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. Bone marrow DNA was sequenced for mutations in 22 frequently mutated myeloid malignancy genes. Somatic mutations were identified in 71% of MDS patients, 62% of patients with ICUS and some dysplasia, and 20% of ICUS patients and no dysplasia. In total, 35% of ICUS patients carried a somatic mutation or chromosomal abnormality indicative of clonal hematopoiesis. We validated these results in a cohort of 91 lower-risk MDS and 249 ICUS cases identified over a 6-month interval. Mutations were found in 79% of those with MDS, in 45% of those with ICUS with dysplasia, and in 17% of those with ICUS without dysplasia. The spectrum of mutated genes was similar with the exception of SF3B1 which was rarely mutated in patients without dysplasia. Variant allele fractions were comparable between clonal ICUS (CCUS) and MDS as were mean age and blood counts. We demonstrate that CCUS is a more frequent diagnosis than MDS in cytopenic patients. Clinical and mutational features are similar in these groups and may have diagnostic utility once outcomes in CCUS patients are better understood.

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. Although epithelioid blue nevi are considered blue nevic variants, no GNAQ or GNA11 mutations have been reported. Classification of deep penetrating nevi as blue nevic variants has also been proposed, however, no GNAQ or GNA11 mutations have been reported and none have been tested for HRAS mutations. To better characterize these tumors, we performed mutational analysis for GNAQ, GNA11, and HRAS, with blue nevi and Spitz nevi as controls. Within deep penetrating nevi, none demonstrated GNAQ or GNA11 mutations (0/38). However, 6% revealed HRAS mutation (2/32). Twenty percent of epithelioid blue nevi contained a GNAQ mutation (2/10), while none displayed GNA11 or HRAS mutation. Eighty-seven percent of blue nevi contained a GNAQ mutation (26/30), 4% a GNA11 mutation (1/28), and none an HRAS mutation. Within Spitz nevi, none demonstrated GNAQ or GNA11 mutations (0/30). Seventeen percent contained an HRAS mutation (5/30). All GNAQ and GNA11 mutations were p.Q209L (c.626A>T) point mutations, except 2 GNAQ mutations, which contained novel c.625_626CA>TT double mutations. Four HRAS mutations were in exon 2, and three in exon 3. This is the first study to identify HRAS mutations in deep penetrating nevi. The presence of HRAS mutations and absence of GNAQ or GNA11 mutations in deep penetrating nevi suggests classification of these unusual nevi within the Spitz nevus category of melanocytic tumors, rather than the blue nevus category.

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. Individuals with CD were identified as those who tested positive for anti-endomysial immunoglobulin A (EMA+) in an immunofluorescence assay. RESULTS: Samples homozygous for DQ2.5 (HLA-DQA1 05-DQB1 02) or DQ2.2/DQ2.5 (HLA-DQA1 05-DQB1 02 and HLA-DQA1 0201-DQB1 02) comprised 5.38% of the total; 28.28% of these were EMA+ (95% confidence interval [CI], 24.55-32.26). Of the samples that were DQ2.5 heterozygous (HLA-DQA1 05-DQB1 02); 9.09% were EMA+ (95% CI, 7.82-10.51). Among samples in which HLA-DQ8 (HLA-DQA1 03-DQB1 0302) was detected, 8.42% of homozygotes (95% CI, 3.71-15.92) and 2.11% of heterozygotes (95% CI, 1.43-3.00) were EMA+. Samples with DQ2.2/DQ8 or DQ2.5/DQ8 comprised 5.08% of the total, and 11.78% of these were EMA+ (95% CI, 9.13-14.87). HLA-DQ2 and HLA-DQ8 were absent in 4283 samples (42.03% of the total); 0.16% of these samples were EMA+ (95% CI, 0.07-0.34). CONCLUSIONS: High-resolution, sequence-specific oligonucleotide probe typing with 35 DQA1-specific and 37 DQB1-specific probes of DNA from more than 10,000 subjects was used to stratify risk of CD in an at-risk U.S. population. DQ2 homozygosity (DQ2.5/DQ2.2+2.5) increased risk for CD, estimated by the rate of EMA positivity, compared with the entire sample population and other DQ genotypes. These data suggest a quantitative relationship between the type/proportion of DQ heterodimers and the risk of CD and identify potential immunotherapeutic targets.

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. With one exception, all rearrangements reported thus far are single or multiple exon deletions, whereas only one report has described a large duplication. We describe here the detection and characterization of a novel large duplication in the CFTR gene. This duplication, referred to as gIVS6a + 415_IVS10 + 2987Dup26817bp, was detected in a classic CF female patient whose other mutation was DeltaF508. The duplication was inherited paternally. The duplication encompassed exons 6b to 10 and occurred on the IVS8-11TG/IVS8-7T/G1540 haplotype. This large duplication is predicted to result in the production of a truncated CFTR protein lacking the terminal part of NBD1 domain and beyond and thus can be considered a null allele. The combination of the DeltaF508 and gIVS6a + 415_IVS10 + 2987Dup26817bp mutation probably causes the severe CF phenotype in this patient. We designed a simple polymerase chain reaction test to detect the duplication, and we further detected the same duplication from another independent laboratory. The duplication breakpoint is identical in all three patients, suggesting a likely founder mutation.

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. This tool is used to produce plots of all high-volume molecular genetic assays (>50 samples per month). A single web page contains pull-down menus, enabling the user to select the type of chart to be generated (genotype or allele frequency), the molecular genetic assays to chart (from one to all), the ending date for data in the chart (month and year), and the duration of the time period to plot (1 to 12 months). The rendered graphical and textual frequency data can then be viewed or printed. This tool can be used by any laboratory and interfaced with a standard laboratory information system. Monthly quality control charts and tables are now generated in minutes compared with the hours it took using manual charting applications. This simplified process enables timely compliance with a College of American Pathologists checklist item.

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. Here, we present a conservation of amino acid properties method for mutation prediction, Multiple Properties Tolerance Analysis (MuTA), and a new strategy, MuTA/S, to incorporate the solvent accessible surface (SAS) property into MuTA. Instead of combining multiple features by machine learning or mathematical methods, an intuitive strategy is used to divide the residues of a protein into different groups, and in each group the properties used is adjusted.The results for LacI, lysozyme, and HIV protease show that MuTA performs as well as the widely used SIFT algorithm while MuTA/S outperforms SIFT and MuTA by 2%-25% in terms of prediction accuracy. By incorporating the SAS term alone, the alignment dependency of overall prediction accuracy is significantly reduced. MuTA/S also defines a new way to incorporate any structural features and knowledge and may lead to more accurate predictions.

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. Almost all Delta F508 mutation alleles occur in a 10TG-9T-470M haplotype. Therefore, it is possible to determine the haplotype of the 5T variant in trans. RESULTS: Of the 74 samples analyzed, 41 (55%) were 11TG-5T-470M, 31 (42%) were 12TG-5T-470V, and 2 (3%) were 13TG-5T-470M. Of the 49 cases for which we had clinical information, 17.6% of females (6/34) and 66.7% of males (10/15) showed symptoms resembling atypical cystic fibrosis. The haplotype with the highest penetrance in females (42% or 5/12) and more than 80% (5/6) in males is 12TG-5T-470V. We also evaluated 12 males affected with congenital bilateral absence of vas deferens and positive for the 5T variant; 10 of 12 had the 12TG-5T-470V haplotype. CONCLUSION: Overall, the 5T variant has a milder clinical consequence than previously estimated in females. The clinical presentations of the 5T variant are associated with the 5T-12TG-470M haplotype.

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. A deletion encompassing the CFTR promoter and exons 1 and 2 was detected in a sample from one proband, and in the maternal DNA as well. In another family, a deletion of the promoter and exon 1 was detected in three siblings. In both of these cases, the families were African American and the 3120+1G > A splice site mutation was also identified. These promoter deletions have not been previously described. In a third case, a deletion of exons 17a, 17b, and 18 was identified in a Caucasian female and the same mutation was detected in the paternal DNA. In the other seven cases, we identified the following deletions: exons 2 and 3 (n = 2); exons 4, 5, and 6a; exons 17a and 17b; exons 22 and 23; and exons 22, 23, and 24 (n = 2). In our series, the frequency of CFTR rearrangements in classic CF patients, when only one mutation was identified by extensive DNA sequencing, was >60% (10/16). Screening for exon deletions and duplications in the CFTR gene would be beneficial in classic CF cases, especially when only one mutation is identified by standard methodologies.

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. The 820 samples were then stripped of identifiers, coded, and reanalyzed using the Tm Biosciences (Toronto, Canada) Ashkenazi Jewish panel analyte-specific reagent in a blinded fashion. For the controls, comparisons were made with their known genotypes. For the patient samples, the results of the Tm assay were compared with the results of our current assay. For 24 of the 30 mutations, we had genomic DNA controls or detected patients' samples heterozygous for these mutations. RESULTS: There were no discrepant results in the control or patient samples. In the patient samples, 19,680 genotyping reactions were performed without error in both our laboratory-developed single-disease assays and the Tm multiplex assay. Including the controls, 22,296 genotypes were determined without error. CONCLUSION: The Tm Biosciences Ashkenazi Jewish analyte-specific reagent is capable of performing accurate analyses of 24 different mutations in eight different genes in a single multiplex reaction and can be used with confidence in the clinical molecular genetics laboratory.

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. The objective of this report is to summarize the findings of extensive CFTR sequencing from our first 157 consecutive patient samples. In most patients with classic CF symptoms (18/24, 75%), extensive CFTR sequencing confirmed the diagnosis by finding two disease-associated mutations. In contrast, only 5 of 75 (7%) patients with atypical CF had been identified with two CFTR mutations. A diagnosis of CF was confirmed in 10 of 17 (58%) newborns with either positive sweat chloride readings or positive immunoreactive trypsinogen (IRT) screen results. We ascertained ten novel sequence variants that are potentially disease-associated: two deletions (c.1641AG>T, c.2949_2853delTACTC), seven missense mutations (p.S158T, p.G451V, p.K481E, p.C491S, p.H949L, p.T1036N, p.F1099L), and one complex allele ([p.356_A357del; p.358I]). We ascertained three other apparently novel complex alleles. Finally, several patients were found to carry partial CFTR gene deletions. In summary, extensive CFTR gene sequencing can detect rare mutations which are not found with other screening and diagnostic tests, and can thus establish a definitive diagnosis in symptomatic patients with previously negative results. This enables carrier detection and prenatal diagnosis in additional family members.

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. RESULTS: The MTHFR frequencies of the 677CT/1298AA, 677CC/1298AC, 677CT/1298AC, 677CC/1298AA, 677TT/1298AA, 677CC/1298CC, 677TT/1298AC, and 677CT/1298CC genotypes were 0.228, 0.208, 0.198, 0.153, 0.122, 0.088, 0.0005, and 0.0003, respectively. CONCLUSIONS: Individuals containing double variant MTHFR mutations on one allele (cis) cannot be distinguished between compound heterozygotes (trans) for 677CT and 1298AC mutations in routine clinical testing, a genotype associated with thrombophilia. Such patients could be inappropriately counseled for being at high risk for thrombotic episodes. Until information regarding prevalence and the clinical consequences of this double variant (cis) allele becomes available, caution should be used in interpreting the genotyping results of compound heterozygosity for 677CT and 1298AC.

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. RESULTS: As expected, we observed lower carrier frequencies in a general, US population than those reported in literature. In 2427 patients tested for a panel of 8 AJ diseases, 20 (1:121) were carriers of two diseases and 331 (1:7) were carriers of a single disease. Fifty-three of 7184 (1:306) individuals tested for Gaucher disease had 2 Gaucher Disease mutations indicating a potentially affected phenotype. CONCLUSIONS: As the number of AJ diseases increases, progressively more individuals will be identified as carriers of at least one disease.

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. The added ability to analyze other large molecules such as peptides and proteins further underlines the versatility and usefulness of this technology. We describe the use of mass spectrometry for screening of sickle cell disease at the level of DNA and protein. CONCLUSION: Analytic methods are needed for large-scale screening and diagnostic applications that are highly accurate, fully automated and cost-effective. The precise measurement of molecular weights and the use of high fidelity enzymes to produce diagnostic products make mass spectrometry the method of choice for DNA and protein screening procedures.

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. RESULTS: Targeted sequencing of the 39 electrophoretic variants from SSCP analysis revealed eight novel and deleterious mutations and 31 with previously described mutations. Six novel mutations were found in non-Jewish carriers, and two were found in two patients with infantile Tay-Sachs disease. CONCLUSION: Identification of these eight novel mutations provides additional insight to the mutational spectrum for the HEXA gene. Furthermore, this knowledge should enhance diagnosis and prognosis for Tay-Sachs disease, carrier identification, and fundamental studies in structure/function relationships between this gene and its enzymatic product.