Factor XIII deficiency causing mutation, Ser295Arg, in exon 7 of the factor XIIIA gene.

Inherited factor XIII (FXIII) deficiency is an autosomal recessive disorder which results in a serious bleeding diathesis, problems with wound healing and a very high risk of recurrent miscarriage in deficient females. We have analysed the molecular basis of factor XIII deficiency in two patients and their parents, who originate from the North of Pakistan. Four sequence changes were identified: an AGC-->AGG (Ser-->Arg) FXIII deficiency-causing mutation in codon 295; G-->A at position -246 upstream of exon 1; T-->C and C-->T at positions -23 and -24, respectively, in intron 9. Using molecular modelling we predict that the Ser295Arg mutation would prevent the FXIIIA molecule from folding correctly and thus result in an unstable FXIIIA mutant polypeptide. The sequence changes -246G-->A, -23T-->C and -24C-->T are normal polymorphisms. RT-PCR analysis demonstrates that the intronic sequence changes do not appear to affect the accuracy of FXIIIA RNA processing.

Splicing and missense mutations in the human FXIIIA gene causing FXIII deficiency: effects of these mutations on FXIIIA RNA processing and protein structure.

We have investigated the molecular basis of factor XIII (FXIII) deficiency in a family from the north-west region of the U.K. and identified two sequence changes in the FXIII subunit A (FXIIIA) gene. We report a novel Asn to Lys mutation at codon 541, and a g-->a mutation at the intron 5/exon 6 splice junction in the FXIIIA gene. The splicing mutation results in two abnormal FXIIIA transcripts. The Asn541 residue is important for stabilizing an external fold in the FXIIIA barrel 1 domain. The Asn541Lys mutation is expected to result in inappropriate folding and therefore an unstable FXIIIA molecule.

New splicing mutations in the human factor XIIIA gene, each producing multiple mutant transcripts of varying abundance.

Coagulation factor XIII, a transglutaminase which stabilises blood clots by covalently cross-linking fibrin, is essential for normal haemostasis. FXIII deficiency results in a life-long bleeding disorder with added complications in wound healing and tissue repair. Sequence changes in the human FXIIIA gene, largely missense mutations, are primarily responsible for inherited FXIII deficiency. We have carried out molecular analysis of the FXIIIA gene in two unrelated FXIII deficient individuals and identified three splice site mutations; a g-->a at the exon 6 acceptor splice site, a g-->a at the exon 7 donor splice site and a coding sequence T-->G at the exon 8 donor splice site. We have also examined the FXIIIA mRNA in these patients and find that each mutation gives rise to multiple transcripts which vary in their relative abundance. The precise molecular mechanisms which result in these variant transcripts, and their relative abundance in our FXIII deficient patients, are discussed.

Identification of a large deletion, spanning exons 4 to 11 of the human factor XIIIA gene, in a factor XIII-deficient family.

Inherited deficiency of factor XIIIA subunit (FXIIIA) is an autosomal recessive disorder that is characterized by a life-long bleeding tendency and complications in wound healing. Molecular genetic studies have shown the deficiency can be due to small sequence changes within the FXIIIA gene, such as point mutations or microdeletions. On molecular analysis of the FXIIIA gene in an FXIII-deficient patient, of United Kingdom origin, we identified a putative homozygous missense mutation, Arg408Gln. However, the father of this patient is homozygous normal for arginine at codon 408. Having proved paternity in this pedigree by microsatellite analysis, we examined the FXIIIA RNA of the patient by reverse transcriptase-polymerase chain reaction and found the paternal allele to lack exons 4 through 11 inclusive. Hence, a huge deletion extending from intron 3 to intron 11 and the Arg408Gln mutation are jointly responsible for FXIIIA deficiency in this family. This is the first finding of such a large deletion in the FXIIIA gene.

Molecular basis of inherited factor XIII deficiency: identification of multiple mutations provides insights into protein function.

Factor XIII (FXIII) is a zymogen essential for normal haemostasis. In inherited FXIII deficiency the majority of cases show absence of the FXIIIa subunit. Molecular analysis of PCR-amplified FXIIIa subunit exonic regions, and of RT-PCR amplified cDNA from six patients with FXIIIa subunit deficiency, from five unrelated families, has revealed 10 sequence changes: three mutations resulting in abnormal splicing of pre-mRNA, one nonsense mutation, one deletion/insertion change, three point mutations producing Val34Leu, Asn60Lys and Arg408Gln changes, and two silent mutations. In three families the patients are homozygous for a specific deficiency causing mutation, and patients from the remaining two families are compound heterozygotes. Understanding the molecular pathology of the disorder provides insights into the structure-function relationships of the various domains within the FXIII protein. From a clinical point of view, it enables direct diagnosis at the DNA level and may aid the development of FXIII analogues to promote wound healing.

Factor XIII: inherited and acquired deficiency.

Factor XIII (XIII), an enzyme found in plasma (present as a pro-enzyme), platelets and monocytes, is essential for normal haemostasis. It may also have a role to play in the processes of wound healing and tissue repair. Inherited XIII deficiency results in a life-long, severe bleeding diathesis which, if untreated, carries a very high risk of death in early life from intracranial bleeding. XIII is a zymogen requiring thrombin and calcium for activation. In plasma, XIII has two subunits: the 'a' subunit, which is the active enzyme, and the 'b' subunit which is a carrier protein. Activated XIII modifies the structure of clot by covalently crosslinking fibrin through an epsilon (gamma-glutamyl)lysine link. It also crosslinks other proteins, including fibronectin and alpha-2-plasmin inhibitor (alpha-2PI), into the clot through the same link. Clot modified by XIII is physically stronger, relatively more resistant to fibrinolysis and may be a more suitable medium for the ingrowth of fibroblasts. Inheritance of factor XIII is autosomal recessive. The majority of patients with the inherited defect show no XIII activity and absence of 'a' subunit protein in plasma, platelets and monocytes. At the molecular level, the defect is not a major gene rearrangement or deletion, but most likely a single point mutation which may be different in each family. Because of the severity of the bleeding diathesis, prophylaxis is desirable and has been shown to be very effective as the in vivo half-life of plasma XIII is long, and low plasma levels are sufficient for haemostasis. Acquired inhibitors have been reported in only two cases with inherited XIII deficiency. Acquired XIII deficiency has been described in a variety of diseases and bleeding has been controlled by therapy with large doses of XIII in such conditions as Henoch-Schönlein purpura, various forms of colitis, erosive gastritis and some forms of leukaemia. Large dose XIII therapy has also been used in an endeavour to promote wound healing after surgery and bone union in non-healing fractures. The use of XIII in these conditions remains controversial. Very rarely a bleeding diathesis results from the development of a specific inhibitor to XIII arising de novo, often as a complication in the course of a disease or in association with long-term drug therapy. The bleeding diathesis in these patients is difficult to treat.

Palliative removal of a giant polypoid 'carcinosarcoma' of the oesophagus by YAG laser photocoagulation of the tumour stalk.

Dysphagia in a 79 year old lady was caused by a giant polypoid tumour in mid-oesophagus. Surgery was not appropriate. Shrinkage of the tumour and its eventual detachment were achieved by stopping its blood supply by YAG laser photocoagulation of the tumour stalk. Good, temporary palliation of the dysphagia was achieved.

Changes in intracranial pressure during machine and continuous haemofiltration.

Nine consecutive patients with both fulminant hepatic failure and acute oliguric renal failure were treated either by daily machine haemofiltration (MHF), or by continuous arterio-venous haemofiltration (CAVHF). Six patients received a total of twenty treatments by MHF and four CAVHF, mean duration of treatment 56 hours, range 24-160. Intracranial pressure (ICP) was measured using a subdural catheter. During treatment with MHF, the mean ICP increased from 8.9 +/- 1.4 mmHg at the start of filtration to 14.8 +/- 2.1 mmHg at the end of treatment (p less than 0.05), whereas there was no corresponding increase during the same period of time with CAVHF treatment, the mean ICP fell, but not significantly from 19.4 +/- 4.8 mmHg to 11.2 +/- 2.3 mmHg. The mean ICP increased to greater than 25 mmHg on eleven occasions during treatment with MHF, requiring treatment with bolus mannitol or propofol, during the same period of treatment with CAVHF no such surges in ICP were recorded. This suggests that continuous haemofiltration is to be preferred to intermittent machine haemofiltration in the management of patients with acute hepatorenal failure.

Changes in intracranial pressure during haemofiltration in oliguric patients with grade IV hepatic encephalopathy.

Seven consecutive patients with grade IV hepatic encephalopathy, due to fulminant hepatic failure complicated by oliguric renal failure were allocated at random to treatment with daily machine haemofiltration (MHF) or continuous arteriovenous haemofiltration (CAVHF). Intracranial pressure (ICP) was continuously monitored using a subdural catheter. Four patients received 17 treatments by MHF, and ICP increased from 8.4 +/- 1.5 mm Hg (mean + SEM) prior to treatment to 12.6 +/- 1.8 mm Hg on completion (p less than 0.05). Active intervention was required on twenty occasions to treat sustained increases in ICP (greater than 25 mm Hg maintained for 5 min or longer). A total of 12 haemofilters were used in the treatment of 3 patients by CAVHF. The ICP showed greater stability during CAVHF therapy, the mean pressure prior to treatment was 15.6 +/- 5.2 mm Hg and fell to 11.7 +/- 2.3 mm Hg at 4 h. Sustained increases in ICP occurred in only 1 patient as a preterminal event. These findings suggest that CAVHF is the preferred method of treatment in patients with fulminant hepatic failure complicated by oliguric renal failure who are at risk of developing cerebral oedema.

The prognosis of elderly subjects with oesophageal varices.

Discharge survival following variceal bleeding was similar in 27 elderly subjects, aged 65-93 years, and 125 younger subjects, aged under 65 (63% versus 70%, p greater than 0.05). Of 39 elderly subjects with varices which were not bleeding on admission, 82% were discharged alive but subsequent mortality was higher and long-term survival was similar (median 1 year) on life-table analysis. The overall long-term survival in the 66 elderly subjects was far worse than in the comparable group of 269 subjects under 65 (p less than 0.001). However, the excess deaths were from causes unrelated to hepatic failure or bleeding (e.g. stroke). Considering only deaths from hepatic failure or bleeding, long-term survival in the elderly and young was similar (p greater than 0.5). We conclude that elderly subjects with varices should be managed as actively as younger subjects since the majority are discharged alive and prolonged survival is common (25% at 3 years).

Comparison of the effectiveness of midazolam and diazepam in lipid emulsion as sedation during upper gastrointestinal endoscopy.

In a study of 101 patients undergoing upper gastrointestinal endoscopy, 90% of patients had complete amnesia for the procedure after intravenous midazolam (average dose 10 mg), but only 61% had complete amnesia after intravenous diazepam in lipid emulsion (average dose 18.4 mg) (P = 0.0006). However, when assessed by two different tests, recovery within the first hour was significantly more rapid after diazepam (P less than 0.0001). Prolonged sedation (over 20 hours after injection) was reported occasionally by patients who had received either drug. Thus, as with patients who have been sedated with diazepam, those who have been sedated with midazolam should also be advised to avoid driving or operating machinery for at least 24 hours after injection.

Raised parathyroid hormone levels in the milk alkali syndrome: an appropriate response?

A case of the 'milk alkali syndrome' associated with grossly elevated levels of amino terminal parathyroid hormone is described. The hypercalcaemia (calcium 4.09 mmol/l) and hyperparathyroidism settled on conservative measures. Factors in the milk alkali syndrome which might stimulate the release of parathyroid hormone include parathyroid gland hyperplasia secondary to suppression of ionized calcium, alteration in sensitivity of calcium receptors on the cells of the parathyroid glands, the stimulation of an intermittent alkaline tide in the blood and the high intake of phosphate and bicarbonate. We suggest that high levels of parathyroid hormone in the milk alkali syndrome may be appropriate rather than paradoxical.

Localisation of factor XIII in human tissues using an immunoperoxidase technique.

An immunoperoxidase technique has been used to localise clotting factor XIII subunits A and S in human tissues. The presence of factor XIII in placenta and megakaryocytes was confirmed. Factor XIII was also found in fibroblasts, a hitherto unreported finding. Factor XIII subunits were not detected in hepatocytes, although factor XIII was found in fibroblasts in portal tracts. These findings suggest that factor XIII is not synthesised in the liver as previously thought.

An acquired inhibitor of factor XIII with a qualitative abnormality of fibrin cross-linking.

A patient with an acquired inhibitor to factor XIII is reported. The patient's plasma produced a profound inhibition of factor XIII activity in normal plasma measured by a dansylcadaverine casein assay and stimulated a very abnormal pattern of fibrin cross-linking, not normally seen with factor XIII. Partial characterisation of the inhibitor suggests that it is heat stable and not an immunoglobulin.

Factor XIII levels in five families of patients with inherited factor XIII deficiency: support for an autosomal recessive inheritance.

Using quantitative methods, f.XIII activity and levels of subunits a and b have been measured in 5 families of 6 patients with inherited XIII deficiency, including 2 children of a XIII deficient male. The parents, as a group, and the children, individually, have low XIII activity and low levels of subunit A when compared to controls. These findings provide further support for an autosomal inheritance of f.XIII deficiency. The measurements, however, did not allow confident selection of individual heterozygotes as has been previously suggested and an explanation of this finding is offered.