Improved biochemical and neurodevelopmental profiles with high-dose hydroxocobalamin therapy in cobalamin C defect.

Cobalamin C (Cbl-C) defect causes methylmalonic acidemia, homocystinuria, intellectual disability and visual impairment, despite treatment adherence. While international guidelines recommend parenteral hydroxocobalamin (OH-Cbl) as effective treatment, dose adjustments remain unclear. We assessed OH-Cbl therapy impact on biochemical, neurocognitive and visual outcomes in early-onset Cbl-C patients treated with different OH-Cbl doses over 3 years. Group A (n = 5), diagnosed via newborn screening (NBS), received high-dose OH-Cbl (median 0.55 mg/kg/day); Group B1 (n = 3), NBS-diagnosed, received low-dose OH-Cbl (median 0.09 mg/kg/day); Group B2 (n = 12), diagnosed on clinical bases, received low-dose OH-Cbl (median 0.06 mg/kg/day). Biochemical analyses revealed better values of homocysteine, methionine and methylmalonic acid in Group A compared to Group B1 (p < 0.01, p < 0.05 and p < 0.01, respectively) and B2 (p < 0.001, p < 0.01 and p < 0.001, respectively). Neurodevelopmental assessment showed better outcome in Group A compared to low-dose treated Groups B1 and B2, especially in Developmental Quotient, Hearing and Speech and Performance subscales without significant differences between Group B2 and Group B1. Maculopathy was detected in 100%, 66% and 83% of patients in the three groups, respectively. This study showed that "high-dose" OH-Cbl treatment in NBS-diagnosed children with severe early-onset Cbl-C defect led to a significant improvement in the metabolic profile and in neurocognitive outcome, compared to age-matched patients treated with a "low-dose" regimen. Effects on maculopathy seem unaffected by OH-Cbl dosage. Our findings, although observed in a limited number of patients, may contribute to improve the long-term outcome of Cbl-C patients.

Deep phenotyping of the neuroimaging and skeletal features in KBG syndrome: a study of 53 patients and review of the literature.

BACKGROUND: KBG syndrome is caused by haploinsufficiency of ANKRD11 and is characterised by macrodontia of upper central incisors, distinctive facial features, short stature, skeletal anomalies, developmental delay, brain malformations and seizures. The central nervous system (CNS) and skeletal features remain poorly defined. METHODS: CNS and/or skeletal imaging were collected from molecularly confirmed individuals with KBG syndrome through an international network. We evaluated the original imaging and compared our results with data in the literature. RESULTS: We identified 53 individuals, 44 with CNS and 40 with skeletal imaging. Common CNS findings included incomplete hippocampal inversion and posterior fossa malformations; these were significantly more common than previously reported (63.4% and 65.9% vs 1.1% and 24.7%, respectively). Additional features included patulous internal auditory canal, never described before in KBG syndrome, and the recurrence of ventriculomegaly, encephalic cysts, empty sella and low-lying conus medullaris. We found no correlation between these structural anomalies and epilepsy or intellectual disability. Prevalent skeletal findings comprised abnormalities of the spine including scoliosis, coccygeal anomalies and cervical ribs. Hand X-rays revealed frequent abnormalities of carpal bone morphology and maturation, including a greater delay in ossification compared with metacarpal/phalanx bones. CONCLUSION: This cohort enabled us to describe the prevalence of very heterogeneous neuroradiological and skeletal anomalies in KBG syndrome. Knowledge of the spectrum of such anomalies will aid diagnostic accuracy, improve patient care and provide a reference for future research on the effects of ANKRD11 variants in skeletal and brain development.

Successful treatment with MEK-inhibitor in a patient with NRAS-related cutaneous skeletal hypophosphatemia syndrome.

Cutaneous skeletal hypophosphatemia syndrome (CSHS) is caused by somatic mosaic NRAS variants and characterized by melanocytic/sebaceous naevi, eye, and brain malformations, and FGF23-mediated hypophosphatemic rickets. The MEK inhibitor Trametinib, acting on the RAS/MAPK pathway, is a candidate for CSHS therapy. A 4-year-old boy with seborrheic nevus, eye choristoma, multiple hamartomas, brain malformation, pleural lymphangioma and chylothorax developed severe hypophosphatemic rickets unresponsive to phosphate supplementation. The c.182A > G;p.(Gln61Arg) somatic NRAS variant found in DNA from nevus biopsy allowed diagnosing CSHS. We administered Trametinib for 15 months investigating the transcriptional effects at different time points by whole blood RNA-seq. Treatment resulted in prompt normalization of phosphatemia and phosphaturia, catch-up growth, chylothorax regression, improvement of bone mineral density, reduction of epidermal nevus and hamartomas. Global RNA sequencing on peripheral blood mononucleate cells showed transcriptional changes under MEK inhibition consisting in a strong sustained downregulation of signatures related to RAS/MAPK, PI3 kinase, WNT and YAP/TAZ pathways, reverting previously defined transcriptomic signatures. CSHS was effectively treated with a MEK inhibitor with almost complete recovery of rickets and partial regression of the phenotype. We identified "core" genes modulated by MEK inhibition potentially serving as surrogate markers of Trametinib action.

The diagnostic challenge of mild citrulline elevation at newborn screening.

Citrulline is a target analyte measured at expanded newborn screening (NBS) and its elevation represents a biomarker for distal urea cycle disorders and citrin deficiency. Altered ratios of citrulline with other urea cycle-related amino acids are helpful for the differential diagnosis. However, the use of cut-off values in screening programmes has raised the issue about the interpretation of mild elevation of citrulline levels detected at NBS, below the usual range observed in the "classical/severe" forms of distal urea cycle disorders and in citrin deficiency. Herein, we report ten subjects with positive NBS for a mild elevation of citrulline (<100 µmol/L), in whom molecular investigations revealed carriers status for argininosuccinate synthase deficiency, a milder form of argininosuccinate lyase deficiency and two other diseases, lysinuric protein intolerance and dihydrolipoamide dehydrogenase deficiency, not primarily affecting the urea cycle. To guide the diagnostic process, we have designed an algorithm for mild citrulline elevation (<100 µmol/L) at NBS, which expands the list of disorders to be included in the differential diagnosis.

Clinical delineation, sex differences, and genotype-phenotype correlation in pathogenic KDM6A variants causing X-linked Kabuki syndrome type 2.

PURPOSE: The variant spectrum and the phenotype of X-linked Kabuki syndrome type 2 (KS2) are poorly understood. METHODS: Genetic and clinical details of new and published individuals with pathogenic KDM6A variants were compiled and analyzed. RESULTS: Sixty-one distinct pathogenic KDM6A variants (50 truncating, 11 missense) from 80 patients (34 males, 46 females) were identified. Missense variants clustered in the TRP 2, 3, 7 and Jmj-C domains. Truncating variants were significantly more likely to be de novo. Thirteen individuals had maternally inherited variants and one had a paternally inherited variant. Neonatal feeding difficulties, hypoglycemia, postnatal growth retardation, poor weight gain, motor delay, intellectual disability (ID), microcephaly, congenital heart anomalies, palate defects, renal malformations, strabismus, hearing loss, recurrent infections, hyperinsulinism, seizures, joint hypermobility, and gastroesophageal reflux were frequent clinical findings. Facial features of over a third of patients were not typical for KS. Males were significantly more likely to be born prematurely, have shorter stature, and severe developmental delay/ID. CONCLUSION: We expand the KDM6A variant spectrum and delineate the KS2 phenotype. We demonstrate that the variability of the KS2 phenotypic depends on sex and the variant type. We also highlight the overlaps and differences between the phenotypes of KS2 and KS1.

First prenatal case of Noonan syndrome with SOS2 mutation: Implications of early diagnosis for genetic counseling.

RASopathies are a group of syndromes with partially overlapping clinical features caused by germline mutations of the RAS/MAPK signaling pathway genes. The most common disorder is Noonan syndrome (NS; MIM 163950). We report the first prenatal case of NS with SOS2 (NM_006939.4) mutation in a euploid fetus with a severe increase in nuchal translucency (NT > 12 mm). Trio-based custom next-generation sequencing detected a de novo heterozygous missense mutation in the SOS2 gene: c.800 T > A (p.Met267Lys). Owing to the marked variable expressivity of NS and the scarcity of SOS2 mutation-related NS cases reported in the literature, it is difficult to provide appropriate genetic counseling. Several issues such as the best management technique and optimal NT cutoff have been discussed. In addition, in general, the fine balance between the advantages of an early prenatal diagnosis and the challenge of determining if the detected gene variant is pathogenic and, primarily, the stress of the counselees when providing a genetic counseling with limited information on the prenatal phenotype have been discussed. A prenatal path comprising examinations and multidisciplinary counseling is essential to support couples in a shared decision-making process.

Co-occurrence of mutations in KIF7 and KIAA0556 in Joubert syndrome with ocular coloboma, pituitary malformation and growth hormone deficiency: a case report and literature review.

BACKGROUND: Joubert syndrome is a recessive neurodevelopmental disorder characterized by clinical and genetic heterogeneity. Clinical hallmarks include hypotonia, ataxia, facial dysmorphism, abnormal eye movement, irregular breathing pattern cognitive impairment and, the molar tooth sign is the pathognomonic midbrain-hindbrain malformation on magnetic resonance imaging. The disorder is predominantly caused by biallelic mutations in more than 30 genes encoding proteins with a pivotal role in morphology and function of the primary cilium. Oligogenic inheritance or occurrence of genetic modifiers has been suggested to contribute to the variability of the clinical phenotype. We report on a family with peculiar clinical spectrum Joubert syndrome molecularly and clinically dissecting a complex phenotype, in which hypogonadism, pituitary malformation and growth hormone deficiency occur as major features. CASE PRESENTATION: A 7 year-old male was enrolled in a dedicated "Undiagnosed Patients Program" for a peculiar form of Joubert syndrome complicated by iris and retinochoroidal coloboma, hypogonadism pituitary malformation, and growth hormone deficiency. The molecular basis of the complex phenotype was investigated by whole exome sequencing. The concomitant occurrence of homozygosity for mutations in KIF7 and KIAA0556 was identified, and the assessment of major clinical features associated with mutations in these two genes provided evidence that these two independent events represent the cause underlying the complexity of the present clinical phenotype. CONCLUSION: Beside the clinical variability of Joubert syndrome, co-occurrence of mutations in ciliopathy-associated genes may contribute to increase the clinical complexity of the trait.

LTBP2-related "Marfan-like" phenotype in two Roma/Gypsy subjects with the LTBP2 homozygous p.R299X variant.

Recessive variants in LTBP2 are associated with eye-restricted phenotypes including (a) primary congenital glaucoma and (b) microspherophakia/megalocornea and ectopia lentis with/without secondary glaucoma. Nosology of LTBP2 pathology in humans is apparently in contrast with the consolidated evidence of a wide expression of this gene in the developing embryo. Accordingly, in previously published patients with LTBP2-related eye disease, additional extraocular findings have been occasionally reported and include, among others, high-arched palate, tall stature, and variable cardiac involvement. Anyway, no emphasis was put on such systemic manifestations. Here, we report two unrelated Roma/Gypsy patients first ascertained for a multisystem disorder mainly characterized by primary congenital glaucoma, complex congenital heart defect, tall stature, long fingers, skin striae and dystrophic scarring, and resembling Marfan syndrome. Heart involvement was severe with polyvalvular heart dysplasia in one, and transposition of great arteries, thoracic arterial tortuosity, polyvalvular heart dysplasia, and neo-aortic root dilatation in the other. Both patients were homozygous for the recurrent c.895C>T[p.(R299X)] variant, typically found in individuals of Roma/Gypsy descent with an eye-restricted phenotype. Our findings point out LTBP2 as responsible of a systemic phenotype coherent with the community of syndromes related to anomalies in genes involved in the TGFß-pathway. Among these disorders, LTBP2-related systemic disease emerges as a distinct condition with expanding prognostic implications and autosomal recessive inheritance.

SOS1 mutations in Noonan syndrome: Cardiomyopathies and not only congenital heart defects! Report of six patients including two novel variants and literature review.

Noonan syndrome (NS) is caused by mutations in more than 10 genes, mainly PTPN11, SOS1, RAF1, and RIT1. Congenital heart defects and cardiomyopathy (CMP) are associated with significant morbidity and mortality in NS. Although hypertrophic CMP has "classically" been reported in association to RAF1, RIT1, and PTPN11 variants, SOS1 appears to be poorly related to CMP. Patients with NS attending our Center from January 2013 to June 2018 were eligible for inclusion if they carried SOS1 variants and presented with-or developed-CMP. Literature review describing the co-existence of SOS1 mutation and CMP was also performed. We identified six patients with SOS1 variants and CMP (male to female ratio 2:1) including two novel variants. CMP spectrum encompassed: (a) dilated CMP, (b) nonobstructive hypertrophic CMPs, and (c) obstructive hypertrophic CMPs. Survival is 100%. Literature review included 16 SOS1 mutated in CMP. CMP, mainly hypertrophic, has been often reported in association to RAF1, RIT1, and PTPN11 variants. Differently from previous reports, due to the frequent association of SOS1 variants and CMP in our single center experience, we suggest potential underestimated proportion of SOS1 in pediatric CMPs.

Identification of a Novel PROP1 Mutation in a Patient with Combined Pituitary Hormone Deficiency and Enlarged Pituitary.

Growth hormone deficiency (GHD) can be present from the neonatal period to adulthood and can be the result of congenital or acquired insults. In addition, GHD can be classified into two types: isolated growth hormone deficiency (IGHD) and combined pituitary hormone deficiency (CPHD). CPHD is a disorder characterized by impaired production of two or more anterior and/or posterior pituitary hormones. Many genes implicated in CPHD remain to be identified. Better genetic characterization will provide more information about the disorder and result in important genetic counselling because a number of patients with hypopituitarism represent familial cases. To date, PROP1 mutations represent the most common known genetic cause of CPHD both in sporadic and familial cases. We report a novel mutation in the PROP1 gene in an infant with CPHD and an enlarged pituitary gland. Close long-term follow-up will reveal other possible hormonal defects and pituitary involution.

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.

Persistent Hypoglycemia in Children: Targeted Gene Panel Improves the Diagnosis of Hypoglycemia Due to Inborn Errors of Metabolism.

OBJECTIVES: To evaluate the role of next generation sequencing in genetic diagnosis of pediatric patients with persistent hypoglycemia. STUDY DESIGN: Sixty-four patients investigated through an extensive workup were divided in 3 diagnostic classes based on the likelihood of a genetic diagnosis: (1) single candidate gene (9/64); (2) multiple candidate genes (43/64); and (3) no candidate gene (12/64). Subsequently, patients were tested through a custom gene panel of 65 targeted genes, which included 5 disease categories: (1) hyperinsulinemic hypoglycemia, (2) fatty acid-oxidation defects and ketogenesis defects, (3) ketolysis defects, (4) glycogen storage diseases and other disorders of carbohydrate metabolism, and (5) mitochondrial disorders. Molecular data were compared with clinical and biochemical data. RESULTS: A proven diagnosis was obtained in 78% of patients with suspicion for a single candidate gene, in 49% with multiple candidate genes, and in 33% with no candidate gene. The diagnostic yield was 48% for hyperinsulinemic hypoglycemia, 66% per fatty acid-oxidation and ketogenesis defects, 59% for glycogen storage diseases and other carbohydrate disorders, and 67% for mitochondrial disorders. CONCLUSIONS: This approach provided a diagnosis in ~50% of patients in whom clinical and laboratory evaluation did not allow identification of a single candidate gene and a diagnosis was established in 33% of patients belonging to the no candidate gene class. Next generation sequencing technique is cost-effective compared with Sanger sequencing of multiple genes and represents a powerful tool for the diagnosis of inborn errors of metabolism presenting with persistent hypoglycemia.

Prominent and elongated coccyx, a new manifestation of KBG syndrome associated with novel mutation in ANKRD11.

KBG syndrome is characterized by short stature, distinctive facial features, and developmental/cognitive delay and is caused by mutations in ANKRD11, one of the ankyrin repeat-containing cofactors. After the advent of whole exome sequencing, the number of clinical reports with KBG diagnosis has increased, leading to a revision of the phenotypic spectrum associated with this syndrome. Here, we report a female child showing clinical features of the KBG syndrome in addition to a caudal appendage at the coccyx with prominent skin fold and a peculiar calcaneus malformation. Exons and exon-intron junctions targeted resequencing of SH3PXD2B and MASP1 genes, known to be associated with prominent coccyx, gave negative outcome, whereas sequencing of ANKRD11 whose mutations matched the KBG phenotype of the proband showed a de novo heterozygous frameshift variant c.4528_4529delCC in exon 9 of ANKRD11. This report contributes to expand the knowledge of the clinical features of KBG syndrome and highlights the need to search for vertebral anomalies and suspect this condition in the presence of a prominent, elongated coccyx.

Aberrant HRAS transcript processing underlies a distinctive phenotype within the RASopathy clinical spectrum.

RASopathies are a group of rare, clinically related conditions affecting development and growth, and are caused by germline mutations in genes encoding signal transducers and modulators with a role in the RAS signaling network. These disorders share facial dysmorphia, short stature, variable cognitive deficits, skeletal and cardiac defects, and a variable predisposition to malignancies. Here, we report on a de novo 10-nucleotide-long deletion in HRAS (c.481_490delGGGACCCTCT, NM_176795.4; p.Leu163ProfsTer52, NP_789765.1) affecting transcript processing as a novel event underlying a RASopathy characterized by developmental delay, intellectual disability and autistic features, distinctive coarse facies, reduced growth, and ectodermal anomalies. Molecular and biochemical studies demonstrated that the deletion promotes constitutive retention of exon IDX, which is generally skipped during HRAS transcript processing, and results in a stable and mildly hyperactive GDP/GTP-bound protein that is constitutively targeted to the plasma membrane. Our findings document a new mechanism leading to altered HRAS function that underlies a previously unappreciated phenotype within the RASopathy spectrum.

Clinical and Neurobehavioral Features of Three Novel Kabuki Syndrome Patients with Mosaic KMT2D Mutations and a Review of Literature.

Kabuki syndrome (KS) is a rare disorder characterized by multiple congenital anomalies and variable intellectual disability caused by mutations in KMT2D/MLL2 and KDM6A/UTX, two interacting chromatin modifier responsible respectively for 56-75% and 5-8% of the cases. To date, three KS patients with mosaic KMT2D deletions in blood lymphocytes have been described. We report on three additional subjects displaying KMT2D gene mosaics including one in which a single nucleotide change results in a new frameshift mutation (p.L1199HfsX7), and two with already-known nonsense mutations (p.R4484X and p.R5021X). Consistent with previously published cases, mosaic KMT2D mutations may result in mild KS facial dysmorphisms and clinical and neurobehavioral features, suggesting that these characteristics could represent the handles for genetic testing of individuals with slight KS-like traits.

Metastatic Group 3 Medulloblastoma in a Patient With Tuberous Sclerosis Complex: Case Description and Molecular Characterization of the Tumor.

Medulloblastoma is the most common pediatric brain tumor. We describe a child with tuberous sclerosis complex that developed a Group 3, myc overexpressed, metastatic medulloblastoma (MB). Considering the high risk of treatment-induced malignancies, a tailored therapy, omitting radiation, was given. Based on the evidence of mammalian target of rapamycin mTORC, mTOR Complex; RAS, Rat sarcoma; RAF, rapidly accelerated fibrosarcoma (mTOR) pathway activation in the tumor, targeted therapy was applied resulting in complete remission of disease. Although the PI3K/AKT/mTOR signaling pathway plays a role in MB, we did not find TSC1/TSC2 (TSC, tuberous sclerosis complex) mutation in our patient. We speculate that a different pathway resulting in mTOR activation is the basis of both TSC and MB in this child; H&E, haematoxilin and eosin; Gd, gadolinium.

CHARGE syndrome due to deletion of region upstream of CHD7 gene START codon.

BACKGROUND: CHARGE syndrome is an autosomal dominant disorder, characterized by ocular Coloboma, congenital Heart defects, choanal Atresia, Retardation, Genital anomalies and Ear anomalies. Over 90 % of typical CHARGE patients are mutated in the CHD7 gene, 65 %-70 % of the cases for all typical and suspected cases combined. The gene encoding for a protein involved in chromatin organization. The mutational spectrum include nonsense, frameshift, splice site, and missense mutations. Large deletions and genomic rearrangements are rare. CASE PRESENTATION: We report here on a 5.9 years old male of Moroccan origin displaying classic clinical features of CHARGE syndrome. Using CGH array and NGS analysis we detected a microdeletion (184 kb) involving the promoter region and exon 1 of CHD7 gene and the flanking RAB2 gene. CONCLUSION: The present observation suggests that deletion limited to the regulatory region of CHD7 is sufficient to cause the full blown CHARGE phenotype. Different size of deletions can result in different phenotypes, ranging from a milder to severe CHARGE syndrome; this is based on a combination of major and minor diagnostic characteristics, therefore to a more variable clinical features, likely due to the additive effect of other genetic imbalances. MLPA and CGH techniques should be considered in the diagnostic protocol of individuals with a clinical suspect of CHARGE syndrome.

Spinal ependymoma in a patient with Kabuki syndrome: a case report.

BACKGROUND: Kabuki syndrome is a rare disorder characterized by the association of mental retardation and postnatal growth deficiency with distinctive facial appearance, skeletal anomalies, cardiac and renal malformation. Two causative genes have been identified in patients with Kabuki syndrome. Mutation of KMT2D (MLL2) was identified in 55-80% of patients, while 9-14% of KMT2D negative patients have mutation in KDM6A gene. So far, few tumors have been reported in patients with Kabuki syndrome. We describe the first case of a patient with spinal ependymoma and Kabuki syndrome. CASE PRESENTATION: A 23 years old girl followed at our Center for KMT2D mutated Kabuki syndrome since she was 4 years old presented with acute lumbar pain and intermittent tactile hyposthenia of the feet. Spine magnetic resonance revealed a lumbar endocanalar mass. She underwent surgical resection of the lesion and histologic examination showed a tanycytic ependymoma (WHO grade II). CONCLUSION: Kabuki syndrome is not considered a cancer predisposition syndrome. Nonetheless, a number of tumors have been reported in patients with Kabuki syndrome. Spinal ependymoma is a rare disease in the pediatric and young adult population. Whereas NF2 mutations are frequently associated to ependymoma such an association has never been described in Kabuki syndrome. To our knowledge this is the first case of ependymoma in a KMT2D mutated Kabuki syndrome patient. Despite KMT2D role in cancer has previously been described, no genetic data are available for previously reported Kabuki syndrome patients with tumors. Nonetheless, the association of two rare diseases raises the suspicion for a common determinant.

Advantages of a next generation sequencing targeted approach for the molecular diagnosis of retinoblastoma.

BACKGROUND: Retinoblastoma (RB) is the most common malignant childhood tumor of the eye and results from inactivation of both alleles of the RB1 gene. Nowadays RB genetic diagnosis requires classical chromosome investigations, Multiplex Ligation-dependent Probe Amplification analysis (MLPA) and Sanger sequencing. Nevertheless, these techniques show some limitations. We report our experience on a cohort of RB patients using a combined approach of Next-Generation Sequencing (NGS) and RB1 custom array-Comparative Genomic Hybridization (aCGH). METHODS: A total of 65 patients with retinoblastoma were studied: 29 cases of bilateral RB and 36 cases of unilateral RB. All patients were previously tested with conventional cytogenetics and MLPA techniques. Fifty-three samples were then analysed using NGS. Eleven cases were analysed by RB1 custom aCGH. One last case was studied only by classic cytogenetics. Finally, it has been tested, in a lab sensitivity assay, the capability of NGS to detect artificial mosaicism series in previously recognized samples prepared at 3 different mosaicism frequencies: 10, 5, 1 %. RESULTS: Of the 29 cases of bilateral RB, 28 resulted positive (96.5 %) to the genetic investigation: 22 point mutations and 6 genomic rearrangements (four intragenic and two macrodeletion). A novel germline intragenic duplication, from exon18 to exon 23, was identified in a proband with bilateral RB. Of the 36 available cases of unilateral RB, 8 patients resulted positive (22 %) to the genetic investigation: 3 patients showed point mutations while 5 carried large deletion. Finally, we successfully validated, in a lab sensitivity assay, the capability of NGS to accurately measure level of artificial mosaicism down to 1 %. CONCLUSIONS: NGS and RB1-custom aCGH have demonstrated to be an effective combined approach in order to optimize the overall diagnostic procedures of RB. Custom aCGH is able to accurately detect genomic rearrangements allowing the characterization of their extension. NGS is extremely accurate in detecting single nucleotide variants, relatively simple to perform, cost savings and efficient and has confirmed a high sensitivity and accuracy in identifying low levels of artificial mosaicisms.