Chromosomal Localization in Mouse and Human of the Vasoactive Intestinal Peptide Receptor Type 2 Gene: A Possible Contributor to the Holoprosencephaly 3 Phenotype

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) have been shown to act on a wide range of tissue and cell types, both in the central nervous system and in the periphery. Two distinct receptors for VIP, the VIP receptor type 1 (VIPR1) and the VIP receptor type 2 (VIPR2), have recently been cloned, each of which binds PACAP and VIP with equal affinity. We report here the chromosomal mapping of the human and mouse VIPR2 genes by fluorescencein situhybridization. The VIPR2 gene maps to the human chromosomal region 7q36.3 and to the F2 region of mouse chromosome 12. Our localization of the human gene places it in the region where the locus for the craniofacial defect holoprosencephaly type 3 (HPE3) maps. Further mapping experiments, carried out on cell lines derived from patients with HPE or HPE microforms and associated 7q deletions, have led us to redefine the distal extent of the HPE3 minimal critical region, originally characterized by Gurrieriet al.(1993,Nature Genet.3: 247–251.) The VIPR2 gene lies within this new HPE3 minimal critical region. Our results suggest that deletion of the VIPR2 gene is not the sole factor responsible for the HPE3 phenotype. However, it is possible that monosomy at the VIPR2 locus may contribute to the phenotype observed in many cases of HPE3.     

Physical Mapping of the Holoprosencephaly Critical Region in 21q22.3, Exclusion of SIM2 as a Candidate Gene for Holoprosencephaly, and Mapping of SIM2 to a Region of Chromosome 21 Important for Down Syndrome

We set out to define the holoprosencephaly (HPE) critical region on chromosome 21 and also to determine whether there were human homologues of the Drosophila single-minded (sim) gene that might be involved in HPE. Analysis of somatic cell hybrid clones that contained rearranged chromosomes 21 from HPE patients defined the HPE minimal critical region in 21q22.3 as D21S113 to qter. We used established somatic cell hybrid mapping panels to map SIM2 to chromosome 21 within subbands q22.2-q22.3. Analysis of the HPE patient–derived somatic cell hybrids showed that SIM2 is not deleted in two of three patients and thus is not a likely candidate for HPE1, the HPE gene on chromosome 21. However, SIM2 does map within the Down syndrome critical region and thus is a candidate gene that might contribute to the Down syndrome phenotype.     

Truncating loss-of-function mutations of <Emphasis Type="Italic">DISP1</Emphasis> contribute to holoprosencephaly-like microform features in humans

Defective function of the Sonic Hedgehog (SHH) signaling pathway is the most frequent alteration underlying holoprosencephaly (HPE) or its various clinical microforms. We performed an extensive mutational analysis of the entire human DISP1 gene, required for secretion of all hedgehog ligand(s) and which maps to the HPE 10 locus of human chromosome 1q41, as a HPE candidate gene. Here, we describe two independent families with truncating mutations in human DISP1 that resemble the cardinal craniofacial and neuro-developmental features of a recently described microdeletion syndrome that includes this gene; therefore, we suggest that DISP1 function contributes substantially to both of these signs in humans. While these clinical features are consistent with common HPE microforms, especially those linked to defective signaling by Sonic Hedgehog, we have insufficient evidence so far that functionally abnormal DISP1 alleles will commonly contribute to the more severe features of typical HPE.     

Defining a holoprosencephaly locus on human chromosome 14q13 and characterization of potential candidate genes

Holoprosencephaly (HPE) is the most common developmental field defect in patterning of the human prosencephalon and associated craniofacial structures. The genetics is complex, with 12 loci defined on 11 chromosomes. We defined a locus for HPE (HPE8) on human chromosome 14q13 between markers D14S49 and AFM205XG5, by mapping deletion intervals of affected subjects with proximal chromosome 14q interstitial cytogenetic deletions. A 35-BAC contig was built by chromosome walking. By annotation of the 2.82-Mb minimal critical region, we identified 28 possible genes. Seven genes were expressed in human fetal brain: NPAS3, SNX6, C14ORF11, C14ORF10, PAX9, NKX2.1, and C14ORF19, the last an apparent gene fragment. Molecular embryology, animal modeling, and human mutation studies were reported elsewhere for PAX9 and NKX2.1. We focused on three genes, SNX6, NPAS3, and C14ORF11, as potential candidates for HPE. Genomic structure, human expression patterns, protein cellular localization, and embryonic expression patterns of orthologous murine genes were determined, showing that the three genes have properties similar to those of known HPE genes.     

Identification of Two Critically Deleted Regions within Chromosome Segment 7q35-q36 in EVI1 Deregulated Myeloid Leukemia Cell Lines

Chromosomal rearrangements involving the EVI1 proto-oncogene are a recurrent finding in myeloid leukemias and are indicative of a poor prognosis. Rearrangements of the EVI1 locus are often associated with monosomy 7 or cytogenetic detectable deletions of part of 7q. As EVI1 overexpression alone is not sufficient to induce leukemia, loss of a 7q tumour suppressor gene might be a required cooperating event. To test this hypothesis, we performed high-resolution array comparative genomic hybridization analysis of twelve EVI1 overexpressing patients and three EVI1 deregulated cell lines to search for 7q submicroscopic deletions. This analysis lead to the delineation of two critical regions, one of 0.39 Mb on 7q35 containing the CNTNAP2 gene and one of 1.33 Mb on chromosome bands 7q35–q36 comprising nine genes in EVI1 deregulated cell lines. These findings open the way to further studies aimed at identifying the culprit EVI1 implicated tumour suppressor genes on 7q.     

Holoprosencephaly: the Maastricht experience.

Holoprosencephaly (HPE) is a developmental field defect with impaired cleavage of the embryonic forebrain as the cardinal feature. The prevalence is about 1 in 11.000-20.000 in live births and 1 in 250 during embryogenesis. In most cases, craniofacial abnormalities are associated and reflect in 80% of cases the degree of severity. The severity is of marked variability and ranges from cyclopia to minimal craniofacial dysmorphism, such as mild microcephaly with a single central incisor. The etiology of HPE is very heterogeneous and comprises environmental factors (e.g. maternal diabetes) and genetic causes. Approximately 50% of HPE cases are associated with a cytogenetic abnormality (the most common of which is trisomy 13) or a monogenic syndrome. Based on recurrent cytogenetic abnormalities, there are at least 12 genetic loci that likely contain genes implicated in the pathogenesis of HPE. Currently, four human HPE genes are known: SHH at 7q36, ZIC2 at 13q32, SIX3 at 2p21 and TGIF at 18p11.3. Over the past 13 years, 16 patients with HPE have been observed at the Department of Clinical Genetics at Maastricht. Some of them are briefly presented in order to emphasize the spectral nature of HPE and the etiological heterogeneity. One patient appeared to have a partial 18p deletion due to a maternal cryptic translocation t(1:18) and, in addition, a SHH mutation. The mildest affected patient presented with microcephaly and a single maxillary incisor; she had a submicroscopic 7q deletion. Finally, we propose a protocol of etiological work-up of HPE cases.     

Physical mapping of the holoprosencephaly critical region in 18p11.3.

Holoprosencephaly (HPE) is a common developmental defect that results in a spectrum of craniofacial malformations. HPE is genetically heterogeneous, some cases being associated with deletions of the short arm of chromosome 18. In order to map the putative HPE gene located on 18p (HPE4) more precisely, six patients with various cytogenetic 18p deletions and clinical features of HPE have been characterized by using a combination of somatic cell hybrid analysis and FISH. By using a set of 27 chromosome 18p-specific markers, the deletion in each patient was characterized. The HPE minimal critical region on 18p was defined on a molecular level, localizing the HPE4 gene to 18p11.3.     

Structure of the human Lanosterol Synthase gene and its analysis as a candidate for holoprosencephaly (HPE1)

Holoprosencephaly (HPE) is the most common birth defect of the brain in humans. It involves various degrees of incomplete separation of the cerebrum into distinct left and right halves, and it is frequently accompanied by craniofacial anomalies. The HPE1 locus in human chromosome 21q22.3 is one of a dozen putative genetic loci implicated in causing HPE. Here, we report the complete gene structure of the human lanosterol synthase (LS) gene, which is located in this interval, and present its mutational analysis in HPE patients. We considered LS an excellent candidate HPE gene because of the requirement for cholesterol modification of the Sonic Hedgehog protein for the correct patterning activity of this HPE-associated protein. Despite extensive pedigree analysis of numerous polymorphisms, as well as complementation studies in yeast on one of the missense mutations, we find no evidence that the LS gene is in fact HPE1, implicating another gene located in this chromosomal region in HPE pathogenesis.     

Chromosomal Localization of the Human and Mouse Hyaluronan Synthase Genes

We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designatedHAS1, HAS2,andHAS3in humans andHas1, Has2,andHas3in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to theStreptococcus pyogenesHA synthase, HasA. Furthermore, expression of any oneHASgene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the threeHASgenes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes.HAS1was localized to the human chromosome 19q13.3–q13.4 boundary andHas1to mouse Chr 17.HAS2was localized to human chromosome 8q24.12 andHas2to mouse Chr 15.HAS3was localized to human chromosome 16q22.1 andHas3to mouse Chr 8. The map position forHAS1reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17.HAS2mapped outside the predicted critical region delineated for the Langer–Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome.     

A Novel Imprinted Gene, HYMAI, Is Located within an Imprinted Domain on Human Chromosome 6 Containing ZAC

Transient neonatal diabetes mellitus (TNDM) is a rare disease characterized by intrauterine growth retardation, dehydration, and failure to thrive due to a lack of normal insulin secretion. This disease is associated with paternal uniparental disomy or paternal duplication of chromosome 6, suggesting that the causative gene(s) for TNDM is imprinted. Recently, Gardner et al. (1999, J. Med. Genet. 36: 192–196) proposed that a candidate gene for TNDM lies within chromosome 6q24.1–q24.3. To find human imprinted genes, we performed a database search for EST sequences that mapped to this region, followed by RT-PCR analysis using monochromosomal hybrid cells with a human chromosome 6 of defined parental origin. Here we report the identification of a novel imprinted gene, HYMAI. This gene exhibits differential DNA methylation between the two parental alleles at an adjacent CpG island and is expressed only from the paternal chromosome. A previously characterized imprinted gene, ZAC/LOT1, is located 70 kb downstream of HYMAI and is also expressed only from the paternal allele. In the pancreas, both genes are moderately expressed. HYMAI and ZAC/LOT1 are therefore candidate genes involved in TNDM. Furthermore, the human chromosome 6q24 region is syntenic to mouse chromosome 10 and represents a novel imprinted domain.     

Clinical characterization of individuals with deletions of genes in holoprosencephaly pathways by aCGH refines the phenotypic spectrum of HPE

Holoprosencephaly (HPE) is the most common developmental forebrain anomaly in humans. Both environmental and genetic factors have been identified to play a role in the HPE phenotype. Previous studies of the genetic bases of HPE have taken a phenotype-first approach by examining groups of patients with HPE for specific mutations or deletions in known or candidate HPE genes. In this study, we characterized the presence or absence of HPE or a microform in 136 individuals in which microarray-based comparative genomic hybridization (aCGH) identified a deletion of one of 35 HPE loci. Frank holoprosencephaly was present in 11 individuals with deletions of one of the common HPE genes SHH, ZIC2, SIX3, and TGIF1, in one individual with a deletion of the HPE8 locus at 14q13, and in one individual with a deletion of FGF8, whereas deletions of other HPE loci and candidate genes (FOXA2 and LRP2) expressed microforms of HPE. Although individuals with deletions of other HPE candidates (DISP1, LSS, HHIP, SMO, BMP4, CDON, CDC42, ACVR2A, OTX2, and WIF1) had clinically significant features, none had frank HPE or a microform. A search for significant aCGH findings in individuals referred for testing for HPE revealed a novel association of a duplication involving GSK3B at 3q13.33 with HPE or a microform, seen in two unrelated individuals.     

The Mouse Region Syntenic for Human Spinal Muscular Atrophy Lies within theLgn1Critical Interval and Contains Multiple Copies ofNaipExon 5

Spinal muscular atrophy (SMA) is a relatively common, autosomal recessively inherited neurodegenerative disorder that maps to human chromosome 5q13. This region of the human genome has an intricate genomic structure that has complicated the evaluation of SMA candidate genes. We have chosen to study the mouse region syntenic for human SMA in the hope that the homologous mouse interval would contain the same genes as human 5q13 on a simpler genomic background. Here, we report the mapping of such a region to mouse chromosome 13 and to the critical interval forLgn1,a mouse locus responsible for modulating the intracellular replication and pathogenicity of the bacteriumLegionella pneumophila.We have generated a mouse YAC contig across theLgn1/Smainterval and have mapped the two flanking gene markers for the human SMA locus, MAP1B and CCNB1, onto this contig. In addition, we have localized the two SMA candidate genes, SMN and NAIP, to theLgn1critical region, making these two genes candidates for theLgn1phenotype. Upon subcloning of the YAC contig into P1s and BACs, we have detected a large, low copy number repeat that contains at least one copy ofNaipexon 5. Identification of theLgn1gene will either provide a novel function for SMN or NAIP or reveal the existence of another, yet uncharacterized gene in the SMA critical region. Mutations in such a gene might help to explain some of the phenotypic variability among the human SMAs.     

Chromosomal map of human brain malformations

The etiology of most central nervous system (CNS) malformations remains unknown. We have utilized the fact that autosomal chromosome aberrations are commonly associated with CNS malformations to identify new causative gene loci. The human cytogenetic database, a computerized catalog of the clinical phenotypes associated with cytogenetically detectable human chromosome aberrations, was used to identify patients with 14 selected brain malformations including 541 with deletions, and 290 carrying duplications. These cases were used to develop an autosomal deletion and duplication map consisting of 67 different deleted malformation associated bands (MABs) in 55 regions and 88 different duplicated MABs in 36 regions; 31 of the deleted and 8 duplicated MABs were highly significantly associated (P < 0.001). All holoprosencephaly MABs found in the database contained a known HPE gene providing some level of validation for the approach. Significantly associated MABs are discussed for each malformation together with the published data about known disease-causing genes and reported malformation-associated loci, as well as the limitations of the proposed approach.     

Mapping the human acetylcholinesterase gene to chromosome 7q22 by fluorescent in situ hybridization coupled with selective PCR amplification from a somatic hybrid cell panel and chromosome-sorted DNA libraries

To establish the chromosomal location of the human ACHE gene encoding the acetylcholine hydrolyzing enzyme acetylcholinesterase (ACHE, acetylcholine acetylhydrolase, E.C. 3.1.1.7), a human-specific polymerase chain reaction (PCR) procedure that supports the selective amplification of ACHE DNA fragments from human genomic DNA was employed with 19 human-hamster somatic cell hybrids carrying one or more human chromosomes. Informative ACHE-specific PCR fragments were produced from two cell lines, both of which include human chromosome 7, but not with DNA from 17 cell hybrids carrying various combinations of all human chromosomes other than 7. Fluorescent in situ hybridization of biotinylated ACHE DNA with metaphase chromosomes from human peripheral blood lymphocytes revealed prominent labeling on the 7q22 position. Therefore, further tests were performed to confirm the chromosome 7 location. DNA samples from the two cell lines including chromosome 7 and the ACHE gene were positive with PCR primers informative for the human cystic fibrosis CFTR gene, known to reside at the 7q31.1 position, but negative for the ACHE-related butyrylcholinesterase (BCHE, acylcholine acylhydrolase, E.C. 3.1.1.8) gene, mapped at the 3q26-ter position, confirming that these lines contain chromosome 7 but not chromosome 3. In contrast, three other cell lines including chromosome 3, but not 7, were BCHE-positive and ACHE-negative. In addition, genomic DNA from a sorted chromosome 7 library supported the production of ACHE- but not BCHE-specific PCR products, whereas with DNA from a sorted chromosome 3 library, the BCHE but not the ACHE fragment was amplified. These findings assign the human ACHE gene to a single locus on chromosome 7q22 and should assist in establishing linkage between the in vivo amplification of the ACHE gene in ovarian tumors and leukemias and the phenomenon of tumor-related breakage in the long arm of chromosome 7.     

Shh and Gli3 regulate formation of the telencephalic-diencephalic junction and suppress an isthmus-like signaling source in the forebrain

In human holoprosencephaly (HPE), the forebrain does not separate fully into two hemispheres. Further, the border between the telencephalon and diencephalon, the telencephalic/diencephalic junction (TDJ), is often indistinct, and the ventricular system can be blocked at the third ventricle, creating a forebrain ‘holosphere’. Mice deficient in Sonic Hedgehog (Shh) have previously been described to show HPE and associated cyclopia. Here we report that the third ventricle is blocked in Shh null mutants, similar to human HPE, and that characteristic telencephalic and diencephalic signaling centers, the cortical hem and zona limitans intrathalamica (ZLI), are merged, obliterating the TDJ. The resulting forebrain holosphere comprises Foxg1-positive telencephalic- and Foxg1-negative diencephalic territories. Loss of one functional copy of Gli3 in Shh nulls rescues ventricular collapse and substantially restores the TDJ. Characteristic regional gene expression patterns are rescued on the telencephalic side of the TDJ but not in the diencephalon.Further analysis of compound Shh;Gli3 mutants revealed an unexpected type of signaling center deregulation. In Shh;Gli3 mutants, adjacent rings of Fgf8 and Wnt3a expression are induced in the diencephalon at the ZLI, reminiscent of the Fgf8/Wnt1-expressing isthmic organizer. Neither Shh nor Gli3 single mutants show this forebrain double ring of Fgf/Wnt expression; thus both Shh and Gli3 are independently required to suppress it. Adjacent tissue is not respecified to a midbrain/hindbrain fate, but shows overgrowth, consistent with ectopic mitogen expression.Our observations indicate that the separation of the telencephalon and diencephalon depends on interactions between Shh and Gli3, and, moreover, demonstrate that both Shh and Gli3 suppress a potential Fgf/Wnt signaling source in the forebrain. That optional signaling centers are actively repressed in normal development is a striking new insight into the processes of vertebrate brain development.     

Autistic Disorder and Chromosome 15q11–q13: Construction and Analysis of a BAC/PAC Contig

Autistic disorder (AD) is a neurodevelopmental disorder that affects approximately 2–10/10,000 individuals. Chromosome 15q11–q13 has been implicated in the genetic etiology of AD based on (1) cytogenetic abnormalities; (2) increased recombination frequency in this region in AD versus non-AD families; (3) suggested linkage with markers D15S156, D15S219, and D15S217; and (4) evidence for significant association with polymorphisms in the γ-aminobutyric acid receptor subunit B3 gene (GABRB3). To isolate the putative 15q11–q13 candidate AD gene, a genomic contig and physical map of the approximately 1.2-Mb region from the GABA receptor gene cluster to the OCA2 locus was generated. Twenty-one bacterial artificial chromosome (BAC) clones, 32 P1-derived artificial chromosome (PAC) clones, and 2 P1 clones have been isolated using the markers D15S540, GABRB3, GABRA5, GABRG3, D15S822, and D15S217, as well as 34 novel markers developed from the end sequences of BAC/PAC clones. In contrast to previous findings, the markers D15S822 and D15S975 have been localized within the GABRG3 gene, which we have shown to be approximately 250 kb in size. NotI and numerous EagI restriction enzyme cut sites were identified in this region. The BAC/PAC genomic contig can be utilized for the study of genomic structure and the identification and characterization of genes and their methylation status in this autism candidate gene region on human chromosome 15q11–q13.     

Identification of the Human βA2 Crystallin Gene (CRYBA2): Localization of the Gene on Human Chromosome 2 and of the Homologous Gene on Mouse Chromosome 1

By using primers synthesized on the basis of the bovine βA2 crystallin gene sequence, we amplified exons 5 and 6 of the human gene (CRYBA2). CRYBA2 was assigned to human chromosome 2 by concordance analysis in human × rodent somatic cell hybrids using the amplified PCR products as probe. Regional localization to 2q34-q36 was established by hybridizing the CRYBA2 probe to microcell and radiation hybrids containing defined fragments of chromosome 2 as the only human contribution. The CRYBA2 probe was also used to localize, by interspecific backcross mapping, the mouse gene (Cryba2) to the central portion of chromosome 1 in a region of known human chromosome 2 homology. Finally, we demonstrate that in both species the βA2 crystallin gene is linked but separable from the γA crystallin gene. The βA2 crystallin gene is a candidate gene for human and mouse hereditary cataract.     

Control of tissue culture response in wheat (Triticum aestivum L.)

Summary The ability of immature embryos of wheat (Triticum aestivum L.) to respond to tissue culture has been shown to involve the group 2 chromosomes. The available group 2 ditelosomic and nullisomic-tetrasomic lines of Chinese Spring wheat were used to determine the chromosome arm location and chromosome dosage effect associated with the expression of tissue culture response (TCR). Significant differences were found between the aneuploid lines and the euploid control for the expression of both regenerable callus formation and callus growth rate. A model is proposed suggesting that a major TCR gene is located on 2DL and that 2AL and 2BS possess minor TCR genes. Furthermore, a major regulatory gene controlling the expression of TCR genes may be located on chromosome 2BL.