Oxford Nanopore Technology and its Application in Liquid Biopsies.

Advanced medical technologies are transforming the future of healthcare, in particular, the screening and detection of molecular-genetic changes in patients suspected of having a neoplasm. They are based on the assumption that neoplasms release small amounts  of  various  neoplasm-specific molecules, such as tumor DNA, called circulating DNA (cirDNA), into the extracellular space and subsequently into the blood. The detection of tumor-specific molecules and specific molecular changes in body fluids in a noninvasive or minimally invasive approach is known as "liquid biopsy." The aim of this review is to summarize the current knowledge of the application of ONT for analyzing circulating DNA in the field of liquid biopsies among cancer patients. Databases were searched using the keywords "nanopore" and "liquid biopsy" and by applying strict inclusion criteria. This technique can be used for the detection of neoplastic disease, including metastases, guiding precision therapy, and monitoring its effects. There are many challenges, however, for the successful implementation of this technology into the clinical practice. The first one is the low amount of tumor-specific molecules in the body fluids. Secondly, a tumor molecular signature should be discriminated from benign conditions like clonal hematopoiesis of unknown significance. Oxford Nanopore Technology (ONT) is a third-generation sequencing technology that seems particularly promising to complete these tasks. It offers rapid sequencing thanks to its ability to detect changes in the density of the electric current passing through nanopores. Even though ONT still needs validation technology, it is a promising approach for early diagnosis, therapy guidance, and monitoring of different neoplasms based on analyzing the cirDNA.

Autosomal-recessive congenital cerebellar ataxia is caused by mutations in metabotropic glutamate receptor 1.

Autosomal-recessive congenital cerebellar ataxia was identified in Roma patients originating from a small subisolate with a known strong founder effect. Patients presented with global developmental delay, moderate to severe stance and gait ataxia, dysarthria, mild dysdiadochokinesia, dysmetria and tremors, intellectual deficit, and mild pyramidal signs. Brain imaging revealed progressive generalized cerebellar atrophy, and inferior vermian hypoplasia and/or a constitutionally small brain were observed in some patients. Exome sequencing, used for linkage analysis on extracted SNP genotypes and for mutation detection, identified two novel (i.e., not found in any database) variants located 7 bp apart within a unique 6q24 linkage region. Both mutations cosegregated with the disease in five affected families, in which all ten patients were homozygous. The mutated gene, GRM1, encodes metabotropic glutamate receptor mGluR1, which is highly expressed in cerebellar Purkinje cells and plays an important role in cerebellar development and synaptic plasticity. The two mutations affect a gene region critical for alternative splicing and the generation of receptor isoforms; they are a 3 bp exon 8 deletion and an intron 8 splicing mutation (c.2652_2654del and c.2660+2T>G, respectively [RefSeq accession number NM_000838.3]). The functional impact of the deletion is unclear and is overshadowed by the splicing defect. Although ataxia lymphoblastoid cell lines expressed GRM1 at levels comparable to those of control cells, the aberrant transcripts skipped exon 8 or ended in intron 8 and encoded various species of nonfunctional receptors either lacking the transmembrane domain and containing abnormal intracellular tails or completely missing the tail. The study implicates mGluR1 in human hereditary ataxia. It also illustrates the potential of the Roma founder populations for mutation identification by exome sequencing.

Characterization of Bulgarian Copper Mine Tailing as a Precursor for Obtaining Geopolymers.

Valorization of high-volume mine tailings could be achieved by the development of new geopolymers with a low CO2 footprint. Materials rich in aluminum and silicon with appropriate solubility in an alkaline medium can be used to obtain a geopolymer. This paper presents a study of copper mine tailings from Bulgaria as precursors for geopolymers. Particle size distribution, chemical and mineralogical composition, as well as alkaline reactivity, acidity and electroconductivity of aqueous slurry are studied. The heavy metal content and their mobility are studied by leaching tests. Sequential extraction was applied to determine the geochemical phase distribution of heavy metals. The studied samples were characterized by high alkalinity, which could favor the geopolymerization process. The water-soluble sulphates were less than 4%. The Si/Al ratio in mine tailing was found to be 3. The alkaline reactivity depended more so on the time of extraction than on the concentration of NaOH solution. The main part of the heavy metals was found in the residual fraction; hence, in high alkaline medium during the geopolymerization process, they will stay fixed. Thus, the obtained geopolymers could be expected to exert low environmental impact. The presented results revealed that studied copper mine tailing is a suitable precursor for geopolymerization.

CFTR gene variants as a reason for impaired spermatogenesis: a pilot study and a Meta-analysis of published data.

There is increasing data that IVS8-5T variand and TG repeats could lead to impaired spermatogenesis. To investigate this we performed Sanger sequencing on 50 Bulgarian men with a sperm count below 5 × 106/mL and 20 normal fertile men. Frequencies of the results were compared among the two groups. A meta-analysis was perfomed by using the data for 6,423 patients and 5,834 control subjects, tested for the IVS8-5T polymorphism. One case subject (2.0%) was homozygote for the 5 T/5T variant whereas two (4.0%) were heterozygotes for the 5 T/7T variant. No 5 T alleles were found in the control group. The genotypes of the two groups showed a statistically significant difference (p = 0.04, α < 0.05). Also, the odds ratio was 3.73, but this was unsignificant (p = 0.38). All control subjects had 11 TG repeats and for the test group: 47 (94.0%) men with 11 TG repeats and three (6.00%) with 10 TG repeats. Fisher's test showed no significant difference (p = 0.55). The meta-analysis showed that IVS8-5T variant was a risk factor for impaired spermatogenesis (OR = 2.84, p < 0.05) and this was more prominent for non-European (OR = 4.50, p < 0.05) compared to European (OR = 1.28, p < 0.05) men. The IVS8 - 5 T variant could be associated with disorders of sperm production.

Potential role of dynein-related genes in the etiology of male infertility: A systematic review and a meta-analysis.

BACKGROUND: The dynein-related genes may have a role in the etiology of male infertility, particularly in cases of impaired sperm motility. OBJECTIVES: The goal of this review is to compile a list of the most important dynein-related candidate genes that may contribute to male factor infertility. MATERIALS AND METHODS: Databases were searched using the keywords "dynein," "male," "infertility," and by applying strict inclusion criteria. A meta-analysis was also performed by using the eligible case-control studies. The odd ratios (ORs), the Z-test score, and the level of significance were determined using a fixed model with a p value of 0.05. Funnel plots were used to check for publication bias. RESULTS: There were 35 studies that met the inclusion criteria. There were a total of 15 genes responsible for the production of dynein structural proteins, the production of dynein assembling factors, and potentially associated with male infertility. A total of five case-control studies were eligible for inclusion in the meta-analysis. Variants in the dynein-related genes were linked to an increased the risk of male infertility (OR = 21.52, 95% confidence interval 8.34-55.50, Z test = 6.35, p < 0.05). The percentage of heterogeneity, I2 , was 47.00%. The lack of variants in the dynein genes was an advantage, and this was statistically significant. DISCUSSION: The results from the present review illustrate that pathogenic variants in genes both for dynein synthesis and for dynein assembly factors could be associated with isolated cases of male infertility without any other symptoms. CONCLUSIONS: The genes addressed in this study, which are involved in both the production and assembly of dynein, could be used as molecular targets for future research into the etiology of sperm motility problems.

Variants of uncertain significance in the era of next-generation sequencing.

ABSTRACT: Next-generation sequencing (NGS) is now widely used in diagnosing rare diseases. However, it has some limitations, such as variants of uncertain significance (VUS). This can present difficulties even for nurse practitioners involved in clinical genetics. We present three cases from our clinical practice: two targeted panel testing and one exome sequencing. Whole blood samples were collected and sent for NGS analysis. In case 1, a VUS was found in the LITAF gene, which is associated with autosomal dominant Charcot-Marie-Tooth disease type 1C. In case 2, a VUS was reported in the MEFV gene, which is associated with autosomal recessive and autosomal dominant familial Mediterranean fever. In these cases, the reported VUS corresponded to the clinical diagnosis. In case 3, two variants in the heterozygous state were found in the ATP7B gene, which is associated with Wilson disease, and the disorder was later clinically recognized. According to the published guidelines, VUSs should not be discussed as a cause for an observed genetic condition. Nevertheless, if the reported variant is in a gene associated with the clinically diagnosed disorder, and there is a strong genotype-phenotype correlation, it could be suggestive of the etiological role of this variant.

Molecular screening for fragile X syndrome in children with unexplained intellectual disability and/or autistic behaviour.

INTRODUCTION: Fragile X syndrome (FXS, OMIM #300624) is the most common inherited form of intellectual disability and the leading monogenic cause of autism.

16p11.2 Duplication Syndrome - a Case Report.

16p11.2 duplication syndrome is a rare disorder, often associated with intellectual disability, attention deficit, hyperactivity disorder, and a predisposition to epilepsy and schizophrenia. There are no specific dysmorphic features for this genetic condition, but micro-cephaly, micrognathia and hypertelorism could be present. We report a case of 16p11.2 duplication syndrome which has the typical clinical presentation - slight facial dysmorphism, impaired intellectual development, and autistic behavior. Whole-exome sequencing was performed, but no pathogenic or likely pathogenic mutations were identified. Array comparative genomic hybridization analysis established the diagnosis of 16p11.2 duplication syndrome, which illustrates the importance of this method when diagnosing children with unexplained intellectual disability.

Association between polymorphic markers human leucocyte antigen-G and tumour necrosis factor alpha and susceptibility to recurrent miscarriages among Bulgarian women.

OBJECTIVE: To analyze the role of 14 base pair (bp) insertion (ins)/deletion (del) and tumour necrosis factor alpha (TNF-α) G/A polymorphisms as risk factors for spontaneous miscarriage in patients with two or more unsuccessful pregnancies and a group of control women with at least two normal live births. MATERIALS AND METHODS: To investigate the role of these mutations, 50 patients with two or more idiopathic recurrent miscarriages and 50 normal fertile women were tested for the presence of human leucocyte antigen-G (HLA-G) 14 bp ins/del and TNF-α -308 G/A variants. The frequencies of the studied polymorphisms were compared between the two groups. RESULTS: Individuals with a history of miscarriages had a significantly higher prevalence of 14 bp insertion alleles compared with control patients (p=0.04). There was also a two times higher relative risk for miscarriage among carriers of this variant. No statistical difference in allele frequencies of the TNF-α -308 G/A polymorphism was established between controls and study patients (p=0.78). CONCLUSION: The 14 bp ins HLA-G variant could be associated with a higher risk for unsuccessful pregnancy according to the results from the study. There is no association between the studied TNF-α -308 GA polymorphism and rate of spontaneous abortions.

Comparison between thrombophilic gene polymorphisms among high risk patients.

INTRODUCTION: The purpose of this study was to compare the role of the thrombophilic variants among two groups of high risk patients with vascular disorders and recurrent pregnancy loss. METHODS: 200 patients, including 76 with thrombotic accidents and 124 with two or more idiopathic recurrent miscarriage during the first trimester, were tested for the presence of Factor V (F V) Leiden G1691A, Factor II (F II) G20210A, plasminogen activator inhibitor (PAI) 4G/5G, and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms using Real time polymerase chain reaction (RT - PCR) in the Laboratory of Medical Genetics, Varna, Bulgaria between June 2016 and May 2019. Frequencies of thrombophilic gene polymorphisms were compared among the two populations and to the expected genotype frequencies. RESULTS: Individuals with a history of vascular disorders had a significantly higher frequency of F V Leiden variant compared to women with recurrent miscariage. There was no statistical difference between the analyzed patients for the other three thrombophilic polymorphisms. The allelic frequencies and the expected genotype frequencies of the F V, F II and MTHFR polymorphisms were calculated according to Hardy-Weinberg equilibrium. The percentages of the homozygotes for F V and F II were higher than expected in the two groups of patients. For the MTHFR there was no difference. CONCLUSION: F V Leiden remains the strongest risk factor for vascular disorders and recurrent pregnancy loss. Screening for this variant should be recommended to patients with thrombotic accidents and women with repeated miscarriage. The role of F II, PAI and MTHFR remains controversial.

Application of chemical and biological tests for estimation of current state of a tailing dump and surrounding soil from the region of Tarnita, Suceava, Romania.

This paper presents the results from a study on the current state of tailing dump, surrounding soil and water in the region of Tarnita-Suceava, Romania. A number of chemical analyses and germination tests were applied in an attempt to estimate the ability of soil to maintain the plants growing, the bioavailability, and heavy metals uptake. Total heavy metals, exchangeable metals, acidity, and carbon and nitrogen content were determined. A modified sequential extraction method was used to determine geochemical phase distribution of heavy metals. The most abundant heavy metals in the studied samples were Cu, Zn, and Pb. Elevated concentrations of As were also found. The results from sequential extraction revealed that up to 51% of copper was retained by amorphous and crystalline iron oxides in soil. Higher content of lead was noticed in amorphous iron oxide fraction. The heavy metal concentration in river water during dry season varied from 0.13 mg/L (Fe) to 4.2 mg/L (Zn) and was below the maximum contamination level for drinking water. The soil toxicity and heavy metal bioavailability of tailing dump material and surrounding soils were studied by germination tests. The germinated plantlets on the studied soils were found to accumulate elevated concentrations of heavy metals thus indicating the bioavailability of soil contaminants. Soil decontamination by distilled water or magnesium nitrate solution was found to be efficient enough to improve the capability of the studied soils to support the germination process.

GNE myopathy in Roma patients homozygous for the p.I618T founder mutation.

GNE myopathy is an autosomal-recessive disorder caused by mutations in the GNE gene, encoding the key enzyme in the sialic acid biosynthetic pathway, UDP-N-acetylglucosamine 2-epimerase/N-acetyl mannosamine kinase. We studied 50 Bulgarian Roma patients homozygous for p.I618T, an ancient founder mutation in the kinase domain of the GNE gene, dating before the Gypsy exodus from North West India. The clinical features in the Bulgarian GNE group can be described with disease onset mostly in the third decade, but in individual cases, onset was as early as 10 years of age. The majority of patients had foot drop as the first symptom, but three patients developed hand weakness first. Muscle weakness was early and severe for the tibialis anterior, and minimal or late for quadriceps femoris, and respiratory muscles were only subclinically affected even in the advanced stages of the disease. During a 15-year follow-up period, 32 patients became non-ambulant. The average period between disease onset and loss of ambulation was 10.34 ± 4.31 years, ranging from 3 to 20 years. Our analysis of affected sib pairs suggested a possible role of genetic modifying factors, accounting for significant variation in disease severity.

LTBP2 and CYP1B1 mutations and associated ocular phenotypes in the Roma/Gypsy founder population.

Primary congenital glaucoma (PCG) is a genetically heterogeneous autosomal recessive disorder, which is an important cause of blindness in childhood. The first known gene, CYP1B1, accounts for a variable proportion of cases in most populations. A second gene, LTBP2, was recently reported in association with a syndrome, in which glaucoma is secondary to lens dislocation. We report on the molecular and clinical profile of 34 families diagnosed as PCG, all originating from the Roma/Gypsy founder population. Comprehensive sequencing analysis revealed a level of heterogeneity unusual for this population, with five CYP1B1 and one ancestral LTBP2 mutation accounting for ∼70% of patients (25 out of 37) and the remainder still unexplained. Homozygosity for the founder LTBP2 p.R299X mutation resulted in a more severe clinical phenotype and poorer outcome despite a markedly higher number of surgical interventions. The genetically homogeneous group of p.R299X homozygotes showed variable phenotypes (presumably also underlying pathogenetic mechanisms), wherein PCG proper with primary dysgenesis of the trabecular meshwork, and Marfan syndrome-like zonular disease with ectopia lentis and later onset secondary glaucoma are two extremes. The spectrum manifestations may occur in different combinations and have a different evolution even within the same sibship or a single patient. Preliminary observations on compounds with mutations in both CYP1B1-LTBP2 suggest that the observed combinations are of no clinical significance and digenic inheritance is unlikely. We provide a population genetics perspective to explain the allelic heterogeneity, comparing the history and geographic distribution of the two major founder mutations--p.R299X/LTBP2 and p.E387K/CYP1B1.