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CAP Home > CAP Reference Resources and Publications > CAP TODAY > CAP TODAY 2004 Archive > Case-control studies of disease association markers
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  Clinical Abstracts





cap today

December 2004

Michael Bissell, MD, PhD, MPH, Professor and Director of Clinical Services and Vice Chair, Department of Pathology, Ohio State University Medical Center, Columbus
Ronald Domen, MD, Professor of Pathology, Medicine, and Humanities, Penn State University College of Medicine, Hershey, Pennsylvania

Case-control studies of disease association markers
Leukemia in the vicinity of nuclear waste
Perinatal transmission of HIV
Urinary free sialic acid
Genetics of rheumatoid arthritis
Media for transporting and storing Streptococcus pneumoniae

Case-control studies of disease association markers

Genetic mapping of complex disease often involves case-control disease-marker association studies in which a sample of affected cases is compared with a suitable control group to test for an association between allelic variants and disease status. These studies are conducted in candidate genes and linkage candidate regions. As our understanding of variations in the genome improves, these studies may be conducted genomewide. Many markers typically must be genotyped to adequately canvas a region of interest for association. The density of the markers required to adequately sample the variation depends on the extent and pattern of linkage disequilibrium in the region. In addition, because modest effect sizes are expected for loci predisposing to complex diseases, hundreds or even thousands of individuals may be required to obtain sufficient power to detect association. Thus, depending on the size of the region, association studies may require the genotyping of hundreds of thousands of markers in thousands of individuals. Although much progress has been made in developing accurate, high-throughput SNP genotyping, association studies can be costly and require substantial effort, making efficient study design important. One approach to improving the power of genetic studies is to select individuals who are most likely to carry genetic risk factors. These genetically loaded individuals or families presumably provide a stronger signal and facilitate identification of the variants responsible for disease. Standard designs include choosing individuals with an early age at onset, a more severe form of the disease, or a family history of disease. Identifying genetically loaded individuals in this manner will continue to be an attractive approach for disease-marker association studies in particular. Affected individuals with at least one affected sibling have been shown to carry more copies of the disease allele, on average, than singleton cases, which increases the power of association tests when cases with affected siblings are chosen for study. Chromosomes that occur in multiple affected individuals are more likely to carry disease alleles. An even more effective approach to obtaining a case sample enriched for genetic risk factors may be to select individuals who carry these shared chromosomes. Allele-sharing information could be used to identify families showing excess allele sharing in a region of interest or to identify individuals within each family who show the most allele sharing with other affected family members. Carefully selecting families or individuals on the basis of increased allele sharing might increase the frequency of the disease-associated allele in the case sample and thereby increase the power of association studies. Using allele-sharing information may also be a more efficient use of genotyping resources. In addition to defining a potentially more informative sample, a strategy that uses individuals only from the families with excess allele sharing leads to fewer individuals being genotyped. The authors of this study compared three case-selection strategies that use allele-sharing information with a strategy that selects a single individual from each family at random. They found that by choosing the affected sibling who shows the most evidence for pair-wise allele sharing with the other affected siblings in families, the test statistic is increased by more than 20 percent, on average, for additive models with modest genotype relative risks.

Fingerlin TE, Boehnke M, Abecasis GR. Increasing the power and efficiency of disease-marker case-control association studies through use of allele-sharing information. Am J Hum Genet. 2004;74:432-443.

Reprints: Dr. Tasha E. Fingerlin, Dept. of Preventive Medicine and Biometrics, University of Colorado Health Sciences Center, Box B119, 4200 E. Ninth Ave., Denver, CO 80262;

Leukemia in the vicinity of nuclear waste

The increased risks of leukemia in young people in France living in the vicinity of the nuclear waste reprocessing plants at Sellafield, Dounreay, and La Hague has been the subject of extensive scientific study. Radiological assessments concluded that the levels of radiation in these areas were far below those necessary to account for the observed excesses. An association between cases of leukemia and preconceptional irradiation of these young peoples' fathers while working at Sellafield was hypothesized, but this could not account for the excesses in Dounreay and La Hague. The areas near Sellafield and Dounreay are remarkable because of their geographic isolation and large population influx. It has been postulated that in such areas, the herd immunity to an unknown and widespread infectious agent could be lower than average, and a large population influx might have been conducive to epidemics. Childhood leukemia could be a rare response to this common infection. This hypothesis, known as the population mixing hypothesis, is supported by a series of studies. The Nord Cotentin region of France shares characteristics with the Sellafield and Dounreay regions: It used to be an isolated and rural area, and it experienced a large population increase between 1978 and 1992 with the construction of the Flamanville nuclear power station and a new nuclear waste reprocessing unit on the La Hague site. The authors investigated whether there was an association between population mixing and leukemia among young people living in the Nord Cotentin region. They conducted a geographical study for 1979 to 1998 in Nord Cotentin. A population mixing index was defined on the basis of the number of workers born outside the French department of "La Manche" and living in each "commune," the basic geographical unit under study. The analyses were done with indirect standardization and Poisson regression model, allowing or not for extra-Poisson variation. Urban communes were considered the reference population. The incidence rate ratio was 2.7 in rural communes belonging to the highest tertile of population mixing (95 percent Bayesian credible interval, 95 percent BCI=1.2-5.9). A positive trend was observed among rural strata with increasing population mixing index (IRR for trend=2.4, 95 percent BCI=1.1-1.8). The risk became stronger for acute lymphoblastic leukemia in children one to six years old in the highest tertile of population mixing (IRR=5.5, 95 percent BCI=1.4-23.3). These findings provide further support for a possible infective basis for childhood leukemia.

Boutou O, Guizard A-V, Slama R, et al. Population mixing and leukaemia in young people around the La Hague nuclear waste reprocessing plant. B J Cancer. 2002;87:740-745.

Reprints: O. Boutou, Ecole Nationale de la Sante Publique, deartment EGERIES, avenue du Professeur Leon Bernard, CS 74312, 35043 Rennes cedex, France;

Perinatal transmission of HIV

Maternal plasma HIV-1 RNA from the blood and genital fluids of HIV-1-positive mothers has been documented to correlate strongly with mother-to-child transmission, but the RNA viral load threshold for perinatal transmission is not clearly defined. Improved understanding of viral and host determinants for perinatal transmission of HIV may help predict the likelihood of transmission and could assist in making treatment decisions. The authors of this study measured several virus markers in a drug-naïve population of HIV-1-infected mothers in Botswana. Maternal plasma HIV-1 RNA load, peripheral blood mononuclear cell-associated blood HIV-1 DNA load, and cervicovaginal fluid HIV-1 DNA load were determined using quantitative real-time polymerase chain reaction. The overall rate of transmission among these mother-infant pairs was 35.7 percent. Median infant age was 2.5 months. An association between increased plasma HIV-1 RNA load and perinatal transmission was observed (odds ratio, 2.20; 95 percent confidence interval, 1.15-4.18). However, the association between increased blood HIV-1 DNA load and perinatal transmission was stronger (odds ratio, 10.30; 95 percent confidence interval, 2.11-50.38). When blood HIV-1 DNA load was combined with cervicovaginal fluid HIV-1 DNA load, the association with transmission increased (odds ratio, 25.0; 95 percent confidence interval, 2.73-228.6).

Montano M, Russell M, Gilbert P, et al. Comparative prediction of perinatal human immunodeficiency virus type 1 transmission, using multiple virus load markers. J Infect Dis. 2003;188:406-413.

Reprints: Dr. Monty Montano, Boston University School of Medicine, 650 Albany St., Room 646, Boston, MA 02118; mmontano@

Urinary free sialic acid

Sialic acid storage diseases are autosomal recessive neurodegenerative disorders that may present as a severe infantile form, called infantile sialic acid storage disease, or as a slowly progressive adult form, called Salla disease, that is prevalent in the Finnish population. Clinical features of infantile sialic storage disease include visceromegaly, coarse facial features, failure to thrive, and early death. Salla disease is characterized by psychomotor developmental delay, hypotonia, and ataxia, usually appearing during the first year of life. The known forms of sialic acid storage disease (SSD) are primarily caused by a transport defect across the lysosomal membrane, which leads to the accumulation of free sialic acid [N-acetylneuraminic acid (NANA)] in tissues, fibroblasts, and urine. The origin of the transport defect lies in a mutation in the SLC17A5 gene, which encodes for sialin protein. Defective free sialic acid transport can be established by quantitative analysis of NANA in urine relative to the concentration of creatinine. Several types of analyses are available to quantify NANA, but the main disadvantages of current methods are a lack of selectivity or lack of speed, or both. The authors described and validated a simple, rapid, selective, and sensitive method for analyzing NANA in urine samples based on high-pressure liquid chromatography combined with tandem mass spectrometry (HPLC-tMS). The deaminated sialic acid 2-keto-3-deoxy-D-glycero-D-galactononic acid (KDN) was used as an internal standard. Urine sample size was adjusted to the equivalent of 100 nmol of creatinine. After adding KDN as an internal standard, samples were diluted with water to an end volume of 250 B5L. The authors used 10 B5L for HPLC-tMS analysis in the negative electrospray ionization mode, monitoring transitions m/z 308.3→m/z 86.9 (sialic acid) and m/z267.2→m/z86.9 (internal standard). The overall method was validated and studied for ion suppression, interfering compounds, and pH effects. Samples from controls (n=72) and SSD patients (n=3) were analyzed. The limit of detection was 3 B5mol/L. Intra-assay imprecision (CV; n=10) was six percent, three percent, and two percent at 30, 130, and 1,000 mmol/mol creatinine, respectively; corresponding inter-assay coefficients of variation (n=10) were five percent, five percent, and two percent. Recovery was 109 percent (100-1,000 mmol/mol creatinine). The mean (standard deviation) [range] excretion rates (mmol/mol creatinine) were 31.3 (16.6) [0.7-56.9] at zero to one year (n=20); 21.2 (9.8) [6.3-38.3] at one to three years (n=15); 14.4 (8.2) [1.7-32.9] at three to 10 years (n=25); and 4.6 (2.6) [0-9.8] above age 10 years (n=12). SSD patients that were 1.2, 3.9, and 12 years of age had concentrations of 111.5, 54.2, and 36.1 mmol/mol creatinine, respectively. The authors concluded that the HPLC-tMS method for free sialic acid in urine is more rapid, accurate, sensitive, selective, and robust than earlier methods and may serve as a candidate reference method for free sialic acid in the diagnosis of SSD.

Valianpour F, Abeling NGGM, Duran M, et al. Quantification of free sialic acid in urine by HPLC-electrospray tandem mass spectrometry: a tool for the diagnosis of sialic acid storage disease. Clin Chem. 2004;50:403-409.

Reprints: Willem Kulik, Laboratory Genetic Metabolic Diseases, FO-224, P.O. Box 22700, 1100 DE Amsterdam, Netherlands;

Genetics of rheumatoid arthritis

Rheumatoid arthritis is associated with inflammation of the synovium and development of RA synovial fibroblasts that undergo hyperplasia and invade cartilage and bone. RA synovial fibroblasts (RASFs) exhibit a decreased ability to undergo apoptosis. They produce pro-inflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α), which provide further stimulation for the inflammation in RA, as well as enzymes, including stromelysin and collagenase, which are capable of invading cartilage and bone. The cytokine TNF-α is central to the development and continued growth and invasion of RASFs. The authors of this study used oligonucleotide microarrays to measure gene expression levels in six independent replicate samples of RASFs. The RASFs were transfected for 18 hours with AdlκB-dominant negative (AdlκB-DN) (n=3) or with control AdTet expressing the reverse tetracycline trans-activator (n=3). The cells were stimulated for three hours with TNF-α and total RNA was prepared. Several novel parametric and nonparametric methods were used to rank genes in terms of the magnitude and significance of intergroup differences. Microarray expression differences were confirmed by real-time quantitative reverse transcription-polymerase chain reaction. Small interfering RNA (siRNA) was used specifically to down-modulate microarray-identified genes to demonstrate their role in promoting apoptosis, proliferation, or matrix metalloproteinase expression. Blocking of NF-κB by AdlκB-DN was associated with a down-modulation of anti-apoptosis genes, including BIRC-3, and several novel genes, including GG2-1, a TNF-α-inducible FLIP-like gene. Other families of genes that were significantly down-regulated by AdlκB-DN included cytokines/chemokines (interleukin-1β [IL-1β], IL-8, IL-15, and RANTES), adhesion molecules (vascular cell adhesion molecule 1, intercellular adhesion molecule 1), and unique genes that have not previously been reported to be regulated by TNF-α in RA. Inhibition of the GG2-1 gene using siRNA technique resulted in significantly enhanced apoptosis, decreased proliferation, and decreased production of MMP-1 in TNF-α-stimulated RASFs. The authors concluded that these studies provide a comprehensive analysis of genes that are differentially regulated by TNF-α signaling and NF-κB nuclear translocation in RASFs and demonstrate methods for confirming the expression and functional significance of such genes.

Zhang H-G, Hyde K, Page GP, et al. Novel tumor necrosis factor alpha-regulated genes in rheumatoid arthritis. Arthritis Rheum. 2004;50:420-431.

Reprints: Dr. John D. Mountz, University of Alabama at Birmingham, 701 S. 19th St., LHRB 473, Birmingham, AL 35294-0007;

Media for transporting and storing Streptococcus pneumoniae

An estimated three to five million deaths occur annually in children under five years of age due to acute respiratory infections, for which Streptococcus pneumoniae is the most important pathogen. Information on the distribution of pneumococcal serotypes and antibiotic sensitivities in developing countries is essential to designing vaccines and treating people in areas where the burden of disease is greatest. For meaningful surveillance studies to be undertaken regarding pneumococcal carriage and disease, it is essential to ensure that primary swab samples are processed efficiently with optimal recovery of pneumococci and that the transport and storage medium maintains the viability of the swab samples without introducing bias that would skew the data. The authors evaluated two liquid media, consisting of skim milk, glycerol, glucose, and tryptone soya broth (STGG), or consisting of skim milk, glycerol, and glucose (SGG) alone, for their ability to maintain pneumococcal viability. Optimal recovery of S. pneumoniae was achieved when swabs were transferred to STGG medium prior to plating onto blood agar-gentamicin selective plates (22 percent), compared to seven percent when plated out directly (P<0.0001 by Fisher’s exact test). Both STGG and SGG media are appropriate for the long-term storage of pneumococci and primary swab samples at -70A1C, with no decrease in viable count observed following repeated freeze-thaw cycles. Samples could be stored refrigerated for up to three days in STGG or SGG medium with no significant loss of viability in the higher temperatures. STGG preserved pneumococci significantly better (about twofold) than SGG medium at 21A1C (P<0.0001) and 30A1C (P<0.0001). Samples can be stored for four and 2.5 days at 6A1C to 8A1C, 28 and 17 hours at 21A1C, and 15 and seven hours at 30A1C in STGG and SGG media, respectively. For field studies undertaken in resource-limited environments, SGG medium can be prepared using locally available materials. The authors concluded that the quantitative data reported in this study will allow researchers to plan appropriate transport and storage protocols.

Charalambous BM, Batt SL, Peek AC, et al. Quantitative validation of media for transportation and storage of Streptococcus pneumoniae. J Clin Microbiol. 2003;41: 5551-5556.

Reprints: S.H. Gillespie, Dept. of Medical Microbiology, University College London, Royal Free Campus, Rowland Hill St., London NW3 2PF, United Kingdom;




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