Background Joubert syndrome (JBTS) is a rare neurodevelopmental disorder with marked phenotypic variability and genetic heterogeneity. Homozygous or compound heterozygous mutations in the KIAA0586 gene on chromosome 14q23 are known to be associated with JBTS-23. The frameshift variant c.428delG is the most frequent KIAA0586 variant reported in JBTS-23; yet, homozygosity of this variant was observed in two patients with JBTS-23. However, homozygosity of the c.428delG variant was recently reported as well in one healthy individual.
Objective To clarify whether the frameshift variant c.428delG in KIAA0586 is pathogenic in the homozygous state.
Methods Whole-exome sequencing as well as RNA analysis were performed.
Results We identified biallelic mutations, including the variant c.428delG and a splice site variant c.1413–1G>C, in KIAA0586 in two siblings with clinical and MRI features of JBTS. The c.1413–1G>C variant was inherited from the healthy father. The c.428delG variant was found in the healthy mother in a homozygous state in blood lymphocytes, hair root cells and buccal epithelial cells. RNA analysis revealed that the transcript harbouring the c.428delG variant was expressed in blood cells from the healthy mother, indicating that transcripts harbouring this variant elude the mechanism of nonsense-mediated mRNA decay.
Conclusion Considering this and the high allele frequency of 0.003117 in the gnomAD database, we conclude that c.428delG represents a JBTS disease-causing variant only if present in compound heterozygous state with a more severe KIAA0586 variant, but not in a homozygous situation.
- clinical genetics
- joubert syndrome
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Joubert syndrome (JBTS) is a rare neurodevelopmental disorder with marked phenotypic variability and genetic heterogeneity. Main clinical features comprise congenital hypotonia, ataxia, motor retardation, cognitive impairment and abnormal eye movements.1–3 A wide range of multisystem organ involvement, including liver and kidney disease, polydactyly, retinal dystrophy, chorioretinal coloboma and encephalocele, may occur. The ‘molar tooth sign’ (MTS) deriving from a distinct malformation of the brainstem and cerebellum constitutes a highly characteristic neuroimaging feature of JBTS.1 4
JBTS was assigned to the group of ciliopathies as the proteins disordered in JBTS were recognised to interfere with the function of non-motile cilia.5 Until now, biallelic or X–linked variants in more than 30 genes were reported to be associated with various subtypes of JBTS.3 Recently, variants in the KIAA0586 gene, the human homologue of the chicken talpid3 gene, were described in patients with JBTS and other ciliopathies.3 6–10 The OMIM catalogue of human genes and genetic disorders designates KIAA0586-associated JBTS as type 23 of JBTS (OMIM # 616490).
Here, we report a family with two affected individuals presenting with clinical and neuroimaging features of JBTS caused by compound heterozygous variants in KIAA0586. Of note, the healthy mother carried one of these variants, c.428delG, in homozygous state leading us to the conclusion that homozygosity for c.428delG is not associated with JBTS. Our finding has important implications for molecular testing and diagnosis in patients with JBTS.
As part of an ongoing study on clinical and genetic features of congenital oculomotor apraxia, we investigated two siblings (a man aged 24 years and a woman aged 21 years at last follow-up) with JBTS. Clinical and neuroimaging details were described previously (patients #8 and #9 in Ref. 11). In brief, both siblings presented in their first year of life with impaired fixation, poor visual pursuit, muscular hypotonia and motor developmental delay. Marked ataxia and cognitive impairment became apparent in the course. Ophthalmological features included ocular apraxia with inability to initiate horizontal saccades and Duane syndrome. Ocular apraxia attenuated over the years and disappeared at 4–5 years of age. MRI of the brain revealed the MTS in both siblings, indicating JBTS (figure 1).
Motor and mental development as well as physical and neurological examination of the mother were normal. She had normal eye movements with normal horizontal saccades. MRI of the brain was normal (figure 1).
Whole-exome sequencing (WES) was performed in both affected siblings and both healthy non-consanguineous parents. Exonic and adjacent intronic sequences were enriched from genomic DNA using the NimbleGen SeqCap EZ Human Exome Library v3.0 Enrichment Kit and were run on an Illumina HiSeq2000 sequencer at the Cologne Center for Genomics (CCG), University of Cologne, Germany. Data analysis and filtering of mapped target sequences were performed with the ‘Varbank’ exome analysis pipeline and data were filtered for high-quality variants. The KIAA0586 variants were confirmed by Sanger sequencing.
For RNA analysis, RNA was extracted from a blood sample of the healthy mother using the PAXgene system and transcribed into cDNA according to standard protocols.
To reveal the expression of KIAA0586 in the healthy mother who carried the homozygous variant c.428delG, three pairs of primers were designed according to the reference sequence (NM_001244189.1). The primers of amplicon 1 were located in E4 (forward) and E7 (reverse). This amplicon includes the exon 5 where the variant c.428delG exists. The primers of amplicon 2 were located in E15 (forward) and E18 (reverse). The primers of amplicon 3 were located in E30 (forward) and E33 (reverse) (figure 2). The amplified product was Sanger sequenced.
The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki. Written informed consent was obtained from all patients.
Written informed consent was obtained for WES from both affected siblings and both healthy parents. Data were generated at the CCG. WES data analysis and filtering of variants were carried out using the exome analysis pipeline ‘Varbank’ (CCG). We applied the following criteria for filtering of the WES variants: coverage of more than 6 reads, a minimum quality score of 10, an allele frequency ≥25% and a minor allele frequency <0.1% in the various databases. We identified two variants in KIAA0586 in DNA derived from blood lymphocytes from both siblings in a compound heterozygous state, c.428delG (p.Arg143Lysfs*4), inherited from the mother, and c.1413–1G>C, inherited from the father. The c.428delG (p.Arg143Lysfs*4) variant was found in the healthy mother in a homozygous state in blood lymphocytes. No pathogenic variants in other ciliopathy genes could be observed by exome sequencing in the healthy mother. Sequencing of the c.428delG variant in KIAA0586 was also performed in hair root cells and in buccal epithelial cells from the mother, which again revealed the homozygous c.428delG variant as well. Thus, no evidence for mosaicism was found. RNA analysis with three amplicons revealed that the transcript harbouring the c.428delG variant was expressed in blood cells from the healthy mother (figure 3).
The c.428delG variant is listed in the gnomAD database (http://gnomad.broadinstitute.org/) in 769 of 246.736 alleles (2 times in homozygous state) with a frequency of 0.003117.
Both siblings affected by JBTS carry a combination of a frameshift variant c.428delG (p.Arg143Lysfs*4) and a splice site variant c.1413–1G>C in the KIAA0586 gene in a compound heterozygous state. This genotype was reported before in patients with JBTS.7 9 10 Furthermore, the c.428delG (p.Arg143Lysfs*4) variant was identified in a homozygous state in two patients with clinical and neuroimaging features of JBTS.9 10 This variant represents the most frequent KIAA0586 variant reported yet.
Thus, the homozygous occurrence of the frameshift variant c.428delG in the healthy mother was initially puzzling. However, homozygosity of this variant in a healthy subject was reported before. In a study analysing the whole genomes of 2636 healthy Icelanders, the KIAA0586 c.428delG variant was observed in a homozygous state in one healthy individual aged 57 years.12 A possible explanation for these findings is that c.428delG is a hypomorphic variant, which in the homozygous state is not sufficient to cause the clinical phenotype of JBTS. The variant c.428delG leads to a frameshift and a predicted premature stop codon (p.Arg143Lysfs*4). In the healthy individual reported here carrying the homozygous c.428delG variant, RNA analysis revealed that transcripts harbouring this variant are still expressed and elude the mechanism of nonsense-mediated mRNA decay (NMD). NMD is a mechanism that selectively degrades mRNAs containing premature termination codons, if these codons are located more than 50–55 nt upstream of an exon–exon junction.13 The premature stop codon produced by the variant c.428delG is located only 7–10 bp upstream of the exon–exon junction. Therefore, it is not surprising that the transcript evades the NMD mechanism.
KIAA0586 encodes a centrosomal protein required for ciliogenesis and hedgehog signalling.14–16 Eleven protein-coding isoforms of the gene are described (NCBI, ENSEMBL and UCSC). The full-length isoform contains 34 exons and initiates in exon 1 (NM_001244189.1), the other transcripts have different initiation sites and partially lack exons. The shortest protein-coding transcript has the start codon in exon 7, lacks exons 32, 33 and 34 and uses an alternative exon for termination (NM_001244193.1).10 The variant c.428delG affects nine of the 11 transcripts. A truncated protein would lack the coiled-coil domain, which was shown to be important for the mediation of centrosomal localisation and function of the KIAA0586 protein.6 14 One explanation of the hypomorphic behaviour of the c.428delG variant in a homozygous state might be that the existence of the short isoform (NM_001244193.1) prevents causing the phenotype. It is also conceivable that an alternative start codon is used for the initiation of the isoforms affected by the variant c.428delG. The coexistence of the c.428delG variant with a more severe mutation in trans like the splice site mutation c.1413–1G>C, which affects all isoforms seems to be sufficient to result in JBTS.
The putative conflicting observation of homozygosity of the KIAA0586 c.428delG variant in both, patients with JBTS and healthy individuals, was discussed previously.6 Homozygous occurrence of this variant in healthy individuals was assumed to ‘either be due to protective modifiers or a low mutational load in the ciliome of the respective person’.6 Based on the frequency data of 0.003117 for the c.428delG variant obtained from the gnomAD database—which also includes two homozygous carriers—we conclude that the c.428delG variant likely represents a mild hypofunctional allele, which is not disease causing in a homozygous situation.
The c.428delG variant was already considered a hypomorphic allele that might boost susceptibility for the clinical phenotype of JBTS. It was presumed that in heterozygous carriers of this variant, as in the siblings reported here or in several patients described previously,9 10 a more severe mutation in the other KIAA0586 allele is necessary to develop JBTS. In homozygous carriers, mutations in other ciliopathy genes are supposed to be mandatory for disease manifestation. A previously reported 4-year-old patient with JBTS and homozygosity of the KIAA0586 c.428delG variant carried an additional heterozygous p.T1598K variant in the C5ORF42 (CPLANE1) gene associated with JBTS-17.9 In a second patient with JBTS and the homozygous c.428delG variant, no data concerning additional variants in ciliopathy genes were provided.10 As an alternative explanation, the c.428delG variant in these homozygous patients could be located on the same allele with the ‘true’ disease-causing variant not being identified during molecular analysis.
Taken together, the assumption of the KIAA0586 c.428delG variant being a hypomorphic allele is confirmed by our findings. This result is of importance for genetic testing of patients with JBTS as well as counselling of families with KIAA0586 c.428delG variant carriers.
The authors would like to thank the family for participating and supporting this study. We thank Karin Boss for kindly reviewing the manuscript.
Contributors SP and KB provided clinical data and wrote the manuscript, JA, SS, AO, HT, CB, PN and YL provided molecular genetic data, SD-K provided neuroimaging data, SP, PN, BW and KB supervised the study.
Funding This work was supported by a grant from the Niedersächsisches Ministerium für Wissenschaft und Kultur, grant no.74ZN1284 (to KB).
Competing interests None declared.
Patient consent Obtained.
Ethics approval The study was approved by the ethics committee of the Faculty of Medicine, University of Göttingen (file no. 19/5/14).
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement Additional unpublished data from the whole-exome sequencing of both patients and their parents are available.