Defective airway intraflagellar transport underlies a combined motile and primary ciliopathy syndrome caused by IFT74 mutations

Ciliopathies are inherited disorders caused by defective cilia. Mutations affecting motile cilia usually cause the chronic muco-obstructive sinopulmonary disease primary ciliary dyskinesia (PCD) and are associated with laterality defects, while a broad spectrum of early developmental as well as degenerative syndromes arise from mutations affecting signalling of primary (non-motile) cilia. Cilia assembly and functioning requires intraflagellar transport of cargos assisted by IFT-B and IFT-A adaptor complexes. Within IFT-B, the N-termini of partner proteins IFT74 and IFT81 govern tubulin transport to build the ciliary microtubular cytoskeleton. We detected a homozygous 3 kb intragenic IFT74 deletion removing the exon 2 initiation codon and 40 N-terminal amino acids in two affected siblings. Both had clinical features of PCD with bronchiectasis, but no laterality defects. They also had retinal dysplasia and abnormal bone growth, with a narrowed thorax and short ribs, shortened long bones and digits and abnormal skull shape. This resembles short-rib thoracic dysplasia, a skeletal ciliopathy previously linked to IFT defects in primary cilia, not motile cilia. Ciliated nasal epithelial cells collected from affected individuals had reduced numbers of shortened motile cilia with disarranged microtubules, some mis-orientation of the basal feet, and disrupted cilia structural and IFT protein distributions. No full length IFT74 was expressed, only truncated forms that were consistent with N-terminal deletion and inframe translation from downstream initiation codons. In affinity purification mass spectrometry, exon 2-deleted IFT74 initiated from the nearest inframe downstream methionine 41 still interacts as part of the IFT-B complex, but only with reduced interaction levels and not with all its usual IFT-B partners. We propose that this is a hypomorphic mutation with some residual protein function retained, that gives rise to a non-lethal primary skeletal ciliopathy combined with defective motile cilia and PCD

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

Risk factors for situs defects and congenital heart disease in primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is associated with abnormal organ positioning (situs) and congenital heart disease (CHD). This study investigated genotype–phenotype associations in PCD to facilitate risk predictions for cardiac and laterality defects. This retrospective cohort study of 389 UK patients with PCD found 51% had abnormal situs and 25% had CHD and/or laterality defects other than situs inversus totalis. Patients with biallelic mutations in a subset of nine PCD genes had normal situs. Patients with consanguineous parents had higher odds of situs abnormalities than patients with non-consanguineous parents. Patients with abnormal situs had higher odds of CHD and/or laterality defects

1,3-Butadiene: Biomarkers and application to risk assessment

1,3-Butadiene (BD) is a known rodent and human carcinogen that is metabolized mainly by P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB) and 1,2-epoxy-3,4-butanediol (EB-diol). The individual epoxides vary up to 200-fold in their mutagenic potency, with DEB being the most mutagenic metabolite. It is important to understand the internal formation of the individual epoxides to assign the relative risk for each metabolite and to understand the molecular mechanisms responsible for major species differences in carcinogenicity. We have conducted extensive exposure-biomarker studies on mice, rats and humans. Using low exposures that range from current occupational levels to human exposures from tobacco smoke has provided evidence that mice are very different from humans, with mice forming ~200 times more DEB than humans at exposures of 0.1–1.5 ppm BD. While no gender differences have been noted in mice and rats for globin adducts or N-7 guanine adducts, female rats and mice had 2–3-fold higher Hprt mutations and DNA-DNA cross-links, suggesting a gender difference in DNA repair. Numerous molecular epidemiology studies have evaluated globin adducts and Hprt mutations, SCEs and chromosomal abnormalities. None of the blinded studies have shown evidence of human genotoxicity at current occupational exposures and studies of globin adducts have shown similar or lower formation of adducts in females than males. If one calculates the EB dose-equivalents for the three species, mice clearly differ from rats and humans, being ~44 and 174 times greater than rats and humans, respectively. These data provide a scientific basis for improved risk assessment of BD

Clinical utility of NGS diagnosis and disease stratification in a multiethnic primary ciliary dyskinesia cohort

Background Primary ciliary dyskinesia (PCD), a genetically heterogeneous condition enriched in some consanguineous populations, results from recessive mutations affecting cilia biogenesis and motility. Currently, diagnosis requires multiple expert tests.Methods The diagnostic utility of multigene panel next-generation sequencing (NGS) was evaluated in 161 unrelated families from multiple population ancestries.Results Most (82%) families had affected individuals with biallelic or hemizygous (75%) or single (7%) pathogenic causal alleles in known PCD genes. Loss-of-function alleles dominate (73% frameshift, stop-gain, splice site), most (58%) being homozygous, even in non-consanguineous families. Although 57% (88) of the total 155 diagnostic disease variants were novel, recurrent mutations and mutated genes were detected. These differed markedly between white European (52% of families carry DNAH5 or DNAH11 mutations), Arab (42% of families carry CCDC39 or CCDC40 mutations) and South Asian (single LRRC6 or CCDC103 mutations carried in 36% of families) patients, revealing a striking genetic stratification according to population of origin in PCD. Genetics facilitated successful diagnosis of 81% of families with normal or inconclusive ultrastructure and 67% missing prior ultrastructure results.Conclusions This study shows the added value of high-throughput targeted NGS in expediting PCD diagnosis. Therefore, there is potential significant patient benefit in wider and/or earlier implementation of genetic screening

Mutations in CCDC 39 and CCDC 40 are the Major Cause of Primary Ciliary Dyskinesia with Axonemal Disorganization and Absent Inner Dynein Arms

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder caused by cilia and sperm dysmotility. About 12% of cases show perturbed 9+2 microtubule cilia structure and inner dynein arm (IDA) loss, historically termed ‘radial spoke defect’. We sequenced CCDC39 and CCDC40 in 54 ‘radial spoke defect’ families, as these are the two genes identified so far to cause this defect. We discovered biallelic mutations in a remarkable 69% (37/54) of families, including identification of 25 (19 novel) mutant alleles (12 in CCDC39 and 13 in CCDC40). All the mutations were nonsense, splice and frameshift predicting early protein truncation, which suggests this defect is caused by ‘null’ alleles conferring complete protein loss. Most families (73%; 27/37) had homozygous mutations, including families from outbred populations. A major putative hotspot mutation was identified, CCDC40 c.248delC, as well as several other possible hotspot mutations. Together, these findings highlight the key role of CCDC39 and CCDC40 in PCD with axonemal disorganisation and IDA loss, and these genes represent major candidates for genetic testing in families affected by this ciliary phenotype. We show that radial spoke structures are largely intact in these patients and propose this ciliary ultrastructural abnormality be referred to as ‘IDA and nexin-dynein regulatory complex (N-DRC) defect’, rather than ‘radial spoke defect’

Connecting with Country in Mungo National Park, Australia: a case study to measure the emotional dimension of experience and place attachment

Connecting people with nature improves well-being, but how people connect with natural places is not well documented. We asked 43 people (19 Aboriginal Australians, 24 non-Aboriginal people) about the messages they received from Country during an interactive experience in the remote Mungo National Park, Australia, and analysed the physical senses, emotions and cognitive processes they mentioned. The physical
 senses mentioned by most respondents were sight, hearing and motion (particularly walking). These senses helped people receive messages from Country and connect with place. We used the primary-process emotional systems of Panksepp [2010. Dialogues in Clinical Neuroscience, 12 (4), 533–545] as a framework to capture the emotional dimension of experience. Most people reported positive emotions; they spoke about being nurtured by the group and the land (CARE), and the intense joy (PLAY) of being part of the community, being on Country and being accompanied by Aboriginal people. However, our results indicate the framework does not capture the breadth of positive emotions, particularly those associated with connection to place and spiritual experiences. Both groups mentioned cognitive processes reflecting their beliefs, existing knowledge, or sharing and acquiring new knowledge. Our results indicate that the emotional dimension of experience has the potential to measure connection to place, and provide a subjective measure of well-being. More research is needed to document this dimension of experience, and how it changes with context. Our case study provides further insight for those who manage protected areas and seek to enrich the experience of visitors

Accuracy of diagnostic testing in primary ciliary dyskinesia

Diagnosis of primary ciliary dyskinesia (PCD) lacks a "gold standard" test and is therefore based on combinations of tests including nasal nitric oxide (nNO), high-speed video microscopy analysis (HSVMA), genotyping and transmission electron microscopy (TEM). There are few published data on the accuracy of this approach.Using prospectively collected data from 654 consecutive patients referred for PCD diagnostics we calculated sensitivity and specificity for individual and combination testing strategies. Not all patients underwent all tests.HSVMA had excellent sensitivity and specificity (100% and 93%, respectively). TEM was 100% specific, but 21% of PCD patients had normal ultrastructure. nNO (30?nL·min(-1) cut-off) had good sensitivity and specificity (91% and 96%, respectively). Simultaneous testing using HSVMA and TEM was 100% sensitive and 92% specific.In conclusion, combination testing was found to be a highly accurate approach for diagnosing PCD. HSVMA alone has excellent accuracy, but requires significant expertise, and repeated sampling or cell culture is often needed. TEM alone is specific but misses 21% of cases. nNO (?30?nL·min(-1)) contributes well to the diagnostic process. In isolation nNO screening at this cut-off would miss ?10% of cases, but in combination with HSVMA could reduce unnecessary further testing. Standardisation of testing between centres is a future priority

Microvillous tip vesicles may be an origin of placental extracellular vesicles

INTRODUCTION: Extracellular vesicles are now believed to be important mediators of placental-maternal communication. However, little is known about the formation of extracellular vesicles by human placenta. This study uses nanoscale three-dimensional imaging to investigate how and where placental extracellular vesicles form.METHODS: Term and first trimester human placental villi were imaged by serial block face scanning electron microscopy. These images were analysed to quantify vesicle surface density. Segmentation was performed to reconstruct three-dimensional images of extracellular vesicles. Live imaging light microscopy of first trimester villous explants was performed.RESULTS: Vesicles were observed on the tips of placental microvilli in term and first trimester placenta. In term placenta these microvillous tip vesicles had a median size of 0.55 μm and their surface area density exceeded 22000 per mm2. Microvillous tip vesicle membranes had a lower electron density than the microvillous plasma membrane. Thirty seven percent of vesicles had a complex membrane structure including double membranes, internal vesicles and vesicle chains. Budding of smaller secondary vesicles from microvillous tip vesicle membranes was observed. Live imaging of a first trimester villus explant observed formation of vesicles which were larger but visually similar to the secondary vesicles observed by electron microscopy.DISCUSSION: These observations suggest that extracellular vesicles are forming on the tips of placental microvilli prior to release into maternal blood. However, it cannot be discounted that there are maternal extracellular vesicles that have bound to microvilli. In either case, the high surface area density of microvillous tip vesicles is consistent with an important role in placental-maternal signalling.</p