rs138089183

Positions:

chr1-45332174-G-A

Variant summary

Our verdict is Likely benign. Variant got -2 ACMG points: 2P and 4B. PM2BP4_Strong

The ENST00000456914.7(MUTYH):c.841C>T(p.Arg281Cys) variant causes a missense change involving the alteration of a non-conserved nucleotide. The variant allele was found at a frequency of 0.000694 in 1,614,204 control chromosomes in the GnomAD database, including 1 homozygotes. In-silico tool predicts a benign outcome for this variant. 13/21 in silico tools predict a benign outcome for this variant. Variant has been reported in ClinVar as Conflicting classifications of pathogenicity (no stars). Another variant affecting the same amino acid position, but resulting in a different missense (i.e. R281H) has been classified as Likely benign.

Frequency

Genomes: 𝑓 0.00047 ( 0 hom., cov: 33)

Exomes 𝑓: 0.00072 ( 1 hom. )

Consequence

MUTYH
ENST00000456914.7 missense

Scores

Clinical Significance

Conflicting classifications of pathogenicity criteria provided, conflicting classifications U:16B:5O:1

Conservation

PhyloP100: -0.212

Variant links:

Genes affected

MUTYH (HGNC:7527): (mutY DNA glycosylase) This gene encodes a DNA glycosylase involved in oxidative DNA damage repair. The enzyme excises adenine bases from the DNA backbone at sites where adenine is inappropriately paired with guanine, cytosine, or 8-oxo-7,8-dihydroguanine, a major oxidatively damaged DNA lesion. The protein is localized to the nucleus and mitochondria. This gene product is thought to play a role in signaling apoptosis by the introduction of single-strand breaks following oxidative damage. Mutations in this gene result in heritable predisposition to colorectal cancer, termed MUTYH-associated polyposis (MAP). Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Apr 2017]

MUTYH links:

Genome browser will be placed here

ACMG classification

Classification made for transcript

Verdict is Likely_benign. Variant got -2 ACMG points.

PM2

Very rare variant in population databases, with high coverage;

BP4

Computational evidence support a benign effect (MetaRNN=0.059933275).

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	Protein	UniProt
MUTYH	NM_001128425.2 linkuse as main transcript	c.925C>T	p.Arg309Cys	missense_variant	10/16	ENST00000710952.2	NP_001121897.1
MUTYH	NM_001048174.2 linkuse as main transcript	c.841C>T	p.Arg281Cys	missense_variant	10/16	ENST00000456914.7	NP_001041639.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Protein	Appris	UniProt
MUTYH	ENST00000710952.2 linkuse as main transcript	c.925C>T	p.Arg309Cys	missense_variant	10/16		NM_001128425.2	ENSP00000518552
MUTYH	ENST00000456914.7 linkuse as main transcript	c.841C>T	p.Arg281Cys	missense_variant	10/16	1	NM_001048174.2	ENSP00000407590	A1	Q9UIF7-6

Frequencies

GnomAD3 genomes

AF:

0.000466

AC:

AN:

152206

Hom.:

Cov.:

show subpopulations

Gnomad AFR

AF:

0.000145

Gnomad AMI

AF:

0.00

Gnomad AMR

AF:

0.000327

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:

0.000882

Gnomad OTH

AF:

0.00

GnomAD3 exomes

AF:

0.000454

AC:

114

AN:

251334

Hom.:

AF XY:

0.000442

AC XY:

AN XY:

135892

show subpopulations

Gnomad AFR exome

AF:

0.000123

Gnomad AMR exome

AF:

0.0000868

Gnomad ASJ exome

AF:

0.0000992

Gnomad EAS exome

AF:

0.00

Gnomad SAS exome

AF:

0.0000327

Gnomad FIN exome

AF:

0.0000462

Gnomad NFE exome

AF:

0.000889

Gnomad OTH exome

AF:

0.000815

GnomAD4 exome

AF:

0.000718

AC:

1050

AN:

1461880

Hom.:

Cov.:

AF XY:

0.000714

AC XY:

519

AN XY:

727244

show subpopulations

Gnomad4 AFR exome

AF:

0.0000896

Gnomad4 AMR exome

AF:

0.000201

Gnomad4 ASJ exome

AF:

0.0000383

Gnomad4 EAS exome

AF:

0.00

Gnomad4 SAS exome

AF:

0.0000348

Gnomad4 FIN exome

AF:

0.000169

Gnomad4 NFE exome

AF:

0.000883

Gnomad4 OTH exome

AF:

0.000679

GnomAD4 genome

AF:

0.000466

AC:

AN:

152324

Hom.:

Cov.:

AF XY:

0.000470

AC XY:

AN XY:

74476

show subpopulations

Gnomad4 AFR

AF:

0.000144

Gnomad4 AMR

AF:

0.000327

Gnomad4 ASJ

AF:

0.00

Gnomad4 EAS

AF:

0.00

Gnomad4 SAS

AF:

0.00

Gnomad4 FIN

AF:

0.00

Gnomad4 NFE

AF:

0.000882

Gnomad4 OTH

AF:

0.00

Alfa

AF:

0.000632

Hom.:

Bravo

AF:

0.000431

TwinsUK

AF:

0.000270

AC:

ALSPAC

AF:

0.000259

AC:

ESP6500AA

AF:

0.000227

AC:

ESP6500EA

AF:

0.000698

AC:

ExAC

AF:

0.000436

AC:

Asia WGS

AF:

0.000289

AC:

AN:

3478

EpiCase

AF:

0.000709

EpiControl

AF:

0.00130

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Uncertain:16Benign:5Other:1

Revision: criteria provided, conflicting classifications

LINK: link

Submissions by phenotype

Familial adenomatous polyposis 2

Uncertain:5Benign:1Other:1

Uncertain significance, no assertion criteria provided	clinical testing	Department of Pathology and Laboratory Medicine, Sinai Health System	-	The MUTYH p.Arg309Cys variant was identified in 11 of 6552 proband chromosomes (frequency: 0.002) from individuals or families with MAP, colorectal cancer, or breast cancer and was present in 9 of 3332 control chromosomes (frequency: 0.003) from healthy individuals (Aretz 2006, Halford 2003, Jones 2009, Nielsen 2009, Olschwang 2007, Out 2012, Rohlin 2017, Sieber 2003, Vogt 2009). Note that the variant was identified as c.883C>T (NM_001048171.1), p.Arg295Cys (NP_001041636.1) and p.Arg281Cys (NP_001041638.1) in the literature (Goto 2010, Komine 2015, Brinkmeyer 2015). The variant was also identified in dbSNP (ID: rs138089183) as "With Uncertain significance allele", and in ClinVar (classified as likely benign by Ambry Genetics; as uncertain significance by GeneDx, Invitae, and three clinical laboratories). The variant was not identified in COGR, Cosmic, or the UMD-LSDB database. The variant was identified in control databases in 129 of 277082 chromosomes at a frequency of 0.0005 increasing the likelihood this could be a low frequency benign variant (Genome Aggregation Database Feb 27, 2017). The variant was observed in the following populations: African in 1 of 24006 chromosomes (freq: 0.00004), Other in 5 of 6466 chromosomes (freq: 0.0008), Latino in 4 of 34414 chromosomes (freq: 0.0001), European in 115 of 126606 chromosomes (freq: 0.0009), Finnish in 3 of 25792 chromosomes (freq: 0.0001), and South Asian in 1 of 30780 chromosomes (freq: 0.00003); it was not observed in the Ashkenazi Jewish, or East Asian populations. The p.Arg309 residue is not conserved in mammals and computational analyses (PolyPhen-2, SIFT, AlignGVGD, BLOSUM, MutationTaster) provide inconsistent predictions regarding the impact to the protein; this information is not very predictive of pathogenicity. The variant occurs outside of the splicing consensus sequence and 1 of 4 in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer) predict a greater than 10% difference in splicing; this is not very predictive of pathogenicity. The variant exhibited low levels of active enzyme (5%) compared to WT MUTYH and displayed a significant decrease in substrate binding affinity relative to the WT enzyme (Brinkmeyer 2015). In addition, in an in vitro glycosylase assay two studies displayed the variant having normal glycosylase activity (Goto 2010, Komine 2015) but a third showed that glycosylase activity was less efficient (Brinkmeyer 2015). In summary, based on the above information the clinical significance of this variant cannot be determined with certainty at this time. This variant is classified as a variant of uncertain significance. -
not provided, no classification provided	phenotyping only	GenomeConnect - Invitae Patient Insights Network	-	Variant interpreted as Uncertain significance and reported on 05-19-2020 by Invitae. GenomeConnect-Invitae Patient Insights Network assertions are reported exactly as they appear on the patient-provided report from the testing laboratory. Registry team members make no attempt to reinterpret the clinical significance of the variant. Phenotypic details are available under supporting information. -
Uncertain significance, criteria provided, single submitter	clinical testing	Labcorp Genetics (formerly Invitae), Labcorp	Nov 02, 2022	This sequence change replaces arginine, which is basic and polar, with cysteine, which is neutral and slightly polar, at codon 309 of the MUTYH protein (p.Arg309Cys). This variant is present in population databases (rs138089183, gnomAD 0.09%), and has an allele count higher than expected for a pathogenic variant. This missense change has been observed in individual(s) with denomatous polyps and colorectal cancer, and breast cancer (PMID: 12606733, 12707038, 16408224, 16557584, 17949294, 19032956, 19394335, 19732775, 22297469; Invitae). In at least one individual the data is consistent with being in trans (on the opposite chromosome) from a pathogenic variant. This variant is also known as c.883C>T (Arg295Cys) and c.841C>T (Arg281Cys). ClinVar contains an entry for this variant (Variation ID: 41765). Algorithms developed to predict the effect of missense changes on protein structure and function (SIFT, PolyPhen-2, Align-GVGD) all suggest that this variant is likely to be tolerated. Experimental studies have shown that this missense change does not substantially affect MUTYH function (PMID: 20848659, 26377631). In summary, the available evidence is currently insufficient to determine the role of this variant in disease. Therefore, it has been classified as a Variant of Uncertain Significance. -
Uncertain significance, criteria provided, single submitter	clinical testing	Illumina Laboratory Services, Illumina	Mar 08, 2018	This variant was observed as part of a predisposition screen in an ostensibly healthy population. A literature search was performed for the gene, cDNA change, and amino acid change (where applicable). Publications were found based on this search. However, the evidence from the literature, in combination with allele frequency data from public databases where available, was not sufficient to rule this variant in or out of causing disease. Therefore, this variant is classified as a variant of unknown significance. -
Uncertain significance, criteria provided, single submitter	clinical testing	St. Jude Molecular Pathology, St. Jude Children's Research Hospital	Aug 10, 2021	The MUTYH c.925C>T (p.Arg309Cys) missense change has a maximum subpopulation frequency of 0.091% in gnomAD v2.1.1 (https://gnomad.broadinstitute.org/variant/1-45797846-G-A). Both in silico tools and functional assays are not in agreement about the effect of this variant on protein function. Functional studies have demonstrated a significant decrease in enzyme activity, impaired binding affinity to damaged DNA, and reduced rates of base excision repair (PMID: 26377631), as well as glycosylase activity similar to the wildtype (PMID: 20848659). A complementation assay evaluating functional deficiency in E.coli showed partially defective activity based on spontaneous mutation rates (PMID: 25820570). This variant has been reported in one individual with 10-20 colorectal polyps at age 58 and without colorectal cancer who was compound heterozygous for this variant and the pathogenic p.Tyr179Cys (PM3; PMID: 19732775). It has also been reported in individuals with MUTYH-associated polyposis or referred for cancer genetic testing (PMID: 19032956, 27153395, 31159747) as well as in control individuals (PMID: 22703879). This variant is also known as c.883C>T (Arg295Cys) and c.841C>T (Arg281Cys) in the literature. In summary, this variant meets criteria to be classified as of uncertain significance based on the ACMG/AMP criteria: PM3. -
Likely benign, criteria provided, single submitter	clinical testing	All of Us Research Program, National Institutes of Health	Sep 26, 2024	- -
Uncertain significance, criteria provided, single submitter	clinical testing	MGZ Medical Genetics Center	May 25, 2022	- -

not provided

Uncertain:5Benign:1

Uncertain significance, criteria provided, single submitter	clinical testing	Eurofins Ntd Llc (ga)	Feb 27, 2018	- -
Uncertain significance, criteria provided, single submitter	clinical testing	GeneDx	Jun 27, 2024	Observed with MUTYH European founder variants, phase unknown, in individuals with phenotypes consistent with MAP at this laboratory and in the published literature as well as in individuals without MAP at an outside laboratory (PMID: 19394335; ClinVar); Identified as homozygous in two individuals with colorectal cancer whose tumors demonstrated a mutational signature profile which was apparently not characteristic of biallelic MUTYH inactivation (PMID: 35668106); In silico analysis indicates that this missense variant does not alter protein structure/function; Also known as Arg295Cys (c.883C>T), Arg281Cys (c.841C>T), and Arg306Cys (c.916C>T); This variant is associated with the following publications: (PMID: 23507534, 24252905, 16408224, 22297469, 16557584, 20848659, 25820570, 25980754, 21235684, 27829682, 31159747, 25318351, 22703879, 12606733, 12707038, 17949294, 19032956, 19732775, 26377631, 19394335, 27696107, 23605219, 27153395, 28087410, 28108460, 19527492, 15465463, 29684080, 32615015, 34426522, 33471991, 28135145, 28726808, 35545820, 35668106, 35534704, 34326862, Bayraktar2023[article], 20816984, 16879101, 11160897, 11092888, 37937776) -
Uncertain significance, criteria provided, single submitter	clinical testing	Institute for Clinical Genetics, University Hospital TU Dresden, University Hospital TU Dresden	Nov 03, 2021	- -
Uncertain significance, no assertion criteria provided	research	Biesecker Lab/Clinical Genomics Section, National Institutes of Health	Jul 13, 2012	- -
Likely benign, criteria provided, single submitter	clinical testing	CeGaT Center for Human Genetics Tuebingen	Jul 01, 2024	MUTYH: BP2, BS3:Supporting -
Uncertain significance, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Aug 11, 2023	In the published literature, the variant has been reported in individuals affected with breast cancer (PMID: 33471991 (2021), 31159747 (2019)), pancreatic cancer (PMID: 28726808 (2018)), colorectal cancer (PMID: 28135145 (2017), 16408224 (2006), 12707038 (2003) 12606733 (2003)), MUTYH-associated polyposis (MAP) (PMID: 19732775 (2009)), and familial adenomatous polyposis (FAP) (PMID: 17949294 (2007)). Additionally, this variant has been observed in unaffected individuals (PMID: 33471991 (2021), 22703879 (2012)). Functional studies have reported conflicting accounts that this variant has no effect or a partial defect on MUTYH glycosylase activity and function (PMID: 20848659 (2010), 26377631 (2015), and 25820570 (2015)). The frequency of this variant in the general population, 0.001 (52/50772 chromosomes, http://gnomad.broadinstitute.org), is uninformative in assessment of its pathogenicity. Analysis of this variant using bioinformatics tools for the prediction of the effect of amino acid changes on protein structure and function yielded conflicting predictions that this variant is deleterious or benign. Based on the available information, we are unable to determine the clinical significance of this variant. -

Hereditary cancer-predisposing syndrome

Uncertain:2Benign:2

Uncertain significance, criteria provided, single submitter	clinical testing	GeneKor MSA	Aug 01, 2018	- -
Likely benign, criteria provided, single submitter	clinical testing	Ambry Genetics	Aug 28, 2018	This alteration is classified as likely benign based on a combination of the following: seen in unaffected individuals, population frequency, intact protein function, lack of segregation with disease, co-occurrence, RNA analysis, in silico models, amino acid conservation, lack of disease association in case-control studies, and/or the mechanism of disease or impacted region is inconsistent with a known cause of pathogenicity. -
Benign, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Jun 22, 2016	- -
Uncertain significance, criteria provided, single submitter	curation	Sema4, Sema4	Feb 15, 2022	- -

not specified

Uncertain:3

Uncertain significance, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	Aug 01, 2024	Variant summary: MUTYH c.925C>T (p.Arg309Cys) results in a non-conservative amino acid change in the encoded protein sequence. Four of five in-silico tools predict a benign effect of the variant on protein function. The variant allele was found at a frequency of 0.0007 in 1617344 control chromosomes in the gnomAD database, including 1 homozygote. This frequency is not significantly higher than estimated for a pathogenic variant in MUTYH causing MUTYH-Associated Polyposis (0.0007 vs 0.0046), allowing no conclusion about variant significance.c.925C>T has been reported in the literature among individuals affected with APC-negative adenomatous polyposis, colorectal cancer, gastric cancer, with personal/family history of breast cancer, or with other tumor phenotypes without strong evidence for causality (e.g. Bhai_2021, deOliveira_2022, Bedics_2022, Aretz_2006, Niessen_2006, Halford_2003, Calvello_2023, Martin_2024). Thus, these report(s) do not provide unequivocal conclusions about association of the variant with MUTYH-associated Polyposis. Several publications have reported conflicting experimental evidence evaluating an impact on protein function. Multiple studies showed discordant results for adenine glycosylase activity with values similar to the wild-type enzyme (Goto_2010) while another report demonstrated low fractions of active enzyme, compromised affinity for damaged DNA and reduced rates for adeninine excision (Brinkmeyer_2015). The variant did not affect binding of MUTYH with the Hus1 subunit of the 9-1-1 heterotrimeric complex, Rad9-Hus1-Rad1, which is thought to stimulate the glycosylase activity of the protein (Brinkmeyer_2015). Lastly, a complementation assay evaluating the functional deficiency in the E Coli by monitoring spontaneous mutation rates showed a partially defective activity (Komine_2015). The following publications have been ascertained in the context of this evaluation (PMID: 15465463, 34326862, 32615015, 31159747, 27829682, 28726808, 28135145, 27153395, 27696107, 12606733, 12707038, 35534704, 35545820, 19732775, 19032956, 17949294, 26377631, 16557584, 16408224, 25820570, 22297469, 22703879, 20848659, 37239438, 38566764). ClinVar contains an entry for this variant (Variation ID: 41765). Based on the evidence outlined above, the variant was classified as uncertain significance -
Uncertain significance, criteria provided, single submitter	clinical testing	Genetic Services Laboratory, University of Chicago	Nov 05, 2021	DNA sequence analysis of the MUTYH gene demonstrated a sequence change, c.925C>T, in exon 10 that results in an amino acid change, p.Arg309Cys. This sequence change has been described in the gnomAD database with a frequency of 0.09% in the European population (dbSNP rs138089183). The p.Arg309Cys change affects a poorly conserved amino acid residue located in a domain of the MUTYH protein that is known to be functional. In-silico pathogenicity prediction tools (SIFT, PolyPhen2, Align GVGD, REVEL) provide contradictory results for the p.Arg309Cys substitution. The c.925C>T sequence change has been identified in individuals with colorectal polyps and/or colorectal cancer (PMIDs: 19732775; 19527492, 27829682). Out et al., 2012, identified the p.Arg309Cys change in individuals with a personal and/or family history of breast cancer and in controls, without a statistically significant difference in the rates of detection between the two populations (PMID: 22297469). In vitro analyses of the p.Arg309Cys change have shown contradictory results. Goto et al., 2010, demonstrated that the variant retains normal glycosylase activity in vitro (PMID: 20848659). However, Brinkmeyer et al., 2015, and Komine et al., 2015, performed in vitro assays that demonstrated significant decreases in enzyme activity, binding activity to damaged DNA, and defective base excision repair activity (PMIDs: 26377631, 25820570). Due to these contrasting evidences, the clinical significance of the p.Arg309Cys change remains unknown at this time. -
Uncertain significance, criteria provided, single submitter	clinical testing	Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital	Jul 31, 2024	- -

MUTYH-related disorder

Uncertain:1

Uncertain significance, no assertion criteria provided

clinical testing

PreventionGenetics, part of Exact Sciences

Aug 19, 2024

The MUTYH c.925C>T variant is predicted to result in the amino acid substitution p.Arg309Cys. This variant has been identified in individuals with adenomatous polyposis and/or colorectal cancer; in some individuals a second MUTYH variant was also reported or an additional missense variant in PALB2 was identified (Supplementary Table 1, Jones et al. 2009. PubMed ID: 19394335; Supplementary Table 1, Vogt et al. 2009. PubMed ID: 19732775; Nielsen et al. 2009. PubMed ID: 19032956; Sieber et al. 2003. PubMed ID: 12606733; Table 2, Aretz et al. 2006. PubMed ID: 16557584; Subject 1095188515, Table S2, Yurgelun et al. 2015. PubMed ID: 25980754). At least one study identified this variant in individuals with a history of breast cancer, but also at an equal frequency in the unaffected control cohort (Out et al. 2012. PubMed ID: 22297469). Functional studies are conflicting regarding this variant’s impact on MUTYH function. Some studies have revealed this variant does not impact MUTYH glycosylase function in vitro (Table 1, Goto et al. 2010. PubMed ID: 20848659), while others have reported a decrease in enzyme activity, binding activity to damaged DNA, and impaired base excision repair activity (Komine et al. 2015. PubMed ID: 25820570; Brinkmeyer and David. 2015. PubMed ID: 26377631). This variant has conflicting interpretations regarding its pathogenicity in ClinVar, ranging from benign to uncertain significance (https://www.ncbi.nlm.nih.gov/clinvar/variation/41765/). This variant is reported in 0.091% of alleles in individuals of European (Non-Finnish) descent in gnomAD. At this time, the clinical significance of this variant is uncertain due to the absence of conclusive functional and genetic evidence. -

Hereditary cancer

Benign:1

Likely benign, criteria provided, single submitter

clinical testing

Mendelics

Jan 23, 2024

- -

Computational scores

Source: dbNSFP v4.3

Name

Calibrated prediction

Score

Prediction

AlphaMissense

Benign

0.10

BayesDel_addAF

Benign

-0.094

BayesDel_noAF

Uncertain

0.030

CADD

Benign

DANN

Benign

0.78

DEOGEN2

Benign

0.11

.;.;.;.;.;T;.;.;.;T;.

Eigen

Benign

-0.76

Eigen_PC

Benign

-0.84

FATHMM_MKL

Benign

0.031

LIST_S2

Benign

0.84

.;T;.;T;T;T;T;T;T;T;T

M_CAP

Benign

0.062

MetaRNN

Benign

0.060

T;T;T;T;T;T;T;T;T;T;T

MetaSVM

Uncertain

-0.042

MutationAssessor

Uncertain

2.1

.;.;.;.;.;M;.;.;.;.;.

MutationTaster

Benign

1.0

N;N;N;N;N;N;N;N;N;N;N;N;N;N

PrimateAI

Benign

0.20

PROVEAN

Benign

-1.4

N;N;N;N;N;N;N;N;N;.;N

REVEL

Uncertain

0.59

Sift

Benign

0.12

T;T;T;T;T;T;T;T;T;.;T

Sift4G

Benign

0.11

T;T;T;T;T;T;T;T;T;T;T

Polyphen

0.054, 0.020, 0.012

.;.;.;.;.;B;B;.;B;.;.

Vest4

0.20

MVP

0.82

MPC

0.16

ClinPred

0.017

GERP RS

-2.1

Varity_R

0.13

gMVP

0.67

Splicing

Name

Calibrated prediction

Score

Prediction

SpliceAI score (max)

0.010

Details are displayed if max score is > 0.2

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

Publications

LitVar

Below is the list of publications found by LitVar. It may be empty.

Variant summary

Frequency

Consequence

Scores

Clinical Significance

Conservation

ACMG classification

Transcripts

RefSeq

Ensembl

Frequencies

ClinVar

Submissions by phenotype

Computational scores

Splicing

Publications

LitVar

Other links and lift over