Mutations in intermediary metabolism

M.R. Seashore

REFERENCES:

Gelehrter, Collins, and Ginsburg, Chapter 7

Seashore, MR, and Wappner, R. Genetics in Primary Care and Clinical Practice, 1st edition, Appleton&Lange, 1995, various chapters on inborn errors of metabolism

Take a look at some websites: http://www.franken.de/lists/metab-l/nonmembers/links.html

SUMMARY:

The first inherited disorders of metabolism were described by Sir Archibald Garrod, physician to the Hospital for Sick Children at Great Ormond Street. In 1901 he first observed that some human traits behaved as if they followed the principles set forth by Mendel. He published these ideas in 1908 as the Croonian Lectures and developed the notion of "inborn errors of metabolism". The original disorder which he described, alcaptonuria, is recognized today as an inherited deficiency in homogentisic acid oxidase. The other three, albinism, cystinuria, and pentosuria are all now recognized inherited biochemical disorders. Much of the refinement of the original general principles set forth by Beadle and Tatum has been possible because of the study of human mutations in intermediary metabolism. The idea of one gene-one enzyme has given way to one gene-one polypeptide. Similarly, the idea of one gene-one pathway has been modified to explain how one gene can affect multiple pathways. The explanations for that phenomenon include a common subunit of a holoenzyme complex, a common coenzyme, multisubstrate enzymes. The phenomenon of genetic heterogeneity, several different genetic ways to lead to a similar phenotype is illustrated by these conditions.

The general paradigm for an inherited disorder of intermediary metabolism is illustrated below. The sites at which genetic control can operate include the following:

Transport into the cell

Transport into mitochondria

Mitochondrial metabolism

Lysosomal transport

Lysosomal metabolism

Peroxisomal function

Cytosolic metabolism

Mechanisms of genetic control include the following:

Apoenzyme biosynthesis

Coenzyme biosynthesis

Membrane structure

Phenylketonuria (PKU) can be taken as a prototype of these basic principles. First identified in Norway, in 1934, by Asbjorn Følling, this recessively inherited inborn error of metabolism occurs in about 1/15,000 births in the US. Untreated, it leads to severe mental retardation, seizures, eczema, and agitation. Following Følling's identification of phenylpyruvic acid in the urine of affected patients, attention turned to the pathway of phenylalanine degradation and investigation led in 1944 to the identification of tyrosine as a direct metabolite of phenylalanine.

The defect in phenylalanine metabolism leads to accumulation of phenylalanine and its metabolic by-products in the blood and urine of affected individuals; deficiency of tyrosine, the normal product of the pathway also results. Abnormal brain myelination is seen, which may be related to the mental retardation and seizures.

By the early 1950's experimental dietary treatment aimed at reducing the phenylalanine load was being tried, and by 1964 methods for screening presymtomatic newborn infants were developed. Such newborn screening is performed now in all 50 states in the US and in most of the developed countries around the world. Treatment is highly successful in preventing mental retardation, but it is an unpalatable diet at present, thus stimulating research directed toward gene therapy.

Biochemical and molecular genetic studies have refined the understanding of the pathophysiology of PKU.

Enzymology:

Human phenylalanine hydroxylase, mw 100,000-110,000 daltons, shows two polypeptides on SDS gel and a tertiary structure of 265,000 MW. Normal activity produces 48-96 µmol tyr/gm protein/hr.   Holoenzyme complex is made of:

apoenzyme protein

biopterin cofactor

phe-hydroxylase stimulating protein

lysolecithin

In classical PKU, the hydroxylase deficiency is severe: 0 µmol tyr/gm pro/hr; in the variant forms, the deficiency is milder: 3-30 µmol tyr/gm pro/hr.  Cofactor abnormalities have been identified, both deficiency of dihydropteridine reductase and a defect in dihydropteridine synthesis. 

Molecular genetics:

The human phenylalanine hydroxylase gene has been cloned and sequenced by Woo and colleagues. The locus has been assigned to chromosome 12q. The gene contains 13 exons and 12 introns.Data from Woo, of 66 normal and 66 mutant alleles in 33 Danish kindreds studied, show linkage disequilibrium between the mutation and the different haplotypes. A single haplotype, haplotype 3, which is rare among the normal population, accounts for 38% of the mutant alleles. Haplotype 3 has been shown to be due to a splicing abnormality at the 5'donor site of intron 12. This results in the production of an abnormal mRNA which is translated into a truncated unstable protein. Haplotype 2 results from a point mutation in exon 12 in which a single base substitution leads to the substitution of tryptophan for arginine; the mutant protein has no activity.  More than 50 different mutations have now been identified in the phenylalanine hydroxylase gene. These include single base substitutions, splice-site mutations, codon deletions, and single base deletions. The geographical distribution of these mutations has provided some information about their history and fits with described migrations in early human history. In vitro expression correlates with enzyme activity. Specific mutation analysis can be used for diagnosis.

Genetic heterogeneity in disorders of intermediary metabolism

One of the perplexing features of the early study of the inborn errors of metabolism has been the clinical variability among patients who seem to have the same biochemical disorder. As the case reports accumulate, it has become clear in many of these, that some patients seem to do very well with therapy directed at the biochemical pathology, and others, who seem to show the same metabolic derangements, do poorly, even die. These kinds of observations make evaluation of clinical intervention and therapeutic efforts difficult, and they lead to prognostic dilemmas. While it is tempting to ascribe such differences to therapeutic vigor, early recognition, and variations in treatment strategy and administration, molecular genetic explanations have been more fruitful. One such example of the role of genetic heterogeneity in explaining clinical and therapeutic variability is provided by the disorders in metabolism of methylmalonic acid, branched chain amino acid metabolism, and in homocysteine metabolism.

The pathophysiology of any of these disorders determines treatment. The general principles of therapy include the following:

Restriction of precursor : PKU is an example. Disorders of essential amino acid metabolism and transport often fall into this category.

Replacement of deficient product: The inherited disorders of hormone biosynthesis are examples of this principle

Coenzyme replacement: The vitamin B₁₂ responsive methylmalonicacidurias fit this description, as do the thiamine responsive branched chain ketoacidurias. Folate-responsive methylene tetrahydrofolate reductase deficiency may also fit this prototype.

Enzyme replacement:  Has not yet proved practical in most disorders. There are a few examples, such as Gaucher disease, in which intravenous enzyme is taken up by liver cells and results in clinical improvement in cerebroside storage in the liver.

Gene replacement: Still a major hope for the future