ClinVar Genomic variation as it relates to human health

This missense variant replaces lysine with asparagine at codon 666 of the RET protein. Computational prediction 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). A functional study has reported that the variant protein, p.Lys666Asn, resulted in elevated phosphorylation of the ERK protein in transiently-transfected at a level that is intermediate between the wild-type protein and the pathogenic control p.Cys634Arg (PMID: 20103606). The protein variant p.Lys666Asn, caused by c.1998G>T or c.1998G>C, has been reported in more than 10 heterozygous individuals affected with medullary thyroid cancer and/or pheochromocytoma (PMID: 20103606, 22865907, 26269449, 27673361, 28946813, 29408964) and a homozygous carrier affected with bilateral medullary thyroid cancer and pheochromocytoma (PMID: 29408964). The protein change was also reported in an individual with Cowden syndrome clinical features (macrocephaly, lipoma, renal cancer, thyroid cancer, goiter and Hashimoto's thyroiditis) and pheochromocytoma (PMID: 29684080), and it was observed to segregate with medullary thyroid cancer, C-cell hyperplasia, elevated calcitonin and/or pheochromocytoma (PMID: 27673361, 29408964). This variant has been identified in 7/282120 chromosomes in the general population by the Genome Aggregation Database (gnomAD). Based on the available evidence, this variant is classified as Likely Pathogenic.

This variant is considered likely pathogenic. Functional studies indicate this variant impacts protein function [PMID: 20103606]. This variant has been reported in multiple individuals with clinical features of gene-specific disease [PMID: 27673361, 29408964, 20103606].

Variant summary: RET c.1998G>C (p.Lys666Asn) results in a non-conservative amino acid change in the encoded protein sequence. Four of five in-silico tools predict a damaging effect of the variant on protein function. The variant was absent in 250810 control chromosomes (gnomAD). c.1998G>C has been reported in the literature in multiple individuals affected with Multiple Endocrine Neoplasia Type 2 (e.g. Boichard_2012, Curras_2015, Jaber_2018, Rosen_2022). These data indicate that the variant is very likely to be associated with disease. A different variant resulting in the same amino acid consequence has been classified as pathogenic by our lab (c.1998G>T), supporting the pathogenicity of this variant. The following publications have been ascertained in the context of this evaluation (PMID: 26269449, 29408964, 22865907, 35304457). ClinVar contains an entry for this variant (Variation ID: 230926). Based on the evidence outlined above, the variant was classified as pathogenic.

The inherited c.1998G>C,p.Lys666Asn missense variant identified in RET has been reported to segregate with medullary thyroid carcinoma, C-cell dysplasia, and elevated calcitonin levels in several families (PMID:27673361, 20103606). This variant has also been reported in individuals with pheochromocytoma (PMID:26269449) and breast cancer (PMID:26687385). This variant is present in population database (gnomAD v2.1) with a frequency of 0.002%. Experimental studies have shown that this missense variant results in increased RET phosphorylation activity and increased transformation potential in cell culture (PMID:20103606). Rare missense variants at the same codon (p.Lys666Glu, p.Lys666Met, p.Lys666Arg) have been reported in the individuals with medullary thyroid carcinoma and multiple endocrine neoplasia type II and suggests that the lysine residue is critical for RET protein function (PMID:15858153, 21690267, 21678021, 20516206, 25319874). Based on the available evidence, the inherited missense variant c.1998G>C,p.Lys666Asn in the RET gene is classified as Pathogenic.

ACMG classification criteria: PS1 strong, PS4 moderated, PM1 moderated, PM2 moderated

The p.K666N mutation (also known as c.1998G>C) is located in coding exon 11 of the RET gene. This alteration results from a G to C substitution at nucleotide position 1998. The lysine at codon 666 is replaced by asparagine, an amino acid with similar properties. This specific alteration was identified in one patient from a screen of 329 patients with sporadic paraganglioma or pheochromocytoma (Curr&aacute;s-Freixes M et al. J. Med. Genet., 2015 Oct;52:647-56). A similar alteration causing the same amino acid change (c.1998G>T p.K666N) was identified in eight unrelated index cases with medullary thyroid carcinoma (MTC) (Xu JY et al. Thyroid, 2016 Oct, epub). Analysis of the families from these 8 cases showed several additional family members who were carriers of the variant that also had either MTC or C-cell hyperplasia. Three carriers were shown to have normal pathology at ages 21, 30 and 30 years of age. Xu et al. conclude that this alteration is low penetrance MTC allele, with no evidence for association with other MEN2A pathogenic features of pheochromocytoma and parathyroid abnormalities. This same c.1998G>T p.K666N alteration was reported in a case of sporadic medullary thyroid cancer in a 65 year old female (Muzza M et al. Eur J Endocrinol. 2010 Apr;162(4):771-7). Further analysis by Muzza et al. demonstrated increased oncogenic potential as compared to wild type, as well as significant structural impact that was predicted to alter the transmembrane &alpha;-helix, likely changing the secondary structure of the protein. In addition, several other alterations at this same position (p.K666E, p.K666R, and p.K666M) have been identified in sporadic cases of MTC (Yamazaki M et al. Endocr. J. 2014 Nov;61(11):1141-4; Borrello MG et al. Endocr. Relat. Cancer, 2011 Aug;18:519-27), or in large pedigrees demonstrating segregation with MTC or C-cell hyperplasia (Ahmed SA et al. J Mol Diagn. 2005 May;7(2):283-8; Mastroianno S et al. Endocrine, 2011 Dec;40:481-5). Of note, The American Thyroid Association has designated p.K666E as a mutation with moderate risk for MTC,10% incidence of pheochromocytomas and no incidence of hyperparathyroidism (Wells SA et al. Thyroid 2015 Jun;25(6):567-610). As observed in the literature and Ambry internal data, p.K666N, is primarily associated with MTC and not other features of MEN2A. Based on the available evidence, this alteration is classified as a pathogenic mutation with moderate risk.

This sequence change replaces lysine, which is basic and polar, with asparagine, which is neutral and polar, at codon 666 of the RET protein (p.Lys666Asn). This variant is not present in population databases (gnomAD no frequency). This missense change has been observed in individual(s) with medullary thyroid carcinoma, C-cell dysplasia, and elevated calcitonin levels and pheochromocytoma (PMID: 20103606, 26269449, 26687385, 27673361). It has also been observed to segregate with disease in related individuals. ClinVar contains an entry for this variant (Variation ID: 230926). 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 have shown that this missense change affects RET function (PMID: 20103606). This variant disrupts the p.Lys666 amino acid residue in RET. Other variant(s) that disrupt this residue have been determined to be pathogenic (PMID: 15858153, 21690267). This suggests that this residue is clinically significant, and that variants that disrupt this residue are likely to be disease-causing. For these reasons, this variant has been classified as Pathogenic.

The RET c.1998G>C variant is predicted to result in the amino acid substitution p.Lys666Asn. This variant has been reported in the heterozygous state in several individuals with a personal and family history of medullary thyroid cancer (MTC), and an in vitro assay showed it displayed high kinase and transforming activity (Muzza et al. 2010. PubMed ID: 20103606; Xu et al. 2016. PubMed ID: 27673361). This variant was also reported in a cohort of individuals with phaeochromocytoma (Curras-Freixes et al. 2015. PubMed ID: 26269449, supplementary data) and in cohorts of individuals with breast cancer (Bernstein-Molho et al. 2019. PubMed ID: 30980208; Yablonski-Peretz et al. 2016. PubMed ID: 26687385). Lastly, this variant was reported in the homozygous state in an individual with medullary thyroid cancer and bilateral pheochromocytoma; the patient's son, who carried this variant in the heterozygous state, also had MTC (Jaber et al. 2018. PubMed ID: 29408964). However, not all carriers of this variant were affected at the time of testing, suggesting potential incomplete penetrance or variable age at onset. Other amino acid substitutions at this position have been reported in individuals with MTC or pheochromocytoma, suggesting that this amino acid residue may be critical for function (Human Gene Mutation Database; https://www.hgmd.cf.ac.uk/). This variant has not been reported in a large population database, indicating this variant is rare. This variant is interpreted as pathogenic in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/variation/230926/?new_evidence=true). This variant is interpreted as likely pathogenic.

Variant interpreted as Pathogenic and reported on 11-17-2020 by Lab or GTR ID New York Genome Center. Assertions are reported exactly as they appear on the patient provided laboratory report. GenomeConnect does not attempt to reinterpret the variant. The IDDRC-CTSA National Brain Gene Registry (BGR) is a study funded by the U.S. National Center for Advancing Translational Sciences (NCATS) and includes 13 Intellectual and Developmental Disability Research Center (IDDRC) institutions. The study is led by Principal Investigator John Constantino MD PhD from Washington University. The BGR is a data commons of gene variants paired with subject clinical information. This database helps scientists learn more about genetic changes and their impact on the brain and behavior. Participation in the Brain Gene Registry requires participation in GenomeConnect. More information about the Brain Gene Registry can be found on the study website - https://braingeneregistry.wustl.edu/.