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.

CATSHL syndrome, a new family and phenotypic expansion.

We report the case of a 12-year-old girl and her father who both had marked postnatal tall stature, camptodactyly and clinodactyly, scoliosis and juvenile-onset hearing loss. The CATSHL (CAmptodactyly - Tall stature - Scoliosis - Hearing Loss syndrome) syndrome was suspected, and molecular analysis revealed a hitherto unreported, monoallelic variant c.1861C>T (p.Arg621Cys) in FGFR3. This variant affects the same residue, but is different than, the variant p.Arg621His reported in the two families with dominant CATSHL described so far. Interestingly, peg-shaped incisors were observed in the proband, a feature never reported in CATSHL but typical of another FGFR3-related condition, LADD (Lacrimo - Auricolo - Dento - Digital) syndrome. The FGFR3 p.Arg621Cys variant seems to be a newly identified cause of CATSHL syndrome with some phenotypic overlap with the LADD syndrome.

New insights into the molecular basis of spinal neurofibromatosis type 1.

Spinal neurofibromatosis (SNF) is a form of neurofibromatosis type 1 (NF1) characterized by bilateral neurofibromas involving all spinal roots. The pathogenic mechanisms determining the SNF form are currently unknown. To verify the presence of genetic variants possibly related to SNF or classic NF1, we studied 106 sporadic NF1 and 75 SNF patients using an NGS panel of 286 genes encoding RAS pathway effectors and neurofibromin interactors and evaluated the expression of syndecans (SDC1, SDC2, SDC3, SDC4), the NF1 3' tertile interactors, by quantitative real-time PCR. We previously identified 75 and 106 NF1 variants in SNF and NF1 cohorts, respectively. The analysis of the distribution of pathogenic NF1 variants in the three NF1 tertiles showed a significantly higher prevalence of NF1 3' tertile mutations in SNF than in the NF1 cohort. We hypothesized a potential pathogenic significance of the 3' tertile NF1 variants in SNF. The analysis of syndecan expression on PBMCs RNAs from 16 SNF, 16 classic NF1 patients and 16 healthy controls showed that the expression levels of SDC2 and SDC3 were higher in SNF and NF1 patients than in controls; moreover, SDC2, SDC3 and SDC4 were significantly over expressed in patients mutated in the 3' tertile compared to controls. Two different mutational NF1 spectra seem to characterize SNF and classic NF1, suggesting a pathogenic role of NF1 3' tertile and its interactors, syndecans, in SNF. Our study, providing new insights on a possible role of neurofibromin C-terminal in SNF, could address effective personalized patient management and treatments.

Congenital diaphragmatic hernia in Coffin Siris syndrome: Further evidence from two cases.

Coffin-Siris Syndrome (CSS) is a rare multi-system dominant condition with a variable clinical presentation mainly characterized by hypoplasia/aplasia of the nail and/or distal phalanx of the fifth digit, coarse facies, hirsutism/hypertrichosis, developmental delay and intellectual disability of variable degree and growth impairment. Congenital anomalies may include cardiac, genitourinary and central nervous system malformations whereas congenital diaphragmatic hernia (CDH) is rarely reported. The genes usually involved in CSS pathogenesis are ARID1B (most frequently), SMARCA4, SMARCB1, ARID1A, SMARCE1, DPF2, and PHF6. Here, we present two cases of CSS presenting with CDH, for whom Whole Exome Sequencing (WES) identified two distinct de novo heterozygous causative variants, one in ARID1B (case 1) and one in SMARCA4 (case 2). Due to the rarity of CDH in CSS, in both cases the occurrence of CDH did not represent a predictive sign of CSS but, on the other hand, prompted genetic testing before (case 1) or independently (case 2) from the clinical hypothesis of CSS. We provide further evidence of the association between CSS and CDH, reviewed previous cases from literature and discuss possible functional links to related conditions.

Prenatal ultrasound findings associated with PIGW variants: One more piece in the FRYNS syndrome puzzle? PIGW-related prenatal findings.

OBJECTIVE: We describe the prenatal ultrasound findings and autopsy of three fetuses with multiple congenital anomalies (MCA) whose diagnostic workup suggested the same genetic etiology. We conducted a literature review to corroborate the molecular results and find evidence that the identified variants are responsible for the phenotype seen. METHODS: Trio-based Exome Sequencing (ES) analysis was performed on chorionic villus samples. We reviewed available reports dealing with prenatal manifestations of genes involved in the Glycosylphosphatidylinositols (GPI) biosynthesis defects (GPIBDs). RESULTS: Prenatal findings shared by all the three pregnancies included facial dysmorphisms, brain malformations of the posterior fossa, skeletal and genitourinary anomalies. ES analysis identified homozygous variants of uncertain significance in PIGW in the three fetuses. Prenatal findings of the three pregnancies overlapped with those previously described for PIGW variants and with those associated with PIGN, PIGV and PIGA variants. CONCLUSION: Based on the phenotypic overlap between the prenatal findings in our three cases and other cases with pathogenic variants in other genes involved in GPIBDs, we speculate that the variants identified in the three fetuses are likely causal of their phenotype and that the PIGWclinical spectrum might extend to MCA, mainly involving brain, skeletal and genitourinary systems. Moreover, we suggest that also PIGW could be involved in Fryns/Fryns-like phenotypes.

Family history is key to the interpretation of exome sequencing in the prenatal context: unexpected diagnosis of Basal Cell Nevus Syndrome.

OBJECTIVES: To reach a molecular diagnosis for a family with two consecutive fetuses presenting with multiple congenital anomalies. METHODS: The two fetuses underwent prenatal ultrasound, autopsy, radiologic, and genetic investigation. Genetic analysis included karyotype and array-CGH for both fetuses and trio-based whole exome sequencing (WES) only for the second fetus. RESULTS: WES results, initially focusing on recessive or dominant de novo variants, were negative.However, as a result of new relevant information regarding family history, the variant c.648_651dup in the PTCH1 gene was identified as causative of the fetal phenotype. CONCLUSIONS: This case further highlights how WES data analysis and interpretation strongly rely on family history and robust genotype-phenotype correlation. This is even more relevant in the prenatal setting, where access to fetal phenotype is limited and prenatal recognition of many morbid genes is not fully explored. We also provide a detailed description of the prenatal manifestations of Basal Cell Nevus Syndrome.

A Translational Approach to Spinal Neurofibromatosis: Clinical and Molecular Insights from a Wide Italian Cohort.

Spinal neurofibromatosis (SNF), a phenotypic subclass of neurofibromatosis 1 (NF1), is characterized by bilateral neurofibromas involving all spinal roots. In order to deepen the understanding of SNF's clinical and genetic features, we identified 81 patients with SNF, 55 from unrelated families, and 26 belonging to 19 families with at least 1 member affected by SNF, and 106 NF1 patients aged >30 years without spinal tumors. A comprehensive NF1 mutation screening was performed using NGS panels, including NF1 and several RAS pathway genes. The main features of the SNF subjects were a higher number of internal neurofibromas (p < 0.001), nerve root swelling (p < 0.001), and subcutaneous neurofibromas (p = 0.03), while hyperpigmentation signs were significantly less frequent compared with the classical NF1-affected cohorts (p = 0.012). Fifteen patients underwent neurosurgical intervention. The histological findings revealed neurofibromas in 13 patients and ganglioneuromas in 2 patients. Phenotypic variability within SNF families was observed. The proportion of missense mutations was higher in the SNF cases than in the classical NF1 group (21.40% vs. 7.5%, p = 0.007), conferring an odds ratio (OR) of 3.34 (CI = 1.33−10.78). Two unrelated familial SNF cases harbored in trans double NF1 mutations that seemed to have a subclinical worsening effect on the clinical phenotype. Our study, with the largest series of SNF patients reported to date, better defines the clinical and genetic features of SNF, which could improve the management and genetic counseling of NF1.

Case Report: The Association Between Chromosomal Anomalies and Cluster A Personality Disorders: The Case of Two Siblings With 16p11.2 Deletion and a Review of the Literature.

Although several studies have shown the correlation between chromosomal rearrangements and the risk of developing psychotic disorders, such as schizophrenia, little attention has been given to identifying the genetic basis of pre-disposing personality so far. In this regard, a limited but significant number of studies seem to indicate an association between chromosomal anomalies and cluster A personality disorders (CAPD). Starting from the clinical description of two brothers affected by familial 16p11 deletion syndrome (OMIM #611913), both sharing cluster A and C personality traits, the aim of the present study is to critically review the literature regarding the correlation between chromosomal rearrangements and CAPD. A bibliographic search on PubMed has been conducted, and eight studies were finally included in our review. Most of the studies highlight the presence of schizotypal personality disorder in the 22q11.2 deletion syndrome, whose evolutionary course toward psychotic pictures is well-known. One study also identified a paranoid personality disorder in a patient with a deletion on chromosome 7q21.3. No studies have so far identified the presence of paranoid personality disorder in 16p11 deletion, as in the case of the two siblings we report, while its association with psychosis and autism is already known. Although further epidemiologic studies on broader populations are indicated, our observations might pave the way for the definition of new diagnostic subgroups of CAPD and psychotic disorders, in order to implement the clinical management of such complex conditions.

Simultaneous Detection of NF1, SPRED1, LZTR1, and NF2 Gene Mutations by Targeted NGS in an Italian Cohort of Suspected NF1 Patients.

Neurofibromatosis type 1 (NF1) displays overlapping phenotypes with other neurocutaneous diseases such as Legius Syndrome. Here, we present results obtained using a next generation sequencing (NGS) panel including NF1, NF2, SPRED1, SMARCB1, and LZTR1 genes on Ion Torrent. Together with NGS, the Multiplex Ligation-Dependent Probe Amplification Analysis (MLPA) method was performed to rule out large deletions/duplications in NF1 gene; we validated the MLPA/NGS approach using Sanger sequencing on DNA or RNA of both positive and negative samples. In our cohort, a pathogenic variant was found in 175 patients; the pathogenic variant was observed in NF1 gene in 168 cases. A SPRED1 pathogenic variant was also found in one child and in a one year old boy, both NF2 and LZTR1 pathogenic variants were observed; in addition, we identified five LZTR1 pathogenic variants in three children and two adults. Six NF1 pathogenic variants, that the NGS analysis failed to identify, were detected on RNA by Sanger. NGS allows the identification of novel mutations in five genes in the same sequencing run, permitting unambiguous recognition of disorders with overlapping phenotypes with NF1 and facilitating genetic counseling and a personalized follow-up.

Unexpected Genotype in a Non-Transfusion Dependent Thalassemia Family.

Non-transfusion dependent thalassemia (NTDT) is an inherited hemoglobin disorder characterized by an α/non-α globin chain imbalance of variable severity, resulting in a wide spectrum of clinical manifestations. The coinheritance of additional α genes with a beta-thalassemia heterozygous mutation has a well-known negative effect. Triplication or quadruplication alone are mostly found by chance, but the coinheritance with ß mutations can worsen the very mild anemia to a more severe hematological and clinical phenotype causing NTDT, depending on the severity of beta mutations. We describe a case of a 38-year-old ß-thalassemia trait, pregnant woman at 33 weeks of gestation with supernumerary α-globin genes and two ß-globin defects.

Risk of Optic Pathway Glioma in Neurofibromatosis Type 1: No Evidence of Genotype-Phenotype Correlations in A Large Independent Cohort.

The occurrence of optic pathway gliomas (OPGs) in children with neurofibromatosis type 1 (NF1) still raises many questions regarding screening and surveillance because of the lack of robust prognostic factors. Recent studies of an overall cohort of 381 patients have suggested that the genotype may be the main determinant of the development of OPG, with the risk being higher in patients harbouring NF1 mutations in the 5' tertile and the cysteine/serine-rich domain. In an attempt to confirm this hypothesis, we used strict criteria to select a large independent cohort of 309 NF1 patients with defined constitutional NF1 mutations and appropriate brain images (255 directly enrolled and 54 as a result of a literature search). One hundred and thirty-two patients had OPG and 177 did not. The association of the position (tertiles and functional domains) and type of NF1 mutation with the development of OPG was analysed using the χ2 test and Fisher's exact probability test; odds ratios (ORs) with 95% confidence intervals were calculated, and Bonferroni's correction for multiple comparisons was applied; multiple logistic regression was also used to study genotype-phenotype associations further. Our findings show no significant correlation between the site/type of NF1 mutation and the risk of OPG, and thus do not support the hypothesis that certain constitutional mutations provide prognostic information in this regard. In addition, we combined our cohort with a previously described cohort of 381 patients for a total of 690 patients and statistically re-analysed the results. The re-analysis confirmed that there were no correlations between the site (tertile and domain) and the risk of OPG, thus further strengthening our conclusions.

Hb Milano [α109(G16)Leu→Pro (CTG>CCG); HBA1: c.329T>C]: A Novel Variant on the α1-Globin Gene in an Italian Family.

Interest in α-globin point mutations has increased in the past few years because nondeletional variations can affect protein function and stability, giving rise to hemoglobin (Hb) variants that present a wide spectrum of phenotypes, from asymptomatic forms to hemolytic anemia. We describe a novel α1-globin gene variant, which we have named Hb Milano [α109(G16)Leu→Pro (CTG>CCG); HBA1: c.329T>C]. We performed high performance liquid chromatography (HPLC) to carry out Hb analysis, capillary electrophoresis (CE) for Hb separation and quantitation of Hb subtypes, two tests on stroma-free lysates for evaluating Hb stability, multiplex ligation-dependent probe amplification (MLPA) to detect deletions/duplications within the α gene cluster and Sanger sequencing of the α-globin genes. No abnormal Hb variants were identified by HPLC and CE. Isopropanol and stability tests were negative. The peripheral blood film showed no inclusions such as Hb H or Heinz bodies. Multiplication ligation-dependent probe amplification of the α-globin gene cluster detected a heterozygosity for the -α3.7 (rightward) deletion. Direct sequencing of the α-globin genes identified the Hb Milano variant on the HBA1 gene. No mutations were found on the HBA2 gene. The clinical consequences of the Hb Milano variant differ based on the genotype: according to our study, the hematological parameters range from a marked microcythemia with mild anemia if the variant is coinherited with an α gene deletion, to mild microcytosis when the variant is not associated with α gene deletions.

Correction: The absence that makes the difference: choroidal abnormalities in Legius syndrome.

Correction to: Journal of Human Genetics advance online publication 27 July 2017; https://doi.org/10.1038/jhg.2017.78.

The absence that makes the difference: choroidal abnormalities in Legius syndrome.

Neurofibromatosis type 1 (NF1) is an hereditary disorder characterized by abnormal proliferation of multiple tissues of neural crest origin, and presents mainly with multiple café-au-lait macules, axillary freckling and neurofibromas. Choroidal involvement in NF1 patients has been studied, thanks to the development of non-invasive tools such as infrared monochromatic light during fundus examination, which showed bright patchy lesions consistent with choroidal nodules. Choroidal abnormalities identified with near-infrared reflectance have reported with a frequency of up to 100% in NF1, and have been recently been proposed as a novel diagnostic criterion for NF1. Legius syndrome can be clinically indistinguishable from NF1 and results in a small percentage of individuals being misdiagnosed. We investigated the presence of choroidal abnormalities in Legius syndrome to determine their specificity to NF1 and their potential usefulness as a novel diagnostic criterion for NF1. We examined the fundus of 16 eyes by confocal scanning laser ophthalmoscopy with infrared monochromatic light in eight patients with molecularly confirmed Legius syndrome. No abnormalities were observed, confirming the diagnostic value of choroidal abnormalities for the diagnosis of NF1.

SMCHD1 mutations associated with a rare muscular dystrophy can also cause isolated arhinia and Bosma arhinia microphthalmia syndrome.

Arhinia, or absence of the nose, is a rare malformation of unknown etiology that is often accompanied by ocular and reproductive defects. Sequencing of 40 people with arhinia revealed that 84% of probands harbor a missense mutation localized to a constrained region of SMCHD1 encompassing the ATPase domain. SMCHD1 mutations cause facioscapulohumeral muscular dystrophy type 2 (FSHD2) via a trans-acting loss-of-function epigenetic mechanism. We discovered shared mutations and comparable DNA hypomethylation patterning between these distinct disorders. CRISPR/Cas9-mediated alteration of smchd1 in zebrafish yielded arhinia-relevant phenotypes. Transcriptome and protein analyses in arhinia probands and controls showed no differences in SMCHD1 mRNA or protein abundance but revealed regulatory changes in genes and pathways associated with craniofacial patterning. Mutations in SMCHD1 thus contribute to distinct phenotypic spectra, from craniofacial malformation and reproductive disorders to muscular dystrophy, which we speculate to be consistent with oligogenic mechanisms resulting in pleiotropic outcomes.

Prenatal detection of 5q14.3 duplication including MEF2C and brain phenotype.

The 5q14.3 duplication is a rare condition comprising speech and developmental delay, microcephaly, and mild ventriculomegaly. The region 5q14.3 contains several genes but the predominant role for the onset of the neurodevelopmental phenotype has been attributed to MEF2C. We describe the prenatal identification of 5q14.3 duplication, including MEF2C, in a monochorionic twin pregnancy with corpus callosum anomalies, confirmed by autopsy. To the best of our knowledge, this cerebral finding has been observed for the first time in 5q14.3 duplication patients, possibly widening the neurological picture of this scarcely known syndrome. A pathogenetic role of MEF2C overexpression in brain development may be assumed, but further studies are needed.

Expanding the spectrum of human ganglionic eminence region anomalies on fetal magnetic resonance imaging.

INTRODUCTION: Ganglionic eminence (GE) is a transient fetal brain structure that harvests a significant amount of precursors of cortical GABA-ergic interneurons. Prenatal magnetic resonance (MR) imaging features of GE anomalies (i.e., cavitations) have already been reported associated with severe micro-lissencephaly. The purpose of this report was to illustrate the MR imaging features of GE anomalies in conditions other than severe micro-lissencephalies. METHODS: Among all the fetuses submitted to prenatal MR imaging at our center from 2005 to 2014, we collected eight cases with GE anomalies and only limited associated brain anomalies. The median gestational age at the time of MR imaging was 21 weeks ranging from 19 to 29 weeks. Two senior pediatric neuroradiologists categorized the anomalies of the GE region in two groups: group one showing cavitation in the GE region and group two showing enlarged GE region. For each fetal case, associated cranial anomalies were also reported. RESULTS: Five out of the eight cases were included in group one and three in group two. Besides the GE region abnormality, all eight cases had additional intracranial anomalies, such as mild partial callosal agenesis, vermian hypoplasia and rotation, cerebellar hypoplasia, ventriculomegaly, enlarged subarachnoid spaces, molar tooth malformation. Ultrasound generally detected most of the associated intracranial anomalies, prompting the MR investigation; on the contrary in none of the cases, GE anomalies had been detected by ultrasound. CONCLUSIONS: Our observation expands the spectrum of human GE anomalies, demonstrating that these may take place also without associated severe micro-lissencephalies.

Prenatal MR imaging features of isolated cerebellar haemorrhagic lesions.

OBJECTIVE: Prenatal features of isolated cerebellar haemorrhagic lesions have not been sufficiently characterised. We aimed to better define their MR imaging characteristics, documenting the location, extension, evolution stage and anatomic sequelae, and to better understand cerebellar haemorrhage pathophysiology. MATERIALS AND METHODS: We screened our foetal MR imaging database (3200 cases) for reports of haemorrhagic lesions affecting only the cerebellum (without any supratentorial bleeding or other clastic lesions), defined as one of the following: T2-weighted hypointense or mixed hypo-/hyperintense signal; rim of T2-weighted hypointense signal covering the surface of volume-reduced parenchyma; T1-weighted hyperintense signal; increased DWI signal. RESULTS: Seventeen cases corresponded to the selection criteria. All lesions occurred before the 26th week of gestation, with prevalent origin from the peripheral-caudal portion of the hemispheres and equal frequency of unilateral/bilateral involvement. The caudal vermis appeared affected in 2/3 of cases, not in all cases confirmed postnatally. Lesions evolved towards malformed cerebellar foliation. The aetiology and pathophysiology were unknown, although in a subset of cases intra- and extracranial venous engorgement seemed to play a key role. CONCLUSIONS: Onset from the peripheral and caudal portion of the hemispheres seems characteristic of prenatal cerebellar haemorrhagic lesions. Elective involvement of the peripheral germinal matrix is hypothesised. KEY POINTS: • The cerebellum can be vulnerable to bleeding during foetal development. • Isolated cerebellar haemorrhages can be seen on prenatal MRI. • In our cohort, isolated foetal cerebellar haemorrhages occurred before the 26th gestational week. • Haemorrhagic lesions happening in utero could look like malformations on post-natal MRI. • Venous engorgement could have a role in causing cerebellar haemorrhagic lesions.