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Human Molecular Genetics Pages 1207-1214  

Inter- and intrachromosomal sub-telomeric rearrangements on 4q35: implications for facioscapulohumeral muscular dystrophy (FSHD) aetiology and diagnosis
Introduction
Results
Discussion
Materials And Methods
   Controls
   Family Rf100
   Pulsed field gel electrophoresis analysis
   Haplotype analysis
   Fluorescence in situ hybridization
Acknowledgements
References

Inter- and intrachromosomal sub-telomeric rearrangements on 4q35: implications for facioscapulohumeral muscular dystrophy (FSHD) aetiology and diagnosis

Inter- and intrachromosomal sub-telomeric rearrangements on 4q35: implications for facioscapulohumeral muscular dystrophy (FSHD) aetiology and diagnosis

Richard J. L. F. Lemmers1,+, Silvère M. van der Maarel1,*,+, Judith C. T. van Deutekom1, Michiel J. R. van der Wielen1, Giancarlo Deidda2,§, Hans G. Dauwerse1, Jane Hewitt3, Marten Hofker1, Egbert Bakker1, George W. Padberg4, Rune R. Frants1

1MGC Department of Human Genetics, Leiden University Medical Center, Wassenaarseweg 72, 2300 RA Leiden, The Netherlands, 2Institute of Cell Biology, CNR, Rome, Italy, 3School of Biological Sciences, University of Manchester, Manchester, UK and 4Department of Neurology, University of Nijmegen, Nijmegen, The Netherlands

Received November 24, 1997; Revised and Accepted May 9, 1998

The autosomal dominant myopathy facioscapulohumeral muscular dystrophy (FSHD) is causally related to a short EcoRI fragment detected by probe p13E-11. This remnant fragment is the result of a deletion of an integral number of tandemly arrayed 3.3 kb repeat units (D4Z4) on 4q35. Despite intensive efforts, no transcribed sequences have been identified within this array. Previously, we have shown that these repeats on 4q35 have been exchanged for a similar highly homologous repeat locus on 10q26 in 20% of the population and that a short chromosome 10-like array on 4q35 also results in FSHD. Here, we describe the hybrid structure of some of these repeat arrays, reflecting additional sub-telomeric instability. In three healthy individuals carrying a 4-like repeat on chromosome 10 or vice versa, one repeat array was shown to consist of hybrid clusters of 4-derived and 10-derived repeat units. Moreover, employing pulsed field gel electrophoresis analysis, we identified two unrelated individuals carrying deletions of a chromosomal segment (p13E-11) proximal to the repeat locus. These deletions were not associated with FSHD. In one of these cases, however, an expansion of the deletion into the repeat array was observed in one of his children suffering from FSHD. These data provide additional evidence for instability of this sub-telomeric region and suggests that the length of the repeat, and not its intrinsic properties, is crucial to FSHD. Moreover, they are in agreement with the hypothesis that FSHD is caused by a position effect in which the repeat structure influences the expression of genes nearby. Therefore, the region deleted proximal to the repeat locus in healthy individuals can be instrumental to refine the critical region for FSHD1.

INTRODUCTION

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder mainly characterized by asymmetrical weakness of the facial, shoulder girdle and upper arm muscles (1,2). The disorder is clinically and genetically heterogeneous and its major locus (FSHD1) has been mapped to 4q35 by linkage analysis (3-7). The disorder has been shown to be causally related to a short repeat array that remains after deletion of an integral number of tandemly arrayed 3.3 kb repeat units (D4Z4). The size of this polymorphic locus usually varies between 35 and 300 kb, while patients consistently show a fragment <35 kb (8,9). Despite extensive efforts, no transcript has been identified within the deleted region (10-13). Together with its heterochromatic structure, these data are suggestive of a position effect in which the partial deletion of the repeat array may disturb proper expression of genes nearby.

The probe p13E-11 (D4F104S1) detects the polymorphic EcoRI locus on which the repeat units reside and is generally used for diagnostic purposes. However, the probe also recognizes a highly polymorphic repeat locus of similar size on 10q26 consisting of highly homologous repeat units (14). The presence of this homologous locus has complicated FSHD1 molecular diagnosis, since a short fragment on chromosome 10 does not cause FSHD. Recently, a unique chromosome 10-specific BlnI site within the repeat unit has been identified which makes it possible to discriminate between chromosome 4 and chromosome 10 alleles (15). Generally, after digestion with EcoRI and pulsed field gel electrophoresis (PFGE), the probe will recognize four alleles, two from chromosome 4 and two from chromosome 10. However, double digestion with EcoRI and BlnI will digest the chromosome 10 alleles into small fragments while the chromosome 4 copies will remain undigested, except for one BlnI site 3 kb from the proximal EcoRI site.

Recently, we presented data implying sub-telomeric exchanges between the repeat loci on chromosomes 10 and 4 (16). These exchanges result in the presence of chromosome 4 repeats on chromosome 10 and, vice versa, chromosome 10 repeats on chromosome 4. The prevalence of individuals having one (monosomy) or three (trisomy) chromosome 4 alleles in the population as a result of these interchromosomal exchanges is 20% (16).

To analyse the structure of these homologous and polymorphic repeat loci and their behaviour in more detail, we hybridized DNA of apparent monosomic or trisomic individuals with probe 9B6A (D4Z4), recognizing the repeat units themselves. Here, we report on the hybrid structure of some of these repeat arrays and on the deletions proximal to the D4Z4 repeat array observed in patients and controls.

RESULTS

From the 50 healthy unrelated male individuals of a control population, five showed a hybridization pattern compatible with a chromosome 4 repeat on one of their chromosomes 10 and five were presumed to carry a chromosome 10 repeat on one of their chromosomes 4, after hybridization with probe p13E-11 (16). To investigate the nature of these apparently translocated repeats in more detail, we subsequently hybridized DNA from these individuals with 9B6A, which recognizes the repeat itself and not, like p13E-11, a unique region proximal to the repeat. Of these 10 carriers of apparently translocated alleles, seven showed a hybridization pattern compatible with a homogeneous repeat structure, i.e. either a homogeneous stretch of chromosome 4-derived 3.3 kb repeat units on one of the chromosomes 10 or a contiguous stretch of chromosome 10-derived repeat units on one of their chromosomes 4. However, in three cases, a more complex hybridization pattern consistent with a hybrid structure of the repeat locus itself was observed (Fig. 1).


Figure 1. (A) Southern blot analysis of DNA of three unrelated individuals digested with EcoRI (E) and EcoRI/BlnI (E/B). The blot was hybridized with p13E-11 and subsequently with 9B6A. After hybridization with p13E-11, individual 252 most likely carries a 10-like repeat on chromosome 4 based on the BlnI differentiation. However, after hybridization with 9B6A, a previously undetected fragment of 130 kb (arrow) is detected, suggestive of an interjection of one or a few 10-like repeats between p13E-11 and a large cluster of 4-like repeats on chromosome 4. The same holds true for individual 325 carrying a 96 kb cluster (arrow) of chromosome 4 repeats separated from p13E-11 by a cluster of chromosome 10-type repeats. Individual 530 exhibits an additional 23 kb fragment after hybridization with p13E-11. Hybridization with 9B6A reveals the presence of a second fragment of 30 kb. These fragments are most likely derived from a chromosome 10 carrying a repeat array that consists of a 4-like (23 kb), a 10-like and a 4-like (30 kb) repeat cluster respectively. Note that due to the repetitiveness of probe 9B6A, cross-hybridizing fragments are present in the lower part of the blot. (B) Schematic representation of these hybrid chromosomes for each individual. Black arrows represent 4-like repeat units, while shaded arrows represent 10-like repeat units. EcoRI (E) and BlnI (B) sites are depicted. Probe p13E-11 recognizes a sequence proximal to the repeat units, 9B6A recognizes each individual repeat unit as indicated with the dashed boxes.

After hybridization of p13E-11 to genomic DNA digested with EcoRI and BlnI, individual 252 (case 4 in ref. 16) exhibits three chromosome 10-like alleles and one chromosome 4-like allele by inference. On hybridization with 9B6A, the emergence on EcoRI/BlnI digestion of a 130 kb fragment, shortened from the 136 kb EcoRI fragment, implies a cluster of chromosome 4 repeat units separated from the p13E-11 site by one or more BlnI sites, most likely due to the interjection of one chromosome 10-type repeat unit (Fig. 1). Likewise, individual 325 (case 3 in ref. 16) has a similar composition on one of his chromosomes 4, i.e. an interjection of several chromosome 10-type repeat units between the p13E-11 site and the 96 kb cluster of chromosome 4-like repeat units (Fig. 1). Since we cannot determine from which of the two largest EcoRI fragments (120 or 135 kb) this 96 kb repeat cluster is derived, we are not able to estimate the number of interjected chromosome 10-like repeat units (24 or 39 kb). One of the chromosome 10 alleles in individual 530 seems to exhibit an even more complex structure (Fig. 1). Hybridization of p13E-11 shows, apart from the normal chromosome 4 repeat arrays, an additional 23 kb EcoRI/BlnI repeat fragment. Subsequent hybridization with 9B6A reveals a second 30 kb repeat fragment. These results imply a chromosome 10 carrying alternating segments of chromosome 4 (23 kb), 10 and 4 (30 kb) repeated units in the telomeric direction respectively. Again, since we cannot estimate the size of the chromosome 10-type repeat clusters on this allele, it is unclear whether these 23 and 30 kb fragments reside on the 70 or 150 kb EcoRI fragment.

Additionally, another unrelated healthy male individual (547) was identified carrying three alleles that hybridized with p13E-11, two derived from chromosome 10 and only one derived from chromosome 4 (Fig. 2). After hybridization with 9B6A, an additional fragment was observed indicative of a chromosome 4 carrying a deletion of p13E-11.


Figure 2. Hybridization of EcoRI (E)- and EcoR/BlnI (E/B)-digested DNA of individual 547 with probes p13E-11 and 9B6A respectively. This healthy individual carries a deletion of p13E-11 on one of his chromosomes 4 (arrow).

A deletion of p13E-11 was also observed in a family (Rf100) with a sporadic case of FSHD (Fig. 3). After hybridization with p13E-11, the mother was shown to be tetrasomic, i.e. carrying four chromosome 4-like repeat arrays of which two are 65 kb in size and therefore co-migrate. However, only three alleles were observed in the father (I-1), the affected son (II-1) and one unaffected son (II-2), indicative of a deletion of p13E-11. Two of these alleles were derived from chromosome 10 and one from chromosome 4, based on differential BlnI digestion. Confirming a deletion of p13E-11, rather than an additional EcoRI site polymorphism between p13E-11 and the D4Z4 repeats, similar results were obtained with HindIII instead of EcoRI digestion (data not shown). HindIII has no restriction sites in the repeat array and may be used as an alternative for EcoRI. This deletion of p13E-11 was also observed by fluorescence in situ hybridization (FISH) analysis. First, the 4.9 kb KpnI fragment on which p13E-11 resides was hybridized to metaphase chromosomes of I-1, I-2 and II-1 (Fig. 4). This probe contains 900 bp of the D4Z4 repeat and shows an intense and a weak hybridizing chromosome 4 in the father I-1 (Fig. 4A), equally intense hybridizing chromosomes 4 in the mother I-2 (Fig. 4C) and only one chromosome 4 hybridizing in the affected son II-1 (Fig. 4E). Since the father carries repeats of 140 and 60 kb, the mother two repeat arrays of 65 kb and the affected son an array of 65 and 9 kb (not shown) on their chromosomes 4 respectively, these hybridization results suggest a linear correlation between the length of the repeat array and the signal intensity obtained. Digestion of this probe with NaeI results in a 4.1 kb KpnI-NaeI fragment which lacks most of the repeat array sequence. Subsequent hybridization of this probe to metaphase chromosomes of the same individuals shows only one hybridizing chromosome 4 in the father (Fig. 4B) and the affected son (Fig. 4F) and two hybridizing chromosomes 4 in the unaffected mother (Fig. 4D).


Figure 3. (Top) Southern blot of DNA digested with EcoRI (E) or double digested with EcoRI and BlnI (E/B) of family Rf100 hybridized with p13E-11 and 9B6A respectively. Paternal alleles from chromosome 4 and 10 are indicated by P4 and P10 (a and b) respectively. Maternal alleles by M4 and M10 (a and b). The allele carrying a deletion of p13E-11 detected with 9B6A (P4a) is marked with an arrow. This allele is not detected in the affected individual (II-1), indicative of an additional rearrangement resulting in expansion of the deletion into the repeat array. Note that probe 9B6A recognizes a low copy repeat dispersed throughout the genome and will therefore hybridize to multiple fragments (e.g. asterisk) and that I-2 is tetrasomic (chromosome 4-like repeat arrays on both chromosomes 4 and 10, of which the chromosome 4 alleles co-migrate at 65 kb). (Bottom) Schematic representation of the familial deletion. Arrows represent the repeat units, the dashed lines the deletions (not drawn to scale). On top is the control allele (C).


Figure 4. Fluorescence in situ hybridization of the 4.9 kb KpnI fragment (A, C and E) and of the 4.1 kb KpnI-NaeI fragment (B, D and F), on which p13E-11 resides, to metaphase chromosomes of individuals I-1, I-2 and II-1 of family Rf100 respectively. Both probes are labelled in red, while the chromosomes 4 are labelled in green. The 4.9 kb KpnI fragment containing 900 bp of the D4Z4 repeat recognizes all chromosomes 4 in these individuals, except for the affected son, in which only one chromosome 4 hybridizes (E, arrow). An apparent linear correlation between the strength of the signals and the number of repeat units within one array seems evident, since the mother (I-2) shows equally intense signals (two alleles of 65 kb) (C), the father (I-1) exhibits a strong and a weaker signal (140 and 60 kb) (A) while the affected son (II-1) shows only one hybridizing chromosome (65 and 9 kb) (E). The 4.1 kb KpnI-NaeI probe, which lacks most of the D4Z4 repeat sequence, fails to hybridize to one of the chromosomes 4 in the father and affected son, indicative of the deletion (B and F, arrows). Note that the 4.9 kb KpnI fragment hybridizes to all 3.3 kb repeat units dispersed over the genome while the 4.1 kb KpnI-NaeI probe only recognizes chromosomes 4 and 10.

Haplotype analysis in this family of chromosomes 4 and 10 is in agreement with these findings except for the paternal chromosome 10 haplotype in II-2, most likely due to a recombination between D10S590 and the repeat array on chromosome 10. The two eldest sons (II-1 and II-2) have inherited the same deletion-carrying chromosome P sub 4 sup a from the father (I-1) (Fig. 5).


Figure 5. Haplotype analysis of family Rf100. Paternal alleles from chromosomes 4 and 10 are indicated by P4 and P10 (a and b) respectively, maternal alleles by M4 and M10 (a and b). The markers D4S163, D4S139, D10S555 and D10S590 have been used for haplotype analysis. Deletion of p13E-11 (D4F104S1) is indicated by -, its presence by +. The size of the repeat array (D4Z4) is indicated in kb. For the homologous regions on chromosome 10, D4F104S1(10) and D4Z4(10), their presence and sizes are also indicated. The deletion-carrying haplotype is shaded, while the aberrant paternal chromosome 10 haplotype in II-2, possibly due to a recombination event, is marked with an arrow.

After hybridization of the EcoRI- and EcoRI/BlnI-digested DNA with 9B6A, the deletion-carrying allele is present in both the father (I-1) and the unaffected son (II-2) (Fig. 3, arrows). This hybridization reveals in the EcoRI lanes, apart from the deletion-carrying P sub 4 sup a allele co-migrating with the P sub {1 0} sup b allele, an a-specific fragment of 60 kb in I-1, II-1, II-2 and II-3 (Fig. 3, asterisk). However, the probe fails to recognize the P sub 4 sup a allele in the affected son (II-1), indicative of an additional rearrangement expanding the deletion into the repeat array (Fig. 3, arrows). To demonstrate the deletion-carrying P sub 4 sup a allele more clearly, we subsequently hybridized 9B6A to a PFGE blot containing DNA of this family digested with Tru9I, which does not cut in the D4Z4 repeat array itself, while most of the non 4qter and 10qter repeat arrays are fragmented (L. Felicetti, in preparation). The father I-1 and son II-2 show the same P sub 4 sup a allele (Fig. 6), while the a-specific fragment in Figure 3 is not resistant to this enzyme.


Figure 6. Southern blot of DNA from family Rf100 digested with Tru9I and hybridized with 9B6A. Tru9I does not cut in the sub-telomeric D4Z4 repeat arrays from chromosome 4q and 10q, while other D4Z4-like repeat arrays are mostly fragmented by this enzyme. The p13E-11 deletion-carrying P4a allele is detected in the father I-1 and the unaffected son II-2 (arrows). However, due to the additional deletion of most of the D4Z4 repeat units from this allele in the affected son II-1, this probe fails to recognize the P4a allele in II-1. The faintly hybridizing P4b allele in II-3 is marked with an asterisk. Note that the a-specific fragment (marked with an asterisk in Fig. 3) has disappeared upon Tru9I digestion and that the maternal chromosome 10 repeat arrays contain a Tru9I polymorphism resulting in a 10 kb reduction.

To estimate the size of the deletion expansion, we made use of the most distal NotI recognition site in the CpG island of FRG1, which is located ~100 kb proximal of the repeat locus (17). PFGE analysis of NotI-digested DNA after hybridization with probe E8, a single copy probe derived from a Sau3A fragment in the fourth intron of FRG1, showed only the chromosome 4 alleles. Indeed, the affected son (II-1) showed in the paternally derived allele P sub 4 sup a (200 kb) an additional 50 kb deletion of the repeat arrays (Fig. 7, [Delta]). Moreover, probe p13E-11 failed to recognize the deletion-carrying alleles (Fig. 7, asterisk and [Delta]). These data imply that the affected son has retained two repeat units. Due to its small size, this allele is barely detectable between the cross-hybridizing fragments after EcoRI or Tru9I digestion and hybridization with probe 9B6A (data not shown).


Figure 7. Southern blot of NotI-digested DNA of family Rf100 after hybridization with probes E8 and p13E-11 respectively. The small, p13E-11 deletion-carrying fragment (P4a) (200 kb) in the father (I-1) and unaffected son (II-2) is marked with an asterisk. The affected son (II-1) carries a fragment of 150 kb indicative of an extra 50 kb expansion of the deletion and is marked with [Delta]. These alleles are only recognized when using probe E8 and not probe p13E-11. Maternal and paternal chromosome 4 and 10 alleles recognized by p13E-11 are marked M4, M10, P4, and P10 (a and b) respectively. The numbers on the side refer to a [lambda] ladder as size marker.

DISCUSSION

It has been shown that repeat arrays on chromosome 4 <35 kb cause FSHD1 (8,9). Interestingly, short repeat arrays consisting of chromosome 10-like repeats and residing on chromosome 4 are also associated with FSHD1 (16). Therefore, only the length of the repeat locus on chromosome 4, and not its intrinsic properties, is causally related to the disease. Identification of the hybrid repeat arrays in healthy individuals described here makes it less likely that disruption of a gene spanning this repeat locus is the cause of FSHD. Moreover, they substantiate the hypothesis that FSHD1 is caused by a position effect in which the deletion influences regulation of expression of nearby genes. Despite extensive efforts to detect transcriptional activity in the repeats, we cannot formally rule out that these hybrid repeat loci disturb transcribed sequences within the repeat unit itself, since the chromosome 4 and 10 repeats are highly homologous.

Although sub-telomeric exchanges between chromosomes 4 and 10 are relatively common in the population (20%), they may complicate FSHD1 diagnosis in only 5% of cases (18). Nevertheless, diagnosis so far has relied on differentiation between the repeat structures on chromosomes 4 and 10 by the chromosome 10-specific BlnI restriction site. Identification of the hybrid repeat structures described here (Fig. 1) may complicate molecular FSHD diagnosis. Performing conventional gel electrophoresis and Southern blotting will not enable visualization of all four alleles, due to the size of the hybridizing fragments. The occurrence of apparently translocated or hybrid alleles as described here and previously (16) may therefore hamper proper FSHD diagnosis in these 5% of FSHD1 cases. Although one FSHD1 family carrying a short 10-like repeat array on chromosome 4 has been identified (16), the actual occurrence of these 10-like repeat arrays on chromosome 4 in the FSHD1 population still has to be determined. The same holds true for family Rf100, carrying a deletion of p13E-11 (Figs 3-7), in which diagnosis would have failed if not all alleles had been visualized. Therefore, PFGE should routinely be used for otherwise inconsistent results.

The mechanism behind these putative sub-telomeric rearrangements is unknown. Based on these and previous findings (16), two models can be envisaged: intrachromosomal and interchromosomal exchange of material. Sequence analysis has revealed that the sub-telomeric regions of 4q and 10q are >95% homologous up to 50 kb proximal of the repeat loci. Moreover, two distinct chromosome 4q telomeres have been identified which differ from each other by three insertion/deletion polymorphisms (J. Hewitt, in preparation). During meiosis, telomeres attach to the nuclear membrane and subsequently migrate along this membrane together, resulting in bouquet formation, a common feature of meiotic prophase which may increase the probability that homologues recognize each other (19). It has been proposed that during this bouquet formation homologous chromosomes align and start initiation of synaptonemal complexes (SC) (20). These SCs are initiated at the telomeres of the homologous chromosomes and the sub-telomeric regions are believed to play an important role in initiation. Therefore, it is conceivable that during meiosis chromosome 4q and 10q telomeres line up adjacently and exchange material, eventually resulting in monosomic, trisomic or hybrid karyotypes as described here. This crosstalk may be facilitated by the presence of the two different chromosome 4 telomeres. In this situation, one of the chromosome 4 telomeres may be more homologous to the chromosome 10 telomeres than to its actual chromosome 4 homologue. In fact, during meiosis of yeast cells carrying one chromosome II and one chromosome III respectively, synaptonemal-like complexes in which chromosome II and III physically interact have been identified (21). Additionally, the variable distribution and copy number of sub-telomeric repeat sequences, mainly found at the chromosome ends, are suggestive of rare non-homologous unequal chromosome exchanges (22).

Thus, the presence of these karyotypes in the population may either be the result of a very old mutation segregating through the population, the result of rare de novo exchanges during meiosis or mitosis as described above or reflect a combination of the two. Since the individuals described here are from a random control population, we are currently unable to pursue these possibilities by testing the karyotypes of the parents of these individuals.

Performing PFGE electrophoresis also enables us to identify all four alleles. Its necessity for diagnostic purposes is demonstrated by the identification of healthy individuals carrying a deletion of p13E-11. In particular, expansion of the deletion of p13E-11 into the repeat array in family Rf100 is of importance. This expansion demonstrates again that only the length of the repeat array contributes to the FSHD phenotype and that the deleted segment proximal to these repeat units in the healthy father and healthy son is probably not involved in FSHD.

Recently, sub-telomeric chromosomal rearrangements have been implicated in some cases of ideopathic mental retardation (IMR) (23). It was suggested that these rearrangements may account for at least 6-8% of the mentally retarded. Therefore, much emphasis has been placed on the generation of human sub-telomeric probe panels and their use for detection of rearrangements in sub-telomeric regions in patients with IMR, congenital anomalies or cancer (24-26). The results presented here indicate that these sub-telomeric rearrangements may also occur without pathological consequences. Therefore, more understanding of the frequency of sub-telomeric rearrangements, their pathological consequences and their underlying mechanisms will be required. In this light, the exchanges described here may prove to be a unique tool to explore human sub-telomeric chromosome behavior.

MATERIALS AND METHODS

Controls

The 50 unrelated healthy male individuals reported here have been described elsewhere and have been shown to carry either a chromosome 4-specific repeat on chromosome 10 or vice versa (16). The healthy individual 547 carrying a deletion of p13E-11 is also part of this random control group.

Family Rf100

Family Rf100 was ascertained via one of the diagnostic centres in The Netherlands. The proband was clinically diagnosed with FSHD (G.W.P.).

Pulsed field gel electrophoresis analysis

Genomic DNA isolated from fresh blood (27) was digested with either EcoRI (E) (Pharmacia) or double digested with EcoRI and BlnI (E/B) (TaKaRa) according to the manufacturer's instructions. The same holds true for digestion with Tru9I (Eurogentec). An aliquot of 5 µg digested DNA was loaded on a 1% agarose gel (Seakem) in 0.5× TBE and separated by PFGE for 30 h at 8.5 V/cm. Conditions for PFGE have been described elsewhere (16). After electrophoresis, the gel was stained with ethidium bromide and the DNA was fragmented with a UV crosslinker (Stratagene) at 180 000 J/cm3. After denaturation, the DNA was transferred to a Hybond N+ membrane (Amersham) and hybridized with p13E-11 (D4F104S1; 8) and 9B6A (D4Z4; 8) respectively. These probes were random primed labeled using the megaprime DNA labeling kit (Amersham). Hybridizations were performed for at least 16 h at 65°C in a buffer containing 0.125 M Na2HPO4 (pH 7.2), 0.25 M NaCl, 1 mM EDTA and 7% SDS. After hybridization, blots were washed with 2×SSC, 0.1% SDS at 65°C, followed by autoradiography.

For PFGE of NotI-digested DNA, genomic DNA embedded in agarose plugs was digested overnight with 24 U NotI according to the manufacturer's instructions (Pharmacia). An additional digestion was performed with 12 U enzyme for 6 h. DNA was separated on a 0.8% agarose (Seakem) gel for 38 h at 8.5 V/cm. In four cycles, switch times were increased linearly from 3 to 35 s for each cycle. A pause interval of 2% of the forward switch time was included. DNA was hybridized with probes p13E-11 and E8, a single copy chromosome 4-specific Sau3A clone derived from the fourth intron of FRG1, ~100 kb proximal of p13E-11 (28).

Haplotype analysis

Haplotype analysis for family Rf100 was done with markers D4S163, D4S139, D10S555 and D10S590 as described previously (18).

Fluorescence in situ hybridization

Metaphase chromosome spreads of cultured lymphoblastoid cell lines of the father (I-1), mother (I-2) and affected son (II-2) were hybridized with a 4.9 kb KpnI fragment and a 4.1 kb KpnI-NaeI fragment, on which p13E-11 resides, respectively. The probes were labelled by nick-translation with biotin 11-dUTP (Sigma) (29). To identify the chromosomes 4, a chromosome 4-specific paint (30) was accordingly labelled with digoxigenin 16-dATP (Boehringer). Hybridization, washing and staining were performed as described previously (31).

ACKNOWLEDGEMENTS

This study was funded by The Prinses Beatrix Fonds, The Netherlands Organization for Scientific Research (NWO), The Muscular Dystrophy Association (USA), The Dutch FSHD Foundation and the Association Française contre les Myopathies (AFM).

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*To whom correspondence should be addressed. Tel: +31 715 276107/6085; Fax: +31 715 276075; Email: maarel@ruly46.medfac.leidenuniv.nl
+The first two authors contributed equally to this work
§Present address: MGC Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands

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