Autosomal dominant craniometaphyseal dysplasia is caused by mutations in the transmembrane protein ANK.

Craniometaphyseal dysplasia (CMD) is a rare skeletal disorder characterized by progressive thickening and increased mineral density of craniofacial bones and abnormally developed metaphyses in long bones. Linkage studies mapped the locus for the autosomal dominant form of CMD to an approximately 5-cM interval on chromosome 5p, which is defined by recombinations between loci D5S810 and D5S1954. Mutational analysis of positional candidate genes was performed, and we describe herein three different mutations, in five different families and in isolated cases, in ANK, a multipass transmembrane protein involved in the transport of intracellular pyrophosphate into extracellular matrix. The mutations are two in-frame deletions and one in-frame insertion caused by a splicing defect. All mutations cluster within seven amino acids in one of the six possible cytosolic domains of ANK. These results suggest that the mutated protein has a dominant negative effect on the function of ANK, since reduced levels of pyrophosphate in bone matrix are known to increase mineralization.

The Treacher Collins syndrome (TCOF1) gene product, treacle, is targeted to the nucleolus by signals in its C-terminus.

The TCOF1 gene product, treacle, responsible for the craniofacial disorder Treacher Collins syndrome, has been predicted to be a member of a class of nucleolar phosphoproteins based on its primary amino acid sequence. Treacle is a low complexity protein with ten repeating units of acidic and basic residues, each of which contains a large number of putative casein kinase 2 and protein kinase C phosphorylation sites. In addition, the C-terminus of treacle contains multiple putative nuclear localization signals. The overall structure of treacle, as well as sequence similarity to several nucleolar phosphoproteins, predicts that treacle is a member of this class of proteins. Using green fluorescent protein fusion constructs with the full-length and deleted domains of the murine homolog of treacle, we demonstrate that the cellular localization of treacle is nucleolar. This localization is mediated by the last 41 residues of the C-terminus (residues 1262-1302). At least two functional nuclear localization signals have been identified in the protein, one between residues 1176 and 1270 and the second within the last 32 residues of the protein (1271-1302). The nucleolar localization signal is disrupted by two constructs that split the C-terminal region between residues 1270 and 1271. This study provides the first direct analysis of treacle and demonstrates that the protein involved in TCOF1 is a nucleolar protein.

Hyperekplexia-like syndromes without mutations in the GLRA1 gene.

Hyperekplexia (MIM: 149400), or startle disease, is an autosomal dominant neurological disorder characterized by an extreme generalized stiffness immediately after birth, normalizing during the first years of life. Other features of this disorder are excessive startle reactions to unexpected, particularly auditory, stimuli together with a short period of generalized stiffness during which voluntary movements are impossible. Linkage analysis mapped a gene for this disorder to chromosome 5q33-q35. Subsequently, mutations in the GLRA1 gene encoding the alpha 1-subunit of the glycine receptor proved to be causally related to the disease. In the present study, mutation analysis of all exon and flanking intron sequences of this gene was performed in sporadic patients and their parents. Moreover, a branch of the original Dutch hyperekplexia family with a very severely affected individual was screened for an additional mutation in the GLRA1 gene. Except for two polymorphisms, of which one results in an amino acid change, no potentially disease causing mutations were found in the alpha 1-subunit of the glycine receptor. Together with haplotype analysis these results exclude a recessive inheritance or new mutation etiology in these hyperekplexia-like syndrome and emphasize that hyperekplexia-like syndromes can be caused by other genetic factors. The involvement of other genes encoding subunits of the functional glycine receptor complex has not been excluded.

A high-resolution physical and transcript map of the Cri du chat region of human chromosome 5p.

A high-resolution physical and transcription map has been generated of a 3.5-Mb region of 5p15.2 that is associated with the Cri du chat (CDC) syndrome. Utilizing a variety of resources including a natural deletion panel, a chromosome specific radiation hybrid panel, and YAC, PAC, and BAC genomic clones we have ordered > 60 STSs within this region. Approximately 45% of these STSs were obtained from publicly available whole genome maps, thus allowing for integration of this map with currently available resources. Thirteen of these markers were ESTs. In addition, > 70 exon trapped products have been mapped on the natural deletion panel and bacterial clone resource. The combination of these resources has allowed for the identification of 17 transcripts within this region, all of which represent candidate genes for CDC. Further characterization of the genomic contig also revealed that this region of 5p15 contains a large number of repetitive elements.

Sequence analysis, identification of evolutionary conserved motifs and expression analysis of murine tcof1 provide further evidence for a potential function for the gene and its human homologue, TCOF1.

The gene mutated in Treacher Collins syndrome, an autosomal dominant disorder of facial development, has recently been cloned. While the function of the predicted protein, Treacle, is unknown, it has been shown to share a number of features with the highly phosphorylated nucleolar phosphoproteins, which play a role in nucleolar-cytoplasmic transport. In the current study, the murine homologue of the Treacher Collins syndrome gene has been isolated and shown to encode a low complexity, serine/alanine-rich protein of 133 kDa. Interspecies comparison indicates that the proteins display 61.5% identity, with the level of conservation being greatest in the regions of acidic/basic amino acid repeats and nuclear localization signals. These features are shared with the nucleolar phosphoproteins. Confirmation that the gene isolated in the current study is orthologous with the Treacher Collins syndrome gene was provided by the demonstration that it mapped to central mouse chromosome 18 in a conserved syntenic region with human chromosome 5q21-q33. Expression analysis in the mouse indicated that the gene was expressed in a wide variety of embryonic and adult tissues. Peak levels of expression in the developing embryo were observed at the edges of the neural folds immediately prior to fusion, and also in the developing branchial arches at the times of critical morphogenetic events. These observations support a role for the gene in the development of the craniofacial complex and provide further evidence that the gene encodes a protein which may be involved in nucleolar-cytoplasmic transport.

Studies of the candidate genes TGFB2, MSX1, TGFA, and TGFB3 in the etiology of cleft lip and palate in the Philippines.

Population-based candidate-gene studies can be an effective strategy for identifying genes involved in the etiology of disorders where family-based linkage studies are compromised by lack of access to affected members, low penetrance, and/or genetic heterogeneity. We evaluated association data for four candidate genes using a population from the Philippines that is genetically separate from previously studied Caucasian populations. Case ascertainment was made possible by collaboration with Operation Smile, a volunteer medical organization, which facilitated identification of a large number of cases for study. A new allelic variant of transforming growth factor-beta 3 was identified to use in these studies. After exclusion of syndromic cases of cleft lip and palate, no evidence for association with previously reported allelic variants of transforming growth factor-beta 2 (TGFB2), homeobox 7 (MSX1), or transforming growth factor-alpha (TGFA), or with the new TGFB3 variant was detected. Previous association studies using Caucasian populations of nonsyndromic cleft lip and/or palate (CL/P) and cleft palate only (CPO) have strongly suggested a role for TGFA in the susceptibility of clefting in humans. Exclusion of significant association in a non-Caucasian population for TGFA suggests that TGFA plays less of a role than it does in Caucasians. This may be due to multiple or different genetic and/or environmental factors contributing to the etiology of this most common cranio-facial anomaly in the Philippine population.

Mutational analysis of familial and sporadic hyperekplexia.

Hyperekplexia is a rare, autosomal dominant neurological disorder characterized by hypertonia, especially in infancy, and by an exaggerated startle response. This disorder is caused by mutations in the alpha 1 subunit of the inhibitory glycine receptor (GLRA1). We previously reported two GLRA1 point mutations detected in 4 unrelated hyperekplexia families; both mutations were at nucleotide 1192 and resulted in the replacement of Arg271 by a glutamine (R271Q) in one case and a leucine (R271L) in the other. Here, 5 additional hyperekplexia families are shown to have the most common G-to-A transition mutation at nucleotide 1192. Haplotype analysis using polymorphisms within and close to the GLRA1 locus suggests that this mutation has arisen at least twice (and possibly four times). In 2 additional families, a third mutation is also presented that changes a tyrosine at amino acid 279 to a cysteine (Y279C). Five patients with atypical clinical features and equivocal or absent family history of hyperekplexia and 1 patient with a classical presentation but not family history are presented in whom a mutation in the GLRA1 gene was not detected. Thus, only clinically typical hyperekplexia appears to be consistently associated with GLRA1 mutations, and these affect a specific extracellular domain of the protein.

Molecular genetic reevaluation of the Dutch hyperekplexia family.

OBJECTIVES: To confirm linkage of the locus of the major form of hyperekplexia to markers on chromosome 5q, to screen for a point mutation in the gene encoding the alpha 1 subunit of the glycine receptor, and to investigate whether the putative "minor" form of hyperkeplexia consisting of an excessive startle response without stiffness, is based on the same genetic defect as the major form. DESIGN: A survey of various symptoms of hyperekplexia was performed in the Dutch pedigree. Linkage studies were performed for these symptoms. SETTING: Subjects were visited at home, and the genetic study was performed at University Hospital Leiden, (the Netherlands). PATIENTS: A history was taken from 76 subjects in the pedigree, and neurologic examinations were performed on 61 subjects from four generations of the pedigree. MAIN OUTCOME MEASURES: The main outcome measures were lod scores for markers on chromosome 5q for the major and minor forms of hyperekplexia and periodic leg movements during sleep. Mutations in the alpha 1 subunit of the glycine receptor were detected by screening the exons with denaturing gradient gel electrophoresis. RESULTS: Exaggerated startle responses were reported in 44 patients. The major form consisted of stiffness in addition to the excessive startle reaction and occurred in 28 subjects. Sixteen of 44 subjects had startle responses without stiffness, indicating the minor form. Linkage was found between markers CSF1-R, D5S209, and D5S119 and the disease locus for the major form, but not for the minor form. The alpha 1 subunit of the glycine receptor showed a G to A transition mutation in codon 271 for the major form, but not for the minor form. CONCLUSIONS: Linkage and an abnormal glycine receptor were found only in the major form of hyperekplexia. Recognition of a major form is based on additional stiffness. This is therefore the most important diagnostic symptom. The minor form is not a different expression of the same genetic defect and may represent a normal but pronounced startle response.

Thanatophoric dysplasia (types I and II) caused by distinct mutations in fibroblast growth factor receptor 3.

Thanatophoric dysplasia (TD), the most common neonatal lethal skeletal dysplasia, affects one out of 20,000 live births. Affected individuals display features similar to those seen in homozygous achondroplasia. Mutations causing achondroplasia are in FGFR3, suggesting that mutations in this gene may cause TD. A sporadic mutation causing a Lys650Glu change in the tyrosine kinase domain of FGFR3 was found in 16 of 16 individuals with one type of TD. Of 39 individuals with a second type of TD, 22 had a mutation causing an Arg248Cys change and one had a Ser371Cys substitution, both in the extracellular region of the protein. None of these mutations were found in 50 controls showing that mutations affecting different functional domains of FGFR3 cause different forms of this lethal disorder.

Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia.

Achondroplasia (ACH) is the most common genetic form of dwarfism. This disorder is inherited as an autosomal dominant trait, although the majority of cases are sporadic. A gene for ACH was recently localized to 4p16.3 by linkage analyses. The ACH candidate region includes the gene encoding fibroblast growth factor receptor 3 (FGFR3), which was originally considered as a candidate for the Huntington's disease gene. DNA studies revealed point mutations in the FGFR3 gene in ACH heterozygotes and homozygotes. The mutation on 15 of the 16 ACH-affected chromosomes was the same, a G-->A transition, at nucleotide 1138 of the cDNA. The mutation on the only ACH-affected chromosome 4 without the G-->A transition at nucleotide 1138 had a G-->C transversion at this same position. Both mutations result in the substitution of an arginine residue for a glycine at position 380 of the mature protein, which is in the transmembrane domain of FGFR3.

A missense mutation in the gene encoding the alpha 1 subunit of the inhibitory glycine receptor in the spasmodic mouse.

Hereditary hyperekplexia, an autosomal dominant neurologic disorder characterized by an exaggerated startle reflex and neonatal hypertonia, can be caused by mutations in the gene encoding the alpha 1 subunit of the inhibitory glycine receptor (GLRA1). Spasmodic (spd), a recessive neurologic mouse mutant, resembles hyperekplexia phenotypically, and the two disease loci map to homologous chromosomal regions. Here we describe a Glra1 missense mutation in spd that results in reduced agonist sensitivity in glycine receptors expressed in vitro. We conclude that spd is a murine homologue of hyperekplexia and that mutations in GLRA1/Glra1 can produce syndromes with different inheritance patterns.

Genes encoding adrenergic receptors are not clustered on the long arm of human chromosome 5.

The distal portion of the long arm of chromosome 5 (5q) contains a large number of genes encoding membrane receptors belonging to various gene families, including G protein-coupled adrenergic receptors. Previous reports indicated that the genes for two of the adrenergic receptors, ADRB2 and ADRA1B, were within 300 kb of one another on 5q. In an effort to determine if a third adrenergic receptor assigned to 5q, ADRA1A, was physically close to the genes encoding the other adrenergic receptors, we attempted to place all three loci on a radiation hybrid map of 5q. The results conflicted with previous mapping results in two ways. First, ADRA1B is on 5q but is several million bases, rather than a few hundred thousand bases, from ADRB2. Second, ADRA1A is not on chromosome 5, but rather on chromosome 20. Thus, even though 5q contains an extraordinary number of genes encoding receptors for various hormones, growth factors, and neurotransmitters, there is no particular clustering of genes encoding adrenergic receptors in this region.

Mutations in the alpha 1 subunit of the inhibitory glycine receptor cause the dominant neurologic disorder, hyperekplexia.

Hereditary hyperekplexia, or familial startle disease (STHE), is an autosomal dominant neurologic disorder characterized by marked muscle rigidity of central nervous system origin and an exaggerated startle response to unexpected acoustic or tactile stimuli. Linkage analyses in several large families provided evidence for locus homogeneity and showed the disease gene was linked to DNA markers on the long arm of chromosome 5. Here we describe the identification of point mutations in the gene encoding the alpha 1 subunit of the glycine receptor (GLRA1) in STHE patients from four different families. All mutations occur in the same base pair of exon 6 and result in the substitution of an uncharged amino acid (leucine or glutamine) for Arg271 in the mature protein.

Association of transforming growth-factor alpha gene polymorphisms with nonsyndromic cleft palate only (CPO).

Genetic analysis and tissue-specific expression studies support a role for transforming growth-factor alpha (TGFA) in craniofacial development. Previous studies have confirmed an association of alleles for TGFA with nonsyndromic cleft lip with or without cleft palate (CL/P) in humans. We carried out a retrospective association study to determine whether specific allelic variants of the TGFA gene are also associated with cleft palate only (CPO). The PCR products from 12 overlapping sets of primers to the TGFA cDNA were examined by using single-strand conformational polymorphism analysis. Four DNA polymorphic sites for TGFA were identified in the 3' untranslated region of the TGFA gene. These variants, as well as previously identified RFLPs for TGFA, were characterized in case and control populations for CPO by using chi 2 analysis. A significant association between alleles of TGFA and CPO was identified which further supports a role for this gene as one of the genetic determinants of craniofacial development. Sequence analysis of the variants disclosed a cluster of three variable sites within 30 bp of each other in the 3' untranslated region previously associated with an antisense transcript. These studies extend the role for TGFA in craniofacial morphogenesis and support an interrelated mechanism underlying nonsyndromic forms of CL/P.

The CEPH consortium linkage map of human chromosome 13.

The CEPH consortium map of chromosome 13 is presented. This map contains 59 loci defined by genotypes generated from CEPH family DNAs with 94 different probe and restriction enzyme combinations contributed by 9 laboratories. A total of 25 loci have been placed on the map with likelihood support of at least 1000:1. The map extends from loci in the centromeric region of chromosome 13 to the terminal band of the long arm. Multipoint linkage analyses provided estimates that the male, female, and sex-averaged maps extend for 158, 203, and 178 cM respectively. The largest interval is 24 cM and is between D13Z1 (alpha RI) and ATP1AL1. The mean genetic distance between the 25 uniquely placed loci is 7 cM.

Genetic and radiation hybrid mapping of the hyperekplexia region on chromosome 5q.

Hyperekplexia, or startle disease (STHE), is an autosomal dominant neurologic disorder characterized by muscular rigidity of central nervous system origin, particularly in the neonatal period, and by an exaggerated startle response to sudden, unexpected acoustic or tactile stimuli. STHE responds dramatically to the benzodiazepine drug clonazepam, which acts at gamma-aminobutyric acid type A (GABA-A) receptors. The STHE locus (STHE) was recently assigned to chromosome 5q, on the basis of tight linkage to the colony-stimulating factor 1-receptor (CSF1-R) locus in a single large family. We performed linkage analysis in the original and three additional STHE pedigrees with eight chromosome 5q microsatellite markers and placed several of the most closely linked markers on an existing radiation hybrid (RH) map of the region. The results provide strong evidence for genetic locus homogeneity and assign STHE to a 5.9-cM interval defined by CSF1-R and D5S379, which are separated by an RH map distance of 74 centirays (roughly 2.2-3.7 Mb). Two polymorphic markers (D5S119 and D5S209) lie within this region, but they could not be ordered with respect to STHE. RH mapping eliminated the candidate genes GABRA1 and GABRG2, which encode GABA-A receptor components, by showing that they are telomeric to the target region.

A detailed multipoint map of human chromosome 4 provides evidence for linkage heterogeneity and position-specific recombination rates.

Utilizing the CEPH reference panel and genotypic data for 53 markers, we have constructed a 20-locus multipoint genetic map of human chromosome 4. New RFLPs are reported for four loci. The map integrates a high-resolution genetic map of 4p16 into a continuous map extending to 4q31 and an unlinked cluster of three loci at 4q35. The 20 linked markers form a continuous linkage group of 152 cM in males and 202 cM in females. Likely genetic locations are provided for 25 polymorphic anonymous sequences and 28 gene-specific RFLPs. The map was constructed employing the LINKAGE and CRIMAP computational methodologies to build the multipoint map via a stepwise algorithm. A detailed 10-point map of the 4p16 region constructed from the CEPH panel provides evidence for heterogeneity in the linkage maps constructed from families segregating for Huntington disease (HD). It additionally provides evidence for position-specific recombination frequencies in the telomeric region of 4p.

Chromosomal localization of the human gene for annexin V (placental anticoagulant protein I) to 4q26----q28.

Annexin V is a member of a new family of calcium-dependent phospholipid-binding proteins. It has been previously isolated as placental anticoagulant protein I, inhibitor of blood coagulation, vascular anticoagulant-alpha, endonexin II, lipocortin V, placental protein 4, and anchorin CII. The human gene encoding annexin V (ANX5) was localized to 4q26----q28 by in situ hybridization with a cDNA probe and polymerase chain-reaction (PCR) analysis of a human x hamster hybrid cell panel. The regional localization to 4q26----q28 was supported by Southern-blot analysis of a human cell line with a deletion in 4q23----q27. This localization overlaps but differs slightly from the previous assignment of ANX5 to 4q28----q32. Digestion with PvuII and TaqI identified polymorphisms at the ANX5 locus; the PvuII polymorphism could also be detected by PCR analysis.

Generation of variability at VNTR loci in human DNA.

Our laboratory has constructed linkage maps of the human chromosomes to use as a tool towards the goal of cloning by position the genes responsible for genetic disorders. Construction of the map required the development of polymorphic marker systems in the form of Restriction Fragment Length Polymorphisms (RFLPs). Work by Yusuke Nakamura in the laboratory led to the identification of more than 200 highly informative Variable Number Tandem Repeat (VNTR) markers. The hypervariable nature of these marker loci has allowed individualization at the DNA level. Techniques for individualization have subsequently been adopted by diverse fields including gene mapping, cancer genetics and forensic biology. These markers have also become a resource to test hypotheses as to how the VNTRs generate their intrinsic variability. We have demonstrated that the hypothesis that VNTRs generate their variability by unequal exchange between homologous chromosomes in incorrect (Wolff et al., 1988; Wolff et al., 1989). Our data are consistent with intrachromosomal models such as unequal sister chromatid exchange and replication slippage. Using DNA derived from nonhuman primate species, we have tested hypotheses that try to explain the sequence relationship at dispersed VNTR loci. Our data reveal that VNTR loci are most likely not related by transposition but rather arose independently at multiple loci.