DNA BASIS FOR PROTEIN VARIATION.

Variations or mutations in DNA can be as small as point mutations, either

transitions (A > G, C > T) or transversions (A > T, C > G), deletions of a single

base, or affect a larger amount of DNA. For example, various amounts of DNA (I

nucleotide to over a million bases) can be either inserted or deleted from a given

sequence. Insertions may involve duplication of DNA or represent an insertion of a

different piece of DNA. Inversions and translocations of pieces of chromosome can also

occur and affect proteins whose coding sequence has been dismpted.

These different types of mutation can cause a wide variety of effects in the

protein, depending on whether they occur in the coding sequence, in the promoter, or

affect processing. Alteration of a single base can change an amino acid, as in the case of

sickle cell anaemia where a glutamate is altered to a valine at amino acid 6 of the P-globin

chain. Alternatively, single base changes can cause premature teiTnination of translation

due to the introduction of premature stop codons. This type of vaiiation is the most

common cause of inborn errors of metabolism. For example, single base changes in the

phenylalanine hydroxylase gene have been shown to cause approximately 97% of

phenylketonuria (PKU) cases in Denmark (Guldberg et a i , 1993). Deletions or insertions can cause the message to be inconectly translated by causing a frameshift or

they can remove or insert specific amino acids if the number of bases is a multiple of 3.

Larger deletions may result in the production of no protein at all, either because the entire

Muscular Dystrophy (Monaco et al., 1988). Insertions or duplications of larger areas of DNA are at present thought to be rare, although a duplication of 1.5 megabases was

found to underlie the condition Charcot-Marie-Tooth disease (type 1 A) on chromosome

17. Interestingly, deletion of this region leads to hereditary neuropathy with liability to

pressure palsies (HNPP) (also known as tomaculous neuropathy) (Chance et at., 1994).

The processing of a messenger RNA can be altered. IncoiTect splicing of the

message can occur if the mutation occurs at a splice donor/acceptor site (for example in

ornithine 0-aminotransferase (Michaud etal., 1992), in p- thallasaemia (Vogel &

Motulsky, 1986)). Polymoiphic vaiiation can also result from alternative splicing. This

has been found, for example, in the red cell acid phosphatase (A CPI) polymorphism,

where the isozymes S and F are detennined by alternative central portions o f the

molecule. Differences in splicing caused by single base changes lead to genetically

detennined differences in the amount of each isozyme produced (giving rise to the B and

C alleles)(Dissing, 1993).

If the sequence change is in the promoter region then transcription of the message

may be affected (for example causing the persistence of fetal haemoglobin (B eny et a i, 1992)). The polyadenylation signal can also be affected, as in a patient with a

thallasaemia where the mutation responsible was found to be a change from AATAAA to

AATAAG in the a 2 globin gene and this inactivated the 3' processing of this message

(Higgs et a i , 1983; Whitelaw & Proudfoot, 1986).

Recently a few conditions have been found to be due to the expansion of a piece

of sequence containing trinucleotide repeats, such that the number of repeat units is

increased. These include Huntington's chorea where an expanded CAG repeat was

found at the 5' end of the gene 'him ting tin' in affected individuals (The Huntington's

Disease Collaborative Research Group, 1993). Such repeats have also been found in the

coding region of the androgen receptor gene (Spinal and Bulbar Muscular Atrophy)

kinase gene of unknown normal function (GCT)(Buxton etal., 1992; Mahadevan etal., 1992; Fu et al., 1992) and in the 5' untranslated region of FMR-1 (CGG) (FraXA) (Fu e t a l , 1991).

Certain severe fornis of Haemophilia A have recently been shown to involve an

inversion of the telomeric end of the X chromosome causing a break in the factor VIII

gene and incoiporation of inappropriate exons into the messenger RNA (Lakich et at., 1993). This inversion has been suggested to be due to a recombination event between

homologous sequences on the X chromosome (Lakich et a l , 1993). Homologous non­ reciprocal recombination has also been shown to cause protein variation in the alpha

globin cluster where recombination between the highly homologous a l , o2 and \\fal sequences has been observed (Figure 1.2.3.1. shows the gene airangements in both

globin gene clusters). The loss of an alpha globin gene causes mild thallassemia, and

indeed this vai iation is estimated to show a heterozygous frequency of up to 33% in parts

of Africa (Vogel and Motulsky, 1986). The conesponding gene duplication chromosome

bearing three alpha genes has been identified but has only been seen much more rarely.

In the beta globin cluster non-reciprocal recombination results in the production of the so

called Lepore haemoglobin which is a fusion of the ô and p stnictural genes. The other

predicted product of such a recombination would be a fusion product p -5 and the

presence of both Ô and p structural gene products, this so called haemoglobin anti-Lepore

5< I V S 1 I VS 2 3 '

a,

5 kb

' 5- E G y 6 p

Figure 1.23.1

The genomic arrangement of the globin gene clusters.

a, the a globin cluster on chromosome 16p and b, the p globin gene cluster on human chromosome 1 Ip Black boxes, exon sequences; white boxes, introns or pseudogenes; IVS, intervening sequence;

chains

100-1

8 0

60

2 0

Diagrammatic representation of the proportion of globin chains of each type produced at different stages of development.