Anna Mikhailov

CC2D2A, Encoding A Coiled-Coil and C2 Domain Protein, Causes Autosomal-Recessive Mental Retardation with Retinitis Pigmentosa

Autosomal-recessive inheritance is believed to be relatively common in mental retardation (MR), although only four genes for nonsyndromic autosomal-recessive mental retardation (ARMR) have been reported. In this study, we ascertained a consanguineous Pakistani family with ARMR in four living individuals from three branches of the family, plus an additional affected individual later identified as a phenocopy. Retinitis pigmentosa was present in affected individuals, but no other features suggestive of a syndromic form of MR were found. We used Affymetrix 500K microarrays to perform homozygosity mapping and identified a homozygous and haploidentical region of 11.2 Mb on chromosome 4p15.33-p15.2. Linkage analysis across this region produced a maximum two-point LOD score of 3.59. We sequenced genes within the critical region and identified a homozygous splice-site mutation segregating in the family, within a coiled-coil and C2 domain-containing gene, CC2D2A. This mutation leads to the skipping of exon 19, resulting in a frameshift and a truncated protein lacking the C2 domain. Conservation analysis for CC2D2A suggests a functional domain near the C terminus as well as the C2 domain. Preliminary functional studies of CC2D2A suggest a possible role in Ca(2+)-dependent signal transduction. Identifying the function of CC2D2A, and a possible common pathway with CC2D1A, in correct neuronal development and functioning may help identify possible therapeutic targets for MR.

Identification of risk genes for autism spectrum disorder through copy number variation analysis in Austrian families

Autism or autism spectrum disorder (ASD) is a range of neurodevelopmental disorders starting in early childhood and is characterized by impairments in communication and reciprocal social interaction and presence of restricted and repetitive patterns of behavior. The contribution of genetic factors to autism is clear in twin and family studies. It is apparent that, overall, ASD is a complex non-Mendelian disorder. Recent studies suggest that copy number variations (CNVs) play a significant role in the etiology of ASD. For the current work, we recruited 245 family members from 73 ASD families from Styria, Austria. The DNA from probands was genotyped with Affymetrix single nucleotide polymorphism (SNP) 6.0 microarrays to screen for CNVs in their genomes. Analysis of the microarray data was performed using three different algorithms, and a list of stringent calls was compared to existing CNV data from over 2,357 controls of European ancestry. For stringent calls not present in controls, quantitative real-time PCR (qRT-PCR) was used to validate the CNVs in the probands and in their family members. Twenty-two CNVs were validated from this set (five of which are apparently de novo), many of which appear likely to disrupt genes that may be considered as good candidates for neuropsychiatric disorders, including DLG2, S100B, ARX, DIP2A, HPCAL1, and GPHN. Several others disrupt genes that have previously been implicated in autism, such as BDNF, AUTS2, DPP6, and C18orf22, and our data add to the growing evidence of their involvement in ASD.

Biallelic Truncating Mutations in FMN2, Encoding the Actin-Regulatory Protein Formin 2, Cause Nonsyndromic Autosomal-Recessive Intellectual Disability

Dendritic spines represent the major site of neuronal activity in the brain; they serve as the receiving point for neurotransmitters and undergo rapid activity-dependent morphological changes that correlate with learning and memory. Using a combination of homozygosity mapping and next-generation sequencing in two consanguineous families affected by nonsyndromic autosomal-recessive intellectual disability, we identified truncating mutations in formin 2 (FMN2), encoding a protein that belongs to the formin family of actin cytoskeleton nucleation factors and is highly expressed in the maturing brain. We found that FMN2 localizes to punctae along dendrites and that germline inactivation of mouse Fmn2 resulted in animals with decreased spine density; such mice were previously demonstrated to have a conditioned fear-learning defect. Furthermore, patient neural cells derived from induced pluripotent stem cells showed correlated decreased synaptic density. Thus, FMN2 mutations link intellectual disability either directly or indirectly to the regulation of actin-mediated synaptic spine density.

Mapping autosomal recessive intellectual disability: combined microarray and exome sequencing identifies 26 novel candidate genes in 192 consanguineous families

Approximately 1% of the global population is affected by intellectual disability (ID), and the majority receive no molecular diagnosis. Previous studies have indicated high levels of genetic heterogeneity, with estimates of more than 2500 autosomal ID genes, the majority of which are autosomal recessive (AR). Here, we combined microarray genotyping, homozygosity-by-descent (HBD) mapping, copy number variation (CNV) analysis, and whole exome sequencing (WES) to identify disease genes/mutations in 192 multiplex Pakistani and Iranian consanguineous families with non-syndromic ID. We identified definite or candidate mutations (or CNVs) in 51% of families in 72 different genes, including 26 not previously reported for ARID. The new ARID genes include nine with loss-of-function mutations (ABI2, MAPK8, MPDZ, PIDD1, SLAIN1, TBC1D23, TRAPPC6B, UBA7 and USP44), and missense mutations include the first reports of variants in BDNF or TET1 associated with ID. The genes identified also showed overlap with de novo gene sets for other neuropsychiatric disorders. Transcriptional studies showed prominent expression in the prenatal brain. The high yield of AR mutations for ID indicated that this approach has excellent clinical potential and should inform clinical diagnostics, including clinical whole exome and genome sequencing, for populations in which consanguinity is common. As with other AR disorders, the relevance will also apply to outbred populations.

Mutations in the histamine N-methyltransferase gene, HNMT, are associated with nonsyndromic autosomal recessive intellectual disability

Histamine (HA) acts as a neurotransmitter in the brain, which participates in the regulation of many biological processes including inflammation, gastric acid secretion and neuromodulation. The enzyme histamine N-methyltransferase (HNMT) inactivates HA by transferring a methyl group from S-adenosyl-l-methionine to HA, and is the only well-known pathway for termination of neurotransmission actions of HA in mammalian central nervous system. We performed autozygosity mapping followed by targeted exome sequencing and identified two homozygous HNMT alterations, p.Gly60Asp and p.Leu208Pro, in patients affected with nonsyndromic autosomal recessive intellectual disability from two unrelated consanguineous families of Turkish and Kurdish ancestry, respectively. We verified the complete absence of a functional HNMT in patients using in vitro toxicology assay. Using mutant and wild-type DNA constructs as well as in silico protein modeling, we confirmed that p.Gly60Asp disrupts the enzymatic activity of the protein, and that p.Leu208Pro results in reduced protein stability, resulting in decreased HA inactivation. Our results highlight the importance of inclusion of HNMT for genetic testing of individuals presenting with intellectual disability.

Over-Expression of Either MECP2_e1 or MECP2_e2 in Neuronally Differentiated Cells Results in Different Patterns of Gene Expression

Mutations in MECP2 are responsible for the majority of Rett syndrome cases. MECP2 is a regulator of transcription, and has two isoforms, MECP2_e1 and MECP2_e2. There is accumulating evidence that MECP2_e1 is the etiologically relevant variant for Rett. In this study we aim to detect genes that are differentially transcribed in neuronal cells over-expressing either of these two MECP2 isoforms. The human neuroblastoma cell line SK-N-SH was stably infected by lentiviral vectors over-expressing MECP2_e1, MECP2_e2, or eGFP, and were then differentiated into neurons. The same lentiviral constructs were also used to infect mouse Mecp2 knockout (Mecp2tm1.1Bird) fibroblasts. RNA from these cells was used for microarray gene expression analysis. For the human neuronal cells, ∼800 genes showed >three-fold change in expression level with the MECP2_e1 construct, and ∼230 with MECP2_e2 (unpaired t-test, uncorrected p value <0.05). We used quantitative RT-PCR to verify microarray results for 41 of these genes. We found significant up-regulation of several genes resulting from over-expression of MECP2_e1 including SRPX2, NAV3, NPY1R, SYN3, and SEMA3D. DOCK8 was shown via microarray and qRT-PCR to be upregulated in both SK-N-SH cells and mouse fibroblasts. Both isoforms up-regulated GABRA2, KCNA1, FOXG1 and FOXP2. Down-regulation of expression in the presence of MECP2_e1 was seen with UNC5C and RPH3A. Understanding the biology of these differentially transcribed genes and their role in neurodevelopment may help us to understand the relative functions of the two MECP2 isoforms, and ultimately develop a better understanding of RTT etiology and determine the clinical relevance of isoform-specific mutations.

Truncation of the E3 ubiquitin ligase component FBXO31 causes non-syndromic autosomal recessive intellectual disability in a Pakistani family

In this study, we have performed autozygosity mapping on a large consanguineous Pakistani family segregating with intellectual disability. We identified two large regions of homozygosity-by-descent (HBD) on 16q12.2–q21 and 16q24.1–q24.3. Whole exome sequencing (WES) was performed on an affected individual from the family, but initially, no obvious mutation was detected. However, three genes within the HBD regions that were not fully captured during the WES were Sanger sequenced and we identified a five base pair deletion (actually six base pairs deleted plus one base pair inserted) in exon 7 of the gene FBXO31. The variant segregated completely in the family, in recessive fashion giving a LOD score of 3.95. This variant leads to a frameshift and a premature stop codon and truncation of the FBXO31 protein, p.(Cys283Asnfs*81). Quantification of mRNA and protein expression suggests that nonsense-mediated mRNA decay also contributes to the loss of FBXO31 protein in affected individuals. FBXO31 functions as a centrosomal E3 ubiquitin ligase, in association with SKP1 and Cullin-1, involved in ubiquitination of proteins targeted for degradation. The FBXO31/SKP1/Cullin1 complex is important for neuronal morphogenesis and axonal identity. FBXO31 also plays a role in dendrite growth and neuronal migration in developing cerebellar cortex. Our finding adds further evidence of the involvement of disruption of the protein ubiquitination pathway in intellectual disability.

New recessive truncating mutation in LTBP3 in a family with oligodontia, short stature, and mitral valve prolapse.

Latent TGFB‐binding protein 3 (LTBP3) is known to increase bio‐availability of TGFB. A homozygous mutation in this gene has previously been associated with oligodontia and short stature in a single family. We report on two sisters with homozygous truncating mutations in LTBP3. In addition to oligodontia and short stature, both sisters have mitral valve prolapse, suggesting a link between truncating LTBP3 mutations and mitral valve disease mediated through the TGFB pathway.

Identification of a homozygous missense mutation in LRP2 and a hemizygous missense mutation in TSPYL2 in a family with mild intellectual disability.

Non-syndromic autosomal recessive intellectual disability (ID) is a genetically heterogeneous disorder with more than 50 mutated genes to date. ID is characterized by deficits in memory skills and language development with difficulty in learning, problem solving, and adaptive behaviors, and affects ∼1% of the population. For detection of disease-causing mutations in such a heterogeneous disorder, homozygosity mapping together with exome sequencing is a powerful approach, as almost all known genes can be assessed simultaneously in a high-throughput manner. In this study, a hemizygous c.786C>G:p.Ile262Met in the testis specific protein Y-encoded-like 2 (TSPYL2) gene and a homozygous c.11335G>A:p.Asp3779Asn in the low-density lipoprotein receptor-related protein 2 (LRP2) gene were detected after genome-wide genotyping and exome sequencing in a consanguineous Pakistani family with two boys with mild ID. Mutations in the LRP2 gene have previously been reported in patients with Donnai–Barrow and Stickler syndromes. LRP2 has also been associated with a 2q locus for autism (AUTS5). The TSPYL2 variant is not listed in any single-nucleotide polymorphism databases, and the LRP2 variant was absent in 400 ethnically matched healthy control chromosomes, and is not listed in single-nucleotide polymorphism databases as a common polymorphism. The LRP2 mutation identified here is located in one of the low-density lipoprotein-receptor class A domains, which is a cysteine-rich repeat that plays a central role in mammalian cholesterol metabolism, suggesting that alteration of cholesterol processing pathway can contribute to ID.

Biallelic truncating SCN9A mutation identified in four families with congenital insensitivity to pain from Pakistan

(a) Homozygosity-mapping-by-descent of four Bhakkar congenital indifference/insensitivity to pain (CIP) families. (b) Identification of mutation Met1190* in SCN9A. (c) SCN9A/NaV1.7 2D structure (as predicted by CCTOP and SMART) and approximate position of known nonsense (*) and missense (M) mutations ( www.hgmd.cf.ac.uk), as well as the Bhakkar mutation (this study) in red.