Estimation of nonpaternity in the Mexican population of Nuevo Leon: A validation study with blood group markers

A method for estimating the general rate of nonpaternity in a population was validated using phenotype data on seven blood groups (A₁A₂BO, MNSs, Rh, Duffy, Lutheran, Kidd, and P) on 396 mother, child, and legal father trios from Nuevo León, Mexico. In all, 32 legal fathers were excluded as the possible father based on genetic exclusions at one or more loci (combined average exclusion probability of 0.694 for specific mother-child phenotype pairs). The maximum likelihood estimate of the general nonpaternity rate in the population was 0.118 ± 0.020. The nonpaternity rates in Nuevo León were also seen to be inversely related with the socioeconomic status of the families, i.e., the highest in the low and the lowest in the high socioeconomic class. We further argue that with the moderately low (69.4%) power of exclusion for these seven blood group systems, the traditional critical values of paternity index (PI ≥ 19) were not good indicators of true paternity, since a considerable fraction (307/364) of nonexcluded legal fathers had a paternity index below 19 based on the seven markers. Implications of these results in the context of genetic-epidemiological studies as well as for detection of true fathers for child-support adjudications are discussed, implying the need to employ a battery of genetic markers (possibly DNA-based tests) that yield a higher power of exclusion. We conclude that even though DNA markers are more informative, the probabilistic approach developed here would still be needed to estimate the true rate of nonpaternity in a population or to evaluate the precision of detecting true fathers. Am J Phys Anthropol 109:281–293, 1999. © 1999 Wiley-Liss, Inc.     

Surnames and the Y chromosome

A randomly ascertained sample of males with the surname "Sykes" was typed with four Y-chromosome microsatellites. Almost half the sample shared the same Y-chromosome haplotype, which has not been observed in control samples either from the same geographic region or from the United Kingdom as a whole. This points to a single surname founder for extant Sykes males, even though written sources had predicted multiple origins. The distribution of other Sykes Y-chromosome haplotypes were not significantly different from those in controls and may be accounted for by the historical accumulation of nonpaternity during the past 700 years, in which case the average rate estimate is 1.3%/generation. If this pattern is reproduced with other surnames, it may have important forensic and genealogical applications.     

Low nonpaternity rate in an old Afrikaner family

Extrapair paternity is a crucial parameter for evolutionary explanations of reproductive behavior. Early studies and human testis size suggest that human males secure/suffer frequent extrapair paternity. If these high rates are indeed true, it brings into question studies that use genealogies to infer human life history and the history of diseases since the recorded genealogies do not reflect paths of genetic inheritance. We measure the rate of nonpaternity in an old Afrikaner family in South Africa by comparing Y-chromosome short tandem repeats to the genealogy of males. In this population, the nonpaternity rate was 0.73%. This low rate is observed in other studies that matched genealogies to genetic markers and more recent studies that also find estimates below 1%. It may be that imposed religious morals have led to reduced extrapair activities in some historic populations. We also found that the mutation rate is high for this family, but is unrelated to age at conception.     

Glyoxalase 1: a possible 'null' allele.

A three-generation family, ascertained through the presence of two diabetic sibs, provides segregation data suggestive of the existence of a null allele at the glyoxalase (GLO) locus. This conclusion is supported by the GLO 1 phenotype in two children from a GLO 2 father. These two children inherited the same paternal HLA allele, while two other sibs received GLO 2 with the other paternal HLA haplotype. The rest of the pedigree is in agreement with this suggestion, while the segregation of all other informative markers does not suggest nonpaternity.     

Assigning a probability for paternity in apparent cases of mutation

Any direct estimator of mutation in a human population is subject to error due to nonpaternity. This paper deals with the quantification of this error by producing, under certain assumptions, the probability for paternity. In addition, a new direct estimator of the mutation rate is introduced.     

Evaluating pedigree data. I. The estimation of pedigree error in the presence of marker mistyping

Pedigrees used in the analysis of genetic or medical data are usually ascertained from sources subject to a variety of errors including misidentification of individuals, faults in historical documents or record linkage, nonpaternity, and unidentified adoption. Genetic markers can be used to verify putative family and pedigree data through the search for inconsistencies, or genetic exclusions, between putative parents and offspring. The probability of observing an exclusion given the occurrence of an error depends upon the gene frequencies at the loci under study and the forms of error. In addition, inconsistencies can arise from laboratory errors in marker determination. Together, these problems make the proper statistical analysis of such data desirable. Here we give a model that specifies the combined effects of various kinds of pedigree error along with genetic marker error. This model allows the maximum-likelihood estimation of the rates of various forms of pedigree error and laboratory error from genetic marker data collected on putative families. The method is illustrated by applying it to data obtained from a South Pacific island population, Tokelau. From the observed distribution of genetic marker inconsistencies between the parents and offspring of putative families, derived from the extensive genealogy of this population, we are able to estimate that the error of a paternal link is 4%, the error of a maternal link is zero, and the overall system typing error is 1%.     

A statistical framework for modeling HLA-dependent T cell response data

The identification of T cell epitopes and their HLA (human leukocyte antigen) restrictions is important for applications such as the design of cellular vaccines for HIV. Traditional methods for such identification are costly and time-consuming. Recently, a more expeditious laboratory technique using ELISpot assays has been developed that allows for rapid screening of specific responses. However, this assay does not directly provide information concerning the HLA restriction of a response, a critical piece of information for vaccine design. Thus, we introduce, apply, and validate a statistical model for identifying HLA-restricted epitopes from ELISpot data. By looking at patterns across a broad range of donors, in conjunction with our statistical model, we can determine (probabilistically) which of the HLA alleles are likely to be responsible for the observed reactivities. Additionally, we can provide a good estimate of the number of false positives generated by our analysis (i.e., the false discovery rate). This model allows us to learn about new HLA-restricted epitopes from ELISpot data in an efficient, cost-effective, and high-throughput manner. We applied our approach to data from donors infected with HIV and identified many potential new HLA restrictions. Among 134 such predictions, six were confirmed in the lab and the remainder could not be ruled as invalid. These results shed light on the extent of HLA class I promiscuity, which has significant implications for the understanding of HLA class I antigen presentation and vaccine development.     

HLA-B maternal-fetal genotype matching increases risk of schizophrenia

Schizophrenia and human leukocyte antigen (HLA) matching between couples or between mothers and offspring have independently been associated with prenatal/obstetric complications, including preeclampsia and low birth weight. Here, we report the results of a family-based candidate-gene study that brings together these two disparate lines of research by assessing maternal-fetal genotype matching at HLA-A, -B, and -DRB1 as a risk factor of schizophrenia. We used a conditional-likelihood modeling approach with a sample of 274 families that had at least one offspring with schizophrenia or a related spectrum disorder. A statistically significant HLA-B maternal-fetal genotype-matching effect on schizophrenia was demonstrated for female offspring (P=.01; parameter estimate 1.7 [95% confidence interval 1.22-2.49]). Because the matching effect could be associated with pregnancy complications rather than with schizophrenia per se, these findings are consistent with the neurodevelopmental hypothesis of schizophrenia and with accumulating evidence that the prenatal period is involved in the origins of this disease. Our approach demonstrates how genetic markers can be used to characterize the biology of prenatal risk factors of schizophrenia.     

The importance of HLA typing in cases of disputed paternity

In dispute paternity, the biologists must reply to two questions: 1. Is the paternity excluded or possible? 2. If it is possible, what is its probability? Valid answers can be given, using several genetic markers, among which HLA genes are specially interesting. Looking at HLA-A, B, C, DR typing of child, mother and presumed father, we propose a method which allows a direct calculation of paternity probability. Crossing over between HLA genes in presumed father and in mother are also considered in this method. In our experience, adding the date provided by the HLA genes and other genetic markers, we obtained, either formal exclusions, or possible paternities with a probability almost always higher than 90%.     

Forensic applications of molecular genetic analysis: an Italian collaborative study on paternity testing by the determination of variable number of tandem repeat DNA polymorphisms

Several variable number of tandem repeat (VNTR) DNA polymorphisms detecting different loci (YNH24/MspI or TaqI, CMM101/MspI or MLJ14/MspI, EFD64.2/RsaI or HinfI, YNZ22/TaqI, AW101/EcoRI, EKMDA2.1/PvuII and 3'-HVR/PvuII) were used in the analysis of 27 cases of disputed paternity in the Italian population. Fourteen exclusions and 17 attributions were performed. The results were compared with those obtained with immunohematologic analyses. Four exclusions and 2 attributions were made possible only by the combined use of several DNA polymorphisms, as the analyses of red-blood-cell antigens and isoenzymes, serum proteins and HLA group determinants were inconclusive. With the DNA test, 10 exclusions and 15 attributions were confirmed, with increased overall probability. In conclusion, VNTR polymorphisms were more informative, accurate and sensitive than the immunohematologic tests. Therefore, DNA analysis is the method of choice for testing genetic relationships.     

Pathogen-driven selection and worldwide HLA class I diversity

The human leukocyte antigen (HLA; known as MHC in other vertebrates) plays a central role in the recognition and presentation of antigens to the immune system and represents the most polymorphic gene cluster in the human genome [1]. Pathogen-driven balancing selection (PDBS) has been previously hypothesized to explain the remarkable polymorphism in the HLA complex, but there is, as yet, no direct support for this hypothesis [2 and 3]. A straightforward prediction coming out of the PDBS hypothesis is that populations from areas with high pathogen diversity should have increased HLA diversity in relation to their average genomic diversity. We tested this prediction by using HLA class I genetic diversity from 61 human populations. Our results show that human colonization history explains a substantial proportion of HLA genetic diversity worldwide. However, between-population variation at the HLA class I genes is also positively correlated with local pathogen richness (notably for the HLA B gene), thus providing support for the PDBS hypothesis. The proportion of variations explained by pathogen richness is higher for the HLA B gene than for the HLA A and HLA C genes. This is in good agreement with both previous immunological and genetic data suggesting that HLA B could be under a higher selective pressure from pathogens.     

Donor Exclusion in the National Blood Service Tissue Services Living Bone Donor Programme

National Blood Service (NBS) Tissue Services (TS) operates living donor and deceased donor tissue banking programmes. The living bone donor programme operates in collaboration with 91 orthopaedic departments across the country and collects bone donations, in the form of surgically removed femoral heads (FHs), from over 5000 patients per annum undergoing total hip replacement. Bone donated via the living programme constitutes approximately 55% of the total bone donated to NBS. Non-NBS tissue banks, primarily in hospital orthopaedic departments, also bank donated bone for the UK. A survey of information received from 16 collaborating orthopaedic centres, between April 2003 and August 2004, identified 709 excluded donors. The total number of donations banked from these sites was 1538. Donations can be excluded before collection if there are contraindications noted in a potential donor’s medical history before their operation. Donors may also be excluded after collection of the FH, for instance because of reactive microbiology tests for blood borne viruses, or if the donation storage conditions or related documentation have not met stringent quality requirements. In this survey, bone or joint conditions were the major reasons for excluding potential donors before donation (154 of 709 exclusions, 22%), followed by a current or a past history of malignancy (139 of 709 exclusions, 20%). Local staffing and operational difficulties sometimes resulted in potential donors being missed, or specific reasons for exclusion not being reported (117 exclusions). These out numbered exclusions due to patient refusal (80 exclusions). A small number (< 5) appear to have been excluded erroneously. There was considerable local variation in the reasons given for exclusion and certainly under-reporting. A survey of donations discarded after collection in the same period highlighted that 43% were donor related; 110 of 370 did not provide a follow-up blood sample. More than 30% were due to delays in forwarding blood samples to the microbiological laboratory for testing, resulting in deterioration of the sample quality. Training to ensure that standards are complied with and a firm evidence base for exclusion criteria, applied uniformly, will help focus donor identification efforts on individuals meeting rational criteria so that fewer potential donations are lost.     

Informed consent and the misattributed paternity problem in genetic counseling

When misattributed paternity is discovered in the course of genetic testing, a genetic counselor is presented with a dilemma concerning whether to reveal this information to the clients. She is committed to treating the clients equally and enabling informed decision making, but disclosing the information may carry consequences for the woman that the counselor cannot judge in advance. A frequent suggestion aimed at avoiding this problem is to include the risk of discovering nonpaternity in the informed consent process for counseling. In this paper I argue that such a move does not resolve the problem, because the conflict hinges on the interpretation of equality on which the counselor operates. Given the principles of genetic counseling, neither construal of equality yields a satisfactory solution to the conflict. In fact, I conclude that including nonpaternity in informed consent is not endorsed by either view, and we are still left with the question of what to do should nonpaternity be discovered. I suggest a compromise position concerning disclosure, involving revealing relevant genetic information but withholding nonpaternity when possible.     

No fallacies in the formulation of the paternity index

In a recent publication, Li and Chakravarti claim to have shown that the paternity index is not a likelihood ratio. They present a method of estimating the prior probability of paternity from a sample of previous court cases on the basis of exclusions and nonexclusions. They propose calculating the posterior probability on the basis of this estimated prior and the test result expressed as exclusion/nonexclusion. Their claim is wrong--the paternity index is a likelihood-ratio, that is, the ratio of the likelihood of the observation conditional on the two mutually exclusive hypotheses. Their proposed method of estimating the prior has been long known, has been applied to several samples, and is inferior (in terms of variance of the estimate) to maximum likelihood estimation based on all the phenotypic information available. Their proposed "new method" of calculating a posterior probability is based on the use of a less informative likelihood ratio 1/(1-PE) instead of Gürtler's fully informative paternity index X/Y (Acta Med Leg Soc Liege 9:83-93, 1956), but is otherwise identical to the Bayesian approach originally introduced by Essen-M?ller in 1938.     

HLA associations with obesity

A subgroup of 351 subjects with human leukocyte antigen (HLA) typing were available from the Framingham Heart Study for analyses to identify associations with obesity. The subjects consisted of 143 males and 208 females aged 58-88 years at the 15th biennial examination in 1978. The obese classification was based on maximum body mass index (BMI) over the 16 available biennial examinations of the Framingham Heart Study. The subjects were classified as obese if they exceeded the 95th percentile of BMI for 20- to 29-year-old subjects as described in the NHANES II study; males were obese if BMI greater than 31.1 and females were obese if BMI greater than 32.3. There were 27 obese males (18.9%) and 44 obese females (21.2%) in the sample. Gene frequencies were compared between the nonobese and obese groups for the pooled sample as well as by sex. Among alleles previously shown to be related to obesity, HLA Bw35 appeared to be more frequent in obese females but these data did not confirm a difference for the B18 or Cw4 alleles. More importantly, HLA Aw30 was found to be significantly higher among the obese subjects in both males and females. Further analyses adjusting for potential confounding variables reduced the estimated relative risk for obesity for subjects with the Bw35 allele to approximately 1.30 and no longer significant for this sample size. In contrast, the relative risk for Aw30, while reduced, remained significant after adjustment for confounding variables. Based on these data, individuals with the Aw30 allele have a relative risk of 2.61 for obesity.(ABSTRACT TRUNCATED AT 250 WORDS)     

An Integrated Tool to Study MHC Region: Accurate SNV Detection and HLA Genes Typing in Human MHC Region Using Targeted High-Throughput Sequencing

The major histocompatibility complex (MHC) is one of the most variable and gene-dense regions of the human genome. Most studies of the MHC, and associated regions, focus on minor variants and HLA typing, many of which have been demonstrated to be associated with human disease susceptibility and metabolic pathways. However, the detection of variants in the MHC region, and diagnostic HLA typing, still lacks a coherent, standardized, cost effective and high coverage protocol of clinical quality and reliability. In this paper, we presented such a method for the accurate detection of minor variants and HLA types in the human MHC region, using high-throughput, high-coverage sequencing of target regions. A probe set was designed to template upon the 8 annotated human MHC haplotypes, and to encompass the 5 megabases (Mb) of the extended MHC region. We deployed our probes upon three, genetically diverse human samples for probe set evaluation, and sequencing data show that ∼97% of the MHC region, and over 99% of the genes in MHC region, are covered with sufficient depth and good evenness. 98% of genotypes called by this capture sequencing prove consistent with established HapMap genotypes. We have concurrently developed a one-step pipeline for calling any HLA type referenced in the IMGT/HLA database from this target capture sequencing data, which shows over 96% typing accuracy when deployed at 4 digital resolution. This cost-effective and highly accurate approach for variant detection and HLA typing in the MHC region may lend further insight into immune-mediated diseases studies, and may find clinical utility in transplantation medicine research. This one-step pipeline is released for general evaluation and use by the scientific community.     

A review of theoretical aspects of HLA and disease associations

A number of antigens (alleles) of the human histocompatibility (HLA) system have been shown to be associated with a wide range of chronic diseases. Recently a great deal of interest has been shown in many of the theoretical aspects of HLA disease associations. These range from statistical problems of detecting significant associations to the development of models to determine the mode of inheritance of the disease. These models, as well as other features of HLA disease associations, will be reviewed in this paper.     

Multiple mechanisms generate HLA class I altered phenotypes in laryngeal carcinomas: high frequency of HLA haplotype loss associated with loss of heterozygosity in chromosome region 6p21

Major histocompatibility complex (MHC) class I loss or downregulation in cancer cells is a major immune escape route used by a large variety of human tumors to evade anti-tumor immune responses mediated by cytotoxic T lymphocytes. Multiple mechanisms are responsible for such HLA class I alterations. However, the precise frequency of these molecular defects has not been clearly determined in tumors derived from specific tissues. To analyze such defects we aim to define the major HLA class I-altered phenotypes in different tumor types. In this paper we report on HLA class I expression in 70 laryngeal carcinomas. We used immunohistological techniques with a highly selective panel of anti-HLA monoclonal antibodies (mAb), and polymerase chain reaction (PCR) microsatellite amplification of previously selected microsatellite markers (STR) located in chromosome 6 and 15. DNA was obtained from microdissected tumor tissues and surrounding stroma to define the loss of heterozygosity (LOH) associated with chromosome 6p21. Our results showed that LOH in chromosome 6 produced HLA haplotype loss (phenotype II) in 36% of the tumors. In addition, HLA class I total loss (phenotype I) was found in 11%; HLA A or B locus downregulation (phenotype III) was detected in 20%; and HLA class I allelic loss (phenotype IV) in 10% of all cases. We sometimes observed two or more associated mechanisms in the same HLA-altered phenotype, such as LOH and HLA total loss in phenotype I. In only 23% of tumors it was not possible to identify any HLA class I alteration. We conclude that the combination of immunohistological techniques and molecular analysis of tumor DNA obtained from microdissected tumor tissues provides a means for the first time of determining the actual frequency of the major HLA class I-altered phenotypes in laryngeal carcinomas.     

HLA class I allele promiscuity revisited

The peptide repertoire presented on human leukocyte antigen (HLA) class I molecules is largely determined by the structure of the peptide binding groove. It is expected that the molecules having similar grooves (i.e., belonging to the same supertype) might present similar/overlapping peptides. However, the extent of promiscuity among HLA class I ligands remains controversial: while in many studies T cell responses are detected against epitopes presented by alternative molecules across HLA class I supertypes and loci, peptide elution studies report minute overlaps between the peptide repertoires of even related HLA molecules. To get more insight into the promiscuous peptide binding by HLA molecules, we analyzed the HLA peptide binding data from the large epitope repository, Immune Epitope Database (IEDB), and further performed in silico analysis to estimate the promiscuity at the population level. Both analyses suggest that an unexpectedly large fraction of HLA ligands (>50%) bind two or more HLA molecules, often across supertype or even loci. These results suggest that different HLA class I molecules can nevertheless present largely overlapping peptide sets, and that “functional” HLA polymorphism on individual and population level is probably much lower than previously anticipated.