PrP Gene Research Articles

Novel Single-Nucleotide Polymorphisms (SNPs) and Genetic Studies of the Shadow of Prion Protein (SPRN) in Quails.

Prion diseases are a group of deadly neurodegenerative disorders caused by the accumulation of the normal prion protein (PrPC) into misfolding pathological conformations (PrPSc). The PrP gene is essential for the development of prion diseases. Another candidate implicated in prion pathogenesis is the shadow of the prion protein (SPRN) gene. To date, genetic polymorphisms of the SPRN gene and the structure of the Sho protein have not been explored in quails. We used polymerase chain reaction (PCR) to amplify the SPRN gene sequence and then conducted Sanger DNA sequencing to identify the genetic polymorphisms in quail SPRN. Furthermore, we examined the genotype, allele, and haplotype frequencies, and assessed the linkage disequilibrium among the genetic polymorphisms of the SPRN gene in quails. Additionally, we used in silico programs such as MutPred2, SIFT, MUpro, AMYCO, and SODA to predict the pathogenicity of non-synonymous single-nucleotide polymorphisms (SNPs). Alphafold2 predicted the 3D structure of the Sho protein in quails. The results showed that a total of 13 novel polymorphisms were found in 106 quails, including 4 non-synonymous SNPs. Using SIFT and MUpro in silico programs, three out of the four non-synonymous SNPs (A68T, L74P, and M105I) were predicted to have deleterious effects on quail Sho. Furthermore, the 3D structure of quail Sho was predicted to be similar to that of chicken Sho. To our knowledge, this is the first report to investigate the genetic and structural properties of the quail SPRN gene.

Familial Creutzfeld-Jakob disease, compatible with PRNP c.532G>A (p.Asp178sn) gene mutation

Background: Prion disease is a rare entity; a prevalence between 0.32-1.73 per million people is estimated. The familial form corresponds to 10% of the total cases, with a peak of presentation between 40-50 years. Over fourty known germline mutations have been described, the most frequent being c.598G>Ap.Glu200Lys (E200K). Case presentation: A 41-year-old man who began in November 2021 with progressive memory impairment. In April 2022 tremor was added in all four limbs, with balance disturbances. A neurological examination with data compatible with dementia, pancerebellar and parkinsonian syndromes. Magnetic resonance imaging showed symmetrical and bilateral hyperintensities of the basal ganglia. Due to the findings and family history, a sequencing search for the PrP gene was performed, resulting in a mutation of the PrPSc gene c.532G>A (p. Asp178sn), compatible with a familial variant of Creutzfeldt Jacob Disease. Conclusions: Prionopathy should be considered as a diagnosis to rule out in people with rapidly progressive dementia. Although there are both clinical and paraclinical diagnostic criteria, diagnosis through DNA sequencing is necessary to determine de novo or autosomal dominant hereditary mutations.

PMCA-Based Detection of Prions in the Olfactory Mucosa of Patients With Sporadic Creutzfeldt-Jakob Disease.

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder caused by the conformational conversion of the prion protein (PrPC) into an abnormally folded form, named prion (or PrPSc). The combination of the polymorphism at codon 129 of the PrP gene (coding either methionine or valine) with the biochemical feature of the proteinase-K resistant PrP (generating either PrPSc type 1 or 2) gives rise to different PrPSc strains, which cause variable phenotypes of sCJD. The definitive diagnosis of sCJD and its classification can be achieved only post-mortem after PrPSc identification and characterization in the brain. By exploiting the Real-Time Quaking-Induced Conversion (RT-QuIC) assay, traces of PrPSc were found in the olfactory mucosa (OM) of sCJD patients, thus demonstrating that PrPSc is not confined to the brain. Here, we have optimized another technique, named protein misfolding cyclic amplification (PMCA) for detecting PrPSc in OM samples of sCJD patients. OM samples were collected from 27 sCJD and 2 genetic CJD patients (E200K). Samples from 34 patients with other neurodegenerative disorders were included as controls. Brains were collected from 26 sCJD patients and 16 of them underwent OM collection. Brain and OM samples were subjected to PMCA using the brains of transgenic mice expressing human PrPC with methionine at codon 129 as reaction substrates. The amplified products were analyzed by Western blot after proteinase K digestion. Quantitative PMCA was performed to estimate PrPSc concentration in OM. PMCA enabled the detection of prions in OM samples with 79.3% sensitivity and 100% specificity. Except for a few cases, a predominant type 1 PrPSc was generated, regardless of the tissues analyzed. Notably, all amplified PrPSc were less resistant to PK compared to the original strain. In conclusion, although the optimized PMCA did not consent to recognize sCJD subtypes from the analysis of OM collected from living patients, it enabled us to estimate for the first time the amount of prions accumulating in this biological tissue. Further assay optimizations are needed to faithfully amplify peripheral prions whose recognition could lead to a better diagnosis and selection of patients for future clinical trials.

Gene-Edited Cell Models to Study Chronic Wasting Disease

Prion diseases are fatal infectious neurodegenerative disorders affecting both humans and animals. They are caused by the misfolded isoform of the cellular prion protein (PrPC), PrPSc, and currently no options exist to prevent or cure prion diseases. Chronic wasting disease (CWD) in deer, elk and other cervids is considered the most contagious prion disease, with extensive shedding of infectivity into the environment. Cell culture models provide a versatile platform for convenient quantification of prions, for studying the molecular and cellular biology of prions, and for performing high-throughput screening of potential therapeutic compounds. Unfortunately, only a very limited number of cell lines are available that facilitate robust and persistent propagation of CWD prions. Gene-editing using programmable nucleases (e.g., CRISPR-Cas9 (CC9)) has proven to be a valuable tool for high precision site-specific gene modification, including gene deletion, insertion, and replacement. CC9-based gene editing was used recently for replacing the PrP gene in mouse and cell culture models, as efficient prion propagation usually requires matching sequence homology between infecting prions and prion protein in the recipient host. As expected, such gene-editing proved to be useful for developing CWD models. Several transgenic mouse models were available that propagate CWD prions effectively, however, mostly fail to reproduce CWD pathogenesis as found in the cervid host, including CWD prion shedding. This is different for the few currently available knock-in mouse models that seem to do so. In this review, we discuss the available in vitro and in vivo models of CWD, and the impact of gene-editing strategies.

Virus Infection, Genetic Mutations, and Prion Infection in Prion Protein Conversion

Conformational conversion of the cellular isoform of prion protein, PrPC, into the abnormally folded, amyloidogenic isoform, PrPSc, is an underlying pathogenic mechanism in prion diseases. The diseases manifest as sporadic, hereditary, and acquired disorders. Etiological mechanisms driving the conversion of PrPC into PrPSc are unknown in sporadic prion diseases, while prion infection and specific mutations in the PrP gene are known to cause the conversion of PrPC into PrPSc in acquired and hereditary prion diseases, respectively. We recently reported that a neurotropic strain of influenza A virus (IAV) induced the conversion of PrPC into PrPSc as well as formation of infectious prions in mouse neuroblastoma cells after infection, suggesting the causative role of the neuronal infection of IAV in sporadic prion diseases. Here, we discuss the conversion mechanism of PrPC into PrPSc in different types of prion diseases, by presenting our findings of the IAV infection-induced conversion of PrPC into PrPSc and by reviewing the so far reported transgenic animal models of hereditary prion diseases and the reverse genetic studies, which have revealed the structure-function relationship for PrPC to convert into PrPSc after prion infection.

Scrapie Resistance Gene Identification using Optimized Taqman Test qPCR Method in Sheep on the Territory of the Republic of Serbia

Abstract Scrapie is an infectious neurodegenerative disease affecting the central nervous system of sheep and goats that belongs to transmissible spongiform encephalopathies. The disease is caused by the accumulation of proteinase-resistant isoform of the prion protein. The sheep predisposition to scrapie is associated with polymorphisms of the PrP gene. Genetic susceptibility to scrapie is mainly related to codons 136, 154, and 171. ARR sheep are strongly scrapie resistant and VRQ genotype is the most susceptible. Many countries have scrapie eradication programs based on using rams with resistant genotype. The eradication program has not yet been implemented in the Republic of Serbia. To examine the genetic makeup of sheep in Serbia related to scrapie, we optimized TaqMan probes of real-time polymerase chain reaction (qPCR) technique for three codons. Blood samples from 100 sheep were analyzed by qPCR and the majority of the examined sheep were AA homozygous for the 136 codon. For codon 154 the most frequent genotype was RR and for codon 171 the most frequent genotype was QQ.

Intrinsic disorder and phase transitions: Pieces in the puzzling role of the prion protein in health and disease.

After four decades of prion protein research, the pressing questions in the literature remain similar to the common existential dilemmas. Who am I? Some structural characteristics of the cellular prion protein (PrPC) and scrapie PrP (PrPSc) remain unknown: there are no high-resolution atomic structures for either full-length endogenous human PrPC or isolated infectious PrPSc particles. Why am I here? It is not known why PrPC and PrPSc are found in specific cellular compartments such as the nucleus; while the physiological functions of PrPC are still being uncovered, the misfolding site remains obscure. Where am I going? The subcellular distribution of PrPC and PrPSc is wide (reported in 10 different locations in the cell). This complexity is further exacerbated by the eight different PrP fragments yielded from conserved proteolytic cleavages and by reversible post-translational modifications, such as glycosylation, phosphorylation, and ubiquitination. Moreover, about 55 pathological mutations and 16 polymorphisms on the PrP gene (PRNP) have been described. Prion diseases also share unique, challenging features: strain phenomenon (associated with the heterogeneity of PrPSc conformations) and the possible transmissibility between species, factors which contribute to PrP undruggability. However, two recent concepts in biochemistry-intrinsically disordered proteins and phase transitions-may shed light on the molecular basis of PrP's role in physiology and disease.

Prion protein polymorphisms in Michigan white-tailed deer (Odocoileus virginianus)

ABSTRACT Chronic Wasting Disease (CWD), a well-described transmissible spongiform encephalopathy of the Cervidae family, is associated with the aggregation of an abnormal isoform (PrPCWD) of the naturally occurring host prion protein (PrPC). Variations in the PrP gene (PRNP) have been associated with CWD rate of infection and disease progression. We analysed 568 free-ranging white-tailed deer (Odocoileus virginianus) from 9 CWD-positive Michigan counties for PRNP polymorphisms. Sampling included 185 CWD-positive, 332 CWD non-detected, and an additional 51 CWD non-detected paired to CWD-positives by sex, age, and harvest location. We found 12 polymorphic sites of which 5 were non-synonymous and resulted in a change in amino acid composition. Thirteen haplotypes were predicted, of which 11 have previously been described. Using logistic regression, consistent with other studies, we found haplotypes C (OR = 0.488, 95% CI = 0.321–0.730, P < 0.001) and F (OR = 0.122, 95% CI = 0.007–0.612, P < 0.05) and diplotype BC (OR = 0.340, 95% CI = 0.154–0.709, P < 0.01) were less likely to be found in deer infected with CWD. As has also been documented in other studies, the presence of a serine at amino acid 96 was less likely to be found in deer infected with CWD (P < 0.001, OR = 0.360 and 95% CI = 0.227–0.556). Identification of PRNP polymorphisms associated with reduced vulnerability to CWD in Michigan deer and their spatial distribution can help managers design surveillance programmesand identify and prioritize areas for CWD management.

Monitoring of chronic wasting disease using real-time quaking-induced conversion assay in Japan.

There has been no report on Chronic wasting disease (CWD) cases in Japan to date; however, there is concern about the geographic spread of CWD. To clarify the CWD status in Japan, we conducted CWD monitoring using real-time quaking-induced conversion (RT-QuIC) assay which can detect the low level of CWD prions. A total of 690 obex samples collected from sika deer and Reeves’s muntjac in Hokkaido and Honshu was tested for CWD prions. No CWD-positive cases were found, suggesting that CWD is nonexistent in Japan. Our results also indicate that RT-QuIC assay is useful for continuous monitoring of CWD. Furthermore, nucleotide sequence analysis of the PrP gene revealed sika deer in Japan harbor CWD susceptible allele.

Co-existence of PrPD types 1 and 2 in sporadic Creutzfeldt-Jakob disease of the VV subgroup: phenotypic and prion protein characteristics

We report a detailed study of a cohort of sporadic Creutzfeldt-Jakob disease (sCJD) VV1–2 type-mixed cases (valine homozygosity at codon 129 of the prion protein, PrP, gene harboring disease-related PrP, PrPD, types 1 and 2). Overall, sCJDVV1–2 subjects showed mixed clinical and histopathological features, which often correlated with the relative amounts of the corresponding PrPD type. However, type-specific phenotypic characteristics were only detected when the amount of the corresponding PrPD type exceeded 20–25%. Overall, original features of types 1 (T1) and 2 (T2) in sCJDVV1 and -VV2, including rostrocaudal relative distribution and conformational indicators, were maintained in sCJDVV1–2 except for one of the two components of T1 identified by electrophoretic mobility as T121. The T121 conformational characteristics shifted in the presence of T2, inferring a conformational effect of PrPD T2 on T121. The prevalence of sCJDVV1–2 was 23% or 57% of all sCJDVV cases, depending on whether standard or highly sensitive type-detecting procedures were adopted. This study, together with previous data from sCJDMM1–2 (methionine homozygosity at PrP gene codon 129) establishes the type-mixed sCJD variants as an important component of sCJD, which cannot be identified with current non-tissue based diagnostic tests of prion disease.

PrP gene polymorphism and its influence on some productive traits of sheepbreeds reared in Bulgaria

The rapiddissemination of scrapie over the past few decades led to development of aspecific eradication programme, based on the polymorphisms within the prionprotein gene (PRNP). Current approach encourages the selection ofanimals carrying the resistant ARR/ARR genotype, while other genotypes areconsidered less preferable. Although the strategy seems to be working quitewell, farmers are concerned whether this will affect sheep productivity andsubsequently decrease net profits. The current study was aimed to elucidatethe linkage between the PrP gene polymorphism (based on codons 136,154 and 171) and some productive traits (live weight, reproduction, milk andfleece yield) of three sheep breeds reared in Bulgaria – Assaf, NortheastBulgarian Merino and Blackhead Pleven. The total number of detectedgenotypes was six – ARR/ARR, ARR/ARQ, ARR/ARH, ARQ/ARQ, AHQ/ARQ and ARR/VRQ,with different prevalence within each breed. The observed lack ofsignificant differences in the studied performance traits between the PRNP genotypes suggests that PRNP polymorphisms did not influencethe sheep productive performance. Therefore, selection of animals on theresistant genotype (ARR/ARR) would not worsen their productivity. Theobtained results should help the better understanding of scrapie selectionand the positive effect that it would have to both health care and industry.

Gerstmann-Str\xe4ussler-Scheinker disease revisited: accumulation of covalently-linked multimers of internal prion protein fragments

Despite their phenotypic heterogeneity, most human prion diseases belong to two broadly defined groups: Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker disease (GSS). While the structural characteristics of the disease-related proteinase K-resistant prion protein (resPrPD) associated with the CJD group are fairly well established, many features of GSS-associated resPrPD are unclear. Electrophoretic profiles of resPrPD associated with GSS variants typically show 6–8 kDa bands corresponding to the internal PrP fragments as well as a variable number of higher molecular weight bands, the molecular nature of which has not been investigated. Here we have performed systematic studies of purified resPrPD species extracted from GSS cases with the A117V (GSSA117V) and F198S (GSSF198S) PrP gene mutations. The combined analysis based on epitope mapping, deglycosylation treatment and direct amino acid sequencing by mass spectrometry provided a conclusive evidence that high molecular weight resPrPD species seen in electrophoretic profiles represent covalently-linked multimers of the internal ~ 7 and ~ 8 kDa fragments. This finding reveals a mechanism of resPrPD aggregate formation that has not been previously established in prion diseases.

Proteasomal Inhibition Redirects the PrP-Like Shadoo Protein to the Nucleus

The Shadoo protein (Sho) exhibits homology to the hydrophobic region of the cellular isoform of prion protein (PrPC). As prion-infected brains gradually accumulate infectivity-associated isoforms of prion protein (PrPSc), levels of mature endogenous Sho become reduced. To study the regulatory effect of the proteostatic network on Sho expression, we investigated the action of lactacystin, MG132, NH4Cl, and 3-methyladenine (3-MA) in two cell culture models. In primary mixed neuronal and glial cell cultures (MNGCs) from transgenic mice expressing wild-type Sho from the PrP gene promoter (Tg.Sprn mice), lactacystin- and MG132-mediated inhibition of proteasomal activity shifted the repertoire of Sho species towards unglycosylated forms appearing in the nuclei; conversely, the autophagic modulators NH4Cl and 3-MA did not affect Sho or PrPC glycosylation patterns. Mouse N2a neuroblastoma cells expressing Sho under control of a housekeeping gene promoter treated with MG132 or lactacystin also showed increased nuclear localization of unglycosylated Sho. As two proteasomal inhibitors tested in two cell paradigms caused redirection of Sho to nuclei at the expense of processing through the secretory pathway, our findings define a balanced shift in subcellular localization that thereby differs from the decreases in net Sho species seen in prion-infected brains. Our data are indicative of a physiological pathway to access Sho functions in the nucleus under conditions of impaired proteasomal activity. We also infer that these conditions would comprise a context wherein Sho’s N-terminal nucleic acid–binding RGG repeat region is brought into play.

Nucleotide structure of prion protein gene in Egyptian camels

ABSTRACT The prion protein is coded by the PRP gene which is active in the brain and several other tissues. The functions of normal PrPs are related to energy production, protein degradation and DNA replication. Prions are the essential factor for the transmissible spongiform encephalopathies (TSEs) in different mammalian species. The present study aimed to identify the nucleotide characterization of the PRP gene in six camel breeds reared in Egypt. Genomic DNA which was extracted from 80 camels belonging to six breeds reared in Egypt was used to amplify fragments at 725-bp using a specific primer for PRP gene. Alignment of Egyptian camel PRP nucleotide sequences with those of other mammalian species declared the similarity 100% between PRP sequences of Egyptian and Iranian camels and 99.7% similarity with English, German and Chinese camel populations. The nucleotide sequence of Egyptian camel was submitted to GenBank under the accession no.: MF685344. The comparison between PRP amino acids of Camelus species with other mammalian species showed two missing amino acids in Camelus and Lama glama species; Glycine at position 83 and Leucine at position 229 whereas there is a unique amino acid addition – Glycine at position 89 – in Camelus species.

PrP gene polymorphism in the red deer (Cervus elaphus L.)

Transmissible spongiform encephalopathy (TSE) belongs to the group of contagious disease that affects the nervous system with fatal outcome. Chronic wasting disease belongs to this group and is characteristic for animals from the deer family. In this study polymorphisms in eleven codons located in exon 3 of the PrP gene has been investigated red deer (Cervus elaphus L.) population. Exon 3 regions, 628 bp long, were amplified by the PCR method from genomic DNA. Sequencing was performed by applying the Sanger dideoxi method. Results showed presence of eight different genotypes and eight different haplotypes. Susceptible genotypes were not found at a high frequency.

Biodiversity and selection for scrapie resistance in sheep: genetic polymorphism in eight breeds of Algeria

Scrapie is a prion disease that affects the sheep and goats. It belongs to the group of transmissible spongiform encephalopathies (TSE). TSEs are characterized by the accumulation of the pathological form (PrPSc) of the cellular prion protein (PrPC). The susceptibility of sheep to scrapie is influenced by polymorphisms in the PrP gene (PRNP). The aim of this study was to identify the genetic variability of sheep PRNP in Algerian sheep. Two-hundred and thirteen Algerian sheep from eight breeds (Ouled Djellal, Rembi, Hamra, Berbere, Barbarine, Sidaou, Taadmit and Tazegzawt) with no clinical manifestation of scrapie were analysed. Sequencing of the entire coding sequence of PRNP showed four main alleles (ARQ, ARR, AHQ and ARH) based on codons 136, 154 and 171 with different frequencies among the investigated breeds. Moreover, 14 additional nonsynonymous polymorphisms (Q101R, N103K, M112T, A116P, M137I, L141F, I142M, H143R, N146S, R151G, Y172D, N176K, H180Y and S240P) as well as two synonymous polymorphisms at codons 231 and 237 were found in the PRNP gene. Interestingly, the N103K, M137I and I142M polymorphisms were not described in sheep. The ARQ, ARR and ARH haplotypes were present in all breeds with a highest frequency of ARQ in Barbarine. The ARH was absent in Barbarine breed and the VRQ haplotype was absent in all Algerian breeds studied. The ARQ and ARR alleles were the most common with frequencies ranging from 30 to 65% and from 8 to 26%, respectively, in different breeds. These results represent the first study on PRNP variability in Algerian sheep and may serve as a basis for the development of breeding programmes to render national sheep breeds resistant to scrapie.

A novel Gerstmann-Sträussler-Scheinker disease mutation defines a precursor for amyloidogenic 8 kDa PrP fragments and reveals N-terminal structural changes shared by other GSS alleles.

To explore pathogenesis in a young Gerstmann-Sträussler-Scheinker Disease (GSS) patient, the corresponding mutation, an eight-residue duplication in the hydrophobic region (HR), was inserted into the wild type mouse PrP gene. Transgenic (Tg) mouse lines expressing this mutation (Tg.HRdup) developed spontaneous neurologic syndromes and brain extracts hastened disease in low-expressor Tg.HRdup mice, suggesting de novo formation of prions. While Tg.HRdup mice exhibited spongiform change, PrP aggregates and the anticipated GSS hallmark of a proteinase K (PK)-resistant 8 kDa fragment deriving from the center of PrP, the LGGLGGYV insertion also imparted alterations in PrP's unstructured N-terminus, resulting in a 16 kDa species following thermolysin exposure. This species comprises a plausible precursor to the 8 kDa PK-resistant fragment and its detection in adolescent Tg.HRdup mice suggests that an early start to accumulation could account for early disease of the index case. A 16 kDa thermolysin-resistant signature was also found in GSS patients with P102L, A117V, H187R and F198S alleles and has coordinates similar to GSS stop codon mutations. Our data suggest a novel shared pathway of GSS pathogenesis that is fundamentally distinct from that producing structural alterations in the C-terminus of PrP, as observed in other prion diseases such as Creutzfeldt-Jakob Disease and scrapie.

Chapter 13 - Genetic Creutzfeldt–Jakob disease

Genetic Creutzfeldt-Jakob disease (CJD) is associated with mutations in the human PrP gene (PRNP) on chromosome 20p12-pter. Pathogenic mutations have been identified in 10-15% of all CJD patients, who often have a family history of autosomal-dominant pattern of inheritance and variable penetrance. However, the use of genetic tests implemented by surveillance networks all over the world increasingly identifies unexpectedly PRNP mutations in persons apparently presenting with a sporadic form of CJD. A high phenotypic variability was reported in genetic prion diseases, which partly overlap with the features of sporadic CJD. Here we review recent advances on the epidemiologic, clinical, and neuropathologic features of cases that phenotypically resemble CJD linked to point and insert mutations of the PRNP gene. Multidisciplinary studies are still required to understand the phenotypic spectrum, penetrance, and significance of PRNP mutations.

Identification of the internal ribosome entry sites (IRES) of prion protein gene

Many studies demonstrated that there are several type bands of prion protein in cells. However, the formation of different prion protein bands is elusive. After several low molecular weight bands of prion protein appeared in SMB-S15 cells infected with scrapie agent Chandler, we think that IRES-dependent translation mechanism induced by prion is involved in the formation of prion protein bands. Then we designed a series of pPrP-GFP fusing plasmids and bicistronic plasmids to identify the IRES sites of prion protein gene and found 3 IRES sites inside of PrP mRNA. We also demonstrated that cap-independent translation of PrP was associated with the ER stress through Tunicamycin treatment. We still found that only IRE1 and PERK pathway regulated the IRES-dependent translation of PrP in this study. Our results indicated, we found that PrP gene had an IRES-dependent translation initiation mechanism and we successfully identified the IRESs inside of the prion protein gene.

Novel diagnostic technologies for clinical and frontline use: Advanced diagnostics based on molecular markers and analysis technologies has been improving diagnosis across a wide range of diseases.

Misdiagnosis is a major problem at all levels of health care causing unnecessary pain or even death as it prevents efficient, timely, and adequate treatment. In the case of infectious diseases, for instance, identification of the wrong pathogen might lead to incorrect prescription of antibiotics. While reliable data on the full extent of misdiagnosis are elusive, some evidence of the problem's scale came from a meta‐analysis of deaths within US intensive care units (ICUs), which found that 40,500 adult patients in the USA may die each year as a result of a misdiagnosis [1]. An earlier, more comprehensive, study based on searching the MEDLINE (1950–2007) and EMBASE (1980–2007) electronic databases for articles about diagnostic error or delay in primary care also found that misdiagnosis was a significant issue, although the reasons varied with each condition [2]. Moreover, many diseases, in particular rare diseases, are hard to diagnose as physicians are often unfamiliar with the symptoms; alternatively, the symptoms might be vague or untypical, as can happen with some autoimmune disorders. Many cancers in their early stages do not even show any symptoms, which prevents timely and effective treatment. There are also complex disorders that present highly varied symptoms that can lead doctors in the wrong direction, at least at first. Not surprisingly, there is an immense interest in molecular‐level diagnostics that promise to deliver far more accurate and rapid diagnoses without relying on interpreting symptoms. While this can raise new challenges, such as poor‐quality samples or analyzing complex data sets, molecular diagnostics has made progress across a range of clinical conditions, including infectious diseases and some cancers, through identification of characteristic molecular markers. A wide variety of diseases could also be diagnosed using next‐generation sequencing (NGS), including solid cancers or rare diseases of genetic origin. NGS …