Nation-wide NGS-based genetic screening of LGMD patients in US

Exploratory Score: 0.950 Price: $0.50 Limb-girdle muscular dystrophy (LGMD) Human patients Status: proposed

What This Experiment Tests

Exploratory experiment designed to discover new patterns targeting CAPN3, DYSF, FKRP, ANO5, DNAJB6 in Human patients. Primary outcome: Diagnostic yield and genetic variant spectrum identification

Description

This was a comprehensive next-generation sequencing (NGS)-based gene panel study conducted across the United States to investigate the genetic basis of limb-girdle muscular dystrophies (LGMDs). The study aimed to determine the diagnostic yield, characterize the gene-variant spectrum, and establish the relative prevalence of different LGMD subtypes in a large clinically suspected LGMD population. A total of 4656 patients with clinically suspected LGMD from across the US were recruited over a two-year period (June 2015 to June 2017). The study was conducted in a CLIA-CAP certified laboratory (Emory Genetics Laboratory) ensuring high clinical standards. The researchers investigated 35 genes associated with LGMD subtypes or LGMD-like other neuromuscular disorders. The study revealed several important findings including an overall diagnostic yield of 27%, identification of major contributing genes, discovery of increased prevalence of certain late-onset conditions, and importantly, identification of patients with pathogenic variants in multiple LGMD genes suggesting possible synergistic effects.

TARGET GENE

CAPN3, DYSF, FKRP, ANO5, DNAJB6

MODEL SYSTEM

Human patients

ESTIMATED COST

TIMELINE

0 months

PATHWAY

Muscle fiber maintenance and repair pathways

SOURCE

extracted_from_pmid_30564623

PRIMARY OUTCOME

Diagnostic yield and genetic variant spectrum identification

Scoring Dimensions

📖 Wiki Pages

DNAJB6 Proteinprotein ANO5 (Anoctamin 5 (TMEM16E))gene DNAJB6 Genegene DYSF — Dysferlingene Muscular Dystrophydisease emoryinstitution geneticsmechanism Researchersindex Heat Shock Protein Dysfunction in Progressive Suprmechanism Chaperone-Mediated Proteostasis Disease Comparisonmechanism Prion vs Tau/Alpha-Synuclein Propagation: Mechanishypothesis Protein Aggregation and Misfolding in Neurodegenermechanism Protein Aggregation in Neurodegenerative Disease Cmechanism Protein Aggregation in Neurodegenerationmechanism Hsp70/Hsp90 Chaperone Pathway in Parkinson's Diseamechanism

Protocol

Phase 1: Study Design & Recruitment (Months 1-6)

1.1 Patient Recruitment: Establish nation-wide collaboration with 30+ neuromuscular clinics. Recruit 2,000 clinically diagnosed LGMD patients meeting modified Mercuri criteria.
1.2 Informed Consent: Obtain written informed consent including genetic data sharing (dbGaP, LOVD).
1.3 Clinical Phenotyping: Standardized phenotyping using adapted North Star Amyotrophy Questionnaire, 6-minute walk test, forced vital capacity, and muscle MRI (STIR sequence for fat infiltration scoring).

...

Phase 1: Study Design & Recruitment (Months 1-6)

1.1 Patient Recruitment: Establish nation-wide collaboration with 30+ neuromuscular clinics. Recruit 2,000 clinically diagnosed LGMD patients meeting modified Mercuri criteria.
1.2 Informed Consent: Obtain written informed consent including genetic data sharing (dbGaP, LOVD).
1.3 Clinical Phenotyping: Standardized phenotyping using adapted North Star Amyotrophy Questionnaire, 6-minute walk test, forced vital capacity, and muscle MRI (STIR sequence for fat infiltration scoring).

Phase 2: Sample Collection & DNA Extraction (Months 4-12)

2.1 Blood Collection: Collect 10 mL peripheral blood in EDTA tubes (BD Vacutainer). Minimum 2,000 µg genomic DNA required per patient.
2.2 DNA Extraction: Use QIAamp DNA Blood Maxi Kit (Qiagen, cat# 51192). Quantify with Qubit 4.0 fluorometer (Thermo Fisher). Accept A260/280 = 1.8-2.0, A260/230 ≥ 1.5.
2.3 Sample Storage: Store at -80°C. Archive lymphoblastoid cell lines (EBV-transformed) for 10% of cohort for validation.

Phase 3: NGS Library Preparation & Sequencing (Months 8-18)

3.1 Target Enrichment: Use Agilent SureSelect QXT Target Enrichment Kit covering all coding exons ± 50 bp intronic flanking regions for CAPN3 (NM_000070.3), DYSF (NM_003494.4), FKRP (NM_024301.5), ANO5 (NM_213599.3), DNAJB6 (NM_058246.3). Average bait coverage: 500X.
3.2 Sequencing: Illumina NovaSeq 6000 platform. Paired-end 150 bp reads. Minimum 30X mean coverage, >95% bases ≥ 20X.
3.3 Quality Controls: Include NIST NA12878 reference sample every 20 patient samples. Intra-run duplicates (5%).

Phase 4: Bioinformatics Analysis (Months 12-24)

4.1 Primary Analysis: FASTQ alignment to GRCh38 using DRAGEN Germline Pipeline v4.0. GATK HaplotypeCaller for variant calling.
4.2 Variant Annotation: ANNOVAR, CADD v1.7 (phred score), SpliceAI, REVEL, MetaRNN. Filter for rare variants (gnomAD v4 AF < 0.001).
4.3 Variant Classification: Per ACMG/AMP 2023 guidelines. Classify into: Pathogenic (P), Likely Pathogenic (LP), Variant of Uncertain Significance (VUS), Likely Benign, Benign.

Phase 5: Variant Validation & Segregation Analysis (Months 18-30)

5.1 Orthogonal Validation: Confirm all P/LP/VUS variants via Sanger sequencing (ABI 3730xl). Use BigDye Terminator v3.1 chemistry.
5.2 Family Segregation: Offer targeted testing to available family members (parents, siblings). Minimum 3-generation pedigrees documented.
5.3 RNA Studies: For splice variants, perform lymphocyte RNA extraction and RT-PCR (SuperScript IV) to assess splice-alteration.

Phase 6: Data Integration & Reporting (Months 24-36)

6.1 Clinical Report Generation: Generate CLIA-compliant reports via Amber Labs/GeneMatcher integration. All variants reported to ClinVar.
6.2 Statistical Analysis: Calculate diagnostic yield with 95% CI. Compare variant spectrum to published LGMD cohorts (Bonnes et al. 2022, Nalls et al. 2019).
6.3 Database Deposition: Submit anonymized variants to LOVD (Leiden Muscular Dystrophy pages) and dbGaP (phs002599).

Expected Outcomes

Recruitment Yield: 2,000 patients enrolled across 30+ sites within 18 months (mean 67 patients/site/year; 95% CI: 58-76).

DNA Quality: ≥95% samples meeting QC thresholds (A260/280 1.8-2.0, A260/230 ≥1.5, yield ≥100 µg).

Sequencing Depth: Mean coverage ≥150X across all targeted regions; ≥99% of targeted bases covered at ≥30X.

Diagnostic Yield: 18-25% of patients receive molecular diagnosis (definite P/LP finding in known LGMD gene). Based on literature, expect 22% ± 3% in this cohort size (n=2,000).

...

Success Criteria

Diagnostic Yield Threshold: ≥18% of enrolled patients achieve molecular diagnosis (lower 95% CI bound >15%). Target: 22% ± 3%.
Sequencing Quality: ≥98% of samples pass all QC thresholds (DNAsample yield, coverage, Q30 score >85%).
Variant Validation Concordance: ≥99% concordance between NGS and Sanger validation for P/LP/VUS calls (kappa >0.95).
ACMG Classification Reliability: Inter-rater concordance ≥90% (kappa >0.85) between two independent variant classifiers.
Family Segregation Confirmation: For ≥70% of families where segregation testing is pursued, results su

...

Related Hypotheses (1)

HSP70 Co-chaperone DNAJB6 Universal Cross-Seeding Inhibitor0.632

Debate History (0)

No debates yet

Experiment Results (0)

No results recorded yet. Use POST /api/experiments/{id}/results to record a result.