Microtia Associated Large Fragment Chromosome Variations And Relevant Genes

BackgroundMicrotia, which encompasses a spectrum of congenital anomalies of the external and middle ear, involves immature derivatives from the first and second pharyngeal arches. The pathogenesis of microtia is perplexing. Environmental factor such as retinoic acid has been proved to be associated in microtia pathogenesis. Significant genetic contribution is confirmed:(1) Higher concordance in monozygotic twins than in dizygotic twins; (2) Incomplete penetrance and variable expression has been observed in family cases; (3) Family cases ranging from 3 to 34%; (4) Chromosomal aberrations or single-gene defects have been reported; 5) Mutations of specific genes result in microtia in mice. Therefor research on genetic mutation site is of great importance to illuminate pathogenesis of microtia.ObjectiveGenetic disorders associated with microtia was subgrouped into alterations at chromosomal level and gene level. We conduct copy number analysis using the genotype data from the genome-wide SNP array among microtia patients for chromosomal malformations. Furthermore, the neighbouring genes of chromosomal aberrations or genes in the extra or deleted chromosome fragments would be screened to investigate the possible causative genes.MethodsCase control study which included 942 microtia cases and 1802 healthy controls was conducted to screen for large fragments chromosome variations and relevant genes.1. Search for large fragments of chromosome aberration. Copy number analysis was conducted using the genotype data from the genome-wide SNP (Single Nucleotide Polymorphism) array in microtia patients and healthy controls for large fragment chromosomal malformations. The methods were as follows:(1) Determine the variations of chromosome copy number. In the normal diploids of human, CN is equivalent to 2, whereas CN=1 indicates chromosome deletion and CN=3 suggests chromosome duplication; (2) Record the length of the chromosome fragment and the loci of break points referring to B alle frequency. (3) Category the type of chromosome aberration: chromosome structure variation and chromosome number variation.2. Gene analysis in accordance with large fragment for chromosome structure variation. (1) For chromosome deletion, the genes in the deleted segment should be screened for possible causative gene. (2) For chromosome fragment duplication, the neighboring genes in the break point are retrieved to prove the possible involvement, also referred to gene position effect; besides, gene dosage effect caused by the duplication of chromosome is also in consideration.3. Gene analysis in chromosome number variation.Results1. Large chromosomal fragments variations were detected in 5 patients in the case group. No chromosomal abnormality was observed in the control. Fisher’s Exact Test was conducted to calculate the proportion of chromosomal aberrations in the microtia group in comparison to the control group, and P value of is 0.0033, which suggests statistical significance.2.2 cases were proved to be associated with chromosome structural variations. The malformations of the first case presented to be partial duplication of the long arm of 13 and 14 chromosome. In detail,13q14→13qter trisomy、14q13→14q22 trisomy、 14q31.3→14qter trisomy was detected. On screening for causative genes in the neighboring chromosomal break points, OTX2 was detected to be in the break point of chromosome 14. BMP4 and GSC were found to be in the duplication fragments of 13 and 14 chromosomes for possible gene dosage effect. The second case presented to be partial duplication of the long arm of chromosome5. In detail,5q11.2→5q13.1 and 5q13.2→5qter trisomy was detected. No causative gene was involved in the break points. FGF pathway associated genes FGF18, FGFR4, FGF1 and BMP pathway associated genes FST, MSX2, SMAD5 were incorporated in the extra duplicated chromosome for possible gene dosage effect.3.3 cases were found to carry an extra X chromosome. Among the 3 cases, one patient suffered from XXY karyotype and the other 2 patients were of X trisomy.Conclusion1. Fisher’s Exact Test is conducted to compare the probability of chromosomal aberrations between microtia group and control group with the P equivalent to 0.0033, indicating the possible association of chromosome abnormity and microtia.2. OTX2 is involved in break points of chromosome 14 and is suggestive to be the causative gene of microtia in the first case.3. GSC,BMP4 duplication in chromosome 13 and 14 might be associated to microtia pathogenesis.4. Extra copies of FGF pathway associated genes FGF18, FGFR4, FGF1 and BMP pathway associated genes FST, MSX2, SMAD5 are discovered in the duplicated fragment, indicating involvement of the development of microtia in the second case.