Recurrence and Familial Inheritance of Intronic NIPBL Pathogenic Variant Associated With Mild CdLS.

Splicing pathogenic variants account for a notable fraction of NIPBL alterations underlying Cornelia de Lange syndrome but are likely underrepresented, due to overlooking of non-canonical intronic variants by traditional and contemporary sequencing methods. We describe five subjects, belonging to three families, displaying a mild Cornelia de Lange syndrome phenotype who carry the NIPBL pathogenic variant c.5329-15A>G, affecting the IVS27 branch site, yet reported in a single case. By RNA analysis we evidenced two alternative transcripts: the exon 28 in frame skipped transcript, described in the published case and an out-of-frame transcript retaining 14 nucleotides of IVS27 3'end. Even if both aberrant transcripts are at negligible levels, their presence justifies the CdLS phenotype shared by our patients consisting of borderline-mild cognitive impairment and slight but typical facial dysmorphisms. Transmission of the pathogenic variant from pauci-symptomatic mother to her siblings emphasizes the need of molecular diagnosis extended to deep intronic regions in patients with subtle but recognizable CdLS phenotype.

Functional and pharmacological evaluation of novel GLA variants in Fabry disease identifies six (two de novo) causative mutations and two amenable variants to the chaperone DGJ.

BACKGROUND: Allelic heterogeneity is an important feature of the GLA gene for which almost 900 known genetic variants have been discovered so far. Pathogenetic GLA variants cause alpha-galactosidase A (α-Gal A) enzyme deficiency leading to the X-linked lysosomal storage disorder Fabry disease (FD). Benign GLA intronic and exonic variants (e.g. pseudodeficient p.Asp313Tyr) have also been described. Some GLA missense variants, previously deemed to be pathogenetic (e.g. p.Glu66Gln and p.Arg118Cys), they have been reclassified as benign after re-evaluation by functional and population studies. Hence, the functional role of novel GLA variants should be investigated to assess their clinical relevance. RESULTS: We identified six GLA variants in 4 males and 2 females who exhibited symptoms of FD: c.159C>G p.(Asn53Lys), c.400T>C p.(Tyr134His), c.680G>C (p.Arg227Pro), c.815A>T p.(Asn272Ile), c.907A>T p.(Ile303Phe) and c.1163_1165delTCC (p.Leu388del). We evaluated their impact on the α-Gal A protein by bioinformatic analysis and homology modelling, by analysis of the GLA mRNA, and by site-directed mutagenesis and in vitro expression studies. We also measured their responsiveness to the pharmacological chaperone DGJ. CONCLUSIONS: The six detected GLA variants cause deficient α-Gal A activity and impairment or loss of the protein wild-type structure. We found p.Asn53Lys and p.Ile303Phe variants to be susceptible to DGJ.

Italian multicentre study found infectious and vaccine-preventable diseases in children adopted from Africa and recommends prompt medical screening.

AIM: This study evaluated the prevalence of infectious diseases and immunisation status of children adopted from Africa. METHODS: We studied 762 African children referred to 11 Italian paediatric centres in 2009-2015. Clinical and laboratory data were retrospectively collected and analysed. RESULTS: The median age of the children (60.3% males) was 3 years and 6 months, 52.6% came from Ethiopia and 50.1% had at least one infectious disease. Parasitic infections accounted for the majority of the infectious diseases (409 of 715), and the most common were Giardia lamblia (n = 239), Toxocara canis (n = 65) and skin infections (n = 205), notably Tinea capitis/corporis (n = 134) and Molluscum contagiosum (n = 56) Active tuberculosis (TB) was diagnosed in nine children (1.2%). Latent TB infections were diagnosed in 52 (6.8%) children, and only 23 had concordant positive tuberculin skin tests and Quantiferon Gold In-Tube results. Discordant results were associated with Bacille de Calmette-Guérin vaccinations (odd ratio 6.30 and 95% confidence interval of 1.01-39.20, p = 0.011). Nonprotective antitetanus or antihepatitis B antibody titres were documented in 266 (34.9%) and 396 (51.9%) of the 762 children. CONCLUSION: The prevalence of infectious conditions and not-protective titres for vaccine-preventable diseases observed in our population underlines the need for prompt and complete medical screening of children adopted from Africa.

12 year follow up of enzyme-replacement therapy in two siblings with attenuated mucopolysaccharidosis I: the important role of early treatment.

BACKGROUND: Mucopolysaccharidosis type I is an autosomal recessive disorder caused by deficiency of α-L-iduronidase and characterized by a progressive course with multisystem involvement. Clinically, Mucopolysaccharidosis type I is classified into two forms: severe (Hurler syndrome), which presents in infancy and is characterized by rapid progressive neurological involvement and attenuated (Hurler/Scheie and Scheie syndromes), which presents with slower progression and absent to mild nervous system involvement. The specific treatment for attenuated Mucopolysaccharidosis type I consists of enzyme-replacement therapy with laronidase (human recombinant α-L-iduronidase, Aldurazyme). We present here the clinical and laboratory results in an 12-year-old patient affected by the attenuated form of Mucopolysaccharidosis type I treated by enzyme-replacement therapy from the age of 5 months, compared with his 17 year old affected sister, who started therapy at 5 years of age. CASE PRESENTATION: Clinical evaluation of these siblings shows that initiation of therapy prior of the onset of clinically detectable disease resulted in considerable improvement in outcome in the young sibling. After 12 years of enzyme-replacement therapy, facial appearance, linear growth rate, and liver and spleen volumes were normal; moreover, the degree of joint disease, vertebral, and cardiac valvular involvement were only minimal compared with those of his sister. CONCLUSION: This study demonstrates that early diagnosis and early initiation of enzyme-replacement therapy substantially modify the natural history of the attenuated form of Mucopolysaccharidosis type I.

Optimizing the molecular diagnosis of GALNS: novel methods to define and characterize Morquio-A syndrome-associated mutations.

Morquio A syndrome (MPS IVA) is a systemic lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), encoded by the GALNS gene. We studied 37 MPS IV A patients and defined genotype-phenotype correlations based on clinical data, biochemical assays, molecular analyses, and in silico structural analyses of associated mutations. We found that standard sequencing procedures, albeit identifying 14 novel small GALNS genetic lesions, failed to characterize the second disease-causing mutation in the 16% of the patients' cohort. To address this drawback and uncover potential gross GALNS rearrangements, we developed molecular procedures (CNV [copy-number variation] assays, QF-PCRs [quantitative fluorescent-PCRs]), endorsed by CGH-arrays. Using this approach, we characterized two new large deletions and their corresponding breakpoints. Both deletions were heterozygous and included the first exon of the PIEZO1 gene, which is associated with dehydrated hereditary stomatocitosis, an autosomal-dominant syndrome. In addition, we characterized the new GALNS intronic lesion c.245-11C>G causing m-RNA defects, although identified outside the GT/AG splice pair. We estimated the occurrence of the disease in the Italian population to be approximately 1:300,000 live births and defined a molecular testing algorithm designed to help diagnosing MPS IVA and foreseeing disease progression.

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.

Cognitive-motor profile, clinical characteristics and diagnosis of CHARGE syndrome: an Italian experience.

Since 2005, the Pediatric Clinic of Maternal-Infantile Sciences Institute in Ancona, in collaboration with the Lega del Filo d'Oro in Osimo, has been taking care of 35 patients with clinical and molecular diagnosis of CHARGE syndrome. Our investigation is the largest Italian cohort study of CHARGE patients. CHARGE syndrome is a multiple malformation syndrome involving ocular coloboma, heart defects, choanal atresia, retardation of growth and\or development, genital anomalies and\or urinary and ear abnormalities which leads to visual-auditory disabilities, cognitive impairment and behavioral abnormalities. Our purpose is to expand the knowledge of this syndrome by reviewing this group of affected patients in order to delineate in detail the natural history of the disease, and in particular to define the cognitive and motor profiles using an Italian questionnaire called "Progress Guide". Our main results show that Italian CHARGE patients have more delayed development in their physical abilities or skills with respect to normal patients. In particular, the delay is statistically significant in regard to self-care skills (worse toileting, better washing) and the communication skill (language). On the other hand, the expressive skills are still preserved. When patients are considered according to their age (≤3 years) and (>3 years), the older ones have more delayed development than the younger ones when compared with healthy individuals of the same age.

Severe congenital cutis laxa with cardiovascular manifestations due to homozygous deletions in ALDH18A1.

Autosomal recessive cutis laxa (ARCL) type 2 constitutes a heterogeneous group of diseases mainly characterized by lax and wrinkled skin, skeletal anomalies, and a variable degree of intellectual disability. ALDH18A1-related ARCL is the most severe form within this disease spectrum. Here we report on the clinical and molecular findings of two affected individuals from two unrelated families. The patients presented with typical features of de Barsy syndrome and an overall progeroid appearance. However, the phenotype was highly variable including cardiovascular involvement in the more severe case. Investigation of a skin biopsy of one patient revealed not only the typical alterations of elastic fibers, but also an altered structure of mitochondria in cutaneous fibroblasts. Using conventional sequencing and copy number analysis we identified a frameshift deletion of one nucleotide and a microdeletion affecting the ALDH18A1 gene, respectively, in a homozygous state in both patients. Expression analysis in dermal fibroblasts from the patient carrying the microdeletion showed an almost complete absence of the ALDH18A1 mRNA resulting in an absence of the ALDH18A1 protein. So far, only 13 affected individuals from seven unrelated families suffering from ALDH18A1-related cutis laxa have been described in literature. Our findings provide new insights into the clinical spectrum and show that beside point mutations microdeletions are a possible cause of ALDH18A1-ARCL.

Optimal position of a long-term central venous catheter tip in a pediatric patient with congenital diseases.

Progress in medical and scientific research has increased the chances of survival for young patients with congenital diseases, children who, in the past, would not have had any chance of survival. Nowadays, congenital diseases can be treated with appropriate replacement therapies. These treatments can be difficult to administer in young patients because of the high frequency of administration (sometimes more than a dose per week), the use of intravenous infusion and the long-term or life-term requirement.

Brain and spine MRI features of Hunter disease: frequency, natural evolution and response to therapy.

BACKGROUND: Hunter disease is a rare X-linked mucopolysaccharidosis. Despite frequent neurological involvement, characterizing the severe phenotype, neuroimaging studies are scarce. OBJECTIVES: To determine frequency and severity of neuroradiological mucopolysaccharidosis-related features; to correlate them with clinical phenotype; to evaluate their natural evolution and the impact of intravenous enzymatic replacement therapy (ERT). METHODS: Sixty nine brain MRI examinations of 36 Italian patients (mean-age 10.4 years; age-range 2.2-30.8; severe phenotype in 22 patients) were evaluated. Twenty patients had multiple MRIs (median follow-up 3.1 years, range 1-16.9): among them 15 had MRIs before and after ERT, six had repeated MRIs without being on ERT and five while on ERT. Perivascular, subarachnoid and ventricle space enlargement, white matter abnormality (WMA) burden, pituitary sella/skull/posterior fossa abnormalities, periodontoid thickening, spinal stenosis, dens hypoplasia, myelopathy, vertebral and intervertebral disc abnormalities were graded by means of dedicated scales. RESULTS: Perivascular spaces enlargement (89%), WMAs (97%), subarachnoid space enlargement (83%), IIIrd-ventricle dilatation (100%), pituitary sella abnormalities (80%), cranial hyperostosis (19%), craniosynostosis (19%), enlarged cisterna magna (39%), dens hypoplasia (66%), periodontoid thickening (94%), spinal stenosis (46%), platyspondylia (84%) and disc abnormalities (79%) were frequently detected. WMAs, IIIrd-ventricle dilatation and hyperostosis correlated with the severe phenotype (p < 0.05). Subarachnoid spaces and ventricle enlargement, WMAs and spinal stenosis progressed despite ERT, while other MR features showed minimal or no changes. CONCLUSIONS: The spectrum of brain and spine MRI abnormalities in Hunter disease is extremely wide and requires a thorough evaluation. WMAs, atrophy/communicating hydrocephalus and spinal stenosis progress over time and might represent possible disease severity markers for new treatment efficacy assessment.

Oligosaccharides in 4 different milk groups, Bifidobacteria, and Ruminococcus obeum.

OBJECTIVES: The aim of this study was to identify a link between the total amount of breast milk oligosaccharides and faecal microbiota composition of newborns at the end of the first month of life, with special attention paid to bifidobacteria, and establish the role, if any, of the different oligosaccharides in determining the gut microbiota composition. SUBJECTS AND METHODS: Milk oligosaccharide groups were identified by high-performance anion exchange chromatography analysis. DPCRNA from newborns' faecal samples at 30 days of life was isolated and processed by polymerase chain reaction analyses that allow the identification of 6 species of bifidobacteria (adolescentis, bifidum, breve, catenulatum, longum, infantis) and Ruminococcus spp; denaturing gradient gel electrophoresis analysis was also performed. RESULTS: No substantial differences in bifidobacteria species composition within milk groups 1, 2, and 3 were observed; however, infants fed with group 4 milk show a microbiota characterised by a greater frequency of Bifidobacteria adolescentis and the absence of Bifidobacteria catenulatum. For the first time, a high percentage of the Ruminococcus genus in infants fed with all milk groups was found. CONCLUSIONS: Our data show that milk groups 1, 2, and 3, containing an amount of oligosaccharides ranging within 10 to 15 g/L, share a substantially identical composition of the intestinal microbiota in breast-fed infants, despite quali-quantitative difference in oligosaccharides content. Newborns taking milk with only 5 g/L of oligosaccharides (group 4) harbour a different intestinal microbiota.

Mental retardation, facial anomalies, brachydactyly, cerebral angiomas, femoral nucleus necrosis: a new entity or Hall-Riggs syndrome?

We report on a 4-year-old boy with mental retardation, facial and skeletal anomalies, cerebral angiomas, femoral nucleus necrosis, mild biochemical abnormalities. This complex of features resembles the Hall-Riggs syndrome but could represent a novel syndrome.

Comprehensive clinical and molecular assessment of 32 probands with congenital contractural arachnodactyly: report of 14 novel mutations and review of the literature.

Beals-Hecht syndrome or congenital contractural arachnodactyly (CCA) is a rare, autosomal dominant connective tissue disorder characterized by crumpled ears, arachnodactyly, contractures, and scoliosis. Recent reports also mention aortic root dilatation, a finding previously thought to differentiate the condition from Marfan syndrome (MFS). In many cases, the condition is caused by mutations in the fibrillin 2 gene (FBN2) with 26 mutations reported so far, all located in the middle region of the gene (exons 23-34). We directly sequenced the entire FBN2 gene in 32 probands clinically diagnosed with CCA. In 14 probands, we found 13 new and one previously described FBN2 mutation including a mutation in exon 17, expanding the region in which FBN2 mutations occur in CCA. Review of the literature showed that the phenotype of the FBN2 positive patients was comparable to all previously published FBN2-positive patients. In our FBN2-positive patients, cardiovascular involvement included mitral valve prolapse in two adult patients and aortic root enlargement in three patients. Whereas the dilatation regressed in one proband, it remained marked in a child proband (z-score: 4.09) and his father (z-score: 2.94), warranting echocardiographic follow-up. We confirm paradoxical patellar laxity and report keratoconus, shoulder muscle hypoplasia, and pyeloureteral junction stenosis as new features. In addition, we illustrate large intrafamilial variability. Finally, the FBN2-negative patients in this cohort were clinically indistinguishable from all published FBN2-positive patients harboring a FBN2 mutation, suggesting locus heterogeneity.

Two novel and one known mutation of the TGFBR2 gene in Marfan syndrome not associated with FBN1 gene defects.

TGF-beta-receptor 2 (TGFBR2) gene defects have been recently associated with Marfan syndrome (MFS) with prominent cardio-skeletal phenotype in patients with negative fibrillin-1 (FBN1) gene screening. Four mutations have been identified to date in five unrelated families. We screened TGFBR2 gene by direct automated sequencing in two adult patients diagnosed with MFS according to Ghent criteria, and in one girl clinically suspected as affected on the basis of a major cardiovascular criterion and skeletal involvement, all proven not to carry mutations in the exon-intron boundaries of FBN1 gene. We identified two novel and one known TGFBR2 gene mutations in the three unrelated probands. The D446N was identified in a 4-year-old girl with de novo disease characterized by severe cardiovascular disease and skeletal involvement. The M425V and R460H mutations were identified in two familial, autosomal dominant MFSs, both characterized by major cardio-skeletal signs and absence of major ocular signs. The mutation R460H has been recently reported in a family with thoracic aortic aneurysms and dissection. The three mutations are absent in 192 controls and affect evolutionarily conserved residues of the serine/threonine kinase domain (exon 5). Our data support the recently reported association between TGFBR2 gene and MFS without major ocular signs (MFS2). The number of genotyped cases however is too low to confirm that major ocular signs are characteristically absent in MFS2. Accordingly, all patients proven or suspected to be affected by MFS with negative FBN1 gene screening could benefit from rapid investigation of the TGFBR2 gene.