Petra Pennekamp - Academia.edu (original) (raw)

Papers by Petra Pennekamp

Research paper thumbnail of Motility of efferent duct cilia aids passage of sperm cells through the male reproductive system

Molecular Human Reproduction

Motile cilia line the efferent ducts of the mammalian male reproductive tract. Several recent mou... more Motile cilia line the efferent ducts of the mammalian male reproductive tract. Several recent mouse studies have demonstrated that a reduced generation of multiple motile cilia in efferent ducts is associated with obstructive oligozoospermia and fertility issues. However, the sole impact of efferent duct cilia dysmotility on male infertility has not been studied so far either in mice or human. Using video microscopy, histological- and ultrastructural analyses, we examined male reproductive tracts of mice deficient for the axonemal motor protein DNAH5: this defect exclusively disrupts the outer dynein arm (ODA) composition of motile cilia but not the ODA composition and motility of sperm flagella. These mice have immotile efferent duct cilia that lack ODAs, which are essential for ciliary beat generation. Furthermore, they show accumulation of sperm in the efferent duct. Notably, the ultrastructure and motility of sperm from these males are unaffected. Likewise, human individuals wit...

Research paper thumbnail of Correction: Chromosomal evolution of the PKD1 gene family in primates (vol 8, pg 263, 2008)

ABSTRACT ABSTRACT: Correction to Kirsch S, Pasantes J, Wolf A, Bogdanova N, Munch C, Pennekamp P,... more ABSTRACT ABSTRACT: Correction to Kirsch S, Pasantes J, Wolf A, Bogdanova N, Munch C, Pennekamp P, Krawczak M, Dworniczak B, Schempp W: Chromosomal evolution of the PKD1 gene family in primates. BMC Evolutionary Biology 2008, 8:263 (doi:10.1186/1471-2148-8-263).

Research paper thumbnail of Diagnosis of Primary Ciliary Dyskinesia: a multi-center experience

Molecular pathology and functional genomics

Research paper thumbnail of Defects in the cytoplasmic assembly of axonemal dynein arms cause morphological abnormalities and dysmotility in sperm cells leading to male infertility

PLOS Genetics

Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for ... more Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for the generation and regulation of flagellar and ciliary beating. Studies in various ciliated model organisms have shown that axonemal dynein arms are first assembled in the cell cytoplasm and then delivered into axonemes during ciliogenesis. In humans, mutations in genes encoding for factors involved in this process cause structural and functional defects of motile cilia in various organs such as the airways and result in the hereditary disorder primary ciliary dyskinesia (PCD). Despite extensive knowledge about the cytoplasmic assembly of axonemal dynein arms in respiratory cilia, this process is still poorly understood in sperm flagella. To better define its clinical relevance on sperm structure and function, and thus male fertility, further investigations are required. Here we report the fertility status in different axonemal dynein preassembly mutant males (DNAAF2/ KTU, DNAAF4/ DYX1C1...

Research paper thumbnail of Registries and collaborative studies for primary ciliary dyskinesia in Europe

ERJ Open Research

Primary ciliary dyskinesia (PCD) is a rare inherited disease characterised by malfunctioning cili... more Primary ciliary dyskinesia (PCD) is a rare inherited disease characterised by malfunctioning cilia leading to a heterogeneous clinical phenotype with many organ systems affected. There is a lack of data on clinical presentation, prognosis and effectiveness of treatments, making it mandatory to improve the scientific evidence base.This article reviews the data resources that are available in Europe for clinical and epidemiological research in PCD, namely established national PCD registries and national cohort studies, plus two large collaborative efforts (the international PCD (iPCD) Cohort and the International PCD Registry), and discusses their strengths, limitations and perspectives.Denmark, Cyprus, Norway and Switzerland have national population-based registries, while England and France conduct multicentre cohort studies. Based on the data contained in these registries, the prevalence of diagnosed PCD is 3–7 per 100 000 in children and 0.2–6 per 100 000 in adults. All registries...

Research paper thumbnail of Homozygous loss-of-function mutations in MNS1 cause laterality defects and likely male infertility

PLOS Genetics

The clinical spectrum of ciliopathies affecting motile cilia spans impaired mucociliary clearance... more The clinical spectrum of ciliopathies affecting motile cilia spans impaired mucociliary clearance in the respiratory system, laterality defects including heart malformations, infertility and hydrocephalus. Using linkage analysis and whole exome sequencing, we identified two recessive loss-of-function MNS1 mutations in five individuals from four consanguineous families: 1) a homozygous nonsense mutation p.Arg242* in four males with laterality defects and infertility and 2) a homozygous nonsense mutation p.Gln203* in one female with laterality defects and recurrent respiratory infections additionally carrying homozygous mutations in DNAH5. Consistent with the laterality defects observed in these individuals, we found Mns1 to be expressed in mouse embryonic ventral node. Immunofluorescence analysis further revealed that MNS1 localizes to the axonemes of respiratory cilia as well as sperm flagella in human. In-depth ultrastructural analyses confirmed a subtle outer dynein arm (ODA) defect in the axonemes of respiratory epithelial cells resembling findings reported in Mns1deficient mice. Ultrastructural analyses in the female carrying combined mutations in MNS1 and DNAH5 indicated a role for MNS1 in the process of ODA docking (ODA-DC) in the distal respiratory axonemes. Furthermore, co-immunoprecipitation and yeast two hybrid analyses demonstrated that MNS1 dimerizes and interacts with the ODA docking complex component CCDC114. Overall, we demonstrate that MNS1 deficiency in humans causes laterality

Research paper thumbnail of Recessive DNAH9 Loss-of-Function Mutations Cause Laterality Defects and Subtle Respiratory Ciliary-Beating Defects

The American Journal of Human Genetics

Dysfunction of motile monocilia, altering the leftward flow at the embryonic node essential for d... more Dysfunction of motile monocilia, altering the leftward flow at the embryonic node essential for determination of left-right body asymmetry, is a major cause of laterality defects. Laterality defects are also often associated with reduced mucociliary clearance caused by defective multiple motile cilia of the airway and are responsible for destructive airway disease. Outer dynein arms (ODAs) are essential for ciliary beat generation, and human respiratory cilia contain different ODA heavy chains (HCs): the panaxonemally distributed g-HC DNAH5, proximally located b-HC DNAH11 (defining ODA type 1), and the distally localized b-HC DNAH9 (defining ODA type 2). Here we report loss-of-function mutations in DNAH9 in five independent families causing situs abnormalities associated with subtle respiratory ciliary dysfunction. Consistent with the observed subtle respiratory phenotype, high-speed video microscopy demonstrates distally impaired ciliary bending in DNAH9 mutant respiratory cilia. DNAH9-deficient cilia also lack other ODA components such as DNAH5, DNAI1, and DNAI2 from the distal axonemal compartment, demonstrating an essential role of DNAH9 for distal axonemal assembly of ODAs type 2. Yeast two-hybrid and co-immunoprecipitation analyses indicate interaction of DNAH9 with the ODA components DNAH5 and DNAI2 as well as the ODA-docking complex component CCDC114. We further show that during ciliogenesis of respiratory cilia, first proximally located DNAH11 and then distally located DNAH9 is assembled in the axoneme. We propose that the b-HC paralogs DNAH9 and DNAH11 achieved specific functional roles for the distinct axonemal compartments during evolution with human DNAH9 function matching that of ancient b-HCs such as that of the unicellular Chlamydomonas reinhardtii.

Research paper thumbnail of Mutations in C11orf70 Cause Primary Ciliary Dyskinesia with Randomization of Left/Right Body Asymmetry Due to Defects of Outer and Inner Dynein Arms

American journal of human genetics, Jan 3, 2018

Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, an... more Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, and randomization of the left/right body axis as a result of defects of motile cilia and sperm flagella. We identified loss-of-function mutations in the open-reading frame C11orf70 in PCD individuals from five distinct families. Transmission electron microscopy analyses and high-resolution immunofluorescence microscopy demonstrate that loss-of-function mutations in C11orf70 cause immotility of respiratory cilia and sperm flagella, respectively, as a result of the loss of axonemal outer (ODAs) and inner dynein arms (IDAs), indicating that C11orf70 is involved in cytoplasmic assembly of dynein arms. Expression analyses of C11orf70 showed that C11orf70 is expressed in ciliated respiratory cells and that the expression of C11orf70 is upregulated during ciliogenesis, similar to other previously described cytoplasmic dynein-arm assembly factors. Furthermore, C11orf70 shows an interaction with cy...

Research paper thumbnail of Evolutionary Proteomics Uncovers Ancient Associations of Cilia with Signaling Pathways

Developmental cell, Dec 18, 2017

Cilia are organelles specialized for movement and signaling. To infer when during evolution signa... more Cilia are organelles specialized for movement and signaling. To infer when during evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from sea urchins, sea anemones, and choanoflagellates. We identified 437 high-confidence ciliary candidate proteins conserved in mammals and discovered that Hedgehog and G-protein-coupled receptor pathways were linked to cilia before the origin of bilateria and transient receptor potential (TRP) channels before the origin of animals. We demonstrated that candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein identified in the cilia of all three organisms. ENKUR localizes to motile cilia and is required for patterning the left-right axis in vertebrates. Moreover, mutation of ENKUR causes situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancie...

Research paper thumbnail of Expression of the lysosomal membrane protein LMBD1 is essential for the initiation of gastrulation

Molecular Genetics and Metabolism

Research paper thumbnail of Interferon-gamma increases IL-6 production in human glioblastoma cell lines

Anticancer Research, 2000

Research paper thumbnail of Lmbrd1expression is essential for the initiation of gastrulation

Journal of Cellular and Molecular Medicine, 2016

The rare inborn cblF defect of cobalamin metabolism is caused by mutations in the limb region 1 (... more The rare inborn cblF defect of cobalamin metabolism is caused by mutations in the limb region 1 (LMBR1) domain containing 1 gene (LMBRD1). This defect is characterized by massive accumulation of free cobalamin in lysosomes and loss of mitochondrial succinyl-CoA synthesis and cytosolic methionine synthesis. Affected children suffer from heart defects, developmental delay and megaloblastic anemia. LMBRD1 encodes for LMBD1, a predicted lysosomal cobalamin transport protein. In this study, we determine the physiological function of LMBRD1 during embryogenesis by generating Lmbrd1 deficient mice using the Cre/LoxP system. Complete loss of Lmbrd1 function is accompanied by early embryonic death in mice. Whole mount in situ hybridization studies against bone morphogenetic protein 4 and Nodal show that initial formation of the proximal-distal axis is unaffected in early embryonic stages whereas the initiation of gastrulation is disturbed shown by the expression pattern of even skipped homeotic gene 1 and fibroblast growth factor 8 in Lmbrd1 deficient mice. We conclude that intact function of LMBD1 is essential for the initiation of gastrulation.

Research paper thumbnail of DNAH11 Localization in the Proximal Region of Respiratory Cilia Defines Distinct Outer Dynein Arm Complexes

American Journal of Respiratory Cell and Molecular Biology, 2016

Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respira... more Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respiratory disorders owing to impaired mucociliary clearance. Conventional transmission electron microscopy (TEM) is a diagnostic standard to identify ultrastructural defects in respiratory cilia but is not useful in approximately 30% of PCD cases, which have normal ciliary ultrastructure. DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its pathophysiology remains poorly understood. We therefore characterized DNAH11 in human respiratory cilia by immunofluorescence microscopy (IFM) in the context of PCD. We used whole-exome and targeted next-generation sequence analysis as well as Sanger sequencing to identify and confirm eight novel loss-offunction DNAH11 mutations. We designed and validated a monoclonal antibody specific to DNAH11 and performed highresolution IFM of both control and PCD-affected human respiratory cells, as well as samples from green fluorescent protein (GFP)-leftright dynein mice, to determine the ciliary localization of DNAH11. IFM analysis demonstrated native DNAH11 localization in only the proximal region of wild-type human respiratory cilia and loss of DNAH11 in individuals with PCD with certain loss-of-function DNAH11 mutations. GFP-left-right dynein mice confirmed proximal DNAH11 localization in tracheal cilia. DNAH11 retained proximal localization in respiratory cilia of individuals with PCD with distinct ultrastructural defects, such as the absence of outer dynein arms (ODAs). TEM tomography detected a partial reduction of ODAs in DNAH11-deficient cilia. DNAH11 mutations result in a subtle ODA defect in only the proximal region of respiratory cilia, which is detectable by IFM and TEM tomography.

Research paper thumbnail of Identification of distinct ciliary beat pattern abnormalities by high-speed video microscopy in primary ciliary dyskinesia

European Respiratory Journal, 2015

Introduction: Primary Ciliary Dyskinesia (PCD) is a rare, genetically heterogenous disorder leadi... more Introduction: Primary Ciliary Dyskinesia (PCD) is a rare, genetically heterogenous disorder leading to recurrent respiratory tract infections due to abnormal ciliary motility causing impaired mucociliary clearance. High-speed videomicroscopy analysis (HVMA) of ciliary beat pattern (CBP) and frequency as the current first-line diagnostic tool is an increasing challenge, because current studies widely expended the spectrum of HVMA findings from very subtle to markedly abnormal. Objectives: We assigned typical HVMA findings to genetically confirmed PCD individuals in order to identify typical patterns for various PCD variants. Methods: We assessed 1072 videos from nasal brush biopsies of 66 PCD individuals by HVMA as part of routine diagnostic work-up. HVMA findings were subsequently correlated with the genotype (biallelic mutations in 17 genes). Results: Distinct CBP correlated well with genetic findings, which allows the classification of typical HVMA findings for various genetic groups: Respiratory cilia with outer dynein arm defects (ODA) showed minimal residual movements with a minority of cilia being completely immotile. Cilia with combined inner (IDA) and ODA defects were completely immotile. Defects of the central pair apparatus resulted in a rigid and uncoordinated CBP. Combined IDA and microtubular disorganization defects resulted in a hyperkinetic, very stiff and vibratory CBP. Nexin link defects showed an almost regular CBP with only slightly reduced beating amplitude. Conclusion: This study improves clinical PCD diagnostics by classifying different PCD subtypes using HVMA as the first-line diagnostic tool and facilitates the subsequent diagnostics.

Research paper thumbnail of Autophagy Induces Prosenescent Changes in Proximal Tubular S3 Segments

Journal of the American Society of Nephrology : JASN, Jan 20, 2015

Evidence suggests that autophagy promotes the development of cellular senescence. Because cellula... more Evidence suggests that autophagy promotes the development of cellular senescence. Because cellular senescence contributes to renal aging and promotes the progression from AKI to CKD, we investigated the potential effect of tubular autophagy on senescence induction. Compared with kidneys from control mice, kidneys from mice with conditional deletion of autophagy-related 5 (Atg5) for selective ablation of autophagy in proximal tubular S3 segments (Atg5(Δ) (flox/) (Δ) (flox)) presented with significantly less tubular senescence, reduced interstitial fibrosis, and superior renal function 30 days after ischemia/reperfusion injury. To correlate this long-term outcome with differences in the early injury process, kidneys were analyzed 2 hours and 3 days after reperfusion. Notably, compared with kidneys of control mice, Atg5(Δ) (flox/) (Δ) (flox) kidneys showed more cell death in outer medullary S3 segments at 2 hours but less tubular damage and inflammation at day 3. These data suggest tha...

Research paper thumbnail of 509 Noninvasive cardiovascular phenotyping of PKD2 — Knock-out-mice

European Journal of Echocardiography, 1999

Research paper thumbnail of Ciliary beat pattern and frequency in genetic variants of primary ciliary dyskinesia

The European respiratory journal, 2014

Primary ciliary dyskinesia (PCD) is a rare genetic disorder leading to recurrent respiratory trac... more Primary ciliary dyskinesia (PCD) is a rare genetic disorder leading to recurrent respiratory tract infections. High-speed video-microscopy analysis (HVMA) of ciliary beating, currently the first-line diagnostic tool for PCD in most centres, is challenging because recent studies have expanded the spectrum of HVMA findings in PCD from grossly abnormal to very subtle. The objective of this study was to describe the diversity of HVMA findings in genetically confirmed PCD individuals. HVMA was performed as part of the routine work-up of individuals with suspected PCD. Subsequent molecular analysis identified biallelic mutations in the PCD-related genes of 66 individuals. 1072 videos of these subjects were assessed for correlation with the genotype. Biallelic mutations (19 novel) were found in 17 genes: DNAI1, DNAI2, DNAH5, DNAH11, CCDC103, ARMC4, KTU/DNAAF2, LRRC50/DNAAF1, LRRC6, DYX1C1, ZMYND10, CCDC39, CCDC40, CCDC164, HYDIN, RSPH4A and RSPH1. Ciliary beat pattern variations correlated...

Research paper thumbnail of Situs inversus and ciliary abnormalities: 20 years later, what is the connection?

Cilia, 2015

Heterotaxy (also known as situs ambiguous) and situs inversus totalis describe disorders of later... more Heterotaxy (also known as situs ambiguous) and situs inversus totalis describe disorders of laterality in which internal organs do not display their typical pattern of asymmetry. First described around 1600 by Girolamo Fabrizio, numerous case reports about laterality disorders in humans were published without any idea about the underlying cause. Then, in 1976, immotile cilia were described as the cause of a human syndrome that was previously clinically described, both in 1904 by AK Siewert and in 1933 by Manes Kartagener, as an association of situs inversus with chronic sinusitis and bronchiectasis, now commonly known as Kartagener's syndrome. Despite intense research, the underlying defect of laterality disorders remained unclear. Nearly 20 years later in 1995, Björn Afzelius discussed five hypotheses to explain the connection between ciliary defects and loss of laterality control in a paper published in the International Journal of Developmental Biology asking: 'Situs inversus and ciliary abnormalities: What is the connection?'. Here, nearly 20 research years later, we revisit some of the key findings that led to the current knowledge about the connection between situs inversus and ciliary abnormalities.

Research paper thumbnail of Inducible Cre/loxP Recombination in the Mouse Proximal Tubule

Nephron Experimental Nephrology, 2007

Transgenic technologies in mice became invaluable experimental tools to identify the in vivo func... more Transgenic technologies in mice became invaluable experimental tools to identify the in vivo function of proteins. However, conventional knockout technology often results in embryonic lethality and because genes are frequently expressed in multiple cell types, the resulting knockout phenotypes can be complex and difficult or impossible to dissect. These issues are particularly important for gene-targeting strategies used to examine renal function. The kidney contains quite a number of different cell types, the function of many of which impacts that of other renal cells. To avoid these limitations conditional knockout strategies have been designed. As one important part of this system we describe the development of a mouse line expressing the tamoxifen-activatable Cre recombinase Cre-ER(T2) specifically in renal proximal tubules. The expression of Cre-ER(T2) is driven by a promoter fragment of the mouse gamma-glutamyl transpeptidase type II gene resulting in the generation of the activatable recombinase in S3 segments of the proximal tubules from which over 80% were positive for Cre activity. In combination with loxP-based conditional mutant mice as a second tool this tamoxifen-inducible Cre-ER(T2) line allows functional analysis of a variety of genes important for renal development and function in a precisely controlled spatiotemporal manner.

Research paper thumbnail of Cilia at the Node of Mouse Embryos Sense Fluid Flow for Left-Right Determination via Pkd2

Science, 2012

Distinguishing Right from Left In most vertebrates during embryonic development, rotational movem... more Distinguishing Right from Left In most vertebrates during embryonic development, rotational movement of the cilia within a structure in the embryo, known as the node, generates unidirectional flow required for future left-right asymmetry of the internal organs. The flow may transport a determinant molecule or provide mechanical force. However, it is not clear how the flow is sensed. Yoshiba et al. (p. 226 , published online 13 September; see the Perspective by Norris and Grimes ) show that nodal flow in mouse embryos is sensed by the cilia of perinodal cells located at the edge of the node, in a manner dependent on Pkd2, a Ca 2+ -permeable cation channel that has been implicated in polycystic kidney disease in humans.

Research paper thumbnail of Motility of efferent duct cilia aids passage of sperm cells through the male reproductive system

Molecular Human Reproduction

Motile cilia line the efferent ducts of the mammalian male reproductive tract. Several recent mou... more Motile cilia line the efferent ducts of the mammalian male reproductive tract. Several recent mouse studies have demonstrated that a reduced generation of multiple motile cilia in efferent ducts is associated with obstructive oligozoospermia and fertility issues. However, the sole impact of efferent duct cilia dysmotility on male infertility has not been studied so far either in mice or human. Using video microscopy, histological- and ultrastructural analyses, we examined male reproductive tracts of mice deficient for the axonemal motor protein DNAH5: this defect exclusively disrupts the outer dynein arm (ODA) composition of motile cilia but not the ODA composition and motility of sperm flagella. These mice have immotile efferent duct cilia that lack ODAs, which are essential for ciliary beat generation. Furthermore, they show accumulation of sperm in the efferent duct. Notably, the ultrastructure and motility of sperm from these males are unaffected. Likewise, human individuals wit...

Research paper thumbnail of Correction: Chromosomal evolution of the PKD1 gene family in primates (vol 8, pg 263, 2008)

ABSTRACT ABSTRACT: Correction to Kirsch S, Pasantes J, Wolf A, Bogdanova N, Munch C, Pennekamp P,... more ABSTRACT ABSTRACT: Correction to Kirsch S, Pasantes J, Wolf A, Bogdanova N, Munch C, Pennekamp P, Krawczak M, Dworniczak B, Schempp W: Chromosomal evolution of the PKD1 gene family in primates. BMC Evolutionary Biology 2008, 8:263 (doi:10.1186/1471-2148-8-263).

Research paper thumbnail of Diagnosis of Primary Ciliary Dyskinesia: a multi-center experience

Molecular pathology and functional genomics

Research paper thumbnail of Defects in the cytoplasmic assembly of axonemal dynein arms cause morphological abnormalities and dysmotility in sperm cells leading to male infertility

PLOS Genetics

Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for ... more Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for the generation and regulation of flagellar and ciliary beating. Studies in various ciliated model organisms have shown that axonemal dynein arms are first assembled in the cell cytoplasm and then delivered into axonemes during ciliogenesis. In humans, mutations in genes encoding for factors involved in this process cause structural and functional defects of motile cilia in various organs such as the airways and result in the hereditary disorder primary ciliary dyskinesia (PCD). Despite extensive knowledge about the cytoplasmic assembly of axonemal dynein arms in respiratory cilia, this process is still poorly understood in sperm flagella. To better define its clinical relevance on sperm structure and function, and thus male fertility, further investigations are required. Here we report the fertility status in different axonemal dynein preassembly mutant males (DNAAF2/ KTU, DNAAF4/ DYX1C1...

Research paper thumbnail of Registries and collaborative studies for primary ciliary dyskinesia in Europe

ERJ Open Research

Primary ciliary dyskinesia (PCD) is a rare inherited disease characterised by malfunctioning cili... more Primary ciliary dyskinesia (PCD) is a rare inherited disease characterised by malfunctioning cilia leading to a heterogeneous clinical phenotype with many organ systems affected. There is a lack of data on clinical presentation, prognosis and effectiveness of treatments, making it mandatory to improve the scientific evidence base.This article reviews the data resources that are available in Europe for clinical and epidemiological research in PCD, namely established national PCD registries and national cohort studies, plus two large collaborative efforts (the international PCD (iPCD) Cohort and the International PCD Registry), and discusses their strengths, limitations and perspectives.Denmark, Cyprus, Norway and Switzerland have national population-based registries, while England and France conduct multicentre cohort studies. Based on the data contained in these registries, the prevalence of diagnosed PCD is 3–7 per 100 000 in children and 0.2–6 per 100 000 in adults. All registries...

Research paper thumbnail of Homozygous loss-of-function mutations in MNS1 cause laterality defects and likely male infertility

PLOS Genetics

The clinical spectrum of ciliopathies affecting motile cilia spans impaired mucociliary clearance... more The clinical spectrum of ciliopathies affecting motile cilia spans impaired mucociliary clearance in the respiratory system, laterality defects including heart malformations, infertility and hydrocephalus. Using linkage analysis and whole exome sequencing, we identified two recessive loss-of-function MNS1 mutations in five individuals from four consanguineous families: 1) a homozygous nonsense mutation p.Arg242* in four males with laterality defects and infertility and 2) a homozygous nonsense mutation p.Gln203* in one female with laterality defects and recurrent respiratory infections additionally carrying homozygous mutations in DNAH5. Consistent with the laterality defects observed in these individuals, we found Mns1 to be expressed in mouse embryonic ventral node. Immunofluorescence analysis further revealed that MNS1 localizes to the axonemes of respiratory cilia as well as sperm flagella in human. In-depth ultrastructural analyses confirmed a subtle outer dynein arm (ODA) defect in the axonemes of respiratory epithelial cells resembling findings reported in Mns1deficient mice. Ultrastructural analyses in the female carrying combined mutations in MNS1 and DNAH5 indicated a role for MNS1 in the process of ODA docking (ODA-DC) in the distal respiratory axonemes. Furthermore, co-immunoprecipitation and yeast two hybrid analyses demonstrated that MNS1 dimerizes and interacts with the ODA docking complex component CCDC114. Overall, we demonstrate that MNS1 deficiency in humans causes laterality

Research paper thumbnail of Recessive DNAH9 Loss-of-Function Mutations Cause Laterality Defects and Subtle Respiratory Ciliary-Beating Defects

The American Journal of Human Genetics

Dysfunction of motile monocilia, altering the leftward flow at the embryonic node essential for d... more Dysfunction of motile monocilia, altering the leftward flow at the embryonic node essential for determination of left-right body asymmetry, is a major cause of laterality defects. Laterality defects are also often associated with reduced mucociliary clearance caused by defective multiple motile cilia of the airway and are responsible for destructive airway disease. Outer dynein arms (ODAs) are essential for ciliary beat generation, and human respiratory cilia contain different ODA heavy chains (HCs): the panaxonemally distributed g-HC DNAH5, proximally located b-HC DNAH11 (defining ODA type 1), and the distally localized b-HC DNAH9 (defining ODA type 2). Here we report loss-of-function mutations in DNAH9 in five independent families causing situs abnormalities associated with subtle respiratory ciliary dysfunction. Consistent with the observed subtle respiratory phenotype, high-speed video microscopy demonstrates distally impaired ciliary bending in DNAH9 mutant respiratory cilia. DNAH9-deficient cilia also lack other ODA components such as DNAH5, DNAI1, and DNAI2 from the distal axonemal compartment, demonstrating an essential role of DNAH9 for distal axonemal assembly of ODAs type 2. Yeast two-hybrid and co-immunoprecipitation analyses indicate interaction of DNAH9 with the ODA components DNAH5 and DNAI2 as well as the ODA-docking complex component CCDC114. We further show that during ciliogenesis of respiratory cilia, first proximally located DNAH11 and then distally located DNAH9 is assembled in the axoneme. We propose that the b-HC paralogs DNAH9 and DNAH11 achieved specific functional roles for the distinct axonemal compartments during evolution with human DNAH9 function matching that of ancient b-HCs such as that of the unicellular Chlamydomonas reinhardtii.

Research paper thumbnail of Mutations in C11orf70 Cause Primary Ciliary Dyskinesia with Randomization of Left/Right Body Asymmetry Due to Defects of Outer and Inner Dynein Arms

American journal of human genetics, Jan 3, 2018

Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, an... more Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, and randomization of the left/right body axis as a result of defects of motile cilia and sperm flagella. We identified loss-of-function mutations in the open-reading frame C11orf70 in PCD individuals from five distinct families. Transmission electron microscopy analyses and high-resolution immunofluorescence microscopy demonstrate that loss-of-function mutations in C11orf70 cause immotility of respiratory cilia and sperm flagella, respectively, as a result of the loss of axonemal outer (ODAs) and inner dynein arms (IDAs), indicating that C11orf70 is involved in cytoplasmic assembly of dynein arms. Expression analyses of C11orf70 showed that C11orf70 is expressed in ciliated respiratory cells and that the expression of C11orf70 is upregulated during ciliogenesis, similar to other previously described cytoplasmic dynein-arm assembly factors. Furthermore, C11orf70 shows an interaction with cy...

Research paper thumbnail of Evolutionary Proteomics Uncovers Ancient Associations of Cilia with Signaling Pathways

Developmental cell, Dec 18, 2017

Cilia are organelles specialized for movement and signaling. To infer when during evolution signa... more Cilia are organelles specialized for movement and signaling. To infer when during evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from sea urchins, sea anemones, and choanoflagellates. We identified 437 high-confidence ciliary candidate proteins conserved in mammals and discovered that Hedgehog and G-protein-coupled receptor pathways were linked to cilia before the origin of bilateria and transient receptor potential (TRP) channels before the origin of animals. We demonstrated that candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein identified in the cilia of all three organisms. ENKUR localizes to motile cilia and is required for patterning the left-right axis in vertebrates. Moreover, mutation of ENKUR causes situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancie...

Research paper thumbnail of Expression of the lysosomal membrane protein LMBD1 is essential for the initiation of gastrulation

Molecular Genetics and Metabolism

Research paper thumbnail of Interferon-gamma increases IL-6 production in human glioblastoma cell lines

Anticancer Research, 2000

Research paper thumbnail of Lmbrd1expression is essential for the initiation of gastrulation

Journal of Cellular and Molecular Medicine, 2016

The rare inborn cblF defect of cobalamin metabolism is caused by mutations in the limb region 1 (... more The rare inborn cblF defect of cobalamin metabolism is caused by mutations in the limb region 1 (LMBR1) domain containing 1 gene (LMBRD1). This defect is characterized by massive accumulation of free cobalamin in lysosomes and loss of mitochondrial succinyl-CoA synthesis and cytosolic methionine synthesis. Affected children suffer from heart defects, developmental delay and megaloblastic anemia. LMBRD1 encodes for LMBD1, a predicted lysosomal cobalamin transport protein. In this study, we determine the physiological function of LMBRD1 during embryogenesis by generating Lmbrd1 deficient mice using the Cre/LoxP system. Complete loss of Lmbrd1 function is accompanied by early embryonic death in mice. Whole mount in situ hybridization studies against bone morphogenetic protein 4 and Nodal show that initial formation of the proximal-distal axis is unaffected in early embryonic stages whereas the initiation of gastrulation is disturbed shown by the expression pattern of even skipped homeotic gene 1 and fibroblast growth factor 8 in Lmbrd1 deficient mice. We conclude that intact function of LMBD1 is essential for the initiation of gastrulation.

Research paper thumbnail of DNAH11 Localization in the Proximal Region of Respiratory Cilia Defines Distinct Outer Dynein Arm Complexes

American Journal of Respiratory Cell and Molecular Biology, 2016

Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respira... more Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respiratory disorders owing to impaired mucociliary clearance. Conventional transmission electron microscopy (TEM) is a diagnostic standard to identify ultrastructural defects in respiratory cilia but is not useful in approximately 30% of PCD cases, which have normal ciliary ultrastructure. DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its pathophysiology remains poorly understood. We therefore characterized DNAH11 in human respiratory cilia by immunofluorescence microscopy (IFM) in the context of PCD. We used whole-exome and targeted next-generation sequence analysis as well as Sanger sequencing to identify and confirm eight novel loss-offunction DNAH11 mutations. We designed and validated a monoclonal antibody specific to DNAH11 and performed highresolution IFM of both control and PCD-affected human respiratory cells, as well as samples from green fluorescent protein (GFP)-leftright dynein mice, to determine the ciliary localization of DNAH11. IFM analysis demonstrated native DNAH11 localization in only the proximal region of wild-type human respiratory cilia and loss of DNAH11 in individuals with PCD with certain loss-of-function DNAH11 mutations. GFP-left-right dynein mice confirmed proximal DNAH11 localization in tracheal cilia. DNAH11 retained proximal localization in respiratory cilia of individuals with PCD with distinct ultrastructural defects, such as the absence of outer dynein arms (ODAs). TEM tomography detected a partial reduction of ODAs in DNAH11-deficient cilia. DNAH11 mutations result in a subtle ODA defect in only the proximal region of respiratory cilia, which is detectable by IFM and TEM tomography.

Research paper thumbnail of Identification of distinct ciliary beat pattern abnormalities by high-speed video microscopy in primary ciliary dyskinesia

European Respiratory Journal, 2015

Introduction: Primary Ciliary Dyskinesia (PCD) is a rare, genetically heterogenous disorder leadi... more Introduction: Primary Ciliary Dyskinesia (PCD) is a rare, genetically heterogenous disorder leading to recurrent respiratory tract infections due to abnormal ciliary motility causing impaired mucociliary clearance. High-speed videomicroscopy analysis (HVMA) of ciliary beat pattern (CBP) and frequency as the current first-line diagnostic tool is an increasing challenge, because current studies widely expended the spectrum of HVMA findings from very subtle to markedly abnormal. Objectives: We assigned typical HVMA findings to genetically confirmed PCD individuals in order to identify typical patterns for various PCD variants. Methods: We assessed 1072 videos from nasal brush biopsies of 66 PCD individuals by HVMA as part of routine diagnostic work-up. HVMA findings were subsequently correlated with the genotype (biallelic mutations in 17 genes). Results: Distinct CBP correlated well with genetic findings, which allows the classification of typical HVMA findings for various genetic groups: Respiratory cilia with outer dynein arm defects (ODA) showed minimal residual movements with a minority of cilia being completely immotile. Cilia with combined inner (IDA) and ODA defects were completely immotile. Defects of the central pair apparatus resulted in a rigid and uncoordinated CBP. Combined IDA and microtubular disorganization defects resulted in a hyperkinetic, very stiff and vibratory CBP. Nexin link defects showed an almost regular CBP with only slightly reduced beating amplitude. Conclusion: This study improves clinical PCD diagnostics by classifying different PCD subtypes using HVMA as the first-line diagnostic tool and facilitates the subsequent diagnostics.

Research paper thumbnail of Autophagy Induces Prosenescent Changes in Proximal Tubular S3 Segments

Journal of the American Society of Nephrology : JASN, Jan 20, 2015

Evidence suggests that autophagy promotes the development of cellular senescence. Because cellula... more Evidence suggests that autophagy promotes the development of cellular senescence. Because cellular senescence contributes to renal aging and promotes the progression from AKI to CKD, we investigated the potential effect of tubular autophagy on senescence induction. Compared with kidneys from control mice, kidneys from mice with conditional deletion of autophagy-related 5 (Atg5) for selective ablation of autophagy in proximal tubular S3 segments (Atg5(Δ) (flox/) (Δ) (flox)) presented with significantly less tubular senescence, reduced interstitial fibrosis, and superior renal function 30 days after ischemia/reperfusion injury. To correlate this long-term outcome with differences in the early injury process, kidneys were analyzed 2 hours and 3 days after reperfusion. Notably, compared with kidneys of control mice, Atg5(Δ) (flox/) (Δ) (flox) kidneys showed more cell death in outer medullary S3 segments at 2 hours but less tubular damage and inflammation at day 3. These data suggest tha...

Research paper thumbnail of 509 Noninvasive cardiovascular phenotyping of PKD2 — Knock-out-mice

European Journal of Echocardiography, 1999

Research paper thumbnail of Ciliary beat pattern and frequency in genetic variants of primary ciliary dyskinesia

The European respiratory journal, 2014

Primary ciliary dyskinesia (PCD) is a rare genetic disorder leading to recurrent respiratory trac... more Primary ciliary dyskinesia (PCD) is a rare genetic disorder leading to recurrent respiratory tract infections. High-speed video-microscopy analysis (HVMA) of ciliary beating, currently the first-line diagnostic tool for PCD in most centres, is challenging because recent studies have expanded the spectrum of HVMA findings in PCD from grossly abnormal to very subtle. The objective of this study was to describe the diversity of HVMA findings in genetically confirmed PCD individuals. HVMA was performed as part of the routine work-up of individuals with suspected PCD. Subsequent molecular analysis identified biallelic mutations in the PCD-related genes of 66 individuals. 1072 videos of these subjects were assessed for correlation with the genotype. Biallelic mutations (19 novel) were found in 17 genes: DNAI1, DNAI2, DNAH5, DNAH11, CCDC103, ARMC4, KTU/DNAAF2, LRRC50/DNAAF1, LRRC6, DYX1C1, ZMYND10, CCDC39, CCDC40, CCDC164, HYDIN, RSPH4A and RSPH1. Ciliary beat pattern variations correlated...

Research paper thumbnail of Situs inversus and ciliary abnormalities: 20 years later, what is the connection?

Cilia, 2015

Heterotaxy (also known as situs ambiguous) and situs inversus totalis describe disorders of later... more Heterotaxy (also known as situs ambiguous) and situs inversus totalis describe disorders of laterality in which internal organs do not display their typical pattern of asymmetry. First described around 1600 by Girolamo Fabrizio, numerous case reports about laterality disorders in humans were published without any idea about the underlying cause. Then, in 1976, immotile cilia were described as the cause of a human syndrome that was previously clinically described, both in 1904 by AK Siewert and in 1933 by Manes Kartagener, as an association of situs inversus with chronic sinusitis and bronchiectasis, now commonly known as Kartagener's syndrome. Despite intense research, the underlying defect of laterality disorders remained unclear. Nearly 20 years later in 1995, Björn Afzelius discussed five hypotheses to explain the connection between ciliary defects and loss of laterality control in a paper published in the International Journal of Developmental Biology asking: 'Situs inversus and ciliary abnormalities: What is the connection?'. Here, nearly 20 research years later, we revisit some of the key findings that led to the current knowledge about the connection between situs inversus and ciliary abnormalities.

Research paper thumbnail of Inducible Cre/loxP Recombination in the Mouse Proximal Tubule

Nephron Experimental Nephrology, 2007

Transgenic technologies in mice became invaluable experimental tools to identify the in vivo func... more Transgenic technologies in mice became invaluable experimental tools to identify the in vivo function of proteins. However, conventional knockout technology often results in embryonic lethality and because genes are frequently expressed in multiple cell types, the resulting knockout phenotypes can be complex and difficult or impossible to dissect. These issues are particularly important for gene-targeting strategies used to examine renal function. The kidney contains quite a number of different cell types, the function of many of which impacts that of other renal cells. To avoid these limitations conditional knockout strategies have been designed. As one important part of this system we describe the development of a mouse line expressing the tamoxifen-activatable Cre recombinase Cre-ER(T2) specifically in renal proximal tubules. The expression of Cre-ER(T2) is driven by a promoter fragment of the mouse gamma-glutamyl transpeptidase type II gene resulting in the generation of the activatable recombinase in S3 segments of the proximal tubules from which over 80% were positive for Cre activity. In combination with loxP-based conditional mutant mice as a second tool this tamoxifen-inducible Cre-ER(T2) line allows functional analysis of a variety of genes important for renal development and function in a precisely controlled spatiotemporal manner.

Research paper thumbnail of Cilia at the Node of Mouse Embryos Sense Fluid Flow for Left-Right Determination via Pkd2

Science, 2012

Distinguishing Right from Left In most vertebrates during embryonic development, rotational movem... more Distinguishing Right from Left In most vertebrates during embryonic development, rotational movement of the cilia within a structure in the embryo, known as the node, generates unidirectional flow required for future left-right asymmetry of the internal organs. The flow may transport a determinant molecule or provide mechanical force. However, it is not clear how the flow is sensed. Yoshiba et al. (p. 226 , published online 13 September; see the Perspective by Norris and Grimes ) show that nodal flow in mouse embryos is sensed by the cilia of perinodal cells located at the edge of the node, in a manner dependent on Pkd2, a Ca 2+ -permeable cation channel that has been implicated in polycystic kidney disease in humans.