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PubMed1991

Use of 33P for Sanger DNA sequencing.

The use of [alpha-33P]deoxyadenosine 5'-triphosphate ([alpha-33P]dATP) in DNA sequencing has been described. 33P has a maximum beta-emission energy that is 50% stronger than 35S, but fivefold weaker than 32P. As a result, sequences generated using [alpha-33P]dATP have short exposure times like 32P, yet they maintain band resolution similar to 35S. Handling of [alpha-33P]dATP is straightforward because no special lead or Plexiglas shielding is necessary.

Base Sequence↗

PubMed1988

A rapid droplet method for Sanger dideoxy sequencing.

A method for performing the dideoxy sequence reaction on petri dishes is described. It allows rapid manipulation of clones and provides large amounts of sequence information quickly and without the need for elaborate laboratory equipment.

Base Sequence↗

PubMed2004

Unlocking hidden genomic sequence.

Despite the success of conventional Sanger sequencing, significant regions of many genomes still present major obstacles to sequencing. Here we propose a novel approach with the potential to alleviate a wide range of sequencing difficulties. The technique involves extracting target DNA sequence from variants generated by introduction of random mutations. The introduction of mutations does not destroy original sequence information, but distributes it amongst multiple variants. Some of these variants lack problematic features of the target and are more amenable to conventional sequencing. The technique has been successfully demonstrated with mutation levels up to an average 18% base substitution and has been used to read previously intractable poly(A), AT-rich and GC-rich motifs.

AT Rich Sequence↗

PubMed2026

Compound Heterozygous PCDH15 Variants Associated With Cone-Rod Dystrophy in a Chinese Pedigree.

BACKGROUND: This study aimed to characterize the clinical and genetic features of a Chinese family with cone-rod dystrophy in which compound heterozygous PCDH15 variants were identified. METHODS: A Chinese pedigree with autosomal recessive cone-rod dystrophy was investigated. A comprehensive ophthalmic assessment was performed in the proband, a 42-year-old woman, together with genetic evaluation of her family members. Candidate variants were identified using whole-exome sequencing and subsequently assessed by Sanger sequencing and family segregation analysis. RESULTS: Ophthalmoscopic examination revealed pigmentary changes and atrophic lesions affecting the posterior pole and peripapillary area bilaterally. Optical coherence tomography (OCT) demonstrated bilateral outer retinal layer atrophy with disruption of the ellipsoid zone at the posterior pole. Multifocal electroretinography (mfERG) revealed attenuated central responses, while full-field electroretinography (ffERG) documented a more pronounced reduction in cone-mediated (photopic) responses. Two novel compound heterozygous variants in PCDH15, namely c.4903_4906del (p.Glu1635Lysfs*4) and c.3470C>A (p.Ala1157Glu), were identified in this autosomal recessive cone-rod dystrophy pedigree. Family co-segregation analysis provided supportive evidence for their potential association with the disease phenotype. Cross-species analysis revealed high evolutionary conservation of the PCDH15 protein. Three-dimensional structural modeling predicted potential alterations in protein structure. CONCLUSION: To our knowledge, this is the first report describing an association between compound heterozygous PCDH15 variants and cone-rod dystrophy, thereby providing preliminary evidence that may broaden the mutational spectrum associated with this gene.

Adult↗

PubMed2026

Novel avian calicivirus genome with type IV internal ribosomal entry site (IRES) in black-headed gull (Chroicocephalus ridibundus) in Hungary.

In this study, a taxonomically novel avian calicivirus detected and characterized by next generation sequencing, RT-PCR and Sanger sequencing methods in faecal specimen collected from black-headed gull (Chroicocephalus ridibundus) in Hungary. The complete genome length of the calicivirus strain gull/HA15097/HUN/2018 (PZ810127) is remarkably long, 8,845 nucleotides, which had type IV internal ribosomal entry site (IRES) at the 5', and a stem-loop-II-like (s2m) sequence motif at the 3' untranslated regions. The VP1 capsid protein had less than 26% aa identity to the members of the known calicivirus genera. Caliciviruses appear to be widespread not only in mammals including humans but also in various bird species.

Animals↗

PubMed2025

Identification of four novel ACADVL variants in eight Chinese unrelated patients with very long-chain acyl-CoA dehydrogenase deficiency.

BACKGROUND: Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is a disorder of mitochondrial fatty acid oxidation with an autosomal recessive manner and is due to the VLCAD enzyme deficiency which is encoded by the ACADVL gene. The purpose of this study was to elucidate the clinical manifestations and analyze the molecular findings of eight Chinese patients with VLCADD. METHODS: We investigated eight Chinese VLCADD patients (three males, five females) from eight unrelated families. Molecular analysis was performed under the application of next-generation sequencing (NGS) in combination with Sanger sequencing validation to confirm the likely pathogenic variants in these patients. RESULTS: Patient 1 (P1) exhibited the most severe clinical features and passed away 3 h after admission on the second day of life. Unfortunately, P2 succumbed to hypoketotic hypoglycemia at 5 months of age. Except for asymptomatic P7, the remaining patients also developed clinical presentations of varying severity at different ages. Molecular data revealed that all affected individuals were compound heterozygotes for ACADVL variants. A total of 14 variants (4 novel and 10 known) were identified and the pathogenicity was evaluated based on the American College of Medical Genetics and Genomics (ACMG) criteria and different in silico prediction tools. CONCLUSIONS: The analysis of genotype-phenotype relationships preliminarily suggests that the compound heterozygous variants identified in these patients are likely the primary cause of VLCADD. Our study expands the mutation spectrum of ACADVL and highlights the significance of genetic analysis in early diagnosis and therapeutic intervention of monogenic hereditary diseases especially those with rapid disease progression.

Female↗

PubMed2026

Clinical and genetic analysis of a family with 16p11.2 microduplication syndrome and variable multisystem manifestations.

16p11.2 microduplication syndrome (OMIM #614671) is a pathogenic recurrent copy-number gain at the 16p11.2 locus and is associated with variable expressivity across neurodevelopmental, growth, and medical phenotypes. Gastrointestinal symptoms have been reported in carrier cohorts, but detailed documentation of gastrointestinal motility and neuromuscular findings remains limited. We performed clinical and genetic analyses in a multigenerational family in which the proband (III1) presented with limb muscle pain, exercise intolerance, and chronic gastrointestinal symptoms. Next-generation sequencing (NGS), low-pass whole-genome sequencing (lpWGS)-based CNV analysis, Sanger sequencing, and qPCR validation identified a 0.8 Mb microduplication at 16p11.2 (BP4-BP5), involving 44 genes including TBX6, inherited from the mother (II2). The proband's clinical manifestations included developmental delay, pointed chin, low body mass index, gastrointestinal dysfunction (chronic abdominal pain, diarrhea, esophageal motility disorder, and rectal prolapse), forward-leaning gait, mild scoliosis, and limb muscle atrophy with inflammatory muscle involvement. Four family members (II2, III1, III2, and III4) carried the microduplication, but their available clinical features varied in severity and system involvement. The proband's twin brother (III2) had left ear deafness and epilepsy, individual II2 had blindness from cone-rod dystrophy, and III4 showed more pronounced scoliosis. This family provides a detailed clinical and genetic description of 16p11.2 microduplication carriers with prominent gastrointestinal motility and neuromuscular manifestations, thereby enriching the clinical characterization of this recurrent CNV and supporting substantial intrafamilial phenotypic heterogeneity.

16p11.2 microduplication syndrome↗

PubMed2001

DNA sequencing using biotinylated dideoxynucleotides and mass spectrometry.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) has been explored widely for DNA sequencing. The major requirement for this method is that the DNA sequencing fragments must be free from alkaline and alkaline earth salts as well as other contaminants for accurately measuring the masses of the DNA fragments. We report here the development of a novel MS DNA sequencing method that generates Sanger-sequencing fragments in one tube using biotinylated dideoxynucleotides. The DNA sequencing fragments that carry a biotin at the 3'-end are made free from salts and other components in the sequencing reaction by capture with streptavidin-coated magnetic beads. Only correctly terminated biotinylated DNA fragments are subsequently released and loaded onto a mass spectrometer to obtain accurate DNA sequencing data. Compared with gel electrophoresis-based sequencing systems, MS produces a very high resolution of DNA-sequencing fragments, fast separation on microsecond time scales, and completely eliminates the compressions associated with gel electrophoresis. The high resolution of MS allows accurate mutation and heterozygote detection. This optimized solid-phase DNA-sequencing chemistry plus future improvements in detector sensitivity for large DNA fragments in MS instrumentation will further improve MS for DNA sequencing.

Base Sequence↗

PubMed2025

Identifying novel heterozygous PI4KA variants in fetal abnormalities.

BACKGROUND: The clinical manifestations of PI4KA-related disorders are characterized by considerable variability, predominantly featuring neurological impairments, gastrointestinal symptoms, and a combined immunodeficiency. The aim of this study was to delineate the novel spectrum of PI4KA variants detected prenatally and to assess their influence on fetal development. METHODS: A thorough fetal ultrasound screening was conducted, supplemented by both antenatal and post-abortion magnetic resonance imaging (MRI) studies. Novel PI4KA variants were detected through clinical Whole exon sequencing (WES) and validated by Sanger sequencing. The functional consequences of these variants were evaluated using bioinformatics tools. The effects of the identified variants on splicing were analyzed through minigene splicing assays. Subsequently, both wild-type and mutant PI4KA protein fragments were purified, and their enzymatic activities were quantitatively assessed. RESULTS: Ultrasound imaging, MRI scans revealed a dilated small intestine with an obstruction. Compound heterozygous variants (NM_058004.3: c.2802_2863-40del and c.2819 C > T, p.Ala940Val) were identified in the PI4KA of the affected fetus through clinical trio-WES. Both variants were predicted deleterious. The PI4KA variant c.2802_2863-40del resulted in the production of three distinct mRNA isoforms. The PI4KA variant c.2819 C > T (p.Ala940Val) significantly reduced the enzyme activity. CONCLUSIONS: This study extended the mutational spectrum of PI4KA and may provide guidance for genetic counseling. Functional studies confirmed that the identified variant induces alterations in RNA splicing and impairs enzyme activity.

Adult↗

PubMed2002

Mitochondrial sequence analysis for forensic identification using pyrosequencing technology.

Over recent years, requests for mtDNA analysis in the field of forensic medicine have notably increased, and the results of such analyses have proved to be very useful in forensic cases where nuclear DNA analysis cannot be performed. Traditionally, mtDNA has been analyzed by DNA sequencing of the two hypervariable regions, HVI and HVII, in the D-loop. DNA sequence analysis using the conventional Sanger sequencing is very robust but time consuming and labor intensive. By contrast, mtDNA analysis based on the pyrosequencing technology provides fast and accurate results from the human mtDNA present in many types of evidence materials in forensic casework. The assay has been developed to determine polymorphic sites in the mitochondrial D-loop as well as the coding region to further increase the discrimination power of mtDNA analysis. The pyrosequencing technology for analysis of mtDNA polymorphisms has been tested with regard to sensitivity, reproducibility, and success rate when applied to control samples and actual casework materials. The results show that the method is very accurate and sensitive; the results are easily interpreted and provide a high success rate on casework samples. The panel of pyrosequencing reactions for the mtDNA polymorphisms were chosen to result in an optimal discrimination power in relation to the number of bases determined.

Base Sequence↗

PubMed2026

A Novel SLC25A4 Variant Causing Mitochondrial Dysfunction, Myopathy and Cardiomyopathy: A Functional and Molecular Characterization.

SLC25A4, solute carrier family 25 member 4, gene is a member of the mitochondrial carrier subfamily within the solute carrier protein family. Pathogenic variants in SLC25A4 are associated with a spectrum of mitochondrial disorders that exhibit variable inheritance patterns and clinical manifestations. Specifically, dominantly inherited variants are typically associated with progressive external ophthalmoplegia with mitochondrial DNA deletions, recessively inherited variants are linked to myopathy and cardiomyopathy, and de novo variants can result in early-onset fatal disease presentations. In this study, we aimed to identify and characterize the disease-causing mutation(s) in a nine-year-old female patient from a consanguineous Saudi family. The patient was asymptomatic until the age of 3 years, when she presented with cardiomyopathy and myopathy. Comprehensive genetic analysis inclusive of whole exome sequencing and segregation analysis using Sanger sequencing identified an SLC25A4 variant (NM_001151.4: exon 2: c.112-1G>C) as the most likely cause of the disease. To assess transcript-level effects, we performed RT-PCR on RNA extracted from the patient's cultured lymphoblast cell lines (LCLs) and fibroblast cell lines (FCLs). RT-PCR analysis demonstrated that the variant causes aberrant splicing, resulting in a 6 bp in-frame deletion (p.Gln37_Val38del) in the ANT1 protein. Quantitative RT-PCR demonstrated reduced SLC25A4 transcript levels in both FCLs and LCLs. Quantitative PCR analysis of mitochondrial DNA demonstrated a trend toward increased mtDNA copy number in patient-derived FCLs compared with controls, suggesting a possible compensatory response to mitochondrial dysfunction. Furthermore, Seahorse assays revealed marked reductions in both oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in patient-derived FCLs compared with controls. These findings expand the molecular and functional spectrum of SLC25A4-associated disease and may inform clinical practice, including genetic interventions such as preimplantation genetic diagnosis, premarital genetic screening, targeted genetic counseling, and cascade testing of at-risk family members.

Humans↗

PubMed2026

A Case Report of a Pedigree with Distal Hereditary Motor Neuropathy Caused by a Homozygous c.1124G>A Variant an the /*9Vaccinia-Related Kinase 1 Gene.

This study aimed to analyze the clinical phenotypes, neurophysiological characteristics, and pathogenicity of gene variants in a pedigree with distal hereditary motor neuropathy (dHMN) caused by VRK1 variants, and to provide evidence to support clinical diagnosis and genetic counseling for this disease. We report a Chinese consanguineous family with dHMN caused by a homozygous c.1124G>A variant in the VRK1 gene. Clinical and electrophysiological data of the proband were collected. Whole-exome sequencing (WES) and validation by Sanger sequencing were performed to identify the variant site, and pathogenicity interpretation was conducted in accordance with American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines. The proband was a 24-year-old male who presented with 2 years of progressive weakness and atrophy of the distal lower limbs, accompanied by slender upper limbs and no sensory disturbance. Electrophysiological examination showed decreased compound muscle action potential (CMAP) amplitude in motor nerves of both upper and lower limbs, indicating peripheral neurogenic damage, while sensory nerve conduction was normal. Genetic testing detected a homozygous c.1124G>A (p.Trp375Ter) variant in the VRK1 gene. His parents and elder sisters were heterozygous carriers, and the pedigree conformed to autosomal recessive inheritance. According to ACMG guidelines, this variant was classified as pathogenic (evidence: PVS1, PM2, PP1). The homozygous VRK1 c.1124G>A variant causes adult-onset dHMN, rather than pontocerebellar hypoplasia type 1A (PCH1A) as annotated in some genetic databases. This pedigree presents distinctive phenotypes, including slender upper limbs and diffusely decreased CMAP amplitudes in both upper and lower limbs, thereby expanding the clinical and genetic spectrum of VRK1-related dHMN in the Chinese population.

Humans↗

PubMed1996

Affinity methods for purification of DNA sequencing reaction products for mass spectrometric analysis.

Time-of-flight mass spectrometry has the potential to replace gel electrophoresis for rapid and accurate analysis of DNA sequencing mixtures. However, impurities in the Sanger sequencing reaction solutions can complicate and degrade the mass spectrometric performance. Therefore, a fast purification procedure is necessary for mass spectrometric analysis. Two affinity methods were tested for the capture of the target fragments: a probe strand, complementary to the primer used to initiate synthesis of the Sanger fragments, is immobilized to a solid support either before or after hybridization so that the impurities are readily separated by filtering and washing the support material. The approaches were tested using mock sequencing mixtures assembled from synthetic DNA strands, to which representative impurities could be added. The results showed that the latter method has better overall yield. The recovered fragments were analyzed by matrix-assisted laser desorption/ionization.

Bacterial Proteins↗

PubMed2026

Meropenem-Colistin Combination Mitigates Porin-Associated Carbapenem Resistance Development in Ertapenem-Mono-Resistant Enterobacterales.

BACKGROUND: Non-carbapenemase-producing Enterobacterales with isolated ertapenem resistance (ETP-mono-R) may represent an early stage in the evolution toward broader carbapenem resistance, but whether further resistance induction occurs and its underlying mechanisms remain poorly understood. METHODS: Resistance induction was assessed in three Escherichia coli, four Klebsiella pneumoniae, and two Enterobacter cloacae isolates through serial exposure to subinhibitory concentrations of meropenem (MEM), imipenem, ceftazidime-avibactam, or colistin (COL), with antibiotic-free passaging for reversion. Resistance induction under MEM+COL was evaluated separately. Whole-genome sequencing (WGS), targeted porin-gene Sanger sequencing, and transcriptional analysis were used to characterize resistance mechanisms across induction stages. RESULTS: Subinhibitory MEM exposure rapidly selected for carbapenem resistance through porin-associated alterations in a species-specific manner. E. coli accumulated loop-region mutations in ompC, while K. pneumoniae predominantly developed disruptive mutations in ompK36, both accompanied by marked transcriptional downregulation. In contrast, E. cloacae retained wild-type porins but showed increased MEM MICs, suggesting a non-porin-mediated mechanism. Subinhibitory exposure to COL alone rapidly induced colistin resistance but was associated with decreased carbapenem MICs. Co-exposure to MEM and COL significantly delayed resistance development and reduced MIC increases (all P < 0.05). Targeted sequencing of 26 non-carbapenemase-producing K. pneumoniae isolates resistant to all carbapenems revealed widespread disruptive ompK36 alterations, including the S337P substitution identified experimentally, consistent with a shared permeability-loss pathway. CONCLUSIONS: In ETP-mono-R Enterobacterales, subinhibitory carbapenem exposure promotes carbapenem resistance, with porin-associated mechanisms predominating in E. coli and K. pneumoniae. Co-exposure to COL attenuates this process, suggesting a potential strategy to delay the emergence of carbapenem resistance.

Enterobacterales↗

PubMed1992

Partial nucleotide sequence of the enzyme pyruvate, orthophosphate dikinase of Entamoeba histolytica HM1:IMSS.

We isolated a clone from a lambda gt11 cDNA library of E. histolytica, subcloned its insert in the vector pTZ18R and sequenced it by the method of Sanger. Sequence comparison with Genbank and of the corresponding amino acid sequence with the NBRF/PIR data bank using the FastA program, showed homologies between 54.9 and 58.5% for the nucleotides and between 48.6 and 49.4% for the amino acids with pyruvate, orthophosphate dikinases from maize, Flaveria trinervia and Bacteroides symbiosus. The sequence obtained for E. histolytica represents about 20% of the complete gene or protein sequence.

Amino Acid Sequence↗

PubMed2025

Peripheral Macular Endothelial Dystrophy: Clinical, Histopathologic, Genetic and Functional Characterization.

OBJECTIVE: To report a CHST6-associated corneal endothelial dystrophy. DESIGN: Prospective observational case series. PARTICIPANTS: Thirty-five individuals from seven families, including 13 affected individuals exhibiting corneal epithelial and stromal edema, peripheral posterior corneal macular opacities, and endothelial guttae, as well as 22 unaffected family members. METHODS: Whole-exome sequencing was performed in 3 families and Sanger sequencing of CHST6 was performed in all individuals. Histological examination of Descemet membrane (DM) excised at the time of endothelial keratoplasty was performed for three probands. Serum keratan sulfate (KS) levels were measured in members of six families. Functional analysis of identified mutations was performed using CHST6 promoter containing CHST6 expression vector in human keratocytes (HK) and corneal endothelial cells (HCEnC). MAIN OUTCOME MEASURES: Clinical phenotype; genetic analysis; functional analysis of identified CHST6 mutations; serum KS levels; histologic examinations of DM. RESULTS: All affected individuals demonstrated peripheral macular opacities at the level of DM. Visually significant corneal edema in affected individuals was successfully managed by endothelial keratoplasty. Genetic analysis demonstrated a rare CHST6 promoter mutation (c.-690G>C) in the homozygous state in affected individuals from three families and in the compound heterozygous state with a CHST6 coding mutation (p.R211Q, p.Y268C or p.P280L) in affected individuals from the other four families. In silico analysis predicted c.-690G>C to be a regulatory variant, located at the RNA polymerase II binding site. Functional analysis in vitro demonstrated that c.-690G>C leads to increased KS sulfation in the corneal endothelium and DM, with no change of KS sulfation in keratocytes. Histologic examination of DM from affected individuals revealed elevated levels of sulfated and non-sulfated KS in DM and endothelium, consistent with the functional analysis. Minimum changes in serum sulfated KS levels were observed in affected individuals. CONCLUSIONS: We suggest the name Peripheral macular endothelial dystrophy (PMED) to describe this dystrophy that is characterized by peripheral posterior corneal macular opacities and endothelial dysfunction without stromal haze or opacities. Given that both PMED and macular corneal dystrophy are associated with promoter and coding region mutations in CHST6, we propose that they be categorized as CHST6-associated corneal dystrophies.

Humans↗

PubMed2025

Identification of IDH3G, encoding the gamma subunit of mitochondrial isocitrate dehydrogenase, as a novel candidate gene for X-linked retinitis pigmentosa.

PURPOSE: Retinitis pigmentosa (RP) is a genetically heterogeneous group of retinal degenerative disorders characterized by the loss of rod and cone photoreceptors, leading to visual impairment and blindness. To date, to our knowledge, X-linked RP has been associated with variants in 3 genes (RPGR, RP2, and OFD1), whereas genetic defects at 3 loci (RP6, RP24, and RP34) are yet unidentified. The aim of this study was to identify a novel candidate gene underlying X-linked RP. METHODS: Participants were identified from cohorts of genetically unsolved male individuals affected by RP, who underwent genome sequencing, exome sequencing, or candidate gene screening via direct Sanger sequencing at 3 referral centers. Specifically, 2 probands were identified at the National Reference Centre for Rare Retinal Diseases (Paris, France), 2 at the Massachusetts Eye and Ear Hospital (Boston, MA), and 1 at the National Reference Centre for Inherited Sensory Diseases (Montpellier, France). The pathogenicity of the identified variants was assessed using bioinformatic predictions, protein expression analyses, and mitochondrial function assays. RESULTS: We identified 4 rare single-nucleotide variants in IDH3G (HGNC:5386), located at the RP34 locus on the X chromosome, and a complete gene deletion, in 5 unrelated male individuals affected with nonsyndromic RP. The variants segregated with the phenotype in all available family members. In all cases, the disease severity was intermediate. None had high myopia. IDH3G encodes the γ subunit of mitochondrial isocitrate dehydrogenase (IDH3), an enzyme involved in the citric acid cycle, which is expressed in the inner segments of photoreceptors. Variants in IDH3A and IDH3B, encoding the other subunits of IDH3, have already been associated with nonsyndromic autosomal recessive RP. Bioinformatic predictions and functional assays support a pathogenic role for the variants identified in this study, possibly through partial loss of enzymatic activity and mitochondrial function. CONCLUSION: Our findings suggest that variants in IDH3G are a novel cause of X-linked RP.

Humans↗

PubMed1994

Manifold sequencing: efficient processing of large sets of sequencing reactions.

Automated instruments for DNA sequencing greatly simplify data collection in the Sanger sequencing procedure. By contrast, the so-called front-end problems of preparing sequencing templates, performing sequencing reactions, and loading these on the instruments remain major obstacles to extensive sequencing projects. We describe here the use of a manifold support to prepare and perform sequencing reactions on large sets of templates in parallel, as well as to load the reaction products on a sequencing instrument. In this manner, all reaction steps are performed without pipetting the samples. The strategy is applied to sequencing PCR-amplified clones of the human mitochondrial D-loop and for detection of heterozygous positions in the human major histocompatibility complex class II gene HLA-DQB, amplified from genomic DNA samples. This technique will promote sequencing in a clinical context and could form the basis of more efficient genomic sequencing strategies.

Base Sequence↗