1. Results (continued)
References
Acknowledgements
S. M. Naimul Hasan1,4 Biswajit Chowdhury1, Richard Hemming1, Brian Mark3, Barbara Triggs-Raine1,2,4
Conclusion
1 Departments of Biochemistry and Medical Genetics, 2Pediatrics and Child Health, 3 Microbiology, University of Manitoba, 4Children’s Hospital Research Institute of Manitoba
Analysis of two novel mutations in HYAL2 associated with orofacial
clefting in humans
Material and MethodsIntroduction
Orofacial clefts are a common congenital
malformation and include cleft lip (CL)
with/without cleft palate (CLP). Orofacial clefts
are classified as syndromic or non-syndromic
depending on whether, or not, additional
congenital abnormalities are present.1
Although mutations in several genes have been
reported to be associated with orofacial clefting,
the complete etiology remains to be determined.
Recently, through homozygosity mapping followed
by exome sequencing, lysine148 to arginine and
proline250 to serine mutations in HYAL2 were
identified as the putative cause of syndromic
orofacial clefting respectively (Fig1) in Amish and
Saudi Arabian families. 2,3
The affected patients have a broad nose and
increased interorbitary distance. Cleft lip and
palate have been surgically repaired in the patient
in “C”. Most of the patients also exhibited cardiac
abnormalities.
Hyaluronidase 2 is a GPI anchored protein
involved in hyaluronic acid (HA) degradation.
Hyaluronic acid is a large polysaccharide
abundantly present in the extracellular matrix,
making the matrix easy for cells to proliferate and
migrate.4
Homology Modeling
A homology model of HYAL2 was predicted using
the crystal structure of hyaluronidase 1 (2PE4) as
the 3D template with Modeller v9.13. Pymol was
used to introduce Lys148Arg and Pro250Leu
mutation into the predicted HYAL2 model.
Results
Site Directed Mutagenesis
The c.443A>G p.K148R and c.749C>T p.P250L
(NM_003773) mutations were introduced into the
full length human HYAL2 cDNA expression vector
pCMV6-XL5 (Origene, Cat # SC117754) by fusion
PCR followed by sequencing. As a negative
control, a deletion of the 5’ region of HYAL2 cDNA
was made, preventing HYAL2 expression.
Protein Expression
HYAL2 -/- mouse embryonic fibroblasts (MEFs)
were used for the transfection of HYAL2
expression vectors together with a β-gal control
vector. Cells were collected 48 hours post-
transfection, and the β-galactosidase activity was
determined using O-nitrophenol-β-D-
galactopyranoside as a substrate to normalize the
transfection efficiency. The expression level of the
two mutant HYAL2 proteins was analyzed by
western blot with an anti-HYAL2 polyclonal
antibody and detected by enhanced
chemiluminescence. Student’s t test was done
from the average of four experiments to
determine the significance.
Hyaluronan Binding Assay
To compare the binding of hyaluronan to wild-type
and mutant HYAL2, exogenous HA was added
into the solubilized lysate and allowed for binding.
Later, the HYAL2 bound hyaluronan was
precipitated using cetylpyridinium chloride (CPC)
which is a cationic detergent with affinity for
anionic HA. Finally, western blot was used to
determine the presence of HYAL2 in the
precipitate.
Western blot
HA-HYAL2 binding assay
Mutation introduction Transfection
1. Homology Modeling of HYAL2
Homology modeling of mutated HYAL2 shows
that the pro250leu is deeply buried near the
active site of the HYAL (Fig3). Mutation in this
position could interfere with the normal folding
and stability of HYAL2. The lysine148arg mutation
is located near the HA binding site of HYAL2 and
could impact the binding of HYAL2.
2. Protein Expression of the mutated HYAL2
We have done western blot analysis from the
wild type and mutated HYAL2 transfected cell
lysate to detect the HYAL2 expression level using
anti-HYAL2 antibody. Our quantification from the
western blots of the HYAL2 shows that the
lys148arg mutation had 11 fold reduction and
pro250leu had 20 fold reduction in HYAL2
compared to the wild type (Fig4).
Binding of hyaluronic acid to equal amounts of
wild type and Lys148Arg HYAL2 showed no
difference (Fig5).
3. Hyaluronan Binding Assay
We have demonstrated that both the lysine 148
to arginine and proline 250 leucine mutations
decrease HYAL2 stability. Therefore, HYAL2 is
indeed the likely cause of these syndromic cleft
lip and palate cases. It will be interesting to
determine if HYAL2 mutations can be identified in
other patients with syndromic cleft lip and palate.
Future direction
In future we will use a HYAL2 deficient mouse
model to study how the loss of HYAL2 impacts
palate formation at different stages of mouse
development.
Our overall objective was to determine if the
Lys148Arg and Gly250Ser mutations have a
detrimental impact on HYAL2, making them the
likely cause of the syndromic CLP in these
families.
Objective
Fig 4: (A) Western blot analysis of expressed HYAL2
protein. An arrow indicates HYAL2. (B) HYAL2 levels from
WT and mutation transfected
Fig 3: (A) Comparative molecular model of human HYAL2 bound
to a hexamer of hyaluronic acid. (B) Active site of the HYAL2
showing the position of the mutation and HA
Fig 5: (A) Western blot analysis of HA bound HYAL2 from WT and
K148R-HYAL2 transfection lysates. (B) Western blot analysis
comparing input (IP) levels of WT or K148R-HYAL2 compared to
HA bound HYAL2 (+HA). Arrows indicate the position of HYAL2,
showing degradation products in the K148R HYAL2 samples.
Fig 1: Facial phenotype of individuals with HYAL2 deficiency2
(A) (B)
(A) (B)
1. Mossey P a, Little J, Munger RG, Dixon MJ,
Shaw WC. Lancet. 2009;374(9703):1773-1785.
2. Muggenthaler M, Chowdhury B, Hasan S,
Cross H, Mark B, et al. Submitted (2016).
3. Shaheen R, Patel N, Shamseldin H, et al.
Genet Med. 2015;(May):1-10.
4. Chowdhury B, Hemming R, Hombach-Klonisch
S, Flamion B, Triggs-Raine B. J Biol Chem.
2013;288(1):520-528.
This work is part of a collaborative study submitted
for publication with groups from the United
Kingdom and Saudi Arabia.2
Plasmid multiplication
Fig 2: Experimental design