5'UTR mutations of ENG cause hereditary hemorrhagic telangiectasia

<p>Abstract</p> <p>Background</p> <p>Hereditary hemorrhagic telangiectasia (HHT) is a vascular disorder characterized by epistaxis, arteriovenous malformations, and telangiectases. The majority of the patients have a mutation in the coding region of the activin A receptor type II-like 1 (<it>ACVRL1</it>) or Endoglin (<it>ENG</it>) gene. However, in approximately 15% of cases, sequencing analysis and deletion/duplication testing fail to identify mutations in the coding regions of these genes. Knowing its vital role in transcription and translation control, we were prompted to investigate the 5'untranslated region (UTR) of <it>ENG</it>.</p> <p>Methods and Results</p> <p>We sequenced the 5'UTR of <it>ENG </it>for 154 HHT patients without mutations in <it>ENG </it>or <it>ACVRL1 </it>coding regions. We found a mutation (c.-127C > T), which is predicted to affect translation initiation and alter the reading frame of endoglin. This mutation was found in a family with linkage to the <it>ENG</it>, as well as in three other patients, one of which had an affected sibling with the same mutation. <it>In vitro </it>expression studies showed that a construct with the c.-127C > T mutation alters the translation and decreases the level of the endoglin protein. In addition, a c.-9G > A mutation was found in three patients, one of whom was homozygous for this mutation. Expression studies showed decreased protein levels suggesting that the c.-9G > A is a hypomorphic mutation.</p> <p>Conclusions</p> <p>Our results emphasize the need for the inclusion of the 5'UTR region of <it>ENG </it>in clinical testing for HHT.</p

Infection-dependent phenotypes in MHC-congenic mice are not due to MHC: can we trust congenic animals?

BACKGROUND: Congenic strains of mice are assumed to differ only at a single gene or region of the genome. These mice have great importance in evaluating the function of genes. However, their utility depends on the maintenance of this true congenic nature. Although, accumulating evidence suggests that congenic strains suffer genetic divergence that could compromise interpretation of experimental results, this problem is usually ignored. During coinfection studies with Salmonella typhimurium and Theiler's murine encephalomyelitis virus (TMEV) in major histocompatibility complex (MHC)-congenic mice, we conducted the proper F(2 )controls and discovered significant differences between these F(2 )animals and MHC-genotype-matched P(0 )and F(1 )animals in weight gain and pathogen load. To systematically evaluate the apparent non-MHC differences in these mice, we infected all three generations (P(0), F(1 )and F(2)) for 5 MHC genotypes (b/b, b/q and q/q as well as d/d, d/q, and q/q) with Salmonella and TMEV. RESULTS: Infected P(0 )MHC q/q congenic homozygotes lost significantly more weight (p = 0.02) and had significantly higher Salmonella (p < 0.01) and TMEV (p = 0.02) titers than the infected F(2 )q/q homozygotes. Neither weight nor pathogen load differences were present in sham-infected controls. CONCLUSIONS: These data suggest that these strains differ for genes other than those in the MHC congenic region. The most likely explanation is that deleterious recessive mutations affecting response to infection have accumulated in the more than 40 years that this B10.Q-H-2(q )MHC-congenic strain has been separated from its B10-H-2(b )parental strain. During typical experiments with congenic strains, the phenotypes of these accumulated mutations will be falsely ascribed to the congenic gene(s). This problem likely affects any strains separated for appreciable time and while usually ignored, can be avoided with the use of F(2 )segregants

Major Histocompatibility Complex Heterozygosity Reduces Fitness in Experimentally Infected Mice

It is often suggested that heterozygosity at major histocompatibility complex (MHC) loci confers enhanced resistance to infectious diseases (heterozygote advantage, HA, hypothesis), and overdominant selection should contribute to the evolution of these highly polymorphic genes. The evidence for the HA hypothesis is mixed and mainly from laboratory studies on inbred congenic mice, leaving the importance of MHC heterozygosity for natural populations unclear. We tested the HA hypothesis by infecting mice, produced by crossbreeding congenic C57BL/10 with wild ones, with different strains of Salmonella, both in laboratory and in large population enclosures. In the laboratory, we found that MHC influenced resistance, despite interacting wild-derived background loci. Surprisingly, resistance was mostly recessive rather than dominant, unlike in most inbred mouse strains, and it was never overdominant. In the enclosures, heterozygotes did not show better resistance, survival, or reproductive success compared to homozygotes. On the contrary, infected heterozygous females produced significantly fewer pups than homozygotes. Our results show that MHC effects are not masked on an outbred genetic background, and that MHC heterozygosity provides no immunological benefits when resistance is recessive, and can actually reduce fitness. These findings challenge the HA hypothesis and emphasize the need for studies on wild, genetically diverse species

Unlabeled Oligonucleotides as Internal Temperature Controls for Genotyping by Amplicon Melting

Amplicon melting is a closed-tube method for genotyping that does not require probes, real-time analysis, or allele-specific polymerase chain reaction. However, correct differentiation of homozygous mutant and wild-type samples by melting temperature (Tm) requires high-resolution melting and closely controlled reaction conditions. When three different DNA extraction methods were used to isolate DNA from whole blood, amplicon Tm differences of 0.03 to 0.39°C attributable to the extractions were observed. To correct for solution chemistry differences between samples, complementary unlabeled oligonucleotides were included as internal temperature controls to shift and scale the temperature axis of derivative melting plots. This adjustment was applied to a duplex amplicon melting assay for the methylenetetrahydrofolate reductase variants 1298A>C and 677C>T. High- and low-temperature controls bracketing the amplicon melting region decreased the Tm SD within homozygous genotypes by 47 to 82%. The amplicon melting assay was 100% concordant to an adjacent hybridization probe (HybProbe) melting assay when temperature controls were included, whereas a 3% error rate was observed without temperature correction. In conclusion, internal temperature controls increase the accuracy of genotyping by high-resolution amplicon melting and should also improve results on lower resolution instruments