Application of a custom NGS Gene Panel revealed a high diagnostic utility for molecular testing of hereditary ataxias.

Background Hereditary ataxias (HA) are a rare group of heterogeneous disorders. Here, we present results of molecular testing a group of ataxia patients using custom-designed Next Generation Sequencing (NGS) panel. Due to genetic and clinical overlapping of hereditary ataxias and spastic paraplegias (HSP), designed panel encompassing together HA and HSP genes. Methods The NGS libraries comprising coding sequence for 152 genes were performed using KAPA HyperPlus and HyperCap Target Enrichment Kit and sequenced on the MiSeq instrument. Obtained results were analyzed using BaseSpace Variant Interpreter and Integrative Genomics Viewer. All pathogenic and likely pathogenic variants were confirmed using the Sanger sequencing. Results A total of 29 patients with hereditary ataxias were enrolled to the NGS testing, and 16 patients had a confirmed molecular diagnosis with diagnostic efficiency of 55.2%. Pathogenic or likely pathogenic mutations were identified in 10 different genes: POLG (PEOA1, n=3; SCAE, n=2), CACNA1A (EA2, n=2), SACS (ARSACS, n=2), SLC33A1 (SPG42, n=2), STUB1 (SCA48, n=1), SPTBN2 (SCA5, n=1), TGM6 (SCA35, n=1), SETX (AOA2, n=1), ANO10 (SCAR10, n=1), SPAST (SPG4, n=1). Conclusions We demonstrated that approach based on targeted NGS panel can be highly effective and useful tool in the molecular diagnosis of ataxia patients. Furthermore, we highlight that sequencing panel targeted to ataxias together with HSP genes increase the diagnostic success.

Expression Profiling of Heat Shock Protein Genes as Putative Early Heat-Responsive Members in Lettuce

High temperatures due to global warming can cause harmful effects on the productivity of lettuce, a cool-season crop. To identify lettuce heat shock protein (HSP) genes that could be involved in early responses to heat stress in plants, we compared RNA transcriptomes between lettuce plants with and without heat treatment of 37 °C for 1 h. Using transcriptome sequencing analyses, a total of 7986 differentially expressed genes (DEGs) were identified including the top five, LsHSP70A, LsHSP70B, LsHSP17.3A, LsHSP17.9A and LsHSP17.9B, which were the most highly differentially expressed genes. In order to investigate the temporal expression patterns of 24 lettuce HSP genes with a fold-change greater than 100 under heat stress, the expression levels of the genes were measured by qRT-PCR at 0, 1, 4, 8, 14, and 24 h time points after heat treatment. The 24 LsHSP genes were classified into three groups based on the phylogenetic analysis and/or major domains available in each protein, and we provided a potential link between the phylogenetic relationships and expression patterns of the LsHSP genes. Our results showed putative early heat-responsive lettuce HSP genes that could be possible candidates as breeding guides for the development of heat-tolerant lettuce cultivars.

Analysis of proline accumulation, antioxidant capacity and HSP expression in mutant rice lines with different heat tolerance

Three mutant rice (Oryza sativa L.) lines (AG1, AG2 and AG3) were selected as heat tolerant mutants from a gamma-ray-irradiated population of a heat-susceptible line (AG), based on their floret fertility grown under high temperatures. They were subjected to heat stress treatment (45°C, 22 hrs) at the 5-leaf stage, together with a heat-tolerant cultivar N22 and AG. Analysis of seedling root growth by WinRHIZO scanning revealed that N22 and AG3 were more heat-tolerant than the other lines (AG being the most heat susceptible). Following heat stress, a significantly higher level of oxidative damage, as indicated by TBARS, was observed in AG than in N22 and AG3. The proline accumulation was significantly higher in N22 and AG3（12- to 13.5- fold）than AG (2.5- fold). Similarly, significantly greater increases of total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity were observed in N22 and AG3 than in AG. The expression of four heat shock proteins was also investigated using qPCR: OsHSP16.9, OsHSP80.3 and OsHSP100.9were induced by heat stress to various levels while OsHSP72.6was down-regulated in all tested lines. The heat-induced expression of OsHSP16.9 and OsHSP100.9 in N22 and AG3 was about twice that of AG. Higher proline accumulation and expression of the three HSP genes, as well as elevation ofT-AOC and SOD activity were observed in the heat-tolerant N22 and the mutant line AG3 under heat stress

Genome Size Estimation and Full-Length Transcriptome of Sphingonotus tsinlingensis: Genetic Background of a Drought-Adapted Grasshopper

Sphingonotus Fieber, 1852 (Orthoptera: Acrididae), is a grasshopper genus comprising approximately 170 species, all of which prefer dry environments such as deserts, steppes, and stony benchlands. In this study, we aimed to examine the adaptation of grasshopper species to arid environments. The genome size of Sphingonotus tsinlingensis was estimated using flow cytometry, and the first high-quality full-length transcriptome of this species was produced. The genome size of S. tsinlingensis is approximately 12.8 Gb. Based on 146.98 Gb of PacBio sequencing data, 221.47 Mb full-length transcripts were assembled. Among these, 88,693 non-redundant isoforms were identified with an N50 value of 2,726 bp, which was markedly longer than previous grasshopper transcriptome assemblies. In total, 48,502 protein-coding sequences were identified, and 37,569 were annotated using public gene function databases. Moreover, 36,488 simple tandem repeats, 12,765 long non-coding RNAs, and 414 transcription factors were identified. According to gene functions, 61 cytochrome P450 (CYP450) and 66 heat shock protein (HSP) genes, which may be associated with drought adaptation of S. tsinlingensis, were identified. We compared the transcriptomes of S. tsinlingensis and two other grasshopper species which were less tolerant to drought, namely Mongolotettix japonicus and Gomphocerus licenti. We observed the expression of CYP450 and HSP genes in S. tsinlingensis were higher. We produced the first full-length transcriptome of a Sphingonotus species that has an ultra-large genome. The assembly characteristics were better than those of all known grasshopper transcriptomes. This full-length transcriptome may thus be used to understand the genetic background and evolution of grasshoppers.

Current Knowledge of Endolysosomal and Autophagy Defects in Hereditary Spastic Paraplegia

Hereditary spastic paraplegia (HSP) refers to a group of neurological disorders involving the degeneration of motor neurons. Due to their clinical and genetic heterogeneity, finding common effective therapeutics is difficult. Therefore, a better understanding of the common pathological mechanisms is necessary. The role of several HSP genes/proteins is linked to the endolysosomal and autophagic pathways, suggesting a functional convergence. Furthermore, impairment of these pathways is particularly interesting since it has been linked to other neurodegenerative diseases, which would suggest that the nervous system is particularly sensitive to the disruption of the endolysosomal and autophagic systems. In this review, we will summarize the involvement of HSP proteins in the endolysosomal and autophagic pathways in order to clarify their functioning and decipher some of the pathological mechanisms leading to HSP.

Genome-Wide Identification and Characterization of Hsf and Hsp Gene Families and Gene Expression Analysis under Heat Stress in Eggplant (Solanum melongema L.)

Under high temperature stress, a large number of proteins in plant cells will be denatured and inactivated. Meanwhile Hsfs and Hsps will be quickly induced to remove denatured proteins, so as to avoid programmed cell death, thus enhancing the thermotolerance of plants. Here, a comprehensive identification and analysis of the Hsf and Hsp gene families in eggplant under heat stress was performed. A total of 24 Hsf-like genes and 117 Hsp-like genes were identified from the eggplant genome using the interolog from Arabidopsis. The gene structure and motif composition of Hsf and Hsp genes were relatively conserved in each subfamily in eggplant. RNA-seq data and qRT-PCR analysis showed that the expressions of most eggplant Hsf and Hsp genes were increased upon exposure to heat stress, especially in thermotolerant line. The comprehensive analysis indicated that different sets of SmHsps genes were involved downstream of particular SmHsfs genes. These results provided a basis for revealing the roles of SmHsps and SmHsp for thermotolerance in eggplant, which may potentially be useful for understanding the thermotolerance mechanism involving SmHsps and SmHsp in eggplant.

Lipid Droplets in the Pathogenesis of Hereditary Spastic Paraplegia

Hereditary spastic paraplegias (HSPs) are genetically heterogeneous conditions caused by the progressive dying back of the longest axons in the central nervous system, the corticospinal axons. A wealth of data in the last decade has unraveled disturbances of lipid droplet (LD) biogenesis, maturation, turnover and contact sites in cellular and animal models with perturbed expression and function of HSP proteins. As ubiquitous organelles that segregate neutral lipid into a phospholipid monolayer, LDs are at the cross-road of several processes including lipid metabolism and trafficking, energy homeostasis, and stress signaling cascades. However, their role in brain cells, especially in neurons remains enigmatic. Here, we review experimental findings linking LD abnormalities to defective function of proteins encoded by HSP genes, and discuss arising questions in the context of the pathogenesis of HSP.

The association of heat shock protein genetic polymorphisms with age-related hearing impairment in Taiwan

Background Age-related hearing impairment (ARHI) is a major disability among the elderly population. Heat shock proteins (HSPs) were found to be associated with ARHI in animal studies. The aim of this study was to analyze the associations of single nucleotide polymorphisms (SNPs) of HSP genes with ARHI in an elderly population in Taiwan. Methods Participants ≥65 years of age were recruited for audiometric tests and genetic analyses. The pure tone average (PTA) of the better hearing ear was calculated for ARHI evaluation. The associations of HSPA1L (rs2075800 and rs2227956), HSPA1A (rs1043618) and HSPA1B (rs2763979) with ARHI were analyzed in 146 ARHI-susceptible (cases) and 146 ARHI-resistant (controls) participants. Results The “T” allele of HSPA1B rs2763979 showed a decreased risk of ARHI. The “TT” genotype of rs2763979 also showed a decreased risk of ARHI in the dominant hereditary model. For HSPA1L (rs2075800 and rs2227956) and HSPA1A (rs1043618), the haplotype “CAG” was related to a decreased risk of ARHI. Conclusion These findings suggest that HSP70 polymorphisms are associated with susceptibility to ARHI in the elderly population. Graphical abstract