Oral PresentationMolecular diagnosis for paediatric genetic diseases is
important for targeted or tailored treatment, more
informative genetic counselling and guiding the families
for prenatal or pre-implantation diagnosis. Traditionally,
linkage analysis using large multiplex families or multiple
families with the same molecular cause is essential and the
process could take years before a diagnosis can be reached.
Candidate gene screening is usually the only method
available for clinical laboratories for genetic diseases in
Hong Kong.
Next generation sequencing technology has virtually
revolutionised the way genetic studies are conducted and
provides opportunities for molecular diagnosis for genetic
disorders that were never available before. With the
possibility of sequencing the whole genome or almost all
the coding exons of the genome, the method does not require
the availability of large, multiple affected families and prior knowledge of candidate causal genes. It can be applied to a
single patient or, as a usual practice, whole genome or whole
exome sequencing for the patient plus parents. For whole
exome sequencing (WES), it usually produces up to 100
million short sequencing reads of usually 100bp long. These
short reads were firstly compared with sequences of a
reference human genome and mapped to genomic regions
from which they were generated. Each position (base pair)
of a coding exon is usually covered with dozens to hundreds
of sequencing reads. Analysing the sequences of these reads
allows for identification of mutations that are different from
the reference sequences. For WES for a single individual, up to 100,000 variants
can be identified, with some of which are common variants
in a population and some of which rare or private. The
population frequencies of these variants are looked up in
public databases such as those from the 1000 Genome
Project or ESP6500, a project that sequenced 6500
individuals in the US. An internal database is also
established with WES data from 200 samples from the local
population. For rare, severe genetic disorders that are likely
to be caused by mutations from a single gene, we can safely
rule out the common (>1% in a population) variants and
only focus on the rare or private variants. The nature of the
mutations, such as with or without amino acid changes,
changes in the open reading frame of the protein, the nature
of the amino acid changes (similarity of the amino acid
changed to), the conservation of this amino acid in different
species, and the function of the gene in relationship to the
disease phenotype, are considered to help pinpoint the
causal mutations. We will present examples on using WES for molecular
diagnosis for paediatric genetic disorders in our
Department. These include detection of de novo mutations
(mutations that are not detected in parents), somatic
mutations and compound heterozygous mutations, and
mutations missed by traditional laboratory testing, which
demonstrated the power of this new technology in providing
accurate molecular diagnosis.published_or_final_versio