Exon Sequences Research Articles

A novel active transposon creates allelic variation through altered translation rate to influence protein abundance.

Protein translation is tightly and precisely controlled by multiple mechanisms including upstream open reading frames (uORFs), but the origins of uORFs and their role in maize are largely unexplored. In this study, an active transposition event was identified during the propagation of maize inbred line B73. The transposon, which was named BTA for 'B73 active transposable element hAT', creates a novel dosage-dependent hypomorphic allele of the hexose transporter gene ZmSWEET4c through insertion within the coding sequence in the first exon, and results in reduced kernel size. The BTA insertion does not affect transcript abundance but reduces protein abundance of ZmSWEET4c, probably through the introduction of a uORF. Furthermore, the introduction of BTA sequence in the exonof other genes can regulate translation efficiency without affecting their mRNA levels. A transposon capture assay revealed 79 novel insertions for BTA and BTA-like elements. These insertion sites have typical euchromatin features, including low levels of DNA methylation and high levels of H3K27ac. A putative autonomous element that mobilizes BTA and BTA-like elements was identified. Together, our results suggest a transposon-based origin of uORFs and document a new role for transposable elements to influence protein abundance and phenotypic diversity by affecting the translation rate.

Study of the molecular characteristics of a Bweak phenotype due to a novel c.398T>C variant of the ABO gene

To explore the molecular mechanism for an individual with Bweak subtype. Serological methods were used to identify the proband's phenotype. In vitro enzyme activity test was used to determine the activity of B-glycosyltransferase (GTB) in her serum. The genotype was determined by PCR amplification and direct sequencing of exons 5 to 7 and flanking sequences of the ABO gene. T-A cloning technology was used to isolate the haploids. The primary physical and chemical properties and secondary structure of the protein were analyzed with the ProtParam and PSIPRED software. Three software, including PolyPhen-2, SIFT, and PROVEAN, was used to analyze the effect of missense variant on the protein. Serological results showed that the proband's phenotype was Bweak subtype with anti-B antibodies presented in her serum. In vitro enzyme activity assay showed that the GTB activity of the subject was significantly reduced. Analysis of the haploid sequence revealed a c.398T>C missense variant on the B allele, which resulted in a novel B allele. The 398T>C variant has caused a p.Phe133S substitution at position 133 of the GTB protein. Based on bioinformatic analysis, the amino acid substitution had no obvious effect on the primary and secondary structure of the protein, but the thermodynamic energy of the variant protein has increased to 6.07 kcal/mol, which can severely reduce the protein stability. Meanwhile, bioinformatic analysis also predicted that the missense variant was harmful to the protein function. The weak expression of the Bweak subtype may be attributed to the novel allele of ABO*B.01-398C. Bioinformatic analysis is helpful for predicting the changes in protein structure and function.

Identification of two novel FUT1 mutations in people with Bombay phenotype from Iran

Background and purposeBombay and Para-Bombay phenotypes are characterized by FUT1 gene mutation and lack of H antigen expression in red blood cells. ABH antigens are not present in the body secretions of Bombay individuals, while they are expressed in the secretions of para-Bombay. The aim of this study was to investigate the molecular basis of FUT1 and FUT2 genes in Iranians with the Bombay or Para-Bombay phenotype. Materials and MethodsABO phenotype analysis and routine serological tests were performed on 11 people with Bombay and Para-Bombay phenotypes. The coding regions of FUT1 and FUT2 genes were amplified by PCR followed by sequencing. The ABO genotypes were also determined by sequencing exons 6 and 7 of the ABO gene. Results: Serological investigations confirmed the Bombay phenotype in 8 samples and the Para-Bombay phenotype in 3 samples. Family members with the Bombay phenotype had the classic c 0.725 T > G mutation in the FUT1 gene, accompanied by deletion of the FUT2 gene. Other samples had c.653 A>G, c 0.661 C>T, c 0.652 C>G, and c.722 A>C mutations in the FUT1 while FUT2 was silenced by c 0.461 G>A. Conclusion: In this research, we identified two novel mutations in the FUT1 gene in individuals with the Bombay phenotype. This and previous works confirm the variety of FUT1 mutations.

Expanding the phenotypic spectrum of KCNK4: From syndromic neurodevelopmental disorder to rolandic epilepsy.

The KCNK4 gene, predominantly distributed in neurons, plays an essential role in controlling the resting membrane potential and regulating cellular excitability. Previously, only two variants were identified to be associated with human disease, facial dysmorphism, hypertrichosis, epilepsy, intellectual/developmental delay, and gingival overgrowth (FHEIG) syndrome. In this study, we performed trio-based whole exon sequencing (WES) in a cohort of patients with epilepsy. Two de novo likely pathogenic variants were identified in two unrelated cases with heterogeneous phenotypes, including one with Rolandic epilepsy and one with the FHEIG syndrome. The two variants were predicted to be damaged by the majority of in silico algorithms. These variants showed no allele frequencies in controls and presented statistically higher frequencies in the case cohort than that in controls. The FHEIG syndrome-related variants were all located in the region with vital functions in stabilizing the conductive conformation, while the Rolandic epilepsy-related variant was distributed in the area with less impact on the conductive conformation. This study expanded the genetic and phenotypic spectrum of KCNK4. Phenotypic variations of KCNK4 are potentially associated with the molecular sub-regional effects. Carbamazepine/oxcarbazepine and valproate may be effective antiepileptic drugs for patients with KCNK4 variants.

ABO discrepancy due to the para-Bombay phenotype: a case report

In this study, we aimed to determine and analyze the genetic differences of the FUT1 and FUT2 genes within an individual exhibiting the Para-Bombay phenotype. Standard serological tests were conducted to analyze the ABO phenotype. The amplification of FUT1 and FUT2 genes was conducted by polymerase chain reaction and amplicons were sequenced. The determination of the ABO genotype was conducted by sequencing exons 1 to 7 of the ABO gene. The Lewis phenotype was defined as Le(a−b+); Despite this, red blood cells (RBCs) did not show signs of A, B and H antigens using the serological method utilized in this study. However, Analyses of sequencing and cloning demonstrated the existence of a FUT1 mutation at position c.658C>T and an inactive allele FUT1*01N.06 (c.551_552delAG). Genetic testing for ABO blood types was subsequently determined to be ABO*B.01/ABO*B.01 (c.297A>G; c.526C>G; c.657C>T; c.703G>A; c.796C>A; c.803G>C; c.930G>A). Heterozygosity for indel and missense mutation within the coding region of FUT1 are likely to be responsible for development of the Para-Bombay phenotype.

On the retinae of Glis and Graphiurus: photoreceptor and ganglion cell populations, an absence of shortwave-sensitive cones, and some other features (Rodentia: Gliridae)

The retina of the fat dormouse Glis glis was studied histologically. Opsin immunolabeling identified an unusually dense population of rod photoreceptors (ca. 600,000–780,000/mm²) and a low-density population of L cone photoreceptors containing the longwave-sensitive (LWS) cone opsin, with a shallow maximum of ca. 3,300/mm² in temporal retina and ca. 2,000–3,000/mm² in the remaining retina. Hence the cones comprise only 0.25–0.50% of the photoreceptors. There are no S cones expressing the shortwave-sensitive (SWS1) opsin that is the second cone opsin in most mammals, conveying dichromatic colour vision. We conclude that the fat dormouse is a cone monochromat. Sequencing of the tuning-relevant exon 1 of the SWS1 opsin gene revealed that the gene would have coded for a UV-sensitive visual pigment, but that it contains mutational changes making it nonfunctional. Retinal interneurons (rod bipolar cells, horizontal cells, several amacrine cell types) have rodent-typical features. NeuN-labeled presumed retinal ganglion cells have densities between ca. 4000/mm² in temporal central retina and 850–1300/mm² in peripheral retina. The peak ganglion cell density would result in an estimated visual acuity maximum of ca. 1.8 cycles/degree or ca. 33 minutes of arc. Assessment of a further Gliridae species, the African dormouse Graphiurus sp., also revealed a high rod density, low L cone density, and an absence of SWS1 opsin.

Are both distinct epithelial and stromal cells molecular analysis from phyllodes tumors versus fibroadenoma components affected in breast fibroepithelial progression?

To determine molecular events involved in the tumorigenesis of phyllodes tumors (PT) and the role of each stromal (SC) and epithelial (EC) cell. Frozen breast samples enriched with epithelial and stromal cells from three fibroadenomas and 14 PT were retrieved and laser microdissected. Sanger and polymerase chain reaction-based sequencing of exon 2 MED12 and TERT promoter hotspot mutations were performed; 44K microarray platform was used to analyze gene expression. All three fibroadenomas (FAs) presented mutations in MED12, but not in TERT, whose mutation was observed in five of the 14 PTs. EC and SC of each affected tumor displayed identical alterations. Of the total differentially expressed genes (DEG) (EC = 1,543 and SC = 850), 984 were EC-eDEGs and 291 were SC-eDEGs. We found a high similarity of diseases and functions enriched by both cell types, but dissimilarity in the number of enriched canonical pathways. Three signaling canonical pathways overlapping with EC and SC were predicted to be activated in one cell type and inactivated in the other, while no overlap in eDEGs was assigned to them. We also identified 13 EC-eDEGs and five SC-eDEGs enriched networks, in which the SC-eDEGs were able to segregate FA from PT samples. Identical TERT mutations from both SC and ES origins might affect the PTs tumorigenesis. Gene expression differences suggest coordinated molecular processes between these components with determinant differences acquired by SC, able to fully distinguish PTs from FAs lesions.

Molecular Genetic Analysis of Para-Bombay Phenotype in Chinese: Identification of a Novel FUT1 Allele

The goal was to identify a novel FUT1 allele and to study serologic and gene feature of the para-Bombay blood type of one expectant mother in Xinjiang, China. ABO and Lewis groups were recognized by standard serologic techniques in an ABO typing discrepancy specimen from one person at the Tianjin Blood Center. DNA (deoxyribonucleic acid) was collected and polymerase chain reactions with sequence-specific primers (PCR-SSP) were performed to sequence exons 6 and 7 of ABO gene, exon 4 of FUT1 gene, and exon 2 of FUT2. PCR products were sequenced to identify ABO groups and the variation sites. The genotype was determined by family study. In our laboratory testing, erythrocytes from the proposita did not react with anti-A and anti-B reagents. B antigen was discovered only after adsorption and elution. Red cells were nonreactive with monoclonal anti-H. The sera of the proposita contained anti-A and were weakly agglutinated by B cells. The hybrid 902 A>G mutation was detected in the proposita's father and mother. The proposita has the same mutation 902 A>G, which was conjectured as homozygosity for 902 A>G. One novel mutation of FUT1 gene was observed in our laboratory. It has never been reported previously. The para-Bombay phenotype in the proposita originating from Xinjiang (China) results from homozygosity for FUT1 902 A>G, together with 357 C>T of FUT2.

Non-Syndromic Hearing Loss in a Romanian Population: Carrier Status and Frequent Variants in the GJB2 Gene.

The genetic causes of autosomal recessive nonsyndromic hearing loss (ARNSHL) are heterogeneous and highly ethnic-specific. We describe GJB2 (connexin 26) variants and carrier frequencies as part of our study and summarize previously reported ones for the Romanian population. In total, 284 unrelated children with bilateral congenital NSHL were enrolled between 2009 and 2018 in northwestern Romania. A tiered diagnostic approach was used: all subjects were tested for c.35delG, c.71G>A and deletions in GJB6 (connexin 30) using PCR-based methods. Furthermore, 124 cases undiagnosed at this stage were analyzed by multiplex-ligation-dependent probe amplifications (MLPA), probe mix P163, and sequencing of GJB2 exon 2. Targeted allele-specific PCR/restriction fragment length polymorphism (RFLP) established definite ethio-pathogenical diagnosis for 72/284 (25.35%) of the cohort. Out of the 124 further analyzed, in 12 cases (9.67%), we found compound heterozygous point mutations in GJB2. We identified one case of deletion of exon 1 of the WFS1 (wolframin) gene. Carrier status evaluation used Illumina Infinium Global Screening Array (GSA) genotyping: the HINT cohort-416 individuals in northwest Romania, and the FUSE cohort-472 individuals in southwest Romania. GSA variants yielded a cumulated risk allele presence of 0.0284. A tiered diagnostic approach may be efficient in diagnosing ARNSHL. The summarized contributions to Romanian descriptive epidemiology of ARNSHL shows that pathogenic variants in the GJB2 gene are frequent among NSHL cases and have high carrier rates, especially for c.35delG and c.71G>A. These findings may serve in health strategy development.

Increased diagnostic yield in a cohort of hearing loss families using a comprehensive stepwise strategy of molecular testing.

Hearing loss is one of the most common sensory disorders in humans. This study proposes a stepwise strategy of deafness gene detection using multiplex PCR combined with high-throughput sequencing, Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and whole-exome sequencing (WES) to explore its application in molecular diagnosis of hearing loss families. A total of 152 families with hearing loss were included in this study, the highest overall diagnosis rate was 73% (111/152). The diagnosis rate of multiplex PCR combined with high-throughput sequencing was 52.6% (80/152). One families was diagnosed by Sanger sequencing of GJB2 exon 1. Two families were diagnosed by MLPA analysis of the STRC gene. The diagnosis rate with additional contribution from WES was 18.4% (28/152). We identified 21 novel variants from 15 deafness genes by WES. Combining WES and deep clinical phenotyping, we diagnosed 11 patients with syndromic hearing loss (SHL). This study demonstrated improved diagnostic yield in a cohort of hearing loss families and confirmed the advantages of a stepwise strategy in the molecular diagnosis of hearing loss.

A splicing-dependent ER retention signal regulates surface expression of the mechanosensitive TMEM63B cation channel

TMEM63B is a mechanosensitive cation channel activated by hypoosmotic stress and mechanic stimulation. We recently reported a brain-specific alternative splicing of exon 4 in TMEM63B. The short variant lacking exon 4, which constitutes the major isoform in the brain, exhibits enhanced responses to hypoosmotic stimulation compared to the long isoform containing exon 4. However, the mechanisms affecting this differential response are unclear. Here, we showed that the short isoform exhibited stronger cell surface expression compared to the long variant. Using mutagenesis screening of the coding sequence of exon 4, we identified an RXR-type endoplasmic reticulum (ER) retention signal (RER). We found that this motif was responsible for binding to the COPI retrieval vesicles, such that the longer TMEM63B isoforms were more likely to be retrotranslocated to the ER than the short isoforms. In addition, we demonstrated long TMEM63Bs could form heterodimers with short isoforms and reduce their surface expression. Taken together, our findings revealed an ER retention signal in the alternative splicing domain of TMEM63B that regulates the surface expression of TMEM63B protein and channel function.

Missing heritability of Wilson disease: a search for the uncharacterized mutations.

Wilson disease (WD), a copper metabolism disorder caused by mutations in ATP7B, manifests heterogeneous clinical features. Interestingly, in a fraction of clinically diagnosed WD patients, mutations in ATP7B appears to be missing. In this review we discuss the plausible explanations of this missing heritability and propose a workflow that can identify the hidden mutations. Mutation analyses of WD generally includes targeted sequencing of ATP7B exons, exon-intron boundaries, and rarely, the proximal promoter region. We propose that variants in the distal cis-regulatory elements and/or deep intronic variants that impact splicing might well represent the hidden mutations. Heterozygous del/ins that remain refractory to conventional PCR-sequencing method may also represent such mutations. In this review, we also hypothesize that mutations in the key copper metabolism genes, like, ATOX1, COMMD1, and SLC31A1, could possibly lead to a WD-like phenotype. In fact, WD does present overlapping symptoms with other rare genetic disorders; hence, the possibility of a misdiagnosis and thus adding to missing heritability cannot be excluded. In this regard, it seems that whole-genome analysis will provide a comprehensive and rapid molecular diagnosis of WD. However, considering the associated cost for such a strategy, we propose an alternative customized screening schema of WD which include targeted sequencing of ATP7B locus as well as other key copper metabolism genes. Success of such a schema has been tested in a pilot study.

Uncovering single‐nucleus RNA velocity variations in neuropathologic Alzheimer’s disease

AbstractBackgroundDifferential single‐nucleus (snRNA‐seq) gene expression analyses in Alzheimer’s disease (AD) provide fixed snapshots of cellular alterations, failing to detect the temporal dynamics of genes at individual cells. To overcome this limitation, here we analyze single‐nucleus RNA velocities (RNA‐vel) from the prefrontal cortex to characterize dynamic genetic and cellular differences in neuropathological AD progression. RNA‐vel is the rate of change of gene expression obtained by comparing intronic and exonic sequence counts. Comparison of AD pathology associated gene expression with parallel RNA‐vel differences reveals sets of genes and molecular pathways that underlie the static and putative dynamic regimes of cell type‐specific dysregulations underlying the disease.MethodWe used snRNA‐seq data from the prefrontal cortex of 48 subjects1 with varied levels of AD pathology from ROSMAP2. We then calculated both the expression and RNA‐vel differences between low AD‐pathology and mild‐to‐severe AD‐pathology. These differences were cell‐specific across six major cell types: excitatory neurons, inhibitory neurons, astrocytes, microglia, oligodendrocytes, and oligodendrocyte progenitor cells.1. We characterized RNA‐vel differences associated with four AD neuropathology traits: neuritic plaque, neuronal neurofibrillary tangle, overall β‐amyloid load, and PHF tau tangle density.2. We demonstrated the reproducibility of the RNA‐vel differences in two independent datasets: dorsolateral prefrontal cortex (N = 24) from ROSMAP3 and superior frontal gyrus (N = 6) from GEO4.ResultOnly 10 of 843 (1.2%) genes overlap between the differential RNA expression and velocity analyses, suggesting substantial AD‐pathology related differences between these two RNA descriptors. The genes with only velocity differences relate to cell developmental and synaptic processes. Conversely, the genes with only differential expression are majorly associated with mitochondrial activity and ribosomal processes. Many genes were reproduced in the independent datasets; these overlapping genes are again associated with neural development and synaptic activities across many cell types, as well as vascular‐ and immune‐related processes in astrocytes and microglia, respectivelyConclusionWe use RNA velocity to characterize, for the first time to our knowledge, the dynamical multicellular processes underlying neuropathological AD progression. The results support the validity of the novel RNA‐vel concept for achieving a complementary molecular characterization of AD, which may be obscured by typical analysis of RNA abundance alone.

Identification of B(A) Blood Group Serology Combined with Molecular Biology and Blood Transfusion Strategy

To analyze the reasons for ABO blood group forward typing and reverse typing incompatibility of the proband by serological and molecular biological tests, so as to clarify the blood group and genetic rules and determine the reasonable transfusion strategy. On the basis of serological testing results, PCR-SSP method was utilized for the ABO exon sequencing in the peripheral blood of the proband and 5 family members, and blood group results were comprehensively analyzed. The serological results of ABO blood group of the proband were incompatibility, and the molecular biological test results showed that there was a c.700C>G mutation compared with B101, which was consistent with B(A)02 subtype, and the genotype was B(A)02/O02. The results of the elder brother was the same as the proband. The nephew of the proband also detected c.700C>G, genotype A102/B(A) 02. When the result of standard serological test for ABO blood group incompatibility occurs, using molecular biology detection technology to explore mutation is an effective method to confirm ABO subtype, and ensures the safety of clinical blood transfusion.

Identification and Molecular Biology of Variant D Blood Group of RHD*95A Genotype

To explore the molecular biology of D variant blood group with RHD*95A genotype and the genetic mechanism of its generation. A total of 6 samples from 3 generations of a family were analyzed. RHD blood group was identified by saline test tube and microcolumn gel card method. 10 exons of RHD gene were amplified by Polymerase Chain Reaction-Sequence Specific Primer (PCR-SSP) and analyzed by direct sequencing. Homology modeling was used to compare the structural differences between mutant RHD protein and wild-type RHD protein. The proband was identified as D variant by serological identification, RHD gene sequencing directly detected a c. 95 c > A mutation in exon 1 that leads to encoding the 32-bit amino acids by threonine Thr (T) into aspartic acid Asn (N), the rest of the exon sequences were normal compared with the normal RHD*01 gene. In the family, the proband's father, grandmather and uncle were all carried the same RHD*95A allele. Protein modeling results suggested that the hydrogen chain connected to the 32nd amino acid residue was changed after p.T32N mutation, which affected the structural stability of RHD protein. The first genetic lineage of the RHD*95A gene was identified in a Chinese population. The c.95C>A mutation in RHD gene was found in the family, which resulted in reduced expression of RHD antigen and showed D variant, the mutation could be stably inheritable. Gene identification and protein structure analysis of D variant population is helpful to explore the molecular mechanism of its formation and ensure the safety of blood transfusion.

KBTBD11, encoding a novel PPARγ target gene, is involved in NFATc1 proteolysis by interacting with HSC70 and HSP60

We previously revealed that Kbtbd11 mRNA levels increase during 3T3-L1 differentiation and Kbtbd11 knockdown suppresses whereas its overexpression promotes adipogenesis. However, how Kbtbd11 mRNA is regulated during adipocyte differentiation and how the KBTBD11 protein functions in adipocytes remain elusive. This study aimed to examine the transcriptional regulatory mechanism of Kbtbd11 during adipocyte differentiation, KBTBD11-interacting protein functions, and elucidate the role of KBTBD11 in adipocytes. First, we identified the PPRE consensus sequences in the Kbtbd11 exon 1- and intron 1-containing region and demonstrated that PPARγ acts on this region to regulate Kbtbd11 expression. Next, we purified the KBTBD11 protein complex from 3T3-L1 adipocytes and identified heat shock proteins HSC70 and HSP60 as novel KBTBD11-interacting proteins. HSC70 and HSP60 inhibition increased KBTBD11 protein levels that promoted NFATc1 ubiquitination. These data suggest that HSC70 and HSP60 are involved in KBTBD11 stabilization and are responsible for NFATc1 regulation on the protein level. In summary, this study describes first the protein regulatory mechanism of NFATc1 through the HSC70/HSP60-KBTBD11 interaction that could provide a potential new target for the differentiation and proliferation of various cells, including adipocytes and tumors.

Detection and Characterization of a De Novo Alu Retrotransposition Event Causing NKX2-1-Related Disorder.

Heterozygous NKX2-1 loss-of-function variants cause combinations of hyperkinetic movement disorders (MDs, particularly childhood-onset chorea), pulmonary dysfunction, and hypothyroidism. Mobile element insertions (MEIs) are potential disease-causing structural variants whose detection in routine diagnostics remains challenging. To establish the molecular diagnosis of two first-degree relatives with clinically suspected NKX2-1-related disorder who had negative NKX2-1 Sanger (SS), whole-exome (WES), and whole-genome (WGS) sequencing. The proband's WES was analyzed for MEIs. A candidate MEI in NKX2-1 underwent optimized SS after plasmid cloning. Functional studies exploring NKX2-1 haploinsufficiency at RNA and protein levels were performed. A 347-bp AluYa5 insertion with a 65-bp poly-A tail followed by a 16-bp duplication of the pre-insertion wild-type sequence in exon 3 of NKX2-1 (ENST00000354822.7:c.556_557insAlu541_556dup) segregated with the disease phenotype. We identified a de novo exonic AluYa5 insertion causing NKX2-1-related disorder in SS/WES/WGS-negative cases, suggesting that MEI analysis of short-read sequencing data or targeted long-read sequencing could unmask the molecular diagnosis of unsolved MD cases. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Alkali cation-mediated topology displayed by an exonic G-rich sequence of TRPA1 gene

G-rich sequences are intrinsic parts of the genome, widespread in promoters, telomeres, or other regulatory regions. The in vivo existence and biological significance have established the functional aspect of G-quadruplex structures and thus have developed immense interest in exploring their therapeutic aspects. Herein, using biophysical methods, we examined the structural status and comprehensive cation-dependence of a 17-bp G-rich genomic sequence (SKGT17) located in the coding region of the human TRPA1 gene, known to be associated with various neurovascular, cardiovascular, and respiratory conditions. TRPA1 is primarily seen as a therapeutic target for the development of novel analgesics. Bioinformatics analysis has suggested that 17-bp quadruplex motif is a binding site for transcription factor 'Sp1'. The formation and recognition of SKGT17 G-quadruplex might impact its regulatory functioning. Biophysical studies confirmed that the presence of alkali metal ions facilitated the formation of G-quadruplex in parallel topology. Native gel further substantiated the formation of a biomolecular species. Circular dichroism (CD), UV-thermal melting (Tm), and CD melting confirmed the formation of parallel G-quadruplex with metal ion-dependent stability. The stability of the G-quadruplex formed is found to be significantly high in the presence of K+ ions than that of other ions. Intriguingly, we have also established that this segment of the TRAP1 gene favors G-quadruplex formation over its participation in the corresponding duplex formation under K+ ions conditions. This study attempts to explain the rationale for the stabilization of G-quadruplex in the presence of alkali metal ions and may add to a better understanding and insights into DNA-metal ions interactions.

293 Application of nanopore sequencing in identifying null mutations and intragenic copy number variations in filaggrin gene

Mutations in the gene encoding filaggrin (FLG) have been identified as the cause of ichthyosis vulgaris (IV) and shown to be major predisposing factors for atopic dermatitis. The profilaggrin protein consists of 10-12 filaggrin monomer repeats arranged in tandem, which arise from an ultra-long sequence of exon 3 comprised of near-indistinguishable repeats, demonstrating intragenic copy number variations (CNVs). As such, it was not until 2006 that null mutations in FLG could be detected using the newly developed long-range polymerase chain reaction (PCR) technique.

Molecular and serological analysis of the D variant in the Chinese population and identification of seven novel RHD alleles.

The molecular basis of the D variant phenotype in the Chinese differs greatly from that of the Caucasian. Adapting a specific D typing strategy to the spectrum of prevalent RHD variant alleles is necessary. Blood samples with ambiguous D phenotypes were collected in the Southern Chinese population. A special three-step typing strategy was applied. First, the common DVI type 3 was identified from epitope profiles of D antigen. Then, another common weak D type 15 (RHD*845A) was identified by epitope profiles of D antigen and Sanger sequencing of RHD exon 6. Finally, the remaining D variants were genotyped mainly by Sanger sequencing. For the novel RHD alleles in the coding region and exon-intron junction, in vitro transfection and minigene splicing assays were performed, respectively. The anti-D investigation was performed. DVI type 3 (65/253, 25.7%) and weak D type 15 (62/253, 24.5%) were common Chinese D variants, and RHD*960A, DFR, RHD*weak D type 25, 72, and 136 were frequent variant RHD alleles. Besides, twenty-two sporadic and seven novel RHD alleles (RHD*188A; RHD*688C; RHD*782 T; RHD*1181C; RHD*165 T, 993A; RHD*148 + 3G > T and RHD*1227 + 5G > C) were identified. The deleterious effect of the novel RHD alleles on D antigen or mRNA expression was confirmed. Anti-D was detected in two DVI type 3 pregnant women. The three-step typing strategy provides an effective approach for Chinese D variant typing. It can be anticipated that commercially available RHD genotyping kits have limitations for testing Chinese D variants, as some of the frequent variants are not interrogated.