RESUMO
BACKGROUND: Adoption of new technology in both basic research and clinical settings requires rigorous validation of analytical performance. The OncoScan® FFPE Assay is a multiplexing tool that offers genome-wide copy number and loss of heterozygosity detection, as well as identification of frequently tested somatic mutations. METHODS: In this study, 162 formalin fixed paraffin embedded samples, representing six different tumour types, were profiled in triplicate across three independent laboratories. OncoScan® formalin fixed paraffin embedded assay data was then analysed for reproducibility of genome-wide copy number, loss of heterozygosity and somatic mutations. Where available, somatic mutation data was compared to data from orthogonal technologies (pyro/sanger sequencing). RESULTS: Cross site comparisons of genome-wide copy number and loss of heterozygosity profiles showed greater than 95% average agreement between sites. Somatic mutations pre-validated by orthogonal technologies showed greater than 90% agreement with OncoScan® somatic mutation calls and somatic mutation concordance between sites averaged 97%. CONCLUSIONS: Reproducibility of whole-genome copy number, loss of heterozygosity and somatic mutation data using the OncoScan® assay has been demonstrated with comparatively low DNA inputs from a range of highly degraded formalin fixed paraffin embedded samples. In addition, our data shows examples of clinically-relevant aberrations that demonstrate the potential utility of the OncoScan® assay as a robust clinical tool for guiding tumour therapy.
Assuntos
Técnicas de Laboratório Clínico/normas , Perfilação da Expressão Gênica/métodos , Genoma Humano , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fixação de Tecidos/métodos , Análise Mutacional de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Perda de Heterozigosidade , Masculino , Mutação , Neoplasias/metabolismo , Inclusão em Parafina , Controle de Qualidade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
The frequency of killer immunoglobulinlike receptors (KIR) genes was examined in type 1 diabetes mellitus patients and controls from Finland. The KIR gene 2DS5 was significantly decreased in patients versus controls, but this was no longer significant after correction for the number of comparisons made.
Assuntos
Diabetes Mellitus Tipo 1/genética , Frequência do Gene , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação de Sentido Incorreto , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/imunologia , Feminino , Finlândia , Frequência do Gene/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Monoméricas de Ligação ao GTP/imunologia , Mutação de Sentido Incorreto/imunologiaRESUMO
This review updates the on-going investigations into KIR genes and their alleles with the main emphasis on what has taken place in this laboratory over the last 3 years.
Assuntos
Proteínas Imediatamente Precoces/genética , Células Matadoras Naturais/fisiologia , Proteínas Monoméricas de Ligação ao GTP/genética , Alelos , Animais , Haplótipos , HumanosRESUMO
Killer-cell Ig-like receptors (KIR) are structurally and functionally diverse, and enable human NK cells to survey the expression of individual HLA class I molecules, often altered in infections and tumors. Multiple events of non-reciprocal recombination have contributed to the rapid diversification of KIR. We show that approximately 4.5% of the individuals of a Caucasoid population bear a recombinant allele of KIR3DP1, officially designed KIR3DP1*004, that associates tightly with gene duplications of KIR3DP1, KIR2DL4 and KIR3DL1/KIR3DS1. The KIR3DP1 gene is normally silent, but the recombinant allele carries a novel promoter sequence and, as a consequence, is transcribed in all tested individuals. Messenger RNA of KIR3DP1*004 is made up of six exons; of these, exons 1-5 are similar to, and spliced like, those encoding the leader peptide and Ig-domains of KIR3D. By contrast, exon 6 is homologous to no other human KIR sequence, but only to possible homologs in chimpanzees and rhesus macaques, and encodes a short hydrophilic tail. The putative KIR3DP1*004 product, like those of the related genes LAIR-2 and LILRA3/ILT6/LIR4, is predicted to be secreted to the extracellular medium rather than anchored to the cell membrane.
Assuntos
Receptores Imunológicos/genética , Alelos , Sequência de Aminoácidos , Duplicação Gênica , Inativação Gênica , Haplótipos , Humanos , Células Matadoras Naturais/imunologia , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Receptores Imunológicos/biossíntese , Receptores Imunológicos/química , Receptores KIR , Receptores KIR2DL4 , Receptores KIR3DL1 , Receptores KIR3DS1 , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Recombinação Genética , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
The polymorphic nature of the KIR3DL1/S1 gene complex has been investigated through the development of a polymerase chain reaction-sequence-specific oligonucleotide probes (PCR-SSOP) allele typing system. The KIR3DL1/S1 system was applied to a healthy group of Northern Irish individuals to establish allele frequencies within this Caucasian population. Additionally, cell line DNA and CEPH families, both from the 13th International Histocompatibility Workshop, and local families have been investigated. The generated data emphasize the complexity and highly polymorphic nature of this KIR gene complex; 11 allelic variations were identified, 2 of which are novel to this study. Use of the PCR-SSOP system has confirmed the presence of multiple copies of KIR3DL1/S1 alleles in a number of individuals.
Assuntos
Alelos , Frequência do Gene/genética , Polimorfismo Genético , Receptores Imunológicos/genética , Frequência do Gene/imunologia , Humanos , Irlanda do Norte , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Polimorfismo Genético/imunologia , Receptores Imunológicos/imunologia , Receptores KIR , Receptores KIR3DL1 , População BrancaRESUMO
Multiple copies of the killer immunoglobulin-like receptor gene, 3DL/S1, have been identified in certain individuals. Additionally, allele determination of the killer immunoglobulin-like receptor gene (KIR), 2DL4, has identified three alleles of this gene present in these same individuals. This event has been confirmed by isolating three distinct KIR2DL4 allele clones in each individual, which sequenced as the alleles identified by the allele identification technique. It is our assumption that an unequal crossover event has occurred between differing KIR haplotypes resulting in the duplication of the 2DL4, 3DS1/3DL1 genes on the newly formed haplotype(s).
