RASopathies and hemostatic abnormalities: key role of platelet dysfunction.

BACKGROUND: Bleeding anomalies have been reported in patients affected by Noonan syndrome. No study has been performed in patients with molecularly confirmed RASopathy. We aimed to characterize the frequency and types of bleeding disorders in patients with RASopathies and evaluate any significant association with laboratory findings. PATIENTS AND METHODS: Forty-nine individuals (PTPN11, n = 27; SOS1, n = 7; RIT1, n = 3; SPRED1, n = 1; LZTR1, N = 3; RAF1, n = 2; BRAF, n = 4; MEK1, n = 1; MEK2, n = 1), and 49 age- and sex-matched controls were enrolled. The "Paediatric Bleeding Questionnaire Scoring Key" was administered to patients and families. Laboratory screening tests including clotting factors dosing, platelet count, Prothrombin Time and Partial Thromboplastin Time, were employed both in patients and controls to characterize the bleeding diathesis. A subgroup of 29/49 patients and 29/49 controls was also tested for platelet function. RESULTS: Regardless of the gene involved, pathological paediatric bleeding scores were recorded in 14/49 (28.5%) patients. Indeed, 7 were mutated in PTPN11, 3 in SOS1, 2 in RIT1, 1 in BRAF, and 1 in MEK1. Compared to patients with normal bleeding scores, those with pathologic bleeding score showed higher prevalence of splenomegaly (p = 0.006), prolonged aPTT (p = 0.04), lower levels of coagulation factor V (FV, p = 0.001), FVII (p = 0.003), FX (p = 0.0008) and FXIII (p = 0.002), higher vWAg (p = 0.04), and lower platelet sensitivity to Ristocetin (p = 0.001), arachidonic acid (AA) (p = 0.009) and collagen (p = 0.01). The presence of hematomas inversely correlated with factor V (p = 0.002), factor VII (p = 0.003), factor X (p = 0.002) and factor XIII (p = 0.004) levels, and directly correlated with platelet response to collagen (p = 0.02) and AA (p = 0.01). The presence of splenomegaly directly correlated with the presence of hematoma (p = 0.006), platelet response to Ristocetin (p = 0.04) and AA (p = 0.04), and inversely correlated with factor V levels (p = 0.03). CONCLUSIONS: Patients with RASopathies and a bleeding tendency exhibit multiple laboratory abnormalities, including platelet-related disorders. Splenomegaly is frequently detected and might be a suggestive sign for qualitative platelet dysfunction. A comprehensive clinical assessment should be carried out at diagnosis, during the follow-up and before any surgical procedures. Since there is currently no consensus on management of bleeding complications, it is important that physicians closely monitor these patients.

Plasma phospholipid dysregulation in patients with cystathionine-ß synthase deficiency.

BACKGROUND & AIMS: Patients with cystathionine ß-synthase deficiency (CBSD) exhibit high circulating levels of homocysteine and enhanced lipid peroxidation. We have characterized the plasma lipidome in CBSD patients and related lipid abnormalities with reactions underlying enhanced homocysteine levels. METHODS AND RESULTS: Using an ultra-high-performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry method, plasma lipids were determined with an untargeted lipidomics approach in 11 CBSD patients and 11 matched healthy subjects (CTRL). Compared to CTRL, CBSD patients had a higher medium and long-chain polyunsaturated fatty acids (PUFA) content in phosphatidylethanolamine (PE) and lysophosphatidylethanolamine (LPE) species (p < 0.02), and depletion of phosphatidylcholine (PC; p = 0.02) and of lysophosphatidylcholine (LPC; p = 0.003) species containing docosahexaenoic acid (DHA), suggesting impaired phosphatidylethanolamine-N-methyltransferase (PEMT) activity. PEMT converts PE into PC using methyl group by S-adenosylmethionine (SAM) thus converted in S-adenosylhomocysteine (SAH). Whole blood SAM and SAH concentrations by liquid chromatography tandem mass spectrometry were 1.4-fold (p = 0.015) and 5.3-fold (p = 0.003) higher in CBSD patients than in CTRL. A positive correlation between SAM/SAH and PC/PE ratios (r = 0.520; p = 0.019) was found. CONCLUSIONS: A novel biochemical abnormality in CBSD patients consisting in depletion of PC and LPC species containing DHA and accumulation of PUFA in PE and LPE species is revealed by this lipidomic approach. Changes in plasma SAM and SAH concentrations are associated with such phospholipid dysregulation. Given the key role of DHA in thrombosis prevention, depletion of PC species containing DHA in CBSD patients provides a new direction to understand the poor cardiovascular outcome of patients with homocystinuria.

Association of human leukocyte antigen-G 14 bp polymorphism with recurrent pregnancy loss in European countries: a meta-analysis of literature studies.

OBJECTIVE: To study the controversial association between human leukocyte antigen-G (HLA-G) 14 bp polymorphism and recurrent pregnancy loss (RPL). We performed a meta-analysis of studies in the literature that enrolled only women of European countries who experienced RPL spontaneously or after undergoing IVF. DESIGN: Systematic meta-analysis of articles published before January 2019 pertaining the association of HLA-G genotype and RPL. The search was performed in electronic databases (PubMed, Web of Science, Scopus, EMBASE), without any language or publication year restriction. SETTING: Academic hospitals and private clinics. PATIENT(S): Women who experienced RPL spontaneously or after undergoing IVF. INTERVENTION(S): Genotyping of 14 bp polymorphism (insertion/insertion, insertion/deletion, deletion/deletion) in exon 8 of the HLA-G gene. MAIN OUTCOME MEASURE(S): Meta-analyses of the association between HLA-G 14 bp polymorphism in homozygosis (insertion/insertion) and heterozygosis (insertion/deletion) in women with RPL compared with pregnant controls with at least one live birth and no history of RPL. RESULT(S): Ten studies were analyzed comprising 1,091 women with RPL and 808 controls without RPL. Women with RPL showed significantly higher prevalence of HLA-G 14 bp insertion/insertion genotype compared with women without RPL (19.8% vs. 14.1%; odds ratio = 1.562; 95% confidence interval, 1.203-2.027), and this result was also confirmed when separately analyzing women with RPL during a spontaneous pregnancy (odds ratio, 1.562; 95% confidence interval, 1.203-2.027) and those undergoing IVF (odds ratio, 1.990; 95% confidence interval, 0.978-4.051). CONCLUSION(S): Women of European countries with the HLA-G 14 bp insertion/insertion genotype have a significantly higher prevalence of RPL.

Identification of the first dominant mutation of LAMA5 gene causing a complex multisystem syndrome due to dysfunction of the extracellular matrix.

BACKGROUND: The laminin alpha 5 gene (LAMA5) plays a master role in the maintenance and function of the extracellular matrix (ECM) in mammalian tissues, which is critical in developmental patterning, stem cell niches, cancer and genetic diseases. Its mutations have never been reported in human disease so far. The aim of this study was to associate the first mutation in LAMA5 gene to a novel multisystem syndrome. METHODS: A detailed characterisation of a three-generation family, including clinical, biochemical, instrumental and morphological analysis, together with genetics and expression (WES and RNAseq) studies, was performed. RESULTS: The heterozygous LAMA5 mutation c.9418G>A (p.V3140M) was associated with skin anomalies, impaired scarring, night blindness, muscle weakness, osteoarthritis, joint and internal organs ligaments laxity, malabsorption syndrome and hypothyroidism. We demonstrated that the mutation alters the amount of LAMA5 peptides likely derived from protein cleavage and perturbs the activation of the epithelial-mesenchymal signalling, producing an unbalanced expression of Sonic hedgehog and GLI1, which are upregulated in cells from affected individuals, and of ECM proteins (COL1A1, MMP1 and MMP3), which are strongly inhibited. Studies carried out using human skin biopsies showed alteration of dermal papilla with a reduction of the germinative layer and an early arrest of hair follicle downgrowth. The knock-in mouse model, generated in our laboratory, shows similar changes in the tissues studied so far. CONCLUSIONS: This is the first report of a disease phenotype associated with LAMA5 mutation in humans.

Cardiovascular risk factors and outcome in patients with retinal vein occlusion.

The pathogenesis and treatment of retinal vein occlusions (RVO) are largely unclear. Prevalence of cardiovascular risk factors and of thrombophilic abnormalities was evaluated in 117 patients (61 M, 56 F; mean age 51 +/- 13 years) with a history of RVO (62 central, CRVO; 48 branch, BRVO; 7 both) and in 202 age- and sex-matched control subjects. Cardiovascular outcome after a mean 8.2 year follow-up was recorded for 90 patients. Arterial hypertension was significantly more frequent in patients than in controls (64.9 vs. 28.2%; adjusted OR 4.5 95% CI 2.4-7.9; P < 0.0001), as well diabetes mellitus (17.9 vs. 7.9%; P < 0.05). Antithrombin, Protein C, Protein S and homocysteine levels, lupus anticoagulant, anticardiolipin antibodies, FV G1691A and prothrombin G20210A polymorphisms were comparable in the two groups, nor were different according to RVO localization or to the age at event. BRVO patients were significantly older (55 +/- 9 vs. 47 +/- 15 years; P = 0.002) and had higher prevalence of diabetes, overweight and hypertension (29.2 vs. 8.1%; 83.3 vs. 58.1%, 79.2 vs. 56.5%; P always <0.05). In 58/90 (64%) patients for whom clinical follow-up was available, new vascular events were recorded (coronary/cerebral, n = 38); only 22 patients (24%) received long-term antiplatelet agents (mostly aspirin 100 mg/d), with lower, but not statistically significant, prevalence of overall vascular recurrence (45.4 vs. 70.6%, P = 0.06). High rate of vascular recurrence is shown in patients with previous RVO, in which conventional cardiovascular risk factors play a major role, especially in BRVO and in older patients.

Ramipril in post-renal transplant erythrocytosis.

BACKGROUND: Posttransplant erythrocytosis (PTE; i.e., hematocrit [Ht] >=51%) may be responsible for cardiovascular events. Angiotensin-converting enzyme inhibitors (ACEIs) are increasingly employed in PTE treatment. Diverse ACEIs have been administered at variable doses and with erratic follow-up. In addition, guidelines recommend the administration of ACEIs as first-line therapy for PTE but do not give information on dosage. In this study the dose-response of a single ACEI was assessed, and patients were followed up for 1 year. The role of ACE gene polymorphism in both prevalence of PTE and successful response to ACEI therapy was also tested. METHODS: At study entry, blood chemistry and ACE-gene polymorphism were measured. ACEI (ramipril) was initiated at 1.25 mg/day; if Ht was still >=51%, ramipril was increased every 6 weeks to ensuing greater dosages. Scheduled dosages were 1.25, 2.5, 5.0, 7.5 and 10 mg/day. Blood chemistry was repeated every 6 weeks. Serum erythropoietin (EPO) concentration was assayed at the start and end of the study. Follow-up was extended for 1 year. RESULTS: PTE developed 12.6 +/- 16.0 months after transplantation in 40 out of 400 patients; 27 patients completed the study. Initial Ht was not correlated with any variable. Final Ht appeared normalized in 26 out of 27 patients. Mean dose (+/- SD) of ramipril was 4.6 +/- 3.6 mg. Mean time for correction of PTE was 135 days, and was not dependent on baseline Ht, hemoglobin or EPO. PTE relapsed in 4 patients. Prevalence of PTE and successful response to ramipril was not dependent on ACE-gene polymorphism. CONCLUSION: Ramipril was effective in PTE; low doses normalized Ht in most patients. No clinical characteristics or biochemical variables predicted the response to ramipril. PTE may relapse; thus long-term follow-up is mandatory.

Hyperhomocysteinemia and other inherited prothrombotic conditions in young adults with a history of ischemic stroke.

BACKGROUND AND PURPOSE: The mechanisms of ischemic stroke in young adults are poorly understood. During the last years, several studies suggested a role for genetic factors predisposing to thrombophilia and for moderate hyperhomocysteinemia in this setting. METHODS: We evaluated in 132 consecutive patients (66 males, 66 females; mean+/-SD age, 38.4+/-11.7 years; mean+/-SD age at first event, 34.8+/-10.9 years; range, 6 months to 50 years) referred to our center between January 1997 and December 1999 for a history of young adult ischemic stroke (age at first event, <51 years) the prevalence of factor V (FV) Leiden, prothrombin (FII) G20210A, and C677T and 5,10-methylenetetrahydrofolate reductase (MTHFR) gene mutations and fasting serum total homocysteine levels. Two hundred sixty-two apparently healthy subjects (117 males, 145 females; mean+/-SD age, 36+/-13.2 years) served as controls. RESULTS: Total homocysteine levels differed significantly (P=0.004, t test) between patients and controls: 13.03+/-18.61 versus 10.75+/-6.24 micromol/L (mean+/-SD), respectively. In contrast, homozygosity for the TT mutation of the MTHFR gene was 30 of 132 (22.7%) in patients and 45 of 262 (17.2%) in controls; this difference was not statistically significant (P>0.05, chi(2) test). However, when we stratified the whole population according to genotype, fasting serum homocysteine levels were significantly higher in TT patients than in TT controls (25.3+/-36.8 versus 15+/-11.6 micromol/L; P=0.02, t test). Mutations of FV Leiden and of FII G20210A gene are currently reported to be associated with a tendency toward ischemic stroke. Their frequencies were not statistically significantly different between patients and controls in this setting: 7 of 132 (5.3%) versus 17 of 262 (6.5%) for FV Leiden and 10 of 132 (7.6%) versus 16 of 262 (6.1%) for FII G20210A, respectively (all P>0.05, chi(2) test). CONCLUSIONS: In the present cohort of patients, moderate hyperhomocysteinemia is the only variable that helps to identify young adults with a history of ischemic stroke.