Timothy M. Cox

Classification and genetic features of neonatal haemochromatosis: a study of 27 affected pedigrees and molecular analysis of genes implicated in iron metabolism

Neonatal haemochromatosis (NH) is a severe and newly recognised syndrome of uncertain aetiology, characterised by congenital cirrhosis or fulminant hepatitis and widespread tissue iron deposition. NH occurs in the context of maternal disease including viral infection, as a complication of metabolic disease in the fetus, and sporadically or recurrently, without overt cause, in sibs. Although an underlying genetic basis for NH has been suspected, no test is available for predictive analysis in at risk pregnancies.  As a first step towards an understanding of the putative genetic basis for neonatal haemochromatosis, we have conducted a systematic study of the mode of transmission of this disorder in a total of 40 infants born to 27 families. We have moreover carried out a molecular analysis of candidate genes (β2-microglobulin,HFE, and haem oxygenases 1 and 2) implicated in iron metabolism. No pathogenic mutations in these genes were identified that segregate consistently with the disease phenotype in multiplex pedigrees. However, excluding four pedigrees with clear evidence of maternal infection associated with NH, a pedigree showing transmission of maternal antinuclear factor and ribonucleoprotein antibodies to the affected infants, and two families with possible matrilineal inheritance of disease in maternal half sibs, a large subgroup of the affected pedigrees point to the inheritance of an autosomal recessive trait. This included 14 pedigrees with affected and unaffected infants and a single pedigree where all four affected infants were the sole offspring of consanguineous but otherwise healthy parents.  We thus report three distinct patterns of disease transmission in neonatal haemochromatosis. In the differentiation of a large subgroup showing transmission of disease in a manner suggesting autosomal recessive inheritance, we also provide the basis for further genome wide studies to define chromosomal determinants of iron storage disease in the newborn.

RIPK3 as a potential therapeutic target for Gaucher's disease

Gauchers disease (GD), an inherited metabolic disorder caused by mutations in the glucocerebrosidase gene (GBA), is the most common lysosomal storage disease. Heterozygous mutations in GBA are a major risk factor for Parkinsons disease. GD is divided into three clinical subtypes based on the absence (type 1) or presence (types 2 and 3) of neurological signs. Type 1 GD was the first lysosomal storage disease (LSD) for which enzyme therapy became available, and although infusions of recombinant glucocerebrosidase (GCase) ameliorate the systemic effects of GD, the lack of efficacy for the neurological manifestations, along with the considerable expense and inconvenience of enzyme therapy for patients, renders the search for alternative or complementary therapies paramount. Glucosylceramide and glucosylsphingosine accumulation in the brain leads to massive neuronal loss in patients with neuronopathic GD (nGD) and in nGD mouse models. However, the mode of neuronal death is not known. Here, we show that modulating the receptor-interacting protein kinase-3 (Ripk3) pathway markedly improves neurological and systemic disease in a mouse model of GD. Notably, Ripk3 deficiency substantially improved the clinical course of GD mice, with increased survival and motor coordination and salutary effects on cerebral as well as hepatic injury.

Molecular analysis of aldolase B genes in hereditary fructose intolerance

The molecular basis of hereditary fructose intolerance (HFI) was studied in 50 subjects (41 pedigrees, 82 apparently independent mutant alleles of aldolase B) by direct analysis of aldolase B genes amplified by means of the polymerase chain reaction. The mutation A149P (ala 149----pro) was found in 67% of alleles but was significantly more common in patients from northern than from southern Europe. Two other point mutations of aldolase B were identified. A174D (C----A; ala 174----asp) was found in subjects from Italy, Switzerland, and Yugoslavia (overall frequency 16%) but not in those from the United Kingdom, France, or the United States. L288 delta C carried a single base-pair deletion causing frameshift at codon 288 and was restricted to Sicilian subjects. By testing for these mutations in amplified DNA with a limited panel of allele-specific oligonucleotides, more than 95% of HFI patients will be susceptible to genetic diagnosis.

Catalytic deficiency of human aldolase B in hereditary fructose intolerance caused by a common missense mutation

Hereditary fructose intolerance (HFI) is a human autosomal recessive disease caused by a deficiency of aldolase B that results in an inability to metabolize fructose and related sugars. We report here the first identification of a molecular lesion in the aldolase B gene of an affected individual whose defective protein has previously been characterized. The mutation is a G----C transversion in exon 5 that creates a new recognition site for the restriction enzyme Ahall and results in an amino acid substitution (Ala----Pro) at position 149 of the protein within a region critical for substrate binding. Utilizing this novel restriction site and the polymerase chain reaction, the patient was shown to be homozygous for the mutation. Three other HFI patients from pedigrees unrelated to this individual were found to have the same mutation: two were homozygous and one was heterozygous. We suggest that this genetic lesion is a prevailing cause of hereditary fructose intolerance.

Neonatal screening for hereditary fructose intolerance: frequency of the most common mutant aldolase B allele (A149P) in the British population.

Hereditary fructose intolerance (HFI) causes severe and sometimes fatal metabolic disturbances in infants and children but responds to dietary treatment. To determine the practicability of screening newborn infants for HFI, we have investigated the frequency of the most common and widespread mutant allele of aldolase B, A149P, in the neonatal population. The polymerase chain reaction was used to amplify aldolase B exon 5 genomic sequences in DNA present in dried blood specimens preserved on Guthrie cards. The A149P mutation was identified by discriminatory hybridisation to allele specific oligonucleotides and confirmed independently by digestion with the restriction endonuclease BsaHI. Twenty-seven A149P heterozygotes were identified by the molecular analysis of aldolase B genes in blood samples obtained from a random cohort of 2050 subjects born in 1994 and 1995, 1.32 +/- 0.49% (95% confidence level). Although no A149P homozygotes were identified, the data allow the frequency of 1 in 23,000 homozygotes for this allele to be predicted. Our findings have implications for establishing an interventional mass screening programme to identify newborn infants with HFI in the UK.

Relative acidic compartment volume as a lysosomal storage disorder–associated biomarker

Lysosomal storage disorders (LSDs) occur at a frequency of 1 in every 5,000 live births and are a common cause of pediatric neurodegenerative disease. The relatively small number of patients with LSDs and lack of validated biomarkers are substantial challenges for clinical trial design. Here, we evaluated the use of a commercially available fluorescent probe, Lysotracker, that can be used to measure the relative acidic compartment volume of circulating B cells as a potentially universal biomarker for LSDs. We validated this metric in a mouse model of the LSD Niemann-Pick type C1 disease (NPC1) and in a prospective 5-year international study of NPC patients. Pediatric NPC subjects had elevated acidic compartment volume that correlated with age-adjusted clinical severity and was reduced in response to therapy with miglustat, a European Medicines Agency–approved drug that has been shown to reduce NPC1-associated neuropathology. Measurement of relative acidic compartment volume was also useful for monitoring therapeutic responses of an NPC2 patient after bone marrow transplantation. Furthermore, this metric identified a potential adverse event in NPC1 patients receiving i.v. cyclodextrin therapy. Our data indicate that relative acidic compartment volume may be a useful biomarker to aid diagnosis, clinical monitoring, and evaluation of therapeutic responses in patients with lysosomal disorders.

Pseudodominant transmission of fructose intolerance in an adult and three offspring. Heterozygote detection by intestinal biopsy

HEREDITARY fructose intolerance is an inborn error of metabolism characterized by abdominal pain, vomiting, and hypoglycemia, which follow the ingestion of fructose.1 The disorder is associated wit...

Aldolase B mutations in Italian families affected by hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is an inborn error of metabolism caused by aldolase B deficiency. The aldolase B gene has been cloned and the following mutations causing HFI have been identified: A149P (a G----C transversion in exon 5), A174D (a C----A transversion in exon 5), L288 delta C (a base pair deletion in exon 8), and N334K (a G----C transversion in exon 9). We have investigated the occurrence of these mutations in 11 Italian patients affected by HFI using PCR and hybridisation to specific oligomers. We found that four patients were homozygous for the A149P mutation, two patients were homozygous for the A174D mutation, three patients were compound heterozygotes for both the A149P and A174D mutations, one patient was homozygous for the N334K mutation, and one patient did not show any of the reported mutations (HFI diagnosis carried out by aldolase B assay). The L288 delta C mutation has not been found in this survey.

Biosynthesis of the transferrin receptor in rabbit reticulocytes

These studies were performed to determine whether the reticulocyte can synthesize its own transferrin receptor and, if so, whether synthesis is subject to translational control by intracellular heme. Reticulocytosis (20-35%) was produced by bleeding rabbits and the washed cells were incubated for 1-4 h at 37 degrees C in buffered nutritional medium containing L-[35S]methionine. After washing and detergent lysis in the presence of protease inhibitors, supernatant reticulocyte extracts were analyzed for transferrin receptors by immunoprecipitation with specific ovine receptor antibody raised against denatured rabbit transferrin receptor. Immunoprecipitates were analyzed by SDS-gel electrophoresis and fluorography. Antibody, but not preimmune sheep immunoglobin, consistently precipitated a 35S-labeled protein with an Mr of 90,000 (reduced), coincident with bona fide receptor subunits purified by ligand-affinity chromatography. Incorporation of radioactive methionine was exclusively associated with receptor in reticulocyte stroma, and nascent receptor was not detected on free polyribosomes. Incorporation of radioactivity in the receptor moiety accounted for 0.1-0.2% of total incorporation into TCA insoluble cell protein. Treatment of the cells with 40 micrograms/ml cycloheximide markedly inhibited amino acid incorporation into the receptor, thus indicating de novo synthesis of receptor protein. On treatment of reticulocytes with 4,6 dioxoheptanoate to induce heme deficiency by diminishing the formation of intracellular heme, synthesis of the receptor was inhibited by greater than 50%; synthesis was restored to control rates on addition of 50 microM exogenous hemin. These findings indicate that the reticulocyte retains receptor mRNA and that synthesis of the receptor in erythroid cells is subject to translational regulation by intracellular heme.

Hereditary fructose intolerance.

Abstract Hereditary fructose intolerance (HFI) is an inborn error of carbohydrate metabolism that is inherited as an autosomal recessive condition. The disease is caused by a catalytic deficiency of aldolase B and is characterized by severe abdominal symptoms and hypoglycaemia which follow the ingestion of fructose, sucrose or sorbitol. The exact prevalence of HFI in different populations is unknown but studies from Switzerland suggest a disease frequency of about 1 in 20 000 live births, thus predicting a carrier frequency of greater than 1% and a gene prevalence that approaches polymorphic frequency. It is notable that many patients who endure severe symptoms during early infancy develop a marked aversion to harmful foodstuffs and thereby survive to adulthood. Although exposure to fructose may prove to be fatal in this disorder, institution of a strict exclusion diet is curative. HFI, when treated rigorously after diagnosis, is thus compatible with a long and healthy life. HFI vividly illustrates the interplay of dietary factors and heredity in the development of human disease. The recent identification of genetic lesions that cause this disorder further demonstrates the remarkable clinical benefits that may accrue from the study of the molecular basis of inherited diseases and its population genetics: it is now possible to detect asymptomatic disease carriers and diagnose the disorder in affected families by non-invasive analysis of small samples of genomic DNA. Moreover, the systematic investigation of natural mutations in the human gene for aldolase B has allowed regions that are critical for catalytic function of this enzyme to be identified as part of an extended study of its molecular biology.