TP53 germline mutation testing in early-onset breast cancer: findings from a nationwide cohort.

Early-onset breast cancer may be due to Li-Fraumeni Syndrome (LFS). Current national and international guidelines recommend that TP53 genetic testing should be considered for women with breast cancer diagnosed before the age of 31 years. However, large studies investigating TP53 mutation prevalence in this population are scarce. We collected nationwide laboratory records for all young breast cancer patients tested for TP53 mutations in the Netherlands. Between 2005 and 2016, 370 women diagnosed with breast cancer younger than 30 years of age were tested for TP53 germline mutations, and eight (2.2%) were found to carry a (likely) pathogenic TP53 sequence variant. Among BRCA1/BRCA2 mutation negative women without a family history suggestive of LFS or a personal history of multiple LFS-related tumours, the TP53 mutation frequency was < 1% (2/233). Taking into consideration that TP53 mutation prevalence was comparable or even higher in some studies selecting patients with breast cancer onset at older ages or HER2-positive breast cancers, raises the question of whether a very early age of onset is an appropriate single TP53 genetic testing criterion.

Genotype-specific differences in the tumor metabolite profile of pheochromocytoma and paraganglioma using untargeted and targeted metabolomics.

CONTEXT AND OBJECTIVE: Pheochromocytomas and paragangliomas (PGLs) are neuroendocrine tumors of sympathetic or parasympathetic paraganglia. Nearly 40% of PGLs are caused by germline mutations. The present study investigated the effect of genetic alterations on metabolic networks in PGLs. DESIGN: Homogenates of 32 sporadic PGLs and 48 PGLs from patients with mutations in SDHB, SDHD, SDHAF-2, VHL, RET, and NF-1 were subjected to proton ((1)H) nuclear magnetic resonance (NMR) spectroscopy at 500 MHz for untargeted and HPLC tandem mass spectrometry for targeted metabolite profiling. RESULTS: (1)H NMR spectroscopy identified 28 metabolites in PGLs of which 12 showed genotype-specific differences. Part of these results published earlier reported low complex II activity (P < .0001) and low ATP/ADP/AMP content (P < .001) in SDH-related PGLs compared with sporadics and PGLs of other genotypes. Extending these results, low levels of N-acetylaspartic acid (NAA; P < .05) in SDH tumors and creatine (P < .05) in VHL tumors were observed compared with sporadics and other genotypes. Positive correlation was observed between NAA and ATP/ADP/AMP content (P < .001) and NAA and complex II activity (P < .0001) of PGLs. Targeted purine analysis in PGLs showed low adenine in cluster 1 compared with cluster 2 tumors (SDH P < .0001; VHL P < .05) whereas lower levels (P < .05) of guanosine and hypoxanthine were observed in RET tumors compared with SDH tumors. Principal component analysis (PCA) of metabolites could distinguish PGLs of different genotypes. CONCLUSIONS: The present study gives a comprehensive picture of alterations in energy metabolism in SDH- and VHL-related PGLs and establishes the interrelationship of energy metabolism and amino acid and purine metabolism in PGLs.

Fourfold increased detection of Lynch syndrome by raising age limit for tumour genetic testing from 50 to 70 years is cost-effective.

BACKGROUND: Recognising colorectal cancer (CRC) patients with Lynch syndrome (LS) can increase life expectancy of these patients and their close relatives. To improve identification of this under-diagnosed disease, experts suggested raising the age limit for CRC tumour genetic testing from 50 to 70 years. The present study evaluates the efficacy and cost-effectiveness of this strategy. METHODS: Probabilistic efficacy and cost-effectiveness analyses were carried out comparing tumour genetic testing of CRC diagnosed at age 70 or below (experimental strategy) versus CRC diagnosed at age 50 or below (current practice). The proportions of LS patients identified and cost-effectiveness including cascade screening of relatives, were calculated by decision analytic models based on real-life data. RESULTS: Using the experimental strategy, four times more LS patients can be identified among CRC patients when compared with current practice. Both the costs to detect one LS patient (€9437/carrier versus €4837/carrier), and the number needed to test for detecting one LS patient (42 versus 19) doubled. When family cascade screening was included, the experimental strategy was found to be highly cost-effective according to Dutch standards, resulting in an overall ratio of €2703 per extra life-year gained in additionally tested patients. CONCLUSION: Testing all CRC tumours diagnosed at or below age 70 for LS is cost-effective. Implementation is important as relatives from the large number of LS patients that are missed by current practice, can benefit from life-saving surveillance.

Easy-to-use online referral test detects most patients with a high familial risk of colorectal cancer.

AIM: Currently only 12-30% of individuals with a high risk of Lynch syndrome, the most common hereditary colorectal cancer (CRC) syndrome, are referred for genetic counselling. We assessed the sensitivity, usability and user experiences of a new online referral test aimed at improving referral of high-risk individuals for genetic counselling. METHOD: Sensitivity was assessed by entering pedigree data from high-risk individuals (i.e. Lynch syndrome mutation carriers) into the referral test to determine whether genetic counselling was recommended. For usability, we assessed nonmedical staff members' ability to determine referral, according to guidelines, in seven fictive clinical cases using the referral test after minimal training. Real-life users answered questions about their experience with the referral test. RESULT: Sensitivity of the referral test was 91% for mutation carriers with CRC (n = 164) and 73% for all affected and nonaffected mutation carriers (n = 420). Nonmedical staff members (n = 20) determined referral according to guidelines in 84% of cases using the referral test. Ten per cent (256/2470) of real-life users provided feedback about experiences; of those, 71% reported that the referral test increased reassurance, certainty about their familial risk and/or certainty about referral. CONCLUSION: The referral test has a high sensitivity in detecting individuals with a high risk of Lynch syndrome and is suitable for use in clinical practice. Widespread use of the referral test should improve cancer prevention in high-risk patients and their relatives.

ATL1 and REEP1 mutations in hereditary and sporadic upper motor neuron syndromes.

SPAST mutations are the most common cause of autosomal dominant hereditary spastic paraplegias (AD-HSPs), but many spastic paraplegia patients are found to carry no mutations in this gene. In order to assess the contribution of ATL1 and REEP1 in AD-HSP, we performed mutational analysis in 27 SPAST-negative AD-HSP families. We found three novel ATL1 mutations and one REEP1 mutation in five index-patients. In 110 patients with sporadic adult-onset upper motor neuron syndromes, a novel REEP1 mutation was identified in one patient. Apart from a significantly younger age at onset in ATL1 patients and restless legs in some, the clinical phenotype of ATL1 and REEP1 was similar to other pure AD-HSPs.

Hereditary leiomyomatosis and renal cell cancer in families referred for fumarate hydratase germline mutation analysis.

Heterozygous fumarate hydratase (FH) germline mutations cause hereditary leiomyomatosis and renal cell cancer (HLRCC), an autosomal dominant syndrome characterized by multiple cutaneous piloleiomyomas, uterine leiomyomas and papillary type 2 renal cancer. The main objective of our study was to evaluate clinical and genetic data from families suspected of HLRCC on a nationwide level. All families referred for FH mutation analysis in the Netherlands were assessed. We performed FH sequence analysis and multiplex ligation-dependent probe amplification. Families with similar FH mutations were examined for haplotype sharing. In 14 out of 33 families, we identified 11 different pathogenic FH germline mutations, including 4 novel mutations and 1 whole-gene deletion. Clinical data were available for 35 FH mutation carriers. Cutaneous leiomyomas were present in all FH mutation carriers older than 40 years of age. Eleven out of 21 female FH mutation carriers underwent surgical treatment for symptomatic uterine leiomyomas at an average of 35 years. Two FH mutation carriers had papillary type 2 renal cancer and Wilms' tumour, respectively. We evaluated the relevance of our findings for clinical practice and have proposed clinical diagnostic criteria, indications for FH mutation analysis and recommendations for management.

Hereditary spastic paraplegia due to SPAST mutations in 151 Dutch patients: new clinical aspects and 27 novel mutations.

BACKGROUND: In the clinically and genetically heterogeneous group of the hereditary spastic paraplegias (HSPs), mutations in the SPAST gene are most frequently found and cause a pure autosomal dominant form. OBJECTIVE: To provide the clinical and genetic characteristics of Dutch patients with HSP due to a SPAST mutation (SPG4). METHODS: SPAST mutation carriers were identified through a comprehensive national database search. Available medical records were reviewed. RESULTS: 151 mutation carriers carried 60 different changes in the SPAST gene, of which one was a known polymorphism, and 27 were novel. Missense mutations were most frequently found (39%). Clinical information was available from 72 mutation carriers. Age at onset ranged from 1 to 63 years with a bimodal peak distribution in the first decade and above age 30. The predominantly pure spastic paraplegia was accompanied by deep sensory disturbances and sphincter problems in almost 50%. An additional hand tremor was found in 10%. Patients with missense mutations and exon deletions did not reveal a distinctive phenotype. CONCLUSIONS: Dutch SPAST mutation carriers show a broad mutation spectrum, with 27 novel mutations in the present series. A bimodal peak distribution in age at onset was found and an accompanying tremor as peculiar feature of SPG4. The pathogenicity of S44L, the first exon 4 mutation, and a possible autosomal recessive mode of inheritance are discussed.

TRPV5, the gateway to Ca2+ homeostasis.

Ca2+ homeostasis in the body is tightly controlled, and is a balance between absorption in the intestine, excretion via the urine, and exchange from bone. Recently, the epithelial Ca2+ channel (TRPV5) has been identified as the gene responsible for the Ca2+ influx in epithelial cells of the renal distal convoluted tubule. TRPV5 is unique within the family of transient receptor potential (TRP) channels due to its high Ca2+ selectivity. Ca2+ flux through TRPV5 is controlled in three ways. First, TRPV5 gene expression is regulated by calciotropic hormones such as vitamin D3 and parathyroid hormone. Second, Ca2+ transport through TRPV5 is controlled by modulating channel activity. Intracellular Ca2+, for example, regulates channel activity by feedback inhibition. Third, TRPV5 is controlled by mobilization of the channel through trafficking toward the plasma membrane. The newly identified anti-aging hormone Klotho regulates TRPV5 by cleaving off sugar residues from the extracellular domain of the protein, resulting in a prolonged expression of TRPV5 at the plasma membrane. Inactivation of TRPV5 in mice leads to severe hypercalciuria, which is compensated by increased intestinal Ca2+ absorption due to augmented vitamin D3 levels. Furthermore, TRPV5 deficiency in mice is associated with polyuria, urine acidification, and reduced bone thickness. Some pharmaceutical compounds, such as the immunosuppressant FK506, affect the Ca2+ balance by modulating TRPV5 gene expression. This underlines the importance of elucidating the role of TRPV5 in Ca(2+)-related disorders, thereby enhancing the possibilities for pharmacological intervention. This chapter describes a unique TRP channel and highlights its regulation and function in renal Ca2+ reabsorption and overall Ca2+ homeostasis.

Stimulation of the in vivo production of very low density lipoproteins by apolipoprotein E is independent of the presence of the low density lipoprotein receptor.

Apolipoprotein (apo) E stimulates the secretion of very low density lipoproteins (VLDLs) by an as yet unknown mechanism. Recently, a working mechanism for apoE was proposed (Twisk, J., Gillian-Daniel, D. L., Tebon, A., Wang, L., Barrett, P. H., and Attie, A. D. (2000) J. Clin. Invest. 105, 521-532) in which apoE prevents the inhibitory action of the low density lipoprotein receptor (LDLr) by binding to it. We have first tested whether this newly described effect of the LDLr on VLDL secretion, obtained in vitro, is also observed in vivo. In LDLr knockout mice (LDLr-/-), the production of VLDL triglycerides and apoB was 30% higher than that in controls. Also the ratio of apoB100:apoB48 secretion was increased in the LDLr-/- mice. The composition of nascent VLDL was similar in both strains. To test whether the action of apoE depends on the presence of the LDLr, VLDL production was measured in LDLr-/- and apoE-/- LDLr-/- mice. Deletion of apoE on a LDLr-/- background still caused a 50% decrease of VLDL triglycerides and apoB production. The composition of nascent VLDL was again similar for both strains. We conclude that the effect of apoE on hepatic VLDL production is independent of the presence of the LDLr.

Mice expressing only the mutant APOE3Leiden gene show impaired VLDL secretion.

Apolipoprotein E (apoE)-deficient mice develop hepatic steatosis and show impaired very low density lipoprotein (VLDL)-triglyceride (TG) secretion. These effects are normalized on the introduction of the human APOE3 gene. To assess whether this apoE effect is isoform specific, we studied hepatic lipid metabolism in mice expressing either APOE3 or the mutant APOE3Leiden on apoe-/- or apoe+/- backgrounds. The transgenes were expressed mainly in periportal hepatocytes, as revealed by in situ hybridization. Mice expressing APOE3Leiden, on the apoe-/- and apoe+/- backgrounds, had fatty livers, which were absent in APOE3/apoe-/- mice. APOE3Leiden/apoe-/- mice showed a strongly reduced VLDL-TG secretion compared with APOE3/apoe-/- mice (48+/-14 versus 82+/-10 micromol/kg per hour, respectively). The presence of a single mouse apoe allele increased VLDL-TG secretion in APOE3Leiden/apoe+/- mice (121+/-43 micromol/kg per hour) compared with APOE3Leiden/apoe-/- mice. These results show that APOE3Leiden does not prevent development of a fatty liver and does not normalize VLDL-TG secretion in mice with an apoE-deficient background. The presence of a single mouse apoe allele is sufficient to normalize the APOE3Leiden-associated reduction of VLDL-TG secretion but does not prevent steatosis. We conclude that apoE-mediated stimulation of VLDL secretion is isoform specific.

Hepatic lipid accumulation, altered very low density lipoprotein formation and apolipoprotein E deposition in apolipoprotein E3-Leiden transgenic mice.

BACKGROUND/AIM: Apolipoprotein (apo) E-deficiency leads to hepatic steatosis and impaired Very Low Density Lipoprotein (VLDL)-triglyceride production rates in mice. A mutant apoE isoform, apoE3-Leiden, is associated with a dominantly inherited form of dysbetalipoproteinemia in humans. The aim of this study was to evaluate the effects of APOE*3-Leiden expression on hepatic lipid content, VLDL formation and liver morphology in mice. METHODS: Comparison of lipid parameters and liver morphology in mouse strains with different expression of the APOE*3-Leiden transgene with and without co-expression of human APOCI. RESULTS: Hepatic triglyceride content was increased to maximally 233% of control values, depending on hepatic APOE*3-Leiden expression. Hepatic secretion of VLDL-associated triglycerides was impaired (-20%) in high-expressing transgenics, with a concomitant increase from 1.6 to 8.1 of the apoB48/ apoB100 ratio in newly-formed VLDL. Hepatocytes of the transgenic mice contained characteristic inclusions, up to 20 microm in diameter, in numbers dependent on APOE*3-Leiden expression and independent of APOCI expression. These inclusions contained material positively reacting with antihuman apoE antibodies. Immunogold-labeling confirmed the presence of apoE3-Leiden within these inclusions and also revealed the presence of the mutant protein on sinusoidal membranes, in multivesicular bodies and in peroxisomes, i.e., a distribution pattern similar to that of endogenous apoE in rodents. Nascent VLDL particles associated with the Golgi apparatus were also labeled. CONCLUSION: This study has demonstrated that introduction of human apoE3-Leiden in mice, in addition to its reported effects on lipolysis and lipoprotein clearance, leads to hepatic deposition of the mutant apolipoprotein, development of fatty liver and to altered hepatic VLDL secretion. The latter findings are consistent with a role of apoE in the regulation of intrahepatic lipid metabolism.

Apolipoprotein E participates in the regulation of very low density lipoprotein-triglyceride secretion by the liver.

ApoE-deficient mice on low fat diet show hepatic triglyceride accumulation and a reduced very low density lipoprotein (VLDL) triglyceride production rate. To establish the role of apoE in the regulation of hepatic VLDL production, the human APOE3 gene was introduced into apoE-deficient mice by cross-breeding with APOE3 transgenics (APOE3/apoe-/- mice) or by adenoviral transduction. APOE3 was expressed in the liver and, to a lesser extent, in brain, spleen, and lung of transgenic APOE3/apoe-/- mice similar to endogenous apoe. Plasma cholesterol levels in APOE/apoe-/- mice (3.4 +/- 0.5 mM) were reduced when compared with apoe-/- mice (12.6 +/- 1.4 mM) but still elevated when compared with wild type control values (1.9 +/- 0.1 mM). Hepatic triglyceride accumulation in apoE-deficient mice was completely reversed by introduction of the APOE3 transgene. The in vivo hepatic VLDL-triglyceride production rate was reduced to 36% of control values in apoE-deficient mice but normalized in APOE3/apoe-/- mice. Hepatic secretion of apoB was not affected in either of the strains. Secretion of (3)H-labeled triglycerides synthesized from [(3)H]glycerol by cultured hepatocytes from apoE-deficient mice was four times lower than by APOE3/apoe-/- or control hepatocytes. The average size of secreted VLDL particles produced by cultured apoE-deficient hepatocytes was significantly reduced when compared with those of APOE3/apoe-/- and wild type mice. Hepatic expression of human APOE3 cDNA via adenovirus-mediated gene transfer in apoE-deficient mice resulted in a reduction of plasma cholesterol depending on plasma apoE3 levels. The in vivo VLDL-triglyceride production rate in these mice was increased up to 500% compared with LacZ-injected controls and correlated with the amount of apoE3 per particle. These findings indicate a regulatory role of apoE in hepatic VLDL-triglyceride secretion, independent from its role in lipoprotein clearance.