Correlation Between Children’s External Ear Development And Autism Spectrum Disorder And Analysis Of Gene Differential Expression

Background:Autism spectrum disorder（ASD）is a developmental neurological disorder in childhood and has poor prognosis.Its core characteristics are social dysfunction and limited and repetitive behaviors,interests or activities.Early diagnosis of ASD is a feasible and useful step to improve the degree of injury in children and to improve,alleviate or even prevent medical problems related to ASD symptoms.However,since the current diagnosis mainly depends on behavioral performance,most of the ASD diagnosis is late and missed the best intervention time.Early diagnosis requires more biological characteristics and laboratory indicators.Recent studies have shown that ASD is caused by neural crest dysfunction,and defects in neural crest development can also cause abnormalities in the size and shape of the external ear and head circumference.This is found in many cases of syndromic ASD which only accounts for less than 1%of all ASD cases.Although the mutated genes are variable,all ASDs tend to be consistent in common symptomology.Studies have found that common molecular pathways are connected,and many molecular pathways are related to the development of the external ear.Therefore,children with non-syndromic ASD may also have abnormal external ear and head circumference development patterns.Whether it can be used as a biological feature for early screening and diagnosis of ASD still needs further research.In this study,we first developed a parameter model of normal children’s external ear development by analyzing the size and shape data of the external ear of normal Han children in the Children’s Health Clinic of Second Hospital of Shandong University.Then compare the developmental patterns of the external ear in children with nonsyndromic ASD and normal children.And study the influence of factors during pregnancy and childbirth on non-syndromic ASD,external ear and head development.The third part compares the differential expression of developmental genes in peripheral blood of children with non-syndromic ASD and normal children.Analyze the correlation between external ear development and non-syndromic ASD.Part Ⅰ A cross-sectional study on the development of external ears in normal childrenObjective:By analyzing the external ear parameters of normal Han children in the Children’s Health Clinic of Second Hospital of Shandong University,a model of normal Han children’s external ear development was established.Objects and Methods:1.Research objects:This study selected Han children who were examined in the Children’s Health Clinic of Second Hospital of Shandong University from March 2016 to February 2018 as the main research objects.After screening according to the established enrollment criteria,they were divided into 14 groups according to their age（1-14 years old）,and selected 18-year-old Han undergraduate from Shandong University School of Medicine as the reference standard for adult external ear data.Each group includes 30 males and 30 females,a total of 900 subjectsInclusion criteria:（1）Physical development is within the normal range,refer to the physical development indicators of children in 9 cities in China in 2005;（2）Normal mental development;（3）No history of external ear disease.Exclusion criteria:（1）Physical development is not within the normal range;（2）Abnormal mental development;（3）Have a history of external ear disease.2.Research method:Measure physical development and the ear length and ear width of the left and right external ears of all research subjects.This study introduces auricular index to better describe the shape and proportion of the external ear.Auricular index（AI）=ear width/ear length×100.Compare the differences in the size of the external ears between the groups.Observe the change trend of external earshape with age.Results:1.The ear length of boys increased from 4.86 cm at the age of 1 to 6.67 cm at the age of 14,and the ear length of girls increased from 4.69 cm at the age of 1 to 6.40 cm at the age of 14.The ear width of boys increased from 3.00cm at the age of 1 to 3.64cm at the age of 14,and for girls from 2.80cm at the age of 1 to 3.47cm at the age of 14.The ear length and ear width of boys in each age group are larger than those of girls in the same age group.The difference in ear length after 12 years old is statistically significant,and the difference in ear width after 6 years old is statistically significant（all P≤0.05）.2.Ear length and ear width have a positive linear correlation trend with age,linear regression equation:male ear length（cm）=4.618+0.143 × age（years）,female ear length（cm）=4.54+0.136 × age（years）;Male ear width（cm）=3.210+0.037 × age（years）,female ear width（cm）=3.133+0.029 × age（years）.3.The AI value reaches its highest value at 3 years old and 67.03 for boys while 67.78 for girls.4.Ear width and head circumference were linearly correlated in different genders and different age groups.The correlation coefficient ranged from 0.46 to 0.81.5.There was a normal distribution of head circumference/ear width between different genders.The value of males were 14.68 and females were 14.95.6.There are differences in comparison with the external ear measurement data of the population in different regions at home and abroad.Conclusions:1.This study shows that the ear length and ear width data of the external ear of Han children of different ages and genders in this region are different from the data of the external ear of the population in different regions at home and abroad.2.There is a linear correlation trend between ear length and ear width and age,linear regression equation:male ear length（cm）=4.618+0.143 × age（years）,female ear length（cm）=4.54+ 0.136 × age（years）;Male ear width（cm）=3.210+0.037× age（years）,female ear width（cm）=3.133+0.029 × age（years）.3.The ear index peaked at 3 years of age and then declined.It is suggested that the external ear is closest to a round shape when it is 3 years old,and then gradually becomes a narrow and long shape.4.Ear width and head circumference are positively correlated in different genders and different age groups.5.The head circumference/ear width values of different genders are relatively fixed in different age groups.The male is 14.68 and the female is 14.95.Part Ⅱ Correlation analysis of children’s external ear development and ASD:a case-control studyObjective:Compare the difference between the external ear development of non-syndromic ASD Han children and the normal control Han children.Analyze the influence of pregnancy and childbirth factors on non-syndromic ASD and the influence of external ear abnormalities and abnormal head circumference.Objects and Methods:1.Research objects:The ASD group collected 41 Han children with nonsyndromic ASD who attended the Children’s Health Clinic of Second Hospital of Shandong University from March 2018 to February 2019,including 30 boys and 11 girls,aged 3-7 years old.Inclusion criteria:（1）Accord the diagnostic criteria of ASD in DSM-5;（2）Tests such as MRI,electroencephalogram,chromosome,metabolic screening,and secondgeneration sequencing were all negative.Exclusion criteria:（1）Persons with other forms of general developmental disorders;（2）Persons with obvious chromosomal aberrations or abnormal metabolic screening or abnormal second-generation sequencing;（3）Persons with delayed motor development;（4）Persons with a history of underlying diseases or congenital diseases.The control group collected 264 normally-developed children of Han nationality who visited the children’s health clinic of this hospital during the same period,including 149 boys and 115 girls,aged between 3 and 7 years old.Inclusion criteria:（1）There was no delay in neuromotor development at all stages;（2）No family history of ASD.Exclusion criteria:（1）Persons with any form of general developmental disorder;（2）Persons with delayed motor development;（3）Persons with a history of underlying diseases or congenital diseases.2.Research method:Based on interviews with parents and maternal health records,collect the general information of the parents during pregnancy（such as age,education,whether the parents have autoimmune disease history,etc.）,the mother’s condition during pregnancy,such as whether folic acid is supplemented,whether there are complications（including respiratory infection,anemia,abnormal thyroid function,hypertension,hyperglycemia,etc.）and whether premature delivery.Measure the height,weight,head circumference,left and right ear length and width of all research subjects.The collection methods for height,weight,head circumference,left and right ear length,and width are the same as in the first part.According to the Propensity Score Matching（PSM）analysis plan,grouping variables（0=normal control children,1=ASD children）are used as dependent variables.Children’s gender and age are used as matching variables.SPSS 22.0 software automatically uses logistic regression to the independent variables and dependent variables are fitted and the propensity points of each sample are calculated.The ASD child group and the normal control child group are matched by the nearest neighbor matching method at a ratio of 1:1,and the caliper value is set to 0.02.Use the nearest neighbor matching method to match the ASD child group and the normal child group at a ratio of 1:1.After the matching is completed,the normal child group with successful matching is removed.Continue the above process until a complete match is not possible.A total of 1:4 ASD group and normal control group were matched.The development patterns of the external ear and head circumference of children in the ASD group and the control group were compared after matching,and the influence of pregnancy and childbirth factors was analyzed.Results:1.The incidence of macrotia in the ASD group was 17.1%（n=7）,and the incidence of microtia was 7.3%（n=3）.The incidence of macrotia in the control group was 1.8%（n=3）,and the incidence of microtia was 0.6%（n=1）.The differences between the ASD group and the control group were statistically significant（P<0.01）.2.The incidence of macrocephaly in the ASD group was 22.0%（n=9）,and the incidence of microcephaly was 17.1%（n=7）.The incidence of macrocephaly in the control group was 1.8%（n=3）,and the incidence of microcephaly was 1.8%（n=3）.The differences between the ASD group and the control group were statistically significant（P<0.05）.3.The AI of the ASD group and the control group were 52.56 and 65.99,and the head circumference/ear width were 16.27 and 14.83,respectively.The differences between the ASD group and the control group were statistically significant（P<0.01）.4.There was a statistically significant correlation between ASD and whether the mother was supplemented with folic acid during pregnancy（P<0.05）,and the OR（95%CI）was 3.69（1.76,7.76）.5.Whether the mother supplemented with folic acid during pregnancy was statistically significant with macrotia and macrocephaly（P<0.05）,and the OR（95%CI）was 8.13（1.63,40.61）and 4.16（1.18,14.60）,respectively.No pregnancy factors were found to be related to microtia and microcephaly.Conclusions:1.The incidence of external ear abnormalities in the ASD group was higher than that in the control group.2.The incidence of abnormal head circumference in the ASD group was higher than that in the control group.3.Children in the ASD group had abnormal developmental patterns of external ears.4.Failure to supplement folic acid during pregnancy is a risk factor for nonsyndromic ASD.5.Failure to supplement folic acid during pregnancy is also a risk factor for macrotia and macrocephaly.No risk factors for microtia and microcephaly were found.Part Ⅲ Analysis of differential expression of genes related to external ear development in children with ASDObjective:This part of the study screens and compares the genes related to the development of the external ear and head.Compare their differential expression in the peripheral blood of children with non-syndromic ASD and normal children.Objects and Methods:1.Research objects:This study recruited 8 Han children with non-syndromic ASD who attended the Children’s Health Clinic of Second Hospital of Shandong University from May 2019 to April 2020,including 7 boys and 1 girl,aged 2-7 years old.In the control group,there were 7 boys and 1 girl matched by age.2.Research method:According to the search method of subject words plus free words,the genes related to ASD,head and external ear were searched in the gene expression database.The genes related to the development of the external ear and the head were screened out using the Venn diagram.Take 5ml of fasting venous blood from two groups of subjects in the morning.Take PBMC and then use qRT-PCR to screen gene expression in children with ASD and normal control group.Results:1.The genes related to external ear and head development in children with ASD are:AKT1,BCL2,HOXA 1,NOS2,TNF and TGFB1.2.The relative expression of AKT1 mRNA in peripheral blood was 6.74 in the ASD group and 3.41 in the control group;the relative expression of BCL2 mRNA was 0.12 in the ASD group and 0.23 in the control group;the relative expression of HOXA1 mRNA in the ASD group was 0.07 and the control group was 0.19;The differences between the ASD group and the control group were statistically significant（all P<0.05）.Conclusions:1.The relative expression of AKT1 mRNA in the PBMC of the ASD group was higher than that of the control group.2.The relative expression of BCL2 mRNA in the PBMC of the ASD group was lower than that of the control group.3.The relative expression of HOXA1 mRNA in PBMC of children in the ASD group was lower than that in the control group.