Neuropathologically directed profiling of PRNP somatic and germline variants in sporadic human prion disease.

Creutzfeldt-Jakob Disease (CJD), the most common human prion disease, is associated with pathologic misfolding of the prion protein (PrP), encoded by the PRNP gene. Of human prion disease cases, < 1% were transmitted by misfolded PrP, ~ 15% are inherited, and ~ 85% are sporadic (sCJD). While familial cases are inherited through germline mutations in PRNP, the cause of sCJD is unknown. Somatic mutations have been hypothesized as a cause of sCJD, and recent studies have revealed that somatic mutations accumulate in neurons during aging. To investigate the hypothesis that somatic mutations in PRNP may underlie sCJD, we performed deep DNA sequencing of PRNP in 205 sCJD cases and 170 age-matched non-disease controls. We included 5 cases of Heidenhain variant sporadic CJD (H-sCJD), where visual symptomatology and neuropathology implicate localized initiation of prion formation, and examined multiple regions across the brain including in the affected occipital cortex. We employed Multiple Independent Primer PCR Sequencing (MIPP-Seq) with a median depth of > 5000× across the PRNP coding region and analyzed for variants using MosaicHunter. An allele mixing experiment showed positive detection of variants in bulk DNA at a variant allele fraction (VAF) as low as 0.2%. We observed multiple polymorphic germline variants among individuals in our cohort. However, we did not identify bona fide somatic variants in sCJD, including across multiple affected regions in H-sCJD, nor in control individuals. Beyond our stringent variant-identification pipeline, we also analyzed VAFs from raw sequencing data, and observed no evidence of prion disease enrichment for the known germline pathogenic variants P102L, D178N, and E200K. The lack of PRNP pathogenic somatic mutations in H-sCJD or the broader cohort of sCJD suggests that clonal somatic mutations may not play a major role in sporadic prion disease. With H-sCJD representing a localized presentation of neurodegeneration, this serves as a test of the potential role of clonal somatic mutations in genes known to cause familial neurodegeneration.

Toward a better definition of focal cortical dysplasia: An iterative histopathological and genetic agreement trial.

OBJECTIVE: Focal cortical dysplasia (FCD) is a major cause of difficult-to-treat epilepsy in children and young adults, and the diagnosis is currently based on microscopic review of surgical brain tissue using the International League Against Epilepsy classification scheme of 2011. We developed an iterative histopathological agreement trial with genetic testing to identify areas of diagnostic challenges in this widely used classification scheme. METHODS: Four web-based digital pathology trials were completed by 20 neuropathologists from 15 countries using a consecutive series of 196 surgical tissue blocks obtained from 22 epilepsy patients at a single center. Five independent genetic laboratories performed screening or validation sequencing of FCD-relevant genes in paired brain and blood samples from the same 22 epilepsy patients. RESULTS: Histopathology agreement based solely on hematoxylin and eosin stainings was low in Round 1, and gradually increased by adding a panel of immunostainings in Round 2 and the Delphi consensus method in Round 3. Interobserver agreement was good in Round 4 (kappa = .65), when the results of genetic tests were disclosed, namely, MTOR, AKT3, and SLC35A2 brain somatic mutations in five cases and germline mutations in DEPDC5 and NPRL3 in two cases. SIGNIFICANCE: The diagnoses of FCD 1 and 3 subtypes remained most challenging and were often difficult to differentiate from a normal homotypic or heterotypic cortical architecture. Immunohistochemistry was helpful, however, to confirm the diagnosis of FCD or no lesion. We observed a genotype-phenotype association for brain somatic mutations in SLC35A2 in two cases with mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy. Our results suggest that the current FCD classification should recognize a panel of immunohistochemical stainings for a better histopathological workup and definition of FCD subtypes. We also propose adding the level of genetic findings to obtain a comprehensive, reliable, and integrative genotype-phenotype diagnosis in the near future.

MIPP-Seq: ultra-sensitive rapid detection and validation of low-frequency mosaic mutations.

BACKGROUND: Mosaic mutations contribute to numerous human disorders. As such, the identification and precise quantification of mosaic mutations is essential for a wide range of research applications, clinical diagnoses, and early detection of cancers. Currently, the low-throughput nature of single allele assays (e.g., allele-specific ddPCR) commonly used for genotyping known mutations at very low alternate allelic fractions (AAFs) have limited the integration of low-level mosaic analyses into clinical and research applications. The growing importance of mosaic mutations requires a more rapid, low-cost solution for mutation detection and validation. METHODS: To overcome these limitations, we developed Multiple Independent Primer PCR Sequencing (MIPP-Seq) which combines the power of ultra-deep sequencing and truly independent assays. The accuracy of MIPP-seq to quantifiable detect and measure extremely low allelic fractions was assessed using a combination of SNVs, insertions, and deletions at known allelic fractions in blood and brain derived DNA samples. RESULTS: The Independent amplicon analyses of MIPP-Seq markedly reduce the impact of allelic dropout, amplification bias, PCR-induced, and sequencing artifacts. Using low DNA inputs of either 25 ng or 50 ng of DNA, MIPP-Seq provides sensitive and quantitative assessments of AAFs as low as 0.025% for SNVs, insertion, and deletions. CONCLUSIONS: MIPP-Seq provides an ultra-sensitive, low-cost approach for detecting and validating known and novel mutations in a highly scalable system with broad utility spanning both research and clinical diagnostic testing applications. The scalability of MIPP-Seq allows for multiplexing mutations and samples, which dramatically reduce costs of variant validation when compared to methods like ddPCR. By leveraging the power of individual analyses of multiple unique and independent reactions, MIPP-Seq can validate and precisely quantitate extremely low AAFs across multiple tissues and mutational categories including both indels and SNVs. Furthermore, using Illumina sequencing technology, MIPP-seq provides a robust method for accurate detection of novel mutations at an extremely low AAF.