College of American Pathologists
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November 2001

Data mining in linkage disequilibrium mapping
Linkage disequilibrium is a tool in genetic epidemiology that is useful for evaluating disease-susceptibility genes. It is evaluated using association analysis, which involves comparing allele or haplotype frequencies between affected and control individuals. One must assume, however, that a reasonable proportion of disease-associated chromosomes have been derived from a common ancestor. Traditional association-analysis methods have been used for a long time to test the involvement of candidate genes in diseases and to fine-map disease loci found by linkage methods. The testing primarily has involved simple two-point measures. Some improved statistical methods to detect linkage disequilibrium have been presented in recent years. Using these methods, one searches for genomic regions, rather than alleles, that are shared among affected individuals. These methods are powerful, but they contain assumptions about the inheritance model of the disease and the structure of the survey population. Violations of these assumptions in real data have, as yet, unknown effects. In addition, these methods are only capable of considering the association of one region at a time, making them better suited to fine-mapping than to genomic screening. The methods are also computationally demanding. The authors introduced haplotype pattern mining, a technique that uses data mining methods in linkage disequilibrium-based gene mapping. This method is based on algorithms developed to find frequent patterns from large databases and uses haplotypes as input. In diseases with a reasonable genetic contribution, affected individuals are likely to have higher frequencies of associated marker alleles near the disease-susceptibility gene than are control subjects. It is these combinations of marker alleles, which are more frequent in the disease-associated chromosomes, that are sought in the data. These combination haplotype patterns are sorted by the strength of their association to the disease, and it is the resulting list of haplotype patterns that is used in localizing the disease-susceptibility gene. The method does not rely on a model of the inheritance of the disease, and the statistical model is nonparametric. The method has good localization power in data sets with large degrees of phenocopies and with a great deal of missing and erroneous data. Its capacity to handle high proportions of phenocopies makes the method promising for mapping complex diseases. The method can be extended to include environmental covariates or phenotype measurements or to find several genes simultaneously.

Toivonen HTT, Onkamo P, Vasko K, et al. Data mining applied to linkage disequilibrium mapping. Am J Hum Genet. 2000;67:133-145.

Reprints: Päivi Onkamo, M.Sc., Rolf Nevanlinna Institute, P.O. Box 4 (Yliopistonkatu 5), FIN-00014, University of Helsinki, Finland;

Impact of genetic carrier testing on young women
A practical need exists to institute genetic carrier testing before people become sexually active. However, childhood testing is sometimes considered inappropriate because it may harm a child's self-esteem, distort a family's perception of a child, or present other adverse consequences. The World Health Organization, American Society of Human Genetics, and American College of Medical Genetics have issued statements that genetic testing should be deferred in cases where the medical or psychosocial benefits of the tests will not accrue until adulthood. When an index of a hereditary disease is found within a family, however, the parents of that child usually want to know about the carrier status of other siblings. They may consider testing in childhood important to help children adjust to the knowledge of their carrier status and to inform them of their genetic risk when marrying. Yet little is known about the experiences of individuals tested during childhood, how well they understand their test results, and how the testing procedure, in general, affects their lives. A study was undertaken to increase understanding in this area. Genetic screening practice in Finland prior to 1990 involved routinely testing siblings of indexed cases. The authors interviewed 66 young females in Finland who had been tested for carriership during childhood, between 1984 and 1988. Of these 66 females, 23 were members of families affected by Duchenne's muscular dystrophy and 23 were members of families affected by hemophilia A. The authors used a questionnaire that included multiple-choice and open-ended questions. Only 65 percent of the Duchenne's muscular dystrophy mothers and 78 percent of the hemophilia A mothers remembered correctly the test results of their daughters. And overall, in both types of families, only 65 percent of the daughters knew their test results. Eighty-three percent of these young women sought no genetic counseling upon reaching adulthood. The reason for this was not determined. Seventy-eight percent of the young women reported that the test result had not influenced their lives, yet some felt relieved to know that they had not been carriers. In general, conversations about hereditary disease between family members and friends had been open, and the results of the carrier test usually had been shared with friends. The authors concluded that such testing caused no serious harm to the women who had been tested, yet there were no obvious benefits from early testing.

Järvinen O, et al. Carrier testing of children for two X-linked diseases: a retrospective study of comprehension of the test results and social and psychological significance of the testing. Pediatrics. 2000;106:1460-1465.

Reprints: Outi Järvinen, MD, Dept. of Medical Genetics, Family Federation of Finland, Box 849 FIN-00101, Helsinki, Finland;

Infection in children with diabetic ketoacidosis
The association between diabetic ketoacidosis and infection in the pediatric patient is unclear. The authors performed a retrospective medical record review of children admitted to a children's hospital with the diagnosis of DKA. The study involved 247 children who were a mean age of 10.7±5.6 years. Patients were divided into three groups: no infection; presumed viral infection; and bacterial infection. The mean admission glucose was 593±280 mg/dL and the mean pH was 7.15±0.12. The mean admission white blood cell count was 17,519±9,582/mm3 and the mean maximum temperature was 37.2±4.0°C. Among the 247 children, 171 (69 percent) had no evidence of infection, 44 (17.8 percent) had presumed viral infection, and 32 (12.9 percent) had bacterial infection. Of those with bacterial infection, 24 (9.7 percent) had minor infection (otitis media, streptococcal pharyngitis, sinusitis, bronchitis, and paronychia) and eight (2.3 percent) had major infection (pneumonia, urinary tract infection, necrotizing fasciitis, phlebitis, cellulitis, and infectious colitis with hemolytic uremic syndrome). No significant differences were noted between the three groups with regard to age, gender, temperature, new onset, total white blood cell count, total neutrophil count, total band count, and total lymphocyte count. The presence of fever, however, was a significant predictor of infection (P<0.001) but was not a significant predictor of bacterial infection. Children younger than three years who had DKA were more likely to have bacterial infection than those older than three years (P=0.03). The authors speculated that the observed leukocytosis in children with DKA may be another indicator of the severity of ketoacidosis rather than a predictor of infection. They concluded that the majority of children with DKA do not have clinical evidence of infection.

Flood RG, Chiang VW. Rate and prediction of infection in children with diabetic ketoacidosis. Am J Emerg Med. 2001;19: 270-273.

Correspondence: Vincent W. Chiang, MD, Division of Emergency Medicine, Children’s Hospital, Harvard Medical School, 300 Longwood Ave., Boston, MA 02115;

Fever in travelers returning from overseas
It is estimated that tens of millions of people from industrialized countries visit developing countries each year and that 15 to 37 percent of short-term travelers experience a health problem related to overseas travel. An estimated 11 percent of “returned” travelers have a febrile illness. A study was conducted to evaluate the causes of fever in returned travelers. The authors studied 232 consecutive patients admitted to a hospital in Australia. The largest proportion of illness occurred in travelers between the ages of 20 and 29 years (42 percent). Regions of acquired illness included Asia (61 percent), the Pacific (20 percent), Africa (15 percent), and Latin America (two percent). The median interval between return and admission to the hospital for 62 percent of the patients was 6.5 days (range, one to 590 days). All patients who presented at least six months after return had malaria. Most patients presented with nonspecific symptoms, such as fever (100 percent), headache (63 percent), myalgia (40 percent), cough (32 percent), diarrhea (31 percent), nausea (27 percent), and abdominal pain (24 percent). Patients with splenomegaly were eight times more likely to have malaria. Malaria was four times more likely to be the cause of fever in patients with hepatomegaly than it was in those without hepatomegaly. Twenty-seven percent of patients had malaria (61 percent had P. vivax and 23 percent had P. falciparum). Respiratory tract infections were seen in 24 percent, gastroenteritis in 14 percent, undiagnosed febrile illness in nine percent, Dengue fever in eight percent, typhoid fever in three percent, and hepatitis A in three percent. One patient acquired HIV infection after traveling to northern Africa. Febrile patients who returned from the Pacific region and Africa were significantly more likely to present with malaria (P<0.001). Patients returning from Africa were six times more likely to present with falciparum than nonfalciparum malaria, and those who returned from the Pacific region were four times more likely to have nonfalciparum malaria. Gastroenteritis was the most common foodborne and waterborne infection. Bacterial diagnoses in gastroenteritis included Campylobacter species (62 percent), Shigella species (23 percent), and nontyphoidal salmonella (15 percent). Typhoid fever presented in 3.5 percent of the patients and was more common in travelers returning from Asia. None of the six patients who acquired hepatitis A virus infection had been vaccinated for it. The authors concluded that a significant proportion of illness acquired in overseas travel is potentially preventable.

O'Brien D, Tobin S, Brown GV, et al. Fever in returned travelers: review of hospital admissions for a three-year period. Clin Infect Dis. 2001;33:603-609.

Correspondence: Joseph Torresi, MD, Victorian Infectious Diseases Service, Royal Melbourne Hospital, Victoria 3050, Australia;

Changes in PSA levels over time in young men
Limited information is available in the literature about reference ranges for serum PSA levels in men aged 40 to 49 years, and little is known about PSA levels in men younger than 40. Understanding the distribution of serum PSA levels in young men would help in screening patients at increased risk of prostate malignancy. The authors used the Department of Defense Serum Repository to evaluate serum PSA levels in white and black men aged 20 to 45 years. Because the repository contains serial specimens from many individuals, the authors were also able to evaluate changes in serum PSA levels over time. The DODrepository is a serum bank that stores all residual serum from the military HIVscreening program. These specimens are stored at -25°C. The serum bank was sampled to obtain 588 black and 588 white subjects. The authors conducted a retrospective study with only demographic data available on the subjects. The samples used for the study were collected between 1988 and 1996. The authors excluded individuals with a history of prostate disease. They selected three serum specimens, approximately evenly distributed over a mean of six years, for each individual. The authors calculated the free and total PSA levels and the PSA velocity on each specimen. The assay methodology was the Hybritech Tandem-E PSA assay for total PSA measurement and the Hybritech Tandem-R assay for free PSA measurement. The median baseline serum PSA level for black and white men aged 20 to 29 was 0.38 ng/mL and for black and white men aged 30 to 39 was 0.45 ng/mL. For black men aged 40 to 45, this value was 0.52 ng/mL, and the corresponding value for white men aged 40 to 45 was 0.40 ng/mL. The PSA velocity was found to be higher in white men than in black men, with a mean of 2.8 percent per year and 1.6 percent per year, respectively.

Preston DM, et al. Prostate-specific antigen levels in young white and black men 20 to 45 years old. Urology. 2000;56:812-816.

Reprints: Dr. Judd W. Moul, Dept. of Surgery, Uniformed Services University of the Health Sciences, 1530 E. Jefferson St., Rockville, MD 20852

Red cell arachidonic acid levels in stone formation
Recent reports in the literature have noted an increased concentration of arachidonic acid and the arachidonic/linoleic acid ratio in red blood cell membranes in individuals known to be stone formers. The changes in fatty acid composition of the red cell membranes has been related to erythrocyte oxalate exchange and oxalate urinary excretions in these patients. This suggested a role for increased arachidonic cell membrane content in the mild hyperoxaluric syndromes. The increased arachidonic acid in cell membranes, however, could also affect calcium excretion by increasing production of the arachidonic acid metabolite PGE2. This, in turn, could increase urinary calcium output, intestinal calcium absorption, and calcium re-absorption from bone. It has been suggested that a change in the fatty acid composition of red cell membranes in stone formers may underlie the most common anomalies in nephrolithiasis—hyperoxaluria and hypercalciuria. The authors tested this hypothesis by examining 21 patients, six of whom were females ranging in age from 25 to 63 years and who had active kidney stone disease—that is, more than two stones formed during the last three years. The patients were treated at a clinic in Italy. Primary parathyroidism, medullary sponge kidney disease, and tubular acidosis were excluded by routine evaluation. All of the patients were free of drug treatments for at least three months preceding the study, and their renal functions were within the normal range as judged by serum creatinine and creatinine clearance measurements. The patients were compared with 40 healthy control subjects. The fatty acid composition of RBC membranes was assessed experimentally in samples from all patients. The following parameters also were evaluated in the stone formers: daily and fasting urinary calcium excretion, fractional calcium intestinal absorption, 1,25-dihydroxy-vitamin D, intact parathyroid hormone, hydroxyproline in fasting urine, daily urinary excretion of oxalate, citrate, urate, electrolytes, urea, sulphate, and relative supersaturation for calcium oxalate monohydrate. The results were that the RBC membrane content of arachidonic acid, linoleic acid, and docosahexaenoic acid was lower for stone formers than for controls. Arachidonic acid was not correlated with any of the parameters studied. When patients were grouped according to the degree of oxalate excretion, hyperoxaluric stone formers had a higher arachidonic acid content and arachidonic linoleic acid ratio than stone formers with normal oxalate excretion. The authors concluded that their results do not confirm the finding of increased arachidonic acid content of RBC membrane in stone formers. On the contrary, the study subjects showed reduced arachidonic acid levels. However, hyperoxaluric stone formers had relatively higher arachidonic acid content than stone formers with normal urinary oxalate excretion.

Messa P, et al. Abnormal arachidonic acid content of red blood cell membranes and main lithogenic factors in stone formers. Nephrol Dial Transplant. 2000;15:1388-1393.

Reprints: Piergiorgio Messa, MD, Nephrology & Dialysis Division, Ospedale S. Martino, I-16132, Genova, Italy;

Isoforms of ACE
The primary action of angiotensin 1-converting enzyme is to convert angiotensin-1 and bradykinin to angiotensin 2. It also may be involved in vivo in the metabolism of substance P3. Two molecular forms of human ACE exist: somatic ACE, with a molecular weight between 150 and 180 kDa, which has two homologous domains, each of which contains a zinc-binding site and an active center, and a low-molecular weight form of 90 to 100 kDa found in testis, which contains only the C-domain. Human plasma ACE levels are high during infancy and, for unknown reasons, decrease to stable values during adulthood. The authors purified and characterized the ACEs from the urine of premature and full-term infants to detect the N-domain form of ACE. They also studied the ACE isoform activity profile in postnatal evolution to verify whether nephrogenesis could influence the secretion of ACE. While healthy subjects have high- and low-molecular weight ACEs, hypertensive individuals have only low-molecular weight ACEs. The renal tubules of premature human infants are not completely mature since nephrogenesis is not complete until week 36 of gestation. Urine from premature and full-term infants was concentrated, dialyzed, and purified by gel filtration. Two peaks with ACE activity were detected. All enzymes were chloride dependent and inhibited by captopril and EDTA. The enzymes P2 and TP2 are N-domain ACE. In premature infants, the P1 activity was 12-fold lower than the P2 activity, but in full-term infants the difference between the TP1 and TP2 activity was 1.6 fold. The results indicate that ACE activity is related to renal development and that N-domain ACE and full-length ACE are present in the urine of premature infants. This may indicate that healthy subjects produce and secrete the N-domain form of ACE even before term development.

Hattori MA, et al. Angiotensin 1-converting enzyme isoforms (high and low molecular weight) in urine of premature and full-term infants. Hypertension. 2000;35:1284-1290.

Reprints: Dulce Elena Casarini, MD, Universidade Federal de São Paulo, Escola Paulista de Medicina, Depto de Medicina, Disciplina de Nefrologia, Rua Botucatu 740, CEP 04023 900, São Paulo, SP, Brazil;

Anti-cardiolipin antibody and coronary calcium levels in angina
Chest pain may be typical anginal pain, which suggests coronary atherosclerosis, or atypical chest pain, which results from noncardiac underlying conditions, including musculoskeletal, gastrointestinal, and other cardiac diseases. Atherosclerosis is increasingly being recognized as an inflammatory process in the arterial wall, including the accumulation of macrophages and activated T-lymphocytes. Oxidized low-density lipoprotein (ox-LDL) and b2-glycoprotein-1 (b2-GP1) are candidate autoantigens that may play a role in that process. Anti-ox-LDL antibodies are elevated in patients with systemic lupus erythematosus who experience premature atherosclerosis, and they cross-react with cardiolipin and b2-GP1. Increased levels of antibodies against ox-LDL and cardiolipin at age 50 have been correlated positively with myocardial infarction and related mortality ten to 20 years later. The authors attempted to determine whether coronary calcium and the autoimmune response associated with atherosclerosis, either solely or together, differ between patients with typical and atypical chest pain. To make this assessment, they compared coronary calcium on spiral computerized tomography and determined the levels of autoantibodies to cardiolipin, ox-LDL, and b2-GP1. The authors studied 52 patients with typical chest pain, 19 patients with atypical chest pain, and 21 patients without chest pain. The patients with typical chest pain had higher mean levels of coronary calcium compared with patients with atypical chest pain and the control subjects. The levels of anticardiolipin were 262, 170, and 230 for patients with typical, atypical, and no chest pains, respectively. No difference was found between the groups regarding anti-ox-LDL and anti-b2-GP1 autoantibody levels. A higher prevalence of total coronary calcium scores and high anticardiolipin levels were found in the typical chest pain group compared with the atypical chest pain group. Nineteen of the 52 patients with typical chest pain had high calcium and high antibody levels. None of the 19 patients who had atypical chest pain, however, had high levels of both, and this difference was highly significant (P=0.003). High coronary calcium scores or high anticardiolipin levels are found more often in typical than atypical chest pain patients, but a combination of high levels of both can better differentiate typical from atypical chest pain.

Sherer Y, Shemesh J, Tenenbaum A, et al. Coronary calcium and anti-cardiolipin antibody are elevated in patients with typical chest pain. Am J Cardiol. 2000;86:1306-1311.

Reprints: Yehuda Shoenfeld, MD, Dept. of Medicine "B", Sheba Medical Center, Tel-Hashomer, 52621, Israel;

Serum immunoglobulin levels in systemic sclerosis
Interstitial lung disease is a major complication of systemic sclerosis and accounts for much of the morbidity and mortality from that disease. Patients with evidence of pulmonary involvement may be good candidates for therapy for interstitial lung disease. Corticosteroids, cyclophosphamide, and D-penicillamine may be useful therapeutic agents for systemic sclerosis that involves the lung. However, these drugs have serious adverse side effects. The finding of increased polymorphonuclear leukocyte counts on bronchoalveolar lavage specimens may be a useful monitor of the condition, but the test is too invasive to be used routinely. A serum marker would be more practical. Serum immunoglobulin levels are commonly elevated in autoimmune diseases and reflect the active phase of the disease. The authors conducted a study to assess whether serum immunoglobulin levels might be useful as a predictor of the course of interstitial lung disease in systemic sclerosis. They studied 24 patients with the diagnosis of systemic sclerosis who were seen at a hospital in Japan between 1980 and 1996 and who had at least two sets of pulmonary functions tests. The rates of change of forced vital capacity (FVC), carbon monoxide diffusing capacity (DLco), and DLco per unit alveolar volume (Kco) were measured. The rates of change of FVC, DLco, and Kco correlated with the annualized rate of change of IgG, and the rate of change of DLco also correlated with the serum IgM level at the first pulmonary function test. The authors concluded that serum Ig levels may be useful prognostically.

Yuhara T, Takemura H, Akama T, et al. The relationship between serum immunoglobulin levels and pulmonary involvement in systemic sclerosis. J Rheumatol. 2000;27:1207-1214.

Reprints: Deborah B. Nelson, PhD, Center for Epidemiology and Biostatistics, University of Pennsylvania, 922 Blockly Hall, 423 Guardian Drive, Philadelphia, PA 19104-6021