Efficient,robust, and unified method for mapping complex traits (I): Two-point linkage analysis

The completion of a preliminary human genome map and development of molecular methods have enabled researchers to assay a large number of polymorphic markers that are evenly spaced along the entire human genome. Among many applications, marker data are valuable for mapping complex traits through linkage or linkage-disequilibrium analysis, the former of which is the focus of this paper, the first in a series on this subject. Formalizing the concept and computation for linkage analysis, Elston and Stewart [1971; Human Heredity 21:523–542] introduced a likelihood function to capture relevant genetic information and a recursive algorithm for computing the likelihood function. However, the computing burden is prohibitive in processing complex pedigrees. Since that fundamental development, improving the computational algorithm and extending the method has been a dynamic area of research. The primary objective of this communication is to introduce a semiparametric method for linkage analysis. It is a particularly suitable approach with desirable properties for mapping complex traits that may be binary, continuous, and partially observed (i.e., censored). It incorporates candidate genes, environmental factors, and their interactions with the putative gene and is expected to be robust and efficient in comparison with likelihood-based methods. The properties of the estimates have been studied in finite samples with a limited simulation study. This method is illustrated with an application to family data contributed to the Breast Cancer Consortium. Am. J. Med. Genet. 77:366–383, 1998. © 1998 Wiley-Liss, Inc.     

Associations of glutathione S-transferase P1, M1, and environmental tobacco smoke with wheezing illness in school children

BACKGROUND: Polymorphisms at the glutathione S-transferase (GST) were associated with asthma-related phenotypes. We hypothesized that the GSTP1 and GSTM1 genotypes could modify the effects of household environmental tobacco smoke (ETS) on childhood wheezing illness. METHODS: We conducted a case-control study comprised of 216 lifetime wheezing children and 185 nonwheezing controls, all of whom were selected from 2524 fourth- to ninth-grade school children in southern Taiwan. RESULTS: Homozygous GSTP1 Ile-105 was significantly associated with current wheezing (OR = 1.78, 95% CI 1.04-3.12), but insignificantly associated with ever wheezing (OR = 1.26, 95% CI 0.82-1.94). The risks of ever or current wheezing on GSTM1 null genotype were positive but not statistically significant. Although household ETS exposure was not associated with wheezing illness, after excluding subjects having in utero ETS or active smoking habits, the adverse effects of household ETS exposure differed significantly by GSTP1-105 genotypes. In children without any ETS exposure at home, GSTP1 Ile-105 homozygosity was significantly related to increased risks for both ever wheezing (OR = 2.29, 95% CI 1.17-4.49) and current wheezing (OR = 4.86, 95% CI 1.86-12.70). In children with household ETS exposure, the risks of wheezing illness did not increase for those carrying two GSTP1 Ile-105 alleles. Children carrying any GSTP1 Val-105 allele were at a significantly greater risk of both ever and current wheezing when exposed to ETS, with a clear dose-response relationship to the number of smokers at home. CONCLUSION: Household ETS exposure is a modifiable cause of wheezing illness in a genetically susceptible subpopulation.     

Sperm-cervical mucus interaction, in particular in the presence of antispermatozoal antibodies

Disturbances of the interaction between spermatozoa and cervicalmucus can cause subfertility or infertility. The diagnosis ofsuch a disturbed interaction is possible with simple laboratorytests. Oligomucorrhoea and dysmucorrhoea are the most frequentcauses of a disturbed sperm-cervical mucus interaction. AntispermatozoalIgA plays a quantitatively limited, but qualitatively importantrole. Sophisticated time-consuming laboratory investigationshave mostly only additional value for the diagnosis.     

Fate of SRY, PABY, DYS1, DYZ3 and DYZ1 loci in Indian patients harbouring sex chromosomal anomalies

We analysed chromosomes, conducted hormonal assays and screened genomic DNA of 34 patients with or without detectable Y chromosome for the presence/absence of SRY, PABY, DYS1, DYZ3 and DYZ1 loci and for mutations in the SRY gene. The samples studied represented cases of oligozoospermia, cryptorchidism, Swyer syndrome, Turner syndrome, male pseudohermaphroditism, XXY female syndrome, Klinefelter's syndrome, repeated abortion and instances of male infertility. Chromosomal constitutions and the level of hormones (FSH, LH, PRL, E2 and TSH) were found to be abnormal in several cases. A phenotypic female (P20) positive for all the Y-linked loci screened, showed mutations upstream of the HMG box in the SRY gene. In addition, one or more of the Y-linked loci were detected in several phenotypic females. Fluorescence in-situ hybridization of metaphase chromosomes and interphase nuclei of an aborted fetus with DYZ1 probe detected signals from normal to low levels to its complete absence confirming a complex Y chromosome mosaicism. Upon DNA analysis, the fetus was found to be positive for all the above-mentioned Y-linked loci. Organizational variation within the DYZ1 arrays and its correlation with recurrent spontaneous abortion may be followed-up in subsequent studies to substantiate this observation. This would augment genetic counselling to the affected couples. Prospects of this approach in the overall management of clinical cases with sex chromosome-related anomalies are discussed.     

The human immunoglobulin VH7 gene family consists of a small,polymorphic group of six to eight gene segments dispersed throughout the VH locus

In this report we describe the analysis and mapping of members of the human immunoglobulin V_H7 gene family. V_H7 and V_H1 gene segments are closely related, with individual gene segments sharing between 78% and 82% sequence identity. Divergence from V_H1 gene sequence occurs as an abrupt event at the boundary between framework region (FR) 2 and complementarity-determining region (CDR) 2 and continues through a major portion of FR 3. We used polymerase chain reaction amplification to create a 162-base pair probe spanning the family-specific region of CDR 2 and FR 3 that proved suitable for standard Southern analysis of genomic DNA. The V_H7 gene family was found to be a small but discrete V_H gene family consisting of five to eight germ-line elements, of which at least three are polymorphic. Four different V_H7 gene segments were cloned from the germ line of a single individual, and assigned to specific restriction fragments by sequence-specific hybridization. Two of the four V_H7 elements were pseudogenes. The pattern of sequence variation in these and other known pseudogenes suggests that these nonfunctional elements may play a role in the evolution of novel V_H families. A combination of one and two-dimensional pulsed field gel electrophoresis was employed to map the chromosomal location of all of these V_H7 elements. Individual V_H7 gene segments were found to be dispersed over a region of at least 940 kb of DNA, and interspersed with members from other V_H gene families. The polymorphism of the V_H7 gene segments and their scattered location throughout the V_H locus makes them potentially useful markers for mapping and linkage studies.     

CYP17, CYP1A1 and COMT polymorphisms and the risk of adenomyosis and endometriosis in Taiwanese women

BACKGROUND: The aim of the study was to test whether the COMT, CYP1A1 and CYP17 genes influence the risk of developing adenomyosis and endometriosis. METHODS: We conducted two case-control studies, where the cases (n = 198) had either of the two diseases, and controls (n = 312) were disease-free women. For the COMT gene, we selected the G/A nonsynonymous single-nucleotide polymorphism (SNP) that leads to valine-to-methionine (Val/Met) substitution. For the CYP1A1 gene, we used a functional T/C SNP in the 3'-noncoding region, and we genotyped a T/C functional SNP in the 5' region of the CYP17 gene for the present study. Hardy-Weinberg equilibrium was checked in both cases and controls. Logistic regression models were used to evaluate the genetic effect, with adjustment for other covariates. RESULTS: We found that the homozygous COMT genotype that encodes low enzyme activity had an increased risk for adenomyosis with an age-adjusted odds ratio of 3.2 (95% confidence interval 1.3-7.8; P = 0.006). The COMT gene, however, was not associated with endometriosis. Neither the CYP1A1 nor CYP17 genes had any significant association with either of the two diseases. CONCLUSION: The COMT gene significantly influences the risk of adenomyosis but not endometriosis. The present study does not provide evidence to support any of the three genes exerting pleiotropic effects on both diseases.     

Human testicular protein TPX1/CRISP-2: localization in spermatozoa, fate after capacitation and relevance for gamete interaction

Testicular protein Tpx-1, also known as CRISP-2, is a cysteine-rich secretory protein specifically expressed in the male reproductive tract. Since the information available on the human protein is limited to the identification and expression of its gene, in this work we have studied the presence and localization of human Tpx-1 (TPX1) in sperm, its fate after capacitation and acrosome reaction (AR), and its possible involvement in gamete interaction. Indirect immunofluorescence studies revealed the absence of significant staining in live or fixed non-permeabilized sperm, in contrast to a clear labelling in the acrosomal region of permeabilized sperm. These results, together with complementary evidence from protein extraction procedures strongly support that TPX1 would be mainly an intra-acrosomal protein in fresh sperm. After in vitro capacitation and ionophore-induced AR, TPX1 remained associated with the equatorial segment of the acrosome. The lack of differences in the electrophoretic mobility of TPX1 before and after capacitation and AR indicates that the protein would not undergo proteolytical modifications during these processes. The possible involvement of TPX1 in gamete interaction was evaluated by the hamster oocyte penetration test. The presence of anti-TPX1 during gamete co-incubation produced a significant and dose-dependent inhibition in the percentage of penetrated zona-free hamster oocytes without affecting sperm motility, the AR or sperm binding to the oolema. Together, these results indicate that human TPX1 would be a component of the sperm acrosome that remains associated with sperm after capacitation and AR, and is relevant for sperm-oocyte interaction.     

Embryo transfer and related techniques in domestic animals, and their implications for human medicine

This report was commissioned by the Canadian government on therelevance of new techniques in animal embryology to the social,ethical and clinical applications of assisted human reproduction.It briefly describes the history of animal breeding, and theregulation of the female reproductive tract, ovulation and fertilizationin laboratory and veterinary species. Embryo transfer is describedin detail, including the synchronization of reproductive cycles,superovulation and embryo growth in vitro. Methods of experimentalembryology, including bisection, sexing of spermatozoa and embryos,cloning and gene therapy are described. The relevance of thesestudies to human IVF are considered briefly.     

Influence of genome imprinting on gene expression, phenotypic variations and development

Genome imprinting confers functional differences on parental chromosomes as a result of the differences in epigenetic inheritance from parental germlines. Repressed and derepressed chromatin structures probably constitute the initial germline-dependent 'imprints'. Any subsequent modifications, such as DNA methylation, will be influenced by these initial epigenetic modifications. Hence, epigenetic modifications of parental alleles probably occur progressively and this will affect their potential for expression. It appears that imprinting of some parental alleles is critical for their dosage, affecting embryonic growth, cell proliferation and differentiation. Genetic studies highlight the influence of subsets of imprinted genes and identify those which are crucial for development. Genomic imprinting also affects some transgene loci and dominant mutations with accompanying variable penetrance and expressivity. The response of transgenes can be influenced by modifier genes whose presence is most readily detected in different inbred backgrounds. The influence of modifier genes can in turn be affected by their parental origin, perhaps partly by the maternally inherited oocyte cytoplasmic factors, as well as by complex interactions between some parental alleles and oocyte cytoplasmic factors. The resulting epigenetic modifications of unlinked loci can result in substantial phenotypic variations.     

SRY-negative 46,XX male with normal genitals, complete masculinization and infertility

XX maleness is a rare syndrome with a frequency of 1 in 20,000-25,000 males. XX males exist in different clinical categories with ambiguous genitalia or partially to fully mature male genitalia, in combination with complete or incomplete masculinization. In this study, we report a case of SRY-negative XX male with complete masculinization but infertility. The patient had fully mature male genitalia with descended but small testes and no signs of undervirilization. PCR analysis for SRY, ZFY, Amelogenin, AZFa, AZFb, AZFc genes, a pair of primers from heterochromatic region and six Y-STRs showed the absence of any Y-chromosome-derived material. Absence of SRY gene was confirmed by three independent PCRs for each of two sets of primers covering an increasing length of the gene. Sequence analysis of the coding regions of SOX9 and DAX1 genes did not reveal any mutation. Real-time PCR assay revealed normal copy number for SOX9 gene. Microsatellite analysis showed no evidence of 17q (SOX9 gene) or 22q duplication. Genotyping with X-STRs ruled out the possibility of any deletion on X chromosome. Development of the male phenotype in the absence of SRY probably resulted from the loss of function mutation in some unknown sex-determining gene, which normally inhibits the male pathway, or from a gain of function mutation in a gene downstream to SRY in male pathway.     

Difference in signalling between various hormone therapies in endometrium, myometrium and upper part of the vagina

BACKGROUND: Combined hormone treatments in post-menopausal women have different clinical responses on uterus and vagina; therefore, we investigated differences in steroid signalling between various hormone therapies in these tissues. METHODS: A total of 30 post-menopausal women scheduled for hysterectomy were distributed into four subgroups: control-group (n = 9), Tibolone-group (n = 8); estradiol (E(2))-group (n = 7); E(2) + medroxyprogesterone acetate (MPA)-group (n = 6). Medication was administered orally every day for 21 days prior to removal of uterus and upper part of the vagina. Tissue RNA was isolated, and gene expression profiles were generated using GeneChip technology and analysed by cluster analysis and significance analysis of microarrays. Apoptosis and cell proliferation assays, as well as immunohistochemistry for hormone receptors were performed. RESULTS: 21-days of treatment with E(2), E(2) + MPA or tibolone imposes clear differential gene expression profiles on endometrium and myometrium. Treatment with E(2) only results in the most pronounced effect on gene expression (up to 1493 genes differentially expressed), proliferation and apoptosis. Tibolone, potentially metabolized to both estrogenic and progestagenic metabolites, shows some resemblance to E(2) signalling in the endometrium and, in contrast, shows significant resemblance to E(2) + MPA signalling in the myometrium. In the vagina the situation is entirely different; all three hormonal treatments result in regulation of a small number (4-73) of genes, in comparison to signalling in endometrium and myometrium. CONCLUSION: Endometrium and myometrium differentially respond to the hormone therapies and use completely different sets of genes to regulate similar biological processes, while in this experiment the upper part of the vagina is hardly hormone responsive.     

Susceptibility to human cancer: From the perspective of a pathologist

The etiologies of human cancer can only be discerned when the genetic clustering of cancer occurs within a family or when cancer occurs endemically in a particular environment. The possible approaches to solving the nature/nurture problem, especially for human carcinogenesis, posit a fascinating challenge for pathologists. This perspective review presents some examples of how clues to human cancer etiologies and/or susceptibilities reside in the realm of pathology practice. These examples using various omics techniques including adductomics, which I would like to highlight in this article, show that the currently available concepts and methods in human pathology can open a path toward the brave new world of a post‐genomic era of medicine for young pathologists, whether their original intention was toward the pursuit of diagnostic or investigative knowledge.     

IL1β, IL18, NFKB1 and IFNG gene interactions are associated with severity of rheumatoid arthritis: A pilot study

Abstract

Rheumatoid arthritis (RA) is an autoimmune disease which can lead to progressive and functional disability. Literature data suggest that some inflammatory proteins are dysregulated in RA patients and its genetic polymorphisms may contribute to the aetiology and pathogenesis of disease in different ethnic groups. Polymorphisms in IL1β, IL18, NFKB1 and IFNG genes were studied in different populations with RA, but the analysis indicated contradictory results. Thereby, we hypothesised that polymorphisms in these genes could have a combined effect on susceptibility to and severity of disease. We evaluated the +3953?C/T IL1β (rs1143634), –137?G/C IL18 (rs187238), –94 ins/del ATTG NFKB1 (rs28362491) and +874?T/A IFNG (rs2430561) polymorphisms in the northeastern Brazilian population. Peripheral blood samples were collected and DNA extraction was conducted. The polymorphisms were evaluated by RFLP and ARMS–PCR. An association was observed in rs1143634 which showed a protective effect against development of RA in carriers of the T allele (OR?=?0.58; 95% CI 0.36–0.92; p?=?.020). In addition, we found an association among genotypes of the rs1143634 with the HAQ index (p?=?.021) and rs2430561 with DAS28 (p?=?.029) and CDAI (p?=?.029). In relation to combined effects of these SNPs (C/C to rs1143634, G/G to rs187238, I/I to rs28362491 and AA to rs2430561) we found a significant association with decreased functional disability (HAQ index p?<?.001) and ESR (p?=?.034), indicating a lower disease activity in carriers of these genotypes. GLM analysis confirmed these associations (HAQ (F?=?5.497; p?<?.001) and ESR (F?=?2.727; p?=?.032)). Our analysis indicated that in the studied population +3953?C/T IL-1β (rs1143634), –137?G/C IL-18 (rs187238), –94 ins/del ATTG NFKB1 (rs28362491) and +874?T/A IFNG (rs2430561) polymorphisms can together contribute to RA severity although they do not individually influence the disease.     

Clinical characterization of 42 oligospermic or azoospermic men with microdeletion of the AZFc region of the Y chromosome,and of 18 children conceived via ICSI

BACKGROUND: Severe spermatogenic compromise may be the result of a Y-chromosomal deletion of the AZFc region. Prior studies are limited to relatively small numbers of AZFc-deleted men. In this study, we have fully characterized 42 infertile men with a Y chromosome microdeletion strictly confined to the AZFc region, and we report on 18 children conceived through the use of ICSI. METHODS: A total of 42 oligospermic or azoospermic men had AZFc deletions. History, physical examination, karyotype, FSH, LH, testosterone, testis histology and results of ICSI using ejaculated or testis sperm were retrospectively accumulated in two academic clinical practices. RESULTS: All men were somatically healthy. Karyotypes were 46,XY in all but two men. FSH, LH, testosterone and testis histology could not differentiate those with oligospermia or azoospermia, nor could they predict whether sperm could be found in harvested testis tissue. Paternal age was not increased. Sperm production appeared stable over time. The results of ICSI were not affected by the AZFc deletion. All but one of the offspring were healthy. The sons inherited the AZFc deletion with no increase in length. CONCLUSIONS: AZFc-deleted men are somatically healthy, will most likely have useable sperm, will have stable sperm production over time and will have a good chance to experience biological paternity, but their sons will also be AZFc-deleted.     

Oestrogen receptor-alpha gene polymorphism is associated with endometriosis, adenomyosis and leiomyomata

Endometriosis, adenomyosis and leiomyomata develop in women of reproductive age and regress after menopause or ovariectomy, suggesting that they grow in an oestrogen-dependent fashion. We investigated whether polymorphism in the oestrogen receptor-alpha (ERalpha) gene is related to oestrogen-dependent benign uterine disease. A total of 203 women with regular menstrual cycles underwent laparotomy or laparoscopy and were diagnosed histologically with endometriosis, adenomyosis and/or leiomyomata. Patients with cervical carcinoma in situ, tubal occlusion or adhesion but no other gynaecological disease were considered to be disease-free. A total of 179 women undergoing annual health examination were grouped as reference population. The distribution of PVUII genotypes (PP, Pp, and pp) of the ERalpha gene was different between each pair of the four groups of endometriosis, adenomyosis/leiomyomata, disease-free, and reference population (P = 0.022-0.0005), except between the former two groups. The PP genotype was less frequent in the groups of endometriosis (P = 0.0002) and adenomyosis/leiomyomata (P = 0.002) as compared to that in the disease-free group. In the endometriosis group, there was no difference in the distribution of PVUII genotypes due to complicating diseases (adenomyosis and/or leiomyomata) or severity of the clinical stages. These results suggest that the PVUII polymorphism of the ERalpha gene is associated with the risk for endometriosis, adenomyosis, and leiomyomata.     

Chlamydia trachomatis infection, Fallopian tube damage and a mannose-binding lectin codon 54 gene polymorphism

BACKGROUND: Mannose-binding lectin (MBL), a component of the innate immune system, provides a first-line defense against invading microorganisms. Polymorphisms in the MBL gene have been associated with increased risk of infection. Chlamydia trachomatis genital tract infections are a major cause of Fallopian tube occlusion. Our objective was to test whether an MBL codon 54 polymorphism might contribute to development of C. trachomatis-associated tubal damage. METHODS: In a case-control study, 97 women with occluded and 104 women with patent Fallopian tubes were tested for a history of chlamydial infection by serology and for their MBL codon 54 genotype by PCR and restriction fragment length polymorphism analysis. Clinical data were blinded to those performing all laboratory analyses. RESULTS: Women with tubal occlusion who also had a positive chlamydial serology had the highest rate of variant MBL B allele carriage (P<0.001). Among women who were chlamydial antibody negative, allele B carriage was also more frequent in those with blocked, as opposed to patent, Fallopian tubes (P<0.01). CONCLUSIONS: Wild-type allele A homozygosity is protective against, while carriage of the variant allele B is a risk factor for, Fallopian tube occlusion in women who are seropositive or seronegative for C. trachomatis.