A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis.

The progressive familial intrahepatic cholestases (PFIC) are a group of inherited disorders with severe cholestatic liver disease from early infancy. A subgroup characterized by normal serum cholesterol and gamma-glutamyltranspeptidase (gammaGT) levels is genetically heterogeneous with loci on chromosomes 2q (PFIC2) and 18q. The phenotype of the PFIC2-linked group is consistent with defective bile acid transport at the hepatocyte canalicular membrane. The PFIC2 gene has now been identified by mutations in a positional candidate, BSEP, which encodes a liver-specific ATP-binding cassette (ABC) transporter, sister of p-glycoprotein (SPGP). The product of the orthologous rat gene has been shown to be an effective bile acid transporter in vitro. These data provide evidence that SPGP is the human bile salt export pump (BSEP).

High frequency of missense mutations in glycogen storage disease type VI.

Deficiency of liver glycogen phosphorylase in glycogen storage disease (GSD) type VI results in a reduced ability to mobilize glucose from glycogen. Six mutations of the PYGL gene, which encodes the liver isoform of the enzyme, have been identified in the literature. We have characterized eight patients from seven families with GSD type VI and identified 11 novel PYGL gene defects. The majority of the mutations were missense, resulting in the substitution of highly conserved residues. These could be grouped into those that were predicted to affect substrate binding (p.V456M, p.E673K, p.S675L, p.S675T), pyridoxal phosphate binding (p.R491C, p.K681T), or activation of glycogen phosphorylase (p.Q13P) or that had an unknown effect (p.N632I and p.D634H). Two mutations were predicted to result in null alleles, p.R399X and [c.1964_1969inv6;c.1969+1_+4delGTAC]. Only 7 of the 23 (30%) reported PYGL alleles carry nonsense, splice site or frameshift mutations compared to 68-80% of affected alleles of the highly homologous muscle glycogen phosphorylase gene, PYGM, that underlie McArdle disease. There was heterogeneity in the clinical symptoms observed in affected individuals. These varied from hepatomegaly and subclinical hypoglycaemia, to severe hepatomegaly with recurrent severe hypoglycaemia and postprandial lactic acidosis. We conclude that deficiency of liver glycogen phosphorylase is predominantly the result of missense mutations affecting enzyme activity. There are no common mutations and the severity of clinical symptoms varies significantly.

Role of copper in Indian childhood cirrhosis.

Of the cirrhoses that affect Indian children, Indian childhood cirrhosis (ICC) is a discrete clinical and histologic entity in which large amounts of copper are deposited in the liver. The evidence linking copper deposition to increased dietary copper intake in infancy was reviewed. Prevention of this feeding pattern prevents ICC, and the disease has now largely disappeared from many parts of India. Penicillamine, if given before the terminal clinical stage of ICC, reduces mortality from 92% to 53%. Long-term survivors show a sequence of histologic resolution, resulting either in inactive micronodular cirrhosis or in virtually normal histologic appearance. Twenty-nine treated ICC patients reexamined at 8.8 y of age (range: 6.3-13 y), 5-12 y after diagnosis, were well and had normal results from liver function tests. Clinical and epidemiologic evidence show that there must be excessive copper ingestion for ICC to develop, but the lack of an animal model, the inconstant relation between liver copper concentrations and liver damage, and the rarity of liver disease in adults suggests that other etiologic factors contribute. Two mechanisms are discussed: 1) that copper may be acting in synergy with a hepatotoxin, or 2) that there may be a genetic predisposition to copper-associated liver damage, as suggested recently for Tyrollean childhood cirrhosis. Although ICC is now rare, sporadic cases of an ICC-like disorder in infants continue to occur. There should be a greater awareness among pediatricians of this disease to enable early diagnosis. Penicillamine should be used early and adverse prognostic factors recognized as indications for early transplantation and unregulated water supplies should not be used to prepare infant feeds.

A comparison of the ontogeny of enterocytic and hepatic cytochromes P450 3A in the rat.

Enzymes of the cytochrome P450 3A (CYP3A) sub-family are abundant in adult liver and gut and contribute significantly to the first-pass metabolism of many orally administered drugs. The development of CYP3A enzymes with regard to their expression and activity in enterocytic and hepatic microsomes from 1-day-old through to adult male and female rats has been studied. Microsomes were prepared by calcium precipitation. Enzyme expression was assessed semi-quantitatively by Western blotting using rat polyclonal CYP3A2 and 2C11 antibodies and peptide antibodies specific to rat CYPs 3A1, 3A2, 2C12, and 2C13. The formation of 6beta-hydroxytestosterone (6OHT), determined by HPLC, was used as a measure of enzyme activity. Formation of 6OHT by enterocytic microsomes was similar for males and females and showed a sharp increase at weaning. This pattern was mirrored by levels of immunoquantifiable CYP3A2 (CYP3A9), but CYP3A1 followed a more gradual development. CYPs 2C11, 2C12, or 2C13 were not detected in gut microsomes. In contrast, CYPs 3A1, 3A2, 2C11, 2C12, and 2C13 were all expressed in hepatic microsomes. There was no surge in hepatic enzyme expression or hepatic 6OHT formation at weaning, and a marked sex difference in 6OHT formation was apparent from day 25. The surge in gut activity at weaning may be a protective mechanism against ingested toxins.

Development of a PCR probe test for identifying Pseudomonas aeruginosa and Pseudomonas (Burkholderia) cepacia.

AIMS: To develop a system of species specific polymerase chain reaction (PCR) and DNA hybridisation based on 16s ribosomal RNA sequences for the identification of Pseudomonas aeruginosa and Pseudomonas (Burkholderia) cepacia in sputum from children with cystic fibrosis. METHODS: Most of the 16s rRNA sequences from strains of Ps aeruginosa, Ps (Burkholderia) cepacia, and Ps putida were determined. PCR primers and DNA probes were synthesised from suitable sequences and then evaluated on bacterial cultures and sputum samples. RESULTS: About 1000 bases of sequence was obtained from strains of Ps aeruginosa, Ps (Burkholderia) cepacia, and Ps putida. PCR of bacterial cultures was species specific, but PCR on sputum resulted in some non-specific amplification products. The subsequent hybridisation reaction was species specific. CONCLUSION: A species specific system of PCR and DNA hybridisation based on 16s rRNA sequences is applicable in clinical practice, and may aid the early diagnosis of respiratory tract infection with small numbers of Ps aeruginosa and Ps (Burkholderia) cepacia in patients with cystic fibrosis.

Recommended approaches for the laboratory measurement of homocysteine in the diagnosis and monitoring of patients with hyperhomocysteinaemia.

Several recent studies have indicated that an increased concentration of plasma homocysteine is an independent risk factor for the premature development of vascular disease. These important findings emphasize the need for careful selection of an appropriate analytical approach to diagnose and treat individuals who may be at risk. We compared the results obtained from the measurement of plasma total homocysteine (free + protein-bound fractions) by high-performance liquid chromatography (HPLC) with the measurement of plasma free homocystine (free fraction) by conventional ion-exchange chromatography in 10 patients with inherited defects of homocysteine metabolism and 13 obligate heterozygote individuals. This study can be used to formulate recommendations on the appropriate use of these assays in different clinical circumstances. Our results show that the concentration of total plasma homocysteine must exceed 60 mumol/L before plasma free homocystine becomes detectable by conventional ion-exchange chromatography. Similarly, assessment of the urinary excretion of homocysteine in these patients indicates that it may not become consistently detectable by conventional ion-exchange chromatography or HPLC until plasma total homocysteine exceeds 150 mumol/L. On this basis, while most patients with classical homocystinuria would be detected by analysis of plasma using conventional ion-exchange chromatography or by measurement of of the urinary homocysteine excretion, occasional patients would be missed. When monitoring patients receiving treatment for classical homocystinuria, in whom metabolic control is good, and when investigating individuals with a suspected inherited defect of cobalamin or folate metabolism, a method which measures plasma total homocysteine should be used. The identification of moderate hyperhomocysteinaemia of undefined cause investigated in relation to a history of early vacsular disease can only be identified by this approach.

Copper-induced hepatotoxicosis with hepatic stellate cell activation and severe fibrosis in North Ronaldsay lambs: a model for non-Wilsonian hepatic copper toxicosis of infants.

Copper-sensitive North Ronaldsay sheep represent a possible model for certain hepatic-overload syndromes of infancy and childhood that are clinically, pathologically and genetically distinct from Wilson's disease. The purpose of this study was to simulate in artificially reared lambs the syndrome produced by copper exposure in susceptible human infants. Twenty four North Ronaldsay lambs were assigned to three groups of eight animals, namely, an unsupplemented control group and two trial groups given milk replacer to which copper (CuSO4) had been added at the rate of 5 mg/litre and 10 mg/litre. Four lambs from each group were killed at 40 or 69 days. Livers were fixed in 10% formalin and analysed for copper by mass spectrometry. Paraffin wax-embedded sections were stained with rhodanine for copper and labelled immunohistochemically for alpha smooth muscle actin (ASMA). At 40 days the maximum amounts of copper in the livers of both copper-supplemented groups was 1466-1605 microg/g dry weight (control group 172-201 microg/g Cu dry weight). Histochemically, copper was demonstrated within hepatocytes, together with marked apoptosis. At 69 days there was a florid pericellular fibrosis complemented by strong ASMA immunolabelling, confirming phenotypic modulation of hepatic stellate cells. Such primary copper-induced fibrogenesis confirms the unique status of this animal model in respect of childhood copper toxicosis.

Copper toxicity affects proliferation and viability of human hepatoma cells (HepG2 line).

In Wilson's disease and Indian childhood cirrhosis (ICC) copper accumulates in the liver resulting in poor hepatocyte regeneration and fibrosis. An inhibition of hepatocyte proliferation and an increase in cell death could account for these outcomes. To establish how the toxicity of this metal ion impacts upon the proliferation and viability of the HepG2 cells they were cultured in 4-32 microM copper(II) sulphate (CuSO4)). These levels were comparable to the circulatory and tissue concentrations of copper recorded for these two diseases. Specific uptake comparable to levels of copper recorded in the livers of patients with Wilson's disease and ICC was measured in the HepG2 cells. After 48 h acid vesicle function increased from 4 to 32 microM Cu2+ but significantly declined at 64 microM compared to the controls. Lysosomal acid phosphatase showed a concentration dependent decline in activity at 72 h. Cellls exposed to 64 microM Cu2+ had a potential doubling time (Tpot) 21 h longer than the control cells due to a prolonged DNA synthesis phase. At 64 microM Cu2+, increases of necrosis up to 18% were seen whereas comparable levels of apoptotic and necrotic cells (<5%) were seen below this concentration. Chronic exposure over 8 weeks impaired colony-forming efficiency at concentrations of 16 microM Cu2+ and above. This study suggests that when liver cells sequester large amounts of copper, the toxic effects include delayed cell-cycle progression, a gradual loss of replicative capacity, and an increased incidence of cell death.

Optic nerve hypoplasia in infancy.

Certain features of optic nerve hypoplasia (ONH), its systemic associations and investigation are exclusive to infancy. These include the facility to use cranial ultrasound, difficulties in assessing ocular features and visual function, and neonatal hypoglycaemia and jaundice. Six infants with ONH are presented; cerebral abnormalities were demonstrated by cranial ultrasound in five. Neonatal cholestatic jaundice and hypoglycaemia occurred in one infant. Two died and represent a group likely to remain undetected unless routine ophthalmic examination of neurologically abnormal neonates is undertaken. In infancy, both ocular and systemic aspects of ONH can be investigated simply and without sedation.