Integrated Rare-Disease Variant Annotation Report

Teaching and research use only. The datasets are synthetic and the results are not clinical diagnoses.

Executive Summary

A reproducible, container-based workflow was developed for the annotation of small variants and copy-number variants in four synthetic rare-disease datasets: Achondroplasia, Klippel–Trénaunay syndrome, McCune–Albright syndrome and Pompe disease.

The small-variant branch uses bcftools, Ensembl VEP 115, SnpEff, ClinVar, SpliceAI and cached gnomAD fields. The CNV branch uses AnnotSV, ClassifyCNV and ISV-CNV with SHAP values.

All four pipelines completed successfully. Every final small-variant VCF retained all 20 input records. The GPT/manual synthetic baseline and pipeline outputs matched for both small variants and CNVs in all four diseases.

Automated Workflow

Small variants

Raw VCF
  → bcftools normalization
  → VEP
  → SnpEff
  → ClinVar
  → SpliceAI
  → cached gnomAD
  → final VCF.gz + index

CNVs

Raw CNV VCF
  → BED conversion
  → AnnotSV
  → ClassifyCNV
  → ISV-CNV probabilities and SHAP values

Tools and Containers

Container	Purpose
core_tools.sif	bcftools, samtools, bgzip, tabix and bedtools
vep.sif	Ensembl VEP 115 annotation
snpeff.sif	SnpEff consequence annotation
spliceai.sif	Splice-effect prediction
annotsv.sif	Structural-variant annotation and ranking
classifycnv.sif	ACMG-style CNV classification
isvcnv.sif	Machine-learning CNV prioritization and SHAP values

Cross-Disease Results

Disease	Gene	ClinVar	AnnotSV	ClassifyCNV	ISV probability	SNV comparison	CNV comparison
Achondroplasia	FGFR3	Pathogenic	3	Uncertain significance	0.006764889229089022	MATCH	MATCH
Klippel–Trénaunay syndrome	PIK3CA	Pathogenic	3	Uncertain significance	0.013497603125870228	MATCH	MATCH
McCune–Albright syndrome	GNAS	Pathogenic/Likely pathogenic	5	Likely pathogenic	0.8791712522506714	MATCH	MATCH
Pompe disease	GAA	Pathogenic	5	Likely pathogenic	0.3094403147697449	MATCH	MATCH

Achondroplasia

Disease and inheritance

Main gene: FGFR3
Inheritance: Autosomal dominant

Main small variant

Variant: chr4:1804392 G>A; rs28931614; p.Gly380Arg
GPT/manual expectation: Pathogenic
Pipeline ClinVar: Pathogenic
Record comparison: MATCH
Records retained: 20 of 20

Main CNV

CNV: chr4:1793293–1808867 DUP; genotype 0/1
Expected class: VUS
AnnotSV: 3
ClassifyCNV: Uncertain significance
ISV probability: 0.006764889229089022
Comparison: MATCH

Integrated educational interpretation

The activating FGFR3 small variant matches the established Achondroplasia mechanism more directly than the synthetic duplication.

Mechanism-fit summary: SNV: High; CNV: Low

Klippel–Trénaunay syndrome

Disease and inheritance

Main gene: PIK3CA
Inheritance: Postzygotic somatic mosaic

Main small variant

Variant: chr3:179234297 A>G; rs121913279; synthetic VAF 8%
GPT/manual expectation: Pathogenic
Pipeline ClinVar: Pathogenic
Record comparison: MATCH
Records retained: 20 of 20

Main CNV

CNV: chr3:179148126–179240093 DUP; genotype 0/1
Expected class: VUS
AnnotSV: 3
ClassifyCNV: Uncertain significance
ISV probability: 0.013497603125870228
Comparison: MATCH

Integrated educational interpretation

The mosaic activating PIK3CA small variant matches the usual disease mechanism more directly than the synthetic duplication.

Mechanism-fit summary: SNV: High; CNV: Low

McCune–Albright syndrome

Disease and inheritance

Main gene: GNAS
Inheritance: Postzygotic somatic mosaic

Main small variant

Variant: chr20:58909366 G>A; rs121913495; synthetic VAF 12%
GPT/manual expectation: Pathogenic or likely pathogenic
Pipeline ClinVar: Pathogenic/Likely pathogenic
Record comparison: MATCH
Records retained: 20 of 20

Main CNV

CNV: chr20:58839748–58911192 DEL; genotype 0/1
Expected class: Pathogenic or likely pathogenic
AnnotSV: 5
ClassifyCNV: Likely pathogenic
ISV probability: 0.8791712522506714
Comparison: MATCH

Integrated educational interpretation

The activating mosaic GNAS small variant matches McCune–Albright syndrome. The deletion may be pathogenic through dosage but is not the typical mechanism of this named disease.

Mechanism-fit summary: SNV: High; CNV: Low for named disease

Pompe disease

Disease and inheritance

Main gene: GAA
Inheritance: Autosomal recessive

Main small variant

Variant: chr17:80104542 T>G; rs386834236; homozygous
GPT/manual expectation: Pathogenic
Pipeline ClinVar: Pathogenic
Record comparison: MATCH
Records retained: 20 of 20

Main CNV

CNV: chr17:80101581–80119881 DEL; genotype 1/1
Expected class: Pathogenic
AnnotSV: 5
ClassifyCNV: Likely pathogenic
ISV probability: 0.3094403147697449
Comparison: MATCH

Integrated educational interpretation

The homozygous small-variant and deletion designs both fit a recessive GAA loss-of-function testing model.

Mechanism-fit summary: SNV: High; CNV: High

GPT/Manual vs Pipeline Comparison

All four small-variant files contained 20 GPT/manual baseline records and 20 pipeline records. All variant identities matched.

The expected main-variant classifications agreed with the relevant pipeline fields. CNV expected classifications also agreed with AnnotSV and ClassifyCNV for every disease.

The GPT/manual files are transparent manually curated synthetic baselines. They are not presented as literal output from an independent clinical annotation engine.

Limitations

The datasets are synthetic and contain no patient phenotype.
Automated classifications are not diagnoses.
Some synthetic CNVs do not represent the usual mechanism of the named disease.
Databases and prediction tools may differ in version and interpretation.
Current gnomAD website checks should supplement cached frequency fields.
GeneHancer annotations were unavailable in the current AnnotSV setup.

Conclusion

A complete two-branch GRCh38 annotation pipeline was successfully built, containerized and validated. The pipeline preserved all background variants and produced complementary small-variant and CNV results for all four diseases.

The final repository includes synthetic inputs, automated pipeline scripts, selected annotated outputs, GPT/manual baseline files, comparison reports and an integrated clinical-style summary.