Comparative study of three diagnostic approaches (FISH, STRs and MLPA) in 30 patients with 22q11.2 deletion syndrome

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1 Clin Genet 2005: 68: Copyright # Blackwell Munksgaard 2005 Printed in Singapore. All rights reserved CLINICAL GENETICS doi: /j x Short Report Comparative study of three diagnostic approaches (FISH, STRs and MLPA) in 30 patients with 22q11.2 deletion syndrome Ferna ndez L, Lapunzina P, Arjona D, Lo pez Pajares I, Garcı a-guereta L, Elorza D, Burgueros M, De Torres ML, Mori MA, Palomares M, Garcı a-alix A, Delicado A. Comparative study of three diagnostic approaches (FISH, STRs and MLPA) in 30 patients with 22q11.2 deletion syndrome. Clin Genet 2005: 68: # Blackwell Munksgaard, 2005 The 22q11.2 deletion syndrome is commonly diagnosed using fluorescence in situ hybridization (FISH) with commercial probes. The chromosomal breakpoints and deletion size are subsequently characterized by short tandem repeat (STR) segregation tests or by further FISH probes. Recently, a multiplex ligation-dependent probe amplification (MLPA) single tube assay was developed to detect deletions of the 22q11.2 region and other chromosomal regions associated with DiGeorge/velocardiofacial syndrome. We have compared the results of these three techniques in a group of 30 patients affected with 22q11.2 deletion syndrome. MLPA correctly called all patients who had been previously diagnosed by FISH. The MLPA results were concordant in all patients with the STR analysis in respect to deletion size. Furthermore, this novel technique resolved seven cases that were undetermined by STR analysis. These results confirm the efficiency of MLPA as a rapid, reliable, economical, high-throughput method for the diagnosis of 22q11.2 deletion syndrome. L Fernández a, P Lapunzina a, D Arjona b,ilópez Pajares a, L García-Guereta c, D Elorza d, M Burgueros c, ML De Torres a, MA Mori a, M Palomares a, A García-Alix d and A Delicado a a Department of Medical Genetics, b Sequencing Unit, c Department of Pediatric Cardiology, d Department of Neonatology, Hospital Universitario La Paz, Madrid, Spain Key words: 22q11.2 deletion syndrome DiGeorge/velocardiofacial syndrome FISH MLPA STR Corresponding author: Luis Fernández García-Moya, Servicio de Genética Médica Hospital Universitario La Paz, Paseo de la Castellana 261, Madrid, Spain. Tel.: þ ; fax: þ ; lfernandez.hulp@salud.madrid.org Received 22 February 2005, revised and accepted for publication 22 June 2005 The 22q11.2 deletion syndrome (del22q11.2) is the most common deletion disorder in humans, with an incidence of approximately 1:4000 births. The majority of patients (90%) present with a typical 3-Mb deletion, while a minority present with either a common approximately 1.5-Mb nested deletion or other atypical deletions (1, 2). Fluorescence in situ hybridization (FISH) with probes at locus D22S75 or TUPLE1 detects virtually all the deleted chromosomes. FISH is widely used as a diagnostic technique for this disorder, and in many centres it is the gold standard for diagnosis of del22q11.2. We routinely also examine the segregation of polymorphic short tandem repeat (STR) markers within the 22q11.2 region in the affected proband and their parents. These analyses not only contribute to map the chromosomal breakpoints and define the parental origin of the deleted chromosome but are also useful to detect atypical, smaller deletions in patients with the DiGeorge syndrome (DGS, OMIM ) or the velocardiofacial syndrome (VCFS) phenotypes. In some laboratories, FISH analysis carried out with non-commercial probes, which hybridize to other specific loci in the region, are also used to characterize the deletions. Recently, a novel method, multiplex ligationdependent probe amplification (MLPA) technique has been designed to detect many structural rearrangements and aneuploidies in a single assay (3). This method allows the quantitative genomic screening at specific target sequences, based on the simultaneous hybridization and one primerpair amplification of up to 40 different probes in 373

2 Fernández et al. a single tube. The SALSA P023 DiGeorge syndrome/vcfs MLPA Kit [Microbiology Research Center (MRC); Holland, the Netherlands] consists of seven critical DiGeorge/VCFS probes, two Cat- Eye syndrome (CES) probes, two control 22q11 and 22q13 probes plus 28 probes specific for other chromosomal regions reported to have overlapping phenotypes with DGS/VCFS, such as 4q, 7p, 8p, 10p, 10q, 17p and 18q (4 7). To the best of our knowledge, no data has been previously reported comparing the sensitivity, specificity and usefulness of MLPA with other conventional methods in patients with or without previously confirmed diagnosis of del22q11.2. In order to evaluate the potential future clinical and diagnostic use of MLPA in routine evaluation of patients with suspected del22q11.2, we analyzed and compared data generated from FISH, STRs and MLPA in 30 patients with 22q11.2 deletion syndrome. Materials and methods We analyzed a cohort of 30 patients in which the 22q11.2 deletion syndrome was previously diagnosed in our hospital during the last few years. FISH had been undertaken to detect the presence of deletions, while STR analysis of the 22q11.2 region allowed the characterization of the deletions. Karyotypes in each patient were performed on peripheral blood lymphocytes by standard chromosome preparations and GTL banding. At least 20 metaphases were analyzed for each patient. FISH protocols were carried out following the manufacturer s instructions, using the D22S75 (Vysis Ò, Oncor Ò ) and occasionally the TUPLE1 (Vysis Ò ) probes. Genomic DNA extraction from peripheral blood lymphocytes was carried out with the PureGene Ò DNA Isolation Kit (Gentra Systems, Minneapolis, MN, USA) from 3 ml peripheral blood in ethylenediaminetetraacetic acid tubes. Twelve polymorphic STR markers, eight of them spanning the 22q11.2 critical region (from centromere to telomere, D22S1638, NLJH1, D22S1648, D22S941, D22S944, D22S1623, D22S264 and D22S311; Fig. 1) and four flanking the typical deleted region (proximally D22S420, D22S427, and distally D22S306, D22S303; Fig. 1) were tested by amplification with specific 5 0 -labelled fluorescent primers [primers and polymerase chain reaction conditions obtained from the National Center for Biotechnology Information web site, except for NLJH1 from Henwood et al. (8)] and analyzed on an ABI Prism Ò 377 or 3100 Avant automatic sequencer (Applied Biosystems Inc, Foster City, CA, USA). Allele sizes and peak areas were determined using GENESCAN, GENOTYPER or GENEMAPPER software (Applied Biosystems). Markers D22S1638-D22S311 span the typical 3-Mb deletions, whereas D22S1638-D22S1623 define the nested proximal segment commonly involved in the 1.5-Mb deletions (Fig. 1). The breakpoints flanking these markers are set within unstable sequences termed low-copy repeats (LCRs; A, B, C and D in Fig. 1) that work as substrates for nonallelic homologous recombinations leading to the loss of chromosomal material (9). 22q11.2 Approximately 3 Mb Approximately 1.5 Mb LCRs FISH probes A B C D N25 TUPLE1 Polymorphic STR markers D22S420 D22S427 D22S1638 NLJH1 D22S1648 D22S941 D22S944 D22S1623 D22S264 D22S311 D22S306 D22S303 MLPA probes HIRA CLDN5 KIAA1652 FLJ14360 PCQAP SNAP29 LZTR1 ARSA Fig. 1. Schematic representation of the relative position of the low-copy repeats (LCRs), fluorescence in situ hybridization (FISH) probes, polymorphic short tandem repeat (STR) markers and multiplex ligation-dependent probe amplification (MLPA) probes in the DiGeorge critical region of the 22q11.2 band. The most commonly deleted segments (3 and 1.5 Mb) are indicated by black bars. Numbers in circles denote the relative order of the seven MLPA probes. ARSA control locus is set in 22q13 region and therefore outside the DiGeorge critical region. The typical 3-Mb deletion spans LCRs A to D, markers D22S1638 to D22S311 and MLPA probes HIRA to LZTR1, whereas the smaller 1.5-Mb deletion spans LCRs A to B, markers D22S1638 to D22S1623 and MLPA probes HIRA to KIAA

3 MLPA diagnosis of 22q11.2 deletion A set of MLPA probes for DGS/VCFS (SALSA P023 DGS/VCFS MLPA Kit; MRC) was used for the screening of the 22q11.2 critical region. The set consists of a pool of 39 cloned probes related with DGS/VCFS and CES hybridizing all over the genome, including seven 22q11.2-specific probes plus a subtelomeric 22q13 control probe. A 94-bp synthetic control probe and four smaller DNA-quantity control probes are also present in the kit. An amount of ng DNA in a final volume of 5 ml was heated at 98 C and mixed with 1.5 ml probe mix and 1.5 ml SALSA hybridization buffer. Probe hybridization, ligation and amplification reactions were carried out according to the protocols supplied by MRC in a standard thermal cycler (Tpersonal, Biometra Ò,Go ttingen, Germany). The amplification products were analyzed by capillary electrophoresis in an ABI 3100 genetic analyzer, using the GENEMAPPER software v3.5 (Applied Biosystems), following the recommended protocol supplied with the MLPA kit. Raw data were exported to an MS Excel Ò spreadsheet for further calculations. Peak areas were first normalized by dividing them by the areas of a median of nine normal individuals that were used as controls. A second normalization vs an internal control was then undertaken, as described by Yobb et al. (10) and Wang et al. (11). Briefly, peak ratios were calculated for the eight 22q test probes by dividing their normalized areas by the median of the four non-22q probes closest in size. This set the value for a diploid gene dosage to 1, with deletion and duplication thresholds established at below 0.75 and above 1.25, respectively. Results MLPA electropherograms of three samples, two patients and a control, are shown in Fig. 2. The 22q11.2 deletions were apparent on qualitative inspection of MLPA peak profiles. According to FISH analysis, all samples carried a deletion in the 22q11.2 region. Table 1 shows the deleted segments detected and their sizes determined after STR markers segregation analysis and MLPA. The series of haploinsufficient markers is indicated as the LCR interval, according to the correlations in Fig. 1. Haploinsufficient MLPA probes are numbered as shown in Fig. 1. STR alleles and quantification values for MLPA probes are not shown. In all cases, the MLPA results were concordant with the STR markers analysis. No rare or atypical deletions were observed by either STR analysis or MLPA. Furthermore, MLPA resolved the deletion size in seven cases that were uncertain by STRs due to amplification problems, non-informative markers or lack of parental data. In summary, both methods were concordant in diagnosing 17 cases of 3-Mb deletions and six cases of 1.5-Mb deletions, including one carrying an unbalanced translocation between chromosomes 6 and 22 (Patient 1502, Table 1), and MLPA resolved seven cases that were uninformative by STRs. Discussion MLPA has been tested as a diagnostic method in several diseases involving chromosomal disorders, such as Rett syndrome, hereditary multiple exostoses, CMT1A and HNPP, neurofibromatosis 2 and Duchenne and Becker muscular dystrophies (12 16), as well as cancer predisposition genes, prenatal screenings and subtelomeric rearrangements (17 19). This is, to our knowledge, the first evaluation of MLPA in the 22q11.2 deletion syndrome compared with STRs and FISH analyses. In our experience, MLPA proved to be an efficient tool in the diagnosis of 22q11.2 deletion syndrome. MLPA was successful in diagnosing all patients previously confirmed to have a deletion by FISH: the corresponding 22q11.2 critical MLPA probes were all shown to be haploinsufficient. The sizes of the deletions determined by the MLPA were in agreement with those generated by STR analyses (Table 1). Moreover, the deletion size was determined by MLPA in patients 1, 2, 5, 9, 10, 12 and 16 (Table 1), in which gene dosage by STRs was non-informative due to the low-marker heterozygosity, absent parental data or amplication failures. Therefore, MLPA is an easy, rapid and economical method for the simultaneous diagnosis of multiple anomalies with a simple kit. Specifically, the SALSA P023 DiGeorge/ VCFS MLPA Kit hybridizes seven probes to the 22q11.2 region in each sample, detecting haploinsufficiency in this critical region and occasionally in other chromosomal regions that have been reported to be involved in overlapping phenotypes (4 7). An advantage of this multiprobe system is that it may detect atypical deletions in regions not covered by the commercially available probes and therefore not routinely tested by FISH. These uncommon deletions are estimated to occur in 2% of 375

4 Fernández et al. MC fsa 1724 (a) SALSA P al 175C al 193 CLDN5 al 220C al 247C al 265C al 292C al 310C al 328C al 346C al 364C al 382C al 400C al 418 LZTR1 al 445 ARSA al 472c al 139 al 157 HIRA al 148C al 184C al 202C al 229C al 256C al 274C al 301C al 337 IL17R al 373 SNAP29 al 409C al 427C al 454c al 481c al 166C al 211C al 238 KIAA 1652 al 283 FLJ14360 al 319 PCQAP al 355 BID al 391C al 436c al 463c MC fsa 1593 (b) SALSA P al 139 al 157 HIRA al 175C al 193 CLDN5 al 220C al 247C al 265C al 292C al 310C al 328C al 346C al 364C al 382C al 400C al 418 LZTR1 al 445 ARSA al 472c al 148C al 166C al 184C al 202C al 256C al 274C al 301C al 337 IL17R al 373 SNAP29 al 409C al 427C al 454c al 481c al 229C al 211C al 283 FLJ14360 al 319 PCQAP al 355 BID al 391C al 436c al 463c al 238 KIAA 1652 MC fsa 2155 (c) SALSA P al 175C al 193 CLDN5 al 220C al 247C al 265C al 292C al 310C al 328C al 346C al 364C al 382C al 400C al 418 LZTR1 al 445 ARSA al 472c al 139 al 157 HIRA al 148C al 166C al 184C al 202C al 229C al 256C al 274C al 301C al 337 IL17R al 373 SNAP29 al 409C al 427C al 454c al 481c al 211C al 238 KIAA1652 al 283 FLJ14360 al 319 PCQAP al 355 BID al 391C al 436c al 463c Fig. 2. Multiplex ligation-dependent probe amplification (MLPA) electropherograms of two samples with clinical diagnosis of 22q11.2 deletion syndrome. Amplified probes are detected as fluorescent signals, the areas of which are compared and normalized to quantify the gene dosage (data not shown). Probes from region 22q11.2 indicate a 3-Mb deletion in sample a (probes 1 7, Fig. 1) and a 1.5-Mb deletion in sample b (probes 1 3, Fig. 1), shown by the half signal intensity of the peaks indicated with arrows, compared with a control sample c. 22q11.2 cases (1, 2) and span five critical regions (20), some of which are detectable by MLPA or STR markers but not by standard FISH. STR and MLPA analyses indicated no uncommon chromosomal breakpoints in our cohort. All the patients with common deletions were shown to have the expected deletion intervals (Table 1). Another important advantage of MLPA is that parental samples are not required. Copy numbers can be determined from only a single sample from the affected individual. In summary, all of the above points, together with the relative low cost of the MLPA, confirm this novel technique as a suitable del22q11.2 screening method. The test includes six 22q11.2 additional loci compared with standard FISH and also allows the determination of the deletion breakpoints in a quantitative way and without the need of pedigree analysis. Our future work will include the screening of patients by MLPA, with a clinical diagnosis of DGS/VCFS in whom no 22q11.2 deletion was detected by FISH. Acknowledgements This work was partially funded by Red de Centros de Gene tica Clı nica y Molecular (C03/07) and supported by the Fondo de Investigacio n Sanitaria, Instituto de Salud Carlos III, Spain and by the Fundacio n de Investigacio n Me dica Mutua Madrilen a Automovilista. The authors thank Cintia Amin oso for technical support at the Sequencing Unit and Rosario Madero for contributing comments at the Biostatistics Unit, Hospital Universitario La Paz, Madrid, Spain. We thank Karen Heath and Juliette Siegfried for language review. 376

5 MLPA diagnosis of 22q11.2 deletion Table 1. Results of the three assays performed in 30 patients STR markers MLPA probes Patient s DNA ID FISH results Markers deleted Deletion Size Probes deleted Deletion size (Mb) 96 D22S75- U U D22S75- U U D22S75- A-D 3 Mb D22S75- A-D 3 Mb D22S75- U U D22S75- A-D 3 Mb D22S75- A-D 3 Mb D22S75- A-D 3 Mb D22S75- U U D22S75- U U D22S75- A-D 3 Mb D22S75- U U D22S75- A-B 1.5 Mb D22S75- A-B 1.5 Mb D22S75- A-D 3 Mb D22S75- U U D22S75- A-D 3 Mb D22S75- A-B 1.5 Mb D22S75- A-D 3 Mb D22S75- A-D 3 Mb TUPLE1- A-B 1.5 Mb D22S75- A-D 3 Mb D22S75- A-B 1.5 Mb D22S75- A-B 1.5 Mb D22S75- A-D 3 Mb D22S75- A-D 3 Mb D22S75- A-D 3 Mb D22S75- A-D 3 Mb D22S75- A-D 3 Mb D22S75- A-D 3 Mb Fluorescence in situ hybridization (probe indicated) showed monosomy at the 22q11.2 region in all patients (D22S75- or TUPLE1-). The deleted segments found by short tandem repeats (STRs) segregation analysis are indicated by their flanking low-copy repeats (A, B, C and D; Fig. 1), as well as their size. The multiplex ligation-dependent probe amplification (MLPA) probes that show haploinsufficiency (1, 2, 3, 4, 5, 6 and 7; Fig. 1) are also indicated as intervals, with the approximated size. U: unknown. References 1. Carlson C, Sirotkin C, Pandita R et al. Molecular definition of 22q11 deletions in 151 Velo-cardio-facial Syndrome patients. Am J Hum Genet 1997: 6: Edelmann L, Pandita RK, Spiteri E et al. A common molecular basis for rearrangement disorders on chromosome 22q11. Hum Mol Genet 1999: 8: Schouten JP, McElgunn CJ, Waaijer R et al. Relative quantification of 40 nucleic acid sequences by multiplex ligationdependent probe amplification. Nucleic Acids Res 2002: 30: Daw SCM, Taylor C, Kraman M et al. A common region on 10p deleted in DiGeorge and velocardiofacial syndromes. Nat Genet 1996: 13: Lichtner P, Konig R, Hasegawa T et al. An HDR (hypoparathyroidism, deafness, renal dysplasia) syndrome locus maps distal to the DiGeorge syndrome region on 10p13/14. J Med Genet 2000: 37: Greenberg F, Courtney KB, Wessels RA et al. Prenatal diagnosis of deletion 17p13 associated with DiGeorge anomaly. Am J Med Genet 1988: 31: Van Essen AJ, Schoots CJ, van Lingen RA et al. Isochromosome 18q in a girl with holoprosencephaly, DiGeorge anomaly, and streak ovaries. Am J Med Genet 1993: 47: Henwood J, Pickard C, Leck JP et al. A region of homozygosity within 22q11.2 associated with congenital heart disease: recessive DiGeorge/velocardiofacial syndrome? J Med Genet 2001: 38: Stankiewicz P, Lupski JR. Genome architecture, rearrangements and genomic disorders. Trends Genet 2002: 18: Yobb TM, Somerville MJ, Willatt L et al. Microduplication and triplication of 22q11.2: a highly variable syndrome. Am J Hum Genet 2005: 76: Wang J, Ban MR, Hegele RA. Multiplex ligationdependent probe amplification of LDLR enhances molecular diagnosis of familial hypercholesterolemia. J Lipid Res 2005: 46: Erlandson A, Samuelsson L, Hagberg B et al. Multiplex ligation-dependent probe amplification (MLPA) detects large deletions in the MECP2 gene of Swedish Rett syndrome patients. Genet Test 2003: 7: White SJ, Vink GR, Kriek M et al. Two-color multiplex ligation-dependent probe amplification: detecting genomic rearrangements in hereditary multiple exostoses. Hum Mutat 2004: 24: Slater H, Bruno D, Ren H et al. Improved testing for CMT1A and HNPP using multiplex ligation-dependent 377

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