Jos Jansen

1 Introduction to hepatic congenital glycosylation disorders 15 Diagnostic techniques in CDG Exome sequencing Filtering steps As described in the first paragraph, exome sequencing allows analysis of all nucleotides in the exome. A nucleotide that differs from the most prevalent nucleotide is called a variant. As contemporary exome sequencing strategies generate ~80.000 variants, the sheer amount of data generated by exome sequencing requires rigorous filtering to identify thepathogenic variant. Themost important filtering step for rare Mendelian disorders is based on the prevalence of a variant in the general population.(43) If a variant is more prevalent in the general population than the disorder itself, pathogenicity is unlikely. Databases with large cohorts of exomes (such as the Exome Aggregation Consortium (ExAC)) have been very important to facilitate this step.(44) After this first step, about hundred variants still remain. In silico tools such as Polyphen 2.0, SIFT and MutationTaster can be used to predict pathogenicity, however validation of these diagnostic tools demonstrated a limited sensitivity and specificity (60-80%). (45) For recessive disorders filtering based on familial segregation is a powerful adjunct. Homozygous or compound heterozygous variants must segregate within the family and preferably in multiple unrelated families.(9, 46) Limitations and challenges of exome sequencing The most obvious limitation of exome sequencing is that only 1% of the genome is sequenced. Leaving out the other 99% undoubtedly results in loss of information. Another limitation is incomplete coverage of the exome due to low call rates. This was particularly the case for earlier kits (call rate of ~85%). (47) However, technical advances increased this percentage to ~95%.(48) Applied whole genome sequencing was first described in 2008 and its utility and technique is rapidly evolving.(49, 50) Whole genome sequencing covers almost the whole genome. Its use has been proven in patients with intellectual disability where exome sequencing was inconclusive.(51) An interesting outcome of a carrier screening study showed that 122 out of 460 (27%) of initially reported pathogenic variants were redefined as common polymorphisms or sequence errors.(52) The high false-positive rate in this study, and of other studies questioning causality of reported variants, led the American