MTHFR Mutation Research Articles

4. Trombophilia and pregnancy – A participation of pai 4 g/5 g

Introduction Pregnancy is hypercoagulable state. The tendency to thrombosis, has been developed rapidly and has been linked to many aspects of pregnancy. It is recently that severe pregnancy complications such as severe preeclampsia, intrauterine growth retardation, abruptio placentae and stillbirth has been shown to be associated with thrombophilia. The obstetrics service of the Federal University of Juiz de Fora has an Ambulatory for the attendance of pregnant women with poor obstetric result and this study was taken. Methods Study was performed when 79 a coort study with poor obstetric results (hypertensive syndromes, intrauterine fetal death, abortium, intrauterina growth retardation, abruption placentae) were evaluated. All those patients were submitted to trombophiilia research (V Leiden factor, mutation of prothrombin, SAAF, MTHFR, protein S, protein C, antithrombin III and PAI 4G/5G mutation). Results The mutation PAI 4G/5G was associated to abortium (p Conclusions The authors conclude that this mutation should be searched in the effective effects of the fetal result.

A pilot trial examining the absorption of oral forms of folate supplementation in a healthy population: A randomised control trial

Background & aimsFolate supplements are commonly prescribed by health professionals for a number of different conditions, however, the absorption of the different derivatives remains unclear. This pilot trial aims to assess the absorption of various forms of folate supplements in a healthy population. MethodsA four-week, single blinded, randomised control trial was conducted on 30 healthy individuals. The intervention included supplementation with 500mcg of either folic acid, folinic acid or 5-Methyltetrahydrofolate (5-MTHF). Fifteen participants were allocated to the intervention and fifteen to the control group for comparison. At baseline (week 0) a case report was completed and pathology tests for serum folate, B12 and MTHFR polymorphisms were conducted. Follow up serum folate blood pathology tests were assessed at week 2 and week 4 along with additional food diaries. ResultsOf the cohort, 87% (n = 26) were found to have a MTHFR polymorphism with both A1298C (n = 12) and C677 T (n = 18) being tested. Only13% (n = 4) of participants had no mutation. The different MTHFR mutations were observed across both the control group and all of the intervention groups. The mean (±SD) baseline folate was 33.7 nmol/L ± 7.55 (Reference range: >9.0). Participants who had both A1298C and C677 T polymorphism had lower baseline folate with a mean (±SD) of 29 nmol/L ± 8.75. During the four weeks an overall increase in mean serum folate was observed in both the folinic acid and 5-MTHF group. The folinic acid intervention saw a mean (±SD) increase of 15.3 nmol/L ± 3.56 and 5-MTHF saw a mean (±SD) increase of 9.1 nmol/L ± 1.67, however a decrease in mean serum folate was detected in the folic acid group. In the folinic acid and 5-MTHF groups, serum folate increases were observed in individuals irrespective of their MTHFR status. ConclusionsThis research has provided a foundation for further work investigating folate absorption. The results of the trial suggest that folinic acid has the best absorption, however, it may not have the best bioavailability. More research is required for greater clarification regarding the absorption and bioavailability of these supplements.

WED 209 A case report of hyperhomocystinemia due to homozygous MTHFR mutation

Homocycteine is metabolized via two pathways, remethylation or trans-sulfuration. The former requires 5-methyltetrahydrofolate reductase (MTHFR) and Vitamin B12, the later requires cystathione–beta-synthetase and Vitamin B6. Mutations in the MTHFR genes affect the enzyme activity to various degree and manifestations can be neuropsychiatric, vascular and neurodegenerative. Here we report a case of severe homocysteinemia secondary to two homozygous MTHFR gene mutations.32 year old pregnant female initially presented in 2014 with grand mal seizure. After being seizure free on antiepileptics, she was admitted again to hospital in January 2017 with headaches, confusion, slurred speech and neuropathic pain in feet. She developed spastic paraparesis and her condition remained critical for several weeks due to refractory seizures and low GCS.She was on warfarin for recurrent DVTs and had family history of mental health problems. Homocysteinemia was discovered when he was screened for metabolic causes and leucodystrophies. MRI of the brain showed cerebral, cerebellar and brain stem atrophy. Neurophysiological studies were normal. Genetic testing found her to be homozygous for 2 MTHFR mutations. She was commenced on calcium folinate and parenteral B12, to which she responded really well.Thorough history, targeted investigations and systematic approach are keys for diagnosis of rare metabolic disorders.

Endometriosis pathogenesis : role played by the oxidative stress due to MTHFR mutations

Endometriosis pathogenesis : role played by the oxidative stress due to MTHFR mutations

P-220 - MTHFR, TSER and DPYD gene mutation is associated with toxicity and response in pre-operative chemo-radiotherapy for local advanced rectal cancer

Introduction: Radiotherapy and 5 FU based chemotherapy is the most common pre-operative regimen used for cT3-T4, N1 rectal cancer (RC). Evaluation of predictive markers of response and toxicity to radio-chemotherapy is a challenging approach for patients (pts) and drug selection. In the present experience we have analyzed the predictive role of the genetic polymorphisms (MTHFR, TSER and DPYD) on toxicity and response to pre-operative radio-chemotherapy. Methods: We have enrolled eighteen patients with locally advanced RC treated with pre-operative radiotherapy and fluoropyrimidines based chemotherapy. Genetic polymorphisms of MTHFR C677T, MTHFRA 1298C, DPYD IVS 14 + 1G>A, DPYDA 2846T, DPYD T 1679 G, TSER 28 bp VNTR were analyzed by PCR and pyrosequencing of genomic DNA extracted from peripheral blood samples. Genetic markers were correlated with toxicity to treatment (chemotherapy and radio-chemotherapy) and clinical response. Results: Patients characteristics were: male 15 pts, female 3 pts, median age 66 years, ECOG PS 0-1 all pts. We found DPYD IVS 14 + 1 G>A G/G homozygous wild type, DPYD A2846T, T/T homozygous wild type and DPYD T1679 g, T/T homozygous wild type in 100% of pts, homozygous wild type MTHFR C677T in 10% of pts, MTHFR C677T homozygous mutated in 50% of pts, heterozygous MTHFR A1298C in 60% of pts and homozygous wild type MTHFR A 1298C in 40% of pts. Adverse events G-3 (diarrhea, neutropenia, asthenia, mucositis) were observed in 60% of pts with heterozygous MTHFR A 1298C and in 10% of pts with homozygous mutated MTHFR C 677t treated with chemo radiotherapy combination. DPYD homozygous wild type was not associated with severe toxicity. Rectal surgery with TME/TEM will be performed 8 weeks after the end of pre-operative chemo-radiotherapy. We obtained 9 pathological complete response and 9 partial pathological response. Adjuvant chemotherapy was well tolerated without G3-G4 adverse events. Five pts with pathological complete response were treated with Transanal Endoscopic Microsurgery (TEM) and they are alive without recurrence to twelve months after surgery. Conclusion: Concomitant assessment of genetic polymorphisms of MTHFR and DPYD is promising to predict severe toxicity during preoperative chemo-radiotherapy approach for pts with locally advanced rectal cancer. This result does not exclude the need to consider other non-genetic factors that might influence the individual enzyme activities.

Choroidal infarction following ophthalmic artery chemotherapy

BackgroundMethylenetetrahydrofolate reductase (MTHFR) genetic mutations and intra-procedural inhaled nitrous oxide (N2O) independently increase blood levels of homocysteine, a compound associated with thrombosis. Patients with MTHFR mutations who also receive N2O during ophthalmic artery chemotherapy (OAC) for retinoblastoma may have a heightened thrombotic risk.Case presentationsSingle-center retrospective review of pediatric patients with advanced retinoblastoma who received OAC and developed choroidal infarcts. Four retinoblastoma patients with advanced intraocular disease (2 males, 2 females: 13–58 months) experienced choroidal infarcts within the one-month period after OAC, in which procedural N2O induction was used (duration between 21 and 58 min). All 4 patients had MTHFR (chromosome 1p, position 36.22) genetic abnormalities: one was homozygous for the C677T mutation, one was C677T heterozygous, one was A1298C heterozygous, and one was heterozygous for both C677T and A1298C. In all 4 patients, indirect ophthalmoscopy and fundus photography showed marked disturbance of the retinal pigment epithelium and optical coherence tomography (OCT) confirmed thinning of the choroid. Follow-up time ranged from 15 to 46 months (median 21 months).ConclusionsChoroidal infarction in eyes treated with OAC developed in children who were both deficient in at least one working allele of the MTHFR gene (heterozygous or homozygous) and received N2O induction during OAC.

MTHFR, TSER and DPYD gene mutation is associated with toxicity and response in pre-operative chemo-radiotherapy for local advanced rectal cancer

Background: Radiotherapy and 5 FU based chemotherapy is the most common pre-operative regimen used for cT3-T4, N1 rectal cancer (RC). Evaluation of predictive markers of response and toxicity to radio-chemotherapy is a challenging approach for patients (pts) and drug selection. in the present experience we have analyzed the predictive role of the genetic polymorfisms (MTHFR, TSER and DPYD) on toxicity and response to pre-operative radio-chemotherapy. Materials and methods: We HAVE enrolled sixteen patients with locally advanced RC treated with pre-operative radiotherapy and fluoropyrimidines base chemotherapy. Genetic polymorphisms of MTHFR C677T, MTHFRA 1298C, DPYD IVS 14 + 1G>A, DPYDA 2846T, DPYD T 1679 G, TSER 28 bp VNTR were analyzed by PCR and pyrosequensing of genomic DNA extracted from peripheral blood samples. Genetics markers were correlated with toxicity to treatment (chemotherapy and radio-chemotherapy) and clinical response. Results: Patients characteristics were: male 13 pts, female 3 pts, median age 66 years, ECOG PS 0-1 all pts. We found DPYD IVS 14 + 1 G>A G/G homozygous wilde type, DPYD A2846T, T/T homozygous wilde type and DPYD T1679 g, T/T homozygous wilde type in 100% of pts, homozygous wilde type MTHFR C677T in 10% of pts, MTHFR C677T homozygous mutated in 50% of pts, heterozygous MTHFR A1298C in 60% of pts and homozygous wilde type MTHFR A 1298C in 40% of pts. G3-G4 advers events (diarrhea, neutropenia, asthenia, mucositis) were observed in 60% of pts with heterozygous MTHFR A 1298C and in 10% of pts with homozygous mutated MTHFR C 677t. treated with chemo-radiotherapy combination. DPYD homozygous wilde type was not associated with severe toxicity. Rectal surgery with TME will be performed 8 weeks after the end of pre-operative chemo-radiotherapy. We obtained 7 patological complete response and nine partial patological response. Adjuvant chemotherapy was well tolerated without G3-G4 adverse events. Three pts with patological complete respose were treated with Transanal Endoscopic Microsurgery (TEM) and they are alive without recurrence to twelve months after surgery. Conclusion: Concomitant assesment of genetic polymorphisms of MTHFR and DPYD is promising to predict severe toxicity during preoperative chemo-radioterapy approach for pts with locally advanced rectal cancer. This result does not exclude the need to consider other non-genetic factors that might influence the individual enzyme activities.

Diagnostic approach to neurotransmitter monoamine disorders: experience from clinical, biochemical, and genetic profiles.

To improve the diagnostic work-up of patients with diverse neurological diseases, we have elaborated specific clinical and CSF neurotransmitter patterns. Neurotransmitter determinations in CSF from 1200 patients revealed abnormal values in 228 (19%) cases. In 54/228 (24%) patients, a final diagnosis was identified. We have reported primary (30/54, 56%) and secondary (24/54, 44%) monoamine neurotransmitter disorders. For primary deficiencies, the most frequently mutated gene was DDC (n=9), and the others included PAH with neuropsychiatric features (n=4), PTS (n=5), QDPR (n=3), SR (n=1), and TH (n=1). We have also identified mutations in SLC6A3, FOXG1 (n=1 of each), MTHFR (n=3), FOLR1, and MTHFD (n=1 of each), for dopamine transporter, neuronal development, and folate metabolism disorders, respectively. For secondary deficiencies, we have identified POLG (n=3), ACSF3 (n=1), NFU1, and SDHD (n=1 of each), playing a role in mitochondrial function. Other mutated genes included: ADAR, RNASEH2B, RNASET2, SLC7A2-IT1 A/B lncRNA, and EXOSC3 involved in nuclear and cytoplasmic metabolism; RanBP2 and CASK implicated in post-traductional and scaffolding modifications; SLC6A19 regulating amino acid transport; MTM1, KCNQ2 (n=2), and ATP1A3 playing a role in nerve cell electrophysiological state. Chromosome abnormalities, del(8)(p23)/dup(12) (p23) (n=1), del(6)(q21) (n=1), dup(17)(p13.3) (n=1), and non-genetic etiologies (n=3) were also identified. We have classified the final 54 diagnoses in 11 distinctive biochemical profiles and described them through 20 clinical features. To identify the specific molecular cause of abnormal NT profiles, (targeted) genomics might be used, to improve diagnosis and allow early treatment of complex and rare neurological genetic diseases.

Maternal methyltetrahydrofolate reductase gene mutation in patients with missed abortions.

BackgroundMissed abortions are a common problem, often caused by thrombophilia in both recurring and non-recurring cases.AimTo determine whether the presence of a mutation in the methyltetrahydrofolate reductase (Mthfr) gene correlates with missed abortions.Patients and methodsWe selected two hundred patients for this study in two groups: the study group, which consisted of one hundred patients with a history of missed abortion; and the control group, which consisted of one hundred patients with no history of missed abortion.ResultsOf the 200 patients, mutations in Mthfr were only found in forty-four patients—thirty-four from the study group and ten from the control group.ConclusionsMthfr gene mutation is a common cause of both recurring and non-recurring missed abortions.

Effect of GSTP1 and MTHFR Gene Polymorphism on Side Effects of HD-MTX in ALL Children

To Study the effect of C677T and MTHFR gene polymorphism on side effects of HD-MTX in ALL children. The gene polymorphism of C677T A303G and MTHFR C677T were detected by PCR in 98 ALL children from January 2014 to January 2016. The side effects during HD-MTX therapy were observed, and the relationship among GSTP1, MTHFR gene polymorphism and incidence of side effect of HD-MTX were analyzed. Among 98 ALL children, the gene variation was observed in 61 ALL children (62.24%). Polymorphism study on C677T A303G showed that the gene frequency of A was 84.69%, while that of G was 15.31%; for polymorphism of MTHFR C677T, gene frequency of C was 66.33%, and that of T was 33.67%. Seven patients(7.14%) experienced with bone marrow supression, 23 patients(23.47%) with liver function damage, 15 patients(15.31%) with renal function damage, 48 patients(48.98%) with gastrointestinal reactions and 46 patients(46.94%) with mucosal lesions. After adjustment of sex, age, risk stratification and dosage of MTX, the gene polymorphism had no significant relationship with bone marrow suppression, gastrointestinal reactions and mucosal lesions(P>0.05). However, the number of the mutant genes had statistically significant relationship with liver and renal function damage(P<0.05). The risk of side effects during HD-MTX therapy increases in ALL children with combined mutation of MTHFR and C677T.

Histology determination of lung cancers: A report on genomic profiling of lung cancer of mixing histology.

8570 Background: Histopathology, largely determined by morphology, plays a critical role in choosing appropriate treatment for lung cancer. The understanding of molecular determination of lung cancer histology is rudimentary. Our recently published data (Zhang, Science, 2014 and Liu, Nature Communications, 2016) have demonstrated that within the same patients with identical genetic background and identical exposure, tumor regions with different morphologic appearances may have very similar genomic profiles while tumors with the same morphology may have distinct genomic landscape. Methods: We collected 12 lung cancers of mixing histology with 2 to 4 histologic components within each tumor. In total, 26 tumor regions including 9 adenocarcinomas, 6 large-cell neuroendocrine carcinoma, 6 small cell carcinomas and 4 squamous cell carcinomas and one poorly differentiated lung carcinoma were microdissected and subjected to whole exome sequencing. Results: A substantial number of identical mutations were shared between different histologic components within the same tumor in all 12 patients. However, the proportion of shared mutations varies in different patients ranging from as little as 4% to as much as 99%. Mutation spectrum is also similar between different histologic components within the same tumors suggesting similar mutational process in place. Identical canonical cancer gene mutations including TP53, KRAS, PIK3CA, SOS1 and STK11 are generally shared between different histologic components within the same tumors. Canonical mutations in FBXW7 and MTHFR were detected in squamous component, but not small-cell component in one patient. Conclusions: Different histologic components of lung cancers of mixing histology are likely derived from the same progenitor cells, but the molecular timing of branch separation of subclones giving rise to different histologic components varies in different tumors. Although genomic aberrations may play a role in a subset of tumors, histologic features may not be determined at genomic level for most lung cancers. Gene expression and methylation analyses from these tumors are underway.

Frequency of Thrombophilic Gene Mutations in Patients with Deep Vein Thrombosis and in Women with Recurrent Pregnancy Loss

AbstractThrombophilia may be anticipated by single or combined hereditary defects in encoding genes factor V, Prothrombin, and MTHFR. The aim of this study was to determine the prevalence and associated risks of V Leiden (G1691A), Prothrombin (G20210A), and MTHFR (C677T) mutations in Saudi women with Deep Vein Thrombosis (DVT) and women with recurrent pregnancy loss (RPL). Protein C and protein S activity were measured to determine combined effects, if any. We examined 60 women with a history of DVT and 60 with RPL, extracted DNA from EDTA blood and determined three mutations by using multiplex PCR reactions followed by Strip Assay KIT. Pro C Global assay was used to determine the cutoff value [PCATNR = 0.80]. Protein C/S chromogenic assay was used to estimate protein C and S percentages. Frequency of Factor V Leiden G/A genotype in patients with DVT 7 (11.6%) had a significant association for DVT χ2 (OR = 5.1, P = 0.03). In women with RPL the three mutations did not show any significant association, levels of Protein C, protein S and PCAT-NR in patient groups not different from controls (P &gt; 0.05). In conclusion, we recommend expanding on these data to provide larger-scale studies.

ГЕН ФОЛАТНОГО ЦИКЛА MTHFR И ПИТАНИЕ

Climatic and geographical features always influenced on adaptation to the environment as well as the development level of economy and culture. The formation of certain food behavior in population determined fixation of specific gene alleles and mutation. They were responsible for digestion a typical food for the population. At the moment there is information about such genetic features as metabolism of proteins, carbohydrates and fats for many races and nations. But a few studies are about causes of prevalence differences of gene polymorphisms of folate cycle in the world's populations. The main objective was to explore 677C>T mutation MTHFR gene among Russian and Buryat to compare it with ones in other populations. The total of399 children and adolescents (200 Russians and 199 Buryats) were involved in this study. Genotyping of MTHFR 677C>T was performed by PCR-RT We used soft STATISTICA 8.0 to compare results. As a result, T-allele frequency was 30 % in Russians and 21,1 % in Buryats. We found significant differences of prevalence of polymorphism 677C>T between studied groups (p = 0.011). Also we found significant difference between Russian and British (p = 0.054), French (p = 0.0263), Spanish (p < 0.0001), Italian (p < 0.0001) and Greek samples (p = 0.0454). The Buryat group had significant differences with Chinese (p < 0.0001), Korean (p < 0.0001), Mongolian (p < 0.004), Japanese (p < 0.0001), Kazakh (p = 0.0198) samples and two samples of Hans (p < 0.0001; p = 0.0390).

Successful management of hereditary angioedema during pregnancy in a patient with heterozygous MTHFR mutation

Successful management of hereditary angioedema during pregnancy in a patient with heterozygous MTHFR mutation

Superficial vein thrombosis in non-varicose veins of the lower limbs and thrombophilia.

Objectives Superficial vein thrombosis in non-varicose veins of the lower limbs is rather frequent and may be underestimated. This study aims to evaluate the prevalence of inherited or acquired thrombophilia in a sample of outpatients with the disease. Method An observational study was conducted on 73 consecutive superficial vein thrombosis patients tested for inherited or acquired thrombophilia. Results Sixty of 73 patients with superficial vein thrombosis completed the testing protocol, while 13 dropped out; 46 of 60 patients were found to have a thrombophilia (76.6%). The types detected were: factor V Leiden (31/60, i.e. 51.6%), prothrombin mutation (2/60, i.e. 3.3%), MTHFR mutation (23/60, i.e. 38.3%), antiphospholipid antibodies (5/60, i.e. 8.3%), protein C deficit (1/60, i.e. 1.6%), protein S deficit (1/60, i.e. 1.6%), and antithrombin deficit (0/60, i.e. 0%). Conclusions Among patients with superficial vein thrombosis in non-varicose veins, testing demonstrated a high prevalence of thrombophilia. The most common form proved to be factor V Leiden. As thrombophilia was found to be a major cause of superficial vein thrombosis in non-varicose veins, the authors recommend that patients with superficial vein thrombosis in non-varicose veins be investigated for thrombophilia.

Diagnostic Yield and Novel Candidate Genes by Exome Sequencing in 152 Consanguineous Families With Neurodevelopmental Disorders

Autosomal recessive inherited neurodevelopmental disorders are highly heterogeneous, and many, possibly most, of the disease genes are still unknown. To promote the identification of disease genes through confirmation of previously described genes and presentation of novel candidates and provide an overview of the diagnostic yield of exome sequencing in consanguineous families. Autozygosity mapping in families and exome sequencing of index patients were performed in 152 consanguineous families (the parents descended from a same ancestor) with at least 1 offspring with intellectual disability (ID). The study was conducted from July 1, 2008, to June 30, 2015, and data analysis was conducted from July 1, 2015, to August 31, 2016. Of the 152 consanguineous families enrolled, 1 child (in 45 families [29.6%]) or multiple children (107 families [70.4%]) had ID; additional features were present in 140 of the families (92.1%). The mean (SD) age of the children was 10.3 (9.0) years, and 171 of 297 (57.6%) were male. In 109 families (71.7%), potentially protein-disrupting and clinically relevant variants were identified. Of these, a clear clinical genetic diagnosis was made in 56 families (36.8%) owing to 57 (likely) pathogenic variants in 50 genes already established in neurodevelopmental disorders (46 autosomal recessive, 2 X-linked, and 2 de novo) or in 7 previously proposed recessive candidates. In 5 of these families, potentially treatable disorders were diagnosed (mutations in PAH, CBS, MTHFR, CYP27A1, and HIBCH), and in 1 family, 2 disease-causing homozygous variants in different genes were identified. In another 48 families (31.6%), 52 convincing recessive variants in candidate genes that were not previously reported in regard to neurodevelopmental disorders were identified. Of these, 14 were homozygous and truncating in GRM7, STX1A, CCAR2, EEF1D, GALNT2, SLC44A1, LRRIQ3, AMZ2, CLMN, SEC23IP, INIP, NARG2, FAM234B, and TRAP1. The diagnostic yield was higher in individuals with severe ID (35 of 77 [45.5%]), in multiplex families (42 of 107 [39.3%]), in patients with additional features (30 of 70 [42.9%]), and in those with remotely related parents (15 of 34 [44.1%]). Because of the high diagnostic yield of 36.8% and the possibility of identifying treatable diseases or the coexistence of several disease-causing variants, using exome sequencing as a first-line diagnostic approach in consanguineous families with neurodevelopmental disorders is recommended. Furthermore, the literature is enriched with 52 convincing candidate genes that are awaiting confirmation in independent families.

The Headaches and Polymorphisms of the Methylenetetrahydrofolate Reductase

The headaches are a heterogeneous clinical manifestations characterized not only by pain but also by a severe multifactorial disabilities. Numerous studies showed the association between the enzyme MTHFR mutations, the T allele and genotype TT and migraine, particularly migraine with aura. Aim of the study: On the basis of the literature data we propose to evaluate the prevalence of the MTHFR mutation in the population of patients admitted to a headache center of the childhood. Materials and methods: we examined 226 children, aged 5-17 years, admitted to Headache Center of Children Neuropsychiatry of Hospital San Salvatore L'Aquila, from 2013 to the year 2015. The diagnosis of headache is made according to ICHD III criteria. The identification of the MTHFR C677T polymorphism of the enzyme was carried out by the Real-time PCR qualitative. The statistical analysis by SAS System. Results: Diagnosis of migraine without aura in 96 patients, migraine with aura in 39, in 18 chronic migraine, frequent episodic tension-type headache in 40, chronic tension type headache in 20 and in 13 other headaches. 61 patients are without mutation, 85 with heterozygous mutation, and 28 homozygous. Therefore, the mutation is present in 147 patients with a percentage of 65%. The heterozygous mutation is present in 50.56% of females and 47.06% males; the homozygous in 10.19% and 12.94% in females than males. In migraineurs the mutation is present in 66.67% compared with patients with tension-type headache (33.33%), homozygous is the 70.37% versus 29.63% (p<0.001). Homocysteine levels are elevated only on the 8.69% of mutated patients, of which 5/12 patients have a mutation in homozygosity. Conclusion: we confirme the statistical association between MTHFR mutation both in the heterozygous and homozygous forms and migraines. The migraines with aura, unlike the literature, does not seem to have an important role.

Common Prothrombotic Gene Mutations in Cerebral Venous Sinus Thrombosis in North-West of Iran

Objective: Cerebral venous sinus thrombosis (CVST) is a life-threatening cerebrovascular disease which has high prevalence and mortality rate in Iran. Thrombophilia caused by gene mutation is a common cause of CVST. The present study aimed at assessing the prevalence of thrombophilic gene mutations in Iranian CVST patients and then comparing it with normal population. Materials and methods: In a case-control study, polymerase chain reaction-restriction fragment length polymorphism (PCR_RFLP) and amplification-refractory mutation system (ARMS-PCR) were carried out to detect common thrombophilic mutations in 70 CVST patients. Next, it was compared with 82 sex- and age-matched healthy controls. Results: Factor-V-Leiden, Factor-V-Leiden HR2, Factor prothrombin II, MTHFR (667C/T) and MTHFR (1298A/C) prevalence were significantly high in cases of CVST as compared to the controls (P values: 0.012, 0.019, 0.007 and 0.036, respectively). However, there was no significant difference between the two groups in plasminogen activator inhibitor (PAI), angiotensin-converting enzyme (ACE), beta-fibrinogen (FGB), Factor VIII, Factor XIII, and tissue plasminogen activator (tPA) mutations. Conclusion: The findings of the present study suggest that Factor V-Leiden, Factor-V-Leiden HR2, prothrombin II (G20210A), and MTHFR (667C/T & 1298A/C) mutations are more frequent in CVST. Detection of these mutations may help clinicians to decide on the duration of treatment and referral to genetic counseling for valuable prevention.

Inverse PAR1 Activity of Hematopoietic Stem Cells and BM Stromal Cells Mediates G-CSF-Induced Mobilization By Regulation of Nitric Oxide Generation

Hematopoietic stem and progenitor cell (HSPC) egress from the bone marrow (BM) to the circulation is tightly regulated and is accelerated during stress conditions, a process utilized for BM harvest. Recently, we demonstrated that mouse long term repopulating hematopoietic stem cell (LT-HSC) BM retention and their rapid release to the blood circulation are governed by a switch in nitric oxide (NO) generation via distinct coagulation-related protease activated receptor 1 (PAR1) cascades (Gur-Cohen S. et al., NM, 2016).Herein we report that surface PAR1 expression can be exploited and serve as a positive predictive marker for the efficiency of human CD34+ HSPC mobilization among healthy donors in clinical G-CSF-induced mobilization for matched allogeneic transplantations. We found that PAR1 expression on circulating leukocytes before G-CSF administration was positively correlated with higher yields of mobilized leukocytes after stimulation (P<0.001). In addition, the percentage of PAR1-expressing CD34+ HSPCs in the blood and their absolute numbers before initiation of mobilization positively correlated with peripheral blood leukocyte counts (P<0.001) and yield of mobilized CD34+ HSPCs (P=0.002). Importantly, tracing the consequences of the follow-up BM transplantation, we found accelerated neutrophil recovery in patients transplanted with mobilized cells expressing higher surface PAR1 levels at baseline (P=0.074), shortening the time for neutrophil recovery (neutrophil count>0.5 x 109/L) from an average of 14.6 days to an average of 11 days post-transplant. In addition, a trend of accelerated platelet production was documented to be related with higher PAR1 expression by circulating leukocytes prior to G-CSF stimulation. Consequently, poor mobilizer donors were characterized by extremely low surface PAR1 expression on circulating CD34+ cells prior to G-CSF stimulations. Herein we present a case report of a thrombophilic donor carrying the MTHFR mutation, expressing exceedingly low PAR1 levels at baseline, with the outcome of inadequate numbers of mobilized CD34+ HSPC in the blood following G-CSF treatments.To further gain insight into the role played by PAR1 signaling in the regulation of G-CSF-induced HSPC mobilization, we used mice as a functional preclinical small animal model. We found that antagonizing PAR1 signaling attenuated both steady state release and G-CSF-induced HSPC mobilization. Furthermore, co-administration of G-CSF with PAR1 antagonist attenuated secretion of BM stromal CXCL12 and abrogated upregulation of surface CXCR4 and PAR1 expression by BM HSPCs, all leading to significantly reduced HSPC migration, differentiation and mobilization. In support, PAR1-/- mice failed to efficiently mobilize HSPCs in response to G-CSF compared to wild type counterparts. Enforced HSPC recruitment by G-CSF treatments dramatically accelerated PAR1-dependent NO production by eNOS, known to promote TACE-mediated EPCR shedding and rapid LT-HSC mobilization. Concomitantly, circulating steady state and G-CSF-mobilized stem cells lack surface EPCR expression. Intriguingly, while EPCR expression by primitive BM stem cells was transiently reduced after G-CSF treatments, antagonizing PAR1 signaling along with G-CSF stimuli blocked NO generation and synchronically expanded BM EPCR+ LT-HSC and their supportive stromal progenitor cells (MSPCs), as confirmed by increased repopulation in transplanted mice. Finally, we report an inverse PAR1 expression and regulation by BM HSPC and stromal MSPCs in mediating G-CSF-induced mobilization. G-CSF induced elevation of PAR1 expression on BM HSPCs, providing the driving force for their enhanced NO mediated migration, proliferation, differentiation and recruitment to the circulation. Contrary, the levels of PAR1 expression were reduced on MSPCs in response to G-CSF treatment, and following NO generation by eNOS activity and CXCL12 secretion, resulted in reduced mesenchymal differentiation leading to accumulated numbers of immature mesenchymal (CFU-F) and osteoblast (CFU-OB) progenitor cells.Taken together, our study identifies and highlights inverse PAR1 signaling and NO generation as essential regulator of G-CSF induced HSPC mobilization and MSPC development opening new avenues to advance therapeutics for enhancing clinical G-CSF induced stem cell mobilization and transplantation protocols. DisclosuresRuf:Iconic Therapeutics: Consultancy.

Thrombophilia in Pathological Pregnancies

Abstract Introduction. Many situations in pregnancy create a thrombophilia, which is believed to have a protective function, which is the result of the hormone influence, primarily by gestagenic hormones. This is a physiological condition and it helps in successful implantation of the embryo and prevention of bleeding. The presence of additional, hereditary or acquired cause of thrombophilia disrupts the natural balance and creates conditions for venous thromboembolism and consequences for pregnancy, such as miscarriage, intrauterine fetal death, delayed intrauterine growth, eclampsia and abruption of the placenta. Methods. The study was designed as a retrospective epidemiological cross-sectional study and was conducted at the Institute of Transfusion Medicine in Skopje in the period from June to October 2016. The study included patients with current pathological pregnancies. The following laboratory analyses were conducted in all patients: hemostasis, D-dimer, antithrombin III, protein C, protein S, antiphospholipid antibodies, lupus anticoagulant, MTHFR, factor V Leiden and prothrombin mutation. Results. The study included 41 patients, of whom 3 were excluded due to an insufficient medical history, 3 who were receiving corticosteroid therapy, 2 due to incomplete laboratory analyses and one patient with proven systemic lupus. Data analysis showed that family history of thrombophilia was present in 25%. The most common MTHFR mutation was recorded in 26 (82%) patients, followed by the factor V mutation present in 18 (55%) of the examined patients. AT III deficiency was detected in only one patient (2%). In 12 patients (35%) a disturbed ratio of DD vs. markedly shortened APTT or inappropriately low levels of DD was registered, which is an indirect indicatorof hypofibrinolysis. Conclusion. Pathologic pregnancy is a condition that is often associated with thrombophilia. Advanced age of a patient, family and personal history of thrombotic conditions indicate a high risk of complications in pregnancy. Due to the current increased frequency of high risk pregnancies associated with circulation problems with the placenta, there is a need for more frequent hemostatic examinations in pregnancy.