Mapping of crown rust (Puccinia coronata f. sp. avenae) resistance gene Pc54 and a novel quantitative trait locus effective against powdery mildew (Blumeria graminis f. sp. avenae) in the oat (Avena sativa) line Pc54

The Pc54 oat line carries the crown rust resistance gene ‘Pc54’ and an unknown gene effective against powdery mildew. In this study two recombinant inbred line populations were developed to identify the genomic locations of the two genes and producing lists of molecular markers with a potential for marker assisted selection. The RILs and parents were phenotyped for crown rust and powdery mildew in a controlled environment. They were also genotyped using the 6K Illumina Infinium iSelect oat SNP chip. Multiple interval mapping placed Pc54 on the linkage group Mrg02 (chromosome 7D) and the novel powdery mildew QTL ‘QPm.18’ on Mrg18 (chromosome 1A) both in the mapping and validating population. A total of nine and 31 significant molecular markers were identified linked with the Pc54 gene and QPm.18, respectively. Reactions to crown rust inoculations have justified separate identity of Pc54 from other genes and QTL that have previously been reported on Mrg02 except for ’qPCRFd’. Pm3 is the only powdery mildew resistance gene previously mapped on Mrg18. However, the pm3 differential line, Mostyn was susceptible to the powdery mildew race used in this study suggesting that Pm3 and QPm.18 are different genes. Determining the chromosomal locations of Pc54 and QPm.18 is helpful for better understanding the molecular mechanism of resistance to crown rust and powdery mildew in oats. Furthermore, SNPs and SSRs that are closely linked with the genes could be valuable for developing PCR based molecular markers and facilitating the utilization of these genes in oat breeding programs.

NGS Evaluation of a Bernese Cohort of Unexplained Erythrocytosis Patients

(1) Background: Clinical and molecular data on patients with unexplained erythrocyto-sis is sparse. We aimed to analyze the clinical and molecular features of patients with congenital erythrocytosis in our tertiary reference center. (2) Methods: In 34 patients with unexplained erythrocytosis, a 13-gene Next-Generation Sequencing erythrocytosis panel developed at our center was conducted. (3) Results: In 6/34 (18%) patients, eight different heterozygous gene variants were found. These patients were, therefore, diagnosed with congenital erythrocytosis. Two patients had two different gene variants each. All variants were characterized as variants of unknown significance as they had not previously been described in the literature. The rest of the patients (28/34, 82%) had no detected gene variants. (4) Conclusions: Our experience shows that the NGS panel can be helpful in determining the reasons for persistent, unexplained erythrocytosis. In our cohort of patients with erythrocytosis, we identified some, thus far unknown, gene variants which may explain the clinical picture. However, further investigations are needed to determine the relationship between the molecular findings and the phenotype.

Genetic and Phenotypic Diversity of Morganella morganii Isolated From Cheese

The bacterium Morganella morganii can produce the biogenic amines (BA) cadaverine, putrescine, and histamine in vitro and is responsible for high histamine concentrations in fish products. These BA can have toxic effects upon ingestion and are undesired in food. The purpose of this study was to characterize the phenotype and genotype of 11 M. morganii isolated from cheese in regard to the BA formation. In addition, we investigated the phylogeny, trehalose fermentation ability, and antibiotic resistance of the cheese isolates. To do so, we sequenced their genomes using both long and short read technologies. Due to the presence of the trehalose operon and the ability to ferment trehalose, the cheese isolates can be assigned to the subsp. sibonii. Comparative genomics with public available M. morganii genomes shows that the genomes of the cheese isolates cluster together with other subsp. sibonii genomes. All genomes between subsp. morganii and subsp. sibonii are separated by an average nucleotide identity (ANI) of less than 95.0%. Therefore, the subspecies could represent two distinct species. Nine of the strains decarboxylated lysine yielding cadaverine in vitro. This metabolic activity is linked to a previously unknown gene cluster comprising genes encoding a lysine-tRNA ligase (lysS), an HTH-transcriptional regulator (argP), a cadaverine-lysine antiporter (cadB), and a lysine decarboxylase (cadA). The formation of putrescine is linked to the speF gene encoding an ornithine decarboxylase. The gene is disrupted in five strains by an insertion sequence, and these strains only exhibit a weak putrescine production. Antimicrobial susceptibility profiling revealed that all cheese strains are resistant to tetracycline, chloramphenicol, tigecycline, colistin, and ampicillin. These phenotypes, except for colistin which is intrinsic, could be linked to antimicrobial resistance genes located on the chromosome.

The Clinical Utility of Optical Genome Mapping for the Assessment of Genomic Aberrations in Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most prevalent type of cancer occurring in children. ALL is characterized by structural and numeric genomic aberrations that strongly correlate with prognosis and clinical outcome. Usually, a combination of cyto- and molecular genetic methods (karyotyping, array-CGH, FISH, RT-PCR, RNA-Seq) is needed to identify all aberrations relevant for risk stratification. We investigated the feasibility of optical genome mapping (OGM), a DNA-based method, to detect these aberrations in an all-in-one approach. As proof of principle, twelve pediatric ALL samples were analyzed by OGM, and results were validated by comparing OGM data to results obtained from routine diagnostics. All genomic aberrations including translocations (e.g., dic(9;12)), aneuploidies (e.g., high hyperdiploidy) and copy number variations (e.g., IKZF1, PAX5) known from other techniques were also detected by OGM. Moreover, OGM was superior to well-established techniques for resolution of the more complex structure of a translocation t(12;21) and had a higher sensitivity for detection of copy number alterations. Importantly, a new and unknown gene fusion of JAK2 and NPAT due to a translocation t(9;11) was detected. We demonstrate the feasibility of OGM to detect well-established as well as new putative prognostic markers in an all-in-one approach in ALL. We hope that these limited results will be confirmed with testing of more samples in the future.

Efficacy of Immunohistochemistry for SDHB in the Screening of Hereditary Pheochromocytoma–Paraganglioma

The most common genetic backgrounds of hereditary paraganglioma and pheochromocytoma (PPGL) are SDHx germline mutations. Given the fact that the immunohistochemistry (IHC) result for SDHB is always negative regardless of the type of SDHx mutation, we aimed to evaluate the efficacy of using SDHB IHC for screening SDHx mutations in PPGL cases. In total, 52 patients who underwent surgery for PPGL treatment between 2006 and 2020 and underwent genetic analysis at diagnosis were included. Tissue microarrays (TMAs) were constructed with PPGL tissues and IHC for SDHB was performed on TMA sections. All 10 patients with SDHB-negative IHC contained SDHB or SDHD mutations. The genetic test results of patients with SDHB-weakly positive IHC varied (one SDHB, two RET, one VHL, and three unknown gene mutations). There were no SDHx mutations in the SDHB-positive IHC group. Six patients with weakly positive SDHB IHC with primarily unknown genetic status were re-called and underwent next-generation sequencing. None of them had SDHx mutations. In conclusion, SDHB-negative IHC is a cost-effective and reliable method to predict SDHx mutations. However, in the case of weakly positive SDHB staining, an additional gene study should be considered.

Clinicopathological and Genomic Profiles of Atypical Fibroxanthoma and Pleomorphic Dermal Sarcoma Identify Overlapping Signatures with a High Mutational Burden

Atypical fibroxanthoma (AFX) and pleomorphic dermal sarcoma (PDS) are rare tumors developing in chronically sun-exposed skin. Clinicopathological features are similar, but they differ in prognosis, while PDS has a more aggressive course with a higher risk for local recurrence and metastases. In current clinical practice, they are diagnosed by exclusion using immunohistochemistry. Thus, stringent diagnostic criteria and correct differentiation are critical in management and treatment for optimal outcomes. This retrospective single-center study collected clinicopathological data and tumor samples of 10 AFX and 18 PDS. Extracted genomic DNA from tumor specimens was analyzed by a next-generation sequencing (NGS) platform (FoundationOne-CDx™). Among 65 identified mutations, TP53 inactivating mutations were observed in all tumor specimens. In both AFX and PDS, the known pathogenic gene alterations in CDKN2A, TERT promoter, and NOTCH1 were frequently present, along with high mutational burden and stable Micro-Satellite Instability status. The mutational profiles differed only in ASXL1, which was only present in AFX. Further differences were identified in likely pathogenic and unknown gene alterations. Similarities in their genomic signatures could help to distinguish them from other malignancies, but they are not distinguishable between each other using the FoundationOne-CDx™ NGS panel. Therefore, histological criteria to determine diagnosis remain valid. For further insight, performing deep tumor profiling may be necessary.

A large chromosomal inversion shapes gene expression in seaweed flies (Coelopa frigida)

Inversions often underlie complex adaptive traits, but the genic targets inside them are largely unknown. Gene expression profiling provides a powerful way to link inversions with their phenotypic consequences. We examined the effects of the Cf-Inv(1) inversion in the seaweed fly Coelopa frigida on gene expression variation across sexes and life stages. Our analyses revealed that Cf-Inv(1) shapes global expression patterns but the extent of this effect is variable with much stronger effects in adults than larvae. Furthermore, within adults, both common as well as sex specific patterns were found. The vast majority of these differentially expressed genes mapped to Cf-Inv(1). However, genes that were differentially expressed in a single context (i.e. in males, females or larvae) were more likely to be located outside of Cf-Inv(1). By combining our findings with genomic scans for environmentally associated SNPs, we were able to pinpoint candidate variants in the inversion that may underlie mechanistic pathways that determine phenotypes. Together the results in this study, combined with previous findings, support the notion that the polymorphic Cf-Inv(1) inversion in this species is a major factor shaping both coding and regulatory variation resulting in highly complex adaptive effects.

Ocular coloboma—a comprehensive review for the clinician

Typical ocular coloboma is caused by defective closure of the embryonal fissure. The occurrence of coloboma can be sporadic, hereditary (known or unknown gene defects) or associated with chromosomal abnormalities. Ocular colobomata are more often associated with systemic abnormalities when caused by chromosomal abnormalities. The ocular manifestations vary widely. At one extreme, the eye is hardly recognisable and non-functional—having been compressed by an orbital cyst, while at the other, one finds minimalistic involvement that hardly affects the structure and function of the eye. In the fundus, the variability involves the size of the coloboma (anteroposterior and transverse extent) and the involvement of the optic disc and fovea. The visual acuity is affected when coloboma involves disc and fovea, or is complicated by occurrence of retinal detachment, choroidal neovascular membrane, cataract, amblyopia due to uncorrected refractive errors, etc. While the basic birth anomaly cannot be corrected, most of the complications listed above are correctable to a great extent. Current day surgical management of coloboma-related retinal detachments has evolved to yield consistently good results. Cataract surgery in these eyes can pose a challenge due to a combination of microphthalmos and relatively hard lenses, resulting in increased risk of intra-operative complications. Prophylactic laser retinopexy to the border of choroidal coloboma appears to be an attractive option for reducing risk of coloboma-related retinal detachment. However, a majority of the eyes have the optic disc within the choroidal coloboma, thus making it difficult to safely administer a complete treatment.

Sex, age, tissue, and disease patterns of matrisome expression in GTEx transcriptome data

The extracellular matrix (ECM) has historically been explored through proteomic methods. Whether or not global transcriptomics can yield meaningful information on the human matrisome is unknown. Gene expression data from 17,382 samples across 52 tissues, were obtained from the Genotype-Tissue Expression (GTEx) project. Additional datasets were obtained from The Cancer Genome Atlas (TCGA) program and the Gene Expression Omnibus for comparisons. Gene expression levels generally recapitulated proteome-derived matrisome expression patterns. Further, matrisome gene expression properly clustered tissue types, with some matrisome genes including SERPIN family members having tissue-restricted expression patterns. Deeper analyses revealed 388 genes varied by age and 222 varied by sex in at least one tissue, with expression correlating with digitally imaged histologic tissue features. A comparison of TCGA tumor, TCGA adjacent normal and GTEx normal tissues demonstrated robustness of the GTEx samples as a generalized control, while also determining a common primary tumor matrisome. Additionally, GTEx tissues served as a useful non-diseased control in a separate study of idiopathic pulmonary fibrosis matrix changes. Altogether, these findings indicate that the transcriptome, in general, and GTEx in particular, has value in understanding the state of organ ECM.

Tobacco rattle virus–induced gene silencing in Hevea brasiliensis

ABSTRACT Virus-induced gene silencing (VIGS) is a powerful gene-silencing tool that has been intensively applied in plants. To data, the application of VIGS in rubber tree has not yet been reported. In this study, we described the efficient gene silencing in rubber tree by VIGS. The gene encoding Hevea brasiliensis phytoene desaturase (HbPDS) was identified in rubber tree genome. Small interfering RNAs from HbPDS and the silencing gene fragment were predicted and a length of 399 bp was selected to be tested. We showed that the tobacco rattle virus (TRV)-VIGS could induce effective HbPDS silencing in rubber tree. This study was the first to report VIGS in rubber tree. The present TRV-VIGS method could be used to perform reverse genetic approaches to identify unknown gene functions and might be further applied to produce gene silenced rubber tree plants, to advance functional gene of rubber tree.