Received: 12 June 2020 Revised: 21 September 2020 Accepted: 26 September 2020

DOI: 10.1002/ajmg.a.61976

RESEARCH LETTER

Empirically downgrading 10 constitutional missense variants of

the NF1 gene based on co-existing truncating variants

Lan Kluwe | Victor F. Mautner

Department of Neurology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Correspondence
Lan Kluwe, Laboratory for tumor genetics, Building O48, 4th floor, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20251 Hamburg.
Email: kluwe@uke.de

Funding information
The German Bundesverband Neurofibromatose

To the Editor
A considerable proportion of missense variants belong to the “variants
of uncertain significance” and therefore pose a challenge for genetic
diagnosis (Richards, Aziz, Bale, et al., 2015). Estimation largely relies
on calculation-based tools. However, empirical, experimental, func-
tional, and clinical data are essential. In the case of the NF1 gene
which is involved in the genetic disorder neurofibromatosis type
1 (NF1, MIM 162200; Ferner et al., 2007), empirical, genetic and clini-
cal data for pathogenicity for some recurrent missense NF1 variants is
accumulating (Jang et al., 2016; Koczkowska et al., 2019; Koczkowska,
Callens, Chen, et al., 2020; Pros et al., 2008). In contrast, empirical
data for downgrading missense NF1 variants is lacking.
We conceived a strategy to accumulate empirical data for down-
grading some missense variants. We searched our dataset for cases
with a missense and a co-existing truncating NF1 variant including non-
sense, frameshift and canonical splice variants, deletions of exons, and
deletions of the whole NF1 gene. A total of 355 such truncating variants
and 72 variants of unknown significance were identified in our cohort.
Among the 72 patients with NF1 variants of unknown significance,
10 had co-existing truncating NF1 variants, providing empirical supporting
data for downgrading these 10 missense variants (Table 1). All these vari-
ants were confirmed by repeated Sanger sequencing for the corresponding
exons using DNA extracted from different batch blood of the patients.
Theoretically, it is possible that some of these 10 missense NF1
variants are pathogenic and coincidently occurred in the same individ-
ual with a truncating variant. However, the probability of such events
should be low for the following reasons. First, we are not aware of
any such cases in the literatures and databases. Second, bi-allelic

TABLE 1 Ten empirically downgraded missense NF1 variants and the co-existing truncating variants

Case Missense variant (protein change) Frequencya Co-existing truncating variant (protein change)
1 c.865G>C (p.Val289Leu), exon 8 0.000191 c.2038delT (p.Cys680fs)
2 c.1166A>G (p.His389Arg), exon 10 0.0000796 c.1541_1542del (p.Gln514fs)
3 c.1756A>C (p.Thr586Pro), exon 16 Not given c.305del (p.Met102fs)
4 c.1975C>T (p.Arg659Trp), exon 17 0.00000797 c.1756_1759del (p.Thr568fs)
5 c.2131C>T (p.Arg711Cys), exon 18 0.00000398 c.6428del (p.Leu2143fs)
6 c.2870A>G (p.Asn957Ser), exon 22 Not given c.2875C>T (p.Gln959*)
7 c.3377A>T (p.Gln1126Leu), exon 26 Not given c.4111-2A>G (splicing)
8 c.5200A>G (p.Ile1734Val), exon 36 0.00000412 c.6888G>A (p.Trp2296*)
9 c.6618C>A (p.Asp2206Glu), exon 43 Not given c.2827A>T (p.Lys943*)
10 c.6913G>C (p.Asp2305His), exon 46 0.00000795 c.79C>T (p.Gln27*)
a
Given in gnomAD (https://gnomad.broadinstitute.org/).

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© 2020 The Authors. American Journal of Medical Genetics Part A published by Wiley Periodicals LLC.

602 wileyonlinelibrary.com/journal/ajmga Am J Med Genet. 2021;185A:602–603.

KLUWE AND MAUTNER
inactivation of the NF1 gene (two pathogenic variants in trans) is lethal
as shown in the mouse studies. Third, not a single case with two trun-
cating NF1 variants has been observed in our patient cohort despite
the fact that truncating variants are far more frequent than missense
variants in the same cohort (355 vs. 72).
A limitation of the present study is the lack of detailed clinical and
family data. In addition, we do not know the cis/trans relation of the
two variants in each case. We will continue to address these issues in
more comprehensive future studies and hope that increasing data will
be accumulated from more laboratories and research groups.
In summary, our findings add empirical supportive data for down-
grading 10 NF1 variants of unknown significance which may contrib-
ute to diagnosis and to improve predicting algorithms.
ACKNOWLEDGMENTS
We thank Mrs. Alster for carrying out the variant analyses. We also thank
Mrs. Eick, who is a native English speaker, for correcting the language.
Open access funding enabled and organized by Projekt DEAL.
CONF LICT OF IN TE RE ST
None to disclose.
ETHI C AN D CONSENT
Written consent from all patients for the genetic diagnosis and for
publishing their data in anonymized form.
DATA AVAI LAB ILITY S TATEMENT
Data are presented in the manuscript. Additional informations such as
experimental conditions are available upon request.
ORCID
Lan Kluwe https://orcid.org/0000-0001-9919-4551
603
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How to cite this article: Kluwe L, Mautner VF. Empirically
downgrading 10 constitutional missense variants of the NF1
gene based on co-existing truncating variants. Am J Med Genet
Part A. 2021;185A:602–603. https://doi.org/10.1002/ajmg.a.
61976