Delineating genotype and parent-of-origin effect on the phenotype in MSH6-associated Lynch syndrome.

BACKGROUND: This study investigates the potential influence of genotype and parent-of-origin effects (POE) on the clinical manifestations of Lynch syndrome (LS) within families carrying (likely) disease-causing MSH6 germline variants. PATIENTS AND METHODS: A cohort of 1615 MSH6 variant carriers (310 LS families) was analyzed. Participants were categorized based on RNA expression and parental inheritance of the variant. Hazard ratios (HRs) were calculated using weighted Cox regression, considering external information to address ascertainment bias. The findings were cross-validated using the Prospective Lynch Syndrome Database (PLSD) for endometrial cancer (EC). RESULTS: No significant association was observed between genotype and colorectal cancer (CRC) risk (HR = 1.06, 95% confidence interval [CI]: 0.77-1.46). Patients lacking expected RNA expression exhibited a reduced risk of EC (Reference Cohort 1: HR = 0.68, 95% CI: 0.43-1.03; Reference Cohort 2: HR = 0.63, 95% CI: 0.46-0.87). However, these results could not be confirmed in the PLSD. Moreover, no association was found between POE and CRC risk (HR = 0.78, 95% CI: 0.52-1.17) or EC risk (Reference Cohort 1: HR = 0.93, 95% CI: 0.65-1.33; Reference Cohort 2: HR = 0.8, 95% CI: 0.64-1.19). DISCUSSION AND CONCLUSION: No evidence of POE was detected in MSH6 families. While RNA expression may be linked to varying risks of EC, further investigation is required to explore this observation.

Discordant Staining Patterns and Microsatellite Results in Tumors of MSH6 Pathogenic Variant Carriers.

Diagnosis of Lynch syndrome (LS) caused by a pathogenic germline MSH6 variant may be complicated by discordant immunohistochemistry (IHC) and/or by a microsatellite stable (MSS) phenotype. This study aimed to identify the various causes of the discordant phenotypes of colorectal cancer (CRC) and endometrial cancer (EC) in MSH6-associated LS. Data were collected from Dutch family cancer clinics. Carriers of a (likely) pathogenic MSH6 variant diagnosed with CRC or EC were categorized based on an microsatellite instability (MSI)/IHC test outcome that might fail to result in a diagnosis of LS (eg, retained staining of all 4 mismatch repair proteins, with or without an MSS phenotype, and other staining patterns). When tumor tissue was available, MSI and/or IHC were repeated. Next-generation sequencing (NGS) was performed in cases with discordant staining patterns. Data were obtained from 360 families with 1763 (obligate) carriers. MSH6 variant carriers with CRC or EC (n = 590) were included, consisting of 418 CRCs and 232 ECs. Discordant staining was reported in 77 cases (36% of MSI/IHC results). Twelve patients gave informed consent for further analysis of tumor material. Upon revision, 2 out of 3 MSI/IHC cases were found to be concordant with the MSH6 variant, and NGS showed that 4 discordant IHC results were sporadic rather than LS-associated tumors. In 1 case, somatic events explained the discordant phenotype. The use of reflex IHC mismatch repair testing, the current standard in most Western countries, may lead to the misdiagnosis of germline MSH6 variant carriers. The pathologist should point out that further diagnostics for inheritable colon cancer, including LS, should be considered in case of a strong positive family history. Germline DNA analysis of the mismatch repair genes, preferably as part of a larger gene panel, should therefore be considered in potential LS patients.

Identification of Medicinal Products: Providing an Educational Framework.

The ISO standards for the Identification of Medicinal Products (IDMP) prove to be difficult to implement. Guidance needs to be provided to those using and further developing IDMP-related standards and terminologies. Providing fitting and structured education would be a step forward. This article describes research of current education and certification on IDMP and the creation of an educational framework for targeted IDMP knowledge transfer. This framework indicates the required level of knowledge for the various identified roles within the organizations working with IDMP. Based on a combination of desk research, a questionnaire, and individual interviews, relevant roles were identified covering the various organizations (Users, Educators, Medicine Authorities, Standard Developing Organizations, and IT-suppliers) and five levels of required knowledge were determined, including applicable roles and educational components. Furthermore, this article lists several recommendations that should be taken into consideration whilst developing content and implementing educational modules for IDMP.

Birt-Hogg-Dubé syndrome in apparent primary spontaneous pneumothorax patients; results and recommendations for clinical practice.

BACKGROUND: Birt-Hogg-Dubé syndrome (BHD) is an inherited disease caused by pathogenic variants in the FLCN gene. One of the characteristics is the increased risk for spontaneous pneumothorax, likely due to the presence of pulmonary cysts mainly distributed under the carina. Due to variable expression and lack of awareness, BHD is likely to be underdiagnosed. We aimed to examine the prevalence of BHD in patients presenting with an apparent primary spontaneous pneumothorax and to evaluate the contribution of chest CT in establishing the diagnosis. METHODS: Patients who presented with apparent primary spontaneous pneumothorax between 2004 and 2017 in a large Dutch teaching hospital were enrolled in this quantitative cross-sectional study. A questionnaire was sent to eligible patients. Patients who completed the questionnaire and consented to further participation were invited to visit the hospital for genetic testing and low dose, volumetric chest CT. RESULTS: Genetic testing was performed in 88 patients with apparent primary spontaneous pneumothorax. Three patients were found to have a pathogenic variant in the FLCN gene (3.4%). No variants of unknown significance were detected. Pulmonary cysts were detected in 14 out of 83 participants with an available chest CT, six had more than one cyst. All three patients with BHD had multiple pulmonary cysts. CONCLUSIONS: Based on previous literature and the present study, we believe that performing a chest CT in every patient presenting with primary spontaneous pneumothorax is justified. Subsequent genetic testing of the FLCN gene should be considered when multiple pulmonary cysts are present. TRIAL REGISTRATION: The study was registered at clinicaltrials.gov with reference NCT02916992. Three out of 88 patients with an apparent primary spontaneous pneumothorax were diagnosed with Birt-Hogg-Dubé syndrome in this study and all three had multiple pulmonary cysts. We believe that performing a chest CT in every patient with an apparent primary spontaneous pneumothorax is justified to identify underlying diseases.

Genetic Evaluation in a Cohort of 126 Dutch Pulmonary Arterial Hypertension Patients.

Pulmonary arterial hypertension (PAH) is a severe, life-threatening disease, and in some cases is caused by genetic defects. This study sought to assess the diagnostic yield of genetic testing in a Dutch cohort of 126 PAH patients. Historically, genetic testing in the Netherlands consisted of the analysis of BMPR2 and SMAD9. These genes were analyzed in 70 of the 126 patients. A (likely) pathogenic (LP/P) variant was detected in 22 (31%) of them. After the identification of additional PAH associated genes, a next generation sequencing (NGS) panel consisting of 19 genes was developed in 2018. Additional genetic testing was offered to the 48 BMPR2 and SMAD9 negative patients, out of which 28 opted for NGS analysis. In addition, this gene panel was analyzed in 56 newly identified idiopathic (IPAH) or pulmonary veno occlusive disease (PVOD) patients. In these 84 patients, NGS panel testing revealed LP/P variants in BMPR2 (N = 4), GDF2 (N = 2), EIF2AK4 (N = 1), and TBX4 (N = 3). Furthermore, 134 relatives of 32 probands with a LP/P variant were tested, yielding 41 carriers. NGS panel screening offered to IPAH/PVOD patients led to the identification of LP/P variants in GDF2, EIF2AK4, and TBX4 in six additional patients. The identification of LP/P variants in patients allows for screening of at-risk relatives, enabling the early identification of PAH.

Declining detection rates for APC and biallelic MUTYH variants in polyposis patients, implications for DNA testing policy.

This study aimed to determine the prevalence of APC-associated familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP) in a large cohort, taking into account factors as adenoma count and year of diagnosis. All application forms used to send patients in for APC and MUTYH variant analysis between 1992 and 2017 were collected (n = 2082). Using the data provided on the application form, the APC and biallelic MUTYH prevalence was determined and possible predictive factors were examined using multivariate multinomial logistic regression analysis in SPSS. The prevalence of disease causing variants in the APC gene significantly increases with adenoma count while MAP shows a peak prevalence in individuals with 50-99 adenomas. Logistic regression analysis shows significant odds ratios for adenoma count, age at diagnosis, and, interestingly, a decline in the chance of finding a variant in either gene over time. Moreover, in 22% (43/200) of patients with FAP-related extracolonic manifestations a variant was identified. The overall detection rates are above 10% for patients with >10 adenomas aged <60 and >20 adenomas aged <70. Patients with variants outside these criteria had FAP-related extracolonic manifestations, colorectal cancer aged <40, somatic KRAS c.34G > T variant in the tumor or a first-degree relative with >10 adenomas. Therefore, APC and MUTYH testing in patients with >10 adenomas aged <60 and with >20 adenomas aged <70 is advised. Almost all FAP and MAP patients not meeting these criteria showed other characteristics that can be used as an indication to prompt genetic testing.

No evidence for increased prevalence of colorectal carcinoma in 399 Dutch patients with Birt-Hogg-Dubé syndrome.

BACKGROUND: Previously, it has been suggested that colorectal polyps and carcinomas might be associated with Birt-Hogg-Dubé syndrome. We aimed to compare the occurrence of colorectal neoplasms between Dutch patients with Birt-Hogg-Dubé syndrome and their relatives without Birt-Hogg-Dubé syndrome. METHODS: In all, 399 patients with a pathogenic FLCN mutation and 382 relatives without the familial FLCN mutation were included. Anonymous data on colon and rectum pathology was provided by PALGA: the Dutch Pathology Registry. RESULTS: No significant difference in the percentage of individuals with a history of colorectal carcinoma was found between the two groups (3.6% vs 2.6%, p = 0.54). There was also no significant difference between the age at diagnosis, diameter, differentiation and location of the colorectal carcinomas. Significantly more individuals with Birt-Hogg-Dubé syndrome underwent removal of colorectal polyps (12.2% vs 6.3%, p = 0.005). However, there was no significant difference between the number of polyps per person, the histology, grade of dysplasia and location of the polyps. CONCLUSION: Our data do not provide evidence for an increased risk for colorectal carcinoma in Birt-Hogg-Dubé syndrome, arguing against the need for colorectal surveillance. The difference in polyps might be due to a bias caused by a higher number of colonoscopies in patients with Birt-Hogg-Dubé syndrome.

IPS Governance Framework: Current Practices in Specification Use and Updates.

The eStandards methodology stressed the importance of trust and flow for health data as a key characteristic of well-functioning health systems. A digital health compass, leveraging perspectives of health systems, digital health markets, citizens, and workforce, drives a process of co-creation, governance and alignment in eStandards. A repository of best practices and common components further advances interoperability, as new projects add their experience. This paper proposes a governance framework for requirements management, intelligence gathering, specification use, and updates to promote sustainable governance for International Patient Summaries. It is based on interviews of 14 patient summary projects and initiatives in Europe and the United States.

Renal imaging in 199 Dutch patients with Birt-Hogg-Dubé syndrome: Screening compliance and outcome.

Birt-Hogg-Dubé syndrome is associated with an increased risk for renal cell carcinoma. Surveillance is recommended, but the optimal imaging method and screening interval remain to be defined. The main aim of our study was to evaluate the outcomes of RCC surveillance to get insight in the safety of annual US in these patients. Surveillance data and medical records of 199 patients with Birt-Hogg-Dubé syndrome were collected retrospectively using medical files and a questionnaire. These patients were diagnosed in two Dutch hospitals and data were collected until June 2014. A first screening for renal cell carcinoma was performed in 172/199 patients (86%). Follow-up data were available from 121 patients. The mean follow-up period per patient was 4.2 years. Of the patients known to be under surveillance, 83% was screened at least annually and 94% at least every two years. Thirty-eight renal cell carcinomas had occurred in 23 patients. The mean age at diagnosis of the first tumour was 51. Eighteen tumours were visualized by ultrasound. Nine small tumours (7-27 mm) were visible on MRI or CT and not detected using ultrasound. Our data indicate that compliance to renal screening is relatively high. Furthermore, ultrasound might be a sensitive, cheap and widely available alternative for MRI or part of the MRIs for detecting clinically relevant renal tumours in BHD patients,but the limitations should be considered carefully. Data from larger cohorts are necessary to confirm these observations.

SNP association study in PMS2-associated Lynch syndrome.

Lynch syndrome (LS) patients are at high risk of developing colorectal cancer (CRC). Phenotypic variability might in part be explained by common susceptibility loci identified in Genome Wide Association Studies (GWAS). Previous studies focused mostly on MLH1, MSH2 and MSH6 carriers, with conflicting results. We aimed to determine the role of GWAS SNPs in PMS2 mutation carriers. A cohort study was performed in 507 PMS2 carriers (124 CRC cases), genotyped for 24 GWAS SNPs, including SNPs at 11q23.1 and 8q23.3. Hazard ratios (HRs) were calculated using a weighted Cox regression analysis to correct for ascertainment bias. Discrimination was assessed with a concordance statistic in a bootstrap cross-validation procedure. Individual SNPs only had non-significant associations with CRC occurrence with HRs lower than 2, although male carriers of allele A at rs1321311 (6p21.31) may have increased risk of CRC (HR = 2.1, 95% CI 1.2-3.0). A polygenic risk score (PRS) based on 24 HRs had an HR of 2.6 (95% CI 1.5-4.6) for the highest compared to the lowest quartile, but had no discriminative ability (c statistic 0.52). Previously suggested SNPs do not modify CRC risk in PMS2 carriers. Future large studies are needed for improved risk stratification among Lynch syndrome patients.

Pediatric Diamond-Blackfan anemia in the Netherlands: An overview of clinical characteristics and underlying molecular defects.

INTRODUCTION: Diamond-Blackfan anemia (DBA) is characterized by hypoplastic anemia, congenital anomalies, and a predisposition for malignancies. Most of our understanding of this disorder stems from molecular studies combined with extensive data input from international patient registries. OBJECTIVES: To create an overview of the pediatric DBA population in the Netherlands. METHODS: Forty-three patients diagnosed with DBA from all Dutch university pediatric hospitals were included in this study, and their clinical and genetic characteristics were collected from patient records. RESULTS: Congenital malformations were present in 24 of 43 patients (55.8%). An underlying genetic defect was identified in 26 of 43 patients (60.5%), the majority of which were found in the RPS19 gene (12 of 43, 27.9%) with 1 patient carrying a mutation in a novel DBA candidate gene, RPL9. In 31 of 35 (88.6%) patients, an initial response to glucocorticoid treatment was observed. Six patients (14.0%) underwent hematopoietic stem cell transplantation, and eleven patients (11 of 43, 25.6%) became treatment-independent spontaneously. CONCLUSION: In agreement with previous reports, the Dutch pediatric DBA population is both clinically and genetically heterogeneous. National and international registries, together with more extensive genetic testing, are crucial to increase our understanding of genotype and phenotype correlations of this intriguing disorder.

Comprehensive Mutation Analysis of PMS2 in a Large Cohort of Probands Suspected of Lynch Syndrome or Constitutional Mismatch Repair Deficiency Syndrome.

Monoallelic PMS2 germline mutations cause 5%-15% of Lynch syndrome, a midlife cancer predisposition, whereas biallelic PMS2 mutations cause approximately 60% of constitutional mismatch repair deficiency (CMMRD), a rare childhood cancer syndrome. Recently improved DNA- and RNA-based strategies are applied to overcome problematic PMS2 mutation analysis due to the presence of pseudogenes and frequent gene conversion events. Here, we determined PMS2 mutation detection yield and mutation spectrum in a nationwide cohort of 396 probands. Furthermore, we studied concordance between tumor IHC/MSI (immunohistochemistry/microsatellite instability) profile and mutation carrier state. Overall, we found 52 different pathogenic PMS2 variants explaining 121 Lynch syndrome and nine CMMRD patients. In vitro mismatch repair assays suggested pathogenicity for three missense variants. Ninety-one PMS2 mutation carriers (70%) showed isolated loss of PMS2 in their tumors, for 31 (24%) no or inconclusive IHC was available, and eight carriers (6%) showed discordant IHC (presence of PMS2 or loss of both MLH1 and PMS2). Ten cases with isolated PMS2 loss (10%; 10/97) harbored MLH1 mutations. We confirmed that recently improved mutation analysis provides a high yield of PMS2 mutations in patients with isolated loss of PMS2 expression. Application of universal tumor prescreening methods will however miss some PMS2 germline mutation carriers.

Analysis of the novel fanconi anemia gene SLX4/FANCP in familial breast cancer cases.

SLX4/FANCP is a recently discovered novel disease gene for Fanconi anemia (FA), a rare recessive disorder characterized by chromosomal instability and increased cancer susceptibility. Three of the 15 FA genes are breast cancer susceptibility genes in heterozygous mutation carriers--BRCA2, PALB2, and BRIP1. To investigate if defects in SLX4 also predispose to breast cancer, the gene was sequenced in a cohort of 729 BRCA1/BRCA2-negative familial breast cancer cases. We identified a single splice site mutation (c.2013+2T>A), which causes a frameshift by skipping of exon 8. We also identified 39 missense variants, four of which were selected for functional testing in a Mitomycin C-induced growth inhibition assay, and appeared indistinguishable from wild type. Although this is the first study that describes a truncating SLX4 mutation in breast cancer patients, our data indicate that germline mutations in SLX4 are very rare and are unlikely to make a significant contribution to familial breast cancer.

Common variants of the BRCA1 wild-type allele modify the risk of breast cancer in BRCA1 mutation carriers.

Mutations in the BRCA1 gene substantially increase a woman's lifetime risk of breast cancer. However, there is great variation in this increase in risk with several genetic and non-genetic modifiers identified. The BRCA1 protein plays a central role in DNA repair, a mechanism that is particularly instrumental in safeguarding cells against tumorigenesis. We hypothesized that polymorphisms that alter the expression and/or function of BRCA1 carried on the wild-type (non-mutated) copy of the BRCA1 gene would modify the risk of breast cancer in carriers of BRCA1 mutations. A total of 9874 BRCA1 mutation carriers were available in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) for haplotype analyses of BRCA1. Women carrying the rare allele of single nucleotide polymorphism rs16942 on the wild-type copy of BRCA1 were at decreased risk of breast cancer (hazard ratio 0.86, 95% confidence interval 0.77-0.95, P = 0.003). Promoter in vitro assays of the major BRCA1 haplotypes showed that common polymorphisms in the regulatory region alter its activity and that this effect may be attributed to the differential binding affinity of nuclear proteins. In conclusion, variants on the wild-type copy of BRCA1 modify risk of breast cancer among carriers of BRCA1 mutations, possibly by altering the efficiency of BRCA1 transcription.

Origin, functional role, and clinical impact of Fanconi anemia FANCA mutations.

Fanconi anemia is characterized by congenital abnormalities, bone marrow failure, and cancer predisposition. To investigate the origin, functional role, and clinical impact of FANCA mutations, we determined a FANCA mutational spectrum with 130 pathogenic alleles. Some of these mutations were further characterized for their distribution in populations, mode of emergence, or functional consequences at cellular and clinical level. The world most frequent FANCA mutation is not the result of a mutational "hot-spot" but results from worldwide dissemination of an ancestral Indo-European mutation. We provide molecular evidence that total absence of FANCA in humans does not reduce embryonic viability, as the observed frequency of mutation carriers in the Gypsy population equals the expected by Hardy-Weinberg equilibrium. We also prove that long distance Alu-Alu recombination can cause Fanconi anemia by originating large interstitial deletions involving FANCA and 2 adjacent genes. Finally, we show that all missense mutations studied lead to an altered FANCA protein that is unable to relocate to the nucleus and activate the FA/BRCA pathway. This may explain the observed lack of correlation between type of FANCA mutation and cellular phenotype or clinical severity in terms of age of onset of hematologic disease or number of malformations.

Hereditary causes of kidney tumours.

BACKGROUND: In most cases of renal cell carcinoma there is no family history of renal cancer and no hereditary cause of the disease. Hereditary renal cancer accounts for about 2-4% of cases. Recognition of this subgroup by clinicians is important because of the possibility of severe medical consequences for patients and their relatives. MATERIALS AND METHODS: We review the latest data about different genetic conditions characterized by an increased risk of developing renal cancer and we formulate tools to recognize high-risk families. RESULTS: In general, a positive family history, young age at diagnosis of renal cancer, multiple and/or bilateral renal tumours and combined occurrence of different histological types of renal tumours should raise suspicion of a hereditary renal tumour syndrome. In addition, the presence of specific extrarenal symptoms in patients could assist in differentiating between tumour syndromes. CONCLUSIONS: A detailed medical and family history, along with physical examination are key factors to diagnose hereditary renal cancer syndromes. When a genetic predisposition for renal cancer is suspected, referral to a Family Cancer Clinic is warranted to initiate genetic examination and counselling on preventive options.

A DGGE system for comprehensive mutation screening of BRCA1 and BRCA2: application in a Dutch cancer clinic setting.

Rapid and reliable identification of deleterious changes in the breast cancer genes BRCA1 and BRCA2 has become one of the major issues in most DNA services laboratories. To rapidly detect all possible changes within the coding and splice site determining sequences of the breast cancer genes, we established a semiautomated denaturing gradient gel electrophoresis (DGGE) mutation scanning system. All exons of both genes are covered by the DGGE scan, comprising 120 amplicons. We use a semiautomated approach, amplifying all individual amplicons with the same PCR program, after which the amplicons are pooled. DGGE is performed using three slightly different gel conditions. Validation was performed using DNA samples with known sequence variants in 107 of the 120 amplicons; all variants were detected. This DGGE mutation scanning, in combination with a PCR test for two Dutch founder deletions in BRCA1 was then applied in 431 families in which 52 deleterious changes and 70 unclassified variants were found. Fifteen unclassified variants were not reported before. The system was easily adopted by five other laboratories, where in another 3,593 families both exons 11 were analyzed by the protein truncation test (PTT) and the remaining exons by DGGE. In total, a deleterious change (nonsense, frameshift, splice-site mutation, or large deletion) was found in 661 families (16.4%), 462 in BRCA1 (11.5%), 197 in BRCA2 (4.9%), and in two index cases a deleterious change in both BRCA1 and BRCA2 was identified. Eleven deleterious changes in BRCA1 and 36 in BRCA2 had not been reported before. In conclusion, this DGGE mutation screening method for BRCA1 and BRCA2 is proven to be highly sensitive and is easy to adopt, which makes screening of large numbers of patients feasible. The results of screening of BRCA1 and BRCA2 in more than 4,000 families present a valuable overview of mutations in the Dutch population.

Leukemia and exposure to ionizing radiation among German uranium miners.

BACKGROUND: It is well known that uranium miners are at an increased risk of lung cancer. Whether they also have an increased risk for other cancer sites remains under discussion. The aim of this study was to examine the leukemia risk among miners. METHODS: An individually matched case-control study of former uranium miners in East Germany was conducted with 377 cases and 980 controls. RESULTS: Using conditional logistic regression models, a dose-response relationship between leukemia risk and radon progeny could not be confirmed. Yet, a significantly elevated risk is seen in the category > or = 400 mSv when combining gamma-radiation and long-lived radionuclides. CONCLUSIONS: The results suggest that an elevated risk for leukemia is restricted to employees with a very long occupational career in underground uranium mining or uranium processing. Moreover, the study does not support the hypothesis of an association between exposure to short-lived radon progeny and leukemia risk.