RESUMO
Mutations in the PAX6 gene that cause aniridia have been identified in various ethnicities but not in the Malaysian population. Therefore, the objective of this study was to investigate the PAX6 mutation in a Malaysian family with congenital aniridia. In this study, a complete ophthalmic examination was performed on a Dusun ethnic family with aniridia. Genomic DNA was extracted from the peripheral blood of the subjects and screened for the PAX6 gene mutation using polymerase chain reaction amplification high-resolution melting curve analysis (PCR-HRM) followed by confirmation via direct DNA sequencing. A heterozygous G deletion (c.857delG) in exon 7 causing a frame shift in PAX6 was identified in all affected family members. Genotype-phenotype correlation analysis revealed congenital cataract and all affected family members showed a similar spectrum of aniridia with no phenotypic variability but with differences in severity that were age-dependent. In summary, by using a PCR-HRM approach, this study is the first to report a PAX6 mutation in a Malaysian family. This mutation is the cause of the aniridia spectra observed in this family and of congenital cataract.
Assuntos
Aniridia/genética , Proteínas do Olho/genética , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Homeodomínio/genética , Fatores de Transcrição Box Pareados/genética , Polimorfismo Conformacional de Fita Simples/genética , Proteínas Repressoras/genética , Aniridia/patologia , Povo Asiático/genética , Feminino , Estudos de Associação Genética , Humanos , Malásia , Masculino , Mutação , Desnaturação de Ácido Nucleico , Fator de Transcrição PAX6 , LinhagemRESUMO
Myostatin, a transforming growth factor-beta superfamily member, has been well documented as a negative regulator of muscle growth and development. Myostatin, which has 376 amino acids, is synthesized as a precursor protein. Polymorphism of the myostatin gene in Makoei sheep was investigated by PCR and single-strand conformation polymorphism technique (SSCP). Genomic DNA of 92 sheep was isolated from whole blood. A 417-bp myostatin intron I segment was amplified by standard PCR, using locus-specific primers. Four SSCP patterns, representing four different genotypes, were identified. The frequencies of the genotypes were 0.413, 0.293, 0.130, and 0.163 for AD, AC, AE, and BC, respectively. Allele frequencies were 0.4185, 0.0815, 0.2283, 0.2065, and 0.0652 for A, B, C, D, and E, respectively. Observed heterozygosity was 0.7192. There was significant deviation from Hardy-Weinberg equilibrium for this locus. Analysis of myostatin gene sequences revealed heterozygous SNPs, which were in agreement with results obtained in the SSCP analysis. We concluded that SSCP analysis is a quick, sensitive and reliable technique for determination of DNA polymorphisms. The effect of these genotypes on some traits was investigated, and the AD genotype was found to be associated with birth weight. No phenotypic associations were detected with the other genotypes. No associations of myostatin variants with weight gain were detected. We conclude that polymorphism in the ovine myostatin gene is associated with birth weight, but not with weight gain in Iranian Makoei sheep.
Assuntos
Peso ao Nascer/genética , Estudos de Associação Genética , Miostatina/genética , Polimorfismo de Nucleotídeo Único/genética , Carneiro Doméstico/genética , Aumento de Peso/genética , Animais , Cruzamento , Loci Gênicos/genética , Genótipo , Irã (Geográfico) , Polimorfismo Conformacional de Fita Simples/genéticaRESUMO
The mannan-binding lectin gene (MBL) participates as an opsonin in the innate immune system of mammals, and single nucleotide polymorphisms (SNPs) in MBL cause various immune dysfunctions. In this study, we detected SNPs in MBL2 at exon 1 using polymerase chain reaction single-strand conformation polymorphism analysis and DNA sequencing techniques in 825 Chinese Holstein cows. Four new SNPs with various allele frequencies were also found. The g.1164 G>A SNP was predicted to substitute arginine with glutamine at the N-terminus of the cysteine-rich domain. In the collagen-like domain, SNPs g.1197 C>A and g.1198 G>A changed proline to glutamine, whereas SNP g.1207 T>C was identified as a synonymous mutation. Correlation analysis showed that the g.1197 C>A marker was significantly correlated to somatic cell score (SCS), and the g.1164 G>A locus had significant effects on SCS, fat content, and protein content (P < 0.05), suggesting possible roles of these SNPs in the host response against mastitis. Nine haplotypes and nine haplotype pairs corresponding to the loci of the 4 novel SNPs were found in Chinese Holsteins. Haplotype pairs MM, MN, and BQ were correlated with the lowest SCS; MN with the highest protein yield; MM with the highest protein rate, and MN with the highest 305- day milk yield. Thus, MM, MN, and BQ are possible candidates for marker-assisted selection in dairy cattle breeding programs.
Assuntos
Bovinos/genética , Estudos de Associação Genética , Lectina de Ligação a Manose/genética , Leite/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Característica Quantitativa Herdável , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , China , Éxons/genética , Frequência do Gene/genética , Haplótipos/genética , Heterozigoto , Análise dos Mínimos Quadrados , Lectina de Ligação a Manose/química , Dados de Sequência Molecular , Polimorfismo Conformacional de Fita Simples/genética , Análise de Sequência de DNA , Coloração pela PrataRESUMO
Osteogenesis imperfecta (OI) is a Mendelian disease with genetic heterogeneity characterized by bone fragility, recurrent fractures, blue sclerae, and short stature, caused mostly by mutations in COL1A1 or COL1A2 genes, which encode the pro-α1(I) and pro-α2(I) chains of type I collagen, respectively. A Brazilian family that showed variable expression of autosomal dominant OI was identified and characterized. Scanning for mutations was carried out using SSCP and DNA sequence analysis. The missense mutation c.3235G>A was identified within exon 45 of the COL1A1 gene in a 16-year-old girl diagnosed as having OI type I; it resulted in substitution of a glycine residue (G) by a serine (S) at codon 1079 (p.G1079S). The proband's mother had the disease signs, but without bone fractures, as did five of nine uncles and aunts of the patient. All of them carried the mutation, which was excluded in four healthy brothers of the patient's mother. This is the first description in a Brazilian family with OI showing variable expression; only one among seven carriers for the c.3235G>A mutation developed bone fractures, the most striking clinical feature of this disease. This finding has a significant implication for prenatal diagnosis in OI disease.
Assuntos
Colágeno Tipo I/genética , Mutação de Sentido Incorreto/genética , Osteogênese Imperfeita/genética , Adolescente , Adulto , Idoso , Substituição de Aminoácidos/genética , Sequência de Bases , Brasil , Criança , Cadeia alfa 1 do Colágeno Tipo I , Família , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Polimorfismo Conformacional de Fita Simples/genéticaRESUMO
Downy mildew (Plasmopara viticola) and anthracnose (Sphaceloma ampelinum) are two major diseases that severely affect most grapevine (Vitis vinifera) cultivars grown commercially in Thailand. Progress of conventional breeding programs of grapevine for improved resistance to these diseases can be speeded up by selection of molecular markers associated with resistance traits. We evaluated the association between 13 resistance gene analog (RGA)-single-strand conformation polymorphism (SSCP) markers with resistance to downy mildew and anthracnose in 71 segregating progenies of seven cross combinations between susceptible cultivars and resistant lines. F(1) hybrids from each cross were assessed for resistance to downy mildew and anthracnose (isolates Nk4-1 and Rc2-1) under laboratory conditions. Association of resistance traits with RGA-SSCP markers was evaluated using simple linear regression analysis. Three RGA-SSCP markers were found to be significantly correlated with anthracnose resistance, whereas significant correlation with downy mildew resistance was observed for only one RGA-SSCP marker. These results demonstrate the usefulness of RGA-SSCP markers. Four candidate markers with significant associations to resistance to these two major diseases of grapevine were identified. However, these putative associations between markers and resistance need to be verified with larger segregating populations before they can be used for marker-assisted selection.
Assuntos
Ascomicetos/fisiologia , Resistência à Doença/genética , Estudos de Associação Genética , Peronospora/fisiologia , Doenças das Plantas/genética , Polimorfismo Conformacional de Fita Simples/genética , Vitis/genética , Cruzamentos Genéticos , Eletroforese em Gel de Ágar , Genes de Plantas/genética , Marcadores Genéticos , Hibridização Genética , Modelos Lineares , Doenças das Plantas/microbiologia , Vitis/microbiologiaRESUMO
The peroxisome proliferator-activated receptor gamma (PPAR-γ) is a key molecule in adipocyte differentitation; it transactivates multiple target genes in lipid metabolic pathways. Using PCR-SSCP and DNA sequencing, we evaluated a potential association of an SNP (72472 G﹥T in exon7) of the bovine PPAR-γ gene with carcass and meat quality traits in 660 individuals from five Chinese indigenous cattle breeds, Qinchuan (QC), Luxi (LX), Nanyang (NY), Jiaxian (JX), and Xianan (XN). This 72472 G﹥T mutation identified a missense mutation, Q448H. Two alleles were named C and D. Allele frequencies of PPAR-γ-C/D in the five breeds were 0.7815/0.2185, 0.9/0.1, 0.7442/0.2558, 0.7051/0.2949, and 0.8333/0.1667 for QC, NY, JX, LX, and XN, respectively. Except for the XN breed, all breeds were in Hardy-Weinberg equilibrium at this locus. The polymorphism information content was low for NY and XN (0.16 and 0.24, respectively), while it was moderately high for QC, JX, and LX (0.28, 0.31 and 0.33, respectively). Correlation analysis showed significant association of this missense mutation with carcass length, backfat thickness and water holding capacity in the QC breed. Animals with the genotype CD had significantly greater carcass length than those with genotypes CC and DD, while animals with genotype CC had significantly greater backfat thickness than those with genotypes CD and DD. Animals with genotype CC had lower water holding capacity than those with the genotypes CD and DD. In conclusion, this locus is a candidate for a major quantitative trait locus affecting production traits and could be used for beef breeding selection.
Assuntos
Cruzamento , Bovinos/genética , Estudos de Associação Genética , Carne/normas , Mutação de Sentido Incorreto/genética , PPAR gama/genética , Característica Quantitativa Herdável , Sequência de Aminoácidos , Animais , Sequência de Bases , China , Éxons/genética , Frequência do Gene/genética , Loci Gênicos/genética , Genótipo , Dados de Sequência Molecular , PPAR gama/química , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples/genética , Análise de Sequência de DNARESUMO
Body measurement traits, influenced by genes and environmental factors, play numerous important roles in the value assessment of productivity and economy. There has been some indication that ANAPC13 influences adult height. We used PCR-SSCP and DNA sequencing technology to identify polymorphisms in the ANAPC13 gene. A polymorphism in intron 1 (A > G at base 17) was identified and an additional polymorphic site (C > T at base 42) was also uncovered, which accompanied the previous polymorphism in more than 98% of the subjects. The two novel polymorphisms in exon 1 were assayed and potential associations with body measurement traits were evaluated in 404 individuals. Three genotypes were detected in the study group, named AACC, AGCT and GGTT. Significant differences were observed between genotypes AACC and AGCT for body length, withers height, hip height, hip width, heart girth, pin bone width. However, no associations were found among any genotypes and chest depth. We conclude that polymorphisms and mutations in non-coding regions of the ANAPC13 gene significantly affect body measurement traits.
Assuntos
Pesos e Medidas Corporais , Bovinos/anatomia & histologia , Bovinos/genética , Estudos de Associação Genética , Polimorfismo de Nucleotídeo Único/genética , Característica Quantitativa Herdável , Complexos Ubiquitina-Proteína Ligase/genética , Ciclossomo-Complexo Promotor de Anáfase , Animais , Sequência de Bases , Distribuição de Qui-Quadrado , China , Éxons/genética , Frequência do Gene/genética , Loci Gênicos/genética , Genótipo , Análise dos Mínimos Quadrados , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples/genética , Análise de Sequência de DNARESUMO
Glucokinase (GCK) plays a key role in glucose homeostasis. Gestational diabetes mellitus increases the risk of gestational complications in pregnant women and fetuses. We screened for mutations in coding and flanking regions of the GCK gene in pregnant women with or without gestational diabetes in a Brazilian population. A sample of 200 pregnant women classified as healthy (control, N = 100) or with gestational diabetes (N = 100) was analyzed for mutations in the GCK gene. All gestational diabetes mellitus patients had good glycemic control maintained by diet alone and no complications during pregnancy. Mutations were detected by single-strand conformation polymorphism and DNA sequencing. Thirteen of the 200 subjects had GCK gene mutations. The mutations detected were in intron 3 (c.43331A>G, new), intron 6 (c.47702T>C, rs2268574), intron 9 (c.48935C>T, rs2908274), and exon 10 (c.49620G>A, rs13306388). None of these GCK mutations were found to be significantly associated with gestational diabetes mellitus. In summary, we report a low frequency of GCK mutations in a pregnant Brazilian population and describe a new intronic variation (c.43331A>G, intron 3). We conclude that mutations in GCK introns and in non-translatable regions of the GCK gene do not affect glycemic control and are not correlated with gestational diabetes mellitus.
Assuntos
Diabetes Gestacional/sangue , Diabetes Gestacional/genética , Glucoquinase/genética , Glicemia/metabolismo , Feminino , Humanos , Mutação , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples/genética , GravidezRESUMO
Infrapopulation genetic variation of the oioxenous, hermaphroditic flatworm Nasicola klawei (Monogenea: Capsalidae) infecting the nasal cavities of nine yellowfin tuna, Thunnus albacares, from the Gulf of Mexico was analyzed using the first internal transcribed spacer (ITS1) single strand conformation polymorphism (SSCP), ITS1 sequencing, and amplified fragment length polymorphism (AFLP). Of a total of 32 worms, six had unique ITS1-SSCP types and the rest was grouped by three types. Two worms of the same infrapopulation shared an ITS1-SSCP type in nine instances but no infrapopulation was monophyletic by ITS1-SSCP analysis. ITS1 sequences (420 bp) varied by 1-11 (0.2-2.6%) nucleotides. Twenty-three AFLP profiles of 80-110 bands failed to support genomic monophyly of any N. klawei infrapopulation. 28S rDNA (990 bp) sequences from four worms representing four infrapopulations were identical and matched conspecific GenBank sequences. Concordant ITS1-SSCP and AFLP analyses indicated that these N. klawei infrapopulations principally resulted from tuna being repeatedly colonized by planktonic, infective larvae (oncomiracidia) rather than by a single host colonization followed by parasite maturation, self-fertilization, and production of auto-infecting progeny.
Assuntos
DNA Espaçador Ribossômico/genética , Doenças dos Peixes/parasitologia , Cavidade Nasal/parasitologia , Polimorfismo Conformacional de Fita Simples/genética , Trematódeos/genética , Atum/parasitologia , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Animais , Impressões Digitais de DNA , DNA Ribossômico/genética , Bases de Dados Genéticas , Golfo do México , México , Filogenia , Autofertilização/fisiologia , Trematódeos/classificação , Trematódeos/isolamento & purificaçãoRESUMO
Heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing were utilised to genotype human parvovirus B19 samples from Brazil and Paraguay. Ninety-seven serum samples were collected from individuals presenting with abortion or erythema infectiosum, arthropathies, severe anaemia and transient aplastic crisis; two additional skin samples were collected by biopsy. After the procedure, all clinical samples were classified as genotype 1.
Assuntos
Análise Heteroduplex/métodos , Infecções por Parvoviridae/virologia , Parvovirus B19 Humano/genética , Polimorfismo Conformacional de Fita Simples/genética , Sequência de Bases , Brasil , Feminino , Genótipo , Humanos , Dados de Sequência Molecular , Paraguai , Parvovirus B19 Humano/isolamento & purificação , Gravidez , Complicações Infecciosas na Gravidez/virologiaRESUMO
Polymorphisms of the CART gene were investigated by PCR-single-strand conformation polymorphism analysis in 540 samples from 10 goat breeds. Ten novel single-nucleotide polymorphisms as well as three microsatellites were detected; a mutation, 77T â C, led to an amino acid change (Leu â Ser). Associations between polymorphic loci and reproductive traits were analyzed in Chuandong White, Guizhou White and Gulin Ma breeds. Mutation at position 524 had no significant effect on litter size in these three goat breeds. The polymorphism 539C â A differed significantly among the three breeds (P < 0.05); C(7)T(8)/C(9)T(8) at 939 was associated with larger litter size (P < 0.05) than genotypes C(7)T(8)/C(7)T(8) and C(7)T(8)/C(8)T(8). No significant association of birth weight was found with gene variation (524C â T, 539C â A and 939 CnTn). These findings could be valuable for marker-assisted selection for goat breeding.
Assuntos
Cabras/genética , Proteínas do Tecido Nervoso/genética , Polimorfismo de Nucleotídeo Único , Polimorfismo Conformacional de Fita Simples/genética , Reprodução/genética , Animais , Sequência de Bases , Biomarcadores , Peso ao Nascer/genética , Cruzamento , Tamanho da Ninhada de Vivíparos/genética , Repetições de Microssatélites/genética , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
PURPOSE: Lynch syndrome is the most common inherited syndrome of colorectal cancer, caused principally by germline mutations in MLH1 and MSH2. We report our experience with genetic screening in the diagnosis of Lynch syndrome in Chile, a country previously underserved in the capacity to diagnose hereditary colorectal cancer. METHODS: Families from our Familial Colorectal Cancer Registry were selected for this study if they fulfilled either Amsterdam I/II or Bethesda criteria for classification of Lynch syndrome. Analysis of colorectal tumors from probands included a microsatellite instability study and immunohistochemical evaluation for MLH1 and MSH2. Screening of germline mutations was performed by single-strand conformation polymorphism analysis and DNA sequencing. RESULTS: A total of 21 families were evaluated, 14 meeting Amsterdam criteria and 7 meeting Bethesda criteria. Tumors in 20 families (95%) showed microsatellite instability (19 high and 1 low) and 9 of these 20 families (45%) harbored a germline mutation (7 of 13 Amsterdam and 2 of 7 Bethesda families). Of the 9 mutations identified, 6 were in MLH1 and 3 in MSH2. Two of the mutations were novel, 3 were previously found in 1 to 2 European populations, and 4 were previously found in various ethnic populations worldwide. Only 2 mutations were previously found in another Latin American population (Colombia). In our probands, colorectal cancer was located mainly (57%) in the right or transverse colon. Pedigree information from 104 family affected members of 21 studied families showed endometrial cancer to be the most frequent primary extracolonic tumor, accounting for 15.1% of total cases, followed by stomach (13.2%) and breast cancer (11.3%). Analysis of mitochondrial DNA haplotypes showed a strong Amerindian genetic component in 15 (71.4%) of the 21 families analyzed. CONCLUSION: The study of Lynch syndrome in families of different ethnic origins contributes to the definition of genetic and clinical differences among populations. Wide distribution in other ethnic populations strongly suggests varying origins of 4 the mutations found. Although cancer phenotype was consistent with those from other Latin American populations, only 2 of 9 mutations were shared with other South American populations and 2 novel mutations were found. The Chilean population is considered to be an admixture of Amerindian and European-mainly Spanish-populations, producing an ethnic group with significant genetic differences from populations previously studied.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/genética , Adolescente , Adulto , Idoso , Sequência de Bases , Chile , Feminino , Predisposição Genética para Doença , Testes Genéticos , Mutação em Linhagem Germinativa , Humanos , Técnicas Imunoenzimáticas , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Fenótipo , Polimorfismo Conformacional de Fita Simples/genética , Sistema de Registros , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Marine turtles are increasingly being threatened worldwide by anthropogenic activities. Better understanding of their life cycle, behavior and population structure is imperative for the design of adequate conservation strategies. The mtDNA control region is a fast-evolving matrilineal marker that has been employed in the study of marine turtle populations. We developed and tested a simple molecular tracing system for Caretta caretta mtDNA haplotypes by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Using this technique, we were able to distinguish the SSCP patterns of 18 individuals of the haplotypes CC-A4, CC-A24 and CCxLO, which are commonly found in turtles sampled on the Brazilian coast. When we analyzed 15 turtles with previously unknown sequences, we detected two other haplotypes, in addition to the other four. Based on DNA sequencing, they were identified as the CC-A17 and CC-A1 haplotypes. Further analyses were made with the sea turtles, Chelonia mydas (N = 8), Lepidochelys olivacea (N = 3) and Eretmochelys imbricata (N = 1), demonstrating that the PCR-SSCP technique is able to distinguish intra- and interspecific variation in the family Cheloniidae. We found that this technique can be useful for identifying sea turtle mtDNA haplotypes, reducing the need for sequencing.
Assuntos
DNA Mitocondrial/química , Polimorfismo Genético , Polimorfismo Conformacional de Fita Simples/genética , Tartarugas/genética , Animais , Geografia , Reação em Cadeia da Polimerase/métodosRESUMO
The Notch signaling pathway (NSP) is an important intercellular communication mechanism that regulates embryo development and adult physiological functions. The Hairless (H) protein is a powerful antagonist of the NSP by its interaction with the Suppressor of Hairless (Su[H]) protein, recruiting the corepressors Gro and CtBP. In the present work, we examined the role of several important amino acids in different H protein domains analyzing four mutant lines of Drosophila melanogaster. The mutant alleles were evaluated by single-strand conformational polymorphism (SSCP) analysis and we located mutated regions at different positions along the sequence of the Hairless gene.