Evolution of genome diagnostics in epidermolysis bullosa: Unveiling the power of next-generation sequencing.

BACKGROUND: Genome diagnostics is considered gold standard diagnostics for epidermolysis bullosa (EB), a phenotypically and genetically heterogeneous group of rare disorders characterized by blistering and wounding of mucocutaneous tissues. EB is caused by pathogenic variants in genes encoding proteins of the dermo-epidermal junction. Accurate genetic diagnosis of EB is crucial for prognostication, counselling and precision-medicine. Genome diagnostics for EB started in 1991 with the introduction of Sanger sequencing (SS), analysing one gene at a time. In 2013, SS was superseded by next-generation sequencing (NGS), that allow for high-throughput sequencing of multiple genes in parallel. Several studies have shown a beneficial role for NGS in EB diagnostics, but its true benefit has not been quantified. OBJECTIVES: To determine the benefit of NGS in EB by systematically evaluating the performance of different genome diagnostics used over time based on robust data from the Dutch EB Registry. METHODS: The diagnostic performances of SS and NGS were systematically evaluated in a retrospective observational study including all index cases with a clinical diagnosis of EB in whom genome diagnostics was performed between 01 January 1994 and 01 January 2022 (n = 308), registered at the Dutch EB Expertise Centre. RESULTS: Over time, a genetic diagnosis was made in 289/308 (94%) EB cases. The diagnostic yield increased from 89% (SS) to 95% (NGS). Most importantly, NGS significantly reduced diagnostic turnaround time (39 days vs. 211 days, p < 0.001). The likelihood of detecting variants of uncertain significance and additional findings increased from 5% and 1% (SS) to 22% and 13% (NGS) respectively. CONCLUSIONS: Our study quantifies the benefit of NGS-based methods and demonstrate they have had a major impact on EB diagnostics through an increased diagnostic yield and a dramatically decreased turnaround time (39 days). Although our diagnostic yield is high (95%), further improvement of genome diagnostics is urgently needed to provide a genetic diagnosis in all EB patients.

Novel insights into the epidemiology of epidermolysis bullosa (EB) from the Dutch EB Registry: EB more common than previously assumed?

BACKGROUND: Epidermolysis bullosa (EB) is a heterogeneous group of rare and incurable genetic disorders characterized by fragility of the skin and mucosae, resulting in blisters and erosions. Several epidemiological studies in other populations have been carried out, reporting varying and sometimes inconclusive figures, highlighting the need for standardized epidemiological analyses in well-characterized cohorts. OBJECTIVES: To evaluate the epidemiological data on EB in the Netherlands, extracted from the molecularly well-characterized cohort in the Dutch EB Registry. METHODS: In this observational study all EB-patients that were based in the Netherlands and captured in the Dutch EB Registry between 1988 and 2018 were included. The epidemiological outcomes were based on complete diagnostic data (clinical features, immunofluorescence, electron microscopy and mutation analysis), with longitudinal follow-up. RESULTS: A total of 464 EB-patients (287 families) were included. The incidence and point-prevalence of EB in the Netherlands were 41.3 per million live births and 22.4 per million population, respectively. EB Simplex (EBS), Junctional EB (JEB), Dystrophic EB (DEB) and Kindler EB were diagnosed in 45.7%, 18.8%, 34.7% and 0.9% of the EB-patients, respectively, with an incidence and point-prevalence of 17.5 and 11.9 (EBS), 9.3 and 2.1 (JEB), 14.1 and 8.3 (DEB), 0.5 and 0.2 (Kindler EB). In 90.5% of the EB-patients the diagnosis was genetically confirmed. During the investigated time period 73 EB-patients died, 72.6% of whom as a direct consequence of their EB. CONCLUSION: The epidemiological outcomes of EB in the Netherlands are high, attributed to a high detection rate in a well-organized set-up, indicating that EB might be more common than previously assumed. These epidemiological data help to understand the extensive need for (specialized) medical care of EB-patients and is invaluable for the design and execution of therapeutic trials. This study emphasizes the importance of thorough reporting systems and registries worldwide.

An import signal in the cytosolic domain of the Neurospora mitochondrial outer membrane protein TOM22.

TOM22 is an integral component of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The protein is anchored to the lipid bilayer by a central trans-membrane segment, thereby exposing the amino-terminal domain to the cytosol and the carboxyl-terminal portion to the intermembrane space. Here, we describe the sequence requirements for the targeting and correct insertion of Neurospora TOM22 into the outer membrane. The orientation of the protein is not influenced by the charges flanking its trans-membrane segment, in contrast to observations regarding proteins of other membranes. In vitro import studies utilizing TOM22 preproteins harboring deletions or mutations in the cytosolic domain revealed that the combination of the trans-membrane segment and intermembrane space domain of TOM22 is not sufficient to direct import into the outer membrane. In contrast, a short segment of the cytosolic domain was found to be essential for the import and assembly of TOM22. This sequence, a novel internal import signal for the outer membrane, carries a net positive charge. A mutant TOM22 in which the charge of the import signal was altered to -1 was imported less efficiently than the wild-type protein. Our data indicate that TOM22 contains physically separate import and membrane anchor sequences.