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. 2025 May 27;20(5):e0308771. doi: 10.1371/journal.pone.0308771

The impact of VUS reclassification on reproductive decision making

Alexandra Peyser 1,*, Kenan Onel 2, Avner Hershlag 3
Editor: Nejat Mahdieh4
PMCID: PMC12111534  PMID: 40424322

Abstract

Introduction

Laboratories will occasionally reclassify a VUS to pathogenic (P) or likely pathogenic (LP), making it clinically actionable. Here, we aim to characterize the frequency of this reclassification in genes tested routinely during preconception counseling in order to help guide reproductive decision making.

Design

Utilizing the American College of Medical Genetics (ACMG) 113-gene pre-conception panel, we conducted data analysis from ClinVar Miner (https://clinvarminer.genetics.utah.edu/). The numbers of VUS’s reported for each gene were recorded over a 3-year period (2019, 2021, & 2022). In addition, data on the number of VUS’s in conflict (VUS/P and VUS/LP) were recorded. The 10 genes with the most VUS’s and the genes with the most frequent reporting discordance were compared over the 3 years,

Results

There was a 103% increase in the number of VUS’s reported (2019: 13,278, 2021: 22,434, 2022: 26,965) and a 235% increase in the number of VUS’s in conflict (2019:387, 2021: 946, 2022:1297). The overall percent conflict increased significantly (2019: 2.9% vs. 2022: 4.8%). Nine genes among the top 10 with the most frequent VUS’s remained the same over the 3-year period, while five out of the ten most frequent genes with VUS’s in conflict remained the same.

Conclusions

The rate of conflicting reporting of a VUS has increased in 3 years and is currently at 4.8%. A potential upgrade of a VUS to pathogenic or likely pathogenic may turn the variant “actionable,” justifying testing embryos for the variant through PGT-M.

Introduction

Due to an increase in preconception genetic testing, laboratories are now detecting novel sequence variants for an increasing number of genes associated with genetic disorders. The significance of these variants falls along a gradient indicating the likelihood that the variant is associated with a disease. In 2015, the American College of Medical Genetics (ACMG) published guidelines to standardize the classification of variants indicating the likelihood of being associated with disease ranging from pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB) to benign (B) [1]. While variants classified as B/LB are not actionable and variants classified as P/LP are actionable, the finding of a VUS poses a dilemma, as its association with disease is unclear and may change over time as more individuals are sequenced. Patients with a family history of a heritable disease with a VUS in a gene associated with that disorder present a unique challenge to the reproductive geneticist: While embryos can be tested for any genetic variant through preimplantation genetic testing (PGT), VUS’s are considered not actionable. Indeed, most PGT labs do not test embryos for a VUS.

Variants may be classified as VUS for several reasons: Either the effect of the specific genetic alteration on gene function is unknown, or there is insufficient genetic data to definitively confirm the pathogenicity of the variant [1]. VUS reporting during the pre-conception period presents a significant challenge to reproductive specialists. This holds true when both partners are carriers for a VUS or if one partners screen positive for an X-linked condition where male offspring will be affected. When a VUS is reported, the question arises whether pre-implantation genetic testing for monogenic disease (PGT-M) of embryos should be recommended to prevent the offspring from inheriting the variant. Since assisted reproductive technology (ART) has the unique capacity to identify genetic variants, in the embryo preconceptionally thus preventing their transmission to the offspring, the common reporting of VUS presents a difficult dilemma, especially due to the lack of specific guidelines by the professional societies [2,3]. Additionally, as data continues to accumulate, laboratories will occasionally reclassify a VUS to P or LP, both of which are considered clinically actionable, allowing the utilization of PGT-M to prevent vertical transmission to the offspring. The ongoing reclassification of VUS, coupled with conflicting reporting by testing laboratories of the pathogenic significance of variants has led to confusion regarding the need for preconception testing of embryos for the variant in question [4]. Since the use of wide carrier screening (CS) is becoming a staple of preconception screening, especially in the field of Assisted Reproductive Technologies (ART), where a couple will be screened for hundreds of disease causing variants, the incidence VUS reporting keeps growing. Providers are facing an increasing need to explain the significance of VUSs and their actionability. There is a dilemma; what should the provider do when the variant is reported as VUS by one or more labs and as P or LP by another lab(s). Should conflicting reports present an opportunity to perform PGT-M? The objective of this study was to explore the extent of VUS reporting over a 3-year period for the recommended pre-conception genetic screening panel, and to specifically determine how many VUS’s are in conflict between laboratories, making them potentially actionable. It is important to note that in this study, we analyzed VUS in conflict that were classified as a VUS by one laboratory and P or LP with another. We did not look at those that were in conflict in being classified as benign or likely benign because they were clinically irrelevant.

Materials and methods

Utilizing the ACMG recommended 113 gene pre-conception panel [5], consisting of 97 autosomal recessive and 16 X-linked genetic conditions, we conducted data analysis from ClinVar Miner (https://clinvarminer.genetics.utah.edu), a web-based platform utilizing data from the National Center for Biotechnology Information’s ClinVar archive (https://www.ncbi.nlm.nig.gov/clinvar) in December 2022. ClinVar, the main community-based repository of genomic knowledge is a shared variant interpretation database that is updated weekly with several thousand modifications of variant classifications.

The number of VUS’s reported for each gene was recorded over a 3-year period (2019, 2021, and 2022) by one author (AP). Data for 2020 was not reported on ClinVar Miner (due to the COVID pandemic). In addition, data on the number of VUS’s in conflict (defined as those reported as VUS by one submitter and as P or LP by another) were obtained. The top 10 genes with the highest number of VUS’s per gene as well as the highest percent in conflict (defined as a VUS in conflict divided by the total number of VUS) were compared over the 3 years.

Results

Over the study period, there was a 103% increase in the number of VUS’s reported from the genes within the ACMG recommended panel (2019: 13,278, 2021: 22,434, 2022: 26,965) and a 235% increase in the number of VUS’s in conflict (2019:387, 2021: 946, 2022: 1297) (Fig 1). The total percentage of VUS’s in conflict increased over time from 2.9% in 2019 to 4.8% in 2022. Of the 113 genes on the panel, there was an increase over time in the number of genes with conflicting reporting (2019: 64, 2021:90, 2022:101) (Fig 2). The top 9 genes with the highest number of VUS’s remained the same over the 3 years (NEB, DMD, USH2A, BLM, PKHD1, CFTR, POLG, GAA, PCDH15) while the 10th gene differed each year (Fig 3). Of the top 10 genes with the highest percent in conflict, 5 remained in the top 10 over the 3-year period (Fig 4).

Fig 1. Total VUS’s in Conflict by Year.

Fig 1

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Fig 2. Total Genes in Conflict by Year.

Fig 2

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Fig 3. Genes with the highest number of VUS by year.

Fig 3

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The top 9 genes with the highest number of VUS’s remained the same over the 3 years while the 10th gene (red) differed each year.

Fig 4. Genes with the highest percent in conflict by year.

Fig 4

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Of the top 10 genes with the highest percent in conflict, the 5 in bold remained in the top 10 over the 3-year period.

Discussion

Over the study period, the number of VUS’s reported for the ACMG-recommended preconception panel has increased significantly. In addition, the rate of conflicting reporting (VUS/P or VUS/LP) has increased by 235% and is currently at 4.8%. There exists no consensus on how reproductive medicine providers should counsel patients regarding VUS results on CS panels [6–8]. While an ever-expanding carrier-screening has become an integral part of preconception testing, we have witnessed an exponential increase in VUS reporting falling into an interpretive vacuum, given the absence of a broad consensus regarding the clinical management to VUS’s and how should patients be counseled. Some argue that a VUS should be ignored during clinical decision making due to misleading consequences [9], while others have called for a more widely accepted consensus regarding management [10].

Currently, there is a lack of guidelines in the United States for determining whether PGT-M should be performed for a VUS. This has resulted in wide discrepancies between PGT-M laboratories as to whether or not they agree to perform PGT-M for a VUS [2]. Porto et al. interviewed nine genetic counselors from 5 different PGT-M laboratories regarding utilization of PGT-M for VUS [2]. The authors concluded that there was a significant variation in the eligibility policy regarding VUS for PGT-M between laboratories, and that there is a perceived lack of specific guidelines by the professional societies regarding counseling patients [2].

The American Society for Reproductive Medicine (ASRM) ethics committee opinion states that PGT-M is ethically justifiable in cases of serious conditions with no known interventions, as well as for cases of less serious or lower penetrance disorders due to reproductive liberty [11]. Additionally, the American College of Obstetricians and Gynecologists (ACOG) Committee Opinion from March 2020 states that PGT-M has clinical utility but did not make specific recommendations as to which variants should be tested for via PGT-M [12]. A recent ASRM committee opinion stated that offering PGT-M for VUS depends on multiple factors including how the VUS was identified, supporting classification evidence, familial penetrance, recurrence risk and supporting documentation. The document does not mention what to do when a VUS is labeled pathogenic one place and a VUS elsewhere [13].

At a single IVF center experience in Israel, data on couples presenting for PGT-M for a VUS was collected and analyzed [14]. From 2014–2019, a total of 45 couples requested PGT-M, of which 24 (52%) had a VUS. Twelve (50%) of the latter couples had an isolated VUS and 12 (50%) had a VUS diagnosed in addition to a disease-causing variant. Nine couples underwent PGT-M for an isolated VUS, 5 couples performed PGT-M for both pathogenic findings and a VUS.12 Another study which looked at 970 prenatal microarrays following invasive diagnostic prenatal testing found a VUS in 55 cases (5.8%) [15]. A VUS result creates challenges for providers to educate and counsel their patients appropriately as well as presenting difficult decisions for parents regarding how to proceed following a VUS result.

Whether VUS results should be disclosed in CS reporting remains an ongoing debate, especially within the prenatal context. VUS disclosure has the potential to increase parental distress as well create difficulties for genetic counselors as to how to advise patients [16]. In fact, reporting of VUS varies globally; the U.K and Belgium withhold VUS reporting whereas the Netherlands and the U.S. routinely report them [16]. Following a diagnosis of a VUS prenatally, patients have expressed concern about the child’s health and development [17]. Furthermore, it has been reported that a substantial proportion of physicians lack a true understanding of the implications of a VUS result and do not feel comfortable disclosing a VUS to a patient [18]. Prior studies have shown that patients often misinterpret VUS results depending on how counseling was performed [19]. Physicians should be educated regarding VUS. Our disease prevention mission is handicapped by the currently inefficient setting. The development of clear guidelines by the professional societies will fill in an important void, enhance communication with patients and dramatically reduce frustration of patients, genetic counselors, and physicians.

Another dilemma reproductive specialists face is what to do when reclassification of a VUS occurs. Studies have reported the median time for reclassification is 39 months [17]. A previous study looked into reclassification data over 3.5 years (2016–2019) and found that 0.95% of VUS’s were reclassified, of them 6% to P and 18% to LP, making these variants clinically actionable, thus enabling the utilization of PGT-M [20]. Many studies have emphasized the clinical impact of reclassification on cancer genes [21–23]. In cases where embryos were frozen, often times following a biopsy for aneuploidy, a delayed reclassification to P or LP would raise the option of embryo thaw and re-biopsy. What happens when there is a discrepancy of the variant classification between laboratories; When one laboratory reports the variant as P or LP and another calls it is a VUS. Is PGT-M warranted in these cases? If conflicting reports exist, who should decide whether a VUS should be actionable? A prior study looked at discordant variant classifications between clinicians and genetic testing laboratories within a single cardiovascular disease clinic and found that 18% of variants were reported differently by the two groups, with 83% affecting the clinical care of the patient [24]. Our study demonstrates that currently, 4.8% of VUS’s from the ACMG prenatal panel are in conflict between genetic testing laboratories, making them clinically actionable.

There is no formal reclassification process published yet by the ACMG or any other professional society. There is a missing link between the lab finding a variant and the clinical background. There is no mechanism set in place, nor a central registry where the familial inheritance of the disease in question is reported. Therefore, the determination of pathogenicity seems at this point to be haphazard in the lack of an organized database. A recent case series discussed 3 pediatric cases of adrenoleukodystrophy in patients conceived via the same oocyte donor, all found to be heterozygous of the same VUS which the donor was not originally screened for [24]. After the death of one child at the age of 5, the two other families from the same donor were notified by their clinic and the children were treated. This case highlights the gap in screening recommendations of gamete donors as well as the lack of standardized protocol for sharing any subsequent reclassification. When new evidence suggests pathogenicity of a VUS, the ACMG guidelines can be used to change the classification. Furthermore, reclassification of a VUS is highly dependent on testing laboratories. Some laboratories have an active variant reclassification process in which periodic updates to VUS classified variants is performed and if reclassification occurs, all providers on record for those affected are informed [25]. Whereas other laboratories use a passive reclassification system where the providers are the ones to inform the lab of any new information with regard to the variant [23]. This variation in reclassification process warrants a uniform system. It is incumbent upon providers to review the literature and rationales presented by their testing laboratory for classification interpretation and reinterpretation.

We may assume that given the exponential increase in VUS and VUS in conflict reporting these numbers are only going to increase several-fold within 5,10 and 20 years [26,3]. Importantly, since the rate of CS is increasing as well as the increase availability (and cost containment) of whole exome and whole genome sequencing is upon us, the absence of clear policy and guidelines is bound to lead to total chaos in the interpretation and clinical utilization of genetic data. We suggest a combined forum of ACMG and ASRM to propose a specific protocol for examining the pathogenicity of VUS’s and a well-defined mechanism to determine, in a timely fashion, which VUS’s should be upgraded.

The strengths of our study include that it is the first to describe the predicament of VUS and VUS in conflict within the field of reproductive medicine and more importantly, within the preconception period. We highlight the need for uniformity in the variant reclassification system in reproductive medicine. Limitations include the ever-changing variant classification and therefore our results may differ by the time this study is completed. In addition, our study was limited to the 3-year time period studied, further hampered by the lack of 2020 data due to the COVID pandemic. Furthermore, prior studies using ClinVar have revealed that variant types, penetrance, and a widely varied testing technology between genetic labs could affect the concordance rates between submitters, which could affect our results [27]. We did not report on those VUS’s in conflict with a benign and likely benign classification as our focus was on those VUS that are actionable leading to potential use of PGT-M.

Conclusion

The reporting of a VUS, especially with significant family history, presents a difficult dilemma to the reproductive physician: should action be taken to prevent inheritance to the offspring through PGT-M. The ACMG has not established a clear reclassification process for VUS’s when conflicting reporting is documented on ClinVar. Therefore, a proactive approach to a VUS may reveal that it has been reported as pathogenic elsewhere, making intervention through PGT-M a reasonable action, without having to wait for an official upgrade. Prior to each subsequent pregnancy, it is important to reevaluate previously reported VUS’s, since they may have been reclassified.

Key Message.

The rate of conflicting reporting of VUS on the pre-conception panel is increasing. A proactive approach to a VUS may reveal it is pathogenic elsewhere, making PGT-M a reasonable action. Prior to each subsequent pregnancy, it is important to reevaluate previously reported VUS’s, since they may have been reclassified.

Supporting information

S1 Table. ClinVar_Data_ASRM22_REAL.

(XLSX)

pone.0308771.s001.xlsx (156.3KB, xlsx)

Data Availability

https://clinvarminer.genetics.utah.edu/ Within clinvarminer, searching by specific gene will display the number of VUS, pathogenic and likely pathogenic variants for each gene for each year of interest can be found.

Funding Statement

The author(s) received no specific funding for this work.

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Decision Letter 0

Nejat Mahdieh

9 Sep 2024

PONE-D-24-31357The Impact of VUS Reclassification on Reproductive Decision MakingPLOS ONE

Dear Dr. Peyser,

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“MINING” VUS’S FOR PATHOGENICITY: CAN INTER-LAB CONFLICTS RENDER VARIANTS OF UNCERTAIN SIGNIFICANCE (VUS’S) PATHOGENIC AND THEREFORE, ACTIONABLE? - https://doi.org/10.1016/j.fertnstert.2022.08.168

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Reviewer #1: The authors of the article entitled: The Impact of VUS Reclassification on Reproductive Decision Making” gives a view of the 3 years of VUS reports and report the increase of VUS in reproductive genes. “When a VUS is reported, the question arises whether pre-implantation genetic testing for monogenic disease (PGT-M) of embryos should be recommended to prevent the offspring from inheriting the variant.” As mentioned the variants with Pathogenic and LP effect are actionable.

Please clarify how many VUS variants of the genes were changed during these years to P/LP. Note that these are actionable for PGT.

Reviewer #2: Variant discovery resulting in VUS during preconception recessive and X-linked carrier testing is an important issue in the medical literature. Defining precisely what the authors consider to be carrier screening would benefit this paper as there are a few areas in which the definitions cross boundaries into other types of genetic testing in the prenatal setting. Prenatal genetic testing differs across jurisdictions – the paper would benefit if the authors state what specific issue within their jurisdiction needs to be resolved. Please find some comments on the proposed paper:

Abstract

“Laboratories will occasionally upgrade a VUS to pathogenic (P) or likely pathogenic (LP), making it clinically actionable.”

- Variant reclassification may result in the changed classification of any variant to any other distinct category and is not restricted to an “upgrade”

Introduction

There are three distinct entities within genetic testing in the prenatal setting that seem to be viewed interchangeably by the authors. The aim of the authors study appears to be point 1 below, and this study conflates an overlap between 2 & 3:

1. Asymptomatic recessive and X-linked carrier testing within prenatal medicine which is indicated for an unaffected (asymptomatic) individual without any unprobed family history of a genetic condition

2. Non-invasive prenatal testing using chromosomal microarray technology

3. VUS that are uncovered during the course of diagnostic testing in an affected individual, thereby leaving the case unsolved and no option for prenatal/preconception genetic testing for a known condition available to closely related family members. (Clinically, risk could be established for an unaffected but closely related couple using carrier frequencies to estimate risks of a rare recessive condition).

e.g. “Patients with a family history of a heritable disease with a VUS in a gene associated with that disorder present a unique challenge to the reproductive geneticist”.

- The proper steps for a genetic diagnosis in a family for which preconception genetic testing can be offered is to make a diagnosis in the affected individual. The focus of the paper appears to be on asymptomatic recessive and X-linked carrier testing in prenatal medicine which is performed independent of a diagnosis in consideration. Therefore this statement appears to confuse diagnostic versus carrier testing. (Screening for autosomal recessive and X-linked conditions during pregnancy and preconception: a practice resource of the American College of Medical Genetics and Genomics (ACMG) )

“VUS’s are generally considered not actionable. Indeed, most PGT labs do not test embryos for a VUS” –

- VUS are always considered not actionable at the point of classification. VUS testing of an embryo may occur in select instances and is not really generalisable to the point of this paper as a whole (Laboratory testing for preconception/prenatal carrier screening: A technical standard of the American College of Medical Genetics and Genomics (ACMG))

“VUS reporting during the pre-conception period presents a significant challenge to reproductive specialists. When a VUS is reported, the question arises whether pre-implantation genetic testing for monogenic disease (PGT-M) of embryos should be recommended to prevent the offspring from inheriting the variant.”

- The authors should emphasise that risk for the genetic variants classified as VUS during prenatal carrier testing is only significant in light of their partner’s status. Therefore, this is a leap to say that a VUS in a recessive gene may have clinical implications when you don’t consider the partner’s risk.

“the common reporting of VUS presents a difficult dilemma”

- Do the authors have a reference for how commonly VUS are reported in the prenatal setting?

“The ongoing reclassification of VUS, coupled with conflicting reporting by testing laboratories of the pathogenic significance of variants has led to confusion regarding the need for preconception testing of embryos for the variant in question (3).”

- The authors repeatedly reference laboratory reporting of discordant classification of the same variant. This is a separate issue to VUS reclassification and although it is examined in the methods of this study, may not contribute to ultimate genetic counselling. Can the authors please define the significance of variant discordance within the context of the study aims?

- “We did not report on those VUS’s in conflict with a benign and likely benign classification as our focus was on those VUS that are actionable leading to potential use of PGT-M.” – I note this as the final statement of the Discussion which provides useful information about the aim of the paper overall and would be important in the Introduction to clarify the aims.

“There is a dual dilemma; first, how should the patient be counselled when the variant is reported as VUS by one or more labs and as P or LP by another lab(s)”

- Genetic counselling is clinically confined to the genetic test report and the consideration of the testing centre and clinician interpretation.

“The second dilemma is how to relate to a VUS reported, for instance, for a cancer gene, where there is a significant familial history of the cancer associated with the gene (e.g., breast/ovarian cancer and BRCA1/2; colon cancer and APC).”

- This would require clarification as the authors previously state that the ACMG recommended gene panel for preconception genetic testing is the foundation of this study. However, cancer associated genes are not included in this panel. Can the authors please clarify this?

Materials and methods:

“The top 10 genes with the highest number of VUS’s per gene as well as the highest percent in conflict (defined as a VUS in conflict divided by the total number of VUS) were compared over the 3 years.”

- Why did the authors restrict their analysis to 10 genes?

- Why did the authors confine the time period to 3 years of data?

- Discordant submissions in ClinVar are not the same as VUS classification of variants and the authors should note the distinction

- the methods could be improved by comprehensively examining VUS detected for genes within the recommended gene panel

Discussion:

“VUS disclosure has the potential to increase parental distress as well create difficulties for genetic counselors as to how to advise patients (15). I”

- This distress seems to arise from the disclosure of CNV VUS detected during NIPT for an active pregnancy. Do the authors feel that this may extend to preconception counselling for what may be non clinically relevant VUS in recessive genes?

“. Our disease (cancer included) prevention mission is handicapped by the currently inefficient setting”

- Again, can the authors please clarify what preconception carrier testing methods report pathogenic or likely pathogenic variants in cancer related genes?

“Reporting to ClinVar is not mandatory”.

- However ClinVar still represents a rich repository of human genetic variation with over 4 million genetic variants submitted. It has proven use in the literature as an adjunct to variant classification and therefore does not represent a limitation of variant classification or reclassification.

“We may assume that given the exponential increase in VUS and VUS-in-conflict reporting, these numbers are only going to increase several-fold within 5,10 and 20 years.”

- Standardised guidelines and improved sequencing and bioinformatic techniques are resolving unsolved variation within the human genome. Do the authors have a reference to support this statement? Otherwise, it is in conflict with the current published literature.

“What will happen if VUS’s in conflict reach 25% of all cases?”

- Can the authors clarify the implications of this statement? What would happen? And is it likely to happen?

“Currently, many genetic labs do not report all VUS’s and therefore, the true incidence of potentially upgradable VUS’s is difficult to determine but likely, involves many more under-reported cases.”

- The study methods use ClinVar miner which is taken from ClinVar. ClinVar is a variant submission database, not a genetic test report database. Therefore, submitting labs and facilities submit any variant uncovered during genetic testing, and not just those that are clinically relevant for the test performed. Therefore this statement is inaccurate as VUS within ClinVar are more likely to closely represent all VUS detected during genetic diagnostic testing and research settings and not just those that reach the eyes of the clinician and patient on the final genetic test report.

**********

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Reviewer #2: No

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Attachment

Submitted filename: Preconception VUS.pdf

pone.0308771.s002.pdf (61.5KB, pdf)
PLoS One. 2025 May 27;20(5):e0308771. doi: 10.1371/journal.pone.0308771.r003

Author response to Decision Letter 1

14 Feb 2025

Thank you for your consideration of our manuscript. We have revised the manuscript to accommodate your suggestions and greatly appreciate each review. Specific responses to comments are itemized below.

Reviewer #1: The authors of the article entitled: The Impact of VUS Reclassification on Reproductive Decision Making” gives a view of the 3 years of VUS reports and report the increase of VUS in reproductive genes. “When a VUS is reported, the question arises whether pre-implantation genetic testing for monogenic disease (PGT-M) of embryos should be recommended to prevent the offspring from inheriting the variant.” As mentioned the variants with Pathogenic and LP effect are actionable.

Please clarify how many VUS variants of the genes were changed during these years to P/LP. Note that these are actionable for PGT.

Thank you for this comment. The main focus of our study was reporting on VUS in conflict (VUS/P and VUS/LP) that are potentially actionable. We did not look specifically at how many of these gene were changed to P/LP. However a prior study did study this and it is quoted in our discussion on page 9: “A previous study looked into reclassification data over 3.5 years (2016-2019) and found that 0.95% of VUS’s were reclassified, of them 6% to P and 18% to LP, making these variants clinically actionable, thus enabling the utilization of PGT-M (19).”

Reviewer #2: Variant discovery resulting in VUS during preconception recessive and X-linked carrier testing is an important issue in the medical literature. Defining precisely what the authors consider to be carrier screening would benefit this paper as there are a few areas in which the definitions cross boundaries into other types of genetic testing in the prenatal setting. Prenatal genetic testing differs across jurisdictions – the paper would benefit if the authors state what specific issue within their jurisdiction needs to be resolved. Please find some comments on the proposed paper:

Thank you. We have added a sentence to clarify that our carrier screening is performed for patients attempting a pregnancy. “Since the use of wide carrier screening (CS) is becoming a staple of preconception screening, especially in the field of Assisted Reproductive Technologies (ART), where a couple will be screened for hundreds of disease causing variants, the incidence of VUS reporting keeps growing.”

Abstract

“Laboratories will occasionally upgrade a VUS to pathogenic (P) or likely pathogenic (LP), making it clinically actionable.”

- Variant reclassification may result in the changed classification of any variant to any other distinct category and is not restricted to an “upgrade”

Thank you for this comment. We have changed the wording “upgrade” to “reclassify.”

Introduction

There are three distinct entities within genetic testing in the prenatal setting that seem to be viewed interchangeably by the authors. The aim of the authors study appears to be point 1 below, and this study conflates an overlap between 2 & 3:

1. Asymptomatic recessive and X-linked carrier testing within prenatal medicine which is indicated for an unaffected (asymptomatic) individual without any unprobed family history of a genetic condition

2. Non-invasive prenatal testing using chromosomal microarray technology

3. VUS that are uncovered during the course of diagnostic testing in an affected individual, thereby leaving the case unsolved and no option for prenatal/preconception genetic testing for a known condition available to closely related family members. (Clinically, risk could be established for an unaffected but closely related couple using carrier frequencies to estimate risks of a rare recessive condition).

e.g. “Patients with a family history of a heritable disease with a VUS in a gene associated with that disorder present a unique challenge to the reproductive geneticist”.

- The proper steps for a genetic diagnosis in a family for which preconception genetic testing can be offered is to make a diagnosis in the affected individual. The focus of the paper appears to be on asymptomatic recessive and X-linked carrier testing in prenatal medicine which is performed independent of a diagnosis in consideration. Therefore this statement appears to confuse diagnostic versus carrier testing. (Screening for autosomal recessive and X-linked conditions during pregnancy and preconception: a practice resource of the American College of Medical Genetics and Genomics (ACMG) )’

Yes, our main focus is point 1. However, often patients present with a family history of disease with a VUS detected and would like to do PGT-M. Therefore, we raise this point in the paper.

“VUS’s are generally considered not actionable. Indeed, most PGT labs do not test embryos for a VUS” –

- VUS are always considered not actionable at the point of classification. VUS testing of an embryo may occur in select instances and is not really generalisable to the point of this paper as a whole (Laboratory testing for preconception/prenatal carrier screening: A technical standard of the American College of Medical Genetics and Genomics (ACMG))

We have removed the word “generally.”

“VUS reporting during the pre-conception period presents a significant challenge to reproductive specialists. When a VUS is reported, the question arises whether pre-implantation genetic testing for monogenic disease (PGT-M) of embryos should be recommended to prevent the offspring from inheriting the variant.”

- The authors should emphasise that risk for the genetic variants classified as VUS during prenatal carrier testing is only significant in light of their partner’s status. Therefore, this is a leap to say that a VUS in a recessive gene may have clinical implications when you don’t consider the partner’s risk.

We have added an additional statement following that sentence explaining that this risk to the offspring is dependent on both a carrier status of both male and female partners. The exception is with X-linked genes where only male offspring are affected regardless.

“the common reporting of VUS presents a difficult dilemma”

- Do the authors have a reference for how commonly VUS are reported in the prenatal setting?

We have cited a reference that states that 41% of patients had a VUS reported.

“The ongoing reclassification of VUS, coupled with conflicting reporting by testing laboratories of the pathogenic significance of variants has led to confusion regarding the need for preconception testing of embryos for the variant in question (3).”

- The authors repeatedly reference laboratory reporting of discordant classification of the same variant. This is a separate issue to VUS reclassification and although it is examined in the methods of this study, may not contribute to ultimate genetic counselling. Can the authors please define the significance of variant discordance within the context of the study aims.

Later on in the introduction, we state “how should the patient be counselled when the variant is reported as VUS by one or more labs and as P or LP by another lab(s)… Should conflicting reports present an opportunity to perform PGT-M?” This is the ultimate conflict that reproductive endocrinologist often have.

- “We did not report on those VUS’s in conflict with a benign and likely benign classification as our focus was on those VUS that are actionable leading to potential use of PGT-M.” – I note this as the final statement of the Discussion which provides useful information about the aim of the paper overall and would be important in the Introduction to clarify the aims.

Thank you for this comment. We have added the statement to further clarify this in the last sentence in the introduction.

“There is a dual dilemma; first, how should the patient be counselled when the variant is reported as VUS by one or more labs and as P or LP by another lab(s)”

- Genetic counselling is clinically confined to the genetic test report and the consideration of the testing centre and clinician interpretation.

We have changed this to what should the provider in this case, as opposed to how the patient should be counselled. Conflicting reports on a variant make genetic counseling difficult, since the provider cannot be certain as to the significant of the variant, therefore inevitably setting the stage for confusion and frustration on the receiving end that is the patient. In the lack of clear genetic authority determining whether the variant is pathogenic (LP or P), this confusion is unsettling.

“The second dilemma is how to relate to a VUS reported, for instance, for a cancer gene, where there is a significant familial history of the cancer associated with the gene (e.g., breast/ovarian cancer and BRCA1/2; colon cancer and APC).”

- This would require clarification as the authors previously state that the ACMG recommended gene panel for preconception genetic testing is the foundation of this study. However, cancer associated genes are not included in this panel. Can the authors please clarify this?

We have removed this statement, as our main focus was on the ACMG recommended panel.

Materials and methods:

“The top 10 genes with the highest number of VUS’s per gene as well as the highest percent in conflict (defined as a VUS in conflict divided by the total number of VUS) were compared over the 3 years.”

- Why did the authors restrict their analysis to 10 genes?

For the sake of high incidence reporting, we chose the genes with the most frequent VUS as we have done so in previous published reports on genetic screening.

- Why did the authors confine the time period to 3 years of data?

The 3 years of data was all the data that was available on Clinvar Miner at the time of collection. The year of the pandemic (2020), there was minimal data collection.

- Discordant submissions in ClinVar are not the same as VUS classification of variants and the authors should note the distinction

This is stated in the material and methods: “data on the number of VUS’s in conflict (defined as those reported as VUS by one submitter and as P or LP by another) were obtained.”

- the methods could be improved by comprehensively examining VUS detected for genes within the recommended gene panel

We did examine the VUS for the recommended 113 pre-conception gene panel.

Discussion:

“VUS disclosure has the potential to increase parental distress as well create difficulties for genetic counselors as to how to advise patients (15). I”

- This distress seems to arise from the disclosure of CNV VUS detected during NIPT for an active pregnancy. Do the authors feel that this may extend to preconception counselling for what may be non clinically relevant VUS in recessive genes?

Similar concerns exist with the presence of the unknown significance of a variant whether in the preconception as well as the prenatal periods. Patients planning on having a child through ART who are being told that a variant is not actionable and may actually be inherited by their offspring may feel distressed and concerned about the genetic health of their baby, especially if that variant is reported as pathogenic by a different lab than they were tested in. This distress can be inherent in them before and during the entire pregnancy.

“. Our disease (cancer included) prevention mission is handicapped by the currently inefficient setting”

- Again, can the authors please clarify what preconception carrier testing methods report pathogenic or likely pathogenic variants in cancer related genes?

We have removed this as we did not look at cancer genes.

“Reporting to ClinVar is not mandatory”.

- However ClinVar still represents a rich repository of human genetic variation with over 4 million genetic variants submitted. It has proven use in the literature as an adjunct to variant classification and therefore does not represent a limitation of variant classification or reclassification.

We have removed this comment.

“We may assume that given the exponential increase in VUS and VUS-in-conflict reporting, these numbers are only going to increase several-fold within 5,10 and 20 years.”

- Standardised guidelines and improved sequencing and bioinformatic techniques are resolving unsolved variation within the human genome. Do the authors have a reference to support this statement? Otherwise, it is in conflict with the current published literature.

Yes, we have references to support the statement and have added them. (References 26 and 27)

“What will happen if VUS’s in conflict reach 25% of all cases?”

- Can the authors clarify the implications of this statement? What would happen? And is it likely to happen?

We have removed this statement as well.

“Currently, many genetic labs do not report all VUS’s and therefore, the true incidence of potentially upgradable VUS’s is difficult to determine but likely, involves many more under-reported cases.”

- The study methods use ClinVar miner which is taken from ClinVar. ClinVar is a variant submission database, not a genetic test report database. Therefore, submitting labs and facilities submit any variant uncovered during genetic testing, and not just those that are clinically relevant for the test performed. Therefore this statement is inaccurate as VUS within ClinVar are more likely to closely represent all VUS detected during genetic diagnostic testing and research settings and not just those that reach the eyes of the clinician and patient on the final genetic test report.

We very much appreciate your insight into the ClinVar reporting. We have removed the statement. This raises a significant clinical and ethical question. First, should a patient be unaware of the existence of a VUS that is not reported to her or him in the lab results but is reported to Clin Var. Secondly, what if the unreported VUS that is sent to ClinVar but is blinded to the patient conflicts with the same variant that is reported as LP or P by another lab. This phenomenon puts both the provider as well as the patient at a disadvantage.

Attachment

Submitted filename: Respose to reviewers_PLOS.docx

pone.0308771.s004.docx (21.8KB, docx)

Decision Letter 1

Nejat Mahdieh

10 Mar 2025

The Impact of VUS Reclassification on Reproductive Decision Making

PONE-D-24-31357R1

Dear Dr. Peyser,

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Academic Editor

PLOS ONE

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Reviewer #3: All comments have been addressed

**********

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Reviewer #3: Yes

**********

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Reviewer #3: Yes

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Reviewer #3: Yes

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Reviewer #3: Yes:  Farnoosh Emami

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Acceptance letter

Nejat Mahdieh

PONE-D-24-31357R1

PLOS ONE

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. ClinVar_Data_ASRM22_REAL.

    (XLSX)

    pone.0308771.s001.xlsx (156.3KB, xlsx)
    Attachment

    Submitted filename: Preconception VUS.pdf

    pone.0308771.s002.pdf (61.5KB, pdf)
    Attachment

    Submitted filename: Respose to reviewers_PLOS.docx

    pone.0308771.s004.docx (21.8KB, docx)

    Data Availability Statement

    https://clinvarminer.genetics.utah.edu/ Within clinvarminer, searching by specific gene will display the number of VUS, pathogenic and likely pathogenic variants for each gene for each year of interest can be found.

    Articles from PLOS One are provided here courtesy of PLOS

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