Allelic diversity within the high frequency Mamu-A2*05/Mane-A2*05 (Mane-A*06)/Mafa-A2*05 family of macaque MHC-A loci.

Macaque species serve as important animal models of human infection and immunity. To more fully scrutinize their potential in both the analysis of disease pathogenesis and vaccine development, it is necessary to characterize the major histocompatibility complex (MHC) class I loci of Macaca mulatta (Mamu), Macaca nemestrina (Mane), and Macaca fascicularis (Mafa) at the genomic level. The oligomorphic Mamu-A2*05/Mane-A2*05 (previously known as Mane-A*06) family of macaque MHC-A alleles has recently been shown to be present at high frequency in both Indian rhesus and pig-tailed macaque populations. Using a locus-specific amplification and direct DNA typing methodology, we have additionally found that the locus encoding this family is very prevalent (75%) among a sampling of 182 Chinese rhesus macaques and has a high prevalence (80%) within a larger, independent cohort of 309 pig-tailed macaques. Interestingly, among the Chinese rhesus macaques, only six alleles previously identified in Indian-origin animals were observed, while three recently identified in Chinese-origin animals and 25 new alleles were characterized. Among the pig-tailed macaques, we observed 1 previously known (Mane-A*06) and 19 new alleles. Examination of the orthologous locus in a preliminary sampling of 30 cynomolgus macaques showed an even higher presence (87%) of Mafa-A2*05 family alleles, with 5 previously identified and 15 new alleles characterized. The continued discovery of novel alleles and thus further diversity within the Mamu-A2*05/Mane-A2*05/Mafa-A2*05 family indicates that this MHC-A locus, although highly conserved across the three species of macaques, has remained a dynamic entity during evolution.

Estrogen receptor beta dinucleotide (CA) repeat polymorphism is significantly associated with bone mineral density in postmenopausal women.

Significant associations between the lengths of a highly polymorphic dinucleotide (CA) repeat located within the human estrogen receptor beta (ESR2) gene on chromosome 14, bone mineral density (BMD) and androgen levels have been reported previously in premenopausal women. We measured the size of this microsatellite repeat in 226 healthy women (60-98 years). After adjustment for age, body mass index, hormone replacement status, and other variables known to influence BMD, women with < 25 CA repeats had significantly higher BMD measured in the total skeleton, lumbar spine, and femoral neck when compared with women having longer alleles. Women with shorter alleles also had higher circulating estrone and estradiol levels that approached statistical significance as compared with women harboring longer alleles after appropriate adjustments were performed in linear regression models. Women having both short and long CA repeats had BMD values in all regions of the skeleton that were midway between those found in women homozygous for longer or shorter repeat sizes. Because the ESR2 CA repeat size was neither associated with change in BMD nor serum levels of biochemical markers of bone turnover, it is likely that ESR2 CA repeat genotype is significantly linked to the attainment of peak bone mass in women.

Identification and sequencing of HLA-B*0714 and B*2718 alleles and novel exon 1 sequences of B*0709 and B*2714 alleles in potential bone marrow donors.

Sequence specific oligonucleotide probe hybridization and sequence specific primer PCR typing of volunteer bone marrow donors suggested the presence of variants of known HLA-B alleles in two individuals. PCR products encompassing HLA-B locus exons 1, 2, and 3 were prepared, subcloned and sequenced. A Hispanic individual had a novel B*07 allele (B*0714) and a Chinese individual had a novel B*27 allele (B*2718). In two other individuals, a previously unknown sequence of exon 1 was determined for HLA-B*0709 (African American) and B*2714 (Native American). These findings further illustrate the substantial genetic variation present at the HLA-B locus within human populations. We discuss the structural variation in the protein sequence for these HLA-B alleles and its potential functional effects.

Diagnostic efficacy of serum cross-linked N-telopeptide (NTx) and aminoterminal procollagen extension propeptide (PINP) measurements for identifying elderly women with decreased bone mineral density.

The purpose of this study was to determine the diagnostic sensitivity, specificity, predictive value and overall efficiency of serum cross-linked N-telopeptides of bone collagen (NTx) and aminoterminal procollagen extension propeptide (PINP) measurements for identifying women with decreased spine, femoral neck and total body bone mineral density (BMD). Serum NTx and PINP levels and dual X-ray absorptiometry were performed on 196 healthy elderly women, aged 60-90 years. Twelve women were classified as having decreased BMD on the basis of regional and total skeletal densitometric values that were 1.5 to 2.5 standard deviations (SD) below the respective, age-stratified means and were compared with 184 women with BMD values greater than 1.5 SD below the mean. The results of receiver operating characteristic analysis revealed that a cutoff level of more than 15.0 nmol BCE/L for serum NTx, as measured by the Osteomark assay (Ostex International, Seattle WA USA) was associated with a 100% sensitivity and 70% specificity rate for identifying postmenopausal women with low BMD. The positive likelihood ratio was 3.3 and the negative predictive value was 1.0 using the 15.0 nmol decision level for NTx. The overall diagnostic efficiency of a single NTx measurement for identifying women with low BMD was 89%. A cutoff level of >45.0 microg/L for PINP as measured by the Orion Diagnostica RIA assay (Espoo, Finland) had a diagnostic sensitivity of 83% and specificity of 64% for identifying women with decreased BMD. The positive likelihood ratio was 2.3. the negative predictive value 0.98 and the overall diagnostic efficiency 73% using the 45.0 microg/L decision level for PINP. These results warrant future studies using larger populations that are inclusive of more women with low bone mineral density.

Novel HLA-B alleles identified in potential marrow donors: B*3917, B*1405 and B*3528.

Sequence-specific oligonucleotide probe hybridization and sequence-specific primer polymerase chain reaction (PCR) typing of volunteer bone marrow donors suggested the presence of variants of known HLA-B alleles in five individuals. PCR products encompassing HLA-B locus exons 1 through 3 were prepared and subcloned. Three African-American individuals had a novel HLA-B*39 allele (B*3917), and another African-American was found to have a novel HLA-B*14 allele (B*1405). In a third individual of Hispanic origin, a novel HLA-B*35 allele (B*3528) was identified. These findings further illustrate the substantial genetic variation present at the HLA-B locus within human populations.

Critical differences in the serial measurement of three biochemical markers of bone turnover in the sera of pre- and postmenopausal women.

OBJECTIVES: The purpose of this investigation was to quantify the biologic, day-to-day variability and critical differences in serum levels of crosslinked collagen N-telopeptides (NTx), procollagen aminoterminal extension propeptides (PINP) and bone specific alkaline phosphatase (bAP) in healthy women. DESIGN AND METHODS: Seven blood samples were collected from 12 pre- and 15 postmenopausal women over 4 to 6 months. NTx, PINP and bAP levels were determined utilizing enzyme- and radioimmunoassay techniques. RESULTS: The within-subject coefficient of variation (C.V.) in serum bAP, NTx and PINP levels was 7.1, 10.6 and 12.4% respectively. These variances did not differ significantly among premenopausal women when compared with postmenopausal subjects. Combining terms for analytical and biologic variability revealed that a critical difference between 2 successive serial measurements is 24% for bAP, 34% for NTx and 38% for PINP. CONCLUSION: Circulating levels of NTx, PINP and bAP are stable over time periods of several months, allowing for the determination of significant changes in skeletal metabolism of women.

Novel HLA-B*15 alleles identified in potential marrow donors.

Several methods for low-resolution class I typing of potential bone marrow donors are available. The National Marrow Donor Program (NMDP) has initiated pilot projects for large-scale DNA-based class I typing to initially characterize donors. Sequence-specific oligonucleotide probe hybridization and sequence-specific primer polymerase chain reaction (PCR) screening of 3,500 NMDP potential donors suggested the presence of variants of known HLA-B*15 variants in 3 donors. PCR products encompassing HLA-B locus exons 1 through 3 were prepared and subcloned. Sequencing revealed 3 alleles differing from known HLA-B*15 alleles by nucleotide substitutions resulting in predicted novel HLA-B antigens. The new alleles occur in distinct ethnic groups. These findings further illustrate the substantial genetic variation present at the HLA-B locus within human populations.

A novel HLA-B*40 allele and novel exon 1 sequences of two B*40 alleles identified in potential marrow donors.

Sequence-specific oligonucleotide probe hybridization and sequence-specific primer polymerase chain reaction (PCR) typing suggested the presence of variants of HLA-B*40 in three individuals. Two were part of 3,500 potential marrow donors being screened for the National Marrow Donor Program, while the third was a clinical specimen. PCR products encompassing HLA-B locus exons 1 through 3 were prepared and subcloned. In one individual, a native of the Pacific Islands, sequencing revealed a novel HLA-B*40 allele (B*4023). In two other individuals, a previously unknown exon 1 sequence was determined for HLA-B*4016 (ethnicity unknown) and B*4020 (Hispanic). These findings further illustrate the substantial genetic variation present at the HLA-B locus within human populations.

Estrogen replacement therapy, serum lipids, and polymorphism of the apolipoprotein E gene.

BACKGROUND: Pharmacogenomics, the study of genetic loci that modulate drug responsiveness, may help to explain why estrogen replacement therapy (ERT) has differential effects on serum lipid and lipoprotein concentrations in postmenopausal women who inherit distinct alleles of the apolipoprotein E gene (APOE). METHODS: We compared total-cholesterol, triglyceride, and lipoprotein (LDL and HDL) concentrations in 66 postmenopausal women receiving ERT ([+]ERT) with 174 postmenopausal women not receiving ERT ([-]ERT), controlling for three APOE genotypes divided into three groups: E2 (epsilon2/epsilon3, n = 31), E3 (epsilon3/epsilon3, n = 160), and E4 (epsilon3/epsilon4 + epsilon4/epsilon4, n = 49). RESULTS: Mean total-cholesterol concentrations were lower in all three [+]ERT groups compared with their [-]ERT counterparts but were statistically significant only for women in group E4 (P = 0.014). The mean LDL-cholesterol concentrations were significantly lower in all three [+]ERT groups compared with their [-]ERT counterparts (P

Serum leptin in elderly people: associations with sex hormones, insulin, and adipose tissue volumes.

OBJECTIVE: There are few data for associations of serum leptin with body fat, fat distribution, sex hormones, or fasting insulin in elderly adults. We hypothesized that the sex difference in serum leptin concentrations would disappear after adjustment for subcutaneous, but not visceral body fat. Serum leptin would not be associated with sex hormone concentrations or serum fasting insulin after adjusting for body fat and fat distribution. RESEARCH METHODS AND PROCEDURES: Subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) volumes were measured using magnetic resonance imaging in a cross-sectional sample of 56 nondiabetic, elderly men and women aged 64 years to 94 years. Serum leptin, sex hormones (testosterone and estrone), sex hormone-binding globulin, and fasting insulin were also measured. Nine women were taking hormone replacement, and five men were clinically hypogonadal. RESULTS: Leptin was significantly associated with both SAT and VAT in each sex. Adjustment for SAT reduced the sex difference in leptin by 56%, but adjustment for VAT increased the difference by 25%. Leptin was not associated with serum estrone or hormone replacement therapy in the women, but had a significant, negative association with testosterone in the men that was independent of SAT, but not VAT. Leptin was significantly associated with fasting insulin in both sexes independent of age, sex hormones, sex hormone-binding globulin, VAT and SAT. DISCUSSION: Sex difference in serum leptin is partly explained by different amounts of SAT. Studies including both men and women should adjust for SAT rather than total body fat that includes VAT. The sex difference in serum leptin is not due to estrogen, but may be partly explained by testosterone. Testosterone is negatively associated with leptin in men, but the association is confounded with VAT. Leptin is associated with fasting insulin in nondiabetic elderly men and women independent of body fat, fat distribution, or sex hormones.

Age-related changes in sex hormones affect the sex difference in serum leptin independently of changes in body fat.

Serum leptin concentrations are highly correlated with body fatness, but there is considerable variability among individuals after adjusting for differences in body fatness. Theoretically, sex hormone levels may influence serum leptin, since the levels are higher in women than in men independently of body fat. Increasing old age is associated with decreases in serum sex hormone concentrations and changes in body fatness that may independently alter serum leptin concentrations. In a cross-sectional sample of 106 men and 166 women aged 62 to 98 years, serum leptin adjusted for total body fat had a significant positive association with age in men and a nonsignificant negative association with age in women. Serum testosterone had a significant negative association with serum leptin in men after adjusting for total body fat, the fasting insulin resistance index (FIRI), and sex hormone-binding globulin (SHBG). In a longitudinal sample of 22 elderly men and 52 women, serum leptin levels increased significantly over a 14-year period in men, but not in women. Increases in serum leptin were significantly associated with decreases in serum testosterone but not with changes in the body mass index (BMI) in men. In contrast, changes in leptin were associated with changes in the BMI but not with changes in serum estrone in women. These results suggest that differences among men and changes with age in serum leptin are associated with circulating levels of testosterone. Elderly men become progressively "hyperleptinemic" with age regardless of changes in body fatness, possibly due to decreasing testosterone levels.

Impact of HLA-H mutations on iron stores in healthy elderly men and women.

The DNA of 287 healthy white elderly volunteers in the New Mexico Aging Process Study, between 63 and 91 years of age, was examined for mutations of the HLA-H gene at nt 845 and nt 187. None were found to be homozygous for the 845A mutation and there were no gender differences in the percentage of the various mutations. The frequency of the 845A mutation was 0.061 resulting in a carrier frequency of 12.2%. The frequency of the 187G mutation was 0.136 resulting in a carrier frequency of 19.9% for a single mutation; 2.4% were compound heterozygous, 834A/187G and 2.4% were homozygous for the 187G mutation. After excluding 5 men and 4 women with microcytic or macrocytic anemia, mean percent transferrin saturation (PSAT) and iron stores, as estimated from serum ferritin concentrations, were calculated for each mutation. Estimated iron stores were normally distributed (range approximately 50 to 1,550 mg) with men (n=111) having significantly higher mean estimated iron stores than women (n=167), 826 +/- 318 and 753 +/- 287 mg, respectively. More men, 15 of 28, (54%) with estimated iron stores in the upper quartile, >/= 1,050 mg, had a HLA-H mutation compared to 25 of 83 (30%) who had a mutation and whose estimated iron stores were < ,050 mg, P<0.05. Seven were heterozygous for the 845A mutation with mean estimated iron stores of 1,300 +/- 127 mg, 7 were heterozygous for the 187G mutation with mean estimated iron stores of 1,439 mg. Similar differences were not noted in women. Even though the potential role of the 187G mutation in the phenotypic expression of HH is less certain than the 845A mutation, the increase in PSAT seen in men with the 187G mutation and the equal distribution of 845A and 187G mutations seen with iron stores >/= 1,050 mg lends support for the involvement of the 187G mutation, or a linked mutation, in iron metabolism. We concluded that men having either a single chromosomal 845A and/or 187G mutation results in higher PSAT's and estimated iron stores than if no HLA-H mutation were present.

Strategy for distinguishing a new DQB1 allele (DQB1*0611) from the closely related DQB1*0602 allele via sequence specific PCR or direct DNA sequencing.

A novel DQ6 allele (DQB1*0611) was identified via direct DNA sequencing in an African-American donor for bone marrow transplantation. The allele was not suspected on the basis of a sequence specific PCR assay which instead indicated the presence of DQB1*0602. DQB1*0602 and DQB1*0611 differ in exon 2 only at codon 9 resulting in a tyrosine substitution for phenylalanine. A modification of current DQB1 sequence specific PCR assays was devised which allows distinction between the closely related DQB1*0602 and DQB1*0611 alleles. Preliminary allele frequency studies suggest that DQB1*0611 is rare both in a non-African American sample and in American of African descent carrying DR11, DQ6 haplotypes. The selection of various DQB1*0611 detection methods is discussed.

Autoantigenic germ cells exist outside the blood testis barrier.

Preleptotene spermatocytes and spermatogonia are germ cells located outside the blood-testis barrier provided by the Sertoli cells. These cells have been found to express autoantigens accessible to circulating antibodies. Mice immunized with syngeneic testis with or without bacterial adjuvant had detectable IgG on cells at the periphery of seminiferous tubules. Sera from orchiectomized but not from testes-intact mice immunized with testis and adjuvants readily transferred similar IgG deposits to testes of normal recipients. When testis-specific antisera from orchiectomized mice and testis-intact mice were compared for their reactivity on prepuberal testicular cells, serum from orchiectomized donors had significantly higher reactivity. Ig was eluted from IgG-positive testes with acid buffer and was shown to be highly enriched in antibody to prepuberal testicular cells, confirming the Ag-specific nature of the IgG deposits. The testis IgG deposits reacted with antisera to IgG1 and IgG3 but not IgG2a or IgG2b. This finding can explain lack of association of C3 in the deposits. Only 30 to 40% of seminiferous tubules had IgG deposits and they coincided with stages 7 to 12 of the spermatogenic cycle. Thus, the expression of the autoantigens is stage specific. The in situ formation of immune complexes by circulating autoantibodies demonstrates conclusively that testis autoantigens are not completely sequestered, and the blood-testis barrier as an immunologic barrier is incomplete.