Coexistence of Multiple Gene Variants in Some Patients with Erythrocytoses.

Background: Erythrocytosis is a relatively common condition; however, a large proportion of these patients (70%) remain without a clear etiologic explanation. Methods: We set up a targeted NGS panel for patients with erythrocytosis, and 118 sporadic patients with idiopathic erythrocytosis were studied. Results: In 40 (34%) patients, no variant was found, while in 78 (66%), we identified at least one germinal variant; 55 patients (70.5%) had 1 altered gene, 18 (23%) had 2 alterations, and 5 (6.4%) had 3. An altered HFE gene was observed in 51 cases (57.1%), EGLN1 in 18 (22.6%) and EPAS1, EPOR, JAK2, and TFR2 variants in 7.7%, 10.3%, 11.5%, and 14.1% patients, respectively. In 23 patients (19.45%), more than 1 putative variant was found in multiple genes. Conclusions: Genetic variants in patients with erythrocytosis were detected in about 2/3 of our cohort. An NGS panel including more candidate genes should reduce the number of cases diagnosed as "idiopathic" erythrocytosis in which a cause cannot yet be identified. It is known that HFE variants are common in idiopathic erythrocytosis. TFR2 alterations support the existence of a relationship between genes involved in iron metabolism and impaired erythropoiesis. Some novel multiple variants were identified. Erythrocytosis appears to be often multigenic.

Relapsing thrombotic thrombocytopenic purpura with low ADAMTS13 antigen levels: An indication for splenectomy?

With more recent modalities of immunosuppression, splenectomy is now rarely considered in refractory/relapsed thrombotic thrombocytopenic purpura (TTP). However, the surgical approach had shown convincing evidences of high efficacy in the pre-rituximab era and therefore may still represent a lifesaving option in selected challenging cases. To define the characteristics of subjects who may benefit from splenectomy may ease clinical decision making. In this paper we describe the clinical and laboratory data of 2 multiple relapsing TTP cases who successfully underwent splenectomy in the pre-rituximab era. Whereas high anti-ADAMTS13 antibody titre and low ADAMTS13 activity never correlated with remission and relapse, a drop in the ADAMTS13 antigen level was always associated with the acute phase, whereas levels consistently returned to normal following splenectomy, heralding long term remission. Splenectomy may therefore be considered in refractory TTP cases associated with increased ADAMTS13 antigen clearance, irrespective of persistence of inhibitory antibodies.

Correlation between ADAMTS13 activity and neurological impairment in acute thrombotic microangiopathy patients.

Differential diagnosis between thrombotic thrombocytopenic purpura (TTP) and other thrombotic microangiopathies (TMA) is usually difficult because of frequently overlapping clinical presentations. Severely depressed ADAMTS13 activity (<10 %) seems distinctive for TTP because of its pathogenetic role. However a long debate exists in the literature about its sensibility and specificity. Our aim was to search for clinical differences between TMA patients referred to our laboratory, comparing them for protease activity <10 versus ≥10 %. ADAMTS13 activity ≥10 % patients (n = 73) showed a higher prevalence of drug- (p = 0.005) and cancer-associated (p < 0.001) TMA. Mean platelet count and renal dysfunction prevalence was lower (p < 0.001), while neurological impairment was more frequent (p = 0.001) in the <10 % ADAMTS13 activity group (n = 109), confirming previous literature findings. When taken neurological manifestations singularly, epilepsy (p = 0.04), focal motor deficit (p < 0.001) and cranial nerve palsy (p = 0.007) were more frequent in the <10 % activity group. In our case series, a <10 % ADAMTS13 activity depicts a group of patients with clinical features similar to TTP patients. Focal motor impairment or epileptic manifestations could further address toward a TTP diagnosis. Studies about treatment efficacy and follow-up are advised to determine whether laboratory findings can guide therapeutic decisions.

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.

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.