Full Gene Sequence Analysis Research Articles

P189 Class II haplotypes associated with DRB1∗12 using full gene sequence analysis by next generation sequencing

Aim There is very limited information on the importance of DRB1 exon 1 variations in transplantation. Routinely, clinical laboratories do not test for exon 1 assuming it codes for the leader peptide which is cleaved off of the mature protein, and therefore not clinically important. Currently there are two CWD DRB1 alleles that differ in exon 1, DRB1∗12:01 and DRB1∗12:10, which can only be resolved by sequencing exon 1. We sought to interrogate exon 1 of DRB1 for the presence of the CWD allele, DRB1∗12:10, and to determine the HLA class II haplotypes associated with it in our local population. Methods The Immucor Mia Fora Flex 11 HLA Typing kit was used to genotype eight HSCT recipients and their unrelated donors, as well as seven additional patients. All 23 of these individuals were previously genotyped as DRB1∗12:01 using SSOP. Results All 23 individuals typed as DRB1∗12:01:01. No DRB1∗12:10 was identified in this limited cohort. Two haplotypes were identified for DRB1∗12:01:01: sixteen individuals (76%) were DRB1∗12:01:01, DRB3∗02:02:01, DQA1∗05:05:01, DQB1∗03:01:01, and five individuals (24%) were DRB1∗12:01:01, DRB3∗01:01:02, DQA1∗05:05:01, DQB1∗03:01:01. Two individuals could not have their DRB3∗01:01:01 and DRB3∗02:02:01 haplotypes identified. Conclusions The Immucor Mia Fora Flex 11 HLA Typing kit is unique among several commercial NGS kits in that it contains primers capable of sequencing exon 1 of DRB1. Traditionally, this region has been avoided because of the difficulty in primer design due to the very large 8000–10,000 bp intron 1. The CWD allele, DRB1∗12:10 originally identified in Korean population, differs from DRB1∗12:01 by one bp in exon 1, but since exon 1 is not routinely typed, the individual frequencies of these alleles are not known. Retrospective studies in Asian populations indicated that 12% of DRB1 alleles previously typed as 12:01 were in Fact 12:10. In our study, 100% of our cohort of 23 patients typed as DRB1∗12:01, which was found to be predominantly associated with DRB3∗02:02:01 (76%) and less frequently with DRB3∗01:01:02 (23%). This is in contrast to the reported preferential association of DRB1∗12:01 with DRB3∗01:01:02 (97%), while association with DRB3∗02:02:01 was very rare (2.1%). Retrospective studies may be helpful to determine the importance of exon 1 mismatches in HSCT.

Type 3 Von Willebrand Disease: A Case Report of a Rare Entity

Background:Von Willebrand Factor (VWF) is essential for proper hemostasis. It serves as a carrier protein for factor VIII and also aids in platelet adherence to damaged endothelium. Von Willebrand Disease (VWD) may result from congenital absence of VWF, structural or functional abnormalities or mutations leading to mild decreases in expression of VWF. Type 3 VWD is an autosomal recessive disorder, which accounts for only 1% of patients with VWD and is characterized by a virtual absence of VWF and low levels of Factor VIII.Objective:To describe a case of Type 3 VWD including the presentation, diagnosis and clinical course.Design/Method: Case Report.A 4 month old female presented with a history of 8 hours of persistent bleeding from an injection site following vaccination administration. At that time, the patient’s platelet count and PT/PTT were normal. She had a few intermittent episodes of persistent bleeding with minor abrasions and had labs repeated at the age of 4 months which were significant for a prolonged PTT of 59.5 seconds with normal PT and platelet count. Factor VIII level was <1% and VWF activity was <15%.Results:A complete Von Willebrand panel was sent to Mayo Medical Labs and showed a Factor VIII level of 2%, VWF antigen 6%, VWF activity of 8, and an absence of VWF multimers. Confirmatory VWF full gene sequence analysis testing was sent to Blood Center of Wisconsin and showed a heterozygous sequence variant, c.2435delC, in the VWF gene consistent with a diagnosis of VWD Type 3. VWF full gene sequence analysis was also sent on both parents. Mother's results showed that she is a carrier of the same VWF gene sequence variant. Father's results were negative. Given the patient only has a heterozygous sequence variant yet still has expression of VWD, gene duplication testing was sent to Blood Center of Wisconsin to look for possible duplication of this area. Results of this testing showed that the patient had two pathogenic variants, one on each allele: heterozygous c.243delC in exon 18 and a deletion involving exon 6 in the VWF gene.Conclusion:This case represents the clinical presentation and diagnosis of a patient with Type 3 VWD. While VWD is not uncommon, diagnosis of VWD Type 3 is quite rare. VWD Type 3 is inherited in an autosomal recessive fashion. In the case of our patient, only one parent was identified as a carrier of the same gene sequence mutation. Therefore, the second pathogenic variant resulting in VWD Type 3 was found to be a de novo deletion, further adding to the uniqueness of this case. DisclosuresNo relevant conflicts of interest to declare.

Pleuropulmonary blastoma: The causative role of germ-line DICER1 mutations.

10024 Background: Pleuropulmonary blastoma (PPB) is a rare, aggressive childhood lung cancer that is often the first manifestation of the PPB-DICER1 familial tumor predisposition which includes other benign and malignant conditions. The initial genetic mutation is inherited by a germ-line loss of function of DICER1 gene which was discovered by Hill et al. It is proposed that loss of DICER1 expression alters miRNA regulation of key regulatory cellular mechanisms which promote tumor growth. PPB can progress from the purely cystic, curable Type I to a high-grade Type III having a dismal prognosis. We sought to determine the frequency of DICER1mutation in the largest cohort of PPB patients to date. Methods: We obtained germ-line DNA from saliva or blood samples from 113 PPB patients collected from 2005-2012. DNA was extracted using the Maxwell 16 Research System (Promega Corporation, Madison WI). Sample quality and quantity was checked using the Nanodrop 2000 (Thermo Scientific, Wilmington, DE). Sequencing was performed using the Sanger sequencer. For a subset of cases we used targeted sequencing services or full gene sequence analysis (Ambry Genetics, Aliso Viejo, CA). Results: Seventy-four (65.5%) PPB patients were found to have deleterious DICER1germ-line mutations. The most common mutation type found was small insertion/deletions. These 74 samples were composed of 7 (9.5%)Type Ir PPBs, 22 (29.7%)Type I PPBs, 24 (32.4%) Type II PPBs, and 21 (28.4%)Type III PPBs. The following Table shows a summary of mutation types identified. Conclusions: Our results confirm that DICER1 germline mutations are the most common genetic alterations in PPB making it critical for genetic testing to be performed at diagnosis. Additionally, this underscores the need for important correlative studies to describe the genotype-phenotype relationship in order for appropriate screening guidelines to be developed and implemented to allow for early detection. While a majority of cases are explained by germline DICER1 mutations using current sequencing methods, further investigation is warranted to elucidate other possible mechanisms in the development of PPB. [Table: see text]

Assessment of clinical analytical sensitivity and specificity of next-generation sequencing for detection of simple and complex mutations

BackgroundDetecting mutations in disease genes by full gene sequence analysis is common in clinical diagnostic laboratories. Sanger dideoxy terminator sequencing allows for rapid development and implementation of sequencing assays in the clinical laboratory, but it has limited throughput, and due to cost constraints, only allows analysis of one or at most a few genes in a patient. Next-generation sequencing (NGS), on the other hand, has evolved rapidly, although to date it has mainly been used for large-scale genome sequencing projects and is beginning to be used in the clinical diagnostic testing. One advantage of NGS is that many genes can be analyzed easily at the same time, allowing for mutation detection when there are many possible causative genes for a specific phenotype. In addition, regions of a gene typically not tested for mutations, like deep intronic and promoter mutations, can also be detected.ResultsHere we use 20 previously characterized Sanger-sequenced positive controls in disease-causing genes to demonstrate the utility of NGS in a clinical setting using standard PCR based amplification to assess the analytical sensitivity and specificity of the technology for detecting all previously characterized changes (mutations and benign SNPs). The positive controls chosen for validation range from simple substitution mutations to complex deletion and insertion mutations occurring in autosomal dominant and recessive disorders. The NGS data was 100% concordant with the Sanger sequencing data identifying all 119 previously identified changes in the 20 samples.ConclusionsWe have demonstrated that NGS technology is ready to be deployed in clinical laboratories. However, NGS and associated technologies are evolving, and clinical laboratories will need to invest significantly in staff and infrastructure to build the necessary foundation for success.

Report of two patients with associated conditions in addition to cystic fibrosis

Purpose To report two patients with associated conditions in addition to cystic fibrosis. Methods We reviewed our database and report two patients with cystic fibrosis who had associated conditions. These patients also had novel disease causing CFTR mutations on full gene sequence analysis. Results We identified 2 patients with novel disease causing cystic fibrosis transmembrane conductance regulator mutations that we report here. A 12-year-old female with cystic fibrosis, diagnosed at 18 months, had normal pulmonary function tests and chest X-ray. Her main cystic fibrosis-related health issue was poor growth. Results of cystic fibrosis transmembrane conductance regulator DNA analysis showed deltaF508; L467P; and 7T/9T. She was later diagnosed with Crohn's disease. An 11-year-old male with Rubinstein–Taybi syndrome, diagnosed with cystic fibrosis at 2 years of age, had minimal findings on chest X-ray and pancreatic insufficiency. Results of his cystic fibrosis transmembrane conductance regulator DNA analysis showed deltaF508; 4329delCT; and 7T/9T. Conclusion We report 2 patients with CF who had associated conditions and also had novel disease causing CFTR mutations. Associated conditions may worsen the clinical manifestations of CF and complicate medical management.

Uptake of BRCA1 rearrangement panel testing: In individuals previously tested for BRCA1/2 mutations

Purpose: Individuals undergoing genetic testing for BRCA1/2 mutations are routinely counseled about the sensitivity and specificity of testing. In August 2002, testing for 5 large genomic rearrangements in the BRCA1 gene that would not have been detected with full gene sequence analysis became commercially available. We present our data on uptake of the BRCA1 rearrangement panel testing in our clinical cancer genetics program.Methods: Women who participated in our clinical genetic testing program and had previously received an uninformative negative or variant of uncertain significance result from BRCA1/2 full gene sequencing were invited to consider BRCA1 rearrangement panel testing.Results: Overall, 18/72 individuals underwent BRCA1 rearrangement panel testing. No significant differences were found in the levels of BRCAPRO scores (P = 0.406), age at testing (P = 0.986), number of children (P = 0.35) or number of siblings (P = 0.4) between individuals who chose to pursue additional testing with the rearrangement panel and those who declined. Fisher's Exact Test analysis showed that there is a negative association between having breast or ovarian cancer and being inclined to undergo rearrangement panel testing (P = 0.013).Conclusion: Individuals who undergo genetic testing will not consistently pursue additional or enhanced genetic testing. Future research is needed to clearly elucidate the factors associated with uptake of additional genetic testing.