Primary Ciliary Dyskinesia Caused by Homozygous Mutation in DNAL1, Encoding Dynein Light Chain 1
2011; Elsevier BV; Volume: 88; Issue: 5 Linguagem: Inglês
10.1016/j.ajhg.2011.03.018
ISSN1537-6605
AutoresM. Mazor, Soliman Alkrinawi, Vered Chalifa‐Caspi, Esther Manor, Val C. Sheffield, Micha Aviram, Ruti Parvari,
Tópico(s)Epigenetics and DNA Methylation
ResumoIn primary ciliary dyskinesia (PCD), genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility. The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Although it has long been suspected that mutations in DNAL1 encoding the ODA light chain1 might cause PCD such mutations were not found. We demonstrate here that a homozygous point mutation in this gene is associated with PCD with absent or markedly shortened ODA. The mutation (NM_031427.3: c.449A>G; p.Asn150Ser) changes the Asn at position150, which is critical for the proper tight turn between the β strand and the α helix of the leucine-rich repeat in the hydrophobic face that connects to the dynein heavy chain. The mutation reduces the stability of the axonemal dynein light chain 1 and damages its interactions with dynein heavy chain and with tubulin. This study adds another important component to understanding the types of mutations that cause PCD and provides clinical information regarding a specific mutation in a gene not yet known to be associated with PCD. In primary ciliary dyskinesia (PCD), genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility. The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Although it has long been suspected that mutations in DNAL1 encoding the ODA light chain1 might cause PCD such mutations were not found. We demonstrate here that a homozygous point mutation in this gene is associated with PCD with absent or markedly shortened ODA. The mutation (NM_031427.3: c.449A>G; p.Asn150Ser) changes the Asn at position150, which is critical for the proper tight turn between the β strand and the α helix of the leucine-rich repeat in the hydrophobic face that connects to the dynein heavy chain. The mutation reduces the stability of the axonemal dynein light chain 1 and damages its interactions with dynein heavy chain and with tubulin. This study adds another important component to understanding the types of mutations that cause PCD and provides clinical information regarding a specific mutation in a gene not yet known to be associated with PCD. Cilia are ancient, evolutionarily conserved organelles that project from the surfaces of most cells to perform diverse biological roles, including whole-cell locomotion; movement of fluid; chemo-, mechano-, and photosensation; and paracrine signal transduction. These organelles can be classified according to the arrangement of their microtubule cytoskeleton core, called an axoneme.1Satir P. Christensen S.T. Overview of structure and function of mammalian cilia.Annu. Rev. Physiol. 2007; 69: 377-400Crossref PubMed Scopus (639) Google Scholar The axoneme consists of nine outer-doublet microtubules, which are connected by nexin links and surround a central pair of microtubules (i.e., 9 + 2 pattern). In some cilia, the central pair of microtubules is absent (i.e., 9 + 0 pattern). The 9 + 0 primary cilia are immotile, except in the embryonic node, where they are involved in left-right asymmetry.2Basu B. Brueckner M. Cilia multifunctional organelles at the center of vertebrate left-right asymmetry.Curr. Top. Dev. Biol. 2008; 85: 151-174Crossref PubMed Scopus (102) Google Scholar The 9 + 2 motile cilia, structurally identical to spermatozoan flagella, are involved in the transport of extracellular fluids, as in the respiratory tract, where they propel mucus and therefore represent the first line of airway defenses.1Satir P. Christensen S.T. Overview of structure and function of mammalian cilia.Annu. Rev. Physiol. 2007; 69: 377-400Crossref PubMed Scopus (639) Google Scholar Motile cilia are powered by outer dynein arms (ODAs) and inner dynein arms (IDAs), multiprotein ATPase complexes that are attached to the peripheral doublets and are essential for ciliary motion.1Satir P. Christensen S.T. Overview of structure and function of mammalian cilia.Annu. Rev. Physiol. 2007; 69: 377-400Crossref PubMed Scopus (639) Google Scholar For most motile cilia, additional accessory structures, for example radial spokes and central pair projections, are involved in regulating dynein-mediated motility. Primary ciliary dyskinesia (PCD [MIM 242650]) refers to a heterogeneous group of genetic disorders characterized by ultrastructural defects in the axonemal structure of the 9 + 2 motile cilia and sperm flagella.3Bush A. Chodhari R. Collins N. Copeland F. Hall P. Harcourt J. Hariri M. Hogg C. Lucas J. Mitchison H.M. et al.Primary ciliary dyskinesia: Current state of the art.Arch. Dis. Child. 2007; 92: 1136-1140Crossref PubMed Scopus (247) Google Scholar The incidence is estimated at 1:15,000–30,000,4Rott H.D. Kartagener's syndrome and the syndrome of immotile cilia.Hum. Genet. 1979; 46: 249-261Crossref PubMed Scopus (86) Google Scholar and there is a higher incidence in certain consanguineous and isolated populations.5Jeganathan D. Chodhari R. Meeks M. Faeroe O. Smyth D. Nielsen K. Amirav I. Luder A.S. Bisgaard H. Gardiner R.M. et al.Loci for primary ciliary dyskinesia map to chromosome 16p12.1-12.2 and 15q13.1-15.1 in Faroe Islands and Israeli Druze genetic isolates.J. Med. Genet. 2004; 41: 233-240Crossref PubMed Google Scholar, 6O'Callaghan C. Innate pulmonary immunity: Cilia.Pediatr. Pulmonol. Suppl. 2004; 26: 72-73Crossref PubMed Scopus (4) Google Scholar Clinical features reflect the distribution of immotile cilia in the body and include neonatal respiratory distress, chronic respiratory infections, sinusitis, and bronchiectasis because of deficient function of motile cilia in the upper and lower airways. Male and female subfertility occur as a result of defective sperm flagella and oviduct cilia, respectively. Occasionally, hydrocephalus occurs as a result of deficient ependymal cilia.7Kosaki K. Ikeda K. Miyakoshi K. Ueno M. Kosaki R. Takahashi D. Tanaka M. Torikata C. Yoshimura Y. Takahashi T. Absent inner dynein arms in a fetus with familial hydrocephalus-situs abnormality.Am. J. Med. Genet. A. 2004; 129A: 308-311Crossref PubMed Scopus (38) Google Scholar, 8Ibañez-Tallon I. Pagenstecher A. Fliegauf M. Olbrich H. Kispert A. Ketelsen U.P. North A. Heintz N. Omran H. Dysfunction of axonemal dynein heavy chain Mdnah5 inhibits ependymal flow and reveals a novel mechanism for hydrocephalus formation.Hum. Mol. Genet. 2004; 13: 2133-2141Crossref PubMed Scopus (248) Google Scholar In most families, there is an apparent randomization of left-right axis development; this randomization is proposed to result from the defective function of the embryonic node. This manifests in about half of patients as situs inversus or more severe laterality defects, such as cardiovascular abnormalities. The association of PCD and situs inversus is also referred to as Kartagener's syndrome (MIM 244400).9Storm van's Gravesande K. Omran H. Primary ciliary dyskinesia: Clinical presentation, diagnosis and genetics.Ann. Med. 2005; 37: 439-449Crossref PubMed Scopus (77) Google Scholar, 10Baker K. Beales P.L. Making sense of cilia in disease: The human ciliopathies.Am. J. Med. Genet. C. Semin. Med. Genet.C Semin Med Genet. 2009; 151C: 281-295Crossref PubMed Scopus (223) Google Scholar There is a large variation in the severity of the clinical phenotype. Clinical features of PCD can also mimic those of other diseases, such as cystic fibrosis, allergies or immunologic disorders. Studying the ciliary-beat pattern and frequency by direct microscopy on transnasal brushings and examining the axonemal structure of respiratory cilia by transmission electron microscopy allows diagnosis of PCD. Typical ultrastructural defects in PCD consist of total or partial absence of dynein arms, absence or dislocation of central tubules, defects of the radial spoke, and peripheral microtubule anomalies. However, in a subset of patients, no ultrastructural defect are observed.11Meeks M. Bush A. Primary ciliary dyskinesia (PCD).Pediatr. Pulmonol. 2000; 29: 307-316Crossref PubMed Scopus (123) Google Scholar, 12MacCormick J. Robb I. Kovesi T. Carpenter B. Optimal biopsy techniques in the diagnosis of primary ciliary dyskinesia.J. Otolaryngol. 2002; 31: 13-17Crossref PubMed Scopus (25) Google Scholar Additionally, in patients with PCD, nasal nitric oxide (nNO) levels are often extremely low in comparison to controls. A nNO concentration of 16 cM and 14.4 Mb) on chromosome 14 was found to be shared by the two affected patients. To confirm linkage to this region, we tested all family members with both known polymorphic microsatellite markers and with additional markers developed for this purpose.27Levy-Litan V. Hershkovitz E. Avizov L. Leventhal N. Bercovich D. Chalifa-Caspi V. Manor E. Buriakovsky S. Hadad Y. Goding J. Parvari R. Autosomal-recessive hypophosphatemic rickets is associated with an inactivation mutation in the ENPP1 gene.Am. J. Hum. Genet. 2010; 86: 273-278Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar Linkage was identified to chromosomal locus 14q24.2-q31.3, chr14: 70,128,727–84,524,979 (NCBI Build 36.1) (Figure 1A). We calculated the multipoint Lod score by using the Pedtool server and assuming recessive inheritance with 99% penetrance and an incidence of 0.01 or 0.001 for the disease allele in the population, was 2.49. The linkage interval contains 84 genes; by using the ciliaproteome server, we found 22 of them. Among these, we considered DNAL1 to be the strongest candidate for PCD because it encodes the ortholog of the Chlamydomonas axonemal dynein light chain 1 (LC1), an ODA component that contains the molecular motors for ATP-dependent cilia movement. Therefore, we analyzed it first. Total RNA was extracted from lymphoblastoid cells of patient IV-1 with the EZ-RNA Reagent (Biologic Industries, Israel). Reverse transcriptase reactions were performed with the SuperScript II Reverse Transcriptase Synthesis Kit (Invitrogen). The whole cDNA coding region for DNAL1 was amplified in overlapping segments by PCR. No splice variants were detected. The PCR products were directly sequenced on an ABI PRISM 3100 DNA Analyzer with the BigDye Terminator v. 1.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA) according to the manufacturer's protocol. The primers that revealed the mutation were primer 5′-CCCTAGCAACCAGAGCAGTGA-3′ (forward) and primer 5′-AGGTTGGCATTACCAGTTTTG-3′ (reverse). We identified a homozygous nonsynonymous variation (NM_031427.3: c.449A>G; NP_113615.2: p.Asn150Ser) (Figure 2A ). Because the patient in family B is from the same population, although not known to be related to family A, we evaluated him for the p.Asn150Ser mutation by sequencing of a PCR product amplified from his genomic DNA (see below) and found to be homozygous for the mutation. His parents, who were similarly analyzed, are heterozygous for this variant. The patient's siblings are either heterozygous (IV-2) or homozygous normal (IV-3). Testing for the variation in all members of both families and controls was performed by restriction analysis because the variation creates a BsmFI restriction site that does not exist in the wild-type sequence. PCR amplification of genomic DNA, around exon 7 of the gene, was performed with the use of the primers 5′- CCTCCCATCCTGTACTGTCTTC-3′ (forward) and 5′- GCTTTTGGTCTAGGGAGAATCTT-3′ (reverse). BsmFI restriction analysis of the PCR amplicons generated differential cleavage products of the wild-type (fragments were 370 bp and, uncut, 495 bp) versus variation (fragments were 370 bp and, cut, 148 bp and 347 bp) allele. Fragments were separated by electrophoresis on 2% agarose gel (Figure 2B). This variation segregates as expected in the families. The mutation was found in the heterozygous state in only one individual and was not found in the homozygous state in any individuals out of 124 healthy Bedouin controls. We sought evidence for the pathogenic relevance of this variation by analyzing the conservation of the protein sequence. Sequence alignment of the human DNAL1 with other species orthologs demonstrated complete conservation of the leucine-rich-repeat (LRR) consensus domain. Asn at position150 in human DNAL1 is one of the LRR consensus residues (Figure 2C); it aligns with the Asn located between the β10 and α7 folds of the sixth LRR domain of the corresponding Chlamydomonas (LC1) ortholog.28Wu H. Maciejewski M.W. Marintchev A. Benashski S.E. Mullen G.P. King S.M. Solution structure of a dynein motor domain associated light chain.Nat. Struct. Biol. 2000; 7: 575-579Crossref PubMed Scopus (78) Google Scholar Human DNAL1 is highly conserved: alignment of its predicted 3D structure to the orthologous Chlamydomonas LC1 show minor differences, a loop instead of α7 helix and shortened C terminus.29Horváth J. Fliegauf M. Olbrich H. Kispert A. King S.M. Mitchison H. Zariwala M.A. Knowles M.R. Sudbrak R. Fekete G. et al.Identification and analysis of axonemal dynein light chain 1 in primary ciliary dyskinesia patients.Am. J. Respir. Cell Mol. Biol. 2005; 33: 41-47Crossref PubMed Scopus (37) Google Scholar We verified the effect of the change on the protein conformation by using the SWISS-MODEL tool and found that the replacement of Asn by Ser at position 150 indeed has a major effect on the structure of the protein, in that it disturbs the folding of the sixth LRR domain (Figure 2D). Next, we studied the effect of the mutation on the protein. We subcloned the mutated and normal protein sequences into the pc3myc plasmid to produce the DNAL1 protein fused at its N terminal to a Myc tag. In brief, we used RNA from lymphoblastoid cells of patient IV-1 in family A and lymphoblastoid cells of a healthy control to reverse transcribe (by using SuperScript II Reverse Transcriptase Synthesis kit [Invitrogen]) and amplify the whole coding sequence of DNAL1 by PCR with the primers EcoRI-F, 5′-GAATTCATGGCGAAAGCAACAACAATCAA-3′, and XhoI-R, 5′-CTCGAGGTTGTCTTCTTCCTCATCCCC-3′, by using the DreamTaq DNA Polymerase (Fermentas). The PCR product was restricted with EcoRI and Xho-I and subcloned into these sites of the plasmid. We sequenced the inserts with the flanking regions with the T7 and SP6 primers to ascertain insertion in frame with the Myc tag and to verify that no PCR mutations were introduced during the cloning process. HEK293T cells were transiently transfected with the constructs by using TransIT-LT1 reagent (Mirus) and lysed with RIPA lysis buffer 48 hr after transfection. The DNAL1-Myc protein levels were studied by immunoblot with a Myc antibody (gift of Noah Isakov). Although the transfection conditions were identical, there was a marked reduction (7-fold) in the quantity of the mutated protein in comparison to the normal amount, as estimated with the GAPDH protein as a loading control (not shown). We tested the possibility that the observed low level of the mutant protein is caused by its instability. Forty-eight hours after transfection of HEK293T with the DNAL1-Myc plasmid, cycloheximide (CMX, Sigma-Aldrich), an inhibitor of protein translational elongation in eukaryotes, was added (to a final concentration of 10 mg/ml). The quantities of mutated and control DNAL1-Myc proteins, starting from approximately equal quantities, were compared by immunoblot analysis at 3 and 6 hr after the addition of CMX. A representative experiment, demonstrating faster reduction in the quantity of the mutant protein than in that of the normal protein, is shown in Figure 3A . To quantify this reduction, we performed three independent experiments. The intensity of the immunoblot signals was measured with the Image Lab software (Bio-Rad). The densitometric ratio of DNAL1-Myc to GAPDH at the time of addition of CMX was established as 100%, and the ratio of the DNAL1-Myc to GAPDH at 3 and 6 hr was compared to time 0. Indeed, the quantity of the mutated protein was reduced by 40% and 94% at 3 and 6 hr, respectively, whereas the normal protein was reduced only by 10% and 30%. The difference between the normal and mutated protein at the two time points was evaluated by a two-tailed Student's t test, under an assumption of unequal variance (p < 0.01 and p < 0.001 at 3 hr and 6 hr, respectively) (Figure 3B). In humans, dynein is composed of two heavy chains (∼500 kD), two intermediate chains (70–125 kD), and several light chains (15–30 kD).30Pazour G.J. Agrin N. Walker B.L. Witman G.B. Identification of predicted human outer dynein arm genes: Candidates for primary ciliary dyskinesia genes.J. Med. Genet. 2006; 43: 62-73Crossref PubMed Scopus (86) Google Scholar The heavy chains form the globular heads and stems of the complex. In Chlamydomonas two molecules of LC1 are tightly associated with AAA nucleotide-binding domains of the γ heavy chain.28Wu H. Maciejewski M.W. Marintchev A. Benashski S.E. Mullen G.P. King S.M. Solution structure of a dynein motor domain associated light chain.Nat. Struct. Biol. 2000; 7: 575-579Crossref PubMed Scopus (78) Google Scholar, 31Benashski S.E. Patel-King R.S. King S.M. Light chain 1 from the Chlamydomonas outer dynein arm is a leucine-rich repeat protein associated with the motor domain of the gamma heavy chain.Biochemistry. 1999; 38: 7253-7264Crossref PubMed Scopus (71) Google Scholar The interaction of the LC1 is mediated through a central LRR section that folds as a cylindrical, right-handed spiral formed from six β-β-α motifs. This central cylinder is flanked by terminal helical subdomains. The C-terminal helical domain juts out from the cylinder and is proposed to interact with the dynein heavy chain. The position of the mutated aspargine is just at the last LRR consensus that stabilizes the turn between the β strand and the α helix. In humans, the ortholog of the γ heavy chain is encoded by two genes, DNAH5 and DNAH8,30Pazour G.J. Agrin N. Walker B.L. Witman G.B. Identification of predicted human outer dynein arm genes: Candidates for primary ciliary dyskinesia genes.J. Med. Genet. 2006; 43: 62-73Crossref PubMed Scopus (86) Google Scholar and a similar association of DNAL1 to DNAH5 was also demonstrated29Horváth J. Fliegauf M. Olbrich H. Kispert A. King S.M. Mitchison H. Zariwala M.A. Knowles M.R. Sudbrak R. Fekete G. et al.Identification and analysis of axonemal dynein light chain 1 in primary ciliary dyskinesia patients.Am. J. Respir. Cell Mol. Biol. 2005; 33: 41-47Crossref PubMed Scopus (37) Google Scholar. It was also shown that Chlamydomonas LC1 interacts directly with tubulin and tethers the γ heavy motor unit to the A tubule of the outer-doublet microtubules within the axoneme31Benashski S.E. Patel-King R.S. King S.M. Light chain 1 from the Chlamydomonas outer dynein arm is a leucine-rich repeat protein associated with the motor domain of the gamma heavy chain.Biochemistry. 1999; 38: 7253-7264Crossref PubMed Scopus (71) Google Scholar, 32Patel-King R.S. King S.M. An outer arm dynein light chain acts in a conformational switch for flagellar motility.J. Cell Biol. 2009; 186: 283-295Crossref PubMed Scopus (44) Google Scholar (schematically presented in Figure 4A ). To verify whether t
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