MicroRNAs (miRNAs) are single-stranded non-coding short ribonucleic acid sequences that take part in many cellular and biological processes. Recent studies have shown that altered expression of miRNAs is involved in pathological processes, and they can thus be considered biomarkers for the early detection of various diseases. Here, we demonstrate a selection and elimination process of fluorescent single-walled carbon nanotube (SWCNT) sensors for miRNA biomarkers based on RNA-DNA hybridization with a complementary DNA recognition unit bound to the SWCNT surface. We use known miRNA biomarkers for acute myocardial infarction (AMI), commonly known as a heart attack, as a case study. We have selected five possible miRNA biomarkers which are selective and specific to AMI and tested DNA-SWCNT sensor candidates with the target DNA and RNA sequences in different environments. Out of these five miRNA sensors, three could recognize the complementary DNA or RNA sequence in a buffer, showing fluorescence modulation of the SWCNT in response to the target sequence. Out of the three working sensors in buffer, only one could function in serum and was selected for further testing. The chosen sensor, SWCNT-miDNA208a, showed high specificity and selectivity toward the target sequence, with better performance in serum compared to a buffer environment. The SWCNT sensor selection pipeline highlights the importance of testing sensor candidates in the appropriate environment and can be extended to other libraries of biomarkers.

OBJECTIVE: To explore the genetic etiology of a patient with epilepsy and provide genetic counseling.
METHODS: A patient who had visited the Center for Reproductive Medicine of Shandong University on November 11, 2020 was selected as the study subject, and her clinic information was collected. Candidate variant was identified through whole exome sequencing (WES), and Sanger sequencing was used for validation. Possible transcriptional changes caused by the variant was detected by reverse transcription-PCR and Sanger sequencing.
RESULTS: The patient was a 35-year-old female with no fever at the onset, loss of consciousness and abnormal firing in the temporal lobe, manifesting predominantly as convulsions and fainting. WES revealed that she had harbored a heterozygous c.2841+5G>A variant of the SCN9A gene, which was verified by Sanger sequencing. cDNA sequencing confirmed that 154 bases were inserted between exons 16 and 17 of the SCN9A gene, which probably produced a truncated protein and affected the normal function of the SCN9A protein. Based on the guidelines from the American College of Medical Genetics and Genomics, the c.2841+5G>A variant was classified as likely pathogenic (PVS1_Strong+PM2_Supporting).
CONCLUSIONS: The c.2841+5G>A variant of the SCN9A gene probably underlay the epilepsy in this patient. Above finding has enriched the variant spectrum of the SCN9A gene and provided a basis for the prenatal diagnosis and preimplantation genetic testing for this patient.

BACKGROUND:  Biallelic pathogenic variants in AIMP1 gene cause hypomyelinating leukodystrophy type 3, a severe neurodegenerative disorder with early onset characterized by microcephaly, axial hypotonia, epilepsy, spasticity, and developmental delay.
METHODS:  Clinical exome sequence was performed on patient\'s DNA and Sanger sequencing was used to confirm the candidate variant. To better characterize the effect of the genetic variant, functional analysis based on Sanger sequencing of the proband\'s complementary DNA (cDNA) was performed.
RESULTS:  We report a case of 2-year-old girl with microcephaly, significant global developmental delay, refractory epilepsy, flaccid paralysis, hypomyelination, leukodystrophy, and cerebral atrophy on brain magnetic resonance imaging (MRI). Clinical exome sequencing revealed a novel splice site variant c.603 + 1G > A in homozygosity in the AIMP1 gene. Studies on patient\'s cDNA showed that the variant disrupts the canonical donor splice site of intron 5, with the recognition of a cryptic splice site within exon 5, leading to the skipping of the last 24 nucleotides of this exon together with the flanking intron. This alteration is predicted to cause an in-frame deletion of eight amino acids (p.Val194_Gln201del) belonging to the tRNA-biding domain of the protein.
CONCLUSIONS:  To the best of our knowledge, this is the first report of a splice site variant in the AIMP1 gene causing hypomyelinating leukodystrophy. The description of this patient not only expands the mutational spectrum of AIMP1 but also provides deeper insights on genotype-phenotype correlation by comparing the clinical features of our patient with previously reported affected individuals.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most widespread and economically devastating diseases in the swine industry. Typing circulating PRRSV strains by means of sequencing is crucial for developing adequate control strategies. Most genetic studies only target the highly variable open reading frame (ORF) 5, for which an extensive database is available. In this study, we performed whole-genome sequencing (WGS) on a collection of 124 PRRSV-1 positive serum samples that were collected over a 5-year period (2015-2019) in Belgium. Our results show that (nearly) complete PRRSV genomes can be obtained directly from serum samples with a high success rate. Analysis of the coding regions confirmed the exceptionally high genetic diversity, even among Belgian PRRSV-1 strains. To gain more insight into the added value of WGS, we performed phylogenetic cluster analyses on separate ORF datasets as well as on a single, concatenated dataset (CDS) containing all ORFs. A comparison between the CDS and ORF clustering schemes revealed numerous discrepancies. To explain these differences, we performed a large-scale recombination analysis, which allowed us to identify a large number of potential recombination events that were scattered across the genome. As PRRSV does not contain typical recombination hot-spots, typing PRRSV strains based on a single ORF is not recommended. Although the typing accuracy can be improved by including multiple regions, our results show that the full genetic diversity among PRRSV strains can only be captured by analysing (nearly) complete genomes. Finally, we also identified several vaccine-derived recombinant strains, which once more raises the question of the safety of these vaccines.

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BACKGROUND: Juvenile neuronal ceroid lipofuscinosis (CLN3 disease) is a hereditary progressive neurodegenerative disease well documented among Caucasians, but such clinical data and genetic characterization is lacking among Asian populations.
METHODS: A 13-year-old Chinese girl presented for diagnostic evaluation with retinitis pigmentosa, generalised tonic-clonic seizure and cerebellar ataxia. Electron microscopy of whole blood and skin biopsy, and mutation analysis of CLN3 gene with genomic DNA and cDNA, were performed.
RESULTS: Electron microscopy showed vacuolated lymphocytes, and characteristic patterns in eccrine glands suggestive of neuronal ceroid lipofuscinosis. Sequencing of genomic DNA showed homozygous splice site variant NM_000086.2(CLN3):c.906+6T>G, and the pathogenicity of which was confirmed by cDNA sequencing to demonstrate the deletion of a transmembrane domain of the CLN3 protein. The mutant protein was predicted to adversely affect ligand binding of CLN3 as a lysosomal membrane protein.
CONCLUSIONS: Here we report the first genetically confirmed CLN3 disease in Chinese, with a novel splice site variant with proposed pathogenetic mechanism relating gene and protein, and highlights the potential ethnic differences in the mutation spectrum. We wish to establish the importance of clinical awareness and laboratory diagnosis of CLN3 disease, especially in the promising age of gene therapy.

BACKGROUND: Primary malignant melanoma of the vagina is extremely rare, with a poorer prognosis than cutaneous malignant melanoma. Previous studies have explored the repurposing of itraconazole, a common oral anti-fungal agent, for the treatment of various cancers. Here, we describe a patient with metastatic, unresectable vaginal malignant melanoma treated with 200 mg oral itraconazole twice a day in a clinical window-of-opportunity trial.
METHODS: A 64-year-old Japanese woman with vaginal and inguinal tumours was referred to our institution. On the basis of an initial diagnosis of vaginal cancer metastatic to the inguinal lymph nodes, we treated her with itraconazole in a clinical trial until the biopsy and imaging study results were obtained. During this period, biopsies were performed three times, and 18F-fluoro-deoxyglucose positron emission tomography (FDG/PET)-computed tomography (CT) was performed twice. Biopsy results confirmed the diagnosis of primary malignant melanoma of the vagina. Imaging studies revealed metastases to multiple sites, including the brain, for which she underwent gamma-knife radiosurgery. During the window period before nivolumab initiation, the patient received itraconazole for 30 days. Within a week of itraconazole initiation, pain in the inguinal nodes was ameliorated. PET-CT on days 6 and 30 showed a reduction in tumour size and FDG uptake, respectively. The biopsied specimens obtained on days 1, 13, and 30 were subjected to cDNA microarray analysis, which revealed a 100-fold downregulation in the transcription of four genes: STATH, EEF1A2, TTR, and CDH2. After 12 weeks of nivolumab administration, she developed progressive disease and grade 3 immune-related hepatitis. Discontinuation of nivolumab resulted in the occurrence of left pelvic and inguinal pain. Following re-challenge with itraconazole, the patient has not reported any pain for 4 months.
CONCLUSIONS: The findings of this case suggest that itraconazole is a potential effective treatment option for primary malignant melanoma of the vagina. Moreover, we identified potential itraconazole target genes, which could help elucidate the mechanism underlying this disease and potentially aid in the development of new therapeutic agents.

The new environmentally responsive fluorescent nucleosides, 3,7-bis-(naphthalen-1-ylethynyl)-8-aza-3,7-dideaza-2\'-deoxyadenosine (3n7nzA, 1) and 7-(naphthalen-1-ylethynyl)-8-aza-3,7-dideaza-2\'-deoxyadenosine (37nzA, 2), have been synthesized. Both 3n7nzA (1) and 37nzA (2) possess large π-conjugated systems which extend into both the minor and major grooves or the major groove alone, respectively. The nucleosides exhibited large solvatochromic shifts (3n7nzA: Δλ = 45 nm, 37nzA: Δλ = 78 nm) and were examined for their ability to fluorimetrically report hybridization events. When incorporated into ODN probes, the bis-substituted 3n7nzA (1) selectively recognized thymidine on target strands which was reported by a distinct change in its emission wavelength in the long wavelength region, whereas 37nzA (2) showed a preference for pairing to cytidine and a smaller wavelength shift. Thus, 3n7nzA (1) has the potential for use as a fluorescent probe for structural studies of DNAs/RNAs including the detection of single-base alterations in target DNA sequences.

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Ascertaining the clinical consequences of BRCA1 and BRCA2 variants of uncertain significance (VUS) is currently indispensable for providing effective genetic counseling and preventive actions for families with hereditary breast and ovarian cancer (HBOC). To this end, we conducted a combination of in silico prediction and cDNA splicing analyses of 13 BRCA1 and 10 BRCA2 VUS identified in our cohort as well as a case-control analysis in a population-based sample of 10 recurrent VUS. We observed consistent results between the in silico predictions and sequencing analyses for all analyzed VUS. An abnormal cDNA pattern was observed for variants c.212+1G>A and c.5278-1G>A in BRCA1 and c.516+2T>A and c.8168A>G in BRCA2 according to in silico splicing prediction. A case-control study of VUS confirmed the polymorphisms of the c.67+62A>G, c.7008-62A>G and c.8851G>A BRCA2 variants previously published. c.4068G>A in the BRCA2 gene can also be considered a polymorphism due to its occurrence at a frequency greater than 1% in our population. Our study shows that employing population-based analysis and a combination of several in silico methods yields highly accurate information, resulting in a reliable tool for selecting variants for cDNA sequencing analysis in routine cancer genetic counseling units.