2. Molecular Genetics and Diagnosis of SSADH deficiency

3. Conflict of Interest Disclosure
Keith Hyland is Executive Vice President of Medical Neurogenetics Laboratories ( MNGlabs ) a commercial company that provides molecular and metabolic testing for SSADH deficiency and other inherited disorders. Any recommendations made in this presentation are on best available evidence or are consistent with generally accepted medical practice”.

4. Biochemistry Diagnosis Genetics Challenges with Molecular Diagnosis
Succinic Semialdehyde Dehydrogenase Deficiency
Biochemistry Diagnosis Genetics Challenges with Molecular Diagnosis

5. SSADH deficiency - Biochemistry
Deficiency of SSADH may lead to: Elevation of neuromodulators: GABA, GHB and 4-HNE SSADH responsible for metabolism of the toxic aldehyde 4-hydroxy-2-nonenal (4-HNE: lipid peroxidation aldehyde) to the non-toxic acid, 4-hydroxy-2-nonenoate.
(4-hydroxybutyric acid -GHB)

6. Diagnostic Methodologies
Analyte: GC/MS ----LC/MS/MS 4-hydroxybutyric acid Urine Blood/Plasma/Dry Blood spots CSF Enzyme Assay: Lymphoblasts Genetic Analysis: Single Gene: ALDH5A1 NGS: Targeted Panels Whole Exome Whole Genome

7. Diagnostic Methodologies
CLINICAL SIGNS AND SYMPTOMS.
Hypotonia,
Developmental delay
Ataxia
Hyporeflexia
Cognitive impairment
Epilepsy
Neuropsychiatric problems
Sleep disturbance (insomnia)
Clinical features are not diagnostic

8. Diagnostic Methodologies
URINE ORGANIC ACID SCREENING:
GHB Measurement by GCMS +/- stable isotope dilution
Normal Values 0 to 7 mmol/mol creatinine
Pathogenic values mmol/mol creatinine.
Need to use selective ion monitoring due to co-elution with urea

9. Diagnostic Methodologies
Possibility for Incorporation into New born Screening.
LC/MS/MS
GHB is present in dried blood spots
Values in patients do not overlap controls
Controls: – 78 nmol/l
SSADH patients: nmol/l
Forni et al

10. Diagnostic Methodologies
Enzyme assay.
Sensitive Fluorescence assay.
Lymphocytes.

11. Diagnostic Methodologies
GHB Measurement in Cerebrospinal Fluid
Why would you want to do this?

12. ARE CASES OF SSADH DEFICIENCY BEING MISSED
ARE CASES OF SSADH DEFICIENCY BEING MISSED? A lesson from a study of adenylosuccinate lyase deficiency
Many cases diagnosed in Europe
Very few cases diagnosed in the US?

13. ARE CASES OF SSADH DEFICIENCY BEING MISSED
ARE CASES OF SSADH DEFICIENCY BEING MISSED? A lesson from a study of adenylosuccinate lyase deficiency
Analyzed 1500 non selected CSF samples from infants and children with neurological
disorders of multiple etiologies (HPLC with UV detection - succinylpurines)
Two had adenylosuccinate lyase deficiency
=0.13%
Selected for seizures/and or autism and/or hypomyelination (535)
= 0.4%
Have detected 20 + cases in the last 15 years.

14. Succinyladenosine appears on the CSF neurotransmitter metabolite chromatogram
HVA
5HIAA

15. Diagnostic Methodologies
GHB in Cerebrospinal Fluid.
GHB Measurement by GCMS
Reference range: < 3 umol/l
SSADH deficiency: umol/l

16. Measurement of GHB in CSF: The MNG experience.
Started in mid 2014
Analyzed 485 samples
(1 sample from USA - positive) – Already identified
(2 samples from Middle East - positive)
= 0.62%

17. Developing new methods for CSF testing: Same reasoning
CSF - thiamine – Thiamine transporter -2 deficiency
(SLC19A3) . Reversible cause of acute dystonia and
Leigh encephalopathy (treated with thiamine and biotin)
CSF – riboflavin – Riboflavin transporter.
Brown-Vialetto-Van Laere Syndrome (SLC52A2; SLC52A3)
. Sensorineural hearing loss with a
variety of cranial nerve palsies (treated with riboflavin)
Many more transporter defects are likely to emerge in the future that may require
CSF testing for diagnosis

18. SSADH gene
Two transcripts encoding two different isoforms have been identified.
10 Exons encoding 535 amino acids (isoform 1)
Alternate splicing inserts an 11th exon (4B) between exon 4 and 5 and may
lead to a 548 amino acid protein (isoform 2)
First 47 residues comprise a mitochondrial targeting peptide
Many possible regulatory sites in both 3’ and 5’ regions.

19. SSADH - Genetics
Located on the short arm of Chromosome 6; 24, to 24,537,207 (GRCh38.P2)
INHERITANCE: Autosomal Recessive

20. Genetic Diagnostic Testing – ALDH5A1
Single Gene Sanger Sequencing:

21. ALDH5A1 Sequencing - Sanger:
27 requests since 2012.
10 negative
5 frameshift (Pathogenic)
3 missense (Pathogenic)
5 termination codons (Pathogenic)
1 splice site (Pathogenic)
3 variants of uncertain significance
HIGH HIT RATE LIKELY DUE TO POSITIVE BIOCHEMICAL DATA

22. SINGLE GENE SANGER SEQUENCING (ALDH5A1)
c.104_127del p.S35Xfs
c.111_121del p.P37fs*53
c.122C>G p.Gln43Glu (VUS)
c.278G>T p.C93F
c.581C>T p.P194L (VUS)
c.612G>A p.W204X
c.608C>T p.P203L
c.664delG p.G224Afs*5
c.842G>A p.G281E
c.858delT p.D287Ifs*27
c A>C
c.1273C>T p.R425X
c.1360G>A p.G454R ?? (VUS)

23. Constitutional genetics NGS test menu
MNG Combined Genetic Panel ~ 2100 genes
(Custom Nimblegen Panel)
NEUROLOGIC DISORDERS
METABOLIC DISORDERS
CARDIAC DISEASE
HEARING & VISION SYNDROMES
AMYLOID RELATED DISEASE
CONNECTIVE TISSUE DISEASE & BONE DISORDERS
FEVER SYNDROMES
KIDNEY DISEASE

24. Next Generation Sequencing: Phenotype Driven Panels
Panels containing ALDH5A1

25. Comparative analysis: SNP catcher
Windows sql server that integrates
Diagnostic tool
Frequency calculations from 1000 genome, EVS, EXAC and internal database
Gene – phenotype associations (Clinvar, OMIM)
Patterns of inheritance (OMIM)
Functional predictions from the internet (Provean)

26. SNP-catcher constitutional output
All varianys in protein coding regions (+/- 5bp) listed in vcf file
(coverage >10 fold; allele frequency >10%)
Known disease mutation filter
Coding region filter
CATEGORY 1
Known pathogenic
mutations
(Clinvar/HGMD)
CATEGORY 2
Known pathogenic
mutations not covered
(Clinvar/HGMD)
CATEGORY 3
Mutations in known
Disease associated genes
(Clinvar/HGMD)
CATEGORY 4
Mutations in
non-disease
associated genes
Frequency filter
(<1% allele frequency in 1000 genome project and EVS and internal database)
CATEGORY 5
Rare known
pathogenic
mutations
(Clinvar/HGMD)
CATEGORY 6
Rare known
pathogenic
mutations not covered
(Clinvar/HGMD)
CATEGORY 7
Rare mutations in known
disease associated genes
CATEGORY 8
Rare mutations in
non-disease
associated genes
Missense
Compound
De novo Unique
SS, FS, SC
Homozygous

27. ALDH5A1 Variants Found in Targeted Sequencing Panels
Over panels requested (mainly comprehensive epilepsy)
ONLY SIX VUS
One pathogenic Gly268Glu (Gly281Glu) (known mutation) (heterozygous)

28. ALDH5A1 variants found in all samples submitted.
Over 5,000 samples have been examined.
80 variants found that were classified as NOT benign.
3 classified as pathogenic (heterozygous)
77 classified as VUS (heterozygous)
Suggests that SSADH deficiency is rarely missed but that carrier frequency may be
higher than the currently accepted rate.

29. Difficulty in Classification
ALDH5A1 Variants
Difficulty in Classification
EXAC Database:
Investigates variants from control populations and provides
frequency data.
161 missense variants with allele frequency suggesting they are rare
(possibility of being pathogenic)
29 had dbSNP ‘s
2 found in ClinVar: 1 labeled pathogenic, 1 labeled benign

30. Questions