Martine J. van Belzen

Phenotype and genotype in 52 patients with Rubinstein–Taybi syndrome caused by EP300 mutations

Rubinstein–Taybi syndrome (RSTS) is a developmental disorder characterized by a typical face and distal limbs abnormalities, intellectual disability, and a vast number of other features. Two genes are known to cause RSTS, CREBBP in 60% and EP300 in 8–10% of clinically diagnosed cases. Both paralogs act in chromatin remodeling and encode for transcriptional co‐activators interacting with >400 proteins. Up to now 26 individuals with an EP300 mutation have been published. Here, we describe the phenotype and genotype of 42 unpublished RSTS patients carrying EP300 mutations and intragenic deletions and offer an update on another 10 patients. We compare the data to 308 individuals with CREBBP mutations. We demonstrate that EP300 mutations cause a phenotype that typically resembles the classical RSTS phenotype due to CREBBP mutations to a great extent, although most facial signs are less marked with the exception of a low‐hanging columella. The limb anomalies are more similar to those in CREBBP mutated individuals except for angulation of thumbs and halluces which is very uncommon in EP300 mutated individuals. The intellectual disability is variable but typically less marked whereas the microcephaly is more common. All types of mutations occur but truncating mutations and small rearrangements are most common (86%). Missense mutations in the HAT domain are associated with a classical RSTS phenotype but otherwise no genotype–phenotype correlation is detected. Pre‐eclampsia occurs in 12/52 mothers of EP300 mutated individuals versus in 2/59 mothers of CREBBP mutated individuals, making pregnancy with an EP300 mutated fetus the strongest known predictor for pre‐eclampsia.

Noninvasive prenatal diagnosis of Huntington disease: detection of the paternally inherited expanded CAG repeat in maternal plasma

With a shift towards noninvasive testing, we have explored and validated the use of noninvasive prenatal diagnosis (NIPD) for Huntington disease (HD).

Archetypal NOTCH3 mutations frequent in public exome: implications for CADASIL

To determine the frequency of distinctive EGFr cysteine altering NOTCH3 mutations in the 60,706 exomes of the exome aggregation consortium (ExAC) database.

CREBBP and EP300 mutational spectrum and clinical presentations in a cohort of Swedish patients with Rubinstein-Taybi syndrome.

Rubinstein–Taybi syndrome (RTS) is a rare autosomal dominant congenital disorder characterized by distinctive facial features, broad thumbs and halluces, growth retardation, and a variable degree of cognitive impairment. CREBBP is the major causative gene and mutations in EP300 are the cause of RTS in a minority of patients. In this study, 17 patients with a clinical diagnosis of RTS were investigated with direct sequencing, MLPA, and array‐CGH in search for mutations in these two genes. Eleven patients (64.7%) had disease‐causing point mutations or a deletion in CREBBP and in one patient (5.9%) a causal de novo frameshift mutation in EP300 was identified. This patient had broad thumbs, mild intellectual disability, and autism. In addition, an inherited missense mutation of uncertain clinical significance was identified in EP300 in one patient and his healthy father, and three patients had intronic nucleotide changes of uncertain clinical significance in CREBBP. Snoring and sleep apnea were common in both groups and four of the patients mothers had preeclampsia during pregnancy. Importantly, difficulties associated with anesthesia were frequently reported and included delayed or complicated emergency in 53.3% of patients.

Making (anti-) sense out of huntingtin levels in Huntington disease

BackgroundHuntington disease (HD) is an autosomal dominant neurodegenerative disorder, characterized by motor, psychiatric and cognitive symptoms. HD is caused by a CAG repeat expansion in the first exon of the HTT gene, resulting in an expanded polyglutamine tract at the N-terminus of the huntingtin protein. Typical disease onset is around mid-life (adult-onset HD) whereas onset below 21xa0years is classified as juvenile HD. While much research has been done on the underlying HD disease mechanisms, little is known about regulation and expression levels of huntingtin RNA and protein.ResultsIn this study we used 15 human post-mortem HD brain samples to investigate the expression of wild-type and mutant huntingtin mRNA and protein. In adult-onset HD brain samples, there was a small but significantly lower expression of mutant huntingtin mRNA compared to wild-type huntingtin mRNA, while wild-type and mutant huntingtin protein expression levels did not differ significantly. Juvenile HD subjects did show a lower expression of mutant huntingtin protein compared to wild-type huntingtin protein. Our results in HD brain and fibroblasts suggest that protein aggregation does not affect levels of huntingtin RNA and protein. Additionally, we did not find any evidence for a reduced expression of huntingtin antisense in fibroblasts derived from a homozygous HD patient.ConclusionsWe found small differences in allelic huntingtin mRNA levels in adult-onset HD brain, with significantly lower mutant huntingtin mRNA levels. Wild-type and mutant huntingtin protein were not significantly different in adult-onset HD brain samples. Conversely, in juvenile HD brain samples mutant huntingtin protein levels were lower compared with wild-type huntingtin, showing subtle differences between juvenile HD and adult-onset HD. Since most HD model systems harbor juvenile repeat expansions, our results suggest caution with the interpretation of huntingtin mRNA and protein studies using HD cell and animal models with such long repeats. Furthermore, our huntingtin antisense results in homozygous HD cells do not support reduced huntingtin antisense expression due to an expanded CAG repeat.

CREBBP mutations in individuals without Rubinstein-Taybi syndrome phenotype

Mutations in CREBBP cause Rubinstein–Taybi syndrome. By using exome sequencing, and by using Sanger in one patient, CREBBP mutations were detected in 11 patients who did not, or only in a very limited manner, resemble Rubinstein–Taybi syndrome. The combined facial signs typical for Rubinstein–Taybi syndrome were absent, none had broad thumbs, and three had only somewhat broad halluces. All had apparent developmental delay (being the reason for molecular analysis); five had short stature and seven had microcephaly. The facial characteristics were variable; main characteristics were short palpebral fissures, telecanthi, depressed nasal ridge, short nose, anteverted nares, short columella, and long philtrum. Six patients had autistic behavior, and two had self‐injurious behavior. Other symptoms were recurrent upper airway infections (nu2009=u20095), feeding problems (nu2009=u20097) and impaired hearing (nu2009=u20097). Major malformations occurred infrequently. All patients had a de novo missense mutation in the last part of exon 30 or beginning of exon 31 of CREBBP, between base pairs 5,128 and 5,614 (codons 1,710 and 1,872). No missense or truncating mutations in this region have been described to be associated with the classical Rubinstein–Taybi syndrome phenotype. No functional studies have (yet) been performed, but we hypothesize that the mutations disturb protein–protein interactions by altering zinc finger function. We conclude that patients with missense mutations in this specific CREBBP region show a phenotype that differs substantially from that in patients with Rubinstein–Taybi syndrome, and may prove to constitute one (or more) separate entities.

High Yield of Pathogenic Germline Mutations Causative or Likely Causative of the Cancer Phenotype in Selected Children with Cancer

Purpose: In many children with cancer and characteristics suggestive of a genetic predisposition syndrome, the genetic cause is still unknown. We studied the yield of pathogenic mutations by applying whole-exome sequencing on a selected cohort of children with cancer. Experimental Design: To identify mutations in known and novel cancer-predisposing genes, we performed trio-based whole-exome sequencing on germline DNA of 40 selected children and their parents. These children were diagnosed with cancer and had at least one of the following features: (1) intellectual disability and/or congenital anomalies, (2) multiple malignancies, (3) family history of cancer, or (4) an adult type of cancer. We first analyzed the sequence data for germline mutations in 146 known cancer-predisposing genes. If no causative mutation was found, the analysis was extended to the whole exome. Results: Four patients carried causative mutations in a known cancer-predisposing gene: TP53 and DICER1 (n = 3). In another 4 patients, exome sequencing revealed mutations causing syndromes that might have contributed to the malignancy (EP300-based Rubinstein–Taybi syndrome, ARID1A-based Coffin–Siris syndrome, ACTB-based Baraitser–Winter syndrome, and EZH2-based Weaver syndrome). In addition, we identified two genes, KDM3B and TYK2, which are possibly involved in genetic cancer predisposition. Conclusions: In our selected cohort of patients, pathogenic germline mutations causative or likely causative of the cancer phenotype were found in 8 patients, and two possible novel cancer-predisposing genes were identified. Therewith, our study shows the added value of sequencing beyond a cancer gene panel in selected patients, to recognize childhood cancer predisposition. Clin Cancer Res; 24(7); 1594–603. ©2018 AACR.

Mosaic CREBBP mutation causes overlapping clinical features of Rubinstein–Taybi and Filippi syndromes

Rubinstein–Taybi syndrome (RTS, OMIM 180849) and Filippi syndrome (FLPIS, OMIM 272440) are both rare syndromes, with multiple congenital anomalies and intellectual deficit (MCA/ID). We present a patient with intellectual deficit, short stature, bilateral syndactyly of hands and feet, broad thumbs, ocular abnormalities, and dysmorphic facial features. These clinical features suggest both RTS and FLPIS. Initial DNA analysis of DNA isolated from blood did not identify variants to confirm either of these syndrome diagnoses. Whole-exome sequencing identified a homozygous variant in C9orf173, which was novel at the time of analysis. Further Sanger sequencing analysis of FLPIS cases tested negative for CKAP2L variants did not, however, reveal any further variants. Subsequent analysis using DNA isolated from buccal mucosa revealed a mosaic variant in CREBBP. This report highlights the importance of excluding mosaic variants in patients with a strong but atypical clinical presentation of a MCA/ID syndrome if no disease-causing variants can be detected in DNA isolated from blood samples. As the striking syndactyly observed in the present case is typical for FLPIS, we suggest CREBBP analysis in saliva samples for FLPIS syndrome cases in which no causal CKAP2L variant is detected.

Early onset dystonia and parkinsonism with abnormal globus pallidal signal in MRI: A diagnostic challenge

Juvenile-onset dystonia parkinsonism is rare and has a long differential diagnosis of mainly genetic disorders. Investigation for dopa-responsive dystonia and Wilson’s disease should primarily be done because these are treatable conditions. The further workup is challenging, but MRI abnormalities can provide strong guidance, particularly when iron accumulation within the basal ganglia is found in this specific clinical setting. The list of diseases grouped under the umbrella of neurodegeneration with brain iron accumulation (NBIA) that can present with a dystonia parkinsonism phenotype includes pantothenate kinaseassociated neurodegeneration (PKAN), PLA2G6-associated neurodegeneration, and Kufor-Rakeb (PARK9). Here we describe a sporadic case of young-onset onset dystonia parkinsonism with brain-iron accumulation on MRI, who turned out to have Huntington’s disease (HD). At the age of 12 years, the parents first noticed slight changes in their daughter’s behavior and psychomotor development. It was not until the age of 15 years that she developed slurred speech, slowness of movement, pain in the arms and legs, and difficulty walking. Family history was unremarkable; particularly, the parents were healthy and well. Neurological examination showed bradyphrenia, masked face, slow saccades, bilateral symmetric bradykinesia and rigidity without tremor, and dystonia of the trunk and the hands. There were no cerebellar or pyramidal signs (Video). An extensive workup focused on juvenile dystonia parkinsonism was done. Cupper and ceruloplasmin levels were normal, and split lamp examination showed no KayserFleischer rings. Cerebrospinal fluid (CSF) showed normal neurotransmitter levels. A screen for acanthocytes was negative. MRI of the brain showed hypointensity bilaterally in the globus pallidus on T2-weighted images, compatible with NBIA (Fig. 1). Dopamine transporter single-photon emission computed tomography (DaT-SPECT) investigation suggested nigrostriatal degeneration. Mutation analysis was requested for PARKIN, DJ-1, PINK, and PLA2G6, but all came back negative. Given the dystonia parkinsonism phenotype, we also requested mutation analysis of the huntingtin (HTT) gene. Although we considered HD unlikely given the negative family history and the MRI features, a CAG repeat expansion of 67 was found, thereby establishing a diagnosis of juvenile HD. On the other chromosome an intermediate allele of 30 repeats was found. Genetic testing of the parents revealed that the intermediate repeat allele was stably transmitted by the mother (she carried 17 repeats on the other allele). The father was found to have 1 repeat in the normal range (17 repeats), and a repeat in the reduced penetrance range (38 repeats). Paternity testing confirmed that they are the biological parents. The most likely explanation for the large repeat found in the daughter is an extreme expansion of the reduced penetrance allele carried by the father. HD is an autosomal dominant disorder due to a trinucleotide (CAG) repeat expansion in the HTT gene. Juvenile HD is defined as a disease onset before the age of 20 years and accounts for 5% to 10% of all HD cases. The clinical picture is dominated by an akinetic rigid syndrome, but other features include epilepsy, dystonia, chorea, eye movement disorders, behavioral problems, cognitive problems and sporadically also cerebellar signs (ataxia). Juvenile HD is predominantly paternally inherited and associated with larger trinucleotide CAG repeat expansions (60–100). The de novo occurrence of juvenile HD in this family is likely to be explained by the presence of a reduced penetrance repeat in -----------------------------------------------------------Additional Supporting Information may be found in the online version of this article.