Sanger DNA Sequencing Research Articles

Association of pituitary neuroendocrine tumors and neurofibromatosis type 1: assessing causation versus coincidence. Case report

IntroductionPatients with neurofibromatosis type 1 (NF1) are at risk for developing various neoplasms. Since the early twentieth century, multiple cases of pituitary neuroendocrine tumors (PitNETs) occurring in this context have been published. Yet, the role of NF1 (17q11.2) loss-of-function (LOF) variants in pituitary tumorigenesis remains unclear.AimWe report the clinical and molecular characterization of a case of PitNET diagnosed in a patient with NF1. We also review the available data for and against a causal association between NF1 defects and pituitary tumors.MethodsOur patient was recruited via an ongoing prospective study of individuals with neuroendocrine neoplasms. Genetic testing was carried out by means of targeted next generation sequencing (NGS) and Sanger sequencing in blood and tumor DNA, respectively. NF1 expression was analyzed via quantitative polymerase chain reaction (qPCR) in blood and tumor cDNA. Similar cases were searched in the literature.ResultsA 54-year-old-man was incidentally diagnosed with a clinically non-functioning PitNET via brain imaging. He had a personal and family history of NF1 and carried the germline pathogenic variant NF1 (NM_001042492.3): c.147C>A, p.Y49*. Via transsphenoidal surgery, a 16 mm lesion was resected, showing strong granular cytoplasmic immunoreactivity with patchy distribution for NF1 and preserved heterozygosity for the NF1 defect. Additional NGS ruled out germline defects in PitNET-associated genes. By qPCR, NF1 was significantly overexpressed in the tumor when compared with another NF-PitNET, but not when compared with a corticotropinoma. We reviewed twenty-three case reports of PitNETs occurring in patients with either clinical NF1 without genetic study, individuals with NF1 germline variants with or without clinical NF1 or associated with somatic NF1 defects. Predominance of GH-secreting and large PitNETs, with young-onset in around half of the cases, were noticed. Two individuals developed multiple endocrine neoplasia-like phenotypes but tested negative for other relevant genetic defects.ConclusionsAlthough the association of NF1 and PitNETs could be coincidental, the clinical characteristics of the reviewed cases differ from those of typical incidentalomas. NF1 could drive pituitary tumorigenesis via haploinsufficiency, but this hypothesis requires further research. Additional clinical and molecular data from large cohorts of affected individuals should help clarify this question.

Genetic Polymorphisms of UGT1A1, ABCG2, NR1I2 and SLC22A2 in Thai People Living with HIV: Comparative Analysis with other Populations

This study investigated the allele frequencies of UGT1A1, ABCG2, NR1I2, and SLC22A2 in Thai people living with HIV (PLWH) and compared the allele frequencies with those of other populations. Four single nucleotide polymorphisms (SNPs); UGT1A1*6 221G>A, ABCG2 421C>A, NR1I2 63396C>T, and SLC22A2 808G>T were genotyped using TaqMan allelic discrimination assays with a real time polymerase chain reaction system. The genotyping of (TA)n repeat polymorphisms, including UGT1A1*28 (TA7), *36 (TA5), and *37 (TA8), was performed by Sanger DNA sequencing. The Chi-square test was used to evaluate the distribution of observed genotypes according to Hardy-Weinberg equilibrium and to compare genetic polymorphisms between populations. A total of 266 PLWH with 266 blood samples were included in this study. The frequencies of the homozygous variants of UGT1A1*6 221G>A, ABCG2 421C>A, NR1I2 63396C>T, and SLC22A2 808G>T were 0.38%, 6.77%, 34.59%, and 4.14%, respectively. UGT1A1*1/*1 (TA6/TA6) was the most frequent genotype (74.6%) among the (TA)n repeat polymorphisms. The frequencies of UGT1A1*6 221G>A, ABCG2 421C>A, and NR1I2 63396C>T observed in this study were consistent with previous studies in the Thai population. However, these polymorphisms, including SLC22A2 808G>T, show significant deviation from those observed in Caucasian populations.

FLCN Variants in Parathyroid Carcinoma and Atypical Parathyroid Tumors.

Parathyroid carcinoma (PC) and atypical parathyroid tumors (APT) are incompletely understood and pose challenges in definitive diagnosis. FLCN sequence variants have recently been linked to PC and APT. Inactivating mutations in the ubiquitously expressed FLCN tumor suppressor gene, encoding folliculin, cause Birt-Hogg-Dubé syndrome (BHD), a rare tumor predisposition syndrome. Germline inactivating FLCN variants, accompanied by somatic allelic loss, were reported in 2 unrelated patents with PC, both with clinical features, but no diagnosis, of BHD. Somatic frameshift variants of likely pathogenicity were reported in 1 patient with PC and 1 with APT. On the other hand, neither PC nor APT has been reported in sizeable BHD series. To better understand the frequency of FLCN variants in PC and APT, we analyzed a series of 10 patients with sporadic PC and 14 with APT by direct Sanger DNA sequencing. We identified no inactivating FLCN mutations in any of the PC or APT samples examined. A germline missense variant (p.Gly325Val), predicted as benign/tolerated, was seen in 1 PC and a synonymous variant in 1 APT. The absence of pathogenic mutations detected in our series of PC and APT further suggests that FLCN variants are rare in these tumors. Nevertheless, the potential roles of FLCN in the pathogenesis of PC and APT merits further consideration and study.

Epidemiology of norovirus disease in the first 2 years of life: A prospective multisite cohort study in Lima, Peru.

Norovirus is associated with 18% of acute gastroenteritis (AGE) cases worldwide. We aim to document the norovirus-related AGE incidence in peri-urban areas of Lima (Peru), evaluating the potential impact of a norovirus vaccine introduction. A prospective, community-based pediatric cohort study was established at two sites in Lima. Healthy children between 5 and 18 months were contacted weekly for AGE detection during a 6-month period. Stool samples from AGE cases were tested for norovirus by RT-PCR. Incidence and coinfection of norovirus-associated AGE were analyzed. All norovirus-positive samples were genotyped by Sanger DNA sequencing. Among 498 enrolled children, 461 (93%) completed the follow-up period. We detected 799 AGE cases, yielding 676 valid stool samples. Norovirus was detected in 216 samples (32%), with an incidence of 7.7 episodes per 100 child-months (95% CI: 6.7-8.8). Genotypes GII.4 (31%) and GII.6 (22%) were frequent. Campylobacter (43%) and Salmonella spp. (15%) were the most common coinfections with norovirus. Ninety-five percent of study children had received an oral rotavirus vaccine. Norovirus was the second most frequent cause of AGE in this Peruvian cohort with high rotavirus vaccine coverage. An effective norovirus vaccine would have an important public health benefit in this population.

Genotype and Haplotype Analysis With In Silico Prediction of TMPRSS2 Gene in Jordanian Population.

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly become a global health concern. The entry of the virus into host cells is facilitated by the transmembrane protease serine 2 (TMPRSS2) receptor, and genetic variations in the TMPRSS2 gene may influence disease susceptibility. However, there is a lack of knowledge regarding TMPRSS2 genetic variants and haplotypes in the Jordanian population. This study aimed to characterize the genotype and haplotype variations in the TMPRSS2 binding domain with SARS-CoV-2 among Jordanian volunteers. The binding domain of TMPRSS2 with SARS-CoV-2 (Exons 9 and 10) was amplified using polymerase chain reaction (PCR) for a random sample of 120 healthy unrelated Jordanian volunteers, followed by Sanger DNA sequencing for the PCR products. The effect of the novel genetic variants on the TMPRSS2 protein structure was predicted using in silico methods. The results showed significant (p<0.05, chi-square) allele frequencies for known TMPRSS2 variants, with c.888C>T being the most prevalent among Jordanian volunteers. Novel genetic variants, including c.869A>G and c.923T>A, were also identified, with the latter being the most common novel variant. Haplotype analysis showed that the most prevalent TMPRSS2 haplotype is c.911G/1051A/1052T/1010+45C/1011-38T/1011-52C/1011-54A. In silico programs predicted that TMPRSS2 c.923T>A and c.1052T>A variants affect transmembrane proteins and catalytic sites. This research provides information about the gene structure of the TMPRSS2 binding domain in Jordanians. Some of the identified variants, especially c.923T>A, may influence protein function, warranting further in vitro and in vivo investigations. In addition, further clinical research studies are needed to link the identified TMPRSS2 variants with COVID-19 susceptibility and severity among Jordanians.

Whole exome sequencing revealed ultra-rare genetic variations in juvenile myoclonic epilepsy.

Juvenile Myoclonic Epilepsy (JME) is the most common adolescent and adult-onset genetic generalized epilepsy. In this study, we aimed to identify all rare variants present in exons and exon-intron junctions in patients who met the criteria of JME, determine potentially pathogenic variants, and find the assumed genotype/phenotype correlation between the identified variants and the JME clinical features. Whole Exome Sequencing (WES) was performed for ten JME patients from different families. Validation, co-segregation and mode of inheritance were determined using Sanger DNA sequencing. Predictable damaging variants were found in six families with positive co-segregation. Eight variants in eight genes (SCN1B, KCNQ2, CACNA1I, GABRA3, BSN, RYR3, SEZ6, and RYR2) and one novel variant in (TNR) gene were found to be associated with JME. All these genes play key roles in the interactions between neurons, neurotransmitter release, and maintenance of the balance between neuronal excitation and inhibition. Since the identified genes are involved in the molecular mechanisms underlying seizures, such variants can potentially be epileptogenic. In conclusion, the identified variants that co-segregate with JME symptoms and likely contribute in creating the adequate genetic background for the JME phenotype.

Forget-me-not phylogenomics: Improving the resolution and taxonomy of a rapid island and mountain radiation in Aotearoa New Zealand (Myosotis; Boraginaceae)

Island and mountain systems represent natural laboratories for studies of species radiations, but they often present several challenges for phylogenetic inference and species delimitation. The southern hemisphere forget-me-nots (Myosotis, Boraginaceae) comprise a geologically recent radiation centred in Aotearoa New Zealand, a mountainous archipelago, with about 50 species that are morphologically and ecologically divergent but lack genetic variation sufficient to resolve phylogenetic relationships and species boundaries using standard DNA Sanger sequencing markers, AFLPs, or microsatellites. Many of these Myosotis species are geographically restricted in alpine areas, uncommon or threatened, have polyploid and dysploid genomes, and are of high taxonomic and conservation priority. Here we present phylogenomic analyses using target-capture of Angiosperms353 baits, and genome skimming of whole plastomes and nrDNA, to improve resolution of the radiation, explore biogeographic and morphological patterns within it, and address specific taxonomic questions for each species. Our comprehensive sampling includes over 300 individuals representing nearly all species from New Zealand and Australia, which is ∼ 2–3 × more taxon sampling and ∼ 80–120 × more molecular data than previously published for Myosotis. Exploration of different data filtering, curation and analyses (coalescent vs. concatenation) improved the resolution of the Angiosperms353 tree, which despite short backbone branches with low support values, showed taxonomic and geographic patterns, including multiple switches between ebracteate and bracteate inflorescences and multiple expansions within New Zealand from Te Waipounamu South Island to Te Ika-a-Māui North Island, Rakiura Stewart Island, subantarctic islands, and Australia. Some of these patterns were also seen in the genome skimming datasets, and comparison of the three datasets was useful for improving our understanding of the taxonomy and resolution of this radiation. Although this phylogenomic study does not fully overcome all of the challenges regarding species delimitation of this rapid island and mountain species radiation, it nevertheless makes an important contribution to an integrative taxonomic revision of the southern hemisphere species of Myosotis.

Students’ Habits and Identification of Bacteria on Inanimate Surface of Educational Setting

Introduction: Diponegoro Clinic reported 29.1% of students seek infectious diseases treatment. Student habits in the classroom were thought to play roles in the presence of bacteria. Surfaces of inanimate objects in the classroom were potential source for bacteria. The research objectives were analyzing student’s habits and identifying bacteria on the surface of inanimate objects in the classroom. Methods: Four types of samples, including the surfaces of tables, chairs, flips of air conditioners, and floors were taken from 13 faculties at Universitas Diponegoro. Plate count agar media were used to isolate bacterial colonies, and PCR analysis was performed for DNA extraction and amplification. DNA Sanger sequencing techniques were used for genetic bacterial identification. The online questionnaire was used to assess student habits in the classroom. Two hundred students responded. Results and Discussion: Acinetobacter baumannii and Staphylococcus aureus were found in classrooms. These bacteria were associated with respiratory tract infections. This study revealed that 86.5% were between the ages of 17-21, 60.81% were from outside Semarang City, and 88.33% lived in Semarang City. About 60.81% of respondents studied in health sciences. Furthermore, it was reported that 66.67% of respondents were sick in the last few weeks, attended class despite being sick (72.52%), and coughed and sneezed in class (40.99%). Conclusion: Bacteria associated with respiratory tract infection were found. Students' habits in the classroom were potentially caused by the presence of these bacteria. The use of antibacterial agents could reduce the presence of bacteria on inanimate object surfaces.

Emergence and spread of resistant and biofilm-forming Acinetobacter baumannii in critically ill COVID-19 patients

The COVID-19 pandemic has raised concerns beyond the viral infection itself. Bacterial co-infections, particularly those involving Acinetobacter baumannii, have become a significant worry in critically ill COVID-19 patients. A. baumannii is an opportunistic pathogen that can cause nosocomial infections, especially in patients with compromised immune systems. This study investigated 36 A. baumannii isolates obtained from COVID-19 patients during a concurrent outbreak. The isolates were collected over two years through routine medical requests sent to the Clinical Microbiology laboratory. Identification of the strains was confirmed through biochemical tests, the Phoenix BD® Automated Microbiology System, and MALDI-TOF Mass Spectrometry. The study assessed the antimicrobial sensitivity of the isolates, with a specific focus on resistance to the beta-lactam group as well as aminoglycosides. The presence of specific antibiotic resistance genes (blaOXA-23, -24, -51 and -58, blaKPC, blaSHV, blaIMP, blaVIM, aac(6′)-Ib, ant(3″)-Ia, and aph(3′)-Ia) was investigated using PCR and Sanger DNA sequencing. Biofilm-forming capabilities of the isolates were also evaluated. The findings revealed diverse resistance profiles, with a high prevalence of resistant strains, including resistance to carbapenems. Genetic analysis suggested potential clonal spread of certain strains within the hospital setting. Moreover, a significant proportion of the isolates demonstrated strong biofilm-forming abilities, which can enhance persistence and antibiotic resistance. In conclusion, this study highlights the need for vigilant monitoring and targeted interventions to address bacterial co-infections in COVID-19 patients. The diversity in resistance patterns, potential clonal spread, and robust biofilm-forming abilities among A. baumannii isolates underscore the importance of addressing this issue to better manage and treat critically ill COVID-19 patients.

Dental abnormalities observed in the oculo-facio-cardio-dental (OFCD) syndrome present in two Czech families bearing novel de novo BCOR pathogenic variants

BackgroundThe oculo-facio-cardio-dental syndrome (OFCD) is an ultra-rare multiple congenital anomaly. This report describes clinical findings emphasising dental phenotype in five, molecularly confirmed, female cases from two Czech families.Case presentationDental examinations were carried out. An orthopantomogram was taken in three patients, and all patients’ intraoral cavities and teeth were photographed. Exome sequencing was performed in both probands. Results were validated by Sanger DNA sequencing which was also used to follow segregation of the variants in first-degree relatives. Dental abnormalities and congenital cataracts were present in all five cases, whilst other signs were variable and included facial dysmorphism, microphthalmia, and cardiac and skeletal abnormalities. Two individuals had cleft lip and/or cleft palate. Radiculomegaly occurred in three patients with permanent teeth and was diagnosed on orthopantomograms. Two patients had agenesis of permanent teeth. Malocclusion was also present in two patients due to crowding and a Class III malocclusion and mandibular overjet. De novo novel pathogenic variants in the BCOR gene were identified; c.2382del p.(Lys795Argfs*12) and c.3914dup p.(Gln1306Alafs*20) and co-segregated with the disease in each family.ConclusionsThe OFCD syndrome has a unique dental phenotype and dentists should be aware of signs of this ultra-rare genetic disorder. All patients with congenital cataracts and dental abnormalities, including those without a family history, should be referred for genetic testing and indicated to specialised dental care.

TFL1-like genes in Vigna unguiculata (L.) Walp. with different growth habit types

Background. Vigna unguiculata (L.) Walp. is among important legume crops. Agricultural producers prefer cultivars suitable for mechanized cultivation, with a determinate growth habit type. Plant architectonics depends on the functioning of the apical meristem, while the transition to the reproductive stage is controlled by a set of genes, including the TFL1 gene. Analyzing the genes responsible for the growth habit type is relevant for more efficient and rapid development of high-tech cultivars. Materials and methods. Using the Sanger DNA sequencing method, the primary structure of TFL1-like genes was studied in six cowpea accessions with different growth habit types and architectonics.Results. Promoter regions and coding parts of TFL1-like genes (VuTFL1.1, VuTFL1.2, VuATC, and VuBFT) were sequenced and analyzed. Information about the genes is available in the NCBI nucleotide sequence database. A comparative study showed that there were no exon differences between different genotypes. Rearrangements were found in the introns and the promoter region, but no relationship was traced between these rearrangements and the phenotype in terms of growth habit types or architectonics.Conclusion. The next step towards understanding the role of TFL1-like genes requires obtaining knockout lines based on these genes and studying their phenotype. Meanwhile, the results of this analysis call for a need to consider a wider range of cowpea genes potentially associated with the variability of stem growth habit types and plant architectonics.

Whole Exome Sequencing and Panel-Based Analysis in 176 Spanish Children with Neurodevelopmental Disorders: Focus on Autism Spectrum Disorder and/or Intellectual Disability/Global Developmental Delay

Background: Neurodevelopmental disorders (NDDs) represent a significant challenge in pediatric genetics, often requiring advanced diagnostic tools for the accurate identification of genetic variants. Objectives: To determine the diagnostic yield of whole exome sequencing (WES) with targeted gene panels in children with neurodevelopmental disorders (NDDs). Methods: This observational, prospective study included a total of 176 Spanish-speaking pediatric patients with neurodevelopmental disorders (NDDs), encompassing intellectual disability (ID), global developmental delay (GDD), and/or autism spectrum disorder (ASD). Participants were recruited from January 2019 to January 2023 at a University Hospital in Madrid, Spain. Clinical and sociodemographic variables were recorded, along with genetic study results. The age range of the subjects was 9 months to 16 years, and the percentage of males was 72.1%. The diagnostic yield of whole exome sequencing (WES) was calculated both before and after parental testing via Sanger DNA sequencing. Results: The study included 176 children: 67 (38.1%) with ID, 62 (35.2%) with ASD, and 47 (26.7%) with ASD + ID. The diagnostic yield of proband-only exome sequencing was 12.5% (22/176). By group, the diagnostic yield of proband-only exome sequencing was 3.2% in the ASD, 12.7% in the ASD + ID, and 20.8% in the ID group. Variants of uncertain significance (VUS) were found in 39.8% (70/176). After parental testing, some variants were reclassified as “likely pathogenic”, increasing the diagnostic yield by 4.6%, with an overall diagnostic yield of 17.1%. Diagnostic yield was higher in patients with syndromic ID (70.6%% vs. 29.4%; p = 0.036). Conclusions: A sequential approach utilizing WES followed by panel-based analysis, starting with the index case and, when appropriate, including the parents, proves to be a cost-effective strategy. WES is particularly suitable for complex conditions, as it allows for the identification of potentially causative genes beyond those covered by targeted panels, providing a more comprehensive analysis. Including parental testing enhances the diagnostic yield and improves accuracy, especially in cases with variants of uncertain significance (VUS), thereby advancing our understanding of NDDs.

Genomic Epidemiology of Extrapulmonary Nontuberculous Mycobacteria Isolates at Emerging Infections Program Sites - United States, 2019-2020.

Nontuberculous mycobacteria (NTM) cause pulmonary and extrapulmonary infections. Although isolation of NTM from clinical specimens has increased nationally, few studies delineated the molecular characteristics of extrapulmonary NTM. Extrapulmonary isolates were collected by four Emerging Infections Program sites from October 2019 to March 2020 and underwent laboratory characterization, including matrix-assisted laser desorption ionization-time of flight mass spectrometry, Sanger DNA sequencing, and whole genome sequencing. Bioinformatics analyses were employed to identify species, sequence types (STs), antimicrobial resistance (AR), and virulence genes; isolates were further characterized by phylogenetic analyses. Among 45 isolates, the predominant species were Mycobacterium avium (n=20, 44%), Mycobacterium chelonae (n=7, 16%), and Mycobacterium fortuitum (n=6, 13%). The collection represented 31 STs across 10 species; the most common ST was ST11 (M. avium, n=7). Mycobacterium fortuitum and Mycobacterium abscessus isolates harbored multiple genes conferring resistance to aminoglycosides, beta-lactams, and macrolides. No known AR mutations were detected in rpoB, 16S, or 23S rRNAs. Slow-growing NTM species harbored multiple virulence genes including type-VII secretion components, adhesion factors, and phospholipase C. Continued active laboratory- and population-based surveillance will further inform the prevalence of NTM species and STs, monitor emerging clones, and allow AR characterization.

7826 The Identification of an Activating ESR1 Mutation in an Aggressive Prolactinoma Enabled the Use of Personalized Treatment

Abstract Disclosure: T. Paes: None. J.B. Mebarak: None. J. C. Magnotto: None. G. A. Stamatiades: None. Y. Kuang: None. C. Paweletz: None. E.R. Laws: None. R.S. Carroll: None. R. Jeselsohn: None. D. R. Mohan: None. A. Marcondes Lerario: None. W. L. Bi: None. D. A. Reardon: None. D. M. Meredith: None. U.B. Kaiser: None. A. Abreu: None. Introduction: Prolactinomas are benign tumors typically treated with dopamine agonists; few progress on medical therapy, through unclear molecular mechanisms. Although the SF3B1 mutation has been identified in one study as a driver of aggressive prolactinomas, in most cases no genetic mutations have been reported. We aimed to identify genetic alterations associated with prolactinomas using a gene panel. Method and Results: Oncopanel, a massively parallel sequencing panel, was performed to identify genomic variants and copy number variation (CNV) in a cohort of 21 patients with prolactinomas. No SF3B1 pathogenic variants were identified in this cohort. A MEN1 mutation was detected in a macroadenoma resistant to dopamine agonist. In a case of an extremely aggressive prolactinoma diagnosed in a 49-year-old woman, a somatic ESR1 [encoding estrogen receptor alpha (ERα)] mutation (ESR1Y537S) was identified in tissue from the patient’s fourth surgery. Using droplet digital PCR (ddPCR), ESR1Y537S was also detected in circulating cell-free DNA. We aimed to investigate whether ESR1Y537S may have driven initial tumor formation, or whether it was acquired as the tumor progressed. To this end, Sanger sequencing and ddPCR analyses of DNA from the first two resected tumors were performed and failed to identify ESR1Y537S. In the CNV analysis, the ESR1-mutated prolactinoma had the highest number of CNVs, compared to the rest of the cohort. Despite efforts to control tumor growth with multiple therapeutic modalities, the mutated prolactinoma had progressively enlarged post-menopausally, with invasion of surrounding structures. ESR1Y537S is a hotspot mutation in metastatic breast cancer. In breast cancer cells, ESR1Y537S activates the ERα independent of ligand binding, resulting in high cell proliferation and conferring resistance to classic hormonal treatment in ER-positive breast cancer. Elacestrant, a second-line ER degrader, increases overall free-survival in patients with resistant breast cancer and ESR1Y537S. Given the lack of response to multimodality therapy, off-label elacestrant was initiated after the third cycle of radiotherapy was completed in the patient with the aggressive prolactinoma. The combination of radiotherapy with this personalized treatment targeting ESR1 activity (elacestrant) was able to control tumor growth and significantly reduce prolactin levels. Conclusion:ESR1 regulates lactotroph differentiation and proliferation, possibly contributing to prolactinoma tumorigenesis. Our data support a role for ESR1Y537S in this prolactinoma’s unusually aggressive behavior in the postmenopausal setting. Molecular profiling revealing the mutation (ESR1Y537S) allowed the patient to receive a targeted therapy which, together with radiotherapy, resulted in a decrease in prolactin levels and a remarkable improvement in disease control. Presentation: 6/2/2024

Acinetobacter baumannii clinical isolates from outbreaks in Erbil hospitals after the COVID-19 pandemic.

Acinetobacter baumannii is endemic in hospital environments, and since the coronavirus disease 2019 (COVID-19) pandemic, multidrug-resistant A. baumannii has become more potent. This potential evolution is driven by the undetectable numbers of gene resistances it has acquired. We evaluated the antibiotic-resistance genes in isolates from patients in Erbil hospitals. This is the first study to demonstrate the antimicrobial resistance epidemic in Erbil, Iraq. A total of 570 patients, including 100 COVID-19 patients were tested. Isolate identification, characterization, antibiotics susceptibility test, polymerase chain reaction (PCR) amplification of the antibiotic resistance genes in both bacterial chromosome and plasmid, 16S-23S rRNA gene intergenic spacer (ITS) sequencing using the Sanger DNA sequencing, and phylogenetic analysis were used in this study. Only 13% of A. baumannii isolates were from COVID-19 patients. All isolates were multi-drug resistant due because of 24 resistance genes located in both the bacterial chromosome or the plasmid. blaTEM gene was detected in the isolates; however, aadB was not detected in the isolated bacteria. New carbapenemase genes were identified by Sanger sequencing and resistance genes were acquired by plasmids. The study identified metabolic differences in the isolates; although all the strains used the coumarate pathway to survive. Several resistance genes were present in the isolates' plasmids and chromosome. There were no strong biofilm producers. The role of the plasmid in A. baumannii resistance development was described based on the results.

Sanger Sequencing Reveals Novel Variants in GLO-1, ACE, and CBR1 Genes in Patients of Early and Severe Diabetic Nephropathy.

Background and Objectives: Diabetes is a global health issue, with approximately 50% of patients developing diabetic nephropathy (DN) and 25% experiencing early and severe forms of the disease. The genetic factors contributing to rapid disease progression in a subset of these patients are unclear. This study investigates genetic variations in the GLO-1, CBR-1, and ACE genes associated with early and severe DN. Materials and Methods: Sanger DNA sequencing of the exons of CBR1, GLO1, and ACE genes was conducted in 113 patients with early and severe DN (defined as occurring within 10 years of the diagnosis of diabetes and with eGFR < 45 mL/min/1.73 m2) and 100 controls. The impact of identified genetic variations was analyzed using computational protein models created in silico with SWISS-Model and SWISS-Dock for ligand binding interactions. Results: In GLO1, two heterozygous missense mutations, c.102G>T and c.147C>G, and one heterozygous nonsense mutation, c.148G>T, were identified in patients. The SNP rs1049346 (G>A) at location 6:38703061 (GRCh38) was clinically significant. The c.147C>G mutation (C19S) was associated with ligand binding disruption in the GLO1 protein, while the nonsense mutation resulted in a truncated, non-functional protein. In CBR1, two heterozygous variations, one missense c.358G>A, and one silent mutation c.311G>C were observed, with the former (D120N) affecting the active site. No significant changes were noted in ACE gene variants concerning protein structure or function. Conclusions: The study identifies four novel and five recurrent mutations/polymorphisms in GLO1, ACE, and CBR1 genes associated with severe DN in Pakistani patients. Notably, a nonsense mutation in GLO1 led to a truncated, non-functional protein, while missense mutations in GLO1 and CBR1 potentially disrupt enzyme function, possibly accelerating DN progression.

Novel Single-Nucleotide Polymorphisms (SNPs) and Genetic Studies of the Shadow of Prion Protein (SPRN) in Quails.

Prion diseases are a group of deadly neurodegenerative disorders caused by the accumulation of the normal prion protein (PrPC) into misfolding pathological conformations (PrPSc). The PrP gene is essential for the development of prion diseases. Another candidate implicated in prion pathogenesis is the shadow of the prion protein (SPRN) gene. To date, genetic polymorphisms of the SPRN gene and the structure of the Sho protein have not been explored in quails. We used polymerase chain reaction (PCR) to amplify the SPRN gene sequence and then conducted Sanger DNA sequencing to identify the genetic polymorphisms in quail SPRN. Furthermore, we examined the genotype, allele, and haplotype frequencies, and assessed the linkage disequilibrium among the genetic polymorphisms of the SPRN gene in quails. Additionally, we used in silico programs such as MutPred2, SIFT, MUpro, AMYCO, and SODA to predict the pathogenicity of non-synonymous single-nucleotide polymorphisms (SNPs). Alphafold2 predicted the 3D structure of the Sho protein in quails. The results showed that a total of 13 novel polymorphisms were found in 106 quails, including 4 non-synonymous SNPs. Using SIFT and MUpro in silico programs, three out of the four non-synonymous SNPs (A68T, L74P, and M105I) were predicted to have deleterious effects on quail Sho. Furthermore, the 3D structure of quail Sho was predicted to be similar to that of chicken Sho. To our knowledge, this is the first report to investigate the genetic and structural properties of the quail SPRN gene.

Expression of Autophagy-Related Proteins in Microlissencephaly Associated with a Novel Variant in the WDR81 Gene

Introduction: Microlissencephaly is a subtype of congenital microcephaly characterized by extreme microcephaly with simplified gyral pattern. Other brain malformations may accompany it. WDR81 encodes a multi-domain transmembrane protein that is predominantly expressed in the brain and is thought to play a role in endolysosomal trafficking and autophagy. Methods: We reported two siblings with microlissencephaly born to consanguineous Turkish parents and reviewed all previously reported patients with WDR81 variants presenting with severe microcephaly accompanied by congenital brain malformations. Whole-exome sequencing was performed on both siblings. Sanger DNA sequencing was performed on the patients’ parents. We also examined LC3, p62, and Beclin 1 mRNA and protein expression levels from blood samples of both siblings using real-time PCR and Western blotting, respectively. Results: Whole-exome sequencing revealed a novel biallelic c.4157+5G>A splice variant in the WDR81 gene in both siblings. In our study, LC3, p62, and Beclin 1 mRNA expression levels were high, LC3 protein expression level was also high and p62 and Beclin 1 protein expression levels were low. Conclusion: High LC3 and low p62 protein expression levels supported studies concluding that WDR81 inhibits autophagy through PI3KC3 inhibition, while low Beclin 1 protein expression level supported studies concluding that autophagy is suppressed in case of loss of function variants in the WDR81 gene. We suggest that due to its role in endolysosomal trafficking, WDR81-related diseases should be included in vesicular trafficking disorders.

Association of the rs1042522 SNP with prostate cancer risk: a study of cancer tissues, primary tumor cultures, and serum samples from a Spanish Caucasian population.

Prostate cancer (PCa) is a leading cause of cancer-related deaths in European men, emphasizing the urgent need for effective risk assessment strategies. The TP53 gene, a tumor suppressor gene frequently mutated in cancer, commonly harbors the rs1042522 single nucleotide polymorphism (SNP), known as the P72R SNP, which may influence PCa susceptibility. This study investigated the prevalence of the P72R SNP in European Caucasian PCa samples and its association with PCa risk. Genotyping was conducted on 12 hormone-naïve aggressive PCa cultures (hnPCs) from untreated patients (Gleason ≥8), 11 radical prostatectomies (RP), and 94 serum samples using DNA Sanger sequencing and melting curve analysis. Comparative analysis utilized data from the GnomAD database's European Caucasian non-cancer population. Our results demonstrate a significantly higher frequency of the P72R SNP in PCa samples and serums compared to the general European non-cancer population. A robust and statistically significant association (p < 0.0001) between the SNP and prostate cancer risk was identified, with an odds ratio of 7.937 (95% CI 5.37-11.00). Notably, the G allele (R72) showed a pronounced prevalence in high Gleason score (≥8) patients, although statistical significance was not reached. These results highlight a potential association with undifferentiated and malignant PCa lesions. The compelling association between the P72R SNP and prostate cancer risk underscores the potential utility of this marker for the early identification of patients at risk of aggressive metastatic prostate cancer. This insight could empower further research to intervene at an early stage by offering enhanced opportunities for timely and targeted interventions.

Genetic Variants in Vitamin-D Metabolism Genes (rs1155563, rs12785878 and rs10500804) among Females with Type-2 Diabetes Mellitus in Saudi Arabia.

Hypovitaminosis D has shown to be linked with T2DM development and control in numerous studies. The association of SNPs in genes related to VitD metabolism with T2DM has not been sufficiently studied. Consequently, our aim in the present study was to explore the association between genetic variants in genes connected with VitD, mainly a SNP in GC (rs1155563), a SNP in DHCR7 (rs12785878) and a SNP in CYP2R1 (rs10500804) with glycaemic parameters in females with T2DM in Saudi Arabia. The cross-sectional study included 149 females (age 38-52 years) with T2DM from Jeddah, Saudi Arabia (September 2022-March 2023). Blood was extracted from the participants for biochemical tests including measuring VitD [25(OH)D] concentration, parameters of glycaemia (HbA1c, insulin, fasting glucose and insulin sensitivity indices including HOMA2-IR and HOMA2-%β), and for genomic DNA isolation. Sanger DNA sequencing was used to screen for VitD genetic polymorphisms (rs1155563, rs12785878 and rs10500804). Minor allele frequency for rs1155563C, rs12785878T and rs10500804G was 0.21, 0.23 and 0.37, respectively. Levels of 25(OH)D and glycaemic parameters as well did not show any significant difference between the genotypes of each SNP. This study showed lack of association of rs1155563 in GC, rs12785878 in DHCR7 and rs10500804 in CYP2R1 with VitD level primarily and with glycaemic parameters secondarily. Additional research is required to explore further other VitD genetic polymorphisms influencing T2DM which might lead consequently to genetically-based personalized management for T2DM.