Identification of a 220-kb insertion into the Duchenne gene in a family with an atypical course of muscular dystrophy

Most known mutations in the gene region responsible for Duchenne or Becker muscular dystrophy are deletions of varying extent. Here we describe a 220-kb insertion within the DMD/BMD gene that cosegregates with a somewhat atypical course of muscular dystrophy in a pedigree. The insertion is demonstrated by field-inversion gel electrophoresis as an enlarged SfiI fragment hybridizing to probe J-Bir, while neighboring SfiI fragments (detected by probes PERT 87 and J-66) are unchanged. Hybridization with DMD c-DNA probes did not reveal alterations in coding sequences. In this pedigree, the altered SfiI fragments provide convenient markers for carrier identification.     

Molecular analysis of hemophilia A mutations in the Finnish population

We have examined the Finnish hemophilia A population for factor VIII gene mutations. This study included 83 unrelated patients and revealed 10 mutations associated with hemophilia. Using cloned cDNA, genomic, and oligonucleotide probes, we have identified three classes of mutations: five mutations causing the loss of TaqI restriction sites, a point mutation resulting in a new TaqI site, and four partial gene deletions. Although exons 5 and 6 were involved in three of the four partial gene deletions, the extent of the DNA lost differs in each case. The fourth deletion was located entirely within intron 1 and segregated with the disease in a large hemophilia pedigree. There was no history of hemophilia in eight of the 10 families. The origin of the mutation was determined in six of these pedigrees, two of which showed evidence for maternal mosaicism.     

High-resolution joint linkage disequilibrium and linkage mapping of quantitative trait loci based on sibship data

This paper proposes variance component models for high resolution joint linkage disequilibrium (LD) and linkage mapping of quantitative trait loci (QTL) based on sibship data; this can include population data if independent individuals are treated as single sibships. One application of these models is late onset complex disease gene mapping, when parental data are not available. The models simultaneously incorporate both LD and linkage information. The LD information is contained in mean coefficients of sibship data. The linkage information is contained in the variance-covariance matrices of trait values for sibships with at least two siblings. We derive formulas for calculating the probability of sharing two trait alleles identical by descent (IBD) for sibpairs in interval mapping of QTL; this is the coefficient of dominant variance of the trait covariance of sibpairs on major QTL. To investigate the performance of the formulas, we calculate the numerical values via the formulas and get satisfactory approximations. We compare the power and sample sizes for both LD and linkage mapping. By simulation and theoretical analysis, we compare the results with those of Fulker and Abecasis "AbAw" approach. It is well known that the resolution of linkage analysis can be low for complex disease gene mapping. LD mapping, on the other hand, can increase mapping precision and is useful in high resolution mapping. Linkage analysis is less sensitive to population subdivisions and admixtures. The level of LD is sensitive to population stratification which may easily lead to spurious association. Performing a joint analysis of LD and linkage mapping can help to overcome the limits of both approaches. Moreover, the advantages of the two complementary strategies can be utilized maximally. In practice, linkage analysis may be performed using pedigree data to identify suggestive linkage between markers and trait loci based on a sparse marker map. In the presence of linkage, joint LD and linkage mapping can be carried out to do fine gene mapping based on a dense genetic map using both pedigree and population data. Population and pedigree data of any type can be combined to perform a joint analysis of high resolution LD and linkage mapping of QTL by generalizing the method.     

Deletions in the Parkin gene and genetic heterogeneity in a Greek family with early onset Parkinson’s disease

Parkinson’s disease is the second most common neurodegenerative disease after Alzheimer’s disease and is manifested as a movement disorder. A positive family history is the second most important risk factor for developing the illness, after age. Both autosomal dominant and recessive forms of the illness have been described. Recently deletions in a novel gene, parkin, have been associated with the autosomal recessive form of the illness in Japanese families. In this study, we demonstrate that deletions of exons 5, 6 and 7 of the parkin gene are present in two affected individuals of a Greek pedigree with early onset Parkinson’s disease. However, no deletions were identified in a different branch of the same pedigree with three affected individuals. These results suggest that deletions in the parkin gene will be found in other families besides those of Japanese origin and that there must be at least one additional locus responsible for early onset autosomal recessive Parkinson’s disease. Received: 9 June 1998 / Accepted: 10 August 1998     

Isolation of Y Chromosome-Specific Sequences from Silene Latifolia and Mapping of Male Sex-Determining Genes Using Representational Difference Analysis

The genomic subtraction method representational difference analysis (RDA) was used to identify male-specific restriction fragments in the dioecious plant Silene latifolia. Male-specific restriction fragments are linked to the male sex chromosome (the Y chromosome). Four RDA-derived male-specific restriction fragments were used to identify polymorphisms in a collection of X-ray-generated mutant plants with either hermaphroditic or asexual flowers. Some of the mutants have cytologically detectable deletions in the Y chromosome that were correlated with loss of male-specific restriction fragments. One RDA-derived probe detected a restriction fragment present in all mutants, indicating that each has retained Y chromosomal DNA. The other three probes detected genomic fragments that were linked in a region deleted in some hermaphroditic and some asexual mutants. Based on the mutant phenotypes and the correlation of cytologically visible deletions with loss of male-specific restriction fragments, these markers were assigned to positions on the Y chromosome close to the carpel suppression locus. This RDA mapping also revealed a Y-linked locus, not previously described, which is responsible for early stamen development.     

A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip whole-genome resequencing platform

DNA resequencing arrays enable rapid acquisition of high-quality sequence data. This technology represents a promising platform for rapid high-resolution genotyping of microorganisms. Traditional array-based resequencing methods have relied on the use of specific PCR-amplified fragments from the query samples as hybridization targets. While this specificity in the target DNA population reduces the potential for artifacts caused by cross-hybridization, the subsampling of the query genome limits the sequence coverage that can be obtained and therefore reduces the technique's resolution as a genotyping method. We have developed and validated an Affymetrix Inc. GeneChip(R) array-based, whole-genome resequencing platform for Francisella tularensis, the causative agent of tularemia. A set of bioinformatic filters that targeted systematic base-calling errors caused by cross-hybridization between the whole-genome sample and the array probes and by deletions in the sample DNA relative to the chip reference sequence were developed. Our approach eliminated 91% of the false-positive single-nucleotide polymorphism calls identified in the SCHU S4 query sample, at the cost of 10.7% of the true positives, yielding a total base-calling accuracy of 99.992%.     

Localization of the Fanconi anemia complementation group D gene to a 200-kb region on chromosome 3p25.3

Fanconi anemia (FA) is a rare autosomal recessive disease manifested by bone-marrow failure and an elevated incidence of cancer. Cells taken from patients exhibit spontaneous chromosomal breaks and rearrangements. These breaks and rearrangements are greatly elevated by treatment of FA cells with the use of DNA cross-linking agents. The FA complementation group D gene (FANCD) has previously been localized to chromosome 3p22-26, by use of microcell-mediated chromosome transfer. Here we describe the use of noncomplemented microcell hybrids to identify small overlapping deletions that narrow the FANCD critical region. A 1.2-Mb bacterial-artificial-chromosome (BAC)/P1 contig was constructed, bounded by the marker D3S3691 distally and by the gene ATP2B2 proximally. The contig contains at least 36 genes, including the oxytocin receptor (OXTR), hOGG1, the von Hippel-Lindau tumor-suppressor gene (VHL), and IRAK-2. Both hOGG1 and IRAK-2 were excluded as candidates for FANCD. BACs were then used as probes for FISH analyses, to map the extent of the deletions in four of the noncomplemented microcell hybrid cell lines. A narrow region of common overlapping deletions limits the FANCD critical region to approximately 200 kb. The three candidate genes in this region are TIGR-A004X28, SGC34603, and AA609512.     

An unknown genetic defect increases venous thrombosis risk, through interaction with protein C deficiency.

We used two-locus segregation analysis to test whether an unknown genetic defect interacts with protein C deficiency to increase susceptibility to venous thromboembolic disease in a single large pedigree. Sixty-seven pedigree members carry a His107Pro mutation in the protein C gene, which reduces protein C levels to a mean of 46% of normal. Twenty-one carriers of the mutation and five other pedigree members had verified thromboembolic disease. We inferred the presence in this pedigree of a thrombosis-susceptibility gene interacting with protein C deficiency, by rejecting the hypothesis that the cases of thromboembolic disease resulted from protein C deficiency alone and by not rejecting Mendelian transmission of the interacting gene. When coinherited with protein C deficiency, the interacting gene conferred a probability of a thrombotic episode of approximately 79% for men and approximately 99% for women, before age 60 years.     

Adrenoleukodystrophy: a complex chromosomal rearrangement in the Xq28 red/green-color-pigment gene region indicates two possible gene localizations.

We have characterized a complex chromosomal rearrangement in band Xq28, in an adrenoleukodystrophy patient who also has blue-cone monochromacy. A 130-kb region upstream from the color-vision pigment genes was isolated as yeast artificial chromosome or cosmid clones. Another Xq28 sequence, not included in the above region, was obtained by cloning a deletion breakpoint from the patient. Using probes derived from the cloned sequences, we have shown that the rearrangement affects the color-pigment genes and includes two deletions, most likely separated by a large (greater than 110-kb) inversion. One deletion encompasses part of the pigment gene cluster and 33 kb of upstream sequences and accounts for the patient's blue-cone monochromacy. If this rearrangement also caused ALD, the disease gene would be expected to lie within or close to one of the deletions. However, deletions were not detected in a 50-kb region upstream of the red-color-pigment gene in 81 other ALD patients. Two CpG islands were mapped, at 46 and 115 kb upstream from the pigment genes.     

Exon array CGH: detection of copy-number changes at the resolution of individual exons in the human genome

The development of high-throughput screening methods such as array-based comparative genome hybridization (array CGH) allows screening of the human genome for copy-number changes. Current array CGH strategies have limits of resolution that make detection of small (less than a few tens of kilobases) gains or losses of genomic DNA difficult to identify. We report here a significant improvement in the resolution of array CGH, with the development of an array platform that utilizes single-stranded DNA array elements to accurately measure copy-number changes of individual exons in the human genome. Using this technology, we screened 31 patient samples across an array containing a total of 162 exons for five disease genes and detected copy-number changes, ranging from whole-gene deletions and duplications to single-exon deletions and duplications, in 100% of the cases. Our data demonstrate that it is possible to screen the human genome for copy-number changes with array CGH at a resolution that is 2 orders of magnitude higher than that previously reported.     

Genetic counseling in rare syndromes: a resampling method for determining an approximate confidence interval for gene location with linkage data from a single pedigree.

Multipoint linkage analysis is a powerful method for mapping a rare disease gene on the human gene map despite limited genotype and pedigree data. However, there is no standard procedure for determining a confidence interval for gene location by using multipoint linkage analysis. A genetic counselor needs to know the confidence interval for gene location in order to determine the uncertainty of risk estimates provided to a consultant on the basis of DNA studies. We describe a resampling, or "bootstrap," method for deriving an approximate confidence interval for gene location on the basis of data from a single pedigree. This method was used to define an approximate confidence interval for the location of a gene causing nonsyndromal X-linked mental retardation in a single pedigree. The approach seemed robust in that similar confidence intervals were derived by using different resampling protocols. Quantitative bounds for the confidence interval were dependent on the genetic map chosen. Once an approximate confidence interval for gene location was determined for this pedigree, it was possible to use multipoint risk analysis to estimate risk intervals for women of unknown carrier status. Despite the limited genotype data, the combination of the resampling method and multipoint risk analysis had a dramatic impact on the genetic advice available to consultants.     

A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip® whole-genome resequencing platform

DNA resequencing arrays enable rapid acquisition of high-quality sequence data. This technology represents a promising platform for rapid high-resolution genotyping of microorganisms. Traditional array-based resequencing methods have relied on the use of specific PCR-amplified fragments from the query samples as hybridization targets. While this specificity in the target DNA population reduces the potential for artifacts caused by cross-hybridization, the subsampling of the query genome limits the sequence coverage that can be obtained and therefore reduces the technique's resolution as a genotyping method. We have developed and validated an Affymetrix Inc. GeneChip® array-based, whole-genome resequencing platform for Francisella tularensis, the causative agent of tularemia. A set of bioinformatic filters that targeted systematic base-calling errors caused by cross-hybridization between the whole-genome sample and the array probes and by deletions in the sample DNA relative to the chip reference sequence were developed. Our approach eliminated 91% of the false-positive single-nucleotide polymorphism calls identified in the SCHU S4 query sample, at the cost of 10.7% of the true positives, yielding a total base-calling accuracy of 99.992%.     

Detecting deletions in families affected by a dominant disease by use of marker data

A method of testing for whether inherited deletions are a cause of a single-locus dominant disease was derived, involving analysis of the marker segregation within the pedigree of a single family that segregates for the disease. It is shown that markers can be used to test deductively for the presence of an inherited deletion. The probabilities of confirming or rejecting the presence of a deletion in an arbitrary pedigree without inbreeding are then derived. The power of the test is shown to be limited in single trios but to increase rapidly as the size of the pedigree increases. For larger pedigrees, the probabilities of confirming or rejecting a deletion are higher than 0.9 for SNPs having a minor allele frequency greater than 0.4. The probabilities are higher using multiallelic markers such as microsatellites, reaching levels as high as 0.9 in even rather small pedigrees. In certain cases the test outcome is not deductive, a deletion being neither confirmed nor rejected. It is shown to still be possible then to employ a statistical test for the presence of a deletion by use of an a priori probability for a deletion.     

Restriction endonuclease mapping of six novel deletions of the factor VIII gene in hemophilia A

Summary Hemophilia A is an X-linked disease of blood coagulation caused by deficiency of factor VIII. Using cloned cDNA, genomic and synthetic oligonucleotide factor VIII probes, we have identified six novel partial gene deletions in patients with severe hemophilia A. We have previously reported six other deletions of the factor VIII gene. The number of gross molecular defects (deletions, insertions) in the factor VIII gene in our series of 240 patients is 17 (3 insertions and 2 complicated deletions will be described elsewhere). No association was observed between the size or location of the deletions and the presence of inhibitors to factor VIII. No deletion breakpoint hotspots have been identified by restriction analysis. The parental origin of several of the deletions was determined.     

New method for generating deletions and gene replacements in Escherichia coli.

We describe a method for generating gene replacements and deletions in Escherichia coli. The technique is simple and rapid and can be applied to most genes, even those that are essential. What makes this method unique and particularly effective is the use of a temperature-sensitive pSC101 replicon to facilitate the gene replacement. The method proceeds by homologous recombination between a gene on the chromosome and homologous sequences carried on a plasmid temperature sensitive for DNA replication. Thus, after transformation of the plasmid into an appropriate host, it is possible to select for integration of the plasmid into the chromosome at 44 degrees C. Subsequent growth of these cointegrates at 30 degrees C leads to a second recombination event, resulting in their resolution. Depending on where the second recombination event takes place, the chromosome will either have undergone a gene replacement or retain the original copy of the gene. The procedure can also be used to effect the transfer of an allele from a plasmid to the chromosome or to rescue a chromosomal allele onto a plasmid. Since the resolved plasmid can be maintained by selection, this technique can be used to generate deletions of essential genes.     

两个DMD cDNA片段在进行性肌营养不良症基因诊断中的应用

本文使用了缺失热点区的两个DMD cDNA片段1b-2a及8为探针检测Duc-henne型及Becker型肌营养不良(DMD/BMD)患者的基因缺失。在34例不相关患者中分别检测到5例及8例基因片段缺失,缺失检测率分别为14.7％及23.5％,总检出率为38.2％。结果表明,中国肌营养不良患者的基因缺失也不是随机分布的,主要集中于基因中心附近,其次在基因5′侧。     

Genotype-phenotype correlations in families with deletions in the von Hippel-Lindau (VHL) gene

Von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome characterized by predisposition for bilateral and multi-centric hemangioblastoma in the retina and central nervous system, pheochromocytoma, renal cell carcinoma, and cysts in the kidney, pancreas, and epididymis. We describe five families for which direct sequencing of the coding region of the VHL gene had failed to identify the family-specific mutation. Further molecular analysis revealed deletions involving the VHL gene in each of these families. In four families, partial deletions of one or more exons were detected by Southern blot analysis. In the fifth family, FISH analysis demonstrated the deletion of the entire VHL gene. Our results show that (quantitative) Southern blot analysis is a sensitive method for detecting germline deletions of the VHL gene and should be implemented in routine DNA diagnosis for VHL disease. Our data support the previously established observation that families with a germline deletion have a low risk for pheochromocytoma. Further unraveling of genotype-phenotype correlations in VHL disease has revealed that families with a full or partial deletion of the VHL gene exhibit a phenotype with a preponderance of central nervous system hemangioblastoma.     

一个可用于复杂大群体的可视化系谱绘制和分析软件

对于在遗传研究和家系研究中大的系谱结构图还很难分析。系谱的绘制通常是遗传性状的分析研究的第一步。系图可以反映整个群体的结构、每个个体之间的相互关系以及基因流的走向,便于理解遗传性状的本质。因为所用家系数目的增大和复杂性的增加,绘制1个清晰的系谱有时变得十分困难。因此开发了1种名为Pedigraph软件,可以解决这个问题。Pedigraph能够完成对于大的复杂的群体的系谱绘制工作,并能进行相应的系谱分析。初步的测试表明这个软件在研究动植物的遗传育种中是1个有用的工具,同时它也可以用于人类的群体和历史等方面的研究。     

Comparisons of substitution, insertion and deletion probes for resequencing and mutational analysis using oligonucleotide microarrays

Although oligonucleotide probes complementary to single nucleotide substitutions are commonly used in microarray-based screens for genetic variation, little is known about the hybridization properties of probes complementary to small insertions and deletions. It is necessary to define the hybridization properties of these latter probes in order to improve the specificity and sensitivity of oligonucleotide microarray-based mutational analysis of disease-related genes. Here, we compare and contrast the hybridization properties of oligonucleotide microarrays consisting of 25mer probes complementary to all possible single nucleotide substitutions and insertions, and one and two base deletions in the 9168 bp coding region of the ATM (ataxia telangiectasia mutated) gene. Over 68 different dye-labeled single-stranded nucleic acid targets representing all ATM coding exons were applied to these microarrays. We assess hybridization specificity by comparing the relative hybridization signals from probes perfectly matched to ATM sequences to those containing mismatches. Probes complementary to two base substitutions displayed the highest average specificity followed by those complementary to single base substitutions, single base deletions and single base insertions. In all the cases, hybridization specificity was strongly influenced by sequence context and possible intra- and intermolecular probe and/or target structure. Furthermore, single nucleotide substitution probes displayed the most consistent hybridization specificity data followed by single base deletions, two base deletions and single nucleotide insertions. Overall, these studies provide valuable empirical data that can be used to more accurately model the hybridization properties of insertion and deletion probes and improve the design and interpretation of oligonucleotide microarray-based resequencing and mutational analysis.