Establish A Collecting System For Birth Defects Pedigrees And Mutation Analysis Of HOXD13 In A Chinese Pedigree With Synpolydactyly

In China, birth defects are a severe public health problem. Every year newborns with birth defects are estimated to take up 4% ~6% of all the live births. Etiological research is very important to the prevention and treatment of birth defects. A great majority of birth defects remain unclear etiologically, and most of them can be grouped into multifactorial disorders or inheritable diseases. Birth defects monitoring and specialized epidemiological study are critical in identifying factors contributing to birth defects and evaluating intervention measures. The completion of Human Genome Project and the development of molecular genetics facilitated the etiological study of birth defects. The combination of these disciplines promoted the development of etiological studies based on family cluster, which aroused increasing concern of many medical researchers. The collection of pedigrees of birth defects is vital to the research. The traditional way of collecting birth defects pedigrees basing on clinical settings and specialized epidemiological study cannot meet the demands, for its limit in disease diversity, ethnic group and geographic area. The collection of birth defects pedigrees provides a sound grand for the research. In addition, by collecting biological specimens of pedigrees of birth defects, human genetic resource can be well protected. A rational, effective and sustainable collection system for birth defect pedigrees is critical for thedevelopment of the birth defects monitoring in China.We established a huge network to collect pedigree data and biological samples of the birth defects, on the base of existing network for birth defects monitoring and the maternal and child heath surveillance, supplemented with other measures. This research is grounded on the practice of birth defects monitoring, and will meet the needs of etiological study and protection of the genetic resource of inherited diseases. A preliminary research has been done on establishing the collecting system for birth defects pedigrees on an epidemiological surveillance network. The major achievements are as follows:(1) We proposed and set up a nationwide network of collecting system for birth defects pedigrees;(2) We designed a systemic protocol for biological specimen collection, processing, storage and management for DNA bank, and set up a DNA bank and a computerized management system simultaneously;(3) The total of biological samples reached 800 over the past one and half year;(4) We obtained some congenital malformation pedigrees with high scientific value, even rare disorders not reported before in China;(5) We explored the possibility of application of the data and biological samples of birth defects pedigrees;(6) We first identified that synpolydactyly in Chinese population can be caused by polyalanine expansion of HOXD13 gene;(7) We discovered a new site of mutation in HOXD13, which has never been reported before.Part ICollection of birth defects pedigrees -to establish a collecting system for birth defects pedigrees and set up a DNA bank on the basis of birth defectsmonitoring network In China, the maternal and child health surveillance network (including BirthDefects Monitoring, Children Death Surveillance under five years old of age, and Maternal Mortality Surveillance) has run effectively for some twenty years. The network is government-supported and well organized, with specially trained health professionals. It is an ideal base on which we can establish an integrated system to collect information and specimens of birth defects pedigrees.Measures: 1) In the existing hospital-based monitoring network for birth defects, establish a nationwide system through trainings and quality control to report possible birth defects pedigrees and to collect pedigree data and biological samples of nuclear birth defects pedigrees. 2) According to case-parent trio design, launch population-based pilot work to collect clinical data and blood samples of all children with birth defects along with those of their parents. 3) Take supplemental measures to collect information of possible birth defects pedigrees: clinical screening, disability rating, and individual reporting. 4) Standardize biological sample collection and DNA banking techniques. 5) Develop an information management system for data management and samples management. Furthermore, the application and future development of the network for pedigree collection and DNA bank have been discussed.Part IIMutation analysis of H0XD13 in a Chinese pedigree with synpolydactylyThis research was carried out using collected data and blood samples of a Synpolydactyly pedigree through field investigation. No aberration was found in both the proband and her mother with routine chromosome karyotype analysis. By studying the clinical manifestation of the affected subjects and the transmission mode in this family and some research documents, we selected H0XD13 as candidate gene to study. PCR method for mutation screening was optimized. For every member of this synpolydactyly pedigree, primers flanking the hot spots of mutation of H0XD13 sequence were used to amplified a piece of fragment.Electrophoretic separation of PCR products showed a mutation in the H0XD13 gene in individuals affected by synpolydactyly.To examine whether there is any other mutation within coding sequence of H0XD13, normal and selected mutant alleles were amplified in three segments. PCR methods were also optimized respectively. Amplified fragments were electrophoresed on 2% agarose gels for analysis, and then mutant fragments were electrophoresed on 5% polyacrylamide gels to be separated. Purified PCR products of normal and selected mutant alleles were directly cycle sequenced. By sequence assembling, a full sequence of exons in H0XD13 were obtained. After comparing the H0XD13 coding sequence of affected individual with H0XD13 sequence in the Genebank and with that of the unaffected, we detected an inserted segment coding 8 alanine residues within H0XD13 segregating with the disorder. This sort of mutation is also termed polyalanine expansion. The 8-alanine expansion can be interpreted as a reduplication of normal alanines 5-12. No other mutation was found in the coding region of H0XD13. Our results suggest for the first time that synpolydactyly in Chinese population can be caused by polyalanine expansion in H0XD13. The site of mutation in H0XD13 has not been reported before. In discussion, we analyzed the relationship between HOX gene mutation and SPD, proposed possible mechanism by which the mutation of HOX gene causes SPD, reviewed the role of HOX genes in limb development, and presented directions for further research.