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C S Richards

Publications and source records attributed to C S Richards.

At least 37 records · Page 2Linked to original sources
PubMed1996

Molecular detection of new mutations, resolution of ambiguous results and complex genetic counseling issues in Huntington disease.

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by expansion of a variable length (CAG)n repeat in the 5' coding region of a novel gene on chromosome 4p16.3. We provide comprehensive molecular analysis of a sporadic case of HD in which a paternally derived normal length allele expanded to an affected length allele. Linkage analysis and paternity testing confirm the paternal origin of the expansion and demonstrate that unequal crossing over during meiosis is an unlikely mechanism for de novo expansion in HD. This case identifies a complex genetic counseling issue for the families of sporadic cases since calculations of recurrence risk are not possible at this time. In addition, we describe utilization of a combination of polymerase chain reaction (PCR) based assays for examination of both the CAG repeat and an adjacent variable length CCG repeat in the huntingtin gene. The combination of these assays can increase the accuracy of molecular diagnosis for HD and may clarify any ambiguous results obtained during molecular testing of HD families.

Female↗
PubMed1996

Population-specific screening by mutation analysis for diseases frequent in Ashkenazi Jews.

We describe a partially automated DNA mutation assay for detecting the most frequent mutations in the alpha-subunit of beta-hexosaminidase A, the acid beta-glucosidase and the cystic fibrosis transmembrane conductance regulator genes for the Ashkenazi Jewish population. The assay detects carriers for Tay-Sachs disease, Gaucher disease, and cystic fibrosis with sensitivities of at least 92%, 96%, and 97%, respectively. Among 1,364 young adults of Ashkenazic ancestry in the Dor Yeshurim community who were tested, 52 were Tay-Sachs carriers, 110 were Gaucher carriers, and 62 were cystic fibrosis carriers. Ten individuals were carriers for two diseases, and three unsuspected cases were diagnosed with Gaucher disease based on mutation test results. In addition to Tay-Sachs mutation data, results for hexosaminidase A activity were also available. All of 1,254 samples normal by enzyme quantitation were also negative for the three alpha-subunit mutations tested, and all of 43 samples with 'inconclusive' enzyme results were negative by DNA. Only 52 of 67 samples positive by enzyme assay were also positive for one of the three mutations tested for Tay-Sachs disease. The data suggest a high degree of false positivity inherent in enzyme identification of carriers. There are no correlative methods to assess the sensitivity of Gaucher and CF carrier testing. The results show that population screening can be carried out efficiently by DNA analysis, with the accrual of carrier information for three separate diseases conducted as a single test. Furthermore, the DNA method for Tay-Sachs screening appears to exceed the specificity of hexosaminidase A enzyme testing.

Adult↗
PubMed1996

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.

Alleles↗
PubMed1996

Pharmacokinetics of reconstituted human high-density lipoprotein in pigs after hemorrhagic shock with resuscitation.

OBJECTIVES: Reconstituted human high-density lipoprotein (HDL) can inhibit lipopolysaccharide effects in vivo. The major objectives of this study were to characterize the pharmacokinetics of reconstituted HDL in a stressed large-animal model and to provide preclinical tolerance information in support of use of reconstituted HDL in humans. DESIGN: A randomized, blinded, placebo-controlled trial where each animal received either reconstituted human HDL at a dose of 100 mg/kg (apolipoprotein A-I) or placebo, immediately after hemorrhagic shock and resuscitation. SETTING: Animal laboratory. SUBJECTS: Twelve immature female swine (18 to 25 kg) were studied. INTERVENTIONS: Six to 8 days before shock and study drug administration, animals were anesthesized and catheters were placed in the external jugular vein and abdominal aorta. These catheters were secured to the dorsal surface. On the day of shock, the animals were sedated (alpha-chloralose) and 50 mL/kg of arterial blood was removed over 0.5 hr. One half hour after blood removal, shed blood was infused, which was immediately followed by study drug (reconstituted HDL or placebo), and then by 1 L of lactated Ringer's solution. MEASUREMENTS AND MAIN RESULTS: Physiologic (arterial blood pressure, heart rate, respiratory rate) and laboratory (serum chemistries, hematologic and coagulation studies, and blood gases) measurements were determined intermittently for 96 hrs after the induction of shock. Blood was collected intermittently for 48 hrs after shock for assay of apolipoprotein A-I and phosphatidylcholine in plasma. Reconstituted HDL was well tolerated and did not appear to alter the physiologic responses to shock and resuscitation. HDL transient increase in aspartate aminotransferase concentration was noted in the reconstituted group but this increase normalized by 24 hrs after drug administration. Mean apolipoprotein A-I pharmacokinetic parameters were as follows: half-life 24.5+/-5.3 (SD) hrs; clearance 41.9+/-10 mL/hr; and volume of distribution 1.39+/-0.08 L. The apparent mean half-life of phosphatidylcholine was 5.4+/-0.8 hrs. CONCLUSIONS: Reconstituted human HDL was well tolerated in animals that had undergone hemorrhagic shock with resuscitation. The apolipoprotein component of reconstituted HDL had a relatively long half-life, with distribution limited to the vascular space. These findings support the investigational use of this product in humans.

Analysis of Variance↗
PubMed1996

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.

Base Sequence↗
PubMed1996

Use of RAPD-PCR to differentiate genetically defined lines of an intermediate host of Schistosoma mansoni, Biomphalaria glabrata.

The genetic differentiation among several laboratory-maintained pedigree snail lines of Biomphalaria glabrata (with different susceptibility phenotypes to Schistosoma mansoni infection) was assessed with the random amplified polymorphic DNA method. Out of the 20 primers tested, 2 (OPA-01 and OPA-06) gave reproducible markers with either individual or bulked DNA samples from resistant (BS-90, 10-R2, LAC-line) and susceptible (M-line) snails. Arbitrary primer, OPA-01, amplification of BS-90 DNA identified a 180-bp strain-specific fragment and a 400-bp marker in the susceptible M-line stock. In the 10-R2 and LAC snail lines, OPA-01 specific markers of 200 bp and 550 bp were identified. Amplification with primer OPA-06 identified several major strain-specific markers in the BS-90 (150 bp, 400 bp, 800 bp) and M-line (1,100 bp) snails. The heritability of the RAPD markers was evaluated in progeny snails derived from a cross between the BS-90 and M-line stocks. Results showed that markers were inherited in a dominant or codominant fashion. The 1,100-bp M-line marker was inherited in all susceptible progeny snails analyzed.

Animals↗
PubMed1995

Epstein-Barr virus-associated leiomyosarcomas in liver transplantation recipients. Origin from either donor or recipient tissue.

BACKGROUND: Leiomyosarcoma, a mesenchymal malignancy with smooth muscle differentiation, is extremely rare in children. Immunosuppression, due to either antirejection medication in organ transplantation recipients or human immunodeficiency virus infection (HIV), appears to constitute a predisposition. METHODS: Two cases of leiomyosarcoma in pediatric liver transplantation recipients were investigated and compared clinically with respect to site of origin and course of the disease and pathologically by routine histology and electron microscopy, by forensic DNA methodology for origin from donor or recipient tissue, and by EBER-1 in situ hybridization for evidence of latent Epstein-Barr virus (EBV) infection. RESULTS: A 9-year-old male developed a high grade, poorly differentiated leiomyosarcoma in his allografted liver 2 years after transplantation, and despite antineoplastic chemotherapy, he died of metastatic disease. The genotype of his tumor indicated an origin from allografted tissue. A 12-year-old female had a low grade retroperitoneal leiomyosarcoma involving the superior mesenteric vein. After resection, she remained disease free without chemotherapy. The genotype of her tumor indicated an origin from native tissue. In both tumors, latent EBV infection was documented. CONCLUSIONS: Neoplastic smooth muscle proliferation in immunosuppressed liver transplantation recipients is analogous to the more common posttransplantation lymphoproliferative disorder in involving transformation of either engrafted donor tissue or recipient tissue elsewhere in the body, in displaying a wide spectrum of histologic differentiation, grade and clinical behavior, and in exhibiting evidence of latent EBV infection.

Base Sequence↗
PubMed1995

Somatic heterogeneity of the CTG repeat in myotonic dystrophy is age and size dependent.

The most common form of adult muscular dystrophy, myotonic dystrophy (DM), is caused by the abnormal expansion of the CTG repeat, located in the 3' UTR of the DM gene. The expanded-CTG allele often presents as a diffused band on Southern blot analysis, suggesting somatic mosaicism. In order to study the somatic instability of the CTG repeat, we have investigated the dynamics of the size heterogeneity of the CTG expansion. Size heterogeneity is shown as a smear on Southern blot and is measured by the midpeak-width ratio of the expanded allele to the normal sized allele. The ratio is also corrected for compression in the higher-molecular-weight region. It is found that the size heterogeneity of the expanded-CTG repeats, of 173 DM patients, correlates well with the age of the patient (r = .81, P << .001). The older patients show larger size variation. This correlation is independent of the sex of either the patient or the transmitting parent. The size heterogeneity of the expansion, based on age groups, is also dependent on the size of the expanded trinucleotide repeat. However, obvious size heterogeneity is not observed in congenital cases, regardless of the size of expansion. Comparison of individual patient samples collected at two different times has confirmed that the degree of size heterogeneity increases with age and has revealed a subtle but definite upward shift in the size of the expanded-CTG allele. The progression of the CTG repeat toward larger expansion with age is further confirmed by small-pool PCR assay that resolved the heterogeneous fragments into discrete bands.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

A robotics-assisted procedure for large scale cystic fibrosis mutation analysis.

We describe a convenient, efficient, semiautomated protocol for assaying large numbers of DNA samples for over 20 mutations causing cystic fibrosis. The protocol uses the following: (1) a programmable robotic workstation to perform rapid pipetting and dot-blotting operations, (2) an allele-specific oligonucleotide hybridization in a single water bath without correcting for G+C content of oligonucleotides, and (3) a combinatorial system that allows direct determination of the genotype for more frequent mutations. We have used this system routinely for 16 months for carrier detection and for diagnosis of cystic fibrosis. The method can be readily applied to any combination of allele-specific oligonucleotide assays whether for multiple alleles at one locus or for a few alleles at multiple loci.

Base Sequence↗
PubMed1994

Schistosoma mansoni: relationship between low fecundity and reduced susceptibility to parasite infection in the snail Biomphalaria glabrata.

Biomphalaria glabrata snails which were not susceptible as juveniles to infection by Schistosoma mansoni were selectively bred (by self-fertilization) from the highly susceptible NMRI laboratory snail stock. The susceptibility rate among juvenile snails derived from interbreeding NMRI parents was initially 85-95%, but after several generations of selection, less than 5% of exposed snails became infected by the parasite. Selection for low susceptibility also resulted in a large proportion of snails that displayed low fecundity and produced abnormal egg masses. Individual adult snails which were isolated from an interbreeding population of nonselected NMRI snails usually produced well-developed egg masses each containing 15-30 embryos. However, when juvenile snails from this same population were reared in isolation and not allowed to cross-fertilize, many displayed a pattern of low fecundity and abnormal egg production similar to that observed in the selected low susceptible line. Furthermore, it was found that many of the isolated snails which exhibited low egg production were also not susceptible to parasitic infection.

Animals↗
PubMed1994

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.

Base Sequence↗
PubMed1994

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.

Alleles↗
PubMed1994

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.

Adult↗
PubMed1994

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.

Adult↗
PubMed1994

Experience of an academic reference laboratory using automation for analysis of cystic fibrosis mutations.

OBJECTIVE: To examine the use of partial automation for molecular analysis of cystic fibrosis and to evaluate the diagnostic experience gained. DESIGN: Twenty-four cystic fibrosis mutations, with cumulative mutation detection of 89% in North American whites and of 97% in the Ashkenazim, were tested by multiplex amplification and allele-specific oligonucleotide hybridization. SETTING: A university-based DNA diagnostic laboratory. SUBJECTS: More than 700 serial specimens were analyzed for cystic fibrosis mutations over a 5-month period. The study included 377 individuals tested for carrier status, of which 288 had no family history for cystic fibrosis; prenatal diagnosis for 17 fetuses at a one in four risk and eight pregnancies at lower risk; fetal or parental samples for 33 pregnancies with fetal ultrasound abnormalities; 40 individuals diagnosed with cystic fibrosis; and 87 individuals with a possible diagnosis of the disease. RESULTS: Automation has permitted increasing numbers of mutations while decreasing personnel time and cost. Mutation testing identified 10 carriers with no family history for cystic fibrosis, four couples at a one in four risk, and five affected fetuses, one ascertained by abnormal fetal ultrasound. Mutation analysis also identified two mutant copies of the gene in 26 of 40 individuals with a clinical diagnosis of cystic fibrosis, and in two of 87 patients with possible cystic fibrosis. CONCLUSION: This partially automated, direct mutation analysis provides DNA diagnostic laboratories with the capacity to process a larger number of samples at lower cost with greater sensitivity for mutation detection. As pilot screening programs are reported, it is appropriate to reevaluate recommendations regarding population-based carrier screening for cystic fibrosis.

Automation↗
PubMed1993

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.

Chromosome Banding↗
PubMed1993

Schistosoma mansoni: analysis of an unusual infection phenotype in the intermediate host snail Biomphalaria glabrata.

Snails from a B. glabrata stock, selected for nonsusceptibility to the NMRI strain of S. mansoni, exhibited unusual schistosome infections, with multiple sporocysts prominent in the head-foot (foot-sporocysts). When F1 progeny from numerous crosses between well-defined susceptible (S) and resistant (R) parental snails were exposed, 60% of the infected snails also exhibited this unusual phenotype. F1 progeny from S x S snail crosses developed normal infections. Foot-sporocysts usually developed much later than secondary sporocysts of normal infections and before 12 weeks postexposure the tissue reaction surrounding foot-sporocysts was less intense than reactions to sporocysts in other tissues of the body. Cercariae were seen emerging directly from foot-sporocysts. Infection of S x R hybrid progeny by three other strains of S. mansoni also resulted in the production of foot-sporocysts. Development of the foot-sporocyst infection phenotype in progeny from many snail crosses suggests that this phenotype is more common than previously recognized. Over 50% of the F1 progeny from S x R parental crosses developed patent infections, a fact relevant to the possible biological control of schistosomiasis by the introduction of refractory snails into endemic areas.

Animals↗