Singleton exome sequencing of 90 fetuses with ultrasound anomalies revealing novel disease-causing variants and genotype–phenotype correlations

Exome sequencing has been increasingly implemented in prenatal genetic testing for fetuses with morphological abnormalities but normal rapid aneuploidy detection and microarray analysis. We present a retrospective study of 90 fetuses with different abnormal ultrasound findings, in which we employed the singleton exome sequencing (sES; 75 fetuses) or to a lesser extent (15 fetuses) a multigene panel analysis of 6713 genes as a primary tool for the detection of monogenic diseases. The detection rate of pathogenic or likely pathogenic variants in this study was 34.4%. The highest diagnostic rate of 56% was in fetuses with multiple anomalies, followed by cases with skeletal or renal abnormalities (diagnostic rate of 50%, respectively). We report 20 novel disease-causing variants in different known disease-associated genes and new genotype–phenotype associations for the genes KMT2D, MN1, CDK10, and EXOC3L2. Based on our data, we postulate that sES of fetal index cases with a concurrent sampling of parental probes for targeted testing of the origin of detected fetal variants could be a suitable tool to obtain reliable and rapid prenatal results, particularly in situations where a trio analysis is not possible.

Can the Synergic Contribution of Multigenic Variants Explain the Clinical and Cellular Phenotypes of a Neurodevelopmental Disorder?

We describe an infant female with a syndromic neurodevelopmental clinical phenotype and increased chromosome instability as cellular phenotype. Genotype characterization revealed heterozygous variants in genes directly or indirectly linked to DNA repair: a de novo X-linked HDAC8 pathogenic variant, a paternally inherited FANCG pathogenic variant and a maternally inherited BRCA2 variant of uncertain significance. The full spectrum of the phenotype cannot be explained by any of the heterozygous variants on their own; thus, a synergic contribution is proposed. Complementation studies showed that the FANCG gene from the Fanconi Anaemia/BRCA (FA/BRCA) DNA repair pathway was impaired, indicating that the variant in FANCG contributes to the cellular phenotype. The patient’s chromosome instability represents the first report where heterozygous variant(s) in the FA/BRCA pathway are implicated in the cellular phenotype. We propose that a multigenic contribution of heterozygous variants in HDAC8 and the FA/BRCA pathway might have a role in the phenotype of this neurodevelopmental disorder. The importance of these findings may have repercussion in the clinical management of other cases with a similar synergic contribution of heterozygous variants, allowing the establishment of new genotype–phenotype correlations and motivating the biochemical study of the underlying mechanisms.

First Insights Into the Virus and Viroid Communities in Hemp (Cannabis sativa)

Hemp (Cannabis sativa L.) production has increased significantly in recent years; however, the crop has been understudied in the U.S. since its production declined in the late 1950s. Disease identification and management is an increasing challenge for hemp growers across the country. In 2019, beet curly top virus (BCTV) was first reported in hemp in Colorado. Hence, we were motivated to understand the diversity and prevalence of BCTV strains infecting hemp in Colorado. We detected BCTV at high incidence rate (81%) in leaf samples from 12 counties. Two different strains of BCTV, Worland (Wor) and Colorado (CO) were present as a single or mixed infection in hemp leaf samples. Phylogenetic analysis revealed BCTV sequences from hemp formed a distinct group along with BCTV strains CO and Wor. To determine other potential viral and viroid pathogens in hemp, we performed next generation sequencing (NGS). Virome analysis revealed the presence of both virus and viroid sequences that had high nucleotide sequence identity with GenBank accessions for cannabis cryptic virus, cannabis sativa mitovirus, citrus yellow vein associated virus, opuntia-like virus and hop latent viroid. In contrast, tobacco streak virus sequences were highly variable compared to sequences in GenBank suggesting a possible new genotype of this virus. The data presented here has important implications for the epidemiology and management of the various diseases of hemp and will lead to the development of integrated pest management strategies designed to interrupt transmission cycles and facilitate efficient crop production.

The genetic diversity of Asplenium viride (Aspleniaceae) fern colonizing heavy metal-polluted sites

Heavy metals can affect the morphology, physiology and evolution of plants. Asplenium viride is a diploid species, belonging to the largest genus of the cosmopolitan fern family Aspleniaceae, and occurring on various types of alkaline rocks. It is known to colonize sites with high concentrations of heavy metals, exhibiting changes in frond morphology. Microevolutionary processes, manifesting as changes in genome size and new genotype formation, can ultimately lead to the formation of new subspecies and speciation. This study aimed to evaluate the morphological and genetic diversity of A. viride, and to test for a potential correlation between variability and heavy metal concentration. Analysis of A. viride specimens, from one metalliferous site and five non-metalliferous localities, showed no apparent variation in genome size between plants from affected and non-affected sites. There was no significant correlation between genetic variability and heavy metal concentration. This was possibly due to intragametophytic selfing, caused by patchy habitats and subsequent founder effects, resulting from long-distance colonization by single spores.

Distribution of Two Strains of Leptoglossus zonatus (Dallas) (Hemiptera: Coreidae) in the Western Hemisphere: Is L. zonatus a Potential Invasive Species in California?

The leaffooted plant bug, Leptoglossus zonatus (Dallas) (Hemiptera: Coreidae) is polyphagous and widely distributed in the Western Hemisphere. Although it has been recorded in California since around 1900, it has become a more common pest in almonds in the last decade. Other studies have shown that an established insect can become a pest when a new genotype is introduced. This study investigated the distribution of two lineages (strains) of L. zonatus in the Western Hemisphere. Specimens from the Leptoglossus collection in the national insect collection in Mexico were used to extract DNA and sequence the mitochondrial DNA cytochrome oxidase I (mtDNA COI) gene, for use in population genetic and phylogenetic analyses. New sequences from Mexico, Central and South America were combined with those available in GenBank, from California and Brazil. Two lineages (strains) of L. zonatus were uncovered. One lineage occurs in California, Mexico and Ecuador. The second lineage is more widespread and found in California, Mexico, Guatemala, Nicaragua, Bolivia and Brazil. The haplotype number and diversity, and nucleotide diversity, were found for samples from California, Mexico, and Brazil, for the two lineages, and for all 118 sequences combined. All sequences combined produced five haplotypes, and a haplotype diversity of 0.54. California and Brazil had 3 haplotypes each, with one haplotype shared (5 total). Haplotype diversity in California and in Brazil were 0.526 and 0.505, respectively. A haplotype network found that one haplotype was most abundant and widespread. The small number of haplotypes, a range expansion, and economic pest status of L. zonatus in California, all contribute to this insect being a potentially invasive insect pest.

MAGRARIO: A NEW GENOTYPE TO PRODUCE QUALITY SHEEP MEAT

Generally there is poor tradition to produce and to commercialize heavy lean lamb carcasses. To achieve a better product for the ovine meat market Ideal (Polwarth) breed ewes were backcrossed to Texel breed rams (breed recognized to reduce carcass fat). Ideal breed (I) is one of the most ordinary breeds in Argentina. However, when their lambs are reared in feed-lot conditions, a high fat content is found in their lamb carcasses. After three generations of backcrosses followed by a breeding program for increase male lamb weaning weight and female fertility a new genotype was obtained for the local ovine meat production systems. This new genotype registered as Magrario (M) was obtained at Villarino Field Station of UNR (Zavalla, Santa Fe, 33º S, 61º W). It was verify that M produced more lean meat than I breed under feet lot conditions. M rams were introduced in flocks of Hampshire Down (HD) breed to evaluate lamb crosses with lean meat. Genotype M was compared under feed lot conditions with HD lambs during two months in the post weaning. Also crosses (MxHD), (MxI) and (IxHD) were evaluated in the same conditions. At the end of the experiment ultrasonic methods were used to evaluate fat depot on Longissimus dorsi. The (MxHD) showed a reduction of 20% respecting to HD. These results suggested that M genotype could be a useful paternal genotype to reduce fat depots when the aim is to produce lamb crosses under feed lot conditions in a short period of time. Key words: ovine, lean meat, feed lot, crossing.

Pathogen Detection and Genotyping With Trace Sample by A Phi29 Based Unbiased Exponential Amplification

Ticks are vectors for many infectious diseases such as spotted fever group (SFG) rickettsioses and borrelioses. Ticks are valuable material for pathogen ecology study. Ticks have several growth stages with significant varying size, and therefore, in most cases, the collected ticks cannot provide sufficient DNA for subsequent studies, particularly for multiple pathogen screening and genotyping. Unbiased pretreatment of the tick samples for subsequent analysis is an urgent need for subsequent ecological survey and other studies. Phi29 DNA polymerase, an enzyme with strand displacement activity, could exponentially amplify DNA randomly and non-biasedly, generating large quantities of DNA. In the present study, we developed a Phi29 based unbiased exponential amplification (PEA) assay for unbiased treatment of sample nucleic acid to provide sufficient DNA for genetic analysis. By using tick borne pathogen detection and genotype as a model, we tested and evaluated the feasibility of the assay. Nucleic acid were extracted from single ticks and subjected to PEA. The results showed that tick DNA could be amplified up to 10 5 folds. The amplified products were successfully used for pathogen screening and genotyping. With the amplified DNA from single tick, Rickettsia was successfully detected and genotyped. A new genotype of Rickettsia was identified from ticks collected from Dandong city, Liaoning province, Northeast China. This PEA assay is universal and can also be extended to other applications where samples are greatly limited.

Modelling the geographical spread of HIV among MSM in Guangdong, China: a metapopulation model considering the impact of pre-exposure prophylaxis

Men who have sex with men (MSM) make up the majority of new human immunodeficiency virus (HIV) diagnoses among young people in China. Understanding HIV transmission dynamics among the MSM population is, therefore, crucial for the control and prevention of HIV infections, especially for some newly reported genotypes of HIV. This study presents a metapopulation model considering the impact of pre-exposure prophylaxis (PrEP) to investigate the geographical spread of a hypothetically new genotype of HIV among MSM in Guangdong, China. We use multiple data sources to construct this model to characterize the behavioural dynamics underlying the spread of HIV within and between 21 prefecture-level cities (i.e. Guangzhou, Shenzhen, Foshan, etc.) in Guangdong province: the online social network via a gay social networking app, the offline human mobility network via the Baidu mobility website, and self-reported sexual behaviours among MSM. Results show that PrEP initiation exponentially delays the occurrence of the virus for the rest of the cities transmitted from the initial outbreak city; hubs on the movement network, such as Guangzhou, Shenzhen, and Foshan are at a higher risk of ‘earliest’ exposure to the new HIV genotype; most cities acquire the virus directly from the initial outbreak city while others acquire the virus from cities that are not initial outbreak locations and have relatively high betweenness centralities, such as Guangzhou, Shenzhen and Shantou. This study provides insights in predicting the geographical spread of a new genotype of HIV among an MSM population from different regions and assessing the importance of prefecture-level cities in the control and prevention of HIV in Guangdong province. This article is part of the theme issue ‘Data science approach to infectious disease surveillance’.

Genetic Transformation of Sugarcane, Current Status and Future Prospects

Sugarcane (Saccharum spp.) is a tropical and sub-tropical, vegetative-propagated crop that contributes to approximately 80% of the sugar and 40% of the world’s biofuel production. Modern sugarcane cultivars are highly polyploid and aneuploid hybrids with extremely large genomes (>10 Gigabases), that have originated from artificial crosses between the two species, Saccharum officinarum and S. spontaneum. The genetic complexity and low fertility of sugarcane under natural growing conditions make traditional breeding improvement extremely laborious, costly and time-consuming. This, together with its vegetative propagation, which allows for stable transfer and multiplication of transgenes, make sugarcane a good candidate for crop improvement through genetic engineering. Genetic transformation has the potential to improve economically important properties in sugarcane as well as diversify sugarcane beyond traditional applications, such as sucrose production. Traits such as herbicide, disease and insect resistance, improved tolerance to cold, salt and drought and accumulation of sugar and biomass have been some of the areas of interest as far as the application of transgenic sugarcane is concerned. Although there have been much interest in developing transgenic sugarcane there are only three officially approved varieties for commercialization, all of them expressing insect-resistance and recently released in Brazil. Since the early 1990’s, different genetic transformation systems have been successfully developed in sugarcane, including electroporation, Agrobacterium tumefaciens and biobalistics. However, genetic transformation of sugarcane is a very laborious process, which relies heavily on intensive and sophisticated tissue culture and plant generation procedures that must be optimized for each new genotype to be transformed. Therefore, it remains a great technical challenge to develop an efficient transformation protocol for any sugarcane variety that has not been previously transformed. Additionally, once a transgenic event is obtained, molecular studies required for a commercial release by regulatory authorities, which include transgene insertion site, number of transgenes and gene expression levels, are all hindered by the genomic complexity and the lack of a complete sequenced reference genome for this crop. The objective of this review is to summarize current techniques and state of the art in sugarcane transformation and provide information on existing and future sugarcane improvement by genetic engineering.

Toxoplasmosis in a free ranging hairy dwarf porcupine ( Sphiggurus spinosus) with a novel genotype

Toxoplasmosis is a zoonotic disease caused by the ubiquitous coccidia Toxoplasma gondii. Rodents play an important role in maintaining its life cycle, as they are one of the main diet sources for felids (wild and domestic), the unique definitive hosts. However, reports of toxoplasmosis in porcupines (Rodentia Order) are uncommon, with gaps concerning its pathophysiology. South America is the continent with the greatest genetic diversity of rodents and T. gondii. A free-ranging hairy dwarf porcupine was admitted to a wildlife rescue center with a history of trauma. During rehabilitation, the animal presented neurological symptoms (sporadic episodes of hind limbs paresis) and died five months later. The main findings during necropsy were brain congestion and severe incisor overgrowth associated with maxillary perforation. The histopathological exam showed moderate encephalitis, with variable-sized round cysts, positive for PAS stain and immunohistochemistry for T. gondii. Additionally, two cysts were observed in the medulla of the adrenal gland. Molecular techniques were performed to characterize the parasite load by qPCR (Cq=30) and the genotype by PCR-RFLP with 11 markers, which revealed a new genotype. This case adds to the body of knowledge in comparative pathology of Neotropical Rodentia and reports a new genotype circulating in South America.