Mutation analysis in Bardet-Biedl syndrome by DNA pooling and massively parallel resequencing in 105 individuals

Bardet–Biedl syndrome (BBS) is a rare, primarily autosomal-recessive ciliopathy. The phenotype of this pleiotropic disease includes retinitis pigmentosa, postaxial polydactyly, truncal obesity, learning disabilities, hypogonadism and renal anomalies, among others. To date, mutations in 15 genes (BBS1–BBS14, SDCCAG8) have been described to cause BBS. The broad genetic locus heterogeneity renders mutation screening time-consuming and expensive. We applied a strategy of DNA pooling and subsequent massively parallel resequencing (MPR) to screen individuals affected with BBS from 105 families for mutations in 12 known BBS genes. DNA was pooled in 5 pools of 21 individuals each. All 132 coding exons of BBS1–BBS12 were amplified by conventional PCR. Subsequent MPR was performed on an Illumina Genome Analyzer II(™) platform. Following mutation identification, the mutation carrier was assigned by CEL I endonuclease heteroduplex screening and confirmed by Sanger sequencing. In 29 out of 105 individuals (28%), both mutated alleles were identified in 10 different BBS genes. A total of 35 different disease-causing mutations were confirmed, of which 18 mutations were novel. In 12 additional families, a total of 12 different single heterozygous changes of uncertain pathogenicity were found. Thus, DNA pooling combined with MPR offers a valuable strategy for mutation analysis of large patient cohorts, especially in genetically heterogeneous diseases such as BBS

Indentifying genetic determinants of colorectal cancer susceptibility in mice

Inbred mice differ with respect to colitis-associated colorectal cancer (CA-CRC) susceptibility due to inherited genetic factors. However, little is known with respect to the precise genetic variants underlying this susceptibility. We hypothesize, that similar to other complex traits, these genetic effects can be fixed by inbreeding and mapped by linkage analysis in informative mouse populations generated between CA-CRC susceptible and resistant mice. To identify novel CA-CRC susceptible and resistant strains for gene mapping, we tested 11 common inbred mouse strains for azoxymethane/dextran sulfate sodium (AOM/DSS)-induced CA-CRC, identifying a bimodal distribution pattern with respect to tumor multiplicity 12.5 weeks post-treatment initiation. This bimodal distribution pattern was not observed with respect to tumor surface area in these inbred strains suggesting that different events may regulate tumor initiation and proliferation. Re-testing of A/J and FVB/NJ CA-CRC susceptible mice at early time points in tumor development identified a unique hyper-susceptibility phenotype with FVB/NJ mice developing 5.5-fold more tumors than A/J, suggesting that CA-CRC susceptibility may be under differential genetic control in these two strains. Subsequent gene mapping identified a novel two-locus system involving mouse chromosome 9 (colon cancer susceptibility 4, Ccs4) and 14 that regulates tumor multiplicity in A/J. CA-CRC hyper-susceptibility is under distinct genetic control (Ccs6, chr 6) in FVB/NJ. These are amongst the first CA-CRC loci identified in mice and highlight an important role for genetic interactions in CA-CRC pathogenesis. Further experiments to characterize the Ccs4 locus in recombinant congenic mice (mixed A/J and B6 background) identified the AcB60 mouse strain as hyper-susceptible with AcB60 mice developing 28-fold more tumors than either of its A/J or B6 parental strains at 7-weeks post-initiation of treatment. This was also associated with increased colitis following both acute and chronic DSS treatments. Murine models of early-onset CA-CRC are rare and therefore both AcB60 and FVB/NJ may offer unique models to the study the mechanisms associated with early-onset CA-CRC possibly leading to new genetic predictors of risk and treatment options in humans.La pré-disposition au développement du cancer colorectal associé à la colite (CA-CRC) est différente chez les lignée des souris consanguines en raison de l'expression de certains facteurs génétiques intrinsèques. Présentement, peu de ces facteurs ont été identifiés. Nous avons donc émis l'hypothèse que, tel que défini dans plusieurs autres maladies complexes, des différences géniques pourraient être révélées par croisement de souris, et les gènes responsables pourraient donc être identifiés par une approche de cartographie génique de populations de souris soit susceptibles ou résistantes au CA-CRC. Nous avons ainsi évalué la pré-disposition au CA-CRC de 11 lignées de souris consanguines en induisant la formation de tumeurs suite à l'administration d'azoxyméthane/ dextran sodium sulfate (AOM/DSS). Le nombre de tumeurs se développant à 12.5 semaines de traitement se répartissait selon une distribution bimodale, ce qui n'était pas par contre le cas pour le volume des tumeurs, suggérant que l’initiation et la prolifération tumorales soient régulées par des évènements génétiques distincts. L'évaluation de la pré-disposition des souris A/J et FVB/NJ au CA-CRC a permis d’identifier une hyper-susceptibilité unique chez les souris FVB/NJ développant 5.5-fois plus de tumeurs que les souris A/J à 8 semaines de traitement: la pré-disposition au CA-CRC est donc contrôlée différemment au niveau génétique dans ces deux lignées. L’analyse par cartographie génique a démontré que la susceptibilité les souris A/J est régulée par un système interactif de deux loci définis sur le chromosome 9 (colon cancer susceptibility 4, Ccs4) et le chromosome 14 respectivement. Chez la souris FVB/NJ, la pré-disposition est sous contrôle d'un nouveau locus Ccs6 défini sur le chromosome 6. Ces deux loci représentent les premiers loci identifiés responsables du développement du CA-CRC et établissent le rôle important que des interactions génétiques jouent dans le développement du CA-CRC. Nous avons aussi défini le locus Ccs4 dans des lignées de souris recombinantes congéniques (RCS, générées par croisement de souris A/J et B6), et identifié la souris AcB60 comme un modèle d'hyper-susceptibilité au CA-CRC. Cette souris développe 28-fois plus de tumeurs suite à 7 semaines de traitement que les souris parentales A/J ou B6. Des traitements chroniques et aigus au DSS ont permis de constater le développement d'une colite sévère chez les souris AcB60. Les lignées AcB60 et FVB/NJ représentent donc des modèles extrêmement utiles permettant de découvrir les mécanismes moléculaires responsables du développement du CA-CRC précoce. Elles pourront aussi servir à l'identification de facteurs pronostiques ou de cibles thérapeutiques pour les patients humains

Colitis-associated colon cancer: Is it in your genes?

Colitis-associated colorectal cancer (CA-CRC) is the cause of death in 10%-15% of inflammatory bowel disease (IBD) patients. CA-CRC results from the accumulation of mutations in intestinal epithelial cells and progresses through a well-characterized inflammation to dysplasia to carcinoma sequence. Quantitative estimates of overall CA-CRC risks are highly variable ranging from 2% to 40% depending on IBD severity, duration and location, with IBD duration being the most significant risk factor associated with CA-CRC development. Recently, studies have identified IBD patients with similar patterns of colonic inflammation, but that differ with respect to CA-CRC development, suggesting a role for additional non-inflammatory risk factors in CA-CRC development. One suggestion is that select IBD patients carry polymorphisms in various low penetrance disease susceptibility genes, which pre-dispose them to CA-CRC development, although these loci have proven difficult to identify in human genome-wide association studies. Mouse models of CA-CRC have provided a viable alternative for the discovery, validation and study of individual genes in CA-CRC pathology. In this review, we summarize the current CA-CRC literature with a strong focus on genetic pre-disposition and highlight an emerging role for mouse models in the search for CA-CRC risk alleles

Inflammation-induced tumorigenesis in mouse colon is caspase-6 independent.

Caspases play an important role in maintaining tissue homeostasis. Active Caspase-6 (Casp6) is considered a novel therapeutic target against Alzheimer disease (AD) since it is present in AD pathological brain lesions, associated with age-dependent cognitive decline, and causes age-dependent cognitive impairment in the mouse brain. However, active Casp6 is highly expressed and activated in normal human colon epithelial cells raising concerns that inhibiting Casp6 in AD may promote colon carcinogenesis. Furthermore, others have reported rare mutations of Casp6 in human colorectal cancers and an effect of Casp6 on apoptosis and metastasis of colon cancer cell lines. Here, we investigated the role of Casp6 in inflammation-associated azoxymethane/dextran sulfate sodium (AOM/DSS) colon cancer in Casp6-overexpressing and -deficient mice. In wild-type mice, AOM/DSS-induced tumors had significantly higher Casp6 mRNA, protein and activity levels compared to normal adjacent colon tissues. Increased human Casp6 or absence of Casp6 expression in mice colon epithelial cells did not change colonic tumor multiplicity, burden or distribution. Nevertheless, the incidence of hyperplasia was slightly reduced in human Casp6-overexpressing colons and increased in Casp6 null colons. Overexpression of Casp6 did not affect the grade of the tumors while all tumors in heterozygous or homozygous Casp6 null colons were high grade compared to only 50% high grade in wild-type mice. Casp6 levels did not alter cellular proliferation and apoptosis. These results suggest that Casp6 is unlikely to be involved in colitis-associated tumors

Casp6 levels are increased in AOM/DSS-induced colon tumors.

<p>(A) Schematic overview of the inflammation-induced cancer model. Age and sex-matched mice were injected with Azoxymethane (AOM) intraperitoneally at a dose of 6 mg/kg body weight. After one week, mice were treated with 2% DSS in the drinking water for 5 days, then followed by 16 days of regular water. This cycle was repeated once. Mice were sacrificed wither 12 or 20 weeks post AOM injection. (B) Casp6 mRNA was assessed by qRT-PCR on isolated RNA from tumors (T) and adjacent normal appearing tissues (N). HPRT was used as a loading control (n = 5). Statistical significance was assessed with a paired t-test. * p<0.05. (C) Casp6 protein expression in colon tissue extracts of WT mice was measured by western blot analysis with actin as a loading control (n = 3). (D) Specific VEIDase Casp6 activity in extracts from WT colons (n = 3). Statistical significance was assessed with a paired t-test. ** p<0.01. (E) Cleavage of lamin A by activated Casp6 was also tested using anti-lamin AΔCasp6 antibody and anti-lamin A+C used as equal loading control in tumors and normal appearing tissue from WT and KO Casp6 mice.</p

Susceptibility of Casp6 overexpressing mice to AOM-DSS treatment.

<p>(<b>A</b>) RT-PCR of human Casp6 and Cre recombinase in colons of KI and Cre mice. (B) Western blot of human Casp6 protein and active subunits in mice colon proteins. Cleavage of lamin A by activated Casp6 was also tested using anti-lamin AΔCasp6 antibody and anti-lamin A+C used as equal loading control. (C) Immunohistochemistry of WT/WT, KI/WT, WT/Cre and KI/Cre mice colons with anti-p20 active Casp6 neoepitope antisera. (D) Percent weight change measured weekly during AOM-DSS treatment. The black boxes represent the period of DSS treatment. (E) Human Casp6 mRNA levels were assessed by qRT-PCR in tumors and adjacent normal appearing tissue from WT/WT, WT/Cre, KI/WT and KI/Cre mice colons. HPRT was used as a loading control (n = 3). One-way ANOVA followed by Tukey-Kramer post hoc analysis was performed to determine statistical significance. * p<0.05, ** p<0.01 comparing with Normal KI/Cre and ### p<0.001 comparing with Tumor KI/Cre. (F) Specific VEIDase Casp6 activity for extracts from WT/WT, WT/Cre, KI/WT and KI/Cre colons (n = 3). One-way ANOVA followed by Tukey-Kramer post hoc analysis was performed to determine statistical significance. * p<0.05 compares normal versus tumor tissues and # p<0.05 compares with tumors from KI/Cre. (G) Western blot analyses for pro-Casp6 and active p20 subunit of Casp6 in colon protein extracts from WT/WT, WT/Cre, KI/WT and KI/Cre mice. (H) Number of hyperplasia per colon induced by AOM-DSS treatment 12 weeks after AOM injection. (I) Number of tumors per colon induced by AOM-DSS treatment 12 weeks after AOM injection. (J) Tumor load per mouse in mice after AOM/DSS treatment. (K) Number of tumors per mouse located in proximal, middle or distal part of colons. (H-K) Statistics were performed using one way ANOVA followed by Tukey-Kramer post hoc analysis.</p

Histological grades of AOM/DSS-induced colon tumors in KI/Cre (A) and KO (B) mice.

<p>Abbreviations: LGD, adenoma with low-grade dysplasia; HGD, adenoma with high-grade dysplasia.</p><p>Histological grades of AOM/DSS-induced colon tumors in KI/Cre (A) and KO (B) mice.</p