Hepatitis B virus infection in children, adolescents, and their relatives: genotype distribution and precore and core gene mutations

INTRODUCTION:The objectives of this study were evaluate hepatitis B virus (HBV) serological markers in children and adolescents followed up at the Child Institute of the Hospital das Clínicas, Faculdade de Medicina de São Paulo, Universidade de São Paulo; identify chronic HBV carriers and susceptible individuals in the intrafamilial environment; characterize HBV genotypes; and identify mutations in the patients and household contacts. METHODS: Ninety-five hepatitis B surface antigen-positive children aged <19 years and 118 household contacts were enrolled in this study. Commercial kits were used for the detection of serological markers, and PCR was used for genotyping. RESULTS: Hepatitis B e antigen (HBeAg) was detected in 66.3% (63/95) of cases. Three of the 30 HBeAg-negative and anti-HBeAg-positive patients presented with precore mutations and 11 presented with mutations in the basal core promoter (BCP). Genotype A was identified in 39 (43.8%) patients, genotype D in 45 (50.6%), and genotype C in 5 (5.6%). Of the 118 relatives, 40 were chronic HBV carriers, 52 presented with the anti-HBc marker, 19 were vaccinated, and 7 were susceptible. Among the relatives, genotypes A, D, and C were the most frequent. One parent presented with a precore mutation and 4 presented with BCP mutations. CONCLUSIONS: Genotypes A and D were the most frequent among children, adolescents, and their relatives. The high prevalence of HBV in the families showed the possibility of its intrafamilial transmission.

The novel proteins Rng8 and Rng9 regulate the myosin-V Myo51 during fission yeast cytokinesis.

The myosin-V family of molecular motors is known to be under sophisticated regulation, but our knowledge of the roles and regulation of myosin-Vs in cytokinesis is limited. Here, we report that the myosin-V Myo51 affects contractile ring assembly and stability during fission yeast cytokinesis, and is regulated by two novel coiled-coil proteins, Rng8 and Rng9. Both rng8Δ and rng9Δ cells display similar defects as myo51Δ in cytokinesis. Rng8 and Rng9 are required for Myo51's localizations to cytoplasmic puncta, actin cables, and the contractile ring. Myo51 puncta contain multiple Myo51 molecules and walk continuously on actin filaments in rng8(+) cells, whereas Myo51 forms speckles containing only one dimer and does not move efficiently on actin tracks in rng8Δ. Consistently, Myo51 transports artificial cargos efficiently in vivo, and this activity is regulated by Rng8. Purified Rng8 and Rng9 form stable higher-order complexes. Collectively, we propose that Rng8 and Rng9 form oligomers and cluster multiple Myo51 dimers to regulate Myo51 localization and functions.

Avaliação de fatores virológicos associados ao desenvolvimento de carcinoma hepatocelular (CHC) em pacientes com hepatite B crônica

O objetivo principal deste estudo foi avaliar fatores virais associados com a evolução para o carcinoma hepatocelular (CHC) em pacientes com hepatite B crônica. Para tanto caracterizamos os subgenótipos do HBV, investigamos a ocorrência de mutações nos genes pré-core/core do HBV associadas à presença de CHC avaliamos por análise filogenética a associação de linhagens virais com a ocorrência de CHC e por fim a associação de outros fatores de risco com o desenvolvimento de CHC. Foram incluídos 119 amostras de soro de pacientes com infecção crônica pelo HBV, destas amostras 60 pertencem ao grupo 1 (CHC), que são pacientes com diagnóstico confirmado de carcinoma hepatocelular e 59 amostras pertencem ao grupo 2 (sem CHC) que são pacientes com hepatite crônica sem detecção prévia de nódulos hepáticos. Foram obtidas informações acerca da idade, sexo e naturalidade. Além disso, os pacientes responderam a um questionário sobre fatores de riscos associados ao desenvolvimento de CHC. Foram realizados exames bioquímicos, sorológicos, determinação da carga viral, e amplificação por nested PCR e sequenciamento das regiões S/polimerase e pré-core/core do genoma viral para posterior caracterização dos genótipos/subgenótipos do HBV e pesquisa de mutações associadas com evolução da doença hepática. Em relação à idade e sexo não houve grande variação entre os grupos. Quanto à naturalidade a maioria era procedente da região sudeste, seguido pela região nordeste; e por fim seis pacientes eram procedentes de outros países. Com base no sobrenome dos pacientes avaliou-se também a frequência de etnia oriental na casuística estudada, que foi similar nos 2 grupos. O perfil sorológico HBeAg negativo foi o mais frequente nos dois grupos de pacientes, assim como níveis de carga viral abaixo de 2.000 UI/mL. Em relação aos exames bioquímicos foram observadas diferenças estatisticamente significantes nos níveis séricos de AFP (p= 0,0013), FA (p= 0,0003) e GGT (p= 0,005). Dentre os fatores de risco analisados neste estudo, o consumo de amendoim foi o único que apresentou significância estatística (p= 0,003). A região S/pol foi amplificada e sequenciada com sucesso em 58 amostras (28 do grupo 1 e 30 do grupo 2). Entre as 58 amostras analisadas 4 genótipos e 8 subgenótipos do HBV foram identificados, sendo o subgenótipo A1 o mais frequente nos dois grupos. Não se observou diferença estatisticamente significante na distribuição dos subgenótipos entre os dois grupos de pacientes. Na topologia da árvore filogenética construída com sequências do HBV isoladas dos pacientes incluídos neste estudo e sequências disponíveis no GenBank não se observou padrões de agrupamento associados com o perfil clinico do paciente (com e sem CHC). Foram obtidas sequências de boa qualidade da região précore/ core em 44 amostras, sendo 20 amostras do grupo 1 e 24 do grupo 2. Diversas das mutações investigadas foram identificadas na região précore/ core, as quais foram avaliadas estatisticamente para verificar a existência de diferença na frequência das mesmas entre os grupos de pacientes estudados. Entre as mutações identificadas se destacaram com significância estatística as seguintes mutações: T1768A (p= 0,006), a combinação das mutações C1766T + T1768A (p= 0,043) e G1888H (p= 0,05). Na análise de regressão logística simples foi possível identificar que a chance de um paciente do grupo 2 desenvolver CHC aumenta 14,7 vezes na presença de infecção por cepas do HBV com a mutação T1768A, enquanto que a infecção com cepas do HBV que albergam a mutação G1888H reduz tal chance 2,5 vezes

Phenotype-genotype analysis of cryopyrin-associated periodic syndromes (CAPS): Description of a rare non-exon 3 and a novel CIAS1 missense mutation

We describe in this paper the phenotype-genotype analysis of a Brazilian cohort of patients with cryopyrin-associated periodic syndromes (CAPS). Patient 1 presented with an urticarial rash and recurrent fever exacerbated by cold weather, arthritis, and anterior uveitis, thus, receiving a clinical diagnosis of familial cold autoinflammatory syndrome. CIAS1 sequencing identified the T436I mutation, previously associated to a clinical phenotype of chronic infantile neurological cutaneous and articular/neonatal onset multisystem inflammatory disease. Patient 2 developed a papular exanthema with daily fever shortly after birth, frontal bossing, patellae enlargement, and cognitive and motor impairments. Sequencing identified the exceedingly rare G755R CIAS1 mutation in exon 4. Patient 3 developed skin rash and articular symptoms 6 h after birth, followed by aseptic meningitis. He was found to have the novel C148Y missense mutation in CIAS1. This report expands the spectrum of CIAS1 mutations associated to clinical disease, suggests that the same mutation can be associated with different clinical syndromes, and supports the evidence that CAPS patients should always be screened for mutations outside exon 3.

. Phenotype/genotype correlation associated with a novel nonsense Glu20stop mutation in the Rp2 gene in a three-generation family with x-linked retinitis pigmentosa

Purpose: To assess the phenotype of patients in a large 3 generation Swiss family with X-linked retinitis pigmentosa (XLRP) due to a novel nonsense mutation Glu20stop in RP2 gene and to correlate with the genotype. Methods: 6 affected patients (1 male, 5 females, age range: 23 - 73 years) were assessed with a complete ophthalmologic examination. All had fundus autofluorescence images, standardised electroretinography, Goldmann visual fields and Optical Coherence Tomography. In addition, medical records of 2 affected male patients were reviewed. Blood sample was taken for molecular analysis. Results: The male patients were severely affected at a young age with early macular involvement. The youngest 23 y old male had also high myopia and vision of less than 0.05 according to Snellen EDTRS chart bilaterally. All 5 female carriers had some degree of rod-cone dystrophy, but no macular involvement. The visual acuity was 1.0 in the younger carriers, while the 73 years old had VA of 0.5. Two females had mild myopia (range -0.75 to -2) and one had anisometropia of 3.5D, with the more severely affected eye being myopic. Three out of 5 female carriers had optic nerve drusen. Conclusions: We report a novel Glu20stop mutation in RP2 gene, which is a rare cause of XLRP. Our description of severe phenotype in male patients with high myopia and early macular atrophy confirms previous reports. Unlike previous reports, all our female carriers had RP, but not macular involvement or high myopia. The identifiable phenotype for RP2-XLRP aids in clinical diagnosis and targeted genetic screening.

Novel Cone Transducin Alpha Subunit Mutation In Tunisian Patients And Genotype-phenotype Correlation In Complete Achromatopsia

Purpose: Complete achromatopsia is a rare autosomal recessive disease due to CNGA3, CNGB3, GNAT2 and PDE6C mutations. We studied a large consanguineous Tunisian family including twelve individuals.Methods: Ophthalmic evaluation included a full clinical examination, color vision testing, optical coherence tomography and electroretinography. Linkage analysis using microsatellite markers flanking CNGA3, CNGB3, GNAT2 and PDE6C genes was performed. Mutations were screened by direct sequencing.Results: In all affected subjects, acuity ranged from 20/50 to 20/200. Fundus examination was normal except for two patients who had respectively 4 mm and 5 mm diameters of peripheral congenital hypertrophy. Likewise retinal layers exploration by OCT revealed no change in the thickness of the central retina. Color Vision with 100 Hue Farnsworth test described a profound color impairment along all three axes of color vision. The haplotype analysis of GNAT2 markers revealed that all affected offspring were homozygous by descent for the four polymorphic markers. The maximum lod score value, 4.33, confirmed the evidence for linkage to the GNAT2 gene.A homozygous novel nonsense mutation R313X was identified segregating with an identical GNAT2 haplotype in all affected subjects. This mutation could interrupt interaction with photoactivated rhodopsin, resulting in a failure of visual transduction. In fact, ERG showed a clearly abolished photopic b-wave and flicker responses with no residual cone function justifying the severe GNAT2 achromatopsia phenotype.Conclusions: This is the first report of the clinical and genetic investigation of complete achromatopsia in North Africa and of the largest family with recessive achromatopsia involving GNAT2, thus providing a unique opportunity for genotype phenotype correlation for this extremely rare condition.

MLL2 mutation detection in 86 patients with Kabuki syndrome: a genotype-phenotype study.

Recently, pathogenic variants in the MLL2 gene were identified as the most common cause of Kabuki (Niikawa-Kuroki) syndrome (MIM#147920). To further elucidate the genotype-phenotype correlation, we studied a large cohort of 86 clinically defined patients with Kabuki syndrome (KS) for mutations in MLL2. All patients were assessed using a standardized phenotype list and all were scored using a newly developed clinical score list for KS (MLL2-Kabuki score 0-10). Sequencing of the full coding region and intron-exon boundaries of MLL2 identified a total of 45 likely pathogenic mutations (52%): 31 nonsense, 10 missense and four splice-site mutations, 34 of which were novel. In five additional patients, novel, i.e. non-dbSNP132 variants of clinically unknown relevance, were identified. Patients with likely pathogenic nonsense or missense MLL2 mutations were usually more severely affected (median 'MLL2-Kabuki score' of 6) as compared to the patients without MLL2 mutations (median 'MLL2-Kabuki score' of 5), a significant difference (p < 0.0014). Several typical facial features such as large dysplastic ears, arched eyebrows with sparse lateral third, blue sclerae, a flat nasal tip with a broad nasal root, and a thin upper and a full lower lip were observed more often in mutation positive patients.

KLF2 mutation is the most frequent somatic change in splenic marginal zone lymphoma and identifies a subset with distinct genotype.

To characterise the genetics of splenic marginal zone lymphoma (SMZL), we performed whole exome sequencing of 16 cases and identified novel recurrent inactivating mutations in Kruppel-like factor 2 (KLF2), a gene whose deficiency was previously shown to cause splenic marginal zone hyperplasia in mice. KLF2 mutation was found in 40 (42%) of 96 SMZLs, but rarely in other B-cell lymphomas. The majority of KLF2 mutations were frameshift indels or nonsense changes, with missense mutations clustered in the C-terminal zinc finger domains. Functional assays showed that these mutations inactivated the ability of KLF2 to suppress NF-κB activation by TLR, BCR, BAFFR and TNFR signalling. Further extensive investigations revealed common and distinct genetic changes between SMZL with and without KLF2 mutation. IGHV1-2 rearrangement and 7q deletion were primarily seen in SMZL with KLF2 mutation, while MYD88 and TP53 mutations were nearly exclusively found in those without KLF2 mutation. NOTCH2, TRAF3, TNFAIP3 and CARD11 mutations were observed in SMZL both with and without KLF2 mutation. Taken together, KLF2 mutation is the most common genetic change in SMZL and identifies a subset with a distinct genotype characterised by multi-genetic changes. These different genetic changes may deregulate various signalling pathways and generate cooperative oncogenic properties, thereby contributing to lymphomagenesis.

Mutation Spectrum in the Large GTPase Dynamin 2, and Genotype-Phenotype Correlation in Autosomal Dominant Centronuclear Myopathy

Centronuclear myopathy (CNM) is a genetically heterogeneous disorder associated with general skeletal muscle weakness, type I fiber predominance and atrophy, and abnormally centralized nuclei. Autosomal dominant CNM is due to mutations in the large GTPase dynamin 2 (DNM2), a mechanochemical enzyme regulating cytoskeleton and membrane trafficking in cells. To date, 40 families with CNM-related DNM2 mutations have been described, and here we report 60 additional families encompassing a broad genotypic and phenotypic spectrum. In total, 18 different mutations are reported in 100 families and our cohort harbors nine known and four new mutations, including the first splice-site mutation. Genotype-phenotype correlation hypotheses are drawn from the published and new data, and allow an efficient screening strategy for molecular diagnosis. In addition to CNM, dissimilar DNM2 mutations are associated with Charcot-Marie-Tooth (CMT) peripheral neuropathy (CMTD1B and CMT2M), suggesting a tissue-specific impact of the mutations. In this study, we discuss the possible clinical overlap of CNM and CMT, and the biological significance of the respective mutations based on the known functions of dynamin 2 and its protein structure. Defects in membrane trafficking due to DNM2 mutations potentially represent a common pathological mechanism in CNM and CMT. Hum Mutat 33: 949-959, 2012. (C) 2012 Wiley Periodicals, Inc.

Frequency of Genotype With ΔF508 Mutation in CFTR Gene Among Iranian Cystic Fibrosis Patients With Pancreatic Insufficiency

Background: Cystic fibrosis (CF) is the most prevalent lethal autosomal recessive disease with a broad spectrum of phenotypes. Mutation of ΔF508 in the CFTR gene is the most important and lethal mutation in CF, which contains 70% of all predisposing mutations for CF worldwide. Objectives: Determining frequency of genotypes with ΔF508 mutation in CFTR gene, and evaluation of correlation between genotype and phenotype of Iranian patients with CF. Patients and Methods: Thirty six patients were included in this cross sectional study. ΔF508 mutations in both alleles of the CFTR gene were checked. Results: Among 36 pediatric patients, ΔF508 mutation was detected in 9 (25%) patients; 2 patients were heterozygous, and 7 patients homozygous for this mutation. From overall 72 tracked alleles, 11 (15.2%) were found to have ΔF508 mutations. Differences in prevalence of dyspnea and bronchiectasis were significant in homozygote group, compared with non-mutated group for ΔF508. Conclusions: It seems that more ΔF508 mutated alleles lead to more severe symptoms of CF.

Genotype-phenotype correlation in Brazillian Rett syndrome patients

BACKGROUND: Rett syndrome (RS) is a severe neurodevelopmental X-linked dominant disorder caused by mutations in the MECP2 gene. PURPOSE: To search for point mutations on the MECP2 gene and to establish a correlation between the main point mutations found and the phenotype. METHOD: Clinical evaluation of 105 patients, following a standard protocol. Detection of point mutations on the MECP2 gene was performed on peripheral blood DNA by sequencing the coding region of the gene. RESULTS: Classical RS was seen in 68% of the patients. Pathogenic point mutations were found in 64.1% of all patients and in 70.42% of those with the classical phenotype. Four new sequence variations were found, and their nature suggests patogenicity. Genotype-phenotype correlations were performed. CONCLUSION: Detailed clinical descriptions and identification of the underlying genetic alterations of this Brazilian RS population add to our knowledge of genotype/phenotype correlations, guiding the implementation of mutation searching programs.

A novel COL1A1 gene-splicing mutation (c. 1875+1G > C) in a Brazilian patient with osteogenesis imperfecta

Osteogenesis imperfecta is a heterogeneous genetic disorder characterized by bone fragility and deformity, recurrent fractures, blue sclera, short stature, and dentinogenesis imperfecta. Most cases are caused by mutations in COL1A1 and COL1A2 genes. We present a novel splicing mutation in the COL1A1 gene (c. 1875+ 1G>C) in a 16-year-old Brazilian boy diagnosed as a type III osteogenesis imperfecta patient. This splicing mutation and its association with clinical phenotypes will be submitted to the reference database of COL1A1 mutations, which has no other description of this mutation.

Iron deficiency and frequency of HFE C282Y gene mutation in Brazilian blood donors

Limited data are available about iron deficiency (ID) in Brazilian blood donors. This study evaluated the frequencies of ID and iron-deficiency anaemia (IDA) separately and according to frequency of blood donations. The protective effect of the heterozygous genotype for HFE C282Y mutation against ID and IDA in female blood donors was also determined. Five hundred and eight blood donors were recruited at the Blood Bank of Santa Casa in Sao Paulo, Brazil. Haemoglobin and serum ferritin concentrations were measured. The genotype for HFE C282Y mutation was determined by polymerase chain reaction followed by restriction fragment length polymorphism analysis. The ID was found in 21 center dot 1% of the women and 2 center dot 6% of the men whereas the IDA was found in 6 center dot 8 and 0 center dot 3%, respectively. The ID was found in 11 center dot 9% of the women in group 1 (first-time blood donors) and the frequency increased to 38 center dot 9% in women of the group 3 (blood donors donating once or more times in the last 12 months). No ID was found in men from group 1; however the ID frequency increased to 0 center dot 9% in group 2 (who had donated blood before but not in the last 12 months) and 5 center dot 0% in group 3. In summary, the heterozygous genotype was not associated with reduction of ID or IDA frequencies in both genders, but in male blood donors it was associated with a trend to elevated ferritin levels (P = 0 center dot 060). ID is most frequent in Brazilian women but was also found in men of group 3.

Structural interpretation of mutations in phenylalanine hydroxylase protein aids in identifying genotype-phenotype correlations in phenylketonuria

Phenylalanine hydroxylase (PAH) is the enzyme that converts phenylalanine to tyrosine as a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. Over 300 mutations have been identified in the gene encoding PAH that result in a deficient enzyme activity and lead to the disorders hyperphenylalaninaemia and phenylketonuria. The determination of the crystal structure of PAH now allows the determination of the structural basis of mutations resulting in PAH deficiency. We present an analysis of the structural basis of 120 mutations with a 'classified' biochemical phenotype and/or available in vitro expression data. We find that the mutations can be grouped into five structural categories, based on the distinct expected structural and functional effects of the mutations in each category. Missense mutations and small amino acid deletions are found in three categories:'active site mutations', 'dimer interface mutations', and 'domain structure mutations'. Nonsense mutations and splicing mutations form the category of 'proteins with truncations and large deletions'. The final category, 'fusion proteins', is caused by frameshift mutations. We show that the structural information helps formulate some rules that will help predict the likely effects of unclassified and newly discovered mutations: proteins with truncations and large deletions, fusion proteins and active site mutations generally cause severe phenotypes; domain structure mutations and dimer interface mutations spread over a range of phenotypes, but domain structure mutations in the catalytic domain are more likely to be severe than domain structure mutations in the regulatory domain or dimer interface mutations.