A novel COLEC10 mutation in a child with 3MC syndrome.

3MC syndrome is an autosomal recessive disorder encompassing four rare disorders previously known as the Malpuech, Michels, Mingarelli and Carnevale syndromes. They are characterized by a variable spectrum of abnormalities, including facial dysmorphisms, along with genital, limb and vesico-renal anomalies. The syndrome was originally attributed to mutations in MASP1 and COLEC11, which code for proteins involved in the lectin complement pathway. More recently, mutations in COLEC10, a third gene coding for collectin CL-L1, were identified in a limited number of patients with 3MC syndrome. Here we describe a 4-years-old patient with typical 3MC phenotypic characteristics, including blepharophimosis, telecanthus, high arched eyebrows, fifth finger clinodactyly, sacral dimple and horseshoe kidney. Initial genetic analysis was based on clinical exome sequencing, where only MASP1 and COLEC11 genes are present, without evidence of pathogenic variants. Sanger sequencing of COLEC10 identified the homozygous frameshift variant c.807_810delCTGT; p.Cys270Serfs*33, which results in the loss of the natural stop codon. The resulting protein is 24 amino acids longer and lacks a conserved cysteine residue (Cys270), which could affect protein folding. Segregation studies confirmed that both parents were carriers for the variant: interestingly they originate from the same area of Apulia in southern Italy. Plasma levels of CL-L1 in the patient and her parents were within normal range, suggesting that this variant does not modify transcription or secretion. However, the variant affects the chemo-attractive feature of CL-L1, as HeLa cells migrate significantly less in response to the mutant protein compared to the wild-type one.

Intrahepatic Administration of Human Liver Stem Cells in Infants with Inherited Neonatal-Onset Hyperammonemia: A Phase I Study.

Previous studies have shown that human liver stem-like cells (HLSCs) may undergo differentiation in vitro into urea producing hepatocytes and in vivo may sustain liver function in models of experimentally induced acute liver injury. The aim of this study was to assess the safety of HLSCs intrahepatic administration in inherited neonatal-onset hyperammonemia. The study was approved by the Agenzia Italiana del Farmaco on favorable opinion of the Italian Institute of Health as an open-label, prospective, uncontrolled, monocentric Phase I study (HLSC 01-11, EudraCT-No. 2012-002120-33). Three patients affected by argininosuccinic aciduria (patient 1) and methylmalonic acidemia (patients 2 and 3) and included in the liver transplantation list were enrolled. In all patients, HLSCs were administered by percutaneous intrahepatic injections (once a week for two consecutive weeks) within the first months of life. The first patient received 125,000 HLSCs x gram of liver/dose while the other two patients received twice this dose. No immunosuppression was administered since HLSCs possess immunomodulatory activities. None of the patients experienced infections, hyperammonemia decompensation, or other adverse events during the whole observation period. No donor specific antibodies (DSA) against HLSCs were detected. Patients were metabolic stable despite an increase (~30%) in protein intake. Two patients underwent liver transplantation after 19 and 11 months respectively, and after explantation, the native livers showed no histological alterations. In conclusion, percutaneous intrahepatic administration of HLSCs was safe in newborn with inherited neonatal-onset hyperammonemia. These data pave the way for Phase II studies in selected inherited and acquired liver disorders.

Metabolic progression to clinical phenotype in classic Fabry disease.

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder due to α-galactosidase A (α-Gal A) deficiency. Clinical onset of Fabry disease is preceded by significant storage of globotriaosylceramide (Gb3) and related glycosphingolipids, but the extent of the metabolic progression before symptoms is unknown. Using a newly recognized effector and marker of Fabry disease, globotriaosylsphingosine (LysoGb3), we aimed to provide a metabolic picture of classic Fabry disease from the neonatal period to childhood. METHODS: LysoGb3 was assessed at different times in two brothers with classic Fabry disease (genotype c. 370-2 A > G). The firstborn was diagnosed after clinical onset at 11 years of age, whereas the second-born was diagnosed in the neonatal period. LysoGb3 was measured in dried blood spots by high-sensitive electrospray ionization liquid chromatography tandem mass spectrometry. RESULTS: Blood LysoGb3 concentrations were consistent with patients' age and clinical picture, with lower levels in the asymptomatic neonate (19.1 ng/ml) and higher levels in the symptomatic child (94.3 ng/ml). In the second-born, LysoGb3 doubled during the first 5 months of life (37.4 ng/ml), reaching ~40% concentration observed in the symptomatic period. The neonatal LysoGb3 concentration in classic Fabry disease exceeded that observed in normal subjects by over 15 times. CONCLUSIONS: A substantial increase of LysoGb3 was documented during the first months of life in classic Fabry disease, suggesting an early plateau during the pre-symptomatic period. Such a progressive metabolic trend during the pre-symptomatic period implies the potential definition of a metabolic threshold useful for a preventive therapeutic approach of classic Fabry disease. Additionally, the consistent increase of LysoGb3 in the neonatal period in classic Fabry disease suggests LysoGb3 as a useful marker for improving the specificity of newborn screening for Fabry disease.

Prevention and management of hearing loss in syndromic craniosynostosis: A case series.

OBJECTIVE: To assess the audiological profile in a cohort of children affected by syndromic craniosynostosis. METHODS: Eleven children with Apert syndrome (n=4), Saethre-Chotzen syndrome (n=3), Muenke syndrome (n=2), Crouzon syndrome (n=1) and Pfeiffer syndrome type 1 (n=1) were submitted to a complete audiologic evaluation including otoscopy, pure-tone audiometry, tympanometry and acoustic reflex testing, ABR, otoacustic emissions, temporal bone High Resolution CT (HRCT) scan. The main outcome measures were prevalence, type and severity of hearing loss, prevalence of chronic otitis media, correlation with the time of first surgical correction. RESULTS: Seven of 11 patients (64%) presented hearing loss (HL), conductive in 3/7 patients (43%) and mixed in 4/7 (57%). No patients showed a purely sensorineural HL. All hearing impaired patients displayed middle ear disorders: the patients with conductive HL had otitis media with effusion (OME) and 3/4 patients with mixed HL showed tympanic alterations or cholesteatoma. A bilateral vestibular aqueduct enlargement was detected by HRCT scan in one normal hearing patient. The ABRs resulted normal in all cases. CONCLUSION: Our study confirms the high prevalence of otologic diseases in such patients. In contrast with previous studies, middle ear disorders were responsible for the hearing impairment also in patients with mixed HL due to secondary inner ear damage. These findings restate the necessity of a close audiologic follow-up. We did not detect the specific ABR abnormalities previously reported, possibly because of an early correction of the cranial vault malformations.

Target sequencing approach intended to discover new mutations in non-syndromic intellectual disability.

The technological improvements over the last years made considerable progresses in the knowledge of the etiology of intellectual Disability (ID). However, at present very little is known about the genetic heterogeneity underlying the non-syndromic form of ID (NS-ID). To investigate the genetic basis of NS-ID we analyzed 43 trios and 22 isolated NS-ID patients using a targeted sequencing (TS) approach. 71 NS-ID genes have been selected and sequenced in all subjects. We found putative pathogenic mutations in 7 out of 65 patients. The pathogenic role of mutations was evaluated through sequence comparison and structural analysis was performed to predict the effect of alterations in a 3D computational model through molecular dynamics simulations. Additionally, a deep patient clinical re-evaluation has been performed after the molecular results. This approach allowed us to find novel pathogenic mutations with a detection rate close to 11% in our cohort of patients. This result supports the hypothesis that many NS-ID related genes still remain to be discovered and that NS-ID is a more complex phenotype compared to syndromic form, likely caused by a complex and broad interaction between genes alterations and environment factors.

Molecular analysis, pathogenic mechanisms, and readthrough therapy on a large cohort of Kabuki syndrome patients.

Kabuki syndrome (KS) is a multiple congenital anomalies syndrome characterized by characteristic facial features and varying degrees of mental retardation, caused by mutations in KMT2D/MLL2 and KDM6A/UTX genes. In this study, we performed a mutational screening on 303 Kabuki patients by direct sequencing, MLPA, and quantitative PCR identifying 133 KMT2D, 62 never described before, and four KDM6A mutations, three of them are novel. We found that a number of KMT2D truncating mutations result in mRNA degradation through the nonsense-mediated mRNA decay, contributing to protein haploinsufficiency. Furthermore, we demonstrated that the reduction of KMT2D protein level in patients' lymphoblastoid and skin fibroblast cell lines carrying KMT2D-truncating mutations affects the expression levels of known KMT2D target genes. Finally, we hypothesized that the KS patients may benefit from a readthrough therapy to restore physiological levels of KMT2D and KDM6A proteins. To assess this, we performed a proof-of-principle study on 14 KMT2D and two KDM6A nonsense mutations using specific compounds that mediate translational readthrough and thereby stimulate the re-expression of full-length functional proteins. Our experimental data showed that both KMT2D and KDM6A nonsense mutations displayed high levels of readthrough in response to gentamicin treatment, paving the way to further studies aimed at eventually treating some Kabuki patients with readthrough inducers.

Progressive extreme heterotopic calcification.

The formation of ectopic calcifications in soft tissues can occur either sporadically or as a genetically determined condition, and is seen only infrequently. We report on a girl in whom widespread, rapidly progressive ectopic calcifications were detected shortly after birth. Calcifications became present around all joints, tendons and ligaments, but did not involve internal organs and skin, and eventually caused almost complete immobility of the child at 2 years. There were no other health problems and cognitive development was normal. We compare the manifestations in the child to the characteristics of known entities causing ectopic calcifications and conclude the child differs to each. Laboratory evaluation failed to identify autoimmune phenomena as well as calcium metabolism or other biochemical abnormalities; molecular studies did not identify occurrence of mutations in disease genes known to be involved in ectopic calcifications. We conclude the manifestations in the child represent an unreported entity of hitherto unknown etiology.

Absence of deletion and duplication of MLL2 and KDM6A genes in a large cohort of patients with Kabuki syndrome.

Kabuki syndrome is a rare, multiple congenital anomaly/mental retardation syndrome caused by MLL2 point mutations and KDM6A microdeletions. We screened a large cohort of MLL2 mutation-negative patients for MLL2 and KDM6A exon(s) microdeletion and microduplication. Our assays failed to detect such rearrangements in MLL2 as well as in KDM6A gene. These results show that these genomic events are extremely rare in the Kabuki syndrome, substantiating its genetic heterogeneity and the search for additional causative gene(s).

A restricted spectrum of mutations in the SMAD4 tumor-suppressor gene underlies Myhre syndrome.

Myhre syndrome is a developmental disorder characterized by reduced growth, generalized muscular hypertrophy, facial dysmorphism, deafness, cognitive deficits, joint stiffness, and skeletal anomalies. Here, by performing exome sequencing of a single affected individual and coupling the results to a hypothesis-driven filtering strategy, we establish that heterozygous mutations in SMAD4, which encodes for a transducer mediating transforming growth factor ß and bone morphogenetic protein signaling branches, underlie this rare Mendelian trait. Two recurrent de novo SMAD4 mutations were identified in eight unrelated subjects. Both mutations were missense changes altering Ile500 within the evolutionary conserved MAD homology 2 domain, a well known mutational hot spot in malignancies. Structural analyses suggest that the substituted residues are likely to perturb the binding properties of the mutant protein to signaling partners. Although SMAD4 has been established as a tumor suppressor gene somatically mutated in pancreatic, gastrointestinal, and skin cancers, and germline loss-of-function lesions and deletions of this gene have been documented to cause disorders that predispose individuals to gastrointestinal cancer and vascular dysplasias, the present report identifies a previously unrecognized class of mutations in the gene with profound impact on development and growth.