| Background:Neu-Laxova syndrome is an autosomal recessive genetic disease,which is characterized by a variety of congenital malformations,involving microcephaly,brain malformation,limb defects,facial and skin characteristics,and other congenital abnormalities.Neu-Laxova syndrome is caused by homozygous or compound heterozygous mutations of PHGDH,PSAT1 and PSPH genes encoding key enzymes in serine biosynthesis.These genes are involved in serine biosynthesis and play an important role in cell proliferation and participate in serine biosynthesis pathway.PHGDH is the key enzyme of L-serine de novo synthesis.Its coding gene PHGDH is located in human chromosome 1p12,which plays an important role in cell and material metabolism and body growth and development.PHGDH is involved in the first step of serine biosynthesis,oxidation of glycolysis intermediate3-phosphoglycerate to phosphohydroxypyruvate,and finally synthesis of serine by amino group and dephosphorylation under the catalysis of a series of enzymes(PSAT1 and PSPH).Serine plays an important role in the synthesis of biological factors such as nucleotide,phospholipid serine and sphingosine,which are necessary for protein and cell proliferation.L-serine is a neurotrophic factor,which deficiency may affect the survival,growth and differentiation of nerve cells,and affect the dendritic elongation and synaptogenesis of neurons,therefore,PHGDH gene mutation may lead to defects in the process of serine biosynthesis and transport,resulting in the lack of L-serine,which may have a harmful impact on brain development.In addition,serine deficiency leads to insufficient synthesis of active tetrahydrofolate,which may affect the division and transformation of epidermal cells,and then lead to the occurrence of Neu-Laxova syndrome skin phenotype.Up to now,15 PHGDH gene mutations related to the disease have been reported in the literature:c.418G> A(p.Gly140Arg),c.488G> A(p.Arg163Gln),c.160C> T(p.Arg54Cys),c.793G> A(p.Glu265Lys),c.856G> C(p.Ala286Pro),c.1297C>T(p.Gln433*),c.1518G>A(p.Trp506*),c.1286G>T(p.Gly429Val),c.399 G>A(p.Trp133*),c.1A>C(p.Met1?),c.1468G>A(p.Val490Met),c.1263C>G(p.Cys421Trp),c.746 T >C(p.Leu249Pro),c.638C>T(p.Thr213Met),c.704C>T(p.Ala235Val)。Objective: In this study,we found a case of induced labor due to multiple malformations.The fetus showed severe intrauterine growth restriction and multiple malformations(micrognathia,abnormal face,small heart,etc),through phenotypic analysis and consulting references,it is considered that the fetus may be Neu-Laxova syndrome.After fully informing the family members,obtaining consent and signing the informed consent form,we performed trios whole exons sequencing(trios WES)for the induced labor fetus and parents in order to clarify the genetic cause of disease.Trios WES results showed that the induced labor fetus carried three variation sites of PHGDH gene: c.488G>A,c.607A>T and c.743C>T.The father carries the c.488 G > A mutation site,which is recorded as a pathogenic mutation in the clinvar database,and the mother carries the c.607A>T and c.743C>T mutation sites,theoretically,the two sites exist on the same chromosome,and there are no database records and literature reports.According to the ACMG rating,the pathogenicity of the two sites is variants of uncertain significance(VUS).In order to confirm the pathogenicity of heterotopic sites of c.607A>T and c.743C>T,and there are few studies on the mechanism of Neu-Laxova syndrome caused by PHGDH gene mutation,this subject plans to prepare PHGDH wild-type and mutant(c.488A>A,c.607A>T and c.743C>T)overexpression lentivirus,construct HEK293 T stable transformation cell line,and confirm the pathogenicity of three heterotopic sites through cell experiment and enzyme activity experiment.To clarify the effect of PHGDH gene compound heterozygous mutation on the function of PHGDH protein and preliminarily explain the molecular mechanism of fetal abnormal phenotype,so that we can better understand the occurrence of Neu-Laxova syndrome,and provide a theoretical basis for genetic counseling,prenatal diagnosis and preimplantation diagnosis of this family.Methods:1.Specimens and cell lines:The muscular tissue of the fetus with suspected Neu-Laxova syndrome and the peripheral blood of its parents were taken for subsequent trios whole exome sequencing(trios WES)analysis.Both parents signed informed consent.Human embryonic kidney cells(HEK293T)were purchased from Institute of Biochemistry and Cell Biology,Chinese Academy of Sciences.2.Experimental methods:(1)Whole exome sequencing was used to screen the gene mutations of the fetus with suspected Neu-Laxova syndrome and its parents,and Sanger sequencing was used to verify the screened gene mutation sites again;(2)The structural differences between wild-type and mutant PHGDH proteins were predicted by SWISS-MODEL workspace;(3)The m RNA and protein expression of PHGDH gene in induced fetus was detected by real time PCR,western blot and immunohistochemistry;(4)The mutant and wild-type overexpression lentivirus of PHGDH gene were prepared,and the stable transformation cell line HEK293 T was constructed.Real-time quantitative PCR was used to detect the m RNA transcription level of PHGDH gene,and western blot was used to detect the protein expression level of PHGDH;(5)Prepare PHGDH gene mutant and wild-type overexpression lentivirus,construct HEK293 T stable transformation cell line,and detect the changes of PHGDH enzyme activity;(6)The biological characteristics of proliferation,apoptosis,cell cycle and migration of HEK293 T cells were observed.Results:1.Three missense mutations of PHGDH gene were found in Neu-Laxova syndrome pedigree.The mutation c.488 G >A carried by father was reported as pathogenic mutation,while the mutation c.607 A > T and c.743 C >T carried by mother have not been reported as pathogenic mutations in previous studies.2.Bioinformatics analysis of the PHGDH mutations c.488 G > A,c.607 A > T and c.743 C > T indicated that the wild type and mutant PHGDH protein structure had significant differences.3.The expression of PHGDH protein in induced labor fetus decreased.4.After the mutant and wild-type expression lentivirus of PHGDH gene were infected into HEK293 T cells,it was observed that the transcription level of PHGDH gene was up-regulated,and there was no significant difference in protein expression.5.The enzyme function test showed that compound mutation of PHGDH gene c.488G>A and c.607A>T decreased the enzyme activity.6.The compound mutation of PHGDH gene c.488G>A and c.607A>T reduced the proliferation,arrested S-phase of cell cycle,increased necrosis and decreased migration ability of HEK293 T cells.Conclusions:1.PHGDH gene c.488 G > A and c.607 A >T compound heterozygous mutations were found to be the cause of Neu-Laxova syndrome;2.PHGDH gene c.488 G > A and c.607 A >T compound heterozygous mutations decreased PHGDH enzyme activity,inhibited the proliferation,arrested S-phase of cell cycle,promoted necrosis and reduced migration of cells. |
