Evidence for subtelomeric exchange of 3.3 kb tandemly repeated units between chromosomes 4q35 and 10q26: implications for genetic counselling and etiology of FSHD1
Evidence for subtelomeric exchange of 3.3 kb tandemly repeated units between chromosomes 4q35 and 10q26: implications for genetic counselling and etiology of FSHD1Judith C. T. van Deutekom1,2, Egbert Bakker1, Richard J. L. F. Lemmers1, Michiel J. R. van der Wielen1, Elsa Bik1, Marten H. Hofker1, George W. Padberg3 and Rune R. Frants1,*
1MGC-Department of Human Genetics, Sylvius Laboratory, Leiden University, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands, 2Department of Neurology, Leiden University, Leiden, The Netherlands and 3Department of Neurology, University of Nijmegen, Nijmegen, The Netherlands
Received August 6, 1996;Revised and Accepted September 30, 1996
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant myopathy, clinically characterized by asymmetric weakness of muscles in the face, shoulder girdle and upper arm. Deletion of an integral number of 3.3 kb repeated units within a highly polymorphic EcoRI fragment at chromosome 4q35, generating a relatively short EcoRI fragment (<35 kb), has been shown to cause FSHD1. Probe p13E-11 detects these short fragments in FSHD1 patients, and has therefore been used for diagnostic DNA analysis. However, the reliability of this analysis has been hampered by cross-hybridization of p13E-11 to chromosome 10q26-linked EcoRI fragments of comparable size, which also contain a variable number of 3.3 kb repeated units. Recently, a BlnI restriction site was identified within each of the repeated units derived from chromosome 10q26, which enables differentiation of the two polymorphic p13E-11 loci in most cases without haplotype analysis. Remarkably, applying the differential analysis to screen DNA of 160 Dutch cases referred to us for FSHD1 diagnosis, we obtained evidence for subtelomeric exchange of 3.3 kb repeated units between chromosomes 4q35 and 10q26 in affected and unaffected individuals. Subsequently, analysis of 50 unrelated control samples indicated such exchange between chromosomes 4q35 and 10q26 in at least 20% of the population. These subtelomeric rearrangements have generated a novel interchromosomal polymorphism, which has implications for the specificity and sensitivity of the differential restriction analysis for diagnostic purposes. Moreover, the high frequency of the interchromosomal exchanges of 3.3 kb repeated units suggests that they probably do not contain (part of) the FSHD1 gene, and supports position effect variegation as the most likely mechanism for FSHD1.
Facioscapulohumeral muscular dystrophy (FSHD) is clinically characterized by progressive weakness of the facial, shoulder-girdle and upper arm muscles, and in some cases, also the abdominal, foot extensor and pelvic girdle muscles (1 ,2 ). The autosomal dominant inherited myopathy shows a variable phenotypic expression with a high penetrance of 95% at the age of 20, and an estimated prevalence of 1 in 20 000 (1 ,3 ).
Linkage analysis assigned the major FSHD locus (FSHD1) to chromosome 4q35 (4 ,5 ) distal to D4S139 (6 -8 ). However, genetic heterogeneity was demonstrated for a small number of FSHD families, not linked to 4q35 (9 -12 ). In patients, the FSHD1 locus on chromosome 4q35 is characterized by a DNA rearrangement within a highly polymorphic EcoRI fragment detectable using probe p13E-11 (D4F104S1) (13 ). In patients, these FSHD1-associated rearrangements generate EcoRI fragments which are shorter than 35 kb (13 -17 ), while the size of the 4q35-linked EcoRI fragments in unaffected individuals varies from 35 to 300 kb (13 ). Analysis of the p13E-11 EcoRI fragments of four unrelated FSHD1 patients and controls revealed that the polymorphic fragment contains a variable number of 3.3 kb repeated units (D4Z4), a critical number of which is deleted in patients (18 ).
As short EcoRI fragments (<35 kb) linked to 4q35 are associated with FSHD1, probe p13E-11 has been used for diagnostic DNA analysis (17 ). However, a straightforward analysis is hampered by cross-hybridization of p13E-11 to another polymorphic locus which has recently been assigned to chromosome 10q26 (12 ,19 ). This locus is similarly characterized by EcoRI fragments containing a variable number of 3.3 kb repeated units. The size of these chromosome 10 fragments can be up to 300 kb, but also less than 35 kb in 10% of the population (12 ,17 ). Hence, short EcoRI fragments derived from 10q26 may incorrectly be interpreted as chromosome 4q35 deletion fragments. Haplotype analysis in FSHD families using chromosome 4q35 and 10q26 markers had to be performed to determine the chromosomal origin of a short (<35 kb) p13E-11 fragment (17 ). However, due to the presence of small families and isolated cases, only about 50% of short p13E-11 fragments could unambiguously be assigned to chromosome 4q35, and subsequently used for diagnostic purposes (17 ).
Recently, comparative sequence analysis of 3.3 kb repeated units derived from chromosomes 4q35 and 10q26 revealed the specific presence of a BlnI site within each repeated unit from chromosome 10q26. As this restriction site is not present in the repeated units from chromosome 4q35, differential restriction analysis using BlnI enabled direct differentiation of the chromosomal EcoRI fragments without haplotype analysis (19 ). By double digestion of genomic DNA with EcoRI and BlnI (or AvrII), the p13E-11 EcoRI fragments from 4q35 are reduced in size by 3 kb, while the EcoRI fragments derived from chromosome 10q26 are fragmented completely. This differential restriction analysis enabled presymptomatic and prenatal diagnosis to be performed with an improved efficiency and reliability.
Here, we report the screening of 160 independent Dutch familial or isolated cases referred to us for FSHD1 diagnosis, using the differential restriction analysis. In most cases an unequivocal diagnosis was achieved. Remarkably, a few individuals appeared to be `monosomic' or `trisomic' for the 4q35-linked p13E-11 fragment. Further analysis of these cases suggested the occurrence of subtelomeric exchanges of 3.3 kb tandemly repeated units between chromosomes 4q35 and 10q26, generating hybrid EcoRI fragments. As interchromosomal exchanges would have implications for the specificity and sensitivity of the diagnostic DNA testing, the frequency of such events in the population was determined by screening 50 unrelated control DNA samples.
EcoRI and EcoRI/BlnI digested DNA of FSHD patients and family members was analyzed by conventional agarose gel electrophoresis and subsequent Southern blot hybridization using probe p13E-11. To illustrate the potency of the differential restriction analysis for diagnostic purposes, the results of family 87 (17 ) are shown in Figure 1 . After EcoRI digestion only, probe p13E-11 detected a fragment of approximately 30 kb in affected as well as in some unaffected individuals. Haplotype analysis using chromosome 4q35 and 10q26 markers revealed that the 30 kb p13E-11 EcoRI fragment does not cosegregate with either the chromosome 4q35 or 10q26 haplotype (17 ) (Fig. 1 ). However, recently, differential restriction analysis using EcoRI and BlnI indicated heterogeneity of the 30 kb EcoRI fragments. In the affected individuals, the 30 kb EcoRI fragment was reduced in size by 3 kb indicating a chromosome 4 origin (Fig. 1 , haplotype `A'), whereas in the unaffected individuals the 30 kb EcoRI fragment was completely fragmented, revealing a chromosome 10 origin. The chromosomal origin of the 30 kb EcoRI fragments as suggested by the differential restriction analysis, was confirmed by haplotype analysis performed previously (17 ). Accordingly, an unequivocal diagnosis was achieved in this family.
In applying the differential restriction analysis we observed several FSHD index cases and unaffected spouses which appeared to be either `monosomic' or `trisomic' for the p13E-11 fragment on chromosome 4q35. To demonstrate such aberrant BlnI restriction patterns, a very interesting family (family 28, Fig. 2 a) was selected. In this family, a 25 kb EcoRI fragment was detected which cosegregates with FSHD1 and which was reduced in size by 3 kb after BlnI digestion (Fig. 2 b). Therefore, a clear diagnosis was achieved. Remarkably, the father (individual 28.1) who is asymptomatic, also carries a second short BlnI-resistant EcoRI fragment (22 kb). As the father is transmitting both his chromosomes 4 and both his chromosomes 10 whereas the 22 kb fragment is not inherited by any of his children (Fig. 2 a), he is considered somatic mosaic for this 22 kb fragment. In addition, the father strikingly showed two other BlnI-resistant, 4q35-like fragments. In his offspring, three BlnI-resistant EcoRI fragments were also detected in sons 28.4 and 28.5, while one BlnI-resistant EcoRI fragment only was observed in the youngest son 28.6. Moreover, the unaffected mother (individual 28.2) also shows one BlnI-resistant EcoRI fragment only. Pulsed-field gel electrophoresis was performed (Fig. 2 b), which confirmed the apparent `mono- and trisomy' observed by conventional gel electrophoresis. We hypothesized that an exchange of 3.3 kb repeated units between chromosomes 4q35 and 10q26 might have occurred, generating hybrid p13E-11 fragments (Fig. 3 ). In cases 28.1, 28.4 and 28.5, one of the chromosome 10q26 fragments contain repeated units without BlnI sites, probably derived from chromosome 4q35. In cases 28.2 and 28.6 one of the chromosome 4q35 fragments contains 10q26-like repeated units including BlnI sites. Haplotype analysis using chromosome 4q35 and 10q26 markers (D4S163-D4S139 and D10S555-D10S590, respectively) was performed to differentiate the chromosomes 4 and 10 segregating in this family (Fig. 2 a). One of the 4q35-like EcoRI/BlnI fragments observed in individuals 28.1, 28.4 and 28.5 indeed segregates with a chromosome 10q26 haplotype indicating it to be a hybrid p13E-11 fragment. In addition, one of the 10q26-like fragments observed in individuals 28.2 and 28.6 segregates with a chromosome 4q35 haplotype, also supporting the interchromosomal exchange hypothesis.
The differential restriction analysis of FSHD cases is based on the presence of a BlnI restriction site in each of the 3.3 kb repeated units derived from chromosome 10q26 (19 ). As this BlnI site is not present in the repeated units of chromosome 4q35, differentiation between the two polymorphic p13E-11 loci can easily be achieved in most cases. Therefore, especially in isolated cases suspected of having FSHD, the differential restriction analysis accounts for an important improvement of the diagnostic DNA analysis. In addition, in some familial cases haplotype analysis may not reveal clear differentiation between chromosome 4q35 and 10q26 derived EcoRI fragments due to either non-informative marker data or short EcoRI fragments of similar size segregating in the family. In this paper, we describe one such family (family 87, Fig. 1 ) which remained diagnostically inconclusive after haplotype analysis, but which could reliably be diagnosed after EcoRI/BlnI double digestion. Applying the differential restriction analysis, most of 160 index cases clinically suspected of having FSHD and referred for DNA analysis, were diagnosed unequivocally.
Although the diagnostic value and the specificity and sensitivity of the differential restriction analysis were found to be high, we observed several FSHD index cases and unaffected spouses exhibiting unanticipated BlnI restriction patterns. In family 28 (Fig. 2 ) apparent `monosomy' and `trisomy' for chromosome 4q35-linked p13E-11 fragments was detected. We hypothesized that an exchange of 3.3 kb repeated units between chromosomes 4q35 and 10q26 might have occurred. This exchange would generate hybrid p13E-11 EcoRI fragments: chromosome 4q35- linked EcoRI fragments containing chromosome 10q26-like or BlnI-sensitive repeats and conversely, chromosome 10q26-linked EcoRI fragments containing chromosome 4q35-like or BlnI- resistant repeats. Further analysis of family 28 using pulsed-field gel electrophoresis to separate the individual chromosomal EcoRI and EcoRI/BlnI restriction fragments, and subsequent haplotype analysis using markers from the chromosome 4q35 and 10q26 regions, supported this subtelomeric exchange hypothesis.
As subtelomeric exchange of the 3.3 kb repeated units between chromosomes 4q35 and 10q26 would have implications for the specificity and sensitivity of the differential restriction analysis, we estimated the frequency of interchromosomal exchange in the population. Apparent `trisomy' or `monosomy' for a 4q35-linked EcoRI fragment was observed in at least 20% of the population. The specificity of the differential restriction analysis is determined by the percentages of unaffected individuals carrying a short 10q26-linked EcoRI fragment which contains BlnI-resistant repeated units (mostly `trisomics'), whereas the sensitivity is determined by the percentages of FSHD patients carrying a short 4q35-linked EcoRI fragment which contains BlnI-sensitive repeated units (mostly `monosomics'). Differential restriction analysis of 50 unrelated controls revealed two `trisomic' samples and one apparent `disomic' sample (Fig. 5 , control 2), exhibiting a short (<35 kb) chromosome 10q26-linked EcoRI fragment containing BlnI-resistant repeated units. Therefore, at present the specificity of the differential restriction analysis is estimated to be 94% (47 of 50). In addition, differential restriction analysis of 160 Dutch FSHD index cases revealed in 12 of 114 Dutch index cases a short (<35 kb) EcoRI fragment containing BlnI-sensitive repeated units. Based on clinical signs, at least six of these cases were unambiguously diagnosed with FSHD. The other six cases were either not analyzed by our neurologist (GWP) and therefore considered to be `clinically uncertain', or possibly misdiagnosed. As an EcoRI fragment which is completely fragmented by BlnI suggests a chromosome 10q26 origin, the six clinically certain FSHD cases would have been predicted to have a low probability of FSHD1. However, pulsed-field gel electrophoresis revealed that these cases are `monosomic' after EcoRI/BlnI double-digestion, and carry a short hybrid chromosome 4q35-linked EcoRI fragment containing BlnI-sensitive repeated units. Therefore, the sensitivity of the differential restriction analysis is estimated to be 95% (108 of 114).
In conclusion, the differential restriction analysis facilitates an efficient and unequivocal diagnosis in approximately 95% of cases. However, index cases which show clear clinical FSHD signs but carry a short EcoRI fragment completely fragmented by BlnI suggesting a chromosome 10q26 origin, should be further analyzed by pulsed-field gel electrophoresis and haplotype analysis to confirm the chromosomal origin of the short fragment, and to prevent misdiagnosis.
The molecular mechanism behind the subtelomeric exchanges between chromosomes 4q35 and 10q26 remains to be elucidated. Chromosomal rearrangements occur more often in (sub)telomeric regions than in other parts of the genome. Translocations of chromosome ends have recently been reported as the cause for idiopathic mental retardation syndrome (21 ), [alpha]-thalassemia mental retardation syndrome (22 ), Wolf-Hirschhorn syndrome (23 ), Miller-Dieker syndrome (24 ) and cri-du-chat syndrome (25 ). However, in this paper we report an interchromosomal polymorphism without phenotype, which is to our knowledge a remarkable novelty in human genetics. Owing to high homology between the chromosome 4q35 and 10q26 subtelomeric regions, it is plausible that there is an interchromosomal `cross-talk' during meiosis. Two types of chromosomal rearrangements might have generated the exchange of 3.3 kb repeated units: either translocations including the particular telomeres, or gene conversions limited to the repeated units only. As the respective telomeric regions of chromosomes 4q and 10q contain non-unique sequences only, it will be difficult to determine whether the telomeres are involved in the interchromosomal exchanges. In addition, in most cases analyzed the p13E-11 EcoRI fragments revealed BlnI restriction patterns as expected; either completely fragmented, or reduced in size by 3 kb. Therefore, probably complete arrays of repeated units have been exchanged, suggesting that in case of telomeric translocations the proximal breakpoint might even be proximal to the repeated units. However, in rare cases the EcoRI fragment was found to be reduced in size by more than 3 kb after BlnI digestion, indicating that hybrid p13E-11 EcoRI fragments may also contain a mixture of BlnI-resistant and BlnI-sensitive repeated units. Finally, whether the hybrid EcoRI fragments are due to a recurrent event of interchromosomal exchange in the population, or part of a polymorphism which is due to one unique interchromosomal exchange event in evolution, remains to be elucidated.
Familial and isolated cases are referred to us through one of the seven Clinical Genetics Centres in the Netherlands. They are referred by neurologists after being diagnosed clinically as possibly having FSHD. In this study, 160 FSHD index cases are included. Further analysis was performed on two particular 4q35-linked families. In family 87 (17 ) a 30 kb EcoRI fragment, and in the Turkish family 28 a 25 kb EcoRI fragment segregates with the disease. DNA isolated from 50 unaffected male individuals was used in the control study.
Genomic DNA was isolated from freshly collected blood as previously described (27 ). DNA was digested with restriction enzymes EcoRI and BlnI according to the manufacturer's instructions (Pharmacia). Conventional gel electrophoresis was performed as reported by Bakker et al. (17 ). DNA was transferred to a Hybond N+ membrane (Amersham). The inserts of probes p13E-11(D4F104S1) (13 ), 9B6A (D4Z4) (13 ) and pLAM1 (D4Z3) (18 ) were labelled by random priming with [alpha]32P-dCTP, using the megaprime DNA labelling system (Amersham). Hybridizations were performed for 16 h at 65oC as described (28 ). The blots were washed at 65oC and at a stringency of 2* SSC/0.1% SDS, followed by autoradiography for 16-36 h at -70oC using Konica AX film with an intensifying screen.
Electrophoresis was performed in 0.5* TBE (45 mM Tris, 45 mM boric acid, 1 mM EDTA, adjusted to pH 8.0) at 18oC. EcoRI and EcoRI/BlnI double digested DNA in solution was run for 30 h at an electric field of 8.5 V/cm. In four identical cycles, switch times were increased linearly from 1 s at the beginning and 20 s at the end of each cycle. A pause interval of 2% of the forward switch time was included. After electrophoresis, gels were stained with ethidium bromide. The high molecular weight DNA molecules were fragmented by a UV-Cross-Linker (Stratagene) at 180 000 J/cm3.
The authors thank Dr J. Hewitt and Dr R. Fodde for comments on the manuscript. This study was funded by the Prinses Beatrix Fonds (the Netherlands), the Muscular Dystrophy Association (USA) and the Association Française contre les Myopathies.
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