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We have generated a transgenic rat model of Charcot-Marie-Tooth disease type 1A (CMT1A) providing formal proof that this neuropathy can be caused by increased expression of peripheral myelin protein-22 (PMP22). Heterozygous PMP22-transgenic rats develop muscle weakness and gait abnormalities as well as reduced nerve conduction velocities and EMG abnormalities, which closely resemble recordings in patients with CMT1A. Dys- and demyelination, Schwann cell hypertrophy, and "onion bulb" formation are also similar to findings in humans. When bred to homozygosity, transgenic rats completely fail to elaborate myelin, but all myelin-forming Schwann cells segregate with axons in the normal one-to-one ratio. Although arrested at this "promyelin" stage, differentiation proceeds in homozygous rats at the molecular level, as demonstrated by high-level expression of myelin structural genes. Intracellular trafficking of the wild-type protein is not visibly impaired, even when strongly overexpressed, suggesting that PMP22 blocks myelin assembly in a late Golgi/cell membrane compartment of the affected Schwann cell.

Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disorder of upper and lower motor neurons. Genetic variants in the paraoxonase gene cluster have been associated with susceptibility to sporadic ALS. Because these studies have yielded conflicting results, we have further investigated this association in a larger data set. Twenty SNPs spanning the paraoxonase gene cluster were genotyped on a panel of 597 case and 692 control samples and tested for association with risk of sporadic ALS and with ALS sub-phenotypes. Our study revealed two SNPs, rs987539 and rs2074351, within the paraoxonase gene cluster that are associated with susceptibility to sporadic ALS (uncorrected p=6.47E-04 and 7.87E-04, respectively). None of the 20 SNPs displayed significant associations with age of onset, site of onset or disease survival. Using a sliding window approach, we have also identified a 5-SNP haplotype that is significantly associated with risk of sporadic ALS (p=2.75E-05). We conclude that a common haplotype within the PON1 promoter region is associated with susceptibility to sporadic ALS.

NR3B is a modulatory subunit of the NMDA receptor, abundantly expressed in both cranial and spinal somatic motoneurons and at lower levels in other regions of the brain as well. Recently, we found the human NR3B gene (GRIN3B) to be highly genetically heterogeneous, and that approximately 10% of the normal European-American population lacks NR3B due to homozygous occurrence of a null allele in the gene. Therefore, it is especially important to understand the phenotypic consequences of the genetic loss of NR3B in both humans and animal models. We here provide results of behavioral analysis of mice genetically lacking NR3B, which is an ideal animal model due to homogeneity in genetic and environmental background. The NR3B(-/-) mice are viable and fertile. Consistent with the expression of NR3B in somatic motoneurons, the NR3B(-/-) mice showed a moderate but significant impairment in motor learning or coordination, and decreased activity in their home cages. Remarkably, the NR3B(-/-) mice showed a highly increased social interaction with their familiar cage mates in their home cage but moderately increased anxiety-like behaviour and decreased social interaction in a novel environment, consistent with the inhibitory role of NR3B on the functions of NMDA receptors. This work is the first reporting of the functional significance of NR3B in vivo and may give insight into the contribution of genetic variability of NR3B in the phenotypic heterogeneity among human population.

We report a patient with autosomal-dominant amyotrophic lateral sclerosis (ALS) and a sequence variation in the SOD1 promoter region, located in the conserved TATA box motif (TATAAA→TGTAAA). Functional promoter studies of this variant in an in vitro system showed moderate reduction in transcriptional activity of SOD1. This variant was present in only two of 301 individuals with sporadic amyotrophic lateral sclerosis, was not detected in 396 matched controls, and was recently reported in dbSNP (rs7277748). Our data suggest that this TATA box defect is not a disease-causing mutation or susceptibility factor for ALS but rather a rare polymorphism with a potential effect on SOD1 gene expression. Muscle Nerve, 2007

Mutations in genes coding for three of the four components of mitochondrial complex II can cause paragangliomas (PGLs)/pheochromocytomas. The three genes include SDHB, -C, and -D. SDHC and SDHD anchor the catalytic subunits SDHA and -B of mitochondrial complex II in the inner mitochondrial membrane. SDHD is maternally imprinted but SDHB and -C are not. While SDHD and -- to a lesser degree -- SDHB mutations have been found in many cases of hereditary PGL, SDHC mutations are rare. This article reviews the SDHC mutations described to date and discusses possible mechanisms of tumorigenesis.

X-linked dystonia parkinsonism (XDP) is an X-linked recessive adult onset movement disorder characterized by both dystonia and parkinsonism. We report delineation of the disease gene within a 300-kb interval of Xq13.1 by allelic association. Sequencing of this region in a patient revealed five disease-specific single-nucleotide changes (here referred to as DSC) and a 48-bp deletion unique to XDP. One of the DSCs is located within an exon of a not previously described multiple transcript system that is composed of at least 16 exons. There is a minimum of three different transcription start sites that encode four different transcripts. Two of these transcripts include distal portions of the TAF1 gene (TATA-box binding protein-associated factor 1) and are alternatively spliced. Three exons overlap with ING2 (a putative tumor suppressor) and with a homologue of CIS4 (cytokine-inducible SH2 protein 4), both of which are encoded by the opposite strand. Although all DSCs are located within this multiple transcript system, only DSC3 lies within an exon. This exon is used by all alternative transcripts making a pathogenic role of DSC3 in XDP likely. The multiple transcript system is therefore referred to as DYT3 (disease locus in XDP).

A chromosomal translocation t(18;21)(q23;q22) is reported in a patient with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We exclude the physical involvement and silencing of the ALS-linked gene for copper/zinc superoxide dismutase (SOD1) on chromosome 21q22.1. The breakpoints are assigned to sequences flanked by the markers ATA1H06, D18S462, D21S1915, and D21S1898. These critical regions may contain susceptibility loci for FTD associated with ALS.

We have generated previously transgenic rats that overexpress peripheral myelin protein 22 (PMP22) in Schwann cells. In the nerves of these animals, Schwann cells have segregated with axons to the normal 1:1 ratio but remain arrested at the promyelinating stage, apparently unable to elaborate myelin sheaths. We have examined gene expression of these dysmyelinating Schwann cells using semiquantitative reverse transcription-PCR and immunofluorescence analysis. Unexpectedly, Schwann cell differentiation appears to proceed normally at the molecular level when monitored by the expression of mRNAs encoding major structural proteins of myelin. Furthermore, an aberrant coexpression of early and late Schwann cell markers was observed. PMP22 itself acquires complex glycosylation, suggesting that trafficking of the myelin protein through the endoplasmic reticulum is not significantly impaired. We suggest that PMP22, when overexpressed, accumulates in a late Golgi–cell membrane compartment and uncouples myelin assembly from the underlying program of Schwann cell differentiation.

We performed a whole genome scan in a family with maternally transmitted paraganglioma (PGL3). The family included five patients with histologically proven paraganglioma and one patient with imaging findings consistent with a paraganglioma. In addition, there were 33 clinically unaffected family members. Of these eight could be examined by magnetic resonance imaging. Our investigations indicate that PGL3 is located in 1q21-q23 for several reasons: 1) two-point linkage analysis yielded the highest LOD score of 2.25 at 1q21-q23 (marker D1S2675); 2) haplotype analysis was most consistent for 1q21-q23 markers; and 3) the locus was excluded from more than 97% of the genome using a total of 381 highly polymorphic markers.

Mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) account for approximately 20% of patients with familial amyotrophic lateral sclerosis (FALS). In this study, sequence analysis of exons 1-5 of SOD1 in a large German cohort with FALS was performed. Among 75 affected patients, who were not obviously related probands with a positive family history, nine had missense mutations in SOD1. Four of the nine probands carry the same R115G mutation in exon 4 of the SOD1 gene. Genotyping with markers from the SOD1 locus revealed a common haplotype and shared allelic characteristics in these patients. These findings suggest that the R115G mutation in the German population originates from a common founder.

Mutations in SDHC cause autosomal dominant paraganglioma, type 3 (PGL3), and have to date been demonstrated in only one family. Here, we report on a novel mutation in a patient with a malignant, catecholamine-producing paraganglioma at the carotid bifurcation. The mutation is a G--T transversion at position +1 of intron 5 of the SDHC gene, leading to the deletion of exon 5 and a shift in the reading frame.

Nonchromaffin paragangliomas (PGLs) are usually benign, neural-crest-derived, slow-growing tumours of parasympathetic ganglia. Between 10% and 50% of cases are familial and are transmitted as autosomal dominant traits with incomplete and age-dependent penetrance.

Purpose: We have shown that DNA methylation of the PITX2 gene predicts risk of distant recurrence in steroid hormone receptor-positive, node-negative breast cancer. Here, we present results from a multicenter study investigating whether PITX2 and other candidate DNA methylation markers predict outcome in node-positive, estrogen receptor-positive, HER-2-negative breast cancer patients who received adjuvant anthracycline-based chemotherapy. Experimental Design: Using a microarray platform, we analyzed DNA methylation in regulatory regions of PITX2 and 60 additional candidate genes in 241 breast cancer specimens. Using Cox regression analysis, we assessed the predictive power of the individual marker/marker panel candidates. Clinical endpoints were time to distant metastasis, disease-free survival, and overall survival. A nested bootstrap/cross-validation strategy was applied to identify and validate marker panels. Results: DNA methylation of PITX2 and 14 other genes was correlated with clinical outcome. In multivariate models, each methylation marker added significant information to established clinical factors. A four-marker panel including PITX2, BMP4, FGF4, and C20orf55 was identified that resulted in improvement of outcome prediction compared with PITX2 alone. Conclusions: This study provides further evidence for the PITX2 biomarker, which has now been successfully confirmed to predict outcome among different breast cancer patient populations. We further identify new DNA methylation biomarkers, three of which can be combined into a panel with PITX2 to increase the outcome prediction performance in our anthracycline-treated primary breast cancer population. Our results show that a well-defined panel of DNA methylation markers enables outcome prediction in lymph node-positive, HER-2-negative breast cancer patients treated with anthracycline-based chemotherapy.

The embryonal carcinoma cell line P19 responds to treatment with retinoid acid by differentiation into neuronal cell types [2]. Using radioactively labeled cDNA derived from differentiating P19 cells we screened an adult mouse brain cDNA library and isolated a gene named shyc for selective hybridizing clone. The encoded protein did not reveal homology to any known protein. We used in situ hybridization on mouse embryonic and adult brain sections to study shyc expression. The developing and embryonic nervous system showed the most prominent hybridization signals. In the adult brain the olfactory pathway was marked by shyc expression.

Tetra-amelia is a rare human genetic disorder characterized by complete absence of all four limbs and other anomalies. We studied a consanguineous family with four affected fetuses displaying autosomal recessive tetra-amelia and craniofacial and urogenital defects. By homozygosity mapping, the disease locus was assigned to chromosome 17q21, with a maximum multipoint LOD score of 2.9 at markers D17S931, D17S1785, D17SS1827, and D17S1868. Further fine mapping defined a critical interval of approximately 8.9 Mb between D17S1299 and D17S797. We identified a homozygous nonsense mutation (Q83X) in the WNT3 gene in affected fetuses of the family. WNT3, a human homologue of the Drosophila wingless gene, encodes a member of the WNT family known to play key roles in embryonic development. The Q83X mutation truncates WNT3 at its amino terminus, suggesting that loss of function is the most likely cause of the disorder. Our findings contrast with the observation of early lethality in mice homozygous for null alleles of Wnt3. To our knowledge, this is the first report of a mutation in a WNT gene associated with a Mendelian disorder. The identification of a WNT3 mutation in tetra-amelia indicates that WNT3 is required at the earliest stages of human limb formation and for craniofacial and urogenital development.

We searched for novel genes as candidates of X-linked dystonia parkinsonism (XDP) in the critical interval of Xq13.1 that harbors the disease locus (DYT3). A gene, ACRC (acidic repeat containing), was discovered by a combination of in silico and "wet" experiments. ACRC is composed of at least 12 exons and 11 introns. It is expressed in all tissues tested, including skeletal muscle, liver, kidney, pancreas, heart, lung, and brain. Highest levels of expression are found in skeletal muscle. The ACRC protein is characterized by a previously undescribed acidic repeat tract of 21 units of 8–10 amino acids. The N-terminal portion of the protein is highly acidic (pI=3.2), and the C-terminal region is basic (pI=10.2). There are nuclear localization signals in its C-terminal portion. Extensive mutation analysis of the transcribed region of the gene, including intron-exon boundaries and the 5' and 3' untranslated intervals, did not reveal a mutation in XDP patients. Exclusion of a mutation in the transcribed portion of this and all other known genes within the DYT3 critical interval suggests that XDP is most likely caused by a mutation in a regulatory region of a gene within the critical interval, or by a structural rearrangement.

Paragangliomas (glomus tumors) are slowly growing, mostly benign tumors of the parasympathetic ganglia which most frequently occur in the head and neck region. Between 10% and 50% of cases are familial and follow an autosomal dominant mode of inheritance. The trait is maternally imprinted and exclusively transmitted through the paternal line. To date, two loci have been implicated in this disorder: one at 11q23 (PGL1), the other one at 11q13 (PGL2). We have analyzed a large German family with hereditary paraganglioma, but no evidence of maternal imprinting. By linkage analysis with markers flanking both PGL1 and PGL2, we demonstrate that the trait does not segregate with either of the loci at 11q. Our findings show that a third locus, PGL3, can be involved in hereditary paraganglioma.

X-linked dystonia-parkinsonism (XDP) is a recessive disorder characterized by generalized dystonia with some patients exhibiting parkinsonism. The disease gene, DYT3, is located between DXS453 (DXS993) and DXS559, and strongest linkage disequilibrium is found distal to DXS7117 and proximal to DXS559. We have isolated and analyzed four novel polymorphic markers between DXS7117 and DXS559 and, by haplotype analysis, have narrowed the candidate interval to

In the mammalian nervous system, the formation of myelin byglial cells enables a rapid saltatory impulse conduction. Myelina-tion depends on the coordinated expression of myelin-specificstructural proteins, based on interactions of axons with oligoden-drocytes in the central nervous system (CNS) and Schwann cells inthe peripheral nervous system (PNS). Mice with defects in myelinassembly comprise a large and genetically heterogeneous group ofspontaneous mutants (Nave 1994), which also serve as models forhuman neurological diseases (Snipes and Suter 1995). Several my-elin-deficient mice carry defects in genes yet unidentified.The murine autosomal mutation claw paw (genotype clp/clp)produces abnormalities of limb posture associated with a myelina-tion disorder of the peripheral nervous system, but not the centralnervous system (Henry et al. 1991). Peripheral nerve myelinationis greatly delayed, and the failure to myelinate results in the per-sistence of promyelin Schwann cell/axon units, involving predomi-nantly small-caliber fibers. The clinical abnormalities, which areobvious within 2 days after birth, include abnormally flexed andextended joints, most obvious in the forelimbs. In human, congen-ital joint contractures (arthrogryposis) may be a presenting featureof congenital hypomyelinating neuropathy (Boylan et al. 1992)and have been associated with mutations in the gene for the majorperipheral myelin protein P0 (Warner et al. 1996). Unlike patientswith arthrogryposis, however, clp/clp mice display nonfixed ab-normal limb postures in early life. Very severely affected mutantsdie within 2–3 days after birth, but those that survive the firstseveral weeks have a normal life span. The claw paw gene, whichis molecularly undefined, has been mapped by linkage analysisclose to the glucose phosphate isomerase-1 locus (Gpi-1)onmouse Chromosome (Chr) 7 (Henry et al. 1991).Myelin-associated glycoprotein (MAG) is a myelin-specificcell adhesion molecule of the immunoglobulin super-family ofmembrane proteins (Arquint et al. 1987; Lai et al. 1987). Themurine Mag gene has been mapped to mouse Chr (Barton et al.1987) where it is also closely linked to the Gpi-1locus. The MAGprotein is a minor constituent of CNS and PNS myelin, and itsdistribution at the glial-axonal interface suggests a specific role inthe early steps of axonal recognition and myelin formation.In myelinating glia, two MAG isoforms (termed S-MAG andL-MAG) are generated by alternative RNA splicing of exon 12,which carries an in-frame termination codon (Lai et al. 1987). Theresulting MAG-mRNAs encode a 67-kDa (S-MAG) and a 72-kDaisoform (L-MAG), which differ by the length of their carboxyltermini (Milner et al. 1990). In the rodent brain, these two MAGisoforms are developmentally regulated, with L-MAG being themajor neonatal form. In the peripheral nervous system, S-MAG isthe major form at all developmental stages.In an attempt to demonstrate the function of MAG in vivo, anull allele of the Mag gene has been created by gene targeting(Montag et al. 1994; Li et al. 1994). Surprisingly, MAG-deficientmouse mutants displayed no obvious developmental defects, ex-cept for subtle ultrastructural abnormalities of myelin, in markedcontrast to the dysmyelinated phenotype of claw paw, which isthus highly unlikely to be a functional Mag null allele. It remainsunknown which myelin proteins are able to compensate for theabsence of MAG in this null allele. In contrast, transgenic over-expression of the murine Mag gene may not be tolerated (M.Klugmann, A. Schneider, D. Montag, and K.-A. Nave, unpub-lished observation). However, even in mice with 8–10 transgeniccopies of the wild-type MAG gene, neurological deficits are rela-tively subtle and the clinical phenotype is transient and differentfrom that of claw paw mice. Nevertheless, MAG overexpression inmice suggested to us that alterations of this protein may result inaberrant gain-of-function effects. A well-documented example thatmissense mutations can underlie a severe dysmyelination (whereasthe complete deletion of the same gene is compatible with myelinassembly) is provided by the proteolipid protein (PLP) gene, en-coding the major component of CNS myelin (Klugmann et al.1997). Given the close linkage between Mag and clp (see below),Mag qualified as a candidate gene for the claw paw mutation:alterations of the MAG coding sequence could, for example, exerta misfolded protein with dominant-negative effects on other mem-brane proteins, yet without having the full penetrance of an auto-somal-dominant mutation. In fact, heterozygous claw paw miceare viable, but some myelin abnormalities have been described(Henry et al. 1991).A search of all linkage data available from the Mouse GenomeDatabase (Jackson Laboratory), comprising a total of 332 mice,did not show any recombination between Mag and Gpi-1(Rudert1992; Saunders 1990; Tong 1993; Fig. 1A). Since clp is alsoclosely linked to Gpi-1(Henry et al. 1991), the physical proximityof Mag and clp can be inferred. To test directly the possible in-volvement of MAG in the claw paw defect, we determined thecDNA-sequence of both MAG-isoforms in mice homozygous forthe clp mutation. Assuming that peripheral and central MAG pro-teins are molecularly indistinguishable, cDNA was reverse-transcribed from total brain RNA of a visibly affected mutant andan age-matched wild-type control. In independent amplifications,which followed standard procedures, three overlapping fragmentsof the MAG cDNA were generated, including 47 bp at the 58 endand 14 bp at the 38 end of the open reading frame (Fujita et al.1989), with different primer pairs corresponding to the knownexon sequences (Fig 1B). The PCR-generated products had theexpected sizes of 433 bp (with primer pair E3-AS6), 394 bp(primer pair S6-AS8), and 980 bp (primer pairS8-E13). RT-PCR

P19 embryonal carcinoma cells differentiate into neuroectodermal derivates upon aggregation and treatment with retinoic acid (RA). Such RA-induced P19 cells, concomitant with neuronal differentiation, produce the neuropeptide head activator (HA) and release HA into the culture medium. The amount of HA produced by such differentiating P19 cells is more than 100-fold higher than that of uninduced P19 cells. RA-induced P19 cells respond to HA treatment with a two-fold increase in total number of neuronally differentiating P19 progeny. As early response to HA, an increase in intracellular calcium concentration is observed.