An allele of COL9A2 associated with intervertebral disc disease

Science

Volumn 285, Issue 5426, 1999, Pages 409-412

  Annunen, Susanna ^a   Paassilta, Petteri ^a   Lohiniva, Jaana ^a   Perälä, Merja ^a   Pihlajamaa, Tero ^a   Karppinen, Jaro ^a   Tervonen, Osmo ^a   Kröger, Heikki ^b   Lände, Seppo ^a   Vanharanta, Heikki ^a   Ryhänen, Lasse ^a   Göring, Harald H H ^c   Ott, Jürg ^c,d   Prockop, Darwin J ^e   Ala Kokko, Leena ^a,e  

^a UNIVERSITY OF OULU   (Finland)

^b UNIVERSITY OF EASTERN FINLAND   (Finland)

^c Columbia University ^*  (United States)

^d ROCKEFELLER UNIVERSITY   (United States)

^e DREXEL UNIVERSITY COLLEGE OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

COLLAGEN TYPE 9; GLUTAMINE; TRYPTOPHAN;

ADULT; AGED; ALLELE; AMINO ACID SUBSTITUTION; ARTICLE; CODON; COMPUTER ASSISTED TOMOGRAPHY; CONTROLLED STUDY; FINLAND; GEL ELECTROPHORESIS; GENE FREQUENCY; GENE LINKAGE DISEQUILIBRIUM; GENETIC VARIABILITY; HUMAN; INTERVERTEBRAL DISK DISEASE; ISCHIALGIA; MAJOR CLINICAL STUDY; NUCLEAR MAGNETIC RESONANCE; NUCLEOTIDE SEQUENCE; PENETRANCE; PHENOTYPE; POLYMERASE CHAIN REACTION; PRIORITY JOURNAL;

ADULT; AGED; ALLELES; AMINO ACID SUBSTITUTION; CASE-CONTROL STUDIES; CODON; COLLAGEN; COLLAGEN TYPE IX; FEMALE; GENETIC PREDISPOSITION TO DISEASE; HUMANS; INTERVERTEBRAL DISK DISPLACEMENT; LINKAGE (GENETICS); LINKAGE DISEQUILIBRIUM; MALE; MIDDLE AGED; MUTATION; PENETRANCE; POLYMORPHISM, GENETIC; SCIATICA; TRYPTOPHAN;

EID: 0033575339     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5426.409     Document Type: Article

References (56)

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20. MRI scans obtained with a 1.5-T imaging system (Signa, General Electric) consisted of sagittal images with TR/ TE of 4000/95 ms and axial images with TR/TE of 640/14 ms. CT images (Hi Speed Advantage, GE Medical Systems) consisted of scans through the L2-L3 interspace to the L5-S1 interspace. All MRI and CT images were read by two experienced neuroradiologists blinded to the results of the mutation analysis and clinical history and physical status of the patient. Clinical assessment was also performed blinded to the results of the mutation analysis. The following radiological findings were considered as indications of intervertebral disc disease: (i) disc extrusion; (ii) any herniation at two or more levels; (iii) endplate degeneration at one or more levels in patients under 30 years old, at two or more levels in patients 30 to 50 years old, or at four or more levels in patients over 50 years old; or (iv) bulging or protrusion (or both) at four or more levels [D. Weishaupt, M. Zanetti, J. Hodler, N. Boos, Radiology 209, 661 (1998); M. C. Jensen et al., N. Engl. J. Med. 331, 69 (1994)].
21. MRI scans obtained with a 1.5-T imaging system (Signa, General Electric) consisted of sagittal images with TR/ TE of 4000/95 ms and axial images with TR/TE of 640/14 ms. CT images (Hi Speed Advantage, GE Medical Systems) consisted of scans through the L2-L3 interspace to the L5-S1 interspace. All MRI and CT images were read by two experienced neuroradiologists blinded to the results of the mutation analysis and clinical history and physical status of the patient. Clinical assessment was also performed blinded to the results of the mutation analysis. The following radiological findings were considered as indications of intervertebral disc disease: (i) disc extrusion; (ii) any herniation at two or more levels; (iii) endplate degeneration at one or more levels in patients under 30 years old, at two or more levels in patients 30 to 50 years old, or at four or more levels in patients over 50 years old; or (iv) bulging or protrusion (or both) at four or more levels [D. Weishaupt, M. Zanetti, J. Hodler, N. Boos, Radiology 209, 661 (1998); M. C. Jensen et al., N. Engl. J. Med. 331, 69 (1994)].
22. Genomic DNA was extracted from blood leukocytes and used to screen for sequence variations in the COL9A2 gene by CSGE. PCR primers to amplify the exonic sequences and the flanking sequences were designed on the basis of the genomic sequences [T. Pihlajamaa et al., Matrix Biol. 17, 237 (1998)], and PCR and CSGE analysis were performed as described [J. Körkkö, S. Annunen, T. Pihlajamaa, D. J. Prockop, L. Ala-Kokko, Proc. Natl. Acad. Sci. U.S.A. 95, 1681 (1998)].
23. Genomic DNA was extracted from blood leukocytes and used to screen for sequence variations in the COL9A2 gene by CSGE. PCR primers to amplify the exonic sequences and the flanking sequences were designed on the basis of the genomic sequences [T. Pihlajamaa et al., Matrix Biol. 17, 237 (1998)], and PCR and CSGE analysis were performed as described [J. Körkkö, S. Annunen, T. Pihlajamaa, D. J. Prockop, L. Ala-Kokko, Proc. Natl. Acad. Sci. U.S.A. 95, 1681 (1998)].
24. PCR products that contained heteroduplexes on the CSGE analysis were sequenced (T7 Sequenase PCR Product Sequencing Kit, USB, or ABI Prism 377 and dRhodamine Terminator Cyde Sequencing Ready Reaction Kit, Perkin Elmer). Some of the PCR products were cloned and sequenced (T7 Sequencing Kit, Pharmacia).
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30. Artificial pedigrees were created by coding two case or control individuals as the parents of a hypothetical offspring of unknown phenotype and genotype (15). Creating such pedigrees (that is, triads) does not create false, artificial information. Such pedigrees do not provide any linkage information whatsoever, but they do give information about linkage disequilibrium, just as if the unrelated case and control individuals were analyzed separately.
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33. Linkage and linkage disequilibrium analyses were performed using the ILINK program of the FASTLINK software package [G. M. Lathrop, J.-M. Lalouel, C. Julier, J. Ott, Proc. Natl. Acad. Sci. U.S.A. 81, 3443 (1984); A. Becker, D. Geiger, A. A. Schaffer, Hum. Hered. 48, 49 (1998)].
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37. Pedigree members who tested positive for the disease in either the clinical or the radiological test were coded as affected, and those testing negative in both tests were coded as unaffected. Individuals who were only examined by one method (clinical or radiological) and were found to be normal were coded as having unknown phenotypic status. Unexamined individuals, including married-in spouses and deceased individuals from earlier generations, were likewise coded as unknown in their phenotype.
38. 1 = log[L(θ̂)/L(θ = 0.5)] is a test for linkage. [L(θ) denotes the likelihood as a function of the recombination fraction θ.] The allele frequencies of the observed polymorphism were treated as nuisance parameters using "profile" likelihoods [R. Royall, Statistical evidence: A Likelihood Paradigm (Chapman & Hall, London, 1997), pp. 158-161];
39. that is, the likelihood was maximized over these allele frequencies independently under the null hypothesis of no linkage and under the alternative hypothesis of linkage [(25), pp. 186-187; H. H. H. Göring and J. D. Terwilliger, in preparation]. A lod score of 3 is generally considered to be significant [N. E. Morton, Am. J. Hum. Genet. 7, 277 (1955)] and asymptotically corresponds to a pointwise P value of 0.0001 [J. Chotai, Ann. Hum. Genet. 48, 359 (1984)].
40. that is, the likelihood was maximized over these allele frequencies independently under the null hypothesis of no linkage and under the alternative hypothesis of linkage [(25), pp. 186-187; H. H. H. Göring and J. D. Terwilliger, in preparation]. A lod score of 3 is generally considered to be significant [N. E. Morton, Am. J. Hum. Genet. 7, 277 (1955)] and asymptotically corresponds to a pointwise P value of 0.0001 [J. Chotai, Ann. Hum. Genet. 48, 359 (1984)].
41. that is, the likelihood was maximized over these allele frequencies independently under the null hypothesis of no linkage and under the alternative hypothesis of linkage [(25), pp. 186-187; H. H. H. Göring and J. D. Terwilliger, in preparation]. A lod score of 3 is generally considered to be significant [N. E. Morton, Am. J. Hum. Genet. 7, 277 (1955)] and asymptotically corresponds to a pointwise P value of 0.0001 [J. Chotai, Ann. Hum. Genet. 48, 359 (1984)].
42. Affecteds-only analysis provides only little evidence for linkage (lod score 0.5), whereas the linkage disequilibrium results conditional on linkage remain significant (lod score 6.3) (data not shown). It is not that surprising, however, that the linkage lod score is reduced, because much linkage information is provided by unaffected individuals when the disease is dominant with high penetrance. To avoid spurious results, we only coded pedigree members as unaffected when they were found to be normal in two different diagnostic schemes, as mentioned above.
43. 1 (20), only written differently], the likelihood is maximized over the marker allele frequencies using ILINK, as before. In the numerator, the likelihood is maximized over the disease-marker haplotype frequencies, keeping the marginal frequency of the disease-predisposing allele constant. This maximization was performed with the EH program [(25), pp. 200-203] using only unrelated individuals, as ILINK would have changed the marginal disease-allele frequency also. Although the haplotype frequencies estimated with the EH program may not be the maximum likelihood estimates, this procedure is conservative in that the true maximum likelihood estimates are used in the denominator. This lod score has two degrees of freedom. (There are five free parameters in the numerator: θ and four haplotype frequencies, the frequencies of the two remaining haplotypes being constrained by the marginal allele frequencies of the disease locus. There are three free parameters in the denominator: θ and two allele frequencies, with the frequency of the remaining allele being constrained because the allele frequencies need to sum to 1.) For interpretation, the P value for the observed lod score is given in the text.
44. The evidence for linkage disequilibrium conditional on linkage using the whole data set (families and unrelated singletons) is much higher than if the case and control data had been analyzed separately. Using Fisher's "exact" test to test for equality of Trp allele frequencies among cases (6 of 314 chromosomes) and controls (0 of 348 chromosomes) only gives a P value of about 0.01. The reason for this apparent discrepancy is that, in joint linkage and linkage disequilibrium analysis, the linkage disequilibrium provides not only phase information for pedigree founders, but also genotype information for untyped founders. For this reason, joint analysis of linkage and linkage disequilibrium is often much more than the sum of the parts, sometimes leading to much higher lod scores (15, 17).
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56. We thank J. D. Terwilliger for helpful comments and A. Harju for her expert technical assistance. Supported by grants from the Academy of Finland (L.A.-K. and M.P.) and the Arthritis Foundation (D.J.P.) and by NIH grants AR39740 (D.J.P.) and HG00008 (J.O.).