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J Maynard

Publications and source records attributed to J Maynard.

At least 19 recordsLinked to original sources
PubMed2002

A technique for the determination of 18O/16O and 17O/16O isotopic ratios in water from small liquid and solid samples.

We have developed a new technique in which a solid reagent, cobalt(III) fluoride, is used to prepare oxygen gas for isotope ratio measurement from water derived either from direct injection or from the pyrolysis of solid samples. The technique uses continuous flow, isotope ratio monitoring, gas chromatography/mass spectrometry (irmGC/MS) to measure the delta18O and delta17O of the oxygen gas. Water from appropriate samples is evolved by a procedure of stepped pyrolysis (0-1000 degrees C, typically in 50 degrees C increments) under a flowing stream of helium carrier gas. The method has considerable advantages over others used for water analysis in that it is quick; requires only small samples, typically 1-50 mg of whole rock samples (corresponding to approximately 0.2 micromol of H2O); and the reagent is easy and safe to handle. Reproducibility in isotope ratio measurement obtained from pyrolysis of samples of a terrestrial solid standard are delta18O +/- 0.54, delta17O +/- 0.33, and delta17O +/- 0.10/1000, 1sigma in all cases. The technique was developed primarily for the analysis of meteorites, and the efficiency of the method is illustrated herein by results from water standards, solid reference materials, and a sample of the Murchison CM2 meteorite.

Meteoroids↗
PubMed2001

Pathological mutations in TSC1 and TSC2 disrupt the interaction between hamartin and tuberin.

Critical functions of hamartin and tuberin, encoded by the TSC1 and TSC2 genes, are likely to be closely linked. The proteins interact directly with one another and mutations affecting either gene result in the tuberous sclerosis phenotype. However, the regions of hamartin and tuberin that interact have not been well defined, and the relationship between their interaction and the pathogenesis of tuberous sclerosis has not been explored. To address these issues a series of hamartin and tuberin constructs were used to assay for interaction in the yeast two-hybrid system. Hamartin (amino acids 302-430) and tuberin (amino acids 1-418) interacted strongly with one another. A region of tuberin encoding a putative coiled-coil (amino acids 346-371) was necessary but not sufficient to mediate the interaction with hamartin, as more N-terminal residues were also required. A region of hamartin (amino acids 719-998) predicted to encode coiled-coils was capable of oligermerization but was not important for the interaction with tuberin. Subtle, non-truncating mutations identified in patients with tuberous sclerosis and located within the putative binding regions of hamartin (N198_F199delinsI;593-595delACT) or tuberin (G294E and I365del), abolished or dramatically reduced interaction of the proteins as assessed by yeast two-hybrid assays and by co-immunoprecipitation of the full-length proteins from Cos7 cells. In contrast, three non-pathogenic missense polymorphisms of tuberin (R261W, M286V, R367Q) in the same region as the disease-causing TSC2 mutations did not. These results indicate a requirement for interaction in critical growth suppressing functions of hamartin and tuberin.

Animals↗
PubMed2001

LD-PCR coupled to long-read direct sequencing: an approach for mutation detection in genes with compact genomic structures.

A number of techniques have been developed as primary screens to scan for DNA sequence variants, including denaturing gradient gel electrophoresis, denaturing high-performance liquid chromatography, single-strand conformation polymorphism and heteroduplex analysis. Variant alleles detected by these assays are subsequently characterised by DNA sequencing. Sequencing itself is rarely used as a primary screen because of labour intensity, cost, and, upon automation, occasional inaccuracy in identifying heterozygous sites. We have previously developed an approach based on coupling long-distance PCR (LD-PCR) to long-read direct sequencing to allow the detection of mutations in the approximately 1.1 kb exon 3 of MECP2. Our use of dye-labelled primers generated high-quality bi-directional sequence runs > 650 bp and allowed easy discrimination of heterozygous bases. We now describe the application of this approach to the detection of mutations in a considerably larger 6.35 kb LD-PCR fragment spanning 10 exons (exons 32-41) of the structurally complex, but genomically compact, TSC2 gene. In a blind analysis, 15/15 previously characterised mutations were successfully identified using seven overlapping bi-directional sequencing reactions. Our approach of long-read sequencing of long-distance PCR products may allow rapid sequencing of multiple exons of compact genes and may be appropriate as a highly sensitive primary screen for mutations.

Base Sequence↗
PubMed2000

Long-read sequence analysis of the MECP2 gene in Rett syndrome patients: correlation of disease severity with mutation type and location.

Mutations in the methyl-CpG-binding protein gene MECP2 at Xq28 cause Rett syndrome (RTT), an X-linked dominant neurodevelopmental disorder characterized by a period of stagnation followed by regression in the development of young girls. Mutations were sought in MECP2 in 48 females with classical sporadic RTT, seven families with possible familial RTT and five sporadic females with features suggestive, but not diagnostic of RTT. Long distance PCR coupled with long-read direct sequencing was employed to sequence the entire MECP2 gene coding region in all cases. Mutations were identified in 44/55 (80%) unrelated classical sporadic and familial RTT patients, but only 1/5 (20%) sporadic cases with suggestive but non-diagnostic features of RTT. Twenty-one different mutations were identified (12 missense, four nonsense and five frame-shift mutations); 14 of these were novel. All missense mutations were located either in the methyl-CpG-binding domain or in the transcription repression domain. Nine recurrent mutations were characterized in a total of 33 unrelated cases (73% of all cases with MECP2 mutations). Significantly milder disease was noted in patients carrying missense mutations as compared with those with truncating mutations ( P = 0. 0023), and milder disease was associated with late as compared with early truncating mutations ( P = 0.0190).

Amino Acid Sequence↗
PubMed2000

Antibody engineering.

Antibodies are unique in their high affinity and specificity for a binding partner, a quality that has made them one of the most useful molecules for biotechnology and biomedical applications. The field of antibody engineering has changed rapidly in the past 10 years, fueled by novel technologies for the in vitro isolation of antibodies from combinatorial libraries and their functional expression in bacteria. This review presents an overview of the methods available for the de novo generation of human antibodies, for engineering antibodies with increased antigen affinity, and for the production of antibody fragments. Select applications of recombinant antibodies are also presented.

Animals↗
PubMed1999

Comprehensive mutation analysis of TSC1 and TSC2-and phenotypic correlations in 150 families with tuberous sclerosis.

Tuberous sclerosis (TSC [MIM 191090 and MIM 191100]) is an autosomal dominant disorder characterized by hamartomas in many organs. Two thirds of cases are sporadic and are thought to represent new mutations. TSC is caused by mutations affecting either of the presumed tumor-suppressor genes, TSC1 and TSC2. Both appear to function as tumor suppressors, because somatic loss or intragenic mutation of the corresponding wild-type allele is seen in the associated hamartomas. Here we report the first comprehensive mutation analysis of TSC1 and TSC2 in a cohort of 150 unrelated TSC patients and their families, using heteroduplex and SSCP analysis of all coding exons and using pulsed-field gel electrophoresis and conventional Southern blot analysis and long PCR to screen for large rearrangements. Mutations were characterized in 120 (80%) of the 150 cases, affecting TSC1 in 22 cases and TSC2 in 98 cases. TSC1 mutations were significantly underrepresented in sporadic cases (P=. 000185). Twenty-two patients had TSC2 missense mutations that were found predominantly in the GAP-related domain (eight cases) and in a small region encoded in exons 16 and 17, between nucleotides 1849 and 1859 (eight cases), consistent with the presence of residues performing key functions at these sites. In contrast, all TSC1 mutations were predicted to be truncating, consistent with a structural or adapter role for the encoded protein. Intellectual disability was significantly more frequent in TSC2 sporadic cases than in TSC1 sporadic cases (P=.0145). These data provide the first representative picture of the distribution and spectrum of mutations across the TSC1 and TSC2 loci in clinically ascertained TSC and support a difference in severity of TSC1- and TSC2-associated disease.

Blotting, Southern↗
PubMed1998

Gross deletions of the neurofibromatosis type 1 (NF1) gene are predominantly of maternal origin and commonly associated with a learning disability, dysmorphic features and developmental delay.

Mutation screening in neurofibromatosis type 1 (NF1) families has long been hampered by the complexity of the NF1 gene. By using a novel multi-track screening strategy, 67 NF1 families (54 two-generation, 13 three-generation) with a de novo mutation in the germline of the first generation were studied with two extragenic and 11 intragenic markers. The pathological lesion was identified in 31 cases. Loss of heterozygosity (LOH) in the affected individual revealed a gross gene deletion in 15 of the two-generation families; in 12 (80%) of them, the deletion was maternally derived. Eleven patients with a gross deletion exhibited developmental delay, ten had dysmorphic features and six manifested a learning disability. No gross deletion was apparent in any of the 13 three-generation families, suggesting that such lesions are subject to more intense selection. In these families, the new mutation was of paternal origin in 11 kindreds and the underlying mutational event could be characterised in three of them.

Adolescent↗
PubMed1997

Mutational and functional analysis of the neurofibromatosis type 1 (NF1) gene.

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders. It is caused by mutations in the NF1 gene which comprises 60 exons and is located on chromosome 17q. The NF1 gene product, neurofibromin, displays partial homology to GTPase-activating protein (GAP). The GAP-related domain (GRD), encoded by exons 20-27a, is the only region of neurofibromin to which a biological function has been ascribed. A total of 320 unrelated NF1 patients were screened for mutations in the GRD-encoding region of the NF1 gene. Sixteen different lesions in the NF1 GRD region were identified in a total of 20 patients. Of these lesions, 14 are novel and together comprise three missense, two nonsense and three splice site mutations plus six deletions of between 1 and 4 bp. The effect of one of the missense mutations (R1391S) was studied by in vitro expression of a site-directed mutant and GAP activity assay. The mutant protein, R1391S, was found to be some 300-fold less active than wild-type NF1 GRD. The mutations reported in this study therefore provide further material for the functional analysis of neurofibromin as well as an insight into the mutational spectrum of the NF1 GRD.

Base Sequence↗
PubMed1997

Characterization and significance of nine novel mutations in exon 16 of the neurofibromatosis type 1 (NF1) gene.

Nine novel mutations have been characterized as the result of screening exon 16 of the human NF1 gene in 465 unrelated neurofibromatosis type 1 patients. These lesions include three nonsense and two missense mutations, two deletions, one duplication, and one mutation in the 5' splice site of intron 16. Although exon 16 is the largest NF1 exon, no mutations have so far been reported in this region. This apparent paucity of lesions may be due either to a reduced functional importance of exon 16 or a screening bias or both. However, consideration of the mutability of exon 16 in comparison with other exons suggests that, at least for single base pair substitutions, no such factors need be invoked. Any previous lack of exon 16 mutations in this category would be explicable in terms of a lower propensity to mutate for codons in this gene region.

Bias↗
PubMed1997

Improved molecular diagnosis of facioscapulohumeral muscular dystrophy (FSHD): validation of the differential double digestion for FSHD.

A major advance in the molecular diagnosis of facioscapulohumeral muscular dystrophy is the recently reported elimination of confounding DNA fragments arising from homologous sequences located at 10q26. In order to evaluate the specificity and sensitivity of this important diagnostic test, we have compared a group of 130 patients fulfilling the diagnostic criteria for FSHD with 200 control subjects not known to have an increased risk of having an FSHD mutation. Among the FSHD cases the smallest BlnI/EcoRI fragment sizes ranged from 10 to > 48 kb with 94.6% (95% CI 89.2-97.8%) of cases having fragment sizes of 34 kb or less. Among the 400 chromosomes from controls the smallest BlnI/EcoRI fragment observed with the EcoRI/BlnI double restriction enzyme digest was 38 kb +/- 2 kb, suggesting a test specificity at a fragment size < 34 kb of or very near to 100% (lower 95% CI 98.2%). Test sensitivity at < 34 kb is estimated at 94.6% (95% CI 89.2-97.8%), all outliers having fragments > 38 kb. The Southern blot analysis with DNA probe p13E-11 has created a valuable molecular diagnostic test for FSHD.

Alleles↗
PubMed1996

Characterization of six mutations in exon 37 of neurofibromatosis type 1 gene.

Neurofibromatosis type 1 (NF1) is one of the most common inherited disorders, with an incidence of 1 in 3,000. We screened a total of 320 unrelated NF1 patients for mutations in exon 37 of the NF1 gene. Six independent mutations were identified, of which three are novel, and these include a recurrent nonsense mutation identified in 2 unrelated patients at codon 2281 (G2281X), a 1-bp insertion (6791 ins A) resulting in a change of TAG (tyrosine) to a TAA (stop codon), and a 3-bp deletion (6839 del TAC) which generated a frameshift. Another recurrent nonsense mutation, Y2264X, which was detected in 2 unrelated patients in this study, was also previously reported in 2 NF1 individuals. All the mutations were identified within a contiguous 49-bp sequence. Further studies are warranted to support the notion that this region of the gene contains highly mutable sequences.

Adult↗
PubMed1996

A cytogenetic deletion, del(17)(q11.22q21.1), in a patient with sporadic neurofibromatosis type 1 (NF1) associated with dysmorphism and developmental delay.

We report the first visible cytogenetic deletion involving the NF1 gene in a patient with sporadic neurofibromatosis, dysmorphic features, and marked developmental delay. The combined evidence of molecular and cytogenetic techniques based on dosage reduction, hemizygosity for microsatellite markers, high resolution G banding, and FISH analysis, predicts this deletion to be approximately 7 Mb in size. Our findings highlight the importance of conducting a detailed cytogenetic and FISH analysis in patients with NF1 who have additional dysmorphic features or particularly severe learning difficulties.

Cells, Cultured↗
PubMed1995

Towards the finer mapping of facioscapulohumeral muscular dystrophy at 4q35: construction of a laser microdissection library.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disorder which has been mapped to the 4q35 region. In order to saturate this distal 4q region with DNA markers, a laser-based chromosomal microdissection and microcloning procedure was used to construct a genomic library from the distal 20% of chromosome 4, derived from a single human metaphase spread. Of the 100 microclones analyzed from this library, 94 clones contained inserts sized from 80-800 bp, with an average size of 340 bp. Less than 20% of these clones hybridized to human repeat sequences. Seventy-two single-copy clones were further characterized by Southern blot hybridization against a DNA panel of somatic cell hybrids, containing various regions of chromosome 4. Forty-two clones mapped to chromosome 4, of which 8 clones mapped into the relevant 4q35 region. Twenty of these chromosome 4-specific clones were screened against "zoo-blots"; 11 clones, of which 3 mapped to 4q35, identified conserved sequences. This is the first report to describe the isolation of potential expressed sequences derived from the FSHD region. These chromosome region-specific microclones will be useful in the construction of the physical map of the region, the positional cloning of potential disease-associated genes, and the identification of additional polymorphic markers from within the distal 4q region.

Biomarkers↗
PubMed1995

Correlation between fragment size at D4F104S1 and age at onset or at wheelchair use, with a possible generational effect, accounts for much phenotypic variation in 4q35-facioscapulohumeral muscular dystrophy (FSHD)

In facioscapulohumeral muscular dystrophy (FSHD), the wide range of clinical severity observed both within and between families has obscured past attempts to identify any phenotypic differences between families from which phenotype-genotype correlation could proposed, although it is noted that age at onset is youngest and severity greatest in isolated cases. From 14/16 large 4q35-linked FSHD families, and 25/34 isolated cases exhibiting a de novo D4F104S1 DNA fragment, we find a significant correlation between proband age at onset and FSHD-associated D4F104S1 fragment size (r = 0.56; p < 0.001), with the smallest fragments occurring in isolated cases. A similar correlation (r = 0.70; p < 0.01) with fragment size is observed for age to loss of ambulation in 16 subjects using a wheelchair. We find also that age at onset appears younger with successive generations in the 4q35 families. We propose that fragment size at D4F104S1, together with a possible generational effect, accounts for a significant part of the wide phenotypic variation in FSHD. Our results predict a more limited range for severity within families, and in one family with a 4q35-linked 38kb fragment support scapulohumeral presentation without facial involvement as a late onset variant of FSHD. We propose that in FSHD, quantitative variation in a uniform mutation mechanism influences age at onset, but by deletion rather than expansion of DNA.

Adolescent↗
PubMed1995

Characterisation of germline mutations in the neurofibromatosis type 1 (NF1) gene.

Neurofibromatosis type 1 is one of the most common inherited disorders with an incidence of 1 in 3000. The search for NF1 mutations has been hampered by the overall size of the gene, the large number of exons, and the high mutation rate. To date, fewer than 90 mutations have been reported to the NF1 mutation analysis consortium and the details on 76 mutations have been published. We have identified five new mutations using single strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) and three intragenic deletions with the microsatellite markers. Of the five new mutations, two were in exon 27a, two in exon 45, and one in exon 49 and these include 4630delA, 4572delC, R7846X, T7828A, and one in the 3' untranslated region (3' UTR). The two nucleotide alterations in exon 27a and the one in exon 45 are predicted to produce a truncated protein.

Adolescent↗
PubMed1995

Phenotypic-genotypic correlation will assist genetic counseling in 4q35-facioscapulohumeral muscular dystrophy.

The wide range of severity in facioscapulohumeral muscular dystrophy (FSHD) complicates genetic advice, although onset age is youngest and severity is greatest in isolated cases. From 14 of 16 large FSHD families which are 4q35 linked, and from 25 of 34 isolated cases exhibiting a de novo D4F104S1 DNA fragment, we find a correlation between proband age at onset and FSHD-associated D4F104S1 fragment size (r = 0.56; P < 0.001), with the smallest fragments occurring in isolated cases. A 4q35-linked 38-kb fragment in one family supports scapulohumeral presentation without facial involvement as a milder late-onset variant of FSHD, and with apparent "unaffected" recombinants in small families, suggests that nonpenetrance is more likely with large fragment sizes. Our results, predicting a more limited range for severity within families, and suggesting > 85% of FSHD maps to 4q35, will facilitate genetic counseling. We propose that quantitative variation in a uniform mutation mechanism influences age at onset, but by deletion rather than expansion of DNA.

Adolescent↗
PubMed1995

Germinal mosaicism in facioscapulohumeral muscular dystrophy (FSHD).

Facioscapulohumeral dystrophy (FSHD) is an autosomal-dominant neuromuscular disorder with a prevalence of 1 in 20,000. The DNA marker p13E-11 (D4F104S1) detects a de novo DNA rearrangement in the majority of sporadic and FSHD cases. These rearrangements consist of deletions of multiple copies of tandem repeat (D4Z4). We have studied 34 new mutation FSHD families of which 26 showed a de novo fragment with p13E-11. In three of the remaining eight families without a de novo fragment, germinal mosaicism was noted. In each case, the proband had inherited a small EcoR1 fragment from the clinically unaffected mother; however, the hybridization signal intensity of this fragment in the mother's DNA was significantly reduced in all three families. This is the first study to describe such mosaicism in FSHD families using DNA analysis and therefore has a considerable significance for genetic counseling and prenatal diagnosis.

Adolescent↗