A novel ANO3 variant in two siblings with different phenotypes.

INTRODUCTION: Dystonia type 24 is due to the mutation of the ANO3 gene. It generally consists of craniocervical dystonia associated with tremor; however, other neurological manifestations may also occur. Scientific literature has been expanding on its phenotype over the past few years. CASE: Here we present two siblings affected by dystonia 24 associated to a novel missense mutation of the ANO3 gene. Description of their phenotype, with regard to motor and non-motor features, may improve the knowledge on DYT 24. Consistent with previous reports, our patients presented with cranio-cervical involvement, and they also exhibited different severity and phenotypes. However non-motor symptoms were present too. CONCLUSION: Dystonia 24 spectrum is continuously expanding. This case suggests that the ANO3 missense mutation should be sought in all cases of dystonia and isolated tremor and that non-motor symptoms are an integral part of dystonic syndromes. It also shows that clinical and treatment features may vary from patient to patient, even if they may present the same mutation.

Clinical variability in DYNC2H1-related skeletal ciliopathies includes Ellis-van Creveld syndrome.

Deleterious variants of DYNC2H1 gene are associated with a wide spectrum of skeletal ciliopathies (SC). We used targeted parallel sequencing to analyze 25 molecularly unsolved families with different SCs. Deleterious DYNC2H1 variants were found in six sporadic patients and two monozygotic (MZ) twins. Clinical diagnoses included short rib-polydactyly type 3 in two cases, and asphyxiating thoracic dystrophy (ATD) in one case. Remarkably, clinical diagnosis fitted with EvC, mixed ATD/EvC and short rib-polydactyly/EvC phenotypes in three sporadic patients and the MZ twins. EvC/EvC-like features always occurred in compound heterozygotes sharing a previously unreported splice site change (c.6140-5A>G) or compound heterozygotes for two missense variants. These results expand the DYNC2H1 mutational repertoire and its clinical spectrum, suggesting that EvC may be occasionally caused by DYNC2H1 variants presumably acting as hypomorphic alleles.

SPRED2 loss-of-function causes a recessive Noonan syndrome-like phenotype.

Upregulated signal flow through RAS and the mitogen-associated protein kinase (MAPK) cascade is the unifying mechanistic theme of the RASopathies, a family of disorders affecting development and growth. Pathogenic variants in more than 20 genes have been causally linked to RASopathies, the majority having a dominant role in promoting enhanced signaling. Here, we report that SPRED2 loss of function is causally linked to a recessive phenotype evocative of Noonan syndrome. Homozygosity for three different variants-c.187C>T (p.Arg63∗), c.299T>C (p.Leu100Pro), and c.1142_1143delTT (p.Leu381Hisfs∗95)-were identified in four subjects from three families. All variants severely affected protein stability, causing accelerated degradation, and variably perturbed SPRED2 functional behavior. When overexpressed in cells, all variants were unable to negatively modulate EGF-promoted RAF1, MEK, and ERK phosphorylation, and time-course experiments in primary fibroblasts (p.Leu100Pro and p.Leu381Hisfs∗95) documented an increased and prolonged activation of the MAPK cascade in response to EGF stimulation. Morpholino-mediated knockdown of spred2a and spred2b in zebrafish induced defects in convergence and extension cell movements indicating upregulated RAS-MAPK signaling, which were rescued by expressing wild-type SPRED2 but not the SPRED2Leu381Hisfs∗95 protein. The clinical phenotype of the four affected individuals included developmental delay, intellectual disability, cardiac defects, short stature, skeletal anomalies, and a typical facial gestalt as major features, without the occurrence of the distinctive skin signs characterizing Legius syndrome. These features, in part, characterize the phenotype of Spred2-/- mice. Our findings identify the second recessive form of Noonan syndrome and document pleiotropic consequences of SPRED2 loss of function in development.

A Novel Triplet-Primed PCR Assay to Detect the Full Range of Trinucleotide CAG Repeats in the Huntingtin Gene (HTT).

The expanded CAG repeat number in HTT gene causes Huntington disease (HD), which is a severe, dominant neurodegenerative illness. The accurate determination of the expanded allele size is crucial to confirm the genetic status in symptomatic and presymptomatic at-risk subjects and avoid genetic polymorphism-related false-negative diagnoses. Precise CAG repeat number determination is critical to discriminate the cutoff between unexpanded and intermediate mutable alleles (IAs, 27-35 CAG) as well as between IAs and pathological, low-penetrance alleles (i.e., 36-39 CAG repeats), and it is also critical to detect large repeat expansions causing pediatric HD variants. We analyzed the HTT-CAG repeat number of 14 DNA reference materials and of a DNA collection of 43 additional samples carrying unexpanded, IAs, low and complete penetrance alleles, including large (>60 repeats) and very large (>100 repeats) expansions using a novel triplet-primed PCR-based assay, the AmplideX PCR/CE HTT Kit. The results demonstrate that the method accurately genotypes both normal and expanded HTT-CAG repeat numbers and reveals previously undisclosed and very large CAG expansions >200 repeats. We also show that this technique can improve genetic test reliability and accuracy by detecting CAG expansions in samples with sequence variations within or adjacent to the repeat tract that cause allele drop-outs or inaccuracies using other PCR methods.

Generation of induced pluripotent stem cell line CSSi008-A (4698) from a patient affected by advanced stage of Dentato-Rubral-Pallidoluysian atrophy (DRPLA).

Dentato-Rubral-pallidoluysian atrophy (DRPLA) is a rare autosomal, dominant, progressive neurodegenerative disease that causes involuntary movements, mental and emotional problems. DRPLA is caused by a mutation in the ATN1 gene that encodes for an abnormal polyglutamine stretch in the atrophin-1 protein. DRPLA is most common in the Japanese population, where it has an estimated incidence of 2 to 7 per million people. This condition has also been seen in families from North America and Europe. We obtained a reprogrammed iPSC line from a Caucasian patient with a juvenile onset of the disease, carrying 64 CAG repeat expansion in the ATN1 gene.

Biological and clinical manifestations of juvenile Huntington's disease: a retrospective analysis.

BACKGROUND: Huntington's disease is a rare, neurodegenerative disease caused by an expanded CAG repeat mutation in the huntingtin gene. Compared with adult-onset Huntington's disease, juvenile Huntington's disease (onset ≤20 years) is even rarer and has not been studied extensively. We aimed to further characterise juvenile Huntington's disease by examining the effect of CAG repeat size on disease presentation, progression, and survival. METHODS: We did a retrospective analysis of patients with juvenile Huntington's disease aged 20 years or younger, according to the length of their CAG repeat and who had disabling psychiatric symptoms (with motor symptoms) or motor symptoms alone, and of patients with adult-onset Huntington's disease manifesting aged 30-60 years with 40 or more CAG repeats, from the REGISTRY and ENROLL-HD platforms and from two institutional databases (Lega Italiana Ricerca Huntington Foundation and the Instituto Neurociencias de Buenos Aires and the Sanatorio de la Trinidad Mitre). Patients with psychiatric but no motor symptoms were excluded. We compared symptoms at onset and longitudinally in patients with juvenile Huntington's disease with highly expanded (HE subgroup) or low expansion (LE subgroup) mutations, grouped by hierarchical clustering analysis. We also compared disease progression (longitudinal change in Unified Huntington's Disease Rating Scale-Total Motor Score) and survival of patients with juvenile and adult-onset Huntington's disease. FINDINGS: We extracted medical records from 580 patients entered into the studies or databases between June 23, 2004, and March 31, 2018, of whom 36 patients met our definition of juvenile Huntington's disease and 197 for adult-onset Huntington's disease. According to caregiver reports, gait disturbance was more often a first presenting symptom in the HE subgroup (eight [80%] of 10 patients) than in the LE subgroup (seven [27%] of 26 patients; p=0·0071), whereas loss of hand dexterity was more common in the LE subgroup (11 [42%] of 26 patients) than in the HE subgroup (0 [0%] of 10 patients; p=0·0160). Compared with the LE subgroup, development delay (0 [0%] in the LE subgroup vs nine [90%] in the HE subgroup; p<0·0001), severe gait impairment (nine [35%] in the LE subgroup vs nine [90%] in the HE subgroup; p=0·0072), and seizures (three [11%] in the LE subgroup vs eight [80%] in the HE subgroup; p<0·0001) prevailed over time in the HE subgroup. Disease progression was more rapid in juvenile Huntington's disease (n=14) than in adult-onset Huntington's disease (n=52; generalised estimating equation model, p=0·0003). Of 121 deceased patients, median survival was shorter in the juvenile Huntington's disease (n=17) cohort than in adult-onset Huntington's disease (n=104) cohort (hazard ratio 2·18 [95% CI 1·08-4·40]; p=0·002). INTERPRETATION: Patients with HE juvenile Huntington's disease differ clinically from patients with LE juvenile Huntington's disease or adult-onset Huntington's disease, suggesting reclassification of this particularly aggressive form of Huntington's disease might be required. FUNDING: Lega Italiana Ricerca Huntington Foundation and IRCCS Ospedale Casa Sollievo della Sofferenza.

Heterozygous missense mutations in NFATC1 are associated with atrioventricular septal defect.

Atrioventricular septal defect (AVSD) may occur as part of a complex disorder (e.g., Down syndrome, heterotaxy), or as isolate cardiac defect. Multiple lines of evidence support a role of calcineurin/NFAT signaling in AVSD, and mutations in CRELD1, a protein functioning as a regulator of calcineurin/NFAT signaling have been reported in a small fraction of affected subjects. In this study, 22 patients with isolated AVSD and 38 with AVSD and heterotaxy were screened for NFATC1 gene mutations. Sequence analysis identified three missense variants in three individuals, including a subject with isolated AVSD [p.(Ala367Val)], an individual with AVSD and heterotaxy [p.(Val210Met)], and a subject with AVSD, heterotaxy, and oculo-auriculo-vertebral spectrum (OAVS) [p.(Ala696Thr)], respectively. The latter was also heterozygous for a missense change in TBX1 [p.(Pro86Leu)]. Targeted resequencing of genes associated with AVSD, heterotaxy, or OAVS excluded additional hits in the three mutation-positive subjects. Functional characterization of NFATC1 mutants documented defective nuclear translocation and decreased transcriptional transactivation activity. When expressed in zebrafish, the three NFATC1 mutants caused cardiac looping defects and altered atrioventricular canal patterning, providing evidence of their functional relevance in vivo. Our findings support a role of defective NFATC1 function in the etiology of isolated and heterotaxy-related AVSD.

Generation of the induced pluripotent stem cell line CSSi006-A (3681) from a patient affected by advanced-stage Juvenile Onset Huntington's Disease.

Juvenile Onset Huntington's Disease (JOHD) is a rare variant of HD withage of onset ≤20 years, accounting for 3-10% of all HD patients. The rarity occurrence of JOHD cases, who severely progress towards mental and physical disability with atypical clinical manifestations compared to classical HD, are responsible of general lack of knowledge about this variant. We obtained a fully reprogrammed iPS cell line from fibroblasts of a JOHD patient carrying 65 CAG repeats and age at onset at age 15. At the biopsy time, the patient showed an advanced stage after 10 years of disease.

Generation of induced pluripotent stem cell line, CSSi004-A (2962), from a patient diagnosed with Huntington's disease at the presymptomatic stage.

Huntington's disease (HD) is an incurable, autosomal dominant, hereditary neurodegenerative disorder that typically manifests itself in midlife. This pathology is linked to the deregulation of multiple, as yet unknown, cellular processes starting before HD onset. A human iPS cell line was generated from skin fibroblasts of a subject at the presymptomatic life stage, carrying a polyglutamine expansion in HTT gene codifying Huntingtin protein. The iPSC line contained the expected CAG expansion, expressed the expected pluripotency markers, displayed in vivo differentiation potential to the three germ layers and had a normal karyotype.

Generation of induced pluripotent stem cell line, CSSi002-A (2851), from a patient with juvenile Huntington Disease.

Huntington Disease (HD) is an autosomal dominant disorder characterized by motor, cognitive and behavioral features caused by a CAG expansion in the HTT gene beyond 35 repeats. The juvenile form (JHD) may begin before the age of 20years and is associated with expanded alleles as long as 60 or more CAG repeats. In this study, induced pluripotent stem cells were generated from skin fibroblasts of a 8-year-old child carrying a large size mutation of 84 CAG repeats in the HTT gene. HD appeared at age 3 with mixed psychiatric (i.e. autistic spectrum disorder) and motor (i.e. dystonia) manifestations.

Paroxysmal Dyskinesias in a PRRT2 Mutation Carrier.

Background: Paroxysmal movement disorders are rare and heterogeneous genetic conditions characterized by the recurrence of transient involuntary movements. Phenomenology Shown: The phenomenology of a paroxysmal kinesigenic dyskinesia in a young professional athlete. Educational Value: Providing basic clinical and genetic elements for the early recognition and diagnosis of a rare movement disorder.

Lack of pathogenic mutations in SOS1 gene in phenytoin-induced gingival overgrowth patients.

OBJECTIVE: Gingival overgrowth is a side effect associated with some distinct classes of drugs, such as anticonvulsants, immunosuppressants, and calcium channel blockers. One of the main drugs associated with gingival overgrowth is the antiepileptic phenytoin, which affects gingival tissues by altering extracellular matrix metabolism. It has been shown that mutation of human SOS1 gene is responsible for a rare hereditary gingival fibromatosis type 1, a benign gingival overgrowth. The aim of the present study is to evaluate the possible contribution of SOS1 mutation to gingival overgrowth-related phenotype. DESIGN: We selected and screened for mutations a group of 24 epileptic patients who experienced significant gingival overgrowth following phenytoin therapy. Mutation scanning was carried out by denaturing high-performance liquid chromatography analysis of the entire coding region of the SOS1 gene. Novel identified variants were analyzed in-silico by using Alamut Visual mutation interpretation software, and comparison with normal control group was done. RESULTS: Mutation scanning of the entire coding sequence of SOS1 gene identified seven intronic variants and one new exonic substitution (c.138G>A). The seven common intronic variants were not considered to be of pathogenic importance. The exonic substitution c.138G>A was found to be absent in 100 ethnically matched normal control chromosomes, but was not expected to have functional significance based on prediction bioinformatics tools. CONCLUSIONS: This study represents the first mutation analysis of the SOS1 gene in phenytoin-induced gingival overgrowth epileptic patients. Present results suggest that obvious pathogenic mutations in the SOS1 gene do not represent a common mechanism underlying phenytoin-induced gingival overgrowth in epileptic patients; other mechanisms are likely to be involved in the pathogenesis of this drug-induced phenotype.

Structural, Functional, and Clinical Characterization of a Novel PTPN11 Mutation Cluster Underlying Noonan Syndrome.

Germline mutations in PTPN11, the gene encoding the Src-homology 2 (SH2) domain-containing protein tyrosine phosphatase (SHP2), cause Noonan syndrome (NS), a relatively common, clinically variable, multisystem disorder. Here, we report on the identification of five different PTPN11 missense changes affecting residues Leu261 , Leu262 , and Arg265 in 16 unrelated individuals with clinical diagnosis of NS or with features suggestive for this disorder, specifying a novel disease-causing mutation cluster. Expression of the mutant proteins in HEK293T cells documented their activating role on MAPK signaling. Structural data predicted a gain-of-function role of substitutions at residues Leu262 and Arg265 exerted by disruption of the N-SH2/PTP autoinhibitory interaction. Molecular dynamics simulations suggested a more complex behavior for changes affecting Leu261 , with possible impact on SHP2's catalytic activity/selectivity and proper interaction of the PTP domain with the regulatory SH2 domains. Consistent with that, biochemical data indicated that substitutions at codons 262 and 265 increased the catalytic activity of the phosphatase, while those affecting codon 261 were only moderately activating but impacted substrate specificity. Remarkably, these mutations underlie a relatively mild form of NS characterized by low prevalence of cardiac defects, short stature, and cognitive and behavioral issues, as well as less evident typical facial features.

Myoclonic status and central fever in Angelman syndrome due to paternal uniparental disomy.

Myoclonic status in nonprogressive encephalopathy (MSNE) is an early-onset, drug-resistant epileptic syndrome characterized by occurrence of continuous diffuse epileptiform abnormalities, associated with positive and/or negative phenomena and accompanied by transient and recurring motor, cognitive, and behavioral impairment. MSNE has been reported in Angelman syndrome (AS) secondary to 15q11-13 deletions or UBE3A mutations but not to paternal uniparental disomy (UPD). We describe the case of a male patient with AS caused by UPD who developed a myoclonic status (MS) associated with long-lasting fever of central origin, both promptly regressed with introduction of levetiracetam. Only three descriptions of thermal dysregulation in AS exist, and none of the previously reported cases were associated with MS or with UPD. Association of MS and central fever expands the spectrum of epileptic and non-epileptic features in UPD-related AS and provides a further evidence of hypothalamus involvement in the pathogenesis of this neurodevelopmental disorder.

Molecular Diversity and Associated Phenotypic Spectrum of Germline CBL Mutations.

Noonan syndrome (NS) is a relatively common developmental disorder with a pleomorphic phenotype. Mutations causing NS alter genes encoding proteins involved in the RAS-MAPK pathway. We and others identified Casitas B-lineage lymphoma proto-oncogene (CBL), which encodes an E3-ubiquitin ligase acting as a tumor suppressor in myeloid malignancies, as a disease gene underlying a condition clinically related to NS. Here, we further explored the spectrum of germline CBL mutations and their associated phenotype. CBL mutation scanning performed on 349 affected subjects with features overlapping NS and no mutation in NS genes allowed the identification of five different variants with pathological significance. Among them, two splice-site changes, one in-frame deletion, and one missense mutation affected the RING domain and/or the adjacent linker region, overlapping cancer-associated defects. A novel nonsense mutation generating a v-Cbl-like protein able to enhance signal flow through RAS was also identified. Genotype-phenotype correlation analysis performed on available records indicated that germline CBL mutations cause a variable phenotype characterized by a relatively high frequency of neurological features, predisposition to juvenile myelomonocytic leukemia, and low prevalence of cardiac defects, reduced growth, and cryptorchidism. Finally, we excluded a major contribution of two additional members of the CBL family, CBLB and CBLC, to NS and related disorders.

p.Arg1809Cys substitution in neurofibromin is associated with a distinctive NF1 phenotype without neurofibromas.

Analysis of 786 NF1 mutation-positive subjects with clinical diagnosis of neurofibromatosis type 1 (NF1) allowed to identify the heterozygous c.5425C>T missense variant (p.Arg1809Cys) in six (0.7%) unrelated probands (three familial and three sporadic cases), all exhibiting a mild form of disease. Detailed clinical characterization of these subjects and other eight affected relatives showed that all individuals had multiple cafè-au-lait spots, frequently associated with skinfold freckling, but absence of discrete cutaneous or plexiform neurofibromas, Lisch nodules, typical NF1 osseous lesions or symptomatic optic gliomas. Facial features in half of the individuals were suggestive of Noonan syndrome. Our finding and revision of the literature consistently indicate that the c.5425C>T change is associated with a distinctive, mild form of NF1, providing new data with direct impact on genetic counseling and patient management.

Alpha-synuclein gene duplication: marked intrafamilial variability in two novel pedigrees.

BACKGROUND: Multiplications of the SNCA gene that encodes alpha-synuclein are a rare cause of autosomal dominant Parkinson's disease (PD). METHODS: Here, we describe 2 novel families in which there is autosomal dominant PD associated with SNCA duplication, and we compare the clinical features of all known patients carrying 3 or 4 SNCA copies. RESULTS: Affected members in family A presented with early onset PD that was variably associated with nonmotor features, such as dysautonomia, cognitive deficits, and psychiatric disturbances. In family B, the clinical presentation ranged from early onset PD-dementia with psychiatric disturbances to late onset PD with mild cognitive impairment. CONCLUSIONS: The presence of 4 SNCA copies is associated with a rich phenotype, characterized by earlier onset of motor and nonmotor features compared with patients who bear 3 SNCA copies. The clinical spectrum associated with SNCA duplications is wide, even within a single family, suggesting a role for as yet unidentified genetic or environmental modifiers.

Congenital heart defects in recurrent reciprocal 1q21.1 deletion and duplication syndromes: rare association with pulmonary valve stenosis.

Microdeletion 1q21.1 (del 1q21.1) and the reciprocal microduplication 1q21.1 (dup 1q21.1) are newly recognized genomic disorders, characterized by developmental delay, dysmorphic features and congenital malformations. Congenital heart defect (CHD) is a major feature of del 1q21.1, and has been occasionally reported in dup 1q21.1. We report here a family segregating del 1q21.1 in 3 members. Two of the affected family members had CHD, including the proband with syndromic atrial septal defect, pulmonary valve stenosis (PVS), and muscular ventricular septal defects, and the maternal uncle with non-syndromic PVS. This finding prompted investigation of the role of recurrent rearrangements of chromosome 1q21.1 in the pathogenesis of PVS. We gathered 38 patients with PVS (11 syndromic and 27 non-syndromic), and searched for genomic rearrangements of 1q21.1. A dup 1q21.1 was detected in a single sporadic non-syndromic patient. Review of the CHDs in published del 1q21.1 and dup 1q21.1 subjects showed a great heterogeneity in anatomic types. In conclusion, the present family illustrates recurrent CHD in del 1q21.1, expressing either as syndromic in one family member or as non-syndromic in the another one. The spectrum of CHDs associated with del 1q21.1 and dup 1q21.1 can occasionally include PVS.

"Double trouble" or an amplification of the triploidy phenotype?

Triploidy occurs in about 1 to 3% of clinically recognizable pregnancies and is typically associated with growth restriction, craniofacial dysmorphisms and congenital anomalies. We report the case of a female fetus with prenatal diagnosis of complete triploidy, polysplenia, bilateral cleft-palate, horseshoe-kidneys and bilateral club-feet. Whereas bilateral cleft-palate, horseshoe-kidneys and bilateral club feet are known to be part of the triploidy-associated malformation spectrum, polysplenia, which usually occurs as part of the heterotaxia spectrum, has never been associated with triploidy. An amplification of the triploidy phenotype or a "double trouble".

Novel and recurrent EVC and EVC2 mutations in Ellis-van Creveld syndrome and Weyers acrofacial dyostosis.

Ellis van Creveld syndrome and Weyers acrofacial dysostosis are allelic disorders caused by mutations in EVC or EVC2 genes. We illustrate the results of direct analysis of whole EVC and EVC2 genes' coding regions in 32 unrelated families with clinical diagnosis of Ellis van Creveld syndrome and in 2 families with Weyers acrofacial dysostosis. We identified mutations in 27/32 (84%) cases with Ellis van Creveld syndrome and 2/2 cases with Weyers acrofacial dysostosis. Of the Ellis van Creveld syndrome cases, 20/27 (74%) had a mutation in EVC and 7/27 (26%) in EVC2 genes. The two subjects with Weyers acrofacial dysostosis had a heterozygous mutation in the last exon of EVC2. In total, we detected 25 independent EVC and 11 independent EVC2 mutations. Nineteen EVC mutations (19/25, 76%) and 4 EVC2 mutations (4/11, 36%) were novel. Also one EVC2 gene mutation found in Weyers acrofacial dysostosis was novel. In 5 unrelated cases with a clinical diagnosis of Ellis van Creveld syndrome, we did not find any mutation in either EVC or EVC2 genes. Current findings expand the Ellis van Creveld syndrome and Weyers acrofacial dysostosis mutation spectra, and provide further evidence that the last exon of EVC2 gene is a hot spot for Weyers acrofacial dysostosis mutations. Accordingly, EVC2 exon 22 should be analyzed with priority by mutation screening in individuals with a suspected diagnosis of Weyers acrofacial dysostosis.