Episodic ataxia/myokymia syndrome is associated with point mutations in the human potassium channel gene, KCNA1.

Episodic ataxia (EA) is a rare, familial disorder producing attacks of generalized ataxia, with normal or near-normal neurological function between attacks. One type of EA is characterized by brief episodes of ataxia with myokymia (rippling of muscles) evident between attacks. Linkage studies in four such families suggested localization of an EA/myokymia gene near the voltage gated K+ channel gene, KCNA1 (Kv1.1), on chromosome 12p. Mutation analysis of the KCNA1 coding region in these families identified four different missense point mutations present in the heterozygous state, indicating that EA/myokymia can result from mutations in this gene.

Homozygosity mapping of Hallervorden-Spatz syndrome to chromosome 20p12.3-p13.

Hallervorden-Spatz syndrome (HSS) (OMIM #234200) is a rare, autosomal recessive neurode-generative disorder with brain iron accumulation as a prominent finding. Clinical features include extrapyramidal dysfunction, onset in childhood, and a relentlessly progressive course. Histologic study reveals massive iron deposits in the basal ganglia. Systemic and cerebrospinal fluid iron levels are normal, as are plasma levels of ferritin, transferrin and ceruloplasmin. Conversely, in disorders of systemic iron overload, such as haemochromatosis, brain iron is not increased, which suggests that fundamental differences exist between brain and systemic iron metabolism and transport. In normal brain, non-haem iron accumulates regionally and is highest in basal ganglia. Pathologic brain iron accumulation is seen in common disorders, including Parkinson's disease, Alzheimer's disease and Huntington disease. In order to gain insight into normal and abnormal brain iron transport, metabolism and function, our approach was to map the gene for HSS. A primary genome scan was performed using samples from a large, consanguineous family (HS1) (see Fig. 1). While this family was immensely powerful for mapping, the region demonstrating homozygosity in all affected members spans only 4 cM, requiring very close markers in order to detect linkage. The HSS gene maps to an interval flanked by D20S906 and D20S116 on chromosome 20p12.3-p13. Linkage was confirmed in nine additional families of diverse ethnic backgrounds.

The entry of granule-associated peroxidase into the phagocytic vacuoles of eosinophils.

The ultrastructural localization of peroxidase was studied in guinea pig eosinophils which had phagocytized zymosan or Escherichia coli. After phagocytosis, the membrane of the granule was joined to the membrane of the phagocytic vacuole, and the enzyme, which is ordinarily restricted to the matrix of the granule, was seen in the vacuole surrounding the ingested particle.

Artificial membranes: a new type of cell for measuring diffusional resistance.

A novel technique allows determination of membrane diffusivities and eliminates the complications of the fluid resistance to mass transfer on either side of the membrane. Results on the permeability of a dialysis membrane of the type used in artificial kidneys agree with previous data and are obtained in much shorter time. The measured activation energy for diffusion demonstrates that the transport of salt through the membrane occurs by the same mechanism as the diffusion of salt in water, but that only 10 percent of the membrane surface is effective.

Histidine transfer RNA levels in Friend leukemia cells: stimulation by histidine deprivation.

Friend leukemia cells incubated with sublethal concentrations of histidinol for 5 to 6 days show up to twofold increases in their relative concentrations of histidine transfer RNA and no change in the relative concentrations of leucine transfer RNA. A similar effect is seen when cells are grown to stationary phase in the presence of 0.2 times the amount of histidine in Eagle's minimum essential medium. These observations support the theory that the concentrations of specific transfer RNA's are regulated by a mechanism that is sensitive to the extent of their aminoacylation.

Correlation between histone lysine methylation and developmental changes at the chicken beta-globin locus.

Methylation of histones at specific residues plays an important role in transcriptional regulation. Chromatin immunoprecipitation of dimethylated lysine 9 on histone H3 across 53 kilobases of the chicken beta-globin locus during erythropoiesis shows an almost complete anticorrelation between regions of elevated lysine 9 methylation and acetylation. Lysine 9 is methylated most over constitutive condensed chromatin and developmentally inactive globin genes. In contrast, lysine 4 methylation of histone H3 correlates with H3 acetylation. These results lead us to propose a mechanism by which the insulator in the beta-globin locus can protect the globin genes from being silenced by adjacent condensed chromatin.

Molecular genetics of human blue cone monochromacy.

Blue cone monochromacy is a rare X-linked disorder of color vision characterized by the absence of both red and green cone sensitivities. In 12 of 12 families carrying this trait, alterations are observed in the red and green visual pigment gene cluster. The alterations fall into two classes. One class arose from the wild type by a two-step pathway consisting of unequal homologous recombination and point mutation. The second class arose by nonhomologous deletion of genomic DNA adjacent to the red and green pigment gene cluster. These deletions define a 579-base pair region that is located 4 kilobases upstream of the red pigment gene and 43 kilobases upstream of the nearest green pigment gene; this 579-base pair region is essential for the activity of both pigment genes.

In vivo footprinting and high-resolution methylation analysis of the mouse hypoxanthine phosphoribosyltransferase gene 5' region on the active and inactive X chromosomes.

To investigate potential mechanisms regulating the hypoxanthine phosphoribosyltransferase (HPRT) gene by X-chromosome inactivation, we performed in vivo footprinting and high-resolution DNA methylation analysis on the 5' region of the active and inactive mouse HPRT alleles and compared these results with those from the human HPRT gene. We found multiple footprinted sites on the active mouse HPRT allele and no footprints on the inactive allele. Comparison of the footprint patterns of the mouse and human HPRT genes demonstrated that the in vivo binding of regulatory proteins between these species is generally conserved but not identical. Detailed nucleotide sequence comparison of footprinted regions in the mouse and human genes revealed a novel 9-bp sequence associated with transcription factor binding near the transcription sites of both genes, suggesting the identification of a new conserved initiator element. Ligation-mediated PCR genomic sequencing showed that all CpG dinucleotides examined on the active allele are unmethylated, while the majority of CpGs on the inactive allele are methylated and interspersed with a few hypomethylated sites. This pattern of methylation on the inactive mouse allele is notably different from the unusual methylation pattern of the inactive human gene, which exhibited strong hypomethylation specifically at GC boxes. These studies, in conjunction with other genomic sequencing studies of X-linked genes, demonstrate that (i) the active alleles are essentially unmethylated, (ii) the inactive alleles are hypermethylated, and (iii) the high-resolution methylation patterns of the hypermethylated inactive alleles are not strictly conserved. There is no obvious correlation between the pattern of methylated sites on the inactive alleles and the pattern of binding sites for transcription factors on the active alleles. These results are discussed in relationship to potential mechanisms of transcriptional regulation by X-chromosome inactivation.

Biosynthesis of mammalian transfer RNA. Evidence for regulation by deacylated transfer RNA.

The rate of tRNA synthesis in cultured Friend leukemia cells has been examined as a function of the variation in polyribosome structure produced by treatment with a variety of inhibitors of protein synthesis. The results indicate, in contrast to the conclusions of Bölcsföldi (Bölcsföldi, G. (1974) Exp. Cell Res., 88, 231--240), that no necessary relationship exists between the ribosome distribution and the rate of tRNA synthesis. Alternatively, it is observed that inhibitors of tRNA aminoacylation cause, in all cases, a decrease in the rate of tRNA synthesis whereas drugs which may stimulate the aminoacylation of tRNA cause, in all cases, an elevation of the rate of tRNA synthesis. It is concluded that tRNA synthesis in mammalian cells may be regulated by the relative levels of acylated and deacylated tRNA.

Effect of mucolytic agents on the rheological properties of tracheal mucus.

Canine tracheal mucus was dissolved by a number of mucolytic agents, including disulfide bond reducing agents, hydrogen bond breaking agents, and chaotropic ions, and their effect on rheological properties was assessed. Sodium thiocyanate led to 85-100% dissolution with the maximum retention of elasticity. Thiocyanate exposure did not result in demonstrable alterations in the size or shape of the mucus glycoproteins. Sodium thiocyanate is therefore recommended as a suitable dispersing agent for physiochemical studies of glycoprotein secretions.

Composition and functioin of human cervical mucus.

Human cervical mucus was collected from seven donors during the follicular, ovulatory and luteal phases of the ovulatory menstrual cycle. Individual mucus samples were solubilized and fractionated on Sepharose columns into excluded mucins and low-molecular-weight proteins. Mucin fractions were highly purified, as evidenced by the presence of a single N-terminal amino acid residue, threonine, and by the absence of contaminating plasma proteins. Amino acid compositions of mucins isolated during different menstrual phases of a single donor or from different donors were similar. Mucin carbohydrate compositions were also similar, except for the sialic acid-to-fucose ratio, which varied significantly between donors but not within the menstrual cycle of a single donor. An analysis of variance was applied to evaluate the contribution of mucin composition to viscoelasticity, as quantitated by microrheometry. Viscoelasticity was dependent on the donor, on the percent nondialyzable solids and on the mucin content, b ut not on the phase of the menstrual cycle during which the sample was collected. These findings suggest that mucus function (viscoelasticity) is reflected in carbohydrate composition and/or structure and that this relationship is unique for each donor. Furthermore, the absence of menstrual phase-dependent differences in mucins suggests that mucin concentration and not composition changes in response to alterations in the hormonal milieu.

Net costs from three perspectives of using low versus high osmolality contrast medium in diagnostic angiocardiography.

OBJECTIVES: We conducted an economic analysis to assess the extent to which a reduction in adverse drug reactions induced by low osmolality compared with high osmolality contrast media during diagnostic angiocardiography would result in savings to hospitals, society and third-party payers that would offset the substantially higher price of low osmolality contrast medium. BACKGROUND: Substitution of low osmolality for high osmolality contrast media in the approximately 1 million diagnostic angiocardiographic procedures performed each year in the United States could substantially increase health care costs. Cost-effectiveness estimates should include savings that might occur through reduced costs of managing adverse drug reactions. METHODS: In a randomized clinical trial of 505 persons under-going diagnostic angiography with either high osmolality or low osmolality contrast medium, we measured and compared 1) material costs of contrast media, and 2) costs from three perspectives of incremental resources used to manage contrast-related adverse drug reactions. We also performed sensitivity analyses to examine the effect of different assumptions with regard to relative risk, absolute risk and costs of adverse drug reactions on estimates of net cost of use of high osmolality and low osmolality contrast media. RESULTS: One-hundred thirty-seven (54.2%) of 253 patients receiving high osmolality contrast medium and 44 (17.5%) of 252 patients receiving low osmolality contrast medium experienced adverse drug reactions. The average cost (from society's perspective) of resources used to manage adverse drug reactions per patient undergoing angiography was significantly (p = 0.0001) greater for high osmolality (mean $249) versus low osmolality (mean $92) contrast medium. Differential costs (from the hospital's perspective) were $67 greater for high osmolality contrast medium. Charges and professional fees (from the payer's perspective) were $182 greater for high osmolality (mean $312) than for low osmolality (mean $130) contrast medium (p = 0.42, NS). The higher differential and average costs of managing adverse drug reactions with high osmolality contrast medium offset 33% and 75%, respectively, of the $207 difference in mean material costs, but these estimates are sensitive to infrequent high cost cases. CONCLUSIONS: Although low osmolality contrast medium is not cost-saving in diagnostic angiocardiography, its higher price is partially offset by lower management costs of adverse drug reactions. The cost offset for the hospital is lower than that for society and may not be realized by third-party payers. These methods and results may be useful in establishing clinical and payment guidelines for use of alternative contrast media in diagnostic angiocardiography.

Types of alcoholics, II. Application of an empirically derived typology to treatment matching.

Data from 79 male alcoholics who were randomly assigned to either coping skills training or interactional group psychotherapy were used to replicate a multidimensional, empirically derived typology and to evaluate the typology's usefulness in matching patients to treatment. Consistent with previous cluster analysis research, indicators of risk for alcoholism, alcohol dependence, drinking history, and psychopathological impairment distinguished alcoholics along two broad dimensions of vulnerability and severity, with one subtype (type B alcoholics) manifesting an earlier onset of problem drinking, more familial alcoholism, greater dependence on alcohol, and more symptoms of antisocial personality than the other subtype (type A alcoholics). Analyses of outcome indicated that type A alcoholics fared better in interactional treatment and more poorly with coping skills training. Conversely, type B alcoholics had better outcomes with the coping skills treatment and worse outcomes with interactional therapy. Differences in treatment response were maintained for 2 years from the beginning of aftercare treatment.

Clouston syndrome (hidrotic ectodermal dysplasia) is not linked to keratin gene clusters on chromosomes 12 and 17.

Clouston syndrome is an hidrotic form of ectodermal dysplasia, inherited as an autosomal dominant trait with high penetrance. The main features of the disorder are alopecia, severe dystrophy of the nails, and palmoplantar hyperkeratosis. A molecular abnormality of keratin has long been hypothesized to be the basic defect in this disorder. We have performed linkage analyses between the disorder and markers close to the keratin gene clusters on chromosomes 12 and 17 and have excluded linkage to these candidate regions in three apparently unrelated families. In addition, linkage has been excluded to four other candidate regions including 1q2l, 17q23-qter, 18q2l, and 2Oql2. These data indicate that Clouston syndrome is not due to a defect in keratin or in a subset of keratin-associated proteins.

Confirmation of linkage of Clouston syndrome (hidrotic ectodermal dysplasia) to 13q11-q12.1 with evidence for multiple independent mutations.

Clouston syndrome (hidrotic ectodermal dysplasia) is an autosomal dominant disorder characterized by the triad of nail dystrophy, alopecia, and palmoplantar hyperkeratosis. Recently, linkage of a Clouston syndrome locus to chromosome 13q11-q12.1 was reported in eight families of French-Canadian descent. We have confirmed linkage to this region in four additional families: two of French-Canadian descent, one of Scottish-Irish descent, and one French family. Multipoint linkage analysis gave a lod score of 5.09 at marker D13S175. The two families of French-Canadian descent share haplotypes with those reported by Kibar et al (1996), indicating a common founder. The French and Scottish-Irish families do not demonstrate the common haplotype, indicating that the mutations in these populations are most likely of different origin.

A 3-Mb region for the familial hemiplegic migraine locus on 19p13.1-p13.2: exclusion of PRKCSH as a candidate gene. Dutch Migraine Genetic Research Group.

Familial hemiplegic migraine (FHM) is an autosomal domianant subtype of migraine with attacks, associated with transient episodes of hemiparesis. One of the genes for FHM has been assigned to chromosome 19p13. Detailed analysis of critical recombinants from two different chromosome 19-linked FHM families, using new markers indicated a 6-cM candidate region on 19p13.1-p13.2 flanked by loci D19S394 and D19S226. Another paroxysmal neurological disorder, episodic ataxia type 2 (EA-2), has also been linked to the same chromosomal region. Most of the interval was completely covered by YAC and cosmid contigs; the physical map yielded approximately 3 Mb encompassing several genes including the protein kinase substrate 80K-H (PRKCSH) gene. Since PRKCSH is involved in neuronal signal transduction, it was considered to be an FHM candidate gene. The genomic structure of this gene was established and mutation analysis for all exon and flanking intron sequences was performed in FHM- and EA-2-affected individuals. Five polymorphisms were identified, including a trinucleotide repeat length variation in the coding sequence. However, no potential disease causing mutation was found and therefore the PRKCSH gene can be excluded for both FHM and EA-2.

Human cytochrome c oxidase subunit VIb: characterization and mapping of a multigene family.

We report the isolation and sequence of a human heart cDNA coding for cytochrome c oxidase (COX) subunit VIb (COX VIb). This cDNA extends 50 bp upstream from the region coding for the mature peptide. By Northern analysis, a single transcript of approx. 550 nucleotides (nt) has been identified in six human tissues. Southern analysis of human genomic DNA demonstrates the presence of multiple loci that show high homology to the cDNA. These loci cosegregate with either five or six different human chromosomes in human-rodent somatic cell hybrids. Using the COX6b cDNA, genomic sequences representing two of these loci have been isolated and characterized. The nt sequence analysis suggests that both loci represent COX6b pseudogenes.

A novel nonsense mutation in CACNA1A causes episodic ataxia and hemiplegia.

OBJECTIVE: To identify the disease-causing mutation and to characterize penetrance and phenotypic variability in a large pedigree with episodic ataxia type 2 (EA-2) previously linked to chromosome 19. BACKGROUND: Mutations in the CACNA1A gene on chromosome 19 encoding a calcium channel subunit cause EA-2, which is characterized by recurrent attacks of imbalance with interictal eye movement abnormalities. METHODS: The authors used single-strand conformation polymorphism (SSCP) analysis to screen for point mutations, and direct sequencing to identify mutations in CACNA1A. Allele-specific oligonucleotides were designed to detect the presence of the diseased allele in members of their pedigree as well as in normal control subjects. RESULTS: Reassessment of members of the pedigree revealed two notable clinical features. Diffuse weakness during attacks of ataxia was a prominent complaint. Two affected individuals had had episodic hemiplegia, one with typical migraine headaches. SSCP analysis revealed aberrant bands in exon 29 in affected members but not in normal control subjects. Direct sequencing of exon 29 identified a C-to-T change at position 4914 of the coding sequence of CACNA1A, predicting an early stop code at codon 1547. Two asymptomatic mutation carriers demonstrated the incomplete penetrance of this mutation. CONCLUSIONS: A nonsense mutation in CACNA1A causes episodic ataxia and complaint of weakness, and may be associated with hemiplegia.

No evidence for association of familial Parkinson's disease with CAG repeat expansion.

In some kindreds, familial Parkinson's disease (PD) exhibits genetic anticipation. Thus, we postulated that familial PD in certain kindreds may be associated with a CAG repeat expansion. However, using the repeat expansion detection method, we found no significant increase in the frequency of CAG repeat expansion among 46 unrelated PD probands compared with controls. Nor did we find evidence for CAG repeat expansion between generations in 11 different PD families that exhibit anticipation in age at onset.

Paroxysmal kinesigenic dyskinesia and infantile convulsions: clinical and linkage studies.

OBJECTIVE: To clinically characterize affected individuals in families with paroxysmal kinesigenic dyskinesia (PKD), examine the association with infantile convulsions, and confirm linkage to a pericentromeric chromosome 16 locus. BACKGROUND: PKD is characterized by frequent, recurrent attacks of involuntary movement or posturing in response to sudden movement, stress, or excitement. Recently, an autosomal dominant PKD locus on chromosome 16 was identified. METHODS: The authors studied 11 previously unreported families of diverse ethnic background with PKD with or without infantile convulsions and performed linkage analysis with markers spanning the chromosome 16 locus. Detailed clinical questionnaires and interviews were conducted with affected and unaffected family members. RESULTS: Clinical characterization and sampling of 95 individuals in 11 families revealed 44 individuals with paroxysmal dyskinesia, infantile convulsions, or both. Infantile convulsions were surprisingly common, occurring in 9 of 11 families. In only two individuals did generalized seizures occur in later childhood or adulthood. The authors defined a 26-cM region using linkage data in 11 families (maximum lod score 6.63 at theta = 0). Affected individuals in one family showed no evidence for a shared haplotype in this region, implying locus heterogeneity. CONCLUSIONS: Identification and characterization of the PKD/infantile convulsions gene will provide new insight into the pathophysiology of this disorder, which spans the phenotypic spectrum between epilepsy and movement disorder.