Juvenile Idiopathic Arthritis Subtypes
Another example of the use of gene-expression profiling is for identifying patient subtypes in juvenile idiopathic arthritis (JIA). “We are studying gene-expression analysis in peripheral blood mononuclear cells (PBMC) in order to identify sets of genes that may help us better understand differences within the patient population,” says Michael G. Barnes, Ph.D., research associate, division of rheumatology, Cincinnati Children’s Hospital Medical Center, speaking on behalf of a large team of researchers involved in the project, which was supported by the NIH.
Juvenile idiopathic arthritis (JIA) encompasses the majority of childhood arthritis. Although seven subtypes have been described, there is increasing evidence for heterogeneity even within these types. “Use of genome-level technologies can provide a comprehensive determination of genetic and genomic biological signatures, giving an unprecedented opportunity to define JIA on the basis of molecular phenotypes and can help us understand disease mechanisms. This may ultimately help improve therapeutic approaches,” Dr. Barnes explains.
To begin the analysis, PBMC are first isolated using Ficoll gradient centrifugation. RNA is immediately stabilized and later isolated and purified. “We assess RNA quality with standard protocols and then label it using NuGEN Ovation (NuGEN Technologies). Next we hybridize the labeled samples to Affymetrix GeneChips (Affymetrix). This array has nearly 55,000 probe sets and can measure up to 47,000 transcripts.”
Processing the monumental amount of data generated into meaningful results requires the use of bioinformatic approaches. “To begin analysis, we import the data we generate into a program called GeneSpring GX (Agilent Technologies). We then adjust batch to batch variation by a process called distance-weighted discrimination. Next we identify genes with different levels between groups. Finally, we perform a functional analysis of the data.”
Employing these approaches, Dr. Barnes and colleagues found substantial PBMC gene-expression differences in patients with early-onset JIA as compared to those with late-onset disease.
“Age of onset may be an important characteristic for classifying certain JIA patient subtypes. Today, differential diagnosis between the oligoarticular and polyarticular JIA subtypes is based, to a large extent, on how many joints are affected in patients. Utilizing molecular approaches in addition to other biologic markers like antinuclear antibodies (ANA) provides great potential to grasp pathologic mechanisms that may help explain the differences between patients with early and late disease onset. Understanding these processes ultimately may lead to better treatments for JIA.”