Mapping the facioscapulohumeral muscular dystrophy gene is complicated by chromsome 4q35 recombination events.

A gene responsible for facioscapulohumeral muscular dystrophy (FSHD) has been linked to polymorphisms on chromosome 4q35. Multipoint linkage analyses have placed this gene distal to all reported genetic markers on the chromosome. By using as a probe a clone isolated from a cosmid containing sequences related to a homeobox domain, de novo DNA rearrangements were reported in sporadic and familial cases of FSHD. Linkage analysis of an EcoRI polymorphism detected by this clone in twenty-four multigenerational FSHD families revealed recombinants between this marker and the disease with a recombination fraction of 0.05. Two families with apparent germline mosaicism were also identified.

The Huntington's disease candidate region exhibits many different haplotypes.

Analysis of 78 Huntington's disease (HD) chromosomes with multi-allele markers revealed 26 different haplotypes, suggesting a variety of independent HD mutations. The most frequent haplotype, accounting for about one third of disease chromosomes, suggests that the disease gene is between D4S182 and D4S180. However, the paucity of an expected class of chromosomes that can be related to this major haplotype by assuming single crossovers may reflect the operation of other mechanisms in creating haplotype diversity. Some of these mechanisms sustain alternative scenarios that do not require a multiple mutational origin for HD and/or its positioning between D4S182 and D4S180.

Cloning of the alpha-adducin gene from the Huntington's disease candidate region of chromosome 4 by exon amplification.

We have applied the technique of exon amplification to the isolation of genes from the chromosome 4p16.3 Huntington's disease (HD) candidate region. Exons recovered from cosmid Y24 identified cDNA clones corresponding to the alpha-subunit of adducin, a calmodulin-binding protein that is thought to promote assembly of spectrin-actin complexes in the formation of the membrane cytoskeleton, alpha-adducin is widely expressed and, at least in brain, is encoded by alternatively spliced mRNAs. The alpha-adducin gene maps immediately telomeric to D4S95, in a region likely to contain the HD defect, and must be scrutinized to establish whether it is the site of the HD mutation.

Characterization of a yeast artificial chromosome contig spanning the Huntington's disease gene candidate region.

The Huntington's disease (HD) gene has been localized by recombination events to a region covering 2.2 megabases (Mb) DNA within chromosome 4p16.3. We have screened three yeast artificial chromosome (YAC) libraries in order to isolate and characterize 44 YAC clones mapping to this region. Approximately 50% of the YACs were chimaeric. Unstable YACs were identified across the whole region, but were particularly prevalent around the D4S183 and D4S43 loci. The YACs have been assembled into a contig extending from D4S126 to D4S98 covering roughly 2 Mb DNA, except for a gap of about 250 kilobases (kb). The establishment of a YAC contig which spans the region most likely to contain the HD mutation is an essential step in the isolation of the HD gene.

Actinin-associated LIM protein: identification of a domain interaction between PDZ and spectrin-like repeat motifs.

PDZ motifs are protein-protein interaction domains that often bind to COOH-terminal peptide sequences. The two PDZ proteins characterized in skeletal muscle, syntrophin and neuronal nitric oxide synthase, occur in the dystrophin complex, suggesting a role for PDZ proteins in muscular dystrophy. Here, we identify actinin-associated LIM protein (ALP), a novel protein in skeletal muscle that contains an NH2-terminal PDZ domain and a COOH-terminal LIM motif. ALP is expressed at high levels only in differentiated skeletal muscle, while an alternatively spliced form occurs at low levels in the heart. ALP is not a component of the dystrophin complex, but occurs in association with alpha-actinin-2 at the Z lines of myofibers. Biochemical and yeast two-hybrid analyses demonstrate that the PDZ domain of ALP binds to the spectrin-like motifs of alpha-actinin-2, defining a new mode for PDZ domain interactions. Fine genetic mapping studies demonstrate that ALP occurs on chromosome 4q35, near the heterochromatic locus that is mutated in fascioscapulohumeral muscular dystrophy.

Genetics of facioscapulohumeral muscular dystrophy: new mutations in sporadic cases.

A gene for facioscapulohumeral muscular dystrophy (FSHD) has been linked to chromosome 4q35 in families with the disease. We have used recently characterized p13E-11/D4S809 probes that map near or within the FSHD gene to investigate eight sporadic cases of FSHD whose parents showed no signs of disease. Probe p13E-11/D4S809 detected novel DNA fragments in seven of the eight sporadic FSHD individuals and not in the parents, substantiating the clinical diagnosis. Two sisters with FSHD whose parents were clinically normal had a novel DNA fragment suggestive of germline mosaicism. Probe p13E-11/D4S809 is potentially helpful in genetic counseling. However, because this probe may also detect a locus unlinked to chromosome 4, because of possible genetic heterogeneity in FSHD, and because of the presence of recombinants in autosomal dominantly inherited families, closer markers or gene definition will be needed for accurate genetic counseling in other situations.

Wolf-Hirschhorn and Pitt-Rogers-Danks syndromes caused by overlapping 4p deletions.

Wolf-Hirschhorn syndrome (WHS), a multiple congenital malformation syndrome, and Pitt-Rogers-Danks syndrome (PRDS), a rare condition with similar anomalies, were previously thought to be clinically distinct conditions. While WHS has long been associated with deletions near the terminus of 4p, several recent studies have shown PRDS is associated with deletions in 4p16.3. In this paper we evaluate three patients, two described as PRDS and one diagnosed as WHS. We demonstrate that the molecular defects associated with the two syndromes show a considerable amount of overlap. We conclude that both of these conditions result from the absence of similar, if not identical, genetic segments and propose that the clinical differences observed between these two syndromes are likely the result of allelic variation in the remaining homologue.

FISH detection of Wolf-Hirschhorn syndrome: exclusion of D4F26 as critical site.

Wolf-Hirschhorn syndrome (WHS) is due to a deletion in the terminal band of 4p16.3. Among loci that have been involved in deletions are D4S98, D4S95, D4S125, D4F26, as shown by PCR typing, Southern blot hybridization, and/or fluorescent in situ hybridization (FISH). Currently, FISH detection of WHS is predicted upon the deletion of the D4F26 locus with failure to hybridize to pC847.351, a commercially available cosmid probe. A WHS patient is shown to have an interstitial deletion, by hemizygosity at D4S98 and D4S95 but not at D4F26. This suggests that the tip of 4p, specifically D4F26, is not a critical deletion site for WHS.

Towards the finer mapping of facioscapulohumeral muscular dystrophy at 4q35: construction of a laser microdissection library.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disorder which has been mapped to the 4q35 region. In order to saturate this distal 4q region with DNA markers, a laser-based chromosomal microdissection and microcloning procedure was used to construct a genomic library from the distal 20% of chromosome 4, derived from a single human metaphase spread. Of the 100 microclones analyzed from this library, 94 clones contained inserts sized from 80-800 bp, with an average size of 340 bp. Less than 20% of these clones hybridized to human repeat sequences. Seventy-two single-copy clones were further characterized by Southern blot hybridization against a DNA panel of somatic cell hybrids, containing various regions of chromosome 4. Forty-two clones mapped to chromosome 4, of which 8 clones mapped into the relevant 4q35 region. Twenty of these chromosome 4-specific clones were screened against "zoo-blots"; 11 clones, of which 3 mapped to 4q35, identified conserved sequences. This is the first report to describe the isolation of potential expressed sequences derived from the FSHD region. These chromosome region-specific microclones will be useful in the construction of the physical map of the region, the positional cloning of potential disease-associated genes, and the identification of additional polymorphic markers from within the distal 4q region.

Delimiting the Wolf-Hirschhorn syndrome critical region to 750 kilobase pairs.

Wolf-Hirschhorn syndrome (WHS) is a multiple anomaly condition characterized by mental and developmental defects, resulting from the absence of the distal segment of one chromosome 4 short arm (4p16.3). Owing to the complex and variable expression of this disorder, it is thought that the WHS is a contiguous gene syndrome with an undefined number of genes contributing to the phenotype. The 2.2 Mbp genomic segment previously defined as the critical region by the analyses of patients with terminal or interstitial deletions is extremely gene dense and an intensive investigation of the developmental role of all the genes contained within it would be daunting and expensive. Further refinement in the definition of the critical region would be valuable but depends on available patient material and accurate clinical evaluation. In this study, we have utilized fluorescence in situ hybridization to further characterize a WHS patient previously demonstrated to have an interstitial deletion and demonstrate that the distal breakpoint occurs between the loci FGFR3 and D4S168. This reduces the critical region for this syndrome to less than 750 kbp. This has the effect of eliminating several genes previously proposed as contributing to this syndrome and allows further research to focus on a more restricted region of the genome and a limited set of genes for their role in the WHS syndrome.

Molecular detection of a 4p deletion using PCR-based polymorphisms: a technique for the rapid detection of the Wolf-Hirschhorn syndrome.

Wolf-Hirschhorn syndrome (WHS) results from a deletion of part of chromosome 4p. The region of 4p consistently deleted in WHS is near the tip of 4p. Two loci in this region D4S95 and D4S125 are associated with highly informative VNTR polymorphisms and were recently converted to allow PCR-based screening. PCR analysis was used successfully to identify a small de novo deletion of 4p in a patient suspected of having WHS. This procedure allows a rapid and accurate confirmation of 4p deletions in cases where cytogenetics alone cannot provide a clear answer.

"Tandem" duplication of 4p16.1p16.3 chromosome region associated with 4p16.3pter molecular deletion resulting in Wolf-Hirschhorn syndrome phenotype.

Chromosome imbalance affecting the short arm of chromosome 4 results in a variety of distinct clinical conditions. Most of them share a number of manifestations, such as mental retardation, microcephaly, pre- and post-natal growth retardation, anteverted and low-set ears, that can be considered as nonspecific signs, generally attributable to gene dosage impairment. On the other hand, more distinctive phenotypic traits correlate with the segmental aneuploidy. Duplications of the distal half of 4p give rise to the partial trisomy 4 syndrome, characterized by a "boxer" nose configuration and deep-set eyes. These signs are usually observed even in cases of small terminal duplications. Haploinsufficiency of 4p16.3 results in the so-called Wolf-Hirschhorn (WH) syndrome, a contiguous gene syndrome characterized by maxillary hypoplasia, large and protruding eyes, high nasal bridge, skeletal abnormalities, and midline defects. The smallest overlapping deletion described so far as a cause of this condition is only 165 kb long, suggesting that one or a few genes in this region act as "master" regulators of different developmental pathways. A "tandem" duplication of 4p16.1p16.3 was detected in association with a subtle deletion of 4p16.3pter on the same chromosome in a patient with the WH phenotype. The 3.2 Mb deletion, spanning the genomic region from the vicinity of D4S43 to the telomere, encompasses the recently delimited "WHS critical region" [Wright et al., 1997: Hum. Mol. Genet. 6:317-324]. This unusual chromosome rearrangement resulted in WH phenotype, clinical manifestations of partial 4p trisomy being mild or absent. This observation led us to speculate that the regulatory gene/genes in the critical WH region affect the expression of other genes in a dose-dependent manner. Haploinsufficiency of this region could be more deleterious than various partial trisomies.

First known microdeletion within the Wolf-Hirschhorn syndrome critical region refines genotype-phenotype correlation.

Deletions within HSA band 4p16.3 cause Wolf-Hirschhorn syndrome (WHS), which comprises mental retardation and developmental defects. A WHS critical region (WHSCR) of approximately 165 kb has been defined on the basis of 2 atypical interstitial deletions; however, genotype-phenotype correlation remains controversial, due to the large size of deletion usually involving several megabases. We report on the first known patient with a small de novo interstitial deletion restricted to the WHSCR who presented with a partial WHS phenotype consisting only of low body weight for height, speech delay, and minor facial anomalies; shortness of stature, microcephaly, seizures and mental retardation were absent. The deletion was initially demonstrated by FISH analysis, and breakpoints were narrowed with a "mini-FISH" technique using 3-5 kb amplicons. A breakpoint-spanning PCR assay defined the distal breakpoint as disrupting the WHSC1 gene within intron 5, exactly after an AluJb repeat. The proximal breakpoint was not found to be associated with a repeated sequence or a known gene. The deletion encompasses 191.5 kb and includes WHSC2, but not LETM1. Thus, manifestations attributable to this deletion are reduced weight for height, minor facial anomalies, ADHD and some learning and fine motor deficiencies, while seizures may be associated with deletions of LETM1.

High resolution characterization of an interstitial deletion of less than 1.9 Mb at 4p16.3 associated with Wolf-Hirschhorn syndrome.

Wolf-Hirschhorn syndrome (WHS) caused by 4p16.3 deletions comprises growth and mental retardation, distinct facial appearance and seizures. This study characterized a subtle interstitial deletion of 4p16.3 in a girl with mild retardation and possessing facial traits characteristic of WHS. The patient had generalized seizures in conjunction with fever at 3 and 5 years of age. Fluorescence in situ hybridization (FISH) with a series of markers in the 4p16.3 region showed that the interstitial deletion in this patient was between the probes D4S96 and D4S182, enabling the size of the deletion to be estimated as less than 1.9 Mb. This is the smallest interstitial deletion of 4p16.3 which has been reported. The patient contributes to a refinement of the phenotypic map of the WHS region in 4p16.3. The critical region for the characteristic facial changes of WHS, failure to thrive and developmental delay is now localized to a region of less than 700 kb. The mental retardation of this patient was mild suggesting that small interstitial deletion may have less severe phenotypic consequences.

Genotype-phenotype correlations and clinical diagnostic criteria in Wolf-Hirschhorn syndrome.

We report on a clinical-genetic study of 16 Wolf-Hirschhorn syndrome (WHS) patients. Hemizygosity of 4p16.3 was detected by conventional prometaphase chromosome analysis (11 patients) or by molecular probes on apparently normal chromosomes (4 patients). One patient had normal chromosomes without a detectable molecular deletion within the WHS "critical region." In each deleted patient, the deletion was demonstrated to be terminal by fluorescence in situ hybridization (FISH). The proximal breakpoint of the rearrangement was established by prometaphase chromosome analysis in cases with a visible deletion. It was within the 4p16.1 band in six patients, apparently coincident with the distal half of this band in five patients. The extent of each of the four submicroscopic deletions was established by FISH analyses with a set of overlapping cosmid clones spanning the 4p16.3 region. We found ample variations in both the size of the deletions and the position of the respective breakpoints. The precise definition of the cytogenetic defect permitted an analysis of the genotype-phenotype correlations in WHS, leading to the proposal of a set of minimal diagnostic criteria, which in turn may facilitate the selection of critical patients in the search for the gene(s) responsible for this disorder. We observed that genotype-phenotype correlations in WHS mostly depend on the size of the deletion, a deletion of <3.5 Mb resulting in a mild phenotype, in which malformations are absent. The absence of a detectable molecular deletion is still consistent with a WHS diagnosis. Based on these observations a "minimal" WHS phenotype was inferred, the clinical manifestations of which are restricted to the typical facial appearance, mild mental and growth retardation, and congenital hypotonia.

In situ studies with membrane diffusion chambers of antibiotic resistance transfer in Escherichia coli.

Coliform bacteria were isolated from raw sewage and sewage effluent-receiving waters and tested for their antibiotic susceptibility patterns and their ability to transfer antibiotic resistance to Escherichia coli K-12 C600. An environmental isolate of E. coli (MA527) capable of transferring antibiotic resistance to C600 was mated, both in vitro and in situ, with an antibiotic-sensitive E. coli environmental isolate (MA728). In situ matings were conducted in modified membrane diffusion chambers, in the degritter tank at the Grant Street (Melbourne, Fla.) sewage treatment facility, and in the sewage effluent-receiving waters in Melbourne, Fla. The transfer frequencies in situ were 3.2 x 10(-5) to 1.0 x 10(-6), compared with 1.6 x 10(-4) to 4.4 x 10(-5) observed in vitro. Transfer was shown to occur in raw sewage but was not detected in the effluent-receiving waters. The presence of a 60-megadalton plasmid species in both donor and transconjugants, but not in the recipients, provided physical evidence for the transfer of antibiotic resistance in situ.

Genomic organization of the mouse fibroblast growth factor receptor 3 (Fgfr3) gene.

The fibroblast growth factor receptor 3 (Fgfr3) protein is a tyrosine kinase receptor involved in the signal transduction of various fibroblast growth factors. Recent studies suggest its important role in normal development. In humans, mutation in Fgfr3 is responsible for growth disorders such as achondroplasia, hypoachondroplasia, and thanatophoric dysplasia. Here, we report the complete genomic organization of the mouse Fgfr3 gene. The murine gene spans approximately 15 kb and consists of 19 exons and 18 introns. One major and one minor transcription initiation site were identified. Position +1 is located 614 nucleotides upstream from the ATG initiation codon. The translation initiation and termination sites are located in exons 2 and 19, respectively. Five Sp1 sites, two AP2 sites, one Zeste site, and one Krox 24 site were observed in the 5'-flanking region. The Fgfr3 promoter appears to be contained within a CpG island and, as is common in genes having multiple Sp1-binding sites, lacks a TATA box.

Genomic organization of the human fibroblast growth factor receptor 3 (FGFR3) gene and comparative sequence analysis with the mouse Fgfr3 gene.

Fibroblast growth factor receptor 3 (FGFR3) is a developmentally regulated transmembrane protein. Three other FGFRs (1, 2, and 4) in conjunction with FGFR3 are part of the receptor tyrosine kinase super-family. Mutations in three of these genes (FGFR1, 2, and 3) have been determined to be the cause of human growth and developmental disorders. We have characterized a 22-kb DNA fragment containing the human FGFR3 gene and determined 11 kb of its nucleotide sequence. The gene consists of 19 exons and 18 introns spanning 16.5 kb, and the boundaries between exons and introns follow the GT/AG rule. The translation initiation and termination sites are located in exon 2 and exon 19 respectively. The sequence of the 5'-flanking region (1.5 kb) lacks the typical TATA or CAAT boxes. However, several putative binding sites for transcription factors SP1, AP2, Krox 24, IgHC.4, and Zeste are present. The 0.77-kb region from position -889 (5'-flanking region) to -119 (intron 1) contains a CpG island. A comparative sequence analysis of the human and mouse FGFR3 genes indicates that the overall genomic structure and organization of the human gene are nearly identical to those of its mouse counterpart. Furthermore, there is a striking similarity in the promoter regions of both genes, and several of the putative transcription factor-binding sites are conserved across species, suggesting a definitive role of these factors in the transcriptional regulation of these genes.

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.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder. The FSHD locus has been linked to the most distal genetic markers on the long arm of chromosome 4. An EcoRI fragment length polymorphism segregates with the disease in most FSHD families. Within the EcoRI fragment lies a tandem array of 3.2 kb repeats. Deletions of integral copies of this repeat have been associated with the disease. The 3.2 kbp repeat has recently been shown to cross-hybridize to several regions of heterochromatin in the human genome and DNA sequence analysis reveals strong homology to a class of heterochromatin repeats, LSau. In this report, we demonstrate that the 3.2 kbp tandem repeat lies adjacent to a subtelomeric sequence, which is within 5-14 kb of the telomeric repeat (TTAGGG)n. Direct visual fluorescence hybridization to linearly extended strands of DNA enabled the visualization of this subtelomeric sequence as a short string of signals at the end of a longer string of signals from the differentially labeled 3.2 kbp tandem repeat. Furthermore, in support of our data showing that the 3.2 kbp repeat lies in close proximity to the telomere of 4q, we demonstrated the lack of hybridization of total human DNA to this same region. Our results indicate that the tandem array of 3.2 kbp repeats, disrupted in FSHD, lies immediately adjacent to the telomere of 4q and that the gene responsible for FSHD is likely located proximal to the tandem repeat.