This study sequenced four genes (TNNT3, TNNI2, TPM2, MYH3) in 19 individuals with distal arthrogryposis (DA) to search for pathogenic mutations. No mutations were found in the sequenced regions for the 13 individuals with unclassified DA or 5 with Sheldon-Hall syndrome. A previously reported pathogenic mutation in MYH3 was identified in the single individual with Freeman-Sheldon syndrome, providing further evidence that mutations in this gene cause this condition. The results suggest that mutations in other regions of the genes or in non-coding regions may be responsible for the unclassified DA cases.

1. Materials and Methods
Sequencing of Distal Arthrogryposis (DA) candidate genes in patients with Freeman-Sheldon,
Sheldon-Hall, and unclassified DA syndromes
Whitney S. Best1, Kathryn M. Bofferding1, Heidi Gildersleeve1 Margaret J. McMillin1,2, Anita E. Beck1,2, PhD MD and Michael J. Bamshad1,2,3, MD
1Department of Pediatrics, University of Washington, Seattle WA
2Seattle Children’s Hospital, Seattle, WA
3Department of Genome Sciences, University of Washington, Seattle WA
Introduction
Figure 1. Schematic of the contractile apparatus of skeletal
muscle. Grey text denotes the names of the protein subunits. Red
text corresponds to the gene screened which encodes the subunit.
Figure 3. Gene schematics, Electropherograms of Exon 17 of MYH3, and FSS pedigree. The
individual affected with FSS has a heterozygous mutation at c.2014C>T. DNA from additional family
members was unavailable for sequencing. Electropherograms identify DNA sequence for FSS mutation
and wild type allele at location c.2014.
Results
Abstract
Distal Arthrogryposis (DA) is a group of congenital disorders
characterized by contractures of distal limbs. The
incidence of multiple congenital contractures is one in
three thousand births. Individuals with DA are cognitively
normal. Previous studies have shown that mutations in
TNNT3, TNNI2, TPM2, and MYH3 collectively explain
roughly 30% SHS cases and nearly 95% of FSS cases. These
four genes each encode protein subunits of skeletal muscle
and are hypothesized to be involved in muscular
contraction. Mutations in TNNI2 and TNNT3 may interfere
with the binding of the troponin complex to the
tropomyosin filament and/or inhibit the conformational
change of the troponin complex necessary for muscle
contraction. Mutations in MYH3 may interfere with the
binding of the myosin head to actin filaments. Mutations in
TPM2 may interfere with the interaction between the
tropomyosin filament and the binding site for the myosin
head on the actin filament. However, the precise
mechanism by which mutations in these genes affect
muscle contraction is unknown.
In this study, TNNT3, TNNI2, TPM2, and MYH3 were
screened in 19 individuals who were categorized into three
phenotype groups: un-classified Distal Arthrogryposis (UN
DA) n=13, Sheldon-Hall syndrome (SHS) n=5, and Freeman-
Sheldon syndrome (FSS) n=1.
•DNA samples were collected from one individual
diagnosed with FSS, five individuals diagnosed with
SHS and thirteen individuals with UN DA.
•Primers were designed to capture each of the 9
exons of TPM2, exons 17 and 18 of MYH3, exon 10 of
TNNT3, and exon 8 of TNNI2 in all nineteen
individuals.
•Sanger sequencing was performed on each sample.
•Electropherograms of sequence reads were aligned
in Codon Code Aligner software.
•Mutations were determined by comparison to a
control DNA and a reference sequence from the
Ensembl database (GRCh37).
Affected female and male Unaffected female and male
Key
Distal Arthrogryposis (DA) is a group of syndromes defined by multiple congenital contractures, primarily of the limbs. DA is divided into
subtypes based on the presence of additional clinical features. DA1 is characterized by isolated contractures of the hands and feet.
Sheldon-Hall syndrome (SHS), also known as DA2B, involves mild facial contractures, small mouth and prominent nasolabial folds.
Freeman-Sheldon syndrome (FSS), also known as DA2A, is distinguished by severe facial contractures with pursed lips and H-shaped
dimpling of the chin. DA1, DA2A and DA2B are all autosomal dominant disorders. Mutations in at least four genes, TNNT3, TNNI2, TPM2,
and MYH3, have been shown to cause DA1 and DA2B, whereas DA2A has only been reported to have mutations in MYH3. Each of these
genes encodes a component of the contractile apparatus of skeletal muscle. We screened nineteen individuals diagnosed with DA2A,
DA2B, or an unclassified type of DA for mutations in TNNT3, TNNI2, TPM2, and MYH3. A missense mutation c.2014C>T (p.R672C) in exon
17 of MYH3 was identified in an individual with FSS. This mutation has been previously reported as pathogenic in individuals with FSS.
Parental DNA was unavailable for sequencing.
I.
II.
Pedigree of Individual with FSS
Exon 17 mutation c.2014C>T in MYH3
Al-Haggar, Mohammad. " CLINICAL BRIEF p.R672C Mutation of
MYH3 Gene in an Egyptian Infant Presented with Freeman-
Sheldon Syndrome." Indian Journal Pediatrics . 17 May
2010. academia.edu. 8 August
2012 <www.mansoura.academia.edu>.
MYH3. 30 July 2012 National Institute of Health. 8 August
2012 <http://ghr.nlm.nih.gov/gene/MYH3>.
myosin, heavy chain 3, skeletal muscle, embryonic. 15 May
2012 Weizmann Institute of Health. 8 August
2012 <http://www.genecards.org/cgi-
bin/carddisp.pl?gene=MYH3>.
Robinson, Paul . "Mutations in fast skeletal troponin I, troponin
T, and β-tropomyosin that cause distal arthrogryposis all
increase contractile function."
The FASEB Journal . 2006. 28 June
2012 <www.faseb.org/content/21/3/896.long>.
Toydemir, Reha. "Mutations in embryonic myosin heavy chain
(MYH3) cause Freeman-Sheldon syndrome and Sheldon-Hall
syndrome." Nature Genetics . 2006. 7 August
2012 <http://www.nature.com/ng/journal/v38/n5/full/ng177
Literature Cited
Acknowledgements
Special thanks to my mentor, Kathryn Bofferding, for
being incredibly patient and guiding me through my
research project. Thank you to Dr. Michael Bamshad (PI)
for granting me the opportunity to work and learn in the
lab. Also, thank you to everyone in the Bamshad lab and
to my lab mates for supporting and aiding me through my
research.
This research was supported by University of Washington
STAR Program (67-3473)
Conclusion
Figure 4. Protein
crystal structure of
MYH3. R672C,
underlined, indicates
the amino acid
position and the
predicted protein
consequence of the
c.2014C>T mutation.
•No mutations were identified in the sequenced exons of
TNNI2, TPM2, TNNT3 or MYH3 in any of the individuals
with UN DA or DA2B.
•It is possible that large-scale deletions or duplications,
which would not be detected in the sequence read, may
have occurred. Such deletions or duplications may be
detected by future research.
•Screening of additional exons, intronic and promoter
regions may also yield causative mutations.
•Re-sequencing of exon 1 of TPM2 may produce mutations
as exon 1 in all samples failed to sequence.
•In the one individual with a diagnosis of FSS, a mutation
was identified in exon 17 of MYH3. Since this mutation
has been previously reported in individuals with FSS, it is
likely that this mutation is pathogenic.
(TNNT3)
(TPM2)
(TNNI2)
(MYH3)
TPM2
TNNT3
TNNI2
MYH3
Gene Schematic Key
Sequenced Exon
Unsequenced Exon
Untranslated Region
A
Figure 2. Photos of FSS and SHS Phenotypes. A) Phenotype of classic Freeman-Sheldon B) Phenotype of
classic Sheldon-Hall C) and D) Muscular limb contractures characteristic of distal arthrogryposis syndromes.
C
D
B
R672C
c.2014C>T  p.R672C
Wild Type
FSS
*These photos are to illustrate the phenotypes of FSS, SHS and limb contractures; they are not the individuals in this study.