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Molecular cytogenetic characterization of a familial pericentric inversion 3 associated with short stature
European Journal of Medical Genetics 58 (2015) 154e159
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European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Clinical research
Molecular cytogenetic characterization of a familial pericentric inversion 3 associated with short stature Usha R. Dutta a, b, *, Ingo Hansmann a, Dietmar Schlote a a b
Institut fuer Humangenetik, Martin Luther University, Halle-Wittenberg, Halle (Saale) 06097, Germany Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad 500 001, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 July 2014 Accepted 5 January 2015 Available online 13 January 2015
Short stature refers to the height of an individual which is below expected. The causes are heterogenous and influenced by several genetic and environmental factors. Chromosomal abnormalities are a major cause of diseases and cytogenetic mapping is one of the powerful tools for the identification of novel disease genes. Here we report a three generation family with a heterozygous pericentric inversion of 46, XX, inv(3) (p24.1q26.1) associated with Short stature. Positional cloning strategy was used to physically map the breakpoint regions by Fluorescence in situ hybridization (FISH). Fine mapping was performed with Bacterial Artificial Chromosome (BAC) clones spanning the breakpoint regions. In order to further characterize the breakpoint regions extensive molecular mapping was carried out with the breakpoint spanning BACs which narrowed down the breakpoint region to 2.9 kb and 5.3 kb regions on p and q arm respectively. Although these breakpoints did not disrupt any validated genes, we had identified a novel putative gene in the vicinity of 3q26.1 breakpoint region by in silico analysis. Trying to find the presence of any transcripts of this putative gene we analyzed human total RNA by RT-PCR and identified transcripts containing three new exons confirming the existence of a so far unknown gene close to the 3q breakpoint. Ó 2015 Elsevier Masson SAS. All rights reserved.
Keywords: BAC clones Breakpoint regions FISH Inversion Physical mapping Short stature
1. Introduction Short stature (SS) refers to the height of an individual which is below expected and is one of the major features found in many syndromic cases. The causes of SS are many and influenced by several genetic and environmental factors hence the systematic classification of the various syndromes involving SS is not possible [Enders, 1992]. The genetic factors may include chromosomal anomalies in both autosomes as well as in sex chromosomes. The most common cytogenetic cause of SS in females is Turner syndrome, with different chromosomal variants affecting X chromosome. The Short stature HOmeobox-containing gene on the short arm of X and Y (SHOX) is an important determining factor for stature phenotype [Musebeck et al., 2001]. Whereas in autosomes; the SHOX2 gene is localized on chromosome 3q25-26 [Baere et al., 1998; Blaschke et al., 1998]. SHOX2 is closely related to SHOX on sex chromosome [Clement-Jones et al., 2000]. But till * Corresponding author. Diagnostics Division, Center for DNA Fingerprinting and Diagnostics, Tuljaguda complex, 4-1-714, Hyderabad 500 001, Andhra Pradesh, India. Tel.: þ91 40 24749337; fax: þ91 40 24749448. E-mail addresses: [email protected], [email protected] (U.R. Dutta). http://dx.doi.org/10.1016/j.ejmg.2015.01.001 1769-7212/Ó 2015 Elsevier Masson SAS. All rights reserved.
date there are only two reports on the chromosomal analyses with SS [Lam et al., 2002; Moreno-Garcia et al., 2005]. In general, the structural rearrangements alter the genome architecture and may result in human disease phenotypes. The patients with translocations and inversions often have breakpoints located within the disease gene, or very close to it [Chen et al., 2010]. In order to identify the disease gene, characterization of the breakpoints has often been a promising start point in the molecular elucidation of early-onset of Mendelian disorders [Kalscheuer et al., 2003; Moller et al., 2008]. The constitutional pericentric inversion on chromosome 3 occurs rarely [Gahrton et al., 2010], accounting for about 4% of the normal population [Soudek and Sroka, 1978]. Most of the inversions are not clearly visualized by GTG banding [Hasle et al., 1992] and therefore not frequently reported [Welborn, 2004]. Nevertheless a few cases of chromosome 3 abnormalities associated with SS were reported in literature [Barajas-Barajas et al., 2001], like a 3q deletion with SS reported by Nguyen [Nguyen et al., 2005] and a partial monosomy 3q with SS reported by Brueton et al., 1989. Most of other cases were associated with other chromosomal rearrangements [Kondo et al., 2006; Stine et al., 1982; Yip et al., 1996]. But to the best of our knowledge there is no case reported with a familial inversion
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3 associated with SS. In this study, we characterized a familial inversion inv(3) (p24.1q26.1) associated with SS and identified a novel putative gene on 3q26.1 breakpoint region. 2. Patient data A 16-year old girl was referred to our center with SS. She was the first child born at term to a non-consanguineous couple after an uneventful pregnancy. Her milestones were normal. She was only 146 cm tall, significantly under the 3rd centile, whereas her 7 year old brother was 125 cm tall, consistent with the 50th centile. The detailed family history and written consent was taken from the patient’s family. 3. Methods
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Heidelberg, Germany) were used. Specific products were amplified with exon 5 specific primers p5 (50 GACATTGTCTGGGAGCAGC-30 ), p5n (50 -AGTAGATCCTGAAGGCGTG-30 ) and exon 11 specific primers p11 (50 -CTTGTCTAAGGTTGCAGACTCA-30 ), p11n (50 GTGAAGCCTCGTTTCATCC-30 ) in a first round of standard PCR and re-amplified using the nested primers. For the splice variant SV2 an exon 9a specific primer p9a (50 -GGAAAGAAGCAGAGGTAGCC-30 ) was used as forward primer. 3.5. Subcloning The breakpoint spanning clone RP11-12N13 on 3q26.1 region was digested with Sac I restriction enzyme and the desired fragments were excised from the gel, eluted and subcloned into pBluescript SK Vector which were further labeled and used as probes for FISH mapping.
3.1. Cytogenetic analyses 3.6. Restriction digestion Chromosomal analyses were carried out on the peripheral blood lymphocytes in the patient, her parents, brother, maternal aunt and maternal grandmother by standard methods. Metaphases were analyzed by G-banding using Trypsin and Giemsa (GTG). 3.2. Fluorescence in situ hybridization Initially 18 Yeast Artificial Chromosomes (YAC) clones were selected based on the chromosomal band position and then 20 Bacterial Artificial Chromosome (BAC) clones were randomly selected using GRCh37/hg19 assembly utilizing the Ensembl and UCSC Genome browser. The international standard nomenclature was used for clone names, which were obtained from ImaGenes, Berlin, Germany [Kent et al., 2002; Pruitt et al., 2007]. BAC DNA was isolated using NucleoBond Plasmid Midi kit (Macherey-Nagel, Dueren, Germany) according to manufacturer’s instructions. The isolated BAC DNA was labeled with biotin-16-dUTP (Roche Diagnostics, Mannheim, Germany) by nick translation and FISH analysis was done on the patient metaphase slides as described by the standard protocols [Langer et al., 1981].
Several different restriction enzymes were selected to generate fragments of desired size for the breakpoint spanning clones. These fragments were gel eluted and directly labeled for further FISH experiments which helped to substantially narrow down the breakpoint region. 3.7. DNA sequencing PCR amplified bands were excised from the gel and purified using a QIAGEN gel extraction kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. DNA sequencing was performed using a BigDye v.1.1 Terminator Cycle sequencing kit (Applied Biosystems, Darmstadt, Germany). The products were analyzed on an ABI PrismÒ 3100 Genetic Analyzer. 3.8. Database searches and sequence alignments Homology searching was performed using the National Center for Biotechnology information BLAST program (http://blast.ncbi. nlm.nih.gov/Blast.cgi) against nt and the dbEST databases.
3.3. PCR amplification 4. Results To narrow down the breakpoints, primers specific for breakpoint spanning clones CTD-2007B5 and RP11-12N13 were designed and amplified by standard PCR. Thus two specific products of size 2.9 kb (50 -CTGATGATTAAAGGGATGAAGAC-30 ; 50 -ACCTGGTTGTTGGAGCTTATC-30 ) with CTD-2007H5 and 5.3 kb (50 -CTCGGTTCGTCTAAAGCTG C-30 ; 50 -ATTAACCCAAGACCTTAGCG-30 ) with RP11-12N13 as templates were amplified. For nested or semi-nested PCR, 2 ml of the first reaction mixture was used as the template for the second amplification. The amplified PCR products were electrophoresed on an agarose gel with a molecular weight DNA standard and stained with ethidium bromide. Long range PCR was performed with Long PCR enzyme mix (Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s instructions. Specific primers (50 -CTGTGTCTTAGGGTCTACTTTGGTCAGAAT-30 ; 50 -AGTGTTGAAATAGTTATCACCATGAGGAC30 ) for the breakpoint spanning BAC clone RP11-12N13 amplified a product of 13.7 kb. 3.4. Reverse Transcription (RT-PCR) Reverse Transcription was performed using the M-MLV Reverse Transcriptase, RNase H Minus (Promega, Mannheim, Germany) according to the specification provided by the supplier. As template; testes, brain, whole embryonic and fetal human total RNA (Biochain,
Cytogenetic analysis of the GTG banded metaphase chromosomes of the patient revealed a karyotype of 46, XX, inv(3) (p23q25q26) (Fig. 1B) according to ISCN (Fig. 1C). The chromosomal analyses of the patient’s father and brother showed a normal 46,XY male karyotype and her maternal aunt also showed a normal 46,XX female karyotype. But the chromosomal analyses of both her mother and maternal grandmother showed the same pericentric inversion 3 (Fig. 1A). The human chromosome 3 is metacentric and the inversion event in the patient is also equidistant from the centromere making it difficult to identify the p and q arms. So to rule out the arm identity one 3p terminal clone RP11-91K04 was used as a reference for all initial FISH experiments. To narrow down the breakpoint regions it was co-hybridized with specific probes to the patients metaphase spreads as double hybridization FISH. Initial FISH with YAC clones helped in the identification of breakpoint spanning YACs which had further helped in the easy selection of the BAC clones (data not shown). Subsequently, breakpoint spanning BAC contigs were assembled by screening genomic libraries. The 3p breakpoint interval was covered by a contig of 4 BAC clones (Fig. 2E), of which RP11-666G20 (186 kb) and CTD-2007B5 (97.5 kb) (Fig. 2A) showed split signals. For further analysis CTD-2007B5 clone was taken as the breakpoint spanning clone.
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Fig. 1. A: The pedigree showing the chromosome 3 inversion in the family. B: GTG banded partial karyogram on the metaphase chromosomes of the patient. C: The Ideogram showing the inversion chromosome 3.
In higher resolution mapping several different restriction enzymes were chosen to produce desired fragments covering the breakpoint spanning CTD-2007B5 clone. Many fragments were generated out of which a 26 kb BamHI fragment showed a split signal. This region was further overlapped by four different fragments i.e., 15.7 kb AccI, 11.7 kb EcoRV, 14.9 kb XbaI and 11.6 kb HincII. All these four fragments showed spilt signals. Subtracting the overlaps of all the four fragments the breakpoint was thus narrowed down to a mere 4 kb region, the distance between the AccI and HincII. An additional 8.6 kb KpnI fragment on the distal end showed signals on the q region only, thus validating our breakpoint region. Further a 2.9 kb PCR product were amplified with CTD-2007B5 as template also showing split signals confirming the breakpoint (Fig. 2C). The detailed map of the restriction fragments is shown in the figures (Fig. 2G). For the 3q breakpoint region also a contig of 4 BAC clones were established (Fig. 2F) and RP11-12N13 (174 kb) showed split signals spanning the breakpoint (Fig. 2B). Subtracting the overlapping distance of 95 kb with the adjacent clone RP11-480L9 (210 kb), it was deduced that the breakpoint region was restricted to 79 kb on 3q26.1 region. To further narrow down the breakpoint region a PCR product of size 13.7 kb was generated by Long range PCR with RP11-12N13 as template which showed split signals (data not shown). Subsequently two restriction fragments of 7.4 kb KpnI and 14.6 kb ScaI were selected covering the 13.7 kb region and FISH with both the fragments showed split signals. Thus, subtracting the overlapping regions of the two restriction fragments the breakpoint region was approximately narrowed down to a region of 3.7 kb region (Fig. 2H). This was also confirmed by a PCR product of 5.3 kb amplified with RP11-12N13 as template showing split signals (Fig. 2D). Parallel analysis with subcloned fragments of Sac I also showed similar results (data not shown). The identification of the junction fragment was not successful due to the presence of 30% repetitive elements on either sides of the breakpoint regions (Fig. 2I and 2J). 4.1. Identification of novel putative gene Neither the breakpoint spanning BAC clone RP11-12N13 nor CTD-2007B5 did contain coding regions of any known human gene.
Despite this, in silico analysis using the UCSC genome browser revealed a Genescan predicted gene NT_005612.1229 spanning the 5 kb breakpoint region on 3q. In order to further study this putative expressed sequence, BLAST analysis was performed and corresponding predicted mRNAs were identified. Covering a genomic size of 730 kb a total of 4 predicted genes (NT_005612.1229, NT_005612.1230 (NCBI36/hg18); XR_015783.1, XR_017393.1 (NCBI35/hg17)) were identified which had given rise to a gene structure with 12 exons (Fig. 3A). The putative mRNA sequence was 1959 bp showing a single ORF (Open Reading Frame) in the NCBI ORF Finder. This ORF predicted a polypeptide sequence of 652 amino acids starting with ATG in the first exon and a promoter sequence (TATAAA) 28 bp upstream of the start codon but a polyadenylation site in the 30 -region could not be identified. The 3q26.1 breakpoint region disrupted the second intron of this putative gene. To provide additional evidence for a possible role of this putative gene in the present case, expression analysis was performed by standard RT-PCR using cDNA libraries of human testes, brain, embryonic and fetal RNA. All of them were tested with different combinations of primers specific for every single exon. Repeated experiments with distinct cDNA samples and varying PCR conditions revealed amplicons with nested primers for exon 5 and exon 11 only in human fetal and embryonic RNA. One of these amplicons from embryonic RNA with a length of approximately 380 bp revealed 3 new exons located between former exons 9 and 10. These 3 exons of sizes 107 bp (9a), 122 bp (9b) and 106 bp (9c) formed one splice variant called SV1. Another splice variant SV2 (Acc. HG933790) of size 256 bp lacks exon 9b and could be verified using an exon 9a specific primer (P9a) in combination with exon 11 specific primer P11 (Fig. 3B). These results demonstrate a so far unknown transcribed sequence close to the 3q breakpoint region whereas no other genes are known in that area. 5. Discussion One method of identifying disease gene is to clone the breakpoint regions of the chromosomal rearrangement associated with the phenotype [Tadin et al., 2004]. In most cases one of the chromosomal breakpoints maps within or close to the respective gene thus disturbing its gene activity. An example is the inversion
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Fig. 2. FISH on lymphocyte metaphase spreads of the patient. A: FISH with 3p BAC clone CTD-2007B5 showing split signals on inverted chromosome 3. B: FISH with RP11-12N13 showing split signals on inverted chromosome 3. C: PCR-fragment (2.9 kb) spanning the breakpoint on 3p showing split signals. D: PCR-fragment (5.3 kb) spanning the 3q breakpoint showing split signals on inverted chromosome 3. E: The physical map representing BAC clones used for mapping the breakpoint region 3p24.1. F: The physical map representing BAC clones used for mapping the breakpoint region 3q26.1. G: The detailed physical map of 3p24.1 with breakpoint spanning BAC clone CTD-2007B5 (black bar) with the restriction fragments (red bars). The BAC clone with corresponding restriction fragments representing the 3p24.1 breakpoint with sizes given above each line. The 26.4 kb BamHI fragment as well as AccI (15.7 kb) and HincII(11.5 kb) showed a split signal. All these fragments anchored the breakpoint and the vertical interrupted line in red encompasses the breakpoint region. The 8.5 kb KpnI fragment (blue bar) showed signals only on the 3q region. The vertical black interrupted lines shows the overlapping region and the arrowhead black line shows the final narrowed down region of 4 kb. Also the 3p spanning PCR fragment is shown at the bottom. H: The detailed physical map of 3p26.1 with breakpoint spanning BAC clone RP11-12N13 (black bar) with the restriction fragments (red bars) and long range PCR fragments (green bar) spanning the breakpoint. The BAC clone RP11-12N13 represents the 3p26.1 breakpoint. The 13.7 kb long range PCR showed split signals in FISH as well as the ScaI and the KpnI restriction fragment narrowing down 3q region to 3.7 kb. The red interrupted vertical line encompasses the breakpoint region. Also in relation to the breakpoint the 3q spanning PCR fragment is shown at the bottom. I: The detailed repetitive elements surrounding the 3p24.1 breakpoint region. J: The detailed repetitive elements surrounding the of 3p26.1 breakpoint region.
event disrupting the factor VIII gene causing severe Haemophilia-A [Lakich et al., 1993]. Here, a familial pericentric inversion of chromosome 3 with SS was characterized by positional cloning strategy. By FISH the breakpoints were localized to 3p24.1 and 3q26.1 regions. Fine mapping in the 3p region with restriction fragments had narrowed the breakpoint region to a mere 4 kb region and on the 3q region with long-range PCR and restriction fragments the region was further narrowed down to 3.7 kb. Alternatively we had generated two fragments 2.9 kb on 3p region and 5.3 kb on 3q region which confirmed the exact breakpoint regions. In the process of identifying the junction fragment, several different products were obtained and to our surprise most of these products showed homology to chromosome 21. This could be best explained by the syntenic association of chromosomes 3 and 21 which may be ancestral for all placental mammals [Mueller et al., 2000]. The formation of these different PCR products hinted the presence of repetitive sequences in the breakpoint regions. As in the human genome it is known that the repeats represent an extraordinary trove of information about biological roles such as gene expression, genome structural stabilization and recombination [Murakami et al., 2004]. So, most of the recurring chromosomal breakpoints occur in
regions of high content of repetitive sequences that function as recombination hotspots mediating the rearrangement event. For example a pericentric inversion breakpoint 8 was characterized and the sequences surrounding the breakpoint regions showed highly repetitive LTR region, which mediated the inversion [Graw et al., 2000]. In our study, the sequences surrounding the 2.9 kb and 5.3 kb breakpoint regions showed 27.5% of repetitive elements with LTR33A, MER67C, L2 and MER67D on 3p region (Fig. 2I) and 26.4% with LTR16C, MER20, (TATG)n, AT rich region, MLT2B1, and LTR1B on 3q region (Fig. 2J). The lack of similarities in the repeat elements suggests that the molecular mechanism of the inversion in this case might have occurred by non-homologous (illegitimate) recombination. The phenotype of SS could be explained by two factors, disruption of a known gene followed by haploinsufficiency or lack of any gene(s) surrounding the breakpoint showing a position effect deregulating genes even at a distance of 1 Mb. A position effect is defined as a deleterious change in the level of gene expression brought about by a change in the position of the gene relative to its normal chromosomal environment, but not associated with an intragenic mutation or deletion [Kleinjan and
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Fig. 3. Mapping the balanced inversion and the putative gene. A: A BAC contig starting with breakpoint spanning RP11-12N13 harboring 4 distinct Genescan sequences representing parts of the putative gene put together as 12 exons. B: Gel picture showing the results of a re-amplification PCR after cDNA synthesis and first round PCR with primers specific for putative exon 5 (p5) and exon 11 (p11). Using nested primers (p5n, p11n) in the second PCR only embryonic (E) and fetal (F) RNA shows PCR fragments. There are no products in brain (B) or testes (T) RNA. Cloning and sequencing of the 380 bp fragments results in a transcript with 3 new exons between exon 9 and 10 representing the splice variant SV1. A second splice variant SV2 could be verified with specific primers p9a and p11 lacking exon 9b. (N ¼ negative control; NR ¼ negative control of reamplification; B ¼ brain; T ¼ testes, E ¼ embryo; F ¼ fetal).
vanHeyningen, 1998]. For instance, position effects were even demonstrated in genes which are located at a maximum distance of 1.3 Mb from the breakpoint region [Velagaleti et al., 2005]. As the stature gene SHOX2 was approximately 8 Mb proximal to the 3q26.1 breakpoint region, it was ruled out as a candidate gene in this patient. Other candidates likely to show position effect are ZBBX (zinc finger B-box domain-containing protein 1), SERPINI2 (serine protease inhibitor, clade I, member 2), PDCD10 (programmed cell death protein 10) and SERPINI1 (serine protease inhibitor, clade I, member 1) which are present distal to the 3q breakpoint region. There is nothing specific known about the function of ZBBX whereas loss of function of PDCD10 results in the onset of Cerebral Cavernous Malformations (CCM) [Bergametti et al., 2004; Chen et al., 2007]. SERPINI1/2 are involved in many cellular processes, SERPINI1 (Neuroserpin) was first identified as a secreted protein from the axons of dorsal root ganglion neurons and is expressed in the late stages of neurogenesis [Stöckli et al., 1989]. The protein encoded by SERPINI2 in contrast plays a central role in the regulation of a wide variety of physiological processes, and has a role in growth control and especially growth suppressing pathway. Due to which it can be speculated that the inversion breakpoint on 3q26.1 might have down regulated the SERPINI2 gene located almost 1 Mb away from the breakpoint showing position effect. Apparently, the inversion rearrangement might also have affected some other gene located far from the breakpoint thus causing the ‘unique’ phenotype of SS in this family. Apparently, in the 3q26.1 breakpoint region we had identified a novel putative gene with 12 exons and the breakpoint disrupted the second intron of this gene. We had seen the expression of parts of this putative gene in human embryonic RNA only. Probably, the putative gene could be expressed early in development lacking transcripts in all other tissues used for cDNA synthesis. The
transcripts we had identified revealed 3 new exons following rules for exon-intron boundaries. In addition we have identified two splice variants of this so far unknown gene. Whether or not these new exons are part of the putative transcript with an ORF predicting a polypeptide of 652 amino acids, is not clear yet. Both exons 9b and 9c exhibits stop codons in all three reading frames which would break the putative polypeptide of the core transcript. On the other side it is not unusual to share exons as part of totally different transcripts like the GNAS locus on human chromosome 20 and mouse chromosome 2, coding for several different gene products like GNAS1, XLas and NESP55 [Weinstein et al., 2002]. Nevertheless we have identified a transcription unit in the vicinity of the 3q26.1 breakpoint whereas no other transcripts or breakpoint spanning product was identified yet. The prediction status of all other exons leaves the possibility of other new exons even beyond the breakpoint. Therefore we focus on 50 -RACE experiments to evaluate any possible transcript targeting the breakpoint which is now about 100 kb away from exon 5. Hence we propose that disruption of this putative gene may have a role in causing the SS in the family. Regarding the rearrangement of this inversion we looked for already known CNVs in the breakpoint regions using the Decipher database (https://decipher.sanger.ac.uk/). In particular, there are 6 gain of functions (DECIPHER case numbers 280373, 2839, 267118, 257117, 250174 and 270613) showing no comparable phenotypic alterations in the 3q24 region. In addition two overlapping deletions were stated (DECIPHER cases numbers 630 and 291507), from which case 291507 exhibit a very complex phenotype with short stature mentioned. However, both deletions were comparable in length with around 9 Mb representing an overlapping area of 6 Mb containing the breakpoint region but the case number 630 shows intellectual disability and a triphalangeal thumb only. On 3q26.1 breakpoint also 6 duplications were
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mentioned without any hint on short stature (DECIPHER cases numbers 289621, 283584, 267682, 1561, 251819 and 250564). Also two deletions spanning the breakpoint were listed (DECIPHER cases numbers 250665 and 257440), from which case 250665 contains 270 genes with a broad phenotypic spectrum whereas case 257440 containing 107 genes shows only broad thumb, intellectual disability and macrocephaly. Another deletion named case number 289184 which is not spanning the breakpoint but deleting most of the putative gene as well as all exons found to be expressed together with the already known gene ZBBX which also shows intellectual disability and psychosis as phenotype. So the DECIPHER database could not confirm any short stature phenotype regarding our breakpoints with the exception of case 291507 which is not that convincing due to the overlap with case 630 and the complex phenotype. In summary, we had characterized the breakpoints of a novel inversion chromosome 3 in a family with SS. The breakpoints had not disrupted any validated genes but disrupted the second intron of a novel gene, from which parts are transcribed in human embryonic RNA. We also speculate the non-homologous recombination as a molecular mechanism for this inversion. We propose that disruption of this putative gene may have a role in causing the short stature in the family. Validation of the identified putative gene, and subsequent functional analysis could provide insights into the inversion breakpoint and its association with the phenotype precisely. Conflict of interest The authors declare no conflict of interest. Acknowledgments We thank Sebastian Werner for the expression analysis of so many different human RNAs. This study was supported by Deutscher Akademischer Austausch Dienst (DAAD) or German Academic Exchange Service sponsored fellowship (Code no: A/04/ 08474 ) to URD. References Baere ED, Speleman F, Van RN, De Paepe A, Messiaen L. Assignment of SHOX2 (alias OG12X and SHOT) to human chromosome bands 3q25/q26.1 by in situ hybridization. Cytogenet Cell Genet 1998;82:228e9. Barajas-Barajas LO, Velarde-Felix S, Elizarraras-Rivas J, Hernandez-Zaragoza G, Vazquez-Herrera JA. De novo ring chromosome 3 in a girl with hypoplastic thumb and coloboma of iris. Genet Couns 2001;12:151e6. Bergametti F, Denier C, Labauge P, Arnoult M, Boetto S, Clanet M, et al. Mutations within the programmed cell death 10 gene cause cerebral cavernous malformations. Am J Hum Genet 2004;76:42e51. Blaschke RJ, Monaghan AP, Schiller S, Schechinger B, Rao E, Padilla-Nash H, et al. SHOT, a SHOX-related homeobox gene, is implicated in craniofacial, brain, heart, and limb development. Proc Natl Acad Sci 1998;95:2406e11. Brueton LA, Barber JC, Huson SM, Winter RM. Partial monosomy 3q in a boy with short stature, developmental delay, and mild dysmorphic features. J Med Genet 1989;26:729e30. Chen PY, Chang WS, Chou RH, Lai YK, Lin SC, Chi CY, et al. Two non-homologous brain diseases-related genes, SERPINI1 and PDCD10, are tightly linked by an asymmetric bidirectional promoter in an evolutionarily conserved manner. BMC Mol Biol 2007;8:2. Chen W, Ullmann R, Langnick C, Menzel C, Wotschofsky Z, Hu H, et al. Breakpoint analysis of balanced chromosome rearrangements by next-generation pairedend sequencing. Eur J Hum Genet 2010;18:539e43.
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Clement-Jones M, Schiller S, Rao E, Blaschke RJ, Zuniga A, Zeller R, et al. The short stature homeoboxgene SHOX is involved in skeletal abnormalities in Turner syndrome. Hum Mol Genet 2000;9:695e702. Enders H. Chromosomal and genetic forms of growth failure. Baillieres Clin Endocrinol Metab 1992;6:621e43. Gahrton C, Nahi H, Jansson M, Wallblom A, Alici E, Sutlu T, et al. Constitutional inv(3) in myelodysplastic syndromes. Leuk Res 2010;34:1627e9. Graw SL, Sample T, Bleskan J, Sujansky E, Patterson D. Cloning, sequencing, and analysis of Inv8 chromosome breakpoints associated with recombinant 8 syndrome. Am J Hum Genet 2000;66:1138e44. Hasle H, Jacobsen BB, Asschenfeldt P, Andersen K. Mediastinal germ cell tumourssociated with Klinefelter syndrome. A report of case and review of the literature. Eur J Pediatr 1992;151:735e9. Kalscheuer VM, Tao J, Donnelly A, Hollway G, Schwinger E, Kübart S, et al. Disruption of the serine/threonine kinase 9 gene causes severe X-linked infantile spasms and mental retardation. Am J Hum Genet 2003;72:1401e11. Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, et al. The human genome browser at UCSC. Genome Res 2002;12:996e1006. Kleinjan DJ, vanHeyningen V. Position effect in human genetic disease. Hum Mol Genet 1998;7:1611e8. Kondo Y, Mizuno S, Ohara K, Nakamura T, Yamada K, Yamamori S, et al. Two cases of partial trisomy 21 (pter-pq22.1) without the major features of Down syndrome. Am J Med Genet 2006;140:227e32. Lakich D, Kazazian Jr HH, Antonarakis SE, Gitschier J. Inversions disrupting the factor VIII gene are a common cause of severe haemoplilia A. Nat Genet 1993;5: 236e41. Lam WF, Hau WL, Lam TS. An evaluation of referrals for genetics investigation of short stature in Hong Kong. Chin Med J 2002;115:607e11. Langer PR, Waldrop AA, Ward DC. Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc Natl Acad Sci 1981;78: 6633e7. Moller RS, Kubart S, Hoeltzenbein M, Heye B, Vogel I, Hansen CP, et al. Truncation of the Down syndrome candidate gene DYRK1A in two unrelated patients with microcephaly. Am J Hum Genet 2008;82:1165e70. Moreno-Garcia M, Fernandez-Martinez FJ, Miranda EB. Chromosomal anomalies in patients with short stature. Pediatr Int 2005;47:546e9. Mueller S, Stanyon R, Finelli P, Archidiacono N, Wienberg J. Molecular cytogenetic dissection of human chromosomes 3 and 21 evolution. Proc Natl Acad Sci 2000;97:206e11. Murakami H, Sugaya N, Sato M, Imaizumi A, Aburatani S, Horimoto K. Detection of inter-spread repeat sequence in genomic DNA sequence. Genome Inform 2004;15:170e9. Musebeck J, Mohnike K, Beye P, Tonnies H, Neitzel H, Schnabel D, et al. Short stature homeobox-containing gene deletion screening by fluorescence in situ hybridization in patients with short stature. Eur J Pediatr 2001;160:561e5. Nguyen T, McDonnell CM, Zacharin MR. Primary ovarian failure and deletions of the long arm of chromosome 3. J Pediatr Endocrinol Metab 2005;18:1013e7. Pruitt KD, Tatusova T, Maglott DR. NCBI Reference Sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res 2007;35:61e5. Soudek D, Sroka H. Inversion of ‘flourescent’ segment in chromosome 3: polymorphic trait. Hum Genet 1978;44:109e15. Stine SB, Clark CE, Telfer MA, Casey PA, Cowell HR. Ullrich-Turner syndrome (45,X/ 46,Xi[Xq]) in a child with a familial inversion of chromosome 3. Am J Med Genet 1982;12:57e62. Stöckli KA, Lottspeich F, Sendtner M, Masiakowski P, Carroll P, Götz R, et al. Molecular cloning, expression, andregional distribution of rat ciliaryneurotrophic factor. Nature 1989;342:920e3. Tadin SM, Warburton D, Baumeister FAM, Fischer SG, Yonan J, Gilliam TC, et al. Cloning of the breakpoints of a de novo inversion of chromosome 8 inv(8)(p11.2q23.1) in a patient with Ambras syndrome. Cytogenet Genome Res 2004;107:68e76. Velagaleti GVN, Bien-Willer GA, Northuo JK, Lockhart LH, Hawkins JC, Jalal SM, et al. Position effects due to chromosome breakpoints that map w900 kb upstream and w1.3 Mb Downstream of SOX9 in two patients with campomelic dysplasia. Am J Hum Genet 2005;76:652e62. Weinstein LS, Chen M, Liu J. Gs(alpha) mutations and imprinting defects in human disease. Ann N Y Acad Sci 2002;968:173e97. Welborn J. Constitutional chromosome aberrations as pathogenetic events in hematologic malignancies. Cancer Genet Cytogenet 2004;149:137e53. Yip MY, MacKenzie H, Kovacic A, Mc Intosh A. Chromosome 3p23 break with ring formation and translocation of displaced 3p23/pter segment to 6pter. J Med Genet 1996;33:789e92.
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European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Clinical research
Molecular cytogenetic characterization of a familial pericentric inversion 3 associated with short stature Usha R. Dutta a, b, *, Ingo Hansmann a, Dietmar Schlote a a b
Institut fuer Humangenetik, Martin Luther University, Halle-Wittenberg, Halle (Saale) 06097, Germany Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad 500 001, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 July 2014 Accepted 5 January 2015 Available online 13 January 2015
Short stature refers to the height of an individual which is below expected. The causes are heterogenous and influenced by several genetic and environmental factors. Chromosomal abnormalities are a major cause of diseases and cytogenetic mapping is one of the powerful tools for the identification of novel disease genes. Here we report a three generation family with a heterozygous pericentric inversion of 46, XX, inv(3) (p24.1q26.1) associated with Short stature. Positional cloning strategy was used to physically map the breakpoint regions by Fluorescence in situ hybridization (FISH). Fine mapping was performed with Bacterial Artificial Chromosome (BAC) clones spanning the breakpoint regions. In order to further characterize the breakpoint regions extensive molecular mapping was carried out with the breakpoint spanning BACs which narrowed down the breakpoint region to 2.9 kb and 5.3 kb regions on p and q arm respectively. Although these breakpoints did not disrupt any validated genes, we had identified a novel putative gene in the vicinity of 3q26.1 breakpoint region by in silico analysis. Trying to find the presence of any transcripts of this putative gene we analyzed human total RNA by RT-PCR and identified transcripts containing three new exons confirming the existence of a so far unknown gene close to the 3q breakpoint. Ó 2015 Elsevier Masson SAS. All rights reserved.
Keywords: BAC clones Breakpoint regions FISH Inversion Physical mapping Short stature
1. Introduction Short stature (SS) refers to the height of an individual which is below expected and is one of the major features found in many syndromic cases. The causes of SS are many and influenced by several genetic and environmental factors hence the systematic classification of the various syndromes involving SS is not possible [Enders, 1992]. The genetic factors may include chromosomal anomalies in both autosomes as well as in sex chromosomes. The most common cytogenetic cause of SS in females is Turner syndrome, with different chromosomal variants affecting X chromosome. The Short stature HOmeobox-containing gene on the short arm of X and Y (SHOX) is an important determining factor for stature phenotype [Musebeck et al., 2001]. Whereas in autosomes; the SHOX2 gene is localized on chromosome 3q25-26 [Baere et al., 1998; Blaschke et al., 1998]. SHOX2 is closely related to SHOX on sex chromosome [Clement-Jones et al., 2000]. But till * Corresponding author. Diagnostics Division, Center for DNA Fingerprinting and Diagnostics, Tuljaguda complex, 4-1-714, Hyderabad 500 001, Andhra Pradesh, India. Tel.: þ91 40 24749337; fax: þ91 40 24749448. E-mail addresses: [email protected], [email protected] (U.R. Dutta). http://dx.doi.org/10.1016/j.ejmg.2015.01.001 1769-7212/Ó 2015 Elsevier Masson SAS. All rights reserved.
date there are only two reports on the chromosomal analyses with SS [Lam et al., 2002; Moreno-Garcia et al., 2005]. In general, the structural rearrangements alter the genome architecture and may result in human disease phenotypes. The patients with translocations and inversions often have breakpoints located within the disease gene, or very close to it [Chen et al., 2010]. In order to identify the disease gene, characterization of the breakpoints has often been a promising start point in the molecular elucidation of early-onset of Mendelian disorders [Kalscheuer et al., 2003; Moller et al., 2008]. The constitutional pericentric inversion on chromosome 3 occurs rarely [Gahrton et al., 2010], accounting for about 4% of the normal population [Soudek and Sroka, 1978]. Most of the inversions are not clearly visualized by GTG banding [Hasle et al., 1992] and therefore not frequently reported [Welborn, 2004]. Nevertheless a few cases of chromosome 3 abnormalities associated with SS were reported in literature [Barajas-Barajas et al., 2001], like a 3q deletion with SS reported by Nguyen [Nguyen et al., 2005] and a partial monosomy 3q with SS reported by Brueton et al., 1989. Most of other cases were associated with other chromosomal rearrangements [Kondo et al., 2006; Stine et al., 1982; Yip et al., 1996]. But to the best of our knowledge there is no case reported with a familial inversion
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3 associated with SS. In this study, we characterized a familial inversion inv(3) (p24.1q26.1) associated with SS and identified a novel putative gene on 3q26.1 breakpoint region. 2. Patient data A 16-year old girl was referred to our center with SS. She was the first child born at term to a non-consanguineous couple after an uneventful pregnancy. Her milestones were normal. She was only 146 cm tall, significantly under the 3rd centile, whereas her 7 year old brother was 125 cm tall, consistent with the 50th centile. The detailed family history and written consent was taken from the patient’s family. 3. Methods
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Heidelberg, Germany) were used. Specific products were amplified with exon 5 specific primers p5 (50 GACATTGTCTGGGAGCAGC-30 ), p5n (50 -AGTAGATCCTGAAGGCGTG-30 ) and exon 11 specific primers p11 (50 -CTTGTCTAAGGTTGCAGACTCA-30 ), p11n (50 GTGAAGCCTCGTTTCATCC-30 ) in a first round of standard PCR and re-amplified using the nested primers. For the splice variant SV2 an exon 9a specific primer p9a (50 -GGAAAGAAGCAGAGGTAGCC-30 ) was used as forward primer. 3.5. Subcloning The breakpoint spanning clone RP11-12N13 on 3q26.1 region was digested with Sac I restriction enzyme and the desired fragments were excised from the gel, eluted and subcloned into pBluescript SK Vector which were further labeled and used as probes for FISH mapping.
3.1. Cytogenetic analyses 3.6. Restriction digestion Chromosomal analyses were carried out on the peripheral blood lymphocytes in the patient, her parents, brother, maternal aunt and maternal grandmother by standard methods. Metaphases were analyzed by G-banding using Trypsin and Giemsa (GTG). 3.2. Fluorescence in situ hybridization Initially 18 Yeast Artificial Chromosomes (YAC) clones were selected based on the chromosomal band position and then 20 Bacterial Artificial Chromosome (BAC) clones were randomly selected using GRCh37/hg19 assembly utilizing the Ensembl and UCSC Genome browser. The international standard nomenclature was used for clone names, which were obtained from ImaGenes, Berlin, Germany [Kent et al., 2002; Pruitt et al., 2007]. BAC DNA was isolated using NucleoBond Plasmid Midi kit (Macherey-Nagel, Dueren, Germany) according to manufacturer’s instructions. The isolated BAC DNA was labeled with biotin-16-dUTP (Roche Diagnostics, Mannheim, Germany) by nick translation and FISH analysis was done on the patient metaphase slides as described by the standard protocols [Langer et al., 1981].
Several different restriction enzymes were selected to generate fragments of desired size for the breakpoint spanning clones. These fragments were gel eluted and directly labeled for further FISH experiments which helped to substantially narrow down the breakpoint region. 3.7. DNA sequencing PCR amplified bands were excised from the gel and purified using a QIAGEN gel extraction kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. DNA sequencing was performed using a BigDye v.1.1 Terminator Cycle sequencing kit (Applied Biosystems, Darmstadt, Germany). The products were analyzed on an ABI PrismÒ 3100 Genetic Analyzer. 3.8. Database searches and sequence alignments Homology searching was performed using the National Center for Biotechnology information BLAST program (http://blast.ncbi. nlm.nih.gov/Blast.cgi) against nt and the dbEST databases.
3.3. PCR amplification 4. Results To narrow down the breakpoints, primers specific for breakpoint spanning clones CTD-2007B5 and RP11-12N13 were designed and amplified by standard PCR. Thus two specific products of size 2.9 kb (50 -CTGATGATTAAAGGGATGAAGAC-30 ; 50 -ACCTGGTTGTTGGAGCTTATC-30 ) with CTD-2007H5 and 5.3 kb (50 -CTCGGTTCGTCTAAAGCTG C-30 ; 50 -ATTAACCCAAGACCTTAGCG-30 ) with RP11-12N13 as templates were amplified. For nested or semi-nested PCR, 2 ml of the first reaction mixture was used as the template for the second amplification. The amplified PCR products were electrophoresed on an agarose gel with a molecular weight DNA standard and stained with ethidium bromide. Long range PCR was performed with Long PCR enzyme mix (Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s instructions. Specific primers (50 -CTGTGTCTTAGGGTCTACTTTGGTCAGAAT-30 ; 50 -AGTGTTGAAATAGTTATCACCATGAGGAC30 ) for the breakpoint spanning BAC clone RP11-12N13 amplified a product of 13.7 kb. 3.4. Reverse Transcription (RT-PCR) Reverse Transcription was performed using the M-MLV Reverse Transcriptase, RNase H Minus (Promega, Mannheim, Germany) according to the specification provided by the supplier. As template; testes, brain, whole embryonic and fetal human total RNA (Biochain,
Cytogenetic analysis of the GTG banded metaphase chromosomes of the patient revealed a karyotype of 46, XX, inv(3) (p23q25q26) (Fig. 1B) according to ISCN (Fig. 1C). The chromosomal analyses of the patient’s father and brother showed a normal 46,XY male karyotype and her maternal aunt also showed a normal 46,XX female karyotype. But the chromosomal analyses of both her mother and maternal grandmother showed the same pericentric inversion 3 (Fig. 1A). The human chromosome 3 is metacentric and the inversion event in the patient is also equidistant from the centromere making it difficult to identify the p and q arms. So to rule out the arm identity one 3p terminal clone RP11-91K04 was used as a reference for all initial FISH experiments. To narrow down the breakpoint regions it was co-hybridized with specific probes to the patients metaphase spreads as double hybridization FISH. Initial FISH with YAC clones helped in the identification of breakpoint spanning YACs which had further helped in the easy selection of the BAC clones (data not shown). Subsequently, breakpoint spanning BAC contigs were assembled by screening genomic libraries. The 3p breakpoint interval was covered by a contig of 4 BAC clones (Fig. 2E), of which RP11-666G20 (186 kb) and CTD-2007B5 (97.5 kb) (Fig. 2A) showed split signals. For further analysis CTD-2007B5 clone was taken as the breakpoint spanning clone.
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Fig. 1. A: The pedigree showing the chromosome 3 inversion in the family. B: GTG banded partial karyogram on the metaphase chromosomes of the patient. C: The Ideogram showing the inversion chromosome 3.
In higher resolution mapping several different restriction enzymes were chosen to produce desired fragments covering the breakpoint spanning CTD-2007B5 clone. Many fragments were generated out of which a 26 kb BamHI fragment showed a split signal. This region was further overlapped by four different fragments i.e., 15.7 kb AccI, 11.7 kb EcoRV, 14.9 kb XbaI and 11.6 kb HincII. All these four fragments showed spilt signals. Subtracting the overlaps of all the four fragments the breakpoint was thus narrowed down to a mere 4 kb region, the distance between the AccI and HincII. An additional 8.6 kb KpnI fragment on the distal end showed signals on the q region only, thus validating our breakpoint region. Further a 2.9 kb PCR product were amplified with CTD-2007B5 as template also showing split signals confirming the breakpoint (Fig. 2C). The detailed map of the restriction fragments is shown in the figures (Fig. 2G). For the 3q breakpoint region also a contig of 4 BAC clones were established (Fig. 2F) and RP11-12N13 (174 kb) showed split signals spanning the breakpoint (Fig. 2B). Subtracting the overlapping distance of 95 kb with the adjacent clone RP11-480L9 (210 kb), it was deduced that the breakpoint region was restricted to 79 kb on 3q26.1 region. To further narrow down the breakpoint region a PCR product of size 13.7 kb was generated by Long range PCR with RP11-12N13 as template which showed split signals (data not shown). Subsequently two restriction fragments of 7.4 kb KpnI and 14.6 kb ScaI were selected covering the 13.7 kb region and FISH with both the fragments showed split signals. Thus, subtracting the overlapping regions of the two restriction fragments the breakpoint region was approximately narrowed down to a region of 3.7 kb region (Fig. 2H). This was also confirmed by a PCR product of 5.3 kb amplified with RP11-12N13 as template showing split signals (Fig. 2D). Parallel analysis with subcloned fragments of Sac I also showed similar results (data not shown). The identification of the junction fragment was not successful due to the presence of 30% repetitive elements on either sides of the breakpoint regions (Fig. 2I and 2J). 4.1. Identification of novel putative gene Neither the breakpoint spanning BAC clone RP11-12N13 nor CTD-2007B5 did contain coding regions of any known human gene.
Despite this, in silico analysis using the UCSC genome browser revealed a Genescan predicted gene NT_005612.1229 spanning the 5 kb breakpoint region on 3q. In order to further study this putative expressed sequence, BLAST analysis was performed and corresponding predicted mRNAs were identified. Covering a genomic size of 730 kb a total of 4 predicted genes (NT_005612.1229, NT_005612.1230 (NCBI36/hg18); XR_015783.1, XR_017393.1 (NCBI35/hg17)) were identified which had given rise to a gene structure with 12 exons (Fig. 3A). The putative mRNA sequence was 1959 bp showing a single ORF (Open Reading Frame) in the NCBI ORF Finder. This ORF predicted a polypeptide sequence of 652 amino acids starting with ATG in the first exon and a promoter sequence (TATAAA) 28 bp upstream of the start codon but a polyadenylation site in the 30 -region could not be identified. The 3q26.1 breakpoint region disrupted the second intron of this putative gene. To provide additional evidence for a possible role of this putative gene in the present case, expression analysis was performed by standard RT-PCR using cDNA libraries of human testes, brain, embryonic and fetal RNA. All of them were tested with different combinations of primers specific for every single exon. Repeated experiments with distinct cDNA samples and varying PCR conditions revealed amplicons with nested primers for exon 5 and exon 11 only in human fetal and embryonic RNA. One of these amplicons from embryonic RNA with a length of approximately 380 bp revealed 3 new exons located between former exons 9 and 10. These 3 exons of sizes 107 bp (9a), 122 bp (9b) and 106 bp (9c) formed one splice variant called SV1. Another splice variant SV2 (Acc. HG933790) of size 256 bp lacks exon 9b and could be verified using an exon 9a specific primer (P9a) in combination with exon 11 specific primer P11 (Fig. 3B). These results demonstrate a so far unknown transcribed sequence close to the 3q breakpoint region whereas no other genes are known in that area. 5. Discussion One method of identifying disease gene is to clone the breakpoint regions of the chromosomal rearrangement associated with the phenotype [Tadin et al., 2004]. In most cases one of the chromosomal breakpoints maps within or close to the respective gene thus disturbing its gene activity. An example is the inversion
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Fig. 2. FISH on lymphocyte metaphase spreads of the patient. A: FISH with 3p BAC clone CTD-2007B5 showing split signals on inverted chromosome 3. B: FISH with RP11-12N13 showing split signals on inverted chromosome 3. C: PCR-fragment (2.9 kb) spanning the breakpoint on 3p showing split signals. D: PCR-fragment (5.3 kb) spanning the 3q breakpoint showing split signals on inverted chromosome 3. E: The physical map representing BAC clones used for mapping the breakpoint region 3p24.1. F: The physical map representing BAC clones used for mapping the breakpoint region 3q26.1. G: The detailed physical map of 3p24.1 with breakpoint spanning BAC clone CTD-2007B5 (black bar) with the restriction fragments (red bars). The BAC clone with corresponding restriction fragments representing the 3p24.1 breakpoint with sizes given above each line. The 26.4 kb BamHI fragment as well as AccI (15.7 kb) and HincII(11.5 kb) showed a split signal. All these fragments anchored the breakpoint and the vertical interrupted line in red encompasses the breakpoint region. The 8.5 kb KpnI fragment (blue bar) showed signals only on the 3q region. The vertical black interrupted lines shows the overlapping region and the arrowhead black line shows the final narrowed down region of 4 kb. Also the 3p spanning PCR fragment is shown at the bottom. H: The detailed physical map of 3p26.1 with breakpoint spanning BAC clone RP11-12N13 (black bar) with the restriction fragments (red bars) and long range PCR fragments (green bar) spanning the breakpoint. The BAC clone RP11-12N13 represents the 3p26.1 breakpoint. The 13.7 kb long range PCR showed split signals in FISH as well as the ScaI and the KpnI restriction fragment narrowing down 3q region to 3.7 kb. The red interrupted vertical line encompasses the breakpoint region. Also in relation to the breakpoint the 3q spanning PCR fragment is shown at the bottom. I: The detailed repetitive elements surrounding the 3p24.1 breakpoint region. J: The detailed repetitive elements surrounding the of 3p26.1 breakpoint region.
event disrupting the factor VIII gene causing severe Haemophilia-A [Lakich et al., 1993]. Here, a familial pericentric inversion of chromosome 3 with SS was characterized by positional cloning strategy. By FISH the breakpoints were localized to 3p24.1 and 3q26.1 regions. Fine mapping in the 3p region with restriction fragments had narrowed the breakpoint region to a mere 4 kb region and on the 3q region with long-range PCR and restriction fragments the region was further narrowed down to 3.7 kb. Alternatively we had generated two fragments 2.9 kb on 3p region and 5.3 kb on 3q region which confirmed the exact breakpoint regions. In the process of identifying the junction fragment, several different products were obtained and to our surprise most of these products showed homology to chromosome 21. This could be best explained by the syntenic association of chromosomes 3 and 21 which may be ancestral for all placental mammals [Mueller et al., 2000]. The formation of these different PCR products hinted the presence of repetitive sequences in the breakpoint regions. As in the human genome it is known that the repeats represent an extraordinary trove of information about biological roles such as gene expression, genome structural stabilization and recombination [Murakami et al., 2004]. So, most of the recurring chromosomal breakpoints occur in
regions of high content of repetitive sequences that function as recombination hotspots mediating the rearrangement event. For example a pericentric inversion breakpoint 8 was characterized and the sequences surrounding the breakpoint regions showed highly repetitive LTR region, which mediated the inversion [Graw et al., 2000]. In our study, the sequences surrounding the 2.9 kb and 5.3 kb breakpoint regions showed 27.5% of repetitive elements with LTR33A, MER67C, L2 and MER67D on 3p region (Fig. 2I) and 26.4% with LTR16C, MER20, (TATG)n, AT rich region, MLT2B1, and LTR1B on 3q region (Fig. 2J). The lack of similarities in the repeat elements suggests that the molecular mechanism of the inversion in this case might have occurred by non-homologous (illegitimate) recombination. The phenotype of SS could be explained by two factors, disruption of a known gene followed by haploinsufficiency or lack of any gene(s) surrounding the breakpoint showing a position effect deregulating genes even at a distance of 1 Mb. A position effect is defined as a deleterious change in the level of gene expression brought about by a change in the position of the gene relative to its normal chromosomal environment, but not associated with an intragenic mutation or deletion [Kleinjan and
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Fig. 3. Mapping the balanced inversion and the putative gene. A: A BAC contig starting with breakpoint spanning RP11-12N13 harboring 4 distinct Genescan sequences representing parts of the putative gene put together as 12 exons. B: Gel picture showing the results of a re-amplification PCR after cDNA synthesis and first round PCR with primers specific for putative exon 5 (p5) and exon 11 (p11). Using nested primers (p5n, p11n) in the second PCR only embryonic (E) and fetal (F) RNA shows PCR fragments. There are no products in brain (B) or testes (T) RNA. Cloning and sequencing of the 380 bp fragments results in a transcript with 3 new exons between exon 9 and 10 representing the splice variant SV1. A second splice variant SV2 could be verified with specific primers p9a and p11 lacking exon 9b. (N ¼ negative control; NR ¼ negative control of reamplification; B ¼ brain; T ¼ testes, E ¼ embryo; F ¼ fetal).
vanHeyningen, 1998]. For instance, position effects were even demonstrated in genes which are located at a maximum distance of 1.3 Mb from the breakpoint region [Velagaleti et al., 2005]. As the stature gene SHOX2 was approximately 8 Mb proximal to the 3q26.1 breakpoint region, it was ruled out as a candidate gene in this patient. Other candidates likely to show position effect are ZBBX (zinc finger B-box domain-containing protein 1), SERPINI2 (serine protease inhibitor, clade I, member 2), PDCD10 (programmed cell death protein 10) and SERPINI1 (serine protease inhibitor, clade I, member 1) which are present distal to the 3q breakpoint region. There is nothing specific known about the function of ZBBX whereas loss of function of PDCD10 results in the onset of Cerebral Cavernous Malformations (CCM) [Bergametti et al., 2004; Chen et al., 2007]. SERPINI1/2 are involved in many cellular processes, SERPINI1 (Neuroserpin) was first identified as a secreted protein from the axons of dorsal root ganglion neurons and is expressed in the late stages of neurogenesis [Stöckli et al., 1989]. The protein encoded by SERPINI2 in contrast plays a central role in the regulation of a wide variety of physiological processes, and has a role in growth control and especially growth suppressing pathway. Due to which it can be speculated that the inversion breakpoint on 3q26.1 might have down regulated the SERPINI2 gene located almost 1 Mb away from the breakpoint showing position effect. Apparently, the inversion rearrangement might also have affected some other gene located far from the breakpoint thus causing the ‘unique’ phenotype of SS in this family. Apparently, in the 3q26.1 breakpoint region we had identified a novel putative gene with 12 exons and the breakpoint disrupted the second intron of this gene. We had seen the expression of parts of this putative gene in human embryonic RNA only. Probably, the putative gene could be expressed early in development lacking transcripts in all other tissues used for cDNA synthesis. The
transcripts we had identified revealed 3 new exons following rules for exon-intron boundaries. In addition we have identified two splice variants of this so far unknown gene. Whether or not these new exons are part of the putative transcript with an ORF predicting a polypeptide of 652 amino acids, is not clear yet. Both exons 9b and 9c exhibits stop codons in all three reading frames which would break the putative polypeptide of the core transcript. On the other side it is not unusual to share exons as part of totally different transcripts like the GNAS locus on human chromosome 20 and mouse chromosome 2, coding for several different gene products like GNAS1, XLas and NESP55 [Weinstein et al., 2002]. Nevertheless we have identified a transcription unit in the vicinity of the 3q26.1 breakpoint whereas no other transcripts or breakpoint spanning product was identified yet. The prediction status of all other exons leaves the possibility of other new exons even beyond the breakpoint. Therefore we focus on 50 -RACE experiments to evaluate any possible transcript targeting the breakpoint which is now about 100 kb away from exon 5. Hence we propose that disruption of this putative gene may have a role in causing the SS in the family. Regarding the rearrangement of this inversion we looked for already known CNVs in the breakpoint regions using the Decipher database (https://decipher.sanger.ac.uk/). In particular, there are 6 gain of functions (DECIPHER case numbers 280373, 2839, 267118, 257117, 250174 and 270613) showing no comparable phenotypic alterations in the 3q24 region. In addition two overlapping deletions were stated (DECIPHER cases numbers 630 and 291507), from which case 291507 exhibit a very complex phenotype with short stature mentioned. However, both deletions were comparable in length with around 9 Mb representing an overlapping area of 6 Mb containing the breakpoint region but the case number 630 shows intellectual disability and a triphalangeal thumb only. On 3q26.1 breakpoint also 6 duplications were
U.R. Dutta et al. / European Journal of Medical Genetics 58 (2015) 154e159
mentioned without any hint on short stature (DECIPHER cases numbers 289621, 283584, 267682, 1561, 251819 and 250564). Also two deletions spanning the breakpoint were listed (DECIPHER cases numbers 250665 and 257440), from which case 250665 contains 270 genes with a broad phenotypic spectrum whereas case 257440 containing 107 genes shows only broad thumb, intellectual disability and macrocephaly. Another deletion named case number 289184 which is not spanning the breakpoint but deleting most of the putative gene as well as all exons found to be expressed together with the already known gene ZBBX which also shows intellectual disability and psychosis as phenotype. So the DECIPHER database could not confirm any short stature phenotype regarding our breakpoints with the exception of case 291507 which is not that convincing due to the overlap with case 630 and the complex phenotype. In summary, we had characterized the breakpoints of a novel inversion chromosome 3 in a family with SS. The breakpoints had not disrupted any validated genes but disrupted the second intron of a novel gene, from which parts are transcribed in human embryonic RNA. We also speculate the non-homologous recombination as a molecular mechanism for this inversion. We propose that disruption of this putative gene may have a role in causing the short stature in the family. Validation of the identified putative gene, and subsequent functional analysis could provide insights into the inversion breakpoint and its association with the phenotype precisely. Conflict of interest The authors declare no conflict of interest. Acknowledgments We thank Sebastian Werner for the expression analysis of so many different human RNAs. This study was supported by Deutscher Akademischer Austausch Dienst (DAAD) or German Academic Exchange Service sponsored fellowship (Code no: A/04/ 08474 ) to URD. References Baere ED, Speleman F, Van RN, De Paepe A, Messiaen L. Assignment of SHOX2 (alias OG12X and SHOT) to human chromosome bands 3q25/q26.1 by in situ hybridization. Cytogenet Cell Genet 1998;82:228e9. Barajas-Barajas LO, Velarde-Felix S, Elizarraras-Rivas J, Hernandez-Zaragoza G, Vazquez-Herrera JA. De novo ring chromosome 3 in a girl with hypoplastic thumb and coloboma of iris. Genet Couns 2001;12:151e6. Bergametti F, Denier C, Labauge P, Arnoult M, Boetto S, Clanet M, et al. Mutations within the programmed cell death 10 gene cause cerebral cavernous malformations. Am J Hum Genet 2004;76:42e51. Blaschke RJ, Monaghan AP, Schiller S, Schechinger B, Rao E, Padilla-Nash H, et al. SHOT, a SHOX-related homeobox gene, is implicated in craniofacial, brain, heart, and limb development. Proc Natl Acad Sci 1998;95:2406e11. Brueton LA, Barber JC, Huson SM, Winter RM. Partial monosomy 3q in a boy with short stature, developmental delay, and mild dysmorphic features. J Med Genet 1989;26:729e30. Chen PY, Chang WS, Chou RH, Lai YK, Lin SC, Chi CY, et al. Two non-homologous brain diseases-related genes, SERPINI1 and PDCD10, are tightly linked by an asymmetric bidirectional promoter in an evolutionarily conserved manner. BMC Mol Biol 2007;8:2. Chen W, Ullmann R, Langnick C, Menzel C, Wotschofsky Z, Hu H, et al. Breakpoint analysis of balanced chromosome rearrangements by next-generation pairedend sequencing. Eur J Hum Genet 2010;18:539e43.
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