Isolation of genes from complex sources of mammalian genomic DNA using exon amplification.

Modifications to exon amplification have been instituted that increase its speed, efficiency and reliability. Exons were isolated from target human or mouse genomic DNA sources ranging from 30 kilobases (kb) to 3 megabases (Mb) in complexity. The efficiency was dependent upon the amount of input DNA, and ranged from isolation of an exon for every 20 kb to an exon for every 80 kb of target genomic DNA. In these studies, several novel genes and a smaller number of genes isolated previously that reside on human chromosome 9 have been identified. These results indicate that exon amplification is presently adaptable to large scale isolation of exons from complex sources of genomic DNA.

Localization of the gene for familial dysautonomia on chromosome 9 and definition of DNA markers for genetic diagnosis.

Familial dysautonomia (DYS), the Riley-Day syndrome, is an autosomal recessive disorder characterized by developmental loss of neurons from the sensory and autonomic nervous system. It is limited to the Ashkenazi Jewish population, where the carrier frequency is 1 in 30. We have mapped the DYS gene to chromosome 9q31-q33 by linkage with ten DNA markers in 26 families. The maximum lod score of 21.1 with no recombinants was achieved with D9S58. This marker also showed strong linkage disequilibrium with DYS, with one allele present on 73% of affected chromosomes compared to 5.4% of controls (chi 2 = 3142, 15 d.f. p < 0.0001). D9S53 and D9S105 represent the closest flanking markers for the disease gene. This localization will permit prenatal diagnosis of DYS in affected families and aid the isolation of the disease gene.

Mutations in transcript isoforms of the neurofibromatosis 2 gene in multiple human tumour types.

The neurofibromatosis 2 gene (NF2) has recently been isolated and predicted to encode a novel protein related to the moesin-ezrin-radixin family of cytoskeleton-associated proteins. Here we describe a novel isoform of the NF2 transcript that shows differential tissue expression and encodes a modified C terminus of the predicted protein. Mutations affecting both isoforms of the NF2 transcript were detected in multiple tumour types including melanoma and breast carcinoma. These findings provide evidence that alterations in the NF2 transcript occur not only in the hereditary brain neoplasms typically associated with NF2, but also as somatic mutations in their sporadic counterparts and in seemingly unrelated tumour types. The NF2 gene may thus constitute a tumour suppressor gene of more general importance in tumorigenesis.

Hyperkalemic periodic paralysis and the adult muscle sodium channel alpha-subunit gene.

Hyperkalemic periodic paralysis (HYPP) is an autosomal dominant disorder characterized by episodes of muscle weakness due to depolarization of the muscle cell membrane associated with elevated serum potassium. Electrophysiological studies have implicated the adult muscle sodium channel. Here, portions of the adult muscle sodium channel alpha-subunit gene were cloned and mapped near the human growth hormone locus (GH1) on chromosome 17. In a large pedigree displaying HYPP with myotonia, these two loci showed tight linkage to the genetic defect with no recombinants detected. Thus, it is likely that the sodium channel alpha-subunit gene contains the HYPP mutation.

New quantitative trait loci for the genetic variance in circadian period of locomotor activity between inbred strains of mice.

Provisional quantitative trait loci (QTL) for circadian locomotor period and wheel-running period have been identified in recombinant inbred (RI) mouse strains. To confirm those QTL and identify new ones, the genetic component of variance of the circadian period was partitioned among an F2 intercross of RI mouse strains (BXD19 and CXB07). First, a genomic survey using 108 SSLP markers with an average spacing of 15 cM was carried out in a population of 259 (BXD19 x CXB07)F2 animals. The genome-wide survey identified two significant QTL for period of locomotor activity measured by infrared photobeam crossings on mouse chromosomes 1 (lod score 5.66) and 14 (lod score 4.33). The QTL on distal chromosome 1 confirmed a previous report based on congenic B6.D2-Mtv7a/Ty mice. Lod scores greater than 2.0 were found on chromosomes 1, 2, 6, 12, 13, and 14. In a targeted extension study, additional genotyping was performed on these chromosomes in the full sample of 341 F2 progeny. The 6 chromosome-wide surveys identified 3 additional QTL on mouse chromosomes 6, 12, and 13. The QTL on chromosome 12 overlaps with circadian period QTL identified in several prior studies. For wheel-running period, the chromosome-wide surveys identified QTL on chromosomes 2 and 13 and one highly suggestive QTL on proximal chromosome 1. The results are compared to other published studies of QTL of circadian period.

New variant in exon 3 of the proteolipid protein (PLP) gene in a family with Pelizaeus-Merzbacher disease.

A C--greater than G transversion has been found in exon 3 of the PLP gene of affected males and their mother in a single sibship with Pelizaeus-merzbacher disease (PMD). The transversion should not result in an amino acid change in the protein but it does result in the loss of a HaeIII restriction endonuclease cleavage site. It is concordant with the disease in this family. One-hundred-ten unrelated X chromosomes are negative for this mutation. No other sequence defect was found in the PLP exons of the affected males. The cause of disease in this family remains unknown, but the association between this rare mutation and PMD is intriguing. The mutation can serve as a marker for following segregation of the PLP gene.

Localization of the gene for X-linked nephrogenic diabetes insipidus to Xq28.

X-linked nephrogenic diabetes insipidus (NDI) was segregating in a large Indiana family. It was tested for linkage of the NDI gene to X-chromosome molecular markers. Maximum lod scores of 3.15 and 3.01 (theta = 0) obtained for the molecular markers F8A (F8C) and DXS15 (DX13) respectively, indicate that the NDI gene is located in Xq28. A lod score of 3.61 (theta = 0) was obtained with multipoint linkage analysis of F8A and DXS15.

In-frame deletion in the proteolipid protein gene of a family with Pelizaeus-Merzbacher disease.

We describe an in-frame deletion of parts of exons 3 and 4 of the proteolipid protein gene (PLP), with all of the intervening sequence, in a 3-generation family with Pelizaeus-Merzbacher disease. The mutation removes 49 amino acids of the PLP.

A new mutation in the proteolipid protein (PLP) gene in a German family with Pelizaeus-Merzbacher disease.

A C-to-T transition in exon 4 of the PLP gene was found in 2 affected males and two obligate carriers in a German family with Pelizaeus-Merzbacher disease. The mutation, which causes loss of an HphI site and changes amino acid 155 from threonine to isoleucine, was absent from 108 normal chromosomes. There are 5 concordances and 1 discrepancy between these results and those obtained by magnetic resonance imaging in this family.

Multipoint linkage analysis of spinocerebellar ataxia and markers on chromosome 6.

Heterogeneity among the spinocerebellar ataxias (SCA) has been shown on clinical, biochemical, and genetic criteria. Among the autosomal dominant SCAs, several kindreds have shown loose linkage to the HLA loci on chromosome 6, while linkage in other kindreds has been rejected. The advent of multipoint linkage analysis allows the use of several marker loci simultaneously, thus increasing the amount of usable information. We have reanalyzed linkage data from a large kindred with SCA and provide evidence for a telomeric location of the SCA gene in this family. Knowledge of the relative gene location will ease the identification of the SCA gene by reducing the size of the chromosomal regions that must be examined.

Mapping of the human TATA-binding protein gene (TBP) to chromosome 6qter.

TATA-binding protein (TBP) is a general transcription factor involved in transcriptional initiation. We have used oligonucleotide primers flanking a polymorphic stretch of 38 glutamine codons in the 5' coding region of the TBP gene to genetically map this gene. We report the location of the human TBP gene to be at 6qter.

Pelizaeus-Merzbacher disease: tight linkage to proteolipid protein gene exon variant.

Pelizaeus-Merzbacher disease (PMD) is a human X chromosome-linked dysmyelination disorder of the central nervous system for which the genetic defect has not yet been established. The jimpy mutation jp of the mouse is an X chromosome-linked disorder of myelin formation. The mutation is at an intron/exon splice site in the mouse gene for proteolipid protein (PLP). With the jimpy mouse mutation as a precedent, we focused our attention on the human PLP gene, which is found at Xq22. The polymerase chain reaction was used to amplify the exons of the PLP gene of an affected male from a large Indiana PMD kindred. DNA sequencing showed a C----T transition at nucleotide 40 of the second exon. An affected third cousin also showed this sequence variation, while two unaffected male relatives (sons of an obligate carrier female) had the normal cytidine nucleotide. Allele-specific oligonucleotides were used to generate data for linkage studies on the above mentioned PMD kindred. Our results show tight linkage (theta = 0) of PMD to PLP with a lod (logarithm of odds) score of 4.62. In six other unrelated PMD kindreds, only the normal-sequence oligonucleotide hybridized, which indicates genetic heterogeneity. The radical nature of the predicted amino acid change (proline to leucine), suggests that the PMD-causing defect may have been delineated in one kindred.

Cloning and characterization of a novel human clathrin heavy chain gene (CLTCL).

An exon representing a novel clathrin heavy chain gene (CLTCL) was isolated during gene identification studies and transcription mapping of human chromosome 22. Isolation and sequencing of cDNA clones corresponding to this exon revealed extensive similarity of the predicted amino acid sequence of this gene product to those of clathrin heavy chain genes of other species. Northern blot analysis has revealed an apparent developmental expression pattern of an approximately 6-kb mRNA. The gene appears to be expressed ubiquitously in the limited number of fetal tissues that were tested, but is selectively expressed in certain adult tissues, particularly in skeletal muscle. In addition, alternative splicing of an exon was observed near the carboxyl terminus of the predicted gene product. Its location overlaps the domain putatively involved in clathrin light chain binding and is adjacent to the heavy chain self-assembly (or trimerization) region, suggesting that alternative splicing may be involved in regulating one or both of these interactions. The expression pattern of this gene, in addition to its potential role in receptor-mediated endocytosis and signal transduction, suggests that it may be important in some developmental processes. The location of CLTCL on human chromosome 22 near the region commonly deleted in DiGeorge and other apparent haploinsufficiency syndromes warrants further investigation into its relationship with these developmental disorders.

Onset symptoms in 510 patients with Huntington's disease.

The onset of Huntington's disease (HD) is preceded or accompanied by events and symptoms which contribute to the natural history of the disease. Data obtained from the first 510 completed 'Questionnaires for Affected Individuals', recorded by the National Huntington's Disease Research Roster (NHDRR) were analysed. The following features were evaluated: (1) neurological and psychiatric onset symptoms; (2) the precipitating effect of stressful events and drugs; (3) the modification after onset of smoking and alcohol consumption. The most frequent psychiatric onset symptom was depression. Stressful events in the year before onset occurred in 43% of patients. However, onset age was the same in patients with and without previous stressful events. Smoking and especially alcohol consumption showed a decreasing trend after onset.

Suicide risk in Huntington's disease.

In order to evaluate the relevance of suicide risk in families affected by Huntington's disease (HD), 2793 subjects registered with the National Huntington's Disease Research Roster were studied. Suicide was the reported cause of death in 205 subjects (7.3%). This group included affected and possibly affected subjects, subjects at 50% and 25% risk, possibly at risk subjects, and normal relatives. In all categories suicide was more frequent than in the general US population. The data suggest that suicide is quite frequent in some families with HD. This increased suicide risk must be carefully considered in planning genetic counselling for predictive testing in HD.

The murine NF2 homologue encodes a highly conserved merlin protein with alternative forms.

The recently isolated gene for neurofibromatosis type 2 (NF2) encodes a 595 amino acid protein, named merlin, which is related to the cytoskeleton-associated proteins moesin, ezrin and radixin. To identify evolutionarily conserved regions and to provide sequence information necessary for the establishment of a mouse model for NF2, we have determined the cDNA sequence of the mouse NF2 tumor suppressor gene, and mapped it in the mouse genome. Mouse merlin is a 596 amino acid protein, 98% identical to human merlin, but one amino acid longer due to the insertion of a proline residue near the C-terminus. Of the nine amino acid differences between mouse and humans, seven occur in the C-terminal 20% of the protein, far from the protein 4.1 domain that defines this family. Two of the NF2 cDNA clones reveal evidence of alternative splicing events that alter the predicted merlin product, one removing a 45 amino acid segment from the middle section of the protein and the other changing the C-terminus. The existence of several different forms of merlin potentially with different primary roles will complicate the identification of the precise function that must be disrupted to cause the NF2-associated tumors. The mouse NF2 homologue maps to Chr 11, in a region homologous to human Chr 22, but devoid of any mouse mutations which could be models of the human disorder.

A highly polymorphic dinucleotide repeat on the proximal short arm of the human X chromosome: linkage mapping of the synapsin I/A-raf-1 genes.

A compound (AC)n repeat located 1,000 bp downstream from the human synapsin I gene and within the last intron of the A-raf-1 gene has been identified. DNA data-base comparisons of the sequences surrounding the repeat indicate that the synapsin I gene and the A-raf-1 gene lie immediately adjacent to each other, in opposite orientation. PCR amplification of this synapsin I/A-raf-1 associated repeat by using total genomic DNA from members of the 40 reference pedigree families of the Centre d'Etude du Polymorphisme Humaine showed it to be highly polymorphic, with a PIC value of .84 and a minimum of eight alleles. Because the synapsin I gene has been mapped previously to the short arm of the human X chromosome at Xp11.2, linkage analysis was performed with markers on the proximal short arm of the X chromosome. The most likely gene order is DXS7SYN/ARAF1TIMPDXS255DXS146, with a relative probability of 5 x 10(8) as compared with the next most likely order. This highly informative repeat should serve as a valuable marker for disease loci mapped to the Xp11 region.

Myelin protein zero gene mutated in Charcot-Marie-tooth type 1B patients.

Autosomal dominant of Charcot-Marie-Tooth disease (CMT), whose gene is type 1B (CMT1B), has slow nerve conduction with demyelinated Schwann cells. In this study the abundant peripheral myelin protein zero (MPZ) gene, MPZ, was mapped 130 kb centromeric to the Fc receptor immunoglobulin gene cluster in band 1q22, and a major MPZ point mutation was found to cosegregate with CMT1B in one large CMT1B family. The MPZ point mutation in 18 of 18 related CMT1B pedigree 1 patients converts a positively charged lysine in codon 96 to a negatively charged glutamate. The same MPZ locus cosegregates with the CMT1B disease gene in a second CMT1B family [total multipoint logarithm of odds (lod) = 11.4 at theta = 0.00] with a splice junction mutation. Both mutations occur in MPZ protein regions otherwise conserved identically in human, rat, and cow since these species diverged 100 million years ago. MPZ protein, expressed exclusively in myelinated peripheral nerve Schwann cells, constitutes > 50% of myelin protein. These mutations are anticipated to disrupt homophilic MPZ binding and result in CMT1B peripheral nerve demyelination.

An expression-independent catalog of genes from human chromosome 22.

To accomplish large-scale identification of genes from a single human chromosome, exon amplification was applied to large pools of clones from a flow-sorted human chromosome 22 cosmid library. Sequence analysis of more than one-third of the 6400 cloned products identified 35% of the known genes previously localized to this chromosome, as well as several unmapped genes and randomly sequenced cDNAs. Among the more interesting sequence similarities are those that represent novel human genes that are related to others with known or putative functions, such as one exon from a gene that may represent the human homolog of Drosophila Polycomb. It is anticipated that sequences from at least half of the genes residing on chromosome 22 are contained within this exon library. This approach is expected to facilitate fine-structure physical and transcription mapping of human chromosomes, and accelerate the process of disease gene identification.