Maria Francesca Bedeschi

13q Deletion and central nervous system anomalies: further insights from karyotype–phenotype analyses of 14 patients

Background: Chromosome 13q deletion is associated with varying phenotypes, which seem to depend on the location of the deleted segment. Although various attempts have been made to link the 13q deletion intervals to distinct phenotypes, there is still no acknowledged consensus correlation between the monosomy of distinct 13q regions and specific clinical features. Methods: 14 Italian patients carrying partial de novo 13q deletions were studied. Molecular–cytogenetic characterisation was carried out by means of array-comparative genomic hybridisation (array-CGH) or fluorescent in situ hybridisation (FISH). Results: Our 14 patients showed mental retardation ranging from profound–severe to moderate–mild: eight had central nervous system (CNS) anomalies, including neural tube defects (NTDs), six had eye abnormalities, nine had facial dysmorphisms and 10 had hand or feet anomalies. The size of the deleted regions varied from 4.2 to 75.7 Mb. Conclusion: This study is the first systematic molecular characterisation of de novo 13q deletions, and offers a karyotype–phenotype correlation based on detailed clinical studies and molecular determinations of the deleted regions. Analyses confirm that patients lacking the 13q32 band are the most seriously affected, and critical intervals have been preliminarily assigned for CNS malformations. Dose-sensitive genes proximal to q33.2 may be involved in NTDs. The minimal deletion interval associated with the Dandy–Walker malformation (DWM) was narrowed to the 13q32.2–33.2 region, in which the ZIC2 and ZIC5 genes proposed as underlying various CNS malformations are mapped.

FAM111A mutations result in hypoparathyroidism and impaired skeletal development

Kenny-Caffey syndrome (KCS) and the similar but more severe osteocraniostenosis (OCS) are genetic conditions characterized by impaired skeletal development with small and dense bones, short stature, and primary hypoparathyroidism with hypocalcemia. We studied five individuals with KCS and five with OCS and found that all of them had heterozygous mutations in FAM111A. One mutation was identified in four unrelated individuals with KCS, and another one was identified in two unrelated individuals with OCS; all occurred de novo. Thus, OCS and KCS are allelic disorders of different severity. FAM111A codes for a 611 amino acid protein with homology to trypsin-like peptidases. Although FAM111A has been found to bind to the large T-antigen of SV40 and restrict viral replication, its native function is unknown. Molecular modeling of FAM111A shows that residues affected by KCS and OCS mutations do not map close to the active site but are clustered on a segment of the protein and are at, or close to, its outer surface, suggesting that the pathogenesis involves the interaction with as yet unidentified partner proteins rather than impaired catalysis. FAM111A appears to be crucial to a pathway that governs parathyroid hormone production, calcium homeostasis, and skeletal development and growth.

Molecular Analysis, Pathogenic Mechanisms, and Readthrough Therapy on a Large Cohort of Kabuki Syndrome Patients

Kabuki syndrome (KS) is a multiple congenital anomalies syndrome characterized by characteristic facial features and varying degrees of mental retardation, caused by mutations in KMT2D/MLL2 and KDM6A/UTX genes. In this study, we performed a mutational screening on 303 Kabuki patients by direct sequencing, MLPA, and quantitative PCR identifying 133 KMT2D, 62 never described before, and four KDM6A mutations, three of them are novel. We found that a number of KMT2D truncating mutations result in mRNA degradation through the nonsense‐mediated mRNA decay, contributing to protein haploinsufficiency. Furthermore, we demonstrated that the reduction of KMT2D protein level in patients’ lymphoblastoid and skin fibroblast cell lines carrying KMT2D‐truncating mutations affects the expression levels of known KMT2D target genes. Finally, we hypothesized that the KS patients may benefit from a readthrough therapy to restore physiological levels of KMT2D and KDM6A proteins. To assess this, we performed a proof‐of‐principle study on 14 KMT2D and two KDM6A nonsense mutations using specific compounds that mediate translational readthrough and thereby stimulate the re‐expression of full‐length functional proteins. Our experimental data showed that both KMT2D and KDM6A nonsense mutations displayed high levels of readthrough in response to gentamicin treatment, paving the way to further studies aimed at eventually treating some Kabuki patients with readthrough inducers.

(Epi)genotype–phenotype correlations in Beckwith–Wiedemann syndrome

Beckwith–Wiedemann syndrome (BWS) is characterized by cancer predisposition, overgrowth and highly variable association of macroglossia, abdominal wall defects, nephrourological anomalies, nevus flammeus, ear malformations, hypoglycemia, hemihyperplasia, and organomegaly. BWS molecular defects, causing alteration of expression or activity of the genes regulated by two imprinting centres (IC) in the 11p15 chromosomal region, are also heterogeneous. In this paper we define (epi)genotype–phenotype correlations in molecularly confirmed BWS patients. The characteristics of 318 BWS patients with proven molecular defect were compared among the main four molecular subclasses: IC2 loss of methylation (IC2-LoM, n=190), IC1 gain of methylation (IC1-GoM, n=31), chromosome 11p15 paternal uniparental disomy (UPD, n=87), and cyclin-dependent kinase inhibitor 1C gene (CDKN1C) variants (n=10). A characteristic growth pattern was found in each group; neonatal macrosomia was almost constant in IC1-GoM, postnatal overgrowth in IC2-LoM, and hemihyperplasia more common in UPD (P<0.001). Exomphalos was more common in IC2/CDKN1C patients (P<0.001). Renal defects were typical of UPD/IC1 patients, uretheral malformations of IC1-GoM cases (P<0.001). Ear anomalies and nevus flammeus were associated with IC2/CDKN1C genotype (P<0.001). Macroglossia was less common among UPD patients (P<0.001). Wilms’ tumor was associated with IC1-GoM or UPD and never observed in IC2-LoM patients (P<0.001). Hepatoblastoma occurred only in UPD cases. Cancer risk was lower in IC2/CDKN1C, intermediate in UPD, and very high in IC1 cases (P=0.009). In conclusion, (epi)genotype–phenotype correlations define four different phenotypic BWS profiles with some degree of clinical overlap. These observations impact clinical care allowing to move toward (epi) genotype-based follow-up and cancer screening.

Loss of function of the E3 ubiquitin-protein ligase UBE3B causes Kaufman oculocerebrofacial syndrome

Background Kaufman oculocerebrofacial syndrome (KOS) is a developmental disorder characterised by reduced growth, microcephaly, ocular anomalies (microcornea, strabismus, myopia, and pale optic disk), distinctive facial features (narrow palpebral fissures, telecanthus, sparse and laterally broad eyebrows, preauricular tags, and micrognathia), mental retardation, and generalised hypotonia. KOS is a rare, possibly underestimated condition, with fewer than 10 cases reported to date. Here we investigate the molecular cause underlying KOS. Methods An exome sequencing approach was used on a single affected individual of an Italian consanguineous family coupled with mutation scanning using Sanger sequencing on a second unrelated subject with clinical features fitting the disorder. Results Exome sequencing was able to identify homozygosity for a novel truncating mutation (c.556C>T, p.Arg186stop) in UBE3B, which encodes a widely expressed HECT (homologous to the E6-AP carboxyl terminus) domain E3 ubiquitin-protein ligase. Homozygosity for a different nonsense lesion affecting the gene (c.1166G>A, p.Trp389stop) was documented in the second affected subject, supporting the recessive mode of inheritance of the disorder. Mutation scanning of the entire UBE3B coding sequence on a selected cohort of subjects with features overlapping, in part, those recurring in KOS did not reveal disease-causing mutations, suggesting phenotypic homogeneity of UBE3B lesions. Discussion Our data provide evidence that KOS is caused by UBE3B loss of function, and further demonstrate the impact of misregulation of protein ubiquitination on development and growth. The available clinical records, including those referring to four UBE3B mutation-positive subjects recently described as belonging to a previously unreported entity, which fits KOS, document the clinical homogeneity of this disorder.

Cytogenetic, FISH and array-CGH characterization of a complex chromosomal rearrangement carried by a mentally and language impaired patient.

We describe a patient with an abnormal phenotype and a de novo CCR consisting of a reciprocal translocation between chromosomes 1 and 15 and an insertion of an interstitial segment from chromosome 2 within chromosome 1. The CCR was studied by QFQ banding and FISH. The apparently balanced rearrangement was further investigated with array-CGH, that uncovered three cryptic deletions on chromosome 2q. By means of BAC-FISH two deletions were located at the breakpoints of the insertion, at 2q14.3 and 2q31.2, and one at 2q22.2, in the region of 2q translocated on derivative 1. Consequently, in silico analysis of the deleted regions was performed. Among deleted genes, particularly interesting seems to be CNTNAP5, encoding a member of the neurexin superfamily. The three mouse orthologues are highly expressed in adult brain tissues. We speculate that loss of CNTNAP5 might contribute to the developmental language delay of this patient, similar to CNTNAP2, another member of the same protein family, whose alterations have been recently associated with delay in the age at first word in autistic patients. At clinical patients evaluation, a Mowat-Wilson syndrome (MWS) like appearance was noted. The disease is caused by mutation or deletion of ZEB2 gene, located in our patient 794Kb distally to the 2q22.2 deletion, in the chromosome 2 segment inserted into the derivative 1. The loss of the gene has been excluded by the array-CGH results, but its proximity to the deleted segment and/or its insertion in a different genetic context might influence and consequently impair its expression. Our study confirms that array-CGH is a precious method to identify cryptic imbalances in CCR carriers and underlie the essential role of BAC-FISH as second step of analysis to assess the reciprocal position of the chromosomal segments involved in CCRs and the exact mapping of the imbalances.

Association of syndromic mental retardation with an Xq12q13.1 duplication encompassing the oligophrenin 1 gene

OPHN1 mutations cause a syndromic form of mental retardation (MR) characterized by cerebellar hypoplasia, early hypotonia, motor and speech delay, with occasional seizures and strabismus. Here we report on a familial chromosome duplication spanning about 800 Kb of Xq12q13.1, associated with MR and a distinctive phenotype in the affected male, but not in his heterozygous mother. The parents were healthy and non‐consanguineous with a history of three pregnancies. The first resulted in the birth of a boy with MR, motor impairment and seizures. The second pregnancy was terminated because of trisomy 18. At the time of the third, the first affected boy was analyzed by array‐CGH, which revealed a 800 Kb duplication at Xq12q13.1, encompassing three genes, including OPHN1. This mutation was inherited from his healthy mother and was not present in any of the three maternal brothers. To our knowledge this is the first report of a clinical phenotype associated with duplication of Xq12q13.

Electroclinical pattern in MECP2 duplication syndrome: Eight new reported cases and review of literature

Purpose:  Duplications encompassing the MECP2 gene on the Xq28 region have been described in male patients with moderate to severe mental retardation, absent speech, neonatal hypotonia, progressive spasticity and/or ataxia, recurrent severe respiratory infections, gastrointestinal problems, mild facial dysmorphisms (midface hypoplasia, depressed nasal bridge, large ears) and epilepsy. Epilepsy can occur in >50% of cases, but the types of seizures and the electroclinical findings in affected male individuals have been poorly investigated up to the present. Herein we describe eight patients with MECP2 duplication syndrome and a specific clinical and electroencephalographic pattern.

Cornelia de Lange syndrome: Extending the physical and psychological phenotype†‡

Cornelia de Lange Syndrome: Extending the Physical and Psychological Phenotype Chris Oliver,* Maria Francesca Bedeschi, Natalie Blagowidow, Cheri S. Carrico, Anna Cereda, David R. FitzPatrick, Cristina Gervasini, Gemma M. Griffith, Antonie D. Kline, P. Marchisio, Joanna Moss, Feliciano J. Ramos, Angelo Selicorni, Penny Tunnicliffe, Jolanta Wierzba, and Raoul C.M. Hennekam School of Psychology, University of Birmingham, Birmingham, UK Department of Maternal and Pediatric Sciences, Milan, Italy Harvey Institute of Human Genetics, Greater Baltimore Medical Center, Baltimore, Maryland Speech-Language-Hearing Clinic, Elmhurst College, Elmhurst, Illinois Department of Pediatrics, Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milan, Italy MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK Medical Genetics, San Paolo School of Medicine, Milan, Italy School of Psychology, Bangor University, Bangor, UK Institute of Psychiatry, Kings College, London, UK Laboratorio de Gen etica Cl ınica y Gen omica Funcional, Facultad de Medicina, Zaragoza, Spain Department of General Nursery and Department of Pediatrics, Hematology, Oncology and Endocrinology, Medical University, Gdansk, Poland Department of Pediatrics, Academic Medical Centre, UVA, The Netherlands

Germline mosaicism in achondroplasia detected in sperm DNA of the father of three affected sibs

We describe a sib recurrence for achondroplasia with parents of average stature. The three sibs shared the paternal allele and all carried the same causal mutation in the fibroblast growth factor receptor 3 gene (FGFR3): G > A nt1138 (Gly380Arg). We were able to identify this mutation on sperm DNA confirming paternal germinal mosaicism. Our family shows that a more precise definition of the recurrence risk is feasible using this approach, based on a single DNA test, which could be offered in selected cases.