| Background:Schwachman-Diamond syndrome(SDS)is a rare autosomal recessive disorder that belongs to the group of congenital bone marrow failure disorders commonly seen in children and is characterized clinically by pancreatic exocrine insufficiency,congenital somatoform anomalies(mainly abnormal development of the long bone epiphysis)and bone marrow hematopoietic failure,with the development of myelodysplastic syndrome/acute Myeloid leukemia(MDS/AML)is at a very high risk and is the leading cause of death in adulthood.The most significantly associated mutated gene in SDS is the Schwachman-Bodian-Diamond gene(SBDS),located on chromosome 7q11,and mutations in this gene are present in more than 90%of clinical patients.SBDS proteins play an important role in ribosome synthesis and spindle stabilization,in addition to being involved in cellular stress response,cell chemotaxis and apoptosis Therefore,SDS is considered to be primarily a ribosomal synthesis deficiency disease.In addition to SBDS mutations that can lead to the development of the disease,there are still about 10%of patients who do not have SBDS mutations in clinical practice.The three genes that have been well studied are EFL1,DNAJC21(DnaJ heat shock protein family(Hsp40)member C21)and SRP54(Signal recognition particle 54).EFL1 is involved in the biogenesis of the 60S ribosomal subunit and translational activation of ribosomes.Together with SBDS,it triggers the GTP-dependent release of EIF6 from 60S pre-ribosomes in the cytoplasm,thereby activating ribosomes for translation competence by allowing 80S ribosome assembly and facilitating EIF6 recycling to the nucleus,where it is required for 60S rRNA processing and nuclear export.EFL1 has low intrinsic GTPase activity,but its GTPase activity can be increased by contact with 60S ribosome subunits.DNAJC21 is a member of the heat shock protein family,and is similar to SBDS,DNAJC21 is involved in the transfer and maturation of pre-60S from the nucleus to the cytoplasm,leaving the pre-60S upon completion.If a mutation in the gene results in the protein not being able to dissociate from the pre-60S,it will result in the inability of the pre-60S to mature and thus to assemble into the 80S with protein synthesis SRPs are ribosomal protein complexes that mediate the targeting of peptides carried by nascent signal sequences to translocons on the surface of the endoplasmic reticulum,and a mutation in this gene would prevent the protein from reaching the endoplasmic reticulum and affect protein synthesis.Previous studies have suggested a role for the myocyte Enhancer Factor 2(MEF2)protein family in heart and muscle development.Recent reports suggest that they are also closely associated with the onset and development of many human diseases.MEF2 transcription factors play critical roles in physiological and pathological processes.Members of the MEF2 family are primarily involved in neurodevelopment,muscle formation,cardiac development,and tumor carcinogenesis.Although their role in cancer is well established,the molecular mechanisms of their action are not clear.There are growing evidences that the MEF2 family contributes to the development of various human diseases.For example,relevant to this study,in the area of hematological diseases,MEF2B,MEF2C and MEF2D mutations causing malignant hematological diseases have all been reported previously,with the most elaborated functional studies of MEF2C among MEF2 family in hematopoietic system.MEF2C is also found being associated with the Notch signaling pathway,which is an important signaling pathway for hematopoietic cell differentiation and development.Therefore,MEF2C has an important relevance to hematopoietic cell development.In addition,there are studies showing that abnormal expression of MEF2C is associated with the prognosis of AML.MEF2A is the only MEF2 family gene that has not been reported being associated with hematologic disorders.More previous studies on MEF2A have been focused on cardiovascular and muscular diseases.However,in an evolutionary point of view,MEF2A and MEF2C are the most similar and MEF2B is the most distant of the whole family.The two are also the most similar in terms of genetic structure.It is reasonable to speculate that MEF2A should have a similar role to MEF2C in the blood system.Whole-exome sequencing(WES)is the most frequently used genome sequencing method.Exons are protein-coding regions of the human genome,and their DNA can be captured and enriched using sequencing capture techniques.Although exonic regions possess only about 1%of the whole genome,they contain 85%of disease-causing mutations.Compared to whole genome sequencing,whole exome sequencing is more economical and efficient.Whole-exome sequencing is mainly used to identify and study variants within coding regions and UTR regions associated with diseases and population evolution.Combined with the exome data provided by public databases,it is beneficial to better interpret the relationship between the mutations and diseases.The colony-forming unit(CFU)assay,also known as the colony forming cell(CFC)assay,is the most commonly-used way in vitro differentiation assay for hematopoietic progenitor cells.density deposition of single cell suspensions.These conditions support the proliferation and differentiation of individual progenitor cells,resulting in the formation of discrete colonies.Colonies from different types of progenitor cells can be sorted and counted using morphological and phenotypic criteria based on the number and type of mature cells they contain.CFU assays are the most commonly-used ways to detect multipotent progenitor cells from erythroid,granulocyte and macrophage lines,while megakaryocytes and B-lymphocyte progenitors can also be detected.Although purified hematopoietic stem cells can form colonies under appropriate culture conditions,the majority of CFUs detected in bone marrow,blood and other tissues are progenitor cells with limited self-renewal and limited in vivo hematopoietic repopulation potential.However,the CFU test can be a useful hematopoietic stem cell replacement test in cases where long-term transplantation trials are too expensive or impractical.Conditional knock-out mice(also called Flox mice)are the mice that contain paired loxp loci in the target gene and can be mated with Cre mice to knock out the target gene in specific tissues or cells.Site-specific recombination techniques are mediated by recombinase systems such as Cre-loxP,a recombinase(38kDa)that recognizes the 34bp long DNA sequence loxP.LoxP constitutes an palindrome structure at 13bp on each side and a non-palindrome structure at 8bp in the middle,thus making loxP directional.(when two isotropic loxP sequences are presented on a DNA molecule,Cre cuts it out,circularizes the DNA fragment between the two loxP sequences and joins the sequences on both sides of the loxP;when two loxP sequences of opposite orientation are presented on a DNA molecule,Cre causes the sequence between the loxPs to reverse.)The target gene for a conditional knockout must have a loxP sequence that can be recognized by Cre recombinase and this gene is called a floxed gene.Mice with a floxed target gene are called flox mice.In such mice,a homologous DNA recombination method is usually used to place a homologous loxP site on each side of the proposed knockout gene fragment.LoxP sites should be presented without affecting the function of the gene,so flox/flox mice are chosen as controls.In addition to flox mice,recombinase system-mediated conditional knockout requires the involvement of another important type of genetically engineered mice,which is called the Cre mice.In Cre mice,the sequence coding for Cre recombinase is placed under a specific gene promoter and the expression characteristics of Cre determine the occurrence of spatial-temporal knockout of the target gene.The level of Cre expression will affect the efficiency of target gene modification in certain tissue or cells.Study objectives:1.To explore the causative genes in the patient’s family line using whole exon sequencing technology and reverse validation by Sanger sequencing.2.To do functional experimental validation analysis of the screened genes,explaining the pathogenesis of diseases suffered by clinical patients.Study methods;1.Collecting bone marrow samples from the family line,approximately 3 mL per case.2.Isolation of bone marrow single nucleated cells.3.Extraction of single nucleus cell DNA.4.Whole exon sequencing.5.Cleaning,filtering and screening of candidate genes from sequencing data.6.Adding DNA from another person in the family and Sanger sequencing to verify candidate genes and further narrow down the scope.7.Identification of study genes.8.Searching for information about the gene to be studied.9.Isolation hematopoietic stem progenitor cells from umbilical cord blood.10.Encapsulating the lentivirus and infect the hematopoietic stem progenitor cells with the lentivirus.11.Culturing and sorting GFP-positive cells for in vitro CFU assay.12.Counting colony size and number after 2 weeks of culture and performing statistical analysis.13.Construction conditional knockout MEF2A mice using C57BL/6J and Vav-Cre mice.14.Verifying the success of the mouse constructs.15.Detecting the hematological changes in mice.Study results:1.The patient’s clinical manifestations and family histories are consistent with Schwachman-Diamond syndrome,which can be clinically diagnosed as Schwachman-Diamond syndrome according to the existing diagnostic consensus.Next,we need to make molecular biological diagnosis of the patient which using Whole-exome sequencing to sequence the DNA of bone marrow mononuclear cells in the family.2.After data filtering and cleaning,there were 159 SNPs and 85 INDELs candidate mutation loci,respectively,118 SNPs followed a compound heterozygous inheritance pattern,38 INDELs mutations followed an autosomal dominant pattern,22 SNP mutations followed an autosomal dominant pattern,47 INDELs mutations followed an autosomal recessive pattern and 19 SNP mutations followed an autosomal recessive pattern.Finally,a phenotype database was introduced to assist in further narrowing the scope.Phenotype databases included OMIM and ClinVar,and others included DisGeNet,HPO,and PheGenI.Through this filtering steps,four candidate mutant genes were finally selected,namely MEF2A,which conforms to an autosomal recessive inheritance pattern,KRAS,which conforrms to an autosomal dominant inheritance pattern,and rs147640812,rs117497991,which conform to a compound heterozygous inheritance pattern.3.By targeting design of the mutant genes,we added the DNA of the patient’s brother for auxiliary validation.MEF2A was eventually selected as the candidate mutant gene for study under two reasons:(i)the mutant locus was only present in the DNA of the proband and his brother,not in their healthy parents;(ii)In the genotype statistics,rs147640812 and rs117497991 were excluded because the mismatch genotypes of both the proband and his brother;KRAS was excluded because it should conform to autosomal dominant mode of inheritance,so theoretically both homozygous and heterozygous children should develop the disease,which is not in line with the family status;MEF2A was identified as the candidate gene because it conforms to the autosomal recessive mode of inheritance,and both proband and his brother are heterozygous for the mutation,while the parents do not contain the mutation,which is in accord with the inheritance pattern.4.The results of colony-forming unit assay showed that MEF2A mutant overexpression,MEF2A wild-type overexpression,and MEF2A knocking-down expression all had inhibitory effects on normal hematopoiesis,which mainly inhibited colony production in the erythroid and granular cells.5.The MEF2Afl/fl;Vav-Cre+/-and MEF2Afl/fl;Vav-Cre-/-mice were successful constructed and the MEF2A mRNA expression level in hematopoietic system were much lower in MEF2Afl/fl;Vav-Cre+/-mice compared with MEF2Afl/fl;Vav-Cre-/-mice,with statistically significant differences.6.The peripheral blood cell count and ratio were not statistically different between experimental and control group mice,indicating that knock-down expression of MEF2A in bone marrow does not affect normal hematopoietic function. |
