ARTICLE Submicroscopic Deletion in Patients with Williams-Beuren Syndrome Influences Expression Levels of the Nonhemizygous Flanking Genes
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector ARTICLE Submicroscopic Deletion in Patients with Williams-Beuren Syndrome Influences Expression Levels of the Nonhemizygous Flanking Genes Giuseppe Merla, Ce´dric Howald, Charlotte N. Henrichsen, Robert Lyle, Carine Wyss, Marie-The´re`se Zabot, Stylianos E. Antonarakis, and Alexandre Reymond Genomic imbalance is a common cause of phenotypic abnormalities. We measured the relative expression level of genes that map within the microdeletion that causes Williams-Beuren syndrome and within its flanking regions. We found, unexpectedly, that not only hemizygous genes but also normal-copy neighboring genes show decreased relative levels of expression. Our results suggest that not only the aneuploid genes but also the flanking genes that map several megabases away from a genomic rearrangement should be considered possible contributors to the phenotypic variation in genomic disorders. Segmental aneuploidies (i.e., gain or loss of subchromo- Bioanalyzer (Agilent Technologies). Total RNA was converted to somic DNA fragments) are important contributors to hu- cDNA with the use of Superscript II (Invitrogen) primed with poly man diseases1 and, potentially, to phenotypic variation,2,3 d(T). For each cell line included in the study, 4.5 mg of total RNA as well as a major force of evolutionary changes.4–9 There was converted to cDNA in three individual reactions; these were then pooled and were diluted 1:14. is evidence that such genomic insertions and deletions contribute to phenotypic differences by modifying the ex- Sample Population pression levels of genes within the aneuploid segments.10– 13 We hypothesize that these rearrangements also induce Lymphoblastoid cell lines from 10 individuals with WBS and from altered expression of the genes that lie near the break- 40 control individuals, as well as skin fibroblasts from 7 control points, although these do not vary in copy number; this individuals, were acquired from the cell culture collection of the effect could be mediated by disturbances of the copy num- Coriell Institute for Medical Research, and skin fibroblasts from ber of long-range cis regulatory elements.14–17 14 individuals with WBS and from 6 control individuals were To test this hypothesis, we assessed whether the human obtained from the cell culture collections of the Centre de Bio- technologie Cellulaire, Hospices Civils de Lyon, Hoˆpital De- chromosome 7 (HSA7) recurrent DNA deletion causing brousse, in Lyon, France. One more control was received from Williams-Beuren syndrome (WBS [MIM 194050])18 influ- the Galliera Genetic Bank in Genova, Italy (table 1). Appropriate ences the transcription levels of both the hemizygous informed consent was obtained for each sample by the physicians genes within the deleted region and the nonhemizygous in charge. DNA was extracted from each cell line of the sample genes in the WBS flanking regions. WBS is a neurodevel- population, with the use of PureGene (Gentra Systems), in ac- opmental disorder characterized by numerous clinical as- cordance with the manufacturer’s instructions. We assayed each pects, including mental retardation with a unique cogni- DNA with a quantitative PCR approach, using SybrGreen dye and tive and personality profile.19 Its incidence is estimated to probes, mapping the region from the BAZ1 locus to the CYLN2 be between 1:7,500 and 1:20,000, and sporadic de novo locus and the flanks of the commonly deleted region,23 to deter- inheritance is usual.20–22 mine the size of the deletions and to ensure (1) that none of the patients with WBS presented an atypical deletion23–29 or an in- 18,30 Material and Methods version at 7q11.23 and (2) that none of the controls were hemizygous for that same region. The results are presented in Cell Culture, RNA, and cDNA Preparations table 2. Human skin fibroblasts and lymphoblastoid cell lines were grown To make sure that differences in expression levels measured in in HAM F-10 or RPMI 1640 media, respectively, supplemented lymphoblastoid cells were not merely due to transformation, we with 10% fetal bovine serum and 1% antibiotics (Invitrogen). established six lymphoblastoid cell lines from two blood samples Total RNA was prepared from logarithmic growth–phase cells, collected at 1-wk intervals from the same individual, after in- with the use of RNeasy Mini Kit (Qiagen), in accordance with the formed consent. We measured expression levels of 25 HSA21 manufacturer’s instructions. After DNAse treatment (Qiagen), the genes (ITBG2, CBS, APP, PFKL, U2AF1, PRDM15, LSS, PDXK, quality of all RNA samples were checked using an Agilent 2100 SLC19A1, SLC37A1, PWP2H, MCM3AP, GART, CBR1, TMEM1, From the Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland (G.M.; C.H.; R.L.; C.W.; S.E.A.; A.R.); Servizio Genetica Medica, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy (G.M.); Centre de Biotechnologie Cellulaire, Hospices Civils de Lyon, Hoˆpital Debrousse (M.-T.Z.), and Interdisciplinary Group to Study the ELN Gene (M.-T.Z.), Lyon, France; and Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland (C.N.H; A.R.) Received April 26, 2006; accepted for publication May 31, 2006; electronically published June 23, 2006. Address for correspondence and reprints: Dr. Alexandre Reymond, Center for Integrative Genomics, Genopode Building, University of Lausanne, Lausanne, Switzerland. E-mail: [email protected] Am. J. Hum. Genet. 2006;79:332–341. ᭧ 2006 by The American Society of Human Genetics. All rights reserved. 0002-9297/2006/7902-0015$15.00 332 The American Journal of Human Genetics Volume 79 August 2006 www.ajhg.org Table 1. Cell Lines Employed Table 3. Assayed Genes, Primers, Probes, The table is available in its entirety in the online and Efficiencies edition of The American Journal of Human Genetics. The table is available in its entirety in the online edition of The American Journal of Human Genetics. BTG3, DSCR1, ETS2, IFNAR2, ANKRD3, WRB, GABPA, SON, replicates per sample and were set up in a 384-well plate format, IFNAR1, and CCT8) that show a large variation in transcript levels with the use of a Biomek 2000 robot (Beckman). They were run in the normal population and found no significant differences in in an ABI 7900 Sequence Detection System (Applied Biosystems) their expression levels in the assayed samples (Pearson 0.8 ! r ! with the following amplification conditions: 50ЊC for 2 min, 95ЊC 0.92 [mean 0.87];P ! .001 ). The observed differences correspond for 10 min, and 50 cycles at 95ЊCfor15sand60ЊCfor1min. to the experimental variation we measure between replicates. Each plate contained the appropriate normalization genes to con- trol for any variability between the different plate runs. Real-Time Quantitative PCR and Data Analysis Raw threshold-cycle (CT) values were obtained using SDS2.2 We opted for Taqman real-time quantitative PCR, to measure any (Applied Biosystems). To calculate the normalized relative ex- small differences in gene expression levels. Primers and probes pression ratio between individuals with WBS and controls, we were designed using the PrimerExpress program (Applied Biosys- followed methods described elsewhere.31 We exploited the ge- tems), with default parameters in every case for all the confirmed Norm method32 to select the three normalization genes: AGPAT1, genes mapping on HSA7, from the centromere to the beginning EEF1A1, and PSMA5. They were used to normalize input cDNA of band 7q21.11. They can be divided into three groups of genes: for each sample, whereas mixes of 40 lymphoblastoid and 12 23 HSA7 test genes that are hemizygous in patients with WBS, 2 fibroblast control-cell-line cDNA samples (table 1) were used to genes mapping in the low-copy repeats (LCRs) flanking the WBS define normal RELs. deletion, and 24 HSA7 test genes that are nonhemizygous in pa- tients with WBS. We also designed assays in 2 HSA7p genes, in Results 13 control genes, and in 3 normalization genes. The complete list of tested genes, their accession numbers and mapping posi- We used the high sensitivity of real-time quantitative PCR tions, and the primers and probes used are indicated in table 3. to accurately measure the expression of all the HSA7q Amplicon sequences were checked by both BLAST and BLAT genes mapping in the region 11.9 Mb downstream (band against the human genome, to ensure specificity. Whenever pos- 7q21.11) to 8.4 Mb upstream (centromere position) of the sible (in 94% of cases), oligos were designed to span an intron. WBS deleted region, in which we could design a working reverse ע Non–intron-spanning assays were tested in standard у р transcriptase reactions of RNA samples for genomic contamina- Taqman assay (i.e., efficiency 0.95 and 1.05 for ex- tion; in all cases, no amplification was observed in the absence pression in fibroblasts and/or lymphoblastoid cell lines) of reverse transcriptase. High-performance liquid chromato- (see “Material and Methods” and table 3 for details). These graphy–purified, FAM-TAMRA–labeled, double-dye Taqman genes can be divided into 17 HSA7q hemizygous genes probes and qPCR mastermix (RT-QP2X-03) were obtained from that map within the WBS deletion, 14 HSA7q nonhemi- Eurogentec. zygous genes, and 2 genes that map within the LCR flank- The efficiency of each Taqman assay was measured using a ing the deletion. This panel of genes was completed with dilution series of fibroblast cDNA and lymphoblastoid cells or a 2 HSA7 nonhemizygous genes that map on the short arm pool of cDNA samples of brain, liver, and testis, as described else- of the chromosome (band 7p11.2) and 19 control, non- where31 (see table 3 for results). A working Taqman assay was HSA7 genes. We compared the mRNA expression levels of obtained for 57 (85%) of the 67 assayed genes.