Nadira Yuldasheva

The Eurasian Heartland: A continental perspective on Y-chromosome diversity

The nonrecombining portion of the human Y chromosome has proven to be a valuable tool for the study of population history. The maintenance of extended haplotypes characteristic of particular geographic regions, despite extensive admixture, allows complex demographic events to be deconstructed. In this study we report the frequencies of 23 Y-chromosome biallelic polymorphism haplotypes in 1,935 men from 49 Eurasian populations, with a particular focus on Central Asia. These haplotypes reveal traces of historical migrations, and provide an insight into the earliest patterns of settlement of anatomically modern humans on the Eurasian continent. Central Asia is revealed to be an important reservoir of genetic diversity, and the source of at least three major waves of migration leading into Europe, the Americas, and India. The genetic results are interpreted in the context of Eurasian linguistic patterns.

The Genetic Legacy of the Mongols

We have identified a Y-chromosomal lineage with several unusual features. It was found in 16 populations throughout a large region of Asia, stretching from the Pacific to the Caspian Sea, and was present at high frequency: approximately 8% of the men in this region carry it, and it thus makes up approximately 0.5% of the world total. The pattern of variation within the lineage suggested that it originated in Mongolia approximately 1,000 years ago. Such a rapid spread cannot have occurred by chance; it must have been a result of selection. The lineage is carried by likely male-line descendants of Genghis Khan, and we therefore propose that it has spread by a novel form of social selection resulting from their behavior.

Piezo1 integration of vascular architecture with physiological force

The mechanisms by which physical forces regulate endothelial cells to determine the complexities of vascular structure and function are enigmatic. Studies of sensory neurons have suggested Piezo proteins as subunits of Ca2+-permeable non-selective cationic channels for detection of noxious mechanical impact. Here we show Piezo1 (Fam38a) channels as sensors of frictional force (shear stress) and determinants of vascular structure in both development and adult physiology. Global or endothelial-specific disruption of mouse Piezo1 profoundly disturbed the developing vasculature and was embryonic lethal within days of the heart beating. Haploinsufficiency was not lethal but endothelial abnormality was detected in mature vessels. The importance of Piezo1 channels as sensors of blood flow was shown by Piezo1 dependence of shear-stress-evoked ionic current and calcium influx in endothelial cells and the ability of exogenous Piezo1 to confer sensitivity to shear stress on otherwise resistant cells. Downstream of this calcium influx there was protease activation and spatial reorganization of endothelial cells to the polarity of the applied force. The data suggest that Piezo1 channels function as pivotal integrators in vascular biology.

A Genetic Landscape Reshaped by Recent Events: Y-Chromosomal Insights into Central Asia

Sixteen Y-chromosomal microsatellites and 16 binary markers have been used to analyze DNA variation in 408 male subjects from 15 populations in Central Asia. Large genetic differences were found between populations, but these did not display an obvious geographical or linguistic pattern like that usually seen for Y-chromosomal variation. Nevertheless, an underlying east-west clinal pattern could be detected by the Autocorrelation Index for DNA Analysis and admixture analysis, and this pattern was interpreted as being derived from the ancient peopling of the area, reinforced by subsequent migrations. Two particularly striking features were seen: an extremely high level of Y-chromosomal differentiation between geographically close populations, accompanied by low diversity within some populations. These were due to the presence of high-frequency population-specific lineages and suggested the occurrence of several recent bottlenecks or founder events. Such events could account for the lack of a clear overall pattern and emphasize the importance of multiple recent events in reshaping this genetic landscape.

A Novel Y-Chromosome Variant Puts an Upper Limit on the Timing of First Entry into the Americas

To the Editor: The Americas were the last continents to be settled by humans, yet many details of the earliest occupation remain poorly understood. Proposals for the date of first entry fall into two ranges, one suggesting a very early occupation ∼30,000–40,000 years before present (BP), and the other favoring dates ∼13,000 years BP, when the polar climate was again hospitable. We present Y-chromosomal data that support strongly the latter dates. Recent activity in archeology has prompted a reinterpretation of the economy and culture of the earliest Americans (Dillehay 2000). Although this has revolutionized thinking about American prehistory, it has not pushed dates for the earliest human entry substantially backward in time. Indeed, the paucity of sites and skeletal material credibly dated to >14,000 years BP has been a consistent puzzle for those who would posit an extremely ancient history of human occupation in the Americas. Only two Y-chromosome haplotypes seem to have reached the Americas from Asia before colonial contacts began in the 15th century. The most frequent haplotype—reaching a frequency of 100% in some populations—was identified by hypervariable markers (Pena et al. 1995) and was later confirmed by the discovery of two SNPs known as “M3” and “M45” (Underhill et al. 1996, 2000). A second haplotype, marked by a nucleotide substitution in the RPS4Y gene, was subsequently discovered at lower frequencies (Bergen et al. 1999), presumably reaching the Americas more recently. This pattern of limited haplotype diversity has also been observed in mtDNA (Torroni et al. 1994). Putting a date on the earliest human entry into the continent has been hampered by the absence of a known mutation occurring before—but very close to—the time that the resulting haplotype entered the Americas. The M3/DYS199 polymorphism occurred after the first populations entered the Americas (Underhill et al. 1996), so knowing when it arose would not put an upper bound on the time of first colonization. Here, we describe a novel Y-chromosome SNP that can be reliably dated and that occurred before—but sufficiently close in time to—the initial human radiation into the Americas, so as to provide a meaningful upper bound on the time of entry. The polymorphism, which we call “M242” in the framework established by Underhill et al. (2000) (dbSNP accession number ss9805824), is a C→T transition residing in intron 1 (IVS-866) of the DBY gene. The M242-T allele is found only on chromosomes bearing the derived alleles at M45 and M74 (fig. 1). In addition, the derived allele (M242-T) is found on every chromosome bearing the M3 mutation, but not exclusively. Thus, as shown in figure 1, the M242 mutation arose after the M45/M74 mutations but before M3. This places it within a crucial gap that is very close in time to the entry of the first modern humans into the American continents. In the standard nomenclatural system of the Y Chromosome Consortium (2002), chromosomes bearing this polymorphism would be denoted as Q* in the absence of the Qa-defining mutation. The derived allele of M242 occurs at a frequency of 100% in indigenous Y chromosomes that do not carry the RPS4Y mutation (Bortolini et al. 2003 [in this issue]; P.U., unpublished data). It also occurs at a low but appreciable frequency (0%–17%; average ∼5%) in central Asian, Indian, and Siberian populations (table 1). On the basis of its widespread Eurasian distribution and its position within the Y-chromosome phylogeny (fig. 1), it is clear that the M242 mutation occurred before the first migration into the Americas. Thus, determining its age in Asian populations will establish the earliest time at which the first migrants could have reached the Americas. Figure 1 Partial revision of the Y-chromosome phylogeny presented by Underhill et al. (2000), indicating the placement of M242 relative to other Y-chromosome polymorphisms in haplogroups IX and X, arising in central Asia and the Americas. As can be seen, M242 ... Table 1 Frequency of M242 in 49 Populations Examined[Note] We applied several approaches to dating this mutation in 69 Eurasian samples—out of a total 1,935 individuals tested—observed to have the M242-T allele (table 2 and Wells et al. 2001). These methods are based on the accumulation of variation at 15 microsatellite loci. One approach is a widely used, model-based technique (Su et al. 1999), which was corroborated independently by a technique less dependent on assumptions about a population’s demographic history (Stumpf and Goldstein 2001). Guided by the original description of the method (Su et al. 1999), the initial effective population size (Ne) for the Asian male population was assumed to fall between a low of 1,111 and a high of 1,500. If a Y-chromosome microsatellite mutation rate of 0.18% and a human generation time (g) of 25 years are used, this leads to an estimate of 9,200–9,700 years BP, when the average variance across all microsatellite loci is taken (0.589). Although a male generation time of 25 years is frequently assumed, there is now compelling evidence that the male generation time—at least within modern times—is closer to 32 or 35 years (Tremblay and Vezina 2000; Helgason et al. 2003). When a generation time of 35 years is applied, the age estimates range from 13,000 to 14,000 years BP (table 3). As can be seen from the table, the estimates of effective population size have a relatively minor effect on age estimates, whereas mutation rates have a somewhat greater impact. A solid consensus regarding an average Y-chromosome microsatellite mutation rate, for mostly the same loci used in the present study, is developing around a rate of 0.18%–0.20% per generation (Heyer et al. 1997; Kayser et al. 2000). The best estimates of the age of the M242 mutation when this method is used appear to center on the 14,000–15,000-year range. A significant drawback of this method is its lack of a clear approach for calculating standard errors. Table 2 Microsatellite Genotypes for 69 M242-Bearing Chromosomes Identified in the Populations Listed in Table 1 Table 3 Estimated Ages of M242 for a Range of Male Generation Times, Y-Chromosome Microsatellite Mutation Rates, and Effective Population Sizes, Calculated Using the Method of Su et al. (1999) Although this method is now well established for a range of demographic scenarios, we wanted to compare these estimates with those from an entirely different method—one that is insensitive to assumptions of Ne and population growth rate (Stumpf and Goldstein 2001). This approach relies on an inference of the ancestral microsatellite haplotype, which we identified as the haplotype with the most frequent allele at each locus. Again using a mutation rate of 0.18% per generation, we estimate the age of the M242 mutation to be 15,000 years BP, averaged over each of the 15 loci (table 4). The SD for this average estimate is only 1,700 years, which definitively precludes an arrival time of ⩾30,000 years BP. Table 4 shows the results obtained when a variety of mutation rates and generation times are used, and here, too, our best estimate of the mutation’s age is ∼18,000 years BP. This establishes a fairly solid upper bound on the time of first entry into the Americas that seems to preclude a time of entry >20,000 years BP, and our best guess would be closer to 15,000–18,000 years BP. Since our estimate derives solely from the ancestral Asian populations, it is unaffected by changes of diversity occurring after European conquest of the Americas. Even demographic changes that occurred in central Asia and Siberia after Russians established greater sway in the region are likely to have had a minor effect, because we have sampled haplotype diversity over such a huge geographic area. Furthermore, microsatellite diversity on the M242-bearing haplotype in several Native American populations (Bortolini et al. 2003 [in this issue]) is nearly the same as in our Asian sample, substantiating our result and suggesting that the M242-T haplotype entered the Americas very soon after it arose. Table 4 Estimated Age of M242 for a Range of Male Generation Times and Y-Chromosome Microsatellite Mutation Rates, Calculated Using the Method of Stumpf and Goldstein (2001) This discovery, which indicates a rather more recent entry into the Americas than suggested by previous genetic studies (Cavalli-Sforza et al. 1994; Torroni et al. 1994), places the DNA evidence more in line with archeological data, which is characterized by a clear dearth of sites credibly dated beyond 14,000 years BP. Our results do not contradict earlier studies of mtDNA (Torroni et al. 1994) and the autosomes (Cavalli-Sforza et al. 1994), whose standard errors were large and whose authors noted several reasons to expect their dates to overestimate the timing of the first human arrivals to the Americas. In addition, a more recent time of entry into the continent makes the proposal of the Amerind language family more plausible; or, conversely—given the rapidity of linguistic change—the existence of a unified Amerind family would itself imply a fairly recent settling of the Americas, as we have suggested here.

Nox2 NADPH Oxidase Has a Critical Role in Insulin Resistance–Related Endothelial Cell Dysfunction

Insulin resistance is characterized by excessive endothelial cell generation of potentially cytotoxic concentrations of reactive oxygen species. We examined the role of NADPH oxidase (Nox) and specifically Nox2 isoform in superoxide generation in two complementary in vivo models of human insulin resistance (endothelial specific and whole body). Using three complementary methods to measure superoxide, we demonstrated higher levels of superoxide in insulin-resistant endothelial cells, which could be pharmacologically inhibited both acutely and chronically, using the Nox inhibitor gp91ds-tat. Similarly, insulin resistance–induced impairment of endothelial-mediated vasorelaxation could also be reversed using gp91ds-tat. siRNA-mediated knockdown of Nox2, which was specifically elevated in insulin-resistant endothelial cells, significantly reduced superoxide levels. Double transgenic mice with endothelial-specific insulin resistance and deletion of Nox2 showed reduced superoxide production and improved vascular function. This study identifies Nox2 as the central molecule in insulin resistance–mediated oxidative stress and vascular dysfunction. It also establishes pharmacological inhibition of Nox2 as a novel therapeutic target in insulin resistance–related vascular disease.

Pregnenolone Sulphate- and Cholesterol-Regulated TRPM3 Channels Coupled to Vascular Smooth Muscle Secretion and Contraction

Rationale: Transient receptor potential melastatin (TRPM)3 is a calcium-permeable ion channel activated by the neurosteroid pregnenolone sulfate and positively coupled to insulin secretion in &bgr; cells. Although vascular TRPM3 mRNA has been reported, there is no knowledge of TRPM3 protein or its regulation and function in the cardiovascular system. Objective: To determine the relevance and regulation of TRPM3 in vascular biology. Methods and Results: TRPM3 expression was detected at mRNA and protein levels in contractile and proliferating vascular smooth muscle cells. Calcium entry evoked by pregnenolone sulfate or sphingosine was suppressed by TRPM3 blocking antibody or knock-down of TRPM3 by RNA interference. Low-level constitutive TRPM3 activity was also detected. In proliferating cells, channel activity was coupled negatively to interleukin-6 secretion via a calcium-dependent mechanism. In freshly isolated aorta, TRPM3 positively modulated contractile responses independently of L-type calcium channels. Concentrations of pregnenolone sulfate required to evoke responses were higher than the known plasma concentrations of the steroids, leading to a screen for other stimulators. &bgr;-Cyclodextrin was one of few stimulators of TRPM3, revealing the channels to be partially suppressed by endogenous cholesterol, the precursor of pregnenolone. Elevation of cholesterol further suppressed channel activity and loading with cholesterol to generate foam cells precluded observation of TRPM3 activity. Conclusions: The data suggest functional relevance of TRPM3 in contractile and proliferating phenotypes of vascular smooth muscle cells, significance of constitutive channel activity, regulation by cholesterol, and potential value of pregnenolone sulfate in therapeutic vascular modulation.

Insulin Resistance Impairs Circulating Angiogenic Progenitor Cell Function and Delays Endothelial Regeneration

OBJECTIVE Circulating angiogenic progenitor cells (APCs) participate in endothelial repair after arterial injury. Type 2 diabetes is associated with fewer circulating APCs, APC dysfunction, and impaired endothelial repair. We set out to determine whether insulin resistance adversely affects APCs and endothelial regeneration. RESEARCH DESIGN AND METHODS We quantified APCs and assessed APC mobilization and function in mice hemizygous for knockout of the insulin receptor (IRKO) and wild-type (WT) littermate controls. Endothelial regeneration after femoral artery wire injury was also quantified after APC transfusion. RESULTS IRKO mice, although glucose tolerant, had fewer circulating Sca-1+/Flk-1+ APCs than WT mice. Culture of mononuclear cells demonstrated that IRKO mice had fewer APCs in peripheral blood, but not in bone marrow or spleen, suggestive of a mobilization defect. Defective vascular endothelial growth factor–stimulated APC mobilization was confirmed in IRKO mice, consistent with reduced endothelial nitric oxide synthase (eNOS) expression in bone marrow and impaired vascular eNOS activity. Paracrine angiogenic activity of APCs from IRKO mice was impaired compared with those from WT animals. Endothelial regeneration of the femoral artery after denuding wire injury was delayed in IRKO mice compared with WT. Transfusion of mononuclear cells from WT mice normalized the impaired endothelial regeneration in IRKO mice. Transfusion of c-kit+ bone marrow cells from WT mice also restored endothelial regeneration in IRKO mice. However, transfusion of c-kit+ cells from IRKO mice was less effective at improving endothelial repair. CONCLUSIONS Insulin resistance impairs APC function and delays endothelial regeneration after arterial injury. These findings support the hypothesis that insulin resistance per se is sufficient to jeopardize endogenous vascular repair. Defective endothelial repair may be normalized by transfusion of APCs from insulin-sensitive animals but not from insulin-resistant animals.

Increasing Circulating IGFBP1 Levels Improves Insulin Sensitivity, Promotes Nitric Oxide Production, Lowers Blood Pressure, and Protects Against Atherosclerosis

Low concentrations of insulin-like growth factor (IGF) binding protein-1 (IGFBP1) are associated with insulin resistance, diabetes, and cardiovascular disease. We investigated whether increasing IGFBP1 levels can prevent the development of these disorders. Metabolic and vascular phenotype were examined in response to human IGFBP1 overexpression in mice with diet-induced obesity, mice heterozygous for deletion of insulin receptors (IR+/−), and ApoE−/− mice. Direct effects of human (h)IGFBP1 on nitric oxide (NO) generation and cellular signaling were studied in isolated vessels and in human endothelial cells. IGFBP1 circulating levels were markedly suppressed in dietary-induced obese mice. Overexpression of hIGFBP1 in obese mice reduced blood pressure, improved insulin sensitivity, and increased insulin-stimulated NO generation. In nonobese IR+/− mice, overexpression of hIGFBP1 reduced blood pressure and improved insulin-stimulated NO generation. hIGFBP1 induced vasodilatation independently of IGF and increased endothelial NO synthase (eNOS) activity in arterial segments ex vivo, while in endothelial cells, hIGFBP1 increased eNOS Ser1177 phosphorylation via phosphatidylinositol 3-kinase signaling. Finally, in ApoE−/− mice, overexpression of hIGFBP1 reduced atherosclerosis. These favorable effects of hIGFBP1 on insulin sensitivity, blood pressure, NO production, and atherosclerosis suggest that increasing IGFBP1 concentration may be a novel approach to prevent cardiovascular disease in the setting of insulin resistance and diabetes.

Human Immunodeficiency Virus in Uzbekistan: Epidemiological and Genetic Analyses

This study investigates the molecular epidemiology of HIV in Uzbekistan--a former Soviet Union (FSU) country located in central Asia. A total of 18,910,370 subjects were involved in an HIV serological examination through a population survey conducted in 1987-2002. Rapid changes in epidemiological dynamics and transmission modes have been observed since 1999: incidence rose from 25 newly HIV-infected subjects per year to more than 100 new cases per month within the first half of 2002, and the rate of intravenous drug use (IVDU)-associated HIV infection increased to 75% per year during the same period. Thirty HIV-1 strains, isolated from specimens obtained in 1999-2000, were directly sequenced in the env region. Phylogenetic analysis revealed a relationship to genotype A in 56.7%, and to 03_CRFAB in 13.3%; both variants have been previously reported in the FSU. The majority (85.7%) of these strains were isolated from IVDUs. The demographic history of the most prevalent HIV strain in Uzbekistan was inferred from reconstructed molecular phylogenies; exponential growth of the viral population size was thus observed to occur after the mid-1990s. In summary, detectable HIV seroprevalence remains low in the general population of Uzbekistan. However, the current study demonstrates a substantially increasing number of new infections, in association with IVDU, and an exponentially growing effective population size of the IVDU-associated HIV strain.