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Characterization of a Novel Gene,PNUTL2,on Human Chromosome 17q22–q23 and Its Exclusion as the Meckel Syndrome Gene
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SHORT COMMUNICATION Characterization of a Novel Gene, PNUTL2, on Human Chromosome 17q22– q23 and Its Exclusion as the Meckel Syndrome Gene Paulina Paavola,* ,† ,1 Nina Horelli-Kuitunen,‡ Aarno Palotie,‡ and Leena Peltonen* ,† *Department of Human Molecular Genetics, National Public Health Institute, Helsinki, Finland; and †Department of Medical Genetics and ‡Department of Clinical Chemistry and Biomedicine and Laboratory Department of Helsinki University Central Hospital, University of Helsinki, Helsinki, Finland Received July 1, 1998; accepted September 28, 1998
We assigned two human expressed sequence tags (ESTs), WI-15444 and SGC32067, homologous to mouse brain protein h5, to the critical region for Meckel syndrome (MKS) on 17q22– q23. For the sequence analyses in MKS patients, we isolated the corresponding human gene, PNUTL2, by analyzing an Image cDNA clone that contained these ESTs. Based on sequence homologies, the gene belongs to an expanding family of GTP-binding proteins, septins, that are involved in cytokinesis. In Northern analysis, PNUTL2 is ubiquitously expressed as a 1.7-kb transcript in adult and fetal tissues with particularly high expression in the heart, liver, and adrenal gland. Mutation analysis using sequencing of RT-PCR products and Northern blot analysis in MKS patients exclude PNUTL2 as the gene for MKS. © 1999 Academic Press
Meckel syndrome (MKS; MIM 249000) is an autosomal recessive, congenital malformation syndrome characterized by a central nervous system malformation, polycystic kidneys with fibrotic changes of the liver, and in most cases polydactyly. We previously assigned the MKS locus to 17q21– q24 (5) and recently refined its localization between markers D17S1606 and D17S1604 (17q22– q23) based on novel recombinations and allelic association (Paavola et al., unpublished results; Fig. 1d). The two human ESTs SGC32067 (GenBank Accession No. T64878) and WI15444 (R44887), both homologous to mouse brain protein h5, have previously been roughly localized to the chromosomal region overlapping the MKS locus (Human transcript map: http://www.ncbi.nlm.nih.gov/ science96). We have now PCR-mapped these ESTs in the critical MKS region to YAC clones 791A5 and 925H8, to PAC clone 95i19, and to BAC clone 12j2 (Fig. 1d). Based on both physical position and assumed function, a human gene homologous to the mouse h5 gene, 1 To whom correspondence should be addressed. Telephone: 358 9 4744 8496. Fax: 358 9 4744 8480. E-mail: [email protected].
Genomics 55, 122–125 (1999) Article ID geno.1998.5612 0888-7543/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
which was reported to be expressed in specific regions of the brain (4), represented a good candidate gene for MKS. For mutation screening in MKS patients, we isolated the corresponding human gene by analyzing an Image cDNA clone, 33869 (Clone ID), the partial 39-sequence of which was represented by EST WI15444 and by SGC32067. We also used these EST sequences to perform a search in the TIGR database (http://www.tigr.org). A contig of overlapping ESTs (and the cDNA clones from which the partial sequences originated) for the 39-end of the gene was found, comprising 922 bp of sequence. Of the cDNA clones that represented the human h5 homolog, we chose the cDNA clone 33869 (GenBank Accession No. R44887), as a BLAST search with the mouse h5 gene showed us that a 59-EST (R24290) for 33869 contained the translation initiation site. The cDNA clone 33869 was obtained from Genome Systems Inc. (St. Louis, MO). We confirmed that the clone had the right insert by sequencing the ends of the insert with universal vector primers (M13F, M13R). The sequence obtained matched both the ESTs and the mouse h5 gene sequences. Furthermore, we were able to amplify the cDNA clone with the EST primers. In addition to ESTbased PCR, we confirmed the localization of the human homolog of mouse brain protein h5 by visual mapping. In the metaphase-FISH (3), the 33869 clone demonstrated a specific assignment to 17q22– q23 (Fig. 1a). Using the fiber-FISH with tyramide-based detection (3, 6), the cDNA was more precisely located to PAC 95i19 and BAC 12j2, which were also PCR-positive with the EST sequences (Fig. 1b; BAC 12j2 not shown). We sequenced the Image cDNA clone 33869, which was found to encompass both the complete putative coding region and the 39-untranslated region (UTR), which was predicted by comparison to the mouse nucleotide sequence. The 59-RACE method was used to extend the sequence further 59 upstream of the cDNA clone. A specific antisense primer covering the translation initiation site (59-tcc cag tga acg gtc cat gtc cca
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FIG. 1. (A) Initial chromosomal assignment of the PNUTL2 gene was confirmed by metaphase-FISH with a cDNA probe, 33869, to chromosome 17q22– q23 [detected by 40% (24/60) hybridization efficiency]. (B) A fiber-FISH image of the assignment of the PNUTL2 gene (cDNA, red) to a central position on the PAC clone 95i19 telomeric to the previously mapped PAC clone 53n20. (C) The line measurement option of the IPLab software makes it possible to determine precisely the increased signal intensity produced by the cDNA probe. The positive signal of the cDNA clone was detected as one or two signals (red) on top of the reference PAC clone 95i19 signal (green; ;165 kb). (D) Part of the physical contig covering the MKS region. l values, calculated using the DISLAMB program (9), for the polymorphic markers in the most critical region (Paavola et al., unpublished results) are shown in parentheses. SFRS1, serine–arginine-rich splicing factor 1; MPO, myeloperoxidase; n.s., not significant; -`, obligatory recombination.
cgc ttc-39) and a nested primer (59-cca gtg aac ggt cca tgt cc-39; also used in sequencing of the 59-RACE PCR product) were employed with Clontech human fetal brain Marathon Ready cDNA as the template, in accordance with the manufacturer’s instructions. We obtained 99 bp of sequence upstream of the putative translation initiation codon. The size of the cDNA obtained, 1763 bp, was in accordance with the transcript size of ;1.7 kb observed in Northern analysis (see below). The cDNA sequence (GenBank Accession No. AF073312) contained an open reading frame from nu-
cleotide (nt) 125 to 1561 capable of coding for a 55-kDa polypeptide comprising 478 amino acids. Computerbased analysis of the protein sequence revealed a GTPbinding domain in position 151–158. Database searches also revealed that this cDNA shows considerable sequence homology to an expanding family of genes coding for GTP-binding proteins. The greatest similarity at the protein level was observed with the mouse brain protein h5 (88% identity) and human Peanut-like protein 1 (PNUTL1) (76%), which is located in the critical region for DiGeorge syndrome (MIM
FIG. 2. (A) Multiple-tissue Northern blot analysis of PNUTL2. A 708-bp RT-PCR product was used as a probe. Blots 1 and 2 were Northern blots (Clontech) made from multiple tissue poly(A) RNAs. Blot 3 was made from poly(A) RNAs of a Meckel patient’s fibroblasts and control fetus brain. The same blots were rehybridized with b-actin cDNA. (B) The tissue distribution of PNUTL2 was studied using human RNA master blot. The blot has been normalized for eight housekeeping genes (Clontech).
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188400) on 22q11 (10). Less homologous were murine Diff6 protein (60%), Drosophila Septin1 (59%), the human NEDD5 homolog (59%), and human DIFF6 (50%). These proteins are all related to the yeast cell division cycle (CDC) proteins, also referred to as septins. Yeast and Drosophila septins have been reported to be involved in cytokinesis and development (1, 2). Our gene was given the name PNUTL2, for peanut-like (Drosophila) 2, due to its homology to PNUTL1. Using the PNUTL2 sequence, a homologous cDNA sequence was later identified in GenBank (Accession No. AF035811) which differed from our sequence in its 59-UTR and 39-UTR regions, and in G 1198, which was replaced by A. Nucleotides 1–25 of our sequence differed from nucleotides 6 –29 of AF035811, and RT-PCR experiments failed to produce any product with a forward primer consisting of the first 20 nucleotides of AF035811. In the 39-UTR region of our sequence, nt 1633 and nt 1635 were cytosines, whereas they are thymidines in AF035811. We analyzed the expression of PNUTL2 in both fetal and adult tissues. Human adult and fetal multipletissue Northern blots and human RNA master blot were obtained from Clontech (Palo Alto, CA) and hybridized in accordance with manufacturer’s instructions. A random prime-labeled 708-bp RT-PCR fragment of PNUTL2 was used as a probe in these experiments (primers: 59-ggc aga cac act gac acc tc-39 and 59-ctc tga gtc tct ggg cag tca-39). In Northern analysis, a single, approximately 1.7-kb transcript was observed in all the fetal and adult tissues analyzed, but less abundantly in adult lung and placenta (Fig. 2a). Human RNA master blot analysis revealed ubiquitous expression of the PNUTL2 gene. The most abundant expression was observed in adult heart, liver, and adrenal gland and in fetal heart, kidney, liver, and lung (Fig. 2b). We searched for mutations in MKS patients by sequencing the coding region of PNUTL2 in five Meckel fetuses (four homozygous for the common haplotype and one heterozygote for rare disease chromosomes; Paavola et al., unpublished results) as well as in a control fetus. Poly(A) RNAs were isolated from different tissues of MKS patients using FastTrack 2.0 Kit (Invitrogen) in accordance with the manufacturer’s instructions. Biotinylated RT-PCR products were sequenced by the solid-phase method (8). Two silent substitutions, which were not analyzed further, were found: G 11983 A (both CGG and CGA coding for arginine) and A 13213 G (both ACA and ACG coding for threonine). The reading frame was confirmed by its similarity to the mouse codons. At nucleotide position 1198, the MKS patient with the heterozygote haplotype was homozygous for A, and the four MKS patients with the homozygote haplotype and the control were homozygous for G. At nucleotide position 1321, the MKS patients with the homozygote haplotype were
homozygous for G, the heterozygote patient was homozygous for A, and the control was heterozygous A/G. No other nucleotide variations were found in the coding region of the PNUTL2 gene. For steady-state mRNA analyses in patients and controls, Northern blotting was performed using standard procedures (7), and hybridizations were carried out as described above. The results showed that PNUTL2 was expressed abundantly as a 1.7-kb transcript in all the MKS tissues studied (brain, liver, kidney, and fibroblasts) with no obvious difference in the transcript size level (also observed in RT-PCR experiments) between the patients and the controls (Fig. 2a). Consequently, mutations in the regulatory regions of PNUTL2 affecting the level of transcription are unlikely. These results provide evidence that the PNUTL2 gene is not affected in MKS, although as a developmental gene expressed in the brain it appeared to be a good functional and positional candidate for this lethal malformation syndrome. ACKNOWLEDGMENTS This work was financially supported by the Academy of Finland, the Research and Science Foundation of Farmos, and the Ulla Hjelt Fond of the Pediatric Research Foundation.
REFERENCES 1.
2.
3.
4. 5.
6.
7.
8.
9.
10.
Fares, H., Peifer, M., and Pringle, J. R. (1995). Localization and possible function of Drosophila septins. Mol. Cell Biol. 6: 1843– 1859. Hartwell, L. H. (1971). Genetic control of the cell division cycle in yeast. IV. Genes controlling bud emergence and cytokinesis. Exp. Cell Res. 69: 265–271. Heiskanen, M., Kallioniemi, O. P., and Palotie, A. (1996). FiberFISH: Experiences and a refined protocol. Genet. Anal. Biomol. Eng. 12: 179 –184. Kato, K. (1991). A collection of cDNA clones with specific expression patterns in mouse brain. Eur. J. Neurosci. 2: 704 –711. Paavola, P., Salonen, R., Weissenbach, J., and Peltonen, L. (1995). The locus for Meckel syndrome with multiple congenital anomalies maps to chromosome 17q21– q24. Nat. Genet. 11: 213–215. Raap, A. K., van de Corput, M. P. C., Vervenne, R. A. W., van Gijlswijk, R. P. M., Tanke, H. J., and Wiegant, J. (1995). Ultrasensitive FISH using oproxidase-mediated deposition of biotinor fluorechrome tyramides. Hum. Mol. Gen. 4: 529 –534. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Suomalainen, A., and Syva¨nen, A.-C. (1996). Affinity capture and solid-phase sequencing of biotinylated PCR products. In “Methods in Molecular Biology: PCR Sequencing Protocols” (R. Rapley, Ed.), pp. 67–72, Humana, Totowa, NJ. Terwilliger, J. D. (1995). A powerful likelihood method for the analysis of linkage disequilibrium between trait loci and one or more polymorphic marker loci. Am. J. Hum. Genet. 56: 777–787. Zieger, B., Hashimoto, Y., and Ware, J. (1997). Alternative expression of platelet glycoprotein Ibb mRNA from an adjacent 59 gene with an imperfect polyadenylation signal sequence. J. Clin. Invest. 99: 520 –525.
SHORT COMMUNICATION Characterization of a Novel Gene, PNUTL2, on Human Chromosome 17q22– q23 and Its Exclusion as the Meckel Syndrome Gene Paulina Paavola,* ,† ,1 Nina Horelli-Kuitunen,‡ Aarno Palotie,‡ and Leena Peltonen* ,† *Department of Human Molecular Genetics, National Public Health Institute, Helsinki, Finland; and †Department of Medical Genetics and ‡Department of Clinical Chemistry and Biomedicine and Laboratory Department of Helsinki University Central Hospital, University of Helsinki, Helsinki, Finland Received July 1, 1998; accepted September 28, 1998
We assigned two human expressed sequence tags (ESTs), WI-15444 and SGC32067, homologous to mouse brain protein h5, to the critical region for Meckel syndrome (MKS) on 17q22– q23. For the sequence analyses in MKS patients, we isolated the corresponding human gene, PNUTL2, by analyzing an Image cDNA clone that contained these ESTs. Based on sequence homologies, the gene belongs to an expanding family of GTP-binding proteins, septins, that are involved in cytokinesis. In Northern analysis, PNUTL2 is ubiquitously expressed as a 1.7-kb transcript in adult and fetal tissues with particularly high expression in the heart, liver, and adrenal gland. Mutation analysis using sequencing of RT-PCR products and Northern blot analysis in MKS patients exclude PNUTL2 as the gene for MKS. © 1999 Academic Press
Meckel syndrome (MKS; MIM 249000) is an autosomal recessive, congenital malformation syndrome characterized by a central nervous system malformation, polycystic kidneys with fibrotic changes of the liver, and in most cases polydactyly. We previously assigned the MKS locus to 17q21– q24 (5) and recently refined its localization between markers D17S1606 and D17S1604 (17q22– q23) based on novel recombinations and allelic association (Paavola et al., unpublished results; Fig. 1d). The two human ESTs SGC32067 (GenBank Accession No. T64878) and WI15444 (R44887), both homologous to mouse brain protein h5, have previously been roughly localized to the chromosomal region overlapping the MKS locus (Human transcript map: http://www.ncbi.nlm.nih.gov/ science96). We have now PCR-mapped these ESTs in the critical MKS region to YAC clones 791A5 and 925H8, to PAC clone 95i19, and to BAC clone 12j2 (Fig. 1d). Based on both physical position and assumed function, a human gene homologous to the mouse h5 gene, 1 To whom correspondence should be addressed. Telephone: 358 9 4744 8496. Fax: 358 9 4744 8480. E-mail: [email protected].
Genomics 55, 122–125 (1999) Article ID geno.1998.5612 0888-7543/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
which was reported to be expressed in specific regions of the brain (4), represented a good candidate gene for MKS. For mutation screening in MKS patients, we isolated the corresponding human gene by analyzing an Image cDNA clone, 33869 (Clone ID), the partial 39-sequence of which was represented by EST WI15444 and by SGC32067. We also used these EST sequences to perform a search in the TIGR database (http://www.tigr.org). A contig of overlapping ESTs (and the cDNA clones from which the partial sequences originated) for the 39-end of the gene was found, comprising 922 bp of sequence. Of the cDNA clones that represented the human h5 homolog, we chose the cDNA clone 33869 (GenBank Accession No. R44887), as a BLAST search with the mouse h5 gene showed us that a 59-EST (R24290) for 33869 contained the translation initiation site. The cDNA clone 33869 was obtained from Genome Systems Inc. (St. Louis, MO). We confirmed that the clone had the right insert by sequencing the ends of the insert with universal vector primers (M13F, M13R). The sequence obtained matched both the ESTs and the mouse h5 gene sequences. Furthermore, we were able to amplify the cDNA clone with the EST primers. In addition to ESTbased PCR, we confirmed the localization of the human homolog of mouse brain protein h5 by visual mapping. In the metaphase-FISH (3), the 33869 clone demonstrated a specific assignment to 17q22– q23 (Fig. 1a). Using the fiber-FISH with tyramide-based detection (3, 6), the cDNA was more precisely located to PAC 95i19 and BAC 12j2, which were also PCR-positive with the EST sequences (Fig. 1b; BAC 12j2 not shown). We sequenced the Image cDNA clone 33869, which was found to encompass both the complete putative coding region and the 39-untranslated region (UTR), which was predicted by comparison to the mouse nucleotide sequence. The 59-RACE method was used to extend the sequence further 59 upstream of the cDNA clone. A specific antisense primer covering the translation initiation site (59-tcc cag tga acg gtc cat gtc cca
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FIG. 1. (A) Initial chromosomal assignment of the PNUTL2 gene was confirmed by metaphase-FISH with a cDNA probe, 33869, to chromosome 17q22– q23 [detected by 40% (24/60) hybridization efficiency]. (B) A fiber-FISH image of the assignment of the PNUTL2 gene (cDNA, red) to a central position on the PAC clone 95i19 telomeric to the previously mapped PAC clone 53n20. (C) The line measurement option of the IPLab software makes it possible to determine precisely the increased signal intensity produced by the cDNA probe. The positive signal of the cDNA clone was detected as one or two signals (red) on top of the reference PAC clone 95i19 signal (green; ;165 kb). (D) Part of the physical contig covering the MKS region. l values, calculated using the DISLAMB program (9), for the polymorphic markers in the most critical region (Paavola et al., unpublished results) are shown in parentheses. SFRS1, serine–arginine-rich splicing factor 1; MPO, myeloperoxidase; n.s., not significant; -`, obligatory recombination.
cgc ttc-39) and a nested primer (59-cca gtg aac ggt cca tgt cc-39; also used in sequencing of the 59-RACE PCR product) were employed with Clontech human fetal brain Marathon Ready cDNA as the template, in accordance with the manufacturer’s instructions. We obtained 99 bp of sequence upstream of the putative translation initiation codon. The size of the cDNA obtained, 1763 bp, was in accordance with the transcript size of ;1.7 kb observed in Northern analysis (see below). The cDNA sequence (GenBank Accession No. AF073312) contained an open reading frame from nu-
cleotide (nt) 125 to 1561 capable of coding for a 55-kDa polypeptide comprising 478 amino acids. Computerbased analysis of the protein sequence revealed a GTPbinding domain in position 151–158. Database searches also revealed that this cDNA shows considerable sequence homology to an expanding family of genes coding for GTP-binding proteins. The greatest similarity at the protein level was observed with the mouse brain protein h5 (88% identity) and human Peanut-like protein 1 (PNUTL1) (76%), which is located in the critical region for DiGeorge syndrome (MIM
FIG. 2. (A) Multiple-tissue Northern blot analysis of PNUTL2. A 708-bp RT-PCR product was used as a probe. Blots 1 and 2 were Northern blots (Clontech) made from multiple tissue poly(A) RNAs. Blot 3 was made from poly(A) RNAs of a Meckel patient’s fibroblasts and control fetus brain. The same blots were rehybridized with b-actin cDNA. (B) The tissue distribution of PNUTL2 was studied using human RNA master blot. The blot has been normalized for eight housekeeping genes (Clontech).
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188400) on 22q11 (10). Less homologous were murine Diff6 protein (60%), Drosophila Septin1 (59%), the human NEDD5 homolog (59%), and human DIFF6 (50%). These proteins are all related to the yeast cell division cycle (CDC) proteins, also referred to as septins. Yeast and Drosophila septins have been reported to be involved in cytokinesis and development (1, 2). Our gene was given the name PNUTL2, for peanut-like (Drosophila) 2, due to its homology to PNUTL1. Using the PNUTL2 sequence, a homologous cDNA sequence was later identified in GenBank (Accession No. AF035811) which differed from our sequence in its 59-UTR and 39-UTR regions, and in G 1198, which was replaced by A. Nucleotides 1–25 of our sequence differed from nucleotides 6 –29 of AF035811, and RT-PCR experiments failed to produce any product with a forward primer consisting of the first 20 nucleotides of AF035811. In the 39-UTR region of our sequence, nt 1633 and nt 1635 were cytosines, whereas they are thymidines in AF035811. We analyzed the expression of PNUTL2 in both fetal and adult tissues. Human adult and fetal multipletissue Northern blots and human RNA master blot were obtained from Clontech (Palo Alto, CA) and hybridized in accordance with manufacturer’s instructions. A random prime-labeled 708-bp RT-PCR fragment of PNUTL2 was used as a probe in these experiments (primers: 59-ggc aga cac act gac acc tc-39 and 59-ctc tga gtc tct ggg cag tca-39). In Northern analysis, a single, approximately 1.7-kb transcript was observed in all the fetal and adult tissues analyzed, but less abundantly in adult lung and placenta (Fig. 2a). Human RNA master blot analysis revealed ubiquitous expression of the PNUTL2 gene. The most abundant expression was observed in adult heart, liver, and adrenal gland and in fetal heart, kidney, liver, and lung (Fig. 2b). We searched for mutations in MKS patients by sequencing the coding region of PNUTL2 in five Meckel fetuses (four homozygous for the common haplotype and one heterozygote for rare disease chromosomes; Paavola et al., unpublished results) as well as in a control fetus. Poly(A) RNAs were isolated from different tissues of MKS patients using FastTrack 2.0 Kit (Invitrogen) in accordance with the manufacturer’s instructions. Biotinylated RT-PCR products were sequenced by the solid-phase method (8). Two silent substitutions, which were not analyzed further, were found: G 11983 A (both CGG and CGA coding for arginine) and A 13213 G (both ACA and ACG coding for threonine). The reading frame was confirmed by its similarity to the mouse codons. At nucleotide position 1198, the MKS patient with the heterozygote haplotype was homozygous for A, and the four MKS patients with the homozygote haplotype and the control were homozygous for G. At nucleotide position 1321, the MKS patients with the homozygote haplotype were
homozygous for G, the heterozygote patient was homozygous for A, and the control was heterozygous A/G. No other nucleotide variations were found in the coding region of the PNUTL2 gene. For steady-state mRNA analyses in patients and controls, Northern blotting was performed using standard procedures (7), and hybridizations were carried out as described above. The results showed that PNUTL2 was expressed abundantly as a 1.7-kb transcript in all the MKS tissues studied (brain, liver, kidney, and fibroblasts) with no obvious difference in the transcript size level (also observed in RT-PCR experiments) between the patients and the controls (Fig. 2a). Consequently, mutations in the regulatory regions of PNUTL2 affecting the level of transcription are unlikely. These results provide evidence that the PNUTL2 gene is not affected in MKS, although as a developmental gene expressed in the brain it appeared to be a good functional and positional candidate for this lethal malformation syndrome. ACKNOWLEDGMENTS This work was financially supported by the Academy of Finland, the Research and Science Foundation of Farmos, and the Ulla Hjelt Fond of the Pediatric Research Foundation.
REFERENCES 1.
2.
3.
4. 5.
6.
7.
8.
9.
10.
Fares, H., Peifer, M., and Pringle, J. R. (1995). Localization and possible function of Drosophila septins. Mol. Cell Biol. 6: 1843– 1859. Hartwell, L. H. (1971). Genetic control of the cell division cycle in yeast. IV. Genes controlling bud emergence and cytokinesis. Exp. Cell Res. 69: 265–271. Heiskanen, M., Kallioniemi, O. P., and Palotie, A. (1996). FiberFISH: Experiences and a refined protocol. Genet. Anal. Biomol. Eng. 12: 179 –184. Kato, K. (1991). A collection of cDNA clones with specific expression patterns in mouse brain. Eur. J. Neurosci. 2: 704 –711. Paavola, P., Salonen, R., Weissenbach, J., and Peltonen, L. (1995). The locus for Meckel syndrome with multiple congenital anomalies maps to chromosome 17q21– q24. Nat. Genet. 11: 213–215. Raap, A. K., van de Corput, M. P. C., Vervenne, R. A. W., van Gijlswijk, R. P. M., Tanke, H. J., and Wiegant, J. (1995). Ultrasensitive FISH using oproxidase-mediated deposition of biotinor fluorechrome tyramides. Hum. Mol. Gen. 4: 529 –534. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Suomalainen, A., and Syva¨nen, A.-C. (1996). Affinity capture and solid-phase sequencing of biotinylated PCR products. In “Methods in Molecular Biology: PCR Sequencing Protocols” (R. Rapley, Ed.), pp. 67–72, Humana, Totowa, NJ. Terwilliger, J. D. (1995). A powerful likelihood method for the analysis of linkage disequilibrium between trait loci and one or more polymorphic marker loci. Am. J. Hum. Genet. 56: 777–787. Zieger, B., Hashimoto, Y., and Ware, J. (1997). Alternative expression of platelet glycoprotein Ibb mRNA from an adjacent 59 gene with an imperfect polyadenylation signal sequence. J. Clin. Invest. 99: 520 –525.