Skewed X-chromosome inactivation drives the proportion of DNAAF6-defective airway motile cilia and variable expressivity in primary ciliary dyskinesia.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare airway disorder caused by defective motile cilia. Only male patients have been reported with pathogenic mutations in X-linked DNAAF6, which result in the absence of ciliary dynein arms, whereas their heterozygous mothers are supposedly healthy. Our objective was to assess the possible clinical and ciliary consequences of X-chromosome inactivation (XCI) in these mothers. METHODS: XCI patterns of six mothers of male patients with DNAAF6-related PCD were determined by DNA-methylation studies and compared with their clinical phenotype (6/6 mothers), as well as their ciliary phenotype (4/6 mothers), as assessed by immunofluorescence and high-speed videomicroscopy analyses. The mutated X chromosome was tracked to assess the percentage of cells with a normal inactivated DNAAF6 allele. RESULTS: The mothers' phenotypes ranged from absence of symptoms to mild/moderate or severe airway phenotypes, closely reflecting their XCI pattern. Analyses of the symptomatic mothers' airway ciliated cells revealed the coexistence of normal cells and cells with immotile cilia lacking dynein arms, whose ratio closely mirrored their XCI pattern. CONCLUSION: This study highlights the importance of searching for heterozygous pathogenic DNAAF6 mutations in all female relatives of male PCD patients with a DNAAF6 defect, as well as in females consulting for mild chronic respiratory symptoms. Our results also demonstrate that about one-third-ranging from 20% to 50%-normal ciliated airway cells sufficed to avoid severe PCD, a result paving the way for gene therapy.

High risk of lung cancer in surfactant-related gene variant carriers.

BACKGROUND: Several rare surfactant-related gene (SRG) variants associated with interstitial lung disease are suspected to be associated with lung cancer, but data are missing. We aimed to study the epidemiology and phenotype of lung cancer in an international cohort of SRG variant carriers. METHODS: We conducted a cross-sectional study of all adults with SRG variants in the OrphaLung network and compared lung cancer risk with telomere-related gene (TRG) variant carriers. RESULTS: We identified 99 SRG adult variant carriers (SFTPA1 (n=18), SFTPA2 (n=31), SFTPC (n=24), ABCA3 (n=14) and NKX2-1 (n=12)), including 20 (20.2%) with lung cancer (SFTPA1 (n=7), SFTPA2 (n=8), SFTPC (n=3), NKX2-1 (n=2) and ABCA3 (n=0)). Among SRG variant carriers, the odds of lung cancer was associated with age (OR 1.04, 95% CI 1.01-1.08), smoking (OR 20.7, 95% CI 6.60-76.2) and SFTPA1/SFTPA2 variants (OR 3.97, 95% CI 1.39-13.2). Adenocarcinoma was the only histological type reported, with programmed death ligand-1 expression ≥1% in tumour cells in three samples. Cancer staging was localised (I/II) in eight (40%) individuals, locally advanced (III) in two (10%) and metastatic (IV) in 10 (50%). We found no somatic variant eligible for targeted therapy. Seven cancers were surgically removed, 10 received systemic therapy, and three received the best supportive care according to their stage and performance status. The median overall survival was 24 months, with stage I/II cancers showing better survival. We identified 233 TRG variant carriers. The comparative risk (subdistribution hazard ratio) for lung cancer in SRG patients versus TRG patients was 18.1 (95% CI 7.1-44.7). CONCLUSIONS: The high risk of lung cancer among SRG variant carriers suggests specific screening and diagnostic and therapeutic challenges. The benefit of regular computed tomography scan follow-up should be evaluated.

Similarities and differences of interstitial lung disease associated with pathogenic variants in SFTPC and ABCA3 in adults.

BACKGROUND AND OBJECTIVE: Variants in surfactant genes SFTPC or ABCA3 are responsible for interstitial lung disease (ILD) in children and adults, with few studies in adults. METHODS: We conducted a multicentre retrospective study of all consecutive adult patients diagnosed with ILD associated with variants in SFTPC or ABCA3 in the French rare pulmonary diseases network, OrphaLung. Variants and chest computed tomography (CT) features were centrally reviewed. RESULTS: We included 36 patients (median age: 34 years, 20 males), 22 in the SFTPC group and 14 in the ABCA3 group. Clinical characteristics were similar between groups. Baseline median FVC was 59% ([52-72]) and DLco was 44% ([35-50]). An unclassifiable pattern of fibrosing ILD was the most frequent on chest CT, found in 85% of patients, however with a distinct phenotype with ground-glass opacities and/or cysts. Nonspecific interstitial pneumonia and usual interstitial pneumonia were the most common histological patterns in the ABCA3 group and in the SFTPC group, respectively. Annually, FVC and DLCO declined by 1.87% and 2.43% in the SFTPC group, respectively, and by 0.72% and 0.95% in the ABCA3 group, respectively (FVC, p = 0.014 and DLCO , p = 0.004 for comparison between groups). Median time to death or lung transplantation was 10 years in the SFTPC group and was not reached at the end of follow-up in the ABCA3 group. CONCLUSION: SFTPC and ABCA3-associated ILD present with a distinct phenotype and prognosis. A radiologic pattern of fibrosing ILD with ground-glass opacities and/or cysts is frequently found in these rare conditions.

TTC12 Loss-of-Function Mutations Cause Primary Ciliary Dyskinesia and Unveil Distinct Dynein Assembly Mechanisms in Motile Cilia Versus Flagella.

Cilia and flagella are evolutionarily conserved organelles whose motility relies on the outer and inner dynein arm complexes (ODAs and IDAs). Defects in ODAs and IDAs result in primary ciliary dyskinesia (PCD), a disease characterized by recurrent airway infections and male infertility. PCD mutations in assembly factors have been shown to cause a combined ODA-IDA defect, affecting both cilia and flagella. We identified four loss-of-function mutations in TTC12, which encodes a cytoplasmic protein, in four independent families in which affected individuals displayed a peculiar PCD phenotype characterized by the absence of ODAs and IDAs in sperm flagella, contrasting with the absence of only IDAs in respiratory cilia. Analyses of both primary cells from individuals carrying TTC12 mutations and human differentiated airway cells invalidated for TTC12 by a CRISPR-Cas9 approach revealed an IDA defect restricted to a subset of single-headed IDAs that are different in flagella and cilia, whereas TTC12 depletion in the ciliate Paramecium tetraurelia recapitulated the sperm phenotype. Overall, our study, which identifies TTC12 as a gene involved in PCD, unveils distinct dynein assembly mechanisms in human motile cilia versus flagella.

RNF213-associated urticarial lesions with hypercytokinemia.

BACKGROUND: Urticarial lesions are observed in both cutaneous and systemic disorders. Familial forms of urticarial syndromes are rare and can be encountered in systemic autoinflammatory diseases. OBJECTIVE: We sought to investigate a large family with dominantly inherited chronic urticarial lesions associated with hypercytokinemia. METHODS: We performed a genetic linkage analysis in 14 patients from a 5-generation family, as well as whole-exome sequencing, cytokine profiling, and transcriptomic analyses on samples from 2 patients. The identified candidate protein was studied after in vitro expression of the corresponding normal and mutated recombinant proteins. An unsupervised proteomic approach was used to unveil the associated protein network. RESULTS: The disease phenotype of the most affected family members is characterized by chronic urticarial flares associated with extremely high plasma levels of proinflammatory (IL-1ß, IL-6, and TNF-α) and anti-inflammatory (IL-10 and IL-1 receptor antagonist [IL-1RA]) cytokines, with no secondary organ dysfunction, no susceptibility to infections, no fever, and normal C-reactive protein levels. Monocyte transcriptomic analyses identified an immunotolerant profile in the most affected patient. The affected family members carried a loss-of-function mutation in RNF213 that encodes mysterin, a protein with a poorly known physiologic role. We identified the deubiquitinase CYLD, a major regulator of inflammation, as an RNF213 partner and showed that CYLD expression is inhibited by wild-type but not mutant RNF213. CONCLUSION: We identified a new entity characterized by chronic urticarial lesions associated with a clinically blunted hypercytokinemia. This disease, which is due to loss of function of RNF213, reveals mysterin's key role in the complex molecular network of innate immunity.

Lack of GAS2L2 Causes PCD by Impairing Cilia Orientation and Mucociliary Clearance.

Primary ciliary dyskinesia (PCD) is a genetic disorder in which impaired ciliary function leads to chronic airway disease. Exome sequencing of a PCD subject identified an apparent homozygous frameshift variant, c.887_890delTAAG (p.Val296Glyfs∗13), in exon 5; this frameshift introduces a stop codon in amino acid 308 of the growth arrest-specific protein 2-like 2 (GAS2L2). Further genetic screening of unrelated PCD subjects identified a second proband with a compound heterozygous variant carrying the identical frameshift variant and a large deletion (c.867_∗343+1207del; p.?) starting in exon 5. Both individuals had clinical features of PCD but normal ciliary axoneme structure. In this research, using human nasal cells, mouse models, and X.laevis embryos, we show that GAS2L2 is abundant at the apical surface of ciliated cells, where it localizes with basal bodies, basal feet, rootlets, and actin filaments. Cultured GAS2L2-deficient nasal epithelial cells from one of the affected individuals showed defects in ciliary orientation and had an asynchronous and hyperkinetic (GAS2L2-deficient = 19.8 Hz versus control = 15.8 Hz) ciliary-beat pattern. These results were recapitulated in Gas2l2-/- mouse tracheal epithelial cell (mTEC) cultures and in X. laevis embryos treated with Gas2l2 morpholinos. In mice, the absence of Gas2l2 caused neonatal death, and the conditional deletion of Gas2l2 impaired mucociliary clearance (MCC) and led to mucus accumulation. These results show that a pathogenic variant in GAS2L2 causes a genetic defect in ciliary orientation and impairs MCC and results in PCD.

Mutations in DNAH17, Encoding a Sperm-Specific Axonemal Outer Dynein Arm Heavy Chain, Cause Isolated Male Infertility Due to Asthenozoospermia.

Motile cilia and sperm flagella share an evolutionarily conserved axonemal structure. Their structural and/or functional defects are associated with primary ciliary dyskinesia (PCD), a genetic disease characterized by chronic respiratory-tract infections and in which most males are infertile due to asthenozoospermia. Among the well-characterized axonemal protein complexes, the outer dynein arms (ODAs), through ATPase activity of their heavy chains (HCs), play a major role for cilia and flagella beating. However, the contribution of the different HCs (γ-type: DNAH5 and DNAH8 and ß-type: DNAH9, DNAH11, and DNAH17) in ODAs from both organelles is unknown. By analyzing five male individuals who consulted for isolated infertility and displayed a loss of ODAs in their sperm cells but not in their respiratory cells, we identified bi-allelic mutations in DNAH17. The isolated infertility phenotype prompted us to compare the protein composition of ODAs in the sperm and ciliary axonemes from control individuals. We show that DNAH17 and DNAH8, but not DNAH5, DNAH9, or DNAH11, colocalize with α-tubulin along the sperm axoneme, whereas the reverse picture is observed in respiratory cilia, thus explaining the phenotype restricted to sperm cells. We also demonstrate the loss of function associated with DNAH17 mutations in two unrelated individuals by performing immunoblot and immunofluorescence analyses on sperm cells; these analyses indicated the absence of DNAH17 and DNAH8, whereas DNAH2 and DNALI, two inner dynein arm components, were present. Overall, this study demonstrates that mutations in DNAH17 are responsible for isolated male infertility and provides information regarding ODA composition in human spermatozoa.

Combining RSPH9 founder mutation screening and next-generation sequencing analysis is efficient for primary ciliary dyskinesia diagnosis in Saudi patients.

Primary ciliary dyskinesia (PCD) is a clinically and genetically heterogeneous ciliopathy. Dysfunction of motile respiratory and nodal cilia results in sinopulmonary symptoms associated with laterality defects (LD) found in half of the patients. The molecular basis of the disease is insufficiently investigated in patients originating from the Arabian Peninsula. In a group of 16 unrelated Saudi patients clinically suspected of PCD and among whom only 5 (31%) had LD, we first screened by PCR-RFLP two founder mutations, RSPH9 c.804_806del and CCDC39 c.2190del previously identified in patients from the Arabian Peninsula and Tunisia, respectively. When negative, targeted panel or whole-exome sequencing was performed. Three patients were homozygous for the mutation in RSPH9, which encodes an axonemal protein that is absent from nodal cilia. None of the patients carried the CCDC39 founder mutation frequent in Tunisia. NGS analysis showed that nine patients had homozygous mutations in PCD genes. In total, sequential RFLP and NGS analysis solved 75% (12/16) of cases and identified ten distinct mutations, among which six are novel, in nine different genes. These results, which highlight the genetic heterogeneity of PCD in Saudi Arabia, show that the RSPH9 c.804_806del mutation is a prevalent mutation among Saudi patients, whereas the CCDC39 c.2190del ancestral allele is most likely related to the Berber population. This study shows that RSPH9 founder mutation first-line screening and NGS analysis is efficient for the genetic exploration of PCD in Saudi patients. The RSPH9 founder mutation accounts for the low rate of LD among Saudi patients.

Mosaic variants in TNFRSF1A: an emerging cause of tumour necrosis factor receptor-associated periodic syndrome.

OBJECTIVE: To identify the molecular basis of a systemic autoinflammatory disorder (SAID) evocative of TNF receptor-associated periodic syndrome (TRAPS). METHODS: (i) Deep next generation sequencing (NGS) through a SAID gene panel; (ii) variant allele distribution in peripheral blood subpopulations; (iii) in silico analyses of mosaic variants using TNF receptor superfamily 1A (TNFRSF1A) crystal structure; (iv) review of the very rare TNFRSF1A mosaic variants reported previously. RESULTS: In a 36-year-old man suffering from recurrent fever for 12 years, high-depth NGS revealed a TNFRSF1A mosaic variant, c.176G>A p.(Cys59Tyr), which Sanger sequencing failed to detect. This mosaic variant displayed a variant allele fraction of 14% in whole blood; it affects both myeloid and lymphoid lineages. p.(Cys59Tyr), a recurrent germline pathogenic variant, affects a crucial cysteine located in the first cysteine-rich domain (CRD1) and involved in a disulphide bridge. Introduction of a tyrosine at this position is expected to disrupt the CRD1 structure. Review of the three previously reported TNFRSF1A mosaic variants revealed that they are all located in a small region of CRD2 and that germinal cells can be affected. CONCLUSION: This study expands the localization of TNFRSF1A mosaic variants to the CRD1 domain. Noticeably, residues involved in germline TNFRSF1A mutational hot spots can also be involved in post-zygotic mutational events. Including our study, only four patients have been thus far reported with TNFRSF1A mosaicism, highlighting the need for a high-depth NGS-based approach to avoid the misdiagnosis of TRAPS. Genetic counselling has to consider the potential occurrence of TNFRSF1A mosaic variants in germinal cells.

Impact of Gender on the Characteristics of Patients with Idiopathic Pulmonary Fibrosis Included in the RaDiCo-ILD Cohort.

BACKGROUND: There is growing evidence of gender-specific phenotypic differences among patients with idiopathic pulmonary fibrosis (IPF), which may affect patient outcomes. OBJECTIVES: We present the characteristics of patients with IPF at inclusion in the French Rare Disease Cohort - Interstitial Lung Disease (RaDiCo-ILD) with the aim of characterizing gender-specific phenotypic differences. METHODS: Patients with IPF who were enrolled in the national, multicentre RaDiCo-ILD cohort were included. Demographic characteristics, comorbidities, health-related quality of life (HRQoL) scores, pulmonary function, chest imaging, and IPF treatment were collected at inclusion and described by gender. RESULTS: The cohort included 724 patients with IPF (54% of RaDiCo-ILD cohort), of whom 82.9% were male. The proportion of male and female patients with a prior history of smoking was 75.0% and 26.8%, respectively. Emphysema was present in 17.0% (95% confidence interval [CI]: 10.0, 24.0) of men and 5.4% (95% CI: 1.2, 9.6) of women. At inclusion, females had poorer HRQoL than males based on St. George's Respiratory Questionnaire scores (48.5 [95% CI: 43.9, 53.0] and 41.5 [39.4, 43.6], respectively). The mean forced vital capacity per cent predicted was 77.7% (95% CI: 76.2, 79.3) and 87.4% (83.4, 91.4) for males and females, respectively. Honeycombing on high-resolution computed tomography (HRCT) was present in 70.8% (95% CI: 61.0, 80.6) of males and 45.8% (95% CI: 35.1, 56.5) of females. CONCLUSIONS: This analysis of patients with IPF at inclusion in the RaDiCo-ILD cohort provides evidence that comorbid emphysema, lung volume reduction, and honeycombing on HRCT are more common characteristics of males than females.

Mutations in Outer Dynein Arm Heavy Chain DNAH9 Cause Motile Cilia Defects and Situs Inversus.

Motile cilia move body fluids and gametes and the beating of cilia lining the airway epithelial surfaces ensures that they are kept clear and protected from inhaled pathogens and consequent respiratory infections. Dynein motor proteins provide mechanical force for cilia beating. Dynein mutations are a common cause of primary ciliary dyskinesia (PCD), an inherited condition characterized by deficient mucociliary clearance and chronic respiratory disease coupled with laterality disturbances and subfertility. Using next-generation sequencing, we detected mutations in the ciliary outer dynein arm (ODA) heavy chain gene DNAH9 in individuals from PCD clinics with situs inversus and in one case male infertility. DNAH9 and its partner heavy chain DNAH5 localize to type 2 ODAs of the distal cilium and in DNAH9-mutated nasal respiratory epithelial cilia we found a loss of DNAH9/DNAH5-containing type 2 ODAs that was restricted to the distal cilia region. This confers a reduced beating frequency with a subtle beating pattern defect affecting the motility of the distal cilia portion. 3D electron tomography ultrastructural studies confirmed regional loss of ODAs from the distal cilium, manifesting as either loss of whole ODA or partial loss of ODA volume. Paramecium DNAH9 knockdown confirms an evolutionarily conserved function for DNAH9 in cilia motility and ODA stability. We find that DNAH9 is widely expressed in the airways, despite DNAH9 mutations appearing to confer symptoms restricted to the upper respiratory tract. In summary, DNAH9 mutations reduce cilia function but some respiratory mucociliary clearance potential may be retained, widening the PCD disease spectrum.

MUC5B Promoter Variant and Rheumatoid Arthritis with Interstitial Lung Disease.

BACKGROUND: Given the phenotypic similarities between rheumatoid arthritis (RA)-associated interstitial lung disease (ILD) (hereafter, RA-ILD) and idiopathic pulmonary fibrosis, we hypothesized that the strongest risk factor for the development of idiopathic pulmonary fibrosis, the gain-of-function MUC5B promoter variant rs35705950, would also contribute to the risk of ILD among patients with RA. METHODS: Using a discovery population and multiple validation populations, we tested the association of the MUC5B promoter variant rs35705950 in 620 patients with RA-ILD, 614 patients with RA without ILD, and 5448 unaffected controls. RESULTS: Analysis of the discovery population revealed an association of the minor allele of the MUC5B promoter variant with RA-ILD when patients with RA-ILD were compared with unaffected controls (adjusted odds ratio, 3.8; 95% confidence interval [CI], 2.8 to 5.2; P=9.7×10-17). The MUC5B promoter variant was also significantly overrepresented among patients with RA-ILD, as compared with unaffected controls, in an analysis of the multiethnic case series (adjusted odds ratio, 5.5; 95% CI, 4.2 to 7.3; P=4.7×10-35) and in a combined analysis of the discovery population and the multiethnic case series (adjusted odds ratio, 4.7; 95% CI, 3.9 to 5.8; P=1.3×10-49). In addition, the MUC5B promoter variant was associated with an increased risk of ILD among patients with RA (adjusted odds ratio in combined analysis, 3.1; 95% CI, 1.8 to 5.4; P=7.4×10-5), particularly among those with evidence of usual interstitial pneumonia on high-resolution computed tomography (adjusted odds ratio in combined analysis, 6.1; 95% CI, 2.9 to 13.1; P=2.5×10-6). However, no significant association with the MUC5B promoter variant was observed for the diagnosis of RA alone. CONCLUSIONS: We found that the MUC5B promoter variant was associated with RA-ILD and more specifically associated with evidence of usual interstitial pneumonia on imaging. (Funded by Société Française de Rhumatologie and others.).

Tumour necrosis factor receptor-1 associated periodic syndrome (TRAPS)-related AA amyloidosis: a national case series and systematic review.

OBJECTIVES: TNF receptor-1-associated periodic syndrome (TRAPS) is a rare autosomal dominant autoinflammatory disorder associated with mutations in the TNF receptor super family 1 A (TNFRSF1A) gene. AA amyloidosis (AA) is the most severe complication of TRAPS. To study the occurrence and prognosis of AA in TRAPS, we conducted a retrospective study of all French cases and a systematic literature review. METHODS: This case series includes TRAPS patients followed by our centre from 2000 to 2020 presenting with histologically confirmed AA. We conducted a systematic literature review on the PubMed and EMBASE databases for articles published up to February 2021 following the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines and using the keywords: amyloidoisis, amyloid, TNF receptor-associated periodic syndrome, TNF receptor-associated periodic syndrome, tumor necrosis factor receptor-associated periodic syndrome, TRAPS, TNFRSF1A, familial hibernian fever and hibernian familial fever. RESULTS: A total of 41 TRAPS with AA were studied: three new patients and 38 cases from the literature. AA diagnosis preceded that of TRAPS in 96% of cases, and 17/36 (47%) required renal replacement therapy. Death occurred in 5/36 (14%) with a median follow-up of 23 months. Effect of biologics on AA were available for 21 regimens in 19 patients: 10 improved renal function, seven stabilized and four worsened. Four patients (36% of transplanted patients) relapse AA on kidney graft (only one under etanercept). CONCLUSION: TRAPS is revealed by AA in most cases. Therefore, clinical features of TRAPS should be screened for in AA patients. IL-1 antagonist can help to normalize inflammation and to preserve renal function.

Chronic hepatic involvement in the clinical spectrum of A20 haploinsufficiency.

BACKGROUND & AIMS: Secondary to tumour necrosis factor-alpha induced protein 3 (TNFAIP3) mutations, A20 haploinsufficiency (HA20) is a recently described autoinflammatory disease with clinical features similar to those of Behçet's and Crohn's diseases but with a constantly expanding clinical spectrum. Here, we describe HA20 liver involvement in three new patients from the same family. METHODS: We retrospectively assessed clinical, biological and/or histological findings for eight patients over three generations of the same family with heterozygous mutations in the TNFAIP3 gene (c.259C > T, p.Arg87*). RESULTS: The eight patients exhibited the following: aphthous ulcers (8/8, bipolar in 7), autoimmune features (6/8, including 5 with definitive autoimmune disease diagnoses, ie, type I diabetes, Hashimoto thyroiditis, pernicious anaemia, and/or 5 with antinuclear antibodies ≥320), pustulosis/folliculitis (5/8), abdominal pain (4/8), arthralgia (3/8), enlarged cervical lymph nodes (3/8) and pericarditis (1/8). In addition, three patients (twin sisters and their grandmother aged 23 and 70 years, respectively) exhibited persistent mild hepatic cytolysis associated with splenomegaly (n = 3), hepatomegaly (n = 1) and/or liver atrophy (n = 1) on echography. We could not detect any other causes of chronic liver diseases. Liver biopsies from three patients displayed hepatic fibrosis, hepatocyte injury and/or CD4+ /CD8+ T lymphocyte infiltration, and patterns of inflammatory cells and NLRP3 or NF-κB immunostaining differed from the predominant neutrophil infiltration observed in skin or some digestive tract biopsies. CONCLUSIONS: This study reinforces the dual involvement of innate and adaptive immunity in HA20 according to both acute and chronic injury and the organ involved and widens its clinical spectrum to include chronic hepatic involvement.

"Helicobacter pylori in familial mediterranean fever: A series of 120 patients from literature and from france".

INTRODUCTION: Familial Mediterranean Fever (FMF), the most common monogenic auto-inflammatory disease, is characterized by recurrent febrile abdominal pain. Helicobacter pylori infection (HPI), one of the most frequent infections worldwide, can mimic an FMF attack. OBJECTIVES: Identify FMF patients with HPI in a cohort of French FMF patients and the literature and identify features allowing to distinguish HPI from an FMF attack. METHODS: A retrospective study of all HPI cases was performed on the cohort of FMF patients fulfilling the Livneh criteria from the French Reference Center for rare Auto-Inflammatory Diseases and Amyloidosis (CEREMAIA). A systematic literature review of HPI in FMF patients was conducted according to the PRISMA guidelines. RESULTS: Eight French patients developed HPI, whose symptoms of epigastralgia, diarrhea, anorexia/weight loss, and nausea/vomiting differed from their typical abdominal FMF attacks. A total of 112 FMF patients with HPI have been described in the literature, including 61 adults. Diagnosis of HPI was made by gastroscopy (n = 43), labelled urea test (n = 55) or IgG serology by ELISA (n = 12). When performed, C-reactive protein was always elevated. Ten cases of interaction between colchicine and antibiotic therapy for HPI (clarithromycin (n = 9) and azithromycin (n = 1)) were reported. CONCLUSION: We described a total of 120 patients with typical FMF and HPI. When FMF patients develop atypical abdominal symptoms, upper gastrointestinal endoscopy with biopsies is essential to eliminate underlying HPI. Untreated HPI can lead to misdiagnosis of colchicine resistance with inappropriate prescription of an interleukin-1 inhibitor at a non-negligible cost.

Deep phenotyping, including quantitative ciliary beating parameters, and extensive genotyping in primary ciliary dyskinesia.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare genetic disorder resulting in abnormal ciliary motility/structure, extremely heterogeneous at genetic and ultrastructural levels. We aimed, in light of extensive genotyping, to identify specific and quantitative ciliary beating anomalies, according to the ultrastructural phenotype. METHODS: We prospectively included 75 patients with PCD exhibiting the main five ultrastructural phenotypes (n=15/group), screened all corresponding PCD genes and measured quantitative beating parameters by high-speed video-microscopy (HSV). RESULTS: Sixty-eight (91%) patients carried biallelic mutations. Combined outer/inner dynein arms (ODA/IDA) defect induces total ciliary immotility, regardless of the gene involved. ODA defect induces a residual beating with dramatically low ciliary beat frequency (CBF) related to increased recovery stroke and pause durations, especially in case of DNAI1 mutations. IDA defect with microtubular disorganisation induces a low percentage of beating cilia with decreased beating angle and, in case of CCDC39 mutations, a relatively conserved mean CBF with a high maximal CBF. Central complex defect induces nearly normal beating parameters, regardless of the gene involved, and a gyrating motion in a minority of ciliated edges, especially in case of RSPH1 mutations. PCD with normal ultrastructure exhibits heterogeneous HSV values, but mostly an increased CBF with an extremely high maximal CBF. CONCLUSION: Quantitative HSV analysis in PCD objectives beating anomalies associated with specific ciliary ultrastructures and genotypes. It represents a promising approach to guide the molecular analyses towards the best candidate gene(s) to be analysed or to assess the pathogenicity of the numerous sequence variants identified by next-generation-sequencing.

NLRP3-associated autoinflammatory diseases: Phenotypic and molecular characteristics of germline versus somatic mutations.

BACKGROUND: NLRP3-associated autoinflammatory diseases (NLRP3-AIDs) include conditions of various severities, due to germline or somatic mosaic NLRP3 mutations. OBJECTIVE: To identify mosaic- versus germline-specific NLRP3 mutations' characteristics, we reinterpreted all the mutations reported in NLRP3-AIDs and performed an in-depth study of 3 novel patients. METHODS: The pathogenicity of all reported mosaic/germline mutations was reassessed according to international recommendations and their location on the NLRP3 3-dimensional structure. Deep-targeted sequencing and NLRP3-inflammasome-activation assays were used to identify the disease-causing mutation in 3 patients. RESULTS: We identified, in 3 patients, mosaic mutations affecting the same NLRP3 amino acid (Glu569). This residue belongs to 1 of the 2 mosaic mutational hot spots that face each other in the core of the NLRP3 ATPase domain. The review of the 90 NLRP3 mutations identified in 277 patients revealed that those hot spots account for 68.5% of patients (37 of 54) with mosaic mutations. Glu569 is affected in 22% of the patients (12 of 54) with mosaic mutations and in 0.4% of patients (1 of 223) with germline mutations. Only 8 of 90 mutations were found in mosaic and germinal states. All of the germline mutations were associated with a severe phenotype. These data suggest that mutations found only in mosaic state could be incompatible with life if present in germinal state. None of the 5 most frequent germline mutations was identified in mosaic state. Mutations found only in germinal state could, therefore, be asymptomatic in mosaic state. CONCLUSIONS: The phenotypic spectrum of NLRP3-AIDs appears to be related to the germinal/mosaic status and localization of the underlying mutations.

Primary ciliary dyskinesia gene contribution in Tunisia: Identification of a major Mediterranean allele.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disease of motile cilia. Even though PCD is widely studied, North-African patients have been rarely explored. In this study, we aim at confirming the clinical diagnosis and explore the genetic spectrum of PCD in a cohort of Tunisian patients. Forty clinically diagnosed patients with PCD belonging to 34 families were recruited from Tunisian pediatric departments. In each proband, targeted capture PCD panel sequencing of the 40 PCD genes was performed. PCD panel sequencing identified bi-allelic mutations in 82% of the families in eight PCD genes. Remarkably, 23.5% of patients carried the same c.2190del CCDC39 mutation. Single nucleotide polymorphism profiling in six unrelated patients carrying this mutation has revealed a founder effect in North-African patients. This mutation is estimated to date back at least 1,400-1,750 years ago. The identification of this major allele allowed us to suggest a cost-effective genetic diagnostic strategy in North-African patients with PCD.

Functional characterization of tektin-1 in motile cilia and evidence for TEKT1 as a new candidate gene for motile ciliopathies.

A child presenting with Mainzer-Saldino syndrome (MZSDS), characterized by renal, retinal and skeletal involvements, was also diagnosed with lung infections and airway ciliary dyskinesia. These manifestations suggested dysfunction of both primary and motile cilia, respectively. Targeted exome sequencing identified biallelic mutations in WDR19, encoding an IFT-A subunit previously associated with MZSDS-related chondrodysplasia, Jeune asphyxiating thoracic dysplasia and cranioectodermal dysplasia, linked to primary cilia dysfunction, and in TEKT1 which encodes tektin-1 an uncharacterized member of the tektin family, mutations of which may cause ciliary dyskinesia. Tektin-1 localizes at the centrosome in cycling cells, at basal bodies of both primary and motile cilia and to the axoneme of motile cilia in airway cells. The identified mutations impaired these localizations. In addition, airway cells from the affected individual showed severe motility defects without major ultrastructural changes. Knockdown of tekt1 in zebrafish resulted in phenotypes consistent with a function for tektin-1 in ciliary motility, which was confirmed by live imaging. Finally, experiments in the zebrafish also revealed a synergistic effect of tekt1 and wdr19. Altogether, our data show genetic interactions between WDR19 and TEKT1 likely contributing to the overall clinical phenotype observed in the affected individual and provide strong evidence for TEKT1 as a new candidate gene for primary ciliary dyskinesia.

Homozygous missense mutation L673P in adenylate kinase 7 (AK7) leads to primary male infertility and multiple morphological anomalies of the flagella but not to primary ciliary dyskinesia.

Motile cilia and sperm flagella share an extremely conserved microtubule-based cytoskeleton, called the axoneme, which sustains beating and motility of both organelles. Ultra-structural and/or functional defects of this axoneme are well-known to cause primary ciliary dyskinesia (PCD), a disorder characterized by recurrent respiratory tract infections, chronic otitis media, situs inversus, male infertility and in most severe cases, hydrocephalus. Only recently, mutations in genes encoding axonemal proteins with preferential expression in the testis were identified in isolated male infertility; in those cases, individuals displayed severe asthenozoospermia due to Multiple Morphological Abnormalities of the sperm Flagella (MMAF) but not PCD features. In this study, we performed genetic investigation of two siblings presenting MMAF without any respiratory PCD features, and we report the identification of the c.2018T > G (p.Leu673Pro) transversion in AK7, encoding an adenylate kinase, expressed in ciliated tissues and testis. By performing transcript and protein analyses of biological samples from individual carrying the transversion, we demonstrate that this mutation leads to the loss of AK7 protein in sperm cells but not in respiratory ciliated cells, although both cell types carry the mutated transcript and no tissue-specific isoforms were detected. This work therefore, supports the notion that proteins shared by both cilia and sperm flagella may have specific properties and/or function in each organelle, in line with the differences in their mode of assembly and organization. Overall, this work identifies a novel genetic cause of asthenozoospermia due to MMAF and suggests that in humans, more deleterious mutations of AK7 might induce PCD.