Linkage analysis was developed to detect excess co-segregation of the putative alleles underlying a phenotype with the alleles at a marker locus in family data. Many different variations of this analysis and corresponding study design have been developed to detect this co-segregation. Linkage studies have been shown to have high power to detect loci that have alleles (or variants) with a large effect size, i.e. alleles that make large contributions to the risk of a disease or to the variation of a quantitative trait. However, alleles with a large effect size tend to be rare in the population. In contrast, association studies are designed to have high power to detect common alleles which tend to have a small effect size for most diseases or traits. Although genome-wide association studies have been successful in detecting many new loci with common alleles of small effect for many complex traits, these common variants often do not explain a large proportion of disease risk or variation of the trait. In the past, linkage studies were successful in detecting regions of the genome that were likely to harbor rare variants with large effect for many simple Mendelian diseases and for many complex traits. However, identifying the actual sequence variant(s) responsible for these linkage signals was challenging because of difficulties in sequencing the large regions implicated by each linkage peak. Current ‘next-generation’ DNA sequencing techniques have made it economically feasible to sequence all exons or the whole genomes of a reasonably large number of individuals. Studies have shown that rare variants are quite common in the general population, and it is now possible to combine these new DNA sequencing methods with linkage studies to identify rare causal variants with a large effect size. A brief review of linkage methods is presented here with examples of their relevance and usefulness for the interpretation of whole-exome and whole-genome sequence data.
© 2011 S. Karger AG, Basel
- DNA sequence
- Whole-genome sequence
- Whole-exome sequence
- Tsui LC, et al: Cystic fibrosis locus defined by a genetically linked polymorphic DNA marker. Science 1985;230:1054–1057.
- Riordan JR, et al: Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989;245:1066–1073.
- Gusella JF, et al: A polymorphic DNA marker genetically linked to Huntington’s disease. Nature 1983;306:234–238.
- The Huntington Disease Collaborative Research Group: A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. Cell 1993;72:971–983.
- Brant SR, et al: American families with Crohn’s disease have strong evidence for linkage to chromosome 16 but not chromosome 12. Gastroenterology 1998;115:1056–1061.
- Ogura Y, et al: A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature 2001;411:603–606.
- Hugot JP, et al: Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature 2001;411:599–603.
- Hampe J, et al: Association between insertion mutation in NOD2 gene and Crohn’s disease in German and British populations. Lancet 2001;357:1925–1928.
- Goldin LR, et al: Segregation and linkage studies of plasma dopamine-beta-hydroxylase (DBH), erythrocyte catechol-O-methyltransferase (COMT), and platelet monoamine oxidase (MAO): possible linkage between the ABO locus and a gene controlling DBH activity. Am J Hum Genet 1982;34:250–262.
- Asamoah A, et al: Segregation and linkage analyses of dopamine-beta-hydroxylase activity in a six-generation pedigree. Am J Med Genet 1987;27:613–621.
- Wilson AF, et al: Linkage of a gene regulating dopamine-beta-hydroxylase activity and the ABO blood group locus. Am J Hum Genet 1988;42:160–166.
- Craig SP, et al: Localization of the human dopamine beta hydroxylase (DBH) gene to chromosome 9q34. Cytogenet Cell Genet 1988;48:48–50.
- Zabetian CP, et al: A quantitative-trait analysis of human plasma-dopamine beta-hydroxylase activity: evidence for a major functional polymorphism at the DBH locus. Am J Hum Genet 2001;68:515–522.
- Cubells JF, et al: Linkage analysis of plasma dopamine-Β-hydroxylase in families of patients with schizophrenia. Hum Genet 2011;130:635–643.
- Ioannidis JP: Why most published research findings are false. PLoS Med 2005;2:e124.
- Moonesinghe R, Khoury MJ, Janssens AC: Most published research findings are false – but a little replication goes a long way. PLoS Med 2007;4:e28.
- Sobreira NL, et al: Whole-genome sequencing of a single proband together with linkage analysis identifies a Mendelian disease gene. PLoS Genet 2010;6:e1000991.
- Bowden DW, et al: Molecular basis of a linkage peak: exome sequencing and family-based analysis identify a rare genetic variant in the ADIPOQ gene in the IRAS Family Study. Hum Mol Genet 2010;19:4112–4120.
- Guo X, et al: Genome-wide linkage of plasma adiponectin reveals a major locus on chromosome 3q distinct from the adiponectin structural gene: the IRAS family study. Diabetes 2006;55:1723–1730.
- Sutton WS: On the morphology of the chromosome group in Brachystola magna. Biol Bull 1902;4:24–39.
- Sutton WS: The chromosomes in heredity. Biol Bull 1903;4:231–251.
- Correns C: Uber Vererbungsgesetze. Berlin, G. Borntrager, 1905.
- Bateson W, Punnett RC: Experimental studies in the physiology of heredity. Reports of the Evolution Committee. Roy Soc 1906;3:1–53.
- Muller HJ: The mechanism of crossing over. Am Nat 1916;50:193–221.
- Morgan TH: Random segregation versus coupling in Mendelian inheritance. Science 1911;34:384.
- Sturtevant AH: The linear arrangement of six sex-linked factors in Drosophila as shown by their mode of association. J Exp Zool 1913;14:43–59.
- Wilson AF, et al: Stepwise oligogenic segregation and linkage analysis illustrated with dopamine-beta-hydroxylase activity. Am J Med Genet 1990;35:425–432.
- Fisher RA: The detection of linkage. Ann Eugen 1935;6:187–201.
- Penrose LS: The detection of autosomal linkage in data which consists of pairs of brothers and sisters of unpecified parentage. Ann Eugen 1935;6:133–138.
- Finney DJ: The detection of linkage. Ann Eugen 1940;10:171–214.
- Finney DJ: The detection of linkage. II. Further mating types, scoring of Boyd’s data. Ann Eugen 1941;11:10–30.
- Finney DJ: The detection of linkage. III. Incomplete parental testing. Ann Eugen 1941;11:115–135.
- Finney DJ: The detection of linkage. IV. Lack of parental records and the use of empirical information. J Hered 1942;33:157–160.
- Finney DJ: The detection of linkage. V. Supplementary tables. Ann Eugen 1942;11:224–232.
- Finney DJ: The detection of linkage. VII. Combination of data from matings of known and unknown phase. Ann Eugen 1943;12:31–43.
- Haldane JB, Smith CA: A new estimate of the linkage between the genes for colourblindness and haemophilia in man. Ann Eugen 1947;14:10–31.
- Smith CAB: The detection of linkage in human genetics. J Roy Stat Soc B 1953;15:153–184.
- Wald A: Sequential Analysis. New York, Wiley, 1947.
- Morton NE: Sequential tests for the detection of linkage. Am J Hum Genet 1955;7:277–318.
- Bernard GA: Statistical inference. J R Stat Soc B 1949;11:115–140.
- Morton NE: Significance levels in complex inheritance. Am J Hum Genet 1998;62:690–697.
- Morton NE: Erratum, significance levels in complex inheritance. Am J Hum Genet 1998;63:1252.
- Rao DC, et al: Variability of human linkage data. Am J Hum Genet 1978;30:516–529.
- Morton NE: The detection and estimation of linkage between the genes for elliptocytosis and the Rh blood type. Am J Hum Genet 1956;8:80–96.
- Morton NE, Steinberg AG: Sequential test for linkage between cystic fibrosis of the pancreas and the MNS locus. Am J Hum Genet 1956;8:177–189.
- Morton NE: Further scoring types in sequential linkage tests, with a critical review of autosomal and partial sex linkage in man. Am J Hum Genet 1957;9:55–75.
- Elston RC, Stewart J: A general model for the genetic analysis of pedigree data. Hum Hered 1971;21:523–542.
- Mather K, Jinks JL: Biometrical Genetics; the Study of Continuous Variation, ed 2, revised. Ithaca, Cornell Univ Press, 1971, p 382.
- Lange K, Elston RC: Extensions to pedigree analysis I. Likehood calculations for simple and complex pedigrees. Hum Hered 1975;25:95–105.
- Mather K, Jinks JL: Introduction to Biometrical Genetics. Ithaca, Cornell Univ Press, 1977, p 231.
- Elston RC: Segregation analysis. Adv Hum Genet 1981;11:63–120, 372–373.
- Mather K, Jinks JL: Biometrical Genetics: The Study of Continuous Variation, ed 3. London, New York, Chapman and Hall, 1982, p 396.
- Lalouel JM, et al: A unified model for complex segregation analysis. Am J Hum Genet 1983;35:816–826.
- Elston RC: Segregation and linkage analysis. Anim Genet 1992;23:59–62.
- Bonney GE: Regressive logistic models for familial disease and other binary traits. Biometrics 1986;42:611–625.
- Bonney GE: A note on the basis of regressive models for genetic analysis. Genet Epidemiol Suppl 1986;1:37–42.
- Bonney GE, Lathrop GM, Lalouel JM: Combined linkage and segregation analysis using regressive models. Am J Hum Genet 1988;43:29–37.
- Bonney GE, Dunston GM, Wilson J: Regressive logistic models for ordered and unordered polychotomous traits: application to affective disorders. Genet Epidemiol 1989;6:211–215.
- Demenais FM, Bonney GE: Equivalence of the mixed and regressive models for genetic analysis. I. Continuous traits. Genet Epidemiol 1989;6:597–617.
- Bonney GE: Compound regressive models for family data. Hum Hered 1992;42:28–41.
- Stricker C, Fernando RL, Elston RC: Segregation analysis under an alternative formulation for the mixed model. Genet Epidemiol 1993;10:653–658.
- Falconer DS, Mackay TFC: Introduction to Quantitative Genetics, ed 4. Essex, Longman, 1996, p 464.
- Elston RC, Olson JM, Palmer L: Biostatistical Genetics and Genetic Epidemiology. Wiley reference series in biostatistics. Chichester, New York, Wiley, 2002, p 831.
- Wilson AF, Elston RC: Linkage analysis in the study of the genetics of alcoholism; in Begleiter H, Kissin B (eds): The Genetics of Alcoholism. Oxford University Press, 1995, pp 353–376.
- Ott J: Estimation of the recombination fraction in human pedigrees: efficient computation of the likelihood for human linkage studies. Am J Hum Genet 1974;26:588–597.
- Lander E, Botstein D: Mapping Mendelian factors underlying quantitative traits using RFLP linkage maps. Genetics 1989;121:185–199.
- Lander ES, Green P: Construction of multilocus genetic linkage maps in humans. Proc Natl Acad Sci USA 1987;84:2363–2367.
- Ott J: Linkage analysis and family classification under heterogeneity. Ann Hum Genet 1983;47:311–320.
- Smith CAB: Homogeneity test for linkage data. 2nd International Congress on Human Genetics, 1961.
- Cannings C, Thompson EA, Skolnick MH: Probability functions on complex pedigrees. Adv Appl Prob 1978;10:26–61.
- Meyers DA, et al: Linkage group I: the simultaneous estimation of recombination and interference. Cytogenet Cell Genet 1976;16:335–339.
- Lathrop GM, et al: Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci USA 1984;81:3443–3446.
- Lathrop GM, et al: Multilocus linkage analysis in humans: detection of linkage and estimation of recombination. Am J Hum Genet 1985;37:482–498.
- Cottingham RW Jr, Idury RM, Schaffer AA: Faster sequential genetic linkage computations. Am J Hum Genet 1993;53:252–263.
- Hodge SE, et al: Age-of-onset correction available for linkage analysis (LIPED). Am J Hum Genet 1979;31:761–762.
- Lander ES, et al: MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1987;1:174–181.
- Kruglyak L, et al: Parametric and nonparametric linkage analysis: a unified multipoint approach. Am J Hum Genet 1996;58:1347–1363.
- Sobel E, Lange K: Descent graphs in pedigree analysis: applications to haplotyping, location scores, and marker-sharing statistics. Am J Hum Genet 1996;58:1323–1337.
- Forabosco P, Falchi M, Devoto M: Statistical tools for linkage analysis and genetic association studies. Expert Rev Mol Diagn 2005;5:781–796.
- Ott J: Analysis of Human Genetic Linkage, ed 3. Baltimore, Johns Hopkins University Press, 1999.
- Dudbridge F: A survey of current software for linkage analysis. Hum Genomics 2003;1:63–65.
- Elston RC, Anne Spence M: Advances in statistical human genetics over the last 25 years. Stat Med 2006;25:3049–3080.
- Terwilliger JD, Goring HH: Gene mapping in the 20th and 21st centuries: statistical methods, data analysis, and experimental design. Hum Biol 2000;72:63–132.
- Strug LJ, Hodge SE: An alternative foundation for the planning and evaluation of linkage analysis. I. Decoupling ‘error probabilities’ from ‘measures of evidence’. Hum Hered 2006;61:166–188.
- Strug LJ, Hodge SE: An alternative foundation for the planning and evaluation of linkage analysis. II. Implications for multiple test adjustments. Hum Hered 2006;61:200–209.
- Freimer N, Sabatti C: The use of pedigree, sib-pair and association studies of common diseases for genetic mapping and epidemiology. Nat Genet 2004;36:1045–1051.
- Conneally PM, Rivas ML: Linkage analysis in man. Adv Hum Genet 1980;10:209–266.
- Lander E, Kruglyak L: Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat Genet 1995;11:241–247.
- Simonsen KL, McIntyre LM: Using alpha wisely: improving power to detect multiple QTL. Stat Appl Genet Mol Biol 2004;3:1.
- Cheverud JM: A simple correction for multiple comparisons in interval mapping genome scans. Heredity 2001;87:52–58.
- Curtis D: Genetic dissection of complex traits. Nat Genet 1996;12:356–357.
- Risch N, Botstein D: A manic depressive history. Nat Genet 1996;12:351–353.
- Witte JS, Elston RC, Schork NJ: Genetic dissection of complex traits. Nat Genet 1996;12:355–356.
- Rao DC: CAT scans, PET scans, and genomic scans. Genet Epidemiol 1998;15:1–18.
- Fernando RL, et al: Controlling the proportion of false positives in multiple dependent tests. Genetics 2004;166:611–619.
- Risch N: A note on multiple testing procedures in linkage analysis. Am J Hum Genet 1991;48:1058–1064.
- Hodge SE, Abreu PC, Greenberg DA: Magnitude of type I error when single-locus linkage analysis is maximized over models: a simulation study. Am J Hum Genet 1997;60:217–227.
- Greenberg DA, et al: Power, mode of inheritance, and type I error in LOD scores and affecteds-only methods: reply to Kruglyak. Am J Hum Genet 1998;62:202–204.
- Greenberg DA, Abreu PC: Determining trait locus position from multipoint analysis: accuracy and power of three different statistics. Genet Epidemiol 2001;21:299–314.
- Hodge SE, et al: Multipoint lods provide reliable linkage evidence despite unknown limiting distribution: type I error probabilities decrease with sample size for multipoint lods and mods. Genet Epidemiol 2008;32:800–815.
- Camp NJ, Farnham JM: Correcting for multiple analyses in genomewide linkage studies. Ann Hum Genet 2001;65:577–582.
- Vieland VJ, Logue M: HLODs, trait models, and ascertainment: implications of admixture for parameter estimation and linkage detection. Hum Hered 2002;53:23–35.
- Abreu PC, Hodge SE, Greenberg DA: Quantification of type I error probabilities for heterogeneity LOD scores. Genet Epidemiol 2002;22:156–169.
- Hodge SE, Vieland VJ, Greenberg DA: HLODs remain powerful tools for detection of linkage in the presence of genetic heterogeneity. Am J Hum Genet 2002;70:556–559.
- Xing C, Morris N, Xing G: Distribution of model-based multipoint heterogeneity lod scores. Genet Epidemiol 2010;34:912–916.
- Huang Q, et al: Examining the effect of linkage disequilibrium on multipoint linkage analysis. BMC Genet 2005;6(suppl 1):S83.
- Boyles AL, et al: Linkage disequilibrium inflates type I error rates in multipoint linkage analysis when parental genotypes are missing. Hum Hered 2005;59:220–227.
- Kim Y, et al: Examining the effect of linkage disequilibrium between markers on the Type I error rate and power of nonparametric multipoint linkage analysis of two-generation and multigenerational pedigrees in the presence of missing genotype data. Genet Epidemiol 2008;32:41–51.
- Penrose LS: Genetic linkage in graded human characters. Ann Eugen 1938;8:233–237.
- Haseman JK, Elston RC: The investigation of linkage between a quantitative trait and a marker locus. Behav Genet 1972;2:3–19.
- Suarez BK, Rice J, Reich T: The generalized sib pair IBD distribution: its use in the detection of linkage. Ann Hum Genet 1978;42:87–94.
- Amos CI, Dawson DV, Elston RC: The probabilistic determination of identity-by-descent sharing for pairs of relatives from pedigrees. Am J Hum Genet 1990;47:842–853.
- Kruglyak L, Daly MJ, Lander ES: Rapid multipoint linkage analysis of recessive traits in nuclear families, including homozygosity mapping. Am J Hum Genet 1995;56:519–527.
- Kruglyak L, Lander ES: Complete multipoint sib-pair analysis of qualitative and quantitative traits. Am J Hum Genet 1995;57:439–454.
- Amos CI, Elston RC: Robust methods for the detection of genetic linkage for quantitative data from pedigrees. Genet Epidemiol 1989;6:349–360.
- Elston RC: The genetic dissection of multifactorial traits. Clin Exp Allergy 1995;25(suppl 2):103–106.
- Tiwari HK, Elston RC: Linkage of multilocus components of variance to polymorphic markers. Ann Hum Genet 1997;61:253–261.
- Tiwari HK, Elston RC: Restrictions on components of variance for epistatic models. Theor Popul Biol 1998;54:161–174.
- Goldgar DE: Multipoint analysis of human quantitative genetic variation. Am J Hum Genet 1990;47:957–967.
- Schork NJ: Extended multipoint identity-by-descent analysis of human quantitative traits: efficiency, power, and modeling considerations. Am J Hum Genet 1993;53:1306–1319.
- Amos CI: Robust variance-components approach for assessing genetic linkage in pedigrees. Am J Hum Genet 1994;54:535–543.
- Blangero J, Almasy L: SOLAR: Sequential Oligogenic Linkage Analysis Routines. Technical notes. 1996, Southwest Foundation for Biomedical Research. Population Genetics Laboratory: San Antonio, TX.
- Elston RC, et al: Haseman and Elston revisited. Genet Epidemiol 2000;19:1–17.
- Elston RC, Cordell HJ: Overview of model-free methods for linkage analysis. Adv Genet 2001;42:135–150.
- Almasy L, Blangero J: Contemporary model-free methods for linkage analysis. Adv Genet 2008;60:175–193.
- Almasy L, Blangero J: Human QTL linkage mapping. Genetica 2009;136:333–340.
- Almasy L, Blangero J: Variance component methods for analysis of complex phenotypes. Cold Spring Harb Protoc 2010;2010:pdb.top77.
- Penrose LS: The general purpose sib pair linkage test. Ann Eugen London 1953;18:120–124.
- Day NE, Simons MJ: Disease susceptibility genes – their identification by multiple case family studies. Tissue Antigens 1976;8:109–119.
- Fishman PM, et al: A robust method for the detection of linkage in familial disease. Am J Hum Genet 1978;30:308–321.
- Blackwelder WC, Elston RC: A comparison of sib-pair linkage tests for disease susceptibility loci. Genet Epidemiol 1985;2:85–97.
- Knapp M, Seuchter SA, Baur MP: Linkage analysis in nuclear families. 1. Optimality criteria for affected sib-pair tests. Hum Hered 1994;44:37–43.
- Schaid DJ, Nick TG: Sib-pair linkage tests for disease susceptibility loci: common tests vs. the asymptotically most powerful test. Genet Epidemiol 1990;7:359–370.
- Feingold E, Siegmund DO: Strategies for mapping heterogeneous recessive traits by allele-sharing methods. Am J Hum Genet 1997;60:965–978.
- Whittemore AS, Halpern J: A class of tests for linkage using affected pedigree members. Biometrics 1994;50:118–127.
- Whittemore AS, Halpern J: Probability of gene identity by descent: computation and applications. Biometrics 1994;50:109–117.
- Whittemore AS, Tu IP: Simple, robust linkage tests for affected sibs. Am J Hum Genet 1998;62:1228–1242.
- Holmans P: Asymptotic properties of affected-sib-pair linkage analysis. Am J Hum Genet 1993;52:362–374.
- Faraway JJ: Improved sib-pair linkage test for disease susceptibility loci. Genet Epidemiol 1993;10:225–233.
- Knapp M, Seuchter SA, Baur MP: Two-locus disease models with two marker loci: the power of affected-sib-pair tests. Am J Hum Genet 1994;55:1030–1041.
- Dupuis J, Brown PO, Siegmund D: Statistical methods for linkage analysis of complex traits from high-resolution maps of identity by descent. Genetics 1995;140:843–856.
- Farrall M: Affected sibpair linkage tests for multiple linked susceptibility genes. Genet Epidemiol 1997;14:103–115.
- Olson JM: Likelihood-based models for genetic linkage analysis using affected sib pairs. Hum Hered 1997;47:110–120.
- Cordell HJ, et al: Multilocus linkage tests based on affected relative pairs. Am J Hum Genet 2000;66:1273–1286.
- Davis S, et al: Nonparametric simulation-based statistics for detecting linkage in general pedigrees. Am J Hum Genet 1996;58:867–880.
- Kong A, Cox NJ: Allele-sharing models: LOD scores and accurate linkage tests. Am J Hum Genet 1997;61:1179–1188.
- McPeek MS: Optimal allele-sharing statistics for genetic mapping using affected relatives. Genet Epidemiol 1999;16:225–249.
- Olson JM: A general conditional-logistic model for affected-relative-pair linkage studies. Am J Hum Genet 1999;65:1760–1769.
- Bailey-Wilson JE: Parametric versus nonparametric and two-point versus multipoint: controversies in gene mapping; in Dunn M, et al. (eds): Encyclopedia of Genomics, Proteomics and Bioinformatics. John Wiley & Sons, 2005.
- Miki Y, et al: A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 1994;266:66–71.
- Hall JM, et al: Linkage of early-onset familial breast cancer to chromosome 17q21. Science 1990;250:1684–1689.
- Walsh T, et al: Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing. Proc Natl Acad Sci USA 2010;107:12629–12633.
- Suarez BK, Hampe CL, Van Eerdewegh P: Problems of replicating linkage claims in psychiatry; in Gershon ES, Cloninger CR (eds): Genetic Approaches to Mental Disorders. Washington, American Psychiatric Press, 1994, pp 23–46.
- Wilson AF, Ziegler A: Lessons learned from genetic analysis workshop 17: transitioning from genome-wide association studies to whole-genome statistical genetic analysis. Genet Epidemiol 2011, in press.
Joan E. Bailey-Wilson, PhD
333 Cassell Drive, Suite 1200
Baltimore, MD 21224 (USA)
Tel. +1 443 740 2921, E-Mail email@example.com
Published online: December 23, 2011
Number of Print Pages : 9
Number of Figures : 0, Number of Tables : 0, Number of References : 154
Human Heredity (International Journal of Human and Medical Genetics)
Vol. 72, No. 4, Year 2011 (Cover Date: December 2011)
Journal Editor: Devoto M. (Philadelphia, Pa./Rome)
ISSN: 0001-5652 (Print), eISSN: 1423-0062 (Online)
For additional information: http://www.karger.com/HHE
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.