Wild-type Sequence Research Articles

Peptide nucleic acid-immobilised paper combined with multiplex recombinase polymerase amplification for the ultrasensitive and rapid detection of rifampicin-resistant tuberculosis

Rifampicin-resistant tuberculosis (RR-TB) is a critical issue with significant implications for patient care, public health, and TB control efforts that necessitate comprehensive strategies for detection. This study presents a novel point-of-care diagnostic tool for RR-TB detection employing a peptide nucleic acid (PNA)-paper-based sensor combined with isothermal recombinase polymerase amplification (RPA). The sensor targets mutations in codons 516, 526, and 531 of the rpoB gene, the top three common mutations associated with rifampicin-resistant strains. PNA probes specifically recognised wild-type sequences, generating a visual signal through a reverse hybridisation assay. The absence of a signal was observed when the mutant strains were detected because of the inability to bind the mutant sequence. Our proof-of-concept assay displayed high accuracy (100% for detecting mutations at codons 516, 526, and 531), a short turnaround time (110 min), no cross-reactivity with other bacterial pathogens, and ultrasensitivity. This PNA-paper-based sensor model can be a valuable diagnostic tool for RR-TB detection, providing an accessible diagnostic platform that can be advantageous in resource-limited settings where sophisticated laboratory infrastructure may be lacking.

Metabolic Engineering of Escherichia coli BL21(DE3) for 2'-Fucosyllactose Synthesis in a Higher Productivity.

2'-Fucosyllactose (2'-FL) is the most abundant human milk oligosaccharides (HMOs). 2'-FL exhibits great benefits for infant health, such as preventing infantile diarrhea and promoting the growth of intestinal probiotics. The microbial cell factory technique has shown promise for the massive production of 2'-FL. Here, we aimed to construct a recombinant E. coli BL21(DE3) strain for the hyperproduction of 2'-FL. Initially, multicopy genomic integration and expression of the lactose permease gene lacY reduced the formation of byproducts. Furthermore, a more efficient Shine-Dalgarno sequence was used to replace the wild-type sequence in the manC-manB and gmd-wcaG gene clusters, which significantly increased the 2'-FL titer. Based on these results, we overexpressed the sugar efflux transporter SetA and knocked out the pgi gene. This further improved 2'-FL synthesis when glycerol was used as the sole carbon source. Finally, a new α-1,2-fucosyltransferase was identified in Neisseria sp., which exhibited a higher capacity for 2'-FL production. Fed-batch fermentation produced 141.27 g/L 2'-FL in 45 h with a productivity of 3.14 g/L × h. This productivity rate achieved the highest recorded 2'-FL levels, indicating the potential of engineered E. coli BL21 (DE3) strains for use in the industrial production of 2'-FL.

Development and Evaluation of the Immunogenic Potential of an Unmodified Nucleoside mRNA Vaccine for Herpes Zoster.

Approved mRNA vaccines commonly use sequences modified with pseudouridine to enhance translation efficiency and mRNA stability. However, this modification can result in ribosomal frameshifts, reduced immunogenicity, and higher production costs. This study aimed to explore the potential of unmodified mRNA sequences for varicella-zoster virus (VZV) and evaluate whether codon optimization could overcome the limitations of pseudouridine modification. We utilized artificial intelligence (AI) to design several unmodified gE mRNA sequences for VZV, considering factors such as codon preference and secondary structure. The optimized mRNA sequences were assessed for protein expression levels in vitro and were subsequently used to develop a vaccine, named Vac07, encapsulated in a lipid nanoparticle (LNP) delivery system. The immunogenicity of Vac07 was evaluated in mice. Codon-optimized mRNA sequences showed significantly higher protein expression levels in vitro compared to wild-type (WT) sequences. Vaccination with Vac07 demonstrated immunogenicity in mice that was comparable to, or even superior to, the licensed Shingrix vaccine, characterized by a stronger Th1-biased antibody response and a slightly more robust Th1-type cellular response. Codon-optimized unmodified mRNA sequences may also represent a viable approach for mRNA vaccine development. These optimized sequences have the potential to lower production costs while possibly enhancing the immunogenicity of mRNA vaccines. Vac07, developed using this method, shows promise as a potentially more efficient and cost-effective mRNA vaccine candidate for VZV.

Whole Genome Sequence Analysis of Terbinafine Resistant and Susceptible Trichophyton Isolates from Human and Animal Origin.

Trichophyton indotineae, first identified in India, has increasingly been reported in Asia, the Middle East, Europe, and recently in the USA. The global spread of terbinafine-resistant T. indotineae underscores the urgency of the issue. With its ability for human-to-human transmission, it can be considered anthropophilic. However, its highly virulent nature suggests a possible link to zoophilic species, raising the potential for disease transmission from animals. In this study, we have performed whole genome sequencing (WGS) of terbinafine susceptible and resistant Trichophyton species from animal and human origin to understand transmission dynamics of this species. Thirteen isolates of Trichophyton spp. from human (n = 9) and canine (n = 4) origin, respectively from Chandigarh and Bareilly, India, were included in this study. Isolate identification based on ITS extracted from WGS data identified six T. indotineae (ITS genotype VIII) and seven T. interdigitale (ITS genotype II) isolates. WGS single nucleotide polymorhpism (SNP) analysis separated the isolates into two distinct groups, T. indotineae and T. interdigitale and showed the clonal nature of both species. For both species, low SNP differences between isolates from humans and dogs were observed as well as low differences between isolates from Chandigarh and Bareilly, cities >350km apart from each other. These findings suggest zoonotic transmission, next to fast spread across large distances. The T. indotineae terbinafine-resistant strains exhibitedthe SQLEF397L substitution while susceptible strains had the SQLES395P substitution or demonstrated a wild-type (WT) SQLE sequence. However, all T. interdigitale strains displayed a WT SQLE sequence despite terbinafine minimum inhibitory concentrations (MICs) ranging between 0.031 to64µg/mL.

Genetic diversity of dengue virus circulating in the Philippines (2014-2019) and comparison with dengue vaccine strains.

Dengue virus has four distinct serotypes and the genetic diversity within each of the four serotypes contribute to its complexity. An important aspect of dengue molecular evolutionary studies has been the dissection of the extent and structure of genetic variation among major genotypes within each serotype. It is important to understand the role of dengue genetic variability and its potential role and impact in the effectiveness of the dengue vaccine. Demographic data and blood were collected from patients seen at a tertiary hospital in the Philippines and clinically diagnosed with dengue from 2014-2019. Dengue virus (DENV) RT-PCR was used to confirm infection and positive samples underwent whole genome sequencing. Phylogenetic analysis was performed on 127 samples (25 DENV-1, 19 DENV-2, 70 DENV-3, and 13 DENV-4). We observed a serotype shift in 2014 and 2022. We detected the following genotypes per serotype for the wild-type (WT) DENV sequences: genotype IV (DENV-1), cosmopolitan (DENV-2), genotype I (DENV-3) and genotype IIa (DENV-4). WT DENV belonged to different genotypes versus the QDENGA strains and except for DENV-4, belonged to different genotypes versus the Dengvaxia strains. Comparing Dengvaxia vaccine sequences with WT DENV, we observed 23, 24, 34, and 9 positions with amino acid changes in the entire envelope protein, with 1, 5, 1, and 2 positions with amino acid changes identified among the important human monoclonal antibodies (mAbs) targeted epitope positions. We detected 24, 25, 36 and 12 positions with amino acid changes in the E protein with 0, 5, 1, and 2 positions with amino acid changes among the important mAbs targeted epitope positions for DENV-1, DENV-2, DENV-3, and DENV-4, respectively when comparing QDENGA vaccine sequences with the WT DENV. We showed low genotype complexity, genetically distinct clades and local evolution for DENV circulating in the Philippines.

TAC1b mutation in Candida auris decreases manogepix susceptibility owing to increased CDR1 expression.

Candida auris is an emerging pathogenic fungus that is highly resistant to existing antifungal drugs. Manogepix is a novel antifungal agent that exerts antifungal activity by inhibiting glycosylphosphatidylinositol anchor biosynthesis. Although the mechanisms of resistance of Candida species to manogepix have been reported previously, those of C. auris are yet to be studied. To investigate the resistance mechanisms of C. auris, we exposed a clinical isolate (clade I) to manogepix in vitro and generated strains with reduced susceptibility to manogepix. A search for gain-of-function mutations that upregulate efflux pump expression confirmed the presence of the D865N amino acid mutation in TAC1b. We used the clustered regularly interspaced short palindromic repeats-Cas9 system to create a recovery strain (N865D) in which only this single nucleotide mutation was returned to the wild-type sequence. We generated a mutant strain by introducing only the D865N mutation into the parent strain and a different clade strain (clade III). The D865N mutant strains were clearly less susceptible to manogepix than the parental strains and exhibited high CDR1 expression. Moreover, we generated a strain deficient in CDR1 and confirmed that this strain had significantly increased susceptibility to manogepix. Thus, the present study demonstrated that the TAC1b mutation in C. auris upregulates CDR1 expression and decreases its susceptibility to manogepix.

In silico splicing analysis of the PMS2 gene: exploring alternative molecular mechanisms in PMS2-associated Lynch syndrome

Lynch syndrome (LS) is one of the most common hereditary cancer syndrome in human populations, associated with germline variants in MLH1, MSH2/EPCAM, MSH6 and PMS2 genes. The advent of next generation sequencing has proven a significant impact in germline variant detection in the causative genes; however, a large proportion of patients with clinical criteria still receive uncertain or negative results. PMS2 is the least frequent reported gene, associated with up to 15% of LS cases with late-onset disease and low penetrance phenotype; however, the proportion of PMS2-LS cases is considered to be highly underestimated. In this context, our analysis aimed to improve the current diagnostic yield by focusing on missense and intronic PMS2 variants available in public clinical databases (ClinVar, LOVD). We performed an in silico assessment of the wild-type DNA sequence and the reported genetic variants, employing splicing bioinformatics tools known for their effectiveness in other genes. Splicing variants were predicted in silico and using GTEx short-read RNA expression data. Out of the 2384 missense variants discovered, 90% were classified with uncertain significance (VUS). 4.9% of missense variants were shown to have a potential splicing consequence (DS > 0.2) using SpliceAI. As described in the original publication, SpliceAI-visual was proven effective in annotation of short intronic variants (< 50 bp). Four short intronic variants were identified using SpliceAI-visual as potentially splicing disturbing, in spite of using a lower threshold (DS > 0.1). Exons 2, 3, 4, 5, 6, 7, 8, 11, 12 and 14 were consistently predicted in at least three out of eight software with weak canonical splice sites. Additionally, we noted that both Exonic Splicing Enhancers (ESEs) and Exonic Splicing Silencers (ESSs) contribute significantly to alternative splicing and exonic selection in PMS2 gene. Specifically, ESE motifs were consistently more abundant in highly expressed exons 5, 11 and 14, while ESS motifs played a fundamental role in exons 6, 7 and 10. Computational analysis performed in our study serves as a valuable filtering step for guiding further RNA experiments. Additional functional data is necessary to validate our findings.

Kv3.3 Expression Enhanced by a Novel Variant in the Kozak Sequence of KCNC3.

Pathogenic variants in KCNC3, which encodes the voltage-gated potassium channel Kv3.3, are associated with spinocerebellar ataxia type 13. SCA13 is a neurodegenerative disease characterized by ataxia, dysarthria and oculomotor dysfunction, often in combination with other signs and symptoms such as cognitive impairment. Known disease-causing variants are localized in the protein coding regions and predominantly in the transmembrane and voltage sensing domains. In a patient with an ataxic movement disorder and progressive cognitive decline, the c.-6C>A variant was detected in the Kozak sequence of KCNC3. The Kozak sequence is responsible for efficient initiation of translation. Functional analysis of the new c.-6C>A variant and the upstream 5'-UTR region of KCNC3 by luciferase assays, quantitative PCR and methylation analysis shows increased protein expression but no effect on transcription rate. Therefore, increased translation initiation of KCNC3 transcripts compared to wild-type Kozak sequence seems to be the cause of the increased expression. Variants in the regulatory elements of disease-causing genes probably play an underestimated role.

Bacterial expression, purification, and characterization of human cytochrome P450 3A4 without N-terminal modifications

In this communication we reported a bacterial system that over-expressed full-length wild-type (WT) human CYP3A4 in Escherichia coli (E. coli) at a level of 495 nmol/L culture. This level of expression was achieved by cloning the cDNA sequence of CYP3A4 WT to a pLW01-P450 vector and co-expressing it with chaperones GroEL/ES in bacterial C41(DE3) cells. Aided with a C-terminal His5-tag, the expressed CYP3A4 WT was purified to homogeneity with a specific content of 14.3 ± 2.0 nmole P450/mg protein using a single Ni-Penta agarose column. Like the N-terminal modified form (CYP3A4-NF14), CYP3A4 WT binds substrate testosterone with a typical sigmoidal feature at slightly higher affinity. Functional characterization revealed that CYP3A4 WT exhibited lower testosterone 6β-hydroxylase activities than CYP3A4-NF14 in reconstituted phospholipid systems. In addition, it was found that the 6β-hydroxylase activity of CYP3A4 WT was less dependent on excess cytochrome P450 oxidoreductase (POR), compared with CYP3A4-NF14. These results suggest that the N-terminal membrane anchor of CYP3A4 WT enhances its interactions with POR and marginally increases testosterone binding.

High-Resolution Melting Assay to Detect the Mutations That Cause the Y132F and G458S Substitutions at the ERG11 Gene Involved in Azole Resistance in Candida parapsilosis.

Candida parapsilosis is a pathogenic yeast that has reduced susceptibility to echinocandins and ranks as the second or third leading cause of candidaemia, depending on the geographical region. This yeast often causes nosocomial infections, which are frequently detected as outbreaks. In recent years, resistance to azoles in C. parapsilosis has increased globally, primarily due to the accumulation of mutations in the ERG11 gene. In this study, we have developed an assay based on real-time PCR and high-resolution melting (HRM) curve analysis to detect two of the most prevalent mutations at ERG11 that confer resistance to fluconazole (Y132F and G458S). We designed allele-specific oligonucleotides that selectively bind to either the wild type or mutated sequences and optimised the conditions to ensure amplification of the specific allele, followed by detection via high-resolution melting (HRM) analysis. The designed oligonucleotides to detect the Erg11Y132F and Erg11G458S mutations produced specific amplification of either WT or mutated alleles. We conducted a duplex real-time PCR combining oligonucleotides for the wild-type sequences in one mix, and oligonucleotides for the mutated alleles in another. Following this, we performed an analysis of the HRM curve to identify the amplified allele in each case. This technique was blindly evaluated on a set of 114 C. parapsilosis isolates, all of which were unequivocally identified using our approach. This technique offers a new method for the early detection of azole resistance mechanism in C. parapsilosis.

Precise template-independent correction of c.1278insTATC frameshift mutation in Tay-Sachs disease restores full gene function, while gene reframing yields negligible recovery

Tay-Sachs disease is a fatal neurodegenerative disorder caused by HEXA mutations inactivating the metabolic enzyme HexA. The most common mutation is c.1278insTATC, a tandem 4 base pair (bp) duplication disrupting HEXA expression by frameshift. In an engineered cell model, we explore the use of CRISPR/Cas9 for therapeutic editing of c.1278insTATC. Within genomic microduplications, the microhomology-mediated end joining (MMEJ) pathway is favoured to repair double-stranded breaks with collateral deletion of one repeat. Protospacer adjacent motif (PAM) constraints on Cas9 endonuclease activity prevented cleavage at the duplication centre – the optimal position for MMEJ initiation. Rather, cleavage 1 bp from the c.1278insTATC duplication centre spontaneously reconstructed the wildtype sequence at ∼14.7% frequency, with concomitant restoration of normal cellular HexA activity. As an alternative to perfect correction, short insertions/deletions were serially introduced to restore an open reading frame across a 19 bp sequence encompassing c.1278insTATC. Frame-restored variants did not recover significant HexA function, presumably due to structural incompatibility of incurred amino acid insertions. Hence, precise correction of c.1278insTATC is the only therapeutically relevant outcome achieved in this study, with MMEJ highlighted as a potential template-free CRISPR/Cas9 modality to that end.

Distinct Effects of SARS-CoV-2 Protein Segments on Structural Stability, Amyloidogenic Potential, and α-Synuclein Aggregation.

Amyloidosis is characterized by the abnormal accumulation of misfolded proteins, called amyloid fibrils, leading to diverse clinical manifestations. Recent studies on the amyloidogenesis of SARS-CoV-2 protein segments have raised concerns on their potential link to post-infection neurodegeneration, however, the mechanisms remain unclear. Herein, we investigated the structure, stability, and amyloidogenic propensity of a nine-residue segment (SK9) of the SARS-CoV-2 envelope protein and their impact on neuronal protein α-synuclein (αSyn) aggregation. Specifically, the amino acid sequence of the SK9 wildtype has been modified from a basic and positively charged peptide (SFYVYSRVK), to a nearly neutral and more hydrophobic peptide (SAAVASAVK, labelled as SK9 var1), and to an acidic and negatively charged peptide (SDAVANAVK, labelled as SK9 var2). Our findings reveal that the SK9 wildtype exhibited a pronounced amyloidogenic propensity due to its disordered and unstable nature, while the SK9 variants possessed more ordered and stable structures preventing the amyloid formation. Significantly, the SK9 wildtype demonstrated distinct effect on αSyn aggregation kinetics and aggregate morphology to facilitate the formation of αSyn aggregates with enhanced resistance against enzymatic degradation. This study highlights the potential of modifying short peptide sequences to fine-tune their properties, providing insights into understanding and regulating viral-induced amyloid aggregations.

Cryo-EM structure of a natural prion: chronic wasting disease fibrils from deer

Chronic wasting disease (CWD) is a widely distributed prion disease of cervids with implications for wildlife conservation and also for human and livestock health. The structures of infectious prions that cause CWD and other natural prion diseases of mammalian hosts have been poorly understood. Here we report a 2.8 Å resolution cryogenic electron microscopy-based structure of CWD prion fibrils from the brain of a naturally infected white-tailed deer expressing the most common wild-type PrP sequence. Like recently solved rodent-adapted scrapie prion fibrils, our atomic model of CWD fibrils contains single stacks of PrP molecules forming parallel in-register intermolecular β-sheets and intervening loops comprising major N- and C-terminal lobes within the fibril cross-section. However, CWD fibrils from a natural cervid host differ markedly from the rodent structures in many other features, including a ~ 180° twist in the relative orientation of the lobes. This CWD structure suggests mechanisms underlying the apparent CWD transmission barrier to humans and should facilitate more rational approaches to the development of CWD vaccines and therapeutics.

MSH6-proficient crypt foci in MSH6 constitutional mismatch repair deficiency: reversion of a frameshifted coding microsatellite to its wild-type sequence.

The discovery of "mismatch repair deficient (MMRd)-crypt foci" in non-neoplastic intestinal mucosa in Lynch syndrome (LS) has significantly enhanced our understanding of how tumors and tumor immunity form and evolve in LS. In this study, we report the frequent presence of "mismatch repair proficient (MMRp)-crypt foci" in both non-neoplastic and neoplastic intestinal mucosa in a patient with constitutional MMR deficiency (CMMRD), who carried a germline MSH6 pathogenic variant (c.3261dupC) in trans with an MSH6 likely pathogenic variant (c.3724_3726del) and whose tissues were otherwise deficient in MMR globally. The MMRp-crypts occurred at a rate of 1.1/100 crypts in non-neoplastic intestinal mucosa and were readily discernible in adenomas > 1cm. Sequencing analysis revealed normalization of the MSH6c.3261dupC variant in MMRp-adenoma crypts, indicating reverse frameshifting of the exon 5 C8 microsatellite. Interestingly but not surprisingly, the MMRp-adenoma crypts remained microsatellite-instability-high (MSI-H), and shared oncogenic APC mutations with the background MMRd-adenoma. Contrasting with MSH6-CMMRD, no PMS2-CMMRD individuals (0/5) harbored MMRp-crypts. In conclusion, our study documents distinct MMRp-crypts in MSH6-CMMRD, a phenomenon in keeping with MSH6 being a frequent target of MSI-H due to its coding microsatellite and suggesting that MSH6-CMMRD can potentially serve as a unique model system to further our understanding of MSH6's role in MSI-H tumor formation and evolution. Our findings also bear diagnostic implications; when using MMR immunohistochemistry as an ancillary tool in detecting CMMRD, awareness of these MMRp crypts can help avoid diagnostic pitfalls.

CRISPR/Cas9-mediated CHS2 mutation provides a new insight into resveratrol biosynthesis by causing a metabolic pathway shift from flavonoids to stilbenoids in Vitis davidii cells.

Resveratrol is an important phytoalexin that adapts to and responds to stressful conditions and plays various roles in health and medical therapies. However, it is only found in a limited number of plant species in low concentrations, which hinders its development and utilization. Chalcone synthase (CHS) and stilbene synthase (STS) catalyze the same substrates to produce flavonoids and resveratrol, respectively. However, it remains unclear how CHS and STS compete in metabolite synthesis. In this study, two CHS2 mutant cell lines (MT1 and MT2) were generated using CRISPR/Cas9 genome editing. These CHS2 mutant cell lines exhibited abundant mutations in CHS2, leading to the premature termination of protein translation and subsequent CHS2 knockout. Amplicon sequencing confirmed comprehensive CHS2 knockout in MT1, whereas the wild-type sequence remained predominant in the MT2 cell line. Transcriptome and RT-qPCR results showed a significant downregulation of genes involved in flavonoid biosynthesis, including CHS2, CHS3, F3H, F3'H, DFR, FLS, LDOX, among others, resulting in decreased flavonoid accumulation, such as anthocyanins, proanthocyanidins, quercetin, and kaempferol. Conversely, STS genes involved in stilbenoid biosynthesis were upregulated competing with the flavonoid pathway. Consequently, there was a marked increase in stilbenoids, including resveratrol, piceatannol, piceid, and pterostilbene, with a 4.1-fold increase in resveratrol and a 5.3-fold increase in piceid (a derivative of resveratrol) observed in CHS2 mutant cell lines. This research demonstrates that CHS2 mutation induces a shift from flavonoid biosynthesis towards stilbenoid biosynthesis, offering new insights into metabolite biosynthesis and regulation, as well as an alternative solution for natural resveratrol production, and a novel breeding approach for eliminating non-target agronomic traits using CRISPR-Cas9.

The use of CRISPR-Cas9 technology for targeted gene therapy in Parkinson's disease focusing on LRRK2 and SNCA genes

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta, leading to the start of motor and non-motor symptoms. These include bradykinesia, resting tremor, rigidity, and postural instability, alongside cognitive impairments, mood disorders, and autonomic dysfunctions. The hallmark of PD is the accumulation of α-synuclein protein aggregates, forming Lewy bodies within the neurons. Given the numerous causes of Parkinson’s, involving genetic, epigenetic, and environmental factors, therapeutic treatments are hard to come by. Traditional pharmacological treatments, such as levodopa and dopamine agonists, primarily offer symptomatic relief and are associated with diminishing efficacy over time. Therefore, there is a need for new therapeutic approaches that target the disease at its roots. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, with the RNA-guided Cas9 endonuclease enables precise, targeted modifications at the genomic level. This technology offers potential for terminating the genetic factors causing parkinsons and aid in developing gene-based therapies. By attempting to regulate gene expression, CRISPR holds promise for addressing the fundamental causes of neurodegeneration in Parkinsons. One of the key genetic targets in PD is the leucine-rich repeat kinase 2 (LRRK2) gene, where specific gain-of- function mutations, such as G2019S, lead to hyperactivation of its kinase activity, contributing to neurotoxicity. CRISPR-Cas9 can be employed to introduce precise double-strand breaks at the mutation site, followed by homology-directed repair (HDR) to restore the wild-type sequence, thereby normalizing LRRK2 function and preventing neuronal death. Similarly, CRISPR technology can target the SNCA gene, which encodes α-synuclein. Pathogenic duplicates or triplicates of SNCA result in overproduction of α-synuclein and subsequent aggregation. Utilizing CRISPR to disrupt these extra copies can reduce α-synuclein levels, alleviating its toxic effects.CRISPR activation (CRISPRa) and interference (CRISPRi) systems enable fine-tuned upregulation or downregulation of gene expression, offering strategies to increase the expression of neuroprotective factors or suppress the expression of deleterious genes.

B-247 A Missing Peptide Link: A Case Study Involving Detection of an Unusual IGF-1 Protein Form Using High-Resolution Mass Spectrometry

Abstract Background Previously, we described a method for monitoring insulin-like growth factor-1 (IGF-1) variants using liquid chromatography coupled with high-resolution, accurate-mass, mass spectrometry (LC-HRAM-MS). The method split s variants in 4 Variant Groups (VG) and characterizes them by the Isotopic Peak index (IPi) and relative retention time (rRT). Uncommon variants are analyzed by tandem mass-spectrometry (MS/MS) and specimens suspected to have a novel variant are deidentified and routed for DNA sequencing. A potentially novel species was detected that did not match characteristics of any previously identified IGF-1 variant. Methods LC-HRAM-MS was used for the detection of wild type (WT) IGF-1 protein and its variants in a serum specimen. Results The patient’s IGF-1 WT concentration was 210 ng/mL and z-score was -1.5, which were within normal reference ranges values for their sex and age. LC-HRAM-MS also revealed the isotopic distribution and charge for the variant, but with new characteristics (VG2:IP2, rRT= 0.01 min). The area under curve for the variant was 36% of that of the WT, which is a stark contrast to the usual 100% found in a typical heterozygous patient. MS/MS of the suspected variant showed 2 sets of b- and y-ions, which has not been seen in our laboratory. Sequencing of the fragments revealed that one set of C- and N-terminal amino acids were from the WT (C-terminus GPETL sequence and N-terminus PLKPAKSA sequence). The intact molecular weight (MW) of the species (monoisotopic m/z 1095.5250 at charge 7+) was 18 Da heavier than that of the WT (monoisotopic m/z 1092.9482 at charge 7+), so further investigation was warranted. The other set of b- and y-ions sequenced were manually identified as GL(or I)VD and PTGYG, respectively. BLAST search did not identify relevant proteins with these N- and C-termini. However, closer inspection revealed that identified sequences were found in the WT protein, albeit not at its termini. The rest of the amino acids were found that complete termini: C-terminal b-ions sequence RAPQTGIVD corresponded to positions 37-45 of the WT sequence, while N-terminal y-ions sequence KPTGYGSSSR corresponded to positions 27-36. All evidence indicates that the species is the WT protein, but with loss of a peptide bond between 2 arginines at positions 36-37 resulting in a gain of a water molecule to “cap” the newly formed termini, which is confirmed by its observed intact m/z. Conclusions We identified an extremely rare IGF-1 species formed through loss of a peptide bond between arginines at positions 36 and 37 resulting in a “missing link” in IGF-1 amino acid sequence. Despite the cleavage, the variant was a single protein molecule with intact disulfide bonds and 2 sets of C- and N-termini. The processes involved in generating this species (e.g., hydrolysis or enzymatic cleavage) and potential consequences for the patient are unknown. Because binding to receptors and binding proteins are potentially affected, and the total level of active WT IGF-1 is reduced, we are working with patient’s physician to establish whether this unusual form of IGF-1 protein may contribute to the etiology of the patient’s condition.

A novel platform for mutation detection in colorectal cancer using a PNA-LNA molecular switch

Detection of KRAS mutation in colorectal cancer (CRC) is important in the prediction of response to target therapy. The study aims to develop a novel mutation detection platform called the “PNA-LNA molecular switch” for the detection of KRAS mutation in CRC. We employed the enhanced binding specificity of peptide nucleic acid (PNA) and locked nucleic acid (LNA) in conjunction with a loop-mediated isothermal amplification (LAMP) approach to identify the mutation status of KRAS oncogene codon 12 (c.35G>T/G12V and c.35G>A/G12D) using synthetic oligonucleotides and colon cancer cell lines (Caco-2 and SW480). This method specifically blocked the amplification of the wild-type sequences while substantially amplifying the mutated ones, which was visualized by both colorimetric and fluorescence assays. We then checked the mutation profile of KRAS codon 12 in the DNA derived from tumor tissue samples (number of samples, n = 30) and circulating tumor cells (n = 24) from CRC patients. Finally, we validated the results by comparing them with the data obtained from DNA sequencing of colon tumors (n = 21) of the same CRC patients. This method showed excellent sensitivity (1 DNA copy/μl), reproducibility [relative standard deviation (%RSD) < 5%, for n = 3], and linear dynamic range (1 ag/μl-10 pg/μl, R2 = 0.94). This platform is significantly faster, relatively cheaper, has superior sensitivity and specificity, and does not require any high-end equipment. To conclude, this method has the potential to be translated into clinical settings for the detection of mutations in diverse diseases and conditions.

Whole Genome Sequences of the Wildtype AU-1 Rotavirus A Strain: The Prototype of the AU-1-like Genotype Constellation.

Most human rotaviruses belong to the Wa-like, DS-1-like, or AU-1-like genotype constellation. The AU-1-like constellation, albeit minor, captured attention because its prototype strain AU-1 originated from feline rotavirus, leading to the concept of interspecies transmission of rotavirus. The AU-1 genome sequence determined by various laboratories over the years has documented two conflicting VP7 sequences in the GenBank. As culture-adaptation may introduce changes in the viral genome, the original fecal (wild-type) and the seed stock of culture-adapted AU-1 genomes were sequenced using the Illumina's MiSeq platform to determine the authentic AU-1 sequence and to identify what mutational changes were selected during cell-culture adaptation. The wild-type and culture-adapted AU-1 genomes were identical except for one VP4-P475L substitution. Their VP7 gene was 99.9% identical to the previously reported AU-1 VP7 under accession number AB792641 but only 92.5% to that under accession number D86271. Thus, the wild-type sequences determined in this study (accession numbers OR727616-OR727626) should be used as the reference. The VP4-P475L mutation was more likely incidental than inevitable during cell-culture adaptation. This was the first study in which the whole genomes of both wild-type and cultured RVA strains were simultaneously determined by deep sequencing.

Somatic reversion in Wiskott-Aldrich syndrome: Case reports and mechanistic insights.

This report describes two brothers from India and a Chinese patient with somatic reversion of an inherited deleterious mutation in the WAS gene. Both the Indian siblings had inherited a single nucleotide deletion causing a frameshift mutation (c.1190del, p.Pro397Argfs*48) (variant 1: marked in blue) from the mother. Another variant (variant 2: marked in red), a 12-nucleotide deletion at position 1188-1199 (c.1188_1199del, p.P401_P404del) was also found, which resulted in restoration of the frame and subsequent rescue of the protein sequence. DNA sequencing from buccal mucosal cells revealed only the inherited variant (variant 1), while no reversion mutation was identified in the mucosal cells. Similarly, the Chinese patient was found to have a novel germline 14-base duplication (ACGAAAATGCTTGG) c.120_132 + 1dup (variant 1). This resulted in abolishment of the original splice junction coupled with the creation of a new junction 14 bases 3' and a frameshift mutation with predicted protein truncation p. Thr45Aspfs*. DNA from the patient's PBMC showed co-existence of wild-type and mutated sequences, but only the mutant was present in the buccal cells. Genomic and mRNA analysis of the isolated CD3+ T lymphocytes, CD3- mononuclear cells, and EBV-transformed B lymphocytes indicated that the reverant variant (germline variant was restored to wild-type sequence) were selectively found in CD3+ T lymphocytes.