Current advances in Holt-Oram syndrome
Taosheng Huang, MD, PhD
Holt-Oram syndrome is an autosomal-dominant condition
characterized by congenital cardiac and forelimb anomalies. It
is caused by mutations of the TBX5 gene, a member of the
T-box family that encodes a transcription factor. Molecular
studies have demonstrated that mutations predicted to create
null alleles cause substantial abnormalities in both the limbs
and heart, and that missense mutations of TBX5 can produce
distinct phenotypes. One class of missense mutations causes
significant cardiac malformations but only minor skeletal
abnormalities; others might cause extensive upper limb
malformations but less significant cardiac abnormalities.
Intrafamilial variations of the malformations strongly suggest
that genetic background or modifier genes play an important
role in the phenotypic expression of HOS. Efforts to
understand the intracellular pathway of TBX5 would provide a
unique window onto the molecular basis of common
congenital heart diseases and limb malformations. Curr Opin
Pediatr 2002, 14:691–695 © 2002 Lippincott Williams & Wilkins, Inc.
Clinical features
Holt and Oram first described this syndrome when they
reported on a family with atrial septal defects and congenital anomalies of the thumbs [1]. Since then, about
200 clinical papers have been published that further delineate the clinical features of Holt-Oram syndrome
(HOS). The prevalence of HOS is 1 of 100,000 live
births, and it occurs with wide ethnic and geographic
distribution. Its clinical manifestations have proved to be
variable [2,3•,4•], but with complete penetrance. All patients with HOS have upper limb anomaly and about
85% to 95% have cardiac malformation. On the basis of
these findings, the criteria for diagnosis include either
the presence of cardiac malformations, conduction defects and radial ray abnormalities (or both) in an individual, or the presence of radial ray abnormalities with or
without cardiac malformations or conduction defects in
individuals with a family history of HOS [5••]. The family history should be consistent with autosomal-dominant
inheritance.
Cardiac defects
Division of Human Genetics, Department of Pediatrics, University of California,
Irvine, California, USA.
Correspondence to Taosheng Huang, MD, PhD, Division of Genetics, Department
of Pediatrics, Med-Sci, C202, University California, College of Medicine, Irvine, CA
92697, USA; e-mail: [email protected]
Current Opinion in Pediatrics 2002, 14:691–695
Abbreviations
ASD
HOS
VSD
atrial septal defect
Holt-Oram syndrome
ventricular septal defect
ISSN 1040–8703 © 2002 Lippincott Williams & Wilkins, Inc.
Secundum-type atrial septal defect (ASD) and ventricular septal defect (VSD) are the most common heart defects. Other cardiac defects range from asymptomatic
conduction disturbances (first-degree heart block) to
multiple structural defects. Almost every type of cardiac
anomaly has been reported, either singly or as part of a
group of multiple defects [6–8]. Sudden death from heart
block has been reported. Bruneau et al. summarize the
defects in 240 patients [9••]. Among these patients, 58%
had ASD, and 28% have VSD. Less common anomalies,
such as conduction defect, truncus arteriosus, mitral
valve defect, patent ductus arteriosus, and tetralogy of
Fallot, occur in 18%, 8%, 4%, 4%, and 3%, respectively.
In an earlier series of studies [3•], heart defects in
189 patients were classified by severity. Among these
patients, 66% had single abnormalities, including isolated conduction defects; 16% had “mild” combinations
consisting of two or three malformations (eg, ASD, VSD);
11% had “moderate” combinations that required more
complicated surgical repair (eg, tetralogy of Fallot and
endocardial cushion defect); and 6% had “severe” combinations with life-threatening defects, including hypoplastic left heart, total anomalous pulmonary venous return, and truncus arteriosus.
Diagnosis of heart defects requires electrocardiography
and two-dimensional echocardiography with doppler.
691
692 Genetics
Cardiac catheterization may be required to fully define a
defect.
Upper limb anomalies
Skeletal abnormalities affect the upper limbs exclusively; lower limb abnormalities have not been reported.
The abnormalities are always bilateral and often asymmetric, predominantly involving the radial ray. The
thumb is the most commonly affected structure and can
be triphalangeal, hypoplastic, or completely absent. Abnormalities range from minor (clinodactyly of the fingers,
limited supination of the forearms, and sloping shoulders) to severe (reduction deformities, including phocomelia and ectromelia). Clinical recognition of subtle limb
anomalies in patients with HOS can require both physical examination and radiographs of the upper extremities.
Poznanski et al. demonstrated that carpal abnormalities
are more specific for HOS than are changes in the thumb
[10]. Other radiographic abnormalities include posteriorly and laterally protuberant medial epicondyles of the
humerus, hypoplastic clavicles, shortened radii, and
ulnar hypoplasia (occurring only in patients with radial
defects).
Overlapping conditions and differential diagnosis
Other congenital malformations reported with cardiac
malformation and upper limb anomalies, include lung
hypoplasia and cardiomyopathy, postaxial or central
polydactyly, arachnodactyly, thoracic scoliosis, hemiatrophy of the body, high myopia, Hirschsprung disease,
malformations of the urinary system, the RokitanskyKuster-Hauser syndrome, cryptorchidism, malformations
of renal and cerebral arteries, hypoplastic peripheral upper extremity vasculature, hypoplasia of the left radial
artery, pulmonary hypertension, multiple strokes and
end-stage renal failure, and malignant tumors [11–21].
These reports probably reflect fortuitous occurrences or
represent different conditions. To date, no mutations in
TBX5 have been found in individuals with “atypical”
phenotypes (Huang et al., unpublished data).
The following autosomal-dominant conditions need to
be considered for differential diagnosis:
• Fanconi anemia syndrome is characterized by congenital abnormalities. These abnormalities include
malformations of the thumbs, forearms, and heart;
progressive bone marrow failure with pancytopenia,
typically in the first decade; and increased risk for
myelodysplasia or acute myelogenous leukemia. The
diagnosis of Fanconi anemia syndrome relies on detection of chromosomal breakage or rearrangements
in the presence of diepoxybutane or mitomycin C.
• Thrombocytopenia-absent radius: both radii are always absent; the thumbs are always present. By contrast, radial aplasia in HOS is invariably associated
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with hypoplasia or absence of the thumb [4]. Phocomelia occasionally occurs. The lower limbs can be
involved, including club foot and instability of the
knee. Thrombocytopenia, present in infancy, generally improves with time. Heart defects can be
present.
Heart-hand syndrome II (Tabatznik): type D brachydactyly (shortening of the distal phalanx of the thumb
with or without shortening of the fourth and fifth
metacarpals), sloping shoulders, short upper limbs,
bowing of the distal radii, and absence of the styloid
process of the ulna with supraventricular tachycardia.
Patients can also have mild facial dysmorphism and
mild mental retardation [22].
Heart-hand syndrome III: type C brachydactyly
(shortening of the middle phalanges) with an accessory wedged-shaped ossicle on the proximal phalanx
of the index fingers with sick sinus syndrome [23].
Okihiro syndrome: Duane syndrome (a congenital
eye-movement disorder resulting from abnormal development of cranial nerve VI and characterized by
absence of abduction of the globe and narrowing of
the palpebral fissure on adduction of the globe), upper extremity reduction defects, and cardiac malformation [24].
Long thumb brachydactyly syndrome: elongation of
the thumb distal to the proximal interphalangeal
joint, often associated with index finger brachydactyly, clinodactyly, narrow shoulders, secondary short
clavicles, and pectus excavatum. Occasionally, rhizomelic limb shortening occurs. The cardiac abnormality is often a conductive defect [25].
Vertebral, anal, cardiac, tracheal, esophageal, renal,
and limb (VACTERL) anomalies association: radial
defects are usually unilateral and accompanied by
characteristic other malformations (ie, imperforate
anus, tracheoesophageal [TE] fistula).
Genetic counseling and management
Genetic counseling should be provided to all patients
with HOS. Of probands, 60% to 70% have an affected
parent, and 30% to 40% have a de novo mutation. Evaluation of both parents is recommended, including physical examination and radiographs of the upper extremities
to detect subtle changes of the thumb and carpal bones,
and examination of the heart, including electrocardiogram and echocardiogram are recommended.
Risk to siblings depends on the genetic status of the
parents. If one of the parents is affected, the siblings of
a proband have a 50% risk of inheriting the diseasecausing mutation. When the parents are clinically unaffected, the risk to the siblings of a proband appears to be
low. Each individual with HOS has a 50% chance of
inheriting the disease-causing mutation.
Current advances in Holt-Oram syndrome Huang 693
For individuals with conduction defects, regular electrocardiograms are recommended, as conduction defects
can get worse with time. Many patients with severe atrioventricular block will need pacemakers. Antiarrhythmic
drugs have been used for patients with atrioventricuolar
block, but not for prophylaxis.
Figure 1. Three-dimensional structure of T-box and the
missense mutations found in HOS
Molecular studies
The disease gene for HOS, which was linked to the
chromosome 12q2 region by studying multiple unrelated
families [26•,27•,28•], was identified as TBX5, a member of the T-box gene family [29•,30•]. Mutations in the
TBX5 gene were demonstrated in many affected individuals and families [5••,29•,30•, 31•,32•]. The coding
region of TBX5 cDNA is 1.5 kb with 8 exons. The DNAbinding domain of a TBX5 protein is composed of 180
amino acid residues (amino acid residues 56 to 236) and
binds to a 24-nucleotide palindromic DNA duplex in
vitro [33•]. It is possible that the protein binds as a dimer
interacting with the major and minor grooves of the
DNA [34].
Molecular genetic testing of TBX5 is currently available
only on a research basis. Using gene sequencing of the
TBX5 coding regions or mutation scanning (single-strand
conformation polymorphism [SSCP] followed by sequencing of exons with abnormal band patterns), the
mutation detection rate in the TBX5 coding region
ranges from 20% to 55% in familial cases and from 15%
to 40% in isolated cases with apparently negative family
history [31•] (Huang et al., unpublished data). Mutations
are distributed throughout the gene, with a few “hot
spots” (codon 273) [5••,29•] and nucleotide 824 [30•].
Most mutations lead to premature termination of the
protein product. The disease is probably caused by haploinsufficiency of TBX5 [5••,35].
Some tentative genotype-phenotype correlations have
been established. Mutations predicted to create null alleles caused substantial abnormalities both in limb and
heart [5••]. In one study, patients with frameshift mutations had severe phecomelia, whereas those with missense mutations had normal arms and absent or hypoplastic thumbs [32•]. Missense mutations of TBX5 may
have distinct phenotypes. G80R caused significant cardiac malformations but only minor skeletal abnormalities, whereas mutations at amino acid 237 cause severe
skeletal abnormalities, but minor cardiac malformations
[5••] (Fig. 1). However, intrafamilial variations do exist.
Basson et al. [26• ]reported two large families with different mutations and showed an interfamilial pattern and
intrafamilial variations. It seems that TBX5 mutations
serve as gross tune and that other factors (eg, genetic
background) serve as a “finer tuning” for the phenotypic
expression of HOS.
To determine the roles of background modifier genes
and environment in the phenotypic expression of HOS,
Three-dimensional structure of the Xbra T-box bound to a 24 bp DNA target, as
a model for the human TBX5 gene. The mutations found in human HOS are I54
(purple), G169 (red), G80 (yellow), and R237 (green). Some missense
mutations may interfere with protein-DNA interactions, others may interrupt
protein-protein interactions.
we identified a pair of identical twins affected with HOS
and compared the clinical features in such genetically
identical individuals (monozygotic twins) (Huang et al.,
unpublished data). Such a comparison provides a tremendous lens onto the role of genetic background and
other factors that might contribute to the phenotypic
expression of HOS. The twins were first diagnosed
based on their hand and heart abnormalities, and their
monozygosity was confirmed by genotyping. The same
1-base pair (bp) deletion was detected in both twins. The
deletion was predicted to cause a frameshift in the TBX5
coding region, which would be truncated at amino acid
residue 263. The defective allele probably encoded an
inactive TBX5. Then we analyzed their clinical features.
Both twins had similar complex cardiac defects, including secundum ASD, a large membranous VSD, and multiple muscular defects. They also had identical forearm
defects: hypoplastic bilateral radial bones, radial club and
delayed carpal ossification. However, the hands of the
twins were not identical: one twin lacked both thumbs,
whereas the other had a remnant of the distal and proximal phalanges of the right thumb and distal phalanx hypoplasia of the left thumb. Thus, although the clinical
features of the twins were strikingly similar compared
with the wide range of phenotypes observed among individuals bearing the same TBX5 mutations, the discordant features do exist, suggesting that genetic background alone cannot explain discordant features in
monozygotic twins. Furthermore, most patients with
HOS show asymmetric limb anomalies. Thus far, it
seems that all genes identified to be involved in limb
development are bilaterally expressed in the developing
694 Genetics
Smith AT, Sack GH Jr, Taylor GJ: Holt-Oram syndrome. J Pediatr 1979,
95:538–543.
limb. These observations suggest that factors other than
the TBX5 mutation itself and genetic background might
contribute to this phenotypic variability.
2
TBX5 intracellular pathway and prospects
4
Newbury-Ecob RA, Leanage R, Raeburn JA, et al.: Holt-Oram syndrome: a
clinical genetic study. J Med Genet 1996, 33:300–307.
•
Summarizes the clinical spectrum of HOS in more than 200 patients.
It is likely that elucidation of the TBX5 intracellular
pathway will open a new venue to study the causes of
common congenital heart and limb anomalies. TBX5 was
found to bind to the T-box binding elements in vitro
[33•]. It is possible to identify the candidate genes that
might be regulated by TBX5 by searching the human
genome database using consensus binding sequences.
The genes, particularly those expressed in the developing heart and limbs, would be promising candidates for
congenital heart disease and limb malformation. Because
TBX5 binding specificity could be determined by a complex of molecules and such cofactors could be heterogeneous, such a nextwork could be complex.
By analyzing Tbx5 knockout mice, Bruneau et al. have
shown that several genes are regulated by Tbx5, including atrial natriuretic factor (ANF) and connexin 40
(Cx40) [36••]. Availability of such an animal model allows us to analyze the gene expression pattern using a
DNA chip and, therefore, to identify the TBX5 targets
that might play important roles in heart development
and even be involved in human congenital heart diseases.
TBX5 as a transcription factor was found to interact with
NKx2.5 and to synergetically regulate other genes
[36••,37•]. However, TBX5 may have other partners,
some of them tissue-specific and others spatially regulated. Understanding its protein-protein interactions in a
developing heart can also lead to the identification of
candidate genes involved in common congenital heart
diseases.
The availability of a TBX5 promoter DNA sequence
would facilitate isolation of the gene(s) regulating TBX5
expression. We believe these efforts will lead to the
identification of the molecular cascades of TBX5 and
could provide a unique window onto the causes of
more common congenital heart diseases and limb
malformations.
Acknowledgments
This work is supported by NIH grant M01 RR00827–28S2. Support is also acknowledged from the Howard Hughes Medical Institute’s Biomedical Research
Support Program.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
•
Of special interest
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Of outstanding interest
1
Holt M, Oram S: Familial heart disease with skeletal malformations. Br Heart
J 1960, 22:236–242.
3
Sletten LJ, Pierpont ME: Variation in severity of cardiac disease in Holt-Oram
syndrome. Am J Med Genet 1996, 65:128–132.
•
Describes the spectrum of heart defects in 189 patients by severity.
Basson CT, Huang T, Lin RC, et al.: Different TBX5 interactions in heart and
limb defined by Holt-Oram syndrome mutations. Proc Natl Acad Sci U S A
1999, 96:2919–24.
This article established the initial genotype-phenotype correlations. Ten different
TBX5 mutations were reported. Defects predicted to create null alleles caused
substantial abnormalities in both the limbs and heart. In contrast, missense mutations produced distinct phenotypes: G80R caused significant cardiac malformations but only minor skeletal abnormalities, and R237Q and R237Y caused extensive upper limb malformations but less significant cardiac abnormalities. Amino
acids altered by missense mutations were located on the three-dimensional structure of a related T-box transcription factor, Xbra, bound to DNA. Residue 80 is
highly conserved within T-box sequences that interact with the major groove of
target DNA; residue 237 is located in the T-box domain that selectively binds to the
minor groove of DNA.
5
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6
Glauser TA, Zackai E, Weinberg P, et al.: Holt-Oram syndrome associated
with the hypoplastic left heart syndrome. Clin Genet 1989, 36:69–72.
7
Sahn DJ, Goldberg SJ, Allen HD, et al.: Cross-sectional echocardiographic
imaging of supracardiac total anomalous pulmonary venous drainage to a
vertical vein in a patient with Holt-Oram syndrome. Chest 1981, 79:113–
115.
8
Wu JM, Young ML, Wang TR, et al.: Unusual cardiac malformations in HoltOram syndrome: report of two cases. Zhonghua Min Guo Xiao Er Ke Yi Xue
Hui Za Zhi 1991, 32:100–104.
Bruneau BG, Logan M, Davis N, et al.: Chamber-specific cardiac expression
of Tbx5 and heart defects in Holt-Oram syndrome. Dev Biol 1999, 211:100–
108.
The authors examine TBX5 expression pattern in the developing mouse and chick
heart and find that TBX5 expression pattern correlates well with cardiac defects
reported in more than 200 patients with HOS in the literature.
9
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10
Poznanski AK, Gall JC Jr, Stern AM: Skeletal manifestations of the Holt-Oram
syndrome. Radiology 1970, 94:45–53.
11
Moens P, De Smet L, Fabry G, et al.: Severe Holt-Oram syndrome with pulmonary hypertension. Am Heart J 1991, 122:250–252.
12
Fryns JP, Bonnet D, De Smet L: Holt-Oram syndrome with associated postaxial and central polydactyly. Further evidence for genetic heterogeneity in the
Holt-Oram syndrome. Genet Couns 1996, 7:323–324.
13
Sunday QB, Zhang KZ, Cheng TO: Holt-Oram syndrome with high myopia. A
hitherto unreported association in a Chinese patient. Am J Med 1987,
82:326–328.
14
Frontera-Izquierdo P, Cabezuelo-Huerta G: Severe Holt-Oram syndrome with
pulmonary hypertension. Am Heart J 1991, 122:250–252.
15
Dogliani P, De Sanctis C, Balocco A: Holt-Oram syndrome with malformations of the urinary system. Minerva Pediatr 1973, 25:1002–1006.
16
Fakih MH, Williamson HO, Seymour EQ, et al.: Concurrence of the Holt-Oram
syndrome and the Rokitansky-Kuster-Hauser syndrome. A case report. J Reprod Med 1987, 32:549–550.
17
Koutlas ED, Papageorgiou AA, Athyros VG: Holt-Oram syndrome with malformations of renal and cerebral arteries. Acta Cardiol 1996, 51:373–376.
18
DuPre CT, Fincher RM: Holt-Oram syndrome associated with hypoplastic
peripheral vasculature and midsystolic click. South Med J 1993, 86:453–
456.
19
Mittal SR, Sethi A, Verma GL: An unusual case of Holt-Oram syndrome. Int J
Cardiol 1984, 6:740–742.
20
Rabinowitz JG, Camera A, Oran E: Holt Oram syndrome associated with
carcinoma. Clin Radiol 1971, 22:346–349.
21
Nik-Akhtar B, Khakpour M, Rashed MA, et al.: Association of Holt-Oram syndrome and lymphosarcoma. Chest 1974, 66:729–731.
22
Silengo MC, Biagioli M, Guala A, et al.: Heart-hand syndrome II. A report of
Tabatznik syndrome with new findings. Clin Genet 1990, 38:105–113.
23
Ruiz de la Fuente S, Prieto F: Heart-hand syndrome. III. A new syndrome in
three generations. Hum Genet 1980, 55:43–47.
24
Okihiro MM, Tasaki T, Nakano KK, et al.: Duane syndrome and congenital
Current advances in Holt-Oram syndrome Huang 695
upper-limb anomalies. A familial occurrence. Arch Neurol 1977, 34:174–
179.
25
Hollister DW, Hollister WG: The “long-thumb” brachydactyly syndrome. Am J
Med Genet 1981, 8:5–16.
Basson CT, Cowley GS, Solomon SD, et al.: The clinical and genetic spectrum of the Holt-Oram syndrome (heart-hand syndrome) N Engl J Med 1994,
330:885–891.
The disease locus was linked to human chromosome 12q24.1 in two large families,
which showed that an intrafamilial pattern of abnormalities does exist. Family A has
19 affected individuals. Clinical evaluation showed that many of them had different
cardiac defects. Some individuals had multiple defects, whereas others show vascular hypoplasia without defects in the heart itself. The skeletal anomalies were also
variable. In contrast to family A, family B showed severe skeletal abnormalities but
milder cardiac malformations. These results strongly suggest that genetic background or modifier genes play an important role in the phenotypic expression
of HOS.
26
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Terrett JA, Newbury-Ecob R, Cross GS, et al.: Holt-Oram syndrome is a genetically heterogeneous disease with one locus mapping to human chromosome 12q. Nat Genet 1994, 6:401–404.
Placed HOS locus in a 21 cM interval in the distal region of chromosome 12q.
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31 Cross SJ, Ching YH, Li QY, et al.: The mutation spectrum in Holt-Oram syndrome. J Med Genet 2000, 37:785–787.
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This article reports additional mutations of TBX5 in HOS patients.
32 Yang J, Hu D, Xia J, et al.: Three novel TBX5 mutations in Chinese patients
with Holt-Oram syndrome. Am J Med Genet 2000, 92:237–240.
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This article reported four mutations in TBX5 and further supports a genotypephenotype correlation in HOS patients with TBX5 mutations.
Ghosh TK, Packham EA, Bonser AJ, et al.: Characterization of the TBX5 binding site and analysis of mutations that cause Holt-Oram syndrome. Hum Mol
Genet 2001, 10:1983–1994.
Using an in vitro oligo binding site selection assay, the authors demonstrate that
TBX5 binds to an 8 bp core sequence that is part of the Brachyury consensusbinding site and that TBX5 also binds to the full palindromic brachyury binding site
and to the half-palindrome. Missense mutations that arise in patients with HOS
indicate that G80R and R237Q eliminate binding to the target site.
33
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34
Muller CW, Herrmann BG: Crystallographic structure of the T domain-DNA
complex of the Brachyury transcription factor. Nature 1997, 389:884–888.
35
Hatcher CJ, Basson CT. Getting the T-box dose right. Nat Med 2001,
7:1185–1186.
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Bruneau BG, Nemer G, Schmitt JP, et al.: A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis
and disease. Cell 2001, 106:709–721.
The authors generated a knockout mouse model. Tbx5-deficient mice (Tbx5 del/del)
have severe hypoplasia of the posterior domains in the developing heart. Several
cardiac-specific genes are markedly decreased, including atrial natriuretic factor
and connexin 40.
Li QY, Newbury-Ecob RA, Terrett JA, et al.: Holt-Oram syndrome is caused by
mutations in TBX5, a member of the Brachyury (T) gene family. Nat Genet
1997, 15:21–29.
This article demonstrates that mutations of TBX5 are a cause of HOS. Six mutations were identified, three in HOS families and three in sporadic HOS cases.
Hiroi Y, Kudoh S, Monzen K, et al.: Tbx5 associates with Nkx2–5 and synergistically promotes cardiomyocyte differentiation. Nat Genet 2001, 28:276–
280.
Using the yeast two-hybrid system with Nkx2.5 as the “bait,” TBX5 and NKX2–5
were found to form a complex. The TBX5/NKX2.5 complex bound to the promoter
of the gene for atrial natriuretic factor, and both transcription factors showed synergistic activation. A G80R mutation of TBX5 did not activate the atrial natriuretic
factor or show synergistic activation, whereas R237Q, which causes upper-limb
malformations without cardiac abnormalities, activated the Nppa promoter to an
extent like that of wild-type TBX5.
28 Bonnet D, Pelet A, Legeai-Mallet L, et al.: A gene for Holt-Oram syndrome
maps to the distal long arm of chromosome 12. Nat Genet 1994, 6:405–408.
•
This article establishes the linkage of the disease locus to chromosome 12q21qter.
Basson CT, Bachinsky DR, Lin RC, et al.: Mutations in human TBX5 (corrected) cause limb and cardiac malformation in Holt-Oram syndrome. Nat
Genet 1997, 15:30–35.
This article demonstrates that mutations in the human TBX5 gene cause HOS.
TBX5 was cloned from the disease locus on human chromosome 12q24.1. A nonsense mutation in TBX5 was identified in affected members of one family, and a
missense mutation was identified in affected members of another.
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