Ultrastructural Defects Research Articles

TU45. SYSTEMATIC ANNOTATION OF BIOLOGICAL NETWORKS ASSOCIATED WITH DEPRESSION AND OBESITY OF THE GENOMIC REGIONS COVERED IN A TARGETED SEQUENCING PANEL

Would the use of genome-wide genotyping be an advantageous strategy to identify the molecular aetiology of two brothers from a non-consanguineous family, clinically diagnosed with total globozoospermia?Two related Spanish globozoospermic patients were studied. Eight first- and second-degree family members were also included in the study. The clinical procedure included anamnesis, physical examination and semen analyses. Acrosome visualization was performed by fluorescein isothiocyanate–Pisum sativum agglutinin labelling and ultrastructural electron microscope sperm analysis. Sperm DNA fragmentation was determined by TUNEL and SCD. Molecular analysis included: the detection of deletion of the DPY19L2 gene by a BPa (break point "a") gap-polymerase chain reaction, and genotyping by using a high-throughput genome-wide genotyping platform and a genotype imputation strategy.The biological characteristics of the two globozoospermic siblings included round-headed spermatozoa without an acrosome; ultrastructural defects in spermatozoa; increased sperm fragmentation and aneuploidies, inability of spermatozoa to activate oocytes (correctable with artificial activation) and good developmental potential of embryos generated by IVF/intracytoplasmic sperm injection. This genetic study focused on a genome-wide compound heterozygote analysis that identified two deleterious rare coding variants in the DPY19L2 gene [rs771726551 (c.431T>A exon 3) and rs147579680 (c.869G>A exon 8)].A genome-wide compound heterozygote analysis strategy should be considered for molecular screening in globozoospermia and other rare congenital diseases, particularly in cases from non-consanguineous families.

Genetic diagnosis, sperm phenotype and ICSI outcome in case of severe asthenozoospermia with multiple morphological abnormalities of the flagellum.

Are ICSI outcomes impaired in cases of severe asthenozoospermia with multiple morphological abnormalities of the flagellum (MMAF phenotype)? Despite occasional technical difficulties, ICSI outcomes for couples with MMAF do not differ from those of other couples requiring ICSI, irrespective of the genetic defect. Severe asthenozoospermia, especially when associated with the MMAF phenotype, results in male infertility. Recent findings have confirmed that a genetic aetiology is frequently responsible for this phenotype. In such situations, pregnancies can be achieved using ICSI. However, few studies to date have provided detailed analyses regarding the flagellar ultrastructural defects underlying this phenotype, its genetic aetiologies, and the results of ICSI in such cases of male infertility. We performed a retrospective study of 25 infertile men exhibiting severe asthenozoospermia associated with the MMAF phenotype identified through standard semen analysis. They were recruited at an academic centre for assisted reproduction in Paris (France) between 2009 and 2017. Transmission electron microscopy (TEM) and whole exome sequencing (WES) were performed in order to determine the sperm ultrastructural phenotype and the causal mutations, respectively. Finally 20 couples with MMAF were treated by assisted reproductive technologies based on ICSI. Patients with MMAF were recruited based on reduced sperm progressive motility and increased frequencies of absent, short, coiled or irregular flagella compared with those in sperm from fertile control men. A quantitative analysis of the several ultrastructural defects was performed for the MMAF patients and for fertile men. The ICSI results obtained for 20 couples with MMAF were compared to those of 378 men with oligoasthenoteratozoospermia but no MMAF as an ICSI control group. TEM analysis and categorisation of the flagellar anomalies found in these patients provided important information regarding the structural defects underlying asthenozoospermia and sperm tail abnormalities. In particular, the absence of the central pair of axonemal microtubules was the predominant anomaly observed more frequently than in control sperm (P < 0.01). Exome sequencing, performed for 24 of the 25 patients, identified homozygous or compound heterozygous pathogenic mutations in CFAP43, CFAP44, CFAP69, DNAH1, DNAH8, AK7, TTC29 and MAATS1 in 13 patients (54.2%) (11 affecting MMAF genes and 2 affecting primary ciliary dyskinesia (PCD)-associated genes). A total of 40 ICSI cycles were undertaken for 20 MMAF couples, including 13 cycles (for 5 couples) where a hypo-osmotic swelling (HOS) test was required due to absolute asthenozoospermia. The fertilisation rate was not statistically different between the MMAF (65.7%) and the non-MMAF (66.0%) couples and it did not differ according to the genotype or the flagellar phenotype of the subjects or use of the HOS test. The clinical pregnancy rate per embryo transfer did not differ significantly between the MMAF (23.3%) and the non-MMAF (37.1%) groups. To date, 7 of the 20 MMAF couples have achieved a live birth from the ICSI attempts, with 11 babies born without any birth defects. The ICSI procedure outcomes were assessed retrospectively on a small number of affected subjects and should be confirmed on a larger cohort. Moreover, TEM analysis could not be performed for all patients due to low sperm concentrations, and WES results are not yet available for all of the included men. An early and extensive phenotypic and genetic investigation should be considered for all men requiring ICSI for severe asthenozoospermia. Although our study did not reveal any adverse ICSI outcomes associated with MMAF, we cannot rule out that some rare genetic causes could result in low fertilisation or pregnancy rates. No external funding was used for this study and there are no competing interests. N/A.

CONCURRENT ULTRASTRUCTURAL ANALYSIS AND GENOMIC ASSESSMENT OF SPERMATOZOA TO ACCURATELY DIAGNOSE RARE AND SEVERE FORMS OF MALE INFERTILITY

In men with ART failure, sperm DNA sequencing with ultrastructural analysis can identify specific sperm defects and guide ART treatment. In the past 2 years, 22 consenting men who failed IVF cycles due to severe astheno-/teratozoospermia were recruited; specimens from 3 fertile men were used as controls. To assess ultrastructure by transmission electron microscopy (TEM), pelleted spermatozoa were fixed, embedded, sectioned to 100-nm slides by ultramicrotome, and viewed with an electron microscope (JEOL-1400) at 300,000X magnification. Sperm head morphology, fibrous sheath intactness, and axonemal/periaxonemal structure were examined on at least 100 spermatozoa to diagnose specific sperm organelle defects. DNA was extracted and amplified from at least 500 spermatozoa for next-generation sequencing (NGS) to identify mutations contributing to the ultrastructural abnormalities. Four types of sperm ultrastructural defects were identified: globozoospermia, dysplasia of fibrous sheath (DFS), proximal centriole defect, and primary ciliary dyskinesia (PCD). One case of combined globozoospermia and DFS and one case of combined DFS and proximal centriole defect were identified. In 13 globozoospermic cases, 97–100% of the spermatozoa displayed complete spherical heads with the absence of acrosomes, perinuclear theca deformities, and dispersed chromatin. Microtubular arrangement of centrosome and flagellum was conserved. NGS identified deletions (DPY19L, PICK1, SPATA16) directly responsible for the globozoospermic phenotype. Based on the findings, assisted oocyte activation was performed for 7 couples with an oocyte-activating agent, boosting fertilization from 5.7% to 40.2% (p<0.00001), resulting in 5 pregnancies and 4 deliveries. In patients with DFS (n=6), flagellar deformities were observed in 90–100% of spermatozoa with mitochondria disorganization, disarranged microtubules, and cytoplasmic remnant enveloping coiled flagella, indicating incomplete late spermiogenesis. Normal acrosomes and nuclei were seen. NGS identified deletions (AKAP4, SPAG16, CATSPER1) involved in flagellar development and function. For patients with proximal centriole defects (n=2), the sperm nuclei and fibrous sheath appeared normal. Approximately 90% of proximal centrioles exhibited microtubular disorganization, confirmed by an ODF2 mutation identified by NGS. For a patient with PCD, chaotic flagellar microtubular arrangement and the absence of outer dynein arms prevailed in 90% of the axonemal cross-sections examined, confirmed by a DNAH5 gene deletion. Nonetheless, a few motile spermatozoa were identified and used for ICSI, yielding 80% fertilization and the delivery of a healthy twin. Concurrent ultrastructural and genomic analysis can pinpoint sperm organelle disruption and lead to successful ART cycles.

O-094 Utilization of ultrastructural analysis and genomics of spermatozoa to better characterize subtle forms of male factor infertility

Abstract Study question Can sequencing the sperm genome provide insight into the various forms of male factor infertility caused by sperm organelle ultrastructural defects? Summary answer A comprehensive genomic assessment of spermatozoal DNA is able to identify genetic causes of ultrastructural defects visualized by transmission electron microscopy (TEM). What is known already To evaluate a man’s reproductive potential, a conventional semen analysis through the assessment of concentration, motility, and morphology can indicate the proficiency of male gametes. Among those, conventional morphology assay can only provide indirect information on the different components of the sperm cell. The assessment of nanoscopic details such as chromatin, centriolar, mitochondrial, and axonemal components can only be observed by TEM. Indeed, TEM has been used to identify defects in the acrosome, chromatin compaction, and axonemal/periaxonemal structures. Furthermore, exome sequencing of spermatozoal DNA may identify novel causes and candidate genes for these ultrastructural defects. Study design, size, duration In the past 2 years, 20 men with history of fertilization failure or severe astheno-/terato-zoospermia were selected for TEM analysis of their spermatozoa, while 3 fertile men served as controls. Sperm head characteristics, intactness of fibrous sheath, and axonemal/periaxonemal structure were examined by diagnostic TEM. For consenting patients, NGS assessment was concurrently performed to identify mutations responsible for the structural abnormalities identified by TEM. Participants/materials, setting, methods TEM was performed on the ejaculates of 20 infertile patients and 3 fertile controls. Post-centrifugation cell pellets were resuspended, fixed, and dehydrated to be infiltrated and embedded onto the resin. Fixed specimens were sliced by ultramicrotome to 100-nm sections, then viewed by JEOL-1400 electron microscope at 300,000X magnification. At least 100 spermatozoa were evaluated by TEM. For consenting patients, DNA was extracted and amplified from at least 500 spermatozoa for concurrent NGS analysis. Main results and the role of chance Four types of sperm ultrastructural defects were observed, including globozoospermia, dysplasia of fibrous sheath (DFS), proximal centriole defect, and primary ciliary dyskinesia (PCD). One combined case of globozoospermia and DFS was identified. In globozoospermic patients (n = 13), 97-100% of the spermatozoa displayed characteristic spherical heads with absence of acrosomes, dispersed chromatin, and perinuclear theca deformities. Centrosomal and axonemal structures were conserved. NGS identified gene deletions (DPY19L, PICK1, SPATA16) directly related to the globozoospermic phenotype. In patients with DFS (n = 4), complete absence of flagellum was observed in 90-100% of spermatozoa. These defective gametes also displayed mitochondria disorganization, microtubular deformities, and cytoplasmic residues containing coiled flagellum with deformed capitulum within the plasma membrane. Contrary to the globozoospermia, acrosomes and nuclei appeared normal, indicating incomplete late spermiogenesis. Indeed, NGS confirmed gene deletions involved in flagellar development/function (AKAP4, SPAG16, CATSPER1). For the patient with proximal centriole defect (n = 1), sperm nucleus, fibrous sheath, and flagellar structure were conserved. However, 90% of proximal centrioles assessed exhibited microtubular disorganization, confirmed by ODF2 mutation per NGS. In the PCD patient (n = 1), chaotic flagellar microtubule arrangement and absence of outer dynein arms were prevalent in 90% of axonemal cross-sections examined, which was explained by a DNAH5 gene deletion. Limitations, reasons for caution While TEM can overcome the limitations of conventional semen analysis by providing direct visualization of the inner organelle arrangement of spermatozoa to accurately diagnose rare sperm pathologies, it is not routinely applied in clinics due to its high cost and technical specifications. Therefore, confirmatory NGS can provide additional diagnostic value. Wider implications of the findings Ultrastructural analysis with a concurrent genomic assessment characterized phenotypes and genotypes of rare sperm pathologies in infertile men. The utilization of TEM, corroborated by genomic assay, is therefore crucial for clinical and translational reproductive medicine to better characterize male factor infertility. Trial registration number N/A

O-092 Sperm phenotype, ICSI outcome and genetic diagnosis in case of severe asthenozoospermia with multiple morphological abnormalities of the flagellum

Abstract Study question What are the feasibility and outcome of ICSI in case of presumably genetic severe asthenozoospermia with Multiple Morphological Abnormalities of the Flagellum (MMAF phenotype)? Summary answer ICSI outcome for couples with MMAF phenotype does not differ from that of other couples requiring ICSI, regardless to the genetic etiology What is known already Severe asthenozoospermia, especially when associated with multiple morphological abnormalities of the sperm flagellum (MMAF phenotype), results in male infertility. Recent findings confirm that a genetic etiology is frequently responsible for this phenotype. In such situations, pregnancies can be obtained using ICSI. However, few studies have provided detailed analyses of the flagellar ultrastructural defects underlying this phenotype, of its genetic etiologies and of the results of ICSI in such cases of male infertility. Study design, size, duration We performed a retrospective study including 25 infertile men showing severe asthenozoospermia associated with a MMAF phenotype identified through standard semen analysis. These men were recruited from an academic center for Assisted Reproduction in Paris between 2009 and 2017. Transmission electron microscopy (TEM) and Whole Exome Sequencing (WES) were performed in order to precise the sperm ultra-structural phenotype and identify causal mutations, respectively. Twenty of the 25 patients benefited from assisted reproductive therapy by ICSI. Participants/materials, setting, methods MMAF patients were recruited based on reduced sperm progressive motility and increased frequencies of absent, short, coiled or irregular flagella, in comparison with fertile control men. A quantified analysis of the ultrastructural defects was performed for the MMAF patients and for fertile control men. ICSI results for the MMAF patients were compared to those of 528 ICSI attempts performed for non-MMAF individuals considering the sperm parameters and the distribution of ultrastructural axonemal anomalies. Main results and the role of chance Thorough categorization by TEM analysis of the flagellar anomalies found in these patients brought important precisions about the structural defects underlying asthenozoospermia and sperm tail abnormalities detectable through standard microscopy. In particular, absence of the central pair of axonemal microtubules was the predominant anomaly, observed significantly more frequently than in control men (p &amp;lt; 0.01). Exome sequencing performed for 24 of the 25 patients (96%), identified in ten of them homozygous or compound heterozygous mutations that were described to be pathogenic (CFAP43, CFAP44, CFAP69, DNAH1, DNAH8, AK7, TTC29, MAATS1). A majority of those patients (55.5%,5/9) displayed the most severe ultra-structural defects of the axoneme. Forty ICSI attempts were performed for 20 MMAF patients. A hypo-osmotic swelling (HOS) test was required in 13 cycles (5 couples). Fertilization rate in MMAF group (65.7%) was not statistically different from the rate obtained for non-MMAF patients (66.0%) and did not differ according to the flagellar phenotype, nor to the use of HOS test, nor to the genotype. Clinical pregnancy rate per embryo transfer did not significantly differ between the MMAF group (23.3%) and the ICSI control group (37.1%). To date, 11 healthy babies were born among 20 MMAF patients. Limitations, reasons for caution The outcome of ICSI procedure was retrospectively assessed on a small sample and may be susceptible to recall bias. Moreover, TEM analysis was not available for some of the patients due to too low sperm concentration, and WES results are not yet available for all men included. Wider implications of the findings Couples requiring ICSI for presumably genetic severe asthenozoospermia should benefit precociously from appropriate phenotypic and genetic investigations. So far ICSI results appear similar to those observed in other ICSI indications. Identifying a genetic etiology and its mode of inheritance allows providing to these couples a most often reassuring genetic counseling. Trial registration number Not applicable

Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is an inherited cause of bronchiectasis. The estimated PCD prevalence in children with bronchiectasis is up to 26% and in adults with bronchiectasis is 1 to 13%. Due to dysfunction of the multiple motile cilia of the respiratory tract patients suffer from poor mucociliary clearance. Clinical manifestations are heterogeneous; however, a typical patient presents with chronic productive cough and rhinosinusitis from early life. Other symptoms reflect the multiple roles of motile cilia in other organs and can include otitis media and hearing loss, infertility, situs inversus, complex congenital heart disease, and more rarely other syndromic features such as hydrocephalus and retinitis pigmentosa. Awareness, identification, and diagnosis of a patient with PCD are important for multidisciplinary care and genetic counseling. Diagnosis can be pursued through a multitest pathway which includes the measurement of nasal nitric oxide, sampling the nasal epithelium to assess ciliary function and structure, and genotyping. Diagnosis is confirmed by the identification of a hallmark ultrastructural defect or pathogenic mutations in one of > 45 PCD causing genes. When a diagnosis is established management is centered around improving mucociliary clearance through physiotherapy and treatment of infection with antibiotics. The first international randomized controlled trial in PCD has recently been conducted showing azithromycin is effective in reducing exacerbations. It is likely that evidence-based PCD-specific management guidelines and therapies will be developed in the near future. This article examines prevalence, clinical features, diagnosis, and management of PCD highlighting recent advances in basic science and clinical care.

Bi-allelic truncating variants in CFAP206 cause male infertility in human and mouse.

Spermatozoa are polarized cells with a head and a flagellum joined together by the connecting piece. Flagellum integrity is critical for normal sperm function, and flagellum defects consistently lead to male infertility. Multiple morphological abnormalities of the flagella (MMAF) is a distinct sperm phenotype consistently leading to male infertility due to a reduced or absent sperm motility associated with severe morphological and ultrastructural flagellum defects. Despite numerous genes recently described to be recurrently associated with MMAF, more than half of the cases analyzed remain unresolved, suggesting that many yet uncharacterized gene defects account for this phenotype. By performing a retrospective exome analysis of the unsolved cases from our initial cohort of 167 infertile men with a MMAF phenotype, we identified one individual carrying a homozygous frameshift variant in CFAP206, a gene encoding a microtubule-docking adapter for radial spoke and inner dynein arm. Immunostaining experiments in the patient's sperm cells demonstrated the absence of WDR66 and RSPH1 proteins suggesting severe radial spokes and calmodulin and spoke-associated complex defects. Using the CRISPR-Cas9 technique, we generated homozygous Cfap206 knockout (KO) mice which presented with male infertility due to functional, structural and ultrastructural sperm flagellum defects associated with a very low rate of embryo development using ICSI. Overall, we showed that CFAP206 is essential for normal sperm flagellum structure and function in human and mouse and that bi-allelic mutations in CFAP206 cause male infertility in man and mouse by inducing morphological and functional defects of the sperm flagellum that may also cause ICSI failures.

Mitochondrial autophagy and cell survival is regulated by the circadian Clock gene in cardiac myocytes during ischemic stress

ABSTRACT Cardiac function is highly reliant on mitochondrial oxidative metabolism and quality control. The circadian Clock gene is critically linked to vital physiological processes including mitochondrial fission, fusion and bioenergetics; however, little is known of how the Clock gene regulates these vital processes in the heart. Herein, we identified a putative circadian CLOCK-mitochondrial interactome that gates an adaptive survival response during myocardial ischemia. We show by transcriptome and gene ontology mapping in CLOCK Δ19/Δ19 mouse that Clock transcriptionally coordinates the efficient removal of damaged mitochondria during myocardial ischemia by directly controlling transcription of genes required for mitochondrial fission, fusion and macroautophagy/autophagy. Loss of Clock gene activity impaired mitochondrial turnover resulting in the accumulation of damaged reactive oxygen species (ROS)-producing mitochondria from impaired mitophagy. This coincided with ultrastructural defects to mitochondria and impaired cardiac function. Interestingly, wild type CLOCK but not mutations of CLOCK defective for E-Box binding or interaction with its cognate partner ARNTL/BMAL-1 suppressed mitochondrial damage and cell death during acute hypoxia. Interestingly, the autophagy defect and accumulation of damaged mitochondria in CLOCK-deficient cardiac myocytes were abrogated by restoring autophagy/mitophagy. Inhibition of autophagy by ATG7 knockdown abrogated the cytoprotective effects of CLOCK. Collectively, our results demonstrate that CLOCK regulates an adaptive stress response critical for cell survival by transcriptionally coordinating mitochondrial quality control mechanisms in cardiac myocytes. Interdictions that restore CLOCK activity may prove beneficial in reducing cardiac injury in individuals with disrupted circadian CLOCK. Abbreviations: ARNTL/BMAL1: aryl hydrocarbon receptor nuclear translocator-like; ATG14: autophagy related 14; ATG7: autophagy related 7; ATP: adenosine triphosphate; BCA: bovine serum albumin; BECN1: beclin 1, autophagy related; bHLH: basic helix- loop-helix; CLOCK: circadian locomotor output cycles kaput; CMV: cytomegalovirus; COQ5: coenzyme Q5 methyltransferase; CQ: chloroquine; CRY1: cryptochrome 1 (photolyase-like); DNM1L/DRP1: dynamin 1-like; EF: ejection fraction; EM: electron microscopy; FS: fractional shortening; GFP: green fluorescent protein; HPX: hypoxia; i.p.: intraperitoneal; I-R: ischemia-reperfusion; LAD: left anterior descending; LVIDd: left ventricular internal diameter diastolic; LVIDs: left ventricular internal diameter systolic; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFN2: mitofusin 2; MI: myocardial infarction; mPTP: mitochondrial permeability transition pore; NDUFA4: Ndufa4, mitochondrial complex associated; NDUFA8: NADH: ubiquinone oxidoreductase subunit A8; NMX: normoxia; OCR: oxygen consumption rate; OPA1: OPA1, mitochondrial dynamin like GTPase; OXPHOS: oxidative phosphorylation; PBS: phosphate-buffered saline; PER1: period circadian clock 1; PPARGC1A/PGC-1α: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; qPCR: quantitative real-time PCR; RAB7A: RAB7, member RAS oncogene family; ROS: reactive oxygen species; RT: room temperature; shRNA: short hairpin RNA; siRNA: small interfering RNA; TFAM: transcription factor A, mitochondrial; TFEB: transcription factor EB; TMRM: tetra-methylrhodamine methyl ester perchlorate; WT: wild -type; ZT: zeitgeber time

Cytoplasmic FUS triggers early behavioral alterations linked to cortical neuronal hyperactivity and inhibitory synaptic defects

Gene mutations causing cytoplasmic mislocalization of the RNA-binding protein FUS lead to severe forms of amyotrophic lateral sclerosis (ALS). Cytoplasmic accumulation of FUS is also observed in other diseases, with unknown consequences. Here, we show that cytoplasmic mislocalization of FUS drives behavioral abnormalities in knock-in mice, including locomotor hyperactivity and alterations in social interactions, in the absence of widespread neuronal loss. Mechanistically, we identified a progressive increase in neuronal activity in the frontal cortex of Fus knock-in mice in vivo, associated with altered synaptic gene expression. Synaptic ultrastructural and morphological defects were more pronounced in inhibitory than excitatory synapses and associated with increased synaptosomal levels of FUS and its RNA targets. Thus, cytoplasmic FUS triggers synaptic deficits, which is leading to increased neuronal activity in frontal cortex and causing related behavioral phenotypes. These results indicate that FUS mislocalization may trigger deleterious phenotypes beyond motor neuron impairment in ALS, likely relevant also for other neurodegenerative diseases characterized by FUS mislocalization.

Alpha-Tubulin Acetylation in Trypanosoma cruzi: A Dynamic Instability of Microtubules Is Required for Replication and Cell Cycle Progression.

Trypanosomatids have a cytoskeleton arrangement that is simpler than what is found in most eukaryotic cells. However, it is precisely organized and constituted by stable microtubules. Such microtubules compose the mitotic spindle during mitosis, the basal body, the flagellar axoneme and the subpellicular microtubules, which are connected to each other and also to the plasma membrane forming a helical arrangement along the central axis of the parasite cell body. Subpellicular, mitotic and axonemal microtubules are extensively acetylated in Trypanosoma cruzi. Acetylation on lysine (K) 40 of α-tubulin is conserved from lower eukaryotes to mammals and is associated with microtubule stability. It is also known that K40 acetylation occurs significantly on flagella, centrioles, cilia, basal body and the mitotic spindle in eukaryotes. Several tubulin posttranslational modifications, including acetylation of K40, have been cataloged in trypanosomatids, but the functional importance of these modifications for microtubule dynamics and parasite biology remains largely undefined. The primary tubulin acetyltransferase was recently identified in several eukaryotes as Mec-17/ATAT, a Gcn5-related N-acetyltransferase. Here, we report that T. cruzi ATAT acetylates α-tubulin in vivo and is capable of auto-acetylation. TcATAT is located in the cytoskeleton and flagella of epimastigotes and colocalizes with acetylated α-tubulin in these structures. We have expressed TcATAT with an HA tag using the inducible vector pTcINDEX-GW in T. cruzi. Over-expression of TcATAT causes increased levels of the alpha tubulin acetylated species, induces morphological and ultrastructural defects, especially in the mitochondrion, and causes a halt in the cell cycle progression of epimastigotes, which is related to an impairment of the kinetoplast division. Finally, as a result of TcATAT over-expression we observed that parasites became more resistant to microtubule depolymerizing drugs. These results support the idea that α-tubulin acetylation levels are finely regulated for the normal progression of T. cruzi cell cycle.

Clinical and molecular characteristics of primary ciliary dyskinesia: A tertiary care centre experience.

BackgroundPrimary ciliary dyskinesia (PCD) is a ciliopathy with diverse clinical and genetic findings caused by abnormal motile cilia structure and function. In this study, we describe the clinical characteristics of confirmed PCD cases in our population and report the radiological, genetic, and laboratory findings.MethodsThis was a retrospective, observational, single-centre study. We enrolled 18 patients who were diagnosed with confirmed PCD between 2015 and 2019. We then analyzed their data, including clinical findings and workup.ResultsIn our cohort, 56% of patients had molecularly confirmed PCD, and RSPH9 was the most common gene identified. Transmission electron microscopy (TEM) showed an ultrastructural defect in 64% of samples, all of which matched the genetic background of the patient. Situs inversus (SI) was observed in 50% of patients, and congenital heart disease was observed in 33%. The median body mass index (BMI) was 15.87 kg/m2, with a median z score of -1.48. The median FEV1 value was 67.6% (z score - 2.43). Radiologically, bronchiectasis was noted in 81% of patients at a variable degree of severity. Lung bases were involved in 91% of patients. We were unable to correlate the genotype-phenotype findings.ConclusionWe describe the clinical and molecular characteristics of patients with confirmed PCD in a tertiary centre in Saudi Arabia and report 9 new pathogenic or likely pathogenic variants in one of the PCD-associated genes.

Missense mutation of Fmr1 results in impaired AMPAR-mediated plasticity and socio-cognitive deficits in mice

Fragile X syndrome (FXS) is the most frequent form of inherited intellectual disability and the best-described monogenic cause of autism. CGG-repeat expansion in the FMR1 gene leads to FMR1 silencing, loss-of-expression of the Fragile X Mental Retardation Protein (FMRP), and is a common cause of FXS. Missense mutations in the FMR1 gene were also identified in FXS patients, including the recurrent FMRP-R138Q mutation. To investigate the mechanisms underlying FXS caused by this mutation, we generated a knock-in mouse model (Fmr1R138Q) expressing the FMRP-R138Q protein. We demonstrate that, in the hippocampus of the Fmr1R138Q mice, neurons show an increased spine density associated with synaptic ultrastructural defects and increased AMPA receptor-surface expression. Combining biochemical assays, high-resolution imaging, electrophysiological recordings, and behavioural testing, we also show that the R138Q mutation results in impaired hippocampal long-term potentiation and socio-cognitive deficits in mice. These findings reveal the functional impact of the FMRP-R138Q mutation in a mouse model of FXS.

Primary ciliary dyskinesia: A neonatal disease?

In this volume of The Journal, Horani and Ferkol update our current understanding of primary ciliary dyskinesia and other ciliopathies. Although neonatologists may be tempted to bypass this article, it is relevant to newborn patients because ciliary function and diseases are of increasing importance in fetal development (eg, determination of body laterality) and neonatal illnesses. The authors report that the majority of infants with primary ciliary dyskinesia present with early and prolonged neonatal respiratory distress or with laterality disorders. Although laterality disorders are readily apparent on radiographs, it is not yet clear how neonatologists should think about these diseases in tachypneic infants because primary ciliopathies are rare diseases and tachypnea is a very common problem in newborn infants. Most neonatologists can recall unusual patients with prolonged tachypnea of undetermined etiology. These patients may be good candidates for further research so we can begin to identify distinguishing neonatal clinical characteristics that would allow for targeted follow up and early diagnosis of affected infants. Article page 15 ▸ Understanding Primary Ciliary Dyskinesia and Other CiliopathiesThe Journal of PediatricsVol. 230PreviewCiliopathies are a collection of disorders related to cilia dysfunction. Cilia are specialized organelles that project from the surface of most cells. Motile and primary (sensory) cilia are essential structures and have wide ranging functions. Our understanding of the genetics, pathophysiology, and clinical manifestations of motile ciliopathies, including primary ciliary dyskinesia (PCD), has rapidly advanced since the disease was linked to ciliary ultrastructural defects nearly 5 decades ago. We will provide an overview of different types of cilia, their role in child health and disease, focusing on motile ciliopathies, and describe recent advances that have led to improved diagnostics and may yield therapeutic targets to restore ciliary structure and function. Full-Text PDF

Topological data analysis reveals genotype-phenotype relationships in primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a heterogeneous inherited disorder caused by mutations in approximately 50 cilia-related genes. PCD genotype-phenotype relationships have mostly arisen from small case series because existing statistical approaches to investigating relationships have been unsuitable for rare diseases. We applied a topological data analysis (TDA) approach to investigate genotype-phenotype relationships in PCD. Data from separate training and validation cohorts included 396 genetically defined individuals carrying pathogenic variants in PCD genes. To develop the TDA models, 12 clinical and diagnostic variables were included. TDA-driven hypotheses were subsequently tested using traditional statistics. Disease severity at diagnosis, measured by forced expiratory volume in 1 s (FEV1) z-score, was significantly worse in individuals with CCDC39 mutations (compared to other gene mutations) and better in those with DNAH11 mutations; the latter also reported less neonatal respiratory distress. Patients without neonatal respiratory distress had better preserved FEV1 at diagnosis. Individuals with DNAH5 mutations were phenotypically diverse. Cilia ultrastructure and beat pattern defects correlated closely to specific causative gene groups, confirming these tests can be used to support a genetic diagnosis. This large scale, multi-national study presents PCD as a syndrome with overlapping symptoms and variations in phenotype according to genotype. TDA modelling confirmed genotype-phenotype relationships reported by smaller studies (e.g. FEV1 worse with CCDC39 mutation) and identified new relationships, including FEV1 preservation with DNAH11 mutations and diversity of severity with DNAH5 mutations.

Genetic Analysis of Korean Adult Patients with Nontuberculous Mycobacteria Suspected of Primary Ciliary Dyskinesia Using Whole Exome Sequencing.

PurposeNontuberculous mycobacteria (NTM) is ubiquitous in the environment, but NTM lung disease (NTM-LD) is uncommon. Since exposure to NTM is inevitable, patients who develop NTM-LD are likely to have specific susceptibility factors, such as primary ciliary dyskinesia (PCD). PCD is a genetically heterogeneous disorder of motile cilia and is characterized by chronic respiratory tract infection, organ laterality defect, and infertility. In this study, we performed whole exome sequencing (WES) and investigated the genetic characteristics of adult NTM patients with suspected PCD.Materials and MethodsWES was performed in 13 NTM-LD patients who were suspected of having PCD by clinical symptoms and/or ultrastructural ciliary defect observed by transmission electron microscopy. A total of 45 PCD-causing genes, 23 PCD-candidate genes, and 990 ciliome genes were analyzed.ResultsFour patients were found to have biallelic loss-of-function (LoF) variants in the following PCD-causing genes: CCDC114, DNAH5, HYDIN, and NME5. In four other patients, only one LoF variant was identified, while the remaining five patients did not have any LoF variants.ConclusionAt least 30.8% of NTM-LD patients who were suspected of having PCD had biallelic LoF variants, and an additional 30.8% of patients had one LoF variant. Therefore, PCD should be considered in patients with NTM-LD with symptoms or signs suspicious of PCD.

Two novel mutations in the DNAH11 gene in primary ciliary dyskinesia (CILD7) with considerable variety in the clinical and beating cilia phenotype

BackgroundDiagnosis of primary ciliary dyskinesia (PCD) still remains a challenge, especially with mutations in the Dynein Arm Heavy Chain 11 (DNAH11) gene. Classical diagnostic measures like Transmission Electron Microscopy (TEM) are not applicable for mutations in the DNAH11 gene since ultrastructural defects of the ciliary apparatus are absent. Novel mutations encoding for PCD appear all the time with considerable variation in the clinical picture, making it necessary to update data bases and guidelines for PCD diagnostics.MethodsIn this study we examined two unrelated, Finnish families with symptoms of PCD applying the clinical scoring system: Primary ciliary dyskinesia Rule (PICADAR), high speed video microscopy analysis (HSVMA) for ciliary movement, a commercially available gene panel analysis and nasal Nitric Oxide (nNO) measurements if applicable.ResultsTwo, likely pathogenic variants in the DNAH11 gene (c.2341G > A, p. (Glu781Lys) ja c.7645 + 5G > A) were detected. In the first family, compound heterozygous mutations led to disease manifestation in two of 4 children, which showed a similar phenotype of cilia beating pattern but marked differences in disease severity. In the second family, all three children were homozygotes for the c.2341G > A p.(Glu781Lys) mutation and showed a similar degree of disease severity. However, the phenotype of cilia beating pattern was different ranging from stiff, static cilia to a hyperkinetic movement in one of these children.ConclusionsIn this study we describe two Finnish families with PCD, revealing two novel mutations in the DNAH11 gene which show considerable variety in the clinical and beating cilia phenotype. The results of this study show the clinician that PCD can be much milder than generally expected and diagnosis demands a combination of measures which are only successful in experienced hands. Chronic and repeatedly treated wet cough should raise suspicion of PCD, referring the patient for further diagnostics to a specialised PCD centre.

Understanding Primary Ciliary Dyskinesia and Other Ciliopathies

Ciliopathies are a collection of disorders related to cilia dysfunction. Cilia are specialized organelles that project from the surface of most cells. Motile and primary (sensory) cilia are essential structures and have wide ranging functions. Our understanding of the genetics, pathophysiology, and clinical manifestations of motile ciliopathies, including primary ciliary dyskinesia (PCD), has rapidly advanced since the disease was linked to ciliary ultrastructural defects nearly 5 decades ago. We will provide an overview of different types of cilia, their role in child health and disease, focusing on motile ciliopathies, and describe recent advances that have led to improved diagnostics and may yield therapeutic targets to restore ciliary structure and function. Primary ciliary dyskinesia: A neonatal disease?The Journal of PediatricsVol. 230PreviewIn this volume of The Journal, Horani and Ferkol update our current understanding of primary ciliary dyskinesia and other ciliopathies. Although neonatologists may be tempted to bypass this article, it is relevant to newborn patients because ciliary function and diseases are of increasing importance in fetal development (eg, determination of body laterality) and neonatal illnesses. The authors report that the majority of infants with primary ciliary dyskinesia present with early and prolonged neonatal respiratory distress or with laterality disorders. Full-Text PDF

Abstract 13562: COQ8B : A Candidate Genetic Modifier of Thoracic Aortic Aneurysm Severity

Thoracic aortic aneurysm (TAA) is a latent aortopathy that predisposes to life-threatening aortic dissection. Personalized prediction of the risk for progressive aortic dilation and dissection is currently limited. The identification of genetic modifiers may facilitate more precise risk prediction and optimize clinical decisions. We previously identified COQ8B , a gene important for mitochondrial biosynthesis of coenzyme Q, as a candidate modifier of TAA severity. Biological evidence for a mitochondrial role in TAA pathogenesis includes severe mitochondrial ultrastructural defects in aortic smooth muscle cells (SMCs) and nitrated tyrosine residues in TAA tissues consistent with oxidative stress. In this study, we hypothesized that a common single nucleotide polymorphism (SNP) rs3865452 in COQ8B (c.521A&gt;G, p.H174R) modifies the rate of aortic root dilation. Forty-eight patients with TAA and a mean age at first echocardiogram of 10 ± 5 years were genotyped for rs3865452. Echocardiograms (mean 6.8 ± 4.0 per patient) were performed clinically over a mean interval of 5.3 ± 4.1 years. In mixed model analysis of longitudinal aortic root Z-scores, the c.521G allele was associated with a decreased rate of aortic root dilation in univariate analysis (p=0.0003) and multivariate analysis (p=0.03) that included sex and genetic syndrome as covariates. Hypothesizing a modifier role for COQ8B , we analyzed a separate cohort of 230 individuals with early onset thoracic aortic dissection. The c.521G allele was less frequent in dissection cases than controls (p=0.04), also consistent with a protective effect for this SNP. To better understand the mechanism of COQ8B effect in TAA, RNA levels of COQ8B in SMCs cultured directly from the aorta were assayed, demonstrating levels 2-fold lower in TAA compared with controls. siRNA knockdown of COQ8B in aortic SMCS resulted in decreased mitochondrial respiration, increased lipid peroxidation, and altered expression of SMC contractile genes. In conclusion, the findings of this study support a modifier role for the rs3865452 SNP in COQ8B in TAA, which may involve mitochondrial pathology and oxidative stress. This suggests that this SNP may be useful for clinical risk prediction.

A Human Induced Pluripotent Stem Cell-Derived Isogenic Model of Huntington's Disease Based on Neuronal Cells Has Several Relevant Phenotypic Abnormalities.

Huntington’s disease (HD) is a severe neurodegenerative disorder caused by a CAG triplet expansion in the first exon of the HTT gene. Here we report the introduction of an HD mutation into the genome of healthy human embryonic fibroblasts through CRISPR/Cas9-mediated homologous recombination. We verified the specificity of the created HTT-editing system and confirmed the absence of undesirable genomic modifications at off-target sites. We showed that both mutant and control isogenic induced pluripotent stem cells (iPSCs) derived by reprogramming of the fibroblast clones can be differentiated into striatal medium spiny neurons. We next demonstrated phenotypic abnormalities in the mutant iPSC-derived neural cells, including impaired neural rosette formation and increased sensitivity to growth factor withdrawal. Moreover, using electron microscopic analysis, we detected a series of ultrastructural defects in the mutant neurons, which did not contain huntingtin aggregates, suggesting that these defects appear early in HD development. Thus, our study describes creation of a new isogenic iPSC-based cell system that models HD and recapitulates HD-specific disturbances in the mutant cells, including some ultrastructural features implemented for the first time.

Functional analysis and evaluation of respiratory cilia in healthy Chinese children

BackgroundTo aid in the diagnosis of Primary Ciliary Dyskinesia (PCD) and to evaluate the respiratory epithelium in respiratory disease, normal age-related reference ranges are needed for ciliary beat frequency (CBF), beat pattern and ultrastructure. Our aim was to establish reference ranges for healthy Chinese children.MethodsCiliated epithelial samples were obtained from 135 healthy Chinese children aged below 18 years by brushing the inferior nasal turbinate. CBF and beat pattern were analysed from high speed video recordings. Epithelial integrity and ciliary ultrastructure were assessed using transmission electronic microscopy.ResultsThe mean CBF from 135 children studied was 10.1 Hz (95% CI 9.8 to 10.4). Approximately 20% (ranged 18.0–24.2%) of ciliated epithelial edges were found to have areas of dyskinetically beating cilia. Normal beat pattern was observed in ciliated epithelium from all subjects. We did not find any effect of exposure to second hand smoke on CBF in our subjects. Microtubular defects were found in 9.3% of all of the cilia counted in these children, while other ciliary ultrastructural defects were found in less than 3%.ConclusionsWe established the reference range for CBF, beat pattern and ultrastructure in healthy Chinese children. Using similar methodology, we found a lower overall mean CBF than previously obtained European values. This study highlights the need to establish normative data for ciliary function in different populations.