Reaching 95 years of age, George M. Martin died on December 17, 2022. His death was not only untimely but viewed within the frame of his career, certainly too early. After a long and impressive series of publications in which he developed many groundbreaking concepts, he had just set out to develop two novel lines of thought.

Being born in New York, George Martin moved at the age of 19 to Alaska. After a stint as a Gandy dancer on the railroad tracks, there he moved to Seattle to study chemistry and subsequently medicine. In 1952, he received his MD from the University of Washington. He took an internship at the Montreal General Hospital and a residency in anatomic pathology at the University of Chicago. In 1957, he joined the newly founded Department of Pathology at the University of Washington. There he performed a thorough review of the symptomatology, natural history, anatomical-pathological, and histopathological features of a patient with Werner syndrome [1]. In 1904, this syndrome was described by an ophthalmologist at the Royal Christian Albrecht University of Kiel in Germany. Otto Werner noted that his patients showed, in addition to bilateral ocular cataracts, a degenerative skin disease, which he termed “sclerodermia.” Holger Hoehn, MD, then a postdoctoral fellow with George Martin translated the dissertation of Otto Werner into English [2]. During his anatomical-pathological examination, George Martin noted numerous signs of accelerated ageing, such as arteriosclerosis, osteoporosis, truncal obesity versus thin limbs, loss of muscle mass, and prominent skin alterations, including thinning, loss of firmness, wrinkling, loss of fat in the upper layers of the skin, greying and loss of hair, discolouration of the nails, and patches of skin pigmentation. Other organ systems, noteworthy the brain and the spinal cord, appeared to be not affected. Therefore, George Martin termed Werner syndrome a “ segmental progeroid syndrome.”

Thereupon, George Martin decided to go beyond a merely observational study. To investigate possible causes of the skin-related phenomena, he took skin biopsies from the Werner syndrome patients and brought those in culture. In vitro these fibroblasts showed a significantly shortened replicative lifespan (roughly 15 population doublings vs. 60 or more for healthy, age-matched controls) [3]. Thus, in cultures of Werner syndrome-derived cells one could observe “ageing in the act.”

Ageing is considered a universal, intrinsic, progressive, and deleterious process, inherent to all life [4]. Thus, ageing is a condition that affects all living organisms resulting from inherent, not accidental, causes. In genetic terms, ageing is a multi-factorial, dominant disorder. Werner syndrome, in stark contrast, is a rare, autosomal recessive disorder. This apparent contradiction was reconciled by observations by George Martin, Thomas Norwood, William Pendergrass, and Curtis Sprague in experiments where they fused senescent (no longer proliferating) and young (readily proliferating) cells in culture [5]. These experiments showed that the finite replicative lifespan of cells was dominant in culture. Thus, the finite replicative lifespan of Werner syndrome-derived cultured cells indeed reflects ageing.

Subsequently, Holger Hoehn found in the karyotypes of Werner syndrome-derived cultured cells a highly peculiar form of genomic instability, which he termed “variegated translocation mosaicism” [6, 7]. This consisted of a suddenly appearing translocation between two chromosomes, which then was carried on for several population doublings, after which it disappeared and a novel translocation emerged, which after several population doublings also vanished. Analysis of cell cycle kinetics of cultured cells from Werner syndrome cells revealed a prolonged S phase and an elevated fraction of cells that arrested permanently in the S phase [8]. Surprisingly, the fraction of cells responding to mitogenic stimulation was not affected. This indicated that the clinical phenotypes of Werner syndrome patients did not result from defective growth factor responses. The cause of the diminished replicative lifespan of Werner syndrome-derived cells apparently resided within the DNA replicative process itself, or in its regulation and surveillance.

Meanwhile, George Martin decided to pursue a completely different approach to elucidate the nature of the defect(s) in Werner syndrome and their relevance to ageing in general. He travelled once around the globe to collect peripheral blood samples from Werner syndrome patients, their healthy siblings, and to the extent that they were still available, also their parents. Since Werner syndrome is generally diagnosed in the fourth or fifth decade of life few parents, let alone grandparents were available for classical linkage analysis. Focussing on consanguineous families (mostly from Japan) George Martin and colleagues used microsatellite markers to narrow down the genomic region in which the Werner syndrome gene would reside. Eventually, a small homozygous interval was defined and the WRN gene isolated [9]. Functional analysis showed that this gene encodes a 1,432-amino-acid protein consisting of two enzymes working in opposite directions, a 3′-> 5′ RECQ-type helicase and a 3′-> 5′ exonuclease [10, 11].

This finding prompted the question as to whether defects in either the helicase, or the exonuclease, both activities simultaneously, or a as yet unknown non-enzymatic activity would be responsible for the accelerated ageing phenotypes observed with Werner syndrome patients [12]. Observational studies provided some clues, which by themselves were not definitive. Peripheral blood lymphocytes from Werner syndrome were found to harbour elevated rates of deletions at the 6-thioguanine resistance locus [13]. In a plasmid-based host cell reactivation assay cells from Werner syndrome patients were as effective as normal cells in ligating cut DNA ends, albeit that Werner syndrome cell lines introduced significantly more mutations at the ligation sites than cell lines from healthy donors [14]. These mutations in plasmids ligated during passage through Werner syndrome cells were mainly due to deletions. These two independent observations pointed towards the participation of helicase and exonuclease activities but did not allow to distinguish between the two.

By comparing survival and proliferation rates of cells form Werner syndrome patients and healthy donors after exposure to drugs that introduced specific forms of DNA damage (e.g., DNA adducts, double-strand breaks, interstrand cross links), one could infer in what kind of DNA repair pathway the WRN protein would be involved [15‒17]. Firstly, cells from Werner syndrome patients showed hypersensitivity to DNA cross links and double-strand breaks-inducing drugs [16]. Subsequently, WRN deficiency conferred hypersensitivity to arabinofuranosyl-cytidine and bleomycin-induced DNA damage [17]. These drugs induce DNA double-strand breaks at which ends modified bases emerge. Removal of this type of lesion requires exonuclease activity. Additional support for the hypothesis that the exonuclease activity of the WRN protein is key to the Werner syndrome phenotypes came from patients who carry the WRN helicase-inactivating variant R834C in the homozygous form [18]. Both studies indicate that the WRN-encoded helicase activity is dispensable for the clinical phenotypes of Werner syndrome.

While this narrowed the scope of WRN-encoded activities an independent study found that cells from WRN patients and their unaffected, heterozygous parents showed hypersensitivity to camptothecin, a drug that covalently traps DNA topoisomerase I to a specific DNA sequence such that a DNA double-strand crosslink next to a stretch of single-stranded DNA was formed [15]. This indicates that in addition to the enzymatic functions the dose of WRN-encoded protein is critical for cell survival. In addition, the WRN-encoded protein may serve as a “molecular switch” between DNA damage response pathways [12, 19, 20]. Probably, in cells deficient for WRN function double-strand breaks can no longer be repaired by the canonical non-homologous end joining system, and the cell has to resort to the error-prone alternative end-joining pathway. Then, the MRE11-encoded 3′ ->5′ exonuclease degrades the DNA at double-strand breaks [21]. This is consistent with the losses of stretches of DNA at ligation sites of double-strand breaks found in WRN-deficient cells [14]. Since the thus resected DNA ends do no longer contain their original DNA motifs, they can conceivably be (mis)joined such that translocations may result [22]. This mechanism may underlie the variegated translocation mosaicism in cells from patients with Werner syndrome [6, 7].

Meanwhile, George Martin took the journey toward the elucidation of ageing one step further by establishing a worldwide comprehensive registry of clinical and molecular information on Werner syndrome (www.wernersyndrome.org). The clinical phenotypes of patients were divided into 5 cardinal signs and 9 additional features. According to the number phenotypes found, patients were grouped as definite (showing all cardinal signs and two further signs), probable (showing the first three cardinal signs and any two other symptoms), and possible (either cataracts or dermatological alterations and any four other features). Patients who showed signs and symptoms before adolescence (except for short stature) were excluded from consideration as possible Werner syndrome. Then patients in this database who shared cardinal signs of Werner syndrome but did not carry mutations in the WRN gene were further genotyped. Since these patients showed a close phenotypical resemblance to patients with the childhood progeroid disorder Hutchinson-Gilford syndrome (OMIM 176670), which is due to mutations in the LMNA gene, this gene was the first to be analysed. From a cohort of 129 index patients with wild-type WRN genes, 26 carried mutations in LMNA [23]. All these patients with adult-onset atypical Werner syndrome showed mutations in the N-terminal part of the LMNA gene, while patients with the childhood Hutchinson-Gilford syndrome typically had a 50 amino acid deletion within the C-terminal domain. As a result a truncated form of laminin, called progerin, accumulated in cells from patients with Hutchinson-Gilford syndrome. Interestingly, these cells also showed a brittle nuclear lamina, which impaired their progression through the cell cycle.

Following this approach soon thereafter variants in genes involved in either DNA repair (POLD1, SPRTN) cell cycle checkpoint control (MDM2), nucleotide pool regulation (SAMHD1), and telomere maintenance (CTC1) were identified [24] [Martin et al., 2021]. POLD1 encodes polymerase delta, which consists of a DNA polymerase and a 3′->5′ exonuclease. Mutations in this gene are associated with the MDPL syndrome (OMIM 615381), typically showing mandibular hypoplasia, deafness, progeroid features, lipodystrophy, and insulin resistance. SPRTN is associated with Ruijs-Aalfs syndrome (OMIM 616200), which is characterized by low body weight, micrognathia, and hepatocellular carcinoma. MDM2 is a co-factor of TP53 and found mutated in Lessel-Kubisch syndrome (OMIM 618681). Patients with this disorder are relatively short and show severe hypertension, renal failure, a scleroderma-like skin, and premature greying. The CTC1 gene encodes a protein involved in telomere maintenance. Mutated forms of this gene were found in patients with Coats plus syndrome (OMIM 612199), which involves cerebro retinal angiography, calcification, cysts, and osteopenia.

Finally, a patient with atypical Aicardi-Goutieres syndrome 5 (OMIM 612952), who was homozygous for a mutation in the SAMHD1 gene and an inherited heterozygous mutation in the WRN gene was described [25]. In addition to an aged appearance with prematurely grey hair and scleroderma-like skin, he showed spastic paraplegia and apparent disability. Patients with mutant forms of the SAMHD1-encoded triphosphohydrolase, which participates in the regulation of intracellular dNTP pools, typically show microcephaly with basal ganglia calcification, spasticity, developmental delay, and chilblains. Nevertheless, a patient with mutations in the SAMHD1 gene did not present with chilblains and mouth ulcers [26]. These two case reports widen the clinical spectrum resulting from SAMHD1 mutations, which thus far have been considered a neonatal and childhood progressive neurological disorder. On the other hand, these syndromes also widen the clinical spectrum of segmental progeroid disorders to also include neuronal and sensoneuronal phenotypes.

Interestingly, mutations in the TREX1-encoded 3′-> 5′ exonuclease also provoke a form of Aicardi-Goutieres syndrome with deep white matter cysts of neonatal and infantile onset hypertrophic cardiomyopathy. TREX1 is known to resolve anaphase bridges formed by dicentric chromosomes [27]. Dicentric chromosomes may form once the telomeres of chromosomes have shortened considerably and have been implicated in cellular ageing [28]. Telomere erosion leads to a specific form of chromothripsis in which only a single arm of two chromosomes is affected. Thus, telomere shortening, TREX1 action, and chromothripsis appear to be connected. On the other hand, altered LMNA proteins may lead to formation of micronuclei and brittle nuclei, which in turn have been implicated in another form of chromothripsis [29, 30].

These lines of evidence share a common denominator: genomic instability leading to impaired cell proliferation and probably cell death during the process of ageing. Decades of research from the discovery of variegated translocation mosaicism have led us to chromothripsis as a probably underlying mechanism. Yet, the possible relationship(s) between translocations and cessation of cell proliferation are not fully understood. Still unresolved is why most chromosome rearrangements do not elicit an organismal phenotype [31]. Possibly, the most damaged cells may arrest permanently during the S phase of the cell cycle and thus would be not appear in the karyotype [8]. On the other hand, sequencing translocation breakpoints revealed that these may not be as simple as initially thought [32]. While the hypothesis that cellular ageing may result from the ravages caused by genomic instability appears to be supported by a wealth of data, George Martin opened two new avenues of ageing research: epigenetic silencing of gene expression and a search for “antigeroid” gene loci.

Based on a highly selected subset of arrays of CpG sites Horvath and colleagues defined an “epigenetic clock” [33]. This epigenetic clock consists of increasing methylation and consequential silencing of genome-wide gene expression. Comparing DNAs isolated from whole blood samples of 18 Werner syndrome patients with WRN mutations and DNAs of 24 age- and sex-matched healthy controls Maierhofer and colleagues found no association with either age-related accelerated global losses of ALU, LINE1, and α-satellite DNA methylations or gains of rDNA methylation [34]. In contrast, they identified 659 differentially methylated regions (DMRs) comprising 3,656 CpG sites and 613 RefSeq genes. The top DMR was located in the HOXA4 promoter. The HOXA4 gene belongs to a family of homeodomain-containing transcription factors involved in developmental processes and haematopoietic differentiation. Additional DMR genes included LMNA, POLD1, which both had been associated with atypical Werner Syndrome. Finally, they found DMRs for 132 genes which have been reported to be differentially expressed in WRN-mutant/depleted cells. This study suggests that expression levels of certain genes, even if they are not affected by sequence variants, like in atypical Werner Syndrome, may contribute to the rate of ageing.

Another novel concept of George Martin was the notion of “antigeroid syndromes” [24]. By studying individuals who are particularly resilient to the ravages of ageing such as diabetes mellitus type 2, arteriosclerosis, osteoporosis, etc. George Martin inferred that we would be able to identify hitherto neglected variants that would be beneficial, rather than harmful. The major intention of clinical genetics is to identify variants that exert detrimental effects giving rise to the clinical phenotypes in a given patient. In George Matin’s view, the intent of ageing research should be to pinpoint genetic factors that would be beneficial to the population at large.

On December 17, 2022, we lost a brilliant, inquisitive, astute, and articulate mind who enriched the field of ageing research with a continuous flow of novel ideas and concepts. Now it is up to the “younger” generations of scientists and clinicians to pursue the paths outlined by George Martin, or even better and in his vein, to search for yet uncharted areas of research.

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