Molecular analysis of FVIII gene in severe HA patients of Costa Rica.

UNLABELLED: Haemophilia A (HA) is X-chromosome linked bleeding disorders caused by deficiency of the coagulation factor VIII (FVIII). It is caused by FVIII gene intron 22 inversion (Inv22) in approximately 45% and by intron 1 inversion (Inv1) in 5% of the patients. Both inversions occur as a result of intrachromosomal recombination between homologous regions, in intron 1 or 22 and their extragenic copy located telomeric to the FVIII gene. The aim of this study was to analyze the presence of these mutations in 25 HA Costa Rican families. PATIENTS, METHODS: We studied 34 HA patients and 110 unrelated obligate members and possible carriers for the presence of Inv22or Inv1. Standard analyses of the factor VIII gene were used incl. Southern blot and long-range polymerase chain reaction for inversion analysis. RESULTS: We found altered Inv22 restriction profiles in 21 patients and 37 carriers. It was found type 1 and type 2 of the inversion of Inv22. During the screening for Inv1 among the HA patient, who were Inv22 negative, we did not found this mutation. DISCUSSION: Our data highlight the importance of the analysis of Inv22 for their association with development of inhibitors in the HA patients and we are continuous searching of Inv1 mutation. This knowledge represents a step for genetic counseling and prevention of the inhibitor development.

Factor VII deficiency: clinical manifestation of 717 subjects from Europe and Latin America with mutations in the factor 7 gene.

The congenital FVII deficiency (FVIID) is a rare haemorrhagic disorder with an autosomal recessive pattern of inheritance. Data on phenotype and the genotype from 717 subjects in Central Europe (six countries), Latin America (Costa Rica, Venezuela) and United States, enrolled in the Greifswald Registry of FVII Deficiency were analysed. We detected 131 different mutations in 73 homozygous, 145 compound heterozygous and 499 heterozygous subjects. Regional differences were observed in the mutation pattern and the clinical profile of the evaluated patients. Seventy-one per cent of homozygous and 50% of compound heterozygous subjects were symptomatic. The clinical manifestations of the homozygous subjects were characterized by intracranial haemorrhage (2%), gastrointestinal bleeding (17%), haemarthrosis (13%), epistaxis (58%), gum bleeding (38%), easy bruising (37%), haematoma (15%), haematuria (10%) and menorrhagia (19 of 26 females, 73%). The clinical variability and genotype-phenotype correlation was evaluated in the homozygous subjects. The pattern of bleeding symptoms among compound heterozygous patients was severe and similar to that of the homozygous patients. The large-scale analysis of 499 heterozygous subjects shows that 93 (19%) presented with spontaneous bleeding symptoms such as haemarthrosis (4%), epistaxis (54%), gum bleeding (14%), easy bruising (38%), haematoma (23%), haematuria (5%) and menorrhagia (19 of 45 females; 42%). The severe haemorrhages - intracranial and gastrointestinal - were not reported in heterozygous subjects. The clinical variability and the regional differences in the mutation pattern are discussed regarding care and treatment.

Factor X deficiency: clinical manifestation of 102 subjects from Europe and Latin America with mutations in the factor 10 gene.

Inherited factor X deficiency (FXD) is a rare (1:1,000,000) recessive bleeding disorder. The clinical and laboratory phenotypes of FXD are poorly correlated and few regional studies on the genotype and the clinical manifestations of FXD are known. To understand the association between clinical manifestations and causative genotype, detailed evaluation of bleeding pattern in a high number of patients is needed. This international study analysed the phenotype and genotype of 102 subjects from Central Europe (Germany, Poland and Slovakia) and Latin America (Costa Rica and Venezuela) with causative mutations in the F10 gene, via sequencing. Twenty-eight homozygous, seven compound-heterozygous and 67 heterozygous FXD subjects were characterized. Twenty-nine different causative mutations, including 15 novel mutations, were analysed. Spontaneous bleeding symptoms in 42 symptomatic individuals (26 homozygous, seven compound heterozygous and nine heterozygous) comprised easy bruising (55%), haematoma (43%), epistaxis (36%), haemarthrosis (33%), intracranial haemorrhage (ICH; 21%), and gastrointestinal (GI) haemorrhage (12%). The manifestation of bleeding symptoms in 9 of 67 (13%) symptomatic heterozygous subjects is described. The bleeding patterns of the enrolled patients showed differences that are associated with the types of F10 mutation, and the corresponding genotypes. The homozygous patients were evaluated for genotype-phenotype correlation. The results suggested that ICH seems to be associated with the F10 mutation Gly380Arg, and possibly with the mutations IVS7-1G>A and Tyr163delAT. A tentative association of other mutations to severe symptoms such as haemarthrosis and GI haemorrhage is reported. The severity of FXD, the genotype-phenotype association, and the results of regional studies are discussed.

Long-term FVII substitution in a preterm infant with severe gastrointestinal bleeding and FVII deficiency due to a homozygous donor splice mutation IVS4+1G-->A.

Congenital FVII deficiency is a rare bleeding disorder. Clinical complications are similar to those seen in hemophilia A, and an increased incidence of intracerebral hemorrhage related to birth trauma has been reported. The authors report on an infant who presented at the second day of life with melaena and hematemesis caused by congenital FVII deficiency with minimal activity of 4%. A homozygous mutation IVS4+G-->A, formerly described in 2 siblings, who died of brain hemorrhage within the first month of life, was identified. Severe bleeding events were prevented with prophylactic treatment. Early identification of the underlying mutation helps to assess the risk of hemorrhage and prevent severe bleeding by prophylactic FVII therapy.

Biochemical indicators of nutritional status and dietary intake in Costa Rican Cabécar Indian adolescents.

The purpose of this study was to determine the blood levels of selected nutritional status indicators and the dietary intake of Costa Rican Cabécar Indians aged 10 to 16 years. The results showed that 65% of the adolescents had an adequate body mass index (BMI) for their age, and 32% had a BMI < 5th percentile. Likewise, the study revealed a high prevalence of anemia (57%), deficient serum folate levels (54%), deficient vitamin B12 levels (31%), and subclinical vitamin A deficiency (10%). Additionally, the youngsters had elevated prevalences of high triglyceride levels (77%), borderline high-density lipoprotein (HDL) cholesterol levels (46%), homocysteine levels > 10 micromol/L (29%), and homozygous mutation of methylenetetrahydrofolate reductase (MTHFR) (49%). The diet was poor, being high in saturated fat and low in polyunsaturated fat, fiber, and several micronutrients. The dietary intakes of more than 55% of the adolescents did not meet 50% of the estimated average requirements (EAR) for zinc, vitamin A, vitamin C, vitamin B12, vitamin B2, and folate. Furthermore, a high prevalence of parasitosis was found (68%). Our results show an adolescent Cabécar population with a mosaic of nutritional deficiencies and cardiovascular risk factors.

Molecular diagnosis of hemophilia A and B. Report of five families from Costa Rica

Hemophilia A and B are X-chromosome linked bleeding disorders caused by deficiency of the respective coagulation factor VIII and IX. Affected individuals develop a variable phenotype of hemorrhage caused by a broad range of mutations within the Factor VIII or Factor IX gene. Here, were report the results of the molecular diagnosis in a five Costa Rican families affected with Hemophilia. Methods of indirect and direct molecular diagnosis are applied in three Hemophilia A and two Hemophilia B families from Costa Rica as well as preconditions, practicability and facilities of this diagnosis. In two families with Hemophilia A and both families with Hemophilia B the causative mutation could be detected by Southern blotting, polymerase chain reaction or sequence analysis. One Hemophilia A family could only analyzed by linkage analysis using genomic markers.

[Factors VII, VIII, IX, and X: molecular genetics and gene diagnosis]. / Gerinnungsfactoren VII, VIII, IX und X. Ausgewählte Aspekte zur Molekulargenetik und Gendiagnostik.

Molecular genetic aspects of the inherited bleeding disorders haemophilia A and B, deficiencies of factor VII and X are described. The spectrum of the mutations is characterized. For genetic counselling the X-chromosomal inheritance of haemophilia and the principles of the indirect and direct genomic diagnosis are explained. Clinics and genetics of the rare inherited bleeding disorders known as factor VII and factor X deficiency are summarized. The mutations spectrum, the role of polymorphisms, the bleeding pattern and genotype-phenotype relations are described.

Molecular diagnosis of hemophilia A and B. Report of five families from Costa Rica.

Hemophilia A and B are X-chromosome linked bleeding disorders caused by deficiency of the respective coagulation factor VIII and IX. Affected individuals develop a variable phenotype of hemorrhage caused by a broad range of mutations within the Factor VIII or Factor IX gene. Here, were report the results of the molecular diagnosis in a five Costa Rican families affected with Hemophilia. Methods of indirect and direct molecular diagnosis are applied in three Hemophilia A and two Hemophilia B families from Costa Rica as well as preconditions, practicability and facilities of this diagnosis. In two families with Hemophilia A and both families with Hemophilia B the causative mutation could be detected by Southern blotting, polymerase chain reaction or sequence analysis. One Hemophilia A family could only analyzed by linkage analysis using genomic markers.

Thrombosis in inherited factor VII deficiency.

Thrombosis in congenital factor (F) VII deficiency was investigated through extensive phenotypic and molecular-genetic studies. Patients with a history of thrombosis among 514 entries in the FVII Deficiency Study Group database were evaluated. Thrombotic events were arterial in one case, disseminated intravascular coagulation in another and venous in seven. Gene mutations were characterized in eight patients: three were homozygous, three compound heterozygous and two heterozygous. FXa and IIa generation assays were consistent with the genetic lesions. One patient was heterozygous for the FV Leiden and one for the FIIG20210A mutation. In seven patients, surgical interventions and/or replacement therapies had a close temporal relationship with thrombosis, while in the remaining, events were apparently spontaneous. Thromboses were not associated with any specific age, phenotype, mutation zygosity or thrombophilic abnormalities. In particular, severe FVII deficiency did not seem to offer protection from strong thrombosis risk factors such as surgery and replacement therapy.

The interleukin-1 polymorphism, smoking, and the risk of periodontal disease in the population-based SHIP study.

Several studies have shown a role for interleukin-1 gene cluster polymorphisms in the risk assessment for periodontal diseases. In the Study of Health in Pomerania (SHIP), 3148 subjects were randomly selected from the population and assessed for a broad range of diseases and environmental/behavioral risk factors. From the complete study group in the age 40 to 60 years, N = 1085 subjects were genotyped for the interleukin-1 genotype composite polymorphism in relation to periodontal parameters. The study objective was to elucidate the gene-environment interaction between the risk factors smoking and IL-1 polymorphism. An increased risk of periodontal disease was found for IL-1 genotype-positive smokers: odds ratio adjusted for age, sex, education, and plaque OR = 2.50 (95% C.I. 1.21 to 5.13; p = 0.013). This was not the case with subjects who never smoked: OR = 1.09 (0.73-1.62; p = 0.676). These results support the hypothesis of gene-environmental interaction in periodontitis.

Estudio de factores trombogénicos en pacientes menores de 45 años con infarto de miocardio / Study of thrombogenic factors in patient under 45 year with myocardium infarct

Introducción: El estudio se orientó a buscar trombofilias en pacientes jóvenes con infarto de miocardio tanto genéticas, como las de las proteínas anticoagulantes. Material y métodos: Las pruebas de laboratorio que se usaron para la medición del fibrinógeno fue el método de Clauss, para la proteína C y Antitrombina III (AIII) se utilizó un método cromogénico, para la S un método coagulométrico y las pruebas genéticas por Reacción de Cadena Polimerasa (PCR). Se seleccionaron 51 pacientes jóvenes menores de 45 años con infarto agudo del miocardio y 43 pacientes sanos de grupo control de las mismas edades. Resultados: Se encontró un 85 por ciento de pacientes masculinos y 15 por ciento femeninos. Las variables que fueron factores de riesgo estadísticamente significativas fueron el sexo P = 0.0023 demostrando que por cada 4.19 hombres sufren infarto existe una mujer. El índice de masa corporal fue significativamente mayor en los pacientes infartados con un valor de P = 0.002733 y existió asociación estadísticamente significativa con obstrucciones coronarias. La hipertensión también fue estadísticamente significativa valor de P = 0.00167. Para el nivel de colesterol total la medida en los pacientes sanos fue de 166mg/dl contra 213mg/dl en los pacientes con infarto con un valor de P = 0.000527mg/dl. Para el HDL colsesterol el 78 por ciento de los pacientes infartados tenían niveles iguales o menores de 35mg/dl y existió asociación estadísticamente significativa con la presencia de obstrucciones coronarias. El nivel de triglicéridos mostró una asociación estadísticamente significativa con un valor de P= 0.000161mg/dl. El 58 por ciento de los pacientes infartados tenína un nivel elevado de LDL colesterol. En el anális de las proteínas anticoagulantes la población enferma tuvo niveles significativamente menores que el grupo control tanto para la proteína S y la C. En el análisis de cruces hay una asociación estadísticamente significativa entre la frecuencia alélica de la mutación de la enzima metiltetrahidrofolato reductasa y tener niveles menores de las proteínas C y S tanto en los pacientes enfermos como en los sanos que son homocigotas a la mutación. No hubo asociación estadísticamente significativa para el factor V Leyden y la variable 20210 de Protombina. Conclusión: En los pacientes menores de 45 con infarto del miocardio existen claros marcadores de riesgo para enfermedad coronaria al compararlos con sujetos sanos de una edad similar.

Influence of the dopamine D2 receptor (DRD2) exon 8 genotype on efficacy of tiapride and clinical outcome of alcohol withdrawal.

We tested the hypothesis that allelic variants of the human dopamine D2 receptor E8 genotype are associated with (i) dopamine D2 antagonist tiapride dose in treatment of alcohol withdrawal (n = 50) and (ii) with anxiety and depression in patients during alcoholism detoxification therapy (admission n = 87; discharge n = 50). DRD2 E8 A/A genotype was associated with increased dose of tiapride during a 9-day detoxification therapy and with increased anxiety and depression scores on admission and 2 weeks later. The findings suggest a pharmacogenetic influence of DRD2 E8 genotype on tiapride efficacy in alcohol withdrawal. In an earlier report, DRD2 E8 A/A genotype was associated with reduced responsiveness to the dopamine D2 agonist apomorphine; however, it is not clear whether both findings share the same biological basis. Earlier findings concerning association of DRD2 E8 A/A with increased anxiety and depression are replicated for the first time.

Factor V Leiden, prothrombin gene G20210A variant, and methylenetetrahydrofolate reductase C677T genotype in young adults with ischemic stroke.

Ischemic stroke in young adults is a well-known disease, but despite extensive clinical and laboratory investigations, its etiology remains unclear in approximately half of the cases. We examined the prevalence of factor V Leiden, the prothrombin G20210A genotype, and the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene in 100 patients (51 males and 49 females) who survived an ischemic stroke without a cardiac embolic source at an age < or = 45 years, and in 238 healthy control subjects from the same geographic area. The patients were selected for study only if the diagnosis of stroke was documented by computed tomography scan or nuclear magnetic resonance (NMR) of the brain, or both. Heterozygosity for the FV Leiden mutation was found in 3 patients (3.0%) and in 10 control subjects (4.2%). Two patients (2.0%) and five control subjects (2.1%) were heterozygous for the prothrombin G20210A mutation. The frequencies of the MTHFR 677TT, CT, and CC genotypes in the patient group were 12%, 37%, and 51%, respectively, and were not significantly different from those in control subjects (11%, 40%, and 49%, respectively). In conclusion, our results indicate that FV Leiden mutation, prothrombin G20210A genotype, and homozygosity for the C677T mutation in the MTHFR gene are not associated with an increased risk for ischemic stroke in young adults.

Lithuanian haemophilia A and B registry comprising phenotypic and genotypic data.

Haemophilia represents the most common hereditary severe bleeding disorder in humans. About 100 families with this condition live in Lithuania, one of the Baltic states with a population of 3.7 million. Haemophilia care and genetic counselling are still rendered difficult owing to limited availability of clotting factor concentrate and molecular genetic diagnosis. In the present study, a haemophilia registry, comprising phenotypic and genotypic data of the majority of Lithuanian haemophilia A and B patients, was established. The phenotype includes the degree of severity, factor VIII:C, factor VIII:Ag, factor IX:C, von Willebrand factor and antigen (VWF:RiCoF, vWF:Ag) and inhibitor status. Genotyping of the factor VIII and IX genes was performed using mutation screening methods and direct sequencing. In 61 out of 63 patients with haemophilia A (96.8%) and all eight patients with haemophilia B (100%), the causative mutations could be detected. Nineteen of the factor VIII gene defects and two of the factor IX gene mutations are reported for the first time. Identified mutations allowed direct carrier diagnosis in 83 female relatives revealing 44 carriers, 38 non-carriers and one somatic mosaicism. The information provided by this registry will be helpful for monitoring the treatment of Lithuanian haemophilia patients and also for reliable genetic counselling of the affected families in the future.

Factor VIII gene polymorphisms in the Asian Indian population.

Little is known about the heterozygous frequency of factor VIII gene markers in the Asian Indian population. The objective of this study was to establish the heterozygous frequency of polymorphic markers within and flanking the factor VIII gene in Indians and identify those most informative for carrier screening and prenatal diagnosis. Factor VIII gene polymorphism analysis at intragenic and extragenic sites was carried out by the polymerase chain reaction (PCR) method and Southern blot procedure. Sixty-three Asian Indian haemophiliacs and their families were screened. A control group of 150 women from nonhaemophilic families were screened for two markers, HindIII and BclI. Among the intragenic markers studied, the HindIII restriction fragment length polymorphism (RFLP) showed the highest heterozygous frequency (0.52) followed by the intron 13 (0.47) and intron 22 (0. 44) short tandem repeats (STRs). Among extragenic markers, TaqI had the highest heterozygous frequency (0.75) followed by BglII (0.54). The intron 22 inversion mutation was observed in eight (40%) of 20 severe cases. In the population studied the most diagnostic polymorphisms were the intragenic markers, intron 22 (70%) STR followed by the intron 13 (52%) STR and HindIII (52%) RFLP, and the TaqI (50%) extragenic marker. Application of HindIII, BclI and the intron 22 dinucleotide repeat combined were diagnostic in 87.2% of haemophilia A families studied.

Molecular biology and clinical manifestation of hereditary factor VII deficiency.

Inherited factor VII (FVII) deficiency is a rare autosomal recessive disorder. Mutations and polymorphisms of the FVII gene were characterized in more than 40 unrelated patients with FVII deficiency. Among the 29 different mutations, the most frequent were Ala294 Val, Ala294Val;404delC, IVS7+7, and Val281 Phe. Four novel mutations (IVS2+1G>C, Arg247 Cys, Glu265 Lys, Asp343 His) were detected. The relationships between genotypes of mutations and polymorphisms of the FVII gene, FVII deficiency, and clinical phenotype were investigated. Homozygosity of the Phe4 Leu, IVS4+1G>A, Cys135 Arg, Ala244 Val, and Ala294 Val;404delC and the double heterozygosity of Tyr68 Cys / IVS3-1G>A, Val252 Met / IVS2+5G>T, Val281 Phe / Cys135 Arg, Ala294 Val / Val281 Phe, Ala294 Val;404delC / Val281Phe, Ala294 Val;404delC / Arg152 stop, Ala294Val;404delC / Gln(-35) stop, Ala294 Val / Val252 Met, Ala294 Val / Gly156 Asp, and Thr359 Met / Asp242 His were related to clinical symptoms. Double heterozygotes for Arg247 Cys / IVS2+1G>C, Ala206 Thr / Pro303 Arg, Leu(-20) Pro / Val252 Met as well as IVS7+7 /Ala294 Val, IVS7+7 /Ala206 Thr, and IVS7+7 / Met298 Ile were asymptomatic. The clinical symptomatology is rather poor in correlation with the FVII activity. Concerning the clinical phanotype, a correlation seems to exist between specific mutations and clinical symptoms.

First hints for a correlation between amplification of the Int-2 gene and infection with human papillomavirus in head and neck squamous cell carcinomas.

Infection with human papillomavirus (HPV) and alterations in certain genes have frequently been proposed as mechanisms in the pathogenesis of head and neck squamous cell carcinoma (HNSCC). Here, we investigated 47 HNSCC for the presence of HPV and, by fluorescence in situ hybridisation, for amplification of Int-2 and Hst-1 in the search for a possible correlation. The highest frequency of HPV infection was found in hypopharyngeal carcinomas, while amplification of Int-2 or Hst-1 was distributed more equally among the different localisations. Amplification of Int-2 was detectable (7 of 9 cases) in 78% of the HPV-positive carcinomas, whereas no virus infection could be found in the five cases with amplified Hst-1 only. In spite of the rather low number of infected tumour samples, our results suggest a correlation between HPV infection and amplification of Int-2.