I. The study screened the CHRNG gene in 29 individuals with distal limb contractures and various diagnoses like Multiple Pterygium Syndrome (MPS), Sheldon Hall Syndrome (SHS), distal arthrogryposis type 5D (DA5D), and neurologic abnormalities and distal contractures not otherwise specified (NAC). II. Two mutations were found in the CHRNG gene in two individuals with MPS - a single-base pair insertion and a two-base pair deletion. It is unclear if these mutations are pathogenic. III. No mutations were found in the individuals with DA5D, SHS or NAC, indicating CHRNG is unlikely responsible for clinical overlap between

1. Materials and Methods
Novel mutation found in CHRNG gene in an individual with Multiple Pterygium Syndrome
Whitney S. Best1
, Kathryn M. Bofferding1
, Margaret J. McMillin1,2
, Anita E. Beck1,2
, PhD MD and Michael J. Bamshad1,2,3
, MD
1
Department of Pediatrics, University of Washington, Seattle WA
2
Seattle Children’s Hospital, Seattle, WA
3
Department of Genome Sciences, University of Washington, Seattle WA
The gene Acetylcholine Receptor Gamma Subunit (CHRNG) encodes for the gamma (γ) subunit of the acetylcholine receptor (AChR).
The AChR is a cell membrane receptor located in cells of the neuromuscular junction. The γ subunit is present during fetal
development and is displaced by the episilon (ε) subunit at thirty-three weeks gestation. Disruption of the γ subunit inhibits
controlled muscle contractions of the developing fetus. Previous studies of individuals with Multiple Pterygium Syndrome (MPS), also
called Escobar syndrome, have reported nonsense, missense, and frameshift mutations in CHRNG. MPS is typically inherited in an
autosomal recessive manner; affected offspring inherit two mutations, one from each parent, who are both carriers. In this study, a
family with a known CHRNG mutation in exon 5 was used as a positive control. We found a previously unreported single-base pair
insertion (c.1083-1084insC, p.361fs*55) in exon 10 in the MPS individual from family one. This change was inherited from the
individual’s unaffected father, and it is unclear if the mutation may be pathogenic. Additionally, we identified a previously reported
two-base pair deletion (c.753-754delCT, p.251fs*46) in the MPS individual of family two. The possible inheritance of this mutation is
unable to be verified due to lack of parental DNA available for sequencing. In both affected individuals only a single mutation was
found, but further screening of the promoter or intronic region of CHRNG may lead to the finding of a second causative mutation.
Figure 2. Pterygia and muscle contractures in Multiple Pterygium Syndrome individuals
(A) Pterygia on the underside of both arms. (B) Pterygia located behind the knee. (C) Muscle contractures in all ten digits of individual.*The photos above are
of individuals diagnosed with MPS. However, they are not the individuals in whom mutations were found in this experiment. They are to illustrate the pterygium phenotype and
muscle contractures.
B C
Acknowledgements
Introduction
Hoffman, Katrin. "Escobar Syndrome is a Prenatal
Myasthenia caused by Disruption of the Acetylcholine
Receptor Fetal Gamma Subunit." The American Journal
of Human Genetics 8 2006. 10. www.ajhg.org.
McKinley , O'Loughlin. Breakdown of ATP and Cross-
bridge Movement during Muscle Contraction. McGraw-
Hill Higher
Education. 8/2/11 <http://highered.mcgraw-
hill.com/novella/MixQuizProcessingServlet>.
Vogt, Julie. "Mutation Analysis of CHRNA1, CHRNB1,
CHRND, and RAPSN Genes in Multiple Pterygium
Syndrome/Fetal Akinesia Patients ." The American
Journal of Human Genetics 01 2008: 222-227.
Morgan, Neil. "Mutations in the Embryonal Subunit of
the Acetylcholine Receptor (CHRNG) Cause Lethal and
Escobar Variants of Multiple Pterygium Syndrome." The
American Journal of Human Genetics 08 2006: 390-
395.
Literature Cited
Figure 1. Structure of Acetylcholine Receptor (AChR) in fetal and
adult muscle cells. The γ subunit in fetal AChR is replaced by, and
will remain, the ε subunit after thirty-weeks of gestation. CHRNG
encodes the gamma subunit of the acetylcholine receptor.
Conclusion
Figure 3. Family 1-MPS pedigree and exon 10
electropherograms Child affected with MPS and unaffected
father each have the heterozygous insertion
c.1083_1084insC. No mutations were identified in the
unaffected mother. DNA was not available from unaffected
sibling.
I-2
I-1
II-11
1 2
I.
II.
+/-
+/- -/-
Family 1
Exon 10 c.1083_1084insC
Figure 5. Family 3-Escobar pedigree and exon 5
electropherograms Unaffected father and mother are
heterozygous while both affected children are homozygous for the
single base-pair insertion c.312_313insA.
Family 3 – (CHRNG positive control)
Exon 5 c.312_313insA
II.
I-1
II-1
I-2
II-2
Figure 4. Family 2-MPS pedigree and exon 7
electropherograms Child affected with MPS and
heterozygous for c.753-754delCT. DNA was not available
from both unaffected parents. A control DNA chromatogram
is shown for comparison.
Family 2
Exon 7 c.753-754delCT
II-1
Control
Results
Abstract
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
Bamshad 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)
•No mutations were identified in the exons of
CHRNG in any of the individuals with DA5D, SHS
or NAC. Therefore, it is unlikely that CHRNG is
responsible for the overlap in clinical features of
these diagnoses and features of MPS.
•Of the nine individuals with MPS, mutations
were identified in two individuals. It is unclear if
these mutations are pathogenic.
•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, and screening of intronic and
promoter regions may also yield causative
pterygia mutations.
Multiple Pterygium Syndrome (MPS), also known as
Escobar Syndrome, is a disorder characterized by
muscle contractures, pterygia, and respiratory
complications. Mutations in the gene CHRNG, which
encodes the gamma (γ) subunit of the acetylcholine
receptor, have been reported in individuals with
MPS and in 30% of cases of the lethal form of MPS. In
this experiment, CHRNG was screened in 29
individuals with distal limb contractures and
variable diagnoses: MPS n=10, Sheldon Hall
Syndrome (SHS) n=6, distal arthrogryposis type 5D
(DA5D) n=8, and individuals with neurologic
abnormalities and distal contractures that do not
have a specific diagnosis (NAC) n=5. Each of these
diagnoses have features that overlap with other case
reports in which mutations in CHRNG have been
identified.
•DNA samples were collected from ten MPS
individuals, eight DA5D, six SHS individuals and five
NAC.
•Primers were designed to capture each of the 13
exons of CHRNG in all twenty-nine individuals.
•Sanger sequencing was performed on each sample
for the 13 exons of CHRNG.
•Electropherograms of sequence reads were aligned
in the computer software Codon Code.
•Mutations were determined by comparison to a
control DNA and a reference sequence from the
Ensembl database (GRCh37)
1
1 2
I.
II.
+/-
1
2
I.
+/+
+/- +/-
+/+
2
1
A B C
c.1083-1084insCc.753-754delCT
1 112 104 12953 76 8 13

CHRNG Gene
Figure 6. Diagram of CHRNG Exons 1-11 are shown in blue. Untranslated exons 12-13 are
shown in orange. Mutations found in this screen of CHRNG are indicated above their
corresponding exons.
Affected female and male
Unaffected female and male
Homozygous for mutation
Heterozygous for mutation
Homozygous for wild type
Key
+/-
-/-
+/+