Detection Of Inherited Diseases In Critically Ill Neonates Using Whole Exome Sequencing Technology

Research backgroundInherited diseases are divided into chromosomal diseases,monogenic diseases,polygenic diseases and mitochondrial diseases.There are many types of Inherited diseases,and their clinical manifestations are not typical,especially in the newborn period.Most inherited disease are critically ill,and their prognosis is poor。Clinical diagnosis of inherited diseases is not easy,and treatment is also quite difficult。Inherited diseases are an important cause of death in infants and toddlers or perinatal period,and the number of children with genetic diseases is increasing year by year.However,traditional cytogenetic technology cannot accurately detect gene mutations at all.85%of human pathogenic gene variants occur in the exon region.WES can accurately detect most（about 98%）point mutations and most copy number variations in the coverage area.WES can efficiently and quickly identify disease-causing genes and mutation sites,which is helpful for rapid screening of genetic diseases.ObjectiveTo study the application value of whole exome sequencing（WES）in critically ill neonates with inherited diseases by collecting clinical data of critically ill newborns with suspected genetic diseases or unclear diagnosis admitted to our hospital in the past 3 years.Materials and Methods1 Research objectResearch object Critically ill neonates with suspected inherited diseases or unclear clinical diagnosis who were admitted to the Neonatal Intensive Care Unit of Zhongshan People’s Hospital from January 2018 to June 2020 were enrolled as subjects.A total of 66 newborns were included in the study,including 34 males（52%）and 32 females（48%）,with gestational age of 28 weeks+1～41 weeks+3,birth weight0.97kg～3.9kg,age of onset 1～28 d.Clinical manifestations The onset of the disease in all children began in the neonatal period.The main clinical manifestations and positive auxiliary examination results of the study subjects were:13 cases of persistent jaundice and cholestasis,4cases of feeding difficulties,4 cases of repeated convulsions,7 cases of skin abnormalities,3 cases of abnormal muscle tone,3 cases of unexplained dyspnea,1case of visceral ectopic,1 case of giant tongue,14 cases of oral and appearance abnormalities,3 cases of severe abnormal heart structure,7 cases of abnormal screening results of genetic metabolic diseases of blood or urine tandem mass spectrometry,1 case of fetal chromosome inheritance,repeated hypoglycemia And 3cases of metabolic acidosis,and 2 cases of repeated three-line reduction.Medical ethics approval The Medical Ethics Committee of Zhongshan People’s Hospital has approved.The guardians of the newborns all signed an informed consent form.The doctor has filled in the basic information,clinical manifestations and related auxiliary examination results of the child and his parents.2 Methods2.1 Patient screeningInclusion criteria:（1）According to clinical symptoms,signs,and other auxiliary examinations,children with genetic diseases were highly suspected,or they have a positive family history,and have close relatives married.（2）The clinical diagnosis was clear and the genetic specificity of the disease is high or the phenotype of the child is non-specific.Exclusion criteria:（1）The child’s guardian did not agree with the signature.（2）The pathogenic gene of the target disease was not in the scope of WES detection.2.2 Specimen collection2～3 m L of venous blood samples were collected from the neonates and their parents,anticoagulated with EDTA/citrate,and stored under refrigeration at2～8℃.We needed to fill in the general information,clinical manifestations and related auxiliary examination results of the child and his parents,and express the specimens to the Guangzhou Golden Mile Medical Laboratory Center for high-throughput WES testing.The guardians of the newborn all signed the informed consent form.2.3 Detection methods2.3.1 WES After the blood samples are delivered to the inspection center in cold storage,the DNA from the peripheral blood is extracted with QIAamp DNA Mini KIT reagent.DNA is fragmented by enzyme digestion method.Fragmented DNA plus a linker of specific sequence.The x Gen Exome Research panel v1 probe produced by IDT manufacturer captures the target gene.The Next Seq 500 second-generation sequencer produced by Illumina performs sequencing of the whole exome.2.3.2 Verification The genetic mutation sites and parent-related gene sites detected by WES were verified by first-generation Sanger sequencing.2.3.3 Database comparison A very large amount of data was used as the control,which contains not only the data of many foreign populations,but also the data of a large number of Chinese populations.The mutation/variation databases used by the Golden Field Laboratory mainly include:Golden Field Database,mainly for Chinese patients with genetic diseases.The Human Gene Mutation Database（HGMD,Professional Edition）is a patient-oriented mutation database.Clin Var database（comprehensive）is a database of human genome variants related to diseases.The ESP6500 database（Exome Sequencing Program of the National Heart,Lung,and Blood Institute of the United States）is mainly for the general population,mainly used for the research of heart,lung,and blood-related diseases;G1000 database（Thousand Genome Project）.The database of single nucleotide polymorphisms（db SNP）is based on the general population and is a resource library for single base substitutions and short insertion and deletion polymorphisms.2.3.4 Interpretation We needed to comprehensively consider various evidences to assess whether the detected genetic variants have clinical significance.The clinician combined the phenotype and comprehensive analysis of the child to interpret the WES test results,and finally gave a report with reference to the2016 version 2American Society of Medical Genetics and Genome（ACMG）guidelines.Pathogenic genetic variation:Refers to the pathological variation that a specific patient carries that causes the disease state.Suspected pathogenic genetic variants:Refers to variants that are likely to be pathogenic,but the evidence is not sufficient.Variations of Unexplained Clinical Significance:As there is no report at present,there is no relevant database showing its clinical significance,but it may be meaningful in the clinical process in the future.Moreover,whether the clinically significant mutations will cause the patients’diseases still needs scientific development,data accumulation and more in-depth functional research.2.4 Other auxiliary examinationsBlood routine,urine routine,liver and kidney function,blood sugar,blood gas analysis,blood ammonia,blood lactate,insulin,homocysteine,tandem mass spectrometry,X-ray,cardiac color Doppler ultrasound,head MRI,EEG,etc.Results1General situation66 critically ill neonates with suspected inherited diseases or unclear clinical diagnosis were enrolled in our study,including 34 boys and 32 girls.The age of onset was the first to 28^th day.15 cases（23%）were diagnosed with inherited diseases,including 11 males and 4 females.Mean age at diagnosis was the 37^th days.2 Clinical informationAmong the 66 neonates,14 cases（21.21%）with gene mutations were found by WES.One newborn had no gene mutation detected by WES but was highly suspected of pigment incontinence based on clinical manifestations,and multiplex ligation-dependent probe amplification detected a heterozygous deletion mutation in exons 4-10 of the IKBKG gene.15 cases（23%）were diagnosed with inherited diseases,according to the correlation between the detected gene mutation sites and the disease,combined with the first-generation Sanger sequencing to verify,supplemented by Clin Var database,OMIM database,domain analysis of gene-encoded protein,family history,and mutation frequency obtained by large sample size investigation and bioinformatics software evaluated the analysis results of some mutation sites,combined with the clinician’s phenotype and comprehensive analysis of the children to interpret the WES test results.Among the 15 neonates with gene mutations,5 cases diagnoses with genetic diseases of the skin,skull and nerve;7 cases with endocrine and metabolic system diseases;2 cases with cardiovascular diseases;1 case with chromosomal disease.Chromosome karyotyping was performed in 13 of 15 cases,and only 1 case had abnormal result.3 Genotype in neonates with gene mutations10（67%）in 15 neonates with gene mutations had pathogenic gene mutations,1（7%）case was suspected of pathogenic gene mutation,and 4（27%）cases had gene mutations with unknown significance.One neonate had no gene mutation detected by WES but was highly suspected of pigment incontinence based on clinical manifestations,and multiplex ligation-dependent probe amplification detected a heterozygous deletion mutation in exons 4-10 of the IKBKG gene.Variants with unclear clinical significance were currently not reported,and there was no relevant database showing their clinical significance,but they may be meaningful in the clinical process in the future.Further scientific verification is needed to determine whether the variants of unclear clinical significance is pathogenic.4（40%）in 10 neonates with pathogenic gene variants were diagnosed with autosomal recessive diseases,3（30%）cases with autosomal dominant diseases,and 1（10%）case with X-linked dominant disease,2（20%）cases with missing gene fragments.Among them,3 of neonates with autosomal recessive inheritance had heterozygous parents who carried the disease-causing gene.The parents of 2 neonates with autosomal dominant inherited disease did not carry the pathogenic gene,and the mutation may be a new mutation.At the same time,it was not ruled out that their parents were carriers of germ cell mosaicism.The parents of the other 5 cases did not have the corresponding genetic examination.The source of the pathogenic variant genes inchildren cannot be further verified.Conclusions1.WES detection technology is an important tool to find inherited diseases in critically ill neonates with suspected inherited diseases or unclear clinical diagnosis.2.WES technology has some limitation and it is thus necessary to combine with other sequencing methods to achieve a correct diagnosis.