Motivation: Most genotyping technologies for single nucleotide polymorphism (SNP) markers use standard clustering methods to ‘call’ the SNP genotypes. These methods are not always optimal in ...distinguishing the genotype clusters of a SNP because they do not take advantage of specific features of the genotype calling problem. In particular, when family data are available, pedigree information is ignored. Furthermore, prior information about the distribution of the measurements for each cluster can be used to choose an appropriate model-based clustering method and can significantly improve the genotype calls. One special genotyping problem that has never been discussed in the literature is that of genotyping of trisomic individuals, such as individuals with Down syndrome. Calling trisomic genotypes is a more complicated problem, and the addition of external information becomes very important. Results: In this article, we discuss the impact of incorporating external information into clustering algorithms to call the genotypes for both disomic and trisomic data. We also propose two new methods to call genotypes using family data. One is a modification of the K-means method and uses the pedigree information by updating all members of a family together. The other is a likelihood-based method that combines the Gaussian or beta-mixture model with pedigree information. We compare the performance of these two methods and some other existing methods using simulation studies. We also compare the performance of these methods on a real dataset generated by the Illumina platform (www.illumina.com). Availability: The R code for the family-based genotype calling methods (SNPCaller) is available to be downloaded from the following website: http://watson.hgen.pitt.edu/register. Contact: liny@upmc.edu Supplementary information: Supplementary data are available at Bioinformatics online.
Myotonic dystrophy type 1 (DM1) is a form of muscular dystrophy causing progressive muscle loss and weakness. Although clinical features can manifest at any age, it is the most common form of ...muscular dystrophy with onset in adulthood. DM1 is an autosomal dominant condition, resulting from an unstable CTG expansion in the 3′-untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. The age of onset and the severity of the phenotype are roughly correlated with the size of the CTG expansion. Multiple methodologies can be used to diagnose affected individuals with DM1, including polymerase chain reaction, Southern blot, and triplet repeat-primed polymerase chain reaction. Recently, triplet repeat interruptions have been described, which may affect clinical outcomes of a fully-variable allele in DMPK. This document supersedes the Technical Standards and Guidelines for Myotonic Dystrophy originally published in 2009 and reaffirmed in 2015. It is designed for genetic testing professionals who are already familiar with the disease and the methods of analysis.
The diagnosis of many genetic disorders relies on a combination of clinical suspicion and confirmatory genetic testing. Our laboratory uses a standard methylation-sensitive PCR (MSP) to target the ...differentially methylated SNRPN gene to test for Prader-Willi syndrome (PWS) and Angelman syndrome. One patient, a 27-month-old female, who lacked the classical clinical features of PWS, but had a molecular diagnosis of PWS by MSP by another laboratory, had repeat testing in our laboratory. Testing by MSP in our laboratory also identified an apparent loss of the unmethylated paternal allele, consistent with a diagnosis of PWS. Confirmatory testing using Southern blot analysis with a methylation-sensitive restriction enzyme showed a normal pattern of methylation, detecting both the methylated maternal and unmethylated paternal alleles. To investigate these discrepant results, we amplified and sequenced the SNRPN locus in this patient and identified a single nucleotide change within the binding site for the unmethylated DNA-specific primer. These results indicate this nucleotide change led to allelic dropout in the MSP analysis, yielding the false-positive result. Subsequently, MSP analysis using an alternate primer set that was developed by our laboratory detected both methylated and unmethylated alleles. These findings illustrate that allelic dropout due to the presence of rare polymorphisms can cause false-positive results in commonly used MSP assays and lead to molecular misdiagnosis.
To develop a high resolution microarray based method to detect single- and multiexons gene deletions and duplications.
We have developed a high-resolution comparative genomic hybridization array to ...detect single- and multiexon deletions and duplications in a large set of genes on a single microarray, using the NimbleGen 385K array with an exon-centric design.
We have successfully developed, validated, and implemented a targeted gene comparative genomic hybridization arrays for detecting single- and multiexon deletions and duplication in autosomal and X-linked disease-associated genes.
The comparative genomic hybridization arrays can be adopted readily by clinical molecular diagnostic laboratories as a rapid, cost-effective, highly sensitive, and accurate approach for the detection of single- and multiexon deletions or duplications, particularly in cases where direct sequencing fails to identify a mutation.
Changes in the phosphorylation state of p53 are important in increasing its half-life and potency as a transcription factor. To investigate their roles, serine residues 15 and 37 were mutated to ...alanines and the mutated proteins were expressed stably at low basal levels in Li-Fraumeni-derived p53-null human fibroblasts. The accumulation of p53 after DNA damage was analysed quantitatively in multiple clones. Mutation of serine 15, serine 37 or both impaired the accumulation of the protein after exposing the cells to ultraviolet radiation (50-100% increase for the mutant proteins, 500% increase for wild-type p53) but not after treatment with adriamycin. The diminished accumulation of mutant p53 protein is due to a reduction of basal HDM association. Analysis of p53-dependent transcription revealed that phosphorylation of serine 15 is required to maintain basal levels of p21 mRNA. These results provide new evidence for an important function of serine 37 phosphorylation, clearly distinguish the pathways of p53 activation in response to ultraviolet radiation or DNA damage inflicted by adriamycin, and reveal that serine 15 is crucial to support the p53-mediated basal expression of p21.
Characterizing heterozygous insertions or deletions in genes by PCR and Sanger sequencing can be a challenge due to overlapping sequencing traces produced by overlapping templates. This is ...particularly problematic for clinical diagnostic laboratories, because mutations must be precisely characterized. Although the mutation detection software used by clinical diagnostic laboratories reliably identifies small insertions and deletions, overlapping deletions and insertions on opposite chromosomes, complex rearrangements, and insertions or deletions close to the primer sites may be missed. Here we describe a rapid, simple method to confirm and precisely characterize deletions and insertions using a capillary-based gel electrophoresis system. This technique has been applied to a series of patients with deletion, duplication, or insertion mutations identified by sequencing, as well as to patients with repeat tract polymorphisms, to demonstrate the utility of this method.
Gene sequencing panels are a powerful diagnostic tool for many clinical presentations associated with genetic disorders. Advances in DNA sequencing technology have made gene panels more economical, ...flexible, and efficient. Because the genes included on gene panels vary widely between laboratories in gene content (e.g., number, reason for inclusion, evidence level for gene–disease association) and technical completeness (e.g., depth of coverage), standards that address technical and clinical aspects of gene panels are needed. This document serves as a technical standard for laboratories designing, offering, and reporting gene panel testing. Although these principles can apply to multiple indications for genetic testing, the primary focus is on diagnostic gene panels (as opposed to carrier screening or predictive testing) with emphasis on technical considerations for the specific genes being tested. This technical standard specifically addresses the impact of gene panel content on clinical sensitivity, specificity, and validity—in the context of gene evidence for contribution to and strength of evidence for gene–disease association—as well as technical considerations such as sequencing limitations, presence of pseudogenes/gene families, mosaicism, transcript choice, detection of copy-number variants, reporting, and disclosure of assay limitations.
PurposeThe ability of a single technology, next-generation sequencing, to provide both sequence and copy number variant (CNV) results has driven the merger of clinical cytogenetics and molecular ...genetics. Consequently, the distinction between the definition of a sequence variant and a CNV is blurry. As the 2015 American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) standards and guidelines for interpretation of sequence variants address CNV classification only sparingly, this study focused on adapting ACMG/AMP criteria for single-gene CNV interpretation.MethodsCNV-specific modifications of the 2015 ACMG/AMP criteria were developed and their utility was independently tested by three diagnostic laboratories. Each laboratory team interpreted the same 12 single-gene CNVs using three systems: (1) without ACMG/AMP guidance, (2) with ACMG/AMP criteria, and (3) with new modifications. A replication study of 12 different CNVs validated the modified criteria.ResultsThe adapted criteria system presented here showed improved concordance and usability for single-gene CNVs compared with using the ACMG/AMP interpretation guidelines focused on sequence variants.ConclusionThese single-gene CNV criteria modifications could be used as a supplement to the ACMG/AMP guidelines for sequence variants, allowing for a streamlined workflow and a step toward a uniform classification system for both sequence and copy number alterations.
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