A subset of patients with Angelman and Prader-Willi syndrome have apparently normal chromosomes of biparental origin, but abnormal DNA methylation at several loci within chromosome 15q11-13, and probably have a defect in imprinting. Using probes from a newly established 160-kb contig including D15S63 (PW71) and SNRPN, we have identified inherited microdeletions in two AS families and three PWS families. The deletions probably affect a single genetic element that we term the 15q11-13 imprinting centre (IC). In our model, the IC regulates the chromatin structure, DNA methylation and gene expression in cis throughout 15q11-13. Mutations of the imprinting centre can be transmitted silently through the germline of one sex, but appear to block the resetting of the imprint in the germline of the opposite sex.

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Emery-Dreifuss muscular dystrophy (EDMD) is a rare disorder characterized by early joint contractures, muscular dystrophy, and cardiac involvement with conduction defects and arrhythmias. So far, only 35% of EDMD cases are genetically elucidated and associated with EMD or LMNA gene mutations, suggesting the existence of additional major genes. By whole-genome scan, we identified linkage to the Xq26.3 locus containing the FHL1 gene in three informative families belonging to our EMD- and LMNA-negative cohort. Analysis of the FHL1 gene identified seven mutations, in the distal exons of FHL1 in these families, three additional families, and one isolated case, which differently affect the three FHL1 protein isoforms: two missense mutations affecting highly conserved cysteines, one abolishing the termination codon, and four out-of-frame insertions or deletions. The predominant phenotype was characterized by myopathy with scapulo-peroneal and/or axial distribution, as well as joint contractures, and associated with a peculiar cardiac disease characterized by conduction defects, arrhythmias, and hypertrophic cardiomyopathy in all index cases of the seven families. Heterozygous female carriers were either asymptomatic or had cardiac disease and/or mild myopathy. Interestingly, four of the FHL1-mutated male relatives had isolated cardiac disease, and an overt hypertrophic cardiomyopathy was present in two. Expression and functional studies demonstrated that the FHL1 proteins were severely reduced in all tested patients and that this was associated with a severe delay in myotube formation in the two patients for whom myoblasts were available. In conclusion, FHL1 should be considered as a gene associated with the X-linked EDMD phenotype, as well as with hypertrophic cardiomyopathy.

Imprinting on human chromosome 15 is regulated by an imprinting centre, which has been mapped to a 100-kb region including exon 1 of SNRPN. From this region we have identified novel transcripts, which represent alternative transcripts of the SNRPN gene. The novel exons lack protein coding potential and are expressed from the paternal chromosome only. We have also identified intragenic deletions and a point mutation in patients who have Angelman or Prader-Willi syndrome due to a parental imprint switch failure. This suggests that imprint switching on human chromosome 15 may involve alternative SNRPN transcripts.

A de novo 9q33.3-q34.11 microdeletion involving STXBP1 has been found in one of four individuals (group A) with early-onset West syndrome, severe hypomyelination, poor visual attention, and developmental delay. Although haploinsufficiency of STXBP1 was involved in early infantile epileptic encephalopathy in a previous different cohort study (group B), no mutations of STXBP1 were found in two of the remaining three subjects of group A (one was unavailable). We assumed that another gene within the deletion might contribute to the phenotype of group A. SPTAN1 encoding alpha-II spectrin, which is essential for proper myelination in zebrafish, turned out to be deleted. In two subjects, an in-frame 3 bp deletion and a 6 bp duplication in SPTAN1 were found at the initial nucleation site of the alpha/beta spectrin heterodimer. SPTAN1 was further screened in six unrelated individuals with WS and hypomyelination, but no mutations were found. Recombinant mutant (mut) and wild-type (WT) alpha-II spectrin could assemble heterodimers with beta-II spectrin, but alpha-II (mut)/beta-II spectrin heterodimers were thermolabile compared with the alpha-II (WT)/beta-II heterodimers. Transient expression in mouse cortical neurons revealed aggregation of alpha-II (mut)/beta-II and alpha-II (mut)/beta-III spectrin heterodimers, which was also observed in lymphoblastoid cells from two subjects with in-frame mutations. Clustering of ankyrinG and voltage-gated sodium channels at axon initial segment (AIS) was disturbed in relation to the aggregates, together with an elevated action potential threshold. These findings suggest that pathological aggregation of alpha/beta spectrin heterodimers and abnormal AIS integrity resulting from SPTAN1 mutations were involved in pathogenesis of infantile epilepsy.

Patients with disorders involving imprinted genes such as Angelman syndrome (AS) and Prader-Willi syndrome (PWS) can have a mutation in the imprinting mechanism. Previously, we identified an imprinting center (IC) within chromosome 15q11-ql3 and proposed that IC mutations block resetting of the imprint, fixing on that chromosome the parental imprint (epigenotype) on which the mutation arose. We now describe four new microdeletions of the IC, the smallest (6 kb) of which currently defines the minimal region sufficient to confer an AS imprinting mutation. The AS deletions all overlap this minimal region, centromeric to the PWS microdeletions, which include the first exon of the SNRPN gene. None of five genes or transcripts in the 1.0 Mb vicinity of the IC (ZNF127, SNRPN, PAR-5, IPW, and PAR-1), each normally expressed only from the paternal allele, was expressed in cells from PWS imprinting mutation patients. In contrast, AS imprinting mutation patients show biparental expression of SNRPN and IPW but must lack expression of the putative AS gene 250-1000 kb distal of the IC. These data strongly support a model in which the paternal chromosome of these PWS patients carries an ancestral maternal epigenotype, and the maternal chromosome of these AS patients carries an ancestral paternal epigenotype. The IC therefore functions to reset the maternal and paternal imprints throughout a 2-Mb imprinted domain within human chromosome 15q11-q13 during gametogenesis.

Recent studies have established important roles of de novo mutations (DNMs) in autism spectrum disorders (ASDs). Here, we analyze DNMs in 262 ASD probands of Japanese origin and confirm the "de novo paradigm" of ASDs across ethnicities. Based on this consistency, we combine the lists of damaging DNMs in our and published ASD cohorts (total number of trios, 4,244) and perform integrative bioinformatics analyses. Besides replicating the findings of previous studies, our analyses highlight ATP-binding genes and fetal cerebellar/striatal circuits. Analysis of individual genes identified 61 genes enriched for damaging DNMs, including ten genes for which our dataset now contributes to statistical significance. Screening of compounds altering the expression of genes hit by damaging DNMs reveals a global downregulating effect of valproic acid, a known risk factor for ASDs, whereas cardiac glycosides upregulate these genes. Collectively, our integrative approach provides deeper biological and potential medical insights into ASDs.

Polycistronic transcripts are common in prokaryotes but rare in eukaryotes. Phylogenetic analysis of the SNRPN (SmN) mRNA in five eutherian mammals reveals a second highly conserved coding sequence, termed SNURF (SNRPN upstream reading frame). The vast majority of nucleotide substitutions in SNURF occur in the wobble codon position, providing strong evolutionary evidence for selection for protein-coding function. Because SNURF-SNRPN maps to human chromosome 15q11-q13 and is paternally expressed, each cistron is a candidate for a role in the imprinted Prader-Willi syndrome (PWS) and PWS mouse models. SNURF encodes a highly basic 71-aa protein that is nuclearlocalized (as is SmN). Because SNURF is the only proteincoding sequence within the imprinting regulatory region in 15q11-q13, it may have provided the original selection for imprinting in this domain. Whereas some human tissues express a minor SNURF-only transcript, mouse tissues express only the bicistronic Snurf-Snrpn transcript. We show that both SNURF and SNRPN are translated in normal, but not PWS, human, and mouse tissues and cell lines. These findings identify SNURF as a protein that is produced along with SmN from a bicistronic transcript; polycistronic mRNAs therefore are encoded in mammalian genomes where they may form functional operons.