Current Protocols in Human Genetics

Harvard Medical School - Partners Healthcare Center for Genetics and Genomics, Boston, Massachusetts, USA.
Current protocols in human genetics / editorial board, Jonathan L. Haines ... [et al.] 05/2008; Chapter 7:Unit7.9. DOI: 10.1002/0471142905.hg0709s57
Source: PubMed


The development of high-throughput DNA sequencing techniques has made direct DNA sequencing of PCR-amplified genomic DNA a rapid and economical approach to the identification of polymorphisms that may play a role in disease. Point mutations as well as small insertions or deletions are readily identified by DNA sequencing. The mutations may be heterozygous (occurring in one allele while the other allele retains the normal sequence) or homozygous (occurring in both alleles). Sequencing alone cannot discriminate between true homozygosity and apparent homozygosity due to the loss of one allele due to a large deletion. In this unit, strategies are presented for using PCR amplification and automated fluorescence-based sequencing to identify sequence variation. The size of the project and laboratory preference and experience will dictate how the data is managed and which software tools are used for analysis. A high-throughput protocol is given that has been used to search for mutations in over 200 different genes at the Harvard Medical School - Partners Center for Genetics and Genomics (HPCGG,

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    • "[14]. 3. Sequencing of the whole dystrophin gene was performed in those patients who had no deletion or duplication by the 2 previous methods using ABI 3037 [15]. "
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    ABSTRACT: Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophies affecting approximately 1:3500 male live births. Deletion of the dystrophin gene accounts for approximately 65% of mutations, duplications occur in 6–10% while the remaining 20–30% are point mutations, small deletion/insertions, or splicing mutations. Aim To study non-deletion mutations in a sample of Egyptian patients with DMD as most previous studies focused on deletion mutations. Patients and methods The study included 25 patients with DMD from 18 different families from the genetics clinic, Children’s Hospital, Ain Shams University. Diagnosis was made based on typical clinical findings, high CPK and EMG result. Molecular analysis included Polymerase Chain Reaction (PCR) followed by multiplex ligation-dependent probe amplification (MLPA) to those patients with no deletion by PCR. Direct sequencing of the whole dystrophin gene was done to those patients who had no deletion or duplication by the previous 2 methods. Results Non-deletion mutation included duplications (5 families (27.8%)) which are higher than previously reported and point mutation (c.583C>T) in only one family. Deletion mutations were found in 9 families (50%) and no mutation found in 3 families (16.7%). Interestingly, 60% of the duplications were located in the distal region of the dystrophin gene. A frame shift mutation was identified in most patients (93%) except one with duplication of exons 50–51 who had an unexpected severe disease with an early age of onset. Also, an intragenic deletion involving the 5′ end of the dystrophin gene (deletion of muscle protomor and exon 1) was found in another patient with severe disease without cardiac involvement. Conclusion The relative higher frequency of duplication mutations in Egyptian patients with DMD may indicate that MLPA and not PCR should be preferred for molecular testing of Egyptian patients with DMD.
    Egyptian Journal of Medical Human Genetics 07/2014; 15(3). DOI:10.1016/j.ejmhg.2014.03.004
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    • "In 2006, Tjeldhorn et al. demonstrated a reduction from 72 hours manual labor in the fully manual mutation detection processing for 16 Marfan syndrome patients to 23 hours of manual work in an automated process reducing testing time from 145 total hours to 67 hours[22]. In addition in 2008, protocols were published for mutation detection using automated fluorescence-based sequencing[23,24]. With more than 40 different primary muscle disorders, a different approach to sequencing and diagnostics was desperately needed. "
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    ABSTRACT: Background One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive. These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels. The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. Results An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other patients). Methods and assay sequences are reported in this paper. Conclusion This automated process allows laboratories to discover DNA variations in a short time and at low cost.
    BMC Genetics 10/2009; 10(1). DOI:10.1186/1471-2156-10-66 · 2.40 Impact Factor
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    ABSTRACT: The immense molecular diversity of neurons challenges our ability to understand the genetic and cellular etiology of neuropsychiatric disorders. Leveraging knowledge from neurobiology may help parse the genetic complexity: identifying genes important for a circuit that mediates a particular symptom of a disease may help identify polymorphisms that contribute to risk for the disease as a whole. The serotonergic system has long been suspected in disorders that have symptoms of repetitive behaviors and resistance to change, including autism. We generated a bacTRAP mouse line to permit translational profiling of serotonergic neurons. From this, we identified several thousand serotonergic-cell expressed transcripts, of which 174 were highly enriched, including all known markers of these cells. Analysis of common variants near the corresponding genes in the AGRE collection implicated the RNA binding protein CELF6 in autism risk. Screening for rare variants in CELF6 identified an inherited premature stop codon in one of the probands. Subsequent disruption of Celf6 in mice resulted in animals exhibiting resistance to change and decreased ultrasonic vocalization as well as abnormal levels of serotonin in the brain. This work provides a reproducible and accurate method to profile serotonergic neurons under a variety of conditions and suggests a novel paradigm for gaining information on the etiology of psychiatric disorders.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 02/2013; 33(7):2732-2753. DOI:10.1523/JNEUROSCI.4762-12.2013 · 6.34 Impact Factor
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