grossi, Alice Grossi Laboratory of Human Genetics–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorIsabella Ceccherini, Isabella Ceccherini Laboratory of Human Genetics–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorMaurizio Miano, Maurizio Miano orcid.org/0000-0002-9816-1704 Hematology Unit–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this authorCarlo Dufour, Carlo Dufour Hematology Unit–IRCCS Istituto Giannina Gaslini, Genoa, ItalySearch for more papers by this author First published: 10 June 2022 https://doi.org/10.1002/ajh.26625Citations: 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 To the Editor: Dr Bergman and colleagues have recently described a case of refractory autoimmune neutropenia (AINP) in a patient carrying the p.A181E (NM_012452) variant of the TACI gene.1 The patient had a very severe clinical manifestation, including AINP refractory to granulocyte colony-stimulating factor (G-CSF), which was complicated by pure red cell aplasia (PRCA). The authors have highlighted the rarity of the association between an apparent acquired disorder (AINP) and an inherited condition of immune dysregulation.1 The transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), a member of the tumor necrosis factor receptor superfamily (TNF(R)SF), promotes T-cell-independent antibody responses and plasma cell differentiation and counteracts BAFF-driven B cells. Defects in TNF(R)SF members were first described in common variable immune deficiency and selective immunoglobulin A deficiency, but the spectrum of disorders attributed to TNF(R)SF has widened, involving benign lymphoproliferation and an increased autoimmunity rate.2 Abnormal TACI signaling is frequently related to autoimmune "signature," as shown by the tendency to develop autoimmune cytopenia, which is 3.34-fold higher in patients with TACI mutation than that in the healthy population.2, 3 Increasing evidence suggests that some autoimmune disorders, including cytopenia, represent an early sign of primary immune dysregulation disorders (PIRD). In pediatric patients, a subgroup of inborn errors of immunity, recently named PIRD, has been shown to play a relevant role in the development of autoimmune cytopenia, which is characterized by abnormal functioning of the immune system without severe immunodeficiency, lymphoproliferation, autoimmune disorders, and predisposition to malignancies.4, 5 In the case of an attenuated clinical phenotype, these disorders may be easily underestimated, particularly in adults.6 However, in pediatric patients, AINP or other mono-/multi-lineage autoimmune cytopenias appear to be epiphenomena of immunedysregulation.7 For example, cytopenia can be caused by impaired marrow cell production, as observed in aplastic anemia (AA) and PRCA, or increased destruction of peripheral blood cells, as seen in Evans' syndrome.8, 9 The case reported by Bergman gives us an opportunity to share the experience of the Pediatric Hematology Unit of the IRCCS, G. Gaslini Research Hospital on autoimmune/idiopathic neutropenia, which was initially considered an acquired disorder but was eventually shown to be an immune dysregulation associated with TACI variants. From 2012 to 2022, 58 patients affected by chronic AINP underwent complete workup, including a dedicated genetic panel. Four of them (one female) were found to have mutated TACI variants. The median age at first observation was 109 months (range, 108–144 months). All patients were in good clinical condition after a follow-up of 30 months (range, 20–90 months) and are currently being followed up at a median age of 174 months (range, 156–324 months). Data were collected according to the Italian Neutropenia Registry (INR) rules approved by the local ethics committee. Three of four patients had "classical" AINP with positivity for indirect anti-neutrophil antibody. Patient 2 tested negative on the indirect anti-neutrophil antibody test using two consecutive samples. Therefore, according to the INR definition, the patient was considered to have "idiopathic neutropenia." Given the low sensitivity of the method used (indirect antibodies tests by GIFT), Patient 2 could have been diagnosed with classical AINP on repeat testing.10 All patients presented with mild or moderate clinical phenotypes. In Patient 2, osteomyelitis in the absence of immunoglobulin depletion or other immunodeficiency features was the reason for the first hospital admission. Patients 3 and 4 had a history of mild recurrence of upper respiratory tract infections and diarrhea during early infancy (Table 1). TABLE 1. Clinical, immunological, and genetic features of the cohort Patient no. PT1 PT2 PT3 PT4 Sex Male Male Male Female Date of birth July 31, 1995 May 28, 2007 June 10, 2009 February 20, 2008 Age at first observation (years, months) 9 years 12 years 9 years 2 months 9 years Length of follow-up (years) 7.5 2.4 2.7 1.7 Neutropenia type Autoimmune Np Idiopathic Np Autoimmune Np Autoimmune Np Clinical signs No clinical signs Osteomyelitis Recurrent URTIa in infancy Episodes of diarrhea Genotype/variant found in heterozigosity c.579C>A p.Cys193Ter c.260T>A p.Ile87Asn c.118T>C p.Trp40Arg c.542C>A p.Ala181Glu Median leukocytes value ×103/μl 1.84 3.50 3.83 2.85 Median neutrophils value ×103/μl 0.44 1.22 1.47 1.335 Lymphocytes median values ×103/μl 1.0 1370 1.65 1.123 Total lymphocytes ×103/μl 1.18 1.47 1.65 1.25 B-lymphocytes (%) 9.7↓ 21.44 8.09↓ 17.8 Transitional (CD27−CD10++CD38++) 0.5↓ 0.7↓ 11.2↑ – Naive (CD27−CD10+CD38+IgD+) 54.9↓ 84.6↑ 72.6 – Marginal zone (CD27+IgD+IgM+) 24.6↑ 10.3↑ 10.1↑ – Switched memory (CD27+IgD−IgM−) 5.8↓ 5.4↓ 0.4↓ – Preswitched memory (CD27+IgD−IgM+) 0 0.1 0.4 – IgD memory (CD27+IgD+IgM−) 0 0.4 0 – Antibody-secreting cells 0.5↓ 1 0.2↓ – Double negative (DN) (CD27−IgD−) 13.5↑ 13.8↑ 4.4 – B memory (%) 31.3 9.9↓ 7.2↓ 17.9 T lymphocytes (%) 78.8 73.8 77,62 68.8 Absolute CD4+ 550 545 699 456 Absolute CD8+ 238 373 465 208↓ T reg (%) 0.5↓ 0.5↓ 0.3↓ 0.7 NK cells (%) 9.3↓ 6.28↓ 12.1 14.2 Immunoglobulin levels (mg/dl) IgG 1056 724 371↓ 1301 IgA 217 130 32↓ 218 IgM 78 68 15↓ 77 IgG1 593 436 278 1050 IgG2 451 230 81 357 IgG3 41.9 28.9 43.1 63.2 IgG4 426 412 22.2 47.1 Marrow cellularity/maturation Normal Not available Not done Normal Status/current therapy Alive/none Alive/none Alive/none Alive/none a URTI: upper respiratory tract infections; in all cases, G-CSF is recommended only in cases of severe infections. Neutropenia was mild in all patients, except in Patient 1, who had severe neutropenia (0.44 × 103/μl). Leukopenia was present in all patients since the beginning of their clinical history (median white blood cell count: 3.17 × 103/μl, range, 1.84–3.83 × 103/μl) and was associated with borderline low lymphocyte count in Patient 1. No association with thrombocytopenia or hemolytic anemia was observed over time. G-CSF and other immunosuppressants were never administered. No association with other signs or biomarkers of autoimmunity or subsequent appearance of other cytopenias has been observed in this cohort to date. The immunological pattern of our cohort did not show the expected exhaustion of immunoglobulins; however, lower levels of absolute B cell count were observed in two of four patients. In the three patients in whom B cell subpopulations were studied, the number of marginal zone cells (CD27+IgD+IgM+) was increased compared to reference values, while the number of switched memory cells (CD27+IgD−IgM−) and memory B cells were below the normal range. The number of double-negative (DN) B cells (CD27−IgD−) was increased compared to normal values in two of three patients. The number of CD4+ and CD8+ T cells was normal, whereas that of T regs (CD3+CD4+ CD25bright+) was reduced in three of four patients (Table 1). In patients who underwent bone marrow, maturation was normal with no block at the promyelocyte stage. Cytogenetic abnormalities were not observed. Genetic analysis using an NGS panel including 161 genes involved in immunodeficiency/immunedysregulations, autoimmune disorders, and marrow failures showed that Patients 1, 2, and 3 had heterozygous pathogenic variants (p.Cys193Ter, p.Ile87Asn, and p.Trp40 Arg, respectively), previously described as risk-increasing TNFRSF13B variants in antibody deficiency syndromes,3 while Patient 4 had the same variant p.Ala181Glu as that reported in Bergman et al.'s case (Table 1). Overall, our cohort presented with isolated neutropenia with no additional autoimmune markers, mild-to-moderate infection profile (the right ischial branch osteomyelitis of Patient 2 resolved completely after the standard long-term prolonged antibiotic treatment with no recurrence), no substantial limitations of the quality of life, lack of immunoglobulin exhaustion typical of TACI disease but consistent with the young age of the cohort, tendency to have fewer B cells, abnormal switched B cells, and memory B cell depletion, as reported in patients with TACI mutations.1, 2 Pathogenic variants of TACI have incomplete penetrance and variable expression. The presence of a homozygous or heterozygous variant, the possible role of additional genetic variants, or exposure to environmental factors (i.e., microbial exposure) could be responsible for this heterogeneity1, 2 and the wide spectrum of clinical features ranging from severe to silent/mild phenotypes.1, 2 It is important to highlight the presentation of TACI disorders with mild symptomatic neutropenia that may further evolve in the long term to severe/refractory mono-/multi-lineage cytopenia, as shown by the case report by Bergman et al.1 Even if the patient reported by Dr Bergman et al.1 in good clinical condition up to adulthood, he might have carried some asymptomatic immunological disturbance long before his first admission, consistent with the long time required for the progressive exhaustion of cellular reserves, typical of the natural history of TACI disease. Differentiating TACI-related neutropenia from a typical primary autoimmune dysfunction may be challenging in infancy.10 Factors such as older age of presentation, longer duration of neutropenia, leukopenia, and lymphopenia could be signs, as we already described, of "atypical" AINPs.7 Moreover, a reduced number of total B cells, B switched cells, and B memory cells with an increase in the number of DN B cells should raise the suspicion of an underlying immune dysregulation. In particular, an increase in DN B cells has been described in several autoimmune disorders (such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, neuromyelitis optica, myasthenia gravis, and Guillain–Barré syndrome); however, their role in these diseases is unclear. One hypothesis (also seen in a vaccination response model) is that DN B cells may represent a source of disease-relevant immunoglobulins.11 These four cases together with the one described by Bergman et al. highlight that autoimmune cytopenias that show nontypical features or those that are refractory to common therapies require special immunologic and genetic investigation. In our patients, the identification of pathogenic mutations in TACI, one of the most common drivers of PIRD (but not the unique) suggests that the mechanism of neutropenia is likely sustained by dysregulation of the central B cell tolerance with missing self-antigen recognition. In these particular cases tailored follow-up including strict monitoring of immunoglobulin levels/lymphocyte subsets to document any immune cell exhaustion and the clinical surveillance for the appearance of autoimmune signs/surrogate markers is highly recommended. Early recognition of the type of AINP is critical to offer the best monitoring program and optimal therapy possibly tailored to the underlying disorder (i.e., immunoglobulin replacement), particularly in light of severe clinical findings such as PRCA, which may appear over the course of the disease. This specific type of evolution, as indicated by recent findings from our group,8, 9 outlines the need to investigate the underlying PID/PIRD in childhood marrow failure. Finally, the occurrence of PID/PIRD-related neutropenia in late childhood/young adulthood highlights the importance of establishing good collaborations between pediatricians and hematologists to share diagnostic algorithms and treatment protocols. ACKNOWLEDGMENT The authors acknowledge Editage for the editorial revision of the paperand Dr Elena Ricolfi for secretarial assistance. CONFLICT OF INTEREST The authors declare no conflict of interest. Open Research DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. REFERENCES 1Bergman P, Broliden PA, Ratcliffe P, et al. Mutation in the TACI gene and autoimmune neutropenia: a case report. Am J Hematol. 2022; 97: E207- E210. 2Salzer U, Grimbacher B. TACI deficiency—a complex system out of balance. Curr Opin Immunol. 2021; 71: 81- 88. 3Salzer U, Bacchelli C, Buckridge S, et al. Relevanceof biallelic versus monoallelic TNFRSF13B mutations in distinguishing disease-causing from risk-increasing TNFRSF13B variants in antibody deficiency syndromes. Blood. 2009; 113: 1967- 1976. 4Kolukısa B, Barış S. Primary immune regulatory disorders and targeted therapies. Turk J Haematol. 2021; 38: 1- 14. 5Seidel MG. Treatment of immune-mediated cytopenias in patients with primary immunodeficiencies and immune regulatory disorders (PIRDs). Hematol Am Soc Hematol Educ Program. 2020; 2020: 673- 679. 6Walter JE, Ayala IA, Milojevic D. Autoimmunity as a continuum in primary immunodeficiency. Curr Opin Pediatr. 2019; 31: 851- 862. 7Fioredda F, Rotulo GA, Farruggia P, et al. Late-onset and long-lasting autoimmune neutropenia: an analysis from the Italian Neutropenia Registry. Blood Adv. 2020; 4: 5644- 5649. 8Miano M, Grossi A, Dell'Orso G, et al. Genetic screening of children with marrow failure. The role of primary immunodeficiencies. Am J Hematol. 2021; 96: 1077- 1086. 9Miano M, Guardo D, Grossi A, et al. Underlying inborn errors of immunity in patients with Evans syndrome and multilineage cytopenias: a single-centre analysis. Front Immunol. 2022; 13:869033. 10Farruggia P, Fioredda F, Puccio G, et al. Primary autoimmune neutropenia in children data from the Italian Neutropenia Registry. Am J Hematol. 2015; 9: 546- 549. 11Yuzi L, Zhanguo L, Fanlei H. Double-negative (DN) B cells: an under-recognized effector memory B cell subset in autoimmunity. Clin Exp Immunol. 2021; 205: 119- 127. Citing Literature Volume97, Issue9September 2022Pages E328-E331 ReferencesRelatedInformation

We report a novel mutation on the beta-globin gene in a 68-year-old woman of Sicilian origin living in Alessandria, Italy. This mutation produces a hemoglobin (Hb) variant of Hb A that was detected by the capillary electrophoresis (CE) method during measurement of Hb A(1c). The variant Hb did not separate from Hb A using different high performance liquid chromatography (HPLC) instruments. Direct DNA sequencing revealed a G>T transversion at codon 37 and subsequent substitution of a tryptophan residue for a leucine residue. The new Hb variant was named Hb Alessandria [beta 37(C3)Trp -> Leu; HBB: c.113G>T]. The p50 value was slightly decreased while the stability test at 37 degrees C in isopropyl alcohol and the main erythrocyte parameters were normal. Overall, the patient appeared clinically normal.

Background: Hemoglobin A (Hb A) (α2β2) in the normal adult subject constitutes 96–98% of hemoglobin, and Hb F is normally less than 1%, while for hemoglobin A2 (Hb A2) (α2δ2), the normal reference values are between 2.0 and 3.3%. It is important to evaluate the presence of possible delta gene mutations in a population at high risk for globin gene defects in order to correctly diagnose the β-thalassemia carrier. Methods: The most used methods for the quantification of Hb A2 are based on automated high performance liquid chromatography (HPLC) or capillary electrophoresis (CE). In particular Hb analyses were performed by HPLC on three dedicated devices. DNA analyses were performed according to local standard protocols. Results: Here, we described eight new δ-globin gene variants discovered and characterized in some laboratories in Northern Italy in recent years. These new variants were added to the many already known Hb A2 variants that were found with an estimated frequency of about 1–2% during the screening tests in our laboratories. Conclusions: The knowledge recognition of the delta variant on Hb analysis and accurate molecular characterization is crucial to provide an accurate definitive thalassemia diagnosis, particularly in young subjects who would like to ask for a prenatal diagnosis or preimplantation genetic diagnosis.

Abstract Objectives Artifactually altered glycated hemoglobin (HbA1c) concentrations are frequently linked to hemoglobin (Hb) variants. Their expression and detection require in-depth analysis. Methods Cation exchange high performance liquid chromatography (HPLC) (Bio-Rad Variant™ II; Trinity Biotech Premier Hb9210 Resolution), capillary electrophoresis (CE) (Sebia Capillarys 2 Flex Piercing) and mass spectrometry (MS) (Waters) were used for variant detection; Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA) and next generation sequencing (NGS) were used for DNA analysis; HbA1c was measured with cation exchange HPLC (Bio-Rad Variant™ II; Arkray Adams HA-8180V; Tosoh HLC-723 G7), CE (Sebia Capillarys 2 Flex Piercing), boronate affinity HPLC (Trinity Biotech Hb9210 Premier), immunoassay (Cobas c501 Tina-quant HbA1c Gen. 3; Nihon Kohden CHM-4100 Celltac chemi HbA1c HA-411V) and enzymatic assay (Abbott Architect c 8000 HbA1c). Results Hb Yamagata [β132(H10)Lys→Asn; (HBB: c.399A>T)] was identified in the proband by MS after the observation of an abnormal peak in HPLC and CE. A mosaic expression of this variant was detected by NGS (mutant: 8%; wild type: 92%), after negative results in Sanger sequencing. Hb Yamagata interfered with HbA1c measurements by cation exchange HPLC and CE whereas immuno and enzymatic assay values showed good agreement with boronate affinity HPLC measurement. Conclusions A mosaicism of Hb Yamagata was found in a patient with altered HbA1c values. This rare gene variant was detected only by advanced technologies as MS and NGS. The variant interfered with common HbA1c determination methods.

We describe a new hemoglobin (Hb) variant, found in a 6-year-old Italian male living in Pistoia, Italy. An abnormal pattern compatible with a Hb A2 variant was observed on capillary electrophoresis (CE); direct sequencing revealed a transition at codon 89 of the δ gene (HBD: c.269G>A) changing serine into asparagine. The variant was also identified as Hb A2-Pistoia according to the traditional nomenclature and no other globin defect was present. The observation and description of this Hb A2 variant contributes to the number and heterogeneity of mutations of the δ-globin gene in the Mediterranean Area.

Thalassemia is one of the genetic diseases most prevalent in the world, mainly in Mediterranean countries, Africa, the Middle East, India and Southeast Asia. Although Europe no longer has a more ur...

Negli ultimi anni il ruolo del Laboratorio (di 1° livello o di livello intermedio), dedicato alla diagnostica dei difetti dell’emoglobina, sta diventando sempre più centrale e impegnativo a causa dell’aumentato numero di difetti che possono essere osservati in una popolazione sempre più eterogenea. Gli esami per le emoglobinopatie a volte richiesti con modalità non appropriate rispetto alle raccomandazioni specifiche delle Società scientifiche necessitano di un percorso diagnostico appropriato, che comporta l’utilizzo di più strumenti, con principi di funzionamento diversi, tali da fornire informazioni utili e suggerire percorsi di diagnosi molecolare più mirati. Ne è un esempio il caso di una rara variante emoglobinica, identificata come Hb Hekinan.

Two different perspectives are the main focus of this book chapter: (1) A perspective that looks to the future, with the goal of devising rational associations of targeted inhibitors against distinct altered signaling-network pathways. This goal implies a sufficiently in-depth molecular diagnosis of the personal cancer of a given patient. A sufficiently robust and extended dynamic modeling will suggest rational combinations of the abovementioned oncoprotein inhibitors. The work toward new selective drugs, in the field of medicinal chemistry, is very intensive. Rational associations of selective drug inhibitors will become progressively a more realistic goal within the next 3-5 years. Toward the possibility of an implementation in standard oncologic structures of technologically sufficiently advanced countries, new (legal) rules probably will have to be established through a consensus process, at the level of both diagnostic and therapeutic behaviors. (2) The cancer patient of today is not the patient of 5-10 years from now. How to support the choice of the most convenient (and already clinically allowed) treatment for an individual cancer patient, as of today? We will consider the present level of artificial intelligence (AI) sophistication and the continuous feeding, updating, and integration of cancer-related new data, in AI systems. We will also report briefly about one of the most important projects in this field: IBM Watson US Cancer Centers. Allowing for a temporal shift, in the long term the two perspectives should move in the same direction, with a necessary time lag between them.

Current colorectal cancer (CRC) treatment guidelines are primarily based on clinical features, such as cancer stage and grade. However, outcomes may be improved using molecular treatment guidelines. Potentially useful biomarkers include driver mutations and somatically inherited alterations, signaling proteins (their expression levels and (post) translational modifications), mRNAs, micro-RNAs and long noncoding RNAs. Moving to an integrated system is potentially very relevant. To implement such an integrated system: we focus on an important region of the signaling network, immediately above the G1-S restriction point, and discuss the reconstruction of a Molecular Interaction Map and interrogating it with a dynamic mathematical model. Extensive model pretraining achieved satisfactory, validated, performance. The model helps to propose future target combination priorities, and restricts drastically the number of drugs to be finally tested at a cellular, in vivo, and clinical-trial level. Our model allows for the inclusion of the unique molecular profiles of each individual patient's tumor. While existing clinical guidelines are well established, dynamic modeling may be used for future targeted combination therapies, which may progressively become part of clinical practice within the near future. WIREs Syst Biol Med 2016, 8:314-336. doi: 10.1002/wsbm.1342 For further resources related to this article, please visit the WIREs website.

Transcription factors (TFs) represent key regulators of gene-expression patterns controlling cell behavior. TFs are active at nuclear – chromatin levels. TFs do not act in isolation; small sets of TFs cooperate toward the transcription of sets of mRNAs and consequently the translation of new proteins (the molecular phenotypes of a cell). Most TFs are activated through a cascade of biochemical reactions mediated by receptors expressed on the target cell surface. Nuclear Receptors (NRs) are transcription factors activated instead by small hydrophobic molecules capable of crossing the plasma membrane. The convergence of different pathways on TFs and their posttranslational modifications ensure that the external stimuli generate appropriate and integrated responses. The reconstruction of the molecular anatomy of these pathways through Molecular Interactions Maps (MIMs) can depict these intricate interactions. A mathematical modeling approach simulates/mimics their mechanism of action in normal and pathological conditions. We can simulate the effect of virtual hits in neoplastic transformation as mutations/alterations in these pathways. We can also simulate the effect of targeted inhibitors on these deregulated pathways. This strategy can help to guide an appropriate combination of targeted drugs in the treatment of a cancer patient, a major innovative perspective of incoming years.