Anand K. Srivastava

Alterations in CDH15 and KIRREL3 in Patients with Mild to Severe Intellectual Disability

Cell-adhesion molecules play critical roles in brain development, as well as maintaining synaptic structure, function, and plasticity. Here we have found the disruption of two genes encoding putative cell-adhesion molecules, CDH15 (cadherin superfamily) and KIRREL3 (immunoglobulin superfamily), by a chromosomal translocation t(11;16) in a female patient with intellectual disability (ID). We screened coding regions of these two genes in a cohort of patients with ID and controls and identified four nonsynonymous CDH15 variants and three nonsynonymous KIRREL3 variants that appear rare and unique to ID. These variations altered highly conserved residues and were absent in more than 600 unrelated patients with ID and 800 control individuals. Furthermore, in vivo expression studies showed that three of the CDH15 variations adversely altered its ability to mediate cell-cell adhesion. We also show that in neuronal cells, human KIRREL3 colocalizes and interacts with the synaptic scaffolding protein, CASK, recently implicated in X-linked brain malformation and ID. Taken together, our data suggest that alterations in CDH15 and KIRREL3, either alone or in combination with other factors, could play a role in phenotypic expression of ID in some patients.

Sequence feature-based prediction of protein stability changes upon amino acid substitutions

BackgroundProtein destabilization is a common mechanism by which amino acid substitutions cause human diseases. Although several machine learning methods have been reported for predicting protein stability changes upon amino acid substitutions, the previous studies did not utilize relevant sequence features representing biological knowledge for classifier construction.ResultsIn this study, a new machine learning method has been developed for sequence feature-based prediction of protein stability changes upon amino acid substitutions. Support vector machines were trained with data from experimental studies on the free energy change of protein stability upon mutations. To construct accurate classifiers, twenty sequence features were examined for input vector encoding. It was shown that classifier performance varied significantly by using different sequence features. The most accurate classifier in this study was constructed using a combination of six sequence features. This classifier achieved an overall accuracy of 84.59% with 70.29% sensitivity and 90.98% specificity.ConclusionsRelevant sequence features can be used to accurately predict protein stability changes upon amino acid substitutions. Predictive results at this level of accuracy may provide useful information to distinguish between deleterious and tolerant alterations in disease candidate genes. To make the classifier accessible to the genetics research community, we have developed a new web server, called MuStab (http://bioinfo.ggc.org/mustab/).

RAI1 Transcription Factor Activity Is Impaired in Mutants Associated with Smith-Magenis Syndrome

Smith-Magenis Syndrome (SMS) is a complex genomic disorder mostly caused by the haploinsufficiency of the Retinoic Acid Induced 1 gene (RAI1), located in the chromosomal region 17p11.2. In a subset of SMS patients, heterozygous mutations in RAI1 are found. Here we investigate the molecular properties of these mutated forms and their relationship with the resulting phenotype. We compared the clinical phenotype of SMS patients carrying a mutation in RAI1 coding region either in the N-terminal or the C-terminal half of the protein and no significant differences were found. In order to study the molecular mechanism related to these two groups of RAI1 mutations first we analyzed those mutations that result in the truncated protein corresponding to the N-terminal half of RAI1 finding that they have cytoplasmic localization (in contrast to full length RAI1) and no ability to activate the transcription through an endogenous target: the BDNF enhancer. Similar results were found in lymphoblastoid cells derived from a SMS patient carrying RAI1 c.3103insC, where both mutant and wild type products of RAI1 were detected. The wild type form of RAI1 was found in the chromatin bound and nuclear matrix subcellular fractions while the mutant product was mainly cytoplasmic. In addition, missense mutations at the C-terminal half of RAI1 presented a correct nuclear localization but no activation of the endogenous target. Our results showed for the first time a correlation between RAI1 mutations and abnormal protein function plus they suggest that a reduction of total RAI1 transcription factor activity is at the heart of the SMS clinical presentation.

Detection of classical 17p11.2 deletions, an atypical deletion and RAI1 alterations in patients with features suggestive of Smith–Magenis syndrome

Smith–Magenis syndrome (SMS) is a complex disorder whose clinical features include mild to severe intellectual disability with speech delay, growth failure, brachycephaly, flat midface, short broad hands, and behavioral problems. SMS is typically caused by a large deletion on 17p11.2 that encompasses multiple genes including the retinoic acid induced 1, RAI1, gene or a mutation in the RAI1 gene. Here we have evaluated 30 patients with suspected SMS and identified SMS-associated classical 17p11.2 deletions in six patients, an atypical deletion of ∼139u2009kb that partially deletes the RAI1 gene in one patient, and RAI1 gene nonsynonymous alterations of unknown significance in two unrelated patients. The RAI1 mutant proteins showed no significant alterations in molecular weight, subcellular localization and transcriptional activity. Clinical features of patients with or without 17p11.2 deletions and mutations involving the RAI1 gene were compared to identify phenotypes that may be useful in diagnosing patients with SMS.

Autism and Intellectual Disability-Associated KIRREL3 Interacts with Neuronal Proteins MAP1B and MYO16 with Potential Roles in Neurodevelopment

Cell-adhesion molecules of the immunoglobulin superfamily play critical roles in brain development, as well as in maintaining synaptic plasticity, the dysfunction of which is known to cause cognitive impairment. Recently dysfunction of KIRREL3, a synaptic molecule of the immunoglobulin superfamily, has been implicated in several neurodevelopmental conditions including intellectual disability, autism spectrum disorder, and in the neurocognitive delay associated with Jacobsen syndrome. However, the molecular mechanisms of its physiological actions remain largely unknown. Using a yeast two-hybrid screen, we found that the KIRREL3 extracellular domain interacts with brain expressed proteins MAP1B and MYO16 and its intracellular domain can potentially interact with ATP1B1, UFC1, and SHMT2. The interactions were confirmed by co-immunoprecipitation and colocalization analyses of proteins expressed in human embryonic kidney cells, mouse neuronal cells, and rat primary neuronal cells. Furthermore, we show KIRREL3 colocalization with the marker for the Golgi apparatus and synaptic vesicles. Previously, we have shown that KIRREL3 interacts with the X-linked intellectual disability associated synaptic scaffolding protein CASK through its cytoplasmic domain. In addition, we found a genomic deletion encompassing MAP1B in one patient with intellectual disability, microcephaly and seizures and deletions encompassing MYO16 in two unrelated patients with intellectual disability, autism and microcephaly. MAP1B has been previously implicated in synaptogenesis and is involved in the development of the actin-based membrane skeleton. MYO16 is expressed in hippocampal neurons and also indirectly affects actin cytoskeleton through its interaction with WAVE1 complex. We speculate KIRREL3 interacting proteins are potential candidates for intellectual disability and autism spectrum disorder. Moreover, our findings provide further insight into understanding the molecular mechanisms underlying the physiological action of KIRREL3 and its role in neurodevelopment.

AT2 Receptor-Interacting Proteins ATIPs in the Brain.

A complete renin-angiotensin system (RAS) is locally expressed in the brain and fulfills important functions. Angiotensin II, the major biologically active peptide of the RAS, acts via binding to two main receptor subtypes designated AT1 and AT2. The present paper focuses on AT2 receptors, which have been reported to have neuroprotective effects on stroke, degenerative diseases, and cognitive functions. Our group has identified a family of AT2 receptor interacting proteins (ATIPs) comprising three major members (ATIP1, ATIP3, and ATIP4) with different intracellular localization. Of interest, all ATIP members are expressed in brain tissues and carry a conserved domain able to interact with the AT2 receptor intracellular tail, suggesting a role in AT2-mediated brain functions. We summarize here current knowledge on the ATIP family of proteins, and we present new experimental evidence showing interaction defects between ATIP1 and two mutant forms of the AT2 receptor identified in cases of mental retardation. These studies point to a functional role of the AT2/ATIP1 axis in cognition.

Differential diagnosis of Smith-Magenis syndrome: 1p36 deletion syndrome

Smith–Magenis syndrome (SMS; OMIM 182290) is a complexdisorder with an estimated incidence of approximately1:15,000–25,000 births [Greenberg et al., 1991]. The syndrome istypically caused by a large deletion on 17p11.2 that encompassesmultiplegenesincludingtheretinoicacid-induced1,RAI1,geneoramutationintheRAI1gene[Slageretal.,2003;Vlangosetal.,2003].The phenotype associated with SMS is characterized by a specificcombination of clinical features including variable intellectualdisability, sleep disturbance, craniofacial and skeletal anomalies,self-injurious and attention-seeking behaviors, and speech andmotor delay [Smith et al., 2005, 2010; Elsea and Girirajan, 2008].SMS is often under-diagnosed as its clinical features overlap withother intellectual disability syndromes as Prader–Willi, Williams,and Down syndromes, and 1p36 deletion syndrome.The 1p36 deletion syndrome was described in 1997 by Shapiraetal.Ithasanestimatedfrequencyof1in5,000–10,000birthsandiscaused by monosomy at chromosome 1p36 [Shapira et al., 1997;Shaffer and Lupski, 2000]. The phenotype associated with thiscontiguousgenedeletionsyndromeischaracterizedbyintellectualdisability, hypotonia, distinctive facies (deep-set eyes, prominentchin, flat nasal bridge, and asymmetric ears), and growth retarda-tion.Thesizeofthedeletionandthelocationofbreakpointsvaryineachfamilyandseemtobecorrelatedwiththephenotype[Heilstedtet al., 2003; Yu et al., 2003; Battaglia et al., 2008; Tsuyusaki et al.,2010].Recently, monosomy of 1p36.32–p36.33 was detected in apatient with Smith–Magenis-like phenotype without a deletion ormutationoftheRAI1gene[Williamsetal.,2010].Thepatientwiththe 1p36 deletion reportedly had sleep difficulties, learning andbehavioralproblemsaswellasshortstature,obesity,prognathism,dental abnormalities, brachydactyly, scoliosis, eye abnormalities,chronicearandrespiratoryinfections,andself-injuriousbehavior.SMSand1p36deletionsyndromeshowanoverlapofsomeclinicalfeatures, such as midface hypoplasia, deep-set eyes, intellectualdisability, speech delay, hypotonia, and behavior problems. How-ever,thereisnopreviousreportintheliteratureofthetypeofsleepdisordersnotedcommonlyinSMScasesoccurringinpatientswiththe 1p36 deletion. A comparison between SMS features and 1p36deletion syndrome features is shown (Table I).We present a female case (Fig. 1) with a 1p36 deletion whoseclinical features are consistent with SMS, but lacked 17p11.2deletion or a mutation in the RAI1 gene (data not shown). ThisstudywasapprovedbytheResearchEthicsCommitteeofBotucatuMedical School, S~ao Paulo State University/UNESP, Brazil.The patient is a 15-year-old female, born at term after anuncomplicatedpregnancytohealthy,nonconsanguineousparents.Her birth weight was 3,180g (50th centile) and birth length was50cm(50thcentile).Herheadcircumferencewasnotrecorded.Thepatienthaddysmorphiccraniofacialfeatures,developmentaldelay,speechdelay,infantilehypotonia,sleepdisturbance,seizures,dentalanomalies, and behavior problems. The diagnosis of SMS in thispatient was initially considered because the patient has a broad,

Microdeletion at 4q21.3 is associated with intellectual disability, dysmorphic facies, hypotonia, and short stature

Chromosomal imbalances are a major cause of intellectual disability (ID) and multiple congenital anomalies. We have clinically and molecularly characterized two patients with chromosome translocations and ID. Using whole genome array CGH analysis, we identified a microdeletion involving 4q21.3, unrelated to the translocations in both patients. We confirmed the 4q21.3 microdeletions using fluorescence in situ hybridization and quantitative genomic PCR. The corresponding deletion boundaries in the patients were further mapped and compared to previously reported 4q21 deletions and the associated clinical features. We determined a common region of deletion overlap that appears unique to ID, short stature, hypotonia, and dysmorphic facial features.

Candidate Agtr2 influenced genes and pathways identified by expression profiling in the developing brain of Agtr2−/y mice

Intellectual disability (ID) is a common developmental disability observed in 1 to 3% of the human population. A possible role for the Angiotensin II type 2 receptor (AGTR2) in brain function, affecting learning, memory, and behavior, has been suggested in humans and rodents. Mice lacking the Agtr2 gene (Agtr2(-/y)) showed significant impairment in their spatial memory and exhibited abnormal dendritic spine morphology. To identify Agtr2 influenced genes and pathways, we performed whole genome microarray analysis on RNA isolated from brains of Agtr2(-/y) and control male mice at embryonic day 15 (E15) and postnatal day one (P1). The gene expression profiles of the Agtr2(-/y) brain samples were significantly different when compared to profiles of the age-matched control brains. We identified 62 differently expressed genes (p< or =0.005) at E15 and in P1 brains of the Agtr2(-/y) mice. We verified the differential expression of several of these genes in brain samples using quantitative RT-PCR. Differentially expressed genes encode molecules involved in multiple cellular processes including microtubule functions associated with dendritic spine morphology. This study provides insight into Agtr2 influenced candidate genes and suggests that expression dysregulation of these genes may modulate Agtr2 actions in the brain that influences learning and memory.

Biological Features for Sequence-Based Prediction of Protein Stability Changes upon Amino Acid Substitutions

Protein destabilization is a common mechanism by which amino acid substitutions cause human diseases. In this study, a new machine learning method has been developed for sequence-based prediction of protein stability changes upon single amino acid substitutions. Support vector machines were trained with data from experimental studies on the free energy change of protein stability upon mutations. To construct accurate classifiers, twenty biological features were examined for input vector encoding. It was shown that classifier performance varied significantly by the use of different features. The most accurate classifier was constructed using a combination of several biological features. This classifier achieved an overall accuracy of 82.24% with 75.24% sensitivity and 85.36% specificity. Predictive results at this level of accuracy may be used in human genetic studies to distinguish between deleterious and tolerant alterations in disease candidate genes.