Disruption of a synaptotagmin (SYT14) associated with neurodevelopmental abnormalities.

Abstract

We report cytogenetic and molecular studies of a de novo, apparently balanced t(1;3)(q32.1;q25.1) identified in a 12-year-old female (designated DGAP128) with cerebral atrophy, macrocephaly seizures, and developmental delay. A combination of fluorescence in situ hybridization (FISH) and Southern blot analysis demonstrated disruption of a synaptotagmin gene (SYT14) at the 1q32 breakpoint. Expression of SYT14 in human brain was confirmed using Northern analysis. Because members of the synaptotagmin family of proteins function as sensors that link changes in calcium levels with a variety of biological processes, including neurotransmission and hormone-responsiveness, SYT14 is an intriguing candidate gene for the abnormal development in this child. This is the first known constitutional rearrangement of SYT14, and further systematic genetic analysis and clinical studies of DGAP128 may offer unique insights into the role of SYT14 in neurodevelopment.

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? 2007 Wiley-Liss, Inc.American Journal of Medical Genetics Part A 143A:558–563 (2007)
Disruption of a Synaptotagmin (SYT14) Associated
With Neurodevelopmental Abnormalities
Fabiola Quintero-Rivera,1,4,5Alicia Chan,2Diana J. Donovan,3
James F. Gusella,1,4and Azra H. Ligon3,4*
1Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts
2Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada
3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts
4Harvard Medical School, Boston, Massachusetts
5Department of Pathology, The David Geffen School of Medicine at UCLA, Los Angeles, California
Received 28 July 2006; Accepted 6 November 2006
We report cytogenetic and molecular studies of a de novo,
apparently balanced t(1;3)(q32.1;q25.1) identified in a 12-
year-old female (designated DGAP128) with cerebral atro-
phy, macrocephaly seizures, and developmental delay. A
combination of fluorescence in situ hybridization (FISH) and
Southern blot analysis demonstrated disruption of a synapto-
tagmin gene (SYT14) at the 1q32 breakpoint. Expression of
SYT14 in human brain was confirmed using Northern
analysis. Because members of the synaptotagmin family of
proteins function as sensors that link changes in calcium
levels with a variety of biological processes, including neuro-
transmission and hormone-responsiveness, SYT14 is an
intriguing candidate gene for the abnormal development in
this child. This is the first known constitutional rearrangement
of SYT14, and further systematic genetic analysis and clinical
studies of DGAP128 may offer unique insights into the role of
SYT14 in neurodevelopment. ? 2007 Wiley-Liss, Inc.
Key words: reciprocal translocation; neurodevelopment;
seizures; synaptotagmin
How to cite this article: Quintero-Rivera F, Chan A, Donovan DJ, Gusella JF, Ligon AH. 2007.
Disruption of a synaptotagmin (SYT14) associated with neurodevelopmental abnormalities.
Am J Med Genet Part A 143A:558–563.
INTRODUCTION
The cytogenetic and molecular study of rare
individuals with apparently balanced chromosomal
rearrangements has been a successful approach to
identify genes causing an abnormal phenotype
[Krantz et al., 2004; Abelson et al., 2005]. Recently,
identification of constitutional rearrangements caus-
ing gene disruption or dysregulation also has
provided insight into potentially novel pathways
for known genes [Ligon et al., 2005].
DGAP128 is a 12-year-old female who carries a
de novo, apparently balanced rearrangement, initi-
ally reported as t(1;3)(q32.1;q25.1). Her clinical
evaluation was significant for macrocephaly, cere-
bral atrophy, seizures, and developmental delay.
Cytogeneticandmolecularapproacheswereusedto
map the translocation breakpoints in DGAP128, and
subsequently to uncover disruption of a known
gene, SYT14, which we propose contributes to the
abnormal phenotype.
CLINICAL BACKGROUND
DGAP128 was born at 39 weeks gestation by
normal spontaneous vaginal delivery, weighing
3,005 g and without any neonatal complications.
Her mother had two prior spontaneous pregnancy
losses and one healthy son. Her maternal grand-
mother and great aunts had a history of learning
difficulties and childhood seizures, the latter of
which resolved. Her mother’s occipital-frontal cir-
cumference (OFC) was enlarged at 58 cm (þ2 SD),
and this woman has mild bilateral 2–3 syndactyly of
Grant sponsor: NIH; Grant number: GM61354.
*Correspondence to: Azra H. Ligon, Ph.D., Brigham and Women’s
Hospital, Department of Pathology, Boston, MA 02115.
E-mail: aligon@rics.bwh.harvard.edu
DOI 10.1002/ajmg.a.31618

Page 2

the toes. Her father is 182.9 cm tall, weighs 99.8 kg,
and has an OFC within normal range.
Previously, both height and weight parameters for
DGAP met or exceeded the 95th centile, though on
most recent examination she was 160.2 cm (90th
centile) and her weight was 56.7 kg (90th centile).
She had an OFC of 55.5 cm (>þ2 SD). She was not
dysmorphic. A single telangiectactic lesion measur-
ing 3?1.5 cm was identified over her right flank.
There was mild bilateral 2–3 syndactyly of the toes,
identical to that of her mother. Deep tendon reflexes
were slightly brisk. The physical examination was
otherwise unremarkable.
The following investigations were all normal:
plasma amino acids, urine organic acids, liver
enzymes, cholesterol, 7-dehydrocholesterol, uric
acid, and molecular analysis for fragile X syndrome.
Her bone age was appropriate for her chronolo-
gical age. A head MRI showed diffuse atrophy,
especially of the frontal and parietal lobes with
possible mild cerebellar and vermian atrophy.
Ophthalmologic assessment revealed mild conver-
gence insufficiency type of exotropia requiring
no intervention. WISC III scores were 58, 57, and
64 for full scale, verbal, and performance IQ,
respectively. Her weak areas noted by her school-
teacher were in logic, analysis, planning, sequenc-
ing, and abstract thinking.
METHODS
Fluorescence In Situ Hybridization Analysis
Breakpoint mapping studies were initiated to map
precisely the chromosomal rearrangement using
fluorescence in situ hybridization (FISH). For this
purpose, a peripheral blood specimen was received
after informed consent was obtained in accordance
with institutional policies and procedures for
human research at Partners HealthCare System. Cell
transformation was performed at the Massachusetts
GeneralHospitalCenter
Research tissue culture resource using standard
protocols. Transformed lymphoblastoid cells were
thenculturedtogeneratesufficientmaterialforGTG-
banding and FISH-mapping using standard proto-
cols (see below).
Metaphase chromosome spreads were prepared
using conventional cytogenetic protocols [Ney et al.,
1993]. FISH was performed with directly labeled
cosmid or BAC probes. BAC or fosmid clones were
selectedusingtheUniversityofCaliforniaSantaCruz
Genome Browser (http://genome.ucsc.edu/; May
2004 freeze used for probe selection, March 2006
freeze used to confirm results) and were hybridized
simultaneously in pairs (SpectrumGreen and Spec-
trumRed/Orange; Abbott Molecular/Vysis, Inc., Des
Plaines, IL). At least 10 metaphases were studied per
hybridization.
forHumanGenetic
Southern Blot Analysis
Southern blotting was performed using standard
methods.RepeatMasker(http://www.repeatmasker.
org) was used to identify single copy sequences
for probe design. Genomic DNA from DGAP128
and from a phenotypically and karyotypically
normal control was digested with several restric-
tion enzymes (StuI, BglII, BlnI, EcoRI, HpaI, NcoI,
NsiI, SspBI; Webcutter: http://rna.lundberg.gu.se/
cutter2/)and hybridizedto a700bpprobeamplified
from within the sequence of BAC RP11-168F20. The
following primers were used to generate the probe:
(F) 50-TCTTCCCGATTTAGGGCATT-30and (R): 50-
CAAATGCAACTACAGCACATCA-30(Primer design:
http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_
www.cgi; http://www.ncbi.nlm.nih.gov/BLAST/).
Probe labeling was performed using the MegaPrime
labeling kit (Amersham/GE Healthcare, Piscataway,
NJ), following the manufacturer’s instructions.
Northern Blot Analysis
AcommerciallypreparedNorthernblot(Clontech,
MountainView, CA)ofpolyadenylated RNAisolated
from multiple regions of human adult brain (amyg-
dala, caudate nucleus, corpus callosum, hippocam-
pus, thalamus, and whole brain), was hybridized
with a probe representing a 683 bp region of the
30UTR of SYT14. Labeling, hybridization, and blot
washes were performed using standard methods, as
described above. The blot was normalized with
b-actin as a control (data not shown).
RESULTS
Translocation Breakpoint Mapping
FISH was performed with BAC and fosmid clones
(Table I, also see Supplementary Material) mapping
in the vicinity of the 1q32 breakpoint. Ultimately,
hybridization of BAC RP11-168F20 showed a signal
split between the derivative chromosomes 1 and 3
(Fig. 1), indicating that the 1q32 breakpoint maps
withinthisclone.RP11-168F20spans synaptotagmin
14 (SYT14), which is transcribed in a centromere to
telomere orientation and has two alternative pro-
moters that can produce five transcripts via alter-
native splicing [Craxton, 2004]. A relatively weaker
hybridization to the der(1) is consistent with the
majority of SYT14 being translocated to the der(3)
(Fig. 1). The 29 kb FISH-defined 1q32 breakpoint
interval was refined by Southern blot analysis
(Fig. 2A) to a 2,880 bp interval within intron 3 of
SYT14 (Fig. 2B).
Similarly, the 3q25 breakpoint was studied by
FISH (data not shown) and the critical interval
narrowed to ?383 kb, flanked proximally by RP11-
372M20 and distally by RP11-664C7, and including
no known genes or conserved non-genic (CNG)
SYT14 IN ABNORMAL NEURODEVELOPMENT
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sequences; Southern blot analysis was not per-
formedforthisbreakpoint.FollowingFISH-mapping
of DGAP128, the GTG-banded breakpoints were
revised and the rearrangement was designated as
t(1;3)(q32.2;q25.2)dn.
SYT14 Expression
Based upon previous mouse studies it has been
suggested that SYT14, unlike other synaptotagmins,
is expressed mainly in non-neural tissues [Fukuda,
2003]. Consequently, we examined the expression
pattern of SYT14 in human brain using a commer-
cially prepared Northern blot and a specific cDNA
probe amplified from within the 30UTR of SYT14. A
?2.9 kb full-length transcript (NM_153262) was
identified, as well as a smaller (?2.5 kb) related
transcript derived from a synaptotagmin pseudo-
gene(notshown),inthecorpuscallosum,amygdala,
caudate nucleus, hippocampus, and thalamus
(Fig. 3), supporting the view that SYT14 may
be responsible for the phenotype observed in
DGAP128.
DISCUSSION
The incidence of apparently balanced reciprocal
translocations is reported to be approximately 1/
500–1/2,000 live births, with a risk for congenital
anomalies twice that of an unselected population of
newborns [Warburton, 1991]. This increased risk is
generally attributed to disruption or dysregulation
of genes mapping at or near the individual break-
pointsor,insomecases,tosubmicroscopicdeletions
or duplications that occur in the vicinity of the
breakpoints [Kumar et al., 1998; Gribble et al.,
2005]. We report here on the results of molecular
cytogenetic analysis of DGAP128, a 12-year-old
female with multiple neurological abnormalities
including cerebral atrophy, absence seizures, and
developmental delay. Breakpoint-mapping of the
t(1;3)(q32.2;q25.2)demonstrateddirectdisruptionof
SYT14.
SYT14 belongs to a family of at least 15 related
mammalian genes [Andrews and Chakrabarti, 2005],
some of which are conserved in Drosophila mela-
nogaster, Caenorhabditis elegans, and Arabidopsis
thaliana [Craxton, 2004]. Synaptotagmins include
a transmembrane domain, linker domain, and a
cytoplasmic region comprised of tandem C2A and
C2B domains [Rickman et al., 2004]. As a group,
synaptotagmins can undergo both homo- and
hetero-oligomerization, either in a calcium depen-
dent or independent manner [Perin et al., 1991;
Littleton et al., 1994; Bai et al., 2000; Sudhof, 2002].
Calcium-dependent phospholipid binding specifi-
cally involves the C2 domains, both of which show
homologytotheregulatorydomainofproteinkinase
C and, in the case of SYT1, affect calcium-dependent
activation [Perin et al., 1990; Fernandez-Chacon
et al., 2001].
Fukuda[2003]reviewedsynaptotagminfunctionin
processes as diverse as peptide hormone secretion
[Gao et al., 2000], fertilization [Roggero et al., 2005],
and neuronal communication [Geppert et al., 1994;
Chapman, 2002; Sudhof, 2002]. While some synap-
totagmins show ubiquitous expression, vertebrate
synaptotagmins are expressed mainly in neurons
andneuroendocrinecells[Sudhof,2002].Expression
of synaptotagmins in such a diverse range of
organisms is consistent with evolution of a role that
may extend beyond neurotransmission [Craxton,
2004].
The best-described member of this family, SYT1,
encodes a synaptic vesicle membrane protein that
responds to calcium influx and mediates the rapid
TABLE I. BAC and Fosmid Clones Used to Map DGAP128 Breakpoints
BreakpointCloneAccession number Signal position
1q32RP11-148K15
RP11-57I17
RP11-45F21
RP11-167J2
RP11-168F20
RP11-216F1
RP11-91C15
RP11-123O6
RP11-91I7
G248P85272B11
G248P88070E8
RP11-362A9
RP11-89L22
RP11-372M20
RP11-664C7
RP11-451C20
RP11-270G15
AQ374947
AL137789
AL355533
AQ421322
AL355335
BZ749095
AZ518690
BZ749030
AL691441
—
—
AC026347
AQ286615
AC107423
AQ433955
AQ583106
AC106724
Centromeric to break
Centromeric to break
Centromeric to break
Centromeric to break
CROSSES break
Telomeric to break
Telomeric to break
Telomeric to break
Telomeric to break
Telomeric to break
Telomeric to break
Centromeric to break
Centromeric to break
Centromeric to break
Telomeric to break
Telomeric to break
Telomeric to break
3q25
Representative BAC and fosmid clones used to map the translocation breakpoints in DGAP128. Accession numbers are
indicated, where available, as well as the relative map location of each clone with respect to a given breakpoint. Note that
BAC RP11-168F20 crosses the 1q32 breakpoint.
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release phase of presynaptic vesicle exocytosis,
thus serving a critical function in neurotransmission
[Perin et al., 1990; Geppert et al., 1994]. An essen-
tial step in neuronal communication is release of
neurotransmitters from presynaptic vesicles, a pro-
cess mediated by specific synaptotagmins and
associated proteins [Davis et al., 1999; Fernandez-
Chacon et al., 2001; Bai et al., 2004].
Study of murine and rat Syt proteins has provided
additional insight into synaptotagmin function
and plasticity. For certain synaptotagmins (e.g.,
Syt1 and Syt7), alternative splicing has been demon-
strated [Perin et al., 1990; Sugita et al., 2001]. Syt7
encodes a thyroid hormone-responsive protein
that may play a role in thyroid-regulated develop-
ment of the central nervous system [Thompson,
1996]. Interestingly, Syt4 has been implicated in
hippocampal-based memory mediated by calcium-
dependent glutamate release [Zhang et al., 2004].
Though Syt4?/? mice lack any gross phenotypic
abnormalities, hippocampal-dependent
and memory, as well as fine motor coordination,
appear to be compromised.
Murine Syt14 can undergo calcium-independent
oligomerization and phospholipid association, and
structural analyses suggest a role in membrane
transport that is conserved across several phyla
[Fukuda, 2003]. Additional diversity might be
achieved through differential SYT14 splicing [Crax-
ton,2004].Becausebraintissuewasunavailablefrom
DGAP128, it was not feasible to assess SYT14
expression in this individual. However, a Northern
blot analysis of normal human brain demonstrated
expression of a 2.9 kb SYT14 mRNA (Refseq:
NM_153262) in adult amygdala, caudate nucleus,
corpus callosum, hippocampus, and thalamus.
Interestingly, microarray data (U133A and GNF1H
chips, UCSC Genome Browser) indicate SYT14
expression in the atrioventricular node, trachea,
salivary gland, ovary and fetal liver, suggesting a
function for SYT14 that may not limited to brain
[Fukuda, 2003; Su et al., 2004].
Given the critical role of certain synaptotagmins in
neurotransmissionandthedemonstratedexpression
of SYT14 in human brain, the possibility that
disruption of one SYT14 allele contributes to the
neurodevelopmental abnormalities in DGAP128 is
intriguing. Potential mechanisms by which the
translocation might contribute to the DGAP128
phenotypeincludehaploinsufficiencyorunmasking
of a recessive allele. Furthermore, the mother’s large
OFC suggests that macrocephaly in DGAP128 likely
reflects a familial trait that is unrelated to the
chromosomal translocation. Those maternal rela-
tives with seizures reportedly did not have absence
seizures,asdocumentedforDGAP128,andsowould
not exclude a role for disruption of a neurodeve-
lopmentally critical gene by the t(1;3) in pathogen-
esis of the DGAP128 seizure phenotype. Currently
learning
FIG. 1. Metaphase FISH with RP11-168F20 showing hybridization to the
normal chromosome 1, der(1) and der(3).
SYT14 IN ABNORMAL NEURODEVELOPMENT
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there are no reports of individuals with 1q32
deletions known to include SYT14, with similar or
overlapping phenotypes. Similarly, though a murine
knockoutmodelforSYT14doesnotcurrentlyexist,it
willbe animportantstepinfurtherassessing therole
of SYT14 in this neurodevelopmental phenotype.
Finally,thelackofanyknowngenesdisruptedbythe
3q25.2 breakpoint does not exclude possible dis-
ruption of a regulatory element or of position effect.
As such, our data provide an impetus for further
studiesexploringtheroleofSYT14inneurodevelop-
ment and for seeking potential disruption of this
locusinotherindividualswithsimilaroroverlapping
phenotypes.
ACKNOWLEDGMENTS
The authors wish to acknowledge the patient and
her family for their participation in the Develop-
mental Genome Anatomy Project (DGAP), and the
assistanceofHeatherFerguson,M.S.,C.G.C.,Chantal
Kelly, M.S., Tara Dzwiniel, M.S., C.G.C., Robert
Eisenmann, B.S., David J. Harris, M.D., and Gail
Bruns, M.D., Ph.D.
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