Stephan Zuckner - 'Neuropatías periféricas hereditarias'
The Molecular Basis of Chondrodysplasia Punctata in an Affected Female and her Unaffected Parents
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CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
Molecular Basis of Chondrodysplasia Punctata in an Affected Female and her
Unaffected Parents
By
Danielle Robinson
May 2011
Table of Contents
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Abstract………………………………………………………………………………3
Chapter #1 Introduction
Chondrodysplaysia Punctata (CDP)……………………………………..5
History of the X-linked Chondrodysplaysia Punctat………………….6
Clinical Presentation of X-linked Chondrodysplaysia Punctata……8
The EBP Gene………………………………………………………………..8
Treatment for X-Linked Dominant Chondrodysplaysia Punctata…..10
Objective………………………………………………………………….......10
Chapter # 2 Materials and Methods
Patients Sample……………………………………………………………..11
Resuspending and Diluting Primers……………………………………..12
Amplification of Exons for Analysis………………………………………12
PCR Amplicon Purification………………………………………………...17
Chapter #3 Results
Exon 3………………………………………………………………………….18
Exon 4 & 5……………………………………………………………………..19
Exon 2………………………………………………………………………….20
Chapter #4 Discussion
Amplication of 3 Exons of the EBP Gene……………………………….20
Next Step……………………………………………………………………...21
X-Inactivation………………………………………………………………...22
Limitations….………………………………………………………………...23
Future Research……………………………………………………………..23
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ABSTRACT
The Molecular Basis of Chondrodysplasia Punctata in an Affected Female and
her Unaffected Parents
Danielle Robinson
Chondrodysplasia Punctata (CDP) is a heritable disorder that results in skeletal
abnormalities. Chondrodysplasia Punctata is characterized by the formation of small,
hardened spots of calcium on the "growing portion" of the long bones (stippled
epiphyses) or inside other areas of cartilage in the body. X-linked CDP, also known
as Conradi-Hunermann syndrome, is the most well-characterized form of CDP, and
is the most severe form (male-lethal). Chrondrodysplaysia Punctata is caused by
mutation in the EBP gene. EBP gene stands for emopamil binding protein (sterol
isomerase). The DNA of an Italian woman with skeletal abnormalities and skin
discoloration was tested. Her mother and father were not affected by similar
symptoms. Our hypothesis was that the families X-linked Chondrodysplaysia
Punctata is caused by a mutation in the EBP gene.
To test this hypothesis we will clone the exons of the EBP gene in the affected
woman, including the splice junctions, and determine the DNA sequence (I did this
work in collaboration with another student, Joaquin Zuluaga, and the work was
directed by Dr. Aida Metzenberg). We are studying more than just the coding
regions We are studying exons and part of the introns. Then we are going to
determine the nucleotide sequence and compare to the wild-type.
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Thus far, we have isolated 2 of 3 amplicons that would amplify the coding region
and splice junctions. The next step will be to amplify the third amplicon, the one we
are having trouble with, and then clone each amplicon into pGem 3 vector. This will
be sequenced by the CSUN DNA sequencing facility using universal primers .
Afterwards, we will be doing bioinformatics comparing our sequence to the
sequence in the Gene Bank database. Since we are looking at a female we will be
looking for an unknown mutation in a female. We expect this affected female to be
heterozygous for an EBP mutation. To test our prediction we are going to have to
sequence multiple clones. Each clone has a 50% chance of being wild-type and a
50% chance of having the mutation. Therefore if we sequence multiple clones we
expect at least one to have the mutation, which is what we are looking for. We are
not sequencing amplicons directly, because the quality of sequence will be much
better if we clone the amplicons first (A. Metzenberg personal communication).
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Figure 1: Punctate epiphyses contain small epiphyseal
calcifications, are present at birth, and are classically
associated with chondrodysplasia punctata.
Source: Poznanski AK. Punctate epiphyses: a radiological
sign not a disease. Pediatr Radiol. 1994;24(6):418-24, 436.
Chapter #1: Introduction
Chondrodysplaysia Punctata (CDP)
Chondrodysplaysia Punctata, also known as CDP, is a collection of hereditary
disorders that result in skeletal abnormalities. The main characteristic of CDP is
punctuate calcification, which is the accumulation of hard calcium spots in the cartilage
of, the growing ends of long bones.
These alternate ends are known as
stippled epiphyses. Calcifications are
also found in other areas of the body
including the larynx or trachea (Mueller
et. al, 1985). Symptoms of CDP include
formation of cataracts, large skin pores,
growth retardation, limb shortening,
patches of coarse dry hair, and dry,
scaly skin.
There are four forms of CDP :
1) autosomal recessive form, 2)
autosomal dominant form, 3) X-
linked recessive form, and 4) X-
linked dominant form. The
autosomal recessive form, also known as Rhizomelic Chondrodysplasia Punctata
(RCDP1), is a peroxisome biogenesis disorder (PBD) that is characterized by severe
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growth and mental deficiency (White, 2003). The autosomal dominant form, is the most
common form of CDP that results in vitamin K deficiency or warfarin teratogenicity (Hall
et al., 1980) . However this one is not well understood. The X-linked recessive form is
known as, Brachytelephalangic Chondrodysplasia Punctata , and is characterized by
underdevelopment of the hands and feet , and behavioral problems(Lachman,2007) .
The X-linked dominant form is known as Conradi- Hunermann- Happle Syndrome or
CDPX2. It is the most understood form of this rare disorder, but is also the most severe,
because it is male lethal (Mueller et al., 1985). The X-linked dominant form is what this
paper will be focusing on. Since the effects of X-inactivation on the phenotype of an X-
linked disorder are better understood now that in the past, it is more common to refer to
these as “X-linked” rather than “X-linked recessive” or “X-linked dominant” disorders.
History of X-linked Chondrodysplaysia Punctata
The first case of chondrodysplaysia punctata was described in 1914 by E.
Conradi. Chondrodysplaysia punctata became known as “Condradi Syndrome”. In
1931 new findings by C. Hunermann in the disease led the changing of the name to
“Condradi-Hunnermann Syndrome” (Gorlin , 2001). For many years chondrodysplaysia
punctata was thought to be a single disorder.
In 1971 , Spranger et al, suggested that there may be two different forms of
chondrodysplaysia punctata. They proposed separating CDP into two groups, the mild
form which we now call Conradi- Hunermann type and the lethal rhizomelic type
(Gorlin, 2001). Rhizomelic form was considered lethal due to the the fact that this form
resulted in death within the first year of the sufferers life (Spranger, 1971).
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1977 R. Happle made an additional contribution, when he saw the variation in
phenotypes among the patients, with chondrodysplasia punctata, and proposed that it
was limited to females. He termed it Chondrodysplaysia Punctata Type B and
proposed in 1979 , that this type was inherited in an X-linked manner. Happle reached
this conclusion after he found a 35:0 female versus male ratio in those that had the
characteristic skin lesions of this form of chondrodysplaysia punctata. In1980 Happle
observed that chondrodysplaysia punctata type B is lethal in hemizygous males, which
is why it occurs exclusively in females (Happle, 1985).
In1995, the first surviving male with X-linked dominant Chrondrodysplaysia
Punctata is described. However he is found to have Klinefelter syndrome which results
in the presence of an extra X chromosome, resulting in a 47, XXY genotype. His ability
to survive was due to X-inactivation (Happle ,1995). X-inactivation is not restricted to
females but, can also occur in males with Klinefelter syndrome who have more than one
X chromosome. He was heterozygous and not hemizygous for the X-linked dominant
form of chrondrodysplaysia punctata. There have also been cases reported of 46,XY
males being affected with this disease, which is why many researchers do not consider
it an X-linked dominant form of inheritance anymore.
Conradi- Hunermann Syndrome was soon renamed to Conradi- Hunermann-
Happle Syndrome, to acknowledge Happle for his significant contribution to the
description of the disorder.
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Figure 2:
(A) showing the right leg with erythroderma
and linear ichthyosiform lesions. (B, C)
showing asymmetric shortening of legs and
numerous punctate calcifications at the
thoracic and lumbar spines, respectively.
Lower panel, photograph of patient 2 at 13
years old. (D) showing bilateral cataracts, a
flat nasal bridge and coarse hair with patchy
alopecia. (E, F) showing postaxial polydactyly
on the left hand and marked scoliosis,
respectively.
Clinical Presentation of X-linked Dominant Chondrodysplaysia Punctata
X-linked dominant chondrodysplaysia punctata (CDP) is characterized by growth
retardation, shortening of limbs, cataracts , dry and scaly skin, large skin pores,
alopecia, and patches of coarse, dry hair (Gorlin, 2001). What separates X-linked
dominant chondrodysplaysia punctata from the other forms of chondrodysplaysia
punctata is the presence of hyperkeratotic lesions (Blaschko, 1901). Examples of these
skin lesions are shown in Figure2.
The EBP Gene
Cholesterol Is the basis of many important hormones, in the body. Some
cholesterol is obtained from one’s diet, but when one cannot synthesize the rest of the
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cholesterol then one is in trouble . CDP is usually caused by mutations in the
emopamil binding protein ( EBP) gene, which is a sterol isomerase. EBP alleles are
also known as CDPX2, CHO2, CPX, or CPXD. The protein encoded by this gene is a
multipass protein found on the surface of the endoplasmic reticulum. The EBP gene is
located on the short arm of the X chromosome and
located between positions 11.23 and 11.22, as shown in Figure 3
The function of EBP protein is to catalyze the conversion of Delta(8)-sterols to
their corresponding Delta(7)-isomers (Braverman, 1999). Delta8-delta7 sterol isomerase
is an enzyme that catalyzes one step of eight in the biochemical synthetic process of
making cholesterol. Defects of this enzyme lead to the accumulation of 8-
dehydrocholesterol and cholest-8(9)-en 3-beta-ol in plasma and tissues. Therefore,
chondrodysplaysia punctata is biochemically characterized by a defect in the cholesterol
biosynthesis pathway. How mutation in EBP causes chondrodysplaysia punctata isn’t
completely worked out , figure 4 shows the mutations in the EBP gene that have been
characterized to date.
Figure 3: The EBP gene is located on the short (p) arm of the X chromosome between
positions 11.23 and 11.22.
http://ghr.nlm.nih.gov/gene/EBP
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Investigations into patients that had point mutations in there EBP gene were
shown to have defect in Delta(8), Delta(7) sterol isomerase (Braverman, 1999).
However, their symptoms varied leading researchers to believe that X-inactivation
occurs at random in affected individuals.
Treatment for X-Linked Dominant Chondrodysplaysia Punctata
There is currently no cure for X-linked dominant chondrodysplaysia punctata.
Treatment is based on controlling the symptoms shown. Common treatments include
dermatological care, to help with rough skin areas, operations to lengthen leg bones, via
surgery, or genetic counseling, and prenatal diagnosis.
Objective
To learn about the molecular basis of chondrodysplasia punctata in an Italian
woman with skeletal abnormalities and skin discoloration. Her mother and father were
Figure 4:
Mutations in CDPX2. Schematic
diagram of the emopamil-binding
protein with the mutations
characterized to date.
Source:
Novel Mutations in X-Linked
Dominant Chondrodysplasia
Punctata (CDPX2) Neil V Whittock et
al.
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not affected by similar symptoms. Gene mutation
analysis is in the process of being performed on exons
2, 3, 4, and 5 of the EBP gene . The overall goal of the
project was to clone exons of the EBP gene. We intend
to clone splice junctions as well as coding sequence. We
propose to sequence the cloned exons to look for a
mutation in the EBP gene of the proposita. The
nucleotide sequences will be compared tothat of allele.
Our hypothesis was that X-linked
Chondrodysplaysia Punctata in this family is caused by
a mutation in the EBP gene.
Chapter # 2 : Materials and Methods
Patients Sample
DNA of Italian woman suffering from skeletal abnormalities and skin discoloration
was isolated and shipped. Her parents’ DNA was also collected even though neither
one of them was shown to have any of their daughter’s symptoms. This study was
approved by California State University Northridge Standing Committee for the
Protection of Human Subjects (IRB).
Figure 5:
Image of patient depicting skeletal
abnormalities and skin discoloration
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Resuspending and Diluting Primers
IDT primers arrived desiccated, and were resuspended in 100 microliters H2O.
The concentration of each primer is listed as follows:
ISO2F = 27,800 pmol
ISO2R = 26,000 pmol
ISO3F2 = 19,300 pmol
ISO3R2 = 25,800 pmol
ISO4F = 20,700 pmol
ISO5R = 29,800 pmol
We then made the stock solution by adding :
5 μl ISO2F + 273 nH2O = 5 pmol / μl
5 μl ISO2R + 255 nH2O = 5 pmol / μl
5 μl ISO3F2 + 188 nH2O = 5 pmol / μl
5 μl ISO3R2 + 202 nH2O = 5 pmol / μl
5 μl ISO4F + 293 nH2O = 5 pmol / μl
5 μl ISO5R + 253 nH2O = 5 pmol / μl
Working dilution for PCR = 5 pmol / μl . A 1 to 50 dilution of stock , therefore for a 25uL
reaction, we used 1uL. The sequencing dilution 1 pmol/microliter = 1:5 dilution of
working dilution for PCR (exactly). We then stored all at -20 degrees.
Amplification of Exons for Analysis
The coding exons 2,3,4,and 5 of the EBP gene were amplified using the
Polymerase Chain Reaction (PCR). Oligonucleotide primers that were complementary
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to coding exons and splice junctions of the EBP gene were designed to carry out the
PCR protocol.
PRIMER SEQUENCE (5’3’) Tm
ISO2F ATA AGC TTA TTC GGT CCA TTT ACA TTT CTC 55.5̊C
ISO2R ATG GAT CCC AAA TCC CAT CCC ACA GC 62.8 ̊C
ISO3F2 ATA AGC TTG ACC CCC AGC TCT CAC AAG 63 ̊C
ISO3R2 ATG GAT CCG ATG GTT TCC ATG CAC ACT G 62.6 ̊C
ISO4F ATA AGC TTG GTG GTG AGT TGG GGA GCA 64.0 ̊C
ISO5R ATG GAT CCA CCC CTG GAA GGG CAC CGT T 68.6 ̊C
Table #1 Sequence of primers and melting points used in the PCR protocol for the coding exons
of the EBP gene.
The genomic sequence of the emopamil-binding protein gene is shown below.
1 cggagccagc gtgggaggcc gctgccgtcg cgcgccttgg tgagtgccct ccacccggcc
61 cctgctccct cccccagctc tccccggcta cgcggccagc cctcggcgtg ccagcgcgag
121 accctttgcc acccgccccc cccaccgccc ttttgcgcct gcgcgagacc cccagctacc
181 gcacggttgt ccagaggaca gaagatccgt cttctcattg ggcagcggga ctggagggtt
241 ctggttcgga ttgaccggct ttgtgttccg ttctagcgct gcacgccaga caccggcctt
301 tcaatatccg tctcttccct gcaggttgac ggcgttgcac gctctcgcgg ggaggctctg
361 gctttccaaa cgctggcacc gagggttgta gttctgattc gttttctttc ctctgtcccc
421 cagttgcagt tttcaggact acgtggggga gggaatagct ttttgttgaa cggtttaaga
481 cgctgacctg tgcaactgga ccacgcctgg ttcctgtgtc tcttaccaaa ccgtgacccc
541 ggagcacagg actgggtttg gtggtcgtga gcagtttgtg acccttgaag gaagacacca
601 ggcctggtcc tgtatctccc tcgccagact gtgaccaggg agggctggga tcaggtgttg
661 ttcccatgtg cctcccacca gactttgacc cccgagggct gagaccggga ctgtttctac
721 cacatcataa tatggcttct aaagggctgg aacaagcttt cagcctttct gtggccttgt
781 gtttcagtca tgttcttttt cttttctttt ttattttatt ttgagacaga gtctcgctct
841 gttgcccagg ctggagtgca gtggcatgac cccggctcac tgcagcctct gcctcccagg
901 ttcaagcgat tctcctgcct cagcctcctg agtagctgta actacaggcg cacgccacca
961 cacctggcta atttttgtat ttttagtaga gacggggttt tgccatgttg gccgggctgg
1021 tcttgaactc ctgagctcaa gtggtccact cgtcttggcc tcccaaagtg ctgggattac
1081 aggcgtgagc caccgcgccc ggccttcttt tttagaggca gagtctcaga ctggagggca
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6301 tagagaattg gcgaaagtgt ccccttcctc actggggctt ctccttcccc tcctgccacc
6361 cacaggccag atctatgggg atgtgctcta cttcctgaca gagcaccgcg acggattcca
6421 gcacggagag ctgggccacc ctctctactt ctggttttac tttgtcttca tgaatgccct
6481 gtggctggtg ctgcctggag tccttgtgct tgatgctgtg aagcacctca ctcatgccca
6541 gagcacgctg gatgccaagg ccacaaaagc caagagcaag aagaactgag gagtggtgga
6601 ccaggctcga acactggccg aggaggagct ctctgcctgc cagaagagtc tagtcctgct
6661 cccacagttt ggagggacaa agctaattga tctgtcacac tcaggctcat gggcaggcac
6721 aagaagggga ataaaggggc tgtgtgaagg cactgctggg agccattaga acacagatac
6781 aagagaagcc aggaggtcta tgatggtgac gatttttaaa atcaggaaat aaaagatctt
6841 gactctaa
Each PCR tube contained .5 μl of dNTPs (10mM), .5 μl of Taq Polymerase (5units/ μL),
2.5 μl of 10x Taq Buffer (Mg + 2 Free), 2.5 μl of (5pmol/ μl) of each forward and reverse
primer. The experimental tubes received 1 μl of genomic DNA, while the controls had 1
μl of nH2O.
The concentration of magnesium chloride + nano pure water varied between 1.5M –
4M, in order to determine the optimal conditions for amplification. The amount of
magnesium chloride + nano pure water added to each tube was 14.5 μl. All of these
were added together bringing the total reaction volume to 25 μl. The PCR reactions
were then placed in the PCR machine, the PCR conditions varied between exons.
Each PCR reaction was set to 40 cycles.
Figure#6. Genomic sequence of the emopamil-binding protein gene. Sequences in green =
exons, in red = intron/exon boundaries. The intron/exon splicing sites (ag/gt) were configured
by Bravermann et al. 1999. Exons 4 and 5 were amplified together.
http://www.ncbi.nlm.nih.gov/nuccore/NC_000023.9?&from=48265201&report=genban
&to=48272048
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Exon 2
Temperature Time Cycles Holds
95̊ C 2 min 1 hold
96̊ C 1 min
52̊ C 1 min
72̊ C 1 min
72̊ C 10 min 1 hold
Exon 3, Exon 4& 5
Temperature Time Cycles Holds
95̊ C 2 min 1 hold
94̊ C 30 sec
52̊ C 30 sec
72̊ C 1.5 min
72̊ C 10 min 1 hold
After completing all 40 cycles the reaction was held at 4̊C for convenience.
PCR Amplicon Purification
Electrophoresis was performed to see if we amplified the amplicons and to
isolate the desired PCR fragments. A 7.5% polyacrylamide gel was electrophesed
(PAGE). Each well contained 10 μl of PCR product and 2 μl of 6 X Dye. The first well
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was loaded with 1 μl of Phi X 174 DNA digested with HaeIII to produce size markers.
These fragments range from 72 base pairs to 1353 base pairs in length. The gel was
submerged in 1x TAE buffer and was electrophoresed for an hour and forty-five minutes
to two hours at 94V to 96V. After electrophoresis, it was placed in ethidium bromide for
staining, for 5 minutes, and then removed and placed in deionized water for another 5
minutes, for de-staining. The gels were then photographed using a UV transilluminator .
The bands of interest were then cut out and placed into a 1.5ml tube containing, 350 μl
of 100mM NaCL/1 mM EDTA. The samples set at room temperature overnight.
Chapter #3: Results
So far we have isolated 2 of 3 amplicons that would amplify the coding region
and splice junctions. These amplified amplicons, are exons 3 and exon 4 & 5.
Exon 3
Figure 7:
7.5% acrylamide gel showing successful amplification
of exon 3. Gel was stained with ethidium bromide. Band in
lane 2 shows amplification of exon 2
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Exon 3 was successfully amplified and is denoted here by the yellow box. Its
nanodrop results were
260/.280 ratio = 1.84
Nucleic acid concentration = 17.7 ng/ μl
We determined that concentration of the PCR products was not adequate enough to
allow for sequencing.
Exon 4 & 5
Exon 4 & 5 was successfully amplified and is denoted here by the yellow box. Exons
4 and 5 were amplified together since they are located very close to each other on the
gene. Its nanodrop results were
260/.280 ratio= 1.19
Nucleic acid concentration = 29.7 ng/ μl
Figure 8:
7.5% acrylamide gel showed using the gradient
of magnesium concentrations in amplification of
exons 4 and 5. Gel was stained with ethidium
bromide.
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We determined that concentration of the PCR products was not adequate to allow DNA
sequencing.
Exon 2
Exon 2 was successfully amplified and is denoted here by the yellow box. Its
nanodrop results were
260/.280 ratio = 1.84
Nucleic acid concentration = 17.7 ng/ μl
However, we determined that the concentration of the PCR products were too low to
allow for sequencing. In future, it will be important to determine conditions for
amplifying this fragment more effectively.
Chapter #4: Discussion
Amplification of 3 exons of the EBP gene
The PCR results differed. Cloning exon 2 was particularly difficult due to the fact
that we have yet to figure out the optimal PCR conditions. Although we were
Figure 9:
7.5% acrylamide gel showing the
gradient of magnesium concentrations
used to amplify exon2. Gel was stained
with ethidium bromide.
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successfully able to amplify exon 2, we determined, by measuring the concentration of
the PCR products, that the concentration of the exon 2 PCR product was too low to
allow for sequencing. We then attempted to optimize PCR conditions by altering the
magnesium concentrations, in our PCR master mix, and altering the annealing
temperatures in the experiments. We are currently working very hard at improving the
yield of the amplicon which includes exon 2 of the EBP gene in this individual with CDP.
It was also speculated that the reason this amplicon was especially difficult could
be due to the run of thymine’s near the beginning of the exon 2. This could be due to
the fact that DNA polymerase , stuttered, and was unable to pinpoint where it is
suppose to be when the same nucleotides are in a row.
Next step
Once we have worked out amplification of exon2 we will be doing restriction
digest and ligating all of the amplicons into the multiple cloning sites on the pGEM3
vector. We will then perform bacterial transformation, grow up the clones and then
isolate the DNA. Sequencing will be carried out by the CSUN DNA sequencing facility,
using universal primers. We expect the affected individual to be heterozygous for an
EBP mutation. We will be sequencing multiple clones so that the chances for missing a
mutation is smaller, since each clone has a 50% chance of being wild type and a 50%
chance of having the mutation.
Bioinformatics will be used to compare our sequence with the wild type
sequences in the GenBank database. We expect that one of the clones will have a
mutation. If we do find a mutation, we will try to confirm that it is a deleterious mutation.
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Two ways to go about doing this are to, 1) See if someone else with X-linked dominant
chondrodysplaysia punctata has the same one, 2) Seeing whether the mutation is
present in 100 random X chromosomes of normal individuals, using a database, or
doing the work ourselves. Polymorphism is defined as more than 3% of the population
having nucleotide changes that do not cause CDP. If a mutation is not present in the
samples studied, it is likely to be a polymorphism rather a deleterious mutation. A
mutation is defined as a deleterious nucleotide change. If we do find a mutation this
does not prove we have a deleterious mutation, but it does support it.
X-inactivation
Mary Lyons’ “X-inactivation Theory” is a phenomenon when one of a females
paternally or maternally derived X-chromosome is randomly inactivated in a cell. Since
females affected with X-linked dominant chondrodysplaysia punctata are
heterozygous, they are only affected if cells with the mutation outnumber the cells
without the mutation. This might be responsible for observations that, if a woman is an
obligate heterozygote for a mutation, then the phenotype can range from absent to
severe. It all depends upon the degree of X-inactivation in the individual.
Even if the mom was unaffected, she can still have a mutation that’s not active in
other cells, which is why she has a normal phenotype. Skewed inactivation results
when most cells with the active x, is the one without the mutation, and the inactive x is
the one with the mutation. Skewed X-inactivation is defined as the state where 95% of
cells only express wild-type, while the other 5% express mutant. Thus, phenotype can
be normal even though the person is heterozygous, if it is skewed toward wild- type.
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Skewing is possible in either direction (wild-type of mutant, so the phenotype of a
heterozygote ranges from normal to severely affected).
In theory, the affected individual could have gotten a mutation from either parent.
Her father would have had to have a new mutation occur well after the first cell division
of the fertilized egg that became him. However, a new mutation might not show up in his
blood DNA. The mother could have random or skewed x-inactivation.
Another possibility is that the daughter could have a new mutation not inherited
from parents. Resulting in a redundancy seeing that there might be another gene
involved that can do similar work as the EBP gene.
Limitations
We are only sequencing the exons in the splice junctions and coding regions.
The mutation in this family could be due to a mutation that is not in either region. For
example, the mutation could be in the promoter region upstream of the gene itself.
Future Research
What we have been doing is molecular diagnosis. We would like there to be a
molecular treatments which might involve 1) enzyme replacement therapy 2) gene
therapy or 3) stem cell therapy. Gene therapy is an ultimate goal, however it is not
efficient right now, and is only used for a few disorders. Since the affected individual will
already have problems by the time he or she is born, the current treatment is focused
on making everything better going forward.
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