Transcriptome of iPSC-derived neuronal cells reveals a module of co-expressed genes consistently associated with autism spectrum disorder.

Evaluation of expression profile in autism spectrum disorder (ASD) patients is an important approach to understand possible similar functional consequences that may underlie disease pathophysiology regardless of its genetic heterogeneity. Induced pluripotent stem cell (iPSC)-derived neuronal models have been useful to explore this question, but larger cohorts and different ASD endophenotypes still need to be investigated. Moreover, whether changes seen in this in vitro model reflect previous findings in ASD postmortem brains and how consistent they are across the studies remain underexplored questions. We examined the transcriptome of iPSC-derived neuronal cells from a normocephalic ASD cohort composed mostly of high-functioning individuals and from non-ASD individuals. ASD patients presented expression dysregulation of a module of co-expressed genes involved in protein synthesis in neuronal progenitor cells (NPC), and a module of genes related to synapse/neurotransmission and a module related to translation in neurons. Proteomic analysis in NPC revealed potential molecular links between the modules dysregulated in NPC and in neurons. Remarkably, the comparison of our results to a series of transcriptome studies revealed that the module related to synapse has been consistently found as upregulated in iPSC-derived neurons-which has an expression profile more closely related to fetal brain-while downregulated in postmortem brain tissue, indicating a reliable association of this network to the disease and suggesting that its dysregulation might occur in different directions across development in ASD individuals. Therefore, the expression pattern of this network might be used as biomarker for ASD and should be experimentally explored as a therapeutic target.

Zebrafish sp7 mutants show tooth cycling independent of attachment, eruption and poor differentiation of teeth.

The capacity to fully replace teeth continuously makes zebrafish an attractive model to explore regeneration and tooth development. The requirement of attachment bone for the appearance of replacement teeth has been hypothesized but not yet investigated. The transcription factor sp7 (osterix) is known in mammals to play an important role during odontoblast differentiation and root formation. Here we study tooth replacement in the absence of attachment bone using sp7 zebrafish mutants. We analysed the pattern of tooth replacement at different stages of development and demonstrated that in zebrafish lacking sp7, attachment bone is never present, independent of the stage of tooth development or fish age, yet replacement is not interrupted. Without bone of attachment we observed abnormal orientation of teeth, and abnormal connection of pulp cavities of predecessor and replacement teeth. Mutants lacking sp7 show arrested dentinogenesis, with non-polarization of odontoblasts and only a thin layer of dentin deposited. Osteoclast activity was observed in sp7 mutants; due to the lack of bone of attachment, remodelling was diminished but nevertheless present along the pharyngeal bone. We conclude that tooth replacement is ongoing in the sp7 mutant despite poor differentiation and defective attachment. Without bone of attachment tooth orientation and pulp organization are compromised.

Richieri-Costa-Pereira syndrome: Expanding its phenotypic and genotypic spectrum.

Richieri-Costa-Pereira syndrome is a rare autosomal recessive acrofacial dysostosis that has been mainly described in Brazilian individuals. The cardinal features include Robin sequence, cleft mandible, laryngeal anomalies and limb defects. A biallelic expansion of a complex repeated motif in the 5' untranslated region of EIF4A3 has been shown to cause this syndrome, commonly with 15 or 16 repeats. The only patient with mild clinical findings harbored a 14-repeat expansion in 1 allele and a point mutation in the other allele. This proband is described here in more details, as well as is his affected sister, and 5 new individuals with Richieri-Costa-Pereira syndrome, including a patient from England, of African ancestry. This study has expanded the phenotype in this syndrome by the observation of microcephaly, better characterization of skeletal abnormalities, less severe phenotype with only mild facial dysmorphisms and limb anomalies, as well as the absence of cleft mandible, which is a hallmark of the syndrome. Although the most frequent mutation in this study was the recurrent 16-repeat expansion in EIF4A3, there was an overrepresentation of the 14-repeat expansion, with mild phenotypic expression, thus suggesting that the number of these motifs could play a role in phenotypic delineation.

Impact of rare variants in ARHGAP29 to the etiology of oral clefts: role of loss-of-function vs missense variants.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a prevalent, complex congenital malformation. Genome-wide association studies (GWAS) on NSCL/P have consistently identified association for the 1p22 region, in which ARHGAP29 has emerged as the main candidate gene. ARHGAP29 re-sequencing studies in NSCL/P patients have identified rare variants; however, their clinical impact is still unclear. In this study we identified 10 rare variants in ARHGAP29, including five missense, one in-frame deletion, and four loss-of-function (LoF) variants, in a cohort of 188 familial NSCL/P cases. A significant mutational burden was found for LoF (Sequence Kernel Association Test, p = 0.0005) but not for missense variants in ARHGAP29, suggesting that only LoF variants contribute to the etiology of NSCL/P. Penetrance was estimated as 59%, indicating that heterozygous LoF variants in ARHGAP29 confer a moderate risk to NSCL/P. The GWAS hits in IRF6 (rs642961) and 1p22 (rs560426 and rs4147811) do not seem to contribute to the penetrance of the phenotype, based on co-segregation analysis. Our data show that rare variants leading to haploinsufficiency of ARHGAP29 represent an important etiological clefting mechanism, and genetic testing for this gene might be taken into consideration in genetic counseling of familial cases.

Modeling non-syndromic autism and the impact of TRPC6 disruption in human neurons.

An increasing number of genetic variants have been implicated in autism spectrum disorders (ASDs), and the functional study of such variants will be critical for the elucidation of autism pathophysiology. Here, we report a de novo balanced translocation disruption of TRPC6, a cation channel, in a non-syndromic autistic individual. Using multiple models, such as dental pulp cells, induced pluripotent stem cell (iPSC)-derived neuronal cells and mouse models, we demonstrate that TRPC6 reduction or haploinsufficiency leads to altered neuronal development, morphology and function. The observed neuronal phenotypes could then be rescued by TRPC6 complementation and by treatment with insulin-like growth factor-1 or hyperforin, a TRPC6-specific agonist, suggesting that ASD individuals with alterations in this pathway may benefit from these drugs. We also demonstrate that methyl CpG binding protein-2 (MeCP2) levels affect TRPC6 expression. Mutations in MeCP2 cause Rett syndrome, revealing common pathways among ASDs. Genetic sequencing of TRPC6 in 1041 ASD individuals and 2872 controls revealed significantly more nonsynonymous mutations in the ASD population, and identified loss-of-function mutations with incomplete penetrance in two patients. Taken together, these findings suggest that TRPC6 is a novel predisposing gene for ASD that may act in a multiple-hit model. This is the first study to use iPSC-derived human neurons to model non-syndromic ASD and illustrate the potential of modeling genetically complex sporadic diseases using such cells.

A review of craniofacial disorders caused by spliceosomal defects.

The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNA transcripts. Mutations in EFTUD2, encoding a component of the major spliceosome, have recently been identified as the cause of mandibulofacial dysostosis, Guion-Almeida type (MFDGA), characterized by mandibulofacial dysostosis, microcephaly, external ear malformations and intellectual disability. Mutations in several other genes involved in spliceosomal function or linked aspects of mRNA processing have also recently been identified in human disorders with specific craniofacial malformations: SF3B4 in Nager syndrome, an acrofacial dysostosis (AFD); SNRPB in cerebrocostomandibular syndrome, characterized by Robin sequence and rib defects; EIF4A3 in the AFD Richieri-Costa-Pereira syndrome, characterized by Robin sequence, median mandibular cleft and limb defects; and TXNL4A in Burn-McKeown syndrome, involving specific craniofacial dysmorphisms. Here, we review phenotypic and molecular aspects of these syndromes. Given the apparent sensitivity of craniofacial development to defects in mRNA processing, it is possible that mutations in other proteins involved in spliceosomal function will emerge in the future as causative for related human disorders.

Autosomal recessive limb-girdle muscular dystrophy, LGMD2F, is caused by a mutation in the delta-sarcoglycan gene.

Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of inherited neuromuscular disorders characterized by proximal muscular weakness of the pelvic and shoulder girdles and a variable progression with symptoms, ranging from very severe to mild. One autosomal dominant (LGMD1A, at chromosome 5q22.3-31.3) (ref. 3) and five autosomal recessive (AR) loci responsible for this phenotype have been identified: LGMD2A at 15q (ref. 4); LGMD2B at 2p (ref. 5), LGMD2C at 13q (ref. 6), LGMD2D at 17q (ref. 7) and LGMD2E at 4q (refs 8,9). In the muscle membrane, dystrophin associates with several proteins and glycoproteins organized in two main subcomplexes: the dystroglycan (DG) and sarcoglycan (SG) complexes. The genes for LGMD2C, LGMD2D and LGMD2E code for proteins of the SG complex. We recently mapped a sixth AR form of LGMD, LGMD2F, to chromosome 5q33-34 in two Brazilian families. In the same chromosomal interval we also mapped the delta SG gene, encoding a novel 35-kD component of the sarcoglycan (SG) complex. We now show that a homozygous mutation in the delta SG gene (a single nucleotide deletion that alters its reading frame) is the cause of LGMD2F.

Limb-girdle muscular dystrophy type 2G is caused by mutations in the gene encoding the sarcomeric protein telethonin.

Autosomal recessive limb-girdle muscular dystrophies (AR LGMDs) are a genetically heterogeneous group of disorders that affect mainly the proximal musculature. There are eight genetically distinct forms of AR LGMD, LGMD 2A-H (refs 2-10), and the genetic lesions underlying these forms, except for LGMD 2G and 2H, have been identified. LGMD 2A and LGMD 2B are caused by mutations in the genes encoding calpain 3 (ref. 11) and dysferlin, respectively, and are usually associated with a mild phenotype. Mutations in the genes encoding gamma-(ref. 14), alpha-(ref. 5), beta-(refs 6,7) and delta (ref. 15)-sarcoglycans are responsible for LGMD 2C to 2F, respectively. Sarcoglycans, together with sarcospan, dystroglycans, syntrophins and dystrobrevin, constitute the dystrophin-glycoprotein complex (DGC). Patients with LGMD 2C-F predominantly have a severe clinical course. The LGMD 2G locus maps to a 3-cM interval in 17q11-12 in two Brazilian families with a relatively mild form of AR LGMD (ref. 9). To positionally clone the LGMD 2G gene, we constructed a physical map of the 17q11-12 region and refined its localization to an interval of 1.2 Mb. The gene encoding telethonin, a sarcomeric protein, lies within this candidate region. We have found that mutations in the telethonin gene cause LGMD 2G, identifying a new molecular mechanism for AR LGMD.

A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B.

The limb-girdle muscular dystrophies are a genetically heterogeneous group of inherited progressive muscle disorders that affect mainly the proximal musculature, with evidence for at least three autosomal dominant and eight autosomal recessive loci. The latter mostly involve mutations in genes encoding components of the dystrophin-associated complex; another form is caused by mutations in the gene for the muscle-specific protease calpain 3. Using a positional cloning approach, we have identified the gene for a form of limb-girdle muscular dystrophy that we previously mapped to chromosome 2p13 (LGMD2B). This gene shows no homology to any known mammalian gene, but its predicted product is related to the C. elegans spermatogenesis factor fer-1. We have identified two homozygous frameshift mutations in this gene, resulting in muscular dystrophy of either proximal or distal onset in nine families. The proposed name 'dysferlin' combines the role of the gene in producing muscular dystrophy with its C. elegans homology.

New SMS mutation leads to a striking reduction in spermine synthase protein function and a severe form of Snyder-Robinson X-linked recessive mental retardation syndrome.

We report the identification of a novel mutation at a highly conserved residue within the N-terminal region of spermine synthase (SMS) in a second family with Snyder-Robinson X-linked mental retardation syndrome (OMIM 309583). This missense mutation, p.G56S, greatly reduces SMS activity and leads to severe epilepsy and cognitive impairment. Our findings contribute to a better delineation and expansion of the clinical spectrum of Snyder-Robinson syndrome, support the important role of the N-terminus in the function of the SMS protein, and provide further evidence for the importance of SMS activity in the development of intellectual processing and other aspects of human development.

High frequency of submicroscopic chromosomal imbalances in patients with syndromic craniosynostosis detected by a combined approach of microsatellite segregation analysis, multiplex ligation-dependent probe amplification and array-based comparative genome hybridisation.

We present the first comprehensive study, to our knowledge, on genomic chromosomal analysis in syndromic craniosynostosis. In total, 45 patients with craniosynostotic disorders were screened with a variety of methods including conventional karyotype, microsatellite segregation analysis, subtelomeric multiplex ligation-dependent probe amplification) and whole-genome array-based comparative genome hybridisation. Causative abnormalities were present in 42.2% (19/45) of the samples, and 27.8% (10/36) of the patients with normal conventional karyotype carried submicroscopic imbalances. Our results include a wide variety of imbalances and point to novel chromosomal regions associated with craniosynostosis. The high incidence of pure duplications or trisomies suggests that these are important mechanisms in craniosynostosis, particularly in cases involving the metopic suture.

MRPL53, a New Candidate Gene for Orofacial Clefting, Identified Using an eQTL Approach.

A valuable approach to understand how individual and population genetic differences can predispose to disease is to assess the impact of genetic variants on cellular functions (e.g., gene expression) of cell and tissue types related to pathological states. To understand the genetic basis of nonsyndromic cleft lip with or without cleft palate (NSCL/P) susceptibility, a complex and highly prevalent congenital malformation, we searched for genetic variants with a regulatory role in a disease-related tissue, the lip muscle (orbicularis oris muscle [OOM]), of affected individuals. From 46 OOM samples, which are frequently discarded during routine corrective surgeries on patients with orofacial clefts, we derived mesenchymal stem cells and correlated the individual genetic variants with gene expression from these cultured cells. Through this strategy, we detected significant cis-eQTLs (i.e., DNA variants affecting gene expression) and selected a few candidates to conduct an association study in a large Brazilian cohort (624 patients and 668 controls). This resulted in the discovery of a novel susceptibility locus for NSCL/P, rs1063588, the best eQTL for the MRPL53 gene, where evidence for association was mostly driven by the Native American ancestry component of our Brazilian sample. MRPL53 (2p13.1) encodes a 39S protein subunit of mitochondrial ribosomes and interacts with MYC, a transcription factor required for normal facial morphogenesis. Our study illustrates not only the importance of sampling admixed populations but also the relevance of measuring the functional effects of genetic variants over gene expression to dissect the complexity of disease phenotypes.

Decreased cellular uptake and metabolism in Allan-Herndon-Dudley syndrome (AHDS) due to a novel mutation in the MCT8 thyroid hormone transporter.

We report a novel 1 bp deletion (c.1834delC) in the MCT8 gene in a large Brazilian family with Allan-Herndon-Dudley syndrome (AHDS), an X linked condition characterised by severe mental retardation and neurological dysfunction. The c.1834delC segregates with the disease in this family and it was not present in 100 control chromosomes, further confirming its pathogenicity. This mutation causes a frameshift and the inclusion of 64 additional amino acids in the C-terminal region of the protein. Pathogenic mutations in the MCT8 gene, which encodes a thyroid hormone transporter, results in elevated serum triiodothyronine (T3) levels, which were confirmed in four affected males of this family, while normal levels were found among obligate carriers. Through in vitro functional assays, we showed that this mutation decreases cellular T3 uptake and intracellular T3 metabolism. Therefore, the severe neurological defects present in the patients are due not only to deficiency of intracellular T3, but also to altered metabolism of T3 in central neurones. In addition, the severe muscle hypoplasia observed in most AHDS patients may be a consequence of high serum T3 levels.

Whole-genome array-CGH screening in undiagnosed syndromic patients: old syndromes revisited and new alterations.

We report array-CGH screening of 95 syndromic patients with normal G-banded karyotypes and at least one of the following features: mental retardation, heart defects, deafness, obesity, craniofacial dysmorphisms or urogenital tract malformations. Chromosome imbalances not previously detected in normal controls were found in 30 patients (31%) and at least 16 of them (17%) seem to be causally related to the abnormal phenotypes. Eight of the causative imbalances had not been described previously and pointed to new chromosome regions and candidate genes for specific phenotypes, including a connective tissue disease locus on 2p16.3, another for obesity on 7q22.1-->q22.3, and a candidate gene for the 3q29 deletion syndrome manifestations. The other causative alterations had already been associated with well-defined phenotypes including Sotos syndrome, and the 1p36 and 22q11.21 microdeletion syndromes. However, the clinical features of these latter patients were either not typical or specific enough to allow diagnosis before detection of chromosome imbalances. For instance, three patients with overlapping deletions in 22q11.21 were ascertained through entirely different clinical features, i.e., heart defect, utero-vaginal aplasia, and mental retardation associated with psychotic disease. Our results demonstrate that ascertainment through whole-genome screening of syndromic patients by array-CGH leads not only to the description of new syndromes, but also to the recognition of a broader spectrum of features for already described syndromes. Furthermore, on the technical side, we have significantly reduced the amount of reagents used and costs involved in the array-CGH protocol, without evident reduction in efficiency, bringing the method more within reach of centers with limited budgets.

Effect of polymorphisms of the MTHFR and APOE genes on susceptibility to diabetes and severity of diabetic retinopathy in Brazilian patients.

Diabetes mellitus (DM) is a highly prevalent complex genetic disorder. There has been a worldwide effort in the identification of susceptibility genes for DM and its complications, and the 5-10-methylenetetrahydrofolate reductase (MTHFR) and apolipoprotein-E (APOE) genes have been considered good candidate susceptibility genes to this condition. The objectives of the present study were to determine if the 677T MTHFR and epsilon2/epsilon3/epsilon4 APOE alleles are risk factors for DM and for severity of diabetic retinopathy (DR). A total of 248 individuals were studied: 107 healthy individuals and 141 diabetic patients (46 with type 1 diabetes and 95 with type 2 diabetes), who also had DR (81 with non-proliferative DR and 60 with proliferative DR). The polymorphisms were analyzed by PCR followed by digestion with restriction enzyme or the single-nucleotide primer extension method. No evidence of association between the 677TT genotype of MTHFR gene and DM [cases: TT = 10/95 (10.6%); controls: TT = 14/107 (13%)] or with severity of DR was observed [cases: TT = 5/60 (8.5%); controls: TT = 9/81 (11.1%); P > 0.05]. We also did not find evidence of an association between APOE alleles and proliferative DR (epsilon2, epsilon3 and epsilon4 in cases: 9, 76, and 15%, and in controls: 5, 88, and 12%, respectively) but the carriers of epsilon2 allele were more frequent among patients with type 2 DM and DR than in controls [cases: 15/95 (15.8%); controls: 7/107 (6.5%); P < 0.05]. Therefore, our results suggest that the epsilon2 allele/APOE might be a risk factor for diabetes in the Brazilian population.

A polymorphism in endostatin, an angiogenesis inhibitor, predisposes for the development of prostatic adenocarcinoma.

We have performed association studies between a novel coding single nucleotide polymorphism (D104N) in endostatin, one of the most potent inhibitors of angiogenesis, and prostate cancer. We observed that heterozygous N104 individuals have a 2.5 times increased chance of developing prostate cancer as compared with homozygous D104 subjects (odds ratio, 2.4; 95% confidence interval, 1.4-4.16). Modeling of the endostatin mutant showed that the N104 protein is stable. These results together with the observation that residue 104 is evolutionary conserved lead us to propose that: (a) the DNA segment containing this residue might contain a novel interaction site to a yet unknown receptor; and (b) the presence of N104 impairs the function of endostatin.

High mutation detection rate in TCOF1 among Treacher Collins syndrome patients reveals clustering of mutations and 16 novel pathogenic changes.

Twenty-eight families with a clinical diagnosis of Treacher Collins syndrome were screened for mutations in the 25 coding exons of TCOF1 and their adjacent splice junctions through SSCP and direct sequencing. Pathogenic mutations were detected in 26 patients, yielding the highest detection rate reported so far for this disease (93%) and bringing the number of known disease-causing mutations from 35 to 51. This is the first report to describe clustering of pathogenic mutations. Thirteen novel polymorphic alterations were characterized, confirming previous reports that TCOF1 has an unusually high rate of single-nucleotide polymorphisms (SNPs) within its coding region. We suggest a possible different mechanism leading to TCS or genetic heterogeneity for this condition, as we identified two families with no apparent pathogenic mutation in the gene. Furthermore, our data confirm the absence of genotype-phenotype correlation and reinforce that the apparent anticipation often observed in TCS families is due to ascertainment bias.

Further evidence for the organisation of the four sarcoglycans proteins within the dystrophin-glycoprotein complex.

Based on the pattern of distribution of the SG proteins in patients with LGMD2C and 2D, and on the observed decreased abundance of dystrophin through WB in some sarcoglycans (SG) patients, we have recently suggested that alpha, beta and delta subunits of sarcoglycan complex might be more closely associated and that gamma-SG might interact more directly with dystrophin. Two additional SG patients here reported give further support to these suggestions: an LGMD2F patient showed patchy labelling for gamma-SG, despite the lack of staining of the other three SG proteins; an LGMD2C boy showed deficiency in dystrophin by means of WB and IF, comparable with an DMD manifesting carrier. These two patients represent further evidence of a closer relation of alpha, beta and delta-SG than of gamma-SG and of the possible association of gamma-SG with dystrophin. In addition the LGMD2C patient illustrates the potential risk of misdiagnosis using only dystrophin analysis, in cases with no positive family history, or when DNA analysis is not informative.

High serum endostatin levels in Down syndrome: implications for improved treatment and prevention of solid tumours.

We report here a comparison of serum endostatin levels in Down syndrome patients to normal control subjects. We analysed serum samples from 35 patients with Down syndrome and 54 normal control subjects and found that although serum levels of endostatin vary widely in a normal human population, serum endostatin levels are significantly elevated in patients with Down syndrome. This result may explain the relative decrease in incidence of various solid tissue tumours observed in Down syndrome, given the role of endostatin as a potent inhibitor of tumour-induced angiogenesis in both human and animal models. Based upon these data, we propose that an increase of about one-third of normal endostatin serum levels may represent an effective therapeutic dose to significantly inhibit many solid tumours.