9-21974682-A-G

Variant summary

Our verdict is Uncertain significance. Variant got 0 ACMG points: 5P and 5B. PM1 PM5 PP5 BP4 BS2

The NM_000077.5(CDKN2A):c.146T>C(p.Ile49Thr) variant causes a missense change. The variant allele was found at a frequency of 0.00011 in 1,613,956 control chromosomes in the GnomAD database, with no homozygous occurrence. In-silico tool predicts 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. I49M) has been classified as Uncertain significance.

• Frequency:
  • Genomes: 𝑓 0.000072 (0 hom., cov: 32)
  • Exomes 𝑓: 0.00011 (0 hom.)
• Consequence: CDKN2A NM_000077.5 missense
• Clinical Significance: Conflicting classifications of pathogenicity criteria provided, conflicting classifications P:3 U:11 O:1
• Conservation: PhyloP100: 5.56

Genes affected

CDKN2A (HGNC:1787): (cyclin dependent kinase inhibitor 2A) This gene generates several transcript variants which differ in their first exons. At least three alternatively spliced variants encoding distinct proteins have been reported, two of which encode structurally related isoforms known to function as inhibitors of CDK4 kinase. The remaining transcript includes an alternate first exon located 20 Kb upstream of the remainder of the gene; this transcript contains an alternate open reading frame (ARF) that specifies a protein which is structurally unrelated to the products of the other variants. This ARF product functions as a stabilizer of the tumor suppressor protein p53 as it can interact with, and sequester, the E3 ubiquitin-protein ligase MDM2, a protein responsible for the degradation of p53. In spite of the structural and functional differences, the CDK inhibitor isoforms and the ARF product encoded by this gene, through the regulatory roles of CDK4 and p53 in cell cycle G1 progression, share a common functionality in cell cycle G1 control. This gene is frequently mutated or deleted in a wide variety of tumors, and is known to be an important tumor suppressor gene. [provided by RefSeq, Sep 2012]

ACMG classification

Verdict is Uncertain_significance. Variant got 0 ACMG points.

• PM1: In a hotspot region, there are 7 aminoacids with missense pathogenic changes in the window of +-8 aminoacids around while only 1 benign, 17 uncertain in NM_000077.5
• PM5: Other missense variant is known to change same aminoacid residue: Variant chr9-21974682-A-C is described in ClinVar as [Likely_pathogenic]. Clinvar id is 430217.Status of the report is criteria_provided_multiple_submitters_no_conflicts, 2 stars.
• PP5: Variant 9-21974682-A-G is Pathogenic according to our data. Variant chr9-21974682-A-G is described in ClinVar as [Conflicting_classifications_of_pathogenicity]. Clinvar id is 127523.We mark this variant Likely_pathogenic, oryginal submissions are: {Uncertain_significance=10, Likely_pathogenic=3, not_provided=1}.
• BP4: Computational evidence support a benign effect (MetaRNN=0.063048065).. Strength limited to SUPPORTING due to the PP5.
• BS2: High AC in GnomAd at 11 AD gene.

Transcripts

RefSeq

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	MANE	UniProt
CDKN2A	NM_000077.5	c.146T>C	p.Ile49Thr	missense_variant	1/3	ENST00000304494.10
CDKN2A	NM_058195.4	c.194-3474T>C		intron_variant		ENST00000579755.2

Ensembl

Gene	Transcript	HGVSc	HGVSp	Effect	#exon/exons	TSL	MANE	Appris	UniProt
CDKN2A	ENST00000304494.10	c.146T>C	p.Ile49Thr	missense_variant	1/3	1	NM_000077.5	P2
CDKN2A	ENST00000579755.2	c.194-3474T>C		intron_variant		1	NM_058195.4

Frequencies

GnomAD3 genomes AF: 0.0000723 AC: 11 AN: 152096 Hom.: 0 Cov.: 32

Gnomad AFR AF: 0.0000241

Gnomad AMI AF: 0.00

Gnomad AMR AF: 0.000655

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: 0.00

Gnomad OTH AF: 0.00

GnomAD3 exomes AF: 0.000625 AC: 156 AN: 249454 Hom.: 0 AF XY: 0.000489 AC XY: 66 AN XY: 135104

Gnomad AFR exome AF: 0.00

Gnomad AMR exome AF: 0.00451

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: 0.00

Gnomad OTH exome AF: 0.00

GnomAD4 exome AF: 0.000114 AC: 167 AN: 1461744 Hom.: 0 Cov.: 32 AF XY: 0.000102 AC XY: 74 AN XY: 727178

Gnomad4 AFR exome AF: 0.00

Gnomad4 AMR exome AF: 0.00369

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: 0.00

Gnomad4 OTH exome AF: 0.0000331

GnomAD4 genome AF: 0.0000723 AC: 11 AN: 152212 Hom.: 0 Cov.: 32 AF XY: 0.000121 AC XY: 9 AN XY: 74428

Gnomad4 AFR AF: 0.0000241

Gnomad4 AMR AF: 0.000654

Gnomad4 ASJ AF: 0.00

Gnomad4 EAS AF: 0.00

Gnomad4 SAS AF: 0.00

Gnomad4 FIN AF: 0.00

Gnomad4 NFE AF: 0.00

Gnomad4 OTH AF: 0.00

Alfa AF: 0.0000844 Hom.: 0

Bravo AF: 0.000298

ExAC AF: 0.000436 AC: 53

ClinVar

Significance: Conflicting classifications of pathogenicity

Submissions summary: Pathogenic:3 Uncertain:11 Other:1

Revision: criteria provided, conflicting classifications

Submissions by phenotype

Hereditary cancer-predisposing syndrome Pathogenic:1 Uncertain:2

Likely pathogenic, criteria provided, single submitter	clinical testing	Ambry Genetics	Jul 28, 2023	The p.I49T variant (also known as c.146T>C), located in coding exon 1 of the CDKN2A gene, results from a T to C substitution at nucleotide position 146. The isoleucine at codon 49 is replaced by threonine, an amino acid with similar properties. This variant has been seen in multiple individuals with personal and/or family histories of pancreatic cancer (Ambry internal data). This variant has been reported in patients with no family history of melanoma but who had a single primary melanoma or multiple primary melanomas (Berwick M et al. Eur. J. Cancer Prev. 2004 Feb;13:65-70; Begg CB et al. J. Natl. Cancer Inst. 2005 Oct;97:1507-15). It has also been reported in multiple familial melanoma cases, however, in one case, it did not completely segregate with disease (Hussussian CJ et al. Nat. Genet. 1994 Sep;8:15-21; Puig S et al. Genet. Med. 2016 Jul;18:727-36). Multiple functional studies have demonstrated that this variant diminishes the binding activity of p16 to CDK4 and CDK6 and displays reduced ability to effect Rb dephosphorylation, however the degree of effect is shown to be more intermediate than other known pathogenic variants (Ranade K et al. Nat. Genet. 1995 May;10:114-6; Reymond A et al. Oncogene. 1995 Sep;11:1173-8; Yang R et al. Biochem. Biophys. Res. Commun. 1996 Jan;218:254-9; Walker GJ et al. Int. J. Cancer. 1999 Jul;82:305-12). Cell cycle inhibition assays have demonstrated that this variant is functionally deleterious (Miller, PJ et al. Hum Mutat 2011 Aug;32(8):900-11; Kimura H et al. Elife 2022 Jan;11:). Internal structural analysis predicts that this amino acid position is involved in CDK4 binding and while other pathogenic missense mutations are also found at this location, the substitution of threonine at this position induces a milder perturbation on the structure than those variants (Ambry internal data). Based on the majority of available evidence to date, this variant is likely to be pathogenic with moderate risk. -
Uncertain significance, criteria provided, single submitter	clinical testing	Color Diagnostics, LLC DBA Color Health	Dec 06, 2023	This variant replaces isoleucine with threonine at codon 49 of the CDKN2A (p16INK4A) protein. Computational prediction tool is inconclusive regarding the impact of this variant on protein structure and function (internally defined REVEL score threshold 0.5 < inconclusive < 0.7, PMID: 27666373). Functional studies have reported the mutant protein to be partially to fully functional in CDK4 and CDK6 binding in yeast and mammalian assays (PMID: 7566978, 7647780, 8573142, 10389768, 10719365, 21462282). Cell cycle and growth arrest assays have produced inconsistent results with the mutant protein showing normal activity (PMID: 33823155) or significantly reduced activity (PMID: 10389768, 21462282, 35001868). This variant has been reported in multiple individuals and families affected with melanoma (PMID: 7987387, 11687599, 15075790, 16234564, 16896043, 17218939, 18335566, 21085193, 21462282, 26681309). This variant has also been identified in 158/280824 chromosomes (157/35432 Latino chromosomes) by the Genome Aggregation Database (gnomAD). Although this variant has been observed in multiple individuals affected with melanoma, it occurs at a high allele frequency in the general population (0.44% in the Latino population). The available evidence is insufficient to determine the role of this variant in disease conclusively. Therefore, this variant is classified as a Variant of Uncertain Significance. -
Uncertain significance, criteria provided, single submitter	curation	Sema4, Sema4	Jan 24, 2022	- -

not specified Uncertain:3

Uncertain significance, criteria provided, single submitter	clinical testing	ARUP Laboratories, Molecular Genetics and Genomics, ARUP Laboratories	Dec 19, 2018	The CDKN2A c.146T>C; p.Ile49Thr variant (rs199907548) is reported in the literature in individuals affected with melanoma or breast cancer (Hussussian 1994, Kurian 2014, Orlow 2007, Puig 2016, Taylor 2017). However, in one family this variant did not segregate with disease (Hussussian 1994), while melanoma was not reported in nine first-degree relatives of another carrier (Orlow 2007). This variant is found in the Latino population with an overall allele frequency of 0.44% (157/35432 alleles) in the Genome Aggregation Database. The isoleucine at codon 49 is moderately conserved, computational analyses (SIFT, PolyPhen-2) predict that this variant is deleterious, and another variant at this codon (p.Ile49Ser) has been reported in melanoma patients and is considered pathogenic (Orlow 2007, Taylor 2017). Functional analyses indicate that the p.Ile49Thr variant fails to arrest growth of melanoma or osteosarcoma cells like wildtype protein (Miller 2011, Walker 1999), and it exhibits roughly half of wildtype activity in an in vitro assay for inhibition of cyclin D1-CDK signaling (Ranade 1995). However, due to conflicting information, the clinical significance of the p.Ile49Thr variant is uncertain at this time. References: Hussussian CJ et al. Germline p16 mutations in familial melanoma. Nat Genet. 1994 Sep;8(1):15-21. Kurian AW et al. Clinical evaluation of a multiple-gene sequencing panel for hereditary cancer risk assessment. J Clin Oncol. 2014 Jul 1;32(19):2001-9. Miller PJ et al. Classifying variants of CDKN2A using computational and laboratory studies. Hum Mutat. 2011 Aug;32(8):900-11. Orlow I et al. CDKN2A germline mutations in individuals with cutaneous malignant melanoma. J Invest Dermatol. 2007 May;127(5):1234-43. Puig S et al. Characterization of individuals at high risk of developing melanoma in Latin America: bases for genetic counseling in melanoma. Genet Med. 2016 Jul;18(7):727-36. Ranade K et al. Mutations associated with familial melanoma impair p16INK4 function. Nat Genet. 1995 May;10(1):114-6. Taylor NJ et al. Germline Variation at CDKN2A and Associations with Nevus Phenotypes among Members of Melanoma Families. J Invest Dermatol. 2017 Dec;137(12):2606-2612. Walker GJ et al. Functional reassessment of P16 variants using a transfection-based assay. Int J Cancer. 1999 Jul 19;82(2):305-12. -
Uncertain significance, no assertion criteria provided	clinical testing	Genetic Services Laboratory, University of Chicago	Nov 15, 2022	DNA sequence analysis of the CDKN2A gene demonstrated a sequence change, c.146T>C, in exon 1 that results in an amino acid change, p.Ile49Thr. This sequence change has been described in the gnomAD database with a frequency of 0.44% in the Latino subpopulation (dbSNP rs199907548). The p.Ile49Thr change affects a highly conserved amino acid residue located in a domain of the CDKN2A protein that is known to be functional. In-silico pathogenicity prediction tools (SIFT, PolyPhen2, Align GVGD, REVEL) provide contradictory results for the p.Ile49Thr substitution. This sequence change has been reported in individuals with melanoma (PMID: 7987287, 17218939, 26681309). Experimental studies provide conflicting evidence on the effect of this sequence change on protein function (PMID: 8573142, 10389768, 21462282). Due to insufficient evidences, the clinical significance of the p.Ile49Thr change remains unknown at this time. -
Uncertain significance, criteria provided, single submitter	clinical testing	Women's Health and Genetics/Laboratory Corporation of America, LabCorp	Oct 02, 2023	Variant summary: CDKN2A c.146T>C (p.Ile49Thr) results in a non-conservative amino acid change located in the Ankyrin repeat-containing domain (IPR020683) of the encoded protein sequence. Five of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 0.00063 in 249454 control chromosomes (gnomAD), predominantly at a frequency of 0.0045 within the Latino subpopulation in the gnomAD database. The observed variant frequency within Latino control individuals in the gnomAD database is approximately 15 fold of the estimated maximal expected allele frequency for a pathogenic variant in CDKN2A causing Cutaneous Malignant Melanoma (0.0003), strongly suggesting that the variant is a benign polymorphism found primarily in populations of Latino origin. c.146T>C has been reported in the literature in individuals affected with Melanoma (e.g. Hussussian_1994, Begg_2005, Capanu_2008, Miller_2011, Puig_2016). Analysis of a multi-generation family did not show complete co-segregation of this variant and the disease (Hussussian_1994). In addition, this variant was also found in patients with HBOC, CRC, LS (e.g. Yurgelun_2015, Ricker_2017, Slavin_2018, Oliver_2019). These reports do not provide unequivocal conclusions about association of the variant with Cutaneous Malignant Melanoma. At-least one co-occurrence with another pathogenic variant has been observed at our laboratory (TP53 c.818G>A, p.Arg273His), providing supporting evidence for a benign role. Multiple experimental studies showed conflicting results including: decreased inhibition of cyclinD1/CDK4 and cyclin D1/CDK6 activity (Ranade_1995), decreased binding activity to CDK4 (Yang_1996, Reymond_1995), no effect on binding to the kinases but substantially diminished ability to inhibit cell growth and mis-localization (Walker_1999), loss of function in a cell cycle arrest assay (Miller_2011) and most recently, cell cycle suppression that was similar to WT (Horn_2021). The following publications have been ascertained in the context of this evaluation (PMID: 16234564, 27756164, 18335566, 27960642, 28765326, 33823155, 7987387, 9166859, 21462282, 16818274, 18519632, 31921681, 26681309, 7647780, 7566978, 28640387, 7718873, 30339520, 10389768, 8573142, 25980754). Twelve ClinVar submitters have assessed the variant since 2014: nine classified the variant as uncertain significance and three as likely pathogenic. Based on the evidence outlined above, the variant was classified as uncertain significance. -

not provided Uncertain:2

Uncertain significance, criteria provided, single submitter	clinical testing	GeneDx	May 08, 2023	Variant has been reported in individuals with sporadic and familial melanoma, but the variant did not completely segregate with disease in at least one family (Hussussian et al., 1994; Berwick et al., 2004; Orlow et al., 2007; Kurian et al., 2014; McDaniel et al., 2015; Tung et al., 2015; Puig et al., 2016; Goldstein et al., 2018); Published functional studies are inconclusive regarding effect on binding to CDK4 and ability to inhibit cell growth (Ranade et al., 1995; Reymond et al., 1995; Walker et al., 1999; Lal et al., 2000; Miller et al., 2011); In silico analysis supports that this missense variant has a deleterious effect on protein structure/function; This variant is associated with the following publications: (PMID: 11687599, 15075790, 7647780, 10389768, 21680795, 11518711, 9823374, 24733792, 26670561, 26667234, 28454591, 10719365, 8573142, 28231819, 21085193, 29415044, 30207590, 25980754, 16896043, 26681309, 16234564, 17218939, 25186627, 7566978, 21462282, 28830827, 31921681, 32191290, 35001868, 7987387, 35366121) -
Uncertain significance, criteria provided, single submitter	clinical testing	Quest Diagnostics Nichols Institute San Juan Capistrano	Apr 17, 2023	In the published literature, the variant has been reported in individuals with melanoma (PMIDs: 26681309 (2016), 17218939 (2007), 16234564 (2005), 15075790 (2004), 7987387 (1994)), hereditary breast and/or ovarian cancer (PMIDs: 31921681 (2019), 30339520 (2018), 24733792 (2014)), and unspecified cancers (PMID: 30339520 (2018)). Some functional studies suggest this variant may impair CDKN2A binding and cell cycle arrest function (PMIDs: 7566978 (1995), 10389768 (1999), 10719365 (2000), and 21462282 (2011)), but other studies conflict with this data (PMID: 33823155 (2020)). The frequency of this variant in the general population, 0.0044 (157/35432 chromosomes in Latino/Admixed American subpopulation, http://gnomad.broadinstitute.org), is higher than would generally be expected for pathogenic variants in this gene. Analysis of this variant using bioinformatics tools for the prediction of the effect of amino acid changes on protein structure and function yielded predictions that this variant is damaging. Based on the available information, we are unable to determine the clinical significance of this variant. -

Melanoma and neural system tumor syndrome Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Baylor Genetics

Jan 07, 2020

This variant was determined to be likely pathogenic according to ACMG Guidelines, 2015 [PMID:25741868]. This variant has been previously reported in multiple patients with melanoma [PMID: 7987387, 17218939, 26681309, 28830827] Functional assays also showed impaired protein function caused by this variant [PMID: 7647780, 10389768, 21462282] -

Familial melanoma Pathogenic:1

Likely pathogenic, criteria provided, single submitter

clinical testing

Invitae

Jan 30, 2024

This sequence change replaces isoleucine, which is neutral and non-polar, with threonine, which is neutral and polar, at codon 49 of the CDKN2A (p16INK4a) protein (p.Ile49Thr). This variant is present in population databases (rs199907548, gnomAD 0.4%), and has an allele count higher than expected for a pathogenic variant. This missense change has been observed in individual(s) with melanoma and/or pancreatic cancer (PMID: 7987387, 16896043, 21462282, 26681309; Invitae). It has also been observed to segregate with disease in related individuals. This variant is also known as c.140T>C (p.Ile41Thr). ClinVar contains an entry for this variant (Variation ID: 127523). An algorithm developed to predict the effect of missense changes on protein structure and function (PolyPhen-2) suggests that this variant is likely to be disruptive. Experimental studies are conflicting or provide insufficient evidence to determine the effect of this variant on CDKN2A (p16INK4a) function (PMID: 7566978, 7647780, 8573142, 10389768, 10719365, 21462282). In summary, the currently available evidence indicates that the variant is pathogenic, but additional data are needed to prove that conclusively. Therefore, this variant has been classified as Likely Pathogenic. -

Acute lymphoid leukemia Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Genomic Diagnostics Laboratory, National Institute of Medical Genomics

Sep 23, 2023

- -

CDKN2A-related disorder Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

PreventionGenetics, part of Exact Sciences

Feb 18, 2024

The CDKN2A c.146T>C variant is predicted to result in the amino acid substitution p.Ile49Thr. This variant has been reported in a family with melanoma but did not segregate with disease in the family (Hussussian et al. 1994. PubMed ID: 7987387; Table S3, Taylor et al. 2017. PubMed ID: 28830827). This variant was also reported in an individual with lung cancer (Kurian et al. 2014. PubMed ID: 24733792). Functional studies have suggested that this variant may impact enzyme function (Ranade et al. 1995. PubMed ID: 7647780; Walker et al. 1999. PubMed ID: 10389768). We have also observed this variant in a patient with cancer who has a pathogenic BRCA1 variant. Of note, the CDKN2A variant has been reported in approximately 0.44% of individuals of Latino background in a large population database and has conflicting interpretations of pathogenicity in ClinVar of uncertain and likely pathogenic (https://www.ncbi.nlm.nih.gov/clinvar/variation/127523/). At this time, the clinical significance of this variant is uncertain due to the absence of conclusive functional and genetic evidence. -

Osteoblastic osteosarcoma Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

St. Jude Molecular Pathology, St. Jude Children's Research Hospital

Apr 03, 2017

- -

Melanoma-pancreatic cancer syndrome Uncertain:1

Uncertain significance, criteria provided, single submitter

clinical testing

Counsyl

Jul 12, 2016

- -

Melanoma and neural system tumor syndrome;C1838547:Melanoma-pancreatic cancer syndrome Other:1

not provided, no classification provided

phenotyping only

GenomeConnect - Invitae Patient Insights Network

-

Variant interpreted as Uncertain significance and reported on 05-13-2020 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. -

Computational scores

Source: dbNSFP v4.3

Name	Calibrated prediction	Score	Prediction
AlphaMissense	Pathogenic	0.96
BayesDel_addAF	Benign	-0.11	T
BayesDel_noAF	Uncertain	0.070
Cadd	Uncertain	25
Dann	Uncertain	0.99
DEOGEN2	Uncertain	0.47	T;T;.
Eigen	Uncertain	0.51
Eigen_PC	Uncertain	0.46
FATHMM_MKL	Uncertain	0.97	D
LIST_S2	Uncertain	0.93	D;D;D
M_CAP	Pathogenic	0.58	D
MetaRNN	Benign	0.063	T;T;T
MetaSVM	Uncertain	0.0011	D
MutationTaster	Benign	0.92	D;D;D;D;D;D;D;D;D
PrimateAI	Pathogenic	0.80	T
PROVEAN	Uncertain	-3.8	D;.;D
REVEL	Uncertain	0.60
Sift	Uncertain	0.0010	D;.;D
Sift4G	Uncertain	0.0030	D;D;D
Polyphen		1.0	D;.;.
Vest4		0.70
MVP		0.99
MPC		1.4
ClinPred		0.25	T
GERP RS		4.9
Varity_R		0.49
gMVP		0.98

Splicing

Name	Calibrated prediction	Score	Prediction
SpliceAI score (max)		0.0		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

Other links and lift over

dbSNP: rs199907548; hg19: chr9-21974681; COSMIC: COSV58687879; COSMIC: COSV58687879;