Acta Dermato-Venereologica 98-4CompleteContent | Page 13
INVESTIGATIVE REPORT
437
The Position of Targeted Next-generation Sequencing in Epidermo
lysis Bullosa Diagnosis
Cristina HAS 1 , Julia KÜSEL 2# , Antonia REIMER 1,3# , Julia HOFFMANN 1 , Franziska SCHAUER 1 , Andreas ZIMMER 2 and Judith
FISCHER 2
Department of Dermatology, 2 Institute for Human Genetics, Medical Center, University of Freiburg, and 3 Berta-Ottenstein-Program, Faculty
of Medicine, University of Freiburg, Freiburg, Germany
#
These authors contributed equally to this paper.
1
The precise classification of epidermolysis bullosa (EB)
into 4 main types and more than 30 subtypes is based
on the level of skin cleavage, as well as clinical and
molecular features, and is crucial for early prognosti-
cation, case management, genetic counselling and
prenatal or pre-implantation diagnosis. We report here
the molecular pathology of 40 consecutive cases of su-
spected EB, which were investigated by immunofluo-
rescence mapping (IFM) and/or by a targeted next-
generation sequencing (NGS) multi-gene panel. IFM
correctly established the EB subtype in 76% of cases,
while the molecular pathology was completely eluci-
dated in 90% of cases by the targeted NGS multi-gene
panel. Thirteen previously unreported mutations in
EB genes were identified. In cases with unclear clini-
cal and IFM findings, mutations were found by NGS in
previously unexpected genes. IFM was useful in deli-
vering fast results in newborns, and in indicating the
consequences of the variants of uncertain significance
on protein level. This study underscores the efficacy of
the strategy of combining targeted NGS with IFM in re-
solving unusual EB phenotypes. It also suggests that,
despite technological advances, careful clinical eva-
luation and deep phenotyping remains a crucial factor
that dictates successful diagnosis of EB.
Key words: epidermolysis bullosa; adhesion; blistering; skin
fragility; mutation; keratin; collagen.
Accepted Dec 14, 2017; Epub ahead of print Dec 15, 2017
Acta Derm Venereol 2018; 98: 437–440.
Corr: Cristina Has, Department of Dermatology, Medical Center, Universi
ty of Freiburg, Faculty of Medicine, University of Freiburg, Hauptstrasse 7,
DE-79104 Freiburg, Germany. E-mail: [email protected]
E
pidermolysis bullosa (EB) is a clinically and geneti-
cally heterogeneous group of disorders characterized
by skin fragility and mechanically induced skin blistering.
The genetic basis of EB is well-established (1), yet new
genes and mutational mechanisms still emerge. Clas-
sification of EB into 4 main types (simplex, junctional,
dystrophic and Kindler syndrome) and more than 30 sub-
types is based on immunofluorescence mapping (IFM) and
mutation analysis (2). Precise classification is crucial for
early prognostication, case management, genetic counsel-
ling and prenatal or preimplantation diagnosis (2). Clas-
sical types of EB, such as severe generalized dystrophic
or junctional EB, can be recognized based on clinical
criteria as soon as the clinical picture is fully manifest (3).
In newborns, infants and in individuals with moderate or
mild skin fragility, clinical assessment usually does not
allow precise classification. Such cases require molecular
and genetic diagnosis.
We report here the molecular pathology of 40 conse-
cutive suspected cases of EB, which were investigated
by IFM and/or by a targeted next-generation sequencing
(NGS) multi-gene panel since July 2016, when NGS was
approved as a diagnostic method in Germany.
METHODS
IFM was performed with a panel of 17 antibodies to components
of the dermal–epidermal junction, as described previously (4). The
multi-gene panel included 49 genes: 19 genes in which mutations
were shown to be disease-causing in EB (2), and 30 additional ge-
nes functionally related to epidermal adhesion or potential genetic
modifiers (Table SI 1 ). NGS was performed using a custom Agilent
Haloplex panel (Agilent, Santa Clara, CA, USA) and sequences
were determined with an Illumina MiSeq (2 × 150 base pairs;
Illumina, San Diego, CA, USA). Paired-end sequencing reads
were aligned using Burrows-Wheeler Alignment tool (5) (version
0.7.15) against the human reference genome sequence GRCh37
(hg19). Variants were called using FreeBayes (6) (v1.1.0). AN-
NOVAR (ANNOtate VARiation) (7) was applied to annotate the
function of genetic variants and to cross-reference the variants with
databases. Only variants with a frequency of < 1% and homozy-
gous absence in ExAC (8) v0.3 were considered. Mutations were
confirmed by Sanger sequencing, and whenever possible, segrega-
tion was verified in the families (primer sequences available on
request). Direct and indirect immunofluorescence were performed
as described previously (9, 10). Fluorescein (FITC)-labelled anti-
bodies used for direct immunofluorescence were anti-human IgG,
IgA, IgM and C3c (Dako, Hamburg, Germany) at a dilution of
1:200, 1:50, 1:50 and 1:500. For IIF on salt-split skin, patient sera
were diluted 1:10, secondary antibodies used were FITC-labelled
anti-human IgG (Dako, Hamburg, Germany) at a dilution of 1:100.
Commercial enzyme-linked immunoassay (ELISA) kits for the
detection of NC16A domain of BP180- and bullous pemphigoid
230-kDa antigen (BP230)-specific antibodies (MBL) were used
as per manufacturer’s protocol, with cut-off at 9 U/ml.
RESULTS
Analysis of a skin biopsy by IFM was performed in 25
cases, and correctly established the EB subtype in 19 of
these (76%). In 36 cases (90%) the molecular pathology
https://www.medicaljournals.se/acta/content/abstract/10.2340/00015555-2863
1
This is an open access article under the CC BY-NC license. www.medicaljournals.se/acta
Journal Compilation © 2018 Acta Dermato-Venereologica.
doi: 10.2340/00015555-2863
Acta Derm Venereol 2018; 98: 437–440