Risk factors for inhibitor formation in haemophilia: a prevalent case-control study.

Inhibitor formation is a major complication of haemophilia treatment. In a prevalent case-control study, we evaluated blood product exposure, genotype and HLA type on haemophilia A inhibitor formation. Product exposure was extracted from medical records. Genotype was determined on stored DNA samples by detection of virtually all mutations-SSCP (DOVAM-S) and subcycling PCR. HLA typing was performed by PCR amplification and exonuclease-released fluorescence. Cases experienced higher intensity factor, 455 vs. 200 U per exposure, P < 0.005, more frequent central nervous system (CNS) bleeding, seven of 20 (35.0%) vs. one of 57 (1.7%), P = 0.001 and more commonly from inhibitor families, seven of 20 (35.0%) vs. zero of 57 (0%), P < 0.001, and African-American, 12 of 63 (19.0%) vs. six of 117 (5.1%), P = 0.015. Among the latter, CNS bleeding was more commonly the initial bleed, 60% vs. 0%, P < 0.001, and survival was shorter, 14 vs. 38 yr, P = 0.025. Inhibitor formation was uncommon in those with missense mutations, two of 65 (3.1%) vs. 31 of 119 (26.0%), P = 0.008, and unrelated to factor VIII immunogenic epitope, P = 0.388, or HLA type, P > 0.100. Genotype was not associated with race. Time to immune tolerance was shorter for titres <120 vs. > or = 120 BU/mL, six vs. 16 months, P < 0.01, but unaffected by tolerizing dose regimen, P > 0.50. Inhibitor formation is associated with high intensity product exposure, CNS bleeding, African-American race and low frequency of missense mutations. The ideal time to initiate prophylaxis to reduce CNS bleeding and inhibitor formation will require prospective studies.

High frequency of de novo mutations in Li-Fraumeni syndrome.

BACKGROUND: Li-Fraumeni syndrome is an autosomal dominant cancer predisposition syndrome caused by germline mutations in the TP53 gene. The frequency of germline de novo TP53 mutations is largely unknown; few unequivocal de novo mutations have been reported. METHODS AND RESULTS: Of 341 patients with early onset cancer sent for clinical testing to a national reference laboratory, 75 patients had TP53 germline mutations. Five (7%) de novo mutations were identified, as well as an additional 10 TP53 germline mutations likely to be de novo by family history. The frequency of de novo TP53 mutations in this patient sample is at least 7% and may be as high as 20%. CONCLUSIONS: The possibility that de novo germline TP53 mutations are relatively common has implications for testing and the identification of potential Li-Fraumeni syndrome in patients with little or no family history of cancer.

EGFR somatic doublets in lung cancer are frequent and generally arise from a pair of driver mutations uncommonly seen as singlet mutations: one-third of doublets occur at five pairs of amino acids.

Doublet mutations in cancer are not well studied. We find that allelic somatic doublet mutations are present at high frequency in the epidermal growth factor receptor (EGFR) tyrosine kinase (TK) domain in lung cancers. When doublets from the literature are added, a total of 96 doublets are available for analysis. The frequency of doublets overall is 6%, which is sevenfold greater than that observed in normal tissue in mouse. All characterized doublets are allelic, and silent mutations occur rarely. About half of all doublets contain one or two of 12 distinct missense mutations at five amino acids: E709, G719, S768, T790 and L861. The mutations at these five amino acids are seldom reported as singlets. Moreover, when the common L858 target is included, more than one-third of EGFR doublets are one of five specific missense pairs: G719/E709, G719/S768, G719/L861, L858/E709 and L858/T790. Structure suggests function: The data imply that most EGFR doublets are NOT consistent with a 'driver and passenger' mutation mechanism. EGFR doublets are highly skewed relative to singlets, consistent with functional selection of two individually suboptimal mutations that, in combination, have enhanced oncogenic potential.

PAP-LMPCR for improved, allele-specific footprinting and automated chromatin fine structure analysis.

The analysis of chromatin fine structure and transcription factor occupancy of differentially expressed genes by in vivo footprinting and ligation-mediated-PCR (LMPCR) is a powerful tool to understand the impact of chromatin on gene expression. However, as with all PCR-based techniques, the accuracy of the experiments has often been reduced by sequence similarities and the presence of GC-rich or repeat sequences, and some sequences are completely refractory to analysis. Here we describe a novel method, pyrophosphorolysis activated polymerization LMPCR or PAP-LMPCR, which is capable of generating accurate and reproducible footprints specific for individual alleles and can read through sequences previously not accessible for analysis. In addition, we have adapted this technique for automation, thus enabling the simultaneous and rapid analysis of chromatin structure at many different genes.

The incidence of venous thromboembolism among Factor V Leiden carriers: a community-based cohort study.

While Factor V (FV) Leiden is a risk factor for venous thromboembolism (VTE), the incidence of VTE among FV Leiden carriers is uncertain. The objective of the study was to estimate the overall age-specific and pregnancy-related VTE incidence and the relative risk among FV Leiden carriers. In a community-based sample of 3424 south-eastern Minnesota residents, 230 (6.7%) were genotyped as FV Leiden carriers; 220 carriers (mean age = 68 years) could be matched to a non-carrier on age, gender, ethnicity and length of medical history. We performed a retrospective cohort study of carriers and non-carriers by reviewing the complete medical records in the community for demographic and baseline characteristics, pregnancies and live births, and first lifetime VTE. Over 14 722 person-years, 24 (10.9%) carriers developed VTE [overall incidence = 163 (95% CI 104, 242) per 100,000 person-years]. VTE incidence rates for ages 15-29, 30-44, 45-59 and > or = 60 years were 0, 61, 244 and 764 per 100,000 person-years, respectively (cumulative VTE incidence at age 65 years = 6.3%). VTE incidence for carriers did not differ significantly from that for non-carriers except for those > or = 60 years old (relative risk = 3.6; 95% CI 2.0, 6.0). There were 311 live births among 130 women carriers; no VTE events occurred during pregnancy or postpartum [incidence = 0 (95% CI 0, 1186) per 100,000 women-years]. Most FV Leiden carriers do not develop VTE. Among all carriers, those > or = 60 years old are at the highest risk for VTE. The incidence of VTE among asymptomatic women carriers during pregnancy is low and insufficient to warrant prophylaxis.

Human germline mutation in the factor IX gene.

The molecular epidemiology of factor IX germline mutations in patients with hemophilia B has been studied in detail because it is an advantageous model for analyzing recent germline mutations in humans. It is estimated that mutations have been defined in the majority of nucleotides that are the target for mutation. The likelihood that a factor IX missense mutation will cause disease correlates with the degree of evolutionary conservation of the amino acid. Mutation rates per base-pair have been estimated after careful consideration and correction for biases, predicting about 76 de novo mutations per generation per individual resulting in 0.3 deleterious changes. The male-to-female sex ratio of mutation varies with the type of mutation. There is evidence for a maternal age effect and an excess of non-CpG G:C to A:T transitions. The factor IX mutation pattern is similar among geographically, racially and ethnically diverse human populations. The data support primarily endogenous mechanisms of germline mutation in the factor IX gene. Mutations at splice junctions are compatible with simple rules for predicting disease causing mutations.

Diagnosis of Duchenne dystrophy by enhanced detection of small mutations.

OBJECTIVE: To determine whether detection of small mutations of the dystrophin gene can be increased using an enhanced method of single-strand conformation polymorphism analysis. BACKGROUND: Usual methods of DNA analysis for Duchenne dystrophy cannot identify mutations in one-third of cases. Muscle biopsy, with its inherent risks and added liability for patients with Duchenne dystrophy, becomes the sole method of diagnosis. Even with a tissue diagnosis of dystrophin deficiency, many families are excluded from carrier detection and prenatal diagnosis. METHODS: Genomic DNA from a cohort of 93 patients with Duchenne dystrophy without identifiable gene mutations was screened for mutations. In each case, 22 kilobases of genomic DNA were scanned, including all 79 exons of the dystrophin gene, adjacent intronic regions, and six alternative exons 1. RESULTS: Sixty-eight (73%) had small mutations, including 34 nonsense mutations, 27 microdeletions and insertions, and 7 splice site mutations. No missense mutations were found. One nonsense mutation in exon 59 was detected in four patients. Most mutations were new; 54 of 62 different small mutations have not been reported. Mutations were found throughout the gene: 24% in the first quartile, 31% in the second, 16% in the third, and 29% in the fourth. CONCLUSIONS: A highly sensitive single-strand conformation polymorphism method substantially increased detection of small dystrophin gene mutations and made it possible to diagnose approximately 90% of patients with Duchenne dystrophy by DNA analysis. These findings, combined with cost savings and safety issues, provide compelling reasons to consider DNA analysis as the initial diagnostic test for the suspected dystrophin-deficient patient.

Spontaneous microdeletions and microinsertions in a transgenic mouse mutation detection system: analysis of age, tissue, and sequence specificity.

A total of 3497 independent spontaneous mutations were examined using the Big Blue transgenic mouse mutation detection system. Base substitutions predominate, although 16% of somatic and germline mutations are microdeletions, microinsertions, or deletions combined with insertions. The pattern of microdeletions and microinsertions is similar in both the lacI transgene and the human p53 gene. Single-base deletions (D1) and insertions (I1) are evenly distributed in the lacI transgene, whereas microdeletions from 2 to 50 bp are clustered at two regions (bp 129-228 and 529-628). The pattern of microdeletions and microinsertions is similar between young (< or =3 months) and old (25 months) mice. Brain tissue has a paucity of deletions combined with insertions when compared with that of thymus and nine other tissues (P = 0.01). A 16-bp deletion at lacI base position 272 is a tissue-specific hotspot preferentially occurring in brain. Approximately 68 and 93% of D1 and I1, respectively, occur at mononucleotide repeats. The frequencies of D1 and I1 in mononucleotide repeats increase in an exponential manner with the length of the repeat. The lacI transgene shows similarity to the human p53 gene in the pattern of microdeletions and microinsertions and the size distribution of microdeletions.

Systematic screening for mutations in the glycine receptor alpha2 subunit gene (GLRA2) in patients with schizophrenia and other psychiatric diseases.

The glycine receptor, which is a member of the ligand-gated ion channel superfamily, mediates synaptic inhibition in the spinal cord and other brain regions. This superfamily has been implicated in the pathogenesis of schizophrenia and other psychiatric diseases. The complete coding sequence and splice junctions of the GLRA2 gene were scanned by DOVAM-S, a form of SSCP analysis with sufficient redundancy to detect virtually all mutations. Those analyses were performed in 113 patients with schizophrenia, and in pilot studies of patients with bipolar illness, alcoholism, puerperal psychosis, autism, and attention-deficit hyperactivity disorder (533 kb total scanned sequences). We detected three sequence changes in the coding region, all resulting in silent mutations: C894T in exon 5, C1134T in exon 7, and C1476T in exon 9. These do not alter the structure or the expression of the protein. It is unlikely that mutations in the coding region and splice junction of GLRA2 gene are associated with schizophrenia and other psychiatric diseases.

Frequency of recent retrotransposition events in the human factor IX gene.

Two germline retrotransposition mutations of recent origin were observed in 727 independent mutations (0.28%) in the human factor IX gene (F9) of patients with hemophilia B: 1) a 279 bp insertion in exon H originating from an Alu family of short interspersed elements not previously known to be active and, 2) a 463 bp insertion in exon E of a LINE1 element originating in the maternal grandmother. If the rates of recent germline mutation in F9 are typical of the genome, a retrotransposition event is estimated to occur somewhere in the genome of about one in every 17 children born. Analysis of other estimates for retrotransposition frequency and overall mutation rates suggests that the actual rate of retrotransposition is likely to be in the range of one in every 2.4 to 28 live births.

Scanning of estrogen receptor alpha (ERalpha) and thyroid hormone receptor alpha (TRalpha) genes in patients with psychiatric diseases: four missense mutations identified in ERalpha gene.

Estrogen and thyroid hormones exert effects on growth, development, and differentiation of the nervous system. Hormone administration can lead to changes in behavior, suggesting that genetic variants of the estrogen receptor alpha (ERalpha) and the thyroid hormone receptor alpha (TRalpha) genes may predispose to psychiatric diseases. To investigate this possibility, regions of likely functional significance (all coding exons and flanking splice junctions) of the ERalpha and TRalpha genes were scanned in patients with schizophrenia (113), along with pilot studies in patients with bipolar illness (BPI), puerperal psychosis, autism, attention-deficit hyperactivity disorder (ADHD), and alcoholism. A total of 1.18 megabases of the ERalpha gene and 1.16 megabases of the TRalpha gene were scanned with Detection of Virtually All Mutations-SSCP (DOVAM-S), a method that detects virtually all mutations. Four missense mutations, seven silent mutations and one deletion were identified in the ERalpha gene, while only four silent mutations were present in the TRalpha gene. Two of the missense mutations in ERalpha are conserved in the six available mammalian and bird species (H6Y, K299R) and a third sequence variant (P146Q) is conserved in mammals, birds, and Xenopus laevis, hinting that these sequence changes will be of functional significance. These changes were found in one patient each with BPI, puerperal psychosis, and alcoholism, respectively. Analysis of the ERalpha and TRalpha genes in 240 subjects reveals that missense changes and splice site variants are uncommon (1.7% and 0%, respectively). Further analyses are necessary to determine if the missense mutations identified in this study are associated with predisposition or outcome for either psychiatric or nonpsychiatric diseases.

An in-frame deletion in the alpha(2C) adrenergic receptor is common in African--Americans.

alpha(2) adrenergic receptors are activated by adrenaline and noradrenaline, and three subtypes (ie, A, B, C) have differential affinities for antagonists and medications. The alpha(2c) adrenergic receptor (ADRA2C), located on chromosome 4p16.3, is a candidate gene for schizophrenia because it binds clozapine, an atypical neuroleptic useful for treatment-resistant schizophrenia. In addition, ADRA2C binds clonidine which is prescribed for three psychiatric diseases. This report communicates the findings of the genetic scanning of this gene of very tough GC content. The complete coding sequences and splice junctions were scanned with [DOVAM]-S in 104 schizophrenics, and pilot probes of patients with alcoholism (41 patients), cocaine abuse (25 patients), puerperal psychosis (30 patients), attention deficient/hyperactivity disorder (25 patients) and autism (25 patients). Six sequence variants were found, including five silent polymorphisms (allele frequencies 0.6--25%) and an in-frame deletion of a homologous repeat at nucleotides 967--978 (ie, TIDRU(1)). Genotyping of the normal two repeat unit of the Third Intracytoplasmic Domain Repeat Unit (TIDRU(2)) and the deleted variant (TIDRU(1)) revealed that TIDRU(1) had allelic frequencies of 39% (11/28) and 3.5% (6/172) in African-American and Caucasian schizophrenics, respectively, and it occurred with equal frequency in controls (44%, 31/70 and 3.0%, 6/198). TIDRU(1) occurs at a location similar to the third intracytoplasmic 48-nucleotide repeat unit in the DRD4 that is associated with ADHD. Although these data do not suggest an association of TIDRU(1) with schizophrenia, additional studies are needed to see whether TIDRU(1) confers a clinical phenotype.

Mutation frequency is reduced in the cerebellum of Big Blue mice overexpressing a human wild type SOD1 gene.

Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder caused by motor neuron degeneration. A similar disease phenotype is observed in mice overexpressing a mutant human hSOD1 gene (G93A, 1Gurd(1)). Mice transgenic for lacI (Big Blue) and human mutant (1Gurd(1), Mut hSOD1) or wild type (2Gur, Wt hSOD1) SOD1 genes were used to examine spontaneous mutation, oxidative DNA damage, and neurodegeneration in vivo. The frequency and pattern of spontaneous mutation were determined for forebrain (90% glia), cerebellum (90% neurons) and thymus from 5-month-old male mice. Mutation frequency is not elevated significantly and mutation pattern is unaltered in Mut hSOD1 mice compared to control mice. Mutation frequency is reduced significantly in the cerebellum of Wt hSOD1 mice (1.6x10(-5); P=0.0093; Fisher's Exact Test) compared to mice without a human transgene (2.7x10(-5)). Mutation pattern is unaltered. This first report of an endogenous factor that can reduce in vivo, the frequency of spontaneous mutation suggests potential strategies for lowering mutagenesis related to aging, neurodegeneration, and carcinogenesis.

Detection of mutations by single-strand conformation polymorphism (SSCP) analysis and SSCP-hybrid methods.

Single-strand conformation polymorphism (SSCP) analysis detects mutations based on the fact that single-nucleotide changes in DNA sequences alter the mobility of single-stranded DNA in nondenaturing gels. Four methods for detecting mutations based on SSCP are described here. (1) Traditional SSCP analysis is technically easy and can be used for screening large numbers of samples. SSCP-hybrid methods detect mutations based on either an SSCP effect or an altered component independent of the SSCP effect. (2) Dideoxy fingerprinting (ddF) involves PCR amplification of the target and creation of a set of dideoxy-terminated strands with the mutation. (3) Bi-directional dideoxy fingerprinting (Bi-ddF) involves production of two sets of dideoxy-terminated strands that are generated from two different primers. (4) Restriction endonuclease fingerprinting (REF) involves cleavage of the amplified target with five to six groups of restriction endonucleases.

Pyrophosphorolysis-activated polymerization (PAP): application to allele-specific amplification.

To measure mutation load or to detect minimal residual disease, a robust method for identifying one mutant allele in the range of 10(6)-10(9) wild-type alleles would be advantageous. Herein, we present evidence that pyrophosphorolysis-activated polymerization (PAP) has the potential to provide a highly specific and robust method of allele-specific amplification if DNA polymerases with higher pyrophosphorolysis activity can be found or engineered. In PAP, pyrophosphorolysis and polymerization by DNA polymerase are coupled serially by utilizing a pyrophosphorolysis-activatable oligonucleotide (P*). P*, which is an allele-specific oligonucleotide with a dideoxynucleotide at the 3' terminus, can be activated by pyrophosphorolysis to remove the 3' terminal dideoxynucleotide in the presence of pyrophosphate (PPi) and the complementary strand of the allelic template; then the activated P* can be extended by DNA polymerization. Specificity results from both pyrophosphorolysis and polymerization because significant nonspecific amplification requires the combination of mismatch pyrophosphorolysis and misincorporation by the DNA polymerase, which is an extremely rare event. Proof of principle has been achieved with a polymorphic site within the human D1 dopamine receptor gene. The effects of the dideoxyoligonucleotide sequences, DNA polymerases, PPi concentrations, allele-specific templates, pH and dNTP concentrations were examined.

Evidence that proximal multiple mutations in Big Blue transgenic mice are dependent events.

To characterize the nature of multiple mutations in the tissues of an intact animal, the Big Blue transgenic mouse mutation detection system was used to examine 1459 mutants from eight normal tissues and 507 mutants from 11 tumors. Multiple mutations occurred and predominantly doublet mutants were identified (i.e. two mutations within one mutant lacI gene), but multiplets of up to five mutations were observed. The frequency of doublets in normal tissues and spontaneous tumors from p53-deficient mice was enhanced to the same degree (660 and 667 fold, respectively) over that expected for two independent mutational events. Doublets, multiplets and singlets have similar patterns of mutation. The distance between mutations in doublets fits an exponential distribution, not that expected for randomly spaced events, suggesting that many doublets occur in rapid succession within the same cell cycle.

Factor IX mutations in South Africans and African Americans are compatible with primarily endogenous influences upon recent germline mutations.

Similar patterns of germline mutations in the factor IX gene (F9) have been observed in certain geographically and racially diverse populations. Germline mutation data have not been available from any region of Africa or from the Black race. Analysis of mutation data for Blacks is of interest, since this race has a high frequency of polymorphism compared to other races. This high frequency has been interpreted as evidence for the "out of Africa" hypothesis for the origin of humans, but it is possible that Blacks have a higher mutation rate due to genetic differences or environmental exposures. We report 26 independent mutations that were detected in patients of mixed races with hemophilia B from South Africa. The pattern of mutation in patients from this African country was similar to that of U.S. Caucasians. In addition, 22 independent mutation were detected in African American patients. The patterns of independent germline mutation in 22 African Americans (and in a combination 34 North American and African Blacks) is similar to that of U.S. Caucasians. Neither genetic differences between the Black and Caucasian races nor environmental and cultural differences between South Africa and the U.S. alter the germline pattern of mutation observed in F9. Hum Mutat 16:372, 2000.

High plating density improves Big Blue system efficiency without loss of sensitivity.

To increase efficiency in the Big Blue system, the plating density was increased from 15000 to 30000 or 45000 plaque forming units (pfus) per plate by increasing the density of the E. coli lawn and decreasing individual plaque size. Small plaque size ensured minimal overlap of the plaques. Liver from one 3- and one 25-month-old mouse (low and high mutation frequencies, respectively) was analyzed and neither plating density nor plaque size affected mutant/mutation frequency and pattern. The color intensity of particular mutant plaques was not affected by plaque size or plating density. Optimal sensitivity is achieved by sequencing mutants to calculate the mutation frequency from the mutant frequency and to identify altered patterns of mutation. Detailed effort and cost accounting of the Big Blue system (including mouse handling, DNA extraction, plaque screening, plaque purification, and DNA sequencing) reveals that one-quarter of the total effort is devoted to plating and screening of plates. This effort is reduced two fold with high plating density. The total cost of the Big Blue system is reduced by 17%. The total cost of the High Plating Density Big Blue system is now only 12% more costly than a selectable assay and offers an extensively validated system with a large mutation database representing a decade of effort.