Dual Molecular Effects of Dominant RORA Mutations Cause Two Variants of Syndromic Intellectual Disability with Either Autism or Cerebellar Ataxia.

RORα, the RAR-related orphan nuclear receptor alpha, is essential for cerebellar development. The spontaneous mutant mouse staggerer, with an ataxic gait caused by neurodegeneration of cerebellar Purkinje cells, was discovered two decades ago to result from homozygous intragenic Rora deletions. However, RORA mutations were hitherto undocumented in humans. Through a multi-centric collaboration, we identified three copy-number variant deletions (two de novo and one dominantly inherited in three generations), one de novo disrupting duplication, and nine de novo point mutations (three truncating, one canonical splice site, and five missense mutations) involving RORA in 16 individuals from 13 families with variable neurodevelopmental delay and intellectual disability (ID)-associated autistic features, cerebellar ataxia, and epilepsy. Consistent with the human and mouse data, disruption of the D. rerio ortholog, roraa, causes significant reduction in the size of the developing cerebellum. Systematic in vivo complementation studies showed that, whereas wild-type human RORA mRNA could complement the cerebellar pathology, missense variants had two distinct pathogenic mechanisms of either haploinsufficiency or a dominant toxic effect according to their localization in the ligand-binding or DNA-binding domains, respectively. This dichotomous direction of effect is likely relevant to the phenotype in humans: individuals with loss-of-function variants leading to haploinsufficiency show ID with autistic features, while individuals with de novo dominant toxic variants present with ID, ataxia, and cerebellar atrophy. Our combined genetic and functional data highlight the complex mutational landscape at the human RORA locus and suggest that dual mutational effects likely determine phenotypic outcome.

De Novo Disruption of the Proteasome Regulatory Subunit PSMD12 Causes a Syndromic Neurodevelopmental Disorder.

Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological process in the development of eukaryotic organisms. Dysregulation of this mechanism leads to numerous human neurodegenerative or neurodevelopmental disorders. Through a multi-center collaboration, we identified six de novo genomic deletions and four de novo point mutations involving PSMD12, encoding the non-ATPase subunit PSMD12 (aka RPN5) of the 19S regulator of 26S proteasome complex, in unrelated individuals with intellectual disability, congenital malformations, ophthalmologic anomalies, feeding difficulties, deafness, and subtle dysmorphic facial features. We observed reduced PSMD12 levels and an accumulation of ubiquitinated proteins without any impairment of proteasome catalytic activity. Our PSMD12 loss-of-function zebrafish CRISPR/Cas9 model exhibited microcephaly, decreased convolution of the renal tubules, and abnormal craniofacial morphology. Our data support the biological importance of PSMD12 as a scaffolding subunit in proteasome function during development and neurogenesis in particular; they enable the definition of a neurodevelopmental disorder due to PSMD12 variants, expanding the phenotypic spectrum of UPS-dependent disorders.

A founder noncoding GALT variant interfering with splicing causes galactosemia.

Galactosemia is a rare, treatable hereditary disorder of carbohydrate metabolism. We investigated the etiology of decreased GALT enzyme activity in a cohort of newborns referred by the Florida Newborn Screening Program with no detectable GALT variants in diagnostic molecular tests. Six affected individuals from four families with Guatemalan heritage were included. GALT enzyme activity ranged from 20% to 34% of normal. Clinical findings were unremarkable except for speech delay in two children. Via genome sequencing followed by Sanger confirmation we showed that all affected individuals were homozygous for a deep intronic GALT variant, c.1059+390A>G, which segregated as an autosomal recessive trait in all families. The intronic variant disrupts splicing and leads to a premature termination and is associated with a single haplotype flanking GALT, suggesting a founder effect. In conclusion, we present a deep intronic GALT variant leading to a biochemical variant form of galactosemia. This variant remains undiagnosed until it is specifically targeted in molecular testing.

Arnold-Chiari type 1 malformation in Potocki-Lupski syndrome.

Potocki-Lupski syndrome (PTLS) is a genetic disorder that results from an interstitial duplication within chromosome 17p11.2. Children with PTLS typically present with infantile hypotonia, failure to thrive, and global developmental delay with or without major organ system involvement. Systematic clinical studies regarding growth, cardiovascular disease, and neurocognitive profiles have been published; however, systematic evaluation of central nervous system structure by magnetic resonance imaging (MRI) of the brain has not been reported. Herein, we describe three patients with PTLS who were found-in the course of routine clinical care-to have a type 1 Arnold-Chiari malformation (CM-1). This finding raises the question of whether the incidence of CM-1 is increased in PTLS, and hence, if an MRI of the brain should be considered in the evaluation of all patients with this chromosomal duplication syndrome.

MPV17-related mitochondrial DNA maintenance defect: New cases and review of clinical, biochemical, and molecular aspects.

Mitochondrial DNA (mtDNA) maintenance defects are a group of diseases caused by deficiency of proteins involved in mtDNA synthesis, mitochondrial nucleotide supply, or mitochondrial dynamics. One of the mtDNA maintenance proteins is MPV17, which is a mitochondrial inner membrane protein involved in importing deoxynucleotides into the mitochondria. In 2006, pathogenic variants in MPV17 were first reported to cause infantile-onset hepatocerebral mtDNA depletion syndrome and Navajo neurohepatopathy. To date, 75 individuals with MPV17-related mtDNA maintenance defect have been reported with 39 different MPV17 pathogenic variants. In this report, we present an additional 25 affected individuals with nine novel MPV17 pathogenic variants. We summarize the clinical features of all 100 affected individuals and review the total 48 MPV17 pathogenic variants. The vast majority of affected individuals presented with an early-onset encephalohepatopathic disease characterized by hepatic and neurological manifestations, failure to thrive, lactic acidemia, and mtDNA depletion detected mainly in liver tissue. Rarely, MPV17 deficiency can cause a late-onset neuromyopathic disease characterized by myopathy and peripheral neuropathy with no or minimal liver involvement. Approximately half of the MPV17 pathogenic variants are missense. A genotype with biallelic missense variants, in particular homozygous p.R50Q, p.P98L, and p.R41Q, can carry a relatively better prognosis.

Causes of death in 184 patients with type 1 Gaucher disease from the United States who were never treated with enzyme replacement therapy.

Treatment for type 1 Gaucher disease (GD1) decreases morbidity from hematological cytopenias, hepatosplenomegaly and bone complications. Consequently, untreated symptomatic patients for study of late outcomes are hard to find. We identified 184 untreated GD1 patients (67.4% Ashkenazi; splenectomy 51.1%) who died between 1950 and 2010. Here, we report confirmed causes of death for these patients compared with the overall US population. Median age of death 66years (2-97years); causes of death (COD) with a high proportional mortality rate (PMR) included malignancies (PMR 1.57), suicide/drug overdose (PMR 3.86), liver disease (PMR 4.76) and septicemia (PMR 9.22). PMRs for CNS/gastrointestinal bleeding, pulmonary hypertension, post-splenectomy complications and Parkinsonism were also increased. PMR for heart disease (0.33) was significantly decreased. Average age at death was normal for heart disease, septicemia, suicide, and malignancies but younger for liver disease and Parkinsonism. COD more prevalent in splenectomy patients included liver disease, septicemia, pulmonary hypertension and GI bleeding. With timely diagnosis, improved risk assessment and obsolescence of splenectomy, GD1-associated malignancies, liver disease, septicemia, pulmonary hypertension, suicide and drug dependency may decrease with early institution of appropriate treatment. Our population of untreated patients is a valuable historical control for studies of the effect of GD1 treatment on premature mortality.

Comprehensive analysis of the transcriptional landscape of the human FMR1 gene reveals two new long noncoding RNAs differentially expressed in Fragile X syndrome and Fragile X-associated tremor/ataxia syndrome.

The majority of the human genome is transcribed but not translated, giving rise to noncoding RNAs (ncRNAs), including long ncRNAs (lncRNAs, >200 nt) that perform a wide range of functions in gene regulation. The Fragile X mental retardation 1 (FMR1) gene is a microsatellite locus that in the general population contains <55 CGG repeats in its 5'-untranslated region. Expansion of this repeat region to a size of 55-200 CGG repeats, known as premutation, is associated with Fragile X tremor and ataxia syndrome (FXTAS). Further expansion beyond 200 CGG repeats, or full mutation, leads to FMR1 gene silencing and results in Fragile X syndrome (FXS). Using a novel technology called "Deep-RACE", which combines rapid amplification of cDNA ends (RACE) with next generation sequencing, we systematically interrogated the FMR1 gene locus for the occurrence of novel lncRNAs. We discovered two transcripts, FMR5 and FMR6. FMR5 is a sense lncRNA transcribed upstream of the FMR1 promoter, whereas FMR6 is an antisense transcript overlapping the 3' region of FMR1. FMR5 was expressed in several human brain regions from unaffected individuals and from full and premutation patients. FMR6 was silenced in full mutation and, unexpectedly, in premutation carriers suggesting abnormal transcription and/or chromatin remodeling prior to transition to the full mutation. These lncRNAs may thus be useful as biomarkers, allowing for early detection and therapeutic intervention in FXS and FXTAS. Finally we show that FMR5 and FMR6 are expressed in peripheral blood leukocytes and propose future studies that correlate lncRNA expression with clinical outcomes.

ADAMTSL2 mutations determine the phenotypic severity in geleophysic dysplasia.

Geleophysic dysplasia-1 (GD1) is an autosomal recessive disorder caused by ADAMTS-like 2 (ADAMTSL2) variants. It is characterized by distinctive facial features, limited joint mobility, short stature, brachydactyly, and life-threatening cardiorespiratory complications. The clinical spectrum spans from perinatal lethality to milder adult phenotypes. We developed and characterized cellular and mouse models, to replicate the genetic profile of a patient who is compound heterozygous for 2 ADAMTSL2 variants, namely p.R61H and p.A165T. The impairment of ADAMTSL2 secretion was observed in both variants, but p.A165T exhibited a more severe impact. Mice carrying different allelic combinations revealed a spectrum of phenotypic severity, from lethality in knockout homozygotes to mild growth impairment observed in adult p.R61H homozygotes. Homozygous and hemizygous p.A165T mice survived but displayed severe respiratory and cardiac dysfunction. The respiratory dysfunction mainly affected the expiration phase, and some of these animals had microscopic post-obstructive pneumonia. Echocardiograms and MRI studies revealed a significant systolic dysfunction, accompanied by a reduction of the aortic root size. Histology verified the presence of hypertrophic cardiomyopathy with myocyte hypertrophy, chondroid metaplasia, and mild interstitial fibrosis. This study revealed a substantial correlation between the degree of impaired ADAMTSL2 secretion and the severity of the observed phenotype in GD1.

Adeno-associated virus-mediated gene therapy in a patient with Canavan disease using dual routes of administration and immune modulation.

Gene replacement therapy is a rational therapeutic strategy and clinical intervention for neurodegenerative disorders like Canavan disease, a leukodystrophy caused by biallelic mutations in the aspartoacylase (ASPA) gene. We aimed to investigate whether simultaneous intravenous (i.v.) and intracerebroventricular (i.c.v.) administration of rAAV9-CB6-ASPA provides a safe and effective therapeutic strategy in an open-label, individual-patient, expanded-access trial for Canavan disease. Immunomodulation was given prophylactically prior to adeno-associated virus (AAV) treatment to prevent an immune response to ASPA or the vector capsid. The patient served as his own control, and change from baseline was assessed by clinical pathology tests, vector genomes in the blood, antibodies against ASPA and AAV capsids, levels of cerebrospinal fluid (CSF) N-acetylaspartate (NAA), brain water content and morphology, clinical status, and motor function tests. Two years post treatment, the patient's white matter myelination had increased, motor function was improved, and he remained free of typical severe epilepsy. NAA level was reduced at 3 months and remained stable up to 4 years post treatment. Immunomodulation prior to AAV exposure enables repeat dosing and has prevented an anti-transgene immune response. Dual-route administration of gene therapy may improve treatment outcomes.

Genotype-phenotype analysis of 4q deletion syndrome: proposal of a critical region.

Chromosome 4q deletion syndrome (4q- syndrome) is a rare condition, with an estimated incidence of 1 in 100,000. Although variable, the clinical spectrum commonly includes craniofacial, developmental, digital, skeletal, and cardiac involvement. Data on the genotype-phenotype correlation within the 4q arm are limited. We present detailed clinical and genetic information by array CGH on 20 patients with 4q deletions. We identified a patient who has a ∼465 kb deletion (186,770,069-187,234,800, hg18 coordinates) in 4q35.1 with all clinical features for 4q deletion syndrome except for developmental delay, suggesting that this is a critical region for this condition and a specific gene responsible for orofacial clefts and congenital heart defects resides in this region. Since the patients with terminal deletions all had cleft palate, our results provide further evidence that a gene associated with clefts is located on the terminal segment of 4q. By comparing and contrasting our patients' genetic information and clinical features, we found significant genotype-phenotype correlations at a single gene level linking specific phenotypes to individual genes. Based on these data, we constructed a hypothetical partial phenotype-genotype map for chromosome 4q which includes BMP3, SEC31A, MAPK10, SPARCL1, DMP1, IBSP, PKD2, GRID2, PITX2, NEUROG2, ANK2, FGF2, HAND2, and DUX4 genes.

Genome sequencing reveals novel noncoding variants in PLA2G6 and LMNB1 causing progressive neurologic disease.

Neurodegenerative disorders and leukodystrophies are progressive neurologic conditions that can occur following the disruption of intricately coordinated patterns of gene expression. Exome sequencing has been adopted as an effective diagnostic tool for determining the underlying genetic etiology of Mendelian neurologic disorders, however genome sequencing offer advantages in its ability to identify and characterize copy number, structural, and sequence variants in noncoding regions. Genome sequencing from peripheral leukocytes was performed on two patients with progressive neurologic disease of unknown etiology following negative genetic investigations including exome sequencing. RNA sequencing from peripheral blood was performed to determine gene expression patterns in one of the patients. Potential causative variants were matched to the patients' clinical presentation. The first proband was found to be heterozygous for a likely pathogenic missense variant in PLA2G6 (c.386T>C; p.Leu129Pro) and have an additional deep intronic variant in PLA2G6 (c.2035-926G>A). RNA sequencing indicated this latter variant created a splice acceptor site leading to the incorporation of a pseudo-exon introducing a premature termination codon. The second proband was heterozygous for a 261 kb deletion upstream of LMNB1 that included an enhancer region. Previous reports of copy number variants spanning this region of cis-acting regulatory elements corroborated its pathogenicity. When combined with clinical presentations, these findings led to a definitive diagnosis of autosomal recessive infantile neuroaxonal dystrophy and autosomal dominant adult-onset demyelinating leukodystrophy, respectively. In patients with progressive neurologic disease of unknown etiology, genome sequencing with the addition of RNA analysis where appropriate should be considered for the identification of causative noncoding pathogenic variants.

Expanded newborn screening in Puerto Rico and the US Virgin Islands: education and barriers assessment.

PURPOSE: The implementation of the expanded newborn screening panel of 29 disorders recommended by the American College of Medical Genetics in Puerto Rico and United States Virgin Islands is still in development or in early stages. Efforts in the territories are complicated by educational and resource barriers that generate a wide gap between the islands and the US mainland. METHODS: To meet immediate educational needs, we conducted in-services for local newborn screening professionals. The efficacy of the educational intervention was measured by pre and posttest scores and a seminar evaluation. An assessment was obtained to document local newborn screening needs and barriers, with focus on human resources, intervention, language, social issues, education, and communication. RESULTS: Statistical significance was found (P value < or =0.05) between pre and posttest scores of the educational intervention. Needs and barriers associated with expanded newborn screening were also documented. CONCLUSION: Puerto Rico and United States Virgin Islands face different challenges in their implementation of expanded newborn screening. The data obtained in the present study serves as foundation for the development of public policy and long-term educational programs.

Macrocephaly-capillary malformation: a report of three cases and review of the literature.

Macrocephaly-Cutis Marmorata Telangiectatica Congenita (M-CMTC) is a rare syndrome that was first delineated as distinct from Cutis Marmorata Telangiectatica Congenita (CMTC) in 1997. Since that time, there have been over 75 cases reported in the literature, though few are in the dermatology literature. The syndrome is characterized by macrocephaly, neonatal hypotonia, developmental delay, segmental overgrowth, syndactyly, asymmetry, connective tissue defects, and vascular stains. We report three new patients seen at the University of Miami Genodermatoses Clinic with features of M-CMTC. We believe the skin findings in our patients and in the previously published cases of M-CMTC are more consistent with capillary malformations rather than true CMTC. Therefore, we agree with recent publications that this condition be renamed Macrocephaly-Capillary Malformation (M-CM). The differential diagnoses for patients with M-CMTC include Klippel Trenaunay Syndrome (KTS) and Proteus or Proteus-like syndromes. Given the significant prognostic and likely genetic differences among these conditions it is important to distinguish M-CMTC from these syndromes.

Early-Onset Marfan Syndrome: A Case Series.

Mutations in fibrillin 1 cause Marfan syndrome (MFS), an autosomal dominant disorder of the connective tissue, with multisystem manifestations. In early-onset MFS, the physical characteristics are expressed much earlier than the classical MFS. Those affected by this form generally have their mutations restricted to the gene "hotspot" region of exons 24 to 32. Historically, affected individuals usually die within the first few years of life due to heart failure secondary to severe valvular insufficiency. We report three patients with early-onset MFS, whose clinical evolution has been remarkably positive, when compared with other reported cases in the literature.

Infantile Onset Hypertrophic Cardiomyopathy Secondary to PRKAG2 Gene Mutation is Associated with Poor Prognosis.

Hypertrophic cardiomyopathy (HCM) is the second most prevalent form of cardiomyopathy in children. The etiology of the HCM is heterogeneous, so is the age of onset of symptoms. The HCM associated with metabolic disorders and genetic syndromes presents early in childhood. There are very few case reports of early-onset infantile HCM secondary to the PRKAG2 gene. Here, we report a case of HCM in a neonate diagnosed prenatally and eventually diagnosed with a missense mutation in the PRKAG2 gene.

Detection of pathogenic gene copy number variations in patients with mental retardation by genomewide oligonucleotide array comparative genomic hybridization.

Genomic imbalance is a major cause of developmental disorders. Microarray-based comparative genomic hybridization (aCGH) has revealed frequent imbalances associated with clinical syndromes, but also a large number of copy number variations (CNVs), which have complicated the interpretation of results. We studied 100 consecutive patients with unexplained mental retardation and a normal karyotype using several platforms of CGH arrays. A genomewide array with 44,290 oligonucleotide probes (OaCGH44K) detected imbalances in 15% of cases studied with sizes ranged from 459 kb to 19 Mb while revealing a small number of CNVs (0.72/individual). Another platform with approximately 240,000 oligonucleotide probes (OaCGH244K) revealed a large number of CNVs (20/individual) in selected cases and their normal parents. We used a comprehensive approach for interpreting the results of aCGH, including consideration of the size, inheritance and gene content of CNVs, and consultation with an online Database of Genomic Variants (DGV) and Online Mendelian Inheritance in Men (OMIM) for information on the genes involved. Our study suggests that genomewide oligonucleotide arrays such as the OaCGH44K platform can be used as a powerful diagnostic tool for detection of genomic imbalances associated with unexplained mental retardation or syndromic autism spectrum disorders. It is interesting to note that a small number of common variants were revealed by OaCGH244K in some study subjects but not in their parents and that some inherited CNVs had altered breakpoints. Further investigations on these alterations may provide useful information for understanding the mechanism of CNVs.

Klinefelter Syndrome in Association with Tetralogy of Fallot and Congenital Diaphragmatic Hernia.

Klinefelter syndrome (KS) is the most common sex chromosomal aneuploidy in males. Major cardiovascular and diaphragmatic anomalies are uncommon in this syndrome. Here we report an infant with KS who had tetralogy of Fallot and congenital diaphragmatic hernia, all of which were identified prenatally and managed successfully after birth. Microarray analysis did not reveal any deletions or duplications other than the additional X-chromosome, to account for the additional abnormalities in this infant. To the authors' knowledge, this is the first such report of major cardiac and diaphragm anomaly occurring together, in an infant with KS.