Preliminary Study Of Molecular Diagnosis In Some Rare Genetics Disorders Of Neonatal

Objects:Genetic factor is an important factor related to the birth defects; while congenital multiple anomalies (CMA) is the most common type of birth defects. About 25% MCA newborns carry chromosome aberration so it is an urgent need for cytogenetics analysis to be implemented in the clinical diagnosis. The brain damage due to persistent hypoglycemia in neonatal period is given more and more attention and congenital hyerinsulinism is one of the major causes of persistent hypoglycemia in neonates. HADH gene mutation, one of the genetic causes of the congenital hyperinsulinism, has not been studied within nationwide. Cytogenetics Whole-Genome 2.7M Array was used in this study to identify copy number variations of the CMA newborns to inspect the relation between the genotype, CNVs with clinical potential value and the phenotype of the CMA newborns. Sequencing of HADH gene's coding region and bioinformatics analysis of HADH mutation sites was done in congenital hyperinsulinism patients in order to identify HADH mutations. These studies provide preliminary clinical approach to molecular diagnosis for rare neonatal genetic disorders.Methods:Genomic DNA was extracted from the peripheral whole blood of the CMA newborns admitted into this study that showed congenital anomalies affecting two or more systems. After quality of genomic DNA was assessed, whole genome genotyping was done by using Affymetrix Cytogenetics Whole-Genome 2.7M Array. CYCHP files were generated after the whole genome cytogenetics analysis and were analyzed through ChAS software. False positive CNVs were excluded and the remaining CNVs were compared to the CNVs reported in the DGV database. Benign CNVs defined as CNVs reported in the control populations in the DGV database was excluded and the CNVs with potential clinical significance were revealed. The CNVs with potential clinical significance was defined as CNVs affected regions of the common deletion or duplication syndromes such as the 22q11 deletion reported in DiGeorge syndrome; duplication or deletion region size>2Mb; rare CNVs not reported in the DGV database. The genotype-phenotype relation of the CNVs with potential clinical significance in the MCA newborns was analyzed and compared to the reported patients with chromosome aberrations in similar regions in order to diagnose the genetics-related MCA newborns. Coding regions of HADH gene was amplified and sequenced using the genomic DNA of the 12 congenital hyerinsulinism patients and the sequence files were manually analyzed through the Mutation Surveyor Val V3.25 software in reference to the NCBI HADH sequence NT₀16354. Bioinformatics analysis, including sequence conservation analysis of HADH mutations through the Shannon entropy and the impact of the HADH mutations, identified in this study and reported before, towards the protein structure, was done on the basis of the alignment obtained in the PSI-BLAST alignment.Results:1.123 CNVs identified in the 42 CMA newborns. There were 450 CNVs identified in 61 MCA newborns with mean region size 1.73Mb. Among these,25(6%) CNVs were false positive CNVs,302(67%) were benign CNVs and 123 CNVs(27%) was potentially significant.123 CNVs with potential clinical significance were found in 42 CMA newborns with detection rate of 68.85%. The CNVs with potential clinical significance was mostly identified in the newborns with both dysmorphic facial features and congenital heart defects and the detection rate was 85.71%, then was in the MCA newborns with congenital heart defects and the detection rate was 75%. The recurrent regions of the 123 CNVs with potential clinical significance includes 3 cases of 2q31.1 duplication,3 cases of 5p 15.33 deletion,2 cases of 13q21.1-q34 duplication,4 cases of 7p15.2 duplication and 1 case of deletion,1 deletion and 1 duplication case of 11q24-q25,3 cases of 15q11-q13 deletion,7 cases of 16p12.3 deletion and lease of deletion,2 cases of 22q11.2 deletion and 1 case of duplication and 8 cases of Xq21 duplication.2.12 CMA newborns carry CNVs affected the critical region of common mircro-deletion/duplication syndromes including 15q11-q13 deletion of Prader-Willi syndrome,7q36 deletion/duplication syndrome,18 trisomy,5p deletion of Cri-du-chat syndrome and 13q duplication syndrome, which accounts for 19.7% of the total CNVs identified in this study and could be diagnosed through CNVs analysis.3 of the 12 cases was identified through karyotyping and 2 was diagnosed by clinical presentations, accounting for 0.05% and 0.03% respectively. In Cri-du-chat Syndrome patients,5p15.33-15.31 was found to be related to the characteristic cry and 5p15.1-15.2 was related to the facial features of the Cri-du-chat Syndrome.6 CMA newborns carry 7 CNVs with potential clinical significance, who are affected by the Pierre Robin Sequence and present micrognathia, cleft palate and glossoptosis. The 7 CNVs include the duplication of 1p36.23-p26.22,14q11.1-q11.2 and 20p13 and the deletion of 4q23, 1q43-q44 and 14q32.31, among which the region 1q43-q44 and 14q32.31 might be involved in the development of PRS.3. A novel T238A point mutation was identified in one CHI patients leading to the corresponding V30E change in the HADH protein structure. This site was highly conserved and located in aβ-sheet and close to the NAD+ binding site. The substitution of a non-polar residue Val with an acidic residue Glu may affect the stability of the protein structure, which then would affect HADH's function.Conclusion:1. The diagnostic use of whole genome cytogenetics array in CMA newborns would greatly increase the detection rate of the chromosome aberrations. The atypical or complicated clinical presentations of CMA newborns are influenced by the single or multiple CNVs especially with extending size. The whole genome cytogenetics arrays provide a great possibility of the early diagnosis for the CMA newborns and assist in the clinical diagnosis. Through the copy number variation analysis, regions possible related to Cri-du-chat Syndrome and Pierre Robin Sequence were identified.2. Val30Glu mutation is responsible for the development of congenital hyperinsulinism in the patient. The clinical presentations of the CHI patients were greatly influenced by the conservation degree of HADH mutations and their location in the HADH protein structure. The more impact to the HADH enzyme function due to the mutated HADH site with higher conservation degree leaded to the more complicated clinical symptoms.