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.     

Genetic Linkage Mapping of the Dehydroepiandrosterone Sulfotransferase (STD) Gene on the Chromosome 19q13.3 Region

In the human liver and adrenal, there is a single hydroxysteroid sulfotransferase, which catalyzes the transformation of dehydroepiandrosterone to dehydroepiandrosterone sulfate, the most abundantly circulating steroid in humans, and also catalyzes the sulfation of a series of other 3β-hydroxysteroids as well as cholesterol. Dehydroepiandrosterone sulfate serves as precursor for the formation of active androgens and estrogens in several peripheral tissues, indicating that hydroxysteroid sulfotransferase plays a pivotal role in controlling the hormonal action of sex steroids by regulating their bioavailability. We recently elucidated the structure of the gene encoding hydroxysteroid sulfotransferase (STD), also designated dehydroepiandrosterone sulfotransferase, which spans 17 kb and contains six exons. The STD gene was preliminarily assigned to chromosome 19 by polymerase chain reaction (PCR) amplification of DNA from a panel of human/rodent somatic cell hybrids. To locate the STD gene, the novel biallelic polymorphism found in intron 2 was genotyped in eight CEPH reference families by direct sequencing of PCR products. Two-point linkage analysis was first performed between the latter polymorphism and chromosome 19 markers from Généthon and NIH/CEPH. The closest linkage was observed with D19S412 (Z_max= 9.23; θ_max0.038) and HRC (Z_max= 5.95; θ_max0.036), located on the 19q13.3 region. A framework map including six Généthon markers flanking the polymorphic STD gene was created by multipoint linkage analysis. Thereafter, a high-resolution genetic map of the region was constructed, yielding to the following order: qter–D19S414–D19S224–D19S420–D19S217–(APOC2–D19S412)–(STD–HRC)– KLK–D19S22–D19S180–PRKCG–D19S418–tel.     

The Gene for Human Glutaredoxin (GLRX) Is Localized to Human Chromosome 5q14

Glutaredoxin is a small protein (12 kDa) catalyzing glutathione-dependent disulfide oxidoreduction reactions in a coupled system with NADPH, GSH, and glutathione reductase. A cDNA encoding the human glutaredoxin gene (HGMW-approved symbol GLRX) has recently been isolated and cloned from a human fetal spleen cDNA library. The screening of a human genomic library in Charon 4A led to the identification of three genomic clones. Using fluorescencein situhybridization to metaphase chromosomes with one genomic clone as a probe, the human glutaredoxin gene was localized to chromosomal region 5q14. This localization at chromosome 5 was in agreement with the somatic cell hybrid analysis, using DNA from a human–hamster and a human–mouse hybrid panel and using a human glutaredoxin cDNA as a probe.     

Human Rod Monochromacy: Linkage Analysis and Mapping of a Cone Photoreceptor Expressed Candidate Gene on Chromosome 2q11

We have performed linkage analysis in eight families with rod monochromacy, an autosomal recessively inherited condition with complete color blindness. Significant linkage was found with markers located at the pericentromeric region of chromosome 2. A maximum lod score of 5.36 was obtained for marker D2S2333 at θ = 0.00. Mapping of meiotic breakpoints localized the disease gene between markers D2S2187 and D2S2229. Homozygosity for a number of subsequent markers indicating identity by descent was found in two families and provides evidence for a further refinement of the locus proximal to D2S373. This defines an interval of ≈3 cM covering theACHM2locus for rod monochromacy. Radiation hybrid mapping of theCNGA3gene encoding the α-subunit of the cGMP gated cation channel in human cone photoreceptors resulted in a maximum lod score of 16.1 with marker D2S2311 combined with a calculated physical distance of 6.19cR_10,000. Screening of the CEPH YAC library and subsequent STS mapping indicated the physical order cen–D2S2222–D2S2175–(D2S2187/D2S2311)–qtel ofmarkers on 2q11 and showed that theCNGA3gene maps most closely to D2S2187 and D2S2311. These data indicate that theCNGA3gene maps within the critical interval of theACHM2locus for rod monochromacy and thus is a candidate gene for this disease.     

Molecular characterization of a patient with del(1)(q23–q25)

Summary We report a patient (S.T.) with multiple congenital anomalies and developmental delay associated with an interstitial deletion of 1q23–1q25. Molecular analysis of the deletion was performed using DNA markers that map to 1q. Five DNA markers, MLAJ-1 (D1S61), CRI-L1054 (D1S42), HBI40 (D1S66), OS-6 (D1S75), and BH516 (D1S110), were demonstrated to be deleted. Informative polymorphisms demonstrated this to be a de novo deletion of the maternally derived chromosome. Deletion status was determined using restriction fragment length polymorphism (RFLP) analysis supplemented with densitometry in the experiments where RFLP analysis was not fully informative. Deletions were confirmed by Southern analysis using genomic DNA from a somatic cell hybrid retaining the del(1)(q23–q25) chromosome that was constructed from patient S.T. Flow karyotyping confirmed the deletion and estimated that the deletion encompassed 11,000–16,000 kb. The clinical and cytogenetic characteristics of S.T. are compared with those of ten previously described patients with monosomy 1q21–1q25.     

The murine homolog of the human breast and ovarian cancer susceptibility geneBrca1 maps to mouse chromosome 11D

The recently cloned human breast and ovarian cancer suseptibility gene,BRCA1, is located on human chromosome 17q21. We have isolated murine genomic clones containingBrca1 as a first step in generating a mouse model for the loss ofBRCA1 function. A mouse genomic library was screened using probes corresponding to exon 11 of the humanBRCA1 gene. Two overlapping mouse clones were identified that hybridized to humanBRCA1 exons 9–12. Sequence analysis of 1.4 kb of the region of these clones corresponding to part of human exon 11 revealed 72% nucleic acid identity but only 50% amino acid identity with the human gene. The longest of the mouseBrca1 genomic clones maps to chromosome 11D, as determined by two-color fluorescence in situ hybridization. The synteny to human chromosome 17 was confirmed by cohybridization with the mouse probe for the NF1-gene. This comparative study confirms that the relative location of theBRCA1 gene has been conserved between mice and humans.     

Four restriction fragment length polymorphisms revealed by probes from a single cosmid map to human chromosome 12q

Summary Human gene mapping would be greatly facilitated if marker loci with sufficient polymorphism information content were generally available. As a source of such markers, we have used cosmids from a human genomic library. We have used a rapid method for screening random cosmids to identify those homologous to genomic regions especially rich in restriction fragment length polymophisms (Litt and White 1985). This method allows whole cosmids to be used as probes against Southern transfers of genomic DNA; regions of cosmid probes homologous to repeated genomic sequences are rendered unable to anneal with Southern transfers by prerendered of the probes with a vast excess of non-radioactive genomic DNA. From one cosmid (C1-11) identified by this procedure, we have isolated four single-copy probes, each of which identifies a polymorphic locus. Despite the existence of some linkage disequilibrium in this system, the polymorthism information content was computed as 0.73. Using a somatic cell hybrid mapping panel, we have mapped probes from cosmid 1–11 to human chromosome 12q. Additionally, in situ hybridization of the whole cosmid to metaphase spreads allowed more precise assignment of the locus to the region 12cenq13. The locus revealed by probes from cosmid 1–11 has been designated D12S6.     

Direct regional assignment of the gene for vitamin D binding protein (Gc-globulin) to human chromosome 4q11-q13 and identification of an associated DNA polymorphism

Summary Using a characterized human vitamin D binding protein (DBP) cDNA probe and a panel of rodent X human somatic cell hybrids, we established the chromosomal location of the structural gene for DBP on human chromosome 4. In situ hybridization of ³H-labeled DBP cDNA to human metaphase chromosomes confirmed this assignment and allowed regional localization to bands 4q11–4q13. A restriction fragment length polymorphism associated with the DBP gene that should prove useful in future linkage studies was identified with the enzyme BamHI.     

Subregional localization of the chromosome 21 loci D21S24 and D21S26 using physical mapping techniques

Summary We were able to refine the chromosomal position of two existing marker loci, using an extended chromosome 21 somatic cell hybrid panel. The locus D21S26 mapped in the region 21q11.2–q21.1, and the locus D21S24 in 21q22.1–q22.2. Physical and genetic analysis indicated that D21S26 is tightly linked to D21S13 and D21S16, two markers previously linked to familial Alzheimer's disease.     

Chromosomal localization of ARSB,the gene for human N-acetylgalactosamine-4-sulphatase

Summary A deficiency of N-acetylgalactosamine-4-sulphatase (G4S, gene symbol ARSB), results in the accumulation of undegraded substrate and the lysosomal storage disorder, Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI). In situ hybridization using an ³H-labelled human G4S genomic DNA fragment to human metaphase chromosomes localized ARSB to chromosome 5q13–5q14. This location is consistent with, an refines, previous chromosomal assignments based on the expression of human G4S in somatic cell hybrids.     

Fine mapping of the 2p11 dyslexia locus and exclusion of TACR1 as a candidate gene

Developmental dyslexia, or reading disability, is a multigenic complex disease for which at least five loci, i.e. DYX1–3 and DYX5–6, have been clearly identified from the human genome. To date, DYX1C1 is the only dyslexia candidate gene cloned. We have previously reported linkage to 2p11 and 7q32 in 11 Finnish pedigrees. Here, we report the fine mapping of the approximately 40-cM linked region from chromosome 2 as we increased marker density to one per 1.8 cM. Linkage was supported with the highest NPL score of 3.0 (P=0.001) for marker D2S2216. Association analysis using the six pedigrees showing linkage pointed to marker D2S286/rs3220265 (P value <0.001) in the near vicinity of D2S2216. We went on to further characterise this ~15-cM candidate region (D2S2110-D2S2181) by adding six SNPs covering ~670 kb centred at D2S286/rs3220265. A haplotype pattern could no longer be observed in this region, which was therefore excluded from the candidate area. This also excluded the TACR1 (tachykinin receptor 1) gene, located at marker D2S286. The dyslexia candidate region on 2p11 is, therefore, now limited to the chromosomal area D2S2116-D2S2181, which is ~12 Mbp of human sequence and is at a distinct location from the previously reported DYX3 locus, raising the possibility of two distinct loci on chromosome 2p.H. Anthoni and P. Onkamo contributed equally to this work     

Chromosomal Localization and Genomic Organization of Genes Encoding Guanylyl Cyclase Receptors Expressed in Olfactory Sensory Neurons and Retina

We recently cloned three membrane guanylyl cyclases, designated GC-D, GC-E, and GC-F, from rat olfactory tissue and eye. Amino acid sequence homology suggests that they may compose a new gene subfamily of guanylyl cyclase receptors specifically expressed in sensory tissues. Their chromosomal localization was determined by mouse interspecific backcross analysis. The GC-D, GC-E, and GC-F genes (Gucy2d, Gucy2e,andGucy2f) are dispersed through the mouse genome in that they map to chromosomes 7, 11, and X, respectively. Close proximity of the mouse GC-D gene toOmp(olfactory marker protein) andHbb(hemoglobin β-chain complex) suggests that the human homolog gene maps to 11p15.4 or 11q13.4–q14.1. The human GC-F gene was localized to the long arm of chromosome Xq22 by fluorescencein situhybridization. The genomic organization of the mouse GC-E gene was determined and compared to other guanylyl cyclase genes. The mouse GC-D, GC-E, and GC-F genomic clones contain identical exon–intron boundaries within their extracellular and cytoplasmic domains, demonstrating the conservation of the gene structures. With respect to human genetic diseases, GC-E mapped to mouse chromosome 11 within a syntenic region on human chromosome 17p13 that has been linked with loci for autosomal dominant retinitis pigmentosa and Leber congenital amaurosis. No apparent disease loci have been yet linked to the locations of the GC-D or GC-F genes.     

Cloning of the Human Monocarboxylate Transporter MCT3 Gene: Localization to Chromosome 22q12.3–q13.2

Lactate transport across cell membranes is mediated by a family of proton-coupled monocarboxylate transporters (MCTs). The retinal pigment epithelium (RPE) expresses a unique member of this family, MCT3. A portion of the human MCT3 gene was cloned by polymerase chain reaction using primers designed from rat RPE MCT3 cDNA sequence. The human genomic sequence was used to design primers to clone human MCT3 cDNA and to identify a bacterial artificial chromosome clone containing the human MCT3 gene. The human MCT3 cDNA contained a 1512-nucleotide open reading frame with a deduced amino sequence 85% identical to rat MCT3. Comparison of the cDNA and genomic sequences revealed that the MCT3 gene was composed of five exons distributed over 5 kb of DNA. The exon–intron borders were conserved between the human and the chicken MCT3 genes. Using radiation hybrid mapping, the MCT3 gene was mapped to chromosome 22 between markers WI11639 and SGC30687. A search of chromosome 22 in the Sanger Centre database confirmed the location of the human MCT3 gene at 22q12.3–q13.2.     

Structure and Chromosomal Localization of the Human Salivary Mucin Gene, MUC7

We have isolated and characterized several MUC7 genomic clones encoding the human low-molecular-weight salivary mucin, MG2. The MUC7 gene spans ∼10.0 kb and comprises of three exons and two introns. Intron 1 is ∼1.7 kb long and is located in the 5′-untranslated region of the corresponding MUC7 cDNA. Intron 2 spans ∼6.0 kb and is located close to the boundary of the putative leader peptide and secreted protein. The entire region encoding the secreted peptide is located on exon 3, spanning ∼2.2 kb. The nucleotide sequence of sections of the MUC7 gene, including 1500 bp of the 5′-flanking region, was determined and analyzed for motifs identical or homologous to other known response elements. A modified RACE procedure was used to determine the 5′-end of the MUC7 mRNA. PCR, the human–hamster somatic cell hybrid panel PCRable DNAs kit, and anin situhybridization analysis on the complete metaphase chromosome spreads were used for the chromosomal localization of the MUC7 gene. It was mapped to chromosome 4q13–q21.     

The Gene Encoding Protein Kinase SEK1 Maps to Mouse Chromosome 11 and Human Chromosome 17

We report the mapping of the human and mouse genes encoding SEK1 (SAPK/ERK kinase-1), a newly identified protein kinase that is a potent physiological activator of the stress-activated protein kinases. The human SERK1 gene was assigned to human chromosome 17 using genomic DNAs from human–rodent somatic cell hybrid lines. A specific human PCR product was observed solely in the somatic cell line containing human chromosome 17. The mouseSerk1gene was mapped to chromosome 11, closely linked toD11Mit4,using genomic DNAs from a (C57BL/6J ×Mus spretus)F₁×M. spretusbackcross.     

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.