Patterns of DNA Methylation Across the Leptin Core Promoter in Four Diverse Asian and North American Populations.

DNA methylation is the most widely studied of epigenetic mechanisms, with environmental effects recorded through patterned attachments of methyl groups along the DNA that are capable of modifying gene expression without altering the DNA sequencing. The degree to which these patterns of DNA methylation are heritable, the expected range of normality across populations, and the phenotypic relevance of pattern variation remain unclear. Genes regulating metabolic pathways appear to be vulnerable to ongoing nutritional programming over the life course, as dietary nutrients are significant environmental determinants of DNA methylation, supplying both the methyl groups and energy to generate the methylation process. Here we examine methylation patterns along a region of the metabolic gene leptin (LEP). LEP's putative functions include regulation of energy homeostasis, with its signals affecting energy intake and expenditure, adipogenesis and energy storage, lipid and glucose metabolism, bone metabolism, and reproductive endocrine function. A pattern of differential methylation across CpG sites of the LEP core promoter has been previously identified; however, any consistency of pattern or its phenotypic significance is not fully elucidated among populations. Using DNA extracted from unfractionated white blood cells of peripheral blood samples, our pilot study, divided into two parts, examined the significance of variation in DNA methylation patterns along the leptin core promoter in four populations (phase 1) and used biomarkers reflecting leptin's functional process in two of those populations, western Buryat of Siberia and the Mennonite of central Kansas, to investigate the relevance of the ethnic variation identified in the DNA methylation (phase 2). LEP's core promoter region contains both the binding site for C/EBPα (CCAAT/enhancer binding protein alpha), which tempers the final step in adipocyte maturity and capacity to synthesize leptin, and the TATA motif controlling leptin synthesis. Previous studies report that increased methylation in this region is correlated to decreased gene expression, suggesting tissue-specific methylation variation at this region ( Melzner et al. 2002 ). We hypothesized that evidence of nutritional epigenetic programming would be identified through variation in patterns of DNA methylation and that functional relevance of that variation among populations would be identified through biomarkers that reflect leptin's metabolic signals: serum leptin levels, lipoproteins of the lipid transport system, and anthropometric measures. In phase 1, our combined analyses of 313 individuals documented a distinct and consistent overall pattern of differential DNA methylation across seven CpG sites of LEP core promoter in all ethnicities and both sexes. This pattern replicates those identified in previous studies, suggesting a conserved core promoter region across populations. Phase 2 analyses of two of the four populations (n = 239), correlating methylation at the C/EBPα transcription binding site (TBS) with metabolic and anthropometric biomarkers reflecting LEP roles, showed that stature, which reflects bone growth and remodeling, was significantly and inversely correlated with the percentage of DNA methylation at this site in both sexes. We suggest that variation in DNA methylation along the LEP core promoter plays a substantial role in energy signals affecting both adipogenesis and bone metabolism.

Gluten-sensitive enteropathy. Immunoglobulin G heavy-chain (Gm) allotypes and the immune response to wheat gliadin.

Anti-gliadin antibody was measured by radioimmunoassay in 30 Caucasians with gluten-sensitive enteropathy (GSE). 22 GSE patients maintained on a gluten-free diet for 1.5 to 20 yr (mean duration 76 mo) had elevated serum concentrations of IgG antigliadin antibody. Among GSE patients on a gluten-free diet, antigliadin antibody was seen only in those having the chromosome 14-encoded IgG immunoglobulin heavy chain allotype marker G2m(n). IgG antigliadin antibody was found in GSE patients with G2m(n) regardless of whether the HLA-B8 and/or -DR3 major histocompatibility complex antigens that occur frequently in GSE were present. No patient lacking G2m(n) had significant levels of antigliadin antibody. The association between antigliadin antibody and the immunoglobulin heavy chain allotype marker G2m(n) in GSE patients likely reflects the presence of Gmn-linked variable region genes or Gmn-linked genes that regulate variable region gene expression.

Native American mitochondrial DNA analysis indicates that the Amerind and the Nadene populations were founded by two independent migrations.

Mitochondrial DNAs (mtDNAs) from 167 American Indians including 87 Amerind-speakers (Amerinds) and 80 Nadene-speakers (Nadene) were surveyed for sequence variation by detailed restriction analysis. All Native American mtDNAs clustered into one of four distinct lineages, defined by the restriction site variants: HincII site loss at np 13,259, AluI site loss at np 5,176, 9-base pair (9-bp) COII-tRNA(Lys) intergenic deletion and HaeIII site gain at np 663. The HincII np 13,259 and AluI np 5,176 lineages were observed exclusively in Amerinds and were shared by all such tribal groups analyzed, thus demonstrating that North, Central and South American Amerinds originated from a common ancestral genetic stock. The 9-bp deletion and HaeIII np 663 lineages were found in both the Amerinds and Nadene but the Nadene HaeIII np 663 lineage had a unique sublineage defined by an RsaI site loss at np 16,329. The amount of sequence variation accumulated in the Amerind HincII np 13,259 and AluI np 5,176 lineages and that in the Amerind portion of the HaeIII np 663 lineage all gave divergence times in the order of 20,000 years before present. The divergence time for the Nadene portion of the HaeIII np 663 lineage was about 6,000-10,000 years. Hence, the ancestral Nadene migrated from Asia independently and considerably more recently than the progenitors of the Amerinds. The divergence times of both the Amerind and Nadene branches of the COII-tRNA(Lys) deletion lineage were intermediate between the Amerind and Nadene specific lineages, raising the possibility of a third source of mtDNA in American Indians.

Autologous marrow recovery and sensitization to non-HLA antigens after HLA-identical marrow transplantation for aplastic anemia.

One hundred seventy-five patients with severe aplastic anemia were treated by high-dose cyclophosphamide and HLA-A, -B, and -D-identical sibling marrow transplants. Thirty-eight patients rejected their grafts. Four of the 38 showed autologous marrow recovery as determined by blood genetic markers. The remarkable feature of one case following autologous marrow recovery was the presence of unidirectional proliferative and cytotoxic responses of circulating host lymphocytes to marrow donor lymphocytes in mixed lymphocyte culture and cell-mediated lympholysis. Presumably these responses were the result of in vivo sensitization to those non-HLA antigens for which donor and recipient differed.

Immunoglobulin production of donor origin after marrow transplantation for acute leukemia or aplastic anemia.

Four patients with acute leukemia and one with aplastic anemia were not transfused within 90 days before marrow transplantation from the HLA-identical sibling. When studied 4 to 12 1/2 months after transplantation their immunoglobulin allotypes were those of their donors.

Familial rheumatoid arthritis: a kindred identified through a proband with seronegative juvenile arthritis includes members with seropositive, adult-onset disease.

Segregation of chromosome #6 markers has been studied in a large family, identified by a proband with seronegative, juvenile-onset rheumatoid arthritis, which contains four other individuals with adult-onset rheumatoid arthritis (RA). Two of the adult-onset patients have classical seropositive RA. Sera from two healthy members of this family also contain rheumatoid factors (RF). Six family members had crossovers in the short arm of chromosome #6. Three individuals with recombinants between HLA-B and HLA-D were identified; three others had identifiable crossovers between HLA-D and GLO (Glyoxylase 1). Linkage analysis suggested that susceptibility to RA in this family was influenced by a dominant gene located centromeric to HLA-B. The highest lod score (1.64) was obtained for linkage to GLO at a recombination rate of zero. Inheritance of specific chromosome #6 or Gm immunoglobulin allotype markers did not appear to influence serum RF. These results agree with previous family studies which suggest that acquisition of childhood- and adult-onset RA is influenced by a common disease susceptibility gene, linked to the major histocompatibility gene complex.

Blood groups, immunoglobulin allotypes and dermatoglyphic features of patients with amyotrophic lateral sclerosis and parkinsonism-dementia of Guam.

Blood group frequencies, immunoglobulin allotypes, and dermatoglyphic patterns were determined on patients with amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia (PD), two chronic, degenerative, neurologic disorders of unknown cause found commonly among the Chamorros of the Mariana Islands, in an attempt to identify a specific genetic or phenetic marker associated with either disorder. With the exception of the Kidd system, no significant differences were found in blood group frequencies nor in immunoglobulin allotypes between ALS patients, PD patients, and unaffected controls. The dermatoglyphic analysis demonstrated that ALS patients had higher frequencies of palmar patterns and accessory triradii in the IV interdigital area, and PD patients had significantly higher frequencies of complete simian creases and of palmar patterns in the thenar/I interdigital area than unaffected controls. The frequencies of the remaining dermatoglyphic traits showed no significant differences. We conclude that none of the marker systems tested show a particular pattern of association in patients and controls or a genetic predisposition to either disorder, and that early identification of at-risk individuals remains elusive.

Gm and Km allotypes in disputed parentage.

Immunoglobulin allotyping in 925 cases of disputed paternity provided evidence of exclusion for 70 alleged fathers. Combining results from erythrocytic antigen, enzyme, and serum protein tests, 230 men were proven to be falsely accused; in 15 cases the immunoglobulin allotype provided the only evidence for exclusion, in 67 no exclusion would have been identified if testing had been limited to ABO, Rh, and MNSs. Various cases in this study illustrate the importance of using an extensive battery of immunoglobulin reagents in order maximally to exclude falsely accused men, and to identify infrequent haplotypes, which might erroneously be interpreted as indirect exclusions or may indicate a high likelihood of paternity. The problems of Gm typing of very young children are described. When child is less than 6 months of age, direct exclusions may be missed by the Gm allotypes; indirect exclusions are valid only when the phenotype differs from that of the mother. Km allotypes are not age-dependent.

DNA interpopulational variation in Siberian indigenous populations: The mountain Altai.

Using mtDNA and classical markers, previous studies have found that the Altai are genetically divergent from the rest of Siberia. This study uses five variable number tandem repeat (VNTR) loci to examine the relationship of the Altai to other indigenous Siberian populations. Frequencies of VNTR fragments have been obtained from the DNA of 95 individuals living in the Altai village of Mendur-Sokkon. A Kolmogorov-Smirnov test shows that the Altai are significantly different from the Evenki village of Surinda at loci D11S129 and D20S15. In addition, the Altai are also statistically different from the Evenki village of Poligus at locus D11S129. The test reveals no differences between the Ket village of Sulamai and Mendur-Sokkon. The GST value obtained for Siberia is significant and is almost equal to that found for the GST of American ethnic groups. The significance of the GST values was verified through random resampling of the data. The GST value is an effect of the relative isolation of the Evenki as well as gene flow into the Kets and Altai; this is shown in a plot of rn versus mean heterozygosity. Although genetic differentiation between the Siberian groups is significant, an R matrix analysis, which uses American and Siberian ethnic groups, shows that the Siberians form a tight cluster. When the R matrix, GST , and the Kolmogorov-Smirnov results are combined, the Altai appear to be genetically different among Siberian populations, yet they are not as genetically divergent as previous studies have shown. © 1996 Wiley-Liss, Inc.

Analysis of human specificity in AFLP systems APOB, PAH, and D1S80.

We have previously characterized and databased three human amplified fragment length polymorphism (AFLP) loci: the hypervariable regions 3' to apolipoprotein B (APOB), phenylalanine hydroxylase (PAH) and at locus D1S80. The analysis utilized polymerase chain reaction (PCR) technology for human identification in forensic and paternity testing. This study extended that work by assessment of specificity of amplicons produced with non-human and human control DNAs for APOB, PAH and D1S80 under high and low stringency PCR conditions. It was seen that primate and other animal templates (with the exception of chimpanzee) yielded products below the human allele range under high stringency PCR parameters. Under reduced stringency PCR with animal and primate samples, reproducible genetic fingerprints were generated spanning the human allele range. The patterns were produced with defined human AFLP primer pairs under specifically relaxed PCR reaction and thermalcycling parameters. They showed genetic relationships between species at the DNA level. Amplicon patterns were compared for band size and intensity matches within the PCR synthesis range defined by the conditions used. This technique could become a useful tool in species identification and molecular evolutionary studies.

The identification of maternity in an unusual pregnancy-related homicide.

A recent bizarre homicide which culminated in the delivery of a live-born infant necessitated careful determination of true maternal origin. A 23-year-old pregnant woman was abducted, subdued, strangled, and delivered of a term infant by a crude Cesarean section. The infant was stolen and subsequently presented to physicians by a woman posing as the mother. Methods used to help confirm the surviving infant's parentage involved red cell antigen and enzyme system evaluations as well as immunoglobulin allotyping, which ultimately proved to be the most effective serologic test performed. The forensic science investigation of this unusual case also used bite mark analysis and patterned injury interpretation. Immunoglobulin allotyping is specifically discussed as a forensic serology test which is currently available and particularly applicable in cases involving parentage determination.

Simultaneous subtyping of group specific component and esterase D by isoelectric focusing on agarose gels.

A quick, sensitive and economical technique has been developed to subtype GC and ESD simultaneously on the same agarose IEF gel. This method could be a useful tool for forensic application.

Identification of war victims from mass graves in Croatia, Bosnia, and Herzegovina by use of standard forensic methods and DNA typing.

The postmortem remains of sixty-one war victims were excavated from 6 mass graves in Bosnia and Herzegovina one and a half years after interment Using standard identification methods, including the matching of medical and dental records, the recognition of distinguishing characteristics such as the use of clothing and belongings, and video superimposition, 35 persons were identified. For the remaining 26 persons identification efforts continue. DNA typing was performed at the HLA DQA1 locus and five PM system loci. Results from DNA typing were confirmed by other methods. DNA profiles of family members of 150 missing persons are now being developed using the 6 loci. These DNA profiles will then be compared with those generated from the bone and teeth remains of the unidentified victims.

Applications of pooled DNA samples to the assessment of population affinities: short tandem repeats.

Pooled DNA samples have been used in association studies of Mendelian disease genes. This method involves combining equal quantities of DNA from patients and control subjects into separate pools and comparing the pools for distributions of genetic markers. In this study identical quantities of DNA from 300 individuals representing 6 populations were pooled and amplified for 296 loci using the touchdown polymerase chain reaction (PCR) method. The purpose of this study is to test the efficacy of pooled DNA markers in the reconstruction of the genetic structure of human populations. The populations sampled included Chuvash, Buryats, Kizhi, Native Americans, South Africans, and New York City whites. To test the accuracy of the allele-frequency distributions, we genotyped the Buryats and New York samples individually for six microsatellite markers and compared their frequencies to the allele frequencies derived from the electropherogram peak heights for the pooled DNA, producing a correlation of 0.9811 with a variance of less than 0.04. Two-dimensional scaling of genetic distances among the six populations produced clusters that reflected known historical relationships. A distance matrix was created using all 296 loci, and matrices based on individual chromosomes were correlated against the total matrix. As expected, the largest chromosomes had the highest correlations with the total matrix, whereas one of the smallest chromosomes, chromosome 22, had the lowest correlation and differed most from the combined STR distance matrix.

Application of immunoglobulin heavy chain (GM, AM) and light chain (KM) allotypes to cases of disputed paternity.

To evaluate the usefulness of extended immunoglobulin allotyping compared to the conventional number of reagents in general use, 1,896 cases of disputed paternity tested for HLA, immunoglobulin allotypes (IGH, [GM, AM] and KM) and red blood cell markers (RBC) (ABO, RH, MNS, Kidd, Duffy, Kell, Colton, Lutheran, and Lewis) were analyzed. There were 1,289 cases in which both the mother and alleged father were Black, 548 cases in which the mother and alleged father were White and 59 cases that were either mixed or of different ethnic groups. A total of 691 exclusions were observed (533 Black, 143 White and 15 other). The observed exclusion rates for the HLA system in Blacks (93.9%) and in Whites (94.9%) were similar to previous estimates of the HLA exclusion rates, indicating that these cases appear to be consistent with other studies. The observed exclusion rates for the IGH haplotypes (alleles) were the highest single system exclusion rates beside the HLA system (54.08% in Blacks and 31.47% in Whites). Further, the values were higher than any single blood group system, or electrophoretic system in common usage. The combined IGH and KM observed exclusion rates were 57.50% for Black cases and 37.06% for White cases. The value of the combined immunoglobulin allotypes in Blacks almost exceeds the combined exclusion rate for all RBC antigens tested. The importance of doing extended immunoglobulin allotyping in Black cases is demonstrated by the fact that 76.5% of the exclusions would have been missed if only G1M A, F and X and G3M B0 and G had been used in the Black cases. The inclusion of additional markers in the White cases increased the observed IGH exclusion rate from 23.78 to 31.47%. The increase was primarily due to the addition of G2M N.

Immunoglobulin allotypes (GM and KM) indicate multiple founding populations of Native Americans: evidence of at least four migrations to the New World.

On the basis of GM and KM typing and language, approximately 28,000 Amerindians were divided into 4 groups of populations: non-Nadene South American (8 groups), non-Nadene North American (7 groups), Nadene (4 groups), and Eskaleuts (6 groups). These groups were compared to four groups of Asian populations. The distribution of GM haplotypes differed significantly among and within these groups as measured by chi-square analysis. Furthermore, as reflected in a maximum linkage cluster analysis, Amerindian populations in general cluster along geographic divisions, with Eskaleuts and Nadenes clustering with the Asian populations and non-Nadene North American and non-Nadene South American populations forming two additional clusters. Based on GM haplotype data and other genetic polymorphisms, the divisions appear to reflect populations that entered the New World at different times. It appears that the South American non-Nadene populations are the oldest, characterized by the haplotypes GM*A G and GM*X G, whereas later North American non-Nadene populations are characterized by high frequencies of GM*A G and low frequencies of GM*X G and GM*A T. In contrast, Eskaleuts appear to have only GM*A G and GM*A T. The Nadene speakers have GM*X G and GM*A T in higher and approximately equal frequencies. Maximum linkage cluster analysis places the Alaskan Athapaskans closest to northwestern Siberian populations and the Eskaleuts closest to the Chukchi, their closest Asian neighbor. These analyses, when combined with other data, suggest that, in the peopling of the New World, at least four separate migrant groups crossed Beringia at various times. It appears likely that the South American non-Nadene entered the New World before 17,000 years B.P. and that the North American non-Nadene entered in the immediate postglacial period, with the Eskaleut and Nadene arriving at a later date.

Application of forensic DNA testing in the legal system.

DNA technology has taken an irreplaceable position in the field of the forensic sciences. Since 1985, when Peter Gill and Alex Jeffreys first applied DNA technology to forensic problems, to the present, more than 50,000 cases worldwide have been solved through the use of DNA based technology. Although the development of DNA typing in forensic science has been extremely rapid, today we are witnessing a new era of DNA technology including automation and miniaturization. In forensic science, DNA analysis has become "the new form of scientific evidence" and has come under public scrutiny and the demand to show competence. More and more courts admit the DNA based evidence. We believe that in the near future this technology will be generally accepted in the legal system. There are two main applications of DNA analysis in forensic medicine: criminal investigation and paternity testing. In this article we present background information on DNA, human genetics, and the application of DNA analysis to legal problems, as well as the commonly applied respective mathematics.

The distribution of F13A subtypes in four populations using agarose isoelectric focusing and Western Blot detection.

Using an isoelectric focusing gel containing agarose, urea, a separator and a narrow range ampholyte of pH 5-6, a system was designed to split the F13A*1 and F13A*2 alleles into the subtypes 1A, 1B, 2A and 2B. Four population groups (European-Americans, African-Americans, Mexican-Americans and Native-Americans) were data based. Subtyping F13A increased the information content significantly over the previous F13A typing system by doubling the number of alleles and increasing the number of phenotypes from three to ten. The new system has proved to be of value in parentage testing by increasing exclusionary power in cases of non-paternity and increasing the paternity index in non-exclusionary cases. Though there does not appear to be any significant variation among U.S. populations, published data on Japanese populations suggests that significant differences among populations may exist, leading to an anthropological usefulness as well.