Case Report: Identification of likely recurrent CEP290 mutation in a child with Joubert syndrome and cerebello-retinal-renal features.

Background. Joubert syndrome (JS) is a rare autosomal recessive ciliopathy with an estimated prevalence of 1 in 100,000. JS is characterized by hyperpnoea, hypotonia, ataxia, developmental delay and various neuropathological abnormalities in the brain including cerebellar hypoplasia and cerebellar vermis aplasia. JS can also have variable multi-organ involvement, including the retina, kidneys, liver, and musculoskeletal system. Methods and Results. Here we report a clinical description of two-year-old girl presenting with breathing difficulties, hyperechoic kidneys with loss of corticomedullary differentiation. Brain magnetic resonance imaging revealed the typical molar tooth sign consistent with a clinical diagnosis of JS and retinal examination showed severe retinal dystrophy leading to blindness. Molecular genetic analysis using whole exome sequencing and Sanger sequence confirmation demonstrated a homozygous mutation (c.5493delA, p.(A1832fs*19) in CEP290 which segregated from either parent and was consistent with the multisystem ciliopathy phenotype. This precise variant has been described previously in 2 families from the Kosovar-Albanian region suggesting this allele is a recurrent mutation in this population. Conclusions. Mutations in CEP290 lead to multisystem ciliopathy syndromes and molecular genetic diagnostics of such cases allows precise diagnosis, screening of at risk relatives and appropriate management.


Introduction
Joubert syndrome (JS, MIM 213300) is a rare heterogeneous neurodevelopmental disease, being associated with an autosomal or X-chromosomal recessive inheritance. There are at least 34 genes (OMIM PS213300) associated with different subtypes of JS (Parisi and Glass 1993). JS can present clinically with different symptoms including hypotonia progressing to ataxia, global development delay, eye movement abnormalities (nystagmus and ocular molar apraxia) and dysregulation of breathing. Other clinical features found in JS patients depends upon other systemic involvement but includes retinal dystrophy, hepatic fibrosis, polycystic kidney disease and polydactyly (Brancati et al. 2010;Parisi and Glass 1993;Romani et al. 2013;Sayer et al. 2006;Valente et al. 2013). The prevalence of JS is approximately 1 in 100,000 of live births worldwide. From the currently known JS genes, TMEM67 is the most frequent disease causing gene in European and Japanese patients (Bachmann-Gagescu et al. 2015;Kroes et al. 2016;Suzuki et al. 2016) and is one of the most frequent cause of JS with renal involvement (Fleming et al. 2017). CEP290 is also a frequent cause of JS associated with kidney phenotypes (Bachmann-Gagescu et al. 2015;Vilboux et al. 2017). Here we report a patient from Kosovo with a clinical diagnosis of JS with kidney and retinal involvement in whom we identified a homozygous frameshift mutation in CEP290.

Case presentation
The Institutional Review Board of Department of Paediatrics, Clinical University Center, Kosovo, approved this work. The parents of the patient provided their written informed consent before clinical and laboratory examinations and for publication of the case. Blood from the patient and their healthy parents were obtained to allow DNA extraction and genetic studies. All the procedures performed in the study were in accordance with the Helsinki Declaration.
The female patient (JBTS-2) was born to a not knowingly consanguineous couple from Kosovo. The patient had three healthy siblings, two older sisters and one younger brother. During antenatal scans, ill-defined brain morphology changes were noted. Following birth, via Caesarean section at 38 weeks gestation, the birth weight was 3460 g, length 50 cm, and head circumference 35 cm. Following birth, the baby became dyspnoeic and cyanotic, and was treated with oxygen therapy (FiO 2 30%). Externally, there were no dysmorphic signs. At age of 1 year, the child had evidence of poor growth: weight 7610 g (below 5 th percentile), height 75 cm (below 25 th percentile), head circumference 48 cm (below 5 th percentile). At 2 years of age, the child demonstrated features of developmental delay and was unable to sit or walk. She had started mumbling at the age of 18 months but did not develop any coherent speech. She had horizontal gaze nystagmus and ptosis in right eye. Abdominal ultrasound scanning showed enlarged, hyperechoic kidneys with loss of corticomedullary differentiation, with a number of small cysts up to 12 mm in diameter. There was evidence of progressive chronic kidney disease with serum creatinine 243 μmol/L. Magnetic resonance imaging (MRI) of the head and neck suggested a Dandy Walker anomaly and a typical "molar tooth sign". Eye examination revealed severe retinal dystrophy and ptosis of the right eyelid (Table 1). Molecular genetic analysis using exome sequencing and Sanger sequence confirmation revealed a homozygous frameshift mutations c.5493delA; p.A1832fs*19 in CEP290 (Figure 1). This variant segregated from each parent.

REVISED Amendments from Version 1
The manuscript has been updated with a new table (Table 1) to describe the clinical features of presented case in a clearer manner. Table 2 has been updated to allow the reported case to be compared to cases carrying the same CEP290 allele. We have also now included a detailed discussion of the problems in describing genotype-phenotype correlation within CEP290associated disease cases.
Any further responses from the reviewers can be found at the end of the article Here we report a patient with a molar tooth sign on brain MRI imaging with retinal and kidney involvement in whom we found using exome sequencing a homozygous frameshift mutation in CEP290 (NM_025114.4 c.5493delA; p.A1832fs*19, ClinVar https://www.ncbi.nlm.nih.gov/clinvar/variation/56739/). This variant, located in exon 29 and predicted to truncate the C-terminus of the CEP290 protein (Figure 1 (Table 2). Interestingly, of these families with Meckel syndrome and the CEP290 c.5493delA allele, one consanguineous multiplex family and a second consanguineous family, not knowingly related to the first, were from the Kosovo-Albanian region suggesting this allele might be a recurrent or founder allele from this population. The lack of genotype-phenotype even with this exact variant in families from the same geographical region is noteworthy. The previously reported cases with the homozygous c.5493delA allele in CEP290 had a classical Meckel syndrome phenotype whilst the case we present here is typical for a multisystem JS. Clearly, there may be modifier alleles or unknown factors influencing the phenotype, and CEP290 provides a good example of a gene where genotype-phenotype correlations are absent (Coppieters et al. 2010). There is some evidence of a modifier allele for the kidney phenotype relating to CEP290 mutations (Ramsbottom et al. 2020), but the neurological variability remains unexplained.

Discussion
In conclusion, JS is often a multi-organ primary ciliopathy syndrome and patients should undergo a clinical and imaging diagnostic protocol to evaluate possible different abnormalities (Bachmann-Gagescu et al. 2020) and genetic testing to allow identification of the underlying cause. Our results highlight the importance of combining clinical and radiological features of JS with molecular genetic analysis to allow a precise diagnosis of JS. An accurate diagnosis helps in genetic counselling, early management of genetic disorders and offers prenatal diagnosis as an option for future pregnancies. It also allows genotype-phenotype correlations to be determined and allows new information to be gained regarding population specific disease alleles.

Data availability statement
The original contributions presented in the study are publicly available on LOVD (Fokkema et al. 2011). This data can be found here: https://databases.lovd.nl/shared/individuals/00402012.
Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Ethics statement
The Institutional Review Board of Department of Paediatrics, Clinical University Center, Kosovo, approved this work. The studies involving human participants were reviewed and approved by North-East Newcastle and North Tyneside 1 Research Ethics Committee (18/NE/350). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.

Author contributions
The study was conceived and designed by JS. JS, LS, EB, TL and GT contributed to the acquisition, analysis of data, and writing the first draft. LS contributed to collecting the data and communicated with the patient's family. JS contributed to molecular genetic studies and in silico analysis. The final version was edited by JS. All authors edited and approved the final manuscript. Open Peer Review and brain. The "molar tooth sign" was observed on MRI and she was diagnosed with Joubert Syndrome. Exome sequencing revealed she has a homozygous mutation in CEP290(c.5493delA/p.A1832fs*19) that she inherited from both parents. This variant has previously been identified in 2 other families from the Kosovo-Albanian region suggesting it may be a recurrent allele in this population. The clinical description of the patient is thorough and the observation that this may be a founder allele in this population is important.
I appreciate that the authors include a table which highlights the spectrum of clinical phenotypes associated with this specific CEP290 variant. It is intriguing that previous reports of individuals that were homozygous for this allele were diagnosed with perinatal lethal Meckel Syndrome while the homozygous patient in this case study is has Joubert Syndrome. It would be worthwhile to add a sentence in the discussion to speculate on why this might be.
I have some minor recommendations that would help with clarity: "Blood from the patient and his healthy parents" should be her/their. ○ "no clear signs of foramina magnum obstruction and a typical "molar tooth sign" This should be reworded because I interpreted this as no MTS was observed (even though it is clearly shown in Figure 1C) Thank you, this is a helpful suggestion. We have added some more detailed discussion comparing our case with other cases with loss of function variants in CEP290 gene. There is no real genotype-phenotype with variants in this gene, even with LoF alleles. 3.

Competing Interests:
No competing interests were disclosed.
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