Molecular Genetics and Diagnosis of SSADH deficiency

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”.

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

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)

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

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

Diagnostic Methodologies URINE ORGANIC ACID SCREENING: GHB Measurement by GCMS +/- stable isotope dilution Normal Values 0 to 7 mmol/mol creatinine Pathogenic values 100-1200 mmol/mol creatinine. Need to use selective ion monitoring due to co-elution with urea

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: 0 – 78 nmol/l SSADH patients: 124 -4851 nmol/l Forni et al - 2013

Diagnostic Methodologies Enzyme assay. Sensitive Fluorescence assay. Lymphocytes.

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

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?

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.

Succinyladenosine appears on the CSF neurotransmitter metabolite chromatogram HVA 5HIAA

Diagnostic Methodologies GHB in Cerebrospinal Fluid. GHB Measurement by GCMS Reference range: < 3 umol/l SSADH deficiency: 100-850 umol/l

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%

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

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.

SSADH - Genetics Located on the short arm of Chromosome 6; 24,494969 to 24,537,207 (GRCh38.P2) INHERITANCE: Autosomal Recessive

Genetic Diagnostic Testing – ALDH5A1 Single Gene Sanger Sequencing:

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

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.1054-2A>C c.1273C>T p.R425X c.1360G>A p.G454R ?? (VUS)

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

Next Generation Sequencing: Phenotype Driven Panels Panels containing ALDH5A1

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)

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

ALDH5A1 Variants Found in Targeted Sequencing Panels Over 1000 panels requested (mainly comprehensive epilepsy) ONLY SIX VUS One pathogenic Gly268Glu (Gly281Glu) (known mutation) (heterozygous)

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.

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

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