Alternative splicing of the tyrosinase gene transcript in normal human melanocytes and lymphocytes.

We have identified and isolated ectopically expressed tyrosinase transcripts in normal human melanocytes and lymphocytes and in a human melanoma (MNT-1) cell line to establish a baseline for the expression pattern of this gene in normal tissue. Tyrosinase mRNA from human lymphoblastoid cell lines was reverse transcribed and amplified using specific "nested" primers. This amplification yielded eight identifiable transcripts; five that resulted from alternative splicing patterns arising from the utilization of normal and alternative splice sequences. Identical splicing patterns were found in transcripts from human primary melanocytes in culture and a melanoma cell line, indicating that lymphoblastoid cell lines provide an accurate reflection of transcript processing in melanocytes. Similar splicing patterns have also been found with murine melanocyte tyrosinase transcripts. Our results demonstrate that alternative splicing of human tyrosinase gene transcript produces a number of predictable and identifiable transcripts, and that human lymphoblastoid cell lines provide a source of ectopically expressed transcripts that can be used to study the biology of tyrosinase gene expression in humans.

Mutations in the human orthologue of the mouse underwhite gene (uw) underlie a new form of oculocutaneous albinism, OCA4.

Oculocutaneous albinism (OCA) affects approximately 1/20,000 people worldwide. All forms of OCA exhibit generalized hypopigmentation. Reduced pigmentation during eye development results in misrouting of the optic nerves, nystagmus, alternating strabismus, and reduced visual acuity. Loss of pigmentation in the skin leads to an increased risk for skin cancer. Two common forms and one infrequent form of OCA have been described. OCA1 (MIM 203100) is associated with mutations of the TYR gene encoding tyrosinase (the rate-limiting enzyme in the production of melanin pigment) and accounts for approximately 40% of OCA worldwide. OCA2 (MIM 203200), the most common form of OCA, is associated with mutations of the P gene and accounts for approximately 50% of OCA worldwide. OCA3 (MIM 203290), a rare form of OCA and also known as "rufous/red albinism," is associated with mutations in TYRP1 (encoding tyrosinase-related protein 1). Analysis of the TYR and P genes in patients with OCA suggests that other genes may be associated with OCA. We have identified the mouse underwhite gene (uw) and its human orthologue, which underlies a new form of human OCA, termed "OCA4." The encoded protein, MATP (for "membrane-associated transporter protein") is predicted to span the membrane 12 times and likely functions as a transporter.

Psychosocial and risk behavior correlates of youth suicide attempts and suicidal ideation.

OBJECTIVE: To identify the independent psychosocial and risk behavior correlates of suicidal ideation and attempts. METHOD: The relationships between suicidal ideation or attempts and family environment, subject characteristics, and various risk behaviors were examined among 1,285 randomly selected children and adolescents, aged 9 through 17 years, of whom 42 (3.3%) had attempted suicide and 67 (5.2%) had expressed suicidal ideation only. The youths and their parents were enumerated and interviewed between December 1991 and July 1992 as part of the NIMH Methods for the Epidemiology of Child and Adolescent Mental Disorders (MECA) Study. RESULTS: Compared with subjects with suicidal ideation only, attempters were significantly more likely to have experienced stressful life events, to have become sexually active, to have smoked more than one cigarette daily, and to have a history of ever having smoked marijuana. After adjusting for sociodemographic characteristics, a statistically significant association was found between suicidal ideation or attempt and stressful life events, poor family environment, parental psychiatric history, low parental monitoring, low instrumental and social competence, sexual activity, marijuana use, recent drunkenness, current smoking, and physical fighting. Even after further adjusting for the presence of a mood, anxiety, or disruptive disorder, a significant association persisted between suicidal ideation or attempts and poor family environment, low parental monitoring, low youth instrumental competence, sexual activity, recent drunkenness, current smoking, and physical fighting. CONCLUSION: Low parental monitoring and risk behaviors (such as smoking, physical fighting, alcohol intoxication, and sexual activity) are independently associated with increased risk of suicidal ideation and attempts, even after adjusting for the presence of psychiatric disorder and sociodemographic variables.

Risk behavior in a community sample of children and adolescents.

OBJECTIVES: First, to investigate whether there is covariation between risk behaviors, including suicidality, in a community probability sample of children and adolescents; and second, to investigate whether risk behavior is associated with selected potential correlates. METHOD: A sample of 9- to 17-year-old youths (N = 1,285) and their caretakers were interviewed in the Methods for the Epidemiology of Child and Adolescent Mental Disorders (MECA) Study. The risk behaviors were marijuana smoking, alcohol use, intercourse, fighting, cigarette smoking, and suicidal ideation/attempts. Relationships between the risk behaviors were described using odds ratios. Linear regression analyses of an index of risk behavior on the selected potential correlates of risk behavior were conducted. RESULTS: There were significant relationships between all pairs of risk behaviors. The score on the index of risk behavior was associated with stressors, lack of resources, family psychiatric disorder, psychopathology, and functional impairment. CONCLUSIONS: Clinicians should be alerted to the possibility of risk behaviors, especially in children and adolescents engaging in other risk behaviors and those with inadequate resources, stressors, functional impairment, or psychopathology.

BRCA1 susceptibility markers and postmenopausal breast cancer: the Iowa Women's Health Study.

Much research on early-onset breast cancer families has been performed and has shown that breast cancer in many of these families is linked to either BRCA1 or BRCA2. Fewer studies have examined the role of genetic predisposition in postmenopausal breast cancer. A nested case-control family study of breast cancer was conducted within the Iowa Women's Health Study, a population-based prospective study of 41,836 postmenopausal women. Probands were 251 incident cases diagnosed between 1988 and 1989. Three-generation pedigrees were developed through mailed questionnaires. From this collection of pedigrees, thirteen were identified for more detailed genetic analysis. Sibling-pair linkage analyses were performed using polymorphic markers in candidate regions in these 13 families with multiple cases of breast and other cancers. Four of the DNA markers are located on chromosome 17, and two of these (D17S579 and THRA1) flank the BRCA1 locus. Significant evidence for linkage to D17S579 was obtained in the total sample, in a model without inclusion of covariates or age at onset (P = 0.005), and in a model adjusted for five measured covariates and for variable age at onset (P = 0.008). Complete sequencing of the BRCA1 gene in these families, including all intron/exon boundaries, failed to reveal any mutations in 24 women with breast cancer from the 13 families. These data suggest that in some families identified by postmenopausal breast cancer cases, breast cancer risk may be mediated by a gene (or genes) in the BRCA1 region, but not BRCA1 itself.

Fifty-year follow-up of cancer incidence in a historical cohort of Minnesota breast cancer families.

A family history of breast cancer is well established as a risk factor for the disease. Because family history is a dynamic rather than a static characteristic, longitudinal studies of entire families can be very instructive in quantifying the significance of risk classification. The Minnesota Breast Cancer Family Study is a historical cohort study of relatives of a consecutive series of 426 breast cancer cases (probands) identified between 1944 and 1952. The incidence of cancer and the measurement of risk factors in sisters, daughters, granddaughters, nieces, and marry-ins was determined through telephone interviews and mailed questionnaires. Ninety-eight percent of eligible families were recruited, and 93% of members participated. A total of 9073 at-risk women were studied: 56% were biological relatives of the case probands, whereas the others were related through marriage. Through 1996, 564 breast cancers were identified in nonprobands. Compared to the rate of breast cancer among marry-ins (188 cases), sisters and daughters of the probands were at a 1.9-fold greater age-adjusted risk (128 cases; 95% confidence interval, 1.4-2.4); granddaughters and nieces were at a 1.5-fold greater risk (248 cases, 95% confidence interval, 1.2-1.8). The breast cancer risk since 1952 was not distributed equally across families: although all biological relatives had a family history of breast cancer, 166 families (39%) experienced no additional cases. Most of the cases occurred among a subset of families: 21 families had 5 breast or ovarian cancers, 8 had 6, 2 had 7, and 4 had > or =8. There was no evidence of significantly increased risk for cancer at other sites, including the ovaries, cervix, uterus, colon, pancreas, stomach, or lymphatic tissue, although there was some evidence that stomach cancer in previous generations may help define the susceptible subset. These families contain four to five generations of validated occurrences of cancer, thus minimizing the uncertainty of genetic risk inherent in a disease with a late and variable age at onset. The patterns of breast cancer in these multigeneration families is consistent with the influence of autosomal dominant susceptibility in a subset, low penetrance genes in another, and purely environmental influences in the remainder.

Genetic anticipation and breast cancer: a prospective follow-up study.

Genetic anticipation is characterized by an earlier age of disease onset, increased severity, and a greater proportion of affected individuals in succeeding generations. The discovery of trinucleotide repeat expansion (TRE) mutations as the molecular correlate of anticipation in a number of rare Mendelian neurodegenerative disorders has led to a resurgence of interest in this phenomenon. Because of the difficulties presented to traditional genetics by complex diseases, the testing for genetic anticipation coupled with TRE detection has been proposed as a strategy for expediting the identification of susceptibility genes for complex disorders. In the case of breast cancer, a number of previous studies found evidence consistent with genetic anticipation. It is known that a proportion of such families are linked to either BRCA1 or BRCA2, but no TRE mutations have been identified. It has been shown that the typical ascertainment employed in studies purporting to demonstrate genetic anticipation combined with unadjusted statistical analysis can dramatically elevate the type I error. We re-examine the evidence for anticipation in breast cancer by applying a new statistical approach that appears to have validity in the analysis of anticipation to data ascertained from a recent follow-up of a large prospective cohort family study of breast cancer. Using this approach, we find no statistically significant evidence for genetic anticipation in familial breast cancer. We discuss the limitations of our analysis, including the problem of adequate sample size for this new statistical test.

Inclusion of risk factor covariates in a segregation analysis of a population-based sample of 426 breast cancer families.

Although many segregation analyses of breast cancer have been published, few have included risk factor covariates. Maximum likelihood segregation analyses examining age-at-onset (model 1) and susceptibility (model 2) models of breast cancer were performed on 426 four-generation families originally ascertained between 1944 and 1952 through a breast cancer proband. Cancer status and risk factor data were collected through interviews of participants or surrogates. When segregation analyses were performed on 10,791 women, without estimation of any covariates, all hypotheses under both models were rejected. Model 1, which required estimation of fewer parameters than model 2, provided a better fit to the data according to Akaike's Information Criterion. Further segregation analyses were performed under model 1 on a subset of women with complete data on education, age at first birth (nulliparous women included), and alcohol use, covariates that were found to significantly (P<0.05) improve the fit over the addition of exam age alone in logistic regression models. All three covariates improved the fit of the models, as did year of birth, but at all stages of model building, all of the hypotheses were still rejected. After the allele frequency was fixed at 0.0033, a subset of families appeared to fit a dominant model. Using this model, risk estimates were calculated based on inferred genotype, age, and covariate values. The penetrance was estimated to be 0.15, much lower than previous estimates based on families ascertained through breast cancer probands with early onset. Moreover, the estimates of penetrance were not greatly influenced by incorporation of the measured risk factors.

Does tyrosinase exist in neuromelanin-pigmented neurons in the human substantia nigra?

It is controversial whether tyrosinase is involved in the neuromelanin-biosynthetic pathway. We examined tyrosinase-immunoreactivity in human substantia nigra neurons which contain neuromelanin pigments, using antibodies against human tyrosinase and human tyrosine hydroxylase. In human melanoma, the antibody to tyrosinase showed intense immunoreactivity while there was no immunoreactivity with antibody to tyrosine hydroxylase. In the human midbrain pigmented neurons, however, we could detect no tyrosinase-immunoreactivity while the neurons were strongly immunoreactive to tyrosine hydroxylase. The present results suggest that tyrosinase is not involved in the main pathway of neuromelanin biosynthesis.

Identification of a novel transcript produced by the gene responsible for the Hermansky-Pudlak syndrome in Puerto Rico.

Hermansky-Pudlak Syndrome (HPS) is a rare, autosomal recessive disorder that is characterized by oculocutaneous albinism, a predisposition to mild bleeding caused by storage-pool deficient platelets, and a ceroid storage disorder. A gene responsible for HPS in Puerto Rico maps to chromosome 10q2 and isolation of the gene has been reported. We have now identified a variant HPS cDNA that contains the same 5' sequence as the published HPS gene and a unique 3' sequence. Analysis of genomic DNA suggests that the two cDNA are derived from alternative transcripts of a single gene; two polyadenylated transcripts were found in normal human melanocytes, human bone marrow cells, human melanoma cells, lymphoblastoid cell lines, and megakaryocytic leukemia cells by reverse transcriptase polymerase chain reaction and northern analysis. The splicing exhibited by this gene is identical to the splicing found to produce two alternative transcripts of the Chediak-Higashi Syndrome gene, another pigment disorder exhibiting platelet storage pool deficiency. These studies show that the HPS gene on chromosome 10 is complex and may have more than one biologically active transcript.

Plasma and urinary kinetics of the isoflavones daidzein and genistein after a single soy meal in humans.

The aim of this study was to determine the pharmacokinetics and urinary excretion patterns of the soy isoflavones daidzein and genistein in humans. Six healthy men with a mean age of 37 y and a mean body mass index (in kg/m2) of 24 consumed a soybean flour-based meal on two occasions approximately 6 d apart. Blood samples and total urine were collected at intervals for the measurement of daidzein and genistein with HPLC. Isoflavone concentrations rose slowly and reached maximum values of 3.14 +/- 0.36 micromol/L at 7.42 +/- 0.74 h for daidzein and 4.09 +/- 0.94 micromol/L at 8.42 +/- 0.69 h for genistein. Elimination half-lives were 4.7 +/- 1.1 and 5.7 +/- 1.3 h for daidzein and genistein, respectively. The slow increase in plasma concentrations is consistent with the facilitation of absorption by hydrolysis in the small and large intestines of the glycosidic forms of the isoflavones present in soybean-containing foods to their corresponding aglycones. The rate of urinary excretion of daidzein was greater than that of genistein throughout the postmeal period, with mean recoveries of 62 +/- 6% and 22 +/- 4% (P < 0.001) for daidzein and genistein, respectively. However, the ratio of the areas under the plasma concentration versus time curves for genistein and daidzein was equal to the ratio of the concentrations of the respective isoflavones in the soy meal. It is concluded that the bioavailabilities of daidzein and genistein are similar, not withstanding the difference in urinary excretion.

Genistein inhibits growth of B16 melanoma cells in vivo and in vitro and promotes differentiation in vitro.

Consumption of soy products has been linked to a reduced mortality and morbidity from a number of cancers. Genistein, one of the principal soy isoflavones, has been shown to inhibit the growth of a number of tumour cell lines in vitro; however, a role of genistein in retarding tumour growth in vivo is less well documented. In this study, in addition to examining the effects of genistein on the growth of murine B16 melanoma cells in vitro, we have examined the effects of feeding a genistein-rich diet on s.c. growth of these tumour cells in mice. In vitro, the melanoma cells showed an increase in sensitivity to genistein with increasing time of exposure, culminating in a 50% growth inhibition (IC50) at 12.5 microM after 7 days. Genistein at 25 microM induced micronucleus formation after 24 hr and at concentrations as low as 2.5 microM induced morphological changes indicative of differentiation. Growth of solid tumours implanted into female C57BL/6J mice was inhibited by 50% when mice were fed genistein for 1 week before and for 1 week after inoculation with B16 melanoma cells. Plasma genistein concentrations at the time of tumour removal were 1.1 microM, which is similar to levels reported in humans consuming diets high in soybeans or soybean products, while control animals had no detectable genistein in plasma. Our results provide additional in vivo evidence suggesting that genistein retards the growth of implanted tumours, adding further to studies suggesting that this isoflavonoid is a biologically active component of soy foods.

Genetic analysis of mammographic breast density in adult women: evidence of a gene effect.

BACKGROUND: The appearance of the female breast viewed by mammography varies considerably from one individual to another because of underlying differences in the relative proportions of fat, connective tissue, and glandular epithelium that combine to create a characteristic pattern of breast density. An association between mammographic patterns and family history of breast cancer has previously been reported. However, this association has not been found in all studies, and few data are available on possible genetic components contributing to mammographic breast density. PURPOSE: Our purpose was to estimate familial correlations and perform complex genetic segregation analyses to test the hypothesis that the transmission of a major gene influences mammographic breast density. METHODS: As part of a cohort study (initiated in 1944) of families with a history of breast cancer, the probands' female relatives who were older than 40 years were asked to obtain a routine mammogram. The mammograms of 1370 women from 258 independent families were analyzed. The fraction of the breast volume occupied by radiographically dense tissue was estimated visually from video displays of left or right mediolateral oblique views by one radiologist experienced in mammography who had no knowledge of individual relationships to the probands. Data on breast cancer risk factors were obtained through telephone interviews and mailed questionnaires. Unadjusted and adjusted familial correlations in breast density were calculated, and complex genetic segregation analyses were performed. RESULTS: Sister-sister correlations in breast density (unadjusted and adjusted for age and either body mass index, menopausal status, hormone replacement therapy, waist-to-hip ratio, number of live births, alcohol consumption, or cigarette smoking status) were all statistically significant (r = .16-.27; all P<.05 [two-sided]). Estimated mother-daughter correlations were smaller in magnitude (r = .01-.17) and not statistically significant. Segregation analyses indicate that a major autosomal gene influences breast density. The mendelian transmission of a dominant gene provided the best fit to the data; however, hypotheses involving the inheritance of either a recessive gene or a codominant gene could not be ruled out. The mendelian dominant hypothesis, accounting for 29% of the variability in breast density, suggests that approximately 12% of the population would be expected to carry at least one variant allele of this putative gene. Women who inherit the variant allele would have a mean breast density about twice that of the rest of the population. CONCLUSIONS: Our preliminary findings suggest that, in this cohort of women at risk of breast cancer, mammographic breast density may be genetically influenced.

Obsessive-compulsive characteristics: from symptoms to syndrome.

OBJECTIVE: To assess the distribution and severity of obsessions and compulsions in a nonclinical adolescent population. METHOD: During preinduction military screening, 861 sixteen-year-old Israelis completed a questionnaire regarding the lifetime presence of eight obsessive-compulsive (OC) symptoms and three severity measures. The presence or absence of obsessive-compulsive disorder (OCD) or subclinical OCD was ascertained by an independent interview. RESULTS: Although only 8.0% and 6.3% of respondents reported disturbing and intrusive thoughts, respectively, 27% to 72% of subjects endorsed the six remaining OCD symptoms. Twenty percent of subjects regarded the symptoms they endorsed as senseless and 3.5% found them disturbing; 8% reported spending more than an hour daily on symptoms. OCD and subclinical OCD cases differed significantly from non-OCD cases, but not from each other, in distress and mean number of symptoms. Although the distribution of nine of the items differed for noncases, compared with OCD and subclinical OCD cases, the distributions for all items overlapped markedly across the three groups. CONCLUSIONS: OC phenomena appear to be on a continuum with few symptoms and minimal severity at one end and many symptoms and severe impairment on the other. Defining optimal cutoff points for distinguishing between psychiatric disorder and OC phenomena that are common in the general population remains an open question.

Segregation analysis of breast cancer: a comparison of type-dependent age-at-onset versus type-dependent susceptibility models.

Most segregation analyses of breast cancer susceptibility have modeled the effect of the major gene on the age-at-onset distribution. However, in families linked to BRCA1 or BRCA2, there is wide variation in the age-at-onset among gene carriers. We performed a segregation analysis of 544 Minnesota breast cancer families using models which parameterized the putative major gene effect in two ways: earlier age-at-onset, with a common level of susceptibility (model I), and greater susceptibility, with a common mean age-at-onset (model II). Five hypothetical modes of transmission and an unrestricted general hypothesis were fitted to the data. Twice the difference between the log(e) likelihood for the data under the specified hypothesis (recessive, no major gene, etc.) and the log(e) likelihood under the general hypothesis is distributed asymptotically as a chi-square statistic with the degrees of freedom equal to the difference in the number of parameters estimated. This difference was compared to the critical value for the chi-square distribution to assess goodness-of-fit. Under model I, both Mendelian and non-Mendelian hypotheses were rejected. When model II was used, the non-Mendelian hypotheses were rejected whereas all Mendelian hypotheses were not. Mendelian recessive inheritance of a common allele (qA = 0.11) with a high penetrance (87%) provided the best fit to the data. We then stratified the families into two subsets based on the age at diagnosis of the proband [< or = 55 years (n = 265) versus > 55 years (n = 279)]; there was no evidence of heterogeneity under either model (I or II). These data suggest that, in some breast cancer families, the effect of the putative susceptibility gene is better represented as increasing overall susceptibility to breast cancer rather than as a shift in the age-at-onset distribution.

Absorption and excretion of the soy isoflavone genistein in rats.

Rodent models have been used to study the anticarcinogenic properties of the soy isoflavones, particularly genistein, but there is little information regarding the pharmacokinetics of the absorption and excretion of genistein. In this study, rats were given a single oral dose of genistein (20 mg/kg body weight) or an equivalent dose of its glycone forms, as an isoflavone-rich soy extract. Concentrations of genistein were measured in plasma, urine and feces at intervals up to 48 h after dosing. Plasma genistein concentration at 2 h after dosing was 11.0 +/- 2.3 mumol/L in genistein-treated rats compared with 4.93 +/- 0.22 mumol/L (P = 0.025) in soy extract-treated rats, but there were no significant differences at 8 h and later times. The mean urinary excretion rate during the first 2 h after dosing was more than 10 times higher in the genistein group compared with the soy extract group (0.27 +/- 0.08 mumol/h and 0.020 +/- 0.011 mumol/h, respectively, P = 0.017) but the percentage of dose recovered in urine over 48 h was not different between groups (19.9 +/- 2.4% genistein treated; 17.5 +/- 1.1% soy extract treated). There were no significant differences between groups in the recovery of genistein in feces (21.9 +/- 2.8% and 21.1 +/- 2.5% of dose, respectively). Only 6.1 +/- 0.9% of the daidzein from the soy extract was recovered in the feces. The results suggest that the extent of absorption of genistein is similar for the glycone and aglycone forms. Although higher initial plasma concentrations may be achieved with the aglycone, similar long-term concentrations exist for both forms of isoflavone.

Induction of micronucleus formation in mouse splenocytes by the soy isoflavone genistein in vitro but not in vivo.

The effects of genistein (one of the major soybean isoflavones), genistein (the glucosylated form of genistein) and etoposide (a topoisomerase 11 inhibitor) have been studied in mouse splenocytes in culture. Genistein (25 microM), genistein (25 microM) and etoposide (0.1 microM) all induced the production of large numbers of micronuclei; however, genistein at 12.5 or 2.5 microM had no clastogenic effect. In a second study, mice were gavaged with 20 mg genistein/kg body weight/day for 5 days (approximately equivalent to a 70 kg human consuming 2.8 kg soybeans/day) and the micronucleus frequency was determined. There was no observable increase in the micronucleus frequency even though the plasma genistein levels in the treated animals were found to be 9.2 +/- 2.0 microM compared with 0.1 +/- 0.0004 microM in the control animals. The results show that even though genistein is capable of inducing micronucleus formation, an event associated with genetic damage, plasma levels are unlikely to be sufficiently elevated to produce such an effect.