Insertion Deletion Research Articles

Factor VII Augusta (FVII G395D/G395N):Â a Compound Heterozygous Deletion Insertion and Missense Variant Manifesting As Moderate Bleeding Disorder

Coagulation Factor VII occupies a key role in the coagulation system in that it activates Factor X to Xa when bound to Tissue Factor and also activates IX to IXa that in turn leads to Xa activation via an augmentation pathway of blood coagulation. Factor VII deficiency is usually transmitted in an autosomal recessive fashion and predisposition for bleeding manifestations may be highly variable and does not correlate with plasma factor VII activity levels. More than often patients are compound heterozygous for two different amino acid substitutions and it is difficult to predict the impact that each one of these mutations may have on a patients bleeding predisposition. In this report, we describe a patient with a compound heterozygous deletion insertion and missense variant, manifesting as moderate bleeding disorder.The patient is a 20 year old woman with a significant history of dysmenorrhea and menorrhagia. The patient complains of easy bruising, but so far has not had muscle or joint bleeds. Laboratory evaluation showed the patient to have a only slightly prolonged PT time of 14 sec and decreased Factor VII activity in vitro to 38%.Factor VII gene sequencing revealed that the patient has a nucleotide substitution 1184G>A on one allele resulting in AA substitution Glycin >Aspartate in position 395 (G395D) and 1183_1184 deletion on the other allele resulting in a Glycine to Asparagine substitution at same position (G395N). Both AA substitutions FVII G395D/G395N are located in the carboxy-terminus of the serum protease region of the protein. In both instances glycine a small non-polar AA is replaced with two larger, polar amino-acids (Aspartate and Asparagine). We predict that the G395D is pathogenic, since Asp D is highly negativly charged and the G395N acid substitution would likewise lead to the polar Asn N to interact with other charged AA. The replacement of Glycine, an amino acid which is small and with its two flanking hydrogen side chains can allow for a large conformational flexibility in the structure of proteins, by two highly polar AA predictably may have incurred conformational structural changes on FVII G395D/G395N protease domain that affected Factor VII activity but not antigen. Since FVII G395D/G395N has so far not been described we would like to refer to it as FVII Augusta. DisclosuresNo relevant conflicts of interest to declare.

Sublinear update time randomized algorithms for dynamic graph regression

A well-known problem in data science and machine learning is linear regression, which is recently extended to dynamic graphs. Existing exact algorithms for updating the solution of dynamic graph regression require at least a linear time (in terms of n: the size of the graph). However, this time complexity might be intractable in practice.In the current paper, we utilize subsampled randomized Hadamard transformand CountSketchto propose the first sublinear update time randomized algorithms for regression of general dynamic graphs. Suppose that we are given a n×d matrix embedding M of the graph, where d≪n and M has certain properties. Let r be the number of samples required by subsampled randomized Hadamard transform for a 1±ϵ approximation, which is a sublinear of n. Our first algorithm supports edge insertion and edge deletion and updates the approximate solution in O(rd) time. Our second algorithm is based on CountSketchand supports edge insertion, edge deletion, node insertion and node deletion. It updates the approximate solution in O(qd) time, where q=O(d2ϵ2log6(d/ϵ)).

A new method to accurately identify single nucleotide variants using small FFPE breast samples.

Most tissue collections of neoplasms are composed of formalin-fixed and paraffin-embedded (FFPE) excised tumor samples used for routine diagnostics. DNA sequencing is becoming increasingly important in cancer research and clinical management; however it is difficult to accurately sequence DNA from FFPE samples. We developed and validated a new bioinformatic pipeline to use existing variant-calling strategies to robustly identify somatic single nucleotide variants (SNVs) from whole exome sequencing using small amounts of DNA extracted from archival FFPE samples of breast cancers. We optimized this strategy using 28 pairs of technical replicates. After optimization, the mean similarity between replicates increased 5-fold, reaching 88% (range 0–100%), with a mean of 21.4 SNVs (range 1–68) per sample, representing a markedly superior performance to existing tools. We found that the SNV-identification accuracy declined when there was less than 40 ng of DNA available and that insertion–deletion variant calls are less reliable than single base substitutions. As the first application of the new algorithm, we compared samples of ductal carcinoma in situ of the breast to their adjacent invasive ductal carcinoma samples. We observed an increased number of mutations (paired-samples sign test, P < 0.05), and a higher genetic divergence in the invasive samples (paired-samples sign test, P < 0.01). Our method provides a significant improvement in detecting SNVs in FFPE samples over previous approaches.

High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion.

Precise and efficient insertion of large DNA fragments into somatic cells using gene editing technologies to label or modify endogenous proteins remains challenging. Non-specific insertions/deletions (INDELs) resulting from the non-homologous end joining pathway make the process error-prone. Further, the insert is not readily removable. Here, we describe a method called CRISPR-mediated insertion of exon (CRISPIE) that can precisely and reversibly label endogenous proteins using CRISPR/Cas9-based editing. CRISPIE inserts a designer donor module, which consists of an exon encoding the protein sequence flanked by intron sequences, into an intronic location in the target gene. INDELs at the insertion junction will be spliced out, leaving mRNAs nearly error-free. We used CRISPIE to fluorescently label endogenous proteins in mammalian neurons in vivo with previously unachieved efficiency. We demonstrate that this method is broadly applicable, and that the insert can be readily removed later. CRISPIE permits protein sequence insertion with high fidelity, efficiency, and flexibility.

A critical role of PvFtsH2 in the degradation of photodamaged D1 protein in common bean

Light is required for initiating chloroplast biogenesis and photosynthesis; however, the photosystem II reaction center (PSII RC) can be photodamaged. In this study, we characterized pvsl1, a seedling-lethal mutant of Phaseolus vulgaris. This mutant showed lethality when exposed to sunlight irradiation and a yellow-green leaf phenotype when grown in a growth chamber under low-light conditions. We developed 124 insertion/deletion (INDEL) markers based on resequencing data of Dalong1 and PI60234, two local Chinese common bean cultivars, for genetic mapping. We identified Phvul.002G190900, which encodes the PvFtsH2 protein, as the candidate gene for this pvsl1 mutation through fine-mapping and functional analysis. A single-base deletion occurred in the coding region of Phvul.002G190900 in the pvsl1 mutant, resulting in a frameshift mutation and a truncated protein lacking the Zn2+ metalloprotease domain. Suppressed expression of Phvul.002G190900 at the transcriptional level was detected, while no change in the subcellular localization signal was observed. The seedlings of pvsl1 exhibited hypersensitivity to photoinhibition stress. In the pvsl1 mutant, abnormal accumulation of the D1 protein indicated a failure to rapidly degrade damaged D1 protein in the PSII RC. The results of this study demonstrated that PvFtsH2 is critically required for survival and maintaining photosynthetic activity by degrading photodamaged PSII RC D1 protein in common bean.

Characterization of a novel CSF1R mutation causing hereditary diffuse leukoencephalopathy with spheroids in a case presenting with young-onset dementia

ObjectiveThis poster aims to report an unregistered mutation CSF1R gene in a patient presenting young-onset dementia.Hypothesis: Novel heterozygous deletion–insertion mutation in the Colony-Stimulating Factor 1 Receptor (CSF1R) gene is linked to a case of hereditary diffuse leukoencephalopathy with spheroids (HDLS), presenting with young-onset dementia.BackgroundCSF1R mediates proliferation, differentiation, and survival of monocytes/ macrophages and microglia. Pathogenic variants in the CSF1R gene cause autosomal dominant diffuse hereditary leukoencephalopathy with spheroids characterized by variable behavioural, cognitive, and motor changes, usually presenting with young-onset dementia. The average lifespan after the start of the symptoms is often 6 years.Case reportMolecular genetic analysis of whole-exome sequencing (WES) was carried out for a 49-year-old male patient presenting with rapid cognitive decline, behavioural symptoms and impaired sphincteric control.DiscussionWES identified the heterozygous deletion–insertion variant c.2356_2357delinsAC p.(Leu786Thr) (chr5:149435867-49435868; hg19) in the CSF1R gene. To the best of our knowledge the variant has not been described in the literature so far (HGMD 2019.3). No allele frequencies in the general population have been documented.ConclusionWe believe that we have identified a novel mutation in the CSF1R gene. This mutation is likely to be linked to this patient presenting with young-onset dementia.

Long-read nanopore sequencing enables accurate confirmation of a recurrent PMS2 insertion–deletion variant located in a region of complex genomic architecture

Targeted next generation sequencing (NGS) is the predominant methodology for the molecular genetic diagnosis of inherited conditions. In many laboratories, NGS-identified variants are routinely validated using a different method, to minimize the risk of a false-positive diagnosis. This can be particularly important when pathogenic variants are located in complex genomic regions. In this situation, new long-read sequencing technologies have potential advantages over existing alternatives. However, practical examples of their utility for diagnostic purposes remain scant. Here, we report the use of nanopore sequencing to validate a PMS2 mutation refractory to conventional methods. In a patient who presented with colorectal cancer and loss of PMS2 immunostaining, short-read NGS of Lynch syndrome-associated genes identified the recurrent PMS2 insertion-deletion variant, c.736_741delinsTGTGTGTGAAG (p.Pro246Cysfs*3). Confirmation of this variant using bidirectional Sanger sequencing was impeded by an upstream intron 6 poly(T) tract. Using a locus-specific amplicon template, we undertook nanopore long-read sequencing in order to assess the diagnostic accuracy of this platform. Pairwise comparison between a curated benchmark allele (derived from short-read NGS and unidirectional Sanger sequencing) and the consensus nanopore dataset revealed 100% sequence identity. Our experience provides insight into the robustness and ease of deployment of “third-generation” sequencing for accurate characterisation of pathogenic variants.

Internal Validation of Global Filer PCR Amplification Kit in DNA Section of Punjab Forensic Science Agency, Lahore

Background: Global Filer PCR Amplification Kit is a 6-dye amplification assay, which amplifies 24 genetic loci simultaneously in a single reaction. The amplification system encompasses both Combined DNA Index System loci and European standard set loci along with a highly polymorphic short tandem repeat (STR) locus SE33. The addition of a Y-STR marker and an insertion deletion marker of Y chromosome has provided additional aid in the gender identification of Amelogenin-Y null males. Global Filer PCR Amplification Kit has provided understandably more discrimination power compared with Identifiler Plus PCR Amplification Kit currently being used in Punjab Forensic Science Agency (PFSA). Methodology: In this study, Global Filer PCR Amplification Kit was internally validated in PFSA following the Scientific Working Group on DNA Analysis Methods laid principles to establish quality assurance parameters and interpretation guidelines. Known nonprobative DNA samples were utilized in this study to determine the sensitivity, establishment of proper thresholds for analysis, mixture analysis to determine detectable mixture ratios, contamination assessment, and reproducibility for the new method. Conclusion: The results of this internal validation strongly suggest the use of Applied Biosystems Global Filer PCR Amplification Kit in forensic casework analysis.

A 7-nt nucleotide sequence variant within the sheep KDM3B gene affects female reproduction traits

Lysine demethylase 3B (KDM3B) gene is a histone demethylase, demonstrating specific demethylation of the histone H3 lysine 9. It was detected as a sheep reproductive candidate gene by genome-wide scans, and related studies also showed its significance in female reproductive process. However, rare study researched its polymorphism. Herein, we hypothesized that the polymorphisms of KDM3B gene were associated with sheep reproduction traits. A 7-nt nucleotide sequence variant (rs1088697156) within KDM3B gene was identified in a total of 888 individuals, including the Australian White (AUW) sheep and Lanzhou Fat-tailed (LFT) sheep. II (insertion/insertion) and ID (insertion/deletion) genotypes of 7-nt variant were detected, which were at Hardy-Weinberg equilibrium (HWE) in detected breeds. Association analysis illustrated the 7-nt variant was significantly associated with the litter size, duration of pregnancy, live lamb number, live lamb rate, stillbirth number, stillbirth rate of average and different parity (P < 0.05) in AUW sheep. Moreover, ‘ID’ was the dominant genotype with excellent consistency in reproductive traits. It is instrumental to select individuals with ID genotype for improving the sheep reproduction traits. These findings suggest that the 7-nt variant within KDM3B gene can be used as a candidate marker of reproduction traits for sheep breeding improvement by marker-assisted selection.

CNGB1-related rod-cone dystrophy: A mutation review and update.

Cyclic nucleotide‐gated channel β1 (CNGB1) encodes the 240‐kDa β subunit of the rod photoreceptor cyclic nucleotide‐gated ion channel. Disease‐causing sequence variants in CNGB1 lead to autosomal recessive rod‐cone dystrophy/retinitis pigmentosa (RP). We herein present a comprehensive review and analysis of all previously reported CNGB1 sequence variants, and add 22 novel variants, thereby enlarging the spectrum to 84 variants in total, including 24 missense variants (two of which may also affect splicing), 21 nonsense, 19 splicing defects (7 at noncanonical positions), 10 small deletions, 1 small insertion, 1 small insertion–deletion, 7 small duplications, and 1 gross deletion. According to the American College of Medical Genetics and Genomics classification criteria, 59 variants were considered pathogenic or likely pathogenic and 25 were variants of uncertain significance. In addition, we provide further phenotypic data from 34 CNGB1‐related RP cases, which, overall, are in line with previous findings suggesting that this form of RP has long‐term retention of useful central vision despite the early onset of night blindness, which is valuable for patient counseling, but also has implications for it being considered a priority target for gene therapy trials.

Genomic and transcriptomic analysis of Bacillus subtilis JNFE1126 with higher nattokinase production through ultraviolet combined 60Co-γ ray mutagenesis

Nattokinase is a serine protease which is produced by Bacillus subtilis and exhibits vigorous fibrinolytic activity. Nattokinase is useful for preventing and treating thromboembolic diseases. In this study, the effect of ultraviolet combined 60Co-γ ray on B. subtilis were investigated, and the mechanism of NK production enhancement was elucidated at molecular level. There was obvious differences of NK production and growth characteristic between mutant (JNFE1126) and wild type (JNFE0126) B. subtilis strains, the NK production of B. subtilis JNFE1126 increased by 2 times compared to the wild strain. By comparative genomics analysis, 28 single nucleotide polymorphisms (SNPs), and 15 insertion deletions (Indel) were found. In terms of the transcriptional data, the results revealed there were 1425 genes with differential expression of fold change>=2. Functional categorization and analysis of modulated genes of interlinked pathways pointed out plausible nattokinase production in JNFE1126. Such as a dependency on amino acid metabolism for generating energy, and promoted transcription and extracellular transport of nattokinase. The upregulation expression of NadB, the positive regulatory genes (ComA and ComP), SecY and YidC in JNFE1126. Overall, the study provided an understanding of the molecular basis linked to high nattokinase production in JNFE1126.

Abstract PO-021: Single-cell DNA sequencing as a means to directly examine the size and frequency of radiation-induced mutations - An exploratory study

Abstract This study examines if single-cell DNA sequencing may be used to study the mutational effects of ionizing radiation. As the action of ionizing radiation is a stochastic process, each cell in an irradiated sample experiences its own unique radiation-induced DNA damage. As a result, conventional sequencing methods such as bulk cell sequencing cannot be used to identify individual mutations. In this work, Epstein-Barr virus (EBV) transformed B-lymphoblastoid cells were irradiated with 6 MV X-ray radiation using a medical linear accelerator. Four samples of cells from the same population (400,000 cells/ml) were exposed to sham irradiation (0 Gray [Gy]; control), 0.5 Gy, 1.5 Gy and 3.0 Gy respectively at a common dose rate of about 600 cGy/min. Irradiated samples were incubated for 24 hrs and subsequently underwent single-cell whole-genome DNA sequencing to characterize the radiation impact. Mutational profiles of approximately 500 cells, randomly selected from each sample, were individually analyzed and compared to identify the variation of several mutational parameters as a function of dose. We quantified the copy number variations (CNV) for each cell in our samples. Additionally, we segregated insertion CNVs and deletion CNVs and independently analyzed their dose dependences. We found that the total number of CNVs (insertion and deletion combined) increased with dose, and the number of deletion CNVs consistently increased most. We have repeated the experiment and a new round of single-cell DNA sequencing is underway. If confirmed, our results will demonstrate that the mutational effects of ionizing radiation may be examined directly using single-cell sequencing. Citation Format: Felix Mathew, Jonathan Yeo, Luc Galarneau, Norma Ybarra, Patricia Tonin, Yu Chang Wang, Ioannis Ragoussis, John Kildea. Single-cell DNA sequencing as a means to directly examine the size and frequency of radiation-induced mutations - An exploratory study [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-021.

Whole-genome re-sequencing reveals genome-wide variations between the peach variety Green No. 9 and its bud mutant Daifei

Whole-genome sequencing technologies provide opportunities to further understand genetic variation among different varieties. Some related genes that are useful for the breeding process could be identified by sequencing technologies. In this study, two peach varieties, Green No. 9 (LH) and its bud mutant Daifei (DT), were analyzed with whole-genome re-sequencing. Approximately 109 million total reads were generated, which covered ∼89% of the peach reference genome. A total of 1 143 757 single nucleotide polymorphisms (SNPs), 169 827 insertion–deletion mutations (InDels), 17 132 structural variations (SVs) and 5 040 copy number variations (CNVs) were detected in Green No. 9 and Daifei. Variant genes were classified by Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway database. Detected genes such as LOC18768059 (carbohydrate metabolism) and LOC18785045 (anthocyanin biosynthesis), were good candidate genes for exploring the phenotypic variations between Green No. 9 and Daifei. Green No. 9 and its bud mutant Daifei showed obvious differences in phenotypes and variant genetic loci. The detected genomic variations will contribute to explorations of important functional genes and to understanding the genetic basis of peach bud mutations.

Analysis of DNA variants in miRNAs and miRNA 3ʼUTR binding sites in female infertility patients

Early human embryogenesis relies on maternal gene products accumulated during oocyte growth and maturation, until around day-3 post-fertilization when human zygotic genome activation occurs. The maternal-to-zygotic transition (MZT) is a tightly coordinated process of selective maternal transcript clearance and new zygotic transcript production. If MZT is disrupted, it will lead to developmental arrest and pregnancy loss. It is well established that microRNA (miRNA) mutations disrupt regulation of their target transcripts. We hypothesize that some cases of embryonic arrest and pregnancy loss could be explained by the mutations in the maternal genome that affect miRNA-target transcript pairs. To this end, we examined mutations within miRNAs or miRNA binding sites in the 3ʼ untranslated regions (3ʼUTR) of target transcripts. Using whole-exome sequencing data from 178 women undergoing in vitro fertilization (IVF) procedures, we identified 1197 variants in miRNA genes, including 93 single nucleotide variants (SNVs) and 19 small insertions/deletions (INDELs) within the seed region of 100 miRNAs. Eight miRNA seed-region variants were significantly enriched among our patients when compared to a normal population. Within predicted 3ʼUTR miRNA binding sites, we identified 7393 SNVs and 1488 INDELs. Between our patients and a normal population, 52 SNVs and 30 INDELs showed significant association in the single-variant testing, whereas 51 genes showed significant association in the gene-burden analysis for genes that are expressed in preimplantation embryos. Interestingly, we found that many genes with disrupted 3ʼUTR miRNA binding sites follow gene expression patterns resembling MZT. In addition, some of these variants showed dramatic allele frequency difference between the patient and the normal group, offering potential utility as biomarkers for screening patients prior to IVF procedures.

Rare, Protein-Altering Variants in AS3MT and Arsenic Metabolism Efficiency: A Multi-Population Association Study.

Background:Common genetic variation in the arsenic methyltransferase (AS3MT) gene region is known to be associated with arsenic metabolism efficiency (AME), measured as the percentage of dimethylarsinic acid (DMA%) in the urine. Rare, protein-altering variants in AS3MT could have even larger effects on AME, but their contribution to AME has not been investigated.Objectives:We estimated the impact of rare, protein-coding variation in AS3MT on AME using a multi-population approach to facilitate the discovery of population-specific and shared causal rare variants.Methods:We generated targeted DNA sequencing data for the coding regions of AS3MT for three arsenic-exposed cohorts with existing data on arsenic species measured in urine: Health Effects of Arsenic Longitudinal Study (HEALS, ), Strong Heart Study (SHS, ), and New Hampshire Skin Cancer Study (NHSCS, ). We assessed the collective effects of rare (allele frequency ), protein-altering AS3MT variants on DMA%, using multiple approaches, including a test of the association between rare allele carrier status (yes/no) and DMA% using linear regression (adjusted for common variants in 10q24.32 region, age, sex, and population structure).Results:We identified 23 carriers of rare-protein-altering AS3MT variant across all cohorts (13 in HEALS and 5 in both SHS and NHSCS), including 6 carriers of predicted loss-of-function variants. DMA% was 6–10% lower in carriers compared with noncarriers in HEALS [ (95% CI: , )], SHS [ (95% CI: , )], and NHSCS [ (95% CI: , )]. In meta-analyses across cohorts, DMA% was 8.7% lower in carriers [ (95% CI: , )].Discussion:Rare, protein-altering variants in AS3MT were associated with lower mean DMA%, an indicator of reduced AME. Although a small percentage of the population (0.5–0.7%) carry these variants, they are associated with a 6–10% decrease in DMA% that is consistent across multiple ancestral and environmental backgrounds. https://doi.org/10.1289/EHP8152

Genotype to Phenotype: CRISPR Gene Editing Reveals Genetic Compensation as a Mechanism for Phenotypic Disjunction of Morphants and Mutants.

Forward genetic screens have shown the consequences of deleterious mutations; however, they are best suited for model organisms with fast reproductive rates and large broods. Furthermore, investigators must faithfully identify changes in phenotype, even if subtle, to realize the full benefit of the screen. Reverse genetic approaches also probe genotype to phenotype relationships, except that the genetic targets are predefined. Until recently, reverse genetic approaches relied on non-genomic gene silencing or the relatively inefficient, homology-dependent gene targeting for loss-of-function generation. Fortunately, the flexibility and simplicity of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has revolutionized reverse genetics, allowing for the precise mutagenesis of virtually any gene in any organism at will. The successful integration of insertions/deletions (INDELs) and nonsense mutations that would, at face value, produce the expected loss-of-function phenotype, have been shown to have little to no effect, even if other methods of gene silencing demonstrate robust loss-of-function consequences. The disjunction between outcomes has raised important questions about our understanding of genotype to phenotype and highlights the capacity for compensation in the central dogma. This review describes recent studies in which genomic compensation appears to be at play, discusses the possible compensation mechanisms, and considers elements important for robust gene loss-of-function studies.

Syllable Structure and Word Stress in Central Kerala Konkani Variety: An OT Approach

This paper addresses the possible phonological patterns governing the syllable structures and word stress in Central Kerala Konkani. Unlike other northern dialects spoken in Kerala, Central Konkani variety does not allow any consonant clusters; instead it makes use of repair strategies such as vowel insertion and consonant deletion which result in resyllabification. The resulting words can be explained with the following constraints couched in Optimality Theory in the order: *CC &gt;&gt; NoCoda &gt;&gt; DEP-IO. Here we have observed that stress in Central Konkani is placed on the heaviest syllable. However, in cases where a word has two heavy syllables or only light syllables, stress is on the leftmost syllable. This is explained by the constraints ranked as WSP &gt;&gt; Edgemost (pk;L).

The MinION as a cost-effective technology for diagnostic screening of the SCN1A gene in epilepsy patients

The MinION is a portable DNA sequencer that allows real time sequencing at low capital cost investment. We assessed accuracy and cost-effectivess of the MinION for genetic diagnostic testing of known SCN1A mutations that cause Dravet Syndrome (DS). DNA samples (n = 7) from DS patients previously shown to carry SCN1A mutations via Ion Torrent and Sanger sequencing were sequenced using the MinION. SCN1A amplicons for 8 exons were sequenced using the MinION with 1D chemistry on an R9.4 flow cell. All known missense mutations were detected in all samples showing 100 % concordance with results from other methods. However, the MinION failed to detect the insertions/deletions (INDELs) present in these patients. Nevertheless, these results indicate that MinION is a cost-effective platform for use as an initial screening step in the detection of nucleotide substitution mutations in in SCN1A, especially in under-resourced laboratories or hospitals. Further improvements are required to reliably detect INDELS in this gene.

Fine mapping and candidate gene analysis of the orange petal colour gene Bnpc2 in spring rapeseed (Brassica napus)

AbstractPetal colour is considered as an important trait and major visual signal for insect‐pollinated plants and landscaping. In previous study, we isolated and identified two genes (BnPC1 and BnPC2) that control the orange petal colour (OPC) in rapeseed. To identify the molecular markers linked to the orange petal colour, we constructed a B. napus linkage map with four newly developed simple sequence repeat (SSR) markers and nine insertion–deletion (InDel) markers using BC5II, a near isogenic line population segregated at the BnPC2 locus. The BnPC2 gene was flanked by Indel‐3 and Indel‐20 and was found to represent a 27‐kb (3.721–3.748 Mb) region on A09 of B. napus. High‐pressure liquid chromatography analysis revealed that, compared to yellow petals, orange petals exhibit significantly increased zeaxanthin, β‐carotene and lutein contents and a relatively decreased violaxanthin content. Expression profiling confirmed that BnaA09.ZEP (zeaxanthin epoxidase) is an important candidate gene for BnPC2 and is predominantly expressed in floral tissues. Overall, our results provide a basis for understanding the molecular mechanisms underlying the orange petal colour trait in B. napus.

CHM mutation spectrum and disease: An update at the time of human therapeutic trials.

Choroideremia is an X-linked inherited retinal disorder (IRD) characterized by the degeneration of retinal pigment epithelium, photoreceptors, choriocapillaris and choroid affecting males with variable phenotypes in female carriers. Unlike other IRD, characterized by a large clinical and genetic heterogeneity, choroideremia shows a specific phenotype with causative mutations in only one gene, CHM. Ongoing gene replacement trials raise further interests in this disorder. We describe here the clinical and genetic data from a French cohort of 45 families, 25 of which carry novel variants, in the context of 822 previously reported choroideremia families. Most of the variants represent loss-of-function mutations with eleven families having large (i.e. ≥6 kb) genomic deletions, 18 small insertions, deletions or insertion deletions, six showing nonsense variants, eight splice site variants and two missense variants likely to affect splicing. Similarly, 822 previously published families carry mostly loss-of-function variants. Recurrent variants are observed worldwide, some of which linked to a common ancestor, others arisen independently in specific CHM regions prone to mutations. Since all exons of CHM may harbor variants, Sanger sequencing combined with quantitative polymerase chain reaction or multiplex ligation-dependent probe amplification experiments are efficient to achieve the molecular diagnosis in patients with typical choroideremia features.