Oliver Bartsch

DNA sequencing of CREBBP demonstrates mutations in 56% of patients with Rubinstein–Taybi syndrome (RSTS) and in another patient with incomplete RSTS

Rubinstein–Taybi syndrome (RSTS) is a distinct dominant disorder characterized by short stature, typical face, broad angulated thumbs and halluces, and mental retardation. The RSTS can be caused by chromosomal microdeletions and molecular mutations in the CREBBP gene; however, relatively few mutations have been reported to date. Here, we aimed to determine the rate of point mutations and other small molecular lesions in true RSTS and possible mild variants, by using genomic DNA sequencing. A consecutive series of patients including 17 patients from our previous study was investigated. We identified 19 causative mutations of CREBBP in a total of 45 patients representing three different diagnostic groups: (a) 17 mutations in 30 patients with unequivocal RSTS (detection rate 56.6%), (b) two mutations in eight patients with features suggestive of RSTS (“moderate or incomplete RSTS”, detection rate 25%), and (c) no mutation in seven patients with undiagnosed syndromes and isolated features of RSTS. In general, the mutations were distributed without hot spots and most were unique; however, three recurrent mutations (R370X, R1664H, and N1978S) were identified. Furthermore, we detected 15 different intragenic polymorphisms, including two non-synonymous coding polymorphisms, L551I and Q2208H. We report not only the highest detection rate (56.6%) of CREBBP mutations in patients with RSTS to date, but also the second missense mutation (N1978S) in a patient with moderate or incomplete RSTS. Previous studies have identified cytogenetic deletions in the CREBBP gene in eight to 12% of patients and very recently, Roelfsema et al. reported EP300 gene mutations in three of 92 (3.3%) patients with either true RSTS or different syndromes resembling RSTS. Our 56.6% detection rate of molecular mutations in CREBBP in patients with unequivocal RSTS supports the new concept that RSTS is a genetically heterogeneous disorder and furthermore, indicates that RSTS may be caused by gene/s other than CREBBP in up to 30% of cases.

Confirmation of EP300 gene mutations as a rare cause of Rubinstein-Taybi syndrome.

The Rubinstein–Taybi syndrome (RSTS, MIM 180849), a dominant Mendelian disorder with typical face, short stature, skeletal abnormalities, and mental retardation, is usually caused by heterozygous mutations of the CREBBP gene, but recently, EP300 gene mutations were reported in three individuals. Using quantitative PCR (for the CREBBP and EP300 genes) and genomic sequencing (for the EP300 gene), we studied here 13 patients who had shown no mutation after genomic sequencing of the CREBBP gene in a previous investigation. Two new disease-causing mutations were identified, including a partial deletion of CREBBP and a 1-bp deletion in EP300, c.7100delC (p.P2366fsX2401). The 1-bp deletion represents the fourth EP300 mutation reported to date and was identified in a patient with non-classical RSTS. Based on the very similar structure of the CREBBP and EP300 genes and the higher rate of single-nucleotide polymorphisms in EP300 (2.23 per individual) as compared to CREBBP (0.71 per individual) (P>0.001, Wilcoxon test), it may be assumed that EP300 gene mutations should be as frequent as CREBBP gene mutations. Based on the location of the EP300 gene mutations identified so far (outside the histone acetyl transferase domain) and the observed (although not very striking) phenotypical differences with the EP300 mutations, we propose that most EP300 mutations could be associated with other phenotypes, not classical RSTS.

Three patients with 9p deletions including DMRT1 and DMRT2: A girl with XY complement, bilateral ovotestes, and extreme growth retardation, and two XX females with normal pubertal development

It is well documented that distal 9p monosomy can be associated with XY sex reversal. Recently, the possibility of DMRT1 and/or DMRT2 (the genes for doublesex and mab‐3 related transcription factor 1 and 2) being the sex determining genes(s) at 9p has been raised. DMRT1 and DMRT2 map near the 9p telomere, distal of marker D9S1779. We describe here three unrelated females with distal 9p monosomy, one with XY complement and two with XX complements. In each individual, fluorescent in situ hybridization predicted the loss of the DMRT genes. Patient 1, an XY individual with monosomy 9pter → p24.1 ∼ 24.2 and trisomy 7q32 → qter had normal female external genitalia, a blind ending vagina, no uterus, a Fallopian tube on the right, and bilateral ovotestes with primitive ovarian tissue. She also had extreme growth retardation. Around 80 cases of distal 9p monosomy have been reported previously, but there has been no report of ovotestes or gonads comprising ovarian tissue in a XY patient with 9p deletion. Findings in Patient 1 suggest that the phenotypic spectrum of the heterozygous DMRT deletion may include true hermaphroditism. Patients 2 and 3 were 12‐ and 14‐year‐old females with XX complements, normal external genitalia, and normal pubertal development. Patient 2 had pure monosomy 9pter → p23 and Patient 3 had monosomy 9pter → p22.3 ∼ 23 combined with trisomy 3pter → p23 ∼ 24. To date, detailed reports on the gonadal status of XX 9p‐females have been limited to prepubertal girls. Patients 2 and 3 are the first females reported to have distal 9p monosomy and a normal puberty.

DNA, FISH and complementation studies in ICF syndrome : DNA hypomethylation of repetitive and single copy loci and evidence for a trans acting factor

ICF syndrome (ICFS) is a rare immunodeficiency disorder characterized by instability of the pericentromeric heterochromatin predominantly of chromosomes 1 and 16. DNA methylation studies in two unrelated ICFS patients provide further evidence for a marked hypomethylation of satellite 2 DNA. The ICFS-specific disturbances of chromatin structure take place within the satellite 2 DNA regions, as demonstrated by fluorescence in situ hybridization analysis. Moreover, methylation studies of genomic imprinted loci D15S63, D15S9, and H19 have revealed hypomethylation to different degrees in both patients; this provides evidence for hypomethylation at autosomal single copy loci in ICFS. Cell fusion experiments have revealed a distinct reduction of chromosomal abnormalities in ICFS cells after fusion with normal cells, suggesting that the abnormalities are caused by the loss of function of an as yet unknown trans acting factor. Although it is now clear that wide-spread DNA hypomethylation is a characteristic feature of ICFS, neither the cause and mechanism of hypomethylation nor their relationship to the clinical symptoms is known. We speculate that a phenotypic effect might result from tissue-dependent abnormal gene expression and/or from a possible structural disturbance of DNA domains, which, with respect to the immunodeficiency, partially prevents the normal somatic recombinations in immunologically active cells.

DiGeorge/velocardiofacial syndrome: FISH studies of chromosomes 22q11 and 10p14, and clinical reports on the proximal 22q11 deletion

DiGeorge anomaly/velocardiofacial syndrome (DG/VCFS) occurs with different deletion intervals on chromosomes 22q11, while the DiGeorge anomaly (with other findings) is seen in patients with deletions of 10p14. The clinical outcome with the common 22q11 deletion (90% of cases) is well known, but the outcome with the less frequent deletion types has not been well documented. Using cytogenetic and fluorescence in situ hybridization (FISH) analysis we studied a series of 295 patients with suspected DG/VCFS. We identified 58 subjects with a 22q11 deletion, and none with a 10p deletion. Fifty‐two subjects had the common deletion, five had the proximal deletion, and one had an atypical proximal deletion due to a 1;22 translocation. We report clinical data of four subjects with the proximal 22q11 microdeletion, and of one patient with the atypical proximal deletion. The anomalies observed with the proximal 22q11 microdeletion fell within the DG/VCFS spectrum. Two females, 6 and 25 years old, had normal mental development. Normal development has been reported with the common 22q11 deletion, but only in a minority of cases. This study may indicate a better intellectual and/or behavioral outcome with the proximal vs. the common 22q11 deletion, rather than a chance finding.

Two Patients With EP300 Mutations and Facial Dysmorphism Different From the Classic Rubinstein-Taybi Syndrome

Rubinstein–Taybi syndrome (RTS) is characterized by mental retardation, broad thumbs and great toes and a recognizable craniofacial phenotype. Causative mutations have been described in the CREBBP and EP300 genes. Here we present a 19‐year‐old woman and an unrelated 3‐year‐old boy, both with broad thumbs and halluces, but with facial aspects distinct from those of typical RTS. The woman had a marked learning disability, but no mental retardation. We identified a de novo c.7100delC mutation in EP300 (which predicts p.P2366RfsX35) in the woman and an apparently de novo c.638delG mutation in the boy, which predicts p.G213EfsX6. Mutations in EP300 are a known but rare cause of RTS. Only five other patients have been reported. We propose that individuals with EP300 mutations may exhibit a slightly different phenotype compared to individuals with CREBBP mutations, with milder cognitive impairment, more pronounced microcephaly, absent or mild downslanting of palpebral fissures, distinct arched eyebrows, and greater degree of retrognathia.

Targeted next-generation sequencing of deafness genes in hearing-impaired individuals uncovers informative mutations

Purpose:Targeted next-generation sequencing provides a remarkable opportunity to identify variants in known disease genes, particularly in extremely heterogeneous disorders such as nonsyndromic hearing loss. The present study attempts to shed light on the complexity of hearing impairment.Methods:Using one of two next-generation sequencing panels containing either 80 or 129 deafness genes, we screened 30 individuals with nonsyndromic hearing loss (from 23 unrelated families) and analyzed 9 normal-hearing controls.Results:Overall, we found an average of 3.7 variants (in 80 genes) with deleterious prediction outcome, including a number of novel variants, in individuals with nonsyndromic hearing loss and 1.4 in controls. By next-generation sequencing alone, 12 of 23 (52%) probands were diagnosed with monogenic forms of nonsyndromic hearing loss; one individual displayed a DNA sequence mutation together with a microdeletion. Two (9%) probands have Usher syndrome. In the undiagnosed individuals (10/23; 43%) we detected a significant enrichment of potentially pathogenic variants as compared to controls.Conclusion:Next-generation sequencing combined with microarrays provides the diagnosis for approximately half of the GJB2 mutation–negative individuals. Usher syndrome was found to be more frequent in the study cohort than anticipated. The conditions in a proportion of individuals with nonsyndromic hearing loss, particularly in the undiagnosed group, may have been caused or modified by an accumulation of unfavorable variants across multiple genes.Genet Med 16 12, 945–953.

Minimal clinical expression of the holoprosencephaly spectrum and of currarino syndrome due to different cytogenetic rearrangements deleting the Sonic Hedgehog gene and the HLXB9 gene at 7q36.3

We report clinical, cytogenetic, and molecular cytogenetic studies on four patients with subtle or submicroscopic 7q36 deletions either of de novo origin or resulting from a cryptic parental translocation. Fluorescence in situ hybridization (FISH) studies indicated that in all four patients, the Sonic Hedgehog gene (SHH) and the homeobox gene HLXB9, among others, are comprised in the deletions. Besides mental retardation and short stature, all patients showed only minimal manifestations of the holoprosencephaly (HPE) spectrum and only one displayed symptoms of the Currarino syndrome. Patient 1 had a de novo 7q36.1‐qter deletion and showed microcephaly, ptosis, sacral agenesis, tethered cord, but no structural brain anomaly. Patient 2 had a submicroscopic de novo 7q36 deletion detected by FISH, and showed facial and cerebral microsigns of the HPE spectrum. Patient 3 had a 7q36 deletion found by subtelomere FISH testing that was the unbalanced product of a subtle maternal 7q;10q translocation. She presented facial and ocular microsigns, but no structural abnormality of the brain. Patient 4 showed no specific syndromal pattern and was found to have a cryptic unbalanced de novo translocation of the terminal parts of chromosomes 7q and 9p by subtelomere FISH. Patients 2, 3, and 4 represent the first report of a de novo submicroscopic 7q36 deletion, the second report of a familial subtle translocation of 7q36, and the first report of an unbalanced de novo submicroscopic translocation of 7q36, respectively. Our results stress the importance of 7q36 deletion studies by FISH in patients with microsigns of the HPE spectrum.

Head and neck paragangliomas: Report of 175 patients (1989–2010)

Attention of the otorhinolaryngologist needs to be drawn to the versatile aspects of head and neck paragangliomas (PGLs).

Molecular studies in 10 cases of Rubinstein-Taybi syndrome, including a mild variant showing a missense mutation in codon 1175 of CREBBP

The genetic analysis of the Rubinstein-Taybi syndrome (RTS, OMIM 180849) may shed light on mechanisms of transcription, brain function, keloid formation, and cancer.1–6 RTS can be caused by heteroallelic mutations of CREBBP (the gene for cAMP responsive element binding (CREB) binding protein).2 Human CREBBP resides on chromosome 16p13.3,7–9 spans approximately 150 kb at the genomic level, and comprises a coding sequence of 7329 bp.10 The protein (2442 amino acids) is conserved (human v mouse, 94% amino acid identity)10 and a transcriptional coactivator.4–6 Predicted domains include the nuclear receptor binding and receptor interacting domain (aa 1-170), the amino-terminal transactivation domain (N-terminal TAD, aa 228-461), the Cys/His rich region (aa 363-496), the CREB binding domain (aa 452-682), the bromodomain (aa 1108-1170), the histone acetyltransferase domain (aa 1173-1849), the trithorax consensus finger and Cys/His rich region (aa 1232-1487), the E1A oncoprotein binding domain (aa 1679-1732), the protein kinase A phosphorylation site (aa 1771), the Gln rich region (aa 1849-1999), and the C-terminal TAD (aa 1960-2162).10 Mutations of CREBBP reported in RTS have included chromosomal translocations, deletions at the microscopic and submicroscopic level, and molecular mutations.2,7–9,11–14 Apart from a recent report,14 previous studies on RTS used the protein truncation test before molecular analysis, thereby limiting the spectrum of observed molecular mutations. To date, 11 small mutations of CREBBP have been reported, comprising truncating mutations and one missense mutation.2,13,14 Two modes of action of how CREBBP mutations may cause RTS have been discussed, haploinsufficiency and dominant negative effects. The haploinsufficiency mechanism is made likely by the observation that some 10% of subjects with RTS exhibit deletion of one CREBBP allele, and that deletions of different domains of CREBBP were found with the same RTS phenotype, without …