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Autosomal dominant acute necrotizing encephalopathy maps to 2q12.1-2q13

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Autosomal Dominant Acute
Necrotizing Encephalopathy
Maps to 2q12.1-2q13
Derek E. Neilson, MD,1–3 Heidi S. Feiler, PhD,4
Kirk C. Wilhelmsen, MD, PhD,4,5 Audrey Lynn, PhD,2
Robert M. Eiben, MD,6 Douglas S. Kerr, MD, PhD,1
and Matthew L. Warman, MD1–3
In autosomal dominant acute necrotizing encephalopathy
(ADANE), apparently healthy children develop necrotizing lesions in their thalami and brainstems in the course
of febrile illnesses. We used DNA from affected subjects
and obligate carriers to map ADANE to a 6.5Mb region
on chromosome 2. Sequencing of four candidate genes in
the interval (BCL2L11, ST6GalII, CHT1, and
FLJ20019), involved in apoptosis, viral recognition, choline transport, and electron transport, showed no disease
causing mutations.
Ann Neurol 2004;55:291–294
Autosomal dominant acute necrotizing encephalopathy
(ADANE) is an incompletely penetrant genetic disorder that primarily affects children.1 Those manifesting
the disorder develop normally until they experience a
sudden-onset encephalopathy 2 or 3 days after the onset of a febrile illness. Coma, seizures, and other neurological deficits comprise the initial presenting signs.
The disorder is fatal in some patients; however, the
majority recover from the coma and may experience
mild to severe developmental regression. Although
complete recovery of neurological function is possible,
significant disability such as spastic quadriplegia and
mental retardation may persist. Half of the survivors
experience a recurrent encephalopathy that results in a
worse outcome. During the acute episode, magnetic
resonance imaging detects fluid-attenuated inversion
recovery and T2-weighted hyperintensities distributed
primarily in the thalamus and brainstem (Fig 1). These
signal abnormalities correlate with neuropathological
findings of necrotizing encephalopathy (cellular necrosis, gliosis, hemorrhage, and capillary dilation and proliferation). Elevated cerebrospinal fluid protein is the
only consistent abnormal clinical laboratory finding
among affected subjects during the acute illness. Pleiomorphic mitochondria associated with cytoplasmic
vacuoles and loose coupling of oxidative phosphorylation have been demonstrated by muscle biopsy in one
patient. Infectious triggers have included influenza A
and parainfluenza II, but most cases have had no organisms identified. Unaffected obligate carriers (based
on position with the pedigree) have no apparent phenotype.1
To date, ADANE has been described in only one
large kindred. As a first step toward finding the cause
of ADANE, we performed whole genome linkage analysis and mapped the disease interval to human chromosome 2q12.1-2q13.
Subjects and Methods
Clinical Description and Collection of DNA Samples
This study was approved by the institutional review board at
the University Hospitals of Cleveland. Clinical findings from
this kindred have been reported recently.1 Blood samples
were drawn from 11 affected persons, 5 obligate carriers, 17
unaffected first-degree relatives of affected subjects, and 5
second-degree or higher relatives who displayed neurological
symptoms (seizures, tremor, gait alteration) or had a sibling
From the Departments of 1Pediatrics and 2Genetics, Case Western
Reserve University School of Medicine and Rainbow Babies and
Children’s Hospital; 3Center for Human Genetics, University Hospitals of Cleveland, Cleveland, OH; 4Ernest Gallo Clinic and Research Center and 5Department of Neurology, University of California San Francisco School of Medicine, Emeryville, CA; and
Department of Pediatrics, MetroHealth Medical Center, Cleveland, OH.
Received Aug 19, 2003, and in revised form Oct 24. Accepted for
publication Oct 28, 2003.
Address correspondence to Dr Neilson, Department of Genetics,
BRB 747B, Case Western Reserve University, 2109 Adelbert Road,
Cleveland, OH 44106. E-mail:
Fig 1. T2-weighted magnetic resonance image of the brain
demonstrating bithalamic hyperintensities during an acute encephalopathy in a 3-year-old boy.
© 2004 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
or child with neurological symptoms. Persons were considered affected if they experienced the typical coma and developmental regression after mild infectious symptoms. Obligate carriers were defined as unaffected persons who had
affected descendants.
DNA Isolation and Genetic Mapping
DNA was extracted from whole blood using the PureGene
DNA isolation kit (Gentra Systems, Minneapolis, MN).
Genome-wide linkage analysis was performed using an 811
marker set of fluorescently labeled primers (HD5 panel from
Applied Biosystems [ABI], Foster City, CA), having an average spacing of 5cM. Amplimers were separated on an ABIPRISM 3700 sequencer and scored using the Genotyper
software package (ABI). Simwalk2 was used to detect and
suppress probable genotype errors and perform whole chromosome multipoint segregation analysis.
Fine mapping of the interval on human chromosome 2
was performed using simple sequence repeat polymorphisms
that were between markers D2S2264 and D2S160 (based on
the University of California Santa Cruz [UCSC] genome
browser April 2003 build2) and spaced at approximately
1Mb intervals (Fig 2). Polymerase chain reactions (PCRs)
were performed using 33P end-labeled primers, products were
resolved by denaturing gel electrophoresis, and alleles were
visualized by autoradiography.
Multipoint logarithm of odds (LOD) score analysis was
performed using the GENEHUNTER3 program, and twopoint LOD scores were determined using the MLINK4 program. For these calculations, the penetrance of ADANE was
estimated at 50%, the mutant allele frequency was estimated
at 0.0001, and marker alleles were assigned equal frequency,
except for marker D2S293 for which the frequencies were
derived from the CEPH DNA collection. Map distances
were taken from the Marshfield map5 with the exception of
markers D2S299 (116.26cM), GATA13H01 (115.97cM),
and D2S135 (116.02cM) for which the recombination map
order did not correlate with the sequenced physical order.
These markers were assigned the correct physical order with
an estimated distance of 0.1cM between them.
Fig 2. Simplified pedigree demonstrating locus haplotypes for 14 studied markers between D2S2264 (centromeric) and D2S1896
(telomeric). The physical distances correspond to the distance from the p telomere. Allele numbers are assigned based on relative
polymerase chain reaction product size. The conserved haplotypes are circled and dashed lines delineate the maximum inclusion region between D2S135 and BCL2L11-SNP. Filled symbols represent affected persons. Half-filled symbols are obligate carriers. Bracketed symbols indicate persons not available for study.
Annals of Neurology
Vol 55
No 2
February 2004
Gene Sequencing
Candidate genes BCL2L11, ST6GALII, CHT1, and
FLJ20019 were identified based on their position using the
UCSC genome browser and their known, or putative, function. Exon-containing regions, including at least 60bp of intronic sequence, were amplified by PCR. Excess primer and
deoxynucleotide triphosphates in the PCR were degraded using ExoSap-IT enzyme (USB, Cleveland, OH). The resultant
PCR products were sequenced using the BigDye 1.1 cycle
terminator kit (ABI), and the sequencing products were analyzed using an ABI Prism model 3100. Sequence analysis
was performed using the Consed software package.6
Whole genome mapping was performed with an average resolution of 5cM between markers. Two loci
yielded positive LOD scores. D2S293 on human chromosome 2q yielded a two-point LOD score of 3.39 at
␪ ⫽ 0. A multipoint LOD score of 0.9 was obtained
near marker D13S1284 located at the telomere of human chromosome 13p. We excluded linkage to human
chromosome 13p by demonstrating that closely flanking markers did not cosegregate with ADANE (data
not shown). More importantly, we strengthened the assignment of ADANE to human chromosome 2 by
demonstrating cosegregation of a five-marker haplotype, which yielded a multipoint LOD score of 3.6
across the interval. The marker D2S135 defines the
centromeric breakpoint because it has apparently recombined with the disease phenotype in Subject II:2.
Initially, the marker D2S1896 set the telomeric breakpoint because it recombined with the disease phenotype in Subject III:3 (see Fig 2), but, after sequencing
the BCL2L11 gene, we found an intronic single nucleotide polymorphism (SNP) that also did not segregate
with the disease in this subject. This C/T SNP is located 2,590bp centromeric to D2S1892 and therefore
refines the genetic interval (see Fig 2). The ADANE
candidate interval between D2S135 and the BCL2L11
SNP contains 33 RefSeq genes, among which were included BCL2L11 (an apoptosis facilitator), ST6GALII
(a brain-specific 2,6-sialyltransferase), CHT1 (the highaffinity choline transporter), and FLJ20019 (a predicted protein that appears related to known electron
transporters). Sequencing the coding regions for each
of these genes showed no mutations.
Having relied on clearly affected and obligate carriers
to assign the ADANE locus to human chromosome 2,
we next performed haplotype analysis for 17 subjects
who were at risk for inheriting the disease-predisposing
gene based on their having an affected or obligate carrier, sibling, or parent. Seven of these 17 family members shared the ADANE-linked haplotype. Among
these seven subjects, one had mild mental retardation
with no history of acute encephalopathy. Of five additional, more distant relatives, one shared the affected
haplotype and was asymptomatic; two of this group
had idiopathic epilepsy but did not share the haplotype, indicating that epilepsy is not useful for the ascertainment of carriers.
The identification of a genetic locus for ADANE provides the framework for further studies to identify the
causative gene. Of the 33 RefSeq genes contained
within the linked region, none have been associated
with neurodegeneration, either acute or chronic. There
are also no genes previously known to be involved in
the uncoupling of oxidative phosphorylation. Many of
the genes are of unknown function.
Necrotizing encephalopathy has been associated previously with three conditions: acute necrotizing encephalopathy (ANE), Leigh syndrome, and Wernicke
encephalopathy. ANE has been described mainly in Japan and Taiwan,7 but recent case reports suggest a
global distribution.8,9 It is clinically identical to
ADANE but has no defined genetic component. Leigh
syndrome differs by its neurodegenerative course, persistent lactic acidosis, and predilection for the basal
ganglia.10 Its heterogeneous genetic basis, which disrupts the mitochondrial energy production system,
provides a potential link with ADANE. Wernicke encephalopathy is caused by thiamine deficiency, a state
that the ADANE patients do not share, but the pathological distribution of lesions in the thalamus and mamillary bodies overlap those observed in ADANE.11
Multiple theories for the pathogenic action of thiamine
deficiency in Wernicke encephalopathy exist, including
metabolic disturbances,12 excitoxicity,12 and blood–
brain barrier disruption.13 Although the pathogenesis
of each disorder is different, the rarity of their shared
phenotype suggests that they may participate in different aspects of a common degenerative pathway. In this
way, these encephalopathies may provide useful comparisons for the understanding of ADANE.
We tested four genes with potential causative roles,
based on comparison with the above disorders, but
have found no mutations. However, this does not fully
exclude these candidates, because the methods utilized
did not assess all possible gene regulatory elements. In
addition, our screening method will miss heterozygous
whole-gene or exon deletions, because the wild-type allele alone will still amplify with PCR and provide a
normal sequence. Studying other families with this disorder may best facilitate discovery of the ADANE gene
by narrowing the candidate region. A broader phenotype may be associated with ADANE, as suggested by
the apparently isolated finding of mental retardation in
a disease haplotype carrier, and that may help clinicians
ascertain new families who segregate this disease.
Neilson et al: ADANE Maps to 2q12.1-2q13
This work was supported by a grant from the French Foundation
(K.C.W.) and a Burroughs Wellcome Fund Clinical Scientist Award
in Translational Research (M.L.W.). M.L.W. is an assistant investigator with the Howard Hughes Medical Institute.
We thank the patients and their extended family for participating in
this research and J. V. Lee for technical support.
1. Neilson D, Eiben R, Waniewski S, et al. Autosomal dominant
acute necrotizing encephalopathy. Neurology 2003;61:
226 –230.
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3. Kruglyak L, Daly MJ, Reeve-Daly MP, Lander ES. Parametric
and nonparametric linkage analysis: a unified multipoint approach. Am J Hum Genet 1996;58:1347–1363.
4. Lathrop GM, Lalouel JM, Julier C, Ott J. Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci USA
5. Broman KW, Weber JL. Method for constructing confidently
ordered linkage maps. Genet Epidemiol 1999;16:337–343.
6. Gordon D, Abajian C, Green P. Consed: a graphical tool for
sequence finishing. Genome Res 1998;8:195–202.
7. Mizuguchi M. Acute necrotizing encephalopathy of childhood:
a novel form of acute encephalopathy prevalent in Japan and
Taiwan. Brain Dev 1997;19:81–92.
8. Campistol J, Gassio R, Pineda M, Fernandez-Alvarez E. Acute
necrotizing encephalopathy of childhood (infantile bilateral thalamic necrosis): two non-Japanese cases. Dev Med Child Neurol 1998;40:771–774.
9. Bassuk AG, Burrowes DM, McRae W. Acute necrotizing encephalopathy of childhood with radiographic progression over
10 hours. Neurology 2003;60:1552–1553.
10. DeVivo DC. Leigh syndrome: historical perspective and clinical
variations. Biofactors 1998;7:269 –271.
11. Victor M, Adams RD, Collins GH. The Wernicke-Korsakoff
syndrome and related neurologic disorders due to alcoholism
and malnutrition. 2nd ed. Philadelphia: F.A. Davis, 1989:231.
12. Hazell AS, Todd KG, Butterworth RF. Mechanisms of neuronal cell death in Wernicke encephalopathy. Metab Brain Dis
13. Langlais PJ, McRee RC, Nalwalk JA, Hough LB. Depletion of
brain histamine produces regionally selective protection against
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2002;17:199 –210.
Prion Deposition in
Olfactory Biopsy of Sporadic
Creutzfeldt–Jakob Disease
Massimo Tabaton, MD,1 Salvatore Monaco, MD,2
Maria Paola Cordone, MD,3 Monica Colucci, MD,1
Giorgio Giaccone, MD,4 Fabrizio Tagliavini, MD,4
and Gianluigi Zanusso, MD, PhD2
Currently, definite peripheral markers for the in vivo diagnosis of sporadic Creutzfeldt–Jakob disease (CJD) are
not available. Here, we report the presence of pathological prion protein in the olfactory mucosa of a case with
sporadic Creutzfeldt–Jakob disease. Prion protein immunoreactivity was detected in an olfactory biopsy performed 45 days after the disease onset, suggesting that
the involvement of olfactory epithelium is an early event
in sporadic Creutzfeldt–Jakob disease.
Ann Neurol 2004;55:294 –296
The central pathogenic event of human spongiform encephalopathies, also termed prion diseases, is the conformational conversion of the cellular prion protein
(PrPC) into protease-resistant forms, named PrPres.1
Sporadic Creutzfeldt–Jakob disease (CJD), the most
common human prion disease, is characterized by rapidly progressing dementia, pyramidal, extrapyramidal,
cerebellar and visual signs, myoclonus, and periodic
electroencephalographic sharp-wave complexes. The diagnosis of probable CJD is based on these features and
the presence of 14-3-3 protein in the cerebrospinal
fluid (CSF).2 In sporadic CJD, PrPres is exclusively detected within central nervous system, and the definite
diagnosis needs the postmortem identification of PrPres
in the brain by immunocytochemistry and immunoblotting.3 We recently reported the postmortem detection of PrPres in the olfactory epithelium of sporadic
CJD cases, suggesting that biopsy of olfactory mucosa
can allow the definite diagnosis in vivo.4
From the 1Department of Neurosciences, Ophthalmology, and Genetic, University of Genoa, Genoa; 2Department of Neurologic and
Visual Sciences, University of Verona, Verona; 3Department of
Otorhinolaryngology, University of Genoa, Genoa; and 4Neurological Institute C. Besta, Milan, Italy.
Received Aug 17, and in revised form Oct 20, 2003. Accepted for
publication Nov 11, 2003.
Address correspondence to Dr Tabaton, Department of Neurosciences, University of Genoa, Via De Toni 5, 16132 Genoa, Italy.
© 2004 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
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autosomal, 2q13, maps, encephalopathy, dominantly, 2q12, necrotizing, acute
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