Systematic Phenotypic Analysis Research Articles

The phenotypic spectrum and genotype-phenotype correlations in 106 patients with variants in major autism gene CHD8

CHD8, a major autism gene, functions in chromatin remodelling and has various roles involving several biological pathways. Therefore, unsurprisingly, previous studies have shown that intellectual developmental disorder with autism and macrocephaly (IDDAM), the syndrome caused by pathogenic variants in CHD8, consists of a broad range of phenotypic abnormalities. We collected and reviewed 106 individuals with IDDAM, including 36 individuals not previously published, thus enabling thorough genotype–phenotype analyses, involving the CHD8 mutation spectrum, characterization of the CHD8 DNA methylation episignature, and the systematic analysis of phenotypes collected in Human Phenotype Ontology (HPO). We identified 29 unique nonsense, 25 frameshift, 24 missense, and 12 splice site variants. Furthermore, two unique inframe deletions, one larger deletion (exons 26–28), and one translocation were observed. Methylation analysis was performed for 13 patients, 11 of which showed the previously established episignature for IDDAM (85%) associated with CHD8 haploinsufficiency, one analysis was inconclusive, and one showing a possible gain-of-function signature instead of the expected haploinsufficiency signature was observed. Consistent with previous studies, phenotypical abnormalities affected multiple organ systems. Many neurological abnormalities, like intellectual disability (68%) and hypotonia (29%) were observed, as well as a wide variety of behavioural abnormalities (88%). Most frequently observed behavioural problems included autism spectrum disorder (76%), short attention span (32%), abnormal social behaviour (31%), sleep disturbance (29%) and impaired social interactions (28%). Furthermore, abnormalities in the digestive (53%), musculoskeletal (79%) and genitourinary systems (18%) were noted. Although no significant difference in severity was observed between males and females, individuals with a missense variant were less severely affected. Our study provides an extensive review of all phenotypic abnormalities in patients with IDDAM and provides clinical recommendations, which will be of significant value to individuals with a pathogenic variant in CHD8, their families, and clinicians as it gives a more refined insight into the clinical and molecular spectrum of IDDAM, which is essential for accurate care and counselling.

High-throughput discovery of genetic determinants of circadian misalignment.

Circadian systems provide a fitness advantage to organisms by allowing them to adapt to daily changes of environmental cues, such as light/dark cycles. The molecular mechanism underlying the circadian clock has been well characterized. However, how internal circadian clocks are entrained with regular daily light/dark cycles remains unclear. By collecting and analyzing indirect calorimetry (IC) data from more than 2000 wild-type mice available from the International Mouse Phenotyping Consortium (IMPC), we show that the onset time and peak phase of activity and food intake rhythms are reliable parameters for screening defects of circadian misalignment. We developed a machine learning algorithm to quantify these two parameters in our misalignment screen (SyncScreener) with existing datasets and used it to screen 750 mutant mouse lines from five IMPC phenotyping centres. Mutants of five genes (Slc7a11, Rhbdl1, Spop, Ctc1 and Oxtr) were found to be associated with altered patterns of activity or food intake. By further studying the Slc7a11tm1a/tm1a mice, we confirmed its advanced activity phase phenotype in response to a simulated jetlag and skeleton photoperiod stimuli. Disruption of Slc7a11 affected the intercellular communication in the suprachiasmatic nucleus, suggesting a defect in synchronization of clock neurons. Our study has established a systematic phenotype analysis approach that can be used to uncover the mechanism of circadian entrainment in mice.

The role of Pannexin gene variants in schizophrenia: systematic analysis of phenotypes.

Pannexins are a group of brain-expressed channel proteins thought to be regulators of schizophrenia-linked pathways including glutamate release, synaptic plasticity and neural stem proliferation. We got evidence for linkage of a catatonic phenotype to the PANX2 locus in a family study. Aim of our study was to evaluate the role of Pannexins in schizophrenia and clinical phenotypes, particularly with regard to periodic catatonia. We genotyped six single-nucleotide polymorphisms at PANX1, five at PANX2 and three at PANX3 in 1173 German cases with schizophrenia according to DSM-5 and 480 controls. Our sample included 338 cases with periodic catatonia corresponding to Leonhard's classification. Association with schizophrenia according to DSM-5 was limited to genotype rs4838858-TT [p=0.02, odds ratio (OR) 3.1] and haplotype rs4838858T-rs5771206G (p=0.02, OR 2.7) at PANX2. We found no significant association with clinical phenotypes. Our limited findings do not support a major contribution of PANX1-3 to disease risk of schizophrenia according to DSM-5. We cannot confirm an association of the PANX2 loci at chromosome 22q13 with periodic catatonia.

Disrupting the male germ line to find infertility and contraception targets

Genetically-manipulated mouse models have become indispensible for broadening our understanding of genes and pathways related to male germ cell development. Until suitable in vitro systems for studying spermatogenesis are perfected, in vivo models will remain the gold standard for inquiry into testicular function. Here, we discuss exciting advances that are allowing researchers faster, easier, and more customizable access to their mouse models of interest. Specifically, the trans-NIH Knockout Mouse Project (KOMP) is working to generate knockout mouse models of every gene in the mouse genome. The related Knockout Mouse Phenotyping Program (KOMP2) is performing systematic phenotypic analysis of this genome-wide collection of knockout mice, including fertility screening. Together, these programs will not only uncover new genes involved in male germ cell development but also provide the research community with the mouse models necessary for further investigations. In addition to KOMP/KOMP2, another promising development in the field of mouse models is the advent of CRISPR (clustered regularly interspaced short palindromic repeat)-Cas technology. Utilizing 20 nucleotide guide sequences, CRISPR/Cas has the potential to introduce sequence-specific insertions, deletions, and point mutations to produce null, conditional, activated, or reporter-tagged alleles. CRISPR/Cas can also successfully target multiple genes in a single experimental step, forgoing the multiple generations of breeding traditionally required to produce mouse models with deletions, insertions, or mutations in multiple genes. In addition, CRISPR/Cas can be used to create mouse models carrying variants identical to those identified in infertile human patients, providing the opportunity to explore the effects of such mutations in an in vivo system. Both the KOMP/KOMP2 projects and the CRISPR/Cas system provide powerful, accessible genetic approaches to the study of male germ cell development in the mouse. A more complete understanding of male germ cell biology is critical for the identification of novel targets for potential non-hormonal contraceptive intervention.

High Mobility Group N Proteins Modulate the Fidelity of the Cellular Transcriptional Profile in a Tissue- and Variant-specific Manner

The nuclei of most vertebrate cells contain members of the high mobility group N (HMGN) protein family, which bind specifically to nucleosome core particles and affect chromatin structure and function, including transcription. Here, we study the biological role of this protein family by systematic analysis of phenotypes and tissue transcription profiles in mice lacking functional HMGN variants. Phenotypic analysis of Hmgn1(tm1/tm1), Hmgn3(tm1/tm1), and Hmgn5(tm1/tm1) mice and their wild type littermates with a battery of standardized tests uncovered variant-specific abnormalities. Gene expression analysis of four different tissues in each of the Hmgn(tm1/tm1) lines reveals very little overlap between genes affected by specific variants in different tissues. Pathway analysis reveals that loss of an HMGN variant subtly affects expression of numerous genes in specific biological processes. We conclude that within the biological framework of an entire organism, HMGNs modulate the fidelity of the cellular transcriptional profile in a tissue- and HMGN variant-specific manner.

Inducible genetic system for the axolotl

Transgenesis promises a powerful means for assessing gene function during amphibian limb regeneration. This approach is complicated, however, by the need for embryonic appendage development to proceed unimpeded despite the genetic alterations one wishes to test later in the context of regeneration. Achieving conditional gene regulation in this amphibian has not proved to be as straightforward as in many other systems. In this report we describe a unique method for obtaining temporal control over exogenous gene expression in the axolotl. Based on technology derived from the Escherichia coli Lac operon, uninduced transgenes are kept in a repressed state by the binding of constitutively expressed Lac repressor protein (LacI) to operator sequences within the expression construct. Addition of a lactose analog, IPTG, to the swimming water of the axolotl is sufficient for the sugar to be taken up by cells, where it binds the LacI protein, thereby inducing expression of the repressed gene. We use this system to demonstrate an in vivo role for thrombospondin-4 in limb regeneration. This inducible system will allow for systematic analysis of phenotypes at defined developmental or regenerative time points. The tight regulation and robustness of gene induction combined with the simplicity of this strategy will prove invaluable for studying many aspects of axolotl biology.

The Chloroplast Function Database: a large‐scale collection of Arabidopsis Ds/Spm‐ or T‐DNA‐tagged homozygous lines for nuclear‐encoded chloroplast proteins, and their systematic phenotype analysis

A majority of the proteins of the chloroplast are encoded by the nuclear genome, and are post-translationally targeted to the chloroplast. From databases of tagged insertion lines at international seed stock centers and our own stock, we selected 3246 Ds/Spm (dissociator/suppressor-mutator) transposon- or T-DNA-tagged Arabidopsis lines for genes encoding 1369 chloroplast proteins (about 66% of the 2090 predicted chloroplast proteins) in which insertions disrupt the protein-coding regions. We systematically observed 3-week-old seedlings grown on agar plates, identified mutants with abnormal phenotypes and collected homozygous lines with wild-type phenotypes. We also identified insertion lines for which no homozygous plants were obtained. To date, we have identified 111 lines with reproducible seedling phenotypes, 122 lines for which we could not obtain homozygotes and 1290 homozygous lines without a visible phenotype. The Chloroplast Function Database presents the molecular and phenotypic information obtained from this resource. The database provides tools for searching for mutant lines using Arabidopsis Genome Initiative (AGI) locus numbers, tagged line numbers and phenotypes, and provides rapid access to detailed information on the tagged line resources. Moreover, our collection of insertion homozygotes provides a powerful tool to accelerate the functional analysis of nuclear-encoded chloroplast proteins in Arabidopsis. The Chloroplast Function Database is freely available at http://rarge.psc.riken.jp/chloroplast/. The homozygous lines generated in this project are also available from the various Arabidopsis stock centers. We have donated the insertion homozygotes to their originating seed stock centers.

Phenome Analysis in Plant Species Using Loss-of-Function and Gain-of-Function Mutants

Analysis of genetic mutations is one of the most effective ways to investigate gene function. We now have methods that allow for mass production of mutant lines and cells in a variety of model species. Recently, large numbers of mutant lines have been generated by both 'loss-of-function' and 'gain-of-function' techniques. In parallel, phenotypic information covering various mutant resources has been acquired and released in web-based databases. As a result, significant progress in comprehensive phenotype analysis is being made through the use of these tools. Arabidopsis and rice are two major model plant species in which genome sequencing projects have been completed. Arabidopsis is the most widely used experimental plant, with a large number of mutant resources and several examples of systematic phenotype analysis. Rice is a major crop species and is used as a model plant, with an increasing number of mutant resources. Other plant species are also being employed in functional genetics research. In this review, the present status of mutant resources for large-scale studies of gene function in plant research and the current perspective on using loss-of-function and gain-of-function mutants in phenome research will be discussed.

Genomewide Analysis of Secretory Activation in Mouse Models

Mouse models have been widely used to elucidate the biology of mammary gland development and secretory activation. Recent advances in the availability of genomic resources for mice will generate a renewed effort to define the genetic basis of lactation phenotypes and help identify candidate gene pathways. Specific aspects of these advances are relevant to the dairy industry and may provide a rationale for improving milk production in the dairy cow. Differences are evident in mammary gland morphology and various characteristics of milk production of inbred mouse strains, but few studies have undertaken any systematic phenotypic analysis of the different inbred strains of mice for lactation performance. Whole genome association analysis using recent strain-specific genotype data and detailed phenotype measurements from available inbred strains, along with transcript profiling of divergent inbred strains for lactation performance, provides a valuable approach to identify putative candidate genes and associated pathways underlying dairy QTL intervals. Here we discuss the utility of integrating mouse phenomic and genomic resources for understanding secretory activation in the mammary gland.

IN SILICO PHENOTYPIC SCREENING METHOD OF MUTANTS BASED ON STATISTICAL MODELING OF GENETICALLY MIXED SAMPLES

In comprehensive functional genomics projects, systematic analysis of phenotypes is vital. However, conventional phenotypic screening is done mainly by imprecise visual observation of qualitative traits, and, therefore, in silico screening techniques for quantitative traits are required. In this report, we propose in silico phenotypic screening method that utilizes a Gaussian mixture model for the trait distribution in the offspring of a mutagenized line and the likelihood ratio test between the estimated Gaussian mixture model and the wild-type single Gaussian model. In order to evaluate the proposed method, we performed a screening experiment using real trait data of Arabidopsis. In this experiment, the proposed screening method properly distinguished the mutant line from the wild-type line. Furthermore, we conducted power analysis of the proposed method and two conventional methods under various simulated conditions of sample size and distribution of trait frequency. The result of the power analysis confirmed the effectiveness of the proposed method compared to the conventional methods.

A collection of 11 800 single-copy Ds transposon insertion lines in Arabidopsis.

More than 10 000 transposon-tagged lines were constructed by using the Activator (Ac)/Dissociation (Ds) system in order to collect insertional mutants as a useful resource for functional genomics of Arabidopsis. The flanking sequences of the Ds element in the 11 800 independent lines were determined by high-throughput analysis using a semi-automated method. The sequence data allowed us to map the unique insertion site on the Arabidopsis genome in each line. The Ds element of 7566 lines is inserted in or close to coding regions, potentially affecting the function of 5031 of 25 000 Arabidopsis genes. Half of the lines have Ds insertions on chromosome 1 (Chr. 1), in which donor lines have a donor site. In the other half, the Ds insertions are distributed throughout the other four chromosomes. The intrachromosomal distribution of Ds insertions varies with the donor lines. We found that there are hot spots for Ds transposition near the ends of every chromosome, and we found some statistical preference for Ds insertion targets at the nucleotide level. On the basis of systematic analysis of the Ds insertion sites in the 11 800 lines, we propose the use of Ds-tagged lines with a single insertion in annotated genes for systematic analysis of phenotypes (phenome analysis) in functional genomics. We have opened a searchable database of the insertion-site sequences and mutated genes (http://rarge.gsc.riken.go.jp/) and are depositing these lines in the RIKEN BioResource Center as available resources (http://www.brc.riken.go.jp/Eng/).

A large-scale sonication assay for cell wall mutant analysis in yeast

The complete yeast genome contains a large number of genes of unknown biological function. Simple, rapid and reliable specific screens are valuable tools in exploring gene function via systematic phenotypic analysis of large mutant collections. This report provides a new approach for monitoring changes in cell wall strength, based on the deleterious effects caused by ultrasound on the yeast cell surface. Sonication can thus be used for the screening of mutants affected in the architecture or stability of the cell wall, since such mutants are expected to have an altered sensitivity to this treatment compared to that of a wild-type. The experimental procedure, consisting in the quantification of damaged cells after a mild sonication treatment, by means of flow cytometry, can be applied on a large scale. The usefulness of the sonication assay as a primary screen for cell wall-related mutants is evaluated on the collection of calcofluor white-hypersensitive and -resistant mutants obtained by Lussier et al. (1997). A further phenotypic characterization of the sonication-hypersensitive mutants within the calcofluor white collection is also presented.

Disruption of six novel yeast genes located on chromosome II reveals one gene essential for vegetative growth and two required for sporulation and conferring hypersensitivity to various chemicals

A PCR-based method for targeted gene deletion by kanMX4 module was used to construct complete deletion mutants of six individual open reading frames from chromosome II: YBR128c, YBR131w, YBR133c, YBR137w, YBR138c and YBR142w. The ORFs were deleted in two diploid strains, FY1679 and W303. Sporulation and tetrad analysis revealed that only one ORF, YBR142w, encoding a putative DEAD-box RNA helicase, is an essential gene. A systematic phenotypic analysis of the deleted mutants was carried out. Homozygous diploids ybr128cDelta/ybr128cDelta and ybr131wDelta/ybr131wDelta did not sporulate. The ybr131cDelta mutant whether haploid or homozygous diploid, in addition displayed an increased sensitivity to Caffeine, Calcium and Zinc, and to emphasize this phenotype we named the gene CCZ1. ORF YBR133c was independently reported by others as Histone Synthetic Lethal (HSL7) (Ma et al., 1996). We found that the aberrant morphology characteristic for ybr133cDelta (hsl7Delta) cells was observed in W303 but not in FY1679 genetic background. Furthermore, we observed that deletion of YBR133c had a pleiotropic effect under a wide range of conditions, including increased sensitivity to calcium, caffeine, calcofluor white, vanadate and verapamil. The effects of the deletion were reinforced in W303 background. We found no phenotypic effects of the two remaining deletions, ybr137wDelta and ybr138cDelta.

A large‐scale sonication assay for cell wall mutant analysis in yeast

The complete yeast genome contains a large number of genes of unknown biological function. Simple, rapid and reliable specific screens are valuable tools in exploring gene function via systematic phenotypic analysis of large mutant collections. This report provides a new approach for monitoring changes in cell wall strength, based on the deleterious effects caused by ultrasound on the yeast cell surface. Sonication can thus be used for the screening of mutants affected in the architecture or stability of the cell wall, since such mutants are expected to have an altered sensitivity to this treatment compared to that of a wild-type. The experimental procedure, consisting in the quantification of damaged cells after a mild sonication treatment, by means of flow cytometry, can be applied on a large scale. The usefulness of the sonication assay as a primary screen for cell wall-related mutants is evaluated on the collection of calcofluor white-hypersensitive and -resistant mutants obtained by Lussier et al. (1997). A further phenotypic characterization of the sonication-hypersensitive mutants within the calcofluor white collection is also presented. Copyright © 1999 John Wiley & Sons, Ltd.

Large‐scale phenotypic analysis—the pilot project on yeast chromosome III

In 1993, a pilot project for the functional analysis of newly discovered open reading frames, presumably coding for proteins, from yeast chromosome III was launched by the European Community. In the frame of this programme, we have developed a large-scale screening for the identification of gene/protein functions via systematic phenotypic analysis. To this end, some 80 haploid mutant yeast strains were constructed, each carrying a targeted deletion of a single gene obtained by HIS3 or TRP1 transplacement in the W303 background and a panel of some 100 growth conditions was established, ranging from growth substrates, stress to, predominantly, specific inhibitors and drugs acting on various cellular processes. Furthermore, co-segregation of the targeted deletion and the observed phenotype(s) in meiotic products has been verified. The experimental procedure, using microtiter plates for phenotypic analysis of yeast mutants, can be applied on a large scale, either on solid or in liquid media. Since the minimal working unit of one 96-well microtiter plate allows the simultaneous analysis of at least 60 mutant strains, hundreds of strains can be handled in parallel. The high number of monotropic and pleiotropic phenotypes (62%) obtained, together with the acquired practical experience, have shown this approach to be simple, inexpensive and reproducible. It provides a useful tool for the yeast community for the systematic search of biochemical and physiological functions of unknown genes accounting for about a half of the 6000 genes of the complete yeast genome. © 1997 John Wiley & Sons, Ltd.

Large-scale phenotypic analysis--the pilot project on yeast chromosome III.

In 1993, a pilot project for the functional analysis of newly discovered open reading frames, presumably coding for proteins, from yeast chromosome III was launched by the European Community. In the frame of this programme, we have developed a large-scale screening for the identification of gene/protein functions via systematic phenotypic analysis. To this end, some 80 haploid mutant yeast strains were constructed, each carrying a targeted deletion of a single gene obtained by HIS3 or TRP1 transplacement in the W303 background and a panel of some 100 growth conditions was established, ranging from growth substrates, stress to, predominantly, specific inhibitors and drugs acting on various cellular processes. Furthermore, co-segregation of the targeted deletion and the observed phenotype(s) in meiotic products has been verified. The experimental procedure, using microtiter plates for phenotypic analysis of yeast mutants, can be applied on a large scale, either on solid or in liquid media. Since the minimal working unit of one 96-well microtiter plate allows the simultaneous analysis of at least 60 mutant strains, hundreds of strains can be handled in parallel. The high number of monotropic and pleiotropic phenotypes (62%) obtained, together with the acquired practical experience, have shown this approach to be simple, inexpensive and reproducible. It provides a useful tool for the yeast community for the systematic search of biochemical and physiological functions of unknown genes accounting for about a half of the 6000 genes of the complete yeast genome.