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Background Genome-wide gene expression profiling analysis of FFPE tissue samples is indispensable for cancer research and provides the opportunity to evaluate links between molecular and clinical information, however, working with FFPE samples is challenging due to extensive cross-linking, fragmentation and limited quantities of nucleic acid. Thus, processing of FFPE tissue samples from RNA extraction to microarray analysis still needs optimization. Materials and methods In this study, a modified deparaffinization protocol was conducted prior to RNA isolation. Trizol, Qiagen RNeasy FFPE and Arcturus PicoPure RNA Isolation kits were used in parallel to compare their impact on RNA isolation. We also evaluated the effect of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) on the percentage of present calls. Results Our optimization study shows that the Qiagen RNeasy FFPE kit with modified deparaffinization step gives better results (RNA quantity and quality) than the other two isolation kits. The Ribo-SPIA protocol gave a significantly higher percentage of present calls than the 3′ IVT cDNA amplification and labeling system. However, no significant differences were found between the two array platforms. Conclusion Our study paves the way for future high-throughput transcriptional analysis by optimizing FFPE tissue sample processing from RNA isolation to microarray analysis.

YILMAZ, Engin/0000-0001-8873-7645; Akarsu, Nurten/0000-0001-5432-0032; Ozdag, Hilal/0000-0001-7940-2499; Liehr, Thomas/0000-0003-1672-3054; Alanay, Yasemin/0000-0003-0683-9731; von Eggeling, Ferdinand/0000-0002-8062-6999 WOS: 000278045300015 PubMed: 20451171 We present an autosomal-recessive frontonasal dysplasia (FND) characterized by bilateral extreme microphthalmia, bilateral oblique facial cleft, complete cleft palate, hypertelorism, wide nasal bridge with hypoplasia of the ala nasi, and low-set, posteriorly rotated ears in two distinct families. Using Affymetrix 250K SNP array genotyping and homozygosity mapping, we mapped this clinical entity to chromosome 12q21. In one of the families, three siblings were affected, and CNV analysis of the critical region showed a homozygous 3.7 Mb deletion containing the ALX1 (CART1) gene, which encodes the aristaless-like homeobox 1 transcription factor. In the second family we identified a homozygous donor-splice-site mutation (c.531+1G > A) in the ALX1 gene, providing evidence that complete loss of function of ALX1 protein causes severe disruption of early craniofacial development. Unlike loss of its murine ortholog, loss of human ALX1 does not result in neural-tube defects; however, it does severely affect the orchestrated fusion between frontonasal, nasomedial, nasolateral, and maxillary processes during early-stage embryogenesis. This study further expands the spectrum of the recently recognized autosomal-recessive ALX-related FND phenotype in humans. Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [108S420]; European Research Area Network (E-RARE) [R07197KS] We are grateful to the families for their participation in the study. We thank Han Brunner for critical reading and comments, Ebru Oralli Bircan for illustrations, Hacettepe University Craniofacial Surgery Study Group members Yucel Erk, Emin Mavili, Aycan Kayikcioglu (Plastic and Reconstructive Surgery), Kemal Benli (Neurosurgery), Aysenur Cila (Radiology), Tulin Taner, and liken Kocadereli (Orthodonty) for evaluating the frontonasal malformation cases in the registry. This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) (grant numbers 108S420 to N.A.A), and the overall consortium (CRANIRARE) was supported by the European Research Area Network (E-RARE) (project number R07197KS).

WOS: 000278045300016, PubMed: 20451170, More than 270 million people worldwide have hearing loss that affects normal communication. Although astonishing progress has been made in the identification of more than 50 genes for deafness during the past decade, the majority of deafness genes are yet to be identified. In this study, we mapped a previously unknown autosomal-recessive nonsyndromic sensorineural hearing loss locus (DENB91) to chromosome 6p25 in a consanguineous Turkish family. The degree of hearing loss was moderate to severe in affected individuals. We subsequently identified a nonsense mutation (p.E245X) in SERPINB6, which is located within the linkage interval for DENB91 and encodes for an intracellular protease inhibitor. The p.E245X mutation cosegregated in the family as a completely penetrant autosomal-recessive trait and was absent in 300 Turkish controls. The mRNA expression of SERPINB6 was reduced and production of protein was absent in the peripheral leukocytes of homozygotes, suggesting that the hearing loss is due to loss of function of SERPINB6. We also demonstrated that SERPINB6 was expressed primarily in the inner ear hair cells. We propose that SERPINB6 plays an important role in the inner ear in the protection against leakage of lysosomal content during stress and that loss of this protection results in cell death and sensorineural hearing loss., The Scientific AMP; Technological Research Council of Turkey [105S464, 108S045]; University of Miami; NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R01 DC006908]; National Natural Science Foundation of ChinaNational Natural Science Foundation of China [30728030]; Federick and Ines Yeatts [R01 DC005575]; Oticon Fonden, Denmark, We are thankful to all participating families. We thank Professor Phillip I. Bird from Monash University, Australia, for helpful suggestions and critical reading of the manuscript. This study was partially supported by The Scientific & Technological Research Council of Turkey Grants (105S464 and 108S045), funds from the University of Miami to M.T., NIH R01 DC006908 to Z.-Y.C., National Natural Science Foundation of China to Z.-Y.C. (30728030), the Federick and Ines Yeatts inner ear hair cell regeneration fellowship to M.H., R01 DC005575 to X.Z.L., and a grant from Oticon Fonden, Denmark, to M.B.P.

Rational drug discovery relies on pathognomonic molecular reporters of disease or biomarkers. Therefore biomarkers contain relational or contextual information about disease pathophysiology. Two broad pathways can be taken to identify biomarkers: a 'top-down', holistic approach that makes no assumptions about biomarker type, or the 'bottom-up' approach, which is hypothesis driven and relies on a priori information. Both approaches involve parallel or sequential methods that include genomic and proteomic profiling. Biomarker discovery and translational medicine owe much to isotopic techniques because these provide near-real-time information about disease status as diagnostics, in drug delivery and for monitoring treatment. Here, we provide an overview of recent developments and some insight into the future role of isotopes in biomarker discovery and disease therapy.

Genome wide homozygosity mapping using Affymetrix 10K arrays revealed the DFNB7/11 locus including the TMC1 gene in 5 of 35 Turkish families with autosomal recessive nonsyndromic severe to profound congenital or prelingual-onset sensorineural hearing loss (SNHL). Additional 51 families were later screened for co-segregation of the locus with the phenotype using microsatellite markers. GJB2 and mtDNA A1555G mutations were negative in probands from each family. Mutation analysis was performed in families showing co-segregation Of autosomal recessive SNHL with haplotypes at the DFNB7/11 locus. A total of six different mutations in seven families were identified, including novel missense alterations, p.G444R (c.1330G > A), p.R445C (c.1333C > T), and p.1677T (c.2030T > C), one novel splice site mutation IVS6+2 T > A (c.64+2T > A), and a novel large deletion ofapproximately 31 kb at the 3' region of the gene including exons 19-24, as well as a previously reported nonsense mutation, p.R34X (c.100C > T). All identified Mutations co-segregated with autosomal recessive SNHL in all families and were not found in Turkish hearing controls. These results expand the mutation spectrum of TMC1 with five novel mutations and provide data for the significant contribution of TMC1 mutations in hearing loss. (c) 2009 Elsevier Ireland Ltd. All rights reserved.

Germ line BRCA1 and/or BRCA2 mutations were screened in 50 Turkish breast and/or ovarian cancer patients composed of hereditary, familial, early onset and male cancer groups. Genomic DNA samples were tested by heteroduplex analysis and DNA sequencing. Two truncating BRCA2 mutations, one novel (6880 insG) and one previously reported (3034 delAAAC), were found in two out of six (33%) hereditary breast and/or ovarian cancer patients. A novel truncating (1200 insA) and a missense (2080A!G) BRCA1 mutation was found in two of 27 (7%) individuals in the early onset group. A total of four (8%) disease-causing mutations in 50 breast cancer patients were identified in BRCA1 and BRCA2 genes. In addition, five BRCA1 sequence variants have been identified in 23 patients. These results indicate that BRCA1 and BRCA2 genes are involved in some, but not all, forms of hereditary predisposition to breast cancer in the Turkish population. # 2000 Published by Elsevier Science Ltd.