Somatic and Germline PRNP Variants in Sporadic Human Prion Disease: A Neuropathological Study

Introduction

Prion diseases are a class of infectious, progressive, and fatal neurodegenerative diseases characterized by the pathological folding and aggregation of prion protein (PrP). Prion protein is encoded by the PRNP gene, with normal cellular prion protein (PrPc) acting as the substrate for the disease-specific conformation (PrPSc). Prion diseases are easily transmissible among different mammals, such as scrapie in sheep and goats, chronic wasting disease in deer and elk, mad cow disease in cattle, and Kuru in humans. Although prion diseases are notorious for their infectious nature, other etiologies also significantly contribute to the disease burden.

The incidence rate is 1–2 people per million, including infectious, familial, and sporadic origins. Infectious cases (including Kuru, iatrogenic infections, and zoonotic mad cow disease) account for less than 1%. The majority of cases (about 85%) are sporadic prion diseases (sCJD), while familial prion diseases account for about 15%. These cases exhibit three modes of causation: infectious cases peak around 30 years of age, genetic cases around 50 years, and sporadic cases mostly occur over 60 years of age. While familial cases provide some insights, the cause of sporadic prion diseases remains unknown.

Familial prion diseases are caused by autosomal dominant mutations (gain-of-function mutations) in the PRNP gene. Three fully penetrant missense mutations delineate specific clinical pathological syndromes: P102L causing Gerstmann-Sträussler-Scheinker syndrome (GSS), D178N leading to fatal familial insomnia (FFI) and familial CJD (fCJD), and E200K also causing fCJD. Other variants like V210I and V180I increase the risk of the disease. Truncating mutations that produce early stop codons, sometimes located at the N-terminus of the prion protein, can truncate most of the protein, providing a protective effect.

Since PRNP germline mutations can cause early-onset disease, we hypothesize that late-onset sporadic prion diseases may be caused by somatic mutations, which arise during an individual’s development and aging, thus present in only a subset of cells. Although somatic mutations are drivers of tumors, they also accumulate in non-tumor tissues, including neurons in aging and neurodegenerative diseases. In some pathological contexts, somatic variant-induced diseases are more late-onset or locally distributed than germline variant-induced diseases, as illustrated by TP53 mutations in Li-Fraumeni syndrome or more widespread mTOR pathway variants in the brain. Although early studies failed to demonstrate widespread pathogenic mutations in Alzheimer’s disease (AD) and prion diseases, the Heidenhain variant of sporadic CJD (h-sCJD) shows localized presentation, providing an opportunity to study somatic mosaicism in localized neurodegenerative pathology.

Research Sources

This paper was authored by Gannon A. McDonough et al., involving collaboration among multiple institutions, including Brigham and Women’s Hospital, Boston Children’s Hospital, Case Western Reserve University School of Medicine, and Howard Hughes Medical Institute. The paper was accepted for publication in the journal “Acta Neuropathologica” in July 2024.

Research Process

The study employed deep DNA sequencing technology to sequence the PRNP gene in 205 confirmed sporadic CJD cases and 170 age-matched non-disease controls. The study specifically included 5 cases of Heidenhain variant sporadic CJD (h-sCJD), which exhibit visual symptoms and neuropathology suggesting localized prion protein formation. The research team used multiplexed independent primer PCR sequencing (MIPP-seq) technology, with a median coverage depth exceeding 5000x, and used MosaicHunter for variant analysis. By conducting allele mixing experiments on large batches of DNA, they demonstrated that variant allele fractions as low as 0.2% could be detected.

Samples and Methods

The study used two independent sample cohorts, including sporadic CJD patients and control individuals without any diagnosed neurological disease. The 205 sporadic CJD samples were collected and confirmed by the National Prion Disease Pathology Surveillance Center, with ages ranging from 45 to 86 years, including 95 females and 110 males. All CJD samples were diagnosed based on histopathology, immunohistochemistry, and prion protein (PrPSc) immunoblotting. Familial prion disease cases with known pathogenic PRNP mutations were excluded to ensure all samples were sporadic CJD.

The control group included 170 individuals, age-matched to the sporadic group, ranging from 35 to 89 years, with 55 females and 115 males, all collected by the NIH Neurobiobank.

For all samples, including both CJD patients and non-disease controls, cerebellar cortex samples were prioritized for analysis. Additionally, for h-sCJD cases, samples from three visual cortex regions (BA 17, 18, and 19), frontal cortex (BA 46), parietal cortex, temporal cortex, thalamus, and dentate nucleus were also included.

Somatic Variant Detection

The PRNP gene was subjected to multiplexed independent primer PCR sequencing (MIPP-seq), generating multiple amplicons covering the target regions, followed by a dual-indexed PCR step to produce amplicon libraries with sample-specific barcodes. MosaicHunter was used for the detection and confirmation of somatic and germline variants, with ultra-deep sequencing data (>5000X) used to identify and confirm low-frequency variant alleles. The lowest detected allele fraction was 0.2%, verified through dilution experiments.

Main Results

The study results indicated that, despite detecting multiple polymorphic germline variants in the study subjects, no true somatic variants were identified in the PRNP gene of sCJD cases. Specifically, no somatic variants were detected in various brain regions (including the affected occipital cortex) of h-sCJD cases and control groups. This indicates that the risk in sporadic CJD does not manifest as somatic mutations of known pathogenic germline variants (such as P102L, D178N, and E200K).

Conclusion and Significance

Through high-coverage PRNP gene sequencing analysis, this study could not demonstrate a major role for somatic mutations in the PRNP gene in sporadic prion disease (especially h-sCJD). The findings challenge the previous hypothesis that somatic variants are a key pathogenic mechanism in sCJD, suggesting that sCJD may result from the natural misfolding of prion protein rather than specific somatic mutations. However, since even a small amount of prion protein misfolding can propagate throughout the brain, a single mutated cell might be sufficient to trigger the disease, making the identification of somatic mutations very complex and difficult.

The study provides new insights into sporadic prion disease: the role of somatic variants in this neurodegenerative disease may be overestimated, indicating future research should focus on exploring other potential pathogenic mechanisms such as the natural misfolding of prion proteins. Furthermore, this finding has public health implications, potentially affecting the development of targeted therapeutic strategies for patients with pathogenic mutations, such as techniques for degrading prion proteins.