Long-range polymerase chain reaction for specific full-length amplification of the human keratin 14 gene and novel keratin 14 mutations in epidermolysis bullosa simplex patients.

Epidermolysis bullosa simplex (EBS) is an autosomal dominant genetic skin disorder characterized by intraepidermal blistering in response to mild physical trauma (Irvine and McLean, 1999). In EBS, histologic and/or ultrastructural examination shows that blistering occurs in the subnuclear cytoplasm of basal keratinocytes. The disorder can be subdivided into three subgroups depending on the severity of the disease (Fine et al, 2000). The EBS Dowling^Meara variant is the most severe variant with widespread herpetiform blistering occurring within the ¢rst week following birth. EBS Weber^Cockayne and EBS K ̨bner are milder forms of the disease where blistering is mainly localized to hands and feet. These forms of EBS are predominantly caused by dominant-negative mutations, either missense or small in-frame insertion^deletion mutations, in keratin 5 (K5) or K14 (Irvine and McLean, 1999). K5 and K14 polymerize to form the intermediate ¢lament cytoskeleton of basal keratinocytes (Coulombe and Omary, 2002). Structural weakening of this cytoskeletal system due to genetic mutations leads to fragility of basal keratinocytes and therefore to hereditary skin blistering. Mutation detection strategies for the K5 gene (KRT5) have been described previously (Stephens et al, 1997; Whittock et al, 2000) but reliable mutation detection for the K14 gene (KRT14) has proved to be more di⁄cult due to the presence of pseudogenes. One intron-containing pseudogene for K14 was reported some time ago (Savtchenko et al, 1988); however, a more recent survey of the emerging human genome data reported a second partial pseudogene for K14 (Hesse et al, 2001). Mutational analysis for K14 has previously been carried out using cDNA prepared from skin biopsies or, more often, using pairs of polymerase chain reaction (PCR) primers speci¢c for each exon of the functional K14 gene (Chen et al, 1995; Mˇller et al, 1998; Shemanko et al, 1998). Another strategy based on long-range ampli¢cation of KRT14 and its pseudogene(s) involved restriction digestion of genomic DNA (Hut et al, 2000). Here, we present a robust longrange PCR method that speci¢cally ampli¢es the whole of the K14 gene from genomic DNA.This allows sequencing of the entire K14 gene from one PCR product using internal primers. Using this method, we have identi¢ed three novel mutations in K14 in EBS patients. The reported K14 genomic sequence was used for homology searching of the April 2002 version of the human genome sequence, via the UCSC website (http://genome.ucsc.edu). This revealed two full-length intron-containing pseudogenes, which were 94.9% and 94.8% identical to the functional gene. This was consistent with the recent survey of keratin genes in the emerging genome data (Hesse et al, 2001). These pseudogenes appear to be located several megabases of DNA centromeric to the type I keratin gene cluster on chromosome 17, assuming the current genome assembly to be correct in this region (data not shown). Alignment of the £anking regions of the K14 gene and its two pseudogenes identi¢ed a number of places where primers might be placed to speci¢cally amplify the functional gene. A number of these were tested for ease of ampli¢cation and speci¢city, of which the following primers and conditions were found to be optimal. Forward primer K14spf2 (50 -ATT TCA GGG ATG GGA CAG AC-30) and reverse primer K14spr2 (50 -GAG GGG ATC TTC CAG TGG GAT CT-30) were used in 50 ml reactions containing 100 ng genomic DNA, 2 mg per ml each primer, 250 mM of each dNTP and Expand LongTemplate PCR system, 0.2 ml Long Template enzyme mix, and Long Template reaction bu¡er 1 (Roche Diagnostics, Lewes, U.K.). A ‘‘touchdown’’ PCR program was used. The PCRs were initially denatured at 941C for 2 min followed by 941C 30 s, 621C 30 s, 721C 4 min for two cycles; then 941C 30 s, 601C 30 s, 721C 4 min for two cycles; 941C 30 s, 591C 30 s, 721C 4 min for 25 cycles; followed by 721C for 5 min. PCR was performed in a computer-controlled ThermoHybaid MBS thermocycler with heated lid, without use of mineral oil. Simulated tube control with a 50 ml volume setting were used for the PCR program within the MBS software. The resultant 5099 bp PCR product (Fig 1) spans all of the KRT14 gene including 624 bp upstream of the initiation codon ATG of K14 and 69 bp downstream of the TGA codon. Using this method we identi¢ed three novel EBS mutations found at either end of the K14 gene, by sequencing with internal primers (Fig 1). Two mutations, R148C and S128del, were found in the 1A domain, encoded by exon 1 of KRT14. The third mutation, R416P, was found in the helix termination motif at the end of the 2B domain, encoded by exon 6 (Fig 1). The mutations were con¢rmed by tried and tested PCRs speci¢c for the individual exons concerned, exons 1 and 6 (Shemanko et al, 1998). The same mutations were found by sequencing of these fragments (data not shown). Mutation R148C creates a novel Bgl II site, S128del ablates an Mnl I site, and R416P destroys an Fnu4H I site. All three novel mutations were excluded from 50 normal ethnically matched controls by the appropriate restriction digests (data not shown). Mutations S128del and R416P are located in the helix initiation and termination motifs of K14, respectively. These are known mutation hotspots for EBS Dowling^Meara (Irvine and McLean, 1999), consistent with the observed phenotypes of these two patients. Mutation R148C is located in the second half of the 1A domain, a known hotspot associated with milder phenotypes (Irvine and McLean, 1999), consistent with the diagnosis of EBS Weber^Cockayne in the patient examined here. Clinical details of these patients will be reported elsewhere. Avoiding pseudogene ampli¢cation is a particular problem in the molecular diagnosis of keratin disorders as many keratins have multiple pseudogenes. A recent survey of the human genome revealed that, of 4250 intermediate ¢lament genes, about 75% are keratin-related pseudogenes (Hesse et al, 2001). The problem is particularly prominent in diagnosis of the two most common types of pachyonychia congenita (PC), where the PC-1 phenotype is caused by mutations in K6a or K16 and the PC-2 variant Reprint requests to: Professor Irwin McLean, Human Genetics Unit, Ninewells Hospital and Medical School, Dundee DD1 9SY, U.K.; Email: wmclean@hgmp.mrc. ac.uk Manuscript received July 1, 2002; revised July 31, 2002; accepted for publication September 21, 2002