Differences in SMN1 allele frequencies among ethnic groups within North America.

BACKGROUND: Spinal muscular atrophy (SMA) is the most common inherited lethal disease of children. Various genetic deletions involving the bi-allelic loss of SMN1 exon 7 are reported to account for 94% of affected individuals. Published literature places the carrier frequency for SMN1 mutations between 1 in 25 and 1 in 50 in the general population. Although SMA is considered to be a pan-ethnic disease, carrier frequencies for many ethnicities, including most ethnic groups in North America, are unknown. OBJECTIVES AND METHODS: To provide an accurate assessment of SMN1 mutation carrier frequencies in African American, Ashkenazi Jewish, Asian, Caucasian, and Hispanic populations, more than 1000 specimens in each ethnic group were tested using a clinically validated, quantitative real-time polymerase chain reaction (PCR) assay that measures exon 7 copy number. RESULTS: The observed one-copy genotype frequency was 1 in 37 (2.7%) in Caucasian, 1 in 46 (2.2%) in Ashkenazi Jew, 1 in 56 (1.8%) in Asian, 1 in 91 (1.1%) in African American, and 1 in 125 (0.8%) in Hispanic specimens. Additionally, an unusually high frequency of alleles with multiple copies of SMN1 was identified in the African American group (27% compared to 3.3-8.1%). This latter finding has clinical implications for providing accurate adjusted genetic risk assessments to the African American population. CONCLUSIONS: Differences in the frequency of SMA carriers were significant among several ethnic groups. This study provides an accurate assessment of allele frequencies and estimates of adjusted genetic risk that were previously unavailable to clinicians and patients considering testing.

In-frame deletions of BRCA1 may define critical functional domains.

The identification of genomic rearrangements involving more than 0.5 kb of the BRCA1 gene has confirmed a more complex mutation spectrum than was initially appreciated. Genomic rearrangements in BRCA1 represent 15% of all mutations in a group of French and American breast and ovarian cancer families and 36% of all mutations in a group of Dutch families. The rearrangements described to date range in size from 510 bp to 23.8 kb, are found throughout the gene, and are most frequently attributable to homologous recombination. We describe the identification of rearrangements in two breast and ovarian cancer families that involve 3.4 and 11.5 kb of the BRCA1 gene and span multiple exons but maintain the reading frame. Both gene rearrangements appear to result from Alu-mediated homologous recombination and have been detected by using a combination of protein truncation analysis and Southern blot analysis. These rearrangements result in the loss of amino acids that lie at the carboxy-terminus of the protein and that have previously been shown to have functional significance. Because these rearrangements result in the deletion of exons but maintain the reading frame, they may provide insights into specific regions and amino acids that have functional significance for the BRCA1 protein.

An Alu-mediated 7.1 kb deletion of BRCA1 exons 8 and 9 in breast and ovarian cancer families that results in alternative splicing of exon 10.

Constitutive large deletions and duplications of BRCA1 resulting from Alu-mediated recombination account for a significant proportion of disease-causing mutations in breast and/or ovarian cancer families. Using Southern blot analysis and a protein truncation test (PTT), we have identified a 7.1 kb germline deletion in two families with breast and ovarian cancer. This deletion, which includes exons 8 and 9 and leads to a frameshift at the mRNA level, appears to result from homologous recombination between closely related Alu repeats, one in intron 7 and one in intron 9. In addition to the transcript without exons 8 and 9, analysis of RNA by protein truncation test from individuals with the deletion also identified the presence of alternative splicing of exon 10 from the mutant allele, which results in a transcript that lacks exons 8, 9, and 10. Of interest is that the two American families who carry this deletion are of northern European ancestry and share a common haplotype, suggesting that this deletion may represent a founder mutation. Genes Chromosomes Cancer 28:300-307, 2000.

Survey of the fragile X syndrome CGG repeat and the short-tandem-repeat and single-nucleotide-polymorphism haplotypes in an African American population.

Previous studies have shown that specific short-tandem-repeat (STR) and single-nucleotide-polymorphism (SNP)-based haplotypes within and among unaffected and fragile X white populations are found to be associated with specific CGG-repeat patterns. It has been hypothesized that these associations result from different mutational mechanisms, possibly influenced by the CGG structure and/or cis-acting factors. Alternatively, haplotype associations may result from the long mutational history of increasing instability. To understand the basis of the mutational process, we examined the CGG-repeat size, three flanking STR markers (DXS548-FRAXAC1-FRAXAC2), and one SNP (ATL1) spanning 150 kb around the CGG repeat in unaffected (n=637) and fragile X (n=63) African American populations and compared them with unaffected (n=721) and fragile X (n=102) white populations. Several important differences were found between the two ethnic groups. First, in contrast to that seen in the white population, no associations were observed among the African American intermediate or "predisposed" alleles (41-60 repeats). Second, two previously undescribed haplotypes accounted for the majority of the African American fragile X population. Third, a putative "protective" haplotype was not found among African Americans, whereas it was found among whites. Fourth, in contrast to that seen in whites, the SNP ATL1 was in linkage equilibrium among African Americans, and it did not add new information to the STR haplotypes. These data indicate that the STR- and SNP-based haplotype associations identified in whites probably reflect the mutational history of the expansion, rather than a mutational mechanism or pathway.

HFE genotyping using multiplex allele-specific polymerase chain reaction and capillary electrophoresis.

CONTEXT: Hereditary hemochromatosis is recognized as one of the most common autosomal recessive disorders, with a prevalence of 1 in 200 to 400 in the white population. Early detection and treatment are completely effective in preventing pathology. It is anticipated that testing for hereditary hemochromatosis will increase, as will the need for a technology that can handle the demand. OBJECTIVE: To describe a high-throughput, single-tube, allele-specific multiplex polymerase chain reaction assay for identifying the 2 mutations in the HFE gene associated with hereditary hemochromatosis. DESIGN: Fluorescence-labeled polymerase chain reaction products from a multiplex polymerase chain reaction are analyzed by automated capillary electrophoresis. DATA ANALYSIS: The assay was validated by analysis of 25 blinded samples, and results were concordant with an established laboratory assay. CONCLUSION: The assay described offers a significant improvement over manual laboratory assays in throughput, reduced technologist time, and cost.

Depressed expression of adipocyte beta-adrenergic receptors is a common feature of congenital and diet-induced obesity in rodents.

OBJECTIVE: To determine whether the dramatic reduction in expression and functional activity of the beta3-adrenergic receptor (AR) and beta1AR subtypes originally observed in adipose tissue of the C57BL/6J Lep(ob)/Lep(ob) ('obese') mouse are general features of all models of obesity, and whether obesity-related differences in betaAR subtype expression occur between adipose depots. DESIGN: Survey of adipose tissue betaAR expression from four mouse models of congenital obesity: the 'obese' mouse (C57BL/6J Lep(ob)/Lep(ob)), the 'diabetic' mouse (C57BL/KsJ LepRdb/LepRdb), the 'tubby' mouse (C57BL/6J tub/tub) and the 'fat' mouse (C57BL/KsJ Cpe(fat)/Cpe(fat)), and in a model of high-fat diet-induced obesity in C57BL/6J mice. MEASUREMENTS: Expression of the betaAR subtypes was measured by Northern blot hybridization in white and brown adipose depots. RESULTS: In the severely obese Lep(ob) and LepRdb mice, mRNA concentrations of beta3AR and beta1AR in white adipose tissue (WAT) were decreased by > 99% and by > 70%, respectively. More modest effects on beta3AR expression were observed in brown adipose tissue (BAT, decreased by 20 - 30%). In less severe forms of obesity, as found in the tubby and carboxypeptidase (Cpe)fat mice, and in diet-induced obese B6 mice, beta3AR expression was decreased in WAT by up to 90%, with more modest decreases in interscapular BAT (IBAT). Changes in beta1AR mRNA concentrations were more variable. Beta2AR mRNA levels did not differ in most cases, with the exception that there was a 3-5-fold increase in BAT for both Lep(ob) and LepRdb mice. CONCLUSIONS: Impaired expression of adipocyte betaAR subtypes is a general feature of both genetic and dietary obesity in mice. The degree of obesity is correlated with the extent of loss of beta3AR and beta1AR expression in WAT. The distinct endocrine abnormalities associated with these obesity models may be responsible for the degree of impaired adipocyte betaAR expression.

Is the hemochromatosis gene a modifier locus for cystic fibrosis?

The variable clinical manifestations of cystic fibrosis (CF) suggest the influence of modifier genes. For example, meconium ileus is present in approximately 10-15% of neonates with cystic fibrosis; however, the genetic and, or environmental factors that determine whether an individual will develop this complication have not been determined. We propose the HFE gene as a candidate modifier locus for CF based on (1) the suggestion of an association between the HLA loci and CF phenotypes; (2) the location of the HFE gene near the HLA loci and; (3) the similarity between the gastrointestinal manifestations of hereditary hemochromatosis and CF. We have determined the frequency of the C282Y and H63D mutations in a group of 89 CF patients who were homozygous for delta F508 and for whom meconium ileus status was known. The carrier frequency of C282Y among the CF patients with meconium ileus was significantly different from that of our unaffected control group (19.4% versus 7.7%). However, the difference between the meconium ileus and the nonmeconium ileus groups was not significant (19.4% versus 10.3%). There was no difference in the frequency of the H63D among the three groups that were studied. These data are suggestive of a relationship between the development of meconium ileus or other gastrointestinal diseases in CF and the HFE gene. Further study of a larger group of patients is warranted.

Direct detection of mutations in the breast and ovarian cancer susceptibility gene BRCA1 by PCR-mediated site-directed mutagenesis.

The tumor suppressor genes BRCA1 and BRCA2, which confer increased susceptibility to breast and (or) ovarian cancer, were recently identified. Mutation analysis of BRCA1 has demonstrated significant allelic heterogeneity; however, some distinct mutations have been detected in unrelated individuals. The most notable is the 185delAG mutation, which occurs at an estimated frequency of approximately 1% in individuals of Ashkenazi Jewish descent [1]. Although consensus has not been reached regarding clinical testing for mutations in BRCA1, a tiered strategy may be appropriate, in which direct testing for the more common mutations is one component. Specific alleles can be detected by using PCR-mediated site-directed mutagenesis (PSM), which alters the PCR products derived from either the wild-type or mutant allele to create or destroy a restriction endonuclease recognition site. Recognition sites are introduced by a base substitution in one of the primers. The alleles are then resolved by electrophoresis of the digested PCR products. We have applied this technique to the detection of four BRCA1 mutations: 185delAG, 5382insC, E1250X, and R1443X. Another mutation, 1294de140, can be resolved from the wild-type allele by high-resolution gel electrophoresis alone. The PSM technique is sensitive, does not require radioactivity, and is specific for individual mutations.

Analytical and clinical evaluation of refractive index-matched anomalous diffraction (RIMAD) for assessment of fetal lung maturation.

We have evaluated refractive index-matched anomalous defraction (RIMAD) (Dubin SB, Clin Chem 1988;34:938-43) as a potential method for assessment of fetal lung maturity (FLM). This method determines the total light scattered by the surfactant-containing lamellar bodies by subtraction of the A650 from amniotic fluid diluted in glycerol from that of amniotic fluid diluted in distilled water. It is not significantly affected by such contaminating chromogens as hemoglobin and bilirubin up to 2.0 g/L and 11.0 mg/L, respectively. However, the addition of as little as 2.5 microL of erythrocytes as whole blood resulted in significant interference. RIMADs for normal respiratory outcomes (n = 78) ranged from 0.018 to 0.471. RIMADs for respiratory distress syndrome (RDS) outcomes (n = 8) ranged from 0.004 to 0.036. Use of a RIMAD referent value of > 0.040 to indicate maturity yielded sensitivity, specificity, predictive value (PV)RDS, and PVmaturity of 100%, 96.2%, 72.2%, and 100%, respectively. The areas under the receiver-operating characteristic curves were 0.997 for the RIMAD assay, 0.993 (P = 0.3) for the TDx-FLM assay, 0.89 (P = 0.017) for the lecithin/sphingomyelin ratio, and 0.87 (P = 0.023) for the foam stability index.

Age-dependent changes in beta-adrenergic receptor subtypes and adenylyl cyclase activation in adipocytes from Fischer 344 rats.

Epididymal adipocytes were isolated from Fischer 344 rats aged 3, 6, 12, and 24 months, to study the mechanisms responsible for age-dependent diminution in cellular adrenergic responsiveness. Messenger RNA (mRNA) levels for the beta 1-, beta 2-, and beta 3-adrenergic receptors (ARs) were compared across age groups and related to adenylyl cyclase activation by selective receptor agonists in adipocyte plasma membranes and activation of lipolysis in intact cells. mRNA levels for the beta 1-AR decreased by 60% between 3-6 months and remained at this reduced level through 12 and 24 months. A modest increase in beta 2-AR mRNA was noted between 3-12 months, but decreased between 12-24 months to levels seen in the 3-month-old group. mRNA for the beta 3-AR did not change between 3-6 months, but decreased by about 40% between 6-12 months, and by a further 50% between 12-24 months. Lipolytic responsiveness also diminished with age, and regardless of whether beta 3-selective or beta 1/beta 2-selective agonists were used, the maximal release of glycerol was most severely blunted in adipocytes from 24-month-old rats. The age-dependent changes in adenylyl cyclase activation by beta-adrenergic agonists mirrored the observed changes in lipolytic responsiveness with respect to diminished efficacy. These results together with the similar forskolin-stimulated adenylyl cyclase activity among the groups suggest age-dependent changes in activation of adenylyl cyclase at a prior step. This suggestion is also supported by the comparable inhibitory capacities of the alpha 2-adrenergic and A1-adenosine signaling systems among the age groups. In view of the similar levels of Gs alpha, the age-dependent decrease in adrenergic responsiveness in rat adipocytes appears to result primarily from specific decreases in the expression of both beta 3-AR and beta 1-ARs.

Regulation of the uncoupling protein gene (Ucp) by beta 1, beta 2, and beta 3-adrenergic receptor subtypes in immortalized brown adipose cell lines.

Immortalized brown adipocyte cell lines derived from a mouse hibernoma express all three beta-adrenergic receptor subtypes, including beta 3-adrenergic receptor (AR). In response to norepinephrine, cAMP production by plasma membranes from four clonal cell lines was stimulated to levels comparable with brown adipocytes isolated from interscapular brown adipose tissue (72.8-89.6 versus 97.8 pmol cAMP/min/mg of protein, respectively). All cell lines responded to the highly selective beta 3-adrenergic receptor agonist CL316,243 by stimulating adenylyl cyclase activity (3-10-fold over basal). beta 1-, beta 2-, and beta 3-adrenergic receptor mRNA was detected by Northern blotting and/or reverse transcriptase-polymerase chain reaction. Competition binding assays with the antagonists CGP20712A and 125I-cyanopindolol showed the proportions of beta 1AR and beta 2AR in immortalized cells to be similar to brown adipocytes from tissue (cells: 35% beta 1AR, 65% beta 2AR; brown adipocytes from tissue: beta 1AR 41%, 59% beta 2AR). Expression of brown fat-specific mitochondrial uncoupling protein (Ucp) was stimulated by beta-adrenergic agonists in two of the four cell lines. The ability of individual beta AR subtypes to regulate Ucp expression was examined with combinations of selective beta-adrenergic agonists and antagonists. Expression of Ucp could be induced by any of the beta-adrenergic receptor subtypes. However, the greatest response was obtained by stimulating all three beta-adrenergic receptor subtypes simultaneously (100 microM isoproterenol). Incubation of membranes from cultured cells or brown adipocytes from tissue with CL316,243 at an optimal concentration (5 microM) did not prevent norepinephrine from further stimulating adenylyl cyclase activity, suggesting that the combined activation of beta 1AR/beta 2AR, plus beta 3AR, together produced an additive cAMP response. Multiple forms of adenylyl cyclase were identified in brown and white adipocyte cell lines and tissues. Northern blot analysis detected adenylyl cyclase types 5, 6, and 10. Screening of reverse transcriptase-PCR products by DNA sequencing confirmed the identities of these forms and lower levels of additional isoforms, raising the possibility that beta-adrenergic receptor subtypes in adipocytes couple to distinct adenylyl cyclases. Because these cell lines display functional and phenotypic similarities to interscapular brown adipocytes, they will be a useful model to study the regulation of beta-adrenergic receptor expression and function, and the control of Ucp expression and activity.

Impaired expression and functional activity of the beta 3- and beta 1-adrenergic receptors in adipose tissue of congenitally obese (C57BL/6J ob/ob) mice.

Adipocytes from genetically obese (ob/ob) mice display an impaired response to beta-adrenergic stimulation, but the molecular defects have not been unequivocally identified. The expression and functional activity of the beta 1-, beta 2-, and beta 3-adrenergic receptor (AR) subtypes in white and brown adipose tissue from genetically lean and obese (ob/ob) mice were compared. Three beta 3AR transcripts of 2.1, 2.6, and 3.5 kilobases were identified in adipose tissue from lean mice by Northern blotting. All three beta 3AR mRNA species were dramatically reduced (by approximately 300-fold) in 12-week-old obese mice compared to those in lean animals. beta 1AR mRNA levels were also reduced (by approximately 4-fold) in obese mice, whereas beta 2AR mRNA levels were not significantly changed. The functional consequences of these changes in beta 3AR and beta 1AR expression were assessed by measuring beta-agonist-stimulated adenylyl cyclase activity in adipocyte plasma membranes with subtype-selective beta-adrenergic agonists and antagonists. Dose-response curves with epinephrine from lean mice were best fit to a two-component model comprised of 23% high affinity (K(act) = 1.42 x 10(-7) M) and 77% low affinity (K(act) = 1.67 x 10(-5) M) components, corresponding to activation of beta 1AR and beta 2AR conjointly, and beta 3AR, respectively. The beta 1AR-selective antagonist CGP20712A reduced the high affinity component to about 10%, whereas the nonselective beta-antagonist propranolol eliminated the high affinity component. The beta 3AR-selective agonist BRL37344 stimulated adenylyl cyclase activity in lean membranes to a slightly lesser extent than epinephrine, but was more potent (73% high affinity component; K(act) = 3.61 x 10(-8) M). In obese mice, stimulation of adenylyl cyclase by all agonists was severely blunted and was best fit to a single class of sites. Studies with CGP20712A or the beta 2AR-selective antagonist ICI118,551 indicated that this residual response was predominantly beta 2AR in character. Expression of beta AR subtypes in both brown and white adipose tissue of weanling obese mice (4-5-weeks of age) was also affected, but to a lesser extent, consistent with the progressive severity of obesity with age. Together the reduction in expression of the beta 3AR and beta 1AR impairs the beta-agonist-stimulated adenylyl cyclase response over a broad concentration range by greatly lowering the maximum stimulation and shifting the adrenergic sensitivity at low concentrations from a mixed beta 1AR/beta 2AR response to predominantly beta 2AR.

Lipid synthesis and secretion by primary cultures of rat mammary epithelial cells.

Lipid synthesis and secretion was measured in primary rat mammary epithelial cells cultured on basement matrix in medium supplemented with lactogenic hormones. The cells grew and differentiated to form alveolar-like structures reminiscent of lactating mammary gland. They synthesized abundant triacylglycerol, containing fatty acids characteristic of rat milk (C10:0-C14:0), using 14C-glucose, 14C-oleic acid or 14C-glycerol as precursors. Basal levels of triacylglycerol secretion were measured using 14C-oleic acid labeling; 1.3 +/- 0.3% of the labeled cellular triacylglycerol was secreted into the medium in 24 hours. Secreted lipid droplets were surrounded by a bilayer membrane with an electron-dense inner coat characteristic of fat globules secreted by the mammary gland. The rate of triglycerol secretion was increased to 998 +/- 98% of control (P < 0.01) by the addition of phorbol 12-myristate 13-acetate (PMA) in combination with staurosporine, a protein kinase inhibitor. Several other protein kinase inhibitors, when combined with PMA, also markedly stimulated secretion. Effective protein kinase inhibitors included sphingosine (has diverse cellular effects including the inhibition of protein kinase C; 13-fold increase in secretion), and KT5823 (a cGMP dependent protein kinase inhibitor; 5-fold increase). KT5720 (a cAMP-dependent protein kinase inhibitor) did not alter secretion. Kinase inhibitors were effective only in the presence of a phorbol ester. 4 alpha-phorbol-12,13-didecanoate, a phorbol ester which does not activate protein kinase C (PKC), could substitute for PMA. Lipid release was not mediated by disruption of cell-cell tight junctions, as EGTA did not release lipid. Based on these observations we suggest that two signals are needed to enable or stimulate lipid secretion in cultured rat mammary epithelial cells: 1) inhibition of a protein kinase and 2) a PKC-independent effect of phorbol ester. We have, for the first time, characterized a cell culture model suitable for studying lipid synthesis and secretion by mammary epithelial cells.

Glycerophosphocholine and phosphocholine are the major choline metabolites in rat milk.

Choline is a constituent of cell membranes, surfactant and acetylcholine and is also a major source of methyl groups for the regeneration of methionine from homocysteine. Previous analyses of rat, human and bovine milk measured only choline, phosphatidylcholine and sphingomyelin. Choline-containing compounds in milk from rats lactating for 15 d were measured by HPLC and gas chromatograph-mass spectrometry. In addition to the previously reported choline metabolites, substantial concentrations of glycerophosphocholine (3.7 mmol/L) and phosphocholine (653 mumol/L) were also detected. At 1 h after oral administration of [methyl-14C]choline to lactating rats, the major labeled metabolites were phosphocholine (91% of label in milk) and betaine (9%). Twenty-four hours after the dose, glycerophosphocholine was the major labeled metabolite (69% of label in milk). Rat mammary epithelial cells, in primary culture, synthesized and secreted phosphatidylcholine, phosphocholine, glycerophosphocholine and betaine. Thus, the mammary gland was able to synthesize the choline metabolites found in milk, but these metabolites may not be derived exclusively from uptake from maternal blood. We have established that the total choline concentration in rat milk is sevenfold higher than previously reported, with > 80% present as glycerophosphocholine and phosphocholine.