Human Congenital Disorders: A study of underlying genetic changes, manifestation, & mechanism

Abstract

Background: Intellectual disability, also referred to as mental retardation, is one of the common conditions associated with the genetic abnormalities and is characterized by limitation in intellectual functioning and adaptive behaviour. Human structural malformation can manifest at a range of severity, and hence need differential diagnosis to optimize clinical management, enable prenatal detection, and genetic counselling. The crucial challenge with genetic disease is inaccurate or delayed diagnosis which leads to unnecessary diagnostic tests and delayed treatment. The important part in clinical examination of genetic diseases in addition to genetic analysis is description of phenotype which can be subjective and imprecise based on the expertise of the scientist involved. The current methods of high throughput advanced molecular techniques also require next generation phenotyping to match with for better interpretation of genotypic and phenotypic heterogeneity commonly observed. In addition to identification, study of mechanism underlying genetic aberration is also focus of our research which is chromosome translocations. The role of recurrent chromosomal translocations in pathogenesis is well characterized in many acquired genetic diseases, predominately leukemia; however the factors leading to such preferential gene fusions are yet to be studied. Eukaryotic genome consists of chromosomes non-randomly positioned within the nuclear space in discrete regions or “chromosome territories”. The proximity of the genetic regions is considered important for genetic exchange, and interphase molecular cytogenetic methods can be employed to measure the same. Aim: The present study was done to indentify the genetic changes in uncharacterized or idiopathic birth defect cases. To unravel the cause of intellectual disability various techniques including Karyotype, Fluorescence In Situ Hybridization, CGH microarray, SNP array, Whole Exome Sequencing were applied along with detailed phenotypic characterization using standard terminology. The findings of molecular studies were further assessed using Bioinformatics tools for possible implications on phenotype. We also addressed the possible mechanism underlying genetic alteration using recurrent chromosomal translocations as a case. Methods: 125 patients with largely uncharacterized birth defects were assessed as per inclusion and exclusion criteria. A predefined questionnaire was used to collect detailed family, clinical and birth history, phenotypic characterization including morphological anomalies and dysmorphic features in befitting patients. The above work was done in consultation with a clinical geneticist by interview and clinical examination of probands with parents. The minor and major abnormalities were described using matching Human Phenotype Ontology terms which were used to query web-based tool Phenomizer. According to the diagnostic clue, the blood samples were collected and karyotype was done to rule out the presence of any known chromosomal abnormality. Based on the normal karyotype and possible clues from the Phenomizer query results, further molecular genetic analysis was attempted in 10 cases to identify submicroscopic genetic aberration. A novel approach of functional and systems biology assessment, and key hub protein identification was also used by various tools including DAVID, PANTHER, Cytoscap, and cytohubba in four probands (two cases including a familial case of three siblings). In addition, in the familial case of three siblings we also tried to find the expression of associated genes in different organs as per developmental phase. In addition to above, the mechanism of genetic alteration was also addressed in the interphase genomic location of gene pairs taking part in translocations which are non-randomly associated with leukemia subtypes was studied for extent of proximity by measuring relative distance and radial location. The fluorescence in situ hybridization signals corresponding to gene pairs were scored for distance measurements and percentage of translocation pairs showing proximity were calculated in cases of BCR-ABL, PML-RARA, and AML-ETO. Results and Conclusions: In present study we could detect genetic changes possibly associated with intellectual disability in 6 out of 10 idiopathic cases, and cause of spontaneous abortions in a mother which was earlier unknown. The cases include two polymorphic variants, Mosaic Trisomy 13, Apert syndrome and t(4p;8p). The two polymorphic variants cases included, a familial case of inversion Y i.e. 46,XY,inv(Y)(p11q11) and a case of prominent satellite i.e. 46,XY,14ps+. In the case of mosaic trisomy 13, cytogenetic analysis of blood lymphocyte demonstrated trisomy 13 mosaicism in 25% cells i.e. 47,XX,+13(9)/46,XX(27), the interphase FISH in lymphocytes showed trisomy 13 in 15%, whereas in buccal epithelial cells it was nearly 6%. In the case of Apert syndrome, the genomic DNA sequencing detected mutation on FGFR2 gene at c.755C>G [p.Ser252Trp]. Our study also includes a rare familial case of partial monosomy 8p along with partial trisomy 4p in three siblings which was attributed to balanced translocation in mother by meiotic adjacent-1-segregation. The genomic sequencing data in probands were assessed bioinformatically. The biological functional assessments of clinically relevant genes in all four probands (two cases including a familial case of three siblings) showed high association with metabolic or cellular processes whereas molecular functions were mainly ion binding and catalytic activity. The pathway analysis and system biology studies showed that the altered genes were amongst the vital hub proteins which could be important for the proper functioning of entire biological system. The combined approach for naming the genetic condition classified as idiopathic can be significant for unveiling the incidence of various genetic conditions. The result of relative distance and percentage of possible translocation pairs showing proximity was higher for BCR-ABL, PML-RARA, and AML-ETO. The radial position of the gene pairs was also recorded to see if there is any preferred location in terms of nuclear centre or periphery for the translocation partners. The results suggested no preferential location of any of the gene pairs, rather random distribution was observed for all the three cases.