American Journal of Medical Genetics 109:121–124 (2002)

Refinement of the Genomic Structure of STX1A and Mutation Analysis in Nondeletion Patients

Yuan-Qing Wu,1 Bassem A. Bejjani,1 Lap-Chee Tsui,2,3 Ariane Mandel,4 Lucy R. Osborne,4 and Lisa G. Shaffer1* 1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 2Department of Genetics and Genomic Biology, The Hospital for Sick Children, Toronto, Ontario, Canada 3Department of Molecular and Medical Genetics, The University of Toronto, Ontario, Canada 4Department of Medicine, The University of Toronto, Ontario, Canada

Williams syndrome (WS) is a contiguous INTRODUCTION deletion disorder in which the com- Williams syndrome (WS) is a neurodevelopmental monly deleted region contains at least 17 disorder characterized by mental disability with unique . One of these genes, 1A cognitive and personality profiles, distinctive facial (STX1A), codes for a that is highly features, vascular and abnormalities, expressed in the and is and infantile hypercalcemia [Francke, 1999; Osborne, essential for the docking of synaptic vesicles 1999]. WS is a contiguous gene deletion disorder in- with the presynaptic plasma membrane. volving at least 17 genes within a commonly deleted In this study, we refine the complete geno- region on 7q11.23. Hemizygosity for elas- mic structure of the human STX1A gene tin has been shown to be responsible for several features by direct sequencing and primer walking of WS, such as supravalvular aortic stenosis and pos- of bacterial artificial chromosome (BAC) sibly the connective tissue abnormalities [Ewart clones and show that STX1A contains at et al., 1993; Li et al., 1997; Tassabehji et al., 1997; least 10 exons and 9 introns. The length of Tassabehji et al., 1998], but no other gene has been exons range from 27 bp to 138 bp and all definitely implicated in the WS phenotype. splice sites conform to the GT-AG rule. Syntaxin (STX1A) was originally described as a neu- Investigation of the STX1A gene sequence ronal-specific protein that co-immunoprecipitated with in five WS patients without detectable dele- the tions did not identify any point mutations. [Bennett et al., 1992; Yoshida et al., 1992]. Described Although the regulatory elements that con- as an integral membrane protein, STX1A was localized trol STX1A transcription were not exam- to the plasma membrane of and presynaptic ined, these results do not support a role for terminals in the nervous system [Inoue et al., 1992; Koh STX1A in the WS phenotype. et al., 1993; Sollner et al., 1993]. STX1A is found almost ß 2002 Wiley-Liss, Inc. exclusively in , where it is a component of KEY WORDS: Williams syndrome; STX1A; the pre-assembled vesicle docking and genomic structure; point machinery which is essential for mutation release [Bennett et al., 1993]. We had previously mapped human STX1A to within the common WS deletion region [Osborne et al., 1997] and determined that it contained at least 7 exons [Osborne et al., 1997, GenBank Accession numbers U87310–U87314], unlike the Syntaxin 1a, which has only a single exon [Schulze et al., 1995]. Because STX1A is a neuronally expressed gene that is located within the *Correspondence to: Dr. Lisa G. Shaffer, Department of common WS deletion region [Nakayama et al., 1997], Molecular and Human Genetics, Baylor College of Medicine, we hypothesized that STX1A may have a role in certain One Baylor Plaza, Room S801, Houston, TX 77030. behavioral characteristics observed in individuals with E-mail: [email protected] WS [Osborne et al., 1997]. Received 27 April 2001; Accepted 23 November 2001 The identification of individuals with features of WS DOI 10.1002/ajmg.10321 and mutations in a specific gene from the common WS

ß 2002 Wiley-Liss, Inc. 122 Wu et al. deletion region would firmly implicate that gene in the respectively, and he has characteristic facial features, pathophysiology of WS. To facilitate the evaluation of with prominent periorbital tissue, anteverted nares, the role of STX1A in WS, we have refined its genomic prominent lips with a long philtrum, and a large- structure and used this information to develop oligo- appearing mouth. A geneticist gave him the clinical nucleotide primers suitable for mutation analysis. We diagnosis of WS when he was a toddler but he was not have then proceeded to analyze the STX1A gene in seen in the Texas Children’s Genetics Clinic until age several individuals who have clinical WS but do not 12 years. A cardiac echocardiogram, serum , a harbor the common deletion. chromosome analysis, and a FISH study with a probe containing the elastin gene were normal. MATERIALS AND METHODS Prior to entering the study, molecular analyses of the WS region was performed on all individuals, using 10 Genomic Screening polymorphic markers within the common deletion In order to determine the complete genomic structure region and FISH analyses with a ELN-50 probe, LIMK1 of STX1A, a human genomic bacterial artificial chro- probe [Wu et al., 1998] and a STX1A probe [Osborne mosome (BAC) library (Research Genetics) was screen- et al., 1997]. No deletions were detected in any of the ed by PCR using primers designed from the published five study subjects. STX1A exon 5 DNA sequence (Table I, newly desig- nated exon 8 primer set). Two positive human BAC Point Mutation Analysis clones, 137N23 and 137N19, were isolated. Fluores- Each STX1A exon, including the exon-intron bound- cence in situ hybridization (FISH) was performed to aries, was amplified from genomic DNA from the five confirm that the clones mapped to 7q11.23 (data not WS individuals and from unrelated controls. The pro- shown). To confirm the exon-intron boundaries of the ducts were purified and sequenced as described above. STX1A gene, PCR primers were designed using the previously published sequence as a starting point. Products from both BACs and total human genomic RESULTS AND DISCUSSION DNA were amplified according to a published protocol [Wu et al., 1998], sized by agarose gel electrophoresis, Complete Genomic Structure purified by GeneClean Kit (Bio 101), and sequenced Based on the published sequence, amplification directly in the Baylor College of Medicine sequencing products of the expected size were found for exons 2, facility. 5, 6, and 7, but larger products were obtained for exons 1, 3, and 4. Using primer walking in the BAC clones to define the boundaries of exons 1 (STX1A-OF AAG GAC WS Patients CGA ACC CAG GAG; STX1A-OR CTC CTG GGT TCG In order to attempt to implicate STX1A in the fea- GTC CTT), 3 (STX1A-gap3 CTA TCC ACC TTC CCA tures of WS, we chose five unrelated individuals with CAT CC) and 4 (STX1A-gap4 CAC CAC ACA GCG TCA clinical features of WS and no identifiable deletion in CAG), we found that each could be separated into two the WS critical region for STX1A mutation screening. distinct exons. Primers were subsequently designed for All individuals met the clinical criteria for WS by the amplification of the newly defined exons and are history and physical findings, but had normal karyo- shown in Table I. Ten exons, ranging in size from 27 to types. Four of the individuals had been reported 138 bp, and the complete genomic sequence, spanning previously [Nickerson et al., 1995; Wu et al., 1998] exons 4 to 7, have been elucidated. Exon-intron boun- and the other was newly enrolled for the present study daries for exons 1, 2, 4, 5, 6, and 7 have been newly (Fig. 1). This individual was a 14-year-old Hispanic defined with GenBank accession numbers: AF297001, male with several clinical features suggestive of WS, AF297002, and AF297003, and the boundaries for including developmental delay, mild hyperactivity and exons 3, 8, 9, and 10 were described previously extreme sociability. His head circumference, height, [accession number: U87311, U87314, U87315, Osborne and weight are at the 5th, 35th, and 25th centile, et al., 1997]. Table II summarizes the splice junctions

TABLE I. PCR Primers for Mutation Analysis of the Human STX1A Gene (50 to 30)

Annealing Exon Forward Reverse (8C)

1 GGCGCGTTGGCGCTGTCG GTCGCGCATGCGGGGCTCAC 68 2 AGGTGGGTCCTGTACATACGTCC CCTCAAAACGGTTCATTCGTTG 60 3 GCAGCCCTAATTTGGGCC CATGAAGGCTGCAGAGGTCC 56 4 CTTCCCAGAGCCTCCCAC GCTGCTGAGTGAATGGGC 60 5 GTGGATAGATGCTGGGTG ACCGACCCAGAGACTCAG 60 6 GCTCCTCCGCTGACCTGA CTCGGGCCCCTAACTAAG 57 7 CAATGCTGCTGCTGAACT CCACCTTTGCCGCTGACA 57 8 TCTGCCCTGCCCTTCAGCAAGC GAGGTTAGTGCAGCCCTG 58 9 CGAGCCCCAACCCAACCGTGAG CGATGCCCAGGATCACACAGCA 56 10 CCCCGAATGTCCAGTCCTCAGC GCTGCCTCCCTCTGCCTCTTCC 56 Genomic Structure of STX1A 123

put database (accession number NT_023557) agrees with our sequence. However, there is a gap in this deposited sequence around exon 1. Our study has completed the genomic structure for STX1A.

Point Mutation Analysis This analysis uncovered two common variants in the exon 3 coding region that do not change the sequence (Table III). The two polymorphic nucleotides were 150C/T and 204T/C (Table III). Aside from these polymorphisms, no other sequence variations were identified in any of the individuals tested, either control or WS. Thus, no point mutations were found in these five nondeleted WS patients.

CONCLUSIONS Our previous studies showed that the majority of WS carry a similar sized deletion spanning an identical number of genes [Wu et al., 1998]. This renders the characterization of genes that contribute to specific features difficult. A small number of individuals with smaller deletions of the region have been identified, including two children with apparently classical WS [Botta et al., 1999]. These two patients, plus the results presented here, suggest that STX1A does not play a major role in the cognitive and behavioral features of Fig. 1. Patient 5. Note periorbital fullness. WS. Although these results do not support a role of STX1A in the WS phenotype, analysis of the promoter and surrounding sequence of the newly defined STX1A region or continued investigation of other non-deletion exons. All splice sites conform to the GT-AG rule patients may help to elucidate any potential role of [Green, 1991]. The 864 bp coding sequence of this newly STX1A in WS. Additionally, it is possible that these constructed genomic region agrees with that originally nondeletion patients represent phenocopies of a differ- published by Zhang et al. [1995] (GenBank L37792) and ent etiology. Therefore, the possible effects of hemi- with that previously published by us [Osborne et al., zygosity of STX1A on the WS phenotype still need to be 1997]. Newly deposited sequence in the high through- examined further.

TABLE II. STX1A Splice Junctions and Surrounding Sequence*

Splice acceptor Flanking exon sequence (50 ! 30) Splice donor ATG AAG GAC----CTC CGC ACG gtgagtccggcc aaccccgcacag GCC AAG GAC----TTT GAG CAG gtgggagccagc cttggtccgcag GTG GAG GAG----CCC GAT GAG A gtgagtgtgtgg ctacctccccag AG ACG AAG----AAG TTA AAG A gtgagtcaggct ctgtgcccccag GC ATC GAG----AAG ACA CAG gtgcggccacgg tcgggcccccag CAC TCC ACG----CTG GAG ATC A gtgagctggggg actcgcttgcag CC GGC AGG----GCC TCT GGG gtgagtgtaggc tcccacccccag ATC ATC ATG----GAG AGC CAG gtgagtgccggg ccggccccgcag GGA GAG ATG----GCG CGC CGG gtcagtagccca cttcccatgcag AAG AAA ATC----ATC TTC GCC

*Bold indicates splice sites.

TABLE III. Polymorphisms Observed in STX1A

Control Non-deletion WS Deletion WS Position Nucleotide chromosomes chromosomes

150 C 27 10 22 150 T 5 0 4 204 C 17 4 11 204 T 15 6 15

Controls, n ¼ 16; non-deletion WS individuals, n ¼ 5; WS individuals, n ¼ 26. 124 Wu et al.

ACKNOWLEDGMENTS Nakayama T, Fuijiwara T, Miyazawa A, Asakawa S, Shimizu N, Shimizu Y, Mikoshiba K, Akagawa K. 1997. Mapping of the human HPC-1/ The authors thank Dr. L. Potocki (Baylor College of syntaxin 1A gene (STX1A) to band q11.2. Genomics Medicine) for supplying Figure 1 and the Patients for 42:173–176. their Participation in this study. Nickerson E, Greenberg F, Keating MT, McCaskill C, Shaffer LG. 1995. Deletions of the elastin gene at 7q11.23 occur in 90% of patients with Williams syndrome. Am J Hum Genet 56:1156–1161. Osborne LR. 1999. Williams-Beuren syndrome—unraveling the mysteries REFERENCES of a microdeletion disorder. Mol Genet Metab 67:1–10. Osborne LR, Soder S, Shi XM, Pober B, Costa T, Scherer SW, Tsui LC. Bennett MK, Calakos N, Scheller RH. 1992. Syntaxin: a synaptic protein 1997. Hemizygous deletion of the syntaxin 1A gene in individuals with implicated in docking of synaptic vesicles at presynaptic active zones. Williams syndrome. Am J Hum Genet 61:449–452. Science 257:255–259. Schulze KL, Broadie K, Perin MS, Bellen HJ. 1995. Genetic and elec- Bennett MK, Garcia-Arraras JE, Elferink LA, Peterson K, Fleming AM, trophysiological studies of Drosophila syntaxin-1A demonstrate its role Hazuka CD, Scheller RH. 1993. The syntaxin family of vesicular in nonneuronal secretion and . 80:311–320. transport receptors. Cell 74:863–873. Sollner T, Bennett MK, Whiteheart SW, Scheller RH, Rothman JE. 1993. Botta A, Novelli G, Mari A, Novelli A, Sabani M, Korenberg J, Osborne LR, A protein assembly-disassembly pathway in vitro that may correspond Diglio MC, Dallapiccola B. 1999. Detection of an atypical 7q11.23 to sequential steps of synaptic vesicle docking, activation, and fusion. deletion in Williams syndrome patients which does not include the Cell 75:409–418. STX1A and FZD3 genes. J Med Genet 36:478–480. Tassabehji M, Metcalfe K, Donnai D, Hurst J, Reardon W, Burch M, Read Ewart AK, Morris CA, Atkinson D, Jin W, Sternes K, Spallone P, Stock AD, AP. 1997. Elastin: genomic structure and point mutations in patients Leppert M, Keating MT. 1993. Hemizygosity at the elastin locus in a with supravalvular aortic stenosis. Hum Mol Genet 6:1029–1036. developmental disorder, Williams syndrome. Nat Genet 5:11–16. Tassabehji M, Metcalfe K, Hurst J, Ashcroft GS, Kielty C, Wilmot C, Francke U. 1999. Williams-Beuren syndrome: genes and mechanisms. Donnai D, Read AP, Jones CJP. 1998. An elastin gene mutation pro- Hum Mol Genet 8:1947–1954. ducing abnormal tropoelastin and abnormal elastic fibres in a patient Green MR. 1991. Biochemical mechanisms of constitutive and regulated with autosomal dominant cutis laxa. Hum Mol Genet 7:1021–1028. pre-mRNA splicing. Annu Rev Cell Biol 7:559–599. Wu YQ, Sutton VR, Nickerson E, Lupski JR, Potocki L, Korenberg JR, Inoue A, Obata K, Akagawa K. 1992. Cloning and sequence analysis of Greenberg F, Tassabehji M, Shaffer LG. 1998. Delineation of the cDNA for a neuronal antigen, HPC-1. J Biol Chem 267: common critical region in Williams syndrome and clinical correlation of 10613–10619. growth, heart defects, ethnicity, and parental origin. Am J Med Genet Koh S, Yamamoto A, Inoue A, Inoue Y, Akagawa K, Kawamura Y, 78:82–89. Kawamoto K, Tashiro Y. 1993. Immunoelectron microscopic loca- Yoshida A, Oho C, Omori A, Kuwahara R, Ito T, Takahashi M. 1992. HPC-1 lization of the HPC-1 antigen in rat cerebellum. J Neurocytol 22:995– is associated with synaptotagmin and omega-conotoxin receptor. J Biol 1005. Chem 267:24925–24928. Li DY, Toland AE, Boak BB, Atkinson DL, Ensing GJ, Morris CA, Keating Zhang R, Maksymowych AB, Simpson LL. 1995. Cloning and sequence MT. 1997. Elastin point mutations cause an obstructive vascular analysis of a cDNA encoding human syntaxin 1A, a polypeptide essen- disease, supravalvular aortic stenosis. Hum Mol Genet 6:1021–1028. tial for . Gene 159:293–294.