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%.      

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.      

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.     

A simple intra-operative maneuver to decrease a duodenal ulcer hemorrhage temporarily: description and anatomical bases

Intraoperative hemostatic suture to treat a bleeding duodenal ulcer is sometimes difficult when there is massive hemorrhage. The aims of this paper are: (1) to describe a practical and easy intraoperative procedure which quickly decreases a massively bleeding duodenal ulcer, allowing the surgeon to identify the bleeding site clearly and obtain definitive hemostasis by suturing the involved vessels with a low risk of common bile duct lesion; and (2) to study in cadavers the anatomical basis of this surgical procedure already successfully performed on patients. Fourteen patients with massive duodenal ulcer bleeding, after unsuccessful endoscopic hemostasis, were operated on and included in this study. After surgical anterior gastroduodenotomy, the surgeon introduced a finger in a downward and forward direction in the bursa omentalis vestibule through the omental foramen. This simple and quick procedure decreased hemorrhage by compressing the gastroduodenal artery against the first part of the posterior surface of the duodenum. Twenty-four fresh blocks of normal tissue were removed from cadavers and were injected with silicone rubber through the common hepatic artery. The distance between the gastroduodenal artery and the omental foramen was measured. With this maneuver the surgeon can clearly see the exact bleeding site and perform an adequate suture with a minor risk of common bile duct lesion.