Cloning and Characterization of an Inversion Breakpoint at 6Q23.3 Suggests a Role for Map7 in Sacral Dysgenesis
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Gene Mapping, Cloning and Sequencing Cytogenet Genome Res 106:61–67 (2004) DOI: 10.1159/000078563 Cloning and characterization of an inversion breakpoint at 6q23.3 suggests a role for Map7 in sacral dysgenesis R. Sood,a P.I. Bader,b M.C. Speer,c Y.H. Edwards,d E.M. Eddings,a R.T. Blair,e P. Hu,a M.U. Faruque,a C.M. Robbins,a H. Zhang,a J. Leuders,a K. Morrison,d D. Thompson,f P.L. Schwartzberg,e P.S. Meltzer,a and J.M. Trenta a Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD; b Northeast Indiana Genetic Counseling Center, Fort Wayne, IN; c Duke University Medical Center, Durham, NC (USA); d MRC Human Biochemical Genetics Unit, Biology Department, University College London, London (UK); e Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD (USA); f Department of Neurosurgery, Great Ormond Street Hospital for Children, London (UK) Abstract. Here we report on a male patient with sacral dys- breakpoint localized in the ninth intron of the MAP7 gene. We genesis (SD) and constitutional pericentric inversion of chro- then evaluated the involvement of MAP7 in SD by further mosome 6 (p11.2;q23.3). SD is a heterogeneous group of con- screening of the gene in several patients with a similar pheno- genital anomalies with complex genetic etiology. Previously, a type. Two nucleotide changes causing Ile257Asn and patient with sacral abnormalities and an interstitial deletion of Glu571Ala substitutions in the protein, both affecting amino 6q23→q25 region has been described. We speculated that a acid residues conserved in the mouse homolog, were identified susceptibility gene for SD lies in 6q23.3 region (disrupted in in two patients. Both changes are either very rare polymor- both patients), and therefore, cloning of the breakpoint in our phisms or true mutations, since they were not detected in 167 patient would lead to the identification of the disrupted gene. normal individuals nor found in the SNP database. Therefore, We performed FISH analysis followed by Southern blot analy- our study suggests MAP7 as a candidate gene for SD. However, sis and inverse PCR to clone the breakpoint. The 6p11.2 break- we were unable to detect any sacral defects in the MAP7 knock- point mapped very close to the centromere, and the 6q23.3 out mice. Copyright © 2004 S. Karger AG, Basel Sacral dysgenesis (SD), also known as sacral agenesis or cau- Pang, 1993). It can occur as an isolated anomaly or with other dal regression syndrome, is characterized by the congenital malformations, such as Currarino syndrome (O’Riordain et al., absence of all or part of the sacrum with contiguous caudal 1991; Lynch et al., 2000). Embryologically caudal regression or defects and associated anomalies (Welch and Aterman, 1984; dysgenesis has been ascribed to defective blastogenesis, and there has been overlap between caudal dysgenesis and other conditions believed to be due to errors in blastogenesis such as the VACTERL association (Cuschieri, 2002). The degree of neurologic involvement usually depends on the amount of The authors gratefully acknowledge support from grants HD33400 and NS39818. sacrum present, most often presenting as a neurogenic bladder Received 17 December 2003; manuscript accepted 11 February 2004. due to the absence of the corresponding sacral motor nerves Request reprints from: Dr. Raman Sood (Lotan et al., 1981). Skeletal defects often go undetected unless NHGRI, NIH, Building 49, Room 3A30 X-rays are performed due to other symptoms, such as bowel 9000 Rockville Pike, Bethesda, MD 20892 (USA) and urine incontinency (Kaneoya et al., 1990; Unluer and telephone: +1-301-435-5746; fax: +1-301-402-4929 e-mail: [email protected]. Bulut, 1991). Most cases of SD are sporadic, although familial Current address of J.M.T.: Translational Genomics Research Institute (TGen) cases have also been reported (Sarica et al., 1998). Sporadic 400 North Fifth Street, Suite 1600, Phoenix AZ 85004 (USA). cases of SD are often associated with maternal diabetes (White Fax + 41 61 306 12 34 © 2004 S. Karger AG, Basel Accessible online at: ABC E-mail [email protected] www.karger.com/cgr www.karger.com and Klauber, 1976; Dunn et al., 1981; Sonek et al., 1990). interval. DNA from YAC clones was isolated by spheroplasting as described Familial cases display autosomal dominant inheritance with by Carpten et al. (1994). DNA from BAC and PAC clones was purified using the Autogen 850 automated DNA isolation system using the manufacturer’s incomplete penetrance (Borrelli et al., 1985; O’Riordain et al., recommended protocol (Autogen). 1991). Cytogenetic studies (Morichon-Delvallez et al., 1993; Savage et al., 1997) and linkage analysis (Lynch et al., 1995) Polymerase chain reaction (PCR) and mutation analysis followed by positional cloning efforts has led to the identifica- PCR reactions were performed using 10 ng of template DNA with ++ tion of a gene, HLXB9 at 7q36 with mutations in several fami- 2.25 mM Mg , 250 nM dNTPs, 333 nM each forward and reverse primer, PCR buffer II (Perkin-Elmer), and 0.6 units of AmpliTaq Gold Polymerase lies with sacral agenesis (Ross et al., 1998). HLXB9 is a homeo- (Perkin-Elmer) in a 15-Ìl total PCR reaction volume. All of the PCR reac- box gene that codes for the nuclear protein HB9. The exact tions were carried out in a model 9700 Thermocycler (Perkin-Elmer) using mechanism by which mutations in HLXB9 cause defects in the following cycling conditions: initial denaturation of 94 ° C for 12 min; caudal development is currently unknown, although most mu- 94 °C for 15 s, 55 ° C for 15 s, 72 ° C for 15 s for 35 cycles; followed by a final extension at 72 °C for 10 min. Mutations in HLXB9 were analyzed by single- tations have been observed in the homeobox domain (Belloni strand conformation polymorphism (SSCP) using nine overlapping primer et al., 2000; Hagan et al., 2000; Kochling et al., 2001). Mice pairs that covered the entire coding sequence. The PCR template was cDNA homozygous for a null mutation in HLXB9 gene do not display synthesized from patient RNA using reverse transcriptase AMV and oligo- any detectable sacral defects (Harrison et al., 1999; Li et al., (dT)15 primer in the presence of RNase-inhibitor (Boehringer Mannheim). TM 1999). Mutations in HLXB9 have been detected in most cases Radiolabeled PCR products were electrophoresed in 0.5× MDE (FMC Bio Products) gels with and without glycerol at room temperature with 0.6× TBE of hereditary sacral agenesis but not in sporadic cases with a at 5 W for 16 h. Mutation analysis of MAP7 was performed on genomic DNA similar phenotype (Lynch et al., 2000). from patients using SSCP, denaturing high performance liquid chromatogra- A lack of mutations in HLXB9 in sporadic cases and other phy (dHPLC, Varian), and bidirectional sequencing technologies. Primers reports of non-7q cytogenetic abnormalities and linkage analy- were designed to cover all 16 coding exons of the gene and primer sequences are available upon request. Most of the samples were analyzed by dHPLC for sis suggest genetic heterogeneity. McLeod and colleagues 12 exons and sequencing for four exons numbered 10, 13, 15, and 16. A small (McLeod et al., 1990) reported a patient with an interstitial subset of patients was screened by SSCP for all exons. PCR products repre- deletion of 6q23 to q25 region and sacral agenesis. Involvement senting heteroduplex samples from dHPLC analysis, conformers from SSCP of the T-locus (Brachyury) was also suggested based on the sim- gels, and direct sequencing exons were sequenced using an Applied Biosys- ilarities in defects caused by T-locus mutations in the mouse to tems 377 XL automated DNA sequencer (Perkin-Elmer). Sequences were analyzed using Sequencher v4.1 (Gene Codes) to identify nucleotide substi- spina bifida and sacral agenesis in human (Fellous et al., 1982). tutions. However, any major role of the T-locus in the etiology of SD was excluded by mutation screening in 28 patients (Papapetrou Fluorescence in situ hybridization (FISH) et al., 1999). These studies indicate complex genetic etiology Slides with metaphase chromosome spreads were prepared from the patient’s lymphoblastoid cell line. All probes were labeled with Spectrum- for SD. In an effort to identify additional candidate genes for Orange conjugated dUTP by degenerate oligonucleotide primed-PCR. The sacral dysgenesis, we performed molecular delineation of a con- slides were treated with RNase for 2 h at 37 °C and were denatured in 70 % stitutional chromosomal aberration involving chromosome 6, formamide/2× SSC at 72 °C for 2 min. Hybridization was performed in a inv (6) (p11.2;q23.3) in a patient with clinical symptoms of SD. humidity chamber at 37 °C overnight followed by washing and counter- staining with DAPI in antifade. The rearrangement appeared to be a balanced inversion with no detectable loss of chromosomal material based on cytogenet- Southern blot analysis ic analysis using FISH. Here we report the molecular cloning Southern blots were prepared in the laboratory using standard methods. and characterization of the 6q23.3 breakpoint in this patient. Briefly, 5 Ìg of DNA from patient and controls was digested with selected restriction enzymes, electrophoresed on 0.8% agarose gels, and transferred to GeneScreen plus nylon membranes (Dupont). Probes were labeled using 32P- dCTP using a Prime-it II kit (Stratagene). Hybridization was performed Materials and methods overnight at 65 ° C in RapidHyb buffer (Amersham). Blots were washed in 2× SSC, 0.1 % SDS twice at room temperature for 10 min each, followed by a Case report 20-min wash in 0.1× SSC, 0.1 % SDS at 65 °C.