FAMILIAL INHERITED MICROTIA CAUSED BY 4PTER OLFACTORY RECEPTOR GENE CLUSTER AMPLIFICATION
Joris Robert Vermeesch1, Kevin Martens1, Nicole Maas1, Cindy Melotte1, Steven Van Vooren5, Bart De Moor5, John Engelen2, Heike Starke3, David Vetrie4, Heike Fiegler4, Gert Matthijs1, Jean-Pierre Fryns1, Ingele
Casteels1 Koen Devriendt1
1Center for Human Genetics and Department of Ophthalmology (IC) University of Leuven, Belgium.
2Research institute for Growth and Development, Department of Clinical Genetics, University of Maastricth, the Netherlands (JE)
3Institute of Human Genetics and Anthropology and Departments of University of Jena, Jena (Germany)
4Human Genetics, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambs., UK
5 ESAT-SISTA K.U. Leuven, Belgium
The olfactory receptor (OR) genes form the largest known multigene family.
Here, we present a family with autosomal dominant inheritance of microtia, eye coloboma and lacrimal duct obstruction linked with a cytogenetically visible alteration at 4pter caused by the amplification of the 4pter OR gene cluster.
This observation pinpoints the amplification of larger genomic regions and, more specifically amplification of OR genes, as an hitherto unsuspected cause of inherited genetic disorders.
We examined a large family with autosomal dominant inheritance of microtia associated with variable degrees of eye coloboma and lacrimal duct obstruction (figure 1, table 1). High resolution cytogenetic analysis revealed an aberrant karyotype 46,XY,add(4)(pter) in all affected family members absent in unaffected family members (figures 2 and 3).
To confirm that the phenotypic anomalies are caused by the 4pter alteration and to delineate the region causing the phenotype, linkage analysis was performed with a series of polymorphic markers derived from the 4 pter region (figure 2). A three point Lod score of 3.215 was obtained between marker D4S2960 and D4S2946. Two point analysis resulted in a Lod score of 2.107 between markers D4S2983 and D4S2994 (figure 2). None of the informative markers were duplicated or deleted.
Multicolor FISH using the 24 human whole chromosome painting probes and FISH using a chromosome 4 paint showed that all chromosomal material in the 4pter region was derived from chromosome 4 (figures 3b). Subsequent FISH with 4 pter region specific probes and multicolor banding analysis revealed an increased signal intensity, suggesting that the apparent rearrangement on chromosome 4 was caused by a duplication or amplification within 4p15-4p16 (figure 3c,d). Fine mapping of this aberration was initially done by FISH using a series of 11 BACs spaced 2 Mb apart as probes. Analysis of both metaphase and interphase chromosomes showed that none of these BAC clones used were duplicated or amplified (data not shown).
To increase the resolution of the analysis, microarray CGH was performed using a genomic array of 1 Mb resolution (Fiegler et al., 2003) and a contiguous 4pter specific tiling path array. While on the 1 Mb array intensity ratios for all loci were normal (figure 3e), four from six contiguous clones on the 4pter array presented with intensities slightly above threshold (figure 3f). Within this region, a single clone, RP11-637J21, has a higher intensity ratio with a log2 value of 0.93 while five flanking clones have an average log2 value of 0.4. FISH with all clones within the region, including the clones with normal average log2 values, show amplification at
4pter in the affected patients and all but RP11-637J21 hybridized at multiple genomic loci (figure 4a). In addition, the proximal flanking clone, RP11-751L19, with an log2
intensity ratio of 0.23 was also shown to be amplified. FISH analysis with the distal and proximal clones with normal log2 values were not visibly amplified. This 4pter region contains the olfactory receptor gene cluster. For clones located within a second 4pter ORGC located at 4 Mb also slightly higher than normal intensity ratios were observed.
Sequence alignment of the amplified region with the 8pter ORGC regions confirms that the amplification coincides almost exclusively with this ORGC (figure 4b). Both the distal (RP11-637J21) and proximal (RP11-751L19) clone within the amplified region do contain sequences not paralogous with the 8pter ORGC. To delineate the size of and determine the number of copies present in the amplified region, quantitative PCR was performed. Within both the distal and proximal amplified clones, quantitative PCR assays were developed. Within RP11-637J21, CPZ and an anonymous locus (which we term 637J21P) flanking a “gap” in the genome sequence and within RP11-751L19 , a locus proximal of DRD5 and within SLC2A9, were amplified. Analysis of the DNA of a 4pter deletion carrier, a normal control individual and a 4pter duplication carrier unambiguously identifies a single, two or three copies at all four loci (figure 4d). For the non-affected family members all values are within the normal range. In contrast, for all affected family members a normal range is detected for the CPZ locus and the SLC2A9 locus , but an average of three fold (six alleles) amplification was detected for 637J21P and DRD5.
Olfactory receptors (ORs)comprise the largest gene family in the human genome, with approximately 900 members, and encode the proteins responsible for odorant binding an discrimination (Buck and Axel, 1991; Malnic et al., 2004). These ORs are often organized in gene clusters and are scattered across the human genome (Rouquier et al., 1998; Glusman et al., 2000) and can mediate several chromosomal disorders by causing meiotic misalignment causing either intra or interchromosomal ectopic recombination (Giglio et al., 2001; Giglio et al., 2002). Here we show that a chromosomal aberration linked with an autosomal dominant phenotype is caused by the amplification of the ORGC.
How the amplification of the ORGC causes the observed phenotype is not obvious.
Multiple copies of the ORGC are scattered across the genome (Newman and Trask, 2003). However, it is unknown whether copy number differences exist within the human population. Microtia and lacrimal duct anomalies have not been observed in patients with partial 4pter trisomy (Schinzel, 2001 and references therein). A single gene, the dopamine receptor D5, is shown to be co-amplified. This gene is structurally and functionally similar to the D1 dopamine receptor. High levels of mRNA were found in discrete cortical areas (layers II,IV,VI), the dentate gyrus and hippocampal subfields (Tiberi et al., 1991; Beischlag et al., 1995). It is not obvious how amplification of DRD5 might cause the observed phenotype. Various hypotheses to explain the phenotype can be put forward: (1) Since a gap in the human draft sequence is located within the amplified region, it could be that unique genes are located within this region which can cause this phenotype when amplified.
(2) An hitherto undetected gene is localized within the unique sequences of the amplified region flanking the ORGC . (3) A three fold amplification of the ORGC
causes over-expression of the OR genes which in turn causes the phenotype. (4) The amplification causes long range chromosomal effects on nearby gene expression. We did show that the region is heavily methylated (data not shown) and changes in gene expression of nearby genes as a result of a chromosomal anomaly have been described (Kleinjan and van Heyningen, 2005). (5) The rearrangement causes ectopic gene expression which may interfere with normal development.
At present it remains unclear whether the ORGC low copy repeats mediated the amplification process or whether the ORGC was fortuitously co-amplified. While amplifications causing cytogenetically recognizable chromosome aberrations are well known in cancer cytogenetics, only ample examples of inheritable amplifications of non-heterochromatic regions exist. Benign copy number variations of the chromosome 9 pericentromeric region and the defensin gene clusters located near the proximal 8p ORGC appear to cause the cytogenetically visible variations at chromosome band 8p23.1 (Hollox et al., 2004). By FISH analysis using an 8p specific BAC (RP11-739E3) containing defensins, we did show the presence of a paralogous defensin locus at 4pter which was amplified in the patients, despite its absence in the draft sequence (data not shown). It may well be that both the amplification of the defensin cluster and the amplification described in this family are mechanistically related.
In conclusion, we demonstrate that a novel private syndrome defined by autosomal dominant inheritance of microtia, eye coloboma and lacrimal duct obstruction is caused by a three fold amplification of an approximately 1 Mb region (0.7Mb plus a gap of unknown length) existing almost exclusively of the proximal 4pter olfactory
receptor gene cluster. This finding implicates the ORGC in a novel type of chromosomal rearrangement and pinpoints amplification of the olfactory receptor gene cluster as a novel genetic disease causing mechanism. However, the presence of a gap in the near-finished human genome sequence in the amplified region confounds the interpretation of the results. This finding underscores the need to continue efforts to completely finish the human genome sequence to enable a full understanding of human genetic disease processes. Amplification of a genomic region as the cause of an inherited disorder is only rarely reported. Recently, triplication of a region of 1.4 Mb including SNCA was shown to be linked with autosomal dominant Parkinsons disease (Singleton et al., 2003). Both SNCA and ORGC amplifications as the cause of AD disorders may well represent the first examples of a group of inherited disorders caused by the amplification of larger genomic regions.
Materials and methods Cytogenetic studies
EBV cell lines were made following standard procedures. High-resolution G-, R- and C- banded chromosomes were prepared from peripheral white blood cells, according to standard procedures and was performed on nine family members (II:3, II:4, II:8, II:5, III:3, III:4,III:5,III:6,III:7).
Fluorescent in situ hybridization (FISH)
Fluorescence in situ hybridization (FISH) and BAC DNA labeling was performed as described (Vermeesch et al., 2003). Chromosome paint 4 (Vysis, Inc.; Downers Grove, IL) and MFISH (Metasystems) was performed following the procedures provided by the company. BACs were obtained from CHORI (Oakland, CA) and the physical distances and cytogenetic location are derived from the Ensembl database (July, 2002 release).
The 1 Mb genome wide array-CGH was performed as described (Fiegler et al., 2003). A high resolution chromosome 4p16 array was constructed using DOP-PCR products from 196 BAC and PAC clones, covering a 20 Mb subtelomeric region with all clones represented in the november 2002 release of the Golden Path and a subsequent 27 Mb region with an average spacing of one clone per 700 kb.
Clones were obtained from CHORI (Oakland, CA) and the Sanger institute. In addition, the array contained 31 X chromosome derived clones and 82 clones derived from different autosomes. BAC DNA preparation, amplification and spotting were performed as described (Vermeesch et al, in press).
The products were arrayed on CodeLink Bioarray System slides (Amersham Pharmacia Biotech, Buckinghamshrine, UK) using a Molecular Dynamics Generation III printer (Amersham Pharmacia Biotech, Piscataway, NJ). The clones were printed in six replicates. Before hybridization, the slides were humidified, dried and target DNA was cross-linked using UV-Stratalinker at 50 mJ (Stratagene, Amsterdam, The Netherlands).
Genomic DNA was extracted from lymphocytes with the Qiagen DNA extraction kit (Qiagen N.V., Venlo, The Netherlands) followed by a phenol/chloroform extraction. Test and reference DNA
was labeled by random prime labeling method modified in our laboratory (Bioprime DNA Labeling System, Invitrogen, Carlsbad, CA) using Cy3/ Cy5-dCTP (Amersham Pharmacia Biotech). Six hundred nanograms of DNA was mixed with random primer solution supplied by the manufacturer and heat denatured for 10 min at 100 °C. Subsequently, the mixture was incubated overnight with 0.2 mM dATP, 0.2 mM dGTP, 0.2 mM dTTP, 0.05 mM dCTP, 0.04 mM Cy3-dCTP or Cy5-dCTP and 80 U Klenow fragment at 37°C. The probes were then purified by Qiaquick columns (Qiagen). The labeled DNA was mixed with 200 g Human Cot-1 DNA (Roche), 400 g yeast tRNA (Invitrogen) and precipitated with ethanol. The precipitated DNA was reconstituted in 5.4 µl 20% SDS, 6.6 µl water, 28
l of master hybridization mix (70% formamide, 2.8XSSC, 7% dextran sulphate), denatured at 70°C for 15 min, immediately followed by incubation at 37°C for 60 min to allow the blocking of repetitive DNA sequence. Slide pretreatment and probe hybridization were performed as described (Vermeesch et al., in press). Arrays were scanned at 532nm (Cy3) and 635nm (Cy5) using the GenIII scanner (Amersham) for the mirror slides or the Agilent G2565BA MicroArrayScanner System (Agilent Inc., Palo Alto, CA). Image analysis was done using Array Vision (Imaging Research Inc, St.Catharines, Ontario, Canada). Spot intensities were background corrected.
Calculations
For each clone the average ratio and standard deviation was calculated over 6 replicas. The
fluorescence intensities measured were first background subtracted. Background values were obtained by the averaging four loci surrounding each spot (Arrayvision). Consistent BAC/PAC clones, for which standard deviation was larger than 20%, were excluded from data analysis. Normalization was determined based on mean values of all clones.
CA repeats spaced along the short arm of chromosome 4 were amplified by PCR, using a fluorescein-isothiocyanate (FITC) label on one of the primers. The fragments were sized on an A.L.F. DNA sequencer (Amersham Pharmacia Biotech). Primer
sequences were as published (http://gdbwww.gdb.org/). Three point and two point Lod scores were calculated using the Fastlink package.
Real-Time PCR was carried out using the ABI Prism 7700 Sequence Detection System (Applied Biosystems) and was performed using the qPCR Master Mix for SYBR green I detection (Eurogentec) according to the manufacturer’s protocol.
Primers were designed by Primer Express 2.0 (Applied Biosystems). CPZ forward and revers primers are F 5’-AGGCAATCATGAAGTGGATGC-3’ and 5’- GGTAGGACACCACCAGGTCG-3’. GAP forward and reverse primers are 5’- CTCCCAAAGCAAAGCTTGTG-3’ and 5’-CCACCCATTGGTCAGAGAAC-3’.
DRD5 forward and reverse primers 5’- GCTACGGGCTGAGCCAGTT-3’ and 5’- TTTTCATCTCAATGCCCTCTGA-3’. SLC2A9 forward and reverse primers 5’- GCTGGCTCAATAAGGCAAGTGT-3’ and
5’-TTTCCCAGAGTTTGAAGCTGATT-3’. P53 (reference gene) forward and reverse primers 5’- CCCAAGCAATGGATGATTTGA-3’ and 5’- GAGCTTCATCTGGACCTGGGT-3’. The number of alleles present was quantified using the comparative threshold cycle (Ct) method (Applied Biosystems).
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FIGURES
Figure 1 Phenotype of the patients. (A) Left and right microtia, (B)left and right coloboma of the iris and (C) choroids.
Figure 2 Linkage of the phenotype with 4p15-4p16.1. Patients with eye coloboma, absent canaliculi and/or microtia are indicated by resepectively a filled left upper square, left lower square and/or right upper and lower square.
The red line indicates the region linked with the phenotype.
Figure 3 (A) Partial karyotype of the patient showing the G-banded normal and aberrant chromosome 4. On the right, an ideogram of a normal chromosome 4 is presented. (B) MFISH analysis showing that only genetic material from chromosome 4 is present in the 4p+. (C) Multicolour banding FISH shows that the chromosome 4p cytogenetic anomaly is caused by the 4p15-16 region and (D) a region specific painting probe from 4pter confirms the chromosomal 4pter origin of the chromosomal anomaly. (E) Genome wide array CGH at 1 Mb resolution can not demonstrate any chromosomal anomaly. Chromosome 4result of the 1 Mb array. (F) Tiling path array of chromosome 4pter demonstrates the amplification of a region located at 8 Mb from the telomere.
The red line indicates the 4xSD threshold.
Figure 4 FISH confirmation of the array CGH data and delineation of the ORGC. (A) FISH using (I) RP11-689P11 (not amplified), (II) RP11-637J21 and (V) RP11-751L19 (amplified at 4pter) , (III) RP11-264E23 and (IV) RP11-180A12 (amplified at 4p and multiple loci scattered across the genome).
(B) Ensembl view from the december 2004 freeze showing both the DNA contigs, the tiling path clones and the Ensembl genes. Also the location of the Taqman PCR loci are indicated. The color bars indicate the colors used for the graph in panel D. Below, the region with six fold amplification is indicated by the red bar. The dotted red line indicates the region potentially amplified.
At the bottom, pairwise FLAG analysis of the proximal 4pter ORGC and the proximal 8pter ORGC is shown. The red dots indicate paralogous loci. (C) Close-up of the region with altered intensity ratios within the 4p tiling path array CGH data (from figure 3e). The latin numbers refer to the clones used for the FISH analysis in panel A. (D) Dosage analysis of four loci bordering the amplified region. In the Y axis the copy number and in the X axis the family members are indicated as represented in figure 2.
