ClinVar Genomic variation as it relates to human health

This sequence change replaces Serine with Phenylalanine at codon 428 of the CHEK2 protein. The serine residue is moderately conserved in the protein kinase domain of the CHEK2 protein and there is a large physiochemical difference between serine and phenylalanine (Grantham Score 155).This variant is present in population databases at a very low frequency (rs137853011, ExAC 0.06%) and is listed in the mutation database ClinVar (Variation ID:5603). This variant has been described in the literature in patients with breast cancer and prostate cancer (PMID: 31159747,18571837) and has been shown in individuals of Ashkenazy Jewish descent to increase the risk of breast cancer approximately 2-fold (PMID:15649950 ). No similar studies have been carried out in other populations. Furthermore, experimental studies in yeast have shown that this variant disrupts CHEK2 protein function (PMID: 15649950, 22419737).

The p.Ser428Phe variant in CHEK2 has been reported in 21 individuals with CHEK2-related cancers including breast, ovarian and testicular germline cell cancer and segregated with disease in 12 affected individuals from seven families (Shaag 2005 PMID: 15649950, Laitman 2007 PMID: 18085035, Roeb 2012 PMID: 22419737, Desmond 2015 PMID: 26270727, Susswein 2015 PMID: 26681312, Cox 2018 PMID: 30152102 and AlDubayan 2019 PMID: 30676620). It has also been identified in 1.1% of Ashkenazi Jewish chromosomes by gnomAD (http://gnomad.broadinstitute.org) as is thought to be a founder mutation in this population (Shaag 2005 PMID: 15649950). Also, please note that for diseases with clinical variability or reduced penetrance, pathogenic variants may be present at a low frequency in the general population. This variant has also been reported in ClinVar (Variation ID 5603). Computational prediction tools and conservation analyses suggest that this variant may impact the protein, though this information is not predictive enough to determine pathogenicity. In vivo functional studies in yeast support an impact on protein function (Shaag 2005 PMID: 15649950, Roeb 2012 PMID: 22419737). Furthermore, loss of heterozygosity was observed in breast tumors from two individuals heterozygous for this variant, suggesting it may be involved in tumor growth (Shaag 2005 PMID: 15649950). In summary, this variant meets criteria to be classified as pathogenic for autosomal dominant hereditary CHEK2-related cancers. ACMG/AMP Criteria applied: PS4, PP1_Strong, PS3_Moderate, PP3.

Variant summary: CHEK2 c.1283C>T (p.Ser428Phe) results in a non-conservative amino acid change located in the Protein kinase domain (IPR000719) of the encoded protein sequence. Three of five in-silico tools predict a damaging effect of the variant on protein function. The variant allele was found at a frequency of 0.00059 in 272936 control chromosomes, predominantly within the Ashkenazi Jewish subpopulation at a frequency of 0.011 in the gnomAD database, including 1 homozygote. The observed variant frequency within Ashkenazi Jewish control individuals in the gnomAD database is approximately 35 fold of the estimated maximal expected allele frequency for a pathogenic variant in CHEK2 causing Hereditary Breast and Ovarian Cancer phenotype (0.00031), suggesting that the variant is a benign polymorphism found primarily in populations of Ashkenazi Jewish origin. In addition, the variant was reported in 10/7325 European American women (i.e. with a frequency of 0.00136), who were older than 70 years of age, and never had cancer (FLOSSIES database). However, the variant is located in a region that is highly homologous to CHEK2 pseudogenes and the technology utilized for these datasets does not rule out pseudogene interference, therefore these data might not be reliable. c.1283C>T has been reported in the literature in multiple individuals affected with Hereditary Breast and Ovarian Cancer and other tumor phenotypes who were primarily of Ashkenazi Jewish origin (e.g. Shaag_2005, Laitman_2007, Tischkowitz_2008, Walsh_2017). These data indicate that the variant is very likely to be associated with disease. One case-control study estimated that this variant confers a moderate risk (odds ratio: 2.13) for developing breast cancer in Ashkenazi Jewish individuals (Shaag_2005). Although two research groups indicated that this variant failed to segregate with the disease in one breast cancer and two prostate cancer families, this variant has been identified in affected individuals (predominantly of AJ ancestry) presenting with HBOC and was reported to segregate with the disease in three families, where at least three affected individuals per family carried the variant (Tischkowitz 2008 and Roeb 2012). Co-occurrences with other pathogenic variants were also noted in the literature, (e.g. BRCA1 185delAG, BRCA2 6174delT and CHEK2 c.1100delC, reported by Walsh 2017). However, the ages at diagnosis of some of these patients were among the youngest, thus likely indicating an additive effect rather than an evidence against pathogenicity (Walsh_2017). In functional studies by multiple independent research groups this variant showed a reduced ability to complement the lethality of a Rad53 deletion in S. cerevisiae (Shaag_2005, Tischkowitz_2008, Roeb_2012, Yilmaz_2014). Twenty-four assessments submitted to ClinVar after 2014 cite the variant as uncertain significance (n=1), likely pathogenic (n=5) and pathogenic (n=18) . Based on the evidence outlined above, the variant was classified as pathogenic.

BP4

The c.1283C>T (p.Ser428Phe) variant is well described in the literature, and is thought to be a founder variant in the Ashkenazi Jewish population (Shaag et al. 2005). This variant is a low penetrance allele, resulting in a two-fold increase in breast cancer risk for women over 50. The p.Ser428Phe variant was first identified in a study by Shaag et al. (2005) in which it was detected in a heterozygous state in 47 out of 1632 probands, and in 23 of the 1673 controls. The association with breast cancer was statistically significant (OR = 2.13, p=0.004). The variant was found in two additional studies in at least 12 probands with breast cancer, ten of whom also carried an intronic variant that is thought to be benign (Walsh et al. 2006; Laitman et al. 2007). Segregation with disease was demonstrated within three families. Roeb et al. (2012) identified the variant in six individuals in one family, where two carriers had breast cancer and one had esophageal cancer. Two of the other unaffected carriers were younger than 50. The variant is reported at a frequency of 0.00055 in the European (Non-Finnish) population of the Exome Aggregation Consortium. The p.Ser428Phe variant occurs at a position that is conserved in mammals and is located within the kinase domain (Roeb et al. 2012). Functional studies in yeast based on a lack of complementation of RAD53 demonstrated that the variant abrogates CHEK2 function (Shaag et al. 2005; Tischkowitz et al. 2008; Roeb et al. 2012). Based on the evidence, the p.Ser428Phe variant is classified as pathogenic for breast cancer but is considered to be a low penetrance risk allele.

The c.1283C>T (p.Ser428Phe) variant in the CHEK2 gene has been reported in patients with breast cancer [PMID 15649950, 22419737]. The variant was detected at a frequency of about 3% among Ashkenazi-Jewish female patients with breast cancer as opposed to 1.4% in controls (odds ratio of 2.13) [PMID 15649950]. The study concluded that this specific variant increases the breast cancer risk by 2-fold among Ashkenazi Jewish women. A family was also reported with two breast cancer female siblings heterozygous for the variant while two additional family members, also heterozygous for the variant, were asymptomatic at 58 and 60 years old and one heterozygous female member had esophageal cancer [PMID 22419737]. This variant is located in the kinase domain and functional assays in yeast showed that the mutant allele disrupt the the normal function of CHEK2 [PMID 15649950]. This variant was reported in 37 heterozygous European and South Asian individuals in ExAC (http://exac.broadinstitute.org/variant/22-29091207-G-A). Serine at amino acid position 428 of the CHEK2 protein is conserved in mammals. While not validated for clinical use, computer-based algorithms (SIFT and Polyphen-2) predict this p.Ser428Phe change to be deleterious. It is thus classified as a pathogenic variant.

The CHEK2 c.1283C>T (p.Ser428Phe) missense variant is well described in the literature, and is thought to be a founder variant in the Ashkenazi Jewish population (Shaag et al. 2005). This variant is a low penetrance allele, expecting to result in a two-fold increase in breast cancer risk for women over 50. The p.Ser428Phe variant was first identified in a study by Shaag et al. (2005) in which it was detected in a heterozygous state in 47 out of 1632 female Ashkenazi Jewish probands with breast cancer (unselected for family history or age at diagnosis), and in 23 of the 1673 controls. The association with breast cancer was statistically significant (OR = 2.13, p=0.004). The variant was found in two additional studies in at least 12 female probands with breast cancer, ten of whom also carried an intronic variant that is thought to be benign (Walsh et al. 2006; Laitman et al. 2007). Segregation with disease was demonstrated within three families. Roeb et al. (2012) identified the variant in six individuals in one family, where two of these individuals had breast cancer and one had esophageal cancer. Two of the other unaffected carriers were younger than 50 years old. The variant is reported at a frequency of 0.00055 in the European (non-Finnish) population of the Exome Aggregation Consortium. The p.Ser428Phe variant occurs at a position that is conserved in mammals and is located within the kinase domain (Roeb et al. 2012). Functional studies in yeast based on a lack of complementation of RAD53 demonstrated that the variant abrogates CHEK2 function (Shaag et al. 2005; Tischkowitz et al. 2008; Roeb et al. 2012). Based on the evidence, the p.Ser428Phe variant is classified as pathogenic for CHEK2-related cancer susceptibility, but is considered to be a low penetrance risk allele. This variant was observed by ICSL as part of a predisposition screen in an ostensibly healthy population.

_x000D_ Criteria applied: PS3, PS4, PP1, PP3

S428F has a reduced penetrance compared to the pathogenic CHEK2 mutation, c.1100delC, and is estimated to confer approximately a two-fold increased risk of breast cancer in the Ashkenazi Jewish population.

Case control studies suggest this variant is associated with breast cancer, although some odds ratios are only modestly elevated (<1.5) (Shaag et al., 2005; Hu et al., 2021; Bychkovsky et al., 2022); Published functional studies demonstrate a damaging effect: loss of CHEK2-mediated DNA damage response and impaired protein function in yeast complementation assays (Shaag et al., 2005; Tischkowitz et al., 2008; Roeb et al., 2012); 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: 18571837, 25674498, 16998506, 27621404, 29086229, 29109859, 27433846, 36136322, 33471974, 24763289, 25629968, 27900359, 27153395, 27751358, 27798748, 18706089, 27296296, 28495237, 28281021, 28452373, 28135137, 28135139, 26270727, 28340968, 28008555, 28981386, 28526081, 28873162, 28727877, 28944238, 28135145, 26681312, 26556299, 29560538, 27806230, 30858171, 29520813, 30067863, 30152102, 29506128, 29961768, 29978187, 30322717, 30309722, 31159747, 30676620, 31360903, 34622392, 32805687, 31447099, 31948886, 28104920, 32885271, 31980526, 29625052, 34308366, 29922827, 35264596, 31794323, 32986223, 34654685, 28888541, 35174967, 32782288, 32923877, 34148862, 22419737, 15649950, 18085035, 33471991, 35626031, 34308104, 19782031)

PM1, PS3

This missense variant replaces serine with phenylalanine at codon 428 in the kinase domain of the CHEK2 protein. Computational prediction suggests that this variant may not impact protein structure and function (internally defined REVEL score threshold <= 0.5, PMID: 27666373). Functional studies have shown that the mutant protein is partially defective in DNA damage repair function (PMID: 15649950, 18571837, 22419737, DOI:10.4236/abb.2014.54046). This variant has been reported in individuals affected with breast cancer (PMID: 15649950, 16551709, 18085035, 22419737, 28727877, 31159747, 33471991, 34622392, 35264596). In a large case-control study of Ashkenazi Jewish women, this variant was observed at a higher frequency in breast cancer cases than in unaffected controls (OR=2.13, 95% CI 1.26 to 3.69, p-value=0.004; PMID: 15649950). In another case-control study conducted in an Ashkenazi Jewish population, this variant was reported in 162/6420 breast cancer cases and 47/2335 controls (OR=1.2536, 95%CI 0.9029 to 1.7406, p-value=0.176; PMID: 34622392). In a large international case-control study, this variant was reported in 11/60466 breast cancer cases and 14/53461 controls (OR=0.695, 95%CI 0.315 to 1.53, p-value=0.425; PMID: 33471991). This variant has been identified in 131/282204 chromosomes in the general population by the Genome Aggregation Database (gnomAD), almost exclusively in the Ashkenazi Jewish population (116/10362, 1.1%). The variant has also been observed in 10/7325 European American women older than 70 years of age that never had cancer (FLOSSIES; whi.color.com), indicating the variant is likely to be of reduced or low penetrance. The available evidence indicates that this variant is associated with disease and therefore is classified as Pathogenic. This variant appears to show reduced penetrance compared to typical pathogenic variants in the CHEK2 gene.

This sequence change replaces serine, which is neutral and polar, with phenylalanine, which is neutral and non-polar, at codon 428 of the CHEK2 protein (p.Ser428Phe). This variant is present in population databases (rs137853011, gnomAD 1.1%), and has an allele count higher than expected for a pathogenic variant. In a large case-control study of Ashkenazi Jewish women, this variant was found in 2.9% of individuals with breast cancer and 1.4% of individuals with no prior indication of cancer. This translates to ~2-fold increased risk (OR=2.13, 95% CI [1.26, 3.69], P=0.004) of breast cancer for c.1283C>T carriers in this population (PMID: 15649950). Outside of the Ashkenazi Jewish population this variant is very rare (<0.05%) and the cancer risks have not been well established. It has been reported in two sisters with breast cancer (ages 41 and 51), although their mother did not develop cancer before passing away at 83 (PMID: 22419737). Their aunt and two female cousins also carried this variant; the aunt had a history of esophageal cancer (at age 76) while the cousins (ages 58 and 60) did not have any prior indication of cancer (PMID: 22419737). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 5603). 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 in yeast have shown that the Ser428Phe substitution abrogates CHEK2 protein function to a similar extent as other partially damaging alleles (PMID: 15649950, 22419737). Furthermore, in two individuals with invasive breast cancer, this missense variant was found to be heterozygous in the germline and hemizygous in the tumors (PMID: 15649950). Altogether, these results suggest that the Ser428Phe change is deleterious and contributes to tumorigenesis. RNA analysis performed to evaluate the impact of this missense change on mRNA splicing indicates it does not significantly alter splicing (Invitae). In summary, this variant is reported to cause disease. However, as this variant is associated with a lower penetrance than other pathogenic alleles in the CHEK2 gene, it has been classified as Pathogenic (low penetrance).

The p.S428F variant (also known as c.1283C>T), located in coding exon 11 of the CHEK2 gene, results from a C to T substitution at nucleotide position 1283. The serine at codon 428 is replaced by phenylalanine, an amino acid with highly dissimilar properties. This alteration has been detected in multiple unrelated patients with personal and/or family histories of breast cancer (Desmond A et al. JAMA Oncol. 2015 Oct;1:943-51; Maxwell KN et al. Am. J. Hum. Genet. 2016 May;98:801-17; Moran O et al. Breast Cancer Res. Treat. 2017 Jan;161:135-142; Walsh T et al. JAMA Oncol. 2017 12;3:1647-1653), men with metastatic prostate cancer (Pritchard CC et al. N. Engl. J. Med. 2016 Aug;375:443-53), and patients with colorectal cancer (DeRycke MS et al. Mol Genet Genomic Med. 2017 Sep;5:553-569). Of note, this variant has been described as a founder mutation in the Ashkenazi Jewish population, and at least one study reported an approximately 2-fold increased risk of breast cancer among Ashkenazi Jewish women carrying this alteration (Shaag A et al. Hum. Mol. Genet. 2005 Feb;14:555-63). However, additional case-control studies have reported that the odds ratio of developing breast cancer for individuals carrying this variant are in the range of 1.08 to 1.26 (Dorling et al. N Engl J Med. 2021 02;384:428-439; Hu C et al. N Engl J Med. 2021 02;384(5):440-451; Bychkovsky BL et al. JAMA Oncol. 2022 Sep; Laitman Y et al. Fam Cancer. 2022 07;21(3):305-308). A yeast-based functional study has indicated that this alteration abolishes normal CHK2 activity (Roeb W et al. Hum. Mol. Genet. 2012 Jun;21:2738-44). This amino acid position is not well conserved in available vertebrate species. In addition, the in silico prediction for this alteration is inconclusive. Based on the majority of available evidence to date, this variant is interpreted as a moderate risk mutation, also referred to as an established risk allele.

CHEK2: PM1:Strong, PS4:Moderate, PS3:Supporting, BP4

DNA sequence analysis of the CHEK2 gene demonstrated a sequence change, c.1283C>T, in exon 12 that results in an amino acid change, p.Ser428Phe. This sequence change has previously been described in individuals and families with breast cancer (PMIDs: 15649950, 18085035, 22419737). One study reported the p.Ser428Phe variant in 47/1,632 Ashkenazi Jewish women with breast cancer and 23/1,673 unaffected controls, conferring a 2-fold increased risk for breast cancer in Ashkenazi Jewish women with this variant (PMID: 15649950). The p.Ser428Phe change affects a moderately conserved amino acid residue located in the kinase domain of the CHEK2 protein. Functional studies show p.Ser428Phe disrupts the function of the CHEK2 protein (PMIDs: 15649950, 22419737)

The CHEK2 c.1283C>T variant is predicted to result in the amino acid substitution p.Ser428Phe. This variant has previously been reported in control populations (<1.4%); however, this variant has been associated with an increased breast cancer risk (~2-fold) among Ashkenazi women. In addition, this variant was shown to abrogate CHEK2 function in yeast complementation assays (Shaaq et al. 2005. PubMed ID: 15649950; Roeb et al. 2012. PubMed ID: 22419737). To our knowledge, increased cancer risks with other types of cancers or in non-Ashkenazi individuals are not well established. This variant is reported as pathogenic or likely pathogenic by several genetic testing laboratories in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/variation/5603/). This variant is reported in 1.1% of alleles in individuals of Ashkenazi Jewish descent in gnomAD. We classify this variant as pathogenic.

The CHEK2 p.Ser428Phe variant was identified in 65 of 4516 proband chromosomes (frequency: 0.014) from Ashkenazi Jewish individuals or families with breast, ovarian, cancer and French Canadian patients with prostate cancer (Laitman 2007, Shaag 2005, Walsh 2006, Tischkowitz 2008). The variant was also identified in the following databases: dbSNP (ID: rs137853011) as “With Pathogenic allele”, ClinVar (7x, as pathogenic by GeneDx, Ambry Genetics, Invitae, University of Washington, Color Genomics, Illumina and 1x, as uncertain significance by Illumina), Clinvitae (6x, as pathogenic, 1x as uncertain significance), Cosmic (1x urinary tract carcinoma), Zhejiang Colon Cancer Database (3x). The variant was not identified in MutDB. The variant was identified in control databases in 127 of 276646 (1 homozygous) chromosomes at a frequency of 0.0005 in the following populations: other in 5 of 6452 chromosomes (freq. 0.0008), European in 9 of 126256 chromosomes (freq. 0.00007), Ashkenazi Jewish in 112 of 10144 (1 homozygous) chromosomes (freq. 0.001), and South Asian in 1 of 30732 chromosomes (freq. 0.00003) (Genome Aggregation Consortium Feb 27, 2017). Population studies have identified the p.Ser428Phe variant as a founder allele in the AJ population; one study concluded that the CHEK2, p.Ser428Phe variant increases breast cancer risk by two fold among Ashkenazi Jewish women (Shaag 2005) though it has been noted that the control group in this study included a large proportion (43.3%) of adults who underwent pre-marriage counseling in Jerusalem and were likely much younger than the cases (Mundt 2017). Functional studies revealed this variant to have loss of CHEK2-mediated DNA damage response (Roeb 2012) and to impair protein function in yeast complementation assays (Shaag 2005, Tischkowitz 2008); however, these studies are not considered sufficient evidence of pathogenicity because of the known variability of yeast assays for CHEK2 (Mundt 2017). Co-segregation with breast cancer has been reported in at least three Ashkenazi Jewish families with the CHEK2 p.Ser428Phe variant (Laitman 2007, Roeb 2012). Loss of heterozygosity was observed in breast tumors from two individuals heterozygous for CHEK2 p.Ser428Phe, suggesting this variant may be involved in tumor growth (Shaag 2005). The p.Ser428Phe residue is 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 in silico or computational prediction software programs (SpliceSiteFinder, MaxEntScan, NNSPLICE, GeneSplicer, HumanSpliceFinder) do not predict a difference in splicing. The variant is located within the kinase domain increasing the likelihood that it may have clinical significance. The challenge of interpreting these data has been highlighted by two papers (Mundt 2017, Balmana 2016). Conflicting interpretation of functional data as well as interpretation of the variant’s relatively high allele frequency in control databases are thought to reflect the challenges of describing a so-called low-penetrance susceptibility allele in a format designed for high-penetrance alleles and rather than being universally low-penetrance, variants in this category may be associated with an increased risk for a subset of individuals, especially for those with a personal or family history of early-onset breast cancer (Byrnes 2008). Given the high frequency of this variant in the Ashkenazi Jewish population and the apparent low penetrance with this variant, a classification of likely pathogenic is conservative. 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 likely pathogenic.

According to the ACMG SVI adaptation criteria we chose this criterion: PS4 (strong pathogenic): Risikoallel; funktionelle Analysen jetzt wiederspüchlich, Stolarova nicht damaging, ältere Analysen eher damaging => kein PS3, kein PM2, PS4-str: OR 2,13; in vielen Frauen >70 ohne

Variant reported in multiple Invitae PIN participants. Variant interpreted as Pathogenic and reported most recently on 1/20/2019 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.