Rare GABRA3 variants are associated with epileptic seizures, encephalopathy and dysmorphic features.

Genetic epilepsies are caused by mutations in a range of different genes, many of them encoding ion channels, receptors or transporters. While the number of detected variants and genes increased dramatically in the recent years, pleiotropic effects have also been recognized, revealing that clinical syndromes with various degrees of severity arise from a single gene, a single mutation, or from different mutations showing similar functional defects. Accordingly, several genes coding for GABAA receptor subunits have been linked to a spectrum of benign to severe epileptic disorders and it was shown that a loss of function presents the major correlated pathomechanism. Here, we identified six variants in GABRA3 encoding the α3-subunit of the GABAA receptor. This gene is located on chromosome Xq28 and has not been previously associated with human disease. Five missense variants and one microduplication were detected in four families and two sporadic cases presenting with a range of epileptic seizure types, a varying degree of intellectual disability and developmental delay, sometimes with dysmorphic features or nystagmus. The variants co-segregated mostly but not completely with the phenotype in the families, indicating in some cases incomplete penetrance, involvement of other genes, or presence of phenocopies. Overall, males were more severely affected and there were three asymptomatic female mutation carriers compared to only one male without a clinical phenotype. X-chromosome inactivation studies could not explain the phenotypic variability in females. Three detected missense variants are localized in the extracellular GABA-binding NH2-terminus, one in the M2-M3 linker and one in the M4 transmembrane segment of the α3-subunit. Functional studies in Xenopus laevis oocytes revealed a variable but significant reduction of GABA-evoked anion currents for all mutants compared to wild-type receptors. The degree of current reduction correlated partially with the phenotype. The microduplication disrupted GABRA3 expression in fibroblasts of the affected patient. In summary, our results reveal that rare loss-of-function variants in GABRA3 increase the risk for a varying combination of epilepsy, intellectual disability/developmental delay and dysmorphic features, presenting in some pedigrees with an X-linked inheritance pattern.

Variants in CUL4B are associated with cerebral malformations.

Variants in cullin 4B (CUL4B) are a known cause of syndromic X-linked intellectual disability. Here, we describe an additional 25 patients from 11 families with variants in CUL4B. We identified nine different novel variants in these families and confirmed the pathogenicity of all nontruncating variants. Neuroimaging data, available for 15 patients, showed the presence of cerebral malformations in ten patients. The cerebral anomalies comprised malformations of cortical development (MCD), ventriculomegaly, and diminished white matter volume. The phenotypic heterogeneity of the cerebral malformations might result from the involvement of CUL-4B in various cellular pathways essential for normal brain development. Accordingly, we show that CUL-4B interacts with WDR62, a protein in which variants were previously identified in patients with microcephaly and a wide range of MCD. This interaction might contribute to the development of cerebral malformations in patients with variants in CUL4B.

Alteration of fatty-acid-metabolizing enzymes affects mitochondrial form and function in hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) is considered one of the most heterogeneous groups of neurological disorders, both clinically and genetically. The disease comprises pure and complex forms that clinically include slowly progressive lower-limb spasticity resulting from degeneration of the corticospinal tract. At least 48 loci accounting for these diseases have been mapped to date, and mutations have been identified in 22 genes, most of which play a role in intracellular trafficking. Here, we identified mutations in two functionally related genes (DDHD1 and CYP2U1) in individuals with autosomal-recessive forms of HSP by using either the classical positional cloning or a combination of whole-genome linkage mapping and next-generation sequencing. Interestingly, three subjects with CYP2U1 mutations presented with a thin corpus callosum, white-matter abnormalities, and/or calcification of the basal ganglia. These genes code for two enzymes involved in fatty-acid metabolism, and we have demonstrated in human cells that the HSP pathophysiology includes alteration of mitochondrial architecture and bioenergetics with increased oxidative stress. Our combined results focus attention on lipid metabolism as a critical HSP pathway with a deleterious impact on mitochondrial bioenergetic function.

Complex rearrangements in patients with duplications of MECP2 can occur by fork stalling and template switching.

Duplication at the Xq28 band including the MECP2 gene is one of the most common genomic rearrangements identified in neurodevelopmentally delayed males. Such duplications are non-recurrent and can be generated by a non-homologous end joining (NHEJ) mechanism. We investigated the potential mechanisms for MECP2 duplication and examined whether genomic architectural features may play a role in their origin using a custom designed 4-Mb tiling-path oligonucleotide array CGH assay. Each of the 30 patients analyzed showed a unique duplication varying in size from approximately 250 kb to approximately 2.6 Mb. Interestingly, in 77% of these non-recurrent duplications, the distal breakpoints grouped within a 215 kb genomic interval, located 47 kb telomeric to the MECP2 gene. The genomic architecture of this region contains both direct and inverted low-copy repeat (LCR) sequences; this same region undergoes polymorphic structural variation in the general population. Array CGH revealed complex rearrangements in eight patients; in six patients the duplication contained an embedded triplicated segment, and in the other two, stretches of non-duplicated sequences occurred within the duplicated region. Breakpoint junction sequencing was achieved in four duplications and identified an inversion in one patient, demonstrating further complexity. We propose that the presence of LCRs in the vicinity of the MECP2 gene may generate an unstable DNA structure that can induce DNA strand lesions, such as a collapsed fork, and facilitate a Fork Stalling and Template Switching event producing the complex rearrangements involving MECP2.

Severe mental retardation, seizures, and hypotonia due to deletions of MEF2C.

We present four patients, in whom we identified overlapping deletions in 5q14.3 involving MEF2C using a clinical oligonucleotide array comparative genomic hybridization (CGH) chromosomal microarray analysis (CMA). In case 1, CMA revealed an approximately 140 kb deletion encompassing the first three exons of MEF2C in a 3-year-old patient with severe psychomotor retardation, periodic tremor, and an abnormal motor pattern with mirror movement of the upper limbs observed during infancy, hypotonia, abnormal EEG, epilepsy, absence of speech, autistic behavior, bruxism, and mild dysmorphic features. MRI of the brain showed mild thinning of the corpus callosum and delay of white matter myelination in the occipital lobes. In case 2, an approximately 1.8 Mb deletion of TMEM161B and MEF2C was found in a child with severe developmental delay, hypotonia, and seizures. Patient 3 had epilepsy, hypotonia, thinning of the corpus callosum, and developmental delay associated with a de novo approximately 2.4 Mb deletion in 5q14.3 including MEF2C and five other genes. In case 4, a de novo approximately 5.7 Mb deletion of MEF2C and five other genes was found in a child with truncal hypotonia, intractable seizures, profound developmental delay, and shortening of the corpus callosum on brain MRI. These deletions further support that haploinsufficiency of MEF2C is responsible for severe mental retardation, seizures, and hypotonia. Our results, in combination with previous reports, imply that exon-targeted oligo array CGH, which is more efficient in identifying exonic copy number variants, should improve the detection of clinically significant deletions and duplications over arrays with probes spaced evenly throughout the genome.

Novel mutation of IL1RAPL1 gene in a nonspecific X-linked mental retardation (MRX) family.

Mental retardation (MR) affects approximately 2% of the population. About 10% of all MR cases result from defects of X-linked genes. Mutations in most of more than 20 known genes causing nonspecific form of X-linked MR (MRX) are very rare and may account for less than 0.5-1% of MR. Linkage studies in extended pedigrees followed by mutational analysis of known MRX genes in the linked interval are often the only way to identify a genetic cause of the disorder. We performed linkage analysis in several MRX families, and in one family with four males with MR we mapped the disease to an interval encompassing Xp21.2-22.11 (with a maximum LOD score of 2.71). Subsequent mutation analysis of genes located in this interval allowed us to identify a partial deletion of the IL1RAPL1 gene. Different nonoverlapping deletions involving IL1RAPL1 have been reported previously, suggesting that this region could be deletion-prone. In this report, we present the results of the molecular analyses and clinical examinations of four affected family members with the deletion in IL1RAPL1. Our data further confirm the importance and usefulness of linkage studies for gene mapping in MRX families and demonstrate that IL1RAPL1 plays an important role in the etiology of MRX. With the development of new methods (aCGH, MLPA), further rearrangements in this gene (including deletions and duplications) might be discovered in the nearest future.

Molecular analysis of defects in the CFTR gene and AZF locus of the Y chromosome in male infertility.

OBJECTIVE: To investigate the frequency and potential impact of mutations and polymorphisms in the CFTR gene and deletions in AZF locus of the Y chromosome in patients with azoospermia (AZOO), cryptozoospermia (CRYPTO) or oligoasthenoteratozoospermia (OAT) who were to be included in an assisted reproductive technologies (ART) program. STUDY DESIGN: A total of 188 infertile men were enrolled in the study: 100 patients with AZOO, 38 with CRYPTO and 50 with OAT. RESULTS: The CFTR gene mutations or IVS8-5T variant in at least 1 allele was identified with similar frequencies among the AZOO (33%) and CRYPTO (21%) patients; 55% of the AZOO patients with normal spermatogenesis (NS) had mutations in 1 or 2 alleles. The novel R810G mutation in exon 13 was identified in 1 NS patient. The OAT or AZOO patients with Sertoli cell only syndrome (SCO) had mutations in the CFTR gene with similar frequencies to that in the general Polish population. The deletions in the AZF locus were detected in 20% of SCO patients, 11.5% of AZOO patients with maturation arrest and in 5% of CRYPTO patients. The other groups (NS, OAT) did not carry deletions in the region studied. CONCLUSION: Molecular diagnosis of the CFTR gene, Y chromosome deletion analysis and genetic counseling are necessary diagnostic elements for patients with male infertility, especially if the are included in an ART program.

Aerobic and anaerobic NAD+ metabolism in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the nicotinic acid moiety of NAD+ can be synthesized from tryptophan using the kynurenine pathway or incorporated directly using nicotinate phosphoribosyl transferase (NPT1). We have identified the genes that encode the enzymes of the kynurenine pathway and for BNA5 (YLR231c) and BNA6 (YFR047c) confirmed that they encode kynureninase and quinolinate phosphoribosyl transferase respectively. We show that deletion of genes encoding kynurenine pathway enzymes are co-lethal with the Deltanpt1, demonstrating that no other pathway for the synthesis of nicotinic acid exists in S. cerevisiae. Also, we show that under anaerobic conditions S. cerevisiae is a nicotinic acid auxotroph.

KIF1A missense mutations in SPG30, an autosomal recessive spastic paraplegia: distinct phenotypes according to the nature of the mutations.

The hereditary spastic paraplegias (HSPs) are a clinically and genetically heterogeneous group of neurodegenerative diseases characterised by progressive spasticity in the lower limbs. The nosology of autosomal recessive forms is complex as most mapped loci have been identified in only one or a few families and account for only a small percentage of patients. We used next-generation sequencing focused on the SPG30 chromosomal region on chromosome 2q37.3 in two patients from the original linked family. In addition, wide genome scan and candidate gene analysis were performed in a second family of Palestinian origin. We identified a single homozygous mutation, p.R350G, that was found to cosegregate with the disease in the SPG30 kindred and was absent in 970 control chromosomes while affecting a strongly conserved amino acid at the end of the motor domain of KIF1A. Homozygosity and linkage mapping followed by mutation screening of KIF1A allowed us to identify a second mutation, p.A255V, in the second family. Comparison of the clinical features with the nature of the mutations of all reported KIF1A families, including those reported recently with hereditary sensory and autonomic neuropathy, suggests phenotype-genotype correlations that may help to understand the mechanisms involved in motor neuron degeneration. We have shown that mutations in the KIF1A gene are responsible for SPG30 in two autosomal recessive HSP families. In published families, the nature of the KIF1A mutations seems to be of good predictor of the underlying phenotype and vice versa.

[Gene mapping in 14 families with X-linked nonspecific mental retardation]. / Mapowanie genów w 14 rodzinach z niespecyficzna niepelnosprawoscia intelektualna sprzezona z chromosomem X.

UNLABELLED: Mental retardation affects 2-3% of the population. The identification of nonspecific X-linked mental retardation genes represents a challenge of considerable medical and scientific importance. AIM: An attempt to identify new genes and mutations in known genes in 14 families with nonspecific X-linked mental retardation. MATERIAL AND METHODS: Linkage analysis with microsatellite markers was performed in 14 families with mental retardation segregating as an X-linked feature. Significant lod score (> 2) was obtained only for 2 families, due to insufficient number of analyzed families' members. Known MRX genes located in the linkage intervals were analysed. RESULTS: Analysis of selected known MRX genes enabled identification of pathogenic mutations in 3 out of 14 families. Sequencing of further candidate genes is in progress. In all families the critical region and the number of genes to analyze was significantly narrowed. CONCLUSIONS: Linkage analysis in families with mental retardation segregating as an X-linked feature is still a considerable approach leading to identification of new genes, and mutations in known genes. It is a first step of identification of disease background, even in small families with lod score < 2.

[Attention deficit hyperactivity disorder (ADHD)--molecular and genetic aspects]. / Nadpobudliwosc psychoruchowa z zaburzeniami uwagi (ADHD)--aspekty genetyczne i molekularne.

Attention deficit hyperactivity disorder (ADHD) is a common neurobehavioral disorder of childhood, affecting approximately 5-10% of children. ADHD is considered to be a multifactorial disorder because both genetic and environmental components may contribute to its progress. The etiology of attention deficit hyperactivity disorder (ADHD) is unknown, however family, twin and adoption studies have suggested that genetic factors are very important in its etiopathogenesis. The research of genetic basis of ADHD consists of linkage analysis, candidate gene approach and association studies. These analyses and also investigations on animal models of disease suggest that mutations in genes involved in dopaminergic, serotonergic and adrenergic systems are likely to be responsible for ADHD.

The ARX mutations: a frequent cause of X-linked mental retardation.

The ARX gene mutations have been demonstrated to cause different forms of mental retardation (MR). Beside FMR1, in families with X-linked mental retardation (XLMR), the ARX dysfunction was demonstrated to be among the most frequent causes of this heterogeneous group of disorders. Nevertheless, in sporadic cases of MR, ARX mutations are extremely rare. In order to evaluate the frequency of ARX mutation in XLMR, we performed mutational analysis of ARX in 165 mentally retarded probands negative for FRAXA and belonging to families in which the condition segregates as an X-linked condition. The same recurrent mutation, an in frame 24 bp insertion (c.428-451 dup (24 bp)), was identified in five patients. In one family, the mother of two affected boys was found not to carry the mutation detected in her sons. These data suggest the presence of germline mosaicism for the mutation in the mother. Our results confirm the significant contribution of ARX mutations in the etiology of MR, especially in this group of patients selected for XLMR (3%). These data, together with those reported in the literature, imply that screening for c.428-451 dup (24 bp) mutation should be recommended in all patients with suspected XLMR.

[I. Single nucleotide polymorphism in human genetic analyses]. / I. Polimorfizm pojedynczego nukleotydu w badaniach genetycznych czlowieka.

The new era of human genetic analyses has been began by finishing of The Human Genome Project. Discovery of the almost complete sequence of human DNA showed surprisingly small differences between the genetic materials of randomly chosen people. Genetists pay intensive attention to the very small part of nucleotide sequence - 0.1% - which contains polymorphic changes. The most frequent type of these changes is polymorphism of a single nucleotide (SNP). It makes up about 90% of all molecular differences in human DNA sequence. There are about 3 mln positions in DNA sequence containing SNPs. Polymorphic changes serve as genetic markers and they enable to map genes or to follow their inheritance. Changes of this kind seem also to be the possible cause of the remarkable variety of susceptibility to many common diseases e.g.: diabetes, cancer and cardiovascular disorders. SNPs are also the object of interest of intensively developing scientific domain - pharmacogenetics. Scientists working on this interdisciplinary field - connecting pharmacology and genetics - try to find out the reason of great variety of response to medicines and their side effects in case of patients belonging to the same therapeutical groups. Progress in this kind of research in near future will enable a significant improvement of pharmacological therapy, which will be based on matching the drug to genetically determined traits of patients.

[Monogenic causes of nonspecific X-linked mental retardation molecular aspects]. / Izolowane postaci niepelnosprawnosci intelektualnej sprzezonej z chromosomem X-aspekty molekularne.

Mental retardation (MR) is a symptom in a large group of clinical conditions and affects around 3% of the population. MR is divided into syndromic, if it is characterized by distinctive clinical features and nonspecific when mental retardation is the only defining manifestation. Although genetic causes of X-linked mental retardation (XLMR) are heterogenous and complex, recent findings have led to the identification of an increasing number of genes involved in these conditions. Eight genes involved in nonspecific X-linked mental retardation have been identified so far, including FMR2, GDI1, OPHN1, PAK3, ARHGEF6, IL1RAPL, TM4SF2, and FACL4. Four other MECP2, RSK2, ARX, ATR-X are involved in syndromic and nonspecific forms of MR. Recent research has shown that these genes encode for proteins involved in signaling pathways which regulate cytoskeleton organization, synaptic vesicle transport and establishment of connections between neuronal cells. These findings provide insight into the molecular mechanisms of crucial processes for the development of intellectual and cognitive functions.