author: Corresponding author: David M. Nathan, <firstname.lastname@example.com>.[^1]
date: 2009-07
references:
title: International Expert Committee Report on the Role of the A1C Assay in the Diagnosis of Diabetes

*An International Expert Committee with members appointed by the American Diabetes Association, the European Association for the Study of Diabetes, and the International Diabetes Federation was convened in 2008 to consider the current and future means of diagnosing diabetes in nonpregnant individuals. The report of the International Expert Committee represents the consensus view of its members and not necessarily the view of the organizations that appointed them. The International Expert Committee hopes that its report will serve as a stimulus to the international community and professional organizations to consider the use of the A1C assay for the diagnosis of diabetes.*

Diabetes is a disease characterized by abnormal metabolism, most notably hyperglycemia, and an associated heightened risk for relatively specific long-term complications affecting the eyes, kidney, and nervous system. Although diabetes also substantially increases the risk for cardiovascular disease, cardiovascular disease is not specific to diabetes and the risk for cardiovascular disease has not been incorporated into previous definitions or classifications of diabetes or of subdiabetic hyperglycemia.

# Background

## Diagnosing diabetes based on the distribution of glucose levels

Historically, the measurement of glucose has been the means of diagnosing diabetes. Type 1 diabetes has a sufficiently characteristic clinical onset, with relatively acute, extreme elevations in glucose concentrations accompanied by symptoms, such that specific blood glucose cut points are not required for diagnosis in most clinical settings. On the other hand, type 2 diabetes has a more gradual onset, with slowly rising glucose levels over time, and its diagnosis has required specified glucose values to distinguish pathologic glucose concentrations from the distribution of glucose concentrations in the nondiabetic population. Virtually every scheme for the classification and diagnosis of diabetes in modern times has relied on the measurement of plasma (or blood or serum) glucose concentrations in timed samples, such as fasting glucose; in casual samples independent of prandial status; or after a standardized metabolic stress test, such as the 75-g oral glucose tolerance test (OGTT).

Early attempts to standardize the definition of diabetes relied on the OGTT, but the performance and interpretation of the test were inconsistent and the number of subjects studied to define abnormal values was very small (1–6). Studies in the high-risk Pima Indian population that demonstrated a bimodal distribution of glucose levels following the OGTT (7,8) helped establish the 2-h value as the diagnostic value of choice, even though most populations had a unimodal distribution of glucose levels (9). Of note, a bimodal distribution was also seen in the fasting glucose samples in the Pimas and other high-risk populations (10,11). However, a discrete fasting plasma glucose (FPG) or 2-h plasma glucose (2HPG) level that separated the bimodal distributions in the Pimas was difficult to identify, with potential FPG and 2HPG cut points ranging from 120 to 160 mg/dl (6.7–8.9 mmol/l) and from 200 to 250 mg/dl (11.1–13.9 mmol/l), respectively.

In 1979, the National Diabetes Data Group (NDDG) provided the diagnostic criteria that would serve as the blueprint for nearly two decades (12). The NDDG relied on distributions of glucose levels, rather than on the relationship of glucose levels with complications, to diagnose diabetes despite emerging evidence that the microvascular complications of diabetes were associated with a higher range of fasting and OGTT glucose values (11,13–15). The diagnostic glucose values chosen were based on their association with decompensation to "overt" or symptomatic diabetes.

When selecting the threshold glucose values, the NDDG acknowledged that "there is no clear division between diabetics and nondiabetics in the FPG concentration or their response to an oral glucose load," and consequently, "an arbitrary decision has been made as to what level justifies the diagnosis of diabetes." The diagnosis of diabetes was made when *1*) classic symptoms were present; *2*) the venous FPG was ≥140 mg/dl (≥7.8 mmol/l); or *3*) after a 75-g glucose load, the venous 2HPG and levels from an earlier sample before 2 h were ≥200 mg/dl (≥11.1 mmol/l). An intermediate group was classified as having "impaired glucose tolerance" (IGT) with FPG \<140 mg/dl (7.8 mmol/l) and a 2HPG value between 140 and 200 mg/dl (7.8–11.1 mmol/l). IGT was identified on the basis of its relatively higher risk of progression to diabetes compared with that of "normal" glucose tolerance, low frequency of "diabetic symptoms," high probability of reverting to normal glucose tolerance or continuing to have IGT, and rarity of "clinically significant" microvascular disease. The NDDG recommendations were also promulgated by the contemporaneous report of the World Health Organization (WHO) (16).

## Diagnosing diabetes based on the relationship between glucose levels and long-term complications

In 1997, the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus (17) reexamined the basis for diagnosing diabetes. This committee made two seminal contributions: First, they refocused attention on the relationship between glucose levels and the presence of long-term complications as the basis for the diagnosis of diabetes. Second, they summarized data negating the widespread hypothesis that the 2HPG was the gold-standard test for diagnosing diabetes. The committee examined data from three cross-sectional epidemiological studies that included an Egyptian population (*n* = 1,018), Pima Indians (*n* = 960), and the U.S. National Health and Nutrition Examination Survey (NHANES) population (*n* = 2,821). Each assessed retinopathy with fundus photography or direct ophthalmoscopy and measured glycemia as FPG, 2HPG, and A1C. These studies demonstrated glycemic levels below which there was little prevalent retinopathy and above which the prevalence of retinopathy increased in an apparently linear fashion (Fig. 1). When the prevalence of retinopathy was expressed by deciles of glycemia for each of the three measures, the deciles at which retinopathy began to increase were the same for each measure within each population. Moreover, the glycemic values above which retinopathy increased were similar among the populations. These data showed a clear relationship between glycemia and the risk for retinopathy that would supplant the previous notion of risk for progression to overt, symptomatic diabetes as the basis for diagnosing diabetes.

In comparing the relationship between FPG and 2HPG values and retinopathy, it was apparent that the previous FPG cut point of ≥140 mg/dl (7.8 mmol/l) was substantially above the glucose level at which the prevalence of retinopathy began to increase. As a result, the committee recommended that the FPG cut point be lowered to ≥126 mg/dl (7.0 mmol/l) so that this cut point would represent a degree of hyperglycemia that was "similar" to the 2HPG value and diagnosis with either measure would result in a similar prevalence of diabetes in the population. The 1997 committee report acknowledged that even at the lower FPG cut point, the FPG and OGTT (2HPG) were not perfectly concordant. An individual could have diabetes using one test but not the other. This discrepancy has been confirmed in numerous subsequent reports and may be due, in part, to the fact that although both tests are measures of glycemia, they reflect different physiological measures of acute glucose metabolism (18). The debate regarding the relative roles of FPG and 2HPG in the diagnosis of diabetes in the nonpregnant adult has continued (19–21).

The 1997 report also recommended that the FPG level, rather than the 2HPG, be the preferred test to diagnose diabetes because it was more convenient for patients and less costly and time consuming and the repeat-test reproducibility was superior (17). In addition, the committee introduced the term "impaired fasting glucose" (IFG) to differentiate the metabolic state between a normal state (FPG \<110 mg/dl or \<6.1 mmol/l) and diabetes (≥126 mg/dl or ≥7.0 mmol/l) when the FPG test was used. If an OGTT was performed, the intermediate glycemic state continued to be called IGT, with the 2HPG (between 140 and 200 mg/dl \[7.8 and 11.1 mmol/l\]) the same as that as in the NDDG report. A WHO consultation (22) adopted most of the above recommendations except they concluded that, whenever feasible, individuals with IFG should be given an OGTT to exclude the presence of diabetes that would otherwise be missed and that the OGTT should remain the "gold standard." A 2003 follow-up report from the expert committee refined the fasting glucose value range for IFG from ≥110 but \<126 mg/dl to ≥100 but \<126 mg/dl (≥6.1 but \<7.0 mmol/l to ≥5.6 but \<7.0 mmol/l) to make it more comparable with the IGT value (21). The WHO did not change its previous recommendations (23).

# Can the A1C test be used to diagnose diabetes?

If chronic hyperglycemia sufficient to cause diabetes-specific complications is the hallmark of diabetes, common sense would dictate that laboratory measures that capture long-term glycemic exposure should provide a better marker for the presence and severity of the disease than single measures of glucose concentration. Observational studies that have assessed glycemia with measures that capture longer-term exposure (i.e., A1C) or with single or longitudinal measurements of glucose levels have consistently demonstrated a strong correlation between retinopathy and A1C (24–26) but a less consistent relationship with fasting glucose levels (27). In one study that measured both FPG and A1C, there was a stronger correlation between A1C and retinopathy than between fasting glucose levels and retinopathy (25). The correlation between A1C levels and complications has also been shown in the setting of controlled clinical trials in type 1 (28) and type 2 (29) diabetes, and these findings have been used to establish the widely accepted A1C treatment goals for diabetes care (30).

All of these observations suggest that a reliable measure of chronic glycemic levels such as A1C, which captures the degree of glucose exposure over time (31,32) and which is related more intimately to the risk of complications than single or episodic measures of glucose levels, may serve as a better biochemical marker of diabetes and should be considered a diagnostic tool. Although the 1997 expert committee report considered this option, it recommended against using A1C values for diagnosis in part because of the lack of assay standardization (17). The 2003 follow-up report noted that, while the National Glycohemoglobin Standardization Program (33) had succeeded in standardizing the vast majority of assays used in the U.S., the use of A1C for diagnosis still had "disadvantages," and it reaffirmed the previous recommendation that A1C not be used to diagnose diabetes (21).

An updated examination of the laboratory measurements of glucose and A1C by the current International Expert Committee indicates that with advances in instrumentation and standardization, the accuracy and precision of A1C assays at least match those of glucose assays. The measurement of glucose itself is less accurate and precise than most clinicians realize (34). A recent analysis of the performance of a variety of clinical laboratory instruments and methods that measure glucose revealed that 41% of instruments have a significant bias from the reference method that would result in potential misclassification of \>12% of patients (35). There are also potential preanalytic errors owing to sample handling and the well-recognized lability of glucose in the collection tube at room temperature (36,37). Even when whole blood samples are collected in sodium fluoride to inhibit in vitro glycolysis, storage at room temperature for as little as 1 to 4 h before analysis may result in decreases in glucose levels by 3–10 mg/dl in nondiabetic individuals (36–39).

By contrast, A1C values are relatively stable after collection (40), and the recent introduction of a new reference method to calibrate all A1C assay instruments should further improve A1C assay standardization in most of the world (41–43). In addition, between- and within-subject coefficients of variation have been shown to be substantially lower for A1C than for glucose measurements (44). The variability of A1C values is also considerably less than that of FPG levels, with day-to-day within-person variance of \<2% for A1C but 12–15% for FPG (45–47). The convenience for the patient and ease of sample collection for A1C testing (which can be obtained at any time, requires no patient preparation, and is relatively stable at room temperature) compared with that of FPG testing (which requires a timed sample after at least an 8-h fast and which is unstable at room temperature) support using the A1C assay to diagnose diabetes.

In summary, compared with the measurement of glucose, the A1C assay is at least as good at defining the level of hyperglycemia at which retinopathy prevalence increases; has appreciably superior technical attributes, including less preanalytic instability and less biologic variability; and is more clinically convenient. A1C is a more stable biological index than FPG, as would be expected with a measure of chronic glycemia levels compared with glucose concentrations that are known to fluctuate within and between days (Table 1).

Advantages of A1C testing compared with FPG or 2HPG for the diagnosis of diabetes

Standardized and aligned to the DCCT/UKPDS; measurement of glucose is less well standardized

Better index of overall glycemic exposure and risk for long-term complications

Substantially less biologic variability

Substantially less preanalytic instability

No need for fasting or timed samples

Relatively unaffected by acute (e.g., stress or illness related) perturbations in glucose levels

Currently used to guide management and adjust therapy

# What is the most appropriate A1C cut point for the diagnosis of diabetes?

As shown in the 1997 committee report, the prevalence of retinopathy increases substantially at A1C values starting between 6.0 and 7.0% (17) (Fig. 1). A recent analysis derived from DETECT-2 (48) and including the 3 that were included in the 1997 report examined the association between A1C and retinopathy, objectively assessed and graded by fundus photography (S. Colagiuri, personal communication). This analysis included ∼28,000 subjects from nine countries and showed that the glycemic level at which the prevalence of "any" retinopathy begins to rise above background levels (any retinopathy includes minor changes that can be due to other conditions, such as hypertension), and for the more diabetes-specific "moderate" retinopathy, was 6.5% when the data were examined in 0.5% increments (Fig. 2). Among the \>20,000 subjects who had A1C values \<6.5%, "moderate" retinopathy was virtually nonexistent. The receiver operating characteristic curve analysis of the same data indicated that the optimal cut point for detecting at least moderate retinopathy was an A1C of 6.5%.

In summary, the large volume of data from diverse populations has now established an A1C level associated with an increase in the prevalence of moderate retinopathy and provides strong justification for assigning an A1C cut point of ≥6.5% for the diagnosis of diabetes. A recently published population-based study of 3,190 adults of Malay ethnicity independently concluded that A1C levels "in the range 6.6 to 7% were optimal for detecting microvascular complications" (26).

Any suggestion that the relationship between chronic glycemic levels and the long-term complications of diabetes may be better expressed as a continuum, rather than as a strictly dichotomous relationship, is belied by the retinopathy findings presented herein. There is a low prevalence of "any" retinopathy at A1C levels \<6.5% that may reflect a continuum of risk; alternatively, retinopathy related to conditions other than diabetes (e.g., hypertension) or inaccurate assessment of long-term glycemic levels with a single A1C measurement may contribute to this observation. However, the substantial increase in the prevalence of moderate retinopathy at A1C levels ≥6.5% supports a threshold level of glycemia that results in retinopathy most characteristic of diabetes.

This cut point should not be construed as an absolute dividing line between normal glycemia and diabetes; however, the A1C level of 6.5% is sufficiently sensitive and specific to identify individuals who are at risk for developing retinopathy and who should be diagnosed as diabetic. The A1C level is at least as predictive as the current FPG and 2HPG values. In selecting a diagnostic A1C level ≥6.5%, the International Expert Committee balanced the stigma and costs of mistakenly identifying individuals as diabetic against the minimal clinical consequences of delaying the diagnosis in someone with an A1C level \<6.5%. The committee agreed to emphasize specificity rather than sensitivity. This decision was aided by the parallel decision to recommend effective prevention strategies for the highest at-risk group with an A1C between 6.0 and 6.5%. (See below.)

# Limitations of A1C as the recommended means of diagnosing diabetes

The A1C assay is the test of choice for the chronic management of diabetes and is now being recommended for its diagnosis; however, there are parts of the world where the costs of providing the assay preclude its routine use. In such circumstances, clinicians should continue to use the previously recommended approaches to diagnose diabetes based on glucose measurements. The International Expert Committee encourages clinicians worldwide to move as quickly as possible to A1C testing using standardized methods and instrumentation. However, the decision to change to A1C assays as the means of diagnosing diabetes should take into account the performance of local A1C assays and the local prevalence of conditions that may interfere with the assay. (See below.)

Although the discussion above argues for using the A1C assay for the diagnosis of diabetes in nonpregnant individuals, there are patient conditions that either will require a specific A1C assay method or will preclude A1C testing. First, some hemoglobin traits, such as HbS, HbC, HbF, and HbE, interfere with some A1C assay methods (49). Currently, many assay methods can correct for the presence of the most common hemoglobin traits ([www.ngsp.org](http://www.ngsp.org)), and affinity assays that are unaffected by hemoglobin traits may be used (49). Second, any condition that changes red cell turnover, such as hemolytic anemia, chronic malaria, major blood loss, or blood transfusions, will lead to spurious A1C results. Clinicians must be aware of these conditions, particularly in populations in which they are more prevalent. As in the setting where A1C assays are unavailable, the traditional diagnostic tests (e.g., FPG, 2HPG) must be used in individuals in whom interpreting the A1C is problematic. Third, A1C levels appear to increase with age (50), but the extent of the change, whether it relates to factors other than glucose metabolism, and the effect of the age-related increases on the development of complications are not sufficiently clear to adopt age-specific values in a diagnostic scheme. Similarly, racial disparities in A1C, based on putative differences in the relationship between glucose levels and A1C, have been suggested (51); however, here too, their etiology and significance are unclear, and it is premature to establish race-specific diagnostic values. Finally, there are rare clinical settings, such as rapidly evolving type 1 diabetes, where the A1C level will not have had time to "catch up" with the acute elevations in glucose levels; however, in these very rare cases, diabetes should be diagnosable with typical symptoms and casual glucose levels \>200 mg/dl (11.1 mmol/l) despite a nondiagnostic A1C level.

Notwithstanding the above limitations of A1C testing, the assay has numerous important advantages compared with the currently used laboratory measurements of glucose (Table 1). The prevalence of diabetes in some populations may not be the same when diagnosis is based on A1C compared with diagnosis with glucose measurements, and one method may identify different individuals than the other. Because the measurements of glucose levels and A1C reflect different aspects of glucose metabolism, this is to be expected. However, establishing identical prevalences should not be the goal in defining a new means of diagnosing diabetes. The ultimate goal is to identify individuals at risk for diabetes complications so that they can be treated. The A1C diagnostic level of 6.5% accomplishes this goal.

# Can A1C measurements define a specific subdiabetic "high-risk" state?

The 2003 International Expert Committee report reduced the lower bound of IFG from 110 mg/dl (6.1 mmol/l) to 100 mg/dl (5.6 mmol/l) on the grounds that the lower level optimized the sensitivity and specificity for predicting future diabetes and also increased the proportion of those with IGT who could be identified with an FPG test (21). While previous studies have shown a powerful effect of IFG and/or IGT on the subsequent development of diabetes diagnosed with glucose values (52–54), recent reports have demonstrated a graded risk of diabetes development at glycemic levels well within what was previously considered "normal," i.e., FPG \<100 mg/dl (5.6 mmol/l) and A1C \<6.0% (55,56). In addition, metabolic derangements related to diabetes have been documented at similarly low glycemic levels, increasing in severity with higher glucose values within the nondiabetic range (57,58).

As with measures of glucose, a continuum of risk for the development of diabetes based on A1C levels has been demonstrated (59–61). Thus, while there appears to be an approximate glycemic threshold above which the risk for retinopathy escalates, there does not appear to be a specific level at which risk for diabetes clearly begins. A continuum of risk for the development of diabetes across a wide range of subdiabetic A1C levels may make the classification of individuals into categories similar to IFG and IGT equally problematic for A1C, as it implies that we actually know where risk begins or becomes clinically important. The continuum of risk in the subdiabetic glycemic range argues for the elimination of dichotomous subdiabetic classifications, such as "pre-diabetes," IFG, and IGT. However, as A1C levels approach the diagnostic level for diabetes, the risk of developing diabetes becomes greatest (59,60,62).

# Should A1C testing be used to identify individuals at high risk for diabetes?

The screening tests to identify individuals at elevated risk for diabetes are the same as the diagnostic tests; therefore, the technical advantages of A1C testing compared with glucose testing apply to the detection of individuals at high risk as they do to the diagnosis of diabetes. Therapeutic decisions should be based on how close A1C levels are to the diagnosis of diabetes. In the absence of a specific identifiable lower threshold defining when prevention efforts should be implemented, and with potentially limited resources taken into consideration, individuals whose A1C values are close to the 6.5% A1C threshold of diabetes (i.e., ≥6.0%) should receive demonstrably effective interventions (63,64). By identifying this very high-risk subdiabetic group, the International Expert Committee is implying not that populations at lower A1C levels are not at risk but, rather, that they are at lower risk. All individuals at risk for diabetes should receive counseling to maintain normal weight, lose weight if necessary, and become more physically active.

Other risk factors for diabetes development in addition to A1C have been identified, including elevated levels of triglycerides, blood pressure, BMI, and family history of diabetes (59,60), and these should be taken into account in determining when to initiate interventions in individuals with A1C \<6.0%. The use of well-validated risk assessment tools may be valuable in that regard. At the population level, the A1C value at which prevention services are provided will depend on the resources available, the size of the target population, and the nature of the intervention.

# What are the practical issues related to A1C testing?

A1C tests to diagnose diabetes should be performed using clinical laboratory equipment. Point-of-care instruments have not yet been shown to be sufficiently accurate or precise for diagnosing diabetes. Although this International Expert Committee has concluded that the attributes of the A1C assay with regard to diagnosing diabetes and detecting individuals at high risk support its use over the FPG or 2HPG tests, the superiority of A1C testing does not invalidate the diagnostic criteria based on glucose testing. In circumstances when A1C testing cannot be performed, the diagnostic glucose tests are acceptable alternatives.

Whichever of the three different tests now available to diagnose diabetes (A1C, FPG, and 2HPG) is used, both initial and confirmatory testing should be performed with the same test. As the three tests are not completely concordant, using different tests could easily lead to confusion. The only exception to the need to confirm the diagnosis of diabetes with the same test would be the presence of clinical symptoms characteristic of diabetes and glucose levels \>200 mg/dl (\>11.1 mmol/l). Confirmatory testing is also not required to establish risk status in individuals identified as in the highest-risk group for diabetes (A1C of 6.0 to \<6.5%).

Most cases of type 1 diabetes, particularly in children and adolescents, are diagnosed by the classical symptoms of polyuria, polydipsia, polyphagia, unexplained weight loss, and a casual glucose \>200 mg/dl. If diabetes is suspected in the absence of those conditions, A1C testing is warranted.

# Recommendations and conclusions

Based on the above discussion, the International Expert Committee has concluded that the best current evidence supports the following recommendations, summarized in Table 2.

Recommendation of the International Expert Committee

For the diagnosis of diabetes:

The A1C assay is an accurate, precise measure of chronic glycemic levels and correlates well with the risk of diabetes complications.

The A1C assay has several advantages over laboratory measures of glucose.

Diabetes should be diagnosed when A1C is ≥6.5%. Diagnosis should be confirmed with a repeat A1C test. Confirmation is not required in symptomatic subjects with plasma glucose levels >200 mg/dl (>11.1 mmol/l).

If A1C testing is not possible, previously recommended diagnostic methods (e.g., FPG or 2HPG, with confirmation) are acceptable.

A1C testing is indicated in children in whom diabetes is suspected but the classic symptoms and a casual plasma glucose >200 mg/dl (>11.1 mmol/l) are not found.

For the identification of those at high risk for diabetes:

The risk for diabetes based on levels of glycemia is a continuum; therefore, there is no lower glycemic threshold at which risk clearly begins.

The categorical clinical states pre-diabetes, IFG, and IGT fail to capture the continuum of risk and will be phased out of use as A1C measurements replace glucose measurements.

As for the diagnosis of diabetes, the A1C assay has several advantages over laboratory measures of glucose in identifying individuals at high risk for developing diabetes.

Those with A1C levels below the threshold for diabetes but ≥6.0% should receive demonstrably effective preventive interventions. Those with A1C below this range may still be at risk and, depending on the presence of other diabetes risk factors, may also benefit from prevention efforts.

The A1C level at which population-based prevention services begin should be based on the nature of the intervention, the resources available, and the size of the affected population.

## For the diagnosis of diabetes

-   There is no single assay related to hyperglycemia that can be considered the gold standard, as it relates to the risk for microvascular or macrovascular complications.

-   A measure that captures chronic glucose exposure is more likely to be informative regarding the presence of diabetes than is a single measure of glucose.

-   The A1C assay provides a reliable measure of chronic glycemia and correlates well with the risk of long-term diabetes complications.

-   The A1C assay (standardized and aligned with the Diabetes Control and Complications Trial/UK Prospective Diabetes Study assay) has several technical, including preanalytic and analytic, advantages over the currently used laboratory measurements of glucose.

-   For the reasons above, the A1C assay may be a better means of diagnosing diabetes than measures of glucose levels.

-   The diagnosis of diabetes is made if the A1C level is ≥6.5%. Diagnosis should be confirmed with a repeat A1C test unless clinical symptoms and glucose levels \>200 mg/dl (\>11.1 mmol/l) are present.

-   If A1C testing is not possible owing to patient factors that preclude its interpretation (e.g., hemoglobinopathy or abnormal erythrocyte turnover) or to unavailability of the assay, previously recommended diagnostic measures (e.g., FPG and 2HPG) and criteria should be used. Mixing different methods to diagnose diabetes should be avoided.

-   In children and adolescents, A1C testing is indicated when diabetes is suspected in the absence of the classical symptoms or a plasma glucose concentration \>200 mg/dl (\>11.1 mmol/l).

-   The diagnosis of diabetes during pregnancy, when changes in red cell turnover make the A1C assay problematic, will continue to require glucose measurements.

## For the identification of individuals at high risk for diabetes

-   Individuals with an A1C level ≥6% but \<6.5% are likely at the highest risk for progression to diabetes, but this range should not be considered an absolute threshold at which preventative measures are initiated.

-   The classification of subdiabetic hyperglycemia as pre-diabetes is problematic because it suggests that all individuals so classified will develop diabetes and that individuals who do not meet these glycemia-driven criteria (regardless of other risk factor values) are unlikely to develop diabetes—neither of which is the case. Moreover, the categorical classification of individuals as high risk (e.g., IFG or IGT) or low risk, based on any measure of glycemia, is less than ideal because the risk for progression to diabetes appears to be a continuum. The glucose-related terms describing subdiabetic hyperglycemia will be phased out of use as clinical diagnostic states as A1C measurements replace glucose measurements for the diagnosis of diabetes.

-   When assessing risk, implementing prevention strategies, or initiating a population-based prevention program, other diabetes risk factors should be taken into account. In addition, the A1C level at which to begin preventative measures should reflect the resources available, the size of the population affected, and the anticipated degree of success of the intervention. Further analyses of cost-benefit should guide the selection of high-risk groups targeted for intervention within specific populations.

# Supplementary Material

###### Accompanying Editorial

## Acknowledgments

No potential conflicts of interest relevant to this article were reported.

### APPENDIX

International Expert Committee members: David M. Nathan, MD (Chair); Beverly Balkau, PhD; Enzo Bonora, MD, PhD; Knut Borch-Johnsen, MD, DMSc; John B. Buse, MD, PhD; Stephen Colagiuri, MD; Mayer B. Davidson, MD; Ralph DeFronzo, MD; Saul Genuth, MD; Rury R. Holman, FRCP; Linong Ji, MD; Sue Kirkman, MD; William C. Knowler, MD, Dr PH; Desmond Schatz, MD; Jonathan Shaw, MD; Eugene Sobngwi, MD; Michael Steffes, MD, PhD; Olga Vaccaro, MD; Nick Wareham, MD; Bernard Zinman, MD; and Richard Kahn, PhD.

# References

[^1]: \*A list of members of the International Expert Committee can be found in the appendix.


abstract: A multi-parametric genetic screening approach sheds light on integrated control of the endocytic pathway in mammalian cells.
author: Peter Hein; Mark von Zastrow
date: 2010
institute: 1Department of Psychiatry, Department of Cellular and Molecular Pharmacology and Program in Cell Biology, University of California, N212 Genentech Hall, 600 16th Street, San Francisco, CA 94158-2140, USA
references:
title: Membrane traffic in the post-genomic era

# Research highlight

A defining feature of eukaryotic cells is the presence of an elaborate network of internal membrane compartments that communicate between themselves and with the cell surface via specific membrane fission and fusion reactions \[1,2\]. Such 'membrane trafficking' processes can be viewed as a network of intracellular transport pathways, whose operation is critical to normal physiology and disturbed in disease. A major goal in the field of cell biology, therefore, is to elucidate the mechanistic basis of these fundamental membrane trafficking processes and how they are regulated. Historically, genetic approaches have been instrumental in this effort, particularly forward genetic screens in model eukaryotes, such as budding yeast, by the traditional route of mutagenesis, phenotype selection, and subsequent identification of the affected gene. Such screens have led to the identification of a variety of essential proteins mediating membrane traffic in the biosynthetic pathway of yeast, many of which have orthologs in mammals \[3,4\].

A long-standing barrier to more comprehensive analysis of membrane-trafficking processes in mammalian cells has been the relative intractability of these cells to forward genetic analysis. The main barrier is that mammalian cell culture lines, unlike yeast, cannot be maintained in a haploid state. Therefore, traditional genetic methods based on mutations in the genome, because they typically disrupt only a single copy of a particular gene, rarely produce a screenable phenotype. This barrier is beginning to break down, however, based on the development of alternative methods. The sequencing and annotation of animal genomes, combined with the use of RNA interference (RNAi) to knock down specific gene expression, are ushering in a new era of forward genetic analysis that extends to mammalian cells \[5\]. A recent study published in *Nature* from Marino Zerial's group in Dresden (Collinet *et al.*) \[6\], illustrates how such approaches are beginning to be applied to study the integrated function of the endocytic pathway in human cells.

Major gaps exist in our understanding of membrane traffic in mammalian cells compared with that in yeast. Such gaps quickly become evident when one begins to consider how membrane trafficking is integrated with other essential cellular processes. Endocytic membrane traffic is essential not only for 'classical' functions such as nutrient uptake from the extracellular milieu, but also plays critical roles in a wide range of superficially unrelated processes. One of the best recognized of these relationships is with cellular signal transduction. Multicellular life is dependent on a diversity of receptor-mediated signaling mechanisms, and animals have greatly expanded the representation of signaling receptors in their genome compared with yeast. Membrane trafficking of many signaling receptors in the endocytic pathway is essential for the proper organization and regulation of downstream information transfer. Such effects are not only critical for organized cell-cell communication under normal physiological conditions, but disturbances in the endocytic trafficking of receptors play a causative or supporting role in disease states such as cancer. There is also compelling and accumulating evidence for regulation in the converse direction - of the membrane machinery by signaling - at multiple stages of both the membrane-biosynthetic and endocytic pathways \[7,8\].

# Analysis of endocytic pathways in mammalian cells

The main new advance introduced in the study of Collinet *et al.* is automated phenotyping of the endocytic pathway, using quantitative fluorescence microscopy. The investigators applied this method to carry out unbiased analysis of the phenotypes produced by knocking down gene expression using RNAi. Using the HeLa human cell line, Collinet *et al.* monitored two receptor-mediated endocytic processes - the uptake of the iron-transport protein transferrin bound to its receptor and the uptake of epidermal growth factor (EGF) bound to its receptor (a receptor tyrosine kinase), which are important to cellular nutrition and cellular signaling, respectively (Figure 1, which also illustrates the core membrane-trafficking pathways in the cell). Endocytosis of these two receptor-ligand complexes is thought to utilize much of the same 'core' endocytic machinery, yet each pathway differs significantly in its regulation, and in the specificity with which internalized ligands are trafficked to different internal membrane compartments (Figure 1). These two processes are also a good choice from the experimental perspective, because fluorochrome-conjugated ligands enable the visualization of these processes by fluorescence microscopy.

Fifty-eight different parameters describing, for example, vesicle amount, size and intracellular distribution, were extracted using a computer-controlled algorithm from automatic confocal images of HeLa cells. The investigators screened multiple libraries of synthetic small interfering RNAs (siRNAs) and an endoribonuclease-prepared siRNA (esiRNA) library, each covering every human gene several-fold. Cluster analysis of these 58 parameters led to 10 parameter groups describing distinct classes of endocytosis phenotypes. This approach resulted in 161,492 knockdowns and around 2.5 × 10^6^ cofocal microscope images, requiring 4.5 × 10^6^ computing hours on a 2,584-core computer cluster to analyze. This is, first, an approach that excludes the subjective bias of a human observer. Second, the multi-parametric description of phenotypes potentially allows the detection of effects on endocytosis that would be missed by more conventional approaches, which are typically limited to relatively severe (or lethal) phenotypes. Taking advantage of their multi-parametric analysis, combined with deep coverage of the expressed genome, the authors have developed an impressively rich database of the effects of genetic disruption on the endocytic pathway in a human cell line.

What emerges from this analysis is both exciting and cautionary. On the exciting side, the authors identified a remarkably large number of genes - more than 4,000 - whose knockdown reliably affected some parameter of the endocytic analysis. On the cautionary side, this is a remarkably high hit rate - around 15% of the coding genome. The authors emphasize that their goal was not to identify particular genes that directly mediate a particular trafficking step or pathway but, instead, to develop a larger genetic profile that would enable appreciation of integrated 'design principles' of the endocytic pathway in mammalian cells. From this perspective, the list of implicated genes supports the existence of exquisitely close relationships, both direct and indirect, between the endocytic pathway and diverse cellular processes.

Returning to the question of how membrane trafficking is related to signal transduction, Collinet *et al.* identified a particularly large number of genes that encode signaling receptors and mediator proteins. For example, the primary hit list includes a large number of seven-pass transmembrane receptors, including 'orphan' receptors whose physiological significance is currently not established. The potential of this forward genetic screening approach to reveal new links in the signaling-endocytosis nexus is indeed very exciting. On the cautionary side, some of the identified hits (such as several neuropeptide receptors) are thought not to be expressed at significant levels in HeLa cells. Thus, despite the careful attention paid to verifying hits with multiple siRNA targets, the possibility that the current list still includes a number of false positives must be kept in mind. All in all, the recent work by Collinet *et al*. represents a bold and interesting effort, with great potential but also significant challenges.

In future studies we can anticipate integration of the strategy used by Collinet *et al*. with proteomics and protein biochemical methods, which will help distinguish direct from indirect genetic effects and provide insight into biochemical mechanisms. Further advances in automation and computational power may allow practical genetic analysis of endocytic effects produced by changes in the cellular environment or the activation of particular signaling pathways. We can also look forward to extension of the genetic approach to paired or combinatorial knockdowns, which may help organize the large number of hits identified into coherent genetic pathways. Such analysis could also provide crucial insight to the significance of hits representing the remarkably large number of human disease-linked genes identified, most of which have not been implicated previously in endocytosis. The paper by Collinet *et al*. indeed makes a bold step into the future, and provides an intriguing preview of a new era in cell biological research.


abstract: Dogs were present in pre-Columbian America, presumably brought by early human migrants from Asia. Studies of free-ranging village/street dogs have indicated almost total replacement of these original dogs by European dogs, but the extent to which Arctic, North and South American breeds are descendants of the original population remains to be assessed. Using a comprehensive phylogeographic analysis, we traced the origin of the mitochondrial DNA lineages for Inuit, Eskimo and Greenland dogs, Alaskan Malamute, Chihuahua, xoloitzcuintli and perro sín pelo del Peru, by comparing to extensive samples of East Asian (*n* = 984) and European dogs (*n* = 639), and previously published pre-Columbian sequences. Evidence for a pre-Columbian origin was found for all these breeds, except Alaskan Malamute for which results were ambigous. No European influence was indicated for the Arctic breeds Inuit, Eskimo and Greenland dog, and North/South American breeds had at most 30% European female lineages, suggesting marginal replacement by European dogs. Genetic continuity through time was shown by the sharing of a unique haplotype between the Mexican breed Chihuahua and ancient Mexican samples. We also analysed free-ranging dogs, confirming limited pre-Columbian ancestry overall, but also identifying pockets of remaining populations with high proportion of indigenous ancestry, and we provide the first DNA-based evidence that the Carolina dog, a free-ranging population in the USA, may have an ancient Asian origin.
author: Barbara van Asch; Ai-bing Zhang; Mattias C. R. Oskarsson; Cornelya F. C. Klütsch; António Amorim; Peter Savolainene-mail: <firstname.lastname@example.com>[^1]
date: 2013-09-07
institute: 1IPATIMUP, Rua Dr Roberto Frias s / n, 4200-465 Porto, Portugal; 2Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal; 3Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, KTH-Royal Institute of Technology, SE-171 65 Solna, Sweden; 4Bethany College, Lindsborg, KS 67456, USA; 5College of Life Sciences, Capital Normal University, Beijing 100048, People's Republic of China
references:
title: Pre-Columbian origins of Native American dog breeds, with only limited replacement by European dogs, confirmed by mtDNA analysis

# Introduction

The dog is now well established as the most ancient domestic animal and is unique as the only domesticate present in human societies on every continent in ancient times \[1\]. Previous studies of mitochondrial (mt) DNA have indicated southern East Asia as the geographical centre for the origin of dogs \[2–5\]. This has been largely supported by analyses of Y-chromosomal DNA \[5,6\], while a recent single nucleotide polymorphism (SNP)-based study argued that Middle Eastern wolves primarily contributed to the extant dog gene pool \[7\]. Regardless of the exact geographical origin of domestic dogs, it seems clear that American dogs originate from the Old World since analysis of ancient samples from several pre-Columbian archaeological sites were shown to have the same mtDNA haplogroups as Old World dogs \[8\].

A remaining question is if today's American dogs trace their origin to the dogs originally introduced from Asia via the Bering Straits in pre-Columbian time, or if this ancestry has been diluted or even completely erased by European dogs brought across the Atlantic following the arrival of Europeans in America. Several studies of mtDNA have attempted to elucidate this question, but did not include comprehensive samples across American breeds and/or of reference samples from the Old World, necessary for a comprehensive analysis \[8–11\]. Importantly, with the detailed knowledge of the worldwide dog mtDNA diversity presented recently \[4\], an opportunity has opened up to identify whether mtDNA haplotypes carried by American dogs originate from East Asia in ancient times or from Europe in the post-Columbian era.

Archaeological data and historic records have provided abundant evidence that dogs in pre-Columbian times were part of native cultures in the American continent long before the dawn of transoceanic travel in the fifteenth century \[12\]. Dogs were used for a large number of different purposes, for example, for hunting, sledging, freighting, protection and company, for religious and medicinal purposes and as a food resource. The earliest archaeological evidence for presence of dogs in the Americas has been dated to 10 000–8500 years ago, the dog thus being the sole domesticate in America during several thousand years \[13,14\]. According to current hypotheses, the Americas started to be colonized by south Siberian peoples at least 15 000 years ago immediately after deglaciation of the Pacific coastal corridor \[15\]. Thus, pre-Columbian dogs must have been brought along by Paleo-Indians of Asian origin in their expansions throughout the American continent, although not necessarily in connection with the first waves of humans.

A small number of extant breeds in the American continent, for example, the Mexican Chihuahua, the xoloitzcuintli (Mexican hairless dog) and the Peruvian perro sín pelo (Peruvian hairless dog) have been claimed to descend from pre-Columbian populations. Archaeological findings in South America are abundant in evidence of dogs increasingly entwined in human societies \[16\]. In the Arctic region, the presence of well-adapted dogs and their use by Native American peoples prior to 1492 is also well recorded \[17\]. Many of those dogs, such as the hare Indian dog and the Tahltan bear dog have disappeared as the aboriginal hunting methods declined. Modern Arctic breeds thought to be indigenous include the Inuit sled dog, the Canadian Eskimo dog and the Greenland dog (all three thought to share the same origin) and the Alaskan Malamute. Some modern American breeds such as the dogo Argentino, the fox Paulistinha, the fila brasileiro and the cimarrón Uruguayo have historical origins in European dogs and are not putatively indigenous. Similarly, the Alaskan husky and the American Eskimo dog have a known origin from Siberian spitzes and European dogs.

Besides breed dogs, free-ranging dogs are abundantly found across the American continent. These dogs commonly have heterogeneous morphologies suggesting an origin predominantly from a mix of European breeds. However, in remote areas of southeastern USA (South Carolina and Georgia) there is a group of free-ranging dogs (called the Carolina dog) which morphologically resemble the Australian dingo and South Asian pariah dogs. Based on this resemblance to 'primitive' dogs, an origin from indigenous pre-Columbian dogs rather than from run-away breed dogs of European origin has been suggested \[18\].

The strong impact of the introduction of domestic species by European settlers and explorers, and its deleterious effects on Native American cultures, is indisputable. The replacement or dilution of Native American dog populations with European dogs is thought to have been severe, with consequent loss of historic Native breeds \[19\]. However, the extent of this replacement or admixture remains largely undisclosed. Did the ancient migrants leave descendants in the modern American gene pool or were they completely erased by European dogs brought across the Atlantic in the post-Columbian era, and are extant dog populations direct descendents of the ancient populations in the same geographical region?

A very recent study of mtDNA in pre-Columbian dogs from Alaska and Greenland \[9\] showed that the ancient population and the modern Inuit sled dog population in Greenland carry predominantly the unique haplotype A31, strongly indicating local ancestry for this group of Arctic dogs. By contrast, a study of mtDNA among village dogs and street dogs from across America indicated that the maternal lineages of the indigenous American dog population have been almost completely replaced with European dogs \[10\]. However, this study did not include American breed dogs. Furthermore, the results were based on comparisons with a restricted source of information, that is, with the haplotypes found among 19 ancient American dog samples analysed in one previous study \[8\].

Another approach, possibly allowing for a more comprehensive picture of the geographical ancestry of mtDNA lineages among American dogs, is to compare extant American dogs with European and East Asian dogs, assuming that today's populations are good approximations for the ancient ones. The possibility of tracing the geographical origin of mtDNA lineages has opened up with the comprehensive picture of the worldwide dog mtDNA diversity presented recently \[4\]. This study showed distinct differences between the European and East Asian mtDNA gene pools, offering the possibility to trace whether mtDNA haplotypes among American dogs may have originated from Europe or East Asia. All populations across the Old World share a common gene pool of three major phylogenetic groups called clades A, B and C. In East Asia, the full diversity of these genetic groups is represented, but European dogs (together with all other populations west of the Himalayas and the Urals) have a limited diversity. Out of totally 206 mtDNA haplotypes identified globally \[3\], there are 15 haplotypes which are represented in virtually every dog population across the Old World, called universal types (UTs). In Europe, 76.7% of the dogs carry one of these 15 haplotypes and totally 92.6% carry a haplotype which is a UT or differs by a single substitution from one of the UTs. In addition, haplogroup D (which is absent in East Asia) has been found in Europe, in 5.9% of the dogs. Thus, totally 98.5% of European dogs carry an mtDNA which is a UT, differs by one substitution from a UT or belongs to haplogroup D. Consequently, if an American dog carries a haplotype which does not belong to this group of haplotypes, it is unlikely that its mtDNA lineage derives from Europe. In East Asia and Siberia, only 54.9% and 50% of the dogs, respectively, carry a UT; instead, there is a large number of unique haplotypes distinct from those in Europe. Therefore, it may be assumed that approximately 50% of the dogs that entered America via Bering Strait in pre-Columbian time carried a haplotype which was not a UT or belonged to haplogroup D and is therefore potentially informative for excluding a European origin.

Importantly, the high frequency of the 15 UTs and haplotypes differing by a single substitution from the UTs is a universal phenomenon west of the Himalayas, with for example 94.5% of dogs in southwest Asia and 93.1% in Africa, carrying such haplotypes \[3,4\]. Therefore, the high proportion of these haplotypes in today's European population most probably reflects the genetic make-up across western Eurasia in ancient times, and thus also in Europe at the time of European colonization of America. The assumption that today's European population can be used to represent the situation 500 years ago therefore seems justified, offering a firm basis for the analyses in this study.

In this study, the origins of American dogs were addressed by comparison of mtDNA sequences from American, European and East Asian dogs. This dataset includes new samples for several American breeds and free-ranging dog populations and an extensive collection of European dogs. Together with previous data for extant dogs across the world \[4\] and sequences derived from ancient American samples \[8\], we obtained a comprehensive picture of the mtDNA gene pools in America, Europe and East Asia. Based on this, we identified the sharing of haplotypes and haplogroups among these regions and between ancient and modern samples. Through this analysis, we traced the maternal ancestry of the modern New World dog populations, assessing for the first time the extent to which American dog breeds descend from the original pre-Columbian population, and reanalysing the ancestry of free-ranging populations in greater phylogeographical detail.

# Material and methods

## Samples, PCR amplification and sequencing

A sample of American dogs, of supposedly indigenous breeds as well as street dogs from both North and South America, was analysed by comparison with a comprehensive sample of European and East Asian dogs (see the electronic supplementary material, table S1). The Old World samples (total, *n* = 1872) were from Europe (*n* = 639), East Asia excluding Siberia (*n* = 889), Siberia (*n* = 95), Africa (*n* = 57), southwest Asia (*n* = 133) and India (*n* = 59). The New World samples (total, *n* = 347) included the Arctic breeds Alaskan Malamute (*n* = 9), Canadian Eskimo dog (*n* = 9), Greenland dog (*n* = 11) and Inuit sled dog (*n* = 18), the North American breeds Chihuahua (*n* = 14) and xoloitzcuintli (*n* = 43), and the South American breed perro sín pelo (Peru, *n* = 53). The American breeds were sampled from pure-bred dogs avoiding known common female ancestry among individuals. Sampling was obtained from European as well as American lineages of the breeds, and for the perro sín pelo, 19 samples were obtained directly from the Peruvian stock. Two American breeds of known Old World origin were also studied: fox Paulistinha (Brazil, *n* = 16) and dogo Argentino (Argentina, *n* = 40). In addition, free-ranging dogs were studied: from North America (USA), the pariah-like Carolina dog \[20\] (*n* = 19; captive dogs descending from free-ranging dogs); from South America, dogs collected at rural locations (total, *n* = 108); Argentina (Cordova and Buenos Aires regions, *n* = 54; Brazil (Rio de Janeiro and Rio Grande do Norte regions), *n* = 19; Colombia (Bucaramanga region), *n* = 35).

The samples were collected in the form of buccal cells or total blood. Buccal cells were collected by swabbing the inside of the cheek using sponge swabs, and applied to Whatman FTA cards (Whatman International Ltd., Maidstone, UK), according to the manufacturer's instructions. Total blood was also sampled on Whatman FTA cards. DNA extractions, PCR amplifications and sequence analysis were performed as previously described \[2\]. A 582 base-pair fragment of the mt control region (CR) was surveyed using previously published and newly sequenced samples (see the electronic supplementary material, table S1). All novel haplotypes identified in this study have been deposited in GenBank under accession numbers [HQ452424](HQ452424)–[HQ452430](HQ452430) and [HQ452435](HQ452435)–[HQ452438](HQ452438). In a previous study \[4\], complete mt genomes of 169 individuals representing almost all parts of the CR networks resulted in the identification of subclades within the original clades A, B and C, which are informative since some of them have not been found in European dogs (figure 1). Individuals sequenced only for the CR can be assigned to subclades by mutations within the CR which are diagnostic for each subclade \[4\].

We also compared our dataset with published data from ancient American dog samples \[8\] and modern American non-breed dogs \[10\]. Since the sequences of these two datasets overlapped only partly with our sequences, comparisons were performed separately from the main analysis. Thus, our modern sequences were compared with 19 ancient DNA sequences obtained from dog remains from South America (Bolivia, Peru and Mexico) and Alaska (Fairbanks area) \[8\]. These sequences overlap by 244 bp with our modern sequence data. We also compared our samples with sequences from Castroviejo-Fisher *et al*. \[10\] which consisted of mtDNA sequences from '400 dogs from rural and isolated areas as well as street dogs' from all across America. These sequence overlap by 342 bp with our sequences.

## Phylogenetic analysis

Sequences were aligned with the MUSCLE tool implemented in Geneious v. 5.5.3 Pro \[21\]. Comparisons of sequences and identification of haplotypes were performed with DnaSp v. 5 \[22\]. Minimum-spanning networks and trees were constructed manually based on the minimum distances between haplotypes calculated with Arlequin v. 3.5 software \[23\] ignoring indels. Unique and shared haplotypes among dog populations or breeds from different geographical regions were identified using the program UPST.EXE (available by request to <firstname.lastname@example.com>).

# Results

Among the American samples, all haplotypes belonged to the universally occurring clades A, B and C, indicating an Old World origin for all maternal lineages. Among the Old World samples, 38 haplotypes were unique to Europe, 121 unique to East Asia (excluding Siberia) and 12 unique to Siberia (figure 1). This dataset included 302 new European samples relative to a previous study \[4\], but the large difference between European and East Asian samples observed previously did not alter. Thus, the diversity among European dogs was very limited, with practically all haplotypes centred around the 15 UTs (figure 1). By contrast, approximately 50% of East Asian dogs carried haplotypes on large distance from those found in Europe, many of which belonging to sub-haplogroups not detected in Europe, implying the possibility of tracing the geographical origins of a large proportion of the American maternal lineages.

## Breed dogs

### Arctic America

Inuit sled dogs, Canadian Eskimo dogs and Greenland dogs had similar mtDNA gene pools, in that the most frequent haplotype for all three breeds was A31, unique to this group of breeds (table 1 and figure 1). The Inuit sled dog and the Greenland dog also shared a haplotype unique to the two breeds (A124). In addition, the Inuit sled dog and the Eskimo dog had one unique haplotype each (A181 and A63, respectively). Three of these four private haplotypes differ by at least two substitutions from any of the European haplotypes. In total, 30/38 individuals in this group of dogs had haplotypes unique to the group, and the remaining eight dogs had universally occurring haplotypes, present in both Europe and East Asia. Importantly, a very recent article \[9\] showed that pre-Columbian samples from Greenland had a high frequency of A31, strongly suggesting ancestry from the pre-Columbian population for this group of dogs.

mtDNA control region haplotypes found among American breed dogs and Carolina dog, and their geographical distribution in both the Old World and the New World. Subclade refers to the 10 subclades of clades A, B and C; only haplotypes belonging to subclades other than the four universal subclades a1, b1, c1 and c2 are indicated. UT, universal types of DNA haplotype found worldwide \[4\]; PT, private types found only in a specific breed, population and/or region.

| breed (code) | *n* | haplotype (subclade) | frequency (*n*) | geographical distribution |
|:---|:---|:---|:---|:---|
| Inuit sled dog (ISD) | 18 | A181 | 6% (1) | PT |
|  |  | A124 | 6% (1) | PT in Arctic America (ISD, GD) |
|  |  | A31 | 78% (14) | PT in Arctic America (ED, GD, ISD) |
|  |  | A20 | 11% (2) | UT |
| Eskimo dog (ED) | 9 | A31 | 67% (6) | PT in Arctic America (ED, GD, ISD) |
|  |  | A63 | 11% (1) | PT |
|  |  | A18 | 22% (2) | UT |
| Greenland dog (GD) | 11 | A31 | 55% (6) | PT in Arctic America (ED, GD, ISD) |
|  |  | A124 | 9% (1) | PT in Arctic America (ISD, GD) |
|  |  | A11; A17; A20 | 36% (4) | UT |
| Alaskan Malamute (AM) | 9 | A29 (a2) | 78% (7) | Siberia, Japan, China, Indonesia |
|  |  | A11; A17 | 22% (2) | UT |
| Chihuahua (CH) | 14 | A185 (a4) | 36% (5) | PT |
|  |  | C16 | 7% (1) | South America (CH and PSP), Siberia |
|  |  | A11; A17; B1 | 57% (8) | UT |
| xoloitzcuintle (Xo) | 43 | A24 | 2% (1) | Europe, Colombia |
|  |  | B12 | 2% (1) | Europe, Japan |
|  |  | A2; A16; A17; B1; C1; C2; C3 | 95% (41) | UT |
| Carolina dog (CD) | 19 | A184 (a5) | 37% (7) | PT |
|  |  | A39 (a2) | 5% (1) | Japan, China |
|  |  | A16; A18; A19; B1 | 58% (11) | UT |
| perro sin pelo Del Peru (PSP) | 53 | C16 | 62% (33) | Siberia, South America (CH, PSP) |
|  |  | A1 | 2% (1) | Europe , China, Korea, Brazil |
|  |  | B8 | 2% (1) | Europe |
|  |  | C8 | 2% (1) | Europe |
|  |  | A2; A11; A17; A18; A19; B1 | 32% (17) | UT |

Among the Alaskan Malamute, 7/9 individuals had haplotype A29, which belongs to subclade a2 (not represented in Europe) and otherwise found only in East Asia (including Siberia) and island Southeast Asia. The two other individuals had universally occurring haplotypes. Haplotype A29 was also found in pre-Columbian samples from Alaska \[8,9\]. This suggests ancestry of modern Alaskan Malamute from the pre-European Alaskan population (figure 2) but, since A29 is also commonly found among Siberian husky with which Alaskan huskys were interbred in the early 1900s, the evidence for this is not clear cut. The Alaskan Malamute and the Eskimo/Greenland/Inuit group had almost totally different mtDNA gene pools, only 2/9 and 2/38 individuals, respectively, sharing haplotypes between the groups.

### North America

For the Mexican breed Chihuahua, the most frequent haplotype (carried by 5/14 individuals) was A185, which was unique to this breed among the modern dogs. Importantly, this haplotype was also found in one pre-Columbian sample from Mexico (figure 2), suggesting direct ancestry of Chihuahua from ancient Mexican dogs. One individual had haplotype C16, the most common type among the South American breed perro sín pelo del Peru. C16 was also reported for xolo at a frequency of 16% in a previous study \[24\]. Among the Old World samples, C16 was found only among Siberian dogs. The remaining Chihuahua had universally occurring haplotypes. The Mexican 'naked breed' xoloitzcuintle (xolo) had only haplotypes occurring universally and two haplotypes found in Europe. However, as noted above, C16 was found in a previous study of xolo, at a frequency of 16% \[24\]. Notably, 79% of the xolo carried haplotypes shared with perro sín pelo del Peru, the South American 'naked breed'.

### South America

For the Peruvian 'naked breed' perro sín pelo del Peru, the majority of dogs (33/53) had haplotype C16, absent among the European samples and shared only with the other 'naked breed' xolo, and with Chihuahua and Siberian dogs. Two individuals had haplotypes otherwise unique to Europe, and the remaining individuals had universally occurring haplotypes. Eighty-one per cent of the individuals shared a haplotype with xolo.

In conclusion, all investigated breeds of presumed indigenous origin across America carried haplotypes indicating pre-European origin, except for Alaskan Malamute for which the results were ambigous. All these breeds except xolo had a high frequency of haplotypes absent in Europe: 79% of the Arctic Inuit, Eskimo and Greenland dogs, and a total of 35% of dogs belonging to the Meso/South American breeds Chihuahua, xolo and perro sín pelo del Peru. Only half of the lineages introduced to America from Asia are expected to differ from European sequences, since the frequency of UTs is approximately 50% in Siberia and East Asia. Therefore, even if 100% of the lineages would have an ancient origin from Asia, only 50% of the individuals would be expected to carry haplotypes absent in Europe. Hence, our data indicates that the Arctic breeds Inuit, Eskimo and Greenland dog (having a frequency above 50% of haplotypes absent in Europe) have remained practically uninfluenced by European lineages, and that among the Meso/South American breeds (35% of the dogs carrying haplotypes distinct from European haplotypes), 70% of the maternal lineages originate from the pre-Columbian population. Thus, replacement of indigenous American breeds by European dogs seems to have been relatively limited.

We also analysed dogs belonging to the fox Paulistinha and dogo Argentino breeds. These breeds originate from dogs of known European ancestry. In accordance, these dogs carried only haplotypes that are frequently found in Europe, five universal haplotypes and one other haplotype frequent in Europe (see the electronic supplementary material, table S2).

## Comparison of pre-Columbian sequences with a comprehensive modern sample

We compared our dataset with ancient American mtDNA sequences from Mexico, Peru, Bolivia and Alaska reported in an earlier study (\[8\]; figure 2). This analysis is the first comparison of these ancient American sequences with a comprehensive collection of sequences from modern East Asian dogs and American breed dogs, instead of predominantly European breeds as in earlier studies. This allowed us to investigate possible geographical continuity over time among American breed dogs and to explore the possibility of an East Asian origin of the ancient American dog mtDNA lineages.

We found two cases where ancient and modern samples from the same geographical region had the same non-universal haplotype, suggesting continuity since pre-European time for these populations. In one case, haplotype A185 was found exclusively in ancient Mexican samples (D25) and in modern samples of the Mexican Chihuahua, strongly indicating direct ancestry of Chihuahua from Mexican pre-Columbian dogs. Furthermore, a haplotype corresponding to A29 (D18), otherwise found only in Siberia, East Asia and Oceania, was identified in ancient Alaskan samples as well as in the modern Arctic breed Alaskan Malamute but, as related above, the presence of A29 also among Siberian husky offers an alternative to genetic continuity in Alaska. Notably, a very recent study of pre-Columbian dogs from Alaska and Greenland \[9\] showed that, in Greenland, both the ancient population and the modern Inuit sled dog population carry predominantly the unique haplotype A31, strongly indicating ancient local ancestry for this group of Arctic dogs.

Importantly, Leonard *et al*. reported one phylogenetic group of haplotypes (containing haplotypes D27, D28, D29, D30, D31 and D33) to be unique for the ancient South/Meso American samples (figure 2). However, we found the central haplotype D28 in East Asia (in Korea, haplotype A161), thus placing the origins of these sequences in East Asia. Eight of the 19 ancient haplotypes correspond to haplotypes found in the modern samples, all of which, except the American specific A185, found in East Asia. The remaining haplotypes were unique to the ancient samples, but differ by one or two substitutions from extant haplotypes found in East Asia, except one ancient haplotype (D40). D40 is separated by at least four substitutions from any other dog haplotype and seemingly represents a separate phylogenetic clade (figure 2). Therefore, D40 is possibly the result of a crossbreeding between a dog and a female wolf in America.

In conclusion, we find a strong indication that the Mexican breed Chihuahua has a direct ancestry from Mexican pre-Columbian dogs, and a similar link was recently shown for Inuit dogs and pre-Columbian samples in Greenland \[9\]. Importantly, except D40, all ancient American samples can be linked to haplotypes found among East Asian dogs.

## Free-ranging dogs

Among the 'free-ranging' Carolina dogs of southern USA, the most frequent haplotype was A184, which was unique to these dogs (it is a novel halpotype not previously reported in GenBank) and belongs to the East Asian-specific phylogenetic subclade a5 (figure 1). One individual had haplotype A39, otherwise found only among Chinese non-breed dogs and the Japanese breed shiba inu. The remaining dogs had universal haplotypes. This gives a strong support to the hypothesis that the Carolina dog has indeed originated from pre-Columbian dogs.

By contrast, South American free-ranging dogs from Argentina, Brazil and Colombia had mainly universally occurring haplotypes (83.3% carrying UTs) and several European specific haplotypes (see the electronic supplementary material, table S2). There were also three unique haplotypes (A189, A190, B45), a single step from haplotypes found also in Europe, and three haplotypes (A38, B2 and B5) absent from Europe but present in East and West Asian breeds (e.g. chow chow, Afghan hound and akita) and rural non-breed dogs. Thus, South American free-ranging dogs in our sample seem to originate mainly from European dogs, although traces of native dogs cannot be totally excluded.

This is in good agreement with a previous study by Castroviejo-Fisher *et al*. \[10\] of 400 dogs from rural and isolated areas, as well as street dogs from across America. This study reported that a very low proportion of the mtDNA lineages among these modern American non-breed dogs (less than 10%) derive from the pre-European dog population, based on a comparison with the 19 ancient American dog samples reported by Leonard *et al.* \[8\]. This indicated that almost all indigenous dogs were replaced by European dogs. However, comparing the dataset in Castroviejo-Fisher *et al*. with our comprehensive sample of modern dogs, we found that these dogs carry several potentially indigenous mtDNA haplotypes which are unique to America or otherwise found exclusively in East Asia (see the electronic supplementary material, figure S1 and table S3). These haplotypes were carried by 7.8% of all dogs, which indicates that approximately 15% of the lineages have pre-European ancestry, considering that at least 50% of indigenous American dogs are expected to carry non-informative haplotypes (UTs) also found in Europe. More importantly, in a detailed analysis of the different subpopulations, we also noted that the putatively indigenous haplotypes were not evenly distributed. Thus, 9.8% of the Mexican dogs and 14.8% of the Bolivian dogs carry these haplotypes (indicating 20–30% Asian ancestry), but only 2.3% of the dogs from Argentina. This agrees with our findings that a large proportion of Carolina dogs carry seemingly indigenous American haplotypes, whereas our sample of free-ranging Argentinan dogs has almost exclusively European haplotypes. Therefore, it seems that while free-ranging American dogs generally derive from European dogs, there are pockets of rural populations, for example, the Carolina dog and populations in Mexico and Bolivia, with a high proportion of pre-Columbian ancestry. Importantly, comparing with a dataset from Europe \[25\], only two of 94 dogs of European origin (2.1%) carried a unique haplotype, confirming that the majority of the unique American haplotypes have an indigenous origin and are not merely European haplotypes not yet sampled.

Castroviejo-Fisher *et al.* \[10\] also reported that a large proportion of haplotypes found in the dataset (23 out of 40) were unique to the American dogs compared with dogs of the Old world. However, the comparison was performed against a limited dataset of non-American dogs \[26\], and comparing instead to our more comprehensive dataset of dogs from across the Old World, we identified the majority of these 23 haplotypes. For the 342 bp region overlapping with our dataset, there were 34 haplotypes in the dataset of Castroviejo-Fisher *et al*. Of these, 27 were shared with dogs in Eurasia, leaving only seven haplotypes unique to America.

# Discussion

This study provides clear evidence for the ancient Asian ancestry of extant American dog breeds. All breeds of presumed indigenous American origin carried haplotypes absent in a comprehensive sample of European dogs, and our data indicates that only 30% or less of the female lineages in indigenous American breeds have a European origin. Importantly, the Mexican breed Chihuahua shared a haplotype uniquely with Mexican pre-Columbian samples, showing genetic continuity over time and geographical region and corroborating the Mexican origins of the Chihuahua. The data also once more confirmed that American dogs have a common origin with Old World dogs, since no distinct haplogroups unique to American dogs were found and all haplotypes fell into the previously described universal phylogenetic clades A, B and C. However, we note that one pre-European Alaskan dog reported by Leonard *et al*. \[8\] had a haplotype (D40) separated by four substitutions from the closest modern dog haplotype, which we believe may derive from a dog–wolf hybridization.

An important finding is that not only American breed dogs but also some populations of free-ranging dogs seem to stem from indigenous American dogs. Thus, we here give genetic evidence that feral dogs from the USA, the so-called Carolina dog, may have an indigenous American origin and are not just 'run-away' dogs of European descent. The dingo-like appearance of the Carolina dogs may therefore be a remnant of their ancient past. The most frequent haplotype (A184) is unique to Carolina dogs and belongs to an East Asian-specific phylogenetic subclade, offering a clear indication of an East Asian origin for these dogs. Also haplotype A39, otherwise found only among East Asian dogs including the shiba inu, may have an ancient East Asian origin, but the possibility remains that this haplotype derives from abandoned shiba. The South American free-ranging dogs carried mainly universal or typically European haplotypes, but a few individuals had haplotypes carried by East Asian dogs and absent among European breeds. However, all these haplotypes are also carried by East Asian breeds present in the Western world dog population and could have resulted from integration of abandoned Asian breed dogs. We conclude that the South American free-ranging dogs mainly originate from dogs brought by the Europeans, although traces of native dog cannot be totally excluded.

In general agreement with our data, the study by Castroviejo-Fisher *et al.* \[10\] reported that at most 10% of the mtDNA lineages among American rural and street dogs have indigenous American origin. However, in a more detailed phylogeographical analysis of these samples, we note that in the subsamples from Mexico and Bolivia approximately 25% of the lineages seem to retain mtDNA haplotypes with an Asian origin. Therefore, pockets of remaining populations with high proportion of indigenous pre-European origin seem to persist among American non-breed dogs, as well as among the indigenous American breeds.

We conclude that the ancestry of the American dog population is complex. There are several different types of populations of American dogs of possibly pre-Columbian origin, and these populations retain different proportions of pre-Columbian ancestry. The dog breeds of supposedly indigenous American origin carry predominantly mtDNA haplotypes of pre-Columbian origin. By contrast, free-ranging dogs, including dogs from rural areas as well as street dogs, generally show no or very little pre-Columbian ancestry, but also among these dogs there remain populations with considerable proportions of indigenous American ancestry.

The studied Arctic American dogs seem to consist of two separate populations. The Alaskan Malamute is thought to descend from dogs bred by the Mahlemut people of the upper-western Alaska. A majority of these dogs carried haplotype A29, which seemingly presents both spacial and temporal clues to the origins of the breed. A29 (absent in Europe) is found among East Asian dogs and, significantly, among Siberian husky, a sled dog originally bred by the Chukchi people on the northeastern tip of Siberia. This indicates genetic links between East Asia, Siberia and Arctic America. A29 was also found in ancient samples from Alaska, indicating the presence of the Alaskan Malamute ancestors in the pre-Columbian era. However, a complicating factor is that Alaskan Malamutes were interbred with Siberian husky in the gold rush era; analyses of additional markers will be necessary to establish how the modern Alaskan Malamutes relates to the dogs of the Mahlemut people. The other modern Arctic breeds (Inuit, Eskimo and Greenland dogs) seem to constitute a separate genetic group, as only 2/38 individuals shared haplotypes with Alaskan Malamutes. The three breeds share a common gene pool including a unique haplotype (A31) found in the majority of individuals. These three breeds, in accordance with the mtDNA data, are often considered a single breed and are thought to descend from sled dogs bred by Thule people, the ancestors of the modern Inuits. This culture developed in coastal Alaska by 1000 years ago and rapidly reached Greenland a few centuries later, having presented the first evidence of consistent use of sled dogs in the Arctic \[27\]. Importantly, a very recent study of pre-Columbian arctic dogs \[9\] showed that the related ancient dog population in Greenland carried almost exclusively A31. Thus, the ancient and modern populations share the unique haplotype A31 at high frequency, confirming the ancestry of modern Inuit, Eskimo and Greenland dogs from the local ancient population. The almost total lack of shared haplotypes between these breeds and the Alaskan Malamute possibly reflects sequential arrival to America of the related human populations \[15\].

Overlap was also minimal between the Arctic breeds and other dogs across America. By contrast, North American (excluding the Arctic) and South American breeds showed genetic links. Chihuahua shared one haplotype (C16) exclusively with perro sín pelo and Siberian dogs. This haplotype was absent from our xolo sample but was found in this breed in a previous study \[24\]. Furthermore, around 80% of the individuals of the Mexican 'naked breed' xolo and the Peruvian 'naked dogs' shared haplotypes, indicating a geographical link connected with the shared morphology, in agreement with all naked breeds carrying the same causative mutation \[28\]. It is notable that genetic data for both dogs and humans indicate an initial migration forming the North and South American populations, followed by subsequent waves into the Arctics \[15\].

Comparison of the modern data with ancient sequences suggested genetic continuity from pre-Columbian into modern times in Mexico and, possibly, Alaska, adding to the evidence for genetic continuity in Greenland presented very recently by Brown *et al.* \[9\]. Modern Chihuahua and ancient Mexico had the same unique haplotype (A185 and D25, respectively), and ancient Alaska and modern Alaskan Malamute shared a non-European haplotype (D18 and A29, respectively). Interestingly, A185 and A29 have been reported also for modern Puerto Rican 'street dogs' \[29\], but the possible link to ancient American samples was not noted in that study. This suggests that A185 and A29 may have been widespread across ancient America but, since the two haplotypes are found among modern Chihuahua and Siberian husky, there remains the possibility that A185 and A29 in Puerto Rico originate from abandoned dogs of these breeds. An important finding when comparing the new modern sequences presented in this study with the ancient DNA samples was that the central haplotype (D28) in a formerly 'ancient America-specific' clade \[8,10\] was found among East Asian dogs (haplotype A161). This implies that all ancient American sequences except one (D40; possibly the result of dog–wolf crossbreeding) can now be linked to haplotypes present in East Asia or Siberia. We also note a large proportion of unique haplotypes among the ancient samples. However, the ancient sequences harbour an unusual amount of transversions, suggesting that these may be single base substitution artefacts, a known problem in the sequencing of ancient samples. For example, the five unique haplotypes surrounding D28 (D27, D29, D30, D31 and D33) are defined by three transversions (in previously non-variable positions) and two transitions, compared with six transversions and 50 transitions found among the 1555 modern samples in clade A. It is therefore possible that several of the haplotypes surrounding D28 are in reality D28 (modern A161), and thus identical to modern East Asian samples.

In the modern sample, we identified nine haplotypes, five in the Arctic and four in North and South America, which were distinct from haplotypes found among European dogs. Only 50% of the pre-European dogs are expected to have carried haplotypes absent in Europe, implying that several more of the American lineages probably have an Asian origin. There were also at least four additional haplotypes found in the ancient American samples. Thus, it seems that the American dog population was not formed through a severe genetic bottleneck, but that several lineages were brought to America from Asia in the pre-Columbian era, probably in several waves of migration. The analyses in this study are based on mtDNA, which is a single genetic marker inherited maternally, putting obvious limitations to the inferences. To assess fully the pre- and post-Columbian contribution of genetic material to the American breeds, as well as the possibility of hybridization between American dogs and wolves, studies based on additional markers, suggestively the Y-chromosome as well as autosomal markers will be necessary. Possibly, improved phylogeographic fine mapping of both American humans and dogs may show whether the genetic diversity of domestic dogs mirrors that of humans and provide clues for understanding the colonization of the New World.

## Acknowledgements

We thank numerous donors of samples, especially Vet. Dr Liliana Arbulu Perez Vargas, Sue Hamilton, Marina Kuzina, Gunvor Bergström, Sonja Kolijn, Martin Whittle, Carlos Vullo, Eliseu Fagundes de Carvalho, Pedro Balonas, Adriana Castillo, Eduardo Arroyo and Jane Gunnell for organizing of sampling.

## Funding statement

This study was supported by grants from the Swedish Research Council, OE and Edla Johanssons Scientific Foundation, the Wenner-Gren Foundations and by a research grant to B.v.A. (SFRH/BDE/15581/2006) from Fundação para a Ciência e a Tecnologia. P.S. is a Royal Swedish Academy of Sciences Research Fellow supported by a grant from the Knut and Alice Wallenberg Foundation and was during the work with this study, a Pearson Distinguished Professor at Bethany College supported by a grant from Gerald Pearson. IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Ministry of Science, Technology and Higher Education and is partially supported by FCT, the Portuguese Foundation for Science and Technology.

# References

[^1]: These authors contributed equally to this study.