Reclassification of Two MLH1 Variants of Uncertain Significance Utilizing Clinical and Functional Data

Jane Hübertz Frederiksen; Ulf Birkedal; Sarah Bachmann; Elisabeth Victoria Eliesen; Lene Juel Rasmussen; Katja Venborg Pedersen; Lana Al-Zehhawi; Susanne E Boonen; Lotte Krogh; Karina Rønlund; Lise Graversen; Jannie Assenholt; Kjeld Schmiegelow; Karin Wadt; Anne-Marie Gerdes; Thomas V O Hansen

doi:10.1002/mgg3.70026

Reclassification of Two MLH1 Variants of Uncertain Significance Utilizing Clinical and Functional Data

Jane Hübertz Frederiksen^*
, Ulf Birkedal
, Sarah Bachmann
, Elisabeth Victoria Eliesen
, Lene Juel Rasmussen
, Katja Venborg Pedersen
, Lana Al-Zehhawi
, Susanne E Boonen
, Lotte Krogh
, Karina Rønlund
, Lise Graversen
, Jannie Assenholt
, Kjeld Schmiegelow
, Karin Wadt
, Anne-Marie Gerdes
, Thomas V O Hansen

^*Corresponding author for this work

Research output: Contribution to journal › Article › Research › peer-review

Abstract

BACKGROUND: Pathogenic variants in the mismatch repair genes are associated with an elevated lifetime risk of colorectal cancer (CRC). We previously identified two variants of uncertain significance (VUS) in the MLH1 gene, c.696_698del, p.(Cys233del) and c.1919C > G, p.(Pro640Arg), in Danish families with numerous occurrences of CRC.

METHODS: To reclassify the variants we collected clinical data, initiated tumor and co-segregation analysis, and performed RNA splicing analysis, subcellular localization, and protein stability studies.

RESULTS: The functional analysis revealed that the c.696_698del, p.(Cys233del) variant had an effect at the RNA level, on subcellular localization, and on protein stability, while the c.1919C > G, p.(Pro640Arg) variant showed decreased expression in localization studies and decreased protein stability. These results suggest both variants disrupt DNA mismatch repair.

CONCLUSION: By applying all collected data and functional results we propose to reclassify the c.696_698del, p.(Cys233del) and the c.1919C > G, p.(Pro640Arg) variants as likely pathogenic (class 4) using MMR gene-specific ACMG/AMP guidelines. Consequently, the two MLH1 variants can now be used for risk assessment of variant carriers, while family members without the variants can be excluded from intensified cancer surveillance and follow population recommendations.

Original language	English
Article number	e70026
Number of pages	7
Journal	Molecular genetics & genomic medicine
Volume	12
Issue number	11
DOIs	https://doi.org/10.1002/mgg3.70026
Publication status	Published - Nov 2024

Keywords

Humans
MutL Protein Homolog 1/genetics
DNA Mismatch Repair
Female
Male
Protein Stability
Middle Aged
Colorectal Neoplasms/genetics
RNA Splicing
Colorectal Neoplasms, Hereditary Nonpolyposis/genetics
Mutation
Nuclear localization
RNA splicing analysis
ACMG/AMP classification
Protein stability
Mlh1
Lynch syndrome

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Frederiksen, J. H., Birkedal, U., Bachmann, S., Eliesen, E. V., Rasmussen, L. J., Pedersen, K. V., Al-Zehhawi, L., Boonen, S. E., Krogh, L., Rønlund, K., Graversen, L., Assenholt, J., Schmiegelow, K., Wadt, K., Gerdes, A.-M., & Hansen, T. V. O. (2024). Reclassification of Two MLH1 Variants of Uncertain Significance Utilizing Clinical and Functional Data. Molecular genetics & genomic medicine, 12(11), Article e70026. https://doi.org/10.1002/mgg3.70026

@article{7e87c321f32c46cf8b5b8eb7856a78a3,

title = "Reclassification of Two MLH1 Variants of Uncertain Significance Utilizing Clinical and Functional Data",

abstract = "BACKGROUND: Pathogenic variants in the mismatch repair genes are associated with an elevated lifetime risk of colorectal cancer (CRC). We previously identified two variants of uncertain significance (VUS) in the MLH1 gene, c.696\_698del, p.(Cys233del) and c.1919C > G, p.(Pro640Arg), in Danish families with numerous occurrences of CRC.METHODS: To reclassify the variants we collected clinical data, initiated tumor and co-segregation analysis, and performed RNA splicing analysis, subcellular localization, and protein stability studies.RESULTS: The functional analysis revealed that the c.696\_698del, p.(Cys233del) variant had an effect at the RNA level, on subcellular localization, and on protein stability, while the c.1919C > G, p.(Pro640Arg) variant showed decreased expression in localization studies and decreased protein stability. These results suggest both variants disrupt DNA mismatch repair.CONCLUSION: By applying all collected data and functional results we propose to reclassify the c.696\_698del, p.(Cys233del) and the c.1919C > G, p.(Pro640Arg) variants as likely pathogenic (class 4) using MMR gene-specific ACMG/AMP guidelines. Consequently, the two MLH1 variants can now be used for risk assessment of variant carriers, while family members without the variants can be excluded from intensified cancer surveillance and follow population recommendations.",

keywords = "Humans, MutL Protein Homolog 1/genetics, DNA Mismatch Repair, Female, Male, Protein Stability, Middle Aged, Colorectal Neoplasms/genetics, RNA Splicing, Colorectal Neoplasms, Hereditary Nonpolyposis/genetics, Mutation, Nuclear localization, RNA splicing analysis, ACMG/AMP classification, Protein stability, Mlh1, Lynch syndrome",

author = "Frederiksen, \{Jane H{\"u}bertz\} and Ulf Birkedal and Sarah Bachmann and Eliesen, \{Elisabeth Victoria\} and Rasmussen, \{Lene Juel\} and Pedersen, \{Katja Venborg\} and Lana Al-Zehhawi and Boonen, \{Susanne E\} and Lotte Krogh and Karina R{\o}nlund and Lise Graversen and Jannie Assenholt and Kjeld Schmiegelow and Karin Wadt and Anne-Marie Gerdes and Hansen, \{Thomas V O\}",

note = "{\textcopyright} 2024 The Author(s). Molecular Genetics \& Genomic Medicine published by Wiley Periodicals LLC.",

year = "2024",

month = nov,

doi = "10.1002/mgg3.70026",

language = "English",

volume = "12",

journal = "Molecular genetics \& genomic medicine",

issn = "2324-9269",

publisher = "John Wiley \& Sons Inc.",

number = "11",

}

Frederiksen, JH, Birkedal, U, Bachmann, S, Eliesen, EV, Rasmussen, LJ, Pedersen, KV, Al-Zehhawi, L, Boonen, SE, Krogh, L, Rønlund, K, Graversen, L, Assenholt, J, Schmiegelow, K, Wadt, K, Gerdes, A-M & Hansen, TVO 2024, 'Reclassification of Two MLH1 Variants of Uncertain Significance Utilizing Clinical and Functional Data', Molecular genetics & genomic medicine, vol. 12, no. 11, e70026. https://doi.org/10.1002/mgg3.70026

TY - JOUR

T1 - Reclassification of Two MLH1 Variants of Uncertain Significance Utilizing Clinical and Functional Data

AU - Frederiksen, Jane Hübertz

AU - Birkedal, Ulf

AU - Bachmann, Sarah

AU - Eliesen, Elisabeth Victoria

AU - Rasmussen, Lene Juel

AU - Pedersen, Katja Venborg

AU - Al-Zehhawi, Lana

AU - Boonen, Susanne E

AU - Krogh, Lotte

AU - Rønlund, Karina

AU - Graversen, Lise

AU - Assenholt, Jannie

AU - Schmiegelow, Kjeld

AU - Wadt, Karin

AU - Gerdes, Anne-Marie

AU - Hansen, Thomas V O

PY - 2024/11

Y1 - 2024/11

N2 - BACKGROUND: Pathogenic variants in the mismatch repair genes are associated with an elevated lifetime risk of colorectal cancer (CRC). We previously identified two variants of uncertain significance (VUS) in the MLH1 gene, c.696_698del, p.(Cys233del) and c.1919C > G, p.(Pro640Arg), in Danish families with numerous occurrences of CRC.METHODS: To reclassify the variants we collected clinical data, initiated tumor and co-segregation analysis, and performed RNA splicing analysis, subcellular localization, and protein stability studies.RESULTS: The functional analysis revealed that the c.696_698del, p.(Cys233del) variant had an effect at the RNA level, on subcellular localization, and on protein stability, while the c.1919C > G, p.(Pro640Arg) variant showed decreased expression in localization studies and decreased protein stability. These results suggest both variants disrupt DNA mismatch repair.CONCLUSION: By applying all collected data and functional results we propose to reclassify the c.696_698del, p.(Cys233del) and the c.1919C > G, p.(Pro640Arg) variants as likely pathogenic (class 4) using MMR gene-specific ACMG/AMP guidelines. Consequently, the two MLH1 variants can now be used for risk assessment of variant carriers, while family members without the variants can be excluded from intensified cancer surveillance and follow population recommendations.

AB - BACKGROUND: Pathogenic variants in the mismatch repair genes are associated with an elevated lifetime risk of colorectal cancer (CRC). We previously identified two variants of uncertain significance (VUS) in the MLH1 gene, c.696_698del, p.(Cys233del) and c.1919C > G, p.(Pro640Arg), in Danish families with numerous occurrences of CRC.METHODS: To reclassify the variants we collected clinical data, initiated tumor and co-segregation analysis, and performed RNA splicing analysis, subcellular localization, and protein stability studies.RESULTS: The functional analysis revealed that the c.696_698del, p.(Cys233del) variant had an effect at the RNA level, on subcellular localization, and on protein stability, while the c.1919C > G, p.(Pro640Arg) variant showed decreased expression in localization studies and decreased protein stability. These results suggest both variants disrupt DNA mismatch repair.CONCLUSION: By applying all collected data and functional results we propose to reclassify the c.696_698del, p.(Cys233del) and the c.1919C > G, p.(Pro640Arg) variants as likely pathogenic (class 4) using MMR gene-specific ACMG/AMP guidelines. Consequently, the two MLH1 variants can now be used for risk assessment of variant carriers, while family members without the variants can be excluded from intensified cancer surveillance and follow population recommendations.

KW - Humans

KW - MutL Protein Homolog 1/genetics

KW - DNA Mismatch Repair

KW - Female

KW - Male

KW - Protein Stability

KW - Middle Aged

KW - Colorectal Neoplasms/genetics

KW - RNA Splicing

KW - Colorectal Neoplasms, Hereditary Nonpolyposis/genetics

KW - Mutation

KW - Nuclear localization

KW - RNA splicing analysis

KW - ACMG/AMP classification

KW - Protein stability

KW - Mlh1

KW - Lynch syndrome

U2 - 10.1002/mgg3.70026

DO - 10.1002/mgg3.70026

M3 - Article

C2 - 39548353

SN - 2324-9269

VL - 12

JO - Molecular genetics & genomic medicine

JF - Molecular genetics & genomic medicine

IS - 11

M1 - e70026

ER -

Reclassification of Two MLH1 Variants of Uncertain Significance Utilizing Clinical and Functional Data

Abstract

Keywords

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