Whole exome sequencing reveals novel CEP104 mutations in a Chinese patient with Joubert syndrome

Abstract Background Joubert syndrome (JS, OMIM: 213300) is a recessive developmental disorder characterized by cerebellar vermis hypoplasia and a distinctive mid‐hindbrain malformation called the “molar tooth sign” on axial magnetic resonance imaging. To date, more than 35 ciliary genes have been identified as the causative genes of JS. Methods Whole exome sequencing was performed to detect the causative gene mutations in a Chinese patient with JS followed by Sanger sequencing. RT‐PCR and Sanger sequencing were used to confirm the abnormal transcript of centrosomal protein 104 (CEP104, OMIM: 616690). Results We identified two novel heterozygous mutations of CEP104 in the proband, which were c.2364+1G>A and c.414delC (p.Asn138Lysfs*11) (GenBank: NM_014704.3) and consistent with the autosomal recessive inheritance mode. Conclusion Our study reported the fourth case of JS patients with CEP104 mutations, which expands the mutation spectrum of CEP104 and elucidates the clinical heterogeneity of JS.

In 2015, four mutations of CEP104 (OMIM: 616690) were found in three JS patients, which elucidated that CEP104 is one of the causative genes of JS (Srour et al., 2015). Here, we report that a Chinese boy was diagnosed with JS features and carried novel compound heterozygous mutations in the CEP104.

| Ethical compliance
This project was approved by Ethics Committee of the National Research Institute for Family Planning. The written informed consent was obtained from the proband's parents. Blood samples were collected from the proband and his parents and sibling after receiving written consent.

| Whole exome sequencing and variants analysis
The blood samples were collected using EDTA anticoagulant tube and processed for genomic DNA isolation by QIAamp DNA Blood MiNi Kit (Qiagen) following the standard procedures. We performed the whole exome sequencing using the proband's DNA. In brief, the whole exome library was prepared using Agilent SureSelect Human All Exon V6 kit (Agilent Technologies Inc.) according to manufacturer's standard protocol. Sequencing was performed on Illumina Novaseq 6000 platform (Illumina Inc.) with 150 bp paired-end reads. Reads were aligned to reference genome hg19 (GRCh37) by   PCR was performed using specific primer pairs followed by Sanger sequencing on ABI3730xl Genetic analyzer (Life Technologies) following the manufacturer's protocol for variants validation in the proband and his parents. Primer 4F (5′-CTGTTGATCCTGCATAGGGG-3′) and primer 4R (5′-TCAGCAGTCTCCCAGAAGAGAT-3′) were used for exon 4 amplification, while primer 18F (5′-TTTCAGGCACCTCCTTGGTG-3′) and 18R (5′-TATGGAATGACTCGCACGCA-3′) were used for exon 18 amplification.

| RNA extraction and reverse transcription
The blood samples of proband and his parents were collected using Tempus ™ Blood RNA Tube (SKU #4342792, Invitrogen). Tempus ™ Spin RNA Isolation Kit (4380204, Invitrogen) was used for RNA extraction from whole blood cells of the proband and his parents. One microgram of RNA was reverse transcribed into cDNA using SuperScript ™ IV First-Strand Synthesis System Kit (Thermo Fisher Scientific, Invitrogen). The primer sequences used for cDNA amplification were 5′-GAATCAGGACATTCAAGGAGGGA-3′ (forward, across the junction of exon 16 and exon 17) and 5′-TTTCCATGCCTCTTCTCCAGG-3′ (reverse, across the junction of exon 20 and exon 21). The PCR amplification products were analyzed by agarose gel electrophoresis. The purified DNA bands were cloned into pMD19-T Vector (TaKaRa) and validated by Sanger sequencing.

| Clinical report
The patient is a 3-year-old boy, who is presented with hypotonia and psychomotor developmental delay. He is the second child of an unrelated couple without personal or familial medical history. Born by caesarean section at 39 week gestation, the patient showed normal birth measurements: weight of 3.05 kg and height of 48 cm. On examination, he has lowset ears, epicanthus, and strabismus. Hypotonia and psychomotor developmental delay was obvious in the patient: he was unable to hold his neck until 10 months and sit unaided at 12 months, and he cannot stand or walk independently by 3 years. He presented with speech delay and can only speak monosyllabic words. The developmental quotients of motor, object, adaptability, language, and social abilities were evaluated by Gesell Developmental Schedules (GDS). Mild-tosevere retardation of the GDS, especially language and motor abilities, was observed. Brain magnetic resonance imaging | 3 of 6 LUO et aL.

| Genetic analysis
Compound heterozygous variants in CEP104 (GenBank: NM_014704.3) (c.414delC [p.Asn138Lysfs*11] and c.2364+1G>A) were found in the proband (Figure 1c). The frameshift variant, p.Asn138Lysfs*11, in the exon 4 was predicted to the generation of a truncated protein. This variant is a novel variant, which is not found in dbSNP, ExAC, or gnomAD. This variant was inherited from his mother and also presented in his brother. The c.2364+1G>A variant led to a nucleotide exchange at an obligatory splice site (NM_014704.3: c.2364+1G>A). This position is 100% conserved in the canonical sequence of mammalian splice sites, mutations of which affect the donor splice site of intron 18. This variant is extremely rare in the gnomAD with 2 of 246,124 alleles bearing this mutation (allele frequency 0.000008126) (Lek et al., 2016). This variant was inherited from his father and absent in his sibling. According to the ACMG guidelines, both of the frameshift and splicing site variants were classified as pathogenic (Richards et al., 2015). Of note, another heterozygous rare damaging variant was detected in centrosomal protein 290 (CEP290, OMIM: 610142), which was c.6012-2A>G, but no other susceptive pathogenic variant in CEP290 was found.

| Confirmation for an abnormal transcript of CEP104
To evaluate the mutational effect of c.2364+1G>A in CEP104, spanning exon amplification product was analyzed by agarose gel electrophoresis. Four bands were observed in the PCR products of the samples from the proband and his father, while only one band (400 bp) was detected in the products from his mother, brother and the healthy control ( Figure  1d). Direct sequencing showed that the 906 bp PCR product was caused by the failure of correct splicing, which inserted 506 bp nucleotides from the adjacent intron into the mRNA (Figure 1e,f). This insertion led to incorrect translation and early translational termination of CEP104.

| DISCUSSION
In this study, we report a Chinese boy with CEP104 mutations presenting with symptoms consistent with JS, displaying global developmental delay, facial dysmorphism, oculomotor apraxia, and hypotonia. Brain MRI showed MTS, which is typical in JS patients. Consistent with the previous reported CEP104-mutated patients, the proband reported here also presented with MTS, developmental delay, and oculomotor apraxia (Srour et al., 2015) (Table 1). Our patient was noticed with hypotonia, which is reported in two of the three other patients (Srour et al., 2015). Respiratory abnormality was not a common symptom of CEP104-mutated patients and only one patient was observed with this defect (Srour et al., 2015). In this case, respiratory abnormality was not noticed as well.
Different from JS patients caused by CEP290 or TMEM67 (OMIM: 609884) mutations (Brooks et al., 2018;Fleming et al., 2017;Strongin et al., 2018), none of CEP104-mutated patients displayed renal or liver involvement (Srour et al., 2015). However, the risk of renal or liver failure of CEP104mutated patients cannot be excluded, since all the patients are younger than 4 years old. Limb anomalies such as polydactyly were not observed in all of the patients (Srour et al., 2015). We failed to evaluate the retinal phenotype of the proband because of the difficulty of cooperation (Table 1).
Exome sequencing revealed two novel compound heterozygous variants of CEP104 (c.2364+1G>A and c.414delC) in the proband which are the cause of the disease. Four pathogenic variants were previously reported in three patients from different families, including two splicing site mutations, one nonsense mutation and one frameshift mutation (Srour et al., 2015). Together with our finding, there are six pathogenic variants were reported, and all of them result in a truncated protein of CEP104 (Figure 2b). It reminds us that CEP104 might be like two other known JS genes, CEP290 and CSPP1 (OMIM: 611645) (Bachmann-Gagescu et al., 2015), in which the pairing of truncating variants seem to be the most frequent mutation type causing JS.
CEP104 was identified as a ciliary tip protein by a comparative, quantitative proteomic study in Chlamydomonas T A B L E 1 Clinical features and genotype of our patient and those described in Srour et al. (2015) (Figure 2a,b). The mutation of c.2364+1G>A is at the second ZNP repeats, which are showed as the interacting domain of CEP104 with CP110 (Al-Jassar et al., 2017; Rezabkova et al., 2016) (Figure 2a,b). Of note, it cannot be excluded that the mutations not only affect the functions of the proteins, but also reduce the stability of the protein, which have been reported in other proteins. In summary, this JS patient has two novel mutations in CEP104, which expands the mutation spectrum of CEP104 and elucidates the clinical heterogeneity of JS. Future description of other patients with mutations in CEP104 and the following studies of their underlying cell biology and physiology will define new mechanisms on the role of CEP104 and cilia in brain development, especially JS.