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1.  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
2.  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
3.  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
4.  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)
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
15.  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
16.  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
17.  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
18.  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
19.  Multilocus loss of DNA methylation in individuals with mutations in the histone H3 Lysine 4 Demethylase KDM5C 
A number of neurodevelopmental syndromes are caused by mutations in genes encoding proteins that normally function in epigenetic regulation. Identification of epigenetic alterations occurring in these disorders could shed light on molecular pathways relevant to neurodevelopment.
Using a genome-wide approach, we identified genes with significant loss of DNA methylation in blood of males with intellectual disability and mutations in the X-linked KDM5C gene, encoding a histone H3 lysine 4 demethylase, in comparison to age/sex matched controls. Loss of DNA methylation in such individuals is consistent with known interactions between DNA methylation and H3 lysine 4 methylation. Further, loss of DNA methylation at the promoters of the three top candidate genes FBXL5, SCMH1, CACYBP was not observed in more than 900 population controls. We also found that DNA methylation at these three genes in blood correlated with dosage of KDM5C and its Y-linked homologue KDM5D. In addition, parallel sex-specific DNA methylation profiles in brain samples from control males and females were observed at FBXL5 and CACYBP.
We have, for the first time, identified epigenetic alterations in patient samples carrying a mutation in a gene involved in the regulation of histone modifications. These data support the concept that DNA methylation and H3 lysine 4 methylation are functionally interdependent. The data provide new insights into the molecular pathogenesis of intellectual disability. Further, our data suggest that some DNA methylation marks identified in blood can serve as biomarkers of epigenetic status in the brain.
PMCID: PMC3573947  PMID: 23356856
KDM5C; DNA methylation; H3K4 methylation; Intellectual disability
20.  17p13.3 microduplications are associated with split-hand/foot malformation and long-bone deficiency (SHFLD) 
European Journal of Human Genetics  2011;19(11):1144-1151.
Split-hand/foot malformation with long-bone deficiency (SHFLD) is a relatively rare autosomal-dominant skeletal disorder, characterized by variable expressivity and incomplete penetrance. Although several chromosomal loci for SHFLD have been identified, the molecular basis and pathogenesis of most SHFLD cases are unknown. In this study we describe three unrelated kindreds, in which SHFLD segregated with distinct but overlapping duplications in 17p13.3, a region previously linked to SHFLD. In a large three-generation family, the disorder was found to segregate with a 254 kb microduplication; a second microduplication of 527 kb was identified in an affected female and her unaffected mother, and a 430 kb microduplication versus microtriplication was identified in three affected members of a multi-generational family. These findings, along with previously published data, suggest that one locus responsible for this form of SHFLD is located within a 173 kb overlapping critical region, and that the copy gains are incompletely penetrant.
PMCID: PMC3198152  PMID: 21629300
split-hand/foot malformation; SHFM; SHFLD; microduplication; microarray; conserved regulatory element
21.  A Missense Mutation in CLIC2 Associated with Intellectual Disability is Predicted by In Silico Modeling to Affect Protein Stability and Dynamics 
Proteins  2011;79(8):2444-2454.
Large-scale next generation resequencing of X chromosome genes identified a missense mutation in the CLIC2 gene on Xq28 in a male with X-linked intellectual disability (XLID) and not found in healthy individuals. At the same time, numerous nsSNPs (nonsynonomous SNP) have been reported in the CLIC2 gene in healthy individuals indicating that the CLIC2 protein can tolerate amino acid substitutions and be fully functional. To test the possibility that p.H101Q is a disease-causing mutation, we performed in silico simulations to calculate the effects of the p.H101Q mutation on CLIC2 stability, dynamics and ionization states while comparing the effects obtained for presumably harmless nsSNPs. It was found that p.H101Q, in contrast with other nsSNPs, (a) lessens the flexibility of the joint loop which is important for the normal function of CLIC2, (b) makes the overall 3D structure of CLIC2 more stable and thus reduces the possibility of the large conformational change expected to occur when CLIC2 moves from a soluble to membrane form and (c) removes the positively charged residue, H101, which may be important for the membrane association of CLIC2. The results of in silico modeling, in conjunction with the polymorphism analysis, suggest that p.H101Q may be a disease-causing mutation, the first one suggested in the CLIC family.
PMCID: PMC3132293  PMID: 21630357
CLIC2; missense mutations; mental disorder; energy calculations; pKa calculations; electrostatics; molecular dynamics simulations
22.  DRD2 and PPP1R1B (DARPP-32) polymorphisms independently confer increased risk for autism spectrum disorders and additively predict affected status in male-only affected sib-pair families 
The neurotransmitter dopamine (DA) modulates executive functions, learning, and emotional processing, all of which are impaired in individuals with autism spectrum disorders (ASDs). Our previous findings suggest a role for dopamine-related genes in families with only affected males.
We examined two additional genes which affect DA function, the DRD2 and PPP1R1B (DARPP-32) genes, in a cohort of 112 male-only affected sib-pair families. Selected polymorphisms spanning these genes were genotyped and both family-based and population-based tests were carried out for association analysis. General discriminant analysis was used to examine the gene-gene interactions in predicting autism susceptibility.
There was a significantly increased frequency of the DRD2 rs1800498TT genotype (P = 0.007) in affected males compared to the comparison group, apparently due to over-transmission of the T allele (P = 0.0003). The frequency of the PPP1R1B rs1495099CC genotype in affected males was also higher than that in the comparison group (P = 0.002) due to preferential transmission of the C allele from parents to affected children (P = 0.0009). Alleles rs1800498T and rs1495099C were associated with more severe problems in social interaction (P = 0.0002 and P = 0.0016, respectively) and communication (P = 0.0004 and P = 0.0046), and increased stereotypic behaviours (P = 0.0021 and P = 0.00072). General discriminant analysis found that the DRD2 and PPP1R1B genes additively predicted ASDs (P = 0.00011; Canonical R = 0.26) and explain ~7% of the variance in our families. All findings remained significant following corrections for multiple testing.
Our findings support a role for the DRD2 and PPP1R1B genes in conferring risk for autism in families with only affected males and show an additive effect of these genes towards prediction of affected status in our families.
PMCID: PMC3479424  PMID: 22559203
Autism spectrum disorders; Dopamine receptors; DARPP-32; Association study; Candidate gene
23.  Disruption of RAB40AL function leads to Martin–Probst syndrome, a rare X-linked multisystem neurodevelopmental human disorder 
Journal of Medical Genetics  2012;49(5):332-340.
Background and aim
Martin–Probst syndrome (MPS) is a rare X-linked disorder characterised by deafness, cognitive impairment, short stature and distinct craniofacial dysmorphisms, among other features. The authors sought to identify the causative mutation for MPS.
Methods and results
Massively parallel sequencing in two affected, related male subjects with MPS identified a RAB40AL (also called RLGP) missense mutation (chrX:102,079,078-102,079,079AC→GA p.D59G; hg18). RAB40AL encodes a small Ras-like GTPase protein with one suppressor of cytokine signalling box. The p.D59G variant is located in a highly conserved region of the GTPase domain between β-2 and β-3 strands. Using RT-PCR, the authors show that RAB40AL is expressed in human fetal and adult brain and kidney, and adult lung, heart, liver and skeletal muscle. RAB40AL appears to be a primate innovation, with no orthologues found in mouse, Xenopus or zebrafish. Western analysis and fluorescence microscopy of GFP-tagged RAB40AL constructs from transiently transfected COS7 cells show that the D59G missense change renders RAB40AL unstable and disrupts its cytoplasmic localisation.
This is the first study to show that mutation of RAB40AL is associated with a human disorder. Identification of RAB40AL as the gene mutated in MPS allows for further investigations into the molecular mechanism(s) of RAB40AL and its roles in diverse processes such as cognition, hearing and skeletal development.
PMCID: PMC3350147  PMID: 22581972
Genetics; guidelines; molecular genetics; clinical genetics; microarray; copy-number; complex traits; developmental; epigenetics; academic medicine; genome-wide; genetic epidemiology; chromosomal
24.  Structural Assessment of the Effects of Amino Acid Substitutions on Protein Stability and Protein-Protein Interaction 
A structure-based approach is described for predicting the effects of amino acid substitutions on protein function. Structures were predicted using a homology modelling method. Folding and binding energy differences between wild-type and mutant structures were computed to quantitatively assess the effects of amino acid substitutions on protein stability and protein–protein interaction, respectively. We demonstrated that pathogenic mutations at the interaction interface could affect binding energy and destabilise protein complex, whereas mutations at the non-interface might reduce folding energy and destabilise monomer structure. The results suggest that the structure-based analysis can provide useful information for understanding the molecular mechanisms of diseases.
PMCID: PMC3319068  PMID: 21297231
Amino acid substitutions; homology modeling; folding energy; binding energy; protein stability; protein-protein interaction
25.  Characterization of novel SLC6A8 variants with the use of splice-site analysis tools and implementation of a newly developed LOVD database 
The X-linked creatine transporter defect is caused by mutations in the SLC6A8 gene. Until now, 66 synonymous and intronic variants in SLC6A8 were detected in our laboratory. To gain more insight in the effect of the detected variants, we applied five free web-based splice-site analysis tools to 25 published variants that were stratified as (non-)disease causing. All were correctly predicted to have no effect (n=18) or to cause erroneous splicing (n=7), with the exception of a pathogenic de novo 24 bp intronic deletion. Second, 41 unclassified variants, including 28 novel, were subjected to analysis by these tools. At least four splice-site analysis tools predicted that three of the variants would affect splicing as the mutations disrupted the canonical splice site. Urinary creatine/creatinine and brain MRS confirmed creatine transporter deficiency in five patients (four families), including one female. Another variant was predicted to moderately affect splicing by all five tools. However, transient transfection of a minigene containing the variant in a partial SLC6A8 segment showed no splicing errors, and thus was finally classified as non-disease causing. This study shows that splice tools are useful for the characterization of the majority of variants, but also illustrates that the actual effect can be misclassified in rare occasions. Therefore, further laboratory studies should be considered before final conclusions on the disease-causing nature are drawn. To provide an accessible database, the 109 currently known SLC6A8 variants, including 35 novel ones, are included in a newly developed LOVD DNA variation database.
PMCID: PMC3039501  PMID: 20717164
SLC6A8; XLMR; splicing; LOVD

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