Vol. 133 No.

5 May 2022

Oral manifestations associated with inherited

hyperhomocysteinemia: A first case description
Bachar Husseini, DDS, MSc,a,b Edgard Nehme, DDS, MSc,c Karim Senni, PhD, DSc,d
Claude Sader Ghorra, MD, PhD,e Khalil Younes, PharmD,f Sandrine Roffino, PhD,g Pierre Ghorra, MD,h
Sylvie Changotade, PhD,i and Ronald Younes, DDS, MSc, PhDa,b

Hyperhomocysteinemia is a rare disease caused by nutritional deficiencies or genetic impairment of cysteine metabolism. To date, no
oral manifestations of hyperhomocysteinemia have been described in humans. Therefore, to our knowledge, the present case report is
the first description of a hyperhomocysteinemic patient showing oral tissue alterations leading to both early tooth loss and failed
implant osseointegration. The patient presented with a methylenetetrahydrofolate reductase gene mutation (677T polymorphism) lead-
ing to mild hyperhomocysteinemia. The radiologic analysis showed hyperdense lesions scattered in the maxillae. The histologic
observations indicated alterations in both collagen and elastic networks in the gingiva and dermis. Interestingly, the presence of
ectopic mineralized inclusions was noted in both periodontal ligament and gingiva. Strong osteoclastic activity was associated with
abnormal calcification of trabecular spaces. Uneven oral tissue remodeling due to high tissue levels of homocysteine could explain
the pathologic manifestations observed in this case. (Oral Surg Oral Med Oral Pathol Oral Radiol 2022;133:e105 e112)

Hyperhomocysteinemia (HHcy) is a rare metabolic dis-
order caused by nutritional deficiencies in folates and B
vitamins or by mutations of enzymes involved in cysteine
metabolism. This could lead to the accumulation of homo-
cysteine (Hcy) in both blood and connective tissues.1
Hcy is a nonproteogenic sulfur-containing amino acid
that is an intermediate metabolic form of the methionine
and cysteine standing at the intersection of transsulfuration
to cystathionine and remethylation to methionine path-
ways. Hcy accumulation causes several biological altera-
tions leading to systemic manifestations1 (Figure 1A).
HHcy genetic alterations are caused by recessive
mutations of the methylenetetrahydrofolate reductase
a
Department of Oral Surgery, Faculty of Dental Medicine, Saint
Joseph University of Beirut, Beirut, Lebanon.
b
Cranio-Facial Research Laboratory, Faculty of Dental Medicine,
Saint Joseph University of Beirut, Beirut, Lebanon.
c
Department of Oral Pathology, Faculty of Dental Medicine, Saint
Joseph University of Beirut, Beirut, Lebanon.
d

Laboratoire EBInnov, Ecole de Biologie Industrielle, Cergy, France.
e
Department of Pathology, Faculty of Medicine, Lebanese Univer-
sity, Beirut, Lebanon.
f
Department of Biochemistry, H^opital Kremlin Bic^etre AP-HP, Paris,
France.
g
Institute of Movement Science E.J. Marey, Aix-Marseille Univer-
sity, CNRS, Marseille, France.
h
Flow Cytometry-HLA Laboratory, Hotel Dieu de France Hospital,
Beirut, Lebanon.
i
Unit
e de Recherche Biomat
eriaux Innovants et Interfaces, URB2I,
UR 4462, F-93000, Universit
e Sorbonne Paris Nord, UFR SMBH,
Bobigny France.
Corresponding author: Bachar Husseini, DDS, MSc, Oral Surgery
Faculty of Dental Medicine, Saint Joseph University of Beirut, P.O.
Box 11-5076, Riad El Solh, Beirut 1107 2180, Lebanon E-mail
address: bachar.husseini@net.usj.edu.lb
Received for publication Jun 8, 2021; returned for revision Aug 9,
2021; accepted for publication Sep 9, 2021.
Ó 2021 Elsevier Inc. All rights reserved.
2212-4403/$-see front matter
https://doi.org/10.1016/j.oooo.2021.09.007
gene (MTHFR; OMIM 236250) located on chromo-
some 1 or cystathionine b-synthase gene (CBS; OMIM
236200) located on chromosome 21.2 In fact, both
enzymes play a crucial role in sulfur amino acid metab-
olism. MTHFR is considered a critical enzyme for Hcy
remethylation and folate extraction from ingested pro-
teins.3 Between 1994 and 2007, 34 rare mutations and
9 polymorphisms of the MTHFR gene were discov-
ered.4 The C677T polymorphism was pointed out
owing to its impact on the enzyme structure and activ-
ity, resulting in a significant accumulation of Hcy in
blood and tissues.2
The severity of the clinical manifestations was
linked to Hcy blood levels classifying HHcy into mild
(16-30 mmol/L), moderate (31-100 mmol/L), and
severe categories (>100 mmol/L).5 Severe forms of
HHcy are associated with hyperhomocysteinuria.6 The
latter is mainly marked by cardiovascular alterations
and vein thrombosis. Moreover, lens disruption, osteo-
porosis, and cleft lip/palate were also reported in some
cases.7 Previous reports revealed that HHcy was further
associated with periodontitis.8 Interestingly, the devel-
opment of periodontitis was recently reported in the
HHcy animal model;8 yet, there are limited data
regarding the effect of HHcy in the oral cavity.
To our knowledge, the present case report is the first
detailed description of oral manifestations in a patient
with inherited HHcy due to a homozygous MTHFR
gene mutation C!T at the 677 nucleotide verified by
two different polymerase chain reaction (PCR) tests.
CASE REPORT
This case report was approved by the university institu-
tional review board (USJ-2020-116). In addition, the
procedure was explained to the patient, who then
signed an informed consent allowing surgical proce-
dures and publication of her data.

e105
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e106 Husseini et al. May 2022

Fig. 1. (A) Homocysteine methylation and transsulfuration pathways highlighting MTHFR mutation (C677T) of the patient.
Enzymes: MTHFR, methylenetetrahydrofolate reductase; CBS, cystathionine b-synthase; MS, methionine synthase. Substrates:
Met, methionine; SAM, S-adenosyl methionine; SAH, S-adenosyl homocysteine; MTHF, methylene tetrahydrofolate, THF, tetra-
hydrofolate; Me-X, methyl acceptor; Ser, serine; Gly, glycine; Hser, homoserine; B6, vitamin B6; B12, vitamin B12. (B) Polymer-
ase chain reaction (PCR) sequence-specific oligonucleotide test strip revealing a stain on the mutant line of the MTHFR C677T
and no stain on the wild type.

Clinical presentation Clinical examination

A 45-year-old woman with a genetic HHcy disorder pre-
sented to the oral surgery department for teeth extrac-
tions followed by implant placement. The patient’s
clinical history revealed an early tooth loss in her mid-
20s and 2 previous early dental implant failures. The
unsuccessful attempts concerned the left mandibular and
right maxillary quadrants. The patient’s main concern
was to receive an implant rehabilitation to restore her
early edentulism and hence improve her life quality.
The patient had no history of systemic diseases
except for HHcy. Her medication history included sali-
cylic acid prescribed by her physician as prophylaxis
for potential thrombosis that might be caused by HHcy
because it has been proved that elevated Hcy blood lev-
els are directly related to a higher incidence of throm-
bosis.1 She reported receiving no other medications.
The patient had class I Kennedy edentulism at both the
maxilla and mandible concomitant with poor oral
hygiene (plaque index 2). Her lower right first premolar
no. 44 and her left first and second premolars nos. #34
and 35 were severely decayed and required extraction;
the remaining teeth were crowned. Her clinical exami-
nation showed moderate gingival inflammation associ-
ated with a heterogeneous color and texture
(Figure 2A C). A thorough examination of the
attached gingiva revealed an irregularly both stippled
(orange peel graining) and smooth surface texture. In
addition, its color was coral pink in some regions and
reddish in others. Concerning the alveolar mucosa, the
examination showed a smooth and shiny texture with
exaggerated, unequally distributed reddish zones
(Figure 2B, C).

Fig. 2. (A-C) Intraoral photos of the patient’s oral cavity.

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Volume 133, Number 5
Radiologic examination
Cone beam computed tomography revealed well-delin-
eated hyperdense lesions scattered on both the left and
right posterior edentulous quadrants of the maxilla and
the mandible more precisely at the molar region
(Figure 3A, C), as well as on the anterior hard palate
midline (Figure 3B) and in the alveolar bone next to
dentate sites in the lower left premolar area
(Figure 3D). Moreover, some of the lesions extended
to reach the buccal and palatal/lingual bony plates,
especially at maxillary and mandibular posterior eden-
tulous sites. A thickened periosteum was also detected
at the bony margins of those lesions.
Genetic analysis
The MTHFR C677T mutation was identified by PCR
sequence-specific oligonucleotide and reverse hybrid-
ization using the ViennaLab CVD StripAssay, which
identifies 12 mutations associated with cardiovascular
disease, 1 of which is the MTHFR C677T mutation.
The procedure includes three steps: DNA isolation
from an ethylenediaminetetraacetic acid (EDTA) blood
sample, PCR amplification using biotinylated primers,
and hybridization of amplification products to a test
strip containing allele-specific oligonucleotide probes
immobilized as an array of parallel lines. Bound bioti-
nylated sequences are detected using streptavidin-alka-
line phosphatase and color substrates. The genotype of
the sample is determined by analyzing the stains
obtained on the test strip (Figure 1B). A sole stain on
Fig. 3. (A-C) Cone beam computed tomography axial slices
showing the hyperdense ossifications in (A) the mandible,
(B) the hard palate, and (C) the maxilla (arrow). (D) Cross-
sectional slice at the lower left first premolar site showing the
extension of the hyperdense ossification starting from the cor-
tical bone toward the periodontal ligament (arrow).
CASE REPORT
Husseini et al. e107
the mutant line was obtained, thus indicating the pres-
ence of the homozygous mutant of the MTHFR C677T.
To confirm this result, the authors performed real-
time PCR using the SMD Thrombophilia Real Time
PCR Kit Multiplex kit according to the technique
described by Elke Wagner,9 confirming the presence of
the MTHFR C677T mutation in a homozygous state.
Interestingly, the 43-year-old patient’s sister was
also screened as a carrier of the C677T and A1298C
mutant alleles of the MTHFR gene. Although heterozy-
gous, her clinical history revealed previous events of
cerebral thrombosis and fetal miscarriages. However,
the aforementioned patient did not disclose any altered
clinical oral signs except for tooth loss in her 30s.
The MTHFR C677T mutation (OMIM: 607093.0003),
also known as rs1801133, is a single-nucleotide polymor-
phism on the MTHFR gene located on chromosome
1:11796321.10 It is a cytosine-to-thymine substitution at
nucleotide 677, resulting in alanine-to-valine (p.
Ala222Val or A222V) substitution in the N-terminal cata-
lytic domain of the MTHFR protein.10 It is a missense
mutation that induces the formation of a thermolabile var-
iant of the MTHFR enzyme, thus reducing its activity and
increasing plasma Hcy levels, particularly in the homozy-
gous state.10
Laboratory investigations
After the discovery of the genetic disorder, the patient
underwent hematological monitoring to assess Hcy lev-
els and other vital elements. Her laboratory test results
were all within normal ranges, except for vitamin B12
levels, for which a deficiency was noted. As for the
Hcy levels, the registered values varied between 15.5
and 21 mmol/L above the normal average. The mildly
high HHcy blood level was coupled to a persisting vita-
min B12 deficiency (70 pg/mL).
Clinical decision
After the clinical and radiologic examinations
described above, a clinical decision was made to per-
form a tissue biopsy from the gingiva that was retrieved
from the edentulous crest and a bone biopsy collected
from the area showing radiologically scattered hyper-
dense lesions. Moreover, the extracted teeth were proc-
essed for histologic analysis. Later, and based on the
oral investigations conducted above, a skin biopsy was
performed to investigate the presence of an elastic or
collagenous network abnormality to identify a potential
generalized elastic disorder that might affect blood
vessels.11
Histologic observations
Gingival, dermal, and tooth biopsies were first fixed in
4% paraformaldehyde + 0.1 mol/L phosphate buffer,
pH 7.2, for 48 hours. The biopsies were dehydrated
ORAL AND MAXILLOFACIAL PATHOLOGY OOOO
e108 Husseini et al. May 2022

gradually in ascending concentrations of ethanol. Sub-

sequently, an automated tissue processor was used to
embed the tissues in paraffin before performing a serial
section (3-5 mm). Furthermore, the tooth was decalci-
fied in EDTA before the dehydration process. Later,
the obtained slices were stained using hematoxylin and
eosin, picrosirius red,12 and (+)catechin fuchsin dyes13
to explore, respectively, the global tissue structure,
fibrillary collagen, and elastic network. A Von Kossa
reaction was performed to investigate the calcified
structures within the gingival tissue.
The bone cores were fixed in 4% buffered parafor-
maldehyde solution, then dehydrated and directly
embedded in polymethyl methacrylate at 4˚C. Longitu-
dinal thin sections (5 mm) were obtained. Toluidine
blue staining was performed to assess the bone archi-
tecture. A tartrate-resistant acid phosphatase enzymatic
reaction14 was used to observe osteoclastic activity. A
Von Kossa reaction was performed on the gingiva to
assess calcifications.
Fig. 4. Characterization of dense osseous inclusions embed-
Alveolar bone. The hyperdense osseous inclusions ded in the alveolar bone. (A and B) Osteoclast activities (tar-
embedded in the alveolar bone of the patient displayed trate-resistant acid phosphatase stained in red). (C and D)
significant osteoclastic activity (Figure 4A, B). The Fibrillar collagen organization (toluidine blue staining under
inclusions presented a dense lamellar structure result- polarized light). (E and F) Mineralization of inclusions (Von
ing in numerous incomplete osteons, as observed Kossa reaction). Arrow: Mineralization of medullary spaces.
throughout the toluidine blue staining under polarized
light (Figure 4C, D). In addition, the Von Kossa stain uneven with hardly distinguishable cementous lines
demonstrated complete mineralization of these inclu- (Figure 5A-C).
sions, as well as partial mineralization of the peripheral
medullar spaces of the surrounding alveolar bone Gingiva. The gingiva presented various tissue struc-
(Figure 4E, F). tural abnormalities. The hematoxylin and eosin stain
revealed a dense collagenous matrix and mixed inflam-
Tooth. Figure 4A illustrates the patient’s healthy tooth matory infiltrates (Figure 6A, B). Elongated fibroblasts
extracted for prosthetic reasons. Hematoxylin and eosin were evenly distributed in the whole gingiva, out of
staining showed a typical fibrous periodontal ligament which some were agglomerated around amorphous
containing numerous fibroblasts. The cementoblasts nodules (Figure 6B). The hematoxylin staining and
were stretched in a parallel manner to the cementum sur- Von Kossa reaction proved the mineralized nature of
face. The pathologic tooth exhibited an unstructured these nodules, which were heavily surrounded by fibro-
periodontal ligament comprising seemingly mineralized blastic cells (Figure 6C). The picrosirius red staining,
dense inclusions. The cementodentinal junction seemed under polarized light, brought out a highly dense

Fig. 5. Histologic observation of the patient’s tooth after hematoxylin and eosin staining. (A) Healthy tooth extracted for prostho-
dontic purposes. (B and C) Pathologic tooth. Ce, cementum; De, dentine; PDL, periodontal ligament. Arrow: Mineralized inclu-
sions.
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Volume 133, Number 5 Husseini et al. e109

Fig. 6. Histologic observation of the patient’s gingiva. (A) Global structure (hematoxylin and eosin staining). (B) Structural
anomalies in gingival connective tissue (hematoxylin staining). Arrow: Nodular structure. (C) Mineralization of nodular struc-
tures (arrow) present in the structural anomalies (hematoxylin staining and Von Kossa reaction).

collagen matrix comprising numerous thick and
crimped fibers dispersed all over the matrix. It is also
worth mentioning that a reduced interfibrillar space
was noted when we compared it with a healthy gingiva
(Figure 7A, B).
Moreover, compared with a healthy network, the
entire elastic network appeared to be particularly
dense. Shortened and thick oxytalan fibers were clearly
observed between gingival rete pegs (Figure 7C, D).
The superficial gingival connective tissue was charac-
terized by uncommonly oriented, numerous, thick, and
fragmented elaunin fibers. In addition, deep gingival
connective tissue was marked by a high density of thick
and shortened elastic fibers without any precise orienta-
tion (Figure 7C, D).
Skin. Hematoxylin and eosin staining revealed a mixed
inflammatory infiltrate around dermal blood vessels
(Figure 8A). The dermal collagen network was charac-
terized by shortened fibers and numerous interfibrillar
spaces (Figure 8B). Both mature and elaunin skin elas-
tic fibers were observed, but the dermis was marked by
rarefied oxytalan fibers. Mature elastic fibers, as well
as elaunin fibers, were numerous and well oriented per-
pendicularly to the dermoepidermal junction, but they
were also highly fragmented (Figure 8C).
DISCUSSION
HHcy is regarded as a metabolic disease and a major
risk factor for thrombosis and cardiovascular dysfunc-
tion. It is particularly associated with proteolytic

Fig. 7. Histologic observation of gingival fibrillar collagen and elastic networks. (A and C) Gingiva of a healthy subject. (B and
D) Gingiva of the patient. (A and B) Collagen network (picrosirius red staining under polarized light). (C and D) Elastic network
[(+)catechin fuchsin staining]. Ep, epithelium; Ox, oxytalan fibers; Elau, elaunin fibers; EF, mature elastic fibers. A and C were
provided by R.Y., photographed by him during his thesis preparation that led to publication of Younes et al.32
ORAL AND MAXILLOFACIAL PATHOLOGY
e110 Husseini et al.
Fig. 8. Histologic observation of the patient’s skin. (A) Global structure (hematoxylin and eosin staining). (B) Fibrillar collagen
network (picrosirius red staining under polarized light). (C) Elastic network [(+)catechin fuchsin staining]. Ox, oxytalan fibers;
Elau, elaunin fibers; EF, mature elastic fibers.
fragmentation of the vascular elastic network15,16
involving a potential direct effect of Hcy on the endo-
thelium.11 Besides, high blood levels of Hcy could also
be involved in osteoporosis and other skeletal malfor-
mations.17 To date, few articles have focused on oral
manifestations due to HHcy.7 The present case report
describes early tooth loss in addition to recurrent
implant failures in a patient with inherited HHcy asso-
ciated with vitamin B12 deficiency that was unrelated
to an unbalanced nutritional diet.
In fact, it has previously been reported that HHcy is
a direct cause of vitamin B12 deficiency.18,19 However,
despite the proven potential role of vitamin B12 in peri-
odontal disease,20 there was no clear relationship with
the observed pathologic mechanisms in the present
case. The reason for this is that the histologic similari-
ties between the gingival biopsy taken from the edentu-
lous maxilla of the patient and her skin as well as the
absence of periodontal pockets and clinical attachment
loss discarded the hypothesis of a vitamin B12 origin of
the observed abnormalities.
The unexpected clinical, radiologic, and histologic
observations in this case led us to the assumption of
possible molecular mechanisms involving high blood
and tissue Hcy levels. The patient displayed alveolar
bone abnormalities marked by the presence of hyper-
dense osseous inclusions at both edentulous and den-
tate sites. The fact that these inclusions were in tight
contact with the periodontal ligament and not restricted
to the teeth apices (Figure 3D), in addition to the
absence of any infectious disease history affecting
bone, excluded the diagnosis of a reactive condensing
osteitis. Neighboring alveolar bone was characterized
by unevenly structured bone collagen apposition and
medullary space hypermineralization. In addition, the
noticeable strong osteoclastic activity could imply
altered bone remodeling. Alveolar bone abnormalities
seemed to go along with the formation of ectopic hard
tissue in both gingiva and periodontal ligament.
Regarding the gingiva, ectopic nodular mineralization
was associated with an amorphous area surrounded by
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May 2022
grouping fibroblastic cells. Moreover, some periodon-
tal ligament ectopic inclusions similar to cementum-
like islets displaying partial incremental lines were
also observed, as previously shown in rats after immu-
nosuppressive treatment.21 Interestingly, other inclu-
sions resembling bone structures containing osteocyte
lacunae were also present in the periodontal ligament.
The imbalance in bone remodeling is associated with
a resorbed cementum, the deposition of an amorphous
tissue replacing the periodontal ligament, and gingival
ectopic mineralization. It can be hypothesized that
alterations in tissue remodeling trigger modifications
of the mesenchymal cell differentiation program.22
Direct activation of osteoclasts by Hcy through
increase of reactive oxygen species (ROS) obviously
explains the observed alveolar bone active resorption.23
Nevertheless, the ectopic bone formation and hypermi-
neralization of alveolar bone medullary spaces is still
not well explained, but it has been demonstrated that
increased Hcy concentrations can stimulate in vitro
osteoblastic activity.24
Tissue accumulation of Hcy causes numerous soft
tissue alterations, particularly in elastic structures, as
was observed in large arteries.15 The patient’s gingiva
and skin were characterized by elastic network altera-
tions and pertinent elastic fiber fragmentations. This
could be explained by ROS-dependent Hcy activation
of the proteolysis process, which was also previously
observed in vascular tissue.16,25 Thus, proteolysis sus-
ceptibility of fibrillins, a major glycoprotein-associated
elastic fiber, is increased by the direct interaction with
Hcy.26 In return, both fibrillins and elastin proteolytic
fragments have demonstrated their ability to upregulate
extracellular matrix proteolysis.27
Furthermore, fibrillins have a major role in trans-
forming growth factor (TGF)-b and bone morphoge-
netic protein (BMP) bone-mediated metabolism.28
Indeed, it is reported that the interaction between Hcy
and epidermal growth factor like domains of fibrillins
disturb the interaction between elastic fibers and TGF-
b or BMPs.29,30 High Hcy tissue levels could then
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Volume 133, Number 5
modify BMP and TGF-b signals and thus could have a
direct effect on bone metabolism leading to ectopic
osseous or cementum formation. As observed in Mar-
fan syndrome, alteration of fibrillin functions during
HHcy leads to vascular defects and periodontal mani-
festations;31 yet, to date, only the our present patient
with HHcy displayed specific bone-remodeling defects
leading to tooth loss and dental implant failures.
Finally, owing to this pathology, implant osseointe-
gration was not achievable; therefore, a removable
prosthesis was delivered to rehabilitate a functional
dentition. In the future, long-term management of pro-
teolytic elastic network hydrolysis could be a solution
to avoid oral tissue impairments. In addition, special
attention should be paid to the nutritional balance and
vitamin B12 deficiency, which is also reported as a con-
sequence of MTHFR (C677T) genetic alteration. Thus,
a folate-rich diet3 may be recommended.
However, further studies should assess this hypothe-
sis using a larger patient cohort that includes patients’
family members.
CONCLUSION
For the first time, to our knowledge, we have shown
that inherited HHcy is associated with a defective
remodeling of the oral tissues, resulting in early tooth
loss and implant failures. It also can be assumed that
the accumulation of Hcy in oral tissues could lead to
altered bioavailability of bone growth factors due to a
destabilized and hydrolyzed elastic network.
ACKNOWLEDGMENTS
The authors thank Dr Fadi Sleilati, Prof Georges Hilal,
and Prof Georges Aftimos for their contribution and
the patient for her valuable cooperation.
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