A Novel Homozygous Variant in CPLANE1 Gene in a Patient with Developmental Deficits

Abstract

Background

Oral-facial-digital syndrome (OFDS) type 6 is a rare subtype of Joubert syndrome characterized by orofacial anomalies and polydactyly with neurological features of Joubert syndrome. This rare syndrome is divided into thirteen subtypes, all of which demonstrate autosomal recessive inheritance, except for OFDS type 1 which demonstrates X-linked dominant inheritance.

Case Presentation

A 19-year-old man with mild developmental delay was brought to a rural community clinic, as he had become irritable and angry, in the recent past. There was no history of prior medical conditions. In view of orofacial anomalies, and developmental deficits, a genetic analysis was requested. Karyotype analysis revealed a normal male karyotype (46,XY) in all 30 metaphase spreads analyzed. No numerical or structural chromosomal abnormalities were observed. Clinical exome sequencing and chromosomal microarray detected a variant of uncertain significance in exon 5 of CPLANE1 gene c.365T>G (p.Val122Gly) leading to substitution of Glycine for Valine. This was confirmed by Sanger sequencing. Parents were heterozygous, and the unaffected sibling was homozygous for the wild-type allele. This variant has not been reported earlier in the mutation databases or gnomAD. Runs of homozygosity (ROH) analysis showed a 3.2 Mb ROH around the CPLANE1 gene in the proband, which was absent in both parents and the unaffected sibling.

Conclusion

We find a novel homozygous mutation in the CPLANE1 gene in a patient of non-consanguineous parentage with atypical orofacial features. This suggests that potentially deleterious, rare variants may occur in the heterozygous state in the population. Hence, sequencing of population samples might help understand the genetic epidemiology of rare syndromes.

Established Facts

• Mutations in CPLANE1 (Ciliogenesis and Planar Polarity Effector Complex Subunit 1) gene leads to change in the encoding pattern of ciliary proteins affecting ciliary functions.

• Most subtypes of oro-facio-digital defect rare syndrome (oro-facio-digital defect rare syndrome) are inherited in an autosomal recessive manner.

• Variants in the CPLANE1 gene, including 19 homozygous and 39 compound heterozygous variants, linked to OFD VI.

Novel Insights

• This specific variant chr5:g.37244580A>C, reported here has not been previously reported in the clinical (ClinVar) or population (GnomAD) databases.

• The mutated residue (p.Val122Gly) is not located in a protein binding domain, but is predicted to be deleterious and the patient is homozygous for this variant. This suggests that the function of the protein may be compromised by this substitution; and contribute to the phenotype observed in Mr. A.

Introduction

The CPLANE1 (Ciliogenesis And Planar Polarity Effector Complex Subunit 1) gene on chromosome 5p13.2 has been linked to oro-facio-digital defect rare syndrome (OFDS) VI and Joubert Syndrome 17. It has at least 53 exons, and is widely expressed, especially in the brain and testis. The 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 [1]. Mutations in CPLANE1 gene leads to change in the encoding pattern of ciliary proteins affecting ciliary functions [2]. Joubert syndrome with orofacio-digital defect (OMIM # 277170; OFDS) is a rare syndrome (OFDS), characterized by orofacial anomalies and polydactyly, with neurological deficits. Most subtypes of OFDS are inherited in an autosomal recessive manner [3].

A 19-year-old man with mild developmental delay was brought by his parents to the Chittadhama center (a rural community outreach clinic, Mysore, India) for review. He had become more angry and irritable over a few weeks, over a background of delayed development since birth. We discuss the clinical, genetic, and epidemiological correlates of this syndrome (OFDS VI), with a particular focus on a rare novel variant identified in this patient.

Case Report

Mr A 19-year-old man with mild developmental delay was born to non-consanguineous parents, who are farm laborers. Mr A was conceived when the mother was 29 years, and is the older of two siblings (the other being normal). There was no history of prior medical conditions, hospitalizations, stillbirths, miscarriages, or abortions in the mother. Antenatal evaluation were unremarkable, and folic acid, iron, and calcium supplementation were continued during pregnancy. The mother continued to work as a farm laborer and had numerous instances of exposure to the insecticide Dimethoate 30% E.C. During the 4th month of gestation, on routine ultrasound, a cleft lip was detected in the fetus, and the mother was notified. Fetal movements were first appreciated in the fifth month of pregnancy, and continued well into the third trimester. The antenatal period was otherwise uneventful, and at full term, Mr. A was born by normal delivery, and weighed 3.5 kg.

Due to the presence of both cleft lip and cleft palate there was initial difficulty in latching and suckling, but these defects were corrected. A global developmental delay was observed, as the child grew up. No seizures were reported. No other family members were identified within the three-generation pedigree with clinical findings similar to the ones noted with Mr. A.

General Physical Examination

Mr A is moderately built and nourished, conscious, and cooperative. He communicates by gestures, follows simple commands, and is able to show and respond to emotional cues, but needs assistance for using the toilet, dressing and other daily activities. On the Vineland social maturity scale (VSMS), his social quotient is 29, corresponding to severe intellectual disability (Table 1). However, he is able to attend to personal care, and do simple tasks, under supervision. He weighed 47 kg (BMI 17.5) falling in the underweight range. His anthropometric measurements (Table 2) arm span/height ratio >1.05 and his upper segment/lower segment ratio <0.86 are suggestive of Marfanoid habitus. Lower segment examination shows presence of polydactyly with syndactyly of great toe, sandal gap deformity, and loss of medial longitudinal arch of both feet (Fig. 1b, c). He has frontal bossing, increased intercanthal distance, and bilateral divergent squint (Fig. 1e, f). Ear, nose, and throat examination showed a depressed nasal tip, low set ears, prognathia (Fig. 1g), and dysphasia. He has a broadened chest, mild sternal asymmetry, gynecomastia, and a hypopigmented patch over the abdomen. Steinberg thumb sign and Walker-Murdoch wrist sign are noted on both upper limbs (Fig. 1d).

Table 1.

Vineland social maturity scale (VSMS) was applied to understand his intellectual disability

Social maturity category	Level of social maturity attained (age)
Self-help general	4 years 4 months
Self-help eating	7 years 4 months
Self-help dressing	7 years
Self-direction	5 years 7 months
Occupation	5 years
Communication	3 years
Locomotor	4 years 4 months
Socialization	3 years 10 months

Table 2.

Anthropometric measurements of the patient

Anthropometric Category	Measurements
Height	163 cm
Arm span	171 cm
Arm span/height ratio	1.05
Lower segment (pubic symphysis to floor)	90 cm
Upper segment (pubic symphysis to vertex)	74 cm
Upper segment: lower segment ratio	0.82
Head circumference	56 cm

Fig. 1.

a MRI of the brain showing “molar tooth sign.” b Foot X-ray showing (c) polydactyly with syndactyly of great toe, sandal gap deformity. d Arachnodactyly. e Craniofacial dysmorphism with frontal bossing and increased intercanthal distance. f Bilateral divergent squint. g Depressed nasal tip, low set ears, and prognathia. h Pedigree of the patient family with genotypes at the variant site. i Lollipop plot of CPLANE1 gene showing position of the unique missense variation currently not reported in ClinVar in exon 5 (chr5:g.37244580A>C p.Val122Gly; ENST00000651892.2). j Result of Sanger sequencing. MRI, magnetic resonance imaging.

Investigations

Karyotype analysis revealed a normal male karyotype (46,XY) in all 30 metaphase spreads analyzed. No numerical or structural chromosomal abnormalities were observed. Hematological evaluations demonstrated no clinically pertinent findings, except for a diminished serum testosterone level corresponding to Tanner Stage 4.

Radiological imaging of the thoracic spine revealed thoracic kyphosis and protrusion of mid sternum, concomitant with depression of the Xiphisternum (Pectum Carinatum). The thoracic vertebra, including the lamina, pedicles, transverse, and spinous processes, exhibited normal morphology, with no evidence of vertebral destruction or osteolytic lesions. The thoracolumbar intervertebral discs were normal. Abdominal ultrasonography yielded no discernible anomalies or organomegaly.

Magnetic resonance imaging of the brain revealed hypoplasia of the cerebellar vermis, deep interpeduncular fossa, and elongation of the superior cerebellar peduncles and Molar Tooth Sign (Fig. 1a). Brain stem, basal ganglia, thalami, and corpus did not exhibit any remarkable findings. The vessels of the circle of Willis and cerebral venous sinuses demonstrated normal flow. Visualized portions of the cranial nerves were normal in size and signal intensity.

Genetic Testing and Analysis

Chromosomal microarray analysis revealed no structural variations.

Chromosomal microarray analysis was performed using an Affymetrix CytoScanTM 750K array. This microarray consists of 750K oligonucleotide probes across the genome, including 550K unique non-polymorphic probes, and 200K bi-allelic SNP (single nucleotide polymorphism) probes. Data were analyzed using Chromosome Analysis Suite (ChAS) version 4.2.1. The analysis is based on the Human Reference Genome (GRCh37/hg 19).

The germline variants identified in the sample are deeply annotated using VariMAT pipeline. Gene annotation of the variants are performed using VEP program [4] against the Ensembl release 99 human gene model [5]. In addition to SNVs and small Indels, copy number variants (CNVs) are detected from targeted sequence data using the ExomeDepth method [6]. This algorithm detects CNVs based on comparison of the read-depths in the sample of interest with the matched aggregate reference dataset.

Clinical exome sequencing revealed the presence of a homozygous, novel variant of uncertain significance, in the fifth exon of CPLANE1 gene (chr5:g.37244580A>C; Depth: 32x, c.365T>G; p.Val122Gly; ENST00000651892.2). No other CNVs or single nucleotide variants were found in other genes of interest. The variant has not been reported earlier in GnomAD or 1000 genome databases but has been detected in the local population with Minor Allele Frequency (MAF <0.0061%). The codon is conserved across species, and the variant was predicted to be deleterious by SIFT and PolyPhen-2 (HumDiv) in silico prediction tools.

Genotyping and Analysis of Runs of Homozygosity

We performed genome wide microarray genotyping from DNA obtained from index proband, his unaffected parents and the younger sibling. Runs of homozygosity (ROH) were called with a sliding window approach, and default parameters using the “slidingruns” function in the “Detectruns” package in R 4.2.3. The patient was noted to carry a large 3.2 Mb ROH around the CPLANE1 gene coordinates that was absent in both parents and the unaffected sibling. None of the other genes included within the boundaries of the ROH had been previously implicated in OFDS VI. We have genotype data from the GSAV3 array for the parents. We computed the relationship between the parents using plink – genome function. The PI_HAT values for this pair was <0.01 suggesting a very low kinship coefficient confirming self-reported history of non-consanguinity. It may be an inbred community where this variant is prevalent leading to an incidental homozygous occurrence in the patient.

Validation of Novel CPLANE1 Variant with Sanger Sequencing

We carried out Sanger sequencing of the family to validate the novel variant of uncertain significance identified in this patient and characterize the pattern of inheritance. We also sequenced six unrelated population controls. PCR Primers used were FP – 5′TAT​GAC​CTG​GGA​CCA​CCG​AC3′ and RP – 5′TGG​GGG​GAA​TTC​AGA​CTT​CAG​AGG3′ The forward primer was used as a sequencing primer. The observed genotypes at c.365T>G are represented in the Figure 1h. Sanger sequencing confirmed the presence of homozygous GG genotype in the proband. Both parents were noted to be heterozygous while the unaffected sibling and the six unrelated controls were noted to have TT genotype at the same position.

Discussion

Joubert syndrome is a rare syndrome (<1/1,000,000) linked to mutations in more than 30 genes, and several forms are described. Mutations in the CPLANE1 have been linked to OFDS VI and Joubert Syndrome 17. Several cases (N = 65) of CPLANE1-related OFDS VI have been reported, including nine fetuses [7, 8]. Diagnostic criteria for OFDS VI have been often revised based on clinical presentation of these patients. They have included the most frequent and specific signs of the disease, which include mesoaxial polydactyly of hands, preaxial polydactyly of feet, syndactyly and/or bifid toe, and hypothalamic hamartoma [9]. Mr. A had many clinical features to support a clinical impression of OFDS VI.

Variants in the CPLANE1 gene, including 19 homozygous and 39 compound heterozygous variants, linked to OFD VI have been described [10]. A homozygous nonsense variant c.7817T>A, p.(Leu2606*) in the CPLANE1 (C5ORF42) gene, as well as a heterozygous frameshift variant (c.1819del, p.(Tyr607Thrfs*6) within the same gene was identified in a child of a non-consanguineous marriage [11]. Surisetti et al. [12], reported a heterozygous variant (p.R2467Kfs*7;p.W24R) in the CPLANE1 gene in a patient who had abnormalities with typical Joubert Syndrome and OFDS in a person from southern India.

This specific variant chr5:g.37244580A>C, reported here has not been previously reported in the clinical (ClinVar) or population (GnomAD) databases. The variant may have a low frequency in the local background population, as it was noted at a frequency of MAF – 0.006 in the internal database (Medgenome). While the mutated residue (p.Val122Gly) is not located in a protein binding domain, its predicted deleteriousness in silico, and homozygosity, suggest that the function of the protein may be compromised by this substitution. The impact of this variant on protein structure was estimated using AlphaMissense, and this suggested that the consequences were ambiguous (score 0.53).

Interestingly, unlike most other cases reported in the literature, Mr. A has survived to adulthood, despite the intellectual handicap and dysplasias. Perhaps the mutation was not severe enough to lead to high morbidity, or mortality; and contributed to an attenuation of the syndrome. Alternatively, it is possible that the presence of other cytoprotective variants might have modified the adverse effects of a CPLANE homozygous deleterious variant.

The genotype of CPLANE1 and its correlation with the phenotype of Joubert syndrome remains contentious. While numerous studies have identified variants in children [13], we detected a novel variant of CPLANE1 in Mr. A, who has survived into adulthood. Matoba et al. [14], reported two siblings, both aged 60 years, who exhibited a mild phenotype of Joubert syndrome, with a CPLANE1 novel compound heterozygous missense variant. Considering this, Mr. A may represent yet another case, with a missense homozygous variant who has survived into adulthood with a mild phenotype.

Though the parents were non-consanguineous by self-report, a large run of homozygosity was observed in Mr A. As mentioned above, the variant, though absent in other populations, has been detected, at a low frequency, in southern India. It is likely that in the heterozygous state, this variant may not contribute to disease. Even non-consanguineous marriages could thus have a risk, though infinitesimal, of homozygosity of rare variants, which then lead to disease [15]. Sequencing of population samples, and a better understanding of the mechanisms that contribute to pathogenicity, in both a heterozygous and homozygous state, could help understand the genetic epidemiology and the biology of this rare syndrome better.

Acknowledgments

We acknowledge Clinicians and staff of Chittadhama and the family for considering to be part of study.

Statement of Ethics

This study protocol was reviewed and approved by [M S Ramaiah Medical College Ethics Committee], reference number [DRP ID: DRP/IFP916/2022]. Written informed consent was obtained from patient’s parents to participate in the study. Written informed consent was obtained from patient’s parents to publish the details of their son’s medical case and accompanying images.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This study was partly funded by the Molecular Genetics Lab, National Institute of Mental Health and Neurosciences (NIMHANS) and a grant from the Department of Science and Technology (DST), Department of Biotechnology, Government of India. MQ Mental Health, UK, Rohini Nilekani Philanthropies, SKAN.

Author Contributions

M.T., S.K., R.S.R., S.J., and S.G. conducted clinical assessment and follow up. B.S. conducted the Sanger sequencing experiment. P.P. performed mutation in silico analysis under the guidance of A.S.N.M. S.G. performed ROH analysis. M.P., B.S., S.J., and S.G. drafted manuscript. S.J., M.P., B.V., and S.G. critically reviewed the manuscript.

Funding Statement

This study was partly funded by the Molecular Genetics Lab, National Institute of Mental Health and Neurosciences (NIMHANS) and a grant from the Department of Science and Technology (DST), Department of Biotechnology, Government of India. MQ Mental Health, UK, Rohini Nilekani Philanthropies, SKAN.

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.