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The aim of this study was to identify the genetic basis of two female siblings - born to consanguineous Sudanese parents - diagnosed clinically as having the rare condition of 25-hydroxylase deficiency (vitamin D-dependent rickets type 1B). The initial diagnosis was established based on clinical data, laboratory and radiological findings retrospectively. Primers for all exons (5) of human CYP2R1 (NM_024514) were generated followed by Sanger sequencing on exons 1–5 for both girls and their parents. Homozygosity for a point mutation (c.85C > T) was detected, leading to a nonsynonymous variant at position 29 in exon 1, resulting in a premature stop codon (p.Q29X). This is a previously unknown variant that leads to a severely truncated protein and predicted to be among the 0.1 % most deleterious genomic variants(CADD score 36). To our knowledge, this family represents the first case series from Sudan with a confirmed CYP2R1 gene mutation and the 6th world-wide. With the lack of genetic facilities, diagnosis should be suspected by the persistently low 25 hydroxyvitamin D level in spite of proper treatment and after ruling out liver disease and malabsorption. Patients in this case series showed healing of rickets when treated with high doses of 1,25-dihydroxyvitamin D3 (1,25(OH)D3; calcitriol) and oral calcium.

Drug resistance via drug-resistant mutations in the human immunodeficiency virus (HIV) genome is the primary cause of antiviral therapy failure. Consequently, HIV drug resistance genotyping has become a critical approach in HIV prevention and control. Compared to the Sanger sequencing technology, high-throughput sequencing (HTS) technology has superior sensitivity and timeliness, with strong detection capabilities for low-frequency mutations. With the continued advancement of HTS technologies, their prominence in HIV drug resistance detection techniques has increased accordingly. This article will review the latest developments in HTS technology and its applications in HIV drug resistance testing.

We present MultiEditR (Multiple Edit Deconvolution by Inference of Traces in R), the first algorithm specifically designed to detect and quantify RNA editing from Sanger sequencing (z.umn.edu/multieditr). Although RNA editing is routinely evaluated by measuring the heights of peaks from Sanger sequencing traces, the accuracy and precision of this approach has yet to be evaluated against gold standard next-generation sequencing methods. Through a comprehensive comparison to RNA sequencing (RNA-seq) and amplicon-based deep sequencing, we show that MultiEditR is accurate, precise, and reliable for detecting endogenous and programmable RNA editing., Graphical abstract, RNA editing allows cells to transiently rewrite the genetic code. However, detecting and quantifying RNA editing relies on RNA-seq, which is expensive and suffers from poor local resolution. Here, we develop MultiEditR (https://moriaritylab.shinyapps.io/multieditr/) to analyze RNA editing from Sanger sequencing for a fraction of the cost while maintaining a comparable fidelity to RNA-seq.

G6PD deficiency c563T is the most common inherent blood disease among the Mediterranean populations and its molecular diagnosis is critical as the enzyme assay fails for heterozygous individuals. The purpose of the study is to estimate the ubiquity of the heterozygous G6PD Med (c563T) variants among Egyptians and UAE nationals living in Dubai. We validated two molecular methods, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and qPCR allelic discrimination assay for detection of G6PD Med variants. Among 100 screened individuals, G6PD c563T variants are 30% of whom 15% are carriers. Sanger sequencing validated the qPCR discrimination assays. In search of a phenotypic marker to detect G6PD heterozygous variants, inheritance of G6PD locus and red-green color vision genes is studied in 1 Egyptian and 2 Emirati families. Among the 3 families, G6PD is polymorphic, displaying 4 phenotypes: in phenotype-1, person is normal, in phenotype-2 the person has no G6PD deficiency but with deuteranopia/deuteranomaly, in phenotype-3 the person is G6PD Med variant with deuteranopia/deuteranomaly and in phenotype 4 the person is G6PD Med variant has normal vision. Based on the molecular analysis of G6PD and Ishihara vision test it can be concluded that the two mutations at the two loci arose independent of each other without any interaction (epistatic effect) between them. Following the pedigree analysis of the two genes for 4 generations it is presumed that it is infeasible to use “deuteranopia /deuteranomaly” as a phenotypic marker to detect G6PD c563T heterozygous individuals among the Egyptian populations.

Background Primary open angle glaucoma (POAG) occurs due to the discrepancies in the angle of anterior chamber characterized by the alterations in intraocular pressure, optic nerves head changes and central loss of visual field. In molecular research, CYP1B1 mutations modulates an integral role in association with glaucoma. Current study was undertaken to reveal the homozygous and heterozygous patterns of CYP1B1 c.1169 G > A variant (rs56010818) in POAG patients of Pakistan. Methods After consent, total n = 88 POAG patients undergone through standard ophthalmological investigations before their recruitment in this study. The blood samples were utilized for DNA isolation. The genotyping of CYP1B1 c.1169 G > A variant was carried out by Sanger sequencing. The mutational patterns and its association with clinical variables were demonstrated by statistical and bioinformatic tools. Results It was evident that the frequencies of heterozygous G/A and homozygous mutants A/A genotypes were higher in males (36.5%, 7.7%) than females (30.6%, 2.8%) of POAG population. Furthermore, the juvenile patients exhibit high manifestation of carrier genotype (66.6%) in comparison to adult patients (31.7%). The results also indicated the significant relationship of intraocular pressure with homozygous mutant A/A genotype of CYP1B1 variant in POAG patients (p Conclusions Our study provided the mutational data of CYP1B1 R390H variant and the patterns of homozygosity and heterozygosity along with clinical associations. Overall, this study revealed the genetic predisposition of CYP1B1 c.1169 G > A variant in the patients of POAG in Pakistan. The findings could be helpful for genetic screening and in-depth understanding of underlying causes in the pathogenesis of POAG.

Objective Several microRNA (miRNA) polymorphisms have been associated with susceptibility to specific health disorders, including cardiovascular diseases. The aim of the present study was to investigate whether four well-studied miRNA polymorphisms in non-Caucasian populations, namely miR146a G>C (rs2910164), miR149 C>T (rs2292832), miR196a2 C>T (rs11614913) and miR499 A>G (rs3746444), contribute to the risk for the development of premature Coronary Artery Disease (CAD) in the Greek population. Methods We used a case-control study to examine these associations in 400 individuals: 200 CAD patients [including a subgroup of myocardial infraction (MI) patients] and 200 healthy controls, all of Greek origin. MiRNA polymorphisms were genotyped using three different assays: Polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP), High resolution Melting (HRM) and Sanger sequencing. Results Two of these polymorphisms, miR196a2 C>T (rs11614913) and miR499 A>G (rs3746444) were found to be strongly associated with increased risk for CAD (p=0.0388 and p=0.0013, respectively) and for MI (p=0.0281 and p=0.0273, respectively). Furthermore, miR146C-miR149C-miR196T-miR499G allele combination appeared to be significantly related to CAD (p=0.0185) and MI (p=0.0337) prevalence. Conclusions Our results suggest that at least two of the studied polymorphisms, miR196a2 C>T (rs11614913) and miR499 A>G (rs3746444), as well as the miR146C-miR149C-miR196T-miR499G allele combination could represent useful biomarkers of CAD and/or MI susceptibility in the Greek population. These special genetic characteristics, in combination with environmental factors and personal habits, might contribute to CAD and/or MI prevalence.

All children, who were born in 2004 and had undergone surgical treatment for recurrent acute tonsillitis and/or acute otitis media at the ear, nose and throat clinic (ENT) between 2004 and 2010, were called on dental examination and blood sampling. Out of 441 invitees, 113 children and their parents/legal guardians agreed to participate. The following data from this group of subjects are presented: the presence of clinical signs of molar–incisor hypomineralisation (MIH), the distribution of human leukocyte antigen (HLA) alleles DQ2 and DQ8 and eight single nucleotide polymorphisms (SNPs) located in amelogenesis-related genes (rs3796704 in the ENAM gene, rs546778141 in the AMBN gene, rs2106416 in the AMELX gene, rs7660807 and rs35286445 in the AMTN gene, rs4870723 in the COL14A1 gene, rs2245803 in the MMP20 gene, and rs3828054 in the TUFT1 gene). Data on clinical signs of MIH were collected in accordance with the recommendation and on the proposed MIH clinical data recording sheet [1] , and with appropriate preliminary training and calibration. Data on HLA DQ2 and DQ8 haplotypes and on SNPs of amelogenesis-related genes were obtained using DNA isolated from blood samples taken from subjects. The HLA DQ2 and DQ8 alleles were determined using the EliGene® Coeliac RT Kits (90,048-RT; Elisabeth Pharmacon spol. s.r.o., Brno-Židenice, Czech Republic) on a 7500 Fast RT-PCR System (Applied Biosystems, Waltham, MA, USA). The distributions of SNPs in the amelogenesis-related genes were determined using high resolution melting (HRM) using the Type-IT HRM Master Mix (Qiagen), TaqMan genotyping assays (ID: C__25766207_10; Thermo Fisher Scientific, Waltham, MA, USA) with the TaqMan Universal Master Mix II, or Sanger sequencing using sequencing master mix BigDye® Terminator v3.1 (Applied Biosystems) and ABI 3500 Genetic Analyser (Applied Biosystems). L. Hocevar, J. Kovac, K. Trebusak Podkrajsek, S. Battelino, A. Pavlic, 2020. The possible influence of genetic aetiological factors on molar–incisor hypomineralisation, Arch. Oral. Biol. 118, 104848. https://doi.org/10.1016/j.archoralbio.2020.104848 .

SARS-CoV-2 has kept the world in suspense for almost 2 years now. The virus spread rapidly worldwide and several variants of concern have emerged: Alpha, Beta, Gamma, Delta and recently Omicron. A rapid method to detect key mutations is needed because these variants may jeopardize the effectiveness of immune protection following vaccination or past infection. This article describes an easy, cheap and fast method for the detection of mutations in the spike protein that are indicative for specific variants. This method can easily distinguish Omicron from other variants. ispartof: JOURNAL OF VIROLOGICAL METHODS vol:304 ispartof: location:Netherlands status: published

Background/Purpose Occult HBV infection (OBI) could have serious clinical consequences in patients receiving immunosuppressive therapy. We aimed to investigate the prevalence of OBI in Chinese patients with autoimmune hepatitis (AIH) and to analyze its clinical and virological features. Methods 103 AIH cases were enrolled. Hepatitis B virus (HBV) serological markers were screened by chemiluminescence. HBV-DNA were detected by nest-PCR and real-time PCR. HBV genotyping and mutation analysis were performed by Sanger sequencing. Results Twenty-four out of 103 (23.30%) AIH patients had OBI as evidenced by positive HBV-DNA and negative hepatitis B surface antigen (HBsAg). HBV genotype C is the predominant genotype (57.89%), which had more amino acid (AA) substitutions in S region than that of B-genotype group (P = 0.001). The distribution of AA substitution in the ‘α’ determinant region between genotype C and B were significantly different (P = 0.042). In addition to those already reported OBI-associated AA substitutions (e.g., sG145R and sV184A), some new OBI-associated AA substitutions (e.g., sV106A, sC137* and sL176P) were found in AIH patients in our study. Three out of 24 (12.50%) OBI patients were diagnosed as decompensated cirrhosis, one patient with S deletion mutation and two patients with HBV extensive AA substitutions. Conclusions There was a higher proportion of AIH patients with OBI than the general population in China, which can be either seropositive or seronegative-OBI in AIH patients is associated with some specific AA substitutions. The presence of deletion mutations and the extent of AA substitutions in the HBV S region may have predictive clinical implications.

Conventional nested PCR and Sanger sequencing methods are currently the gold standards for detecting trypanosomes in wildlife. However, these techniques are time-consuming and can often overlook mixed infections. True trypanosome prevalence can thus be underrepresented. Here, we designed an 18S rDNA-based real-time quantitative PCR (qPCR) assay coupled with High-Resolution Melting Analysis (HRMA) to detect and discriminate three Trypanosoma species (T. copemani, T. noyesi, and T. vegrandis) commonly infecting Australian marsupials. A total of 68 genetically characterised samples from blood and tissue were used to validate the High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR) assay. A further 87 marsupial samples consisting of blood, tissue and in vitro cultures derived from wildlife blood samples, were screened for the first time using this assay, and species identity confirmed using conventional PCR and Sanger sequencing. All three Trypanosoma species were successfully detected in pure cultures using the HRM-qPCR assay, and in samples containing mixed trypanosome infections. Of the 87 marsupial samples screened using the HRM-qPCR assay, 93.1% were positive for trypanosomes, and 8.0% contained more than one trypanosome species. In addition to the three targeted Trypanosoma species, this assay was also able to detect and identify other native and exotic trypanosomes. The turnaround time for this assay, from sample preparation to obtaining results, was less than 2 h, with a detection limit of 10 copies of the amplicon in a reaction for each of the targeted trypanosome species. This more rapid and sensitive diagnostic tool provides a high throughput platform for the detection, identification and quantification of trypanosome infections. It will also improve understanding of host diversity and parasite relationships and facilitate conservation management decisions., Graphical abstract Image 1

Objective/background Retinoblastoma (RB), the most common intraocular malignancy in children, is caused by biallelic inactivation of the human retinoblastoma susceptibility gene (RB1). We are evaluating the impact of the type of RB1 gene mutation on clinical presentation and management outcome. Methods A retrospective case series of 50 patients with RB. Main outcomes were clinical and pathologic features and types of RB1 gene mutations detected using quantitative multiplex polymerase chain reaction (PCR), allele-specific PCR, next-generation sequencing analysis, and Sanger sequencing. Results Twenty (40%) patients had unilateral RB and 30 (60%) had bilateral RB. Overall, 36 (72%) patients had germline disease, 17 (47%) of whom inherited the disease. Of these 17 inherited cases, paternal origin of the RB1 mutation was seen in 15 (88%). The overall eye salvage rate was 74% (n = 49/66; 100% for Groups A + B + C, and 79% for Group D eyes). The most frequent type of mutation was a nonsense mutation generating a stop codon (15/36, 42%). Other mutations that result in a premature stop codon due to deletions or insertions with donor splice site or receptor splice site mutations were detected in 7/36 (19%), 10/36 (28%), and 2/26 (6%) patients, respectively. The remaining two (6%) patients had frameshift mutation. Patients with deletion, acceptor splice site, and frameshift mutations presented with more advanced ICRB (International Classification of Retinoblastoma) stage (75% diagnosed with Group D or E), even though there was no significant difference in eye salvage rate or tumor invasiveness between patients with different types of mutations. Conclusion Despite the heterogeneous nature of RB1 gene mutations, tumor stage remains the most important predictive factor for clinical presentation and outcome. Furthermore, acceptor splice site and frameshift mutations are associated with more advanced tumor stage at diagnosis.

Intellectual developmental disorder with abnormal behavior, microcephaly and short stature (IDDABS), (OMIM# 618342) is an autosomal recessive condition described as developmental delay, poor or absent speech, intellectual disability, short stature, mild to progressive microcephaly, delayed psychomotor development, hyperactivity, seizure, along with mild to swear aggressive behavior. Homozygous frameshift mutation in Pseudouridine Synthase 7, Putative; (PUS7) OMIM# 616,261 NM_019042.3 and splice acceptor variants in Alpha-Aminoadipic Semialdehyde Synthase; (AASS) OMIM# 605,113 NM_005763.3 was funded. Whole exome sequencing (WES) technique was used as tool to identify the molecular diagnostic test. Different bioinformatics analysis done for WES data and we identified two novel mutations one as frameshift mutation c.606_607delGA, p.Ser282CysfsTer9 in the PUS7 gene and splice acceptor variants c.1767–1 G > A in the AASS gene has been reported. The pattern of family segregation maintained the pathogenicity of this variation associated with abnormal behavior, intellectual developmental disorder, microcephaly along with short stature IDDABS. Further, the WES data was validated in the family having other affected individuals and healthy controls (n = 100) was done using Sanger sequencing. Finally, our results further explained the role of WES in the disease diagnosis and elucidated that the mutation in PUS7 and AASS genes may lead an important role for the development of IDDABS in Saudi family.

Background In about 5% of patients with hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE) no mutation in the SERPING1 gene is detected. Methods C1-INH-HAE cases with no mutation in the coding region of SERPING1 after conventional genotyping were examined for defects in the intronic or untranslated regions of the gene. Using a next-generation sequencing (NGS) platform targeting the entire SERPING1, 14 unrelated C1-INH-HAE patients with no detectable mutations in the coding region of the gene were sequenced. Detected variants with a global minor allele frequency lower than the frequency of C1-INH-HAE (0.002%), were submitted to in silico analysis using ten different bioinformatics tools. Pedigree analysis and examination of their pathogenic effect on the RNA level were performed for filtered in variants. Results In two unrelated patients, the novel mutation c.-22-155G > T was detected in intron 1 of the SERPING1 gene by the use NGS and confirmed by Sanger sequencing. All bioinformatics tools predicted that the variant causes a deleterious effect on the gene and pedigree analysis showed its co-segregation with the disease. Degradation of the mutated allele was demonstrated by the loss of heterozygosity on the cDNA level. According to the American College of Medical Genetics and Genomics 2015 guidelines the c.-22-155G > T was curated as pathogenic. Conclusions For the first time, a deep intronic mutation that was detected by NGS in the SERPING1 gene, was proven pathogenic for C1-INH-HAE. Therefore, advanced DNA sequencing methods should be performed in cases of C1-INH-HAE where standard approaches fail to uncover the genetic alteration.

Background and objective: The human ficolin-2, encoded by FCN2, recognizes pathogen-associated acetylated residues on their cell surfaces and activates the lectin complement cascade. This study aimed to investigate the contribution of human ficolin-2 and the functional FCN2 genetic variants in dengue virus (DENV) infection and in clinical progression. Methods: FCN2 genetic polymorphisms in the promoter, intron 7 and exon 8 were genotyped in 279 patients with dengue fever and in 200 healthy controls by direct Sanger sequencing. The ficolin-2 levels were measured in serum samples by ELISA and correlated with clinical data. Results: The frequencies of +6031GG, +6220GG and +6424TT genotypes were significantly higher in dengue patients compared to healthy controls indicating an increased risk of dengue fever. The SNPs rs11103563 (+6031A/G), rs7872508 (+6220 T/G), and rs7851696 (+6424G/T) significantly regulated ficolin-2 levels in dengue patients (P < 0.0001). Ficolin-2 levels were increased in patients with dengue and Dengue with Warning Signs (DWS) compared to healthy controls (P < 0.0001 and P = 0.038, respectively). Ficolin-2 levels were significantly increased after 10–14 days of admission in both dengue and DWS patients and then slightly decreased after three weeks of discharge, indicating that ficolin-2 levels were modulated during the progression of dengue fever. In addition, ficolin-2 levels were negatively correlated with AST levels and positively correlated with platelet counts. Conclusions: FCN2 polymorphisms are associated with dengue fever in the Vietnamese population. Ficolin-2 levels are modulated during the progression of dengue fever and correlated with clinical parameters and thus may play a possible role in the pathogenesis of DENV infection.

Celiac disease (CD) is a gastrointestinal disorder whose genetic basis is not fully understood. Therefore, we studied a Saudi family with two CD affected siblings to discover the causal genetic defect. Through whole exome sequencing (WES), we identified that both siblings have inherited an extremely rare and deleterious CPED1 genetic variant (c.241 A > G; p.Thr81Ala) segregating as autosomal recessive mutation, suggesting its putative causal role in the CD. Saudi population specific minor allele frequency (MAF) analysis has confirmed its extremely rare prevalence in homozygous condition (MAF is 0.0004). The Sanger sequencing analysis confirmed the absence of this homozygous variant in 100 sporadic Saudi CD cases. Genotype-Tissue Expression (GTEx) data has revealed that CPED1 is abundantly expressed in gastrointestinal mucosa. By using a combination of systems biology approaches like protein 3D modeling, stability analysis and nucleotide sequence conservation analysis, we have further established that this variant is deleterious to the structural and functional aspects of CPED1 protein. To the best of our knowledge, this variant has not been previously reported in CD or any other gastrointestinal disease. The cell culture and animal model studies could provide further insight into the exact role of CPED1 p.Thr81Ala variant in the pathophysiology of CD. In conclusion, by using WES and systems biology analysis, present study for the first-time reports CPED1 as a potential causative gene for CD in a Saudi family with potential implications to both disease diagnosis and genetic counseling.

Background: Diabetic Nephropathy (DN) is a major complication of Type 2 Diabetes Mellitus (T2DM) with high morbidity rates worldwide. Objective: To determine the association of PPARγ rs1801282 polymorphism in T2DM and DN in south Indian population. Methods: We have conducted a case–control study to test the association of rs1801282 polymorphism with T2DM and DN in 424 subjects (DN = 128; T2DM = 148 and controls = 148) belonging to the south Indian population using ARMS-PCR and Sanger sequencing method. Further, a meta-analysis was performed for rs1801282 polymorphism from the published literature retrieved from various electronic databases to determine the susceptibility among T2DM and DN across various ethnic populations under five genetic models. Results: The genotyping of rs1801282 polymorphism showed significant (p-value 0.05) was noticed for rs1801282 with DN vs. controls in homozygote, heterozygote, allelic, recessive and dominant genetic models. However, a significant association was observed between rs1801282 SNP and T2DM under heterozygote (Jj vs JJ) genetic model with OR = 0.56, (95%CI [0.43–0.74]), p ≤ 0.0001 of Asian and Caucasian populations. Conclusion: Overall analysis suggests that the rs1801282 polymorphism might be associated with DN and T2DM. More case–control studies on the PPARγ gene with a larger sample size including all the confounding factors are required to corroborate the findings from this meta-analysis. Resumen: Antecedentes: La nefropatía diabética (ND) es una complicación importante de la diabetes mellitus de tipo 2 (DMT2) con altas tasas de morbilidad mundial. Objetivo: Determinar la asociación del polimorfismo rs1801282 de PPARγ en la DMT2 y la ND en la población del sur de India. Métodos: Hemos llevado a cabo un estudio de casos y controles para analizar la asociación del polimorfismo rs1801282 con la DMT2 y la ND en 424 sujetos (ND = 128; DMT2 = 148 y controles = 148) pertenecientes a la población del sur de India mediante RCP-ARMS y método de secuenciación de Sanger. Además, se realizó un metaanálisis para el polimorfismo de rs1801282 a partir de la literatura publicada en varias bases de datos electrónicas para determinar la sensibilidad entre la DMT2 y la ND en varias poblaciones étnicas con 5 modelos genéticos. Resultados: El genotipado de polimorfismo rs1801282 demostró una asociación significativa (valor de p 0,05) de rs1801282 con la ND frente a los controles en modelos genéticos homocigóticos, heterocigóticos, alélicos, recesivos y dominantes. Sin embargo, se observó una asociación significativa entre el polimorfismo de nucleótido único (PNU) rs1801282 SNP y la DMT2 en el modelo genético heterocigótico (Jj frente a JJ) con OR = 0,56, (IC del 95%: 0,43-0,74; p ≤ 0,0001 de poblaciones asiáticas y caucásicas. Conclusión: El análisis general sugiere que el polimorfismo rs1801282 puede asociarse a ND y a DMT2. Se precisan más estudios de casos y controles sobre el gen PPARγ con un tamaño de la muestra mayor que incluya todos los factores de confusión para corroborar los resultados de este metaanálisis.

Background: HIV drug resistance (HIVDR) testing can assist clinicians in selecting treatments. However, high complexity and cost of genotyping assays limit routine testing in settings where HIVDR prevalence has reached high levels. Methods: The oligonucleotide ligation assay (OLA)-Simple kit was developed for detection of HIVDR against first-line non-nucleoside/nucleoside reverse transcriptase inhibitors and validated on 672 codons (168 specimens) from subtypes A, B, C, D, and AE. The kit uses dry reagents to facilitate assay setup, lateral flow devices for visual HIVDR detections, and in-house software with an interface for guiding users and analyzing results. Findings: HIVDR analysis of specimens by OLA-Simple compared to Sanger sequencing revealed 99.6 ± 0.3% specificity and 98.2 ± 0.9% sensitivity, and compared to high-sensitivity assays, 99.6 ± 0.6% specificity and 86.2 ± 2.5% sensitivity, with 2.6 ± 0.9% indeterminate results. OLA-Simple was performed more rapidly compared to Sanger sequencing (

Severe hypertriglyceridemia (HTG) is a relatively common form of dyslipidemia with a complex pathophysiology and serious health complications. HTG can develop in the presence of rare genetic factors disrupting genes involved in the triglyceride (TG) metabolic pathway, including large-scale copy-number variants (CNVs). Improvements in next-generation sequencing technologies and bioinformatic analyses have better allowed assessment of CNVs as possible causes of or contributors to severe HTG. We screened targeted sequencing data of 632 patients with severe HTG and identified partial deletions of the LPL gene, encoding the central enzyme involved in the metabolism of TG-rich lipoproteins, in four individuals (0.63%). We confirmed the genomic breakpoints in each patient with Sanger sequencing. Three patients carried an identical heterozygous deletion spanning the 5′ untranslated region (UTR) to LPL exon 2, and one patient carried a heterozygous deletion spanning the 5′UTR to LPL exon 1. All four heterozygous CNV carriers were determined to have multifactorial severe HTG. The predicted null nature of our identified LPL deletions may contribute to relatively higher TG levels and a more severe clinical phenotype than other forms of genetic variation associated with the disease, particularly in the polygenic state. The identification of novel CNVs in patients with severe HTG suggests that methods for CNV detection should be included in the diagnostic workup and molecular genetic evaluation of patients with high TG levels.

Some sea cucumbers are economically and ecologically important, but they are threatened by thermal and hypoxic stress in changing oceanographic conditions. We construct circRNAs profiles, reveal circRNAs characters, and illustrate the potential regulatory roles of circRNAs in one commercially important species of sea cucumber, Apostichopus japonicus. Reads are distributed in intergenic (44.14%), exonic (48.26%) and intronic (7.60%) regions of the genome. A total of 1684 circRNAs were identified, and the most common spliced length is 269 nt in the present study. In three treatments (HT [thermal stress], LO [hypoxic stress], and HL [combined thermal and hypoxic stress]), 24, 27 and 27 differentially expressed (DE) circRNAs were identified, respectively. Five novel DE-circRNAs commonly occur in these treatments (novel_circ_0003311, novel_circ_0000229, novel_circ_0003944, novel_circ_0001458 and novel_circ_0000707), and based on them, potential circRNA-miRNA binding pairs were predicted. Sanger sequencing, RNase R treatment experiment and qPCR validation identified the accuracy of the circRNAs. Key circRNAs identified in the present study were covalently closed and were more stable under RNase R treatment than linear RNAs. Based on function analysis, circRNAs could regulate metabolic process, signal transduction, and ion responses in A. japonicus when exposed to thermal and hypoxic stress, and ‘regulation of response to stimulus’ is a common gene ontology (GO) term that is significantly enriched in each treatment; GO terms for ‘DNA’ and ‘stress’ are commonly enriched in heat-related treatments (HT and HL); and GO terms for ‘protein’ are commonly enriched in hypoxia-related treatments (LO and HL). When environmentally stressed, ‘metabolism,’ ‘transport and catabolism,’ ‘membrane transport,’ and ‘signal transduction’ were significantly responded in sea cucumber based on KEGG analysis. We provide insights into circRNA functions in stress regulation and lay a foundation for invertebrate circRNA research.

Highlights • GPC3 mutation in primary osteosarcoma becomes abundant in its metastasis. • Mutant GPC3 is over-produced in metastatic spheroids with multidrug resistance. • Anti-GPC3 antibody effectively commits metastatic spheroids to apoptosis. • GPC3 would be a promising therapeutic target of osteosarcomas., Background Drug resistance and the lack of molecular therapeutic target are the main challenges in the management of osteosarcomas (OSs). Identification of novel genetic alteration(s) related with OS recurrence and chemotherapeutic resistance would be of scientific and clinical significance. Methods To identify potential genetic alterations related with OS recurrence and chemotherapeutic resistance, the biopsies of a 20-year-old male osteosarcoma patient were collected at primary site (p-OS) and from its metastatic tumor (m-OS) formed after 5 months of adjuvant chemotherapy. Both OS specimens were subjected to cancer-targeted next generation sequencing (NGS) and their cell suspensions were cultured under three-dimensional condition to establish spheroid therapeutic model. Transcript-oriented Sanger sequencing for GPC3, the detected mutated gene, was performed on RNA samples of p-OS and m-OS tissues and spheroids. The effects of anti-GPC3 antibody and its combination with cisplatin on m-OS spheroids were elucidated. Results NGS revealed 4 mutations (GPC3, SOX10, MDM4 and MAPK8) and 6 amplifications (MDM2, CDK4, CCND3, RUNX2, GLI1 and FRS2) in p-OS, and 3 mutations (GPC3, SOX10 and EGF) and 10 amplifications (CDK4, CCND3, MDM2, RUNX2, GLI1, FRS2, CARD11, RAC1, SLC16A7 and PMS2) in m-OS. Among those alterations, the mutation abundance of GPC3 was the highest (56.49%) in p-OS and showed 1.54 times increase in m-OS. GPC3 transcript-oriented Sanger sequencing confirmed the mutation at 1046 in Exon 4, and immunohistochemical staining showed increased GPC3 production in m-OS tissues and its spheroids. EdU cell proliferation and Calcein/PI cell viability assays revealed that of the anti-OS first line drugs (doxorubicin, cisplatin, methotrexate, ifosfamide and carboplatin), 10 μM carboplatin exerted the best inhibitory effects on the p-OS but not the m-OS spheroids. 2 μg/mL anti-GPC3 antibody effectively committed m-OS spheroids to death by itself (76.43%) or in combination with cisplatin (92.93%). Conclusion This study demonstrates increased abundance and up-regulated expression of mutant GPC3 in metastatic osteosarcoma and its spheroids with multidrug resistance. As GPC3-targeting therapy has been used to treat hepatocellular carcinomas and it is also effective to OS PDSs, GPC3 would be a novel prognostic parameter and therapeutic target of osteosarcomas.

Neonatal diabetes mellitus (NDM) is noted as a genetic, heterogeneous, and rare disease in infants. NDM occurs due to a single-gene mutation in neonates. A common source for developing NDM in an infant is the existence of mutations/variants in the KCNJ11 and ABCC8 genes, encoding the subunits of the voltage-dependent potassium channel. Both KCNJ11 and ABCC8 genes are useful in diagnosing monogenic diabetes during infancy. Genetic analysis was previously performed using first-generation sequencing techniques, such as DNA-Sanger sequencing, which uses chain-terminating inhibitors. Sanger sequencing has certain limitations; it can screen a limited region of exons in one gene, but it cannot screen large regions of the human genome. In the last decade, first generation sequencing techniques have been replaced with second-generation sequencing techniques, such as next-generation sequencing (NGS), which sequences nucleic-acids more rapidly and economically than Sanger sequencing. NGS applications are involved in whole exome sequencing (WES), whole genome sequencing (WGS), and targeted gene panels. WES characterizes a substantial breakthrough in human genetics. Genetic testing for custom genes allows the screening of the complete gene, including introns and exons. The aim of this review was to confirm if the 22 genetic variations previously documented to cause NDM by Sanger sequencing could be detected using second generation sequencing techniques. The author has cross-checked global studies performed in NDM using NGS, ES/WES, WGS, and targeted gene panels as second-generation sequencing techniques; WES confirmed the similar variants, which have been previously documented with Sanger sequencing. WES is documented as a powerful tool and WGS as the most comprehensive test for verified the documented variants, as well as novel enhancers. This review recommends for the future studies should be performed with second generation sequencing techniques to identify the verified 22 genetic and novel variants by screening in NDM (PNDM or TNMD) children., Neonatal diabetes mellitus (NDM), First-generation sequencing, DNA-Sanger sequencing, Second-generation sequencing, Next-generation sequencing (NGS), Exome sequencing (ES), Whole exome sequencing (WES), Whole genome sequencing (WGS), Targeted gene panels

Objectives Monitoring the spread of the G614 in specific locations is critical as this variant is highly transmissible and can trigger the emergence of other mutations. Therefore, a rapid and accurate method that can reliably detect the D614G mutation will be beneficial. This study aims to analyze the potential use of the two-step Reverse Transcriptase quantitative polymerase chain reaction - high resolution melting analysis (RT-qPCR-HRM) to detect a specific mutation in the SARS-CoV-2 genome. Methods Six SARS-CoV-2 RNA samples were synthesized into cDNA and analyzed with the qPCR-HRM method in order to detect the D614G mutation in Spike protein of SARS-CoV-2. The primers are designed to target the specific Spike region containing the D614G mutation. The qPCR-HRM analysis was conducted simultaneously, and the identification of the SARS-CoV-2 variant was confirmed by conventional PCR and Sanger sequencing methods. Results The results showed that the melting temperature (Tm) of the D614 variant was 79.39 ± 0.03 °C, which was slightly lower than the Tm of the G614 variant (79.62 ± 0.015 °C). The results of the HRM analysis, visualized by the normalized melting curve and the difference curve were able to discriminate the D614 and G614 variant samples. All samples were identified as G614 variants by qPCR-HRM assay, which was subsequently confirmed by Sanger sequencing. Conclusions This study demonstrated a sensitive method that can identify the D614G mutation by a simple two-step RT-qPCR-HRM assay procedure analysis, which can be useful for active surveillance of the transmission of a specific mutation., SARS-CoV-2; D614G; Spike; qPCR; HRM analysis

Protozoan parasites in food or water samples are generally detected using microscopy or PCR followed by Sanger sequencing. However, microscopy is subjective, requires a high degree of expertise and has limited sensitivity, while DNA sequencing requires expensive and specialized equipment and facilities. This study describes a cloth-based hybridization array system (CHAS) that is an alternative to Sanger sequencing to confirm PCR-positive samples. CHAS is an inexpensive, rapid and reliable method for the simultaneous detection of multiple protozoan parasite species based on the colorimetric detection of PCR amplicons on a polyester cloth. PCR primers and CHAS hybridization probes were developed to detect the protozoan parasites Giardia duodenalis, Cryptosporidium spp. and Toxoplasma gondii. In addition, CHAS probes were designed for the differentiation of G. duodenalis Assemblages A and B. In artificially contaminated fresh produce (lettuce, parsley) and water samples (river water, wastewater), this CHAS assay allowed for the successful detection of G. duodenalis, Cryptosporidium spp., and T. gondii. The present study demonstrates that the CHAS detection method is a simple and inexpensive alternative to DNA sequencing for the confirmation of PCR-positive results in laboratories testing for parasites in food or water samples. This assay may also be beneficial in developing countries, where DNA sequencing facilities may not be readily available.

Trio-next generation sequencing is useful to identify undiagnosed inherited diseases. We have attended a patient with trigenic ADH5/ALDH2/ADGRV1 pathogenic variants, which caused two distinct diseases, myelodysplastic syndrome and Usher syndrome. Whole genome sequencing of peripheral blood from the patient and his parents were applied to identify disease-causing genes. Sanger sequencing was performed to validate the identified ADH5/ALDH2/ADGRV1 variants. Our results identified disease-associated variants in ADGRV1 (disease inheritance autosomal recessive) and in ADH5 (disease inheritance also autosomal recessive) and a variant in ALDH2 (disease inheritance autosomal dominant). Although the variants identified in ADH5 and ALDH2 have been reported, their co-existence in association with disease-causing variation in a third gene has not. They broaden the spectrum of ADGRV1 in Usher syndrome. Findings on next generation sequencing guided rapid and accurate diagnosis, resulting in patient-tailored therapeutic intervention., Highlights • Trigenic ADH5 / ALDH2 / ADGRV1 ​variants in myelodysplastic syndrome with Usher syndrome were identified. • Two novel pathogenic frameshift variants in ​ADGRV1 ​in compound heterozygous state with Usher syndrome type II were described. • Findings on next generation sequencing guided rapid and accurate diagnosis, resulting in patient-tailored therapy., ADH5/ALDH/ADGRV1 variants, Myelodysplastic syndrome, Trigenic mutations, Trio-next generation sequencing, Usher syndrome.

PBMCs were collected from a patient with a novel GLA gene mutation (c.140G > A) which contributed to Fabry disease. Subsequently, an induced pluripotent stem cell (iPSC) line was derived using an episomal reprogramming method that transfer the reprogramming plasmids expressing OCT3/4, SOX2, KLF4, LIN28 and L-MYC into the PBMCs. The expected mutation in the iPSC line was confirmed by Sanger sequencing, while the pluripotency status was validated by immunofluorescence assay and flow cytometry for pluripotency markers, as well as teratoma formation.

The control of HIV transmission is a high priority for Africa, in line with Agenda 2063 of the African Union. The development of HIV drug resistance poses a significant challenge in maintaining suppressed viral load in the management of patients. Next generation sequencing (NGS) is a more sensitive approach to determine the burden of HIV drug resistance. We aimed to describe the uptake of NGS in HIV drug resistance studies in Africa. An electronic search was conducted for studies published between 2005 and 2019, from three databases: PubMed, Web of Science and Google scholar. The search approach was carried out according to PRISMA guidelines. Studies included in the analysis were those that reported the use of NGS on HIV drug resistance using samples from Africa or in which the studies were done in Africa. A total of 4180 articles were identified according to the search criteria. Out of these, 237 studies (5.7%), all of which were in English, met the inclusion criteria and were included in the analysis. Of the total number of studies, 29 (12.2%) used NGS only, 194 (81.9%) used Sanger sequencing, and 14 (5.9%) used both techniques. Evidence of in-country application of NGS was observed in six studies (14.0%), all from South Africa. In other studies, NGS was either done outside of Africa using samples obtained from Africa, or the country in which NGS was done was not indicated. From 2005 to 2012, only one study was reported on HIV drug resistance using NGS; however, 43 studies were published by 2019. Out of the 54 African countries, investigators from 13 countries (24.1%) published data on HIV drug resistance using NGS, as at the end of 2019. Illumina platform was the most preferred NGS platform (p

Background: Circular RNAs (circRNAs) are involved in inflammation; however, their role in allergic rhinitis (AR) remains unclear. In this study, we analyzed circRNA expression and identified a circRNA-miRNA-mRNA network through which circRNAs regulate AR pathogenesis. Methods: We analyzed circRNA, miRNA, and mRNA expression profiles in the nasal mucosa by high-throughput sequencing (HTS), using a fold-change >1.5 and p-value

Graphical abstract, Highlights • The mutations in two different genes should be sought in the patients with complex phenotypes. • The c.1531G>T in COL1A2 leading to OI and c.364C>T (p.R122W) in LHX4 to CPHD were found in a Thai boy. • The incomplete penetrance and loss-of-function are the features of p.R122W mutation in LHX4. • The mutation spectra of COL1A2 and LHX4 and pathomechanism of LHX4 are expanded., Genetic disorders have been shown to co-occur in individual patient. A Thai boy with features of osteogenesis imperfecta (OI) and combined pituitary hormone deficiency (CPHD) was identified. The causative mutations were investigated by whole exome and Sanger sequencing. Pathogenicity and pathomechanism of the variants were studied by luciferase assay. The proband was found to harbor a novel de novo heterozygous missense mutation, c.1531G > T (p.G511C), in COL1A2 leading to OI and a heterozygous missense variant, c.364C > T (p.R122W), in LHX4. The LHX4 p.R122W has never been reported to cause CPHD. The variant was predicted to be deleterious and found in the highly conserved LIM2 domain of LHX4. The luciferase assays revealed that the p.R122W was unable to activate POU1F1, GH1, and TSHB promoters, validating its pathogenic effect in CPHD. Moreover, the variant did not alter the function of wild-type LHX4, indicating its hypomorphic pathomechanism. In conclusion, the novel de novo heterozygous p.G511C mutation in COL1A2 and the heterozygous pathogenic p.R122W mutation in LHX4 were demonstrated in a patient with OI and CPHD. This study proposes that the mutations in two different genes should be sought in the patients with clinical features unable to be explained by a mutation in one gene.

Background: Breast cancer is a multifactorial disease with the highest frequency in females. Genetic and environmental factors can cause mutation in several genes like tyrosine kinase, JAK2 gene which may initiate cancer. Molecular analysis of mutations in the JAK2 gene along with determination of environmental, clinical and haematological risk factors associated with breast cancer patients is need of hour to improve patient's healthcare. Somatic JAK2 valine-to-phenylalanine (617 codon) mutation is one of the widely prevalent mutations. Methods: Blood was collected from seventy breast cancer patients after their consent. The questionnaire included risk factors, age group, locality, number of children, tumor type, family history, time of initial diagnosis, no of cycles/month, water conditions and exposure to radiations. Molecular analysis were carried out from genomic DNA using Sanger sequencing and allele-specific PCR to check the V617F point mutation. Results: The breast cancer risk factors includes unfiltered water (68.57%), urban (58.57%), menopause (55.71%), family history of cancer (18.57%), tumor grades (II, 37.14% and III, 35.71%), consanguineous marriages (44.28%) and having more than 3–4 children (45.71%). Prevalence of breast cancer was higher after the age of 35 and maximum at 35–50. In allele-specific PCR of 70 patients, 25 patients were wild type (229 bp), 25 patients were with partially deleted gene (200 bp), and 20 patient had shown no or less than 40 bp size fragments. In Sanger's sequencing of 70 BC cases, 18% were found to be positive for V617F point mutation, including 6 homozygous (T/T) and 7 heterozygous (G/T) mutations at nucleotide position 1849 in exon 14 of the JAK2 gene. Conclusions: Environmental and clinical risk factors were associated with breast cancer which can be overcome by improving awareness of associated risks, health facilities and reducing stress. Keywords: Breast cancer, JAK2, V617F mutation, Allele-specific PCR, Sanger’s sequencing

Wilson’s disease (WD), resulting from homozygote and compound heterozygote mutations in ATB7B, is an autosomal recessive disease. WD associated acute liver failure (ALF) is fatal, and a revised Wilson’s disease prognostic index (RWPI) > 11 is a reliable indication of liver transplantation (LT) or artificial liver support (ALS). We described a WD patient who initially presented with ALF and severe hemolytic anemia. A single heterozygote c.2333G > T mutation (p. Arg778Leu, R778L) in ATP7B was screened by whole exome sequencing and validated by Sanger sequencing. Rapid diagnostic criteria (ALP/TBIL < 4 and AST/ALT > 2.2) are suitable for early diagnosis. Although the RWPI amounted to 15, the patient recovered after intermittent plasma transfusion and subsequent chelating therapy without LT or ALS. In conclusion, WD patients with a single R778L heterozygote mutation can present with ALF as the initial clinical manifestation, and intermittent plasma transfusion combined with chelating therapy may alleviate fulminant WD without LT or ALS.