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August 2004

Editors:
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

Familial co-occurrence of clinical features of SLE
Evolutionary relations of Mycobacterium tuberculosis
Prenatal diagnosis of Smith-Lemli-Opitz syndrome
Effects of L-arginine on exercise-induced lactate and ammonia levels
Urinary albumin/creatinine ratio in sickle cell disease
Aberrations of chromosomes 16 and 17 in breast cancer

Familial co-occurrence of clinical features of SLE

Susceptibility to systemic lupus erythematosus has been attributed to complex interactions between multiple genetic and environmental factors. The influential effect of a genetic predisposition on susceptibility to systemic lupus erythematosus (SLE) is supported by its familial aggregation. Siblings of SLE patients have a 20- to 40-fold greater risk of developing SLE than the general population (two percent versus 0.05 percent to 0.1 percent). Studies of monozygotic twins show a concordance rate for SLE ranging from 24 percent to 57 percent. Because most twins share similar environments, the 10-fold lower concordance rate for SLE in dizygotic twins (two percent to five percent) compared with that in monozygotic twins strongly suggests that genes predispose to SLE. Candidate gene studies have shown associations between SLE and many genetic factors, including complement gene deficiency (C1q, C2, or C4) and polymorphic alleles or major histocompatibility class II, Fcγ receptors, mannose-binding ligand, interleukin-10, and tumor necrosis factor alpha. In recent years, collections of multiplex families for linkage analyses have facilitated studies of familial SLE. In several such collections, no differences in clinical manifestations and laboratory findings between familial and sporadic cases of SLE were observed. Strategies to facilitate the transition from linkage analyses to the identification of susceptibility alleles are being developed. One approach is to minimize the phenotypic heterogeneity of the disorder. Patients with SLE present with a diverse array of clinical manifestations, including variable serology and organ involvement, leading to fulfillment of at least four of the 11 criteria for classifying SLE. The authors evaluated sibling concordance of 15 SLE manifestations in 159 SLE-affected sibpairs as well as correlations between the age at diagnosis of SLE in 125 affected sibpairs and 37 affected parent-offspring pairs. The authors used concordance rates and sibling risk ratios as indicators of familiality for 15 manifestations of SLE. Pearson's correlations and paired t-tests were used to compare the age at SLE diagnosis in affected sibpairs and parent-offspring pairs. Increased sibling risk ratios (1.9 to 3.9) for thrombocytopenia, discoid rash, neurologic disorder (defined as seizure or psychosis), and hemolytic anemia were observed in 159 SLE-affected sibpairs. Among these clinical features, paired expression of hemolytic anemia plus thrombocytopenia and hemolytic anemia plus neurologic disorder appeared to be more frequent in 709 SLE patients than would be expected by chance (P=0.00001 and P=0.007, respectively). The ratio of the presence of hemolytic anemia and neurologic disorder was approximately 13 times higher in the younger affected siblings than in the older affected siblings (P=0.02). Familiality of patient age at SLE diagnosis, as observed by relative correlations, was greater in 125 affected sibpairs than in 37 affected parent-offspring pairs (r=0.47; P=0.003). The median age at SLE diagnosis was significantly lower in offspring than in their parents but did not differ in sibpairs. The combined non-Caucasian sibpairs had a younger mean age at SLE diagnosis than the Caucasian sibpairs. The authors concluded that stratifying families by subphenotypes that are familial may reduce heterogeneity and facilitate identification of genetic risk factors for SLE.

Tsao BP, Grossman JM, Riemekasten G, et al. Familiality and co-occurrence of clinical features of systemic lupus erythematosus. Arthritis Rheum. 2002;46:2678-2685.

Reprints: Dr. Betty P. Tsao, Division of Rheumatology, Dept. of Medicine, Rehabilitation Center, Room 32-59, 1000 Veteran Ave., UCLA School of Medicine, Los Angeles, CA 90095-1670; btsao@mednet.ucla.edu

Evolutionary relations of Mycobacterium tuberculosis

Whether Mycobacterium tuberculosis should be treated as a gram-positive bacterium is a controversial issue. Phylogenetic analysis based on molecular markers provides inconsistent results concerning its evolutionary relationships with other bacteria. This organism belongs in the branch of Actinomycetes, which traditionally join the low G+C gram-positive bacteria such as Staphylococci and Streptococci to form a monophyletic group, according to the analysis based on 16S ribosomal RNA. This arrangement suggests that M. tuberculosis is closer to gram-positive than gram-negative bacteria like Enterobacteria and Neisserias, which are classified as Proteobacteria. In the latest taxonomy tree, the class Actinobacteria is elevated to the rank of phylum because the phylogenetic depth in this lineage is equivalent to that of existing phyla. This change raises the question about the evolutionary affinity between M. tuberculosis and gram-positive bacteria. Genome-based analysis offers a new hope for resolving this issue since genomes potentially store much larger information on evolutionary changes than a single or a small ensemble of molecules. The authors reported the results of applying genome comparison for analyzing bacterial evolutionary relationships in an attempt to clarify the phylogenetic position of M. tuberculosis relative to gram-positive and gram-negative bacteria. The genome-based analysis indicates that M. tuberculosis is more closely related to gram-negative bacteria. This conclusion may be supported by another analysis that showed that M. tuberculosis shares relatively more orthologous genes for energy production and conversion with gram-negative bacteria, in particular Escherichia coli and Pseudomonas aeruginosa, than with gram-positive bacteria. The findings can perhaps provide new insights into the pathogenic and pharmacological nature of M. tuberculosis.

Fu LM, Fu-Liu S. Is Mycobacterium tuberculosis a closer relative to gram-positive or gram-negative bacterial pathogens? Tuberculosis. 2002;82:85-90.

Reprints: Dr. Li M. Fu, Bioinformatics & Genomics Group, CISE Dept., CSE 301, P.O. Box 116120, University of Florida, Gainesville, FL 32611; fu@cise.ufl.edu

Prenatal diagnosis of Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome is an autosomal recessive disorder of cholesterol biosynthesis comprising microcephaly, growth retardation, dysmorphic facial features, polydactyly, soft tissue syndactyly of the second and third toes, and various internal and genital abnormalities. The spectrum of severity ranges from isolated cleft palate or minimal 2/3 toe syndactyly associated with developmental delay to severely affected newborns with multiple congenital defects incompatible with life, and sex reversal in males. The incidence has been estimated to range from one in 10,000 to one in 60,000 live births, making it a common, metabolic, multiple congenital anomalies syndrome. The underlying metabolic defect affects the final step of cholesterol synthesis, and the pathognomonic biochemical markers are an abnormally low plasma or tissue cholesterol level combined with a markedly elevated plasma or tissue concentration of precursors, mainly 7-dehydrocholesterol (DHC) and 8-dehydrocholesterol. Prenatal diagnosis of Smith-Lemli-Opitz syndrome (SLOS) has been based on quantifying 7-DHC by gas chromatographic/mass spectrometric analysis in chorionic villus cells or amniotic fluid. SLOS is caused by mutations of the 7-dehydrocholesterol reductase gene (DHCR7) on chromosome 11. The gene for DHCR7 recently was cloned, and mutations causing SLOS were identified, paving the way for molecular prenatal diagnosis, with a mutation detection rate among biochemically proven SLOS patients reported to be more than 96 percent. The authors presented their experience with molecular prenatal diagnosis of SLOS. Mutation analysis of the DHCR7 gene was performed in chorionic villus samples of 13 pregnancies of couples with a family history of SLOS and known SLOS genotypes. This approach is accurate and reliable. The authors concluded that if facilities for biochemical analysis are not available, or in cases with ambiguous biochemical patterns, molecular prenatal diagnosis is an attractive alternative.

Loeffler J, Utermann G, Witsch-Baumgartner M. Molecular prenatal diagnosis of Smith-Lemli-Opitz syndrome is reliable and efficient. Prenat Diagn. 2002;22:827-830.

Reprints: Martina Witsch-Baumgartner, Institute of Medical Biology and Human Genetics, University of Innsbruck, Schoepfstrasse 41, A 6020 Innsbruck, Austria; witsch-baumgartner@uibk.ac.at

Effects of L-arginine on exercise-induced lactate and ammonia levels

Metabolic changes accompanying muscular activity are usually considered important peripheral limiting factors for physical exercise. Besides energy substrate depletion or poor oxygen supply, ammonia and lactate, studied through their plasma accumulation during exhaustive exercise, are important factors in muscular fatigue mechanisms. Lactate and ammonia accumulation increase muscular hydrogen ion concentration and acidity, depressing the force-generating capacity of the working muscle. Some attempts, therefore, have been made to reduce the accumulation of these metabolites during exercise using supplementation of amino acids known to induce beneficial metabolic changes. Among them is L-arginine (L-ARG), an intermediate of the urea cycle that was found to play a precursor role in the formation of nitric oxide. Chronic treatment (four to eight weeks) with oral L-arginine-aspartate salts has been shown to reduce blood lactate or ammonia after maximal or submaximal exercise. To investigate the effect of L-ARG supplementation on physiological and metabolic changes during exercise, the authors determined in a double-blind study the cardiorespiratory (heart rate, oxygen consumption [VO2], and carbon dioxide production [VCO2]) and metabolic (lactate and ammonia) responses to maximal exercise after an intravenous L-ARG hydrochloride salt or placebo load in eight healthy subjects. Exercise-induced increases in heart rate, VO2, and VCO2 were not significantly different after L-ARG or placebo. In contrast, peak plasma ammonia and lactate were significantly decreased after L-ARG load. Plasma L-citrulline increased significantly during exercise only after L-ARG load, despite a concomitant decrease in plasma L-ARG. Furthermore, a significant inverse relationship was observed between changes in lactate and L-citrulline concentrations after L-ARG load (r=-0.84; P=0.009). These results demonstrate that intravenous L-ARG significantly reduces exercise-induced increases in plasma lactate and ammonia. Taken together, the specific L-citrulline increase and the inverse relationship between L-citrulline and plasma lactate after L-ARG might support that L-ARG supplementation enhances the L-arginine-nitric oxide pathway during exercise.

Schaefer A, Piquard F, Geny B, et al. L-arginine reduces exercise-induced increase in plasma lactate and ammonia. Int J Sports Med. 2002; 23:403-407.

Reprints: A. Schaefer, Institut de Physiologie, Faculté de Médecine, 67085 Strasbourg Cedex, France; Adrien.Schaefer@physio-ulp.u-strasbg.fr

Urinary albumin/creatinine ratio in sickle cell disease

Renal involvement similar to that seen in diabetic nephropathy is common in sickle cell disease. Because microalbuminuria is a preclinical indicator of glomerular damage, measurement of urinary albumin excretion (UAE) has become a routine procedure in these patients. Proteinuria has been determined by overnight or 24-hour urine collections, but this procedure is inconvenient and frequently unreliable due to collection errors. Several reports suggest that the measurement of the protein to creatinine ratio (P/C) in single random urine specimens can be used as an alternative to 24-hour urine collection to predict urinary protein excretion in various diseases. In this study, the authors attempted to assess the usefulness of the albumin to creatinine ratio (A/C) in predicting 12-hour urinary albumin excretion (12UA) in patients with sickle cell disease. They also evaluated the usefulness of A/C as a method for selecting patients with normal and elevated UAE values. A/C and 12UA were measured in the nocturnal urine collections and random morning urine samples, respectively, of 72 patients with sickle cell disease. The correlation of A/C values with 12UA values did not provide support for the use of random urine specimens for predicting UAE in these patients. However, values of A/C greater than or equal to 0.45 and less than 0.45 were indicative of raised and normal UAE, respectively. The sensitivity, specificity, and accuracy of the test were 100 percent, 87.2 percent, and 91.7 percent, respectively. The authors concluded that this method cannot be recommended for predicting 12UA in patients with sickle cell disease, but it is useful for selecting patients who should collect 12-hour urine for estimating UAE.

Lima CSP, Bottini PV, Garlipp CR, et al. Accuracy of the urinary albumin to creatinine ratio as a predictor of albuminuria in adults with sickle cell disease. J Clin Pathol. 2002;55: 973-975.

Reprints: Dr. C.S.P. Lima, Hemocentro-UNICAMP, CP: 6198; Cep: 13081-970 Campinas, São Paulo, Brazil; carmenl@obelix.unicamp.br

Aberrations of chromosomes 16 and 17 in breast cancer

It has been suggested that the biological behavior of breast cancer is dictated by its specific pattern of genetic abnormalities. In clinical practice, however, estrogen receptor (ER) expression and amplification of HER2 inform patient management, but the main prognostic indicators remain tumor size, tumor grade, and node status. Comparative genomic hybridization (CGH) technique allows the whole genome of a test sample to be analyzed for amplifications or deletions through comparison with normal reference DNA. A deleted region must be at least 5 Mb to be detectable, but high-level amplifications of as little as 50 kb have been detected. Chromosomal changes such as these have been shown to occur in many malignant solid tumors, and CGH may identify linked patterns of copy number changes peculiar to a histological subtype. Furthermore, by observing several tumors with a deletion or amplification on the same chromosome arm, CGH can identify a minimum region of overlap. These minimum regions of overlap represent targets for more detailed molecular analysis. CGH has been used previously to study breast cancer. All of these reports found that amplifications of 1q and 8q are among the most common amplifications, with 8p and 17p being the regions most commonly deleted. The authors performed a comparative genomic hybridization analysis of 40 primary breast cancers with survival data at a mean of eight to four years. The mean number of aberrations was 9·0, with a mean of 5·5 gains and 3·5 deletions per tumor. The most common aberrations were gain of 1q (27 of 40), 8q (19 of 40), and 17q (13 of 40), and deletion of 17p (12 of 40) and 8p (11 of 40). These results are consistent with a distinctive pattern of large-scale (karyotypic) genetic change in primary breast cancer. The authors noted that only women who were disease-free had loss of 16q (E-cadherin) in association with a gain of 16p, and 17p13 (p53) loss combined with 17q12 (HER2) amplification was found only in the cancers of women who developed recurrent disease. The karyotypic changes seen in primary breast cancer seem to be associated with outcome and point to the underlying genetic events.

Hislop RG, Pratt N, Stocks SC, et al. Karyotypic aberrations of chromosomes 16 and 17 are related to survival in patients with breast cancer. Br J Surg. 2002;89:1581-1586.

Reprints: A.M. Thompson, Dept. of Surgery and Molecular Oncology, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, United Kingdom; a.m.thompson@dundee.ac.uk