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Hydrocephalus in bgh mice. A) Gross hydrocephalus in bgh homozygote. B) Graph showing brain to body ratios for wild type and bgh mutants. Ratios are plotted as (mg brain)/(g body). The ratio for bgh mutants is approximately 2.2 times greater than that of wild-type animals (n ¼ 8 bgh and 4 wild type; P , 0.005). C-F) Coronal sections of wild-type (C) and bgh (D-F) brains through the lateral ventricles. Original magnification is 43 (C, D) or 403 (E, F). High magnification images show that cilia are present on intact bgh ependyma (E) and that substantial gliosis has occurred in the bgh white matter (F). All the sections are stained with hematoxylin and eosin.
Source publication
Primary ciliary dyskinesia (PCD) results from defects in motile cilia function. Mice homozygous for the mutation big giant head (bgh) have several abnormalities commonly associated with PCD, including hydrocephalus, male infertility, and sinusitis. In the present study, we use a variety of histopathological and cell biological techniques to charact...
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... mutation causing hydrocephalus occurred in mice on a mixed C57BL/6J;C57BL/10J background (Fig. 1). The muta- tion was heritable, and the number of affected animals was consistent with an autosomal recessive mode of inheritance. Because of the dramatic hydrocephalus, this mutant was named big giant head (bgh). Homozygous mutants had an enlarged cranial vault indicative of ventricular dilatation (Fig. 1A), which is typically caused ...
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... on a mixed C57BL/6J;C57BL/10J background (Fig. 1). The muta- tion was heritable, and the number of affected animals was consistent with an autosomal recessive mode of inheritance. Because of the dramatic hydrocephalus, this mutant was named big giant head (bgh). Homozygous mutants had an enlarged cranial vault indicative of ventricular dilatation (Fig. 1A), which is typically caused by accumulating cerebrospinal fluid in the ventricular system. As a result, the average brain:body ratio for a 3-wk-old bgh animal on the B6 background was approximately 2.2 times greater than that of unaffected littermates (Fig. 1B). Histological analysis demonstrated extensive dilatation of the lateral ...
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... Homozygous mutants had an enlarged cranial vault indicative of ventricular dilatation (Fig. 1A), which is typically caused by accumulating cerebrospinal fluid in the ventricular system. As a result, the average brain:body ratio for a 3-wk-old bgh animal on the B6 background was approximately 2.2 times greater than that of unaffected littermates (Fig. 1B). Histological analysis demonstrated extensive dilatation of the lateral ventricles in bgh mutants (Fig. 1, C and D). Intact ependymal cells lining the mutant ventricles possessed histologically normal cilia (Fig. 1E). However, there was evidence of extensive ependymal sloughing and substantial gliosis in the underlying white matter ...
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... typically caused by accumulating cerebrospinal fluid in the ventricular system. As a result, the average brain:body ratio for a 3-wk-old bgh animal on the B6 background was approximately 2.2 times greater than that of unaffected littermates (Fig. 1B). Histological analysis demonstrated extensive dilatation of the lateral ventricles in bgh mutants (Fig. 1, C and D). Intact ependymal cells lining the mutant ventricles possessed histologically normal cilia (Fig. 1E). However, there was evidence of extensive ependymal sloughing and substantial gliosis in the underlying white matter (Fig. 1F). This damage was likely a secondary effect of excessive pressure from accumulating ...
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... ratio for a 3-wk-old bgh animal on the B6 background was approximately 2.2 times greater than that of unaffected littermates (Fig. 1B). Histological analysis demonstrated extensive dilatation of the lateral ventricles in bgh mutants (Fig. 1, C and D). Intact ependymal cells lining the mutant ventricles possessed histologically normal cilia (Fig. 1E). However, there was evidence of extensive ependymal sloughing and substantial gliosis in the underlying white matter (Fig. 1F). This damage was likely a secondary effect of excessive pressure from accumulating ...
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... (Fig. 1B). Histological analysis demonstrated extensive dilatation of the lateral ventricles in bgh mutants (Fig. 1, C and D). Intact ependymal cells lining the mutant ventricles possessed histologically normal cilia (Fig. 1E). However, there was evidence of extensive ependymal sloughing and substantial gliosis in the underlying white matter (Fig. 1F). This damage was likely a secondary effect of excessive pressure from accumulating ...
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... spermiogenesis is divided into 16 steps, with steps 1-8 comprising the round spermatid phase and steps 9-16 comprising spermatid elongation [95]. Detailed living cell analysis of the squash preparations under phase contrast microscopy confirmed the normal associations of spermato- genic cell types in various stages of bgh tubules (Supplemental Fig. S1; all the supplemental data are available online at www. biolreprod.org). Acrosome formation in round spermatids in the early steps of spermiogenesis appeared unaffected (Sup- plemental Fig. S1). Elongating spermatid bundles were present in stage IV-V tubules, but the orientation of the spermatids inside the bundles was disrupted and a ...
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... preparations under phase contrast microscopy confirmed the normal associations of spermato- genic cell types in various stages of bgh tubules (Supplemental Fig. S1; all the supplemental data are available online at www. biolreprod.org). Acrosome formation in round spermatids in the early steps of spermiogenesis appeared unaffected (Sup- plemental Fig. S1). Elongating spermatid bundles were present in stage IV-V tubules, but the orientation of the spermatids inside the bundles was disrupted and a dramatic reduction in the number of bundle-associated elongating spermatids (Sup- plemental Fig. S1). Stage VII-VIII tubules contained very few mature spermatozoa, and those that were present ...
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... formation in round spermatids in the early steps of spermiogenesis appeared unaffected (Sup- plemental Fig. S1). Elongating spermatid bundles were present in stage IV-V tubules, but the orientation of the spermatids inside the bundles was disrupted and a dramatic reduction in the number of bundle-associated elongating spermatids (Sup- plemental Fig. S1). Stage VII-VIII tubules contained very few mature spermatozoa, and those that were present had abnormally formed flagella (Supplemental Fig. S1). All the bgh flagella appeared to be short and disorganized (Supple- mental Fig. ...
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... present in stage IV-V tubules, but the orientation of the spermatids inside the bundles was disrupted and a dramatic reduction in the number of bundle-associated elongating spermatids (Sup- plemental Fig. S1). Stage VII-VIII tubules contained very few mature spermatozoa, and those that were present had abnormally formed flagella (Supplemental Fig. S1). All the bgh flagella appeared to be short and disorganized (Supple- mental Fig. ...
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... was disrupted and a dramatic reduction in the number of bundle-associated elongating spermatids (Sup- plemental Fig. S1). Stage VII-VIII tubules contained very few mature spermatozoa, and those that were present had abnormally formed flagella (Supplemental Fig. S1). All the bgh flagella appeared to be short and disorganized (Supple- mental Fig. ...
Similar publications
Primary ciliary dyskinesia (PCD) is a rare genetic disorder of ciliary structure and function. The diagnosis can be challenging, particularly when using nongenetic assays. The "gold standard" diagnostic test is ultrastructural analysis of respiratory cilia obtained by nasal scrape or brush biopsy. A few specialized centers use high-speed videomicro...
Citations
... In Finnish Yorkshire boars, SPEF2 mutations resulted in immotile short-tailed sperm defects [20], while in Holstein bulls, SPEF2 mutation induced sperm malformation and a sperm hypomotility phenotype [26], both without mentioning PCD-related phenotypes. In the mice model, in addition to male infertility with sperm defects, Spef2-deficient mice had PCD phenotypes such as hydrocephalus, sinusitis, and decreased cilia function [23,27,28]. Among the reported human patients with SPEF2 mutations, parts of male patients with SPEF2 mutations exhibit only male infertility without the PCD phenotypes [24,25,29]. ...
This study aimed to identify the genetic causes of male infertility and primary ciliary dyskinesia (PCD)/PCD-like phenotypes in three unrelated Han Chinese families.
We conducted whole-exome sequencing of three patients with male infertility and PCD/PCD-like phenotypes from three unrelated Chinese families. Ultrastructural and immunostaining analyses of patient spermatozoa and respiratory cilia and in vitro analyses were performed to analyze the effects of SPEF2 variants. Intracytoplasmic sperm injection (ICSI) was administered to three affected patients.
We identified four novel SPEF2 variants, including one novel homozygous splicing site variant [NC_000005.10(NM_024867.4): c.4447 + 1G > A] of the SPEF2 gene in family 1, novel compound heterozygous nonsense variants [NC_000005.10(NM_024867.4): c.1339C > T (p.R447*) and NC_000005.10(NM_024867.4): c.1645G > T (p.E549*)] in family 2, and one novel homozygous missense variant [NC_000005.10(NM_024867.4): c.2524G > A (p.D842N)] in family 3. All the patients presented with male infertility and PCD/likely PCD. All variants were present at very low levels in public databases, predicted to be deleterious in silico prediction tools, and were further confirmed deleterious by in vitro analyses. Ultrastructural analyses of the spermatozoa of the patients revealed the absence of the central pair complex in the sperm flagella. Immunostaining of the spermatozoa and respiratory cilia of the patients validated the pathogenicity of the SPEF2 variants. All patients carrying SPEF2 variants underwent one ICSI cycle and delivered healthy infants.
Our study reported four novel pathogenic variants of SPEF2 in three male patients with infertility and PCD/PCD-like phenotypes, which not only extend the spectrum of SPEF2 mutations but also provide information for genetic counseling and treatment of such conditions.
... The gene annotation analysis of genomic regions under selection found some candidate genes involved in respiratory system functions ( Table 2). SPEF2 has been postulated to play an essential role in cilia assembly 73 . AD with fatal respiratory distress and fulminant interstitial pneumonia mainly occurs in American mink kits due to permissive and cytopathic replication of the virus in the lung type II pneumocytes 12,13 . ...
Aleutian disease (AD) is a multi-systemic infectious disease in American mink (Neogale vison) caused by Aleutian mink disease virus (AMDV). This study aimed to identify candidate regions and genes underlying selection for response against AMDV using whole-genome sequence (WGS) data. Three case–control selection signatures studies were conducted between animals (N = 85) producing high versus low antibody levels against AMDV, grouped by counter immunoelectrophoresis (CIEP) test and two enzyme-linked immunosorbent assays (ELISA). Within each study, selection signals were detected using fixation index (FST) and nucleotide diversity (θπ ratios), and validated by cross-population extended haplotype homozygosity (XP-EHH) test. Within- and between-studies overlapping results were then evaluated. Within-studies overlapping results indicated novel candidate genes related to immune and cellular responses (e.g., TAP2, RAB32), respiratory system function (e.g., SPEF2, R3HCC1L), and reproduction system function (e.g., HSF2, CFAP206) in other species. Between-studies overlapping results identified three large segments under strong selection pressure, including two on chromosome 1 (chr1:88,770–98,281 kb and chr1:114,133–120,473) and one on chromosome 6 (chr6:37,953–44,279 kb). Within regions with strong signals, we found novel candidate genes involved in immune and cellular responses (e.g., homologous MHC class II genes, ITPR3, VPS52) in other species. Our study brings new insights into candidate regions and genes controlling AD response.
... CFTR (1) Fig. 1 Genes governing different semen quality traits in bulls identified by candidate gene approach. The number within parentheses signifies the number of mutation(s) in a gene spermatogenesis to be compromised, as well as the formation of tail abnormalities and, as a result, sperm motility (Sironen et al., 2011). TNP2 is a transitional protein that plays a role in histone removal and chromatin condensation during sperm production and maturation (Gao et al., 2014). ...
Poor semen profile reflected by suboptimum fertility statistics is a concern in bulls reared for breeding purpose. A critical review of research on candidate genes and proteins associated with semen quality traits will be useful to understand the progress of molecular marker development for bull semen quality traits. Here, we have tabulated and classified candidate genes and proteins associated with bull semen quality based on a literature survey. A total of 175 candidate genes are associated with semen quality traits in various breeds of cattle. Several studies using candidate gene approach have identified 26 genes carrying a total of 44 single nucleotide polymorphisms. Furthermore, nine genome-wide association studies (GWASes) have identified 150 candidate genes using bovine single nucleotide polymorphisms (SNP) chips. Three genes, namely membrane-associated ring-CH-type finger 1 (MARCH1), platelet-derived growth factor receptor beta, and phosphodiesterase type 1, were identified commonly in two GWASes, which, especially MARCH1, are required to explore their regulatory roles in bull semen quality in in-depth studies. With the advancement of high-throughput-omic technologies, more candidate genes associated with bull semen quality may be identified in the future. Therefore, the functional significance of candidate genes and proteins need to be delved further into future investigations to augment bull semen quality.
... Having observed differences in cilia ultrastructure in the airway, we examined the tissue specificity of phenotypes in mice with different Dnaaf5 genotypes. Motile cilia dysfunction is associated with upper airway sinus disease in patients and mice with knockout of PCD gene (37)(38)(39)(40)(41). Unlike humans, mice deficient in PCD genes are not reported to develop lung disease. ...
... Survival served as a quantifiable measure of allelic fitness. Mice with PCD gene knockout uniformly develop early hydrocephalus and death by two months of age (or earlier) (39)(40)(41). The onset of hydrocephalus occurred in mice by three weeks of age, and newborn animals did not show significant differences in lateral ventricle volumes, though there was a trend of increased volumes in MIS/MIS animals. ...
DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partial preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. While transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. Together, these findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies.
... Having observed differences in cilia ultrastructure in the airway, we examined the tissue specificity of phenotypes in mice with different Dnaaf5 genotypes. Motile cilia dysfunction is associated with upper airway sinus disease in patients and mice with knockout of PCD gene (37)(38)(39)(40)(41). Unlike humans, mice deficient in PCD genes are not reported to develop lung disease. ...
... Survival served as a quantifiable measure of allelic fitness. Mice with PCD gene knockout uniformly develop early hydrocephalus and death by two months of age (or earlier) (39)(40)(41). While rare in humans with PCD, development of hydrocephalus is a well-established phenotype in mice, which we confirm is consistent with defective function and ultrastructure of cilia on the ventricular ependyma (48). ...
DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift null deletion in Dnaaf5 . Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partial preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. While transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. Together, these findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies.
Brief Summary
A mouse model of human DNAAF5 primary ciliary dyskinesia variants reveals gene dosage effects of mutant alleles and tissue-specific molecular requirements for cilia motor assembly.
... When we focused on the DEGs in Cluster 7, we found a testis development-related gene, SOX9, and a luteinizing hormone synthesis regulation gene, NR5A2 [69,70,78]. Interestingly, we identified a set of spermatogenesis-related genes, DNAI1, SPEF2, and TACR3, that maintained low expression levels in the sexreversed chickens [79][80][81][82][83]. Considering the function of DNAI1 and SPEF2, which regulate the development and movement of flagella, we speculated that the low expression of these spermatogenesis-related genes caused the maldevelopment of germ cells in the sex-reversed chickens [79,82]. ...
... When we focused on the DEGs in Cluster 7, we found a testis development-related gene, SOX9, and a luteinizing hormone synthesis regulation gene, NR5A2 [69,70,78]. Interestingly, we identified a set of spermatogenesis-related genes, DNAI1, SPEF2, and TACR3, that maintained low expression levels in the sexreversed chickens [79][80][81][82][83]. Considering the function of DNAI1 and SPEF2, which regulate the development and movement of flagella, we speculated that the low expression of these spermatogenesis-related genes caused the maldevelopment of germ cells in the sex-reversed chickens [79,82]. ...
Background
Sex determination and differentiation are complex and delicate processes. In female chickens, the process of sex differentiation is sensitive and prone to be affected by the administration of aromatase inhibitors, which result in chicken sex reversal and infertility. However, the molecular mechanisms underlying sex differentiation and infertility in chicken sex reversal remain unclear. Therefore, we established a sex-reversed chicken flock by injecting an aromatase inhibitor, fadrozole, and constructed relatively high-resolution profiles of the gene expression and chromatin accessibility of embryonic gonads.
Results
We revealed that fadrozole affected the transcriptional activities of several genes, such as DMRT1 , SOX9 , FOXL2 , and CYP19A1 , related to sex determination and differentiation, and the expression of a set of gonadal development-related genes, such as FGFR3 and TOX3 , by regulating nearby open chromatin regions in sex-reversed chicken embryos. After sexual maturity, the sex-reversed chickens were confirmed to be infertile, and the possible causes of this infertility were further investigated. We found that the structure of the gonads and sperm were greatly deformed, and we identified several promising genes related to spermatogenesis and infertility, such as SPEF2 , DNAI1 , and TACR3 , through RNA-seq.
Conclusions
This study provides clear insights into the exploration of potential molecular basis underlying sex differentiation and infertility in sex-reversed chickens and lays a foundation for further research into the sex development of birds.
... IFT20, IFT52, IFT122 and SPEF2 were selected for immunofluorescence experiments due to their potential relations with CFAP54 revealed by STRING website and their essential roles for axoneme assembly in spermatogenesis according to several previous studies. [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] ...
... SPEF2 plays a key role in flagella assembly, according to the STRING database and previous studies. [22][23][24][25][26][27][28][29] We found that the SPEF2 signal was almost absent in the spermatozoa from individuals harbouring biallelic CFAP54 variants (figure 3). IFT20, IFT52 and IFT122 were also examined due to their important roles in the intraflagellar transport (IFT) system, which were responsible for the deformation of sperm cells. ...
Background
Spermatogenic impairments can lead to male infertility by different pathological conditions, such as multiple morphological abnormalities of the sperm flagella (MMAF) and non-obstructive azoospermia (NOA). Genetic factors are involved in impaired spermatogenesis.
Methods and results
Here, we performed genetic analyses through whole-exome sequencing in a cohort of 334 Han Chinese probands with severe MMAF or NOA. Biallelic variants of CFAP54 were identified in three unrelated men, including one homozygous frameshift variant (c.3317del, p.Phe1106Serfs*19) and two compound heterozygous variants (c.878G>A, p.Arg293His; c.955C>T, p.Arg319Cys and c.4885C>T, p.Arg1629Cys; c.937G>A, p.Gly313Arg). All of the identified variants were absent or extremely rare in the public human genome databases and predicted to be damaging by bioinformatic tools. The men harbouring CFAP54 mutations exhibited abnormal sperm morphology, reduced sperm concentration and motility in ejaculated semen. Significant axoneme disorganisation and other ultrastructure abnormities were also detected inside the sperm cells from men harbouring CFAP54 mutations. Furthermore, immunofluorescence assays showed remarkably reduced staining of four flagellar assembly-associated proteins (IFT20, IFT52, IFT122 and SPEF2) in the spermatozoa of CFAP54- deficient men. Notably, favourable clinical pregnancy outcomes were achieved with sperm from men carrying CFAP54 mutations after intracytoplasmic sperm injection treatment.
Conclusion
Our genetic analyses and experimental observations revealed that biallelic deleterious mutations of CFAP54 can induce severe MMAF and NOA in humans.
... SPEF2 is a component of the central pair complex (CPC), which has been confirmed by flagella lacking CP microtubules in SPEF2deficient patients (Cindrić et al., 2020). SPEF2 was initially found to be related to PCD (Sironen et al., 2011), and several studies have then revealed its relationship with MMAF Sha et al., 2019;Liu et al., 2020a). As a central pair complex protein, SPEF2 has been reported to be localized in the C1b projection (Zhang and Mitchell, 2004) and to interact with other CPC proteins, such as CFAP221 and CFAP54 (McKenzie and Lee, 2020). ...
Sperm carries male genetic information, and flagella help move the sperm to reach oocytes. When the ultrastructure of the flagella is abnormal, the sperm is unable to reach the oocyte and achieve insemination. Multiple morphological abnormalities of sperm flagella (MMAF) is a relatively rare idiopathic condition that is mainly characterized by multiple defects in sperm flagella. In the last decade, with the development of high-throughput DNA sequencing approaches, many genes have been revealed to be related to MMAF. However, the differences in sperm phenotypes and reproductive outcomes in many cases are attributed to different pathogenic genes or different pathogenic mutations in the same gene. Here, we will review information about the various phenotypes resulting from different pathogenic genes, including sperm ultrastructure and encoding proteins with their location and functions as well as assisted reproductive technology (ART) outcomes. We will share our clinical detection and diagnosis experience to provide additional clinical views and broaden the understanding of this disease.
... Targeted immunostaining showed strong endogenous expression and ciliary localization of key motile cilia proteins (Fig. 1, D and E). These include dynein arm components dynein axonemal intermediate chain DNAI1, light intermediate chain DNALI1, and heavy chain DNAH5 (24)(25)(26), which are distributed continuously but non-uniformly along the length of the cilium; growth arrest-specific protein GAS8 that forms the nexindynein regulatory complex (N-DRC) (27,28) and is detected in the axonemal lumen; and the spermatogenesis and flagellar assembly protein SPEF2 (29,30) and kinesin family member KIF9 (31,32), which are known to associate with the central pair and were found in the axoneme and base. The differential enrichment of these proteins in different ciliary compartments likely reflects their unique roles in motile cilia and flagellar assembly and function. ...
Primary cilia are specialized cell-surface organelles that mediate sensory perception and, in contrast to motile cilia and flagella, are thought to lack motility function. Here, we show that primary cilia in human and mouse pancreatic islets exhibit movement that is required for glucose-dependent insulin secretion. Islet primary cilia contain motor proteins conserved from those found in classic motile cilia, and their three-dimensional motion is dynein-driven and dependent on adenosine 5´-triphosphate and glucose metabolism. Inhibition of cilia motion blocks beta cell calcium influx and insulin secretion. Human beta cells have enriched ciliary gene expression, and motile cilia genes are altered in type 2 diabetes. Our findings redefine primary cilia as dynamic structures having both sensory and motile function and establish that pancreatic islet cilia movement plays a regulatory role in insulin secretion.
... Like individuals with HYDIN and SPEF2 mutations, all individuals with CFAP74 mutations display no laterality defects, which is consistent with findings in the corresponding mouse models. In all mouse models for CP defects, no situs abnormalities have been reported, but they showed airway disease consistent with PCD (13). The lack of laterality defects in CFAP74 mutations makes it even more difficult to consider an underlying PCD diagnosis. ...
