A genetic variant in the MAST1 gene is associated with mega-corpus-callosum syndrome with hypoplastic cerebellar vermis, in a fetus

Abstract

Background

Mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations is a rare neurological disorder that is associated with typical clinical and imaging features. The syndrome is caused by pathogenic variants in the MAST1 gene, which encodes a microtubule-associated protein that is predominantly expressed in postmitotic neurons in the developing nervous system.

Methods

Fetal DNA from umbilical cord blood samples and genomic DNA from peripheral blood lymphocytes were subjected to whole-exome sequencing. The potential causative variants were verified by Sanger sequencing.

Results

A 26-year-old primigravid woman was referred to our prenatal center at 25 weeks of gestation due to abnormal ultrasound findings in the brain of the fetus. The brain abnormalities included wide cavum septum pellucidum, shallow and incomplete bilateral lateral fissure cistern, bilateral dilated lateral ventricles, hyperplastic corpus callosum, lissencephaly, and cortical dysplasia. No obvious abnormalities were observed in the brainstem or cerebellum hemispheres, but the cerebellum vermis was small. Whole-exome sequencing identified a de novo, heterozygous missense variant, c.695T>C(p.Leu232Pro), in the MAST1 gene and a genetic diagnosis of mega-corpus-callosum syndrome was considered.

Conclusion

This study is the first prenatal case of MAST1-related disorder reported in the Chinese population and has expanded the mutation spectrum of the MAST1 gene.

INTRODUCTION

The corpus callosum is the largest white matter tract of the human brain and mediates bilateral integration of sensory, motor, and cognitive inputs. Malformations of this brain structure are known to be associated with a wide range of neurodevelopmental disorders (Parrini et al., 2016; Paul et al., 2007). Mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations (MCCCHCM, MIM#618273) is a rare neurological disorder that is associated with typical clinical and imaging features. It is characterized by moderate-to-severe global developmental delay, muscular hypotonia, and poor to no verbal abilities, related to oromotor dysfunction. The characteristic brain abnormalities include an enlarged corpus callosum in the absence of megalencephaly, cerebellar hypoplasia, ventricular dilation, gyral abnormalities, and cortical malformations (Tripathy et al., 2018). The syndrome is an autosomal-dominant disorder that is caused by pathogenic variants in the MAST1 gene (MIM#612256). This gene belongs to the microtubule-associated serine/threonine kinase (MAST) family, which is characterized by a central microtubule-associated protein serine/threonine kinase domain (STK) (Garland et al., 2008). To date, only 12 different variants have been characterized in 14 variably affected individuals (Ben-Mahmoud et al., 2020; Bowling et al., 2017; Gilissen et al., 2014; Hecher et al., 2020; McMichael et al., 2015; Rodríguez-García et al., 2020; Tripathy et al., 2018). Three of these individuals harbored a recurrent mutation and eight had a thick corpus callosum and cortical dysgenesis (Hecher et al., 2020; Rodríguez-García et al., 2020; Tripathy et al., 2018). Seven additional MAST1 variants have been listed in the ClinVar database or Leiden Open Variation Database as pathogenic or likely pathogenic.

Here, we described a fetus, from a Chinese family who presented with neuron migration anomalies and hyperplastic corpus callosum. Whole-exome sequencing (WES) identified a de novo missense variant, c.695T>C(p.Leu232Pro), in the MAST1 gene. This prenatal case highlights the importance of prenatal ultrasound and genetic testing for the diagnosis and management of the MAST1-related disorder.

MATERIALS AND METHODS Prenatal ultrasonography

The patient underwent ultrasound screening performed with Toshiba Aplio500 Color Doppler ultrasound diagnostic instruments (Toshiba, Tokyo, Japan) with a 3–6 MHz transabdominal probe. Ultrasound settings for pregnancy examination were set according to the parameters recommended by the manufacturer. The ultrasound examinations were performed by radiologists with more than 5 years of experience in fetal ultrasound evaluation. The measurements were taken by two consultant radiologists, each measurement was checked three times by an examiner, and the average value was recorded as final data.

Fetal magnetic resonance imaging (MRI)

Fetal MRI was performed on a 1.5-T MRI System (Philips Medical Systems, Best, The Netherlands) using a 16-channel coil placed around the maternal abdomen. The pregnant women received an MRI scan in the supine position. Sedation was not used for either the fetus or mother and the total scan time was restricted to 30 min. Fetal T2-weighted images were acquired in the transverse, sagittal, and coronal plane using single-shot turbo spin echo (ssTSE) sequences with the following scanning parameters: repetition time (TR) = 15 s, echo time (TE) = 175 ms, slice thickness = 2.5 mm with a slice overlap = 1.5 mm, acquisition matrix = 188 × 147, and flip angle = 10°. The brain was oversampled by acquiring multiple overlapping single-shot T2-weighted images to ensure complete coverage of all regions of interest within the brain.

Genetic analysis

Fetal DNA from umbilical cord blood samples was extracted with QIAamp DNA Mini Kit Tissue kit (Qiagen, Germany). Genomic DNA was extracted from peripheral blood lymphocytes of all subjects using a Lab-Aid DNA kit (Zeesan Biotech Co., Ltd, Xiamen, China). For WES, genomic DNA samples were captured to build a sequencing library by Agilent SureSelect Human All Exon V6 Kit (Agilent Technologies, Santa Clara, CA). The library preparations were sequenced on an Illumina HiSeq2500 platform (Illumina, San Diego, CA). Sequence alignment and variant calling against the human GRCh37 reference genome were performed with BWA and the Genome Analysis Toolkit (GATK HaplotypeCaller; McKenna et al., 2010). Copy number variants (CNVs) analysis was performed using an in-house pipeline, and CNVs of significant interest were further visually inspected using the Integrative Genomics Viewer. Single nucleotide variants and indels were annotated and prioritized by the TGex software (LifeMap Sciences, Alameda, CA). All the potential causative variants were verified by Sanger sequencing in patients and their family members. Identified variants were assessed for pathogenicity according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines (Richards et al., 2015).

RESULTS Case presentation

A 26-year-old primigravid woman was referred to our prenatal center at 25 weeks of gestation (by LMP) for abnormal ultrasound findings in the brain of the fetus. The pregnant woman and her husband were not consanguineous and there was no family history of neurological disease. The pregnancy was proceeding with medical monitoring and she was not taking any medication.

Ultrasonography showed that the biparietal diameter was 6.3 cm (+0.2 SD), the head circumference was 22.5 cm (−0.3 SD), the fetal abdominal circumference was 18.6 cm (−1.6 SD), the femur length was 4.4 cm (+0.3 SD), and the transverse diameter of cerebellum was 2.6 cm (reference range, 2.4–3.1 cm). Brain abnormalities included wide cavum septum pellucidum (length of 24.7 mm and width of 6.2 mm, reference range at 25 weeks: 3.96 ~ 7.16 mm), bilateral cerebral ventriculomegaly (10.5 mm, normal range < 10.0 mm), a thick corpus callosum (body height 6.0 mm, +12.6 SD, reference range: 1.3–0.6 mm), shallow lateral fissures and absence of parieto-occipital sulcus and fissura calcarina (Figure 1). The fetal brain MRI showed a shallow and incomplete bilateral lateral fissure cistern, absence of central sulcus, lissencephaly, cortical dysplasia, bilateral dilated lateral ventricles (10.0 mm and 10.8 mm, normal range < 10.0 mm), and a thick corpus callosum. No obvious abnormalities were observed in the brainstem or cerebellum hemispheres, but the cerebellum vermis (craniocaudal diameter 9.98 mm, −2.5 SD; anteroposterior diameter 9.30 mm, normal) was small (Figure 2).

FIGURE 1. Ultrasound images of the fetus at 25 gestation weeks. (a) Wide cavum septum pellucidum, (b) mild cerebral ventriculomegaly and shallow bilateral lateral fissures (white arrow), (c) absence of parieto-occipital sulcus and fissura calcarina, and (d) thick corpus callosum.

FIGURE 2. MRI from the fetus at 25 weeks of gestation. (a) Sagittal T2-weighted MRI showing the hyperplastic corpus callosum (white star). The cerebellum hemispheres and brainstem appear normal. The cerebellum vermis was small (white arrow). (b, c) MRI showing shallow and incomplete bilateral lateral fissure cistern, lissencephaly and cortical dysplasia, and dilated lateral ventricles. (d) Transversal T2-weighted MRI showing enlarged ventricles. (e, f) There were no obvious abnormalities in the cerebellar hemisphere.

Genetic analysis

Through WES analysis, a total of 870 variants were selected in protein-coding regions and splice sites, after filtering out synonymous variants and variants with minor allele frequencies (MAF) > 3% in local and commercial databases. After clinical phenotype analysis, we did not identify any homozygous or compound heterozygous variants in genes that are known to be linked to neurological abnormalities. However, two heterozygous candidate variants were identified in the CHD7 and MAST1 genes, with subsequent validation by Sanger sequencing. The CHD7 variant (c.6109C>T) was inherited from the unaffected mother, while the c.695T>C(p.Leu232Pro) variant in MAST1 (NM_014975.3) was absent from both parents (Figure 3a). Paternity was confirmed by short tandem repeat genotyping. The MAST1 missense variant was not listed in any of the general population databases, such as dbSNP, gnomAD, and the 1000 Genomes Project. Multiple sequence alignments using Clustal X revealed that the Leu232 residue is highly conserved among mammalian species and some nonmammalian species (Figure 3b). In the EBI database, The Leu232 residue also appeared to be conserved (Figure 3c). Additionally, bioinformatics analysis predicted that c.695T>C(p.Leu232Pro) to be disease-causing [SIFT: damaging (0.00); PROVEAN: deleterious (−6.39); Polyphen2: probably damaging (0.998/0.990); Mutation Taster: disease causing (98)]. As the MAST1 gene is intolerant to missense variants (overall missense variants z-score in gnomAD: 5.77), the substitution was classified as likely pathogenic (PS2 + PM2_supporting + PP2), in accordance with the ACMG/AMP guidelines (Richards et al., 2015).

FIGURE 3. Genetic analysis of the fetus and the spectrum of MAST1 mutations. (a) Sanger sequencing analyses of MAST1 gene. The red frame represents variant site (c.695T). The fetus was heterozygous for a missense variation, c.695T>C (p.Leu232Pro) (top), and the transition could not be detected in the couple. (b) Multispecies alignment of MAST1 protein. The red box represents variant site (p.L232P). Multiple sequence alignments revealed that the Leu232 residue is highly conserved. (c) Residue conservation of MAST1 protein from the EBI database. (d) The spectrum of MAST1 mutations. Mutations reported in the literature are shown above the schematic diagram of protein domain structures. Mutations listed in the ClinVar database or LOVD are shown in gray under the schematic diagram. Missense mutations are indicated by dots. The novel variant (c.695T>C) in the fetus is highlighted in red. In-frame deletions are depicted by triangles. Nonsense mutations are represented by squares.

DISCUSSION

In this report, we identified a fetus with a heterozygous missense variation in the MAST1 gene. The fetus presented with wide cavum septum pellucidum, shallow and incomplete bilateral lateral fissure cistern, bilateral dilated lateral ventricles, hyperplastic corpus callosum, lissencephaly, cortical dysplasia, and hypoplastic cerebellum vermis. To the best of our knowledge, this is the first work to describe an MAST1 variant in a fetus with characteristic brain imaging.

The human brain undergoes critical biological processes of development during the embryonic period (Silbereis et al., 2016), and the overall architecture of the human brain is formed in the first 6 months of fetal life (Bakken et al., 2016). The proliferation of neurons occurs predominantly in the germinal matrix, and neuronal precursors migrate radially or tangentially from these zones toward their final destination in the cortical plate (Vasung et al., 2019). When the surface area of the cortical plate grows rapidly, the appearance of cortical convolution follows (Vasung et al., 2016). Numerous genes are involved in the complex processes related to fetal brain development (Kang et al., 2011).

Members of the MAST kinase family (MAST1-4) are comprised of several conserved protein domains, which include a domain of unknown function (DUF), an STK domain, and a postsynaptic density protein-95/discs large/zona occludens-1 domain (PDZ). The MASTL protein contains two STK domains (Garland et al., 2008; Hain et al., 2014). This family is widely expressed in several organs and tissues, and expression is particularly enhanced in the brain (Spinelli et al., 2021). It appears to have an essential role in critical neuronal function and normal cell division (Jing et al., 2020). Genetic defects in the MAST family have been reported to cause several different mitotic abnormalities (Oishi et al., 2016). The STK domain is the characteristic feature of this protein family, and pathogenic MAST3 variants in this domain are associated with epilepsy (Spinelli et al., 2021).

The MAST1 gene (NM_014975.3) contains 26 exons and encodes a 1570 amino acid protein, which consists of an N-terminal domain of DUF1908, followed by an STK domain and a PDZ domain. The MAST1 protein is a microtubule-associated protein predominantly found in post-mitotic neurons in the developing nervous system and is present in both dendritic and axonal compartments (Tripathy et al., 2018). It interacts with the tumor suppressor protein PTEN (phosphatase and tensin homolog), through the PDZ domain, to facilitate the phosphorylation of interacting proteins (Valiente et al., 2005). Epigenetic changes in the malignant transformation of Pheochromocytoma (PCC)/paraganglioma (PGL) are associated with the hypomethylated overexpression of the MAST1 gene (Oishi et al., 2016). Tripathy et al. showed that Mast1 binds to microtubules in a MAP-dependent manner and found that mutations were able to perturb this interaction (Tripathy et al., 2018).

Germline mutations in the MAST1 gene can give rise to a variety of neurological abnormalities, which include MCCCHCM, microcephaly and cerebellar hypoplasia, autism spectrum disorder (ASD), microcephaly and dysmorphia (M-D) and diplegia (Ben-Mahmoud et al., 2020; Hecher et al., 2020; Rodríguez-García et al., 2020; Tripathy et al., 2018). Affected individuals presented with moderate to severe intellectual disability, developmental delay, and characteristic brain abnormalities (Ben-Mahmoud et al., 2020; Hecher et al., 2020; Rodríguez-García et al., 2020; Tripathy et al., 2018). Other symptoms may include severe speech and language impairment, motor dysfunctions, gait instability, short stature, hypotonia, seizures, facial dysmorphism, and visual and hearing impairment. Recombinant human growth hormone (rhGH) therapy can be effective in improving short stature in patients with MAST1-related disorders (Rodríguez-García et al., 2020). Characteristic brain abnormalities were observed in patients with MAST1-related disorders, which included enlargement of the corpus callosum (8 patients, 57%), enlarged ventricles (8 patients, 57%), gyral abnormalities (7 patients, 50%), cerebellar hypoplasia (9 patients, 64%), and brainstem hypoplasia (5 patients, 36%). In addition, five patients presented with pontine hypoplasia (36%).

Despite the typical core symptoms, MAST1-related disorders can be clinically heterogeneous. Structural brain abnormalities are seen in most patients (12 in 14), but normal brain images have been reported in two patients (Table S1). More than half of patients (8 in 14) have thickening of the corpus callosum. However, two patients have shown hypoplasia of the corpus callosum. Muscular hypotonia and hypertonia have been described, and patients can present with either microcephaly or macrocephaly (Table S1). Differences in phenotype have been noted in patients with the same MAST1 mutation (c.1549G>A). Hence, understanding the exact correlation between defects in this gene and the clinical manifestations requires further work.

According to the Swiss-Prot database and Missense3D web server, substitution of Leucine with Proline, at amino acid residue 232, does not lead to a substantial change in the MAST1 protein structure. Ben-Mahmoud et al. reported that MAST1 missense mutant proteins (four missense variants) exhibit similar subcellular localization to the wild-type protein, in HeLa/HEK-293 cells (Ben-Mahmoud et al., 2020). These data suggest that missense mutations do not significantly alter the protein structure but some caution is required in interpreting the in vitro results in cultured cells. The Leu232 residue is located in the domain of unknown function (DUF1908), which is found at the N-terminal of MAST proteins. In accordance with the interPro database, molecular functions and biological processes of this domain are mainly involved in ATP binding, protein serine/threonine kinase activity, magnesium ion binding, and protein phosphorylation. Almost half of the detected mutations (9 in 19) occur in this domain, while 37% (7 in 19) of such mutations are found in the STK domain (Figure 3d).

Mutations in MAST1 usually occur de novo and most occur sporadically. Tripathy et al. showed that Mast1 null mice are phenotypically normal but the deletion of a single amino acid recapitulates the characteristic neurological phenotype observed in patients (Tripathy et al., 2018). In mice with Mast1 microdeletions, the PI3K/AKT3/mTOR pathway is not perturbed, but Mast2 and Mast3 levels are diminished. These findings suggest that the dominant negative effect is the main pathogenic mechanism of MAST1 variants (Tripathy et al., 2018). To date, all patients with MAST1-related disorders had a single amino acid substitution or an in-frame deletion. This gene is indicated to be intolerant of loss-of-function variants, in the gnomAD database (pLI = 1), but only one nonsense variant has been classified as pathogenic (VCV001319774.3) in the ClinVar database. The biological functions and potential pathogenesis of the MAST1 gene need to be further explored.

In the developing human brain, the major neuronal migration occurs between the 12th and the 24th week of gestation, and high levels of MAST1 protein are found at 13th and 22nd weeks of gestation. Cortical development and late migration continue until 5 months after birth (Toi et al., 2004; Tripathy et al., 2018). In 2020, Rodríguez-García et al. reported a patient with global developmental delay, whose brain MRI showed a thickened corpus callosum, cortical malformations, and dilated and abnormal configuration of the lateral ventricles, without cerebellar hypoplasia (Rodríguez-García et al., 2020). The MRI findings of this fetus described here were similar to those reported by Rodríguez-García et al. but in addition, our patient shows a hypoplastic vermis. After genetic counseling, the pregnant woman chose to terminate her pregnancy. We cannot know the exact postnatal abnormalities caused by the mutation in this case. Due to the technical limitations of prenatal imaging and the ongoing process of neuronal migration after 25 weeks gestational age, the structure of the mature brain cannot be predicted more accurately. Given the severity and representative brain imaging features of MAST1-related disorders, clinicians should be aware of this condition.

AUTHOR CONTRIBUTIONS

All authors have contributed sufficiently to the project to be included as authors. Sheng Yi, Xianglian Tang, Junjie Chen, Linlin Wang, Zuojian Yang, Shuihua Yang, Pingshan Pan, and Jingsi Luo acquired clinical data. Sheng Yi, Xianglian Tang, Fei Chen, Minpan Huang, Qi Yang, Limei Huang, Shang Yi, and Zailong Qin performed the experiments and molecular analyses. Jingsi Luo, Zailong Qin, Sheng Yi, and Xianglian Tang designed the study and raised funds. Sheng Yi and Xianglian Tang drafted the manuscript and compiled the revisions. All authors read and approved the final manuscript.

ACKNOWLEDGMENTS

The authors appreciate the participating patient and their families.

FUNDING INFORMATION

This work is funded by the Health Department of Guangxi Zhuang Autonomous Region (Z20200678), the Open Project Funding of Guangxi Key Laboratory of Birth Defects and Stem Cell Biobank (GXWCH-ZDKF-2022-13), Guangxi Medical and Health Appropriate Technology Development and Application Project (S2020060), and Guangxi Clinical Research Center for Pediatric Diseases (AD22035121).

CONFLICT OF INTEREST STATEMENT

The authors report there are no competing interests to declare.

DATA AVAILABILITY STATEMENT

All data generated or analyzed during this study are included in this published article and its supporting information files.

ETHICS APPROVAL

The study was approved by the Ethics committee of the Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region. Written informed consent was obtained from the family members.