Lidia Larizza

X-linked Cornelia de Lange syndrome owing to SMC1L1 mutations.

Cornelia de Lange syndrome is a multisystem developmental disorder characterized by facial dysmorphisms, upper limb abnormalities, growth delay and cognitive retardation. Mutations in the NIPBL gene, a component of the cohesin complex, account for approximately half of the affected individuals. We report here that mutations in SMC1L1 (also known as SMC1), which encodes a different subunit of the cohesin complex, are responsible for CdLS in three male members of an affected family and in one sporadic case.

Hypomethylation at multiple maternally methylated imprinted regions including PLAGL1 and GNAS loci in Beckwith–Wiedemann syndrome

Genomic imprinting is an epigenetic phenomenon restricting gene expression in a manner dependent on parent of origin. Imprinted gene products are critical regulators of growth and development, and imprinting disorders are associated with both genetic and epigenetic mutations, including disruption of DNA methylation within the imprinting control regions (ICRs) of these genes. It was recently reported that some patients with imprinting disorders have a more generalised imprinting defect, with hypomethylation at a range of maternally methylated ICRs. We report a cohort of 149 patients with a clinical diagnosis of Beckwith–Wiedemann syndrome (BWS), including 81 with maternal hypomethylation of the KCNQ1OT1 ICR. Methylation analysis of 11 ICRs in these patients showed that hypomethylation affecting multiple imprinted loci was restricted to 17 patients with hypomethylation of the KCNQ1OT1 ICR, and involved only maternally methylated loci. Both partial and complete hypomethylation was demonstrated in these cases, suggesting a possible postzygotic origin of a mosaic imprinting error. Some ICRs, including the PLAGL1 and GNAS/NESPAS ICRs implicated in the aetiology of transient neonatal diabetes and pseudohypoparathyroidism type 1b, respectively, were more frequently affected than others. Although we did not find any evidence for mutation of the candidate gene DNMT3L, these results support the hypotheses that trans-acting factors affect the somatic maintenance of imprinting at multiple maternally methylated loci and that the clinical presentation of these complex cases may reflect the loci and tissues affected with the epigenetic abnormalities.

Rothmund-Thomson syndrome

Rothmund-Thomson syndrome (RTS) is a genodermatosis presenting with a characteristic facial rash (poikiloderma) associated with short stature, sparse scalp hair, sparse or absent eyelashes and/or eyebrows, juvenile cataracts, skeletal abnormalities, radial ray defects, premature aging and a predisposition to cancer. The prevalence is unknown but around 300 cases have been reported in the literature so far. The diagnostic hallmark is facial erythema, which spreads to the extremities but spares the trunk, and which manifests itself within the first year and then develops into poikiloderma. Two clinical subforms of RTS have been defined: RTSI characterised by poikiloderma, ectodermal dysplasia and juvenile cataracts, and RTSII characterised by poikiloderma, congenital bone defects and an increased risk of osteosarcoma in childhood and skin cancer later in life. The skeletal abnormalities may be overt (frontal bossing, saddle nose and congenital radial ray defects), and/or subtle (visible only by radiographic analysis). Gastrointestinal, respiratory and haematological signs have been reported in a few patients. RTS is transmitted in an autosomal recessive manner and is genetically heterogeneous: RTSII is caused by homozygous or compound heterozygous mutations in the RECQL4 helicase gene (detected in 60-65% of RTS patients), whereas the aetiology in RTSI remains unknown. Diagnosis is based on clinical findings (primarily on the age of onset, spreading and appearance of the poikiloderma) and molecular analysis for RECQL4 mutations. Missense mutations are rare, while frameshift, nonsense mutations and splice-site mutations prevail. A fully informative test requires transcript analysis not to overlook intronic deletions causing missplicing. The diagnosis of RTS should be considered in all patients with osteosarcoma, particularly if associated with skin changes. The differential diagnosis should include other causes of childhood poikiloderma (including dyskeratosis congenita, Kindler syndrome and Poikiloderma with Neutropaenia), other rare genodermatoses with prominent telangiectasias (including Bloom syndrome, Werner syndrome and Ataxia-telangiectasia) and the allelic disorders, RAPADILINO syndrome and Baller-Gerold syndrome, which also share some clinical features. A few mutations recur in all three RECQL4 diseases. Genetic counselling should be provided for RTS patients and their families, together with a recommendation for cancer surveillance for all patients with RTSII. Patients should be managed by a multidisciplinary team and offered long term follow-up. Treatment includes the use of pulsed dye laser photocoagulation to improve the telangiectatic component of the rash, surgical removal of the cataracts and standard treatment for individuals who develop cancer. Although some clinical signs suggest precocious aging, life expectancy is not impaired in RTS patients if they do not develop cancer. Outcomes in patients with osteosarcoma are similar in RTS and non-RTS patients, with a five-year survival rate of 60-70%. The sensitivity of RTS cells to genotoxic agents exploiting cells with a known RECQL4 status is being elucidated and is aimed at optimizing the chemotherapeutic regimen for osteosarcoma.

Targeted next-generation sequencing appoints c16orf57 as clericuzio-type poikiloderma with neutropenia gene.

Next-generation sequencing is a straightforward tool for the identification of disease genes in extended genomic regions. Autozygosity mapping was performed on a five-generation inbred Italian family with three siblings affected with Clericuzio-type poikiloderma with neutropenia (PN [MIM %604173]), a rare autosomal-recessive genodermatosis characterised by poikiloderma, pachyonychia, and chronic neutropenia. The siblings were initially diagnosed as affected with Rothmund-Thomson syndrome (RTS [MIM #268400]), with which PN shows phenotypic overlap. Linkage analysis on all living subjects of the family identified a large 16q region inherited identically by descent (IBD) in all affected family members. Deep sequencing of this 3.4 Mb region previously enriched with array capture revealed a homozygous c.504-2 A>C mismatch in all affected siblings. The mutation destroys the invariant AG acceptor site of intron 4 of the evolutionarily conserved C16orf57 gene. Two distinct deleterious mutations (c.502A>G and c.666_676+1del12) identified in an unrelated PN patient confirmed that the C16orf57 gene is responsible for PN. The function of the predicted C16orf57 gene is unknown, but its product has been shown to be interconnected to RECQL4 protein via SMAD4 proteins. The unravelled clinical and genetic identity of PN allows patients to undergo genetic testing and follow-up.

Mental retardation and cardiovascular malformations in NF1 microdeleted patients point to candidate genes in 17q11.2

Neurofibromatosis type 1 ( NF1 [MIM 162200]) is a common autosomal dominant disorder that affects 1/3500 individuals and is caused by deletion or point mutations of NF1 , a tumour suppressor gene mapping to 17q11.2. Its main features include cafe au lait spots, axillary and inguinal freckling, iris Lisch nodules, neurofibromas, and an increased risk of benign and malignant tumours, particularly optic glioma, neurofibrosarcoma, malignant peripheral nerve sheath tumours (MPNSTs),1 and childhood myeloid leukaemia.2 Over 70% of NF1 germline mutations cause truncation or loss of the encoded protein. Approximately 5–20% of all NF1 patients carry a heterozygous deletion of usually 1.5 Mb involving the NF1 gene and contiguous genes lying in its flanking regions,3,4 which is caused by unequal homologous recombination of NF1 repeats (REPs).5 Known as the “ NF1 microdeletion syndrome,” this condition is often characterised by a more severe phenotype than is observed in the general NF1 group. In particular, NF1 microdeleted patients often show variable facial dysmorphisms, mental retardation, developmental delay, and an excessive number of neurofibromas for age.3,6–12 The severe phenotype of microdeleted patients may be explained by variations in the expression of the genes involved in the rearrangement, which may be caused by different mechanisms, such as gene interruptions, position effects, and decreased gene dosages. Although NF1 microdeleted patients generally have different characteristics from those of classic NF1 patients, it remains difficult to foresee the presence of the deletion at an individual level on the basis of clinical observations. Various studies have reported the clinical characterisation of NF1 deleted patients and the precise extent of the deletion has been characterised in a subset.3–5,13,14 However, no study comparing the incidence of specific clinical signs in NF1 deleted and classical NF1 patients has yet been published. …

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.

NF1 Microdeletion Syndrome: Refined FISH Characterization of Sporadic and Familial Deletions with Locus-Specific Probes

Two familial and seven sporadic patients with neurofibromatosis 1-who showed dysmorphism, learning disabilities/mental retardation, and additional signs and carried deletions of the NF1 gene-were investigated by use of a two-step FISH approach to characterize the deletions. With FISH of YAC clones belonging to a 7-Mb 17q11.2 contig, we estimated the extension of all of the deletions and identified the genomic regions harboring the breakpoints. Mosaicism accounted for the mild phenotype in two patients. In subsequent FISH experiments, performed with locus-specific probes generated from the same YACs by means of a novel procedure, we identified the smallest region of overlapping (SRO), mapped the deletion breakpoints, and identified the genes that map to each deletion interval. From centromere to telomere, the approximately 0.8-Mb SRO includes sequence-tagged site 64381, the SUPT6H gene (encoding a transcription factor involved in chromatin structure), and NF1. Extending telomerically from the SRO, two additional genes-BLMH, encoding a hydrolase involved in bleomycin resistance, and ACCN1, encoding an amiloride-sensitive cation channel expressed in the CNS-were located in the deleted intervals of seven and three patients, respectively. An apparently common centromeric deletion breakpoint was shared by all of the patients, whereas a different telomeric breakpoint defined a deletion interval of 0.8-3 Mb. There was no apparent correlation between the extent of the deletion and the phenotype. This characterization of gross NF1 deletions provides the premise for addressing correctly any genotype-phenotype correlation in the subset of patients with NF1 deletions.

Epigenetic modulation of the IGF2/H19 imprinted domain in human embryonic and extra-embryonic compartments and its possible role in fetal growth restriction

Genomic imprinting, resulting in parent-of-origin-dependent gene expression, is mainly achieved by DNA methylation. IGF2 and H19, belonging to the same cluster of imprinted genes and regulated by ICR1, DMR2 and H19 promoter elements, play a major role in fetal/placental growth. Using quantitative approaches, we explored the epigenetic modulation of IGF2/H19 during human development in 60 normal and 66 idiopathic IUGR (Intrauterine Growth Restriction) pregnancies, studying embryonic (cord blood) and extraembryonic (placenta and umbilical cord) tissues. We found ICR1 normal methylation levels (~50%) and H19 promoter/DMR2 hypomethylation in extra-embryonic tissues. In contrast, in embryonic samples the three loci displayed normal methylation values comparable to those in postnatal blood. This feature is stably maintained throughout gestation and does not vary in IUGR cases. We reported asymmetric allelic expression of H19 and IGF2 as a common feature in pre- and post-natal tissues, independent of H19 promoter and DMR2 methylation levels. In addition, we excluded in IUGR posttranscriptional IGF2 interference possibly related to miRNA 483-3p (IGF2, intron 2) expression defects. Through LINE1 methylation analysis, we observed a methylation gradient with increasing methylation from pre- to post-natal life. The involvement of UPD (Uniparental Disomy) in IUGR aetiology was excluded. Our data indicate that: i) ICR1 3 methylation status is a necessary and sufficient condition to drive the imprinting of IGF2 and H19 present in embryonic as well as in extra-embryonic tissues; ii) hypomethylation of H19 promoter and DMR2 does not influence the expression pattern of IGF2 and H19; iii) there is a gradient of global methylation, increasing from extra-embryonic to embryonic and adult tissues. Finally, because of placental hypomethylation, cautions should be exercised in diagnosis of imprinting diseases using chorionic villi.

Clinical score of 62 Italian patients with Cornelia de Lange syndrome and correlations with the presence and type of NIPBL mutation

Cornelia de Lange syndrome (CdLS) is a rare multisystem disorder characterized by facial dysmorphisms, upper limb abnormalities, growth and cognitive retardation. About half of all patients with CdLS carry mutations in the NIPBL gene. The first Italian CdLS cohort involving 62 patients (including 4 related members) was screened for NIPBL mutations after a clinical evaluation using a quantitative score that integrates auxological, malformation and neurodevelopmental parameters. The patients were classified as having an overall ‘severe’, ‘moderate’ or ‘mild’ phenotype. NIPBL screening showed 26 mutations so classified: truncating (13), splice‐site (8), missense (3), in‐frame deletion (1) and regulatory (1). The truncating mutations were most frequently found in the patients with a high clinical score, whereas most of the splice‐site and all missense mutations clustered in the low‐medium score groups. The NIPBL‐negative group included patients covering the entire clinical spectrum. The prevalence of a severe phenotype in the mutated group and a mild phenotype in the non‐mutated group was statistically significant. In terms of the isolated clinical signs, the statistically significant differences between the mutation‐positive and mutation‐negative individuals were pre‐ and post‐natal growth deficits, limb reduction, and delayed speech development. The proposed score seems to be a valuable means of prioritizing the patients with CdLS to undergo an NIPBL mutation test.

Evaluation of autism traits in Angelman syndrome: a resource to unfold autism genes

Linkage and cytogenetics studies have found the Angelman syndrome (AS) chromosomal region to be of relevance to autism disorder (AD) or autism spectrum disorder (ASD). Autism is considered part of the behavioural phenotype in AS based on formal autism assessments (autism diagnostic interview—revised [ADI-R] and autism diagnostic observation schedule [ADOS]), which have mainly addressed the deleted AS group. We explored 23 AS patients including all genetic subtypes and made a co-morbid diagnosis of AD/ASD in 14/23 (61%), which does not include 4 cases classified within the broader autism spectrum disorder (bASD). Deletions accounted for the main fraction (35%), ubiquitin-protein ligase E3A (UBE3A) mutation represented 13%, imprinting defects and uniparental disomy 9 and 4%, respectively. UBE3A mutations due to lack of the homologous to the E6-associated protein carboxyl terminus domain (n = 3) were associated with the ASD, while more distal mutations (n = 3) seem to escape from a co-morbid diagnosis of autism/autism spectrum. Differences in severity of autistic features were seen across subtypes of AS, with some behavioural features being unique to AS and some representing all forms of developmental disability. Autism signs (poor/lack of eye contact, showing, spontaneous initiation of joint attention, social quality of overtures [ADOS algorithm items for Diagnostic and Statistical Manual of Mental Disorders—IV (DSM-IV)/International Statistical Classification of Diseases and Related Health Problems—10 (ICD-10) autism diagnosis belonging to the reciprocal social interaction domain]) discriminating all the co-morbid AS categories from non-autistic AS belonged to the social interaction domain. Impairments in the communication domain (gestures, pointing, use of another’s body, frequency of vocalisation towards others [ADOS algorithm items for DSM-IV/ICD-10 autism diagnosis belonging to the communication domain]) justified classification of co-morbid AD/ASD vs the classification of less affected bASD. Evaluation of the behaviour domain suggested that repetitive sensory and motor behaviours correlate with a low developmental profile rather than being specific to autism.