A 27-bp deletion from one allele of the type III collagen gene (COL3A1) in a large family with Ehlers-Danlos syndrome type IV

Summary A large family with Ehlers-Danlos syndrome type IV (EDS IV) has previously been described. Unlike most cases of EDS IV, fibroblasts from affected members secreted near normal amounts of type III collagen. We have localised the mutation in this family to the CB5 peptide of type III collagen, by using both protein and cDNA mapping techniques. Sequence analysis of cDNA revealed a 27-bp deletion within exon 37, a deletion that removed nine amino acids and maintained the Gly-X-Y repeat of the collagen helix. Further sequencing of genomic DNA confirmed its location, and amplification of DNA from family members showed that it was absent in unaffected individuals but present in all the affected individuals tested. This deletion is flanked by two short direct repeats of CTCC; it may have arisen by slipped mispairing, and has subsequently been transmitted to all affected family members.     

Effect of knee musculature on anterior cruciate ligament strain in vivo

Squatting is a commonly prescribed exercise following reconstruction of the anterior cruciate ligament (ACL). The objective of this paper was to measure the in vivo strain patterns of the normal ACL and the load at the knee for the simple squat and for squatting with a “sport cord”. A sport cord is a large elastic rubber tube used for added resistance. Strain patterns were deduced using displacement data from a Hall Effect Strain Transducer (HEST), while joint loads were determined by a mathematical model with inputs from a force plate and electrogoniometers. ACL strain for the free squat in one subject had a maximum of 2% at a knee angle of 10° and was slack for knee angles >17°. In squatting with a sport cord, peak strain was 1% at 10° and was slack at knee angles >14°. Since these peak strains are low, squatting appears to be a safe exercise for conservative rehabilitation of ACL reconstruction patients. In addition, the sport cord is a recommended augmentation to the activity. We believe that the decrease in strain with the sport cord results from added joint stiffness due to greater compressive forces at the tibiofemoral joint. This greater compressive force results from the approximately 10% increase in quadriceps activity. From shear force data predicted by the mathematical model, the maximum anterior drawer force for free squatting (50 N) was considerably less than for sport cord squatting (430 N). Therefore, the value of shear force at the tibiofemoral joint only partially determines the load placed on the ACL.     

Substitution of aspartate for glycine 1018 in the type III procollagen (COL3A1) gene causes type IV Ehlers-Danlos syndrome: the mutated allele is present in most blood leukocytes of the asymptomatic and mosaic mother.

A proband with arterial ruptures and skin changes characteristic of the type IV variant of Ehlers-Danlos syndrome was found to have a single-base mutation in the type III procollagen gene, which converted the codon for glycine at amino acid position 1018 to a codon for aspartate. (Amino acid positions are numbered by the standard convention in which the first glycine of the triple-helical domain of an alpha chain is number 1. The numbers of positions in the alpha 1(III) chains can be converted to positions in the human pro alpha(III) chain by adding 167.) Nucleotide sequencing of overlapping PCR products in which the two alleles were distinguished demonstrated that the mutation of glycine 1018 was the only mutation that changed the primary structure of type III procollagen. The glycine substitution markedly decreased the amount of type III procollagen secreted into the medium by cultured skin fibroblasts from the proband. It is surprising that the same mutation was found in about 94% of the peripheral blood leukocytes from the proband's asymptomatic 72-year-old mother. Other tissues from the mother contained the mutated allele; it was present in 0%-100% of different samples of hair cells and in about 40% of cells from the oral epithelium. Therefore, the mother was a mosaic for the mutation. Since the mutated allele was present in cells derived from all three germ layers, the results indicated that the mutation arose by the late blastocyst stage of development. The results also indicate that assays of blood leukocytes do not always reveal mosaicism or predict phenotypic involvement of tissues, such as blood vessels, that are derived from the same embryonic cells as are leukocytes.     

Reducing bias in bacterial community analysis of lower respiratory infections

High-throughput pyrosequencing and quantitative PCR (Q-PCR) analysis offer greatly improved accuracy and depth of characterisation of lower respiratory infections. However, such approaches suffer from an inability to distinguish between DNA derived from viable and non-viable bacteria. This discrimination represents an important step in characterising microbial communities, particularly in contexts with poor clearance of material or high antimicrobial stress, as non-viable bacteria and extracellular DNA can contribute significantly to analyses. Pre-treatment of samples with propidium monoazide (PMA) is an effective approach to non-viable cell exclusion (NVCE). However, the impact of NVCE on microbial community characteristics (abundance, diversity, composition and structure) is not known. Here, adult cystic fibrosis (CF) sputum samples were used as a paradigm. The effects of PMA treatment on CF sputum bacterial community characteristics, as analysed by pyrosequencing and enumeration by species-specific (Pseudomonas aeruginosa) and total bacterial Q-PCR, were assessed. At the local community level, abundances of both total bacteria and of P. aeruginosa were significantly lower in PMA-treated sample portions. Meta-analysis indicated no overall significant differences in diversity; however, PMA treatment resulted in a significant alteration in local community membership in all cases. In contrast, at the metacommunity level, PMA treatment resulted in an increase in community evenness, driven by an increase in diversity, predominately representing rare community members. Importantly, PMA treatment facilitated the detection of both recognised and emerging CF pathogens, significantly influencing ‘core'' and ‘satellite'' taxa group membership. Our findings suggest failure to implement NVCE may result in skewed bacterial community analyses.