Leukocyte Chromosomes Research Articles

Disruption of the Uty epigenetic regulator locus in hematopoietic cells phenocopies the profibrotic attributes of Y chromosome loss in heart failure.

Heart failure affects millions of people worldwide, with men exhibiting a higher incidence than women. Our previous work has shown that mosaic loss of the Y chromosome (LOY) in leukocytes is causally associated with an increased risk for heart failure. Here, we show that LOY macrophages from the failing hearts of humans with dilated cardiomyopathy exhibit widespread changes in gene expression that correlate with cardiac fibroblast activation. Moreover, we identify the ubiquitously transcribed t et ratricopeptide Y-linked (Uty) gene in leukocytes as a causal locus for an accelerated progression of heart failure in male mice with LOY. We demonstrate that Uty disruption leads to epigenetic alterations in both monocytes and macrophages, increasing the propensity of differentiation into profibrotic macrophages. Treatment with a transforming growth factor-β-neutralizing antibody prevented the cardiac pathology associated with Uty deficiency in leukocytes. These findings shed light on the mechanisms that contribute to the higher incidence of heart failure in men.

Abstract 16532: Mosaic Loss of Y Chromosome in Leukocytes Predicts Death in Patients With Chronic Heart Failure Across the Whole Spectrum of Ejection Fraction

Aims: Mosaic loss of Y chromosome (LOY) in men increases with age and correlates with clonal expansion of myeloid cells. Experimental modeling demonstrated that LOY in hematopoietic cells leads to diffuse cardiac fibrosis and subsequent development of heart failure in mice. Cardiac fibrosis is an important pathogenic feature in heart failure irrespective of etiology. Therefore, we investigated the prognostic significance of LOY in men with chronic heart failure. Methods and Results: In a total of 705 male patients with chronic heart failure with reduced and preserved ejection fraction, LOY was determined by digital PCR of peripheral blood cells by assessing the 6bp sequence difference between the AMELX and AMELY genes. ROC analysis relating the extent of LOY to mortality revealed a LOY of 17% as the optimal cut-off value to predict death during follow-up. LOY >17% was observed in 18.6% of all patients, increased with age and was associated with increased mortality after three years (13,7% in patients without LOY compared to 29.0% in patients with LOY, p<0.001, Figure 1). LOY remained an independent predictor of death in multivariate analysis including, age, LVEF and nt-pro BNP serum levels (HR 1,67; 95% CI 1.08-2.57; p=0.017). Importantly, LOY was associated with increased mortality in patients with heart failure with reduced ejection fraction defined as LVEF < 50% (p<0.001) as well as in patients with preserved LVEF ≥ 50% (p=0.01). Conclusion: Among patients with heart failure, LOY in blood cells is associated with increased risk of death across the complete range of ejection fraction. Further studies are warranted to discern if LOY associates with cardiac fibrosis in men with heart failure and triggers increased profibrotic signaling. The results of this study may inform trials evaluating precision-targeted anti-fibrotic therapies in patients with chronic heart failure.

The State of the LPO-AOD System and the Relative Length of Telomeric Repeats in the Chromosomes of Blood Leukocytes in Obstructive Sleep Apnea Syndrome.

We analyzed the relative length of telomeric repeats in peripheral blood leukocytes and indicators of oxidative stress in 32 men (mean age 51.2±3.1 years) with obstructive sleep apnea syndrome (OSAS). The control group consisted of volunteers without OSAS. The relative length of telomeres was determined in a DNA sample isolated from venous whole blood samples. The length of telomeric repeats of chromosomes was determined using quantitative real-time PCR (3 times for each DNA sample); albumin served as an internal control. The intensity of LPO processes and antioxidant protection was assessed by conventional spectrophotometric methods. It was found that in patients with OSAS, the relative length of telomeric repeats was lower by 48%, the levels of substrates and products of LPO were higher (double bonds and diene conjugates by 34 and 19%, respectively), and antioxidant protection indicators were lower (concentrations of fat-soluble vitamins α-tocopherol and retinol by 18 and 19.3%, respectively, total antioxidant activity by 32.9%) than in volunteers of the control group. Thus, we can conclude that the nucleotide sequences are reduced in patients with OSAS under conditions of intensified free radical oxidation.

Mosaic loss of the Y chromosome in human neurodegenerative and oncological diseases.

The development of new biomarkers for prediction and early detection of human diseases, as well as for monitoring the response to therapy is one of the most relevant areas of modern human genetics and genomics. Until recently, it was believed that the function of human Y chromosome genes was limited to determining sex and controlling spermatogenesis. Thanks to occurance of large databases of the genome-wide association study (GWAS), there has been a transition to the use of large samples for analyzing genetic changes in both normal and pathological conditions. This has made it possible to assess the association of mosaic aneuploidy of the Y chromosome in somatic cells with a shorter lifespan in men compared to women. Based on data from the UK Biobank, an association was found between mosaic loss of the Y chromosome (mLOY) in peripheral blood leukocytes and the age of men over 70, as well as a number of oncological, cardiac, metabolic, neurodegenerative, and psychiatric diseases. As a result, mLOY in peripheral blood cells has been considered a potential marker of biological age in men and as a marker of certain age-related diseases. Currently, numerous associations have been identified between mLOY and genes based on GWAS and transcriptomes in affected tissues. However, the exact cause of mLOY and the impact and consequences of this phenomenon at the whole organism level have not been established. In particular, it is unclear whether aneuploidy of the Y chromosome in blood cells may affect the development of pathologies that manifest in other organs, such as the brain in Alzheimer's disease, or whether it is a neutral biomarker of general genomic instability. This review examines the main pathologies and genetic factors associated with mLOY, as well as the hypotheses regarding their interplay. Special attention is given to recent studies on mLOY in brain cells in Alzheimer's disease.

Loss of Y chromosome in leukocytes can be regarded as a male-specific age predictor for age group estimation in forensic genetics.

Age prediction is an important field in forensic and aging research. Traditional methods used DNA methylation, telomere shortening, and mitochondrial DNA mutations to conduct age prediction models. Sex chromosomes, like the Y chromosome, have a significant role in aging as previously reported in hematopoietic disease and many non-reproductive cancers. Until now, there is no age predictor based on the percentage of loss of Y chromosome (LOY). LOY has been previously revealed to be correlated with Alzheimer's disease, short survival, and higher risk of cancer. The possible correlation of LOY between normal aging was not fully explored. In this study, we conducted age prediction by measuring LOY percentage by droplet digital PCR (ddPCR), based on 232 healthy male samples, including 171 blood samples, 49 saliva samples, 12 semen samples. The age group of samples ranges from 0 to 99years, with two individuals in almost every single age. Pearson correlation method was performed to calculate the correlation index. The result indicated a correlation index of 0.21 (p = 0.0059) between age and LOY percentage in blood samples, with the regression formula being y = -0.016823 + 0.001098x. The correlation between LOY percentage and age is obvious only when the individuals were divided into different age groups (R = 0.73, p = 0.016). In the studied saliva and semen samples, p-values of the correlation are 0.11 and 0.20, respectively, showing no significant association between age and LOY percentage in these two biological materials. For the first time, we investigated male-specific age predictor based on LOY. The study showed that LOY in leukocytes can be regarded as a male-specific age predictor for age group estimation in forensic genetics. This study might be indicative for forensic applications and aging research.

Age-related loss of Y chromosome in leukocytes linked to cardiac fibrosis.

Age-related loss of Y chromosome in leukocytes linked to cardiac fibrosis.

Methylation of three genes encoded by X chromosome in blood leukocytes and colorectal cancer risk.

X chromosome change has been proved to be associated with carcinogenesis and related to gender differences in cancer risk. If aberrant methylation of genes encoded by X chromosome involve in the risk and prognosis of cancers, including colorectal cancer (CRC), remain unclear. We conducted a case–control study consisted of 432 CRC cases and 434 controls, detecting the methylation levels of FAM156B, PIH1D3, and PPP1R3F in the X chromosome in blood leukocytes using methylation‐sensitive high‐resolution melting (MS‐HRM). We analyzed the relationship between the methylation levels and CRC susceptibility and then explored the interactions with environmental factors on CRC risk with logistics regression. Moreover, we conducted a follow‐up study containing 225 CRC patients to explore the associations between the methylation of FAM156B, PPP1R3F, and PIH1D3 and CRC prognosis. The hypermethylation of FAM156B, PPP1R3F, and PIH1D3 was related to increased CRC risk (ORPS‐adj = 2.932, 95% confidence interval [CI]: 2.029–4.237; ORPS‐adj = 1.602, 95% CI: 1.078–2.382; ORPS‐adj = 1.628, 95% CI: 1.065–2.490, respectively). In the multiple CpG site methylation (MCSM) analysis, compared with non‐MCSM, a significant relationship between MCSM and increased CRC risk was found (ORPS‐adj = 2.202, 95% CI: 1.512–3.208). We observed synergistic interaction between PPP1R3F hypermethylation and fried food consumption on CRC risk (ORi = 2.682, 95% CI: 1.321–5.446). However, there were no associations between the methylation of FAM156B, PPP1R3F, and PIH1D3 and CRC prognosis (p > 0.05). In conclusion, the methylation of FAM156B, PPP1R3F, and PIH1D3 genes in blood leukocytes is significantly related to CRC risk and may be potential biomarkers for CRC risk but not prognosis.

Telomere length as a biomarker of the risk of cardiovascular complications in patients with coronary heart disease

To study the effect of oxidative stress and telomere length in the chromosomes of blood leukocytes in patients with coronary heart disease (CHD) on the development of cardiovascular complications. In 119 patients with CHD, the level of oxidatively modified low-density lipoproteins (ox-LDL) in blood plasma and the length of telomeres in nuclear blood cells were determined during the examination. After 5 years, a telephone survey of patients (or their relatives) was conducted to obtain data on the presence of cardiovascular complications. Telomere length was determined using quantitative real-time PCR, and the level of ox-LDL was determined by immunochemical method. It was found that reducing the length of telomeres in patients with CHD increases the risk of subsequent development of cardiovascular complications. A strong negative correlation was found between the level of ox-LDL and telomere length in the group of examined CHD patients who had cardiovascular complications after 5 years. CHD patients with short telomere length and high levels of ox-LDL have an increased risk of cardiovascular complications during 5 years.

Association between telomere length in peripheral blood leukocytes and risk of ischemic stroke in a Han Chinese population: a linear and non-linear Mendelian randomization analysis

BackgroundMany contradictory conclusions pertaining to the telomere length in peripheral leukocyte chromosomes as a potential biomarker for ischemic stroke (IS) risk have been reported by the various observational studies in previous years. This study aims to investigate whether the leukocyte telomere length is associated with an increased IS risk or not, based on the Mendelian randomization (MR) approach.MethodsBased on the NHGRI-EBI GWAS Catalog database, the Chinese online genetic database as well as the previous published studies, twelve single nucleotide polymorphisms (SNPs) with minor allele frequency ≥ 0.05 were selected and the leukocyte telomere length was measured in 431 first-ever IS patients and 304 healthy controls (quantitative polymerase chain reaction). To explore linear and non-linear effect of telomere length on the IS risk, we preformed the linear MR analysis (the inverse-variance weighted method, the maximum likelihood method, and the mode-based estimation method), and the non-linear MR analysis (semiparametric method with three tests for non-linearity, including the quadratic test, Cochran’s Q test, and the fractional polynomial test).ResultsTwo verified SNPs (rs11125529 and rs412658) were chosen as instrumental variables. In linear MR analysis, the adjusted odds ratios and 95% confidence intervals of IS for genetically predicted telomere lengths, based on the two SNPs, were 1.312 (0.979 to 1.759), 1.326 (0.932 to 1.888) and 1.226 (0.844 to 1.781) for the inverse-variance weighted method, the maximum likelihood method, and the mode-based estimation method, respectively. Three tests for nonlinearity failed to reject the null exactly, indicating that the relationship between telomere length and IS risk is unlikely to be non-linear.ConclusionThis MR study based on individual data does not provide strong evidence for a positive linear or non-linear effect of telomere length on the IS risk.

Mosaic Y Loss Is Moderately Associated with Solid Tumor Risk.

Mosaic loss of the Y chromosome (mLOY) in peripheral leukocytes is a somatic event in which a fraction of leukocytes have lost the entire Y chromosome. The frequency of mLOY increases with age and may reflect poor genomic maintenance as well as clonal imbalances in normal immune function, making mLOY an attractive candidate marker for cancer risk. Here, we investigated the relationship between mLOY and incident cancer in a large sample of 207,603 cancer-free men from the UK Biobank, in which 13,895 men developed an incident solid tumor during follow-up. We identified mLOY by scanning for deviations in genotyping array log R intensity ratios across the male-specific chromosome Y region. Overall, we detected low proportions of cells with mLOY in 3,358 (1.6%) men and high proportions of mLOY in 524 (0.3%) men. We found an association of mLOY with overall solid tumor incidence using both low and high mLOY thresholds [HRlow = 1.18; 95% confidence interval (CI)low, 1.07-1.30; P low = 0.001; HRhigh = 1.36; 95% CIhigh, 1.09-1.71; P high = 0.007] and more specifically we observed an association with lung cancer (HRhigh = 2.25; 95% CIhigh, 1.36-3.71; P high = 0.002). Stronger associations were observed without adjustment for smoking, suggesting that smoking is an important confounder of tumor incidence. It is unlikely that mLOY is a major mediator of the effect of cigarette smoking on cancer risk, as mLOY was observed in only a small fraction of smokers who developed cancer. In summary, mLOY was modestly associated with incidence of solid tumors in the UK Biobank, although for some cancer subtypes these findings may reflect residual confounding by smoking. SIGNIFICANCE: Evidence from the UK Biobank indicates mosaic chromosome Y loss in leukocytes is moderately associated with increased incidence of select solid tumors.

Abstract 3379: Predictors of mosaic chromosome Y loss and associations with mortality in 223,338 men of the UK Biobank

Abstract Mosaic loss of the Y chromosome (mLOY) in peripheral leukocytes is an age-related event commonly observed in men older than 50 years of age in which a fraction of leukocytes loses the Y chromosome. In addition to age, previous studies have indicated an association between smoking and increased risk of mLOY. The relationship between mLOY and other health-related exposures (e.g., obesity and alcohol consumption) and with disease outcomes (e.g., cancer risk and overall mortality) remains unclear. We therefore investigated the causes and consequences of mLOY in a large collection of 223,338 men from the UK Biobank. We scanned Y chromosomes for deviations in genotyping array log R ratio intensities for evidence of mLOY. A total of 3,789 (1.7%) men showed evidence for mLOY with median chromosome Y LRR values less than -0.15 and 596 (0.3%) men had high proportions of cells affected with mLOY with median LRR values less than -0.40. Our analysis robustly confirms prior associations of increasing mLOY risk with increasing age (Padj<4.9×10-324). In addition, we observe a strong association between mLOY and smoking (Padj=7.1×10-193), with a higher percentage of current smokers (3.4%) affected by mLOY than former smokers (2.1%) and non-smokers (0.9%). As reported previously for autosomal mosaicism, we observed less mLOY in men of African ancestry (0.4%) compared to men of European ancestry (1.8%, Padj=0.002). Surprisingly, obese men were less likely to have mLOY, with a lower percentage of men with a BMI≥35 having mLOY than men in the normal range (1.3% vs 1.7%, Padj=0.0007). Self-reported health was also associated with mLOY, with a higher percentage of mLOY among men reporting poor health than men reporting excellent health (2.1% vs 1.4%, Padj=0.008). Although no associations were observed between mLOY and prevalent non-melanoma skin cancer (Padj=0.54), suggestive associations were observed with prevalent diabetes (Padj=0.003) and prevalent cardiovascular disease (Padj=0.01). With respect to mortality, we observed an association with mLOY in men with high proportions of cells affected only (LRR<-0.40, Padj=0.002). By cause of death, we noted an association for cancer-specific mortality (HR=1.53, 95% CI=1.13-2.06, Padj=0.0056) in men with high proportions of cells affected, but not for cardiovascular disease mortality, although the number of deaths among men with mLOY was modest (LRR<-0.15= 334 deaths, LRR<-0.40= 75 deaths). Overall, we see evidence for an association of mLOY with several health-related exposures as well as with mortality among men with high proportions of cells affected. Future functional studies related to the physiologic effects of chromosome Y loss in circulating leukocytes are needed to better understand how mLOY may impact disease risk. Citation Format: Erikka Loftfield, Weiyin Zhou, Meredith Yeager, Stephen J. Chanock, Neal D. Freedman, Mitchell J. Machiela. Predictors of mosaic chromosome Y loss and associations with mortality in 223,338 men of the UK Biobank [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3379.

The influence of oxidative stress induced by iron on telomere length.

Oxidative stress can be induced by increased concentrations of iron in the body and consequently can cause shortening of telomeres. Telomeres, called mitotic clocks, are non-coding fragments at the end of chromosomes. During the replication of genetic material they are shortened, playing the role of ageing biomarkers in eukaryotes. In human endothelial cells, oxidative stress causes a decrease in telomerase activity. Shortening of chromosomes in telomeric parts was found in patients with primary hemochromatosis and in patients taking supplements containing iron. Increased level of transferrin saturation is associated with the presence of shorter telomeres in the chromosomes of leukocytes. The relationship between iron status and telomere length is still not fully understood.

Primary biliary cirrhosis and autoimmunity: molecules and mimics

There have been significant advances both in humans and experimental models that relate to the etiopathogenesis of primary biliary cirrhosis. Many of these advances are based on the rigorous definition of the antimitochondrial response, the serologic signature of PBC. First, it is well established that AMA are directed against members of the 2-oxoacid dehydrogenase complexes (2-OADC), among which the major epitopes are within the lipoylated domains of the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2). Second, autoreactive CD4+ and CD8+ T cells can be detected in PBC peripheral blood, regardless of the AMA status, and the infiltration of autoreactive T cells in the liver and periductular spaces is one of the most prominent immune features. Autoreactive T cells of both subtypes recognize PDC-E2 sequences overlapping with the AMA epitopes. An increase in cytotoxic T cell precursors in the blood in the early stages of the disease compared to the advanced ones and a 10-fold increase of specific liver CD8+ T cells compared to peripheral blood have been demonstrated. Third, additional data on the immunobiology components of PBC autoimmunity has been recently obtained in CD4+CD25 high natural regulatory T cells which appear to be numerically reduced in PBC. PBC bile duct cells manifest unique features during apoptosis while co-culture experiments do not support a direct role for these cells in determining their immune –mediated injury. Apoptotic cells are phagocytosed by BECs and consequently are an exogenous source of autoantigens in cholangiocytes, possibly through anti-CD16. As a result, the impact of putative changes in apoptosis and autophagy specific to BEC remains to be fully determined in PBC. Fifth, the innate immune compartment has been recently investigated in PBC with promising results. PBC monocytes manifest an increased response to pathogen associated stimuli, as indicated by higher levels of pro-inflammatory cytokines. Further, the hyper-IgM associated with PBC is secondary to an aberrant innate immune response, potentially induced by stimulation of toll like receptor 9 by bacterial CpG-B. The female preponderance may hold an important key to PBC etiology. X-linked genes determine gender-related characteristics at different levels while also regulating the immune function, particulalry to maintain tolerance. Major X chromosome defects such as those leading to Turner’s syndrome or premature ovarian failure are commonly characterized by autoimmune comorbidities (particularly thyroid disease) and, less frequently, cholestasis. Our group first determined a significantly higher frequency of monosomy of the X chromosome in peripheral leukocytes (particularly those of the adaptive immune response, i.e. T and B cells) in women PBC compared to age-matched control women. Monosomy frequency correlated with age in all three groups, as expected but monosomic cells were not microchimeric cells. We further demonstrated that the X loss in PBC affected was not random but affected more frequently one parentally-inherited chromosome. Several key animal models of autoimmune cholangitis have now been described. First, a genomic variant of the non obese diabetic (NOD) mouse (NOD.c3c4) has been observed to manifest autoimmune cholestasis with AMA and ANA positivities in 50%–60% and 80%–90%, respectively. Liver histology demonstrated portal lymphocyte infiltration with chronic non-suppurative cholangitis and PBC-like granulomas. Second, a dominant negative form of trasforming growth factor β (TGFβ) receptor II (dnTGFβRII) mouse develop serum AMA in 100% of mice. The TGFβ receptor II regulates lymphocyte activation and the appearance of PBC in this model suggests a specific condition of T cells with impaired TGFβ signaling in the presence or absence of B cells is involved. Third, the knockout of interleukin 2 receptor α leads to a murine phenotype with 100% serum AMA positivity, 80% serum ANA positivity, and portal lymphocyte infiltration and vanishing bile ducts. This model is of particular interest based on the report of autoimmune cholangitis in a pediatric case of IL2Rα deficiency. Fourth, Ae2a,b also develop autoimmune phenomenon and a PBC-like disease. Finally, immunization of mice with chemical xenobiotics has also been shown to lead to a PBC-like disease. These data and observations will be put in the context of the key mechanisms, including the role of TLRs in modulating these responses.

Genome instability in AZFc region on Y chromosome in leukocytes of fertile and infertile individuals following exposure to gamma radiation

Men are exposed to various doses of ionizing radiation due to living in regions with high natural background radiation, accidentally, occupationally or for cancer treatment. To study genomic instability of AZFc region to gamma radiation, blood samples from normal, oligozoospermia, and azoospermia individuals were irradiated by a Co-60 source. Irradiated cells were kept for 48 h in order to repair initial DNA damages. Real time PCR was performed for three markers (SY 1206, SY 1197, SY 579) for testing copy number variation before and after irradiation. Copy number variations were compared by calculation of cycle threshold comparative method. Copy number variations of studied markers in AZFc region (microdeletion and duplication) in all samples after exposure to radiation increased with a dose dependent fashion. The frequency of instability was significantly higher in samples from infertile men in comparison with fertile ones (p < 0.001). No significant difference was seen between the two infertile groups (p > 0.05). This observation might be a possible explanation for induction of azoospermia and oligozoospermia after radiotherapy. Increased frequency of induced microdeletion and duplication in infertile men compared with normal might be attributed to the deficiency in repair systems and the genetic factors involved in incomplete spermatogenesis of infertile men.

The effect of deoxyadenosine and cytosine arabinoside on the chromosomes of human leukocytes in vitro

The effect of deoxyadenosine and cytosine arabinoside on the chromosomes of human leukocytes in vitro

Chromosome damage in human cells induced by hydroxylamine

The action of hydroxylamine upon human leukocyte chromosomes has been studied as regards the amount of the different morphological types of damage. Constrictions and gaps predominate, whereas few breaks and very few exchanges are observed. The distribution of the aberrations among the chromosomes is non-random. The observations indicate that the important reaction of HA as regards chromosome damage is with the protein moiety of the chromosome. HA does not induce unscheduled DNA synthesis in human leukocytes or HeLa cells.

Leukocytes of exceptionally old persons display ultra-short telomeres

With a view to understanding the association between leukocyte telomere length and the human lifespan, we performed genome-wide telomere length analyses by the terminal restriction fragment length (TRFL) and single molecule telomere length analysis (STELA) of the X and Y chromosomes in leukocytes of exceptionally old (aged 90-104 yr) and younger (aged 23-74 yr) individuals. We found that the mean TRFL of 82 exceptionally old individuals was within a range projected by age-dependent TRFL attrition of 99 younger individuals. However, compared with the younger individuals, exceptionally old persons exhibited peaking of the TRFL distribution with overrepresentation of ultra-short telomeres. These findings were confirmed by the STELA. Women had longer mean TRFL than men (6.10 vs. 5.86 kb), and exceptionally old women exhibited fewer ultra-short telomeres than exceptionally old men. Our results have implications for gerontological studies of the limitation of lifespan in humans.

Age related rejoining of broken chromosomes in human leukocytes following x-irradiation

One theory of aging is that the ability to repair DNA or chromosome damage decreases with age. This study made use of a method which allows the measurement of chromatid repair in interphase nuclei, the technique of premature chromosome condensation. The number of excess chromosome fragments per cell remaining after 24 h of incubation at 37°C was determined and used as a measure of the ability of leukocytes to repair. The leukocytes from male donors of several ages were used and the cells from older donors showed greater residual damage and, therefore, less repair.

Bullous pemphigoid antigen (BPAG1): cDNA cloning and mapping of the gene to the short arm of human chromosome 6

Bullous pemphigoid antigen (BPAG1), an integral component of the cutaneous basement membrane zone, serves as autoantigen in a blistering disease, bullous pemphigoid. In this study, we have generated cDNAs corresponding to human BPAG1 sequences. Two cDNAs, a 0.45-kb PCR product synthesized with human keratinocyte RNA as template and a 2.2-kb cDNA isolated from human keratinocyte λt11 library, were utilized for chromosomal in situ hybridizations to establish the genomic location of the BPAG1 gene. Metaphase chromosomes of phytohemagglutinin-stimulated human peripheral blood leukocytes were examined by hybridizations with 3H-labeled cDNAs, and the chromosomes were identified by R-banding (fluorochrome-photolysis-Giemsa method). The results indicated that the human BPAG1 gene is at locus 6p11-6p12. This conclusion was supported by hybridizations with a panel of human × rodent hybrid cell DNA, which indicated concordance with human chromosome 6. Because different genes encoding human basement membrane components have been mapped previously to chromosomes other than 6, the results further indicate that human basement membrane zone genes are widely dispersed within the human genome.

Videomicroscopic Comparison of Bull Sperm and Leukocyte Chromosome Areas as Related to Gender

Chromosomal areas from metaphase spreads of male bovine leukocytes were digitized and sex chromosomes identified using videomicroscopy. Autosomal areas were ranked in descending order within a cell and assigned to two categories based on alternating rank. X and Y chromosome areas were assigned to respective categories. Areas were divided by 4 to make their sum equivalent to sperm DNA content. Data were analyzed before and after inclusion of sex chromosomal areas. Before X and Y inclusion, rank contributed to difference in chromosomal areas. Rank by category interaction and category effects did not contribute to area variation. After X and Y inclusion, area variation was due to rank by category interaction, rank, and category. Differences between sums of chromosomal areas across categories was 3.57%. Head areas of morphologically normal sperm with intact acrosomes were digitized using the same optics as chromosomal areas. Sum of corrected chromosomal areas per category was used in discriminant analysis to assign sperm head areas to two categories with .5 prior probabilities. Assignment resulted in 1037 sperm in one category and 1177 in the other. Difference between largest sperm head area classes across categories was 3.2%. Discriminatian of sperm head areas, based on sum of chromosomal area and measured with computerized videomicroscopy, may be used to evaluate sex of bovine spermatozoa.