Engineering of Structural Variants using CRISPR/Cas in Mice

Structural variations (SVs) contribute to the variability of our genome and are often associated with disease. Their study in model systems was hampered until now by labor-intensive genetic targeting procedures and multiple mouse crossing steps. Here we present the use of CRISPR/Cas for the fast (10 weeks) and efficient generation of SVs in mice. We specifically produced deletions, inversions, and also duplications at six different genomic loci ranging from 1.1 kb to 1.6 Mb with efficiencies up to 42%. After PCR-based selection, clones were successfully used to create mice via aggregation. To test the practicability of the method, we reproduced a human 500 kb disease-associated deletion and were able to recapitulate the human phenotype in mice. Furthermore, we evaluated the regulatory potential of a large genomic interval by deleting a 1.5 Mb fragment. The method presented permits rapid in vivo modeling of genomic rearrangements

CRISPR/Cas9 - eine Technology um menschliche Fehlbildungen in der Maus wiederzuspiegeln

For decades geneticist have been investigating the human genome and revealed that mutations can cause congenital disorders. The current challenge is to distinguish between pathogenic and nonpathogenic mutations to enable precise diagnostic and risk estimation as well as to determine the choice of efficient treatment. Nevertheless, the effect of coding and noncoding mutations is often not predictable. Therefore, mouse models are needed to verify their pathogenicity. For many years it was extremely time consuming to recapitulate human mutations in mice due to enormous cloning efforts. Additionally, structural variations (SVs) of several hundred kbs could not be generated. Now, CRISPR/Cas9 allows rapid genome editing by sequence specific induction of double strand breaks that facilitate structural rearrangements of more than one Mb and also the integration of point mutations. This work shows that CRISPR/Cas9 can be used in murine ESC for rapid recapitulation of human mutations and generation of viable chimeric mice with duplications, deletions and inversions of several hundred kbs or single nucleotide substitutions. Both, introducing of SVs and point mutations are marked by variable efficiency that seem to be caused by locus specific factors. CRISPR/Cas9 was used to generated six mouse models that helped to verify the pathogenicity of Laf4 truncation, Bhlha9 deletion and Shh duplication while the known pathogenicity of Bhlha9 duplication could not been proved. The effect of the duplication of the enhancer hs1428 at the Tbx15 locus and of the Tyr1096Cys mutation in FNDC3A still remains unclear. Intragenic inframe truncation of LAF4 was found in a patients presenting Nievergelt-like syndrome that is characterized by triangular shaped tibia which was recapitulated in mouse by Laf4 truncation while the Laf4-KO mouse was normal suggesting a dominant gain-offunction effect of intragenic Laf4 truncation. BHLHA9 duplications are found in many patient with SHFM or SHFLD. Nevertheless, overexpression of Bhlha9 does not result in SHFM in mouse. Even the recapitulation of a specific patient duplication did not cause any limb abnormalities. Thus, it is assumed that BHLHA9 and/or its interaction network differs between human and mouse. Contrarily, the deletion of Bhlha9 leads to syndactyly as it was already published for mouse and human. SHH duplication was found in a patient with muscle hypertrophy and in another patient with synpolydactyly. The generation of the corresponding duplication in mouse clearly shows that the tandem duplication leads to soft tissue hypertrophy and not synpolydactyly. A duplication of the enhancer hs1428 at the TBX15 locus was found in a case of radial aplasia. In mouse, the heterozygous recapitulation of the mutation did not affect skeletal development. Now, homozygous mice are needed to clarify if the mutation is pathogenic. In a consanguineous family the homozygous Tyr1096Cys mutation in FNDC3A was found to segregate with recessive SHFM. Since functional investigation could not detect any changes, a mouse models was produced that will help to reveal the effect on limb development. The unsuccessful generation of SHFM in mouse by Bhlha9 duplication shows that mouse models do not always recapitulate human physiology and developmental process. Since only disorders with potential regulative pathomechanism could not be reproduced it is likely that inter species difference on the regulative level prevents the emergence of the expected phenotype. Consequently, mouse model can prove the pathogenicity of a mutation but never its insignificance.Durch Jahrzehntelange Forschung am menschlichen Genom ist inzwischen bekannt, dass Mutationen potentiell Krankheiten verursachen. Für die humangenetische Diagnostik ist es essentiell pathogene von nicht-pathogenen Varianten zu differenzieren. Daher ist es problematisch, dass der Effekt von codierenden und nicht codierenden Mutationen nicht vorhersehbar ist. Um die Pathogenität zu verifizieren werden humane Mutationen in der Maus reproduziert. Dies war bisher jedoch sehr aufwendig und dadurch limitiert, dass Struktur Veränderungen (SVs) von mehreren kbs nicht generiert werden konnten. Das kürzlich entwickelte CRISPR/Cas9 System erlaubt nun durch sequenzspezifische Doppelstrangbrüche die exakte Generierung von großen SVs und Punktmutationen. Diese Arbeit zeigt, dass CRISPR/Cas9 die Generierung von humanen Mutationen wie Deletionen, Duplikationen, Inversionen bzw. Punktmutationen in murinen ESCs ermöglicht, aus denen chimäre Mäuse erzeugt werden. Die Effizienz der Erzeugung von SVs und Punktmutationen hängt jedoch stark von Lokus spezifischen Faktoren ab. Mit CRISPR/Cas9 wurden sechs unterschiedliche Mauslinien erzeugt und die Pathogenität der intragenen LAF4 Deletion, der BHLHA9 Deletion und der SHH Duplikation nachgewiesen. Die bereits bekannte Pathogenität der BHLHA9 Duplikation konnte das Mausmodel nicht bestätigen. Der Effekt der Tyr1096Cys Mutation in FNDC3a und der Duplikation des Enhancers hs1428 am TBX15 Locus ist immer noch unklar. Die intragene inframe-Deletion in LAF4 wurde in einem Patienten mit Nievergelt-ähnlichem Syndrom gefunden. Sowohl der Patient als auch das Mausmodel zeigen die typische dreieckige Verformung der Tibia. Da Laf4-KO keine Extremitätenveränderung verursacht, wird von einem gain-of-function Mechanismus ausgegangen. Aufgrund von zahlreichen klinischen Fällen sind BHLHA9 Duplikationen dafür bekannt SHFM bzw. SHFLD auszulösen. In der Maus haben jedoch weder die Überexpression noch die Duplikation von Bhlha9 einen Effekt auf die Extremitätenentwicklung. Daher wird vermutet, dass die Funktionalität des menschlichen BHLHA9 sich vom murinen Ortholog signifikant unterscheidet. Die Duplikation von SHH wurde in einem Patienten mit Synpolydactyly gefunden wie auch in einem Fall mit Muskelhypertrophy. Das entsprechende Mausmodel zeigt jedoch klar, dass die in- tandem Duplikation zu Hypertrophy von weichem Gewebe und nicht zu Synpolydactyly führt. Die Duplikation des Enhancer hs1428 am TBX15 Locus wurde in einem Patienten mit radialer Aplasie gefunden. Die gleiche heterozygote Mutation hat in der Maus keinen Effekt. Für eine endgültige Beurteilung werden jedoch homozygote Tiere benötigt. Die Missense Mutation Tyr1096Cys in FNDC3A segregiert innerhalb einer konsanguinen Familien mit rezessivem SHFM. Da funktionelle Untersuchungen nicht ausreichten, um die Pathogenität der Mutation zu beurteilen, wurde sie im Mausmodel nachgestellt. Es lässt sich jedoch noch keine abschließende Aussage treffen, weil die Tiere noch zur Homozygotie gezüchtet werden müssen. Obwohl bekannt ist, dass BHLHA9 Duplikationen SHFM erzeugen, konnte dies in der Maus nicht nachgewiesen werden. Dies verdeutlicht, dass die Maus die humane Physiologie und Entwicklung nicht perfekt wiederspiegelt. Da vor allem Mutationen mit einem potentiell regulativen Pathomechanismus in der Maus nicht das menschliche Krankheitsbild reproduzierten, wird angenommen, dass dies vor allem durch Spezies bedingte Unterschiede in der Genregulation verursacht wurde. Folglich ist die Maus ein gutes Model um die Pathogenität von Mutationen nachzuweisen, jedoch nicht um selbige auszuschließen

Association of the Recurrent Rare Variant c.415T>C p.Phe139Leu in CLN5 with a Recessively Inherited Macular Dystrophy

Importance: Homozygous variants in the neuronal ceroid lipofuscinosis type 5 (CLN5) gene are associated with neuronal ceroid lipofuscinosis, a progressive neurologic disorder that leads to ataxia, seizures, and early death. The association between a homozygous variant in this gene and a macular dystrophy is described here. Objective: To describe an autosomal recessive macular dystrophy associated with a recurrent variant in CLN5. Design, Setting, and Participants: This cohort study took place at a national referral center and had a follow-up duration ranging between 1 and 5 years. All patients who were identified to carry a specific homozygous missense variant in CLN5, among more than 2000 patients who were diagnosed with or suspected to have retinal dystrophies, who did not carry this variant, were included. Data were collected between June 2014 and September 2020. Exposures: All patients who were sampled for DNA analysis due to molecularly unconfirmed retinal dystrophy and who were subsequently identified to carry the homozygous missense variant c.415T>C (p.Phe139Leu) in CLN5 were included, while patients who did not carry the variant were excluded. Main Outcomes and Measures: Retinal phenotype associated with this specific homozygous missense variant in CLN5. Results: Seven affected patients (mean [SD] age, 43 [18] years; age range, 33-52 years; 5 male) carried the homozygous missense in CLN5. All patients were diagnosed as having a macular dystrophy. Four patients had mild electroretinographic alterations. All patients had hypoautofluorescent maculas with retinal thinning (central subfield thickness, 80 µm). Visual acuity ranged between 2/200 and 20/100. Neurologic symptoms were mild (dizziness) in 5 patients and absent in 2 patients. Neuroimaging demonstrated cerebellar atrophy and white matter lesions, respectively, in 2 patients. Conclusions and Relevance: These results suggest that CLN5, similar to CLN7, may be associated with isolated macular dystrophy as well as neuronal ceroid lipofuscinosis. The variant c.415T>C p.Phe139Leu does not seem to be associated with any prominent neurologic disease at least until the fourth to sixth decades of life. These findings may imply a specific role of CLN5 in macular neurons. Additional study is suggested, such as molecular screening for this variant in cohorts of patients with undiagnosed macular dystrophies and biological studies of its molecular effects.

ECM alterations in fndc3a (fibronectin domain containing protein 3A) deficient zebrafish cause temporal fin development and regeneration defects

Fin development and regeneration are complex biological processes that are highly relevant in teleost fish. They share genetic factors, signaling pathways and cellular properties to coordinate formation of regularly shaped extremities. Especially correct tissue structure defined by extracellular matrix (ECM) formation is essential. Gene expression and protein localization studies demonstrated expression of fndc3a (fibronectin domain containing protein 3a) in both developing and regenerating caudal fins of zebrafish (Danio rerio). We established a hypomorphic fndc3a mutant line (fndc3a$^{wue1/wue1}$) via CRISPR/Cas9, exhibiting phenotypic malformations and changed gene expression patterns during early stages of median fin fold development. These developmental effects are mostly temporary, but result in a fraction of adults with permanent tail fin deformations. In addition, caudal fin regeneration in adult fndc3a$^{wue1/wue1}$ mutants is hampered by interference with actinotrichia formation and epidermal cell organization. Investigation of the ECM implies that loss of epidermal tissue structure is a common cause for both of the observed defects. Our results thereby provide a molecular link between these developmental processes and foreshadow Fndc3a as a novel temporal regulator of epidermal cell properties during extremity development and regeneration in zebrafish

Squalene synthase deficiency: clinical, biochemical, and molecular characterization of a defect in cholesterol biosynthesis

Mendelian disorders of cholesterol biosynthesis typically result in multi-system clinical phenotypes, underlining the importance of\ua0cholesterol in embryogenesis and development. FDFT1 encodes for an evolutionarily conserved enzyme, squalene synthase (SS, farnesyl-pyrophosphate farnesyl-transferase 1), which catalyzes the first committed step in cholesterol biosynthesis. We report three individuals with profound developmental delay, brain abnormalities, 2-3 syndactyly of the toes, and facial dysmorphisms, resembling Smith-Lemli-Opitz syndrome, the most common cholesterol biogenesis defect. The metabolite profile in plasma and\ua0urine suggested that their defect was at the level of squalene synthase. Whole-exome sequencing was used to identify recessive disease-causing variants in FDFT1. Functional characterization of one variant demonstrated a partial splicing defect and altered promoter and/or enhancer activity, reflecting essential mechanisms for regulating cholesterol biosynthesis/uptake in steady\ua0state

Noncoding copy-number variations are associated with congenital limb malformation

PurposeCopy-number variants (CNVs) are generally interpreted by linking the effects of gene dosage with phenotypes. The clinical interpretation of noncoding CNVs remains challenging. We investigated the percentage of disease-associated CNVs in patients with congenital limb malformations that affect noncoding cis-regulatory sequences versus genes sensitive to gene dosage effects.MethodsWe applied high-resolution copy-number analysis to 340 unrelated individuals with isolated limb malformation. To investigate novel candidate CNVs, we re-engineered human CNVs in mice using clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing.ResultsOf the individuals studied, 10% harbored CNVs segregating with the phenotype in the affected families. We identified 31 CNVs previously associated with congenital limb malformations and four novel candidate CNVs. Most of the disease-associated CNVs (57%) affected the noncoding cis-regulatory genome, while only 43% included a known disease gene and were likely to result from gene dosage effects. In transgenic mice harboring four novel candidate CNVs, we observed altered gene expression in all cases, indicating that the CNVs had a regulatory effect either by changing the enhancer dosage or altering the topological associating domain architecture of the genome.ConclusionOur findings suggest that CNVs affecting noncoding regulatory elements are a major cause of congenital limb malformations

De Novo Truncating Mutations in the Last and Penultimate Exons of PPM1D Cause an Intellectual Disability Syndrome

Contains fulltext : 174535.pdf (publisher's version ) (Closed access)Intellectual disability (ID) is a highly heterogeneous disorder involving at least 600 genes, yet a genetic diagnosis remains elusive in approximately 35%-40% of individuals with moderate to severe ID. Recent meta-analyses statistically analyzing de novo mutations in >7,000 individuals with neurodevelopmental disorders highlighted mutations in PPM1D as a possible cause of ID. PPM1D is a type 2C phosphatase that functions as a negative regulator of cellular stress-response pathways by mediating a feedback loop of p38-p53 signaling, thereby contributing to growth inhibition and suppression of stress-induced apoptosis. We identified 14 individuals with mild to severe ID and/or developmental delay and de novo truncating PPM1D mutations. Additionally, deep phenotyping revealed overlapping behavioral problems (ASD, ADHD, and anxiety disorders), hypotonia, broad-based gait, facial dysmorphisms, and periods of fever and vomiting. PPM1D is expressed during fetal brain development and in the adult brain. All mutations were located in the last or penultimate exon, suggesting escape from nonsense-mediated mRNA decay. Both PPM1D expression analysis and cDNA sequencing in EBV LCLs of individuals support the presence of a stable truncated transcript, consistent with this hypothesis. Exposure of cells derived from individuals with PPM1D truncating mutations to ionizing radiation resulted in normal p53 activation, suggesting that p53 signaling is unaffected. However, a cell-growth disadvantage was observed, suggesting a possible effect on the stress-response pathway. Thus, we show that de novo truncating PPM1D mutations in the last and penultimate exons cause syndromic ID, which provides additional insight into the role of cell-cycle checkpoint genes in neurodevelopmental disorders

Variants in the degron of AFF3 are associated with intellectual disability, mesomelic dysplasia, horseshoe kidney, and epileptic encephalopathy

International audienceThe ALF transcription factor paralogs, AFF1, AFF2, AFF3, and AFF4, are components of the transcriptional super elongation complex that regulates expression of genes involved in neurogenesis and development. We describe an autosomal dominant disorder associated with de novo missense variants in the degron of AFF3, a nine amino acid sequence important for its binding to ubiquitin ligase, or with de novo deletions of this region. The sixteen affected individuals we identified, along with two previously reported individuals, present with a recognizable pattern of anomalies, which we named KINSSHIP syndrome (KI for horseshoe kidney, NS for Nievergelt/Savarirayan type of mesomelic dysplasia, S for seizures, H for hypertrichosis, I for intellectual disability, and P for pulmonary involvement), partially overlapping the AFF4-associated CHOPS syndrome. Whereas homozygous Aff3 knockout mice display skeletal anomalies, kidney defects, brain malformations, and neurological anomalies, knockin animals modeling one of the microdeletions and the most common of the missense variants identified in affected individuals presented with lower mesomelic limb deformities like KINSSHIP-affected individuals and early lethality, respectively. Overexpression of AFF3 in zebrafish resulted in body axis anomalies, providing some support for the pathological effect of increased amount of AFF3. The only partial phenotypic overlap of AFF3-and AFF4-associated syndromes and the previously published transcriptome analyses of ALF transcription factors suggest that these factors are not redundant and each contributes uniquely to proper development