I. Cortella

Functional analysis of two novel splice site mutations of APOB gene in familial hypobetalipoproteinemia.

Familial hypobetalipoproteinemia (FHBL) is a co-dominant disorder characterized by reduced plasma levels of low density lipoprotein cholesterol (LDL-C) and its protein constituent apolipoprotein B (apoB), which may be due to mutations in APOB gene, mostly located in the coding region of this gene. We report two novel APOB gene mutations involving the acceptor splice site of intron 11 (c.1471-1G>A) and of intron 23 (c.3697-1G>C), respectively, which were identified in two patients with heterozygous FHBL associated with severe fatty liver disease. The effects of these mutations on APOB pre-mRNA splicing were assessed in COS-1 cells expressing the mutant APOB minigenes. The c.1471-1G>A APOB minigene generated two abnormal mRNAs. In one mRNA the entire intron 11 was retained; in the other mRNA exon 11 joined to exon 12, in which the first nucleotide was deleted due to the activation of a novel acceptor splice site. The predicted products of these mRNAs are truncated proteins of 546 and 474 amino acids, designated apoB-12.03 and apoB-10.45, respectively. The c.3697-1G>C APOB minigene generated a single abnormal mRNA in which exon 23 joined to exon 25, with the complete skipping of exon 24. This abnormal mRNA is predicted to encode a truncated protein of 1220 amino acids, designated apoB-26.89. These splice site mutations cause the formation of short truncated apoBs, which are not secreted into the plasma as lipoprotein constituents. This secretion defect is the major cause of severe fatty liver observed in carriers of these mutations.

Spectrum of 5’UTR mutations in ANKRD26 gene in patients with inherited thrombocytopenia: c.-140C>G mutation is more frequent than expected

Inherited thrombocytopenia secondary to mutations in the ankyrin repeat domain 26 gene (ANKRD26) (THC2 locus mapped to chromosome 10p11.1-p12, MIM 188000). Clinical features of the patients with these defects are a moderate form of autosomal dominant thrombocytopenia, reduction in platelet α-granules, normal in vitro platelet aggregation, normal platelet size but a prevalence of acute leukemia more than 30-fold higher than in the normal population. This last observation raises the suspicion that mutations in the ANKRD26 5’UTR lead to a risk of hematologic malignancies. This suspicion is increased still further by the identification of large amounts of particulate cytoplasmic structures in platelets and megakaryocytes of patients with ANKRD26-RT consisting of polyubiquitinated proteins and proteasomes as observed in a number of solid cancers [1]. Noris P. et al. presented the first population with this genetic abnormality in 2011, showing that 11 heterozygous single-nucleotide substitutions identified in a short stretch of 22 nucleotides of the 5’UTR of the ANKRD26 gene, six of the mutations including c.-127A>G, c.-126T>G, c.-121A>C, c.-119C>A, c.-118C>A, and c.-113A>C, were novel mutations [2]. We screened 50 patients with inherited thrombocytopenia of unknown origin with mild thrombocytopenia, normal platelet size, family history of neoplasm, and a clinical phenotype compatible with ANKRD26-related disorders, belonging to a cohort of 120 patients who had not been characterized for the genetic defect. All patients, coming from North and Central Italy, had inherited thrombocytopenia and were being observed at the Padua University Medical Centre. Bleeding tendency was measured by the World Health Organization (WHO) bleeding scale. The 5’UTR of the ANKRD26 gene was screened for mutations using genomic DNA extracted from Buccal Swabs with QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Mutational analysis was performed by polymerase chain reaction (PCR) amplification using oligo 1F (5’-CATGGAGCACACTTGACCAC-3’) and 1R (5’-TACTCCAGTGGCACTCAGTC-3’). PCR was carried out in a total volume of 15μL with 50ng of genomic DNA, 10mM of each primer, and 9μL of mastermix (Promega, Madison, Wisconsin, USA). After an initial denaturation step at 96°C for 12 minutes, amplification was performed for 30 cycles (denaturation at 96°C for 30 seconds, annealing at 62°C for 45 seconds, and extension at 72°C for 50 seconds). PCR products were sequenced using ABI310 genetic analyzer (Applied Biosystems, Foster City, Ca, USA). We found four sporadic patients and three pedigrees presenting with mutations of ANKRD26 gene. Single-nucleotide substitutions have been identified in the 5’UTR region of the ANKRD26 gene: c.-128G>A, c.-134G>A, and c.-140C>G. The first two mutations have been extensively described by Noris et al, but the c.-140C>G was recently described only by H. Boutroux et al. 2015 [2,3]. In both sporadic and familial cases, patients are not related. Clinical features and laboratory characteristics are reported in Table 1 Subjects 1 and 2 have parents with thrombocytopenia. Subjects 2 and 4 have a family history for visceral malignancies. Family n°5, which shows a heterozygous mutation c.128G>A, is reported in Figure 1. The mother (III, 1) and the first daughter (IV, 1) present 68 and 35 × 10/l platelets, respectively. The first daughter had mild ecchymosis, rash, and hematoma. The grandfather (II, 2), the sister of the grandfather (II, 3), and the great grandfather (I, 1) had moderate thrombocytopenia; moreover, the grandfather died from acute myeloid leukemia, and the sister of the grandfather was treated for myelodysplastic syndrome, whose biological characteristics are unknown. Pedigree 6 is also reported in Figure 1. This family had been misdiagnosed in 2007 as May Hegglin form, without a genetic analysis. The mother (III, 16) had a marked thrombocytopenia 19 × 10/l, while her son (IV, 4) showed a platelet count of 70 × 10/ l. She presented only gengival bleeding, and at childbirth, she was treated with platelets concentrates. Her brother (III, 14) and her father (II, 7) had also thrombocytopenia (30–40 × 10/l). An aunt (II, 5) had thrombocytopenia and died from uterine cancer. Her daughter (III, 7) had thrombocytopenia and an eye neoplasia. The granddaughter (IV, 1) of the proposita, had Correspondence: Dott.ssa Silvia Ferrari, Department of Medicine, University of Padua Medical School, Via Ospedale 105, Padua 35128, Italy. E-mail: silvia.fer30@gmail.com http://www.tandfonline.com/iplt ISSN: 0953-7104 (print), 1369-1635 (electronic)

Decreased VLDL-Apo B 100 Fractional Synthesis Rate Despite Hypertriglyceridemia in Subjects With Type 2 Diabetes and Nephropathy

CONTEXT Subjects with type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN) often exhibit hypertriglyceridemia. The mechanism(s) of such an increase are poorly known. OBJECTIVE We investigated very low-density lipoprotein (VLDL)-Apo B 100 kinetics in T2DM subjects with and without DN, and in healthy controls. DESIGN Stable isotope (13)C-leucine infusion and modeling analysis of tracer-to-tracee ratio dynamics in the protein product pool in the 6-8-h period following tracer infusion were employed. SETTING Male subjects affected by T2DM, either with (n = 9) or without (n = 5) DN, and healthy male controls (n = 6), were studied under spontaneous glycemic levels in the post-absorptive state. RESULTS In the T2DM patients with DN, plasma triglyceride (TG) (mean ± SD; 2.2 ± 0.8 mmol/L) and VLDL-Apo B 100 (17.4 ± 10.4 mg/dL) concentrations, and VLDL-Apo B 100 pool (0.56 ± 0.29 g), were ∼60-80% greater (P < .05 or less) than those of the T2DM subjects without DN (TG, 1.4 ± 0.5 mmol/L; VLDL-Apo B 100, 9.9 ± 2.5 mg/dL; VLDL-Apo B 100 pool, 0.36 ± 0.09 g), and ∼80-110% greater (P < .04 or less) than those of nondiabetic controls (TG, 1.2 ± 0.4 mmol/L; VLDL-Apo B 100, 8.2 ± 1.7 mg/dL; VLDL-Apo B 100, 0.32 ± 0.09 g). In sharp contrast however, in the subjects with T2DM and DN, VLDL-Apo B 100 fractional synthesis rate was ≥50% lower (4.8 ± 2.2 pools/d) than that of either the T2DM subjects without DN (9.9 ± 4.3 pools/d; P < .025) or the control subjects (12.5 ± 9.1 pools/d; P < .04). CONCLUSIONS The hypertriglyceridemia of T2DM patients with DN is not due to hepatic VLDL-Apo B 100 overproduction, which is decreased, but it should be attributed to decreased apolipoprotein removal.

New heterozygous variant in GP1BB gene is responsible for an inherited form of macrothrombocytopenia

Ezio Zanon Samantha Pasca Cristina Santoro Gabriella Gamba Sergio M. Siragusa Angiola Rocino Isabella Cantori Augusto B. Federici Luciana Mameli Gaetano Giuffrida Anna Falanga Corrado Lodigiani Rita C. Santoro Marta Milan Chiara Ambaglio Mariasanta Napolitano Maria G. Mazzucconi Haemophilia Centre, University Hospital of Padua, Padova, Italy, Cellular Biotechnology and Haematology Department, Umberto I University Hospital, Roma, Italy, Haemophilia Centre, S. Matteo Hospital, Pavia, Italy, Centre of Haemorrhagic and Thrombotic Diseases, University of Palermo, Palermo, Italy, Haemophilia and Thrombosis Centre, S. Giovanni Bosco Hospital, Napoli, Italy, Centre of Coagulation Diseases, Hospital of Macerata, Macerata, Italy, Haematology and Transfusion Medicine Department, Luigi Sacco Hospital, Milano, Italy, Center of Coagulation Diseases, SS Annunziata Hospital, Sassari, Italy, Haematology Department, VE Ferrarotto and S. Bambino University Hospital, Catania, Italy, Transfusion Medicine and Immune-haematology Department, Giovanni XXIII Hospital, Bergamo, Italy, Medicine Department, Humanitas Clinical Institute, Rozzano (Milano), Italy and Centre of Haemorrhagic and Thrombotic Diseases, Pugliese-Ciaccio Hospital, Catanzaro, Italy. E-mail: zanezio61@gmail.com