NM_005359.6:c.424+5G>A

Variant names:

• chr18-51048865-G-A
• rs200772603
• NM_005359.6:c.424+5G>A

Variant summary

Our verdict is Benign. The variant received -8 ACMG points: 1P and 9B. PP3 BP6 BS1 BS2

The NM_005359.6(SMAD4):​c.424+5G>A variant causes a splice region, intron change. The variant allele was found at a frequency of 0.000185 in 1,608,638 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts a benign outcome for this variant. 2/3 splice prediction tools predicting alterations to normal splicing. Variant has been reported in ClinVar as Conflicting classifications of pathogenicity (no stars).

• Frequency:
  • Genomes: 𝑓 0.00013 (0 hom., cov: 33)
  • Exomes 𝑓: 0.00019 (0 hom.)
• Consequence: SMAD4 NM_005359.6 splice_region, intron
• Scores: Splicing: ADA: 0.9973
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications U:7 B:17
• Conservation: PhyloP100: 5.70
• Publications: 6 publications found

Genes affected

SMAD4 (HGNC:6770): (SMAD family member 4) This gene encodes a member of the Smad family of signal transduction proteins. Smad proteins are phosphorylated and activated by transmembrane serine-threonine receptor kinases in response to transforming growth factor (TGF)-beta signaling. The product of this gene forms homomeric complexes and heteromeric complexes with other activated Smad proteins, which then accumulate in the nucleus and regulate the transcription of target genes. This protein binds to DNA and recognizes an 8-bp palindromic sequence (GTCTAGAC) called the Smad-binding element (SBE). The protein acts as a tumor suppressor and inhibits epithelial cell proliferation. It may also have an inhibitory effect on tumors by reducing angiogenesis and increasing blood vessel hyperpermeability. The encoded protein is a crucial component of the bone morphogenetic protein signaling pathway. The Smad proteins are subject to complex regulation by post-translational modifications. Mutations or deletions in this gene have been shown to result in pancreatic cancer, juvenile polyposis syndrome, and hereditary hemorrhagic telangiectasia syndrome. [provided by RefSeq, May 2022]

SMAD4 Gene-Disease associations (from GenCC):

• juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome

  Inheritance: AD Classification: DEFINITIVE, STRONG Submitted by: Labcorp Genetics (formerly Invitae), ClinGen, Genomics England PanelApp, G2P, PanelApp Australia
• Myhre syndrome

  Inheritance: AD Classification: DEFINITIVE, STRONG, SUPPORTIVE Submitted by: ClinGen, Orphanet, G2P, Labcorp Genetics (formerly Invitae)
• generalized juvenile polyposis/juvenile polyposis coli

  Inheritance: AD Classification: STRONG, SUPPORTIVE Submitted by: Orphanet, Genomics England PanelApp
• juvenile polyposis syndrome

  Inheritance: AD Classification: STRONG Submitted by: Labcorp Genetics (formerly Invitae)
• familial thoracic aortic aneurysm and aortic dissection

  Inheritance: AD Classification: SUPPORTIVE Submitted by: Orphanet
• hereditary hemorrhagic telangiectasia

  Inheritance: AD Classification: SUPPORTIVE Submitted by: Orphanet
• pulmonary arterial hypertension

  Inheritance: AD Classification: NO_KNOWN Submitted by: ClinGen

ENSG00000267699 (HGNC:):

ACMG classification

Our verdict: Benign. The variant received -8 ACMG points.

• PP3: Splicing predictors support a deleterious effect. Scorers claiming Pathogenic: dbscSNV1_ADA, dbscSNV1_RF. No scorers claiming Uncertain. Scorers claiming Benign: max_spliceai.
• BP6: Variant 18-51048865-G-A is Benign according to our data. Variant chr18-51048865-G-A is described in ClinVar as Conflicting_classifications_of_pathogenicity. ClinVar VariationId is 127950.
• BS1: Variant frequency is greater than expected in population sas. GnomAd4 allele frequency = 0.000125 (19/151952) while in subpopulation SAS AF = 0.000621 (3/4834). AF 95% confidence interval is 0.000169. There are 0 homozygotes in GnomAd4. There are 10 alleles in the male GnomAd4 subpopulation. Median coverage is 33. This position passed quality control check.
• BS2: High AC in GnomAd4 at 19 AD gene.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	MANE	Protein	UniProt
SMAD4	NM_005359.6	c.424+5G>A		splice_region_variant, intron_variant	Intron 3 of 11	ENST00000342988.8	NP_005350.1

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	Exon rank	TSL	MANE	Protein	Appris	UniProt
SMAD4	ENST00000342988.8	c.424+5G>A		splice_region_variant, intron_variant	Intron 3 of 11	5	NM_005359.6	ENSP00000341551.3
ENSG00000267699	ENST00000590722.2	n.*447+5G>A		splice_region_variant, intron_variant	Intron 4 of 8	2		ENSP00000465737.1

Frequencies

GnomAD3 genomes AF: 0.000125 AC: 19 AN: 151834 Hom.: 0 Cov.: 33

Gnomad AFR AF: 0.00

Gnomad AMI AF: 0.00

Gnomad AMR AF: 0.0000659

Gnomad ASJ AF: 0.00

Gnomad EAS AF: 0.00

Gnomad SAS AF: 0.000620

Gnomad FIN AF: 0.00

Gnomad MID AF: 0.00

Gnomad NFE AF: 0.000221

Gnomad OTH AF: 0.00

GnomAD2 exomes AF: 0.000219 AC: 55 AN: 250792 AF XY: 0.000229

Gnomad AFR exome AF: 0.000123

Gnomad AMR exome AF: 0.000145

Gnomad ASJ exome AF: 0.00

Gnomad EAS exome AF: 0.00

Gnomad FIN exome AF: 0.00

Gnomad NFE exome AF: 0.000379

Gnomad OTH exome AF: 0.000164

GnomAD4 exome AF: 0.000191 AC: 278 AN: 1456686 Hom.: 0 Cov.: 31 AF XY: 0.000212 AC XY: 154 AN XY: 724816

African (AFR) AF: 0.00 AC: 0 AN: 33410

American (AMR) AF: 0.000112 AC: 5 AN: 44474

Ashkenazi Jewish (ASJ) AF: 0.0000384 AC: 1 AN: 26022

East Asian (EAS) AF: 0.00 AC: 0 AN: 39680

South Asian (SAS) AF: 0.000267 AC: 23 AN: 86186

European-Finnish (FIN) AF: 0.00 AC: 0 AN: 52996

Middle Eastern (MID) AF: 0.000558 AC: 3 AN: 5376

European-Non Finnish (NFE) AF: 0.000213 AC: 236 AN: 1108424

Other (OTH) AF: 0.000166 AC: 10 AN: 60118

GnomAD4 genome AF: 0.000125 AC: 19 AN: 151952 Hom.: 0 Cov.: 33 AF XY: 0.000135 AC XY: 10 AN XY: 74308

African (AFR) AF: 0.00 AC: 0 AN: 41554

American (AMR) AF: 0.0000658 AC: 1 AN: 15200

Ashkenazi Jewish (ASJ) AF: 0.00 AC: 0 AN: 3456

East Asian (EAS) AF: 0.00 AC: 0 AN: 5186

South Asian (SAS) AF: 0.000621 AC: 3 AN: 4834

European-Finnish (FIN) AF: 0.00 AC: 0 AN: 10528

Middle Eastern (MID) AF: 0.00 AC: 0 AN: 294

European-Non Finnish (NFE) AF: 0.000221 AC: 15 AN: 67878

Other (OTH) AF: 0.00 AC: 0 AN: 2110

Alfa AF: 0.000258 Hom.: 0

Bravo AF: 0.000132

EpiCase AF: 0.000273

EpiControl AF: 0.000415

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Uncertain:7 Benign:17

Revision: criteria provided, conflicting classifications

Submissions by phenotype

not provided Uncertain:2 Benign:4

-

Clinical Genetics DNA and cytogenetics Diagnostics Lab, Erasmus MC, Erasmus Medical Center

Significance: Likely benign

Review Status: no assertion criteria provided

Collection Method: clinical testing

- -

Jan 31, 2021

ARUP Laboratories, Molecular Genetics and Genomics, ARUP Laboratories

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The SMAD4 c.424+5G>A variant (rs200772603) is reported in the literature in an individual with suspected Lynch syndrome (Yurgelun 2015) and another individual referred for hereditary cancer testing (Tsaousis 2019). This variant is found in the non-Finnish European population with an overall allele frequency of 0.04% (47/128762 alleles) in the Genome Aggregation Database. This is an intronic variant in a highly conserved nucleotide, and computational analyses (Alamut v.2.11) predict that this variant may impact splicing by weakening the nearby canonical donor splice site. However, given the lack of clinical and functional data, the significance of the c.424+5G>A variant is uncertain at this time. References: Tsaousis et al. Analysis of hereditary cancer syndromes by using a panel of genes: novel and multiple pathogenic mutations. BMC Cancer. 2019 Jun 3;19(1):535. Yurgelun MB et al. Identification of a Variety of Mutations in Cancer Predisposition Genes in Patients With Suspected Lynch Syndrome. Gastroenterology. 2015 Sep;149(3):604-13.e20. -

Apr 19, 2023

Quest Diagnostics Nichols Institute San Juan Capistrano

Significance: Benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

The frequency of this variant in the general population, 0.00037 (47/128762 chromosomes in European (Non-Finnish) subpopulation, http://gnomad.broadinstitute.org), is higher than would generally be expected for pathogenic variants in this gene. In the published literature, the variant has been reported in individuals with breast and/or ovarian cancer (PMIDs: 31159747 (2019), 30426508 (2018), and 25186627 (2015)), pancreatic cancer (PMID: 28726808 (2018)), gastric cancer (PMID: 32066632 (2021)), colon cancer (PMID: 25980754 (2015)), and hamartomatous polyposis syndrome (PMID: 2714957 (2016)). Analysis of this variant using software algorithms for the prediction of the effect of nucleotide changes on splicing yielded predictions that this variant may affect proper SMAD4 mRNA splicing . Based on the available information, we are unable to determine the clinical significance of this variant. -

-

Joint Genome Diagnostic Labs from Nijmegen and Maastricht, Radboudumc and MUMC+

Significance: Likely benign

Review Status: no assertion criteria provided

Collection Method: clinical testing

- -

Jan 01, 2025

CeGaT Center for Human Genetics Tuebingen

Significance: Likely benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

SMAD4: BP4, BS2 -

Mar 11, 2025

GeneDx

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

In silico analysis supports that this variant does not alter splicing; however, published functional studies demonstrate evidence of aberrant splicing (Shiraishi Y et al. Systematic identification of intron retention associated variants from massive publicly available transcriptome sequencing data. bioRxiv. October 7, 2021); Observed in individuals with Lynch syndrome-associated cancer, familial gastric cancer, pancreatic cancer, breast cancer, or polyps, but has not been published in individuals with connective tissue disorders to our knowledge (PMID: 25186627, 27146957, 28726808, 30426508, 32066632, 34326862, 35534704); This variant is associated with the following publications: (PMID: 32066632, 25980754, 28873162, 31159747, 27146957, Shiraishi2021, 25186627, 28726808, 30426508, 35534704, 34326862) -

Juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome Uncertain:2 Benign:2

Jun 17, 2024

Molecular Pathology, Peter Maccallum Cancer Centre

Significance: Likely benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Sep 25, 2023

Department of Pathology and Laboratory Medicine, Sinai Health System

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Jan 13, 2018

Illumina Laboratory Services, Illumina

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant was observed in the ICSL laboratory as part of a predisposition screen in an ostensibly healthy population. It had not been previously curated by ICSL or reported in the Human Gene Mutation Database (HGMD: prior to June 1st, 2018), and was therefore a candidate for classification through an automated scoring system. Utilizing variant allele frequency, disease prevalence and penetrance estimates, and inheritance mode, an automated score was calculated to assess if this variant is too frequent to cause the disease. Based on the score, this variant could not be ruled out of causing disease and therefore its association with disease required further investigation. A literature search was performed for the gene, cDNA change, and amino acid change (if applicable). No publications were found based on this search. This variant was therefore classified as a variant of unknown significance for this disease. -

Apr 10, 2023

Myriad Genetics, Inc.

Significance: Likely benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant is considered likely benign. This variant is intronic and is not expected to impact mRNA splicing. Homozygosity has been confirmed in one or more individuals. As homozygosity for pathogenic variants in this gene is generally assumed to result in embryonic lethality, this variant is unlikely to be pathogenic. -

not specified Uncertain:1 Benign:3

May 03, 2024

Women's Health and Genetics/Laboratory Corporation of America, LabCorp

Significance: Likely benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

Variant summary: SMAD4 c.424+5G>A alters a conserved nucleotide located close to a canonical splice site and therefore could affect mRNA splicing, leading to a significantly altered protein sequence. Several computational tools predict a significant impact on normal splicing: Two predict the variant weakens a 5' donor site. One predict the variant abolishes a 5 splicing donor site. One predict the variant no significant impact on splicing. However, these predictions have yet to be confirmed by functional studies. The variant allele was found at a frequency of 0.00022 in 250792 control chromosomes. The observed variant frequency is approximately 110 fold of the estimated maximal expected allele frequency for a pathogenic variant in SMAD4 causing Juvenile Polyposis Syndrome phenotype (2e-06), strongly suggesting that the variant is benign. c.424+5G>A has been reported in the literature in individuals affected with cancer including colorectal cancer, hereditary breast and ovarian cancer, pancreatic ductal adenocarcinoma and familial intestinal gastric cancer (e.g. Tung_2015, Yurgelun_2015, Mandelker_2017, Chaffee_2018, Schubert_2019, Tsaousis_2019, Carvalho_2021). These reports do not provide unequivocal conclusions about association of the variant with Juvenile Polyposis Syndrome. To our knowledge, no experimental evidence demonstrating an impact on protein function has been reported. The following publications have been ascertained in the context of this evaluation (PMID: 25980754, 25186627, 27146957, 28873162, 28726808, 30426508, 31159747, 32066632). ClinVar contains an entry for this variant (Variation ID: 127950). Based on the evidence outlined above, the variant was classified as likely benign. -

Feb 16, 2025

Laboratory of Genetics, Children's Clinical University Hospital Latvia

Significance: Benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Aug 31, 2021

Genetic Services Laboratory, University of Chicago

Significance: Likely benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Mar 04, 2025

Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Hereditary cancer-predisposing syndrome Uncertain:1 Benign:2

Aug 01, 2018

GeneKor MSA

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Nov 12, 2020

Sema4, Sema4

Significance: Likely benign

Review Status: criteria provided, single submitter

Collection Method: curation

- -

Nov 17, 2020

Color Diagnostics, LLC DBA Color Health

Significance: Likely benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Juvenile polyposis syndrome Benign:2

Jan 28, 2025

Labcorp Genetics (formerly Invitae), Labcorp

Significance: Likely benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

May 23, 2018

Counsyl

Significance: Likely benign

Review Status: no assertion criteria provided

Collection Method: clinical testing

This submission and the accompanying classification are no longer maintained by the submitter. For more information on current observations and classification, please contact variantquestions@myriad.com. -

Familial thoracic aortic aneurysm and aortic dissection Uncertain:1

Nov 25, 2016

CHEO Genetics Diagnostic Laboratory, Children's Hospital of Eastern Ontario

Significance: Uncertain significance

Review Status: criteria provided, single submitter

Collection Method: clinical testing

- -

Myhre syndrome Benign:1

Jan 13, 2018

Illumina Laboratory Services, Illumina

Significance: Benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant was observed in the ICSL laboratory as part of a predisposition screen in an ostensibly healthy population. It had not been previously curated by ICSL or reported in the Human Gene Mutation Database (HGMD: prior to June 1st, 2018), and was therefore a candidate for classification through an automated scoring system. Utilizing variant allele frequency, disease prevalence and penetrance estimates, and inheritance mode, an automated score was calculated to assess if this variant is too frequent to cause the disease. Based on the score and internal cut-off values, a variant classified as benign is not then subjected to further curation. The score for this variant resulted in a classification of benign for this disease. -

SMAD4-related disorder Benign:1

Jun 10, 2024

PreventionGenetics, part of Exact Sciences

Significance: Likely benign

Review Status: no assertion criteria provided

Collection Method: clinical testing

This variant is classified as likely benign based on ACMG/AMP sequence variant interpretation guidelines (Richards et al. 2015 PMID: 25741868, with internal and published modifications). -

Hereditary cancer-predisposing syndrome;C4707243:Familial thoracic aortic aneurysm and aortic dissection Benign:1

Jan 02, 2025

Ambry Genetics

Significance: Likely benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

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. -

Generalized juvenile polyposis/juvenile polyposis coli Benign:1

Jan 13, 2018

Illumina Laboratory Services, Illumina

Significance: Likely benign

Review Status: criteria provided, single submitter

Collection Method: clinical testing

This variant was observed in the ICSL laboratory as part of a predisposition screen in an ostensibly healthy population. It had not been previously curated by ICSL or reported in the Human Gene Mutation Database (HGMD: prior to June 1st, 2018), and was therefore a candidate for classification through an automated scoring system. Utilizing variant allele frequency, disease prevalence and penetrance estimates, and inheritance mode, an automated score was calculated to assess if this variant is too frequent to cause the disease. Based on the score and internal cut-off values, a variant classified as likely benign is not then subjected to further curation. The score for this variant resulted in a classification of likely benign for this disease. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
BayesDel_noAF	Benign	-0.29
CADD	Uncertain	24
DANN	Benign	0.96
PhyloP100		5.7
RBP_binding_hub_radar		0.0
RBP_regulation_power_radar		1.1
Mutation Taster		=15/85	disease causing

Splicing

Name	Calibrated prediction	Score	Prediction
dbscSNV1_ADA	Pathogenic	1.0
dbscSNV1_RF	Pathogenic	0.93
SpliceAI score (max)		0.020		Details are displayed if max score is > 0.2

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

Other links and lift over

dbSNP: rs200772603; hg19: chr18-48575235; COSMIC: COSV105241308;