Two Different NF1 Pathogenic Variants in a Family With Neurofibromatosis Type 1

Abstract

Background/Aim

Neurofibromatosis type 1 (NF1) is a genetic disorder with an incidence of approximately one in 3,000. More than half of the patients have new de novo pathogenic variants of the NF1 gene. In most family cases, all family members share an identical NF1-variant. The aim of the study was to investigate the very rare phenomenon of de novo variants in cases of familial neurofibromatosis type 1 and highlight its implications for genetic testing and counseling.

Patients and Methods

Patients underwent clinical examination in our NF outpatient clinic and genetic testing for the NF1-gene was performed by targeted sequencing. All family members were profiled by short-tandem repeat marker analysis. Additionally, a probability calculation was performed for this extremely rare event.

Results

In one NF1 family consisting of mother, father, and two sons, two different pathogenic variants of the NF1 gene were found. The father and one son share one NF1-variant and the other son carries a different de novo NF1-variant. Neither of these two NF1-variants was found in the unaffected mother. Short-tandem repeat analysis confirmed the paternity and revealed that the two sons inherited two different NF1-alleles from their father. The probability of two different NF1-variants occurring in one family is calculated as 1:9,000,000.

Conclusion

Two different NF1-variants in one family is an extremely rare phenomenon: yet its occurrence is not impossible and therefore should be considered in genetic diagnosis and counselling. For an offspring with the indication for neurofibromatosis type 1, but lacking the familial pathogenic variant, a screening of the whole NF1-gene is necessary to detect potential new pathogenic variants and for exact diagnosis.

Neurofibromatosis type 1 (NF1) is an autosomal dominantly inherited tumor predisposition syndrome with an incidence of approximately one in 3,000 newborns (1,2). It is characterized by multiple café-au-lait macules, freckling of skin folds, Iris Lisch nodules and tumors of the central and peripheral nervous system (e.g., neurofibromas) (3,4), whereby the expression of clinical features varies considerably (5). Cutaneous and subcutaneous neurofibromas, the hallmark of neurofibromatosis type 1, usually start to develop rapidly in puberty (6-8). Therefore, in early childhood, NF1-stigmata are limited. Current diagnostic criteria recommends a genetic test for the NF1 gene (9).

More than half of the neurofibromatosis type 1 patients do not have a family history and therefore carry de novo pathogenic variants in the NF1 gene (10-13). For them, a genetic testing has to cover the entire NF1 gene. Other neurofibromatosis type 1 patients have a family history, meaning that either the mother or father is affected by the disease. For families with a known pathogenic NF1-variant in an index patient, other members usually only need to be tested for this familial variants in a certain exon of the NF1 gene. A full screening of the whole NF1 gene is not the routine practice.

However, there are very few cases in which two different pathogenic NF1-variants were observed in one family (14-17). The present study reports another such family where two independent, disease-causing NF1-variants were detected in two siblings. Our finding underlines the importance of screening for the whole NF1 gene at the slightest clinical suspicion to find potential new pathogenic variant and for exact diagnosis.

Patients and Methods

Patients were examined in our NF outpatient clinic at the Department of Neurology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Clinical examination was carried out by a board certified neurologist. Diagnosis of neurofibromatosis type 1 was made on the basis of clinical data collected in 2023 and 2024 and with regard to the revised diagnostic criteria for neurofibromatosis type 1 from Legius et al. 2021 (9).

Panel sequencing. The NF1 gene was sequenced using our custom panel with amplicons designed on the Illumina (Cambridge, UK) platform for AmpliSeq (https://designstudio.illumina.com/). Libraries containing the amplicons were prepared using the AmpliSeq Library PLUS kit and sequenced on an iSeq-100 (both from Illumina, Cambridge, UK). The resulting data were evaluated by an integrated “amplicon analysis module” which gave variants that deviated from the reference sequence. After subsequent manual evaluation, recurrent polymorphisms were excluded and potentially pathogenic variants were identified. These potential pathogenic variants were confirmed by means of Sanger sequencing (18) for corresponding exons. The genomic position is given according to the GRch37 genome and the variants are given in the HGVS nomenclature (http://hgvs.org/mutnomen). For a known variant in a family, only the corresponding exon was sequenced by Sanger method (18).

Short-tandem repeat marker analysis. To confirm the paternity and maternity of the family, all four members were profiled for a total of 16 short-tandem repeat markers (13 CODIS core loci plus D2S1338, D19S433 and Amelogenin) using an investigator IDplex kit (Qiagen, Hilden, Germany) (19,20). In brief, DNA from the 4 family members were amplified with fluorescence-labeled primer pairs in the kit which were optimized for the 16 markers. The amplified markers were separated on a SeqStudio Genetic Analyzer (ThermoFisher Scientific, Waltham, MA, USA).

In addition, to examine whether or not the two sons inherited the same NF1-allele from the affected father, the family members were also profiled for three short-tandem repeat markers within and therefore linked to the NF1 gene (13,21). Primers for amplifying these markers were designed by ourselves and the amplified markers were analyzed on the SeqStudio as described above.

Probability calculation. The de novo mutation rate of the NF1 gene is set as 1/3,000. Two different de novo variants were considered as independent events. Also inheriting a variant and acquiring new variant were considered as independent events.

Consent. Written informed consent was obtained from the patients. The study was carried out in compliance with the Helsinki Declaration.

Results

The index patient, the elder son (age 12) was diagnosed as having neurofibromatosis type 1 clinically in his early childhood (Table I). He showed multiple café-au-lait macules, bilateral freckling and clinical investigation showed incipient growth of cutaneous and subcutaneous neurofibromas. In 2019, a genetic testing was carried out and found a stop-gaining pathogenic variant in the NF1 gene in exon 42. In 2023, the younger son (age five) of the family exhibited typical childhood signs of neurofibromatosis type 1 (Table I), meaning multiple café-au-lait macules, bilateral freckling and incipient cutaneous neurofibromas. Therefore, he was clinically diagnosed as also having the disease. However, the subsequent genetic testing regarding the familial pathogenic NF1-variant in exon 42 was negative. Consequently, the genetic and clinical findings did not match for the younger son.

Table I. Clinical and genetic characterization of patients included in the study.

To exclude potential mix-up of the samples, blood was taken anew from all four family members and DNA from this blood was sequenced for all NF1-exons using our custom panel. The NF1-variant in exon 42 was confirmed in the index patient (the elder son) with a variant-frequency of 50%. However, it was again not found in the younger son. Interestingly, a different pathogenic variant p.(Leu585fs) in exon 16 of the NF1-Gene was found in the younger son with an allele frequency of 53% (Table I).

Clinically, the parents have not been previously examined for neurofibromatosis type 1. To clarify the situation in the two sons and possible inheritance, DNA of both parents was also included in the panel-sequencing. The father turned out to have the pathogenic NF1-variant in exon 42, same of that in the elder son. Subsequent clinical examination of the father found multiple café-au-lait macules, bilateral freckling, cutaneous and plexiform neurofibromas, and confirmed the diagnosis of neurofibromatosis type 1 (Table I). As for the mother, none of the two variants was found in her DNA and clinical findings were also negative for neurofibromatosis type 1. According to the parents, they both are the biological parents of two sons. Results of short-tandem repeat analysis for CORDIS also supported their paternity and maternity. Three markers within the NF1-gene revealed that the two sons inherited different NF1-alleles from their father. The younger son inherited the intact NF1-allele from his father. The de novo NF1-variant in exon 16 occurred newly in him, either on the paternal or maternal NF1-allele.

Assuming that inheritance and a de novo variant are independent events, and considering the incidence of neurofibromatosis type 1 as approximately 1:3,000 in newborns (1), the probability of two different pathogenic NF1-variants within one family can be estimated as 1/3,000 * 1/3,000=1/9,000,000.

Discussion

We hereby described our finding of two different pathogenic NF1-variants in a family with neurofibromatosis type 1. The first variant in exon 42 of the NF1 gene was already in the father and was inherited by the elder son (the index patient). The second variant in exon 16 of the NF1 gene in the younger son was a newly occurred de novo variant.

Two distinct pathogenic NF1-variants within one family is a statistically highly unlikely condition with a probability of one in nine million. This is in concordance with our own experience in genetic diagnosis over decades, since we have never observed such a situation before.

However, though extremely rare, this condition can still happen. There are also few previous described cases of similar situations (14-17). These findings highlight that genetic data needs to be interpreted together with clinical ones. Particularly in early childhood, when the disease-related stigmata are limited (8), a negative finding of familial variant can lead to a false negative diagnosis. In this familial case, the younger sibling was already five years old and showed relatively clear clinical features of neurofibromatosis type 1. Significantly younger offspring of NF1 patients often may not fulfill the clinical diagnostic criteria of NF1 (8). Therefore, should a negative finding of familial variant not match the clinical indication, a full sequencing of the entire NF1 gene is necessary. Also repeated clinical examination several years later is recommended. Generally, a genetic report for a family member not having the familial NF1-variant, the statement “the disease neurofibromatosis type 1 can be excluded” is not proper. Instead, “a disease neurofibromatosis type 1 caused by the tested pathogenic NF1-variant can be excluded” is recommended.

Though fulfilling the diagnostic criteria and having multiple disease-related symptoms, the father has never been diagnosed for NF1 before. This is likely because his symptoms did not cause serious clinical deficits. Nevertheless, a proper diagnosis is important for monitoring the clinical course and detect severe manifestations, such as malignant peripheral nerve sheath tumors, pheochromocytomas or breast cancer, at an early stage. As for example pheochromocytomas are a rare entity, but occur more often in neurofibromatosis type 1 (22), screening and monitoring for these entities is linked to exact diagnosis of NF1. Also plexiform neurofibromas are possible precancerous lesions in NF1. These mostly benign nerve sheath tumors can show morphological differences which may reflect in part biological differences (23) and can turn into malignant peripheral nerve sheath tumors. Therefore, NF1-related tumor manifestations should be monitored consequently and genetic diagnosis in unclear clinical cases is of great importance.

For ascertained children, a simple anamnesis may also be addressed to their parents, such as regarding café-au-lait, freckling and lesions that are suspicious for neurofibromas type 1 which they can even easily check by themselves. By finding any indication, further professional diagnosis and genetic testing can then be recommended.

Conclusion

Two NF1-variants in one family is extremely rare yet can happen and should be considered in genetic diagnosis and counselling. This finding is particularly important as it underscores the complexity of genetic variability in neurofibromatosis type 1. For an offspring with indication for neurofibromatosis type 1 but not having the familial pathogenic variant, a screening of the whole NF1 gene is necessary for finding potential new pathogenic variants, especially in early-childhood, when few cutaneous stigmata of neurofibromatosis type 1 are present and clinical diagnostic criteria are not fulfilled. During this period, it can be challenging to establish a definitive diagnosis, which is why identifying new pathogenic variants is of great importance, even in familial neurofibromatosis type 1. Only an exact diagnosis enables medical monitoring of patients and the identification of NF1-specific risks.

Conflicts of Interest

None to declare.

Authors’ Contributions

Tabea I. Hartung: Conceptualization, Investigation, Methodology, Project administration, Writing – original draft, Writing – Review & Editing. Lan Kluwe: Conceptualization, Methodology, Writing - Review & Editing. Reinhard E. Friedrich: Writing – Review & Editing. Said C. Farschtschi: Conceptualization, Writing - Review & Editing.

Acknowledgements

The Authors thank Ina Alster from the Laboratory for tumor genetics (Department of Neurology, University Medical Center Hamburg-Eppendorf) for her help in sample processing and Susan Eick (Department of Neurology, University Medical Center Hamburg-Eppendorf) for critically reading the manuscript as a native speaker. Furthermore, we thank Hans Pinnschmidt from the Center for Experimental Medicine (Institute of Medical Biometry and Epidemiology, University Medical Center Hamburg-Eppendorf) for his consultation in probability calculation.

Funding

The iSeq-S100 was donated by the “Bundesverband Neuro-fibromatose”.