Braida C, Stefanatos RK, Adam B, Mahajan N, Smeets HJ, Niel F, Goizet C, Arveiler B, Koenig M, Lagier-Tourenne C, Mandel JL, Faber CG, de Die-Smulders CE, Spaans F, Monckton DG. Variant CCG and GGC repeats within the CTG expansion dramatically modify mutational dynamics and likely contribute toward unusual symptoms in some myotonic dystrophy type 1 patients. Hum Mol Genet. 2010 Apr 15;19(8):1399-412. IF 7.249

Gerards M, Sluiter W, van den Bosch BJ, de Wit E, Calis CM, Frentzen M, Akbari H, Schoonderwoerd K, Scholte HR, Jongbloed RJ, Hendrickx AT, de Coo IF, Smeets HJ. Defective complex I assembly due to C20orf7 mutations as a new cause of Leigh syndrome. J Med Genet. 2010 Aug;47(8):507-12. IF 5.713.

Gerards M, van den Bosch B, Calis C, Schoonderwoerd K, van Engelen K, Tijssen M, de Coo R, van der Kooi A, Smeets H. Nonsense mutations in CABC1/ADCK3 cause progressive cerebellar ataxia and atrophy. Mitochondrion. 2010 Aug;10(5):510-5. IF: 4.145

Harssel JJ van, van Roozendaal CE, Detisch Y, Brandăo RD, Paulussen AD, Zeegers M, Blok MJ, Gomez Garcia EB. Efficiency of BRCAPRO and Myriad II mutation probability thresholds versus cancer history criteria alone for BRCA1/2 mutation detection. Fam Cancer. 2010 Jun;9(2):193-201. IF 2.052

Korsten A, de Coo IF, Spruijt L, de Wit LE, Smeets HJ, Sluiter W. Patients with Leber hereditary optic neuropathy fail to compensate impaired oxidative phosphorylation. Biochim Biophys Acta. 2010 Feb;1797(2):197-203. IF 4.579.

Lipoldová M, Havelková H, Badalová J, Vojtíšková J, Quan L, Krulová M, Sohrabi Y, Stassen AP, Demant P.Loci controlling lymphocyte production of interferon c after alloantigen stimulation in vitro and their co-localization with genes controlling lymphocyte infiltration of tumors and tumor susceptibility. Cancer Immunol Immunother 2010  59,203-213. IF 3.804. 
GROW (oncologie)

Martherus RS, Vanherle SJ, Timmer ED, Zeijlemaker VA, Broers JL, Smeets HJ, Geraedts JP, Ayoubi TA. Electrical signals affect the cardiomyocyte transcriptome independently of contraction. Physiol Genomics. 2010 Nov 29;42A(4):283-9. IF:

Martherus RS, Sluiter W, D J Timmer E, Vanherle SJ, Smeets HJ, Ayoubi TA. Functional annotation of heart enriched mitochondrial genes GBAS and CHCHD10 through guilt by association. Biochem Biophys Res Commun. 2010 Nov 12;402(2):203-8. IF: 2.548

Martherus RS, Zeijlemaker VA, Ayoubi TA. Electrical stimulation of primary neonatal rat ventricular cardiomyocytes using pacemakers. Biotechniques. 2010 Jan;48(1):65-7.  IF 2.548

Neeve VCM, Van den Bosch B, Van Goethem G, Bindoff L, Smeets B, Lombes A, Hirano M, DiMauro S, De Vries M, Smeitink J, Czermin B, Holinski-Feder E, Hudson G, Turnbull DM, Taylor RW, Chinnery PF, Horvath R What modifies the clinical presentation of the common homozygous p.A467T POLG mutation?Conference Proceedings (inc. Abstract) United Kingdom Neuromuscular Translational Research Conference. Oxford 2010.

Paulussen AD, Schrander-Stumpel CT, Tserpelis DC, Spee MK, Stegmann AP, Mancini GM, Brooks AS, Collée M, Maat-Kievit A, Simon ME, van Bever Y, Stolte-Dijkstra I, Kerstjens-Frederikse WS, Herkert JC, van Essen AJ, Lichtenbelt KD, van Haeringen A, Kwee ML, Lachmeijer AM, Tan-Sindhunata GM, van Maarle MC, Arens YH, Smeets EE, de Die-Smulders CE, Engelen JJ, Smeets HJ, Herbergs J.  The unfolding clinical spectrum of holoprosencephaly due to mutations in SHH, ZIC2, SIX3 and TGIF genes. Eur J Hum Genet. 2010 Sep;18(9):999-1005.  IF 3.925
GROW (ontwikkelingsbiologie)

Sinnema M, van Roozendaal KE, Maaskant MA, Smeets HJ, Engelen JJ, Jonker-Houben N, Schrander-Stumpel CT, Curfs LM. Different distribution of the genetic subtypes of the Prader-Willi syndrome in the elderly. Eur J Hum Genet. 2010 Sep;18(9):993-8. IF 3.564

Solomon BD, Lacbawan F, Mercier S, Clegg NJ, Delgado MR, Rosenbaum K, Dubourg C, David V, Olney AH, Wehner LE, Hehr U, Bale S, Paulussen A, Smeets HJ, Hardisty E, Tylki-Szymanska A, Pronicka E, Clemens M, McPherson E, Hennekam RC, Hahn J, Stashinko E, Levey E, Wieczorek D, Roeder E, Schell-Apacik CC, Booth CW, Thomas RL, Kenwrick S, Keaton A, Balog JZ, Hadley D, Zhou N, Long R, Velez JI, Pineda-Alvarez DE, Odent S, Roessler E, Muenke M. Mutations in ZIC2 in Human Holoprosencephaly: Description of a Novel ZIC2-Specific Phenotype and Comprehensive Analysis of 157 Individuals. J Med Genet. 2010 Aug;47(8):513-24. IF 1.857

Tienen FH van, Lindsey PJ, van der Kallen CJ, Smeets HJ. Prolonged Nrf1 overexpression triggers adipocyte inflammation and insulin resistance. J Cell Biochem. 2010 Dec 15;111(6):1575-85. IF: 2.935
GROW(ontwikkelingsbiologie), CARIM, NUTRIM

Visser NA, Braun KP, van den Bergh WM, Leijten FS, Willems CR, Ramos L, van den Bosch BJ, Smeets HJ, Wokke JH. Juvenile-onset Alpers syndrome: interpreting MRI findings. Neurology. 2010 Apr 13;74(15):1231-3. IF 7.043

Zoer B, Been JV, Jongen E, Debeer A, Hendrickx A, Smeets HJ, Zimmermann LJ, Villamor E. Mitochondrial DNA damage analysis in bronchoalveolar lavage cells of preterm infants. Front Biosci (Elite Ed). 2010 Jan 1;2:361-8.  IF 3.308

Zwaag PA van der, Cox MG, van der Werf C, Wiesfeld AC, Jongbloed JD, Dooijes D, Bikker H, Jongbloed R, Suurmeijer AJ, van den Berg MP, Hofstra RM, Hauer RN, Wilde AA, van Tintelen JP. Recurrent and founder mutations in the Netherlands : Plakophilin-2 p.Arg79X mutation causing arrhythmogenic right ventricular cardiomyopathy/dysplasia. Neth Heart J. 2010 Dec;18(12):583-91. IF: 1.392





CHIP-based sequence analysis of 34 cardiomyopathy genes reveals new genes involved in HCM and DCM and multiple pathogenic mutations in single patients
I. Krapels1, W. van Dijk1, A. Stassen1, P. Lindsey1, Y. Arens1, A. Helderman1,  C. Marcelis2, J. van der Smagt3, S. Heymans1, P. Volders1, R. Jongbloed1, H. Smeets1, A. van den Wijngaard1; 1Maastricht University Medical Center, Maastricht, Netherlands, 2University Medical Center St Radboud Nijmegen, Netherlands, 3Utrecht Medical Center
Utrecht, Utrecht, Netherlands.
Inherited cardiomyopathy is a common cardiac disease with a prevalence of 1:500 for hypertrophic cardiomyopathy (HCM) and 1: 2500 for dilated cardiomyopathy (DCM). The large genetic and clinical heterogeneity and the laborious screening methods, hamper rapid detection of the genetic cause in all cases. Current diagnostic screening solves only 60-70% of the families by testing a limited number of genes and usually stops when a pathogenic mutation is found. However, double pathogenic mutations seem to be present in 5-10% of the familial cases. In order to create a fast, parallel genetic screening pipeline for inherited cardiomyopathy, we designed a resequencing array (Cardio- CHIP) covering 34 genes in duplicate (300Kb). Genes known to be involved in DCM, HCM, non compaction cardiomyopathy, Limb Girdle Muscular Dystrophy and candidate genes based on their presence in the sarcomere and Z-disc were included, as also exons and flanking introns (38bp) covering the heart- and muscle-specific RNA-isoforms were included. The 5’UTR and 3’UTR regions and, for a selection of genes, the promoter regions were included as well. So far, 250 patients were sequenced for all 34 cardiomyopathy genes. The mutation detection rate is around 99% and about 98% of the novel exonic variants can be confirmed by conventional sequence analysis. In addition to mutations detected in the 13 genes routinely tested for HCM or DCM, we identified mutations in the 21 additional genes, some of which were the first for those new candidate genes. We present several families in which up to 4 pathogenic mutations were identified.  

RT-PCR Analysis of putative BRCA1/2 Splice Variants
R. D. Brandao1,2, K. van Roozendaal1, D. Tserpelis1, E. Gómez García1, M. J.Blok1;1Maastricht University Hospital, Maastricht, Netherlands, 2GROW - Schoolfor Oncology and DevelopmentalBiology, University Maastricht, Maastricht,Netherlands.Introduction: A subset of the unclassified variants (UVs) found in theBRCA genes may affect splicing. Twelve putative BRCA1/2 splice variantsprior selected upon in silico analysis were studied experimentallyusing RT-PCR.Methods: The variants selected were: in BRCA1 c.693G>A and inBRCA2 c.68-7T>A, c.425G>T, c.794-11T>C, c.6935A>T, c.6842-3T>C,c.6943A>G, c.7976+3del2, c.8350C>T, c.8953+13A>G, c.8662C>Tand c.8754+3G>C. We performed short-term lymphocyte cultures, in the absence or presence of puromycin, a nonsense mediated mRNAdecay inhibitor. PCRs using primers flanking the region of interest were performed to look for aberrant cDNA. This was followed by allelespecific PCRs in order to analyze the contribution of each allele to the relative expression of the wild-type and aberrant transcripts found. Results: The variants BRCA2 c.425G>T, c.7976+3del2, and c.8754+3G>C result in aberrant splicing, i.e. exon 4, exon 17 skipping and retention of 46bp of intron 22, respectively. The BRCA1 variant c.693G>A results in exon 11 skipping, which is a normal isoform in breast- and ovarian tissue but not in lymphocytes. BRCA2 variants c.68-7T>A and c.6935A>T induce higher expression of the BRCA2Δ3 and BRCA2Δ12 isoforms, which are also expressed in controls, whereas the level of expression of the full-length transcript is the same as from the WT allele. For the other variants no splice aberrations were detected. Conclusion: The BRCA2 variants c.425G>T, c.7976+3del2, and c.8754+3G>C could be classified as pathogenic. The clinical relevance of variants that induce higher expression of normal isoforms is difficult to evaluate as their function is currently unknown.  



 Riboflavin responsive OXPHOS complex I deficiency caused by defective ACAD9: new function for an old gene.
M. Gerards, B. J. C. van den Bosch, K. Danhauser, V. Serre, M. van Weeghel, R. J. A. Wanders, G. A. F. Nicolaes, W. Sluiter, K. Schoonderwoerd, H. R. Scholte, H. Prokisch, A. Rötig, I. F. M. de Coo, H. J. M. Smeets.
  Mitochondrial complex I deficiency is the most common OXPHOS defect. Mutations have been detected in mitochondrial and nuclear genes, but many patients remain unresolved and new genes are likely involved. In a consanguineous family, patients presented since early childhood with easy fatigability, exercise intolerance and lactic acidosis in blood. In muscle, subsarcolemmal mitochondrial proliferation and a severe complex I deficiency were observed. Exercise intolerance and complex I activity improved by supplementation with a high dosage of riboflavin. Homozygosity mapping revealed a candidate region on chromosome three containing six mitochondria-related genes. Four genes were screened for mutations and a homozygous substitution was identified in ACAD9 (c.1594C>T), changing the highly conserved arginine-532 into tryptophan. This mutation was absent in 188 ethnically matched controls. Protein modelling suggested a functional effect due to loss of a stabilizing hydrogen bond in an α-helix and a local flexibility change. To test whether the ACAD9 mutation caused the complex I deficiency, we transduced fibroblasts of patients with wild type and mutant ACAD9. Wild type ACAD9, but not mutant ACAD9, restored complex I activity. An unrelated patient with the same phenotype carried a homozygous ACAD9 c.1405C>T mutation, changing arginine-469 into tryptophan, which was not present in controls. Our data support a new function for ACAD9 in complex I function, making this gene an important new candidate for patients with complex I deficiency, which could be improved by riboflavin treatment.

CHIP-based sequence analysis of 34 cardiomyopathy genes reveals new genes involved in HCM and DCM and multiple pathogenic mutations in single patients.
A. van den Wijngaard, I. Krapels, W. Van Dijk, A. Stassen, P. Lindsey, Y. Arens, P. Helderman, C. Marcelis, S. Heymans, P. Volders, R. Jongbloed, H. Smeets
 Purpose: Inherited cardiomyopathy is a cardiac disease with an estimated prevalence of 1:500 for hypertrophic cardiomyopathy (HCM) and 1: 2500 for dilated cardiomyopathy (DCM). Both cardiomyopathies show large genetic and clinical heterogeneity and require high-throughput and affordable mutation detection technologies to efficiently integrate molecular screening into clinical practice. Current diagnostic screening of a limited number of genes solves about 60-70% of the familial cases. However, double pathogenic mutations seem to be present in 5-10% of the familial cases. Methods: To create a fast genetic screening method for inherited cardiomyopathy, we designed a DNA resequencing array (CardioCHIP 150K34) covering 34 genes in duplicate. We included genes involved in DCM, HCM, non compaction cardiomyopathy, Limb Girdle Muscular Dystrophy and candidate genes based on their presence or protein-protein interaction in the sarcomere. All exons, flanking introns (38bp), the 5’UTR and 3’UTR regions, covering heart- and muscle-specific RNA-isoforms are included. Genomic regions were amplified in 152 LR-PCR’s covering 395 exons. Results: Recently 250 patients were resequenced for all 34 genes. The mutation detection accuracy is >99%. About 98% of the novel exonic variants were confirmed by conventional sequence analysis. In addition to the mutations detected in the 13 routinely tested genes, we also identified mutations in the additional genes and candidate genes. In several patients up to 4 pathogenic mutations were identified, involved in different pathogenic processes. Conclusion: Our data indicate that parallel analysis of multiple genes is a prerequisite for genetic testing in HCM and DCM to rapidly identify the underlying genetic defect(s).