Novel Pathogenic Biallelic Variants in KIAA0586 Expand the Variant Spectrum of Ciliopathies

ABSTRACT

Joubert syndrome (JBTS) is a group of recessive neurodevelopmental disorders classified as a specific type of ciliopathy with genetic heterogeneity. JBTS23, a subtype of Joubert syndrome, is caused by variations in the KIAA0586 gene. In this study, we report a 9‐month‐old boy diagnosed with JBTS based on the presence of the molar tooth sign in the midbrain and global developmental delay. Whole‐exome sequencing identified two pathogenic variants in KIAA0586 (c.3944 T>G and c.3686 + 3A>G), consistent with an autosomal recessive inheritance pattern. These findings were confirmed through Sanger sequencing of the proband and his parents. This study identifies two novel pathogenic variants in KIAA0586, provides a genetic diagnosis for this patient as JBTS23, and expands the variant spectrum of KIAA0586 associated with JBTS.

1. Introduction

Cilia are microtubule‐based organelles present on the surface of most eukaryotic cells, playing vital roles in various physiological processes, including cellular signaling, motility, and the coordination of embryonic development (Bisgrove and Yost 2006; Sreekumar and Norris 2019). They are categorized as primary (non‐motile) or motile cilia. Primary cilia serve as crucial hubs for sensing extracellular signals and regulating developmental pathways such as Hedgehog signaling (Sreekumar and Norris 2019). Defects in cilia structure or function give rise to a broad spectrum of disorders collectively termed ciliopathies, which are characterized by genetic heterogeneity and multisystem involvement (Fliegauf et al. 2007). Ciliopathies can affect the central nervous system, kidneys, liver, retina, and skeletal system, leading to complex clinical manifestations (Cardenas‐Rodriguez and Badano 2009; Hildebrandt et al. 2011; Shaheen et al. 2016).

Joubert syndrome (JBTS, OMIM#PS213300) is a prominent ciliopathy primarily involving the central nervous system. It is clinically defined by developmental delay, hypotonia, and a distinctive midbrain‐hindbrain malformation known as the “molar tooth sign” on brain imaging (Maria et al. 1997; Parisi et al. 2007; Poretti et al. 2017). JBTS is genetically heterogeneous, with over 40 causative genes identified to date, reflecting the complexity of its molecular underpinnings (Bachmann‐Gagescu, Dempsey, et al. 2015; Vilboux et al. 2017). Among these genes, KIAA0586, encoding the centrosomal protein TALPID3, is implicated in JBTS23 (OMIM#616490), a subtype of JBTS (Stephen et al. 2013). In humans, chickens, mice, and zebrafish, the KIAA0586 (TALPID3) protein is localized to the basal body, a component of the centrosome (Ben et al. 2011; Kobayashi et al. 2014; Wu et al. 2014; Yin et al. 2009), and is essential for centrosomal migration prior to ciliogenesis (Stephen et al. 2013). Yan et al. further refined the function of talpid3 to the specific regulation of ciliary gating and identified its key partner ANKRD26 in C. elegans (Yan et al. 2020). Wang et al. demonstrated that genetic silencing of Talpid3 leads to abnormal delamination of radial glial cells, impairs interkinetic nuclear migration, and results in synchronized, ectopic mitosis of neural progenitors and dysregulated neurogenesis. In Talpid3‐null mice, brains exhibit dysregulated neurogenesis and progressive hydrocephaly (Wang et al. 2020). Variants in KIAA0586 disrupt cilia assembly and function, contributing to the clinical spectrum of ciliopathies (Andersen et al. 2003; Stephen et al. 2013; Yin et al. 2009). Elucidating the genetic basis of JBTS not only enhances diagnostic accuracy but also provides insights into the role of cilia in neurodevelopment and disease pathogenesis. In affected individuals from 9 unrelated families with JBTS23, Bachmann‐Gagescu et al. identified homozygous or compound heterozygous variations in the KIAA0586 gene (Bachmann‐Gagescu, Phelps, et al. 2015). In another study, we reported a case of a JBTS patient caused by a compound heterozygote of a CNV and a synonymous variant of KIAA0586 (Shen et al. 2023).

In this report, we found two novel pathogenic single nucleotide variants in KIAA0586 on a JBTS subject using whole‐exome sequencing (WES).

2. Results

2.1. Clinical Summary

A boy of 9 months old was born after normal delivery with a birth weight of 2.77 kg at 35 weeks and 1 day of gestation. His parents were healthy and unrelated. The boy's global development was delayed. He was unable to raise his head at 4 months and could not turn over and sit independently at 8 months. He spoke with only monosyllables unconsciously. The boy exhibited moderate delay for object, adaptability, language, and social abilities, and severe delay for motor evaluated by the Gesell Developmental Schedules (GDS). The boy was hospitalized many times because of respiratory tract infection. The boy had a height of 71 cm (median), weight of 12.2 kg (+3 SD), and occipitofrontal circumference of 46 cm (+1 SD). Physical examination showed strabismus, hyperopia, and small penis. Ultrasonic examination showed normal heart, liver, gallbladder, spleen, kidneys, ureter, bladder, testis, and epididymis. Magnetic resonance imaging (MRI) of the brain revealed enlarged bilateral lateral ventricles. The brain stem was thin, and the cerebellum vermis was smaller. The superior cerebellar peduncle was thickened and elongated (molar tooth sign), and the ventricles resembled bat wings (Figure 1A). Thus, the boy was diagnosed with JBTS (Detailed clinical features were listed in Table 1).

FIGURE 1.

Identification of novel KIAA0586 variants in a patient with Joubert syndrome. (A) Brain MRI of the proband showing the typical molar tooth sign and the lengthened and thickened superior cerebellar peduncles. (B) Pedigree of the Joubert syndrome family with KIAA0586 variants. (C) Chromatogram of the genomic DNA sequences showing the KIAA0586 variants of the proband and his parents.

TABLE 1.

Clinical features of the patient.

Age	9 months
MRI	Cerebellar vermis hypoplasia, the superior cerebellar peduncle was thickened and lengthened (Molar Tooth Sign)
Neurodevelopmental delay evaluation	Moderate retardation for object, adaptability, language and social abilities; severe retardation for motor.
Ophthalmological findings	Strabismus, hyperopia
Renal involvement	Normal USS
Liver involvement	Normal USS for liver, gallbladder, pancreas and spleen
Skeletal changes	Not detected
Others	Abnormal hearing in the left, small penis

Abbreviations: MRI, magnetic resonance imaging; USS, ultrasound scan.

2.2. Genetic Finding

Two compound heterozygous variants in the KIAA0586 gene (NM_001244189.1, c.3944 T>G [p.Leu1315Ter] and c.3686 + 3A>G) were found in the proband. Validated by direct Sanger sequencing, variants c.3944 T>G (p.Leu1315Ter) and c.3686 + 3A>G were inherited from the father and mother, respectively (Figure 1B,C). The nonsense variant, c.3944 T>G (p.Leu1315Ter) in exon 27 was predicted to generate a truncated protein. This variant is a novel variant, which was neither found in gnomAD nor 1000G. The c.3686 + 3A>G was at an intronic position 3 of intron 25 in the KIAA0586 gene (NM_001244189), and 100% conserved in the canonical sequence of mammalian splice sites. This variant might cause aberrant splicing predicted by online software SpliceAI (https://spliceailookup.broadinstitute.org). The experiments of RNA extraction and T‐clone sequencing validated this hypothesis (Figure 2). While DNA transcribing, the variant c.3686 + 3A>G disrupted the normal splicing in the junction of exon 25 and intron 25, and made six mutant transcripts: transcript 1, extended the length of exon 25 by inserting intron 25; transcript 2, extended the length of exon 25 by inserting 216 bp from 5′ of intron 25; transcript 3, extended the length of exon 25 by inserting 28 bp from 5′ of intron 25; transcript 4, extended the length of exon 25 by inserting 22 bp from 5′ of intron 25; transcript 5, extended the length of exon 25 by inserting 8 bp from 3′ of intron 25; transcript 6, shortened the length of exon 25 by deleting 100 bp from 3′ of exon 25 (Figure 2). The mutant transcripts led to an amino acid change at positions 1229, 1230, or 1196 of the KIAA0586 protein, then made an early translational termination ([p.Ile1229MetfsTer, p.Val1230PhefsTer4, and p.Val1196CysfsTer6]) (Figure 2 and Table S2). According to the ACMG guidelines, these two variants were classified as pathogenic (Richards et al. 2015). No additional deleterious variants of other Joubert syndrome‐ or ciliopathy‐related genes were identified in the WES data of the proband.

FIGURE 2.

Sanger chromatograms for cDNA validation of c.3686 + 3A>G by T‐clone sequencing and predicted amino acid changes. (A) The reference sequence. (B) The variant sequences. *Indicates the mutation site.

To determine the effect of variants on KIAA0586 protein level, we analyzed the whole blood protein extracted from the patient, patient's parents, and an unaffected individual by Western analysis using a KIAA0586‐specific antibody. The level of KIAA0586 protein in the patient sample was significantly reduced compared to the control (p < 0.05) and showed a decrease compared to his parents, though this difference was not statistically significant (Figure 3).

FIGURE 3.

Western blot analysis of KIAA0586 protein levels in whole blood lysates. (A) KIAA0586 protein expression in the whole blood. Lanes are non‐contiguous and were digitally rearranged for clarity. (B) Histogram of KIAA0586 protein expression with densitometry. The asterisk *represents statistically significant differences (p < 0.05). F161C: Proband. F161M: Mother. F161F: Father. Con: Unaffected control.

3. Discussion

In this study, we identified two novel pathogenic variants in the KIAA0586 gene through WES in a patient with JBTS. The two variants are KIAA0586 (NM_001244189.1) c.3944 T>G [p.Leu1315Ter] and c.3686 + 3A>G [p.Ile1229MetfsTer, p.Val1230PhefsTer4, and p.Val1196CysfsTer6]. The c.3944 T>G variant was inherited paternally, while the c.3686 + 3A>G variant was inherited maternally. Both variants were present in a compound heterozygous state in the affected individual, consistent with the autosomal recessive inheritance pattern typical of JBTS.

The KIAA0586 gene, located on chromosome 14q23.1, spans 123,898 base pairs and consists of 34 exons and encodes a centrosomal protein, TALPID3, which is essential for ciliogenesis and Hedgehog signaling (Bangs et al. 2011; Nagase et al. 1998; Yin et al. 2009). Variants in KIAA0586 have been implicated in a range of ciliopathies, including JBTS23, preaxial polydactyly, and short rib thoracic dysplasia 14 (SRTD14; OMIM#616546) with polydactyly (Alby et al. 2015; Bachmann‐Gagescu, Phelps, et al. 2015; Cocciadiferro et al. 2020; Wang et al. 2019). Neurological, skeletal, renal, and ocular abnormalities are frequently observed in JBTS patients with KIAA0586 mutations (Bachmann‐Gagescu, Phelps, et al. 2015). The proband in this study, a 9‐month‐old boy, was initially diagnosed with JBTS based on hindbrain malformations, including the characteristic molar tooth sign and other clinical features. WES confirmed the diagnosis of JBTS23 by identifying two pathogenic variants in KIAA0586. Additional clinical findings in this patient included strabismus, hyperopia, and a small penis, with no abnormalities detected in the heart, liver, gallbladder, spleen, kidneys, ureter, bladder, testis, or epididymis. These observations are consistent with the reported genotype–phenotype correlations in JBTS caused by KIAA0586 variants.

JBTS23 is a rare autosomal recessive neurodevelopmental disorder. To date, 52 cases of JBTS attributed to pathogenic KIAA0586 variants have been reported in the literature, involving homozygous or compound heterozygous states (Akawi et al. 2015; Bachmann‐Gagescu, Phelps, et al. 2015; Malicdan et al. 2015; Roosing et al. 2015; Stark et al. 2016; Stephen et al. 2015; Sumathipala et al. 2020; Summers et al. 2017). The types of pathogenic variants in KIAA0586 are diverse, including missense, nonsense, splice‐site, small insertions/deletions, and gross deletions. Synonymous variants affecting splice sites often result in premature stop codons. Most of the variants are located in the TALPID3 region (Figure 4). Among 28 reported pathogenic KIAA0586 variants from these 52 JBTS patients, 22 are definitely truncating mutations (Figure 4 and Table S3), and the combination of two truncating variants is the most frequently observed genotype in JBTS patients with KIAA0586 mutations. Consistently, the patient in our study harbored two truncating variants (c.3944 T>G [p.Leu1315Ter] and c.3686 + 3A>G [p.Ile1229MetfsTer, p.Val1230PhefsTer4, and p.Val1196CysfsTer6]). By western blotting, we observed that the level of KIAA0586 protein was significantly reduced in the patient's sample compared to the control (p < 0.05) and was also decreased compared to his parents' sample, although not significantly. This is likely because each parent has one wild‐type allele capable of expressing fully functional protein, whereas the patient has inherited two mutant alleles predicted to produce truncated proteins.

FIGURE 4.

Structure of KIAA0586 protein (NP_001231118.1) and JBTS23 associated variants. Colored bars indicate protein functional regions. Blue bar, TALPID3 region. Green bar, low complexity region. Orange bar, coiled coil region. There is overlap between TALPID3 and other regions. The brown line indicates gross deletion. Truncated variants are indicated in red, and others in black. The red star indicates the variants in this study.

In conclusion, we identified two novel variants in KIAA0586 in a 9‐month‐old patient, thereby expanding the spectrum of pathogenic variations associated with JBTS23 caused by KIAA0586. This work contributes to a better understanding of the genotype–phenotype correlations in Joubert syndrome. Further functional studies are needed to elucidate the pathogenic mechanisms of KIAA0586 in Joubert syndrome.

4. Methods

4.1. Ethics Approval and Consent to Participate

Written informed consent was obtained from all participants (the patient's consent was signed by the parents). This study was approved by the ethical committees of the National Research Institute for Family Planning and clearly stated that the blood samples would be used for scientific research purposes, including genetic studies.

4.2. Whole‐Exome Sequencing and Variants Analysis

DNA extraction and sequencing: 2 mL of EDTA‐anticoagulated venous blood sample was obtained from all affected and unaffected family members. Genomic DNAs were extracted from whole blood using QIAamp DNA Blood mini Kit (QIAGEN, Germany) according to the manufacturer's protocol. Exome sequencing was performed on the proband. Agilent SureSelect Human All Exon V6 kit (Agilent Technologies Inc., USA) was used for the preparation of exome library by using 3 μg of genomic DNA. Exome library was sequenced to mean 100 × coverage on Illumina Novaseq 6000 platform (Illumina Inc., USA). The average target coverage depth was 126×, with > 97% of the bases covered at > 20×, and a sensitivity of > 98%.

WES and SNVs/InDel analysis were performed for the proband as previously described (Shen et al. 2020). Online software SpliceAI (https://spliceailookup.broadinstitute.org) was used to predict the splicing effect of each of the variants.

4.3. Sanger Sequencing

PCR was performed using specific primer pairs followed by Sanger sequencing on ABI3730xl Genetic Analyzer (Life Technologies, USA) following the manufacturer's protocol for candidate variants validation screened from Whole Exome Sequencing in the proband and his parents. The Table S1 lists the primer sequences.

4.4. Reverse Transcription and T‐Clone Sequencing

The blood samples of the proband and his parents were collected using Tempus Blood RNA Tube (Invitrogen, USA). Tempus Spin RNA Isolation Kit (Invitrogen, USA) was used for RNA extraction from whole blood cells of the proband and his parents. 1 μg RNA was reverse transcribed into cDNA using SuperScript IV First‐Strand Synthesis System Kit (Invitrogen, USA). PCR was performed with specific primer pairs for cDNA and then the target products were linked to the T‐vector (TransGen Biotech, Beijing, China). The ligation reaction contained 4 μL Solution I (Takara, Shiga, Japan), 1 μL pMD19‐T vector, and 5 μL purified PCR product. DNA Sanger sequencing was performed, followed by vector ligation, E. coli transformation, and bacterial culturing. The primer sequences for cDNA are in Table S1.

4.5. Western Blotting

Total protein was extracted from whole blood from the patient, the patient's parents, and an unaffected control individual according to the manufacturer's instructions (Catalog No. EX1200, Solarbio Co. Ltd., Beijing, China). BCA Protein Colorimetric Assay Kit (Beyotime Biotech Inc., Shanghai, China) was used to quantify the total protein. Samples with an equal amount of protein were loaded and separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE). The proteins were transferred to the appropriate polyvinylidene fluoride membrane (Millipore, USA), blocked with 5% skim milk for 1 h at room temperature, and then incubated with diluted antibodies at 4°C overnight. Rabbit anti‐TALPID3 (1:500) and anti‐GAPDH (1:5000) antibodies were purchased from Proteintech (Wuhan, China). Then, the membranes were washed and incubated with secondary antibodies (1:5000) for 2 h at room temperature. The Goat Anti‐Rabbit IgG (H + L) HRP Secondary Antibody was from OriGene Inc., China. After further washing with Tris‐Buffered Saline with Tween (TBST) buffer, the membranes were incubated with Enhanced Chemiluminescence (ECL) reagent to enable the detection of the protein bands. The immunoblotting experiment was repeated three times. ImageJ software was used to perform densitometric analysis of the protein electrophoresis images, with GAPDH as the loading control, and bar graphs were generated by Prism 8.0 (Graphpad, USA) to compare the differences in KIAA0586 protein levels among the patient, the parents, and the controls.

Author Contributions

Y.S., R.H., and M.L. designed, performed the experiments, and analyzed the data. C.L. and Z.W. performed the experiments. Z.C. and Y.Y. analyzed the data. Y.S., R.H., and M.L. conceived the idea, analyzed the data, and wrote the manuscript. All authors approved the final manuscript.

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Primers used for variants validation.

Table S2: Sequence changes in cDNA and amino acids caused by c.3686 + 3A>G.

S3. Pathogenic variants in KIAA0586 associated with JBTS23.

Shen, Y. , He R., Lu C., et al. 2026. “Novel Pathogenic Biallelic Variants in KIAA0586 Expand the Variant Spectrum of Ciliopathies.” Cytoskeleton 83, no. 8: 626–632. 10.1002/cm.70047.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.