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Purpose: The β3 subunit of the γ-aminobutyric acid type A receptors (GABA(A) -Rs) is an essential component of GABA(A) -Rs in fetal, perinatal, and adult mammalian brain. Various transcripts of the β3 subunit gene (GABRB3) produce various proteins with different N-termini. Rare variants in this N-terminus (exon 1A and exon 2) of GABRB3 protein segregate in affected family members of two multigeneration-multiplex families with remitting childhood absence epilepsy (rCAE), suggesting GABRB3 is a major Mendelian epilepsy gene for rare families with CAE. Therefore, the N-terminus of GABRB3 could be important for GABRB3 regulation in development, and its alteration could produce rCAE. Herein we determine if single nucleotide polymorphisms (SNPs) within the 1,148-bp region upstream from exon 1A influence the expression of GABRB3., Methods: We studied luciferase reporter expression for promoter activity, 1,148-bp upstream from exon 1A, using human embryonic kidney 293 cells. We generated constructs of the promoter region and compared different SNP haplotypes in 48 patients with rCAE. Next, we compared frequencies of rs20317, located in the core promoter region, and rs4906902, located in the enhancer region between 48 patients with rCAE and >500 healthy controls matched for ethnicity and ancestral origin., Key Findings: Highest luciferase expression occurred 230-bp upstream of exon 1A. The construct that excluded this region lost luciferase activity. Therefore, this region contains the core promoter of exon 1A. Allele C but not allele G (rs20317) significantly increased luciferase expression activity. Allele C creates binding motifs for cMYB and EGR-3. Longer constructs overlapping this region have a binding motif for REST (RE1-silencing transcription factor), a critical epigenetic modulator for neuronal genes. REST represses expression of neuronal genes in nonneuronal tissues, resulting in reduced luciferase expression activity. Even in the suppressed condition, the longer construct enhanced luciferase expression activity of the shorter construct, which excluded the distal end containing rs4906902. However, allele frequencies of rs20317 and rs4906902 were not significantly associated with 48 rCAE patients in comparison to >500 controls matched for ethnicity and ancestral origin., Significance: Common SNPs in the promoter region increase luciferase expression activity. An epigenetic modulator, REST, specifically alters expression of GABRB3 exon 1A transcripts, suggesting epigenetic regulation by REST dominantly controls the expression of GABRB3 variant 2 transcript in early life GABA(A) signaling. Abnormal epigenetic regulation could be involved in absence seizures., (Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.)

J-domains are widespread protein interaction modules involved in recruiting and stimulating the activity of Hsp70 family chaperones. We have determined the crystal structure of the J-domain of auxilin, a protein which is involved in uncoating clathrin-coated vesicles. Comparison to the known structures of J-domains from four other proteins reveals that the auxilin J-domain is the most divergent of all J-domain structures described to date. In addition to the canonical J-domain features described previously, the auxilin J-domain contains an extra N-terminal helix and a long loop inserted between helices I and II. The latter loop extends the positively charged surface which forms the Hsc70 binding site, and is shown by directed mutagenesis and surface plasmon resonance to contain side chains important for binding to Hsc70.

Developmental dyslexia is defined as a specific and significant impairment in reading ability that cannot be explained by deficits in intelligence, learning opportunity, motivation or sensory acuity. It is one of the most frequently diagnosed disorders in childhood, representing a major educational and social problem. It is well established that dyslexia is a significantly heritable trait with a neurobiological basis. The etiological mechanisms remain elusive, however, despite being the focus of intensive multidisciplinary research. All attempts to map quantitative-trait loci (QTLs) influencing dyslexia susceptibility have targeted specific chromosomal regions, so that inferences regarding genetic etiology have been made on the basis of very limited information. Here we present the first two complete QTL-based genome-wide scans for this trait, in large samples of families from the United Kingdom and United States. Using single-point analysis, linkage to marker D18S53 was independently identified as being one of the most significant results of the genome in each scan (P< or =0.0004 for single word-reading ability in each family sample). Multipoint analysis gave increased evidence of 18p11.2 linkage for single-word reading, yielding top empirical P values of 0.00001 (UK) and 0.0004 (US). Measures related to phonological and orthographic processing also showed linkage at this locus. We replicated linkage to 18p11.2 in a third independent sample of families (from the UK), in which the strongest evidence came from a phoneme-awareness measure (most significant P value=0.00004). A combined analysis of all UK families confirmed that this newly discovered 18p QTL is probably a general risk factor for dyslexia, influencing several reading-related processes. This is the first report of QTL-based genome-wide scanning for a human cognitive trait.

The loci involved in several X-linked mental retardation syndromes have been linked to the pericentromeric region of the X chromosome long arm (Xq12-q21). To isolate candidate genes for these diseases, we set up the construction of YAC contigs spanning this region. Two of these syndromes (the Juberg-Marsidi syndrome and the alpha-thalessemia mental retardation syndrome) have been recently linked, with high lod scores, to polymorphic probes previously assigned to Xq13.3. We therefore constructed a first YAC contig, encompassing this band, from DXS441 to PGK1. The physical map, deduced from the isolated clones, extends over 2.1 Mb of genomic DNA. Restriction analysis of the YAC contig allowed us to map precisely the loci previously assigned to that chromosomal region and to define their relative order. The validity of this physical map has been checked by comparing Sfi I digests of the YACs to genomic fragments obtained with the same enzyme. A cDNA selection approach, already performed with a previous partial contig, has been extended to cover the whole region.

We report a method to analyse multiple samples by fluorescence in situ hybridisation on a single glass microscope slide. Wells were formed in which independent hybridisation reactions could proceed by sealing a silicon rubber gasket to the slide. In the largest format tested, different probes were hybridised simultaneously by applying them directly from a 96-well microtitre dish which was inverted on a glass plate. This technique will increase the rate of analysis of multiple probes against a standard set of chromosomes and could also be used to analyse different karyotypes using a panel of probes such as single chromosome paints during a single operation. It should be useful for both chromosomal mapping projects and screening for chromosome abnormalities in clinical diagnostic laboratories.

We have found that the microsatellite marker AFM207zg5 (DXS995) maps to all previously described deletions which are associated with X-linked mixed deafness (DFN3) with or without choroideremia and mental retardation. Employing this marker and pHU16 (DXS26) we have identified two partially overlapping yeast artificial chromosome clones which were used to construct a complete 850 kb cosmid contig. Cosmids from this contig have been tested by Southern blot analysis on DNA from 16 unrelated males with X-linked deafness. Two novel microdeletions were detected in patients which exhibit the characteristic DFN3 phenotype. Both deletions are completely contained within one of the known DFN3-deletions, but one of them does not overlap with two previously described deletions in patients with contiguous gene syndromes consisting of DFN3, choroideremia, and mental retardation. Assuming that only a single gene is involved, this suggests that the DFN3 gene spans a chromosomal region of at least 400 kb.

The human Xp21.3-p22.1 region is poorly mapped relative to other X chromosome regions. To target cosmid and YAC clones specifically from Xp21.3-p22.1 for rapid contig construction, a hybridization-based screening approach using irradiation hybrids has been used. Alu-PCR products generated from hybrid lines containing small overlapping fragments from Xp21-p22 were hybridized to an X chromosome cosmid library, and cosmids predicted by their hybridization pattern to map to the region of interest were analyzed by fluorescence in situ hybridization (FISH). Hybridization of the cosmids in pools to gridded YAC libraries identified 15 YACs, which were verified and tested for chimerism by FISH. Cosmid content analysis of the YACs defined two contigs, one with 12 YACs covering about 1.5 Mb and one with 3 YACs. Five YACs from the 12-YAC cluster had been previously recognized by DNA polymerase alpha (POLA). ZFX identified a single YAC; hence, the physical linkage of ZFX and POLA was demonstrated within the contig. Four YACs had been isolated previously with ZFX and these extend the contig to 2 Mb. Restriction mapping of several YACs demonstrates that ZFX and POLA are about 700 kb apart, a distance similar to that reported in the mouse between Zfx and Pola. The order of these two loci and two additional loci identified by homologous mouse linking clones was found to be conserved between human and mouse: tel-ZFX-DXCrc57-DXCrc140-POLA-cen. We have shown that YAC contigs can be rapidly constructed from targeted regions without the need for time-consuming YAC end rescue and chromosomal walking.(ABSTRACT TRUNCATED AT 250 WORDS)

Menkes disease is a lethal-X linked recessive disorder associated with copper metabolism disturbance. We have recently mapped two chromosome breakpoints related to this disease in a 1 megabase yeast artificial chromosome contig at Xq13.3. We now report the construction of a phage contig and the isolation of candidate partial cDNAs for the Menkes disease gene. The candidate gene expresses an 8 kb message in all investigated tissues, and deletions were detected in 16% of 100 unrelated Menkes patients. The deduced partial protein sequence shared the GMTCXXC motif with bacterial metal resistance operons, suggesting a potential heavy metal binding protein. These findings should lead to more accurate prenatal diagnosis of this severe disease and a better understanding of the cellular homeostasis of essential heavy metals.

We have localized several markers in the Xq24-25 region containing DXS12, DXS42 and DXS37 which are closely linked to the X-linked lymphoproliferative syndrome (XLP) locus. A 850-kb restriction map has been established by mapping overlapping YACs and showed that DXS12 and DXS42 are physically linked within about 50 kb. DXS37 is separated from these two loci at a maximum distance of 3,700 kb. Several new probes have been generated which will contribute to further physical mapping of this region.

Menkes disease, an X-linked recessive disorder of copper metabolism, has recently been mapped to Xq13.3 by two Menkes patients carrying chromosome rearrangements within this region. The breakpoints have been investigated by nonisotopic in situ suppression hybridization using YACs isolated from this region with the flanking markers DXS56 and PGK1. Three YACs were extending over the breakpoints at Xq13.3 and were shown to be overlapping by partial digest restriction maps, IRS-PCR fingerprinting and by the presence of common cosmid clones. These cosmids were subcloned and one of the single copy probes detected both breakpoints using rare-cutting restriction enzyme digests of the patients. All the results together localize the breakpoints to about 100 kb within the overlapping region of the YACs. Mapping of both breakpoints in a 1 Mb YAC contig implies that these YACs contain at least partially, the gene responsible for Menkes disease.