| Craniofacial deformity is one of the common human congenital diseases affeting the developmentof head, face and neck, which takes up three quarters of congenital birth defects. As a feature of craniofacial defects, orbital hypertelorism is often accompanied by other abnormal symptoms such as proptosis, syndactyly, mental retardation, etc.To date, it has been known that a great deal of craniofacial disorders such as craniofacial cleft and craniosynostosis can manifest with orbital hypertelorism. Although some kinds of orbital hypertelorism can be caused by chromosomal aberrations or genetic mutation, most of the pathogenic mechanismsare still unknown.After detailedly collecting clinical information about two families with hereditary orbital hypertelorism, we studied systematically on the families from chromosomal to genetic level. Through exploring the nosogenesis of two pedigrees with hereditary orbital hypertelorism, our study could lay theoretical foundation on the diagnosis and clinical intervention treatment of the same disorder. After getting genetic diagnosis result, we can provide onset risk for patients and their family members, even guidance to their prenatal diagnosis.Part I Clinical examination and genetic research on family 11 Materials and methods1.1 Clinical examinationAll information about family history and affection status were collected. Patients of the family were arranged to do physical and specialized examinations.1.2 Extraction of genomic DNA of whole bloodPeripheral blood specimens of affected and unaffected individuals were collected and their genomic DNA was extracted.1.3 Karyotype analysisG-banding karyotype analysis was carried out to observe chromosomal structure and numbers of affected family members.1.4 Array-CGH examinationArray-comparative genome hybridization was applied to identify whether affected family members carried chromosome anomaly or not.1.5 Verification of copy number variantCorresponding primers for the CNV found by aCGH were designed to carry out fluorogenic quantitative PCR for co-segregation analysis. In order to clarify the inheritance pattern of the CNV, we conducted loss of heterozygosity analysis according to the deleted fragment of STS region.2 ResultsThe proband had mild to moderate intellectual disability and facial anomalies including hypertelorism, prominent forehead and broad nasal root, etc. Patient III-3 had similar clinical features but without distinct mental retardation. Two brothers of the proband had serious intellectual disability and developmental disorder. However, other unaffected family members were healthy without any mental problems. The G-banding karyotype analysis of patients was normal. Through array-comparative genomic hybridization analysis, it was found that proband II-3 and patient III-3 respectively carried three copy number variants. After comparison by the database of DGV and ISCA and a CGH results of the unaffected, chr14q32.32q32.33 duplication and chr17p13.3 microdeletion were identified as pathogenic CNVs of the family. Quantitative Real-time PCR and LOH analysis indirectly verified the pathogenicity and inheritance pattern of the CNVs.3 ConclusionIn this study, the affected simultaneously carried chr14q32.32-q32.3 duplication and chr17p13.3 microdeletion and manifested clinical characteristics of twosyndromes as well. The copy number variants were the cause of family 1 with hereditary orbital hypertelorism. It was the first time to find that two kinds of CNV were simultaneously carried in affected person and appeared in the same syndrome.Part II Clinical examination and genetic research on family 21 Materials and methods1.1 Clinical examinationAll information about family history and affection status were collected. Patients of the family were arranged to do physical and specialized examinations.1.2 Extraction of genomic DNA of whole bloodPeripheral blood specimens of affected and unaffected individuals was collected and their genomic DNA were extracted.1.3 Gene screeningGenetic testing was carried out by Sanger sequencing, which detected exons and its flanking regions associated with pathogenic genes of candidate diseases.1.4 Karyotype analysisG-banding karyotype analysis was carried out to observe chromosomal structure and numbers of affected family members.1.5 Array-CGH examinationArray-comparative genome hybridization were applied to detect chromosomal condition of affected family members.1.6 Next generation sequencing for mutationThe sequencing data was analysed after the whole exome sequencing for suitable samples we picked out. According to the genetic characteristics of the family with hereditary orbital hypertelorism, personalized screening strategy was made to screen out candidate genes and functional enrichment analysis was carried out on the base of gene ontology.2 ResultsAll patients in three successive generations of pedigree 2 presented serious hereditary orbital hypertelorism, which showed a dominant inheritance pattern. Some of them manifestated other special symptoms while other members in the family were in health. The relevant pathogenic genic mutation and chromosomal aberration were not discovered by screening for known pathogenic gene, chromosomal karyotype analysis and a CGH inspectation. By the whole exome sequencing and analysis of the sequencing data, 96 candidate genes were obtained. GO analysis showed the candidate genes with high degree enrichment in functional field of "peptidase activity" and "cell adhesion".3 ConclusionPathogenic cause of the affected in family 2 was complicated. Neitther mutational variants nor chromosomal aberrations were found by gene screening, karyotype analysis and a CGH examination, which indicating the causse of the deisease was not associated with chromosome anomaly, Pferiffer syndrome, Crouzon syndrome, Apert syndrome or Craniofrontonasal syndrome. Next generation sequecing and related statistical analysis indicated the pathogenic gene of the family may be related to the function of "peptidase activity" and "cell adhesion". |
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