Elizabeth C. Engle, MD
Associate Professor
of Neurology
Harvard Medical School/Children's Hospital
MRRC Project(s)
NIH/NEI R01 EY12498
Molecular Basis of Congenital Strabismus
NIH/NEI R01 EY13583
Genetic and Anatomic Basis of the Fibrosis Syndromes
NIH/NEI R01 EY15298
Genetic Etiologies of Horizontal Strabismus
NIH/NINDS P01 NS40828
Core Project, Gene Expressing in Normal and Diseased Muscle during
Development
Isolated
strabismus affects 1-5% of the general population. Most forms of strabismus
are multifactorial in origin and, while there is probably an inherited
component, the genetics of these disorders remain unclear. Prior to our
work, several forms of strabismus had been categorized together as the
'congenital fibrosis syndromes' based on their common presentation as
congenital, nonprogressive ophthalmoplegias with active limitation and
passive restriction of globe movement. These disorders included congenital
fibrosis of the extraocular muscles (CFEOM), Duane syndrome, strabismus
fixus, vertical retraction syndrome, and Brown syndrome. The restrictive
ophthalmoplegia, 'tight' feel of the extraocular muscles (EOMs) at surgery,
and finding of connective tissue on EOM biopsies led previous investigators
to propose that these disorders resulted from primary EOM fibrosis. Our
work focuses on those congenital fibrosis syndromes inherited as single
gene defects and is establishing that these disorders actually result
from distinct developmental defects of the oculomotor (nIII), trochlear
(nIV) and/or abducens (nVI) nuclei and their corresponding cranial nerves.
We have renamed these disorders the 'Congenital Cranial Dysinnervation
Disorders' or CCDDs.
Research Description and Major Results:
PRIMARILY VERTICAL CCDDs:
1. Congenital fibrosis of the extraocular muscles (CFEOM).
Individuals with CFEOM have restrictive ophthalmoplegia in the nIII and/or
nVI distribution. We have defined three inherited CFEOM phenotypes (CFEOM1,
CFEOM2, CFEOM3), mapped three CFEOM genetic loci (FEOM1, FEOM2, FEOM3),
and identified KIF21A and PHOX2A as the FEOM1 and FEOM2 genes, respectively.
a. CFEOM2 maps to chromosome 11 and result from mutations in PHOX2A
(ARIX). Our studies of CFEOM2 are based on four consanguineous Middle
Eastern pedigrees with autosomal recessive CFEOM. Affected members of
these pedigrees are born with bilateral ptosis with their eyes fixed primarily
in an exotropic position, with or without secondary hypertropia or hypotropia.
We first conducted a genome-wide linkage screen that led to the identification
of the FEOM2 locus on chromosome 11q13. Linkage refinement, homozygosity
mapping, and construction of a bacterial artificial chromosome physical
map by members of the lab allowed them to identify candidate genes in
the FEOM2 region. Sequence analysis of one of these genes, PHOX2A, revealed
three distinct mutations in the CFEOM2 pedigrees. Two mutations disrupt
splicing of PHOX2A, and the third mutation results in a conserved amino
acid substitution. Subsequently, we have identified a fourth mutation
that results in a nonsense change and is predicted to truncate the PHOX2A
protein just at the start of the homeodomain region. Other investigators
had established that, in mice and zebrafish, Phox2a encodes a homeodomain
transcription factor that plays a primary role in the development of nIII
and nVI alpha motor neurons. These findings correspond to the CFEOM2 phenotype,
and provided the first genetic proof that a congenital fibrosis syndrome
results from aberrant development of brainstem motor nuclei. This work
also represented the first report of a gene causing isolated monogenic
strabismus and emphasizes an important function of PHOX2A in human midbrain
motor neuron development.
b. CFEOM1 maps to chromosome 12 and results from mutations in KIF21A.
The CFEOM1 phenotype is the most prevalent of the CFEOM phenotypes. In
families with CFEOM1, all affected members are born with bilateral ptosis
and restrictive ophthalmoplegia. The primary vertical position of each
eye is downward and cannot be elevated above the midline. The results
of our postmortem exam of an affected member of a CFEOM1 family suggested
that the FEOM1 gene is necessary for normal development of a subset of
oculomotor subnuclei and/or the superior division of the oculomotor nerve,
and accounts for the CFEOM1 phenotype. To identify the CFEOM1 gene, we
conducted a genome wide screen of a large CFEOM1 family and mapped the
disorder to the centromeric region of chromosome 12, referred to as the
FEOM1 locus. Subsequently, we reported many additional CFEOM1 families
and refined the region to < 3-cM region. We have now identified the CFEOM1
gene as the developmental kinesin, KIF21A.
Kinesins are molecular motors responsible for microtubule-dependent transport
of cargo; in neurons, they are responsible for anterograde axonal transport.
We identified six different KIF21A mutations in 44 probands with CFEOM1.
Surprisingly, five of the six KIF21A CFEOM1 mutations alter only three
amino acid residues, each located in the 'a' position of a heptad repeat
within a coiled-coil region of the KIF21A stalk. The stalk is the site
of kinesin dimerization, and the alpha-helical heptad coiled-coil regions
are critical for the association and stability of the dimeric structure,
with the intertwined molecules touching at the 'a' and 'd' positions.
The location and recurrence of these KIF21A mutations suggest that they
may have a dominant negative effect by interfering with KIF21A's interaction
with itself or its unidentified partner(s). Therefore, CFEOM1 likely results
from the inability of mutated KIF21A to successfully deliver a cargo critical
to the development of the oculomotor axons, neuromuscular junction, or
extraocular muscles. We are now embarking on further studies of KIF21A,
including the identification of its cargo and interacting proteins. These
experiments should help elucidate why the oculomotor axis is selectively
vulnerable to these mutations and lead to insights into the role of the
kinesin stalk in health and disease.
c. CFEOM3 maps to chromosome 16. We have defined the CFEOM3 phenotype
based on our studies of families with autosomal dominant CFEOM that do
not meet CFEOM1 criteria. These individuals have restrictive ophthalmoplegia
in the nIII and/or nVI distribution but, unlike those families with CFEOM1,
one or more affected family members are able to raise one or both eyes
above the horizontal midline or have unilateral rather than bilateral
ptosis or ophthalmoplegia. We established one locus for CFEOM3, FEOM3,
located within a 5-cM region of chromosome 16qter, but have not yet identified
this disease gene. In addition, we identified one large CFEOM3 pedigree
whose phenotype maps to the FEOM1 locus.
Recently, we screened 17 of our CFEOM3 probands for KIF21A mutations,
and identified mutations in 2 CFEOM3 pedigrees. One pedigree harbored
a novel KIF21A mutation (2841G'A, M947I) and one harbored the most common
and recurrent of the CFEOM1 mutations we identified previously (2860C'T,
R954W). None of CFEOM3 pedigrees or sporadic individuals harbored mutations
in PHOX2A. This study demonstrated that KIF21A mutations are a rare cause
of CFEOM3 and that KIF21A mutations can be non-penetrant. Although KIF21A
is the first gene to be associated with CFEOM3, our results imply that
mutations in the unidentified FEOM3 gene will be the more common cause
of this phenotype.
2. Congenital Ptosis.
a. Congenital ptosis maps to chromosome 1. Congenital ptosis is a
common and selective neuromuscular defect with isolated levator dysfunction.
Linkage analysis of a family with 147 members, 61 of whom are affected
by congenital ptosis, led to the identification of the PTOS1 locus on
chromosome 1p. We have not identified the PTOS1 gene.
PRIMARILY HORIZONTAL CCDDs
1. Duane syndrome.
Duane syndrome is the most common of the CCDDs, and primarily affects
horizontal eye movement. Neuropathologic studies of sporadic cases of
Duane syndrome have been conducted by other investigators and demonstrate
absence or hypoplasia of nVI and the abducens nerve, with aberrant innervation
of the lateral rectus muscle. These findings suggest that genes mutated
in Duane syndrome are necessary for nVI development or axonal targeting.
Although Duane syndrome is the most common of the congenital fibrosis
syndromes, there are only rare families who clearly transmit the disorder
as a Mendelian trait.
a. Duane syndrome with radial ray anomalies maps to chromosome 20 and
results from mutations in SALL4. Duane syndrome can occur with associated
anomalies, and we ascertained three families with Duane syndrome associated
with thumb and forearm anomalies (Duane radial ray syndrome or Okihiro
syndrome). We mapped this disease gene to chromosome 20 and recently identified
the disease gene as SALL4, a previously unpublished gene that becomes
the fourth member of the SAL family of C2H2-type zinc finger proteins.
b. Isolated Duane syndrome that maps to the DURS2 locus. In 1999,
Appukuttan et al ascertained a 4-generation family with fully penetrant
isolated Duane syndrome and mapped their phenotype to a 17.8-cM region
of chromosome 2q31. Of the 25 affected participants, 80% had DS-I and
20% DS-III, 96% had bilateral DS and 76% had strabismus in primary gaze
(10 esotropic, 1 exotropic, 8 manifest hypertropia, 4 dissociated vertical
deviation). In addition, 48% had amblyopia, 12% had trochlear nerve palsy,
and a majority had vertical as well as horizontal movement abnormalities.
Two (8%) did not have retraction (similar to 5% in Duane's original study).
In 2000, Evans et al analyzed a 4-generation British pedigree with fully
penetrant isolated DS and confirmed linkage to the DURS2 locus, and a
recombination event reduced the critical region to 8.8 cM. The HOXD gene
cluster falls within the DURS2 region but no mutations of HOXD1, HOXD3,
and HOXD4 were identified in either family. We are now studying several
pedigrees that map to this locus in an attempt to identify the DURS2 gene.
2. Horizontal gaze palsy.
Horizontal gaze palsy is rarely reported in isolation and may be misdiagnosed
as Duane syndrome type III. It can occur together with facial weakness
and be classified as Mńbius syndrome. The only consistently inherited
form of congenital horizontal gaze palsy, however, is when is when it
is co-inherited with progressive scoliosis as HGPPS.
a. Horizontal gaze palsy with progressive scoliosis. HGPPS is a
rare recessive CCDD that has been reported in several dozen consanguineous
families of various ethnic backgrounds. Affected individuals are born
with horizontal gaze palsy and, during the first decade of life, develop
progressive scoliosis. Jen et al identified two HGPPS pedigrees and mapped
their disease gene by homozygosity to a 30 cM region on chromosome 11q23-q25.
We have identified five HGPPS pedigrees and all are consistent with linkage
to this chromosome 11 locus. Dr. Mac Bosley has conducted physiologic
studies of individuals with HGPPS and determined that their descending
corticospinal pathway and ascending sensory projections appear to be uncrossed.
In collaboration with Drs. Jen and Bosley, we have identified mutations
in the gene ROBO3 in 10 HGPPS pedigrees. The ROBO3 gene shares homology
with the roundabout genes that are important in axon guidance in developing
Drosophila, zebrafish, and mouse. The human ROBO3 protein is predicted
to be most similar to its mouse homolog, rig1, and unlike other robo proteins,
ROBO3 and rig1 appear to be necessary for hindbrain axon midline crossing.
HGPPS represents the first human disorder to be attributed to a gene that
functions in axon guidance within the central nervous system, and ROBO3
is the first gene to be implicated in scoliosis. Future studies should
help determine how ROBO3 mutations lead to horizontal gaze palsy and scoliosis.
Publications
Engle
EC, Kunkel LM, Specht LA, Beggs AH. Linkage mapping of a gene for congenital
fibrosis of the extraocular muscles to the centromeric region of chromosome
12. Nature Genet 1994; 7:69-73.
Khurana TS, Engle EC, Bennett RR, Silverman G, Selig S, Bruns GAP, Kunkel
LM. (CA) repeat polymorphism in the chromosome 18 encoded dystrophin-like
protein. Hum Molec Genet 1994; 3:841.
Engle EC, Hedley-Whyte ET. A 29-month-old girl with worsening ataxia,
nystagmus, and subsequent opsoclonus and myoclonus. Case record 27-1995
of the Massachusetts General Hospital. N Eng J Med 1995; 333:579-586.
Engle EC, Marondel I, Houtman WA, deVries B, Loewenstein A, Lazar M, Ward
DC, Kucherlapati R, Beggs AH. Congenital fibrosis of the extraocular muscles
(autosomal dominant congenital external ophthalmopleigia): Genetic homogeneity,
linkage refinement, and physical mapping on chromosome 12. Am J Hum
Genet 1995 57:1086-1094.
Engle EC, Holtzman D. Benign intracranial hypertension. In Burg FD, Ingelfinger
JR, Wald ER. (Eds.) Gellis and Kagan's Current Pediatric Therapy 15. Philadelphia:
WB Suanders, Inc. 1996; 109-110.
Engle EC, Goumernov BC, McKeown CA, Schatz M, Johns DR, Porter JD, Beggs
AH. Oculomotor nerve and muscle abnormalities in congenital fibrosis of
the extraocular muscles. Ann Neurol 1997; 41:314-325.
Engle EC, Castro AE, Macy ME, Knoll JHM, Beggs AH. A gene for isolated
congenital ptosis maps to a 3cM region within 1p32-p34.1. Am J Hum
Genet 1997; 60:1150-1157.
Swoboda KJ, Engle EC, Scheindlin B, Anthony DC, Jones HR. Mutilating hand
syndrome in an infant with familial carpal tunnel syndrome. Nerve &
Muscle 1998;21, 104-11.
Wang SM, Zwann J, Mullaney PB, Jabak MH, Al-Awad A, Beggs AH, Engle EC.
Congenital fibrosis of the Extraocular Muscles type 2, an inherited exotropic
strabismus fixus, maps to distal 11q13. Am J Hum Genet 1998;63(2):517-25.
Engle EC. The genetics of strabismus: Duane, Moebius and Fibrosis syndromes.
In: Traboulsi EI. (Ed). Genetic Diseases of the Eye: A Textbook and Atlas.
NY: Oxford University Press 1998; 477-512.
Doherty EJ, Macy ME, Wang SM, Dykeman CP, Melanson MT, Engle EC. CFEOM3:
a new extraocular fibrosis syndrome that maps to 16q24.2-q24.3. IOVS
1999;40(8):1687-1694.
Engle E. A genetic approach to congenital extraocular muscle disorders.
J Child Neurol 1999; 14:34-37.
Traboulsi EI, Lee BA, Mousawi A, Khamis AR, Engle EC. Evidence of genetic
heterogeneity in autosomal recessive Congenital Fibrosis of the Extraocular
Muscles (CFEOM). Am J Ophthalmol 2000;129:658-662.
Sener EC, Lee BA, Turgut B, Akarsu AN, Engle EC. A clinically variant
fibrosis syndrome in a Turkish family maps to the CFEOM1 locus on chromosome
12. Arch Ophthalmol 2000, 118:1090-1097.
Engle EC, Al-Braradie R, McIntosh N, Fain J, and Martonyi J. Identifying
the genetic locus for Duane syndrome with radial ray anolmalies (Okihiro
syndrome). Am J Human Genet 2001;67(4S):307.
Flaherty MP, Grattan-Smith P, Steinberg A, Jemieson R, Engle EC. Congenital
fibrosis of the extraocular muscles associated with cortical dysplasia
and maldevelopment of the basal ganglia. Ophthalmology 2001;08:1313-1322.
Shivaram SM, Engle EC, Petersen RA, Robb RM. Congenital Fibrosis Syndromes.
Int Ophthalmol Clin 2001 Fall;41(4):105-13.
Nakano M, Yamada K, Fain J, Sener EC, Selleck CJ, Awad AH, Zwaan J, Mullaney
PB, Bosley TM, Engle EC. Homozygous mutations in ARIX (PHOX2A) result
in congenital fibrosis of the extraocular muscles type 2 (CFEOM2). Nature
Genetics 2001;29:315-320.
Engle EC, McIntosh N, Yamada K, Lee BA, Johnson R, O'Keefe M, Letson R,
London A, Ballard E, Ruttum M, Matsumoto N, Saito N, Collins MLZ, Morris
L, Del Monte M, Magli A, de Berardinis T. CFEOM1, the classical form of
congenital fibrosis of the extraocular muscles, is genetically heterogeneous
but does not result from mutations in ARIX. BMC Genetics, BMC Genetics
2002, 3:3.
Mackey DA, Chan, W-M, Chan C, Gillies WE, Brooks AMV, O'Day J, Engle EC.
Congenital fibrosis of the vertically acting extraocular muscles maps
to the FEOM3 locus. Human Genetics 2002, 110:510-512.
Engle EC Applications of molecular genetics to understanding congenital
ocular motility disorders. NY Acad Sci 2002;956:55-63.
Engle EC, Leigh RJ. Genes, brainstem development, and eye movements. Neurology
2002, 13;59(3):304-5.
Engle EC. The molecular basis of the congenital fibrosis syndromes. In:
Eds B Lorenz and J.H. Simonsz, Proceedings of the 8th Annual Meeting of
the Bielschowsky Gesellschaft, Regensburt, Germany, July 6th to 8th 2001.
Strabismus 2002;10(2):125-128.
Al-Baradie R, Yamada K, St Hilaire C, Chan WM, Andrews C, McIntosh N,
Nakano M, Martonyi EJ, Raymond WR, Okumura S, Okihiro MM, Engle EC. Duane
radial ray syndrome (Okihiro syndrome) maps to 20q13 and results from
mutations in SALL4, a new member of the SAL family. Am J Hum Genet
2002;71(5):1195-9.
Iannaccone A, McIntosh N, Ciccarelli ML, Baldi A, Mutolo PA, Tedesco SA,
Engle EC. Familial unilateral Brown syndrome. Ophthalmic Genet
2002;23(3):175-84.
Engle EC. The molecular basis of the congenital fibrosis syndromes. Strabismus
2002;10(2):125-8.
Engle EC. Applications of molecular genetics to the understanding of congenital
ocular motility disorders. Ann NY Acad Sci 2002;956:55-63.
Ryan MM, Stasheff SF, Engle EC. Disorders of the ocular motor cranial
nerves and extraocular muscles. In: Neuromuscular Disease of Infancy
and Childhood: A Clinician's Approach. Jones RH, DeVivo D, Darras
BT. (eds). Boston: Butterworth-Heinemann, 2002;1075-1126.
Gutowski NJ, Bosley TM, Engle EC. 110th ENMC International Workshop: the
congenital cranial dysinnervation disorders (CCDDs). Naarden, The Netherlands,
25-27 October, 2002. Neuromuscul Disord 2003;13(7-8):573-8.
Gottlob I, Jain S, Engle EC. Elevation of one eye during tooth brushing.
Am J Ophthalmol 2002;134(3):459-60.
Yazdani A, Chung DC, Abbaszadegan MR, Al-Khayer K, Chan WM, Yazdani M,
Ghodsi K, Engle EC, Traboulsi EI. A novel PHOX2A/ARIX mutation in an Iranian
family with congenital fibrosis of extraocular muscles type 2 (CFEOM2).
Am J Ophthalmol 2003;136(5):861-5.
Yamada K, Andrews C, Chan WM, McKeown CA, Magli A, de Berardinis T, Loewenstein
A, Lazar M, O'Keefe M, Letson R, London A, Ruttum M, Matsumoto N, Saito
N, Morris L, Del Monte M, Johnson RH, Uyama E, Houtman WA, de Vries B,
Carlow TJ, Hart BL, Krawiecki N, Shoffner J, Vogel MC, Katowitz J, Goldstein
SM, Levin AV, Sener EC, Ozturk BT, Akarsu AN, Brodsky MC, Hanisch F, Cruse
RP, Zubcov AA, Robb RM, Roggenkaemper P, Gottlob I, Kowal L, Battu R,
Traboulsi EI, Franceschini P, Newlin A, Demer JL, Engle EC. Heterozygous
mutations of the kinesin KIF21A in congenital fibrosis of the extraocular
muscles type 1 (CFEOM1). Nat Genet 2003;35(4):318-21.
Uyama E, Yamada K, Kawano H, Chan WM, Andrews C, Yoshioka M, Uchino M,
Engle EC. A Japanese family with FEOM1-linked congenital fibrosis of the
extraocular muscles type 1 (CFEOM1) associated with spinal canal stenosis
and refinement of the FEOM1 critical region. Neuromuscl Disord
2003;13(6):472-8.
Ryan MM, Engle EC. Acute ataxia in childhood. J Child Neurol 2003;18(5):309-16.
Pieh C, Goebel HH, Engle EC, Gottlob I. Congenital fibrosis syndrome associated
with central nervous system abnormalities. Graefes Arch Clin Exp Ophthalmol
2003;241(7):546-53.
Yamada K, Chan W-M, Andrews C, Bosley TM, Sener EC, Zwaan JT, Mullaney
TZ, —zt˝rk BT, Akarsu AN, Sabol LJ, Demer JL, Sullivan TJ, Gottlob I,
Roggenkîemper P, Mackey DA, de Uzcategui CE, Uzcategui N, Ben-Zeev B,
Traboulsi EI, Magli A, de Berardinis T, Gagliardi V, Awasthi-Patney S,
Vogel MC, Rizzo JF, Engle EC. KIF21A mutations are a rare cause of congenital
fibrosis of the extraocular muscles type 3 (CFEOM3). Invest Ophthalmol
Vis Sci 2004, in press.
Jen JC, Chan W-M, Bosley TM, Wan J, Carr JR, R˝b U, Shattuck D, Salamon
G, Kudo L, Ou J, Lin DDM, Salih MAM, Kansu T, Dhalaan H, Al Zayed Z, MacDonald
DB, Stigsby B, Plaitakis A, Dretakis EK, Gottlob I, Pieh C, Traboulsi
EI, Wang Q, Wang L, Andrews C, Yamada K, Demer JL, Karim S, Alger J, Geschwind
DH, Deller T, Sicotte NL, Nelson SF, Baloh RW, Engle EC. Mutations in
a human robo gene disrupt hindbrain axon pathway crossing and morphogenesis.
Science 2004, in press.
See Dr. Engle's publications via PubMed.
Contact Information
E-mail:
Elizabeth C. Engle, MD
Associate Professor of Neurology
Harvard Medical School/Children's Hospital
Lab Members:
Caroline Andrews -
candrews@enders.tch.harvard.edu
Wai-Man (Jo) Chan - wchan@enders.tch.harvard.edu
Prashant Dilwali
Cynthia St. Hilaire - csthilai@genetics.med.harvard.edu
Krystal Law - klaw@fas.harvard.edu
Carlos Miranda
Maria Pia Rogines Velo - piasardi@enders.tch.harvard.edu
Koki Yamada - kyamada@enders.tch.harvard.edu
