Low-expression Allele Research Articles

The BDNF Val66Met polymorphism moderates the relationship between Posttraumatic Stress Disorder and fear extinction learning

The low expression Met allele of the BDNF Val66Met polymorphism is associated with impaired fear extinction in healthy controls, and poorer response to exposure therapy in patients with Posttraumatic Stress Disorder (PTSD). Given that fear extinction underlies exposure therapy, this raises the question of the impact of BDNFVal66Met polymorphism on fear extinction in PTSD, yet this question has not yet been examined. One hundred and six participants (22 PTSD, 46 trauma-exposed controls (TC) and 38 non-trauma exposed controls (NTC)) completed a fear conditioning and extinction task and saliva samples were taken for DNA extraction and genotyped for the BDNF Val66Met polymorphism. Moderation analyses using PROCESS examined whether BDNF genotype (Val–Val vs Met carriers) moderated the relationship between PTSD symptom severity (and diagnostic status) and skin conductance response (SCR) amplitude during fear extinction. The PTSD group displayed significantly slower fear extinction learning compared to TC and NTC in the early extinction phase. The BDNF Val66Met polymorphism moderated the relationship between PTSD and fear extinction learning, such that poorer fear extinction learning was associated with greater PTSD symptom severity (and PTSD diagnostic status) in individuals with the low-expression Met allele, but no relationship was demonstrated in individuals with the Val–Val allele. This study reveals that impaired fear extinction learning is particularly evident in individuals with PTSD who carry the low-expression BDNF Met allele and importantly not in those with the Val–Val allele. This provides novel evidence of a link between BDNF and impaired fear extinction learning in PTSD, which may contribute to poorer response to exposure therapy.

Stability and Expression Levels of HLA-C on the Cell Membrane Modulate HIV-1 Infectivity.

ABSTRACTHLA-C expression is associated with a differential ability to control HIV-1 infection. Higher HLA-C levels may lead to better control of HIV-1 infection through both a higher efficiency of antigen presentation to cytotoxic T lymphocytes and the triggering of activating killer immunoglobulin-like receptors on NK cells, whereas lower levels may provide poor HIV-1 control and rapid progression to AIDS. We characterized the relative amounts of HLA-C heterotrimers (heavy chain/β2 microglobulin [β2m]/peptide) and HLA-C free heavy chains on peripheral blood mononuclear cells (PBMCs) from healthy blood donors harboring both alleles with stable or unstable binding to β2m/peptide. We analyzed the stability of HLA-C heterotrimers of different allotypes and the infectivity of HIV-1 virions produced by PBMCs with various allotypes. We observed significant differences in HLA-C heterotrimer stability and in expression levels. We found that R5 HIV-1 virions produced by PBMCs harboring unstable HLA-C alleles were more infectious than those produced by PBMCs carrying the stable variants. We propose that HIV-1 infectivity might depend both on the amounts of HLA-C molecules and on their stability as trimeric complex. According to this model, individuals with low-expression HLA-C alleles and unstable binding to β2m/peptide might have worse control of HIV-1 infection and an intrinsically higher capacity to support viral replication.IMPORTANCE Following HIV-1 infection, some people advance rapidly to AIDS while others have slow disease progression. HLA-C, a molecule involved in immunity, is a key determinant of HIV-1 control. Here we reveal how HLA-C variants contribute to the modulation of viral infectivity. HLA-C is present on the cell surface in two different conformations. The immunologically active conformation is part of a complex that includes β2 microglobulin/peptide; the other conformation is not bound to β2 microglobulin/peptide and can associate with HIV-1, increasing its infectivity. Individuals with HLA-C variants with a predominance of immunologically active conformations would display stronger immunity to HIV-1, reduced viral infectivity and effective control of HIV-1 infection, while subjects with HLA-C variants that easily dissociate from β2 microglobulin/peptide would have a reduced immunological response to HIV-1 and produce more infectious virions. This study provides new information that could be useful in the design of novel vaccine strategies and therapeutic approaches to HIV-1.

P137 Next generation sequencing reveals HLA and KIR susceptibility alleles for rheumatoid arthritis

Aim Previous associations of KIR with rheumatoid arthritis, RA, have been reported in some but not all populations studied, possibly due to limited genotyping in some studies, and gene content homogeneity in some populations. In this study, HLA and KIR typing was carried out, including KIR haplotype and allele typing, using state of the art sequencing methodologies in a re-examination of the association of these gene families with RA in a Japanese cohort. An additional cohort of pollen allergy patients was examined in an effort to distinguish common genetic elements in a phenotype functionally reciprocal to RA. Methods DNA from 116 RA patients, 167 pollen allergy patients, and 185 healthy controls were examined for KIR haplotype, allele type and HLA class I and II allele types using next generation sequencing. Association analysis was carried out with healthy controls classified into two groups, positive and negative for allergen specific IgE antibodies, including a pollen allergy group for comparison with RA. Results Significant results were observed with allele types KIR2DS4∗007 and KIR3DL1∗00501, which strongly associated with disease, while KIR 3DL1∗001 and 3DL1∗02901 associated with a protective phenotype. These findings were significant when compared with the IgE positive control group while the IgE-negative group did not demonstrate significance. Conclusions Given that KIR3DL1 is an inhibitory receptor and the KIR3DL1∗00501 allele has been reported as a low expression allele, these findings are consistent with a model of weak suppression of NK cytotoxic activity as a contributing factor in RA. Further support for this model was observed from the reciprocity of the genetic associations between RA and pollen allergy.

Clinical, Biochemical, and Genetic Characterization of North American Patients With Erythropoietic Protoporphyria and X-linked Protoporphyria

ImportanceAutosomal recessive erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP) are rare photodermatoses presenting with variable degrees of painful phototoxicity that markedly affects quality of life. The clinical variability, determinants of severity, and genotype/phenotype correlations of these diseases are not well characterized.ObjectiveTo describe the baseline clinical characteristics, genotypes, and determinants of disease severity in a large patient cohort with EPP or XLP.Design, Setting, and ParticipantsA prospective observational study was conducted among patients with confirmed diagnoses of EPP or XLP from November 1, 2010, to December 6, 2015, at 6 academic medical centers of the Porphyrias Consortium of the National Institutes of Health Rare Diseases Clinical Research Network. Detailed medical histories, including history of phototoxicity and treatment, were collected on standardized case report forms. Patients underwent baseline laboratory testing, total erythrocyte protoporphyrin (ePPIX) testing, and molecular genetic testing. Data were entered into a centralized database.Main Outcomes and MeasuresResults of biochemical and genetic tests were explored for association with clinical phenotype in patients with EPP or XLP.ResultsOf the 226 patients in the study (113 female and 113 male patients; mean [SD] age, 36.7 [17.0] years), 186 (82.3%) had EPP with a FECH (OMIM 612386) mutation and the common low-expression FECH allele IVS3–48T>C, and only 1 patient had 2 FECH mutations. Twenty-two patients had XLP (9.7%; 10 male and 12 female patients), and 9 patients (4.0%) had elevated ePPIX levels and symptoms consistent with protoporphyria but no detectable mutation in the FECH or ALAS2 (OMIM 301300) gene. Samples of DNA could not be obtained from 8 patients. Patients’ mean (SD) age at symptom onset was 4.4 (4.4) years. Anemia (107 [47.3%]), history of liver dysfunction (62 [27.4%]), and gallstones (53 [23.5%]) were commonly reported. Higher ePPIX levels were associated with earlier age of symptom onset (median ePPIX levels for those who developed symptoms before vs after 1 year of age, 1744 vs 1567 µg/dL; P = .02), less sun tolerance (median ePPIX levels for those reporting symptoms before vs after 10 minutes of sun exposure, 2233 vs 1524 µg/dL; P ≤ .001), and increased risk of liver dysfunction (median ePPIX levels for those with liver dysfunction vs normal liver function, 2016 vs 1510 µg/dL; P = .003). Patients with EPP and FECH missense mutations had significantly lower ePPIX levels than those with other mutations (1462 vs 1702 µg/dL; P = .01). Male patients with XLP had significantly higher ePPIX levels, on average, than did patients with EPP (3574 vs 1669 µg/dL; P < .001). Marked clinical variability was seen in female patients with XLP owing to random X-chromosomal inactivation.Conclusions and RelevanceThese data suggest that higher ePPIX levels are a major determinant of disease severity and risk of liver dysfunction in patients with EPP or XLP. These findings provide a framework for clinical monitoring and management of these disorders.

Influence of IL-18 and IL-10 Polymorphisms on Tacrolimus Elimination in Chinese Lung Transplant Patients.

Aims. The influence of interleukin-10 (IL-10) and interleukin-18 (IL-18) polymorphisms on tacrolimus pharmacokinetics had been described in liver and kidney transplantation. The expression of cytokines varied in different kinds of transplantation. The influence of IL-10 and IL-18 genetic polymorphisms on the pharmacokinetic parameters of tacrolimus remains unclear in lung transplantation. Methods. 51 lung transplant patients at Shanghai Pulmonary Hospital were included. IL-18 polymorphisms (rs5744247 and rs1946518), IL-10 polymorphisms (rs1800896, rs1800872, and rs3021097), and CYP3A5 rs776746 were genotyped. Dose-adjusted trough blood concentrations (C/D ratio, mg/kg body weight) in lung transplant patients during the first 4 postoperative weeks were calculated. Results. IL-18 rs5744247 allele C and rs1946518 allele A were associated with fast tacrolimus metabolism. Combined analysis showed that the numbers of low IL-18 mRNA expression alleles had positive correlation with tacrolimus C/D ratios in lung transplant recipients. The influence of IL-18 polymorphisms on tacrolimus C/D ratios was observed in CYP3A5 expresser recipients, but not in CYP3A5 nonexpresser recipients. No clinical significance of tacrolimus C/D ratios difference of IL-10 polymorphisms was found in our data. Conclusions. IL-18 polymorphisms may influence tacrolimus elimination in lung transplantation patients.

Extravascular hemolysis and complement consumption in Paroxysmal Nocturnal Hemoglobinuria patients undergoing eculizumab treatment

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by complement-mediated intravascular hemolysis that is effectively treated with eculizumab. However, treatment responses are reported heterogeneous with some patients presenting residual hemolysis and requiring RBC transfusions. Recent reports have shown that both extravascular hemolysis and incomplete C5 blockade can explain these suboptimal hematological responses. Here we have tested our eculizumab-treated PNH patients (n=12) for signs of hemolysis and assessed complement biomarkers. Patients were also genotyped for complement receptor 1 (CR1, CD35) and C5 polymorphisms and evaluated for free eculizumab in plasma. We report that 10 patients (83%) present parameters suggesting persistent hemolysis, although they did not require additional transfusions. Seven of them (58%) become direct Coombs-test positive as a consequence of treatment, including all patients carrying the low-expression CR1-L allele. CH50 and sC5b-9 assays demonstrate that the persistent low-level hemolysis identified in our treated patients is not a consequence of incomplete C5 blockade, supporting that this hemolysis, as has been suggested previously, results from the extravascular removal of C3 opsonized PNH erythrocytes. We also show that continuous alternative pathway activation in eculizumab-treated individuals carrying the CR1-L allele results in abnormally decreased levels of C3 in plasma that could, potentially, increase their susceptibility to bacterial infections. Finally, we encourage a routine evaluation of free eculizumab levels and terminal pathway activity to personalize eculizumab administration.

PTSD, alcohol dependence, and conduct problems: Distinct pathways via lability and disinhibition

This study tested the role of affect lability and disinhibition in mediating associations between PTSD symptoms and two forms of alcohol-related problems, dependence syndrome symptoms (e.g., impaired control over consumption) and conduct problems (e.g., assault, risk behaviors). Genotype at the serotonin transporter linked polymorphic region (5-HTTLPR) was hypothesized to moderate associations between traumatic stress and PTSD symptoms. In addition, the study tested whether childhood traumatic stress moderated associations between combat trauma and PTSD symptoms. Participants were 270 OIF/OEF/OND veterans. The hypothesized model was largely supported. Participants with the low expression alleles of 5-HTTLPR (S or LG) exhibited stronger associations between childhood (but not combat) traumatic stress and PTSD symptoms. Affect lability mediated the associations between PTSD symptoms and alcohol dependence symptoms. Behavioral disinhibition mediated associations between PTSD symptoms and conduct related problems. Conditional indirect effects indicated stronger associations between childhood traumatic stress and lability, behavioral disinhibition, alcohol consumption, AUD symptoms, and associated conduct problems via PTSD symptoms among those with the low expression 5-HTTLPR alleles. However, interactions between combat trauma and either childhood trauma or genotype were not significant. The results support the hypothesis that affect lability and behavioral disinhibition are potential intermediate traits with distinct associations with AUD and associated externalizing problems.

Aggregate temperament scores from multiple countries: Associations with aggregate personality traits, cultural dimensions, and allelic frequency

Aggregate temperament scores were calculated across 17 studies for 18 countries. These scores were analyzed in relation to aggregate personality scores, cultural orientation, and allelic distribution reported in prior studies. Cross-culture patterns were largely consistent with those previously reported for personality: countries high on Surgency (SUR) were high on Extraversion, high Negative Affectivity (NEG) was consistent with Neuroticism, and Regulatory Capacity (RC) with Agreeableness. Regarding cultural orientation, aggregate SUR was associated with low Power Distance and Long-Term Orientation; NEG with low Individualism and high Masculinity and Uncertainty Avoidance; and RC with low Power Distance and Masculinity. Higher proportion of MAOA-uVNTR low expression alleles was associated with low SUR and low RC; and A118G G-form allele proportion with low RC.

Next generation sequencing reveals HLA and KIR susceptibility alleles for rheumatoid arthritis

Abstract Previous associations of KIR with rheumatoid arthritis (RA) have been reported in some but not all populations studied, possibly due to limited genotyping in some studies, and gene content homogeneity in some populations. In this study, HLA and KIR typing was carried out, including KIR haplotype and allele typing, using state of the art sequencing methodologies in a re-examination of the association of these gene families with RA in a Japanese cohort. An additional cohort of pollen allergy patients was examined in an effort to distinguish common genetic elements in a phenotype functionally reciprocal to RA. DNAs from 116 RA patients, 167 pollen allergy patients, and 185 healthy controls were examined for KIR haplotype, allele type and HLA class I and II allele types using next generation sequencing (NGS). Association analysis was carried out with healthy controls classified into two groups, positive and negative for allergen specific IgE antibodies, including a pollen allergy group for comparison with RA. Significant results were observed with allele types KIR2DS4*007 and KIR3DL1*00501, which strongly associated with disease, while KIR 3DL1*001 and 3DL1*02901 associated with a protective phenotype. These findings were significant when compared with the IgE positive control group while the IgE-negative group did not demonstrate significance. Given that KIR3DL1 is an inhibitory receptor and the KIR3DL1*00501 allele has been reported as a low expression allele, these findings are consistent with a model of weak suppression of NK cytotoxic activity as a contributing factor in RA. Further support for this model was observed from the reciprocity of the genetic associations between RA and pollen allergy.

A Comprehensive Next Generation Sequencing Gene Panel Focused on Unexplained Anemia

Congenital anemia is difficult to diagnose once common causes have been excluded; for example 80% cases of congenital non-spherocytic hemolytic anemia are undiagnosed once pyruvate kinase and G6PD deficiencies have been excluded using phenotypic analysis. We describe a next generation sequencing strategy, targeting 147 genes, to facilitate the diagnosis of these conditions. The coding regions, splice sites and 200 bp into the untranslated regions were examined in each gene. All clinically significant variants were confirmed by Sanger sequencing, including confirmation in any appropriate family members.Illumina MiSeq data was analysed using a bespoke bioinformatics pipeline, which has been validated to a UK certified standard. The pipeline implements detection of genetic variants using multiple base callers and discovery of copy number variants based on sequencing depth. Variants are annotated with information from ClinVar, and population frequency data from ExAC and 1000 genomes project. All genes are sequenced in every individual but data analysis can easily be restricted to virtual subpanels, excluding analysis of genes not requested. Here we present three cases, highlighting the diagnostic utility of the panel as well as the underlying bioinformatics analysis.Case 1. A male Caucasian child of <1 year, presented with haemolysis (LDH 539 IU/L, total bilirubin 39 umol/L), haematology (Hb 92g/L, MCV 84.4, MCH 28.9, absolute retic count 313.8x109/L); his film showed marked anisopoikilocytes, microspherocytes and polychromasia. He had frontal bossing and a palpable spleen and had suffered several infections, the child was transfused once. His father's film showed elliptocytes, FBC (Hb 127g/L, MCV 89.6, MCH 30.6, absolute retic count 230.5x109/L) but he had never been transfused. The mother's FBC was normal (Hb 113g/L, MCV 87.0, MCH 29.2, absolute retic count 48.4x109/L) but her film also showed elliptocytes. Analysis using the red cell panel found the child to be compound heterozygous for c.83G>A; p.Arg28His and c.[5572C>G; 6531-12C>T]; p.[Leu1858Val;?] in the SPTA1 gene, suggesting the diagnosis of hereditary pyropoikilocytosis. The c.83G>A; p.Arg28His mutation was inherited from the father and the c.[5572C>G; 6531-12C>T]; p.[Leu1858Val;?] low expression allele was inherited from the mother, who was homozygous.Case 2. The post mortem report from a hydropic still birth (36/40) showed extensive extramedullary hematopoiesis and severe anemia. A DNA sample was sent to the laboratory accompanied by blood samples from both parents whose hematology was normal. The DNA sample from the proband was relatively small so only the parental samples were analyzed using the red cell panel. Sequence analysis identified the mother to carry the c.3173dupG; p.Gln1659fs pathogenic variant and the father carried the c.2867_2868+1dupCCG pathogenic variant in the CDAN1 gene. Sanger Sequencing showed that the child had inherited both mutations from the parents. Variants in CDAN1 are associated with CDA type 1 which is documented to be a rare form of anemia which can be lethal.Case 3. An Italian girl carrying a paternally inherited c.118C>T β0 thalassemia variant presented with a severe form of microcytic anemia (FBC, Hb 86g/L, RBC 4.87 x1012/L, MCV 55.2, MCH 17.7 and HbA2=5%). The severity of her anemia (not transfused) and palpable spleen suggested she had an additional pathogenic variant that had not been identified. Her mother had normal hematology FBC, Hb 133g/L, RBC 4.82x1012/L, MCV 79.9, MCH 27.6. After sequencing, Exome Depth analysis of the proband's LCR identified a novel deletion which removed the 5' HS1 and HS2 sites but left HS3-5 intact (confirmed by MLPA in the mother and proband). The combination of this mild down regulation of the beta globin locus in combination with the c.118C>T β0 thalassemia variant caused her phenotype to be more severe than just a beta thalassemia carrier.Identifying pathogenic variants in these families is important as it facilitates prognosis and treatment, and allows prenatal diagnosis to be offered in future. To date the panel has assessed 10 cases of anemia with unknown cause and has made a definitive diagnosis in 8 (80%). Of the two undiagnosed, one was a child that died at 3 weeks and received multiple intrauterine and neonatal transfusions and had severe anemia and the other was a suspected case of CDA with little associated phenotype. DisclosuresNo relevant conflicts of interest to declare.

Genotype-Phenotype Correlations in Hereditary Elliptocytosis (HE) and Hereditary Pyropoikilocytosis (HPP)

▪Hereditary elliptocytosis (HE) results from defects in the erythrocyte membrane proteins α-spectrin, β-spectrin or protein 4.1R that weaken the ÒhorizontalÓ cytoskeletal associations causing decreased mechanical stability and deformability of the red blood cell (RBC). The hallmark of HE is the presence of elliptical RBCs on peripheral blood smears. Diseases within the spectrum of HE include classic HE and hereditary pyropoikilocytosis (HPP). The traditional diagnosis of HE relies on visualization of elliptocytes on the blood smear, while HPP is characterized by significant poikilocytosis and abnormally shaped RBCs. In vitro assessment of RBC membrane biomechanical properties using ektacytometry is characteristic. However, this evaluation requires a pure RBC population, which may not be available in patients requiring frequent transfusions and does not predict disease course or severity.Using a Next-Generation sequencing panel for 12 genes associated with RBC membrane disorders, we identified the causative genetic mutations in 8 patients with clinically diagnosed or suspected HE or HPP (3 with HE and 5 with HPP). We correlated the identified genetic mutations with clinical presentation, blood smear findings, and ektacytometry results.HE patients: 2 patients with HE had heterozygous mutations in SPTA1 (p.L154_L155insL) resulting in spectrin self-association defect, while the third one had a novel nonsense heterozygous mutation in EPB41 (p.R262*) resulting in defective horizontal interaction between spectrin and actin in the RBC cytoskeleton due to decreased band 4.1R. The same novel mutation was identified in the patient's mother. Both, the patient and the mother, had smear findings and ektacytometry profiles consistent with the diagnosis of HE (Fig. 1A).HPP patients: 3 out of 5 HPP patients were found to be compound heterozygous for a SPTA1 mutation at the self-association site (p.R28H or p.L154_L155insL) and a low-expression SPTA1 allele (c.6531-12C>T known as α-LELY or Low Expression LYon) which allows for the expanded expression of the HE-causing mutation located in trans, resulting in a more severe phenotype. Of note, two siblings carried the same SPTA1 mutation (p.L154_L155insL); one was heterozygous and had mild HE while the other sibling carried α-LELY in trans to the HE-mutation causing a more severe anemia with poikilocytosis and worsened deformability consistent with HPP (Fig. 1B). The ektacytometry profile of these two siblingsÕ RBCs demonstrates the role of α-LELY in modifying the phenotype of elliptocytic disorders. A fourth patient with HPP was found to have compound heterozygosity for mutations in SPTA1 (p.L154_L155insL) and SPTB (p.W2024R) both in the self association sites between α- and β-spectrin. The fifth patient was a 3 y.o. boy with transfusion-dependent hemolytic anemia since infancy and therefore his erythrocyte phenotype could not be evaluated. He was homozygous for a novel mutation in SPTB (p.F2014V); both of his parents were heterozygous for the same mutation. The parents were asymptomatic but their ektacytometry profiles and blood smears were consistent with HE (Fig. 1C), indicating that this mutation in the b-spectrin dimerization domain alters the α- and β-spectrin interaction causing HE in heterozygous state and HPP in the homozygous state.Next-Generation sequencing for genetic diagnosis in HE and HPP can confirm or identify the diagnosis and may be helpful to predict the phenotype and clinical severity in these heterogeneous disorders. Combining clinical data, ektacytometry and family studies is important to understand the relevance of new genetic variants to pathology of RBC cytoskeletal disorders, provide insight into the genotype-phenotype correlation, and improve the genetic counseling and clinical care for these disorders.Figure1. Ektacytometry profiles of : A) a patient and his mother heterozygous for a novel nonsense mutation in EPB41 (p.R262*) causing HE, B) a patient with SPTA1 mutation (p.L154_L155insL) and α-LELY causing HPP compared to a sibling with the same heterozygous mutation in SPTA1 without α-LELY causing HE, C) novel mutation in SPTB (p.F2014V) causing mild HE to heterozygous parents but severe transfusion-dependent anemia in the homozygous child. Ektacytometry was not performed in the child since he was chronically transfused. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Spectrum of Alpha-Spectrin Associated Hereditary Spherocytosis

▪Hereditary spherocytosis (HS) is a genetically and phenotypically heterogeneous hemolytic anemia caused by deficiency in red blood cell (RBC) cytoskeleton proteins leading to disruptions in the vertical association of the cytoskeleton with the RBC lipid bilayer. Monoallelic mutations in the genes encoding ankyrin (ANK1), beta-spectrin (SPTB) and band 3 (SLC4A1) or biallelic mutations in the genes encoding alpha-spectrin (SPTA1), ankyrin, and protein 4.2 (EPB42) result in HS. Autosomal recessive HS due to compound heterozygous defects in SPTA1 is typically severe and diagnosis based on phenotypic assays like RBC morphology, osmotic fragility or ektacytometry is complicated by transfusion dependence resulting in most of the circulating RBCs to be of donor origin. We have developed a rapid comprehensive next-generation sequencing-based assay that evaluates 27 genes with published disease-causing mutations for RBC cytoskeletal disorders, enzymopathies, and CDAs. We describe here patients with hemolytic anemia due to SPTA1 mutations, identified utilizing this assay, and their phenotype-genotype correlation. Each of these cases, when possible, has been also evaluated with ektacytometry and immunoblotting of RBC ghosts for alpha-spectrin quantitation.Wichterle et al in 1996 had estimated that alphaLEPRA(Low Expression PRAgue) mutation (c.4339-99C>T) occurs in SPTA1 gene in about 5% of Caucasians. This mutation leads to activation of an alternate acceptor splice site at position -70 of intron 30, causing frame shift and premature termination, thereby leading to decrease in alpha-spectrin production in this allele to about 16% of normal. We have found a cohort of three transfusion-dependent hereditary hemolytic anemia cases where a nonsense mutation in SPTA1 gene has occurred in trans to alphaLEPRA mutation, resulting in premature termination (see Table 1). Transfusion dependence was alleviated in two of these patients after splenectomy; the third one did not have splenectomy yet. RBC phenotype explored after splenectomy revealed an ektacytometry curve indicating spherocytosis (Figure 1A) and severely decreased alpha-spectrin on immunoblotting along with significant decrease of the associated beta-spectrin (Figure 1B).A patient with moderately severe form of HS, maintaining a hemoglobin value greater than 7 g/dL and requiring only occasional transfusions during periods of illness or stress, was found to have alphaLEPRA occurring in trans to an intronic splicing mutation c.1351-1G>TG where there is substitution at nucleotide-1 of intron positioned between nucleotides 1350 and 1351 of the SPTA1 mRNA. This splicing mutation may allow for some expression of functional alpha-spectrin protein from this allele in contrast to no protein expression in the previous cases of premature termination. Alternatively, other gene mutations, not identified by the next-generation sequencing panel we used, may contribute to this patient's milder phenotype.A couple with history of two fetal losses associated with hydrops fetalis seeked genetic counseling and gave consent to have diagnostic evaluation of genes associated with non-immune hemolytic anemia using targeted next-generation sequencing. Results of the panel revealed a heterozygous frameshift SPTA1 mutation in each of the parents (c.4206delG in the father and c.4180delT in the mother). These mutations in compound heterozygous state in the offspring likely caused total absence of alpha spectrin and fatal hemolytic anemia by the time of birth.Hereditary Spherocytosis is characterized by wide phenotypic variability that will be better understood with studies of genotype-phenotype association. While complete absence of alpha-spectrin expression due to null mutations of both SPTA1 alleles is incompatible with life, a nonsense or splicing SPTA1 mutation in trans to an alphaLEPRA low expression allele causes severe or moderately severe recessive HS, respectively. Targeted next-generation sequencing can be an effective diagnostic tool particularly for patients requiring frequent transfusions that preclude meaningful phenotypical testing of their red blood cells. [Display omitted] [Display omitted] DisclosuresBegtrup:GeneDx: Employment.

FRAS1-related extracellular matrix 3 (FREM3) single-nucleotide polymorphism effects on gene expression, amygdala reactivity and perceptual processing speed: An accelerated aging pathway of depression risk.

The A allele of the FRAS1-related extracellular matrix protein 3 (FREM3) rs7676614 single nucleotide polymorphism (SNP) was linked to major depressive disorder (MDD) in an early genome-wide association study (GWAS), and to symptoms of psychomotor retardation in a follow-up investigation. In line with significant overlap between age- and depression-related molecular pathways, parallel work has shown that FREM3 expression in postmortem human brain decreases with age. Here, we probe the effect of rs7676614 on amygdala reactivity and perceptual processing speed, both of which are altered in depression and aging. Amygdala reactivity was assessed using a face-matching BOLD fMRI paradigm in 365 Caucasian participants in the Duke Neurogenetics Study (DNS) (192 women, mean age 19.7 ± 1.2). Perceptual processing speed was indexed by reaction times in the same task and the Trail Making Test (TMT). The effect of rs7676614 on FREM3 mRNA brain expression levels was probed in a postmortem cohort of 169 Caucasian individuals (44 women, mean age 50.8 ± 14.9). The A allele of rs7676614 was associated with blunted amygdala reactivity to faces, slower reaction times in the face-matching condition (p < 0.04), as well as marginally slower performance on TMT Part B (p = 0.056). In the postmortem cohort, the T allele of rs6537170 (proxy for the rs7676614 A allele), was associated with trend-level reductions in gene expression in Brodmann areas 11 and 47 (p = 0.066), reminiscent of patterns characteristic of older age. The low-expressing allele of another FREM3 SNP (rs1391187) was similarly associated with reduced amygdala reactivity and slower TMT Part B speed, in addition to reduced BA47 activity and extraversion (p < 0.05). Together, these results suggest common genetic variation associated with reduced FREM3 expression may confer risk for a subtype of depression characterized by reduced reactivity to environmental stimuli and slower perceptual processing speed, possibly suggestive of accelerated aging.

High HLA-DP Expression and Graft-versus-Host Disease

BackgroundTransplantation of hematopoietic cells from unrelated donors can cure blood disorders but carries a significant risk of acute graft-versus-host disease (GVHD). The risk is higher when the recipient and donor are HLA-DPB1–mismatched, but the mechanisms leading to GVHD are unknown. The HLA-DPB1 regulatory region variant rs9277534 is associated with HLA-DPB1 expression. We tested the hypothesis that the GVHD risk correlates with the rs9277534 allele linked to the mismatched HLA-DPB1 in the recipient.MethodsWe genotyped rs9277534 in 3505 persons to define rs9277534-DPB1 haplotypes. Among 1441 recipients of transplants from HLA-A,B,C,DRB1,DQB1–matched unrelated donors with only one HLA-DPB1 mismatch, linkage of the rs9277534 A and G alleles to the mismatched HLA-DPB1 was determined. HLA-DPB1 expression was assessed by means of a quantitative polymerase-chain-reaction assay. The risk of acute GVHD among recipients whose mismatched HLA-DPB1 allele was linked to rs9277534G (high expression) was compared with the risk among recipients whose mismatched HLA-DPB1 allele was linked to rs9277534A (low expression).ResultsThe mean HLA-DPB1 expression was lower with rs9277534A than with rs9277534G. Among recipients of transplants from donors with rs9277534A-linked HLA-DPB1, the risk of acute GVHD was higher for recipients with rs9277534G-linked HLA-DPB1 mismatches than for recipients with rs9277534A-linked HLA-DPB1 mismatches (hazard ratio, 1.54; 95% confidence interval [CI], 1.25 to 1.89; P<0.001), as was the risk of death due to causes other than disease recurrence (hazard ratio, 1.25; 95% CI, 1.00 to 1.57; P=0.05).ConclusionsThe risk of GVHD associated with HLA-DPB1 mismatching was influenced by the HLA-DPB1 rs9277534 expression marker. Among recipients of HLA-DPB1–mismatched transplants from donors with the low-expression allele, recipients with the high-expression allele had a high risk of GVHD. (Funded by the National Institutes of Health and others.)

Heterochrony underpins natural variation in Cardamine hirsuta leaf form

A key problem in biology is whether the same processes underlie morphological variation between and within species. Here, by using plant leaves as an example, we show that the causes of diversity at these two evolutionary scales can be divergent. Some species like the model plant Arabidopsis thaliana have simple leaves, whereas others like the A. thaliana relative Cardamine hirsuta bear complex leaves comprising leaflets. Previous work has shown that these interspecific differences result mostly from variation in local tissue growth and patterning. Now, by cloning and characterizing a quantitative trait locus (QTL) for C. hirsuta leaf shape, we find that a different process, age-dependent progression of leaf form, underlies variation in this trait within species. This QTL effect is caused by cis-regulatory variation in the floral repressor ChFLC, such that genotypes with low-expressing ChFLC alleles show both early flowering and accelerated age-dependent changes in leaf form, including faster leaflet production. We provide evidence that this mechanism coordinates leaf development with reproductive timing and may help to optimize resource allocation to the next generation.

Shame and Guilt-Proneness in Adolescents: Gene-Environment Interactions.

Rooted in people’s preoccupation with how they are perceived and evaluated, shame and guilt are self-conscious emotions that play adaptive roles in social behavior, but can also contribute to psychopathology when dysregulated. Shame and guilt-proneness develop during childhood and adolescence, and are influenced by genetic and environmental factors that are little known to date. This study investigated the effects of early traumatic events and functional polymorphisms in the brain-derived neurotrophic factor (BDNF) gene and the serotonin transporter gene promoter (5-HTTLPR) on shame and guilt in adolescents. A sample of N = 271 healthy adolescents between 14 and 17 years of age filled in measures of early traumatic events and proneness to shame and guilt, and were genotyped for the BDNF Val66Met and 5-HTTLPR polymorphisms. Results of moderator analyses indicated that trauma intensity was positively associated with guilt-proneness only in carriers of the low-expressing Met allele of BDNF Val66Met. This is the first study that identifies a gene-environment interaction that significantly contributes to guilt proneness in adolescents, with potential implications for developmental psychopathology.

Effects of methylated CpG islands in promoter region on the expression of KIR3DL1 protein

Objective To investigate the effects of methylated CpG islands in the promoter region on the expression of killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1). Methods Three voluntary unpaid blood donors carrying high expression allele KIR3DL1*01502 and three donors carrying low expression allele KIR3DL1*005 were recruited in this study. The nucleotide sequences and the methylated CpG islands in the promoter regions of KIR3DL1*01502 allele and KIR3DL1*005 allele were analyzed. The NK cells expressing KIR3DL1*01502 and KIR3DL1*005 were respectively treated with 5-aza for the demethylation of CpG islands within the promoters. The expression of KIR3DL1 protein on the surface of NK cells was measured with flow cytometer. Results Two differences at nucleotide sites -65 and -269 were detected within the promoter regions of KIR3DL1*01502 and KIR3DL1*005, resulting in two distinct CpG islands. The CpG islands within the promoter of KIR3DL1*01502 allele were highly methylated. The expression of KIR3DL1 protein on NK cells which carried KIR3DL1*01502 allele was significantly increased after the demethylation of CpG islands. However, the treatment of demethylation had no significant effects on the expression KIR3DL1 protein on NK cells harboring KIR3DL1*005 allele. Conclusion The methylated CpG islands within the promoter of KIR3DL1*01502 allele affected the antigen expression on the surface of NK cells. Different KIR3DL1 alleles might show different mechanisms in regulating antigen expression. Key words: Killer cell immunoglobulin like receptor; Methylation; Promoter

WWOX, the chromosomal fragile site FRA16D spanning gene: its role in metabolism and contribution to cancer.

The WWOX gene spans the common chromosomal fragile site FRA16D that is located within a massive (780 kb) intron. The WWOX gene is very long, at 1.1 Mb, which may contribute to the very low abundance of the full-length 1.4 kb mRNA. Alternative splicing also accounts for a variety of aberrant transcripts, most of which are devoid of C-terminal sequences required for WWOX to act as an oxidoreductase. The mouse WWOX gene also spans a chromosomal fragile site implying some sort of functional relationship that confers a selective advantage. The encoded protein domains of WWOX are conserved through evolution (between humans and Drosophila melanogaster) and include WW domains, an NAD -binding site, short-chain dehydrogenase/reductase enzyme and nuclear compartmentalization signals. This homology has enabled functional analyses in D. melanogaster that demonstrate roles for WWOX in reactive oxygen species regulation and metabolism. Indeed the human WWOX gene is also responsive to altered metabolism. Cancer cells typically exhibit altered metabolism (Warburg effect). Many cancers exhibit FRA16D DNA instability that results in aberrant WWOX expression and is associated with poor prognosis for these cancers. It is therefore thought that aberrant WWOX expression contributes to the altered metabolism in cancer. In addition, others have found that a specific (low-expression) allele of WWOX genotype contributes to cancer predisposition.

A Novel Mutation in the FECH Gene in a Czech Family with Erythropoietic Protoporphyria and a Population Study of IVS3-48C Variant Contributing to the Disease

Erythropoietic protoporphyria (EPP), a chronic erythropoietic porphyria, is characterized by excess accumulation of protoporphyrin, particularly in erythroid cells. EPP inheritance is complex, almost always associated with two molecular defects. In most EPP patients, clinical expression requires coinheritance of a private ferrochelatase (FECH) mutation trans- to a hypomorphic FECH*IVS3-48C allele. This leads to a decrease of FECH activity below the critical threshold. This is characterized by cutaneous photosensitivity in early childhood such as itching, burning, swelling and redness in sun-exposed areas. Hepatic failure occurs in some patients (about 1–10 % of EPP patients), which may necessitate liver transplantation. We investigated a Czech family with two patients with manifested EPP in four generations. We found a novel mutation, c.84G &gt;A, in the FECH gene in four individuals including proband and his mother (G84A transition in exon 2; p.W28*). Both clinically manifested probands inherited the hypomorphic IVS3-48C allele as well, while two clinically latent individuals with FECH mutation did not. To address the question whether the relatively low incidence of EPP in the Czech Republic might be due to lower frequency of the IVS3-48C allele, we screened for the frequency of the low expression allele in a control Czech (West Slaves) Caucasian population. Such study has not been performed in any Slavic population. Among 312 control individuals, there were no IVS3-48C/C (c.68-23C-T) homozygotes; 35 IVS3-48C/T heterozygous individuals were detected. The frequency of IVS3-48C allele was thus found to be 5.5 % in the Czech population, comparable to most West Caucasian populations.

A Functional Polymorphism of the MAOA Gene Is Associated with Neural Responses to Induced Anger Control

Aggressiveness is highly heritable. Recent experimental work has linked individual differences in a functional polymorphism of the monoamine oxidase-A gene (MAOA) to anger-driven aggression. Other work has implicated the dorsal ACC (dACC) in cognitive-emotional control and the amygdala in emotional arousal. The present imaging genetics study investigated dACC and amygdala reactivity to induced anger control as a function of MAOA genotype. A research assistant asked 38 healthy male undergraduates to control their anger in response to an insult by a rude experimenter. Men with the low-expression allele showed increased dACC and amygdala activation after the insult, but men with the high-expression allele did not. Both dACC and amygdala activation independently mediated the relationship between MAOA genotype and self-reported anger control. Moreover, following the insult, men with the high-functioning allele showed functional decoupling between the amygdala and dACC, but men with the low-functioning allele did not. These results suggest that heightened dACC and amygdala activation and their connectivity are neuroaffective mechanisms underlying anger control in participants with the low-functioning allele of the MAOA gene.