Informe de evaluación científica basada en la evidencia disponible: examen genético molecular / Scientific evaluation report based on available evidence: molecular genetic examination

INTRODUCCIÓN: Las enfermedades de depósito lisosomal son un grupo heterogéneo de errores metabólicos poco frecuentes, congénitos, de origen genético, caracterizados por la deficiencia funcional de los lisosomas y por la acumulación intralisosomal de sustratos. Se han caracterizado cerca de 50 desórdenes metabólicos de este origen, causados por alteraciones genéticas a nivel enzimático, de receptores, proteínas activadoras, de membrana o transportadora, causando la acumulación lisosomal de sustratos, específicos para cada enfermedad. La acumulación es progresiva, causando finalmente el deterioro funcional celular y tisular. Varios de estos desórdenes afectan el sistema nervioso central, y la mayoría de los pacientes tienen disminución de la esperanza de vida y morbilidad asociada. Para efectos del presente informe de evaluación se consideraron las patologías Mucopolisacaridosis I; Mucopolisacaridosis II; Mucopolisacaridosis VI; Enfermedad de Gaucher y Enfermedad de Fabry. TECNOLOGÍAS SANITARIAS EVALUADAS: Examen genético molecular: secuenciación y/o genotipificación. EFICACIA DE LAS INTERVENCIONES: La intervención es eficaz, cumpliendo con lo dispuesto en la letra a), del artículo 2, del decreto supremo N°13, de 2017, del Ministerio de Salud, que aprueba Reglamento que Establece el Proceso destinado a determinar los Diagnósticos y Tratamientos de Alto Costo con Sistema de Protección Financiera, según lo establecido en los artículos 7° Y 8° de la ley N° 20.850. ANÁLISIS ECONÓMICO: El costo total de implementación de los exámenes es de $ 2.037.565 anuales en el año 2018 y hasta $ 6.883.384 en el 2022. CONCLUSIÓN: Para dar cumplimiento al artículo 28° del Reglamento que establece el proceso destinado a determinar los diagnósticos y tratamientos de alto costo con Sistema de Protección Financiera, según lo establecido en los artículos 7°y 8° de la ley N°20.850, aprobado por el decreto N°13 del Ministerio de Salud, se concluye que el presente informe de evaluación se considera no favorable, de acuerdo a lo establecido en el Título III. de las Evaluaciones Favorables de la Norma Técnica N° 0192 de este mismo ministerio.

QTL mapping for physiology, yield and plant architecture traits in cotton (Gossypium hirsutum L. ) grown under well-watered versus water-stress conditions

Increasing scarcity of irrigation water is a major threat to sustainable production of cotton (Gossypium hirsutum L.). Identifying genomic regions contributing to abiotic stress tolerance will help develop cotton cultivars suitable for water-limited regions through molecular marker-assisted breeding. A molecular mapping F2 population was derived from an intraspecific cross of the drought sensitive G. hirsutum cv. FH-901 and drought tolerant G. hirsutum cv. RH-510. Field data were recorded on physiological traits (osmotic potential and osmotic adjustment); yield and its component traits (seedcotton yield, number of bolls/plant and boll weight); and plant architecture traits (plant height and number of nodes per plant) for F2, F2:3 and F2:4 generations under well-watered versus water-limited growth conditions. The two parents were surveyed for polymorphism using 6500 SSR primer pairs. Joinmap3.0 software was used to construct linkage map with 64 polymorphic markers and it resulted into 35 markers mapped on 12 linkage groups. QTL analysis was performed by composite interval mapping (CIM) using QTL Cartographer2.5 software. In total, 7 QTLs (osmotic potential 2, osmotic adjustment 1, seedcotton yield 1, number of bolls/plant 1, boll weight 1 and plant height 1) were identified. There were three QTLs (qtlOP-2, qtlOA-1, and qtlPH-1) detected only in water-limited conditions. Two QTLs (qtlSC-1 and qtlBW-1) were detected for relative values. Two QTLs (qtlOP-1 and qtlBN-1) were detected for well-watered treatment. Significant QTLs detected in this study can be employed in MAS for molecular breeding programs aiming at developing drought tolerant cotton cultivars.

Seleção, acasalamento e genotipagem seletiva e outras estratégias de amostragem na detecção de QTL / Selection, mating, and selective genotyping and other strategies of sampling on QTL detection

Foram simuladas diferentes estratégias de seleção para estimar o desempenho fenotípico e a endogamia média na seleção assistida por marcadores, em características quantitativas com valores de herdabilidade de 0,10; 0,40 e 0,70. O sistema de simulação genética (Genesys) foi utilizado para a simulação de três genomas (cada qual constituído de uma única característica cuja distinção estava no valor da herdabilidade), e das populações base e inicial. Cada população inicial foi submetida à seleção assistida por marcadores por 20 gerações consecutivas. Avaliaram-se estratégias de acasalamento entre os genitores selecionados, em diferentes intensidades de seleção (tamanhos populacionais), por meio do acasalamento seletivo entre os melhores e os piores, acasalamento apenas entre os melhores e/ou entre os piores e acasalamento aleatório. Em todos os cenários combinando herdabilidade e intensidade, o acasalamento estratégico utilizando a metodologia da genotipagem seletiva foi superior aos demais, tornando-se mais eficaz na detecção de QTL e, consequentemente, no incremento do valor fenotípico e na minimização das médias endogâmicas ao longo das gerações. Ao utilizar a estratégia seletiva de amostragem, menor tamanho populacional é requerido para otimizar a detecção de QTL à medida que o valor da herdabilidade da característica aumenta.

Different strategies of selection were simulated to estimate the phenotypic performance and average inbreeding in selection assisted by markers, for quantitative traits with heritability values of 0.10, 0.40, and 0.70. The genetic simulation system (Genesys) was used for the simulation of three genomes (each one consisting of a single characteristic which distinction was the value of heritability) and base and original populations. Each initial population was subjected to selection assisted by markers for 20 consecutive generations. Strategies of mating between the parents selected in different intensities of selection (population size) were evaluated by selective mating between the best and worst, mating only among the best and/or among the worst and random mating. In all scenarios combining heritability and intensity, the strategic mating using the methodology of selective genotyping was superior to the others, becoming more effective in detecting QTL and, consequently, in the increase of phenotypic value and in the minimization of the inbred average over the generations. By using the selective sampling strategy, smaller population size to optimize the detection of QTL is required, since the value of the heritability of the characteristic increases.

Utilizing linkage disequilibrium information from Indian Genome Variation Database for mapping mutations: SCA12 case study.

Stratification in heterogeneous populations poses an enormous challenge in linkage disequilibrium (LD) based identification of causal loci using surrogate markers. In this study, we demonstrate the enormous potential of endogamous Indian populations for mapping mutations in candidate genes using minimal SNPs, mainly due to larger regions of LD. We show this by a case study of the PPP2R2B gene (approximately 400 kb) that harbours a CAG repeat, expansion of which has been implicated in spinocerebellar ataxia type 12 (SCA12). Using LD information derived from Indian Genome Variation database (IGVdb) on populations which share similar ethnic and linguistic backgrounds as the SCA12 study population, we could map the causal loci using a minimal set of three SNPs, without the generation of additional basal data from the ethnically matched population. We could also demonstrate transferability of tagSNPs from a related HapMap population for mapping the mutation.

Mapeamento de locos de características quantitativas associados à composição de carcaça, no cromossomo seis de suíno / Mapping of quantitative trait loci for carcass composition on swine chromosome six

Uma população de suínos, composta de 550 animais F2, foi produzida a partir do intercruzamento da geração F1, obtida pelo cruzamento divergente de dois machos da raça nativa brasileira Piau com 18 fêmeas comerciais. O objetivo do trabalho foi mapear locos de características quantitativas (QTL) associados a cortes de carcaça. Os animais foram genotipados para 13 marcadores microssatélites, distribuídos no cromossomo 6 de suínos. As características avaliadas foram: peso total do pernil, peso do pernil sem pele e sem capa de gordura, peso total da copa, peso da copa sem pele e sem capa de gordura, peso total da paleta, peso da paleta sem pele e sem capa de gordura, peso total do carré, peso do lombo, peso total do bacon, peso das costelas, peso total da papada, peso do filezinho e peso da banha rama. Utilizou-se o método de regressão por intervalo de mapeamento por meio do programa QTL Express. Foram encontrados indicativos de QTL para peso de pernil limpo, peso de paleta, peso de lombo e peso de filezinho. A região genômica deve ser saturada com marcadores adicionais para confirmar a presença de QTL reais.

A swine population of 550 F2 animals was produced by outbred cross using two sires of the native Brazilian breed Piau and 18 commercial dams. The animals were genotyped for 13 microsatellite markers. The evaluated composition traits of carcass were: ham weight, skinless and fatless ham weight, boston shoulder weight, skinless and fatless boston shoulder weight, picnic shoulder weight, skinless and fatless picnic shoulder weight, total loin (bone-in) weight, loin weight, bacon weight, rib weight, jowl weight, sirloin weight, and belly fat weight. Data were analyzed by multiple regression interval mapping, using the QTL Express software. Suggestive QTL were found for skinless and fatless ham weight, picnic shoulder weight, loin weight, and sirloin weight. However, the genomic region should be saturated with additional markers in order to confirm the presence of real QTL.

Heritability and linkage study on heart rates in a Mongolian population

Elevated heart rate has been proposed as an independent risk factor for cardiovascular diseases, but their interrelationships are not well understood. In this study, we performed a genome-wide linkage scan in 1,026 individuals (mean age 30.6 years, 54.5% women) from 73 extended families of Mongolia and determined quantitative trait loci that influence heart rate. The DNA samples were genotyped using deCODE 1,039 microsatellite markers for 3 cM density genome-wide linkage scan. Correlation analysis was carried out to evaluate the correlation of the covariates and the heart rate. T-tests of the heart rate were also performed on sex, smoking and alcohol intake. Consequently, this model was used in a nonparametric genome-wide linkage analysis using variance component model to create a multipoint logarithm of odds (LOD) score and a corresponding P value. In the adjusted model, the heritability of heart rate was estimated as 0.32 (P<.0001) and a maximum multipoint LOD score of 2.03 was observed in 77 cM region at chromosome 18. The second largest LOD score of 1.52 was seen on chromosome 5 at 216 cM. Genes located on the specified locations in chromosomes 5 and 18 may be involved in the regulation of heart rate.

A theoretical treatment of interval mapping of a disease gene using transmission disequilibrium tests.

The genetic basis of the transmission disequilibrium test (TDT) for two-marker loci is explored from first principles. In this case, parents doubly heterozygous for a given haplotype at the pair of marker loci that are each in linkage disequilibrium with the disease gene with the further possibility of a second-order linkage disequilibrium are considered. The number of times such parents transmit the given haplotype to their affected offspring is counted and compared with the frequencies of haplotypes that are not transmitted. This is done separately for the coupling and repulsion phases of doubly heterozygous genotypes. Expectations of the counts for each of the sixteen cells possible with four-marker gametic types (transmitted vs not transmitted) are derived. Based on a test of symmetry in a square 4 x 4 contingency table, chi-square tests are proposed for the null hypothesis of no linkage between the markers and the disease gene. The power of the tests is discussed in terms of the corresponding non-centrality parameters for the alternative hypothesis that both the markers are linked with the disease locus. The results indicate that the power increases with the decrease in recombination probability and that it is higher for a lower frequency of the disease gene. Taking a pair of markers in an interval for exploring the linkage with the disease gene seems to be more informative than the single-marker case since the values of the non-centrality parameters tend to be consistently higher than their counterparts in the single-marker case. Limitations of the proposed test are also discussed.

Estudo genético da doença de Parkinson / Genetical study of Parkinson's disease

A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum com uma prevalência aproximada de 3 por cento em pacientes com mais de 65 anos. É doença esporádica, mas o parkinsonismo primário (PP) familiar, decorrente de defeitos genéticos específicos, tem sido encontrado em cerca de 10 por cento dos casos diagnosticados como DP. Este estudo teve por fim o rastreamento de mutações em genes relacionados ao parkinsonismo em pacientes com PP de início precoce (< 40a) ou com história familiar positiva. Investigamos 53 probandos do Grupo de Estudo de Distúrbios do Movimento da Clínica Neurológica do HCFMUSP. Foram encontradas 4 famílias portadoras de mutações no gene PARK2, duas no PARK8 e uma no PARK9...

Parkinson disease (PD) is the second most common neurodegenerative disorder affecting approximately 3 per cent of the population over age 65. Most cases of PD manifest in sporadic form, but familial primary parkinsonism (PP) due genetical abnormalities has been found in about 10 per cent of cases diagnosed as PD. The aim of this study was to analyze the DNA of PP patients seen at the Group for the Study of Movement Disorders of the Neurology Department of HCFMUSP who presented early onset of the disease (< 40 years of age) or positive family history, with the purpose of screening candidate mutations for the disease. We found four families carrying mutations in gene PARK2, two in PARK8, and one in PARK9...

Methylation Profiles of CpG Island Loci in Major Types of Human Cancers

Several reports have described aberrant methylation in various types of human cancers. However, the interpretation of methylation frequency in various human cancers has some limitations because of the different materials and methods used for methylation analysis. To gain an insight into the role of DNA hypermethylation in human cancers and allow direct comparison of tissue specific methylation, we generated methylation profiles in 328 human cancers, including 24 breast, 48 colon, 61 stomach, 48 liver, 37 larynx, 24 lung, 40 prostate, and 46 uterine cervical cancer samples by analyzing CpG island hypermethylation of 13 genes using methylation-specific PCR. The mean numbers of methylated genes were 6.5, 4.4, 3.6, 3.4, 3.1, 3.1, 3.1, and 2.1 in gastric, liver, prostate, larynx, colon, lung, uterine cervix, and in breast cancer samples, respectively. The number of genes that were methylated at a frequency of more than 40% in each tumor type ranged from nine (stomach) to one (breast). Generally genes frequently methylated in a specific cancer type differed from those methylated in other cancer types. The findings indicate that aberrant CpG island hypermethylation is a frequent finding in human cancers of various tissue types, and each tissue type has its own distinct methylation pattern.

Relationship Between the Extent of Chromosomal Losses and the Pattern of CpG Methylation in Gastric Carcinomas

The extent of unilateral chromosomal losses and the presence of microsatellite instability (MSI) have been classified into high-risk (high- and baseline-level loss) and low-risk (low-level loss and MSI) stem-line genotypes in gastric carcinomas. A unilateral genome-dosage reduction might stimulate compensation mechanism, which maintains the genomic dosage via CpG hypomethylation. A total of 120 tumor sites from 40 gastric carcinomas were examined by chromosomal loss analysis using 40 microsatellite markers on 8 chromosomes and methylation analysis in the 13 CpG (island/non-island) regions near the 10 genes using the bisulfite-modified DNAs. The high-level-loss tumor (four or more losses) showed a tendency toward unmethylation in the Maspin, CAGE, MAGE-A2 and RABGEF1 genes, and the other microsatellite-genotype (three or fewer losses and MSI) toward methylation in the p16, hMLH1, RASSF1A, and Cyclin D2 genes (p<0.05). The non-island CpGs of the p16 and hMLH1 genes were hypomethylated in the high-level-loss and hypermethylated in the non-high-level-loss sites (p<0.05). Consequently, hypomethylation changes were related to a high-level loss, whereas the hypermethylation changes were accompanied by a baseline-level loss, a low-level loss, or a MSI. This indicates that hypomethylation compensates the chromosomal losses in the process of tumor progression.

Hereditary motor and autonomic neuronopathy 1 maps to chromosome 20q13.2-13.3

The spinal muscular atrophies (SMA) or hereditary motor neuronopathies result from the continuous degeneration and death of spinal cord lower motor neurons, leading to progressive muscular weakness and atrophy. We describe a large Brazilian family exhibiting an extremely rare, late-onset, dominant, proximal, and progressive SMA accompanied by very unusual manifestations, such as an abnormal sweating pattern, and gastrointestinal and sexual dysfunctions, suggesting concomitant involvement of the autonomic nervous system. We propose a new disease category for this disorder, `hereditary motor and autonomic neuronopathy', and attribute the term, `survival of motor and autonomic neurons 1' (SMAN1) to the respective locus that was mapped to a 14.5 cM region on chromosome 20q13.2-13.3 by genetic linkage analysis and haplotype studies using microsatellite polymorphic markers. This locus lies between markers D20S120 and D20S173 showing a maximum LOD score of 4.6 at D20S171, defining a region with 33 known genes, including several potential candidates. Identifying the SMAN1 gene should not only improve our understanding of the molecular mechanisms underlying lower motor neuron diseases but also help to clarify the relationship between motor and autonomic neurons.

Alu and L1 Retroelements Are Correlated with the Tissue Extent and Peak Rate of Gene Expression, Respectively

We exploited the serial analysis of gene expression (SAGE) libraries and human genome database in silico to correlate the breadth of expression (BOE; housekeep-ing versus tissue-specific genes) and peak rate of expression (PRE; high versus low expressed genes) with the density distribution of the retroelements. The BOE status is linearly associated with the density of the sense Alus along the 100 kb nucleotides region upstream of a gene, whereas the PRE status is inversely correlated with the density of antisense L1s within a gene and in the up- and downstream regions of the 0-10 kb nucleotides. The radial distance of intranuclear position, which is known to serve as the global domain for transcription regulation, is reciprocally correlated with the fractions of Alu (toward the nuclear center) and L1 (toward the nuclear edge) elements in each chromosome. We propose that the BOE and PRE statuses are related to the reciprocal distribution of Alu and L1 elements that formulate local and global expression domains.

Molecular cytogenetic assignment of genes to bovine chromosome 5

Seven genes were assigned by molecular cytogenetic methods to bovine chromosome 5. To accomplish this, specific primers were either publicly available or were designed from highly conserved regions of the publicly available mammalian gene sequences. The identity of the amplified segments was verified by sequencing and alignment with the published sequences. The optimized primers that amplified the desired bovine genes were used for screening a bovine bacterial artificial chromosome library. The positive clones were localized to a specific band of bovine chromosome 5 by fluorescence in situ hybridization. The genes HOXC4, SP1 and IGFBP6 were localized to band q21, COL2A1 was localized to bands q21-q23, IGF1 was localized to band q26, MB to band q31 and the gene CYP2D6 was localized to band q35. The cytogenetic assignment of SP1, IGFBP6, COL2A1, IGF1, MB and CYP2D6 is first reported here and the assignment of HOXC4 refines the previous assignment of this gene. The identification and localization of these genes further support the development of the human to bovine comparative map through characterizing the homologous segments conserved in the evolution of these species. This information will be useful for the future localization of genes that affect economically important traits in bovines

The human genome sequence: impact on health care.

The recent sequencing of the human genome, resulting from two independent global efforts, is poised to revolutionize all aspects of human health. This landmark achievement has also vindicated two differeint methodologies that can now be used to target other important large genomes. The human genome sequence has revealed several novel/surprising features notably the probable presence of a mere 30-35,000 genes. In depth comparisons have led to classification of protein families and identification of several orthologues and paralogues. Information regarding non-protein coding genes as well as regulatory regions has thrown up several new areas of research. Although still incomplete, the sequence is poised to become a boon to pharmaceutical companies with the promise of delivering several new drug targets. Several ethical concerns have also been raised and need to be addressed in earnest. This review discusses all these aspects and dwells on the possible impact of the human genome sequence on human health, medicine and also health care delivery system.

Heterogeneidade genética em atrofia óptica autossômica dominante / Genetic Heterogeneity in autosomal dominant optic atrophy

Objetivos: A atrofia óptica autossômica dominante, tipo Kjer ou juvenil, é neuropatia óptica hereditária que causa perda de acuidade visual, anormalidades da visão de cores e defeitos do campo visual, caracterizada por palidez do disco óptico. O gene desta doença foi mapeado por análise de ligação genética em um intervalo de 1,4 cM no cromossomo 3q28-29 entre os marcadores microssatélites D3S3669 e D3S3562. Embora a maioria das famílias estudadas tenha mostrado ligação para a região cromossômica 3q28-29, uma família foi mapeada no cromossomo 18q12.2-12.3. Este trabalho analisa a ligação da atrofia óptica em três famílias com marcadores polimórficos para os cromossomos 3q28-29 e 18q12.2-12.3. Métodos: Cinqüenta e sete indivíduos de três famílias foram submetidos a exame oftalmológico e coleta de sangue. O DNA foi extraído e amplificado em reações de polimerase em cadeia (PCR) com marcadores polimórficos para os cromossomos 3q28-29 e 18q12.2-12.3. Os fragmentos de PCR foram mensurados em seqüenciador automático (373 DNA sequencer). Estes -números foram utilizados como alelos para análise de haplótipos. Os "lod scores" foram calculados pelo programa MLINK. Resultados: Na primeira família houve suspeita da atrofia óptica mapear para o cromossomo 3q28-29, mas sem significância estatística no valor do "lod score". Na segunda família a atrofia óptica apresentou ligação para este locus. Os eventos de recombinação nesta família localizaram o gene num intervalo de 2 cM entre os marcadores D3S3669 e D3S2305. O "lod score" máximo obtido foi de 3,56 no theta de 0,00 com o marcador D3S3669. A terceira família não apresentou ligação nos cromossomos 3q28-29 e 18q12.2-12.3. Conclusão: O fato da terceira família não mapear para nenhum dos dois loci já descritos é indicativo de que existe heterogeneidade genética na atrofia óptica autossômica dominante e levanta a possibilidade de existir um terceiro locus para esta doença.

Importância do gene conexina 26 na etiologia da deficiência auditiva sensorioneural não-sindrômica / Role of connexin 26 gen in etiology of non-syndromic sensorioneral hearing loss

Cerca de 60 por cento de todas as causas de surdez pré-lingual podem ser atribuídas a fatores genéticos. A análise molecular do gene conexina 26, para a identificação da mutação que ocasiona a deficiência auditiva sensorioneural não-sindrômica, têm extrema relevância para o diagnóstico precoce, tratamento adequado e para a implantação de programas de prevenção.

Geraçäo de ®etiquetas de sequências expressas¼ dirigidas para porçöes codificadoras dos genes (Orestes): identificaçäo de novos genes humanos expressos em câncer de mama / Generation of ®expressed sequence tags¼ directed to genes coders portions (Orestes): identification of new human genes expressed in breast tumor

Etiquetas de sequências expressas (ESTs) são fundamentais para a identificação de genes no genoma humano e para definir características de expressão gênica. Neste trabalho, descrevemos uma nova abordagem para a geração de bibliotecas de cDNA, utilizando iniciadores arbitrários para a produção, por PCR, de mini-bibliotecas a partir de mRNA derivado de câncer de mama. Clones destas bibliotecas foram sequenciadas para gerar 6029 ESTs. Utilizando esta abordagem, foi possível observar uma significante normalização das diferentes sub-populações de mRNA e amplificação preferencial de porções centrais dos genes. Análise bioinformática destas sequências mostra que 3.350 ESTs (56 por cento) tem similaridade significante a sequências de DNA e/ou cDNA já conhecidas (sequências anotadas) descritas em diferentes organismos, e 1509 ESTs (25 por cento) não possuem qualquer similaridade a diferentes bancos de dados...