Epilepsia, **(*):1–10, 2009

doi: 10.1111/j.1528-1167.2009.02066.x

FULL-LENGTH ORIGINAL RESEARCH

Partial epilepsy syndrome in a Gypsy family linked to

5q31.3-q32
*1Dora Angelicheva, yz1Ivailo Tournev, yVelina Guergueltcheva, yVioleta Mihaylova,
*Dimitar N. Azmanov, *Bharti Morar, yMelania Radionova, xShelagh J. Smith,
{Dora Zlatareva, xJohn M. Stevens, #Radka Kaneva, yVeneta Bojinova, **Kim Carter,
yyMatthew Brown, zzAssen Jablensky, *1Luba Kalaydjieva, and
x,xx1Josemir W. Sander
*Laboratory for Molecular Genetics, Centre for Medical Research and Western Australian Institute for Medical
Research, The University of Western Australia, Perth, Australia; yDepartment of Neurology, Medical University,
Sofia, Bulgaria; zDepartment of Cognitive Science and Psychology, New Bulgarian University, Sofia, Bulgaria;
xDepartment of Clinical and Experimental Epilepsy, UCL Institute of Neurology, London, United Kingdom;
{Department of Radiology, Tokuda Hospital, Sofia, Bulgaria; #Molecular Medicine Centre, Medical University,
Sofia, Bulgaria; **Western Australian Cardiovascular Genetics Group, Telethon Institute for Child Health
Research, Perth, Australia; yyCentre for Immunology and Cancer Research, Princess Alexandra Hospital,
The University of Queensland, Brisbane, Australia; zzSchool of Psychiatry and Clinical Neurosciences,
The University of Western Australia, Perth, Australia; and xxSEIN – Epilepsy Institute of The Netherlands
Foundation, Heemstede, The Netherlands

Results: We observed an early-onset partial epi-

SUMMARY
Purpose: The restricted genetic diversity and
homogeneous molecular basis of Mendelian disor-
ders in isolated founder populations have rarely
been explored in epilepsy research. Our long-term
goal is to explore the genetic basis of epilepsies in
one such population, the Gypsies. The aim of this
report is the clinical and genetic characterization
of a Gypsy family with a partial epilepsy syndrome.
Methods: Clinical information was collected using
semistructured interviews with affected subjects
and informants. At least one interictal electroen-
cephalography (EEG) recording was performed
for each patient and previous data obtained from
records. Neuroimaging included structural mag-
netic resonance imaging (MRI). Linkage and haplo-
type analysis was performed using the Illumina IVb
Linkage Panel, supplemented with highly informa-
tive microsatellites in linked regions and Affyme-
trix SNP 5.0 array data.
lepsy syndrome with seizure semiology strongly
suggestive of temporal lobe epilepsy (TLE), with
mild intellectual deficit co-occurring in a large
proportion of the patients. Psychiatric morbidity
was common in the extended pedigree but did
not cosegregate with epilepsy. Linkage analysis
definitively excluded previously reported loci, and
identified a novel locus on 5q31.3-q32 with an loga-
rithm of the odds (LOD) score of 3 corresponding
to the expected maximum in this family.
Discussion: The syndrome can be classified as
familial temporal lobe epilepsy (FTLE) or possibly
a new syndrome with mild intellectual deficit. The
linked 5q region does not contain any ion channel–
encoding genes and is thus likely to contribute new
knowledge about epilepsy pathogenesis. Identifi-
cation of the mutation in this family and in addi-
tional patients will define the full phenotypic
spectrum.
KEY WORDS: Founder populations, Partial epi-
lepsy, Linkage analysis.

Accepted January 6, 2009; Early View publication Xxxxx xx, 200x

Address correspondence to Prof. Ley Sander, Box 29, Clinical Neuro-
sciences Centre, UCL Institute of Neurology, Queen Square, London
WC1N 3BG, United Kingdom. E-mail: lsander@ion.ucl.ac.uk
1
These authors have contributed equally to the work.
Wiley Periodicals, Inc.
ª 2009 International League Against Epilepsy
Epilepsy is a common neurologic disorder, with preva-
lence in some resource-poor settings of up to 1% (Sander,
2003; Duncan et al., 2006). Although exposure to pre-,
peri- and postnatal risk factors is likely to contribute to an
increased prevalence in some communities, there is

1
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D. Angelicheva et al.
growing interest in the role of specific genetic factors.
Current understanding of the molecular basis of epilepsies
is mostly confined to studies of rare large pedigrees with
Mendelian forms of the disorder and mutations of large
effect (recently reviewed by Helbig et al., 2008).
Isolated founder populations have contributed to the
understanding of the molecular basis of hereditary disor-
ders (Peltonen et al., 1999; Ostrer, 2001; Arcos-Burgos &
Muenke, 2002), especially of autosomal recessive condi-
tions; their potential has, however, rarely been explored in
genetic studies of epilepsy. Mendelian forms of epilepsy
are mostly autosomal dominant; however, the lack of
effect on reproductive fitness makes it likely that founder
mutations do exist and are shared between ostensibly
‘‘unrelated’’ affected families, as documented for other
autosomal dominant disorders (Risch et al., 1995; John
et al., 2007). We are investigating the genetics of epilepsy
in the Bulgarian Roma/Gypsies, whose genetic structure,
single-gene disorders, and founder mutations we have
studied for more than a decade (Kalaydjieva et al., 2001,
2005). The Gypsies are a young isolate of Asian ancestry
with a history of demographic bottlenecks, strong founder
effects, and a preserved tradition of large extended fami-
lies, all of which facilitates genetic research (Kalaydjieva
et al., 2005). Our previous findings of restricted diversity
led us to hypothesize that the genetic basis of epilepsy may
be less heterogeneous than in outbred populations, with a
limited spectrum of founder mutations accounting for a
large proportion of affected multiplex families and possi-
bly also for seemingly unrelated ‘‘sporadic’’ cases, usually
classified as genetically complex forms. Here we report
the first results of this study—a focal epilepsy syndrome in
a Gypsy family, linked to a novel locus on chromosome 5q.
Subjects and Methods
General study design
Case ascertainment relied on hospital admissions to
neurology departments (adult and pediatric) of the Medi-
cal University in Sofia, general practitioners working in
Gypsy neighborhoods, and the network of health media-
tors—members of Gypsy communities trained in primary
health care. Probands with a documented or suspected
family history of epilepsy were recruited to the study and
other family members were approached. Written informed
consent was obtained from all adult subjects and from
guardians of minors. The project has been approved by,
and complies with, the guidelines of the Ethics Commit-
tees of the Medical University in Sofia, the University of
Western Australia, and University College London.
Data on group (subisolate) identity of affected families
were collected through interviews with key informants.
Family trees were based on multiple interviews, with
particular attention to consanguinity, possible multilineal
inheritance, and relatedness between affected families.
Epilepsia, **(*):1–10, 2009
doi: 10.1111/j.1528-1167.2009.02066.x
Phenotype characterization relied on hospital admis-
sions of key cases from each family, and extensive field-
work involving face-to-face interviews with patients and
at least one key informant for each affected individual.
Information on unaffected family members was also
collected from key informants. Repeat interviews were
conducted to check for consistency and to update informa-
tion. Data on seizure history, developmental history, risk
factors, and medical and psychiatric comorbidity were
collected using a semistructured interview. Affected indi-
viduals were screened for current and past psychopathol-
ogy (anxiety and panic disorders, depression, mania,
psychotic symptoms, and disorders of memory and senso-
rium). Cognitive ability was assessed using a modified
version of the Mini Mental State Examination (MMSE),
adapted for population groups with high rates of social
and educational deprivation. All data were evaluated by
consensus between the two senior clinical investigators.
At least one awake interictal electroencephalography
(EEG) recording with hyperventilation was obtained for
each affected individual, and previous findings were
retrieved from medical records. Digital EEGs were
acquired in accordance with international standards on the
PL-EEG Wavepoint Medtronic (Minneapolis, MN,
U.S.A.) system with 25 electrode inputs (21 scalp
electrodes, system reference, ground, A1 and A2), and
analyzed independently by two neurophysiologists.
Brain magnetic resonance imaging (MRI) was per-
formed using a 1.5T MR imager (MR Signa, GE Health-
care, Milwaukee, WI, U.S.A.). The examination protocol
consisted of five series: sagittal T1-weighted fluid-attenu-
ated inversion recovery (FLAIR); coronal T1-weighted
three-dimensional (3D) fast spoiled gradient echo
(FSPGR), T2 FLAIR, and T2-weighted fast relaxation fast
spin echo sequence (FRFSE) and axial T2-weighted
FRFSE. The images were independently reviewed by two
radiologists.
The family reported in this study
The S1 family (Fig. 1) is the first family enrolled in
the study and has been followed since 2004. A total of
21 subjects from two generations participated, of whom
10 (seven males and three females, aged 10–40 years)
were affected. Several additional members of this
extended family (unaffected by epilepsy), for example,
2–2, 2–3, and so on (Fig. S1), declined participation in
the genetic study. In the previous generation, the father
(deceased) was reported by several informants to have
been affected. Other relatives, in more distantly related
branches along the paternal line, were also described as
affected (including one who died in status epilepticus)
but were not available for investigation. No consanguin-
eous marriages were reported. The pedigree structure
indicated autosomal dominant inheritance with incom-
plete penetrance.
 3
Partial Epilepsy in a Gypsy Family

Figure 1.
Pedigree structure of the S1 family with 5q31.3–q32 haplotypes. The drawing includes only individuals participating in
the genetic study. Affected subjects are represented with shaded symbols, and the legend defines the shading code.
For deidentification purposes, the numbering does not follow birth order. The haplotypes in the figure include
selected microsatellites and single nucleotide polymorphisms (SNPs), illustrating the shared region and the key
recombinations.
Epilepsia ILAE

Genetic analyses
A genome-wide scan was performed with the Illumina
Linkage Panel IVb, comprising 6,008 single nucleotide
polymorphisms (SNPs), 5,663 of which are autosomal.
Genotyping data quality was checked using the Illumina
BeadStudio software (Illumina Inc., San Diego, CA,
U.S.A.), with additional visual inspection of markers with
GenTrain or ClusterSeparation scores £0.75. Mendelian
inconsistencies and improbable recombinations were
checked using the error detection function of Merlin v1.0
(Abecasis et al., 2002), and markers showing genotyping
errors were excluded from subsequent analyses.
For the fine mapping of linked regions, we genotyped
18 highly informative microsatellite markers (Table S1).
Polymerase chain reactions (PCRs) were performed under
standard conditions, with length separation of products on
an ABI377 DNA Analyzer or a Hitachi FMBI0II fluores-
cent scanner (Hitachi Software Engineering America,
Ltd., San Francisco, CA, U.S.A.). Further fine mapping of
the 5q region was done with 23 SNPs, genotyped with the
Affymetrix Genome-Wide SNP5.0 GeneChip array in
samples representing the conserved and the recombinant
haplotypes.
Linkage analysis was performed using Merlin v1.0
(Abecasis et al., 2002) under a dominant model with
incomplete (0.7) penetrance, gene frequency 0.0001, and
marker allele frequencies calculated with the Mendel
program (Lange et al., 2001) by maximum likelihood esti-
mates from the genotyping data. The calculations were
done with phenocopy rates varying between 0.001 and
0.01. The genetic map was as provided by Illumina
(11186808_Linkage_IV_B). In the linkage analysis,
family members were defined as affected based on pres-
ence of seizures. Unaffected subjects, including spouses,
were considered unknown. The expected maximum two-
point LOD score in the family, calculated for the same

Epilepsia, **(*):1–10, 2009

doi: 10.1111/j.1528-1167.2009.02066.x
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D. Angelicheva et al.
model parameters through general simulation program for
linkage analysis (SLINK) simulations (Ott, 1989; Weeks
et al., 1990) was 2.96 for multiallelic markers (lower for
the less informative SNPs). Haplotypes were constructed
using the Merlin haplotyping function and visualized with
HaploPainter (Thiele & Nurnberg, 2005). The relative
physical positions of markers from the three datasets
(Illumina, microsatellites and Affymetrix) were verified
in the reference sequence (NCBI NT_029289), and
genetic positions were obtained from public databases or
determined by interpolation (for new short tandem repeat
[STR] markers).
Sequencing of candidate genes was performed in three
affected subjects (2–8, 3–22, and 3–23), an unaffected
spouse (2–19), and two normal controls from the same
population, using BigDye Terminator v3.1 (Applied
Biosystems, Foster City, CA, U.S.A.). Exons, flanking
introns, and untranslated regions of brain-specific trans-
cripts were PCR-amplified and sequenced with flanking
and internal primers (Table S1). Sequencing reactions
were resolved on an ABI377 DNA Analyzer and examined
using the BioEdit software (http://www.mbio.ncsu.edu/
BioEdit/bioedit.html).
Results
Phenotype description
The clinical data on the 10 affected individuals are sum-
marized in Table 1, with detailed descriptions available as
Supporting Information on-line. The age at onset of con-
vulsions was in childhood (1–12 years), consistently
between 1 and 3 years in the third generation. In all cases,
the first seizure was described as convulsive, although
other seizure types may have gone unrecognized. Uncer-
tain data on febrile illness during initial convulsions were
obtained in three patients. Consistent information on com-
plex partial seizures, commonly characterized by oroali-
mentary and motor automatisms, was obtained in eight
affected individuals. Secondary generalization was
observed in all 10 individuals (description uncertain in
one), with a frequency of 1–3 per month. Aura was auto-
nomic in five cases, epigastric in four, and somatosensory
in four; cephalic, dyscognitive, illusory, and auditory
warning signs were also described. Interictal EEGs were
abnormal in five patients, with localization mostly sug-
gesting temporal lobe involvement, and abnormalities also
recorded in the centroparietal and frontal regions (Fig. 2,
Table 1 and Supporting Information). Antiepileptic drugs
had been prescribed, but it is probable that adherence to
treatment had been poor and some patients had never been
treated. Neuroimaging revealed no structural lesions or
asymmetries. Developmental delay and/or learning diffi-
culties (commonly attributed to memory problems and
school drop-out) were reported in seven patients, of whom
four (subjects 2–8, 3–18, 3–23, and 3–26) had mild to
Epilepsia, **(*):1–10, 2009
doi: 10.1111/j.1528-1167.2009.02066.x
moderate intellectual deficit. Psychiatric comorbidity was
present in five subjects: bipolar affective disorder (1),
panic disorder and mild recurrent depression (1), alcohol
dependence with subdelirious psychotic episodes and
organic personality change (1), and attention deficit
hyperactivity disorder (ADHD) (2). Alcohol or drug
abuse, anxiety and depression, and ADHD were also pres-
ent in several unaffected members of the pedigree
(Fig. S1) and did not show a pattern of cosegregation with
epilepsy.
Genetic analyses
The average genotype call rate for the genome scan was
0.992. Forty-six markers (0.8% of the total number of
autosomal markers) were removed from further analysis,
based on call rates <0.9 and/or problematic clustering (21
markers) and Mendelian inconsistencies and/or double
recombinations (25 markers).
Linkage analysis excluded all previously reported epi-
lepsy loci (Fig. 3 and Table S2) and identified two positive
regions, on chromosomes 5q31.3-q32 and 10p12 (Fig. 3).
In the 5q region, spanning 10.6 cM (7.88 Mb), defined by
rs2053055 on the centromeric and rs1016256 on the telo-
meric side, the highest LOD score was 3.003 at rs33388,
located at 144.52 cM. This corresponds to the maximum
LOD score expected in this family under the genetic
model and family structure described. Variations in the
phenocopy rate from 0.1% to 1% did not change signifi-
cantly the LOD score (difference of 0.05). Independent
linkage analysis of the microsatellite markers, genotyped
in the region, produced LOD scores of 3.00–3.01 for all
markers in the 141.8–148.82 cM interval between
5q31_28TG and D5S638 (map positions specified in
Table S1).
The combined SNP and microsatellite haplotype, co-
segregating with the disease, was inherited by all affected
individuals in family S1, as well as by individual 3–17,
who had mental retardation but no seizures. It was not
found in subject 2–4, diagnosed as having organic brain
disorder (Supporting Information). Haplotype examina-
tion identified a key centromeric recombination in indi-
vidual 3–25 and a telomeric recombination in individual
3–27 (Figs. 1 and 4). Fine mapping placed the centromeric
recombination in the interval between 5q31_18CA and
5q31_28TG, and the telomeric between rs728937 and
D5S434, reducing the size of the critical gene region to
8.47 cM or 5.7 Mb. Further breakpoint mapping, using
the Affymetrix SNP5.0 array, shifted the telomeric bound-
ary proximal to rs7709303 and decreased slightly the size
of the interval to 7.80 cM or 5.6 Mb (Fig. 4), but did not
contribute new information for the centromeric recombi-
nation.
A second, minor linkage peak was observed on chromo-
some 10p12, spanning 6.34 cM (3.9 Mb) between
rs1928365 and rs913034 (both flanked by uninformative
 Table 1. Clinical characteristics of the partial epilepsy syndrome in family S1
Year Seizure Simple
Case of onset partial Complex partial Brain
ID birth Sex (age) FS seizures seizures Aura features Generalization Semiology EEG imaging Comorbidity
2–6 1968 F 7 years None None None reported Epigastric 1–2/month (daytime) T 2006 & 2007 normal MRI normal Hypertension,
reported reported obesity, panic
attacks
2–8 1972 M 2 years None None Ambulatory automatisms Somatosensory, 2/month (daytime) T/E-T 2007 normal MRI normal Alcohol abuse,
reported reported (runs out into the street, autonomic, Mixed subdelirious
urinates; disoriented) dyscognitive partial episodes; organic
personality
change; mild ID
2–10 1975 F 12 None None Ambulatory (ran out into Somatosensory, 1–3/month (daytime, T/E-T 2001 F-T sharp-slow CT normal Bipolar disorder;
years reported reported the street naked); epigastric, possibly also Mixed wave discharges; obsessive-
oroalimentary auditory nocturnal) partial generalization 2006 compulsive
automatisms R&L F sharp waves features
3–16 1989 M 3 years None None Manual automatisms (pulls Autonomic, 1/month (daytime) T 2004 R P-T spike-slow CT&MRI None reported
reported reported down curtains, breaks epigastric, waves 2006 L F-T normal
glasses, grabs things); dyscognitive theta; R & L F spikes
dysphasic automatisms
(talks incomprehensively)
3–18 1988 M 2 years Possible None Ambulatory automatisms None reported Uncertain T 2006 normal MRI normal Moderate ID,
reported (runs around, waving congenital limb
his hands), disoriented malformation
3–22 1995 F 1½ Possible Cephalic, Orofacial and mimetic Somatosensory 2–3/month (both T 2006 R&L C-P sharp CT & MRI Delayed
year epigastric, automatisms (dizziness), daytime and waves 2007 R T normal developmental
illusory oroalimentary nocturnal) sharp–slow waves milestones;
stammer
3–23 1997 M 1 year Possible None Uncertain Aura present, < 1/month (daytime T 2006 R C-T focal CT & MRI Delayed
reported description and nocturnal) abnormalities; 2006 normal developmental
uncertain & 2007 normal milestones.
Mild ID
3–25 1998 M 1 year None None Manual and ambulatory Autonomic 1/week E-T 2006 Bilateral CT & MRI ADHD (constant
reported reported automatisms (running and polyspike waves; R F normal agitation,
screaming, pulling objects) focus 2007 normal aggressive
behaviour)
3–26 1995 M 1 year None None Ambulatory, (‘‘running Autonomic, 1/week T 2005, 2006, 2007 MRI normal Mild ID
reported reported around like a horse’’) and epigastric, normal
vocal (screaming) mimetic
automatisms
3–27 1992 M Infancy None None Dyscognitive (incoherent Somatosensory 1–2/month (both Mixed 2004, 2006, 2007 CT & MRI Learning
reported reported speech); interactive cephalic, daytime and partial normal normal (memory)
automatisms (talks to self, illusory, nocturnal) difficulties
grimacing) autonomic
Partial Epilepsy in a Gypsy Family

T, temporal; E-T, extratemporal; R, right; L, left; C, central; F, frontal; P, parietal; ID, intellectual deficit; FS, febrile seizures.

doi: 10.1111/j.1528-1167.2009.02066.x
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D. Angelicheva et al.

Figure 2.
(A) Case 3–25. Age 8 at time of interictal electroencephalography (EEG) recording. The EEG shows an isolated burst
of generalized spike-wave discharge. There were no focal findings in this trace. (B) Case 3–22. Age 13 years.
Interictal recording showing biparietal focal abnormalities: paroxysmal slow complex on the left (P3), and low
amplitude sharp transients on the right (P4).
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Partial Epilepsy in a Gypsy Family

Figure 3.
Genome-wide multipoint parametric linkage analysis results. Individual chromosomes are separated with dashed
vertical lines. Genotyping was done using the Illumina Linkage Panel IVb. Linkage analysis was performed with Merlin
v1.0.
Epilepsia ILAE

Figure 4.
The region on 5q31.3–q32, showing linkage to the partial epilepsy syndrome in family S1. Markers are presented
following their physical map order from centromere to telomere. Single nucleotide polymorphism (SNP) alleles
correspond to the variant on the positive strand, as shown in dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/).
Microsatellite alleles are numbered in ascending size order. The key recombinations in subjects 3–25 and 3–27 are
shown as filled bars. The 13 brain-expressed genes, contained in the linked region, are shown at the bottom of the
figure.
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D. Angelicheva et al.
markers). The highest LOD score was 0.94, at rs997462
(51.93 cM). Genotyping and independent linkage analysis
of more informative microsatellite markers resulted in
negative LOD scores ()1.7 to )3.8), excluding 10p12 as a
potential modifying locus.
According to the current build 36.3 of the human gen-
ome, the 5.6 Mb interval on 5q31.3–32 contains 31
validated and predicted genes, of which 13 are expressed
in brain (Fig. 4). Three were ranked as the best functional
candidates: Potassium Channel Tetramerization Domain
16 (KCTD16), Fibroblast Growth Factor (FGF1) and
Sprouty homologue 4 (SPRY4). Sequencing analysis of
the coding and untranslated regions of KCTD16 identified
10 known polymorphisms listed in the public databases
(rs3797084, rs187034, rs349704, rs349703, rs358651,
rs349701, rs349700, rs349699, rs349698, rs349697) and
three novel sequence variants—a synonymous coding
SNP (S303S; c.909C>T) and two changes in the 3¢UTR
(c. 1287 + 15C>T and c. 1287 + 3273 G>A). Lack of co-
segregation with the disease phenotype and presence
in the unaffected controls ruled out their role as disease-
causing mutations.
Discussion
Epilepsy is among the best examples of extensive
genetic and clinical heterogeneity, with multiple loci,
incomplete penetrance, low-grade genotype–phenotype
correlations, and a limited number of large informative
pedigrees, all contributing to the difficulty in elucidating
its molecular basis. Founder populations could facilitate
research efforts if their restricted diversity and shared
ancestral mutations also apply to epilepsy. Our project
aims to test this possibility in the Gypsy population, where
our previous studies have shown a strong founder effect
and allowed assessment of the spectrum of clinical mani-
festations in genetically homogeneous groups (Tournev
et al., 1999; Thomas et al., 2001; Colomer et al., 2006;
Mihaylova et al., 2007). The well-known constraints on
conducting genetic research into epilepsy, such as wide-
spread fear of stigmatization and the difficulty in eliciting
‘‘crisp’’ phenotypes, were also encountered in this study.
They were augmented by the social marginalization of the
Gypsies, and the need for adapting the clinical investiga-
tions to the cultural tradition, concepts, and beliefs of this
population, as well as educational and economic depriva-
tion. In the course of the project, we have improved data
collection by fine-tuning the interview and adapting it to
the cultural idiom, and by using multiple sources of infor-
mation and repeat interviews on each affected subject.
The quality of information remained uncertain regarding
several specific clinical parameters: a history of febrile
convulsions has proved very difficult to verify, and the
timing of specific events, particularly age at onset of sei-
zures, was occasionally discrepant, with a difference of
Epilepsia, **(*):1–10, 2009
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several years in the reports of different informants.
Another feature that requires cautious interpretation is
intellectual disability, where an adverse social environ-
ment and lack of culturally fair psychological tests may
significantly affect diagnostic assessment. We are aware
of the risk of such bias and have conservatively
constrained our interpretation of those aspects of the
phenotype to verifiable facts. The data on the remaining
key aspects of the clinical phenotypes, however, were
usually consistent and reliably reproduced in several
interviews.
To summarize, we have identified a Gypsy family with
an epilepsy syndrome of autosomal dominant inheritance
and reduced penetrance, with onset in the first decade of
life, complex partial seizures, and frequent secondary
generalization. The complex partial seizures had mostly
temporal lobe characteristics, but in some cases extra-
temporal phenomenology was also observed. Interictal
EEG abnormalities were present in half of the patients
(Fig. 2), and at some stage localized in the temporal or
adjacent areas. Discharges with frontal localization or a
frontal maximum were observed in three patients, two of
whom (2–10 and 3–16) had frontotemporal and parieto-
temporal abnormalities in previous EEGs and seizure
semiology most characteristic of temporal lobe epilepsy
(Table 1 and Supporting Information). Brain imaging
showed no structural abnormalities or asymmetries.
Intellectual disability in the mild to moderate range
meeting ICD-10 criteria was established in 4 of 10
subjects with epilepsy, and, with certainty, in only one of
the unaffected individuals, thus suggesting a possible
cosegregation with the epileptic syndrome. Other psychi-
atric and behavioral morbidity in this family, although
common, did not appear to cosegregate with seizure
disorders (Fig. S1).
This condition does not resemble partial epilepsy with
pericentral spikes (PEPS) (Kinton et al., 2002) and the
differential diagnosis includes familial temporal lobe
epilepsy (FTLE) and familial partial epilepsy with vari-
able foci (FPEVF), both showing clinical as well as
genetic heterogeneity (Berkovic et al., 1996, 2004; Cen-
des et al., 1998; Scheffer et al., 1998; Xiong et al., 1999;
Gambardella et al., 2000; Picard et al., 2000; Callenbach
et al., 2003; Claes et al., 2004). Individual members in
FPEVF families are described as having a distinct elec-
troclinical phenotype, with congruence between clinical
and EEG localization. The most common seizure type in
some families appears to be nocturnal FLE. Here the
affected individuals do not have such distinct electro-
clinical phenotypes; some may have mixed seizure types
(extratemporal and temporal) and their EEG focus tends
not to be specifically congruent with seizure semiology.
The lack of ictal EEG recordings makes it difficult to
establish a more definitive syndromic diagnosis.
Although the preceding differential diagnoses refer to

forms of idiopathic partial epilepsy, it is possible that
the syndrome in family S1 also includes a raised inci-
dence of intellectual deficit. The interpretation of our
findings should take into account a number of possible
confounders: the generally high frequency of affective
and anxiety disorders in epilepsy patients (Kanner, 2007,
2008), a social environment that could lead to dysthymic
depression and dropping out of school, and widespread
alcohol abuse in this population. The identification of
the disease mutation and of additional patients carrying
the same mutation should contribute to a more precise
definition of the full phenotypic spectrum.
Our genetic analysis excluded all previously reported
loci, including those linked to FTLE and FPEVF (10q24,
4q13.2-q21.3, 12q22-q23.3, 18qter and 1q25-q31, 2qter,
22q11-q12) with LOD scores lower than )3 (Fig. 3 and
Table S2).
The results tentatively place the disease gene in a
5.6 Mb interval on 5q31.3-q32, centromeric of a cluster of
GABA-A (c-aminobutyric acid A) receptor genes, the
involvement of which was excluded by multiple recombi-
nations in the S1 family. The best candidate genes within
the 5q locus include KCTD16, FGF1, and SPRY4. The
choice of Potassium Channel Tetramerization Domain 16
gene (KCTD16) as a candidate gene is based on its pattern
of brain expression in the amygdalae, caudate nucleus,
and hippocampus (Nagase et al., 2000) and on the involve-
ment of another member of the same gene family, KCTD7,
in autosomal recessive progressive myoclonic epilepsy
with dementia (Van Bogaert et al., 2007). Although no
disease-causing mutation was identified in the KCTD16
coding sequence, our current data do not rule out the possi-
bility of an intronic mutation causing aberrant splicing or
affecting a regulatory sequence. Further analysis is in pro-
gress. The acidic Fibroblast Growth Factor (FGF1) has
been found to suppress convulsions in kainate-induced
epilepsy in rats (Cuevas & Gimenez-Gallego, 1997), pro-
mote the survival of hippocampal pyramidal neurons after
ischaemia (Sasaki et al., 1992), and enhance long-term
potentiation (Sasaki et al., 1994; Reuss & von Bohlen und
Halbach, 2003). The Sprouty 4 gene (SPRY4) is involved
in the regulation of FGF function via inhibition of the
downstream ras/MAP kinase signaling pathway (Leeksma
et al., 2002). Given the lack in the 5q locus of obvious epi-
lepsy candidate genes encoding ion channels, the identifi-
cation of the novel gene has the potential of contributing
new information about the molecular pathogenesis of
epilepsy.
Acknowledgments
We wish to thank the families participating in this study, the Neurode-
generative Disorders Centre, QEII Medical Centre for the Affymetrix
SNP5.0 array genotyping, and the Western Australian Genetic Epidemi-
ology Resource for bioinformatics support. The study was funded by
grant 458736 of the National Health and Medical Research Council of
9
Partial Epilepsy in a Gypsy Family
Australia and by UCLH/UCL, which received a proportion of funding
from the Department of Health’s NIHR Biomedical Research Centre’s
funding scheme.
We confirm that we have read the Journal’s position on issues involved in
ethical publication and affirm that this report is consistent with those
guidelines.
Disclosure: None of the authors has any conflict of interest in regard to
this work to disclose.
References
Abecasis GR, Cherny SS, Cookson WO, Cardon LR. (2002) Merlin –
rapid analysis of dense genetic maps using sparse gene flow trees.
Nat Genet 30:97–101.
Arcos-Burgos M, Muenke M. (2002) Genetics of population isolates.
Clin Genet 61:233–247.
Berkovic SF, McIntosh A, Howell RA, Mitchell A, Sheffield l, Hopper
JL. (1996) Familial temporal lobe epilepsy: a common disorder iden-
tified in twins. Ann Neurol 40:227–235.
Berkovic SF, Serratosa JM, Phillips HA, Xiong L, Andermann E,
Diaz-Otero F, Gomez-Garre P, Martin M, Fernandez-Bullido Y,
Andermann F, Lopez-Cendes I, Dubeau F, Desbiens R, Scheffer I,
Wallace RH, Mulley JC, Pandolfo M. (2004) Familial partial epilepsy
with variable foci: clinical features and linkage to chromosome
22q12. Epilepsia 45:1054–1060.
Callenbach PM, van der Maagdenberg AM, Hottenga JJ, van den
Boogerd EH, de Coo RF, Lindhout D, Frants RR, Sandkuijl LA,
Brouwer OF. (2003) Familial partial epilepsy with variable foci in a
Dutch family: clinical characteristics and confirmation of linkage to
chromosome 22q. Epilepsia 44:1298–1305.
Cendes F, Lopes-Cendes I, Andermann E, Andermann F. (1998) Familial
temporal lobe epilepsy: a clinically heterogeneous syndrome. Neuro-
logy 50:554–557.
Claes L, Audenaert D, Deprez L, Van Paesschen W, Depondt C,
Goossens D, Del-Favero J, Van Broeckhoven C, De Jonghe P. (2004)
Novel locus on chromosome 12q22–q23.3 responsible for familial
temporal lobe epilepsy associated with febrile seizures. J Med Genet
41:710–714.
Colomer J, Gooding R, Angelicheva D, King RH, Guillen-Navarro
E, Parman Y, Nascimento A, Conill J, Kalaydjieva L. (2006)
Clinical spectrum of CMT4C disease in patients homozygous for
the p.Arg1109X mutation in SH3TC2. Neuromuscul Disord 16:449–
453.
Cuevas P, Gimenez-Gallego G. (1997) Role of fibroblast growth factors
in neural trauma. Neurol Res 19:254–256.
Duncan JS, Sander JW, Sisodiya SM, Walker MC. (2006) Seminar: adult
epilepsy. Lancet 367:1087–1100.
Gambardella A, Messina D, Le Piane E, Oliveri RL, Annesi G, Zappia
M, Andermann E, Quattrone A, Aguglia U. (2000) Familial temporal
lobe epilepsy autosomal dominant inheritance in a large pedigree
from southern Italy. Epilepsy Res 38:127–132.
Helbig I, Scheffer IE, Mulley JC, Berkovic SF. (2008) Navigating the
channels and beyond: unravelling the genetics of the epilepsies.
Lancet Neurol 7:231–245.
John EM, Miron A, Gong G, Phipps AI, Felberg A, Li FP, West DW,
Whittemore AS. (2007) Prevalence of pathogenic BRCA1 mutation
carriers in 5 US racial/ethnic groups. JAMA 298:2869–2876.
Kalaydjieva L, Gresham D, Calafell F. (2001) Genetic studies of the
Roma (Gypsies): a review. BMC Med Genet 2:5.
Kalaydjieva L, Morar B, Chaix R, Tang H. (2005) A newly discovered
founder population: the Roma/Gypsies. BioEssays 27:1084–1094.
Kanner AM. (2007) Epilepsy and mood disorders. Epilepsia 48(Suppl
9):20–22.
Kanner AM. (2008) Mood disorder and epilepsy: a neurobiologic per-
spective of their relationship. Dialogues Clin Neurosci 10:39–45.
Kinton L, Johnson MR, Smith SJ, Farrell F, Stevens J, Rance JB,
Claudino AM, Duncan JS, Davis MB, Wood NW, Sander JW. (2002)
Partial epilepsy with pericentral spikes: a new familial epilepsy syn-
drome with evidence for linkage to chromosome 4p15. Ann Neurol
51:740–749.
Epilepsia, **(*):1–10, 2009
doi: 10.1111/j.1528-1167.2009.02066.x
10
D. Angelicheva et al.
Lange K, Cantor R, Horvath S, Perola M, Sabatti C, Sinsheimer J, Sobel
E. (2001) Mendel version 4.0: A complete package for the exact
genetic analysis of discrete traits in pedigree and population data sets.
Am J Hum Genet 69(Suppl):A1886.
Leeksma OC, Van Achterbetg TA, Tsumura Y, Toshima J, Eldering E,
Kroes WG, Mellink C, Spaargaren M, Mizuno K, Pannekoek H, de
Vries CJ. (2002) Human sprouty 4, a new ras antagonist on 5q31,
interacts with the dual specificity kinase TESK1. Eur J Biochem
269:2546–2556.
Mihaylova V, Hantke J, Sinigerska I, Cherninkova S, Raicheva M,
Bouwer S, Tincheva R, Khuyomdziev D, Bertranpetit J, Chandler D,
Angelicheva D, Kremensky I, Seeman P, Tournev I, Kalaydjieva L.
(2007) Highly variable neural involvement in sphingomyelinase-
deficient Niemann-Pick disease caused by an ancestral Gypsy muta-
tion. Brain 130:1050–1061.
Nagase T, Kikuno R, Ishikawa KI, Hirosawa M, Ohara O. (2000) Predic-
tion of the coding sequences of unidentified human genes XVI. The
complete sequences of 150 new cDNA clones from brain which code
for large proteins in vitro. DNA Res 7:65–73.
Ostrer H. (2001) A genetic profile of contemporary Jewish populations.
Nat Rev Genet 2:891–898.
Ott J. (1989) Computer-simulation methods in human linkage analysis.
Proc Natl Acad Sci U S A 86:4175–4178.
Peltonen L, Jalanko A, Varilo T. (1999) Molecular genetics of the Finnish
disease heritage. Hum Mol Genet 8:1913–1923.
Picard F, Baulac S, Kahane P, Hirsch E, Sebastianelli R, Thomas P,
Vigevano F, Genton P, Guerrini R, Gericke CA, An I, Rudolf G, Her-
mann A, Brice A, Marescaux C, LeGuern E. (2000) Dominant partial
epilepsies. A clinical, electrophysiological and genetic study of 19
European families. Brain 123:1247–1262.
Reuss B, von Bohlen und Halbach O. (2003) Fibroblast growth factors
and their receptors in the central nervous system. Cell Tissue Res
313:139–157.
Risch N, de Leon D, Ozelius L, Kramer P, Almasy L, Singer B, Fahn S,
Breakefield X, Bressman S. (1995) Genetic analysis of idiopathic tor-
sion dystonia in Ashkenazi Jews and their recent descent from a small
founder population. Nat Genet 9:152–159.
Sander JW. (2003) The epidemiology of the Epilepsies revisited. Curr
Opin Neurol 16:165–170.
Sasaki K, Oomura Y, Suzuki K, Hanai K, Yagi H. (1992) Acidic fibro-
blast growth factor prevents death of hippocampal CA1 pyramidal
cells following ischemia. Neurochem Int 21:397–402.
Sasaki K, Oomura Y, Figurov A, Yagi H. (1994) Acidic fibroblast growth
factor facilitates generation of long-term potentiation in rat hippo-
campal slices. Brain Res Bull 33:505–511.
Scheffer IE, Phillips HA, O’Brien CE, Saling MM, Wrennall JA, Wallace
RH, Mulley JC, Berkovic SF. (1998) Familial partial epilepsy with
variable foci: a new partial epilepsy syndrome with suggestion of
linkage to chromosome 2. Ann Neurol 44:890–899.
Thiele H, Nurnberg P. (2005) HaploPainter: a tool for drawing pedigrees
with complex traits. Bioinformatics 21:1730–1732.
Thomas PK, Kalaydjieva L, Youl B, Rogers T, Angelicheva D, King RH,
Guergueltcheva V, Colomer J, Lupu C, Corches A, Popa G, Merlini
Epilepsia, **(*):1–10, 2009
doi: 10.1111/j.1528-1167.2009.02066.x
L, Shmarov A, Muddle JR, Nourallah M, Tournev I. (2001) Heredi-
tary motor and sensory neuropathy-Russe: new autosomal recessive
neuropathy in Balkan Gypsies. Ann Neurol 50:452–457.
Tournev I, Kalaydjieva L, Youl B, Ishpekova B, Guerguelcheva V,
Kamenov O, Katzarova M, Kamenov Z, Raicheva-Ternzieva M,
King RHM, Petkov R, Shmarov A, Dimitrova G, Popova N, Uzunova
M, Milanov S, Petrova J, Petkov Y, Kolarov G, Anev L, Radeva O,
Thomas PK. (1999) Congenital Cataracts Facial Dysmorphism
Neuropathy (CCFDN) syndrome, a novel complex genetic disease in
Balkan Gypsies: clinical and electrophysiological observations. Ann
Neurol 45:742–750.
Van Bogaert P, Azizieh R, Desir J, Aeby A, De Meirleir L, Laes JF,
Christiaens F, Abramowicz MJ. (2007) Mutation of a potassium
channel-related gene in progressive myoclonic epilepsy. Ann Neurol
61:579–586.
Weeks DE, Ott J, Lathrop GM. (1990) SLINK: a general simulation pro-
gram for linkage analysis. Am J Hum Genet 47(Suppl 3):A204.
Xiong L, Labuda M, Li DS, Hudson TJ, Desbiens R, Patry G, Verret S,
Langevin P, Mercho S, Seni M-H, Scheffer I, Dubeau F, Berkovic
SF, Andermann F, Andermann E, Pandolfo M. (1999) Mapping of a
gene determining familial partial epilepsy with variable foci to chro-
mosome 22q11–q12. Am J Hum Genet 65:1698–1710.
Supporting Information
Additional supporting information may be found in the
online version of this article:
Figure S1: Pedigree structure of the extended S1 family
(including the branches with epilepsy analyzed in this
study), with different symbols indicating the phenotypes
of epilepsy and comorbidities: mild intellectual deficit
and psychiatric disorders [mood disorders, attention defi-
cit hyperactivity disorder (ADHD), alcohol and drug
abuse].
Table S1: Details on primers used for fine mapping and
candidate gene sequencing.
Table S2: Known epilepsy loci with LOD scores in the
investigated Gypsy family S1.
Please note: Wiley-Blackwell is not responsible for the
content or functionality of any supporting information
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.