Annemarie H. van der Hout

TP53 germline mutation testing in 180 families suspected of Li–Fraumeni syndrome: mutation detection rate and relative frequency of cancers in different familial phenotypes

Background Li–Fraumeni syndrome (LFS) is a rare autosomal dominant cancer predisposition syndrome. Most families fulfilling the classical diagnostic criteria harbour TP53 germline mutations. However, TP53 germline mutations may also occur in less obvious phenotypes. As a result, different criteria are in use to decide which patients qualify for TP53 mutation analysis, including the LFS, Li–Fraumeni-like (LFL) and Chompret criteria. We investigated which criteria for TP53 mutation analysis resulted in the highest mutation detection rate and sensitivity in Dutch families. We describe the tumour spectrum in TP53-positive families and calculated tumour type specific relative risks. Method A total of 180 Dutch families referred for TP53 mutation analysis were evaluated. Tumour phenotypes were verified by pathology reports or clinical records. Results A TP53 germline mutation was identified in 24 families. When the Chompret criteria were used 22/24 mutations were detected (sensitivity 92%, mutation detection rate 21%). In LFS and LFL families 18/24 mutations were found (sensitivity 75%). The two mutations detected outside the ‘Chompret group’ were found in a child with rhabdomyosarcoma and a young woman with breast cancer. In the mutation carriers, in addition to the classical LFS tumour types, colon and pancreatic cancer were also found significantly more often than in the general population. Conclusion We suggest TP53 mutation testing for all families fulfilling the Chompret criteria. In addition, TP53 mutation testing can be considered in the event of childhood sarcoma and breast cancer before 30 years. In addition to the risk for established LFS tumour types, TP53-positive individuals may also have an elevated risk for pancreatic and colon cancer.

Mutation screening of the Ectodysplasin-A receptor gene EDAR in hypohidrotic ectodermal dysplasia

Hypohidrotic ectodermal dysplasia (HED) can be caused by mutations in the X-linked ectodysplasin A (ED1) gene or the autosomal ectodysplasin A-receptor (EDAR) and EDAR-associated death domain (EDARADD) genes. X-linked and autosomal forms are sometimes clinically indistinguishable. For genetic counseling in families, it is therefore important to know the gene involved. In 24 of 42 unrelated patients with features of HED, we found a mutation in ED1. ED1-negative patients were screened for mutations in EDAR and EDARADD. We found mutations in EDAR in 5 of these 18 patients. One mutation, p.Glu354X, is novel. In EDARADD, a novel variant p.Ser93Phe, probably a neutral polymorphism, was also found. Clinically, there was a difference between autosomal dominant and autosomal recessive HED patients. The phenotype in patients with mutations in both EDAR alleles was comparable to males with X-linked HED. Patients with autosomal dominant HED had features comparable to those of female carriers of X-linked HED. The teeth of these patients were quite severely affected. Hypohidrosis and sparse hair were also evident, but less severe. This study confirms Chassaing et als earlier finding that mutations in EDAR account for approximately 25% of non-ED1-related HED. Mutations leading to a premature stop codon have a recessive effect except when the stop codon is in the last exon. Heterozygous missense mutations in the functional domains of the gene may have a dominant-negative effect with much variation in expression. Patients with homozygous or compound heterozygous mutations in the EDAR gene have a more severe phenotype than those with a heterozygous missense, nonsense or frame-shift mutation.

A method to assess the clinical significance of unclassified variants in the BRCA1 and BRCA2 genes based on cancer family history

IntroductionUnclassified variants (UVs) in the BRCA1/BRCA2 genes are a frequent problem in counseling breast cancer and/or ovarian cancer families. Information about cancer family history is usually available, but has rarely been used to evaluate UVs. The aim of the present study was to identify which is the best combination of clinical parameters that can predict whether a UV is deleterious, to be used for the classification of UVs.MethodsWe developed logistic regression models with the best combination of clinical features that distinguished a positive control of BRCA pathogenic variants (115 families) from a negative control population of BRCA variants initially classified as UVs and later considered neutral (38 families).ResultsThe models included a combination of BRCAPRO scores, Myriad scores, number of ovarian cancers in the family, the age at diagnosis, and the number of persons with ovarian tumors and/or breast tumors. The areas under the receiver operating characteristic curves were respectively 0.935 and 0.836 for the BRCA1 and BRCA2 models. For each model, the minimum receiver operating characteristic distance (respectively 90% and 78% specificity for BRCA1 and BRCA2) was chosen as the cutoff value to predict which UVs are deleterious from a study population of 12 UVs, present in 59 Dutch families. The p.S1655F, p.R1699W, and p.R1699Q variants in BRCA1 and the p.Y2660D, p.R2784Q, and p.R3052W variants in BRCA2 are classified as deleterious according to our models. The predictions of the p.L246V variant in BRCA1 and of the p.Y42C, p.E462G, p.R2888C, and p.R3052Q variants in BRCA2 are in agreement with published information of them being neutral. The p.R2784W variant in BRCA2 remains uncertain.ConclusionsThe present study shows that these developed models are useful to classify UVs in clinical genetic practice.

A simple method for co-segregation analysis to evaluate the pathogenicity of unclassified variants; BRCA1 and BRCA2 as an example

BackgroundAssessment of the clinical significance of unclassified variants (UVs) identified in BRCA1 and BRCA2 is very important for genetic counselling. The analysis of co-segregation of the variant with the disease in families is a powerful tool for the classification of these variants. Statistical methods have been described in literature but these methods are not always easy to apply in a diagnostic setting.MethodsWe have developed an easy to use method which calculates the likelihood ratio (LR) of an UV being deleterious, with penetrance as a function of age of onset, thereby avoiding the use of liability classes. The application of this algorithm is publicly available http://www.msbi.nl/cosegregation. It can easily be used in a diagnostic setting since it requires only information on gender, genotype, present age and/or age of onset for breast and/or ovarian cancer.ResultsWe have used the algorithm to calculate the likelihood ratio in favour of causality for 3 UVs in BRCA1 (p.M18T, p.S1655F and p.R1699Q) and 5 in BRCA2 (p.E462G p.Y2660D, p.R2784Q, p.R3052W and p.R3052Q). Likelihood ratios varied from 0.097 (BRCA2, p.E462G) to 230.69 (BRCA2, p.Y2660D). Typing distantly related individuals with extreme phenotypes (i.e. very early onset cancer or old healthy individuals) are most informative and give the strongest likelihood ratios for or against causality.ConclusionAlthough co-segregation analysis on itself is in most cases insufficient to prove pathogenicity of an UV, this method simplifies the use of co-segregation as one of the key features in a multifactorial approach considerably.

Direct molecular analysis of a deletion of 3p in tumors from patients with sporadic renal cell carcinoma

Abstract Normal and tumorous nephrectomy specimens from seven renal cell carcinoma patients were subjected to a Southern analysis using chromosome #3-specific polymorphic probes. Three patients were not informative because of homozygosity at all loci studied. One patient showing heterozygosity at 3q in normal tissue had a tumor that remained heterozygous. In three patients the tumor showed loss of heterozygosity for a short arm marker at 3p21. In one of them heterozygosity for a second short arm marker was also lost. Another of these three patients retained heterozygosity for this second short arm marker, as well as for a long arm marker, suggesting a chromosomal breakpoint between the loci for the two short arm markers. Our results demonstrate that the known involvement of a short arm region of chromosome #3 in the development of renal cell carcinoma can readily be further evaluated by direct molecular methods.

A Study of the Clinical and Radiological Features in a Cohort of 93 Patients with a COL2A1 Mutation Causing Spondyloepiphyseal Dysplasia Congenita or a Related Phenotype

Type 2 collagen disorders encompass a diverse group of skeletal dysplasias that are commonly associated with orthopedic, ocular, and hearing problems. However, the frequency of many clinical features has never been determined. We retrospectively investigated the clinical, radiological, and genotypic data in a group of 93 patients with molecularly confirmed SEDC or a related disorder. The majority of the patients (80/93) had short stature, with radiological features of SEDC (n = 64), others having SEMD (n = 5), Kniest dysplasia (n = 7), spondyloperipheral dysplasia (n = 2), or Torrance‐like dysplasia (n = 2). The remaining 13 patients had normal stature with mild SED, Stickler‐like syndrome or multiple epiphyseal dysplasia. Over 50% of the patients had undergone orthopedic surgery, usually for scoliosis, femoral osteotomy or hip replacement. Odontoid hypoplasia was present in 56% (95% CI 38–74) and a correlation between odontoid hypoplasia and short stature was observed. Atlanto‐axial instability, was observed in 5 of the 18 patients (28%, 95% CI 10–54) in whom flexion‐extension films of the cervical spine were available; however, it was rarely accompanied by myelopathy. Myopia was found in 45% (95% CI 35–56), and retinal detachment had occurred in 12% (95% CI 6–21; median age 14 years; youngest age 3.5 years). Thirty‐two patients complained of hearing loss (37%, 95% CI 27–48) of whom 17 required hearing aids. The ophthalmological features and possibly also hearing loss are often relatively frequent and severe in patients with splicing mutations. Based on clinical findings, age at onset and genotype–phenotype correlations in this cohort, we propose guidelines for the management and follow‐up in this group of disorders.

Detection of point mutation in dystrophin gene reveals somatic and germline mosaicism in the mother of a patient with Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most frequent heritable muscle disease in children with a birth prevalence of around 1 in 4,000 live born males [van Essen et al., 1992b]. The milder Becker muscular dystrophy (BMD) has a lower birth prevalence of around 1 in 18,500 live born [Bushby et al., 1991]. DMD and BMD are allelic X-linked recessive diseases caused by mutations in the dystrophin gene located in chromosome band Xp21. The dystrophin gene spans 2.4 Mb and contains 79 exons coding for a 14 kb mRNA [Monaco et al., 1992]. Gross rearrangements in the dystrophin gene are the main cause of both DMD and BMD. These rearrangements are found in about 65–70% of the patients [Koenig et al., 1987; den Dunnen et al., 1989; Hu et al., 1990]. Therefore, detection of small (point) mutations is important as it enables accurate carrier detection in all families. Single strand conformational analysis [Kneppers et al., 1995], heteroduplex analysis [Prior et al., 1995] and the protein truncation test [Roest et al., 1993] have been used for detection of point mutations in the dystrophin gene. In these studies analysis was mostly restricted to exons preferentially deleted in DMD and BMD. However, microlesions do not cluster in hotspots [Roberts et al., 1994; Prior et al., 1995]. Mosaicism for gross rearrangements in the dystrophin gene is a frequent finding [Bakker et al., 1989]. After identification of an apparently de novo rearrangement in an affected boy, there still is a risk of 15–20% that the risk haplotype contains the same mutation in subsequent siblings [van Essen et al., 1992a]. Data on mosaicism for point mutations in the dystrophin gene are still scarce as only two families have been reported [Wilton et al., 1994; Smith et al., 1999]. We report a DMD family in which the mother was a somatic and germline mosaic for a pathogenic point mutation in the dystrophin gene. This patient developed motor skills late and first walked independently at 22 months but was never able to run. Diagnosis was made at 5 years. He lost ambulation at 10 years. His serum creatine kinase (CK) activities were ten times the upper limit of normal levels. Muscle biopsy findings were compatible with muscular dystrophy. Dystrophin analysis was not possible during the time of the muscle biopsy. He has a healthy brother and no other affected family members. His mother had normal serum CK activities. Southern blot analysis using cDNA probes covering the coding region of the dystrophin gene [Darras and Francke, 1988; Mao and Cremer, 1989] did not reveal a rearrangement. Flanking and intragenic microsatellite markers DXS84 (1–2 cM 50 of the dystrophin gene) and DXS206 (located in intron 7 of the dystrophin gene) were used for linkage analysis and showed that the patient and his healthy brother have the same haplotype (Fig. 1) suggesting a de novo mutation in the patient or germline mosaicism in the mother. Subsequently, denaturing gradient gel electrophoresis (DGGE) for all dystrophin exons was done. The technical details of the DGGE analysis will be published separately (Hofstra et al., in preparation). Information concerning gel conditions and primers used to perform a full mutation screen of the dystrophin gene are available on request (e-mail: R.M.W.Hofstra@medgen.azg.nl). An aberrant band pattern was identified for exon 6 (Fig. 1B). Subsequently, this exon was sequenced using the following primers DMD-6AF 50-GGTTCTTGCTCAAGGAATGC-30 and DMD-6AR 50-GCTCAGGAGAATCTTTTCAC-30. The PCR products were purified using the High Pure PCR Product Purification Kit from Boehringer Mannheim and sequenced on an automated sequencer using the Thermo Sequenase Kit (Amersham Life Science) or the Sequenase kit (USB). Direct sequencing revealed a single base pair deletion of adenosine 377 (377delA) in both the patient and the mother, but not in his brother. This deletion causes a Grant sponsor: Princess Beatrix Fund; Grant number: 96-0109.

RB1 mutation spectrum in a comprehensive nationwide cohort of retinoblastoma patients

Background Retinoblastoma (Rb) is a childhood cancer of the retina, commonly initiated by biallelic inactivation of the RB1 gene. Knowledge of the presence of a heritable RB1 mutation can help in risk management and reproductive decision making. We report here on RB1 mutation scanning in a unique nationwide cohort of Rb patients from the Netherlands. Methods From the 1173 Rb patients registered in the Dutch National Retinoblastoma Register until January 2013, 529 patients from 433 unrelated families could be included. RB1 mutation scanning was performed with different detection methods, depending on the time period. Results Our mutation detection methods revealed RB1 mutations in 92% of bilateral and/or familial Rb patients and in 10% of non-familial unilateral cases. Overall an RB1 germline mutation was detected in 187 (43%) of 433 Rb families, including 33 novel mutations. The distribution of the type of mutation was 37% nonsense, 20% frameshift, 21% splice, 9% large indel, 5% missense, 7% chromosomal deletions and 1% promoter. Ten per cent of patients were mosaic for the RB1 mutation. Six three-generation families with incomplete penetrance RB1 mutations were found. We found evidence that two variants, previously described as pathogenic RB1 mutations, are likely to be neutral variants. Conclusions The frequency of the type of mutations in the RB1 gene in our unbiased national cohort is the same as the mutation spectrum described worldwide. Furthermore, our RB1 mutation detection regimen achieves a high scanning sensitivity.

High resolution SNP array profiling identifies variability in retinoblastoma genome stability

Both hereditary and nonhereditary retinoblastoma (Rb) are commonly initiated by loss of both copies of the retinoblastoma tumor suppressor gene (RB1), while additional genomic changes are required for tumor initiation and progression. Our aim was to determine whether there is genomic heterogeneity between different clinical Rb subtypes. Therefore, 21 Rb tumors from 11 hereditary patients and 10 nonhereditary Rb patients were analyzed using high‐resolution single nucleotide polymorphism (SNP) arrays and gene losses and gains were validated with Multiplex Ligation‐dependent Probe Amplification. In these tumors only a few focal aberrations were detected. The most frequent was a focal gain on chromosome 2p24.3, the minimal region of gain encompassing the oncogene MYCN. The genes BAZ1A, OTX2, FUT8, and AKT1 were detected in four focal regions on chromosome 14 in one nonhereditary Rb. There was a large difference in number of copy number aberrations between tumors. A subset of nonhereditary Rbs turned out to be the most genomic unstable, while especially very young patients with hereditary Rb display stable genomes. Established Rb copy number aberrations, including gain of chromosome arm 1q and loss of chromosome arm 16q, turned out to be preferentially associated with the nonhereditary Rbs with later age of diagnosis. In contrast, copy number neutral loss of heterozygosity was detected mainly on chromosome 13, where RB1 resides, irrespective of hereditary status or age. Focal amplifications and deletions and copy number neutral loss of heterozygosity besides chromosome 13 appear to be rare events in retinoblastoma.

Mutation-based growth charts for SEDC and other COL2A1 related dysplasias

From data collected via a large international collaborative study, we have constructed a growth chart for patients with molecularly confirmed congenital spondylo‐epiphyseal dysplasia (SEDC) and other COL2A1 related dysplasias. The growth chart is based on longitudinal height measurements of 79 patients with glycine substitutions in the triple‐helical domain of COL2A1. In addition, measurements of 27 patients with other molecular defects, such as arginine to cysteine substitutions, splice mutations, and mutations in the C‐terminal propeptide have been plotted on the chart. Height of the patients progressively deviate from that of normal children: compared to normal WHO charts, the mean length/height is −2.6 SD at birth, −4.2 SD at 5 years, and −5.8 SD in adulthood. The mean adult height (male and female combined) of patients with glycine substitutions in the triple‐helical region is 138.2 cm but there is a large variation. Patients with glycine to cysteine substitutions tend to cluster within the upper part of the chart, while patients with glycine to serine or valine substitutions are situated between +1 SD and −1 SD. Patients with carboxy‐terminal glycine substitutions tend to be shorter than patients with amino‐terminal substitutions, while patients with splice mutations are relatively tall. However, there are exceptions and specific mutations can have a strong or a relatively mild negative effect on growth. The observation of significant difference in adult height between affected members of the same family indicates that height remains a multifactorial trait even in the presence of a mutation with a strong dominant effect.