De novo variants in MED12 cause X-linked syndromic neurodevelopmental disorders in 18 females.

PURPOSE: MED12 is a subunit of the Mediator multiprotein complex with a central role in RNA polymerase II transcription and regulation of cell growth, development, and differentiation. This might underlie the variable phenotypes in males carrying missense variants in MED12, including X-linked recessive Ohdo, Lujan, and FG syndromes. METHODS: By international matchmaking we assembled variant and clinical data on 18 females presenting with variable neurodevelopmental disorders (NDDs) and harboring de novo variants in MED12. RESULTS: Five nonsense variants clustered in the C-terminal region, two splice variants were found in the same exon 8 splice acceptor site, and 11 missense variants were distributed over the gene/protein. Protein truncating variants were associated with a severe, syndromic phenotype consisting of intellectual disability (ID), facial dysmorphism, short stature, skeletal abnormalities, feeding difficulties, and variable other abnormalities. De novo missense variants were associated with a less specific, but homogeneous phenotype including severe ID, autistic features, limited speech and variable other anomalies, overlapping both with females with truncating variants as well as males with missense variants. CONCLUSION: We establish de novo truncating variants in MED12 as causative for a distinct NDD and de novo missense variants as causative for a severe, less specific NDD in females.

Correction: Exome sequencing of Pakistani consanguineous families identifies 30 novel candidate genes for recessive intellectual disability.

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Exome sequencing of Pakistani consanguineous families identifies 30 novel candidate genes for recessive intellectual disability.

Intellectual disability (ID) is a clinically and genetically heterogeneous disorder, affecting 1-3% of the general population. Although research into the genetic causes of ID has recently gained momentum, identification of pathogenic mutations that cause autosomal recessive ID (ARID) has lagged behind, predominantly due to non-availability of sizeable families. Here we present the results of exome sequencing in 121 large consanguineous Pakistani ID families. In 60 families, we identified homozygous or compound heterozygous DNA variants in a single gene, 30 affecting reported ID genes and 30 affecting novel candidate ID genes. Potential pathogenicity of these alleles was supported by co-segregation with the phenotype, low frequency in control populations and the application of stringent bioinformatics analyses. In another eight families segregation of multiple pathogenic variants was observed, affecting 19 genes that were either known or are novel candidates for ID. Transcriptome profiles of normal human brain tissues showed that the novel candidate ID genes formed a network significantly enriched for transcriptional co-expression (P<0.0001) in the frontal cortex during fetal development and in the temporal-parietal and sub-cortex during infancy through adulthood. In addition, proteins encoded by 12 novel ID genes directly interact with previously reported ID proteins in six known pathways essential for cognitive function (P<0.0001). These results suggest that disruptions of temporal parietal and sub-cortical neurogenesis during infancy are critical to the pathophysiology of ID. These findings further expand the existing repertoire of genes involved in ARID, and provide new insights into the molecular mechanisms and the transcriptome map of ID.

X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes.

X-linked intellectual disability (XLID) is a clinically and genetically heterogeneous disorder. During the past two decades in excess of 100 X-chromosome ID genes have been identified. Yet, a large number of families mapping to the X-chromosome remained unresolved suggesting that more XLID genes or loci are yet to be identified. Here, we have investigated 405 unresolved families with XLID. We employed massively parallel sequencing of all X-chromosome exons in the index males. The majority of these males were previously tested negative for copy number variations and for mutations in a subset of known XLID genes by Sanger sequencing. In total, 745 X-chromosomal genes were screened. After stringent filtering, a total of 1297 non-recurrent exonic variants remained for prioritization. Co-segregation analysis of potential clinically relevant changes revealed that 80 families (20%) carried pathogenic variants in established XLID genes. In 19 families, we detected likely causative protein truncating and missense variants in 7 novel and validated XLID genes (CLCN4, CNKSR2, FRMPD4, KLHL15, LAS1L, RLIM and USP27X) and potentially deleterious variants in 2 novel candidate XLID genes (CDK16 and TAF1). We show that the CLCN4 and CNKSR2 variants impair protein functions as indicated by electrophysiological studies and altered differentiation of cultured primary neurons from Clcn4(-/-) mice or after mRNA knock-down. The newly identified and candidate XLID proteins belong to pathways and networks with established roles in cognitive function and intellectual disability in particular. We suggest that systematic sequencing of all X-chromosomal genes in a cohort of patients with genetic evidence for X-chromosome locus involvement may resolve up to 58% of Fragile X-negative cases.

X-linked congenital ptosis and associated intellectual disability, short stature, microcephaly, cleft palate, digital and genital abnormalities define novel Xq25q26 duplication syndrome.

Submicroscopic duplications along the long arm of the X-chromosome with known phenotypic consequences are relatively rare events. The clinical features resulting from such duplications are various, though they often include intellectual disability, microcephaly, short stature, hypotonia, hypogonadism and feeding difficulties. Female carriers are often phenotypically normal or show a similar but milder phenotype, as in most cases the X-chromosome harbouring the duplication is subject to inactivation. Xq28, which includes MECP2 is the major locus for submicroscopic X-chromosome duplications, whereas duplications in Xq25 and Xq26 have been reported in only a few cases. Using genome-wide array platforms we identified overlapping interstitial Xq25q26 duplications ranging from 0.2 to 4.76 Mb in eight unrelated families with in total five affected males and seven affected females. All affected males shared a common phenotype with intrauterine- and postnatal growth retardation and feeding difficulties in childhood. Three had microcephaly and two out of five suffered from epilepsy. In addition, three males had a distinct facial appearance with congenital bilateral ptosis and large protruding ears and two of them showed a cleft palate. The affected females had various clinical symptoms similar to that of the males with congenital bilateral ptosis in three families as most remarkable feature. Comparison of the gene content of the individual duplications with the respective phenotypes suggested three critical regions with candidate genes (AIFM1, RAB33A, GPC3 and IGSF1) for the common phenotypes, including candidate loci for congenital bilateral ptosis, small head circumference, short stature, genital and digital defects.

Phenotype and genotype in 101 males with X-linked creatine transporter deficiency.

BACKGROUND: Creatine transporter deficiency is a monogenic cause of X-linked intellectual disability. Since its first description in 2001 several case reports have been published but an overview of phenotype, genotype and phenotype--genotype correlation has been lacking. METHODS: We performed a retrospective study of clinical, biochemical and molecular genetic data of 101 males with X-linked creatine transporter deficiency from 85 families with a pathogenic mutation in the creatine transporter gene (SLC6A8). RESULTS AND CONCLUSIONS: Most patients developed moderate to severe intellectual disability; mild intellectual disability was rare in adult patients. Speech language development was especially delayed but almost a third of the patients were able to speak in sentences. Besides behavioural problems and seizures, mild to moderate motor dysfunction, including extrapyramidal movement abnormalities, and gastrointestinal problems were frequent clinical features. Urinary creatine to creatinine ratio proved to be a reliable screening method besides MR spectroscopy, molecular genetic testing and creatine uptake studies, allowing definition of diagnostic guidelines. A third of patients had a de novo mutation in the SLC6A8 gene. Mothers with an affected son with a de novo mutation should be counselled about a recurrence risk in further pregnancies due to the possibility of low level somatic or germline mosaicism. Missense mutations with residual activity might be associated with a milder phenotype and large deletions extending beyond the 3' end of the SLC6A8 gene with a more severe phenotype. Evaluation of the biochemical phenotype revealed unexpected high creatine levels in cerebrospinal fluid suggesting that the brain is able to synthesise creatine and that the cerebral creatine deficiency is caused by a defect in the reuptake of creatine within the neurones.

A 380-kb Duplication in 7p22.3 Encompassing the LFNG Gene in a Boy with Asperger Syndrome.

De novo genomic aberrations are considered an important cause of autism spectrum disorders. We describe a de novo 380-kb gain in band p22.3 of chromosome 7 in a patient with Asperger syndrome. This duplicated region contains 9 genes including the LNFG gene that is an important regulator of NOTCH signaling. We suggest that this copy number variation has been a contributive factor to the occurrence of Asperger syndrome in this patient.

Periventricular heterotopia in common microdeletion syndromes.

Periventricular heterotopia (PH) is a brain malformation characterised by heterotopic nodules of neurons lining the walls of the cerebral ventricles. Mutations in FLNA account for 20-24% of instances but a majority have no identifiable genetic aetiology. Often the co-occurrence of PH with a chromosomal anomaly is used to infer a new locus for a Mendelian form of PH. This study reports four PH patients with three different microdeletion syndromes, each characterised by high-resolution genomic microarray. In three patients the deletions at 1p36 and 22q11 are conventional in size, whilst a fourth child had a deletion at 7q11.23 that was larger in extent than is typically seen in Williams syndrome. Although some instances of PH associated with chromosomal deletions could be attributed to the unmasking of a recessive allele or be indicative of more prevalent subclinical migrational anomalies, the rarity of PH in these three microdeletion syndromes and the description of other non-recurrent chromosomal defects do suggest that PH may be a manifestation of multiple different forms of chromosomal imbalance. In many, but possibly not all, instances the co-occurrence of PH with a chromosomal deletion is not necessarily indicative of uncharacterised underlying monogenic loci for this particular neuronal migrational anomaly.

Screening of ARX in mental retardation families: Consequences for the strategy of molecular diagnosis.

Mutations in the human ARX gene have been shown to cause nonsyndromic X-linked mental retardation (MRX) as well as syndromic forms such as X-linked lissencephaly with abnormal genitalia (XLAG), Partington syndrome and X-linked infantile spasm. The most common causative mutation, a duplication of 24 bp, was found in families with a variety of phenotypes, but not in the more severe XLAG phenotypes. The aim of the study was to access the frequency of ARX mutations in families with established or putative X-linked mental retardation (XLMR) collected by the European XLMR Consortium. We screened the entire coding region of ARX for mutations in 197 novel XLMR families by denaturing high-performance liquid chromatography, and we identified eight mutations (six c.428_451dup24, one insertion and one novel missense mutation p.P38S). To better define the prevalence of ARX mutations, we included previously reported results of 157 XLMR families. Together, these data showed the relatively high rate (9.5%) of ARX mutations in X-linked MR families and an expectedly low rate in families with affected brother pairs (2.2%). This study confirms that the frequency of ARX mutations is high in XLMR, and the analysis of ARX in MRX should not be limited to duplication.

Chromosomal copy number changes in patients with non-syndromic X linked mental retardation detected by array CGH.

Several studies have shown that array based comparative genomic hybridisation (CGH) is a powerful tool for the detection of copy number changes in the genome of individuals with a congenital disorder. In this study, 40 patients with non-specific X linked mental retardation were analysed with full coverage, X chromosomal, bacterial artificial chromosome arrays. Copy number changes were validated by multiplex ligation dependent probe amplification as a fast method to detect duplications and deletions in patient and control DNA. This approach has the capacity to detect copy number changes as small as 100 kb. We identified three causative duplications: one family with a 7 Mb duplication in Xp22.2 and two families with a 500 kb duplication in Xq28 encompassing the MECP2 gene. In addition, we detected four regions with copy number changes that were frequently identified in our group of patients and therefore most likely represent genomic polymorphisms. These results confirm the power of array CGH as a diagnostic tool, but also emphasise the necessity to perform proper validation experiments by an independent technique.

A novel D458V mutation in the SANS PDZ binding motif causes atypical Usher syndrome.

Homozygosity mapping and linkage analysis in a Turkish family with autosomal recessive prelingual sensorineural hearing loss revealed a 15-cM critical region at 17q25.1-25.3 flanked by the polymorphic markers D17S1807 and D17S1806. The maximum two-point lod score was 4.07 at theta=0.0 for the marker D17S801. The linkage interval contains the Usher syndrome 1G gene (USH1G) that is mutated in patients with Usher syndrome (USH) type 1g and encodes the SANS protein. Mutation analysis of USH1G led to the identification of a homozygous missense mutation D458V at the -3 position of the PDZ binding motif of SANS. This mutation was also present homozygously in one out of 64 additional families from Turkey with autosomal recessive nonsyndromic hearing loss and heterozygously in one out of 498 control chromosomes. By molecular modeling, we provide evidence that this mutation impairs the interaction of SANS with harmonin. Ophthalmologic examination and vestibular evaluation of patients from both families revealed mild retinitis pigmentosa and normal vestibular function. These results suggest that these patients suffer from atypical USH.

Four novel TMC1 (DFNB7/DFNB11) mutations in Turkish patients with congenital autosomal recessive nonsyndromic hearing loss.

Mutations in the transmembrane channel-like gene 1 (TMC1) cause prelingual autosomal recessive (DFNB7/11) and postlingual progressive autosomal dominant (DFNA36) nonsyndromic hearing loss. To determine the genetic causes of autosomal recessive nonsyndromic hearing loss (ARNSHL) in the northeast and east of Turkey, 65 unrelated families without mutations in the protein coding region of the GJB2 (GJB2-negative) were analyzed. A genomewide scan for homozygosity and linkage analysis in one of these families revealed a 13.2 cM critical region between D9S273 and D9S153 at chromosome 9p13.2-q21.31 with a maximum two-point lod score of 4.00 at theta=0.0 for marker D9S175. TMC1 is in this critical region. Homozygosity screening with intragenic markers for TMC1 in the remaining 64 families suggested involvement of this gene in three additional families. Subsequent sequencing of TMC1 in these four families revealed four novel homozygous mutations, c.776A>G [p.Tyr259Cys], c.821C>T [p.Pro274Leu], c.1334G>A [p.Arg445His], and c.1083_1087delCAGAT [p.Arg362ProfrX6]. Our results indicate that TMC1 mutations account for at least 6% (4/65) of ARNSHL in GJB2-negative Turkish families from the northeast and east of Turkey.

Disruption of the gene Euchromatin Histone Methyl Transferase1 (Eu-HMTase1) is associated with the 9q34 subtelomeric deletion syndrome.

BACKGROUND: A new syndrome has been recognised following thorough analysis of patients with a terminal submicroscopic subtelomeric deletion of chromosome 9q. These have in common severe mental retardation, hypotonia, brachycephaly, flat face with hypertelorism, synophrys, anteverted nares, thickened lower lip, carp mouth with macroglossia, and conotruncal heart defects. The minimum critical region responsible for this 9q subtelomeric deletion syndrome (9q-) is approximately 1.2 Mb and encompasses at least 14 genes. OBJECTIVE: To characterise the breakpoints of a de novo balanced translocation t(X;9)(p11.23;q34.3) in a mentally retarded female patient with clinical features similar to the 9q- syndrome. RESULTS: Sequence analysis of the break points showed that the translocation was fully balanced and only one gene on chromosome 9 was disrupted--Euchromatin Histone Methyl Transferase1 (Eu-HMTase1)--encoding a histone H3 lysine 9 methyltransferase (H3-K9 HMTase). This indicates that haploinsufficiency of Eu-HMTase1 is responsible for the 9q submicroscopic subtelomeric deletion syndrome. This observation was further supported by the spatio-temporal expression of the gene. Using tissue in situ hybridisation studies in mouse embryos and adult brain, Eu-HMTase1 was shown to be expressed in the developing nervous system and in specific peripheral tissues. While expression is selectively downregulated in adult brain, substantial expression is retained in the olfactory bulb, anterior/ventral lateral ventricular wall, and hippocampus and weakly in the piriform cortex. CONCLUSIONS: The expression pattern of this gene suggests a role in the CNS development and function, which is in line with the severe mental retardation and behaviour problems in patients who lack one copy of the gene.

A novel locus for autosomal dominant non-syndromic hearing loss, DFNA31, maps to chromosome 6p21.3.

BACKGROUND: Non-syndromic hearing loss is the most genetically heterogeneous trait known in humans. To date, 51 loci for autosomal dominant non-syndromic sensorineural hearing loss (NSSHL) have been identified by linkage analysis. OBJECTIVE: To investigate the genes involved in a Dutch family with NSSHL. METHODS: Linkage analysis in a large Dutch pedigree with progressive bilateral loss of the mid and high frequencies, in which a novel dominant locus for postlingual NSSHL (DFNA31) has been identified. RESULTS: DFNA31 was found to be located in a 7.5 cM region of chromosome 6p21.3 between D6S276 (telomeric) and D6S273 (centromeric), with a maximum two point LOD score of 5.99 for D6S1624. DNA sequencing of coding regions and exon/intron boundaries of two candidate genes (POU5F1, GABBR1) in this interval did not reveal disease causing mutations. CONCLUSIONS: Haplotype analysis indicated that the genetic defect in this family does not overlap the DFNA13 and DFNA21 regions that are also located on 6p. Identification of the disease gene will be of major importance in understanding the pathophysiology of hearing impairment.

Determination of the stability of the noncovalent phospholipid transfer protein-lipid complex by electrospray time-of-flight mass spectrometry.

Phosphatidylcholine transfer protein (PC-TP) containing different molecular species of PC and phosphatidylinositol transfer protein alpha (PI-TPalpha) containing either a PI, PC, or PG molecule were identified as intact complexes by nano-electrospray ionization time-of-flight mass spectrometry. The stability of these complexes in the gas phase was determined by elevating the cone voltage (cv) resulting in the appearance of the protein void of lipid. PC-TP containing a PC species carrying an sn-1 palmitoyl chain was less stable than PC-TP containing a PC species carrying an sn-1 stearoyl chain given that these complexes were dissociated for 50% at a cv of roughly 30 and 45 V, respectively. Different acyl chains on the sn-2 position did not lead to significant changes in stability of the complex. In the case of PI-TPalpha, the complexes containing PI and PG were dissociated for 50% at a cv of 100 V as compared to a cv of 40 V for the complex containing PC. We propose that this difference in stability is due to hydrogen bonds between the polar headgroup of PI and PG and the lipid-binding site of PI-TPalpha. This may explain why PI-TPalpha preferentially binds PI from a membrane interface.

Clofibrate-induced relocation of phosphatidylcholine transfer protein to mitochondria in endothelial cells.

The phosphatidylcholine transfer protein (PC-TP) is a specific transporter of phosphatidylcholine (PC) between membranes. To get more insight into its physiological function, we have studied the localization of PC-TP by microinjection of fluorescently labeled PC-TP in foetal bovine heart endothelial (FBHE) cells and by expression of an enhanced yellow fluorescent protein-PC-TP fusion protein in FBHE cells, human umbilical vein endothelial cells, and HepG2 cells. Analysis by confocal laser scanning microscopy showed that PC-TP was evenly distributed throughout the cytosol with an apparently elevated level in nuclei. By measuring the fluorescence recovery after bleaching it was established that PC-TP is highly mobile throughout the cell, with its transport into the nucleus being hindered by the nuclear envelope. Given the proposed function of PC-TP in lipid metabolism, we have tested a number of compounds (phorbol ester, bombesin, A23187, thrombin, dibutyryl cyclic AMP, oleate, clofibrate, platelet-derived growth factor, epidermal growth factor, and hydrogen peroxide) for their ability to affect intracellular PC-TP distribution. Only clofibrate (100 microM) was found to have an effect, with PC-TP moving to mitochondria within 5 min of stimulation. This relocation did not occur with PC-TP(S110A), lacking the putative protein kinase C (PKC)-dependent phosphorylation site, and was restricted to the primary endothelial cells. Relocation did not occur in HepG2 cells, possibly due to the fact that clofibrate does not induce PKC activation in these cells.

The binding of phosphatidylcholine to the phosphatidylcholine transfer protein: affinity and role in folding.

Bovine liver phosphatidylcholine transfer protein (PC-TP) has been expressed in Escherichia coli and purified to homogeneity from the cytosol fraction at a yield of 0.45 mg PC-TP per 10 mg total cytosolic protein. In addition, active PC-TP was obtained from inclusion bodies. An essential factor in the activation of PC-TP was phosphatidylcholine (PC) present in the folding buffer. PC-TP from the cytosol contains phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) with a preference for the di-monounsaturated species over the saturated species as determined by fast atom bombardment mass spectrometry (FAB-MS). By incubation with microsomal membranes the endogenous PE and PG were replaced by PC. Relative to the microsomal PC species composition, PC-TP bound preferentially C16:0/C20:4-PC and C16:0/C18:2-PC (twofold enriched) whereas the major microsomal species C18:0/C18:1-PC and C18:0/C18:2-PC were distinctly less bound. PC-TP is structurally homologous to the lipid-binding domain of the steroidogenic acute regulatory protein (Nat. Struct. Biol. 7 (2000) 408). Replacement of Lys(55) present in one of the beta-strands forming the lipid-binding site, with an isoleucine residue yielded an inactive protein. This suggests that Lys(55) be involved in the binding of the PC molecule.