Molecular confirmation of a clinical diagnosis of an inherited disease or of congenital malformations is of paramount importance for patients and their families. It is the conclusion of the... Show moreMolecular confirmation of a clinical diagnosis of an inherited disease or of congenital malformations is of paramount importance for patients and their families. It is the conclusion of the differential diagnostic process, and provides information on the prognosis, in some cases on the therapeutic options, and on the recurrence risk. Currently, targeted sequencing of gene (s) of interest is the preferred approach for searching for small pathogenic mutations. The work presented in this thesis describes the application of new techniques for detecting small variations (mutations) in genomic DNA that underlie various disorders. These techniques include High Resolution Melting Curve Analysis (HR-MCA) followed by Sanger sequencing, targeted, X-exome and whole exome capture followed by Next Generation Sequencing (NGS). We have optimized, tested and applied the different new molecular techniques mentioned above to 1) faci litate the detection of disease causing mutations in several disorders with suspected Mendelian inheritance, 2) to speed up the identification of disease genes, 3) to provide a systematic tool for classifying previously intractable genetic diseases Show less
Aarskog-Scott syndrome (ASS) is a rare disorder with characteristic facial, skeletal, and genital abnormalities. Mutations in the FGD1 gene (Xp11.21) are responsible for ASS. However, mutation... Show moreAarskog-Scott syndrome (ASS) is a rare disorder with characteristic facial, skeletal, and genital abnormalities. Mutations in the FGD1 gene (Xp11.21) are responsible for ASS. However, mutation detection rates are low. Here, we report a family with ASS where conventional Sanger sequencing failed to detect a pathogenic change in FGD1. To identify the causative gene, we performed whole-exome sequencing in two patients. An initial analysis did not reveal a likely candidate gene. After relaxing our filtering criteria, accepting larger intronic segments, we unexpectedly identified a branch point (BP) variant in FGD1. Analysis of patient-derived RNA showed complete skipping of exon 13, leading to premature translation termination. The BP variant detected is one of very few reported so far proven to affect splicing. Our results show that besides digging deeper to reveal nonobvious variants, isolation and analysis of RNA provides a valuable but under-appreciated tool to resolve cases with unknown genetic defects. Show less
Santen, G.W.E.; Aten, E.; Sun, Y.; Almomani, R.; Gilissen, C.; Nielsen, M.; ... ; Kriek, M. 2012
Although sequencing of a human genome gradually becomes an option, zooming in on the region of interest remains attractive and cost saving. We performed array-based sequence capture using 385K... Show moreAlthough sequencing of a human genome gradually becomes an option, zooming in on the region of interest remains attractive and cost saving. We performed array-based sequence capture using 385K Roche NimbleGen, Inc. arrays to zoom in on the protein-coding and immediate intron-flanking sequences of 112 genes, potentially involved in mental retardation and congenital malformation. Captured material was sequenced using Illumina technology. A data analysis pipeline was built that detects sequence variants, positions them in relation to the gene, checks for presence in databases (eg, db single-nucleotide polymorphism (SNP)) and predicts the potential consequences at the level of RNA splicing and protein translation. In the samples analyzed, all known variants were reliably detected, including pathogenic variants from control cases and SNPs derived from array experiments. Although overall coverage varied considerably, it was reproducible per region and facilitated the detection of large deletions and duplications (copy number variations), including a partial deletion in the B3GALTL gene from a patient sample. For ultimate diagnostic application, overall results need to be improved. Future arrays should contain probes from both DNA strands, and to obtain a more even coverage, one could add fewer probes from densely and more probes from sparsely covered regions. European Journal of Human Genetics (2011) 19, 50-55; doi:10.1038/ejhg.2010.145; published online 24 November 2010 Show less
Sun, Y.; Almomani, R.; Aten, E.; Celli, J.; Heijden, J. van der; Venselaar, H.; ... ; Breuning, M.H. 2010
Terminal osseous dysplasia (TOO) is an X-linked dominant male-lethal disease characterized by skeletal dysplasia of the limbs, pigmentary defects of the skin, and recurrent digital fibroma with... Show moreTerminal osseous dysplasia (TOO) is an X-linked dominant male-lethal disease characterized by skeletal dysplasia of the limbs, pigmentary defects of the skin, and recurrent digital fibroma with onset in female infancy. After performing X-exome capture and sequencing, we identified a mutation at the last nucleotide of exon 31 of the FLNA gene as the most likely cause of the disease. The variant c.5217G>A was found in six unrelated cases (three families and three sporadic cases) and was not found in 400 control X chromosomes, pilot data from the 1000 Genomes Project, or the FLNA gene variant database. In the families, the variant segregated with the disease, and it was transmitted four times from a mildly affected mother to a more seriously affected daughter. We show that, because of nonrandom X chromosome inactivation, the mutant allele was not expressed in patient fibroblasts. RNA expression of the mutant allele was detected only in cultured fibroma cells obtained from 15-year-old surgically removed material. The variant activates a cryptic splice site, removing the last 48 nucleotides from exon 31. At the protein level, this results in a loss of 16 amino acids (p.Val1724_Thr1739del), predicted to remove a sequence at the surface of filamin repeat 15. Our data show that TOD is caused by this single recurrent mutation in the FLNA gene. Show less
