title: Minimally invasive genetic screen for GJB2 related deafness using dried blood spots.
abstract: OBJECTIVE
  Nonsyndromic hearing loss is one of the most abundant human sensory disorders, and can be found in 1 out of 1000 newborns. In 60-70% of the cases this disorder is hereditary. The phenotype varies from moderate hearing loss to almost complete deafness, often only revealed in late childhood. Early detection of hearing related genetic variations in the first few weeks of life would allow planning of the audiological and logopedical procedures to maintain the children's normal audiological and speech development, and if required a cochlear implantation can be planned in time. We wanted to evaluate, whether the blood samples collected from neonates onto Guthrie cards (dried blood spots, or DBS), and blood collected from people of various ages into blood collecting tubes is equally usable for genetic testing. The quality of the samples on DBS's for genetic tests after an extended period of storage was evaluated. The methods for sample preparation and analysis were also evaluated.
  
  
  METHODS
  Two DNA extraction methods were compared on the samples. We extracted DNA from whole blood with the Versagene Blood Kit from Gentra, and from DBS's with boiling. Allele-specific PCRs (AS-PCR) were carried out on each sample. Samples were analyzed with AS-PCR and sequencing, for the 35delG mutation in the GJB2 (Cx26) gene. Freshly drawn and dried blood spot samples stored for several years were used in the experiments.
  
  
  RESULTS
  An AS-PCR method for detecting 35delG mutation on DNA extracted from Guthrie cards was validated. Blood samples up to 10 years of storage were applicable in the screen. 84 patients were found with 35delG mutations, both heterozygous (with no detected hearing related phenotypical discrepancies), and homozygous (phenotipically with moderate to severe hearing loss) forms.
  
  
  CONCLUSIONS
  The dried blood spots on Guthrie cards require only three drops of blood to be collected from children, which causes less stress than taking 3 ml of blood. The blood stored on Guthrie cards can be used to store DNA samples for at least 10 years. Even under suboptimal storage conditions the samples' DNA remains intact for genetic testing. Compared to blood collection tubes Guthrie cards cost less, are easier to transport and store.
authors: University of Szeged, Albert Szent-Györgyi Pharmaceutical and Medical Centre, Department of Oto-rhino-laryngology and Head and Neck Surgery, 6725, Tisza Lajos krt. 111, Szeged, Hungary. attila@orl.szote.u-szeged.hu
authors: Attila L
authors: AL
authors: Nagy
authors: Róbert
authors: R
authors: Csáki
authors: József
authors: J
authors: Klem
authors: László
authors: L
authors: Rovó
authors: Ferenc
authors: F
authors: Tóth
authors: Gyula
authors: G
authors: Tálosi
authors: József
authors: J
authors: Jóri
authors: Kornél
authors: K
authors: Kovács
authors: József Géza
authors: JG
authors: Kiss
mesh_terms: D000072259:Connexin 26; D017630:Connexins / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D003638:Deafness / Q000097:blood* / Q000235:genetics*; D003890:Desiccation / Q000379:methods; D004311:Double-Blind Method; D005820:Genetic Testing / Q000379:methods*; D006403:Hematologic Tests / Q000379:methods*; D006801:Humans; D007231:Infant, Newborn; D015997:Neonatal Screening / Q000379:methods; D016133:Polymerase Chain Reaction
chemical_list: D017630:Connexins; D017931:DNA Primers; C000607042:GJB2 protein, human; D000072259:Connexin 26