AdrenoLeukoDystrophy Blong Yang Molecular Genetics Fall 2004 University of Wisconsin – Eau Claire Fall 2004

AdrenoLeukoDystropAdrenoLeukoDystrophyhy

Blong YangBlong Yang

Molecular GeneticsMolecular Genetics

Fall 2004Fall 2004

University of Wisconsin – Eau Claire Fall 2004

Adrenoleukodystrophy (ALD)Adrenoleukodystrophy (ALD)

What is Adrenoleukodystrophy:What is Adrenoleukodystrophy:

• X-linked genetic disease that is a peroxisomal disorder with impaired X-linked genetic disease that is a peroxisomal disorder with impaired beta-oxidation of saturated Very Long Chain Fatty Acids (VLCFA’s).beta-oxidation of saturated Very Long Chain Fatty Acids (VLCFA’s).

- First recognized in 1923 – Schindler’s Disease Siemerling and - First recognized in 1923 – Schindler’s Disease Siemerling and

CreutzfeldtCreutzfeldt - 1970 – Dr. Michael Blaw first coined the term Adrenoleukodystrophy- 1970 – Dr. Michael Blaw first coined the term Adrenoleukodystrophy

AdrenoAdreno = Referring to adrenal glands = Referring to adrenal glands Leuko Leuko = Referring to white matter of the brain = Referring to white matter of the brain DystrophyDystrophy = Referring to imperfect growth/development = Referring to imperfect growth/development

• Caused by the X-ALD gene that encodes the ALD proteinCaused by the X-ALD gene that encodes the ALD protein - Peroxisomal membrane protein- Peroxisomal membrane protein

- Member of the ATP binding cassette transporter protein - Member of the ATP binding cassette transporter protein superfamily.superfamily. - Demonstrated using mouse model for X-linked ALD- Demonstrated using mouse model for X-linked ALD - Function that contributes to ALD is still unknown- Function that contributes to ALD is still unknown

University of Wisconsin – Eau Claire Fall 2004Lombard-Platet et al. Heinzer et al.

X-ALD PhenotypesX-ALD Phenotypes

Frequency of ALD in human population:Frequency of ALD in human population:• Affects 1/20,000 malesAffects 1/20,000 males

There are 2 Major disease Phenotypes:There are 2 Major disease Phenotypes:

1.1. Childhood onset ALDChildhood onset ALD Most severe form of ALDMost severe form of ALD Affects boys between the age of 4 – 6 yearsAffects boys between the age of 4 – 6 years Accounts for ~35% of ALD patientsAccounts for ~35% of ALD patients Involves inflammatory demyelination of nerve cellsInvolves inflammatory demyelination of nerve cells Leads to a vegetable stateLeads to a vegetable state Death within a few yearsDeath within a few years

University of Wisconsin – Eau Claire Fall 2004Lombard-Platet et al.

Meet Alex……….Meet Alex……….

Born May 1993 and appears as a Born May 1993 and appears as a normal healthy boy.normal healthy boy.

Age 3, difficulties Potty training.Age 3, difficulties Potty training.

Age 7, has difficulties at school, Age 7, has difficulties at school, fighting, constantly getting lost, fighting, constantly getting lost, difficulty learning material and difficulty learning material and keeping attention in class.keeping attention in class.

MRI diagnose Alex with ALDMRI diagnose Alex with ALD

Age 8, Alex can no longer walk. Age 8, Alex can no longer walk. Loses sight, muscle coordination, Loses sight, muscle coordination, ability to speak and swallow.ability to speak and swallow.

Currently, Alex is still alive and Currently, Alex is still alive and undergoing muscle therapy and on undergoing muscle therapy and on muscle relaxants, baclofen and muscle relaxants, baclofen and Clonidine.Clonidine.


X-ALD PhenotypesX-ALD Phenotypes

2.2. Adult onset ALD or AdrenomyeloneuropathyAdult onset ALD or Adrenomyeloneuropathy Occurrence at a mean age of 27.6 +/- 8.7 years.Occurrence at a mean age of 27.6 +/- 8.7 years. Most common, accounting for ~ 65% of ALD patientsMost common, accounting for ~ 65% of ALD patients Progresses slowlyProgresses slowly Adrenal impairment Adrenal impairment Involves mainly long tracts of the spinal cord and peripheral nervesInvolves mainly long tracts of the spinal cord and peripheral nerves No involvement of the Brain No involvement of the Brain Little or No inflammationLittle or No inflammation Learning disabilities, perceptual problems, attention deficit disorder, Learning disabilities, perceptual problems, attention deficit disorder,

short and long-term memory loss, impaired vision, coordination or short and long-term memory loss, impaired vision, coordination or gait, and various personality and behavioral changes. gait, and various personality and behavioral changes.

3.3. Addison DiseaseAddison Disease Impaired Adrenal FunctionImpaired Adrenal Function Adrenocortical Insufficiency with no detectable neurological Adrenocortical Insufficiency with no detectable neurological

involvementinvolvement ““Bronzing” of the skinBronzing” of the skin

University of Wisconsin – Eau Claire Fall 2004Lombard-Platet et al.

Location and Cloning of X-ALD GeneLocation and Cloning of X-ALD Gene

First cloned in 1993First cloned in 1993 Cloned by Mosser Cloned by Mosser et al.et al. Method: Positional CloningMethod: Positional Cloning

Approach:Approach:– Hypothesize that the location of X-ALD gene must be Hypothesize that the location of X-ALD gene must be Xq28Xq28, where the , where the

red/green color pigment gene (R/GPG) resides. On the basis of the high red/green color pigment gene (R/GPG) resides. On the basis of the high incidence of colour vision anomalies in AMN and ALD patients, the ALD incidence of colour vision anomalies in AMN and ALD patients, the ALD and R/GPG must be close.and R/GPG must be close.

– Identify a patient Identify a patient (Patient O)(Patient O) with blue-monochromatic colour vision with blue-monochromatic colour vision with a rearrangement located 5’ of Re-Colour pigment (RCP) gene, with a rearrangement located 5’ of Re-Colour pigment (RCP) gene, including two deletions separated by a large (>100 kb) inversion.including two deletions separated by a large (>100 kb) inversion.

– Only the RPC gene is found on the first deletion (88 kb). This deletion Only the RPC gene is found on the first deletion (88 kb). This deletion was not found in 81 other ALD patients, therefore they postulated that was not found in 81 other ALD patients, therefore they postulated that the inverted repeat or the second deletion were candidate loci for the the inverted repeat or the second deletion were candidate loci for the ALD gene.ALD gene.

University of Wisconsin – Eau Claire Fall 2004Mosser Mosser et al.et al.

Location and Cloning of X-ALD Location and Cloning of X-ALD GeneGene

BP4-BP2 and BP3-BP4-BP2 and BP3-BP1BP1 shows breakpoint shows breakpoint 4 and 2 and of points 3 4 and 2 and of points 3 and 1.and 1.

Dotted lineDotted line represents the represents the breakpoint junctions breakpoint junctions cloned.cloned.

Use a probe 4 kb Use a probe 4 kb proximal to proximal to Fr15 Fr15 deleted in Patient O deleted in Patient O ((1515) to obtain clones ) to obtain clones from a Xq28 cosmid from a Xq28 cosmid library.library.

3 overlapping clones 3 overlapping clones were obtained were obtained spanning 90 kb spanning 90 kb (cosQc11H12, (cosQc11H12, cosQc8F3 and cosQc8F3 and cosQc14A11)cosQc14A11)

In parallel, an Xba1 In parallel, an Xba1 junction fragment (X-8) junction fragment (X-8) was cloned was cloned corresponding to BP4-corresponding to BP4-BP2 in patient O. BP2 in patient O. University of Wisconsin – Eau Claire Fall 2004

A Map of the ALD gene

Mosser Mosser et al.et al.


Restriction map of X-8 Restriction map of X-8 showed a 1.8 kb showed a 1.8 kb XbalXbal--EcoEcoR1 (X2) fragment. It R1 (X2) fragment. It contained a 1.6 kb contained a 1.6 kb segment on the BP4 segment on the BP4 side and is within the side and is within the Fr15 contig.Fr15 contig.

Use Taq1 to generate Use Taq1 to generate probes from digestion probes from digestion of cosQc11h12.of cosQc11h12.

Probes generated are Probes generated are Ta25Ta25 (2.1 kb), (2.1 kb), Ta4Ta4 (3.6 (3.6 kb), kb), Ta1Ta1 (1.0 kb), (1.0 kb), Ta18Ta18 (0.85 kb), (0.85 kb), Ta13aTa13a (935 (935 bp), bp), Ta13bTa13b (252 bp). (252 bp).

Probes detected Probes detected deletions in 5 patients. deletions in 5 patients. Patient O, B, R, L and Patient O, B, R, L and Family Ma.Family Ma.


Generating Probes

Mosser Mosser et al.et al.


Figure Figure dd shows shows segregation of an abnormal segregation of an abnormal junction fragment detected junction fragment detected by probe by probe X2X2 in family. in family.

Here, you can see a 14.4 Here, you can see a 14.4 abnormal fragment was abnormal fragment was detected in an affected detected in an affected male as well as male as well as heterozygous females.heterozygous females.

Figure Figure ee shows 3 brothers shows 3 brothers whose DNA is digested with whose DNA is digested with HindIII and probed with HindIII and probed with X2X2. . The affected brother, filled The affected brother, filled square shows an abnormal square shows an abnormal 22 kb fragment. The 22 kb fragment. The brother affected shows the brother affected shows the same 22 kb fragment. same 22 kb fragment. Unaffected brother doesn’t Unaffected brother doesn’t show 22 kb fragment.show 22 kb fragment.

Southern Blot


Sequencing the ALD geneSequencing the ALD gene

Use probes TA25, TA18, TA13a and TA13b to probe for Use probes TA25, TA18, TA13a and TA13b to probe for transcripts in several tissues.transcripts in several tissues.

Discovered a 4.2 kb transcript.Discovered a 4.2 kb transcript. The sequences for these probes determined (Using a The sequences for these probes determined (Using a

computer program based on a multiple sensor-neural computer program based on a multiple sensor-neural network approach.)network approach.)

Sequencing revealed a large 700 bp putative protein coding Sequencing revealed a large 700 bp putative protein coding region for TA25 and between 300 – 400 bp regions for TA18, region for TA25 and between 300 – 400 bp regions for TA18, TA13b and TA13aTA13b and TA13a

Discovered that the deduced amino-acid sequence from Discovered that the deduced amino-acid sequence from these sequences showed significant sequence identity with these sequences showed significant sequence identity with collinearly positioned regions of human and rat 70 K collinearly positioned regions of human and rat 70 K peroxisomal membrane protein (PMP).peroxisomal membrane protein (PMP).


Using Nested - P.C.R reactions with primers from putative exons produced two fragments. Ex13 andEx3. These fragments where then used to screen a random-primed HeLa cell cDNA library to obtain6 independent overlapping clones, Producing a 2,751 bp sequence encoding a 745 aa protein.

Protein HomologyProtein Homology

Alignment of the c-terminal amino-acid sequence of ALDProtein with human and rat 70 K PMP and other members ofThe ATP-binding protein family. Identical are in black, Conservative substitutions are in gray. The two ATP bindingDomains are underlined, the segment highly conserved in ABC proteins are indicated by asterisks.


Conclusions drawn by J. Mosser Conclusions drawn by J. Mosser et et alal..

The putative location of the ALD gene is in the distal part of The putative location of the ALD gene is in the distal part of Xq28.Xq28.

The ALD gene does not code for VLCDF – CoA synthetase which The ALD gene does not code for VLCDF – CoA synthetase which was once considered to be the cause of ALD.was once considered to be the cause of ALD.

The ALD gene codes for a protein that is almost identical to The ALD gene codes for a protein that is almost identical to human and rat 70 K Peroxisome membrane protein.human and rat 70 K Peroxisome membrane protein.

The ALD protein is a member of the ATP – Binding Cassette The ALD protein is a member of the ATP – Binding Cassette (ABC) transporter protein, which are involved in the transport of (ABC) transporter protein, which are involved in the transport of proteins, amino acids, inorganic ions and peptides.proteins, amino acids, inorganic ions and peptides.

The ALD must either nonfunctional in transport of VLCFA or it The ALD must either nonfunctional in transport of VLCFA or it might be inhibited from activating VLCFA – CoA synthetase.might be inhibited from activating VLCFA – CoA synthetase.

It is still unresolved why the brain and adrenal glands are so It is still unresolved why the brain and adrenal glands are so sensitive to the buildup of VLCFA.sensitive to the buildup of VLCFA.

University of Wisconsin – Eau Claire Fall 2004Mosser Mosser et al.et al. Heinzer et al.

Map of the ALD geneMap of the ALD gene

The X-ALD gene is approximately 20 kb long and consists of 10 exons.

Mutations to the ALD gene that have been discovered can befound at http://www.x-ald.nl/ . To date, there are 703 mutations in their Database.

Mouse model for X-linked ALDMouse model for X-linked ALD

NO!!! YES!!!

- J.F. Lu et al. In 1997-Use mouse model to show that the ALD gene leads to the accumulation of VLCFA.-Method – Disruption of the ALD gene with Neo-R gene.


Disruption of the X-ALD mouse Disruption of the X-ALD mouse genegene

Depiction of X-ALD gene exons1-4. Gene targeting of the X-ALD locus used a replacement-Type vector covering betweenDashed lines.

Homologous recombinationCauses disruption in the X-ALDGene in ES cells.

Grown on selective medium containingGangcyclovir.

Select surviving cells.

Use XbaI to digest and runOn southern blot and ProbeFor sequences.Wildtype ES has 7 kb sequence,Mutant has 8 kb sequence.

University of Wisconsin – Eau Claire Fall 2004J.F. Lu et al.

Disruption of the X-ALD mouse Disruption of the X-ALD mouse genegene

Using neo-r cassette-specific primers (e or b)in combination with external primers (a or f)ES cells 1-3 generated a 4 kb PCR product but no results for normal ES cell.

Using internal primers (d or c) in combinationwith external (a or f), wildtype produced a 3 kb PCR product while mutant ES 1-3 cells produced the 4 kb (included the neo-r gene).


Production of Chimeras and Production of Chimeras and transmittance of X-ALD genetransmittance of X-ALD gene

The targeted ES cells are injected into blastocysts derived from The targeted ES cells are injected into blastocysts derived from C57bl/6 strain and the blastocysts were transferred into pseudo-C57bl/6 strain and the blastocysts were transferred into pseudo-pregnant CD-1 female mice to generate chimeras.pregnant CD-1 female mice to generate chimeras.

Female gave birth to 9 chimeras (6 males, 3 females)Female gave birth to 9 chimeras (6 males, 3 females)

Males are crossed to C57BL/6 to transmit X-ALD- allele to progeny Males are crossed to C57BL/6 to transmit X-ALD- allele to progeny giving rise to heterozygous female carrier mice. giving rise to heterozygous female carrier mice.

Heterozygous female mice crossed with either 129/Sv strain or Heterozygous female mice crossed with either 129/Sv strain or C57bl/6 strain to generate hemizygous male mice (C57bl/6 strain to generate hemizygous male mice (X-ALD-/ X-ALD-/ 00 ).).

Among the progeny:Among the progeny: Normal sex ratio (Male:Female = 110:102) indicating hemizygotes are not Normal sex ratio (Male:Female = 110:102) indicating hemizygotes are not

embryonic lethal.embryonic lethal. Out of 110 male mice, 54 were Out of 110 male mice, 54 were X-ALD-/ X-ALD-/ 00 . .


There is a great difference in the amount of total VLCFAs in normal and X-ALD mice and humans as well, especially when the FAs contain more than26 carbon units.


Histological AnalysisHistological Analysis

Performed at 3 months.

Slide A shows healthy normaladrenocortical cells.

Slide B shows an X-ALD-/ X-ALD-/ 00 mouse adrenocortical cells withclear lipid clefts (indicated by arrows).

Slide C shows the lipid cleft admixed with spicular lamellarmaterial at high magnification.(x 15,000)


Conclusion of Mouse StudyConclusion of Mouse Study

The X-ALD gene leads to the accumulation of VLCFAs in The X-ALD gene leads to the accumulation of VLCFAs in various tissues including the brain, kidneys, pancreas, liver various tissues including the brain, kidneys, pancreas, liver and heart. But the level of accumulation of each tissue and heart. But the level of accumulation of each tissue varies.varies.

The oldest mice at the end of the study were 6 months old The oldest mice at the end of the study were 6 months old and did not show any symptoms of adrenal insufficiency or and did not show any symptoms of adrenal insufficiency or nervous system dysfunction. It is yet too early to conclude nervous system dysfunction. It is yet too early to conclude that the X-ALD will not have any neurological involvement that the X-ALD will not have any neurological involvement equivalent to that of human X-ALD.equivalent to that of human X-ALD.

Biochemically, the X-ALD mouse model represents a valid Biochemically, the X-ALD mouse model represents a valid animal model for X-ALD. Most importantly, X-ALD in both animal model for X-ALD. Most importantly, X-ALD in both mouse and humans result in elevated tissue levels of mouse and humans result in elevated tissue levels of saturated VLCFAs, with the most severe abnormalities saturated VLCFAs, with the most severe abnormalities localized to the brain and adrenal gland.localized to the brain and adrenal gland.University of Wisconsin – Eau Claire Fall 2004J.F. Lu et al.

Diagnosis and Treatment of ALDDiagnosis and Treatment of ALD

Diagnosis:Diagnosis:– Simple blood tests can be done to analyze the levels of Simple blood tests can be done to analyze the levels of

VLCFAs in males. This test is not very accurate for VLCFAs in males. This test is not very accurate for female carries.female carries.

– Genetic testing is available through a DNA-based blood Genetic testing is available through a DNA-based blood test. This test is accurate in testing for carriers. The test. This test is accurate in testing for carriers. The entire testing protocol is available through the United entire testing protocol is available through the United Leukodystrophy Foundation (ULF).Leukodystrophy Foundation (ULF).

Treatment:Treatment:– There is no definitive treatment to ALDThere is no definitive treatment to ALD– Lorenzo’s Oil diet (not FDA approved) Lorenzo’s Oil diet (not FDA approved) – Bone Marrow TransplantBone Marrow Transplant– Gene therapy has been developed in animal models, but Gene therapy has been developed in animal models, but

is currently not available to human patients.is currently not available to human patients.


One more thing……..One more thing……..

Reviews:

“Ebert and Roeper”

- Two thumbs up!-


References:References:

Heinzer, A.K., Kemp, Stephan., Lu, J.F., Watkins, P.A., Smith, K.D. “Mouse Heinzer, A.K., Kemp, Stephan., Lu, J.F., Watkins, P.A., Smith, K.D. “Mouse Very Long-chain Acyl-CoA Synthetase in X-Linked Adrenoleukodystrophy.” Very Long-chain Acyl-CoA Synthetase in X-Linked Adrenoleukodystrophy.” J. J. Biol. Chem. Biol. Chem. Vol 277, Issue 32Vol 277, Issue 32, 28765-28773 (2002), 28765-28773 (2002)

Heinzer, A.K., Watkins, P.A., Lu, J.F., Kemp, Stephan., Moser, A.B., Li, Y.Y., Heinzer, A.K., Watkins, P.A., Lu, J.F., Kemp, Stephan., Moser, A.B., Li, Y.Y., Mihalik, S., Powers, J.M., Smith, K.D. “A Very Long-chain Acyl-CoA Synthetase Mihalik, S., Powers, J.M., Smith, K.D. “A Very Long-chain Acyl-CoA Synthetase Deficient Mouse and Its Relevance to X-linked Adrenoleukodystrophy.” Deficient Mouse and Its Relevance to X-linked Adrenoleukodystrophy.” Human Molecular GeneticsHuman Molecular Genetics Vol. 12 No. 10, Vol. 12 No. 10, 1145-1154 (2003)1145-1154 (2003)

Lombard-Platet, Gael., Savary, Stephane., Sarde, C.O., Mandel, J.L., Chimini, Lombard-Platet, Gael., Savary, Stephane., Sarde, C.O., Mandel, J.L., Chimini, G. “A Close Relative of the Adrenoleukodystrophy Gene Codes for a G. “A Close Relative of the Adrenoleukodystrophy Gene Codes for a Peroxisomal Protein With a Specific Expression Pattern.” Peroxisomal Protein With a Specific Expression Pattern.” Proc. Natl. Acad. Sci. Proc. Natl. Acad. Sci. USA USA 94,94, 1265-1269 (1996) 1265-1269 (1996)

Lu, J.F., Lawler, A.M., Watkins, P.A., Powers, J.M., Moser, A.B., Moser, H.W., Lu, J.F., Lawler, A.M., Watkins, P.A., Powers, J.M., Moser, A.B., Moser, H.W., Smith, K.D. “A Mouse Model for X-linked Adrenoleukodystrophy.” Smith, K.D. “A Mouse Model for X-linked Adrenoleukodystrophy.” Proc. Natl. Proc. Natl. Acad. Sci. USA Acad. Sci. USA 94,94, 9366-9371 (1997) 9366-9371 (1997)

Mosser, J., Douar. A., Sarde, O., Petra, K., Feils, R., Moser, H., Poustka, A.M., Mosser, J., Douar. A., Sarde, O., Petra, K., Feils, R., Moser, H., Poustka, A.M., Mandel, J.L., Aubourg, P.Mandel, J.L., Aubourg, P. “Putative X-linked Adrenoleukodystrophy Gene “Putative X-linked Adrenoleukodystrophy Gene Shares With Unexpected homology with ABC Transporters.” Shares With Unexpected homology with ABC Transporters.” Nature Nature 361361, 726-, 726-730 (1993)730 (1993)


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AdrenoLeukoDystrophy Blong Yang Molecular Genetics Fall 2004 University of Wisconsin – Eau Claire Fall 2004