Charcot-Marie-Tooth type 1A duplication appears to arise from recombination at repeat sequences flanking the 1.5 Mb monomer unit.

We have constructed a 3.1 megabase (Mb) physical map of chromosome 17p11.2-p12, which contains a submicroscopic duplication in patients with Charcot-Marie-Tooth disease type 1A (CMT1A). We find that the CMT1A duplication is a tandem repeat of 1.5 Mb of DNA. A YAC contig encompassing the CMT1A duplication and spanning the endpoints was also developed. Several low copy repeats in 17p11.2-p12 were identified including the large (> 17 kb) CMT1A-REP unit which may be part of a mosaic repeat. CMT1A-REP flanks the 1.5 Mb CMT1A monomer unit on normal chromosome 17 and is present in an additional copy on the CMT1A duplicated chromosome. We propose that the de novo CMT1A duplication arises from unequal crossing over due to misalignment at these CMT1A-REP repeat sequences during meiosis.

Mapping of the congenital generalized hypertrichosis locus to chromosome Xq24-q27.1.

Congenital generalized hypertrichosis (CGH) is a rare, fully penetrant X-linked dominant trait previously described in a single, multigenerational Mexican family. CGH is a visually striking phenotype characterized by excessive facial and upper torso hair in males and by less severe asymmetric hairiness in females. We have found significant evidence for linkage with several markers from the long arm of the X chromosome. Recombinant chromosomes place the CGH gene within a 22 cM interval between DXS425 and DXS1227 in Xq24-Xq27.1. The localization of a gene for CGH represents the first step towards the isolation of genes involved in hair growth pattern, particularly those involved in restriction of areas in humans.

Gene dosage is a mechanism for Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited peripheral neuropathy in humans, characterized electrophysiologically by decreased nerve conduction velocities (NCVs). CMT1A is associated with a large submicroscopic DNA duplication in proximal 17p. In this report we demonstrate that a patient with a cytogenetically visible duplication, dup(17)(p11.2p12), has decreased NCV. Molecular analysis demonstrated this patient was duplicated for all the DNA markers duplicated in CMT1A as well as markers both proximal and distal to the CMT1A duplication. These data support the hypothesis that the CMT1A phenotype can result from a gene dosage effect.

Evidence for a recessive PMP22 point mutation in Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is an autosomal dominant neuropathy that can be caused by dominant point mutations in PMP22 which encodes a peripheral nerve myelin protein. Usually, CMT1A is caused by the duplication of a 1.5-megabase (Mb) region on chromosome 17p11.2-p12 containing PMP22. Deletion of a similar 1.5-Mb region is associated with hereditary neuropathy with liability to pressure palsies (HNPP), a clinically distinct neuropathy. We have identified a severely affected CMT1 patient who is a compound heterozygote for a recessive PMP22 point mutation, and a 1.5 Mb deletion in 17p11.2-p12. A son heterozygous for the PMP22 point mutation had no signs of neuropathy, while two others heterozygous for the deletion had HNPP, suggesting that point mutations in PMP22 can result in dominant and recessive alleles contributing to CMT1A.

The gene for the peripheral myelin protein PMP-22 is a candidate for Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is an autosomal dominant peripheral neuropathy associated with a large DNA duplication on the short arm of human chromosome 17. The trembler (Tr) mouse serves as a model for CMT1A because of phenotypic similarities and because the Tr locus maps to mouse chromosome 11 in a region of conserved synteny with human chromosome 17. Recently, the peripheral myelin gene Pmp-22 was found to carry a point mutation in Tr mice. We have isolated cDNA and genomic clones for human PMP-22. The gene maps to human chromosome 17p11.2-17p12, is expressed at high levels in peripheral nervous tissue and is duplicated, but not disrupted, in CMT1A patients. Thus, we suggest that a gene dosage effect involving PMP-22 is at least partially responsible for the demyelinating neuropathy seen in CMT1A.

A novel g.-1258G>A mutation in a conserved putative regulatory element of PAX9 is associated with autosomal dominant molar hypodontia.

Mutations in the transcription factor PAX9 which plays a critical role in the switching of odontogenic potential from the epithelium to the mesenchyme during tooth development cause autosomal dominant non-syndromic hypodontia primarily affecting molars. Linkage analysis on a family segregating autosomal dominant molar hypodontia with markers flanking and within PAX9 yielded a maximum multipoint LOD score of 3.6. No sequence variants were detected in the coding or 5'- and 3'-untranslated regions (UTRs) of PAX9. However, we identified a novel g.-1258G>A sequence variant in all affected individuals of the family but not in the unaffected family members or in 3088 control chromosomes. This mutation is within a putative 5'-regulatory sequence upstream of PAX9 highly conserved in primates, somewhat conserved in ungulates and carnivores but not conserved in rodents. Bioinformatics analysis of the sequence determined that there was no abolition or creation of a putative binding site for known transcription factors. Based on our previous findings that haploinsufficiency for PAX9 leads to hypodontia, we postulate that the g.-1258G>A variant reduces the expression of PAX9 which underlies the hypodontia phenotype in this family.

Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.

Friedreich's ataxia (FRDA) is an autosomal recessive, degenerative disease that involves the central and peripheral nervous systems and the heart. A gene, X25, was identified in the critical region for the FRDA locus on chromosome 9q13. This gene encodes a 210-amino acid protein, frataxin, that has homologs in distant species such as Caenorhabditis elegans and yeast. A few FRDA patients were found to have point mutations in X25, but the majority were homozygous for an unstable GAA trinucleotide expansion in the first X25 intron.

Charcot-Marie-Tooth disease type 1A: mutational mechanisms and candidate gene.

Charcot-Marie-Tooth disease type 1A, the most common inherited peripheral neuropathy, is associated with a submicroscopic DNA duplication of 1.5 Mb that can arise de novo, and which is flanked by a > 17 kb mosaic repeat. The PMP22 gene, encoding a peripheral myelin protein, maps within the duplication. In a subset of Charcot-Marie-Tooth patients, point mutations can occur within the gene. Thus, the alternative mechanisms of overexpression of PMP22 and structural alterations in the protein encoded can cause the disease phenotype.

Charcot-Marie-Tooth disease: a new paradigm for the mechanism of inherited disease.

Recent work has identified the genes and mutational mechanisms that underlie several inherited diseases of the peripheral nervous system and has provided both the first genetic rationale for classification of these disorders and an insight into their biological basis. These studies have yielded some surprising findings, including the discovery that two very different mutational mechanisms (duplication and point mutation) can result in a similar clinical phenotype in Charcot-Marie-Tooth disease type 1A, and that mutations involving the same gene can give rise to different clinical phenotypes.

Functional characterization of the human hypoxanthine phosphoribosyltransferase gene promoter: evidence for a negative regulatory element.

The enzyme hypoxanthine phosphoribosyltransferase (HPRT) catalyzes the metabolic salvage of the purine bases hypoxanthine and guanine. We previously characterized the genomic structure of the human HPRT gene and described its promoter sequence. In this report, we identify cis-acting transcriptional control regions of the human HPRT gene by linking various 5'-flanking sequences to the bacterial chloramphenicol acetyltransferase gene. The sequence from positions -219 to -122 relative to the translation initiation site is required for maximal expression of this gene, and it functions equally in both normal and reverse orientations. In addition, a cis-acting negative element is present in the region spanning from positions -570 to -388. This negative element can also repress promoters of heterologous genes, such as those of adenosine deaminase and dihydrofolate reductase, which are structurally and functionally similar to the human HPRT promoter. Furthermore, this repressor element functions independently of its orientation but appears to be distance dependent. In vivo competition assays demonstrated that the trans-acting factor(s) that binds to this negative element specifically inhibits human HPRT promoter activity. Taken together, these data localize cis-acting sequences important in the regulation of human HPRT gene expression and should allow the study of protein-DNA interactions which modulate the transcription of this gene.

Fine structure of the human hypoxanthine phosphoribosyltransferase gene.

The human hypoxanthine phosphoribosyltransferase (HPRT) gene has been characterized by molecular cloning, mapping, and DNA sequencing techniques. The entire gene, which is about 44 kilobases in length, is composed of nine exon elements. The positions of the introns within the coding sequence are identical to those of the previously-characterized mouse HPRT gene, although there are significant differences between intron sizes for the two genes. HPRT minigenes have been used in a transient expression assay involving microinjection into HPRT- cells to demonstrate functional promoter activity within a 234-base-pair region upstream from the ATG codon. The promoter of this gene resembles those of other recently characterized "housekeeping" genes in that it lacks CAAT- and TATA-like sequences, but contains several copies of the sequence GGGCGG. Both RNase protection and primer extension analysis indicate that human HPRT mRNA is heterogeneous at the 5' terminus, with transcription initiation occurring at sites located congruent to 104 to congruent to 169 base pairs upstream from the ATG codon. Comparison of the mouse and human HPRT 5'-flanking sequences indicates that there are only limited stretches of conserved sequence, although there are other shared features, such as an extremely high density of potential methylation sites, that may have functional significance.

Ubiquitous and neuronal DNA-binding proteins interact with a negative regulatory element of the human hypoxanthine phosphoribosyltransferase gene.

The hypoxanthine phosphoribosyltransferase (HPRT) gene is constitutively expressed at low levels in all tissues but at higher levels in the brain; the significance and mechanism of this differential expression are unknown. We previously identified a 182-bp element (hHPRT-NE) within the 5'-flanking region of the human HPRT (hHPRT) gene, which is involved not only in conferring neuronal specificity but also in repressing gene expression in nonneuronal tissues. Here we report that this element interacts with different nuclear proteins, some of which are present specifically in neuronal cells (complex I) and others of which are present in cells showing constitutive expression of the gene (complex II). In addition, we found that complex I factors are expressed in human NT2/D1 cells following induction of neuronal differentiation by retinoic acid. This finding correlates with an increase of HPRT gene transcription following neuronal differentiation. We also mapped the binding sites for both complexes to a 60-bp region (Ff; positions -510 to -451) which, when analyzed in transfection assays, functioned as a repressor element analogous to the full-length hHPRT-NE sequence. Methylation interference footprintings revealed a minimal unique DNA motif, 5'-GGAAGCC-3', as the binding site for nuclear proteins from both neuronal and nonneuronal sources. However, site-directed mutagenesis of the footprinted region indicated that different nucleotides are essential for the associations of these two complexes. Moreover, UV cross-linking experiments showed that both complexes are formed by the association of several different proteins. Taken together, these data suggest that differential interaction of DNA-binding factors with this regulatory element plays a crucial role in the brain-preferential expression of the gene, and they should lead to the isolation of transcriptional regulators important in neuronal expression of the HPRT gene.

Genomic organisation of the approximately 1.5 Mb Smith-Magenis syndrome critical interval: transcription map, genomic contig, and candidate gene analysis.

Smith-Magenis syndrome (SMS) is a multiple congenital anomalies/mental retardation syndrome associated with an interstitial deletion of chromosome 17 involving band p11.2. SMS is hypothesised to be a contiguous gene syndrome in which the phenotype arises from the haploinsufficiency of multiple, functionally-unrelated genes in close physical proximity, although the true molecular basis of SMS is not yet known. In this study, we have generated the first overlapping and contiguous transcription map of the SMS critical interval, linking the proximal 17p11.2 region near the SMS-REPM and the distal region near D17S740 in a minimum tiling path of 16 BACs and two PACs. Additional clones provide greater coverage throughout the critical region. Not including the repetitive sequences that flank the critical interval, the map is comprised of 13 known genes, 14 ESTs, and six genomic markers, and is a synthesis of Southern hybridisation and polymerase chain reaction data from gene and marker localisation to BACs and PACs and database sequence analysis from the human genome project high-throughput draft sequence. In order to identify possible candidate genes, we performed sequence analysis and determined the tissue expression pattern analysis of 10 novel ESTs that are deleted in all SMS patients. We also present a detailed review of six promising candidate genes that map to the SMS critical region.

Familial essential tremor in 4 kindreds. Prospects for genetic mapping.

OBJECTIVES: To describe 4 large families with essential tremor (ET) to draw attention to the marked clinical heterogeneity of ET. To use computer simulation analysis to provide information about the power of the family material for future linkage studies. SUBJECTS: We examined a total of 251 members from 4 kindreds with ET. The mean (+/-SD) age at onset of ET varied among the 4 kindreds between 19.0 +/- 11.4 years and 45.6 +/- 7.4 years. Three of the kindreds had a total of 41 members with the combination of ET and dystonia, typically manifested as torticollis or dystonic writers' cramp. In 1 of the kindreds, ET seemed to be associated with malignant hyperthermia. One kindred represented "pure" ET without any associated disorders. METHODS: In addition to detailed clinical assessments, we conducted computer simulations on the families' pedigrees using a model that presumed an autosomal dominant inheritance pattern with high penetrance. RESULTS: Although there was evidence of clinical heterogeneity between the families, the duration of symptoms directly correlated with the severity of disease. The computer simulations indicated that 3 of the 4 pedigrees had enough power to generate a significant linkage result in a total genome search with highly polymorphic markers. CONCLUSIONS: This study confirms the frequent coexistence of ET and dystonia in individual families. Computer simulations can be used to determine the power of the family to detect a linked marker. Identification of the defective gene(s) will enable a better understanding and classification of these common movement disorders.

Very late-onset Friedreich ataxia despite large GAA triplet repeat expansions.

BACKGROUND: Most patients with Friedreich ataxia (FRDA) have abnormal GAA triplet repeat expansions in both X25 genes. The size of the GAA expansion in the shorter of the 2 expanded alleles correlates significantly with parameters of clinical severity and is inversely related to the age at onset. OBJECTIVES: To describe the clinical and molecular genetic findings in a patient with very late-onset FRDA and to review the literature. PATIENT AND METHODS: A 58-year-old white woman with mild progressive gait disturbance of 15 years' duration whose examination revealed mild incoordination was analyzed for mutations in the X25 gene. A combination of long-range polymerase chain reaction and genomic Southern blot analyses were used to identify GAA expansions in intron 1 of the X25 gene. To uncover evidence of somatic variability in triplet repeat length, DNA isolated from several tissue samples was similarly analyzed. Single-strand conformational polymorphism analysis was used to screen for mutations spanning the entire coding sequence of frataxin and all intron-exon junctions of the X25 gene. RESULTS: DNA isolated from blood leukocytes revealed GAA triplet repeat expansions in both X25 genes, which were estimated to contain 835 and 1200 repeats. Similar expansions were detected in DNA isolated from lymphoblasts, fibroblasts, buccal cells, and sural nerve, with estimated mean (+/- SD) lengths of the shorter and longer expansions being 854 (+/-69) and 1283 (+/-72) triplets, respectively. A review of reported cases of late-onset Friedreich ataxia (25-39 years) and very late-onset Friedreich ataxia (> or =40 years) demonstrated that this is the first instance of a patient presenting with very late-onset FRDA despite carrying more than 800 GAA repeats in both expanded X25 alleles. CONCLUSIONS: This unique case of very late-onset FRDA highlights a limitation in our ability to accurately predict the phenotype in FRDA based solely on the size of the GAA expansion. Other genetic or environmental factors may significantly modify disease severity in FRDA.

Unusual EEG pattern linked to chromosome 3p in a family with idiopathic generalized epilepsy.

OBJECTIVE: To map the gene causing an unusual EEG pattern of delta bursts that appears to segregate as an autosomal dominant trait in an Italian family. The EEG pattern was observed in four family members affected by idiopathic generalized epilepsy (IGE) and in six other clinically unaffected members. METHODS: All available family members underwent clinical and EEG examination. DNA samples were obtained and used to perform a whole-genome scan with 270 microsatellite markers. After the first linked marker was identified, 12 additional markers in the same chromosomal region were tested to confirm linkage and define a candidate interval. RESULTS: The gene responsible for the EEG trait was mapped to an 11-cM interval on the proximal short arm of chromosome 3 (3p14.2-p12.1). CONCLUSION: In this family, a characteristic EEG activity is due to the effect of a single gene on chromosome 3p. A gene encoding a Ca2+ channel subunit maps in the interval and is a potential candidate for the trait. The clinical expression of epilepsy in four family members may reflect the interaction of additional genes, though environmental or other factors cannot be excluded.

Stable inheritance of the CMT1A DNA duplication in two patients with CMT1 and NF1.

Charcot-Marie-Tooth disease type 1A (CMT1A) was recently demonstrated to be associated with a large DNA duplication in 17p11.2p12. The gene for neurofibromatosis type 1 (NF1) or von Recklinghausen disease maps to 17q11.2. We have identified 2 unrelated patients who were diagnosed with both CMT1 and NF1. Molecular analysis of these patients demonstrated the presence of the CMT1A duplication and inheritance of this DNA rearrangement from a parent affected with CMT. Analysis of genomic DNA isolated from the neurofibroma removed from one of these patients showed the same 500 kb SacII junction fragment associated with the CMT1A duplication that was found in genomic DNA isolated from the blood. These results lend further support to the hypothesis that the CMT1A duplication is a stable DNA rearrangement. In addition, the molecular analysis of these 2 patients suggests that 2 common autosomal dominant conditions (CMT1 and NF1) can occur in the same individual, not because of an underlying single molecular defect, but rather, secondary to a chance phenomenon.

Smith-Magenis syndrome resulting from a de novo direct insertion of proximal 17q into 17p11.2.

We report on a de novo intrachromosomal rearrangement of chromosome 17 in a patient with Smith-Magenis syndrome (SMS). This 11-year-old boy had short stature, midfacial hypoplasia, and behavioral problems characteristic of this syndrome. Cytogenetic analysis showed that the proximal long arm of a chromosome 17 (q11.2-q21.3) was inserted into its short arm at p11.2, resulting in an apparent deletion of the SMS critical region [ins(17)(p11.2q11.2q21.3)]. Fluorescence in situ hybridization studies (FISH) demonstrated that the inserted segment included both the ERBB2 and RARA loci, and dual color hybridizations defined the insertion as direct, with ERBB2 located more proximally on the short arm of the der(17). The resulting deletion of the short arm included loci c130G3, D17S258, FLI, and D17S29, while the more proximal loci, D17S446 and D17S58, remained apparently unaffected and in their native locations. The CMT1A locus also remained in its native location on the short arm of the metacentric der(17) chromosome. A de novo intrachromosomal insertional rearrangement of chromosome 17 in a case of SMS has not been reported previously and further illustrates the instability of this chromosomal region.