Using next generation sequencing to find causative genes in patients with epidermolysis bullosa

Epidermolysis bullosa (EB) is a heterogeneous group of inherited skin diseases
characterized by trauma induced blistering and erosions of the skin and mucous
membranes presenting directly after birth. There are over 25 different subtypes
of highly varied clinical severity caused by mutations in at least 18 different
genes.
Inherited mutations in 1 of 18 genes cause absence or reduction in proteins
stabilizing the dermal-epidermal junction. Depending on the protein affected by
the genetic mutation, skin cleavage occurs at different levels ranging from the
epidermis (EB Simplex) to underneath the basement membrane (Dystrophic EB).


Aside from the clinical assessment, diagnostic evaluation of a child born with
EB consists of molecular analysis of skin biopsies used for immune-antigen
mapping and electron microscopy followed by genetic analysis. With these
methods one can assess where the level of skin cleavage occurs, which proteins
are affected and eventually which subtype of EB a patient is most likely to be
afflicted with.Within the Netherlands, diagnostic DNA-testing of EB is offered
for almost all of the known genes. For example, in 17% of the cases with
clinically suspected EBS, no mutation is found. In most cases, genes are tested
one by one, which leads to long evaluation times and high costs.

Establishing a specific (genetic) diagnosis in a child with epidermolysis
bullosa is relevant because it provides valuable information regarding
aetiology, prognosis, potential associated disorders and recurrence risk. The
identification of a specific (genetic) diagnosis is also important for family
planning and reproductive choices, and may allow prenatal diagnostic testing or
pre-implantation genetic diagnosis (PGD).

The occurrence of EB in the Netherlands is approximately 20 newborns per year.
There is a need for a rapid and broad approach to quicken and simplify the
diagnostic process, increase the diagnostic yield and to identify the other,
yet unknown, genes involved. For these reasons we want to explore the use of
exome sequencing in the diagnostic evaluation of patients with epidermolysis
bullosa.

Exome sequencing allows the analysis of all exons of all known genes of one
individual in a single test. This technique thus allows the parallel analysis
of all genes known to be related to epidermolysis bullosa. This can be achieved
by either capturing all known genes related to epidermolysis bullosa and
subsequent sequencing of only these genes (= targeted exome sequencing).
Alternatively, all genes can be captured and sequenced and a filter for
epidermolysis bullosa genes can be applied during the data-analysis process (=
whole exome sequencing with targeted analysis). The latter approach also allows
the identification of new genes involved in epidermolysis bullosa, because the
sequence data of all other genes remains available. Moreover, the data-analysis
process can easily be updated when new information on a gene causing EB becomes
available.
Currently, the departments of Genetics & Dermatology are working together to
set up a Skin Panel using next generation sequencing. This panel will include
all of the EB genes known in order to screen each new patient for mutations in
all known genes. Whole exome sequencing will further add to this Skin Panel if
new disease causing genes are discovered.

Exome sequencing has a significantly higher rate of false negative or false
positive findings when compared to classic Sanger sequencing. The coverage
varies over exons, and some exons may not be covered at all, especially if
whole exome sequencing is applied. The greatest advantage, however, is that
exome sequencing allows the analysis of all EB genes in parallel and in a
single test, therefore the expected diagnostic yield is much higher than with
the currently used strategy of sequential and targeted genetic analysis. By
using this method as a standard genetic diagnostic tool for EB, we expect a
shorter evaluation time and a reduction in costs. A comparable study, using
non-targeted exome sequencing and performed in our centre has shown that exome
sequencing can identify pathogenic mutations in already known and novel genes
in patients with microcephaly, a condition known to be extremely heterogeneous.
In a substantial number of patients, most of whom had already been waiting for
a diagnosis for years, a genetic diagnosis could be achieved.

Recently, McGrath et al used genome wide whole exome sequencing in a family
clinically diagnosed with EBS. They identified a homozygous frameshift mutation
in 3 individuals with an atypical clinical phenotype bringing to light a new
role for the Slab2b effector protein in skin fragility. They were able to
confirm their exome data findings by using the immune-antigen mapping and
electron microscopy. McGrath et al. not only identified a new gene, i.e.
exophilin 5, involved in EB thereby furthering diagnostics, they also were able
to counsel this large family properly on risk of recurrence. This study
confirms that exome sequencing is a valuable tool in finding new EB genes.

A great concern to both the dermatology clinicians as well as the clinical
geneticists is that there exists a chance of finding non EB relevant mutations
during whole exome sequencing.
The primary focus of this study is to find new disease causing genes and to
correlate their phenotypic significance (see primary and secondary endpoints).
The chances of finding variants in genes not related to EB exists but is very
small. Many techniques and strategies are carried out in order to decrease the
chance of this happening:

• Linkage analysis either prior to or as first step after whole exome
sequencing in families with multiple affected individuals may direct towards
one or more genetic regions of interest. The variants in the genes in these
region(s) found by exome sequencing will be prioritized for further analysis,
i.e. assessment of relevance to the disease based on their function and
localization of protein expression and then confirmation by Sanger sequencing.

• Trio-analysis of the exome sequencing data will be applied in families with
only one affected individual. In trio analysis the variants found in the
proband are filtered against the variants in the healthy parents. Effectively,
the variants inherited from the parents which are present in the affected child
will be filtered out, resulting in a selection of de novo variants in the
child. The remaining variants, usually 1 to 6 variants will remain by using
this strategy, will be further selected based in the respective gene function.
Genes related to the skin or genes leading to protein expression in the skin
and mucous membranes will be further studied.

Both linkage analysis and Trio analysis directs our focus to the most probable
disease causing locus within each investigated EB family and decreases the
chance of finding mutations in non-EB related disease causing genes.

To explore the use of whole exome sequencing in the diagnostic evaluation of
patients with epidermolysis bullosa

Observation research with invasive measuring.

The burden and risks associated with participation are negligible. Our research
is an extension of the regular diagnostic care patients have been receiving. A
blood sample is taken only when insufficient DNA is available.
If a causative mutation is found, the patient and family are counselled by the
dermatologist and an experienced clinical geneticist. In the unlikely, but as
yet not excluded chance that an unsolicited finding occurs, the family will be
counselled by a clinical geneticist with relevant experience and after
inter-collegial consultation, which is part of routine procedures within the
department of Genetics.

The identification of the genetic mutation within an individual benefits the
patient and family in many ways. If an affected individual can be informed
about his or her disease causing mutation this is helpful in many respects:
-it may improve coping with this devastating disease
-it results in better information on the prognosis.
-it may however reduce the number of other diagnostic evaluations in the future
-It may benefit the family by improving genetic counselling, aid in decision
making related to reproduction, enable carrier testing in family members and
offer possibilities for prenatal diagnosis.