1. Genetic Diagnostic of Leukodystrophy
Massoud Houshmand (Ph.D)
Special Medical Center
Massoud Houshmand 1

2. Clinical Presentations
Intoxication
Urea cycle defects
Energy Failure
Mitochondrial disease
Glycogen storage disease
Complex Molecule
Lysosomal storage disease
Peroxisomal storage disorders

3. Diseases that Cause Leukodystrophy
Some examples
Adrenoleukodystrophy
Metachromatic leukodystrophy
Tay-Sachs
Krabbe
Multiple Sulfatase Deficiency (Austin Disease)
Canavan
Alexsander
Pelizaeus-Merzbacher
Mitochondrial

4. Adrenoleukodystrophy/ Adrenomyeloneuropathy

5. Adrenoleukodystrophy/ Adrenomyeloneuropathy
Most common peroxisomal disorder (1/20,000)
Mutation in ABCD on Xq28 leads to defect in peroxisomal uptake of VLCFA
ALD: progressive neurologic disorder that begins at 5-12 years
Boys with new onset school difficulties & ADHD
Visuo-spatial deficits and hearing loss
Spasticity, ataxia, maybe seizures
Hypoglycemia, salt losing, hyperpigmentation
Rx: steroids, presymptomatic stem cell transplant, Lorenzo’s oil ineffective (oleic and erucic acids)
AMN: early adulthood progressive spastic paraparesis, cerebral demyelination (males)

7. Gene 
Test Method
Mutations Detected 
Mutation Detection Frequency by Test Method 
Males
Heterozygous Females
ABCD1
Sequence analysis
Sequence variants 
99% 
93% 
Del/dup analysis 
(Multi)exonic or whole-gene del/dup
~6% 

8. Gene Symbol
Chromosomal Locus
Protein Name
Locus Specific
HGMD
ABCD1
Xq28
ATP-binding cassette sub-family D member
LOVD ALD mutation database (ABCD1) X-linked Adrenoleukodystrophy Database (ABCD1)

9. DNA Nucleotide Change
Protein Amino Acid Change
c.1415_1416delAG (c.1801_1802delAG)
p.Gln472ArgfsTer83 (fsGlu471)

10. Metachromatic Leukodystrophy

11. Metachromatic Leukodystrophy
AR defect of arylsulfatase-A
Leukodystrophy as well as disease of adrenal glands, kidneys, pancreas, liver

12. Metachromatic Leukodystrophy
3 Presentations
Late infantile (18-24 months)
Gait disturbance, hypotonia to hypertonia, regression, involuntary movements, neuropathy, cherry red spot
Juvenile (4-10 years)
Bradykinesia, poor school performance, ataxia, movement disorder, neuropathy, slower progression
Adult
After puberty get personality and mental changes, cortical and cerebellar regression to frank dementia in third to fourth decade

14. Metachromatic Leukodystrophy

15. Metachromatic Leukodystrophy Familial Lipogranulomaltosis
Sandhoff
GM 1 Gangliosidosis
Tay-sachs
Fabry
Lactosyl Ceramidosis
اینجا ببینید آنزیم آریل سولفاتاز آ می خواهد سولفات را جدا کند. تجمع سولفات باعث بیماری می شود
Gaucher
Krabbe
Metachromatic Leukodystrophy
Niemann-Pick
Familial Lipogranulomaltosis

16. Neurocognitive Deficit
Progression
Neurocognitive Deficit
Frequency
Age at Onset (yrs)
Form
Death within 5-6 yrs
Motor milestones lost, neurocognitive functions lost
Most common
<4
Late infantile
Death within yrs
Motor milestones lost, learning and behavior impaired
Less common
4-6
Early juvenile
Slow
Personality changes, behavioral changes, dementia, psychosis, decreased school or work performance
Rare
6-16
Late juvenile
>16
Adult

17. Molecular Genetic Testing Used in Arylsulfatase A Deficiency
Test Methods
Mutations Detected
Mutation Detection Rate
Infantile onset
Juvenile onset
Adult onset
Targeted mutation analysis
mld alleles  
36% to 50%  
40% to 50%  
73% to 90%  
Pd allele
>90%
Sequence analysis/mutation scanning
ARSA sequence alterations
90-95%  

18. Molecular Genetics of Arylsulfatase A Deficiency
Gene Symbol
Chromosomal Locus
Protein Name
ARSA
22q13.3-qter
Arylsulfatase A

19. Amino Acide
Proteins kDa
RNA bp
Exon
DNA kb
Location
Gene symbol
Gene Name
507
53.6
2039  8
3,12 
22q13.33
ARSA
arylsulfatase A

20. Because of the interference of the enzyme arylsulfatase B (ARSB) in the most commonly used assays of ARSA enzymatic activity , an ARSA enzyme activity in the 5-20% range cannot establish or eliminate the diagnosis of MLD; therefore, one or more of the following additional tests is necessary:

21. Molecular genetic testing of the ARSA gene
Urinary sulfatide excretion
Other evidence of sulfatide storage, such as

22. Tay-Sachs

23. Gm2 Gangliosidosis Tay-sachs disease – hexosaminidase A
Sandhoff disease – hexosaminidase
A & B

24. Metachromatic Leukodystrophy Familial Lipogranulomaltosis
Sandhoff
GM 1 Gangliosidosis
Tay-sachs
Fabry
Lactosyl Ceramidosis
در بیماری بعدی قراره یک استیل گالاكتوز امین جدا بشه این اتفاق بکمک آنزیم هگزوامیداز می افته در صورت نقص این گانگلوسایت تجمع پیدا می کنه.تایپ یک بنام تی ساکس است و تایپ 2 ساندهوف بر خلاف موگوپلی ساکاردیوز که پیشرونده است و سالیان سال طول می کشد در بیماری لیزوزومی این دسته رگررشن را می بینیم که در ماههای اول زندگی خودش را نشان می دهد.
Gaucher
Krabbe
Metachromatic Leukodystrophy
Niemann-Pick
Familial Lipogranulomaltosis

26. Hexosaminidase A Deficiency

27. Metachromatic Leukodystrophy Familial Lipogranulomaltosis
Sandhoff
GM 1 Gangliosidosis
Tay-sachs
Fabry
Lactosyl Ceramidosis
Gaucher
Krabbe
Metachromatic Leukodystrophy
Niemann-Pick
Familial Lipogranulomaltosis

28. Gene
HEXA, the gene encoding the alpha subunit of the HEX A enzyme, is the only gene associated with hexosaminidase A deficiency.

29. Molecular genetic testing: Clinical uses
Confirmatory diagnosis in symptomatic individuals with borderline enzyme activity
Prenatal diagnosis when both parental mutations are known

30. Targeted mutation analysis
Heterozygotes
Mutations Detected
Test Method
Screening  
Obligate  
Non- Jewish
Jewish 8%
80%
32%
81%
+TATC1278
Targeted mutation analysis 9%
15%
+1 IVS 12
10%  
14%
+1 IVS 9 5% 3% 2%
G269S
R247W 4%
R249W
59%  
94%
46%
98%
All of the above

31. Beta-hexosaminidase alpha chain
Protein Name
Chromosomal Locus
Gene Symbol
Beta-hexosaminidase alpha chain
15q23-q24
HEXA

32. Gene Review HEXA Amino Acide Protein RNA Exon DNA Location Gene symbol
Gene Name
529
60.7 kDa
2255 bp 14
32,6 kb
15q23-q24
HEXA
hexosaminidase A
HEXA

34. In Tay-Sachs a mutation occurs which inhibits the productio of necessary proteins by Hex A
The loss of these necessary enzymes will cause brain damage

35. Krabbe Disease

36. Krabbe
AR defect of galactocerebroside-beta-galactosidase on chromosome 14
Pure neurologic condition
Onset at 3-8 months of age
Irritability, intermittent fevers, heightened startle reflex, feeding problems
Develop seizures, opisthotonus
Deafness and blindness by 9 months
MRI:

37. KRABBE DISEASE

38. Metachromatic Leukodystrophy Familial Lipogranulomaltosis
Sandhoff
GM 1 Gangliosidosis
Tay-sachs
Fabry
Lactosyl Ceramidosis
Gaucher
Krabbe
Metachromatic Leukodystrophy
Niemann-Pick
Familial Lipogranulomaltosis

39. Mutation Detection Rate
Mutations Detected
Test Method
Infantile Krabbe disease: varies by ethnicity
GALC 30-kb deletion
Targeted mutation analysis
Late-onset Krabbe disease: approximately 50% have one copy of the mutation
GALC 809G>A mutation
~100%
GALC sequence variants
Sequence analysis

40. Galactocerebrosidase
Protein Name
Chromosomal Locus
Gene Symbol
Galactocerebrosidase
14q31
GALC

41. Percent of All Alleles
Amino Acid Change
Nucleotide Change  
4%-5%
p.R168C
502C>T
8%-10%
p.D232N
694G>A
30%-40%
p.I546T
1637T>C
<2%
p.R168C + p.I546T
502C>T T>C  

42. Percent of Mutant Alleles
Nucleotide Change  
Percent of Mutant Alleles
30-kb deletion
40%-50%
1538C>T
5%-8%
1652A>C
1424delA
2%-5%
809G>A
1%-2%
32%-47%

43. Multiple Sulfatase Deficiency (Austin Disease)
Proptosis
Ichthyosis
hepatosplenomegaly

44. ممکن است کرتوز بدهد بعلت استروئید سولفاتاز و یا همه با همدیگر که نقص مولتیپل سولفاتاز باشد به چشمهای بیمار که بیرون زده نگاه کنید بعلت رسوبی است که در چشم بوجود آمده است. و

45. Multiple Sulfatase Deficiency
AR, mutations in sulfatase-modifying factor-1 gene (SUMF1) on 3p26
Austria: 1 in 1.4 million individuals
Affects 12 sulfatase enzymes
Post-translation modification defect in which cystein residue of enzyme is not activated
Defect in enzyme that causes oxidation of a thiol group in cysteine to generate an alpha-formylglycine residue
Alpha-formylglycine residue may accept the sulfate during sulfate ester cleavage by hydrolysis
Examples: arylsulfatase, steroid sulfatase, heparan sulfatase, N-acetylglucosamine-6-sulfatase

46. Multiple Sulfatase Deficiency
3 phenotypes
Neonatal MSD: severe mucopolysaccharidosis
Late infantile MSD: late-onset MLD
Juvenile MSD
Combined features of MLD, Hunter, Sanfilippo A, Morquio, Maroteaux-Lamy, X-linked ichthyosis

47. Canavan

48. Canavan AR deficiency of asparto-acylase
Macrocephaly, lack of head control, and developmental delays by the age of three to five months
Develop severe hypotonia and failure to achieve independent sitting, ambulation, or speech
Hypotonia eventually changes to spasticity
Life expectancy is usually into the teens
Diagnosis of Canavan disease relies upon demonstration of very high concentration of N-acetyl aspartic acid (NAA) in the urine

49. Canavan disease
Courtesy Dr Isabelle Desguerre, Paris Necker Hospital

51. gene Symbol
Test Method
Mutations Detected
Mutation Detection Frequency by Test Method 
Ashkenazi Jewish
Non-Ashkenazi Jewish
ASPA
Targeted mutation analysis
Panel 
p.Glu285Ala, p.Tyr231X
98% 3%
p.Ala305Glu 1%
30%-60%
Sequence analysis
Sequence variants  NA
87%
Deletion / duplication analysis 
Large genomic deletions/duplications comprising one or more exons
Unknown (<10%)

52. DNA Nucleotide Change
Protein Amino Acid Change
c.433-2A>G --
c.693C>A
p.Tyr231X
c.854A>C
p.Glu285Ala
c.863A>G
p.Tyr288Cys
c.914C>A
p.Ala305Glu

53. Alexander Disease AD mutation in GFAP at 17q21.31
Onset at around 6 months (birth – 2 yrs)
Psychomotor regression, spasticity and seizures
Juvenile patients have ataxia and spasticity
Adult patients have MS-like presentation
Diffuse demyelination, especially in frontal lobes

54. Alexander Disease

56. Gene 
Test Method
Mutations Detected 
Mutation Detection Frequency by Test Method 
GFAP
Sequence analysis
Sequence variants 
97% 

57. Gene Symbol
Chromosomal Locus
Protein Name
Locus Specific
HGMD
GFAP
17q21​.31
Glial fibrillary acidic protein
Human Intermediate Filament Database GFAP GFAP database

58. DNA Nucleotide Change (Alias 1)
Protein Amino Acid Change
c.209G>A
p.Arg70Gln
c.208C>T
p.Arg70Trp
c.218T>C
p.Met73Thr
c.226C>T
p.Leu76Phe
c.230A>G
p.Asn77Ser
c.235C>T
p.Arg79Cys
c.236G>A
p.Arg79His
c.256_259delinsGAGT
p.Glu86_Glu87delinsGluGlu
c.290T>C
p.Leu97Pro
c.376_381dupGCGGCT
p.Arg126_Leu127dup
c.613G>A
p.Glu205Lys
c.628G>A
p.Glu210Lys
c.715C>T
p.Arg239Cys
c.716G>A
p.Arg239His
c.716G>C
p.Arg239Pro
c.731C>T
p.Ala244Val
c.1047_1048insCACTTG
p.Tyr349_Gln350insHisLeu
c.1055T>C
p.Leu352Pro
c.1076T>C
p.Leu359Pro
c.1117G>A
p.Glu373Lys
c.1178G>T
p.Ser393Ile
c.1249delG (1247_1249delGGGinsGG)
p.Asp417MetfsTer15

59. Pelizaeus-Merzbacher

60. Pelizaeus-Merzbacher
Xq22 mutation in proteolipid protein 1 (PLP1)
Onset in first few months of life with rotary head movements, rotary nystagmus, & motor delay
Then ataxia, tremor, choreoathetosis, spasticity
Seizures
Optic atrophy and ocular impairments
MRI: Reversal of gray-white signal due to diffuse dymyelination

62. Pelizaeus-Merzbacher

63. for your patients and attention
Thanks
for your patients and attention 63