Identification of GLI Mutations in Patients With Hirschsprung Disease That Disrupt Enteric Nervous System Development in Mice.

BACKGROUND & AIMS: Hirschsprung disease is characterized by a deficit in enteric neurons, which are derived from neural crest cells (NCCs). Aberrant hedgehog signaling disrupts NCC differentiation and might cause Hirschsprung disease. We performed genetic analyses to determine whether hedgehog signaling is involved in pathogenesis. METHODS: We performed deep-target sequencing of DNA from 20 patients with Hirschsprung disease (16 men, 4 women), and 20 individuals without (controls), and searched for mutation(s) in GLI1, GLI2, GLI3, SUFU, and SOX10. Biological effects of GLI mutations were tested in luciferase reporter assays using HeLa or neuroblastoma cell lines. Development of the enteric nervous system was studied in Sufu(f/f), Gli3(Δ699), Wnt1-Cre, and Sox10(NGFP) mice using immunohistochemical and whole-mount staining procedures to quantify enteric neurons and glia and analyze axon fasciculation, respectively. NCC migration was studied using time-lapse imaging. RESULTS: We identified 3 mutations in GLI in 5 patients with Hirschsprung disease but no controls; all lead to increased transcription of SOX10 in cell lines. SUFU, GLI, and SOX10 form a regulatory loop that controls the neuronal vs glial lineages and migration of NCCs. Sufu mutants mice had high Gli activity, due to loss of Sufu, disrupting the regulatory loop and migration of enteric NCCs, leading to defective axonal fasciculation, delayed gut colonization, or intestinal hypoganglionosis. The ratio of enteric neurons to glia correlated inversely with Gli activity. CONCLUSIONS: We identified mutations that increase GLI activity in patients with Hirschsprung disease. Disruption of the SUFU-GLI-SOX10 regulatory loop disrupts migration of NCCs and development of the enteric nervous system in mice.

GATES: a rapid and powerful gene-based association test using extended Simes procedure.

The gene has been proposed as an attractive unit of analysis for association studies, but a simple yet valid, powerful, and sufficiently fast method of evaluating the statistical significance of all genes in large, genome-wide datasets has been lacking. Here we propose the use of an extended Simes test that integrates functional information and association evidence to combine the p values of the single nucleotide polymorphisms within a gene to obtain an overall p value for the association of the entire gene. Our computer simulations demonstrate that this test is more powerful than the SNP-based test, offers effective control of the type 1 error rate regardless of gene size and linkage-disequilibrium pattern among markers, and does not need permutation or simulation to evaluate empirical significance. Its statistical power in simulated data is at least comparable, and often superior, to that of several alternative gene-based tests. When applied to real genome-wide association study (GWAS) datasets on Crohn disease, the test detected more significant genes than SNP-based tests and alternative gene-based tests. The proposed test, implemented in an open-source package, has the potential to identify additional novel disease-susceptibility genes for complex diseases from large GWAS datasets.

A comprehensive framework for prioritizing variants in exome sequencing studies of Mendelian diseases.

Exome sequencing strategy is promising for finding novel mutations of human monogenic disorders. However, pinpointing the casual mutation in a small number of samples is still a big challenge. Here, we propose a three-level filtration and prioritization framework to identify the casual mutation(s) in exome sequencing studies. This efficient and comprehensive framework successfully narrowed down whole exome variants to very small numbers of candidate variants in the proof-of-concept examples. The proposed framework, implemented in a user-friendly software package, named KGGSeq (http://statgenpro.psychiatry.hku.hk/kggseq), will play a very useful role in exome sequencing-based discovery of human Mendelian disease genes.

[Genetic polymorphisms of ten X chromosome STR loci in Hunan Tujia population and their forensic evaluation].

Genomic DNA was extracted from whole blood of 198 unrelated health individuals of Tujia ethnic group from south China's Hunan Province. Genotyping and detection of PCR products were carried out on denaturing polycrylamide gel electrophoresis followed by silver staining. Allele frequencies and genotype frequencies were computed. Deviation from Hardy-Weinberg equilibrium and differences of gene distribution were examined by SPSS 13.0. Fixation index, genetic polymorphism and indices of forensic application were calculated using Fstat and Powerstats, respectively. The results revealed that the frequencies of 65 alleles were distributed from 0.0048 to 0.6170. Among the ten X-STR loci, DXS6789, DXS6799 and HPRTB presented lower diversity and differentiation, while DXS7133 and DXS7423 showed lower value in forensic application. Results of multiple comparisons by loci showed that difference between German, Italian and Tujia population were the most dominant, and it suggested that great genetic differences did exist between Caucasian and Mongo-lian. In conclusion, DXS6804, DXS7132, DXS7130, DXS8378, DXS6789, DXS6799, DXS7424 and HPRTB had a good value in forensic identification, paternity testing of female and disease related study for Tujia population.

[Genetic polymorphisms of 9 X-chromosome short tandem repeat loci in a Inner Mongolia Ewenki population and their forensic evaluation].

OBJECTIVE: To investigate the alleles and genotypes frequency of 9 short tandem repeat (STR) loci on the X chromosome (DXS6789, DXS101, DXS8378, DXS7132, DXS7133, DXS7423, DXS6804, DXS6799, HPRTB) of Ewenki individuals living in Inner Mongolia Autonomous Region of China. METHODS: The 9 X-chromosomal STR loci were analyzed with polymerase chain reation (PCR), followed by polyacylamide gel electrophoresis and silver staining. Software SPSS13.0, Genepop, Fstat and Powerstats were used to evaluate their polymorphism diversity and potential forensic application. RESULTS: Allele frequencies and genotype frequencies of 99 unrelated Ewenki individuals were obtained. Among the 9 loci, DXS6789, HPRTB showed less polymorphism and diversity in the population. The diversity of DXS7132 has no statistical difference between Ewenki population and other 4 Asian populations. CONCLUSION: Except DXS6789, HPRTB, the other 7 X-chromosomal STR loci are appropriate for individual identification, paternity test involving a female child, and studies on related disease. DXS7132 should be excluded when being used to distinguish diversity difference among populations.

[Study on SNP polymorphism of mitochondrial DNA D-loop region from Nu ethnic population in Yunnan of China].

OBJECTIVE: To investigate the mitochondrial single nucleotide polymorphism (SNP)of Chinese Nu ethnic population from Yunnan region of China and to provide basic database for ethnic origin investigation and forensic purpose. METHODS: Genomic DNA from the whole blood of 87 unrelated individuals was extracted by standard chelex-100. The sequence polymorphism was analyzed by PCR-based assay and using ABI 3730 Analyzer to detect many number of relatively common point mutations. RESULTS: Sixty-two SNP loci were observed among them with 492 point mutations and 59 haploids identified in mitochondrial DNA hypervariable region I (mtDNA HVSI). The gene diversity was estimated to be 0.9675,and the random match probability was calculated to be 0.0437. CONCLUSION: The result suggests that mtDNA HVSISNP database of Nu ethnic population can be a useful tool for forensic identity and original research.

[The application of statistical approaches in studying population genetics with STR data].

As high polymorphism markers, STR loci have been widely used in studying population genetics. This review summarizes various genetic diversity parameters and analysis methods regarding to genotype frequency and allele frequency, including heterozygosity, polymorphism information content (PIC), linkage disequilibrium coefficients, inbreeding coefficients, genetic distance and fixation indices. The methods of statistical analysis included principal component analysis, phylogenetic trees, analysis of molecular variance (AMOVA), R matrix, GIS and spatial autocorrelation. By means of various parameters and methods, graphically and scientifically, we can describe and interpret several key issues in the study of population genetics, such as population genetic structure, population differentiation and human evolution.

[Validation the haplotype polymorphisms of the DXS7424-DXS101 on X-chromosome].

OBJECTIVE: To validate the genetic characteristics and distribution of DXS7424-DXS101 on X chromosome in Han population. METHODS: DXS7424 and DXS101 loci were genotyped by PCR, PAGE and silvers stain methods. Their genetic parameters were analyzed by Arlequin software. RESULTS: There were 37 haplotypes detected in 151 Han unrelated males. The frequencies ranged from 0.0066 to 0.1391, with a GD value of 0.9453 and a DP value of 0.9389. Haplotypes 16-23 were the most common haplotypes in Han population. CONCLUSION: Analysis of combined DXS7424 and DXS101 haplotypes appears to be a powerful means in population genetics and forensic practice for determination of identity and paternity.

Depletion of the IKBKAP ortholog in zebrafish leads to hirschsprung disease-like phenotype.

AIM: To investigate the role of IKBKAP (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein) in the development of enteric nervous system (ENS) and Hirschsprung disease (HSCR). METHODS: In this study, we injected a morpholino that blocked the translation of ikbkap protein to 1-cell stage zebrafish embryos. The phenotype in the ENS was analysed by antibody staining of the pan-neuronal marker HuC/D followed by enteric neuron counting. The mean numbers of enteric neurons were compared between the morphant and the control. We also studied the expressions of ret and phox2bb, which are involved in ENS development, in the ikbkap morpholino injected embryos by quantitative reverse transcriptase polymerase chain reaction and compared them with the control. RESULTS: We observed aganglionosis (χ2, P<0.01) and a reduced number of enteric neurons (38.8±9.9 vs 50.2±17.3, P<0.05) in the zebrafish embryos injected with ikbkap translation-blocking morpholino (morphant) when compared with the control embryos. Specificity of the morpholino was confirmed by similar results obtained using a second non-overlapping morpholino that blocked the translation of ikbkap. We further studied the morphant by analysing the expression levels of genes involved in ENS development such as ret, phox2bb and sox10, and found that phox2bb, the ortholog of human PHOX2B, was significantly down-regulated (0.51±0.15 vs 1.00±0, P<0.05). Although we also observed a reduction in the expression of ret, the difference was not significant. CONCLUSION: Loss of IKBKAP contributed to HSCR as demonstrated by functional analysis in zebrafish embryos.

A three-stage genome-wide association study combining multilocus test and gene expression analysis for young-onset hypertension in Taiwan Han Chinese.

BACKGROUND: Although many large-scale genome-wide association studies (GWASs) have been performed, only a few studies have successfully identified replicable, large-impact hypertension loci; even fewer studies have been done on Chinese subjects. Young-onset hypertension (YOH) is considered to be a more promising target disorder to investigate than late-onset hypertension because of its stronger genetic component. METHODS: To map YOH genetic variants, we performed a 3-stage study combining 1st-stage multilocus GWASs, 2nd-stage gene expression analysis, and 3rd-stage multilocus confirmatory study. RESULTS: In the 1st stage, Illumina550K data from 400 case-control pairs were used, and 22 genes flanked by 14 single nucleotide polymorphism (SNP) septets (P values adjusted for false discovery rate (pFDR) < 3.16×10(-7)) were identified. In the 2nd stage, differential gene expression analysis was carried out for these genes, and 5 genes were selected (pFDR < 0.05). In the 3rd stage, we re-examined the finding with an independent set of 592 case-control pairs and with the joint samples (n = 992 case-control pairs). A total of 6 SNP septets flanking C1orf135, GSN, LARS, and ACTN4 remained significant in all 3 stages. Among them, the same septet flanking ACTN4 was also associated with blood pressure traits in the Hong Kong Hypertension Study (HKHS) and in the Wellcome Trust Case-Control Consortium Hypertension Study (WTCCCHS). LARS was detected in the HKHS, but not in the WTCCCHS. GSN may be specific to Taiwanese individuals because it was not found by either the HKHS or the WTCCCHS. CONCLUSIONS: Our study identified 4 previously unknown YOH loci in Han Chinese. Identification of these genes enriches the hypertension susceptibility gene list, thereby shedding light on the etiology of hypertension in Han Chinese.