LYSOSOMAL STORAGE DISORDERS: FROM BASIC SCIENCE TO PUBLIC
HEALTH
by
Anatália Labilloy
MD, Universidade Federal do Piauí, Brazil, 2010
Submitted to the Graduate Faculty of
Department of Human Genetics
Graduate School of Public Health in partial fulfillment
of the requirements for the degree of
Master of Public Health
University of Pittsburgh
2013
University of Pittsburgh
Graduate School of Public Health
This essay is submitted
by
Anatália Labilloy
on
April 8th 2013
and approved by
Essay Advisor:
David Finegold, MD
Professor
Department of Human Genetics
Graduate School of Public Health
University of Pittsburgh
________________________
Essay Reader:
Karen Cuenco, PhD
Assistant Professor
Department of Oral Biology
Center for Craniofacial Human Genetics
School of Dental Medicine
University of Pittsburgh
________________________
ii
Copyright © by Anatália Labilloy
2013
iii
David Finegold, MD
LYSOSOMAL STORAGE DISORDERS: FROM BASIC SCIENCE TO PUBLIC
HEALTH
Anatália Labilloy, MPH
University of Pittsburgh, 2013
ABSTRACT
According to the first Principle of the Ethical Practice of Public Health by the
Public Health Leadership Society, “Humans have a right to the resources necessary for
health.” When dealing with rare inheritable conditions, oftentimes disease mechanisms
remain largely unknown. The uniqueness of rare conditions render healthcare
professionals unaware of their existence or of ways to deliver best medical care
adapted to individual patients’ needs. Lysosomal Storage Disorders constitute a group
of more than 50 rare heritable conditions that share some pathological and biochemical
features
and
result in
significant morbidity and
early death.
Most of
the
pathophysiological aspects of individual disorders of this group remain uncharacterized.
Advances in clinical development of therapies in the past two decades have improved
clinical outcomes for patients with some of these diseases, but progress still needs to
be made for better management of some of the complications of these disorders. The
cost associated with some of these therapeutic options still represents a limiting factor
to access, especially in developing countries. This study’s primary aim was to foster
basic science research in the field of lysosomal storage disorders by developing and
iv
characterizing a cell model for Fabry disease that can be used to understand disease
pathogenesis for drug development. An additional aim was to develop a strategic plan
for a public health genetics program with focus on Lysosomal Storage Disorders in an
underprivileged state in a developing country. This study’s public health significance lies
in the continuation of efforts to understand disease mechanisms, which potentially allow
development of new therapeutic approaches through basic and clinical science
research. In addition, efforts to increase awareness of these disorders among
healthcare professionals, and to integrate research findings into clinical practice,
fostering the delivery of evidence-based medical care in the field are of public health
relevance and necessary to improve health of patients with Lysosomal Storage
Disorders.
v
TABLE OF CONTENTS
ACKNOWLEDGEMENTS…………………………………………….………………………XII
1. DEVELOPMENT OF A CELL MODEL OF FABRY DISEASE IN RENAL TUBULAR
EPITHELIAL CELLS……………………………………………………………….……………1
1.1 INTRODUCTION AND BACKGROUND………….………..………………………….1
1.1.1 FABRY DISEASE DIAGNOSIS AND TREATMENT…………………………..9
1.1.2 MODELS OF FABRY DISEASE…………………….………………………....12
1.1.3 PUBLIC HEALTH RELEVANCE……………….………………………………14
1.2 METHODS……………………………………….……………………………………..16
1.3 RESULTS……………………………………………………………………………….18
1.4 DISCUSSION……………………………….…………………………………..………21
2. A PUBLIC HEALTH GENETICS PLAN FOR PIAUÍ STATE WITH FOCUS ON
LYSOSOMAL STORAGE DISORDERS…………………………………………………….23
2.1INTRODUCTION AND BACKGROUND……………………………………………...23
2.1.1 HEALTHCARE IN BRAZIL……………..….…………………………………...23
2.1.2 PIAUÍ STATE DEMOGRAPHICS…….………………………………………..24
2.1.3 PIAUÍ STATE HEALTH DEPARTMENT……………..……………………….26
2.1.4 GENETICS IN BRAZIL……………….………………..……………………….28
2.1.4.1 NEWBORN SCREENING IN BRAZIL………………………………29
2.1.4.2 CURRENT GENETICS SERVICES IN PIAUÍ STATE………….…32
2.1.4.3 LYSOSOMAL STORAGE DISORDERS IN BRAZIL AND PIAUÍ
STATE….........................................................................................................................33
2.2 NEEDS ASSESSMENT AND DEVELOPMENT OF A PUBLIC HEALTH
GENETICS STATE PLAN IN LYSOSOMAL STORAGE DISORDERS………...……….34
2.2.1 MISSION………………....…………………………………………………..…...34
vi
2.2.2 ASSUMPTIONS……………………………………………….…………………35
2.2.3 NEEDS ASSESSMENT……………………………………………………………..35
2.2.4 GOALS, OBJECTIVES AND ACTIVITIES…..…………………………………….37
2.2.4.1 GOAL 1…………………………………………………………………….......37
2.2.4.2 GOAL 2…………………………………………………………………………39
2.2.4.3 GOAL 3…………………………………………………………………………40
2.2.5 PROGRAM DEVELOPMENT STRATEGY………………………………………..40
2.2.6 EVALUATION PLAN…………………………………………………..…………….41
2.2.7 EXPECTED OUTCOMES AND IMPACT………………………………………….44
2.2.8 CHALLENGES……………………………………………………………………….45
3. FINAL CONSIDERATIONS AND FUTURE DIRECTIONS……..……………………..45
APPENDIX A: LYSOSOMAL STORAGE DISORDERS……………………………….….48
APPENDIX B: GOVERNMENTAL AND NON-GOVERNMENTAL ORGANIZATIONS
PROVIDING CARE FOR INDIVIDUALS WITH DISABILITIES/SPECIAL NEEDS IN
PIAUÍ STATE, BRAZIL………………………………………………………………………..50
BIBLIOGRAPHY…………………………………………………………………………….....52
vii
LIST OF ABBREVIATIONS
α-Gal A
Alpha-galactosidase A
CNS
Central Nervous System
eGFR
estimated Glomerular Filtration Rate
ERT
Enzyme replacement therapy
Gb3
Globlotriaosylceramide
GSL
Glycosphingolipid
HIV
human immunodeficiency virus
IFN
Interferon
LSD
Lysosomal Storage Disorder
M6P
Mannose-6-phosphate
MDCK
Madin-Darby canine kidney
MHC
major histocompatibility complex
MRI
Magnetic Resonance Imaging
mRNA
messenger ribonucleic acid
siRNA
small interfering ribonucleic acid
SRT
Substrate Reduction Therapy
SUS
Brazilian Unified Health System
TIA
Transient Ischemic attack
viii
NPHPSP
National Public Health Performance Standards Program
ix
LIST OF TABLES
Table 1: Lysosomal Storage Disorders with available treatment ………………………….6
Table 2: The Essential Public Health Services………..……………………………………14
x
LIST OF FIGURES
Figure 1: Fabry disease progresses with age, resulting in multisystem involvement and
early death. ...............................................................................………..………………….2
Figure 2: Disorders of GSL and ganglioside metabolism and their relationship. .......... …5
Figure 3: Efficient silencing of α-Gal A expression in MDCK cells by siRNA transfection
..................................................................................................................................... 18
Figure 4: α-Gal A silenced MDCK cells have increased levels of Gb3......................... 19
Figure 5: Electron micrographs of MDCK cells treated with control and α-Gal A siRNA.20
Figure 6: Accumulation of "zebra bodies in α-Gal A siRNA treated MDCK cells............ 21
Figure 7: Strategy for the Program Development phase of a Public Health Genetics Plan
for Piauí State with Focus on LSDs. .............................................................................. 40
xi
ACKNOWLEDGEMENTS
Another cycle has ended for others to begin. The accomplishments that I have
achieved during my academic studies leading to a Master in Public Health in Public
Health Genetics at the University of Pittsburgh were unparalleled. The difficulties and
barriers existed, but with determination, hard work and full support from the professors
of the Department of Human Genetics and from my wonderful classmates the path was
smooth and the professional and personal achievements were very fruitful.
I would like to express my sincere gratitude to my thesis advisor Dr David
Finegold for guiding and supporting me over the progress of my graduate studies, and
for being an example of passionate scientist and caring doctor. I would also like to thank
Karen Cuenco for accepting to be part of my thesis committee and her helpful advices,
Dr Robert Ferrell for inspiring conversations and my academic advisor Dr Candance
Kammerer for the patience and invaluable guidance.
I am especially thankful to Dr Ora Weisz for introducing me to key concepts in
basic science research in cell biology, a field that was new to me. She has encouraged
me and served as a role model of a successful scientist, leader and woman. Jennifer
Bruns was also essential to this work for her friendship and technical support with the
experiments in developing the renal cell model. I couldn’t have worked in a better
environment than the one I found at Dr Weisz’s lab.
xii
Foremost I would like to thank my family for always being there for me, my
husband Guillaume for his love, patience and understanding and my parents for having
always invested in my education and for showing full support even being so far away.
You are the foundation for my academic success.
Lastly, I would also like to acknowledge the financial support from Shire HGT as
a grant to the Graduate School of Public Health for a scholarship in the field of
Lysosomal Storage Disorders.
xiii
1. DEVELOPMENT OF A CELL MODEL OF FABRY DISEASE IN RENAL
TUBULAR EPITHELIAL CELLS
1.1 Introduction and Background
Fabry disease is an X-linked lysosomal storage disorder (LSD) caused by
abnormal function of the lysosomal hydrolase α-galactosidase A (α-Gal A),
encoded by the gene GLA. [1] Deficiency of α-Gal A leads to progressive
accumulation of its substrates, neutral glycosphingolipids (GSLs) with terminal αD-galactosyl residues, mainly globotriaosylceramide (Gb3), in a variety of cell
types. [2] Fabry disease occurs in both males and females, and classic clinical
presentation of the disease includes renal, cardiovascular, cerebrovascular,
neurological, gastroenterological, dermatological, ocular, and auditory findings,
among others, resulting in significant morbidity and early death. [3, 4] Figure 1
shows a diagram with some of the clinical findings of Fabry disease and its
disease progression with age.
The first signs and symptoms of Fabry disease often appear in the first
decade of life, mostly presenting as neuropathic pain, gastrointestinal symptoms,
and impaired quality of life. Some pediatric patients may also present with
serious complications of Fabry disease and multi-system organ involvement.
Data from Fabry registry have show a mean age of onset of symptoms of 6 years
for boys and 9 years for girls. [5-7]
1
alpha-galactosidase A deficiency
Progression with age
glycosphingolipid accumulation
neuropathic pain, exercise intolerance,
gastrointestinal symptoms, cornea verticillata
and angiokeratomas
microalbuminur
ia, proteinuria
hyperfiltration,
isosthenuria
End-Stage
renal Disease
hypertrophic
cardiomyopath
y, short PR
interval,
arrhythmias
Systolic and
diastolic
dysfunction,
myocardial
infarction
MRI white
matter lesions,
vertebrobasilar
dolichoectasia,
pulvinar sign
TIA and strokes
Considerable morbidity and mortality
Figure 1: Fabry disease progresses with age, resulting in multisystem involvement and early death.
Progression in any organ system can proceed independently from the others. MRI: magnetic resonance
imaging; TIA: transient ischemic attack. Adapted from [8]
Some patients may never display the classic phenotype, presenting a
milder form of the disease. In fact, “cardiac variants” and “renal variants” of Fabry
disease have been described, where patients present disease limited to only one
or few organs. [9]
The kidney is one of the most affected organs in individuals with Fabry
disease. While advanced stages of renal chronic disease usually present in
middle adulthood, the first histological signs of Fabry nephropathy have been
seen as early as in utero. [5] Additionally, children with Fabry disease can
2
present
early
signs
of
kidney
involvement
such
as
hyperfiltration,
microalbuminuria, and overt proteinuria [10, 11]. Within the kidney, podocytes
seem to be the most involved cell type, showing a greater degree of
vacuolization. However, Gb3 inclusions are also observed in proximal and distal
tubules as well as mesangial, endothelial, and vascular smooth muscle cells. As
Fabry nephropathy progresses, mesangial expansion, interstitial fibrosis, tubular
atrophy, and glomerulosclerosis are observed. [12].
Fabry disease is the second most common disease of its group of LSDs,
behind only Gaucher disease, an autosomal recessive form of LSD characterized
in its most common form mainly by spleen, liver, lungs, bone and bone marrow
involvement. [13] Currently, more than 50 disorders have been clinically
categorized as LSDs, resulting from deficient activity of specific lysosomal
enzymes or abnormalities in transmembrane proteins as well as in proteins
involved in lysosomal biogenesis. [14] Appendix A presents a comprehensive list
of LSDs. Although individually rare, the estimated combined prevalence of this
group of conditions ranges from 1:4,000 to 1:8,000 births. [15] In spite of being
considered panethnic, Individual estimates of incidence vary from study
population and some disorders of this group are thought to be underdiagnosed;
some disorders of this group such as Gaucher disease, mucolipidosis type IV,
Tay-Sachs disease and Niemann-Pick disease have increased incidence in
Ashkenazi Jews, attributed to historical founder effects and genetic drift.[16]
In spite of their genetic heterogeneity, LSDs share the phenotype of
accumulation of one or several substrates within lysosomes and in non-
3
lysosomal compartments, as well as some clinical and biochemical features [17,
18]. Figure 2 shows the biochemical link among several LSDs. As lysosomal
sphingolipid degradation occurs in a step-wise manner, the product of
degradation of one enzyme serves as the substrate for the subsequent reaction.
In addition, two or more pathways can be interconnected, presenting the same
final product. As an example, lactosylceramide acts as a product of both alphagalactosidase A degradation of Gb3 and of sialidase degradation of GM3.
Moreover, the same sphingolipid activator protein (SAP), which are proteins
required for hydrolysis of sphingolipids, can participate in different steps of GSL
metabolism. [19]
Specific treatment is available for several LSDs, and early diagnosis and
management translates into better outcomes for patients. Treatment options
either clinically available or under research development for disorders of this
group include enzyme replacement therapy (ERT), substrate reduction therapy
(SRT), hematopoietic stem cell therapy (HSCT), chemical chaperones, cell
therapy and gene therapy. [27] Table 1 shows a list of treatable LSDs with their
respective estimated incidence and treatment options available or under
development.
4
Figure 2: Disorders of GSL and ganglioside metabolism and their relationship. Abbreviations: DHCer
(dihydroceramide), S1P (sphingosine 1-phosphate), SM (sphingomyelin) . [19]
Several studies have estimated the prevalence of Fabry disease to range
between 1:40,000 and 1:117,000 live births. [20] With recent advances in highthroughput multiplex enzyme assays using tandem mass spectrometry, allowing
feasibility for larger scale screening tests, some countries have started pilot
5
newborn screening programs for some LSDs, including Fabry disease. [21, 22]
Surprisingly, newborn screening data suggests a much higher incidence of Fabry
disease than initially thought, ranging from 1:3,859 newborns in Austria [23] and
1:3,100 in Italy [24] to as high as 1:557 births in Taiwan [25]. This suggests that
the disease might be greatly under-diagnosed worldwide. In the United States,
pilot newborn screening studies for Fabry disease and other LSDs are currently
being performed. If expanded to the whole country, more than 1,000 babies are
expected to be diagnosed with the disease each year in the United States, based
upon estimates from European countries [23-24]. However, newborn screening
for LSDs remains controversial, since specific treatment is unavailable for some
conditions of the group. In addition, the onset of symptoms for some LSDs may
not occur until adulthood, in those circumstances neonatal diagnosis may not
interfere with the clinical course of the disease. [26]
Table 1: Lysosomal Storage Disorders with available treatment.
Disease
Treatment available
Aspartylglucosaminuria
HSCT available
Fabry disease
ERT available, Chaperones in CD
Fucosidosis
HSCT available
Gaucher disease
ERT, SRT and HSCT available
Krabbe disease
HSCT available
α-Mannosidosis
HSCT available, ERT in CD
Metachromatic
leukodystrophy
IT-ERT in CD, gene therapy in CD
MPS I
ERT and HSCT available
MPS II
ERT available; IT-ERT in CD
MPS III (all types)
IT- ( in CD or MPS IIIA; SRT in CD; GT in CD
6
Table 1 continued
MPS IV A
ERT in CD
MPS VI
ERT available, HSCT available
Mucolipidosis II/III
HSCT in CD
Neuronal ceroid
lipofuscinosis
Cell therapy in CD, gene therapy in CD
Niemann-Pick B
ERT in CD
Niemann-Pick C
SRT available, HSCT in CD for NPC2
Pompe disease
ERT available
Sandhoff disease
Chaperones in CD for late-onset forms
Tay-Sachs disease
Chaperones in CD for late-onset forms
Adapted from ref. [27]
The lysosomal hydrolase α-Gal A enzyme is encoded by the gene GLA,
located in Xq22. The GLA gene is comprised of seven exons and 12,436 base
pairs. [1] In the lysosome lumen, α-Gal A is found as a homodimer, each
monomer presenting a N-terminal, which contains the active site, and a Cterminal. [28] According to the Human Gene Mutation Database, over 600
mutations have been described for Fabry disease. (http://www.hgmd.org). The
majority of mutations in GLA gene leading to Fabry disease are missense and
translate into a disruption of the hydrophobic core of α-Gal A, which makes Fabry
disease primarily a disease of protein-folding. [29]
Gb3, also known as CD77 and Pk, is the primary glycosphingolipid
degraded by α-Gal A. Biosynthesis of Gb3 from lactosylceramide is performed by
the type II membrane protein alpha-1,4-galactosyltransferase (A4GALT) in the
Golgi apparatus. [30] After its synthesis, Gb3 is then incorporated into the plasma
membrane and intracellular membranes, with a preference for sphingolipid and
7
cholesterol enriched microdomains (aka, lipid rafts). This glycosphingolipid can
also shed from the plasma membrane, being found in the plasma associated with
lipoproteins, preferentially LDL. [31] Gb3’s deacylated form, globotriaosylsphingosine, aka lyso-Gb3, is also elevated in Fabry disease. Lyso-Gb3 is
degraded by α-Gal A at a rate 50 times lower than Gb3, and presents inhibitory
properties over both native and recombinant α-Gal A. [32] Thus, lyso-Gb3 might
have additional direct contribution to the pathogenesis of the disease and in
organ-response to therapy.
Gb3 has been implicated in a variety of cellular processes. As an
example, Gb3 is the receptor for the subunit B of Shiga and Shiga-like toxins
(Stx) produced by bacteria as Escherichia coli and Citrobacter freundii. [33, 34]
Gb3 is also one of the several glycolipids that act as co-receptor for gp120 in HIV
entry, being required in CD4/CXCR4-dependent fusion. [35, 36] In fact, this
interaction is being further explored in the development of new therapies for HIV,
as the Gb3 analog adamantylGb3 inhibits HIV infection in vitro. [37]
As a component of sphingolipid- cholesterol enriched microdomains, Gb3
is thought to play a role in signal transduction, with major involvement in
inflammation and immune responses. Gb3 is involved in CD19-mediated
adhesion and is required for both interferon-alpha induced growth inhibition and
for the plasma membrane localization of Hsp70 [38, 39]. In addition, MHC class II
and type 1 IFN receptor molecules present binding domains for Gb3,
corroborating with its role in immune response [40, 41]. Moreover, Gb3, along
8
with other globo series glycosphingolipids, can be a mediator of the cSrc kinase,
beta-catenin, and caspase signaling pathways [42].
Increase in levels of Gb3 is not a specific finding of Fabry disease.
Although to a lesser extent than in Fabry disease, Gb3 levels are elevated in
Niemann-Pick C, another LSD, which is consistent with the fact that these
conditions share some of their pathophysiology. [43] Greater Gb3 levels can also
be seen in more common disorders. Cells in pre-cancerous and cancerous
states, such as colorectal adenoma, Burkitt’s lymphoma, breast cancer, and
testicular carcinoma may also present increased levels of Gb3. [44] Interestingly,
this increase in Gb3 levels is considered a sign of bad prognosis in these
conditions, being associated with multidrug resistance and greater invasiveness.
[45] However, both the underlying mechanism for such an increase in Gb3 levels
and the cellular consequences culminating in greater tumor aggressiveness
remain unclear. Some cytokines such as TNF-alpha and Interleukin-1 are shown
to up-regulate Gb3 levels [46, 47] nevertheless the pathophysiological impact of
such increases also needs to be better understood.
1.1.1 Fabry disease Diagnosis and Treatment
Due to the presence of several signs and symptoms that mimic common
disorders, the absence of pathognomonic clinical features, and marked
phenotypical heterogeneity, clinical diagnosis of Fabry disease is usually delayed
and involve visits to several medical specialists and misdiagnoses. [48] After
onset of clinical manifestation, overall diagnostic delays are about 15 years for
both genders [49], with possiblly even longer delays in developing countries.
9
Some of the misdiagnoses include rheumatoid arthritis, rheumatic fever, systemic
lupus erythematous, Raynaud’s disease, celiac disease, and multiple sclerosis,
among others. [9] After clinical suspicion is raised, demonstration of deficient αGal A in leukocytes in males is sufficient to establish a diagnosis of Fabry
disease in males, and may be followed by mutational analysis of GLA gene. An
initial screening through plasma enzyme activity in dried blood spots can be
performed
in
males,
but
should
be
confirmed
by leukocyte
enzyme
activity/genotyping. For females, disease status should be determined by
mutation analysis of GLA since α-Gal A enzyme activity may be within the normal
range in females even in the presence of overt disease. [50]
In the recent years, there have been increased screening efforts for
diagnosis of Fabry disease in high-risk adult populations. These efforts included
screening for Fabry disease among patients with end-stage renal disease [51],
unexplained hypertrophic cardiomyopathy [52, 53] and young patients presenting
with stroke but no apparent predisposing factor [54]. Most of the studies focused
in male patients due to the low sensitivity of the enzyme activity analysis in
females, who usually require genotyping, a more expensive and laborious
method, for definite diagnosis. These studies have shown prevalence of Fabry
disease in high-risk populations ranging from 0 to 12 percent.
Although the presence of residual α-Gal A activity is associated with
slower progression of Fabry nephropathy, α-Gal A in plasma or leukocyte
enzyme activity levels do not always correlate with disease phenotype. Similarly,
conservative amino acid changes in GLA gene may be associated with slower
10
progression of the disease, but genotype-phenotype correlations have not been
fully established for Fabry disease. Phenotype heterogeneity may play a role
even within a single family of individuals sharing the same genotype. [55]
Currently, the only commercially available treatment option for patients
with Fabry disease consists of recombinant enzyme replacement therapy (ERT)
administered intravenously. There are two approved forms of recombinant
enzyme: agalsidase alfa and agalsidase beta. Agalsidase alfa is engineered in a
human fibroblast cell line and presents amino acid sequence identical to that of
native human α-Gal A, being glycosylated with both sialic acid and mannose-6phosphate (M6P) residues. Its administration occurs intravenously at a dose of
0.2 mg/Kg every other week. [56] Agalsidase beta is produced in Chinese
hamster ovary cells, also presenting the same amino acid sequence as the
native human α-Gal A. The approved dose for its intravenous administration is 1
mg/Kg every other week. [57] The pattern of glycosylation as well as M6P
receptor binding and M6P receptor mediated endocytosis slightly differs between
the two recombinant enzymes. Agalsidase beta presents a higher number of fully
sialylated oligossacharides and a higher level of phosphorylation in comparison
to agalsidase alfa. However, these discrepancies do not seem to translate in
functionally relevant differences. [58, 59]
Long-term ERT significantly improves overall clinical status of individuals
with
the
disease,
mainly
by
reducing
neuropathic
pain,
decreasing
cardiomyopathy, and improving quality of life. [60-62] However, both ERT
regimens are only able to slightly retard the progression of chronic kidney
11
disease, and a steady decline of eGFR is still observed despite long-term
treatment. [63, 64] Moreover, as the intravenously administered enzyme does not
cross the blood brain barrier, disease progression in the CNS resulting in white
matter lesions and strokes remains unchanged in spite of ERT. [65] A better
comprehension of the pathophysiology of Fabry disease could provide the means
to develop better clinical approaches and novel pharmacological targets in the
management of Fabry disease patients.
Other treatment options are currently under investigation. Results of a
phase II clinical trial for the pharmacological chaperone Migalastat HCl were
recently published, showing that its oral administration in patients with responsive
mutations increased blood α-Gal A activity by 50 percent. [66] The iminosugar
binds and stabilizes the nascent enzyme in the ER, restoring its efficient
trafficking to lysosomes in the cases where proper trafficking of the misfolded
enzyme is the main contributing factor for enzyme deficiency. [67] A phase III
clinical trial for migalastat is currently ongoing.
A plant cell-expressed recombinant α-Gal A, PRX-102, is also under a
phase I/II clinical trial study for treatment of Fabry disease. The recombinant
enzyme has undergone chemical modifications to improve enzyme activity and
stability and to decrease its immunogenicity. [68]
1.1.2 Models of Fabry Disease
Although naturally occurring animal models have been reported for other
LSDs, no animals have been identified with Fabry disease. A knockout mouse
12
model for Fabry disease with lack of α-Gal A activity was developed and
described by Oshima et al. [69] However, use of this knockout mice model in
translational research has been controversial; while the mice present some
histological changes compatible with Fabry disease, both male and female mice
appear to be clinically normal,[69] or present mild phenotype as they age [70,
71], not reflecting what occurs in humans with classic phenotype.
Establishment of cell models have also been an useful as a tool for testing
hypotheses of disease pathogenesis, for identifying biomarkers of disease
progression and clinical prognosis, and for early-stages drug development and
testing for several human diseases.
One of the technologies being used to study protein function in the context
of a living cell and to generate knockdown of proteins of interest for human
disease is RNA interference (RNAi). Small interfering RNA (siRNA) molecules
serve as a template to the multi-protein complex RISC (RNA induced silencing
complex) for recognition of complementary mRNA of interest, which results in
activation of RNAse, culminating in cleavage and degradation of the mRNA and
consequent loss of protein translation. [72] siRNA mediated transient silencing of
α-Gal A could mimic the loss of protein activity observed in Fabry disease in
different cell types. This technology would be of particular significance for kidney
cells, as the pathophysiology of Fabry nephropathy is poorly understood and the
kidneys of Fabry patients have little response to therapy, making kidney cells a
good target for the studies on disease pathogenesis and drug discovery.
13
Madin-Darby canine kidney (MDCK) cells, isolated from dog kidney cortex
and easily cultivated as polarized monolayers in vitro, have been studied and
characterized extensively as a model for the study of renal cell function. [73].
In this study, we have designed, developed and characterized a renal
epithelial tubular cell model for the study of the effects of deficiency of α-Gal A.
1.1.3 Public Health Relevance
The National Public Health Performance Standards Program (NPHPSP)
through the Core Public Health Functions Steering Committee has established in
1994 the “Ten Essential Public Health Services”, which constitute the essential
framework for the core public health activities. Error! Reference source not
found. shows a list of the Essential Public Health Services. The 10th essential
public health service, “research for new insights and innovative solutions to
health problems” can be interpreted as the responsibility of public health systems
to investigate human diseases and their mechanisms, search for elucidation of
their pathophysiology as well as promote means of developing new therapies.
Table
The Essential Public Health Services
Table
2 2:
continued
1.
Monitor health status to identify and solve community health problems
2.
Diagnose and investigate health problems and health hazards in the community
3.
Inform, educate, and empower people about health issues
4.
Mobilize community partnerships and action to identify and solve health problems
5.
Develop policies and plans that support individuals and community health efforts
6.
Enforce laws and regulations that protect health and ensure safety
7.
Link people to needed personal health services and assure the provision of
health care when otherwise available
8.
Assure competent public and personal health care workforce
14
Progress in basic science research involving the development of cell
9.
Evaluate effectiveness, accessibility, and quality of personal and populationbased health services
10.
Research for new insights and innovative solutions to health problems
models for human diseases are an essential first step for understanding the
cellular dysfunctions underlying disease mechanisms, for identifying prognostic
factors and biomarkers of disease progression, and for evaluating therapeutic
approaches to cure diseases. These models are especially crucial in cases
where mouse models do not replicate human disease phenotypes, such as in
Fabry disease.
Moreover, a better understanding of the pathophysiology of this relatively
rare condition can potentially help unravel pathogenic mechanisms underlying
some common diseases. In fact, Fabry nephropathy resembles diabetic
nephropathy in humans. [74] Moreover, a link between glycosphingolipid
metabolism and development and progression of diabetic nephropathy and
metabolic syndrome has been suggested. [75] Furthermore, as previously
mentioned, Gb3 is also elevated in certain cancers. This increase is associated
with metastasis and tumor aggressiveness and a decreased response to
chemotherapy drugs. In addition, some LSDs within the group of sphingolipidosis
may also play a role in complex disorders. For instance, mutations in GBA, the
gene encoding beta-glucocerebrosidase and causing Gaucher’s disease, have
been found to be the most common risk factor ever described for Parkinson’s
disease. [76] As this disease also presents elevated levels of sphingolipids,
elucidation of disease mechanisms for Fabry disease may lead to new avenues
15
of investigation of the link between Parkinson disease and the LSD Gaucher
disease.
1.2 Methods
Cell culture. Madin-Darby canine kidney (MDCK) cells were incubated on 10 cm
plastic dishes in MEM with 10% fetal bovine serum (FBS) at 37 oC with 5% CO2.
Nonpolarized cells were cultured on 12 mm permeable supports with 0.4 m
pores for four to six days prior to the experiments.
siRNA knockdown. We designed a small interfering RNA (siRNA) for transient
knockdown of the canine α-Gal A targeting the following sequence 5’GATAGATCTGCTGAAATT-3’. siRNA against firefly luciferase targeting the
sequence 5’-GAATATTGTTGCACGATTT-3’ was used as a control. The custom
siRNA duplex and control siRNA constructs were purchased from Dharmacon.
MDCK cells were transfected with either α-Gal A or control siRNA using
Lipofectamine™ 2000 transfection reagent (Invitrogen, Inc) and Opti-MEM® I
Reduced Serum Media (Invitrogen, Inc). Briefly, 2.6 μg of either α-Gal A or
control siRNA were incubated with 10 μL of lipofectamine and 250 μL of OptiMEM for 30 minutes at room temperature. 125 μL of the transfection mix and 5 x
105 subconfluent MDCK cells suspended in 333 μL of MEM were added to upper
chamber of a 12-well transwell filter and briefly homogenized. Growth media was
replaced within 6 to 8 hours of transfection and every other day subsequently.
Experiments were performed three to six days later.
16
RT-PCR. Isolation of total RNA was performed using kit RNAquous (Ambion)
according to manufacture’s recommendations. Purified total RNA underwent
reverse transcription by incubation with Moloney murine leukemia virus reverse
transcriptase (Ambion) at 42°C for 1 hour. PCR for canine alpha-galactosidase
was carried out using Phusion ® High-Fidelity PCR kit (New England BioLabs,
Inc.), having as sense primer 5’-TGTGCAACGTTGACTGCCAAGAAG-3’ and the
anti-sense primer 5 ’- TCCTGCAGGTTTACCATAGCCACA-3’. As a control, RTPCR for canine beta-actin was also carried out using the sense primer 5’CTGCTGGAAGGTGGACAG-3’
and
the
anti-sense
primer
5’-
ACCTTCAACTCCATCATGAAG-3’. Denaturing temperature was 95oC, annealing
temperature was 58oC, and extension temperature of 68oC, with 25 cycles of
amplification.
Indirect Immunofluorescence and confocal microscopy. Immunofluorescence was performed according to the protocol described by Mattila et al. [77]
Anti-CD77 monoclonal antibody (clone 38-13, Abcam), which recognizes Gb3,
was used at a dilution of 1:10. Goat anti-rat secondary antibodies (Alexa 488 and
Cy5) were used at a dilution of 1:500. Capture of images was performed on an
Olympus Fluoview FV1000 laser-scanning confocal microscope in the RenalElectrolyte Division, University of Pittsburgh School of Medicine.
Electron
microscopy.
Membranes
containing
monolayers
of
α-Gal A
knockdown and control MDCK cells were fixed in 2.5% of glutaraldehyde, 2% of
paraformaldehyde in PBS and post-fixed in 1% Osmium tetroxide. Samples were
17
then dehydrated in graded series of ethanol solutions and embedded in EPON
according to standard procedures. Ultra-thin sections were stained with uranyl
acetate and lead citrate and examined using a JEOL electron microscope, in
collaboration with Dr Willi Halfter, University of Pittsburgh.
1.3 Results
Silencing of α-Gal A in MDCK cells
In order to confirm knockdown of the protein of interest at the mRNA level,
RT-PCR for α-Gal A was performed. Canine beta-actin was used as a PCR
control. Significant reduction of α-Gal A mRNA was observed in the α-Gal A
silenced MDCK cells compared to control, without significant differences in betaactin levels (Figure 3).
Figure 3: Efficient silencing of α-Gal A expression in MDCK cells by siRNA transfection. MDCK cells
were transfected with siRNA targeting canine α-Gal A or firefly luciferase (control) and processed for RTPCR after three days to determine the extent of GLA mRNA reduction.
Accumulation of Gb3 in MDCK cells silenced for α-Gal A
18
In order to evaluate the effect of α-Gal A silencing in MDCK cells on
metabolism of neutral D-α-galactosyl GSL, levels of Gb3 were investigated using
specific monoclonal antibody against CD77, its membrane-bound form, followed
by indirect immunofluorescence. Intensity and pattern of Gb3 staining were
compared between cells transfected with either α-Gal A or non-silencing siRNA
using fluorescence microscopy. A drastic increase in Gb3 staining intensity, in a
punctate pattern, was observed in α-Gal A silenced cells after three days of
transfection compared to non-silenced MDCK cells (
Figure 4).
Figure 4 α-Gal A silenced MDCK cells have increased levels of Gb3.. MDCK cells were treated with
control (left panel) or α-Gal A (right panel) siRNA for six days, then processed for indirect
immunofluorescence with anti-CD77 (Gb3) antibody (red). Immunofluorescence microscopy images were
acquired and processed using identical settings.
Ultrastructural changes induced by α-Gal A silencing
Light and electron microscopy of biopsied tissues of patients with Fabry disease usually show celldisease usually show cell-type specific ultra-structural changes that are characteristic of LSDs. In
characteristic of LSDs. In cell types with greater degree of lipid deposition, electron microscopy
electron microscopy shows several degrees of intracellular vacuolization and the presence of
19
presence of membranous concentric or parallel shapes of packing of lipid aggregates in the
aggregates in the lysosomes, also called “zebra bodies”, are especially found. [78, 79] To further
[78, 79] To further investigate ultrastructural changes induced by α-Gal A transient silencing,
transient silencing, electron microscopy was performed after six days of transient α-Gal A
α-Gal A knockdown. We observed a greater number of round electron-dense structures in α-Gal A
structures in α-Gal A silenced MDCK cells, corresponding to lysosomes (Figure 5). Furthermore, We
have visualized in the α-Gal A silenced cells some lamellar myelin-like structures, corresponding to
“zebra bodies” (
Figure 6).
Figure 5: Electron micrographs of MDCK cells treated with control and α-Gal A siRNA. MDCK cells
were transfected with control (left panel) or α-Gal A siRNA (right panel) and plated on permeable support for
3 days before processing for transmission electron microscopy. Increased osmiophilic bodies (arrows) are
seen in the GLA-siRNA treated MDCK cells. 6,000x magnification.
20
Figure 6: Accumulation of "zebra bodies in α-Gal A siRNA treated MDCK cells. Electron micrograph of
a lysosome containing transversely-stacked, osmiophilic myelin-like membranes, also called a “zebra body”,
in MDCK cells transiently silenced with α-Gal A siRNA. 60,000x magnification.
1.4 Discussion
In this study, we have developed an in vitro model of Fabry disease in
renal tubular epithelial cells using RNAi technology. The α-Gal A silenced cells
present a dramatic increase in membrane-bound Gb3 levels, as well as ultrastructural changes compatible with Fabry disease, such as increased number of
electron-dense structures corresponding to lysosomes and the characteristic
presence of “zebra bodies”. Due to its effectiveness in showing disease
phenotype in a short period of time, as well as its reproducibility, this model might
be of great utility for testing hypotheses on disease pathogenesis and in highthroughput initial screening in drug development not only for Fabry disease, but
also for common disorders in which overexpression or decreased metabolism of
the GSL may also be found, such as certain types of cancer.
21
Post-transcriptional gene silencing using RNAi technologies has proven to
be effective and easily reproducible. In the past few years, it has been widely
used for developing disease models such as well as for targeted therapy for a
variety of disorders. [80] However, some limitations are foreseen. Although
presenting less potential biological risks than the use of lentiviral vectors for
stable expression, silencing for a particular gene using siRNA molecules remains
effective only for up to few days after the administration of their transfection in
cell culture systems due to successive cell divisions and the action of cellular
nucleases. [80] Therefore, for maintenance of disease status and study of effects
of chronic exposure to substrates, successive transfections might be necessary.
The extensive use of MDCK cells in research has made them a wellcharacterized cell line in the study of intracellular events and renal cell function.
However, a foreseen limitation in the use of MDCKs in translational research, as
it is with other species, is that although a considerable homology between the
human and canine genome exists, there might be some traits that are speciesspecific and cannot be extrapolated to humans. [81]
Although presenting limitations, cell models continue to represent essential tools
for understanding the fundamental bases of disease, for identifying prognostic
factors and biomarkers of disease progression, and for the initial steps in the
development and evaluation of therapeutic approaches to treat human disease.
These models are especially crucial in cases where knockout mice do not
present the same phenotype as humans, presenting challenges for translational
research.
22
2. A PUBLIC HEALTH GENETICS PLAN FOR PIAUI STATE WITH FOCUS ON
LYSOSOMAL STORAGE DISORDERS
2.1 Introduction and Background
2.1.1 Healthcare in Brazil
Brazil is the fifth most populous country in the world, and the sixth biggest
world economy by nominal GDP, according to the United Nations. According to
the World Bank, Brazil’s gross national income per capita is 11,420 USD,
(compared to 48,820 USD in the United States) which categorizes Brazil as an
upper middle-income country by the World Bank. In spite of the recent economic
growth, the country’s development indicators still classify it as a developing
economy according to the International Monetary Fund. [82, 83]
Provision of public health care in Brazil is considered a right to all citizens
and a responsibility and obligation of the State, being provided in public
institutions free of charge. The national health policy had its principles and
directives set by the Constitution of 1988, which established the Brazilian Unified
Health System (in Portuguese, SUS). Although private hospitals and private
health insurances are available in the country, according to the National Health
Agency, 74.9 percent of the Brazilian population rely only on SUS to receive
medical care. [84, 85]
Some of the difficulties faced by the population in regard to public
healthcare services in Brazil include: lack of access to services in remote areas
23
due to scarce number of healthcare professionals, delays in obtaining medical
appointments with specialists, lower number of beds than the demand in tertiary
care hospitals, especially at Intensive Care Units and significant overcrowding of
Emergency Care Units. [86]
In 1998, the Brazilian National Health Council approved a National Drug
Policy Act that aims to promote access to and rational use of medicines that are
considered essential. The National Drug Policy Act sets which drugs classify as
essential, as well as the standards for policies and regulation in drug discovery,
clinical trials and drug approval in the country. The act assigns individual states
the responsibility of promoting access to medications that are considered
exceptional, which are medicines that are the standard of care for a condition but
constitute an economic burden to individual patients. [87]
2.1.2 Piauí State Demographics
Piauí State is composed of 224 counties and covers an area of
251,577.738 square kilometers (equivalent to 97,134.7 square miles) with a
density of 32.1 persons per square mile. According to the Brazilian Institute of
Statistics and Geography (IBGE) demographic census, the 2010 population for
Piauí State was 3,118,360. The economy is primarily based on the teritary sector
(services), industry (chemical and textile), agriculture (soybean, cotton, rice,
sugar cane, and cassava) and free-range livestock farming. Teresina is the
capital of the state and the most populous city. Other major cities include
Parnaíba, Picos, Piripiri and Floriano.[88]
24
Piauí state has the lowest gross state domestic product in Brazil. Data
from 2009 show that the per capita gross state domestic product for Piaui was
6,051.10 Brazilian reais (BRL), which currently corresponds to 2,964.63 USD1.
Data from 2003 by IBGE have shown that 53.11 percent of the population in
Piaui state lives below poverty level taking into account the micro-level estimation
of poverty and inequality by Elbers, Lanjouw and Lanjouw [89]. In 2010, median
household income for Piauí state was 900 BRL per month, corresponding to a
yearly income of 5,296.2 USD per household1. In rural areas of the state, the
median monthly household income is 518 BRL (3,045.24 USD/year1). In 79.41
percent of the households in Piauí state, per capita income was less than
minimal wage for Brazil, which was 510 BRL per month (2,987.52 USD/year) for
the country in 2010. [88]
The majority of the population (69 percent) is multi-ethnic, mainly due to
miscegenation between individuals with European lineage and Amerindians
(originating “caboclos”). Caucasians represent 24 percent of the population.
Black individuals represent only seven percent of the population, mainly due to
the economic activity during the colonization period, which did not favor African
slavery.[88]
According to IBGE in 2009 there were 2,093 healthcare units in Piaui
state, 75.5 percent of which had public healthcare coverage through SUS. There
are 3,125 registered physicians in the State, representing a physician density
rate of ten physicians for each 10,000 people. 83.1 percent of the households
1
Convertion based on exchange rate data obtained on 01/02/2013, 1 BRL = 0.49 USD. Provided
by Citibank N/A.
25
rely on the state for healthcare, and only 12.6 percent of state population has
some sort of private health insurance. [88]
2.1.3 Piauí State Health Department
According to the Brazilian National Health Council, the following actions
constitute duties of Brazilian State Health Departments, according to the Basic
Operational Norms of the Brazilian Unified Health System (published in the
Official Gazette on 11/06/1996) [90]:
-
Planning and implementation of a State Health Plan containing strategies,
priorities and goals of actions and services, including the integration of
plans of the several county health departments;
-
Structuring and operation of the state component of the National Audit
Office;
-
Structuring and operation of epidemiological data processing systems for
delivery of services and for critical inputs;
-
Structuring and operation of epidemiological surveillance systems,
including also health surveillance and monitoring food and nutrition;
-
Structuring and operation of human resources and of science and
technology;
-
Elaboration of the state component of nationwide public health programs
for health threats that have a risk of spreading beyond its territorial
boundary;
-
Elaboration of the state component of the network of public health
laboratories;
26
-
Structuring and operation of the state component of pharmaceutical care;
-
State responsibility regarding the provision of outpatient and hospital
services of high cost, treatment away from home and the availability of
exceptional medicines and supplies;
-
Setting and operating policies regarding the use of blood and blood
components,
-
Hiring and support of qualified permanent technical staff, able to fully
accomplish the duties of the state health department;
-
Implementation of mechanisms to integrate the policies and actions of
relevance to the health of the population; examples of which are those
related to sanitation, water, housing and environmental health.
The Piauí State Health Department stated mission is to coordinate and
implement the operations of the National Health System in the State in order to
ensure increased and timely access to healthcare services and appropriate and
humanized care in the context of health promotion, prevention and recovery, as
well as to ensure quality management of local health departments, aiming to
improve health indicators and quality of life of the state population.[91]
The Piauí State Health Department stated values include [91]:
-
Assurance of universality, thoroughness, equity and accessibility to
healthcare
services
and
actions,
humanization and commitment;
27
with
continuity,
responsibility,
-
Exercise of participatory management, fostering transversality of health
policies, programs, actions and services and strengthening the social
Participation.
2.1.4 Genetics in Brazil
Genetic diseases and birth defects are currently the second most common
cause of infant mortality in Brazil. As health indicators consistent with developing
countries have significantly improved in the last few years, the country has
observed a drastic decrease in infant morbidity and mortality related to poor
sanitary conditions, malnutrition and infectious diseases. Therefore, genetic
diseases arose as a significant public health issue in the country. [86]
Genetics as a medical specialty is still considerably young in the country.
The first Medical Genetics residency was established in 1977 at Hospital das
Clínicas de Ribeirão Preto, University of São Paulo. In 1983, the Brazilian
Federal Council of Medicine recognized Medical Genetics as a medical specialty.
Currently, there are 11 Medical Genetics residency programs in Brazil, offering a
total of 21 new positions per year. There are no genetic counseling programs in
the country, and medical geneticists perform this role. However, there are only
121 medical geneticists in Brazil, 85 percent of which are concentrated in the
South and South East regions. [86] Dr Raskin, president of the Brazilian Society
of Medical Genetics, estimates that the waiting time for a first time appointment in
medical genetics in the country can be as long as one year. [92]
In October of 2004 the Brazilian Ministry of Health established a
workgroup in Clinical Genetics, which was responsible to plan and implement a
28
National Public Health Policy in Clinical Genetics and to incorporate genetics
within the Brazilian public health system, which became effective in 2009. [93,
94]
2.1.4.1 Newborn Screening in Brazil
On June 6th 2001, the Brazilian Ministry of Health implemented the
National Newborn Screening Program, through the act GM/MS n. 822. The
specific aims of the program included [95]:
-
Detection of metabolic conditions in a pre-symptomatic stage;
-
Coverage of 100 percent of live newborns;
-
Active search of screened newborns in the case of a positive initial result;
-
Diagnostic confirmation, as well as follow-up and appropriate early
intervention measures of all identified newborns, in order to minimize their
morbidity and mortality.
In order to select which conditions should be screened for, the Brazilian
Ministry of Health considered the need to select conditions that could be
screened and managed taking into account the health system diversity
throughout the country. Conditions that could be diagnosed early in their course,
through screening tests with high sensitivity and confirmatory tests with high
specificity should be priority. Additionally conditions should have an economically
feasible and cost-effective available treatment that positively interferes with the
disease natural history. [96]
29
The Ministry of Health also established that the program should be carried
out and articulated in a partnership with the State Health Departments.
Consequently, at least one Newborn Screening Reference Center was
implemented in each of the 27 states in Brazil. These centers are responsible for
coordinating the local networks of newborn screening present in each state and
are composed of a multi-disciplinary team for medical care, composed of
physicians, dietitians, psychologists, and social workers. As different states
presented different healthcare system organizations, the Ministry of Health felt
the need to implement the national program in phases. Consequently, the
following phases of operation were established [96]:
● Phase I: screening, confirmatory tests, follow-up and management of PKU
and congenital hypothyroidism;
● Phase II: screening, confirmatory tests, follow-up and management of
PKU,
congenital
hypothyroidism,
sickle
cell
disease
and
other
hemoglobinopathies.
● Phase III: screening, confirmatory tests, follow-up and management of
PKU,
congenital
hypothyroidism,
sickle
cell
disease
and
other
hemoglobinopathies and cystic fibrosis.
Private laboratories also provide newborn screening, however, these are
not integrated into a public health program, thus lacking the coordinated structure
for follow-up and management of newborns with positive screening tests. There
are three different groups of screening tests: basic, plus and master. The basic
screening comprises the conditions covered by the phase III of the National
30
Newborn Screening Program, the plus performs screening for ten conditions
while the master performs screening of 46 conditions. Health insurance
reimbursement policies are not uniform. The cost of the screening for phase I is
approximately 7.50 US dollars per sample, while for phases II and III cost 12
USD and 20 USD per sample, respectively. For confirmatory exams, the cost can
vary from 15 to 40 USD per sample. [96] The cost to the parents for the master
screening in case of non-coverage by private health insurance is about 120 USD.
In 2005, through the act 6197/05, the screening for biotinidase deficiency
was also introduced in the public health system. [97] However, the screening for
this condition, although established by law, has not yet been introduced in most
of the states in Brazil.
The
Newborn
screening
program
in
Brazil
has
still
suboptimal
achievements. Only three out of 27 states have achieved phase III program,
which allows screening for only four conditions. Newborn screening coverage
varies from 52.41 to 100 percent, with an average of 78.92 percent for the year of
2007. In the same year, for 3,035,096 live-births and approximately 2,400,000
screened newborns, 108 cases of PKU were detected, 1,231 cases of congenital
hypothyroidism, 1,140 cases of hemoglobinopathies and 58 cases of cystic
fibrosis, totalizing 2,537 cases. Data from 2007 also showed that 16,408 children
and adults have been followed so far after a diagnosis was received through the
newborn screening program, but those values can be underestimate since they
represent only results from the governmental funded healthcare and no
integrative effort exist with private newborn screening. [98]
31
2.1.4.2 Current Genetics Services in Piauí State
According to the Brazilian Society of Medical Genetics, currently there no
medical geneticists or clinical genetics laboratories are presented in Piaui State.
[99] The genetic-related resources available in Piauí State are the following:
-
Hospital Infantil Lucidio Portela (Lucidio Portela Children’s Hospital),
located in Teresina, serves as the Newborn Screening Reference Center
for the State and is overseen by the State Office for Healthcare for
Individuals with Disabilities (CAPD).
-
Central Laboratory of Piauí State (LACEN) located in Teresina, is the only
laboratory responsible for running the laboratory tests for the State
Newborn screening program, and is overseen by the State Office for
Healthcare for Individuals with Disabilities (CAPD).
A comprehensive list of not-for-profit organizations in the state that provide
care and services to people with mental and physical disabilities, including those
with hereditary causes, can be seen in Appendix B.
2.1.4.3 Lysosomal Storage Disorders in Brazil and Piauí State
The exact prevalence of LSDs in Brazil and Piauí State are not known due
to the lack of an integrated epidemiological surveillance service for genetic
diseases in the country. Gaucher disease, the most prevalent LSD, affects more
than 600 individuals in Brazil according to the Brazilian Gaucher Disease Patient
Association. [100]
32
Several individual states, through resolution by the Brazilian Ministry of
Health, have included imiglucerase, one of the treatment options of ERT for
Gaucher disease, in the list of treatments covered by the Brazilian Unified Health
System, which ensures treatment for this condition without any additional cost to
the patients and their families. Some individual states, such as Sao Paulo State,
have set up guidelines for indications of therapy and follow-up evaluations and
tests for patients with the disease. In case of no appropriate follow-up
insignificant clinical response to the medication, the patient undergoes additional
evaluation prior to continue with therapy, in an attempt to optimize the treatment
since the cost is considerable for Brazilian’s public health system. [101, 102]
At present, treatments for other LSDs have not been included in the list of
covered “exceptional drugs”. Besides Gaucher disease, specific treatment
through either ERT or substrate reduction therapy (SRT) is available for Fabry
disease,
Mucopolysaccharidosis
type
I,
Mucopolysaccharidosis
type
II,
Mucopolysaccharidosis type IV, Pompe Disease and Niemann-Pick disease type
C in developed countries. [103] Some of the diagnosed patients have been
seeking access to these treatments through legal measures.
According to the Fabry Registry 2007 Annual Report, age of diagnosis for
Fabry patients in Brazil averages 53 years old in males and 70 years old in
females, which represents an extremely high frequency of late diagnosis of the
disease in the country.[104]
Screenings for Fabry disease were performed in high-risk populations in
Brazil in some individual states, with special focus on individuals with end-stage
33
renal disease of undetermined cause. [105, 106] In Piauí State, screening among
350 male hemodialysis patients resulted in diagnosis of Fabry disease in two
individuals. The age of the patients was 57 and 74 years, and the first signs and
symptoms of Fabry disease started decades prior to the diagnosis. One of the
individuals had a classic form of Fabry disease and died due to a massive
myocardial infarction several days after receiving the diagnosis of Fabry disease.
A diagnosis of Fabry disease was not suspected in any of the individuals [107]
Further family screening in one of the individuals resulted in diagnosis of Fabry
disease in additional 61 individuals, the majority of them living in a remote village
in the South of Piaui State with scarce access to medical care [unpublished data
from the author].
2.2 Needs Assessment and Development of a Public Health Genetics State
Plan in Lysosomal Storage Disorders
2.2.1 Mission
Our mission is to improve health and quality of life of individuals with LSDs
living in Piaui State, an underprivileged state in Brazil, by developing the needed
infrastructure for proper clinical management and prompt diagnosis of patients
with LSD, delivering state-of-the-art medical care, and by educating physicians
and the public in genetics and LSDs.
2.2.2 Assumptions
-
A significant delay of diagnosis and marked underdiagnosis of LSDs occur
in Piaui State, resulting in preventable morbidity and mortality;
34
-
This delay in diagnosis occurs in part due to:
o
Lack of awareness of LSDs among healthcare professionals and of
proper training of primary care physicians and specialists in
recognizing disease phenotypes that could be compatible with
LSDs.
o Lack of
genetics laboratory and clinic for diagnosis and
management of patients with LSDs
o
2.2.3 Needs Assessment
Some of the following facts were observed by analyzing the current
healthcare situation in Piauí State and in Brazil, with focus on the provision of
genetics services, especially the ones with direct or indirect impact in delivery of
medical care for patients with LSDs:
-
Lack of coordinated national genetics infrastructure
-
Lack of genetics infrastructure in the State,
-
Although some genetics-related activities take place in Piauí state, there is
no centralized state genetics department or program.
-
Genetics expertise among healthcare providers is insufficient to meet
patients’ needs.
-
Lack of effective public policies in genetics that take place uniformly
across the country, including appropriate coverage by public or private
health insurances for diagnostic and follow-up tests, medical appointments
35
and genetic counseling, as well as for treatment of genetic disorders,
when indicated.
A thorough needs assessment will be performed through focus group
interviews involving healthcare professionals, social workers, patients with LSD
and their family members. Additionally, surveys disseminated to physicians of
different specialties will be conducted prior to the implementation of the plan and
after its establishment, and these resources will be used to guide program
planning and implementation.
2.2.4 Goals, Objectives and Activities:
2.2.4.1 Goal 1 - Build the public health infrastructure needed to diagnose and
provide appropriate clinical care to individuals with LSDs in Piauí State, Brazil
-
Objective 1.1: Engage key stakeholders
o Some of the foreseen stakeholders for the development of a LSD
program in Piauí State include: patients with LSDs and their
families, healthcare professionals, hospitals, clinical laboratories,
schools of Medicine, Piauí State Medical Council, Piauí State
Health Department, Brazilian Ministry of Health, health insurance
companies and SUS (Unified Healthcare System).
-
Objective 1.2: Build State Capacity and Self-Efficacy
o Workforce: recruit and train staff and volunteers
36
o Seek for short and long-term funding for program staff and activities
o Ensure quality and sustainability of LSD genetics services

Consult with LSD experts to set standards of care

Consult with policy makers
o Establish and coordinate partnerships

Partner with existing public clinical laboratory in establishing
the required equipment and supplies for running diagnostic
and follow-up tests for the different LSDs.

Partner with reference university hospital for creating and
establishing a LSD medical genetics clinic and a program
office
o Reimbursement and coverage – Seek for implementation of the
legislation that regulates the coverage of genetics services by both
public and private healthcare systems.
o Promote universal access for those in need

Establish regular outreach clinic in key cities of the state
Facilitate scheduling of medical appointments through the
use of a 1-800 hotline and online appointments
-
Objective 1.3: Surveillance
37
o Obtain data from the laboratories that perform clinical
diagnosis of LSDs in Brazil to estimate data on the current
prevalence of the disease in Piauí State.
o Annually assess incidence data for LSDs in Piauí State by
number of individuals diagnosed with LSD in Piauí State
both through and outside the program, as well as monitor
health outcomes for individuals being followed through the
program.
o Maintain accessible epidemiological data in the program
website
2.2.4.2 Goal 2: Increase awareness and foster knowledge in the field of LSD
among healthcare professionals and priority populations in Piaui State, Brazil
-
Objective 2.1: Educate physicians
o Develop and maintain a program website, where we will maintain
up-to-date information on recommendations and guidelines in
diagnosis, follow-up and treatment of patients with LSD.
o Deliver talks in medical conferences and meetings
-
Objective 2.2: Educate medical and allied health sciences students
o Deliver talks as part of the current academic curriculum of the
schools
38
o Discuss with course directors the need of the introduction of a
Medical Genetics course or of improvement of the syllabus in the
curriculum of Schools of Medicine.
-
Objective 2.3: Educate laboratory personnel in technical performance of
LSD diagnostic and follow-up tests
o Provide workshops to laboratory personnel in biochemical and
molecular approaches for the diagnosis of LSDs
-
Objective 2.4: Educate patients and family members
o Provide culturally appropriate learning material in the disease
o Provide genetic counseling
o
2.2.4.3 Goal 3: Reduce mortality and morbidity from LSDs in Piauí State
-
Objective 3.1: improve access to genetics services
o Establish the first genetics clinics with focus on LSD in Piauí State
-
Objective 3.2: promote early and appropriate diagnosis of LSDs
o Establish a Biochemical and Molecular Genetics Laboratory with
focus on LSDs
-
Objective 3.3: promote use of up-to-date evidence-based medical practice
in the management of LSDs.
2.2.5 Program Development Strategy
Figure 7
represents a diagram with a summary of the planned strategy for the
development phase of the LSD program in Piauí State.
39
Formation of
working group
Determination
of Program
Strategy
Stakeholders
and Needs
Assessment
Program logic
and evaluation
plan
Seek funding
Medical Talks
on LSD
Establishment
of Genetics
laboratory
Establishment
of Genetics
clinic
Figure 7. Strategy for the Program Development phase of a Public Health Genetics Plan for
Piauí State with Focus on LSDs. Adapted from [108]
2.2.6 Evaluation plan
We will perform both process and outcome evaluations throughout the
establishment of the program, in the following issues: relevance/needs
assessment of LSDs in the State, design and delivery of the program and
success of the program and its activities. Some of the evaluation questions to be
addressed, as well as the indicators and data sources that will be utilized in the
evaluation plan are described bellow.
Evaluation issues:
1. Relevance/Needs assessment
a. What is the magnitude of LSDs in Piaui State?
40
i. We will investigate along with the only two clinical
laboratories that perform diagnostic tests in LSD in Brazil the
actual number of individuals diagnosed with LSDs in the past
ten years.
b. Is the current prevalence along with what is expected based in
worldwide prevalence data?
i. Based on reported data of incidence for the different LSDs,
we will compare obtained prevalence with expected
prevalence
for
LSDs
based
on
reports
from
other
populations.
c. What is the existing laboratory structure for running LSDs diagnosis
and follow-up tests in Piaui State?
i. In consultancy with experienced Biochemical and Molecular
Genetics Laboratory directors, we will determine the current
structure and what are the needs in terms of equipment,
supplies and specialized training for running diagnostic and
follow-up tests in LACEN, the reference laboratory for
newborn screening in Piauí State.
d. What is the current level of knowledge in LSDs among physicians
and students in the health sciences?
i. Prior to the delivery of medical conferences for healthcare
professionals and students in the health sciences, we will
administer pre-tests with general questions in genetics,
41
diagnosis and treatment of lysosomal storage disorders in
order to obtain a baseline on the general knowledge among
these professionals in regard to proper diagnosis and
management of LSDs.
ii. We will also administer pre-tests along with the surveys to
physicians from different specialties during the needs
assessment process.
2. Design and delivery
a. Has the program changed awareness of relevance of LSDs?
i. Immediately after the delivery of medical conferences for
healthcare
professionals
and
students
in
the
health
sciences, we will administer post-tests to evaluate the
acquired knowledge in the field of LSD through our talks. If
agreed, we will also contact the professionals by email one
year after the participation in our talks and re-administer the
post-test to these professionals in order to determine a
longer-term assimilated knowledge.
b. Does the program follow updated recommendations and guidelines
for diagnosis, follow-up and treatment of LSDs?
i. We will have external consultancy from LSD experts, which
will evaluate the recommendations and guidelines found in
our website, and will also observe and evaluate the activities
42
of the clinic during one week a year to ensure delivery of
updated medical care.
c. Is the program integrated to healthcare systems and adequate to
reality and community needs?
i. We will evaluate the incorporation of the genetic clinic into
the health system of the state based on the number of
referrals from different medical specialties and also evaluate
the number and quality of the outreach clinic services.
d. Which improvements could be made to the program to better serve
the community and healthcare professionals?
i. We will perform periodic surveys among program staff,
patients and family members and healthcare professionals
and gather information on what could be improved in the
delivery of care in LSD.
3. Success
a. Were the expected outcomes achieved?
i. We will annually assess and publically report:
1. Attendance rate to education sessions performed
throughout the program
2. Pre- and post-test results applied before and after
each education session
3. Number of screening and diagnosis tests run in the
biochemical and molecular genetics laboratory
43
4. Number of referral visits to the Medical Genetics LSD
clinic
5. Number of diagnosed individuals with LSDs
6. Health outcomes for individuals being followed
through the program, with elaboration of periodic
reports
2.2.7 Expected Outcomes and Impact
Short-Term Outcomes:
-
Infra-structure for a Medical Genetics clinic and a Biochemical and
Molecular Genetics Laboratory with focus on LSD
-
Increased awareness in the relevance of the field of LSDs among the
medical community
-
Increased knowledge in current recommendations on the diagnosis and
management of patients with LSDs among the medical community
Medium-Term Outcomes:
-
Increased screening and diagnosis of patients with LSDs
-
Early diagnosis and intervention of patients with LSDs
-
Proper management of the comorbidities frequently found in patients with
LSDs
-
Proper genetic counseling of patients with LSD and their family members
at risk
-
Patient and family education on the disease
44
Long-Term Outcomes:
-
Decreased morbidity and mortality and increased quality of life of patients
with manageable LSDs in Piauí State
2.2.8 Challenges
Some of the foreseen challenges in the development and execution of a
Public Health Genetics Plan with Focus on LSDs include:
-
Scarcity of public funding for program development and implementation in
genetics in Brazil
o We will also seek funding through public-private partnerships and
apply for international grants, always optimizing the use of already
existing resources in the state in order to reduce costs.
-
Scarcity of well-trained professionals in the field of LSD and genetics in
general to compose the program staff
o Healthcare professionals recruited for the program will be trained in
a reference center in LSD prior to the start of the activities
-
Some of the remote areas of the State have difficult access (i.e. crumbling
roads and highways)
The outreach clinics will be planned ahead, allowing extra time for
possible intercurrences.
3.0 Final Considerations and Future Directions
In this study, we developed and fully characterized a cell model for Fabry
disease, a Lysosomal Storage Disorder, in renal tubular epithelial cells. Further
45
studies that could be performed in these cells include proteomics and cell
signaling arrays to investigate possible qualitative and quantitative changes in
protein expression and signal transduction in renal tubular cells silenced for αGal A. We could also study intracellular events and function of specific organelles
in the lieu of Fabry disease. As the methods become fully optimized and
expertise becomes available, other cell models of interest could also be
developed, especially from the main organs of involvement of the disease, such
as cardiomyocytes for the study of cardiomyopathy and glial cells for the study of
the CNS involvement. The effect of novel forms or SRTs or ERTs or manipulation
of new pathways could also be studied in a high-throughput manner.
Additionally, here we design the framework for a Public Health Genetics
Plan for Piauí State, the lowest income state in Brazil, a developing country. We
have discussed relevant background information, and important policy issues.
We purpose a comprehensive program aiming to increase awareness and
knowledge in the field, promptly diagnose and promote health and quality of life
for individuals with LSD in Piauí State, including components of needs
assessment and a robust evaluation plan. A Public Health Genetics Plan for
Piauí State could serve as a pilot for implementation of similar programs in
developing countries.
46
Appendix A – Lysosomal Storage disorders by subgroup
Protein
DEFECTS IN GLYCAN DEGRADATION
Defects in glycoprotein degradation
α-Sialidase
Galactosialidosis
α-Mannosidase
β-Mannosidase
Glycosylasparaginase
α-Fucosidase
α-N-Acetylglucosaminidase
Defects in glycolipid degradation
A. GM1 Ganglioside
β-Galactosidase
β-Hexosaminidase α-subunit
β-Hexosaminidase β-subunit
GM2 activator protein
Glucocerebrosidase
Saposin C
B. Defects in the degradation of
sulfatide
Arylsulfatase A
Saposin B
Formyl-Glycin generating enzyme
Disease
Sialidosis
Cathepsin A
α-Mannosidosis
β-Mannosidosis
Aspartylglucosaminuria
Fucosidosis
Schindler
GM1 gangliosidosis / MPS IVB
GM2-gangliosidosis (Tay-Sachs)
GM2-gangliosidosis (Sandhoff)
GM2 gangliosidosis
Gaucher disease
Gaucher disease
Metachromatic leukodystrophy
Metachromatic leukodystrophy
Multiple sulfatase deficiency
47
β-Galactosylceramidase (Krabbe)
C. Defects in degradation of
globotriaosylceramide
α-Galactosidase A
Defects in degradation of
Glycosaminoglycan
(Mucopolysaccharidoses)
A. Degradation of heparan sulphate
Iduronate sulfatase
α-Iduronidase
Heparan N-sulfatase
Acetyl-CoA transferase
N-acetyl glucosaminidase
β-glucuronidase
N-acetyl glucosamine 6-sulfatase
Degradation of other
mucopolysaccharides
N-Acetylgalactosamine 4-sulfatase
Galactose 6-sulfatase
Hyaluronidase
Defects in degradation of glycogen
α-Glucosidase
DEFECTS IN LIPID DEGRADATION
Defects in degradation of
sphingomyelin
Acid sphingomyelinase
Acid ceramidase
Defects in degradation of triglycerides
and cholesteryls ester
Acid lipase
DEFECTS IN PROTEIN DEGRADATION
Cathepsin K
Tripeptidyl peptidase
Palmitoyl-protein thioesterase
DEFECTS IN LYSOSOMAL
TRANSPORTERS
Cystinosin (cystin transport)
Sialin (sialic acid transport)
DEFECTS IN LYSOSOMAL
TRAFFICKING PROTEINS
UDP-N-acetylglucosamine
Phosphotransferase γ-subunit
Mucolipin-1(cation channel)
LAMP-2
NPC1
CLN3
CLN 6
Globoid cell leukodystrophy
Fabry
MPS II (Hunter)
MPS 1 (Hurler, Scheie)
MPS IIIa (Sanfilippo A)
MPS IIIc (Sanfilippo C)
MPS IIIb (Sanfilippo B)
MPS VII (Sly)
MPS IIId (Sanfilippo D)
MPS VI
MPS IVA (Morquio A)
MPS IX
Pompe
Niemann Pick type A and B
Farber lipogranulomatosis
Wolman and cholesteryl ester storage
disease
Pycnodystostosis
Ceroide lipofuscinosis 2
Ceroide lipofuscinosis 1
Cystinosis
Salla disease
Mucolipidosis III (I-cell)
Mucolipidosis IV
Danon
Niemann Pick type C
Ceroid lipofuscinosis
Ceroid lipofuscinosis 6
48
CLN 8
LYST
MYOV
RAB27A
Melanophilin
AP3 β-subunit
Ceroid lipofuscinosis 8
Chediak-Higashi
Griscelli Type 1
Griscelli Type 2
Griscelli Type 3
Hermansky Pudliak 2
Adapted from ref [109]
Appendix B – Governmental and non-governmental organizations providing care
for individuals with disabilities/special needs in Piauí state, Brazil.*
Special need
Autism
Blindness
Physical
disability
Intellectual
disability
Resource
Associação de amigos dos autistas do Piauí
Associação dos cegos do Piauí
CAP - Centro de apoio pegagógico às pessoas
portadoras de deficiência visual
ASCEB - Núcleo Associação dos Cegos de Água
Branca
Associação dos deficientes físicos de Teresina
City
Teresina
Teresina
Teresina
Sociedade de Apoio ao Deficiente Físico
Associação dos Cadeirantes do Município de
Teresina
SADEFINP - Sociedade de Apoio
ao Deficiente Físico do Norte do
Piauí
CIES - Centro integrado de educação especial
Teresina
Teresina
Fundação Viver com digndade - Lar Renascer Pastoral da Esperança
APAE - Centro de Recuperação e Profissionalização
Integrado Cristina Leite
CHAC - Centro de Habilitação Ana Cordeiro
Instituto Panda - Núcleo de Apoio à Pessoa com
Teresina
49
Água
Branca
Teresina
Barras
Teresina
Teresina
Teresina
Teresina
Deficiência/Paralisia Cerebral
Associação Pestalozzi "Casa Odylo Costa Filho"
Escola de Educação Especial Prof Cordão
Deafness
Others
APADA - Associação de pais e amigos dos
deficientes auditivos
ASTE - Associação dos surdos de Teresina
CAS - Centro de Apoio Pedagógico ao Surdo
APAE - Escola de Educação Especial Prof Consuelo
Pinheiro
AMH - Associação de mielomeningocele e/ou
hidrocefalia de Teresina
AOSEPI - Associação dos ostomizados do Estado do
Piauí
FCD - Fraternidade Cristã de Doentes e Deficientes
Pastoral da Amizade
CEPI - Centro de Profissionalização Integrado
UEESPI - Unidade de Educação Especial
PNES - Programa TEC NEP - Educação, tecnologia e
profissionalização para pessoas com necessidades
especiais
SINE - Serviço Nacional de Empregos
CONEDE-PI Conselho Estadual de Defesa dos
Direitos da Pessoa com Deficiência
CONEDE-TE Conselho Municipal de Defesa dos
Direitos da Pessoa Com Deficiência
FUNDECAP - Fundação dos Deficientes de Capitão
de Campos
CIEE- Centro de Integração Empresa Escola
Núcleo de Reabilitação Profissional
SEINT - Ministério do Trabalho e Emprego - Seção
de Inspeção do Trabalho - Grupo Pro Igualdade
Instituto Professor Magalhães
IEDE - Instituto Educacional de Desenvolvimento
Integrado
Escola Viva
Escola Castelinho
Colégio Base Dez
*Adapted from: http://www.seid.pi.gov.br/entidades.php
50
Teresina
Campo
Maior
Teresina
Teresina
Teresina
Teresina
Teresina
Teresina
Teresina
Teresina
Teresina
Teresina
Teresina
Teresina
Teresina
Teresina
Capitão de
Campos
Teresina
Teresina
Teresina
Teresina
Teresina
Teresina
Teresina
Teresina
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