Study Of Two Genetic Birth Defect Model Families Established By Bama Miniature Pig ENU Mutagenesis

BackgroundBirth defect(BD) was structural, functional or metabolic abnormalities occurring during the development process of the embryo or fetus, including anatomical structure malformation, functional defect, growth retardation or congenital metabolic diseases. Birth defect seriously affects children’s life and health, which is the main cause of long-term disability in infants and young children, and has brought heavy mental and economic burden to family and society. There are a total of about 300~400 thousand newborns suffering from birth defect in China, accounting for 4%~6% of the total newly-born population. Birth defect has seriously affected the quality of newly-born population and has become a major public health problem in china. Genetic and environmental factors are the main causes of birth defect. It is generally believed that 20%~30% of birth defects is caused by simple genetic factors, 5%~10% is caused by simple environmental factor, and 60%~70% is caused by the interaction of genetic and environmental factors. Therefore, genetic factors influence on the occurrence of birth defects to a very great degree. In human beings, the study on birth defect has been restricted by many factors, such as ethics problems, long research period and many other factors. Meanwhile, there is a great majority of birth defects remain unclear etiologically until now. Thus, the animal model is still the effective material to study the mechanisms of various birth defects.The chemical N-ethyl-N-nitrosourea(ENU) is the most potent mutagen that can generate random mutations in the animal genome. Following an ENU-mutagenesis screen for dominant and recessive mutations,a large number of mutants with a variety of phenotypes were recovered for the study of gene function and the generation of human disease models. In the past few years, the benefits of ENU have ranged widely, from the generation of novel models of human disorders to a better understanding of gene function and complex biological systems. One of the main advantages of ENU mutagenesis is its versatility: ENU mutagenesis generates point mutations that have the potential to reveal a wide array of mutant effects. Moreover, ENU mutagenesis can be used in forward and reverse genetics approaches. Ultimately, ENU mutagenesis can lead to the production of an allelic series of mutants in any gene.ENU mutagenesis has been widely used in small animals such as Drosophila, zebrafish and mouse. However, it is still a blank for ENU-induced mutation on large animals to create new gene resource in the world. The pig genome is more close to human compared with mice. Through ENU mutagenesis in pigs, we hypothesized that we can find some genes which related to human genetic diseases and are difficult to be discovered in mice.Bama miniature pig is a family of minipig breeds, native to Guangxi Province of China, has been bred in this region for over 500 years. In recent decades, Bama miniature pig has been bred as an experimental animal because it has the characteristics of small body size, genetically stable and highly inbred. Currently, it finds widespread used in human medical studies on dermatology, surgery, drug metabolism, male reproductive studies and etc. In this study, ENU mutagenesis were conducted in the Bama miniature pigs.ObjectivesObjectives of this study are to establish the technical systems of ENU mutagenesis in the Bama miniature pig, to mainly screen birth defect phenotype and establish the family with stable inheritance through phenotype identification, and to provide stable and reliable animal models and related materials for studying the mechanism of action of the corresponding human birth defect diseases.MethodsPart 1 ENU mutagenesis of Bama miniature pigsEighty-one healthy adult Bama boars, 4-12 months, were divided into 7 groups according to different ENU doses: 4 boars in 45 mg/kg group; 9 boars in 65 mg/kg group, 11 boars in 75 mg/kg, 35 boars in 85 mg/kg, 11 boars in 90 mg/kg; 7 boars in 95 mg/kg and 4 boars in 105 mg/kg. ENU was injected via ear vein. Each boar was injected with the corresponding dose for 3 times, once a week, for consecutive three weeks. After the mutagenic agent was injected, the boar seminal fluid was artificially collected every 1 week so as to monitor the sperm quality. After the sperm viability recovered to more than 30%, the mutagenic boars as F0 generation were mated with wild type sows, so as to generate F1 generation offsprings. Two mutagenic families were selected from mutagenic F0 boars(including the F0 parents and 5 F1 offsprings. The ENU injected dose of family I was 65 mg/kg and the injected dose of family II was 85 mg/kg). The ENU mutation and frequency were analyzed and evaluated using simplified Restriction site Associated DNA Sequencing(RAD-Seq). The phenotype identification of F1 generation was conducted so as to mainly screen the mutated phenotypes of birth defect. The mutant F1 boars(or sows) who could survive to adulthood were hybridized with wild type sows(or boars). The phenotypic identification of F2 generation offsprings was conducted. If the same phenotype with F1 generation mutations appeared, the further expanding propagation should be conducted according to the dominant family; If the mutant phenotypes failed to inherit in F2 generation, the family was established according to the recessive family scheme. After a certain F1 boars without mutant phenotype were selected and remained to directly establish the recessive family: F1 boars and wild type sows were copulated, 4 sows were selected and remained in F2 generation so as to backcross with F1 boars. The phenotype identification of F3 generation offsprings was conducted to mainly screen the mutant phenotypes of birth defect.Part 2 Research of an inguinal hernia Bama miniature pig family combined with congenital heart diseaseThe phenotypic identification of obtained mutant family was conducted in detail and accurately: The inguinal hernia phenotypes were repeatedly identified and confirmed in combination with surgical anatomy; The inguinal hernia site, size, type and complications were recorded in detail. Combined with family pedigree chart, the possible genetic model of inguinal hernia was analyzed according to the proportion of affected individuals. The whole-genome scan analysis of the family pig was conducted using Illumiina 60 K pig SNP chip. The possible susceptible loci of inguinal hernia were determined. The genes near susceptible loci were identified through bioinformatics analysis. The susceptible candidate genes were set for exon sequencing. The blood physiological and biochemical indexes, ECG and B ultrasound of all F3 generation pigs in family were detected so as to obtain a detailed clinical basic data related to inguinal hernia. The pathological sections and immunohistochemical analysis of hernial ring and hernial sac tissues in the operation process were conducted. The relevance between inguinal hernia and congenital heart disease was analyzed according to the results of ECG, B ultrasound and cardiac anatomy.Part 3 Establishment and research on an albino Bama miniature pig family combined with growth retardation and hind legs paralysisThe genetic analysis of albinism, growth retardation and rear limbs paralysis was conducted respectively. The mutation detections of albinism, growth retardation and rear limbs paralysis related pathogenic genes were analyzed through candidate gene exon direct sequencing.ResultsPart 1 ENU mutagenesis of Bama miniature pigs1.1 Recovery and reproduction situation of boars mutagenesis: After ENU mutagenesis treatment, boar aspermia time was increased with the increase of the dose, but which was not exactly consistent and there were individual differences; The seminal fluid quality recovery rate(sperm motility rate recovered to more than 70%, density > 107/ml) was increased with the increase of dose, but not exactly consistent and there were individual differences; The fertility of partial high dose individuals was unable to be restored; There was no significant difference in reproductive performance(estrus rate, pregnancy rate and mean clutch litter size) between various dose groups after mutagenesis restored. The reason might be variety factors including seasons, breeding sows and statistical error etc.1.2 F1 generation ENU mutation analysis: The average frequencies of F1 generation ENU mutation were 65 mg/kg and 85 mg/kg. The doses were 1.63×10-6 and 5.86×10-6; The mutation type was mainly G-A type. Transition mutation accounted for 71.2%; The mutation distribution accorded with genome composition, namely a large number of ENU mutation was distributed in intergenic region and intron region. The coding region accounted for less in genome. So the mutation induced by mutagenesis was decreased accordingly.1.3 Establishment and mutant screening of mutagenesis family: 126 mutants were screened in F1 generation. Birth defects included toe malformation, short tail, abdominal wall defect, curved tail, limbs developmental abnormalities, skull dysplasia, encephalocele and crooked nose. Most of them were fatal. The family of 51 mutant individuals who could survive to adulthood was established to verify the heredity. The others had no heredity except for 2 hair albinism individual inheritance.There are 7 families had mutation phenotype in 11 recessive families. Among them, 6 families showed inguinal hernia, and another 1 family showed hair albinism combined with growth retardation phenotype.Part 2 Establishment and research on an inguinal hernia Bama miniature pig family combined with congenital heart disease2.1 Genetic model analysis of inguinal hernia: The family obtaining inguinal hernia was dominant character. The morbidity of F3 generation was 65%, which was far higher than 25% of single-gene recessive genetic theory. The genetic model was more complex. However it was not clear and required for further study.2.2 Illumiina 60 K pig SNP chip whole-genome scan showed there was a strong signal locus in No. 10 chromosome(P=0.00022).2.3 The mutation was not discovered according with inguinal hernia phenotype through exon sequencing with SPATA17 as a candidate gene.2.4 The pathological sections showed that amyotrophy around abdominal ring, myofiber interstitial inflammatory cell infiltration, vascular congestion, nerve fiber degeneration, muscle tissue, fat tissue malnutrition in muscular tissue and other pathological changes in inguinal hernia pigs; The transmission electron microscopy showed collagen fiber denaturation, atrophy, fracture and degradation in hernial sac of sicken pigs; The immunohistochemical analysis showed that the contents of type I and type III collagen were lower than the corresponding fascia trans versalis of normal pigs(P < 0.05), and the content of type III collagen was higher than that of normal pig(P < 0.05). There was no significant difference in total content of type I and type III collagen between two groups(P > 0.05).2.5 The morbidity of congenital heart disease was as high as 20.6% in F3 generation hernia pigs and 13.5% in total boars. The congenital heart disease was not detected in hernia pigs; Although sows did not suffer from hernia, the prevalence rate of congenital heart disease was 16.3%, which was close to the total incidence(14.9%) in family F3 population and whole prevalence rate of congenital heart disease in F3 generation boars.Part 3 Establishment and research of an albino Bama miniature pig family combined with growth retardation and hind legs paralysis3.1 The MC1 R candidate gene exon sequencing for F3 generation albinism phenotype showed that the two CC basic groups were inserted in the 894 th base of mutant individual.3.2 The MC3 R and MC4 R candidate gene exons sequencing showed no mutation according with the growth retardation phenotype.3.3 SMN1 candidate gene exon sequencing showed no gene mutation in F4 generation rear limbs paralysis phenotype.Conclusions1. Bama miniature pig ENU mutagenesis system was successfully established in this study. The suitable dosage and method of miniature pig ENU mutagenesis were explored and determined. The feasibility of ENU mutagenesis applied in miniature pig was confirmed.2. The large animal model family of inguinal hernia with genetic stability was established by Bama miniature pig ENU mutagenesis. Study showed that there was great relevance between inguinal hernia and congenital heart disease. Its pathogenesis may be related to abnormal collagen metabolism; Despite the genetic model was complex and the exact molecular genetic mechanism also required further study, the acquisition and establishment of the family could provide stable and reliable animal model and research materials for the pathological mechanism of inguinal hernia.3. The acquisition and establishment of albinism combined with growth retardation and rear limbs paralysis complex disease family suggested that ENU mutation might lead to multiple bases mutation in the genome.