Defining response to TNF-inhibitors in rheumatoid arthritis: the negative impact of anti-TNF cycling and the need for a personalized medicine approach to identify primary non-responders.

Current guidelines recommend treating rheumatoid arthritis (RA) patients to reach low disease activity or remission, however, most biologic-naive RA patients fail to reach treatment targets on their first biologic therapy. Approximately 90% of biologic-naive RA patients receive a tumor necrosis factor alpha inhibitor (anti-TNF) as their first biologic treatment, even though several alternative mechanism of action (MOA) therapies are approved as first-line options. After 3 months of therapy, patients may remain on anti-TNF therapy even if they fail to achieve the treatment target, mainly due to formulary structures. This means patients have to endure a second and even a third ineffective anti-TNF-called anti-TNF cycling-before changing MOA. This significantly delays patients from reaching their treatment targets. All anti-TNF drugs target the same molecular and inflammatory pathways; thus, it is not surprising that most patients who are primary non-responders to their initial anti-TNF therapy fail to achieve their treatment targets when cycled through alternative anti-TNFs. This suggests that primary non-responders should be switched to an alternative MOA therapy rather than enduring anti-TNF cycling. Avoiding anti-TNF cycling would prevent disease progression and improve quality of life for RA patients who are primary non-responders to anti-TNFs. The development of a personalized medicine approach to identify primary non-responders to anti-TNFs prior to treatment would allow significantly more patients to reach their treatment target by treating them with alternative MOA therapies as first-line therapies.

Identifying genetic risk variants for coronary heart disease in familial hypercholesterolemia: an extreme genetics approach.

Mutations in the low-density lipoprotein receptor (LDLR) gene cause familial hypercholesterolemia (FH), a disorder characterized by coronary heart disease (CHD) at young age. We aimed to apply an extreme sampling method to enhance the statistical power to identify novel genetic risk variants for CHD in individuals with FH. We selected cases and controls with an extreme contrast in CHD risk from 17,000 FH patients from the Netherlands, whose functional LDLR mutation was unequivocally established. The genome-wide association (GWA) study was performed on 249 very young FH cases with CHD and 217 old FH controls without CHD (above 65 years for males and 70 years of age for females) using the Illumina HumanHap550K chip. In the next stage, two independent samples (one from the Netherlands and one from Italy, Norway, Spain, and the United Kingdom) of FH patients were used as replication samples. In the initial GWA analysis, we identified 29 independent single nucleotide polymorphisms (SNPs) with suggestive associations with premature CHD (P<1 × 10(-4)). We examined the association of these SNPs with CHD risk in the replication samples. After Bonferroni correction, none of the SNPs either replicated or reached genome-wide significance after combining the discovery and replication samples. Therefore, we conclude that the genetics of CHD risk in FH is complex and even applying an 'extreme genetics' approach we did not identify new genetic risk variants. Most likely, this method is not as effective in leveraging effect size as anticipated, and may, therefore, not lead to significant gains in statistical power.

Gene expression profiling of immunomagnetically separated cells directly from stabilized whole blood for multicenter clinical trials.

BACKGROUND: Clinically useful biomarkers for patient stratification and monitoring of disease progression and drug response are in big demand in drug development and for addressing potential safety concerns. Many diseases influence the frequency and phenotype of cells found in the peripheral blood and the transcriptome of blood cells. Changes in cell type composition influence whole blood gene expression analysis results and thus the discovery of true transcript level changes remains a challenge. We propose a robust and reproducible procedure, which includes whole transcriptome gene expression profiling of major subsets of immune cell cells directly sorted from whole blood. METHODS: Target cells were enriched using magnetic microbeads and an autoMACS® Pro Separator (Miltenyi Biotec). Flow cytometric analysis for purity was performed before and after magnetic cell sorting. Total RNA was hybridized on HGU133 Plus 2.0 expression microarrays (Affymetrix, USA). CEL files signal intensity values were condensed using RMA and a custom CDF file (EntrezGene-based). RESULTS: Positive selection by use of MACS® Technology coupled to transcriptomics was assessed for eight different peripheral blood cell types, CD14+ monocytes, CD3+, CD4+, or CD8+ T cells, CD15+ granulocytes, CD19+ B cells, CD56+ NK cells, and CD45+ pan leukocytes. RNA quality from enriched cells was above a RIN of eight. GeneChip analysis confirmed cell type specific transcriptome profiles. Storing whole blood collected in an EDTA Vacutainer® tube at 4°C followed by MACS does not activate sorted cells. Gene expression analysis supports cell enrichment measurements by MACS. CONCLUSIONS: The proposed workflow generates reproducible cell-type specific transcriptome data which can be translated to clinical settings and used to identify clinically relevant gene expression biomarkers from whole blood samples. This procedure enables the integration of transcriptomics of relevant immune cell subsets sorted directly from whole blood in clinical trial protocols.

Genetic diversity in black South Africans from Soweto.

BACKGROUND: Due to the unparalleled genetic diversity of its peoples, Africa is attracting growing research attention. Several African populations have been assessed in global initiatives such as the International HapMap and 1000 Genomes Projects. Notably excluded, however, is the southern Africa region, which is inhabited predominantly by southeastern Bantu-speakers, currently suffering under the dual burden of infectious and non-communicable diseases. Limited reference data for these individuals hampers medical research and prevents thorough understanding of the underlying population substructure. Here, we present the most detailed exploration, to date, of genetic diversity in 94 unrelated southeastern Bantu-speaking South Africans, resident in urban Soweto (Johannesburg). RESULTS: Participants were typed for ~4.3 million SNPs using the Illumina Omni5 beadchip. PCA and ADMIXTURE plots were used to compare the observed variation with that seen in selected populations worldwide. Results indicated that Sowetans, and other southeastern Bantu-speakers, are a clearly distinct group from other African populations previously investigated, reflecting a unique genetic history with small, but significant contributions from diverse sources. To assess the suitability of our sample as representative of Sowetans, we compared our results to participants in a larger rheumatoid arthritis case-control study. The control group showed good clustering with our sample, but among the cases were individuals who demonstrated notable admixture. CONCLUSIONS: Sowetan population structure appears unique compared to other black Africans, and may have clinical implications. Our data represent a suitable reference set for southeastern Bantu-speakers, on par with a HapMap type reference population, and constitute a prelude to the Southern African Human Genome Programme.

Robust and tissue-independent gender-specific transcript biomarkers.

CONTEXT: Correct gender assignment in humans at the molecular level is crucial in many scientific disciplines and applied areas. MATERIALS AND METHODS: Candidate gender markers were identified through supervised statistical analysis of genome wide microarray expression data from human blood samples (N = 123, 58 female, 65 male) as a training set. The potential of the markers to predict undisclosed tissue donor gender was tested on microarray data from 13 healthy and 11 cancerous human tissue collections (internal) and external datasets from samples of varying tissue origin. The abundance of some genes in the marker panel was quantified by RT-PCR as alternative analytical technology. RESULTS: We identified and qualified predictive, gender-specific transcript markers based on a set of five genes (RPS4Y1, EIF1AY, DDX3Y, KDM5D and XIST). CONCLUSION: Gene expression marker panels can be used as a robust tissue- and platform-independent predictive approach for gender determination.

LMX1B mutations cause hereditary FSGS without extrarenal involvement.

LMX1B encodes a homeodomain-containing transcription factor that is essential during development. Mutations in LMX1B cause nail-patella syndrome, characterized by dysplasia of the patellae, nails, and elbows and FSGS with specific ultrastructural lesions of the glomerular basement membrane (GBM). By linkage analysis and exome sequencing, we unexpectedly identified an LMX1B mutation segregating with disease in a pedigree of five patients with autosomal dominant FSGS but without either extrarenal features or ultrastructural abnormalities of the GBM suggestive of nail-patella-like renal disease. Subsequently, we screened 73 additional unrelated families with FSGS and found mutations involving the same amino acid (R246) in 2 families. An LMX1B in silico homology model suggested that the mutated residue plays an important role in strengthening the interaction between the LMX1B homeodomain and DNA; both identified mutations would be expected to diminish such interactions. In summary, these results suggest that isolated FSGS could result from mutations in genes that are also involved in syndromic forms of FSGS. This highlights the need to include these genes in all diagnostic approaches to FSGS that involve next-generation sequencing.

The virtuous technology cycle concept and its application in next-generation sequencing.

External access to scientific technology plays an increasingly important part in pharmaceutical R&D. One advantage of accessing technology externally is the avoidance of costs associated with purchase and the reduced time required for developing new methods; in addition, access to external scientific expertise can be beneficial. However, few conceptual frameworks exist for achieving an optimal mix of internal and external technology access. In this review, we describe the virtuous technology cycle (VTC) concept and exemplify its application to next-generation sequencing (NGS). Based on selected examples, we show that the VTC concept can greatly enhance the number of technologies accessed and thus significantly increase flexibility and efficiency in drug discovery. We also discuss the challenges of externally accessing NGS technologies.

PhRMA white paper on ADME pharmacogenomics.

Pharmacogenomic (PGx) research on the absorption, distribution, metabolism, and excretion (ADME) properties of drugs has begun to have impact for both drug development and utilization. To provide a cross-industry perspective on the utility of ADME PGx, the Pharmaceutical Research and Manufacturers of America (PhRMA) conducted a survey of major pharmaceutical companies on their PGx practices and applications during 2003-2005. This white paper summarizes and interprets the results of the survey, highlights the contributions and applications of PGx by industrial scientists as reflected by original research publications, and discusses changes in drug labels that improve drug utilization by inclusion of PGx information. In addition, the paper includes a brief review on the clinically relevant genetic variants of drug-metabolizing enzymes and transporters most relevant to the pharmaceutical industry.

A physical map of the genomic region on mouse chromosome 3 containing the hindshaker (hsh) mutation.

Hindshaker (hsh), a spontaneous, autosomal recessive mouse mutation, displays a developmentally dependent tremor of the hindquarters due to hypomyelination in the CNS. This myelin deficit is followed by progressive, but incomplete, recovery by postnatal day 42. Herein we describe the construction of a genomic contig spanning the interval between the markers D3Mit187 (42.4 cM) and D3Mit232 (45.2 cM) on mouse chromosome 3, which we have previously shown to contain the hsh mutation. A physical map, covering approximately 3.5 Mb, was constructed from a series of overlapping yeast and bacterial artificial chromosomes. A 1.2- to 1.4-Mb segment central to the contig was compared extensively with the syntenic regions in human (chromosome 1q21-q23) and rat (chromosome 2). We present new data on 10 genes erroneously assigned to this area and on another 6 genes previously assigned elsewhere. For absent genes, our work suggests that they are telomeric to the region encompassed in our map. Accordingly, our findings both map the area surrounding the hsh mutation and present important corrections to the current maps in an area rich in genes related to the nervous system.

Abnormal contractile activity and calcium cycling in cardiac myocytes isolated from DMPK knockout mice.

Dysfunction of the gene encoding DMPK (myotonic dystrophy protein kinase) has been implicated in the human neuromuscular disease myotonic dystrophy (DM1). The cardiac features of the disease include progressive conduction defects and ventricular arrhythmias. These defects have been observed in hearts of mice deficient for DMPK function. We have investigated the role of DMPK in the function of ventricular cardiomyocytes using dmpk knockout (KO) mice. A deficit in DMPK caused enhanced basal contractility of single cardiomyocytes and an associated increase in intracellular Ca(2+), measured using fura-2. Biochemical measurements indicated hyperphosphorylation of phospholamban (PLB) in KO mice. This suggests increased Ca(2+) uptake into the sarcoplasmic reticulum (SR) as the underlying cause of enhanced contractility. This conclusion was supported by the larger amplitude of caffeine-induced Ca(2+) release from the SR in KO cardiomyocytes. Concurrent with hyperphosphorylated PLB, the response to isoprenaline was reduced. These observations suggest dmpk has a modulatory role in the control of intracellular Ca(2+) concentration in mouse ventricular cardiomyocytes, loss of which may contribute to cardiac dysfunction in DM1.

Genetics (molecular biology) and Meniere's disease.

COCH is not the FMD gene detected in our linkage study; furthermore, COCH and FMD are not allelic. The indications are that FMD is heterogenetic. The linkage analysis points to the possibility of one FMD mutation in one of the neighboring candidate genes on chromosome 14, and, with anticipation, possibly a triple repeat amplification. Recently, the myotonic dystrophy type 2 locus has been shown to contain an expanded tetranucleotide repeat [46], so the search for a similar repeat on 14q is indicated.

Mapping of the dysmyelinating murine Hindshaker mutation to a 1.2-cM interval on chromosome 3.

Hindshaker (hsh) is a novel, spontaneous, autosomal recessive mouse mutation displaying a myelin deficit, predominantly in the spinal cord. It is characterized by developmentally dependent hypomyelination, first evident at postnatal day (P) 10, followed by progressive but incomplete recovery by P42. Hypomyelination is associated with a decreased number of mature oligodendrocytes, which fail to form complete myelin sheaths. Heterozygotes are phenotypically normal, and the hsh mutation shows considerable variation in penetrance and expression depending on genetic background, indicating the influence of modifying loci. Here, we followed an outcross/backcross breeding strategy in conjunction with genotyping for microsatellites and a novel marker for the gene S100a4. We describe the genomic mapping of the hsh mutation to within a 1.2-cM region near the centromere of mouse chromosome 3. We found that hsh is flanked between D3Mit187 proximally and S100a4 distally. The area containing hsh is gene-rich, with a high proportion of the genes specific to nervous tissue. Identification of the hsh mutation will aid our understanding of processes important in regional control of oligodendrocyte development and myelination.