Primary ciliary dyskinesia caused by homozygous mutation in DNAL1, encoding dynein light chain 1

Abstract

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.

Figure 1

PCD Bedouin Pedigrees, Fine Mapping of the Chromosome 14 Locus in Pedigree A, and Absence of the ODAs in Patient Cilia (A) The haplotype analysis based on microsatellite markers from 14q24.2-q31.3 revealed a founder haplotype (gray bar) for which the patients are homozygous in the critical region harboring DNAL1. The numbers of the patients correspond to those presented in Table 1. Patient IV-1 of family B was identified by analysis of the mutation. Filled symbols represent the homozygote for the mutation and a double line indicates a consanguineous union. The two lines indicate the crossing events delimiting the homozygous interval shared by the two patients. Markers rs17176306 and rs41471545 define the minimal homozygosity locus associated with the disease. (B) Transmission electron micrograph of nasal ciliary epithelium from patient IV-1 of family A in comparison to a control (sibling IV-3 of family A). The arrows in the micrographs point to the ODA that is missing in the patient.

Figure 2

Identification of the DNAL1 Mutation (A) Sequence of the genomic DNA corresponding to the c.449A>G mutation resulting in p.Asn150Ser. Patients were homozygous for the mutation; parents and siblings carrying the founder haplotype were heterozygous, and the healthy siblings without the founder haplotype were normal. (B) Restriction analysis for family A with BsmFI enzyme. The 148 bp band (marked with a red arrow) is apparent only in the mutated allele. The wild-type (fragments were 370 bp and, uncut, 495 bp) versus mutation (fragments were 370 bp and, cut, 148 bp and 347 bp) alleles. (C) Evolutionary conservation of the sixth LRR domain (conserved residues defining the LRR consensus are in bold). Position 150 of the mutated Asn is shown on top. (D) Structural prediction of the mutated LC1 protein (MUT) in comparison to a normal (WT) structure performed by the SWISS-MODEL tool. The change in structure is marked with a red arrow.

Figure 3

Effect of the Mutation on the Stability of the DNAL1 Protein (Top panel) A representative immunoblot analysis presenting DNAL1 protein at 0, 3, and 6 hr after addition of CMX. GAPDH is shown as a loading control. (Bottom panel) Quantification of the DNAL1-Myc stability relative to GAPDH for three experiments. 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 to GAPDH at 3 and 6 hr is compared to time 0. The data is presented as mean ± standard deviation. The difference between the normal and mutated protein at the two time points was evaluated by a two-tailed Student t test, assuming unequal variance. ^∗∗p = 0.006, ^∗∗∗p = 0.001. The following abbreviations are used: MUT, mutated DNAL1 protein; and WT, normal DNAL1 protein.

Figure 4

Interaction of the Mutated and Normal DNAL1 Protein with the Dynein Heavy Chain and with Tubulin (A) Schematic representation of the interaction of DNAL1 with the dynein heavy chain (DNAH) and tubulin. (B) Immunoprecipitation of dynein heavy chain. (a) Immunoprecipitation of the dynein heavy chain with the mutated (MUT) and normal (WT) DNAL1-Myc by Myc antibody probed with an anti-dynein heavy chain antibody. (b) The beads were incubated solely with the lysate mixture and axoneme and probed with an anti-dynein heavy chain antibody, thus presenting the background dynein heavy chain signal in the immunoprecipitation. (c) Input quantity of DNAL1-Myc for the reaction. (d) Input quantity of dynein heavy chain extracted from rat tracheas. The percentage in brackets refers to the loading on the gel of the lysate mixture with axoneme relative to the quantity used for the immunoprecipitation. The antibody used for the Immunoblot analysis is denoted at the right side. (C) Immunopercipitation of α-tubulin. (a) IP of α-tubulin with the mutated (MUT) and normal (WT) DNAL1-Myc by Myc antibody probed with an anti-α-tubulin antibody. (b) The beads were incubated solely with the lysate mixture and axoneme and probed with an anti-α-tubulin antibody, thus presenting the background of α-tubulin signal in the immunoprecipitation. (c) Input quantity of α-tubulin for the reaction. (d) Input quantity of DNAL1. The percentage in brackets refers to the loading on the gel of the lysate mixture with axoneme relative to the quantity used for the immunoprecipitation. The antibody used for the Western analysis is denoted at the right side. The stability of the mutated DNAL1 after the incubation with the axonemal extracts and with the primary antibody at 4°C was comparable to the normal DNAL1 (not shown).