Probing the Role(s) of Bbs1 with CRISPR/Cas9 Gene Editing

BBS1 is the most commonly mutated of 21 genes which cause Bardet-Biedl syndrome, a rare, autosomal recessive “ciliopathy”. It results from dysfunction of an antenna-like organelle called the primary cilium, which participates in signalling events during development and homeostasis. Bardet-Biedl syndrome is considered a model ciliopathy in that it exhibits cardinal, multi-systemic features of ciliopathies, including kidney disease, retinal degeneration, polydactyly and obesity. There is, however, a disconnect in our comprehension of how these phenotypes manifest from mutations in ciliopathy genes. The ways in which BBS1 functions within the cell remain unclear and may extend beyond the cilium. One obstacle to understanding how mutations in BBS genes lead to disease is the great phenotypic variability that exists between patients, even siblings with the same causative variants. This project probed the cellular roles of Bbs1 in mouse kidney epithelial cells using the ground-breaking gene editing technique, CRISPR/Cas9. Clonal cell lines carrying biallelic indel mutations in Bbs1 were developed and their genomic DNA, transcripts and protein characterised. Although clones revealed an inconsistent ability to form cilia, an entirely unexpected, novel cell-cell adhesion phenotype was identified in Bbs1-/-. Consistent with recent discoveries of genetic compensation following gene knockout, the severity of this phenotype correlated inversely with Bbs1 transcript instability. Transcriptomics suggested Bbs1 knockout caused a loss of epithelial identity associated with an upregulation of mesenchymal biomarkers and, in the absence of genetic compensation, the downregulation of epithelial markers, culminating in disrupted apical-basal polarity, defective cell-cell adhesion and loss of epithelial barrier integrity. Whether genetic compensation contributes to the phenotypic variability seen in Bardet-Biedl syndrome patients remains to be seen and might provide valuable opportunities for novel therapeutics. This work suggests Bbs1 may have roles beyond the cilium and the Bbs1-/- cell models generated herein will provide a useful tool for future investigations

CFAP300 mutation causing primary ciliary dyskinesia in Finland

Primary ciliary dyskinesia (PCD) is a rare genetic condition characterized by chronic respiratory tract infections and in some cases laterality defects and infertility. The symptoms of PCD are caused by malfunction of motile cilia, hair-like organelles protruding out of the cell that are responsible for removal of mucus from the airways and organizing internal organ positioning during embryonic development. PCD is caused by mutations in genes coding for structural or assembly proteins in motile cilia. Thus far mutations in over 50 genes have been identified and these variants explain around 70% of all known cases. Population specific genetics underlying PCD has been reported, thus highlighting the importance of characterizing gene variants in different populations for development of gene-based diagnostics. In this study, we identified a recurrent loss-of-function mutation c.198_200delinsCC in CFAP300 causing lack of the protein product. PCD patients homozygous for the identified CFAP300 mutation have immotile airway epithelial cilia associated with missing dynein arms in their ciliary axonemes. Furthermore, using super resolution microscopy we demonstrate that CFAP300 is transported along cilia in normal human airway epithelial cells suggesting a role for CFAP300 in dynein complex transport in addition to preassembly in the cytoplasm. Our results highlight the importance of CFAP300 in dynein arm assembly and improve diagnostics of PCD in Finland.publishedVersionPeer reviewe

De-Suppression of Mesenchymal Cell Identities and Variable Phenotypic Outcomes Associated with Knockout of Bbs1

Bardet–Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate

The Palestinian primary ciliary dyskinesia (PCD) cohort: clinical, diagnostic and genetic spectrum

Background: Diagnostic testing for PCD started in 2013 in Palestine. We aimed to describe the clinical, diagnostic and genetic spectrum of the Palestinian PCD cohort. Methods: 390 individuals with symptoms suggestive of PCD and 74 family members underwent nasal nitric oxide (nNO); and/or transmission electron microscopy (TEM); and/or PCD genetic panel or whole exome testing. Clinical characteristics were collected close to diagnosis including FEV1 GLI z-scores and BMI z-scores. Results: 82 had a definite positive PCD diagnosis (TEM and/or genetics) and 103 were highly likely (Kartagener’s and/or low nNO). Positive cases (n=82) had median age of 13.5 years (range 0-43), were highly consanguineous (95%) and 100% Arabic descent. Clinical features included persistent wet cough (95%), neonatal respiratory distress (79%), clubbing (21%) and situs inversus (41%). Lung function at diagnosis was already impaired FEV1 z-score mean -1.49 (sd=1.79) and BMI z-score mean -0.30 SD=1.4. 69 families were genotyped. 59 individuals from 42 families (60%) had mutations in 14 PCD-genes; CCDC39 (26% of families), DNAH11 (17%) and LRRC6 (12%) were the most common. 16% had mutations in candidate genes, 24% had no variants identified. 100% of variants were homozygous. TEM defects and genotype associations were as expected. Conclusions: Despite limited local resources, collaborations during the last 7-years have facilitated detailed geno- and phenotyping of one of the largest PCD cohorts globally. nNO identifies likely cases and targeted genetic testing, conducted locally, can now identify specific mutations in known families

The Palestinian primary ciliary dyskinesia population: first results of the diagnostic, and genetic spectrum

BACKGROUND: Diagnostic testing for primary ciliary dyskinesia (PCD) started in 2013 in Palestine. We aimed to describe the diagnostic, genetic and clinical spectrum of the Palestinian PCD population. METHODS: Individuals with symptoms suggestive of PCD were opportunistically considered for diagnostic testing: nasal nitric oxide (nNO) measurement, transmission electron microscopy (TEM) and/or PCD genetic panel or whole-exome testing. Clinical characteristics of those with a positive diagnosis were collected close to testing including forced expiratory volume in 1 s (FEV1) Global Lung Index z-scores and body mass index z-scores. RESULTS: 68 individuals had a definite positive PCD diagnosis, 31 confirmed by genetic and TEM results, 23 by TEM results alone, and 14 by genetic variants alone. 45 individuals from 40 families had 17 clinically actionable variants and four had variants of unknown significance in 14 PCD genes. CCDC39, DNAH11 and DNAAF11 were the most commonly mutated genes. 100% of variants were homozygous. Patients had a median age of 10.0 years at diagnosis, were highly consanguineous (93%) and 100% were of Arabic descent. Clinical features included persistent wet cough (99%), neonatal respiratory distress (84%) and situs inversus (43%). Lung function at diagnosis was already impaired (FEV1 z-score median −1.90 (−5.0–1.32)) and growth was mostly within the normal range (z-score mean −0.36 (−3.03–2.57). 19% individuals had finger clubbing. CONCLUSIONS: Despite limited local resources in Palestine, detailed geno- and phenotyping forms the basis of one of the largest national PCD populations globally. There was notable familial homozygosity within the context of significant population heterogeneity

De-Suppression of Mesenchymal Cell Identities and Variable Phenotypic Outcomes Associated with Knockout of <i>Bbs1</i>

Bardet–Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate

The Palestinian primary ciliary dyskinesia population: first results of the diagnostic and genetic spectrum

Background Diagnostic testing for primary ciliary dyskinesia (PCD) started in 2013 in Palestine. We aimed to describe the diagnostic, genetic and clinical spectrum of the Palestinian PCD population. Methods Individuals with symptoms suggestive of PCD were opportunistically considered for diagnostic testing: nasal nitric oxide (nNO) measurement, transmission electron microscopy (TEM) and/or PCD genetic panel or whole-exome testing. Clinical characteristics of those with a positive diagnosis were collected close to testing including forced expiratory volume in 1 s (FEV1) Global Lung Index z-scores and body mass index z-scores. Results 68 individuals had a definite positive PCD diagnosis, 31 confirmed by genetic and TEM results, 23 by TEM results alone, and 14 by genetic variants alone. 45 individuals from 40 families had 17 clinically actionable variants and four had variants of unknown significance in 14 PCD genes. CCDC39, DNAH11 and DNAAF11 were the most commonly mutated genes. 100% of variants were homozygous. Patients had a median age of 10.0 years at diagnosis, were highly consanguineous (93%) and 100% were of Arabic descent. Clinical features included persistent wet cough (99%), neonatal respiratory distress (84%) and situs inversus (43%). Lung function at diagnosis was already impaired (FEV1 z-score median −1.90 (−5.0–1.32)) and growth was mostly within the normal range (z-score mean −0.36 (−3.03–2.57). 19% individuals had finger clubbing. Conclusions Despite limited local resources in Palestine, detailed geno- and phenotyping forms the basis of one of the largest national PCD populations globally. There was notable familial homozygosity within the context of significant population heterogeneity

The Palestinian primary ciliary dyskinesia population: first results of the diagnostic, and genetic spectrum

Diagnostic testing for primary ciliary dyskinesia (PCD) started in 2013 in Palestine. We aimed to describe the diagnostic, genetic, and clinical spectrum of the Palestinian PCD population.Individuals with symptoms suggestive of PCD were opportunistically considered for diagnostic testing: nasal nitric oxide (nNO) measurement, transmission electron microscopy (TEM), and/or PCD genetic panel or whole exome testing. Clinical characteristics of those with a positive diagnosis were collected close to testing including FEV1 GLI z-scores, and BMI z-scores.Sixty-eight individuals had a definite positive PCD diagnosis, 31 confirmed by genetic and TEM results, 23 by TEM results alone, and 14 by genetic variants alone. Forty-five individuals from 40 families had seventeen clinically actionable variants, and 4 had variants of unknown significance in 14 PCD-genes. CCDC39, DNAH11, and DNAAF11 were the most commonly mutated genes. 100% of variants were homozygous. Patients had median age of 11.2 years at diagnosis, were highly consanguineous (93%) and 100% of Arabic descent. Clinical features included persistent wet cough (99%), neonatal respiratory distress (84%), and situs inversus (43%). Lung function at diagnosis was already impaired (FEV1 z-score median -1.90 (-5.0 to 1.32)) and growth was mostly within the normal range (z-score mean= -0.36 (-3.03 to 2.57). 19% individuals had finger clubbing. Despite limited local resources, detailed geno- and phenotyping forms the basis of one of the largest national PCD populations globally. There was notable familial homozygosity within the context of significant population heterogeneity. <br/