Baudhuin, Linnea M.

Background: Genetic variants resulting in quantitative defects in the production of α-spectrin are associated with autosomal recessive hereditary spherocytosis (HS). Because erythroid cells produce α-spectrin in a 3-4:1 ratio in relation to β-spectrin, one defective allele is typically well-tolerated and a single null allele combined with a second partially defective allele may display a sub-clinical phenotype. Protein expression can be reduced by nonsense, frameshift, missense or splicing variants but reported large deletion events are extraordinarily rare (n = 1). Splicing variants include the low expression alleles αLELY - Low Expression LYon (SPTA1:c.6531-12C>T, p.?) and αLEPRA-Low Expression PRAgue (SPTA1:c.4339-99C>T, p.?) which reduce protein expression by 50% and 16%, respectively. Here, we present a family study with a whole gene SPTA1 deletion that characterizes the patient phenotype when a full gene mutation is co-inherited with the αLEPRA and αLELY low expression alleles. Methods: The 6 month-old male proband had a history of congenital Coombs negative hemolytic anemia requiring transfusion from 3 months of age and in whom a red cell membrane disorder was clinically suspected. Pretransfusion CBC and peripheral blood smear results were unknown. Transfused CBC indices were as follows: RBC 4 x 1012/L, HGB 11 g/dL, MCV 76 fL, MCH 27 pg, MCHC 36 g/dL and a reticulocyte count of 1.8%. The proband's mother, of Cuban descent, and father, of European descent, had no history of anemia or HS. Targeted high throughput sequencing of genes related to hereditary hemolytic anemia was performed for the detection of single nucleotide variants (SNVs) and small insertion/deletion variants (indels). Relative quantitative real time PCR (RT-PCR) was utilized to confirm large structural variants. Results: Sequencing identified a large heterozygous deletion that spanned the entire SPTA1 gene (NC_000001.11:g.(?_158656337)_(158581024_?) in the proband and his mother. In addition, the proband had inherited hemizygous αLEPRA from his father. The mother had hemizgous αLELY. The father showed heterozygous αLEPRA and no other SPTA1 variants (Table 1). Conclusion: To our knowledge, only Iolascon et al in 2011 has reported a SPTA1 gene large deletion (SpectrinExeter, exon 11-exon 12 deletion, loss of 83 amino acids) which resulted in a structurally abnormal protein and ellipto-poikilocytosis phenotype when in trans to αLELY. Our current case is the first reported SPTA1 full gene deletion and our family study allows comparison of the effects of coinheritance of the low expression alleles αLEPRA and αLELY. When in combination with complete absence of the SPTA1 gene in trans configuration, αLELY manifests in subclinical effects and αLEPRA causes a recessive HS phenotype, which resulted in severe anemia. This contrast further demonstrates that, unlike αLEPRA, αLELY does not decrease the α-spectrin concentration sufficiently to contribute to the baseline phenotype regardless of the severity of the second allele mutation for quantitative disorders such as HS. Disclosures No relevant conflicts of interest to declare.

Common genetic variation in CYP2C19 (cytochrome P450, family 2, subfamily C, polypeptide 19) *2 and *3 alleles leads to a loss of functional protein, and carriers of these loss-of-function alleles when treated with clopidogrel have significantly reduced clopidogrel active metabolite levels and high on-treatment platelet reactivity resulting in increased risk of major adverse cardiovascular events, especially after percutaneous coronary intervention. The Food and Drug Administration has issued a black box warning advising practitioners to consider alternative treatment in CYP2C19 poor metabolizers who might receive clopidogrel and to identify such patients by genotyping. However, routine clinical use of genotyping for CYP2C19 loss-of-function alleles in patients undergoing percutaneous coronary intervention is not recommended by clinical guidelines because of lack of prospective evidence. To address this critical gap, TAILOR-PCI (Tailored Antiplatelet Initiation to Lessen Outcomes due to Decreased Clopidogrel Response After Percutaneous Coronary Intervention) is a large, pragmatic, randomized trial comparing point-of-care genotype-guided antiplatelet therapy with routine care to determine whether identifying CYP2C19 loss-of-function allele patients prospectively and prescribing alternative antiplatelet therapy is beneficial.

Objective— Ceramides are sphingolipids involved with cellular signaling. Synthesis of ceramides occurs in all tissues. Ceramides accumulate within tissues and the blood plasma during metabolic dysfunction, dyslipidemia, and inflammation. Elevations of ceramides are predictive of cardiovascular mortality. We sought to verify the utility of plasma concentrations of 4 ceramides: N-palmitoyl-sphingosine [Cer(16:0)], N-stearoyl-sphingosine [Cer(18:0)], N-nervonoyl-sphingosine [Cer(24:1)], and N-lignoceroyl-sphingosine [Cer(24:0)] in predicting major adverse cardiovascular events in a diverse patient population referred for coronary angiography. Approach and Results— Plasma ceramides were measured in 495 participants before nonurgent coronary angiography. Coronary artery disease, defined as >50% stenosis in ≥1 coronary artery, was identified 265 (54%) cases. Ceramides were not significantly associated with coronary artery disease. Patients were followed for a combined primary end point of myocardial infarction, percutaneous intervention, coronary artery bypass, stroke, or death within 4 years. Ceramides were significantly predictive of outcomes after adjusting for age, sex, body mass index, hypertension, smoking, LDL (low-density lipoprotein) cholesterol, HDL (high-density lipoprotein) cholesterol, triglycerides, serum glucose, and family history of coronary artery disease. The fully adjusted per SD hazard ratios (95% confidence interval) were 1.50 (1.16–1.93) for Cer(16:0), 1.42 (1.11–1.83) for Cer(18:0), 1.43 (1.08–1.89) for Cer(24:1), and 1.58 (1.22–2.04) for the ceramide risk score. Conclusions— Elevated plasma concentrations of ceramides are independently associated with major adverse cardiovascular events in patients with and without coronary artery disease.

Background— Postmortem genetic testing for heritable cardiovascular (CV) disorders is often lacking because ideal specimens (ie, whole blood) are not retained routinely at autopsy. Formalin-fixed paraffin-embedded tissue (FFPET) is ubiquitously collected at autopsy, but DNA quality hampers its use with traditional sequencing methods. Targeted next-generation sequencing may offer the ability to circumvent such limitations, but a method has not been previously described. The primary aim of this study was to develop and evaluate the use of FFPET for heritable CV disorders via next-generation sequencing. Methods and Results— Nineteen FFPET (heart) and blood (whole blood or dried blood spot) specimens underwent targeted next-generation sequencing using a custom panel of 101 CV-associated genes. Nucleic acid yield and quality metrics were evaluated in relation to FFPET specimen age (6 months to 15 years; n=14) and specimen type (FFPET versus whole blood and dried blood spot; n=12). Four FFPET cases with a clinical phenotype of heritable CV disorder were analyzed. Accuracy and precision were 100% concordant between all sample types, with read depths >100× for most regions tested. Lower read depth, as low as 40×, was occasionally observed with FFPET and dried blood spot. High-quality DNA was obtained from FFPET samples as old as 15 years. Genomic analysis of FFPET from the 4 phenotype-positive/genotype unknown cases all revealed putative disease-causing variants. Conclusions— Similar performance characteristics were observed for next-generation sequencing of FFPET, whole blood, and dried blood spot in the evaluation of inherited CV disorders. Although blood is preferable for genetic analyses, this study offers an alternative when only FFPET is available.