Tracing the Origin of the RSPO2 Long-Hair Allele and Epistatic Interaction between FGF5 and RSPO2 in Sapsaree Dog

Genetic analysis of the hair-length of Sapsaree dogs, a Korean native dog breed, showed a dominant mode of inheritance for long hair. Genome-Wide Association Study (GWAS) analysis and subsequent Mendelian segregation analysis revealed an association between OXR1, RSPO2, and PKHD1L1 on chromosome 13 (CFA13). We identified the previously reported 167 bp insertion in RSPO2 3’ untranslated region as a causative mutation for hair length variations. The analysis of 118 dog breeds and wolves revealed the selection signature on CFA13 in long-haired breeds. Haplotype analysis showed the association of only a few specific haplotypes to the breeds carrying the 167 bp insertion. The genetic diversity in the neighboring region linked to the insertion was higher in Sapsarees than in other Asian and European dog breeds carrying the same variation, suggesting an older history of its insertion in the Sapsaree genome than in that of the other breeds analyzed in this study. Our results show that the RSPO2 3’ UTR insertion is responsible for not only the furnishing phenotype but also determining the hair length of the entire body depending on the genetic background, suggesting an epistatic interaction between FGF5 and RSPO2 influencing the hair-length phenotype in dogs.

A Decade of Molecular Preimplantation Genetic Diagnosis of 350 Blastomeres for Beta-Thalassemia Combined with HLA Typing, Aneuploidy Screening and Sex Selection in Iran

Background: Preimplantation genetic diagnosis (PGD) has been developed to detect genetic disorders before pregnancy which is usually done on blastomeres biopsied from 8-cell stage embryos obtained from in vitro fertilization method (IVF).Here we report molecular PGD results for diagnosing of beta thalassemia (beta-thal) which are usually accompanied with evaluating chromosomal aneuploidies, HLA typing and sex selection.Methods: In this study, haplotype analysis was performed using short tandem repeats (STRs) in a multiplex nested PCR and the causative mutation was detected by Sanger sequencing.Results: We have performed PGDs on 350 blastomeres from 55 carrier couples; 142 blastomeres for beta-thal only, 75 for beta-thal and HLA typing, 76 for beta-thal in combination with sex selection, and 57 for beta-thal and aneuploidy screening. 150 blastomeres were transferable, 15 pregnancies were happened, and 11 babies born.We used 6 markers for beta-thal, 36 for aneuploidy screening, 32 for sex selection, and 35 for HLA typing. To our knowledge combining all these markers together and the number of STR markers are much more than any other studies which have ever done.Conclusions: PGD is a powerful diagnostic tool for carrier couples who desire to have a healthy child and wish to avoid medical abortion.

MLL1 is required for maintenance of intestinal stem cells

Epigenetic mechanisms are gatekeepers for the gene expression patterns that establish and maintain cellular identity in mammalian development, stem cells and adult homeostasis. Amongst many epigenetic marks, methylation of histone 3 lysine 4 (H3K4) is one of the most widely conserved and occupies a central position in gene expression. Mixed lineage leukemia 1 (MLL1/KMT2A) is the founding mammalian H3K4 methyltransferase. It was discovered as the causative mutation in early onset leukemia and subsequently found to be required for the establishment of definitive hematopoiesis and the maintenance of adult hematopoietic stem cells. Despite wide expression, the roles of MLL1 in non-hematopoietic tissues remain largely unexplored. To bypass hematopoietic lethality, we used bone marrow transplantation and conditional mutagenesis to discover that the most overt phenotype in adult Mll1-mutant mice is intestinal failure. MLL1 is expressed in intestinal stem cells (ISCs) and transit amplifying (TA) cells but not in the villus. Loss of MLL1 is accompanied by loss of ISCs and a differentiation bias towards the secretory lineage with increased numbers and enlargement of goblet cells. Expression profiling of sorted ISCs revealed that MLL1 is required to promote expression of several definitive intestinal transcription factors including Pitx1, Pitx2, Foxa1, Gata4, Zfp503 and Onecut2, as well as the H3K27me3 binder, Bahcc1. These results were recapitulated using conditional mutagenesis in intestinal organoids. The stem cell niche in the crypt includes ISCs in close association with Paneth cells. Loss of MLL1 from ISCs promoted transcriptional changes in Paneth cells involving metabolic and stress responses. Here we add ISCs to the MLL1 repertoire and observe that all known functions of MLL1 relate to the properties of somatic stem cells, thereby highlighting the suggestion that MLL1 is a master somatic stem cell regulator.

The MADS-box gene FveSEP3 plays essential roles in flower organogenesis and fruit development in woodland strawberry

Flower and fruit development are two key steps for plant reproduction. The ABCE model for flower development has been well established in model plant species; however, the functions of ABCE genes in fruit crops are less understood. In this work, we identified an EMS mutant named R27 in woodland strawberry (Fragaria vesca), showing the conversion of petals, stamens, and carpels to sepaloid organs in a semidominant inheritance fashion. Mapping by sequencing revealed that the class E gene homolog FveSEP3 (FvH4_4g23530) possessed the causative mutation in R27 due to a G to E amino acid change in the conserved MADS domain. Additional fvesep3CR mutants generated by CRISPR/Cas9 displayed similar phenotypes to fvesep3-R27. Overexpressing wild-type or mutated FveSEP3 in Arabidopsis suggested that the mutation in R27 might cause a dominant-negative effect. Further analyses indicated that FveSEP3 physically interacted with each of the ABCE proteins in strawberry. Moreover, both R27 and fvesep3CR mutants exhibited parthenocarpic fruit growth and delayed fruit ripening. Transcriptome analysis revealed that both common and specific differentially expressed genes were identified in young fruit at 6–7 days post anthesis (DPA) of fvesep3 and pollinated wild type when compared to unpollinated wild type, especially those in the auxin pathway, a key hormone regulating fruit set in strawberry. Together, we provided compelling evidence that FveSEP3 plays predominant E functions compared to other E gene homologs in flower development and that FveSEP3 represses fruit growth in the absence of pollination and promotes fruit ripening in strawberry.

Huntington disease update: new insights into the role of repeat instability in disease pathogenesis

The causative mutation for Huntington disease (HD), an expanded trinucleotide repeat sequence in the first exon of the huntingtin gene (HTT) is naturally polymorphic and inevitably associated with disease symptoms above 39 CAG repeats. Although symptomatic medical therapies for HD can improve the motor and non-motor symptoms for affected patients, these drugs do not stop the ongoing neurodegeneration and progression of the disease, which results in severe motor and cognitive disability and death. To date, there is still an urgent need for the development of effective disease‐modifying therapies to slow or even stop the progression of HD. The increasing ability to intervene directly at the roots of the disease, namely HTT transcription and translation of its mRNA, makes it necessary to understand the pathogenesis of HD as precisely as possible. In addition to the long-postulated toxicity of the polyglutamine-expanded mutant HTT protein, there is increasing evidence that the CAG repeat-containing RNA might also be directly involved in toxicity. Recent studies have identified cis- (DNA repair genes) and trans- (loss/duplication of CAA interruption) acting variants as major modifiers of age at onset (AO) and disease progression. More and more extensive data indicate that somatic instability functions as a driver for AO as well as disease progression and severity, not only in HD but also in other polyglutamine diseases. Thus, somatic expansions of repetitive DNA sequences may be essential to promote respective repeat lengths to reach a threshold leading to the overt neurodegenerative symptoms of trinucleotide diseases. These findings support somatic expansion as a potential therapeutic target in HD and related repeat expansion disorders.

Fibrodysplasia Ossificans Progressiva: What Have We Achieved and Where Are We Now? Follow-up to the 2015 Lorentz Workshop

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare progressive genetic disease effecting one in a million individuals. During their life, patients with FOP progressively develop bone in the soft tissues resulting in increasing immobility and early death. A mutation in the ACVR1 gene was identified as the causative mutation of FOP in 2006. After this, the pathophysiology of FOP has been further elucidated through the efforts of research groups worldwide. In 2015, a workshop was held to gather these groups and discuss the new challenges in FOP research. Here we present an overview and update on these topics.

Base Editing Repairs the HbE Mutation Restoring the Production of Normal Globin Chains in Severe HbE/β-Thalassemia Patient Hematopoietic Stem Cells and Erythroid Cells

HbE/β-thalassemia is the commonest form of severe β-thalassemia, and comprises approximately 50% of all cases worldwide. HbE/β-thalassemia is caused by the HbE codon 26 G>A mutation on one allele and any severe β 0-thalassemia mutation on the other. These mutations lead to a reduction in β-globin production, resulting in a relative excess in α-globin chains that go on to cause ineffective erythropoiesis. Importantly, individuals with a mutation on one, but not two, alleles have β-thalassemia trait, a carrier state with a normal phenotype. Recent gene therapy and gene editing approaches have been developed to treat β-thalassemia but do not directly repair the causative mutation in-situ. Gene replacement approaches rely on lentiviral vector-based sequence insertion or homology directed repair (HDR). HbF induction strategies also rely on non-homologous end joining (NHEJ) targeting of enhancers in-trans. These approaches, whilst variably successful, are associated with potential safety concerns. Adenine base editors (ABEs) potentially circumvent these problems by directly repairing pathogenic variants in-situ through deamination. ABEs catalyse A-T to G-C conversions through targeting with a Cas9-nickase and single-guide RNA (sgRNA). Conversion of the HbE codon to normal through base editing is an attractive strategy to recapitulate the phenotypically normal β-thalassemia trait state without potentially harmful double-strand breaks or random vector insertions (Figure 1A). ABEs are able to convert the HbE codon (AAG, lys) to wild-type (GAG, glu), but also to GGG (gly) or AGG (arg). GGG at codon 26 is found in a naturally occurring hemoglobin, Hb Aubenas. Heterozygotes have normal red cell indices and are phenotypically normal. We electroporated the latest generation of ABE8 editors (ABE8e, ABE8.13 and ABE8 V106W) as mRNA into WT CD34+ hematopoietic stem and progenitor cells (HSPCs) with sgRNAs targeting the middle A of the WT GAG codon. These had similar editing efficiencies although ABE8 V106W had marginally higher on-target efficiency. V106W has been evolved to have a favourable off-target profile. V106W mRNA/sgRNA was electroporated into 3 different severe HbE/β-thalassemia donor HSPCs. The HbE codon was converted to WT with a mean 28.7% efficiency, to Hb Aubenas 48.6% and to an undescribed AGG codon 2.1%. The mean conversion from HbE to a normal or normal variant was 78.7±8.7% (Figure 1B). The indel rate from inadvertent on-target Cas9 cleavage was below 0.5%. Edited cells did not show any perturbations in erythroid differentiation as assessed by Immunophenotyping and cellular morphology. In differentiated erythroid cells, RT-qPCR showed a mean fall in the α/β mRNA ratio to 0.65±0.08 (unedited patient cells normalised to 1, n=5, Figure 1C), indicating a reduction in the relative excess α-globin gene expression. Protein analysis by CE-HPLC showed a 3.6-fold reduction in HbE levels (SD±1.3) and a 13.5-fold increase in HbA/Hb Aubenas (SD±2.4, Figure 1C and D). To prove that base editing using mRNA was possible in long-term HSCs, CD34+ cells from 4 WT cord blood donors were edited using ABEmax. Mice were culled after 16 weeks, and human cells were collected and transplanted into 7 secondary mice, which were also culled after 16 weeks. Each secondary mouse showed the presence of hCD45+ cells, indicating engraftment of LT-HSCs. All secondary replicates showed editing, with a mean editing efficiency of 34.5% (initial editing 46.3%). In both rounds of mice, there was robust lymphoid and myeloid engraftment and expected levels of erythroid engraftment for the NSG model in bone marrow and spleen. Potential off-target effects were assessed in-vitro by CIRCLE-seq in triplicate. These sites were assessed by targeted oligonucleotide capture of DNA from mRNA edited patient cells to detect in-vivo editing. Together these data provide robust evidence for base editing as an effective and safe therapeutic strategy for HbE/β-thalassemia. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Distinct Morphological and Behavioural Alterations in ENU-Induced Heterozygous Trpc7K810Stop Mutant Mice

Trpc7 (transient receptor potential cation channel, subfamily C, member 7; 862 amino acids) knockout mice are described showing no clear phenotypic alterations, therefore, the functional relevance of the gene remains unclear. A complementary approach for the functional analysis of a given gene is the examination of individuals harbouring a mutant allele of the gene. In the phenotype-driven Munich ENU mouse mutagenesis project, a high number of phenotypic parameters was used for establishing novel mouse models on the genetic background of C3H inbred mice. The phenotypically dominant mutant line SMA002 was established and further examined. Analysis of the causative mutation as well as the phenotypic characterization of the mutant line were carried out. The causative mutation was detected in the gene Trpc7 which leads to the production of a truncated protein due to the novel stop codon at amino acid position 810 thereby affecting the highly conserved cytoplasmic C terminus of the protein. Trpc7 heterozygous mutant mice of both sexes were viable and fertile, but showed distinct morphological and behavioural alterations which is in contrast to the published phenotype of Trpc7 knockout mice. Thus, the Trpc7K810Stop mutation leads to a dominant negative effect of the mutant protein.

A human multi-lineage hepatic organoid model for liver fibrosis

To investigate the pathogenesis of a congenital form of hepatic fibrosis, human hepatic organoids were engineered to express the most common causative mutation for Autosomal Recessive Polycystic Kidney Disease (ARPKD). Here we show that these hepatic organoids develop the key features of ARPKD liver pathology (abnormal bile ducts and fibrosis) in only 21 days. The ARPKD mutation increases collagen abundance and thick collagen fiber production in hepatic organoids, which mirrors ARPKD liver tissue pathology. Transcriptomic and other analyses indicate that the ARPKD mutation generates cholangiocytes with increased TGFβ pathway activation, which are actively involved stimulating myofibroblasts to form collagen fibers. There is also an expansion of collagen-producing myofibroblasts with markedly increased PDGFRB protein expression and an activated STAT3 signaling pathway. Moreover, the transcriptome of ARPKD organoid myofibroblasts resemble those present in commonly occurring forms of liver fibrosis. PDGFRB pathway involvement was confirmed by the anti-fibrotic effect observed when ARPKD organoids were treated with PDGFRB inhibitors. Besides providing insight into the pathogenesis of congenital (and possibly acquired) forms of liver fibrosis, ARPKD organoids could also be used to test the anti-fibrotic efficacy of potential anti-fibrotic therapies.

Warmblood fragile foal syndrome causative single nucleotide polymorphism frequency in horses in Ireland

Background Warmblood Fragile Foal Syndrome (WFFS) is an autosomal recessive disorder caused by a mutation in the procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1) gene. Homozygosity for the mutation results in defective collagen synthesis which clinically manifests as the birth of non viable or still born foals with abnormally fragile skin. While the mutation has been identified in non Warmblood breeds including the Thoroughbred, to date all homozygous clinically affected cases reported in the scientific literature are Warmblood foals. The objective of this study was to investigate the carrier frequency of the mutation in the Thoroughbred and sport horse populations in Ireland. Methods A test was developed at the UCD School of Veterinary Medicine using real-time PCR to amplify the PLOD1 gene c.2032G > A variant. A subset of the samples was also submitted to an external laboratory with a licensed commercial WFFS genetic test. Results Warmblood Fragile Foal Syndrome genotyping was performed on hair samples from 469 horses representing 6 different breeds. Six of 303 (1.98%) sport horses tested and three of 109 (2.75%) Thoroughbreds tested were heterozygous for the WFFS polymorphism (N/WFFS). The WFFS polymorphism was not identified in the Standardbred, Cob, Connemara, or other pony breeds. Conclusions The study identified a low frequency of the WFFS causative mutation in sport horses and Thoroughbreds in Ireland, highlighting the importance of WFFS genetic testing in order to identify phenotypically normal heterozygous carriers and to prevent the birth of nonviable foals.