Boricua Founder Variant in FRRS1L Causes Epileptic Encephalopathy With Hyperkinetic Movements.

OBJECTIVE: To describe a founder mutation effect and the clinical phenotype of homozygous FRRS1L c.737_739delGAG (p.Gly246del) variant in 15 children of Puerto Rican (Boricua) ancestry presenting with early infantile epileptic encephalopathy (EIEE-37) with prominent movement disorder. BACKGROUND: EIEE-37 is caused by biallelic loss of function variants in the FRRS1L gene, which is critical for AMPA-receptor function, resulting in intractable epilepsy and dyskinesia. METHODS: A retrospective, multicenter chart review of patients sharing the same homozygous FRRS1L (p.Gly246del) pathogenic variant identified by clinical genetic testing. Clinical information was collected regarding neurodevelopmental outcomes, neuroimaging, electrographic features and clinical response to antiseizure medications. RESULTS: Fifteen patients from 12 different families of Puerto Rican ancestry were homozygous for the FRRS1L (p.Gly246del) pathogenic variant, with ages ranging from 1 to 25 years. The onset of seizures was from 6 to 24 months. All had hypotonia, severe global developmental delay, and most had hyperkinetic involuntary movements. Developmental regression during the first year of life was common (86%). Electroencephalogram showed hypsarrhythmia in 66% (10/15), with many older children evolving into Lennox-Gastaut syndrome. Six patients demonstrated progressive volume loss and/or cerebellar atrophy on brain magnetic resonance imaging (MRI). CONCLUSIONS: We describe the largest cohort to date of patients with epileptic encephalopathy. We estimate that 0.76% of unaffected individuals of Puerto Rican ancestry carry this pathogenic variant due to a founder effect. Children homozygous for the FRRS1L (p.Gly246del) Boricua variant exhibit a very homogenous phenotype of early developmental regression and epilepsy, starting with infantile spasms and evolving into Lennox-Gastaut syndrome with hyperkinetic movement disorder.

Missense variant contribution to USP9X-female syndrome.

USP9X is an X-chromosome gene that escapes X-inactivation. Loss or compromised function of USP9X leads to neurodevelopmental disorders in males and females. While males are impacted primarily by hemizygous partial loss-of-function missense variants, in females de novo heterozygous complete loss-of-function mutations predominate, and give rise to the clinically recognisable USP9X-female syndrome. Here we provide evidence of the contribution of USP9X missense and small in-frame deletion variants in USP9X-female syndrome also. We scrutinise the pathogenicity of eleven such variants, ten of which were novel. Combined application of variant prediction algorithms, protein structure modelling, and assessment under clinically relevant guidelines universally support their pathogenicity. The core phenotype of this cohort overlapped with previous descriptions of USP9X-female syndrome, but exposed heightened variability. Aggregate phenotypic information of 35 currently known females with predicted pathogenic variation in USP9X reaffirms the clinically recognisable USP9X-female syndrome, and highlights major differences when compared to USP9X-male associated neurodevelopmental disorders.

A case of 5,10-methenyltetrahydrofolate synthetase deficiency due to biallelic null mutations with novel findings of elevated neopterin and macrocytic anemia.

We describe a case of 5,10-methenyltetrahydrofolate synthetase (MTHFS) deficiency characterized by microcephaly, global developmental delay, epilepsy, and cerebral hypomyelination. Whole exome sequencing (WES) demonstrated homozygosity for the R74X mutation in the MTHFS gene. The patient had the unexpected finding of elevated cerebrospinal fluid (CSF) neopterin. The novel finding of macrocytic anemia in this patient may provide a clue to the diagnosis of this rare neurometabolic disorder.

Neuronal mechanisms of mutations in SCN8A causing epilepsy or intellectual disability.

Ion channel mutations can cause distinct neuropsychiatric diseases. We first studied the biophysical and neurophysiological consequences of four mutations in the human Na+ channel gene SCN8A causing either mild (E1483K) or severe epilepsy (R1872W), or intellectual disability and autism without epilepsy (R1620L, A1622D). Only combined electrophysiological recordings of transfected wild-type or mutant channels in both neuroblastoma cells and primary cultured neurons revealed clear genotype-phenotype correlations. The E1483K mutation causing mild epilepsy showed no significant biophysical changes, whereas the R1872W mutation causing severe epilepsy induced clear gain-of-function biophysical changes in neuroblastoma cells. However, both mutations increased neuronal firing in primary neuronal cultures. In contrast, the R1620L mutation associated with intellectual disability and autism-but not epilepsy-reduced Na+ current density in neuroblastoma cells and expectedly decreased neuronal firing. Interestingly, for the fourth mutation, A1622D, causing severe intellectual disability and autism without epilepsy, we observed a dramatic slowing of fast inactivation in neuroblastoma cells, which induced a depolarization block in neurons with a reduction of neuronal firing. This latter finding was corroborated by computational modelling. In a second series of experiments, we recorded three more mutations (G1475R, M1760I, G964R, causing intermediate or severe epilepsy, or intellectual disability without epilepsy, respectively) that revealed similar results confirming clear genotype-phenotype relationships. We found intermediate or severe gain-of-function biophysical changes and increases in neuronal firing for the two epilepsy-causing mutations and decreased firing for the loss-of-function mutation causing intellectual disability. We conclude that studies in neurons are crucial to understand disease mechanisms, which here indicate that increased or decreased neuronal firing is responsible for distinct clinical phenotypes.

Refining the phenotype associated with GNB1 mutations: Clinical data on 18 newly identified patients and review of the literature.

De novo germline mutations in GNB1 have been associated with a neurodevelopmental phenotype. To date, 28 patients with variants classified as pathogenic have been reported. We add 18 patients with de novo mutations to this cohort, including a patient with mosaicism for a GNB1 mutation who presented with a milder phenotype. Consistent with previous reports, developmental delay in these patients was moderate to severe, and more than half of the patients were non-ambulatory and nonverbal. The most observed substitution affects the p.Ile80 residue encoded in exon 6, with 28% of patients carrying a variant at this residue. Dystonia and growth delay were observed more frequently in patients carrying variants in this residue, suggesting a potential genotype-phenotype correlation. In the new cohort of 18 patients, 50% of males had genitourinary anomalies and 61% of patients had gastrointestinal anomalies, suggesting a possible association of these findings with variants in GNB1. In addition, cutaneous mastocytosis, reported once before in a patient with a GNB1 variant, was observed in three additional patients, providing further evidence for an association to GNB1. We will review clinical and molecular data of these new cases and all previously reported cases to further define the phenotype and establish possible genotype-phenotype correlations.

PUF60 variants cause a syndrome of ID, short stature, microcephaly, coloboma, craniofacial, cardiac, renal and spinal features.

PUF60 encodes a nucleic acid-binding protein, a component of multimeric complexes regulating RNA splicing and transcription. In 2013, patients with microdeletions of chromosome 8q24.3 including PUF60 were found to have developmental delay, microcephaly, craniofacial, renal and cardiac defects. Very similar phenotypes have been described in six patients with variants in PUF60, suggesting that it underlies the syndrome. We report 12 additional patients with PUF60 variants who were ascertained using exome sequencing: six through the Deciphering Developmental Disorders Study and six through similar projects. Detailed phenotypic analysis of all patients was undertaken. All 12 patients had de novo heterozygous PUF60 variants on exome analysis, each confirmed by Sanger sequencing: four frameshift variants resulting in premature stop codons, three missense variants that clustered within the RNA recognition motif of PUF60 and five essential splice-site (ESS) variant. Analysis of cDNA from a fibroblast cell line derived from one of the patients with an ESS variants revealed aberrant splicing. The consistent feature was developmental delay and most patients had short stature. The phenotypic variability was striking; however, we observed similarities including spinal segmentation anomalies, congenital heart disease, ocular colobomata, hand anomalies and (in two patients) unilateral renal agenesis/horseshoe kidney. Characteristic facial features included micrognathia, a thin upper lip and long philtrum, narrow almond-shaped palpebral fissures, synophrys, flared eyebrows and facial hypertrichosis. Heterozygote loss-of-function variants in PUF60 cause a phenotype comprising growth/developmental delay and craniofacial, cardiac, renal, ocular and spinal anomalies, adding to disorders of human development resulting from aberrant RNA processing/spliceosomal function.

Gain-of-Function Mutations in RARB Cause Intellectual Disability with Progressive Motor Impairment.

Retinoic acid (RA) signaling plays a key role in the development and function of several systems in mammals. We previously discovered that the de novo mutations c.1159C>T (p.Arg387Cys) and c.1159C>A (p.Arg387Ser) in the RA Receptor Beta (RARB) gene cause microphthalmia and diaphragmatic hernia. However, the natural history of affected subjects beyond the prenatal or neonatal period was unknown. Here, we describe nine additional subjects with microphthalmia who have de novo mutations in RARB, including the previously described p.Arg387Cys as well as the novel c.887G>C (p.Gly296Ala) and c.638T>C (p.Leu213Pro). Moreover, we review the information on four previously reported cases. All subjects who survived the neonatal period (n = 10) displayed severe global developmental delay with progressive motor impairment due to spasticity and/or dystonia (with or without chorea). The majority of subjects also showed Chiari type I malformation and severe feeding difficulties. We previously found that p.Arg387Cys and p.Arg387Ser induce a gain-of-function. We show here that the p.Gly296Ala and p.Leu213Pro RARB mutations further promote the RA ligand-induced transcriptional activity by twofold to threefold over the wild-type receptor, also indicating a gain-of-function mechanism. These observations suggest that precise regulation of RA signaling is required for brain development and/or function in humans.

Deletion of and novel missense mutation in POU3F4 in 2 families segregating X-linked nonsyndromic deafness.

OBJECTIVE: To analyze the physical manifestations and genetic features of 2 families segregating X-linked deafness, which is most commonly reported to be caused by mutations of the POU domain gene POU3F4 at the DFN3 locus. DESIGN: Computed tomographic study of the temporal bone in probands from each family, followed by mutation screening and deletion mapping of POU3F4 in family members. SETTING: Two midwestern genetics clinics. PARTICIPANTS: Two families with X-linked deafness. MAIN OUTCOME MEASURES: Anomalies of the inner ear in the probands; results of gene mapping and severity and effects of hearing loss in the family members. RESULTS: In the first family, a large deletion was identified that includes POU3F4 and extends upstream approximately 530 kilobases; in the second family, a novel serine-to-leucine (S228L) amino acid mutation was identified in the POU-specific domain of POU3F4. Both the deletion and the missense mutation segregate with the clinical phenotype and are causally related to the deafness in these families. CONCLUSIONS: Deafness related to the POU3F4 gene is associated with dilation of the internal auditory canal and a spectrum of other temporal bone anomalies that range in severity from mild to severe dysplasia of the cochlea and semicircular canals. The consequence of these anomalies is a congenital mixed hearing loss, the sensorineural component of which progresses over time. Affected males can also present with vestibular dysfunction that is associated with delayed developmental motor milestones. Intrafamilial variability occurs.

Activation of p21-activated kinase 6 by MAP kinase kinase 6 and p38 MAP kinase.

The p21-activated kinases (PAKs) contain an N-terminal Cdc42/Rac interactive binding domain, which in the group 1 PAKs (PAK1, 2, and 3) regulates the activity of an adjacent conserved autoinhibitory domain. In contrast, the group 2 PAKs (PAK4, 5, and 6) lack this autoinhibitory domain and are not activated by Cdc42/Rac binding, and the mechanisms that regulate their kinase activity have been unclear. This study found that basal PAK6 kinase activity was repressed by a p38 mitogen-activated protein (MAP) kinase antagonist and could be strongly stimulated by constitutively active MAP kinase kinase 6 (MKK6), an upstream activator of p38 MAP kinases. Mutation of a consensus p38 MAP kinase target site at serine 165 decreased PAK6 kinase activity. Moreover, PAK6 was directly activated by MKK6, and mutation of tyrosine 566 in a consensus MKK6 site (threonine-proline-tyrosine, TPY) in the activation loop of the PAK6 kinase domain prevented activation by MKK6. PAK6 activation by MKK6 was also blocked by mutation of an autophosphorylated serine (serine 560) in the PAK6 activation loop, indicating that phosphorylation of this site is necessary for MKK6-mediated activation. PAK4 and PAK5 were similarly activated by MKK6, consistent with a conserved TPY motif in their activation domains. The activation of PAK6 by both p38 MAP kinase and MKK6 suggests that PAK6 plays a role in the cellular response to stress-related signals.

Large germline deletions of mitochondrial complex II subunits SDHB and SDHD in hereditary paraganglioma.

More than 30% of adrenal pheochromocytomas are hereditary. These neuroendocrine tumors are major components of three inherited cancer syndromes: multiple endocrine neoplasia type 2, von Hippel-Lindau disease (VHL), and pheochromocytoma/paraganglioma syndrome (PC/PGL). Germline mutations in RET; VHL; and SDHB, SDHC, and SDHD are associated with multiple endocrine neoplasia type 2, VHL, and PC/PGL, respectively. The majority (>70%) of hereditary extraadrenal PCs [catecholamine-secreting paragangliomas (PGL)] are accounted for by germline intragenic mutations in SDHB, SDHC, or SDHD. Therefore, a subset of hereditary PGL is not accounted for. Here we report two unrelated hereditary PGL families, one with a germline whole-gene deletion of SDHD (family 4194), the other a partial deletion of SDHB (family BRZ01). Although they were initially designated mutation negative for all of the PC-associated genes after PCR-based analysis, we suspected that a large deletion or rearrangement might be present. Genotyping around the PC-associated genes demonstrated that both families were consistent with linkage with one of these genes. Using fine structure genotyping and semiquantitative duplex PCR analysis, we identified an approximately 96-kb deletion spanning SDHD in family 4194 and an approximately 1-kb deletion involving the 5' end of SDHB in family BRZ01. Thus, including SDHB and SDHD deletion analysis could increase gene-testing sensitivity for PGL patients, which would aid in genetic counseling and management of patients and families.

Temtamy-like syndrome associated with translocation of 2p24 and 9q32.

We describe the phenotype of a 5 year old girl with features resembling Temtamy syndrome, including agenesis of the corpus callosum, ventriculomegaly, frontal bossing, peaked eyebrows, ptosis, malformed and low set ears, a depressed nasal bridge, a long philtrum, and iris and chorioretinal colobomas. Features unique to this child include profound mental retardation, bilateral sensorineural hearing loss, agenesis of the corpus callosum, patent ductus arteriosus, ventricular septal defect, unilateral renal agenesis, neurogenic bladder and hydronephrosis. High resolution chromosome analysis demonstrated a de novo, balanced translocation [46,XX,t(2;9)(p24;q32)]; and her case has some overlapping phenotypic features with cases of monosomy for 2p. This is the first documented case of Temtamy syndrome with a specific chromosomal anomaly, and will assist with the elucidation of the syndrome's underlying genetic defect.

Deoxyribonuclease I-like III is an inducible macrophage barrier to liposomal transfection.

Extra- and intracellular nucleases are predicted to decrease the in vivo efficiency of liposomal transfection. DNASE1 (D1) has been proposed as the main nuclease barrier, yet liposome-complexed DNA and in vitro lipofection are generally immune to D1. In contrast, medium conditioned by the macrophage enzyme DNASE1-like 3 (DNASE1L3 or D3) erects a potent in vitro barrier to liposomal transfection. Although homologous to D1 over its amino-terminal sequence, D3 has a distinct, highly basic carboxy terminus, which resembles polylysine stretches often found in polycationic liposomal reagents. If this domain is truncated from D3, the resulting enzyme has more nuclease activity against naked DNA ("free DNA"-nuclease activity), yet does not block transfection. C-terminal fusion of this domain to D1 forms a chimeric protein able to block transfection. D3 can be immunodetected in both serum and macrophage lysates. Macrophage-conditioned medium contains both "free DNA"-nuclease activity and the ability to block transfection, and by zymogram only a 28-kDa DNASE, consistent by size with D3, is present. Thus, medium containing D3 confers to cells an in vivo shield to the nuclear acquisition of exogenous DNA. Modulation and further elucidation of this activity are likely to have importance for both gene therapy and autoimmune disorders.