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Benign familial neonatal convulsions Evidence for clinical and genetic heterogeneity.

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Benign F d d Neonatal
Convulsions: Evidence for Clinical
and Genetic Heterogeneity
Stephen G. Ryan, MD,* Max Wiznitzer, MDJ Charlotte Hollman, MD,$
hf. Cristina Torres, BS,“ Maria Szekeresova, &IS,? and Sandra Schneider, P h D i
The gene for autosomal dominant “benign” familial neonatal convulsions, a transient, primary epilepsy of infancy, has
recently been assigned to chromosome 20q. To determine whether this disorder is genetically heterogeneous, we
performed linkage analysis in two previously unreported pedigrees with benign familial neonatal convulsions in which
clinical heterogeneity was evident. There were 14 affected persons in the first family, and none had seizures (febrile
or afebrile) after the age of 2 months. The second family had 13 affected individuals and 2 obligate carriers; seizures
frequently did not remit until 6 to 24 months, febrile convulsions occurred in at least 2 patients, apparent audiogenic
seizures occurred in 4 patients, and 1 individual had refractory epilepsy until late adolescence. Linkage studies with
the chromosome 20 markers D20S19 and D20S20 were performed in both families. The resulting data favored linkage
of the disease and marker loci in Family 2 by a maximum odds ratio of 45:l at 6% recombination. In Family 1,
however, the odds were greater than 20,OOO:l against linkage at 10% recombination or less. We conclude that the
syndrome of benign familial neonatal convulsions is clinically and genetically heterogeneous. Further study will be
necessary to clarify the relationship between phenotype and genotype in this disorder.
Ryan SG, Wiznitzer M, Hollman C, Torres MC, Szekeresova M, Schneider S. Benign familial neonatal
convulsions: evidence for clinical and genetic heterogeneity. Ann Neurol 1991;29:469-473
Genetic influences are thought to contribute to many
forms of human epilepsy [l, 2}. Indeed, among the
so-called “secondary” epilepsies are numerous mendelian or single-gene disorders in which seizures are
symptomatic of a fixed or progressive encephalopathy
El}. Most primary epilepsies, however, are probably
multifactorial in etiology [ 3 } , and the mechanisms
whereby genetic factors cause seizures in these disorders are not understood.
The syndrome of benign familial neonatal convulsions (BFNC) is a rare example of a primary epilepsy
{4} in which inheritance is unequivocally autosomal
dominant 15-91. BFNC is characterized by the occurrence of unprovoked partial or generalized c h i c seizures in the neonatal period or early infancy. Results of
routine diagnostic studies (including cranial computed
tomography, interictal electroencephalography (EEG),
and serum chemistry determinations) and subsequent
intellectual development are normal. Seizures usually
remit by the age of 6 months, but chronic childhood
or adult epilepsy has been reported in about 10% of
patients [7, 91.
Recently, the gene for BFNC was assigned to chromosome 20q by the detection of tight linkage between
the BFNC locus and two polymorphic D N A markers,
D20S19 and D20S20, in a single large pedigree [lo}.
Subsequent studies in two additional families supported this finding, and raised the odds ratio favoring
linkage between the disease and marker loci to greater
than 8,000,000:l Ell}.
We recently identified two large and previously unreported BFNC pedigrees whose clinical features suggested that the disorder was heterogeneous. We therefore performed linkage analysis in these families to
determine whether distinct genetic loci were involved.
From the Departments of *Pediatrics and tperiodontics, The University of Texas Health Science Center, San Antonio, TX, and the
$Departments of Pediatrics and Neurology, Case Western Reserve
University, Cleveland, OH.
Received Sep 11, 1990, and in revked form Nov 1. Accepted for
publication Nov 4,1990.
Materials and Methods
The families were identified through the probands whose
case histories are provided below. The parents (where available) of all individuals “at risk’ for BFNC were interviewed.
Medical records were available in only a few instances, and in
most cases the age at onset and remission were not precisely
recalled. Nonetheless, for most individuals, we were able to
obtain a clear history regarding: ( 1 ) the presence or absence
Address correspondence to Dr Ryan, Department of Pediatrics, The
University of Texas Health Science Center at San Antonio, 7703
Floyd Curl Drive, San Antonio, TX 78284.
Copyright 0 1991 by the American Neurological Association 469
of seizures in the neonatal period or in infancy, (2) whether
seizures persisted past the ages of 2 or 12 months, and ( 3 )
whether or not at least one febrile seizure had occurred.
Genomic D N A was prepared by phenolichloroform extraction of lysed, Epstein-Barr virus-transformed peripheral
blood lymphoblastoid cell lines El2, 131. The samples were
digested with the restriction enzyme TaqI (New England
Biolabs, Beverly, MA) and the resulting fragments were fractionated by 0.7% agarose gel electrophoresis and transferred
by capillary action to nylon membranes (Nytran, Schleicher
and Schuell, Keene, NH) according to protocols supplied by
the manufacturers. Following prehybridization, the blots
were hybridized with the radiolabelled [ 141 markers D20S 19
(pCMM6) [15] and D20S20 (pRMR6) 1161(purchased from
the American Type Culture Collection, Rockville, MD) and
washed in solutions of increasing stringency as suggested by
the manufacturer. These two probes reveal D N A restriction
fragment polymorphisms due to a variable number of tandem
repeats of a short D N A sequence (D20S19) and the presence
or absence of a TayI restriction site (D20S20). Because polymorphisms were poorly demonstrated with the latter probe,
we tested fragments obtained from simultaneous digestion of
the pRMR6 plasmid with Hind111 and EcoRI. A 0.7-kb fragment revealed the polymorphic system clearly and was used
for typing. Autoradiograms of the membranes were then prepared [12), and marker genotypes assigned to each individual.
D20S19 and D20S20 are closely linked, with an estimated
recombination fraction of 0.016 [171. Since no recombination between the two markers was observed in our families,
it was possible to assign a combined marker haplotype to
each individual. Two-point linkage analysis was performed
with the program LIPED [IS] on an IBM-XT compatible
personal computer. A penetrance of 90% was assumed for
the BFNC allele, based on a segregation ratio of 45% reported previously [lo].
Family 1
The proband was a healthy, term neonate of MexicanAmerican ancestry, whose vaginal delivery followed an uncomplicated gestation. O n the ninth postnatal day, she had
four clonic convulsions, each about 2 minutes long: Two
were generalized, one was left-sided, and one was right-sided.
Physical examination results, blood glucose levels, routine
serum electrolyte levels (including calcium and magnesium),
and an EEG were all normal. Neuroimaging was deferred
because at least 3 affected relatives had had normal findings
on cranial computed tomography. Phenobarbital was begun
at a total daily dose of 5 mgikg, and was discontinued by the
mother a month later. At the time of writing, the patient was
9 months old and developmentally normal, and had had no
recurrence of seizures.
Thirteen of the proband's relatives had neonatal seizures
beginning between the second and fourteenth postnatal day
(Fig 1, Table 1). No one in Generations I and 11 received
anticonvulsant therapy, but all of the proband's affected cousins (Generation 111) received phenobarbital for up to 4
months. None had seizures while taking phenobarbital, and
no seizures occurred in any family member after the age of
2 months, regardless of treatment status. Intellectual development appeared normal in all family members.
470 Annals of Neurology
V a l 29 N o 5
May 1991
Fig 1. Pedigree and genotypic data for Family 1 (arrow indicates
proband). The D20S19 alleles are denoted by letters and the
D20S20 alleles by numbers. Numerous instances of recombination
between the disease and marher alleles are &dent.
Table 1. Clinical Features Suggestive of Heterogeneity in
Two Pedigrees with Benign Familial Neonatal Convulsions
Persistence of seizures beyond
12 months
Epilepsy in late childhoodiadolescence
Audiogenic seizures
Febrile seizures
Asymptomatic obligate carriers
Family 1
(n = 14)
Family 2
(n = 15)
Family 2
The proband for Family 2 was born to parents of northern
European ancestry via term, spontaneous vaginal delivery following an uncomplicated gestation. O n the second postnatal
day, she had 11 partial clonic seizures. A loading dose of
phenytoin was administered, and maintenance phenobarbital
was begun at a dose of 5 mgikgiday. Physical examination
findings, cranial computed tomograms, serum chemistry measurements, and EEGs were all normal. At least two additional
seizures occurred during the next 3 weeks despite phenobarbital treatment (serum levels were not measured), but subsequently she was seizure-free. At 10 months old she was
developmentally normal, and continued to take phenobarbital.
Twelve relatives of the proband had a clear history of idiopathic neonatal convulsions (Fig 2;see Table 1).There were
also two obligate carriers: Patients 11-9 and either 1-1 or 1-2.
One infant (IV-3) had severe birth asphyxia due to placental
abruption. Neither his neonatal seizures nor his subsequent
microcephaly and developmental delay were thought to be
manifestations of BFNC, but he was omitted from the linkage analysis because affectation status for BFNC was deemed
One relative (111-3) continued to have frequent convulsions until late adolescence despite phenobarbital therapy;
his management was complicated by noncompliance and at
least three episodes of convulsive status epilepticus. Although of borderline intelligence, he was fully employed at
the time of writing. All other family members (except IV-3)
appeared to be intellectually normal.
Table 2. Results oJ Linkage Analysis
h d Scores
Fig 2.Pedigree and genotypic data for Family 2 (arrow indicates
proband). Marker alleles are denoted as in Figure I . Recombination between the disease and muker loci appears to have occurred
in individuals 111-1 and III-16, but nonpenetrance could a h
explain their marher genotypes. Patient IV-3 was omitted from
the analysis because of uncertainty regarding his phenotype (see
Four affected patients (11-14, 111-3, 111-15, and IV-6) had
a history strongly suggestive of audiogenic seizures. For example, in one 2’/2-month-old infant (IV-6) a prolonged
(longer than 10 minutes) clonic seizure was apparently precipitated by the explosion of a firecracker. Seizures induced
by such stimuli occurred in both sleeping and walung states.
At least 4 affected relatives (11-14, 111-3, 111-6, and 111-8)
continued to have afebrile seizures until the age of 12 months
or beyond. At least 2 affected relatives (111-3 and IV-5) also
had o n e or more febrile convulsions between the ages of 6
and 18 months. EEG results were available only for IV-5,
IV-6, and IV-7; all three tracings were normal.
The marker genotypes are provided in Figs 1 and 2,
and lod scores* from two-point linkage analysis are
presented in Table 2. Both markers were informative
in Family 1, but only D20S19 was informative in Pamily 2, as no affected parents were heterozygous for
D20S20. The results were not influenced by the population frequencies of the marker alleles except for
slight variation, in Family 2, as a function of the frequency of D20S19 allele B (data not shown). A value
of 0.33 for this parameter, based on a survey of 27
unrelated individuals, was employed for the lod score
computations in Table 2.
For Family 1, the data strongly favored nonlinkage
over linkage of the disease and marker loci; the odds
against linkage at 10% recombination, for example, are
greater than 21,OOO:l (lod score, -4.34).
For Family 2, a maximal odds ratio of 45: 1 (lod score,
1.66) favoring linkage occurred at 6% recombina-
‘A Iod (“logarithm of odds”) score is a measure of the likelihood
that a set of genotypic observations results froni linkage as opposed
to chance. A lod score of 3.0, corresponding to a 1,000:1 odds ratio,
is generally accepted as establishing linkage.
Family 1
Family 2
tion, with a one-lod confidence interval of 1 to 37%.
When sex-specific recombination fractions were allowed to vary independently, a maximum lod score of
1.67 occurred at male and female rates of 10 and 396,
Numerous instances of recombination between the
disease and marker loci are evident in Family 1. In
Family 2, only 2 individuals (aside from the obligate
carriers) failed to display the BFNC phenotype despite
inheritance of the linked haplotype. The carrier status
of these individuals (111-1 and 111-16) is uncertain, since
nonpenetrance cannot be distinguished from recombination between the disease and marker loci. No a€fected individuals lacked the linked haplotype.
Numerous reports of BFNC have appeared since the
original description of Rett and TeubelT5-9, 191. The
clinical picture is distinctive and relatively uniform, although it has been suggested that differences among
pedigrees with regard to risk for epilepsy in later life
might reflect genetic heterogeneity. Shevell and associates {9] reviewed the clinical features of 113 patients
with BFNC in 15 families; 11 of 62 individuals in 6
families experienced nonfebrile convulsions beyond infancy, while the 5 1 individuals in the remaining 9 families experienced early and complete remission of seizures. They observed that the data were compatible
with “two forms [of BFNC] with sharply different risks
for subsequent epilepsy.” Similarly, Leppert and associates {lo] noted that the development of nonfebrile
seizures after the age of 6 months may be restricted to
certain pedigrees.
Unfortunately, most published reports of BFNC do
not provide detailed information on age at remission of
seizures, presumably because of difficulty in obtaining
reliable data. We encountered uncertainty with regard
to age at remission as wel, but were nonetheless able
to detect a trend toward later remission in Family 2
than in Family 1.
Ryan et al: Familial Neonatal Convulsions 471
Our linkage data strongly support the existence of
two distinct genetic loci for BFNC. The maximum lod
score of 1.66 obtained for Family 2 suggests, but does
not prove, that the BFNC locus in this kindred is
linked to the marker loci. Given previous reports of
linkage of BFNC to these markers [lo, 1I), however,
it appears quite likely that the disease gene in Family
2 maps to chromosome 20q. In contrast, in Family 1,
the data militate strongly against proximity of the disease and marker loci. Given the magnitude of the negative lod scores in Family 1, it is unlikely that erroneous
phenotype assignment, nonpaternity, or an inaccurate
penetrance estimate could account for these results.
The data further suggest that the BFNC subtype linked
to chromosome 20q may be associated with later remission and a higher risk for subsequent epilepsy (defined as afebrile seizures after age 2 years) than in the
variant of this disorder present in our first family.
Based on the original reports 15-71, late epilepsy occurred in at least 3 of 15, 2 of 14, and none of 6
individuals in the three families studied by Leppert and
associates [lo]. Additional studies are needed to determine whether there is a consistent relationship between the genetic subtype of BFNC and clinical features such as ethnicity, age at remission, response to
anticonvulsant therapy, audiogenic seizures, and the
risk of late epilepsy.
There are several precedents for the discovery of
heterogeneity by linkage analysis of disorders presumed to be uniform. The earliest example is probably
that of hereditary elliptocytosis, for which linkage to
the Rh blood group locus has been established in some
kindreds, but disproved in others r20). Recent examples in clinical neurology include hereditary motor and
sensory neuropathy type I (in which loci on chromosome 1 and 17 have been implicated in different families) 121, 221 and tuberous sclerosis (in which linkage
to the ABO blood group locus on chromosome 9 has
been demonstrated in some families but disproved in
others) [23}.
The presence of heterogeneity in BFNC may complicate efforts to isolate the 20q gene, since a critical
step in such a project will be a search for flanking
markers {24]. This entails a search for individuals who
demonstrate recombination between the disease and a
closely linked marker locus. Given heterogeneity, only
recombinants in pedigrees that unequivocally demonstrate linkage-hence, very large pedigrees-can be
considered. In other words, the potential information
present in small pedigrees is lost because the genetic
type cannot be reliably determined.
The presence of heterogeneity in BFNC, an epilepsy
with highly distinctive clinical features and single-gene
inheritance, suggests that disorders such as childhood
absence and benign partial childhood (rolandicj epilepsy [4], for both of which dominant inheritance has
Annals of Neurology Vol 29
No 5
May 1991
been suggested [25, 261, could also exhibit this phenomenon. If this is the case, then linkage analysis in
these disorders, which will almost certainly involve numerous small families, will be particularly challenging.
Although such an approach may be successful, as in
juvenile myoclonic epilepsy (recently assigned to chromosome Gp) 1271, heterogeneity could easily complicate efforts to assign genes for epilepsies that rarely, if
ever, segregate in extensive pedigrees. Without firm
clinical criteria for classifying families, evidence supporting linkage in some families could be negated by
evidence against it in others.
We have been able to demonstrate heterogeneity in
BFNC because large families were available for study
and because two markers (one moderately and the
other highly polymorphic) mapping to the region of
interest were also available. It is likely that phenotypic
variation among BFNC families is directly related to
genotypic differences, and may be more common than
previously appreciated. Further study should confirm
or refute this hypothesis.
Presented in part at the International Conference on Genetics and
Epilepsy, Minneapolis, MN, July 20-22, 1990, and to the Child
Neurology Society, Atlanta, GA, October 18-20, 1990.
We are indebted to the members of the two families described herein
for their generous cooperation, to Robin Leach, PhD, for many helpful suggestions. and to Sue Wiggins for preparation of the typescript.
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