Genotyping microarray for CSNB-associated genes.
OBJECTIVE
Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous retinal disease. Although electroretinographic (ERG) measurements can discriminate clinical subgroups, the identification of the underlying genetic defects has been complicated for CSNB because of genetic heterogeneity, the uncertainty about the mode of inheritance, and time-consuming and costly mutation scanning and direct sequencing approaches.


METHODS
To overcome these challenges and to generate a time- and cost-efficient mutation screening tool, the authors developed a CSNB genotyping microarray with arrayed primer extension (APEX) technology. To cover as many mutations as possible, a comprehensive literature search was performed, and DNA samples from a cohort of patients with CSNB were first sequenced directly in known CSNB genes. Subsequently, oligonucleotides were designed representing 126 sequence variations in RHO, CABP4, CACNA1F, CACNA2D4, GNAT1, GRM6, NYX, PDE6B, and SAG and spotted on the chip.


RESULTS
Direct sequencing of genes known to be associated with CSNB in the study cohort revealed 21 mutations (12 novel and 9 previously reported). The resultant microarray containing oligonucleotides, which allow to detect 126 known and novel mutations, was 100% effective in determining the expected sequence changes in all known samples assessed. In addition, investigation of 34 patients with CSNB who were previously not genotyped revealed sequence variants in 18%, of which 15% are thought to be disease-causing mutations.


CONCLUSIONS
This relatively inexpensive first-pass genetic testing device for patients with a diagnosis of CSNB will improve molecular diagnostics and genetic counseling of patients and their families and gives the opportunity to analyze whether, for example, more progressive disorders such as cone or cone-rod dystrophies underlie the same gene defects.
Zeitz
Christina
C
Division of Medical Molecular Genetics and Gene Diagnostics, Institute of Medical Genetics, University of Zurich, Zurich, Switzerland. christina.zeitz@inserm.fr
Labs
Stephan
S
Lorenz
Birgit
B
Forster
Ursula
U
Uksti
Janne
J
Kroes
Hester Y
HY
De Baere
Elfride
E
Leroy
Bart P
BP
Cremers
Frans P M
FP
Wittmer
Mariana
M
van Genderen
Maria M
MM
Sahel
José-Alain
JA
Audo
Isabelle
I
Poloschek
Charlotte M
CM
Mohand-Saïd
Saddek
S
Fleischhauer
Johannes C
JC
Hüffmeier
Ulrike
U
Moskova-Doumanova
Veselina
V
Levin
Alex V
AV
Hamel
Christian P
CP
Leifert
Dorothee
D
Munier
Francis L
FL
Schorderet
Daniel F
DF
Zrenner
Eberhart
E
Friedburg
Christoph
C
Wissinger
Bernd
B
Kohl
Susanne
S
Berger
Wolfgang
W
D000293:Adolescent; D020746:Calcium Channels, L-Type / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000235:genetics; D002648:Child; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6 / Q000235:genetics; D004252:DNA Mutational Analysis; D005136:Eye Proteins / Q000235:genetics*; D005260:Female; D020869:Gene Expression Profiling; D005838:Genotype; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics; D006801:Humans; D008297:Male; D009154:Mutation; D009755:Night Blindness / Q000151:congenital* / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D010375:Pedigree; D016133:Polymerase Chain Reaction; D011509:Proteoglycans / Q000235:genetics; D018094:Receptors, Metabotropic Glutamate / Q000235:genetics; D012164:Retinal Diseases / Q000151:congenital / Q000235:genetics*; D012243:Rhodopsin / Q000235:genetics; D015293:Transducin
C514441:CABP4 protein, human; C113330:CACNA1F protein, human; C498838:CACNA2D4 protein, human; D020746:Calcium Channels, L-Type; D002135:Calcium-Binding Proteins; D005136:Eye Proteins; C034809:GNAT1 protein, human; C416505:NYX protein, human; D011509:Proteoglycans; D018094:Receptors, Metabotropic Glutamate; C113897:metabotropic glutamate receptor 6; D012243:Rhodopsin; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; C517431:PDE6B protein, human; D020962:Heterotrimeric GTP-Binding Proteins; D015293:Transducin