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

PRIMARY OUTCOME

Diagnostic yield and genetic variant spectrum identification

EXPECTED OUTCOMES

1. **Recruitment Yield**: 2,000 patients enrolled across 30+ sites within 18 months (mean 67 patients/site/year; 95% CI: 58-76). 2. **DNA Quality**: ≥95% samples meeting QC thresholds (A260/280 1.8-2.0, A260/230 ≥1.5, yield ≥100 µg). 3. **Sequencing Depth**: Mean coverage ≥150X across all targeted regions; ≥99% of targeted bases covered at ≥30X. 4. **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). 5. **Variant Spectrum**: Identify 340-420 unique variants across 5 genes. CAPN3 expected as most frequently mutated (35-40% of molecular diagnoses), followed by DYSF (25-30%), FKRP (15-20%), ANO5 (10-15%), DNAJB6 (5-8%). 6. **Variant Classification Distribution** (of all rare variants found): P: 3-5%, LP: 5-8%, VUS: 45-55%, LB/B: 35-45%. 7. **Novel Variant Discovery**: 40-70 novel variants not previously reported in gnomAD/ClinVar, with highest proportion in DYSF (37%) and CAPN3 (31%). ---

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 support (LOD score ≥1.5) or refute the variant-disease association. - **Novel Variant Functional Validation**: For ≥80% of novel VUS with strong computational support (CADD ≥25, REVEL ≥0.7), functional studies (RNA splice assay or protein expression assay) provide interpretable data. - **Data Sharing Compliance**: 100% of P/LP variants deposited to ClinVar within 90 days of classification; dbGaP submission complete by month 36.

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 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). ---

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[Nation-wide NGS-based genetic screening of LGMD patients in US](http://scidex.ai/artifact/exp-016edb6e-a692-4df5-a2ca-b0e7624cd223)

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