Exome Sequencing as a Discovery and Diagnostic Tool - Next Generation Sequencing Technologies in Medical Genetics - page 72

unrelated affected individuals. For example, four unrelated cases were subjected to
WES and MYH3 was identifi ed as the single candidate gene for Freeman-Sheldon
syndrome with variants that had not been previously reported in dbSNP and HapMap
databases (Ng et al. 2009 ). A similar strategy was used by the same group to iden-
tify the candidate gene for Kabuki syndrome (Ng et al. 2010a ). Specifi cally, the
exomes of ten unrelated individuals affected with Kabuki syndrome were sequenced.
However, to account for genetic heterogeneity, a less stringent strategy was applied
by performing a search of the candidate genes shared among subsets of affected
individuals. Additionally, various ranking and stratifying steps were also taken to
account for phenotypic heterogeneity. These additional strategies fi nally led to the
identifi cation of causal variants in the MLL2 gene. The MLL2 gene of 43 of affected
individuals was screened by Sanger sequencing. Interestingly, 12 individuals had de
novo variants. Similarly, only two out of eight individuals with Sensenbrenner syn-
drome had causal variants in WDR35 (Gilissen et al. 2010 ). These causal variants
were only identifi ed in two unrelated cases with a strikingly similar phenotype. This
highlights the complexity of genetic and phenotypic heterogeneity and implies that
classifying phenotypic heterogeneity helps in identifying the causal variants. Further
studies have also identifi ed a number of novel candidate genes harboring causal
variants for disorders such as Miller syndrome, Fowler syndrome, Perrault syn-
drome, and Schinzel-Giedion syndrome (Ng et al. 2010b ; Hoischen et al. 2010 ;
Lalonde et al. 2010 ; Pierce et al. 2010 ). These studies show the feasibility of apply-
ing WES to identify the candidate genes for Mendelian disorders using unrelated
cases. Furthermore, other studies have applied a combination of WES strategies to
identify candidate genes, namely, WES with linkage or homozygosity analysis.

8.4.2 Sequencing of Family Members

Reported WES strategies have included performing analysis on affected individuals
within a family. Autosomal-dominant spinocerebellar ataxias, previously studied
disorders, have been examined by WES to identify new candidate genes (Ku et al.
2011 ). To date, causal variants in 20 genes have been identifi ed for this disorder
(Wang et al. 2010a ). Authors performed WES in four affected individuals in one
four-generation Chinese family with autosomal-dominant spinocerebellar ataxias.
One of the advantages of this strategy is that it allowed investigators to hypothesize
that all affected individuals should share the same causal variant, as spinocerebellar
ataxia was inherited in an autosomal-dominant pattern in this family, which was
supported by linkage analysis. Spinocerebellar ataxias are also characterized by
clinical and genetic heterogeneity which would benefi t from WES. The sequencing
of affected individuals in one family would offer further advantage to the study
design as unrelated cases from different families are likely to have causal variants in
different genes. Other studies have also performed WES in multiple siblings and
identifi ed causal variants and candidate genes for disorders such as autosomal-
dominant amyotrophic lateral sclerosis, familial combined hypolipidemia, and