The Who’s, What’s, and “Y”s: Y Sex Chromosome Loss and Methylation for Analysis in Male Aging and Mortality and Forensic Science Applications

Abstract

The Y chromosome plays a crucial role in understanding the overall landscape of male health. Incorporating the Y chromosome into genomic and epigenomic research may elucidate the male-specific mechanisms behind aging and the pathogenesis of certain conditions, both acute and chronic. Present epigenetic research focuses on the effects of modifications like methylation on autosomal chromosomes. However, little research has been conducted to further these investigations in sex chromosomes, especially the Y chromosome. Epigenetic analyses can identify age-associated CpG sites that may offer potential biomarkers for age estimation and disease risk assessment, among others. This review emphasizes interdisciplinary efforts that have been made in the construction of an assembly and the application of “epigenetic clocks” to the Y chromosome. The studies reviewed here examined the effects of aging on genes such as NLGN4Y, DDX3Y, and TBL1Y, and on male-specific health disparities and disease etiologies, as well as the potential for the use of these genes to assess the diagnostic and age algorithmic potential of Y-specific genes.

1. Introduction

For decades, genome-wide studies have historically excluded human sex chromosomes [1]. The discrepancy in the study between autosomes and sex chromosomes deepens for the Y chromosome, despite its key role in understanding sex differences, particularly with respect to disease susceptibility [2], infertility [3], aging, and immunity [4].

The genetic content of the Y chromosome is small [5] relative to the rest of the genome, but these genes still appear to have considerable influences on health and wellbeing [6,7,8,9,10,11,12]. Multiple studies have concluded that the influence of these genes is great: variation in the expression of Y-linked genes can lead to deleterious effects on health, ranging from increased risk for different types of cancer [13,14] to Alzheimer’s disease [15], among others.

Until very recently, the Y chromosome had not been completely assembled due to the presence of palindromes and other degenerated regions that hindered the study of several critical regions [7,16,17,18]. New chromosome assemblies and full sequencing performed by Rhie et al. (2023) [17] and Hallast et al. (2023) [18] may signal a new era of research into the Y chromosome to better understand it in its entirety, including Y-specific epigenetic patterns, of which there are currently very few studies.

Present research indicates that this promising field has opportunities for discovering new diagnostic tools, algorithms, and biomarkers in forensics, medicine, and genealogy, among other potential applications. This review examines the state-of-the-art structure of the human Y chromosome and the relationship between the Y chromosome, Y-specific DNA methylation sites, aging, and diseases.

2. Y Chromosome Structure

2.1. Regions of the Y Chromosome

The total Y chromosome based on assembly is believed to be approximately 62.5 Mb long [17] (Figure 1). The chromosome is mostly comprised of the male-specific region, commonly abbreviated as the MSY, which makes up about 95% of the length of the chromosome and consists of well-conserved euchromatic and heterochromatic regions [16]. The remaining 5% is made up of pseudoautosomal regions (PARs). The MSY euchromatin consists of three classes of sequences, which have been identified as the X-transposed, X-degenerate, and ampliconic regions. The X-transposed regions (~3.4 Mb) are the product of an inversion of a duplicative transposition event from the X chromosome. The X-degenerate regions (~8.6 Mb) are remnants of the ancient autosome ancestor of the X and Y chromosomes. The ampliconic region (~9.9 Mb) [18] contains sequences of extremely high identity with other sequences in the MSY; each of these MSY-specific repeat units is thus called an amplicon [16]. Gene annotation performed by Rhie et al. (2023) [17] determined that the Y chromosome contains 693 genes and 883 transcripts, with 106 of these genes predicted to be protein-coding.

2.2. Assemblies and Sequencing

For many years, the dominating opinion of the Y chromosome was that it was a genetic wasteland; it was seen as gene-poor, and the abundance of repetitive elements in the sequence hindered attempts to form assemblies or sequence the full chromosome [16,19,20]. While they were restricted by the limitations of their methodology—specifically, by looking at only cytogenetically detectable deletions—these initial studies yielded preliminary discoveries allowing researchers to link Y-specific genes with spermatogenetic failure [21] and regions that specifically contributed to the determination of sex [22,23].

The first “reference” sequence for a Y chromosome was generated by team Skaletsky et al. in 2003 [16]; they mapped 220 bacterial artificial clones to sequence approximately 97% of the ~23 Mb of the MSY. In this initial assembly, they found that the MSY contained at least 156 transcription units, with about half being protein-coding. The classification of the MSY euchromatin into X-transposed, X-degenerate, and ampliconic comes from this study as well, and it has been used ever since to categorize the MSY [16].

In 2023, the team of Rhie et al. [17] performed a complete sequence of a human Y chromosome. The complete reconstruction of the HG002 Y chromosome from the CHM13 assembly, called T2T-Y by the team, assembled regions of the Y chromosome previously uncategorized and unanalyzed, including pseudoautosomal regions (PARS), ampliconic and palindromic sequences, q-arm heterochromatins, and centromeric satellites. Twenty-nine previously unknown repeats were identified, and their new computational methodology was able to identify over 825,000 repetitive sequence motifs capable of forming alternative DNA structures [17] that are considered to be infamously difficult to sequence [24].

Hallast et al. (2023) [18] created a Y-chromosomal assembly from 43 individuals, “updating” the data to include more diverse genetic information than the Ashkenazi Jewish and European T2T-Y model assembled by Rhie et al. (2023) [17]. Specifically, the Hallast et al. [18] assembly included African origin Y chromosomes [25] in their dataset to create a model with a more comprehensive view of genetic variation in the Y chromosome as a result of diverse human evolution. This assembly exceeds the Skaletsky et al. (2003) [16] predecessor for a multitude of reasons. First, Y chromosomes were assembled from all five continental groups. This de novo assembly also filled in data where sequences were incomplete in the 2003 design. Hallast et al. (2023) [18] also diverged from its predecessor and the Rhie et al. (2023) [17] assembly in its ability to self-validate the quality of its consideration of genetic diversity across lineages by including related African Y chromosomes from the Elblabla-CTS8030 lineage.

2.3. Loss of Y Chromosome

Most studies examining the importance of the Y chromosome for human health have been focused on chromosome and gene content loss. Loss of chromosome Y (LOY) describes the “spread” of mutations in somatic stem cells that are observed with male aging [26]. In single cells, LOY is a binary event. The mosaic loss of Y, commonly abbreviated mLOY, refers to the occurrence of LOY in a subset of cells [27]. Peripheral blood DNA samples can show a continuous mosaicism ranging from 0 to 100% of cells lacking a Y chromosome, and the frequency of LOY in leukocytes dramatically increases with age. In a study examining 1153 elderly males, median survival for males with LOY was 5.5 years shorter [28].

A 2021 study by Dumanski et al. [29] found that approximately 500 different autosomal genes showed dysregulation and abnormal transcriptional effects associated with LOY, many of which are involved in immune function. The same study also found that men diagnosed with Alzheimer’s disease were more likely to be affected with LOY in large granular lymphocytes; likewise, prostate cancer patients displayed LOY more frequently in CD4⁺ T cells and granulocytes [29,30].

The increased presence of LOY in tumor tissue is often correlated with worse prognosis for patients with different kinds of cancers. While some cancers like glioblastoma, glioma, and thyroid carcinoma saw a near-absence of LOY, for others, such as kidney renal papillary cell carcinoma, a frequency of LOY as high as 77% was found [31]. LOY has also been associated by presence in the pathology of diseases such as urothelial bladder cancer [32], pancreatic cancer [33], esophageal cancer [34], head and neck carcinoma [35], clear cell renal cell carcinoma [36], some lines of hepatocellular carcinoma [37], and colorectal and prostate cancer [38], among others.

LOY in aged male blood cells has been linked to the presence of several chronic health issues [39]. Males with autoimmune thyroiditis displayed a significantly increased degree of Y loss relative to healthy controls [40]. Similarly, patients who had suffered major cardiovascular events or who had cardiovascular disease were more likely to present LOY in their blood, possibly due to an immunomodulating role that LOY may play in plaque formation and vascular wall inflammation [41]. It is also likely that the prevalence of LOY in myeloid cells results in interference with cardiac macrophage networks, thereby leading to fibrosis [42,43]. Some additional health risks may arise due to the impact mLOY has on blood cells, including the reduction of erythrocyte counts and elevated leukocyte counts, which may reflect changes of expression in genes vital for immunity [44,45,46].

Among other conditions, a mechanism has been confirmed relating the association of age-related LOY and the etiopathogenesis of primary biliary cirrhosis [47]. Likewise, males who are obese or have a higher BMI, as well as type 2 diabetes patients, have also been observed to have higher levels of mosaic loss of Y (mLOY) than healthy controls [48,49]. Likewise, patients with late-stage age-related macular degeneration were significantly likely to have mLOY, independent of age. However, the strongest correlation between mLOY and macular degeneration was found in individuals aged between 65 and 75 years old [50].

Overall, studies [13,15,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55] on LOY have revealed promising insight into the etiopathogenesis of health conditions disproportionately suffered by males because of changes to the ability to express Y chromosome genes that improve survivability.

2.4. Y Chromosome Haplogroups

The non-recombining region of the Y chromosome (NRY) is an area of the Y chromosome beyond the PARs that fails to undergo meiotic recombination with the X chromosome due to a lack of homology [56]. This region is therefore conserved in patrilineages, passing from father to son; it is also intact between generations, save for accumulating mutations. However, these mutations do not take away from the value of the Y chromosome as a tool for studying male lineages in evolutionary history [46]. In fact, the Y chromosome shows a lower level of DNA polymorphism compared to other chromosomes [57,58]. Thus, these Y chromosome polymorphisms, which are neutral with respect to fitness, have helped answered many questions about the history of human evolution with respect to nucleotide variations [59], including the effects of migration and the founder’s effect on genetic diversity [60,61,62] and molecular variation, even within closely related populations [63]. Genotyping these polymorphisms has allowed researchers to identify specific lineages that have risen out of mutations and group them into haplogroups [55,58,64].

The hypothesis that contemporary humans stem from a common ancestor from Africa about 200,000 years ago is commonly accepted and is supported by mtDNA analysis [65]. Evolutionary data, both genetic and fossil evidence, support multiple origins of modern humans in different parts of Africa [66,67], as well as multiple dispersion routes [68,69,70,71]. Thus, it is common for different haplogroups to be more frequent in certain population areas; haplogroup R is most frequently found in Europe and Central Asia, E is common in Africa, and J is found in western Asia [72]. Subhaplogroups can be used to more specifically distinguish different populations, even within one country [73,74], and thus offer more geographic resolution. For example, R-M479 and R-Z93 are used to distinguish southern and central Asian population haplogroups from R-M412, which is frequently in Western Europe [72]. Similarly, the haplogroup R1b (or R-M343) is commonly found in Europe but is either infrequent or totally absent in other continents [75].

There is considerable value in the study of Y haplogroups. Firstly, they provide useful context about the geography and spread of male lineages in human history [58,76,77,78]. Furthermore, some studies have suggested that there is a possibility of an association between Y haplogroups and health, including the inheritance of coronary artery disease [12], atherosclerotic plaque formation [79], and blood pressure [80], but some of these associations between health and Y haplogroups have been disputed by other studies [75,81,82,83].

3. Y Chromosome DNA Methylation Patterns and Their Applications

3.1. Introduction to DNA Methylation

Cell types are defined often by the specific patterns of gene expression resulting in the non-random production of proteins. However, during the individual’s lifetime, the genomic sequence from which these proteins are transcribed and translated does not change (not including random lifetime-accumulated mutations), but the way that the sequence is “read” by cells varies. The processes that allow for different—and even heritable—changes in gene function that result in different expressions of the same genome are known as epigenetics [84,85,86]. The most commonly studied epigenetic mechanism is DNA methylation: the process by which DNA methyltransferases (DNMTs) covalently transfer a methyl group to the C5 position of the DNA cytosine ring [87]. DNA methylation predominantly occurs in repetitive genomic regions, including satellite DNA, long interspersed transposable elements (LINES), short interspersed transposable elements (SINES), and sites where cytosines are followed by guanine residues, known as CpGs. Once a segment of DNA is methylated, generally, gene expression in that area is suppressed [86,88,89]. The former areas, such as regions of repetitive and parasitic DNA, tend to be hypermethylated, whereas regulatory gene regions tend to be hypomethylated to avoid suppressing essential cellular function [89].

DNA methylation plays a vital role in development and disease. Some inherited conditions occur due to faulty genomic imprinting, where the abnormal phenotype presents itself because of the improper expression of a maternal or paternal copy of a particular gene [90]. Since the 1980s, researchers have been able to link cancer and methylation-related changes, as studies showed that tumor cells were hypomethylated relative to healthy cells [91,92]. Conversely, in some cases, the hypermethylation of tumor-suppressing genes and other regulatory regions can also lead to tumorigenesis [53,93,94].

A ubiquitous focus of study for DNA methylation in autosomes has been age-related changes. A study in 2013 [95] established a strong precedent for determining age across many tissues and cell types through examining DNA methylation levels: by using the weighted average of the 353 “clock” CpGs, a DNA methylation “age” could be determined. Horvath concluded that as an individual ages, their epigenetic “maintenance system” shows greater signs of methylation activity until it reaches a plateau in very late life stages. Horvath’s algorithm correlated with chronological age—measured as the calendar passage of time from birth to old age—well. From this first epigenetic clock, more studies were developed in this field, and a new concept was born: biological age, which reflects the pathophysiological changes that accumulate as a result of an individual’s lifestyle, environment, and disease [96,97,98,99,100,101,102,103,104,105]. Biomarkers for biological and phenotypical age, as determined by “biological clocks” like PhenoAge (which identified 513 CpGs for chronological, physical, and biochemical measures of age) and GrimAge, were better predictors of mortality risk and functional phenotypes, including disease states, polypharmacy, and other factors of physical health [106,107]. Even at the time of Horvath’s 2013 study [95], it had been discovered that diseases, such as certain types of cancer, exhibit signs of significant age acceleration, meaning that the individual’s chronological age was different from their calculated methylation age, which would be referred to as phenotypic or biological ages in more recent studies.

These biological clocks and studies, while extensive in their purview over autosomal CpGs, neglected to discern sex-specific differences in epigenetic mechanisms relating to age and even the pathology of certain diseases observed to have notable sex differences [108]. Aging exhibits sexual dimorphism [109] because of changes in various cellular functions that arise from different mechanisms in expressing genes due to genomic imprinting variations between the X and Y chromosomes [110]. Thus, taking into consideration the value of studying the Y chromosome as an artifact of the evolutionarily conserved haplotype and as a map of CpG sites opens avenues for investigations into male-specific aging epigenetic mechanisms and markers for unique characteristics of health.

3.2. Y Haplogroup Methylation Patterns

Nanopore sequencing [111,112] was utilized to determine the methylation of Y chromosomes from lymphoblastoid cell line HG02982, representing the A0 haplogroup, using Nanopolish models [113] and whole-genome bisulfite sequence data. Low levels of CpG methylation were found at the transcription start sites for genes from the PAR, X-degenerate, and X-transposed regions of the Y chromosome [25], consistent with gene expression patterns discovered nearly a decade earlier [114].

When constructing their Y chromosome assembly, Hallest et al. (2023) [18] utilized Nanopore sequencing data from the study on HG02982 Y to tailor their examination of the base-level epigenetic landscape of Y chromosomes across their sample pool. Subsequently, the team found a significant linear relationship between the length of their Y assembly and the global DNA methylation rates; differences across the haplotypes on 194 genetic variants suggested that the genetic background on the Y chromosome impacted the epigenetic profiles on specific genes.

Nanopore sequencing specifically benefits investigations into the epigenetics of the Y chromosome due to the advantage that the long-read technique has over short-read methods, which typically make regions such as the PAR unreadable and inaccessible. In 2023, a study found consistent methylation groups along Y chromosomes across samples from seven haplogroups. That is, 5-Methylcytosine (5mC) frequency was elevated in the X-degeneration and ampliconic sequences, with methylation specifically being found in the transcription start sites (TSSs), untranslated regions (UTRs), and intragenic CpGs [115]. However, they discovered that the patterns of methylation in their samples were not identical amongst the haplogroups, as has been predicted. Instead, they found that a region with high methylation dispersion spanning NLGN4Y (a protein-coding gene expressed in tissues like the brain) was significantly undermethylated in the A1a haplotype when compared to the others. While Esteller-Cucala et al. (2023) [115] did not examine gene expression data from these cell lines to indicate whether NLGN4Y expression varied between these haplogroups because of methylation variations, their result suggested that DNA methylation levels are in fact conserved within Y chromosome haplogroups.

The question of the conservation of the epigenetic patterns on the human Y chromosome had been posited previously by researchers Zhang et al. in 2016 [116]. Male donors belonging to the O2* haplogroup were found to share specific methylation variations even when they were significantly geographically divorced from one another, having originated from different regions of China. Haplogroup-specific methylation sites were also found between O2* and O3 individuals, suggesting that such patterns were evolutionarily conserved from the O haplogroup. These samples were compared to five samples from Nigeria belonging to the E1b1a1 haplogroup, which had diverged from O over 54 thousand years ago. Gene cg05782707 was found to be a haplogroup E-specific methylation site displaying hypomethylation in the Nigerian samples, whereas they were hypermethylated in the O patients. This revealed that the presence of the site had been conserved despite the huge divergence interval, but the divergence itself had contributed to changes in the methylation pattern [116]. The suggestion that DNA methylation patterns are conserved due to some level of heritability is not entirely novel: previous studies in autosomal chromosomes have revealed that methylation patterns can facilitate the molecular diagnosis of hereditary conditions [117] and that these may contribute to some sex-specific differences due to genomic imprinting [118].

Many of these studies also suggested that Y haplogroup-specific methylation and polymorphisms contributed to differences in health. Differences in cancer prospects, longevity, reproductive success, and other facets of male health were impacted by Y haplogroups [118,119,120,121,122,123,124].

3.3. Target Sites for DNA Methylation Within the Y Chromosome

In a 2020 study, Lund et al. [125] identified between 40 and 219 significant age-associated CpGs on the Y chromosome in four cohorts of whole blood samples. Hypermethylation at these sites was observed in a significant majority of each cohort (≥82%), whereas hypomethylation was observed in less than 20% of the significant CpG sites. They found a tendency of higher methylation (80% or 90% of all significant age-associated CpGs) with age. They postulated that hypermethylation functioned as an active response mechanism to maintain survival at higher ages. Likewise, some autosomal CpGs associated with increased risk for death when hypermethylated showed the opposite: on the Y chromosome, they were hypomethylated, helping shape the study’s conclusion that many of these epigenetic mechanisms work to maintain male survival and successful aging. However, the study was limited in its sampling methodology: the whole blood samples came from individuals between the ages of 56 and 95, meaning that their assessment was only accurate for middle-aged and older-aged males.

In 2021, Vidaki et al. [108] expanded the spectrum of ages for testing of male whole blood samples for age estimation based on Y-chromosomal DNA methylation. The individuals whose blood was tested ranged between 15 and 87 years old, meaning their results could thoroughly validate the accuracy of the relationship between age and DNA methylation due to the presence of younger references for comparison. They confirmed Lund et al.’s (2020) [125] findings that age tended to correlate with increased Y-CpG hypermethylation. Using a Spearman correlation coefficient, Vidaki’s study found that 22 Y-CpGs showed hypermethylation and 6 showed hypomethylation with age. Very young (<20 years old) versus elderly (>70 years old) patient samples had an approximately 15% decrease or increase in methylation in the respective hypomethylated and hypermethylated Y-CpGs. When using the 19 most predictive age-dependent Y-CpGs with the strongest positive/negative age correlation using a support vector machine radial model, the mean absolute deviation (MAD) of age between the chronological and predictive age was 8.46 years [108]. The discrepancy found in the study further emphasized the challenges related to the prediction of biological and chronological age.

Kananen and Marttila (2021) [109] found between 2 and 90 age-CpGs sites across five studied datasets of Y-CpGs, and 76% of the Y-chromosomal age-CpGs sites were hypermethylated, consistent with the results reported by Lund et al. [125]. Age-CpG sites were enriched in gene bodies but depleted in intergenic regions of the Y chromosome. The hypermethylation on the Y chromosome, versus the expected hypomethylation as discovered in autosomes, indicated that these epigenetic patterns were unique to the Y chromosome.

The studies conducted by Lund et al. (2020) [125], Kananen and Marttila (2021) [109], and Vidaki et al. (2021) [108] utilized methylation microarray data exclusively from European males. The distinct disparities in aging patterns between different population groups mean that sample data from non-European populations would be crucial to validate the methylation patterns identified in these studies [126].

In Li et al.’s (2022) [127] non-European study specifically examining 419 and 587 Chinese males in discovery and validation stages, respectively, with ages ranging across the board from 22 to 89 years old, 14 CpGs were identified to have a positive association with age and 4 CpGs were found to be negatively associated. The methylation levels of Y chromosome CpGs displayed more variety compared with those in autosomal and X chromosome counterparts at a p-value level lower than 0.001, and ultimately 18 CpGs were found to be significantly associated with age (p < 0.05). The study also found that heavy smoking activity had a significant association with the methylation level of cg13845221 (one of the 18 CpGs). Conversely, while methylation levels of cg13845521 and cg11816202 were observed to be lower in alcohol drinkers than in non-drinkers, the differences did not reach the multiple comparison threshold. This suggests that drinking status did not seem to have significant modification effects on Y chromosome methylation levels at the chosen CpG sites. Notably, 2 of the 18 sites were specifically linked with an all-cause mortality risk in males: cg03441493 and cg17816615.

Another study conducted in China [128] utilized samples from the Sichuan Han population to build a male-specific model for age prediction for use in forensic science applications. Jiang et al. (2023) [128] examined DNA methylation profiles from blood samples obtained from 15- to 87-year-old males using an Illumina HumanMethylation450 BeadChip array [129]. They used 81 Y-CpGs with /R/ exceeding 0.1 and p < 0.001 as markers for age in a univariate linear regression analysis. Further investigation using PAGE (Polyacrylamide gel electrophoresis) and single-site SBE (Single Based Extension) narrowed the selection of markers to develop a final multiplex methylation SNaPshot assay [130] of 13 Y-CpG markers. However, their results did not show the levels of statistical significance observed by Lund et al. (2020) [125], Vidaki et al. (2021) [108], and Li et al. (2022) [127] among three of their sites: cg178346540, cg11816202, and cg10959847. Their data found that the correlation between age and sample methylation percentages among these 3 sites out of the 13 was weak, with p-values exceeding 0.05. Eliminating these loci from their multiple linear regression and random forest model allowed them to predict the age of each sample with a higher degree of statistical significance and accuracy. While linear regression output significant results (p < 0.001 in training and test sets), the random forest regression model allowed them to improve their predictions more drastically. Specifically, the linear regression-only model for 10 loci had an R² of 0.5103 and a MAD of 12.39 years in the test set, whereas the random forest regression yielded an R² of 0.9341 and a MAD of 4.65 years. They found that SNaPshot systems were suitable to predict the age of male components in single male/female mixtures and bloodstains for the Han population in Sichuan, China [128].

3.4. Age-Related Methylation Patterns on Y Chromosome Genes

The specific CpGs exhibiting statistically significant methylation changes (whether hyper- or hypomethylation throughout different CpG sites) in older males could explain the sexual dimorphism observed with respect to mortality, pathology, and related factors. While Y chromosome genes such as the SRY (the sex-determining region Y gene) have been meticulously studied, research on many other genes, despite the potentially critical roles they may play in male development, is lacking. The following genes represent some of the most frequently identified genes on the Y chromosome that display significant differential methylation levels linked to aging and/or decreased survivability (summarized in

Table 1. Associations between methylation levels (either hyper- or hypomethylation) of significant and/or cross-validated CpGs on the Y chromosome and age.

CpG Site	Hyper/Hypomethylation	Gene
cg17816615 [108,109,127]	Hypo	DDX3Y
cg01988452 [108]	Hypo	EIF1AY
cg13308744 [108,109,127]	Hypo	EIF1AY
cg02233183 [109]	Hyper	NLGN4Y
cg02340092 [109]	Hyper	NLGN4Y
cg03055837 [125]	Hyper	NLGN4Y
cg03278611 [109]	Hyper	NLGN4Y
cg03706273 [127]	Hyper	NLGN4Y
cg04691144 [108,109,127]	Hyper	NLGN4Y
cg06247034 [108,109]	Hyper	NLGN4Y
cg09748856 [109]	Hypo	NLGN4Y
cg27214488 [109]	Hyper	NLGN4Y
cg27443332 [109,127]	Hyper	NLGN4Y
cg18168924 [109]	Hyper	PRKY
cg20401549 [109]	Hypo	PRKY
cg01707559 [109,125,127]	Hyper	TBL1Y
cg02839557 [109]	Hyper	TBL1Y
cg08921682 [108]	Hyper	TBL1Y
cg09728865 [108]	Hyper	TBL1Y
cg14180491 [127]	Hyper	TBL1Y
cg27355713 [109]	Hyper	TBL1Y
cg27611726 [109]	Hyper	TBL1Y
cg00214611 [119]	Hyper	TMSB4Y
cg14463736 [109]	Hyper	TMSB4Y
cg26198148 [127]	Hyper	TMSB4Y
cg00212031 [109]	Hyper	TTTY14
cg03244189 [109,127]	Hyper	TTTY14
cg06628792 [108]	Hyper	TTTY14
cg11816202 [106,127]	Hyper	TTTY14
cg13765957 [109]	Hyper	TTTY14
cg13845521 [109,127]	Hyper	TTTY14
cg15345074 [109,127]	Hyper	TTTY14
cg02616328 [109]	Hypo	ZFY
cg06558765 [109]	Hypo	ZFY
cg14170959 [108]	Hyper	ZFY

and

Table 2. Genes of significant Y chromosome methylation status with age and their impacts on male health.

Gene	Protein	Function	Established Associations
NLGN4Y	neuroligin-4 Y-linked	Neural signal mediator	Neurodevelopmental disease
			Autism spectrum disorder
			Cognitive disease
DDX3Y	DEAD-box helicase 3 Y-linked	(Possibly) RNA metabolism	Male infertility (azoospermia)
		(Possibly) neuronal differentiation	Germ cell reduction
		(Possibly) cell cycle regulation	Cancer (breast, non-seminoma testicular, oral, papillomavirus-associated lung)
TBL1Y	Transducing beta-like 1 Y-linked	Activator of Notch gene	Heart issues (myopathies, coarctation, abnormal contraction)
			Hearing loss
			Cancer (gastric)
TTTY14	Testis-specific transcript Y-linked 14	(Possibly) Cell proliferation	Alcohol drinking
			Tumor cell proliferation (testicular germ cell)
EIF1AY	Eukaryotic translation initiation factor 1A Y-linked	Ribosome dissociation enhancer	Heart failure
		Translation initiation regulator	Post-ischemic stroke recovery
		Antigen production	Transplantation recovery
TMSB4Y	Thymosin beta 4 Y-linked	Tumor suppressing	Cancer (breast, prostate adenocarcinoma, laryngeal)
		Beta actin interacting	Transplantation recovery
ZFY	Zinc finger protein Y-linked	(Possibly) Cellular housekeeping	Unknown
PRKY	Proteinase kinase Y-linked	(Possibly) Kidney development	Prostate cancer
		(Possibly) Immune cell maturation

).

3.4.1. Neuroligin 4 Y-Linked (NLGN4Y)

One of the most prominent genes observed to undergo significant methylation changes relating to age in some studies is NLGN4Y [108,109,125,127]. Most CpG sites within NLGN4Y undergo hypermethylation with age, but some sites, such as cg09748856, have been shown to undergo hypomethylation (see Table 1).

NLGN4Y encodes for neuroligin-4, which belongs to the neuroligin family of neural cell-adhesion molecules. Neuroligins are trans-synaptic cell-adhesion molecules that bind to neurexins to mediate signaling between pre- and post-synaptic specializations [131]. Studies have found that NLGN4Y’s function may be linked to neurodevelopmental disorders. Specifically, the inability of NLGN4Y to induce synapses may be linked to a pathogenic mechanism for male bias in individuals with autism spectrum disorder, which in some cases, has been linked to mutations in the X-linked homolog NLGN4X [132]. The transsynaptic complex formed by neuroligins with neurexins is crucial to the brain’s ability to process information; thus, molecular pathways affecting the interaction between these proteins could result in the pathogenesis of cognitive diseases [131,133].

3.4.2. DEAD-Box Helicase 3 Y-Linked (DDX3Y)

The DDX3 protein, which is encoded by both DDX3X and DDX3Y [134], has been implicated in cell cycle regulation and specifically has been linked to cellular apoptosis and tumorigenesis [135,136]. Some studies suggest that DDX3 contributes to cancer metastasis, particularly in breast cancer, due to its role in Rac1-mediated signaling pathways or by increasing levels of Snail, a transcription factor known to repress cellular adhesion protein expression [137,138,139]. Conversely, some studies have proposed the role of DDX3 as a tumor suppressor gene [140]; for example, low expression of the DDX3Y gene has been observed in differentiated non-seminoma testicular tumors [141] and oral cancer [142].

Likewise, DDX3 is a target of some viruses, specifically hepatitis C virus, hepatitis B virus, West Nile virus, Japanese encephalitis virus, norovirus, pestivirus, vaccinia virus, and cytomegalovirus [143]; as a consequence of being such a strong viral target, alteration of the p53-DDX3 pathway is even associated with poor survival in human papillomavirus-associated lung cancer [144].

Multiple studies also confirmed that a majority of CpG sites within DDX3Y displayed a strong negative association between age and methylation levels, with very few sites showing hypermethylation [108,109,125,127]. Previous studies have implicated that a loss of DDX3Y is linked with male infertility [145,146] due to its role in spermatogenesis [147]; the deletion of the gene leads to germ cell reduction or azoospermia entirely [148]. DDX3Y is known to be a DEAD-box RNA helicase [149], although its exact role in metabolism is unknown [147]. Nonetheless, research shows that when DDX3Y is knocked out, it has a significant effect on the expression of 71 other proteins. Based on the biological functioning of these genes, it is implied that DDX3Y may play a key role in neuronal differentiation in males. The same study also found that DDX3Y downregulation disturbs neural progenitor cell growth, viability, and cell proliferation [149].

Although relatively little is known about DDX3Y, the X-linked homolog is well researched. DDX3X contains a highly conserved helicase core with domains involved in ATP binding and hydrolysis [150]. DDX3X plays a vital role in RNA metabolism: transcription [151,152], splicing [153,154], RNA transport [155], and translation [156].

3.4.3. Transducing Beta-like 1 Protein Y-Linked (TBL1Y)

TBL1Y is a transducing beta-like 1 protein that often presents with hypermethylation at its CpG sites. As a result, TBL1Y exhibits increased expression levels during differentiation, when its X counterpart, TBL1X, shows the exact opposite expression pattern [157]. Meyfour et al. (2017) [157] found that TBL1Y promotes the discharge of CtBP, a Notch gene co-repressor. With age, the gene TBL1Y was found to be hypermethylated [108,109,125,127]. When TBL1Y is expressed, it promotes the activation of genes involved in neurogenesis [158] and cardiogenesis as part of the Notch signaling pathway. This also leads to the cardiac differentiation of mesodermal cells in cardiac tissue [159], and thus, the inhibition of Notch by repressors like CtBP due to TBL1Y underexpression or mutation has been linked to the development of cardiac issues such as myopathies, coarctation, and abnormal contraction patterns relating to spontaneous beating [157,160,161].

There is a possible connection between hearing loss and TBL1Y relating to its interaction with thyroid hormone receptor function [162], and an association between TBL1Y hypermethylation and gastric cancer was found in a longitudinal cohort study of intestinal metaplasia patients [163].

3.4.4. Testis-Specific Transcript Y-Linked 14 (TTTY14)

Although Lund et al. (2020) [125] did not find a statistically significant relationship, several other studies observed a significant positive correlation between TTTY14 methylation and age [108,109,125,127]. TTTY14, or testis-specific transcript Y-linked 14, was linked to alcohol drinking status via the methylation levels of sites cg13845521 and cg11816202, which were negatively associated with TTTY14 expression [127]. A recent study found that TTTY14 expression promotes the malignant transformation of cells; specifically, TTTY14 promotes the proliferation of testicular tumor cells. This results in a significant increase in clonogenesis that can lead to the formation of testicular germ cell tumors [164]. Thus, it is possible that hypermethylation decreases TTTY14 expression as a survival mechanism against natural aging processes.

Other testis-specific transcripts examined in these studies for links between age and methylation included TTTY1, TTTY4 [125], TTTY10 [108], TTTY12 [125], TTTY13 [108,127], TTTY15 [109,125,127], TTTY18 [109,127], TTTY19 [108,125], TTTY20, TTTY21, and TTTY23B [125]. Unfortunately, due to the fact many of these genes are non-protein-coding and speculated to be only regulatory in nature, there is limited research evaluating their biological roles, especially concerning aging and mortality.

3.4.5. Eukaryotic Translation Initiation Factor 1A Y-Linked Protein (EIF1AY)

Although EIF1AY has been observed to show both hypermethylation and hypomethylation throughout its sequence with age, the most commonly presenting and significant CpG sites tend to display hypomethylation [108,109,125,127]. EIF1AY, standing for eukaryotic translation initiation factor 1A Y-linked protein, is one of the genes located on the non-recombining region of the Y chromosome.

EIF1AY codes for an enhancer of ribosome dissociation and plays an important role in the regulation of the initiation of translation; it enhances ribosomal dissociation into subunits and stabilizes the binding of the 33S complex to the 5′ end of capped RNA strands [165]. EIF1AY also encodes a minor histocompatibility antigen that elicits an antibody response for T-cell immunity and plays a role in graft-versus-host disease and immune-mediated disease remission, particularly with hematopoietic stem cell transplantation recovery [166].

Likewise, EIF1AY shows a distinct effect on sex-related differences with respect to cardiovascular accident recovery and overall cardiovascular health, especially heart failure [167]. In patients with new-onset heart failure and dilated cardiomyopathies, EIF1AY was found to be overexpressed [166,168]. Similarly, EIF1AY has been observed to be upregulated in post-ischemic strokes, implicating some role in the immune system for stroke recovery [169].

3.4.6. Thymosin Beta 4 Y-Linked (TMSB4Y)

TMBS4Y (thymosin beta 4 Y-linked) methylation appears to have a positive association with age [108,109,127]. TMSB4Y interacts with beta actin, which acts as a modulator in the progression of the cell cycle and is a primary component of the actin cytoskeleton. Previous studies have found that the region of the Y chromosome where TMSB4Y is located, and the gene itself, has been linked to male breast cancer when deleted (40%) and in metastatic prostate adenocarcinomas (16%) [164,165,166,170,171,172].

The role of TMSB4Y in tumor suppression has also been shown in other varieties of cancer; laryngeal cancer patients with low TMSB4Y expression generally have poorer outcomes regarding recovery relative to those with high expression [173]. As with EIF1AY, evidence suggests that female-to-male hemopoietic stem cell transplantation elicits T-cell responses against the TMSB4Y epitope, contributing to graft-versus-host disease [174].

3.4.7. Zinc Finger Protein Y-Linked (ZFY)

Despite its appearance in studies relating to age-related methylation patterns on the Y chromosome [108,109,125], ZFY is a generally understudied gene. Evidence from studies in the 1990s proved that ZFY does not serve a role as a primary sex-determining factor—which had been hypothesized at the time—but that it still plays a role of some developmental interest for males [175,176]. ZFY is nearly identical in transcript identity and protein homology to the product of the ZFX gene [177,178], and thus, it is speculated that they share similar functionality.

ZFX shows elevated expression in tumors, with patient survivability negatively correlated with expression levels [179,180,181,182,183,184,185,186,187,188]. The deletion of ZFX also causes severe defects in proliferation, implying that ZFY may also be essential for cellular housekeeping functions [189].

3.4.8. Proteinase Kinase Y-Linked (PRKY)

Kananen and Marttila (2021) [109] and Lund et al. (2020) [125] reported hypomethylation associated with aging in the gene PRKY. However, little is known about the cellular functions of PRKY. PRKY encodes a cAMP-dependent serine/threonine protein kinase gene much like its female equivalent, PRKX [190]. In a study evaluating the diagnostic effects of prostate cancer antigen-methylation for urinary biomarkers of prostate cancer, it was found that PRKY expression was significantly higher (p < 0.001) in prostate cancer tissues when compared to adjacent healthy tissues [191]. Otherwise, PRKY is a relatively understudied gene [184,185,186,187,190,191,192,193].

In Madin–Darby canine kidney cells, PRKX expression has been found to be responsible for the activation of branching morphogenesis. This role in epithelial morphogenesis, particularly in the development of the kidneys, is unique to PRKX and has not been found in more classical cAMP-dependent serine/threonine kinase families such as PKA [194]. Other studies have also concluded that PRKX is a key mediator of macrophage and granulocyte maturation [26].

4. The Y Chromosome and DNA Methylation in a Forensic Science Context

Forensic identification is a key part of the forensic investigation process. Historically, other methods of identifying male body-fluid DNA contributions were based on the microscopic identification of spermatozoa [195]. In recent decades, however, use of the Y chromosome has become standard in forensic cases. The Y chromosome contains specific DNA markers in the form of short tandem repeat (STR) patterns, where a unique sequence of nucleotides repeats multiple times throughout the genome. The uniqueness of these repeating patterns can be used, for example, to distinguish a male component in a mixed DNA sample from a sexual assault investigation [196]. This practice is known as Y-STR profiling, and while the practice is similar to autosomal STR profiling, each repeat can only be present in one allele unless locus duplication has occurred due to the singular parent contribution [197]. Y-STR profiling is useful because it can even be applied to DNA samples from azoospermic or vasectomized males, whereas microscopic examination methods cannot identify a contribution [195].

Certain DNA markers tend to be highly localized to particular geographical regions, or they become incredibly common in one place but rare in others. These ancestry-informed DNA markers [198] are passed down from one generation to another; in the case of the Y chromosome, these are preserved from father to son to create a patrilineage that remains mostly intact across generations, with only occasional mutations [46]. These mutations also play an important role in the evolution of human populations and have created unique population group identifiers found on the Y chromosome [55,58,64,198,199].

However, there are limitations when it comes to the use of Y-STRs in forensic investigations. Y-STR profiling requires the use of a reference sample from a suspect for comparison or a profile match from a criminal DNA database. In the absence of a match or comparison, investigations may be stalled. Likewise, rather than identifying a singular individual as autosomal DNA may, Y-STR DNA can only identify a particular lineage, which means that it may be difficult to distinguish a father and a son that may have contributed to a DNA sample [200]. In fact, some methods of analyzing Y-STRs can only distinguish between fathers and sons about 50% of the time [201]. Thus, considerations need to be made about particular case context and whether the results of any such analysis have adequate discriminatory power.

While the use of autosomal DNA is more discriminatory on the individual level, the Y chromosome still holds significant potential for its use in forensic science, particularly in sexual assault investigations where there may be mixed DNA samples with potentially multiple male contributors [200,202]. DNA methylation, however, circumvents issues that Y-STRs may present—methylation is unique among all individuals, and epigenetic differences are even able to distinguish monozygotic twins [203,204].

In the near future, it is possible that the Y chromosome will play a role in the establishment of an “epigenetic fingerprint” for forensic investigations [205]. The epigenetic fingerprint refers to the lifelong molecular responses of an individual to their environment, stress, diet lifestyle, etc. [206], that lead to highly individualized epigenetic variations [205,207,208,209]. While DNA profiling is not new to forensic science, DNA phenotyping is a relatively novel concept that allows researchers to learn more about the features of an individual, such as biogeographic ancestry, appearance, etc., from a DNA sample. The ultimate goal of forensic DNA phenotyping is to narrow down the number of potential suspects for a crime by eliminating suspects that do not have the traits and characteristics inferred from a particular DNA sample [209].

Recent progress within the field of forensic DNA phenotyping has allowed traits [210] such as skin color [211,212,213], eyebrow color [214], freckles [215,216], hair type [217,218,219,220,221,222], height [223,224,225,226,227], and others [210] to be determined from DNA markers by analysts. Age estimation can be a valuable tool in the establishment of a biological profile or the generation of an investigative lead [95,228,229,230]. DNA methylation is perhaps the most extensively studied tool for age prediction in forensics [95,231] with samples collected in saliva [232], teeth [233,234], semen, and, more commonly, blood [235,236,237,238,239]. Based on the concept of epigenetic clocks, methylation levels on different tissues can be used in an algorithm to determine the age of a DNA specimen source. While these epigenetic clock studies were conducted in autosomal chromosomes, further research on the possibility of age estimation based on Y-chromosome DNA methylation is needed to assess the usefulness of this tool in forensic cases. Currently, the studies Lee et al. (2024) [240] and Ji et al. (2024) [241] are some of the only examples of Y-CpG methylation used for male-specific age-prediction modeling for forensic casework in semen [240] and bloodstains [241], respectively.

5. Conclusions and Future Perspectives

In recent years, the perception that the Y chromosome is a “genetic wasteland” has been challenged and refuted. As newer technologies are developed, new avenues for investigating the impact of the Y chromosome on health and mortality are being discovered. While research into the epigenetic map of the Y chromosome is still relatively new and limited, links between extreme Y downregulation and the pathology of diseases like Alzheimer’s, different types of cancer, heart conditions, and other diseases have already been established. In many cases, these conditions are also known to present differently in terms of their prevalence and intensity in males proportionally to females. Studies conducted in autosomes and these initial Y chromosome studies point strongly towards age-related methylation as a possible mechanism for the etiology of many such health issues; however, it is still a question of debate in both autosomes and these Y chromosome sites whether these epigenetic modifications are a consequence of aging leading to higher mortality, or whether they are a biological mechanism to increase survivability by “protecting” against more adverse health issues.

Research into the loss of Y in males currently predominates what is known about the age-associated changes of the Y chromosome. However, many of these studies prove the value of investigating the impact of the age-associated methylation of the Y chromosome and its relevant consequences for disease pathology, survivability, and general aging. Likewise, by investigating these possible correlation and causation relationships between Y chromosome sites and many of these conditions, future research may discover molecular biomarkers on the Y chromosome that could be useful as diagnostic tools and identifiers for health conditions and diseases as they may present, particularly in older male patients.

For many years, the forensic science community has used Y-STR profiling as a tool for investigating DNA mixtures with potential male contributors. However, Y-STR profiling can only provide information about a male lineage rather than a specific individual. The aforementioned studies suggest that there is investigative potential in epigenetic biomarkers of the Y chromosome. Epigenetic fingerprinting introduces the idea of using every individual’s unique epigenetic signatures—which occur as a result of stress, lifestyle, diet, substance abuse, environment, etc.—to characterize them. Though many of the aforementioned CpG sites (Table 1 and Table 2) are associated with particular diseases and/or overall aging, evidence suggests that these CpGs can also be used in the context of DNA phenotyping, particularly as a tool to discriminate between males of the same Y-STR lineage or to infer suspect traits. By examining the methylation levels within key genes of the Y chromosome, forensic analysts may be able to identify new suspects and exclude others by approximating an individual’s age and potential health conditions.