Kamna Das

Mutations in LGI1 cause autosomal-dominant partial epilepsy with auditory features

The epilepsies are a common, clinically heterogeneous group of disorders defined by recurrent unprovoked seizures. Here we describe identification of the causative gene in autosomal-dominant partial epilepsy with auditory features (ADPEAF, MIM 600512), a rare form of idiopathic lateral temporal lobe epilepsy characterized by partial seizures with auditory disturbances. We constructed a complete, 4.2-Mb physical map across the genetically implicated disease-gene region, identified 28 putative genes (Fig. 1) and resequenced all or part of 21 genes before identifying presumptive mutations in one copy of the leucine-rich, glioma-inactivated 1 gene (LGI1) in each of five families with ADPEAF. Previous studies have indicated that loss of both copies of LGI1 promotes glial tumor progression. We show that the expression pattern of mouse Lgi1 is predominantly neuronal and is consistent with the anatomic regions involved in temporal lobe epilepsy. Discovery of LGI1 as a cause of ADPEAF suggests new avenues for research on pathogenic mechanisms of idiopathic epilepsies.

Differential SMN2 expression associated with SMA severity

Recent advances in the molecular genetics of spinal muscular atrophy (SMA) have led to the isolation of the survival motor neuron (SMN) gene1. The SMN gene is present in one telomeric copy, SMN1, and several centromeric copies, SMN2 (refs 2−5). Homozygous deletion or point mutation of SMN1 results in SMA (refs 1,4−12), but the pathogenic role of SMN2 is currently unknown. Recent studies suggest that SMN2 copy number may influence SMA disease severity, and that some SMN2 copies in the milder SMA phenotypes may arise from gene conversions2,4,5,13. The source and function of SMN2 may therefore be quite different among the different SMA subtypes. We report here the first evidence that SMN2 may influence SMA disease severity through preferential expression of an alternative transcript in the more severe phenotypes. We obtained sixty RNA samples (22 severe type I, 14 intermediate type II, 14 mild type III SMA and 10 controls) from human lymphoblastoid cell lines, and found SMN1 to be homozygously deleted in all of them. NAIP, encoding the neuronal apoptosis inhibitory protein, and neighbour to SMN1, was also deleted in 14 type I, 4 type II and none of the type III samples. Quantitative RT-PCR was performed using primers flanking exons 5 and 8 of the SMN gene and primers flanking exon 2 of a single-copy human major histocompatibility complex gene, HLADQA1. The latter was included in the multiplex PCR as an internal standard. The PCR reaction was terminated at 22 cycles to ensure quantitative measurements during the linear phase. Measurements were also taken at 35 and 30 cycles. Results from these higher cycle numbers tended to obliterate the quantitative ratios (data not shown). The PCR products were then separated on a 2% agarose gel containing ethidium bromide. Densitometry analysis was applied using ImageQuant 3.3 software (Molecular Dynamics) to measure the band intensities. This experiment was replicated three times. RT-PCR (Fig. 1) generated a full-length SMN transcript (band a), an alternative spliced variant lacking exon 7 of SMN gene (band b) and the HLA-DQA1 internal standard (band c) in all samples except sample 3761; the latter appears to lack SMN2 but contains an intact SMN1. Its RNA contains no alternative transcript (band b), confirming the previous notion that the exon 7-deleted transcript originates only from the SMN2 copies1. Data collected from all 60 samples are summarized (Table 1). Samples from control individuals give rise to approximately twice as much full-length as the alternative SMN transcript, and we found no significant difference in the amount of full-length transcript produced among the three SMA subtypes; each SMA subgroup produced approximately 40−45% as much Fig. 1 SMN2 transcription pattern among three SMA subtypes. RT-PCR with primers flanking exons 5 and 8 of SMN reveals two SMN transcripts (bands a and b). Band a represents the full-length transcript and band b, the exon 7-deleted splice variant. Band c represents the internal standard derived from the single-copy gene HLA-DQA1. The genomic DNA of sample 3671 retains no SMN2 copy and its RNA shows only a full-length SMN transcript. Table 1 • Densitometry analysis of two SMN2 transcripts in SMA patients

CHARACTERIZATION OF THE INTERACTION BETWEEN THE WILSON AND MENKES DISEASE PROTEINS AND THE CYTOPLASMIC COPPER CHAPERONE, HAH1P

Wilson disease (WD) and Menkes disease (MNK) are inherited disorders of copper metabolism. The genes that mutate to give rise to these disorders encode highly homologous copper transporting ATPases. We use yeast and mammalian two-hybrid systems, along with anin vitro assay to demonstrate a specific, copper-dependent interaction between the six metal-binding domains of the WD and MNK ATPases and the cytoplasmic copper chaperone HAH1. We demonstrate that several metal-binding domains interact independently or in combination with HAH1p, although notably domains five and six of WDp do not. Alteration of either the Met or Thr residue of the HAH1p MTCXXC motif has no observable effect on the copper-dependent interaction, whereas alteration of either of the two Cys residues abolishes the interaction. Mutation of any one of the HAH1p C-terminal Lys residues (Lys56, Lys57, or Lys60) to Gly does not affect the interaction, although deletion of the 15 C-terminal residues abolishes the interaction. We show that apo-HAH1p can bind in vitroto copper-loaded WDp, suggesting reversibility of copper transfer from HAH1p to WD/MNKp. The in vitro HAH1/WDp interaction is metalospecific; HAH1 preincubated with Cu2+ or Hg+ but not with Zn2+, Cd2+, Co2+, Ni3+, Fe3+, or Cr3+ interacted with WDp. Finally, we model the protein-protein interaction and present a theoretical representation of the HAH1p·Cu·WD/MNKp complex.

Results of a genome-wide genetic screen for panic disorder.

Panic disorder is characterized by spontaneous and recurrent panic attacks, often accompanied by agoraphobia. The results of family, twin, and segregation studies suggest a genetic role in the etiology of the illness. We have genotyped up to 23 families that have a high density of panic disorder with 540 microsatellite DNA markers in a first-pass genomic screen. The thirteen best families (ELOD > 6.0 under the dominant genetic model) have been genotyped with an ordered set of markers encompassing all the autosomes, at an average marker density of 11 cM. Over 110,000 genotypes have been generated on the whole set of families, and the data have been analyzed under both a dominant and a recessive model, and with the program SIBPAIR. No lod scores exceed 2.0 for either parametric model. Two markers give lod scores over 1.0 under the dominant model (chromosomes 1p and 20p), and four do under the recessive model (7p, 17p, 20q, and X/Y). One of these (20p) may be particularly promising. Analysis with SIBPAIR yielded P values equivalent to a lod score of 1.0 or greater (i.e., P < .016, one-sided, uncorrected for multiple tests) for 11 marker loci (2, 7p, 8p, 8q, 9p, 11q, 12q, 16p, 20p and 20q).

Selection for contextual fear conditioning affects anxiety-like behaviors and gene expression.

Conditioned fear and anxiety‐like behaviors have many similarities at the neuroanatomical and pharmacological levels, but their genetic relationship is less well defined. We used short‐term selection for contextual fear conditioning (FC) to produce outbred mouse lines with robust genetic differences in FC. The high and low selected lines showed differences in fear learning that were stable across various training parameters and were not secondary to differences in sensitivity to the unconditioned stimulus (foot shock). They also showed a divergence in fear potentiated startle, indicating that differences induced by selection generalized to another measure of fear learning. However, there were no differences in performance in a Pavlovian approach conditioning task or the Morris water maze, indicating no change in general learning ability. The high fear learning line showed greater anxiety‐like behavior in the open field and zero maze, confirming a genetic relationship between FC and anxiety‐like behavior. Gene expression analysis of the amygdala and hippocampus identified genes that were differentially expressed between the two lines. Quantitative trait locus (QTL) analysis identified several chromosomal regions that may underlie the behavioral response to selection; cis‐acting expression QTL were identified in some of these regions, possibly identifying genes that underlie these behavioral QTL. These studies support the validity of a broad genetic construct that includes both learned fear and anxiety and provides a basis for further studies aimed at gene identification.

No Evidence for Significant Linkage between Bipolar Affective Disorder and Chromosome 18 Pericentromeric Markers in a Large Series of Multiplex Extended Pedigrees

Bipolar affective disorder (BP) is a major neuropsychiatric disorder with high heritability and complex inheritance. Previously reported linkage between BP and DNA markers in the pericentromeric region of chromosome 18, with a parent-of-origin effect (linkage was present in pedigrees with paternal transmission and absent in pedigrees with exclusive maternal inheritance), has been a focus of interest in human genetics. We reexamined the evidence in one of the largest samples reported to date (1,013 genotyped individuals in 53 unilineal multiplex pedigrees), using 10 highly polymorphic markers and a range of parametric and nonparametric analyses. There was no evidence for significant linkage between BP and chromosome 18 pericentromeric markers in the sample as a whole, nor was there evidence for significant parent-of-origin effect (pedigrees with paternal transmission were not differentially linked to the implicated chromosomal region). Two-point LOD scores and single-locus sib-pair results gave some support for suggestive linkage, but this was not substantiated by multilocus analysis, and the results were further tempered by multiple test effects. We conclude that there is no compelling evidence for linkage between BP and chromosome 18 pericentromeric markers in this sample.

Fine-mapping of the spinal muscular atrophy locus to a region flanked by MAP1B and D5S6

The microtubule-associated protein 1B (MAP1B) locus has been mapped in close proximity to spinal muscular atrophy (SMA) on chromosome 5q13. We have identified a second microsatellite within a MAP1B intron, which increases the heterozygosity of this locus to 94%. Two unambiguous recombination events establish MAP1B as a closely linked, distal flanking marker for the disease locus, while a third recombinant establishes D5S6 as the proximal flanking marker. The combination of key recombinants and linkage analysis place the SMA gene in an approximately 2-cM interval between loci D5S6 and MAP1B. Physical mapping and cloning locate MAP1B within 250 kb of locus D5S112. The identification and characterization of a highly polymorphic gene locus tightly linked to SMA will facilitate isolation of the disease gene, evaluation of heterogeneity, and development of a prenatal test for SMA.

Assessment of nonallelic genetic heterogeneity of chronic (type II and III) spinal muscular atrophy.

We have previously reported the mapping of the chronic (type II/intermediate and type III/mild/Kugelberg-Welander) form of the childhood-onset spinal muscular atrophies (SMA) to chromosome 5q11.2-13.3, with evidence for nonallelic genetic heterogeneity within a small sample of seven families [Brzustowicz et al., Nature 1990;344:540-541]. We now report the results of linkage analysis and heterogeneity testing on a set of 38 families with chronic SMA. Significant evidence for nonallelic heterogeneity was detected among these families, with the predominant locus for chronic SMA mapping to a 0.51-cM region on 5q, between the loci D5S6 and MAP1B. The estimated proportion of linked families, alpha, was 0.91, with a 2.3-unit support interval of 0.75 to 0.98. The indication that some families diagnosed with chronic SMA are not linked to chromosome 5q must be considered in strategies to map the SMA locus. The relevance of these findings to acute SMA (SMA type I, severe, Werdnig-Hoffmann disease) is still unknown.

Further analysis of the mechanism of copper trafficking pathways using yeast expression arrays

Further analysis of the mechanism of copper trafficking pathways using yeast expression arrays