1 European Journal of Human Genetics 2006 Vol: 14(9):1009-1017. DOI: 10.1038/sj.ejhg.5201661

Simple detection of genomic microdeletions and microduplications using QMPSF in patients with idiopathic mental retardation

In contrast to the numerous well-known microdeletion syndromes, only a few microduplications have been described, and this discrepancy may be due in part to methodological bias. In order to facilitate the detection of genomic microdeletions and microduplications, we developed a new assay based on QMPSF (Quantitative Multiplex PCR of Short fluorescent Fragments) able to explore simultaneously 12 candidate loci involved in mental retardation (MR) and known to be the target of genomic rearrangements. We first screened 153 patients with MR and facial dysmorphism associated with malformations, or growth anomalies, or familial history, with cytogenetically normal chromosomes, and the absence of FRAXA mutation and subtelomeric rearrangements. In this series, we found a 5q35 deletion removing the NSD1 gene in a patient with severe epilepsy, profound MR and, retrospectively, craniofacial features of Sotos syndrome. In a second series, we screened 140 patients with MR and behaviour disturbance who did not fulfil the de Vries criteria for subtelomeric rearrangements and who had a normal karyotype and no detectable FRAXA mutation. We detected a 22q11 deletion in a patient with moderate MR, obesity, and facial dysmorphism and a 4 Mb 17p11 duplication in a patient with moderate MR, behaviour disturbance, strabismus, and aspecific facial features. This new QMPSF assay can be gradually upgraded to include additional loci involved in newly recognised microduplication/microdeletion syndromes, and should facilitate wide screenings of patients with idiopathic MR and provide better estimates of the microduplication frequency in the MR population.

Mentions
Figures
Figure 1: Detection by QMPSF assays of a 5q35 deletion removing the NSD1 locus. (a) Detection of the heterozygous NSD1 deletion using the microdeletion/microduplication QMPSF assay. (b) Confirmation of the rearrangement using a QMPSF assay exploring the 22 coding exons of the NSD1 gene. In each QMPSF panel, the electropherogram of the patient (in red) was superimposed to that of a normal control (in blue) by adjusting to the same level the peaks obtained for the control amplicon. The Y axis displays fluorescence in arbitrary units, and the X axis indicates the size in bp. Heterozygous deletions are easily detected by a 50% reduction of the peaks compared to a normal control. In panel (a), each amplicon corresponds to a single locus. In panel (b), the numbers correspond to the NSD1 exons. In both, C designs the control amplicon. Figure 2: Detection by QMPSF assays of a 22q11.2 deletion. (a) Detection of the 22q11.2 deletion using the microdeletion/microduplication QMPSF assay. (b) Characterisation of the boundaries of the rearrangement using a QMPSF specific of the 22q11.2 and 10p14 loci. In each QMPSF panel, the electropherogram of the patient (in red) was superimposed to that of a normal control (in blue) by adjusting to the same level the peaks obtained for the control amplicon. The Y axis displays fluorescence in arbitrary units, and the X axis indicates the size in bp. In both (a) and (b), C1 and C2 design control amplicons. (c) Schematic representation of the position of the 22q11 amplicons along the chromosome 22.30 The arrow shows the extend of the deletion revealed by QMPSF. (d) Confirmation by FISH of the rearrangement using the Spectrum Orange LSI DiGeorge/VCSF region probe (Vysis, Downers Groove, USA) which encompasses the TUPLE1/HIRA gene and the D22S553, D22S609, D22S942 loci and the Spectrum Green LSI ARSA control probe on 22q13 (Vysis, Downers Groove, USA). Figure 3: Detection by QMPSF assays of a 17p11.2 duplication syndrome. (a) Detection of the 17p11.2 duplication using the microdeletion/microduplication QMPSF assay. (b) Characterisation of the boundaries of the rearrangement using a QMPSF specific of the 17p11.2 locus. In each QMPSF panel, the electropherogram of the patient (in blue) was superimposed to that of a normal control (in red) by adjusting to the same level the peaks obtained for the control amplicon. The Y axis displays fluorescence in arbitrary units, and the X axis indicates the size in bp. In both (a) and (b), C designs the control amplicon. (c) Schematic representation of the position of the 17p11 amplicons along the chromosome 17. The arrow shows the extend of the deletion revealed by QMPSF. (d) Confirmation by FISH of the rearrangement using the Spectrum Orange LSI Smith-Magenis Syndrome critical region probe (Vysis, Downers Groove, USA) and the Spectrum Green LSI Retinoic Acid Receptor Alpha (LSI RARA) control probe on 17q21.1 (Vysis, Downers Groove, USA). (e) Phenotype of the patient with the 17p11.2 duplication.
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References
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    • . . . Mental retardation (MR) occurs in 2–3% of the general population, but its aetiology can be established only in approximately 50% of cases, limiting therefore considerably the efficiency of genetic counselling, detection of carriers, and prenatal diagnosis.1 In this context, the detection and characterisation of deleterious genomic rearrangements, such as microdeletions and microduplications, represents an important challenge . . .
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    • . . . These rearrangements, resulting mainly from abnormal pairing and nonallelic homologous recombination mediated by repeat elements such as Alu repeats and low-copy repeats (LCRs), are the cause of many Mendelian diseases, contiguous gene syndromes, or chromosomal disorders.2, 3, 4 Other uncharacterised recombinational hotspots may also key roles, especially in subtelomeric regions where chromosomal rearrangements are found in about 5% of the patients with idiopathic MR.5, 6, 7 Thus, genome architectural features are involved in the origin of recurrent deleterious DNA rearrangements.3, 8, 9 The use of FISH has significantly improved the diagnosis of microdeletion syndromes suggested by clinical evidence . . .
  3. Ji Y, Eichler EE, Schwartz S, Nicholls RD: Structure of chromosomal duplicons and their role in mediating human genomic disorders. Genome Res 2000; 10: 597-610 , .
    • . . . These rearrangements, resulting mainly from abnormal pairing and nonallelic homologous recombination mediated by repeat elements such as Alu repeats and low-copy repeats (LCRs), are the cause of many Mendelian diseases, contiguous gene syndromes, or chromosomal disorders.2, 3, 4 Other uncharacterised recombinational hotspots may also key roles, especially in subtelomeric regions where chromosomal rearrangements are found in about 5% of the patients with idiopathic MR.5, 6, 7 Thus, genome architectural features are involved in the origin of recurrent deleterious DNA rearrangements.3, 8, 9 The use of FISH has significantly improved the diagnosis of microdeletion syndromes suggested by clinical evidence . . .
  4. Bailey JA, Gu Z, Clark RA et al: Recent segmental duplications in the human genome. Science 2002; 297: 1003-1007 , .
    • . . . These rearrangements, resulting mainly from abnormal pairing and nonallelic homologous recombination mediated by repeat elements such as Alu repeats and low-copy repeats (LCRs), are the cause of many Mendelian diseases, contiguous gene syndromes, or chromosomal disorders.2, 3, 4 Other uncharacterised recombinational hotspots may also key roles, especially in subtelomeric regions where chromosomal rearrangements are found in about 5% of the patients with idiopathic MR.5, 6, 7 Thus, genome architectural features are involved in the origin of recurrent deleterious DNA rearrangements.3, 8, 9 The use of FISH has significantly improved the diagnosis of microdeletion syndromes suggested by clinical evidence . . .
  5. Knight SJ, Regan R, Nicod A et al: Subtle chromosomal rearrangements in children with unexplained mental retardation. Lancet 1999; 354: 1676-1681 , .
    • . . . These rearrangements, resulting mainly from abnormal pairing and nonallelic homologous recombination mediated by repeat elements such as Alu repeats and low-copy repeats (LCRs), are the cause of many Mendelian diseases, contiguous gene syndromes, or chromosomal disorders.2, 3, 4 Other uncharacterised recombinational hotspots may also key roles, especially in subtelomeric regions where chromosomal rearrangements are found in about 5% of the patients with idiopathic MR.5, 6, 7 Thus, genome architectural features are involved in the origin of recurrent deleterious DNA rearrangements.3, 8, 9 The use of FISH has significantly improved the diagnosis of microdeletion syndromes suggested by clinical evidence . . .
  6. Mefford HC, Trask BJ: The complex structure and dynamic evolution of human subtelomeres. Nat Rev Genet 2002; 3: 91-102 , .
    • . . . These rearrangements, resulting mainly from abnormal pairing and nonallelic homologous recombination mediated by repeat elements such as Alu repeats and low-copy repeats (LCRs), are the cause of many Mendelian diseases, contiguous gene syndromes, or chromosomal disorders.2, 3, 4 Other uncharacterised recombinational hotspots may also key roles, especially in subtelomeric regions where chromosomal rearrangements are found in about 5% of the patients with idiopathic MR.5, 6, 7 Thus, genome architectural features are involved in the origin of recurrent deleterious DNA rearrangements.3, 8, 9 The use of FISH has significantly improved the diagnosis of microdeletion syndromes suggested by clinical evidence . . .
  7. Flint J, Knight S: The use of telomere probes to investigate submicroscopic rearrangements associated with mental retardation. Curr Opin Genet Dev 2003; 13: 310-316 , .
    • . . . These rearrangements, resulting mainly from abnormal pairing and nonallelic homologous recombination mediated by repeat elements such as Alu repeats and low-copy repeats (LCRs), are the cause of many Mendelian diseases, contiguous gene syndromes, or chromosomal disorders.2, 3, 4 Other uncharacterised recombinational hotspots may also key roles, especially in subtelomeric regions where chromosomal rearrangements are found in about 5% of the patients with idiopathic MR.5, 6, 7 Thus, genome architectural features are involved in the origin of recurrent deleterious DNA rearrangements.3, 8, 9 The use of FISH has significantly improved the diagnosis of microdeletion syndromes suggested by clinical evidence . . .
  8. Lupski JR: Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits. Trends Genet 1998; 14: 417-422 , .
    • . . . These rearrangements, resulting mainly from abnormal pairing and nonallelic homologous recombination mediated by repeat elements such as Alu repeats and low-copy repeats (LCRs), are the cause of many Mendelian diseases, contiguous gene syndromes, or chromosomal disorders.2, 3, 4 Other uncharacterised recombinational hotspots may also key roles, especially in subtelomeric regions where chromosomal rearrangements are found in about 5% of the patients with idiopathic MR.5, 6, 7 Thus, genome architectural features are involved in the origin of recurrent deleterious DNA rearrangements.3, 8, 9 The use of FISH has significantly improved the diagnosis of microdeletion syndromes suggested by clinical evidence . . .
  9. Stankiewicz P, Lupski JR: Genome architecture, rearrangements and genomic disorders. Trends Genet 2002; 18: 74-82 , .
    • . . . These rearrangements, resulting mainly from abnormal pairing and nonallelic homologous recombination mediated by repeat elements such as Alu repeats and low-copy repeats (LCRs), are the cause of many Mendelian diseases, contiguous gene syndromes, or chromosomal disorders.2, 3, 4 Other uncharacterised recombinational hotspots may also key roles, especially in subtelomeric regions where chromosomal rearrangements are found in about 5% of the patients with idiopathic MR.5, 6, 7 Thus, genome architectural features are involved in the origin of recurrent deleterious DNA rearrangements.3, 8, 9 The use of FISH has significantly improved the diagnosis of microdeletion syndromes suggested by clinical evidence . . .
  10. Moeschler JB, Mohandas TK, Hawk AB, Noll WW: Estimate of prevalence of proximal 15q duplication syndrome. Am J Med Genet 2002; 111: 440-442 , .
    • . . . Although nonallelic homologous recombination is supposed to generate microdeletions as well as microduplications, in the field of MR only four microduplications have clearly been related to phenotypes: a 15q11–q13 duplication has been detected in patients presenting autistic features and its frequency has been estimated to 1/200–600 among patients with developmental delay.10, 11 A 17p11.2 duplication has been associated with moderate MR and behavioural disturbance.12, 13 The 22q11 duplication, initially identified in patients with a clinical presentation similar to the classical 22q11 deletion, has recently been shown to result into a highly variable phenotype.14, 15 A 7q11.23 duplication has been related to severe expressive language delay.16 Among the possible explanations for the lower frequency of observed duplications, compared to deletions, one can speculate that duplications often result in a different or less severe phenotype and/or that a methodological bias contributes to this discrepancy . . .
  11. Thomas JA, Johnson J, Peterson Kraai TL et al: Genetic and clinical characterization of patients with an interstitial duplication 15q11-q13, emphasizing behavioral phenotype and response to treatment. Am J Med Genet A 2003; 119: 111-120 , .
    • . . . Although nonallelic homologous recombination is supposed to generate microdeletions as well as microduplications, in the field of MR only four microduplications have clearly been related to phenotypes: a 15q11–q13 duplication has been detected in patients presenting autistic features and its frequency has been estimated to 1/200–600 among patients with developmental delay.10, 11 A 17p11.2 duplication has been associated with moderate MR and behavioural disturbance.12, 13 The 22q11 duplication, initially identified in patients with a clinical presentation similar to the classical 22q11 deletion, has recently been shown to result into a highly variable phenotype.14, 15 A 7q11.23 duplication has been related to severe expressive language delay.16 Among the possible explanations for the lower frequency of observed duplications, compared to deletions, one can speculate that duplications often result in a different or less severe phenotype and/or that a methodological bias contributes to this discrepancy . . .
  12. Potocki L, Chen KS, Park SS et al: Molecular mechanism for duplication 17p11.2 - the homologous recombination reciprocal of the Smith-Magenis microdeletion. Nat Genet 2000; 24: 84-87 , .
    • . . . Although nonallelic homologous recombination is supposed to generate microdeletions as well as microduplications, in the field of MR only four microduplications have clearly been related to phenotypes: a 15q11–q13 duplication has been detected in patients presenting autistic features and its frequency has been estimated to 1/200–600 among patients with developmental delay.10, 11 A 17p11.2 duplication has been associated with moderate MR and behavioural disturbance.12, 13 The 22q11 duplication, initially identified in patients with a clinical presentation similar to the classical 22q11 deletion, has recently been shown to result into a highly variable phenotype.14, 15 A 7q11.23 duplication has been related to severe expressive language delay.16 Among the possible explanations for the lower frequency of observed duplications, compared to deletions, one can speculate that duplications often result in a different or less severe phenotype and/or that a methodological bias contributes to this discrepancy . . .
    • . . . It is noteworthy that, like in our case, the eight 17p11 duplications reported so far have been identified in the course of wide chromosomal screenings of MR patients.12, 32 The clinical features of these eight patients with 17p11 duplication include mild to borderline MR, behavioural disturbance, short stature, and dental abnormalities, and no specific phenotype could be identified, underlying the importance of wide screenings. . . .
    • . . . We estimated the size of the 17p11 duplication to 4 Mb by a locus-specific QMPSF indicating that this rearrangement, like previously reported cases,12, 37 can be considered as the reciprocal event of the common 4 Mb SMS deletion. (Figure 3b and c) . . .
  13. Moog U, Engelen JJ, Weber BW et al: Hereditary motor and sensory neuropathy (HMSN) IA, developmental delay and autism related disorder in a boy with duplication (17)(p11.2p12). Genet Couns 2004; 15: 73-80 , .
    • . . . Although nonallelic homologous recombination is supposed to generate microdeletions as well as microduplications, in the field of MR only four microduplications have clearly been related to phenotypes: a 15q11–q13 duplication has been detected in patients presenting autistic features and its frequency has been estimated to 1/200–600 among patients with developmental delay.10, 11 A 17p11.2 duplication has been associated with moderate MR and behavioural disturbance.12, 13 The 22q11 duplication, initially identified in patients with a clinical presentation similar to the classical 22q11 deletion, has recently been shown to result into a highly variable phenotype.14, 15 A 7q11.23 duplication has been related to severe expressive language delay.16 Among the possible explanations for the lower frequency of observed duplications, compared to deletions, one can speculate that duplications often result in a different or less severe phenotype and/or that a methodological bias contributes to this discrepancy . . .
  14. Ensenauer RE, Adeyinka A, Flynn HC et al: Microduplication 22q11.2, an emerging syndrome: clinical, cytogenetic, and molecular analysis of thirteen patients. Am J Hum Genet 2003; 73: 1027-1040 , .
    • . . . Although nonallelic homologous recombination is supposed to generate microdeletions as well as microduplications, in the field of MR only four microduplications have clearly been related to phenotypes: a 15q11–q13 duplication has been detected in patients presenting autistic features and its frequency has been estimated to 1/200–600 among patients with developmental delay.10, 11 A 17p11.2 duplication has been associated with moderate MR and behavioural disturbance.12, 13 The 22q11 duplication, initially identified in patients with a clinical presentation similar to the classical 22q11 deletion, has recently been shown to result into a highly variable phenotype.14, 15 A 7q11.23 duplication has been related to severe expressive language delay.16 Among the possible explanations for the lower frequency of observed duplications, compared to deletions, one can speculate that duplications often result in a different or less severe phenotype and/or that a methodological bias contributes to this discrepancy . . .
    • . . . Ensenauer et al14 have screened 653 patients referred for DG/VCFS syndrome testing by FISH, using the TUPLE1 probe on interphase cells, and found a 22q11 microduplication in 13 patients (2%) . . .
  15. Yobb TM, Somerville MJ, Willatt L et al: Microduplication and triplication of 22q11.2: a highly variable syndrome. Am J Hum Genet 2005; 76: 865-876 , .
    • . . . Although nonallelic homologous recombination is supposed to generate microdeletions as well as microduplications, in the field of MR only four microduplications have clearly been related to phenotypes: a 15q11–q13 duplication has been detected in patients presenting autistic features and its frequency has been estimated to 1/200–600 among patients with developmental delay.10, 11 A 17p11.2 duplication has been associated with moderate MR and behavioural disturbance.12, 13 The 22q11 duplication, initially identified in patients with a clinical presentation similar to the classical 22q11 deletion, has recently been shown to result into a highly variable phenotype.14, 15 A 7q11.23 duplication has been related to severe expressive language delay.16 Among the possible explanations for the lower frequency of observed duplications, compared to deletions, one can speculate that duplications often result in a different or less severe phenotype and/or that a methodological bias contributes to this discrepancy . . .
  16. Somerville MJ, Mervis CB, Young EJ et al: Severe expressive-language related to duplication of the Williams-Beuren locus. N Engl J Med 2005; 353: 1694-1701 , .
    • . . . Although nonallelic homologous recombination is supposed to generate microdeletions as well as microduplications, in the field of MR only four microduplications have clearly been related to phenotypes: a 15q11–q13 duplication has been detected in patients presenting autistic features and its frequency has been estimated to 1/200–600 among patients with developmental delay.10, 11 A 17p11.2 duplication has been associated with moderate MR and behavioural disturbance.12, 13 The 22q11 duplication, initially identified in patients with a clinical presentation similar to the classical 22q11 deletion, has recently been shown to result into a highly variable phenotype.14, 15 A 7q11.23 duplication has been related to severe expressive language delay.16 Among the possible explanations for the lower frequency of observed duplications, compared to deletions, one can speculate that duplications often result in a different or less severe phenotype and/or that a methodological bias contributes to this discrepancy . . .
  17. Sharp AJ, Locke DP, McGrath SD et al: Segmental duplications and copy-number variation in the human genome. Am J Hum Genet 2005; 77: 78-88 , .
    • . . . Nevertheless, the recent findings highlighting the previously unsuspected extend of the copy-number polymorphisms in the human genome17, 18, 19 hampers, at the present time, its use on a routine basis in molecular genetics laboratories.20, 21, 22, 23, 24 Therefore, we considered that molecular assays focused on regions that have already been identified as targets for microdeletions and microduplications should be more effective in detecting selectively deleterious rearrangements. . . .
    • . . . Several studies, based on CGH-array at a 1 Mb resolution, reported, in about 25% of MR patients, the presence of genomic imbalances, 30–42% of which corresponding to duplications.20, 21, 22 While CGH-array seems to be the most attractive tool for genomewide screening, its use for guiding genetic counselling is limited by the fact that one cannot differentiate genomic imbalances which cause abnormal phenotypes from variants unrelated to clinical alterations since recent publications have demonstrated the high degree of copy-number polymorphism in the human genome.17, 18, 19 Recently, targeted array-based GCH was developed for medical applications but its cost limits its use in medical genetics laboratories.35 Finally, MLPA (multiplex ligation-dependent probe amplification), represents a powerful technique to detect copy-number changes, including those resulting from subtelomeric rearrangements.36 Therefore, we consider that molecular methods, such as QMPSF or MLPA, represent efficient multilocus diagnostic tools zooming in on regions that have been identified as targets for microdeletions and microduplications involved in MR . . .
  18. Iafrate AJ, Feuk L, Rivera MN et al: Detection of large-scale variation in the human genome. Nat Genet 2004; 36: 949-951 , .
    • . . . Nevertheless, the recent findings highlighting the previously unsuspected extend of the copy-number polymorphisms in the human genome17, 18, 19 hampers, at the present time, its use on a routine basis in molecular genetics laboratories.20, 21, 22, 23, 24 Therefore, we considered that molecular assays focused on regions that have already been identified as targets for microdeletions and microduplications should be more effective in detecting selectively deleterious rearrangements. . . .
    • . . . Several studies, based on CGH-array at a 1 Mb resolution, reported, in about 25% of MR patients, the presence of genomic imbalances, 30–42% of which corresponding to duplications.20, 21, 22 While CGH-array seems to be the most attractive tool for genomewide screening, its use for guiding genetic counselling is limited by the fact that one cannot differentiate genomic imbalances which cause abnormal phenotypes from variants unrelated to clinical alterations since recent publications have demonstrated the high degree of copy-number polymorphism in the human genome.17, 18, 19 Recently, targeted array-based GCH was developed for medical applications but its cost limits its use in medical genetics laboratories.35 Finally, MLPA (multiplex ligation-dependent probe amplification), represents a powerful technique to detect copy-number changes, including those resulting from subtelomeric rearrangements.36 Therefore, we consider that molecular methods, such as QMPSF or MLPA, represent efficient multilocus diagnostic tools zooming in on regions that have been identified as targets for microdeletions and microduplications involved in MR . . .
  19. Sebat J, Lakshmi B, Troge J et al: Large-scale copy number polymorphism in the human genome. Science 2004; 305: 525-528 , .
    • . . . Nevertheless, the recent findings highlighting the previously unsuspected extend of the copy-number polymorphisms in the human genome17, 18, 19 hampers, at the present time, its use on a routine basis in molecular genetics laboratories.20, 21, 22, 23, 24 Therefore, we considered that molecular assays focused on regions that have already been identified as targets for microdeletions and microduplications should be more effective in detecting selectively deleterious rearrangements. . . .
    • . . . Several studies, based on CGH-array at a 1 Mb resolution, reported, in about 25% of MR patients, the presence of genomic imbalances, 30–42% of which corresponding to duplications.20, 21, 22 While CGH-array seems to be the most attractive tool for genomewide screening, its use for guiding genetic counselling is limited by the fact that one cannot differentiate genomic imbalances which cause abnormal phenotypes from variants unrelated to clinical alterations since recent publications have demonstrated the high degree of copy-number polymorphism in the human genome.17, 18, 19 Recently, targeted array-based GCH was developed for medical applications but its cost limits its use in medical genetics laboratories.35 Finally, MLPA (multiplex ligation-dependent probe amplification), represents a powerful technique to detect copy-number changes, including those resulting from subtelomeric rearrangements.36 Therefore, we consider that molecular methods, such as QMPSF or MLPA, represent efficient multilocus diagnostic tools zooming in on regions that have been identified as targets for microdeletions and microduplications involved in MR . . .
  20. Rosenberg C, Knijnenburg J, Bakker E et al: Array-CGH detection of micro rearrangements in mentally retarded individuals: clinical significance of imbalances present both in affected children and normal parents. J Med Genet 2006; 43: 180-186 , .
    • . . . Nevertheless, the recent findings highlighting the previously unsuspected extend of the copy-number polymorphisms in the human genome17, 18, 19 hampers, at the present time, its use on a routine basis in molecular genetics laboratories.20, 21, 22, 23, 24 Therefore, we considered that molecular assays focused on regions that have already been identified as targets for microdeletions and microduplications should be more effective in detecting selectively deleterious rearrangements. . . .
    • . . . Several studies, based on CGH-array at a 1 Mb resolution, reported, in about 25% of MR patients, the presence of genomic imbalances, 30–42% of which corresponding to duplications.20, 21, 22 While CGH-array seems to be the most attractive tool for genomewide screening, its use for guiding genetic counselling is limited by the fact that one cannot differentiate genomic imbalances which cause abnormal phenotypes from variants unrelated to clinical alterations since recent publications have demonstrated the high degree of copy-number polymorphism in the human genome.17, 18, 19 Recently, targeted array-based GCH was developed for medical applications but its cost limits its use in medical genetics laboratories.35 Finally, MLPA (multiplex ligation-dependent probe amplification), represents a powerful technique to detect copy-number changes, including those resulting from subtelomeric rearrangements.36 Therefore, we consider that molecular methods, such as QMPSF or MLPA, represent efficient multilocus diagnostic tools zooming in on regions that have been identified as targets for microdeletions and microduplications involved in MR . . .
  21. Vissers LE, de Vries BB, Osoegawa K et al: Array-based comparative genomic hybridization for the genomewide detection of submicroscopic chromosomal abnormalities. Am J Hum Genet 2003; 73: 1261-1270 , .
    • . . . Nevertheless, the recent findings highlighting the previously unsuspected extend of the copy-number polymorphisms in the human genome17, 18, 19 hampers, at the present time, its use on a routine basis in molecular genetics laboratories.20, 21, 22, 23, 24 Therefore, we considered that molecular assays focused on regions that have already been identified as targets for microdeletions and microduplications should be more effective in detecting selectively deleterious rearrangements. . . .
    • . . . Several studies, based on CGH-array at a 1 Mb resolution, reported, in about 25% of MR patients, the presence of genomic imbalances, 30–42% of which corresponding to duplications.20, 21, 22 While CGH-array seems to be the most attractive tool for genomewide screening, its use for guiding genetic counselling is limited by the fact that one cannot differentiate genomic imbalances which cause abnormal phenotypes from variants unrelated to clinical alterations since recent publications have demonstrated the high degree of copy-number polymorphism in the human genome.17, 18, 19 Recently, targeted array-based GCH was developed for medical applications but its cost limits its use in medical genetics laboratories.35 Finally, MLPA (multiplex ligation-dependent probe amplification), represents a powerful technique to detect copy-number changes, including those resulting from subtelomeric rearrangements.36 Therefore, we consider that molecular methods, such as QMPSF or MLPA, represent efficient multilocus diagnostic tools zooming in on regions that have been identified as targets for microdeletions and microduplications involved in MR . . .
  22. Shaw-Smith C, Redon R, Rickman L et al: Microarray based comparative genomic hybridisation (array-CGH) detects submicroscopic chromosomal deletions and duplications in patients with learning disability/mental retardation and dysmorphic features. J Med Genet 2004; 41: 241-248 , .
    • . . . Nevertheless, the recent findings highlighting the previously unsuspected extend of the copy-number polymorphisms in the human genome17, 18, 19 hampers, at the present time, its use on a routine basis in molecular genetics laboratories.20, 21, 22, 23, 24 Therefore, we considered that molecular assays focused on regions that have already been identified as targets for microdeletions and microduplications should be more effective in detecting selectively deleterious rearrangements. . . .
    • . . . Several studies, based on CGH-array at a 1 Mb resolution, reported, in about 25% of MR patients, the presence of genomic imbalances, 30–42% of which corresponding to duplications.20, 21, 22 While CGH-array seems to be the most attractive tool for genomewide screening, its use for guiding genetic counselling is limited by the fact that one cannot differentiate genomic imbalances which cause abnormal phenotypes from variants unrelated to clinical alterations since recent publications have demonstrated the high degree of copy-number polymorphism in the human genome.17, 18, 19 Recently, targeted array-based GCH was developed for medical applications but its cost limits its use in medical genetics laboratories.35 Finally, MLPA (multiplex ligation-dependent probe amplification), represents a powerful technique to detect copy-number changes, including those resulting from subtelomeric rearrangements.36 Therefore, we consider that molecular methods, such as QMPSF or MLPA, represent efficient multilocus diagnostic tools zooming in on regions that have been identified as targets for microdeletions and microduplications involved in MR . . .
  23. Schoumans J, Ruivenkamp C, Holmberg E, Kyllerman M, Anderlid BM, Nordenskjold M: Detection of chromosomal imbalances in children with idiopathic mental retardation by array based comparative genomic hybridisation (array-CGH). J Med Genet 2005; 42: 699-705 , .
    • . . . Nevertheless, the recent findings highlighting the previously unsuspected extend of the copy-number polymorphisms in the human genome17, 18, 19 hampers, at the present time, its use on a routine basis in molecular genetics laboratories.20, 21, 22, 23, 24 Therefore, we considered that molecular assays focused on regions that have already been identified as targets for microdeletions and microduplications should be more effective in detecting selectively deleterious rearrangements. . . .
  24. de Vries BB, Pfundt R, Leisink M et al: Diagnostic genome profiling in mental retardation. Am J Hum Genet 2005; 77: 606-616 , .
    • . . . Nevertheless, the recent findings highlighting the previously unsuspected extend of the copy-number polymorphisms in the human genome17, 18, 19 hampers, at the present time, its use on a routine basis in molecular genetics laboratories.20, 21, 22, 23, 24 Therefore, we considered that molecular assays focused on regions that have already been identified as targets for microdeletions and microduplications should be more effective in detecting selectively deleterious rearrangements. . . .
  25. Charbonnier F, Raux G, Wang Q et al: Detection of exon deletions and duplications of the mismatch repair genes in hereditary nonpolyposis colorectal cancer families using multiplex polymerise chain reaction of short fluorescent fragments. Cancer Res 2000; 60: 2760-2763 , .
    • . . . QMPSF has been shown to be a sensitive method for the detection of both deletions and duplications25, 26, 27, 28 and is currently used in numerous molecular diagnostic laboratories . . .
  26. Casilli F, Di Rocco ZC, Gad S et al: Rapid detection of novel BRCA1 rearrangements in high-risk breast-ovarian cancer families using multiplex PCR of short fluorescent fragments. Hum Mutat 2002; 20: 218-226 , .
    • . . . QMPSF has been shown to be a sensitive method for the detection of both deletions and duplications25, 26, 27, 28 and is currently used in numerous molecular diagnostic laboratories . . .
  27. Rovelet-Lecrux A, Hannequin D, Raux G et al: APP locus duplication causes autosomal dominant early-onset Alzheimer disease with cerebral amyloid angiopathy. Nat Genet 2006; 38: 24-26 , .
    • . . . QMPSF has been shown to be a sensitive method for the detection of both deletions and duplications25, 26, 27, 28 and is currently used in numerous molecular diagnostic laboratories . . .
  28. Tournier I, Paillerets BB, Sobol H et al: Significant contribution of germline BRCA2 rearrangements in male breast cancer families. Cancer Res 2004; 64: 8143-8147 , .
    • . . . QMPSF has been shown to be a sensitive method for the detection of both deletions and duplications25, 26, 27, 28 and is currently used in numerous molecular diagnostic laboratories . . .
  29. de Vries BB, White SM, Knight SJ et al: Clinical studies on submicroscopic subtelomeric rearrangements: a checklist. J Med Genet 2001; 38: 145-150 , .
    • . . . These patients did not fulfil the de Vries criteria for subtelomeric rearrangements, exhibited normal chromosomes and did not harbour expansions within the FMR1 gene.29 In this second series, 35 cases (25%) presented with a familial history of MR, whereas 104 cases (74%) were sporadic and one patient was adopted . . .
  30. Jacquet H, Raux G, Thibaut F et al: PRODH mutations and hyperprolinemia in a subset of schizophrenic patients. Hum Mol Genet 2002; 11: 2243-2249 , .
    • . . . The DiGeorge QMPSF includes 22 amplicons corresponding to 22 genes localised within the 22q11 region30 and two amplicons within the 10p14 region . . .
    • . . . A 22q11 locus QMPSF analysis30 showed that this patient harbour the classical 3 Mb 22q11 deletion associated with DiGeorge syndrome (Figures 2b and c), and this rearrangement was confirmed by FISH (Figure 2c) . . .
    • . . . In both (a) and (b), C1 and C2 design control amplicons. (c) Schematic representation of the position of the 22q11 amplicons along the chromosome 22.30 The arrow shows the extend of the deletion revealed by QMPSF. (d) Confirmation by FISH of the rearrangement using the Spectrum Orange LSI DiGeorge/VCSF region probe (Vysis, Downers Groove, USA) which encompasses the TUPLE1/HIRA gene and the D22S553, D22S609, D22S942 loci and the Spectrum Green LSI ARSA control probe on 22q13 (Vysis, Downers Groove, USA). . . .
  31. Gratacos M, Nadal M, Martin-Santos R et al: A polymorphic genomic duplication on human chromosome 15 is a susceptibility factor for panic and phobic disorders. Cell 2001; 106: 367-379 , .
    • . . . Six amplicons correspond to genes of interest located within the deleted interval of six genomic disorders, that is: Sotos syndrome (MIM 117550, NSD1 gene), Williams–Beuren syndrome (MIM 194050, ELN gene), Prader–Willi and Angelman syndromes (MIM 176270 and MIM 105830, respectively, SNRPN gene), panic and phobic disorder (LOXL1 gene),31 Smith–Magenis syndrome (SMS, MIM 182290, RAI1 gene) and DiGeorge syndrome/velocardiofacial syndrome (MIM 188400, TBX1 gene) . . .
  32. Kriek M, White SJ, Bouma MC et al: Genomic imbalances in mental retardation. J Med Genet 2004; 41: 249-255 , .
    • . . . It is noteworthy that, like in our case, the eight 17p11 duplications reported so far have been identified in the course of wide chromosomal screenings of MR patients.12, 32 The clinical features of these eight patients with 17p11 duplication include mild to borderline MR, behavioural disturbance, short stature, and dental abnormalities, and no specific phenotype could be identified, underlying the importance of wide screenings. . . .
    • . . . A MAPH (multiple amplifiable probe hybridisation)-based assay, investigating simultaneously 162 loci corresponding to subtelomeric regions or interstitial genomic segments, allowed Kriek et al32 to detect 15 genomic imbalances including seven duplications among 188 patients with MR (8%) . . .
  33. Lese-Martin C, Chung J, Ilkin Y, Geschwind DH, Ledbetter DH: Molecular cytogenetic investigations of multiplex autism families confirm 15q11-q13 duplications as a cause of autism [abstract]. Am J Hum Genet 2004; 73 (Suppl): P975 , .
    • . . . Similarly, Lese-Martin et al33 have analysed, by FISH using a SNRPN probe, 148 patients with autism spectrum disorders and found that two patients (1.4%) harbour a 15q11–q13 microduplication . . .
  34. Keller K, Williams C, Wharton P et al: Routine cytogenetic and FISH studies for 17p11/15q11 duplications and subtelomeric rearrangement studies in children with autism spectrum disorders. Am J Med Genet A 2003; 117: 105-111 , .
    • . . . Finally, Keller et al34 have screened 49 autistic children by FISH, using the D15S10 (15q11.2) and FLII (17p11.2) DNA probes, and detected a single case of 15q11-q13 microduplication (2%) but no 17p11.2 microduplication.34 Thus, FISH can be efficiently used to screen for a specific rearrangement but is not suitable for high throughput diagnostic screening of MR patients . . .
  35. Bejjani BA, Saleki R, Ballif BC et al: Use of targeted array-based CGH for the clinical diagnosis of chromosomal imbalance: is less more? Am J Med Genet 2005; 134: 259-267 , .
    • . . . Several studies, based on CGH-array at a 1 Mb resolution, reported, in about 25% of MR patients, the presence of genomic imbalances, 30–42% of which corresponding to duplications.20, 21, 22 While CGH-array seems to be the most attractive tool for genomewide screening, its use for guiding genetic counselling is limited by the fact that one cannot differentiate genomic imbalances which cause abnormal phenotypes from variants unrelated to clinical alterations since recent publications have demonstrated the high degree of copy-number polymorphism in the human genome.17, 18, 19 Recently, targeted array-based GCH was developed for medical applications but its cost limits its use in medical genetics laboratories.35 Finally, MLPA (multiplex ligation-dependent probe amplification), represents a powerful technique to detect copy-number changes, including those resulting from subtelomeric rearrangements.36 Therefore, we consider that molecular methods, such as QMPSF or MLPA, represent efficient multilocus diagnostic tools zooming in on regions that have been identified as targets for microdeletions and microduplications involved in MR . . .
  36. Northrop EL, Ren H, Bruno DL et al: Detection of cryptic subtelomeric chromosome abnormalities and identification of anonymous chromatin using a quantitative multiplex ligation-dependent probe amplification (MLPA) assay. Hum Mutat 2005; 26: 477-486 , .
    • . . . Several studies, based on CGH-array at a 1 Mb resolution, reported, in about 25% of MR patients, the presence of genomic imbalances, 30–42% of which corresponding to duplications.20, 21, 22 While CGH-array seems to be the most attractive tool for genomewide screening, its use for guiding genetic counselling is limited by the fact that one cannot differentiate genomic imbalances which cause abnormal phenotypes from variants unrelated to clinical alterations since recent publications have demonstrated the high degree of copy-number polymorphism in the human genome.17, 18, 19 Recently, targeted array-based GCH was developed for medical applications but its cost limits its use in medical genetics laboratories.35 Finally, MLPA (multiplex ligation-dependent probe amplification), represents a powerful technique to detect copy-number changes, including those resulting from subtelomeric rearrangements.36 Therefore, we consider that molecular methods, such as QMPSF or MLPA, represent efficient multilocus diagnostic tools zooming in on regions that have been identified as targets for microdeletions and microduplications involved in MR . . .
  37. Chen KS, Manian P, Koeuth T et al: Homologous recombination of a flanking repeat gene cluster is a mechanism for a common contiguous gene deletion syndrome. Nat Genet 1997; 17: 154-163 , .
    • . . . We estimated the size of the 17p11 duplication to 4 Mb by a locus-specific QMPSF indicating that this rearrangement, like previously reported cases,12, 37 can be considered as the reciprocal event of the common 4 Mb SMS deletion. (Figure 3b and c) . . .
  38. Shaw CJ, Bi W, Lupski JR: Genetic proof of unequal meiotic crossovers in reciprocal deletion and duplication of 17p11.2. Am J Hum Genet 2002; 71: 1072-1081 , .
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