| Background and objective:Limb malformations is one of the most common birth defects in our country.The clinical manifestations of acromicric malformations are complex and diverse,including polydactyly,brachydactyly,syndactyly,and other types,and the physical appearance and functional movements of the affected limbs are severely affected.Severe acrodysmorphism can not only lead to the loss of self-care ability and labor ability in patients’ lives,but also seriously affect the mental health of patients.Limb malformations can appear as a kind of disease or a clinical symptom of certain syndromes.Besides,multiple types of limb defects can coexist,such as synpolydactyly.While one gene mutation can result in different types of limb malformations,various mutations can also lead to similar symptoms.Therefore,relying on patients’ clinical manifestations and imaging analysis only is hard to get exact diagnosis and classification.With the advancement of technology and continuous development of molecular biology technolo gy,sequencing technology is also being updated iteratively,next-generation sequencing has a long detection read length,high detection accuracy,and is the gold standard for DNA sequence analysis,but the detection cost is high and throughput is low and can only be tested for known disease causing genes.Generated for high-throughput,rapid detection,and low cost applications,next-generation sequencing(NGS)is an efficient method to detect not only the pathogenic mutations of known genes but also novel pathogenic genes through the analysis of the coding sequences of genomes,which is now widely used in clinical molecular diagnosis of genetic diseases because of its remarkable efficiency in molecular diagnosis.In this study,7 families with different types of limb malformations were collected,and the proband of the families were subjected to whole exon sequencing.Data were filtered and analyzed to screen out relevant pathogenic genes of partial families,then clinical and genotypic diagnosis were further clarified,providing a scientific theoretical basis for family genetic counseling and guidance for next generation fertility.Subject and Methods:subject The subjects of this study were 7 families with limb malformations who were admitted to the Institute of Henan Provincial People’s Hospital from October2018 to May 2020.We collected and sorted out the detailed clinical data and imaging examination results of the proband and some members of the family,then we drew pedigree charts.Among them,three probands were sporadic cases and four patients had a clear family history of hereditary.method After signed informed consent,peripheral venous blood was collected from the proband and some family members,respectively,and genomic DNA was extracted and qualified for DNA quality.The IDT exome capture Kit(xgen (?) exome research panel v1.0)was used for exome sequencing library c onstruction,and whole exome sequencing was performed with the Illumina novaseq6000 high-throughput sequencing platform.Quality control and evaluation of sequencing data was performed using fastp software,BWA software was used for sequence alignment,the reference sequence was GRCh37 / hg19,meanwhile duplicate reads were removed,and variants were annotated using the gatk genome analysis toolkit for SNP,indel,and annovar software.Common single nucleotide polymorphisms from public population databases(db SNP、 1000 Genomes Project、 Ex Ac、 gnom AD)were filtered and synonymous mutations that did not affect splicing were removed.Suspected pathogenic variants were filtered according to the inheritance pattern of the family,combined with variant site reporting in disease-related databases(HGMD,Clin Var)and variant scoring by multiple bioinformatics software(REVEL).The pathogenicity rating of candidate pathogenic variants was comprehensively applied using ACMG guidelines,and for candidate pathogenic mutations,and performed by Sanger sequencing.The HNN line software was used to predict the secondary structure of the unreported candidate mutations.Results:1.Using whole exome sequencing technology to perform genetic diagnosis on 7families with acromia,4 of which have been diagnosed clearly.TNNT3 gene(NM＿006757)c.G188 A heterozygous mutation was detected in family 2,GJA1 gene(NM＿000165)c.G412 A heterozygous mutation was detected in family 5,GNAS gene(NM＿000516.7)c.C121 G was detected in family 6 heterozygous variants,and family7 detected EVC gene(NM＿153717.3)c.C166 T and c.T356 C compound heterozygous variants.Family 1,Family 3 and Family 4 did not detect any acromal deformity related mutations.2.Family 2 was diagnosed with distal arthrogryposis due to a heterozygous mutation in TNNT3 gene,family 5 with oculodentodigital dysplasia due to a heterozygous mutation in GJA1 gene,family 6 with pseudohypoparathyroidism due to a heterozygous mutation in GNAS gene,and family 7 with Ellis van Creveld syndrome due to compound heterozygous mutations in EVC gene.3.After consulting the literature and searching the HGMD and Clin Var databases,there are no related reports and collections about the exon1 c.121C>G p.H41 D missense heterozygous mutations with the GNAS gene(NM＿000516.7)and the EVC gene(NM＿153717.3)C166T p.R56 X,exon3 c.T356 C p.V119 A compound heterozygous nonsense mutations and missense mutations,which enriched the mutation map of acromegaly genes.Conclusion:1.The whole exome sequencing technology is helpful for clinical diagnosis of acromities and screening of pathogenic gene variants,which provides strong evidence for family genetic counseling and and prenatal diagnosis of the family.2.The whole exome sequencing technology is helpful in finding new pathogenicity variants.3.The whole exome sequencing technique has limitations in the detection of the etiology in families with few or sporadic acromities,and trio-WES is recommended to improve the diagnostic rate.If trio-WES is still negative for acrometosis,there is a possibility of abnormal Copy Number Variant(CNV),which requires Chromosome Microarray Analysis(CMA)or Copy Number Variant sequencing(CNV-Seq). |
PDF Full Text Request