The arrhythmogenic cardiomyopathy phenotype associated with PKP2 c.1211dup variant.

BACKGROUND: The arrhythmogenic cardiomyopathy (ACM) phenotype, with life-threatening ventricular arrhythmias and heart failure, varies according to genetic aetiology. We aimed to characterise the phenotype associated with the variant c.1211dup (p.Val406Serfs*4) in the plakophilin­2 gene (PKP2) and compare it with previously reported Dutch PKP2 founder variants. METHODS: Clinical data were collected retrospectively from medical records of 106 PKP2 c.1211dup heterozygous carriers. Using data from the Netherlands ACM Registry, c.1211dup was compared with 3 other truncating PKP2 variants (c.235C > T (p.Arg79*), c.397C > T (p.Gln133*) and c.2489+1G > A (p.?)). RESULTS: Of the 106 carriers, 47 (44%) were diagnosed with ACM, at a mean age of 41 years. By the end of follow-up, 29 (27%) had experienced sustained ventricular arrhythmias and 12 (11%) had developed heart failure, with male carriers showing significantly higher risks than females on these endpoints (p < 0.05). Based on available cardiac magnetic resonance imaging and echocardiographic data, 46% of the carriers showed either right ventricular dilatation and/or dysfunction, whereas a substantial minority (37%) had some form of left ventricular involvement. Both geographical distribution of carriers and haplotype analysis suggested PKP2 c.1211dup to be a founder variant originating from the South-Western coast of the Netherlands. Finally, a Cox proportional hazards model suggested significant differences in ventricular arrhythmia-free survival between 4 PKP2 founder variants, including c.1211dup. CONCLUSIONS: The PKP2 c.1211dup variant is a Dutch founder variant associated with a typical right-dominant ACM phenotype, but also left ventricular involvement, and a possibly more severe phenotype than other Dutch PKP2 founder variants.

Variant Location Is a Novel Risk Factor for Individuals With Arrhythmogenic Cardiomyopathy Due to a Desmoplakin (DSP) Truncating Variant.

BACKGROUND: Truncating variants in desmoplakin (DSPtv) are an important cause of arrhythmogenic cardiomyopathy; however the genetic architecture and genotype-specific risk factors are incompletely understood. We evaluated phenotype, risk factors for ventricular arrhythmias, and underlying genetics of DSPtv cardiomyopathy. METHODS: Individuals with DSPtv and any cardiac phenotype, and their gene-positive family members were included from multiple international centers. Clinical data and family history information were collected. Event-free survival from ventricular arrhythmia was assessed. Variant location was compared between cases and controls, and literature review of reported DSPtv performed. RESULTS: There were 98 probands and 72 family members (mean age at diagnosis 43±8 years, 59% women) with a DSPtv, of which 146 were considered clinically affected. Ventricular arrhythmia (sudden cardiac arrest, sustained ventricular tachycardia, appropriate implantable cardioverter defibrillator therapy) occurred in 56 (33%) individuals. DSPtv location and proband status were independent risk factors for ventricular arrhythmia. Further, gene region was important with variants in cases (cohort n=98; Clinvar n=167) more likely to occur in the regions resulting in nonsense mediated decay of both major DSP isoforms, compared with n=124 genome aggregation database control variants (148 [83.6%] versus 29 [16.4%]; P<0.0001). CONCLUSIONS: In the largest series of individuals with DSPtv, we demonstrate that variant location is a novel risk factor for ventricular arrhythmia, can inform variant interpretation, and provide critical insights to allow for precision-based clinical management.

High Growth Rate of Pancreatic Ductal Adenocarcinoma in CDKN2A-p16-Leiden Mutation Carriers.

CDKN2A-p16-Leiden mutation carriers have a 20% to 25% risk of developing pancreatic ductal adenocarcinoma (PDAC). Better understanding of the natural course of PDAC might allow the surveillance protocol to be improved. The aims of the study were to evaluate the role of cystic precursor lesions in the development of PDAC and to assess the growth rate. In 2000, a surveillance program was initiated, consisting of annual MRI in carriers of a CDKN2A-p16-Leiden mutation. The study cohort included 204 (42% male) patients. Cystic precursor lesions were found in 52 (25%) of 204 mutation carriers. Five (9.7%) of 52 mutation carriers with cystic lesions and 8 (7.0%) of 114 mutation carriers without cystic lesions developed PDAC (P = 0.56). Three of 6 patients with a cystic lesion of ≥10 mm developed PDAC. The median size of all incident PDAC detected between 9 and 12 months since the previous normal MRI was 15 mm, suggesting an annual growth rate of about 15 mm/year. In conclusion, our findings show that patients with and without a cystic lesions have a similar risk of PDAC. However, cystic precursor lesions between 10 and 20 mm increase the risk of PDAC substantially. In view of the large size of the screen-detected tumors, a shorter interval of screening might be recommended for all patients. Cancer Prev Res; 11(9); 551-6. ©2018 AACR.

Lack of evidence for a causal role of CALR3 in monogenic cardiomyopathy.

The pathogenicity of previously published disease-associated genes and variants is sometimes questionable. Large-scale, population-based sequencing studies have uncovered numerous false assignments of pathogenicity. Misinterpretation of sequence variants may have serious implications for the patients and families involved, as genetic test results are increasingly being used in medical decision making. In this study, we assessed the role of the calreticulin-3 gene (CALR3) in cardiomyopathy. CALR3 has been included in several cardiomyopathy gene panels worldwide. Its inclusion is based on a single publication describing two missense variants in patients with hypertrophic cardiomyopathy. In our national cardiomyopathy cohort (n = 6154), we identified 17 unique, rare heterozygous CALR3 variants in 48 probands. Overall, our patient cohort contained a significantly higher number of rare CALR3 variants compared to the ExAC population (p = 0.0036). However, after removing a potential Dutch founder variant, no statistically significant difference was found (p = 0.89). In nine probands, the CALR3 variant was accompanied by a disease-causing variant in another, well-known cardiomyopathy gene. In three families, the CALR3 variant did not segregate with the disease. Furthermore, we could not demonstrate calreticulin-3 protein expression in myocardial tissues at various ages. On the basis of these findings, it seems highly questionable that variants in CALR3 are a monogenic cause of cardiomyopathy.

Benefit of Surveillance for Pancreatic Cancer in High-Risk Individuals: Outcome of Long-Term Prospective Follow-Up Studies From Three European Expert Centers.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis. Hereditary factors play a role in the development of PDAC in 3% to 5% of all patients. Surveillance of high-risk groups, may facilitate detection of PDAC at an early stage. The aim of this study was to assess whether surveillance aids detection of early-stage PDAC or precursor lesions (PRLs) and improves the prognosis. PATIENTS AND METHODS: Screening outcomes were collected from three European centers that conduct prospective screening in high-risk groups including families with clustering of PDAC (familial pancreatic cancer [FPC]) or families with a gene defect that predisposes to PDAC. The surveillance program consisted of annual magnetic resonance imaging, magnetic resonance cholangiopancreatography, and/or endoscopic ultrasound. RESULTS: Four hundred eleven asymptomatic individuals participated in the surveillance programs, including 178 CDKN2A mutation carriers, 214 individuals with FPC, and 19 BRCA1/2 or PALB2 mutation carriers. PDAC was detected in 13 (7.3%) of 178 CDKN2A mutation carriers. The resection rate was 75%, and the 5-year survival rate was 24%. Two CDKN2A mutation carriers (1%) underwent surgical resection for low-risk PRL. Two individuals (0.9%) in the FPC cohort had a pancreatic tumor, including one advanced PDAC and one early grade 2 neuroendocrine tumor. Thirteen individuals with FPC (6.1%) underwent surgical resection for a suspected PRL, but only four (1.9%) had high-risk lesions (ie, high-grade intraductal papillary mucinous neoplasms or grade 3 pancreatic intraepithelial neoplasms). One BRCA2 mutation carrier was found to have PDAC, and another BRCA2 mutation carrier and a PALB2 mutation carrier underwent surgery and were found to have low-risk PRL. No serious complications occurred as consequence of the program. CONCLUSION: Surveillance of CDNK2A mutation carriers is relatively successful, detecting most PDACs at a resectable stage. The benefit of surveillance in families with FPC is less evident.

Functional assessment of potential splice site variants in arrhythmogenic right ventricular dysplasia/cardiomyopathy.

BACKGROUND: Interpretation of genetic screening results in arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) often is difficult. Pathogenicity of variants with uncertain clinical significance may be predicted by software algorithms. However, functional assessment can unambiguously demonstrate the effect of such variants. OBJECTIVE: The purpose of this study was to perform functional analysis of potential splice site variants in ARVD/C patients. METHODS: Nine variants in desmosomal (PKP2, JUP, DSG2, DSC2) genes with potential RNA splicing effect were analyzed. The variants were found in patients who fulfilled 2010 ARVD/C Task Force Criteria (n = 7) or had suspected ARVD/C (n = 2). Total RNA was isolated from fresh blood samples and subjected to reverse transcriptase polymerase chain reaction. RESULTS: An effect on splicing was predicted by software algorithms for all variants. Of the 9 variants, 5 were intronic and 4 exonic. RNA analysis showed a functional effect on mRNA splicing by exon skipping, generation of new splice sites, or activation of cryptic sites in 6 variants. All 5 intronic variants tested severely impaired splicing. Only 1 of 4 exonic potential splice site variants was shown to have a deleterious effect on splicing. The remaining 3 exonic variants had no detectable effect on splicing, and heterozygous presence in mRNA confirmed biallelic expression. CONCLUSION: Six variants of uncertain clinical significance in the PKP2, JUP, and DSG2 genes showed a deleterious effect on mRNA splicing, indicating these are ARVD/C-related pathogenic splice site mutations. These results highlight the importance of functional assessment of potential splice site variants to improve patient care and facilitate cascade screening.

PDZ domain-binding motif regulates cardiomyocyte compartment-specific NaV1.5 channel expression and function.

BACKGROUND: Sodium channel NaV1.5 underlies cardiac excitability and conduction. The last 3 residues of NaV1.5 (Ser-Ile-Val) constitute a PDZ domain-binding motif that interacts with PDZ proteins such as syntrophins and SAP97 at different locations within the cardiomyocyte, thus defining distinct pools of NaV1.5 multiprotein complexes. Here, we explored the in vivo and clinical impact of this motif through characterization of mutant mice and genetic screening of patients. METHODS AND RESULTS: To investigate in vivo the regulatory role of this motif, we generated knock-in mice lacking the SIV domain (ΔSIV). ΔSIV mice displayed reduced NaV1.5 expression and sodium current (INa), specifically at the lateral myocyte membrane, whereas NaV1.5 expression and INa at the intercalated disks were unaffected. Optical mapping of ΔSIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricular conduction. Internalization of wild-type and ΔSIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued ΔSIV INa, suggesting that the SIV motif is important for regulation of NaV1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased NaV1.5 cell surface expression and INa when expressed in HEK293 cells. CONCLUSIONS: Our results demonstrate the in vivo significance of the PDZ domain-binding motif in the correct expression of NaV1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral NaV1.5 in ventricular conduction. Furthermore, we reveal a clinical relevance of the SIV motif in cardiac disease.

Genetic analysis in 418 index patients with idiopathic dilated cardiomyopathy: overview of 10 years' experience.

AIMS: With more than 40 dilated cardiomyopathy (DCM)-related genes known, genetic analysis of patients with idiopathic DCM is costly and time-consuming. We describe the yield from genetic analysis in DCM patients in a large Dutch cohort. METHODS AND RESULTS: We collected cardiological and neurological evaluations, family screenings, and genetic analyses for 418 index patients with idiopathic DCM. We identified 35 (putative) pathogenic mutations in 82 index patients (20%). The type of DCM influenced the yield, with mutations found in 25% of familial DCM cases, compared with 8% of sporadic DCM cases and 62% of cases where DCM was accompanied by neuromuscular disease. A PLN founder mutation (43 cases) and LMNA mutations (19 cases, 16 different mutations) were most prevalent and often demonstrated a specific phenotype. Other mutations were found in: MYH7, DES, TNNT2, DMD, TPM1, DMPK, SCN5A, SGCB (homozygous), and TNNI3. After a median follow-up of 40 months, the combined outcome of death from any cause, heart transplantation, or malignant ventricular arrhythmias in patients with a mutation was worse than in those without an identified mutation (hazard ratio 2.0, 95% confidence interval 1.4-3.0). This seems to be mainly attributable to a high prevalence of malignant ventricular arrhythmias and end-stage heart failure in LMNA and PLN mutation carriers. CONCLUSION: The yield of identified mutations in DCM index patients with clinical clues, such as associated neuromuscular disease or familial occurrence, is higher compared with those without these clues. For sporadic DCM, specific clinical characteristics may be used to select cases for DNA analysis.

Myocardial structural alteration and systolic dysfunction in preclinical hypertrophic cardiomyopathy mutation carriers.

BACKGROUND: To evaluate the presence of myocardial structural alterations and subtle myocardial dysfunction during familial screening in asymptomatic mutation carriers without hypertrophic cardiomyopathy (HCM) phenotype. METHODS AND FINDINGS: Sixteen HCM families with pathogenic mutation were studied and 46 patients with phenotype expression (Mut+/Phen+) and 47 patients without phenotype expression (Mut+/Phen-) were observed. Twenty-five control subjects, matched with the Mut+/Phen- group, were recruited for comparison. Echocardiography was performed to evaluate conventional parameters, myocardial structural alteration by calibrated integrated backscatter (cIBS) and global and segmental longitudinal strain by speckle tracking analysis. All 3 groups had similar left ventricular dimensions and ejection fraction. Basal anteroseptal cIBS was the highest in Mut+/Phen+ patients (-14.0±4.6 dB, p<0.01) and was higher in Mut+/Phen- patients as compared to controls (-17.0±2.3 vs. -22.6±2.9 dB, p<0.01) suggesting significant myocardial structural alterations. Global and basal anteroseptal longitudinal strains (-8.4±4.0%, p<0.01) were the most impaired in Mut+/Phen+ patients as compared to the other 2 groups. Although global longitudinal strain was similar between Mut+/Phen- group and controls, basal anteroseptal strain was lower in Mut+/Phen- patients (-14.1±3.8%, p<0.01) as compared to controls (-19.9±2.9%, p<0.01), suggesting a subclinical segmental systolic dysfunction. A combination of >-19.0 dB basal anteroseptal cIBS or >-18.0% basal anteroseptal longitudinal strain had a sensitivity of 98% and a specificity of 72% in differentiating Mut+/Phen- group from controls. CONCLUSION: The use of cIBS and segmental longitudinal strain can differentiate HCM Mut+/Phen- patients from controls with important clinical implications for the family screening and follow-up of these patients.

Magnetic resonance imaging surveillance detects early-stage pancreatic cancer in carriers of a p16-Leiden mutation.

BACKGROUND & AIMS: Surveillance of high-risk groups for pancreatic cancer might increase early detection and treatment outcomes. Individuals with germline mutations in p16-Leiden have a lifetime risk of 15% to 20% of developing pancreatic cancer. We assessed the feasibility of detecting pancreatic cancer at an early stage and investigated the outcomes of patients with neoplastic lesions. METHODS: Individuals with germline mutations in p16-Leiden (N = 79; 31 male; mean age, 56 years; range, 39-72 years) were offered annual surveillance by magnetic resonance imaging (MRI) and magnetic resonance cholangiopancreatography (MRCP). Those found to have neoplastic lesions were offered options for surgery or intensive follow-up. Individuals found to have possible neoplastic lesions were examined again by MRI/MRCP within 2 to 4 months. RESULTS: After a median follow-up period of 4 years (range, 0-10 years), pancreatic cancer was diagnosed in 7 patients (9%). The mean age at diagnosis was 59 years (range, 49-72 years). Three of the tumors were present at the first examination, and 4 were detected after a negative result in the initial examination. All 7 patients had a resectable lesion; 5 underwent surgery, 3 had an R0 resection, and 2 had lymph node metastases. Possible precursor lesions (ie, duct ectasias, based on MRCP) were found in 9 individuals (11%). CONCLUSIONS: MRI/MRCP detects small, solid pancreatic tumors and small duct ectasias. Although surveillance increases the rate of resectability, carriers of a p16-Leiden mutation develop aggressive tumors.

Genetic testing in familial melanoma: uptake and implications.

OBJECTIVE: We report on the uptake and psychological impact of p16-Leiden genetic testing to contribute to a greater understanding of counseling melanoma families. METHODS: Within a defined research setting, genetic counseling and testing were offered to members of p16-Leiden-positive melanoma pedigrees, at risk of carrying a gene defect associated with an increased risk of melanoma and pancreatic cancer. RESULTS: One hundred and eighty-four individuals sought counseling, of which 141 (77%) opted for genetic testing. Uptake of genetic counseling and testing, and psychological motivation was evaluated in 94 (57%) individuals. Higher pre-test risk of carrying the mutation and older age proved significantly predictive for counseling uptake. Age was predictive for test acceptance, whereas fearful test expectancies predicted test decline. Counselees had lower distress levels than those reported in other oncogenetic testing settings. CONCLUSION: We are the first to report on genetic testing for familial melanoma. Following the first counseling session, we found a relatively high uptake rate for p16-Leiden testing and no clinically worrisome levels of distress.

Homozygosity for a CHEK2*1100delC mutation identified in familial colorectal cancer does not lead to a severe clinical phenotype.

It has recently been suggested that the frequency of the germline CHEK2*1100delC mutation is higher among breast cancer families with colorectal cancer, although the mutation does not seem to be significantly associated with familial colorectal cancer. Five hundred and sixty-four familial colorectal tumours were studied for expression of CHEK2 using tissue microarrays and an antibody against the NH2-terminal SQ regulatory domain of the CHEK2 protein. Normal colonic tissue from patients whose tumours showed loss of CHEK2 expression was investigated further using fragment and sequence analysis for the presence of a CHEK2*1100delC mutation and five other (R117G, R137Q, R145W, I157T, and R180H) known germline variants in CHEK2. Twenty-nine tumours demonstrated loss of expression for CHEK2. Analysis of matched normal colonic tissue from these patients revealed germline CHEK2*1100delC mutation in three cases. In two of these, the mutation was heterozygous but, interestingly, the third patient proved to be homozygous for the deletion, using six different primer pair combinations. None of the other tested germline variants were identified. No CHEK2*1100delC mutations were found in patients whose tumours stained positive. Homozygosity for the CHEK2*1100delC mutation appears not to be lethal in humans. No severe clinical phenotype was apparent, although the patient died from colonic carcinoma at age 52 years. This observation is in line with recent knockout mouse models, although in the latter, cellular defects in apoptosis and increased resistance to irradiation seem to exist. It is also concluded that CHEK2 protein abrogation is not caused by the CHEK2 germline variants R117G, R137Q, R145W, I157T, and R180H in familial colorectal cancer.

Cancer risk in hereditary nonpolyposis colorectal cancer due to MSH6 mutations: impact on counseling and surveillance.

BACKGROUND & AIMS: Hereditary nonpolyposis colorectal carcinoma (HNPCC) is caused by a mutated mismatch repair (MMR) gene. The aim of our study was to determine the cumulative risk of developing cancer in a large series of MSH6 mutation carriers. METHODS: Mutation analysis was performed in 20 families with a germline mutation in MSH6. We compared the cancer risks between MSH6 and MLH1/MSH2 mutation carriers. Microsatellite instability (MSI) analysis and immunohistochemistry (IHC) were performed in the available tumors. RESULTS: A total of 146 MSH6 mutation carriers were identified. In these carriers, the cumulative risk for colorectal carcinoma was 69% for men, 30% for women, and 71% for endometrial carcinoma at 70 years of age. The risk for all HNPCC-related tumors was significantly lower in MSH6 than in MLH1 or MSH2 mutation carriers (P = 0.002). In female MSH6 mutation carriers, the risk for colorectal cancer was significantly lower (P = 0.0049) and the risk for endometrial cancer significantly higher (P = 0.02) than in MLH1 and MSH2 mutation carriers. In male carriers, the risk for colorectal cancer was lower in MSH6 mutation carriers, but the difference was not significant (P = 0.0854). MSI analysis in colorectal tumors had a sensitivity of 86% in predicting a MMR defect. IHC in all tumors had a sensitivity of 90% in predicting a mutation in MSH6. CONCLUSIONS: We recommend starting colonoscopic surveillance in female MSH6 mutation carriers from age 30 years. Prophylactic hysterectomy might be considered in carriers older than 50 years. MSI and IHC analysis are sensitive tools to identify families eligible for MSH6 mutation analysis.