Characterization of a novel HDAC2 pathogenetic variant: a missing puzzle piece for chromatinopathies.

Histone deacetylases (HDACs) are enzymes pivotal for histone modification (i.e. acetylation marks removal), chromatin accessibility and gene expression regulation. Class I HDACs (including HDAC1, 2, 3, 8) are ubiquitously expressed and they often participate in multi-molecular protein complexes. To date, three neurodevelopmental disorders caused by mutations in genes encoding for HDACs (HDAC4, HDAC6 and HDAC8) and thus belonging to the group of chromatinopathies, have been described. We performed whole exome sequencing (WES) for a patient (#249) clinically diagnosed with the chromatinopathy Rubinstein-Taybi syndrome (RSTS) but negative for mutations in RSTS genes, identifying a de novo frameshift variant in HDAC2 gene. We then investigated its molecular effects in lymphoblastoid cell lines (LCLs) derived from the patient compared to LCLs from healthy donors (HD). As the variant was predicted to be likely pathogenetic and to affect the sequence of nuclear localization signal, we performed immunocytochemistry and lysates fractionation, observing a nuclear mis-localization of HDAC2 compared to HD LCLs. In addition, HDAC2 total protein abundance resulted altered in patient, and we found that newly identified variant in HDAC2 affects also acetylation levels, with significant difference in acetylation pattern among patient #249, HD and RSTS cells and in expression of a known molecular target. Remarkably, RNA-seq performed on #249, HD and RSTS cells shows differentially expressed genes (DEGs) common to #249 and RSTS. Interestingly, our reported patient was clinically diagnosed with RSTS, a chromatinopathy which known causative genes encode for enzymes antagonizing HDACs. These results support the role of HDAC2 as causative gene for chromatinopathies, strengthening the genotype-phenotype correlations in this relevant group of disorders.

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.

Double somatic mosaicism in Cornelia de Lange syndrome.

Post-zygotic mosaicism is a well-known biological phenomenon characterized by the presence of genetically distinct lineages of cells in the same individual due to post-zygotic de novo mutational events. It has been identified in about 13% of Cornelia de Lange (CdLS) syndrome patients with a molecular diagnosis, an unusual high frequency. Here, we report the case of a patient affected by classic CdLS harboring post-zygotic mosaicism for two different likely pathogenic variants at the same nucleotide position in NIPBL. Double somatic mosaicism has never been reported in CdLS and only rarely recognized in human diseases. Possible pathogenetic mechanisms are discussed.

De novo variants in CNOT9 cause a neurodevelopmental disorder with or without epilepsy.

PURPOSE: The study aimed to clinically and molecularly characterize the neurodevelopmental disorder associated with heterozygous de novo variants in CNOT9. METHODS: Individuals were clinically examined. Variants were identified using exome or genome sequencing. These variants were evaluated using in silico predictions, and their functional relevance was further assessed by molecular models and research in the literature. The variants have been classified according to the criteria of the American College of Medical Genetics. RESULTS: We report on 7 individuals carrying de novo missense variants in CNOT9, p.(Arg46Gly), p.(Pro131Leu), and p.(Arg227His), and, recurrent in 4 unrelated individuals, p.(Arg292Trp). All affected persons have developmental delay/intellectual disability, with 5 of them showing seizures. Other symptoms include muscular hypotonia, facial dysmorphism, and behavioral abnormalities. Molecular modeling predicted that the variants are damaging and would lead to reduced protein stability or impaired recognition of interaction partners. Functional analyses in previous studies showed a pathogenic effect of p.(Pro131Leu) and p.(Arg227His). CONCLUSION: We propose CNOT9 as a novel gene for neurodevelopmental disorder and epilepsy.

Rock around DYRK1A: Ethnic diversity, clinical challenges.

DYRK1A-related intellectual disability is a recently described syndrome characterized by microcephaly, global developmental delay, impaired speech development, and distinctive facial features, which let to define it as a recognizable syndrome. Here we report four new patients of different ethnicity, broadening the clinical phenotype of the condition and highlighting how ethnic influences in the facial appearance could make it less recognizable.

A novel nonsense and inactivating variant of ST3GAL3 in two infant siblings suffering severe epilepsy and expressing circulating CA19.9.

Three missense variants of ST3GAL3 are known to be responsible for a congenital disorder of glycosylation determining a neurodevelopmental disorder (intellectual disability/epileptic encephalopathy). Here we report a novel nonsense variant, p.Y220*, in two dichorionic infant twins presenting a picture of epileptic encephalopathy with impaired neuromotor development. Upon expression in HEK-293T cells, the variant appears totally devoid of enzymatic activity in vitro, apparently accumulated with respect to the wild-type or the missense variants, as detected by western blot, and in large part properly localized in the Golgi apparatus, as assessed by confocal microscopy. Both patients were found to efficiently express the CA19.9 antigen in the serum despite the total loss of ST3GAL3 activity, which thus appears replaceable from other ST3GALs in the synthesis of the sialyl-Lewis a epitope. Kinetic studies of ST3GAL3 revealed a strong preference for lactotetraosylceramide as acceptor and gangliotetraosylceramide was also efficiently utilized in vitro. Moreover, the p.A13D missense variant, the one maintaining residual sialyltransferase activity, was found to have much lower affinity for all suitable substrates than the wild-type enzyme with an overall catalytic efficiency almost negligible. Altogether the present data suggest that the apparent redundancy of ST3GALs deduced from knock-out mouse models only partially exists in humans. In fact, our patients lacking ST3GAL3 activity synthesize the CA19.9 epitope sialyl-Lewis a, but not all glycans necessary for fine brain functions, where the role of minor gangliosides deserves further attention.

A HS6ST2 gene variant associated with X-linked intellectual disability and severe myopia in two male twins.

X-linked intellectual disability (XLID) refers to a clinically and genetically heterogeneous neurodevelopmental disorder, in which males are more heavily affected than females. Among the syndromic forms of XLID, identified by additional clinical signs as part of the disease spectrum, the association between XLID and severe myopia has been poorly characterized. We used whole exome sequencing (WES) to study two Italian male twins presenting impaired intellectual function and adaptive behavior, in association with severe myopia and mild facial dysmorphisms. WES analysis detected the novel, maternally inherited, mutation c.916G > C (G306R) in the X-linked heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) gene. HS6ST2 transfers sulfate from adenosine 3'-phosphate, 5'-phosphosulfate to the sixth position of the N-sulphoglucosamine residue in heparan sulfate (HS) proteoglycans. Low HS sulfation levels are associated with defective optic disc and stalk morphogenesis during mammalian visual system development. The c.916G>C variant affects the HS6ST2 substrate binding site, and its effect was considered "deleterious" by in-silico tools. An in-vitro enzymatic assay showed that the HS6ST2 mutant isoform had significantly reduced sulphotransferase activity. Taken together, the results suggest that mutant HS6ST2 is possibly involved in the development of myopia and cognitive impairment, characteristics of the probands reported here.

Atypical presentation of pediatric BRAF RASopathy with acute encephalopathy.

We report a 9-year-old girl with hypotonia, severe motor delay, absent speech, and facial dysmorphism who developed acute encephalopathy with severe neurological outcome. Trio-based whole exome sequencing (WES) analysis detected a de novo heterozygous mutation in the BRAF gene leading to the diagnosis of an atypical presentation of cardiofaciocutaneous (CFC) syndrome. This is the second case of CFC syndrome complicated with acute encephalopathy reported in the literature and supports the hypothesis that acute encephalopathy might be one of the complications of the syndrome due to an intrinsic susceptibility to this acute event. The report furthermore highlights the role of WES in providing a fast diagnosis in patients in critical conditions with atypical presentation of rare genetic syndromes.

Sequence variants identification at the KCNQ1OT1:TSS differentially Methylated region in isolated omphalocele cases.

BACKGROUND: Omphalocele is a congenital midline ventral body wall defect that can exist as isolated malformation or as part of a syndrome. It can be considered one of the major and most frequent clinical manifestation of Beckwith-Wiedemann Syndrome (BWS) in case of loss of methylation at KCNQ1OT1: Transcription Star Site-Differentially Methylated Region (TSS-DMR) or in presence of CDKN1C mutations. The isolated form of the omphalocele accounts approximately for about the 14% of the total cases and its molecular etiology has never been fully elucidated. METHODS: Given the tight relationship with BWS, we hypothesized that the isolated form of the omphalocele could belong to the heterogeneous spectrum of the BWS associated features, representing an endophenotype with a clear genetic connection. We therefore investigated genetic and epigenetic changes affecting BWS imprinted locus at 11p15.5 imprinted region, focusing in particular on the KCNQ1OT1:TSS DMR. RESULTS: We studied 21 cases of isolated omphalocele detected during pregnancy or at birth and identified the following rare maternally inherited variants: i) the non-coding variant G > A at nucleotide 687 (NR_002728.3) at KCNQ1OT1:TSS-DMR, which alters the methylation pattern of the imprinted allele, in one patient; ii) the deletion c.624-629delGGCCCC at exon 1 of CDKN1C, with unknown clinical significance, in two unrelated cases. CONCLUSIONS: Taken together, these findings suggest that KCNQ1OT1:TSS-DMR could be a susceptibility locus for the isolated omphalocele.

Is cutis verticis Gyrata-Intellectual Disability syndrome an underdiagnosed condition? A case report and review of 62 cases.

Cutis Verticis Gyrata-Intellectual Disability (CVG-ID) syndrome is a rare neurocutaneous syndrome characterized by intellectual disability and scalp folds and furrows that are typically absent at birth and are first noticed after puberty. First reported in 1893, the syndrome was mainly identified in subjects living in psychiatric institutions, where it was found to have a prevalence of up to 11.4%. Most patients were reported in the literature during the first half of the 20th century. CVG-ID is now a less reported and possibly under-recognized syndrome. Here, we report a patient with CVG-ID that was diagnosed using the novel approach of magnetic resonance imaging and we conduct a systematic review of all patients reported in the last 60 years, discussing the core clinical features of this syndrome. © 2016 Wiley Periodicals, Inc.

A long way to syndromic short stature.

BACKGROUND: Silver-Russell Syndrome (SRS, MIM #180860) is a clinically and genetically heterogeneous disorder characterized by intrauterine and postnatal growth retardation; SRS is also accompanied by dysmorphic features such as triangular facial appearance, broad forehead, body asymmetry and significant feeding difficulties. The incidence is unknown but estimated at 1:30,000-100,000 live births. The diagnosis of SRS is guided by specific criteria described in the Netchine-Harbison clinical scoring system (NH-CSS). CASE PRESENTATION: Hereby we describe four patients with syndromic short stature in whom, despite fitting the criteria for SRS genetic analysis (and one on them even meeting the clinical criteria for SRS), molecular analysis actually diagnosed a different syndrome. Some additional features such as hypotonia, microcephaly, developmental delay and/or intellectual disability, and family history of growth failure, were actually discordant with SRS in our cohort. CONCLUSIONS: The clinical resemblance of other short stature syndromes with SRS poses a risk of diagnostic failure, in particular when clinical SRS only criteria are met, allowing SRS diagnosis in the absence of a positive result of a genetic test. The presence of additional features atypical for SRS diagnosis becomes a red flag for a more extensive and thorough analysis. The signs relevant to the differential diagnosis should be valued as much as possible since a correct diagnosis of these patients is the only way to provide the appropriate care pathway, a thorough genetic counselling, prognosis definition, follow up setting, appropriate monitoring and care of possible medical problems.

Comparison of first-tier whole-exome sequencing with a multi-step traditional approach for diagnosing paediatric outpatients: An Italian prospective study.

BACKGROUND: The recent guidelines suggest the use of genome-wide analyses, such as whole exome sequencing (WES), at the beginning of the diagnostic approach for cases with suspected genetic conditions. However, in many realities it still provides for the execution of a multi-step pathway, thus requiring several genetic tests to end the so-called 'diagnostic odyssey'. METHODS: We reported the results of GENE Project (Genomic analysis Evaluation NEtwork): a multicentre prospective cohort study on 125 paediatric outpatients with a suspected genetic disease in which we performed first-tier trio-WES, including exome-based copy number variation analysis, in parallel to a 'traditional approach' of two/three sequential genetic tests. RESULTS: First-tier trio-WES detected a conclusive diagnosis in 41.6% of patients, way above what was found with routine genetic testing (25%), with a time-to-result of about 50 days. Notably, the study showed that 44% of WES-reached diagnoses would be missed with the traditional approach. The diagnostic rate (DR) of the two approaches varied in relation to the phenotypic class of referral and to the proportion of cases with a defined diagnostic suspect, proving the major difference for neurodevelopmental disorders. Moreover, trio-WES analysis detected variants in candidate genes of unknown significance (EPHA4, DTNA, SYNCRIP, NCOR1, TFDP1, SPRED3, EDA2R, PHF12, PPP1R12A, WDR91, CDC42BPG, CSNK1D, EIF3H, TMEM63B, RIPPLY3) in 19.4% of undiagnosed cases. CONCLUSION: Our findings represent real-practice evidence of how first-tier genome-wide sequencing tests significantly improve the DR for paediatric outpatients with a suspected underlying genetic aetiology, thereby allowing a time-saving setting of the correct management, follow-up and family planning.

Inferring disease course from differential exon usage in the wide titinopathy spectrum.

OBJECTIVE: Biallelic titin truncating variants (TTNtv) have been associated with a wide phenotypic spectrum, ranging from complex prenatal muscle diseases with dysmorphic features to adult-onset limb-girdle muscular dystrophy, with or without cardiac involvement. Given the size and complexity of TTN, reaching an unequivocal molecular diagnosis and precise disease prognosis remains challenging. METHODS: In this case series, 12 unpublished cases and one already published case with biallelic TTNtv were collected from multiple international medical centers between November 2022 and September 2023. TTN mutations were detected through exome or genome sequencing. Information about familial and personal clinical history was collected in a standardized form. RNA-sequencing and analysis of TTN exon usage were performed on an internal sample cohort including postnatal skeletal muscles, fetal skeletal muscles, postnatal heart muscles, and fetal heart muscles. In addition, publicly available RNA-sequencing data was retrieved from ENCODE. RESULTS: We generated new RNA-seq data on TTN exons and identified genotype-phenotype correlations with prognostic implications for each titinopathy patient (whether worsening or improving in prenatal and postnatal life) using percentage spliced in (PSI) data for the involved exons. Interestingly, thanks to exon usage, we were also able to rule out a titinopathy diagnosis in one prenatal case. INTERPRETATION: This study demonstrates that exon usage provides valuable insights for a more exhaustive clinical interpretation of TTNtv; additionally, it may serve as a model for implementing personalized medicine in many other genetic diseases, since most genes undergo alternative splicing.

X-linked reticulate pigmentary disorder with systemic manifestations: a new family and review of the literature.

X-linked reticulate pigmentation disorder with systemic manifestations (XLPDR) is an extremely rare genodermatosis with recessive X-linked inheritance but unknown molecular basis. In males, cutaneous involvement is characterized by reticulate hyperpigmentation of the skin that is associated with a typical facies and severe systemic involvement. In the carrier females, manifestations are apparently limited to the skin with patchy linear hyperpigmentation following the lines of Blaschko that are similar to stage III incontinentia pigmenti. Thus far, only five families affected by this disorder have been described. We report on a new family with clinical features of XLPDR and compare it with those reported in the literature.

Recurring and generalized visceroptosis in Ehlers-Danlos syndrome hypermobility type.

Visceroptosis is described in several heritable connective tissue disorders, including the hypermobility type of Ehlers-Danlos syndrome (hEDS), a.k.a. joint hypermobility syndrome (JHS). Clinical features of hEDS comprise joint hypermobility, mild skin hyperextensibility, joint instability complications, chronic joint/limb pain, and positive family history. Uterine and rectal prolapse has been reported in nulliparous women. We report on a family with two patients with hEDS. The proposita, a 38-year-old woman, present bilateral kidney prolapse requiring three nephropexies, gastric ptosis treated with gastropexy and Billroth I gastrectomy, and liver prolapse treated with a non-codified hepatopexy procedure. Radiological evaluation also showed ovarian and heart prolapse. To our knowledge this is the first case of multiple visceral ptoses in hEDS. Visceral prolapse may lead to severe morbidity, affecting quality of life and a high rate of relapses after surgical procedures. Further investigations are needed to understand the molecular basis of the disease and retrospective studies on surgical outcomes, presentation of case series can be effective in order to offer a better treatment and prevention for hEDS patients.

Nevoid follicular mucinosis: a new type of hair follicle nevus.

Follicular mucinosis represents a term for a histopathologic reaction pattern in follicular epithelium. It is a characteristic of alopecia mucinosa. However, it may also occur in a variety of unrelated conditions. Epidermal nevi are considered to be hamartomatous disorders and they can show a predominant component of non-organoid (keratinocytes) and/or organoid nevi. All the cases of epidermal nevi described with mucin deposits until now are reported as mucinous nevus or mucinous eccrine nevus; in the first type of disorder, diffuse mucin deposition is only seen in the papillary dermis, and in the second type, the mucin is found around the proliferation of eccrine structures. We believe this is the first reported case of epidermal nevus along Blaschko's lines exhibiting typical microscopic findings of mucinosis exclusively distributed inside the follicular epithelia.

SBIDDS Syndrome: A New Spoke of the Epigenetic Machinery Wheel.

Introduction: Mendelian disorders of the epigenetic machinery are a growing group of disorders exhibiting several overlapping clinical features that are probably due to common abnormalities at the epigenomic level, which lead to downstream convergence at the transcriptomic level. Case presentation: Here, we report a new case of short stature, brachydactyly, intellectual developmental disability, and seizures (SBIDDS) syndrome with a severe ocular phenotype and hypogonadism. Conclusion: Similarities and connections with other mendelian disorders of the epigenetic machinery are highlighted, confirming SBIDDS' enrolment as a new spoke of the epigenetic machinery wheel.

Atypical, Composite, or Blended Phenotypes: How Different Molecular Mechanisms Could Associate in Double-Diagnosed Patients.

In the last few years, trio-Whole Exome Sequencing (WES) analysis has revolutionized the diagnostic process for patients with rare genetic syndromes, demonstrating its potential even in non-specific clinical pictures and in atypical presentations of known diseases. Multiple disorders in a single patient have been estimated to occur in approximately 2-7.5% of diagnosed cases, with higher frequency in consanguineous families. Here, we report the clinical and molecular characterisation of eight illustrative patients for whom trio-WES allowed for identifing more than one genetic condition. Double homozygosity represented the causal mechanism in only half of them, whereas the other half showed peculiar multilocus combinations. The paper takes into consideration difficulties and learned lessons from our experience and therefore supports the powerful role of wide analyses for ascertaining multiple genetic diseases in complex patients, especially when a clinical suspicion could account for the majority of clinical signs. It finally makes clear how a patient's "deep phenotyping" might not be sufficient to suggest the presence of multiple genetic diagnoses but remains essential to validate an unexpected multilocus result from genetic tests.

Not Only Diagnostic Yield: Whole-Exome Sequencing in Infantile Cardiomyopathies Impacts on Clinical and Family Management.

Whole-exome sequencing (WES) is a powerful and comprehensive tool for the genetic diagnosis of rare diseases, but few reports describe its timely application and clinical impact on infantile cardiomyopathies (CM). We conducted a retrospective analysis of patients with infantile CMs who had trio (proband and parents)-WES to determine whether results contributed to clinical management in urgent and non-urgent settings. Twenty-nine out of 42 enrolled patients (69.0%) received a definitive molecular diagnosis. The mean time-to-diagnosis was 9.7 days in urgent settings, and 17 out of 24 patients (70.8%) obtained an etiological classification. In non-urgent settings, the mean time-to-diagnosis was 225 days, and 12 out of 18 patients (66.7%) had a molecular diagnosis. In 37 out of 42 patients (88.1%), the genetic findings contributed to clinical management, including heart transplantation, palliative care, or medical treatment, independent of the patient's critical condition. All 29 patients and families with a definitive diagnosis received specific counseling about recurrence risk, and in seven (24.1%) cases, the result facilitated diagnosis in parents or siblings. In conclusion, genetic diagnosis significantly contributes to patients' clinical and family management, and trio-WES should be performed promptly to be an essential part of care in infantile cardiomyopathy, maximizing its clinical utility.