UNASSIGNED: FTO gene belongs to the non-heme Fe (II) and 2 oxoglutarate-dependent dioxygenase superfamily. Polymorphisms within the first intron of the FTO gene have been examined across various populations, yielding disparate findings.The present study aimed to determine the impact of two intronic polymorphisms FTO 30685T/G (rs17817449) and -23525T/A (rs9939609) on the risk of obesity in Punjab, India.
UNASSIGNED: Genotypic and biochemical analysis were done for 671 unrelated participants (obese=333 and non-obese=338) (age≥18 years). Genotyping of the polymorphisms was done by PCR-RFLP method. However, 50% of the samples were sequenced by Sanger sequencing.
UNASSIGNED: Both the FTO variants 30685 (TT vs GG: odds ratio (OR), 2.30; 95% confidence interval (CI), 1.39-3.79) and -23525 (TT vs AA: odds ratio (OR), 2.78; 95% confidence interval (CI), 1.37-5.64) showed substantial risk towards obesity by conferring it 2 times and 3 times, respectively. The analysis by logistic regression showed a significant association for both the variants 30685T/G (rs17817449) and -23525T/A (rs9939609) (OR=2.29; 95%CI: 1.47-3.57) and (OR=5.25; 95% CI: 2.68-10.28) under the recessive genetic model, respectively. The haplotype combination TA (30685; -23525) develops a 4 times risk for obesity (P=0.0001). Among obese, the G allele of 30685T/G and A- allele of -23525T/A showed variance in Body mass index (BMI), waist circumference (WC), waist-to-height ratio(WHtR), systolic blood pressure (SBP), diastolic blood pressure (DBP) and triglyceride(TG).
UNASSIGNED: The present investigation indicated that both the FTO 30685T/G (rs17817449) and -23525T/A (rs9939609) polymorphisms have a key impact on an individual\'s vulnerability to obesity in this population.

The BRAT1 gene plays a crucial role in RNA metabolism and brain development, and mutations in this gene have been associated with neurodevelopmental disorders. The variability in the clinical presentation of BRAT1-related disorders is highlighted, emphasizing the importance of considering this condition in the differential diagnosis of neurodevelopmental disorders. This study aimed to identify a causative variant in an Iranian patient affected by developmental delay, speech delay, seizure, and clubfoot through whole exome sequencing (WES) followed by Sanger sequencing. The WES revealed a novel biallelic variant of the BRAT1, c.398A>G (p.His133Arg), in the patient, which segregated within the family. A literature review suggests that the phenotypic variability associated with BRAT1 mutations is likely due to multiple factors, including the location and type of mutation, the specific functions of the protein, and the influence of other genetic and environmental factors. The phenotypic variability of BRAT1-related disorders underscores the importance of considering BRAT1-related disorders in the differential diagnosis of epileptic encephalopathy with rigidity. These findings provide important insights into the role of BRAT1 in neurodevelopmental disorders and highlight the potential clinical implications of identifying and characterizing novel variants in this gene.

Introduction Thalassemia is a widely prevalent monogenic hematological disorder found worldwide. It exists in two forms: alpha- and beta-thalassemia. Alterations in the hemoglobin subunit beta (HBB) gene cause beta-thalassemia, with missense and point mutations affecting beta-globin synthesis. Consequently, genetic screening for beta-thalassemia is essential for genetic counseling, carrier screening, and prenatal diagnosis. Aim and objective This study aims to examine and identify mutations in the exon 1 region of the HBB gene in beta-thalassemia patients from the Vijayapura region. Methods This study involved 47 clinically diagnosed children with beta-thalassemia from a hospital in Vijayapura, India. Detailed clinical histories of all patients were recorded. Genomic DNA was extracted from the blood samples of these patients and subjected to polymerase chain reaction (PCR) using exon-specific primers for the HBB gene. The PCR products were then sequenced using the capillary-based Sanger sequencing method to identify mutations in the HBB gene. Results A total of 47 clinically diagnosed beta-thalassemia patients were included in the study, comprising 30 males and 17 females, aged between one and 20 years. Sequencing analysis of exon 1 in the beta-globin gene identified 17 beta-thalassemia variants. The most common mutation observed was T>G, G>C, C>A, and C>T in the exon 1 region of the HBB gene.  Conclusion This study identifies the pattern of beta-thalassemia mutations, aiding in the prevention of the disorder through prenatal diagnosis and genetic counseling. Mutations can alter codon sequences, affecting protein production. Research highlights the importance of a primary prevention program to analyze mutations and sequence variations at the molecular level, thereby helping to address numerous genetic disorders.

Cystic fibrosis is caused by biallelic pathogenic variants in the CFTR gene, which contains a polymorphic (TG)mTn sequence (the \"poly-T/TG tract\") in intron 9. While T9 and T7 alleles are benign, T5 alleles with longer TG repeats, e.g., (TG)12T5 and (TG)13T5, are clinically significant. Thus, professional medical societies currently recommend reporting the TG repeat size when T5 is detected. Sanger sequencing is a cost-effective method of genotyping the (TG)mTn tract; however, its polymorphic length substantially complicates data analysis. We developed CFTR-TIPS, a freely available web-based software tool that infers the (TG)mTn genotype from Sanger sequencing data. This tool detects the (TG)mTn tract in the chromatograms, quantifies goodness of fit with expected patterns, and visualizes the results in a graphical user interface. It is broadly compatible with any Sanger chromatogram that contains the (TG)mTn tract ± 15 bp. We evaluated CFTR-TIPS using 835 clinical samples previously analyzed in a CLIA-certified, CAP-accredited laboratory. When operated fully automatically, CFTR-TIPS achieved 99.8% concordance with our clinically validated manual workflow, while generally taking less than 10 s per sample. There were two discordant samples: one due to a co-occurring heterozygous duplication that confounded the tool and the other due to incomplete (TG)mTn tract detection in the reverse chromatogram. No clinically significant misclassifications were observed. CFTR-TIPS is a free, accurate, and rapid tool for CFTR (TG)mTn tract genotyping using cost-effective Sanger sequencing. This tool is suitable both for automated use and as an aid to manual review to enhance accuracy and reduce analysis time.

The Penicillium genus exhibits a broad global distribution and holds substantial economic value in sectors including agriculture, industry, and medicine. Particularly in agriculture, Penicillium species significantly impact plants, causing diseases and contamination that adversely affect crop yields and quality. Timely detection of Penicillium species is crucial for controlling disease and preventing mycotoxins from entering the food chain. To tackle this issue, we implement a novel species identification approach called Analysis of whole GEnome (AGE). Here, we initially applied bioinformatics analysis to construct specific target sequence libraries from the whole genomes of seven Penicillium species with significant economic impact: P. canescens, P. citrinum, P. oxalicum, P. polonicum, P. paneum, P. rubens, and P. roqueforti. We successfully identified seven Penicillium species using the target we screened combined with Sanger sequencing and CRISPR-Cas12a technologies. Notably, based on CRISPR-Cas12a technology, AGE can achieve rapid and accurate identification of genomic DNA samples at a concentration as low as 0.01 ng/µL within 30 min. This method features high sensitivity and portability, making it suitable for on-site detection. This robust molecular approach provides precise fungal species identification with broad implications for agricultural control, industrial production, clinical diagnostics, and food safety.

This case presents a somewhat unique and different phenotype of hereditary spastic paraplegia from previously reported kinase D-interacting substrate of 220 kDa (KIDINS220) gene mutation-related disease. We report a unique putative causative heterozygous mutation in KIDINS220 in a pure hereditary spastic paraplegia (HSP) patient expanding the HSP group further. We also deliberate on how our case was different from prior KIDINS220-related pathologies including spastic paraplegia, intellectual disability, nystagmus, and obesity (SINO) syndrome, and the observation of KIDINS220 and aquaporin-4 (AQP4) downregulation in the ventricular ependymal lining of idiopathic normal pressure hydrocephalus (iNPH) patients. These findings warrant further investigations of the biology of KIDINS220. With the advent of new gene editing technologies like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), variants such as ours provide an opportunity for targeted precision medicine.

UNASSIGNED: Free-living amoebae rarely instigate intracranial infections that may resemble neoplastic conditions on imaging. Naegleria fowleri precipitates an acute, swiftly fatal meningoencephalitis, whereas Acanthamoeba and Balamuthia species typically manifest with a less aggressive onset but carry equally dire consequences.
UNASSIGNED: The case describes a 33-year-old woman with subacute encephalitis caused by Balamuthia mandrillaris. She experienced 2 months of back pain, 1 month of headaches, and 2 weeks of vomiting without fever, recent travel, aquatic activities, or animal exposure. Brain magnetic resonance imaging revealed a sizable, heterogeneous enhancing mass in the right temporal and frontal lobes, accompanied by vasogenic edema and midline shift. Histopathology showed marked inflammation and damage to blood vessels with amoebic trophozoites present. The trophozoites displayed specific characteristics, leading to the diagnosis of amoebic meningoencephalitis. Polymerase chain reaction and Sanger sequencing confirmed B. mandrillaris infection while testing for N. fowleri and Acanthamoeba was negative. Despite antibiotic treatment, the patient\'s condition deteriorated rapidly, resulting in death within 2 weeks of presentation.
UNASSIGNED: This is the first confirmed case of B. mandrillaris central nervous system (CNS) infection from Pakistan. The incidence of this disease is expected to rise due to increasing temperatures due to climate change and the deteriorating quality of the water supply. Balamuthia meningoencephalitis should, therefore be on the differential for non-neoplastic CNS lesions. Furthermore, an atypical histopathologic picture, including the absence of granulomatous inflammation, needs to be recognized.

In recent years, due to the large Romanian community present in Italy, the retail of foods coming from Eastern Europe has increased. The most common type of violation detected in these foods consists of incorrect labeling and species-replacement frauds. In this paper, the compliance of labels of 43 ethnic processed food coming from Eastern Europe and commercialized in Italy was evaluated by means of PCR and Sanger sequencing. Our data revealed 33% of non-compliant labels in samples containing swine, ruminants, and avian ingredients. These results demonstrate that PCR can be easily used for the identification of species in highly processed products, proving to be a rapid, effective, and economic method. On the other hand, samples reporting fish as ingredients highlighted the ineffectiveness of the applied sequencing protocol, due to the low informative property of targeted fragments or to the lack of consensus sequences in the case of uncommon species.

BACKGROUND: The high prevalence of suspected early-onset neonatal sepsis among preterm infants leads to immediate antibiotic administration upon admission. Notably, most blood cultures for suspected early-onset neonatal sepsis do not yield a causative pathogen. This study aimed to assess polymerase chain reaction (PCR) targeting the variable region V4 of the 16S ribosomal gene (16S rDNA) and Sanger sequencing for bacterial identification in preterm infants with suspected early-onset neonatal sepsis.
METHODS: Therefore, this prospective study was conducted. Preterm infants with suspected early-onset neonatal sepsis were included in this study. The three groups were formed based on the risk of infection and clinical sepsis. Blood samples were collected upon admission to the neonatal unit for culture and molecular analysis. PCR amplification and subsequent Sanger sequencing of the V4 region of the 16S rDNA were performed.
RESULTS: Twenty-eight patients were included in this study. Blood cultures were negative in 100% of the patients. Amplification and sequencing of the V4 region identified bacterial genera in 19 patients across distinct groups. The predominant taxonomically identified genus was Pseudomonas.
CONCLUSIONS: Amplifying the 16S rDNA variable region through PCR and subsequent Sanger sequencing in preterm neonates with suspected early-onset neonatal sepsis can enhance the identification of microbial species that cause infection, especially in negative cultures.

Fusarium solani, as an opportunistic pathogen, can infect individuals with immunosuppression, neutropenia, hematopoietic stem cell transplantation (HSCT), or other high-risk factors, leading to invasive or localized infections. Particularly in patients following allogeneic HSCT, Fusarium solani is more likely to cause invasive or disseminated infections. This study focuses on a pediatric patient who underwent HSCT for severe aplastic anemia. Although initial blood cultures were negative, an abnormality was detected in the 1,3-β-D-glucan test (G test) post-transplantation. To determine the causative agent, blood samples were subjected to metagenomic next-generation sequencing (mNGS) and blood cultures simultaneously. Surprisingly, the results of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and mNGS differed slightly, with mNGS identifying Nectria haematonectria, while MALDI-TOF MS based on culture showed Fusarium solani. To clarify the results, Sanger sequencing was performed for further detection, and the results were consistent with those of MALDI-TOF MS. Since the accuracy of Sanger sequencing is higher than that of mNGS, the diagnosis was revised to invasive Fusarium solani infection. With advancements in technology, various detection methods for invasive fungi have been developed in recent years, such as mNGS, which has high sensitivity. While traditional methods may be time-consuming, they are important due to their high specificity. Therefore, in clinical practice, it is essential to utilize both traditional and novel detection methods in a complementary manner to enhance the diagnosis of invasive fungal infections.