Whole genome sequencing identified a novel copy number variant on TPK1 gene in a Chinese family with thiamine metabolism dysfunction syndrome 5

Technical Summary: Whole Genome Sequencing Identifies a Novel CNV in TPK1 for Thiamine Metabolism Dysfunction Syndrome 5

Problem Statement
Thiamine metabolism dysfunction syndrome 5 (THMD5) is a rare autosomal recessive neurometabolic disorder caused by biallelic pathogenic variants in the TPK1 gene, which encodes thiamine pyrophosphokinase (TPK). While over 30 pathogenic variants have been documented, the majority are missense mutations. Copy number variations (CNVs) involving TPK1 are extremely rare and often undetected by standard whole exome sequencing (WES) or targeted gene panels, which may fail to cover coding exons fully or reliably detect larger structural variations, particularly those with breakpoints in non-coding regions. Consequently, diagnosing THMD5 in adult patients or those with atypical presentations remains challenging. This study addresses the need to expand the genetic spectrum of TPK1 and evaluate the diagnostic utility of whole genome sequencing (WGS) in identifying complex genetic lesions.

Methodology
The study analyzed a Chinese family with two affected siblings (a 31-year-old proband and his 33-year-old older brother) and three unaffected family members (parents and a younger brother).

• Clinical Data Collection: Comprehensive clinical histories, neurological examinations, neuroimaging (MRI), laboratory findings (including serum lactate), and cognitive assessments (MMSE, MoCA, ADAS-Cog) were gathered.
• Genetic Analysis: Whole genome sequencing (WGS) was performed on all family members using Illumina TruSeq DNA PCR-Free Library Preparation, achieving an average depth of 30×. Data were aligned to the human reference genome (hg19) using BWA and analyzed with the Gene Analysis ToolKit (GATK).
• Variant Validation and Interpretation: Identified variants were validated via Sanger sequencing. Variants were interpreted using the Variant Effect Predictor (VEP) and screened against databases (ClinVar, OMIM, HGMD, gnomAD). Pathogenicity was assessed using computational algorithms (SIFT, PolyPhen-2, FATHMM) and classified according to ACMG/ClinGen guidelines. CNVs were specifically interpreted based on technical standards for constitutional copy-number variants.
• Phasing: The study utilized split-read sequence analysis of WGS data to determine the cis/trans phasing of compound heterozygous variants.

Key Results

• Clinical Phenotypes:
  • Proband: Onset at age 13 with recurrent episodic encephalopathy, ataxia, and dysarthria. Progressed to spastic gait and limb stiffness by age 19. MRI showed mild cerebral, cerebellar, and brainstem atrophy. Serum lactate was normal. Cognitive function was largely preserved.
  • Older Brother: Onset at age 4 with severe symptoms including symmetric basal ganglia lesions (hyperintensity on T2, hypointensity on T1) and elevated serum lactate (3.65 mmol/L). Progressed to severe spasticity, gait disturbance, and cognitive impairment (MMSE 20/30), rendering him unable to walk independently by his thirties.
• Genetic Findings: WGS identified compound heterozygous variants in TPK1 in both affected siblings:
  1. A known pathogenic missense variant: NM_022445.4:c.382C>T (p.Leu128Phe).
  2. A novel CNV: NC_000007.13:g.144285435_144289647dup, a 4.2 kb tandem duplication involving exon 7 and adjacent introns.
  • Sanger sequencing confirmed the missense variant was inherited from the mother.
  • Split-read analysis confirmed the CNV was inherited from the father and was in trans with the missense variant.
  • The CNV was predicted to cause reading frame disruption and nonsense-mediated decay (NMD).
• Treatment Outcomes:
  • The proband received regular high-dose thiamine (100 mg/day), resulting in symptom stabilization with no further deterioration.
  • The older brother received only a short course of low-dose thiamine (30 mg/day) before discontinuing treatment, leading to progressive neurological decline.

Key Contributions

1. Expansion of Genetic Spectrum: The study reports a novel 4.2 kb tandem duplication in TPK1, expanding the known mutational landscape of THMD5 beyond the predominant missense variants.
2. Diagnostic Superiority of WGS: The research demonstrates that WGS is superior to WES or targeted panels for detecting complex structural variations like intragenic CNVs and determining the phasing of compound heterozygous variants, which is critical for accurate diagnosis.
3. Genotype-Phenotype Correlation: The study highlights significant phenotypic heterogeneity between siblings with identical genotypes. The divergence in clinical trajectories is attributed to treatment adherence rather than genetic differences.

Significance and Claims
The authors claim that their findings underscore the critical importance of lifelong, high-dose thiamine therapy in managing THMD5. The contrasting outcomes of the two siblings—stabilization in the adherent proband versus progressive deterioration in the non-adherent brother—provide compelling evidence that thiamine therapy can arrest neurodegeneration even when initiated later in the disease course.

Furthermore, the paper asserts that WGS should be integrated into the diagnostic workup for inherited metabolic disorders, particularly when initial genetic testing fails to yield a molecular diagnosis. By identifying a novel CNV that would likely have been missed by other methods, the study reinforces the necessity of comprehensive genomic analysis to uncover the full genetic etiology of rare metabolic diseases. The authors advocate for immediate initiation of high-dose thiamine upon clinical or genetic suspicion of THMD5 to prevent irreversible neurological damage.