Biomedical Engineering Reference
had phenotypes that included retinitis pigmentosa, congenital stationary night
blindness, Best disease, early-onset cone dystrophy, and Stargardt disease.
Interestingly, three known and fi ve novel mutations were identifi ed in NR2E3 ,
PRPF3 , EYS , PRPF8 , CRB1 , TRPM1 , and CACNA1F . This unbiased NGS approach
allowed mutation detection in 75 % of control cases and in 57 % of test cases.
Authors found this approach to be time-effi cient as well as novel because it allowed
the possibility of expanding the phenotypes of known gene mutations.
Daiger et al. discuss the development of a VisionCHIP for autosomal dominant
retinitis pigmentosa, which contains 593 genes, to address the issues of genetic
heterogeneity. The selected genes were from the RetNet database of retinal disease
genes as well as EyeSAGE database (Daiger et al. 2010 ). In this approach, a
microarray with oligonucleotides was chosen to capture sheared human DNA, and
eluted DNA was sequenced by NGS technology. To optimize and validate the
VisionCHIP, the focus was on the usage of controls with known adRP mutations,
including deletions, and on 21 families from the adRP cohort without known
mutations. This approach may identify new RP genes and will substantially reduce
the cost per patient.
Primary immunodefi ciencies are fast becoming highlights for policy makers as
SCID is being added to newborn screening in various states in the United States.
Missing or deregulated pathways in the immunological response cascade character-
ize these disorders (Ghosh et al. 2012 ). The clinical presentations tend to be very
similar and, for this reason, diagnosis is challenging. Some traditional techniques
revolve around cytological testing, but cases can be missed. Ghosh et al. designed a
custom microarray to capture exons of 395 human genes, known or predicted to be
associated with primary immunodefi ciency and immune regulation, and sequenced
on a GS FLX Titanium 454 platform (Ghosh et al. 2012 ). Sequencing yielded
152,000-397,000 high-quality reads per patient with coverage of 76-82 % at 5X.
Importantly, this approach found the genetic mutations in two patients with sus-
pected primary immunodefi ciency.
Similar to primary immunodefi ciency, platelet function disorders (PFDs) are
genotypically diverse but present with excessive mucocutaneous bleeding.
Functionally assays may point towards a bleeding diathesis; however, they cannot
pinpoint towards an accurate genetic cause. Jones et al. describe a strategy for
genetic diagnosis of PFDs with Agilent SS in-solution enrichment and Illumina
sequencing of 216 candidate genes (Jones et al. 2012 ). A candidate list of genes
includes HPS1 , HPS1 , VPS33B , NAPA , LYST , HPS4 , VPS18 , VPS16 , SCL3n , and
HPS4 (Jones et al. 2012 ). In ten subjects, approximately 4,500 potential variants, in
the 216 candidate genes, were fi ltered to a shortlist of ten potentially pathogenic
variants. This proof-of-principle study illustrates that NGS enables rapid genetic
diagnosis of a PFD in a single test.