Ashkenazi Jewish population frequencies for common mutations in BRCA1 and BRCA2.

BRCA1 and BRCA2 are the two major identified causes of inherited breast cancer, with mutations in either gene conferring up to 80-90% lifetime risk of breast cancer in carrier females. Mutations in BRCA1 account for approximately 45% of familial breast cancer and 90% of inherited breast/ovarian cancer, whereas mutations in BRCA2 account for a comparable percentage of inherited breast cancer cases. Over 85 distinct BRCA1 mutations and a growing list of BRCA2 mutations have been identified, with the majority resulting in protein truncation. A specific BRCA1 mutation, 185delAG, has a reported increased carrier frequency of approximately 0.9% in the Ashkenazi Jewish population, but is also found in rare non-Jewish patients with a different haplotype. The 6174delT mutation in BRCA2 was recently identified as a frequent mutation in 8 out of 107 Ashkenazi Jewish women diagnosed with breast cancer by age 50 (ref. 8), as well as in three Ashkenazi male breast cancer patients. We have conducted a large-scale population study to investigate the prevalence of specific BRCA1 and BRCA2 mutations in Ashkenazi Jewish individuals who were unselected for breast cancer. BRCA1 mutation screening on approximately 3,000 Ashkenazi Jewish samples determined a carrier frequency of 1.09% for the 185delAG mutation and 0.13% for the 5382insC mutation. BRCA2 analysis on 3,085 individuals from the same population showed a carrier frequency of 1.52% for the 6174delT mutation. This expanded population-based study confirms that the BRCA1 185delAG mutation and the BRCA2 6174delT mutation constitute the two most frequent mutation alleles predisposing to hereditary breast cancer among the Ashkenazim, and suggests a relatively lower penetrance for the 6174delT mutation in BRCA2.

Isolation of a GCC repeat showing expansion in FRAXF, a fragile site distal to FRAXA and FRAXE.

Three folate-sensitive fragile sites, termed FRAXA, FRAXE and FRAXF, have been identified on the distal end of chromosome Xq. The first two contain expanded, hypermethylated and unstable CGG (or GCC) repeats within CpG islands. We now report the isolation of similar sequences responsible for the third fragile site, FRAXF. A 5-kilobase EcoRI fragment derived from a cosmid coincident with the cytogenetic anomaly detects expanded, methylated and unstable sequences in five individuals who exhibit fragile sites in distal Xq; these individuals have normal repeat lengths at both FRAXA and FRAXE. By sequence analysis, the expanded region contains a GCC repeat. PCR and sequence analysis of chromosomes from the general population indicates that the repeat is polymorphic (6 to 29 triplets), and is stable upon transmission.

Length of uninterrupted CGG repeats determines instability in the FMR1 gene.

Analysis of 84 human X chromosomes for the presence of interrupting AGG trinucleotides within the CGG repeat tract of the FMR1 gene revealed that most alleles possess two interspersed AGGs and that the longest tract of uninterrupted CGG repeats is usually found at the 3' end. Variation in the length of the repeat appears polar. Alleles containing between 34 and 55 repeats, with documented unstable transmissions, were shown to have lost one or both AGG interruptions. These comparisons define an instability threshold of 34-38 uninterrupted CGG repeats. Analysis of premutation alleles in Fragile X syndrome carriers reveals that 70% of these alleles contain a single AGG interruption. These data suggest that the loss of an AGG is an important mutational event in the generation of unstable alleles predisposed to the Fragile X syndrome.

Forensic entomology: applications and limitations.

Forensic entomology is the science of collecting and analysing insect evidence to aid in forensic investigations. Its main application is in the determination of the minimum time since death in cases of suspicious death, either by estimating the age of the oldest necrophagous insects that developed on the corpse, or by analysing the insect species composition on the corpse. In addition, toxicological and molecular examinations of these insects may help reveal the cause of death or even the identity of a victim, by associating a larva with its last meal, for example, in cases where insect evidence is left at a scene after human remains have been deliberately removed. Some fly species can develop not only on corpses but on living bodies too, causing myiasis. Analysis of larvae in such cases can demonstrate the period of neglect of humans or animals. Without the appropriate professional collection of insect evidence, an accurate and convincing presentation of such evidence in court will be hampered or even impossible. The present paper describes the principles and methods of forensic entomology and the optimal techniques for collecting insect evidence.

Data quality in thermal summation development models for forensically important blowflies.

To highlight some issues regarding data quality that are significant in estimating post-mortem intervals (PMI) from maggots, the developmental constants of thermal summation models for development of Chrysomya megacephala Fabricius (Diptera: Calliphoridae) were calculated from incidental data gathered from 12 published studies, and from data generated specifically for the purpose in a single experiment. The focused experiment involved measuring the timing of five developmental landmarks at nine constant temperatures with a sampling resolution of 6-12 h, which is characteristic of other published studies. Combining data from different studies produced inconsistent results because of statistical noise introduced by (at least) disparities in temporal precision, descriptive statistics, geographical location and rearing diets. A robust experimental design to estimate a developmental model should involve at least six constant temperatures, starting at about 7 degrees C above the relevant developmental zero (D0) and going almost to the upper critical temperature, and a temporal sampling interval with a relative precision of about 10%, which requires sampling about every 2 h until hatching, about every 3 h until first ecdysis and about every 6 h until second ecdysis.

Models of development for blowfly sister species Chrysomya chloropyga and Chrysomya putoria.

Developmental curves for the sister species Chrysomya chloropyga (Wiedemann, 1818) and Chrysomya putoria (Wiedemann, 1830) (Diptera: Calliphoridae) were established at eight and 10 different constant temperatures, respectively, using developmental landmarks and body length as measures of age. The thermal summation constants (K) and developmental threshold (D(0)) were calculated for five developmental landmarks using a previously described method. Isomorphen and isomegalen diagrams were also constructed for the purpose of estimating postmortem intervals (PMIs). Chrysomya chloropyga had an average developmental threshold value (D(0)) of 10.91 degrees C (standard error [SE] = 0.94 degrees C, n = 5), significantly lower than that of C. putoria (13.42 degrees C, SE = 0.45 degrees C, n = 5) (paired t-test: t = - 4.63, d.f. = 8, P < 0.00). Similarly, K values for C. chloropyga were larger than those for C. putoria for all developmental events except onset of the wandering phase. These are the first data that can be used to calculate minimum PMIs and predict population growth of C. chloropyga and C. putoria in Africa; the data indicate that developmental data for one of these species cannot be used as surrogate data for the sister species.

Correlation of amino acid fucoside labeling patterns with tumorigenicity of mouse mammary epithelial cells.

The amino acid fucosides of tumorigenic and nontumorigenic mouse mammary gland-derived cells were studied. The cells examined included tumorigenic cell lines derived from mammary carcinomas of the following etiologies: induction by hormone; virus; and chemical carcinogen. Also studied were cells derived from normal mammary glands and several clones of cells, which were derived from a mammary carcinoma but were not demonstrably tumorigenic at lower passage levels after cloning, while they were highly tumorigenic at higher passage levels. Cells were cultured in medium supplemented with radiolabeled fucose and extracted, and extracts were analyzed for the amino acid fucosides. Radiolabeled compounds which comigrated with the amino acid fucosides glucosylfucosylthreonine, fucosylthreonine, and fucosylserine were observed. There was a distinctive difference between the tumorigenic and nontumorigenic cells; the ratio of fucosylthreonine plus fucosylserine to glucosylfucosylthreonine was higher in all tumorigenic cells as compared to the ratio observed for the nontumorigenic cells.

Correlation of surface fucopeptide patterns with tumorigenicity of mouse mammary epithelial cells.

The plasma membrane fucopeptides of tumorigenic and nontumorigenic mouse mammary epithelial cells were studied. The types of cells analyzed included (a) cell lines derived from mouse mammary carcinomas of varying etiologies (viral, hormonal, chemical carcinogen), (b) a series of clonal cells lines which were nontumorigenic at lower passage levels and tumorigenic at higher passage levels, (c) normal primary cells derived from the mammary glands of pregnant or lactating animals, and (d) primary cells from tumors produced by s.c. injection of cultured mammary tumor cells into syngeneic animals. A distinctive difference was observed in the size distribution of the trypsin-sensitive surface fucopeptides from tumorigenic and nontumorigenic mammary cells; the tumorigenic cells were relatively enriched in the larger fucopeptides. The size distribution of the trypsin-sensitive surface fucopeptides was not markedly influenced by the physiological state of the cells or by cell population density. It appears that the trypsin-sensitive surface fucopeptide size pattern may be a distinguishing characteristic between tumorigenic and nontumorigenic mouse mammary epithelial cells.

Differential response of cultured mouse mammary cells of varying tumorigenicity to cytochalasin B.

Cultured BALB/c mouse mammary gland epithelial cells of varying oncogenic potential in vivo have been examined for their ability to multinucleate in the presence of cytochalasin B (CB). Highly tumorigenic cell lines derived from mammary tumors with hormonal, viral, or chemical carcinogen etiologies were extensively multinucleated when cultured in CB-supplemented medium. Normal mammary gland cells from either pregnant or lactating animals were predominantly mono- or binucleate under comparable conditions. At low passage levels after cloning, cell lines derived from a chemical carcinogen-induced mammary tumor were weakly oncogenic and remained largely mono-, or binucleated when cultured in CB-supplemented medium. At higher cell passage levels, both the ability to produce tumors in vivo and the degree of multinucleation in CB-supplemented medium increased dramatically with the clonal cell lines. Thus, the response of cultured mouse mammary gland epithelial cells to CB in vitro may be useful as a marker of the oncogenic potential of such cells.

Relevance of RET proto-oncogene mutations in sporadic medullary thyroid carcinoma.

Analysis of peripheral blood or tumor DNA samples from 101 patients with apparent sporadic medullary thyroid carcinoma (MTC) was performed to assess the frequency of RET proto-oncogene mutations in this patient population. Peripheral blood and/or tumor DNA was amplified by polymerase chain reaction. DNA sequence or restriction enzyme analysis was performed to detect mutations of RET proto-oncogene codons 609, 611, 618, 620, 634, 768, and 918. Six of 101 patients with apparent sporadic MTC had peripheral blood DNA mutations more commonly associated with hereditary MTC. In 4 patients, these mutations led to the identification of previously unrecognized kindreds. The remaining 2 patients were examples of de novo mutations. A codon 918 mutation was found in 14 of 57 (approximately 25%) tumor DNA samples. Mutations were not identified in the remaining patients. In this large cancer center population, approximately 6% of patients with sporadic MTC carry peripheral blood DNA mutations, either inherited or de novo, more commonly associated with MEN 2A or familial MTC. Seven additional gene carriers were identified as a direct result of these studies, a 2-fold multiplying effect. We conclude routine application of RET proto-oncogene testing should be included in all cases of apparent sporadic MTC.

Identification of female carriers for Duchenne and Becker muscular dystrophies using a FISH-based approach.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive neuromuscular diseases caused by dystrophin gene mutations. Deletions, or more rarely duplications, of single or multiple exons within the dystrophin gene can be detected by current molecular methods in approximately 65% of DMD patients. Mothers of affected males have a two-thirds chance of carrying a dystrophin mutation, whilst approximately one-third of affected males have de novo mutations. Currently, Southern blot analysis and multiplex PCR directed against exons in deletion hot spots are used to determine female carrier status. However, both of these assays depend on dosage assessment to accurately identify carriers since, in females, the normal X chromosome is also present. To obviate quantitation of gene dosage, we have developed exon-specific probes from the dystrophin gene and applied them to a screen for potential carrier females using fluorescence in situ hybridization (FISH). Cosmid clones, representing 16 exons, were identified and used in FISH analysis of DMD/BMD families. Our preliminary work has identified multiple, informative probes for several families with dystrophin deletions and has shown that a FISH-based assay can be an effective and direct method for establishing the DMD/BMD carrier status of females.

Relative stability of a minimal CTG repeat expansion in a large kindred with myotonic dystrophy.

The genetic basis for myotonic dystrophy (DM) is a CTG trinucleotide repeat expansion. The number of CTG repeats commonly increases in affected individuals of successive generations, in association with anticipation. We identified a large DM family in which multiple members had minimal CTG repeat expansions, and in which the number of CTG repeats remained in the minimally expanded range through at least three, and possibly four, generations. This relative stability of minimal CTG repeat expansions may help to maintain the DM mutation in the population.

Effects of the sex of myotonic dystrophy patients on the unstable triplet repeat in their affected offspring.

The mutation responsible for myotonic dystrophy (DM) is an unstable expansion of the CTG repeat within the myotonin protein kinase gene. To examine whether the parental origin of the expanded repeat influences the repeat size in offspring, we studied 51 father-child and 59 mother-child pairs with DM. Small expansions in fathers resulted in larger size expansions in their offspring, while large paternal expansions resulted in less size change in their offspring. However, there was no correlation between maternal size expansion and size increase in offspring for either congenital or noncongenital DM. These data suggest that the sex of the affected parent influences the unstable expansion of the repeat in DM offspring. While some evidence suggests that DNA methylation status cannot explain this observation, the mechanism for differential maternal/paternal transmission expansion is currently unknown.

DMD-specific FISH probes are diagnostically useful in the detection of female carriers of DMD gene deletions.

The challenge of Duchenne muscular dystrophy (DMD) carrier identification resides in the ability to identify the presence of a mutant gene over the background contributed by the normal allele. Current diagnosis of carrier status when a deletion has been identified in a proband is based on an analysis of a gene dosage. We present a diagnostic strategy that uses fluorescence in situ hybridization (FISH) to detect female carriers with major deletions in the dystrophin gene. We screened a human X-chromosome-derived genomic library with a full-length dystrophin cDNA and isolated 15 dystrophin-specific cosmids that contain DMD gene exons. Six cosmids were further tested as FISH probes in control individuals and subsequently applied on chromosomes from eight males with DMD and known deletions and on samples from three female carriers. As expected, X chromosomes in normal females displayed four signals, two for the DMD-specific probe and two for the X-chromosome centromeric probe. Hybridization on chromosomal spreads from carriers of deletions revealed only one signal from the DMD-specific probe and two from the control centromeric probe. Males carrying deletions showed no DMD-specific signal for the deleted exons tested. Our data indicate that FISH could represent an alternative method for the detection of female carriers with DMD gene deletions.

CAG repeat size and clinical presentation in Huntington's disease.

The specific mutation in Huntington's disease (HD) is an expansion of the unstable CAG trinucleotide repeat in the IT15 gene in chromosome 4p. We examined the relationship between the CAG repeat size and clinical presentation in 36 patients with suspected diagnosis of HD. Twelve patients had no relatives with documented HD, and five of them failed to show the expanded (>37) CAG repeats. The remaining 31 patients, including seven patients with atypical clinical features for HD (three without and four with family history of documented HD), were heterozygotes for the CAG repeat expansion. There were large CAG repeats (50 copies) in paternally transmitted HD cases with early onset (age 30 or earlier). The rate of disease progression was faster in paternally transmitted cases regardless of the CAG repeat length or age of onset. We conclude that (1) patients lacking the family history of HD frequently show no expansion of the CAG repeats, and (2) the sex of the affected parent influences both the CAG repeat size and the phenotypic expression of the HD gene in the offspring.

Congenital myotonic dystrophy pathology and somatic mosaicism.

We present the pathology and molecular genetic analysis of an infant with congenital myotonic dystrophy. The proband/infant, born at 35 weeks' gestational age to a mother with myotonic dystrophy and 750 CTG repeats, was markedly hypotonic and had severe cardiomyopathy. She died after 16 days of life. At autopsy, skeletal and heart muscles were immature and had a decrease in contractile elements. DNA CTG trinucleotide repeat analysis of the proband demonstrated 2,480 repeats in blood and a slightly greater number of repeats in skeletal muscles, viscera, and gray matter. Corresponding to the clinical course and pathology, cardiac tissues displayed somatic mosaicism, with repeats ranging from 2,760 to 3,220.

Amebocytic accumulations in Biomphalaria glabrata: fine structure.

Internal pathologic conditions in four stocks of Biomphalaria glabrata involve amebocytes. Most snails exhibiting these conditions are also nonsusceptible to infection with Schistosoma mansoni. Fine structure studies reveal that periaortic and atrial amebocytic accumulations show two responses that involve heart tissue: a generalized response in which amebocytes have infiltrated the myocardium and focal areas of nodules in which heart tissue is encapsulated and destroyed. In pericardial accumulations amebocytes are subspherical at the periphery with their numerous filopodia intertwining; centrally they elongate and flatten to form a nodule. Hemocoelic accumulations are similar to pericardial ones but more compact. Hyalinocytes and granulocytes were both found in the four types of amebocytic accumulations; each contained phagocytized material. Acid phosphatase reaction product was detected in the Golgi bodies, primary phagosomes, multivesicular bodies and myelin bodies of granulocytes.

Differential effects of 2-deoxy-D-glucose on surface glycoproteins of normal and transformed cells.

Sarcoma virus transformed rat kidney cells, NRK[MSV-MLV] cells, and their non-transformed counterparts, NRK cells, were repeatedly subcultured in medium supplemented with 1 mM 2-deoxy-D-glucose (dGlc). These dGlc-grown cells exhibited alterations in glycosylation of surface proteins. Gel filtration analyses of pronase-treated, trypsin-sensitive, surface fucopeptides indicated a reduction in the average size of the fucopeptides from both dGlc-grown NRK and NRK[MSV-MLV] cells. When the cells were grown in control medium the fucopeptides from the NRK[MSV-MLV] cells were larger than those from NRK cells, i.e. the fucopeptide difference observed in a wide range of transformed versus normal cells. The changes in the fucopeptides of dGlc-grown cells were greater for the NRK cells so that after growth in dGlc-supplemented medium there was an enhancement in the typical fucopeptide differences between normal and transformed cells, namely a larger difference in both size distribution and degree of sialylation. These dGlc-related alterations were readily reversed when the cells were transferred to control medium. The changes in glycosylation were compatible with continued cell replication although there was an increase in doubling time of the dGlc-grown cells, 6-7 h increase for NRK cells, 1-2 h increase for NRK[MSV-MLV] cells.

In situ hybridization shows direct evidence of skewed X inactivation in one of monozygotic twin females manifesting Duchenne muscular dystrophy.

A novel combination of conventional and molecular cytogenetic techniques was used to investigate the expression of an X-linked recessive disorder in one of monozygotic (MZ) twin females. These twins carry a deletion, approximately 300 kb in length, in one of their X chromosomes within the dystrophin gene, which is responsible for Duchenne muscular dystrophy (DMD) in one twin [Richards et al.: Am J Hum Genet 46:672-681, 1990]. A unique DNA fragment generated from an exon within this gene deletion was hybridized in situ to both twins' metaphase chromosomes, a probe which would presumably hybridize only to the normal X chromosome and not to the X chromosome carrying the gene deletion. Chromosomes were identified by reverse-banding (R-banding) and by the addition of 5-bromodeoxyuridine (BrdU) in culture to distinguish early and late replicating X chromosomes, corresponding to active and inactive X chromosomes, respectively. Hybridization experiments showed predominant inactivation of the normal X chromosome in the twin with DMD. This is the first report showing direct evidence at the chromosome level of unequal inactivation of cytogenetically normal X chromosomes resulting in the manifestation of an X-linked recessive disorder in one of monozygotic twin females. This study may now facilitate other research of unequal X inactivation and of females manifesting X-linked recessive disorders.

Prenatal diagnosis of a fetus with a homologous Robertsonian translocation of chromosomes 15.

We present a prenatal diagnosis of a de novo homologous Robertsonian translocation involving both chromosomes 15. Amniocentesis was performed on a 36-year-old woman at 16.5 weeks of gestation. Chromosome analysis documented a 45,XX,der(15;15) (q10;q10) chromosome pattern. No evidence of a deletion was observed by FISH using a SNRPN DNA probe associated with the Prader-Willi/Angelman syndrome critical region. Molecular studies in the family using six polymorphic markers for chromosome 15 and Southern blot analysis of DNA methylation for the CpG island near the SNRPN gene showed normal biparental inheritance of chromosome 15, excluding uniparental disomy. The patient was counseled that her child would not be able to bear off-spring without clinical assistance. Otherwise the health and intellect of her child were not expected to be affected by the translocation. We consider this to be the first prenatal case identified with a balanced der(15;15)(q10;q10) Robertsonian translocation and a phenotypically normal female outcome. Prenatally identified cases of der(15;15)(q10;q10) warrant further investigation by molecular methodology.