The significance of PTEN and AKT aberrations in pediatric T-cell acute lymphoblastic leukemia

textabstractBackground PI3K/AKT pathway mutations are found in T-cell acute lymphoblastic leukemia, but their overall impact and associations with other genetic aberrations is unknown. PTEN mutations have been proposed as secondary mutations that follow NOTCH1-activating mutations and cause cellular resistance to γ-secretase inhibitors. Design and Methods The impact of PTEN, PI3K and AKT aberrations was studied in a genetically well-characterized pediatric T-cell leukemia patient cohort (n=146) treated on DCOG or COALL protocols. Results PTEN and AKT E17K aberrations were detected in 13% and 2% of patients, respectively. Defective PTEN-splicing was identified in incidental cases. Patients without PTEN protein but lacking exon-, splice-, promoter mutations or promoter hypermethylation were present. PTEN/AKTmutations were especially abundant in TAL- or LMO-rearranged leukemia but nearly absent in TLX3-rearranged patients (P=0.03), the opposite to that observed for NOTCH1- activating mutations. Most PTEN/AKT mutant patients either lacked NOTCH1-activating mutations (P=0.006) or had weak NOTCH1-activating mutations (P=0.011), and consequently expressed low intracellular NOTCH1, cMYC and MUSASHI levels. T-cell leukemia patients without PTEN/AKT and NOTCH1-activating mutations fared well, with a cumulative incidence of relapse of only 8% versus 35% for PTEN/AKT and/or NOTCH1-activated patients (P=0.005). Conclusions PI3K/AKT pathway aberrations are present in 18% of pediatric T-cell acute lymphoblastic leukemia patients. Absence of strong NOTCH1-activating mutations in these cases may explain cellular insensitivity to γ-secretase inhibitors

The significance of PTEN and AKT aberrations in pediatric T-cell acute lymphoblastic leukemia

Background PI3K/AKT pathway mutations are found in T-cell acute lymphoblastic leukemia, but their overall impact and associations with other genetic aberrations is unknown. PTEN mutations have been proposed as secondary mutations that follow NOTCH1-activating mutations and cause cellular resistance to γ-secretase inhibitors. Design and Methods The impact of PTEN, PI3K and AKT aberrations was studied in a genetically well-characterized pediatric T-cell leukemia patient cohort (n=146) treated on DCOG or COALL protocols. Results PTEN and AKT E17K aberrations were detected in 13% and 2% of patients, respectively. Defective PTEN-splicing was identified in incidental cases. Patients without PTEN protein but lacking exon-, splice-, promoter mutations or promoter hypermethylation were present. PTEN/AKTmutations were especially abundant in TAL- or LMO-rearranged leukemia but nearly absent in TLX3-rearranged patients (P=0.03), the opposite to that observed for NOTCH1- activating mutations. Most PTEN/AKT mutant patients either lacked NOTCH1-activating mutations (P=0.006) or had weak NOTCH1-activating mutations (P=0.011), and consequently expressed low intracellular NOTCH1, cMYC and MUSASHI levels. T-cell leukemia patients without PTEN/AKT and NOTCH1-activating mutations fared well, with a cumulative incidence of relapse of only 8% versus 35% for PTEN/AKT and/or NOTCH1-activated patients (P=0.005). Conclusions PI3K/AKT pathway aberrations are present in 18% of pediatric T-cell acute lymphoblastic leukemia patients. Absence of strong NOTCH1-activating mutations in these cases may explain cellular insensitivity to γ-secretase inhibitors

Successful Genetic Screening and Creating Awareness of Familial Hypercholesterolemia and Other Heritable Dyslipidemias in the Netherlands

The genetic screening program for familial hypercholesterolemia (FH) in the Netherlands, which was embraced by the Dutch Ministry of Health from 1994 to 2014, has led to twenty years of identification of at least 1500 FH cases per year. Although funding by the government was terminated in 2014, the approach had proven its effectiveness and had built the foundation for the development of more sophisticated diagnostic tools, clinical collaborations, and new molecular-based treatments for FH patients. As such, the community was driven to continue the program, insurance companies were convinced to collaborate, and multiple approaches were launched to find new index cases with FH. Additionally, the screening was extended, now also including other heritable dyslipidemias. For this purpose, a diagnostic next-generation sequencing (NGS) panel was developed, which not only comprised the culprit LDLR, APOB, and PCSK9 genes, but also 24 other genes that are causally associated with genetic dyslipidemias. Moreover, the NGS technique enabled further optimization by including pharmacogenomic genes in the panel. Using such a panel, more patients that are prone to cardiovascular diseases are being identified nowadays and receive more personalized treatment. Moreover, the NGS output teaches us more and more about the dyslipidemic landscape that is less straightforward than we originally thought. Still, continuous progress is being made that underlines the strength of genetics in dyslipidemia, such as discovery of alternative genomic pathogenic mechanisms of disease development and polygenic contribution

Successful genetic screening and creating awareness of familial hypercholesterolemia and other heritable Dyslipidemias in the Netherlands

The genetic screening program for familial hypercholesterolemia (FH) in the Netherlands, which was embraced by the Dutch Ministry of Health from 1994 to 2014, has led to twenty years of identification of at least 1500 FH cases per year. Although funding by the government was terminated in 2014, the approach had proven its effectiveness and had built the foundation for the development of more sophisticated diagnostic tools, clinical collaborations, and new molecular-based treatments for FH patients. As such, the community was driven to continue the program, insurance companies were convinced to collaborate, and multiple approaches were launched to find new index cases with FH. Additionally, the screening was extended, now also including other heritable dyslipidemias. For this purpose, a diagnostic next-generation sequencing (NGS) panel was developed, which not only comprised the culprit LDLR, APOB, and PCSK9 genes, but also 24 other genes that are causally associated with genetic dyslipidemias. Moreover, the NGS technique enabled further optimization by including pharmacogenomic genes in the panel. Using such a panel, more patients that are prone to cardiovascular diseases are being identified nowadays and receive more personalized treatment. Moreover, the NGS output teaches us more and more about the dyslipidemic landscape that is less straightforward than we originally thought. Still, continuous progress is being made that underlines the strength of genetics in dyslipidemia, such as discovery of alternative genomic pathogenic mechanisms of disease development and polygenic contribution

ABCG5 and ABCG8 genetic variants in familial hypercholesterolemia

Background: Familial hypercholesterolemia (FH) is a common inherited disease characterized by elevated low-density lipoprotein cholesterol (LDL-C) plasma levels and increased cardiovascular disease risk. Most patients carry a mutation in the low-density lipoprotein receptor gene (LDLR). Common and rare variants in the genes encoding adenosine triphosphate–binding cassette transporters G5 and G8 (ABCG5 and ABCG8) have been shown to affect LDL-C levels. Objective: The objective of this study was to investigate whether and to which extent heterozygous variants in ABCG5 and ABCG8 are associated with the hypercholesterolemic phenotype. Methods: We sequenced ABCG5 and ABCG8 in a cohort of 3031 clinical FH patients and compared the prevalence of variants with a European reference population (gnomAD). Clinical characteristics of carriers of putative pathogenic variants in ABCG5 and/or ABCG8 were compared with heterozygous carriers of mutations in LDLR. Furthermore, we assessed the segregation of one ABCG5 and two ABCG8 variants with plasma lipid and sterol levels in three kindreds. Results: The frequencies of (likely) pathogenic LDLR, APOB, PCSK9, ABCG5, and ABCG8 variants in our FH cohort were 11.42%, 2.84%, 0.69%, 1.48%, and 0.96%, respectively. We identified 191 ABCG5 and ABCG8 variants of which 53 were classified as pathogenic or likely pathogenic. Of these 53 variants, 51 were either absent from a reference population or more prevalent in our FH cohort than in the reference population. LDL-C levels were significantly lower in heterozygous carriers of a (likely) pathogenic ABCG5 or ABCG8 variant compared to LDLR mutation carriers (6.2 ± 1.7 vs 7.2 ± 1.7 mmol/L, P < .001). The combination of both an ABCG5 or ABCG8 variant and a LDLR variant was found not to be associated with significant higher LDL-C levels (7.8 ± 2.3 vs 7.2 ± 1.7 mmol/L, P = .259). Segregation analysis in three families (nine carriers, in addition to the index cases, and 16 noncarriers) did not show complete segregation of the ABCG5/G8 variants with high LDL-C levels, and LDL-C levels were not different (3.9 ± 1.3 vs 3.5 ± 0.6 mmol/L in carriers and noncarriers, respectively, P = .295), while plasma plant sterol levels were higher in carriers compared to noncarriers (cholestanol: 10.2 ± 1.7 vs 8.4 ± 1.6 μmol/L, P = .007; campesterol: 22.5 ± 10.1 vs 13.4 ± 3.5 μmol/L, P = .008; sitosterol: 17.0 ± 11.6 vs 8.2 ± 2.6 μmol/L, P = .024). Conclusions: 2.4% of subjects in our FH cohort carried putative pathogenic ABCG5 and ABCG8 variants but had lower LDL-C levels compared to FH patients who were heterozygous carriers of an LDLR variant. These results suggest a role for these genes in hypercholesterolemia in FH patients with less severely elevated LDL-C levels. We did not find evidence that these variants cause autosomal dominant FH

Next-generation sequencing to confirm clinical familial hypercholesterolemia

BACKGROUND: Familial hypercholesterolemia is characterised by high low-density lipoprotein-cholesterol levels and is caused by a pathogenic variant in LDLR, APOB or PCSK9. We investigated which proportion of suspected familial hypercholesterolemia patients was genetically confirmed, and whether this has changed over the past 20 years in The Netherlands. METHODS: Targeted next-generation sequencing of 27 genes involved in lipid metabolism was performed in patients with low-density lipoprotein-cholesterol levels greater than 5 mmol/L who were referred to our centre between May 2016 and July 2018. The proportion of patients carrying likely pathogenic or pathogenic variants in LDLR, APOB or PCSK9, or the minor familial hypercholesterolemia genes LDLRAP1, ABCG5, ABCG8, LIPA and APOE were investigated. This was compared with the yield of Sanger sequencing between 1999 and 2016. RESULTS: A total of 227 out of the 1528 referred patients (14.9%) were heterozygous carriers of a pathogenic variant in LDLR (80.2%), APOB (14.5%) or PCSK9 (5.3%). More than 50% of patients with a Dutch Lipid Clinic Network score of 'probable' or 'definite' familial hypercholesterolemia were familial hypercholesterolemia mutation-positive; 4.8% of the familial hypercholesterolemia mutation-negative patients carried a variant in one of the minor familial hypercholesterolemia genes. The mutation detection rate has decreased over the past two decades, especially in younger patients in which it dropped from 45% in 1999 to 30% in 2018. CONCLUSIONS: A rare pathogenic variant in LDLR, APOB or PCSK9 was identified in 14.9% of suspected familial hypercholesterolemia patients and this rate has decreased in the past two decades. Stringent use of clinical criteria algorithms is warranted to increase this yield. Variants in the minor familial hypercholesterolemia genes provide a possible explanation for the familial hypercholesterolemia phenotype in a minority of patients

Enhanced identification of familial hypercholesterolemia using central laboratory algorithms

Background and aims: Familial hypercholesterolemia (FH) is a highly prevalent genetic disorder resulting in markedly elevated LDL cholesterol levels and premature coronary artery disease. FH underdiagnosis and undertreatment require novel detection methods. This study evaluated the effectiveness of using an LDL cholesterol cut-off ≥99.5th percentile (sex- and age-adjusted) to identify clinical and genetic FH, and investigated underutilization of genetic testing and undertreatment in FH patients. Methods:Individuals with at least one prior LDL cholesterol level ≥99.5th percentile were selected from a laboratory database containing lipid profiles of 590,067 individuals. The study comprised three phases: biochemical validation of hypercholesterolemia, clinical identification of FH, and genetic determination of FH.Results: Of 5614 selected subjects, 2088 underwent lipid profile reassessment, of whom 1103 completed the questionnaire (mean age 64.2 ± 12.7 years, 48% male). In these 1103 subjects, mean LDL cholesterol was 4.0 ± 1.4 mmol/l and 722 (65%) received lipid-lowering therapy. FH clinical diagnostic criteria were met by 282 (26%) individuals, of whom 85% had not received guideline-recommended genetic testing and 97% failed to attain LDL cholesterol targets. Of 459 individuals consenting to genetic validation, 13% carried an FH-causing variant, which increased to 19% in clinically diagnosed FH patients.Conclusions: The identification of a substantial number of previously undiagnosed and un(der)treated clinical and genetic FH patients within a central laboratory database highlights the feasibility and clinical potential of this targeted screening strategy; both in identifying new FH patients and in improving treatment in this high-risk population.</p

A Deep Intronic Variant in LDLR in Familial Hypercholesterolemia

BACKGROUND: Familial hypercholesterolemia (FH) is an inherited disorder characterized by high plasma LDL-C (low-density lipoprotein-cholesterol) levels. The vast majority of FH patients carry a mutation in the coding region of LDLR, APOB, or PCSK9. We set out to identify the culprit genetic defect in a large family with clinical FH, in whom no mutations were identified in the coding regions of these FH genes. METHODS: Whole genome sequencing was performed in 5 affected and 4 unaffected individuals from a family with an unexplained autosomal dominant FH trait. The effect on splicing of the identified novel intronic LDLR mutation was ascertained by cDNA sequencing. The prevalence of the novel variant was assessed in 1 245 FH patients without an FH causing mutation identified by Sanger sequencing and in 2 154 patients referred for FH analysis by next-generation sequencing (covering the intronic region). RESULTS: A novel deep intronic variant in LDLR (c.2140+103G>T) was found to cosegregate with high LDL-C in 5 patients, but was not present in 4 unaffected family members. The variant was shown to result in a 97 nucleotides insertion leading to a frameshift and premature stop codon in exon 15 of LDLR. The prevalence of the intronic variant was 0.24% (3/1245) in a cohort of FH patients without a known FH causing mutation and 0.23% (5/2154) in a population of FH patients referred for analysis by next-generation sequencing. Cosegregation analysis of a second family showed full penetrance of the novel variant with the FH phenotype over 3 generations. CONCLUSIONS: The c.2140+103G>T mutation in LDLR is a novel intronic variant identified in FH that cosegregates with the FH phenotype. Our findings underline the need to analyze the intronic regions of LDLR in patients with FH, especially those in whom no mutation is found in the coding regions of LDLR, APOB, or PCSK9

Assessment of practical applicability and clinical relevance of a commonly used LDL-C polygenic score in patients with severe hypercholesterolemia

Background and aims: Low-density lipoprotein cholesterol (LDL-C) levels vary in patients with familial hypercholesterolemia (FH) and can be explained by a single deleterious genetic variant or by the aggregate effect of multiple, common small-effect variants that can be captured in a polygenic score (PS). We set out to investigate the contribution of a previously published PS to the inter-individual LDL-C variation and coronary artery disease (CAD) risk in patients with a clinical FH phenotype. Methods: First, in a cohort of 628 patients referred for genetic FH testing, we evaluated the distribution of a PS for LDL-C comprising 12 genetic variants. Next, we determined its association with coronary artery disease (CAD) risk using UK Biobank data. Results: The mean PS was higher in 533 FH-variant-negative patients (FH/M-) compared with 95 FH-variant carriers (1.02 vs 0.94, p < 0.001). 39% of all patients had a PS equal to the top 20% from a population-based reference cohort and these patients were less likely to carry an FH variant (OR 0.22, 95% CI 0.10–0.48) compared with patients in the lowest 20%. In UK Biobank data, the PS explained 7.4% of variance in LDL-C levels and was associated with incident CAD. Addition of PS to a prediction model using age and sex and LDL-C did not increase the c-statistic for predicting CAD risk. Conclusions: This 12-variant PS was higher in FH/M- patients and associated with incident CAD in UK Biobank data. However, the PS did not improve predictive accuracy when added to the readily available characteristics age, sex and LDL-C, suggesting limited discriminative value for CAD

Use of Lipoprotein(a) to improve diagnosis and management in clinical familial hypercholesterolemia

Background and aims: Lipoprotein(a) (Lp(a)) is an LDL-like particle whose plasma levels are largely genetically determined. The impact of measuring Lp(a) in patients with clinical familial hypercholesterolemia (FH) referred for genetic testing is largely unknown. We set out to evaluate the contribution of (genetically estimated) Lp(a) in a large nation-wide referral population of clinical FH. Methods: In 1504 patients referred for FH genotyping, we used an LPA genetic instrument (rs10455872 and rs3798220) as a proxy for plasma Lp(a) levels. The genetic Lp(a) proxy was used to correct LDL-cholesterol and reclassify patients with clinical FH based on Dutch Lipid Criteria Network (DLCN) scoring. Finally, we used estimated Lp(a) levels to reclassify ASCVD risk using the SCORE and SMART risk scores. Results: LPA SNPs were more prevalent among mutation-negative compared with mutation-positive patients (296/1280 (23.1%) vs 35/224 (15.6%), p = 0.016). Among patients with genetically defined high Lp(a) levels, 9% were reclassified to the DLCN category ‘unlikely FH’ using Lp(a)-corrected LDL-cholesterol (LDL-Ccor) and all but one of these patients indeed carried no FH variant. Furthermore, elevated Lp(a) reclassified predicted ASCVD risk into a higher category in up to 18% of patients. Conclusions: In patients referred for FH molecular testing, we show that taking into account (genetically estimated) Lp(a) levels not only results in reclassification of probability of genetic FH, but also has an impact on individual cardiovascular risk evaluation. However, to avoid missing the diagnosis of an FH variant, clear thresholds for the use of Lp(a)-cholesterol adjusted LDL-cholesterol levels in patients referred for genetic testing of FH must be established