SMAD4 mutations causing Myhre syndrome are under positive selection in the male germline.

While it is widely thought that de novo mutations (DNMs) occur randomly, we previously showed that some DNMs are enriched because they are positively selected in the testes of aging men. These "selfish" mutations cause disorders with a shared presentation of features, including exclusive paternal origin, significant increase of the father's age, and high apparent germline mutation rate. To date, all known selfish mutations cluster within the components of the RTK-RAS-MAPK signaling pathway, a critical modulator of testicular homeostasis. Here, we demonstrate the selfish nature of the SMAD4 DNMs causing Myhre syndrome (MYHRS). By analyzing 16 informative trios, we show that MYHRS-causing DNMs originated on the paternally derived allele in all cases. We document a statistically significant epidemiological paternal age effect of 6.3 years excess for fathers of MYHRS probands. We developed an ultra-sensitive assay to quantify spontaneous MYHRS-causing SMAD4 variants in sperm and show that pathogenic variants at codon 500 are found at elevated level in sperm of most men and exhibit a strong positive correlation with donor's age, indicative of a high apparent germline mutation rate. Finally, we performed in vitro assays to validate the peculiar functional behavior of the clonally selected DNMs and explored the basis of the pathophysiology of the different SMAD4 sperm-enriched variants. Taken together, these data provide compelling evidence that SMAD4, a gene operating outside the canonical RAS-MAPK signaling pathway, is associated with selfish spermatogonial selection and raises the possibility that other genes/pathways are under positive selection in the aging human testis.

Whole-genome sequencing in autism identifies hot spots for de novo germline mutation.

De novo mutation plays an important role in autism spectrum disorders (ASDs). Notably, pathogenic copy number variants (CNVs) are characterized by high mutation rates. We hypothesize that hypermutability is a property of ASD genes and may also include nucleotide-substitution hot spots. We investigated global patterns of germline mutation by whole-genome sequencing of monozygotic twins concordant for ASD and their parents. Mutation rates varied widely throughout the genome (by 100-fold) and could be explained by intrinsic characteristics of DNA sequence and chromatin structure. Dense clusters of mutations within individual genomes were attributable to compound mutation or gene conversion. Hypermutability was a characteristic of genes involved in ASD and other diseases. In addition, genes impacted by mutations in this study were associated with ASD in independent exome-sequencing data sets. Our findings suggest that regional hypermutation is a significant factor shaping patterns of genetic variation and disease risk in humans.

Patterns of de novo tandem repeat mutations and their role in autism.

Autism spectrum disorder (ASD) is an early-onset developmental disorder characterized by deficits in communication and social interaction and restrictive or repetitive behaviours1,2. Family studies demonstrate that ASD has a substantial genetic basis with contributions both from inherited and de novo variants3,4. It has been estimated that de novo mutations may contribute to 30% of all simplex cases, in which only a single child is affected per family5. Tandem repeats (TRs), defined here as sequences of 1 to 20 base pairs in size repeated consecutively, comprise one of the major sources of de novo mutations in humans6. TR expansions are implicated in dozens of neurological and psychiatric disorders7. Yet, de novo TR mutations have not been characterized on a genome-wide scale, and their contribution to ASD remains unexplored. Here we develop new bioinformatics methods for identifying and prioritizing de novo TR mutations from sequencing data and perform a genome-wide characterization of de novo TR mutations in ASD-affected probands and unaffected siblings. We infer specific mutation events and their precise changes in repeat number, and primarily focus on more prevalent stepwise copy number changes rather than large expansions. Our results demonstrate a significant genome-wide excess of TR mutations in ASD probands. Mutations in probands tend to be larger, enriched in fetal brain regulatory regions, and are predicted to be more evolutionarily deleterious. Overall, our results highlight the importance of considering repeat variants in future studies of de novo mutations.

De novo structural mutation rates and gamete-of-origin biases revealed through genome sequencing of 2,396 families.

Each human genome includes de novo mutations that arose during gametogenesis. While these germline mutations represent a fundamental source of new genetic diversity, they can also create deleterious alleles that impact fitness. Whereas the rate and patterns of point mutations in the human germline are now well understood, far less is known about the frequency and features that impact de novo structural variants (dnSVs). We report a family-based study of germline mutations among 9,599 human genomes from 33 multigenerational CEPH-Utah families and 2,384 families from the Simons Foundation Autism Research Initiative. We find that de novo structural mutations detected by alignment-based, short-read WGS occur at an overall rate of at least 0.160 events per genome in unaffected individuals, and we observe a significantly higher rate (0.206 per genome) in ASD-affected individuals. In both probands and unaffected samples, nearly 73% of de novo structural mutations arose in paternal gametes, and we predict most de novo structural mutations to be caused by mutational mechanisms that do not require sequence homology. After multiple testing correction, we did not observe a statistically significant correlation between parental age and the rate of de novo structural variation in offspring. These results highlight that a spectrum of mutational mechanisms contribute to germline structural mutations and that these mechanisms most likely have markedly different rates and selective pressures than those leading to point mutations.

The current 'dramatically' high paternal ages at childbirth are not unprecedented.

There is strong individual-level evidence that late fatherhood is related to a wide range of health disorders and conditions in offspring. Over the last decades, mean paternal ages at childbirth have risen drastically. This has alarmed researchers from a wide range of fields. However, existing studies have an important shortcoming in that they lack a long-term perspective. This article is a step change in providing such a long-term perspective. We unveil that in many countries the current mean paternal ages at childbirth and proportions of fathers of advanced age at childbirth are not unprecedented. Taking the detected U-shaped trend pattern into account, we discuss individual- and population-level implications of the recent increases in paternal ages at childbirth and highlight important knowledge gaps. At the individual level, some of the biological mechanisms that are responsible for the paternal age-related health risk might, at least to some degree, be counterbalanced by various social factors. Further, how these individual-level effects are linked to population health and human cognitive development might be influenced by various factors, including technical advances and regulations in prenatal diagnostics.

Reproductive Ageing: Current insights and a potential role of NAD in the reproductive health of aging fathers and their children.

In brief: In light of the increasing age of first-time fathers, this article summarizes the current scientific knowledge base on reproductive aging in the male, including sperm quality and health impacts for the offspring. The emerging role of NAD decline in reproductive aging is highlighted. Abstract: Over the past decades, the age of first-time fathers has been steadily increasing due to socio-economic pressures. While general mechanisms of aging are subject to intensive research, male reproductive aging has remained an understudied area, and the effects of increased age on the male reproductive system are still only poorly understood, despite new insights into the potential dire consequences of advanced paternal age for the health of their progeny. There is also growing evidence that reproductive aging is linked to overall health in men, but this review mainly focuses on pathophysiological consequences of old age in men, such as low sperm count and diminished sperm genetic integrity, with an emphasis on mechanisms underlying reproductive aging. The steady decline of NAD levels observed in aging men represents one of the emerging concepts in that regard. Because it offers some mechanistic rationale explaining the effects of old age on the male reproductive system, some of the NAD-dependent functions in male reproduction are briefly outlined in this review. The overview also provides many questions that remain open about the basic science of male reproductive aging.

Biodemographic Characteristics of Children with Down Syndrome, Their Siblings, and Their Parents in Moroccan Population.

OBJECTIVE: In this study, we present the results of biodemographic characteristics of households and associated factors with Down syndrome (DS) birth in Morocco. METHODS: We conducted a retrospective survey between 2014 and 2017 addressed to 277 families with 925 siblings and at least one child with DS (279 with DS). The data are collected using a standardized questionnaire in Marrakech-Safi region. Data were entered and analyzed using the statistical program SPSS statistics software for Windows (version 20.0). χ2 and student t tests were used for testing statistical significance. Differences were considered significant when the p value <0.05. RESULTS: The binary logistic regression analysis between DS and non-DS children in their biodemographic characteristics studied (sex, breastfeeding, duration of exclusive breastfeeding, birth weight, maternal age at birth, paternal age at birth, oral contraceptive use, duration of oral contraceptive use before pregnancy, child age, and rank of birth) showed that only maternal age and paternal age at birth, duration of exclusive breastfeeding, birth weight, and child age were associated with DS birth (odds ratio [OR] = 1.08; 95% Cl: 1.04-1.13, OR = 1.04; 95% Cl: 1.00-1.08, OR = 0.95; 95% Cl: 0.92-0.98, OR = 0.31; 95% Cl: 0.22-0.44, and OR = 0.90; 95% CI: 0.87-0.93, respectively). In the other hand, the comparison between some of socio- and biodemographic characteristics of households studied with data from National Population Survey and Family Health (2018) showed a higher level of education in women and men in our sample in comparison with the general population. Similar results were shown in proportion of men and women in paid employment, the proportion of smoking and alcohol consumption among men, and the rate of oral contraceptive use before pregnancy among women. CONCLUSION: Highlighting the biodemographic characteristics of people with DS will help families to take good care of this group.

Paternal age does not jeopardize the live birth rate and perinatal outcomes after in vitro fertilization: an analysis based on 56,113 frozen embryo transfer cycles.

BACKGROUND: The global trend of delaying childbearing has led to an increasing number of couples seeking in vitro fertilization. The adverse effects of advanced maternal age on pregnancy and perinatal outcomes are well documented, regardless of the conception method. In addition, advanced paternal age may contribute to poor reproductive potential because of high levels of sperm DNA fragmentation. However, it remains challenging to guide older men regarding the effect of paternal age on pregnancy and birth outcomes in the field of assisted reproduction. OBJECTIVE: This study aimed to investigate the association of paternal age with live birth and perinatal outcomes following in vitro fertilization-frozen embryo transfer. STUDY DESIGN: A retrospective study was performed at a university-affiliated fertility center, involving women who were younger than 36 years and had undergone frozen embryo transfer from January 2011 to June 2021. Subjects were categorized into 6 groups based on paternal age: <25, 25 to 29, 30 to 34, 35 to 39, 40 to 44, and ≥45 years. A generalized estimating equation logistic regression model was used to account for the clustered nature of data and to adjust for confounders. Paternal age between 25 and 29 years served as the reference group in the logistic regression models. RESULTS: A total of 56,113 cycles who met the inclusion criteria were included in the final analysis. On unadjusted analyses, the reproductive outcome parameters showed a considerable decline with increasing male age. The live birth rate decreased from 47.9% for men aged 25 to 29 years to 40.3% among men aged ≥40 years. Similarly, the clinical pregnancy rate decreased from 54.4% in the reference group to 47.8% in the ≥40 years age group. Conversely, the miscarriage rate increased as male age increased, from 10.2% among men aged 25 to 29 years to 13.5% among men aged ≥45 years. However, the differences in the reproductive outcomes mentioned above were no longer significant in the multivariable models. Compared with the younger controls, advanced paternal age was not associated with a lower chance of live birth (males aged 40-44 years: adjusted odds ratio, 0.94; 95% confidence interval, 0.85-1.04; males aged ≥45 years: adjusted odds ratio, 0.93; 95% confidence interval, 0.79-1.10). In addition, the rates of clinical pregnancy (males aged 40-44 years: adjusted odds ratio, 0.95; 95% confidence interval, 0.85-1.05; males aged ≥45 years: adjusted odds ratio, 0.94; 95% confidence interval, 0.79-1.12) and miscarriage (males aged 40-44 years: adjusted odds ratio, 1.05; 95% confidence interval, 0.85-1.31; males aged ≥45 years: adjusted odds ratio, 1.07; 95% confidence interval, 0.77-1.50) were comparable between the reference and advanced paternal age groups. Furthermore, men in the youngest age group (<25 years) did not have worse pregnancy outcomes than those in the reference group. Regarding perinatal outcomes, there was no difference among the study cohorts in terms of preterm birth, low birthweight, macrosomia, small for gestational age, and large for gestational age, both in the unadjusted and confounder-adjusted models. CONCLUSION: This study did not demonstrate a significant association between paternal age and live birth and perinatal outcomes after in vitro fertilization-frozen embryo transfer when the female partners were younger than 36 years. With the global trend toward delaying childbirth, our findings provide useful information for counseling patients that increasing paternal age may not adversely affect pregnancy and perinatal outcomes in assisted reproduction.

Investigating the impact of paternal age, paternal heat stress, and estimation of non-genetic paternal variance on dairy cow phenotype.

BACKGROUND: Linear models that are commonly used to predict breeding values in livestock species consider paternal influence solely as a genetic effect. However, emerging evidence in several species suggests the potential effect of non-genetic semen-mediated paternal effects on offspring phenotype. This study contributes to such research by analyzing the extent of non-genetic paternal effects on the performance of Holstein, Montbéliarde, and Normande dairy cows. Insemination data, including semen Batch Identifier (BI, a combination of bull identification and collection date), was associated with various traits measured in cows born from the insemination. These traits encompassed stature, milk production (milk, fat, and protein yields), udder health (somatic cell score and clinical mastitis), and female fertility (conception rates of heifers and cows). We estimated (1) the effects of age at collection and heat stress during spermatogenesis, and (2) the variance components associated with BI or Weekly aggregated BI (WBI). RESULTS: Overall, the non-genetic paternal effect estimates were small and of limited biological importance. However, while heat stress during spermatogenesis did not show significant associations with any of the traits studied in daughters, we observed significant effects of bull age at semen collection on the udder health of daughters. Indeed, cows born from bulls collected after 1500 days of age had higher somatic cell scores compared to those born from bulls collected at a younger age (less than 400 days old) in both Holstein and Normande breeds (+ 3% and + 5% of the phenotypic mean, respectively). In addition, across all breeds and traits analyzed, the estimates of non-genetic paternal variance were consistently low, representing on average 0.13% and 0.09% of the phenotypic variance for BI and WBI, respectively (ranging from 0 to 0.7%). These estimates did not significantly differ from zero, except for milk production traits (milk, fat, and protein yields) in the Holstein breed and protein yield in the Montbéliarde breed when WBI was considered. CONCLUSIONS: Our findings indicate that non-genetic paternal information transmitted through semen does not substantially influence the offspring phenotype in dairy cattle breeds for routinely measured traits. This lack of substantial impact may be attributed to limited transmission or minimal exposure of elite bulls to adverse conditions.

Adolescence and the next generation.

Adolescent growth and social development shape the early development of offspring from preconception through to the post-partum period through distinct processes in males and females. At a time of great change in the forces shaping adolescence, including the timing of parenthood, investments in today's adolescents, the largest cohort in human history, will yield great dividends for future generations.

Limiting access to assisted reproductive technologies for males of advanced age-Pros and cons from a Nordic perspective.

It is not controversial to state that parental age is increasing in several countries. But how to deal with this increase might be. Some Nordic countries have set an upper age limit for females seeking assisted reproduction in their national legislation, but none have done so for males. There are also recommendations in place that restrict access to publicly funded assisted reproduction for both females and males of advanced age in some Nordic countries. As recent data now show somatic and psychiatric health risks related to advanced paternal age, we ask if the time has come for countries to set an upper age limit for males seeking assisted reproduction like there already is for females, and summarize some of the risks and rewards involved in treating couples with advanced age in fertility clinics.

Resveratrol intake by males increased the mitochondrial DNA copy number and telomere length of blastocysts derived from aged mice.

The present study examined whether male resveratrol intake affected mitochondrial DNA copy number (mt-cn) and telomere length (TL) in blastocysts fathered by young and aged male mice. C57BL/6N male mice supplied with water or water containing 0.1 mM resveratrol were used for embryo production at 14-23 and 48-58 weeks of age. Two-cell-stage embryos were collected from the oviducts of superovulated female mice (8-15 weeks old) and cultured for 3 days until the blastocyst stage. Mt-cn and TL levels were measured by real-time polymerase chain reaction. Resveratrol intake did not affect body weight or water consumption. Resveratrol intake increased the expression levels of SIRT1 in the liver, the antioxidative ability of serum, and extended TL in the heart, whereas there was no significant difference in mt-cn in the heart or TL in sperm. The rate of blastocyst development was significantly lower in aged male mice than in younger mice, and resveratrol intake increased the total number of blastocysts derived from both young and aged males. Resveratrol intake did not affect mt-cn or TL in blastomeres of blastocyst-stage embryos derived from young mice, but significantly increased both mt-cn and TL in blastomeres of blastocysts derived from aged fathers. In conclusion, resveratrol intake increased mt-cn and TL levels in blastocysts derived from aged male mice.

Paternal age and perinatal outcomes: an observational study.

OBJECTIVES: The study's primary aim was to examine the relationship between paternal age and perinatal outcomes. METHODS: This study used data from two hospital birth registries to examine the association between paternal age and adverse perinatal outcomes. The sample included all live singleton births between 2010 and 2022. The primary exposure was paternal age, and the following perinatal outcomes were considered: mode of conception, mode of delivery, pregnancy complications, and neonatal outcomes. RESULTS: A total of 15,232 pregnant women were considered. Maternal and paternal ages were 31.9 ± 5.3 and 36.5 ± 6.5 years, respectively. Independent of maternal, paternal age was associated with lower odds of spontaneous conceptions (OR 0.930, 95 % CI 0.968/0.993; p=0.003) and higher odds of intracytoplasmatic sperm injection (OR 1.054, 95 % CI 1.045/1.062; p=0.0001), respectively. In contrast to maternal age, paternal age decreased the odds of any (OR 0.922, 95 % CI 0.985/0.999; p=0.032) and urgent/emergent (OR 0.984, 95 % CI 0.975/0.993; p=0.0001) cesarean delivery. Paternal age did not affect the gestation length, placental or neonatal weight, blood loss during delivery, and neonatal 5th-minute Apgar score. CONCLUSIONS: Paternal age is associated with perinatal outcomes. These findings suggest that advanced paternal age may have implications for reproductive counseling and prenatal care.

Against age limits for men in reproductive care.

Almost all countries and fertility clinics impose age limits on women who want to become pregnant through Assisted Reproductive Technologies (ART). Age limits for aspiring fathers, however, are much less common and remain a topic of debate. This article departs from the principle of reproductive autonomy and a conditional positive right to receive ART, and asks whether there are convincing arguments to also impose age limits on aspiring fathers. After considering three consequentialist approaches to justifying age limits for aspiring fathers, we take in a concrete normative stance by concluding that those are not strong enough to justify such cut-offs. We reinforce our position by drawing a comparison between the case of a 39-year-old woman who wants to become a single mother via a sperm donor on the one hand, and on the other hand the same woman who wants to have a child with a 64-year-old man who she loves and who is willing to care for the child as long as he is able to. We conclude that, as long as appropriate precautions are taken to protect the welfare of the future child, couples who want to receive fertility treatment should never be limited on the basis of the age of the (male) partner. An absence of age limits for men would respect the reproductive autonomy of both the man and the woman.

De novo Mutations in Domestic Cat are Consistent with an Effect of Reproductive Longevity on Both the Rate and Spectrum of Mutations.

The mutation rate is a fundamental evolutionary parameter with direct and appreciable effects on the health and function of individuals. Here, we examine this important parameter in the domestic cat, a beloved companion animal as well as a valuable biomedical model. We estimate a mutation rate of 0.86 × 10-8 per bp per generation for the domestic cat (at an average parental age of 3.8 years). We find evidence for a significant paternal age effect, with more mutations transmitted by older sires. Our analyses suggest that the cat and the human have accrued similar numbers of mutations in the germline before reaching sexual maturity. The per-generation mutation rate in the cat is 28% lower than what has been observed in humans, but is consistent with the shorter generation time in the cat. Using a model of reproductive longevity, which takes into account differences in the reproductive age and time to sexual maturity, we are able to explain much of the difference in per-generation rates between species. We further apply our reproductive longevity model in a novel analysis of mutation spectra and find that the spectrum for the cat resembles the human mutation spectrum at a younger age of reproduction. Together, these results implicate changes in life-history as a driver of mutation rate evolution between species. As the first direct observation of the paternal age effect outside of rodents and primates, our results also suggest a phenomenon that may be universal among mammals.

Paternal age in rhesus macaques is positively associated with germline mutation accumulation but not with measures of offspring sociability.

Mutation is the ultimate source of all genetic novelty and the cause of heritable genetic disorders. Mutational burden has been linked to complex disease, including neurodevelopmental disorders such as schizophrenia and autism. The rate of mutation is a fundamental genomic parameter and direct estimates of this parameter have been enabled by accurate comparisons of whole-genome sequences between parents and offspring. Studies in humans have revealed that the paternal age at conception explains most of the variation in mutation rate: Each additional year of paternal age in humans leads to approximately 1.5 additional inherited mutations. Here, we present an estimate of the de novo mutation rate in the rhesus macaque (Macaca mulatta) using whole-genome sequence data from 32 individuals in four large pedigrees. We estimated an average mutation rate of 0.58 × 10-8 per base pair per generation (at an average parental age of 7.5 yr), much lower than found in direct estimates from great apes. As in humans, older macaque fathers transmit more mutations to their offspring, increasing the per generation mutation rate by 4.27 × 10-10 per base pair per year. We found that the rate of mutation accumulation after puberty is similar between macaques and humans, but that a smaller number of mutations accumulate before puberty in macaques. We additionally investigated the role of paternal age on offspring sociability, a proxy for normal neurodevelopment, by studying 203 male macaques in large social groups.

Sex-specific paternal age effects on offspring quality in Drosophila melanogaster.

Advanced paternal age has been repeatedly shown to modulate offspring quality via male- and/or female-driven processes, and there are theoretical reasons to expect that some of these effects can be sex-specific. For example, sex allocation theory predicts that, when mated with low-condition males, mothers should invest more in their daughters compared to their sons. This is because male fitness is generally more condition-dependent and more variable than female fitness, which makes it less risky to invest in female offspring. Here, we explore whether paternal age can affect the quality and quantity of offspring in a sex-specific way using Drosophila melanogaster as a model organism. In order to understand the contribution of male-driven processes on paternal age effects, we also measured the seminal vesicle size of young and older males and explored its relationship with reproductive success and offspring quality. Older males had lower competitive reproductive success, as expected, but there was no difference between the offspring sex ratio of young and older males. However, we found that paternal age caused an increase in offspring quality (i.e., offspring weight), and that this increase was more marked in daughters than sons. We discuss different male- and female-driven processes that may explain such sex-specific paternal age effects.

Paternal age and 13 psychiatric disorders in the offspring: a population-based cohort study of 7 million children in Taiwan.

Although paternal age has been linked to certain psychiatric disorders, the nature of any causal relationship remains elusive. Here, we aimed to comprehensively assess the magnitude of a wide range of offspring's psychiatric risk conferred by paternal age, leveraging a pedigree inferred from covered-insurance relationship (accuracy >98%) in Taiwan's single-payer compulsory insurance program. We also examined whether there is an independent role of paternal age and explored the potential effect of parental age difference. A total cohort of 7,264,788 individuals born between 1980 and 2018 were included; 5,572,232 with sibling(s) were selected for sibling-comparison analyses and 1,368,942 and 1,044,420 children with information of paternal-grandparents and maternal-grandparents, respectively, were selected for multi-generation analyses. Using inpatient/outpatient claims data (1997-2018), we identified schizophrenia, autism, bipolar disorder (BPD), attention deficit-hyperactivity disorder (ADHD), major depressive disorder (MDD), eating disorder (ED), substance use disorder (SUD), mental retardation (MR), tic disorder, obsessive-compulsive disorder (OCD), anxiety, and somatoform disorder. We identified suicides using death certificates. Logistic regression analysis was used to estimate the paternal/maternal/grand-paternal age association with psychiatric risk in the offspring. The total cohort and sibling-comparison cohort resulted in similar estimates. Paternal age had a U-shaped relationship with offspring's MDD, ED, SUD, and anxiety. A very young maternal age (<20 years) was associated with markedly higher risk in offspring's SUD, MR, and suicide. Older paternal age (>25 years) was linearly associated with offspring's schizophrenia, autism, BPD, ADHD, MDD, ED, SUD, MR, OCD, anxiety, and suicide. Older grand-paternal age was linearly associated with offspring's schizophrenia, autism, ADHD, and MR. Dissimilar parental age was positively associated with offspring's ADHD, MDD, SUD, MR, anxiety, and suicide, and negatively associated with offspring's OCD. This comprehensive assessment provides solid evidence for the independent role of paternal age in psychiatric risk in the offspring and clarifies the significance of both early parenthood and delayed paternity.

Paternal age affects offspring via an epigenetic mechanism involving REST/NRSF.

Advanced paternal age can have deleterious effects on various traits in the next generation. Here, we establish a paternal-aging model in mice to understand the molecular mechanisms of transgenerational epigenetics. Whole-genome target DNA methylome analyses of sperm from aged mice reveal more hypo-methylated genomic regions enriched in REST/NRSF binding motifs. Gene set enrichment analyses also reveal the upregulation of REST/NRSF target genes in the forebrain of embryos from aged fathers. Offspring derived from young mice administrated with a DNA de-methylation drug phenocopy the abnormal vocal communication of pups derived from aged fathers. In conclusion, hypo-methylation of sperm DNA can be a key molecular feature modulating neurodevelopmental programs in offspring by causing fluctuations in the expression of REST/NRSF target genes.

Sequencing and de novo assembly of 150 genomes from Denmark as a population reference.

Hundreds of thousands of human genomes are now being sequenced to characterize genetic variation and use this information to augment association mapping studies of complex disorders and other phenotypic traits. Genetic variation is identified mainly by mapping short reads to the reference genome or by performing local assembly. However, these approaches are biased against discovery of structural variants and variation in the more complex parts of the genome. Hence, large-scale de novo assembly is needed. Here we show that it is possible to construct excellent de novo assemblies from high-coverage sequencing with mate-pair libraries extending up to 20 kilobases. We report de novo assemblies of 150 individuals (50 trios) from the GenomeDenmark project. The quality of these assemblies is similar to those obtained using the more expensive long-read technology. We use the assemblies to identify a rich set of structural variants including many novel insertions and demonstrate how this variant catalogue enables further deciphering of known association mapping signals. We leverage the assemblies to provide 100 completely resolved major histocompatibility complex haplotypes and to resolve major parts of the Y chromosome. Our study provides a regional reference genome that we expect will improve the power of future association mapping studies and hence pave the way for precision medicine initiatives, which now are being launched in many countries including Denmark.