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T Rijkers, G Deidda,
S van Koningsbruggen,
M van Geel,
R J L F Lemmers,
J C T van Deutekom,
D Figlewicz,
J E Hewitt,
G W Padberg,
R R Frants,
S M van der Maarel
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ABSTRACT: Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is associated with partial deletion of the subtelomeric D4Z4 repeat array on chromosome 4qter. This chromosomal rearrangement may result in regional chromatin relaxation and transcriptional deregulation of genes nearby.
Here we describe the isolation and characterisation of FRG2, a member of a chromosomally dispersed gene family, mapping only 37 kb proximal to the D4Z4 repeat array. Homology and motif searches yielded no clues to the function of the predicted protein. FRG2 expression is undetectable in all tissues tested except for differentiating myoblasts of FSHD patients, which display low, yet distinct levels of FRG2 expression, partly from chromosome 4 but predominantly originating from its homologue on chromosome 10. However, in non-FSHD myopathy patients only distantly related FRG2 homologues are transcribed, while differentiating myoblasts from healthy controls fail to express any member of this gene family. Moreover, fibroblasts of FSHD patients and control individuals undergoing forced Ad5-MyoD mediated myogenesis show expression of FRG2 mainly originating from chromosome 10. Luciferase reporter assays show that the FRG2 promoter region can direct high levels of expression but is inhibited by increasing numbers of D4Z4 repeat units. Transient transfection experiments with FRG2 fusion-protein constructs reveal nuclear localisation and apparently FRG2 overexpression causes a wide range of morphological changes.
The localisation of FRG2 genes close to the D4Z4 repeats on chromosome 4 and 10, their transcriptional upregulation specifically in FSHD myoblast cultures, potential involvement in myogenesis, and promoter properties qualify FRG2 as an attractive candidate for FSHD pathogenesis.
Journal of Medical Genetics 12/2004; 41(11):826-36. · 6.36 Impact Factor
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Journal of Medical Genetics 05/2004; 41(4):e46. · 6.36 Impact Factor
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ABSTRACT: Chromosomal rearrangements occur more frequently in subtelomeric domains than in other regions of the genome and are often associated with human pathology. To further elucidate the plasticity of subtelomeric domains, we examined the 3.3 kb D4Z4 repeat array on chromosome 4 and its homologue on chromosome 10 in 208 Dutch blood donors by pulsed field gel electrophoresis. These subtelomeric repeats are known to rearrange and partial deletions of this polymorphic array on chromosome 4 are associated with facioscapulohumeral muscular dystrophy (FSHD), an autosomal dominant myopathy. Our results show that mitotic rearrangements occur frequently as 3% of individuals display somatic mosaicism for a repeat expansion or contraction explaining the high variability of subtelomeric repeat array sizes. Translocated 4-type repeat arrays on chromosome 10 and the reverse configuration of 10-type repeat arrays on chromosome 4 are observed in 21% of individuals. The translocated repeat arrays on chromosome 4 tend to be more heterogeneous than 4-type repeats on chromosome 10. The repeat length on chromosome 4 is on average larger than on chromosome 10. But on both chromosomes we observe a multi-modal repeat length distribution with equidistant peaks at intervals of 65 kb, possibly reflecting a higher-order chromatin structure. Interestingly, in as many as six random blood donors (3%) we identified FSHD-sized 4-type repeat arrays. Assuming that these individuals are clinically unaffected, these results imply an incomplete penetrance in the upper range of FSHD alleles. Overall, the observed dynamic characteristics of these homologous domains may serve as a model for subtelomeric plasticity.
Human Molecular Genetics 12/2000; 9(19):2879-84. · 7.64 Impact Factor
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S M van der Maarel, G Deidda,
R J Lemmers,
P G van Overveld,
M van der Wielen,
J E Hewitt,
L Sandkuijl,
B Bakker,
G J van Ommen,
G W Padberg,
R R Frants
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ABSTRACT: Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is caused by deletion of most copies of the 3.3-kb subtelomeric D4Z4 repeat array on chromosome 4q. The molecular mechanisms behind the deletion and the high proportion of new mutations have remained elusive. We surveyed 35 de novo FSHD families and found somatic mosaicism in 40% of cases, in either the patient or an asymptomatic parent. Mosaic males were typically affected; mosaic females were more often the unaffected parent of a nonmosaic de novo patient. A genotypic-severity score, composed of the residual repeat size and the degree of somatic mosaicism, yields a consistent relationship with severity and age at onset of disease. Mosaic females had a higher proportion of somatic mosaicism than did mosaic males. The repeat deletion is significantly enhanced by supernumerary homologous repeat arrays. In 10% of normal chromosomes, 4-type repeat arrays are present on chromosome 10. In mosaic individuals, 4-type repeats on chromosome 10 are almost five times more frequent. The reverse configuration, also 10% in normal chromosomes, was not found, indicating that mutations may arise from transchromosomal interaction, to which the increase in 4-type repeat clusters is a predisposing factor. The somatic mosaicism suggests a mainly mitotic origin; mitotic interchromosomal gene conversion or translocation between fully homologous 4-type repeat arrays may be a major mechanism for FSHD mutations.
The American Journal of Human Genetics 02/2000; 66(1):26-35. · 10.60 Impact Factor
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ABSTRACT: Facioscapulohumeral muscular dystrophy (FSHD) is caused by the size reduction of a polymorphic repeat array on 4q35. Probe p13E-11 recognises this chromosomal rearrangement and is generally used for diagnosis. However, diagnosis of FSHD is complicated by three factors. First, the probe cross hybridises to a highly homologous repeat array locus on chromosome 10q26. Second, although a BlnI polymorphism allows discrimination between the repeat units on chromosomes 4 and 10 and greatly facilitates FSHD diagnosis, the occurrence of translocations between chromosomes 4 and 10 further complicates accurate FSHD diagnosis. Third, the recent identification of deletions of p13E-11 in both control and FSHD populations is an additional complicating factor. Although pulsed field gel electrophoresis is very useful and sometimes necessary to detect these rearrangements, this technique is not operational in most FSHD diagnostic laboratories. Moreover, repeat arrays >200 kb are often difficult to detect and can falsely suggest a deletion of p13E-11. Therefore, we have developed an easy and reliable Southern blotting method to identify exchanges between 4 type and 10 type repeat arrays and deletions of p13E-11. This BglII-BlnI dosage test addresses all the above mentioned complicating factors and can be carried out in addition to the standard Southern blot analysis for FSHD diagnosis as performed in most laboratories. It will enhance the specificity and sensitivity of conventional FSHD diagnosis to the values obtained by PFGE based diagnosis of FSHD. Moreover, this study delimits the FSHD candidate gene region by mapping the 4;10 translocation breakpoint proximal to the polymorphic BlnI site in the first repeat unit.
Journal of Medical Genetics 11/1999; 36(11):823-8. · 6.36 Impact Factor
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G Galluzzi, G Deidda,
S Cacurri,
L Colantoni,
N Piazzo,
E Vigneti,
E Ricci,
S Servidei,
B Merico,
A Pachì,
B Brambati,
F Mangiola,
P Tonali,
L Felicetti
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ABSTRACT: In the majority of facioscapulohumeral muscular dystrophy (FSHD) families (about 95%) the genetic defect has been identified as a deletion of a variable number of KpnI repeats in the 4q35 region, although no specific transcripts from this locus have been isolated so far. Molecular diagnosis is based on the detection by probe p13E-11 of EcoRI small fragments, in the range 10-28 kb, that are resistant to BlnI digestion. In family studies this probe is used with other 4q35 polymorphic markers to assign the haplotype associated with the disease. So far, we performed DNA analysis in 145 FSHD families and identified the 4q35 DNA rearrangement not only in affected individuals, but also in healthy subjects at risk of transmitting the disease, such as non-penetrant gene carriers and somatic mosaics. In addition we applied prenatal tests to 19 fetuses, using DNA extracted from chorionic villi samples (CVS) at 10-11 weeks of gestation. The FSHD status, as determined by the presence of BlnI-resistant small fragments associated with the at risk haplotype, was assessed in nine fetuses; in the remaining 10 cases the disease was excluded. Our results show that molecular analysis of 4q35 rearrangements is a reliable indirect method to perform diagnostic, predictive and prenatal tests in FSHD.
Neuromuscular Disorders 06/1999; 9(3):190-8. · 2.80 Impact Factor
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E Ricci,
G Galluzzi, G Deidda,
S Cacurri,
L Colantoni,
B Merico,
N Piazzo,
S Servidei,
E Vigneti,
V Pasceri,
G Silvestri,
M Mirabella,
F Mangiola,
P Tonali,
L Felicetti
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ABSTRACT: Genotype analysis by using the p13E-11 probe and other 4q35 polymorphic markers was performed in 122 Italian facioscapulohumeral muscular dystrophy families and 230 normal controls. EcoRI-BlnI double digestion was routinely used to avoid the interference of small EcoRI fragments of 10qter origin that were found in 15% of the controls. An EcoRI fragment ranging between 10 and 28 kb that was resistant to BlnI digestion was detected in 114 of 122 families (93%) comprising 76 familial and 38 isolated cases. Among the unaffected individuals, 3 were somatic mosaics and 7, carrying an EcoRI fragment larger than 20 kb, could be rated as nonpenetrant gene carriers. In a cohort of 165 patients with facioscapulohumeral muscular dystrophy we found an inverse correlation between fragment size and clinical severity. A severe lower limb involvement was observed in 100% of patients with an EcoRI fragment size of 10 to 13 kb (1-2 KpnI repeats left), in 53% of patients with a fragment size of 16 to 20 kb (3-4 KpnI repeats left), and in 19% of patients with a fragment size larger than 21 kb (>4 KpnI repeats left). Our results confirm that the size of the fragment is a major factor in determining the facioscapulohumeral muscular dystrophy phenotype and that it has an impact on clinical prognosis and genetic counseling of the disease.
Annals of Neurology 06/1999; 45(6):751-7. · 11.09 Impact Factor
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E. Ricci MD,
G. Galluzzi PhD,
G. Deidda BSc,
S. Cacurri PhD,
L. Colantoni,
B. Merico MD,
N. Piazzo BSc,
S. Servidei MD,
E. Vigneti,
V. Pasceri MD, [......],
S. Cacurri,
B. Merico,
N. Piazzo,
S. Servidei,
V. Pasceri,
G. Silvestri,
M. Mirabella,
F. Mangiola,
P. Tonali,
L. Felicetti
[show abstract]
[hide abstract]
ABSTRACT: Genotype analysis by using the p13E-11 probe and other 4q35 polymorphic markers was performed in 122 Italian facioscapulohumeral muscular dystrophy families and 230 normal controls. EcoRI—BlnI double digestion was routinely used to avoid the interference of small EcoRI fragments of 10qter origin that were found in 15% of the controls. An EcoRI fragment ranging between 10 and 28 kb that was resistant to BlnI digestion was detected in 114 of 122 families (93%) comprising 76 familial and 38 isolated cases. Among the unaffected individuals, 3 were somatic mosaics and 7, carrying an EcoRI fragment larger than 20 kb, could be rated as nonpenetrant gene carriers. In a cohort of 165 patients with facioscapulohumeral muscular dystrophy we found an inverse correlation between fragment size and clinical severity. A severe lower limb involvement was observed in 100% of patients with an EcoRI fragment size of 10 to 13 kb (1–2 KpnI repeats left), in 53% of patients with a fragment size of 16 to 20 kb (3–4 KpnI repeats left), and in 19% of patients with a fragment size larger than 21 kb (>4 KpnI repeats left). Our results confirm that the size of the fragment is a major factor in determining the facioscapulohumeral muscular dystrophy phenotype and that it has an impact on clinical prognosis and genetic counseling of the disease. Ann Neurol 1999;45:751–757
Annals of Neurology 05/1999; 45(6):751 - 757. · 11.09 Impact Factor
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R J Lemmers,
S M van der Maarel,
J C van Deutekom,
M J van der Wielen, G Deidda,
H G Dauwerse,
J Hewitt,
M Hofker,
E Bakker,
G W Padberg,
R R Frants
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ABSTRACT: The autosomal dominant myopathy facioscapulohumeral muscular dystrophy (FSHD) is causally related to a short Eco RI 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.
Human Molecular Genetics 09/1998; 7(8):1207-14. · 7.64 Impact Factor
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ABSTRACT: Physical mapping and in situ hybridization experiments have shown that a duplicated locus with a structural organization similar to that of the 4q35 locus implicated in facioscapulohumeral muscular dystrophy is present in the subtelomeric portion of 10q. We performed sequence analysis of the p13E-11 probe and of the adjacent KpnI tandem-repeat unit derived from a 10qter cosmid clone and compared our results with those published, by other laboratories, for the 4q35 region. We found that the sequence homology range is 98%-100% and confirmed that the only difference that can be exploited for differentiation of the 10qter from the 4q35 alleles is the presence of an additional BlnI site within the 10qter KpnI repeat unit. In addition, we observed that the high degree of sequence homology does facilitate interchromosomal exchanges resulting in displacement of the whole set of BlnI-resistant or BlnI-sensitive KpnI repeats from one chromosome to the other. However, partial translocations escape detection if the latter simply relies on the hybridization pattern from double digestion with EcoRI/BlnI and with p13E-11 as a probe. We discovered that the restriction enzyme Tru9I cuts at both ends of the array of KpnI repeats of different chromosomal origins and allows the use of cloned KpnI sequences as a probe by eliminating other spurious fragments. This approach coupled with BlnI digestion permitted us to investigate the structural organization of BlnI-resistant and BlnI-sensitive units within translocated chromosomes of 4q35 and 10q26 origin. A priori, the possibility that partial translocations could play a role in the molecular mechanism of the disease cannot be excluded.
The American Journal of Human Genetics 08/1998; 63(1):181-90. · 10.60 Impact Factor
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ABSTRACT: The p13E-11 probe has been shown to detect DNA rearrangements in sporadic and familial cases of FSHD. Its use, however, has been hampered by the fact that it detects at least two pairs of EcoRI alleles, one derived from the 4q35 region (D4F104S1), the other from 10q26 (D10F104S2). We have cloned p13E-11 EcoRI fragments from the 4q35 and 10q26 subtelomeric regions and shown the presence of several restriction site differences within the KpnI tandem repeat units. The two loci present a different distribution of restriction sites for the enzyme BlnI which allows differential cleavage of the KpnI units derived from 10q26, leaving intact the 4q35 pair of alleles. This method of differential restriction greatly facilitates the interpretation of Southern blots obtained from affected and unaffected subjects, with an important improvement in reliability for diagnosis and genetic counselling. In addition, this method can be used to investigate the molecular mechanism of the 4q35 rearrangement implicated in the disease and to ascertain whether the rearrangement is because of interchromosomal exchange between 4qter and 10qter KpnI repeats.
Journal of Medical Genetics 06/1996; 33(5):361-5. · 6.36 Impact Factor
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ABSTRACT: Beta-thalassemia mutations were characterized in a sample of 70 patients from United Arab Emirates (U.A.E.), resulting in an enlargement of the spectrum of types found in the country. The complete association between the most common IVS I nt 5 (G-C) mutation and a specific haplotype reveals an independent origin of this mutation in U.A.E.
Human Mutation 02/1995; 5(4):327-8. · 5.69 Impact Factor
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ABSTRACT: p13E-11, a probe (D4F104S1 locus) derived from chromosome 4q35, detects EcoRI-rearranged fragments less than 28 kb in both sporadic and familial cases of facioscapulohumeral muscular dystrophy (FSHD). These fragments are smaller than those observed in healthy individuals. The interpretation of Southern blots is complicated by the fact that p13E-11 reveals two pairs of polymorphic alleles, one 4q35-specific and the other unlinked to 4q35, that sometimes overlap each other. We cloned a non-4q35 13-kb fragment not related to the disease from a sporadic FSHD patient of Italian origin. Haplotype analysis and in situ hybridization experiments showed that this fragment was located on the 10qter region. Restriction mapping of the 10qter clone, when compared with the 4q35 fragment, indicates a similar arrangement of KpnI tandemly repeated units and flanking sequences. However 4q35 and 10q26 EcoRI clones can be distinguished by restriction analysis with SfiI and StyI. This observation could be exploited for future applications in the field of molecular diagnosis and genetic counseling. In addition the isolation of two 10q26 cosmid clones (D10S1484 and D10S1485) from a human genomic library and the construction of a detailed physical map, spanning about 40 kb, showed that the structural homology extended upstream of the EcoRI sites, suggesting that a duplicated FSHD locus resided in the subtelomeric region of the long arm of chromosome 10. We cannot exclude the involvement of the duplicated locus in the molecular mechanism of the disease and in the genetic heterogeneity of FSHD syndromes.
European Journal of HumanGenetics 02/1995; 3(3):155-67. · 4.40 Impact Factor
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ABSTRACT: Four DNA markers on the distal long arm of chromosome 4 have been analyzed for their linkage to facioscapulohumeral muscular dystrophy locus (FSHD) in a series of 16 Italian families. We found that, in two families, the disease is not linked to the 4q35 markers, indicating the presence of genetic heterogeneity among Italian FSHD families. Linkage analysis in the remaining families supports the order cen-D4S171-D4S163-D4S139-D4S810-FSHD-qter, in agreement with the physical map from the literature. EcoRI digestion and hybridization with the distal marker p13E-11 (D4S810)1 detected DNA rearrangements in the affected members of both sporadic and familial cases of FSHD, with family-specific fragments ranging in size between 15 kb and 28 kb. In three sporadic FSHD cases, the appearance of a new "small" fragment not present in either parent was clearly associated with the development of FSHD disease. However, in the familial cases analyzed, we observed two recombinations between all four 4q35 markers and the disease locus in apparently normal subjects, leaving open the possibility of nonpenetrance of the FSHD mutation.
Human Genetics 11/1994; 94(4):367-74. · 5.07 Impact Factor
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[show abstract]
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ABSTRACT: Four DNA markers on the distal long arm of chromosome 4 have been analyzed for their linkage to facioscapulohumeral muscular dystrophy locus (FSHD) in a series of 16 Italian families. We found that, in two families, the disease is not linked to the 4q35 markers, indicating the presence of genetic heterogeneity among Italian FSHD families. Linkage analysis in the remaining families supports the order cen-D4S171-D4S163-D4S139-D4S810-FSHD-qter, in agreement with the physical map from the literature. EcoRI digestion and hybridization with the distal marker p13E-11 (D4S810)1 detected DNA rearrangements in the affected members of both sporadic and familial cases of FSHD, with family-specific fragments ranging in size between 15 kb and 28 kb. In three sporadic FSHD cases, the appearance of a new small fragment not present in either parent was clearly associated with the development of FSHD disease. However, in the familial cases analyzed, we observed two recombinations between all four 4q35 markers and the disease locus in apparently normal subjects, leaving open the possibility of nonpenetrance of the FSHD mutation.
Human Genetics 01/1994; 94(4):367-374. · 5.07 Impact Factor
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ABSTRACT: A previously undescribed mutation (-1, +3, codon 24) causing beta-thalassaemia was identified in an Egyptian patient. It consists in the concomitant deletion of a G in codon 24 and its replacement with the new trinucleotide CAC, thus resulting in the shift of the beta-globin reading frame. The sequence of the chromosome of interest was isolated from the homologous one by means of selective hybridization to an immobilized oligonucleotide. The presence of this mutation in the proband's family was confirmed by dot blot hybridization with an oligonucleotide probe.
British Journal of Haematology 10/1991; 79(1):90-2. · 4.94 Impact Factor
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ABSTRACT: The relative frequency of different beta-thalassemia mutations and their association with beta-globin haplotypes were studied in patients from the Nile delta region, Egypt, by means of the polymerase chain reaction, oligonucleotide hybridization and restriction analysis. We found that 8 mutations account for 77% of beta-thalassemia chromosomes in this population, the commonest being IVS-1 nt 110, IVS-1 nt 6 and IVS-1 nt 1. Each mutation was associated with a specific haplotype, with the exception of IVS-1 nt 110, found on 3 different chromosomal backgrounds. Our data show that testing for the 8 detectable mutations makes feasible prenatal diagnosis in 65% of at risk couples and exclusion testing in an additional 25% of cases.
Human Genetics 09/1990; 85(3):272-4. · 5.07 Impact Factor
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A. Novelletto,
M. Hafez, G. Deidda,
A. Rienzo,
L. Felicetti,
H. El-Tahan,
Z. Morsi,
M. El-Ziny,
Y. Al-Tonbary,
A. Sittien,
L. Terrenato
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ABSTRACT: The relative frequency of different -thalassemia mutations and their association with -globin haplotypes were studied in patients from the Nile delta region, Egypt, by means of the polymerase chain reaction, oligonucleotide hybridization and restriction analysis. We found that 8 mutations account for 77% of -thalassemia chromosomes in this population, the commonest being IVS-1 nt 110, IVS-1 nt 6 and IVS-1 nt 1. Each mutation was associated with a specific haplotype, with the exception of IVS-1 nt 110, found on 3 different chromosomal backgrounds. Our data show that testing for the 8 detectable mutations makes feasible prenatal diagnosis in 65% of at risk couples and exclusion testing in an additional 25% of cases.
Human Genetics 07/1990; 85(3):272-274. · 5.07 Impact Factor
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ABSTRACT: An Egyptian child with thalassemia major was found to carry two different haplotypes (I and VI) associated with two beta-thalassemic chromosomes. Analysis with several oligonucleotides and restriction enzymes, which identify the mutations most common in the Mediterranean area, allowed the identification of only one mutation, namely T----C at position 6 of the first intervening sequence (IVS-I). In order to characterize the other mutation the beta gene was amplified with polymerase chain reaction and sequenced. A G----A substitution was found at position 130 of the IVS-I which alters the conserved dinucleotide AG present in the consensus acceptor sequence, thus producing a beta (0)-thalassemia. This mutation was further confirmed by restriction analysis since it creates a new restriction site for the enzyme Afl II. It is concluded that this subject carries the IVS-I-6 mutation associated with haplotype VI, frequently observed in Mediterranean areas, and a new mutation at the acceptor site of the IVS-I, which has not been described before, associated with haplotype I. This thalassemic gene can be added to the list of mutations that can be identified by Southern analysis using Afl II.
Hemoglobin 02/1990; 14(4):431-40. · 1.30 Impact Factor
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ABSTRACT: The frequency of deletional alpha-thalassemia in the Egyptian population was estimated at 0.08 by DNA analysis of a newborn random sample. No alpha 0 determinants were found. The most frequent alpha+ determinant was the -alpha 3.7 type I in association with the medium allele at inter-zeta HVR. The -alpha 4.2 and alpha alpha alpha anti 3.7 arrangements were found at very low frequencies.
Human Genetics 03/1989; 81(3):211-3. · 5.07 Impact Factor
