Table 3.

Evaluating the clinical validity of BSN–epilepsy associations based on the framework developed by the Clinical Genome Resource

	Evidence type		Case information type (suggested starting score)			Suggested upgrades			Scoring range	Points given	Max score
						Functional data			De novo
Case-level data	Variant evidence		Predicted or proven null variant (1.5 points)			+0.5 points		+0.5 points	0–3 points (per variant)	5.5*	12 points
			Other variant type (0.1 points)			+0.4 points		+0.4 points	0–1.5 points (per variant)	0.9†
	Segregation evidence		Evidence of segregation in one or more families			Sequencing Method			0–3 points	–	3 points
				Total LOD Score		Candidate gene Sequencing		Exome/Genome or all genes sequenced in linkage region
				2–2.99		0.5 points		1 points
				3–4.99		1 points		2 points
				>5		1.5 points		3 points
Case–control data	Case–control study type		Case–control quality criteria			Suggested points/study				Points given	Max score
	Single variant analysis		Variant detection methodology. Power. Bias and confounding factors. Statistical significance.			0–6 points				–	12 points
	Aggregate variant analysis					0–6 points				–
Total allowable points for genetic evidence										6.4	12 points
Evidence category		Evidence type			Suggested points					Points given	Max score
					Default		Range
Function		Biochemical function			0.5		0–2			0.5‡	2
		Protein interaction			0.5		0–2			0.5§
		Expression			0.5		0–2			0.5¶
Functional alteration		Patient cells			1		0–2			0	2
		Non-patient cells			0.5		0–1			0.5**
Models		Non-human model organism			2		0–4			4††	4
		Cell culture model system			1		0–2			0
Rescue		Rescue in human			2		0–4			0
		Rescue in non-human model organism			2		0–4			0
		Rescue in cell culture model			1		0–2			0
		Rescue in patient cells			1		0–2			0
Total allowable points for experimental evidence										6	6
Clinical validity summary matrix										12.4	Strong

*Two de novo null variants and one inherited small deleting variant (2 points/variant×2 variants+1.5 points/variant×1 variant)

†Nine inherited missense variants (0.1 point/variant×9 variants).

‡Neurotransmitter is vital for the neuron excitability. BSN protein participates in the formation of Golgi-derived membranous organelles termed Piccolo-Bassoon transport vesicles that are transported along axons to sites of nascent synaptic contacts. It is crucial for the development and survival of a wide range of neuronal cells (assigned 0.5 point).

§BSN protein is one of the members assembled at the presynaptic cytomatrix at the active zone, in which BSN protein combines with Munc13 proteins. Loss of Munc13-1 function causes cortical hyperexcitability. The mutations in UNC13B gene which encode Munc13-2 protein were associated with epilepsy (assigned 0.5 points).

¶BSN gene is highly expressed in the mammalian brain especially in the cerebral cortex (assigned 0.5 point).

**Experimentally, a genetic deletion of Bassoon or an acute interference with Bassoon-RBP interaction reduces synaptic abundance of CaV2.1, weakens P/Q-type Ca²⁺ current-driven synaptic transmission, and results in higher relative contribution of neurotransmission dependent on CaV2.2. Another experiments show that the absence of Bassoon led to the inactivation of a significant fraction of glutamatergic synapses, induced a reduction in normal synaptic transmission, and resulted in that vesicles were clustered and docked in normal numbers but were unable to fuse (assigned 0.5 point).

††Mice of homozygous BSN null led to partial premature lethality and spontaneously seizures (MGI: 1277955, assigned 4 points).

LOD, log of odds (genetic linkage score).