Targeted long-read sequencing identifies missing disease-causing variation.

Despite widespread clinical genetic testing, many individuals with suspected genetic conditions lack a precise diagnosis, limiting their opportunity to take advantage of state-of-the-art treatments. In some cases, testing reveals difficult-to-evaluate structural differences, candidate variants that do not fully explain the phenotype, single pathogenic variants in recessive disorders, or no variants in genes of interest. Thus, there is a need for better tools to identify a precise genetic diagnosis in individuals when conventional testing approaches have been exhausted. We performed targeted long-read sequencing (T-LRS) using adaptive sampling on the Oxford Nanopore platform on 40 individuals, 10 of whom lacked a complete molecular diagnosis. We computationally targeted up to 151 Mbp of sequence per individual and searched for pathogenic substitutions, structural variants, and methylation differences using a single data source. We detected all genomic aberrations-including single-nucleotide variants, copy number changes, repeat expansions, and methylation differences-identified by prior clinical testing. In 8/8 individuals with complex structural rearrangements, T-LRS enabled more precise resolution of the mutation, leading to changes in clinical management in one case. In ten individuals with suspected Mendelian conditions lacking a precise genetic diagnosis, T-LRS identified pathogenic or likely pathogenic variants in six and variants of uncertain significance in two others. T-LRS accurately identifies pathogenic structural variants, resolves complex rearrangements, and identifies Mendelian variants not detected by other technologies. T-LRS represents an efficient and cost-effective strategy to evaluate high-priority genes and regions or complex clinical testing results.

Characterization of a severe case of PIK3CA-related overgrowth at autopsy by droplet digital polymerase chain reaction and report of PIK3CA sequencing in 22 patients.

PIK3CA-related overgrowth spectrum (PROS) refers to a group of disorders of segmental overgrowth of a wide variety of tissues as well as venous and lymphatic malformations. Clinical and molecular diagnosis can be challenging due to phenotypic heterogeneity and difficulties detecting low-level mosaicism using standard methods. Here, we report a patient with a severe presentation of PIK3CA-related overgrowth with analysis of 27 posthumously collected tissues by droplet digital polymerase chain reaction (PCR) at autopsy. This patient had a complicated medical course, with coagulopathy, ischemic brain injury, and sepsis resulting in multi-organ failure and death at age 2 months despite sirolimus therapy. Five of the 27 tissues analyzed possessed a mosaic PIK3CA mutation (p.E545K), with mutation levels ranging from 3 to 20% across affected tissues. We found no correlation between tissue-specific disease severity and mutation levels, likely reflecting sampling limitations. We also tested a series of 22 individuals with somatic overgrowth and/or vascular-lymphatic malformations using a targeted next generation sequencing panel and found PIK3CA mutations in nine individuals, identifying three novel PIK3CA variants. This report expands the clinical and molecular spectrum of PROS, emphasizes that different molecular methods can be complimentary in the diagnosis of these disorders, and highlights the risk of coagulopathy in a subset of patients with PIK3CA-related overgrowth.

Implementation and Clinical Utility of an Integrated Academic-Community Regional Molecular Tumor Board.

PURPOSE: Precision oncology develops and implements evidence-based personalized therapies that are based on specific genetic targets within each tumor. However, a major challenge that remains is the provision of a standardized, up-to-date, and evidenced-based precision medicine initiative across a geographic region. MATERIALS AND METHODS: We developed a statewide molecular tumor board that integrates academic and community oncology practices. The Precision Medicine Molecular Tumor Board (PMMTB) has three components: a biweekly Web-based teleconference tumor board meeting provided as a free clinical service, an observational research registry, and a monthly journal club to establish and revise evidence-based guidelines for off-label therapies. The PMMTB allows for flexible and rapid implementation of treatment, uniformity in practice, and the ability to track outcomes. RESULTS: We describe the implementation of the PMMTB and its first year of activity. Seventy-seven patient cases were presented, 48 were enrolled in a registry, and 38 had recommendations and clinical follow-up. The 38 subjects had diverse solid tumors (lung, 45%; GI, 21%; breast, 13%; other, 21%). Of these subjects, targeted therapy was recommended for 32 (84%). Clinical trials were identified for 24 subjects (63%), and nontrial targeted medicines for 16 (42%). Nine subjects (28%) received recommended therapy with a response rate of 17% (one of six) and a clinical benefit rate (partial response + stable disease) of 38% (three of eight). Although clinical trials often were identified, patients rarely enrolled. CONCLUSION: The PMMTB provides a model for a regional molecular tumor board with clinical utility. This work highlights the need for outcome registries and improved access to clinical trials to pragmatically implement precision oncology.

A stringent requirement for Plk1 T210 phosphorylation during K-fiber assembly and chromosome congression.

Polo-like kinase 1 (Plk1) is an essential mitotic regulator and undergoes periodic phosphorylation on threonine 210, a conserved residue in the kinase's activation loop. While phosphate-mimicking alterations of T210 stimulate Plk1's kinase activity in vitro, their effects on cell cycle regulation in vivo remain controversial. Using gene targeting, we replaced the native PLK1 locus in human cells with either PLK1 (T210A) or PLK1 (T210D) in both dominant and recessive settings. In contrast to previous reports, PLK1 (T210D) did not accelerate cells prematurely into mitosis, nor could it fulfill the kinase's essential role in chromosome congression. The latter was traced to an unexpected defect in Plk1-dependent phosphorylation of BubR1, a key mediator of stable kinetochore-microtubule attachment. Using chemical genetics to bypass this defect, we found that Plk1(T210D) is nonetheless able to induce equatorial RhoA zones and cleavage furrows during mitotic exit. Collectively, our data indicate that K-fibers are sensitive to even subtle perturbations in T210 phosphorylation and caution against relying on Plk1(T210D) as an in vivo surrogate for the natively activated kinase.