Allele dependent silencing of COL1A2 using small interfering RNAs

Osteogenesis imperfecta (OI) is generally caused by a dominant mutation in Collagen I, encoded by the genes COL1A1 and COL1A2. To date there is no satisfactory therapy for OI, but inactivation of the mutant allele through small interfering RNAs (siRNA) is a promising approach, as siRNAs targeting each allele of a polymorphism could be used for allele-specific silencing irrespective of the location of the actual mutations. In this study we examined the allele dependent effects of several tiled siRNAs targeting a region surrounding an exonic COL1A2 T/C polymorphism (rs1800222) in heterozygous primary human bone cells. Relative abundances of COL1A2 alleles were determined by cDNA sequencing and overall COL1A2 abundance was analyzed by quantitative PCR. One of the siRNAs decreased overall COL1A2 abundance by 71% of which 75% was due to silencing of the targeted T-allele. In conclusion, allele-preferential silencing of Collagen type I genes may be a future therapeutic approach for OI.     

Genotype�Cphenotype correlations in nonlethal osteogenesis imperfecta caused by mutations in the helical domain of collagen type I

Osteogenesis imperfecta (OI) is a heritable disorder with bone fragility that is often associated with short stature, tooth abnormalities (dentinogenesis imperfecta), and blue sclera. The most common mutations associated with OI result from the substitution for glycine by another amino acid in the triple helical domain of either the α1 or the α2 chain of collagen type I. In this study, we compared the results of genotype analysis and clinical examination in 161 OI patients (median age: 13 years) who had glycine mutations in the triple helical domain of α1(I) (n=67) or α2(I) (n=94). Serine substitutions were the most frequently encountered type of mutation in both chains. Compared with patients with serine substitutions in α2(I) (n=40), patients with serine substitutions in α1(I) (n=42) on average were shorter (median height z-score −6.0 vs −3.4; P=0.005), indicating that α1(I) mutations cause a more severe phenotype. Height correlated with the location of the mutation in the α2(I) chain but not in the α1(I) chain. Patients with mutations affecting the first 120 amino acids at the amino-terminal end of the collagen type I triple helix had blue sclera but did not have dentinogenesis imperfecta. Among patients from different families sharing the same mutation, about 90 and 75% were concordant for dentinogenesis imperfecta and blue sclera, respectively. These data should be useful to predict disease phenotype in newly diagnosed OI patients.     

CIAPIN1 gene silencing enhances chemosensitivity in a drug-resistant animal model in vivo

Overexpression of cytokine-induced apoptosis inhibitor 1 (CIAPIN1) contributes to multidrug resistance (MDR) in breast cancer. This study aimed to evaluate the potential of CIAPIN1 gene silencing by RNA interference (RNAi) as a treatment for drug-resistant breast cancer and to investigate the effect of CIAPIN1 on the drug resistance of breast cancer in vivo. We used lentivirus-vector-based RNAi to knock down CIAPIN1 in nude mice bearing MDR breast cancer tumors and found that lentivirus-vector-mediated silencing of CIAPIN1 could efficiently and significantly inhibit tumor growth when combined with chemotherapy in vivo. Furthermore, Western blot analysis showed that both CIAPIN1 and P-glycoprotein expression were efficiently downregulated, and P53 was upregulated, after RNAi. Therefore, we concluded that lentivirus-vector-mediated RNAi targeting of CIAPIN1 is a potential approach to reverse MDR of breast cancer. In addition, CIAPIN1 may participate in MDR of breast cancer by regulating P-glycoprotein and P53 expression.     

Glycine to tryptophan substitution in type I collagen in a patient with OI type III: a unique collagen mutation

We report a unique glycine substitution in type I collagen and highlight the clinical and biochemical consequences. The proband is a 9 year old Turkish boy with severely deforming osteogenesis imperfecta (OI). Biochemical analysis of (pro) collagen type I from a skin fibroblast culture showed both normal and overmodified α chains. Molecular analysis showed a G>T transversion in the COL1A2 gene, resulting in the substitution of glycine by tryptophan at position 277 of the α2(I) collagen chain. Glycine substitutions in type I collagen are the most frequent cause of the severe and lethal forms of OI. The phenotypic severity varies according to the nature and localisation of the mutation. Substitutions of glycine by tryptophan, which is the most voluminous amino acid, have not yet been identified in type I collagen or any other fibrillar collagen. The severe, though non-lethal OI phenotype associated with this mutation may appear surprising in view of the huge size of the tryptophan residue. The fact that the mutation resides within a so called "non-lethal" region of the α2(I) collagen chain supports a regional model in phenotypic severity for α2(I) collagen mutations, in which the phenotype is determined primarily by the nature of the collagen domain rather than the type of glycine substitution involved.


Keywords: osteogenesis imperfecta; COL1A2; tryptophan; collagen     

Gene silencing of EXTL2 and EXTL3 as a substrate deprivation therapy for heparan sulphate storing mucopolysaccharidoses

Neurological pathology is characteristic of the mucopolysaccharidoses (MPSs) that store heparan sulphate (HS) glycosaminoglycan (gag) and has been proven to be refractory to systemic therapies. Substrate deprivation therapy (SDT) using general inhibitors of gag synthesis improves neurological function in mouse models of MPS, but is not specific to an MPS type. We have investigated RNA interference (RNAi) as a method of targeting SDT to the HS synthesising enzymes, EXTL2 and EXTL3. Multiple shRNA molecules specific to EXTL2 or EXTL3 were designed and validated in a reporter gene assay, with four out of six shRNA constructs reducing expression by over 90%. The three EXTL2-specific shRNA constructs reduced endogenous target gene expression by 68, 32 and 65%, and decreased gag synthesis by 46, 50 and 27%. One EXTL3-specific shRNA construct reduced endogenous target gene expression by 14% and gag synthesis by 39%. Lysosomal gag levels in MPS IIIA and MPS I fibroblasts were also reduced by EXTL2 and EXTL3-specific shRNA. Incorporation of shRNAs into a lentiviral expression system reduced gene expression, and one EXTL2-specific shRNA reduced gag synthesis. These results indicate that deprivation therapy through shRNA-mediated RNAi has potential as a therapy for HS-storing MPSs.     

Evidence for autosomal recessive inheritance in SPG3A caused by homozygosity for a novel ATL1 missense mutation

Hereditary spastic paraplegias (HSPs) comprise a heterogeneous group of disorders characterized by progressive spasticity and weakness of the lower limbs. Autosomal dominant and ‘pure'' forms of HSP account for ∼80% of cases in Western societies of whom 10% carry atlastin-1 (ATL1) gene mutations. We report on a large consanguineous family segregating six members with early onset HSP. The pedigree was compatible with both autosomal dominant and autosomal recessive inheritance. Whole-exome sequencing and segregation analysis revealed a homozygous novel missense variant c.353G>A, p.(Arg118Gln) in ATL1 in all six affected family members. Seven heterozygous carriers, five females and two males, showed no clinical signs of HSP with the exception of sub-clinically reduced vibration sensation in one adult female. Our combined findings show that homozygosity for the ATL1 missense variant remains the only plausible cause of HSP, whereas heterozygous carriers are asymptomatic. This apparent autosomal recessive inheritance adds to the clinical complexity of spastic paraplegia 3A and calls for caution using directed genetic screening in HSP.     

Exome sequencing reveals HINT1 mutations as a cause of distal hereditary motor neuropathy

Distal hereditary motor neuropathies (dHMNs) are a heterogenous group of genetic disorders with length-dependent degeneration of motor axons. Obtaining a genetic diagnosis in patients with dHMN remains challenging. We performed exome sequencing in a diagnostic setting in 12 patients with a clinical diagnosis of dHMN. Potential disease-causing variants in genes associated with dHMN and other forms of inherited neuropathies/motor neuron diseases were validated using Sequenom. The coverage in the genes studied was >95% with an average coverage of >50 times. In none of the patients a mutations was found in genes previously reported to be associated with dHMN. However, in 2/12 patients a recessive mutation in histidine triad nucleotide binding protein 1 (HINT1, recently discovered as a cause of axonal neuropathy with neuromyotonia) was identified. Our results demonstrate the diagnostic value of exome sequencing for patients with inherited neuropathies. The phenotypic spectrum of recessive mutations in HINT1 includes dHMN. HINT1 should be added to the list of genes to check for in dHMN.     

PGD for hereditary breast and ovarian cancer: the route to universal tests for BRCA1 and BRCA2 mutation carriers

Preimplantation Genetic Diagnosis (PGD) is a method of testing in vitro embryos as an alternative to prenatal diagnosis with possible termination of pregnancy in case of an affected child. Recently, PGD for hereditary breast and ovarian cancer caused by BRCA1 and BRCA2 mutations has found its way in specialized labs. We describe the route to universal single-cell PGD tests for carriers of BRCA1/2 mutations. Originally, mutation-specific protocols with one or two markers were set up and changed when new couples were not informative. This route of changing protocols was finalized after 2 years with universal tests for both BRCA1 and BRCA2 mutation carriers based on haplotyping of, respectively, 6 (BRCA1) and 8 (BRCA2) microsatellite markers in a multiplex PCR. Using all protocols, 30 couples had a total of 47 PGD cycles performed. Eight cycles were cancelled upon IVF treatment due to hypostimulation. Of the remaining 39 cycles, a total of 261 embryos were biopsied and a genetic diagnosis was obtained in 244 (93%). In 34 of the 39 cycles (84.6%), an embryo transfer was possible and resulted in 8 pregnancies leading to a fetal heart beat per oocyte retrieval of 20.5% and a fetal heart beat per embryonic transfer of 23.5%. The preparation time and costs for set-up and validation of tests are minimized. The informativity of microsatellite markers used in the universal PGD-PCR tests is based on CEPH and deCODE pedigrees, making the tests applicable in 90% of couples coming from these populations.     

Osteogenesis Imperfecta Type VI with Severe Bony Deformities Caused by Novel Compound Heterozygous Mutations in SERPINF1

Osteogenesis imperfecta (OI) comprises a heterogeneous group of disorders characterized by bone fragility, frequent fractures, and low bone mass. Dominantly inherited COL1A1 or COL1A2 mutations appear to be causative in the majority of OI types, but rare recessively inherited genes have also been reported. Recently, SERPINF1 has been reported as another causative gene in OI type VI. To date, only eight SERPINF1 mutations have been reported and all are homozygous. Our patient showed no abnormalities at birth, frequent fractures, osteopenia, and poor response on pamidronate therapy. At the time of her most recent evaluation, she was 8 yr old, and could not walk independently due to frequent lower-extremity fractures, resulting in severe deformity. No clinical signs were seen of hearing impairment, blue sclera, or dentinogenesis imperfecta. In this study, we describe the clinical and radiological findings of one Korean patient with novel compound heterozygous mutations (c.77dupC and c.421dupC) of SERPINF1.     

Penetrance of FMR1 premutation associated pathologies in fragile X syndrome families

Within the past few years, there has been a significant change in identifying and characterizing the FMR1 premutation associated phenotypes. The premutation has been associated with elevated FMR1 mRNA levels and slight to moderate reductions in FMRP levels. Furthermore, it has been established that ∼20% of female premutation carriers present primary ovarian insufficiency (POI) and that fragile X-associated tremor/ataxia syndrome (FXTAS) occurs in one-third of all male premutation carriers older than 50 years. Besides POI and FXTAS, new disorders have recently been described among individuals (especially females) with the FMR1 premutation. Those pathologies include thyroid disease, hypertension, seizures, peripheral neuropathy, and fibromyalgia. However there are few reports related to FXTAS penetrance among female premutation carriers or regarding these disorders recently associated to the FMR1 premutation. Therefore, we have evaluated 398 fragile X syndrome (FXS) families in an attempt to provide an estimation of the premutation associated phenotypes penetrance. Our results show that signs of FXTAS are detected in 16.5% of female premutation carriers and in 45.5% of premutated males older than 50 years. Furthermore, among females with the FMR1 premutation, penetrance of POI, thyroid disease and chronic muscle pain is 18.6, 15.9 and 24.4%, respectively. The knowledge of this data might be useful for accurate genetic counselling as well as for a better characterization of the clinical phenotypes of FMR1 premutation carriers.     

High frequency of COH1 intragenic deletions and duplications detected by MLPA in patients with Cohen syndrome

Cohen syndrome is a rare, clinically variable autosomal recessive disorder characterized by mental retardation, postnatal microcephaly, facial dysmorphisms, ocular abnormalities and intermittent neutropenia. Mutations in the COH1 gene have been found in patients from different ethnic origins. However, a high percentage of patients have only one or no mutated allele. To investigate whether COH1 copy number changes account for missed mutations, we used multiplex ligation-dependent probe amplification (MLPA) to test a group of 14 patients with Cohen syndrome. This analysis has allowed us to identify multi-exonic deletions in 11 alleles and duplications in 4 alleles. Considering our previous study, COH1 copy number variations represent 42% of total mutated alleles. To our knowledge, COH1 intragenic duplications have never been reported in Cohen syndrome. The three duplications encompassed exons 4–13, 20–30 and 57–60, respectively. Interestingly, four deletions showed the same exon coverage (exons 6–16) with respect to a deletion recently reported in a large Greek consanguineous family. Haplotype analysis suggested a possible founder effect in the Mediterranean basin. The use of MLPA was therefore crucial in identifying mutated alleles undetected by traditional techniques and in defining the extent of the deletions/duplications. Given the high percentage of identified copy number variations, we suggest that this technique could be used as the initial screening method for molecular diagnosis of Cohen syndrome.     

Extended extraocular phenotype of PROM1 mutation in kindreds with known autosomal dominant macular dystrophy

Mutations in prominin 1 (PROM1) have been shown to result in retinitis pigmentosa, macular degeneration and cone-rod dystrophy. Because of the putative role of PROM1 in hippocampal neurogenesis, we examined two kindreds with the same R373C PROM1 missense mutation using our established paradigm to study brain structure and function. As the protein encoded by PROM1, known as CD133, is used to identify stem/progenitor cells that can be found in peripheral blood and reflect endothelial reparatory mechanisms, other parameters were subsequently examined that included measures of vascular function, endothelial function and angiogenic capacity. We found that aspects of endothelial function assayed ex vivo were abnormal in patients with the R373C PROM1 mutation, with impaired adhesion capacity and higher levels of cellular damage. We also noted renal infections, haematuria and recurrent miscarriages possibly reflecting consequences of abnormal tubular modelling. Further studies are needed to confirm these findings.     

Impaired osteoblastogenesis in a murine model of dominant osteogenesis imperfecta: a new target for osteogenesis imperfecta pharmacological therapy

The molecular basis underlying the clinical phenotype in bone diseases is customarily associated with abnormal extracellular matrix structure and/or properties. More recently, cellular malfunction has been identified as a concomitant causative factor and increased attention has focused on stem cells differentiation. Classic osteogenesis imperfecta (OI) is a prototype for heritable bone dysplasias: it has dominant genetic transmission and is caused by mutations in the genes coding for collagen I, the most abundant protein in bone. Using the Brtl mouse, a well-characterized knockin model for moderately severe dominant OI, we demonstrated an impairment in the differentiation of bone marrow progenitor cells toward osteoblasts. In mutant mesenchymal stem cells (MSCs), the expression of early (Runx2 and Sp7) and late (Col1a1 and Ibsp) osteoblastic markers was significantly reduced with respect to wild type (WT). Conversely, mutant MSCs generated more colony-forming unit-adipocytes compared to WT, with more adipocytes per colony, and increased number and size of triglyceride drops per cell. Autophagy upregulation was also demonstrated in mutant adult MSCs differentiating toward osteogenic lineage as consequence of endoplasmic reticulum stress due to mutant collagen retention. Treatment of the Brtl mice with the proteasome inhibitor Bortezomib ameliorated both osteoblast differentiation in vitro and bone properties in vivo as demonstrated by colony-forming unit-osteoblasts assay and peripheral quantitative computed tomography analysis on long bones, respectively. This is the first report of impaired MSC differentiation to osteoblasts in OI, and it identifies a new potential target for the pharmacological treatment of the disorder.     

Association of abcb1 polymorphisms and ulcerative colitis susceptibility

Objective: Multi-drug resistance gene 1 (ABCB1) is closely related to bowel diseases. Therefore, our study was aimed to evaluate the correlation between ABCB1 polymorphisms (C1236T and C3435T) and ulcerative colitis (UC) susceptibility. Methods: A total of 61 UC patients and 64 healthy people participated in the study. Genotyping was conducted with the method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). χ² test was used to evaluate the association of ABCB1 gene polymorphisms (C1236T and C3435T) and UC susceptibility. Results: For ABCB1 C1236T polymorphism, the frequencies of CC genotype and C allele were found higher in the cases than those in the controls (CC: 36.1% vs. 20.3%; C: 58.2% vs. 41.4%), which indicated that the CC genotype and C allele might increase the risk for UC (OR = 3.39, 95% CI = 1.28-8.97; OR = 1.97, 95% CI = 1.19-3.26). However, there were no statistical differences in the genotype or allele distribution of ABCB1 C3435T between the case and control group. Conclusion: The CC genotype and C allele of ABCB1 C1236T polymorphisms are significantly associated with UC susceptibility, so we conclude that ABCB1 C1236T polymorphisms might serve as genetic-susceptibility factors for UC. While, no remarkable relationship is observed between ABCB1 C3435T polymorphisms and UC.     

A genome-wide shRNA screen identifies GAS1 as a novel melanoma metastasis suppressor gene

Metastasis suppressor genes inhibit one or more steps required for metastasis without affecting primary tumor formation. Due to the complexity of the metastatic process, the development of experimental approaches for identifying genes involved in metastasis prevention has been challenging. Here we describe a genome-wide RNAi screening strategy to identify candidate metastasis suppressor genes. Following expression in weakly metastatic B16-F0 mouse melanoma cells, shRNAs were selected based upon enhanced satellite colony formation in a three-dimensional cell culture system and confirmed in a mouse experimental metastasis assay. Using this approach we discovered 22 genes whose knockdown increased metastasis without affecting primary tumor growth. We focused on one of these genes, Gas1 (Growth arrest-specific 1), because we found that it was substantially down-regulated in highly metastatic B16-F10 melanoma cells, which contributed to the high metastatic potential of this mouse cell line. We further demonstrated that Gas1 has all the expected properties of a melanoma tumor suppressor including: suppression of metastasis in a spontaneous metastasis assay, promotion of apoptosis following dissemination of cells to secondary sites, and frequent down-regulation in human melanoma metastasis-derived cell lines and metastatic tumor samples. Thus, we developed a genome-wide shRNA screening strategy that enables the discovery of new metastasis suppressor genes.     

Rapid degradation of an active formylglycine generating enzyme variant leads to a late infantile severe form of multiple sulfatase deficiency

Multiple sulfatase deficiency (MSD) is a rare inborn error of metabolism affecting posttranslational activation of sulfatases by the formylglycine generating enzyme (FGE). Due to mutations in the encoding SUMF1 gene, FGE''s catalytic capacity is impaired resulting in reduced cellular sulfatase activities. Both, FGE protein stability and residual activity determine disease severity and have previously been correlated with the clinical MSD phenotype. Here, we report a patient with a late infantile severe course of disease. The patient is compound heterozygous for two so far undescribed SUMF1 mutations, c.156delC (p.C52fsX57) and c.390A>T (p.E130D). In patient fibroblasts, mRNA of the frameshift allele is undetectable. In contrast, the allele encoding FGE-E130D is expressed. FGE-E130D correctly localizes to the endoplasmic reticulum and has a very high residual molecular activity in vitro (55% of wildtype FGE); however, it is rapidly degraded. Thus, despite substantial residual enzyme activity, protein instability determines disease severity, which highlights that potential MSD treatment approaches should target protein folding and stabilization mechanisms.