Epidermolysis Bullosa Panel

Epidermolysis bullosa (EB) is a group of inherited diseases that are characterised by blistering lesions on the skin and mucous membranes, most commonly appearing at sites of friction and minor trauma such as the feet and hands. In some subtypes, blisters may also occur on internal organs, such as the oesophagus, stomach and respiratory tract, without any apparent friction. There are 4 major types of EB based on different sites of blister formation within the skin structure: Epidermolysis bullosa simplex (EBS), Junctional epidermolysis bullosa (JEB), Dystrophic epidermolysis bullosa (DEB), and Kindler syndrome (KS).
EBS is usually characterized by skin fragility and rarely mucosal epithelia that results in non-scarring blisters caused by mild or no trauma. The four most common subtypes of EBS are: 1) localized EBS (EBS-loc; also known as Weber-Cockayne type), 2) Dowling-Meara type EBS (EBS-DM), 3) other generalized EBS(EBS, gen-nonDM; also known as Koebner type) and 4) EBS-with mottled pigmentation (EBS-MP). Skin biopsy from fresh blister is considered mandatory for diagnostics of generalized forms of EBS. The prevalence of EBS is is estimated to be 1:30,000 – 50,000. EBS-loc is the most prevalent, EBS-DM and EBS-gen-nonDM are rare, and EBS-MP is even rarer. Penetrance is 100% for known KRT5 and KRT14 mutations. Location of the mutations within functional domains of KRT5and KRT14 has shown to predict EBS phenotype. JEB is characterized by fragility of the skin and mucous membranes, manifest by blistering with little or no trauma. Blistering may be severe and granulation tissue can form on the skin around the oral and nasal cavities, fingers and toes, and internally around the upper airway. JEB is caused by mutations in LAMB3 (70% of all JEB), COL17A1 (12%), LAMC2 (9%), and LAMA3 (9%). DEB is caused by COL7A1 mutations and can be either dominantly (DDEB) or recessively (RDEB). DDEB is usually mild. Blistering may be localized to the hands, feet, elbows and knees or it may be generalized. RDEB is typically more generalized and severe than DDEB. KS is very rare and involves all layers of the skin with extreme fragility.

The target region for each gene includes coding exons and ±20 base pairs from the exon-intron boundary. In addition, the panel includes non-coding and regulatory variants if listed above (Non-coding variants covered by the panel). Some regions of the gene(s) may be removed from the panel if specifically mentioned in the ‘Test limitations” section above. If the test includes the mitochondrial genome the target region gene list contains the mitochondrial genes. The sequencing data generated in our laboratory is analyzed with our proprietary data analysis and annotation pipeline, integrating state-of-the art algorithms and industry-standard software solutions. Incorporation of rigorous quality control steps throughout the workflow of the pipeline ensures the consistency, validity and accuracy of results. Our pipeline is streamlined to maximize sensitivity without sacrificing specificity. We have incorporated a number of reference population databases and mutation databases including, but not limited, to 1000 Genomes Project, gnomAD, ClinVar and HGMD into our clinical interpretation software to make the process effective and efficient. For missense variants, in silico variant prediction tools such as  SIFT, PolyPhen,MutationTaster are used to assist with variant classification. Through our online ordering and statement reporting system, Nucleus, ordering providers have access to the details of the analysis, including patient specific sequencing metrics, a gene level coverage plot and a list of regions with suboptimal coverage (<20X for nuclear genes and <1000X for mtDNA) if applicable. This reflects our mission to build fully transparent diagnostics where ordering providers can easily visualize the crucial details of the analysis process.

We provide customers with the most comprehensive clinical report available on the market. Clinical interpretation requires a fundamental understanding of clinical genetics and genetic principles. At Blueprint Genetics, our PhD molecular geneticists, medical geneticists, and clinical consultants prepare the clinical statement together by evaluating the identified variants in the context of the phenotypic information provided in the requisition form. Our goal is to provide clinically meaningful statements that are understandable for all medical professionals regardless of whether they have formal training in genetics.

Variant classification is the cornerstone of clinical interpretation and resulting patient management decisions. Our classifications follow the ACMG guideline 2015.

The final step in the analysis is orthogonal confirmation. Sequence and copy number variants classified as pathogenic, likely pathogenic, and variants of uncertain significance (VUS) are confirmed using bi-directional Sanger sequencing or by orthogonal methods such as qPCR/ddPCR when they do not meet our stringent NGS quality metrics for a true positive call.

Our clinical statement includes tables for sequencing and copy number variants that include basic variant information (genomic coordinates, HGVS nomenclature, zygosity, allele frequencies, in silico predictions, OMIM phenotypes, and classification of the variant). In addition, the statement includes detailed descriptions of the variant, gene, and phenotype(s) including the role of the specific gene in human disease, the mutation profile, information about the gene’s variation in population cohorts, and detailed information about related phenotypes. We also provide links to the references, abstracts, and variant databases used to help ordering providers further evaluate the reported findings if desired. The conclusion summarizes all of the existing information and provides our rationale for the classification of the variant.

Identification of pathogenic or likely pathogenic variants in dominant disorders or their combinations in different alleles in recessive disorders are considered molecular confirmation of the clinical diagnosis. In these cases, family member testing can be used for risk stratification. We do not recommend using variants of uncertain significance (VUS) for family member risk stratification or patient management. Genetic counseling is recommended.

Our interpretation team analyzes millions of variants from thousands of individuals with rare diseases. Our internal database and our understanding of variants and related phenotypes increases with every case analyzed. Our laboratory is therefore well-positioned to re-classify previously reported variants as new information becomes available. If a variant previously reported by Blueprint Genetics is re-classified, our laboratory will issue a follow-up statement to the original ordering healthcare provider at no additional cost, according to our latest follow-up reporting policy.