Visual System Homeobox 1 (VSX1) Gene Analysis in Keratoconus: Design of Specific Primers and DNA Amplification Protocols for Accurate Molecular Characterization.

BACKGROUND: Keratoconus is an ocular degeneration characterized by the thinning of corneal stroma that may lead to varying degrees of myopia and visual impairment. Genetic factors have been reported in the pathology of keratoconus where Asians have a higher incidence, earlier onset, and undergo earlier corneal grafts compared to Caucasians. The visual system homeobox 1 (VSX1) gene forms part of a paired-like homeodomain transcription factor which is responsible for ocular development. The gene was marked as a candidate in genetic studies of keratoconus in various populations. Single nucleotide polymorphisms (SNPs) in the VSX1 gene have been reported to be associated with keratoconus. The detection of the SNPs involves DNA amplification of the VSX1 gene followed by genomic sequencing. Thus, the objective of this study aims to establish sensitive and accurate screening protocols for the molecular characterization of VSX1 polymorphisms. METHODS: Keratoconic (n = 74) and control subjects (n = 96) were recruited based on clinical diagnostic tests and selection criteria. DNA extracted from the blood samples was used to genotype VSX1 polymorphisms. In-house designed primers and optimization of PCR conditions were carried out to amplify exons 1 and 3 of the VSX1 gene. PCR conditions including percentage GC content, melting temperatures, and differences in melting temperatures of primers were evaluated to produce sensitive and specific DNA amplifications. RESULTS: Genotyping was successfully carried out in 4 exons of the VSX1 gene. Primer annealing temperatures were observed to be crucial in enhancing PCR sensitivity and specificity. Annealing temperatures were carefully evaluated to produce increased specificity, yet not allowing sensitivity to be compromised. In addition, exon 1 of the VSX1 gene was amplified using 2 different sets of primers to produce 2 smaller amplified products with absence of non-specific bands. DNA amplification of exons 1 and 3 consistently showed single band products which were successfully sequenced to yield reproducible data. CONCLUSIONS: The use of in-house designed primers and optimized PCR conditions allowed sensitive and specific DNA amplifications that produced distinct single bands. The in-house designed primers and DNA amplification protocols established in this study provide an addition to the current repertoire of primers for accurate molecular characterization of VSX1 gene polymorphisms in keratoconus research.

Finite element analysis of posterior cervical fixation.

BACKGROUND CONTEXT: Despite largely, used in the past, biomechanical test, to investigate the fixation techniques of subaxial cervical spine, information is lacking about the internal structural response to external loading. It is not yet clear which technique represents the best choice and whether stabilization devices can be efficient and beneficial for three-column injuries (TCI). HYPOTHESIS: The different posterior cervical fixation techniques (pedicle screw PS, lateral mass screw LS, and transarticular screw TS) have respective indications. MATERIALS AND METHODS: A detailed, geometrically accurate, nonlinear C3-C7 finite element model (FEM) had been successfully developed and validated. Then three FEMs were reconstructed from different fixation techniques after C4-C6 TCI. A compressive preload of 74N combined with a pure moment of 1.8 Nm in flexion, extension, left-right lateral bending, and left-right axial rotation was applied to the FEMs. RESULTS: The ROM results showed that there were obvious significant differences when comparing the different fixation techniques. PS and TS techniques can provide better immediate stabilization, compared to LS technique. The stress results showed that the variability of von Mises stress in the TS fixation device was minimum and LS fixation device was maximum. Furthermore, the screws inserted by TS technique had high stress concentration at the middle part of the screws. Screw inserted by PS and LS techniques had higher stress concentration at the actual cap-rod-screw interface. CONCLUSIONS: The research considers that spinal surgeon should first consider using the TS technique to treat cervical TCI. If PS technique is used, we should eventually prolong the need for external bracing in order to reduce the higher risk of fracture on fixation devices. If LS technique is used, we should add anterior cervical operation for acquire a better immediate stabilization.