MDS-associated somatic mutations and clonal hematopoiesis are common in idiopathic cytopenias of undetermined significance.

Establishing a diagnosis in patients suspected of having a myelodysplastic syndrome (MDS) can be challenging and could be informed by the identification of somatic mutations. We performed a prospective study to examine the frequency and types of mutations encountered in 144 patients with unexplained cytopenias. Based on bone marrow findings, 17% were diagnosed with MDS, 15% with idiopathic cytopenias of undetermined significance (ICUS) and some evidence of dysplasia, and 69% with ICUS and no dysplasia.

Identification of HRAS mutations and absence of GNAQ or GNA11 mutations in deep penetrating nevi.

HRAS is mutated in ∼15% of Spitz nevi, and GNAQ or GNA11 is mutated in blue nevi (46-83% and ∼7% respectively). Epithelioid blue nevi and deep penetrating nevi show features of both blue nevi (intradermal location, pigmentation) and Spitz nevi (epithelioid morphology). Epithelioid blue nevi and deep penetrating nevi can also show overlapping features with melanoma, posing a diagnostic challenge.

Stratifying risk for celiac disease in a large at-risk United States population by using HLA alleles.

Background & Aims: Susceptibility to celiac disease (CD) is related to HLA-DQ2 and DQ8 alleles and the heterodimers they encode. The objective of this study was to stratify risk for CD on the basis of HLA-DQ genotype.

Methods: DNA from 10,191 subjects who are at risk for CD was analyzed for HLA-DQ haplotypes.

Characterization of a recurrent novel large duplication in the cystic fibrosis transmembrane conductance regulator gene.

Recently, DNA rearrangements in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been described with increasing frequency. These large DNA rearrangements are not detected using conventional methods of DNA sequencing, single-strand conformational polymorphism, or denaturing high-performance liquid chromatography. We and others have described methods to detect such rearrangements in the CFTR gene.

Development of a web-based query tool for quality assurance of clinical molecular genetic test results.

The College of American Pathologists molecular pathology checklist item (MOL.20550) calls for periodic review of molecular genetic statistics, including percentages of normal and abnormal findings and allele frequencies. A web-based query tool application for clinical molecular genetic test results was developed to plot dynamically and display genotype and/or allele frequencies for any time period.

CFTR 5T variant has a low penetrance in females that is partially attributable to its haplotype.

Purpose: The study's purpose was to understand the molecular basis for different clinical phenotypes of the 5T variant, a tract of 5 thymidines in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which disrupts processing of CFTR mRNA and reduces synthesis from the corresponding CFTR alleles.

Method: We analyzed the polymorphic TG dinucleotide repeat adjacent to the 5T variant in intron 8 and the codon 470 in exon 10. Patients selected for this study were positive for both the 5T variant and the major cystic fibrosis mutation, Delta F508.

Multiple property tolerance analysis for the evaluation of missense mutations.

Computational prediction of the impact of a mutation on protein function is still not accurate enough for clinical diagnostics without additional human expert analysis. Sequence alignment-based methods have been extensively used but their results highly depend on the quality of the input alignments and the choice of sequences. Incorporating the structural information with alignments improves prediction accuracy.

Novel and recurrent rearrangements in the CFTR gene: clinical and laboratory implications for cystic fibrosis screening.

Because standard techniques used to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene do not detect single or multiple exonic rearrangements, the importance of such rearrangements may be underestimated. Using an in-house developed, single-tube, semi-quantitative fluorescent PCR (SQF PCR) assay, we analyzed 36 DNA samples submitted for extensive CFTR sequencing and identified ten samples with rearrangements. Of 36 patients with classic CF, 10 (28%) harbored various deletions in the CFTR gene, accounting for 14% of CF chromosomes.

Technical validation of a multiplex platform to detect thirty mutations in eight genetic diseases prevalent in individuals of Ashkenazi Jewish descent.

Purpose: This study determines the analytic accuracy of a Luminex bead-based commercial analyte-specific reagent for the simultaneous analysis of 30 mutations prevalent in Ashkenazi Jews at eight genetic disease loci.

Methods: DNA from 20 samples with known abnormal genotypes were run a total of 109 times. DNA from 820 patients with unknown genotypes submitted for Ashkenazi Jewish testing panels were analyzed using our current laboratory techniques.

Extensive sequencing of the CFTR gene: lessons learned from the first 157 patient samples.

Cystic fibrosis (CF) is one of the most common monogenic diseases affecting Caucasians and has an incidence of approximately 1:3,300 births. Currently recommended screening panels for mutations in the responsible gene (CF transmembrane regulator gene, CFTR) do not detect all disease-associated mutations. Our laboratory offers extensive sequencing of the CFTR (ABCC7) gene (including the promoter, all exons and splice junction sites, and regions of selected introns) as a clinical test to detect mutations which are not found with conventional screening.

Detection of 677CT/1298AC "double variant" chromosomes: implications for interpretation of MTHFR genotyping results.

Purpose: Genotyping 37,026 individuals as part of a thrombophilia evaluation, we determined and analyzed the genotypic frequencies of the 677CT and 1298AC mutations in the methylenetetrahydrofolate reductase (MTHFR) gene.

Methods: The 677CT and 1298AC mutations in the MTHFR gene were determined by either a laboratory-developed test involving PCR amplification and restriction digestion utilizing the ABI 3100 capillary electrophoresis apparatus (Applied Biosystems Inc) or by using an Analyte Specific Reagent (ASR) supplied by Third Wave Technologies. The genotype for three specimens with triple variant MTHFR mutations were confirmed by DNA sequencing on the ABI 3100 capillary electrophoresis apparatus.

Molecular screening for diseases frequent in Ashkenazi Jews: lessons learned from more than 100,000 tests performed in a commercial laboratory.

Purpose: To determine the frequency of carriers of Ashkenazi Jewish (AJ) genetic diseases in the US population and compare these numbers with previously published frequencies reported in smaller more isolated cohorts.

Methods: A database containing more than 100,000 genotyping assays was queried. Assays for 10 separate AJ genetic diseases where comparisons were made with published data.

Technology challenges in screening single gene disorders.

Unlabelled: The completion of the human genome project and the accelerated discovery of genes responsible for single gene disorders will allow for the preventive screening of carriers and patients. Accuracy and reliability of analytic tests are major demands on technologies used in a diagnostic screening environment. The consistency of results and the potential of multiplexing suggest mass spectrometry as the method of choice for large-scale genetic screening programs.

Eight novel mutations in the HEXA gene.

Purpose: To characterize novel mutations in the HEXA gene (alpha-subunit beta-hexosaminidase A).

Methods: Subjects included participants in the California Tay-Sachs disease prevention program. DNA samples from 49 subjects (47 enzymatically defined carriers and 2 disease afflicted) who were negative for the four common disease-associated and the two pseudodeficient mutations, were subjected to single-strand conformation polymorphism (SSCP) analysis over 14 exons.