Background

Joubert syndrome (JS, MIM 213300) is an autosomal recessive ciliopathy characterized by specific midbrain-hindbrain malformations, recognizable on axial brain magnetic resonance imaging, known as the “Molar Tooth Sign”, hypotonia and developmental delays. Ciliopathies are a group of genetic disorders that are caused by abnormal formation or function of cellular primary cilia in many organs of the human body. To date, over 35 genes are known to cause autosomal recessive JS and one gene causes X-linked JS. Typical clinical features of JS include neonatal hypotonia, abnormal breathing patterns and eye movements, ataxia and developmental delays. Additionally, patients with JS also exhibit variable associated clinical features including retinal dystrophy, ocular colobomas, congenital heart disease, microcystic kidney disease, liver fibrosis, polydactyly, cleft clip and palate [1]. TMEM67 (MIM *609884) encodes the Frizzled-like Wnt receptor, a transmembrane protein (meckelin) that regulates canonical Wnt/β-catenin signaling pathway in the developing cerebellum. Tmem67−/− mutant mice display cerebellar vermis hypoplasia/aplasia, deep interpeduncular fossa and posterior fossa defects compatible with JS phenotype [2]. Biallelic TMEM67 sequence variations cause a wide range of clinical features observed in ciliopathies with multiorgan involvement and different clinical outcomes including JS (MIM 610688), Meckel-Gruber syndrome (MIM 607361), COACH syndrome (Cerebellar vermis hypoplasia, Oligophrenia, Ataxia, Coloboma and Hepatic fibrosis) (MIM 216360), RHYNS syndrome (Retinitis pigmentosa, Hypopituitarism, Nephronophthisis, Skeletal dysplasia), polycystic kidney disease, nephronophthisis-associated ciliopathy (MIM 613550) [3,4,5,6,7] (Table 1). Moreover, TMEM67-mutated patients with JS are also at increased risk for liver disease development complicated by probable portal hypertension in the second or third decades of life. The most relevant genotype-phenotype correlation has been established between TMEM67 sequence variations and the subtypes of JS with liver disease [29]. We here describe the clinical characteristics and mutational analysis of the first Vietnamese family presenting clinical features of JS. Whole exome sequencing identified a novel compound heterozygous TMEM67 variant. This study expands the mutational spectrum of TMEM67 in JS as well as underscores the importance of molecular diagnosis and genetic counseling in patients initially diagnosed with cerebral palsy in Viet Nam.

Table 1 TMEM67 sequence variations associated with a wide phenotype spectrum previously reported in the medical literature
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Case presentation

This 38 year-old female first came to our clinic because of family history of developmental delay. The fetus (II:2) was the second child of a non-consanguineous Vietnamese healthy couple, mid-trimester prenatal ultrasound at 26 + 6 weeks of gestation showed an abnormal enlarged fourth ventricle with abnormalities of the ventricle floor (Fig. 1a). Moreover, renal hypoplasia and polycystic kidney were also noticed. She already had a first child (II:1) with developmental delay. This 10-year-old-boy was born at term after an uneventful pregnancy. His birth weight, height and head circumference were respectively 3400 g (50-90th centile), 53 cm (90th percentile), 34 cm (50th centile). Hypotonia and abnormal breathing pattern were noted at birth. He was diagnosed with cerebral palsy of unknown origin at 2 years old. Neither genetic counseling nor molecular genetic testing was provided. According to the clinical evaluation conducted at the age of 10, his weight, height and head circumference were respectively 23 kg (5th), 129 cm (5-10th) and 52 cm (10th–25th). The proband had severe psychomotor and language delay, began to walk at the age of 72 months and spoke the first word at the age of 60 months. Clinical physical evaluation showed a prominent forehead with bitemporal narrowing, high arched eyebrows, bilateral ptosis, hypertelorism, lower lip eversion, mild clinodactyly of the fifth finger and tapered fingers (Fig. 1b). The patient also displayed ataxic gait and oculomotor apraxia. He had mild intellectual disability. Family history was unremarkable. His MRI showed a pathognomonic finding of molar tooth sign (Fig. 1c). The couple was concerned about their pregnancy, and thus requested genetic counseling regarding her second pregnancy.

Fig. 1
figure 1

a Ultrasound at 26 + 6 weeks of gestation showing an abnormal enlarged fourth ventricle (red asterisk) and the ventricle floor is abnormal. b Photographs of the face (left image) and right profile (middle image) showing typical facial features of Joubert syndrome including prominent forehead, high arched eyebrows, bilateral ptosis, hypertelorism, lower lip eversion (left and middle image), mild clinodactyly of the fifth finger and tapered fingers (right image). c Axial MRI through the cerebellum and brain stem showing cerebellar vermis hypoplasia, thick and elongated superior cerebellar peduncles (red arrows, left image) and absence of the cerebellar vermis, deep interpeduncular fossa and the fourth ventricle has a bat-wing configuration (red arrows, right image)

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Genetic analysis

Conventional cytogenetics and array-CGH

Blood lymphocytes were cultured in RPMI 1640 supplemental with PHA (Gibco, USA) and chromosomes metaphases were harvested according to the laboratory standard protocol. Conventional cytogenetics showed a normal male karyotype 46,XY. 180 K array-CGH (Agilent Technologies, Santa Clara, USA) with a median probe spacing of 13 kb was carried out according to the manufacturer’s instructions and required at least three consecutive probes to make a call. Array-CGH analysis identified no chromosomal imbalances arr(1–22)×2,(X,Y)×1.

Whole exome sequencing

In order to find causative gene variants, we performed whole exome sequencing in the proband (II:1). Genomic DNA was extracted from whole blood from the family using standard methods (QIAGEN, Germany). Five hundred ng DNA was fragmented with a Biorupter (Diagenode, Seraing, Belgium) and the fragmented DNA quality was checked on a bioanalyser MultiNA (Shimadzu Corporation, Kyoto, Japan). Libraries were performed using the Ultra DNA library preparation kit (NewEngland, Biolabs, UK), exome enrichment was performed using TruSeq Exome Library Prep Kit (Illumina, USA) and IDT’s xGen® Exome Research Panel (Integrated DNA Technologies, USA). Finally, sequencing was done on an Illumina HiSeq® 2500. The exome was covered to a mean depth of 100, data with >10X mean coverage accounted for 95% of the whole data. Variants were validated by Sanger DNA sequencing using the ABI BigDye Terminator v3.1 Cycle Sequencing kit and the ABI PRISM 3130xl genetic analyzer (Applied Biosystems, CA, USA). Sequence alterations were reported according to Human Genome Variation Society guidelines (HGVS) and mapped to Human Genome Build GRCh37/UCSC hg19. Whole exome sequencing showed novel compound heterozygous c.725A > G p.Asn242Ser and c.313-3 T > G p.Lys105Valfs*16 TMEM67 variants in the proband (II:1) (Fig. 2a). No other potentially pathogenic variants in other genes associated with developmental delay were identified. These two variants were interpreted as likely pathogenic according to standards and guidelines from the American College of Medical Genetics and Genomics. The variant c.725A > G has a MAF < 0.01 (PM2: Extremely low frequency). It was previously reported in patients with JS (PM3: For recessive disorders, detected in trans with a pathogenic variant and PP1:Co-segregation with disease in multiple affected family members) and predicted to be deleterious by in silico prediction (PP3: Multiple lines of computational evidence support a deleterious effect on the gene or gene product). The novel splice-site variant has never been documented (PM2: Absent from controls) and produces an aberrant splicing transcript (PVS1: Null variant). Sanger sequencing confirmed that the variant c.725A>G p.Asn242Ser was paternally inherited and the splice-site variant c.313-3T>G p.Lys105Valfs*16 was of maternal origin (Fig. 2b, c). Prenatal diagnosis was performed on genomic DNA extracted from amniocytes and Sanger sequencing identified the same compound heterozygous variant c.725A>G p.Asn242Ser and c.313-3T>G p.Lys105Valfs*16 in the fetus (II:2).

Fig. 2
figure 2

a Familial pedigree with Joubert syndrome shows a novel compound heterozygous TMEM67 variant in the case-index and the fetus while the parents were heterozygous. b, c Sang sequencing showing the proband and his father were heterozygous for TMEM67 c.725A > G p.Asn242Ser variant. Moreover, the proband and his mother were heterozygous for a novel TMEM67 splice-site variant c.313-3 T > G p.Leu105Valfs*16. d Reverse transcriptase PCR showing alternative splicing effect with the deletion of 4 bps in exon 03 in the mRNA resulting in an aberrant transcript with premature codon stop. The last nucleotide in exon 2 is yellow highlighted

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Reverse transcriptase PCR for alternative splicing study (RT-PCR)

Total RNA from blood sample was extracted using Qiagen RNeasy blood mini kit according to the manufacturer’s instruction (Qiagen, Germany). One microgram of RNA was used for Reverse-Transcriptase PCR (RT-PCR) (Quantabio, USA). RT-PCR was performed with primers spanning exons 1 and 5. PCR products were separated on agarose gel 2% and sequenced using the ABI BigDye Terminator v.3.1 Cycle Sequencing kit and the ABIPRISM 3500 XL genetic analyzer (Applied Biosystems, CA, USA). Splicing study demonstrated that the novel splice-site variant produced an aberrant transcript with the loss of four nucleotides of exon 03 (Fig. 2d).

Discussion and conclusion

In the era of clinical genomics in Viet Nam, clinical genetics testing is still relatively new and clinicians have very little knowledge about current approaches to genetic disorders. Many developmentally delayed children suffering from genetic disorders were diagnosed with cerebral palsy and they have not benefited from a modern multi-disciplinary care model. We report a Vietnamese family including a 10-year-old child diagnosed with cerebral palsy without etiologic diagnosis and a fetus with central nervous system malformations. Physical examination of the age of the 10-year old showed typical clinical features of JS and an MRI showing the pathognomonic finding of a molar tooth sign, which confirms the JS diagnosis. Whole exome sequencing identified compound heterozygous TMEM67 variants in the proband (II:1). The variant c.725A > G p.Asn242Ser was previously documented in patients with JS and predicted to be pathogenic by SIFT, Polyphen-2 and Mutation Taster [21]. This variant was considered as founder mutation in Eastern Iranian population. The novel splice-site variant c.313-3 T > G p.Lys105Valfs*16 has never been documented in the medical literature and the nucleotide T at this position is highly conserved across multiple species (Additional file 1: Figure S1). Moreover, alternative splicing study demonstrated that the variant c.313-3 T > G p.Lys105Valfs*16 produced an aberrant transcript with the loss of the first four nucleotides of exon 03 leading to a premature stop codon.

TMEM67 sequence variations were associated with a large clinical spectrum and sequence variants were distributed throughout the entire coding region (Table 1). However, several peculiar phenotypes might be predicted, i.e., TMEM67 missense variants falling in exon 8 to 15, especially combined with a truncating variant would predict to give rise to Meckel-Gruber syndrome. In addition, most of TMEM67 sequence variants were predominantly located in 8 of 28 exons (2, 6, 8, 11, 13, 15, 18, 24) [10]. Based on the review of TMEM67 sequence variations previously recorded in the medical literature, our report also showed several mutational hotspots, which were consistent with the result documented by Lannicelli et al., 2010. The most TMEM67 frequently mutated hotspot was exon 8, followed by exons 24, 18, 6, 13, 11, 2, 15 (Table 1).

Prenatal molecular diagnosis was carried out on DNA extracted from amniocytes and the fetus (II:2) harbored the same compound heterozygous TMEM67 variants found in the 10-year-old sibling. The parents were referred for genetic counseling for the current pregnancy and the pregnancy was terminated at 32 weeks of gestation. Furthermore, the proband was recommended to follow annual surveillance of hepatic functions as well as evaluation of kidney function [30]. The couple was offered pre-implantation genetic diagnosis or prenatal diagnosis options for the future pregnancy. In conclusion, our study reports the first Vietnamese family of JS and expands the TMEM67 mutational spectrum in JS. Furthermore, we also stress the important role of molecular approaches in order to identify the causative gene. Accurate diagnosis would further help in genetic counseling, early management of genetic disorders as well as offer prenatal diagnostic options for future pregnancy.