Clinical And Metabolic Consequences Of Single Gene Defects Biology Essay

Published: Last Edited:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

Phenylketonuria: genetic basis. Phenylketonuria (PKU) was first identified by the Norwegian physician and biochemist Fölling. He followed up his observation that urine of two retard siblings turned green on mixing with an aqueous solution of ferric chloride, by identifying phenylpyruvic acid, and by detecting the condition in eight more retard individuals out of 430 whom he tested. He also noted a tendency for these patients to have a mousy odour, a fair complexion, and dermatitis. Moreover, he showed that overloading rabbits with phenylalanine caused phenylpyruvic acid to appear in their urine, and suggested that the disorder was a genetic defect of phenylalanine metabolism. This was confirmed by Jarvis (1947), who showed that patients were unable to convert phenylalanine into tyrosine. The disorder was named Phenylketonuria by Penrose (1935).The name of the condition derives from the urinary excretion of phenylpyruvic acid, a phenylketone.

Phenylketonuria (PKU, MIM 261600) is the most common inherited disorder of amino acid metabolism in European-descended populations. Phenylketonuria is transmitted by an autosomal recessive mode of inheritance (typical Mendelian trait) caused by mutations of the phenylalanine hydroxylase (PAH) gene (Scriver & Kaufman., 2001).

The phenylalanine hydroxylase (PAH),an enzyme that catalyzes the hydroxylation of the essential amino acid l-phenylalanine resulting in another amino acid, tyrosine, the rate-limiting step in phenylalanine catabolism. This is the major pathway for catabolising dietary l-phenylalanine and accounts for approximately 75% of the disposal of this amino acid. Deficiency in PAH results in hyperphenylalaninemia (a plasma phenylalanine value of more than 120 µM).If undiagnosed and untreated, phenylketonuria can result in impaired postnatal cognitive development resulting from a neurotoxic effect of hyperphenylalaninemia (Zurfluh et al., 2008).Since the introduction of a dietary treatment fifty years ago, PKU has been the prototype for treatable genetic disease and, later for genetic screening in human populations.Phenylketonuria is a "multifactorial" with inherited (genetic) and acquired (dietary) components( Knox.,(1972)as cited by Scriver &Kaufman., (2001), both of which are necessary to establish the variant metabolic phenotype (HPA).

The gene for PAH was isolated in 1986, and subsequently, a striking degree of allelic heterogeneity has been demonstrated in the PAH-deficient population (Scriver et al.,1995).The human PAH gene covers 90 Kb of genomic DNA (Konecki et al., 1991) on chromsome12,band region q22-q24.1.It has 13 exons (Konecki et al., 1991) and a complex 5' untranslated region containing cis-acting,trans-activated regulatory elements. The gene is rich in intragenic polymorphic markers, including biallelic restriction fragment length polymorphism (RFLP) and single nucleotide polymorphism(SNP) alleles, a tetranucleotide short tandem repeat (STR) acting as a fast molecular clock in intron 3,and a variable number of tandem repeats (VNTR,30-bp-long cassettes) in the 3' untranslated region (UTR).The polymorphic sites are in linkage disequilibrium and describe a large series of extended and minihaplotypes.At the molecular level, more than 500 disease associated alleles of the PAH gene have been identified in populations worldwide, nearly 90% of them being point mutations among which only a half-dozen account for the majority of mutant chromosomes in Europeans and Orientals; the remainder are rare, even private, alleles. Deletions in the gene are not common.

Table 1. Class of mutations observed in the PAH Gene

Genetic Mechanism


Deletions (mainly small)





Percentage of mutations







PAHdb knowledgebase

Large deletions account for 2%-3% of mutations (Kazak et al 2006 as cited by Scriver & Mitchell (2007)

Other PAH mutations delete small amounts of DNA from the gene or disrupt the way the gene's instructions are used to make phenylalanine hydroxylase. Although the vast majority of mutations responsible for HPA map to the PAH locus, some occur at loci controlling the synthesis and recycling of tetrahydobiopterin (BH4), the essential cofactor for catalytic activity of phenylalanine enzyme. Each mutation induces a particular alteration in the enzyme resulting in a corresponding quantitative effect on residual enzyme activity ranging from complete absence to 50% of normal values. Pattern of mutations show ethnic variation, a frequent cause among northern Europeans (~40%) is a G-to-A transition at the 5′ donor splice site in intron 12, resulting in absence of the C-terminus. Another common mutation is a C-to-T transition in exon 12, replaces the amino acid arginine with the amino acid tryptophan at position 408 (written as Arg408Trp or R408W). R408W accounts for two-thirds of mutations in eastern Europe compared with 24% in Scotland and a splicing mutation in intron 10 accounts for 40% of Turkish mutations.PKU affects approximately 1 in 10,000 people in the UK and Europe and east Asian (Scriver & Kaufman (2001), for Ireland it's 1 in 4000(DiLella et al (1986) and Turkey, 1 in 3000(Ozalp et al (1986), rare in afro-Caribbean's, Indians and Ashkenazi Jews 1 in 200,000 (Scriver & Kaufman (2001). The wide variability in the common mutations between ethnic groups and geographical areas make PAH deficiency a genetic disease with great allelic heterogeneity. Approximately 98% of PKU cases are caused by defects in the PAH gene. The other 2% are caused by defects in the biosynthesis or reconversion of cofactor of PAH, 6 (R)-L-erythro-tetrahydrobiopterin (BH4). PAHdb, an online relational database was created in 1990s where mutation data on the PAH gene can be centralised (

Biochemistry Phenylketonuria

The biochemical abnormality in PKU is an inability to convert phenylalanine into tyrosine. In normal children, less than 50% of dietary intake of phenylalanine is necessary for protein synthesis. The rest is irreversible converted to tyrosine by PAH in the liver as part of a complex pathway (figure 1).

Phenylalanine hydroxylase requires as cofactors molecular oxygen and reduced pteridine cofactor tetrahydrobiopterin (BH4), which Acts as an electron carrier of this cofactor is dependent either on a regeneration from the quinonoid dihydrobiopterin by the enzyme dihydropteridine reductase (DHPR) or on de novo synthesis from quanosine triphosphate.Tyrosine, quinonoid dihydrobiopterin and water are the reaction products. Intracellular tetrahydobiopterin is regenerated from quinonoid dihydrobiopterin ,firstly by an important pathway tightly coupled with the hydroxylation reaction, catalysed by dihydropteridine reductase and utilizing either NADH or NADPH as a hydrogen donor; and secondly by tautomerization of quinonoid dihydrobiopterin,which is unstable, to the more stable 7,8- dihydrobiopterin,followed by reduction of the last named compound to tetrahydrobiopterin (BH4) catalysed by dihydrofolate reductase with NADPH acting as hydrogen donor.

Figure 2: The phenylalanine hydroxylase (PAH) pathway. Phenylketonuria usually is caused by a congenital deficiency of PAH (reaction 1), but it also can result from defects in the metabolism of biopterin, which is a cofactor for the hydroxylase. Enzymes: 1, phenylalanine hydroxylase; 2, dihydropteridine reductase; 3, GTP cyclohydrolase; 4, 6-pyruvoyltetrahydrobiopterin synthase. QH2, dihydrobiopterin; BH4, tetrahydrobiopterin; DEDT, d-erythro-dihydroneopterin triphosphate.

Several defects in different steps in the metabolism of phenylalanine or its cofactors (BH4) can result in elevated phenylalanine in tissues, plasma and urine. The deficiency of PAH raises plasma phenylalanine from its normal level of 0.5 to 2.0 mg/dL to more than 20 mg/dL.

The accumulated phenylalanine is transminated to phenylpyruvates, normal a minor pathway of phenylalanine metabolism. The phenylypyruvate is converted to phenylacetate, phenyllactate and phenylacetylglutamine, which along with phenylalanine and phenylypyruvates is excreted in the urine (figure 3).

Figure 3 phenylalanine metabolism in phenylketonuria

The metabolic disorders associated with impaired ability to convert phenylalanine to tyrosine can be classified into three groups:

Defects in phenylalanine hydroxylase ("classical" PKU less then 1% activity of PAH).

Deficiency in dihydropteridine reductase;

Disturbed de novo tetrahydrobiopterin biosynthesis

This disturbance in metabolic homeostasis can have clinical consequence depending on its pathogenesis and its degree. The major associated clinical manifestation is impaired cognitive development and function resulting from neurochemical imbalance: postnatally in affected cases and prenatally in the foetus of affected pregnant women.

Clinical presentation of Babies born with PKU

The clinical features (abnormalities) of PKU are absent at birth because phenylalanine and its metabolites are transferred across the placenta. Abnormalities develop within a few days if the newborn is untreated as serum phenylalanine and urinary phenylpyruvate rise; they include the following:

Lower birth weight then normal

No evidence of brain damage at birth (unless born to mother with PKU).

Vomiting, some times misdiagnosed as pyloric stenosis.

Irritability, poor feeding

Seborrheic or eczematoid rash (mild and disappears as child grows)

Unpleasant odour described as a musty or mousy smell of phenylacetic acid

Hypopigmentation (fair skin and blue eyes due to decreased melanin synthesis because of compromised tyrosine formation).

Most infants are hypertonic with hyperactive deep tendon reflexes

Epileptic fits, Seizures (in about ~25%-35%)

Electroencephalographic abnormalities (50%).

Irreversible mental retardation (develops within 3-6 months following birth).

Excess phenylacetic acid in urine/perspiration.

Peculiarities of gait, stance, and sitting posture.

Behaviour problems; autistic-like behaviours, hyperactivity, agitation, aggression (seen in untreated adults).

In untreated children prominent maxilla with widely spaced teeth, enamel hypoplasia, and growth retardation are other common finding. The incidence and severity epilepsy is closely related to the degree of intellectual impairment and is not specific for this condition. Adults with PKU whose condition were not detected (pre PKU neonatal screening in developed world), these patients have impaired intelligence and lose ~50 points off their IQ during the first 12 months (Koch et al.1974) as cited by (Scriver & Kaufman.2001) and often have fair skin and blue eyes, this can be attributed to reduced melanin synthesis because of inhibition of tyrosinase activity by phenylalanine accumulation. The most severely affected adults are microcephalie having a skull circumference of less than 43 cm. Although an infant with untreated PKU is normal at birth, defects in myelination and gliosis with deficient nerve cell maturation occur rapidly if a normal diet is given in first few months. If a phenylalanine-free diet is instituted, further intellectual and neurological deterioration are prevented but if any deterioration has already occurred, there is little recovery to the status quo ante.

Clinical Consequences

The mechanism by which high phe concentration results in intellectual impairment is yet to be clarified, according to Scriver & Kaufman (2001) PKU pathogenesis can be considered from three view points;

Deficiency of tyrosine in the brain

The effect of phenylalanine on transport and distribution of metabolities in the brain

An effect on neurochemical processes.

Tyrosine Deficiency

PKU is a disease of abnormal levels of normal amino acid; tyrosine is promoted to the status of an essential dietary amino acid in PKU because of sever deficiency of the hepatic enzyme phenylalanine hydroxylase. Tyrosine is a precursor of thyroxin, melanin and the neurotransmitter dopamine and noradrenaline and is incorporated in all proteins. Tyr is converted to l-DOPA (3, 4-dihydroxy-l-Phe), a precursor of dopamine and other catecholamine neurochemicals; in the "tyrosine/dopamine" theory (i) the foetus/newborn is unable to obtain tyrosine from phe supply due to the inactive PAH,(ii) the maternal supply of tyrosine is also compromised in carriers of PKU (heterozygosity).this hypothesis is not supported by the following evidence;(1) postnatal tyrosine supplement without reduction of phe alone does not prevent impaired postnatal cognitive development,(2) phe restriction alone should not be beneficial ,according to hypothesis but yet it does prevent neurotoxicity (treatment of PKU).

The effect of phenylalanine on transport and distribution of metabolites in the brain;

Large neutral amino acids (LNAAs) including phe, compete for transport across the blood brain barrier (BBB) via the L-type amino acid carrier. Elevated plasma phe impairs brain uptake of other LNAAs in patients with PKU. Amino acid uptake on system L across the BBB can be measured noninvasively in vivo by positron emission tomography using a labelled [11C] inert substrate .in vivo studies show competition between large neutral amino acids, such as branched chain amino acids, tyrosine and tryptophan and phe on the blood brain barrier transport system. Phenylalanine has the highest affinity for the system. Accordingly elevated concentration of phe could impair uptake of branched chain amino acids (tyrosine and tryptophan) reducing there availability for synthesis of neurotransmitters (cerebral dopamine and serotonin depletion) in the brain in hyperphenylalaninemic state in PKU. Direct effects of elevated Phe concentrations on several enzyme systems, and consequences of a concomitant depletion of other LNAAs in the brain, are thought to be the most important factors for the disturbed brain development in untreated PKU (Pietz et al., 1999).

An effect on neurochemical processes.

The neuropathology seen in treated and untreated PKU appears to involve hypomyelination and demyelination or both. Brain histology and cellular development are altered in human PKU. The number and spread of dendritic basilar processes of large pyramidal cells are reduced by HPA in rat pups, high levels of phe and its metabolites, both in culture and in vivo decrease proliferation and increase loss of neurons. DNA content is decreased in affected brain cells, and its synthesis is impaired. The net effect is impaired brain growth. Long exposure to the deviant metabolic phenotype impairs development of brain architecture in untreated PKU patients, with abnormalities in myelination, width of cortical plate, cell density and organisation, dendritic arborization, and number of synaptic spines. Phenylalanine itself is probably the neurotoxic agent in PKU. Metabolites of phenylalanine are not found in the human (or mouse) disease at sufficiently high concentration to distribute metabolic and chemical relationships in brain.

Diagnosis and Laboratory testing /screening

All infants in the UK are screened for PKU at 6-14 days old, after establishing feeding and protein intake. The test is not done before 3 to 4 days of age because Infants with PKU frequently display normal blood phe at birth. The mother is able to clear excess phe in her affected foetus through the placenta. Phenylalanine level in PKU increases in relation to time after birth in the first week of life. The diagnosis of classical PKU usually depends on demonstration of persistent elevation of plasma phenylalanine concentrations. The concentration of phe must be at least 5 Ã- the upper limit of normal (0.5 to 2.0 mg/dL) before it is associated with retardation .Prior to the newborn screening programmes in the UK for PKU in the 1960; PKU was one of the common causes of mental retardation. Collection of newborn blood by heel prick onto filter paper cards has become an accepted facet of newborn care through out the developed world. Biochemical identification of PKU original was by a colour change reaction of urinary phenylpyruvic acid with ferric chloride. This resulted in too many false negatives and was replaced in UK in the 1960 by Guthrie bacterial inhibition assay. Guthrie test involves determining the ability of plasma to support growth of the bacterium Bacillus subtilis, which can only grow if phenylalanine is present in medium. The bacterial inhibition assay is being replaced by chromatographic, fluorometric or mass spectrometric methods for the estimation of phenylalanine. Fluorometric and HPLC techniques provide high levels of accuracy. Patients with negative Guthrie test are recommended for HPLC and fluorometric for the detection of heterozygous and for determining the extent of hyperphenylalaninaemia in maternal PKU. Absence of dihydropteridine reductase (DHPR) activity results in deficient phenylalanine in the presence of normal phenylalanine hydroxylase enzyme and also in deficient neurotransmitter production. Defects in the synthesis of biopterin, which produce the same effects as DHPR deficiency, are identified by urine assay of biopterin and neopterin.

Molecular Diagnosis of PKU

PKU mutation analysis is important in detection of carriers, for prenatal diagnosis. Polymorphic haplotypes can be analysed at the PAH locus. Molecular genetics techniques utilised include Southern blotting, restriction enzyme digestion, detection of mutation by sequencing and multiplex ligation probe amplification.

Table 2 Investigation of neonatal hyperphenylalaninemia

Quantitative analysis of plasma amino acids

To determine accurately the plasma phe concentration and the concentration of tyrosine in order to distinguish hyperphenylalaninemia caused by transient hereditary defects in tyrosine metabolism

Blood DHPR assay

For the diagnosis of DHPR deficiency. This can be on dried blood spots.

Urinary biopterin/neopterin ratio and percentage BH4

To identify defects in biopterin biosynthesis. Must be done before BH4 loading test.

BH4 loading test

Another approach to the identification of defects in BH4 biosynthesis. Serial measurements of plasma phenylalanine are done immediately before an at 4-hour interval after oral administration of BH4, 20 mg per Kg body weight

PAH mutation analysis

Rarely needed for diagnosis of PKU. However, it is necessary for prenatal diagnosis of the condition in future pregnancies.


Autosomal single gene inheritance

Single gene defects (also known as Mendelian disorders, monogenic disorders or single locus disorders. these are a group of diseased caused by a single mutated gene which alters the coding information and produces a protein which is either defective or no protein at all. The disease symptoms are a direct result of the protein deficiency in function (structural) or absence. Three pattern of inheritance occur:

Autosomal dominant

Autosomal recessive-inborn errors of metabolism


Inherited metabolic disease are inherited in the same manner as Garrod's original inborn errors of metabolism, they are Mendelian, single gene defects, transmitted in an autosomal recessive manner. The autosomes (44) comprise 22 homologus pairs of chromosomes; within the chromosome the gene occupy a specific location or locus. The autosomes are inherited in pairs one from maternal and one from paternal. The individual must be homozygous for the mutation in same gene for disease expression. The parents who are heterozygous for mutant (allele) gene are carriers and pass the mutation to there offspring .Expression of mutation gene (metabolic defect) might not be seen in several generation, autosomal recessive single gene disease often show clear pattern in which the disease "skips" one or more generations.

There is a 25 % (homozygote's) probability of having affected child e.g. with cystic fibrosis, and 50 % (heterozygote's) of offspring to be carriers and 25% that the offspring will not recessive the defective gene and will not be a carrier. If one parent is carrier and the other is normal there is a 50% chance will not recessive the defective gene and 50% chance that there off spring will be a carrier ,there's a 0% chance that there child will have the disease.

Figure 1: Autosomal recessive inheritance

There are more than 6600 human single gene disorders, which collectively affect ~2% of population. The most common autosomal recessive single gene disorder amongst Caucasians is cystic fibrosis, which occurs in a frequency of 1 in 25oo.The ∆F508 mutation is the most common and results in a defective cystic fibrosis transmembrane conductance regulator (CFTR).


Autosomal Recessive Disorders

With autosomal recessive inheritance, the heterozygous carriers of a single mutation usually are clinically normal; rarely, they may show some clinical signs (manifesting heterozygote's). However, persons who have inherited a mutation in the same gene from both parents (homozygote's) will show clinical manifestations of the disease. If both parents are carriers of a mutation in the same gene, then each of their children has a 25% risk for being homozygous for that gene and having the disease. Autosomal recessive disorders usually are seen in only one generation, typically among siblings (Fig. 43-3; see Table 43-1). Both males and females can be affected. In small families, autosomal recessive disorders may appear as isolated or sporadic cases. Autosomal recessive disorders may sometimes appear in multiple generations of highly inbred families with consanguineous marriages. Examples of autosomal recessive neurological disorders are phenylketonuria (PKU), Tay-Sachs disease, Lafora body myoclonic epilepsy, infantile spinal muscular atrophy, Wilson's disease, and Friedreich's ataxia.

Analyses of diploid PAH genotypes and their correlations with

metabolic and clinical phenotypes show that some mutations

(usually null alleles) confer the classical PKU phenotype, whereas

others (usually missense alleles) confer forms of HPA in a

quasicontinuous genotype-phenotype distribution reflecting ''phenogenetic

equivalence'' [Weiss and Buchanan, 2003]. But within

the latter, phenotypes are not always consistent with predictions

from genotype [Kayaalp et al., 1997; Guldberg et al., 1998; Desviat

et al., 1999]. Moreover, patients with similar mutant genotypes

can have dissimilar phenotypes.

A single process can not by it self explain the PKU brain phenotype, a multiple of complex inter connecting biochemical pathways and transport of metabolities (across the blood -brain barrier) defects ,ultimately result in distribution of normal chemical homeostasis of the brain and result in impaired brain development