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Human Immunology (2007) 68, 950 958

Estimation of HLA-A, -B, -DRB1 Haplotype


Frequencies Using Mixed Resolution Data from a
National Registry with Selective Retyping of
Volunteers
Craig Kollmana,*, Martin Maiersb, Loren Gragertb, Carlheinz Mllerc,
Michelle Setterholmb, Machteld Oudshoornd, Carolyn Katovich Hurleye
a

Jaeb Center for Health Research, Tampa, Florida


National Marrow Donor Program, Minneapolis, Minneapolis
c
ZKRD - German National Bone Marrow Donor Registry, Ulm, Germany
d
Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center and Europdonor Foundation,
Leiden, The Netherlands
e
Department of Oncology, Georgetown University Medical Center, Washington, DC
b

Received 13 December 2005; received in revised form 16 September 2007; accepted 5 October 2007

KEYWORDS
EM algorithm;
HLA-DRB1;
Haplotype frequencies

Summary Large registries of volunteer hematopoietic stem cell donors typed for HLA contain
potentially valuable data for studying haplotype frequencies in the general population. However
the usual assumptions for use of the expectation-maximization (EM) algorithm are typically
violated in these registries. To avoid this problem, previous studies using registry data have
reduced the HLA typings to low-resolution and/or excluded subjects who were selected for
testing on behalf of a specic patient (patient-directed typings). These restrictions, added to
avoid bias from selection of nonrepresentative volunteers for higher-resolution typing, have
limited previous results to haplotypes dened at low resolution. In this article we eliminate the
need for such restrictions by formally relaxing the assumptions necessary for the EM algorithm.
We show mathematically and through simulation that varying levels of resolution can be incorporated even if the level of typing resolution is chosen based on the HLA type. This allows use
of intermediate and high resolution data from patient-directed typings to extend haplotype
frequency estimates to the allele level for HLA-DRB1. We demonstrate the feasibility of using
this computationally demanding algorithm on large datasets by applying it to more than 3 million
volunteers listed in the National Marrow Donor Program Registry.
2007 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc.
All rights reserved.

* Corresponding author. Fax: (813) 975-8761.


E-mail address: ckollman@jaeb.org (C. Kollman).

0198-8859/$ -see front matter 2007 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.humimm.2007.10.009

951

HLA-A, -B, -DRB1 haplotype frequencies

ABBREVIATIONS

Table 1 Examples of assignments used to describe


results of HLA testings at varying levels of resolution.

EM
HLA
NMDP

Allele(s) possibly
Assignment present

expectation-maximization
human leukocyte antigen
National Marrow Donor Program

DRB1*0101

0101

DRB1*01ABa 0101 or 0102

Introduction
The National Marrow Donor Program (NMDP) was established
by Congress in 1986 to develop and maintain a registry of
human leukocyte antigen (HLA)typed volunteer unrelated
donors for patients in need of a hematopoietic stem cell
transplant [1,2]. Since then more than 5 million volunteers
have been recruited and typed for HLA polymorphisms, with
a primary focus on HLA-A, -B, and -DRB1. The NMDP Registry
therefore offers a valuable resource for studying allele and
haplotype frequencies of these three loci.
There are limitations to the Registry HLA data, however,
that must be taken into consideration. Typing was done using a variety of techniques at varying levels of resolution
(Table 1) as the technology evolved over the 20-year history
of the NMDP [3 6]. Many of the intermediate- and highresolution typings of volunteer donors were performed because they were potential matches for a patient (patientdirected typings). Volunteers with common HLA types may
therefore be more likely to have allele level data available
so that the level of resolution is not statistically independent
of HLA type.
Previous analyses of HLA frequencies in the NMDP Registry have used a version of the expectation-maximization
(EM) algorithm [7,8] that requires all subjects to be tested
for all three loci (HLA-A, -B, -DRB1) at the same level of
resolution [9 12]. Analyses therefore excluded volunteers
who were not tested for all three loci (introducing a potential selection bias), and DRB1 alleles where known were
collapsed to serologic HLA-DR antigens in these previous
studies.
Adaptations of the EM algorithm to incorporate varying
levels of typing resolution and untyped loci have been previously described [1315], but they have assumed (some implicitly) that the level of typing resolution is statistically
independent of the HLA type. In this article we formally
relax that assumption, specifying conditions under which the
level of typing resolution can depend on the HLA type and
still satisfy the general framework of the EM algorithm. This
justies the use of mixed resolution data that include patient-directed typings to extend haplotype frequency estimates to the allele level.
We use data from more than 3 million volunteer donors
listed in the NMDP Registry to estimate the frequencies of
HLA-A, -B, -DRB1 haplotypes in various racial/ethnic groups.
A simulation is also presented to conrm that the EM algorithm gives valid results even when volunteers with more
common HLA types are disproportionately selected for intermediate- or high-resolution typing.

DRB1*01BV

0101, 0102, 0106


or 0107

DRB1*01XX

0101, 0102, 0103,


0104, 0105,
0106, 0107,
0108, . . . or
0116
0101, 0102, 0103,
0104, 0105,
0106, 0107,
0108, . . . or
0116

DR1

Comment
Allele resolution: Specic
allele is determined
from HLA testing
Intermediate resolution:
HLA testing cannot
distinguish between
these two alleles, but
rules out all other
possibilities.
Intermediate resolution:
Less specic testing
gives a larger list of
possible alleles.
Low resolution: DNA
testing is logically
equivalent to a
serologic typing of DR1
(see next row).
Low resolution: Serologic
testing identies the
protein expressed on
the surface of the cell;
it could be any one of
the 16 DRB1 alleles
that encode the DR1
protein.

Note: HLA typing does not always identify the specic allele(s)
present in an individual. Results are used to narrow the list of
alleles possibly present in the individual. Each of the assignments listed in this table is consistent with the allele DRB1*0101.
An individual with this allele could be reported with any of these
assignments depending on the level of resolution used.
a
The full set of allele letter codes is available online at
http://bioinformatics.nmdp.org/HLA/allele_code_lists.html.
These codes are approved for use in registry typing by the World
Marrow Donor Association [6].

Subjects and methods


Subjects
Analysis included all domestic volunteers recruited into the NMDP
Registry from January 1993 through July 2004 who self-reported
their race/ethnicity as African-American/black, Asian/Pacic Islander, Hispanic, or Caucasian/white. Other groups such as Native
American/Alaska Natives were not analyzed here because of the
relatively smaller sample sizes. All volunteers in the racial/ethnic
groups indicated above were included in the analysis regardless of
the level of resolution of HLA data available.

HLA Typing
All volunteer donors were typed for HLA-A and -B when they joined
the NMDP Registry. Most volunteers were typed using serologic
methods until 1997, when an initiative to use DNA-based testing was
implemented. Starting in 1992 many new volunteers were also typed

952
Table 2
locus.a,b

C. Kollman et al.
Level of resolution available for volunteers in the National Marrow Donor Program (NMDP) Registry at the HLA-DRB1

Level of DRB1 typing


None
Serologyc
Low-resolution DNA
Intermediate-resolution
DNA
Allele(s) determined

African-American/black
(N 412,861)

Asian/Pacic Islander
(N 359,423)

Hispanic
(N 449,844)

37,503
1,600
53,015
283,053

40,601
2,853
44,561
251,433

47,771
2,466
47,518
322,175

(9%)
(1%)
(13%)
(69%)

37,690 (9%)

(11%)
(1%)
(12%)
(70%)

19,975 (6%)

Caucasian/white
(N 2,361,208)

(11%)
773,172
(1%)
68,379
(11%)
171,538
(72%) 1,196,807

29,914 (7%)

(33%)
(3%)
(7%)
(51%)

151,312 (6%)

Total
(N 3,583,336)
899,047
75,298
316,632
2,053,468

(25%)
(2%)
(9%)
(57%)

238,891 (7%)

Numbers do not include 1,259,743 volunteers whose race/ethnicity is unknown and 228,507 volunteers from other racial/ethnic groups
with sample sizes too small for analysis.
b
Each volunteer is classied according to the lowest resolution for the two DRB1 alleles.
c
Serologic testing is considered low resolution.

using molecular biology techniques for HLA-DRB1 at the time of


recruitment. Over time the HLA DNA-based testing has incorporated
additional reagents, and the resolution has increased from low to
intermediate (Table 1).
Subsequent higher-resolution testing of a selected subset of the
volunteer donors was obtained in several scenarios. Transplant centers requested blood samples from volunteers who they believed
may be a potential match for their patient based on the HLA assignments listed in the NMDP Registry (patient-directed typings). These
samples were then retyped by the transplant center choosing the
level of resolution at their discretion subject to minimum requirements set by the NMDP [16]. HLA typing results were reported back
to the NMDP Coordinating Center (regardless of whether the volunteer was considered a suitable match for the patient) where a detailed override algorithm was used to update the information in the
Registry. Other volunteers were typed at high resolution as part of
allele frequency studies or because they carried a novel HLA assignment.
In summary some volunteer donor HLA assignments were obtained by serology at the broad or split level and others by DNAbased testing at low, intermediate, or allele resolution. Because
volunteers with higher-resolution testing are often selected as described above, this subset is not necessarily representative of the
general population (i.e., is not random).
The level of HLA-DRB1 data available varied according to race/
ethnicity (Table 2). Overall 25% of volunteers were tested for only
HLA-A and -B, but more minority individuals have been tested for
HLA-DRB1 because these volunteers were given higher priority. HLADRB1 alleles were determined for 7% of volunteers ranging from 6%
for individuals of Asian/Pacic Islander and Caucasian ethnicities to
9% for those of African-American ethnicity.
Table 3 lists the HLA antigens and alleles that are present within
the HLA assignments in the Registry. Serologic subdivisions of B14
and B70 (i.e., splits) were converted to their broad antigens for
analysis because these antigens were most often reported at this
level. Because of the unreliability of antibody reagents for A69,
serologic A68 and A69 results were converted to the broad A28. For
the analysis described here, cases where HLA-A and/or HLA-B alleles
were known were collapsed to a low-resolution serologic split equivalent [17,18]. The serologic types assigned to alleles are available
online (http://bioinformatics.nmdp.org, listed under the headings
HLA Resources and NMDP Search Determinants).
All 250 HLA-DRB1 alleles present in the NMDP Registry were used
in analysis (Table 3). The NMDP has developed an extensive set of
allele codes to allow HLA testing laboratories to report results of
low- and intermediate-resolution typings [18]. This allows the list of
all possible alleles present in a volunteer donor to be stored in the
computerized Registry. For example, an HLA laboratory will report

the code DRB1*01AB to denote that the individual possesses either


DRB1*0101 or DRB1*0102 (but it is unknown which one). The alleles
included in the code are based on the HLA alleles known at the
time of the typing test interpretation. More examples are given in
Table 1.
The accuracy of the HLA assignments in the Registry has been
evaluated over time. The laboratories performing the testing were
required to be accredited, and the majority of testing incorporated
blinded quality control samples. For DRB1 the accuracy of the quality control samples tested at the time of recruitment has improved
from an overall error rate of 1.6% in 1992 [19] to 0.06% today (data
not shown). Also the accuracy of HLA-A, -B serologic typing compared with DNA-based assignments [20] and a comparison of recruitment typing to higher-resolution typing of volunteers potentially
matching a searching patient have been reported [21].

Estimation of Haplotype Frequencies


The EM algorithm [7] is a very general principle for handling missing
data in statistical analysis. As applied to estimation of multilocus
haplotype frequencies, this algorithm has been described in detail
elsewhere [8] and is briey summarized here (also see Appendix for
more details). This procedure involves two steps at each iteration
and is repeated until convergence of the haplotype frequency estimates is obtained.
Step 1: expectation (E-step). The current haplotype frequency estimates are used to calculate for each subject the conditional probability of each possible genotype (pair of haplotypes) given the
observed HLA data available. These conditional probabilities are
then used to update the current estimates of genotype frequencies.
Step 2: maximization (M-step). The updated genotype frequencies
are used to update the current estimates of haplotype frequencies by
inverting the equation given in Assumption A1 in the Appendix.
For the above procedure to be a valid application of the EM
algorithm, some mathematical assumptions are required about how
the data were obtained. When all subjects are typed at the same
level of resolution, or when the level of typing resolution is statistically independent of the HLA type, it is relatively straightforward
to show that this procedure satises the general framework of the
EM algorithm. However, these assumptions would preclude the use
of allele-level data from patient-directed typings, as volunteers are
not chosen at random. In the Appendix we relax these assumptions
specifying conditions under which the level of typing resolution may
depend on the HLA type without violating the EM principle.

953

HLA-A, -B, -DRB1 haplotype frequencies


Table 3

HLA-A, -B antigens and DRB1 alleles used in the analysis.a,b

HLA-A
1
31 (19)
80
HLA-B
7
41
52 (5)
62 (15)
82
HLA-DR
1
3
4
7
8
9
10
11 (5)
12 (5)
13 (6)
14 (6)
15 (2)
16 (2)

2
32 (19)

3
33 (19)

11
34 (10)

23 (9)
36

24 (9)
43

25 (10)
66 (10)

26 (10)
68 (28)

29 (19)
69 (28)

30 (19)
74 (19)

8
42
53
63 (15)
83

13
44 (12)
54 (22)
67

14
45 (12)
55 (22)
70

18
46
56 (22)
73

27
47
57 (17)
75 (15)

35
48
58 (17)
76 (15)

37
49 (21)
59
77 (15)

38 (16)
50 (21)
60 (40)
78

39 (16)
51 (5)
61 (40)
81

HLA-DRB1 alleles present in NMDP Registryc


01010108 (8 alleles)
03010308; 03100317; 0320 (17 alleles)
04010419; 04220427; 04310436; 0438; 04400441; 04430444; 0450 (37 alleles)
0701; 07030705; 0707 (5 alleles)
08010814; 08170818; 0820, 0822; 0824; 0826 (20 alleles)
09010902 (2 alleles)
1001 (1 allele)
11011134; 11361141; 1143; 1145; 11471148 1151; 11531154 (47 alleles)
12011208 (8 alleles)
13011329; 13311332; 13351343; 1347; 1349, 13511352; 1356; 1359; 13611362 (48 alleles)
14011429; 14321435; 1439; 14411442; 1444; 14471448 (39 alleles)
15011507; 15091512 (11 alleles)
16011605; 16071608 (7 alleles)

Note: There were 21 HLA-A antigens, 42 HLA-B antigens, and 250 HLA-DRB1 alleles from 13 different HLA-DR antigen groups.
a
Numbers in parentheses denote broad antigens. For example a typing result of HLA-A10 denotes the presence of A25, A26, A34, or
A66, but it is unknown which one. Serologic subdivisions of B14 and B70 (i.e., splits) were converted to their broad antigens for analysis
because these antigens were most often reported at this level. A68 and A69 were similarly converted to A28 because of the unreliability
of antibody reagents for A69.
b
More details of HLA nomenclature are available at www.anthonynolan.org.uk/hig
c
Only those alleles that encode different amino acid sequences are included. Each DRB1 allele listed was identied in at least one
volunteer by high-resolution typing results, but many are most commonly listed within intermediate-resolution typing results (i.e.,
included within an allele code).

To account for variations by race/ethnicity [9], haplotype frequency estimates were calculated separately for African-Americans/blacks, Asian/Pacic Islanders, Hispanics, and Caucasians/
whites based on each volunteers self-reported racial/ethnic group
at the time of recruitment.

Simulation
To demonstrate the validity of the EM algorithm when volunteers are selectively chosen for higher-resolution typing
based on their HLA type, we simulated a virtual registry of
volunteer donors. The true haplotype frequencies in this
simulated population were taken from the estimated frequencies for Caucasian individuals (chosen because of the
larger sample size). Two million volunteers were simulated
by randomly selecting two independent haplotypes from the
true distribution.
Simulated volunteers initially had their HLA types
mapped to low resolution (serologic equivalent). Volunteers
were then chosen for intermediate-resolution typing (see
example codes in Table 1) with probability proportional to
their true low-resolution phenotype frequency. The intermediate resolution results were generated by simulating the
diploid probe pattern and resulting allele ambiguities which
would be generated by standard oligo-based HLA typing kits

[22]. A subset of these simulated volunteers was then chosen


for further high-resolution typing (i.e., DRB1 allele(s) determined) with probability proportional to their intermediatelevel resolution typing frequency. The resulting registry
therefore contained a mixture of low- (60%), intermediate(24%), and high-resolution (16%) typings, with the more common HLA types more likely to have intermediate- or highresolution data available.
The EM algorithm as described above was run on this
simulated registry, and the resulting haplotype frequency
estimates were compared with the true frequencies.

Results
Simulated Data
Despite the fact that simulated volunteers with more common HLA types were more likely to have high-resolution
level data available, the EM algorithm did not overestimate
the frequencies of the common haplotypes. There were 178
haplotypes in the simulation with a true frequency of greater
than 1 in 1,000 (common haplotypes), combining to comprise 56.4% of the overall population. Because of the selective retyping, these 178 haplotypes were overrepresented

954

C. Kollman et al.

Figure 1. Simulated registry with selective retyping of volunteers. Common haplotypes with a frequency greater than 1 in
1,000 made up 56% of the simulated population and 90% of the
subgroup chosen for high-resolution typing. Despite this overrepresentation, there was no systematic bias for the expectation-maximization (EM) algorithm to overestimate the frequencies of the common haplotypes. Diagonal represents the line of
identity. Note that both axes are on a reverse logarithmic scale.
Figure excludes less common haplotypes where evaluation of
any bias is confounded by the larger statistical error (variance).

among simulated volunteers with high-resolution typing,


comprising 90% of this subgroup. This overrepresentation did
not bias the results of the EM algorithm, which gave an
estimate of 56.6% for the sum of these 178 haplotype frequencies. The lack of bias for individual haplotypes is shown
in Figure 1.

Actual Data
The complete list of HLA-A, -B, -DRB1 haplotype frequency
estimates by race/ethnicity is available online (http://
bioinformatics.nmdp.org/em-haplotype). Table 4 shows the
10 most common haplotype frequencies in each racial/ethnic group. Although some haplotypes were common to multiple groups, there were considerable differences according

Table 4

to self-reported race/ethnicity. A1-B8-DRB1*0301 was the


most common haplotype in Caucasians and the second most
common in both Hispanics and African-Americans, with estimated frequencies of 1 in 16, 1 in 60, and 1 in 84, respectively. This haplotype was the 36th most common in Asian/
Pacic Islanders with an estimated frequency of 1 in 336.
A3-B7-DRB1*1501 was also common in Caucasians (1 in 33),
Hispanics (1 in 86), and African-Americans (1 in 157) but was
less so in Asian/Pacic Islanders (1 in 463). A33-B58DRB1*0301 was the most common haplotype in Asian Pacic
Islanders, with an estimated frequency of 1 in 52, but occurred in less than 1 in 2,000 haplotypes in each of the other
three groups.
There was also considerable variation in overall HLA polymorphism among the racial/ethnic categories. The median
haplotype frequency (the value at which there is a 50%
chance that a randomly selected haplotype would have a
greater frequency) was approximately 1 in 2,100 for AfricanAmericans, 1 in 1,400 for Hispanics, 1 in 1,100 for Asian/
Pacic Islanders, and 1 in 950 for Caucasians. The distribution of haplotype frequencies by race/ethnicity is shown in
Figure 2. These data show that the HLA-A, -B, -DRB1 polymorphism is highest among African-Americans and lowest
among Caucasians, with Asian/Pacic Islanders and Hispanics falling intermediate.
Extending haplotype frequency estimates to the allele
level for HLA-DRB1 reveals an additional polymorphism
within the previously dened low-resolution HLA-A, -B, -DR
(serologic) groups. One simple way to quantify this increased
polymorphism is by the estimated probability that two randomly chosen haplotypes within the same HLA-A, -B, -DR
group would have different DRB1 alleles. This probability
ranged from 20% (Caucasians) to 28% (Hispanics), with intermediate values for Asian/Pacic Islanders (25%) and AfricanAmericans (27%). The increased allelic polymorphism was
not universal, however, across all haplotypes. Within the
low-resolution level haplotype A1-B8-DR3, for example, the
DRB1*0301 allele comprised more than 99% of the total probability mass in each racial/ethnic group.
Although there were 250 HLA-DRB1 alleles identied in
the NMDP Registry (Table 3), only 81 had an allele frequency
(collapsed over HLA-A and -B) greater than 1 in 10,000 in any

Most common HLA-A, -B, -DRB1 haplotypes according to self-reported racial/ethnic group.a

African-American/black

Asian/Pacic Islander

Hispanic

Rank

DRB1

Frequency

DRB1

Frequency

DRB1

Frequency

DRB1

Frequency

1
2
3
4
5
6
7
8
9
10

30
1
33
68
3
36
34
2
30
68

42
8
53
58
7
53
44
44
42
70

0302
0301
0804
1201
1501
1101
1503
0401
0804
0301

1
1
1
1
1
1
1
1
1
1

33
33
2
24
33
30
33
1
11
24

58
44
46
52
44
13
58
57
75
7

0301
0701
0901
1502
1302
0701
1302
0701
1202
0101

1
1
1
1
1
1
1
1
1
1

29
1
2
3
68
2
33
24
30
2

44
8
35
7
39
39
14
39
18
35

0701
0301
0802
1501
0407
0407
0102
1406
0301
0407

1
1
1
1
1
1
1
1
1
1

1
3
2
2
29
2
1
3
2
2

8
7
44
7
44
62
57
35
8
60

0301
1501
0401
1501
0701
0401
0701
0101
0301
1302

1
1
1
1
1
1
1
1
1
1

in
in
in
in
in
in
in
in
in
in

70
84
138
139
157
163
182
194
204
205

in
in
in
in
in
in
in
in
in
in

52
62
67
82
87
88
89
94
106
123

Caucasian/white

in
in
in
in
in
in
in
in
in
in

54
60
64
86
113
127
129
139
144
152

in
in
in
in
in
in
in
in
in
in

16
33
48
50
64
81
89
89
112
123

A full list of haplotype frequency estimates by race/ethnicity is available online at http://bioinformatics.nmdp.org/em-haplotype

HLA-A, -B, -DRB1 haplotype frequencies

Figure 2. HLA polymorphism by race/ethnicity. Height of


each curve denotes the percentage of HLA-A, -B, -DRB1 haplotypes in the population with frequency less than the value on
the horizontal axis. Note that the horizontal axis is on a reverse
logarithmic scale.

racial/ethnic group, and only 49 had an allele frequency


greater than 1 in 1,000.

Discussion
We have shown, both mathematically and by simulation,
that the EM algorithm can be applied to a dataset of mixed
resolution HLA typings, even when the level of resolution
depends on the HLA type. We intentionally exaggerated the
oversampling of common haplotypes for high-resolution typing in our simulation to demonstrate that this does not skew
the results of the EM algorithm. The 178 haplotypes with a
frequency greater than 1 in 1,000 comprised 57% of the
simulated population but represented 90% of the haplotypes
among individuals selected for high-resolution typing. Despite this oversampling, the EM algorithm did not overestimate the frequencies of these haplotypes (Figure 1).
Algorithms allowing for varying levels of resolution have
been previously described [1315], but the level of resolution was assumed to be independent of the HLA type. Previous results have therefore been limited to haplotypes dened at low resolution. Relaxing this assumption allows the
use of allele-level HLA data available in volunteer registries,
much of which is obtained through patient-directed typings.
We also show that this computationally demanding algorithm can be successfully implemented on large datasets,
even with the greatly expanded list of HLA-DRB1 alleles. The
HAPLO program, for example, is limited to a maximum of
114 haplotypes [13]. In our implementation there are 21
42 250 220,500 total haplotypes. At each iteration, the
EM algorithm requires calculation of the frequency of every
possible genotype for every individual. A single individual
with the low-resolution result A10,19; B15,22 (DRB1 not
tested), for example, has more than 9.5 million possible
genotypes. Nevertheless our program processed a dataset of
3.5 million individuals in 5.5 hours running on a cluster of ve
Sun Fire V100 servers (550 MHz Ultra SPARC IIi 2GB RAM).
This is the rst study to report estimates of multi-locus HLA
haplotype frequencies at the allele level for DRB1 from a dataset of this size. Previous reports have given frequency esti-

955
mates for haplotypes dened at low resolution. Our data reveal
the increased allelic polymorphism within low-resolution
HLA-DR groups. This increased polymorphism is clinically important to identify the optimal stem cell donor [23,24].
One limitation of the EM algorithm is that it is not guaranteed to nd the global maximum likelihood. The algorithm
may settle on a locally maximum likelihood set of frequencies, which is different from global maximum. The problem
has been addressed by varying initial conditions [25,26], but
a satisfactory general solution to this problem has yet to be
established. Recent work has compared the EM algorithm to
Bayesian methods for haplotype estimation [2729].
HLA haplotype population frequency estimates are a
valuable tool in the management of volunteer donor registries. They have been used to project how many volunteer
donors would be needed to achieve a certain probability of
nding an HLA-matched donor. These data have demonstrated the challenges in providing HLA-matched donors for
minority patients and the special need for minority-focused
recruitment in volunteer registries [9,30,31]. The utility of
these tools could be greatly enhanced by using the work
presented here to extend the denition of an HLA match
to the allele level for DRB1.
Another useful application of these haplotype frequencies is to predict the probability that a volunteer typed at
low or intermediate resolution would match a specic patient at high resolution. The initial search of a donor registry
often reveals several potential matches for a patient. The
ability to distinguish which of these volunteers are likely and
which are unlikely to match the patient can be extremely
important when resources and time to perform HLA testing
are limited. Prediction tools at the low-resolution level have
been previously described for unrelated [10] and related
donors within the extended family [32].
Research has also shown that allele level mismatches at
HLA-A, -B, and -C can also affect transplantation outcomes
[24,33,34]. Future research may discover other important
loci. In principle, the algorithm presented here could be
used to extend to the allele level for these loci as well. There
is a practical limit, however, to how far haplotype frequency
estimation can be expanded using this algorithm. The consideration of additional loci dramatically increases the number of possible haplotypes and therefore the amount of data
necessary for reliable estimation.
Although this version of the EM algorithm reduces the
necessary assumptions, there are still limitations that must
be considered in the interpretation of these data. Serologic
typing has a relatively high error rate, especially in minority
populations [20]. Our results may therefore underestimate
the incidence of frequently mistyped antigens (those with
less reliable antisera) and understate the level of polymorphism present. In addition the HLA-DRB1 codes from earlier
typings excluded alleles that were unknown at the time.
These results may therefore underestimate the true frequencies of recently discovered alleles. Without going back
to the original data of tested DNA polymorphisms [22], it is
unclear how these codes could be reinterpreted without
violating Assumption A2 in the Appendix. The fact that these
alleles were only recently discovered suggests they may be
rare in the general population; but this remains to be seen.
Another possible violation of Assumption A2 may occur if
the typing laboratory reported allele codes in an inconsis-

956
tent manner. If, for example, DRB1*0101 were always reported at the allele level, but any other allele in the DR1
family were always reported as DRB1*01XX (a code that includes DRB1*0101 as a possibility), the EM algorithm would
overestimate the frequency of DRB1*0101. The reported
code needs to accurately reect which alleles have been
excluded based on the probes or primers used.
The groupings used in this analysis were based on selfreported race/ethnicity collected at the time of recruitment, but the scientic validity of this categorization has
been questioned [35,36]. The populations dened by these
broad categories are not in exact Hardy-Weinberg equilibrium [11], but the EM algorithm has been shown to be robust
under moderate deviations from this assumption [37]. Nonetheless it is a gross oversimplication to describe the variations
of HLA among human beings in such broadly dened categories.
The presence of low- or intermediate-resolution typings
in the dataset can result in some haplotype frequencies being unidentiable. Suppose, for example, that all subjects
who possess an A1 antigen, a B7 antigen, and a DRB1*0101
and/or 0102 allele were typed with probes that could not
distinguish those two DRB1 alleles (e.g., coded as
DRB1*01AB; see Table 1). Then it would not be possible to
separate the frequencies of A1, B7, DRB1*0101 versus A1,
B7, DRB1*0102. Only the sum of their frequencies would be
identiable. If the EM algorithm were initiated with the uniform distribution, then the nal frequency estimates would
divide this sum equally among the confounded haplotypes.
This could lead to overestimation of the total diversity
present in the population.
As would be the case even if haplotypes could be observed directly, the ability to accurately estimate frequencies of rare haplotypes is limited by the sample size of the
dataset. The statistical margin of error (and therefore the
ability to estimate rare haplotypes) when using the EM algorithm will also depend on the proportion of subjects typed at
low versus intermediate versus high resolution, as well as
the proportion who are typed only for a subset of the loci.
Despite these limitations, the algorithm described here
can be used to approximate the level of polymorphism
present in human populations. This has important implications for the ability of volunteer registries to rapidly identify
HLA-matched, unrelated donors for patients in need of a
transplant.

C. Kollman et al.
general population. Let (i, j) be the genotype comprised of
haplotypes i and j and let gij be its frequency in the general
population. Take Gk to be the genotype of the kth subject. Pr
{} stands for the probability of the event denoted inside the
brackets and E {} denotes the expected value.
Let G be the set of all possible genotypes in a population
and let P be a partition of G. That is, P is a collection of
mutually exclusive sets of genotypes, {S1, . . ., Sp}, whose
union is G. For each subject in the dataset, a partition, Pk, is
chosen and the results of the HLA typing tell us which one of
the sets in that partition contains the subjects true genotype. Thus, we equate each HLA typing result with the set of
genotypes, Sk, possibly present in the kth subject.

Assumptions
The level of typing resolution for the kth subject, Pk, may be
random and can depend on the subjects genotype. Furthermore, the resolution chosen for one subject need not be
independent of those for other subjects. The decision
whether to retype a volunteer donor at higher resolution, for
example, may depend on whether he/she is a potential
match for a patient or whether there are other (potential)
matches in the Registry. We only require that the decision
whether to include a set of possible genotypes in the partition cannot depend on which of those genotypes is actually
present.
More formally, we assume that:
A1. The K genotypes are jointly independent and subjects
are sampled at random from a population in Hardy-Weinberg
equilibrium so that
gij PrGk (i, j)

Notation
Let K be the number of HLA-typed subjects in the dataset
and take hi to be the frequency of the ith haplotype in the

for all k.

Derivation
From Assumption A1, if the genotypes (and therefore haplotypes) were directly observable, the log likelihood would be
that for a multinomial distribution:
L(N, h) log(

Appendix: The expectation-maximization (EM)


Algorithm

hi 2 if i j

A2. The joint probability distribution for the random levK


els of typing resolution, Pkk1
, is such that for any collection
K
of k sets of genotypes, Skk1
, the conditional probability
K
PrSkPk for all kGkk1
is constant over each Sk.

Acknowledgments
We are grateful to each volunteer who has registered to
donate stem cells to offer a stranger a second chance at life.
We thank Bonnie Olson and Chris Hansen for their assistance
in the preparation of this manuscript.

2hi hj if i j

2K
N 1, . . . , N H

N log(h )
i

H
where N Nii1
is the number of occurrences of the ith
haplotype in the dataset i Ni 2K.
K
Instead, we actually observe S Skk1
, a list of possible
genotypes for each subject. Suppose after the nth iteration
H
we have the estimates hn hini1
. The general form of the
EM algorithm is to replace these estimates at the next iterH
ation with the values hn1 hin1i1
that maximize EL
n
N,hS,h over h [7].
This is achieved by taking

hi(n1)

1
2K

E{Ni|S, h(n)}

1
2K

2 Pr{G (i,
k

k1

i)|S, h(n)}

957

HLA-A, -B, -DRB1 haplotype frequencies

Pr{G (i,
k

j)|S, h(n)}

ji

2K

k1

Pr{Gk (i, i) h } Pr{Sl Pl l Gk (i, i)


(n)

Pr{Gk Sk and Sl Pl l|h }

between haplotype and genotype frequencies in Assumption


A1:

(n)

Pr{Gk (i, j)|h(n)} Pr{Sl Pll|Gk (i, j)}


Pr{Gk Sk and Sl Pll|h }
(n)

ji

The conditional probabilities PrGk i,jS,hn are


clearly zero for i,jSk, and must therefore sum to 1 over
i,jSk. By Assumption A2, the quantity PrSlPll|Gk
i,j} is constant for all i,jSk.
The common value of the ratio

PrGk Sk and Sl Pll h(n)


for i,jSk must therefore equal

l,mSk

PrGk l,mhn1.

We therefore have:
hi(n1)

2K k:(i,i)Sk

Pr{Gk (i, i)|h(n)}

Pr{Gk (l, m)|h(n)}

(l,m)Sk

Pr{Gk (i, j)|h(n)}

ji k:(i,j)Sk

Pr{Gk (l, m)|h(n)}

(l,m)Sk

2K k:(i,i)Sk

2 [hi(n)]2

(l,m)Sk

ji k:(i,j)Sk

(n)
lm h(n)
l hm

2 hi(n)h(n)
j

(l,m)Sk

k:(i,j)Sk

(l,m)Sk

where lm

1 if lm
2 if lm

(n)
lm h(n)
l hm

hi(n)h(n)
j

(n)
lm h(n)
l hm

Remarks
1. For subjects who are heterozygous at and fully typed for
all three loci, Sk contains four possible genotypes. In contrast, if the subject is not typed for HLA-DRB1 and is heterozygous at both HLA-A and -B, then Sk contains 125,000
possible genotypes.
2. As is often the case with the EM algorithm, this procedure has an intuitive interpretation. The current haplotype
frequency estimates are used to calculate the conditional
probability of a genotype (i, j) given the observed HLA typing, Sk. The probability mass associated with each individual
(K1) is allocated to each of the possible genotypes (i, j) in
proportion to this estimated conditional probability:
gij(n1)

K k:(i,j)Sk

ij hi(n)h(n)
j

(l,m)Sk

2 ji

(n1)
ij

Note that these two steps are equivalent to the formula in


the nal line of the above derivation.
3. Assumption A2 is a requirement of noninformative censoring and basically states that the event SkPk is conditionally independent of Gk given GkSk. This requires that
reporting of any HLA codes be done in a consistent manner
for each subject.

References

PrSl Pll Gk (i, j)

hi(n1) gii(n1)

(n)
lm h(n)
l hm

The haplotype frequencies are then updated according to


these new genotype frequencies by inverting the relation

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