The Consequences of Abnormal Gene Dosage: Lessons from Chromosome 18.

Accurate interpretation of genomic copy number variation (CNV) remains a challenge and has important consequences for both congenital and late-onset disease. Hemizygosity dosage characterization of the genes on chromosome 18 reveals a spectrum of outcomes ranging from no clinical effect, to risk factors for disease, to both low- and high-penetrance disease. These data are important for accurate and predictive clinical management. Additionally, the potential mechanisms of reduced penetrance due to dosage compensation are discussed as a key to understanding avenues for potential treatment. This review describes the chromosome 18 findings, and discusses the molecular mechanisms that allow haploinsufficiency, reduced penetrance, and dosage compensation.

Genomic evidence supports the genetic convergence of a supergene controlling the distylous floral syndrome.

Heterostyly, a plant sexual polymorphism controlled by the S-locus supergene, has evolved numerous times among angiosperm lineages and represents a classic example of convergent evolution in form and function. Determining whether underlying molecular convergence occurs could provide insights on constraints to floral evolution. Here, we investigated S-locus genes in distylous Gelsemium (Gelsemiaceae) to determine whether there is evidence of molecular convergence with unrelated distylous species. We used several approaches, including anatomical measurements of sex-organ development and transcriptome and whole-genome sequencing, to identify components of the S-locus supergene. We also performed evolutionary analysis with candidate S-locus genes and compared them with those reported in Primula and Turnera. The candidate S-locus supergene of Gelsemium contained four genes, of which three appear to have originated from gene duplication events within Gelsemiaceae. The style-length genes GeCYP in Gelsemium and CYP734A50 in Primula likely arose from duplication of the same gene, CYP734A1. Three out of four S-locus genes in Gelsemium elegans were hemizygous, as previously reported in Primula and Turnera. We provide genomic evidence on the genetic convergence of the supergene underlying distyly among distantly related angiosperm lineages and help to illuminate the genetic architecture involved in the evolution of heterostyly.

Sex chromosome heteromorphism and the Fast-X effect in poeciliids.

Fast-X evolution has been observed in a range of heteromorphic sex chromosomes. However, it remains unclear how early in the process of sex chromosome differentiation the Fast-X effect becomes detectible. Recently, we uncovered an extreme variation in sex chromosome heteromorphism across poeciliid fish species. The common guppy, Poecilia reticulata, Endler's guppy, P. wingei, swamp guppy, P. picta and para guppy, P. parae, appear to share the same XY system and exhibit a remarkable range of heteromorphism. Species outside this group lack this sex chromosome system. We combined analyses of sequence divergence and polymorphism data across poeciliids to investigate X chromosome evolution as a function of hemizygosity and reveal the causes for Fast-X effects. Consistent with the extent of Y degeneration in each species, we detect higher rates of divergence on the X relative to autosomes, a signal of Fast-X evolution, in P. picta and P. parae, species with high levels of X hemizygosity in males. In P. reticulata, which exhibits largely homomorphic sex chromosomes and little evidence of hemizygosity, we observe no change in the rate of evolution of X-linked relative to autosomal genes. In P. wingei, the species with intermediate sex chromosome differentiation, we see an increase in the rate of nonsynonymous substitutions on the older stratum of divergence only. We also use our comparative approach to test for the time of origin of the sex chromosomes in this clade. Taken together, our study reveals an important role of hemizygosity in Fast-X evolution.

Allele-specific knockouts reveal a role for apontic-like in the evolutionary loss of larval melanin pigmentation in the domesticated silkworm, Bombyx mori.

The domesticated silkworm, Bombyx mori, and its wild progenitor, B. mandarina, are extensively studied as a model case of the evolutionary process of domestication. A conspicuous difference between these species is the dramatic reduction in melanin pigmentation in both larval and adult B. mori. Here we evaluate the efficiency of CRISPR/Cas9-targeted knockouts of pigment-related genes as a tool to understand their potential contributions to domestication-associated melanin pigmentation loss in B. mori. To demonstrate the efficacy of targeted knockouts in B. mandarina, we generated a homozygous CRISPR/Cas9-targeted knockout of yellow-y. In yellow-y knockout mutants, black body colour became lighter throughout the larval, pupal and adult stages, confirming a role for this gene in melanin pigment formation. Further, we performed allele-specific CRISPR/Cas9-targeted knockouts of the pigment-related transcription factor, apontic-like (apt-like) in B. mori × B. mandarina F1 hybrid individuals which exhibit B. mandarina-like larval pigmentation. Knockout of the B. mandarina allele of apt-like in F1 embryos results in white patches on the dorsal integument of larvae, whereas corresponding knockouts of the B. mori allele consistently exhibit normal F1 larval pigmentation. These results demonstrate a contribution of apt-like to the evolution of reduced melanin pigmentation in B. mori. Together, our results demonstrate the feasibility of CRISPR/Cas9-targeted knockouts as a tool for understanding the genetic basis of traits associated with B. mori domestication.

The genetic architecture of low-temperature adaptation in the wine yeast Saccharomyces cerevisiae.

BACKGROUND: Low-temperature growth and fermentation of wine yeast can enhance wine aroma and make them highly desirable traits for the industry. Elucidating response to cold in Saccharomyces cerevisiae is, therefore, of paramount importance to select or genetically improve new wine strains. As most enological traits of industrial importance in yeasts, adaptation to low temperature is a polygenic trait regulated by many interacting loci. RESULTS: In order to unravel the genetic determinants of low-temperature fermentation, we mapped quantitative trait loci (QTLs) by bulk segregant analyses in the F13 offspring of two Saccharomyces cerevisiae industrial strains with divergent performance at low temperature. We detected four genomic regions involved in the adaptation at low temperature, three of them located in the subtelomeric regions (chromosomes XIII, XV and XVI) and one in the chromosome XIV. The QTL analysis revealed that subtelomeric regions play a key role in defining individual variation, which emphasizes the importance of these regions' adaptive nature. CONCLUSIONS: The reciprocal hemizygosity analysis (RHA), run to validate the genes involved in low-temperature fermentation, showed that genetic variation in mitochondrial proteins, maintenance of correct asymmetry and distribution of phospholipid in the plasma membrane are key determinants of low-temperature adaptation.

Identification of loci that cause phenotypic variation in diverse species with the reciprocal hemizygosity test.

The reciprocal hemizygosity test is a straightforward genetic test that can positively identify genes that have evolved to contribute to a phenotypic difference between strains or between species. The test involves a comparison between hybrids that are genetically identical throughout the genome except at the test locus, which is rendered hemizygous for alternative alleles from the two parental strains. If the two reciprocal hemizygotes display different phenotypes, then the two parental alleles must have evolved. New methods for targeted mutagenesis will allow application of the reciprocal hemizygosity test in many organisms. This review discusses the principles, advantages, and limitations of the test.

A short story gets longer: recent insights into the molecular basis of heterostyly.

Heterostyly is a fascinating adaptation to promote outbreeding and a classical paradigm of botany. In the most common type of heterostyly, plants either form flowers with long styles and short stamens, or short styles and long stamens. This reciprocal organ positioning reduces pollen wastage and promotes cross-pollination, thus increasing male fitness. In addition, in many heterostylous species selfing and the generation of unfit progeny due to inbreeding depression is limited by a self-incompatibility system, thus promoting female fitness. The two floral forms are genetically determined by the S locus as a complex supergene, namely a chromosomal region containing several individual genes that control the different traits, such as style or stamen length, and are held together by very tight linkage due to suppressed recombination. Recent molecular-genetic studies in several systems, including Turnera, Fagopyrum, Linum, and Primula have begun to identify and characterize the causal heterostyly genes residing at the S locus. An emerging theme from several families is that the dominant S haplotype represents a hemizygous region not present on the recessive s haplotype. This provides an explanation for the suppressed recombination and suggests a scenario for the chromosomal evolution of the S locus. In this review, we discuss the results from recent molecular-genetic analyses in light of the classical models on the genetics and evolution of heterostyly.

Making chromosome abnormalities treatable conditions.

Individuals affected by the classic chromosome deletion syndromes which were first identified at the beginning of the genetic age, are now positioned to benefit from genomic advances. This issue highlights five of these conditions (4p-, 5p-, 11q-, 18p-, and 18q-). It focuses on the increased in understanding of the molecular underpinnings and envisions how these can be transformed into effective treatments. While it is scientifically exciting to see the phenotypic manifestations of hemizygosity being increasingly understood at the molecular and cellular level, it is even more amazing to consider that we are now on the road to making chromosome abnormalities treatable conditions.

Consequences of chromsome18q deletions.

Providing clinically relevant prognoses and treatment information for people with a chromsome18q deletion is particularly challenging because every unrelated person has a unique region of hemizygosity. The hemizygous region can involve almost any region of 18q including between 1 and 101 genes (30 Mb of DNA). Most individuals have terminal deletions, but in our cohort of over 350 individuals 23% have interstitial deletions. Because of this heterogeneity, we take a gene by gene approach to understanding the clinical consequences. There are 196 genes on 18q. We classified 133 of them as dosage insensitive, 15 (8%) as dosage sensitive leading to haploinsufficiency while another 10 (5%) have effects that are conditionally haploinsufficient and are dependent on another factor, genetic or environmental in order to cause an abnormal phenotype. Thirty-seven genes (19%) have insufficient information to classify their dosage effect. Phenotypes attributed to single genes include: congenital heart disease, minor bone morphology changes, central nervous system dysmyelination, expressive speech delay, vesicouretreral reflux, polyposis, Pitt-Hopkins syndrome, intellectual disability, executive function impairment, male infertility, aural atresia, and high frequency sensorineural hearing loss. Additionally, identified critical regions for other phenotypes include: adolescent idiopathic scoliosis and pectus excavatum, Virchow-Robin perivascular spaces, small corpus callosum, strabismus, atopic disorders, mood disorder, IgA deficiency, nystagmus, congenital heart disease, kidney malformation, vertical talus, CNS dysmyelination growth hormone deficiency and cleft palate. Together these findings make it increasingly feasible to compile an individualized syndrome description based on each person's individuated genotype. Future work will focus on understanding molecular mechanisms leading to treatment.

5p deletions: Current knowledge and future directions.

Disorders resulting from 5p deletions (5p-) were first recognized by Lejeune et al. in 1963 [Lejeune et al. (1963); C R Hebd Seances Acad Sci 257:3098-3102]. 5p- is caused by partial or total deletion of the short arm of chromosome 5. The most recognizable phenotype is characterized by a high-pitched cry, dysmorphic features, poor growth, and developmental delay. This report reviews 5p- disorders and their molecular basis. Hemizygosity for genes located within this region have been implicated in contributing to the phenotype. A review of the genes on 5p which may be dosage sensitive is summarized. Because of the growing knowledge of these specific genes, future directions to explore potential targeted therapies for individuals with 5p- are discussed. © 2015 Wiley Periodicals, Inc.

Tiled ChrI RHS collection: a pilot high-throughput screening tool for identification of allelic variants.

Reciprocal hemizygosity analysis is a genetic technique that allows phenotypic determination of the allelic effects of a gene in a genetically uniform background. Expanding this single gene technique to generate a genome-wide collection is termed as reciprocal hemizygosity scanning (RHS). The RHS collection should circumvent the need for linkage mapping and provide the power to identify all possible allelic variants for a given phenotype. However, the published RHS collections based on the existing genome-wide haploid deletion library reported a high rate of false positives. In this study, we report de novo construction of a RHS collection that is not based on the yeast deletion library. This collection has been constructed for the shortest yeast chromosome, ChrI. Using this ChrI RHS collection, we identified 13 allelic variants for the previously mapped loci and novel allelic variants for the growth differences in different environments. A few of these novel variants, which were fine mapped to a gene level, identified novel genetic variation for the previously studied environmental conditions. The availability of a genome-wide RHS collection would thus help us uncover a comprehensive list of allelic variants and better our understanding of the molecular pathways modulating a quantitative trait.

Gene expression, chromosome heterogeneity and the fast-X effect in mammals.

The higher rate of non-synonymous over synonymous substitutions (dN/dS) of the X chromosome compared with autosomes is often interpreted as a consequence of X hemizygosity. However, other factors, such as gene expression, are also known to vary between X and autosomes. Analysing 4800 orthologues in six mammals, we found that gene expression levels, associated with GC content, fully account for the variation in dN/dS between X and autosomes with no detectable effect of hemizygosity. We also report an extensive variance in dN/dS and gene expression between autosomes.

Hemophilia and Other Congenital Coagulopathies in Women.

Deficiencies of factor VIII (FVIII)/von Willebrand factor (VWF) or factor IX (FIX) are underappreciated as potential reasons for heavy menstrual bleeding, recurrent nosebleeds, and easy bruising in girls and women. Bleeding is usually not attributed to hemophilia because clinically significant deficiencies in clotting factors VIII and IX are thought to only affect males. While severe hemophilia is more commonly observed in boys and men, women with mutations in the FVIII or FIX genes (f8 or f9) may have widespread bruising and even joint bleeding. They might be heterozygotes with a hemophilic allele on one X chromosome and a normal allele on the other or rarely homozygotes with hemophilic alleles on both X chromosomes. If most or all of an X chromosome is missing (X-chromosome hemizygosity or Turner syndrome) and a hemophilic mutation is present on the other X chromosome, the affected woman will have a severe bleeding tendency. Other inherited disorders that affect women as well as men are von Willebrand disease, combined deficiencies of factor V (FV) and FVIII, and combined deficiencies of the vitamin K-dependent clotting factors. Women as well as men with autoimmune diseases or even those previously well might acquire a severe hemorrhagic disorder due to autoantibodies directed against FVIII, FIX, or VWF. Lastly, easy bruising and mildly decreased FVIII levels are occasionally observed in both men and women with hypothyroidism or panhypopituitarism. The purpose of this brief review is to increase clinician awareness that these bleeding disorders can affect girls and women. An accurate diagnosis and appropriate therapy will greatly benefit patients and their families.

Genomic analyses elucidate S-locus evolution in response to intra-specific losses of distyly in Primula vulgaris.

Distyly, a floral dimorphism that promotes outcrossing, is controlled by a hemizygous genomic region known as the S-locus. Disruptions of genes within the S-locus are responsible for the loss of distyly and the emergence of homostyly, a floral monomorphism that favors selfing. Using whole-genome resequencing data of distylous and homostylous individuals from populations of Primula vulgaris and leveraging high-quality reference genomes of Primula we tested, for the first time, predictions about the evolutionary consequences of transitions to selfing on S-genes. Our results reveal a previously undetected structural rearrangement in CYPᵀ associated with the shift to homostyly and confirm previously reported, homostyle-specific, loss-of-function mutations in the exons of the S-gene CYPᵀ. We also discovered that the promoter and intronic regions of CYPᵀ in distylous and homostylous individuals are conserved, suggesting that down-regulation of CYPᵀ via mutations in its promoter and intronic regions is not a cause of the shift to homostyly. Furthermore, we found that hemizygosity is associated with reduced genetic diversity in S-genes compared with their paralogs outside the S-locus. Additionally, the shift to homostyly lowers genetic diversity in both the S-genes and their paralogs, as expected in primarily selfing plants. Finally, we tested, for the first time, long-standing theoretical models of changes in S-locus genotypes during early stages of the transition to homostyly, supporting the assumption that two copies of the S-locus might reduce homostyle fitness.

Mexican patient with Ellis-van Creveld syndrome and cleft palate: Importance of functional hemizygosity and phenotype expansion.

BACKGROUND: Ellis-van Creveld syndrome (EvCS) is a chondroectodermal dysplasia caused by germline pathogenic variants in ciliary complex subunit 1 and 2 genes (EVC, EVC2) on chromosome 4p16.2. This disease has a broad phenotype, and there are few described phenotype-genotype correlations. METHODS: Ethical Compliance: Written informed consent was obtained from the parents. Here, we report a genetically confirmed Mexican patient with EvCS having two inherited pathogenic variants in trans in EVC2: c.[1195C>T];[2161delC]. RESULTS: This patient allowed a genotypic-phenotypic comparison with another Mexican subject who presented a more attenuated phenotype; furthermore, our patient also presented cleft palate, a rarely reported feature. CONCLUSION: Our case shows the importance of comparing functional hemizygosity between patient's phenotypes when they share a variant, and our case also supports the association of alterations in the palate as part of the EvCS phenotype.

The homomorphic self-incompatibility system in Oleaceae is controlled by a hemizygous genomic region expressing a gibberellin pathway gene.

In flowering plants, outcrossing is commonly ensured by self-incompatibility (SI) systems. These can be homomorphic (typically with many different allelic specificities) or can accompany flower heteromorphism (mostly with just two specificities and corresponding floral types). The SI system of the Oleaceae family is unusual, with the long-term maintenance of only two specificities but often without flower morphology differences. To elucidate the genomic architecture and molecular basis of this SI system, we obtained chromosome-scale genome assemblies of Phillyrea angustifolia individuals and related them to a genetic map. The S-locus region proved to have a segregating 543-kb indel unique to one specificity, suggesting a hemizygous region, as observed in all distylous systems so far studied at the genomic level. Only one of the predicted genes in this indel region is found in the olive tree, Olea europaea, genome, also within a segregating indel. We describe complete association between the presence/absence of this gene and the SI types determined for individuals of seven distantly related Oleaceae species. This gene is predicted to be involved in catabolism of the gibberellic acid (GA) hormone, and experimental manipulation of GA levels in developing buds modified the male and female SI responses of the two specificities in different ways. Our results provide a unique example of a homomorphic SI system, where a single conserved gibberellin-related gene in a hemizygous indel underlies the long-term maintenance of two groups of reproductive compatibility.

Quantitative genetic analysis of attractiveness of yeast products to Drosophila.

An attractive perfume is a complex mixture of compounds, some of which may be unpleasant on their own. This is also true for the volatile combinations from yeast fermentation products in vineyards and orchards when assessed by Drosophila. Here, we used crosses between a yeast strain with an attractive fermentation profile and another strain with a repulsive one and tested fly responses using a T-maze. QTL analysis reveals allelic variation in four yeast genes, namely PTC6, SAT4, YFL040W, and ARI1, that modulated expression levels of volatile compounds [assessed by gas chromatography-mass spectrometry (GC-MS)] and in different combinations, generated various levels of attractiveness. The parent strain that is more attractive to Drosophila has repulsive alleles at two of the loci, while the least attractive parent has attractive alleles. Behavioral assays using artificial mixtures mimicking the composition of odors from fermentation validated the results of GC-MS and QTL mapping, thereby directly connecting genetic variation in yeast to attractiveness in flies. This study can be used as a basis for dissecting the combination of olfactory receptors that mediate the attractiveness/repulsion of flies to yeast volatiles and may also serve as a model for testing the attractiveness of pest species such as Drosophila suzukii to their host fruit.

Genetic bases of C7 deficiency: systematic review and report of a novel deletion determining functional hemizygosity.

Primary complement system (C) deficiencies are rare but notably associated with an increased risk of infections, autoimmunity, or immune disorders. Patients with terminal pathway C-deficiency have a 1,000- to 10,000-fold-higher risk of Neisseria meningitidis infections and should be therefore promptly identified to minimize the likelihood of further infections and to favor vaccination. In this paper, we performed a systematic review about clinical and genetic patterns of C7 deficiency starting from the case of a ten-year old boy infected by Neisseria meningitidis B and with clinical presentation suggestive of reduced C activity. Functional assay via Wieslab ELISA Kit confirmed a reduction in total C activity of the classical (0.6% activity), lectin (0.2% activity) and alternative (0.1% activity) pathways. Western blot analysis revealed the absence of C7 in patient serum. Sanger sequencing of genomic DNA extracted from peripheral blood of the patient allowed the identification of two pathogenetic variants in the C7 gene: the already well-characterized missense mutation G379R and a novel heterozygous deletion of three nucleotides located at the 3'UTR (c.*99_*101delTCT). This mutation resulted in an instability of the mRNA; thus, only the allele containing the missense mutation was expressed, making the proband a functional hemizygote for the expression of the mutated C7 allele.

Detecting Structural Variants and Associated Gene Presence-Absence Variation Phenomena in the Genomes of Marine Organisms.

As complete genomes become easier to attain, even from previously difficult-to-sequence species, and as genomic resequencing becomes more routine, it is becoming obvious that genomic structural variation is more widespread than originally thought and plays an important role in maintaining genetic variation in populations. Structural variants (SVs) and associated gene presence-absence variation (PAV) can be important players in local adaptation, allowing the maintenance of genetic variation and taking part in other evolutionarily relevant phenomena. While recent studies have highlighted the importance of structural variation in Mollusca, the prevalence of this phenomenon in the broader context of marine organisms remains to be fully investigated.Here, we describe a straightforward and broadly applicable method for the identification of SVs in fully assembled diploid genomes, leveraging the same reads used for assembly. We also explain a gene PAV analysis protocol, which could be broadly applied to any species with a fully sequenced reference genome available. Although the strength of these approaches have been tested and proven in marine invertebrates, which tend to have high levels of heterozygosity, possibly due to their lifestyle traits, they are also applicable to other species across the tree of life, providing a ready means to begin investigations into this potentially widespread phenomena.

Recessive Dystrophic Epidermolysis bullosa due to Hemizygous 40 kb Deletion of COL7A1 and the Proximate PFKFB4 Gene Focusing on the Mutation c.425A>G Mimicking Homozygous Status

Background: Dystrophic Epidermolysis bullosa (DEB) is a rare inherited mechanobullous disease characterised by the hyperfragility of the skin and mucous membranes. It is (typically) caused by (loss-of-function) mutations in the COL7A1 gene that impair the formation of collagen type VII, which represents the major constituent of anchoring fibrils within the basement membrane zone of epithelialised tissues. In a 4-year-old patient diagnosed with the clinical features of recessive DEB, genotyping via Next-Generation EB Panel Sequencing initially revealed the homozygosity of the maternal c.425A>G mutation, while the paternal heterozygosity in exon 3 was lacking. This genetic profile suggested incongruent gene transmission due to uniparental isodisomy (UPD) or the occurrence of a hemizygous deletion of unknown size. Methods: Thus, the EB panel sequencing of genomic DNA, followed by a paternity test and analysis of microsatellite markers, as well as multiplex ligation-dependent probe amplification (MLPA) copy number analysis using patient and parental DNA, were performed. Results: This approach revealed a paternally derived hemizygous deletion spanning from exon 3 to exon 118. Linear amplification-mediated PCR (LAM-PCR) determined the breaking points within intron 2 of the COL7A1 gene, comprising a 40kb segment within intron 1 of the adjacent PFKFB4 gene. Conclusion: This report highlights the relevance of advanced molecular profiling to determine new/exceptional/unusual genotypes and the accurate mode of genetic transmission in DEB.