Meckel Syndrome (MKS, OMIM #249000) is a rare and fatal autosomal recessive ciliopathy with an incidence ranging from 1:13,250 to 1:140,000 (Young et al., 1985; Yaqoubi and Fatema, 2018; Lin et al., 2022). MKS is a highly clinically heterogeneous and multisystem disorder characterised by classic features—occipital encephalocele, polycystic kidneys, postaxial polydactyly and other anomalies (including liver fibrosis, central nervous system malformations and genital anomalies) (Logan et al., 2011; Hartill et al., 2017; Liu et al., 2021). The aetiology of MKS is the dysfunction of the primary cilium during early embryogenesis, and mutations in ciliary-associated proteins result in a group of disorders including Meckel Syndrome (MKS, OMIM #249000), Polycystic kidney disease (PKD, OMIM #173900), nephronophthisis (NPHP, OMIM #256100), Bardet-Biedl Syndrome (BBS, OMIM #209900), Joubert syndrome (JBTS, OMIM #213300) and Alstrom syndrome (ALMS, OMIM #203800) (Badano et al., 2006; Sharma et al., 2008; Yuan and Sun, 2013; McConnachie et al., 2021; Moreno-Leon et al., 2022).

Seventeen genes are found to be linked with MKS, and the MKS1 gene mutations cause 16% of MKS cases (Iannicelli et al., 2010; Shaheen et al., 2013; Wang et al., 2021). The TMEM231 gene, identified in 2013 as a causative gene for MKS, also contributes to JBTS which is another ciliopathy characterised by oculomotor apraxia, abnormal breathing, ataxia, and variable psychomotor developmental delay (Srour et al., 2012; Shaheen et al., 2013; Wang et al., 2021). MKS and JBTS exhibit common clinical features such as polydactyly and renal involvement. A clear clinical diagnosis for MKS is often delayed due to its low frequency and complex phenotype.

With the development of sequencing technologies and the reduction of technology costs, whole-exome sequencing (WES) has been widely applied to reveal the genetic aetiology of congenital diseases, especially in rare diseases with extreme genotypic and phenotypic heterogeneity (Chen et al., 2021; Pan et al., 2022; Wang et al., 2022; Zhang et al., 2022). Here, we collected a pedigree of four siblings affected by MKS and identified two novel splice site variations of the TMEM231 gene (NM_001077418.2; c.583-1G>C and c.583-2_588delinsTCCTCCC) by WES. We used alternative splicing experiments, mRNA expression detection and immunofluorescence techniques to investigate the correlation between genotypes and phenotypes.

TMEM231 is a causative gene for MKS. It encodes a 36 KD two-pass transmembrane protein that is part of the B9 complex and localises to the base of the ciliary axoneme at the TZ (Chih et al., 2011). Mutations in mouse Tmem231 disrupted the ciliary localisation of proteins such as Arl13b and lnpp5e, resulting in the pathogenic features of MKS, and mutations in the TMEM231 gene were first reported in MKS patients in 2013 (Shaheen et al., 2013; Roberson et al., 2015). In this study, we described the clinical features of a Chinese pedigree with four fetuses affected by MKS, and two novel splice site mutations of TMEM231 (c.583-1G>C and c.583-2_588delinsTCCTCCC) were identified in this pedigree by WES.

Only 15 TMEM231-associated MKS cases were reported, and 16 variants (8 splice sites, 7 missense and 1 small deletion) were considered as causative mutations (Supplementary Table S1) (Roberson et al., 2015; Maglic et al., 2016; Li et al., 2020; Watson et al., 2020). The premature stop-gain mutations caused 80% of the MKS cases (12 out of 15). The c.373C>G (p.Pro125Ala) mutation was detected in four cases of northern Europeans, suggesting that this site may be a mutation hotspot in the northern European population (Roberson et al., 2015). The splice site variant of c.583-1G>A (NM_001077418.2) was found to cause loss of exon 5, generating a premature stop codon (Maglic et al., 2016). Our findings on the c.583-1G>C and c.583-2_588delinsTCCTCCC variants confirmed that mutations within this splice site region lead to the deletion of exon 5 during post-transcriptional cleavage of TMEM231. It is speculated that this site may be another mutation hotspot. Actually, the abnormal transcripts were ambiguously observed in the parents (Supplementary Figure S3) and were not detected in the fetus by agarose gel electrophoresis (data not shown). We propose the following hypotheses: (1) a considerable period of time elapsed between the drug-induced abortion and the collection of the fetal sample, during which RNA degradation occurred; (2) the frameshift effect caused by two splicing mutations led to nonsense-mediated mRNA decay (NMD), resulting in a low abundance of RNA that could not be observed in the fetal sample (Amrani et al., 2004; Ivanov et al., 2016).

With the development of next-generation sequencing (NGS), WES has improved the diagnostic rate and increased the knowledge of most disorders (Guo et al., 2022). In particular, WES is the most common approach to identify the candidate gene and disease-causing variants in patients with extreme genotypic and phenotypic heterogeneity disorders. In this study, WES successfully identified disease-causing mutations in the TMEM231 gene. Due to the poor ability of NGS based on short-read sequencing to accurately detect gene conversion events and distinguish pseudogenes, Watson et al. stated that new third-generation long-read sequencing technologies should be considered as a routine clinical approach for genetic diagnosis (Watson et al., 2020). In their study, a splice site mutation and an exon deletion of the TMEM231 gene were detected in MKS patients by long-read nanopore sequencing. Therefore, next-generation sequencing and third-generation sequencing could complement each other in clinical diagnosis to improve diagnostic yield.

MKS is a rare, highly clinically and genetically heterogeneous disease. To date, the diagnostic criteria remain controversial. Salonen et al. showed that the minimum diagnostic criteria for MKS include ① polycystic kidneys, ② fibrotic changes of the liver, ③ occipital encephalocele and ④ malformation of the central nervous system (Salonen, 1984). All 15 reported MKS patients had abnormal fetal features and had a poor prognosis. Cystic dysplasia was a common manifestation in all patients. Hepatic developmental defects were also a common phenotype, but were not manifested in the fetuses in this study. (Supplementary Table S1). However, liver fibrosis and bile duct proliferation have been reported to occur in only 65.5% of MKS cases (Barisic et al., 2015). In this study, we described a pedigree with four fetuses affected by MKS. The proband (Ⅱ-4) not only presented the typical features of MKS, but also showed bilateral talipes equinovarus. Ⅱ-3 presented compatible with typical features of MKS, whereas Ⅱ-1 and Ⅱ-2 did not show polydactyly. In addition, the proband (Ⅱ-4) did not have any liver developmental defects at autopsy. MKS also shares clinical features with several cilia-based disorders such as JTBS and BBS. Due to the complexity of MKS phenotypes, accurate diagnosis of MKS is usually difficult and delayed. Therefore, appropriate ancillary examinations and genetic testing are required.

Genotype-phenotype correlations are essential for clinical research. In this study, these two splice site mutations caused different phenotypes in four affected fetuses, and we hypothesised that these mutations would lead to nonsense-mediated mRNA decay (NMD). Although the pathogenesis of MKS caused by mutations in TMEM231 remains unclear, we have shown that the defective TMEM231 protein results in a developmental defect in primary cilia (Figure 4, Supplementary Figure S4). Accurate splice site recognition is critical for pre-mRNA splicing. RT-PCR followed by Sanger sequencing showed that these two splice site mutations altered the post-transcriptional cleavage of TMEM231. TA cloning sequencing revealed the same alternative transcription in this nonconsanguineous couple. Some researchers have used RNA-seq to explore novel splice sites. On the other hand, RNA-seq can also be used to determine the impact of splicing variants on the transcriptome (Parada et al., 2014). For example, Zhi et al. reported two aberrant transcripts caused by a c.3353 + 5C>A variant of MED12 by RNA-seq (Zhi et al., 2022). Multiple aberrant transcripts caused by transcript splicing may be the reason for the negative genotype-phenotype association in genetic counselling. Although RNA-seq is powerful for detecting different transcripts, the specificity of gene expression in different tissues should be considered before performing RNA-seq.

In conclusion, we reported two novel splice site mutations (c.583-1G>C and c.583-2_588delinsTCCTCCC) of TMEM231 in a Chinese pedigree with four affected fetuses. Considering the clinical phenotype of the pedigree and the functional analysis of two mutations, we believed that all fetuses were affected with MKS and that the mutations were pathogenic mutations causing MKS. The results of RT-PCR and TA clone sequencing showed alternative post-transcriptional cleavage of TMEM231, resulting in a deletion of exon 5 and a premature stop codon in TMEM231 mRNA. The absence of primary cilia in renal tubular epithelial cells partially explained the mechanism by which TMEM231 gene mutations affect primary cilia development and formation. This study also provided a basis for genetic counselling of this pedigree and extended the mutation spectrum of the TMEM231 gene for MKS.