Abstract
Objective
To evaluate the role of COL1A1 gene polymorphism in the etiology of otosclerosis.
Material and Methods
Peripheric blood samples are obtained from 28 patients diagnosed with otosclerosis and 50 control subjects. DNA’s of all samples are isolated and amplified by using the PCR technique. The products are restricted by appropriate enzymes and the allele distributions were compared.
Results
SS (homozygous normal), Ss (heterozygous mutant) and ss (homozygous mutant) alleles of the otosclerotic and control subjects were significantly different from each other.
Conclusion
Otosclerosis is a disease with progressive hearing loss. There are viral, hormonal, immunologic and genetic hypothesis of etiology. In this study, we concluded that the polymorphism seen in the COL1A1 gene resulting in production of excessive type 1 collagen, could play a role in the pathogenesis of otosclerosis.
1
Introductıon
Otosclerosis is a middle ear disorder, leading to progressive hearing loss due to otic capsule involvement. Clinical manifestation is mainly a conductive type of hearing loss and may result in mixed or cochlear type. The definitive etiology of otosclerosis still couldn’t be identified, despite intensive research for many years. Endocrine, enzymatic, autoimmune, genetic and viral hypotheses were investigated. Histopathologic similarity of localized osteogenesis imperfecta or Paget’s disease with otosclerosis were also considered by scientists who were searching possible common genetic origin of these conditions . Hormonal etiology was also investigated due to clinical progression of the disease during pregnancy. 30–60% of women with otosclerosis having at least one pregnancy were found to be clinically worsened before or after delivery . Role of enzymes were also studied, but their initiative effects couldn’t be proved yet. Histiocytes and osteocytes localized in the primary focus thought to be responsible for cellular destruction in different sites of cochlea, by excreting hydrolitic enzymes and proteases .
About 45–50% of individuals diagnosed with otosclerosis have other known affected family members. Most genetic studies among affected families shown otosomal-dominant mode of inheritance . The relation between human leukocyte antigen and otosclerosis couldn’t be revealed and variable conclusions of studies were reported.
COL1A1 gene is responsible for coding the fibrillary component of collagen. Type1 collagen is a protein, abundantly involved in connective tissue of the body, supporting mostly the cartilages, bones, tendons, skin and sclera. This gene codes a chain called pro-alfa1 which is a component of type-1 collagen. Two pro-alfa1 chains and one pro-alfa2 chain unite together producing type-1 collagen fibrile. These molecules are modified by extracellular enzymatic regulations and create longitudinal fibriles around cells. They are connected by tight connections and result in type-1 collagen formation. COL1A1 gene is located in the long arm of chromosome 17, at position 21.33, from base pair 48,261,456 to base pair 48,278,999 .
Recent studies supported remarkable similarity of clinical otological and histopathological findings between otosclerosis and osteogenesis imperfecta. COL1A1 gene mutation is known to be responsible for inadequate collagen type-1 production in case of osteogenesis imperfecta, leading to suspicions of possible same pathophysiologic process which may be seen in otosclerosis . Polymorphism of Sp1 binding site, that is located in the first intron of COL1A1 gene, leads to an increased gene expression three times over normal range. This may play a significant role in etiology of otosclerosis. In this study, our aim is to investigate the genetic polymorphism of this gene in Turkish patients with a diagnosis of otosclerosis.
2
Material and method
Informed written consent was obtained from all patients. This study was performed in two groups with a total number of 78 patients, attended to HNH ENT clinic and HNH Genetic Diagnosis Center, Molecular Genetics Laboratory. First group consisted of 28 patients with otosclerosis and second group consisted of 50 adults with exclusion criteria of not having any type of hearing loss, otosclerosis, osteogenesis imperfecta or any other known collagen tissue disorder. Age range varies between 20–60 years. None of the participants were relatives. Diagnostic criteria for group 1 were conductive hearing loss over at least 30 dB both unilateral and bilaterally, normal tympanic membrane on otoscopy, type As on timpanometry and decreased levels on acoustic reflexometry. Sixteen patients had surgically confirmed otosclerosis whereas remaining 12 had diagnosis by history and related tests. Ten patients had a first degree relative with otosclerosis and were assessed as familial otosclerosis and 18 without any history of blood related family member as sporadic group.
2.1
Genetic analysis
Possible Sp1 binding site polymorphism in COL1A1 gene in patients with otosclerosis was investigated. 2 cc blood samples were collected in EDTA tubes for isolation of DNA for each participant, kept in + 4C overnight. Molecular genetic analysis protocol was performed in order of DNA isolation, PCR (polymerase chain reaction), ASO-PCR (allele specific oligonucleotide), electrophoresis, RFLP (Restriction Fragment Length Polymorphism). Peripheric human leukocytes were used for isolation of DNA, using 200 μl of blood samples with proteinase K and binding buffer added on. Following isolation protocol and PCR amplification with RFLP method, cutting was performed with Van91-I restriction enzyme.
Implemented PCR program was: 3 minutes initiative denaturation at 94 °C, 31 cycles of 50 seconds at 94 °C, 31 cycles of 30 seconds at 55 °C to 67 °C matching, 31 cycles of 15 minutes at 72 °C synthesis, 5 minutes at 72 °C elongation.
Prepared cutting products were evaluated by observing in 4% agarose gel.
2
Material and method
Informed written consent was obtained from all patients. This study was performed in two groups with a total number of 78 patients, attended to HNH ENT clinic and HNH Genetic Diagnosis Center, Molecular Genetics Laboratory. First group consisted of 28 patients with otosclerosis and second group consisted of 50 adults with exclusion criteria of not having any type of hearing loss, otosclerosis, osteogenesis imperfecta or any other known collagen tissue disorder. Age range varies between 20–60 years. None of the participants were relatives. Diagnostic criteria for group 1 were conductive hearing loss over at least 30 dB both unilateral and bilaterally, normal tympanic membrane on otoscopy, type As on timpanometry and decreased levels on acoustic reflexometry. Sixteen patients had surgically confirmed otosclerosis whereas remaining 12 had diagnosis by history and related tests. Ten patients had a first degree relative with otosclerosis and were assessed as familial otosclerosis and 18 without any history of blood related family member as sporadic group.
2.1
Genetic analysis
Possible Sp1 binding site polymorphism in COL1A1 gene in patients with otosclerosis was investigated. 2 cc blood samples were collected in EDTA tubes for isolation of DNA for each participant, kept in + 4C overnight. Molecular genetic analysis protocol was performed in order of DNA isolation, PCR (polymerase chain reaction), ASO-PCR (allele specific oligonucleotide), electrophoresis, RFLP (Restriction Fragment Length Polymorphism). Peripheric human leukocytes were used for isolation of DNA, using 200 μl of blood samples with proteinase K and binding buffer added on. Following isolation protocol and PCR amplification with RFLP method, cutting was performed with Van91-I restriction enzyme.
Implemented PCR program was: 3 minutes initiative denaturation at 94 °C, 31 cycles of 50 seconds at 94 °C, 31 cycles of 30 seconds at 55 °C to 67 °C matching, 31 cycles of 15 minutes at 72 °C synthesis, 5 minutes at 72 °C elongation.
Prepared cutting products were evaluated by observing in 4% agarose gel.
3
Statistical analysis
NCSS (Number Cruncher Statistical System) 2007&PASS 2008 Statistical Software (Utah, USA) program was used for statistical analysis. Student t test was used in comparison of quantitative parameters, the normal distribution of parameters and the two groups as well as the descriptive statistical methods (mean, standard deviation). chi-Squared test was used to compare the qualitative data. Significance was evaluated at p < 0.05 level.
4
Results
Seventy-eight participants were included in this study; 28 (35.9%) cases of otosclerosis (study group) were compared with 50 participants of control group. The study consisted of 45 female (57.7%) and 33 male (42.3%) subjects. Age of patients ranges from 20 to 60 with mean age of 37.77 ± 9.51 years.
The descriptive properties of study group were shown in Table 1 . Ten of 28 patients (35.71%) have a history of a family member diagnosed as otosclerosis (familial cases), whereas 18 (64.29%) cases represent the sporadic form of the disease. 16 (57.1%) patients were diagnosed by surgical findings, remaining 12(42.9%) with clinical findings and audiological tests. 14 (50%) subjects were complaining of tinnitus and 6 (21.4%) subjects had vertigo accompanied with conductive type of hearing loss.
