Novel CPLANE1 c.8948dupT (p.P2984Tfs*7) variant in a child patient with Joubert syndrome

Abstract Joubert syndrome (JBTS) is a class of heterogeneous ciliopathy genetically associated with CPLANE1 mutations. The characteristics of clinical phenotypes and CPLANE1 variants were analyzed in a 2-month-old patient. A 2-month-old patient with JBTS was diagnosed after clinical evaluation including family history, physical examination, cerebral MRI, ultrasonography imaging, VEGG, ocular fundus examination, and comprehensive blood and urine testing. Whole exome sequencing (WES) was performed to detect CPLANE1 variants, and Sanger sequencing was used to confirm the variants. This JBTS patient presented with oculomotor apraxia, dysregulation of breathing pattern, and ataxia. MRI revealed poor continuity of cerebelli, batwing appearance, and molar tooth sign. This patient was noted with abnormal hematology, dysregulation of hepatic function, thyroid function, immunity, and renal function, and encephalopathy. CPLANE1 (c.8948dupT (p.P2984Tfs*7) and c.247G > T (p.G83X)) variants were noticed in the patient as a pathogenic variant and caused autosomal recessive inheritance. The JBTS patient with mutations in CPLANE1 (c.8948dupT (p.P2984Tfs*7) and c.247G > T (p.G83X)) developed JBTS phenotypes. The novel CPLANE1 c.8948dupT (p.P2984Tfs*7) variant will assist clinicians and geneticists in reaching a precise diagnosis for JBTS.

CPLANE1 gene (synonyms: C5orf42, JBTS17, OFD6, and Hug) mutations change the encoding pattern of ciliary proteins affecting ciliary functions [16]. CPLANE1 protein localizes to the ciliary transition zone, mediating the recruitment of peripheral IFT-A proteins to basal bodies, cell polarity and migration, and ciliogenesis [16]. CPLANE1 protein is necessary for the assembly of the cilia transition zone complex [16]. CPLANE1 silence interferes with the pathfinding of commissural axons, retards neural circuit formation, and causes abnormal facial features in chicken embryos [17]. Cell-cycle-dependent proteolysis regulates localization of CPLANE1 to the mitotic kinetochore [18]. CPLANE1 deficiency delays mitosis and chromosome misalignment, and causes defective radial migration of postmitotic cells [18].

Case presentation
The patient was normally delivered. The birth was full term. One day after birth, the patient developed respiratory distress due to choking on milk. The patient was diagnosed with neonatal respiratory distress syndrome, pneumonia, hyperbilirubinemia, myocardial damage, coagulation abnormalities, and atrial septal defect. The patient was then hospitalized for ventilator-assisted ventilation and discharged 9 days after. At 2 months of age, the patient was presented to our hospital. His parents complained that the patient appeared horizontal head tremor during wakefulness without specific inducements 9 days ago. This symptom was especially manifest when crying, while vanished when sleeping or suckling. Since the onset, the patient showed no signs of mental or motor regression and had no problems with sleeping, urination, and defecation. The patient's parents denied the genetic history of the disease and other disorders.
Body temperature (36.7°C), heart rate (130 beats/min), breathing (32 times/min), and body weight (3.98 kg) were normal. The child patient was inclined to tilt his head to the right and look to the right and appeared horizontal head tremor without specific inducements. Ophthalmological examination revealed pupillary isocoria, equal pupils, and normal pupillary light reflex. Patient's performance during the smooth pursuit was poor. Nystagmus was not observed. The patient was not noted to have enlarged superficial lymph nodes, cyanosis, rash, throat congestion, and inspiratory recession of the suprasternal fossa, supraclavicular fossa, and intercostal spaces. An abnormal laryngeal appearance was not observed. Coarse breath sounds were bilaterally noted by auscultation. There were no obvious pulmonary dry crackles and moist crackles. The heart pulsates powerfully with a regular rhythm and no murmur. Clinical abdominal examination showed abdominal softness, no abdominal distension, and no crying episode. Splenomegaly and hepatomegaly were not detected by abdominal palpation. Neurological examination showed no nuchal rigidity. Cranial nerve injury was not noted. The patient had grasp reflex hyperreflexia, and abdominal and patellar reflex. Babinski signs were not evident. There were no limb convulsions. High extremity muscle tensions were noted when the patient was quiet, especially after stimulation. The upper arm slightly rotated inward, and the right hand rotated clockwise with the thumb. Slightly repeated head nodding was occasionally observed. The patient was easily frightened.
Cerebral magnetic resonance imaging (MRI) examinations showed a molar tooth sign (Figure 1a and b). Figure 1c and d show the widened extracerebral spaces of bilateral temporal lobes and poor continuity of cerebellar hemispheres. The fourth ventricle gave rise to the batwing appearance (Figure 1e and f). Morphological and signaling alterations were not observed in brain parenchyma. Diffusion-weighted imaging showed no diffusion restriction. Brain ventricular system showed normal morphology. The subarachnoid space was enlarged. Cisterns, sulci, and fissures were still within the normal range. Brain midline shift was not observed. No abnormality was noted in cerebellum and brainstem. Cranial ultrasound and computed tomographic scans showed normal results. EGG showed delta activity was increased. No significant discharges were noted when the patient shook his head. The patient's head shook vigorously, and the upper limbs stretched a little, and a small number of fast waves were seen on the synchronized EEG.
The sonograms of the liver, bile, spleen, and both kidneys showed no significant abnormalities. Cardiac ultrasound revealed a patent foramen ovale that has been considered a possible risk factor for ischemic stroke. Electrocardiograph suggested sinus tachycardia. Abnormalities in blood and urine chemistries were measured. Table A1 summarizes the abnormal results. Blood investigation of lymphocyte, neutrophil, hemoglobin, and platelet was abnormal. The patient had hepatic problems with increased levels of ALT, TG, ALP, DBil, total bile acid, and GOT. Routine examination revealed abnormalities in thyroid function, immune function, and renal function. This patient exhibited signs of hyoxemia. Elevated plasma ammonia and cerebrospinal fluid (CSF) chloride showed indications of encephalopathy. Hypersensitivity examination of carbamazepine and oxcarbazepine HLA-B was negative in carbamazepine-or oxcarbazepine-induced hypersensitivity. Hepatotoxicity of sodium valproate by genetic variants of POLG was excluded.
WES and Sanger sequencing detected two compound heterozygous variants of CPLANE1 (NM_023073): c.8948dupT (p.P2984Tfs*7) (Figure 2a) and c.247G > T (p.G83X) (Figure 2b). No other potentially pathogenic variants in other genes associated with JBTS were excavated in this study. c.8948dupT in exon48 caused the frameshift in the 8948th base and a substitution of proline by threonine at the 2984th amino acid of the encoded protein. According to the ACMG, CPLANE1 c.8948dupT variant resulted in the loss of function of CPLANE1 (PVS1); this variant was presented with a low frequency in the general population (PM2); CPLANE1 c.8948dupT variant was defined as a pathogenic variant and caused autosomal recessive inheritance (PM3); the patient's phenotype was highly specific to a basic disease with a single genetic inheritance (PM4). This variant has not been recorded in the ClinVar database. c.247G > T in exon4 resulted in a change of G to T that elicited a substitution of glycine at the 83rd amino acid (p.G83X).
The ClinVar database indicated that this variant is pathogenic and caused inborn genetic diseases (PS1). Based on the ACMG guidelines, CPLANE1 c.247G > T was a pathogenic variant (PVS1 + PS1 + PM2 + PM3 + PP4). Sanger sequencing confirmed that c.8948dupT (p.P2984Tfs*7) was of maternal origin, and c.247G > T (p.G83X) was paternally inherited, which was depicted in the pedigrees (Figure 3). Both CPLANE1 c.8948dupT and c.247G > T variants occurred within the conserved protein sequences (Figure 4). Conserved domain analysis revealed that the function of JBTS-associated domain is still unconfirmed [19]. However, mutations of CPLANE1 lead to JBTS, which is related to ciliogenesis [16]. Figure 4 shows the alignment of multiple CPLANE1 sequences. It was found that the two variants c.8948dupT (p.P2984Tfs*7) and c.247G > T (p.G83X) are highly

Discussion
JBTS is typically characterized by cerebellar vermis hypoplasia, developmental delays, and irregular breathing pattern [1]. Retinal degeneration and renal anomalies have also been included in JBTS17 patients [20,21]. CPLANE1 has been predicted to encode a protein with transmembrane domains and coiled-coil domains, which may be involved in ciliogenesis and mitotic process [22,23]. There has been accumulated evidence that numerous CPLANE1 mutations are pathogenic variants in patients with JBTS [24][25][26][27]. In this report, we describe a 2-monthold patient diagnosed with JBTS. Physical examination, laboratory data, and MRI findings revealed the diagnosis of JBTS. The diagnosis was confirmed after the detection of CPLANE1 c.8948dupT (p.P2984Tfs*7) and c.247G > T (p.G83X) variants by WES and Sanger sequencing.
The main oculomotor abnormalities in patients with JBTS include ocular motor apraxia, nystagmus, and strabismus [28,29]. However, our patient presented with strabismus but no nystagmus. Short alternate episodes of apnea and tachypnea are commonly observed [30], which are also noticed in our case. A spectrum of organ defects is demonstrated in JBTS patients commonly retinal defects, renal defects, and congenital liver fibrosis [3,5,20], which is consistent with our case. Other rarely observed features such as midline oral and facial defects, Hirschsprung disease, skeletal dysplasia, severe scoliosis, situs inversus, congenital heart malformations, and optic nerve colobomas were not observed in our patient. Based on the findings of physical examination, laboratory data, and MRI, this patient was presumptively diagnosed with JBTS.
As a genetically heterogeneous primary ciliopathy, JBTS has been detected with pathogenic variants in numerous genes including CPLANE1 [29,32,33]. CPLANE1encoded protein is involved in ciliogenesis, which affects cell polarity and directional cell migration [16]. Cilium assembly or disassembly markedly affects extracellular signals mediating growth and developmental syndromes related to multiple organs and systems such as brain, retina, skeletal system, kidney, liver, skeletal system, and pancreas [34]. It has been confirmed that the knockdown of CPLANE1 caused ciliopathy-related development defects in neural tube closure, Hedgehog signaling, and left-right patterning in Xenopus embryos [35]. The CPLANE1 variants recorded in the ClinVar are classified into five types: 37 frameshift, 153 missense, 28 nonsense, 13 splice site, and 25 untranslated region types. We found a novel CPLANE1 variant c.8948dupT, which has not been documented in the ClinVar and literatures. Protein structure analysis revealed that c.8948dupT and c.247G > T variants change the highly conserved regions, suggesting that the clinical phenotypes may be caused by the two variants. Additionally, couples with a JBTS child have a 25% risk of recurrence. Hence, prenatal diagnosis through chorionic villus sampling at approximately 11 weeks of gestation is necessary and feasible only for proband family with CPLANE1 variants.
Respiratory and feeding problems should be particularly focused to avoid hypotonia and respiratory abnormalities. Intensive monitoring is required to manage renal failure and delay complications. Specific follow-up must be scheduled to treat congestive heart failure. Breathing dysregulation is especially associated with prognosis. Assisted ventilation may reduce the recurrent probabilities of life-threatening prolonged apneas. Dystonia was treated by oral trihexyphenidyl (0.25 mg/time, twice a day during the first to third day; 0.5 mg/time, twice a day from the 4th day). After one week of medication, the patient's tremor gradually subsided, and the head tremor disappeared 1 month after discharge. Compound glycyrrhizin tablets were administered twice a day, and the patient's liver function improved 1 month after discharge. However, the patient's thumbs often retracted inward, and his movements were usually awkward and inflexible, especially in the hands. The patient still has difficulty placing two square toys together.

Conclusions
In this study, we diagnosed an infant patient with JBTS typical of hypoplasia of the cerebellar vermis, irregular breathing pattern, retinal degeneration, and renal anomalies. WES and Sanger sequencing identified 2 mutations in CPLANE1 (c.8948dupT (p.P2984Tfs*7) and c.247G > T (p.G83X)) in an infant patient with JBTS. CPLANE1 c.8948dupT (p.P2984Tfs*7) is a newly reported variant that is not recorded in the ClinVar database or documented in the literature. Further, oral trihexyphenidyl alleviated dystonia and the compound glycyrrhizin tablets improved liver function. Regular follow-up is necessary. This novel finding in CPLANE1 variants will assist clinicians and geneticists in reaching a precise diagnosis for JBTS. Author contributions: Z. Y. X. contributed to the study conception and design. Material preparation, data collection, and analysis were performed by W. H. P., N. W. S., W. C. X., and W. Z. H. The first draft of the manuscript was written by W. H. P. and N. W. S. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.