Novel Biomarkers of Health and Degeneration in Human Intervertebral Discs: In-depth Proteomic Analysis of Collagen Framework of Fetal, Healthy, Scoliotic, Degenerate, and Herniated Discs.

STUDY DESIGN: Profiling proteins expressed in the nucleus pulposus (NP) of intervertebral discs (IVDs) in five different biological states. PURPOSE: To evaluate the molecular complexity of the collagen (COL) framework and its role in the health and disease of human IVDs. OVERVIEW OF LITERATURE: Changes in COL composition have been linked to degenerative disk disease (DDD). Despite the fact that humans have 28 different types of COLs, most of the literature focuses solely on COL-1 and COL-2. This study used high-end proteomic technology to examine the entire COL composition of the human IVD across fetal (developmental-FD), normal (healthy-ND), scoliotic (early degeneration-SD), herniated (degenerate-DH), and degenerated (DD) disk phenotypes. METHODS: Forty NP tissues were snap-frozen in liquid nitrogen (-196°C) immediately before being subjected to proteomic and bioinformatic analyses from five different disk phenotypes (eight each). RESULTS: Tandem mass spectrometric analysis revealed a total of 1,050 proteins in FDs, 1,809 in ND, 1,487 in SD, 1,859 in DH, and 1,538 in the DD group. Of 28 major collagens reported in the human body, this study identified 24 different collagens with 34 subtypes in NP. Fibril-forming collagens (COL-1, 2, and 11A1) and fibril-associated collagens with interrupted triple helices (COL-9A1, 12A1, and 14A1) were abundantly expressed in FDs, representing their role in the development of NP. Multiplexin (COL-15), a hybrid proteoglycan-collagen molecule, was discovered only in FDs. Degeneration was associated with COL2A1 downregulation and COL-10A1 upregulation. CONCLUSIONS: COL10 was discovered to be a new biomarker for disk degeneration. Besides COL-1 and 2, other important COLs (6, 9, 11, 12, 14, 15) with anabolic potential and abundant expression in the fetal phenotype could be investigated for tissue engineering and novel DDD therapy.

Prevalence, Patterns, and Genetic Association Analysis of Modic Vertebral Endplate Changes

STUDY DESIGN: A prospective genetic association study. PURPOSE: The etiology of Modic changes (MCs) is unclear. Recently, the role of genetic factors in the etiology of MCs has been evaluated. However, studies with a larger patient subset are lacking, and candidate genes involved in other disc degeneration phenotypes have not been evaluated. We studied the prevalence of MCs and genetic association of 41 candidate genes in a large Indian cohort. OVERVIEW OF LITERATURE: MCs are vertebral endplate signal changes predominantly observed in the lumbar spine. A significant association between MCs and lumbar disc degeneration and nonspecific low back pain has been described, with the etiopathogenesis implicating various mechanical, infective, and biochemical factors. METHODS: We studied 809 patients using 1.5-T magnetic resonance imaging to determine the prevalence, patterns, distribution, and type of lumbar MCs. Genetic association analysis of 71 single nucleotide polymorphisms (SNPs) of 41 candidate genes was performed based on the presence or absence of MCs. SNPs were genotyped using the Sequenome platform, and an association test was performed using PLINK software. RESULTS: The mean age of the study population (n=809) was 36.7±10.8 years. Based on the presence of MCs, the cohort was divided into 702 controls and 107 cases (prevalence, 13%). MCs were more commonly present in the lower (149/251, 59.4%) than in the upper (102/251, 40.6%) endplates. L4–5 endplates were the most commonly affected levels (30.7%). Type 2 MCs were the most commonly observed pattern (n=206, 82%). The rs2228570 SNP of VDR (p=0.02) and rs17099008 SNP of MMP20 (p=0.03) were significantly associated with MCs. CONCLUSIONS: Genetic polymorphisms of SNPs of VDR and MMP20 were significantly associated with MCs. Understanding the etiopathogenetic mechanisms of MCs is important for planning preventive and therapeutic strategies.

How Reliable Are the Reported Genetic Associations in Disc Degeneration?: The Influence of Phenotypes, Age, Population Size, and Inclusion Sequence in 809 Patients.

STUDY DESIGN: Prospective genetic association study. OBJECTIVE: The aim of this study was to document the variations in the genetic associations, when different magnetic resonance imaging (MRI) phenotypes, age stratification, cohort size, and sequence of cohort inclusion are varied in the same study population. SUMMARY OF BACKGROUND DATA: Genetic associations with disc degeneration have shown high inconsistency, generally attributed to hereditary factors and ethnic variations. However, the effect of different phenotypes, size of the study population, age of the cohort, etc have not been documented clearly. METHODS: Seventy-one single-nucleotide polymorphisms (SNPs) of 41 candidate genes were correlated to six MRI markers of disc degeneration (annular tears, Pfirmann grading, Schmorl nodes, Modic changes, Total Endplate Damage score, and disc bulge) in 809 patients with back pain and/or sciatica. In the same study group, the correlations were then retested for different age groups, different sample, size and sequence of subject inclusion (first 404 and the second 405) and the differences documented. RESULTS: The mean age of population (M: 455, F: 354) was 36.7 ±â€Š10.8 years. Different genetic associations were found with different phenotypes: disc bulge with three SNPs of CILP; annular tears with rs2249350 of ADAMTS5 and rs11247361 IGF1R; modic changes with VDR and MMP20; Pfirmann grading with three SNPs of MMP20 and Schmorl node with SNPs of CALM1 and FN1 and none with Total End Plate Score.Subgroup analysis based on three age groups and dividing the total population into two groups also completely changed the associations for all the six radiographic parameters. CONCLUSION: In the same study population, SNP associations completely change with different phenotypes. Variations in age, inclusion sequence, and sample size resulted in change of genetic associations. Our study questions the validity of previous studies and necessitates the need for standardizing the description of disc degeneration, phenotype selection, study sample size, age, and other variables in future studies. LEVEL OF EVIDENCE: 4.

Genetic susceptibility of lumbar degenerative disc disease in young Indian adults.

PURPOSE: Although the exact mechanisms that lead to degenerative disc disease (DDD) are not well understood, a significant genetic influence has been found. Focusing on DDD that occurs in young adults can be valuable in determining the exact role of genetic predisposition to DDD. METHODS: Patients (<40 years) with lumbar disc degeneration were evaluated with MRI imaging (1.5 Tesla) and genetic association analysis for 58 single nucleotide polymorphism (SNP) of 35 candidate genes was performed. Disc degeneration of individual discs of lumbar spine from L1 to S1 was graded by Pfirrmann's grading. The subjects were stratified into two groups based on Total Disc Degenerative Score (TDDS). Based on TDDS, the severity of DDD was classified as mild (Group A: TDDS <10) and severe (Group B: TDDS >10). RESULTS: 695 Indian subjects including 308 with mild TDDS and 387 with severe TDDS were studied. The mean age of the patients was 29.6 ± 6.9 years in group A and 31.7 ± 6.1 in group B (p < 0.05). Five of the 35 candidate genes viz., rs1337185 of COL11A (p = 0.02), rs5275 (p = 0.03) and rs5277 (p = 0.05) of COX2, rs7575934 of IL1F5 (p = 0.04), rs3213718 of CALM1 (p = 0.04) and rs162509 of ADAMTS5 (p = 0.04) were found to be significantly associated with severe TDDS. CONCLUSION: The study identifies specific SNP associations of five genes in young adults with severe lumbar disc degeneration. These five genes (COL11A1, ADAMTS5, CALM1, IL1F5 and COX2) have different functions in the matrix metabolism, intracellular signalling and inflammatory cascade. This shows that disc degeneration is a complex disease with an intricate interplay of multiple genetic polymorphisms.

Phenotype variations affect genetic association studies of degenerative disc disease: conclusions of analysis of genetic association of 58 single nucleotide polymorphisms with highly specific phenotypes for disc degeneration in 332 subjects.

BACKGROUND CONTEXT: Although the influence of genetics on the process of disc degeneration is well recognized, in recently published studies, there is a wide variation in the race and selection criteria for such study populations. More importantly, the radiographic features of disc degeneration that are selected to represent the disc degeneration phenotype are variable in these studies. The study presented here evaluates the association between single nucleotide polymorphisms (SNPs) of candidate genes and three distinct radiographic features that can be defined as the degenerative disc disease (DDD) phenotype. PURPOSE: The study objectives were to examine the allelic diversity of 58 SNPs related to 35 candidate genes related to lumbar DDD, to evaluate the association in a hitherto unevaluated ethnic Indian population that represents more than one-sixth of the world population, and to analyze how genetic associations can vary in the same study subjects with the choice of phenotype. STUDY DESIGN: A cross-sectional, case-control study of an ethnic Indian population was carried out. METHODS: Fifty-eight SNPs in 35 potential candidate genes were evaluated in 342 subjects and the associations were analyzed against three highly specific markers for DDD, namely disc degeneration by Pfirrmann grading, end-plate damage evaluated by total end-plate damage score, and annular tears evaluated by disc herniations and hyperintense zones. Genotyping of cases and controls was performed on a genome-wide SNP array to identify potential associated disease loci. The results from the genome-wide SNP array were then used to facilitate SNP selection and genotype validation was conducted using Sequenom-based genotyping. RESULTS: Eleven of the 58 SNPs provided evidence of association with one of the phenotypes. For annular tears, rs1042631 SNP of AGC1 and rs467691 SNP of ADAMTS5 were highly significantly associated (p<.01) and SNPs in NGFB, IL1B, IL18RAP, and MMP10 were also significantly associated (p<.05). The rs4076018 SNP of NGFB was highly significant (p<.01) and rs2292657 SNP of GLI1 was significantly (p<.05) correlated to disc degeneration. For end-plate damage, the rs2252070 SNP of MMP 13 showed a significant association (p<.05). Previously associated genes such as COL 9, SKT, CHST 3, CILP, IGFR, SOXp, BMP, MMP 2-12, ADH2, IL1RN, and COX2 were not significantly associated and new associations (NGFB and GLI1) were identified. The validity of all the associations was found to be phenotype dependent. CONCLUSIONS: For the first time, genetic associations with DDD have been performed in an Indian population. Apart from identifying new associations, the highlight of the study was that in the same study population with DDD, SNP associations completely changed when different radiographic features were used to define the DDD phenotype. Our study results therefore indicate that standardization of the phenotypes chosen to study the genetics of disc degeneration is essential and should be strongly considered before planning genetic association studies.