Gloria Negri

Clinical and molecular characterization of Rubinstein-Taybi syndrome patients carrying distinct novel mutations of the EP300 gene.

Rubinstein‐Taybi syndrome (RSTS) is a rare congenital neurodevelopmental disorder characterized by postnatal growth deficiency, skeletal abnormalities, dysmorphic features and cognitive deficit. Mutations in two genes, CREBBP and EP300, encoding two homologous transcriptional co‐activators, have been identified in ˜55% and ˜3–5% of affected individuals, respectively. To date, only eight EP300‐mutated RSTS patients have been described and 12 additional mutations are reported in the database LOVD. In this study, EP300 analysis was performed on 33 CREBBP‐negative RSTS patients leading to the identification of six unreported germline EP300 alterations comprising one deletion and five point mutations. All six patients showed a convincing, albeit mild, RSTS phenotype with minor skeletal anomalies, slight cognitive impairment and few major malformations. Beyond the expansion of the RSTS‐EP300‐mutated cohort, this study indicates that EP300‐related RSTS cases occur more frequently than previously thought (˜8% vs 3–5%); furthermore, the characterization of novel EP300 mutations in RSTS patients will enhance the clinical practice and genotype–phenotype correlations.

Insights into genotype-phenotype correlations from CREBBP point mutation screening in a cohort of 46 Rubinstein-Taybi syndrome patients

The genetic basis of Rubinstein–Taybi syndrome (RSTS), a rare, sporadic, clinically heterogeneous disorder characterized by cognitive impairment and a wide spectrum of multiple congenital anomalies, is primarily due to private mutations in CREBBP (approximately 55% of cases) or EP300 (approximately 8% of cases). Herein, we report the clinical and the genetic data taken from a cohort of 46 RSTS patients, all carriers of CREBBP point mutations. Molecular analysis revealed 45 different gene alterations including 31 inactivating (21 frameshift and 10 nonsense), 10 missense and 4 splicing mutations. Bioinformatic tools and transcript analyses were used to predict the functional effects of missense and splicing alterations. Of the 45 mutations, 42 are unreported and 3 were described previously. Recurrent mutations maybe a key tool in addressing genotype–phenotype correlations in patients sharing the same defects (at the genomic or transcript level) and specific clinical signs, demonstrated here in two cases. The clinical data of our cohort evidenced frequent signs such as arched eyebrows, epicanthus, synophrys and/or frontal hypertrichosis and broad phalanges that, previously overlooked in RSTS diagnosis, now could be considered. Some suggested correlations between organ‐specific anomalies and affected CREB‐binding protein domains broaden the RSTS clinical spectrum and perhaps will enhance patient follow‐up and clinical care.

From Whole Gene Deletion to Point Mutations of EP300‐Positive Rubinstein–Taybi Patients: New Insights into the Mutational Spectrum and Peculiar Clinical Hallmarks

Rubinstein–Taybi syndrome (RSTS) is a rare congenital neurodevelopmental disorder characterized by growth deficiency, skeletal abnormalities, dysmorphic features, and intellectual disability. Causative mutations in CREBBP and EP300 genes have been identified in ∼55% and ∼8% of affected individuals. To date, only 28 EP300 alterations in 29 RSTS clinically described patients have been reported. EP300 analysis of 22 CREBBP‐negative RSTS patients from our cohort led us to identify six novel mutations: a 376‐kb deletion depleting EP300 gene; an exons 17–19 deletion (c.(3141+1_3142‐1)_(3590+1_3591‐1)del/p.(Ile1047Serfs*30)); two stop mutations, (c.3829A>T/p.(Lys1277*) and c.4585C>T/p.(Arg1529*)); a splicing mutation (c.1878‐12A>G/p.(Ala627Glnfs*11)), and a duplication (c.4640dupA/p.(Asn1547Lysfs*3)). All EP300‐mutated individuals show a mild RSTS phenotype and peculiar findings including maternal gestosis, skin manifestation, especially nevi or keloids, back malformations, and a behavior predisposing to anxiety. Furthermore, the patient carrying the complete EP300 deletion does not show a markedly severe clinical picture, even if a more composite phenotype was noticed. By characterizing six novel EP300‐mutated patients, this study provides further insights into the EP300‐specific clinical presentation and expands the mutational repertoire including the first case of a whole gene deletion. These new data will enhance EP300‐mutated cases identification highlighting distinctive features and will improve the clinical practice allowing a better genotype–phenotype correlation.

Novel physiological RECQL4 alternative transcript disclosed by molecular characterisation of Rothmund-Thomson Syndrome sibs with mild phenotype.

Rothmund–Thomson syndrome is a rare genodermatosis caused by biallelic mutations of the RECQL4 gene and is characterised by poikiloderma, sparse hair, eyelashes and/or eyebrows, small stature, skeletal and dental abnormalities and cancer predisposition. Mutations predicted to result in the loss of RECQL4 protein have been associated with osteosarcoma risk, but mutation(s)–phenotype correlations are better addressed by combined DNA and RNA analyses. We describe two siblings with a mild phenotype, mainly restricted to the skin, who carry the unreported paternal c.2272C>T alteration in exon 14 and the previously reported maternal exon 15 c.2492_2493delAT, both predicted to result in premature termination codons (p.(Arg758*), p.(His831Argfs*52)). However real-time and transcript analysis showed, in the carrier father and affected daughter, increased levels of a novel RECQL4 physiological alternative transcript with partial in-frame skipping of exon 14, generated by increased usage of a weak cryptic splice site. This alternative transcript is expressed in all controls and tested tissues, its upregulation is specific to the paternal c.2272C>T mutation and depends on the abrogation of the binding motifs for SF2 and SRp55 serine/arginine-rich proteins with bypass of the mutation site located in the skipped exon 14 portion. Moreover, in the proband the increased levels of the alternative transcript, likely encoding a protein isoform with residual activity, may compensate for the dearth of the canonical transcript with the c.2492_2493delAT, accounting for the mild clinical phenotype of the siblings. Our results emphasise the value of RNA analysis to better predict the effects of RECQL4 mutations on the clinical phenotype.

Clinical utility gene card for: poikiloderma with neutropenia.

1.5 Mutational spectrum Poikiloderma with Neutropenia (PN), a very rare autosomal recessive inherited genodermatosis first described in the Navajo tribe of Native Americans,1 has recently been associated with biallelic mutations in the C16orf57 gene.2 So far, 19 different C16orf57 mutations have been detected in 37 PN patients subjected to molecular-genetic testing. Of these 37 patients, 31 (84%) carry homozygous mutations, whereas the other six are compound heterozygous.3 All identified mutations lead to the generation of truncated and most likely non-functional C16orf57 protein. C16orf57 is a 30–50 exonuclease essential for the biogenesis of the splicing apparatus.4,5 Several classes of mutations have been identified, listed here in order of decreasing prevalence: nonsense mutations (c.232C4T, c.243G4A, c.258T4A, c.267T4A, c.415C4T, c.541C4T, c.673C4T); small out-of-frame deletions (c.176_177delG, c.179delC, c.489_492del4, c.496delA, c.531delA, c.683_893þ 1del12); and splicing alterations, including substitutions at canonical splice junctions or at splice-site consensus sequences (c.265þ 2T4G, c.266 1G4A, c.450 2A4G, c.502A4G, c.504 2A4C, c.693þ 1G4T).2,3,6–10 No missense mutations have yet been found; c.502A4G can be categorised as a splicing alteration because it leads to the excision of the fourth exon from the mature C16orf57-001 transcript.2 The most frequent recurrent mutations, c.531delA, c.496delA and c.179delC, reflect three geographical clusters. c.531delA has been recorded in seven patients from the Caucasus region, two of whom are members of unrelated Turkish families; the second most frequent mutation, c.496delA, has been detected in five patients from the Athabaskan ethnic group; and the third most frequent, c.179delC, was identified in four patients of North African origin. Considering the very low frequency of PN syndrome and the prevalence of patients with homozygous mutations, common ancestry is the hypothesis most likely to explain the recurrence of these mutations in specific ethnic groups.3

Rubinstein Taybi Syndrome in an Indian Child due to EP300 Gene Mutation: Correspondence.

To the Editor: We read with great interest the paper by Tamhankar et al. concerning the identification of an inherited EP300 missense variant in a girl with Rubinstein-Taybi syndrome (RSTS) [1]. To date, only one de novo missense mutation out of 38 described alterations within EP300 is listed in the LOVD database (c.4232C>T, (p.(Thr1411Ile)), while two other inherited missense variants are reported (c.4532A>T, (p.(Asn1511Thr)) and c.5824A>T, (p.(Met1992Leu)) [2, 3]. Tamhankar et al. described a girl with typical RSTS phenotype carrying a missense mutation within exon 5 of EP300 gene (c.1282C>T) leading to a non conservative aminoacidic substitution (p.(P428S)) between the TAZ1/CH1 and KIX domains. Even if this alteration has been inherited from the healthy mother, the authors concluded that it is a pathogenetic sequence change and identified the case as familial. In the same paper, our first EP300mutational scanning [2], where we just mention the presence in a patient of a non conservative missense inherited alteration located in the KAT11 domain (c.4532A>T, (p.(N1511I)), has been incorrectly mentioned by Tamhankar et al. to support their conclusion. We believe that our statement about the inherited c.4532A>T, (p.(N1511I)) EP300 variant has been misunderstood: actually we have neither defined this variation as pathogenic nor described this case as familial, as the mother was unaffected. We have only reported this alteration because of its unclear functional impact and even if different bioinformatic tools (i.e., SIFT, PolyPhen, SNP&GO, MutationTaster) predict a damaging effect on the protein, we minded not to conclude on its pathogenetic nature. To date, this clinically diagnosed RSTS patient remains without any known genetic cause. On the contrary, the same in silico analysis performed for the c.1282C>T, (p.(P428S)) variant described by Tamhankar et al. [1] highlights a very low functional impact on protein and, even if rare, this alteration is present in the ExAC Browser. Furthermore, the c.1282C>T, (p.(P428S)) variant affects the last base of exon 5, not the IVS5 donor splice site as reported [1] (Fig. 1E). We conclude that the role of the c.4532A>T, (p.(N1511I)) EP300 inherited alteration has been incorrectly interpreted by Tamhankar et al. making this citation misleading to support the pathogenetic role of EP300 missense mutations.