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1.  Revealing the Effects of Missense Mutations Causing Snyder-Robinson Syndrome on the Stability and Dimerization of Spermine Synthase 
Missense mutations in spermine synthase (SpmSyn) protein have been shown to cause the Snyder-Robinson syndrome (SRS). Depending on the location within the structure of SpmSyn and type of amino acid substitution, different mechanisms resulting in SRS were proposed. Here we focus on naturally occurring amino acid substitutions causing SRS, which are situated away from the active center of SpmSyn and thus are not directly involved in the catalysis. Two of the mutations, M35R and P112L, are reported for the first time in this study. It is demonstrated, both experimentally and computationally, that for such mutations the major effect resulting in dysfunctional SpmSyn is the destabilization of the protein. In vitro experiments indicated either no presence or very little amount of the mutant SpmSyn in patient cells. In silico modeling predicted that all studied mutations in this work destabilize SpmSyn and some of them abolish homo-dimer formation. Since dimerization and structural stability are equally important for the wild type function of SpmSyn, it is proposed that the SRS caused by mutations occurring in the N-domain of SpmSyn is a result of dysfunctional mutant proteins being partially unfolded and degraded by the proteomic machinery of the cell or being unable to form a homo-dimer.
PMCID: PMC4730321  PMID: 26761001
missense mutation; energy calculations; Snyder-Robinson syndrome; spermine synthase; binding affinity charge
2.  In utero gene therapy rescues microcephaly caused by Pqbp1-hypofunction in neural stem progenitor cells 
Molecular psychiatry  2014;20(4):459-471.
Human mutations in PQBP1 (1), a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain (2–4). Examination of a conditional Pqbp1-knockout (cKO) mouse with microcephaly failed to reveal either abnormal centrosomes or mitotic spindles, increased neurogenesis from the neural stem progenitor cell (NSPC) pool, or increased cell death in vivo. Instead, we observed an increase in the length of the cell cycle, particularly for the M phase in NSPCs. Corresponding to the developmental expression of Pqbp1 (5), the stem cell pool in vivo was decreased at E10 and remained at a low level during neurogenesis (E15) in Pqbp1-cKO mice. The expression profiles of NSPCs derived from the cKO mouse revealed significant changes in gene groups that control the M phase, including anaphase-promoting complex (APC/C) genes, via aberrant transcription and RNA splicing. Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO. These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation. Finally, we demonstrated that in utero gene therapy for Pqbp1-cKO mice by intraperitoneal injection of the PQBP1-AAV vector at E10 successfully rescued microcephaly with preserved cortical structures and improved behavioral abnormalities in Pqbp1-cKO mice, opening a new strategy for treating this intractable developmental disorder.
PMCID: PMC4378255  PMID: 25070536
3.  ZC4H2, an XLID gene, is required for the generation of a specific subset of CNS interneurons 
Human Molecular Genetics  2015;24(17):4848-4861.
Miles–Carpenter syndrome (MCS) was described in 1991 as an XLID syndrome with fingertip arches and contractures and mapped to proximal Xq. Patients had microcephaly, short stature, mild spasticity, thoracic scoliosis, hyperextendable MCP joints, rocker-bottom feet, hyperextended elbows and knees. A mutation, p.L66H, in ZC4H2, was identified in a XLID re-sequencing project. Additional screening of linked families and next generation sequencing of XLID families identified three ZC4H2 mutations: p.R18K, p.R213W and p.V75in15aa. The families shared some relevant clinical features. In silico modeling of the mutant proteins indicated all alterations would destabilize the protein. Knockout mutations in zc4h2 were created in zebrafish and homozygous mutant larvae exhibited abnormal swimming, increased twitching, defective eye movement and pectoral fin contractures. Because several of the behavioral defects were consistent with hyperactivity, we examined the underlying neuronal defects and found that sensory neurons and motoneurons appeared normal. However, we observed a striking reduction in GABAergic interneurons. Analysis of cell-type-specific markers showed a specific loss of V2 interneurons in the brain and spinal cord, likely arising from mis-specification of neural progenitors. Injected human wt ZC4H2 rescued the mutant phenotype. Mutant zebrafish injected with human p.L66H or p.R213W mRNA failed to be rescued, while the p.R18K mRNA was able to rescue the interneuron defect. Our findings clearly support ZC4H2 as a novel XLID gene with a required function in interneuron development. Loss of function of ZC4H2 thus likely results in altered connectivity of many brain and spinal circuits.
PMCID: PMC4527488  PMID: 26056227
4.  Impaired osteoblast and osteoclast function characterize the osteoporosis of Snyder - Robinson syndrome 
Snyder-Robinson Syndrome (SRS) is an X-linked intellectual disability disorder also characterized by osteoporosis, scoliosis, and dysmorphic facial features. It is caused by mutations in SMS, a ubiquitously expressed gene encoding the polyamine biosynthetic enzyme spermine synthase. We hypothesized that the tissue specificity of SRS arises from differential sensitivity to spermidine toxicity or spermine deficiency.
We performed detailed clinical, endocrine, histopathologic, and morphometric studies on two affected brothers with a spermine synthase loss of function mutation (NM_004595.4:c.443A > G, p.Gln148Arg). We also measured spermine and spermidine levels in cultured human bone marrow stromal cells (hBMSCs) and fibroblasts using the Biochrom 30 polyamine protocol and assessed the osteogenic potential of hBMSCs.
In addition to the known tissue-specific features of SRS, the propositi manifested retinal pigmentary changes, recurrent episodes of hyper- and hypoglycemia, nephrocalcinosis, renal cysts, and frequent respiratory infections. Bone histopathology and morphometry identified a profound depletion of osteoblasts and osteoclasts, absence of a trabecular meshwork, a low bone volume and a thin cortex. Comparison of cultured fibroblasts from affected and unaffected individuals showed relatively small changes in polyamine content, whereas comparison of cultured osteoblasts identified marked differences in spermidine and spermine content. Osteogenic differentiation of the SRS-derived hBMSCs identified a severe deficiency of calcium phosphate mineralization.
Our findings support the hypothesis that cell specific alterations in polyamine metabolism contribute to the tissue specificity of SRS features, and that the low bone density arises from a failure of mineralization.
Electronic supplementary material
The online version of this article (doi:10.1186/s13023-015-0235-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4428506  PMID: 25888122
Spermine; Snyder-Robinson syndrome; Osteoblast; Osteoclast; Osteoporosis
5.  Affected Kindred Analysis of Human X Chromosome Exomes to Identify Novel X-Linked Intellectual Disability Genes 
PLoS ONE  2015;10(2):e0116454.
X-linked Intellectual Disability (XLID) is a group of genetically heterogeneous disorders caused by mutations in genes on the X chromosome. Deleterious mutations in ~10% of X chromosome genes are implicated in causing XLID disorders in ~50% of known and suspected XLID families. The remaining XLID genes are expected to be rare and even private to individual families. To systematically identify these XLID genes, we sequenced the X chromosome exome (X-exome) in 56 well-established XLID families (a single affected male from 30 families and two affected males from 26 families) using an Agilent SureSelect X-exome kit and the Illumina HiSeq 2000 platform. To enrich for disease-causing mutations, we first utilized variant filters based on dbSNP, the male-restricted portions of the 1000 Genomes Project, or the Exome Variant Server datasets. However, these databases present limitations as automatic filters for enrichment of XLID genes. We therefore developed and optimized a strategy that uses a cohort of affected male kindred pairs and an additional small cohort of affected unrelated males to enrich for potentially pathological variants and to remove neutral variants. This strategy, which we refer to as Affected Kindred/Cross-Cohort Analysis, achieves a substantial enrichment for potentially pathological variants in known XLID genes compared to variant filters from public reference databases, and it has identified novel XLID candidate genes. We conclude that Affected Kindred/Cross-Cohort Analysis can effectively enrich for disease-causing genes in rare, Mendelian disorders, and that public reference databases can be used effectively, but cautiously, as automatic filters for X-linked disorders.
PMCID: PMC4332666  PMID: 25679214
6.  A mutation in a ganglioside biosynthetic enzyme, ST3GAL5, results in salt & pepper syndrome, a neurocutaneous disorder with altered glycolipid and glycoprotein glycosylation 
Human Molecular Genetics  2013;23(2):418-433.
‘Salt & Pepper’ syndrome is an autosomal recessive condition characterized by severe intellectual disability, epilepsy, scoliosis, choreoathetosis, dysmorphic facial features and altered dermal pigmentation. High-density SNP array analysis performed on siblings first described with this syndrome detected four shared regions of loss of heterozygosity (LOH). Whole-exome sequencing narrowed the candidate region to chromosome 2p11.2. Sanger sequencing confirmed a homozygous c.994G>A transition (p.E332K) in the ST3GAL5 gene, which encodes for a sialyltransferase also known as GM3 synthase. A different homozygous mutation of this gene has been previously associated with infantile-onset epilepsy syndromes in two other cohorts. The ST3GAL5 enzyme synthesizes ganglioside GM3, a glycosophingolipid enriched in neural tissue, by adding sialic acid to lactosylceramide. Unlike disorders of glycosphingolipid (GSL) degradation, very little is known regarding the molecular and pathophysiologic consequences of altered GSL biosynthesis. Glycolipid analysis confirmed a complete lack of GM3 ganglioside in patient fibroblasts, while microarray analysis of glycosyltransferase mRNAs detected modestly increased expression of ST3GAL5 and greater changes in transcripts encoding enzymes that lie downstream of ST3GAL5 and in other GSL biosynthetic pathways. Comprehensive glycomic analysis of N-linked, O-linked and GSL glycans revealed collateral alterations in response to loss of complex gangliosides in patient fibroblasts and in zebrafish embryos injected with antisense morpholinos that targeted zebrafish st3gal5 expression. Morphant zebrafish embryos also exhibited increased apoptotic cell death in multiple brain regions, emphasizing the importance of GSL expression in normal neural development and function.
PMCID: PMC3869362  PMID: 24026681
7.  Missense Mutation R338W in ARHGEF9 in a Family with X-linked Intellectual Disability with Variable Macrocephaly and Macro-Orchidism 
Non-syndromal X-linked intellectual disability (NS-XLID) represents a broad group of clinical disorders in which ID is the only clinically consistent manifestation. Although in many cases either chromosomal linkage data or knowledge of the >100 existing XLID genes has assisted mutation discovery, the underlying cause of disease remains unresolved in many families. We report the resolution of a large family (K8010) with NS-XLID, with variable macrocephaly and macro-orchidism. Although a previous linkage study had mapped the locus to Xq12-q21, this region contained too many candidate genes to be analyzed using conventional approaches. However, X-chromosome exome sequencing, bioinformatics analysis and segregation analysis revealed a novel missense mutation (c.1012C>T; p.R338W) in ARHGEF9. This gene encodes collybistin (CB), a neuronal GDP-GTP exchange factor previously implicated in several cases of XLID, as well as clustering of gephyrin and GABAA receptors at inhibitory synapses. Molecular modeling of the CB R338W substitution revealed that this change results in the substitution of a long electropositive side-chain with a large non-charged hydrophobic side-chain. The R338W change is predicted to result in clashes with adjacent amino acids (K363 and N335) and disruption of electrostatic potential and local folding of the PH domain, which is known to bind phosphatidylinositol-3-phosphate (PI3P/PtdIns-3-P). Consistent with this finding, functional assays revealed that recombinant CB CB2SH3−R338W was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an in vitro clustering assay. Taken together, these results suggest that the R338W mutation in ARHGEF9 is the underlying cause of NS-XLID in this family.
PMCID: PMC4719118  PMID: 26834553
ARHGEF9; collybistin; gephyrin; PH domain; XLID
8.  Protein sector analysis for the clustering of disease-associated mutations 
BMC Genomics  2014;15(Suppl 11):S4.
The importance of mutations in disease phenotype has been studied, with information available in databases such as OMIM. However, it remains a research challenge for the possibility of clustering amino acid residues based on an underlying interaction, such as co-evolution, to understand how mutations in these related sites can lead to different disease phenotypes.
This paper presents an integrative approach to identify groups of co-evolving residues, known as protein sectors. By studying a protein family using multiple sequence alignments and statistical coupling analysis, we attempted to determine if it is possible that these groups of residues could be related to disease phenotypes. After the protein sectors were identified, disease-associated residues within these groups of amino acids were mapped to a structure representing the protein family. In this study, we used the proposed pipeline to analyze two test cases of spermine synthase and Rab GDP dissociation inhibitor.
The results suggest that there is a possible link between certain groups of co-evolving residues and different disease phenotypes. The pipeline described in this work could also be used to study other protein families associated with human diseases.
PMCID: PMC4304181  PMID: 25559331
Co-evolving residues; statistical coupling analysis; protein sectors; disease phenotype
9.  Intellectual Disability and Autism Spectrum Disorders: Causal Genes and Molecular Mechanisms 
Intellectual disability (ID) and Autism Spectrum disorder (ASD) are the most common developmental disorders present in humans. Combined, they affect between 3-5% of the population. Additionally, they can be found together in the same individual thereby complicating treatment.
The causative factors (genes, epigenetic and environmental) are quite varied and likely interact so as to further complicate the assessment of an individual patient. Nonetheless, much valuable information has been gained by identifying candidate genes for ID or ASD. Understanding the etiology of either ID or ASD is of utmost importance for families. It allows a determination of the risk of recurrence, the possibility of other comorbidity medical problems, the molecular and cellular nature of the pathobiology and hopefully potential therapeutic approaches.
PMCID: PMC4185273  PMID: 24709068
Intellectual Disability; Autism Spectrum Disorders; Synaptic plasticity; Neurodevelopmental disorder; Molecular pathways
10.  MED12 Related Disorders 
American journal of medical genetics. Part A  2013;161(11):10.1002/ajmg.a.36183.
MED12 is a member of the large Mediator complex, which has a critical and central role in RNA polymerase II transcription. As a multiprotien complex, Mediator regulates signals involved in cell growth, development and differentiation, and it is involved in a protein network required for extraneuronal gene silencing and also functions as a direct suppressor of Gli3-dependent Sonic hedgehog signaling. This may may explain its role in several different X-linked intellectual disability syndromes that share some overlapping clinical features. This review will compare and contrast four different clinical conditions that have been associated with different mutations in MED12, which is located at Xq13. To date, these conditions include Opitz–Kaveggia (FG) syndrome, Lujan syndrome, Ohdo syndrome (Maat-Kievit-Brunner type, or OSMKB), and one large family with profound X-linked intellectual disability due to a novel c.5898insC frameshift mutation that unlike the other 3 syndromes, resulted in affected female carriers and truncation of the MED12 protein. It is likely that more MED12 mutations will be detected in sporadic patients and X-linked families with intellectual disability and dysmorphic features as exome sequencing becomes more commonly utilized, and this overview of MED12-related disorders may help to correlate MED12 genotypes with clinical findings.
PMCID: PMC3839301  PMID: 24123922
FG syndrome; Opitz-Kaveggia syndrome; Lujan-Fryns syndrome; MED12; X-linked intellectual disability; Ohdo syndrome (Maat-Kievit-Brunner type)
11.  Malformations Among the X-Linked Intellectual Disability Syndromes 
American journal of medical genetics. Part A  2013;161(11):10.1002/ajmg.a.36179.
Malformations are significant contributions to childhood mortality and disability. Their co-occurrence with intellectual disability may compound the health burden, requiring additional evaluation and management measures. Overall, malformations of greater or lesser severity occur in at least some cases of almost half of the 153 XLID syndromes. Genitourinary abnormalities are most common, but tend to contribute little or no health burden and occur in only a minority of cases of a given XLID syndrome. Some malformations (e.g., lissencephaly, hydranencephaly, long bone deficiency, renal agenesis/dysplasia) are not amenable to medical or surgical intervention; others (e.g., hydrocephaly, facial clefting, cardiac malformations, hypospadias) may be substantially corrected.
PMCID: PMC3813298  PMID: 24166814
Malformations; Birth Defects; X Chromosome; X-Linked Intellectual Disability
12.  A Y328C missense mutation in spermine synthase causes a mild form of Snyder–Robinson syndrome 
Human Molecular Genetics  2013;22(18):3789-3797.
Snyder–Robinson syndrome (SRS, OMIM: 309583) is an X-linked intellectual disability (XLID) syndrome, characterized by a collection of clinical features including facial asymmetry, marfanoid habitus, hypertonia, osteoporosis and unsteady gait. It is caused by a significant decrease or loss of spermine synthase (SMS) activity. Here, we report a new missense mutation, p.Y328C (c.1084A>G), in SMS in a family with XLID. The affected males available for evaluation had mild ID, speech and global delay, an asthenic build, short stature with long fingers and mild kyphosis. The spermine/spermidine ratio in lymphoblasts was 0.53, significantly reduced compared with normal (1.87 average). Activity analysis of SMS in the index patient failed to detect any activity above background. In silico modeling demonstrated that the Y328C mutation has a significant effect on SMS stability, resulting in decreased folding free energy and larger structural fluctuations compared with those of wild-type SMS. The loss of activity was attributed to the increase in conformational dynamics in the mutant which affects the active site geometry, rather than preventing dimer formation. Taken together, the biochemical and in silico studies confirm the p.Y328C mutation in SMS is responsible for the patients having a mild form of SRS and reveal yet another molecular mechanism resulting in a non-functional SMS causing SRS.
PMCID: PMC3749864  PMID: 23696453
13.  FG syndrome, an X-linked multiple congenital anomaly syndrome: The clinical phenotype and an algorithm for diagnostic testing 
FG syndrome is a rare X-linked multiple congenital anomaly-cognitive impairment disorder caused by the p.R961W mutation in the MED12 gene. We identified all known patients with this mutation to delineate their clinical phenotype and devise a clinical algorithm to facilitate molecular diagnosis. We ascertained 23 males with the p.R961W mutation in MED12 from 9 previously reported FG syndrome families and 1 new family. Six patients are reviewed in detail. These 23 patients were compared with 48 MED12 mutation-negative patients, who had the clinical diagnosis of FG syndrome. Traits that best discriminated between these two groups were chosen to develop an algorithm with high sensitivity and specificity for the p.R961W MED12 mutation. FG syndrome has a recognizable dysmorphic phenotype with a high incidence of congenital anomalies. A family history of X-linked mental retardation, deceased male infants, and/or multiple fetal losses was documented in all families. The algorithm identifies the p.R961W MED12 mutation-positive group with 100% sensitivity and 90% spec-ificity. The clinical phenotype of FG syndrome defines a recognizable pattern of X-linked multiple congenital anomalies and cognitive impairment. This algorithm can assist the clinician in selecting the patients for testing who are most likely to have the recurrent p.R961W MED12 mutation.
PMCID: PMC4113033  PMID: 19938245
FG syndrome; Opitz-Kaveggia syndrome; MED12; p.R961W; X-linked mental retardation; multiple congenital anomalies; algorithm
14.  Relationships between age and epi-genotype of the FMR1 exon 1/intron 1 boundary are consistent with non-random X-chromosome inactivation in FM individuals, with the selection for the unmethylated state being most significant between birth and puberty 
Human Molecular Genetics  2013;22(8):1516-1524.
Methylation of the fragile X-related epigenetic element 2 (FREE2) located on the exon 1/intron 1 boundary of the FMR1 gene is related to FMRP expression and cognitive impairment in full mutation (FM; CGG>200) individuals. We examined the relationship between age, the size of the FMR1 CGG expansion and the methylation output ratio (MOR) at 12 CpG sites proximal to the exon 1/intron 1 boundary using FREE2 MALDI-TOF MS. The patient cohort included 119 males and 368 females, i.e. 121 healthy controls (CGG<40), 176 premutation (CGG 55–170) and 190 FM (CGG 213–2000). For all CpG units examined, FM males showed a significantly elevated MOR compared with that in hypermethylated FM females. In FM males the MOR for most CpG units significantly positively correlated with both age and CGG size (P< 0.05). In FM females the skewing towards the unmethylated state was significant for half of the units between birth and puberty (P < 0.05). The methylation status of intron 1 CpG10–12 that was most significantly related to cognitive impairment in our earlier study, did not change significantly with age in FM females. These results challenge the concept of fragile X syndrome (FXS)-related methylation being static over time, and suggest that due to the preference for the unmethylated state in FM females, X-inactivation at this locus is not random. The findings also highlight that the prognostic value of FXS methylation testing is not uniform between all CpG sites, and thus may need to be evaluated on a site-by-site basis.
PMCID: PMC3605829  PMID: 23307923
15.  Prevalence of SHANK3 variants in patients with different subtypes of autism spectrum disorders 
Autism spectrum disorders (ASDs) include three main conditions: autistic disorder (AD), pervasive developmental disorder, not otherwise specified (PDD-NOS), and Asperger syndrome. It has been shown that many genes associated with ASDs are involved in the neuroligin–neurexin interaction at the glutamate synapse: NLGN3, NLGN4, NRXN1, CNTNAP2, and SHANK3. We screened this last gene in two cohorts of ASD patients (133 patients from US and 88 from Italy). We found 5/221 (2.3%) cases with pathogenic alterations: a 106 kb deletion encompassing the SHANK3 gene, two frameshift mutations leading to premature stop codons, a missense mutation (p.Pro141Ala), and a splicing mutation (c.1820-4 G>A). Additionally, in 17 patients (7.7%) we detected a c.1304+48C>T transition affecting a methylated cytosine in a CpG island. This variant is reported as SNP rs76224556 and was found in both US and Italian controls, but it results significantly more frequent in our cases than in the control cohorts. The variant is also significantly more common among PDD-NOS cases than in AD cases. We also screened this gene in an independent replication cohort of 104 US patients with ASDs, in which we found a missense mutation (p.Ala1468Ser) in 1 patient (0.9%), and in 8 patients (7.7%) we detected the c.1304+48C>T transition. While SHANK3 variants are present in any ASD subtype, the SNP rs76224556 appears to be significantly associated with PDD-NOS cases. This represents the first evidence of a genotype–phenotype correlation in ASDs and highlights the importance of a detailed clinical-neuropsychiatric evaluation for the effective genetic screening of ASD patients.
PMCID: PMC3573207  PMID: 22892527
autism; SHANK3; PDD-NOS; screening; genotype–phenotype correlation
16.  Is there a Mendelian transmission ratio distortion of the c.429_452dup(24bp) polyalanine tract ARX mutation? 
European Journal of Human Genetics  2012;20(12):1311-1314.
Intellectual disability is common. Aristaless-related homeobox (ARX) gene is one of the most frequently mutated and pleiotropic genes, implicated in 10 different phenotypes. More than half of ∼100 reported cases with ARX mutations are due to a recurrent duplication of 24 bp, c.429_452dup, which leads to polyalanine tract expansion. The excess of affected males among the offspring of the obligate carrier females raised the possibility of transmission ratio distortion for the c.429_452dup mutation. We found a significant deviation from the expected Mendelian 1:1 ratio of transmission in favour of the c.429_452dup ARX mutation. We hypothesise that the preferential transmission of the c.429_452dup mutation may be due to asymmetry of meiosis in the oocyte. Our findings may have implications for genetic counselling of families segregating the c.429_452dup mutation and allude to putative role of ARX in oocyte biology.
PMCID: PMC3499743  PMID: 22490986
intellectual disability; polyalanine tract expansions; Mendelian transmission; ARX; meiotic drive
17.  eIF2γ Mutation that Disrupts eIF2 Complex Integrity Links Intellectual Disability to Impaired Translation Initiation 
Molecular cell  2012;48(4):641-646.
Together with GTP and the initiator methionyl-tRNA, the translation initiation factor eIF2 forms a ternary complex that binds the 40S ribosome and then scans an mRNA to select the AUG start codon for protein synthesis. Here, we show that a human X-chromosomal neurological disorder characterized by intellectual disability and microcephaly is caused by a missense mutation in eIF2γ (encoded by EIF2S3), the core subunit of the heterotrimeric eIF2 complex. Biochemical studies of human cells overexpressing the eIF2γ mutant and of yeast eIF2γ with the analogous mutation revealed a defect in binding the eIF2β subunit to eIF2γ. Consistent with this loss of eIF2 integrity, the mutation in yeast eIF2γ impaired translation start codon selection and eIF2 function in vivo in a manner that was suppressed by overexpression of eIF2β. These findings directly link intellectual disability to impaired translation initiation, and provide a mechanistic basis for the human disease due to partial loss of eIF2 function.
PMCID: PMC3513554  PMID: 23063529
18.  Functional characterization of tissue-specific enhancers in the DLX5/6 locus 
Human Molecular Genetics  2012;21(22):4930-4938.
Disruption of distaless homeobox 5 and 6 (Dlx5/6) in mice results in brain, craniofacial, genital, ear and limb defects. In humans, chromosomal aberrations in the DLX5/6 region, some of which do not encompass DLX5/6, are associated with split hand/foot malformation 1 (SHFM1) as well as intellectual disability, craniofacial anomalies and hearing loss, suggesting that the disruption of DLX5/6 regulatory elements could lead to these abnormalities. Here, we characterized enhancers in the DLX5/6 locus whose tissue-specific expression and genomic location along with previously characterized enhancers correlate with phenotypes observed in individuals with chromosomal abnormalities. By analyzing chromosomal aberrations at 7q21, we refined the minimal SHFM1 critical region and used comparative genomics to select 26 evolutionary conserved non-coding sequences in this critical region for zebrafish enhancer assays. Eight of these sequences were shown to function as brain, olfactory bulb, branchial arch, otic vesicle and fin enhancers, recapitulating dlx5a/6a expression. Using a mouse enhancer assay, several of these zebrafish enhancers showed comparable expression patterns in the branchial arch, otic vesicle, forebrain and/or limb at embryonic day 11.5. Examination of the coordinates of various chromosomal rearrangements in conjunction with the genomic location of these tissue-specific enhancers showed a correlation with the observed clinical abnormalities. Our findings suggest that chromosomal abnormalities that disrupt the function of these tissue-specific enhancers could be the cause of SHFM1 and its associated phenotypes. In addition, they highlight specific enhancers in which mutations could lead to non-syndromic hearing loss, craniofacial defects or limb malformations.
PMCID: PMC3529576  PMID: 22914741
19.  An X-linked channelopathy with cardiomegaly due to a CLIC2 mutation enhancing ryanodine receptor channel activity 
Human Molecular Genetics  2012;21(20):4497-4507.
Chloride intracellular channel 2 (CLIC2) protein is a member of the glutathione transferase class of proteins. Its' only known function is the regulation of ryanodine receptor (RyR) intracellular Ca2+ release channels. These RyR proteins play a major role in the regulation of Ca2+ signaling in many cells. Utilizing exome capture and deep sequencing of genes on the X-chromosome, we have identified a mutation in CLIC2 (c.303C>G, p.H101Q) which is associated with X-linked intellectual disability (ID), atrial fibrillation, cardiomegaly, congestive heart failure (CHF), some somatic features and seizures. Functional studies of the H101Q variant indicated that it stimulated rather than inhibited the action of RyR channels, with channels remaining open for longer times and potentially amplifying Ca2+ signals dependent on RyR channel activity. The overly active RyRs in cardiac and skeletal muscle cells and neuronal cells would result in abnormal cardiac function and trigger post-synaptic pathways and neurotransmitter release. The presence of both cardiomegaly and CHF in the two affected males and atrial fibrillation in one are consistent with abnormal RyR2 channel function. Since the dysfunction of RyR2 channels in the brain via ‘leaky mutations’ can result in mild developmental delay and seizures, our data also suggest a vital role for the CLIC2 protein in maintaining normal cognitive function via its interaction with RyRs in the brain. Therefore, our patients appear to suffer from a new channelopathy comprised of ID, seizures and cardiac problems because of enhanced Ca2+ release through RyRs in neuronal cells and cardiac muscle cells.
PMCID: PMC3459470  PMID: 22814392
21.  Natural History of Christianson Syndrome 
Christianson syndrome is an X-linked mental retardation syndrome characterized by microcephaly, impaired ocular movement, severe global developmental delay, hypotonia which progresses to spasticity, and early onset seizures of variable types. Gilfillan et al. [2008] reported mutations in SLC9A6, the gene encoding the sodium/hydrogen exchanger NHE6, in the family first reported and in three others. They also noted the clinical similarities to Angelman syndrome and found cerebellar atrophy on MRI and elevated glutamate/glutamine in the basal ganglia on MRS. Here we report on nonsense mutations in two additional families. The natural history is detailed in childhood and adult life, the similarities to Angelman syndrome confirmed, and the MRI/MRS findings documented in three affected boys.
PMCID: PMC3698558  PMID: 20949524
X-linked; intellectual disability; SLC9A6; sodium/hydrogen exchanger; Christianson syndrome
22.  Decreased tryptophan metabolism in patients with autism spectrum disorders 
Molecular Autism  2013;4:16.
Autism spectrum disorders (ASDs) are relatively common neurodevelopmental conditions whose biological basis has been incompletely determined. Several biochemical markers have been associated with ASDs, but there is still no laboratory test for these conditions.
We analyzed the metabolic profile of lymphoblastoid cell lines from 137 patients with neurodevelopmental disorders with or without ASDs and 78 normal individuals, using Biolog Phenotype MicroArrays.
Metabolic profiling of lymphoblastoid cells revealed that the 87 patients with ASD as a clinical feature, as compared to the 78 controls, exhibited on average reduced generation of NADH when tryptophan was the sole energy source. The results correlated with the behavioral traits associated with either syndromal or non-syndromal autism, independent of the genetic background of the individual. The low level of NADH generation in the presence of tryptophan was not observed in cell lines from non-ASD patients with intellectual disability, schizophrenia or conditions exhibiting several similarities with syndromal autism except for the behavioral traits. Analysis of a previous small gene expression study found abnormal levels for some genes involved in tryptophan metabolic pathways in 10 patients.
Tryptophan is a precursor of important compounds, such as serotonin, quinolinic acid, and kynurenic acid, which are involved in neurodevelopment and synaptogenesis. In addition, quinolinic acid is the structural precursor of NAD+, a critical energy carrier in mitochondria. Also, the serotonin branch of the tryptophan metabolic pathway generates NADH. Lastly, the levels of quinolinic and kynurenic acid are strongly influenced by the activity of the immune system. Therefore, decreased tryptophan metabolism may alter brain development, neuroimmune activity and mitochondrial function. Our finding of decreased tryptophan metabolism appears to provide a unifying biochemical basis for ASDs and perhaps an initial step in the development of a diagnostic assay for ASDs.
PMCID: PMC3680090  PMID: 23731516
Autism; Biomarker; Tryptophan; Metabolism; Screening
23.  Seizures and X-linked intellectual disability 
Intellectual disability occurs as an isolated X-linked trait and as a component of recognizable X-linked syndromes in the company of somatic, metabolic, neuromuscular, or behavioral abnormalities. Seizures accompany intellectual disability in almost half of these X-linked disorders. The spectrum of seizures found in the X-linked intellectual disability syndromes is broad, varying in time of onset, type of seizure, and response to anticonvulsant therapy. The majority of the genes associated with XLID and seizures have now been identified.
PMCID: PMC3531238  PMID: 22377486
Epilepsy; Seizures; X-chromosome; X-linked; Intellectual disability; Syndrome
24.  Intellectual disability associated with a homozygous missense mutation in THOC6 
We recently described a novel autosomal recessive neurodevelopmental disorder with intellectual disability in four patients from two related Hutterite families. Identity-by-descent mapping localized the gene to a 5.1 Mb region at chromosome 16p13.3 containing more than 170 known or predicted genes. The objective of this study was to identify the causative gene for this rare disorder.
Methods and results
Candidate gene sequencing followed by exome sequencing identified a homozygous missense mutation p.Gly46Arg, in THOC6. No other potentially causative coding variants were present within the critical region on chromosome 16. THOC6 is a member of the THO/TREX complex which is involved in coordinating mRNA processing with mRNA export from the nucleus. In situ hybridization showed that thoc6 is highly expressed in the midbrain and eyes. Cellular localization studies demonstrated that wild-type THOC6 is present within the nucleus as is the case for other THO complex proteins. However, mutant THOC6 was predominantly localized to the cytoplasm, suggesting that the mutant protein is unable to carry out its normal function. siRNA knockdown of THOC6 revealed increased apoptosis in cultured cells.
Our findings associate a missense mutation in THOC6 with intellectual disability, suggesting the THO/TREX complex plays an important role in neurodevelopment.
PMCID: PMC3644499  PMID: 23621916
Intellectual disability; THOC6; THO/TREX complex; mRNA export; Hutterite
25.  Mutations in the guanine nucleotide exchange factor gene IQSEC2 cause nonsyndromic intellectual disability 
Nature genetics  2010;42(6):486-488.
The first family identified as having a nonsyndromic intellectual disability was mapped in 1988. Here we show that a mutation of IQSEC2, encoding a guanine nucleotide exchange factor for the ADP-ribosylation factor family of small GTPases, caused this disorder. In addition to MRX1, IQSEC2 mutations were identified in three other families with X-linked intellectual disability. This discovery was made possible by systematic and unbiased X chromosome exome resequencing.
PMCID: PMC3632837  PMID: 20473311

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