Parental gender, age at birth and expansion length influence GAA ...

 1998 Oxford University Press

Human Molecular Genetics, 1998, Vol. 7, No. 12 1901–1906

Parental gender, age at birth and expansion length influence GAA repeat intergenerational instability in the X25 gene: pedigree studies and analysis of sperm from patients with Friedreich’s ataxia Giuseppe De Michele, Francesca Cavalcanti1, Chiara Criscuolo, Luigi Pianese1, Antonella Monticelli1, Alessandro Filla* and Sergio Cocozza1 Department of Neurological Sciences and 2Department of Molecular and Cellular Biology and Pathology and CEOS, Federico II University, via Pansini 5, 80131 Naples, Italy Received June 1, 1998; Revised and Accepted August 7, 1998

Friedreich’s ataxia is the first known autosomal recessive disease caused by an unstable trinucleotide expansion mutation. The most frequent mutation is expansion of a GAA repeat in the first intron of gene X25. We studied transmission of the expanded GAA repeat in 37 Friedreich’s ataxia pedigrees and analysed blood and sperm alleles in eight patients. We showed intergenerational instability in 84% of the alleles with an overall excess of contractions. Both contractions and expansions of the GAA repeat occurred in maternal transmission with a stronger tendency to expand for smaller repeats and to contract for longer repeats. Paternally transmitted alleles contracted only. Parental age and the intergenerational change in expansion size were directly correlated in maternal transmission and inversely in paternal transmission. The size of the GAA expansion was slightly lower in patients than heterozygous carriers. Sperm analysis confirmed the tendency to contract of paternal alleles, which was more marked with ageing. The degree of contraction of the GAA repeat in sperm was much higher than that found in intergenerational transmission and was directly related to the repeat size. A blood expanded allele reverted to normal size in the sperm of one patient. This study suggests the existence of different mutational mechanisms in Friedreich’s ataxia alleles, which occur both pre- and post-zygotically. INTRODUCTION Trinucleotide repeat expansions are associated with several human genetic diseases. Within them, two classes of diseases may be distinguished (1,2). The first one comprises fragile X syndrome (FRAXA), FRAXE mental retardation, myotonic

dystrophy (MD) and Friedreich’s ataxia (FA). These disorders are caused by large expansions in non-coding regions of a gene and show different patterns of inheritance. With the exception of MD, expansions seem to cause loss of function of the gene affecting both neural and non-neural tissues. Diseases of the second class are represented by X-linked spinobulbar muscular atrophy (SBMA), Huntington’s disease (HD), dentatorubropallidoluysian atrophy and spinocerebellar ataxias (SCA1, SCA2, SCA3, SCA6 and SCA7). Inheritance is autosomal dominant in most of them. Increases in the number of CAG repeats are small and localized in coding regions, probably causing the synthesis of a toxic protein (gain of function) and death of specific neuronal populations. The term dynamic mutation refers to mitotic and meiotic instability of trinucleotide repeats (3). Somatic mosaicism has been shown in FRAXA (4), MD (5,6) and FA (7), whereas it is less evident or absent in SCA1 (8), HD (9) and SBMA (10). Longer repeats are associated with higher instability and, above a threshold of ∼35–50 repeats, they tend to expand over generations. Repeat length is correlated with onset age and disease severity. Anticipation of onset age in generations, associated with parental sex bias, is found. FA is the first autosomal recessive disorder identified among dynamic mutation diseases. The most frequent mutation is expansion of a GAA repeat in the first intron of gene X25, which encodes a 210 amino acid protein named frataxin (11), for which a mitochondrial function has been suggested (12–14). Few patients with point mutations have been described (11,15). Expansion of the GAA repeats leads to a reduction in frataxin mRNA levels which is proportional to the length of the expansion (14). We and other groups also showed a correlation between expansion size and disease onset and severity (7,16,17). Information about repeat instability in triplet diseases may help in understanding the molecular basis of dynamic mutations. We previously demonstrated intergenerational instability of the expanded GAA repeat in the X25 gene in 21 FA pedigrees showing a clear effect of parental gender on trinucleotide stability (18). Normal alleles are stable on transmission, but we reported

*To whom correspondence should be addressed. Tel: +39 81 7462476; Fax: +39 81 5461541; Email: [email protected]

1902 Human Molecular Genetics, 1998, Vol. 7, No. 12

Figure 1. Transmission of the GAA expansion in 13 carrier father– and 19 carrier mother–child pairs. Circles on the line represent no size variation in the GAA repeat transmission.

a rare instance of hyperexpansion of a normal allele (19). Here, we analyse the GAA repeat in the blood and sperm of eight affected individuals. We also compare the results of sperm analysis with those from pedigree studies and investigate the effect of parental age on intergenerational instability. RESULTS Family studies The number of trinucleotide repeats was determined in 32 parent carrier–child pairs from 25 FA pedigrees in which haplotype analysis identified the transmitted allele unambiguously. All the alleles in the normal range (8–23 triplets) were transmitted unchanged. The results concerning transmission of the expanded alleles (range 364–1229) are shown in Figure 1. The FA allele was transmitted unmodified in three out of 19 mother–offspring pairs and in two out of 13 father–offspring pairs. Twenty-seven alleles were unstable on transmission (18 contractions and nine expansions). Maternally transmitted alleles showed both increases (nine alleles) and decreases (seven alleles), whereas paternally transmitted alleles showed only decreases (11 alleles). The distribution of contractions and expansions differed according to parental gender (Fisher’s exact test, P < 0.05), not according to that of the offspring. The mean change in parent–offspring transmission was 137 ± 112 triplets (absolute value), the mean contraction 141 ± 88 and the mean expansion 207 ± 125. The mean number of repeats on expanded alleles was 895 ± 176 in parents and 874 ± 186 in offspring. The difference was not statistically significant by paired t-test. GAA size in mothers was not significantly different from that in their children (894 ± 215 versus 961 ± 164), whereas it was significantly higher in fathers (897 ± 105 versus 748 ± 143, P < 0.001). Parental GAA repeat length was inversely correlated with the intergenerational change in maternal transmission, with a stronger tendency to expand for smaller repeats and to contract for longer repeats (r = –0.656, P < 0.01). No relationship was observed in paternal transmission (r = –0.076).

We analysed the relationship between parental age at birth and intergenerational instability. The absolute value of intergenerational change was correlated with parental age in the whole sample of pairs (r = 0.485, P < 0.005). We also found a direct correlation between parental age and intergenerational change in maternal transmission (r = 0.532, P < 0.05) and an inverse correlation in paternal transmission (r = –0.665, P < 0.05), i.e. a female tendency to expand and a male tendency to contract were more evident in older parents (Fig. 2). In order to evaluate whether the genetic status of the offspring could affect intergenerational instability we also determined the expansion length of both the alleles from the patient and of the single expanded allele from a heterozygous sibling in 60 patient–carrier sibling pairs from 34 families. We found that it was slightly lower in homozygotes (836 ± 162 triplets) than in heterozygotes (873 ± 187, P < 0.05). Sperm analysis Figure 3 shows the PCR amplification of blood and sperm DNA in six FA patients homozygous for the expansion and in two patients compound heterozygous for the GAA expansion and the I154F mutation (11). Patients homozygous for the GAA expansion were selected among those with a large expansion (range 815–1063) on one allele and a relatively small expansion (163–427) on the other. This condition allowed us to better distinguish the alleles in blood and in sperm. The two normal blood alleles did not change in sperm, whereas all the expanded blood alleles resulted in sperm alleles of different size. Furthermore, the smear of the sperm allele appeared slightly broader than the corresponding leukocyte allele. In one case (patient no. 4) we found a sperm allele of normal size. Sequence analysis of the allele showed a pure sequence of eight GAA trinucleotide repeats. To exclude contamination by PCR product derived from another individual, a new semen sample was obtained from the patient and the analysis was repeated. The results of the second experiment were the same. On the basis of the pedigree studies and of a previous sperm analysis of individuals heterozygous for the GAA expansion (18) we assume that an increase in the number of triplets does not

1903 Human Genetics, 1998, 7, No. NucleicMolecular Acids Research, 1994, Vol. Vol. 22, No. 1 12 1903

Figure 2. Correlation between parental age at birth and intergenerational change in GAA expansion size in 13 carrier father– and 19 carrier mother–child pairs.

from the statistical analysis the smallest of the two GAA repeats in blood and sperm. We compared the mean blood/sperm decrease in the GAA repeats (513 ± 112 triplets) with that observed in father–offspring transmission (147 ± 107). The difference was statistically significant (P < 0.001). The difference remained significant (P < 0.05) when the smallest alleles were also included. Table 1. GAA repeat size in blood and sperm in patients with Friedreich’s ataxia Patient

1

Figure 3. PCR amplification of sperm and blood DNA from six Friedreich’s ataxia patients homozygous for the GAA expansion (1–6) and two compound heterozygotes for the GAA expansion and the I154F mutation (7–8). M, molecular weight markers (1 kb ladder); L, leukocytes; S, sperm.

occur in sperm. Following this assumption the largest sperm allele could derive only from the largest blood allele and we were able to match blood and sperm alleles in all patients except patient no. 4 (Table 1). In fact, since sperm alleles of this patient were both of smaller size than blood alleles, blood/sperm matching was not possible and his data were excluded from subsequent statistical calculations. Mean size of the blood FRDA alleles was 666 ± 381 triplets and mean size of the corresponding sperm alleles 298 ± 175, with a mean difference of 348 ± 222 (range 82–683). We found a high direct correlation between the size of blood alleles and their decrease in sperm (r = 0.945, P < 0.001). This correlation completely disappeared when considering the percentage reduction, which was 51 ± 10% with a range of 34–67%. As reported above, blood alleles of patients in the sperm study showed a much larger range (163–1063 triplets) than alleles from male carriers in pedigree analysis (739–1074). Therefore, to compare data from family studies with those from sperm analysis, in the six patients homozygous for the expansions we excluded

Age

59

GAA repeat size

Blood/sperm difference (%)

Blood

Sperm

1022

339

683 (67)

163

81

82 (51)

982

374

608 (62)

2

55

177

86

91 (51)

3

39

945

539

407 (43)

339

223

116 (34)

4

46

1063

276

427

8

815

413

402 (49)

327

113

214 (65)

982

427

555 (57)

207

119

88 (42)

576

407 (41)

5

6

22

46

7

30

982 10

10

8

32

1050

521

8

8

0 529 (65) 0

Patients nos 7 and 8 are compound heterozygotes for the GAA expansion and the I154F mutation. Since both sperm alleles are smaller than those from leukocytes, blood/sperm matching was not possible in patient no. 4.

We also studied the relationship between GAA repeat contraction in sperm and donor age. No correlation was observed considering all sperm alleles, whereas considering only the

1904 Human Molecular Genetics, 1998, Vol. 7, No. 12

Figure 4. Correlation between donor age and sperm/blood GAA expansion size difference.

largest ones the degree of contraction of the GAA repeat in sperm was directly correlated with age of the donor (r = 0.871, P < 0.05; Fig. 4), confirming the results obtained in the pedigree analysis. DISCUSSION In a previous paper we showed intergenerational instability of the expanded FRDA GAA alleles with a bias towards contraction (18). We also showed a strong effect of parental gender, with both contractions and expansions of the GAA allele occurring in maternal transmission but only contractions in paternal transmission. The size of the parental GAA repeat played a role only in maternal transmission, in which large repeats tended to regress in size. The tendency to decrease of paternally transmitted alleles has been confirmed by Monrós et al. (20). These authors also found a direct correlation between paternal expansion and intergenerational variation of the expansion size. In this paper we compared leukocyte and sperm DNA from six individuals homozygous and two heterozygous for the expansion. The results of both sperm analysis and pedigree studies are consistent, showing 100% of contractions in male-derived alleles. However, the degree of contraction obtained in sperm is significantly different from that found in family studies. In fact, length decreases from blood to sperm alleles are more pronounced than those seen in paternal transmission. In one patient we observed a marked reduction in one blood allele which contracted to eight GAA repeats in sperm, which is one of the most frequent sizes among normal alleles (19). This study also demonstrated a role of parental age in intergenerational instability: both the tendency to contract in male transmission and that to expand in female transmission are more evident with ageing. Intergenerational instability in expanded GAA repeats may occur in gametogenesis, as seen in HD (21,22) and SCA1 (23), or post-zygotically, as seen in FRAXA and MD (24,25). In the last two disorders mitotic instability occurs during early postzygotic development with expansion limitation during male transmission, more evident in FRAXA (26).

The different degree of GAA repeat contraction observed from blood to sperm in comparison with that occurring from one generation to another suggests post-zygotic occurrence of instability also in FA. Mitotic instability has recently been demonstrated in FA (7) and a molecular mechanism similar to that shown in FRAXA and MD might be suggested. The tendency for contraction during male transmission appears more marked in FA than in FRAXA or MD. There are some drawbacks in comparing results of sperm and pedigree analyses. We performed a PCR analysis of total sperm which could not show single gamete repeat lengths. In addition, we cannot exclude an effect of allele size on fertility or viability. However, it is not likely that gametes carrying alleles of larger size are at an advantage in fertilization events. Finally, we matched blood and sperm alleles assuming no expansion event. This hypothesis appears the most likely, but exceptions might occur. Also, there was bias ascertainment in the sperm study. In the six homozygous patients who provided sperm for analysis, one of the two alleles was of a size at the lower end of the expanded range. For this reason, to compare sperm and pedigree analyses we considered only large expanded alleles. However, even including all the alleles, contraction was more evident in sperm than in pedigree studies. In the sperm of one patient we found complete normalization of an expanded allele. This raises the possibility of transmission of normal alleles from FA patients and carriers. Two examples of reverse mutation have been described in MD (27). In both cases the allele was inherited from an affected father and contracted to a size within the normal range. In this study the normal range PCR product resulted in a single normal sized allele with eight GAA repeats in two different experiments with sperm samples obtained on different occasions. The presence of the same allele in both experiments makes unlikely a meiotic or late mitotic origin of the allele and favours the occurrence of a mitotic mutation in early embryonic stages. It has been suggested that intergenerational instability in FA may be post-zygotic since affected individuals had shorter expansions than carriers (20). Comparison of expansion length in patients and heterozygotes may be affected by sample ascertainment biases. Molecular studies might not include patients with the largest expansions because of early death. In addition, most heterozygous siblings could originate from the families of the most severely affected patients because they may be more willing to provide blood samples for research. To minimize these biases we used patient–carrier sibling pairs. Only two early deaths of patients with FA occurred in the studied pedigrees. However, we could not exclude a selection against embryos homozygous for large expanded alleles. The different size of expansion found in patients and carrier siblings with a mean difference of 37 repeats may suggest that the genetic status of the offspring may affect GAA repeat instability with a stronger tendency to contract in homozygotes than heterozygotes. The parental sex bias and the correlation between parental age and intergenerational instability, confirmed by sperm data, are in favour of a pre-zygotic occurrence of instability. Oogonia undergo a fixed number of mitotic divisions and cease dividing before the end of fetal gestation, whereas spermatozoa have high turnover rates. The different number of cell divisions in spermatogenesis and oogenesis and/or a specific selection process towards small expansion gametes in spermatogenesis

1905 Human Genetics, 1998, 7, No. NucleicMolecular Acids Research, 1994, Vol. Vol. 22, No. 1 12 1905 could explain the parental bias in FA. The increasing number of mitotic events with age could account for greater instability in older males. An effect of maternal ageing on instability has recently been demonstrated in SCA1 transgenic mice (28). An age-related deficiency in DNA repair mechanisms might be involved. Finally, the pedigree study confirmed an effect of expansion size on the direction of change in the offspring for maternal transmission, with an increased tendency to contract for the largest expanded alleles. The latter finding could be completely related to biological phenomena; however, the hypothesis of regression to the mean must be considered. In the range investigated for paternal alleles (739–1074 triplets) we found no correlation between expansion length and intergenerational change. However, the sperm study, which allowed observation of the behaviour of small expanded alleles, showed a reduced degree of contraction for these alleles, even though the percent value of contraction did not change. This behaviour appears peculiar to FA compared with the other dynamic mutation diseases, which show a tendency to increase in size and might suggest a selection against gametes bearing large expansions. The overall tendency of the FA expanded alleles to contract would lead to their disappearance in the population. Expansion of alleles from the normal range pool to the disease-causing size has recently been reported (19,29,30) and it could balance the process, contributing to maintenance of the FA mutation. It is unlikely that a single molecular mechanism can explain GAA repeat contraction in male transmission, prevalence of an increase in female transmission and hyperexpansion of alleles from the normal pool. Clues from our study suggest that both preand post-zygotic instability mechanisms exist. MATERIALS AND METHODS Study population To evaluate the intergenerational instability of the GAA repeat we examined blood DNA of 121 individuals (44 homozygous and 77 heterozygous for the expansion) from 37 FA kindreds, mostly originating from Southern Italy. Data concerning a subset of families have already been reported in a preliminary paper (18). We also examined the blood and sperm from eight FA patients. Six of them were homozygous for the GAA expansion and the remaining two compound heterozygotes for the GAA expansion (815 and 982 triplets) and the I154F mutation (11). All gave informed consent to the study. Molecular analysis DNA was prepared from fresh blood. In eight patients we also prepared DNA by extracting pelletted sperm from semen samples according to previously reported procedures (9). To analyse the GAA repeat in the first intron of the X25 gene we used the following primers: GAA-104F, 5′-GGCTTAAACTTCCCACACGTGTT-3′; GAA-629R, 5′-AGGACCATCATGGCCACACTT-3′. These primers flank the GAA repeat and generate a PCR product of 500 + 3n bp (n = number of GAA triplets). Amplifications were conducted using the long PCR protocol (Boehringer Mannheim Long Expand) in 10 cycles composed of the following steps: 94
C for 10 s, 60
C for 30 s and 68
C for 3 min, followed by a further 20 cycles in which the length of the 68
C step was increased by 20 s/cycle. Glycerol

(10%) was added to the PCR mixture to better amplify the largest expanded alleles. PCR products were separated on 1% agarose gels. The sizes of the alleles were estimated by a least squares fit of fragment size to gel mobility, using the computer program DNAFRAG v.3.03. The estimated error in size determination was ∼30 triplets for the largest expanded alleles. Statistical analysis Means and frequencies were compared by paired or unpaired t-test and Fisher’s exact test. Correlations were studied by Pearson’s correlation coefficient. All the tests were two-sided. Statistical calculations were performed using the SPSS/PC+ computer package. ACKNOWLEDGEMENTS This work has been supported in part by grant no. 969 from Italian Telethon (S.C.). F.C. is the recipient of a fellowship of the Associazione Italiana Sindromi Atassiche. ABBREVIATIONS FA, Friedreich’s ataxia; FRAXA, fragile X syndrome; HD, Huntington’s disease; MD, myotonic dystrophy; SBMA, Xlinked spinobulbar muscular atrophy; SCA, spinocerebellar ataxia. REFERENCES 1. Bates, G. and Lehrach, H. (1994) Trinucleotide repeat expansions and human genetic disease. Bioessays, 16, 277–284. 2. Mandel, J.L. (1997) Breaking the rule of three. Nature, 386, 767–769. 3. Richards, R.I. and Sutherland, G.R. (1992) Dynamic mutations: a new class of mutation causing human disease. Cell, 70, 709–712. 4. Fu, Y., Kuhl, D.P.A., Pizzuti, A., Pieretti, M., Sutcliffe, J.S., Richards, S., Verkek, A.J.M.H., Hilden, J.J., Fenwick, R.G., Warren, S.T., Oostra, B.A., Nelson, D.L. and Caskey, C.T. (1991) Variation of the CGC repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell, 67, 1047–1058. 5. Brook, J.D., McCurrach, M.E., Harley, H.G., Buckler, A.J., Church, D., Aburatani, H., Hunter, K., Stanton, V.P., Thirion, J.P., Hudson, T., Sohn, R., Zemelman, B., Snell, R.G., Rundle, S.A., Crow, S., Davies, J., Shelbourne, P., Buxton, J., Jones, C., Juvonen, V., Johnson, K., Harper, P.S., Shaw, D.J. and Housman, D.E. (1992) Molecular basis of myotonic distrophy: expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member. Cell, 68, 799–808. 6. Lavedan, C., Hofmann-Radvanyi, H., Shelbourne, P., Rabes, J.P., Duros, C., Savoy, D., Dehaupas, I., Luce, S., Johnson, K. and Junien, C. (1993) Myotonic dystrophy: size and sex-dependent dynamics of CTG meiotic instability and somatic mosaicism. Am. J. Hum. Genet., 52, 875–883. 7. Montermini, L., Richter, A., Morgan, K., Justice, C.M., Julien, D., Castellotti, B., Mercier, J., Poirier, J., Capozzoli, F., Bouchard, J.P., Lemieux, B., Mathieu, J., Vanasse, M., Seni, M.H., Graham, G., Andermann, F., Andermann, E., Melançon, S.B., Keats, B.J.B., Di Donato, S. and Pandolfo, M. (1997) Phenotypic variability in Friedreich ataxia: role of the associated GAA triplet repeat expansion. Ann. Neurol., 41, 675–682. 8. Chung, M.-Y., Ranum, L.P.W., DuvicK, L.A., Servadio, A., Zoghbi, H.Y. and Orr, H.T. (1993) Evidence for a mechanism predisposing to intergenerational CAG repeat instability in spinocerebellar ataxia type I. Nature Genet., 5, 254–258. 9. Telenius, H., Almqvist, E., Kremer, B., Spence, N., Squitieri, F., Nichol, K., Grandell, U., Starr, E., Benjamin, C., Castaldo, I., Calabrese, O., Anvret, M., Goldberg, Y.P. and Hayden, M.R. (1995) Somatic mosaicism in sperm is associated with intergenerational (CAG)n changes in Huntington’s disease. Hum. Mol. Genet., 4, 189–195. 10. Spiegel, R., La Spada, A.R., Kress, W., Fishbeck, K.H. and Schmid, W. (1996) Somatic stability of the expanded CAG trinucleotide repeat in X-linked spinal and bulbar muscular atrophy. Hum. Mutat., 8, 32–37.

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