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Analysis of human alternative first exons and copy number variation of the GJA12 gene in patients with PelizaeusЦMerzbacher-like disease.

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Neuropsychiatric Genetics
Analysis of Human Alternative First Exons and Copy
Number Variation of the GJA12 Gene in Patients With
Pelizaeus–Merzbacher-Like Disease
Nico Ruf1 and Birgit Uhlenberg2*
Department of Neuropediatrics, Charite, University Medical School, Berlin, Germany
Children’s Clinic, Department of Neuropediatrics, Charite, University Medical School, Berlin, Germany
Received 8 February 2008; Accepted 28 April 2008
Pelizaeus–Merzbacher-like disease (PMLD) is a heterogeneous
disease with primary hypomyelination of the central nervous
system. Only the minority of patients have mutations in the
coding region of the GJA12 gene encoding gap junction protein
alpha 12, a subunit of intercellular channels highly expressed by
oligodendrocytes, the myelin forming cells of the central nervous
system. No other gene has been found so far to be mutated in
PMLD besides GJA12. We therefore extended the mutational
screening in the GJA12 gene, searched for alternative first
exons—as described in mice—determined the human 50 -end of
the gene, screened therein for mutations and analyzed for copy
number variations of the GJA12 gene in 14 patients with PMLD.
Unlike in mice we did not find alternative first exons but detected
a unique 79 bp first exon in human adolescent brain and spinal
cord. No mutation in this non-coding region was found in our
cohort. Copy number variation of the GJA12 gene was assessed by
real-time PCR TaqMan gene expression technology, but neither
patient showed an aberrant copy number. These data confirm
that GJA12 alterations are a rare cause of PMLD—even after
extending the screening for copy number variation and for
mutations in the non-coding region of GJA12. Full genome scans
in informative families and further screenings of candidate genes
are feasible approaches to elucidate the genetic background of
the majority of patients with PMLD. 2008 Wiley-Liss, Inc.
Key words: hypomyelination; gap junction proteins; candidate
Myelination defects are clinically represented by leukodystrophies,
which are disorders—mostly inherited—affecting brain white
matter. The pathological process can be either hypo-, de-, or
dysmyelinating, or a combination of these. The term ‘‘hypoelination’’ refers to the fact that a significant and permanent deficit
in the amount of myelin occurs compared with healthy individuals.
There are several different disorders with diffuse hypomyelination,
including Pelizaeus–Merzbacher-disease (PMD [Boulloche
and Aicardi, 1986]), Pelizaeus–Merzbacher-like disease (PMLD
[Uhlenberg et al., 2004]), Salla disease [Sonninen et al.,
2008 Wiley-Liss, Inc.
How to Cite this Article:
Ruf N, Uhlenberg B. 2009. Analysis of Human
Alternative First Exons and Copy Number
Variation of the GJA12 Gene in Patients With
Pelizaeus–Merzbacher-Like Disease.
Am J Med Genet Part B 150B:226–232.
1999], Cockayne syndrome type II [Nishio et al., 1988],
trichothiodystrophy [Ostergaard and Christensen, 1996] and
Wardenburg–Hirschsprung syndrome [Inoue et al., 2002]. Hypomyelination with congenital cataracts [Zara et al., 2006] as well as
leukencephalopathy with ataxia and hypodontia [Wolf et al.,
2005a] are clinically characterized entities that have been described
recently whereas hypomyelination with atrophy of the basal ganglia
and cerebellum (H-ABC) is a neuroradiologically defined disorder
[van der Knaap et al., 2002].
PMD is the prototype of hypomyelinative leukodystrophies,
besides the nervous system no other organ is obviously involved.
Patients with PMD have nystagmus, impaired motor development,
ataxia, choreathetotic movements, dysarthria and progressive spasticity. The disease is caused by mutations in the PLP1 gene and its
This article contains supplementary material, which may be viewed at the
American Journal of Medical Genetics website at http://www.interscience.
Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: SFB
Nico Ruf’s Present address is Laboratory of Developmental Genetics and
Imprinting, The Babraham Institute, Cambridge CB22 3AT, United
*Correspondence to:
Dr. Birgit Uhlenberg, M.D., Children’s Clinic, Department of
Neuropediatrics, Charite, University Medical School, Berlin,
Augustenburger Platz 1, D-13353 Berlin, Germany.
Published online 2 June 2008 in Wiley InterScience
DOI 10.1002/ajmg.b.30792
spliced isoform DM20. PLP1 encodes proteolipidprotein 1, the
major component of CNS myelin that is also expressed in the
peripheral nervous system (for review see Koeppen and Robitaille
[2002]). Its allelic variant is spastic paraplegia type 2 and the disease
clinically differs in the onset and severity of motor disability.
A wide spectrum of PLP1 mutations has been described in animal
models [Nave, 1994] and PMD patients and duplication of the
entire PLP1 gene is the most frequent cause of the disease
[Sistermans et al., 1998]. The disease severity in humans is comparable between patients with missense mutations and duplications of
the PLP1 gene, whereas patients with deletions of the PLP1 gene
have a significantly milder phenotype [Cailloux et al., 2000]. There
is also a clear relationship between the severity of clinical symptoms
and PLP1 gene dosage: three or more copies of the PLP1 gene lead
to a more severe phenotype than two copies. No difference,
however, is found in the severity of clinical symptoms when
comparing patients with three and five copies of the PLP1 gene
[Wolf et al., 2005b].
Patients with the PMD phenotype but without PLP1 alterations
are considered as having PMLD, comprising approximately 20% of
these patients [Schiffmann and Boespflug-Tanguy, 2001]. In a large
consanguineous and in further two families we identified point
mutations in the coding region of the GJA12 gene encoding
gap junction protein alpha 12 as the causative alteration
[Uhlenberg et al., 2004]. Gap junction proteins belong to a family
of at least 20 homologous genes expressed in a wide range
of different tissues in mammals and are made responsible for
intercellular communication. GJA12 is highly—if exclusively—
expressed in oligodendrocytes [Odermatt et al., 2003]. Most gap
junction proteins have two exons and only the larger second
harbors the open reading frame. Whereas there are alternative first
exons in murine Gja12 [Anderson et al., 2005] these circumstances
have not been investigated in human tissue. Functional studies have
shown that certain missense mutated GJA12 proteins are retained in
the endoplasmic reticulum instead of being inserted in the plasma
membrane, leading to a loss-of-function situation [OrthmannMurphy et al., 2007a]. The pathophysiological consequences on
myelination, however, remain unclear.
Mutations in the GJA12 gene as the cause for PMLD could be
confirmed by other research groups [Bugiani et al., 2006; Salviati
et al., 2007; Wolf et al., 2007].
However, in a large multiethnic cohort, GJA12 mutations turned
out to be a rather rare cause of PMLD, namely in 16 out of 193
patients [Henneke et al., 2008].
In order to investigate PMLD patients without mutations in the
coding region of GJA12 we hypothesized and studied the following:
(1) As up to now the transcription start site of the human GJA12
gene has only been predicted and alternative first exons were
demonstrated for the murine orthologue we hypothesized that
human GJA12 transcript might be larger than originally
thought and/or might exist in different isoforms and that
regulatory mutations in this regions might account for another
reasonable amount of patients with PMLD. We therefore
identified the 50 -end in adolescent brain and spinal cord and
reinvestigated patients with the PMLD phenotype but without
mutations in the coding region of GJA12.
(2) Since mutated GJA12 is retained intracellularly instead of being
transported to the plasma membrane we postulated that copy
number variations of the GJA12 gene might also lead to the
pathophysiological phenomenon of ‘‘protein overload’’ of the
oligodendrocyte. We therefore searched for potential copy
number variations of the GJA12 gene in patients without
mutations in this region.
Patients and Human RNA Samples
For DNA mutation screening including copy number variation
analysis DNA samples from peripheral blood specimens from 14
patients with the PMLD phenotype and 15 healthy controls were
collected. The following criteria for the PMLD phenotype had to be
fulfilled: severe and diffuse hypomyelination on brain MRI
(hyperintensity of cerebral white matter compared to gray matter
on T2-weighted scans, connatal nystagmus or manifestation within
the first 4 months, developmental delay followed by progressive
spasticity and ataxia, exclusion of duplications and missense mutations in the PLP1 gene, exclusion of missense mutations in the
coding region of the GJA12 gene). The study was approved by the
local Ethics Committee and met the standards of the declaration of
Helsinki. Total RNA samples from human adult brain (Stratagene,
Amsterdam, Netherlands) and from human adult spinal cord
(Clontech-Takara Bio, Saint-Germain-en-Laye, France) were used
for 50 -RACE.
50 -RACE and Cloning
For determination of the 50 -end of GJA12, the GeneRacer Kit with
Superscript III RT Module (Invitrogen, Karlsruhe, Germany) was
utilized according to the manufacturer’s protocol including all
negative controls (omitting template, gene specific primer or
GeneRacer primer). The following primers were used for the first
combination with the GeneRacer primers. The first PCR contained
1 ml RACE-ready cDNA, 1 ml 5 Buffer A, 4 ml 5 Buffer B, 200 mM
dNTPs, 20 pmol gene specific primer, 20 pmol GeneRacer primer,
and 1 ml Elongase Enzyme Mix from the Elongase Reagent System
in a total volume of 25 ml (Invitrogen). The nested PCR contained
1 ml (diluted 1:10) of the first PCR, 1 ml 5 Buffer A, 4 ml 5 Buffer B,
200 mM dNTPs, 20 pmol nested gene specific primer, 20 pmol
GeneRacer nested primer, and 1 ml Elongase Enzyme Mix. Subsequently, RACE products were cloned into the pCR4-TOPO
plasmid (Invitrogen) and 54 clones were sequenced using T3 or
T7 primers.
Sequence Analysis and Mutational Screening
The DNA sequences of the alternative first exons of murine Gja12
[Anderson et al., 2005] were used for an Ensembl BLAST search to
seek for homologous regions in the human GJA12 gene region
In order to identify transcription factor binding sites in the
putative GJA12 promoter region, DNA sequences upstream of
human and mouse GJA12/Gja12 genes were fed into the web-based
rVISTA software (
Subsequently, a 596 bp fragment of this upstream region including the first exon of GJA12 was amplified by PCR from genomic
DNA with the following primers: forward 50 -AGACAGATGGGTGGGAGAGA-30 and reverse 50 -AAGGAGTGCACCCTCCTATG-30 . The PCR mix contained 50 ng of genomic DNA, 1 U
Taq polymerase, 200 mM dNTPs, 20 pmol of each primer, 1.5 mM
MgCl2 in a total volume of 25 ml. The initial denaturation step of
5 min at 95 C was followed by 45 cycles of 95 C for 30 sec, 60 C for
30 sec, and 72 C for 1 min. Subsequently, PCR products were
sequenced using an ABI3730 DNA Analyzer and a BigDye
Terminator v1.1 Cycle Sequencing Kit according to the information
of the manufacturer (Applied Biosystems, Darmstadt, Germany).
Copy Number Variation Analysis
Copy number variation was assessed using real-time PCR
TaqMan Gene Expression technology (Applied Biosystems). All
real-time PCR assays were able to detect genomic DNA and used
TaqMan minor groove binder (MGB) probes (Applied
Biosystems). The RNAseP control reagent kit was used as endogenous control. The target probes were FAM labeled and the endogenous control was VIC labeled. The GJB1 gene (assay ID
Hs00702141_s1) was used as proof-of-principle, as GJB1 is located
on the human X chromosome which enables to differentiate
between male and female samples by one or two copies of the X
chromosome. GJA12 copy number variation was assessed using a
Custom Taqman Gene Expression assay. The custom assay contained the forward primer 50 -CTGTTGCCCCAGGAGACA-30 , the
reverse primer 50 -TGGCTGGGCCCTAGGAA-30 , and the probe 50
FAM-CACGCTGTGCCCCTTG-30 . Each reaction consisted of
10 ng of genomic DNA, each of the forward and reverse primers
for each the target and the control amplicons, the target probe and
the endogenous control probe, and the TaqMan universal master
mix. The final volume was adjusted to 15 ml. Each sample was
analyzed in triplicates. The real-time PCR was performed using ABI
MicroAmp Optical Fast 96-well Reaction Plates and the ABI 7500
Fast Real-Time PCR System with initial denaturation of 10 min at
95 C and followed by 50 cycles of 95 C for 15 sec and 60 C for
1 min. Copy numbers of the target genes were calculated by the ABI
SDS software 1.3.1 using relative quantification based on the DDCT
method (Livak and Schmittgen, 2001) and control samples as
FIG. 1. Analysis of 50 -RACE by agarose gel electrophoresis. 4 ll of
the products from nested PCR from adult brain and adult spinal
cord were loaded on a 2% agarose gel. The presence of one band
each demonstrates a unique 50 -end of the human GJA12
(Fig. 1) This demonstrates that there are no different isoforms as
previously shown in the mouse [Anderson et al., 2005]. Thus PCR
products were cloned into the pCR4 TOPO vector and sequenced.
In contrast to the current NCBI RefSeq entry (NM_020435), the
analysis showed a 79 bp sized first exon instead of 156 bp and a
transcriptional start site further downstream than predicted (NCBI
GenBank: EU433401). In order to gain more insight into the
structure of the GJA12 promoter region, comparative analysis of
human and murine GJA12 sequences upstream of exon 1 was
undertaken. Interestingly, conserved putative binding sites for the
myelin-associated transcription factors SOX9 (77 to 64 bp) and
SOX10 (53 to 45 bp and 37 to 31 bp, each with respect to the
transcriptional start site) were found (Fig. 2). Both SOX9 and
SOX10 were shown to play a pivotal role in oligodendrocyte
function and myelin formation [Schlierf et al., 2006; Li et al.,
2007; Finzsch et al., 2008].
As the untranslated first exon of GJA12 and the promoter region
were not yet included in previous mutation screenings, it was
feasible to seek for functional mutations in patients with the PMLD
phenotype. Thus, 14 individuals which did not carry mutations in
the coding region of GJA12 were chosen for sequence analysis but
no change in the DNA sequence could be identified in all DNA
samples analyzed.
Copy Number Variation Analysis
50 -RACE and Mutational Analysis
Ensembl BLAST search analysis using the alternative first exons of
the murine Gja12 transcripts detected a human orthologous region
for one alternative exon but not for the others [see Anderson et al.,
2005]. To determine the 50 -end of the GJA12 transcript, 50 -RACE
was performed on total RNA from human adult brain and spinal
cord. The subsequent PCR included forward primers corresponding to the GeneRacer oligo sequence and reverse primers complementary to exon 2 and resulted in a single band in both tissues
As described above, duplication of the entire PLP1 gene is the most
frequent cause in patients with PMD phenotype [Sistermans et al.,
1998]. Therefore, it was hypothesized that changes in copy numbers
of GJA12 might account for patients with the PMLD phenotype as
well. Real-time PCR using TaqMan gene expression technology
which can detect genomic DNA was used in other studies for
detection of copy number variation [Premi et al., 2006; G
omezCuret et al., 2007; Wu et al., 2007]. This method offers the
opportunity of using an endogenous control which enables relative
quantification and reduces the impact of imprecise pipetting and,
thus, was chosen for GJA12 copy number screening. Another
FIG. 2. Schematic structure of the GJA12 gene. The human GJA12 gene is comprised of two exons. Our analysis revealed the transcriptional start
site—shown as white arrow bar—as well as binding sites for the transcription factors SOX9 (77 to 64 bp; dark gray bar) and SOX10 (53 to
45 bp and 37 to 31 bp; light gray bars). GJA12 exons 1 (79 bp) and 2 (2083 bp) are depicted as white boxes. The open reading frame is marked
in black.
advantage in terms of relative quantification is the simple analysis
based on the DDCT method [Livak and Schmittgen, 2001] which is a
feature of the ABI SDS software 1.3.1.
In order to test this approach, copy numbers of the GJB1 gene,
which maps to the human X chromosome, were assessed using each
male and female PMLD patient DNA samples. As shown in Figure 3,
this assay facilitates differentiating individuals with one copy
(male) from those with two copies (female). Therefore, this method
was subsequently applied to the GJA12 gene and the whole cohort of
PMLD patients as well as 15 healthy control individuals. Every
control was used as calibrator for the analysis and in all cases the
results were similar. A typical example is depicted in Figure 4.
Assuming that the control individuals do not have any GJA12 gene
mutations and can be therefore used as calibrator, no copy number
variation was found in any of the PMLD patients.
Leukodystrophies comprise a group of rare central white matter
disorders defined by clinical, pathological, and magnetic resonance
criteria. The so far best understood type of hypomyelinative
leukodystrophies is PMD that is due to alterations in the PLP1
gene coding for the major structural component of myelin. PMLD
is clinically indistinguishable from PMD and by means of a genome
scan in an informative family we in the past detected mutations in
the GJA12 gene as the causative alteration in this disease. GJA12
belongs to the family of gap junction proteins forming intercellular
channels between apposing cells that are expressed in a wide variety
of different tissues. However, GJA12 alterations are only responsible
for a rather small amount of patients with PMLD, as was shown in a
large multiethnic cohort (16 out of 193 patients [Henneke et al.,
FIG. 3. Proof of principle. Copy numbers of the GJB1 gene located on human X chromosome obtained by relative quantification analysis. Samples 408,
426, and 440 are females having two copies whereas samples 417, 425, and 450 are males with one copy of GJB1. The relative quantification was
calculated using sample 425 as calibrator. The results are shown in parentheses.
FIG. 4. Copy numbers in PMLD patients. GJA12 gene copy numbers using relative quantification analysis in PMLD patients (black bars) and compared
with healthy control individuals (gray bars). The relative quantification was calculated using control sample K100 as calibrator. The observed
variation of most of the analyzed samples is between 0.80 and 1.20 regarding to the calibrator sample. All RQ results of this analysis are listed in
Supplementary Table I.
2008]). In the absence of further informative PMLD families the
candidate gene approach seemed a feasible approach to detect other
genes involved but was not successful in the past: mutations in M6B,
another myelin protein, and in the oligodendroglial transcription
factors OLIG1 and OLIG2 were ruled out [Henneke et al., 2004; Ruf
et al., 2007].
In this study we aimed to detect so far uninvestigated alterations
of the GJA12 gene in the larger portion of PMLD patients (90%)
that is not due to mutations in the GJA12 coding region.
Recently it was shown that murine Gja12 displays the most varied
50 -UTR structure of five gap junction proteins studied [Anderson
et al., 2005]. Three out of four different first exons were found in
adult brain, and they were all shown to be spliced to the identical
acceptor site of exon 2. Since the 50 -end was neither determined
in human GJA12 nor was it investigated in recent PMLD studies
we performed 50 -RACE in adult human brain and adult human
spinal cord. In both tissues a unique first exon was identified which
makes the existence of different isoforms in these tissues rather
Since most of the murine Gja12 isoforms do not have a human
orthologue, one might speculate that these isoforms are not likely to
be of functional relevance. Another explanation for the absence of
alternative first exons in humans might be based on the fact that
Anderson et al. used embryonic derived RACE-ready mouse cDNAs
(E10 to E12) whereas we used adult human brain and were not able
to detect a GJA12 transcript in human fetal brain (data not shown).
We included the sequence of the single first exon in the subsequent mutational screening of 14 individuals with the PMLD
phenotype but did not detect any sequence variation. Thus func-
tional mutations in the non-coding region of human GJA12 are not
likely to be associated with the PMLD phenotype.
Since mutations in the coding region of the GJA12 gene are rare
in PMLD patients and mutations in the non-coding sequence
are—as shown—rather unlikely to be causative we aimed to exclude
copy number variations of the GJA12 gene in so far unresolved
patients. This approach was feasible for different reasons. The
prototype of hypomyelinative leukodystrophe—PMD—is mainly
caused by duplications of the PLP1 gene. Pathophysiologically
overexpression of PLP1 leads to the accumulation of cholesterol
and PLP1 in lysosomes and these aggregates disturb membrane
trafficking, possibly by trapping other myelin proteins as well, thus
leading to impairment of the myelination process [Simons et al.,
2002]. A similar cascade might be conceivable for overexpression of
GJA12 which also needs to travel to the oligodendrocytic plasma
membrane. Functional effects have so far only been studied for
certain GJA12 missense mutations [Orthmann-Murphy et al.,
2007a], leading to retainment in the endoplasmic reticulum and
therefore to a loss-of-function situation.
However, our analysis did not reveal any GJA12 mutations in a
cohort of 14 PMLD patients. These findings together with the data
from Henneke et al. [2008] clearly emphasize that another gene
must be involved in the etiology of PMLD. In terms of expression
other gap junction proteins might be good candidates, besides
GJA12 also GJB2 and GJE1 are expressed in oligodendrocytes. GJB2,
however, has been shown to be associated with a peripheral
neuropathy in mice and men, called Charcot-Marie-Tooth, and
this disease is seldom accompanied by transient central nervous
system lesions [Taylor et al., 2003]. GJE1-deficient mice do not
display any abnormalities [Eiberger et al., 2006], and it was shown
that the protein does not form fully functional intercellular
channels [Ahn et al., 2008]. Another gap junction protein seems
to be important for oligodendrocytic homeostasis although not
expressed by oligodendrocytes: GJA1 is expressed by astrocytes, but
was shown to be the interacting hemichannel partner of GJA12
[Orthmann-Murphy et al., 2007b]. Certain missense GJA12 mutations were also shown to lead to impaired GJA1-GJA12 coupling
and it might therefore be possible that the GJA12 phenotype can
also be resembled by mutations in the GJA1 gene.
Pathophysiologically the Kir4.1 potassium channel subunit has
been shown to be crucial for oligodendrocyte development and
myelination [Neusch et al., 2001]. Kir4.1-deficient mice display
motor impairment, the cellular basis is hypomyelination of the
spinal cord with spongiform vacuolization. This phenotype reminds of GJA12-deficient mice in which vacuolization of the optic
nerve is the main feature [Odermatt et al., 2003].
For analysis of GJA12 copy number variations we used the realtime PCR TaqMan gene expression technology and relative
quantification based on the DDCT method. We used the X chromosomal GJB1 gene as a proof-of-principle. By this strategy we were
able to clearly distinguish between one and two copies of the
gene—depending on gender. The lack of patient samples with
confirmed alterations in gene copy numbers and the high standard
in terms of exact pipetting work are usually important premises for
the analysis of copy number variations if real-time PCR is used.
Other methods like southern hybridization are laborious demanding a high amount of patient DNA. These issues can be resolved by
our approach using an endogenous control gene—like single copy
gene RNAseP—as well as healthy control individuals, thus enabling
relative quantification based on the DDCT method. By applying this
method to GJA12 we were, however, not able to detect any
variations in GJA12 copy number. Nevertheless our approach seems
to be simple and reliable and could therefore be applied to larger
scales like regular genetic diagnostics in particular.
The study was supported by a grant from the Deutsche Forschungsgemeinschaft to B.U. (SFB 665). The authors wish to thank Andrea
Toeppel and Juergen Janke for expert help with establishment of the
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patients, variation, disease, alternative, human, like, pelizaeusцmerzbacher, exon, first, gja12, analysis, number, genes
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