ManuscriptbioRxiv preprint doi: https://doi.org/10.1101/288852; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 TITLE

2 Biallelic Mutations in LRRC56 encoding a protein associated with

3 intraflagellar transport, cause mucociliary clearance and laterality

4 defects

5 AUTHORS

6 Serge Bonnefoy,1,10 Christopher M. Watson,2,3,10 Kristin D. Kernohan,4 Moara Lemos,1

7 Sebastian Hutchinson1, James A. Poulter,3 Laura A. Crinnion,2,3 Chris O'Callaghan,5,6

8 Robert A. Hirst,5 Andrew Rutman,5 Lijia Huang,4 Taila Hartley,4 David Grynspan,7

9 Eduardo Moya,8 Chunmei Li,9 Ian M. Carr,3 David T. Bonthron,2,3 Michel Leroux,9

10 Care4Rare Canada Consortium,4 Kym M. Boycott,4 Philippe Bastin,1,10,* and Eamonn G.

11 Sheridan2,3,10,*

12

13 AFFILIATIONS

14 1: Trypanosome Cell Biology Unit & INSERM U1201, Institut Pasteur, 25, rue du Docteur

15 Roux, 75015 Paris, France

16 2: Yorkshire Regional Genetics Service, St. James’s University Hospital, Leeds, LS9 7TF,

17 United Kingdom

18 3: School of Medicine, University of Leeds, St. James’s University Hospital, Leeds, LS9

19 7TF, United Kingdom

20 4: Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa,

21 Ottawa, K1H 8L1, Canada

22 5: Centre for PCD Diagnosis and Research, Department of Infection, Immunity and

23 Inflammation, RKCSB, University of Leicester, Leicester, LE2 7LX, United Kingdom

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24 6: Respiratory, Critical Care & Anaesthesia, Institute of Child Health, University College

25 London & Great Ormond Street Children’s Hospital, 30 Guilford Street, London WC1N

26 1EH, United Kingdom

27 7: Department of Pathology, Children’s Hospital of Eastern Ontario, 401 Smyth Road,

28 Ottawa, K1H 8L1, Canada

29 8: Bradford Royal Infirmary, Bradford, West Yorkshire, BD9 6R, United Kingdom

30 9: Department of Molecular Biology and Biochemistry, and Centre for Cell Biology,

31 Development and Disease, Simon Fraser University, Burnaby, Canada

32 10: These authors contributed equally to this work

33

34 CORRESPONDENCE EMAILS

35 *Correspondence: [email protected] (E.G.S.), [email protected] (P.B)

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36 ABSTRACT

37 Defective motile cilia are responsible for a group of heterogeneous genetic conditions

38 characterised by dysfunction of the apparatus responsible for generating fluid flows.

39 Primary ciliary dyskinesia (PCD) is the prototype for such disorders and presents with

40 impaired pulmonary mucus clearance, susceptibility to chronic recurrent respiratory

41 infections, male infertility and laterality defects in about 50 % of patients. Here we

42 report biallelic variants in LRRC56 (also known as ODA8), identified in two unrelated

43 consanguineous families. The phenotype comprises laterality defects and chronic

44 pulmonary infections. High speed video microscopy of cultured patient epithelial cells

45 showed severely dyskinetic cilia, but no obvious ultra-structural abnormalities on

46 routine transmission electron microscopy (TEM). Further investigation revealed that

47 LRRC56 interacts with the intraflagellar transport (IFT) protein IFT88. The link to IFT

48 was interrogated in Trypanosoma brucei. In this protist, LRRC56 is recruited to the

49 cilium during axoneme construction, where it co-localises with IFT trains and facilitates

50 the addition of dynein arms to the distal end of the flagellum. In T. brucei carrying

51 LRRC56 null mutations, or a mutation (p.Leu259Pro) corresponding to the p.Leu140Pro

52 variant seen in one of the affected families, we observed abnormal ciliary beat patterns

53 and an absence of outer dynein arms restricted to the distal portion of the axoneme.

54 Together, our findings confirm that deleterious variants in LRRC56 result in a human

55 disease, and suggest this protein has a likely role in dynein transport during cilia

56 assembly that is evolutionarily important for cilia motility.

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57 MAIN TEXT

58

59 INTRODUCTION

60 Cilia are highly conserved eukaryotic organelles that are classified into motile and non-

61 motile forms. Defective motile cilia underlie the pathophysiology of patients with

62 impaired mucociliary clearance, which increases susceptibility to respiratory

63 complications, including sinusitis, bronchitis, pneumonia, and otitis media1. Chronic

64 infections frequently lead to progressive pulmonary damage and bronchiectasis.

65 Spermatozoal dysmotility in affected men causes infertility1. Approximately half of all

66 patients with the prototypical disease of motile cilia, Primary Ciliary Dyskinesia ( [MIM:

67 PS244400]), display laterality defects varying from partial to complete situs inversus, a

68 consequence of dysfunctional embryonic nodal cilia1. At least 36 genes currently

69 account for the heterogeneous genetic disorder PCD, which displays mainly autosomal

70 recessive inheritance and is characterised by cilia dyskinesis and structural defects

71 observed by TEM of a nasal biopsy. However this is not always the case. Variants in

72 CCNO ([MIM: 607752]) cause a reduction in the generation of motile cilia and result in a

73 similar phenotype ([CILD29 MIM:615872]), except that it is not associated with

74 laterality defects2. Mutations in DNAH11 ([MIM: 603339]) result in CILD7 ([MIM:

75 611884]) due to an abnormally rapid ciliary beat frequency without a discernible

76 structural defect3. These data highlight the molecular complexity underlying the

77 formation and function of cilia1.

78

79 Motile cilia and flagella, a hallmark of eukaryotes, display remarkable structural and

80 molecular conservation4. Most motile cilia exhibit a 9+2 configuration—a pair of single

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81 microtubules surrounded by 9 peripheral doublets. Connected to each doublet of

82 peripheral microtubules is an inner and outer dynein arm, consisting of multiple dynein

83 chains that provide ATPase-mediated energy for movement1. Dynein arms are

84 preassembled in the cytosol and are transported to an assembly site at the distal end of

85 the growing axoneme. This requires intraflagellar transport (IFT)5,6 7, an evolutionary

86 conserved bidirectional transport system that delivers axoneme building blocks8,9 such

87 as tubulin, to the flagellar tip10.

88

89 Here, we report biallelic variants in LRRC56 in patients with bronchiectasis and

90 laterality defects, and show human LRRC56 interacts with the IFT subunit, IFT88.

91 Functional studies using Trypanosoma brucei reveal that LRCC56 is recruited during

92 axoneme construction, associates with IFT trains, and is required for the addition of

93 dynein arms to the distal segment of the flagellum. Our findings add LRRC56 to an

94 expanding list of genes whose disruption results in an atypical ciliary phenotype and

95 reveal a mechanism whereby disruption of LRRC56 leads to defective IFT-dependent

96 targeting of dynein to cilia, and loss of ciliary motility.

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97 MATERIAL AND METHODS

98 Subject evaluation

99 Two unrelated families were independently ascertained with features suggestive of a

100 ciliopathy (Figure 1A). Family 1 consisted of a single affected female with PCD, whose

101 parents are first cousins of UK Pakistani ethnicity. She presented with chronic chest

102 infections; nasal biopsy was obtained and respiratory epithelial cultures prepared.

103 These were investigated by transmission electron microscopy (TEM) and high-speed

104 video microscopy. Family 2 consisted of two affected individuals, the offspring of first

105 cousin consanguineous parents from Kuwait, ascertained during pregnancy to have

106 lethal congenital heart disease. Both pregnancies were terminated and post mortem

107 pathological investigations were performed. The families provided signed informed

108 consent to participate in studies approved by the Leeds East Research Ethics Committee

109 (07/H1306/113; Family 1) and the Review Ethics Board of the Children’s Hospital of

110 Eastern Ontario (11/04E; Family2).

111

112 Genetic Analysis

113 In view of the consanguinity in both families, genetic analysis was performed under an

114 autosomal recessive model. Whole exome sequencing of genomic DNA was performed

115 at the University of Leeds and the Children’s Hospital of Eastern Ontario. Target

116 enrichment was performed, following manufacturer’s protocols, using SureSelect

117 hybridization capture reagents with v5 and v4 Human All Exon probes for Family 1 and

118 2, respectively (Agilent Technologies). Enriched library preparations were sequenced

119 on either HiSeq 2000/2500 platforms (Illumina) using paired end 100-bp reads.

120 Bioinformatic data processing was performed as previously described, with all variants

121 being reported against human reference genome build hg1911,12. Genomic regions

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122 corresponding to runs of autozygosity were identified from pipeline-produced variant

123 call format (VCF) files using the tool AgileMultiIdeogram (see Web Resources). These

124 were subsequently used to filter Alamut Batch-annotated variant reports. Additional

125 filtering criteria included the exclusion of common variants (those with a minor allele

126 frequency ≥1%) represented in the NHLBI Exome Variant Server (EVS) or an in-house

127 cohort of >1,500 control exomes and the exclusion of genes with biallelic functional

128 variants reported to the Exome Aggregation Consortium (ExAC). Pathogenic variants

129 and segregation in the families were confirmed using Sanger sequencing with an

130 ABI3130xl. Primer sequences and thermocycling conditions are available upon request.

131

132 RNA splicing assay

133 Total RNA was extracted from peripheral blood of the affected proband in Family 1

134 using the QIAamp RNA blood mini kit (Qiagen). A gene-specific primer spanning the

135 boundary of LRRC56 exons 10 and 11 (NM_198075.3) (dCTTGGCCAGCACCATGGGTGAG)

136 was used to perform first-strand cDNA synthesis with a SuperScriptTM II RT kit (Life

137 Technologies). Two PCR amplicons were generated from the resulting cDNA using an

138 exon 6 forward primer (dCAACCTGGACCAACTGAAGC) combined with either an exon 8

139 (dCCTCCAGGGTGAGCATGG) or exon 10 (dCCAGGTCCTCAGAAAGCAGG) reverse primer.

140 PCR products were used to create Illumina-compatible sequencing libraries with

141 NEBNext® UltraTM reagents (New England Biolabs) and sequenced on an Illumina MiSeq

142 using a paired-end 150bp read length configuration.

143

144 Co-IP experiments

145 Human LRRC56 was amplified from an untagged image clone (SC123392, Origene

146 Technologies), inserted into the pDONR201 Gateway cloning vector (Invitrogen) and

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147 subsequently pDEST40 destination vector according to the manufacturer’s instructions.

148 The human IFT88-eYFP construct was provided by Professor Colin Johnson13. Both

149 plasmids were sequenced and maxi-prepped prior to transfection. HEK293 cells were

150 co-transfected with 1.5µg of each plasmid using Lipofectamine 2000 (Invitrogen). Cells

151 were lysed 48 h post transfection,using NP40 cell lysis buffer, containing 1x protease

152 and phosphatase inhibitors, and protein extracted as per standard protocols.

153 Immunoprecipitation (IP) of eYFP was performed with 1000 µg of both transfected and

154 untransfected cell extracts using the GFP-Trap®_M kit (Chromotek) according to the

155 manufacturer’s instructions. Whole cell extracts (WCE) and immunoprecipitates (IP)

156 were blotted using mouse anti-V5 (Invitrogen, 1/2,000) or mouse anti-GFP (Invitrogen,

157 1/1,000). A secondary goat anti-mouse HRP (Dako) was used at 1/10,000 and detected

158 using the SuperSignal West Femto kit (Thermo Fisher Scientific).

159

160 Ciliary function measurements

161 In IV:1, Family 1, ciliary beat frequency (CBF) was measured using a digital high-speed

162 video imaging system, as described previously14,15. Ciliary beat pattern was investigated

163 in three different planes: a sideways profile, beating directly toward the observer and

164 from directly above. Dyskinesia was defined as an abnormal beat pattern that included

165 reduced beat amplitude, stiff beat pattern, flickering motion or a twitching motion.

166 Ciliary beat pattern is associated with specific ultrastructural defects in primary ciliary

167 dyskinesia. Dyskinesia index was calculated as the percentage of dyskinetic cilia within

168 the sample (number of dyskinetic readings/total number of readings for sample ×100).

169 All measurements were taken with the solution temperature between 36.5 and 37.5° C

170 and the pH between 7.35 and 7.45. Normal ranges for CBF and percentage dyskinetic

171 cells are 8-17Hz and 4-49% respectively as previously reported16.

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172

173 Air liquid interface cell culture

174 The modified method we used has previously been described17. Briefly, nasal brush

175 biopsy samples were grown on collagen (0.1%, Vitrogen, Netherlands) coated tissue

176 culture trays (12 well) in Bronchial Epithelial Growth Media (BEGM, Lonza, USA) for 2-7

177 days. The confluent unciliated basal cells were expanded into collagen-coated 80 cm2

178 flasks and the BEGM was replaced every 2-3 days. The basal cells were then seeded at

179 approximately 1-3 x 105 cells per cm2 on a collagen coated 12mm diameter transwell

180 clear insert (Costar, Corning, USA) under BEGM for 2 days. After confluency, the basal

181 cell monolayer was fed on the basolateral side only with ALI-media (50% BEGM and

182 50% Hi-glucose minimal essential medium containing 100 nM retinoic acid). The media

183 was exchanged every 2-3 days and the apical surface liquid was removed by gentle

184 washing with phosphate buffered saline when required. When cilia were observed on

185 the cultures they were physically removed from the transwell insert by gentle scraping

186 with a spatula and washing with 1ml of HEPES (20 mM) buffered medium 199

187 containing penicillin (50 µg/ml), streptomycin (50 µg/ml) and Fungizone (1 µg/ml).

188 The recovered ciliated epithelium was then dissociated by gentle pipetting and 100µl of

189 the cell suspension was examined inside a chamber slide and ciliary beat frequency and

190 pattern assessed as described above 14,15. The remaining 900 µl was fixed in 4%

191 glutaraldehyde for transmission electron microscopy (TEM) analysis of cilia and ciliated

192 epithelium.

193

194 TEM of Human Samples

195 TEM was performed as previously described18, using a Jeol 1200 instrument. For TEM,

196 the ciliated cultures were fixed with glutaraldehyde (4%w/v) in Sorensen’s phosphate

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197 buffered (pH 7.4). After post-fixation in osmium tetroxide (1%w/v), samples were

198 dehydrated through graded ethanol series and immersed in hexamethyldisilazane.

199 Sections were cut at 90nm, with cross-sections categorised for height in the cilium using

200 histological parameters. The bottom (2µm) of the axoneme cross-sections were located

201 because they were associated with microvilli. The middle (2µm) cross sections were

202 identified by wide cross-sections with the outer dynein (ODA) away from the axoneme

203 membrane (with no microvilli present). The top 1um of the cilia was represented by

204 cross-sections which have a slightly smaller diameter compared to the middle sections.

205 In addition, the axonemal membrane was tightly wrapped to the microtubules and the

206 ODA. The tips (0.6µm) appear as thin cross sections and have no dynein arms.

207 208

209

210 Trypanosome cell culture

211 All cloned cell lines used for this work were derivatives of T. brucei strain Lister 427 and

212 were cultured in SDM79 medium supplemented with hemin and 10 % fetal calf serum19.

213 Cell lines IFT88RNAi (targeting an essential protein for anterograde IFT)20, IFT140RNAi

214 (essential protein for retrograde IFT)21, DNAI1RNAi (component of the dynein arm)22, and

215 ODA7RNAi (cytoplasmic assembly machinery of the dynein arm)23 have previously been

216 described. They all express double-stranded RNA under the control of tetracycline-

217 inducible T7 promoters24,25.

218

219 Expression of endogenous LRRC56 fused to YFP

220 Endogenous tagging of the T. brucei LRRC56 gene was carried out by direct integration

221 in the LRRC56 gene using the p2675LRRC56 plasmid. This vector is derived from the

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222 p2675 plasmid and contains a 1495bp fragment of the trypanosome LRRC56 gene

223 sequence (1-1495) downstream of the YFP gene26. Transfection was achieved by

224 nucleofection of T. brucei cells using program X-0014 of the AMAXA Nucleofector®

225 apparatus (Lonza) as previously described 27, with 10 µg plasmid linearized with AfeI in

226 the target LRRC56 gene sequence, for homologous recombination with the target allele.

227 The mutant allele was obtained following T776/C base substitution (Genecust Europe,

228 Luxembourg) to mutate leucine 259 to proline in the p2675TbLRRC56. The resulting

229 p2675LRRC56L259P plasmid was linearized with NheI prior to nucleofection. As a

230 result, the expression of the YFP fluorescent fusion protein is under the control of the

231 endogenous 3′ untranslated region of the LRRC56 gene resulting in a similar expression

232 level as the wild-type allele since most gene expression regulation is determined by

233 3′UTR sequences28. Transgenic cell lines were obtained following puromycin selection

234 and cloning by serial dilution.

235

236 LRRC56 gene deletion or replacement

237 Sequential LRRC56 gene replacement in T. brucei was used to obtain lrrc56-/-

238 cells. Sequences containing either the puromycin (PURO) or the blasticidin (BLA) drug

239 resistance gene flanked by 300 bp long upstream and downstream regions of the

240 LRRC56 gene were synthesized and cloned in a pUC57 plasmid (Genecust Europe,

241 Luxembourg). Amplicons were generated by PCR with primers P1:

242 dTTTGAAGGTGCTGTGTGAGGG and P2 dAGGTAGAGGGAGGCGTTGAG (Eurogentec)

243 which anneal to the sequences 300 bp upstream of the LRRC56 start codon and

244 downstream of LRRC56 stop codon respectively. Prior to nucleofection PCR fragments

245 containing either the blasticidin (BLA) resistance gene or the G418 neomycin (NEO)

246 resistance gene were purified using Nucleospin gel and PCR clean-up kit (Macherey

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247 Nagel). Single allele knockout cells were obtained following nucleofection with the BLA

248 amplicon containing LRRC56 flanking sequences, blasticidin selection and cloning.

249 LRRC56 allele deletion was confirmed using PCR with 5′UTR LRRC56-specific primer P3

250 dTCACCATCACGCCCTTTTGT and BLA-specific primer P4 dCTGGCGACGCTGTAGTCTT.

251 Replacement of the remaining LRRC56 allele was performed with either the

252 YFP::LRRC56 gene or the mutated YFP::LRRC56L259P gene in this single knockout cell

253 line using linearized p2675TbLRRC56 or p2675TbLRRC56L259P plasmid and

254 puromycin selection as described above. Double LRRC56 knockout was achieved

255 following nucleofection of the cell line with a single allele knockout carrying the

256 mutated YFP::LRRC56L259P gene with NEO amplicon containing LRRC56 flanking

257 sequences and validation obtained following PCR analysis of G418-resistant cells with

258 LRRC56-specific primers P5 dCCGTAGCATCATCCGAGACC and P6

259 dACTATTTGCGTCAGGTGGCA. Primers amplifying a 1511-bp region of the unrelated

260 aquaporin 1 gene served as positive controls. For whole-genome sequencing, genomic

261 DNA was extracted using the Qiagen DNeasy kit. Short insert Illumina sequencing

262 libraries were constructed and sequenced at the Beijing Genomics Institute, generating

263 approximately 6.6 million 100-bp paired end reads. Reads were aligned to the T. brucei

264 TREU927 genome (TriTrypDB release 35)29 using bowtie2 in very-sensitive-local

265 alignment mode, with an 88.6% alignment rate29,30. Alignment files were sorted, merged

266 and indexed using Samtools31. Aligned reads were visualised and analysed, including

267 counting reads per CDS using the Artemis pathogen sequence browser32.

268

269 Indirect immunofluorescence assay (IFA)

270 Cultured trypanosomes were washed twice in SDM79 medium without serum and

271 spread directly on poly-L-lysine coated slides (Thermoscientific, Menzel-Gläser) before

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272 fixation. For methanol fixation, cells were air-dried and fixed in methanol at -20°C for 5

273 min followed by a rehydration step in PBS for 15 min. For paraformaldehyde-methanol

274 fixation, cells were left for 10 min to settle on poly-L-lysine coated slides. Adhered cells

275 were washed briefly in PBS before being incubated for 5 min at room temperature with

276 a 4% PFA solution in PBS at pH 7 and fixed with methanol at -20°C for an additional 5

277 min followed by a rehydration step in PBS for 15 min. For immunodetection, slides were

278 incubated for 1 h at 37°C with the appropriate dilution of the first antibody in 0.1% BSA

279 in PBS; mAb25 recognises the axonemal protein TbSAXO133; a monoclonal antibody

280 against the IFT-B protein IFT17221, and a mouse polyclonal antiserum against DNAI1 23.

281 YFP::LRRC56 was observed upon fixation by immunofluorescence using a 1:500

282 dilution of a rabbit anti-GFP antibody that detects YFP (Life Technologies). After several

283 5 min washes in PBS, species and subclass-specific secondary antibodies coupled to the

284 appropriate fluorochrome (Alexa 488, Cy3 or Cy5, Jackson ImmunoResearch) were

285 diluted 1/400 in PBS containing 0.1% BSA and were applied for 1 h at 37°C. After

286 washing in PBS as indicated above, cells were stained with a 1 µg/ml solution of the

287 DNA-dye DAPI (Roche) and mounted with Slowfade antifade reagent (Invitrogen).

288 Slides were either stored at -20°C or immediately observed with a DMI4000 microscope

289 (Leica) with a 100X objective (NA 1.4) using a Hamamatsu ORCA-03G camera with an

290 EL6000 (Leica) as light source. Image acquisition was performed using Micro-manager

291 software and images were analysed using ImageJ (National Institutes of Health,

292 Bethesda, MD)

293

294 Motility analyses

295 Volumes of 250µl at 5x106 trypanosomes/ml in warm medium were distributed in 24

296 well plates. Samples were analysed under 10X objective of a DM IL LED microscope

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297 (Leica) coupled to a DFC3000G camera (Leica). Movies were recorded (200 frames, 50

298 ms of exposure) using LASX Leica software, converted to .avi files and analysed with the

299 medeaLAB CASA Tracking v.5.5 software (medea AV GmbH) 34.

300

301 TEM analysis of trypanosome samples

302 Cells were fixed with 2.5% glutaraldehyde directly in the suspension culture for 10 min

303 and then treated with 4% paraformaldehyde, 2.5% glutaraldehyde in 0.1M cacodylate

304 buffer (pH 7.2) for 1h. Cells were post-fixed for 30 min (in the dark) in 1 % osmium

305 tetroxide (OsO4), in 0.1 M cacodylate buffer (pH 7.2), washed and incubated in 2%

306 uranyl acetate for 1h at room temperature. Samples were washed, dehydrated in a

307 series of acetone solutions of ascending concentrations, and embedded in Polybed 812

308 resin. Cytoskeletons were extracted by treating cells with Nonidet 1% with protease

309 inhibitor cocktail (Sigma, P8340) diluted in PBS for 20 min, washed in PHEM 0.1M

310 pH7.2 for 10min. Fixation was performed in 2.5% glutaraldehyde, 4%

311 paraformaldehyde, and 0.5% tannic acid in 0.1M cacodylate buffer (pH 7.2). Cells were

312 post-fixed, incubated in uranyl acetate, dehydrated and infiltrated as cited above.

313 Ultrathin sections were stained with uranyl acetate and lead citrate.

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314 RESULTS

315 Clinical characterization of families

316 Individual IV:1, Family 1 was born at term by normal vaginal there was no family

317 history of note. After 36 hours, she developed tachypnoea, which was managed with

318 head box oxygen. She was discharged home on day 5 of life. She developed rhinorrheoa

319 and experienced several episodes of documented respiratory tract infection. She

320 suffered from a chronic cough and recurrent middle ear infections. At age 18 months,

321 she developed pneumonia. Chest X-ray and CT scan revealed bronchiectasis and

322 dextrocardia (Figure 1B), suggesting a clinical diagnosis of PCD. However, TEM analysis

323 of nasal biopsy samples along different segments of the cilia (tip, middle and base)

324 showed apparently intact dynein arms (Figure 1C). Direct high-speed videomicroscopy

325 analysis of biopsy material revealed a ciliary beat frequency of 10.87 Hz (10.4 Hz-11.34

326 Hz) and particulate clearance was observed (Supplementary Video 1 and 2). Although

327 cell culture at an air-liquid interface produced a healthy ciliated epithelium, the ciliary

328 beat pattern was in fact dyskinetic (19% twitching, 33% stiff, 48% static), with a beat

329 frequency of 5.39 Hz (4.29 Hz-6.49 Hz) (Supplementary Video 3 and 4).

330

331 Family 2 were from Kuwait and included two affected foetuses. Both pregnancies were

332 terminated because the fetuses were found to have lethal congenital heart disease.

333 Autopsy revealed similar findings in both patients, which included situs inversus of the

334 thoracic and abdominal organs, with a complex congenital heart malformation

335 characterized by double outlet right ventricle (data not shown).

336

337

338

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339 Genetic analyses revealed mutations in LRRC56

340 Autozygosity mapping identified 46 and 33 regions of homozygosity in each proband

341 from Family 1 and 2 respectively (Table S1). Variant filtering, using the autozygous

342 intervals, and public/in-house databases (to eliminate variants with minor allele

343 frequency > 1%) identified a single homozygous variant in each family, located in the

344 same gene, LRRC56 (NM_198075.3: Family 1 c.423+1G>A; Family 2 c.419T>C,

345 p.(Leu140Pro)). Neither variant is recorded in ExAC. A search of an ethnically matched

346 control exome cohort revealed a single heterozygous carrier of the c.423+1G>A

347 mutation among 1,541 normal subjects. Sanger sequencing confirmed the variants as

348 well as segregation in both families. c.423+1G>A is predicted to abrogate the intron 7

349 donor site; high-throughput sequencing analysis of RT-PCR products confirmed that the

350 LRRC56 variant is aberrantly spliced and not predicted to encode a functional protein

351 (Figure S1). The missense variant c.419T>C, p.(Leu140Pro) affects a highly conserved

352 residue (Figure S2), predicted to be deleterious by SIFT (0)35, Polyphen (1.0)36, and

353 CADD (15.72) scores37.

354

355 LRRC56 protein is conserved in eukaryotes with motile cilia constructed by

356 intraflagellar transport (IFT)

357 The human LRRC56 gene encodes a 542 amino acid protein determined from

358 transcriptomic studies and immunohistochemistry to be expressed in many organs,

359 including lung and respiratory epithelial cells, but mostly in testis and pituitary gland38.

360 The protein contains 5 leucine-rich repeat domains conserved across species whose

361 motile cilia are assembled by intraflagellar transport (IFT)39 LRRC56 is the human

362 orthologue of the Chlamydomonas reinhardtii gene ODA8, which is thought to play a role

363 in the maturation and/or transport of outer dynein arm (ODA )complexes during

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364 flagellum assembly, and has a biochemical distribution similar to IFT proteins39. ODA8

365 is proposed to function together with two other proteins termed ODA5 and ODA10 to

366 form an accessory complex involved in ODA docking to microtubules39. However,

367 evidence for a physical interaction is lacking, and the exact role of ODA8 remains to be

368 defined. To evaluate a possible association of LRRC56 with the multi-subunit IFT

369 machinery, HEK293 cells were co-transfected with human LRRC56 tagged with V5 and

370 the reference IFT protein IFT88 fused to eYFP13. Immunoprecipitation using an anti-

371 GFP antibody co-precipitated both IFT88-eYFP and LRRC56-V5, supporting an

372 association of LRRC56 with the IFT machinery (Figure 1D)

373

374 The functional role of LRRC56 was further investigated in T. brucei, an organism that

375 possesses a 9+2 axoneme and is amenable to genetic manipulation23,40. The T. brucei

376 LRRC56 orthologue (Tb427.10.15260) comprises 751 amino acids and shares 42%

377 sequence identity in the conserved leucine-rich repeat (LRR) region (Figure S2).

378

379 Since T. brucei maintains its mature flagellum during formation of the new one, it is

380 possible to monitor both maintenance and assembly in the same cell 41. YFP-tagged

381 LRRC56 (YFP::LRCC56) was expressed in cells upon endogenous tagging and the

382 protein was detected in the distal portion of the new flagellum, whereas the old

383 flagellum lacked the protein (Figure 2A). Following division, cells with one flagellum

384 displayed two different profiles: either a strong signal towards the distal end of the

385 flagellum (Figure 2B) or no signal (not shown). The first scenario reflects daughter cells

386 that inherited a new flagellum, while the latter reflects those daughter cells that

387 inherited an old flagellum. This observation is consistent with LRRC56 being recruited

388 during flagellar assembly before its removal during flagellum maturation, after cell

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389 division. Co-localisation with the IFT protein IFT172, but not with the axoneme marker

390 mAb25 suggests that LRRC56 associates with IFT and not dynein arms. Many, but not

391 all, IFT trains contained LRRC56, suggesting it is a cargo rather than a core component.

392 The LRRC56 signal is lost upon detergent extraction of the cytoskeleton, which strips

393 the membrane and IFT particles but not the dynein arms (data not shown), further

394 supporting association with IFT.

395

396 To further investigate the link with IFT, the distribution of YFP::LRRC56 was studied in

397 trypanosome mutant cell lines in which tetracycline-inducible RNAi knockdown of an

398 IFT-B (IFT88RNAi) or an IFT-A (IFT140RNAi) member results in either defective

399 anterograde or retrograde transport, respectively20,21. In IFT88RNAi cells, the flagellar

400 YFP::LRRC56 signal disappeared when anterograde transport was disrupted and the

401 protein was found predominantly in the cytoplasm (Figure S3A and S3B). In the

402 retrograde mutant IFT140RNAi, trains travel into the new flagellum but fail to be recycled

403 to the base. The YFP::LRRC56 distribution pattern turned out to be more complex

404 (Figure S3C and S3D) and required more detailed investigation. Cultures are not

405 synchronised and RNAi knockdown can impact cells at different stages of flagellum

406 construction. If this happened when flagella started to grow, IFT proteins accumulated

407 in very short flagella, but LRRC56 was rarely detected (Figure 2C). However, when IFT

408 arrest took place at later stages of elongation, LRRC56 was frequently found in high

409 concentration always associated with stalled IFT trains (Figure 2C). These results

410 reflect the association of LRRC56 as cargo of IFT trains, with a progressive increase

411 during flagellar construction. After 2 days of tetracycline-inducible RNAi knockdown,

412 the LRRC56 is no longer detected in the flagellum (Figure S3A and S3B).

413

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414 LRRC56 localisation was unchanged in DNAI1RNAi and ODA7 RNAi mutants that are

415 defective in their dynein arm constitution or cytoplasmic preassembly, respectively

416 (Figure S4). This shows that LRRC56 does not require the presence of dynein arms to be

417 associated with flagella. Together, these findings reveal that LRRC56 likely performs an

418 IFT-associated function in motile cilia.

419

420 Absence or mutation of T. brucei LRRC56 is responsible for motility defects

421 explained by absence of outer dynein arms in the distal segment of the axoneme

422 We next chose to assess any impact caused by the absence, or mutation of LRRC56. An

423 LRCC56 null mutant was generated by double gene knockout (Figure S5A), and whole-

424 genome sequencing demonstrated that the LRRC56 gene had been replaced by the drug

425 resistance cassettes (Figure S5B). Visual examination showed a significant reduction in

426 flagellar beating and cell swimming, with ~10% of cells struggling to complete cell

427 division and remaining attached by their posterior extremities (Figure S5C). These

428 phenotypes indicate slow cytokinesis and increased generation times which, in

429 trypanosomes, is typical of motility defects (Figure S5D)22,42,43. Microscopy revealed

430 that lrrc56-/- cell motility is characterised by an erratic swimming pattern with altered

431 propagation of the tip-to-base wave, increased frequency of base-to-tip waves, and

432 frequent tumbling typical of outer dynein arm mutants (Figure 3) 22,44. Examination of

433 the axoneme structure by TEM revealed that more than one third of sections analysed

434 from lrrc56-/- cells showed absence of 6 to 9 ODAs (Figure 4). This was found in the

435 distal portions of both old and new flagella, as indicated by the presence of a smaller cell

436 body (or no cell body) 45. This suggests a role for LRRC56 in the assembly of dynein

437 arms in the distal portion of the axoneme. Immunostaining with an anti-DNAI1 (dynein

438 arm intermediate chain 1 also known as IC78) antibody22,23 that recognises an essential

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439 structural component of the ODA, confirmed that only the proximal axonemal region

440 stained positively in lrrc56 -/- cells (Figure 4H). Control cells stained from the base to

441 the tip of all axonemes (Figure 4G), confirming that LRRC56 is essential for distal

442 assembly of ODA.

443 We next evaluated in trypanosomes the impact of the p.Leu259Pro mutation which

444 corresponds to the p.Leu140Pro observed in Family 2. One LRRC56 allele was deleted

445 and the other allele was replaced endogenously with a L259P modification in YFP-

446 tagged LRRC56 (YFP::LRRC56L259P cells). The YFP::LRRC56L259P protein localises

447 normally to the distal portion of the new flagellum, showing that this residue is not

448 required for proper expression and localisation of LRRC56 (data not shown). However,

449 cells showed reduced motility, albeit not to the same extent as the double knockout

450 (Figure 3C,D). Ultra-structural analysis of YFP::LRRC56L259P cells by TEM revealed a

451 reduction in the number of ODAs, but that was less frequent compared to lrrc56-/-

452 flagella (Figure 4B,C,F). We conclude that the mutated protein can associate with IFT

453 trains, but functions less efficiently that its normal counterpart in supporting dynein

454 arm assembly.

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455 DISCUSSION

456 Whole exome sequencing in two unrelated consanguineous families enabled us to

457 identify homozygous variants in the LRRC56 gene. The common clinical phenotype in

458 the two families was cardiac laterality defects, while in Family 1 the affected individual

459 also presented with recurrent pulmonary infections, and on investigation was found to

460 have bronchiectasis. This combination of clinical features was suggestive of PCD, but a

461 uniformly dyskinetic beat pattern was not observed. Investigation of cultured nasal

462 epithelial cells from the affected individual in family 1 revealed a dyskinetic ciliary beat

463 pattern, but no structural ciliary defects. We have observed this phenomenon before in

464 our analysis of samples sent for the investigation of PCD (R.Hirst, personal

465 communication).

466 In Family 1, with a homozygous splicing variant in LRRC56, further analysis confirmed

467 that the mutated transcript is aberrantly spliced and is not predicted to encode a

468 functional protein. In Family 2, a homozygous missense variant was identified.

469 Functional investigation demonstrated a role for LRRC56 in the assembly of ODA in the

470 protist T. brucei. We then modelled the effect of a null variant and the homozygous

471 missense variant in this organism. We demonstrate that absence of LRRC56 or the

472 presence of the homozygous missense variant seen in Family 2, both result in motility

473 defects, caused by loss of ODAs in the distal segment of the axoneme. This observation is

474 consistent with our interrogation of the role of LRRC56 in this organism.

475

476 Previous studies have shown that LRRC56 is found only in species with motile cilia that

477 rely on IFT for axonemal assembly39. The reported biochemical distribution of LRRC56

478 is similar to that of IFT subunits, with about 50% of the protein associated with the

479 membrane and matrix fraction39. Our pull-down experiments carried out in human cells

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480 expressing tagged LRRC56 and IFT88 revealed that LRRC56 interacts with the IFT

481 machinery. In T. brucei, LRRC56 is recruited to the flagellar compartment at advanced

482 stages of construction and co-localises with many (but not all) IFT trains. This flagellar

483 localisation is dependent on IFT, as confirmed by analysis of cell lines defective in either

484 anterograde or retrograde IFT. These results support a model in which LRRC56

485 associates with IFT trains and may function as a cargo adaptor to transport dynein arms

486 towards the distal end of cilia and flagella.

487

488 The absence of LRRC56 has an impact on ciliary motility, as observed here in cultured

489 cells from one affected patient and in T. brucei. This is consistent with the oda8 (LRRC56

490 gene homologue) null mutant in Chlamydomonas, although the impact on the presence

491 of dynein arms was remarkably variable. ODAs are absent throughout the length of the

492 flagellum in Chlamydomonas 46 whereas they are missing at only the distal part of the

493 T. brucei flagellum. In both cases, this loss of ODA interferes with proper initiation of

494 flagellum beating resulting in reduced motility and dyskinetic flagellar beating. In

495 samples from the only human patient that were available for analysis, sections were

496 analysed in different segments of the cilia (proximal, intermediate, distal), but dynein

497 arms were not visibly affected.

498

499 Although the central core of the protein containing the leucine-rich repeat domains is

500 well conserved, other segments are variable across species, with large and often unique

501 N- and C-terminal extensions. This may translate into a common central function, such

502 as association of LRRC56 with the IFT machinery for dynein arm transport, but could

503 also result in considerable structural variation, potentially supporting species-specific

504 interaction partners. Indeed, the composition of dynein arms (number and type of

22 bioRxiv preprint doi: https://doi.org/10.1101/288852; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

505 heavy, intermediate and light dynein chains) varies between species, as demonstrated

506 by both biochemical and genetic analysis42. The outer dynein arm composition differs

507 between the Chlamydomonas ODAs that contain 3 heavy dynein chains chains (,  and

508 ), and the human and trypanosome ODA which each contain only 2 heavy chains ( and

509 ).

510

511 In Chlamydomonas, early flagellum growth is very rapid (up to 10 µm per hour)47, and

512 might necessitate a greater contribution from ODA8 to ODA transport compared to the

513 slower growth rate observed in trypanosomes48, and respiratory epithelial cells 49. This

514 could explain the absence of ODA throughout the axoneme compared to only the distal

515 part in trypanosomes. We cannot exclude the possibility that discrete structural defects

516 of dynein arms occur in the human patient whose cilia were analysed. For example,

517 DNAH11 nonsense mutations are associated with subtle dynein arm modifications that

518 can only be detected by advanced TEM imaging and tomography 3. In this regard,

519 approximately 30% of PCD patients showing ciliary dysfunction have no or very subtle

520 ciliary ultrastructure abnormalities, when investigated with standard transmission

521 electron microscopy50

522 Overall, our findings underline the evolutionary complexity of outer dynein arms

523 assembly and trafficking. We have shown that bi-allelic mutations in the LRRC56 gene

524 are responsible for laterality defect in two unrelated families. We provide evidence of

525 PCD-like symptoms caused by mutations in a gene encoding a protein required for ODA

526 transport, rather than composition or assembly. Our work expands the list of ciliary

527 genes involved in human disorders51, while also providing insight into the role of

528 LRRC56 and cilia biology in human development.

23 bioRxiv preprint doi: https://doi.org/10.1101/288852; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

529 530

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531 SUPPLEMENTAL DATA 532 533 Supplementary data consists of six figures, 4 videos and one table 534 535 536 CONFLICT OF INTEREST 537 538 The authors have no conflict of interest to declare. 539 540 541 ACKNOWLEDGEMENTS 542 543 We wish to thank the families reported here for their willingness to participate in these

544 research efforts. We thank the Ultrastructural BioImaging Plateforme for providing

545 access to the TEM equipment. We thank Bruno Louis and Jean-François Papon, Institut

546 National de la Santé et de la Recherche Médicale, U955, Créteil, France, for help in video-

547 microscopy. Research at the Institut Pasteur is funded by an ANR grant (ANR-14-CE35-

548 0009-01), by a French Government Investissement d’Avenir programme, Laboratoire

549 d’Excellence “Integrative Biology of Emerging Infectious Diseases” (ANR-10-LABX-62-

550 IBEID) and by La Fondation pour la Recherche Médicale (Equipe FRM

551 DEQ20150734356). This work was supported, in part, by the Care4Rare Canada

552 Consortium funded by Genome Canada, the Canadian Institutes of Health Research

553 (CIHR), the Ontario Genomics Institute, Ontario Research Fund, Genome Quebec, and

554 Children’s Hospital of Eastern Ontario Foundation and the Canadian Rare Diseases:

555 Models and Mechanisms Network funded by CIHR and Genome Canada. The authors

556 wish to acknowledge the contribution of the high-throughput sequencing platform of

557 the McGill University and Génome Québec Innovation Centre, Montréal, Canada.

558 Research at the University of Leeds was supported by grants MR/M009084/1 and

559 MR/L01629X/1 awarded by the UK Medical Research Council.

560

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561 WEB RESOURCES

562 AgileMultiIdeogram, http://dna.leeds.ac.uk/agile/AgileMultiIdeogram/

563 Burrows-Wheeler Aligner, http://bio-bwa.sourceforge.net/

564 CASA Tracking V.5.5, https://safe.nrao.edu/wiki/bin/view/Software/CASA/WebHome

565 CLUSTAL Omega, https://www.ebi.ac.uk/Tools/msa/clustalo/

566 dbSNP, http://www.ncbi.nlm.nih.gov/projects/SNP/

567 Exome Aggregation Consortium (ExAC) Browser, http://exac. broadinstitute.org/

568 Genome Analysis Toolkit (GATK), https://www.broadinstitute. org/gatk/

569 Human Protein Atlas, https://www.proteinatlas.org

570 ImageJ, https://imagej.nih.gov/ij/

571 NHLBI Exome Variant server, http://evs.gs.washington.edu/EVS/

572 OMIM, http://www.omim.org/

573 Picard, http://broadinstitute.github.io/picard/

574 Protein BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins

575 SAMtools, http://www.htslib.org/

576 TriTrypDB: The Kinetoplastid Genomics Resource, http://tritrypdb.org/tritrypdb/

577 SIFT, http://sift.jcvi.org

578 POLYPHEN2, http://genetics.bwh.harvard.edu/pph2/

579 CADD, http://cadd.gs.washington.edu

580

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695 39. Desai, P.B., Freshour, J.R., and Mitchell, D.R. (2015). Chlamydomonas axonemal dynein 696 assembly locus ODA8 encodes a conserved flagellar protein needed for cytoplasmic 697 maturation of outer dynein arm complexes. Cytoskelet. Hoboken NJ 72, 16–28.

698 40. Vincensini, L., Blisnick, T., and Bastin, P. (2011). 1001 model organisms to study cilia 699 and flagella. Biol. Cell 103, 109–130.

700 41. Sherwin, T., and Gull, K. (1989). The cell division cycle of Trypanosoma brucei brucei: 701 timing of event markers and cytoskeletal modulations. Philos. Trans. R. Soc. Lond. B. Biol. 702 Sci. 323, 573–588.

703 42. Ralston, K.S., Kabututu, Z.P., Melehani, J.H., Oberholzer, M., and Hill, K.L. (2009). The 704 Trypanosoma brucei flagellum: moving parasites in new directions. Annu. Rev. Microbiol. 705 63, 335–362.

706 43. Broadhead, R., Dawe, H.R., Farr, H., Griffiths, S., Hart, S.R., Portman, N., Shaw, M.K., 707 Ginger, M.L., Gaskell, S.J., McKean, P.G., et al. (2006). Flagellar motility is required for the 708 viability of the bloodstream trypanosome. Nature 440, 224–227.

709 44. Baron, D.M., Kabututu, Z.P., and Hill, K.L. (2007). Stuck in reverse: loss of LC1 in 710 Trypanosoma brucei disrupts outer dynein arms and leads to reverse flagellar beat and 711 backward movement. J. Cell Sci. 120, 1513–1520.

712 45. Dawe, H.R., Farr, H., Portman, N., Shaw, M.K., and Gull, K. (2005). The Parkin co- 713 regulated gene product, PACRG, is an evolutionarily conserved axonemal protein that 714 functions in outer-doublet microtubule morphogenesis. J. Cell Sci. 118, 5421–5430.

715 46. Kamiya, R. (1988). Mutations at twelve independent loci result in absence of outer 716 dynein arms in Chylamydomonas reinhardtii. J. Cell Biol. 107, 2253–2258.

717 47. Lefebvre, P.A., and Rosenbaum, J.L. (1986). Regulation of the synthesis and assembly of 718 ciliary and flagellar proteins during regeneration. Annu. Rev. Cell Biol. 2, 517–546.

719 48. Bastin, P., MacRae, T.H., Francis, S.B., Matthews, K.R., and Gull, K. (1999). Flagellar 720 morphogenesis: protein targeting and assembly in the paraflagellar rod of trypanosomes. Mol. 721 Cell. Biol. 19, 8191–8200.

722 49. Hirst, R.A., Jackson, C.L., Coles, J.L., Williams, G., Rutman, A., Goggin, P.M., Adam, 723 E.C., Page, A., Evans, H.J., Lackie, P.M., et al. (2014). Culture of primary ciliary dyskinesia 724 epithelial cells at air-liquid interface can alter ciliary phenotype but remains a robust and 725 informative diagnostic aid. PloS One 9, e89675.

726 50. Shapiro, A.J., and Leigh, M.W. (2017). Value of transmission electron microscopy for 727 primary ciliary dyskinesia diagnosis in the era of molecular medicine: Genetic defects with 728 normal and non-diagnostic ciliary ultrastructure. Ultrastruct. Pathol. 41, 373–385.

729 51. Reiter, J.F., and Leroux, M.R. (2017). Genes and molecular pathways underpinning 730 ciliopathies. Nat. Rev. Mol. Cell Biol. 18, 533–547.

731 52. Hughes, L.C., Ralston, K.S., Hill, K.L., and Zhou, Z.H. (2012). Three-dimensional 732 structure of the Trypanosome flagellum suggests that the paraflagellar rod functions as a 733 biomechanical spring. PloS One 7, e25700.

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734 53. Ishikawa, T. (2017). Axoneme Structure from Motile Cilia. Cold Spring Harb. Perspect. 735 Biol. 9,.

736 737

31 bioRxiv preprint doi: https://doi.org/10.1101/288852; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

738 FIGURES

739 Figure 1. LRRC56 mutations cause PCD and laterality defects

740 (A) The homozygous splice-site mutation (c.423+1G>A, NM_198075.3) disrupts the

741 evolutionarily conserved canonical donor site in Family 1 individual IV:1. The

742 unaffected sibling IV:2 is heterozygous for the mutation. The homozygous missense

743 mutation (c.419T>C, NM_198075.3) was identified in the two affected siblings V:3 and

744 V:4 from Family 2. Consistent with autosomal-recessive inheritance, the mutations

745 described were detected in a heterozygous state in the unaffected parents (data not

746 shown). (B) The Family 1 proband (IV:1) had dextrocardia, documented by chest X-ray

747 (left panel). High-resolution axial computed tomography of the thorax in the same

748 patient demonstrates bronchiectasis, typical of PCD. Dextrocardia is also visible (CT-

749 scan; right panel). (C) Cross section through the axoneme from cultured respiratory

750 cells from Family 1 individual IV:1. The position of the section is indicated, bar =

751 100 nm. Normal axonemal structure is visible, with intact dynein arms. (D)

752 Recombinant human LRRC56 and intraflagellar transport protein IFT88 interact in

753 vitro. HEK293 cells were co-transfected with plasmids encoding human LRRC56 and

754 IFT88 tagged with V5 and YFP respectively (1.5µg of each). After 48 hours,

755 immunoprecipitation (IP) was performed with transfected and untransfected cells using

756 Cell-TRAP magnetic beads bound to an anti-GFP antibody fragment. Protein from input

757 whole cell extracts (WCE, left) and immunoprecipitated proteins (IP, right) were blotted

758 using anti-V5 or anti-GFP. A β-actin control is also shown.

759 Figure 2. LRRC56 associates with IFT trains and not with the axoneme

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760 (A) An YFP::LRRC56 expressing cell that assembles its new flagellum (yellow arrow)

761 shows staining (anti-GFP, green on merged images) in the new flagellum (blue

762 arrowheads) that co-localises with the anti-IFT172 (red on merged images) but not

763 with the anti-axoneme marker (mAb25, blue on merged images). No YFP staining is

764 visible in the old flagellum (white arrow) whereas IFT172 positive trains are clearly

765 present. IFT trains are predominantly found on microtubules doublets 4 and 7,21 hence

766 the visual aspect of two separate tracks around the axoneme. DNA is stained with DAPI

767 (cyan) showing the presence of two kinetoplasts (mitochondrial DNA) and two nuclei

768 typical of cells at late stage of their cycle41. (B) Cytokinesis results in two daughter cells

769 each containing a unique flagellum41. The one inheriting the new flagellum remains

770 positive for YFP::LRRC56 that still shows association with IFT trains (same staining as

771 in A). (C) The same immunofluorescence assay was performed on IFT140RNAi cells

772 expressing YFP::LRRC56 after 24h in RNAi conditions. DNA staining shows that the top

773 cell is mitotic and assembles a new flagellum. The IFT172 staining reveals the presence

774 of a stalled IFT train that contains a considerable quantity of YFP::LRRC56 (arrowhead).

775 The bottom cell is at a further stage of its cell cycle, yet its new flagellum is much

776 shorter, indicating that RNAi knockdown occurred at a very early phase of construction

777 (star). In these conditions, IFT172 staining shows that the very short flagellum contains

778 a large amount of accumulated IFT material, yet no signal is visible for YFP::LRRC56

779 (star).

780 Figure 3. Absence of LRRC56 or expression of the L259P mutation reduces flagellum

781 beating and cell motility

782 (A-C) Tracking analysis34 showing the movement of individual trypanosomes in wild-

783 type control (A), in cells expressing only YFP::LRRC56L259P (B) and in lrrc56-/- cells

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784 (C). Sustained motility is only observed in control conditions. (D) Quantification of the

785 straight-line movement confirms the visual impression that motility was reduced in a

786 statistically significant manner in YFP::LRRC56L259P cells and almost abolished in

787 lrrc56-/-. Total number of cells, mean and standard deviation are indicated on the

788 figure. Statistical analysis was performed using t test.

789 Figure 4. LRRC56 is required for assembly of ODA in the distal portion of the axoneme

790 (A-E) Sections are shown through the flagella of detergent-extracted cytoskeletons from

791 various cell lines. Stripping the flagellum membrane and matrix facilitates the analysis

792 of structures22,52,53. Sections through control YFP::LRRC56-expressing cells (A) possess

793 all 9 ODA, whereas a mixture of sections with normal profiles or with several missing

794 ODA (orange arrowheads) is encountered in YFP::LRRC56L259P-expressing cells (B-C)

795 or lrrc56-/- cells (D-E). (F) Sections were grouped in three categories: defects in 2 ODA

796 or less (blue), in 5 ODA or less (green) and in 6 ODA or more (red). The total counted

797 number of sections is 50 for each sample. Full details are given in Table S2. (G-H) IFA

798 with the anti-DNAI1 antibody stains the whole axoneme of wild-type cells (G) as

799 expected23. However, the staining was limited to the proximal portion in lrrc56-/- cells

800 in both growing (missing portion shown by yellow arrowheads) and mature flagella

801 (white arrowheads) (H).

802 Permissions

803 See email below from RH Hirst re personal communication

34 bioRxiv preprint doi: https://doi.org/10.1101/288852; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/288852; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/288852; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/288852; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/288852; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Supplemental Data

Figure S1. Primer locations for the in vitro splicing assay

(A). The LRRC56 gene structure drawn to-scale for transcript NM_198075.3. (B) The

location of the gene-specific primer (red arrow) used to perform the first-strand cDNA

synthesis for the in vitro splicing assay for Family 1 individual IV:1. (C) Black arrows show

the location of the exon 6 forward primer used in conjunction with either exon 8 or exon

10 reverse primers. Blue text represents the string used to extract LRRC56-specific reads

spanning the exon 6/7 junction. The 2 nucleotides located beyond the red text in each

extracted sequence read were interrogated to determine whether the read represent a

spliced or unspliced transcript. At least 6.9 million reads were generated per primer

combination per sample. Only a small proportion of these were LRRC56-specific, with the

6F/8R primer combination working more effectively than 6F/10R. For the control

sample, these represented 0.46% and 0.08% of total reads respectively. The nucleotides

located beyond the 3′ end of exon 7 were assayed for the correctly spliced exon 8

sequence (GA) and the ratio of spliced to unspliced transcripts was calculated. In the

control sample, 98.4% and 99.9% of detected transcripts were correctly spliced,

suggesting the assay was biologically representative. In contrast, 100.0% and 89.16% of

reads from the affected subject were unspliced.

Figure S2. LRRC56 is conserved in ciliated organisms. (A) Alignment of LRCC56

predicted protein sequences from T. brucei, H. sapiens and C. reinhardtii. While the

leucine-rich domain is evolutionary conserved, the flanking portions are divergent and

vary in length between species. (B) Alignment of the leucine-rich domain in H. sapiens, T.

brucei and three neighbouring species (Trypanosoma congolense, Trypanosoma cruzi and

Leishmania braziliensis), C. reinhardtii and M. musculus. Blue arrows indicate the 5 bioRxiv preprint doi: https://doi.org/10.1101/288852; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

leucine-rich repeat motifs. Conserved residues are highlighted in red. A star (*) marks the

position of the human L140P mutation; the equivalent residue in T. brucei is located at

position 259 due to the N-terminal extension in this organism.

Figure S3. Inhibition of IFT anterograde transport prevents flagellum targeting of

YFP::LRRC56. Upon endogenous tagging, YFP::LRRC56 was expressed in IFT88RNAi cells

or IFT140RNAi cells without induction (A,C) or with (B,D) 48h-induction of RNAi. Whereas

in non-induced conditions the usual pattern of YFP::LRRC56 staining associated with IFT

trains in both cell types is observed in the growing flagellum or in the daughter flagellum

upon cell division (A,C), the staining is virtually absent in flagella of IFT88RNAi induced cells

and the protein is redistributed to the cytoplasm (B). In induced IFT140RNAi cells , the

LRRC56 is no longer detected in the flagellum (D). For each sample, left panels show phase

contrast images with DAPI (blue) and axonemal staining (mAb25, red) whereas right

panels show the immunofluorescence assay staining of YFP::LRRC56 with anti-GFP.

Figure S4. LRRC56 association with IFT trains is independent of the presence of

ODA. Upon endogenous tagging, YFP::LRRC56 was expressed in DNAI1RNAi (A) or ODA7RNAi

(B) cells. DNAI1 (or IC78) is a dynein intermediate chain and an essential component of

the ODA22, and ODA7 (or LRRC50) is required for cytoplasmic pre-assembly of the ODA23.

The YFP::LRRC56 distribution is identical in non-induced (not shown) and induced

conditions for both DNAI1RNAi (A) or ODA7RNAi (B) cells, showing that flagellum targeting

of LRRC56 and its association to IFT trains is independent of the presence of complete

ODA. Left panels show phase contrast images with DAPI (blue) and right panels show the

staining of YFP::LRRC56 with the anti-GFP.

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Figure S5. Double gene knockout of LRRC56 in T. brucei affects cell motility, cell

division and cell growth. (A) Summary of the strategy used for endogenous tagging and

for double gene deletion of LRRC56 in T. brucei. 1. One LRRC56 allele was replaced with a

blasticidin resistance marker. 2. The remaining LRRC56 allele was replaced with the

mutated YFP::LRRC56L259P gene in this single knockout cell line. 3. This tagged allele was

replaced with a G418 drug resistance marker. (B) Next generation sequencing confirmed

lrrc56 knockout in lrrc56-/- strain. Left, scatter plot of non-redundant gene set comparing

reads per coding sequence in wild type and lrrc56-/-strains. Right, visualisation of reads

at the LRRC56 locus on chromosome 10. Black reads (top track): wild type, red reads:

lrrc56-/-. (C) Phase contrast image of fields of cells stained with DAPI (cyan) in wild-type

(WT) or lrrc56-/- cultures. The increased presence of cells at late phase of cytokinesis

(white arrows) is visible. This is explained by the role of motility to complete cell division

and to allow separation of daughter cells 4,22,43. (D) Growth was monitored over 5 days in

wild-type cells and lrrc56-/- cells. Triplicates are shown.

Figure S6. Expression of YFP::LRRC56 bearing the L259P mutation. Localisation by

IFA of LRRC56-/YFP::LRRC56L259P in T. brucei cells obtained using strategy depicted

Figure S5A (steps 1-2). (A & D) methanol-fixed cells stained with anti GFP or anti-

axoneme mAb25 antibody. Left panels show phase contrast images with DAPI (blue), mid

panels are images overlays obtained for GFP (green) mAb25 (red) and DAPI) (blue)

signals and right panels show the IFA staining of YFP::LRRC56L259P with the anti-GFP

(white).

bioRxiv preprint doi: https://doi.org/10.1101/288852; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/288852; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/288852; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/288852; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/288852; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/288852; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/288852; this version posted March 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table S1. Genomic coordinates of the MultiIdeogram identified autozygous intervals. The shared region on chromosome 11 which contains LRRC56 is highlighted in bold Individual Chromosome Start End Length (bp) Family 1, IV:1 1 16,532,803 17,023,209 490,406 Family 1, IV:1 1 24,194,748 38,006,359 13,811,611 Family 1, IV:1 1 94,511,533 101,203,827 6,692,294 Family 1, IV:1 1 117,487,710 120,611,960 3,124,250 Family 1, IV:1 1 145,112,414 147,381,397 2,268,983 Family 1, IV:1 1 149,905,279 211,280,724 61,375,445 Family 1, IV:1 1 222,798,106 241,712,134 18,914,028 Family 1, IV:1 1 248,084,511 248,525,071 440,560 Family 1, IV:1 2 130,832,292 131,674,285 841,993 Family 1, IV:1 2 197,118,729 207,621,759 10,503,030 Family 1, IV:1 2 220,072,766 220,421,417 348,651 Family 1, IV:1 2 228,111,435 231,407,663 3,296,228 Family 1, IV:1 3 49,739,507 52,256,697 2,517,190 Family 1, IV:1 3 105,260,596 113,858,350 8,597,754 Family 1, IV:1 3 195,511,556 195,518,112 6,556 Family 1, IV:1 4 106,474,096 114,177,035 7,702,939 Family 1, IV:1 4 138,449,683 155,407,680 16,957,997 Family 1, IV:1 5 73,930,751 75,427,935 1,497,184 Family 1, IV:1 5 122,685,801 137,507,104 14,821,303 Family 1, IV:1 5 140,228,164 140,531,746 303,582 Family 1, IV:1 6 29,855,570 33,382,288 3,526,718 Family 1, IV:1 6 49,701,523 96,651,740 46,950,217 Family 1, IV:1 7 27,135,314 36,320,821 9,185,507 Family 1, IV:1 7 83,631,376 138,455,988 54,824,612 Family 1, IV:1 9 125,486,968 127,228,596 1,741,628 Family 1, IV:1 9 130,457,332 131,898,880 1,441,548 Family 1, IV:1 10 112,360,315 118,383,463 6,023,148 Family 1, IV:1 10 120,919,246 135,491,083 14,571,837 Family 1, IV:1 11 193,863 1,643,228 1,449,365 Family 1, IV:1 11 56,143,394 56,143,785 391 Family 1, IV:1 11 62,292,882 78,516,318 16,223,436 Family 1, IV:1 11 95,825,374 110,023,628 14,198,254 Family 1, IV:1 12 10,137,557 11,183,451 1,045,894 Family 1, IV:1 12 52,284,233 57,619,209 5,334,976 Family 1, IV:1 12 123,200,247 124,325,977 1,125,730 Family 1, IV:1 13 92,345,579 102,106,276 9,760,697 Family 1, IV:1 14 24,429,093 30,066,976 5,637,883 Family 1, IV:1 14 104,573,690 105,411,781 838,091 Family 1, IV:1 15 52,404,862 91,524,841 39,119,979 Family 1, IV:1 16 88,134,285 88,872,145 737,860

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Family 1, IV:1 17 6,115 970,413 964,298 Family 1, IV:1 17 39,081,713 39,165,174 83,461 Family 1, IV:1 18 44,635,048 60,036,083 15,401,035 Family 1, IV:1 19 47,823,038 48,284,025 460,987 Family 1, IV:1 19 57,175,484 57,909,872 734,388 Family 1, IV:1 20 1,298,794 1,616,206 317,412 Family 2, IV:3 1 22,141,206 22,846,709 705,503 Family 2, IV:3 1 87,549,858 89,652,102 2,102,244 Family 2, IV:3 1 147,121,977 151,396,037 4,274,060 Family 2, IV:3 1 155,026,942 156,280,971 1,254,029 Family 2, IV:3 1 162,344,390 169,272,453 6,928,063 Family 2, IV:3 1 223,900,408 226,074,563 2,174,155 Family 2, IV:3 1 247,921,100 248,487,768 566,668 Family 2, IV:3 2 200,173,684 212,251,864 12,078,180 Family 2, IV:3 2 220,239,561 220,435,034 195,473 Family 2, IV:3 4 17,517,099 36,292,018 18,774,919 Family 2, IV:3 4 10,716,8431 111,398,208 4,229,777 Family 2, IV:3 5 15,858,8633 169,461,547 10,872,914 Family 2, IV:3 6 28,228,342 33,382,288 5,153,946 Family 2, IV:3 6 35,027,927 57,055,354 22,027,427 Family 2, IV:3 6 62,284,247 79,595,167 17,310,920 Family 2, IV:3 7 55,266,417 64,453,136 9,186,719 Family 2, IV:3 7 147,926,734 151,818,777 3,892,043 Family 2, IV:3 8 29,927,300 38,005,835 8,078,535 Family 2, IV:3 8 63,978,658 92,136,728 28,158,070 Family 2, IV:3 9 113,192,655 116,931,737 3,739,082 Family 2, IV:3 9 127,101,924 129,102,840 2,000,916 Family 2, IV:3 9 137,672,012 141,016,262 3,344,250 Family 2, IV:3 11 197,557 7,324,475 7,126,918 Family 2, IV:3 11 56,143,198 56,143,823 625 Family 2, IV:3 11 64,066,859 64,572,557 505,698 Family 2, IV:3 12 10,165,469 12,618,715 2,453,246 Family 2, IV:3 13 103,385,015 109,459,020 6,074,005 Family 2, IV:3 16 16,138,313 33,961,370 17,823,057 Family 2, IV:3 16 46,655,210 72,991,715 26,336,505 Family 2, IV:3 16 89,167,075 89,597,221 430,146 Family 2, IV:3 19 17,306,031 17,547,292 241,261 Family 2, IV:3 20 32,295,541 33,764,632 1,469,091 Family 2, IV:3 20 56,846,580 62,737,318 5,890,738 Positions reported for human genome build hg19.

2