Search Results (14)

Background/Objectives: Pharmacogenomics (PGx) enables personalized therapy by identifying genetic variants that influence drug response. Despite the advantages of next-generation sequencing (NGS), few clinically validated, guideline-aligned panels comprehensively detect common, rare, and structurally complex pharmacogenetic variants. Methods: We developed and analytically validated Action PharmaKitDx, a targeted NGS panel covering 335 pharmacogenes, including all priority genes recommended by CPIC, DPWG, and CPNDS. Performance was assessed using Coriell HapMap and GeT-RM reference materials across multiple library preparation workflows and Illumina platforms. Clinical feasibility was evaluated in 41 patient samples from diverse specialties. Results were compared with established reference methods, including PCR-based assays, STR analysis, Sanger sequencing, and whole-exome sequencing. Results: Analytical validation: More than 99% of target bases achieved ≥30× coverage. Analytical accuracy, sensitivity, specificity, and positive predictive value exceeded 99.3%, with repeatability and reproducibility >99.7%. Concordance with GeT-RM haplotypes reached 98% after star-allele harmonization. The panel accurately detected complex variants, including CYP2D6 copy-number changes and hybrid alleles. Clinical validation: Full concordance with prior genotyping was observed in clinical samples. Beyond the initial testing indication, each sample harbored a mean of six actionable variants (range 2–10). Thirty-six rare (minor allele frequency <1%) potentially actionable variants were additionally identified. Conclusions: Action PharmaKitDx demonstrates high analytical performance and broad clinical applicability, supporting its implementation as a scalable solution for comprehensive pharmacogenetic testing and precision prescribing. Full article

Gliomas are notoriously difficult to treat owing to their pronounced heterogeneity and highly variable treatment responses. This reality drives the development of precise diagnostic and prognostic methods. This review explores the modern arsenal of bioinformatic tools aimed at refining diagnosis and stratifying glioma patients by different malignancy grades and types. We perform a comparative analysis of software solutions for processing whole-exome sequencing data, analyzing DNA methylation profiles, and interpreting transcriptomic data, highlighting their key advantages and limited applicability in routine clinical practice. Special emphasis is placed on the contribution of bioinformatics to fundamental oncology, as these tools aid in the discovery of new biomarker genes and potential targets for targeted therapy. The ninth section discusses the role of computational models in predicting immunotherapy efficacy. It demonstrates how integrative data analysis—including tumor mutational burden assessment, characterization of the tumor immune microenvironment, and neoantigen identification—can help identify patients who are most likely to respond to immune checkpoint inhibitors and other immunotherapeutic approaches. The obtained data provide compelling justification for including immunotherapy in standard glioma treatment protocols, provided that candidates are accurately selected based on comprehensive bioinformatic analysis. The tools discussed pave the way for transitioning from an empirical to a personalized approach in glioma patient management. However, we also note that this field remains largely in the preclinical research stage and has not yet revolutionized clinical practice. This review is intended for biological scientists and clinicians who find traditional bioinformatic tools difficult to use. Full article

Genetic studies and phenotypic expansion of hearing loss (HL) for people living in Africa are greatly needed. We evaluated the clinical phenotypes of three affected siblings presenting non-syndromic (NS) HL and five unaffected members of a consanguineous Ghanaian family. Analysis of exome sequence data was performed for all affected and one unaffected family members. In-depth genetic and cellular characterization studies were performed to investigate biological significance of the implicated variant using bioinformatic tools and cell-based experimentation. Audiological examinations showed severe-to-profound, bilateral, symmetrical, and post-lingual onset. The whole-exome sequencing (WES) identified a homozygous frameshift variant: MARVEL domain containing 2 (MARVELD2):c.1058dup;p.(Val354Serfs*5) in all affected siblings. This frameshift variant leads to an early stop codon insertion and predicted to be targeted by nonsense medicated decay (mutant protein predicted to lack conserved C-terminal domain if translated). Cell immunofluorescence and immunocytochemistry studies exposed the functional impact of the mutant protein’s expression, stability, localization, protein–protein binding, barrier function, and actin cytoskeleton architecture. The identified variant segregates with NSHL in the index Ghanaian family. The data support this nonsense variant as pathogenic, likely to impact the homeostasis of ions, solutes, and other molecules, compromising membrane barrier and signaling in the inner ear spaces. Full article

Background: Pathogenic variants in the Polycystic Kidney Disease 2 (PKD2) gene are associated with Autosomal Dominant Polycystic Kidney Disease (ADPKD) in approximately 30% of cases. In recent years, the high-throughput sequencing techniques have significantly increased the number of variants identified in affected patients. Here, we described the peculiar effect of a PKD2 splicing variant, the c.1717-2A>G, identified in an Italian male patient with ADPKD. This variant led to the unusual and rare skipping of two consecutive exons, causing a large in-frame deletion. Methods: The genetic evaluation of the patient was performed using the Next-Generation Sequencing (NGS) assay Clinical Exome Solution^® (SOPHiA Genetics). Bioinformatics analysis was performed using the SOPHiA DDM platform (SOPHiA Genetics). Prediction of pathogenicity was carried out by integrating several in silico tools. RNA evaluation was performed to test the effect of the variant on the PKD2 splicing using a Reverse-Transcription PCR coupled with cDNA sequencing. Results: NGS revealed the presence of the PKD2 c.1717-2A>G variant that lies in the canonical splice site of intron 7. This rare variant was predicted to have a significant impact on the splicing, proved by the RNA-based analysis. We identified the presence of a transcript characterised by the simultaneous skipping of exons 8 and 9, with a retained reading frame and the merging of exons 7–10. Conclusions: We described for the first time a dual-exon skip event related to the presence of a single-base substitution in the PKD2 gene in an ADPKD-affected patient. We assumed that the molecular basis of such a rare mechanism lies in the specific order of intron removal. The finding represents novel evidence of an alternative and unusual splicing mechanism in the PKD2 gene, adding insights to the pathogenesis of the ADPKD. Full article

The solute carrier organic anion transporter family member 1B1 (SLCO1B1) encodes the organic anion-transporting polypeptide 1B1 (OATP1B1 protein) that transports statins to liver cells. Common genetic variants in SLCO1B1, such as *5, cause altered systemic exposure to statins and therefore affect statin outcomes, with potential pharmacogenetic applications; yet, evidence is inconclusive. We studied common and rare SLCO1B1 variants in up to 64,000 patients from UK Biobank prescribed simvastatin or atorvastatin, combining whole-exome sequencing data with up to 25-year routine clinical records. We studied 51 predicted gain/loss-of-function variants affecting OATP1B1. Both SLCO1B1*5 alone and the SLCO1B1*15 haplotype increased LDL during treatment (beta*5 = 0.08 mmol/L, p = 6 × 10⁻⁸; beta*15 = 0.03 mmol/L, p = 3 × 10⁻⁴), as did the likelihood of discontinuing statin prescriptions (hazard ratio*5 = 1.12, p = 0.04; HR*15 = 1.05, p = 0.04). SLCO1B1*15 and SLCO1B1*20 increased the risk of General Practice (GP)-diagnosed muscle symptoms (HR*15 = 1.22, p = 0.003; HR*20 = 1.25, p = 0.01). We estimated that genotype-guided prescribing could potentially prevent 18% and 10% of GP-diagnosed muscle symptoms experienced by statin patients, with *15 and *20, respectively. The remaining common variants were not individually significant. Rare variants in SLCO1B1 increased LDL in statin users by up to 1.05 mmol/L, but replication is needed. We conclude that genotype-guided treatment could reduce GP-diagnosed muscle symptoms in statin patients; incorporating further SLCO1B1 variants into clinical prediction scores could improve LDL control and decrease adverse events, including discontinuation. Full article

Maturity-onset diabetes of the young (MODY) is a rare form of non-autoimmune diabetes with an autosomal dominant inheritance. To date, 14 genes have been reported as genetic basis of MODY. GCK gene, encoding the glucokinase enzyme, was the first MODY gene to be identified. GCK heterozygous inactivating variants cause the GCK-MODY or MODY2 subtype. However, partial or whole gene deletions have been rarely identified, showing it to be a rare cause of GCK-MODY. We reported the molecular evaluation of a Ukrainian patient with clinical diagnosis of MODY2. We performed the Next generation sequencing of the clinical exome using the Clinical Exome Solution^® kit (SOPHiA Genetics), followed by the design of a 14 genes virtual panel related to the suggestive diagnosis of MODY. Bioinformatics analysis was performed using the SOPHiA DDM platform (SOPHiA Genetics). The SALSA MLPA kit for MODY (MRC-Holland) was used for relative quantification of GCK exons. From the molecular evaluation, no pathogenic sequence variants were detected in the investigated genes. Copy Number Variation analysis was able to identify a large deletion involving the last three exons of the GCK gene. This result was confirmed by MLPA. To the best of our knowledge, the identified rearrangement has never been reported in the literature. Full article

Dilated cardiomyopathy (DCM) is a complex disease affecting young adults. It is a pathological condition impairing myocardium activity that leads to heart failure and, in the most severe cases, transplantation, which is currently the only possible therapy for the disease. DCM can be attributed to many genetic determinants interacting with environmental factors, resulting in a highly variable phenotype. Due to this complexity, the early identification of causative gene mutations is an important goal to provide a genetic diagnosis, implement pre-symptomatic interventions, and predict prognosis. The advent of next-generation sequencing (NGS) has opened a new path for mutation screening, and exome sequencing provides a promising approach for identifying causal variants in known genes and novel disease-associated candidates. We analyzed the whole-exome sequencing (WES) of 15 patients affected by DCM without overloading (hypertension, valvular, or congenital heart disease) or chronic ischemic conditions. We identified 70 pathogenic or likely pathogenic variants and 1240 variants of uncertain clinical significance. Gene ontology enrichment analysis was performed to assess the potential connections between affected genes and biological or molecular function, identifying genes directly related to extracellular matrix organization, transcellular movement through the solute carrier and ATP-binding cassette transporter, and vitamin B12 metabolism. We found variants in genes implicated to a different extent in cardiac function that may represent new players in the complex genetic scenario of DCM. Full article

Autoimmune polyendocrine syndrome (APS) is assumed to involve an immune system malfunction and entails several autoimmune diseases co-occurring in different tissues of the same patient; however, they are orphans of its accurate diagnosis, as its genetic basis and pathogenic mechanism are not understood. Our previous studies uncovered alterations in the ATPase H+/K+ Transporting Subunit Alpha (ATP4A) proton pump that triggered an internal cell acid–base imbalance, offering an autoimmune scenario for atrophic gastritis and gastric neuroendocrine tumors with secondary autoimmune pathologies. Here, we propose the genetic exploration of APS involving gastric disease to understand the underlying pathogenic mechanism of the polyautoimmune scenario. The whole exome sequencing (WES) study of five autoimmune thyrogastric families uncovered different pathogenic variants in SLC4A2, SLC26A7 and SLC26A9, which cotransport together with ATP4A. Exploratory in vitro studies suggested that the uncovered genes were involved in a pathogenic mechanism based on the alteration of the acid–base balance. Thus, we built a custom gene panel with 12 genes based on the suggested mechanism to evaluate a new series of 69 APS patients. In total, 64 filtered putatively damaging variants in the 12 genes of the panel were found in 54.17% of the studied patients and none of the healthy controls. Our studies reveal a constellation of solute carriers that co-express in the tissues affected with different autoimmune diseases, proposing a unique genetic origin for co-occurring pathologies. These results settle a new-fangled genetics-based mechanism for polyautoimmunity that explains not only gastric disease, but also thyrogastric pathology and disease co-occurrence in APS that are different from clinical incidental findings. This opens a new window leading to the prediction and diagnosis of co-occurring autoimmune diseases and clinical management of patients. Full article

SLC25A46 (solute carrier family 25 member 46) mutations have been linked to various neurological diseases with recessive inheritance, including Leigh syndrome, optic atrophy, and lethal congenital pontocerebellar hypoplasia. SLC25A46 is expressed in the outer membrane of mitochondria, where it plays a critical role in mitochondrial dynamics. A deceased 7-month-old female infant was suspected to have Leigh syndrome. Clinical exome sequencing was non-diagnostic, but research reanalysis of the sequencing data identified two novel variants in SLC25A46: a missense (c.1039C>T, p.Arg347Cys; NM_138773, hg19) and a donor splice region variant (c.283+5G>A) in intron 1. Both variants were predicted to be damaging. Sanger sequencing of cDNA detected a single missense allele in the patient compared to control, and the SLC25A46 transcript levels were also reduced due to the splice region variant. Additionally, Western blot analysis of whole-cell lysate showed a decrease of SLC25A46 expression in proband fibroblasts, relative to control cells. Further, analysis of mitochondrial morphology revealed evidence of increased fragmentation of the mitochondrial network in proband fibroblasts, compared to control cells. Collectively, our findings suggest that these novel variants in SLC24A46, the donor splice one and the missense variant, are the cause of the neurological phenotype in this proband. Full article

Mutations in the HSD17B3 gene cause HSD17B3 deficiency and result in 46, XY Disorders of Sex Development (46, XY DSD). The diagnosis of 46, XY DSD is very challenging and not rarely is confirmed only at older ages, when an affected XY female presents with primary amenorrhea or develops progressive virilization. The patient described in this paper represents a case of discrepancies between non-invasive prenatal testing (NIPT) and ultrasound based fetal sex determination detected during prenatal screening. Exome sequencing was performed on the cell free fetal DNA (cffDNA), amniotic fluid, and the parents. Libraries were generated according to the manufacturer’s protocols using TruSight One Kits (Illumina Inc., San Diego, CA, USA). Sequencing was carried out on NEXT Seq 500 (Illumina) to mean sequencing depth of at least 100×. A panel of sexual disease genes was used in order to search for a causative variant. The finding of a mutation (c.645 A>T, p.Glu215Asp) in HSD17B3 gene in amniotic fluid as well as in cffDNA and both parents supported the hypothesis of the HSD17B3 deficiency. In conclusion, we used clinical exome sequencing and non-invasive prenatal detection, providing a solution for NIPT of a single-gene disorder. Early genetic diagnoses are useful for patients and clinicians, contribute to clinical knowledge of DSD, and are invaluable for genetic counseling of couples contemplating future pregnancies. Full article

Background: Approximately fifteen percent of patients with tuberous sclerosis complex (TSC) phenotype do not have any genetic disease-causing mutations which could be responsible for the development of TSC. The lack of a proper diagnosis significantly affects the quality of life for these patients and their families. Methods: The aim of our study was to use Whole Exome Sequencing (WES) in order to identify the genes responsible for the phenotype of nine patients with clinical signs of TSC, but without confirmed tuberous sclerosis complex 1/ tuberous sclerosis complex 2 (TSC1/TSC2) mutations using routine molecular genetic diagnostic tools. Results: We found previously overlooked heterozygous nonsense mutations in TSC1, and a heterozygous intronic variant in TSC2. In one patient, two heterozygous missense variants were found in polycystic kidney and hepatic disease 1 (PKHD1), confirming polycystic kidney disease type 4. A heterozygous missense mutation in solute carrier family 12 member 5 (SLC12A5) was found in one patient, which is linked to cause susceptibility to idiopathic generalized epilepsy type 14. Heterozygous nonsense variant ring finger protein 213 (RNF213) was identified in one patient, which is associated with susceptibility to Moyamoya disease type 2. In the remaining three patients WES could not reveal any variants clinically relevant to the described phenotypes. Conclusion: Patients without appropriate diagnosis due to the lack of sensitivity of the currently used routine diagnostic methods can significantly profit from the wider application of next generation sequencing technologies in order to identify genes and variants responsible for their symptoms. Full article

This review aims at better understanding the genetics of endometriosis. Endometriosis is a frequent feminine disease, affecting up to 10% of women, and characterized by pain and infertility. In the most accepted hypothesis, endometriosis is caused by the implantation of uterine tissue at ectopic abdominal places, originating from retrograde menses. Despite the obvious genetic complexity of the disease, analysis of sibs has allowed heritability estimation of endometriosis at ~50%. From 2010, large Genome Wide Association Studies (GWAS), aimed at identifying the genes and loci underlying this genetic determinism. Some of these loci were confirmed in other populations and replication studies, some new loci were also found through meta-analyses using pooled samples. For two loci on chromosomes 1 (near CCD42) and chromosome 9 (near CDKN2A), functional explanations of the SNP (Single Nucleotide Polymorphism) effects have been more thoroughly studied. While a handful of chromosome regions and genes have clearly been identified and statistically demonstrated as at-risk for the disease, only a small part of the heritability is explained (missing heritability). Some attempts of exome sequencing started to identify additional genes from families or populations, but are still scarce. The solution may reside inside a combined effort: increasing the size of the GWAS designs, better categorize the clinical forms of the disease before analyzing genome-wide polymorphisms, and generalizing exome sequencing ventures. We try here to provide a vision of what we have and what we should obtain to completely elucidate the genetics of this complex disease. Full article

Whole-exome sequencing has become a popular technique in research and clinical settings, assisting in disease diagnosis and increasing the understanding of disease pathogenesis. In this study, we aimed to compare common enrichment capture solutions available in the market. Peripheral blood-purified DNA samples were enriched with SureSelect^QXT V6 (Agilent) and various Illumina solutions: TruSeq DNA Nano, TruSeq DNA Exome, Nextera DNA Exome, and Illumina DNA Prep with Enrichment, and sequenced on a HiSeq 4000. We found that their percentage of duplicate reads was as much as 2 times higher than previously reported values for the previous HiSeq series. SureSelect^QXT and Illumina DNA Prep with Enrichment showed the best average on-target coverage, which improved when off-target regions were included. At high coverage levels and in shared bases, these two solutions and TruSeq DNA Exome provided three of the best performances. With respect to the number of small variants detected, SureSelect^QXT presented the lowest number of detected variants in target regions. When off-target regions were considered, its ability equalized to other solutions. Our results show SureSelect^QXT and Illumina DNA Prep with Enrichment to be the best enrichment capture solutions. Full article

Creatine deficiency syndrome (CDS) comprises three separate enzyme deficiencies with overlapping clinical presentations: arginine:glycine amidinotransferase (GATM gene, glycine amidinotransferase), guanidinoacetate methyltransferase (GAMT gene), and creatine transporter deficiency (SLC6A8 gene, solute carrier family 6 member 8). CDS presents with developmental delays/regression, intellectual disability, speech and language impairment, autistic behaviour, epileptic seizures, treatment-refractory epilepsy, and extrapyramidal movement disorders; symptoms that are also evident in children with autism. The objective of the study was to test the hypothesis that genetic variability in creatine metabolism genes is associated with autism. We sequenced GATM, GAMT and SLC6A8 genes in 166 patients with autism (coding sequence, introns and adjacent untranslated regions). A total of 29, 16 and 25 variants were identified in each gene, respectively. Four variants were novel in GATM, and 5 in SLC6A8 (not present in the 1000 Genomes, Exome Sequencing Project (ESP) or Exome Aggregation Consortium (ExAC) databases). A single variant in each gene was identified as non-synonymous, and computationally predicted to be potentially damaging. Nine variants in GATM were shown to have a lower minor allele frequency (MAF) in the autism population than in the 1000 Genomes database, specifically in the East Asian population (Fisher’s exact test). Two variants also had lower MAFs in the European population. In summary, there were no apparent associations of variants in GAMT and SLC6A8 genes with autism. The data implying there could be a lower association of some specific GATM gene variants with autism is an observation that would need to be corroborated in a larger group of autism patients, and with sub-populations of Asian ethnicities. Overall, our findings suggest that the genetic variability of creatine synthesis/transport is unlikely to play a part in the pathogenesis of autism spectrum disorder (ASD) in children. Full article