Two novel TCTN2 mutations cause Meckel–Gruber syndrome

Abstract

Meckel–Gruber syndrome (MKS) is a clinically and genetically heterogeneous ciliopathy characterized by a triad of occipital encephalocele, polycystic kidneys, and postaxial polydactyly. Pathogenesis of MKS is related to dysfunction of primary cilia. However, reports on MKS caused by Tectonic2 (TCTN2) mutations are scanty whilst. There is no direct evidence of ciliogenesis in such MKS patients. Here, we identified two novel nonsense variants of TCTN2 (c.343G > T, p.E115*; c.1540C > T, p.Q514*) in a Chinese MKS fetus. Compared to reported TCTN2-causing MKS patients, our case represented an endocardial pad defect, which was not reported previously. We also found primary cilia protruded normally from the surface of epithelial cells in the affected fetal kidney tubules compared to controls, indicating TCTN2 is not necessary for ciliogenesis in the kidney. To our knowledge, this is the first case of MKS fetus caused by TCTN2 mutations from China.

MKS is a lethal autosomal recessive ciliopathy, showing highest incidence in Gujurat Indians resulted from consanguineous marriages [1]. The mean prevalence of MKS is 2.6 per 100,000 births in Europe [2], while it is still unclear in China. MKS is characterized by a triad of occipital encephalocele, polycystic kidneys, and postaxial polydactyly [3]. At least 21 genes are known to cause MKS [3, 4]. Overlapping of phenotypes and genes can be observed in MKS and other ciliopathies such as Joubert syndrome (JS), implying the genetic heterogeneity and pleiotropy of ciliapathies.

TCTN2 was discovered in 2006 [5]. Protein encoded by TCTN2 was regarded as membrane protein mainly located in ciliary transition zone (TZ) [6, 7]. However, TCTN2-caused MKS patients were rarely reported. Associations between genotypes in TCTN2 and phenotypes in MKS remain unclear. There is also a lack of direct evidence of ciliogenesis in such MKS patients.

We encountered a healthy mother with a 13-week gestation. Early pregnancy examination results were normal until antenatal ultrasound scan at 12th-week-gestation revealed malformations in the male fetus (Fig. 1a). No family history was found. After genetic and psychological counseling, the parents decided to terminate the pregnancy with postmortem autopsy due to the fatal deformity. Chromosomal microarray analysis showed normal in this MKS fetus and its control. The two couples both agreed to voluntary donations of the fetuses. Written informed consents were obtained from the couples. The procedure above was carried out in accordance with Chinese law and regulation.

Fig. 1
figure1

Clinical features of the patient in this study and pathogenic variants reported in TCTN2-induced MKS and JS to date. a Familial pedigree and prenatal ultrasound images. In a1, the healthy father was 30-year-old, and the mother was 28 years old. Green arrow points to occipital encephalocele of our case in a2. Ultrasound image in a3 shows bilateral polycystic kidneys in transverse section of the MKS fetal abdomen and a4 shows only one group of atrioventricular valves existing in apical four chamber view of our case. P represents proband. b Two novel TCTN2 variants found in this case confirmed by Sanger sequencing which came from his parents respectively. c Sixteen pathogenic variants reported in TCTN2-induced MKS and JS to date. Mutations listed above the gene or protein structure graph were from MKS patients, and the below ones were from JS patients. Two splice-site mutations in record were presented on the top of (c). Other mutaitions were presented at the bottom of (c). Red dot represents nonsence mutation. Yellow dot represents frameshift mutation. Green dot represents missence mutation. d Clinical photographs of TCTN2-induced MKS fetus in this case. No chemical or environmental risk factors of malformation were discovered

We designed a targeted capture sequencing assay to test a panel of 113 known ciliopathy genes on the affected fetus (Table S1). Putative causal variants to the reference sequence (hg19, GRCh37) were validated by Sanger sequencing. We failed in getting brain tissue in MKS fetus. Renal tissues were collected, stained with hematoxylin and eosin (HE) and scanned by electron microscopy (SEM) followed the routine method.

Sequencing revealed the MKS fetus carried two compound heterozygous mutations on TCTN2 (NM_024809 c.343G > T, p.E115*; c.1540C > T, p.Q514*) (Fig. 1b). The longest open reading frame of human TCTN2 gene generates a 697-amino-acid(aa) peptide. The first 25 aa is predicted as signal peptide, followed by a 643-aa-peptide extracellular domain, a 21-aa-peptide transmembrane domain, ended with an 8-aa-peptide cytoplasmic domain (https://www.uniprot.org). The two novel TCTN2-nonsense variants both locate in extracellular domain (Fig. 1c). Nonsense variants causing stop codon will generate truncated proteins which are presumed loss of function.

We summarized TCTN2 mutations reported in MKS and JS patients to date (Fig. 1c). All the mutations are located in the extracellular domain. In the 16 pathogenic alleles, 4 carried a missense mutation, 5 carried a small aa deletion or duplication, 2 carried a splice-site mutation, and 5 carried a nonsense mutation. It seems severe forms of mutations (nonsense and frameshift) were more likely detected in MKS patients. However, a genotype–phenotype correlation is not that obvious in TCTN2. Nonsense mutations or frameshift mutation right after signal peptide sequence in homozygous state could be found in JS patients [8, 9]. As a membrane protein, TCTN2 protein gathered in ciliary TZ, complexed with other proteins, and is also required to localize these complex components [6, 8,10,11,12,13]. TCTN2 may function as a gatekeeper for proteins transported into and out of the cilium. The loss of transmembrane domain or change of key sites in TCTN2 probably break down the protein network in various degree, inducing MKS or JS according to response of different downstream elements.

As primary cilia present on almost each mammalian cell, resulting organs affected in MKS includes brain, kidney, liver, heart, eyes, etc. [14]. Clinical phenotypes associated with TCTN2 variants in MKS patients were listed in Table 1. Autopsy showed features in our case including low-set malformed ears, short neck, micrognathia, apart from the “triad” (Fig. 1d). In comparison, endocardial pad defect was never reported in TCTN2-induced MKS or JS. Whereas, Tctn2−/− mice [6, 8] showed ventricular septal defects, supporting the important role of TCTN2 in heart development. Unfortunately, the information about hearts of MKS patients in Table 1 is unavailable. Hence detailed clinical data from patients with TCTN2-related MKS are needed to depict whether heart defect exists. It also remains to be elucidated whether there are certain genotypes of TCTN2 associated with heart defect in MKS.

Table 1 Summary of the reported information on the clinical phenotypes associated with TCTN2 sequence variants in MKS patients

In Tctn2−/− neural tubes from mouse embryos, cilia were scarce, morphologically defective and failed to elongate axonemes, while in the limb bud mesenchyme, cilia numbers were reduced, but the remaining cilia appeared normal suggesting that TCTN2 protein was required for the ciliogenesis in a tissue-dependent manner [6]. Intriguingly, knockdown of Tctn2 in IMCD3 cells only causes modest ciliation and spheroid defects [8]. However, there were no established TCTN2 causative MKS animal model to date. And no study about primary cilia in polycystic kidney induced by TCTN2 mutations were reported. Morphological observation on the kidney by HE staining here was consistent with previous report (Fig. 2a) [15]. Then we are wondering whether it is the same in kidney of TCTN2-induced MKS fetus. SEM data showed that the primary cilia protruded normally from the surface of epithelial cells in the affected fetal kidney tubules as compared to the controls (Fig. 2b). There was no significant difference between our case and controls on average length of cilia (Fig. 2c). Our data indicated that TCTN2 is not necessary for ciliogenesis in the kidney. As Tctn2 is highly expressed in embryonic brain tissues but below the limit of detection in the kidney [8], the role of TCTN2 in the development of different organs needs to be evaluated furtherly. Cilia were found normal in the cystic kidneys of two affected fetuses with TCTN3 mutations and TCTN3 regulates the key sonic hedgehog signaling pathway (SHH) [16]. The loss of TCTN1 does not disrupt C. elegans ciliary structure [11]. TCTN2 was also found crucial to SHH [7]. It seems pathogenesis of cystic kidney induced by tectonic member mutations is distinguished from other organs. Imbalance of signaling pathways [17], disposition of key proteins of TZ [6] may work together to facilitate the formation of cystic kidney.

Fig. 2
figure2

Phenotypic features and cilia in kidneys of fetus with TCTN2 mutations. a Histology pattern (HE staining) of kidney in TCTN2-mutated fetus (a1–a3) and the control (b1–b3). The corresponding control was from a 13-week healthy male fetus which was aborted due to his mother’s severe heart disease. Magnification: ×100 in a1/b1, ×200 in a2/b2, ×400 in a3/b3.Compared with the control, kidney histology in MKS fetus shows conserved corticomedullary organization with sparsely generation of mature glomeruli. Cysts are found in the deep cortex and medulla, and are smaller at the periphery than in the center (a1–a3). b Cilia in kidneys observed by SEM. a1 and a2: MKS fetus. b1 and b2: control. Magnification: ×5000 in a1 and b1, ×30,000 in a2, ×10,000 in b2. c Cilia are measured by Image J in at least 12 cells in MKS fetus or control fetus. The average length of cilia in MKS fetus with TCTN2 mutations is 8.56 ± 1.62 μm while is 7.35 ± 2.26 μm in control group. There is no significant difference between the two groups

Overall, we identified two novel TCTN2 mutations in a Chinese MKS fetus. SEM data confirmed TCTN2 is not necessary for ciliogenesis in the kidney. Downstream effectors regulated by TCTN2 on cilia in kidney development may become the targets of therapy. Further studies between correlation among TCTN2, cilia function and pathogenesis in cystic kidneys of MKS are required.

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Acknowledgements

We thank all patients and family members involving in this study. This work is supported by the Beijing Natural Science Foundation (7184243) and the Chinese PLA General Hospital Translational Medicine Foundation (2018TM-11) in the design, collection, analysis and the writing of the article. We thank Dr. Junnian Zhou (Beijing Institute of Radiation Medicine; South China Research Center for Stem Cell & Regenerative Medicine, SCIB, China) for editing a draft of the paper.

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Correspondence to Longxia Wang or Yanping Lu.

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Zhang, M., Chang, Z., Tian, Y. et al. Two novel TCTN2 mutations cause Meckel–Gruber syndrome. J Hum Genet (2020). https://doi.org/10.1038/s10038-020-0804-0

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