Clinical and genetic characteristics in a Chinese cohort of complex spastic paraplegia type 4.

Spastic paraplegia type 4 (SPG4), caused by SPAST mutations, is the most predominant subtype of hereditary spastic paraplegia. Most documented SPG4 patients present as pure form, with the complex form rarely reported. We described the clinical and genetic features of 20 patients with complex phenotypes of SPG4 and further explored the genotype-phenotype correlations. We collected detailed clinical data of all SPG4 patients and assessed their phenotypes. SPAST gene mutations were identified by Multiplex ligation-dependent probe amplification in combination with whole exome sequencing. We further performed statistical analysis in genotype and phenotype among patients with various manifestations and different variants. Out of 90 SPG4 patients, 20 patients (male:female = 16:4) with additional neurologic deficits, namely complex form, were included in our study. The bimodal distribution of age of onset at 0-10 and 21-40 years old is concluded. On cranial MRI, obvious white matter lesions can be observed in five patients. We identified 9 novel and 8 reported SPAST mutations, of which 11 mutations were located in AAA (ATPase associated with various cellular activities) domain. The AAA cassette of spastin is the hottest mutated region among complex SPG4. All patients with cognitive impairment (CI) are males (n = 9/9). Additionally, 80% patients with ataxia are due to frameshift mutations (n = 4/5). Overall, our study summarized and analyzed the genetic and phenotypic characteristics of complex SPG4, making up over 1/5 of in-house SPG4 cohort, among which CI and ataxia are the most common features. Further studies are expected to explore the underlying mechanisms.

Evaluating significance of European-associated index SNPs in the East Asian population for 31 complex phenotypes.

BACKGROUND: Genome-wide association studies (GWASs) have identified many single-nucleotide polymorphisms (SNPs) associated with complex phenotypes in the European (EUR) population; however, the extent to which EUR-associated SNPs can be generalized to other populations such as East Asian (EAS) is not clear. RESULTS: By leveraging summary statistics of 31 phenotypes in the EUR and EAS populations, we first evaluated the difference in heritability between the two populations and calculated the trans-ethnic genetic correlation. We observed the heritability estimates of some phenotypes varied substantially across populations and 53.3% of trans-ethnic genetic correlations were significantly smaller than one. Next, we examined whether EUR-associated SNPs of these phenotypes could be identified in EAS using the trans-ethnic false discovery rate method while accounting for winner's curse for SNP effect in EUR and difference of sample sizes in EAS. We found on average 54.5% of EUR-associated SNPs were also significant in EAS. Furthermore, we discovered non-significant SNPs had higher effect heterogeneity, and significant SNPs showed more consistent linkage disequilibrium and allele frequency patterns between the two populations. We also demonstrated non-significant SNPs were more likely to undergo natural selection. CONCLUSIONS: Our study revealed the extent to which EUR-associated SNPs could be significant in the EAS population and offered deep insights into the similarity and diversity of genetic architectures underlying phenotypes in distinct ancestral groups.

Co-Occurrence of Congenital Aniridia Due to Nonsense PAX6 Variant p.(Cys94*) and Chromosome 21 Trisomy in the Same Patient.

This study aims to present a clinical case involving the unique co-occurrence of congenital aniridia and Down syndrome in a young girl and to analyze the combined impact of these conditions on the patient's phenotype. The investigation involved comprehensive pediatric and ophthalmological examinations alongside karyotyping and Sanger sequencing of the PAX6 gene. The patient exhibited distinctive features associated with both congenital aniridia and Down syndrome, suggesting a potential exacerbation of their effects. Cytogenetic and molecular genetic analysis revealed the presence of trisomy 21 and a known pathogenic nonsense variant in exon 6 of the PAX6 gene (c.282C>A, p.(Cys94*)) corresponding to the paired domain of the protein. The observation of these two hereditary anomalies offers valuable insights into the molecular pathogenetic mechanisms underlying each condition. Additionally, it provides a basis for a more nuanced prognosis of the complex disease course in this patient. This case underscores the importance of considering interactions between different genetic disorders in clinical assessments and treatment planning.

Nectary size is a pollination syndrome trait in Penstemon.

Evolution of complex phenotypes depends on the adaptive importance of individual traits, and the developmental changes required to modify traits. Floral syndromes are complex adaptations to pollinators that include color, nectar, and shape variation. Hummingbird-adapted flowers have evolved a remarkable number of times from bee-adapted ancestors in Penstemon, and previous work demonstrates that color over shape better distinguishes bee from hummingbird syndromes. Here, we examined the relative importance of nectar volume and nectary development in defining Penstemon pollination syndromes. We tested the evolutionary association of nectar volume and nectary area with pollination syndrome across 19 Penstemon species. In selected species, we assessed cellular-level processes shaping nectary size. Within a segregating population from an intersyndrome cross, we assessed trait correlations between nectar volume, nectary area, and the size of stamens on which nectaries develop. Nectar volume and nectary area displayed an evolutionary association with pollination syndrome. These traits were correlated within a genetic cross, suggesting a mechanistic link. Nectary area evolution involves parallel processes of cell expansion and proliferation. Our results demonstrate that changes to nectary patterning are an important contributor to pollination syndrome diversity and provide further evidence that repeated origins of hummingbird adaptation involve parallel developmental processes in Penstemon.

Structural mapping: how to study the genetic architecture of a phenotypic trait through its formation mechanism.

Traditional approaches for genetic mapping are to simply associate the genotypes of a quantitative trait locus (QTL) with the phenotypic variation of a complex trait. A more mechanistic strategy has emerged to dissect the trait phenotype into its structural components and map specific QTLs that control the mechanistic and structural formation of a complex trait. We describe and assess such a strategy, called structural mapping, by integrating the internal structural basis of trait formation into a QTL mapping framework. Electrical impedance spectroscopy (EIS) has been instrumental for describing the structural components of a phenotypic trait and their interactions. By building robust mathematical models on circuit EIS data and embedding these models within a mixture model-based likelihood for QTL mapping, structural mapping implements the EM algorithm to obtain maximum likelihood estimates of QTL genotype-specific EIS parameters. The uniqueness of structural mapping is to make it possible to test a number of hypotheses about the pattern of the genetic control of structural components. We validated structural mapping by analyzing an EIS data collected for QTL mapping of frost hardiness in a controlled cross of jujube trees. The statistical properties of parameter estimates were examined by simulation studies. Structural mapping can be a powerful alternative for genetic mapping of complex traits by taking account into the biological and physical mechanisms underlying their formation.

The search for Pleiades in trait constellations: functional integration and phenotypic selection in the complex flowers of Morrenia brachystephana (Apocynaceae).

Pollinator-mediated natural selection on single traits, such as corolla tube or spur length, has been well documented. However, flower phenotypes are usually complex, and selection is expected to act on several traits that functionally interact rather than on a single isolated trait. Despite the fact that selection on complex phenotypes is expectedly widespread, multivariate selection modelling on such phenotypes still remains under-explored in plants. Species of the subfamily Asclepiadoideae (Apocynaceae) provide an opportunity to study such complex flower contrivances integrated by fine-scaled organs from disparate developmental origin. We studied the correlation structure among linear floral traits (i) by testing a priori morphological, functional or developmental hypotheses among traits and (ii) by exploring the organization of flower covariation, considering alternative expectations of modular organization or whole flower integration through conditional dependence analysis (CDA) and integration matrices. The phenotypic selection approach was applied to determine whether floral traits involved in the functioning of the pollination mechanism were affected by natural selection. Floral integration was low, suggesting that flowers are organized in more than just one correlation pleiad; our hypothetical functional correlation matrix was significantly correlated with the empirical matrix, and the CDA revealed three putative modules. Analyses of phenotypic selection showed significant linear and correlational gradients, lending support to expectations of functional interactions between floral traits. Significant correlational selection gradients found involved traits of different floral whorls, providing evidence for the existence of functional integration across developmental domains.

Towards the Genetic Architecture of Complex Gene Expression Traits: Challenges and Prospects for eQTL Mapping in Humans.

The discovery of expression quantitative trait loci (eQTLs) and their target genes (eGenes) has not only compensated for the limitations of genome-wide association studies for complex phenotypes but has also provided a basis for predicting gene expression. Efforts have been made to develop analytical methods in statistical genetics, a key discipline in eQTL analysis. In particular, mixed model- and deep learning-based analytical methods have been extremely beneficial in mapping eQTLs and predicting gene expression. Nevertheless, we still face many challenges associated with eQTL discovery. Here, we discuss two key aspects of these challenges: 1, the complexity of eTraits with various factors such as polygenicity and epistasis and 2, the voluminous work required for various types of eQTL profiles. The properties and prospects of statistical methods, including the mixed model method, Bayesian inference, the deep learning method, and the integration method, are presented as future directions for eQTL discovery. This review will help expedite the design and use of efficient methods for eQTL discovery and eTrait prediction.

A Complex Phenotype of a Patient with Spastic Paraplegia Type 4 Caused by a Novel Pathogenic Variant in the SPAST Gene.

Hereditary spastic paraplegias (HSPs) are rare neurological disorders caused by degeneration of the corticospinal tract. Among the 79 causative genes involved in HSPs, variants in SPAST on chromosome 2p22, which encodes the microtubule-severing protein spastin, are responsible for spastic paraplegia type 4 (SPG4), the most common form of HSPs. SPG4 is characterized by a clinically pure phenotype that is associated with restricted involvement of the corticospinal tract; however, it is often accompanied by additional neurological symptoms such as epilepsy and cognitive impairment. There are few reports regarding the clinical course and treatment of epilepsy associated with SPG4. We describe a 21-year-old male patient with progressive weakness and spasticity of the lower limbs since infancy, which was complicated by epilepsy and cognitive impairment. Magnetic resonance imaging of the brain showed right hippocampal atrophy before the onset of epilepsy. Genetic analysis revealed a novel missense variant (NM_014946.4:c.1330G>C, p.Asp444His) in the SPAST gene. At the age of 13, the patient developed focal epilepsy, characterized by focal onset seizures that were preceded by a sensation of chest tightness. Carbamazepine, levetiracetam, and zonisamide were ineffective in controlling the seizures; however, the use of lacosamide in combination with lamotrigine and valproate was highly effective in improving the seizure symptoms and led to the patient being seizure free for at least 2 years. In conclusion, the missense variant in SPAST may cause a complex SPG4 phenotype accompanied by epilepsy and cognitive impairment, suggesting that the clinical manifestations of this condition do not confine to the motor system.

Combining genetic crosses and pool targeted DNA-seq for untangling genomic variations associated with resistance to multiple insecticides in the mosquito Aedes aegypti.

In addition to combating vector-borne diseases, studying the adaptation of mosquitoes to insecticides provides a remarkable example of evolution-in-action driving the selection of complex phenotypes. Actually, most resistant mosquito populations show multi-resistance phenotypes as a consequence of the variety of insecticides employed and of the complexity of selected resistance mechanisms. Such complexity makes the identification of alleles conferring resistance to specific insecticides challenging and prevents the development of molecular assays to track them in the field. Here we showed that combining simple genetic crosses with pool targeted DNA-seq can enhance the specificity of resistance allele's detection while maintaining experimental work and sequencing effort at reasonable levels. A multi-resistant population of the mosquito Aedes aegypti was exposed to three distinct insecticides (deltamethrin, bendiocarb and fenitrothion), and survivors to each insecticide were crossed with a susceptible strain to generate three distinct lines. F2 individuals from each line were then segregated based on their survival to two insecticide doses. Hundreds of genes covering all detoxifying enzymes and insecticide targets together with more than 7,000 intergenic regions equally spread over mosquito genome were sequenced from pools of F0 and F2 individuals unexposed or surviving insecticide. Differential coverage analysis identified 39 detoxification enzymes showing an increased gene copy number in association with resistance. Combining an allele frequency filtering approach with a Bayesian F ST-based genome scan identified multiple genomic regions showing strong selection signatures together with 50 nonsynonymous variations associated with resistance. This study provides a simple and cost-effective approach to improve the specificity of resistance allele's detection in multi-resistant populations while reducing false positives frequently arising when comparing populations showing divergent genetic backgrounds. The identification of novel DNA resistance markers opens new opportunities for improving the tracking of insecticide resistance in the field.

Novel Type of Complicated Autosomal Dominant Hereditary Spastic Paraplegia Associated with Congenital Distal Arthrogryposis Type I.

Hereditary spastic paraplegia (HSP) is one of the most genetically heterogeneous neurological disorders. HSP is classified as pure when only a spastic weakness of the lower extremities is present. Complex HSP comes with additional neurological or systemic abnormalities. Complex HSP with skeletal abnormalities is rare and mostly seen in autosomal recessive HSP. Autosomal dominant (AD) complex HSP with skeletal abnormalities are consistently seen only in SPG9 (spastic gait type 9). In this paper, we report a kindred condition with AD HSP among four living affected individuals who had progressive, adult onset spastic paraparesis that was associated with a distal arthrogryposis (DA) in every affected individual. They also had episodes of rhabdomyolysis without any clinical signs of myopathy. Exhaustive genetic analysis including targeted sequencing of known HSP and DA genes and whole exome sequencing did not identify the disease-causing gene. It excluded all known HSP and DA genes. We propose that this is a novel genetic type of complex AD HSP. Elucidation of a genetic cause of this type of HSP will further contribute to our understanding of axonal degeneration and skeletal abnormalities.

Insights on Osmotic Tolerance Mechanisms in Escherichia coli Gained from an rpoC Mutation.

An 84 bp in-frame duplication (K370_A396dup) within the rpoC subunit of RNA polymerase was found in two independent mutants selected during an adaptive laboratory evolution experiment under osmotic stress in Escherichia coli, suggesting that this mutation confers improved osmotic tolerance. To determine the role this mutation in rpoC plays in osmotic tolerance, we reconstructed the mutation in BW25113, and found it to confer improved tolerance to hyperosmotic stress. Metabolite analysis, exogenous supplementation assays, and cell membrane damage analysis suggest that the mechanism of improved osmotic tolerance by this rpoC mutation may be related to the higher production of acetic acid and amino acids such as proline, and increased membrane integrity in the presence of NaCl stress in exponential phase cells. Transcriptional analysis led to the findings that the overexpression of methionine related genes metK and mmuP improves osmotic tolerance in BW25113. Furthermore, deletion of a stress related gene bolA was found to confer enhanced osmotic tolerance in BW25113 and MG1655. These findings expand our current understanding of osmotic tolerance in E. coli, and have the potential to expand the utilization of high saline feedstocks and water sources in microbial fermentation.

Overexpression of two stress-responsive, small, non-coding RNAs, 6S and tmRNA, imparts butanol tolerance in Clostridium acetobutylicum.

While extensively studied in several model organisms, the role of small, non-coding RNAs in the stress response remains largely unexplored in Clostridium organisms. About 100 years after the first industrial Acetone-Butanol-Ethanol fermentation process, based on the Weizmann Clostridium acetobutylicum strain, strain tolerance to butanol remains a crucial factor limiting the economics of the process. Several studies have examined the response of this organism to metabolite stress, and several genes have been engaged to impart enhanced tolerance, but no sRNAs have yet been directly engaged in this task. We show that the two stress-responsive sRNAs, 6S and tmRNA, upon overexpression impart tolerance to butanol as assessed by viability assays under process-relevant conditions. 6S overexpression enhances cell densities as well as butanol titres. We discuss the likely mechanisms that these two sRNAs might engage in this tolerance phenotype. Our data support the continued exploration of sRNAs as a basis for engineering enhanced tolerance and enhanced solvent production, especially because sRNA-based strategies impose a minimal metabolic burden on the cells.

Large-scale robot-assisted genome shuffling yields industrial Saccharomyces cerevisiae yeasts with increased ethanol tolerance.

BACKGROUND: During the final phases of bioethanol fermentation, yeast cells face high ethanol concentrations. This stress results in slower or arrested fermentations and limits ethanol production. Novel Saccharomyces cerevisiae strains with superior ethanol tolerance may therefore allow increased yield and efficiency. Genome shuffling has emerged as a powerful approach to rapidly enhance complex traits including ethanol tolerance, yet previous efforts have mostly relied on a mutagenized pool of a single strain, which can potentially limit the effectiveness. Here, we explore novel robot-assisted strategies that allow to shuffle the genomes of multiple parental yeasts on an unprecedented scale. RESULTS: Screening of 318 different yeasts for ethanol accumulation, sporulation efficiency, and genetic relatedness yielded eight heterothallic strains that served as parents for genome shuffling. In a first approach, the parental strains were subjected to multiple consecutive rounds of random genome shuffling with different selection methods, yielding several hybrids that showed increased ethanol tolerance. Interestingly, on average, hybrids from the first generation (F1) showed higher ethanol production than hybrids from the third generation (F3). In a second approach, we applied several successive rounds of robot-assisted targeted genome shuffling, yielding more than 3,000 targeted crosses. Hybrids selected for ethanol tolerance showed increased ethanol tolerance and production as compared to unselected hybrids, and F1 hybrids were on average superior to F3 hybrids. In total, 135 individual F1 and F3 hybrids were tested in small-scale very high gravity fermentations. Eight hybrids demonstrated superior fermentation performance over the commercial biofuel strain Ethanol Red, showing a 2 to 7% increase in maximal ethanol accumulation. In an 8-l pilot-scale test, the best-performing hybrid fermented medium containing 32% (w/v) glucose to dryness, yielding 18.7% (v/v) ethanol with a productivity of 0.90 g ethanol/l/h and a yield of 0.45 g ethanol/g glucose. CONCLUSIONS: We report the use of several different large-scale genome shuffling strategies to obtain novel hybrids with increased ethanol tolerance and fermentation capacity. Several of the novel hybrids show best-parent heterosis and outperform the commonly used bioethanol strain Ethanol Red, making them interesting candidate strains for industrial production.

Complex role of space in the crossing of fitness valleys by asexual populations.

The evolution of complex traits requires the accumulation of multiple mutations, which can be disadvantageous, neutral or advantageous relative to the wild-type. We study two spatial (two-dimensional) models of fitness valley crossing (the constant-population Moran process and the non-constant-population contact process), varying the number of loci involved and the degree of mixing. We find that spatial interactions accelerate the crossing of fitness valleys in the Moran process in the context of neutral and disadvantageous intermediate mutants because of the formation of mutant islands that increase the lifespan of mutant lineages. By contrast, in the contact process, spatial structure can accelerate or delay the emergence of the complex trait, and there can even be an optimal degree of mixing that maximizes the rate of evolution. For advantageous intermediate mutants, spatial interactions always delay the evolution of complex traits, in both the Moran and contact processes. The role of the mutant islands here is the opposite: instead of protecting, they constrict the growth of mutants. We conclude that the laws of population growth can be crucial for the effect of spatial interactions on the rate of evolution, and we relate the two processes explored here to different biological situations.