Advancement in Knowledge of McKusick-Kaufman Syndrome

Martha Holt

October 4, 2021

A paper submitted to the faculty of the Department of Biology, Western Carolina University, in
partial fulfillment of the senior research requirement.
 ABSTRACT

McKusick-Kaufman syndrome (MKS) is an autosomal recessive disorder that affects

many parts of the body including the limbs of the body, the heart, and the reproductive system.
Diagnosis of MKS has improved due to the advancement in the understanding of key mutations
in the DNA sequence. The knowledge of the MKKS gene being highly expressed in skeletal
muscle, heart tissue, and reproductive tissue has led to the advancement of understanding of this
disease. Many studies have contributed to the advancement in knowledge of the gene
transmission, autonomous consequences, and progression of this disease. The MKKS gene is
located on chromosome 20p13) and is caused by mutations in the MKKS gene14). Through multi-
alignment DNA sequencing databases, it is predicted that the MKKS gene is conserved across
species, specifically mammalian species. With the onset of puberty of women, MKS can lead to
further complications in their reproductive system, commonly causing frequent urinary tract
infections and restenosis of the vaginal tract. McKusick-Kaufman syndrome (MKS) and Bardet-
Biedl syndrome (BBS) can present as the same disease in the prenatal and neonatal period. BBS
evolves over time, leading to further complications that typically present themselves at a future
time. Ultrasound prenatal diagnosis is the safest and quickest way to distinguish the two diseases
but can oftentimes be missed and only learned about with the progression of BBS.

INTRODUCTION

McKusick-Kaufman syndrome (MKS) is a condition that is inherited through an

autosomal recessive manner that is characterized by the combination of postaxial polydactyly
(PAP), congenital heart disease (CHD), and hydrometrocolpos (HCM) in females and genital
malformations. For females, hydrometrocolpos (HCM), postaxial polydactyly (PAP), and
congenital heart disease (CHD), for males’ genital malformations (typically hypospadias,
cryptorchidism, and chordee), PAP, and CHD are typical was to diagnose this disease.

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 Figure 1. The symptoms and treatments of McKusick-Kaufman Syndrome.

HCM in children is detected by symptoms of large cystic abdominal masses that protrude
from the pelvis because of the accumulation of cervical secretions from maternal estrogen
stimulation. This causes dilation of the vagina and uterus and is typically caused by the lack of
development in the most distal part of the vagina to develop a transverse vaginal membrane
(imperforate hymen). PAP is characterized by additional digits (fingers/toes) on the ulna side of
the hand and the fibular side of the foot. There have been many congenital heart defects present
in McKusick-Kaufman cases including atrioventricular canal, atrial septal defect, ventricular
septal defect, or a complex congenital heart malformation. These symptoms and conditions
resulting from this disease are typically treated by surgical repair or surveillance of heart defects
to watch for later complications that could arise from these malformations. HMC, PAP, and
CHD can be detected by prenatal ultrasound examinations, but the reliability of prenatal
diagnosis is unknown because the findings during this examination can be variable and most
times the symptoms are not physically apparent until after an individual is born (Slavotinek et al.
2002).
The MKKS gene is a protein coding gene known as the MKKS centrosomal Shuttling
Protein, located on chromosome 20. The MKKS gene instructs proteins associated with the
formation of limbs, heart, and reproductive system. There has been research suggesting that this
protein acts as a chaperonin, assisting in the binding of other proteins.
Since MKS has an autosomal recessive inheritance pattern, if both parents of a particular
individual with MKS are known to be heterozygous for an MKKS pathogenic variant, each
sibling of an affected individual has a 25% chance of being affected, 50% chance of being an
asymptomatic carrier, and a 25% chance of being unaffected and not a carrier. If MKS is a
known concern in an individual, genetic counseling is highly encouraged because of the risk of
the manifestation of Bardet-Biedl syndrome (BBS). This syndrome impacts multiple body
systems ranging in symptoms of retinal degeneration, obesity, reduced kidney function,
polydactyly (extra digits of the hands or feet) among many other features. The phenotype of

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MKS and BBS are indistinguishable at an early age during childhood, multigene panels or
comprehensive genomic testing is encouraged to obtain molecular genetic testing on an
individual. A multigene panel is a cost-efficient test that has the ability to identify the genetic
cause of a condition. This test has limited identification of variants of uncertain significances and
pathogenic variants in genes that do not explain underlying phenotypes, but requires a physician
to determine which gene is likely involved to be able to accurately test using this technique.
Comprehensive genomic testing does not require the clinician to determine which gene is likely
involved (Slavotinek 2020).

METHODS

Study 1
One hundred individuals were screened at birth or in the neonatal period from different
ethnic groups with the MKS phenotype. These were screened for genitourinary malformations in
females, PAP (limbs affected), other digital anomalies, cardiac malformations, renal anomalies,
and GI malformations (Slavotinek 2020).

Study 2
A pedigree study was conducted of affected persons identified in the expanded Old Order
Amish McKusick-Kaufman pedigree. This pedigree was analyzed with the Ped Hunter query
software package applied to the Amish Genealogy Database (AGDB). This is a computerized,
updated and corrected version of the published Old Order Amish genealogy. First, there were
two errors found in the original pedigree that were corrected with the new technology. Second,
PedHunter and AGDB were used to connect the mother of the newly discovered sibship to the
original pedigree and identified that the father was not in the database and cannot be connected
to anyone in the database, leading to the interpretation that their pedigree is incomplete. Third,
PedHunter and AGDB were used to connect the four siblings present in the database into a
pedigree with the minimum number of people required to establish the relationship among
siblings. The small/minimum pedigree allows the researchers to discover the simplest path of
transmission of the disease gene. The pedigree was split into two parts and analyzed separately
since the father of the new sibship cannot be connected to any other person in the pedigree and
three of the original sibships are not in the AGDB.
Two-point LOD scores of MKS are shown in Table 2. LOD scores statistically estimate
whether two genetic loci are physically near each other (Stone et al. 1998).

Study 3
Cells were cultured at 37 degrees Celsius under 5% CO2. The cells were then seeded
onto a lysine-coated glass-bottom dish where they were incubated for 20-24 hours. Following the
incubation time, the cells were analyzed. A polymerase chain reaction (PCR) was used to isolate
the DNA from the cells, which allowed millions of copies for each DNA to be examined. This
DNA sequence was analyzed through ClustalW 2.0.10 software which is a multiple alignment
program for DNA. The human (Homo sapiens) was compared to other species (monkey, mouse,
and rat) (Akimoto et al. 2012).

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Study 4
Complementary DNA (cDNA) is a DNA copy of a messenger RNA (mRNA) molecule
produced by reverse transcriptase which is a DNA polymerase that can use either DNA or RNA
as a template. The cDNA was determined using techniques including alignment of ESTs, full-
length sequencing of associated IMAGE clones, and sequencing of clones isolated from a
gridded cDNA library. Expressed sequence tags (ESTs) is a short sub-sequence of a cDNA
sequence which were used to identify gene transcripts to determine the gene sequence. The
transcript shown in figure 4 has six exons and 570 predicted open reading frames (ORF) with a
start codon in exon 3 and two alternative 5’ terminal exons that are non-coding. A northern
blotting technique was used to detect specific RNA molecules among a mixture of RNA. It was
used to measure the RNA expression of heart, brain, spleen, lung, liver, skeletal muscle, kidney,
and testis tissue (Stone et al. 2000).

Study 5
Studies were performed to identify the correlation between obesity-related genes and
McKusick-Kaufman syndrome to understand the association between MKS genes and metabolic
syndrome. A case-control association study was performed using case-1 and control-1 subjects.
To obtain this information, 85 obesity-related genes that were reported were analyzed. There was
a sample size of 729 people with diagnosed metabolic syndrome tested and a control size of 441
people. The sample of people with metabolic syndrome included people with two or more of
these abnormalities: triglyceride count greater than 150 mg per 100ml and/or high-density
lipoprotein cholesterol less than 1.03 mmoll-1, systolic blood pressure greater than 130 mm Hg
and/or diastolic blood pressure greater than 85 mm Hg, and fasting glucose greater than 6.1
mmol l-1. The control group were people who were not obese and had no metabolic abnormalities
(Hotta et al. 2009).

Study 6
The study includes 350 BBS families, with seven cases from the cohort presented at birth
with hydrometrocolpos and polydactyly who were initially diagnosed with MKKS. Genotyping
was performed on their DNA sequences, exonic sequencing and intron-exon joinings of 12 BBS
genes (BBS1-BBS2) were sequences in the seven patients (Schaefer et al. 2010).

Study 7
Chaperonin genes, that were identified in eukaryotic genomes, were sequenced using
TBLASTN and BLAT. The sequences were aligned in a three-frame-translation of the genomic
sequence to the query sequence with the multiple protein alignment program, ITERALIGN. The
“B-index” was used to obtain the rates of divergence among families of sequences (Mukherjee et
al. 2010).

RESULTS

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Study 1
Table 1. Known MKS diagnosed people who were screened for MKS symptoms
(Slavotinek 2020).
Finding # of Individuals (%)
HMC 42/44 (95%)
Vaginal agenesis 26/44 (59%)
Urogenital sinus 16/44 (36%)
Ectopic urethra 8/44 (18%)
No urethral opening 6/44 (14%)
No vaginal opening 4/44 (9%)
Genitourinary tract fistulae 6/44 (14%)
Hands only 12/42 (29%)
Feet only 6/42 (14%)
Hands & Feet 11/42 (26%)
Four-limb polydactyly 11/42 (26%)
Syndactyly 12/49 (24%)
Metacarpal/tarsal anomalies 8/49 (16%)
Postaxial minimus 6/49 (12%)
Brachydactyly 3/49 (6%)
Absent phalanges 2/49 (4%)
Interstitial polydactyly 0/49 (0%)
Heptadactylia 2/49 (2%)
Various 7/49 (14%)
Hydronephrosis 31/49 (63%)
Hydroureter 12/49 (24%)
Renal cysts 2/49 (4%)
Calyceal dilation 7/49 (14%)
Renal atrophy/hypoplasia 2/49 (4%)
Corticomedullary dysplasia 3/46 (6%)
Nonfunctioning kidney 2/49 (4%)
Imperforate anus 4/49 (8%)
Anal atresia 1/49 (2%)
Hirschsprung disease 6/49 (12%)
Anteriorly placed anus 2/49 (4%)

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Study 2

Figure 2. Pedigrees of extended family structures used in this study. Sibship 7 was added to the
study, but the father was not able to be connected to any known member of the pedigree. The
outlined portion of the pedigree shows a segment that was analyzed separately since the father of
the new sibship cannot be connected to any other person in the pedigree and three of the original
sibships are not in the AGDB (Stone et al. 1998).

Figure 3. Pedigree of sibships 1, 2, 3, and 7 generated by the PedHunter genealogy program.

Determined the most recent common ancestor and minimal looping structure to connect sibships
1, 2, 3, and 7. The relationships specify founder couples that are assumed to carry the abnormal
allele. The shaded symbols (both square (male) and circle (female)) represent individuals who
are present in both this sub-pedigree and the larger pedigree shown in Figure 2 (Stone et al.
1998).

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Table 2. Two-point LOD scores of the disease, phenotypes, and markers in the region. An LOD
score of ~3 or higher is generally understood to mean that two genes are located close to each
other on the chromosome (Stone et al. 1998).

Study 3

Figure 4. Sequence alignment comparison using ClustalW 2.0.10 software of the uMKKS1 and
uMKKS2 amino acid sequences between humans (Homo sapiens), monkeys (Macaca mulatta),
mice (Mus musculus), and rats (Rattus norvegicus) (Akimoto et al. 2012).

Study 4

Figure 5. A diagram of the MKKS gene structure and gene mutations. Exons are shown as
rectangles and altered splicing of exons 1a and 1b (Stone et al. 2000).

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 Figure 6. The expression of MKKS in multiple human tissues using the northern blot
hybridization technique with a probe from exon 3 of MKKS which shows a 2.4-kb transcript in
all tissues and a 7.5-kb transcript in skeletal muscle, heart, and testis (Stone et al. 2000).

Study 5

Table 3. Genotype frequencies and association tests of significant SNPs and replications
(Hotta et al. 2009).

Study 6
Mutations were detected for all seven cases in either MKKS, BBS2, TTC8, or BBS12.
Mutations in BBS2, TTC8, and BBS12 genes are associated with Bardet-Biedl syndrome (BBS).
This disease is characterized with polydactyly and hydrometrocolpos, just as MKS is, so the
results in mutations and phenotypes may not be directly associated to mutations of MKKS, but of
genes related to BBS. Two mutations are recurrent BBS10 mutations. There were frame shift
mutations reported, p.Asn364ThrfsZ5 in BBS10, p.Ala136ArgfsX65, and p.Cys210SerfsX20 in
BBS2. The mutation c.459G>A is a reported splice-site mutation predicted to abolish the splice
the exon TTC8 (Schaefer et al. 2010).

Study 7
Fifty-four chaperonin-like sequences were identified in the human genome and similar
numbers in the genomes of the model organisms (mouse and rat). There are three genes

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associated with the MKKS and BBS pathological conditions. Newly defined chaperonin genes
were named CCT8L and were represented in humans by the CCT8L1 and CCT8L2 sequences.
Vertebrate genomes established the monophyletic origin of chaperonin-like MKKS and BBS
genes from the CCT8 lineage. Sequence analysis and structural predictions of MKKS, BBS, and
CCT8L proteins have a conserved chaperonin-like core structure but are unlikely to form a CCT-
like oligomeric complex. The newly discovered chaperonin pseudogenes uncovered the intense
duplication activity of eukaryotic chaperonin genes (Mukherjee et al. 2010).

DISCUSSION

Case Report 1
A new born weighing 4.3 kg, with a cesarean section at 37 weeks of gestation with gross
anomalies. Her prenatal sonograms showed no abnormalities and were considered normal over
the 35 weeks of being observed by a clinician. She presented mild hydronephrosis of both
kidneys, a 9.5cm cystic mass at the suprapubic area, a transverse vaginal septum, hairy forehead
and ear, bilateral preauricular pits, highly arched palate, soft palate cleft, grade 1 to 2 systolic
murmurs, distended abdomen, collapsed bladder, left foot deformity, anteriorly places stenotic
anus, and asymptomatic pyuria and bacteriuria. During this research, they found that the MKKS
gene is on chromosome 20p13) and is caused by mutations in the MKKS gene14) (Son et al. 2011).

Case Report 2
A case study has been reported, along with many studies, regarding complications for
women that have been diagnosed with MKS at birth at the onset of puberty. This particular case
is a 13-year-old Caucasian female that had been diagnosed with MKS in the neonatal period
presenting with a urogenital sinus, hydrometrocolpos, bilateral hydronephrosis, and postaxial
polysyndactyly. Through the use of computed tomography (CT) scan, physical exam, blood lab
tests, pelvic ultrasound, bimanual pelvic examination, and vaginoscopy.

Table 4. Results from many common reproductive examinations of a 13-year-old Caucasian

female experiencing various symptoms at the onset of puberty (Lueth and Wood 2014).
Test Results
Computed Sonography (CT) scan Enlarged, fluid-filled uterus
Physical exam In pain, hypertensive, tachypneic,
tachycardic, febrile, abdomen distended,
hypoactive bowel sounds, tenderness on
palpation on left side of vaginal wall, hands
and feet cold, strong peripheral pulses,
abnormal left ovary
Blood Test High white blood cell count, high
hemoglobin, high platelet count, elevated
inflammatory markers
Pelvic ultrasound Fluid accumulation in uterus and fallopian
tubes, small dimple at the cervical os
Vaginoscopy Narrow introitus and long vaginal canal

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 From this case report, it shows potential complications that women who are diagnosed
with MKS can experience at the onset of puberty. There have been many case reports discussing
the possible complications of MKS at puberty with recurrent urinary tract infections and re-
stenosis of the vaginal tract. This has led to the conclusion that cervical stenosis, a symptom of
MKS, can lead to the obstruction of menstrual outflow in which hematometra with bilateral
hematosalpinx can potentially be developed. The other concern about the onset of puberty with
women with MKS is the development of endometriosis and risk of associated chronic pain and
infertility (Lueth and Wood 2014).

Study 2
The core of the family was genotyped and three of the affected children were
homozygous for markers at 11 loci (13 markers) in the whole genome scan (385 markers).
Additional affected individuals were added, as well as homozygous markers. This allowed all
regions other than 20p to be eliminated due to heterozygosity in many affected individuals.
Heterozygous markers in the region included D20A162, D20S901, NM1, D20S894,
AFM164TG5, and D20S189. These markers are polymorphic satellite markers that are co-
dominant and track when there is a polymorphic DNA locus containing repeated nucleotide
sequences, generally with 2 to 10 nucleotides per repeated unit. All individuals studied were
homozygous for D20S160 and D20S1154. Ordering of the markers were applied in order based
on physical mapping data in the region (an Alagille syndrome region physical map) (Stone et al.
1998).

Study 3
From figure 4, we can see that there are many amino acids that were highly conserved
over the human, monkey, mouse, and rat genome. This indicates that there is potentially
conservation of biological processes that cause MKS across species, specifically mammals
because they were the ones that matched the DNA sequence on ClustalW 2.0.10 software. There
were partial matches of the -3 position of the initiation codon for the MKKS1 and uMKKS2 start
codon. This start codon is a strong start codon because of the purine being located at -3 and a G
at the +4 position, shown in figure 4. These conclusions lead the researchers to believe that the
uMKKS1 and MKKS2 can be translated into proteins in mammals. This allows the hypothesis
that these proteins that are translated lead to the disruption of normal development of cells and
could potentially cause the MKS condition (Akimoto et al. 2012).

Study 4
Understanding the gene sequence allowed the researchers to improve the diagnoses of the
MKS condition by furthering their understanding of key mutations in DNA sequences. There
were several polymorphisms when sequencing MKKS which indicates that there are two
alternative phenotypes which confirms the recessive manner of transmission of this disease. In
exon 3, G49V is present in 8.3% of normal chromosomes. In exon 6, R517C is present in 6.3%
of normal chromosomes, and G532C is present in 6.4% of normal chromosomes.
The northern blot hybridized, shown in figure 6, with a probe showed a 2.4-kb transcript
in all tissues indicates that there is expression of the MKKS gene in all of these tissues.
Specifically, the MKKS gene is highly expressed in skeletal muscle, the heart, and the testis with

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a 7.5-kb transcript. This supports the hypothesized description of the function of affecting the
hands/feet, heart, and reproductive system (Stone et al. 2000).

Study 5
The case-control association study found that three SNPs (rs1545, rs1547, and
rs2294901) in the MKKS gene were significantly related to metabolic syndrome, while also
applying the Bonferroni’s correlation, shown in table 3. The Q-values of these three SNPs were
significantly small which suggests the MKKS gene is related to metabolic syndrome. A second
set of subjects confirmed the associations of these three SNPs. In case-2 the minor allele
frequency was higher than control-2, this was not considered very significant because of the
small number of control-2. In case-2 significant associations were observed: P = 0.017 in rs1547,
P = 0.022 in rs1545, and P = 0.019 in rs2294901. From these numbers, the conclusion was made
that SNPs in MKKS are significantly associated with metabolic syndrome. Based on this study,
MKKS would be a good candidate gene for the development of metabolic syndrome. The
knowledge of the SNPs that affect the development of metabolic syndrome were needed because
this study has confirmed that the MKKS protein can contribute to the development of metabolic
syndrome (Hotta et al. 2009).

Study 6
From the genotyping performed in this experiment, the conclusion was made that the
MKKS and BBS overlap in the prenatal and neonatal period because of their common
presentation with polydactyly and hydrometrocolpos. The evolution between these two diseases
are different from one another, MKKS patients remain free of late onset complications while
BBS progresses to lead to the development of pigmentosa, obesity, renal dysfunction, and
potentially cognitive impairment. It was reported that nine patients diagnosed with MKKS
initially in infancy developed BBS features. The mean age to make this distinction between these
diseases is nine years of age. To distinguish the difference in MKKS and BBS ultrasound
prenatal diagnosis should be conducted to show hydrometrocolpos with polydactyly and
molecular testing. The mutations associated with MKKS phenotype have been reported only in
the MKKS gene, so this testing should confirm the distinction between MKS and BBS in early
stages. In conclusion, hydrometrocolpos with polydactyly is a good indicator for the BBS
diagnoses compared to the MKKS diagnosis. Patients should potentially get molecular testing for
a prognosis of BBS to follow up with genetic counseling (Schaefer et al. 2010).

Study 7
In vertebrates, chaperonin genes, driven by intense duplication processes, have
diversified into multiple classes and functionalities that extend beyond their well-known protein-
folding role in the oligomeric chaperonin complex. The newly identified chaperonin genes will
need to be further studied through experimental analysis to fully understand their effects
(Mukherjee et al. 2010).

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