Alternative splicing of BUD13 determines the severity of a developmental disorder with lipodystrophy and progeroid features.

PURPOSE: In this study we aimed to identify the molecular genetic cause of a progressive multisystem disease with prominent lipodystrophy. METHODS: In total, 5 affected individuals were investigated using exome sequencing. Dermal fibroblasts were characterized using RNA sequencing, proteomics, immunoblotting, immunostaining, and electron microscopy. Subcellular localization and rescue studies were performed. RESULTS: We identified a lipodystrophy phenotype with a typical facial appearance, corneal clouding, achalasia, progressive hearing loss, and variable severity. Although 3 individuals showed stunted growth, intellectual disability, and died within the first decade of life (A1, A2, and A3), 2 are adults with normal intellectual development (A4 and A5). All individuals harbored an identical homozygous nonsense variant affecting the retention and splicing complex component BUD13. The nucleotide substitution caused alternative splicing of BUD13 leading to a stable truncated protein whose expression positively correlated with disease expression and life expectancy. In dermal fibroblasts, we found elevated intron retention, a global reduction of spliceosomal proteins, and nuclei with multiple invaginations, which were more pronounced in A1, A2, and A3. Overexpression of both BUD13 isoforms normalized the nuclear morphology. CONCLUSION: Our results define a hitherto unknown syndrome and show that the alternative splice product converts a loss-of-function into a hypomorphic allele, thereby probably determining the severity of the disease and the survival of affected individuals.

Adding evidence to the role of NEUROG1 in congenital cranial dysinnervation disorders.

Congenital cranial dysinnervation disorders (CCDDs) are a heterogeneous group of neurodevelopmental phenotypes caused by a primary disturbance of innervation due to deficient, absent, or misguided cranial nerves. Although some CCDDs genes are known, several clinical phenotypes and their aetiologies remain to be elucidated. We describe a 12-year-old boy with hypotonia, developmental delay, sensorineural hearing loss, and keratoconjunctivitis due to lack of corneal reflex. He had a long expressionless face, severe oromotor dysfunction, bilateral agenesis/severe hypoplasia of the VIII nerve with marked atresia of the internal auditory canals and cochlear labyrinth malformation. Trio-exome sequencing identified a homozygous loss of function variant in the NEUROG1 gene (NM_006161.2: c.202G > T, p.Glu68*). NEUROG1 is considered a causal candidate for CCDDs based on (i) the previous report of a patient with a homozygous gene deletion and developmental delay, deafness due to absent bilateral VIII nerves, and severe oromotor dysfunction; (ii) a second patient with a homozygous NEUROG1 missense variant and corneal opacity, absent corneal reflex and intellectual disability; and (iii) the knockout mouse model phenotype which highly resembles the disorder observed in humans. Our findings support the growing compelling evidence that loss of NEUROG1 leads to a very distinctive disorder of cranial nerves development.

Genomic sequencing highlights the diverse molecular causes of Perrault syndrome: a peroxisomal disorder (PEX6), metabolic disorders (CLPP, GGPS1), and mtDNA maintenance/translation disorders (LARS2, TFAM).

Perrault syndrome is a rare heterogeneous condition characterised by sensorineural hearing loss and premature ovarian insufficiency. Additional neuromuscular pathology is observed in some patients. There are six genes in which variants are known to cause Perrault syndrome; however, these explain only a minority of cases. We investigated the genetic cause of Perrault syndrome in seven affected individuals from five different families, successfully identifying the cause in four patients. This included previously reported and novel causative variants in known Perrault syndrome genes, CLPP and LARS2, involved in mitochondrial proteolysis and mitochondrial translation, respectively. For the first time, we show that pathogenic variants in PEX6 can present clinically as Perrault syndrome. PEX6 encodes a peroxisomal biogenesis factor, and we demonstrate evidence of peroxisomal dysfunction in patient serum. This study consolidates the clinical overlap between Perrault syndrome and peroxisomal disorders, and highlights the need to consider ovarian function in individuals with atypical/mild peroxisomal disorders. The remaining patients had variants in candidate genes such as TFAM, involved in mtDNA transcription, replication, and packaging, and GGPS1 involved in mevalonate/coenzyme Q10 biosynthesis and whose enzymatic product is required for mouse folliculogenesis. This genomic study highlights the diverse molecular landscape of this poorly understood syndrome.

WNT10B variants in split hand/foot malformation: Report of three novel families and review of the literature.

Split-hand/foot malformation (SHFM) is a genetically heterogeneous congenital limb malformation typically limited to a defect of the central rays of the autopod, presenting as a median cleft of hands and feet. It can be associated with long bone deficiency or included in more complex syndromes. Among the numerous genetic causes, WNT10B homozygous variants have been recently identified in consanguineous families, but remain still rarely described (SHFM6; MIM225300). We report on three novel SHFM families harboring WNT10B variants and review the literature, allowing us to highlight some clinical findings. The feet are more severely affected than the hands and there is a frequent asymmetry without obvious side-bias. Syndactyly of third-fourth fingers was a frequent finding (62%). Polydactyly, which was classically described in SHFM6, was only present in 27% of patients. No genotype-phenotype correlation is delineated but heterozygous individuals might have mild features of SHFM, suggesting a dose-effect of the WNT10B loss-of-function.

Germline Loss-of-Function Mutations in EPHB4 Cause a Second Form of Capillary Malformation-Arteriovenous Malformation (CM-AVM2) Deregulating RAS-MAPK Signaling.

BACKGROUND: Most arteriovenous malformations (AVMs) are localized and occur sporadically. However, they also can be multifocal in autosomal-dominant disorders, such as hereditary hemorrhagic telangiectasia and capillary malformation (CM)-AVM. Previously, we identified RASA1 mutations in 50% of patients with CM-AVM. Herein we studied non-RASA1 patients to further elucidate the pathogenicity of CMs and AVMs. METHODS: We conducted a genome-wide linkage study on a CM-AVM family. Whole-exome sequencing was also performed on 9 unrelated CM-AVM families. We identified a candidate gene and screened it in a large series of patients. The influence of several missense variants on protein function was also studied in vitro. RESULTS: We found evidence for linkage in 2 loci. Whole-exome sequencing data unraveled 4 distinct damaging variants in EPHB4 in 5 families that cosegregated with CM-AVM. Overall, screening of EPHB4 detected 47 distinct mutations in 54 index patients: 27 led to a premature stop codon or splice-site alteration, suggesting loss of function. The other 20 are nonsynonymous variants that result in amino acid substitutions. In vitro expression of several mutations confirmed loss of function of EPHB4. The clinical features included multifocal CMs, telangiectasias, and AVMs. CONCLUSIONS: We found EPHB4 mutations in patients with multifocal CMs associated with AVMs. The phenotype, CM-AVM2, mimics RASA1-related CM-AVM1 and also hereditary hemorrhagic telangiectasia. RASA1-encoded p120RASGAP is a direct effector of EPHB4. Our data highlight the pathogenetic importance of this interaction and indicts EPHB4-RAS-ERK signaling pathway as a major cause for AVMs.

Phenotype and genotype in 52 patients with Rubinstein-Taybi syndrome caused by EP300 mutations.

Rubinstein-Taybi syndrome (RSTS) is a developmental disorder characterized by a typical face and distal limbs abnormalities, intellectual disability, and a vast number of other features. Two genes are known to cause RSTS, CREBBP in 60% and EP300 in 8-10% of clinically diagnosed cases. Both paralogs act in chromatin remodeling and encode for transcriptional co-activators interacting with >400 proteins. Up to now 26 individuals with an EP300 mutation have been published. Here, we describe the phenotype and genotype of 42 unpublished RSTS patients carrying EP300 mutations and intragenic deletions and offer an update on another 10 patients. We compare the data to 308 individuals with CREBBP mutations. We demonstrate that EP300 mutations cause a phenotype that typically resembles the classical RSTS phenotype due to CREBBP mutations to a great extent, although most facial signs are less marked with the exception of a low-hanging columella. The limb anomalies are more similar to those in CREBBP mutated individuals except for angulation of thumbs and halluces which is very uncommon in EP300 mutated individuals. The intellectual disability is variable but typically less marked whereas the microcephaly is more common. All types of mutations occur but truncating mutations and small rearrangements are most common (86%). Missense mutations in the HAT domain are associated with a classical RSTS phenotype but otherwise no genotype-phenotype correlation is detected. Pre-eclampsia occurs in 12/52 mothers of EP300 mutated individuals versus in 2/59 mothers of CREBBP mutated individuals, making pregnancy with an EP300 mutated fetus the strongest known predictor for pre-eclampsia. © 2016 Wiley Periodicals, Inc.

Loss of function of KIAA2022 causes mild to severe intellectual disability with an autism spectrum disorder and impairs neurite outgrowth.

Existence of a discrete new X-linked intellectual disability (XLID) syndrome due to KIAA2022 deficiency was questioned by disruption of KIAA2022 by an X-chromosome pericentric inversion in a XLID family we reported in 2004. Three additional families with likely pathogenic KIAA2022 mutations were discovered within the frame of systematic parallel sequencing of familial cases of XLID or in the context of routine array-CGH evaluation of sporadic intellectual deficiency (ID) cases. The c.186delC and c.3597dupA KIAA2022 truncating mutations were identified by X-chromosome exome sequencing, while array CGH discovered a 70 kb microduplication encompassing KIAA2022 exon 1 in the third family. This duplication decreased KIAA2022 mRNA level in patients' lymphocytes by 60%. Detailed clinical examination of all patients, including the two initially reported, indicated moderate-to-severe ID with autistic features, strabismus in all patients, with no specific dysmorphic features other than a round face in infancy and no structural brain abnormalities on magnetic resonance imaging (MRI). Interestingly, the patient with decreased KIAA2022 expression had only mild ID with severe language delay and repetitive behaviors falling in the range of an autism spectrum disorder (ASD). Since little is known about KIAA2022 function, we conducted morphometric studies in cultured rat hippocampal neurons. We found that siRNA-mediated KIAA2022 knockdown resulted in marked impairment in neurite outgrowth including both the dendrites and the axons, suggesting a major role for KIAA2022 in neuron development and brain function.

Diversities in Leigh Syndrome Associated with MT-ATP6 Gene Variants.

INTRODUCTION: Leigh syndrome (LS) is clinically and genetically heterogeneous and presents defective mitochondrial bioenergetics. Patients present neurological symptoms and imagiological features that may result in early death [1]. The LS has been associated with mitochondrial DNA (mtDNA) variants, e.g., m.8993T>G (L156R) and m.8993T>C (L156P), in the MT-ATP6 gene. They lead to the substitution of a highly conserved amino acid in subunit 6 of ATP synthase, affecting the F0 domain and ATP synthesis [1-3]. We present five cases with m.8993T>G and a family harbouring m.8993T>C+m.1555A>G (proband and four relatives). METHODS: Our laboratory received 48 samples from LS-suspected patients. The samples (various tissues) were assessed for bioenergetics (activity of mitochondrial respiratory chain (MRC) complexes, ubiquinone content) and genetic analyses (mtDNA copy number, Sequencing and PCR-RFLP) by established protocols. RESULTS/CASE REPORT: Bioenergetics were assessed in 5 patients (various tissues) with varying levels of MRC/ATP synthase impairment. Six cases had a mtDNA pathogenic variant in the 8993 nucleotide associated with LS. Five cases presented the m.8993T>G variant, one of which (P5) possibly de novo. This variant was homoplasmy (P1-3) or very high heteroplasmy (P4/5, 90-95%). Of the four patients with bioenergetics assessment, three (P1/3/4) had deficiencies of MRC complexes, and P5 had small deficits. The other case (familial, proband and 4 relatives) presented a combination of m.1555A>G (homoplasmy) and m.8993T>C (heteroplasmy) variants. The proband presents m.8993T>C in 95% heteroplasmy and 85-35% in three relatives. All have m.1555A>G in homoplasmy, including the fourth relative without m.8993T>C. A deficiency (31%) was found in complex V activity in muscle for proband. CONCLUSION: We present a case series of patients harbouring pathogenic variants in the 8993 nucleotide of mtDNA, which have been associated with LS and impairment of MRC's complex V. These cases highlight the variability in clinical symptoms and their severity, as well as genetic heterogeneity within LS. Many patients will not present a classic pathogenic variant and there are many cases of asymptomatic relatives (carriers). It is important to get a broader view of the cases - classical methods and multiple tissue analysis are still valuable tools for the comprehensive characterization of patients.

Permanent neonatal diabetes mellitus due to KCNJ11 mutation in a Portuguese family: transition from insulin to oral sulfonylureas.

Permanent neonatal diabetes mellitus (PNDM) is a rare form of diabetes diagnosed within the first 6 months of life. Heterozygous activation mutations in KCNJ11, encoding the Kir6.2 subunit of the ATP-sensitive potassium (K(ATP)) channel, which acts as a key role in insulin secretion regulation, account for about half of the cases of PNDM. The majority of the patients represent isolated cases resulting from de novo mutations. Approximately 20% have associated neurologic features: the most severe form, which includes epilepsy and developmental delay, is called developmental delay, epilepsy, and neonatal diabetes (DEND) syndrome and the milder form, with less severe developmental delay and without epilepsy, is designated intermediate DEND syndrome. Individuals with KCNJ11 mutations have been successfully transitioned from insulin to sulfonylurea (SU) therapy. Furthermore, there have been cases reported with variable improvement in neurological function following a successful switching. We describe a 12-year-old Portuguese girl with PNDM due to the previously reported R201C mutation in the KCNJ11 gene. Her medical history includes prematurity and moderate developmental delay. The mutation was inherited from her mother who has isolated PNDM. The patient was successfully transferred from insulin to SU, whereas her mother showed SU resistance. Despite good glycemic control, no improvements in the cognitive performance were verified. We present our experience in switching treatment from insulin to oral SUs in this family, and also discuss whether or not the girl's developmental delay is related with the Kir6.2 mutation. To our knowledge, this is the first Portuguese patient reported with successful transition to SU treatment.

New ocular findings in a patient with a novel pathogenic variant in the FBXO11 gene.

Intellectual developmental disorder with dysmorphic facies and behavioral abnormalities (IDDFBA) is a recently described autosomal dominant entity caused by pathogenic variants, mostly de novo, in the FBXO11 gene. It presents in the first years of life with highly variable clinical manifestations. The main features of IDDFBA include borderline-to-severe intellectual disability, behavioral problems, hypotonia, facial dysmorphisms, minor skeletal abnormalities, and recurrent infections. Although eye problems, such as refractive errors, eye misalignment and minor visual changes, have been described in about 48% of patients, a major ocular defect, namely, bilateral optic nerve hypoplasia, has been reported in the literature only once. We report an 8-year-old boy with a novel de novo pathogenic variant in FBXO11 gene (NM_001190274.1: c.1166dup, p.Cys390Metfs∗3) and a complex ophthalmological phenotype, consisting of right microphthalmia, very shallow anterior chamber, and persistent pupillary membrane, right dense nuclear cataract, bilateral optic nerve hypoplasia, and bilateral horizontal manifest nystagmus.

Loss of FOCAD, operating via the SKI messenger RNA surveillance pathway, causes a pediatric syndrome with liver cirrhosis.

Cirrhosis is usually a late-onset and life-threatening disease characterized by fibrotic scarring and inflammation that disrupts liver architecture and function. While it is typically the result of alcoholism or hepatitis viral infection in adults, its etiology in infants is much less understood. In this study, we report 14 children from ten unrelated families presenting with a syndromic form of pediatric liver cirrhosis. By genome/exome sequencing, we found recessive variants in FOCAD segregating with the disease. Zebrafish lacking focad phenocopied the human disease, revealing a signature of altered messenger RNA (mRNA) degradation processes in the liver. Using patient's primary cells and CRISPR-Cas9-mediated inactivation in human hepatic cell lines, we found that FOCAD deficiency compromises the SKI mRNA surveillance pathway by reducing the levels of the RNA helicase SKIC2 and its cofactor SKIC3. FOCAD knockout hepatocytes exhibited lowered albumin expression and signs of persistent injury accompanied by CCL2 overproduction. Our results reveal the importance of FOCAD in maintaining liver homeostasis and disclose a possible therapeutic intervention point via inhibition of the CCL2/CCR2 signaling axis.

Prenatal diagnosis of persistent urogenital sinus with duplicated hydrometrocolpos and ascites--a case report.

We report a successful case of persistent urogenital sinus associated with a duplicated nonsyndromic form of hydrometrocolpos and ascites diagnosed prenatally. Though urogenital malformations are extremely rare and variable in presentation, the conjugation of those anomalies in a newborn is reported here for the first time. Prenatal ultrasound diagnosis was suspected at 25 weeks' gestation and MRI imaging supported the diagnosis. Periodic ultrasound surveillance was conducted until birth at 35 weeks' gestation by cesarean section. The presumptive diagnosis was confirmed after birth. One month later, the newborn underwent reconstructive surgical intervention with good outcome.

NGLY1 deficiency-A rare congenital disorder of deglycosylation.

Pathogenic variants in the NGLY1 gene are associated with a Congenital Disorder of Deglycosylation (CDDG) characterized by delays in reaching developmental milestones, complex hyperkinetic movement disorder, transient elevation of transaminases, and alacrima or hypolacrima. To date, only few cases of NGLY1 deficiency have been identified and reported in the literature. This report highlights a first child of non-consanguineous parents with no relevant family history who presented with hypotonia and poor weight gain since birth. At 2 months, the child developed paroxysmal cervical dystonia, posteriorly resolving spontaneously by age of 3. Subsequently, delays in reaching developmental milestones, ataxia, dyskinesia, visual impairment due to cone rod retinal dystrophy, low triglycerides, and persistently elevated liver transaminases were observed. Extensive etiological investigation was performed, including array-CGH and metabolic evaluation with no abnormalities to note. Trio whole exome analysis identified a homozygous pathogenic variant of the NGLY1 gene, c.1891del (p.Gln631Serfs*7), consistent with CDDG. Both parents were confirmed to be heterozygous carriers. The authors discuss in this case, the clinical presentation, the diagnostic challenges, and review other relevant NGLY1 deficiency cases previously reported in the literature. This case, along with the previous reported in the literature, indicates that pathogenic variants in NGLY1 cause a recognizable phenotype and should be considered in patients with a typical presentation. It also suggests that decreased sweating is not present universally in these patients.

Short Stature on a Boy: Mosaicism with an Isodicentric Y Chromosome.

Mosaicism brings great variability into the clinical expression of numerical and structural chromosomal abnormalities. The phenotypic variability of 45,X/46,XY mosaicism extends from Turner syndrome to apparently physically normal males. We present a case of a 14-year-old adolescent with short stature and delayed puberty, who was admitted in a Paediatric Endocrinology outpatient clinic. After a careful investigation, he was found to have a 45,X/46,X,idic(Y)(p11.32) mosaicism. This case report emphasizes the wide range of etiologies that can be involved in short stature and that chromosomal study is an important tool when firstly approaching males with short stature, avoiding unnecessary tests. There is an important clinical need for gonadal follow-up in this situation and for support in the decision about sex of rearing and sex orientation, when justifiable.

Targeted next generation sequencing identifies novel pathogenic variants and provides molecular diagnoses in a cohort of pediatric and adult patients with unexplained mitochondrial dysfunction.

Mitochondrial diseases (MD) are a group of rare inherited disorders, characterized by phenotypic heterogeneity, with hitherto no effective therapeutic options. The aim of this study was to develop a next generation sequencing (NGS) strategy, by using a custom gene panel and whole mitochondrial genome, to identify the disease causing pathogenic variants in 146 patients suspicious of MD. The molecular analysis of this cohort revealed six novel and 15 described pathogenic variants, as well as 26 variants of unknown significance. Our findings are expanding the mutational landscape of these disorders and support the use of a NGS strategy for a higher diagnostic yield.

Genomic imbalances defining novel intellectual disability associated loci.

BACKGROUND: High resolution genome-wide copy number analysis, routinely used in clinical diagnosis for several years, retrieves new and extremely rare copy number variations (CNVs) that provide novel candidate genes contributing to disease etiology. The aim of this work was to identify novel genetic causes of neurodevelopmental disease, inferred from CNVs detected by array comparative hybridization (aCGH), in a cohort of 325 Portuguese patients with intellectual disability (ID). RESULTS: We have detected CNVs in 30.1% of the patients, of which 5.2% corresponded to novel likely pathogenic CNVs. For these 11 rare CNVs (which encompass novel ID candidate genes), we identified those most likely to be relevant, and established genotype-phenotype correlations based on detailed clinical assessment. In the case of duplications, we performed expression analysis to assess the impact of the rearrangement. Interestingly, these novel candidate genes belong to known ID-related pathways. Within the 8% of patients with CNVs in known pathogenic loci, the majority had a clinical presentation fitting the phenotype(s) described in the literature, with a few interesting exceptions that are discussed. CONCLUSIONS: Identification of such rare CNVs (some of which reported for the first time in ID patients/families) contributes to our understanding of the etiology of ID and for the ever-improving diagnosis of this group of patients.

Peutz-Jeghers syndrome: early clinical expression of a new STK11 gene variant.

Genetic heterogeneity has been recognised in Peutz-Jeghers syndrome (PJS) (over 230 STK11 gene mutations reported). We report a rare PJS phenotype with early extensive gastrointestinal (GI) presentation and a new genetic variant. The case presented as haematochezia and mucocutaneous pigmentation (the patient was 3 years of age). Endoscopy showed several polyps throughout the stomach/colon (PJ-type hamartomas); the larger polyps were resected. Small bowel imaging detected multiple jejunum/ileum small polyps. During 8 years of follow-up of this asymptomatic patient, an increasing number of diffusely distributed polyps was observed and polypectomies were performed. Subsequently, the patient failed consultations; when the patient was 13 years of age, emergency surgery was required due to small bowel intussusception (ileal polyp). A STK11 gene study identified two missense variants in heterozygous (yet unknown significance but probably pathogenic): c.854T>A (exon 6) and c.446C>T* (exon 2) (*not previously reported). We report two STK11 gene variants (one not previously described) of yet undetermined causality in a paediatric patient presenting with extensive GI involvement at a very early age, with no family medical history. Structural and functional repercussion of the newly described variants should be further investigated.

Two families with sibling recurrence of the 17q21.31 microdeletion syndrome due to low-grade mosaicism.

The 17q21.31 microdeletion syndrome is characterised by intellectual disability, epilepsy, distinctive facial dysmorphism, and congenital anomalies. To date, all individuals reported with this syndrome have been simplex patients, resulting from de novo deletions. Here, we report sibling recurrence of the 17q21.31 microdeletion syndrome in two independent families. In both families, the mother was confirmed to be the parent-of-origin for the 17q21.31 deletion. Fluorescence in situ hybridisation analyses in buccal mucosa cells, of the mother of family 1, identified monosomy 17q21.31 in 4/50 nuclei (8%). In mother of family 2, the deletion was identified in 2/60 (3%) metaphase and in 3/100 (3%) interphase nuclei in peripheral lymphocytes, and in 7/100 (7%) interphase nuclei in buccal cells. A common 17q21.31 inversion polymorphism predisposes to non-allelic homologous recombination and hereby to the 17q21.31 microdeletion syndrome. On the basis of the 17q21.31 inversion status of the parents, we calculated that the probability of the second deletion occurring by chance alone was 1/14,438 and 1/4812, respectively. If the inversion status of the parents of a child with the 17q21.31 microdeletion syndrome is unknown, the overall risk of a second child with the 17q21.31 microdeletion is 1/9461. We conclude that the presence of low-level maternal somatic-gonadal mosaicism is associated with the microdeletion recurrence in these families. This suggests that the recurrence risk for parents with a child with a 17q21.31 microdeletion for future pregnancies is higher than by chance alone and testing for mosaicism in the parents might be considered as a helpful tool in the genetic counselling.