Novel presentation of AADC deficiency as a mild phenotype with exercise-induced dystonic crises: A case report.

Aromatic l-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive neurometabolic disorder caused by biallelic pathogenic variants in the DDC gene; approximately 140 patients have been described worldwide. AADC deficiency is characterised by a combined deficiency of dopamine, serotonin, adrenaline and noradrenaline causing a highly variable phenotype with developmental delay, early-onset hypotonia, movement disorders and autonomic symptoms. We expand the phenotype of this neurometabolic disorder by reporting on a paediatric patient with a mild phenotype with atypical exercise-induced dystonic crises, a feature that has not been described in AADC deficiency up till now. Additionally, we also present a second patient with typical characteristics and a severe phenotype. The diagnosis in both patients was confirmed by the presence of a homozygous pathogenic variant in the DDC gene and reduced AADC enzyme plasma activity. The use of whole exome sequencing-based strategies has played a crucial role in diagnosing these two patients.

Role of CAMK2D in neurodevelopment and associated conditions.

The calcium/calmodulin-dependent protein kinase type 2 (CAMK2) family consists of four different isozymes, encoded by four different genes-CAMK2A, CAMK2B, CAMK2G, and CAMK2D-of which the first three have been associated recently with neurodevelopmental disorders. CAMK2D is one of the major CAMK2 proteins expressed in the heart and has been associated with cardiac anomalies. Although this CAMK2 isoform is also known to be one of the major CAMK2 subtypes expressed during early brain development, it has never been linked with neurodevelopmental disorders until now. Here we show that CAMK2D plays an important role in neurodevelopment not only in mice but also in humans. We identified eight individuals harboring heterozygous variants in CAMK2D who display symptoms of intellectual disability, delayed speech, behavioral problems, and dilated cardiomyopathy. The majority of the variants tested lead to a gain of function (GoF), which appears to cause both neurological problems and dilated cardiomyopathy. In contrast, loss-of-function (LoF) variants appear to induce only neurological symptoms. Together, we describe a cohort of individuals with neurodevelopmental disorders and cardiac anomalies, harboring pathogenic variants in CAMK2D, confirming an important role for the CAMK2D isozyme in both heart and brain function.

Functional and clinical studies reveal pathophysiological complexity of CLCN4-related neurodevelopmental condition.

Missense and truncating variants in the X-chromosome-linked CLCN4 gene, resulting in reduced or complete loss-of-function (LOF) of the encoded chloride/proton exchanger ClC-4, were recently demonstrated to cause a neurocognitive phenotype in both males and females. Through international clinical matchmaking and interrogation of public variant databases we assembled a database of 90 rare CLCN4 missense variants in 90 families: 41 unique and 18 recurrent variants in 49 families. For 43 families, including 22 males and 33 females, we collated detailed clinical and segregation data. To confirm causality of variants and to obtain insight into disease mechanisms, we investigated the effect on electrophysiological properties of 59 of the variants in Xenopus oocytes using extended voltage and pH ranges. Detailed analyses revealed new pathophysiological mechanisms: 25% (15/59) of variants demonstrated LOF, characterized by a "shift" of the voltage-dependent activation to more positive voltages, and nine variants resulted in a toxic gain-of-function, associated with a disrupted gate allowing inward transport at negative voltages. Functional results were not always in line with in silico pathogenicity scores, highlighting the complexity of pathogenicity assessment for accurate genetic counselling. The complex neurocognitive and psychiatric manifestations of this condition, and hitherto under-recognized impacts on growth, gastrointestinal function, and motor control are discussed. Including published cases, we summarize features in 122 individuals from 67 families with CLCN4-related neurodevelopmental condition and suggest future research directions with the aim of improving the integrated care for individuals with this diagnosis.

A Severe Form of Familial Desminopathy Due to a Homozygous Nonsense DES Variant in Two Siblings.

Familial primary desminopathies are usually autosomal dominantly inherited and present at the age of 20 to 40 years with progressive muscle weakness and atrophy, cardiomyopathy, and cardiac arrhythmias. Cardiac features may precede the muscular weakness. Here, we report the rare case of two siblings presenting with a desminopathy at pediatric age, due to homozygous nonsense variations (c.700G > T [p.Glu234Ter]) in DES, representing an autosomal recessive inheritance pattern. The homozygous state of these variants is expected to result in the complete absence of desmin production. Rare autosomal recessive DES variants are associated with an earlier clinical presentation (from childhood to early adulthood) and faster evolution compared with more common autosomal dominant variants. A normal resting electrocardiography (ECG) and cardiac ultrasound can be a pitfall, as seen in our patient who has extensive fibrotic scarring on cardiac magnetic resonance imaging (MRI). We recommend yearly cardiac ultrasound, yearly 24-hour Holter monitoring and 2 yearly cardiac MRI from the age of 10 years in all asymptomatic patients. Heterozygous patients usually have no or only mild complaints but, though not yet reported in autosomal recessive desminopathies, muscular complaints are possible, as seen in the father of our patients. The prognosis for these patients with desminopathy presenting in childhood is unpredictable but anticipated as poor.

L-Serine Treatment is Associated with Improvements in Behavior, EEG, and Seizure Frequency in Individuals with GRIN-Related Disorders Due to Null Variants.

Pathogenic missense variants in GRIN2A and GRIN2B may result in gain or loss of function (GoF/LoF) of the N-methyl-D-aspartate receptor (NMDAR). This observation gave rise to the hypothesis of successfully treating GRIN-related disorders due to LoF variants with co-agonists of the NMDAR. In this respect, we describe a retrospectively collected series of ten individuals with GRIN2A- or GRIN2B-related disorders who were treated with L-serine, each within an independent n-of-1 trial. Our cohort comprises one individual with a LoF missense variant with clinical improvements confirming the above hypothesis and replicating a previous n-of-1 trial. A second individual with a GoF missense variant was erroneously treated with L-serine and experienced immediate temporary behavioral deterioration further supporting the supposed functional pathomechanism. Eight additional individuals with null variants (that had been interpreted as loss-of-function variants despite not being missense) again showed clinical improvements. Among all nine individuals with LoF missense or null variants, L-serine treatment was associated with improvements in behavior in eight (89%), in development in four (44%), and/or in EEG or seizure frequency in four (44%). None of these nine individuals experienced side effects or adverse findings in the context of L-serine treatment. In summary, we describe the first evidence that L-serine treatment may not only be associated with clinical improvements in GRIN-related disorders due to LoF missense but particularly also null variants.

Genetic Testing Contributes to Diagnosis in Cerebral Palsy: Aicardi-Goutières Syndrome as an Example.

Cerebral palsy (CP) is a non-progressive neurodevelopmental disorder characterized by motor impairments, often accompanied by co-morbidities such as intellectual disability, epilepsy, visual and hearing impairment and speech and language deficits. Despite the established role of hypoxic-ischemic injury in some CP cases, several studies suggest that birth asphyxia is actually an uncommon cause, accounting for <10% of CP cases. For children with CP in the absence of traditional risk factors, a genetic basis to their condition is increasingly suspected. Several recent studies indeed confirm copy number variants and single gene mutations with large genetic heterogeneity as an etiology in children with CP. Here, we report three patients with spastic cerebral palsy and a genetically confirmed diagnosis of Aicardi-Goutières syndrome (AGS), with highly variable phenotypes ranging from clinically suggestive to non-specific symptomatology. Our findings suggest that AGS may be a rather common cause of CP, that frequently remains undiagnosed without additional genetic testing, as in only one case a clinical suspicion of AGS was raised. Our data show that a diagnosis of AGS must be considered in cases with spastic CP, even in the absence of characteristic brain abnormalities. Importantly, a genetic diagnosis of AGS may have significant therapeutic consequences, as targeted therapies are being developed for type 1 interferonopathies, the group of diseases to which AGS belongs. Our findings demonstrate the importance of next generation sequencing in CP patients without an identifiable cause, since targeted diagnostic testing is hampered by the often non-specific presentation.

Biallelic MADD variants cause a phenotypic spectrum ranging from developmental delay to a multisystem disorder.

In pleiotropic diseases, multiple organ systems are affected causing a variety of clinical manifestations. Here, we report a pleiotropic disorder with a unique constellation of neurological, endocrine, exocrine, and haematological findings that is caused by biallelic MADD variants. MADD, the mitogen-activated protein kinase (MAPK) activating death domain protein, regulates various cellular functions, such as vesicle trafficking, activity of the Rab3 and Rab27 small GTPases, tumour necrosis factor-α (TNF-α)-induced signalling and prevention of cell death. Through national collaboration and GeneMatcher, we collected 23 patients with 21 different pathogenic MADD variants identified by next-generation sequencing. We clinically evaluated the series of patients and categorized the phenotypes in two groups. Group 1 consists of 14 patients with severe developmental delay, endo- and exocrine dysfunction, impairment of the sensory and autonomic nervous system, and haematological anomalies. The clinical course during the first years of life can be potentially fatal. The nine patients in Group 2 have a predominant neurological phenotype comprising mild-to-severe developmental delay, hypotonia, speech impairment, and seizures. Analysis of mRNA revealed multiple aberrant MADD transcripts in two patient-derived fibroblast cell lines. Relative quantification of MADD mRNA and protein in fibroblasts of five affected individuals showed a drastic reduction or loss of MADD. We conducted functional tests to determine the impact of the variants on different pathways. Treatment of patient-derived fibroblasts with TNF-α resulted in reduced phosphorylation of the extracellular signal-regulated kinases 1 and 2, enhanced activation of the pro-apoptotic enzymes caspase-3 and -7 and increased apoptosis compared to control cells. We analysed internalization of epidermal growth factor in patient cells and identified a defect in endocytosis of epidermal growth factor. We conclude that MADD deficiency underlies multiple cellular defects that can be attributed to alterations of TNF-α-dependent signalling pathways and defects in vesicular trafficking. Our data highlight the multifaceted role of MADD as a signalling molecule in different organs and reveal its physiological role in regulating the function of the sensory and autonomic nervous system and endo- and exocrine glands.

Recurrent NEDD4L Variant in Periventricular Nodular Heterotopia, Polymicrogyria and Syndactyly.

NEDD4L encodes an ubiquitin ligase which is expressed in the cortex and ventricular zone of the fetal brain. Missense variants in NEDD4L have been reported in nine patients with periventricular nodular heterotopia (PNH), polymicrogyria, cleft palate, and syndactyly. All reported variants are located in the HECT domain, causing deregulation of signaling pathways, including the AKT/mTOR pathway. Here we describe a first familial case with four affected members with a high degree of intra-familial phenotypic variability. Phenotypic features in the proband consisted of severe neurodevelopmental delay, refractory seizures, bilateral PNH, and perisylvian polymicrogyria. The other family members were less severely affected with mild developmental delay and isolated bilateral PNH. All family members had syndactyly. An unrelated patient presented with severe neurodevelopmental delay, seizures, and hypospadias, expanding the phenotypic spectrum. MRI revealed bilateral PNH and perisylvian polymicrogyria. All tested patients carry the recurrent variant c.623G > A, p.(Arg208Gln) in the WW domain of NEDD4L. The variant in the unrelated patient occurred de novo. This is the first report of a NEDD4L variant located in the WW domain which is probably involved in the recognition of substrates for ligation suggesting a loss of function variant.

Novel BRPF1 mutation in a boy with intellectual disability, coloboma, facial nerve palsy and hypoplasia of the corpus callosum.

Mutations in the chromatin regulator gene BRPF1 were recently associated with the Intellectual Developmental Disorder With Dysmorphic Facies And Ptosis (IDDDFP). Up till now, clinical data of 22 patients are reported. Besides intellectual disability (ID), ptosis and blepharophimosis are frequent findings, with refraction problems, amblyopia and strabism as other reported ophthalmological features. Animal studies indicate BRPF1 as an important mediator in brain development. However, only 5 of 22 previously reported patients show structural brain abnormalities. We report on an additional patient harboring a novel de novo nonsense mutation p.(Glu219*) in BRPF1. He presented with ID, bilateral iris colobomas, facial nerve palsy and severe hypoplasia of the corpus callosum. Our findings support previous findings of brain abnormalities in BRPF1-mutations and indicates coloboma and facial nerve palsy as possible additional features of IDDDFP syndrome.

Microhomology-mediated mechanisms underlie non-recurrent disease-causing microdeletions of the FOXL2 gene or its regulatory domain.

Genomic disorders are often caused by recurrent copy number variations (CNVs), with nonallelic homologous recombination (NAHR) as the underlying mechanism. Recently, several microhomology-mediated repair mechanisms--such as microhomology-mediated end-joining (MMEJ), fork stalling and template switching (FoSTeS), microhomology-mediated break-induced replication (MMBIR), serial replication slippage (SRS), and break-induced SRS (BISRS)--were described in the etiology of non-recurrent CNVs in human disease. In addition, their formation may be stimulated by genomic architectural features. It is, however, largely unexplored to what extent these mechanisms contribute to rare, locus-specific pathogenic CNVs. Here, fine-mapping of 42 microdeletions of the FOXL2 locus, encompassing FOXL2 (32) or its regulatory domain (10), serves as a model for rare, locus-specific CNVs implicated in genetic disease. These deletions lead to blepharophimosis syndrome (BPES), a developmental condition affecting the eyelids and the ovary. For breakpoint mapping we used targeted array-based comparative genomic hybridization (aCGH), quantitative PCR (qPCR), long-range PCR, and Sanger sequencing of the junction products. Microhomology, ranging from 1 bp to 66 bp, was found in 91.7% of 24 characterized breakpoint junctions, being significantly enriched in comparison with a random control sample. Our results show that microhomology-mediated repair mechanisms underlie at least 50% of these microdeletions. Moreover, genomic architectural features, like sequence motifs, non-B DNA conformations, and repetitive elements, were found in all breakpoint regions. In conclusion, the majority of these microdeletions result from microhomology-mediated mechanisms like MMEJ, FoSTeS, MMBIR, SRS, or BISRS. Moreover, we hypothesize that the genomic architecture might drive their formation by increasing the susceptibility for DNA breakage or promote replication fork stalling. Finally, our locus-centered study, elucidating the etiology of a large set of rare microdeletions involved in a monogenic disorder, can serve as a model for other clustered, non-recurrent microdeletions in genetic disease.

Disease-causing 7.4 kb cis-regulatory deletion disrupting conserved non-coding sequences and their interaction with the FOXL2 promotor: implications for mutation screening.

To date, the contribution of disrupted potentially cis-regulatory conserved non-coding sequences (CNCs) to human disease is most likely underestimated, as no systematic screens for putative deleterious variations in CNCs have been conducted. As a model for monogenic disease we studied the involvement of genetic changes of CNCs in the cis-regulatory domain of FOXL2 in blepharophimosis syndrome (BPES). Fifty-seven molecularly unsolved BPES patients underwent high-resolution copy number screening and targeted sequencing of CNCs. Apart from three larger distant deletions, a de novo deletion as small as 7.4 kb was found at 283 kb 5' to FOXL2. The deletion appeared to be triggered by an H-DNA-induced double-stranded break (DSB). In addition, it disrupts a novel long non-coding RNA (ncRNA) PISRT1 and 8 CNCs. The regulatory potential of the deleted CNCs was substantiated by in vitro luciferase assays. Interestingly, Chromosome Conformation Capture (3C) of a 625 kb region surrounding FOXL2 in expressing cellular systems revealed physical interactions of three upstream fragments and the FOXL2 core promoter. Importantly, one of these contains the 7.4 kb deleted fragment. Overall, this study revealed the smallest distant deletion causing monogenic disease and impacts upon the concept of mutation screening in human disease and developmental disorders in particular.

FOXL2 mutations and genomic rearrangements in BPES.

The FOXL2 gene is one of 10 forkhead genes, the mutations of which lead to human developmental disorders, often with ocular manifestations. Mutations in FOXL2 are known to cause blepharophimosis syndrome (BPES), an autosomal dominant eyelid malformation associated (type I) or not (type II) with ovarian dysfunction, leading to premature ovarian failure (POF). In addition, a few mutations have been described in patients with isolated POF. Here, we review all currently described FOXL2 sequence variations and genomic rearrangements in BPES and POF. Using a combined mutation detection approach, it is possible to identify the underlying genetic defect in a major proportion (88%) of typical BPES patients. Of all genetic defects found in our BPES cohort, intragenic mutations represent 81%. They include missense changes, frameshift and nonsense mutations, in-frame deletions, and duplications, that are distributed along the single-exon gene. Genomic rearrangements comprising both deletions encompassing FOXL2 and deletions located outside its transcription unit, represent 12% and 5% of all genetic defects in our BPES cohort, respectively. One of the challenges of genetic testing in BPES is the establishment of genotype-phenotype correlations, mainly with respect to the ovarian phenotype. Genetic testing should be performed in the context of genetic counseling, however, and should be systematically complemented by a multidisciplinary clinical follow-up. Another challenge for health care professionals involved in BPES is the treatment of the eyelid phenotype and the prevention or treatment of POF.

Identification of 34 novel and 56 known FOXL2 mutations in patients with Blepharophimosis syndrome.

Blepharophimosis syndrome (BPES) is caused by loss-of-function mutations in the single-exon forkhead transcription factor gene FOXL2 and by genomic rearrangements of the FOXL2 locus. Here, we focus on 92 new intragenic FOXL2 mutations, 34 of which are novel. Specifically, we found 10 nonsense mutations (11%), 13 missense mutations (14%), 40 deletions or insertions leading to a frameshift (43%), and 29 in-frame changes (32%), of which 28 (30%) lead to a polyalanine expansion. This study confirms the existence of two previously described mutational hotspots. Moreover, we gained novel insights in genotype-phenotype correlations, emphasizing the need to interpret genotype-phenotype correlations individually and always in the context of further clinical observations.

Missense mutations in the forkhead domain of FOXL2 lead to subcellular mislocalization, protein aggregation and impaired transactivation.

Mutations of the FOXL2 gene have been shown to cause blepharophimosis syndrome (BPES), characterized by an eyelid malformation associated with premature ovarian failure or not. Recently, polyalanine expansions and truncating FOXL2 mutations have been shown to lead to protein mislocalization, aggregation and altered transactivation. Here, we study the molecular consequences of 17 naturally occurring FOXL2 missense mutations. Most of them map to the conserved DNA-binding forkhead domain (FHD). The subcellular localization and aggregation pattern of the mutant FOXL2 proteins in COS-7 cells was variable and ranged from a diffuse nuclear distribution like the wild-type to extensive nuclear aggregation often in combination with cytoplasmic mislocalization and aggregation. We also studied the transactivation capacity of the mutants in FOXL2 expressing granulosa-like cells (KGN). Several mutants led to a loss-of-function, while others are suspected to induce a dominant negative effect. Interestingly, one mutant that is located outside the FHD (S217F), appeared to be hypermorphic and had no effect on intracellular protein distribution. This mutation gives rise to a mild BPES phenotype. In general, missense mutations located in the FHD lead to classical BPES and cannot be correlated with expression of the ovarian phenotype. However, a potential predictive value of localization and transactivation assays in the making of genotype-phenotype correlations is proposed. This is the first study to demonstrate that a significant number of missense mutations in the FHD of FOXL2 lead to mislocalization, protein aggregation and altered transactivation, and to provide insights into the pathogenesis associated with missense mutations of FOXL2 in human disease.

Recurrent mutation in the first zinc finger of the orphan nuclear receptor NR2E3 causes autosomal dominant retinitis pigmentosa.

"Autosomal dominant retinitis pigmentosa" (adRP) refers to a genetically heterogeneous group of retinal dystrophies, in which 54% of all cases can be attributed to 17 disease loci. Here, we describe the localization and identification of the photoreceptor cell-specific nuclear receptor gene NR2E3 as a novel disease locus and gene for adRP. A heterozygous mutation c.166G-->A (p.Gly56Arg) was identified in the first zinc finger of NR2E3 in a large Belgian family affected with adRP. Overall, this missense mutation was found in 3 families affected with adRP among 87 unrelated families with potentially dominant retinal dystrophies (3.4%), of which 47 were affected with RP (6.4%). Interestingly, affected members of these families display a novel recognizable NR2E3-related clinical subtype of adRP. Other mutations of NR2E3 have previously been shown to cause autosomal recessive enhanced S-cone syndrome, a specific retinal phenotype. We propose a different pathogenetic mechanism for these distinct dominant and recessive phenotypes, which may be attributed to the dual key role of NR2E3 in the regulation of photoreceptor-specific genes during rod development and maintenance.

A novel polyalanine expansion in FOXL2: the first evidence for a recessive form of the blepharophimosis syndrome (BPES) associated with ovarian dysfunction.

The blepharophimosis syndrome (BPES) is an autosomal dominant developmental disorder in which craniofacial/eyelid malformations are associated (type I) or not (type II) with premature ovarian failure (POF). Mutations in the FOXL2 gene, encoding a forkhead transcription factor, are responsible for both types of BPES. Heterozygous polyalanine expansions of +10 residues (FOXL2-Ala24) account for 30% of FOXL2 mutations and are fully penetrant for the eyelid phenotype. Here we describe the first homozygous FOXL2 mutation leading to a polyalanine expansion of +5 residues (FOXL2-Ala19). This novel mutation segregates in an Indian family where heterozygous mutation carriers are unaffected whereas homozygous individuals have the typical BPES phenotype, with proven POF in one female. Expression of the FOXL2-Ala19 protein in COS-7 cells revealed a significantly higher cytoplasmic retention compared to the wild-type protein. This is the first study providing genetic evidence for a recessive inheritance of BPES associated with ovarian dysfunction.

Premature ovarian failure and forkhead transcription factor FOXL2: blepharophimosis-ptosis-epicanthus inversus syndrome and ovarian dysfunction.

Recently the molecular basis of the blepharophimosis-ptosis-epicanthus inversus-syndrome (BPES), an autosomal dominant developmental disorder of the eyelids and ovary, was elucidated. This syndromic form of premature ovarian failure (POF) is caused by mutations in the gene encoding the forkhead transcription factor FOXL2. In this manuscript we review the clinical features of BPES, its molecular basis, the structural and functional characteristics of the FOXL2 gene and protein, and known animal models.

The human FOXL2 mutation database.

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES; MIM# 110100) is an autosomal dominant genetic condition in which an eyelid malformation is associated (type I) or not associated (type II) with premature ovarian failure (POF). In 2001, mutations in the FOXL2 gene, encoding a forkhead transcription factor, were shown to cause both BPES type I and II. Since then, a number of reports have appeared that describe intragenic FOXL2 mutations in BPES patients. In addition, a few FOXL2 variants have been reported in isolated POF patients and XX males. Previously, our group has described a large number of FOXL2 mutations, thereby demonstrating the existence of two mutational hotspots in FOXL2, intra- and interfamilial phenotypic variability in BPES families, and genotype-phenotype correlations for a number of mutations in BPES patients. Here we describe a locus-specific Human FOXL2 Mutation Database (http://medgen.ugent.be/foxl2/), created using the MuStaR software. Our database contains general information about the FOXL2 gene, as well as details about 135 intragenic mutations and variants of FOXL2, obtained from published papers, abstracts of meetings, and from unpublished data produced by our group. Not included in the current version of the database are variants residing outside the coding region of FOXL2 and molecular cytogenetic rearrangements of the FOXL2 locus. The Human FOXL2 Mutation Database was created to provide a unique publicly available online resource of information about human FOXL2 mutations/variants associated with BPES and POF. It allows remote users to submit new mutations to the database and to query the database using a web form. It will facilitate evaluation of the pathogenicity of a particular mutation, as it contains data about disease-causing mutations and polymorphisms in BPES and isolated POF patients, and a link to the known FOXL2 orthologs. Moreover, it will allow us to establish more accurate genotype-phenotype correlations, since clinical information is contained in the database.