Disruption of sonic hedgehog signaling in Ellis-van Creveld dwarfism confers protection against bipolar affective disorder.

Ellis-van Creveld syndrome, an autosomal recessively inherited chondrodysplastic dwarfism, is frequent among Old Order Amish of Pennsylvania. Decades of longitudinal research on bipolar affective disorder (BPAD) revealed cosegregation of high numbers of EvC and Bipolar I (BPI) cases in several large Amish families descending from the same pioneer. Despite the high prevalence of both disorders in these families, no EvC individual has ever been reported with BPI. The proximity of the EVC gene to our previously reported chromosome 4p16 BPAD locus with protective alleles, coupled with detailed clinical observations that EvC and BPI do not occur in the same individuals, led us to hypothesize that the genetic defect causing EvC in the Amish confers protection from BPI. This hypothesis is supported by a significant negative association of these two disorders when contrasted with absence of disease (P=0.029, Fisher's exact test, two-sided, verified by permutation to estimate the null distribution of the test statistic). As homozygous Amish EVC mutations causing EvC dwarfism do so by disrupting sonic hedgehog (Shh) signaling, our data implicate Shh signaling in the underlying pathophysiology of BPAD. Understanding how disrupted Shh signaling protects against BPI could uncover variants in the Shh pathway that cause or increase risk for this and related mood disorders.

A new gene, EVC2, is mutated in Ellis-van Creveld syndrome.

Ellis-van Creveld syndrome (EvC; MIM 225500) is an autosomal recessive chondrodysplastic dwarfism. Thus far, the identified mutations in the EVC gene located on chromosome 4p16 have only accounted for illness in a small proportion of affected individuals. In this report we describe a novel gene, EVC2, that is mutated in an Ashkenazi individual with EvC syndrome. Our findings demonstrate for the first time that the heterogeneity observed in this disorder is not solely the result of mutations in a single gene.

Vasopressin V1b receptor knockout reduces aggressive behavior in male mice.

Increased aggression is commonly associated with many neurological and psychiatric disorders. Current treatments are largely empirical and are often accompanied by severe side effects, underscoring the need for a better understanding of the neural bases of aggression. Vasopressin, acting through its 1a receptor subtype, is known to affect aggressive behaviors. The vasopressin 1b receptor (V1bR) is also expressed in the brain, but has received much less attention due to a lack of specific drugs. Here we report that mice without the V1bR exhibit markedly reduced aggression and modestly impaired social recognition. By contrast, they perform normally in all the other behaviors that we have examined, such as sexual behavior, suggesting that reduced aggression and social memory are not simply the result of a global deficit in sensorimotor function or motivation. Fos-mapping within chemosensory responsive regions suggests that the behavioral deficits in V1bR knockout mice are not due to defects in detection and transmission of chemosensory signals to the brain. We suggest that V1bR antagonists could prove useful for treating aggressive behavior seen, for example, in dementias and traumatic brain injuries.

Absence of a significant linkage between Na(+),K(+)-ATPase subunit (ATP1A3 and ATP1B3) genotypes and bipolar affective disorder in the old-order Amish.

Previous studies provide evidence for a genetic component for susceptibility to bipolar affective disorder (BPAD) in the old-order Amish population. El-Mallakh and Wyatt [1995: Biol Psychiatry 37:235-244] have suggested that the Na(+),K(+)-ATPase may be a candidate gene for BPAD. This study examines the relationship between BPAD in the old-order Amish cohort and the Na(+),K(+)-ATPase alpha1 and beta3 subunit genes (ATP1A3, ATP1B3). A total of 166 sibling pairs were analyzed for linkage via nonparametric methods. Suggestive levels of statistical significance were not reached in any stratification model for affective illness. Overall, the results do not support linkage of bipolar disorder to the Na(+),K(+)-ATPase alpha subunit gene (ATP1A3) and beta subunit gene (ATP1B3) in these old-order Amish families and they show that these Na(+),K(+)-ATPase subunit genes are not major effect genes (>or=fourfold increased genetic risk of disease) for BPAD in the old-order Amish pedigrees. We cannot exclude other genetic variants of the Na(+),K(+)-ATPase hypothesis for BPAD, whereby other loci may modifying Na(+),K(+)-ATPase activity.

Childhood-onset schizophrenia/autistic disorder and t(1;7) reciprocal translocation: identification of a BAC contig spanning the translocation breakpoint at 7q21.

Childhood-onset schizophrenia (COS) is defined by the development of first psychotic symptoms by age 12. While recruiting patients with COS refractory to conventional treatments for a trial of atypical antipsychotic drugs, we discovered a unique case who has a familial t(1;7)(p22;q21) reciprocal translocation and onset of psychosis at age 9. The patient also has symptoms of autistic disorder, which are usually transient before the first psychotic episode among 40-50% of the childhood schizophrenics but has persisted in him even after the remission of psychosis. Cosegregating with the translocation, among the carriers in the family available for the study, are other significant psychopathologies, including alcohol/drug abuse, severe impulsivity, and paranoid personality and language delay. This case may provide a model for understanding the genetic basis of schizophrenia or autism. Here we report the progress toward characterization of genomic organization across the translocation breakpoint at 7q21. The polymorphic markers, D7S630/D7S492 and D7S2410/D7S646, immediately flanking the breakpoint, may be useful for further confirming the genetic linkage for schizophrenia or autism in this region. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:749-753, 2000. Published 2000 Wiley-Liss, Inc.

Human glucocerebrosidase: heterologous expression of active site mutants in murine null cells.

Using bioinformatics methods, we have previously identified Glu235 and Glu340 as the putative acid/base catalyst and nucleophile, respectively, in the active site of human glucocerebrosidase. Thus, we undertook site-directed mutagenesis studies to obtain experimental evidence supporting these predictions. Recombinant retroviruses were used to express wild-type and E235A and E340A mutant proteins in glucocerebrosidase-deficient murine cells. In contrast to wild-type enzyme, the mutants were found to be catalytically inactive. We also report the results of various studies (Western blotting, glycosylation analysis, subcellular fractionation, and confocal microscopy) indicating that the wild-type and mutant enzymes are identically processed and sorted to the lysosomes. Thus, enzymatic inactivity of the mutant proteins is not the result of incorrect folding/processing. These findings indicate that Glu235 plays a key role in the catalytic machinery of human glucocerebrosidase and may indeed be the acid/base catalyst. As concerns Glu340, the results both support our computer-based predictions and confirm, at the biological level, previous identification of Glu340 as the nucleophile by use of active site labeling techniques. Finally, our findings may help to better understand the molecular basis of Gaucher disease, the human lysosomal disease resulting from deficiency in glucocerebrosidase.

Glucosylsphingosine accumulation in mice and patients with type 2 Gaucher disease begins early in gestation.

Gaucher disease, the most common of the sphingolipidoses, results from the inherited deficiency of the enzyme glucocerebrosidase (EC 3.2.1.45). Although type 2 (acute neuronopathic) Gaucher disease is associated with rapidly progressive and fatal neurologic deterioration, the pathophysiologic mechanisms leading to the neurologic symptoms and early demise remain uncharacterized. While the pathology encountered in Gaucher disease has been attributed to glucocerebroside storage, glucosylsphingosine (Glc-sph), a cytotoxic compound, also accumulates in the tissues. Elevations of brain Glc-sph have been reported in patients with types 2 and 3 Gaucher disease. In this study, Glc-sph levels were measured using HPLC in tissues from mice with type 2 Gaucher disease created with a null glucocerebrosidase allele. Compared with unaffected littermates, homozygous mice with type 2 Gaucher disease had approximately a 100-fold elevation of Glc-sph in brain, as well as elevated levels in other tissues. This accumulation was detected in utero by E 13 and increased progressively throughout gestation. Similarly, elevated Glc-sph levels were seen in human fetuses with type 2 Gaucher disease, indicating that therapy initiated after birth may be too late to prevent the sequelae of progressive neurologic damage that begins early in gestation. These findings suggest that the accumulation of Glc-sph may be responsible for the rapid demise of mice with type 2 Gaucher disease and the devastating clinical course seen in patients with type 2 Gaucher disease.

Life-threatening splenic hemorrhage in two patients with Gaucher disease.

Massive splenomegaly is a frequent finding in patients with Gaucher disease, the most common of the sphingolipidoses. Even so, the risk for splenic rupture and intracapsular hemorrhage has not been emphasized due to the rarity of this occurrence and the fibrotic, rubbery consistency of splenic tissue in these patients. We report two adult patients with type 1 Gaucher disease who suffered life-threatening splenic bleeds that were not acutely diagnosed. Both patients ultimately required emergent splenectomies. Factors complicating the diagnosis of splenic hemorrhage in patients with Gaucher disease are discussed. Published 2000 Wiley-Liss, Inc.

The structure and expression of the human neuroligin-3 gene.

The neuroligins are a family of proteins that are thought to mediate cell to cell interactions between neurons. During the sequencing at an Xq13 locus associated with a mental retardation syndrome in some studies, we discovered a portion of the human orthologue of the rat neuroligin-3 gene. We now report the structure and the expression of that gene. The gene spans approximately 30kb and contains eight exons. Unlike the rat gene, it codes for at least two mRNAs and at least one of which is expressed outside the CNS. Interestingly, the putative promoter for the gene overlaps the last exon of the neighboring HOPA gene and is located less than 1kb from an OPA element in which a polymorphism associated with mental retardation is found. These findings suggest a possible role for the neuroligin gene in mental retardation and that the role of the gene in humans may differ from its role in rats.

Transgenic expression of green fluorescent protein in mouse oxytocin neurones.

Routine targeting of neurones for expression of exogenous genes would facilitate our ability to manipulate their internal milieu or functions, providing insight into physiology of neurones. The magnocellular neurones of the paraventricular and supraoptic nuclei of the hypothalamus have been the objects of limited success by this approach. Here we report on the placement of the enhanced green fluorescent protein (eGFP) coding sequence at various locations within an oxytocin transgene. Placement within the first exon yielded little to no expression, whereas placement in the third exon (as an in-frame fusion with the carboxyl terminus of the oxytocin preprohormone) resulted in cell-specific expression of eGFP in oxytocin neurones. Furthermore, placement of the eGFP sequence downstream of a picornavirus internal ribosomal entry site (IRES), also in the third exon, allowed expression of the eGFP as a separate protein. Other coding sequences should now be amenable to expression within oxytocin neurones to study their physiology.

The genomic structure and developmental expression patterns of the human OPA-containing gene (HOPA).

We determined the genomic organization of the human OPA-containing gene (HOPA) and characterized its developmental expression. The gene encoding HOPA, which contains a rare polymorphism tightly associated with non-specific mental retardation, is 25 kb in length and consists of 44 exons. A promoter scan analysis demonstrates two possible transcription initiation sites without TATA boxes upstream from the putative translation initiation start site. Several informative polymorphisms are evident in the sequence including a large pentanucleotide repeat. Northern blot analysis of the gene transcript and its murine orthologue, MOPA-1, demonstrates that only one transcript is expressed throughout the soma and the CNS, and that the transcript is highly expressed during early fetal development. We conclude that the delineation of the function of the HOPA gene locus merits further study.

Two novel polymorphic sequences in the glucocerebrosidase gene region enhance mutational screening and founder effect studies of patients with Gaucher disease.

Gaucher disease, an inherited glycolipid storage disorder, is caused by a deficiency of the catabolic enzyme glucocerebrosidase (EC 3.2.1.45). The gene for human glucocerebrosidase is located on chromosome 1q21 and has a highly homologous pseudogene situated 16 kb downstream. We report two novel polymorphic sequences in the glucocerebrosidase gene region: the first consists of a variable number of dinucleotide (CT) repeats located 3.2 kb upstream from the glucocerebrosidase gene, and the second is a tetranucleotide (AAAT) repeat found between the glucocerebrosidase gene and its pseudogene, 9.8 kb downstream from the functional gene. These polymorphic sequences, along with a previously reported PvuII polymorphism in intron 6 of the glucocerebrosidase gene, were analyzed in patients with Gaucher disease (n=106) and in two normal control populations, one of Ashkenazi Jewish ancestry (n=72) and the second comprising non-Jewish individuals (n=46). In these samples, strong linkage disequilibrium was found between mutations N370S, c.84-85insG, and R463C and specific haplotypes; no significant linkage disequilibrium was found when examining haplotypes of patients with the L444P mutation. Studies of these polymorphic sites in several instances also led to the recognition of genotyping errors and the identification of unusual recombinant alleles. These new polymorphic sites provide additional tools for mutational screening and founder effect studies of Gaucher disease.

Monitoring the CNS pathology in aspartylglucosaminuria mice.

Aspartylglucosaminuria (AGU) is a recessively inherited lysosomal storage disorder caused by the deficiency of the aspartylglucosaminidase (AGA) enzyme. The hallmark of AGU is slowly progressing mental retardation but the progression of brain pathology has remained uncharacterized in humans. Here we describe the long-term follow-up of mice carrying a targeted AGU-mutation in both alleles. Immunohistochemistry, histology, electron microscopy, quantitative magnetic resonance imaging (MRI) and behavioral studies were carried out to evaluate the CNS affection of the disease during development. The lysosomal storage vacuoles of the AGA -/- mice were most evident in central brain regions where MRI also revealed signs of brain atrophy similar to that seen in the older human patients. By immunohistochemistry and MRI examinations, a subtle delay of myelination was observed in AGA -/- mice. The life span of the AGA -/- mice was not shortened. Similar to the slow clinical course observed in human patients, the AGA -/- mice have behavioral symptoms that emerge at older age. Thus, the AGU knock-out mice represent an accurate model for AGU, both histopathologically and phenotypically.

A genome-wide search for chromosomal loci linked to mental health wellness in relatives at high risk for bipolar affective disorder among the Old Order Amish.

Bipolar affective disorder (BPAD; manic-depressive illness) is characterized by episodes of mania and/or hypomania interspersed with periods of depression. Compelling evidence supports a significant genetic component in the susceptibility to develop BPAD. To date, however, linkage studies have attempted only to identify chromosomal loci that cause or increase the risk of developing BPAD. To determine whether there could be protective alleles that prevent or reduce the risk of developing BPAD, similar to what is observed in other genetic disorders, we used mental health wellness (absence of any psychiatric disorder) as the phenotype in our genome-wide linkage scan of several large multigeneration Old Order Amish pedigrees exhibiting an extremely high incidence of BPAD. We have found strong evidence for a locus on chromosome 4p at D4S2949 (maximum GENEHUNTER-PLUS nonparametric linkage score = 4.05, P = 5. 22 x 10(-4); SIBPAL Pempirical value <3 x 10(-5)) and suggestive evidence for a locus on chromosome 4q at D4S397 (maximum GENEHUNTER-PLUS nonparametric linkage score = 3.29, P = 2.57 x 10(-3); SIBPAL Pempirical value <1 x 10(-3)) that are linked to mental health wellness. These findings are consistent with the hypothesis that certain alleles could prevent or modify the clinical manifestations of BPAD and perhaps other related affective disorders.

The characterization and sequence analysis of thirty CTG-repeat containing genomic cosmid clones.

We have systematically isolated and characterized DNA containing large CTG (n > 7) repeats from a human cosmid genomic DNA library. Using a CTG10 probe, more than 100 cosmid clones were identified, and 30 of these have been extensively characterized. The sequenced cosmids contain repeats that are between three and 19 perfect units (average 10 perfect repeats). The cosmids map to at least 12 different chromosomes. Sequence analysis of flanking regions suggests that more than one third of the repeats occur in exons, and many share strong sequence identity with databank sequences, including the gene involved in dentatorubral pallidoluysian atrophy (DRPLA). Genotyping of human DNA samples demonstrates that more than half of the repeats are polymorphic. This and similar collections of clones containing trinucleotide repeats should aid in the identification of genes that may contain expansions of trinucleotide repeats involved in human disease.

Lack of an association between a dopamine-4 receptor polymorphism and attention-deficit/hyperactivity disorder: genetic and brain morphometric analyses.

Although the etiology of attention-deficit/hyperactivity disorder (ADHD) is likely multifactorial, family, adoption, and twin studies suggest that genetic factors contribute significantly. Polymorphisms of the dopamine 4 receptor (DRD4) affect receptor binding, and one allele with seven tandem repeats in exon 3 (DRD4*7R) has been associated with ADHD. We examined this putative association in 41 children with severe ADHD and 56 healthy controls who were group matched for ethnicity and sex. The frequency of the DRD4*7R allele did not vary by diagnosis (0.220 vs 0.205 in patients and controls, respectively). Behavioral and brain anatomic MRI measures, previously found to discriminate patients from controls, did not differ significantly between subjects having and those lacking a DRD4*7R allele. These data do not support the reported association between DRD4*7R and the behavioral or brain morphometric phenotype associated with ADHD.

Association of an X-chromosome dodecamer insertional variant allele with mental retardation.

Mental retardation is a prominent feature of many neurodevelopmental syndromes. In an attempt to identify genetic components of these illnesses, we isolated and sequenced a large number of human genomic cosmid inserts containing large trinucleotide repeats. One of these cosmids, Cos-4, maps to the X-chromosome and contains the sequence of a 7.3-kb mRNA. Initial polymorphism analysis across a region of repetitive DNA in this gene revealed a rare 12-bp exonic variation (<< 1% in non-iII males) having an increased prevalence in non-Fragile X males with mental retardation (4%, P < 0.04, n = 81). This variant was not present in the highly conserved mouse homologue that has 100% amino acid identity to the human sequence near the polymorphism. Subsequent screening of two additional independent cohorts of non-Fragile X mentally retarded patients and ethnically matched controls demonstrated an even higher prevalence of the 12-bp variant in males with mental retardation (8%, P < 0.0003, n = 125, and 14%, P < 0.10, n = 36) vs the controls. Multivariate analysis was conducted in an effort to identify other phenotypic components in affected individuals, and the findings suggested an increased incidence of histories of hypothyroidism (P < 0.001) and treatment with antidepressants (P < 0.001). We conclude that the presence of this 12-bp variant confers significant susceptibility for mental retardation.