Susan A. Lagerstedt

Gender Differences in C-Reactive Protein Concentrations - Confirmation with Two Sensitive Methods

Abstract Two new automated, highly sensitive methods for the measurement of C-reactive protein (CRP) were compared using blood samples from active and retired health care workers (116 males, 114 females). The regression equation was y=1.115x−0.0267 for the Kamiya K assay immunoturbidimetric method (x) vs. the Behring BNII nephelometric method (y) with medians (IQR) of 0.156 (0.081 to 0.276) and 0.150 (0.060 to 0.240) mg/dl, respectively. Significant differences in hsCRP values between males and females were observed (p=0.013 and p=0.0018 for Behring and Kamiya methods, respectively). This gender difference in CRP levels was confirmed by analysis of serum samples from 1114 individuals (639 males and 475 females) obtained through Mayo Medical Laboratories reference services, which represents participants from across the United States. The gender difference, which was confirmed (p<0.0001), was independent of age differences as assessed by multiple regression analysis. The observed gender differences have important implications for the establishment of cut-off points for cardiovascular risk stratification.

Preemptive Pharmacogenomic Testing for Precision Medicine: A Comprehensive Analysis of Five Actionable Pharmacogenomic Genes Using Next-Generation DNA Sequencing and a Customized CYP2D6 Genotyping Cascade

Significant barriers, such as lack of professional guidelines, specialized training for interpretation of pharmacogenomics (PGx) data, and insufficient evidence to support clinical utility, prevent preemptive PGx testing from being widely clinically implemented. The current study, as a pilot project for the Right Drug, Right Dose, Right Time-Using Genomic Data to Individualize Treatment Protocol, was designed to evaluate the impact of preemptive PGx and to optimize the workflow in the clinic setting. We used an 84-gene next-generation sequencing panel that included SLCO1B1, CYP2C19, CYP2C9, and VKORC1 together with a custom-designed CYP2D6 testing cascade to genotype the 1013 subjects in laboratories approved by the Clinical Laboratory Improvement Act. Actionable PGx variants were placed in patients electronic medical records where integrated clinical decision support rules alert providers when a relevant medication is ordered. The fraction of this cohort carrying actionable PGx variant(s) in individual genes ranged from 30% (SLCO1B1) to 79% (CYP2D6). When considering all five genes together, 99% of the subjects carried an actionable PGx variant(s) in at least one gene. Our study provides evidence in favor of preemptive PGx testing by identifying the risk of a variant being present in the population we studied.

Confirming Variants in Next-Generation Sequencing Panel Testing by Sanger Sequencing.

Current clinical laboratory practice guidelines for next-generation sequencing (NGS) do not provide definitive guidance on confirming NGS variants. Sanger confirmation of NGS results can be inefficient, redundant, and expensive. We evaluated the accuracy of NGS-detected single-nucleotide variants (SNVs) and insertion/deletion variants (indels) and the necessity of NGS variant confirmation using four NGS target-capture gene panels covering 117 genes, 568 Kbp, and 77 patient DNA samples. Unique NGS-detected variants (1080 SNVs and 124 indels) underwent Sanger confirmation and/or were compared to data from the 1000 Genomes Project (1000G). Recurrent variants in unrelated samples resulted in 919 comparisons between NGS and Sanger, with 100% concordance. In a second comparison, 762 unique NGS results (736 SNVs, 26 indels) from seven 1000G samples were found to have 97.1% concordance with 1000G phase 1 data. Sanger sequencing and 1000G phase 3 data confirmed the accuracy of the NGS results for all 1000G phase 1 discrepancies. In all samples, the depth of coverage exceeded 100× in >99.7% of bases in the target regions. In conclusion, confirmatory analysis by Sanger sequencing of SNVs detected via capture-based NGS testing that meets appropriate quality thresholds is unnecessarily redundant. In contrast, Sanger sequencing for indels may be required for defining the correct genomic location, and Sanger may be used for quality-assurance purposes.

Increased frequency of FBN1 truncating and splicing variants in Marfan syndrome patients with aortic events

Purpose:Marfan syndrome is a systemic disorder that typically involves FBN1 mutations and cardiovascular manifestations. We investigated FBN1 genotype–phenotype correlations with aortic events (aortic dissection and prophylactic aortic surgery) in patients with Marfan syndrome.Methods:Genotype and phenotype information from probands (n = 179) with an FBN1 pathogenic or likely pathogenic variant were assessed.Results:A higher frequency of truncating or splicing FBN1 variants was observed in Ghent criteria–positive patients with an aortic event (n = 34) as compared with all other probands (n = 145) without a reported aortic event (79 vs. 39%; P < 0.0001), as well as Ghent criteria–positive probands (n = 54) without an aortic event (79 vs. 48%; P = 0.0039). Most probands with an early aortic event had a truncating or splicing variant (100% (n = 12) and 95% (n = 21) of patients younger than 30 and 40 years old, respectively). Aortic events occurred at a younger median age in patients with truncating/splicing variants (29 years) as compared with those with missense variants (51 years). A trend toward a higher frequency of truncating/splicing variants in patients with aortic dissection (n = 21) versus prophylactic surgery (n = 13) (85.7 vs. 69.3%; not significant) was observed.Conclusion:These aortic event– and age-associated findings may have important implications for the management of Marfan syndrome patients with FBN1 truncating and splicing variants.Genet Med 17 3, 177–187.

LDLR promoter variant and exon 14 mutation on the same chromosome are associated with an unusually severe FH phenotype and treatment resistance.

Familial hypercholesterolemia (FH) is the most common form of autosomal-dominant hypercholesterolemia, and is caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Heterozygous FH is characterized by elevated low-density lipoprotein (LDL) cholesterol and early-onset cardiovascular disease, whereas homozygous FH results in more severe LDL cholesterol elevation with death by 20 years of age. We present here the case of an African-American female FH patient presenting with a myocardial infarction at the age of 48, recurrent angina pectoris and numerous coronary artery stents. Her pretreated LDL cholesterol levels were more typical of a homozygous FH pattern and she was resistant to conventional lipid-lowering treatment, yet her other clinical parameters were not necessarily consistent with homozygous FH. Genetic testing revealed two LDLR variants on the same chromosome: one a novel missense mutation in exon 14 (Cys681Gly) and the other a promoter variant (IVS1-217C>T) previously shown to result in increased LDLR transcription. Disease-associated PCSK9 or APOB mutations were not identified in this individual. Overall, her genetic and clinical profile suggests that enhanced expression of the mutant LDLR allele resulted in a severe phenotype with characteristics of both heterozygous and homozygous FH.

Decreased frequency of FBN1 missense variants in Ghent criteria-positive Marfan syndrome and characterization of novel FBN1 variants

The diagnosis of Marfan syndrome (MFS) remains challenging despite the 2010 revision to Ghent nosology criteria, and there is a lack of published information regarding FBN1 genotype associations in patients since the update in Ghent criteria. Applying revised Ghent criteria, we reviewed consecutive proband cases (n=292) submitted for FBN1 sequencing. Testing yielded 207 pathogenic or likely pathogenic FBN1 variants, with 114/207 (55%) missense, 67/207 (32%) non-sense or frameshift, and 28/207 (13%) splicing. There were 130 novel FBN1 variants predicted as pathogenic or likely pathogenic (n=109) or variant of undetermined significance (n=21). Of the 104 patients who met 2010 revised Ghent criteria, 87/104 (82%) had a pathogenic or likely pathogenic variant. There was a significantly lower frequency of missense variants (41 vs 89%; P<0.0001) observed in the Ghent-positive (vs Ghent-negative) patients, and this association held true in age-based groupings. Previously described genotype associations with ectopia lentis and early onset/‘neonatal’ MFS were confirmed in our cohort. Overall, our study points to the imperfect nature of relying solely on clinical criteria to diagnose MFS as well as the potential importance of truncating/splicing variants in Ghent-positive cases. Furthermore, the description of numerous novel variants and associated clinical findings may be useful for future clinical interpretation of FBN1 genotype in patients with suspected MFS.

WILD syndrome is GATA2 deficiency: A novel deletion in the GATA2 gene

The clinical syndrome of Warts, Immunodeficiency, Lymphedema, and anogenital Dysplasia has been previously described as WILD syndrome. GATA2 deficiency can have numerous clinical manifestations, some of which can overlap with WILD syndrome. Here, we present a case illustrating that WILD syndrome is GATA2 haploinsufficiency. A 19-year-old man presented with multiple clinical abnormalities including persistent human papillomavirus (HPV)driven warts since age 5 years. There were 2 previous episodes of lymphangitis and/or cellulitis of the right lower extremity with resultant streptococcal bacteremia at the ages of 5 and 9 years. He also had a history of molluscum contagiosum treated with multiple cryotherapy and laser treatments. At the age of 12 years he developed scrotal edema secondary to hydroceles and at the age of 13 years underwent bilateral hydrocelectomy. At 17 years, he developed recurrent scrotal and penile lymphedema and scrotal biopsy showed dilated lymphatic vessels. Based on the constellation of warts and lymphedema, an initial diagnosis of WILD syndrome was made. The patient was also referred to an infectious diseases specialist for evaluation of his infections and further laboratory workup was pursued. Laboratory evaluation was significant for leukopenia (white blood count, 3.3 10 L [3.5-10.5]), normal absolute neutrophil count (1.85 109 L [1.7-7.0]), absolute monocytopenia (monocyte count, 0.01 10 L [0.3-0.9]), and mild thrombocytopenia (platelets, 132 10 L [150-450]). There was also B-cell lymphopenia (CD19þ B cells, 54 cells/mcL [91-409]) and normal T-cell counts (CD3þ T cells, 1103 cells/mcL [550-2202]; CD4þ T cells, 598 cells/mcL [365-1437]; CD8þ T cells, 483 cells/mcL [199-846]). There were reduced total natural killer (NK) cells (89 cells/mcL [103-498]). Based on the above immunophenotyping and the clinical presentation, GATA2 deficiency was suspected and additional flow cytometric analysis was performed. Dendritic cell phenotyping revealed significant abnormalities with absent or reduced dendritic cell subsets (Figure 1, A). Detailed NK-cell subset phenotyping demonstrated normal cytotoxic NK-cell subset (85 cells/mcL [47-395]) and absence of the cytokine-producing subset of NK cells, CD56 NK cells (Figure 1, B). With findings of dendritic cell deficiency, monocytopenia, and B and NK lymphoid deficiency (DCML deficiency), the suspicion for GATA2 deficiency was stronger and genetic testing was undertaken to confirm the diagnosis. Initial sequencing of the GATA2 gene was performed by Sanger methodology and no mutation was identified. Because of high clinical suspicion for GATA2 deficiency, the Sanger sequencing assay was redesigned to identify large deletions (Figure 2, A). Repeat analysis demonstrated a large (354bp), novel deletion from the middle of intron 5 into exon 6: c.1143þ200_1198del (NM_001145661.1) in the GATA2 gene (Figure 2, B). The observed deletion began in the middle of intron 5 and extended 55bp into exon 6. The deletion was also identified by next-generation sequencing (Agilent SureSelect capture reagent on Illumina Hi-Seq platf orm). Genetic analysis of the parents’ DNA did not reveal the same mutation in either (Figure 2, B), indicating this was a de novo mutation causing GATA2 haploinsufficiency. Given the risk for myelodysplastic syndrome in patients with GATA2 deficiency, a bone marrow biopsy was performed. It showed 40% cellularity with minimal atypia in granulocytes and megakaryocytes. No increases in blasts were seen and cytogenetics was normal at 46XY. In addition, he was found to have a somatic ASXL1 mutation, which has been reported in up to onethird of GATA2-haploinsufficient patients and is associated with myeloid transformation, although he has not yet had evidence of this. A donor for peripheral blood stem cell transplant has been identified with plans for the patient to undergo a matched unrelated donor transplant. WILD syndrome or a similar presentation has previously been described in case reports. It has been postulated that WILD syndrome could be a manifestation of GATA2 deficiency given overlapping clinical features. The GATA2 gene encodes a hematopoietic transcription factor that in the context of haploinsufficiency due to monoallelic mutations can result in numerous clinical presentations including B-cell and NK-cell lymphopenia, monocytopenia, and absent dendritic cells. Given this, there can be increased susceptibility to severe infections, including viral infections such as HPV. In addition to its role in hematopoiesis, GATA2 plays a role in lymphatic development, which can result in lymphedema in some GATA2-deficient patients. GATA2 deficiency is a known cause of classic NK-cell deficiency but it is also known to specifically cause NK-cell subset deficiency in CD56 NK cells, which produce cytokines primarily as opposed to the cytotoxic subset (CD16CD56). Multiple syndromes including MonoMAC syndrome, DCML syndrome, Emberger syndrome, and familial myelodysplastic syndrome are now synonymous with GATA2 deficiency. Our patient had persistent and recurrent HPV-driven warts, lymphedema (see Figure E1, A and B, in this article’s Online Repository at www.jaci-inpractice.org), and immunodeficiency and therefore was given a diagnosis of WILD syndrome. However, from an immunologist’s perspective, there was concern for GATA2 deficiency because of the clinical presentation as well as laboratory findings of monocytopenia and

Genetic and biochemical analyses in dyslipidemic patients undergoing LDL apheresis

Objective: Familial hypercholesterolemia (FH) can be due to mutations in LDLR, PCSK9, and APOB. In phenotypically defined patients, a subset remains unresponsive to lipid‐lowering therapies and requires low density‐lipoprotein (LDL) apheresis treatment. In this pilot study, we examined the genotype/phenotype relationship in patients with dyslipidemia undergoing routine LDL apheresis. Design: LDLR, APOB, and PCKS9 were analyzed for disease‐causing mutations in seven patients undergoing routine LDL apheresis. Plasma and serum specimens were collected pre‐ and post‐apheresis and analyzed for lipid concentrations, Lp(a) cholesterol, and lipoprotein particle concentrations (via NMR). Results: We found that four patients harbored LDLR mutations and of these, three presented with xanthomas. While similar reductions in LDL‐cholesterol (LDL‐C), apolipoprotein B, and LDL particles (LDL‐P) were observed following apheresis in all patients, lipid profile analysis revealed the LDLR mutation‐positive cohort had a more pro‐atherogenic profile (higher LDL‐C, apolipoprotein B, LDL‐P, and small LDL‐P) pre‐apheresis. Conclusion: Our data show that not all clinically diagnosed FH patients who require routine apheresis have genetically defined disease. In our small cohort, those with LDLR mutations had a more proatherogenic phenotype than those without identifiable mutations. This pilot cohort suggests that patients receiving the maximum lipid lowering therapy could be further stratified, based on genetic make‐up, to optimize treatment. J. Clin. Apheresis 29:256–265, 2014.