Detection and characterisation of human astroviruses in children with acute gastroenteritis in Blantyre,Malawi

In a 2-year hospital-based study of paediatric gastroenteritis in Blantyre, Malawi, astroviruses were detected by enzyme immunoassay in 15 (1.9%) of 786 inpatients and in 9 (2.3%) of 400 outpatients. Greater disease severity was noted in children coinfected with human immunodeficiency virus (HIV). Six human astrovirus (HAstV) genotypes were identified, including HAstV-1 (25%), HAstV-2 (21%), HAstV-3 (25%), HAstV-4 (13%), HAstV-5 (4%), and HAstV-8 (13%). Although astroviruses are not major causes of gastroenteritis among children admitted to hospital in Blantyre, concomitant HIV infection appears to be a risk factor for increased severity of disease.     

Autosomal glycogenosis of liver and muscle due to phosphorylase kinase deficiency is caused by mutations in the phosphorylase kinase beta subunit (PHKB)

Glycogen storage disease due to phosphorylase kinase deficiency occurs in several variants that differ in mode of inheritance and tissue- specificity. This heterogeneity is suspected to be largely due to mutations affecting different subunits and isoforms of phosphorylase kinase. The gene of the ubiquitously expressed beta subunit, PHKB, was a candidate for involvement in autosomally transmitted phosphorylase kinase deficiency of liver and muscle. To identify such mutations, the complete PHKB coding sequence was amplified by RT-PCR of RNA isolated from blood samples of patients and analyzed by direct sequencing of PCR products. The characterization of mutations was complemented by PCR of genomic DNA. In one female and four male patients, we identified five independent nonsense mutations (Y418ter; R428ter; Y974H+E975ter; Q656ter in two cases), one single-base insertion in codon N421, one splice-site mutation affecting exon 31, and a large deletion involving the loss of exon 8. Although these severe translation-disrupting mutations occur in constitutively expressed sequences of the only known beta subunit gene of phosphorylase kinase, PHKB, they are associated with a surprisingly mild clinical phenotype, affecting virtually only the liver, and relatively high residual enzyme activity of approximately 10%.      

Use of stereolithography to manufacture critical-sized 3D biodegradable scaffolds for bone ingrowth

A novel approach to the manufacture of biodegradable polymeric scaffolds for tissue-engineering utilizing stereolithography (SLA) is presented. SLA is a three-dimensional (3D) printing method that uses an ultraviolet laser to photo-crosslink a liquid polymer substrate. The current generation of SLA devices provide a 3D printing resolution of 0.1 mm. The experiments utilized a biodegradable resin mixture of diethyl fumarate (DEF), poly(propylene fumarate) (PPF), and a photoinitiator, bisacylphosphine oxide (BAPO). The PPF is crosslinked with the use of the SLA's UV laser (325-nm wavelength). An SLA device was retrofitted with a custom fixture build tank enclosing an elevator-driven build table. A 3D prototype model testing the manufacturing control this device provides was created in a computer-aided-design package. The resulting geometric data were used to drive the SLA process, and a DEF/PPF prototype part was successfully manufactured. These scaffolds have application in the tissue engineering of bony substrates.     

Somatic mutation processes at a human minisatellite

Germline instability at human minisatellites frequently involves complex inter-allelic transfers of repeat units usually restricted to one end of the repeat array and apparently regulated by flanking DNA. In contrast, nothing is known about the structural basis of somatic instability at minisatellites. An electrophoretic size-enrichment strategy was therefore developed at minisatellite MS32 (D1S8) to enable rare abnormal-length mutants to be detected, validated and quantitated in blood DNA by single molecule PCR. Structural analysis of rare mutant alleles in blood revealed simple deletions/duplications of repeat unit blocks located at random along the tandem repeat array, a mode of mutation completely different from that seen in sperm. Furthermore, allele-specific suppression of sperm instability at MS32 did not affect somatic instability. These data suggest that conversion-based minisatellite mutation in sperm is completely germline-specific and most likely meiotic in origin. Somatic instability appears to occur by a separate pathway involving replication slippage or, more likely, intra-allelic unequal crossing over.      

Mutations in the TSC2 gene: analysis of the complete coding sequence using the protein truncation test (PTT)

Mutations in the TSC2 gene on chromosome 16p13.3 are responsible for approximately 50% of familial tuberous sclerosis (TSC). The gene has 41 small exons spanning 45 kb of genomic DNA and encoding a 5.5 kb mRNA. Large germline deletions of TSC2 occur in <5% of cases, and a number of small intragenic mutations have been described. We analysed mRNA from 18 unrelated cases of TSC for TSC2 mutations using the protein truncation test (PTT). Three cases were predicted to be TSC2 mutations on the basis of linkage analysis or because a hamartoma from the patient showed loss of heterozygosity for 16p13.3 markers. Three overlapping PCR products, covering the complete coding sequence of mRNA, were generated from lymphoblastoid cell lines, translated into 35S-methionine labelled protein, and analysed by SDS-PAGE. PCR products showing PTT shifts were directly sequenced, and mutations confirmed by restriction enzyme digestion where possible. Six PTT shifts were identified. Five of these were caused by mutations predicted to produce a truncated protein: (i) a sporadic case showed a 32 bp deletion in exon 11, and a mutant mRNA without exon 11 was produced; the normal exon 10 was also spliced out; (ii) a sporadic case had a 1 bp deletion in exon 12 (1634delT); (iii) a TSC2-linked mother and daughter pair had a G-->T transversion in exon 23 (G2715T) introducing a cryptic splice site causing a 29 bp truncation of mRNA from exon 23; (iv) a sporadic case showed a 2 bp deletion in exon 36; (v) a sporadic case showed a 1 bp insertion disrupting the donor splice site of exon 37 (5007+2insA), resulting in the use of an upstream exonic cryptic splice site to cause a 29 bp truncation of mRNA from exon 37. In one case, the PTT shift was explained by in-frame splicing out of exon 10, in the presence of a normal exon 10 genomic sequence. Alternative splicing of exon 10 of the TSC2 gene may be a normal variant. Three 3rd base substitution polymorphisms were also detected during direct sequencing of PCR products. Confirmed mutations were identified in 28% of the families studied and on the assumption that half of the sporadic cases should have TSC2 mutations, a crude estimate of the detection rate would be 60%. This compares favourably with other screening methods used for TSC2, notably SSCP, and since PTT involves much less work it may be the method of choice.      

Genetic linkage analysis identifies new proximal and distal flanking markers for the X-linked agammaglobulinemia gene locus, refining its localization in Xq22

Genetic linkage analysis has been instrumental in mapping thegene for X-linked agammaglobulinemia (XLA) to the proximal longarm of the human X chromosome, to Xq22. Due to the relativerarity of this disease the localization of the gene within Xq22has remained imprecise. We have investigated twenty-nine familiesaffected by XLA and have found no recombinants with the DXS178locus in over 30 informative meioses. DXS178 is now the mostreliable and informative locus for use in pre-natal diagnosisand carrier detection of XLA. In addition, we have identifiednew closely linked proximal and distal flanking markers forXLA, DXS442 and DXS101, respectively. These loci are separatedby 2cM, considerably reducing the extent of DNA within whichthe XLA locus can be contained. This will open up the way formore directed positional cloning efforts for the isolation ofthe XLA gene.     

Detecting pre-ovulatory luteinizing hormone surges in urine

The study objectives were to determine (i) if pre-ovulatory luteinizing hormone (LH) surges, undetected in urine by two immunoradiometric assays (IRMA), were detectable by an ultrasensitive immunofluorometric assay (IFMA) and (ii) the influence of creatinine adjustment on the detection and timing of the urinary LH surges. Daily urine specimens were contributed by healthy 25-36 year old volunteers during 14 ovulatory menstrual cycles for an epidemiological study conducted in 1983-1985. Specimens were selected as having been previously assayed by two IRMA without consistently detecting LH surges. These urine specimens were remeasured using an IFMA and adjusted for creatinine concentration. IFMA measurements revealed unambiguous LH surges in all cycles. Adjusting IRMA urinary LH values for creatinine concentrations revealed previously undetected LH surges in four of eight cycles. Creatinine adjustment also altered the timing of IRMA and IFMA LH surges by 1-5 days. These results demonstrate an IFMA that detects pre- ovulatory LH surges in unpreserved, frozen urine from cycles where such surges were previously undetectable. Further, creatinine adjustment can markedly affect detection and timing of the onset and peak of the urinary LH surge. While our analysis suggests that this adjustment improves the validity of the LH measure, this requires further investigation.      

Tourniquet inflation after intra-articular morphine and bupivacaine administration following knee arthroscopy

We performed a randomized doubled-blind study to evaluate whether there was a benefit in delay in tourniquet deflation with intra-articular administration of morphine and bupivacaine following operative arthroscopic surgery. In 34 patients the tourniquet was deflated immediately and in 38 patients the tourniquet remained inflated for 10 min following injection. The analgesic efficacy was assessed using pain scores and the amount of supplementary analgesia required. The results demonstrate no benefit in delay in tourniquet deflation.     

Sequence variation and size ranges of CAG repeats in the Machado-Joseph disease, spinocerebellar ataxia type 1 and androgen receptor genes

A subgroup of trinucleotide repeat diseases result from abnormalexpansions of CAG repeats which are translated into polyglutaminestretches. As yet there is little understanding of how the polyglutaminesfunction either normally, or when expanded. We have investigatedthese sequences in the Machado-Joseph disease, androgen receptorand spinocerebellar ataxia type 1 genes in humans and otherprimates. None of the 748 normal chromosomes that were examinedhad more than 34 uninterrupted gluta-mine codons in the Machado-Josephdisease gene. Similarly, no normal alleles with more than 39uninterrupted glutamine codons have been reported for the otherdisease genes associated with polyglutamine expansions. Sequenceanalyses of the repeats in primates revealed shorter polyglutaminestretches in some of the non-human primates at all three lociand marked diversions from the expected polyglutamines in theorang-utan Machado-Joseph gene and in the marmoset spinocerebellarataxia type 1 gene. These data suggest that conservation ofthese polyglutamine stretches may not always be necessary fornormal gene function.