Identification of the first gene (FRG1) from the FSHD region on human chromosome 4q35
Identification of the first gene ( FRG1 ) from the FSHD region on human chromosome 4q35Judith C. T. van Deutekom1,2, Richard J. L. F. Lemmers1, Prabhjit K. Grewal3, Michel van Geel1,2,+, Silke Romberg1,2, Hans G. Dauwerse1, Tracy J. Wright3,[Dagger], George W. Padberg4, Marten H. Hofker1, Jane E. Hewitt3 and Rune R. Frants1,*
1MGC-Department of Human Genetics, and 2Department of Neurology, Leiden University, Leiden, The Netherlands, 3School of Biological Sciences, University of Manchester, Manchester, UK and 4Department of Neurology, University of Nijmegen, Nijmegen, The Netherlands
Received January 29, 1996;Revised and Accepted February 23, 1996GenBank accession nos L76159, L76173, L76174
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant, neuromuscular disorder characterized by progressive weakness of muscles in the face, shoulder and upper arm. Deletion of integral copies of a 3.3 kb repeated unit from the subtelomeric region on chromosome 4q35 has been shown to be associated with FSHD. These repeated units which are apparently not transcribed, map very close to the 4q telomere and belong to a 3.3 kb repeat family dispersed over heterochromatic regions of the genome. Hence, position effect variegation (PEV), inducing allele-specific transcriptional repression of a gene located more centromeric, has been postulated as the underlying genetic mechanism of FSHD. This hypothesis has directed the search for the FSHD gene to the region centromeric to the repeated units. A CpG island was identified and found to be associated with the 5' untranslated region of a novel human gene, FRG1 (FSHD Region Gene 1). This evolutionary conserved gene is located about 100 kb proximal to the repeated units and belongs to a multigene family with FRG1 related sequences on multiple chromosomes. The mature chromosome 4 FRG1 transcript is 1042 bp in length and contains nine exons which encode a putative protein of 258 amino acid residues. Transcription of FRG1 was detected in several human tissues including placenta, lymphocytes, brain and muscle. To investigate a possible PEV mechanism, allele-specific FRG1 steady-state transcript levels were determined using RNA-based single-strand conformation polymorphism (SSCP) analysis. A polymorphic fragment contained within the first exon of FRG1 was amplified from reverse transcribed RNA from lymphocytes and muscle biopsies of patients and controls. No evidence for PEV mediated repression of allelic transcription was obtained in these tissues. However, detection of PEV in FSHD patients may require analysis of more specific cell types at particular developmental stages.
Facioscapulohumeral muscular dystrophy (FSHD) is a clinically heterogeneous, autosomal dominant myopathy with an estimated prevalence of 1 in 20 000 (1 ,2 ). Initially, the facial and shoulder-girdle muscles are affected but during progression of the disease the upper-arm, abdominal, pelvic-girdle and foot-extensor muscles can also become involved (1 ,3 ).
The FSHD locus has been assigned to the distal long arm of chromosome 4 (4q35) by linkage analysis (4 ,5 ), with a most likely location distal to D4S139 (6 -8 ). More recently, rearrangements within a polymorphic EcoRI fragment were detected in FSHD patients using probe p13E-11 (D4F104S1) (9 ). These rearrangements were found to be due to deletion of an integral number of 3.3 kb tandemly repeated units (D4Z4) (9 ,10 ). Although these 3.3 kb repeated units contain a double homeobox motif (11 ,12 ), no transcripts derived from this locus could be identified (11 -14 ). In addition, fluorescence in situ hybridization (FISH) experiments showed that the tandem array of 3.3 kb repeats maps very close to the 4q telomere (13 ,15 ,16 ), suggesting a probable location of the FSHD gene proximal to the repeated units. Recent data suggested that the repeated units are members of a 3.3 kb repeat family dispersed over heterochromatic regions of the genome (14 ). This is supported by FISH results demonstrating hybridization of the 3.3 kb repeated unit to the short arms of all acrocentric chromosomes as well as to chromosomes 1q12, 4q35, 10qter, and 10 cen (13 ,16 ). Weak signals were also detected on a large heterochromatic region on chromosome 9q12, and chromosome Y. Therefore, considering that the 3.3 kb repeated unit may be heterochromatin-associated, deletion of a number of these repeated units may lead to position effect variegation (PEV) by alteration of the chromosomal structure. Heterochromatinization or extensive chromatin packaging (17 ) can transcriptionally silence a gene by (i) inducing a later replication time point which will put the gene at a disadvantage in the competition for transcription factors (18 ), or (ii) generating a more tightly condensed structure of the chromatin which itself contributes to its transcriptionally silent state (18 -20 ). A classic example of heterochromatinization in mammals involves transcriptional silencing of genes by X chromosome inactivation (21 -23 ). Furthermore, since telomeres have a functional location in the nucleus (24 ), the FSHD associated deletions may also have implications for the nuclear organization. PEV leading to reduced transcript levels of one allele of a gene has been observed in Drosophila, yeast and mammals (17 ,25 ), and may affect genes over a region of several hundred kb (26 ). When taken into account that the deletion prone EcoRI fragments can be 300 kb in healthy individuals and 10 kb in FSHD patients (27 ), the FSHD-associated deletions of 3.3 kb repeated units may also involve several hundred kb. Therefore, the FSHD candidate gene region subjected to several gene identification methods was chosen to extend about 150 kb proximal to the 3.3 kb repeats.
Here, we report the isolation and characterization of a novel gene cloned from the FSHD region (FRG1). The PEV hypothesis was tested by analysis of allele-specific transcription of this gene in lymphocytes and muscle tissue of patients and controls.
Previous mapping studies (28 ,29 ) showed the presence of a CpG island located within a 6.5 kb EcoRI fragment of cosmid cT171 which was isolated from a cosmid library made from YAC 25C2E (D4S1093) (28 ). A total of 6.2 kb of contiguous sequence from this 6.5 kb EcoRI fragment was determined (GenBank accession number L76174) by sequencing adjacent PstI subclones located within this fragment. The CpG island spans a region of 1 kb showing a G+C content of 63% and a CpG:GpC ratio of 0.8. A 1.2 kb PstI subclone (p10B) encompassing the CpG island (Fig. 1 ), was used as a probe to screen a human skeletal muscle cDNA library. A 640 bp cDNA clone (10B7, Fig. 1 ), was isolated and, after sequencing, shown to contain the 3' end of a cDNA. Clone 10B7 hybridized to a 1.1 kb transcript in total RNA from lymphocytes and fetal muscle, and poly-(A)+ RNA from adult muscle, fetal brain and placenta (Fig. 2 ). Subsequently, two cDNA clones were obtained by screening a total infant brain cDNA library (BS3; 3.5 kb insert), and a fetal brain cDNA library (10B1; 1.3 kb insert), with probe 10B7 (Fig. 1 ). These clones were sequenced and their unexpectedly large cDNA inserts were shown to be due to splicing aberrations and cloning artefacts (see below). Additional cDNA fragments were obtained by amplification of different cDNA libraries using one of the cDNA library vector-specific primers flanking the cloning site, in combination with 10B7-based primers. Two cDNA fragments, PLC2FB22 (700 bp) and ML8RT3 (780 bp) were amplified from the placenta and muscle cDNA library, respectively. By sequence analysis these fragments were found to overlap, spanning 913 bp (Fig. 1 ). To determine the transcription start site of the cDNA, two primers at the 5' end of the cDNA (1ra and 2r) were designed to perform 5' RACE [rapid amplification of cDNA ends (30 )] from human brain cDNA. The obtained RACE products were sequenced and found to start at, or just distal of, position -191 upstream from the putative translation initiation site (Fig. 3 b). Subsequently, primers (1f and 9r) were chosen at positions -134 and 801 to perform RT-PCR on RNA isolated from a chromosome 4 only cell hybrid, muscle tissue, and lymphocytes. In all cases, an identical 935 bp product was obtained which is in accordance with the total transcript length of about 1.1 kb detected by Northern blot analysis. Further sequence analysis confirmed the identity of these RT-PCR products. The complete cDNA sequence of this novel human gene, named FRG1 (FSHD Region Gene 1), was compiled (GenBank accession number L76159); spanning 1042 bp and containing an open reading frame of 258 amino acid residues (Fig. 3 b). The putative translation start site is the first in frame ATG codon located 191 bp downstream of the 5' end of the transcript, which conforms to the Kozak consensus sequence (31 ). The 3' untranslated region is 74 bp and ends with a poly-(A) tail 25 bp downstream of a polyadenylation signal at position 821 (Fig. 3 b). Nucleotide sequence database searches (BLAST-N) identified several highly homologous human EST clones: a human HL60 EST clone (accession number D19665), and from the WashU-Merck EST Project; accession numbers T41211, T51111, T51019; all being 96-99% homologous to the 3' end of the cDNA, and R77057; 95% homologous to the internal part of the cDNA. Furthermore, a mouse EST clone (GEG-123, accession number X71639) showed high homology (86%) to the 3' end of the FRG1 transcript. Southern blot analysis also indicated that FRG1 is evolutionarily conserved (Fig. 4 a). No significant homology to known genes was detected in either the nucleotide sequence databases or the protein databases (BLAST-P). Protein motif searches gave no indication of a possible function for the deduced FRG1 protein.
Southern blot analysis revealed that the FRG1 cDNA is distributed over three EcoRI fragments contained within cosmid cT171 (Fig. 1 , Fig. 4 b). These EcoRI fragments were subcloned and partially sequenced using FRG1 cDNA-based primers. The 1042 bp cDNA sequence was found to be completely identical to the genomic sequence and is composed of nine exons (Fig. 1 ). The sequences of the intron-exon boundaries were determined and shown to be in accordance with splice site consensus sequences (Table 1 ).
The promoter region of FRG1 (Fig. 3 a, GenBank accession number L76173) coincides with the identified CpG island (see above). No TATA or CAAT box motifs were found, but three putative Sp1 binding sites (32 ) are present at positions -743, -403, and -310, which may function as control elements of transcription (33 ). Absence of a TATA box in genes with a 5' CpG island has been reported for many housekeeping genes and appears to be related to the regulation of widely expressed genes (34 ).
The 935 bp chromosome 4 FRG1 RT-PCR product hybridized to eight different human genomic EcoRI fragments with varying intensities (Fig. 4 ). Only three of these fragments are derived from the chromosome 4q35 region (Fig. 1 , Fig. 4 b), suggesting the presence of multiple FRG1-related sequences in the genome. To identify the chromosomal origin of the different members of this novel multigene family, the three EcoRI fragments of cosmid cT171 containing the FRG1 exons were independently used as probes for fluorescent in situ hybridization (FISH) to metaphase spreads. For each fragment an identical hybridization pattern was detected. Hybridization signals were found at chromosome 4q35, at three sites in the pericentromeric region of chromosome 9, at the short arm of all acrocentric chromosomes (13, 14, 15, 21, and 22) and at the centromeric region of chromosome 20 (Fig. 5 ). Subsequent PCR analyses of DNA from a monochromosomal somatic cell hybrid mapping panel using several primer combinations, confirmed the chromosomal dispersion of FRG1-related sequences. With most primer combinations, appropriate PCR products could be amplified from cell hybrids containing one of the human chromosomes 4, 8, 9, 12, 13, 14, 15, 20, 21, and 22. To illustrate this, amplification of a fragment containing part of intron 4 and exon 5 using primers 5fa and 5r, is shown in Figure 6 a. The appropriate 320 bp product was detected in somatic cell hybrids containing chromosomes 4, 8, 9, 12, 20, 21 or 22. However, this product could not be amplified from cell hybrids containing chromosomes 13, 14, or 15, which may be an indication for sequence differences or the presence of processed FRG1-like pseudogenes. Amplification of the 320 bp fragment from cell hybrids containing chromosomes 8 or 12 does not correspond to the negative FISH results, suggesting that these chromosomes may harbor incomplete FRG1-related sequences not detectable by FISH.
Unlike most FRG1 primers, primer 1f derived from the 5' untranslated region of FRG1, enabled chromosome 4 specific amplification of FRG1 fragments, when used in several primer combinations. For example, using primers 1f and 1rb, a 180 bp fragment in exon 1 could only be amplified from DNA of the single chromosome 4 cell hybrid (Fig. 6 b). In addition, RT-PCR analysis of total RNA isolated from somatic cell hybrids specific for chromosome 4, 8, 9, 10, 12, 14, 15, 20, 21, or 22, using primer 1f in combination with primer 9r, revealed the 935 bp cDNA fragment in the chromosome 4 cell hybrid only (Fig. 6 c).
Northern blot analysis of total RNA isolated from lymphocytes of four sporadic FSHD patients with a de novo deletion and their unaffected parents, revealed a 1.1 kb FRG1 transcript in all individuals without significant deviations in the amount of transcript (data not shown). However, possible transcription of FRG1-related sequences may mask any effects on the transcription of the chromosome 4q35 gene. Therefore, detection of alteration of the FRG1 transcript level in FSHD patients compared to unaffected individuals requires the 4q35-locus to be analyzed specifically. As was indicated by the above described genomic and cDNA PCR analyses of the monochromosomal cell hybrids (Fig. 6 b,c), 4q35-specific amplification of FRG1 fragments is possible using primer 1f. Additional evidence for the specificity of this primer was obtained by SSCP-analysis of a Dutch FSHD family (see below).
Since FSHD is caused by a mutation on only one allele, the complete FRG1 cDNA was screened for polymorphisms to enable analysis of allele-specific transcripts. Primary, reverse transcribed lymphocyte RNA of a number of unaffected individuals was amplified using primers 1f and 9r to ensure 4q35-specific products and to avoid amplification of potential DNA contaminations. These 935 bp products were used in secondary, nested PCRs. Three overlapping cDNA fragments were obtained using primer combinations 1f-2r, 2f-6r and 5f-9r (Fig. 1 ) which were analyzed for sequence variation by single-strand conformation polymorphism analysis (SSCP) (35 ,36 ). Subsequent sequence analysis was performed to characterize the variant fragments. So far, five polymorphic sites were observed: three in the 5' untranslated region at positions -82 (C/T), -62 (C/T), and -10 (G/T), one in exon 1 at position 18 (TAT/TAC; Tyr18Tyr), and one in exon 5 at position 321 (ATT/ATC; Ile321Ile). These positions are indicated in Figure 3 b. The polymorphisms at positions -82 and -62, which can be analyzed by amplifying cDNA or DNA using primer combination 1f/1ra, were used to demonstrate co-segregation of the polymorphisms with the chromosome 4q35 haplotype in DNA from part of the large Dutch FSHD family 1 (4 ). Four alleles were observed which segregate in the family in a Mendelian fashion and show complete co-segregation with the FSHD haplotype, as determined by the loci D4S163 and D4S139 (Fig. 7 ).
FSHD has been shown to be associated with deletions within an EcoRI fragment detectable using probe p13E-11 (D4F104S1) (9 ). These deletions involve integral numbers of 3.3 kb repeated units (9 ,10 ) which are located adjacent to the 4q telomere (13 ,15 ,16 ). Since no transcribed sequences were identified within these repeated units (11 -14 ), the FSHD gene search has been focused on the region more centromeric. Deletion of a number of the repeated units may induce position effect variegation (PEV), reducing allelic transcript levels of proximally located genes. Recently, PEV has also been proposed as a potential genetic cause for aniridia (37 ).
The FSHD candidate region was scrutinized for genes using several gene identification methods. However, the region appeared to be comprised of different kinds of repetitive elements and regions homologous to several loci dispersed over the genome (38 ). This property has seriously complicated the search for candidate genes, in particular using hybridization-based gene identification methods such as cDNA selection (38 ). For this reason, other gene identification methods were applied such as exon trapping and screening of cDNA libraries with specifically selected genomic subclones. Several pseudogenes were identified, each originating from a different chromosome (Hewitt, Van Deutekom, unpublished results), which further confirms the complexity of the FSHD region.
By screening a skeletal muscle cDNA library with a genomic subclone encompassing a CpG island, we have identified a FSHD candidate gene. This gene, FRG1, is located approximately 100 kb centromeric to the repeated units involved in the FSHD associated deletions. FISH and PCR analysis indicated the presence of multiple FRG1-related sequences in the genome, some of which may represent FRG1 pseudogenes. Sequence analysis of the cDNA clones 10B7, 10B1 and BS3 showed that these clones contain several cloning artefacts and/or splicing aberrations (Fig. 1 ). Strikingly, multiple EST clones identified by FRG1 exhibit similar splicing aberrations, most of which can be explained by mutations in splicing junctions. The identified splicing aberrations introduce stop codons in the deduced open reading frame, suggesting that these FRG1-related sequences represent pseudogenes. The structure and function of the FRG1-related sequences on the different chromosomes are under further investigation (Van Deutekom, in preparation).
Since PEV was suggested to be the underlying mechanism for FSHD, allele-specific FRG1 transcript levels were analyzed to obtain indication for transcriptional repression. However, quantitative detection of allelic transcripts was complicated by the existence of several potentially transcribed FRG1-related sequences in the genome. Identification of primer 1f was valuable as it enabled 4q35-specific amplification of FRG1 transcripts. For analysis of allele-specific FRG1 transcript levels a RNA-based single-strand conformation polymorphism (SSCP) analysis was applied, using polymorphisms in exon 1. In multiple experiments no significant transcription repression was detected in reverse transcribed RNA isolated from patient lymphocytes and open muscle biopsies shown to contain muscle tissue for at least 95% by histologic analysis (see Materials and Methods). These results suggest that the FSHD associated deletion of repeated units is not influencing the transcript levels of FRG1 in these tissues. However, detection of PEV in FSHD patients may require analysis of more specific cell types at particular developmental stages. Moreover, since severely affected muscle groups mostly contain connective and fatty tissues, biopsies were taken from mildly affected muscle groups in which the percentage of affected muscle cells may have been low. If PEV would induce transcriptional silencing in affected cells only, this would remain undetectable by the abundant presence of unaffected muscle cells in the biopsies analyzed.
Recent data show a correlation between the D4F104S1 fragment size and the FSHD phenotype (39 ) which is supported in our own family panel (Padberg, unpublished results), suggesting that there may be differences in degree of transcript repression; larger fragments would induce weaker position effects. Detection of relatively weak transcription repression may require a more sensitive method. Although we have tested the sensitivity of our RNA-based SSCP analysis approach, we cannot exclude the possibility of a subtle change in transcription levels which we were not able to detect.
Despite application of multiple advanced gene identification methods in order to identify FSHD candidate genes, FRG1 is the first gene cloned from the complicated FSHD gene region since the detection of FSHD associated deletion fragments in 1992. If FRG1 is not the gene responsible for FSHD, it may define the centromeric border of this region assuming that PEV affects all genes in the interval.
Total RNA was isolated from lymphocytes of four sporadic FSHD patients, denoted before by patient numbers 2, 29, 260201, and 42, and their healthy parents (9 ,10 ,40 ). These patients carry de novo rearranged EcoRI fragments of 18 kb (patient 2), 27 kb (patient 29), 15.5 kb (patient 260201) and 13.5 kb (patient 42). Open muscle biopsies were taken from three familial FSHD patients, denoted before by numbers 10403, 10405, and 096021 (4 ), and one sporadic patient (patient number 33). From patients 10403 and 10405 carrying a 25 kb p13E-11 fragment, biopsies were taken from the musculus gastrocnemius and musculus quadriceps, respectively. The biopsy taken from patient 096021 who carries a p13E-11 fragment of 23 kb, was derived from the musculus quadriceps. From patient 33 in whom a de novo rearranged fragment of 16 kb was detected, a biopsy was taken from the musculus biceps brachii. Histologic analysis showed the percentage of muscle tissue in the different biopsies to be 95% (patient 10405), 95% (patient 096021), and 98% (patient 33). For technical reasons, the percentage could not be determined for the biopsy taken from patient 10403. Lymphocytes were also isolated from these four patients.
DNA of part of the large Dutch FSHD family 1 (4 ) was used to study co-segregation of the exon 1 polymorphisms with the chromosome 4 haplotype.
DNA from a human monochromosomal somatic cell hybrid panel was available to determine the chromosomal origin of FRG1-related sequences (UK HGMP Resource Centre). RNA was isolated from somatic cell hybrids specific for chromosome 4, 8, 9, 10, 12, 14, 15, 20, 21, and 22 (41 -44 ).
A human skeletal muscle cDNA library ([lambda]ZAPII, Stratagene) was screened with a [alpha]-32P labelled genomic probe p10B (Fig. 1), which is a 1.2 kb genomic PstI subclone of cosmid cT171 (28 ). Phage clone 10B7 was identified and the insert (640 bp) was recovered by amplification using the T7 and T3 vector primers (T7: 5'-AATACGACTCACTATAG-3', T3: 5'-ATTAACCCTCACTAAAG-3'). The PCR product was cloned into the pCRTMII vector using the TA Cloning Kit (Invitrogen) and subsequently sequenced. A human fetal brain cDNA library (HL1065b, ClonTech) and a total infant brain cDNA library (Soares, LLNL Library id 52.) were screened using [alpha]-32P labelled 10B7 as probe. Positive phage clones 10B1 (fetal brain cDNA) and BS3 (infant brain cDNA) were isolated and the EcoRI inserts (1.3 kb and 3.5 kb, respectively) were subsequently subcloned in pBluescriptIISK+ (Stratagene) and further characterized. For each phage library 5 * 105 plaque forming units were screened. Hybridizations were carried out for 16 h at 65oC as described (45 ). In addition, by amplifying cDNA libraries with cDNA-vector-specific primers in combination with 10B7-specific primers, two cDNA fragments were obtained: (i) PLC2FB22 was amplified from the human placenta cDNA library (HL1008, ClonTech) using 10B7-specific primer 2f (Fig. 1 , Table 1 ) and [lambda]gt11-specific primer B22 (5'-GGTGGCGACGACTCCTGGAG-3'); and (ii) ML8RT3 was amplified from the skeletal muscle cDNA library using 10B7-specific primer 8r (Fig. 1 , Table 1 ) and [lambda]ZAPII-specific primer T3.
To isolate the 5' end of the cDNA, the 5'-RACE-Ready Human Brain cDNA kit (ClonTech) was used in combination with one of the primers 1ra and 2r (Fig. 1 , Table 1 ). The PCR fragments generated were cloned in the TA cloning vector (Invitrogen) and subsequently sequenced.
All sequence reactions were performed according to the dideoxy method (46 ), using either the T7Sequencing Kit (Pharmacia) or Sequenase V2.0 (Amersham). Genomic and cDNA sequences were compared and/or characterized using computer programs BLAST-N (Basic Local Alignment Search Tool) (47 ), GDE V2.2 (Genetic Data Environment, S. Smith, University of Illinois), and the GCG package of programmes [Genetics Computer Group (1994)]. The putative protein sequence was analyzed using BLAST-P.
Table 1 . Sizes, splice junctions, and primers of FRG1 exons
Exon
Nucleotide
Exon
Splice junctions
Forward primer
Reverse primer
no.
position
size (bp)
3' splice site
5' splice site
1
-191*-62
253
-
GAG/gtggg
1f: 5'-TCTACAGAGACGTAGGCTGTCA-3'
1ra: 5'-CCGGTTCTGGACGAGTATGT-3'
1rb: 5'-CTTGAGCACGAGCTTGGTAG-3'
2
63-133
71
tgtag/TAA
TTG/gtgag
2f: 5'-GAAGAAGATGAAGAAACCCAGC-3'
2r: 5'-CAACAATATCAAGCTGGGTTTC-3'
3
134-259
126
tttag/GAA
AAG/gtttg
3f: 5'-GCCATTGAAATGGATAAGGG-3'
3r: 5'-TCCATTTCAATGGCTATGGT-3'
4
260-317
58
aatag/TTG
CAG/gtgag
4f: 5'-GCCCTAGTCCTCCAGAGCAG-3'
4r: 5'-GGAATCAGATAATTTGACAGCC-3'
5
318-432
115
tacag/AAT
AAT/gtaag
5fa[sect]: 5'-AGATGGGGTCTCACTGTGTTG-3'
5r: 5'-TCTTGGTCCAATTGCATCTG-3'
5fb: 5'-AGAGAACAATGGGAACCAGTC-3'
6
433-537
105
cttag/GGG
AAG/gtaat
6f: 5'-GGAAAATGGCTTTGTTGGCCTC-3'
6r: 5'-CTTCTATGTCCCCTGCTTCA-3'
7
538-629
92
tctag/ATT
TGT/gtatg
7f: 5'-CAAGATTAGATCCTGTGCTG-3'
7r: 5'-TTTCCTTTGTCTTCTTCTGGAA-3'
8
630-740
111
tacag/AAA
CAG/gtagc
8f: 5'-GGCTCGGAAAGATGGATTTT-3'
8r: 5'-AAAATCCATCTTTCCGAGCC-3'
9
741-851
111
aacag/GAG
-
9f: 5'-GAGAGCCAAATTGAAAGCCG-3'
9r: 5'-ATAAGGCAGAAACAAAAATCCC-3'
*Putative transcriptional start site[sect]Primer 5fa is derived from intron 4 sequence
The 935 bp FRG1 RT-PCR product, which was amplified from a chromosome 4 only somatic cell hybrid using primers 1f and 9r, was hybridized to Southern blots containing: (i) EcoRI digests of total human (2 [mu]g/lane), monkey, sheep, and rat (8 [mu]g/lane) (`Zoo blot', Fig. 4 a); and (ii) EcoRI and HindIII digests of total human lymphocytes DNA (7 [mu]g/lane), YAC 25C2E (D4S1093) (1.5 [mu]g/lane), and cosmid cT171 (28 ) (1.5 ng/lane) (Fig. 4 b). Digestions with specified restriction enzymes were performed according to the manufacturer's instructions (Pharmacia). Hybridizations were performed for 16 h at 65oC for the Southern blot and at 57oC for the `Zoo' blot, as described (48 ). The blots were washed at 65oC and to a stringency of 0.3* SSC/0.1% SDS for the Southern blot and of 1* SSC/0.1% SDS for the `Zoo' blot and, followed by autoradiography for 16-36 h at -70oC using Konica AX film with an intensifying screen.
Total RNA was isolated from various human tissues and somatic cell hybrids as previously described by Chomczynski and Sacchi (49 ). Poly-(A)+ RNA was isolated using the PolyATtract mRNA Isolation Systems kit (Promega) or purchased from ClonTech. Northern blots were prepared containing 20 [mu]g total RNA or 1 [mu]g poly-(A)+ RNA. Hybridizations were performed in a formamide hybridization mix (45 ) for 16 h at 54oC using [alpha]-32P labelled 10B7 as probe. The blots were washed at 65oC and to 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.
cDNA was synthesized from 5 [mu]g of total RNA isolated from human muscle, human lymphocytes, somatic cell hybrids specific for the particular chromosomes, Chinese hamster lung cells (V79) and mouse A9 cells, using an oligo-dT primer and the AMV Reverse Transcriptase kit from Promega. The primers used for (RT-) PCR analyses are indicated in Figure 1 and Table 1 . All primers were provided by Isogen Bioscience BV, Maarssen, The Netherlands. A total of 35 cycles (94oC for 40 s, 60oC for 60 s and 72oC for 90 s) were run in a 50 [mu]l volume containing 2.5 ng of cDNA, 1.0 U Taq-DNA-polymerase (Perkin-Elmer), 200 [mu]M of each dNTP, 100 ng of each primer and 5* PCR standard-buffer [300 mM Tris-HCl, 75 mM (NH4)2SO4, 17.5 mM MgCl2, pH 8.5].
Metaphase chromosome spreads from normal human lymphocytes were hybridized with the 3 kb, 6.5 kb, and 9.5 kb EcoRI fragments of cosmid cT171. Probes were labelled by nick translation with biotin 11-dUTP (Sigma) (50 ). Hybridization, washing and staining were performed as previously described (8 ).
cDNA of lymphocytes and muscle tissue was primarily amplified using primers 1f and 9r (Fig. 1 , Table 1 ). For the SSCP analysis (35 ,36 ), PCR products were diluted 1:50 of which subsequently 1 [mu]l was used for secondary amplifications with primer combinations 1f/1ra, 1f/2r, 2f/6r or 5f/9r (Fig. 1 , Table 1 ) and with incorporation of [alpha]-32P labelled dCTP. Products were diluted 1:25 in a formamide loading buffer (1:1 mix of 95% formamide/20 mM EDTA and 0.1% SDS/ 10 mM EDTA) and denatured at 100oC for 10 min. Electrophoresis was performed in non-denaturing 0.5* MDE polyacrylamide gels (J.T. Baker) and 0.6* TBE buffer (1*: 0.09 M Tris, 0.09 M Boric acid, 0.002 M EDTA, adjusted to pH 8.0) using a BRL-S2 vertical gel apparatus (Life Technologies, Inc.). Gels of 40 cm were run at 25 Watt for 3-4 h and subsequently dried at 80oC under vacuum, and exposed at -70oC for 12-24 h to Konica AX film with an intensifying screen.
The authors thank R. Lyle and C. Wijmenga for comments on the manuscript. This study was funded by The Prinses Beatrix Fonds, The Muscular Dystrophy Group of Great Britain, The Muscular Dystrophy Association (USA), and the Association Française contre les Myopathies.
2 Lunt, P.W. and Harper, P.S. (1991) Genetic counseling in facioscapulohumeral muscular dystrophy. J. Med. Genet., 28, 655-664.MEDLINE Abstract
3 Munsat, T.L. (1986) Facioscapulohumeral dystrophy and the scapuloperoneal syndrome. In: Myology; McGraw Hill, New York.
4 Wijmenga, C., Frants, R.R., Brouwer, O.F., Moerer, P., Weber, J.L. and Padberg, G.W. (1990) Location of facioscapulohumeral muscular dystrophy gene on chromosome 4. Lancet, 336, 651-653. MEDLINE Abstract
5 Upadhyaya, M., Lunt, P.W., Sarfarazi, M., Broadhead, W., Daniels, J., Owen, M. and Harper, P.S. (1990) DNA marker applicable to presymptomatic and prenatal diagnosis of facioscapulohumeral disease. Lancet, 336, 1320-1321. MEDLINE Abstract
6 Sarfarazi, M., Wijmenga, C., Upadhyaya, M., Weiffenbach, B., Hyser, C., Mathews, K., Murray, J.C., Gilbert, J., Pericak-Vance, M., Lunt, P., Frants, R.R., Jacobsen, S., Harper, P.S., Padberg, G.W. (1992) Regional mapping of facioscapulohumeral muscular dystrophy gene on 4q35. Combined analysis of an international consortium. Am. J. Hum. Genet., 51, 396-403. MEDLINE Abstract
7 Upadhyaya, M., Lunt, P.W., Sarfarazi, M., Broadhead, W., Daniels, J., Owen, M. and Harper, P.S. (1991) A closely linked DNA marker for facioscapulohumeral disease on chromosome 4q. J. Med. Genet., 28, 665-671.MEDLINE Abstract
8 Wijmenga, C., Padberg, G.W., Moerer, P., Wiegant, J., Liem, L., Brouwer, O.F., Milner, E.C.B., Weber, J.L., Van Ommen, G-J., Sandkuijl, L.A. and Frants, R.R. (1991) Mapping of facioscapulohumeral muscular dystrophy gene to chromosome 4q35-qter by multipoint linkage analysis and in situ hybridization. Genomics, 9, 570-575. MEDLINE Abstract
9 Wijmenga, C., Hewitt, J.E., Sandkuijl, L.A., Clark, L.N., Wright, T.J., Dauwerse, J.G., Gruter, A-M., Hofker, M.H., Moerer, P., Williamson, R., Van Ommen, G-J., Padberg G.W. and Frants, R.R. (1992) Chromosome 4q DNA rearrangements associated with facioscapulohumeral muscular dystrophy. Nature Genet., 2, 26-30.MEDLINE Abstract
10 Van Deutekom, J.C.T., Wijmenga, C., Van Tienhoven, E.A.E., Gruter, A-M., Hewitt, J.E., Padberg, G.W., Van Ommen, G-J., Hofker, M.H. and Frants, R.R. (1993) FSHD associa-ted DNA rearrangements are due to deletions of integral copies of a 3.2 kb tandemly repeated unit. Hum. Mol. Genet., 2, 2037-2042.
11 Hewitt, J.E., Lyle, R., Clark, L.N., Valleley, E.M., Wright, T.J., Wijmenga, C., Van Deutekom, J.C.T., Francis, F., Sharpe, P.T., Hofker, M., Frants, R.R. and Williamson, R. (1994) Analysis of the tandem repeat locus D4Z4 associated with facioscapulohumeral muscular dystrophy. Hum. Mol. Genet., 3, 1287-1295.MEDLINE Abstract
12 Lee, J.H, Goto, K., Matsuda, C. and Arahata, K. (1995) Characterization of a tandemly repeated 3.3 kb KpnI unit in the facioscapulohumeral muscular dystrophy (FSHD) gene region on chromosome 4q35. Muscle & Nerve, Suppl 2: S6-S13.
13 Winokur, S.T., Bengtsson, U., Feddersen, J., Mathews, K.D., Weiffenbach, B., Bailey, H., Markovich, R.P., Murray, J.C., Wasmuth, J.J., Altherr, M.R. and Schutte, B.C. (1994) The DNA rearrangement associated with facioscapulohumeral muscular dystrophy involves a heterochromatin-associated repetitive element: implications for a role of chromatin structure in the pathogenesis of the disease. Chromosome Research, 2, 225-234.MEDLINE Abstract
14 Lyle, R., Wright, T.J., Clark, L.N. and Hewitt, J.E. (1995) The FSHD-associated repeat, D4Z4, is a member of a dispersed family of homeobox-containing repeats, subsets of which are clustered on the short arms of the acrocentric chromosomes. Genomics, 28, 389-397.MEDLINE Abstract
15 Bengtsson, U., Altherr, M.R., Wasmuth, J.J. and Winokur, S.T. (1995) High resolution fluorescence in situ hybridization to linearly extended DNA visually maps a tandem repeat associated with facioscapulohumeral muscular dystrophy immediately adjacent to the telomere of 4q. Hum. Mol. Genet., 3, 1801-1805.
16 Altherr, M.R., Bengtsson, U., Markovich, R.P. and Winokur, S.T. (1995) Efforts toward understanding the molecular basis of facioscapulohumeral muscular dystrophy. Muscle & Nerve, Suppl 2: S32-S38.MEDLINE Abstract
17 Hendrich, B.D. and Willard, H.F. (1995) Epigenetic regulation of gene expression: the effect of altered chromatin structure from yeast to mammals. Hum. Mol. Genet., 4, 1765-1777.MEDLINE Abstract
18 Moehrle, A. and Paro, R. (1994) Spreading the silence: epigenetic transcriptional regulation during Drosophila development. Dev. Genet., 15, 478-484.MEDLINE Abstract
20 Braunstein, M., Rose, A.B., Holmes, S.G., Allis, C.D. and Broach, J.R. (1993) Transcriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes Dev., 7, 592-604.MEDLINE Abstract
21 Eicher, E. M. (1970) X-autosome translocations in the mouse: total inactivation versus partial inactivation of the X chromosome. Adv. Genet., 15, 175-259.MEDLINE Abstract
22 Torchia, B.S., Call, L.M. and Migeon, B.R. (1994) DNA replication analysis of FMR1, XIST, and factor 8C loci by FISH shows nontranscribed X-linked genes replicate late. Am. J. Hum. Genet., 55, 96-104.MEDLINE Abstract
23 Jeppesen, P. and Turner, B.M. (1993) The inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene expression. Cell, 74, 281-289.MEDLINE Abstract
24 Gilson, E., Laroche, T. and Gasser, S.M. (1993) Telomeres and the functional architecture of the nucleus. Trends Cell Biol., 3, 128-134.
25 Karpen, G.H. (1994) Position-effect variegation and the new biology of heterochromatin. Curr. Opin. Genet. Dev.,4, 281-291.MEDLINE Abstract
26 Karpen, G.H. and Spradling, A.C. (1992) Analysis of subtelomeric heterochromatin in the Drosophila minichromosome Dp1187 by single P element insertional mutagenesis. Genetics, 132, 737-753.MEDLINE Abstract
27 Lee, J.H, Goto, K., Sahashi, K., Nonaka, I., Matsuda, C., and Arahata, K. (1995) Cloning and mapping of a very short (10-kb) EcoRI fragment associated with facioscapulohumeral muscular dystrophy (FSHD). Muscle & Nerve, Suppl 2: S27-S31.
28 Wright, T.J., Wijmenga, C., Clark, L.N., Frants, R.R., Williamson, R. and Hewitt, J.E. (1993) Fine mapping of the FSHD region orientates the rearranged fragment detected by the probe p13E-11. Hum. Mol. Genet., 2, 1673-1678.MEDLINE Abstract
29 Wijmenga, C., Wright, T.J., Baan, M.J., Padberg, G.W., Williamson, R., Van Ommen, G-J., Hewitt, J.E., Hofker, M.H. and Frants, R.R. (1993) Physical mapping and YAC-cloning connects four genetically distinct 4qter loci (D4S163, D4S139, D4F35S1 and D4F104S1) in the FSHD gene-region. Hum. Mol. Genet., 2, 1667-1672. MEDLINE Abstract
30 Frohman, M.A., Dush, M.K. and Martin, G.R. (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl Acad. Sci. USA, 85, 8998-9002.MEDLINE Abstract
31 Kozak, M. (1984) Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res., 12, 857-872.MEDLINE Abstract
32 Kadonaga, J.T, Jones, K.A. and Tjian, R. (1986) Promoter-specific activation of RNA polymerase II transcription by Sp1. Trends Biochem. Sci., 11, 20-23.
33 Blake, M.C., Jambou, R.C., Swick, A.G., Kahn, J.W. and Azizkhan, J.C. (1990) Transcriptional initiation is controlled by upstream GC-box interactions in a TATAA-less promoter. Mol. Cell. Biol., 10, 6632-6641.MEDLINE Abstract
34 Gardiner-Garden, M. and Frommer, M. (1987) CpG islands in vertebrate genomes. J. Mol. Biol., 196, 261-282.MEDLINE Abstract
35 Orita, M., Suzuki, Y., Sekiya, T. and Hayashi, K. (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics, 5, 874-879.MEDLINE Abstract
36 Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K. and Sekiya, T. (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc. Natl Acad. Sci. USA, 86, 2766-2770.MEDLINE Abstract
37 Fantes, J., Redeker, B., Breen, M., Boyle, S., Brown, J., Fletcher, J., Jones, S., Bickmore, W., Fukushima, Y., Mannens, M., Danes, S., Van Heyningen, V. and Hanson, I. (1995) Aniridia-associated cytogenetic rearrangements suggest that a position effect may cause the mutant phenotype. Hum. Mol. Genet., 4, 415-422. MEDLINE Abstract
38 Van Deutekom, J.C.T., Hofker, M.H., Romberg, S., Van Geel, M., Rommens, J., Wright, T. J., Hewitt, J.E., Padberg, G.W., Wijmenga, C. and Frants, R.R. (1995) Search for the FSHD gene using cDNA selection in a region spanning 100 kb on chromosome 4q35. Muscle & Nerve, Suppl 2: S19-S26.
39 Lunt, P.W., Jardine, P.E., Koch, M.C., Maynard, J., Osborn, M., Williams, M., Harper, P.S. and Upadhyaya, M. (1995) Correlation between fragment size at D4F104S1 and age at onset or at weelchair use, with a possible generational effect, accounts for much phenotypic variation in 4q35-facioscapulohumeral muscular dystrophy (FSHD). Hum. Mol. Genet., 4, 951-958.MEDLINE Abstract
40 Wijmenga, C., Brouwer, O.F., Padberg, G.W. and Frants, R.R. (1992) Transmission of a de novo mutation associated with facioscapulohumeral muscular dystrophy. Lancet, 340, 985-986.MEDLINE Abstract
41 Carlock, L.R., Smith, D. and Wasmuth, J.J. (1986) Genetic counterselective procedure to isolate interspecific cell hybrids containing single human chromosomes: construction of cell hybrids and recombinant DNA libraries specific for human chromosomes 3 and 4. Somat. Cell Mol. Genet., 12, 163-174.MEDLINE Abstract
42 Koi, M., Shimizu, M., Morita, H., Yamada, H. and Oshimura, M. (1989) Construction of mouse A9 clones containing a single human chromosome tagged with neomycin-resistance gene via microcell fusion. Jpn. J. Cancer Res., 80, 413-418.MEDLINE Abstract
43 Athwal, R.S., Smarsh, M., Searle, B.M. and Deo, S.S. (1985) Integration of a dominant selectable marker into human chromosomes and transfer of marked chromosomes to mouse cells by microcell fusion. Somat. Cell Mol. Genet., 11, 177-187.MEDLINE Abstract
44 Cuthbert, A.P., Trott, D.A., Ekong, R.M., Jezzard, S., England, N.L., Themis, M., Todd, C.M. and Newbold, R.F. (1995) Construction and characterization of a highly stable human:rodent monochromosomal hybrid panel for genetic complementation and genome mapping studies. Cytogenet. Cell Genet., 71, 68-76.MEDLINE Abstract
45 Sambrook, J., Fritsch, E. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
46 Sanger F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc. Natl Acad. Sci. USA, 74, 5463-5467.MEDLINE Abstract
47 Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol., 215, 403-410.MEDLINE Abstract
48 Church, G.M. and Gilbert, W. (1984) Genome sequencing. Proc. Natl Acad. Sci. USA, 81, 1991-1995.MEDLINE Abstract
49 Chomczynski, P. and Sacchi, N. (1987) Single step method of RNA isolation by acidguanidinium thiocyanate phenol chloroform extraction. Anal. Biochem., 162, 156-159.MEDLINE Abstract
50 Langer, P.R., Waldrop, A.A. and Ward, D.C. (1981) Enzymatic synthesis of biotin labelled polynucleotides: novel nucleic acid affinity probes. Proc. Natl Acad. Sci. USA, 78, 6633-6637.MEDLINE Abstract
*To whom correspondence should be addressed
+Present address: Department of Human Genetics, Roswell Park Cancer Institute, Buffalo, NY, USA
}Present address: Life Sciences Division LS-3, Los Alamos National Laboratory, Los Alamos, NM, USA
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