Vittoria Longo

Screening of mutations of hemophilia A in 40 Italian patients: a novel G-to-A mutation in intron 10 of the F8 gene as a putative cause of mild hemophilia A in southern Italy.

Hemophilia A is an X-linked bleeding disorder caused by widespread mutations in the human coagulation factor 8 gene. We have searched for mutations in factor 8 gene DNAs from 40 unrelated Italian patients with hemophilia A. All patients came from the same region (Calabria) and were followed-up at the same hemophilia center. Of the 40 patients, 20 (50%) had severe hemophilia A, 19 (47.5%) had moderate hemophilia A, and one (2.5%) had mild hemophilia A. All patients were first screened for the common intron 22 and intron 1 inversions. Inversion-negative samples were screened for point mutations by direct sequencing of all coding regions and intron–exon boundaries of the factor 8 gene. Mutations previously reported as causative of hemophilia A were identified in 14 of the 40 patients. These included five (12.5%) intron 22 inversions, one (2.5%) small deletion, one (2.5%) small insertion and seven (17.5%) point mutations. In all patients with moderate and mild hemophilia A, a nucleotide change in the c.1538 –18G > A in intron 10, not reported in the HAMSTeRS factor 8 mutation database (http://europium.csc.mrc.ac.uk/), was found. The G-to-A change predicts the appearance of a new acceptor splice site. We have also demonstrated that all patients share a common haplotype, suggesting that the mutation probably occurred in a single ancestor. In conclusion, we suggest that the c.1538 –18G > A transition can be the putative mutation, which probably occurred in a common ancestor and then spread in neighbours, in patients with moderate–mild hemophilia A investigated in the present study.

Outcome of patients with splanchnic venous thrombosis presenting without overt MPN: A role for the JAK2 V617F mutation re-evaluation

INTRODUCTION Although investigation for JAK2 V617F mutation is recommended in patients presenting with splanchnic venous thrombosis (SVT), no specific clinical advice is given to SVT patients presenting without myeloproliferative neoplasms (MPN) and JAK2 V617F mutation. In MPN-free SVT patients, to investigate the clinical outcome, the clinical impact of re-evaluation for the JAK2 V617F mutation, and relationships with the occurrence and time to diagnosis of MPN. MATERIALS AND METHODS A cohort of non-cirrhotic SVT patients, enrolled at a single centre and prospectively analyzed. RESULTS In 121 SVT patients prospectively followed from 1994 to 2012, a MPN was present in 28 (23.1%). Additional 13 patients (10.7%) showed only the JAK2 V617F mutation. During the follow-up, the JAK2 V617F mutation and/or MPN were identified in 8 patients (median time of development: 21 months, range 6-120), whereas 72 remained (MPN and JAK2 V617F)-free until the end of the observation. The mortality rate was higher among patients presenting with MPN and/or the JAK2 V617F mutation than in patients who developed later or remained disease-free (p=0.032). The thrombosis-free survival was lower in patients with (p=0.04) or developing later MPN and the JAK2 V617F mutation (p=0.005) than in patients (MPN and JAK2 V617F)-free. The incidence of bleeding was similar among groups. CONCLUSIONS MPN with or without circulating positive clones for JAK2 V617F mutation can occur long after a SVT, identifying at risk patients for new thrombotic events. If confirmed in other studies, re-evaluation for JAK2 V617F mutation may be of help in early MPN detection and clinical management of SVT patients.

Homozygosity by descent of a 3Mb chromosome 17 haplotype causes coinheritance of Glanzmann thrombasthenia and primary ciliary dyskinesia

To the editor: Glanzmann thromboasthenia (GT) is an autosomal recessively inherited bleeding diathesis, and mutations within the genes (ITGA2B and ITGB3) that code for subunits οφ τηe αIIbβ3 integrin have been shown to be responsible for this disorder.[1][1] The GT clinical phenotype can be

Polymorphic miRNA-mediated gene contribution to inhibitor development in haemophilia A

Development of inhibitory antibodies is perhaps the most serious complication of FVIII replacement therapy, precluding efficient clinical management of patients with haemophilia A (HA). The development and function of immune system are also regulated by microRNAs (miRNAs). Mutations and changes in the level of expression of some miRNA genes have been associated with the onset and progression of immunological disorders. The aim of this study was to investigate new genetic polymorphisms in loci for miRNA and their targets to evaluate whether these SNPs may confer susceptibility to inhibitor development in patients with HA. Italian HA patients with and without inhibitors and healthy controls were recruited in this study. For SNP analysis, standard DNA sequencing method was used. We have studied four SNPs, i.e. rs36101366, rs34683807, rs1803603 and rs3024496 located in the 3′UTR of F8 and IL‐10 genes. These SNPs have been checked for their frequencies in patients with and without inhibitors, but no statistically significant differences were found. Then, we have searched for other genetic variants in loci for haematopoietic‐specific miRNAs, i.e. hsa‐mir‐150, hsa‐mir‐155, hsa‐mir‐146a, hsa‐mir‐142, hsa‐mir‐181a and in a specific miRNA, hsa‐mir‐1184, i.e. predicted to be located in the intron 22 of F8 gene. For all miRNAs selected, we did not identify any sequence variation in our study population. This is the first study to demonstrate that there was no association between selected SNPs in miRNAs and their targets and the susceptibility to inhibitor development in people affected by HA.

Coexistence of β-Thalassemia and Hereditary Hemochromatosis in Homozygosity: A Possible Synergic Effect?

A few considerations, which we found in the literature, inspired us to reevaluate patients previously investigated [characterized for β-thalassemia (β-thal) and hereditary hemochromatosis (HH) genes] by our department at Medical Genetics, School of Medicine, University of Foggia, Italy.

Lack of genotypephenotype correlation in congenital adrenal hyperplasia due to a CYP21A2-like gene

CONTEXT Congenital Adrenal Hyperplasia (CAH) due to 21-hydroxylase deficiency, encoded by CYP21A2 gene, is an autosomal recessive disorder. The CYP21A2 gene, localized in a genetic unit defined RCCX module, is considered one of the most polymorphic of human genes. OBJECTIVES We considered new evidences about the presence of a RCCX trimodular haplotype with a CYP21A2-like gene to explain the lack of a genotype-phenotype correlation in individuals of two different families. DESIGN AND METHODS To verify gene duplication we used Multiplex Ligation Probe-Dependent Amplifications (MLPA) and to confirm the presence of a CYP21A2-like gene downstream TNXA gene we used previously described amplification and restriction strategy followed by the sequencing of the CYP21A2 gene downstream TNXB gene. RESULTS The amplification strategy and restriction analysis of CYP21A1P/CYP21A2-TNXA PCR product in association with MLPA assay and sequencing of CYP21A2 gene downstream TNXB were able to identify the presence of the CYP21A2-like gene in healthy subjects of the two families, wherein the direct sequencing of CYP21A2 gene showed genotypes correlated to pathological phenotypes. CONCLUSIONS The strategy suggested is useful to facilitate molecular testing in CAH patients, considering the new evidence about possible different haplotypes.

Intra-familiar discordant PKU phenotype explained by mutation analysis in three pedigrees.

Classical phenylketonuria (PKU) and mild hyperphenylalaninemia (MHP) are two phenotypes of phenylalanine hydroxylase (PAH) deficiency with different degrees of severity. We have analyzed three families in which classical PKU, MHP and a normal phenotype occurred within each family due to the different combinations of three mutations segregating within the family. Indeed, sequence PAH analysis revealed three different alleles segregating in each family. This report suggests that when discordant phenotypes occur in a family, complete analysis of the PAH gene may be performed in order to support the diagnosis and assist in accurate genetic counseling and patient management. We further support the marked heterogeneity of hyperphenylalaninemia primarily due to allelic heterogeneity at the PAH locus.

A Platelet Defect Modulates Bleeding in Mild Hemophilia: The Tale of 2 Brothers

To the Editor: One open issue in the field of hemophilia is the detection of factors influencing the clinical phenotype of the disease. Bleeding tendency in severely affected hemophiliac patients has a dramatic impact on life quality and is the basic parameter to determine mode, dose, and frequency of substitution therapy. In severe hemophiliacs, considerable variation in the frequency and the extent of bleeding is described with consequent large heterogeneity in the rate of musculoskeletal damage. In particular, about 10% of severe hemophiliacs bleed rarely and prophylaxis is not required in such patients. Several factors have been evoked as bleeding modulators in severely affected patients, such as the main thrombophilias, the fibrinolytic activity as well as the levels and the kinetics of other coagulation cascade proteins. Results of such studies are conflicting and have been found to account only for a part of the clinical heterogeneity in severe hemophilia. Less observations exist in both moderate and mild hemophilia. Given the generally acknowledged correlation between severity of the disease and levels of circulating factor VIII (FVIII), mild-to-moderate hemophiliacs present with a variegate clinical picture and worsening in the bleeding tendency are suggestive of inhibitor appearance, which is, in nonsevere hemophilia, still a grey area to explore. In addition, the role of platelets as modifiers of bleeding in hemophiliacs has poorly been considered. An interesting report, though, concerns enhanced bleeding following the intake of herbal moieties disturbing platelet function in hemophiliacs with a low bleeding rate. Primary hemostasis impairment was also suspected to worsen the bleeding pattern in HIV-positive hemophiliacs taking HIV protease inhibitors-based antiretroviral therapy, a finding that has recently been confirmed with platelet aggregation studies performed in nonhemophiliac HIV affected patients. Since our Haemophilia Centre is active in 2005, we have cared for 2 mild hemophiliac brothers (14 and 24 years old, respectively) who were genetically characterized to carry the mutation c.1700 T>C, Ile548Thr in the exon 11 of the FVIII gene, a common mutation in patients with mild hemophilia A. FVIII:C assay was 43% and 37%, respectively. Personal hemorrhagic history was significantly different in the 2 siblings: while the older brother had only 1 bleeding episode all along his life after surgery for a chronic sinusitis, the younger brother frequently referred to our Centre for spontaneous and posttraumatic skeletal muscle bleedings and hemarthroses, the right iliopsoas being the most frequent skeletal muscle location and the left knee the target joint. Annual bleeding rate in this patient was about 6 to 10 bleeding episodes per year. Despite a good laboratory response to desmopressin, which was also From the Centro Emofilia e Trombosi, Università De L’Aquila, Italy (ML, MN, GM); Servizio di Genetica Medica Università degli Studi di Foggia, Italy (RS, VB, VL, MM); and Unità di Aterosclerosi e Trombosi Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy (GF, EG).