{"paper_id":"a05fd8b8-f3d5-440e-bfac-37a0a7eb1570","body_text":"1 \n \nHigh diagnostic rate of whole genome sequencing in primary ciliary 1 \ndyskinesia 2 \n 3 \n 4 \nHolly A Black1,2, Sophie Marion de Proce1*, Jose L Campos3*, Alison Meynert3, Mihail Halachev3, Joseph 5 \nA Marsh3, Robert A Hirst 4, Chris O’Callaghan4, Scottish Genomes Partnership, Javier Santoyo -Lopez5, 6 \nJennie Murray 2,3, Kenneth Macleod 6, Don S Urquhart 6,7, Stefan Unger 6,7, Timothy J Aitman 1^, 7 \nPleasantine Mill3^  8 \n 9 \n1 Centre for Genomic and Experimental Medicine, MRC Institute of Genetics and Cancer, University of Edinburgh, 10 \nEdinburgh, UK 11 \n2 South East of Scotland Genetics Service, Western General Hospital, Edinburgh, UK 12 \n3 MRC Human Genetics Unit, MRC Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, UK 13 \n4 Centre for PCD Diagnosis and Research, Department of Respiratory Sciences, University of Leicester, UK 14 \n5 Edinburgh Genomics, Edinburgh, UK 15 \n6 Department of Paediatric Respiratory and Sleep Medicine, Royal Hospital for Sick Children, Edinburgh, UK 16 \n7 Department of Child Life and Health, University of Edinburgh, Edinburgh, UK 17 \n* These authors contributed equally to the manuscript 18 \n^ Joint senior authors 19 \n 20 \nSummary: Whole genome sequencing (WGS) yielded a high genetic diagnostic rate (100%) in eight Scottish 21 \npatients with clinically diagnosed pr imary ciliary dyskinesia (PCD) by detection of large structural variants, 22 \nhomology modelling and identification of a novel disease gene with a dominant mode of inheritance. Prioritised 23 \nWGS may facilitate early genetic diagnosis in PCD. 24 \n 25 \n  26 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \nNOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.\n\n2 \n \n 27 \n 28 \nAbstract 29 \nAim: Primary ciliary dyskinesia (PCD) is a genetic disorder affecting motile cilia. Most cases 30 \nare inherited recessively, due to variants in more than 50 genes that result in abnormal or 31 \nabsent motile cilia. This leads to chronic upper and lower airway disease, sub-fertility and 32 \nlaterality defects in some cases. Given overlapping clinical features and genetic 33 \nheterogeneity, diagnosis can be difficult and often occurs late. Of those tested, an estimated 34 \n30% of genetically screened PCD patients still lack a molecular diagnosis. Here, we aimed to 35 \nidentify how readily a genetic diagnosis could be made in a clinically diagnosed population 36 \nusing whole genome sequencing (WGS) to facilitate identification of pathogenic variants in 37 \nknown genes as well as identify novel PCD candidate genes. 38 \nMethods: WGS was used to screen for variants causing PCD in 8 clinically diagnosed PCD 39 \npatients, sequenced as trios where parental samples were available.  40 \nResults: Seven of the eight cases (87.5%) had homozygous or biallelic variants in DNAH5, 41 \nDNAAF4 or DNAH11 that were classified as pathogenic or likely pathogenic. Three of the 42 \nvariants were deletions, ranging from 3kb to 13kb, for which WGS identified precise 43 \nbreakpoints, permitting confirmation by Sanger sequencing. WGS yielded a high genetic 44 \ndiagnostic rate from this clinically diagnosed population, in part through detection of 45 \nstructural variants as well as identification of a de novo variant in a novel PCD gene TUBB4B.  46 \nConclusion: A molecular diagnosis allows for appropriate clinical management for cases and 47 \ntheir families, including prediction of phenotypic features correlated to genotype. Here, WGS 48 \nuplifted genetic diagnosis in cases of clinically diagnosed PCD by identifying structural variants 49 \nand novel modes of inheritance in new candidate genes. Our study suggests that WGS could 50 \nbe a powerful part of the PCD diagnostic toolkit to increase the current molecular diagnostic 51 \nyield from 70%. It provides important new insight into our understanding of fundamental 52 \nbiology of motile cilia as well as of variation in the non-coding genome in PCD. 53 \n  54 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n3 \n \n 55 \nIntroduction 56 \nPrimary ciliary dyskinesia (PCD, OMIM: PS244400) is a genetic disorder of motile cilia (1–3), 57 \nhighly structurally organised organelles that project from the surface of the cells. Their 58 \norganised structure allows the cilia to beat in a coordinated manner to effectively propel 59 \nfluids across the surface of the airways, brain ventricles and reproductive tracts. However, in 60 \nPCD, these cilia have an abnormal structure or function, resulting in static cilia, cilia that beat 61 \nin an uncoordinated manner, or a complete absence of cilia (2,3). This can result in a chronic 62 \nrespiratory disease, due to impaired mucociliary clearance, as well as hydrocephaly, laterality 63 \ndefects (e.g. situs inversus) and fertility defects in a subset of patients. PCD often presents at 64 \nbirth as unexplained neonatal respiratory distress. Affected ch ildren then present with 65 \nvariable clinical features including a daily wet cough, chronic respiratory tract infections, 66 \nrhinitis, sinusitis and otitis media. Over time, these can lead to debilitating long -term 67 \ncomplications, including bronchiectasis and hearing impairment (1–6).  68 \n 69 \nHowever, PCD is a heterogeneous disorder that has significant phenotypic overlap with other 70 \ngenetic respiratory diseases, such as cystic fibrosis and primary immunodeficiency. The 71 \ncurrent clinical diagnostic work -up for suspected PCD cases requires a battery of highly 72 \nspecialised tests (7,8). This includes detection of low nasal nitric oxide (nNO), as well as high-73 \nspeed video microscopy (HS VM) to assess cilia beating pattern and frequency and 74 \ntransmission electron microscopy (TEM) to assess the cilia ultrastructure from a nasal brush 75 \nbiopsy. Abnormalities identified in cilia structure and/or beating pattern or in the presence of 76 \nclinical fea tures such as chronic oto -sinopulmonary symptoms can be used to confirm a 77 \ndiagnosis of PCD (1,2,6,8,9). Access to diagnostic testing is limited to a small number of highly 78 \nspecialised units in the UK. Families must travel for testing, often over long distances and 79 \nsometimes on multiple occasions. Even in those patients with a clinical diagnosis of PCD, 21% 80 \nhad normal ciliary ultrastructure (10). Therefore, additional sensitive and accessible 81 \ndiagnostic techniques acceptable to patients of all ages are required. 82 \n 83 \nPCD is a genetically heterogeneous disorder. The majority of cases are autosomal recessively 84 \ninherited, with over 50 genes currently associated with PCD (1,2,6,11,12). Mutations in these 85 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n4 \n \ngenes account for approximately 70% of PCD cases screened, s uggesting additional causal 86 \ngenes exist and/or more complicated structural variants (SVs) are being missed in known 87 \ngenes (13). The genes ass ociated with PCD encode proteins involved in cilia structure, cilia 88 \nassembly or regulatory complexes. There is often a correlation between the affected gene 89 \nand the defects in cilia motility or ultrastructure observed at diagnosis (1,6,7,9). However, this 90 \nis not always the case: it is estimated that 9 -20% of PCD cases have normal or in conclusive 91 \nultrastructure by TEM (14). Similarly normal or non -diagnostic HSVM results complicate 92 \ndiagnosis for a significant portion of kno wn PCD genes (15). nNO is also considered a useful 93 \nscreening test prior to referral for nasal brushing. While low nNO measurements may be 94 \nindicative of PCD in patients lacking clear ultrastructural defects, its use, particularly in young 95 \nchildren, has not been universally rolled out across PCD centres (16,17) and patients with 96 \nmutations in at least some PCD genes show normal nNO levels (18,19). In such suspected 97 \ncases, where a clinical diagnosis is unclear, a genetic diagnosis is an important means to 98 \nconfirm PCD (1,5,6,20). However, genetic testing often occurs late in the diagnostic work -up 99 \nfor PCD (7). Recent guidelines have recommended studies to investigate the utility of genetic 100 \ntesting in the PCD diagnostic pathway, particularl y as next generation sequencing (NGS) is 101 \nincreasingly available (8,21,22).  102 \n 103 \nIn this study, we investi gated the utility of whole genome sequencing (WGS) to detect 104 \ndisease-causing variants in children and young adults with a clinical diagnosis of PCD. We 105 \nselected a non-endogamous population where PCD was clinically confirmed to test whether 106 \nWGS could effect ively improve the molecular diagnostic yield of PCD. WGS would provide 107 \nunbiased genome coverage beyond exons of ~50 genes associated with PCD whilst 108 \nsimultaneously detecting a range of variants, from single nucleotide changes, small indels as 109 \nwell as compl icated SVs, which may be missed by targeted sequencing approaches. Eight 110 \npatients were recruited, alongside parental samples where available. This led to a genetic 111 \ndiagnosis for all eight cases, three of whom had a pathogenic deletion of 3 kb -13 kb and one 112 \npatient had a de novo missense variant p.P259L in a novel candidate gene TUBB4B. 113 \n 114 \n 115 \nMaterials and Methods 116 \nPatient cohort 117 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n5 \n \nEight patients with a confirmed clinical diagnosis of PCD (50% female), aged 6 to 31 years, 118 \nwere recruited to the study.  Signed and in formed consent was obtained from the affected 119 \nindividual as well as relatives through approved protocols. Sample IDs were assigned and the 120 \nkey known only those within the research group. Clinical phenotypes are shown in Table 1. 121 \nFor details of blood samples, DNA extraction, and analysis of sample ethnicity see 122 \nSupplementary Methods and  Supplementary Figure 1 . The study was approved by the 123 \nLondon-West London and Gene Therapy Advisory Committee Research Ethics Committee 124 \n(REC number 11/LO/0883). 125 \n 126 \nWhole Genome Sequencing 127 \nDNA was sequenced by WGS at Edinburgh Genomics. Libraries were prep ared using the 128 \nIllumina TruSeq PCR -free protocol and sequenced on the Illumina HiSeq X platform. The 129 \naverage yield per sample was 136 Gb, with mean coverage of 36x (range 33.9-38.3).  130 \n 131 \nGene Panel, data analysis and variant classification 132 \nA virtual gene pan el of 146 genes was created, which included the known ‘green’ 34 PCD 133 \ngenes plus 107 suspected ciliopathy genes that may have respiratory features based on the 134 \nPCD PanelApp panel (v1.14) with five additional genes identified in the literature ( CFAP300, 135 \nDNAH6, DNAJB13, STK36 and TTC25) (23–25). Analysis of variants was carried out as described 136 \nin Supplementary Methods. Additional analys es were carried out across the FOXJ1 locus for 137 \nCase 3, in whom no diagnostic variants were identified using the virtual gene panel. A 138 \ngenome-wide expanded analysis identified a de novo  missense mutation p.P259L 139 \n(chr9:g.137242994:C>T (hg38)) in the gene TUBB4B only in the patient, and not present in 140 \neither parent (26).  141 \n 142 \nModelling of whole exome sequencing data 143 \nA whole exome sequencing (WES) -like subset of the WGS data was obtained by extracting 144 \nonly the reads mapping to the regions in the TWIST Exome Capture Kit (using samtools v1.6) 145 \nfrom the BAM file for each sample. WES CNV calling was performed by ExomeDepth (v1.1.15) 146 \nas detailed in Supplementary Methods. 147 \n 148 \nHomology modelling of DNAH11 and location of missense variants 149 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n6 \n \nA homology model of the C -terminal region of the DNAH11 motor domain (residues 3348 -150 \n4504) was built using PHYRE2 (27,28). The effects of mutations in the C-terminal domain (CTD) 151 \n(residues 4124-4504) on protein stability were modelled with FoldX (29). Sequences of human 152 \ndynein genes were aligned with MUSCLE (30) and the sequence alignment was visualised with 153 \nMView (31). Further details are given in the Supplementary Methods. 154 \n 155 \n 156 \nResults 157 \nEight PCD patients were recruited to the study as six trios, one proband-mother duo and one 158 \nsingleton (Table 1). All eight cases had a confirmed clinical diagnosis of PCD, following a nasal 159 \nbrush biopsy. In all cases, nasal brushings were undertaken for clinical suspicion of PCD. 160 \nPresentations and phenotype severity were varied and are summarised in Table 1.  161 \n 162 \nUsing a virtual PCD panel approach to initially screen the WGS, we confirmed genetic 163 \ndiagnosis for seven of the eight cases ( Table 2). Three cases had biallelic pathogenic and/or 164 \nlikely pathogenic variants in the outer dynein arm heavy chain subunit gene DNAH5, which 165 \nhas previously been shown to contribute to the largest proportion of PCD cases among 166 \npopulations of European descent (1). Case 1 had a hemizygous nonsense variant (c.5281C>T, 167 \np.(Arg1761Ter)), inherited from the mother and previously reported in PCD cases (32,33), and 168 \nan overlapping 13kb deletion on the other allele, inherited from the father ( Supplementary 169 \nFigure 3). Case 2 had two nonsense variants in DNAH5, c.3949C>T, p.(Gln1317Ter), which was 170 \npreviously reported (34), and the c.5281C>T, p.(Arg1761Ter) variant. ddPCR was used to 171 \nphase the variants in Case 2 (Supplementary Figure 4). Case 4 had a single base pair deletion, 172 \nresulting in a frameshift and premature termination codon (PTC) (c.10815del, 173 \np.(Pro3606Hisfs)), and a single base pair duplication, resulting in direct creation of a PTC 174 \n(c.13458dup, p.(Asn4487Ter)), which were shown to be in trans through trio -based phasing. 175 \nBoth variants have been reported previously in PCD cases (33). All three cases with biallelic 176 \nvariants in DNAH5 were shown to have absence or defects of outer dynein arms on TEM 177 \n(Figure 1), with Case 1 and Case 2 also shown to have static cilia ( Table 1, Supplementary 178 \nVideo 1 ). This is consistent with loss of DNAH5, which has been shown to result in outer 179 \ndynein arm truncation and immotile cilia (33). All three share a similar clinical phenotype, 180 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n7 \n \nwith chronic wet cough, sinus problems and recurrent chest infections. Hearing loss was also 181 \nnoted in cases 2 and 4. 182 \n 183 \nTwo cases had likely pathogenic variants in the cytoplasmic axonemal dynein assembly factor 184 \nDNAAF4 (DYX1C1), necessary for assembly and/or stability of the inner and outer dynein arms 185 \n(Table 2) (35). Case 5 had a homozygous 3.5kb deletion encompassing exon 7 of DNAAF4, 186 \nwhich was a lso heterozygous in the mother. There was no evidence of uniparental disomy 187 \nand, given Case 5 is also reported to have an affected sibling, it is likely the father also carries 188 \nthe 3.5kb deletion, although the father's DNA was unavailable. This variant has  previously 189 \nbeen reported in PCD (35). Ultrastructural analysis was consistent with previous reports for 190 \nDNAAF4 mutations, with reports of absent dynein arms (35) (Figure 1 ) and immotile cilia 191 \nshown on HSVM. Case 6 had a maternally -inherited nonsense va riant (c.856G>T, 192 \np.(Glu286Ter)) and a paternally-inherited 3.1 kb deletion encompassing the last two exons of 193 \nDNAAF4, predicted to disrupt the C -terminal tetratricopeptide -like helical domain region 194 \n(TPR) by deleting the final 71 amino acids, as well as th e 3’UTR. We predict this allele to be 195 \nloss-of-function as the resulting transcript is predicted to be subject to nonsense -mediated 196 \ndecay. Case 6 has bronchiectasis with a recurrent need for antibiotics. He has no ear or 197 \nhearing problems and situs solitus. 198 \n 199 \nA further two cases had pathogenic or likely pathogenic variants in the outer dynein arm 200 \nheavy chain subunit DNAH11. Case 7 had a homozygous missense variant in DNAH11 (c.13373 201 \nC>T, p.(Pro4458Leu)) previously reported in PCD cases (36,37), at a low frequency in gnomAD 202 \nand in silico  predictions support pathogenicity ( Table 2 ). There was sufficient evidence to 203 \nclassify the var iant as likely pathogenic using ACMG guidelines (38,39). Case 8 had a 204 \nheterozygous 2 bp deletion, resulting in a PTC (c.10 221_10222del, p.(Cys3409Trpfs)), which 205 \nwas classified as pathogenic ( Table 2 ). The second variant was a heterozygous missense 206 \n(c.13288G>C, p.(Gly4430Arg)). This variant is at a low frequency in gnomAD and in silico  207 \npredictions support pathogenicity, but it  has not previously been reported in PCD cases. As 208 \nthe variant is in trans with the c.10221_10222del variant, shown by phasing with parental 209 \nsamples, there is sufficient evidence to classify the variant as likely pathogenic (Table 2). Both 210 \nCase 7 and Case 8 were shown to have a normal cilia ultrastructure with a dysmotile 211 \nphenotype ( Figure 1, Supplementary Video 2 ), consistent with loss of DNAH11, which 212 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n8 \n \nlocalises to the proximal portion of respiratory cilia (40) . There is however phenotypic 213 \nvariability between Case 7 (situs inversus with dextrocardia and recurrent chest infections) 214 \nand Case 8 (chronic wet cough and recurrent ear infections, with situs solitus). 215 \n 216 \nThe two missense variants in DNAH11 both reside at the 3 ' C-terminal domain (CTD) of the 217 \naxonemal dynein heavy chain. While there is no structure available for DNAH11, there are 218 \nhomologous structures of other dynein proteins. To investig ate the effects of the DNAH11 219 \nmissense mutations on protein structure, we built a homology model of the DNAH11 motor 220 \ndomain. Both mutations occur within the CTD, on the outer side of the dynein motor dimer 221 \n(Figure 2A) and are intriguingly very close to each other in three -dimensional space (Figure 222 \n2B), separated by only 4.2 Å.  223 \n 224 \nMolecular modelling of the missense mutations using the program FoldX (29) predicts that 225 \nthe Gly4430Arg should be extremely disruptive to protein structure, with a ΔΔG of 10.9 226 \nkcal/mol. We also modelled all other CTD missense variants presented in the gnomAD v2.1 227 \ndatabase (41) . Remarkably, out of 269 variants (Supplementary Table 1), Gly4430Arg has the 228 \nhighest ΔΔG and therefore is predicted to be the most damaging. More over, this position is 229 \nfully conserved across all human dyneins (Figure 2C). This strongly suggests that Gly4430Arg 230 \nis pathogenic due to its disruptive effects on protein structure. 231 \n 232 \nIn contrast to Gly4430Arg, Pro4458Leu is predicted to be relatively mild at a structural level, 233 \nwith a ΔΔG of 1.1 kcal/mol, making it only the 65 th most damaging out of 269 variants 234 \n(Supplementary Table 1 ). However, this residue is highly conserved, existing as a proline 235 \nacross all human dyneins except DNAH1, where it is an alanine. Thus, while Pro4458 is unlikely 236 \nto cause a severe destabilisation of protein structure, its remarkable proximity to Gly4430 237 \ncombined with a moderate structural perturbation and high conservation are supportive of 238 \npathogenicity. 239 \n 240 \nTo assess whether the three deletion variants identified in Cases 1, 5 and 6 would have been 241 \nidentified using WES, as opposed to WGS, the WGS data was subsetted to create a WES -like 242 \ndataset for each sample ( Supplementary Figure 2). CNV calling using ExomeD epth was able 243 \nto identify the DNAH5 variant c.5272-955_6197del p.(?) in both the proband and father for 244 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n9 \n \nFamily 1. However, the deletion, which spans five exons and has a breakpoint within exon 37, 245 \nwas called as covering only 4 of the 5 affected exons in the proband. The single exon deletion 246 \nof DNAAF4 c.784-1037_894-2012del p.(?) was identified for the proband in Family 5, where 247 \nit is homozygous, but not in the heterozygous mother. The DNAAF4 deletion c.1048 -248 \n149_*1048del p.(?) identified in Family 6 was not detected in the proband or father, despite 249 \nboth carrying the variant.  250 \n 251 \nThe phenotype of Case 3 is particularly relevant as ultrastructural analysis revealed ciliary 252 \nagenesis (Supplementary Video 3), sometimes referred to as reduced generation of multiple 253 \nmotile cilia (RGMC), a specific subtype of PCD. In addition, Case 3 has shunted hydrocephalus, 254 \nhaving undergone initial ventriculo -peritoneal (VP) shunt insertion neonatally, and a 255 \nsubsequent VP shunt revision as an adolescent. To date, few genes have been  implicated in 256 \nthis RGMC phenotype by recessive inheritance, CCNO (cyclin O) and MCIDAS (multicilin) 257 \n(42,43) but no pathogenic or potentially pathogenic variants were identified in either gene. 258 \nHeterozygous de novo mutations in the master motile ciliogenesis transcriptional regulator 259 \nFOXJ1 were identified as the first autosomal dominant cause of a distinct PCD-like condition, 260 \nassociated with chronic respiratory disease, laterality defects and hydrocephalus (18). Similar 261 \ncellular defects are observed with reduced apical docking of centrioles and fewer cilia, but a 262 \nfocused analysis revealed no identifiable pathogenic mutations in the FOXJ1 locus. An 263 \nexpanded, whole genome analysis for SNP and indel candidates produced a very limited list 264 \nof variants ( Supplementary Table 2 ).  This included a de novo missense mutation p.P259L 265 \n(chr9:g.137242994:C>T (hg38)) in the gene TUBB4B only in the patient, and not present in 266 \neither parent or foun d on gnomAD 4.0 or other publicly available databases. Whilst 267 \ninterpretation of pathogenicity from a single patient is limiting, as part of a large international 268 \ncollaboration, we were able to identify a further eleven patients with PCD carrying TUBB4B 269 \nvariants identified by next-generation sequencing (NGS) (26). This included five patients with 270 \nPCD-only carrying the identical p.P259L (chr9:g. 137242994:C>T) variant, one carrying a 271 \ndifferent missense p.P259S (chr9:g.137242993:C>T (hg38)) variant and one patient carried an 272 \nin-frame ten amino acid duplication p.F242_R251dup (chr9: g.137242941_137242970dup 273 \n(hg38)) (26). Moreover, we also identified a recurrent de novo TUBB4B variant four patients 274 \nwith a p.P358S (chr9:g.137243290:C>T (hg38)) variant, who presented with features of both 275 \nPCD with Leber congenital amaurosis and sensorineural hearing loss. In -depth functional 276 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n10 \n \nanalyses in cell and animal models confirmed pathogenicity and distinct dominant -negative 277 \nmechanisms of action to cause different disease presentations (26). In summary, our WGS 278 \nstrategy was very effective at identifying a novel PCD disease gene with the first report of 279 \ndominant negative disease mechanisms.    280 \n 281 \n 282 \n 283 \nDiscussion 284 \nIn this study, WGS of affected probands and family members led to a genetic diagnosis in all 285 \neight PCD patients, representing 11 different mutations in 3 autosomal recessive PCD genes 286 \nand 1 de novo mutation in a novel autosomal dominant PC D candidate TUBB4B. In Case 3, a 287 \npatient with features of reduced generation of motile cilia and hydrocephalus, no pathogenic 288 \nvariants would been identified in a panel-based approach, even with targeted sequencing of 289 \npotential candidates for known RGMC loc i. The diagnostic rate of 100% in our small study is 290 \nhigher than previous reports, in which 60-70% of cases received a genetic diagnosis based on 291 \nthe known PCD gene panels (1,5,12,13,20), 75% on extended NGS panels (44) and 68-94% by 292 \nWES (45–48).  293 \n 294 \nNext-generation sequencing (NGS) technologies continue to revolutioni se rare genetics 295 \nresearch and clinical diagnostics, where the advantages of WES versus WGS are often fiercely 296 \ndebated. Cheaper in terms of costs of sequencing, analysis and data storage, WES is generally 297 \npreferred as a front -line diagnostic tool. WES, howe ver, has several issues in terms of 298 \nevenness of genome coverage and sequence bias, particular for copy number variations 299 \n(CNV). In comparison, several studies have found more accurate variant calls as well as even 300 \nand unbiased coverage of coding regions ar e generated by WGS (49,50). Our analysis was 301 \nfocused on known and candidate PCD genes, screening simultaneously for single nucleotide 302 \nvariants (SNVs), small indels and more complex SVs. In three cases, pathogenic deletions were 303 \nidentified, ranging in size from 3kb to 13kb. Modelling of our WGS data to represent WES-like 304 \ndata suggested that WES data would only have identified t he deletion in one of these three 305 \ncases, in which the deletion was homozygous. Since our WES -like model has more uniform 306 \ncoverage than true WES data, our WES-like model could be considered more reliable for CNV 307 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n11 \n \ncalling than real -life WES sequence. However,  we acknowledge that our model is based on 308 \n36X WGS data, whereas WES would be nearer 100X in practice but coverage does vary across 309 \ncommercial platforms. The ability to detect SVs in PCD is of importance and consistent with 310 \nrecent diagnostic guidelines for PCD, which highlighted that causal SVs and intronic mutations 311 \ncan be missed due to the large number and size of PCD genes (8,21). Further, WES would not 312 \nhave provided the precise breakpoint information that allowed confirmation of the three 313 \ndeletion variants by Sanger sequencing.  314 \n 315 \nWhere WGS was clearly advantageous was PCD disease gene discovery in patients without 316 \nbiallelic variants in known genes or in the one recent example of autosomal dominant 317 \ninheritance ( FOXJ1) or in the few cases of X -linked recessive inheritance ( RPGR, PIH1D3, 318 \nOFD1) (12). In Case 3, WES would have likely identified the de novo SNV in TUBB4B, as WES 319 \npanels identified SNVs in the other 11 TUBB4B patients (26).  Here, the clear advantage of 320 \nWGS was to rule out potential non -coding alterations in the known RGMC PCD genes of 321 \nknown inheritance such as FOXJ1 modes as to quickly prioritise novel candidates in our first 322 \nproband. As such, a clear benefit of WGS, similar to WES, is that its findings are future-proof; 323 \nthe data generated can be re -screened for PCD -causing variants identified subsequent to 324 \ngenetic testing, if initial testing is inconclusive. However, only WGS will allow fut ure analysis 325 \nof non-coding genome for variations in regulatory elements such as of transcription factor 326 \nbinding sites that may underlie a subset of unsolved PCD cases. 327 \n 328 \nWhatever the modality, increased genetic testing for PCD is critical. Currently genetic testing 329 \nfor PCD sits as an additional step to confirm diagnosis by both European and American 330 \nguidelines (8,21,22). As shown here, and elsewhere, genetic testing can accurately diagnose 331 \nPCD where standard clinical procedures are unavailable, impractical or inconclusive (45–332 \n47,51) Importantly, a delayed PCD diagnosis is associated with worse progno sis (52,53). A 333 \nclinical diagnosis of PCD requires specialised, invasive testing, and frequently necessitates 334 \ntravel over considerable distances to specialist centres. These tests are unsuitable for critically 335 \nill neonates and for those unable to travel or unwilling to undergo invasive testing. Genetic 336 \ntesting for PCD may therefore have utility in the neonatal period or in i nfancy that may have 337 \nbeen preferred to the diagnostic odyssey of the patients in this study, where the age of 338 \ndiagnosis was between 5 and 12 years. Suggested clinical criteria for genetic testing in PCD 339 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n12 \n \ncould include term babies who become unwell at >12 ho urs of age with respiratory disease, 340 \nlobar collapse, situs inversus and/or an unexpectedly high or prolonged oxygen requirement 341 \n(54,55). Increasing availability, reducing costs and recognised clinical utility of NGS platforms 342 \nsuch as WGS in the neonatal and paediatric intensive care unit setting would be consistent 343 \nwith such indications (50,51,54,56–59). Moreover, either WES or WGS would hel p rule out 344 \nother confounding clinical presentations such as primary immune deficiency disorders (PIDs), 345 \nwith overlapping symptoms of frequent, often severe, airway infections as well as recurrent 346 \notitis media, and sinusitis (60–62). Both platforms are advantageous as they allow analysis of 347 \nall potential causative genes, known and novel, thus faster to keep up with recently reported 348 \ngenes that may not yet be included on PCD-gene panels. Indeed such technologies may prove 349 \nto be more cost - and time -effective as providing a molecular diagnosis test for PCD than 350 \nconventional clinical gene panels (45,46,48,63). A prospective study would appear to be 351 \nwarranted, to define the optimum criteria for genetic testing and diagnostic yield in neonates 352 \nand older infants and children with respiratory symptomatology to help expedite PCD 353 \ndiagnosis.  354 \n 355 \nWhile earlier genetic testing for PCD is clearly a priority, PCD also remains underdiagnosed. 356 \nPCD ha s an estimated incidence of 1 in 7,500 births, rising to 1 in 2,300 in endogamous 357 \npopulations (64,65). In North America,  it is estimated that only 1,000 patients have a 358 \nconfirmed diagnosis of PCD as opposed to the predicted ~25,000 - 50,000 these rates would 359 \nsuggest to be affected with PCD (66). Similar underdiagnosis is reported in the UK, where 360 \nthese prevalence estimates suggest there should be at least 8,900 people with PCD in the UK; 361 \nless than a quarter of these are known to the NHS highly specialised PCD service (67). A large 362 \nportion of these missing patients are likely adult patients within primary care or 363 \nbronchiectasis clinics, as suggested by one study which found 12% of bronchiectasis patients 364 \nhad pathogenic variants in known motile ciliopathy genes (68). Increased genetic testing as a 365 \nfirst-pass diagnostic test in these suspected cases of PCD could be a way of controlling access 366 \nto more labour -intensive clinical and pathological diagnostic work -up in limited specialist 367 \ncentres.  368 \n 369 \nPCD needs to enter the precision medicine era. A genetic diagnosis is key to improved patient 370 \nprognosis as we better understand genotype-phenotype relations (69) and critically to being 371 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n13 \n \ntrial-ready as much-needed genetic therapies come online (70,71). Increased genetic testing 372 \nis also being combined with a curated worldwide database, similar to that of CFTR2 for Cystic 373 \nFibrosis (72), to enable a better understanding of these genotype-phenotype relations in PCD 374 \ncalled CiliaVar (73). Such a database will significantly facilitate interpretation of variants, of 375 \nwhich 21% has been suggested to be variants of unknown significance (VUS) (73). For 376 \nexample, in Cases 7 and 8 in our study, structural modelling of the CTD of DNAH11 aided the 377 \nassignment of pathogenicity of two missense variants, with one predicted to be detrimental 378 \nto protein stability and  the second, in close proximity to the first, shown to be highly 379 \nconserved. 380 \n 381 \nIn conclusion, this study demonstrates the benefits of using WGS to obtain a genetic diagnosis 382 \nfor PCD, through its ability to detect SNVs and SVs simultaneously as well as detecting variants 383 \nin genes outwith current gene panels. The detection of multi -kilobase deletions in three of 384 \nthe seven diagnosed cases highlights the need to detect SVs as part of the genetic testing for 385 \nPCD. It also allowed rapid prioritisation of SNVs in nov el candidate genes with dominant 386 \nmodes of inheritance. Practically and financially, WGS would likely sit behind current standard 387 \nof care panel -based or WES diagnostic platforms. Given the high genetic diagnostic rate 388 \nobserved here and elsewhere (44,45,47,48), we suggest that genetic testing should be an 389 \nearly step in the current diagnostic pathway for PCD, particularly in cases where nasal brush 390 \nbiopsy is unavailable. By moving clinical and genetic diagnostic pathways in PCD earlier, 391 \nideally to early life, we could have a transformative long -term reduction in morbidity with 392 \naccess to specialist care and disease-modifying therapies commenced before permanent lung 393 \ndamage. 394 \n 395 \n 396 \nAcknowledgements 397 \nWe thank the PCD families who participated in this study and the UK PCD Family Support 398 \nGroup for support; Dr Lee Murphy and colleagues at the Wellcome Trust Clinical Research 399 \nFacility; Edinburgh Genomics for sequencing and analysis; and the Brompton Hospital PCD 400 \nDiagnostic Service and South East Scotland Genetics Service for clinical support.  401 \n 402 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n14 \n \nSupport statement: The Scottish Genomes Partnership is funded by the Chief Scientist Office 403 \nof the Scottish Government Health Directorates [SGP_1] and the MRC Whole Genome 404 \nSequencing for Health and Wealth Initiative (MC_PC_15080). We acknowledge support from 405 \nthe MRC (PM: MC_ UU_00007_14, MR_Y015002_1); an MRC Career Development Award 406 \n(MR_M02122X_1) and Lister Prize Fellowship to JAM; an NHS Research Scotland fellowship 407 \nto SU; and an NRS/R+D fellowship from the NHS Lothian R&D office to DU. 408 \n 409 \nCompeting interests: We confirm that no competing interests. 410 \n 411 \n 412 \n 413 \n 414 \n 415 \n  416 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n15 \nFigures: 413 \n414 \n415 \n416 \n417 \n418 \n419 \n420 \n421 \n422 \n423 \n424 \n425 \n426 \n427 \n428 \n429 \n430 \nFigure 1: Ultr astructure analysis supports genetic diagnosis for outer arm dynein variants in PCD. \n431 \n(left) DN AH5 variants disrupt outer dynein arms (red arrowhead = disrupted, black arrowheads \n432 \nnormal) across axonemes of nasal brush samples from cases HG-001,  HG-002 and HG-004. (right) In \n433 \ncontrast, DN AH11 variants do not uniformly disrupt outer dynein arms (black arrowheads) in cilia \n434 \nfrom nasal brush of case HG-007.  Disruption of both inner and outer dynein arms (red arrowheads) \n435 \nis observed in DN AAF4 variants, as shown for HG-005. Scale bars = 100 nm. \n436 \n437 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n16 \n438 \n439 \n440 \n441 \n442 \n443 \n444 \n445 \n446 \n447 \n448 \n449 \n450 \n451 \n452 \n453 \n454 \n455 \nFigure 2: Structural and evolutionary analysis of DNAH11 missense mutations. (A ) Structure of the \n456 \nhuman cytoplasmic dynein-1 dimer (PDB ID: 5NUG), with the location of the C-terminal domain \n457 \ncoloured beige, and the equivalent sites of the DNAH11 mutations highlighted in red. (B) Homology \n458 \nmodel of the DNAH11 C-te rminal domain with the sites of the missense mutations shown in red, \n459 \nalong with the ΔΔG values calculated with FoldX. (C) Multiple sequence alignment of human dynein \n460 \nproteins around the region where the missense mutations occur. \n461 \n462 \n463 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n17 \n \nTable 1: Clinical details of PCD cases.  480 \nCase number \nSamples \nrecruited \nSex \nEthnicity \nAge at \ndiagnosis \nAge at \nrecruitment \nCiliary \nultrastructure \nCilia motility \nnNO (ppb) \nCurrent FEV1 \n(% predicted) \nNRD \nChronic wet \ncough \nRegular IV \nantibiotics \nRecurrent \ninfections \nRhinosinusitis \nBronchiectasis \nRecurrent ear \ninfection \nHearing loss \nSitus inversus \nDextrocardia \n1 Trio F EUR 11-\n15 \n16-20 Absent \nouter \ndynein \narms \nStatic cilia <5 83 U Y N Y Y N Y N N N \n2 Proband \nonly \nM EUR 6-10 11-15 Outer \ndynein \narm defect \nStatic cilia 8.5 90 Y Y Y N Y Y Y Y N N \n3 Trio F EUR 11-\n15 \n11-15 Ciliary \nagenesis \nN/A 15.5 47 Y Y Y Y Y Y N Y N N \n4 Trio M EUR 0-5 6-10 Outer \ndynein \narm defect \nStatic cilia 75 82 Y Y N Y Y N N Y N N \n5 Proband \nand \nMother \nF EUR 11-\n15 \n31-35 Complete \nabsence of \ndynein \narm \nStatic cilia U U N Y U Y Y Y Y N N N \n6 Trio M EUR U 21-25 Unknown Unknown 8.5 62 U Y Y Y Y Y N N N N \n7 Trio M SAS 6-10 6-10 Normal Dysmotile 49.5 87 N N N Y Y U N N Y Y \n8 Trio F EUR 6-10 11-15 Normal Dysmotile 22 75 U Y N Y Y N Y N N N \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n18 \n \nY= yes; N= no; U= unknown; F= female; M= male; NRD= neonatal respiratory distress; nNO= nasal nitric oxide; ppb= parts per bi llion; FEV1= 481 \nforced expiratory volume in one second; IV= intravenous; EUR= European; SAS= South Asian 482 \n  483 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n19 \nTable 2: Genetic variants identified in PCD cases. Variants were classified according to the ACMG/ACGS guidelines (38,39); the criteria met for each variant 468 \nare shown. 469 \nCase \nnumbe\nr \nGene Transcript dbSNP ID Variant(s) Zygosity gnomAD allele \nfrequency \n(v4.0.0) \nIn silico \npredictions \nACMG/ACG\nS \nClassificatio\nn \nClinVar \nAccession \nNumber \n1 DNAH5 NM_001369.3 N/A c.5272-\n955_6197del p.(?)\nCompound \nheterozygous \nAbsenta N/A P (PVS1, \nPM3, PP4) \nSCV001334255 \nrs148891849 c.5281C>T,\np.(Arg1761Ter) (32–\n34)\n6x10-5 N/A P (PVS1, \nPM2, \nPM3_str, \nPP4) \nSCV001334256 \n2 DNAH5 NM_001369.3 rs176950857\n1 \nc.3949C>T,\np.(Gln1317Ter)(74)\nCompound \nheterozygous \n1.6x10-6 N/A P (PVS1, \nPM2, \nPM3_str, \nPP4) \nSCV001334257 \nrs148891849 c.5281C>T,\np.(Arg1761Ter)(32–\n34)\n6x10-5 N/A P (PVS1, \nPM2, \nPM3_str, \nPP4) \nSCV001334256 \n3 TUBB4B NM_006088.6 N/A c.776C>T,\np.(Pro259Leu) (26)\nHeterozygous Absent Grantham \nScore 98 \nAlphaMissens\ne 0.998 (LP) \nP (PS2, PM2, \nPP2, PP3) \nSCV002770069.\n1 \n4 DNAH5 NM_001369.3 rs397515540 c.10815del,\np.(Pro3606Hisfs)\n(33)\nCompound \nheterozygous \n4.1x10-4 N/A P (PVS1, \nPM2, \nPM3_str, \nPP4) \nSCV001334258 \nrs775696136 c.13458dup,\np.(Asn4487Ter) (33)\n1.6x10-4 N/A P (PVS1, \nPM2, \nPM3_str, \nPP4) \nSCV001334259 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n20 \n5 DNAAF4 NM_130810.4 N/A c.784-1037_894-\n2012del p.(?) (35)\nHomozygous 2.5x10-4 b N/A P (PVS1, \nPM3_str, \nPP4) \nSCV001334260 \n6 DNAAF4 NM_130810.4 rs770136467 c.856G>T,\np.(Glu286Ter)\nCompound \nheterozygous \n2.6x10-5 N/A P (PVS1, \nPM2, PP4) \nSCV001334261 \nN/A c.1048-\n149_*1048del p.(?)\nAbsenta N/A P (PVS1, \nPM3, PP4) \nSCV001334262 \n7 DNAH11 NM_00127711\n5.2 \nrs72658835 c.13373C>T,\np.(Pro4458Leu)\n(36,37,47,51)\nHomozygous 8.4x10-5 Grantham \nScore 98 \nREVEL 0.377 \n(Uncertain) \nAlphaMissens\ne 0.625 (LP) \nLP (PM2, \nPM3, PP3, \nPP4) \nSCV001334263 \n8 DNAH11 NM_00127711\n5.2 \nrs145054078\n8 \nc.10221_10222del,\np.(Cys3409Trpfs)\nCompound \nheterozygous \n1.5x10-5 N/A P (PVS1, \nPM2, PP4) \nSCV001334264 \nN/A c.13288G>C,\np.(Gly4430Arg) (75)\n2x10-6 Grantham \nscore 125 \nREVEL 0.641 \n(Damaging) \nAlphaMissens\ne 0.813 (LP) \nLP \n(PM1_sup, \nPM2, \nPM3_str, \nPP3, PP4) \nSCV001334265 \nP= pathogenic; LP= likely pathogenic; N/A= not applicable. aabsent from gnomAD SV and gnomAD CNV. bfrequency from gnomAD SV. 470 \n471 \n472 \n . 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CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\n26 \nsequencing (WES). BMC Med Genomics. 2018 Oct 25;11(1):93. 670 \n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\nHigh diagnostic rate of whole genome sequencing in primary ciliary\ndyskinesia\nSupplementary Material and Methods\nHolly A Black 1,2, Sophie Marion de Proce 1*, Jose L Campos 3*, Alison Meynert 3, Mihail Halachev 3,\nJoseph A Marsh 3, Robert A Hirst 4, Chris O’Callaghan 4, Scottish Genomes Partnership, Javier\nSantoyo-Lopez5, Jennie Murray 2,3, Kenneth Macleod 6, Don S Urquhart 6,7, Stefan Unger 6,7, Timothy J\nAitman1^, Pleasantine Mill3^\n1 Centre for Genomic and Experimental Medicine, MRC Institute of Genetics and Cancer, University of\nEdinburgh, Edinburgh, UK\n2 South East of Scotland Genetics Service, Western General Hospital, Edinburgh, UK\n3 MRC Human Genetics Unit, MRC Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, UK\n4 Centre for PCD Diagnosis and Research, Department of Respiratory Sciences, University of Leicester, UK\n5 Edinburgh Genomics, Edinburgh, UK\n6 Department of Paediatric Respiratory and Sleep Medicine, Royal Hospital for Sick Children, Edinburgh, UK\n7 Department of Child Life and Health, University of Edinburgh, Edinburgh, UK\n* These authors contributed equally to the manuscript\n^ Joint senior authors\n1\n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\nSUPPLEMENTARY MATERIALS\nSupplementary Table 1: Ranked list of most disruptive reported variants ( ΔΔG) in the C-terminal\ndomain (CTD) of DNAH11 as predicted by FoldX. ΔΔG represents the change in free energy by\nmutation/design of proteins as predicted by the FoldX algorithm, where\nΔΔG = ΔGfold(mutation) − ΔGfold(wild type).\nSupplementary Table 2: Genome wide list of variants detected in patient HG-003 by Slivar.\nSupplementary File 1: Gene panel used for variant filtering with G2P under a biallelic inheritance\nmodel\nSupplementary File 2: Gene panel used for variant filtering with G2P under a monoallelic\ninheritance model\nSupplementary File 3 Droplet digital PCR for variant phasing.\nSupplementary Video 1: HSVM for cases 1, 2 and 4, which have a genetic diagnosis in DNAH5,\nshowing static cilia\nSupplementary Video 2: HSVM for cases 7 and 8, which have a genetic diagnosis in DNAH11,\nshowing dysmotile cilia\nSupplementary Video 3: HSVM for case 3, showing ciliary agenesis\n2\n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\nSupplementary Figure 1: Principal component analysis (PCA) of study samples compared to 1000\nGenomes project samples. Peddy was used to predict ancestry of the samples used in the study by\ncomparison with the 1000 Genomes samples. The 1000 Genomes samples (dots) are colour-coded\nby location. The samples in our study are represented by squares. All samples were predicted to be\nof European ancestry (purple squares), except three (orange squares), which were predicted to have\nSouth Asian ancestry. AFR= African; AMR= American; EAS= East Asian; EUR= European; SAS= South\nAsian.\n3\n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\nSupplementary Figure 2: Alignments of the modelled WES data showing sufficient coverage to call\nthe deletion variants identified in Cases 1, 5 and 6. A: Alignment for Family 1, showing coverage of\nexons 32-38 of DNAH5. B: Alignment for Family 5, showing coverage of exons 6 to 8 of DNAAF4. C:\nAlignment for Family 6, showing coverage exons 8-10 ofDNAAF4.\n4\n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\nSupplementary Figure 3: Sanger sequencing confirms 13kb deletion in DNAH5 in Case 1.\nAlignments of the WGS data for Family 1 show a drop in read depth to approximately 50% of that of\nthe surrounding regions across a 13kb region of DNAH5 in the proband and the father. This spans\nfrom intron 32 to exon 37. PCR and Sanger sequencing across the breakpoints confirms this deletion.\n* indicates the position of the c.5281C>T nonsense variant, which is on the maternal haplotype and\nis therefore hemizygous in the proband.\n5\n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\nSupplementary Figure 4: Drop phase results confirms the two DNAH5 nonsense variants are on\ndifferent haplotypes for Case 2 . The c.3949 variant was assayed using FAM probes and the c.5281\nvariant was assayed using HEX probes. For each combination of alleles, a representative result from\ngenomic DNA (left) and PacI-digested DNA (right) is shown. Each figure plots the number of\nFAM-only positive (blue), HEX-only positive (green), FAM and HEX-positive (orange) and negative\n(grey) droplets. The linkage % is shown for each test. (Details see Supplementary File 3).\n6\n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\nSUPPLEMENTARY METHODS\nPatient cohort\nEight young people with PCD (50% female), aged 6 to 31 years (mean=15, SD= 7.9), were\nrecruited to the study within the Department of Paediatric Respiratory and Sleep Medicine\nat the Royal Hospital for Sick Children, Edinburgh, and the South East Scotland Genetics\nservice. All eight cases had a confirmed clinical diagnosis of PCD, following TEM and/or\nHSVM of a nasal brush biopsy sample. The screening and diagnostic testing was performed\naccording to the PCD National Service protocols, with investigations including nNO, nasal\nbrush biopsies analysed by HSVM for ciliary beat frequency and pattern and quantitative\nelectron microscopy for ciliary ultrastructure. Clinical phenotypes are shown in Table 1.\nBlood samples were collected in EDTA tubes from the patients and parents, where available.\nDNA was extracted using the Chemagic DNA blood kit (Chemagen) or the Nucleon Bacc3 kit\n(GE Healthcare). Sample ethnicity was assessed using Peddy (v4.0.6) (1) (Supplementary\nFigure 1).\nGene Panel, sequence data analysis and variant classification\nBCBio-Nextgen (0.9.7) was used for alignment and variant detection. This used bwa mem\n(0.7.13) to align reads to the hg38/GRCh38 reference genome (2), samblaster (0.1.22) to\nmark duplicate fragments (3) and GATK (3.4-0-g7e26428) for indel realignment and base\nrecalibration (4). GATK HaplotypeCaller was used to calculate genotype likelihoods. Joint\ngenotyping and quality control, including kinship estimates to confirm sample relatedness,\nwere performed using in-house pipelines with GATK (4.0.2.1) following the GATK best\npractices. Variants were annotated using Ensembl variant effect predictor (VEP 90) (5).\nA bespoke gene panel of 146 genes was created (Supplementary Files 1 and 2), based on\nthe PCD PanelApp panel (v1.14) and five additional genes identified in the literature\n(CFAP300, DNAH6, DNAJB13, STK36 and TTC25) (6–8). Variants (SNPs and small indels) were\nfiltered to retain only those within genes on the gene panel and then further filtered to\nidentify candidate variants by inheritance model (both biallelic and monoallelic), transcript\nconsequence, and population allele frequency using the G2P plugin for VEP (9). Variants\nwere assessed using Alamut (v2.13) (10) and classified using the ACMG variant\n7\n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\ninterpretation guidelines (11,12). Any variants classified as pathogenic or likely pathogenic\nwere validated using Sanger sequencing and were submitted to ClinVar. Parental samples\nwere used to determine the phase of compound heterozygous variants, except for Case 2,\nwhich used droplet digital PCR (ddPCR) for phasing (Supplementary File 3).\nSVs were called using Manta (13) and Canvas (version 1.38) (14). The detection of SVs can be\nchallenging and it is common practise to use complementary approaches to detect them\n(15). Manta detects SVs using discordant paired-end and split reads, whereas Canvas relies\non changes in read coverage. We searched for any SVs present in our gene panel that were\ninherited from either parent. We also searched for de novo SVs for cases where both parents\nwere available. SVs were confirmed in silico using SV-Plaudit (16), a tool for rapidly curating\nSV predictions, and/or using the Integrative Genomics Viewer tool (17). Candidate variants\nwere confirmed in the laboratory by PCR and Sanger sequencing across the deletion\nbreakpoints.\nFor Case 3, we did not find any diagnostic variants using our gene panel. As FOXJ1 was only\nrecently identified as a PCD gene and hence was not present on the panel, we searched for\nSNVs, indels and SVs in Case 3 in this gene, given the ciliary agenesis phenotype observed in\nthis case is associated with FOXJ1. We also expanded our analysis for this case to a genome\nwide search for SNV and small indel candidates with Slivar (0.1.10), following the protocol\nfor rare diseases (https://github.com/brentp/slivar/wiki/rare-disease Date accessed: January\n2020). A small number of variants was detected (Supplementary Table 2) and none were\nidentified that fitted the current modes of inheritance for PCD.\nModelling of whole exome sequencing data\nA whole exome sequencing (WES)-like subset of the WGS data was obtained by extracting\nonly the reads mapping to the regions in the TWIST Exome Capture Kit (using samtools v1.6)\nfrom the BAM file for each sample. The capture region fully covers the exons affected by\ncopy number variants (CNVs) identified based on the WGS data (Supplementary Figure 2).\nThe WES CNV calling was performed using ExomeDepth (v 1.1.15) separately on each\nindividual from the three families in which a pathogenic CNV was identified (Families 1, 5\n8\n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\nand 6). As controls, we used WES-like subset data from the WGS data for the other samples\nin this project (total of 21), from which we excluded any members of the family currently\nbeing evaluated. As the WGS data was aligned to GRCh38, we generated a custom reference\ndataset (exons.GRCh38) required by ExomeDepth, to replace the dataset currently\ndistributed with the ExomeDepth package (exons.hg19); exons.GRCh38  is based on the\nlatest CCDS release (r 22) available for the GRCh38 human genome reference (Available at:\nwww.ncbi.nlm.nih.gov/projects/CCDS/CcdsBrowse.cgi?REQUEST=SHOW_STATISTICS).\nHomology modelling of DNAH11 and location of missense variants\nA homology model of the C-terminal region of the DNAH11 motor domain (residues\n3348-4504) was built using PHYRE2 (18), based upon the cryo-electron microscopy structure\nof human cytoplasmic dynein-1 (PDB ID: 5NUG) (19). The effects of the mutations in the\nC-terminal domain (CTD) (residues 4124-4504) on protein stability were modelled with FoldX\n(20), using default parameters and calculated over 10 replicates. Sequences of human\ndynein genes were aligned with MUSCLE (21) and the sequence alignment was visualised\nwith MView (22).\n9\n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint \n\nSUPPLEMENTARY REFERENCES\n1. Pedersen BS, Quinlan AR. Who’s Who? Detecting and Resolving Sample Anomalies in Human\nDNA Sequencing Studies with Peddy. Am J Hum Genet. 2017 Mar 2;100(3):406–13.\n2. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.\nBioinformatics. 2009 Jul 15;25(14):1754–60.\n3. Faust GG, Hall IM. SAMBLASTER: fast duplicate marking and structural variant read extraction.\nBioinformatics. 2014 Sep 1;30(17):2503–5.\n4. 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MView: a web-compatible database search or multiple alignment\nviewer. Bioinformatics. 1998;14(4):380–1.\n11\n . CC-BY 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted February 22, 2024. ; https://doi.org/10.1101/2024.02.21.24302995doi: medRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}