Scientific publications

1. Genetic polymorphisms associated with outcome in multiple myeloma patients receiving high-dose melphalan
   Dumontet C, Landi S, Reiman T, Perry T, Plesa A, Bellini I, Barale R, Pilarski LM, Troncy J, Tavtigian S, Gemignani F.
   Bone Marrow Transplantation advance online publication, 7 December 2009
   
   High-dose melphalan (HDM) is an essential component in the treatment of patients with multiple myeloma (MM). Few data are available regarding genetic polymorphisms associated with patient outcome or toxicity in this setting. To identify such polymorphisms, we performed a retrospective analysis, genotyping single nucleotide polymorphisms (SNPs) with the arrayed primer extension (APEX) technology in 169 patients having received HDM for MM. We analyzed 209 SNPs in 95 genes involved in drug metabolism, DNA repair, cell cycle and apoptosis. SNPs in ABCB1, CYP3A4 and TP53BP2 were associated with response to VAD induction therapy (P<0.01). SNPs in ALDH2, GSTT2 and BRCA1 were associated with response to HDM (P<0.01). Polymorphisms in CYP1A1, RAD51 and PARP were associated with disease progression whereas polymorphisms in ALDH2 and CYP1A1 were correlated with OS. Polymorphisms in BRCA1, CDKN1A and XRCC1 were associated with the occurrence of severe mucositis after HDM. These results suggest that SNPs of genes involved in drug metabolism or DNA repair could be used to distinguish MM patient subgroups with different toxicity/efficacy profiles.
   

2. Genotyping microarray for CSNB-associated genes
   Zeitz C, Labs S, Lorenz B, Forster U, Uksti J, Kroes HY, De Baere E, Leroy BP, Cremers FP, Wittmer M, van Genderen MM, Sahel JA, Audo I, Poloschek CM, Mohand-Saïd S, Fleischhauer JC, Hüffmeier U, Moskova-Doumanova V, Levin AV, Hamel CP, Leifert D, Munier FL, Schorderet DF, Zrenner E, Friedburg C, Wissinger B, Kohl S, Berger W.
   Invest Ophthalmol Vis Sci. 2009 Dec;50(12):5919-26. Epub 2009 Jul 2.
   
   PURPOSE: Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous retinal disease. Although electroretinographic (ERG) measurements can discriminate clinical subgroups, the identification of the underlying genetic defects has been complicated for CSNB because of genetic heterogeneity, the uncertainty about the mode of inheritance, and time-consuming and costly mutation scanning and direct sequencing approaches. METHODS: To overcome these challenges and to generate a time- and cost-efficient mutation screening tool, the authors developed a CSNB genotyping microarray with arrayed primer extension (APEX) technology. To cover as many mutations as possible, a comprehensive literature search was performed, and DNA samples from a cohort of patients with CSNB were first sequenced directly in known CSNB genes. Subsequently, oligonucleotides were designed representing 126 sequence variations in RHO, CABP4, CACNA1F, CACNA2D4, GNAT1, GRM6, NYX, PDE6B, and SAG and spotted on the chip. RESULTS: Direct sequencing of genes known to be associated with CSNB in the study cohort revealed 21 mutations (12 novel and 9 previously reported). The resultant microarray containing oligonucleotides, which allow to detect 126 known and novel mutations, was 100% effective in determining the expected sequence changes in all known samples assessed. In addition, investigation of 34 patients with CSNB who were previously not genotyped revealed sequence variants in 18%, of which 15% are thought to be disease-causing mutations. CONCLUSIONS: This relatively inexpensive first-pass genetic testing device for patients with a diagnosis of CSNB will improve molecular diagnostics and genetic counseling of patients and their families and gives the opportunity to analyze whether, for example, more progressive disorders such as cone or cone-rod dystrophies underlie the same gene defects.
   

3. Evaluation of the 124-plex SNP typing microarray for forensic testing
   Krjutskov K, Viltrop T, Palta P, Metspalu E, Tamm E, Suvi S, Sak K, Merilo A, Sork H, Teek R, Nikopensius T, Kivisild T, Metspalu A.
   Forensic Sci Int Genet. 2009 Dec; 4(1): 43-8. Epub 2009 May 15.
   
   Human identification systems such as criminal databases, forensic DNA testing and genetic genealogy require reliable and cost-effective genotyping of autosomal, mitochondrial and Y chromosome markers from different biological materials, including venous blood and saliva. Although many such assays are available, few systems are capable of simultaneously detecting all three targets in a single reaction. Employing the APEX-2 principle, we have characterized a novel 124-plex assay, using specific primer extension, universal primer amplification and single base extension on an oligonucleotide array. The assay has been designed for simultaneous genotyping of SNPs from the single copy loci (46 autosomal and 29 Y chromosomal markers) side by side with SNPs from the mitochondrial genome (49 markers) that appears in up to thousands of copies per cell in certain tissue types. All the autosomal SNPs (from the SNPforID Consortium) included in the multiplex assay are unlinked and are distributed widely across autosomes, enabling genetic fingerprints to be distinguished. Mitochondrial DNA and Y chromosome polymorphisms that define haplogroups common in European populations are included to allow for maternity and paternity testing and for the analysis of genetic genealogies. After assay optimization we estimated the accuracy (99.83%) and call rate (99.66%) of the protocol on 17 mother–father–child/children families and five internal control DNAs. In addition, 79 unrelated Estonian and Swedish DNA samples were genotyped and the accuracy of mtDNA and Y chromosome haplogroup inference by the multiplex method was assessed using conventional genotyping methods and direct sequencing.
   

4. Arrayed primer extension on in situ synthesized 5'-->3' oligonucleotides in microchannels
   Pullat J, Kusnezow W, Jaakson K, Beier M, Hoheisel JD, Metspalu A.
   N Biotechnol. 2008 Oct-Dec; 25(2-3): 133-41. Epub 2008 Aug 14.
   

5. Arrayed Primer Extension for the Noninvasive Prenatal Diagnosis of β-Thalassemia Based on Detection of Single Nucleotide Polymorphisms
   Papasavva T, Kalikas I, Kyrri A, and Kleanthousa M.
   Annals of the New York Academy of Sciences  2008, vol. 1137, pp. 302-308.
   

6. Microarray-based mutation analysis of the ABCA4 gene in Spanish patients with Stargardt disease: evidence of a prevalent mutated allele
   Diana Valverde, R. Riveiro-Alvarez, Sara Bernal, Kaie Jaakson, Montserrat Baiget, Rafael Navarro, Carmen Ayuso
   Molecular Vision 2006; 12:902-908
   
   Purpose:To evaluate, in a pool of affected families, the mutation spectrum in Stargardt patients from Spain, using the ABCR400 microarray that contains described sequence variants in the gene encoding for the photoreceptor specific ATP-binding cassette transporter (ABCA4).
   Methods:We analyzed 76 Spanish patients with STGD1 for a population-specific survey on the sequence variations in the ABCA4gene, using the ABCR400 microarray.
   Results:Potential disease-associated alleles were identified in 91 of the 152 STGD1 chromosomes studied, resulting in a detection rate of 60%. The two mutant alleles were found in 33/76 patients (43%), whereas in 25/76 cases (33%) only one allele could be identified. In the remaining 18 patients no mutations were found. In total, we identified 40 sequence variations that could be related to the disease. The vast majority of these substitutions (35/40) were missense mutations. Three frameshift mutations and two splicing variants were also found.
   Conclusions:We identified a major disease-associated allele, R1129L, which accounted for 24% of the mutated alleles detected, and a high frequency (12%) of complex alleles.
   
   

7. Comprehensive Arrayed Primer Extension Array for the Detection of 59 Sequence Variants in 15 Conditions Prevalent Among the (Ashkenazi) Jewish Population.
   Iris Schrijver, Maigi Külm, Phyllis I. Gardner, Eugene P. Pergament, and Morris B. Fiddle
   Journal of Molecular Diagnostics, Vol. 9, No. 2, April 2007
   
   In the Ashkenazi Jewish population, serious and lethal genetic conditions occur with relatively high frequency. A single test that encompasses the majority of population-specific mutations is not currently available. For comprehensive carrier screening and molecular diagnostic purposes, we developed a population-specific and inclusive microarray. The arrayed primer extension genotyping microarray carries 59 sequence variant detection sites, of which 53 are detectable bi-directionally. These sites represent the most common variants in Tay-Sachs disease, Bloom syndrome, Canavan disease, Niemann-Pick A, familial dysautonomia, torsion dystonia, mucolipidosis type IV, Fanconi anemia, Gaucher disease, factor XI deficiency, glycogen storage disease type 1a, maple
   syrup urine disease, nonsyndromic sensorineural hearing loss, familial Mediterranean fever, and glycogen storage disease type III. Several mutations in the selected disorders that are not prevalent per se in the Ashkenazi Jewish populations, as well pseudodeficiency
   alleles, are also included in the array. The initial technical evaluation of this microarray demonstrates that it is comprehensive, robust, sensitive, specific , and easily modifiable. This cost-effective array is based on a diversely applied platform technology and is suitable for both carrier screening and disease detection in Ashkenazi and Sephardic Jewish populations.
    

8. Association study of sporadic Parkinson's disease genetic risk factors in patients from Russia by APEX technology.
   Shadrina M, Nikopensius T, Slominsky P, Illarioshkin S, Bagyeva G, Markova E, Ivanova-Smolenskaia I, Kurg A, Limborska S, Metspalu A.
   Neurosci Lett. 2006 Sep 25;405(3):212-6. Epub 2006 Jul 28.
   
   Most patients with Parkinson’s disease (PD) have sporadic form of the disease with a multifactorial etiology due to interactions between environmental conditions and the genetic constitution of the individuals.We have analyzed by APEX technology 50 single nucleotide polymorphisms (SNPs) in 19 genes related to cholecystokinin, serotonin, dopamine and opioid neurotransmission. Significant differences in the allele and genotype frequencies between the controls and PD patients were detected for four SNPs from three genes (serotonin 2A receptor (rs6311, P= 0.043), Wolfram syndrome 1 (rs1801211, P= 0.007), proopiomelanocortin (rs28930368, P= 0.026 and rs2071345, P= 0.027) genes). Two SNPs in proopiomelanocortin (POMC) gene were also associated with different clinical forms of PD. Our data suggest that at least three genes involved in neurotransmitter systems may have more specific role in genetic predisposition to PD.
   
   

9. Simultaneous multigene mutation detection in patients with sensorineural hearing loss through a novel diagnostic microarray: a new approach for newborn screening follow-up.
   Gardner P, Oitmaa E, Messner A, Hoefsloot L, Metspalu A, Schrijver I.
   Pediatrics. 2006 Sep;118(3):985-94.
   
   The advent of universal newborn hearing screening in the United States and other countries, together with the identification of genes involved in the process of hearing, have led to an increase in both the need and opportunity for accurate molecular diagnosis of patients with hearing loss. Deafness and hearing impairment have a genetic cause in at least half the cases. The molecular genetic basis for the majority of these patients remains obscure, however, because of the absence of associated clinical features in approximately 70% (ie, nonsyndromic hearing loss) of patients, genetic heterogeneity, and the lack of molecular genetic tests that can evaluate a large number of mutations across multiple genes. DESIGN: We report on the development of a diagnostic panel with 198 mutations underlying sensorineural (mostly nonsyndromic) hearing loss. This panel, developed on a microarray, is capable of simultaneous evaluation of multiple mutations in 8 genes (GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5 and the mitochondrial genes encoding 12S rRNA and tRNA-Ser[UCN]). RESULTS: The arrayed primer extension array for sensorineural hearing loss is based on a versatile platform technology and is a robust, cost-effective, and easily modifiable assay. Because hearing loss is a major public health concern and common at all ages, this test is suitable for follow-up after newborn hearing screening and for the detection of a genetic etiology in older children and adults. CONCLUSIONS: Comprehensive and relatively inexpensive genetic testing for sensorineural hearing loss will improve medical management for affected individuals and genetic counseling for their families.
     

10. Development of a Genotyping Microarray for Usher Syndrome
    Cremers FP, Kimberling WJ, Kulm M, de Brouwer A, van Wijk E, Te Brinke H, Cremers CW, Hoefsloot LH, Banfi S, Simonelli F, Fleischhauer JC, Berger W, Kelley PM, Haralambous E, Bitner-Glindzicz M, Webster AR, Saihan Z, Debaere E, Leroy BP, Silvestri G, McKay G, Koenekoop RK, Millan JM, Rosenberg T, Joensuu T, Sankila EM, Weil D, Weston MD, Wissinger B, Kremer H.
    J Med Genet. 2006 Sep 8
    
    Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, 8 genes have been implicated, which together comprise 347 protein-coding exons. Therefore, sequence analysis and the most routinely used mutation scanning techniques are not cost-effective for molecular diagnostics of Usher syndrome. To improve DNA-diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. METHODS: Allele-specific oligonucleotides corresponding to 298 Usher syndrome-associated sequence variants known to date, 76 of which are novel, were arrayed. The accuracy of the microarray was analysed using DNAs from 158 patients with known mutations; the efficiency of the microarray was analysed using DNAs from 370 novel European and American patients with Usher syndrome. RESULTS: Validation of the microarray yielded an accuracy of >98%. Among the novel patients, sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I (USH1), 45/189 (24%) patients with Usher syndrome type II (USH2), 6/21 (29%) patients with Usher syndrome type III (USH3), and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. DISCUSSION: The Usher genotyping microarray represents a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first-pass screening tool.
    
    

11. Evaluation of arrayed primer extension for TP53 mutation detection in breast and ovarian carcinomas.
    Pedro Kringen, Anna Bergamaschi, Eldri Undlien Due, Yun Wang, Elda Tagliabue, Jahn M. Nesland, Aune Ahman, Neeme Tönisson, and Anne-Lise Børresen-Dale.
    BioTechniques, vol. 39 , no 5 (2005): pp 755-761
    
    Mutations in the tumor suppressor gene TP53 are associated with a wide range of different cancers and may have prognostic and therapeutic implications. Methods for rapid and sensitive detection of mutations in this gene are therefore required. In order to make screening more effective, a commercially available TP53 genotyping microarray from Asper Biotech has been constructed by arrayed primer extension (APEX). The present study is the first report that blindly evaluates the efficiency of the second generation APEX TP53 genotype chip outside the Asper laboratory and compares it to temporal temperature gradient electrophoresis (TTGE) and sequencing of TP53 for mutation detection in ovarian and breast cancer samples. All nucleotides in the TP53 gene from exon 2–9 are included on the chip by synthesis and application of sequence-specific oligonucleotides. The chip was validated by screening 48 breast and 11 ovarian cancer cases, all of which had previously been analyzed by TTGE and sequencing. APEX scored 17 of 20 sequence variants, missing one deletion, one insertion, and a missense mutation. Resequencing efficiency using APEX was 92% for both DNA strands and 99.5% for sense and/or antisense strand. We conclude that the APEX TP53 microarray is a robust, rapid, and comprehensive screening tool for sequence alterations in tumors.

12. Genotyping microarray (disease chip) for leber congenital amaurosis: detection of modifier alleles.
    Zernant J, Kulm M, Dharmaraj S, den Hollander AI, Perrault I, Preising MN, Lorenz B, Kaplan J, Cremers FP, Maumenee I, Koenekoop RK, Allikmets R.
    Invest Ophthalmol Vis Sci. 2005 Sep;46(9):3052-9.
    
    PURPOSE: Leber congenital amaurosis (LCA) is an early-onset inherited disorder of childhood blindness characterized by visual impairment noted soon after birth. Variants in at least six genes (AIPL1, CRB1, CRX, GUCY2D, RPE65, and RPGRIP1) have been associated with a diagnosis consistent with LCA or early-onset retinitis pigmentosa (RP). Genetically heterogeneous inheritance complicates the analyses of LCA cases, especially in patients without a family history of the disorder, and conventional methods are of limited value. METHODS: To overcome these limitations, arrayed primer extension (APEX) technology was used to design a genotyping microarray for early-onset, severe retinal degenerations that includes all of the >300 disease-associated variants currently described in eight genes (in addition to the six just listed, the early-onset RP genes LRAT and MERTK were added). The resultant LCA array allows simultaneous detection of all known disease-associated alleles in any patient with early-onset RP. The array was validated by screening 93 confirmed patients with LCA who had known mutations. Subsequently, 205 novel LCA cases were screened on the array, followed by segregation analyses in families, if applicable. RESULTS: The microarray was >99% effective in determining the existing genetic variation and yielded at least one disease-associated allele in approximately one third of the novel patients. More than two (expected) variants were discovered in a substantial fraction (22/300) of the patients, suggesting a modifier effect from more than one gene. In support of the latter hypothesis, the third allele segregated with a more severe disease phenotype in at least five families. CONCLUSIONS: The LCA genotyping microarray is a robust and cost-effective screening tool, representing the prototype of a disease chip for genotyping patients with a genetically heterogeneous condition. Simultaneous screening for all known LCA-associated variants in large LCA cohorts allows systematic detection and analysis of genetic variation, facilitating prospective diagnosis and ultimately predicting disease progression.
    
    

13. Genotyping Microarray for the Detection of More Than 200 CFTR Mutations in Ethnically Diverse Populations
    Schrijver I, Oitmaa E, Metspalu A, Gardner P. 
    J Mol Diagn. 2005 Aug;7(3):375-87
    
    Cystic fibrosis (CF), which is due to mutations in the cystic fibrosis transmembrane conductance regulator gene, is a common life-shortening disease. Although CF occurs with the highest incidence in Caucasians, it also occurs in other ethnicities with variable frequency. Recent national guidelines suggest that all couples contemplating pregnancy should be informed of molecular screening for CF carrier status for purposes of genetic counseling. Commercially available CF carrier screening panels offer a limited panel of mutations, however, making them insufficiently sensitive for certain groups within an ethnically diverse population. This discrepancy is even more pronounced when such carrier screening panels are used for diagnostic purposes. By means of arrayed primer extension technology, we have designed a genotyping microarray with 204 probe sites for CF transmembrane conductance regulator gene mutation detection. The arrayed primer extension array, based on a platform technology for disease detection with multiple applications, is a robust, cost-effective, and easily modifiable assay suitable for CF carrier screening and disease detection.
    
    

14. Arrayed Primer Extension Resequencing of Mutations in the TP53 Tumor Suppressor Gene: Comparison with Denaturing HPLC and Direct Sequencing.
    
    Le Calvez F, Ahman A,Tonisson N,Lambert J, Temam S, Brennan P, Zaridze DG, Metspalu A, Hainaut P.
    Clin Chem. 2005 Jul;51(7):1284-7.
    
    Mutations of TP53(17p13.1; OMIM 191170; PubMed accession numberX54156) are common in cancers and are typically missense withinexons 4–9, impairing the capacity of p53 to transactivategenes involved in cell cycle arrest, apoptosis, and DNA repair(1). Functionally, mutations may differ according to their natureand position, as well as to the presence of a common polymorphismat codon 72 (arginine or a proline) in the mutant allele (2).KnowingTP53mutation status has potential applications forcancer prognosis (3)(4) and early diagnosis (5), identificationof mutagen "fingerprints" (1)(6), and prediction of therapeuticoutcomes (7)(8). To achieve this purpose, sensitive, fast, andcost-effective methods are needed to assess the whole codingsequence plus exon/intron boundaries. Current approaches arebased on mutation prescreening with single strand conformationalpolymorphism analysis, temporal temperature gradient electrophoresis,or denaturing HPLC (DHPLC) combined with direct sequencing ofrelevant PCR fragments [reviewed in Ref. (9)]. These methodsare labor-intensive, difficult to standardize, and in some cases,of limited sensitivity. In recent years, 2 microarray methodsfor resequencing TP53have been described: the Affymetrix p53GeneChip array, described elsewhere (10)(11), and the ArrayedPrimer Extension (APEX), based on incorporation of 4 dye terminatorsinto oligonucleotide primers that each identify a base in thetarget sequence (12). In 2002, we described an APEX array forresequencingTP53exons 2–9, which contain 95% of knownmutations in TP53(13). Here we compare the sensitivity anddetection limits of APEX with a standard method, DHPLC/directsequencing, and discuss the potential of APEX for applicationto cancer diagnostic or prognostic purposes.

15. Reliable Detection of beeta-Thalasseemia and G6PDMutations by a DNA Microarray
    
    Gemignani, F., Perra, C., Landi, St., Canzian, F., Kurg, A., Tõnisson, N., Galanello, R., Cao, A., Metspalu, A. and Romeo, G.
    Clinical Chemistry 48, No.11, 2002 2051-2054