Box 16.7, Unstable expanding repeats - a novel cause of disease - Human Molecular Genetics

Box 16.7Unstable expanding repeats - a novel cause of disease

Unstable expanding trinucleotide repeats were an entirely novel and unprecedented disease mechanism when first discovered in 1991, and they raise two major questions:

What is the mechanism of the instability and expansion? This is discussed in Section 9.5.2
Why do expanded repeats make you ill? Discussed here.

A hallmark of all these diseases is anticipation - that is, the age of onset is lower and/or the severity worse, in successive generations. Two different classes of expansion have been noted; the currently known examples are tabulated below. In some cases, intermediate-sized alleles are non-pathogenic but unstable, and readily expand to full mutation alleles (e.g. FRAXA repeats of 50–200 units); in other cases such alleles only very occasionally expand (e.g. HD alleles with 29–35 repeats). Data from OMIM and Andrews et al., 1997. The list of diseases is likely to expand in the future. An expanded polyalanine tract in the HOXD13 gene has been found in patients with synpolydactyly (MIM 186000); the normal gene has a run of 15 alanines and the pathogenic forms have 22–29 alanines. However, this does not seem to be another unstable expanding repeat. The expansion is probably the result of unequal crossing over, and at least in one family it has been stable for 7 generations (Akarsu et al., 1996).

Highly expanded repeats cause loss of gene function

In Fragile-X syndrome and Friedreich ataxia, an enormously expanded repeat causes a loss of function by abolishing transcription. The same is true for the expanding 12-mer in juvenile myoclonus epilepsy. In each case, the disease is occasionally caused by different, more conventional, loss of function mutations in the gene. Such mutations produce the identical clinical phenotype to expansions, apart (presumably) from not showing anticipation. Other similar highly expanded repeats, such as FRA16A (expanded CCG repeat) or FRA16B (an expanded 33 bp minisatellite) are nonpathogenic, presumably because no important gene is located nearby.

Myotonic dystrophy may be different because no other mutation has ever been found in a myotonic dystrophy patient, so there must be something quite specific about the action of the CTG repeat. It may affect processing of the primary transcript in a specific way, or it may affect expression of a whole series of genes by altering chromatin structure in this gene-rich chromosomal region. The site of the expansion forms part of the CpG island of an adjacent gene, DMAHP (MIM 600963), and the expansion reduces expression of this gene. SCA8 (Koob et al., 1999) may have a similar mechanism.

The CAG repeats encode polyglutamine tracts within the gene product that cause it to aggregate within certain cells and kill them

Common features of the eight diseases caused by expansion of an unstable CAG repeat within a gene include:

They are all late-onset neurodegenerative diseases, and except for Kennedy disease, are all dominantly inherited.

No other mutation in the gene has been found that causes the disease.

Unstable expanding repeats - a novel cause of disease

Disease	MIM no.	Mode of inheritance	Location of gene	Location of repeat	Repeat sequence	Stable repeat no.	Unstable repeat no.
1. Very large expansions of repeats outside coding sequences
Fragile-X site A (FRAXA)	309550	X	Xq27.3	5′UT	(CGG)_n	6–54	200–>1000
Fragile-X site E (FRAXE)	309548	X	Xq28	Promoter	(CCG)_n	6–25	>200
Friedreich ataxia (FA)	229300	AR	9q13-q21.1	Intron 1	(GAA)_n	7–22	200–1700
Myotonic dystrophy (DM)	160900	AD	19q13	3′UT	(CTG)_n	5–35	50–4000
Spinocerebellar ataxia 8	-	AD	13q21	Untranslated RNA	(CTG)_n	16–37	110–>500
Juvenile myoclonus epilepsy (JME)	254800	AR	21q22.3	Promoter	(CCCCGC CCCGCG)_n	2–3	40–80
2. Modest expansions of CAG repeats within coding sequences
Huntington disease (HD)	143100	AD	4p16.3	Coding	(CAG)_n	6–35	36–>100
Kennedy disease (SBMA)	313200	XR	Xq21	Coding	(CAG)_n	9–35	38–62
Spinocerebellar ataxia 1 (SCA1)	164400	AD	6p23	Coding	(CAG)_n	6–38	39–83
Spinocerebellar ataxia 2 (SCA2)	183090	AD	12q24	Coding	(CAG)_n	14–31	32–77
Machado-Joseph disease (SCA3, MJD)	109150	AD	14q32.1	Coding	(CAG)_n	12–39	62–86
Spinocerebellar ataxia 6 (SCA6)	183086	AD	19p13	Coding	(CAG)_n	4–17	21–30
Spinocerebellar ataxia 7 (SCA7)	164500	AD	3p12-p21.1	Coding	(CAG)_n	7–35	37–200
Dentatorubral-pallidoluysian atrophy (DRPLA)	125370	AD	12p	Coding	(CAG)_n	3–35	49–88

The expanded allele is transcribed and translated.
The trinucleotide repeat encodes a polyglutamine tract in the protein.
There is a critical threshold repeat size, below which the repeat is nonpathogenic and above which it causes disease.
The larger the repeat, above the threshold, the earlier is the age of onset (on average; predictions cannot be made for individual patients, but there is a clear statistical correlation).

Laboratory diagnosis of trinucleotide repeat diseases

(A) Huntington disease. A fragment of the gene containing the (CAG)n repeat has been amplified by PCR and run out on a polyacrylamide gel. Bands are revealed by silver staining. The scale shows numbers of repeats. Lanes 1, 2, 6 and 10 are from unaffected people, lanes 3, 4, 5, 7 and 8 are from affected people. Lane 5 is a juvenile onset case; her father (lane 4) had 45 repeats but she has 86. Lane 9 is an affected fetus, diagnosed prenatally. Courtesy of Dr Alan Dodge, St Mary's Hospital, Manchester. (B) Myotonic dystrophy. Southern blot of DNA digested with EcoRI. Bands of 9 or 10 kb (arrows) are normal variants. The grandfather has cataracts but no other sign of myotonic dystrophy. His 10 kb band appears to be very slightly expanded, but this is not unambiguous on the evidence of this gel alone. His daughter has one normal and one definitely expanded 10 kb band; she has classical adult onset myotonic dystrophy. Her son has a massive expansion and the severe congenital form of the disease. Courtesy of Dr Simon Ramsden, St Mary's Hospital, Manchester.

The androgen receptor mutation in Kennedy disease provides clear evidence that CAG-repeat diseases involve a specific gain of function. Loss of function mutations in this gene are well known and cause androgen insensitivity or testicular feminization syndrome (MIM 300068), a failure of male sexual differentiation. The polyglutamine expansion, by contrast, causes a quite different neurodegenerative disease, although patients often also show minor feminization. The other CAG-repeat disease genes so far identified are widely expressed and encode proteins of unknown function. When the polyglutamine tract exceeds the threshold length the protein aggregates, forming an inclusion body that apparently kills the cell (Kim and Tanzi, 1998). The different clinical features of each disease reflect killing of different cells, presumably because of interactions with other cell-specific proteins. Neuronal cell death caused by protein aggregates is a common thread in the pathology of CAG repeat diseases, Alzheimer disease, Parkinson disease and the prion diseases; the mechanisms and their general significance remain to be discovered.

Laboratory diagnosis of expanded repeats

A single PCR reaction makes the diagnosis in the polyglutamine repeat diseases. Panel (A) in the figure shows an example from Huntington disease. The very large expansions in myotonic dystrophy (B) require Southern blotting.

From: Chapter 16, Molecular pathology