RESUMO
Dental care professionals regularly see patients with hypodontia. Hypodontia can be acquired, for example through chemotherapy or radiation at a young age, but is hereditary in most patients. Due to an error (pathogenic variant) in one of the many genes that control odontogenesis, the formation of the tooth germ is disrupted at an early stage. The genes involved are not only crucial for tooth development, but they also play an important role in other physical processes. This article provides background information on hypodontia. Based on an inventory of gastrointestinal complaints in patients with hypodontia and a case description of the simultaneous occurrence of a coagulation disorder and hypodontia, the importance of a broad view of this patient group is illustrated. It is concluded that, in addition to a dental assessment, examination of these patients should include a limited physical examination and the medical history of the patient and his close relatives.
Assuntos
Anodontia , Dente , Humanos , Anodontia/patologia , OdontogêneseRESUMO
STUDY QUESTION: Are fragile X mental retardation gene 1 (FMR1) CGG repeats in the normal and intermediate range (up to 55 repeats) associated with primary ovarian insufficiency (POI) in a large case-control study? SUMMARY ANSWER: No association was found between CGG repeats of intermediate size and POI compared with controls. WHAT IS KNOWN ALREADY: CGG repeats in the FMR1 gene in the premutation range (55-200 repeats) have consistenly associated with POI. Intermediate range CGG repeats have been considered for a potential association with POI. STUDY DESIGN, SIZE: A case-control study in 375 well-phenotyped Dutch women diagnosed with POI and 3368 controls with natural menopause ≥40 years of age. PARTICIPANTS/MATERIALS, SETTING, METHODS: The FMR1 CGG repeat number was determined by PCR amplification in women diagnosed with POI and women with a known age at natural menopause ≥40 years. The prevalence of intermediate sized CGG repeats (45-54 repeats) was compared between POI cases and controls using Fisher's exact test. Differences in mean CGG repeat lengths on allele 1 and allele 2 between POI cases and controls were tested using analysis of variance. MAIN RESULTS AND THE ROLE OF CHANCE: The frequency of intermediate sized CGG repeats on the allele with the longest triple repeat number was not statistically significantly different between POI cases and controls (2.7 and 3.8%, respectively, odds ratio 0.72, 95% confidence interval: 0.38-1.39, P = 0.38). In women with POI, linear regression analysis for age at POI diagnosis and CGG repeat size also failed to show any association (ß = -0.018, P = 0.74). LIMITATIONS, REASONS FOR CAUTION: FMR1 CGG repeat lengths in POI cases and controls were genotyped in two different laboratories. The distributions of CGG repeats may vary among the different ethnic populations in our study. Also, in our study women with primary amenorrhea (N = 17) were included in the POI group. WIDER IMPLICATIONS OF THE FINDINGS: We found no association between intermediate sized CGG repeats and POI compared with controls. Therefore, a role for FMR1 CGG repeat sizes up to 55 repeats in the ovarian ageing process may be questioned. Moreover, there seems limited value in the evaluation of normal- and intermediate FMR1 repeat size in the diagnostic work-up of women affected by POI, or for prognostic purposes in women at risk of developing POI. STUDY FUNDING/COMPETING INTERESTS: The Prospect-EPIC study was funded by 'Europe Against Cancer' Program of the European Commission (SANCO); the Dutch Ministry of Health; the Dutch Cancer Society; ZonMW the Netherlands Organization for Health Research and Development; World Cancer Research Fund (WCRF) and the Dutch Heart Association.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/genética , Insuficiência Ovariana Primária/genética , Expansão das Repetições de Trinucleotídeos , Repetições de Trinucleotídeos , Adolescente , Adulto , Alelos , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Humanos , Menopausa , Pessoa de Meia-Idade , Razão de Chances , Prognóstico , Adulto JovemRESUMO
STUDY QUESTION: Is there an association between the number of CGG repeats in the FMR1 gene in the normal and intermediate range and age at natural menopause? SUMMARY ANSWER: The number of CGG repeats in the normal and intermediate range in the FMR1 gene was not associated with age at natural menopause. WHAT IS KNOWN ALREADY: Excessive triple CGG repeats in the FMR1 gene have been widely associated with primary ovarian insufficiency. Recently, the number of CGG repeats in the normal and intermediate range (up to 55 repeats) was found to be associated with serum levels of anti-Müllerian hormone and follicle-stimulating hormone, as markers for ovarian ageing. This suggests that repeats in the normal and intermediate range could be involved in the rate of exhaustion of the ovarian primordial follicle pool and ultimately the timing of menopause. STUDY DESIGN, SIZE: Cross-sectional study in a population-based sample of 3611 Caucasian women with natural menopause. PARTICIPANTS/MATERIALS, SETTING, METHODS: The FMR1 CGG repeat number was determined by PCR amplification in 3611 women with a known age at natural menopause. A possible relation between CGG repeats in the normal and intermediate range (up to 55 repeats) and menopausal age were analysed in various ways, including linear regression analysis and analysis of variance. MAIN RESULTS AND THE ROLE OF CHANCE: The number of CGG repeats in the normal and intermediate range in the FMR1 gene was not associated with age at natural menopause. The mean age at menopause was 50.30 (± 4.2) years for women with <45 repeats and 50.64 (± 3.4) years for women with intermediate-sized repeats (P = 0.37). Linear regression analysis of the number of CGG repeats showed no association with menopausal age (ß = 0.019, P = 0.16). LIMITATIONS, REASONS FOR CAUTION: In our cohort, age at menopause was self-reported and determined retrospectively. WIDER IMPLICATIONS OF THE FINDINGS: Earlier observations suggesting that the number of CGG repeats in the normal and intermediate range is associated with the individual variation of the ovarian ageing process could not be confirmed in the current, large sample size study. A relation between the number of CGG repeats in the normal and intermediate range and age at natural menopause appeared to be absent. This finding questions the role of CGG repeat sizes in the ovarian ageing process.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/química , Menopausa/genética , Repetições de Trinucleotídeos , Adulto , Fatores Etários , Análise de Variância , Estudos Transversais , Feminino , Proteína do X Frágil da Deficiência Intelectual/genética , Humanos , Modelos Lineares , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Four neonates with a positive phenylalanine screening test (Phe concentrations between 258 and 1250 micromol/L) were investigated further to differentiate between phenylalanine hydroxylase (PAH) deficiency and variant hyperphenylalaninaemia (HPA) forms. In patients 1 and 2 a tetrahydrobiopterin (BH4) load caused a significant decrease of the plasma Phe levels. A combined phenylalanine/BH4 loading test was performed in patients 2, 3 and 4. In the latter two patients, plasma Phe concentrations completely normalized within 8 h after the BH4 load (20 mg/kg). Basal urinary pterins were normal in all four patients. The activity of dihydropteridine reductase (DHPR) was normal in patients 1, 2 and 3 and 50% of control values in patient 4 (not in the range of DHPR-deficient patients). In patient 3 a subsequent phenylalanine loading test with concomitant analysis of plasma biopterins revealed a normal increase of biopterin, excluding a BH4 biosynthesis defect. Pterins and neurotransmitter metabolites in CSF of patients 1, 3 and 4 were normal. DNA mutations detected in the PAH gene of patients 1-4 were A313T, and L367fsinsC; V190A and R243X; A300S and A403V; R241C and A403V. The results are suggestive for mutant PAH enzymes with decreased affinity for the cofactor BH4.
Assuntos
Biopterinas/análogos & derivados , Biopterinas/uso terapêutico , Fenilalanina Hidroxilase/deficiência , Biopterinas/sangue , Análise Mutacional de DNA , Diagnóstico Diferencial , Di-Hidropteridina Redutase/metabolismo , Feminino , Humanos , Recém-Nascido , Cinética , Mutação , Países Baixos , Fenilalanina/sangue , Fenilalanina Hidroxilase/genética , Polimorfismo Conformacional de Fita Simples , Pterinas/líquido cefalorraquidiano , Pterinas/urinaRESUMO
BACKGROUND: Multiple endocrine neoplasia type 1 (MEN 1) is an autosomal, dominantly inherited cancer syndrome, with tumours in various endocrine glands. In 1997 the responsible tumour suppressor gene was identified. MEN1 gene germ-line mutations are detected in the vast majority of MEN 1 patients, however, with regard to case-finding, unfortunately only at a very low frequency in patients with apparently sporadic MEN 1-related tumours. In order to increase the detection rate of disease gene carriers among patients with apparently sporadic MEN 1-related tumours, clinical criteria were needed. DESIGN AND RESULTS: In this study MEN1 gene germ-line mutations were revealed in 16/16 MEN 1 patients/families (100%). Based on our clinical experience with MEN 1 patients/families we formulated clinical criteria to identify disease gene carriers among patients with apparently sporadic MEN 1-related tumours. The criteria for MEN 1-suspected patients are: young age at onset (< 35 years) and/or multiple MEN 1-related lesions in a single organ or two distinct organs affected. Application of these criteria yielded MEN1 gene germ-line mutations in nine of 15 MEN 1-suspected patients (60%), thus identifying novel MEN 1 families. Follow up was also guaranteed for patients not fulfilling these criteria. CONCLUSIONS: The clinical criteria for MEN 1-suspected patients increase the detection rate of germ-line MEN1 gene mutations among patients with apparently sporadic MEN 1-related tumours. These criteria may be used for (presymptomatic) identification of MEN 1 disease gene-carriers, thus enabling early detection of tumour development and timely treatment, as well as genetic counselling.
Assuntos
Análise Mutacional de DNA/métodos , Testes Genéticos/métodos , Mutação em Linhagem Germinativa , Neoplasia Endócrina Múltipla Tipo 1/diagnóstico , Neoplasia Endócrina Múltipla Tipo 1/genética , Adulto , Idoso , DNA de Neoplasias/isolamento & purificação , Éxons , Saúde da Família , Feminino , Humanos , Íntrons , Masculino , Pessoa de Meia-Idade , RNA Neoplásico/isolamento & purificaçãoRESUMO
To analyze the diagnostic value of various laboratory tests for the confirmation of adult-onset glycogen storage disease type II (GSD II), we performed a clinical, biochemical, and genetic study of 18 patients with this disease. Measurement of acid alpha-glucosidase (GAA) activity in muscle and histopathologic analysis of muscle tissue appeared to have no additional value when GAA activity in leukocytes was clearly deficient. Our study showed that creatine kinase elevation is a sensitive marker of GSD II. A diagnostic protocol is formulated.
Assuntos
Glucosidases/metabolismo , Doença de Depósito de Glicogênio Tipo II/diagnóstico , Idade de Início , Eletromiografia , Doença de Depósito de Glicogênio Tipo II/genética , Humanos , Leucócitos/enzimologia , Músculos/enzimologia , Músculos/fisiopatologiaRESUMO
Hereditary tyrosinaemia type 1 is a rare but serious metabolic disorder with an autosomal recessive mode of inheritance. We describe the prenatal diagnosis of an affected fetus performed by DNA-mutation analysis and a subsequent pregnancy with a healthy child in the same family.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Erros Inatos do Metabolismo dos Aminoácidos/genética , Falência Hepática/genética , Mutação/genética , Tirosina/sangue , Amostra da Vilosidade Coriônica , Heterozigoto , Humanos , Hidrolases/genética , Lactente , Falência Hepática/sangue , MasculinoAssuntos
Substituição de Aminoácidos , Degeneração Hepatolenticular/genética , Histidina , Mutação Puntual , Adolescente , Adulto , Humanos , PolôniaRESUMO
Benign recurrent intrahepatic cholestasis (BRIC) is an autosomal recessive liver disease characterized by multiple episodes of cholestasis without progression to chronic liver disease. The gene was previously assigned to chromosome 18q21, using a shared segment analysis in three families from the Netherlands. In the present study we report the linkage analysis of an expanded sample of 14 BRIC families, using 15 microsatellite markers from the 18q21 region. Obligate recombinants in two families place the gene in a 7-cM interval, between markers D18S69 and D18S64. All intervening markers had significant LOD scores in two-point linkage analysis. Moreover, we identified one family in which the BRIC gene seems to be unlinked to the 18q21 region, or that represents incomplete penetrance of the BRIC genotype.
Assuntos
Colestase Intra-Hepática/genética , Cromossomos Humanos Par 18 , Heterogeneidade Genética , Mapeamento Cromossômico , Feminino , Genótipo , Humanos , Masculino , Repetições de Microssatélites , Linhagem , RecidivaRESUMO
Germ line mutations in one allele of the RET proto-oncogene predispose to the multiple endocrine neoplasia type 2 (MEN 2) syndromes. To investigate whether these inherited mutations alone can cause the development of tumors in vivo (oncogene model) or whether somatic mutations in the homologous RET allele are required for tumorigenesis (tumor suppressor gene model), we analyzed the entire coding region of both alleles of the RET gene in two MEN 2A and two MEN 2B tumors by reverse transcription-PCR and direct sequencing. No tumor-specific mutations could be detected in either allele of the RET gene in these tumors. Unlike the molecular mechanism in other hereditary tumor syndromes, somatic mutations in the homologous allele are apparently not required in MEN 2 tumorigenesis. Thus, RET genes with MEN 2-specific germ line mutations act as dominantly transforming oncogenes in vivo.
Assuntos
Proteínas de Drosophila , Heterozigoto , Neoplasia Endócrina Múltipla Tipo 2a/genética , Mutação , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Receptores Proteína Tirosina Quinases/genética , DNA Complementar/química , Humanos , Masculino , Polimorfismo Genético , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-retRESUMO
We describe two unrelated Dutch patients with typical symptoms of infantile glycogen storage disease type II (GSD II) and virtual absence of acid alpha-glucosidase activity in leukocytes and cultured skin fibroblasts. The patients were identified as homozygotes for a deletion of exon 18 of the acid alpha-glucosidase gene (GAA). The in-frame deletion manifests at the protein level in a characteristic way: the enzyme precursor is smaller than normal and degraded in the endoplasmic reticulum or Golgi complex. These case present an evident example of a genotype-phenotype correlation in glycogen storage disease type II.
Assuntos
Deleção Cromossômica , Precursores Enzimáticos/metabolismo , Éxons , Doença de Depósito de Glicogênio Tipo II/genética , Homozigoto , Lisossomos/enzimologia , alfa-Glucosidases/deficiência , Humanos , Recém-Nascido , Masculino , Linhagem , alfa-Glucosidases/metabolismoRESUMO
Hereditary C-cell carcinoma is encountered in multiple endocrine neoplasia type 2A (MEN 2A), MEN 2B, and familial medullary thyroid carcinoma (FMTC). Mutations of the RET proto-oncogene are associated with all three diseases. To obtain an insight into the molecular heterogeneity of MEN 2 syndromes and FMTC in the Netherlands, probands of 20 MEN 2A families, two FMTC families, and seven MEN 2B families were analyzed by the polymerase chain reaction (PCR), DNA sequencing, and restriction enzyme digestion for abnormalities in the RET proto-oncogene. RET mutations were found in all cases. All MEN 2A families had a mutation involving one of five cysteine codons in exons 10 and 11 of RET. Two novel dinucleotide mutations and a de novo mutation were found. Both FMTC families had a mutation of the Cys at codon 618. All MEN 2B probands carried a Met to Thr mutation in exon 16. All mutations could be confirmed by restriction enzyme digestion of PCR amplicons. Identification of the RET mutation in the Dutch population with hereditary C-cell carcinoma facilitates genetic testing for families or individuals at risk for MEN 2A, FMTC, and MEN 2B.
Assuntos
Carcinoma Medular/genética , Proteínas de Drosophila , Neoplasia Endócrina Múltipla Tipo 2a/genética , Neoplasia Endócrina Múltipla Tipo 2b/genética , Mutação , Mutação Puntual , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Receptores Proteína Tirosina Quinases/genética , Neoplasias da Glândula Tireoide/genética , Sequência de Aminoácidos , Sequência de Bases , Análise Mutacional de DNA , Primers do DNA , Éxons , Humanos , Dados de Sequência Molecular , Países Baixos , Reação em Cadeia da Polimerase , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-retRESUMO
We performed a clinical, biochemical, and genetic study in 16 patients from 11 families with adult-onset acid maltase deficiency. All patients were compound heterozygotes and carried the IVS1(-13T --> G) transversion on one allele; the second allele harbored either a deletion of a T at position 525 in exon 2 (7 probands, 64%) or a deletion of exon 18 (1 proband, 9%). Deterioration of handicap was related to age, and decrease in vital capacity to duration of the symptomatic stage. Respiratory insufficiency was never the first manifestation. The levels of activity of serum creatine kinase and of alpha-glucosidase in peripheral blood cells or muscle were helpful for the diagnosis, but did not have prognostic value. The adult form of acid maltase deficiency appears to be both clinically and genetically rather homogeneous; decrease of alpha-glucosidase activity is the final common pathway leading to destruction of muscle fibers and progression of muscle weakness over a period of years.
Assuntos
Glucana 1,4-alfa-Glucosidase/genética , Doença de Depósito de Glicogênio Tipo II/genética , Adolescente , Adulto , Idade de Início , Alelos , Sequência de Bases , DNA/análise , Feminino , Genótipo , Glucana 1,4-alfa-Glucosidase/deficiência , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Fenótipo , alfa-GlucosidasesRESUMO
Sequencing of the arylsulfatase A gene in a late infantile metachromatic leukodystrophy patient showed the presence of a 12-bp deletion in exon 2. This deletion was found in a compound heterozygous state with the previously described 287 C-->T transition.
Assuntos
Cerebrosídeo Sulfatase/genética , Leucodistrofia Metacromática/genética , Deleção de Sequência/genética , Sequência de Aminoácidos , Sequência de Bases , Cerebrosídeo Sulfatase/química , Cerebrosídeo Sulfatase/metabolismo , Éxons/genética , Genes Recessivos/genética , Humanos , Lactente , Leucodistrofia Metacromática/enzimologia , Masculino , Dados de Sequência Molecular , Mutação Puntual/genética , Polimorfismo Conformacional de Fita Simples , Estrutura Secundária de ProteínaRESUMO
Human protein S interacts noncovalently with human C4b-binding protein (C4BP). We have studied this interaction using deletion variants of recombinant human protein S. Two deletion variants were constructed by restriction enzyme digestion and in vitro site-specific mutagenesis of the human protein S cDNA. The variants were stably expressed in C127 cells. Recombinant proteins were purified using Fast Flow Q anion-exchange chromatography. The activated protein C (APC) cofactor activity, C4BP binding properties and reactivity to different monoclonal antibodies against human protein S were examined. The first variant (E variant), which has a deletion of the third epidermal growth factor (EGF)-like domain (deletion of exon VII, corresponding to amino acid residues ASP-160 to Asp-202) expresses normal APC cofactor activity in a plasma system. This activity was inhibited by the addition of purified C4BP. The second variant (L variant), which has a deletion of the C-terminal loop of the sex hormone binding globulin (SHBG)-like domain (deletion of exon XV, corresponding to amino acid residues Asp-583 to Ser-635) also expresses normal APC cofactor activity in plasma. This activity could only be partially inhibited by the addition of purified C4BP. Binding of the recombinant proteins to C4BP was studied in a system using purified proteins. The E variant binds to C4BP with the same affinity similar as recombinant wild type protein S (apparent Kd approximately 10(-10) M). The L variant, however, shows a markedly reduced affinity for binding to C4BP (apparent Kd approximately 10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Inativadoras do Complemento , Glicoproteínas , Proteína S/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Humanos , Neoplasias Mamárias Animais , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteína C/metabolismo , Proteína S/química , Proteína S/genética , Proteína S/imunologia , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Células Tumorais CultivadasRESUMO
A panel of eight unrelated subjects with inherited type I protein S deficiency was screened for mutations in the PROS1 gene. In five subjects an abnormality was found but mutations were not detected in the remaining three subjects. Two subjects shared a G-->A transition at position +5 of the donor splice site consensus sequence of intron 10. Also in two subjects an A-->T transversion was detected in the stopcodon of the PROS1 gene; this transversion predicts a protein S molecule that is extended by 14 amino acids. The fifth subject was found to possess two sequence abnormalities. One allele carried a G-->A transition near the donor splice junction of intron 2, but this abnormality is probably neutral, since it was inherited from the parent with normal protein S antigen levels. In the other allele a single T insertion in codon -25 was found. Analysis of platelet RNA showed that only the mRNA with the A-->T mutation in the stopcodon is present in amounts comparable to wildtype RNA. mRNA from the alleles with the other two mutations was either undetectable or present in greatly reduced amounts. The latter indicates that a mRNA based approach is not feasible for the genetic analysis of protein S deficiency type I.
Assuntos
Plaquetas/metabolismo , Éxons , Mutação Puntual , Deficiência de Proteína S , Proteína S/genética , Alelos , Sequência de Bases , Primers do DNA , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Mensageiro/sangue , Transcrição GênicaRESUMO
The von Willebrand factor (vWF) genes of nine unrelated, severe, type III von Willebrand's disease (vWD) patients (six of Dutch origin) and four unrelated Dutch type I vWD patients were screened for mutations in exons that contain CGA codons (Arg), which are liable to mutation to TGA stop codons. The nine exons of the vWF gene (3, 8, 9, 10, 28, 31, 32, 43 and 45) that contain all the CGA codons (11 in total) of the vWF cDNA were amplified by the polymerase chain reaction and screened for mutations by single-strand conformation polymorphism analysis, restriction enzyme - and/or nucleotide sequence analysis. Three of the severe vWD patients were found to be heterozygous for a nonsense mutation: CGA Arg 2535-->TGA Stop. Three other severe vWD patients were homozygous for a single nucleotide substitution, AAC Asn 2546-->TAC Tyr. The transcription of these mutated alleles was tested by cDNA dependent amplification of platelet RNA. The level of transcription product was strongly reduced for either mutant allele.
Assuntos
Genética Populacional , RNA Mensageiro/sangue , Doenças de von Willebrand/genética , Fator de von Willebrand/genética , Alelos , DNA/genética , Amplificação de Genes , Regulação da Expressão Gênica/fisiologia , Testes Genéticos , Genoma Humano , Humanos , Mutação/genética , Países Baixos/epidemiologia , Conformação de Ácido Nucleico , Polimorfismo Genético/genéticaRESUMO
Mouse C127 epithelioid cells were genetically engineered to produce biologically active gamma-carboxylated human protein S. A full length human protein S cDNA was cloned into a bovine papilloma virus (BPV) based shuttle vector under the transcriptional control of the Moloney murine sarcoma virus enhancer and the mouse metallothionein promoter. Stable expression was obtained in transfected C127 cells. Expression of gamma-carboxylated protein S was dependent on the presence of vitamin K in the culture medium. Protein sequence analysis showed that recombinant and plasma protein S have the same amino terminal sequence. Analysis of specific post-translationally modified amino acids shows that recombinant protein S is fully gamma-carboxylated and fully beta-hydroxylated. Immunoblotting analysis using polyclonal and monoclonal antibodies shows that recombinant protein S has a slightly higher molecular weight than plasma protein S. After N-Glycanase treatment, identical molecular weights are observed for recombinant and plasma protein S, indicating that the difference is caused by differences in the N-linked carbohydrate side chains. Recombinant protein S also demonstrates normal cofactor activity for activated protein C in a clotting assay. Binding studies with the complement component, C4b-binding protein (C4BP), shows that recombinant protein S binds to C4BP with the same apparent affinity as plasma protein S. Two variant molecules are also tested for their binding to C4BP. The first variant has a replacement of amino acid residue leu-608 by val and was designated B variant. The second variant has three alterations, at positions 609, 611 and 612 where the acidic amino acid residues asp, asp and glu were replaced by asn, asn and gln, respectively and this variant was designated C variant.(ABSTRACT TRUNCATED AT 250 WORDS)