Downloaded from jmg.bmj.com on December 2, 2009 - Published by group.bmj.com
Further clinical and molecular delineation of the
9q Subtelomeric Deletion Syndrome supports a
major contribution of EHMT1 haploinsufficiency
to the core phenotype
Tjitske Kleefstra, Wendy A van Zelst-Stams, Willy M Nillesen, et al.
J Med Genet published online March 4, 2009
doi: 10.1136/jmg.2008.062950
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JMG Online First, published on March 4, 2009 as 10.1136/jmg.2008.062950
1
Further clinical and molecular delineation of the 9q Subtelomeric Deletion
Syndrome supports a major contribution of EHMT1 haploinsufficiency to the core
phenotype
Tjitske Kleefstra1, Wendy A. van Zelst-Stams2, Willy M. Nillesen1, Valerie Cormier-Daire3, Gunnar
Houge4, Nicola Foulds5, Marieke van Dooren6, Marjolein H.Willemsen1, Rolph Pfundt1, Anne Turner7,
Merdith Wilson8, Julie McGaughran9, Anita Rauch10, Martin Zenker10, Margaret P. Adam11, Micheil
Innes12, Christine Davies12, Antonio González-Meneses López 13, Rosario Casalone14, Astrid Weber15,
Louise A. Brueton16, Alicia Delicado Navarro17, Maria Palomares Bralo17, Hanka Venselaar18, Sander P.A.
Stegmann2, Helger G. Yntema1, Hans van Bokhoven1, Han G. Brunner1
1
Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The
Netherlands
2
Department of Human Genetics, Maastricht University Hospital, Maastricht, The Netherlands
3
Department of Medical Genetics and INSERM U781, Hôpital Necker Enfants Malades, Paris, France
4
Center for medical genetics and molecular medicine, Haukeland University Hospital, Bergen, Norway
5
Wessex Clinical Genetics Service, Princess Anne Hospital, Southampton, United Kingdom
6
Department of Clinical Genetics, Erasmus Medical Centre, Rotterdam, The Netherlands
7
Department of Medical Genetics, Sydney Children's Hospital, Randwick, Sydney, Australia
8
Department of Clinical Genetics, Children’s Hospital at Westmead, Sydney, Australia
9
Genetic Health Queensland, Royal Children's Hospital and Health District, Herston, Brisbane,
Queensland, Australia
10
Institute of Human Genetics, University Hospital Erlangen, University of Erlangen-Nuremberg,
Erlangen, Germany
11
Emory University, Atlanta, Georgia, USA
12
Department of Clinical Genetics, Alberta Children's Hospital, Calgary, Canada
13
Unidad de Dismorfología, Servicio de Pediatría,Hospital UniversitarioVirgendel Rocío, Sevilla, Spain
14
Dipartimento di Patologia Clinica, S.S. di Genetica Medica, Azienda Ospedaliero Universitaria,
Ospedale di Circolo e Fondazione Macchi, Varese, Italy
15
Alder Hey Children’s Hospital, Liverpool, Great Britain
16
Clinical Genetics Unit, Birmingham Women’s Hospital, Birmingham, United Kingdom
17
Sección de Genética Médica, Hospital Universitario La Paz, Madrid, Spain
18
Center for Molecular and Biomolecular Informatics, Radboud University Nijmegen, The Netherlands
Corresponding author:
Tjitske Kleefstra MD, PhD
849 Department of Human Genetics
PO Box 9101
6500 HB Nijmegen
The Netherlands
0031-(0)243613946
Key words Chromosome 9q, Subtelomere deletion, 9qSTDS, EHMT1, Histone Methylation
Copyright Article author (or their employer) 2009. Produced by BMJ Publishing Group Ltd under licence.
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2
Abstract
The 9q Subtelomeric Deletion Syndrome (9qSTDS) is clinically characterized by
moderate to severe mental retardation, childhood hypotonia and facial dysmorphisms. In
addition, congenital heart defects, urogenital defects, epilepsy and behavioral problems
are frequently observed. The syndrome can be either caused by a submicroscopic 9q34.3
deletion or by intragenic EHMT1 mutations leading to haploinsufficiency of the EHMT1
gene. So far it has not been established if and to what extent other genes in the 9q34.3
region contribute to the phenotype observed in deletion cases.
Here we report the largest cohort of 9qSTDS cases so far. By a Multiplex Ligation
dependent Probe Amplification approach, we identified and characterized 16 novel
submicroscopic 9q deletions. Direct sequence analysis of the EHMT1 gene in 24 patients
exhibiting the 9qSTD phenotype without such deletion, identified 6 patients with an
intragenic EHMT1 mutation. Five of these mutations predict a premature termination
codon whereas one mutation gives rise to an amino acid substitution in a conserved
domain of the protein.
Our data do not provide any evidence for phenotype-genotype correlations between size
of the deletions or type of mutations and severity of clinical features. Therefore, we
confirm the EHMT1 gene to be the major determinant of the 9qSTDS phenotype.
Interestingly, five of six patients who had reached adulthood had developed severe
psychiatric pathology, which may indicate that EHMT1 haploinsufficiency is associated
with neurodegeneration in addition to neurodevelopmental defect.
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3
Introduction
In the last decade, several microdeletion and duplication syndromes emerged from
routine screening of subtelomere regions by Multiplex Ligation-dependent Probe
Amplification (MLPA) and Fluorescent In situ Hybridization (FISH).[1-4] The
chromosome 9q subtelomeric deletion represents one of the most common subtelomere
deletions (6% of all).[3] In addition, the chromosome 9q subtelomere deletion syndrome
(9qSTDS) is also one of the first clinically recognizable telomeric syndromes.[3,5-9]
Affected individuals invariably have severe hypotonia with speech and gross motor
delay. The facial features comprise micro- or brachycephaly, hypertelorism, synophrys or
arched eyebrows, midface hypoplasia, a short nose with upturned nares, a protruding
tongue with everted lower lip and down-turned corners of the mouth. Approximately half
of affected individuals have congenital heart defects, primarily atrial or ventricular
septum defects. A substantial part (10-20%) have epilepsy and/or behavioral and sleep
disturbances. A variety of other major and minor eye, ear, genital and limb anomalies
have been reported.[7-9] We demonstrated that the syndrome is caused by
haploinsufficiency of Eu-chromatin Histon Methyl Transferase 1 (EHMT1) (Online
Mendelian Inheritance in Man 607001), a gene involved in histone methylation.[10-15]
Since this finding, we have implemented the testing for the 9q subtelomeric deletion
syndrome in a DNA diagnostic setting. This analysis includes a screen for small deletions
by a Multiplex MLPA approach and for intragenic EHMT1 mutations by direct
sequencing of the gene in MLPA negative cases. To date it is not yet known if and to
what extent the size of the deletions or type of EHMT1 mutations are related to particular
features or severity of the 9qSTDS. Therefore we have carefully studied the clinical and
molecular characteristics of a cohort of newly identified cases with the 9qSTDS.
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4
Patients
Patients included in this study were referred by clinical geneticists from the Department
of Human Genetics of the Radboud University Nijmegen Medical Centre and through
collaborating centers. All patients referred had the core phenotype of the 9qSTDS
comprising mental retardation, hypotonia and facial characteristics. The cohort included
16 patients with a known 9q deletion identified through routine subtelomere chromosome
testing or whole genome array, who were referred for confirmation and/or further fine
mapping of the deletion. In 14 cases the deletion was identified by routine subtelomeric
MLPA (P070 MSRC Holland) or FISH (Vysis probe).[1-2] In the other two, a deletion
was identified through whole genome array (Affymetrix 500k SNP array and custom
oligonucleotide array EmArrayCyto6000_version2). The second cohort comprised 24
patients with the typical manifestations of the syndrome but with normal routine
MLPA/FISH results. The selection of patients for this cohort was carried out by two of us
(TK and HGB) based on facial characteristics.
Methods
DNA was obtained from peripheral blood cells and extracted according to standardised
procedures. MLPA and direct sequencing of the EHMT1 gene was carried out as
described previously.[12]
We used the structure of the pre-SET and SET-domains (PDB-file 2rfi) to study the effect
of the missense mutations on the 3D-structure of the protein. The WHAT IF & Yasara
Twinset (www.yasara.org) was used for analysis of the effects of the amino acid
substitution towards the protein structure. A more extensive explanation of the
methodologies and results is available online (http://www.cmbi.ru.nl/~hvensela/EHMT1).
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5
Results
Molecular analysis
The size of 16 novel deletions is schematically shown in figure 1. Deletion fine mapping
was performed in 16 patients in whom presence of a 9q deletion had already been
suggested based on findings by routine telomere studies or genomewide array
approaches. In seven patients the deletion was initially identified by FISH (patients 8, 9,
10, 11, 12, 13 and 14) for another seven patients routine MLPA had been abnormal
(patients 1, 2, 3, 4, 7, 15 and 16) and in two patients interstitial 9q deletions were found
by microarray analysis: patient 5 by custom oligonucleotide array
EmArrayCyto6000_version2 and patient 6 by Affymetrix 500k SNP array. The
breakpoint and sizes of the deletions show a heterogeneous distribution. The most
common deletion was found in six patients and had a size between 700 kb and 1.3Mb
(figure 1). The largest one is 3.1Mb in size extending from CACNA1B to OLFM1 (patient
2). The smallest one is 40 kb, comprising exon 11 to 25 of the EHMT1 gene exclusively
(patient 6).
Further deletion analysis followed by direct sequencing of EHMT1 was performed in 24
patients with normal routine MLPA/FISH results. No additional deletions were identified
by MLPA whereas in total, six novel intragenic mutations were found that are highly
likely pathogenic (table 1). The mutations comprised two nonsense changes, c.778C>T
and c.1717C>T (p.Arg260X, p.Gln573X) and one 4 bp deletion c.1440_1443del
(p.Asp481fs), all three predicted to result in nonsense mediated mRNA decay (NMD).
Two comprised de novo splice site mutations (c.2775-1G>A and c.2100-1G>C ).
6
Table 1: Clinical characteristics of patients with EHMT1 intragenic mutations and (interstitial) subtelomeric deletions
Age*
(y)
Sex
(M/F)
Birth
Weight
(g,
centile)
Present
Weight
(kg,
centile)
Present
Height
(centile)
1
4
F
4500
(>95th)
50th
2-16th
2
4
M
3200
(25th)
3
4
F
3800
(7590th)
>95th
4
52
F
normal
5
8
M
6
7
M
Present
OFC
(centile)
Heart
defect
Epilepsy
innocent
murmur
-
33 cm
at birth,
(2nd)
VSD
aortic
coarctation
-
>>95th
50th
Tetralogy of
Fallot
-
50th
2nd
0.6th
-
3650
(5075th)
99.6th
98th
16th
innocent
murmur
50th
2nd
2nd
5th
-
Other
MRI/CT
cerebrum
Interstitial
Normal (MRI)
micropenis,
cryptorchidism
VUR
hearing loss
inguinal, periumbilical and
epigastric hernia
mixed hearing
loss umbilical
hernia
Mildly dilated ventricles
Reduction in white
matter volume (MRI)
absences
tonic-clonic
insults
scoliosis
complex
partial
seizures
inguinal and
umbilical hernia
-
tracheo/bronchomalacia
chronic renal
insufficiency
small pons, slight
central atrophy of both
hemispheres (P.E.G
examination)
increased CSF spaces
over frontal lobes and
Sylvian fissures
compatible with atrophy
(MRI)
small brainstem (MRI)
no data
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Patient
7
20
M
4350
(.>90th)
50th
8
48
M
4200
(95th)
25-50th
9
33
F
3rd
50th
-
Abnormal
EEG
No epilepsy
hearing loss
small ears
small hands
supernumerary
nipples
cryptorchidism
Normal (MRI)
2nd
-
+
asymmetric face
Normal (CT)
2nd
ASD
+
Telomeric
M
2760
(<5th )
2nd
16th
0.6th
VSD/ASD
-
2300
(<2 th )
3690
(75th )
98th
2nd
2nd
VSD
-
98th
98th
25th
bicuspid aortic
valve
-
no data
tracheomalacia
11
14
F
12
20
M
13
4
M
3600
(75th )
95th
95th
16th
-
-
14
21
M
4390
(>95th )
84th
185 cm
50th
16th
VSD
tonic-clonic
insults
15
12
M
3890
(7590th)
98th
16th
50th
-
-
pyloric stenosis
died age 21, cause
unknown
cryptorchidism
3
F
4380
(95th )
>> 99th
>> 99th
50th
-
-
-
no data
no data
VUR, GER
Retained primary
dentition
pes equinovarus
16
Prominent VirchowRobin spaces (MRI)
Normal
slight asymmetry
anterior horns
arachnoidal cyst,
supratentorial mildly
enlarged CSF spaces (5
yrs)
no data
Intragenic
EHMT1**
c.2100-1G>C
17
abnormal myelination
(MRI)
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7
8
18
13
F
4000
(90th )
95th
50th
50th
Patent FO
(closed
spontaneously)
-
p.Arg260X
(c.778C>T)
19
7
M
3822
(7590th)
90th
90th
16th
-
p.Cys1042Tyr
(c.3125G>A)
20
7
F
4100
(90th )
90th
75th
90th
Pulm stenosis
first at age 7
generalized
with right
posterior focal
onset
-
p.Asp481fs
(c.1440_1443del)
21
2.6
F
2495
(<5th)
50th
84th
<2 nd
-
-
p.Gln573X
(c.1717CT)
22
3
M
2690
(<5th)
<2nd
-
-
constipation
incontinent for
urine/feces
normal
mildly enlarged
ventricles
(MRI)
glue ear requiring
grommets
hypermobile joints
delayed secondary
dentition at age 8
GER
cryptorchidism
(prenatal NT)
normal myelination,
normal ventricles, some
prominence of Sylvian
fissures with frontal
lobe hypoplasia (MRI)
normal
corpus callosum
hypoplasia
(MRI)
VUR= vesico- ureteral reflux; GER= gastro-esophageal reflux; VSD= ventricular septum defect; FO= Foramen Ovale; ASD= atrial septum defect; PEG= pneumoencephalogram;
CSF= cerebral spinal fluid
* Age of examination
** GenBank Accession Number NM_024757.3 was used as a reference sequence. The A at the start codon was designated as nucleotide number 1.
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c.2775-1G>A
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9
These mutations have not been previously reported as pathogenic mutations, they are not
present as non-pathogenic variants (http://www.ncbi.nlm.nih.gov/SNP/). In three cases
the expected de novo occurrence was confirmed by analysing DNA from both parents,
whereas forthree other cases with loss of function mutations, parents could not be
analysed.
Based
on
the
prediction
software
for
splice
sites
(http://www.fruitfly.org/seq_tools/splice.html,
http://www.cbs.dtu.dk/services/NetGene2/,
and
http://www.genet.sickkids.on.ca/~ali/splicesitefinder.html), the c.2775-1G>A and c.21001G>C changes cause aberrant splicing. Finally, one de novo missense change
c.3125G>A (p.Cys1042Tyr) was found. This mutation affects a highly conserved
cysteine residue located in the pre-SET protein domain (fig 2).
Protein modeling
Cysteine 1042 is located in a cysteine cluster surrounding three zinc-ions. This
cysteine/zinc-cluster is important for the local structure of this particular region of the
pre-SET domain. The cysteines make strong interactions with the zinc-ions thereby
placing the intervening loops in a correct position for dimer interactions. (Figure 3 and
supplementary data http://www.cmbi.ru.nl/~hvensela/EHMT1/). Mutation of cysteine
1042 into a tyrosine will abolish the strong cysteine/zinc interaction, causing a less stable
domain. Moreover, the sidechain of tyrosine will not fit at position 1042. Introduction of
this residue will completely disturb the structure of the zinc/cysteine-cluster and displace
the residues in the loops that interact with the other monomer. The mutation will destroy
the conformation of this domain that is needed for dimer-conta
Clinical data
Individual descriptions
of
patients
are
summarized
in
tables
1
and
2.
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10
Table 2: Developmental, behavioural and psychiatric characteristics
Patient
Psychiatric symptoms
behaviour features
Development
1
+
no data
2
frustration and tantrums
3
temper tantrums
severely delayed only a few
words uses signing and picture
cards
speaks some words
4
disturbed sleep pattern
aggressive outbursts
active and passive periods
5
hyperactive, aggressive outbursts
temper tantrums, disturbed sleep
pattern
autistic traits, hand flapping
6
no data
7
psychoses
passiveness
not walking at age 7 yrs
3 words age 7 yrs
temporarily regression at age 16
yrs/dementia like
8
aggressive behavior
regression at age 28 yrs
9
no data
10
mood disorder
self-mutilation
-
no data
11
-
mild mental retardation
12
friendly
since age 14 sudden mood disorder,
withdrawal, catatonic(-like) period
recovering at age 21 yrs
sat at age 10 months, walked at
age 22 months
single words, some phrases since
age 2 yrs
13
-
14
No data
15
friendly boy,
autistic behaviour
16
no data
sat at age 11 months, walked at
age 20 months
climbed stairs up and down age 4
yrs
few single words age 4 yrs
mild- moderate mental
retardation
sat at 10 months, walked at 18
months
speech delay, some writing and
counting up to 20
no data
c.2100-1G>C
17
disturbed sleep pattern
self-mutilation, hyperphagia
walked at 17 months
a few words at 5 yrs
c.2775-1G>A
18
temper tantrums/tics
rigid and ritualistic behavior
severe mental retardation
walked at age 30 months
Interstitial
regression
sat at 18 months, walked at 4 yrs
spoke a few words when young:
papa, mama, car
delayed
sat at 11 months, walked at
2.5yrs
many single words some phrases,
short sentences
Telomeric
Intragenic
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11
apathic/depressive behavior with loss
of all interests since age 13
3-4 words
walked at 2 yrs, jumping,
running etc age 7 yrs
5 words at age 3, >100 words 7
yrs
severe mental retardation
walked at 3yrs, can not run age 7
yrs
unable to ride bicycle
some speech, in general ‘signs’
walked at 22 months
no speech
p.Arg260X
19
poor eye contact, teeth grinding (all
improving)
p.Cys1042Tyr
20
trusting and friendly personality
p.Asp481fs
21
autism spectrum
p.Gln573X
22
autism spectrum
mild mental retardation
Clinical information of affected patients was systematically obtained by a standard
clinical data sheet. No additional clinical data were obtained for patient 16. All patients
showed the core phenotype of the 9qSTDS comprising mental retardation, childhood
hypotonia and recognizable facial features. Photographs showing the facial profile of
several patients are shown in figures 4-7. Figure 4 shows five patients with an intragenic
EHMT1 mutation. Figure 5 shows patients with obesity. When available and appropriate,
series of photographs at different ages are depicted, showing the progression of the facial
phenotype with age (figure 6 and 7).
Birth weight in all patients was within or above normal range. In 4/15 deletion cases and
3/6 EHMT1 intragenic mutation cases, birth weight was at or above 90th centile.
Childhood obesity was present in the same 3 mutated cases and one additional case (19)
and in 5 deletion cases, noteworthy overlapping only with one who had high birth weight
(patient 15). Microcephaly was noted in 6/15 patients with a deletion and 2/6 cases with a
mutation and was already present at birth. No secondary microcephaly was reported. The
degree of mental retardation was moderate to severe. Most did not develop speech,
though language development was usually at a higher level making communication by
other means, such as sign language or pictograms possible. Variable cognitive function
was noted. The best performance was in patient 11, with a deletion of minimal 970 kb,
who can speak complete sentences (Total Intelligence Quotient, TIQ=52).
Motor function was delayed, but all individuals were able to walk, usually independently
between two and three years of age. A notable exception is patient 6 who has an out of
frame intragenic EHMT1 deletion (exons 11-25) and who is not able to walk at present
age (7 years).
Variable congenital heart defects were observed in 7/15 deletion cases (ASD/VSD,
Tetralogy of Fallot, aortic coarctation, bicuspid aortic valve and in patient 20 with the
p.Cys1042Tyr EHMT1 mutation pulmonic stenosis). Genital defects were observed in 2/6
males with a deletion (micropenis, cryptorchidism) and in 1/2 males with a mutation
(cryptorchidism). In addition vesico-ureteral reflux (VUR) was noted in patients 2 and 12
and chronic renal insufficiency was observed in patient 6 (intragenic EHMT1 deletion).
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12
Other anomalies were tracheo-/ bronchomalacia with respiratory insufficiency and
gastro-eosophageal reflux (GER). Epilepsy was present in 6/15 deletion cases comprising
tonic-clonic seizures, absences and complex partial epilepsy and generalized with focal
onset in patient 19 with the p.Arg260X EHMT1 mutation. Results of brain imaging by
CT or MRI performed in 12 individuals were generally normal but individual patients
had corpus callosum hypoplasia (patient 22), enlarged CSF spaces/cortical atrophy
(patients 5,15,19,20), prominent Virchow-Robin spaces (patient 12), brain stem
hypoplasia (patient 6) and abnormal myelinization (patient 17).
Six patients reached adult age and were examined at respective 19, 20, 20, 33, 48 and 51
years of age. One of these died at age 19, probably because of cardiac arrhythmia, but
autopsy was not performed. No behavior characteristics were reported for this patient. In
the other five adult patients an abrupt change in behavior and daily functioning was
observed starting at adolescence but recovered to some extent in adulthood. All five had
been under psychiatric care and used psychotropic medication. Abnormalities included
apathy, aggressive periods, psychosis or (autistic) catatonia, bipolar mood disorder and
regression in daily function and cognitive abilities.
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13
Discussion
In this study the clinical and molecular data of 22 novel identified patients with the
9qSTDS are shown. Six of them had an intragenic EHMT1 mutation and the remaining
16 had submicroscopic 9q deletions of variable size, including 7 interstitial deletions. The
smallest deletion was 40 kb, taking out exons 11 to 25 of the EHMT1 gene exclusively.
The seven interstitial deletions would have remained undetected by routine subtelomeric
FISH analysis, since the probe that is commonly used for that purpose was not deleted.
On the other hand, patient 15 showed abnormal FISH results, but unexpectedly, by
validation with the standard MLPA probe (P070 in exon 9), normal results were found. It
turned out that the proximal border of the deletion was disrupting the EHMT1 gene
between exons 16 (not deleted) and 19 (deleted by MLPA). So neither routine
subtelomeric testing with FISH nor with MLPA can exclude presence of 9q deletions in
all cases. Therefore, we recommend that testing with the more extensive 9q MLPA kit or
high resolution genomic profiling should be used to definitely discard such deletions.
This study reports the smallest deletion reported so far, comprising only part of the
EHMT1 gene (patient 6). Since it appeared that no other genes in the 9q region were
involved, we considered this patient among the EHMT1 mutation cohort. This patient was
known with tracheo-bronchomalacia and chronic renal insufficiency with unknown cause
in addition to the core phenotype. The largest deletion in this study extended into the
TRAF2 gene where the most proximal MLPA probe was located. By additional SNP
array, it was shown that the deletion was 3.1Mb in size. This patient was born with
several congenital abnormalities, encompassing micropenis, undescended testes, vesico
ureteral reflux and aortic coarctation. It is possible that other genes in this larger deletion
size are contributing to the severity of congenital defects seen in this patient, however all
of these features have been observed also in patients with smaller deletions or EHMT1
mutations.[6,9,12] Moreover, a previously reported case with a 2.2 Mb interstitial 9q
deletion, which completely overlaps the proximal part of the 3.1Mb deletion described
here, did not show any other major congenital abnormalities besides the mental
retardation.[11] So this does not support a major contribution of genes proximal of
TRAF2 to the severity of the phenotype.
In 6 of 24 patients (25%) who were selected by us for exhibiting the 9qSTDS core
phenotype, a pathogenic EHMT1 mutation was found. Five of six mutations disrupt the
open reading frame of the EHMT1 gene and are predicted to create premature termination
codons which may lead to nonsense mediated decay (NMD). Remarkably, one missense
mutation was identified. Previously reported missense changes all turned out to be
polymorphisms [12]. However, the p.Cys1042Tyr missense change in the current study is
most likely causal for the phenotype because of the de novo occurrence. Another
argument is that the mutation replaces a highly conserved cysteine residue in the pre-SET
domain of the Eu-chromatin Histon Methyl Transferase 1 protein. As predicted by the
pre-SET domain model, this mutation will strongly influence the local conformation the
pre-SET domain. The phenotype observed in this patient (patient 20) is fully compatible
with the 9qSTDS. She had severe psychomotor retardation, hypotonia and characteristic
facial profile with midface hypoplasia, synophrys and eversion of lower lip. It thus
appears that the p.Cys1042Tyr change has a dramatic effect on the protein function, and
may reflect an EHMT1 null-allele just like mutations in the other patients. A dominantnegative effect is not expected since complete absence of the protein is not believed to be
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14
compatible with life.[15] Further functional studies could give additional proof for
pathogenicity of this mutation.
Another novel category among the mutations, are the two splice site changes. Both
predicted to result in aberrant splicing and consequently, disruption of the open reading
frame. There were no cells available to test this further.
No significant differences were noted between previously reported patients with 9q
subtelomeric deletions and patients with EHMT1 mutations (tables 1 and 3), except for
high birthweight and childhood obesity.
Table 3: Total percentage of features reported in patients with intragenic EHMT1
aberrations reported in the current and in previous papers [10,12], in relation to
patients with deletions extending EHMT1 reported previously in literature.
Total
9q deletions
EHMT1 aberration
N= >50
N=10
%
n (%)
Features
Mental retardation
Hypotonia
Birth weight > P90
Childhood obesity
Heart defect
Genital defect (males)
Renal disorder
(incl. VUR)
Tracheomalacia
GER
Epilepsy
Behavioral abnormality
10 (100%)
10 (100%)
5 (50%)
5 (50%)
3 (30%)
2 (66%)
1 (10%)
100%
100%
10%
20%
30%
33%
14%
0
0
2 (20%)
8 (80%)
2%
2%
30%
10%
VUR= vesico-ureteral reflux; GER= gastro-esophageal reflux
However, the frequency of features such as birth weight and behaviour/psychiatric
aspects are not consistently reported in previous studies. In addition to the previously
recognised childhood obesity that is present in a higher frequency in the 9qSTDS [6], it
seems that birth weight tends to be higher as well. When considering also previously
reported data, high birthweight has been reported in approximately 10% of patients with
small 9q deletions and in 50% of patients with EHMT1 mutations (table 3). The
prevalence of heart defects is 30% in both cohorts. One patient with the p.Cys1042Tyr
had a pulmonic stenosis, one previously reported patient with the p.Arg1137X had a VSD
and the originally reported patient with translocation breakpoint through EHMT1 had an
ASD.[10,12] Another patient with a small deletion of 200kb, comprising only EHMT1
gene and possibly ARRDC1 had a tetralogy of Fallot.
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15
Seizures were present in 20% (mutation cohort) and in 30% (deletion cohort), presenting
in infancy or adolescence with tonic-clonic insults (TCI), absences or grand mal.
Epilepsy can present severely in the first year of life or later in childhood. Other features
mentioned were recurrent ear nose and throat infections (8 patients), persistence of
deciduous teeth (5 patients), pigmentation defect (1 patient), hearing loss (mixed or
perceptive in 6 patients) and pes equinovarus (1 patient). A variable spectrum of
behavioral abnormalities was observed in both patient categories. Frequently reported
behavioral problems in younger children are temper tantrums, severe sleep disturbances
and autistic features. However, sociable skills, friendliness and happiness were also
mentioned. A sudden and dramatic change in behavior and performance was observed
during adolescence in five of the six adult patients leading to psychiatric problems. These
patients were diagnosed with psychosis, bipolar mood disorder and/or autistic-catatonia.
Noteworthy, in two of them a transient or even permanent loss of functioning with loss of
skills as toilet training and the ability to independent eating occurred. Disturbance of
epigenetic gene regulation can result in regression of daily performance and cognitive
abilities as demonstrated in Rett syndrome [MIM_312750]. Regression is one of the
hallmarks of this condition that is caused by disturbance of chromatin modulation,
through mutations in the Methyl-CpG- Binding Protein 1 (MeCP2) gene. [16] It has been
shown that the MeCP2 protein thereby interacts with several interesting proteins, as the
ATRX protein, a SWI2/SNF2 DNA helicase/ATPase [17], and histone methyl transferase
Suv39H1. [16] To our knowledge, there is as yet no direct evidence as that any of these
proteins directly interact with EHMT1. However, it has been demonstrated that Suv39H1
is found in complexes with the paralog of EHMT1, the G9a protein [18], which in turn
forms functional complexes with the EHMT1 protein. [15] This raises the question
whether haploinsufficiency of EHMT1 similarly leads to regression in function and
behavior pattern albeit at a later age. Further detailed neuropsychiatric studies are needed
to get more insight into these features. Such information clearly will be of great
importance to parents and caregivers of 9qSTDS patients.
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16
Resources
Online Mendelian Inheritance in Man (chromosome 9q subtelomere deletion syndrome #610253; EHMT1
#607001):
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM&cmd=Limits
Unique (Rare Chromosome Disorder Support Group in the United Kingdom):
http://www.rarechromo.org/html/home.asp
University of Nijmegen (clinical EHMT1 sequencing and MLPA screening):
http://www.humangenetics.nl/en/diagnostics_en.php
Emory University (clinical EHMT1 sequencing):
http://www.genetics.emory.edu/testing/index.php
USC Genome Browser (Assembly March 2006):
http://genome.ucsc.edu
3-D EHMT1 (pre-)SET domain structure and Cys1042Tyr mutation:
http://www.cmbi.ru.nl/~hvensela/EHMT1/
Informed consent has been obtained for publication of all images present in this paper .
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17
Legends to the figures
Figure 1:
Schematic drawing of the 9q subtelomeric region and the different deletion sizes
identified. Indicated on the right the patients numbered corresponding to table 1, that
were shown to have the respective deletion. Relative positions of routine FISH and P070
MLPA probes are indicated.
Figure 2:
Domain structure of the EHMT1 protein and sequence identity of the pre-SET domain of
EHMT1, the paralogous human gene EHMT2 (G9a), and the mouse and Drosophila
orthologues. Note the highly conserved cysteine (arrow and underlined) that is mutated in
patient 20.
Figure 3:
3A: Location of the Cys1042Tyr mutant. Cysteine 1042 is shown in red and is one of the
cysteines in the cluster of cysteines (shown in yellow) that surround three zinc-ions
(green). Interactions of the cysteines with Zinc are indicated as grey cilinders. The cluster
is important for the correct position of the loops that interact with the other monomer
(light blue surface).
3B: Mutation of Cys1042Tyr. All cysteines in the cluster are coloured yellow, including
cysteine 1042. This residue is mutated into a tyrosine (red), other colours are the same as
the preceeding figure. The sidechain of tyrosine is shown to show the difference in size
with the cysteine sidechain.
Figure 4-7:
Photographs of patients showing the characteristic facial profile comprising
brachycephaly, hypertelorism, synophrys/arched eyebrows, midface hypoplasia,
protruding tongue, eversion of lower lip and prognathism of chin.
Informed consent has been obtained for publication of all images present in this paper .
Figure 4:
patients with EHMT1 mutations; patient 17 (AB), patient 18 (CD)
patient 19 (EF), patient 21 (GH), patient 20 (IJ )
Figure 5:
Patients 11 (ABC), 3 (DE) and 15 (FG) showing childhood obesity
Figure 6 and 7:
patients at different ages showing evolution with age.
Patient 12 (row A), patient 18 (row B), patient 13 (row C), patient 4 (row D), patient 8
(row E) and patient 7 (row F). Note asymmetric face in patient 8
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18
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"The Corresponding Author has the right to grant on behalf of all authors
and does grant on behalf of all authors, an exclusive licence (or non
exclusive for government employees) on a worldwide basis to the BMJ
Publishing Group Ltd to permit this article (if accepted) to be published
in Journal of Medical Genetics and any other BMJPGL products and
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cen
1
M
F
L
O
137.10
14
L
P
R
M
2F
A
R
T
138.90
91
D
N
Y
M
Z
139.56 139.59
P070
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139.85
FISH
140.13
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8,10,11
12,14,16
deleted
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Patients
Figure 2
Ankyrin
repeats
PreSET SET
1267
H.
M.
H.
M.
D.
sapiens
1027-QYCVCIDDCSSSNCMCGQLSMRCW-1050
musculus
1058-QYCVCVDDCSSSTCMCGQLSMRCW-1081
sapiens
971-QHCTCVDDCSSSNCLCGQLSIRCW-994
musculus
933-QHCTCVDDCSSSNCLCGQLSIRCW-956
melanogaster 1396-RICSCLDSCSSDRCQCNGASSQNW-1419
Cys1042Tyr
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EHMT1
Ehmt1
EHMT2
Ehmt2
dEHMT
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