Deeann Wallis

Mutations in the homeodomain of the human SIX3 gene cause holoprosencephaly

Holoprosencephaly (HPE) is a common, severe malformation of the brain that involves separation of the central nervous system into left and right halves. Mild HPE can consist of signs such as a single central incisor, hypotelorism, microcephaly, or other craniofacial findings that can be present with or without associated brain malformations. The aetiology of HPE is extremely heterogeneous, with the proposed participation of a minimum of 12 HPE-associated genetic loci as well as the causal involvement of specific teratogens acting at the earliest stages of neurulation. The HPE2 locus was recently characterized as a 1-Mb interval on human chromosome 2p21 that contained a gene associated with HPE. A minimal critical region was defined by a set of six overlapping deletions and three clustered translocations in HPE patients. We describe here the isolation and characterization of the human homeobox-containing SIX3 gene from the HPE2 minimal critical region (MCR). We show that at least 2 of the HPE-associated translocation breakpoints in 2p21 are less than 200 kb from the 5´ end of SIX3. Mutational analysis has identified four different mutations in the homeodomain of SIX3 that are predicted to interfere with transcriptional activation and are associated with HPE. We propose that SIX3 is the HPE2 gene, essential for the development of the anterior neural plate and eye in humans.

Mutations in holoprosencephaly

Holoprosencephaly (HPE) is the most common developmental defect of the forebrain and midface in humans. In holoprosencephaly the cerebral hemispheres of the brain fail to separate into distinct left and right hemispheres. This malformation is due to the improper specification and formation of the forebrain during early development. When one considers the great number and kinds of genetic interactions that must occur to properly pattern the developing forebrain, it is not surprising that HPE is extremely heterogeneous. In addition to teratogenic agents, several genes are implicated as the cause of HPE. At least 12 different loci have been associated with HPE and now several distinct human genes for holoprosencephaly have been identified. These genes include Sonic Hedgehog (SHH), ZIC2, SIX3, and TG‐interacting factor (TGIF). Here we present an overview of the presently known genes causing human holoprosencephaly. We discuss their functional role in development of the forebrain and summarize the mutations and polymorphisms that have been identified within them. Hum Mutat 16:99–108, 2000. Published 2000 Wiley‐Liss, Inc.

A common variant of the latrophilin 3 gene, LPHN3, confers susceptibility to ADHD and predicts effectiveness of stimulant medication

Attention-Deficit/Hyperactivity Disorder (ADHD) has a very high heritability (0.8), suggesting that about 80% of phenotypic variance is due to genetic factors. We used the integration of statistical and functional approaches to discover a novel gene that contributes to ADHD. For our statistical approach, we started with a linkage study based on large multigenerational families in a population isolate, followed by fine mapping of targeted regions using a family-based design. Family- and population-based association studies in five samples from disparate regions of the world were used for replication. Brain imaging studies were performed to evaluate gene function. The linkage study discovered a genome region harbored in the Latrophilin 3 gene (LPHN3). In the world-wide samples (total n=6360, with 2627 ADHD cases and 2531 controls) statistical association of LPHN3 and ADHD was confirmed. Functional studies revealed that LPHN3 variants are expressed in key brain regions related to attention and activity, affect metabolism in neural circuits implicated in ADHD, and are associated with response to stimulant medication. Linkage and replicated association of ADHD with a novel non-candidate gene (LPHN3) provide new insights into the genetics, neurobiology, and treatment of ADHD.

Review: Genetics of Attention Deficit/Hyperactivity Disorder

OBJECTIVE The intent of this review is to provide an overview for the practicing psychologist/psychiatrist regarding the complexities of and the most recent advances made in the study of the genetic basis of attention-deficit/hyperactivity disorder (ADHD). METHODS We review a variety of concepts including: (a) complexities involved in studying the genetics of ADHD, (b) evidence for a primarily genetic component of ADHD, (c) evidence suggesting that there are only a few genes with major effects contributing to ADHD, (d) identification of the best candidate genes, (e) linkage analysis for the identification of novel candidate genes, and (f) data on gene-environment interactions. RESULTS It is now generally accepted that ADHD has a biological and even primarily genetic basis. However, despite the identification of several candidate genes, none of them seems to have a substantial effect and the exact etiology underlying ADHD has remained elusive. Genome-wide linkage analysis can help in the identification of novel candidate genes. While several independent groups have initiated these studies, we await further details and specific genes from fine-mapping studies. Most recently, researchers have been trying to identify gene by environment interactions to help understand ADHD. Replication of positive findings will be essential in teasing out these combinatorial influences. CONCLUSIONS Ideally, one day specific genes with major effects and specific risk factors with which they interact will be identified and we will be able to implement personalized medicine. Knowledge of such genes will allow us to identify specific diagnostic biological markers. In addition, defining the target genes is the first step in developing novel drug therapies to treat the ADHD symptoms that lead to impairment. Furthermore, such markers could also identify at risk individuals at a younger age in order to implement treatments sooner to decrease the severity of ADHD symptoms or even to prevent future ADHD symptomatology.

Attention-Deficit/Hyperactivity Disorder and Comorbid Disruptive Behavior Disorders: Evidence of Pleiotropy and New Susceptibility Loci

BACKGROUND Attention-deficit/hyperactivity disorder (ADHD) comorbid with oppositional defiant disorder (ODD) or conduct disorder (CD) and substance abuse/dependence seems to represent a specific subset within the phenotypic ADHD spectrum. METHODS We applied complex segregation and linkage analyses in a set of multigenerational families densely segregating ADHD comorbid with ODD, CD, alcohol abuse/dependence, and nicotine dependence. RESULTS Our data suggest that ADHD cosegregates with disruptive behaviors as a unique, phenotypically variable trait as evidenced by highly significant pair-wise linkages among: ADHD and ODD (logarithm of odds [LOD]=14.19), ADHD and CD (LOD=5.34), ODD and CD (LOD=6.68), and CD and alcohol abuse/dependence (LOD=3.98). In addition to previously reported ADHD susceptibility loci, we found evidence of linkage for comorbid ADHD phenotypes to loci at 8q24, 2p21-22.3, 5p13.1-p13.3, 12p11.23-13.3, 8q15, and 14q21.1-22.2. These results were replicated with an affected status phenotype derived from latent class clusters. CONCLUSIONS Patterns of cosegregation of ADHD with comorbidities can inform our understanding of the inheritance patterns not only of ADHD but also of disruptive behavioral disorders and alcohol abuse/dependence. Refining the comorbid ADHD phenotype by determining the cosegregation profile of specific comorbidities might be a powerful tool for defining significant regions of linkage.

Initial characterization of mice null for Lphn3, a gene implicated in ADHD and addiction

The LPHN3 gene has been associated with both attention deficit-hyperactivity disorder (ADHD) and addiction, suggesting that it may play a role in the etiology of these disorders. Unfortunately, almost nothing is known about the normal functions of this gene, which has hampered understanding of its potential pathogenic role. To begin to characterize such normal functions, we utilized a gene-trap embryonic stem cell line to generate mice mutant for the Lphn3 gene. We evaluated differential gene expression in whole mouse brain between mutant and wild type male littermates at postnatal day 0 using TaqMan gene expression assays. Most notably, we found changes in dopamine and serotonin receptors and transporters (Dat1, Drd4, 5Htt, 5Ht2a), changes in neurotransmitter metabolism genes (Th, Gad1), as well as changes in neural developmental genes (Nurr, Ncam). When mice were examined at 4-6 weeks of age, null mutants showed increased levels of dopamine and serotonin in the dorsal striatum. Finally, null mutant mice had a hyperactive phenotype in the open field test, independent of sex, and were more sensitive to the locomotor stimulant effects of cocaine. Considered together, these results suggest that Lphn3 plays a role in development and/or regulation of monoamine signaling. Given the central role for monoamines in ADHD and addiction, it seems likely that the influence of LPHN3 genotype on these disorders is mediated through alterations in monoamine signaling.

Screening of human LPHN3 for variants with a potential impact on ADHD susceptibility

Attention deficit hyperactivity disorder (ADHD) is the most common behavioral disorder in childhood, and often has effects detectable into adulthood. Advances in genetic linkage and association analysis have begun to elucidate some of the genetic factors underlying this complex disorder. Recently, we identified LPHN3, a novel ADHD susceptibility gene harbored in 4q, and showed that a LPHN3 common haplotype confers susceptibility to ADHD and predicts effectiveness of stimulant medication. Here we present the mutational analysis of the entire coding region of LPHN3 in a cohort of 139 ADHD subjects and 52 controls from across the USA. We identified 21 variants, of which 14 have been reported and 7 are novel. These include 5 missense, 8 synonymous, and 8 intronic changes. Interestingly, neither susceptibility nor protective haplotype alleles are associated with obviously significant coding region changes, or canonical splice site alterations, suggesting that non‐coding variations determining the quantity and/or quality of LPHN3 isoforms are the likely contributors to this common behavioral disorder.

Additional EFNB1 mutations in craniofrontonasal syndrome

Deeann Wallis, Felicitas Lacbawan, Mahim Jain, Vazken M. Der Kaloustian, Carlos E. Steiner, John B. Moeschler, H. Wolfgang Losken, Ilkka I. Kaitila, Stephen Cantrell, Virginia K. Proud, John C. Carey, Donald W. Day, Dorit Lev, Ahmad S. Teebi, Luther K. Robinson, H. Eugene Hoyme, Nadia Al-Torki, Jacqueline Siegel-Bartelt, John B. Mulliken, Nathaniel H. Robin, Dolores Saavedra, Elaine H. Zackai, and Maximilian Muenke* Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland McGill University Health Center, Montreal, Canada Department of Medical Genetics, Unicamp, Campinas, Sao Paulo, Brazil Dartmouth Medical Center, Lebanon, New Hampshire University of Pittsburgh, Pittsburgh, Pennsylvania Haartman Institute, University of Helsinki, Helsinki, Finland New Jersey Dental School, Newark, New Jersey Children’s Hospital of the King’s Daughters, Norfolk, Virginia University of Utah Medical Center, Salt Lake City, Utah Private Practice, Hewitt, Texas Institute of Clinical Genetics, Wolfson Medical Genetics Center, Holon, Israel Weill Cornell Medical College, New York, New York Buffalo School of Medicine and Biomedical Sciences, Buffalo, New York Stanford University School of Medicine, Stanford, California Kuwait Medical Genetics Center, Sulibihkhat, Kuwait The Genetics Institute, Pasadena, California Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts University of Alabama, Birmingham, Alabama Hospital General Dr. Manuel Gea Gonzáles, Mexico City, Mexico Division of Human and Molecular Genetics, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania

Development of a Novel Lead that Targets M. tuberculosis Polyketide Synthase 13.

Summary Widespread resistance to first-line TB drugs is a major problem that will likely only be resolved through the development of new drugs with novel mechanisms of action. We have used structure-guided methods to develop a lead molecule that targets the thioesterase activity of polyketide synthase Pks13, an essential enzyme that forms mycolic acids, required for the cell wall of Mycobacterium tuberculosis. Our lead, TAM16, is a benzofuran class inhibitor of Pks13 with highly potent in vitro bactericidal activity against drug-susceptible and drug-resistant clinical isolates of M. tuberculosis. In multiple mouse models of TB infection, TAM16 showed in vivo efficacy equal to the first-line TB drug isoniazid, both as a monotherapy and in combination therapy with rifampicin. TAM16 has excellent pharmacological and safety profiles, and the frequency of resistance for TAM16 is ∼100-fold lower than INH, suggesting that it can be developed as a new antitubercular aimed at the acute infection. PaperClip

Detection of Salmonella enteritidis in equine feces using the polymerase chain reaction and genus-specific oligonucleotide primers

Salmonella was identified in feces from horses, using the polymerase chain reaction (PCR) and genus-specific oligonucleotide primers. Feces from healthy horses were determined to be culture negative and PCR negative for Salmonella. Fecal samples were inoculated with known numbers of colony-forming units (CFU) of S. enteritidis. The fecal samples were enriched overnight in tetrathionate broth, and then DNA was extracted and amplified by PCR using genus-specific primers. Sensitivity of the assay extended to 100 CFU Salmonella enteritidis/g feces; sensitivity of microbiologic culture with enrichment extended to 100 CFU Salmonella enteritidis/g feces. Feces that were not inoculated with S. enteritidis were negative by the PCR. Detection of salmonellae in feces was possible using the PCR within 24 hours from the time of submission of samples. Because samples were enriched, isolates were available for determining antibiograms and serologic grouping or typing.