Dysregulated Urinary Extracellular Vesicle Small RNAs in Diabetic Nephropathy: Implications for Diagnosis and Therapy.

Background: Diabetic nephropathy (DN) represents a major chronic kidney disorder and a leading cause of end-stage renal disease (ESRD). Small RNAs have been showing great promise as diagnostic markers as well as drug targets. Identifying dysregulated micro RNAs (miRNAs) could help in identifying disease biomarkers and investigation of downstream interactions, shedding light on the molecular pathophysiology of DN. In this study, we analyzed small RNAs within human urinary extracellular vesicles (ECVs) from DN patients using small RNA next-generation sequencing. Method: In this cross-sectional study, urine samples were collected from 88 participants who were divided into 3 groups: type 2 diabetes (T2D) with DN (T2D + DN, n = 20), T2D without DN (T2D - DN, n = 40), and healthy individuals (n = 28). The study focused on isolating urinary ECVs to extract and sequence small RNAs. Differentially expressed small RNAs were identified, and a functional enrichment analysis was conducted. Results: The study revealed a distinct subset of 13 miRNAs and 10 Piwi-interacting RNAs that were significantly dysregulated in urinary ECVs of the DN group when compared to other groups. Notably, miR-151a-3p and miR-182-5p exhibited a unique expression pattern, being downregulated in the T2D - DN group, and upregulated in the T2D + DN group, thus demonstrating their effectiveness in distinguishing patients between the 2 groups. Eight driver genes were identified PTEN, SMAD2, SMAD4, VEGFA, CCND2, CDK6, LIN28B, and CHD1. Conclusion: Our findings contribute valuable insights into the pathogenesis of DN, uncovering novel biomarkers and identifying potential therapeutic targets that may aid in managing and potentially decelerating the progression of the disease.

Evaluation of HLA typing content of next-generation sequencing datasets from family trios and individuals of arab ethnicity.

Introduction: HLA typing is a critical tool in both clinical and research applications at the individual and population levels. Benchmarking studies have indicated HLA-HD as the preferred tool for accurate and comprehensive HLA allele calling. The advent of next-generation sequencing (NGS) has revolutionized genetic analysis by providing high-throughput sequencing data. This study aims to evaluate, using the HLA-HD tool, the HLA typing content of whole exome, whole genome, and HLA-targeted panel sequence data from the consanguineous population of Arab ethnicity, which has been underrepresented in prior benchmarking studies. Methods: We utilized sequence data from family trios and individuals, sequenced on one or more of the whole exome, whole genome, and HLA-targeted panel sequencing technologies. The performance and resolution across various HLA genes were evaluated. We incorporated a comparative quality control analysis, assessing the results obtained from HLA-HD by comparing them with those from the HLA-Twin tool to authenticate the accuracy of the findings. Results: Our analysis found that alleles across 29 HLA loci can be successfully and consistently typed from NGS datasets. Clinical-grade whole exome sequencing datasets achieved the highest consistency rate at three-field resolution, followed by targeted HLA panel, research-grade whole exome, and whole genome datasets. Discussion: The study catalogues HLA typing consistency across NGS datasets for a large array of HLA genes and highlights assessments regarding the feasibility of utilizing available NGS datasets in HLA allele studies. These findings underscore the reliability of HLA-HD for HLA typing in underrepresented populations and demonstrate the utility of various NGS technologies in achieving accurate HLA allele calling.

Unraveling Verapamil's Multidimensional Role in Diabetes Therapy: From ß-Cell Regeneration to Cholecystokinin Induction in Zebrafish and MIN6 Cell-Line Models.

This study unveils verapamil's compelling cytoprotective and proliferative effects on pancreatic ß-cells amidst diabetic stressors, spotlighting its unforeseen role in augmenting cholecystokinin (CCK) expression. Through rigorous investigations employing MIN6 ß-cells and zebrafish models under type 1 and type 2 diabetic conditions, we demonstrate verapamil's capacity to significantly boost ß-cell proliferation, enhance glucose-stimulated insulin secretion, and fortify cellular resilience. A pivotal revelation of our research is verapamil's induction of CCK, a peptide hormone known for its role in nutrient digestion and insulin secretion, which signifies a novel pathway through which verapamil exerts its therapeutic effects. Furthermore, our mechanistic insights reveal that verapamil orchestrates a broad spectrum of gene and protein expressions pivotal for ß-cell survival and adaptation to immune-metabolic challenges. In vivo validation in a zebrafish larvae model confirms verapamil's efficacy in fostering ß-cell recovery post-metronidazole infliction. Collectively, our findings advocate for verapamil's reevaluation as a multifaceted agent in diabetes therapy, highlighting its novel function in CCK upregulation alongside enhancing ß-cell proliferation, glucose sensing, and oxidative respiration. This research enriches the therapeutic landscape, proposing verapamil not only as a cytoprotector but also as a promoter of ß-cell regeneration, thereby offering fresh avenues for diabetes management strategies aimed at preserving and augmenting ß-cell functionality.

Global analysis of urinary extracellular vesicle small RNAs in autosomal dominant polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent monogenic renal disease progressing to end-stage renal disease. There is a pressing need for the identification of early ADPKD biomarkers to enable timely intervention and the development of effective therapeutic approaches. Here, we profiled human urinary extracellular vesicles small RNAs by small RNA sequencing in patients with ADPKD and compared their differential expression considering healthy control individuals to identify dysregulated small RNAs and analyze downstream interaction to gain insight about molecular pathophysiology. METHODS: This is a cross-sectional study where urine samples were collected from a total of 23 PKD1-ADPKD patients and 28 healthy individuals. Urinary extracellular vesicles were purified, and small RNA was isolated and sequenced. Differentially expressed Small RNA were identified and functional enrichment analysis of the critical miRNAs was performed to identify driver genes and affected pathways. RESULTS: miR-320b, miR-320c, miR-146a-5p, miR-199b-3p, miR-671-5p, miR-1246, miR-8485, miR-3656, has_piR_020497, has_piR_020496 and has_piR_016271 were significantly upregulated in ADPKD patient urine extracellular vesicles and miRNA-29c was significantly downregulated. Five 'driver' target genes (FBRS, EDC3, FMNL3, CTNNBIP1 and KMT2A) were identified. CONCLUSIONS: The findings of the present study make significant contributions to the understanding of ADPKD pathogenesis and to the identification of novel biomarkers and potential drug targets aimed at slowing disease progression in ADPKD.

Association between alleles, haplotypes, and amino acid variations in HLA class II genes and type 1 diabetes in Kuwaiti children.

Type 1 diabetes (T1D) is a complex autoimmune disorder that is highly prevalent globally. The interactions between genetic and environmental factors may trigger T1D in susceptible individuals. HLA genes play a significant role in T1D pathogenesis, and specific haplotypes are associated with an increased risk of developing the disease. Identifying risk haplotypes can greatly improve the genetic scoring for early diagnosis of T1D in difficult to rank subgroups. This study employed next-generation sequencing to evaluate the association between HLA class II alleles, haplotypes, and amino acids and T1D, by recruiting 95 children with T1D and 150 controls in the Kuwaiti population. Significant associations were identified for alleles at the HLA-DRB1, HLA-DQA1, and HLA-DQB1 loci, including DRB1*03:01:01, DQA1*05:01:01, and DQB1*02:01:01, which conferred high risk, and DRB1*11:04:01, DQA1*05:05:01, and DQB1*03:01:01, which were protective. The DRB1*03:01:01~DQA1*05:01:01~DQB1*02:01:01 haplotype was most strongly associated with the risk of developing T1D, while DRB1*11:04-DQA1*05:05-DQB1*03:01 was the only haplotype that rendered protection against T1D. We also identified 66 amino acid positions across the HLA-DRB1, HLA-DQA1, and HLA-DQB1 genes that were significantly associated with T1D, including novel associations. These results validate and extend our knowledge on the associations between HLA genes and T1D in Kuwaiti children. The identified risk alleles, haplotypes, and amino acid variations may influence disease development through effects on HLA structure and function and may allow early intervention via population-based screening efforts.

Differentially methylated and expressed genes in familial type 1 diabetes.

There has recently been a growing interest in examining the role of epigenetic modifications, such as DNA methylation, in the etiology of type 1 diabetes (T1D). This study aimed to delineate differences in methylation patterns between T1D-affected and healthy individuals by examining the genome-wide methylation of individuals from three Arab families from Kuwait with T1D-affected mono-/dizygotic twins and non-twinned siblings. Bisulfite sequencing of DNA from the peripheral blood of the affected and healthy individuals from each of the three families was performed. Methylation profiles of the affected individuals were compared to those of the healthy individuals Principal component analysis on the observed methylation profiling based on base-pair resolution clustered the T1D-affected twins together family-wide. The sites/regions that were differentially methylated between the T1D and healthy samples harbored 84 genes, of which 18 were known to be differentially methylated in T1D individuals compared to healthy individuals in publicly available gene expression data resources. We further validated two of the 18 genes-namely ICA1 and DRAM1 that were hypermethylated in T1D samples compared to healthy samples-for upregulation in T1D samples from an extended study cohort of familial T1D. The study confirmed that the ICA1 and DRAM1 genes are differentially expressed in T1D samples compared to healthy samples.

Identification of Maturity-Onset-Diabetes of the Young (MODY) mutations in a country where diabetes is endemic.

Genetic variants responsible for Maturity-Onset-Diabetes of the Young (MODY) in Kuwait were investigated. A newly established a National Referral Clinic, the Dasman Diabetes Institute (DDI-NRC), assessed forty-five members from 31 suspected MODY families by whole exome sequencing. Thirty-three of the 45 samples were independently sequenced at the DDI-NRI, Exeter University, UK ( https://www.diabetesgenes.org/ ) using targeted 21-gene panel approach. Pathogenic mutations in GCK, HNF1A, HNF1B, HNF4A, and PDX1 confirmed MODY in 7 families, giving an overall positivity rate of 22.6% in this cohort. Novel variants were identified in three families in PDX1, HNF1B, and HNF1B. In this cohort, Multiplex Ligation-dependent Probe Amplification assay did not add any value to MODY variant detection rate in sequencing negative cases. In highly selected familial autoantibody negative diabetes, known MODY genes represent a minority and 77.3% of the familial cases have yet to have a causal variant described.

Salivary Microbiome Diversity in Kuwaiti Adolescents with Varied Body Mass Index-A Pilot Study.

The potential role of the salivary microbiome in human diseases has increasingly been explored. The salivary microbiome has been characterized in several global populations, except the Arabian Gulf region. Hence, in this pilot study, we profiled the salivary microbiome of Kuwaiti adolescents with varied body mass indexes (BMI). The analyses of core microbiome composition showed Firmicutes, Bacteroidota, Proteobacteria, Patescibacteria, Fusobacteriota, Actinobacteriota, and Campylobacterota as the common phylum found in the Kuwaiti adolescent population. We also illustrated a diverse microbial community among the sampled individuals grouped according to their BMI. Notably, the overweight group was found with a higher number of distinct taxa than other groups. As such, the core microbiome composition was found to be significantly different (p-value < 0.001) across different BMI groups. Overall, this pilot investigation outlined the microbial diversity and suggested that changes in salivary microbiome composition in people with obese or overweight BMI might reflect their susceptibility to oral diseases.

Microarray analysis reveals ONC201 mediated differential mechanisms of CHOP gene regulation in metastatic and nonmetastatic colorectal cancer cells.

The imipramine ONC201 has antiproliferative effects in several cancer cell types and activates integrated stress response pathway associated with the induction of Damage Inducible Transcript 3 (DDIT3, also known as C/EBP homologous protein or CHOP). We investigated the signaling pathways through which ONC201/CHOP crosstalk is regulated in ONC201-treated nonmetastatic and metastatic cancer cell lines (Dukes' type B colorectal adenocarcinoma nonmetastatic SW480 and metastatic LS-174T cells, respectively). Cell proliferation and apoptosis were evaluated by MTT assays and flow cytometry, gene expression was assessed by Affymetrix microarray, signaling pathway perturbations were assessed in silico, and key regulatory proteins were validated by Western blotting. Unlike LS-174T cells, SW480 cells were resistant to ONC201 treatment; Gene Ontology analysis of differentially expressed genes showed that cellular responsiveness to ONC201 treatment also differed substantially. In both ONC201-treated cell lines, CHOP expression was upregulated; however, its upstream regulatory mechanisms were perturbed. Although, PERK, ATF6 and IRE1 ER-stress pathways upregulated CHOP in both cell types, the Bak/Bax pathway regulated CHOP only LS-174T cells. Additionally, CHOP RNA splicing profiles varied between cell lines; these were further modified by ONC201 treatment. In conclusion, we delineated the signaling mechanisms by which CHOP expression is regulated in ONC201-treated non-metastatic and metastatic colorectal cell lines. The observed differences could be related to cellular plasticity and metabolic reprogramming, nevertheless, detailed mechanistic studies are required for further validations.

A 71-year-old man with a rare rhabdoid brain tumour: using a multidisciplinary medical and rehabilitative model of care.

Atypical rhabdoid tumours (AT/RTs) of pineal origin are rare in adults with rapid progression and poor prognosis. We present the case of a 71-year-old man with confusion and memory loss who was diagnosed with a pineal AT/RT after genetic analysis. Due to his limited functional capacity and goal to return home with family, a multidisciplinary care approach was essential for coordination of medical management, radiation treatment and acute inpatient rehabilitation. After diagnosis and rehabilitation, his functional ability improved allowing him to tolerate cranial irradiation, initiate systemic chemotherapy and eventually returned home for a brief period with an improved quality of life. His progress was temporary due to rapid progression of the tumour. He required additional aggressive oncological treatment and was admitted for subsequent inpatient rehabilitation before opting for hospice care. This case underscores the importance of a multidisciplinary approach to cancer treatment in a patient with a rare and aggressive brain tumour, while respecting the individual goals of patients and their families.

Identification of HLA-C*16:46 allele in a Kuwaiti individual.

One nucleotide substitution in codon 90 of HLA-C*16:02:01 results in a novel allele, HLA-C*16:46.

Leptin rs7799039 polymorphism is associated with multiple sclerosis risk in Kuwait.

BACKGROUND: Leptin association with Multiple sclerosis (MS) pathogenesis and MS related clinical characteristics is inconsistent. Here, we investigated whether two common variants in leptin (LEP) and leptin receptor (LEPR) genes influence MS risk and leptin levels in MS patients. METHODS: In a case-control study including 169 MS patients and 100 controls we examined the association of leptin in MS. Blood samples were used for DNA extraction and plasma retrieval. Taqman genotyping assays were used for LEP rs7799039 and LEPR rs1137101 genotyping, and enzyme-linked immunosorbent assay for plasma leptin level. RESULTS: Leptin levels were significantly lower in MS patients compared to controls (ß = 0.157, 95%CI: 0.033-0.26, p = 0.012). LEP rs7799039AA associated with MS risk (OR: 2.52; 95%CI: 1.35-4.67, p = 0.003). None of the assessed markers associated with MS disability, severity or response to treatment. CONCLUSION: LEP rs7799039AA is a risk factor for MS in our Kuwaiti population, and leptin levels are lower in MS patients compared to healthy controls. Our findings suggest future studies must consider all factors influencing leptin levels to resolve its controversial involvement in MS pathogenesis or progression.

PLXNA3 Variant rs5945430 is Associated with Severe Clinical Course in Male Multiple Sclerosis Patients.

Multiple sclerosis (MS) exhibits sex bias in disease clinical course as male MS patients develop severe, progressive clinical course with accumulating disability. So far, no factors have been found associating with this sex bias in MS severity. We set out to determine the genetic factor contributing to MS male-specific progressive disease. This is an MS cross-sectional study involving 213 Kuwaiti MS patients recruited at Dasman Diabetes Institute. Exome sequencing was performed on 18 females and 8 male MS patients' genomic DNA. rs5945430 genotyping was performed using Taqman genotyping assay. Estradiol levels were determined by enzyme-linked immunosorbent assay. Exome analysis revealed a missense variant (rs5945430) in Plexin A3 (PLXNA3) gene (Xq28) associated with male-specific MS severity. Genotyping of 187 MS patients for rs5945430 confirmed the association of rs5945430G with increased disease severity in MS males (p = 0.013; OR 3.8; 95% CI 1.24-11.7) and disability (p = 0.024). Estradiol levels shown to effect PLXNA3 expression were lower in MS males compared to MS females, and they were lower than control rs5945430G males (p = 0.057), whereas MS females had similar estradiol levels to healthy females reducing the level of expressed PLXNA3 GG in MS females. PLXNA3 rs5945430G is associated with increased disease severity in MS male patients. Estradiol is a possible protective factor against the expression of rs5945430G in MS females.

Assessment of plasma biomarkers for their association with Multiple Sclerosis progression.

In a cross-sectional study involving 160 Multiple Sclerosis (MS) patients and 70 healthy controls we set out to determine the association of five blood biomarkers with MS risk and progression scores. High levels of Semaphorin3A (SEMA3A) in females, and low levels of prolactin and estradiol in males associated with MS risk. High MS disability correlated with higher SEMA3A levels in females. Our findings suggest the clinical applicability of SEMA3A, and prolactin as biomarkers for MS progression. However, these biomarkers had sex-specific associations with MS, and any therapeutic approaches utilizing them should take that into consideration.

Gender influence in EBV antibody response in multiple sclerosis patients from Kuwait.

BACKGROUND: Epstein-Barr virus (EBV) infection is implicated with multiple sclerosis (MS) risk, exacerbation, and progression. The HLA-DRB1*1501 haplotype is a strong MS risk factor consistently documented in MS populations. There are no studies of EBV infections and HLA-DRB1*1501 haplotype associating with MS from Kuwait where MS prevalence has increased significantly. OBJECTIVES: To determine the association of EBV infection with MS incidence, and to investigate HLA-DRB1*1501 as a potential genetic risk factor for MS in Kuwait. METHODS: This is a case-control study involving 141 MS patients and 40 healthy controls. Antibody titers against EBV antigens' viral capsid antigen (VCA) and Epstein-Barr nuclear antigen 1 (EBNA1) were measured using enzyme-linked immunosorbent assays. HLA-DRB1*1501 haplotype assessment was done using rs3135005 TaqMan genotyping assay. RESULTS: Antibody titers against EBV were significantly elevated in MS patients compared to healthy controls (anti-EBNA1, p=0.008; anti-VCA, p=0.028). MS males had higher antibody titers to EBNA1 than healthy male controls (p=0.005) and female MS patients (p=0.03). HLA-DRB1*1501 haplotype genotypes failed to generate a risk association with MS or EBV antibody titers (p=0.6). CONCLUSION: An increased immune response to EBV infection is associated with MS incidence influenced by the type of antigen and sex. HLA-DRB1*1501 haplotype is not associated with MS risk in our Kuwaiti MS cohort.

Gender-associated genomic differences in colorectal cancer: clinical insight from feminization of male cancer cells.

Gender-related differences in colorectal cancer (CRC) are not fully understood. Recent studies have shown that CRC arising in females are significantly associated with CpG island methylator phenotype (CIMP-high). Using array comparative genomic hybridization, we analyzed a cohort of 116 CRCs (57 males, 59 females) for chromosomal copy number aberrations (CNA) and found that CRC in females had significantly higher numbers of gains involving chromosome arms 1q21.2-q21.3, 4q13.2, 6p21.1 and 16p11.2 and copy number losses of chromosome arm 11q25 compared to males. Interestingly, a subset of male CRCs (46%) exhibited a "feminization" phenomenon in the form of gains of X chromosomes (or an arm of X) and/or losses of the Y chromosome. Feminization of cancer cells was significantly associated with microsatellite-stable CRCs (p-value 0.003) and wild-type BRAF gene status (p-value 0.009). No significant association with other clinicopathological parameters was identified including disease-free survival. In summary, our data show that some CNAs in CRC may be gender specific and that male cancers characterized by feminization may constitute a specific subset of CRCs that warrants further investigation.

The influence of admixture and consanguinity on population genetic diversity in Middle East.

The Middle East (ME) is an important crossroad where modern humans migrated 'out of Africa' and spread into Europe and Asia. After the initial peopling and long-term isolation leading to well-differentiated populations, the ME also had a crucial role in subsequent human migrations among Africa, Europe and Asia; thus, recent population admixture has been common in the ME. On the other hand, consanguinity, a well-known practice in the ME, often reduces genetic diversity and works in opposition to admixture. Here, we explored the degree to which admixture and consanguinity jointly affected genetic diversity in ME populations. Genome-wide single-nucleotide polymorphism data were generated in two representative ME populations (Arabian and Iranian), with comparisons made with populations worldwide. Our results revealed an overall higher genetic diversity in both ME populations relative to other non-African populations. We identified a much larger number of long runs of homozygosity in ME populations than in any other populations, which was most likely attributed to high levels of consanguineous marriages that significantly decreased both individual and population heterozygosity. Additionally, we were able to distinguish African, European and Asian ancestries in ME populations and quantify the impact of admixture and consanguinity with statistical approaches. Interestingly, genomic regions with significantly excessive ancestry from individual source populations are functionally enriched in olfactory pathways, which were suspected to be under natural selection. Our findings suggest that genetic admixture, consanguinity and natural selection have collectively shaped the genetic diversity of ME populations, which has important implications in both evolutionary studies and medical practices.

Reduced FHIT expression is associated with mismatch repair deficient and high CpG island methylator phenotype colorectal cancer.

Colorectal cancer (CRC) is a heterogeneous disease and a major contributor to world cancer mortality rates. Molecular subtypes of CRC have become standards for CRC classification and have established prognostic potential. Here, we attempt to corroborate and provide further insight pertinent to the fragile histidine triad (FHIT) gene in microsatellite instable (MSI), microsatellite stable (MSS), and CpG island methylator phenotype (CIMP) CRC subtypes. We employed array comparative genomic hybridization and multiplex ligation-dependent probe amplification (MLPA) techniques to survey genomic aberrations in FHIT gene and their effects on FHIT protein expression using immunohistochemistry (IHC) in a CRC cohort. We further studied FHIT protein expression by IHC in a larger CRC cohort defined for its mismatch repair (MMR) protein expression and genomic methylation profiles. Our results show FHIT genomic deletions centered in exons 4 and 5 in most of MSI-CRC samples. Moreover, we confirmed the significant association of FHIT protein expression diminution (p=0.035) with MSI-CRC. In the larger cohort, reduced FHIT protein expression was significantly associated with CIMP-high subtype of CRC (p=0.009) and loss of PMS2 protein expression (p=0.017). We conclude that FHIT expression may be a valuable marker for CRC subtyping, and its diagnostic, prognostic, and therapeutic potential should be perused.

Primary care physician treatment of low HDL: Rational approach or Pandora's Box?

BACKGROUND: Guidelines for treating high low-density lipoproteins are clear, whereas guidelines for treating low high-density lipoproteins (HDL) are less so. Physicians approach to treating low HDL cholesterol is not known. OBJECTIVE: To determine primary care physicians approach to managing low HDL. METHODS: Three-thousand, nine-hundred and nineteen surveys were mailed to all primary care physicians in the State of Indiana, asking questions regarding demographics, case studies to assess the provider's approach to managing low HDL, and direct questions regarding management of HDL levels and general lipid knowledge questions. RESULTS: Seven-hundred and eighty-one surveys were returned, for a response rate of 19.9%. Fifty-eight percent of participants would initiate HDL-raising therapy after achieving the appropriate low-density lipoprotein cholesterol goal. The approaches used to raise HDL included lifestyle changes (diet, exercise, smoking cessation) (85%), niacin (83%), fibric acid derivative (61%), and alcohol (31%). Reasons inhibiting initiating therapy for raising HDL included concern over side effects (56%), perceived lack of effectiveness of currently available drugs (24%), lack of clear guidelines (22%), and lack of evidenced-based trials demonstrating benefit of raising HDL (14%). For men, 40% of physicians use 40 mg/dL as a cutoff for initiating HDL-raising therapy, while 25% using a cutoff of 35 mg/dL. For women, 24% use a cutoff of 50 mg/dL for initiating HDL-raising therapy, while 12% use 45 mg/dL as a cutoff. CONCLUSIONS: The majority of primary care physicians in the State of Indiana treated low HDL with appropriate approaches, although use of alcohol to raise HDL raises concerns.