Association Between IL-6 and MMP3 Common Genetic Polymorphisms and Idiopathic Scoliosis in Bulgarian Patients: A Case-control Study.

STUDY DESIGN: A case-control study was performed on 105 patients with idiopathic scoliosis (IS) and 210 unrelated gender-matched controls from Bulgarian population. OBJECTIVE: Investigation of the association between common genetic polymorphisms of IL-6 and MMP3 genes and the etiology and progression of IS among Bulgarian patients. SUMMARY OF BACKGROUND DATA: The IL-6 and MMP3 genes have been considered as candidate genes of IS in Caucasian population. METHODS: Molecular detection of the promoter polymorphisms of IL-6 and MMP3 was performed by polymerase chain reaction followed by restriction fragment length polymorphism. The statistical analysis was performed by χ test with a value of P < 0.05 as statistically significant. The combinatorial effect of the candidate genes was also examined. RESULTS: This case-control study revealed statistically significant association between the IL-6 (rs1800795) functional polymorphism and susceptibility to IS (χ = 16.055; P < 0.0001). In addition, a significant association between IL-6 (rs1800795) and curve severity was detected (χ = 16.87; P < 0.0001). No genotype or allele of MMP3 (rs3025058) was found to be correlated to the onset or progression of IS (P > 0.05). One IL-6-MMP3 genotype combination was associated with the susceptibility to IS. CONCLUSION: IL-6 gene could be considered as a susceptibility and modifying factor of IS. The identification of molecular markers with diagnostic and prognostic value could be useful for early detection of children at risk for the development of IS and for prognosis of the risk for a rapid deformity progression. That would facilitate the therapy decisions and early stage treatment of the patient with the least invasive procedures. LEVEL OF EVIDENCE: 4.

Association Study between Idiopathic Scoliosis and Polymorphic Variants of VDR, IGF-1, and AMPD1 Genes.

Idiopathic scoliosis (IS) is a complex genetic disorder of the musculoskeletal system, characterized by three-dimensional rotation of the spine with unknown etiology. For the aims of the current study we selected 3 single nucleotide polymorphisms with a low incidence of the polymorphic allele in Bulgarian population, AMPD1 (rs17602729), VDR (rs2228670), and IGF-1 (rs5742612), trying to investigate the association between these genetic polymorphisms and susceptibility to and progression of IS. The polymorphic regions of the genes were amplified by polymerase chain reaction (PCR). The PCR products were cleaved with the appropriate restriction enzymes. The statistical analysis was performed by Pearson's chi-squared test. A value of p < 0.05 was considered to be statistically significant. In conclusion, this case-control study revealed no statistically significant association between the VDR, IGF-1, and AMPD1 polymorphisms and the susceptibility to IS or curve severity in Bulgarian patients. Replication case-control studies will be needed to examine the association between these candidate-genes and IS in different populations. The identification of molecular markers for IS could be useful for early detection and prognosis of the risk for a rapid progression of the curve. That would permit early stage treatment of the patient with the least invasive procedures.

In Search of Biomarkers for Idiopathic Scoliosis: Leptin and BMP4 Functional Polymorphisms.

Idiopathic scoliosis (IS) is the most common spinal disorder in children and adolescents. The current consensus on IS maintains that it has a multifactorial etiology with genetic predisposition factors. In the present study the association of two functional polymorphisms of leptin (rs7799039) and BMP4 (rs4898820) with susceptibility to IS and curve severity was investigated in a Bulgarian population sample. The molecular detection of the genotypes was performed by amplification followed by restriction technology. The statistical analysis was performed by Pearson's chi-squared test. This case-control study revealed no statistically significant association between the functional polymorphisms of leptin and BMP4 and susceptibility to IS or curve progression (p > 0.05). On the basis of these results the examined polymorphic variants of leptin and BMP4 could not be considered as genetic variants with predisposition effect or as risk factors for the progression of the curve. In addition, these results do not exclude a synergistic effect of the promoter polymorphisms of leptin and BMP4 in the etiology and pathogenesis of IS. The identification of molecular markers for IS could be useful for early detection and prognosis of the risk for a rapid progression of the curve. That would permit early stage treatment of the patient with the least invasive procedures.

Spectrum and frequencies of BRCA1/2 mutations in Bulgarian high risk breast cancer patients.

BACKGROUND: About 3885 women are diagnosed with breast cancer and 1285 die from the disease each year in Bulgaria. However no genetic testing to identify the mutations in high-risk families has been provided so far. METHODS: We evaluated 200 Bulgarian women with primary invasive breast cancer and with personal/ family history of breast cancer for the presence of unequivocally damaging germline mutations in BRCA1/2 using Sanger sequencing. RESULTS: Of the 200 patients, 39 (19.5 %) carried a disease predisposing mutation, including 28 (14 %) with a BRCA1 mutation and 11 (5.5 %) with a BRCA2 mutation. At BRCA1, 6 different mutations were identified, including 2 frameshifts, 1 nonsense and 1 missense that had been previously reported (c.5030_5033delCTAA, c.5263_5264insC, c.4603G > T, c.181 T > G), and 2 frameshifts, which were novel to this study (c.464delA, c.5397_5403delCCCTTGG). At BRCA2, 7 different frameshift mutations were identified, including 5 previously reported (5851_5854delAGTT, c.5946delT, c.5718_5719delCT, c.7910_7914delCCTTT,c.9098_9099insA) and 2 novel (c.8532_8533delAA, c.9682delA). A BRCA1 mutation was found in 18.4 % of women diagnosed with breast cancer at/or under the age of 40 compared to 11.2 % of women diagnosed at a later age; a BRCA2 mutation was found in 4 % of women diagnosed at/or under the age of 40 compared to 6.5 % of women diagnosed at a later age. A mutation was present in 26.8 % patients with a positive family history and in 14.4 % of women with a negative family history. The most prevalent mutation observed in 22 patients (11 %) was BRCA1 c.5263_5264insC, a known Slavic mutation with founder effect in Eastern European and AJ communities. Other recurrent mutations were BRCA2 c.9098-9099insA (2 %), BRCA1 c.181T > G (1 %) and BRCA2 c.5851_5854delAGTT (1 %). Notably, BRCA1 c.5263_5264insC represented 56 % of all mutations identified in this series. Of the 22 patients with BRCA1 c.5263_5264insC, 9 were diagnosed with early onset breast cancer, 11 with TNBCs, 4 with bilateral breast cancer, and 6 with both breast and ovarian cancer. CONCLUSIONS: This is the first comprehensive study of the BRCA1/2 mutation spectrum in Bulgaria and will assist the establishment of efficient protocols for genetic testing and individualized risk assessment for Bulgarian breast/ovarian cancer patients and healthy individuals at a high-risk.

Clinical and molecular studies of EXT1/EXT2 in Bulgaria.

EXT1/EXT2-CDG (Multiple cartilagineous exostoses, hereditary multiple osteochondroma (MO); OMIM 133700/133701) are common defects of O-xylosylglycan glycosylation. The diagnostic criteria are at least two osteochondromas of the juxta-epiphyseal region of long bones with in the majority of cases a positive family history and/or mutation in one of the EXT genes. The authors report data on clinical symptoms and complications of 23 patients (from 16 families), discussing the family history, age of diagnosis, new clinical and molecular data. Fifteen mutations and large deletions, of which nine are new, were detected in the EXT1 and EXT2 gene by sequence analysis, FISH and MLPA analysis.

Novel MLH1 frameshift mutation in an extended hereditary nonpolyposis colorectal cancer family.

AIM: To present novel frameshift mutation c.31delC [p.L11X] in the MLH1 gene identified in an extended Bulgarian hereditary non-polyposis colorectal cancer (HNPCC) family and to analyze the molecular and clinical findings within the pedigree concerning the proposal of adequate individual prophylactic strategy for all mutation carriers. METHODS: The pedigree of the family consists of 42 members in four generations. Search for mutations in the MLH1 and hMSH2 genes was performed in the proband. After PCR amplification of all exons including flanking intronic regions, amplicons were directly sequenced. RESULTS: The mutation was found in nine from the thirteen pedigree members who signed informed consent to participate in the study. In three adenocarcinomas, microsatellite instability and lack of the MLH1 protein expression were detected. The only one tubulovillous adenoma analyzed was microsatellite stable and the MLH1 protein showed an intact staining. CONCLUSION: The newly described mutation c.31delC is HNPCC causative. Besides the typical clinical features of the syndrome, we found a specific pathologic manifestation such as moderate to high differentiated adenocarcinomas of the colon. One of the mutation carriers developed a benign giant cell soft tissue tumor. The primary tumor localizations were frequently extracolonic and detailed yearly gastrointestinal and gynecological examinations have been proposed to the mutation carriers. We emphasize the importance of including the HNPCC genetic counseling and testing as well in the following surveillance of all patients at risk in the services covered by the health insurance in Bulgaria.