A clinical and genetic overview of 18 years neurofibromatosis type 1 molecular diagnostics in the Netherlands.

NF1 mutations are the underlying cause of neurofibromatosis type 1 (NF1), a neuro-cardio-facio-cutaneous syndrome (NCFC). Because of the clinical overlap between NCFCs, genetic analysis of NF1 is necessary to confirm a clinical diagnosis NF1. This report describes the clinical and genetic findings of 18 years of NF1 molecular diagnostics in the Netherlands. A pathogenic mutation was found in 59.3% (1178/1985) of the index patients, mostly de novo (73.8%). The majority of the index patients (64.3%) fulfilled the National Institute of Health NF1 criteria, a pathogenic mutation was found in 80.9% of these patients. Seventy-four percent of the index patients with an NF1 pathogenic mutation and not fulfilling the NF1 criteria is <12 years, in agreement with the fact that some NF1 symptoms appear after puberty. Genotype-phenotype correlations were studied for 527 index patients. NF1 patients with a type 1 microdeletion have a sixfold higher risk of special education vs NF1 patients with an intragenic mutation. No evidently milder NF1 phenotype for patients with a missense mutation was observed. Forty-six prenatal analyses were performed in 28 (2.4%) families, of which 29 (63%) showed heterozygosity for the familial pathogenic mutation. This indicates that there is a need for prenatal NF1 testing.

Mutations in the vasopressin type 2 receptor gene (AVPR2) associated with nephrogenic diabetes insipidus.

Nephrogenic diabetes insipidus (DIR) is an X-linked disorder characterized by insensitivity of the distal nephron for the pituitary hormone, vasopressin. The genetic map location of the DIR gene on chromosome Xq28 coincides with the physical map location of the functional vasopressin renal V2-type receptor. Recently, the human and rat cDNAs for the vasopressin V2 receptor (AVPR2) have been identified. We show here that the structural AVPR2 gene is localized between DXS52 and G6PD, which is within the genetic map location of DIR. We also tested eight X-linked DIR probands and their families for mutations in one of the most conserved extracellular regions of AVPR2: in three of them, we have identified point mutations resulting in non-conservative amino acid substitutions which cosegregated with DIR in all families.

Pitfalls in molecular analysis for mismatch repair deficiency in a family with biallelic pms2 germline mutations.

Heterozygous germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 cause Lynch syndrome. Biallelic mutations in the MMR genes are associated with a childhood cancer syndrome [constitutional mismatch repair deficiency (CMMR-D)]. This is predominantly characterized by hematological malignancies and tumors of the bowel and brain, often associated with signs of neurofibromatosis type 1 (NF1). Diagnostic strategies for selection of patients for MMR gene analysis include analysis of microsatellite instability (MSI) and immunohistochemical (IHC) analysis of MMR proteins in tumor tissue. We report the clinical characterization and molecular analyses of tumor specimens from a family with biallelic PMS2 germline mutations. This illustrates the pitfalls of present molecular screening strategies. Tumor tissues of five family members were analyzed for MSI and IHC. MSI was observed in only one of the analyzed tissues. However, IHC analysis of brain tumor tissue of the index patient and his sister showed absence of PMS2 expression, and germline mutation analyses showed biallelic mutations in PMS2: p.Ser46IIe and p.Pro246fs. The same heterozygous mutations were confirmed in the father and mother, respectively. These data support the conclusion that in case of a clinical phenotype of CMMR-D, it is advisable to routinely combine MSI analysis with IHC analysis for the expression of MMR proteins. With inconclusive or conflicting results, germline mutation analysis of the MMR genes should be considered after thorough counselling of the patients and/or their relatives.

Distress in partners of individuals diagnosed with or at high risk of developing tumors due to rare hereditary cancer syndromes.

OBJECTIVE: Li Fraumeni syndrome (LFS) and Von Hippel-Lindau disease (VHL) are two rare hereditary tumor syndromes, characterized by a high risk of developing multiple tumors at various sites and ages for which preventive and treatment options are limited. For partners, it may be difficult to deal with the on-going threat of tumors in both their spouse and children. Therefore, this study aims to evaluate the prevalence of and factors associated with psychological distress among partners of individuals with or at high risk of LFS or VHL. METHODS: As part of a nationwide, cross-sectional study, partners of individuals diagnosed with or at high risk of LFS or VHL were invited to complete a self-report questionnaire assessing distress, worries, and health-related quality of life. RESULTS: Fifty-five (58%) of those high-risk individuals with a partner consented to having their partner approached for the study. In total, 50 partners (91%) completed the questionnaire, of whom 28% reported clinically relevant levels of syndrome-related distress. Levels of distress and worries of the partners and their high-risk spouse were significantly correlated. Younger age and a lack of social support were also associated significantly with heightened levels of distress and worries. The majority of partners (76%) believed that professional psychosocial support should be routinely offered to them. CONCLUSIONS: Approximately one-quarter of the partners exhibit clinically relevant levels of distress that warrant psychological support. The distress levels of the 'patient' could potentially be used to identify partners at risk of developing clinically relevant levels of distress.

Psychosocial impact of Von Hippel-Lindau disease: levels and sources of distress.

Von Hippel-Lindau disease (VHL) is a hereditary tumor susceptibility syndrome, characterized by an increased risk of developing multiple benign and malignant tumors at various sites and ages with limited preventive options. This study evaluates the prevalence of distress among VHL family members and factors associated significantly with such distress. Forty-eight families with a VHL mutation were identified via the nine family cancer clinics in the Netherlands. In total, 171 family members (carriers, 50% at-risk, non-carriers) were approached, of whom 123 (72%) completed a self-report questionnaire. Approximately 40% of the VHL family members reported clinically relevant levels of distress, approaching 50% among the carriers and, possibly even more striking, 36% among the non-carriers. Having lost a first degree relative due to VHL during adolescence (OR 11.2; 95% CI 1.4-86.9) was related significantly to heightened levels of distress. Approximately, only one-third of those who reported heightened levels of distress had received professional psychosocial support. A substantial percentage of family members experience clinically relevant levels of distress. We would recommend the introduction of a procedure for screening for distress in this vulnerable population. Special attention should be paid to those individuals who have lost a close relative due to VHL during adolescence.

A unique case of two somatic APC mutations in an early onset cribriform-morular variant of papillary thyroid carcinoma and overview of the literature.

We report a case of a 22-year-old female patient who was diagnosed with a cribriform-morular variant of papillary thyroid carcinoma (CMV-PTC). While at early ages this thyroid cancer variant is highly suggestive for familial adenomatous polyposis (FAP), there was no family history of FAP. In the tumor biallelic, inactivating APC variants were identified. The patient tested negative for germline variants based on analysis of genomic DNA from peripheral blood leukocytes. Somatic mosaicism was excluded by subsequent deep sequencing of leukocyte and normal thyroid DNA using next generation sequencing (NGS). This report presents a rare sporadic case of CMV-PTC, and to the best of our knowledge the first featuring two somatic APC mutations underlying the disease, with an overview of CMV-PTC cases with detected APC and CTNNB1 pathogenic variants from the literature.

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.

Inflammatory processes in cortical tubers and subependymal giant cell tumors of tuberous sclerosis complex.

Cortical tubers and subependymal giant cell tumors (SGCT) are two major cerebral lesions associated with tuberous sclerosis complex (TSC). In the present study, we investigated immunocytochemically the inflammatory cell components and the induction of two major pro-inflammatory pathways (the interleukin (IL)-1beta and complement pathways) in tubers and SGCT resected from TSC patients. All lesions were characterized by the prominent presence of microglial cells expressing class II-antigens (HLA-DR) and, to a lesser extent, the presence of CD68-positive macrophages. We also observed perivascular and parenchymal T lymphocytes (CD3(+)) with a predominance of CD8(+) T-cytotoxic/suppressor lymphoid cells. Activated microglia and reactive astrocytes expressed IL-1beta and its signaling receptor IL-1RI, as well as components of the complement cascade, such as C1q, C3c and C3d. Albumin extravasation, with uptake in astrocytes, was observed in both tubers and SGCT, suggesting that alterations in blood brain barrier permeability are associated with inflammation in TSC-associated lesions. Our findings demonstrate a persistent and complex activation of inflammatory pathways in cortical tubers and SGCT.

Evaluation of current prediction models for Lynch syndrome: updating the PREMM5 model to identify PMS2 mutation carriers.

Until recently, no prediction models for Lynch syndrome (LS) had been validated for PMS2 mutation carriers. We aimed to evaluate MMRpredict and PREMM5 in a clinical cohort and for PMS2 mutation carriers specifically. In a retrospective, clinic-based cohort we calculated predictions for LS according to MMRpredict and PREMM5. The area under the operator receiving characteristic curve (AUC) was compared between MMRpredict and PREMM5 for LS patients in general and for different LS genes specifically. Of 734 index patients, 83 (11%) were diagnosed with LS; 23 MLH1, 17 MSH2, 31 MSH6 and 12 PMS2 mutation carriers. Both prediction models performed well for MLH1 and MSH2 (AUC 0.80 and 0.83 for PREMM5 and 0.79 for MMRpredict) and fair for MSH6 mutation carriers (0.69 for PREMM5 and 0.66 for MMRpredict). MMRpredict performed fair for PMS2 mutation carriers (AUC 0.72), while PREMM5 failed to discriminate PMS2 mutation carriers from non-mutation carriers (AUC 0.51). The only statistically significant difference between PMS2 mutation carriers and non-mutation carriers was proximal location of colorectal cancer (77 vs. 28%, p < 0.001). Adding location of colorectal cancer to PREMM5 considerably improved the models performance for PMS2 mutation carriers (AUC 0.77) and overall (AUC 0.81 vs. 0.72). We validated these results in an external cohort of 376 colorectal cancer patients, including 158 LS patients. MMRpredict and PREMM5 cannot adequately identify PMS2 mutation carriers. Adding location of colorectal cancer to PREMM5 may improve the performance of this model, which should be validated in larger cohorts.

Human c-fms proto-oncogene: comparative analysis with an abnormal allele.

The organization of the human c-fms proto-oncogene has been determined and compared with an abnormal allele. The human v-fms homologous genetic sequences are dispersed discontinuously and colinearly with the viral oncogene over a DNA region of ca. 32 kilobase pairs. The abnormal c-fms locus contains a small deletion in its 3' portion. DNA sequencing analysis indicated that it was 426 base pairs in size and located in close proximity to a putative c-fms exon.

Interdependence of clinical factors predicting cognition in children with tuberous sclerosis complex.

Cognitive development in patients with tuberous sclerosis complex is highly variable. Predictors in the infant years would be valuable to counsel parents and to support development. The aim of this study was to confirm factors that have been reported to be independently correlated with cognitive development. 102 patients included in this study were treated at the ENCORE-TSC expertise center of the Erasmus Medical Center-Sophia Children's Hospital. Data from the first 24 months of life were used, including details on epilepsy, motor development and mutation status. Outcome was defined as cognitive development (intellectual equivalent, IE) as measured using tests appropriate to the patients age and cognitive abilities (median age at testing 8.2 years, IQR 4.7-12.0). Univariable and multivariable regression analyses were used. In a univariable analysis, predictors of lower IE were: the presence of infantile spasms (ß = -18.3, p = 0.000), a larger number of antiepileptic drugs used (ß = -6.3, p = 0.000), vigabatrin not used as first drug (ß = -14.6, p = 0.020), corticosteroid treatment (ß = -33.2, p = 0.005), and a later age at which the child could walk independently (ß = -2.1, p = 0.000). An older age at seizure onset predicted higher IE (ß = 1.7, p = 0.000). In a multivariable analysis, only age at seizure onset was significantly correlated to IE (ß = 1.2, p = 0.005), contributing to 28% of the variation in IE. In our cohort, age at seizure onset was the only variable that independently predicted IE. Factors predicting cognitive development could aid parents and physicians in finding the appropriate support and schooling for these patients.

Characterization of the feline c-abl proto-oncogene.

Analysis of total feline DNA by genomic blot hybridization, using the viral oncogene of Abelson murine leukemia virus as a specific probe, has led to the identification of multiple v-abl homologous genetic sequences in the cat genome. Upon restriction endonuclease BamHI digestion, the combined size of the v-abl homologous DNA fragments was about 31 kbp. To characterize these sequences further, four independent v-abl homologous cosmid clones with overlapping cellular inserts have been isolated from a gene library of cat lung genomic DNA. These inserts represent a contiguous region of cellular DNA sequences of 56 kbp in length. Within this region of the feline genome, the v-abl homologous sequences are discontinuously dispersed over a region of about 34 kbp. They represent the complete feline v-abl cellular homolog and are colinear with the viral v-abl oncogene. Nine regions of highly repetitive DNA sequences have been mapped in close proximity to v-abl homologous sequences. These results establish the presence of only a single c-abl proto-oncogene in the cat genome and present its genetic organization.

Comparative analysis of the human and feline c-sis proto-oncogenes. Identification of 5' human c-sis coding sequences that are not homologous to the transforming gene of simian sarcoma virus.

Feline and human genetic sequences, homologous to the v-sis gene of simian sarcoma virus, have been isolated from cosmid gene libraries and characterized by restriction endonuclease analysis. Comparison of the two loci revealed their related structural organization. In both loci, similar unique genetic sequences were found upstream of the v-sis homologous region and these hybridized to a 4.2 kbp c-sis transcript in human lung tumor cells. These data establish and map as yet unidentified coding sequences at the 5' part of the c-sis proto-oncogene of both species.

Survival in hereditary breast cancer associated with germline mutations of BRCA2.

PURPOSE: Breast cancer in BRCA1 and BRCA2 gene-mutation carriers may differ from so-called sporadic breast cancer in clinical features and behavior. These potential differences may be of importance for the prevention, screening, and, ultimately, treatment of breast cancer in women with such germline mutations. Thus far, there have been very few studies on the survival of BRCA2-associated breast cancer patients. PATIENTS AND METHODS: We determined the disease-free and overall survival of 28 breast cancer patients from 14 consecutive families with eight different BRCA2 germline mutations. These patients' survival and tumor characteristics were compared with those of a control group of 112 sporadic breast cancer patients matched to them by age and year of diagnosis. RESULTS: The 5-year disease-free survival was 52% for each group (P =.91); the overall survival was 74% for BRCA2 carriers and 75% for sporadic cases (P =.50). At the time of diagnosis, tumors from the BRCA2 carriers were borderline significantly larger in comparison to the tumors in sporadic cases (P =.05), but axillary nodal status was not significantly different in the two groups (node-negativity, 63% v 52. 8%, respectively; P =.34). With respect to steroid receptor status, BRCA2-associated tumors were more likely to be steroid receptor-positive, especially regarding progesterone receptor status (100% v 76.7% positive, respectively; P =.06). Stage-adjusted recurrence and death rates were nonsignificantly better for BRCA2 cases (hazard ratios of 0.84 and 0.59 [P =.61 and P =.19], respectively). In contrast, after 5 years, the rate of metachronous contralateral breast cancer in BRCA2 patients was 12% (v 2% in controls; P =.02). CONCLUSION: Patients with hereditary breast cancer due to BRCA2 have a similar prognosis when compared with age-matched sporadic breast cancer patients. Contrary to our previous observation regarding BRCA1-associated breast cancer, BRCA2 tumors tended to be steroid receptor-positive, instead of steroid receptor-negative.

Hyperechogenic fetal bowel: counseling difficulties.

The detection of echodense fetal bowel on ultrasound examination in the second trimester of pregnancy justifies invasive procedures such as amniocentesis to detect an underlying cause. We present a case in which initial tests identified only one mutation in the cystic fibrosis transmembrane regulator (CFTR)-gene of the fetus, the family history being negative for CF. Strongly reduced intestinal enzyme activities suggested intestinal obstruction and further increased the estimated risk for CF. After the 24th gestational week, a second mutation was found, confirming cystic fibrosis in this child. Problems in counseling in this particular case are discussed.

Large deletion at the TSC1 locus in a family with tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by seizures, mental retardation and the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene on chromosome 9q34, or the TSC2 gene on chromosome 16p13.3. Here we describe a deletion encompassing the TSC1 gene and two neighboring transcripts on chromosome 9q34 in six affected individuals from a family with TSC. To our knowledge, this is the first report of such a large deletion at the TSC1 locus and indicates that screening for similar mutations at the TSC1 locus is warranted in individuals with TSC.

The growth response to GH treatment is greater in patients with SHOX enhancer deletions compared to SHOX defects.

OBJECTIVE: Short stature caused by point mutations or deletions of the short stature homeobox (SHOX) gene (SHOX haploinsufficiency (SHI)) is a registered indication for GH treatment. Patients with a SHOX enhancer deletion (SED) have a similar phenotype, but their response to GH is unknown. It is uncertain if duplications of SHOX or its enhancer (SDUP) cause short stature. This study aimed to describe the clinical characteristics and growth response to GH treatment in patients with aberrations of SHOX and its enhancers. DESIGN: In this retrospective multi-center study (2002-March 2014) clinical information was available from 130 patients (72 SHI, 44 SED, and 14 SDUP) of whom 52 patients were treated with GH. We evaluated height, sitting height (SH), arm span, dysmorphic features and indicators of the growth response to GH (delta height SDS, height velocity, and index of responsiveness). RESULTS: Patients with SEDs showed similar HtSDS to patients with SHI (-2.3 and -2.6, respectively, P=0.2), but they were less disproportionate (SH/height ratio SDS 2.0 vs 3.1 (P<0.01) and extremities/trunk ratio 2.57 vs 2.43 (P=0.03)). The 1st year growth response to GH treatment was significantly greater in prepubertal patients with SEDs than SHI. None of the patients with an SDUP was disproportionate and SDUP cosegregated poorly with short stature; their growth response to GH treatment (n=3) was similar to the other groups. CONCLUSIONS: Patients with SEDs are equally short, but less disproportionate than patients with SHI, and show a greater response to GH.

Analysis of TSC2 stop codon variants found in tuberous sclerosis patients.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations to the TSC1 and TSC2 tumour suppressor genes. We detected two sequence changes involving the TSC2 stop codon and investigated the effects of these changes on the expression of tuberin, the TSC2 gene product, and on the binding between tuberin and the TSC1 gene product, hamartin. While elongation of the tuberin open reading frame by 17 amino acids did not interfere with tuberin-hamartin binding, a longer extension prevented this interaction. Our data illustrate how functional protein assays can assist in the verification and characterisation of disease-causing mutations.

Molecular cloning of the feline c-fes proto-oncogene and construction of a chimeric transforming gene.

The feline c-fes proto-oncogene, different parts of which were captured in feline leukemia virus (FeLV) to generate the transforming genes (v-fes) of the Gardner-Arnstein (GA) strain of feline sarcoma virus (FeSV) and the Snyder-Theilen strain (ST) of FeSV, was cloned and its genetic organization determined. Southern blot analysis revealed that the c-fes genetic sequences were distributed discontinuously and colinearly with the v-fes transforming gene over a DNA region of around 12.0 kb. Using cloned c-fes sequences, complementation of GA-FeSV transforming activity was studied. Upon replacement of the 3' half of v-fesGA with homologous feline c-fes sequences and transfection of the chimeric gene, morphological transformation was observed. Immunoprecipitation analysis of these transformed cells revealed expression of high Mr fusion proteins. Phosphorylation of these proteins was observed in an in vitro protein kinase assay, and tyrosine residues appeared to be involved as acceptor amino acid.

Ipsilateral breast tumour recurrence in hereditary breast cancer following breast-conserving therapy.

The overall rate of an ipsilateral breast tumour recurrence (IBTR) after breast-conserving therapy (BCT) ranges from 1% to 2% per year. Risk factors include young age but data on the impact of BRCA1/2 mutations or a definite positive family history for breast cancer are scarce. We investigated IBTR after BCT in patients with hereditary breast cancer (HBC). Through our family cancer clinic we identified 87 HBC patients, including 26 BRCA1/2 carriers, who underwent BCT between 1980 and 1995 (cases). They were compared to 174 patients with sporadic breast cancer (controls) also treated with BCT, matched for age and year of diagnosis. Median follow up was 6.1 years for the cases and 6.0 years for controls. Patient and tumour characteristics were similar in both groups. An IBTR was observed in 19 (21.8%) hereditary and 21 (12.1%) sporadic patients. In the hereditary patients more recurrences occurred elsewhere in the breast (21% versus 9.5%), suggestive of new primaries. Overall, the actuarial IBTR rate was similar at 2 years, but higher in hereditary as compared to sporadic patients at 5 years (14% versus 7%) and at 10 years (30% versus 16%) (P=0.05). Post-relapse and overall survival was not different between hereditary and sporadic cases. Hereditary breast cancer was therefore associated with a higher frequency of early (2-5 years) and late (>5 years) local recurrences following BCT. These data suggest an indication for long-term follow up in HBC and should be taken into account when additional 'risk-reducing' surgery after primary BCT is eventually considered.