A Twins Case of Lissencephaly With GPR56 Compound Heterozygous Mutations and Literatures Review

Wenxin Lin Nanjing Children's Hospital: Nanjing Medical University Second A liated Hospital https://orcid.org/0000-0003-0591-3880 Yingying Chai Nanjing Medical University A liated Nanjing Children's Hospital: Nanjing Medical University Second A liated Hospital Xia Zhang Nanjing Children's Hospital: Nanjing Medical University Second A liated Hospital Tingting Huang Nanjing Children's Hospital: Nanjing Medical University Second A liated Hospital Guo Zheng Nanjing Children's Hospital: Nanjing Medical University Second A liated Hospital Gang Zhang Nanjing Children's Hospital: Nanjing Medical University Second A liated Hospital Fang Peng Fudan University Huashan Hospital Department of Neurology Yanjun Huang (  njhuang2013@126.com ) Department of Neurology, the A liated Children’s Hospital of Nanjing Medical University https://orcid.org/0000-0001-5270-0867


Introduction
Lissencephaly(LIS) is a group of abnormal cerebral cortical dysplasia caused by the wrong migration of neurons. It can be diagnosed clinically by neuroimaging, presenting with thickening of the cerebral cortex, widening of the gyri, and the disappearance or shallowness of the sulcus. In severe cases, it can be agyria, which is manifested as the complete disappearance of the sulci and gyri, showing smooth surface of the brain [1] . Both agyria and pachygyria belong to LIS. According to neuroimaging, LIS can be divided into 6 grades, ranging from severe agyria (grade 1) to mild subcortical band heterotopias (grade 6), and the severity of nerve damage is widely related to the degree of LIS and cortical thickening, and the mortality rate of severe LIS is high [2] . In the early stage, patients often exhibit developmental delay and hypotonia, followed by seizures, and eventually have a severe intellectual disability. But it can be normal in the neonatal period. The main problems encountered in the neonatal are persistent feeding problems and many different types of epilepsy, which are usually di cult to cure [3] . Rare individuals with mild lissencephaly and normal intelligence have been reported [4] . Up to now, 20 genes have been implicated in LIS, many of which are microtubule genes [5] [6] . GPR56(OMIM#606854, NM_0001145773)encodes an orphan G protein-coupled receptor which is widely expressed in the nervous system and is essential for the normal development of cerebral cortex and cerebellar morphology [7,8,9] . At present, the mutations of GPR56 have been con rmed to be related to bilateral frontoparietal polymicrogyria (BFPP) [10] .
In this study, we reported a case of LIS twins, coming from nonconsanguineous family, both carried a novel compound heterozygous GPR56 mutation (Fig. 1). To our knowledge, they were the rst GRP56related LIS patients. The proband's clinical features were demonstrated retrospectively.

Methods
The probands were collected in the neurology department in Children's Hospital of Nanjing Medical University(Nanjing, PRC). The clinical materials were investigated in proband.
Genomic DNA was extracted from peripheral blood of the twins and their parents using the blood genomic DNA extraction kit (QIAamp DNA Blood Mini Kit, Qiagen). Genetic testing of the proband was performed by high-throughput sequencing of the whole exome. The candidate mutations were further performed by PCR and Sanger sequencing. These mutations were also performed in the parents and her twin sister to further con rm.
Written informed consents were obtained from the patients. This study was approved by the ethics committee of Children's Hospital of Nanjing Medical University.

Results of genetic testing
In the genetic testing, the mean depth of whole exon sequencing was 105 and the coverage was 99.73%. The percentage of the mean depth > 20X was 99.37%. According to the selection criteria for mutation sites: 1) The frequency of the normal population of dominant inheritance is less than 0.0002; the frequency of the normal population of recessive inheritance is less than 0.05; 2) remove synonymous mutations and regulatory region mutation sites. We found suspected pathogenic mutations (compound heterozygous) related to the subject's clinical symptoms ): GPR56 c.228delC (p.F76fs) and c.1820_1821delAT (p.H607fs), two mutations could cause the frame shift mutations, resulting in abnormal protein function.
Both mutation sites have been con rmed in the proband, their parents ,and twin sister by Sanger sequencing. The unaffected father carried a heterozygous c.1820_1821delAT mutation and the unaffected mother carried a heterozygous c.228delC mutation. The twin sister carried both mutations as the proband (Fig. 2).
Both mutation sites have not been reported in the Human Gene Mutation Database(HGMD, Professional edition). According to The American College of Medical Genetics and Genomics(ACMG) guideline rating, both variants were con rmed as PVS1 + PM2, which were suspected pathogenic mutations. And both variants were predicted to be disease causing by mutationtaster (mutationtaster.org) since the frame shift mutation took place in F76 and H607 which might cause nonfunctional protein product or splice site changes.

Clinical characteristics
The proband had a at head deformity and manifested as developmental delay and suddenly generalized tonic-clonic seizure at ve months without any causes. The pregnancy and delivery of proband were uneventful. The neurological examination, electroencephalogram(EEG), and laboratory ndings (full blood count, liver, kidney, and thyroid function tests, creatine kinase, uric acid, metabolic study, and chromosome karyotyping) were normal. Brain Magnetic Resonance Image(MRI)revealed the brain structure was simple with widened and thickened gyrus and shallow sulcus. The white matter of the brain was signi cantly reduced. The patchy long T1 and long T2 signals could be seen around the ventricle which were expanded and the extracerebral space was widened ( Fig. 3 .

Literatures review
All thirty-three families including 58 patients carrying GPR56 mutations reported till recently were reviewed (Table 1). According to the pedigree analysis, all the pedigrees showed a pattern of autosomal recessive inheritance. One case carried a compound heterozygous mutation and the others carried homozygous mutations. In these patients with available clinical data, developmental delay and epilepsy were the predominant symptoms. The brain MRI showed BFPP and white matter abnormalities, which often involving the brainstem and cerebellum, with or without developmental malformations of the corpus callosum.

Discussion
GPR56 spans 45 kb and consists of 14 exons, encoding an orphan G protein-coupled receptor(GPCR) which was constituted by 693 amino acids [7,11] . The G protein-coupled receptor belongs to the adhesion G protein-coupled receptor family, which has an N-and a C-terminal fragmental and a G Protein-Coupled Receptor proteolytic site [12] . In central nervous system, GPR56 plays an important role in the normal development of the cerebral cortex and cerebellar morphogenesis [8] . In the peripheral nervous system, CPR56 can regulate the formation and maintenance of myelin sheath [13] . Therefore, the normal expression of the GPR56 is essential for the function of nervous system. We have already known that the mutations of GPR56 were related to BFPP. The patient's clinical manifestations were overall developmental delay, seizures, and MRI shows symmetrical polygyria (the frontal parietal area is the most serious part), ventricular enlargement, bilateral white matter changes, and 28 pathogenic GPR56 mutations related to the BFPP phenotype have been reported [11,14] . All the individuals inherited in an autosomal recessive mode and almost all missense mutations showed similar clinical symptoms, indicating that the similar phenotype might be caused by the same mechanism, but the mechanism was not clear, which might involve GPR56 tra cking and surface reduction of receptor expression [15,16,17] . Knocking out the GPR56 did not affect the migration of neural progenitor cells, while overexpressing GPR56 could inhibit the migration of neural progenitor cells. This mechanism might be through the reorganization of cerebral cortex actin to change cell morphology to regulate neural progenitor cell behavior [8] . We know that premature stop of neuronal migration could cause LIS, which might explain the mechanism of GPR56 mutations causing the LIS to some extent.
The development of brain is a delicate and complex physiological process, and the proper migration of neurons is one of the most critical steps. LIS is the brain dysplasia caused by the premature stop of neuron migration. The typical lissencephaly (type I LIS) is characterized by the thickened cerebral cortex (10-20 mm, normal 4 mm), and there are no other forms of brain developmental malformations, such as severe congenital microcephaly, corpus callosum hypoplasia, or cerebellar hypoplasia [2] . And it can be observed under the microscope that the cerebral cortex is divided into 4 thick and dysplastic layers: molecular layer, super cial cellular layer, cell spare layer, and deeper cellular layer, while the normal cerebral cortex has 6 layers [1] . Up to now, 20 genes have been considered to be related to LIS, many of which are microtubule genes [5] [6] . In a cohort study of 811 patients with LIS, the overall mutation frequency of the entire cohort was 81%, of which LIS1 accounted for 40%, followed by DCX (23%), TUBA1A (5%), and DYNC1H1 (3%). Other genes account for 1% or less, and 19% of patients still have not found the cause, which indicates that other additional genes need to be discovered [6] . In the past, there have been no reports about the GPR56-related LIS. Therefore, the relationship between LIS and GPR56 still needs further research.
There is no speci c treatment method for LIS, mainly symptomatic treatment such as anti-epileptic treatment and rehabilitation training. Studies in animal models have shown that it might be possible to restart neuronal migration by re-expressing the missing genes after birth. Even if the degree of cortical deformity was partially improved, it could also be signi cantly bene cial by controlling seizures and reducing the clinical severity [2] . Therefore, with all the advances in genetic testing and medical technology, the diagnosis and treatment of LIS will continue to improve and progress.

Conclusion
In this case, the compound mutations of the GPR56 in the LIS twin sisters are both novel mutations, which have not been reported in the HGMD professional database. This enriches the mutation database of GPR56 and also broadened the clinical manifestation, increasing our understanding of GPR56. All these observations indicated that genetic testing is necessary when patients suffering from LIS symptoms. Written informed consents for participation were obtained from their parents.

Consent for publication
Written informed consents for publication were obtained from their parents.

Availability of data and material
Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

Funding
This work was supported by grants from the Six talent peaks project in Jiangsu Province(No.2016-YY-055).

Competing Interests
The authors declare that they have no competing interests in this article.

Authors' contributions
Wenxin Lin performed the data analysis and drafted the manuscript. Yingying Chai conducted the molecular genetic studies and drafted the manuscript. Xia Zhang participated in the design of the study.Tingting Huang participated in the design of the study. Guo Zheng participated in the design of the study.Gang Zhang participated in the design of the study.Fang Peng participated in the design of the study. Yanjun Huang conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the nal manuscript.   Sanger sequencing of proband and her family members.