rs147484110

chr21-43774760-C-G

Variant summary

Our verdict is Pathogenic. Variant got 11 ACMG points: 11P and 0B. PVS1_Strong PM2 PP3_Strong PP5

The NM_000100.4(CSTB):c.67-1G>C variant causes a splice acceptor change. The variant allele was found at a frequency of 0.000298 in 1,612,442 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a pathogenic outcome for this variant. 3/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Conflicting classifications of pathogenicity (no stars).

• Frequency:
  • Genomes: 𝑓 0.00026 (0 hom., cov: 33)
  • Exomes 𝑓: 0.00030 (0 hom.)
• Consequence: CSTB NM_000100.4 splice_acceptor
• Scores: Splicing: ADA: 0.9999
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications P:14 U:1 O:2
• Conservation: PhyloP100: 4.01

Genes affected

CSTB (HGNC:2482): (cystatin B) The cystatin superfamily encompasses proteins that contain multiple cystatin-like sequences. Some of the members are active cysteine protease inhibitors, while others have lost or perhaps never acquired this inhibitory activity. There are three inhibitory families in the superfamily, including the type 1 cystatins (stefins), type 2 cystatins and kininogens. This gene encodes a stefin that functions as an intracellular thiol protease inhibitor. The protein is able to form a dimer stabilized by noncovalent forces, inhibiting papain and cathepsins l, h and b. The protein is thought to play a role in protecting against the proteases leaking from lysosomes. Evidence indicates that mutations in this gene are responsible for the primary defects in patients with progressive myoclonic epilepsy (EPM1). One type of mutation responsible for EPM1 is the expansion in the promoter region of this gene of a CCCCGCCCCGCG repeat from 2-3 copies to 30-78 copies. [provided by RefSeq, Jul 2016]

ACMG classification

Verdict is Pathogenic. Variant got 11 ACMG points.

• PVS1: Splicing variant, NOT destroyed by nmd, known LOF gene, truncates exone, which is 0.34006733 fraction of the gene. No cryptic splice site detected. Exon removal is inframe change.
• PM2: Very rare variant in population databases, with high coverage
• PP3: Splicing scoreres supports a deletorius effect: Scorers claiming Pathogenic: dbscSNV1_ADA, dbscSNV1_RF, max_spliceai. No scorers claiming Uncertain. No scorers claiming Benign.
• PP5: Variant 21-43774760-C-G is Pathogenic according to our data. Variant chr21-43774760-C-G is described in ClinVar as [Conflicting_classifications_of_pathogenicity]. Clinvar id is 8395.We mark this variant Likely_pathogenic, oryginal submissions are: {Pathogenic=12, not_provided=2, Uncertain_significance=1}. Variant chr21-43774760-C-G is described in Lovd as [Pathogenic]. Variant chr21-43774760-C-G is described in Lovd as [Pathogenic].

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
CSTB	NM_000100.4	c.67-1G>C		splice_acceptor_variant		ENST00000291568.7

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
CSTB	ENST00000291568.7	c.67-1G>C		splice_acceptor_variant		1	NM_000100.4	P1
CSTB	ENST00000640406.1	c.67-1G>C		splice_acceptor_variant		2
CSTB	ENST00000639959.1	c.36-430G>C		intron_variant		5
CSTB	ENST00000675996.1	n.492-1G>C		splice_acceptor_variant, non_coding_transcript_variant

Frequencies

GnomAD3 genomes AF: 0.000263 AC: 40 AN: 152218 Hom.: 0 Cov.: 33

Gnomad AFR AF: 0.0000724

Gnomad AMI AF: 0.00

Gnomad AMR AF: 0.000196

Gnomad ASJ AF: 0.00

Gnomad EAS AF: 0.00

Gnomad SAS AF: 0.00

Gnomad FIN AF: 0.00

Gnomad MID AF: 0.00

Gnomad NFE AF: 0.000485

Gnomad OTH AF: 0.000478

GnomAD3 exomes AF: 0.000151 AC: 38 AN: 251426 Hom.: 0 AF XY: 0.000155 AC XY: 21 AN XY: 135906

Gnomad AFR exome AF: 0.0000615

Gnomad AMR exome AF: 0.00

Gnomad ASJ exome AF: 0.00

Gnomad EAS exome AF: 0.00

Gnomad SAS exome AF: 0.00

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.000316

Gnomad OTH exome AF: 0.000163

GnomAD4 exome AF: 0.000302 AC: 441 AN: 1460106 Hom.: 0 Cov.: 29 AF XY: 0.000300 AC XY: 218 AN XY: 726556

Gnomad4 AFR exome AF: 0.0000299

Gnomad4 AMR exome AF: 0.0000447

Gnomad4 ASJ exome AF: 0.00

Gnomad4 EAS exome AF: 0.00

Gnomad4 SAS exome AF: 0.00

Gnomad4 FIN exome AF: 0.00

Gnomad4 NFE exome AF: 0.000374

Gnomad4 OTH exome AF: 0.000381

GnomAD4 genome AF: 0.000263 AC: 40 AN: 152336 Hom.: 0 Cov.: 33 AF XY: 0.000215 AC XY: 16 AN XY: 74488

Gnomad4 AFR AF: 0.0000722

Gnomad4 AMR AF: 0.000196

Gnomad4 ASJ AF: 0.00

Gnomad4 EAS AF: 0.00

Gnomad4 SAS AF: 0.00

Gnomad4 FIN AF: 0.00

Gnomad4 NFE AF: 0.000485

Gnomad4 OTH AF: 0.000473

Alfa AF: 0.000427 Hom.: 0

Bravo AF: 0.000280

TwinsUK AF: 0.000539 AC: 2

ALSPAC AF: 0.000519 AC: 2

ESP6500AA AF: 0.00 AC: 0

ESP6500EA AF: 0.000233 AC: 2

ExAC AF: 0.0000988 AC: 12

EpiCase AF: 0.000273

EpiControl AF: 0.000237

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Pathogenic:14 Uncertain:1 Other:2

Revision: criteria provided, conflicting classifications

Submissions by phenotype

Unverricht-Lundborg syndrome Pathogenic:6 Uncertain:1 Other:2

Uncertain significance, criteria provided, single submitter	clinical testing	New York Genome Center	Apr 29, 2021	- -
Pathogenic, no assertion criteria provided	literature only	OMIM	Mar 22, 1996	- -
Pathogenic, criteria provided, single submitter	clinical testing	Illumina Laboratory Services, Illumina	Aug 31, 2018	The CSTB c.67-1G>C variant occurs in a canonical splice site (acceptor) and is therefore predicted to disrupt or distort the normal gene product. Across a selection of the literature, the c.67-1G>C variant has been identified in a compound heterozygous state in at least seven individuals with Unverricht-Lundborg disease (ULD) from six families and in one additional family where zygosity was not specified (Pennacchio et al. 1996; Joensuu et al. 2007 and Canafoglia et al. 2012). The c.67-1G>C variant was most commonly identified in a compound heterozygous state with the expanded dodecamer repeat variant, which is the most common pathogenic variant associated with ULD. Bespalova et al. (1997) analyzed mRNA transcripts from heterozygous individuals carrying the c.67-1G>C variant. RT-PCR of total mRNA showed an aberrant, shorter transcript created as a result of skipping of exon 2, confirming that the c.67-1G>C variant disrupts normal splicing and results in expression of a truncated protein. The c.67-1G>C variant was absent from at least 95 unaffected control individuals and is reported at a frequency of 0.000347 in the European (non-Finnish) population of the Genome Aggregation Database. Based on the evidence, the c.67-1G>C variant is classified as pathogenic for Unverricht-Lundborg disease. This variant was observed by ICSL as part of a predisposition screen in an ostensibly healthy population. -
not provided, no classification provided	phenotyping only	GenomeConnect - Brain Gene Registry	-	Variant interpreted as Pathogenic and reported on 02-23-2021 by lab or GTR ID 239772. Assertions are reported exactly as they appear on the patient provided laboratory report. GenomeConnect does not attempt to reinterpret the variant. The IDDRC-CTSA National Brain Gene Registry (BGR) is a study funded by the U.S. National Center for Advancing Translational Sciences (NCATS) and includes 13 Intellectual and Developmental Disability Research Center (IDDRC) institutions. The study is led by Principal Investigator John Constantino MD PhD from Washington University. The BGR is a data commons of gene variants paired with subject clinical information. This database helps scientists learn more about genetic changes and their impact on the brain and behavior. Participation in the Brain Gene Registry requires participation in GenomeConnect. More information about the Brain Gene Registry can be found on the study website - https://braingeneregistry.wustl.edu/. -
Pathogenic, criteria provided, single submitter	clinical testing	Revvity Omics, Revvity	Jul 26, 2019	- -
Pathogenic, criteria provided, single submitter	clinical testing	Genetic Services Laboratory, University of Chicago	Dec 06, 2014	- -
Pathogenic, criteria provided, single submitter	clinical testing	Fulgent Genetics, Fulgent Genetics	Oct 31, 2018	- -
Pathogenic, criteria provided, single submitter	research	HudsonAlpha Institute for Biotechnology, HudsonAlpha Institute for Biotechnology	Oct 13, 2016	- -
not provided, no classification provided	literature only	GeneReviews	-	- -

not provided Pathogenic:4

Pathogenic, criteria provided, single submitter	clinical testing	GeneDx	Jan 10, 2022	Published studies in a patient who was compound heterozygous for this variant and an expansion variant demonstrated that the level of CSTB mRNA was diminished compared to controls, and no protein was detected on Western blot (Joensuu et al., 2007); Canonical splice site variant expected to result in aberrant splicing, although in the absence of functional evidence the actual effect of this sequence change is unknown.; This variant is associated with the following publications: (PMID: 18325013, 23205931, 9360639, 9054946, 25525159, 20301321, 9012407, 17003839, 29915382, 32581362, 8596935, 31589614) -
Pathogenic, criteria provided, single submitter	clinical testing	Institute for Clinical Genetics, University Hospital TU Dresden, University Hospital TU Dresden	Nov 03, 2021	- -
Pathogenic, criteria provided, single submitter	clinical testing	Athena Diagnostics	Apr 27, 2022	This variant is expected to severely impact normal RNA splicing, and consequently, protein structure and/or function. The frequency of this variant in the general population is consistent with pathogenicity (http://gnomad.broadinstitute.org). This variant has been identified in at least one individual with clinical features associated with this gene. In multiple individuals, this variant has been seen with a single recessive pathogenic variant in the same gene, suggesting this variant may also be pathogenic. -
Pathogenic, criteria provided, single submitter	clinical testing	Eurofins Ntd Llc (ga)	Sep 15, 2015	- -

Inborn genetic diseases Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Ambry Genetics

May 04, 2018

The c.67-1G>C intronic pathogenic mutation results from a G to C substitution one nucleotide upstream from coding exon 2 of the CSTB gene. This mutation was identified in trans with other pathogenic CSTB alterations in multiple unrelated individuals with progressive myoclonus epilepsy; it was shown to result in a misspliced mRNA product with an aberrant CSTB product lacking 33 amino acids, p.V23_K56del (Pennacchio LA et al. Science, 1996 Mar;271:1731-4; Joensuu T et al. Eur. J. Hum. Genet., 2007 Feb;15:185-93; Canafoglia L et al. Epilepsia, 2012 Dec;53:2120-7). Analysis of cells from affected patients demonstrated reduced mRNA and protein levels compared to wild type (Joensuu T et al. Eur. J. Hum. Genet., 2007 Feb;15:185-93; Canafoglia L et al. Epilepsia, 2012 Dec;53:2120-7). In addition to the clinical data presented in the literature, alterations that disrupt the canonical splice site are expected to cause aberrant splicing, resulting in an abnormal protein or a transcript that is subject to nonsense-mediated mRNA decay. As such, this alteration is classified as a disease-causing mutation. -

Chorea;C0013384:Dyskinesia Pathogenic:1

Pathogenic, no assertion criteria provided

research

NIHR Bioresource Rare Diseases, University of Cambridge

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Encephalopathy;C1854885:Cerebral dysmyelination;C4551563:Microcephaly Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Génétique des Maladies du Développement, Hospices Civils de Lyon

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Progressive myoclonic epilepsy Pathogenic:1

Pathogenic, criteria provided, single submitter

clinical testing

Invitae

Dec 17, 2023

This sequence change affects an acceptor splice site in intron 1 of the CSTB gene. RNA analysis indicates that disruption of this splice site induces altered splicing and likely results in a shortened protein product. This variant is present in population databases (rs147484110, gnomAD 0.03%). Disruption of this splice site has been observed in individuals with Unverricht-Lundborg disease (PMID: 8596935, 9012407, 9054946, 23205931). This variant is also known as 1924G>C. ClinVar contains an entry for this variant (Variation ID: 8395). Studies have shown that disruption of this splice site results in skipping of exon 2, but is expected to preserve the integrity of the reading-frame (PMID: 9360639, 17003839, 23205931). For these reasons, this variant has been classified as Pathogenic. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_addAF	Pathogenic	0.35	D
BayesDel_noAF	Uncertain	0.11
Cadd	Pathogenic	33
Dann	Uncertain	0.99
Eigen	Pathogenic	0.94
Eigen_PC	Pathogenic	0.75
FATHMM_MKL	Uncertain	0.86	D
MutationTaster	Benign	1.0	D
GERP RS		4.7
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.7

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.95
SpliceAI score (max)		1.0		Details are displayed if max score is > 0.2
DS_AG_spliceai		0.88		Position offset: -6
DS_AL_spliceai		1.0		Position offset: -1

Find out detailed SpliceAI scores and Pangolin per-transcript scores at spliceailookup.broadinstitute.org

Publications

LitVar

Other links and lift over

dbSNP: rs147484110; hg19: chr21-45194641;