Search Results (11)

Globally, intron 22 inversions (Inv22s) of the factor VIII gene (F8) are the most frequent pathogenic variants and account for 45–50% of severe hemophilia A (SHA) cases, while intron 1 inversion (Inv1) explains 1–5% of SHA cases. The detection of both inversions by an inverse shifting-polymerase chain reaction (IS-PCR) is the first choice worldwide for the diagnosis of patients and carriers of SHA. To improve its sensitivity and reproducibility in the visualization of PCR products, we approached the IS-PCR with fluorescent capillary electrophoresis instead of agarose electrophoresis. Based on the original protocol, we modified two primers by 5’-end labeling with FAM™ fluorescent dye for the detection of the PCR products by capillary electrophoresis. Additionally, the “fast enzymes” BclI and T4-Ligase were incorporated for work saving in the genomic digestion and ligation reactions, respectively. Once we accomplished the standardization and verified the reproducibility of the modified IS-PCR method, we applied it for the diagnosis of a cohort of SHA patients and carriers. The modified IS-PCR by fluorescent capillary electrophoresis for PCR product detection is more sensitive than agarose electrophoresis. The method was also improved by using the new rapid enzymes to save time in the whole process. Full article

CDKL5 deficiency disorder (CDD) is an X-linked dominant epileptic encephalopathy, characterized by early-onset and drug-resistant seizures, psychomotor delay, and slight facial features. Genomic variants inactivating CDKL5 or impairing its protein product kinase activity have been reported, making next-generation sequencing (NGS) and chromosomal microarray analysis (CMA) the standard diagnostic tests. We report a suspicious case of CDD in a female child who tested negative upon NGS and CMA and harbored an X chromosome de novo pericentric inversion. The use of recently developed genomic techniques (optical genome mapping and whole-genome sequencing) allowed us to finely characterize the breakpoints, with one of them interrupting CDKL5 at intron 1. This is the fifth case of CDD reported in the scientific literature harboring a structural rearrangement on the X chromosome, providing evidence for the hypothesis that this type of anomaly can represent a recurrent pathogenic mechanism, whose frequency is likely underestimated, with it being overlooked by standard techniques. The identification of the molecular etiology of the disorder is extremely important in evaluating the pathological outcome and to better investigate the mechanisms associated with drug resistance, paving the way for the development of specific therapies. Karyotype and genomic techniques should be considered in all cases presenting with CDD without molecular confirmation. Full article

ABCA4 is the most frequently mutated gene leading to inherited retinal disease (IRD) with over 2200 pathogenic variants reported to date. Of these, ~1% are copy number variants (CNVs) involving the deletion or duplication of genomic regions, typically >50 nucleotides in length. An in-depth assessment of the current literature based on the public database LOVD, regarding the presence of known CNVs and structural variants in ABCA4, and additional sequencing analysis of ABCA4 using single-molecule Molecular Inversion Probes (smMIPs) for 148 probands highlighted recurrent and novel CNVs associated with ABCA4-associated retinopathies. An analysis of the coverage depth in the sequencing data led to the identification of eleven deletions (six novel and five recurrent), three duplications (one novel and two recurrent) and one complex CNV. Of particular interest was the identification of a complex defect, i.e., a 15.3 kb duplicated segment encompassing exon 31 through intron 41 that was inserted at the junction of a downstream 2.7 kb deletion encompassing intron 44 through intron 47. In addition, we identified a 7.0 kb tandem duplication of intron 1 in three cases. The identification of CNVs in ABCA4 can provide patients and their families with a genetic diagnosis whilst expanding our understanding of the complexity of diseases caused by ABCA4 variants. Full article

Vernalization plays a crucial role in the flowering and yield of Chinese cabbage, a process intricately influenced by long non-coding RNAs (lncRNAs). Our research focused on lncFLC1, lncFLC2a, and lncFLC2b, which emerged as key players in this process. These lncRNAs exhibited an inverse expression pattern to the flowering repressor genes FLOWERING LOCUS C 1 (BrFLC1) and FLOWERING LOCUS C 2 (BrFLC2) during vernalization, suggesting a complex regulatory mechanism. Notably, their expression in the shoot apex and leaves was confirmed through in fluorescent in situ hybridization (FISH). Furthermore, when these lncRNAs were overexpressed in Arabidopsis, a noticeable acceleration in flowering was observed, unveiling functional similarities to Arabidopsis’s COLD ASSISTED INTRONIC NONCODING RNA (COOLAIR). This resemblance suggests a potentially conserved regulatory mechanism across species. This study not only enhances our understanding of lncRNAs in flowering regulation, but also opens up new possibilities for their application in agricultural practices. Full article

Species within the genus Chenopodium hold significant research interest due to their nutritional richness and salt tolerance. However, the morphological similarities among closely related species and a dearth of genomic resources have impeded their comprehensive study and utilization. In the present research, we conduct the sequencing and assembly of chloroplast (cp) genomes from six Chenopodium and related species, five of which were sequenced for the first time. These genomes ranged in length from 151,850 to 152,215 base pairs, showcased typical quadripartite structures, and encoded 85 protein-coding genes (PCGs), 1 pseudogene, 37 tRNA genes, and 8 rRNA genes. Compared with the previously published sequences of related species, these cp genomes are relatively conservative, but there are also some interspecific differences, such as inversion and IR region contraction. We discerned 929 simple sequence repeats (SSRs) and a series of highly variable regions across 16 related species, predominantly situated in the intergenic spacer (IGS) region and introns. The phylogenetic evaluations revealed that Chenopodium is more closely related to genera such as Atriplex, Beta, Dysphania, and Oxybase than to other members of the Amaranthaceae family. These lineages shared a common ancestor approximately 60.80 million years ago, after which they diverged into distinct genera. Based on InDels and SNPs between species, we designed 12 pairs of primers for species identification, and experiments confirmed that they could completely distinguish 10 related species. Full article

Background and Objectives: Despite the vast heterogeneity in the genetic defects causing hemophilia A (HA), large intron inversions represent a major cause of disease, accounting for almost half of the cases of severe HA worldwide. We investigated the intron 22 and intron 1 inversion status in a cohort of Romanian unrelated patients with severe HA. Moreover, we evaluated the role of these inversions as relative risk factors in inhibitor occurrence. Materials and Methods: Inverse shifting—a polymerase chain reaction method was used to detect the presence of intron 22 and intron 1 inversions in 156 Romanian patients with HA. Results: Intron inversion 22 was found in 41.7% of the patients, while intron 1 inversion was detected in 3.2% of the patients. Overall, large intron inversions represented the molecular defect in 44.9% of the studied patients. Our findings are in accord with previously published reports from Eastern Europe countries and with other international studies. The risk of inhibitor development was higher in patients with inversion 1 compared to the patients with HA without any inversion detected. Conclusions: The current study demonstrates the major causative role of large intron inversions in severe HA in Romanian patients. Moreover, our study confirms the contribution of intron 1 inversion in inhibitor development. Full article

Patent ductus arteriosus (PDA) is frequent in preterm newborns, and its incidence is inversely associated with the degree of prematurity. The first choice of pharmacological treatment is ibuprofen. Several genes, including EPAS1, have been proposed as probable markers associated with a genetic predisposition for the development of PDA in preterm infants. EPAS 1 NG_016000.1:g.84131C>G or rs7557402 has been reported to be probably benign and associated with familial erythrocytosis by the Illumina Clinical Services Laboratory. Other variants of EPAS1 have been previously reported to be benign for familial erythrocytosis because they decrease gene function and are positive for familial erythrocytosis because the overexpression of EPAS1 is a key factor in uncontrolled erythrocyte proliferation. However, this could be inconvenient for ductal closure, since for this process to occur, cell proliferation, migration, and differentiation should take place, and a decrease in EPAS1 gene activity would negatively affect these processes. Single-nucleotide polymorphisms (SNPs) in EPAS1 and TFAP2B genes were searched with high-resolution melting and Sanger sequencing in blood samples of preterm infants with hemodynamically significant PDA treated with ibuprofen at the National Institute of Perinatology. The variant rs7557402, present in the EPAS1 gene eighth intron, was associated with a decreased response to treatment (p = 0.007, OR = 3.53). The SNP rs7557402 was associated with an increased risk of pharmacological treatment failure. A probable mechanism involved could be the decreased activity of the product of the EPAS1 gene. Full article

Current research on the angiotensin-converting-enzyme (ACE) gene has yielded controversial results on whether different ACE polymorphisms are linked with human longevity. ACE polymorphisms are a risk factor for Alzheimer’s disease and age-onset diseases that may contribute to the mortality of older people. Our goal is to consolidate existing studies, using artificial intelligence-assisted software to come to a more precise understanding of the role of the ACE gene in human longevity. The I (insertion) and D (deletion) polymorphisms in the intron are correlated with the levels of circulating ACE; homozygous D (DD) is high, and homozygous I (II) is low. Here, we performed a detailed meta-analysis of the I and D polymorphisms using centenarians (100+ years old), long-lived subjects (85+ years old), and control groups. ACE genotype distribution was analyzed across a total of 2054 centenarians and 12,074 controls, as well as 1367 long-lived subjects between the ages of 85–99, using the inverse variance and random effects methods. The ACE DD genotype was found to be favored in centenarians (OR: 1.41 (95% CI: 1.19–1.67), p < 0.0001) with a heterogeneity of 32%, and the II genotype slightly favored the control groups (OR: 0.81 (95% CI: 0.66–0.98), p = 0.03) with a heterogeneity of 28%, corroborating results from previous meta-analyses. Novel to our meta-analysis, the ID genotype was found to be favored in control groups (OR: 0.86 (95% CI: 0.76–0.97), p = 0.01) with a heterogeneity of 0%. The long-lived group showed a similar positive association between the DD genotype and longevity (OR: 1.34 (95% CI: 1.21–1.48), p < 0.0001) and a negative association between the II genotype and longevity (OR: 0.79 (95% CI: 0.70–0.88), p < 0.0001). The long-lived ID genotype did not show significant findings (OR: 0.93 (95% CI: 0.84–1.02), p = 0.79). In conclusion, the results suggest a significant positive association of the DD genotype with human longevity. However, despite the previous study, the results do not confirm a positive association of the ID genotype with human longevity. We suggest a few important paradoxical implications: (1) inhibition of ACE can increase longevity in model systems from nematodes to mammals, seemingly opposite to the finding in humans; (2) exceptional longevity associated with homozygous DD is also associated with age-related diseases with higher mortality risks in homozygous DD. We discuss ACE, longevity, and age-related diseases. Full article

Hemophilia A (HA) is one of the most widespread, X-linked, inherited bleeding disorders, which results from defects in the F8 gene. Nowadays, more than 3500 different pathogenic variants leading to HA have been described. Mutation analysis in HA is essential for accurate genetic counseling of patients and their relatives. We analyzed patients from 273 unrelated families with different forms of HA. The analysis consisted of testing for intron inversion (inv22 and inv1), and then sequencing all functionally important F8 gene fragments. We identified 101 different pathogenic variants in 267 patients, among which 35 variants had never been previously reported in international databases. We found inv22 in 136 cases and inv1 in 12 patients. Large deletions (1–8 exons) were found in 5 patients, and we identified a large insertion in 1 patient. The remaining 113 patients carried point variants involving either single nucleotide or several consecutive nucleotides. We report herein the largest genetic analysis of HA patients issued in Russia. Full article

Hemophilia A is an X-linked bleeding disorder caused by mutations in the FVIII gene. Genetic factors have been shown to be a risk factor for the development of inhibitors. We aimed to identify the specific variations of the FVIII gene of patients with hemophilia A with inhibitors and their association with the inhibitor titer. Methods: Cross-sectional descriptive study. We included 12 Colombian patients from a health care provider, “Integral Solutions SD”, who underwent analysis of genetic material (DNA), which was reported by the Molecular Hemostasis Laboratory in Bonn, Germany. Results: All of these patients were diagnosed with severe hemophilia A with inhibitors; ages ranged between 6 and 48 years, with a median age of 13.5 years. Molecular analysis showed the inversion of intron 22 in six patients (50.0%), a small duplication in two patients (16.7%), the inversion of intron 1 in one patient (8.3%), a large deletion (8.3%), a nonsense mutation (8.3%) and a splice-site (8.3%), findings similar to those of other studies. A total of 58.3% of the patients presented inversion mutations with a high risk of developing inhibitors A total of 83.3% of the evaluated patients presented null mutations; however the presence of high inhibitor titers was 66.7%. The most frequent mutation was the inversion intron 22. Knowing the type of mutation and its association as a risk factor for generating inhibitors invites us to delve into other outcomes such as residual values of coagulation FVIII as well as its impact on the half-life of the exogenous factor applied in prophylaxis. Full article

Inversions are structural variants that are generally balanced. However, they could lead to gene disruptions or have positional effects leading to diseases. Mutations in the NHS gene cause Nance-Horan syndrome, an X-linked disorder characterised by congenital cataracts and dental anomalies. Here, we aimed to characterise a balanced pericentric inversion X(p22q27), maternally inherited, in a child with syndromic bilateral cataracts by breakpoint mapping using whole-genome sequencing (WGS). 30× Illumina paired-end WGS was performed in the proband, and breakpoints were confirmed by Sanger sequencing. EdU assays and FISH analysis were used to assess skewed X-inactivation patterns. RNA expression of involved genes in the breakpoint boundaries was evaluated by droplet-digital PCR. We defined the breakpoint position of the inversion at Xp22.13, with a 15 bp deletion, disrupting the unusually large intron 1 of the canonical NHS isoform, and also perturbing topologically-associated domains (TADs). Moreover, a microhomology region of 5 bp was found on both sides. RNA analysis confirmed null and reduced NHS expression in the proband and his unaffected mother, respectively. In conclusion, we report the first chromosomal inversion disrupting NHS, fine-mapped by WGS. Our data expand the clinical spectrum and the pathogenic mechanisms underlying the NHS defects. Full article