Torkos Attila, Teschner Magnus, Erfurt Peter, Paasche Gerrit, Lenarz Thomas, Stöver Timo
Clinic of Otorhinolaryngology and Head & Neck Surgery, University of Szeged, Tisza Lajos krt. 111, Szeged 6725, Hungary.
Int J Pediatr Otorhinolaryngol. 2006 Jun;70(6):965-71. doi: 10.1016/j.ijporl.2005.10.007. Epub 2005 Nov 14.
Recent advances in genetic research indicate that about 50% of congenital deaf patients have a genetic background, with mutations in the Connexin-26 gene being the most frequent one. Screening methods for the genetic cause of deafness have so far mostly been based on the use of peripheral whole blood as DNA source. The use of buccal smears for the genetic screening of deaf patients presents an interesting alternative to drawing blood, especially in young children. In order to validate this method, we compared results from buccal smears from very young deaf children (age<or=3 years) and deaf patients older than 3 years with the results from blood samples deriving from the same patients.
The detection of the 35delG mutation in the Connexin-26 gene was chosen to demonstrate the method's feasibility. Blood and buccal smears were collected for genetic analysis from 29 very young deaf children (Group 1: age<or=3 years) and 31 deaf patients older than 3 years (Group 2) during their clinical pre-evaluation for cochlear implantation. Genomic DNA was isolated from blood as well as from buccal smears. Yield of both sources was determined by photometric evaluation of the isolated DNA concentration. Genomic DNA isolated from blood and buccal smears was then submitted to PCR-mediated site-directed mutagenesis followed by BS1 YI restriction and electrophoresis. The results for 35delG detection in DNA originating from blood were compared to those of buccal smears.
Quantitative DNA analysis showed that both sources provided adequate amounts of DNA for PCR. No significant difference was found between Group 1 and Group 2 considering either the DNA amount isolated from blood or from buccal smears. In all 60 patients, DNA isolated from blood revealed the same results concerning the presence of the 35delG mutation as DNA originating from buccal smears.
This study demonstrates that buccal smears are an adequate, reliable source of genomic DNA providing material for genetic tests that can especially help to avoid drawing blood from very young children for the genetic screening of deafness.
基因研究的最新进展表明，约50%的先天性耳聋患者具有遗传背景，其中连接蛋白26基因的突变最为常见。迄今为止，耳聋遗传病因的筛查方法大多基于使用外周全血作为DNA来源。使用口腔拭子对耳聋患者进行基因筛查是一种有趣的替代采血的方法，尤其适用于幼儿。为了验证该方法，我们将非常年幼的耳聋儿童（年龄≤3岁）和3岁以上耳聋患者的口腔拭子检测结果与同一患者的血液样本检测结果进行了比较。
选择检测连接蛋白26基因中的35delG突变来证明该方法的可行性。在29名非常年幼的耳聋儿童（第1组：年龄≤3岁）和31名3岁以上耳聋患者（第2组）进行人工耳蜗植入临床预评估期间，采集血液和口腔拭子进行基因分析。从血液和口腔拭子中分离基因组DNA。通过对分离出的DNA浓度进行光度评估来确定两种来源的产量。然后将从血液和口腔拭子中分离出的基因组DNA进行PCR介导的定点诱变，随后进行BS1 YI酶切和电泳。将血液来源DNA中35delG的检测结果与口腔拭子的结果进行比较。
定量DNA分析表明，两种来源都能提供足够量的DNA用于PCR。考虑从血液或口腔拭子中分离出的DNA量，第1组和第2组之间未发现显著差异。在所有60例患者中，血液中分离出的DNA关于35delG突变的存在情况与口腔拭子来源的DNA结果相同。
本研究表明，口腔拭子是基因组DNA的一种充足、可靠的来源，可为基因检测提供材料，尤其有助于避免为耳聋基因筛查而从幼儿身上采血。
