Identification of compound heterozygous DNAH11 variants in a Han‐Chinese family with primary ciliary dyskinesia

Abstract Primary ciliary dyskinesia (PCD) is a group of genetically and clinically heterogeneous disorders with motile cilia dysfunction. It is clinically characterized by oto‐sino‐pulmonary diseases and subfertility, and half of the patients have situs inversus (Kartagener syndrome). To identify the genetic cause in a Han‐Chinese pedigree, whole‐exome sequencing was conducted in the 37‐year‐old proband, and then, Sanger sequencing was performed on available family members. Minigene splicing assay was applied to verify the impact of the splice‐site variant. Compound heterozygous variants including a splice‐site variant (c.1974‐1G>C, rs1359107415) and a missense variant (c.7787G>A, p.(Arg2596Gln), rs780492669), in the dynein axonemal heavy chain 11 gene (DNAH11) were identified and confirmed as the disease‐associated variants of this lineage. The minigene expression in vitro revealed that the c.1974‐1G>C variant could cause skipping over exon 12, predicted to result in a truncated protein. This discovery may enlarge the DNAH11 variant spectrum of PCD, promote accurate genetic counselling and contribute to PCD diagnosis.


| INTRODUC TI ON
Primary ciliary dyskinesia (PCD, OMIM 244400) is a group of genetically and clinically heterogeneous conditions with ultrastructural and/or functional defects of cilia, which may result in oto-sinopulmonary diseases, subfertility and situs anomalies. 1-3 Estimated PCD prevalence is 1 per 10,000-20,000 with no apparent racial or gender distinctions. [4][5][6] The true prevalence may be higher than estimated due to diagnostic limitations. 1,5 Its earliest description in the medical literature is believed to be in 1933 by M. Kartagener, who reported a triad of chronic sinusitis, bronchiectasis and situs inversus, following termed Kartagener syndrome (KS). 2,7 About 50% of all PCD cases display situs inversus, which is caused by nodal cilia dysfunction during embryogenesis. 8,9 Respiratory distress, nasal and pulmonary symptoms usually appear during neonatal period and then gradually progress and eventually develop into bronchiectasis. 10 Additionally, most PCD patients have non-chest symptoms, such as recurrent otitis media, chronic rhinitis, recurrent sinusitis, ectopic pregnancy and infertility. 3,11 PCD is a genetic disease inherited predominantly in an autosomal recessive fashion. 12 However, X-linked inheritance has occasionally been evidenced. 13,14 To date, over 40 PCD-associated genes have been identified, 15 accounting for more than 70% of all patients. 12 Plenty of studies on the genetics and biology of PCD over the last decade allowed the identification of numerous pathogenic variants in genes encoding proteins essential for cilia motility and helped to reveal the underlying pathogenic mechanisms (Table S1). [16][17][18] Biallelic variants in the dynein axonemal heavy chain 11 gene (DNAH11, OMIM 603339) have been reported as responsible for primary ciliary dyskinesia-7 (OMIM 611884). 15,19 At least 137 PCD-associated DNAH11 variants have been reported, about 70% of which are missense/nonsense variants. The reported DNAH11 variants and their heterogeneous PCD-associated phenotypes were listed in Table S2. 19 In this study, a three-generation Han-Chinese family with PCD, consistent with KS's diagnostic criteria, was recruited. Compound heterozygous variants, c.1974-1G>C and c.7787G>A (p.(Arg-2596Gln)), in DNAH11 gene (NG_012886. 2, NM_001277115.2) were identified as disease-associated variants.

| Subjects and clinical data
A three-generation Chinese family with PCD was recruited from south central China ( Figure 1A), and an unrelated healthy male without related condition and family history was enrolled as a control.
After informed the relevant matters and obtained signed consent from each enrolee, clinical data and peripheral blood specimens were acquired from five available family members, comprised of the

| Whole-exome sequencing
Genomic DNA (gDNA) was extracted from peripheral blood samples via standard procedure previously reported. 21 One microgram of gDNA from the proband (II:3) was randomly broken into fragments by Covaris technology, and 150-250 bp of fragments were selected. 22 Size-selected fragments were subjected to end-repairing, A-tailing and ligating adaptors. Ligation-mediated polymerase chain reaction (PCR) amplified the ligated fragments. Purification and hybridization to the exome array for enrichment were performed. 23 DNA nanoballs were formed via rolling circle amplification using circular single-stranded libraries and then loaded onto a sequencing chip. The captured exome library underwent high-throughput sequencing on the BGISEQ-500 (BGI, Shenzhen, China) platform in accord with the manufacturer's instructions. 24 Sequencing-derived raw image files obtained were transformed into 'raw data' with BGISEQ-500 base calling software.
The SnpEff software (http://snpeff.sourc eforge.net/SnpEff_manual. html) was then used to annotate variants. The filtering flowsheet is presented in Figure S1. All candidate variants were filtered by several public databases, including the NHLBI exome sequencing Detailed clinical characteristics of available members in this family are presented in Table 1.

| Bioinformation analysis
Conservative analysis results revealed that the arginine at site 2596 (p.R2596) was highly conserved in many species ( Figure 1D

| Splicing study of DNAH11 c.1974-1G>C by minigene assay
Minigene analysis was performed to further characterize the ab-

| DISCUSS ION
PCD is a rare genetic disease predominantly inherited in an autosomal recessive mode and resulted from impaired ciliary function. 4,35 At present, specific clinical treatment for PCD is not unravelled, and the clinical curative effect extremely depends on early clinical diagnosis and timely intervention. 5,36 Gene editing in respiratory ciliated cells with the DNAH11 variant, c.6727C>T, p.(R2243*), can restore cilia motility ex vivo, indicating an exploitable strategy for future therapy. 37 The classical PCD diagnosis was cilia ultrastructural defects visualized by TEM. 38 However, normal cilia ultrastructure appeared in about 30% patients, 39 ~20% of which had biallelic DNAH11 variants. 40 The tests of TEM, HSVM and immunofluorescence applied in PCD patients show significant efficacy in pathological changes, as well as prospect in further genotypephenotype relationship with genetic discovery. 41,42 With the emerging molecular genetic analysis, the diagnostic yield can be obviously raised. 38,42 In this study, compound heterozygous variants Further functional studies and co-segregation analyses in more PCD families are warranted to validate the accurate pathogenicity of those variants and genotype-phenotype correlation.
The unimplemented cilia ultrastructural or function assessment may be the limitations of our study. Previous studies showed that in most PCD cases (92%) with DNAH11 variants, no specific ciliary ultrastructural abnormalities were detected by conventional TEM (Table S2). However, subtle outer dynein arm defects in the proximal ciliary part were visualized when re-examined by three-dimensional electron tomography and immunofluorescence microscopy in two studies. 42,46 No significant genotype-phenotype correlation seems to exist in PCD individuals with DNAH11 variants (

ACK N OWLED G EM ENTS
We thank all the participating members for their cooperation and contribution. This work was funded by National Natural Science Province, China. The funders had no role in study design, data collection, data analysis, decision to publish or preparation of the manuscript.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or used during the study are available from the corresponding author upon reasonable request.