PAFAH1B3–CLK2 Fusion Gene in a Female with Mental Retardation, Ataxia and Atrophy of the Brain Hans Gerd Nothwang1,2, H.G
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© 2001 Oxford University Press Human Molecular Genetics, 2001, Vol. 10, No. 8 797–806 Functional hemizygosity of PAFAH1B3 due to a PAFAH1B3–CLK2 fusion gene in a female with mental retardation, ataxia and atrophy of the brain Hans Gerd Nothwang1,2, H.G. Kim1,J.Aoki3,M.Geisterfer4,S.Kübart1,R.D.Wegner5, A. van Moers5,L.K.Ashworth6, T. Haaf1,J.Bell4,H.Arai3,N.Tommerup7,H.H.Ropers1,8,+ and J. Wirth1 1Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin-Dahlem, Germany, 2FB Biologie, Universität Kaiserslautern, Kaiserslautern, Germany, 3Department of Health Chemistry, University of Tokyo, Japan, 4Ottawa Regional Cancer Centre, Ottawa, Canada, 5Institut für Humangenetik, Freie Universität Berlin, Berlin, Germany, 6Lawrence Livermore National Laboratory, Livermore, CA, USA, 7Department of Medical Genetics, IMBG, Copenhagen, Denmark and 8Department of Human Genetics, University Hospital, Nijmegen, The Netherlands Received 30 October 2000; Revised and Accepted 15 February 2001 We report on the molecular characterization of a of several MR disease genes and is further supported by translocation t(1;19)(q21.3;q13.2) in a female with several studies that indicate an increase of balanced structural mental retardation, ataxia and atrophy of the brain. rearrangements among the retarded (1–4). Sequence analysis of the breakpoints revealed an To fully exploit the information provided by MR associated Alu-repeat-mediated mechanism of recombination chromosome rearrangements, the Mendelian Cytogenetic that led to truncation of two genes: the kinase CLK2 Network (MCN) has identified more than 1000 such cases and PAFAH1B3, the gene product of which interacts (N. Tommerup, unpublished data). In addition, a comprehen- sive YAC panel over the entire human genome was recently with LIS1 as part of a heterotrimeric G protein established (5) and this tool, together with the rapidly complex PAF–AH1B. In addition, two reciprocal progressing sequence information of the human genome, fusion genes are present. One expressed fusion allows the characterization of these rearrangements with gene encodes the first 136 amino acids of PAFAH1B3 reasonable effort. Therefore, we initiated a systematic analysis followed by the complete CLK2 protein. Truncated of de novo balanced chromosomal rearrangements associated PAFAH1B3 protein lost its potential to interact with with any kind of MR. LIS1 whereas CLK2 activity was conserved within the Here, we report on a proband with brain dysfunction and fusion protein. These data emphasize the importance dysmorphology, and a translocation t(1;19)(q21.3;q13.2). of PAF–AH1B in brain development and functioning Molecular characterization of this translocation revealed a and demonstrate the first fusion gene apparently not complex genotype with the truncation of two genes and the associated with cancer. formation of two reciprocal fusion genes. Both breakpoints were characterized in detail to unravel the mechanism of the non-homologous rearrangement between chromosomes INTRODUCTION 1q21.3 and 19q13.2. Furthermore, the major fusion protein Approximately 2–3% of the human population present with was functionally analysed. mental retardation (MR, IQ < 70) and it is estimated that 5–10% of mild MR (IQ 50–70) and ~50% of severe MR (IQ < 35) RESULTS have a genetic cause (1). Several genes for X-linked MR have been recently isolated, but elucidation of the molecular defects Delineation of the breakpoint regions on 1q21.3 and underlying autosomal MR remains a major challenge. This is 19q13.2 due to the enormous genetic heterogeneity as demonstrated by the analysis of X-linked forms (1). This hampers the efficient A female patient was reported with MR, ataxia and atrophy of identification of autosomal candidate genes by genome-wide the brain. Cytogenetic analysis revealed an apparently linkage analysis, since most families are too small and their balanced de novo translocation t(1;19)(q21.3;q13.2). Fluo- data cannot be combined with those of other families. A suitable rescence in situ hybridization (FISH) analysis of mega-YAC approach to overcome this problem is the characterization of clones from a genome-wide YAC panel narrowed down the chromosomal rearrangements associated with MR. This breakpoints on both chromosomes to intervals of <3 cM. YAC method has already been instrumental in the positional cloning clone CEPHy904F05930 containing STS D1S2771 (173 cM) +To whom correspondence should be addressed at: Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin-Dahlem, Germany; Tel: +49 30 8413 1240; Fax: +49 30 8413 1383; Email: [email protected] 798 Human Molecular Genetics, 2001, Vol. 10, No. 8 Figure 1. FISH analysis of t(1;19)(q12;q13.2). (A) Ideogram of the chromosomes, involved in the translocation of patient E.B., with arrows at the breakpoint positions. (B) FISH of cosmid F19975 to metaphase spreads of patient E.B. The cosmid clone spans the breakpoint since its hybridization signal is seen on the normal chromosome 19 and the derivative chromosomes 1 and 19 [der (1) and der (19)]. mapped proximal and clone CEPHy904D05873 containing STS on a Southern blot. Again, it detected additional fragments in D1S484 (174 cM) distal to the breakpoint on 1q21.3. The break- patient DNA restricted with four different enzymes: 6 kb and point on chromosome 19 was located between markers ~13kb(Ecl136II), >23 kb (EcoRV),3.5kb(DraI) and 1.1 kb D19S223 (64 cM) and D19S420 (66 cM), since YAC clones (RsaI) (Fig. 2). Thus, the gene PAFAH1B3 is disrupted CEPHyC10875 and CEPHy904C01761 mapped proximally and between exons 4 and 5 by the translocation on chromosome distally, respectively, to the breakpoint (data not shown). Since 19q13.2. The encoded protein forms with two other subunits chromosome 19 is well covered by ordered genomic clones the platelet-activating factor (PAF) acetylhydrolase 1B (PAF– (6), the 19q13 breakpoint was cytogenetically and molecularly AH1B) complex and bears the hydrolytic activity of this characterized in more detail. For high-resolution mapping, enzyme that produces biologically inactive lyso-PAF (1-O- cosmid clones from a chromosome 19 contig in this region alkyl-2-hydroxy-sn-glycero-3-phosphocholine) (7). The disrup- were analysed by FISH. Two clones, F19975 and R31213, tion leads to a truncated allele that encodes a protein with only hybridized to the normal chromosome 19, to der(1) and to the first 136 instead of 232 amino acid residues, lacking amino der(19), and thus spanned the breakpoint on 19q13.2 (Fig. 1). acids Asp192 and His195 (8). Together with Ser47, these two The localization of the breakpoint was confirmed by cosmids amino acids form the catalytic triad and their substitution leads that physically had been mapped adjacent to these cosmids: to loss of hydrolytic activity (8). cosmid R34603 mapped proximally and cosmid R27716 distally to the breakpoint according to FISH results (data not A second gene, CLK2 on 1q12.3, is disrupted shown). To characterize the breakpoint on chromosome 1, the junction fragment was cloned by genome walking. RsaI-restricted PAFAH1B3 on 19q13.2 is truncated by the translocation genomic DNA from the patient was ligated to an adaptor, and The restriction patterns of the breakpoint spanning cosmids nested PCR using adapter- and chromosome 19-specific demonstrated overlap with the sequenced BAC clone CIT-B- primers was performed. Human primers were derived from the 147B23 (GenBank accession no. AC006486) (data not shown). breakpoint-spanning 3 kb RsaI fragment of PAFAH1B3 intron 4. Sequence analysis identified two genes in the breakpoint area: This PCR yielded a product of ~1.1 kb (data not shown). This KIAA0306 and PAFAH1B3 (GenBank accession nos size corresponded well with that of the aberrant RsaI fragment AB002304 and D63391). Southern blot hybridization with seen in the patient lane on the Southern blot (Fig. 2). Sequence EST clones corresponding to KIAA0306 did not detect aberrant analysis of this junction fragment demonstrated identity both bands in patient DNA compared with a control DNA (data not to BAC clone CIT-B-147B23 on chromosome 19 and to a shown). Hybridization of cDNA clones corresponding to sequence on chromosome 1q21 that contains several genes PAFAH1B3, however, resulted in the detection of several addi- (GenBank accession no. AF023268) (9). tional fragments in restricted patient DNA (data not shown). A The breakpoint on chromosome 19 is situated between comparison of the hybridization signals with the restriction nucleotides 91 425 and 91 426 of BAC clone CIT-B-147B23 pattern, obtained by in silico analysis of the sequence, and between nucleotides 606 and 607 of the genomic sequence suggested that the breakpoint is located between exons 4 and 5 on chromosome 1. These data revealed that a second gene, the of PAFAH1B3. To confirm this localization, a 3 kb RsaI frag- dual specificity cdc-like kinase 2 (CLK2) on 1q21, is disrupted mentoftheintron4regionwasamplifiedbyPCRandprobed (Fig. 3) (10,11). The first nucleotide of CLK2 corresponds to Human Molecular Genetics, 2001, Vol. 10, No. 8 799 large fusion gene, PAFAH1B3∆ex5–CLK2∆ex1 (Fig. 3). The latter will give rise to a protein containing amino acids 1–136 of PAFAH1B3, followed by the complete and unmodified CLK2 polypeptide, since exon 2 of CLK2, starting with the initiation codon ATG, is fused in frame to PAFAH1B3.The small fusion gene CLK2∆ex2-13–PAFAH1B3∆ex1-4 on der(19) might be translated into a 42 amino acid long polypeptide, since there is an ATG codon in exon 5 at position 681 of PAFAH1B3, situated within a potential Kozak consensus sequence (GACATG) (12). To corroborate this in silico analysis and to demonstrate the expression of the large fusion gene PAFAH1B3∆ex5–CLK2∆ex1, RT–PCR analysis was performed with PAFAH1B3-derived forward and CLK2-derived reverse primers. A 2.2 kb RT–PCR product could be amplified from an mRNA population that was isolated from a lymphoblastoid cell line of the patient.