Genetic/epigenetic effects in NF1 microdeletion syndrome: beyond the haploinsufficiency, looking at the contribution of not deleted genes.

NF1 microdeletion syndrome, accounting for 5-11% of NF1 patients, is caused by a deletion in the NF1 region and it is generally characterized by a severe phenotype. Although 70% of NF1 microdeletion patients presents the same 1.4 Mb type-I deletion, some patients may show additional clinical features. Therefore, the contribution of several pathogenic mechanisms, besides haploinsufficiency of some genes within the deletion interval, is expected and needs to be defined. We investigated an altered expression of deletion flanking genes by qPCR in patients with type-1 NF1 deletion, compared to healthy donors, possibly contributing to the clinical traits of NF1 microdeletion syndrome. In addition, the 1.4-Mb deletion leads to changes in the 3D chromatin structure in the 17q11.2 region. Specifically, this deletion alters DNA-DNA interactions in the regions flanking the breakpoints, as demonstrated by our 4C-seq analysis. This alteration likely causes position effect on the expression of deletion flanking genes.Interestingly, 4C-seq analysis revealed that in microdeletion patients, an interaction was established between the RHOT1 promoter and the SLC6A4 gene, which showed increased expression. We performed NGS on putative modifier genes, and identified two "likely pathogenic" rare variants in RAS pathway, possibly contributing to incidental phenotypic features.This study provides new insights into understanding the pathogenesis of NF1 microdeletion syndrome and suggests a novel pathomechanism that contributes to the expression phenotype in addition to haploinsufficiency of genes located within the deletion.This is a pivotal approach that can be applied to unravel microdeletion syndromes, improving precision medicine, prognosis and patients' follow-up.

9q34.3 microduplications lead to neurodevelopmental disorders through EHMT1 overexpression.

Both copy number losses and gains occur within subtelomeric 9q34 region without common breakpoints. The microdeletions cause Kleefstra syndrome (KS), whose responsible gene is EHMT1. A 9q34 duplication syndrome (9q34 DS) had been reported in literature, but it has never been characterized by a detailed molecular analysis of the gene content and endpoints. To the best of our knowledge, we report on the first patient carrying the smallest 9q34.3 duplication containing EHMT1 as the only relevant gene. We compared him with 21 reported patients described here as carrying 9q34.3 duplications encompassing the entire gene and extending within ~ 3 Mb. By surveying the available clinical and molecular cytogenetic data, we were able to discover that similar neurodevelopmental disorders (NDDs) were shared by patient carriers of even very differently sized duplications. Moreover, some facial features of the 9q34 DS were more represented than those of KS. However, an accurate in silico analysis of the genes mapped in all the duplications allowed us to support EHMT1 as being sufficient to cause a NDD phenotype. Wider patient cohorts are needed to ascertain whether the rearrangements have full causative role or simply confer the susceptibility to NDDs and possibly to identify the cognitive and behavioral profile associated with the increased dosage of EHMT1.

Segmental Maternal UPD of Chromosome 7q in a Patient With Pendred and Silver Russell Syndromes-Like Features.

Pendred syndrome (PS) is an autosomal recessive disorder due to mutations in the SLC26A4 gene (chr7q22. 3) and characterized by sensorineural hearing loss and variable thyroid phenotype. Silver-Russell syndrome (SRS) is a heterogeneous imprinting disorder including severe intrauterine and postnatal growth retardation, and dysmorphic features. Maternal uniparental disomy of either the whole chromosome 7 (upd(7)mat) or 7q (upd(7q)mat) is one of the multiple mechanisms impacting the expression of imprinted genes in SRS, and is associated with milder clinical features. Here, we report genetic and clinical characterization of a female child with PS, postnatal growth retardation, and minor dysmorphic features. A gross homozygous deletion of SLC26A4 exons 17-20 was suspected by Sanger sequencing and then confirmed by array-CGH. Moreover, an insertion of about 1 kb of the CCDC126 gene (7p15.3), which does not appear to be clinically relevant, was detected. The possible occurrence of a balanced rearrangement between 7p and 7q was excluded. The absence of the deletion in the father led to the investigation of upd, and microsatellite segregation analysis revealed a segmental 7q (upd(7q)mat), leading to SLC26A4 homozygosity and responsible for both PS and SRS-like traits. The proband matched 3 out of 6 major SRS criteria. In conclusion, this is the first report of uniparental isodisomy encompassing almost the whole long arm of chromosome 7 resulting in PS and SRS-like features. Whereas, the inner ear phenotype of PS is typical, the clinical features suggestive of SRS might have been overlooked.

13q mosaic deletion including RB1 associated to mild phenotype and no cancer outcome - case report and review of the literature.

BACKGROUND: The 13q deletion syndrome is a rare chromosome disorder associated with wide phenotypic spectrum, which is related to size and location of the deleted region and includes intellectual disability, growth retardation, craniofacial dysmorphisms, congenital malformations, and increased risk of retinoblastoma. CASE PRESENTATION: Here, we report on a teenage boy with a mild phenotype characterized by obesity, hyperactivity, dysphagia, dysgraphia, sleep disturbance, and minor dysmorphic features (round face, bushy eyebrows, and stubby hands). Array Comparative Genomic Hybridization on blood identified a mosaic 13q14.13-13q31.1 deletion, with a mosaicism rate around 40%, which was confirmed by quantitative PCR and interphase Fluorescent In Situ Hybridization (iFISH) on both blood genomic DNA and cultured/uncultured blood lymphocytes, respectively. Conversely, karyotype analysis on blood estimated a mosaicism rate of 24% and iFISH on buccal smears revealed a borderline value of 0.4%, suggesting the absence of 13q deletion in this cell line. CONCLUSIONS: The comparison with previous patients carrying similar deletions informed that the proband clinical presentation is the mildest reported to date, thus supporting the burden of mosaicism in modulating the phenotype also in case of large chromosomal rearrangements. Characterization of further cases by in-depth mosaicism rate in tissues with different embryonic origins might contribute in the future to a better definition of genotype-phenotype correlation, including tumor risk.

A novel nonsense ATP7A pathogenic variant in a family exhibiting a variable occipital horn syndrome phenotype.

We report on a family with occipital horn syndrome (OHS) diagnosed in the proband's late fifties. A novel ATP7A pathogenic variant (c.4222A > T, p.(Lys1408*)), representing the first nonsense variant and the second late truncation causing OHS rather than classic Menkes disease, was found to segregate in the family. The predicted maintenance of transmembrane domains is consistent with a residual protein activity, which may explain the mild clinical presentation.

Familial intragenic duplication of ANKRD11 underlying three patients of KBG syndrome.

BACKGROUND: KBG syndrome, a rare autosomal disorder characterised by distinctive craniofacial and skeletal features and developmental delay, is caused by haploinsufficiency of the ANKRD11 gene. RESULTS: Here we describe two siblings with multiple symptoms characteristic of KBG and their mother with a milder phenotype. In the siblings, array-based comparative genomic hybridization (array CGH) identified an intragenic microduplication affecting ANKRD11 that was absent from the parents' array CGH profiles. Microsatellite analysis revealed the maternal origin of the rearrangement and interphase fluorescent in situ hybridization (i-FISH) experiments identified the rearrangement in low-level mosaicism in the mother. Molecular characterisation of the duplication allele demonstrated the presence of two mutant ANKRD11 transcripts containing a premature stop codon and predicting a truncated non-functional protein. CONCLUSIONS: Similarly to deletions and point mutations, this novel pathogenetic rearrangement causes haploinsufficiency of ANKRD11, resulting in KBG syndrome.

Complex de novo chromosomal rearrangement at 15q11-q13 involving an intrachromosomal triplication in a patient with a severe neuropsychological phenotype: clinical report and review of the literature.

Interstitial triplications of 15q11-q13, leading to tetrasomy of the involved region, are very rare, with only 11 cases reported to date. Their pathogenicity is independent of the parental origin of the rearranged chromosome. The associated phenotype resembles, but is less severe, than that of patients bearing inv dup(15) marker chromosomes. Here, we describe a boy of 3 years and 9 months of age who exhibited very mild craniofacial dysmorphism (arched eyebrows, hypertelorism, and a wide mouth), developmental delay, generalized hypotonia, ataxic gait, severe intellectual disability, and autism. Array comparative genomic hybridization (CGH) analysis identified a heterozygous duplication of 1.1 Mb at 15q11.2 (between low-copy repeats BP1 and BP2), and a heterozygous triplication of 6.8 Mb at 15q11.2-q13.1 (BP2-BP4). Both acquisitions were de novo and contiguous. Microsatellite polymorphism analysis revealed the maternal origin of the triplication and the involvement of both maternal chromosomes 15. Furthermore, fluorescence in situ hybridization (FISH) analysis using BAC clones revealed that the rearrangement was complex, containing three differently sized tandem repeats of which the middle one was inverted. Our study confirms and extends the model proposed to explain the formation of intrachromosomal triplications through recombination events between non-allelic duplicons. The comparison of the proband's clinical presentation with those of previously described cases attests the existence of endophenotypes due to the parental origin of the 15q11-q13 triplicated segment and suggests a timetable for achievement of developmental milestones, thereby contributing to improved genotype-phenotype correlations.

New case of trichorinophalangeal syndrome-like phenotype with a de novo t(2;8)(p16.1;q23.3) translocation which does not disrupt the TRPS1 gene.

BACKGROUND: Trichorhinophalangeal syndrome (TRPS) is a rare autosomal dominant genetic disorder characterised by distinctive craniofacial and skeletal abnormalities. TRPS is generally associated with mutations in the TRPS1 gene at 8q23.3 or microdeletions of the 8q23.3-q24.11 region. However, three deletions affecting the same chromosome region and a familial translocation t(8;13) co-segregating with TRPS, which do not encompass or disrupt the TRPS1 gene, have been reported. A deregulated expression of TRPS1 has been hypothesised as cause of the TRPS phenotype of these patients. CASE PRESENTATION: We report the clinical and molecular characterisation of a 57-year-old Caucasian woman carrying the t(2;8)(p16.1;q23.3) de novo balanced translocation. The proband presented with peculiar clinical features (severe craniofacial dysmorphism, alopecia universalis, severe scoliosis, mitral valve prolapse, mild mental impairment and normal growth parameters) that partially overlap with TRPS I. Mutational and array CGH analyses ruled out any genetic defect affecting TRPS1 or genomic alteration at the translocation breakpoint or elsewhere in the genome. Breakpoint mapping excluded disruption of TRPS1, and revealed that the chromosome 8q23.3 breakpoint was located within the IVS10 of the long intergenic non-coding RNA LINC00536, at approximately 300 kb from the TRPS1 5' end. Conversely, the 2p16.1 breakpoint mapped within a LINE sequence, in a region that lacks transcriptional regulatory elements. As a result of the translocation, nucleotide base pair additions and deletions were detected at both breakpoint junction fragments, and an evolutionarily conserved VISTA enhancer element from 2p16.1 was relocated at approximately 325 kb from the TRPS1 promoter. CONCLUSIONS: We suggest that the disruption of the genomic architecture of cis regulatory elements downstream the TRPS1 5' region, combined with the translocation of a novel enhancer element nearby TRPS1, might be the pathogenetic mechanism underpinning the proband's phenotype. The clinical and genetic characterisation of the present subject allowed us to make a genetic diagnosis in the context of a known syndrome, contributing to a better comprehension of the complex transcriptional regulation of TRPS1 and TRPS ethiopathogenesis.

Gene dosage as a relevant mechanism contributing to the determination of ovarian function in Turner syndrome.

STUDY QUESTION: What is the burden of X chromosome mosaicism in the occurrence of spontaneous menarche (SM) in Turner syndrome (TS)? SUMMARY ANSWER: SM was significantly associated with X chromosome mosaicism in the TS patients; a mosaicism with around 10% euploid cell line may predict spontaneous pubertal development when determined by molecular-cytogenetic techniques on uncultivated tissues. WHAT IS KNOWN ALREADY: Spontaneous puberty can be observed in a minority of patients with TS, more frequently, but not exclusively, in those with a high level of 46,XX/45,X mosaicism at standard karyotype. The genetic mechanisms contributing to ovarian function in TS patients are still not determined. However, submicroscopic X-linked and autosomal copy number variations (CNVs) have recently emerged as an important genetic risk category for premature ovarian insufficiency and may be involved in modulating the TS ovarian phenotype. STUDY DESIGN, SIZE, DURATION: A group of 40 patients with a diagnosis of TS at conventional karyotyping participated in the study; 6 patients had SM and 34 patients had primary amenorrhoea (PA). All clinical data and the patients' DNA samples were collected over the years at a single paediatric clinic. PARTICIPANTS/MATERIALS, SETTING, METHODS: The patients' samples were used to perform both genetic (Copy Number Assay) and molecular-cytogenetic (array-CGH and iFISH, interphase-FISH) analyses in order to evaluate the X chromosome mosaicism rate and to detect possible rare CNVs of genes with a known or predicted role in female fertility. MAIN RESULTS AND THE ROLE OF CHANCE: All TS patients showed variable percentages of the 46,XX lineage, but these percentages were higher in the SM group (P < 0.01). A mosaicism around 10% for the euploid cell line may predict spontaneous pubertal development when determined by molecular-cytogenetic techniques performed in uncultivated tissues. A few CNVs involving autosomal and X-linked ovary-related loci were identified by array-CGH analysis and confirmed by real-time quantitative PCR, including a BMP15 gene duplication at Xp11.22, a deletion interrupting the PAPPA gene at 9q33.1, and an intragenic duplication involving the PDE8A gene at 15q25.3. LIMITATIONS, REASONS FOR CAUTION: This is a pilot study on a relatively small sample size and confirmation in larger TS cohorts may be required. The ovarian tissue could not be studied in any patients and in a subgroup of patients, the mosaicism was estimated in tissues of different embryonic origin. WIDER IMPLICATIONS OF THE FINDINGS: The combined determination of X chromosome mosaicism by molecular and molecular-cytogenetic techniques may become useful for the prediction of SM in TS. The detection of CNVs in both X-linked and autosomal ovary-related genes further suggests gene dosage as a relevant mechanism contributing to the ovarian phenotype of TS patients. These CNVs may pinpoint novel candidates relevant to female fertility and generate further insights into the mechanisms contributing to ovarian function. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Telethon Foundation (grant no: GGP09126 to L.P.), the Italian Ministry of the University and Research (grant number: 2006065999 to P.F.) and a Ministry of Health grant 'Ricerca Corrente' to IRCCS Istituto Auxologico Italiano (grant number: 08C704-2006). The authors have no conflict of interest to declare.

Design and validation of a pericentromeric BAC clone set aimed at improving diagnosis and phenotype prediction of supernumerary marker chromosomes.

BACKGROUND: Small supernumerary marker chromosomes (sSMCs) are additional, structurally abnormal chromosomes, generally smaller than chromosome 20 of the same metaphase spread. Due to their small size, they are difficult to characterize by conventional cytogenetics alone. In regard to their clinical effects, sSMCs are a heterogeneous group: in particular, sSMCs containing pericentromeric euchromatin are likely to be associated with abnormal outcomes, although exceptions have been reported. To improve characterization of the genetic content of sSMCs, several approaches might be applied based on different molecular and molecular-cytogenetic assays, e.g., fluorescent in situ hybridization (FISH), array-based comparative genomic hybridization (array CGH), and multiplex ligation-dependent probe amplification (MLPA).To provide a complementary tool for the characterization of sSMCs, we constructed and validated a new, FISH-based, pericentromeric Bacterial Artificial Chromosome (BAC) clone set that with a high resolution spans the most proximal euchromatic sequences of all human chromosome arms, excluding the acrocentric short arms. RESULTS: By FISH analysis, we assayed 561 pericentromeric BAC probes and excluded 75 that showed a wrong chromosomal localization. The remaining 486 probes were used to establish 43 BAC-based pericentromeric panels. Each panel consists of a core, which with a high resolution covers the most proximal euchromatic ~0.7 Mb (on average) of each chromosome arm and generally bridges the heterochromatin/euchromatin junction, as well as clones located proximally and distally to the core. The pericentromeric clone set was subsequently validated by the characterization of 19 sSMCs. Using the core probes, we could rapidly distinguish between heterochromatic (1/19) and euchromatic (11/19) sSMCs, and estimate the euchromatic DNA content, which ranged from approximately 0.13 to more than 10 Mb. The characterization was not completed for seven sSMCs due to a lack of information about the covered region in the reference sequence (1/19) or sample insufficiency (6/19). CONCLUSIONS: Our results demonstrate that this pericentromeric clone set is useful as an alternative tool for sSMC characterization, primarily in cases of very small SMCs that contain either heterochromatin exclusively or a tiny amount of euchromatic sequence, and also in cases of low-level or cryptic mosaicism. The resulting data will foster knowledge of human proximal euchromatic regions involved in chromosomal imbalances, thereby improving genotype-phenotype correlations.

A novel mosaic NSD1 intragenic deletion in a patient with an atypical phenotype.

Sotos syndrome, which is characterized by overgrowth, macrocephaly, distinctive facial features, and developmental delay, arises from mutations and deletions of the NSD1 gene at 5q35.3. Sixteen NSD1 intragenic deletions (including one in a mosaic condition) and one partial duplication have been reported in patients with Sotos syndrome. Here, we describe a boy aged 4 years and 10 months that showed facial dysmorphism (including frontal bossing, widely spaced eyes, deeply set eyes, a wide nasal bridge, anteverted nares, and a wide mouth), normal growth, and a psychomotor delay. High-resolution array comparative genomic hybridization (CGH) analysis identified a mosaic heterozygous intragenic NSD1 deletion of 38 kb, which included part of intron 2 and the entire exon 3, and led to NSD1 haploinsufficiency. The deletion somatic mosaicism was subsequently confirmed by fluorescence in situ hybridization (FISH) analysis using fosmid clones. This patient presents the most atypical phenotype thus far associated with NSD1 haploinsufficiency. It is possible that this atypical phenotype may have resulted from the somatic mosaicism of the NSD1 defect. Our study confirms the usefulness of array CGH for increasing the detection rate of NSD1 abnormalities and for diagnosing syndromic patients that do not present an easily recognized phenotype.

Increased loss of the Y chromosome in peripheral blood cells in male patients with autoimmune thyroiditis.

Multiple mechanisms have been proposed to explain the peculiar distribution of autoimmune thyroiditis (AIT) among women and men. Most attention has been focused on the detection of the role of estrogens and the X chromosome. Specifically, a potential role for X haploinsufficiency has been proposed in the female patient population and an association with the disease has been confirmed. Our knowledge of the etiopathogenesis of autoimmunity in male patients remains, however, limited. Next to the possible role of androgens and their imbalances, the Y chromosome appears as a potential candidate for influence of the immune function in men. Herein we analyzed a population of male patients with AIT (n=31) and healthy controls (n=88) to define a potential association of disease and the loss of the Y chromosome. Y chromosome loss increases in AIT compared to unaffected subjects; these phenomenon increases with aging as expected, however, the degree of loss is significantly increased in the patient population compared to the healthy controls. We were, thus, able to confirm the existence of an analogous mechanism in the male population to previously identified X haploinsufficiency in female patients with AIT. We propose that this commonality might represent a relevant feature in the etiopathogenesis of AIT that should be further investigated.

The natural history of Cri du Chat Syndrome. A report from the Italian Register.

The aim of this report is to provide an update on the natural history of the Cri du Chat Syndrome by means of the Italian Register (I.R.). Two hundred twenty patients were diagnosed by standard cytogenetic methods and 112 of these were also characterised by molecular-cytogenetic investigation (FISH). FISH analysis showed interstitial deletions, short terminal deletions and other rare rearrangements not previously correctly diagnosed by standard cytogenetics. The diagnosis was made in the first month of life in 42% and within first year in 82% of cases. The remaining 18% were diagnosed at an age ranging from 13 months to 47 years. At the last follow-up, patient age ranged from 8 months to 61 years. Mortality, already low, has decreased over time as it is lower between 1984-2002 compared to 1965-1983. Mortality was higher in patients with unbalanced translocations resulting in 5p deletions. Our data confirm that the cat-like cry and peculiar timbre of voice are the most typical signs of the syndrome, not only at birth but also later and these are the only signs which might suggest the diagnosis in patients with small deletions and mild clinical picture. A cytogenetic and clinical variability must be underlined. Cardiac, cerebral, renal and gastrointestinal malformations were more frequent in the patients with unbalanced translocations resulting in 5p deletions. Sucking and feeding difficulties and respiratory infections are frequent in the first months or years of life. Intubation difficulties linked to larynx anomalies must be considered. Psychomotor development is delayed in all patients but there is a variability related to deletion size and type as well as other genetic and environmental factors. However, the results showed an improvement in the acquisition of the development skills and progress in social introduction which should encourage caregivers and parents to work together in carrying out the rehabilitative and educational interventions.