Targeted resequencing of 14 individuals with Joubert syndrome identified variants in 26 genes, 9 of which had variants in multiple individuals. This approach seeks to discover novel disease genes contributing to Joubert syndrome by combining targeted DNA capture and massively parallel sequencing. While several genes have been identified as causing Joubert syndrome, they only account for about 50% of cases, so the aim is to identify additional causal genes through this sequencing of protein-coding regions in affected individuals.

1. Targeted resequencing for gene discovery in
Joubert syndrome
Paige Taylor1, Hane Lee1,2, Stanley F. Nelson1,2
1Department of Human Genetics, University of California, Los Angeles, California, USA
2Department of Pathology and Laboratory Medicine, University of California, Los Angeles, California, USA
INTRODUCTION RESULTS
Joubert Syndrome & Related Disorders (JSRD) Results
• Joubert syndrome is a rare, autosomal recessive brain malformation
disorder characterized by a specific congenital malformation of the
hindbrain (known as the molar tooth sign) as well as a broad spectrum of
other phenotypic findings now known to be caused by defects in the
structure and/or function of the primary cilium.
• Although Joubert syndrome is inherited in an autosomal recessive fashion,
it shows marked genetic and phenotypic heterogeneity as several genes
have been identified in recent years.
• At least 10 genes have been
found to cause JSRD, however
these account for only ~50% of all
known cases.
• The genes for other forms of the
disease have not yet been found.
Objective
• To discover novel disease genes
contributing to or causing JSRD
by combining targeted DNA
capture and massively parallel
sequencing in 14 unrelated
affected individuals.
Molar Tooth Sign1
METHODS
We created a custom 1 MB cGH Array containing 180 genes localized to
or affecting the structure/function of primary cilia. Paired-end libraries
were prepared using genomic DNA from 14 affected individuals. Each
sample was tagged using custom 4-bp barcodes. The barcoded libraries
were then pooled, captured, and sequenced to high depth of coverage
using an Illumina HiSeq2000. The de-barcoded sequencing data was
aligned to build 37 of the human genome using Novoalign and variants
were called using the Genome Analysis Tool Kit. Variants present in
dbSNP132 & the 1000 Genomes Project were removed. Variants were
further filtered against publicly available sequencing datasets and in-house
datasets. Because Joubert syndrome is a recessive disorder, our analysis DISCUSSION
was restricted to variants causing changes in the final protein product.
After variant filtering, 36 heterozygous, protein-damaging mutations in 26
genes remained. We were particularly interested in genes that were
Sequencing and Analysis Workflow mutated in multiple individuals. Nine of the 26 genes were mutated in
more than one individual for a total of 19 variants (see table below). For
Indel#Realignment# the gene CCT4, the same mutation was found in two individuals. For the
Add#barcodes#to# Unified#Genotyper
&#Base#Quality#
DNA#samples (Call#SNVs#&#Indels)
RecalibraMon other eight genes with multiple sample hits, each variation was unique.
dbSNP132,#
1KG
Create#180R Merge#Samples#&#
Pool#14#samples Novel#Variants
gene#Array Remove#Duplicates
CodingR
synonymous
Capture#on#a#
Custom#1Mb# Novoalign#(b37) Candidate#Variants
Agilent#cGH#Array
Sequence#on#
HiSeq2000# Novobarcode
100+100#bp#PE
1Brancati et al. Joubert syndrome and related disorders. Orphanet J Rare Dis 2010;5:20