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Objective: To analyze the clinical syndromes and inheritance patterns of multiplex families with epilepsy toward the ultimate aim of uncovering the underlying molecular genetic basis. Methods: Following the referral of families with 2 or more relatives with epilepsy, individuals were classified into epilepsy syndromes. Families were classified into syndromes where at least 2 family members had a specific diagnosis. Pedigrees were analyzed and molecular genetic studies were performed as appropriate. Results: A total of 211 families were ascertained over an 11-year period in Israel. A total of 169 were classified into broad familial epilepsy syndrome groups: 61 generalized, 22 focal, 24 febrile seizure syndromes, 33 special syndromes, and 29 mixed. A total of 42 families remained unclassified. Pathogenic variants were identified in 49/211 families (23%). The majority were found in established epilepsy genes (e.g., SCN1A , KCNQ2 , CSTB ), but in 11 families, this cohort contributed to the initial discovery (e.g., KCNT1 , PCDH19 , TBC1D24 ). We expand the phenotypic spectrum of established epilepsy genes by reporting a familial LAMC3 homozygous variant, where the predominant phenotype was epilepsy with myoclonic-atonic seizures, and a pathogenic SCN1A variant in a family where in 5 siblings the phenotype was broadly consistent with Dravet syndrome, a disorder that usually occurs sporadically. Conclusion: A total of 80% of families were successfully classified, with pathogenic variants identified in 23%. The successful characterization of familial electroclinical and inheritance patterns has highlighted the value of studying multiplex families and their contribution towards uncovering the genetic basis of the epilepsies.

The clinical significance of chromosomal microdeletions and microduplications was predicted based on their gene content, de novo or familial inheritance and accumulated knowledge recorded on public databases. A patient group comprised of 247 cases with epilepsy and its common co-morbidities of developmental delay, intellectual disability, autism spectrum disorders, and congenital abnormalities was reviewed prospectively in a diagnostic setting using a standardized oligo-array CGH platform. Seventy-three (29.6%) had copy number variations (CNVs) and of these 73 cases, 27 (37.0%) had CNVs that were likely causative. These 27 cases comprised 10.9% of the 247 cases reviewed. The range of pathogenic CNVs associated with seizures was consistent with the existence of many genetic determinants for epilepsy. © 2012 Wiley Periodicals, Inc.

We aimed to refine the phenotypic spectrum and map the causative gene in two families with familial focal epilepsy with variable foci (FFEVF). A new five-generation Australian FFEVF family (A) underwent electroclinical phenotyping, and the original four-generation Australian FFEVF family (B) (Ann Neurol, 44, 1998, 890) was re-analyzed, including new affected individuals. Mapping studies examined segregation at the chromosome 22q12 FFEVF region. In family B, the original whole genome microsatellite data was reviewed. Five subjects in family A and 10 in family B had FFEVF with predominantly awake attacks and active EEG studies with a different phenotypic picture from other families. In family B, reanalysis excluded the tentative 2q locus reported. Both families mapped to chromosome 22q12. Our results confirm chromosome 22q12 as the solitary locus for FFEVF. Both families show a subtly different phenotype to other published families extending the clinical spectrum of FFEVF.

Incomplete penetrance and variable phenotypic expression are characteristic of a number of syndromes of familial epilepsy. The purpose of the present investigation is to determine if the 15q13.3 copy number deletion functions as a locus modifying the epilepsy phenotype caused by other known or presumed pathogenic mutations segregating in families with epilepsies. No 15q13.3 microdeletions were detected in 756 affected or definite obligate carrier individuals across 151 families selected on the basis of having multiple members affected with epilepsy and showing a range of seizure types. Therefore, the 15q13.3 microdeletion does not act as a genetic modifier in this cohort of families and is not responsible for any of the genetic heterogeneity hypothesized to account for failure to detect linkage in previous genome-wide scans in five of the larger families included in this study.

Objective: To determine the genetic etiology of the severe early infantile onset syndrome of malignant migrating partial seizures of infancy (MPSI). Methods: Fifteen unrelated children with MPSI were screened for mutations in genes associated with infantile epileptic encephalopathies: SCN1A, CDKL5, STXBP1, PCDH19, and POLG. Microarray studies were performed to identify copy number variations. Results: One patient had a de novo SCN1A missense mutation p.R862G that affects the voltage sensor segment of SCN1A. A second patient had a de novo 11.06 Mb deletion of chromosome 2q24.2q31.1 encompassing more than 40 genes that included SCN1A. Screening of CDKL5 (13/15 patients), STXBP1 (13/15), PCDH19 (9/11 females), and the 3 common European mutations of POLG (11/15) was negative. Pathogenic copy number variations were not detected in 11/12 cases. Conclusion: Epilepsies associated with SCN1A mutations range in severity from febrile seizures to severe epileptic encephalopathies including Dravet syndrome and severe infantile multifocal epilepsy. MPSI is now the most severe SCN1A phenotype described to date. While not a common cause of MPSI, SCN1A screening should now be considered in patients with this devastating epileptic encephalopathy. GLOSSARY: CNV: copy number variation EE: epileptic encephalopathy MPSI: malignant migrating partial seizures of infancy SIMFE: severe infantile multifocal epilepsy.

We set out to review the extent to which molecular karyotyping has overtaken conventional cytogenetics in applications related to epilepsy. Multiplex ligase-dependent probe amplification (MLPA) targeted to predetermined regions such as SCN1A and KCNQ2 has been effectively applied over the last half a decade, and oligonucleotide array comparative genome hybridization (array CGH) is now well established for genome-wide exploration of microchromosomal variation. Array CGH is applicable to the characterization of lesions present in both sporadic and familial epilepsy, especially where clinical features of affected cases depart from established syndromes. Copy number variants (CNVs) associated with epilepsy and a range of other syndromes and conditions can be recurrent due to nonallelic homologous recombination in regions of segmental duplication. The most common of the recurrent microdeletions associated with generalized epilepsy are typically seen at a frequency of ∼ 1% at 15q13.3, 16p13.11, and 15q11.2, sites that also confer susceptibility for intellectual disability, autism, and schizophrenia. Incomplete penetrance and variable expressivity confound the established rules of cytogenetics for determining the pathogenicity for novel CNVs; however, as knowledge is gained for each of the recurrent CNVs, this is translated to genetic counseling. CNVs play a significant role in the susceptibility profile for epilepsies, with complex genetics and their comorbidities both from the "hotspots" defined by segmental duplication and elsewhere in the genome where their location and size are often novel.

We characterized an autosomal-recessive syndrome of focal epilepsy, dysarthria, and mild to moderate intellectual disability in a consanguineous Arab-Israeli family associated with subtle cortical thickening. We used multipoint linkage analysis to map the causative mutation to a 3.2 Mb interval within 16p13.3 with a LOD score of 3.86. The linked interval contained 160 genes, many of which were considered to be plausible candidates to harbor the disease-causing mutation. To interrogate the interval in an efficient and unbiased manner, we used targeted sequence enrichment and massively parallel sequencing. By prioritizing unique variants that affected protein translation, a pathogenic mutation was identified in TBC1D24 (p.F251L), a gene of unknown function. It is a member of a large gene family encoding TBC domain proteins with predicted function as Rab GTPase activators. We show that TBC1D24 is expressed early in mouse brain and that TBC1D24 protein is a potent modulator of primary axonal arborization and specification in neuronal cells, consistent with the phenotypic abnormality described. Refereed/Peer-reviewed

A family with dominantly inherited neonatal seizures and intellectual disability was atypical for neonatal and infantile seizure syndromes associated with potassium (KCNQ2 and KCNQ3) and sodium (SCN2A) channel mutations. Microsatellite markers linked to KCNQ2, KCNQ3, and SCN2A were examined to exclude candidate locations, but instead revealed a duplication detected by observation of three alleles for two markers flanking SCN2A. Characterization revealed a 1.57 Mb duplication at 2q24.3 containing eight genes including SCN2A, SCN3A, and the 3¢ end of SCN1A. The duplication was partially inverted and inserted within or near SCN1A, probably affecting the expression levels of associated genes, including sodium channels. Rare or unique microchromosomal copy number mutations might underlie familial epilepsies that do not fit within the clinical criteria for the established syndromes.

To identify genes involved in idiopathic absence epilepsies by analyzing gene expression using a monozygotic (MZ) twin design.Genome-wide gene expression in lymphoblastoid cell lines (LCLs) was determined using microarrays derived from five discordant and four concordant MZ twin pairs with idiopathic absence epilepsies and five unaffected MZ twin pairs. Gene expression was analyzed using three strategies: discordant MZ twins were compared as matched pairs, MZ twins concordant for epilepsy were compared to control MZ twins, and a singleton design of affected versus unaffected MZ twin individuals was used irrespective of twin pairing. An overlapping gene list was generated from these analyses. Dysregulation of genes recognized from the microarray experiment was validated using quantitative real time PCR (qRT-PCR) in the twin sample and in an independent sample of 18 sporadic absence cases and 24 healthy controls.Sixty-five probe sets were identified from the three combined microarray analysis strategies. Sixteen genes were chosen for validation and nine of these genes confirmed by qRT-PCR in the twin sample. Differential expression for EGR1 (an immediate early gene) and RCN2 (coding for the calcium-binding protein Reticulocalbin 2) were reconfirmed by qRT-PCR in the independent sample.Using a unique sample of discordant MZ twins, our study identified genes with altered expression, which suggests novel mechanisms in idiopathic absence epilepsy. Dysregulation of EGR1 and RCN2 is implicated in idiopathic absence epilepsy.

There have been incredible advances made in human genetics over the past 40 years. I have set out in the next few pages to describe just some of these changes and to illustrate how they unfolded through my own experiences.

Chorionic Villous Biopsy (CVS) for diagnosis of XLP was undertaken at 10 weeks gestation in an obligate carrier. The fetus was found to be male by cytogenetic analysis. XLP (Xq25–q26) is closely linked to the RFLP markers DXS10, DXS37 and DXS42, but only DXS10 (distal to XLP) was informative for prenatal diagnosis in this family. RFLP analysis using this marker gave a 7% risk that the fetus was affected, based on the known recombination frequency between DXS10 and XLP. Further investigation was then undertaken to obtain a rapid and more accurate diagnosis using the three highly polymorphic PCR based markers. These were the AC repeat markers DXS424 (XL5A) and DXS425 (XL90A3) and the tetramer repeat marker within HPRT. DX425 is approximately 10 cM proximal to DXS10 and HPRT but is not known with certainty to map proximal or distal to XLP. DXS424 is proximal to DXS10 and HPRT and was inferred to be proximal to XLP on the basis of map distance from HPRT estimated by linkage analysis of data from CEPH pedigrees. This was confirmed by a recombinant in the XLP family between DXS424 and DXS425, placing DXS424 proximal to XLP. Diagnosis by linkage using DXS424 and DXS425, at least one of which is proximal to XLP, and distal markers DXS10 and HPRT, increased the accuracy of diagnosis using flanking marker analysis to greater than 99% that the fetus was unaffected. HLA DR typing of the CVS showed that the fetus was DR identical to a male sibling with XLP. HLA compatibility was confirmed at delivery by full HLA typing and MLC. Transplantation has been carried out utilising cord and placental blood.

Certain mutations in specific parts of the neuronal nicotinic acetylcholine receptor (nAChR) subunit genes CHRNA4, CHRNB2, and probably CHRNA2, can cause autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). All but one of the known causative mutations are located in the second transmembrane region (TM2), which serves as the major ion poreforming domain of the receptor. Functional characterization of these ADNFLE mutations has shown that although each mutant exhibits specific properties, they all confer a gain of function with increased sensitivity to acetylcholine. In this work, we characterize the second and third ADNFLE-associated mutations that are external to TM2 but affect different amino acid residues within the third transmembrane region (TM3). The two new CHRNB2 mutations were identified in three families of Turkish Cypriot, Scottish, and English origin. These TM3 mutations elicit the same gain of function pathomechanism as observed for the TM2 mutations with enhanced acetylcholine sensitivity, despite their unusual localization within the gene. Electrophysiological experiments, including single channel measurements, revealed that incorporation of these new mutant subunits does not affect the conductance of the ionic pore but increases the probability of opening. Determination of the sensitivity to nicotine for nAChRs carrying mutations in TM2 and TM3 showed clear differences in the direction and the extent to which the window current for nicotine sensitivity was shifted for individual mutations, indicating differences in pharmacogenomic properties that are not readily correlated with increased ACh affinity.

Summary Background The Epilepsy Genetics (EPIGEN) Consortium was established to undertake genetic mapping analyses with augmented statistical power to detect variants that influence the development and treatment of common forms of epilepsy. Methods We examined common variations across 279 prime candidate genes in 2717 case and 1118 control samples collected at four independent research centres (in the UK, Ireland, Finland, and Australia). Single nucleotide polymorphism (SNP) and combined set-association analyses were used to examine the contribution of genetic variation in the candidate genes to various forms of epilepsy. Findings We did not identify clear, indisputable common genetic risk factors that contribute to selected epilepsy subphenotypes across multiple populations. Nor did we identify risk factors for the general all-epilepsy phenotype. However, set-association analysis on the most significant p values, assessed under permutation, suggested the contribution of numerous SNPs to disease predisposition in an apparent population-specific manner. Variations in the genes KCNAB1, GABRR2, KCNMB4, SYN2 , and ALDH5A1 were most notable. Interpretation The underlying genetic component to sporadic epilepsy is clearly complex. Results suggest that many SNPs contribute to disease predisposition in an apparently population-specific manner. However, subtle differences in phenotyping across cohorts, combined with a poor understanding of how the underlying genetic component to epilepsy aligns with current phenotypic classifications, might also account for apparent population-specific genetic risk factors. Variations across five genes warrant further study in independent cohorts to clarify the tentative association.

BACKGROUND: Benign familial neonatal seizures are most often caused by mutations in the voltage-gated potassium channel subunit gene KCNQ2. More than 60 mutations have been described in BFNS families, approximately half of which lead to protein truncation. The hypothesis of this study was that deletion or duplication of >or=1 exons of KCNQ2 could cause BFNS in cases without coding or splicing mutations. METHODS: Multiplex ligation-dependent probe amplification (MLPA) was used to test a group of 21 unrelated patients with clinical features consistent with either BFNS, benign familial neonatal-infantile seizures or sporadic neonatal seizures, for exonic deletions and duplications. RESULTS: Three deletions and one duplication mutation were identified in four familial cases and cascade testing of their available family members showed that the mutations segregated with the phenotype in each family. The junction fragment for one of the deletions was amplified by PCR and sequenced to characterise the breakpoint and verify that a deletion had occurred. CONCLUSIONS: Submicroscopic deletions or duplications of KCNQ2 are seen in a significant proportion of BFNS families: four of nine (44%) cases previously testing negative for coding or splice site mutation by sequencing KCNQ2 and KCNQ3. MLPA is an efficient second-tier testing strategy for KCNQ2 to identify pathogenic intragenic mutations not detectable by conventional DNA sequencing methods.

Seizure susceptibility is high in human infants compared to adults, presumably because of developmentally regulated changes in neural excitability. Benign familial neonatal-infantile seizures (BFNIS), characterized by both early onset and remission, are caused by mutations in the gene encoding a human sodium channel (NaV1.2). We analyzed neonatal and adult splice forms of NaV1.2 with a BFNIS mutation (L1563V) in human embryonic kidney cells. Computer modeling revealed that neonatal channels are less excitable than adult channels. Introduction of the mutation increased excitability in the neonatal channels to a level similar to adult channels. By contrast, the mutation did not affect the adult channel variant. This "adult-like" increased excitability is likely to be the mechanism underlying BFNIS in infants with this mutation. More generally, developmentally regulated NaV1.2 splicing may be one mechanism that counters the normally high excitability of neonatal neurons and helps to reduce seizure susceptibility in normal human infants.

Purpose: Although complex idiopathic generalized epilepsies (IGEs) are recognized to have a significant genetic component, as yet there are no known common susceptibility variants. It has recently been suggested that variation in the BRD2 gene confers increased risk of juvenile myoclonic epilepsy (JME), which accounts for around a quarter of all IGE. Here we examine the association between the candidate causal SNP (the promoter variant rs3918149) and JME in five independent cohorts comprising in total 531 JME cases and 1,390 healthy controls. Methods: The strongest candidate causal variant from the original report (rs3918149) was genotyped across all five cohorts. In an effort to identify novel candidate causal polymorphisms, previously unscreened regions of UTR were resequenced. Results: We observed a significant effect in a small sample recruited in Britain (genotype p = 0.001, allele p = 0.001), a borderline significant effect in a sample recruited in Ireland and no association in larger samples of German, Australian, and Indian populations. There was no association with other common forms of epilepsy or any other clear candidate casual variants in or near the BRD2 region. Conclusions: The replication of an effect in the British cohort and suggestive evidence from that recruited in Ireland but lack of replication from the larger German, Australian, and Indian cohorts is surprising and difficult to explain. Further replication in carefully matched populations is required. Results presented here do not, however, support a strong effect for susceptibility to JME across populations of European descent.

Summary Background Vaccination, particularly for pertussis, has been implicated as a direct cause of an encephalopathy with refractory seizures and intellectual impairment. We postulated that cases of so-called vaccine encephalopathy could have mutations in the neuronal sodium channel α1 subunit gene ( SCN1A ) because of a clinical resemblance to severe myoclonic epilepsy of infancy (SMEI) for which such mutations have been identified. Methods We retrospectively studied 14 patients with alleged vaccine encephalopathy in whom the first seizure occurred within 72 h of vaccination. We reviewed the relation to vaccination from source records and assessed the specific epilepsy phenotype. Mutations in SCN1A were identified by PCR amplification and denaturing high performance liquid chromatography analysis, with subsequent sequencing. Parental DNA was examined to ascertain the origin of the mutation. Findings SCN1A mutations were identified in 11 of 14 patients with alleged vaccine encephalopathy; a diagnosis of a specific epilepsy syndrome was made in all 14 cases. Five mutations predicted truncation of the protein and six were missense in conserved regions of the molecule. In all nine cases where parental DNA was available the mutations arose de novo. Clinical-molecular correlation showed mutations in eight of eight cases with phenotypes of SMEI, in three of four cases with borderline SMEI, but not in two cases with Lennox-Gastaut syndrome. Interpretation Cases of alleged vaccine encephalopathy could in fact be a genetically determined epileptic encephalopathy that arose de novo. These findings have important clinical implications for diagnosis and management of encephalopathy and, if confirmed in other cohorts, major societal implications for the general acceptance of vaccination.

Most human idiopathic generalized epilepsies (IGEs) are polygenic, but virtually nothing is known of the molecular basis for any of the complex epilepsies. Recently, two GABAA receptor delta subunit variants (E177A, R220H) were proposed as susceptibility alleles for generalized epilepsy with febrile seizures plus and juvenile myoclonic epilepsy. In human embryonic kidney 293T cells, recombinant halpha1beta2delta(E177A) and halpha1beta2delta(R220H) receptor currents were reduced, but the basis for the current reduction was not determined. We examined the mechanistic basis for the current reduction produced by these variants using the halpha4beta2delta receptor, an isoform more physiologically relevant and linked to epileptogenesis, by characterizing the effects of these variants on receptor cell surface expression and single-channel gating properties. Expression of variant alpha4beta2delta(R220H) receptors resulted in a decrease in surface receptor proteins, and a smaller, but significant, reduction was observed for variant alpha4beta2delta(E177A) receptors. For both variants, no significant alterations of surface expression were observed for mixed population of wild-type and variant receptors. The mean open durations of alpha4beta2delta(E177A) and alpha4beta2delta(R220H) receptor single-channel currents were both significantly decreased compared to wild-type receptors. These data suggest that both delta(E177A) and delta(R220H) variants may result in disinhibition in IGEs by similar cellular and molecular mechanisms, and in heterozygously affected individuals, a reduction in channel open duration of delta subunit-containing GABAA receptors may be the major contributor to the epilepsy phenotypes.

SCN1A is part of the SCN1A-SCN2A-SCN3A gene cluster on chromosome 2q24 that encodes for alpha pore forming subunits of sodium channels. The 26 exons of SCN1A are spread over 100 kb of genomic DNA. Genetic defects in the coding sequence lead to generalized epilepsy with febrile seizures plus (GEFS+) and a range of childhood epileptic encephalopathies of varied severity (e.g., SMEI). All published mutations are collated. More than 100 novel mutations are spread throughout the gene with the more debilitating usually de novo. Some clustering of mutations is observed in the C-terminus and the loops between segments 5 and 6 of the first three domains of the protein. Functional studies so far show no consistent relationship between changes to channel properties and clinical phenotype. Of all the known epilepsy genes SCN1A is currently the most clinically relevant, with the largest number of epilepsy related mutations so far characterized.

Summary: Success has been achieved in identifying many mutations in rare monogenic epilepsy syndromes by using linkage analysis, but dissecting the genetic basis of common epilepsy syndromes has proven more difficult. Common epilepsies are genetically complex disorders believed to be influenced by variation in several susceptibility genes. Association studies can theoretically identify these genes, but despite more than 50 association studies in epilepsy, no consistent or convincing susceptibility genes have emerged, leading to scepticism about the association-study approach. We review the results of existing association studies in focal epilepsies, generalized epilepsies, febrile seizures, and epilepsy pharmacogenetics. By using an illustrative example, we discuss how methodologic issues of sample size, selection of appropriate controls, population stratification, and significance thresholds can lead to bias and false-positive associations; the importance of biologic plausibility also is emphasized. Newer methodologic refinements for association studies, such as use of two control groups, genomic control, haplotyping, and use of two independent datasets, are discussed. A summary of existing guidelines and a checklist for planning and appraising such association studies in epilepsy is presented. We remain cautiously optimistic that with methodologic refinements and multicenter collaborations with large sample sizes, association studies will ultimately be useful in dissecting the genetic basis of common epilepsy syndromes.

We have identified truncating mutations in the human DLG3 (neuroendocrine dlg) gene in 4 of 329 families with moderate to severe X-linked mental retardation. DLG3 encodes synapse-associated protein 102 (SAP102), a member of the membrane-associated guanylate kinase protein family. Neuronal SAP102 is expressed during early brain development and is localized to the postsynaptic density of excitatory synapses. It is composed of three amino-terminal PDZ domains, an src homology domain, and a carboxyl-terminal guanylate kinase domain. The PDZ domains interact directly with the NR2 subunits of the NMDA glutamate receptor and with other proteins responsible for NMDA receptor localization, immobilization, and signaling. The mutations identified in this study all introduce premature stop codons within or before the third PDZ domain, and it is likely that this impairs the ability of SAP102 to interact with the NMDA receptor and/or other proteins involved in downstream NMDA receptor signaling pathways. NMDA receptors have been implicated in the induction of certain forms of synaptic plasticity, such as long-term potentiation and long-term depression, and these changes in synaptic efficacy have been proposed as neural mechanisms underlying memory and learning. The disruption of NMDA receptor targeting or signaling, as a result of the loss of SAP102, may lead to altered synaptic plasticity and may explain the intellectual impairment observed in individuals with DLG3 mutations.

Background and Objectives: A number of familial temporal lobe epilepsies (TLE) have been recently recognized. Mutations in LGI1 (leucine-rich, glioma-inactivated 1 gene) have been found in a few families with the syndrome of autosomal dominant partial epilepsy with auditory features (ADPEAF). The authors aimed to determine the spectrum of TLE phenotypes with LGI1 mutations, to study the frequency of mutations in ADPEAF, and to examine the role of LGI1 paralogs in ADPEAF without LGI1 mutations.Methods: The authors performed a clinical and molecular analysis on 75 pedigrees comprising 54 with a variety of familial epilepsies associated with TLE and 21 sporadic TLE cases. All were studied for mutations in LGI1. ADPEAF families negative for LGI1 mutations were screened for mutations in LGI2, LGI3, and LGI4.Results: Four families had ADPEAF, 22 had mesial TLE, 11 had TLE with febrile seizures, two had TLE with developmental abnormalities, and 15 had various other TLE syndromes. LGI1 mutations were found in two of four ADPEAF families, but in none of the other 50 families nor in the 21 individuals with sporadic TLE. The mutations were novel missense mutations in exons 1 (c.124T→G; C42G) and 8 (c.1418C→T; S473L). No mutations in LGI2, LGI3, or LGI4 were found in the other two ADPEAF families.Conclusion: In TLE, mutations in LGI1 are specific for ADPEAF but do not occur in all families. ADPEAF is genetically heterogeneous, but mutations in LGI2, LGI3, or LGI4 did not account for families without LGI1 mutations.

We recently reported mutations in the sodium channel gene SCN2A in two families with benign familial neonatal-infantile seizures (BFNISs). Here, we aimed to refine the molecular-clinical correlation of SCN2A mutations in early childhood epilepsies. SCN2A was analyzed in 2 families with probable BFNIS, 9 with possible BFNIS, 10 with benign familial infantile seizures, and in 93 additional families with various early childhood epilepsies. Mutations effecting changes in conserved amino acids were found in two of two probable BFNIS families, in four of nine possible BFNIS families, and in none of the others. Our eight families had six different SCN2A mutations; one mutation (R1319Q) occurred in three families. BFNIS is an autosomal dominant disorder presenting between day 2 and 7 months (mean, 11.2 +/- 9.2 weeks) with afebrile secondarily generalized partial seizures; neonatal seizures were not seen in all families. The frequency of seizures varied; some individuals had only a few attacks without treatment and others had clusters of many per day. Febrile seizures were rare. All cases remitted by 12 months. Ictal recordings in four subjects showed onset in the posterior quadrants. SCN2A mutations appear specific for BFNIS; the disorder can now be strongly suspected clinically and the families can be given an excellent prognosis.

We describe a family of 19 males in five generations with mild to borderline non-syndromic X-linked mental retardation (MRX). There were no clinical manifestations in the affected males other than mental impairment and relatively long ears, with neuropsychiatric problems in some cases. Linkage analysis carried out on part of the pedigree using 34 markers spanning the X chromosome localized the gene between DXS454 and DXS1001 in Xq23. The maximum two-point lod score was 3.21 at DXS1059. PAK3 is a known MRX gene mapping to the same region. The affected males and obligate carrier females were found to have a missense mutation c.1094C > A in exon 10 causing an A365E substitution in a highly conserved region of the protein. The C to A base change abolishes a PvuII restriction enzyme site providing the basis for a simple test, if required, for carrier detection and prenatal diagnosis in the extended family.

The γ-aminobutyric acid type A (GABA A ) receptor mediates fast inhibitory synaptic transmission in the CNS. Dysfunction of the GABA A receptor would be expected to cause neuronal hyperexcitability, a phenomenon linked with epileptogenesis. We have investigated the functional consequences of an arginine-to-glutamine mutation at position 43 within the GABA A γ 2 -subunit found in a family with childhood absence epilepsy and febrile seizures. Rapid-application experiments performed on receptors expressed in HEK-293 cells demonstrated that the mutation slows GABA A receptor deactivation and increases the rate of desensitization, resulting in an accumulation of desensitized receptors during repeated, short applications. In Xenopus laevis oocytes, two-electrode voltage-clamp analysis of steady-state currents obtained from α 1 β 2 γ 2 or α 1 β 2 γ 2 (R43Q) receptors did not reveal any differences in GABA sensitivity. However, differences in the benzodiazepine pharmacology of mutant receptors were apparent. Mutant receptors expressed in oocytes displayed reduced sensitivity to diazepam and flunitrazepam but not the imidazopyridine zolpidem. These results provide evidence of impaired GABA A receptor function that could decrease the efficacy of transmission at inhibitory synapses, possibly generating a hyperexcitable neuronal state in thalamocortical networks of epileptic patients possessing the mutant subunit.

Idiopathic hyperphosphatasia is an autosomal recessive bone disease characterized by deformities of long bones, kyphosis and acetabular protrusion, increasing in severity as affected children pass through adolescence. Biochemical and histological evidence indicate that there is extremely rapid bone turnover, with indices of both bone resorption and formation greatly increased. A genome-wide search, in a family with three children affected by idiopathic hyperphosphatasia, suggested linkage to a locus on the long arm of chromosome 8 (8q24). The gene TNFRSF11B encoding osteoprotegerin (OPG), which lies within this locus, was an obvious candidate, given the critical role of OPG in regulating osteoclast development. All three affected siblings were homozygous for a 3 bp inframe deletion in exon 3 of the TNFRSF11B gene, resulting in the loss of an aspartate residue. Their parents (who were first cousins) were heterozygous for the mutation. Recombinant wild-type and mutant OPG cDNAs were expressed in human epithelial kidney cells, and secreted OPG was collected from the conditioned medium. In vitro measurements of bone resorption showed that wild-type OPG suppressed bone resorption, whereas the mutant form did not, confirming this to be an inactivating mutation. This description of abnormal OPG function in humans expands the spectrum of genetic bone diseases arising from perturbations of the OPG/RANK-L/RANK system that regulates osteoclastogenesis.

Generalized epilepsy with febrile seizures plus (GEFS(+)) is an important childhood genetic epilepsy syndrome with heterogeneous phenotypes, including febrile seizures (FS) and generalized epilepsies of variable severity. Forty unrelated GEFS(+) and FS patients were screened for mutations in the sodium channel beta-subunits SCN1B and SCN2B, and the second GEFS(+) family with an SCN1B mutation is described here. The family had 19 affected individuals: 16 with typical GEFS(+) phenotypes and three with other epilepsy phenotypes. Site-specific mutation within SCN1B remains a rare cause of GEFS(+), and the authors found no evidence to implicate SCN2B in this syndrome.

Recent findings from studies of two families have shown that mutations in the GABA(A)-receptor gamma2 subunit are associated with generalized epilepsies and febrile seizures. Here we describe a family that has generalized epilepsy with febrile seizures plus (GEFS(+)), including an individual with severe myoclonic epilepsy of infancy, in whom a third GABA(A)-receptor gamma2-subunit mutation was found. This mutation lies in the intracellular loop between the third and fourth transmembrane domains of the GABA(A)-receptor gamma2 subunit and introduces a premature stop codon at Q351 in the mature protein. GABA sensitivity in Xenopus laevis oocytes expressing the mutant gamma2(Q351X) subunit is completely abolished, and fluorescent-microscopy studies have shown that receptors containing GFP-labeled gamma2(Q351X) protein are retained in the lumen of the endoplasmic reticulum. This finding reinforces the involvement of GABA(A) receptors in epilepsy.

Objective: Analysis of twins with epilepsy to explore the genetic architecture of specific epilepsies, to evaluate the applicability of the 2010 International League Against Epilepsy (ILAE) organization of epilepsy syndromes, and to integrate molecular genetics with phenotypic analyses. Methods: A total of 558 twin pairs suspected to have epilepsy were ascertained from twin registries (69%) or referral (31%). Casewise concordance estimates were calculated for epilepsy syndromes. Epilepsies were then grouped according to the 2010 ILAE organizational scheme. Molecular genetic information was utilized where applicable. Results: Of 558 twin pairs, 418 had confirmed seizures. A total of 534 twin individuals were affected. There were higher twin concordance estimates for monozygotic (MZ) than for dizygotic (DZ) twins for idiopathic generalized epilepsies (MZ = 0.77; DZ = 0.35), genetic epilepsy with febrile seizures plus (MZ = 0.85; DZ = 0.25), and focal epilepsies (MZ = 0.40; DZ = 0.03). Utilizing the 2010 ILAE scheme, the twin data clearly demonstrated genetic influences in the syndromes designated as genetic. Of the 384 tested twin individuals, 10.9% had mutations of large effect in known epilepsy genes or carried validated susceptibility alleles. Conclusions: Twin studies confirm clear genetic influences for specific epilepsies. Analysis of the twin sample using the 2010 ILAE scheme strongly supported the validity of grouping the “genetic” syndromes together and shows this organizational scheme to be a more flexible and biologically meaningful system than previous classifications. Successful selected molecular testing applied to this cohort is the prelude to future large-scale next-generation sequencing of epilepsy research cohorts. Insights into genetic architecture provided by twin studies provide essential data for optimizing such approaches.

The X-linked form of spondyloepiphyseal dysplasia tarda (SEDL), a radiologically distinct skeletal dysplasia affecting the vertebrae and epiphyses, is caused by mutations in the SEDL gene. To characterize the molecular basis for SEDL, we have identified the spectrum of SEDL mutations in 30 of 36 unrelated cases of X-linked SEDL ascertained from different ethnic populations. Twenty-one different disease-associated mutations now have been identified throughout the SEDL gene. These include nonsense mutations in exons 4 and 5, missense mutations in exons 4 and 6, small (2–7 bp) and large (>1 kb) deletions, insertions, and putative splicing errors, with one splicing error due to a complex deletion/insertion mutation. Eight different frameshift mutations lead to a premature termination of translation and account for >43% (13/30) of SEDL cases, with half of these (7/13) being due to dinucleotide deletions. Altogether, deletions account for 57% (17/30) of all known SEDL mutations. Four recurrent mutations (IVS3+5G→A, 157–158delAT, 191–192delTG, and 271–275delCAAGA) account for 43% (13/30) of confirmed SEDL cases. The results of haplotype analyses and the diverse ethnic origins of patients support recurrent mutations. Two patients with large deletions of SEDL exons were found, one with childhood onset of painful complications, the other relatively free of additional symptoms. However, we could not establish a clear genotype/phenotype correlation and therefore conclude that the complete unaltered SEDL-gene product is essential for normal bone growth. Molecular diagnosis can now be offered for presymptomatic testing of this disorder. Appropriate lifestyle decisions and, eventually, perhaps, specific SEDL therapies may ameliorate the prognosis of premature osteoarthritis and the need for hip arthroplasty.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is an uncommon, idiopathic partial epilepsy characterized by clusters of motor seizures occurring in sleep. We describe a mutation of the beta2 subunit of the nicotinic acetylcholine receptor, effecting a V287M substitution within the M2 domain. The mutation, in an evolutionary conserved region of CHRNB2, is associated with ADNFLE in a Scottish family. Functional receptors with the V287M mutation are highly expressed in Xenopus oocytes and characterized by an approximately 10-fold increase in acetylcholine sensitivity. CHRNB2 is a new gene for idiopathic epilepsy, the second acetylcholine receptor subunit implicated in ADNFLE.

We have recently cloned the mouse activity-dependent neuroprotective protein (ADNP). Here, we disclose the cloning of human ADNP (hADNP) from a fetal brain cDNA library. Comparative sequence analysis of these two ADNP orthologs indicated 90% identity at the mRNA level. Several single nucleotide polymorphic sites were noticed. The deduced protein structure contained nine zinc fingers, a proline-rich region, a nuclear bipartite localization signal, and a homeobox domain profile, suggesting a transcription factor function. Further comparative analysis identified an ADNP paralog (33% identity and 46% similarity), indicating that these genes belong to a novel protein family with a nine-zinc finger motif followed by a homeobox domain. The hADNP gene structure spans approximately 40 kilobases and includes five exons and four introns with alternative splicing of an untranslated second exon. The hADNP gene was mapped to chromosome 20q12-13.2, a region associated with aggressive tumor growth, frequently amplified in many neoplasias, including breast, bladder, ovarian, pancreatic, and colon cancers. hADNP mRNA is abundantly expressed in distinct normal tissues, and high expression levels were encountered in malignant cells. Down-regulation of ADNP by antisense oligodeoxynucleotides up-regulated the tumor suppressor p53 and reduced the viability of intestinal cancer cells by 90%. Thus, ADNP is implicated in maintaining cell survival, perhaps through modulation of p53.

Summary The aim of this study was to determine prospectively the frequency of pathogenic chromosomal microdeletions and microduplications in a large group of referred patients with developmental delay (DD), intellectual disability (ID) or autism spectrum disorders (ASD) within a genetic diagnostic service. First tier testing was applied using a standardised oligo-array comparative genomic hybridization (CGH) platform, replacing conventional cytogenetic testing that would have been used in the past. Copy number variants (CNVs) found to be responsible for the clinical condition on the request form could all be subdivided into three groups: well established pathogenic microdeletion/microduplication/aneuploidy syndromes, predicted pathogenic CNVs as interpreted by the laboratory, and recently established pathogenic disease susceptibility CNVs. Totalled from these three groups, with CNVs of uncertain significance excluded, detection rates were: DD (13.0%), ID (15.6%), ASD (2.3%), ASD with DD (8.2%), ASD with ID (12.7%) and unexplained epilepsy with DD, ID and ASD (10.9%). The greater diagnostic sensitivity arising from routine application of array CGH, compared with previously used conventional cytogenetics, outweighs the interpretative issues for the reporting laboratory and referring clinician arising from detection of CNVs of uncertain significance. Precise determination of any previously hidden molecular defect responsible for the patient’s condition is translated to improved genetic counselling.

Autosomal dominant nocturnal frontal lobe epilepsy is sometimes due to mutations in CHRNA4. The commoner presentation of sporadic nocturnal frontal lobe epilepsy has not been associated with genetic defects. A 30-year-old woman diagnosed as having sporadic nocturnal frontal lobe epilepsy was found to have a de novo Ser252Leu CHRNA4 mutation. A pattern is emerging of site-specific mutation within the second transmembrane domain of CHRNA4 in association with autosomal dominant nocturnal frontal lobe epilepsy and sporadic nocturnal frontal lobe epilepsy in families with different ethnic backgrounds.

Summary: Purpose: To describe the clinical features of a family from Northern Norway in which autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is associated with a Ser248Phe amino acid exchange in the second transmembrane domain of the neuronal nicotinic acetylcholine receptor α4 subunit (CHRNA4). We also tested for evidence of a de novo mutation or founder effect by comparing haplotypes with the original Australian family where the Ser248Phe mutation was first described. Methods: Clinical details were obtained from 19 family members. Personal interviews and genetic analysis were carried out in 17. Parts of the coding region of CHRNA4 were sequenced, and two known polymorphisms (bp555/FokI, bp594/CfoI) were typed by restriction analysis. Results: Eleven individuals had ADNFLE. The haplotypes of the mutation-carrying alleles of affected individuals from the Northern Norwegian and the Australian ADNFLE family are different. The phenotypic expressions are remarkably similar. Conclusions: The Ser248Phe mutation occurred independently in both families. Given the rarity of the disease, this suggests that not only the position of a mutation in the coding sequence but also the type of an amino acid exchange is important for the etiology of ADNFLE. The phenotypic similarity of these two families with different genetic backgrounds suggests that the Ser248Phe mutation largely determines the phenotype, with relatively little influence of other background genes.

No abstract available

Spondyloepiphyseal dysplasia tarda (SEDL; MIM 313400) is an X-linked recessive osteochondrodysplasia that occurs in approximately two of every one million people1. This progressive skeletal disorder which manifests in childhood is characterized by disproportionate short stature with short neck and trunk, barrel chest and absence of systemic complications2,3,4. Distinctive radiological signs are platyspondyly with hump-shaped central and posterior portions, narrow disc spaces, and mild to moderate epiphyseal dysplasia. The latter usually leads to premature secondary osteoarthritis often requiring hip arthroplasty3,4,5. Obligate female carriers are generally clinically and radiographically indistinguishable from the general population4,5, although some cases have phenotypic changes consistent with expression of the gene defect2,4,6,7. The SEDL gene has been localized to Xp22 (Refs 8,9) in the approximately 2-Mb interval between DXS16 and DXS987 (ref. 10). Here we confirm and refine this localization to an interval of less than 170 kb by critical recombination events at DXS16 and AFMa124wc1 in two families. In one candidate gene we detected three dinucleotide deletions in three Australian families which effect frameshifts causing premature stop codons. The gene designated SEDL is transcribed as a 2.8-kb transcript in many tissues including fetal cartilage. SEDL encodes a 140 amino acid protein with a putative role in endoplasmic reticulum (ER)-to-Golgi vesicular transport.

Autosomal dominant periodic fever syndromes are characterized by unexplained episodes of fever and severe localized inflammation. In seven affected families, we found six different missense mutations of the 55 kDa tumor necrosis factor receptor (TNFR1), five of which disrupt conserved extracellular disulfide bonds. Soluble plasma TNFR1 levels in patients were approximately half normal. Leukocytes bearing a C52F mutation showed increased membrane TNFR1 and reduced receptor cleavage following stimulation. We propose that the autoinflammatory phenotype results from impaired downregulation of membrane TNFR1 and diminished shedding of potentially antagonistic soluble receptor. TNFR1-associated periodic syndromes (TRAPS) establish an important class of mutations in TNF receptors. Detailed analysis of one such mutation suggests impaired cytokine receptor clearance as a novel mechanism of disease.

Börjeson-Forssman-Lehmann syndrome (BFLS) is a syndromal X-linked mental retardation, which maps by linkage to the q26 region of the human X chromosome. We have identified a male patient with BFLS-like features and a duplication, 46,Y,dup(X)(q26q28), inherited from his phenotypically normal mother. Fluorescence in situ hybridisation using yeast artificial chromosome clones from Xq26 localised the duplication breakpoint to an approximately 400-kb interval in the Xq26.3 region between DXS155 and DXS294/DXS730. Database searches and analysis of available genomic DNA sequence from the region revealed the presence of the fibroblast growth factor homologous factor gene, FHF2, within the duplication breakpoint interval. The gene structure of FHF2 was determined and two new exons were identified, including a new 5' end exon, 1B. FHF2 is a large gene extending over approximately 200 kb in Xq26.3 and is composed of at least seven exons. It shows tissue-specific alternative splicing and alternative transcription starts. Northern blot hybridisation showed highest expression in brain and skeletal muscle. The FHF2 gene localisation and tissue-specific expression pattern suggest it to be a candidate gene for familial cases of the BFLS syndrome and other syndromal and non-specific forms of X-linked mental retardation mapping to the region.

Summary: Photosensitive epilepsy is less frequent among males than females. Red is the most epileptogenic color. The X-linked red pigment gene contains the polymorphism Ser180Ala; the Ser180 allele increases red sensitivity. We hypothesized that the paucity of males with photosensitive epilepsy is explained by the distribution of this sex-linked allele, and predicted photosensitive males would have a low frequency of this allele. We genotyped 35 males with photosensitive epilepsy and 84 male controls. Allele frequencies did not differ between these groups. The hypothesis was not supported, so alternate reasons for the sex bias in photosensitive epilepsy must be sought.

Establishing an etiologic diagnosis in adults with refractory epilepsy and intellectual disability is challenging. We analyzed the phenotype of 14 adults with severe myoclonic epilepsy of infancy. This phenotype comprised heterogeneous seizure types with nocturnal generalized tonic-clonic seizures predominating, mild to severe intellectual disability, and variable motor abnormalities. The diagnosis was suggested by a characteristic evolution of clinical findings in the first years of life. Ten had mutations in SCN1A and one in GABRG2.

FMR2 is the gene associated with FRAXE mental retardation. It is expressed as an 8.7-kb transcript in placenta and adult brain. A fetal-specific FMR2 transcript of approximately 12 kb was detected in fetal brain and at a lower level in fetal lung and kidney. FMR2 is a large gene composed of 22 exons spanning at least 500 kb on Xq28. Alternative splicing involving exons 2, 3, 5, 7, and 21 was not tissue specific as tested on mRNA from human fetal and infant brain. FMR2 is translated into a 1311-amino-acid nuclear protein with putative transcription transactivation potential. Subcellular localization studies with green fluorescent protein as a reporter show that both nuclear addresses found in the FMR2 sequence are functional and direct the FMR2 protein into the nucleus. FMR2 together with AF4 and LAF4 forms a new family of nuclear proteins with DNA-binding capacity and transcription transactivation potential. BLAST searches of the dbEST database revealed the presence of at least two other groups of nonoverlapping ESTs showing high similarity to the FMR2-related family of proteins. One of them, represented by the EST W26686, maps to chromosome 5q31. Amino acid similarity among the proteins encoded by members of the gene family is high in the NH 2 terminus, low in the middle, and high again in the COOH end. Available information from members of the family shows that genomic organization is conserved. This FMR2-related gene family encodes nuclear proteins with involvement in mental retardation (FMR2), cancer (AF4), and lymphocyte differentiation (LAF4) or with unknown function (EST W26686 and/or AA025630).

The genomic era has enabled the application of molecular tools to the solution of many of the genetic epilepsies, with and without comorbidities. Massively parallel sequencing has recently reinvigorated gene discovery for the monogenic epilepsies. Recurrent and novel copy number variants have given much-needed impetus to the advancement of our understanding of epilepsies with complex inheritance. Superimposed upon that is the phenotypic blurring by presumed genetic modifiers scattering the effects of the primary mutation. The genotype-first approach has uncovered associated syndrome constellations, of which epilepsy is only one of the syndromes. As the molecular genetic basis for the epilepsies unravels, it will increasingly influence the classification and diagnosis of the epilepsies. The ultimate goal of the molecular revolution has to be the design of treatment protocols based on genetic profiles, and cracking the 30% of epilepsies refractory to current medications, but that still lies well into the future. The current focus is on the scientific basis for epilepsy. Understanding its genetic causes and biophysical mechanisms is where we are currently positioned: prizing the causes of epilepsy "out of the shadows" and exposing its underlying mechanisms beyond even the ion-channels. Refereed/Peer-reviewed

Mutations of the SCN1A subunit of the sodium channel is a cause of genetic epilepsy with febrile seizures plus (GEFS+) in multiplex families and accounts for 70–80% of Dravet syndrome (DS). DS cases without SCN1A mutation inherited have predicted SCN9A susceptibility variants, which may contribute to complex inheritance for these unexplained cases of DS. Compared with controls, DS cases were significantly enriched for rare SCN9A genetic variants. None of the multiplex febrile seizure or GEFS+ families could be explained by highly penetrant SCN9A mutations. Refereed/Peer-reviewed

The majority of epilepsies are focal in origin, with seizures emanating from one brain region. Although focal epilepsies often arise from structural brain lesions, many affected individuals have normal brain imaging. The etiology is unknown in the majority of individuals, although genetic factors are increasingly recognized. Autosomal dominant familial focal epilepsy with variable foci (FFEVF) is notable because family members have seizures originating from different cortical regions. Using exome sequencing, we detected DEPDC5 mutations in two affected families. We subsequently identified mutations in five of six additional published large families with FFEVF. Study of families with focal epilepsy that were too small for conventional clinical diagnosis with FFEVF identified DEPDC5 mutations in approximately 12% of families (10/82). This high frequency establishes DEPDC5 mutations as a common cause of familial focal epilepsies. Shared homology with G protein signaling molecules and localization in human neurons suggest a role of DEPDC5 in neuronal signal transduction. Refereed/Peer-reviewed

There are two forms of mental handicap associated with fragile sites on the end of the long arm of the X chromosome. The well known common disorder Fragile X syndrome is associated with FRAXA and a rare non-specific form of mental handicap is associated with FRAXE. The cytogenetics of these fragile sites is considered. For Fragile X syndrome details are given of the molecular genetics, inheritance patterns, genetic counselling, methods for diagnosis of index cases, carrier detection and prenatal diagnosis. Series of prenatal diagnoses are briefly reviewed and technical and biological problems associated with this procedure are considered. Prenatal diagnosis of Fragile X syndrome using molecular genetic techniques is now a well established procedure, with the only significant problem being the inability to accurately predict phenotype in female fetuses with full mutations. Few prenatal diagnoses of Fragile XE non-specific mental retardation have been recorded. In principle the technical aspects of such a prenatal diagnosis should be little different from those for Fragile X syndrome. Incomplete knowledge of the phenotypic effect of the full mutation in males and females would make phenotypic prediction for any fetus shown to have such a mutation very difficult. At this stage all that could be determined with precision is that the mutation was present or absent in the fetus. Possible consequences of this are discussed.

The Seventh International Workshop on the Fragile X and X-linked Mental Retardation was held at the University of Tromso in Norway on August 2-5, 1995. Approximately 120 participants from 20 countries attended the Workshop. By special invitation Dr. Felix de la Cruz, who initiated the first international Workshop on fragile X, attended this Workshop. For the first time, the workshop took place in Scandinavia and was hosted by Lisbeth Tranebjaerg and Herbert Lubs. For most participants this Workshop, held at the northernmost university in the world, presented a unique opportunity to visit this exotic place. Between sessions, the participants had a chance to experience 24 hours of daylight, codfishing, and extreme weather situations with excessive amounts of rain as well as spectacular changes in the light and rainbows. The format of the Workshop was a combination of platform presentations and poster presentations. In contrast to previous meetings, the Workshop opened with syndromal and non-syndromal X-linked mental retardation in order to allow time for discussion. 34 refs., 1 fig.

A detailed map of genetic markers was constructed around the gene for the X-linked mental retardation syndrome of Borjeson-Forssman-Lehmann (BFLS). A multipoint linkage map of framework markers across Xq26-27, based on CEPH families, was integrated with the physical map, based on a YAC contig, to confirm marker order. The remaining genetic markers, which could not be ordered by linkage, were added to create the comprehensive genetic background map, in the order determined by physical mapping, to determine genetic distances between adjacent markers. This background genetic map is applicable to the refinement of the regional localization for any disease gene mapping to this region. The BFLS gene was localized using this background map in an extended version of the family described by Turner et al. The regional localization for BFLS extends between recombination events at DXS425 and DXS105, an interval of 24.6 cM on the background genetic map. The phenotypic findings commonly seen in the feet of affected males and obligate carrier females may represent a useful clinical indicator of carrier status in potential female carriers in the family. Recombination between DXS425 and DXS105 in a female with such characteristic feet suggests that the distal limit of the regional localization formore » the BFLS gene might reasonably be reduced to DXS294 for the purpose of selecting candidate genes, reducing the interval for the BFLS gene to 15.5 cM. Positional candidate genes from the interval between DXS425 and DXS105 include the SOX3 gene, mapped between DXS51(52A) and DXS98(4D-8). SOX3 may have a role in regulating the development of the nervous system. The HMG-box region of this single exon gene was examined by PCR for a deletion and then sequenced. No deviation from normal was observed, excluding mutations in the conserved HMG-box region as the cause of BFLS in this family. 27 refs., 1 fig., 2 tabs.« less