Genetic Markers of a Filipino Population by 4w9DQc

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									                   Genetic Markers of a Filipino Population
                                   Lilian Pagaduan
                               Adivsor: Dr. Douglas Oba
                                  13 December 2002

ABSTRACT

        Although a number of genetic studies have been conducted on Filipino
populations, the number of genetic markers has been limited. The primary purpose of
this study was to strengthen the genetic and genealogical database consisting of DNA
samples representing individuals who have Filipino ancestries. The secondary purpose
was the construction of haplotype histories of a Filipino subset population within and
between Asia and Pacific island populations. One hundred forty-five college students
from the Philippines and Hawaii residents who have Filipino ancestries were recruited to
provide four generations of genealogical information and a blood sample for DNA
isolation. DNA samples were analyzed using polymerase chain reaction (PCR) of
multiplex panels consisting of at least eight short tandem repeat loci. Fluorescent-labeled
PCR products were analyzed on an Applied Biosystem 3100 genetic analyzer for each
given marker. Genotyping was completed by running the data from the ABI Prism
GeneScan Analysis Software through ABI Prism Genotyper Software. The true alleles
which belonged to the standard repeat alleles were binned, and the filtering steps were
processed on the samples through the recorded macro. In addition to genotyping, specific
population haplogroups were determined. Four population haplogroups were tested for
mutated mitochondrial DNA by using restriction enzyme analysis. Haplotyping results
showed that most of the samples containing Asian ancestry were classified into the B
haplogroup. Of 145 DNA samples, thirty-one belonged to the B haplogroup, three to the
D haplogroup, thirty-four to the H haplogroup, and five to the U haplogroup.


INTRODUCTION

       In the prehistory of the Philippines, Zaide and Zaide (1999) commented that the

origin of the first human settlement in this region remains unknown. The migration

theory of Beyer suggests the settlement of the aboriginal pygmy group or the Negritos

was the primitive group and belonged to the old stone age (Zaide and Zaide 1999).

Bugawan et al. (1994) noted that the Filipino population is linked to Indonesian and

Malaysian populations. The contemporary ancestries of Filipinos are mixed with

Chinese, Hindus (Indian-Subcontinent), Indonesians, Spanish, and Euro-Americans

(Bugawan et al. 1994).    In Guam, the Chamorros’ ancestry is traced back to the


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Filipinos (Budolwe et al. 1998). Similar genetic markers for both the Chamorro and

Filipino populations identified more similarity to Chinese and Korean than to U.S.

Caucasians (Budolwe et al. 1998). The studies of the Polynesians’ evolution by Mack et

al. (2000) determined origins of Pacific population in the Eastern regions of Austronesia,

extending from Taiwan and the Philippines.

       Identification of DNA repeated markers in a population are important in

generating a forensic, ethnohistory, and genetic database. Halos et al. (1998) reported the

first Filipinos' DNA loci for forensic purposes. The initial results of DNA typing at the

University of the Philippines enhanced the genetic database used by the Philippines

National Police (PNP) and the National Bureau of Investigations (NBI) (Halos et al.

1998). The allelic data of Chamorro and Filipino populations added useful information

for human identity purposes as well as ethnohistory of human populations ( Budowle et

al. 1998). Population studies conducted in Metro Manila and Davao in the Philippines

resulted in relatively similar alleles along the 18 to 33 base repeats using the D1S180

locus (Alcantara and Halos 1998). Forty-two genotypes and 18 alleles in D1S80 locus

yielded a ratio of match indicating that 92.9% of the time, the allele drawn from the

Metro Manila population would be similar as that drawn from the Davao population.

Alcantara and Halos (1998) suggest that the data from the two populations are the basis

for a single genetic database.

       Larger data sets have been collected and used for determining the worldwide

origins of modern humans. Many of these studies utilize limited mitochondrial and

nuclear genetic loci (Jorde et al. 1995). Human populations have generally been defined

based on geography, language, ethnicity, and religion. Many of these criteria reflect




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barriers to gene flow and contribute to the non-random distribution of alleles among

populations and occasional fluctuations in gene frequencies (Tufto et al. 1996). A

number of different methods have been proposed for determining the relatedness of

individuals within and outside of specific populations including the assignment method

(Rannala and Mountain 1997, Cornuet et al. 1999), maximum-likelihood estimation

(Excoffier and Slatkin1995), and transmission/disequilibrium tests (Zhao et al. 2000).

       In the recent genetic studies of Filipino populations, one of the major concerns

was the small number of genetic markers.     Rannala and Mountain (1997) suggested that

a sample of 60 independent loci is powerful enough to detect the ancestral immigration

up to two generations. In addition, a correlation between the Filipino-specific genetic

markers and written genealogical data is important to confirm potential ancestral origins



PURPOSE OF THE STUDY

       The primary purpose of this study was to construct a correlated genetic and

genealogical database consisting of DNA samples representing individuals who have

Filipino ancestry. The genetic and genealogical data were used to create representative

chromosomal fragments (haplotypes) for the identification of the Filipino subset

population. The secondary purpose of this study was the construction of haplotype

histories of a Filipino subset population within and between Asia and Pacific Island

populations, within the three major islands in the Philippines, and within clan groups in

the Philippines.




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METHODS

       One hundred and forty-five Brigham Young University- Hawaii students of

Filipino ancestry and Hawaii residents were recruited who could provide four generations

of genealogical information and a blood sample for DNA isolation. Individuals whose

parents, grandparents, or great-grandparents were born in the Philippines were considered

to belong to the Filipino population.. Participants signed a consent form approved by the

Human Subjects Institutional Review Board of BYU-Provo.

       A four-generation pedigree from each participant was the major source of

genealogies. This information included the biological line of the individual (proband),

two parents, four grandparents, and eight great grandparents.   Dates and places of birth

of each ancestor were provided for the genealogical data and entered into the database.

Personal names were omitted from the database. Some individuals lacking in complete

four genetic genealogies were included if either parents or grandparents were identified to

belong to a specific populations.

       DNA from whole blood was extracted by following a modified Gentra Systems

protocol (Sambrook et al. 1989). The participants provided a DNA sample in the form of

8-10 ml whole blood. Red blood cells were lysed and centrifuged to isolate white blood

cells. These cells were then lysed and the proteins precipitated with ammonium sulfate

(6M) and removed by centrifugation. DNA was precipitated in isopropyl alcohol and

removed by spooling on a glass rod and resuspended in Tris-EDTA buffer.

               DNA samples were analyzed by the MGRG at Brigham Young
University-Provo at fifty genetic loci using Polymerase Chain Reaction (PCR) amplified


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specifically for each marker. PCR products were analyzed on an Applied Biosystem
3100 genetic analyzer for each given marker. Eighteen loci that were used to genotype
the alleles of the DNA samples were: D4S2948, D4S2970, D5S421, D5S1994, D6S299,
D7S503, D7S1839, D8S1477, D13S158, D13S1323, D14S977, D17S946, D17S1818,
D18S485, D20S112, D20S118, D20S178, and D20S197.
        Raw data were returned and analyzed using the genetic and statistical software,

Structure (Pritchard et al. 2000). Allele frequency at each genetic loci was compared to

the genealogical data to confirm population identity.

       Haplogroups were determined by using the following restriction enzymes.


                         Primers                               Polymorphic site
  Haplogroup                             Enzyme


        B                 B1/B2              N/A           9 bp deletion (8271-8281)
        D                 D1/D2              AluI                    -5176
        C                 C1/C2             HincII                     -
        A                 A1/A2             HaeIII                     +
        H                 H1/H2              AluI                    -7025
        J                  J1/J2            BstNI                   -13704
        U                 U1/U2              HinfI              -12308/ -9052
        K                 K1/K2             HaeII               +12308/ -9052
        V                 V1/V2             NlaIII                   -4577
       X1                IX1/IX2             DdeI                    -1715
       X2                 X3/X4              DdeI           -10394 (use M primers)
        I                  I5/I6             AluI               +10028/ -1715




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RESULTS

       Based on the genealogical data, the 145 DNA samples collected from

BYU-Hawaii represented four major populations (Fig. 1). Of these, 96 were

females and 49 males.




                                                       Philippine
                                                            s
                                                         22%




              USA
              62%                                            Polynesia
                                                               16%




         Fig. 1. The percentage of the individuals in the study representing the
         Philippines, Polynesians, and USA based on the genealogical data.



Genotyping

       Isolated DNA samples from 145 individuals were divided into three populations,

Philippines, United States, and Polynesia Islands based on their genealogical data. The

statistical analysis, Structure, was used to compare the inferred clustering of the alleles

based on sequence data with the four genealogically assigned populations (Table1). The

frequency divergence among the three populations was determined (Table 2).


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Table1. The Proportion of membership of each pre-defined population in each of the
three clusters.
          Given                         Sequence-based Inferred Clusters
          Population
1-- Philippines                     0.970             0.013             0.020
2-- USA                             0.023             0.923             0.046
3 --Polynesians                     0.037             0.005             0.956

Table 2. Allele-Frequency divergence among populations (Kullback-Leibler distance),
computed using point estimates of P.



       The inferred clustering within the Filipino population attempted to find out if there

was a divergence in the population from the recent ancestors. The statistical clustering did

not show high allele-frequency in two populations (Table 3 and Table 4) and three

populations (Table 5).


Table 3. The overall proportion of membership of the sample in each of the three clusters
                                                                                 the
Given                           1                 2                     3
                                                                                 Filipin
Population
                                                                                 o
1-- Philippines              -               0.37                 0.20
                                                                                 popula
 2-- USA                     0.35            -                    0.37
                                                                                 tion.
 3 --Polynesian              0.20            0.39                 -
                                     Inferred Clusters
           1                            2                                   3
      0.298                        0.346                               0.357


Table 4. The allele-frequency divergence in the Filipino population.
  Population                1                     2                             3
       1                        -                    0.380                          0.200
       2                      0.350                    -                            0.360
       3                      0.220                  0.370                            -




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Table 5. The overall proportion of membership of the samples in each of the two clusters
in the Filipino population.

                 Inferred Clusters
         1                          2
       0.500                      0.500

Haplotyping
       In addition to genotyping, specific population haplogroups were determined.
Four population haplogroups were tested for the mutation of mitochondrial DNA by
using restriction enzyme analysis. The haplotyping results showed that most of the
samples containing Asian and Polynesian ancestries were classified into the B
haplogroup (Table 6 and Table 7). Thirty-one out of 145 DNA samples belonged to the
B haplogroup. Three of the DNA samples were in the D haplogroup, thirty-four were in
the H haplogroup, and five were in the U haplogroup.


 Table 6. Percent of individuals within B haplogroup.
Haplotype            B          D             H                U         J            K
Positive            31           3            34               5         4            15
Samples
Percentage in     21.4%        2.6%         29.8%         6.25%        2.76%        10.34%.
Population


Table7. Number and percentage of individuals in each population for each
haplogroup.Table7. zNumber and percentage of indieach haplogroup
Given Population                                   Haplogroup
                          B                      D                    H                            U
                    Ind        %          Ind         %         Ind       %            Ind              %
1-- Philippines      10       6.90         2         1.37         0        0            0               0
2 –USA                6       4.14         0           0         33      22.76          5              3.45


3-- Polynesia         15        10.3         1          0.69       1         0.69          0            0




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Discussion and Conclusion

       The individuals who had Filipino ancestries clustered to 97.0% of membership in

the Philippines, first population. The allele-frequency divergence among the populations

indicated the deviation of one population to the other three assigned populations. The

Filipino population had a 20% of deviation from the Polynesians and a 37% of deviation

from Caucasians.    The smaller divergence of Filipino population from Polynesians

confirmed a more recent common ancestry between the two groups. This was further

confirmed by 10.0% of the B haplogroup had Polynesian ancestry and 6.90% of the same

haplogroup belonged to the Filipino population.

     An attempt to cluster the Filipino population into two and three populations was not

successful. This was possibly due to the small sample test size of the population. An

additional database, 167 individuals who have Filipino ancestries, from the Molecular

Genealogy Research Group will be used to identify clustering within the Filipino

population, and data from an additional 47 markers will be used in a follow-up study.




                              Works Cited

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Budowle, B., F. Samuel Baichtel, &R. Fejeran. 1998. Polymaker, HLA-DQA1, and
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Bugawan, T. L., J.D. Chang, W. Klitz, & H. A. Erlich. 1994. PCR/Oligonucleotide
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     54:331- 340.



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