DYNC2LI1 mutations broaden the clinical spectrum of dynein-2 defects

Skeletal ciliopathies are a heterogeneous group of autosomal recessive osteochondrodysplasias caused by defects in formation, maintenance and function of the primary cilium. Mutations in the underlying genes affect the molecular motors, intraflagellar transport complexes (IFT), or the basal body. The more severe phenotypes are caused by defects of genes of the dynein-2 complex, where mutations in DYNC2H1, WDR34 and WDR60 have been identified. In a patient with a Jeune-like phenotype we performed exome sequencing and identified compound heterozygous missense and nonsense mutations in DYNC2LI1 segregating with the phenotype. DYNC2LI1 is ubiquitously expressed and interacts with DYNC2H1 to form the dynein-2 complex important for retrograde IFT. Using DYNC2LI1 siRNA knockdown in fibroblasts we identified a significantly reduced cilia length proposed to affect cilia function. In addition, depletion of DYNC2LI1 induced altered cilia morphology with broadened ciliary tips and accumulation of IFT-B complex proteins in accordance with retrograde IFT defects. Our results expand the clinical spectrum of ciliopathies caused by defects of the dynein-2 complex.

Cilia play important roles in differentiation, migration, proliferation, determination of left-right asymmetry and are thus important for the embryonic and postnatal development and proper organ function in adulthood 1,14,15 . The non-motile primary cilium consists of two main components, the basal body complex on the cytoplasmic side of the cell membrane, and the ciliary axoneme, surrounded by a cytoplasmic membrane extending into the extracellular space. The axoneme is radially arranged by nine microtubule doublets growing out of the distal end of the basal body (9 × 2 + 0 structure). The basal body complex consists of a mother and daughter centriole embedded in the pericentriolar material (PCM) 3,4,14,16,17 . The junction between the basal body and the axoneme, the transition zone, is a Scientific RepoRts | 5:11649 | DOi: 10.1038/srep11649 specialized structure with a barrier function ("ciliary gate"), regulating the passage of ciliary proteins into or out of the primary cilium 18,19 .
During the cell cycle cells present a primary cilium in G0/G1 phase and before entering mitosis the cilium is disassembled. For assembly, maintenance and dismantling different proteins are required, brought to their destination via the intraflagellar transport (IFT). The IFT is a bidirectional transit system that transports cargos to the tip of the cilium (anterograde transport) and back to the base (retrograde transport) 17,20,21 . Kinesin-2 motors and subunits of the IFT-B complex are integral parts of the anterograde movement, whereas cytoplasmic dynein-2 and IFT-A components are responsible for retrograde transport 16,22,23 . Cell cycle regulatory signals, cytoplasmic vesicle transport systems and recruitment of all required IFT components at the right time and the right place are important for proper ciliogenesis.
Defects of genes encoding a variety of proteins involved in cilia formation, maintenance and function, such as IFT subcomponents and components of the ciliary axoneme, transition zone or basal body, lead to the broad phenotypic spectrum of ciliopathies 15,24 . Many of the associated phenotypes include brain malformations, polydactyly, kidney cysts, retinal degeneration, and skeletal abnormalities. Today, mutations in 14 different genes have been identified to be causative for skeletal ciliopathies. Because of the genetic heterogeneity of skeletal dysplasias, disrupted proteins affect either the dynein motor (DYNC2H1), or the core components of the IFT transport complexes (IFT43, IFT80, IFT122, IFT140, IFT172, WDR19, WDR34, WDR35, WDR60 and TTC21B) or the basal body region (NEK1, EVC, EVC2) [25][26][27][28][29][30] . The phenotypic spectrum of skeletal ciliopathies includes short ribs, narrow thorax, short stature, postaxial polydactyly and other skeletal abnormalities. These phenotypes are shared by the skeletal short-rib thoracic dysplasias (SRTD

Results
Clinical features of patients with DYNC2LI1 mutations. The affected children were the offsprings of non-consanguineous parents ( Supplementary Fig. 1a). The first child (patient 1) developed a respiratory distress syndrome and died at the age of 3 days. Three further pregnancies ended as missed abortions. The second child (patient 2) developed well, but showed dysmorphic features. During the following pregnancy fetal postaxial polydactyly was noticed and the pregnancy terminated at 19 th week of gestation (patient 3). The observed clinical features were classified between Ellis-van-Crefeld syndrome and Jeune syndrome. Although the affected children present with variable features they do share characteristics in common.
Patient 1 showed a polyhydramnion during pregnancy and was born at 38 th weeks of gestation with a weight of 3300 g (0.3 s.d.) and a body length of 50 cm (-0.17 s.d.). Clinical findings included agenesis of the epiglottis, postaxial hexadactyly of the both hands and the right foot, hypoplastic nails, atrioventricular septal defect and hepatosplenomegaly (Supplementary Fig. 1b-d). She presented with distinctive facial features characterized by medial cleft lip, low set ears, asymmetric tongue, hyperplastic gingivae, epicanthal fold and down-slanting palpebral fissures. A hypoplastic thorax led to a respiratory distress syndrome and the patient died two days after birth. Her karyotype was normal (46,XX).
Patient 2 also showed a polyhydramnion during pregnancy and was born at 40 th week of gestation with a weight of 4120 g (1.53 s.d.) and a body length of 53 cm (0.59 s.d.). Her psychomotor development was normal. She spoke first words at 8 month and was able to walk independently at 14 month. She likewise presented with postaxial hexadactyly of the left hand, brachydactyly, a small medial cleft lip, a hypoplastic epiglottis and narrow thorax, in addition we observed a syndactyly VI/VII of the right hand (Supplementary Fig. 1e-g). Her facial feature include a broad and prominent forehead, a depressed nasal bridge, broad and up-slanting nasal tip and low set ears. She also went through respiratory distress syndrome but recovered after 2 month. Beside an episode of pneumonia she showed no signs of increases susceptibility to infections. No further malformation are known.
Patient 3 was the fetus after termination of the pregnancy at 19 th week of gestation. During pregnancy polyhydramnion and postaxial hexadacytly was noticed ( Supplementary Fig. 1h-j). In addition, the fetus presented with a medial cleft lip and a pronounced narrow thorax.
Radiological investigation of both girls were performed and revealed cone-shaped epiphysis of the phalangeal bones, a metaphyseal dysplasia and a narrow thorax with short ribs (personal communication with Prof. F. Majewksi), so an atypical Jeune syndrome was proposed ( Supplementary Fig. 1k,l).
Whole exome sequencing and candidate screening. Exome sequencing has been demonstrated to be highly successful to identify the underlying genetic cause of autosomal recessive inherited entities [36][37][38][39][40][41] . After Agilent sure select capturing and exome sequencing of patient 2 we received 156.9 M single end reads. 85.01% of the reads were mapped to the human genome version hg19 and 88.06% were on-target. 81.5% of the captured target was covered at least 5x with an average depth of 73.3-fold. A total of 52,679 variants were called, of which 2,076 were indels and 50,603 were single nucleotide variants. Based on the supposed rare incidence of the phenotype we excluded all frequent variants (above 0.01%), detected in any of the 666 in-house control individuals as well as all annotated variants of dbSNP132, the 1000genomes project (phase1), and the exome variant server (http://evs.gs.washington.edu/EVS). Excluding intergenic, intronic and synonymous variants and proposing an autosomal recessive inheritance led to 58 remaining, variants of unknown significance (VUS) in 4 genes. Based on the mutational effect of these variants, their conservation (PhyloP, GERP+ + ) and prediction program scores of SIFT, PolyPhen2 and MutationTaster, CADD only the two DYNC2LI1 mutations remained as candidate variants (Supplementary Table 1): A nonsense mutation c.622C > T (p.Arg208Ter; NM_001193464) in exon 8 and the missense mutation c.662C > T (p.Thr221Ile; NM_001193464) in exon 9 of the DYNC2LI1 gene. Nucleotide numbering uses + 1 as the A of the ATG translation initiation codon in the reference sequence, with the initiation codon as codon 1. Both variants could be confirmed by Sanger sequencing (Supplementary Fig. 2) and were not present in 858 control chromosomes of healthy individuals and in the 61,486 exomes of the Exome Aggregation Consortium (Exome Aggregation Consortium (ExAC), Cambridge, MA (URL: http://exac.broadinstitute.org) Nov. 2014). The affected positions are highly conserved on nucleotide and amino acid level. No mutations in any of the known skeletal ciliopathy genes or other potential candidate genes were identified in this individual. The healthy mother, brother and son were carrier for the missense mutation p.Thr221Ile. DNA of the healthy father and the affected patients 2 and 3 was not available. Based on the family history the nonsense mutation is paternally inherited. Therefore, the identified mutations are highly likely to be pathogenic. DYNC2LI1 expression. We were not able to investigate the effect of the mutations on the expression level, as RNA of the patients was not available. We analyzed the expression levels of DYNC2LI1 in different tissues with adult and fetal complementary DNA (cDNA) panels as well as in osteoblasts and chondrocytes. We examined a ubiquitous expression of DYNC2LI1 in all examined tissues ( Supplementary  Fig. 3). However, the overall highest expression levels were measured in chondrocytes, brain and kidney, no differences between fetal and adult tissues were observed except for a higher fetal expression in brain, kidney and lungs. This is in accordance with expression profiles of genes involved in structure and function of the primary cilium. In addition, we observed an expression of all 3 isoforms in the examined tissues with the highest contribution of the 2 longest isoforms.  (Fig. 1a). The most common functions of the P-loop containing nucleoside triphosphate hydrolases domains are the hydrolysis of nucleoside triphosphate 42 . Modelling of the nucleoside triphosphate hydrolase domain revealed that Thr221 (Fig. 1a,b) is close to the nucleoside binding site. An exchange from threonine to isoleucine in the mutant causes steric clashes and decreases the site of the nucleoside binding pocket (Fig. 1b). Therefore, the mutation is expected to impair the hydrolase function of DYNC2LI1.
As RNA was not available, we were not able to test whether the nonsense mutation might lead to mRNA decay or a shorter protein. However, a predicted truncated protein will miss the coiled-coil domain (position aa 315 -340) which might be necessary for interaction with the coiled coil domain of DYNC2H1 of the dynein-2 complex. DYNC2LI1 localizes to the primary cilium and DYNC2LI1 defects affect morphology and cilia length. As the dynein-2 complex is part of the retrograde IFT of the primary cilium we expected the DYNC2LI1 protein to be localized to the primary cilium (Fig. 1c) 35 . Using immunofluorescence analysis in ciliated fibroblast cells we confirmed the localization of the DYNC2LI1 protein to the cytoplasm, centrosomes, as well as around the basal body, and the transition zone of the primary cilium (Fig. 2).
To determine the patient's phenotype on a cellular level we performed immunofluorescence staining on human fibroblasts. As patient-derived fibroblasts were not available, the cellular effects of DYNC2LI1 depletion were examined following siRNA knockdown of DYNC2LI1 and compared to scrambled siRNA. The remaining expression rate of DYNC2LI1 was on average 39%. Cilia were examined after siRNA transfection and following 5 days of serum starvation to induce ciliogenesis. While cilia were detected in the majority of the cells, no significant difference in percentage of ciliated cells was detected (Fig. 3 a,b: 85% ciliated cells in scrambled controls vs. 84% in DYNC2LI1 depleted cells, p = 0.927 χ 2-test). As DYNC2LI1 is part of the dynein-2 complex and defects of other components of this complex have been associated with alterations of cilia length and morphology we performed further immunofluorescence analysis of the DYNC2LI1 depleted cells. Control cilia showed a mean cilia length of median 1.83 μ m, in comparison to cilia in DYNC2LI1 depleted cells, which were only 1.42 μ m long (Fig. 3 c,d). Measured differences were highly significant with a p-value of 1.205 × 10 −6 [Mann-Whitney-U test]. In addition, we observed significantly altered cilia morphology with broadened ciliary tips in 14% of cilia of the  (Fig. 4 a). To study the defect in retrograde IFT due to the abnormal ciliary morphology, we stained components of the IFT-B complex IFT57 and IFT88. We observed a localization of these at the basal body region of the primary cilium in scrambled controls and knockdown cells. Morphological abnormal cilia in DYNC2LI1 depleted cells showed an accumulation of IFT57 and IFT88 in the bulbous ciliary tip confirming a retrograde IFT defect in these cells (Fig. 4 b,c).

Discussion
The primary cilium has a pivotal role in a variety of complex processes during pre-and postnatal development of almost all vertebrate cells 1,14,15 . The assembly, maintenance and disassembly of the primary cilium is highly controlled throughout the cell cycle to ensure proper function of the associated pathways 43 . Several disorders have been associated with defects of the primary cilium 15,24,31 . The observed phenotypes of these ciliopathies represent alteration of the ciliary pathways and can affect nearly every major organ 44 . Thus, these clinical entities have many clinical features in common. This holds true in particular for the group of the skeletal ciliopathies. Here, the most common group constitutes of the short-rib thoracic dysplasias (SRTDs), including the asphyxiating thorax dysplasia / Jeune and Ellis-van Crefeld syndrome 25,32 . Jeune syndrome / ATD is distinguished by severely constricted thorax leading to respiratory insufficiency, shortened tubular bones and inconsistent polydactyly [45][46][47] . Features observed in Elli-van Crefeld syndrome are congenital cardiac defects, dysplastic nails and teeth, and labio-gingival adhesions, as well as skeletal features like polydactyly, short ribs and limbs 48 . These entities show significant clinical overlap and variable phenotypical expression. The affected individuals of the family reported here present with clinical features that include hypoplastic thorax, short stature, polydactyly, hypoplastic nails, and atrioventricular septal defects. In accordance with the taxonomy, our patients have been classified as Jeune-like or EVC.
NGS has been proven to be effective to identify the underlying genetic cause of inherited diseases [36][37][38][39][40][41] . Concerning the genetic heterogeneity of the skeletal ciliopathies we aimed at the identification of the underlying cause in our family by using whole exome sequencing after SureSelect enrichment. After excluding mutations in the known genes associated with skeletal ciliopathies we focused on rare, coding variants in cilia associated genes 5,[49][50][51][52] . This identified the two heterozygous mutations c.622C > T (p.Arg208Ter) in exon 8 and c.662C > T (p.Thr221Ile) in exon 9 of the DYNC2LI1 gene (Fig. 1 a). In agreement with autosomal-recessive inheritance the non-affected family members were heterozygous carriers for the identified mutations. These mutations were confirmed by Sanger sequencing and could be excluded in further control samples. Both mutations were not present in the 61,486 exomes of the Exome Aggregation Consortium and are therefore conserved, rare variants. Mice homozygous for a knockout of the orthologous gene Dync2li1 tm1Aar are not viable and die before embryonic day 11.5 53 . These mice lack monocilia and develop neural tube and cardiac defects, and defects in trunck and tail development. Thus, we considered these mutations as most likely disease causing.   (D2LIC, LIC3) consists of 3 isoforms (NM_001193464; NM_016008; NM_015522) with the longest to be composed of 13 exons encoding a 352-amino-acid-long protein. It is ubiquitously expressed with high expression levels in brain, kidney, lung and testes shown in immunoblots of adult mice 54 . In human tissues we confirmed the highest mRNA expression levels in chondrocytes, brain and kidney ( Supplementary Fig. 3). Significant difference with higher expression levels in fetal tissues was observed in brain, lung and kidney. The two longest isoforms are predominantly expressed in all examined tissues. As our mutations are located in these 2 longest isoforms only, we considered these to have a pathogenic effect on skeletal development in our patients and therefore to be disease causing.
DYNC2LI1 has been identified as part of the dynein-2 complex involved in the retrograde transport (retrograde IFT) 34,35 . Loss of cytoplasmic dynein-2 in dync2-li1 zebrafish morphants leads to shorter and lower amount of cilia with accumulation of IFT88 near the distal end of the connecting cilium 55 . These morphants have a characteristic phenotype with shortening of the body length, small eyes and kidney cysts in 62% as observed in ciliopathies. Interestingly, defects of other components of the dynein-2 complex, DYNC2H1, WDR34, WDR60 in human have been associated with the clinical phenotype of SRPS type III, Jeune syndrome / asphyxiating thoracic dystrophy (SRTD3/8/11) 34 .
Previous studies localized DYNC2LI1 in mammalian cells at the Golgi apparatus and the centrosomal region 54,56,57 . In addition, a localization to the axoneme of ciliated epithelia cells was reported. Our immunocytochemistry using an antibody against DYNC2LI1 supported the localization to the centrosome (Fig. 2c). Moreover, we refined the reported localization in the axoneme to be concentrated at its base and at the region of the transition zone (Fig. 2a,b). Even though Madhivanan et al. 58 speculated that defects of proteins involved in the control of vesicle trafficking to the cilium leads to a more severe phenotype than defects of the IFT only, the pronounced clinical phenotypes of patients with dynein-2 complex defects, Jeune and our patient, do not support this observation.
On the basis of the observed ciliary defects caused by dysfunction of IFT and basal body proteins, we expected loss of function of DYNC2LI1 to cause morphological abnormal and missing cilia in humans. As we were not able to obtain patient cells, we used immunofluorescence in DYNC2LI1 depleted human fibroblasts. We did not observe a significant difference in the percentage of ciliated cells (Fig. 3a,b), a significant average size reduction could be detected (Fig. 3c,d). The observation of shorter cilia confirmed the results in zebrafish morphants and Dync2li1 tm1Aar knockout mice 53,55 . Reduced cilia length with bulbous tip was although reported for defects of DYNC2H1 and WDR34, other components of the dynein-2 complex 28,59 . However, one report showed no significant difference in proportion of ciliated cells and cilia length in Wdr34-knockdown ATDC5 lines 29 . In a further component of the dynein-2 complex, WDR60, a reduced amount of ciliated cells but normal cilia length was observed in cell lines of affected patients 30 . Together, these data indicate that the reduction of ciliated cells as well as of cilia length is a common hallmark of dynein-2 defects. In addition, we showed a higher incidence of cilia with a bulbous tip suggesting a defect in microtubule architecture (Fig. 4 a). This was also observed in Dync2li1 tm1Aar knockout mice where stumpy nodal cilia contain disorganized microtubules, IFT proteins and cellular debris 53,60 . IFT-B proteins to be accumulated in cilia with retrograde IFT transport defects include IFT57 and IFT88 30,61 . Morphological abnormal cilia in our knockdown cells showed a significant accumulation at the ciliary tip confirming the IFT transport defect in DYNC2LI1 deficient cells (Fig. 4 b,c).
As primary cilia act as sensors for different pathways, the defect of the dynein-2 complex leading to retrograde IFT impairment is consistent with impairment of these signaling cascades. Among others, defects of the Sonic Hedgehog pathway have been associated with limb-development defects confirming the skeletal phenotype in our patients 24,62,63 .
In conclusion, mutations in members of the dynein-2 complex result in Jeune syndrome / ATD and other forms of the SRTD spectrum. Different defects of this complex suggest a correlation between the amount of ciliated cells, the resulting cilia length and the functional IFT components with the phenotypically overlapping, but variable clinical features. Thus, the identification of mutations in DYNC2LI1 in our patients expands the phenotypic spectrum of skeletal ciliopathies.

Methods
Subjects and clinical data. Informed consent was obtained from the patients for experimental protocols and displaying participants' facial appearances in publications. Peripheral blood samples were obtained from the patients as well as control individuals. Genomic DNA from all collected samples was extracted according to standard procedures. This study was approved by the Ethical Review Board of the Friedrich-Alexander-Universität Erlangen-Nürnberg.
Whole Exome Sequencing. DNA of patient 2 was enriched using the Agilent's SureSelect Human All Exon Kit V3 (Agilent technologies, Santa Clara, CA) and sequenced in single-end reads on a SOLiD system (Life Sciences, Santa Clara, CA). Sequenced reads were mapped with LifeScope (Life Technologies, Carlsbad, CA) to the reference human genome assembly hg19 (GRCh37). Genotypes were additionally called with GATK 2 and SAMtools [64][65][66]  database consisting of 666 sequenced exomes was also used to exclude sequence errors and frequent variants. Graphical presentation of the mapped sequences was viewed with the Integrative Genomics Viewer (IGV) 72,73 . Variants were selected based on autosomal recessive inheritance, their population frequency, the location within the coding sequence (exon, splice site) and the functional effect. The resulting variants were Sanger sequenced and the segregation in the family tested Protein modelling. The domain architecture of DYNC2LI1 was investigated using the InterPro 74 and Genesilico 75 Meta prediction servers. Molecular modelling of residues 28-241 of DYNC2LI1 was performed with Modeller 9.9 76 using the crystal structure of human RAB5B in complex with GDP (PDB code: 2HEI) as a template. The mutation was modeled using SwissModel 77 and RasMol was used for structure analysis and visualization 78 . DYNC2LI1 expression analysis in human tissues. Relative DYNC2LI1 expression was measured by quantitative real-time PCR using predesigned TaqMan Gene Expression Assays with the TaqMan Gene Expression Mastermix (Life Technologies) specific for all 3 isoforms (Hs00602913_m1, spanning exon 5-6), isoform 4 (Hs01005273_m1, spanning exon 6-7 of NM_001193464) and isoform 1 (Hs01011588_ m1, spanning exon of 6-7 NM_016008). An isoform 2 (NM_015522) specific probe was designed using the Custom TaqMan  We used commercially available cDNA panels (Human Fetal MTC TM Panel", "Human MTC TM Panel I" [Clonetech, Mountain View, USA]) covering fetal (whole brain, heart, liver, lung, kidney) and adult (whole brain, heart, liver, lung, kidney) tissues to determine the expression pattern of DYNC2LI1 in different human tissues. RNA of osteoblasts and chondrocytes were obtained from 2 different (osteoblasts) and 8 different (chondrocytes) adult individuals.