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Reflections About Ludwine Messiaen: A Pioneer Who Advanced NF and Other Women in NF

By September 18, 2024September 20th, 2024Awareness, Featured, Featured EU, Science & Research

Personal Reflections from Four of Her Female Mentees

Discover more articles in the Women in NF series by clicking here.

Introduction

A smiling woman with short gray hair and orange-framed glasses on her head, wearing a pink patterned scarf and a black jacket. The background shows an outdoor, wooded area.

Ludwine Messiaen, PhD

When Dr. Meena Upadhyaya contacted Dr. Ludwine Messiaen and asked her to provide her reflections on NF, she was no longer in a health condition that would have allowed her to provide an essay to this series. After her passing, it was clear to the organizers of this series that the essay series should be dedicated to her to pay tribute to her outstanding contributions to the field. Now her warm and colorful smile heads the essay series, but visitors from outside the NF community would not know what enormous impact Ludwine’s work had on the progress of the NF field. Ludwine has been a role model as a female researcher and mentor of several female researchers, including the four authors of this essay. Hence, we, Kathleen Claes, Alicia Gomes, Magdalena Koczkowska, and Katharina Wimmer, would like to express our gratitude to have the opportunity to become mentees and over the years good friends of Ludwine with personal statements on the impact she had on our careers. We also hope to provide an earnest and appropriate essay summarizing Ludwine’s main contributions to NF and give some insight into her career and personality.

Essay on Luwine Messiaen’s Main Contributions to the NF Field

Even those who collaborated with Dr. Ludwine Messiaen over many years, may not know that Ludwine began her career in social work after receiving a bachelor’s degree in social work. This background speaks to the empathy and compassion that Ludwine continued to carry into her future genetics pursuits. She then moved on to study molecular biology and cancer genetics at Ghent University in Belgium. In her PhD thesis, which she completed in 1990, she studied oncogenic transformation. Thereafter, Ludwine joined the Center for Medical Genetics at Ghent University Hospital where she set up the first laboratory for molecular diagnostics.

At that time, the NF1 gene had just been identified. With her innovative spirit, Ludwine decided to establish diagnostics for this gene because she considered it an intriguing challenge to develop and offer testing for a ‘new gene’ that was complex with an array of mutations. The early 1990s were still the time of pre-screening assays. Transcript analysis using the protein truncation test (PTT) was probably an effective way to circumvent the multiple non-processed NF1 pseudogenes and identify truncating mutations in the NF1 gene. (1) Ludwine understood that she should use this assay and immediately found interesting splice variants with this assay. (2, 3) However, as a meticulous perfectionist, she aspired to identify the underlying variant for all patients. She noticed the obstacles inherent to any mutation analysis protocol that is based on mRNA analysis, i.e. nonsense-mediated decay and illegitimate splicing, and the limitations of the protein truncation test in detecting missense and other non-truncating mutations. Therefore, she developed a comprehensive testing algorithm applying a cascade of complementary assays, with the core assay of the PTT using RNA from puromycin-treated EBV cells. In 2000, she published a hallmark paper showing that with this comprehensive approach, pathogenic NF1 variants were found in 95% of all familial NF1 cases.(4) This was by far the highest mutation detection rate published so far. With all 64 pathogenic variants characterized both at DNA and RNA level, this study also showed that the rate of splice mutations in this gene is 30%, and 2/3 of these mutations are located outside the canonical GT-AG dinucleotides at the 5’ and 3’ splice site, respectively. This still held true 21 years later, when Ludwine retired and her Medical Genomics Laboratory (MGL) at the University of Alabama at Birmingham had identified pathogenic variants in over 10,000 NF1 patients. With this publication, cited 391 times in August 2024, Ludwine set the foundation to further direct not only her future career, but set a compass for many researchers and clinicians to continue to advance the field. It was clear for many researchers and clinicians that her lab needs to be contacted to identify a mutation in any challenging case. Such cases were for instance two large families with familial spinal NF1 in whom her lab identified the underlying NF1 pathogenic variants and, hence, showed that the spectrum of NF1 pathogenic variants causing familial spinal NF1 is diverse.(5)

With her PhD student Ina Vandenbroucke, Ludwine continued to characterize the spectrum of naturally (not mutation induced) NF1 splice variants and found that NF1 exon 52 (historically exon 43) is alternatively spliced and contains a nuclear localisation signal. Interestingly, until now no pathogenic missense variant has been identified in this exon.(6-9)

Performing NF1 molecular diagnostics, Ludwine inevitably got confronted with mosaic NF1, another diagnostic challenge she eagerly started to tackle. For one of her first cases she developed direct cDNA sequencing from three ultra-long RT-PCR products, which at that time allowed to identify a multi-exon intragenic NF1 deletion in an EBV culture from blood leukocytes of a mosaic NF1 patient.(10)  This assay remained for many years the corner-stone of the comprehensive NF1 diagnostics program.(11) However, it became clear that in many mosaic patients, in particular those with segmental NF1, the underlying postzygotic pathogenic NF1 variant could only be uncovered in affected tissues and, in these, probably only in specific cell types. From a seminal finding published in the year 2000, by Drs. Eduard Serra, Thorsten Rosenbaum and Conxi Lázaro -Ludwine had always acknowledged their ground-breaking finding in her presentations- it was known that in neurofibromas, which consist of different cell types, Schwann cells are the true neoplastic cells.(12) This finding was facilitated by the development of specific cultivation conditions that enrich for NF1-/- Schwann cells, which harbour the first and the second NF1 hit, in agreement with Knudson’s two hit hypothesis. Ludwine understood that cultivation of these cells should be an effective way to identify the postzygotic NF1 pathogenic variants underlying mosaic NF1 patients. She also wanted to better understand the pathoetiology of café au lait spots in NF1 patients. Since mosaic NF1 patients may have only café au lait macules, the aim was also to understand which cells are the cells-of-origin that need to be cultivated from café au lait macules to identify the underlying postzygotic NF1 pathogenic variants of mosaic patients with this manifestation only. Therefore, she set up a collaboration with the dermatology department at the Ghent University Hospital and co-promoted the PhD theses of Jo Lambert and Sofie De Schepper, who are now both professors at this Department.

These efforts started around the time when Ludwine was recruited by Prof. Bruce Korf, chairing at that time the Department of Genetics at the University of Alabama in Birmingham (UAB), to build up and direct the Medical Genomics Laboratory (MGL) and join the faculty of UAB as full professor. Together with her husband, she decided to move with the entire family of three young children from small Belgium to the USA. This was not only an enormous change and challenge for the entire family, but also for her co-workers and students in Ghent. Whilst her long-term technician, Tom Callens, decided to move to Alabama and continue to work with her in the MGL, her PhD student at the time, Ophelia Maertens, stayed in Ghent, but a close collaboration was set up with the laboratory of Prof.  Eric Legius at the Catholic University of Leuven in Belgium, who co-supervised her with Ludwine. This was also the starting point of a long and extraordinarily successful collaboration between Ludwine and Eric Legius and the installation of the “Leuven meetings”. In addition to Ludwine’s and Eric’s students, a growing number of additional collaboration partners were invited to these meetings supported by a “Scientific Research Networks” grant from Flander’s research council (FWO) and initially awarded for 5 years. Due to the open, friendly, and scientifically highly stimulating atmosphere created by the organizers, Eric Legius and Hilde Brems, the scientific outcomes were so impressive that they were able to renew this grant twice, allowing them to organise these special and memorable scientific meetings for 15 years.

Two findings with enormous impact ensued from the collaborative efforts lead by Ludwine. First, the molecular dissection of isolated disease features in mosaic NF1, as the title of the manuscript reads, proved that comprehensive NF1 testing in phenotype–related cell types, i.e. Schwann cells cultivated under specific conditions from neurofibromas and melanocytes cultivated from café au lait spots, facilitates clinical diagnosis and further genetic follow-up in mosaic patients.(13) Her new laboratory at UAB subsequently offered this approach as a diagnostic service, the only one in the U.S. Hence, many mosaic NF1 patients from different continents profited from this finding. Secondly, the analysis of different café au lait macules derived cell types showed that melanocytes, but not keratinocytes or fibroblasts, have biallelic NF1 gene inactivation due to second hit-mutations as seen in neoplastic cells of NF1-associated tumours.(14)  This finding lead to a paradigm shift, because previously, it was thought that “non-neoplastic” features of NF1, such as café au lait macules, result from neurofibromin haploinsuficiency. Hence, this finding fostered genetic studies aimed at identifying the cells-of-origin having second hit NF1 mutations also on other “non-neoplastic” NF1 manifestations, such as pseudarthrosis.(15)

Coming to a country with a large and diverse population, Ludwine as an innovative and meticulous planner had the clear vision from the beginning to develop a large database with as many patients as possible, comprehensively and thoroughly characterized both at the genetic and phenotypic levels. Therefore, the MGL at UAB offered comprehensive and individualized NF1 diagnostics from the start at a fair price. In addition, unflinchingly and adamantly Ludwine insisted on including a well thought-out questionnaire for the clinical phenotype of the patients, which became the foundation for several important findings.

In June 2007, Hilde Brems and Eric Legius identified  SPRED1 as a gene that caused an NF1-like syndrome, which we now know as Legius syndrome.(16) Ludwine had already contributed to this work and the MGL at UAB offered SPRED1 testing from August 2007. Most importantly, with nearly 2500 phenotypically well-characterized patients in their database, Ludwine’s lab could now test all patients in whom no pathogenic NF1 variant was identified after comprehensive testing for this possible alternative diagnosis. This seminal study not only swiftly delineated the clinical and mutational spectrum of Legius syndrome in 40 individuals from 22 families carrying a pathogenic SPRED1 loss-of-function (LOF) variant, but also defined the NF1 and SPRED1 mutation detection rates in different phenotypic patient cohorts.(17) Hence, the paper still is the reference paper for diagnostic labs offering NF1 diagnostics.

Ludwine was undoubtedly recognized as a worldwide leader in NF1 genotype-phenotype studies. NF1 is known for its clinical variability, both inter- and intra-familial, its age dependency, and the enormous NF1 allelic heterogeneity, making it challenging to establish new genotype-phenotype correlations. Ludwine understood that to identify mutation-specific, clinically significant genotype-phenotype correlations in NF1, it is pivotal to collect a large datasets of patients, preferably postpubertal, with the same constitutional pathogenic variant, and to record the associated phenotype in a standardized way. Although she initially identified several genotype-phenotype correlations using her extensive UAB database, which at that time included more than 8,000 unrelated individuals molecularly diagnosed with NF1, she sought collaboration with numerous clinicians and molecular geneticists worldwide to enhance the statistical significance of her findings. Her meticulous work style and commitment to having referring physicians carefully collect and verify detailed phenotypic data upon submission of samples for NF1 genetic analysis in her lab, along with her openness to international collaboration, led to the identification of several clinically significant genotype-phenotype correlations.(18-21) This substantial effort was recognized by the Children’s Tumor Foundation, which awarded her the Isaac and Sadie Fuchs Genotype-Phenotype Study grant to support her promising research.

In 2015, research led by Ludwine on a large cohort of 136 NF1-affected patients from 98 unrelated families, each carrying one of five different missense pathogenic variants at the NF1 p.Arg1809 codon, showed that these individuals typically presented with mild phenotype, including the presence of café au lait macules, skinfold freckling and/or Lisch nodules, but without externally visible neurofibromas. However, a high incidence of Noonan syndrome features was observed in these individuals.(21) As Ludwine used to say, “surface has only been scratched”, and indeed, a few years later, together with her postdoctoral fellow Dr. Magdalena Koczkowska, Ludwine published another significant genotype-phenotype correlation in NF1. They discovered that heterozygous missense variants in one of the five neighbouring codons 844-848 in the NF1 gene, located in the cysteine serine rich domain (CSRD) of neurofibromin, were associated with a severe phenotype. This included a higher likelihood of developing malignancies, particularly with the NF1 p.Leu847 missense variants, compared with the general NF1 population.(20) Through this specific genotype-phenotype correlation study, Ludwine also paved the way for other research teams. The specific function of the NF1 codons 844-848 remained unknown until 2023 when it has been shown that dimerization with wild-type neurofibromin might explain why the 844-848 missense variants act as dominant negative mutants and result in a severe phenotype.(22) Ludwine did not stop there. Her subsequent work, published a year later, provided an extended clinical update on a large cohort of NF1-affected patients with the in-frame deletion p.Met992del in the NF1 gene, which had been originally reported in 2007 by Upadhyaya et al (23).(19) Finally, in 2021, she demonstrated that variants at codon positions p.Met1149, p.Arg1276 and p.Leu1423 were associated with an increased chance of Noonan-syndrome clinical features, especially developing pulmonic stenosis, similar to p.Arg1809. Additionally, p.Arg1276 pathogenic variants were found to be frequently associated with spinal neurofibromas. (18)

In addition to her work on NF1, Ludwine also contributed to the field of schwannomatosis. Together with Dr. Arkadiusz (Arek) Piotrowski from the Medical University of Gdansk and Dr. Jing Xie from UAB, Ludwine aimed at identifying the underlying genetic alterations in familial and sporadic, well-characterised patients with schwannomatosis not attributable to (mosaic) pathogenic variants in the known schwannomatosis genes at the time, NF2 and SMARCB1. Given that the schwannomas of these patients show loss of the long arm of chromosome 22, the research hypothesis was that other gene(s) involved in the molecular pathogenesis of schwannomatosis might be located on chromosome 22. Using therefore next-generation-sequencing of evolutionarily highly conserved sequences specifically on chromosome 22, they identified pathogenic variants in LZTR1 in 6/6 familiar and 8/11 sporadic unrelated schwannomatosis patients, demonstrating that LZTR1 is a novel tumor suppressor gene that when having a pathogenic alterations predisposes individuals to multiple schwannomas.(24)

With her large data set of phenotypically and genetically well-characterized patients with NF1 and SPRED1 mutations Ludwine also supported the contributions to the field of many other researchers. Here we can mention only some of these. She continued to support the work of Drs. Akito Yoshimura, Hilde Brems and Eric Legius in defining the functional domains of the SPRED1 protein.(25) Furthermore, she contributed to the efforts of Dr. Hildegard Kehrer-Sawatzki who was the leading person in delineating the spectrum of NF1 microdeletions.(26-34) Dr. Katharina Wimmer will describe in her personal statement below how Ludwine supported her work in characterizing the pathogenetic mechanisms of splice mutations (35-38), developing tools for CNV detection by NGS data (39), characterising functional PMS2 “hybrid” alleles (40), and assessing the prevalence of constitutional mismatch repair deficiency (CMMRD) syndrome in suspected sporadic NF1/LGSS patients without malignancy and without identifiable NF1/SPRED1 pathogenic variants (41).

When CTF decided to support the effort to revise the diagnostic criteria defined in 1987 for NF1 and NF2 by the National Institutes of Health (NIH), Ludwine was one of the main drivers in this huge effort which took several years and included over 90 experts from around the globe. It is not least to her enormous knowledge and expertise as well as her accuracy and endurance that we have now up-to-date diagnostic criteria for NF1 and Legius syndrome and the schwannomatoses.(42, 43) Equally, her expertise and accuracy, as well as her open, fair, considerate, and enjoying personality, contributed much to the success of the first ”Joint Global NF Conference” held in November 2018 in Paris, France, for which she served as one of the chairs in the organization committee. During this conference, she was awarded with the CTF Friedrich Von Recklinghausen award, the highest honour in NF research.

In July 2021, Ludwine retired from her position as head of the MGL at UAB and moved back to Belgium. As a fully dedicated member of the NF community, even in retirement and during burdensome treatment, she provided together with her former mentees, Doctors Magdalena Koczkowska, Yunjia Chen, and Jing Xie, a state-of-the-art summary of the currently 75 unique deep intronic NF1 variants that have a well-documented effect on splicing and account for 2.5% of the pathogenic variants in patients with NF1.(44) They also found that the exonization of intron 31 (formally 23a) sequences due to the recurrent deep intronic variants predominantly affects the NF1 isoform II, not isoform I, and still results in a classic NF1 phenotype. These observations may provide further insights into the alternative splicing and the DNA sequences that are essential to this process.

That was her last published paper. Unfortunately, her disease and much too early passing in February 2024 prevented her from publishing her full data set of NF1 pathogenic variants characterized both at RNA and DNA levels and found in over 8,000 index patients in a reference database. She intended to leave this database as her legacy to the NF family. Even with this last piece missing, her pioneering work enormously contributed to the progress the NF community has witnessed over the past three decades, and her lab, founded on dedication and passion, was the diagnostic epicentre for the NF community. She was an inspiring mentor, colleague, and true friend whose legacy continues to shape the future of NF research. We will undoubtedly miss her, but her memory will forever stay with those of us who had the privilege of working with her. Please see below the personal statements of four of her former mentees: Doctors Kathleen Claes, Alicia Gomes, Magdalena Koczkowska, and Katharina Wimmer.

Personal Statement of Kathleen Claes

I met Ludwine for the first time in November 1996 – I remember her appearance like it happened yesterday: green velvet dress, red lips. I applied for a position as PhD student in her lab to work on hereditary breast and ovarian cancer. I was young and nervous. She made a lasting impression.

I was very proud to be recruited and to start working in her lab. A whole new world opened up to me.

I came from another faculty (bio-engineering) and switched from plant cells to human DNA. Ludwine trained me in human molecular genetics, the protein truncation test, and the difference between RNA and DNA based testing.

I learned from her how important it is to work hard, and to dig deep to find causal mutations. She introduced us, the first generation PhD students, to the concept of multi-omics ‘avant la lettre’, combining the transcript and genomic level of NF1, before this turned out to be a very powerful approach to shorten diagnostic odysseys in rare diseases, now, more than 20 years later.

I learned from her how important it is to be a creative thinker and a problem solver. And that was needed when she announced her departure to the States. I was appointed to take over the supervision of the genetic test for NF1, which she had set up in Gent. Big shoes to fill.

Leaving Belgium with three growing children and a husband, illustrates her sense for adventure and her courage to pursue her dreams. But we stayed in touch and our contacts remained very warm. She was always open to give her expert advice on specific cases, variants, etc.

I am very grateful to her for also inviting her European colleagues to contribute to the extensive genotype-phenotype studies. Our referring clinicians were always very willing to fill in the extensive checklist as they knew they would be recognized for their efforts. Ludwine’s respect and recognition for colleagues’ expertise and input fueled a vast international network, to which she also introduced me and for which I am very thankful.

When she returned to Belgium in fall 2021, our contacts intensified. We shared great moments with lunches, dinners, wine, gossips, etc. Fantastic memories! And Ludwine kept surprising us by her immense creativity, authenticity, passion, and dedication not only for genetics but also for artistic work. Her unconventional angle, which made her such an excellent scientist, also allowed her to discover hidden things in art or nature.

Ludwine did her best in everything, also in the way she dealt with her fate. Colorful, positive, combative, dignified, empathetic. I highly value her mentorship, her (genetic) legacy in the field of neurofibromates and related disorders, her work ethics and her courage to never give up. And I strongly believe that this sentiment is shared by her beloved family, her international colleagues, and many patients for whom she made the difference. A lab technician who worked with Ludwine for several years in the Ghent period put it this way: ‘Ludwine was a remarkable person full of energy who established a fantastic career. At as same time she was a wonderful “boss” from whom I learned a lot.’

We received her irreversible epigenetic imprint, for which we are very grateful.

Four women standing side by side in front of a stone wall, smiling at the camera. They are wearing a variety of colorful and formal outfits.

October 7, 2023 in Leuven at the emeritation of Eric Legius: Great Reunion of the first generation PhD students of Ludwine (from left to right: Kathleen Claes, Ludwine Messiaen, Elfride De Baere, Ophelia Maertens).

Personal Statement of Alicia Gomes

As a new genetic counselor, looking to find my place in the world of clinical genetics, I was privileged with the opportunity to find a role in Ludwine’s laboratory. I remember my first interview with her; she was sitting in front of a large world map that had pins scattered all over the world. “Are these pins for places you have visited?” I asked. “No, these are all of the places that have sent samples to our laboratory over the years.” From this first interview, I got an immediate sense that each sample was not a business transaction for this laboratory; but a mission to help and serve the individuals behind every tube. Working with Ludwine each day, I learned so many scientific lessons. From the need to not just double check, but quadruple check any data that you plan to stand behind to ensuring that you provide the due credit to each person who has been as committed as you have been in advancing science. Beginning my career with Ludwine also provided me with the mind-set that conformity does not ensure success; sometimes you have to think outside of the box. Instead of accepting limitations as a rule, I would watch Ludwine ask her iconic question of “How comes?” and through polite negotiation, the MGL continues to try to move the needle on the types of tests that could be offered within the constraints of an academic setting. Lastly, of all of the lessons learned from Ludwine, I learned the importance of being a mother first and a scientist second. From “snow” storms to sick children, Ludwine ensured that all of our employees were able to meet the needs of their families first while still ensuring that the lab was able to provide the amazing testing everyone was used to. During our career together in the MGL, 13 babies were born to our employees and each one was celebrated and made a priority. In her legacy, I will continue to remember and share with other women considering a role in science that you do not have to decide between being an engaged mother or having an accomplished career. Instead, you continue to think outside the box, triple check everything you do, and don’t be afraid to ask “how comes.”

Two women stand next to each other, smiling. The woman on the left has short gray hair and red glasses, and the woman on the right has curly black hair. Both have an arm around the other's shoulder.

September 2022, Alicia Gomes and Ludwine Messiaen continuing to connect and mentor even after retirement.

Personal Statement of Magdalena Koczkowska

I first met Ludwine in spring 2015, when Arek Piotrowski, with whom I had briefly collaborated during my doctoral studies and who had worked closely with Ludwine, recommended me for a postdoc position in her lab. Initially, I approached the opportunity with some skepticism, as I hadn’t even dreamt of moving to the United States, but I decided to give it a try. At that time, Ludwine was giving a guest lecture at our university, the Medical University of Gdansk, so we arranged to conduct a preliminary interview with me during her visit. Even though we had access to Arek’s office, Ludwine suggested that we should take advantage of the beautiful spring weather and hold the interview in the university’s botanical garden. Immediately after the interview, which felt more like a pleasant conversation than a formal interview, Ludwine informed me that was interested in having me in her research team. I was surprised by how quickly she made her decision, but in the following years, I came to realize that Ludwine had an incredible instinct for recognizing potential in people. That’s how one of the best years of my professional career began.

I was privileged to spend three years in Ludwine’s lab. During that time, I not only gained significant scientific knowledge and experience – we co-authored three research papers focused on genotype-phenotype correlations in NF1 – but I also grew as a researcher, with Ludwine standing by my side as a true mentor. I will always cherish our meetings in her office, where before diving into research discussions, she gradually welcomed me into her private world with stories about her family, children, and passions. She made a point to know each of her team members as more than just employees. Ludwine was incredibly supportive, participating actively in both the good and challenging moments of each team member’s life. When I became a mother, I learned even more from her, as she inspired me with her ability to balance her duties as a loving mom (always putting family first) and as a dedicated researcher. What I also learned from her was the importance of celebrating small things every day.

She was that person who introduced me to the NF community, a world I knew very little about eight years ago. Thanks to her, I now have the privilege of calling myself a part of this world and have met many fantastic people, some of whom I actively collaborate with. She was my ethical role model, and ‘gratitude’ is the word that comes to my mind when I think of her. I am grateful that she trusted me enough to invite me into her circle of close team members. I am grateful to have had the privilege of meeting her on my professional way and working closely with her for almost eight years.

Two women in an office review documents at a glass desk with a world map in the background. Both are smiling and appear engaged in the discussion.

August 2018, Ludwine Messiaen and Magdalena Koczkowska in Ludwine Messiean’s office at UAB after the acceptance of one of the genotype-phenotype studies for online publication.

Personal Statement of Katharina Wimmer

I met Ludwine for the first time in September 1999 at the 8. European NF meeting in Ulm, Germany. Having started exactly a year earlier to set up NF1 diagnostics for Austrian patients, this was the first international NF meeting I attended. My start in NF diagnostics was similar to that of Ludwine several years earlier. Trained during my post-doctoral studies at the University of Michigan in Ann Arbor in cancer genetics, I was asked (in turn of receiving a research assistant position) to set up a molecular diagnostics laboratory at the Institute of Medical Biology at the University Vienna, which is now the Institute of Medical Genetics of the Medical University Vienna. However, in contrast to Ludwine it was not my decision to start with the NF1 gene, but that of the Institute’s head, Prof. Christa Fonatsch, who thought I should start with this more challenging gene. So I began also to use the protein truncation test according to the “Heim protocol”. (1)

Stimulated by a reported splice effect of a recurrent mutation which I could not observe to this extent in the patient of my cohort with this pathogenic variant, I started contemplating on the reasons for this and other splice alterations that I observed in several patients and that seemed not to be related to genomic variants. I noticed that they occurred preferentially in patient samples that were shipped to our department from other parts of Austria and less so in samples collected at our Institute in Vienna. So I reasoned that this “illegitimate splicing” is related to “aging” of the blood sample which was several days on its way until RNA was extracted in our lab. (45, 46) I made this observation just before the meeting in Ulm and was very keen to discuss it with others. However, I found nobody who was interested to talk about it until I approached Ludwine. When I told her about my findings, she immediately took my forearm with her small hand and she said “Let’s sit down and talk.” This was the start of our friendship that lasted almost 25 years until her much too early passing.

Two people wearing jackets and scarves smile at the camera against a backdrop of a partly cloudy sky.

November 2022, Ludwine Messiaen and Katharina Wimmer enjoying together bright weather in Jardin du Luxembourg, Paris, France.

In the beginning I was just happy to have someone to exchange thoughts and whom I could ask for competent and always well-considered advice also on general questions of variant interpretation and reporting in a diagnostic setting. Our closer collaboration started in 2002, when Ludwine invited me to come to her lab in Gent to learn direct cDNA sequencing of NF1 transcripts, which she had developed in her lab. Still today, we use essentially the same protocol.(11) To further intensify our collaboration, I joined the MGL at UAB as visiting professor for the first half year of 2005. During this stay Ludwine and I evaluated the -at that time- novel method of MLPA to effectively identify copy number variations in the NF1 gene.(47) In our Austrian laboratory, direct cDNA sequencing supplemented with MLPA was then the protocol in use until we switched to NGS and we could confirm that this comprehensive approach reaches detection rates of >95%.(36)

As anyone who performed RNA-based mutation analysis of the NF1 gene, I got caught by the amount and the diversity of pathogenic NF1 variants affecting splicing and the possibility to use them to elucidate basic mechanisms of splice site definition and disruption, which in turn helps to develop more general rules to predict which variants have a splice effect.  However, analysing in our lab first in Vienna and then in Innsbruck essentially only Austrian patients, the number of variants in our Austrian cohort were too small to substantiate any hypothesis that seemed to emerge from the limited data set. Hence, our conclusions from the analysis of pathogenic NF1 splice variants would not have a solid basis without Ludwine’s support, who was always willing to share data of pathogenic variants from her data set, and the close collaborations between our laboratories.(35-38)

A suspected NF1 patient with multiple café au lait macules and glioblastoma referred to our laboratory in 2003 lead to a shift of my research focus from NF1 to constitutional mismatch repair deficiency (CMMRD). This recessively inherited childhood cancer syndrome firstly described in 1999 is caused by bi-allelic pathogenic variants in one of the four MMR genes. Patients with CMMRD often show features of NF1 and there is clinical overlap.(48, 49) However, the risk for and the spectrum of associated malignancies is different in CMMRD and NF1, although some overlap exists. When we could not identify a causative pathogenic NF1 variant in this patient, I was sure that our patient aged 10 years had CMMRD. His phenotype and family history resembled the few CMMRD patients described at the time. But, it took several years until we could confirm this. It was key to identify in this and several subsequent CMMRD patients the underlying pathogenic variants that we developed direct cDNA sequencing also for the MMR genes based on our experience with NF1 transcript analysis. This approach allows to identify non-canonical splice mutations and, for the PMS2 gene, it circumvents the pitfalls resulting from the presence of the highly homologous PMS2 pseudogene, PMS2CL.(50) Sequence exchange between PMS2 and PMS2CL results in PMS2 “hybrid” alleles that are functional but compromise gDNA-based PMS2 mutation analysis. Ludwine’s support made it possible to assess the frequency and nature of PMS2 “hybrid” alleles in individuals of different ethnic backgrounds and show that they are very frequent.(40) It was also Ludwine who encouraged and supported me in different ways to consider it my task to make the NF community aware of CMMRD being a rare but relevant differential diagnosis of NF1. Being at the time associate editor of Clinical Genetics, Ludwine invited me to write a review on the connection between CMMRD and NF1 which has implications for patient care and, retrospectively, challenges reported associations of NF1 with childhood malignancies associated also with CMMRD. As it was the case for any article I wrote together with Ludwine, I also could rely here on her so valuable critical questions.(51) She also made sure that this topic was discussed at the first ”Joint Global NF Conference” held in November 2018 in Paris, France. Ludwine’s huge with a vision collect patient cohort was then again one of the cornerstones that made a study possible in which we determined that CMMRD is the diagnosis in only a small proportion (0.41%) of children suspected to have sporadic NF1/Legius syndrome in whom this diagnosis cannot be genetically confirmed.(41) These empirical data provide now reliable numbers for genetic counselling. They also confirm previous prevalence estimations, on which guidelines of the European Care for CMMRD consortium are based that advocate CMMRD testing of preselected patients rather than offering reflex testing to all suspected sporadic NF1 children lacking NF1/SPRED1 PVs. Ludwine’s critical voice and always constructive advice was also important to develop these guidelines.(52)

I am grateful for Ludwine’s support over 25 years which was pivotal to the small contribution I could make to the NF field.  I am also thankful that she introduced me to her international network of close collaborators and to the wonderful “Leuven meetings” which were each year a possibility to meet her at least once in person. Ludwine is a true role model as female researcher for me, who never gave the quick answers or went for the fast success, but was thoroughly considering with a vision where she could contribute to long-lasting progress of NF patient care. She critically scrutinized all results before they were delivered – be it to a patient and their doctor in a genetic report or to the public in a scientific paper. This required tenacity in many respects, but working with her was always pleasurable as she showed great respect for the patients, the subject and the colleagues whose contributions she would always fairly acknowledge in her talks and papers. Most importantly, working and being together with Ludwine was full of fun as she was colourful and cheerful person, who had many interests.

An elderly woman with grey hair works on painting a sculpted bust figure on a table in an art studio, with paint jars, brushes, and glasses nearby. A wall calendar is seen in the background.

January 2024, Ludwine Messiaen working on her big vase.

For many years Ludwine was a passionate and talented ceramicist. At my last visit three weeks before her passing, she showed me a big vase that she had been working on over the last months. This vase tells so much about Ludwine – why and how she pursued a project. She wanted this vase to be a legacy to her daughters. In a very nice booklet she had planned the technically demanding two-bellied construction. She explained to me that it needed multiple steps in-between the clay needed to dry to make this construction stable. In the booklet, she had also sketched the decoration of the vase, which was a floral design made in sgraffito technique. The flowers consisted of matroyshka/babushka nesting dolls and mitochondria symbolizing the pure maternal heritage from one generation to the next. Ludwine told me with so much enthusiasm and joy about one day on which her condition allowed her to work for several hours on the vase and on which she could accomplish a lot of this decoration. The decoration was not full completed when I saw it, one of her daughters finalized this work for her. Having written this essay together with three other mentees of Ludwine, I strongly feel that Ludwine para-genetically passed on some of her mitochondrial DNA also to some matroyshkas outside the family and for this we are very grateful.

Ludwin Messiaen's Literature

Authored Works

  1. Heim RA, Kam-Morgan LN, Binnie CG, Corns DD, Cayouette MC, Farber RA, et al. Distribution of 13 truncating mutations in the neurofibromatosis 1 gene. Hum Mol Genet. 1995;4(6):975-81.
  2. Messiaen L, Callens T, De Paepe A, Craen M, Mortier G. Characterisation of two different nonsense mutations, C6792A and C6792G, causing skipping of exon 37 in the NF1 gene. Hum Genet. 1997;101(1):75-80.
  3. Messiaen LM, Callens T, Roux KJ, Mortier GR, De Paepe A, Abramowicz M, et al. Exon 10b of the NF1 gene represents a mutational hotspot and harbors a recurrent missense mutation Y489C associated with aberrant splicing. Genet Med. 1999;1(6):248-53.
  4. Messiaen LM, Callens T, Mortier G, Beysen D, Vandenbroucke I, Van Roy N, et al. Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects. Hum Mutat. 2000;15(6):541-55.
  5. Messiaen L, Riccardi V, Peltonen J, Maertens O, Callens T, Karvonen SL, et al. Independent NF1 mutations in two large families with spinal neurofibromatosis. J Med Genet. 2003;40(2):122-6.
  6. Vandenbroucke, II, Vandesompele J, Paepe AD, Messiaen L. Quantification of splice variants using real-time PCR. Nucleic Acids Res. 2001;29(13):E68-8.
  7. Vandenbroucke I, Callens T, De Paepe A, Messiaen L. Complex splicing pattern generates great diversity in human NF1 transcripts. BMC Genomics. 2002;3:13.
  8. Vandenbroucke I, Van Oostveldt P, Coene E, De Paepe A, Messiaen L. Neurofibromin is actively transported to the nucleus. FEBS Lett. 2004;560(1-3):98-102.
  9. Vandenbroucke I, Vandesompele J, De Paepe A, Messiaen L. Quantification of NF1 transcripts reveals novel highly expressed splice variants. FEBS Lett. 2002;522(1-3):71-6.
  10. Vandenbroucke I, van Doorn R, Callens T, Cobben JM, Starink TM, Messiaen L. Genetic and clinical mosaicism in a patient with neurofibromatosis type 1. Hum Genet. 2004;114(3):284-90.
  11. Messiaen L, Wimmer K. Mutation analysis of the NF1 gene by cDNA-based sequencing of the coding region. In: Cunha KSG, Geller M, editors. Advances in Neurofibromatosis Research. New York: Nova Science Publishers, Inc.; 2012.
  12. Serra E, Rosenbaum T, Winner U, Aledo R, Ars E, Estivill X, et al. Schwann cells harbor the somatic NF1 mutation in neurofibromas: evidence of two different Schwann cell subpopulations. Hum Mol Genet. 2000;9(20):3055-64.
  13. Maertens O, De Schepper S, Vandesompele J, Brems H, Heyns I, Janssens S, et al. Molecular dissection of isolated disease features in mosaic neurofibromatosis type 1. Am J Hum Genet. 2007;81(2):243-51.
  14. De Schepper S, Maertens O, Callens T, Naeyaert JM, Lambert J, Messiaen L. Somatic mutation analysis in NF1 cafe au lait spots reveals two NF1 hits in the melanocytes. J Invest Dermatol. 2008;128(4):1050-3.
  15. Stevenson DA, Zhou H, Ashrafi S, Messiaen LM, Carey JC, D’Astous JL, et al. Double inactivation of NF1 in tibial pseudarthrosis. Am J Hum Genet. 2006;79(1):143-8.
  16. Brems H, Chmara M, Sahbatou M, Denayer E, Taniguchi K, Kato R, et al. Germline loss-of-function mutations in SPRED1 cause a neurofibromatosis 1-like phenotype. Nat Genet. 2007;39(9):1120-6.
  17. Messiaen L, Yao S, Brems H, Callens T, Sathienkijkanchai A, Denayer E, et al. Clinical and mutational spectrum of neurofibromatosis type 1-like syndrome. JAMA. 2009;302(19):2111-8.
  18. Koczkowska M, Callens T, Chen Y, Gomes A, Hicks AD, Sharp A, et al. Clinical spectrum of individuals with pathogenic NF1 missense variants affecting p.Met1149, p.Arg1276, and p.Lys1423: genotype-phenotype study in neurofibromatosis type 1. Hum Mutat. 2020;41(1):299-315.
  19. Koczkowska M, Callens T, Gomes A, Sharp A, Chen Y, Hicks AD, et al. Expanding the clinical phenotype of individuals with a 3-bp in-frame deletion of the NF1 gene (c.2970_2972del): an update of genotype-phenotype correlation. Genet Med. 2019;21(4):867-76.
  20. Koczkowska M, Chen Y, Callens T, Gomes A, Sharp A, Johnson S, et al. Genotype-Phenotype Correlation in NF1: Evidence for a More Severe Phenotype Associated with Missense Mutations Affecting NF1 Codons 844-848. Am J Hum Genet. 2018;102(1):69-87.
  21. Rojnueangnit K, Xie J, Gomes A, Sharp A, Callens T, Chen Y, et al. High Incidence of Noonan Syndrome Features Including Short Stature and Pulmonic Stenosis in Patients carrying NF1 Missense Mutations Affecting p.Arg1809: Genotype-Phenotype Correlation. Hum Mutat. 2015;36(11):1052-63.
  22. Young LC, Goldstein de Salazar R, Han SW, Huang ZYS, Merk A, Drew M, et al. Destabilizing NF1 variants act in a dominant negative manner through neurofibromin dimerization. Proc Natl Acad Sci U S A. 2023;120(5):e2208960120.
  23. Upadhyaya M, Huson SM, Davies M, Thomas N, Chuzhanova N, Giovannini S, et al. An absence of cutaneous neurofibromas associated with a 3-bp inframe deletion in exon 17 of the NF1 gene (c.2970-2972 delAAT): evidence of a clinically significant NF1 genotype-phenotype correlation. Am J Hum Genet. 2007;80(1):140-51.
  24. Piotrowski A, Xie J, Liu YF, Poplawski AB, Gomes AR, Madanecki P, et al. Germline loss-of-function mutations in LZTR1 predispose to an inherited disorder of multiple schwannomas. Nat Genet. 2014;46(2):182-7.
  25. Hirata Y, Brems H, Suzuki M, Kanamori M, Okada M, Morita R, et al. Interaction between a Domain of the Negative Regulator of the Ras-ERK Pathway, SPRED1 Protein, and the GTPase-activating Protein-related Domain of Neurofibromin Is Implicated in Legius Syndrome and Neurofibromatosis Type 1. J Biol Chem. 2016;291(7):3124-34.
  26. Summerer A, Schafer E, Mautner VF, Messiaen L, Cooper DN, Kehrer-Sawatzki H. Ultra-deep amplicon sequencing indicates absence of low-grade mosaicism with normal cells in patients with type-1 NF1 deletions. Hum Genet. 2019;138(1):73-81.
  27. Summerer A, Mautner VF, Upadhyaya M, Claes KBM, Hogel J, Cooper DN, et al. Extreme clustering of type-1 NF1 deletion breakpoints co-locating with G-quadruplex forming sequences. Hum Genet. 2018;137(6-7):511-20.
  28. Hillmer M, Wagner D, Summerer A, Daiber M, Mautner VF, Messiaen L, et al. Fine mapping of meiotic NAHR-associated crossovers causing large NF1 deletions. Hum Mol Genet. 2016;25(3):484-96.
  29. Kehrer-Sawatzki H, Bengesser K, Callens T, Mikhail F, Fu C, Hillmer M, et al. Identification of large NF1 duplications reciprocal to NAHR-mediated type-1 NF1 deletions. Hum Mutat. 2014;35(12):1469-75.
  30. Vogt J, Bengesser K, Claes KB, Wimmer K, Mautner VF, van Minkelen R, et al. SVA retrotransposon insertion-associated deletion represents a novel mutational mechanism underlying large genomic copy number changes with non-recurrent breakpoints. Genome Biol. 2014;15(6):R80.
  31. Bengesser K, Vogt J, Mussotter T, Mautner VF, Messiaen L, Cooper DN, et al. Analysis of crossover breakpoints yields new insights into the nature of the gene conversion events associated with large NF1 deletions mediated by nonallelic homologous recombination. Hum Mutat. 2014;35(2):215-26.
  32. Vogt J, Mussotter T, Bengesser K, Claes K, Hogel J, Chuzhanova N, et al. Identification of recurrent type-2 NF1 microdeletions reveals a mitotic nonallelic homologous recombination hotspot underlying a human genomic disorder. Hum Mutat. 2012;33(11):1599-609.
  33. Zickler AM, Hampp S, Messiaen L, Bengesser K, Mussotter T, Roehl AC, et al. Characterization of the nonallelic homologous recombination hotspot PRS3 associated with type-3 NF1 deletions. Hum Mutat. 2012;33(2):372-83.
  34. Messiaen L, Vogt J, Bengesser K, Fu C, Mikhail F, Serra E, et al. Mosaic type-1 NF1 microdeletions as a cause of both generalized and segmental neurofibromatosis type-1 (NF1). Hum Mutat. 2011;32(2):213-9.
  35. Wimmer K, Callens T, Wernstedt A, Messiaen L. The NF1 gene contains hotspots for L1 endonuclease-dependent de novo insertion. PLoS Genet. 2011;7(11):e1002371.
  36. Wimmer K, Roca X, Beiglbock H, Callens T, Etzler J, Rao AR, et al. Extensive in silico analysis of NF1 splicing defects uncovers determinants for splicing outcome upon 5′ splice-site disruption. Hum Mutat. 2007;28(6):599-612.
  37. Wimmer K, Schamschula E, Wernstedt A, Traunfellner P, Amberger A, Zschocke J, et al. AG-exclusion zone revisited: Lessons to learn from 91 intronic NF1 3′ splice site mutations outside the canonical AG-dinucleotides. Hum Mutat. 2020;41(6):1145-56.
  38. Zatkova A, Messiaen L, Vandenbroucke I, Wieser R, Fonatsch C, Krainer AR, et al. Disruption of exonic splicing enhancer elements is the principal cause of exon skipping associated with seven nonsense or missense alleles of NF1. Hum Mutat. 2004;24(6):491-501.
  39. Povysil G, Tzika A, Vogt J, Haunschmid V, Messiaen L, Zschocke J, et al. panelcn.MOPS: Copy-number detection in targeted NGS panel data for clinical diagnostics. Hum Mutat. 2017;38(7):889-97.
  40. Ganster C, Wernstedt A, Kehrer-Sawatzki H, Messiaen L, Schmidt K, Rahner N, et al. Functional PMS2 hybrid alleles containing a pseudogene-specific missense variant trace back to a single ancient intrachromosomal recombination event. Hum Mutat. 2010;31(5):552-60.
  41. Perez-Valencia JA, Gallon R, Chen Y, Koch J, Keller M, Oberhuber K, et al. Constitutional mismatch repair deficiency is the diagnosis in 0.41% of pathogenic NF1/SPRED1 variant negative children suspected of sporadic neurofibromatosis type 1. Genet Med. 2020;22(12):2081-8.
  42. Plotkin SR, Messiaen L, Legius E, Pancza P, Avery RA, Blakeley JO, et al. Updated diagnostic criteria and nomenclature for neurofibromatosis type 2 and schwannomatosis: An international consensus recommendation. Genet Med. 2022;24(9):1967-77.
  43. Legius E, Messiaen L, Wolkenstein P, Pancza P, Avery RA, Berman Y, et al. Revised diagnostic criteria for neurofibromatosis type 1 and Legius syndrome: an international consensus recommendation. Genet Med. 2021;23(8):1506-13.
  44. Koczkowska M, Chen Y, Xie J, Callens T, Gomes A, Wimmer K, et al. Analysis of 200 unrelated individuals with a constitutional NF1 deep intronic pathogenic variant reveals that variants flanking the alternatively spliced NF1 exon 31 [23a] cause a classical neurofibromatosis type 1 phenotype while altering predominantly NF1 isoform type II. Hum Genet. 2023;142(7):849-61.
  45. Wimmer K, Eckart M, Rehder H, Fonatsch C. Illegitimate splicing of the NF1 gene in healthy individuals mimics mutation-induced splicing alterations in NF1 patients. Hum Genet. 2000;106(3):311-3.
  46. Wimmer K, Eckart M, Stadler PF, Rehder H, Fonatsch C. Three different premature stop codons lead to skipping of exon 7 in neurofibromatosis type I patients. Hum Mutat. 2000;16(1):90-1.
  47. Wimmer K, Yao S, Claes K, Kehrer-Sawatzki H, Tinschert S, De Raedt T, et al. Spectrum of single- and multiexon NF1 copy number changes in a cohort of 1,100 unselected NF1 patients. Genes Chromosomes Cancer. 2006;45(3):265-76.
  48. Ricciardone MD, Ozcelik T, Cevher B, Ozdag H, Tuncer M, Gurgey A, et al. Human MLH1 deficiency predisposes to hematological malignancy and neurofibromatosis type 1. Cancer Res. 1999;59(2):290-3.
  49. Wang Q, Lasset C, Desseigne F, Frappaz D, Bergeron C, Navarro C, et al. Neurofibromatosis and early onset of cancers in hMLH1-deficient children. Cancer Res. 1999;59(2):294-7.
  50. Etzler J, Peyrl A, Zatkova A, Schildhaus HU, Ficek A, Merkelbach-Bruse S, et al. RNA-based mutation analysis identifies an unusual MSH6 splicing defect and circumvents PMS2 pseudogene interference. Hum Mutat. 2008;29(2):299-305.
  51. Wimmer K, Rosenbaum T, Messiaen L. Connections between constitutional mismatch repair deficiency syndrome and neurofibromatosis type 1. Clin Genet. 2017;91(4):507-19.
  52. Suerink M, Ripperger T, Messiaen L, Menko FH, Bourdeaut F, Colas C, et al. Constitutional mismatch repair deficiency as a differential diagnosis of neurofibromatosis type 1: consensus guidelines for testing a child without malignancy. J Med Genet. 2019;56(2):53-62.
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