Sources of genetic material

Mutational and linkage analyses are performed using DNA from the proband and his or her relatives. Among the latter, the most important samples are those of the patient’s parents. DNA can be extracted from any tissue. In EB diagnostics constitutional DNA is usually obtained from the white cells present in a blood draw, which is stored in tubes containing EDTA. Standard sampling is 5 mL of EDTA blood from adults and children and 2 to 3 mL from infants, although adequate DNA amount can be obtained from 0.2 mL of blood or less. Whenever a blood draw cannot be performed, buccal swabs may represent an alternative source to obtain constitutional DNA. The DNA amount retrieved might be insufficient for the screening of large genes, however, such as COL7A1. DNA can also be extracted from the skin biopsy used for immunomapping investigations or from keratinocyte-fibroblast cell cultures derived from the skin biopsy. Cultured cells also represent a source of messenger RNA (mRNA), which in many circumstances is useful to verify mutation consequences on splicing of the pre-mRNA. In addition, total RNA can be used as the target molecule for mutational screening procedures (see later). DNA extraction from lesional and nonlesional skin can be of interest if revertant or somatic mosaicism is suspected. Sperm is the proper source of constitutional DNA to verify the presence of a known mutation in the proband’s father when the occurrence of a paternal gonadal mosaicism has to be proved.

Screening of unknown mutations

Mutational detection strategies have been developed for all genes involved in EB. Preliminary to mutation search is the choice of the gene to be analyzed. In particular, EB types showing locus heterogeneity, such as JEB and EBS, can require screening of more than one gene. IFM findings can allow identification of the defective protein (eg, laminin-332 or collagen XVII or α6β4 integrin in JEB) and guide mutation search. Nevertheless, IFM is not adequate to distinguish the mutant polypeptide subunit in altered multimeric proteins. In these cases, multistep testing can be devised. When a gene coding for a chain subunit of a multimeric protein is more frequently targeted by mutations compared with the genes coding for the other subunits, then that gene should be screened at first. In JEB with laminin-332 deficiency, molecular analysis should start with LAMB3 testing, thereafter continue with LAMC2, and terminate with LAMA3. In JEB with pyloric atresia, ITGB4 should be screened at first, followed by ITGA6, which is very rarely mutated.

Mutation scanning techniques are based on PCR amplification of target DNA or RNA sequences. Because missense, nonsense, and splice site mutations represent the most common types of nucleotide changes underlying EB, the screening strategies have been designed to detect point mutations. Selective and rapid amplification of each exon and intron-exon boundary is obtained using genomic DNA (gDNA) as a template and specific primer pairs that anneal on flanking intronic sequences. Alternatively, overlapping primer pairs that anneal on exons and cover the entire coding region can be used when reverse transcribed mRNA (complementary DNA [cDNA]) is chosen as a template. The latter strategy depends on the availability of an RNA source, which in turn implies laboratory facilities and tools for setting up primary keratinocyte-fibroblast cell cultures. Because mRNA comprises all the coding regions but does not contain intervening sequences, the advantage of a cDNA-based screening strategy resides in the amplification of a reduced number of PCR fragments.

Conversely, gDNA-based screening strategies imply, in case of genes with many large introns, the amplification of a greater number of PCR products, each encompassing one or a few exons and flanking sequences. Blood from which the gDNA is extracted is easy to obtain, however, whereas procedures for deriving primary cultured cells are time consuming and more expensive. Mutation search on gDNA is favored by most of the diagnostic laboratories.

Genomic DNA-based strategies are available for all genes involved in EB, whereas cDNA-based strategies have been developed for a limited number of genes, in particular the largest ones, such as COL7A1, ITGB4, ITGA6, LAMB3, and LAMC2. Seventy-two primer pairs (72 PCRs) are commonly used to amplify all 118 COL7A1 exons using gDNA, whereas the entire COL7A1 coding region is comprised in only 22 cDNA amplicons. To amplify ITGB4, ITGA6, LAMB3, and LAMC2 exons, 38, 26, 23, and 23 primer pairs are usually used with gDNA as a template, whereas 12, 9, 8, and 8 PCR fragments can be generated from cDNA, respectively.

Molecular testing of keratin 14 gene (KRT14), which is involved in EBS, is complicated by the presence of two inactive pseudogenes, whose sequences could be coamplified with functional KRT14 sequences. Current gDNA-based strategies for KRT14 mutational screening have been conceived to avoid pseudogene amplification. These protocols imply single-step allele-specific PCR with primers that specifically anneal on functional KRT14 sequences, or exploit restriction sites on gDNA to exclude the pseudogene sequences from the subsequent PCR.

Once chosen, the target molecule’s gene segments are amplified using PCR; then, the most accurate way to characterize them is to obtain the exact DNA sequence. In this manner, aberrant variations can be identified and their presence confirmed in family members. Direct sequencing can be labor intensive because of the size of the gene but, especially when a cDNA-based strategy is used, it represents a relatively simple and reliable technology endowed with high sensitivity (<95%). Moreover, if a gene has been extensively screened and a large mutation database is already available, “priority” strategies based on gDNA amplification and direct sequencing of priority regions, which are defined according to the number and frequency of the reported mutations, can be devised. Priority screening is a general principle that also works well in genotyping new patients belonging to specific populations with a well-characterized constellation of recurrent and unique mutations. This strategy may speed diagnosis and improve the mutation detection rate in highly complex genes necessitating the generation and sequencing of many amplicons, such as LAMA3, LAMB3, LAMC2, and COL7A1 genes.

Nevertheless, systematic DNA sequencing has the disadvantage of being expensive and time-consuming for analysis of genes with a large exon number, such as COL7A1. Alternative strategies have been developed over time with the purpose to identify a single amplicon for sequencing. These include single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), conformation sensitive gel electrophoresis (CSGE), denaturing high-performance liquid chromatography (DHPLC), and protein truncation test (PTT). By using adapted apparatus, the first four methods are able to detect local mismatches within heteroduplex DNA molecules that form following hybridization between wild-type and mutant DNA ( Fig. 1 A). Their sensitivities vary greatly depending on the size of the amplicon screened. SSCP and CSGE have low detection levels (<70%–75%), but are easy to use. DGGE and DHPLC are more complex to set up, but once optimized they are very efficient with a mutation detection rate of up to 95%, approaching that of DNA sequencing. PTT is conceived for screening very big genes using either RNA or DNA and can detect only mutations that lead to protein truncation. It is important to consider that these methods allow the identification of point variations, which can be either true mutations or polymorphisms without any relation to the disease. The pathogenic role of a sequence variant can be obvious by looking at its nature, position within the gene, and frequency. If doubts remain, functional tests at the mRNA and protein levels should be considered.

( A ) Schematic representation of heteroduplex formation, which results by mixing, denaturing, and reannealing of PCR fragments amplified from wild-type and mutant alleles that differ in sequence because of a point mutation. Current mutation screening strategies use a range of techniques to detect heteroduplexes. ( B ) Aberrant DHPLC profile of a COL7A1 PCR amplicon harboring the c.7344G>A mutation, which is recurrent in Italian recessive DEB patients. At an optimal running temperature (62.3°C), the PCR product amplified from a standard ( normal ) DNA shows only one chromatographic peak corresponding to homoduplex molecules, whereas a mixture of mutant and standard DNA reveals homoduplex ( right peak ) and early released heteroduplex traces ( left peaks ). ( C ) Subsequent identification of the c.7344G>A mutation by direct DNA sequencing of the aberrant amplicon. Overlapped G/A peaks at nucleotide position 7344 (highlighted by N) indicate the heterozygous status for the mutation.