Kirti Mittal

15q11.2 Duplication Encompassing Only the UBE3A Gene Is Associated with Developmental Delay and Neuropsychiatric Phenotypes

Duplications of chromosome region 15q11‐q13 with the maternal imprint are associated with a wide spectrum of neuropsychiatric disorders, including autism spectrum disorders, developmental delay, learning difficulties, schizophrenia, and seizures. These observations suggest there is a dosage‐sensitive imprinted gene or genes within this region that explains the increased risk for neuropsychiatric phenotypes. We present a female patient with developmental delay in whom we identified a maternally inherited 129‐Kb duplication in chromosome region 15q11.2 encompassing only the UBE3A gene. Expression analysis in cultured fibroblasts confirmed overexpression of UBE3A in the proband, compared with age‐ and sex‐matched controls. We further tested segregation of this duplication in four generations and found it segregated with neuropsychiatric phenotypes. Our study shows for the first time clinical features associated with overexpression of UBE3A in humans and underscores the significance of this gene in the phenotype of individuals with 15q11‐q13 duplication.

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.

Mutations in MECP2 exon 1 in classical rett patients disrupt MECP2_e1 transcription, but not transcription of MECP2_e2

The overwhelming majority of Rett syndrome cases are caused by mutations in the gene MECP2. MECP2 has two isoforms, termed MECP2_e1 and MECP2_e2, which differ in their N‐terminal amino acid sequences. A growing body of evidence has indicated that MECP2_e1 may be the etiologically relevant isoform in Rett Syndrome based on its expression profile in the brain and because, strikingly, no mutations have been discovered that affect MECP2_e2 exclusively. In this study we sought to characterize four classical Rett patients with mutations that putatively affect only the MECP2_e1 isoform. Our hypothesis was that the classical Rett phenotype seen here is the result of disrupted MECP2_e1 expression, but with MECP2_e2 expression unaltered. We used quantitative reverse transcriptase PCR to assay mRNA expression for each isoform independently, and used cytospinning methods to assay total MECP2 in peripheral blood lymphocytes (PBL). In the two Rett patients with identical 11 bp deletions within the coding portion of exon 1, MECP2_e2 levels were unaffected, whilst a significant reduction of MECP2_e1 levels was detected. In two Rett patients harboring mutations in the exon 1 start codon, MECP2_e1 and MECP2_e2 mRNA amounts were unaffected. In summary, we have shown that patients with exon 1 mutations transcribe normal levels of MECP2_e2 mRNA, and most PBL are positive for MeCP2 protein, despite them theoretically being unable to produce the MECP2_e1 isoform, and yet still exhibit the classical RTT phenotype. Altogether, our work further supports our hypothesis that MECP2_e1 is the predominant isoform involved in the neuropathology of Rett syndrome.

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.

A synonymous change, p.Gly16Gly in MECP2 Exon 1, causes a cryptic splice event in a Rett syndrome patient

BackgroundMutations in MECP2 are the main cause of Rett Syndrome. To date, no pathogenic synonymous MECP2 mutation has yet been identified. Here, we investigated a de novo synonymous variant c.48C>T (p.Gly16Gly) identified in a girl presenting with a typical RTT phenotype.MethodsIn silico analyses to predict the effects of sequence variation on mRNA splicing were employed, followed by sequencing and quantification of lymphocyte mRNAs from the subject for splice variants MECP2_E1 and MECP2_E2.ResultsAnalysis of mRNA confirmed predictions that this synonymous mutation activates a splice-donor site at an early position in exon 1, leading to a deletion (r.[=, 48_63del]), codon frameshift and premature stop codon (p.Glu17Lysfs*16) for MECP2_E1. For MECP2_E2, the same premature splice site is used, but as this is located in the 5′untranslated region, no effect on the amino acid sequence is predicted. Quantitative analysis that specifically measured this cryptic splice variant also revealed a significant decrease in the quantity of the correct MECP2_E1 transcript, which indicates that this is the etiologically significant mutation in this patient.ConclusionThese findings suggest that synonymous variants of MECP2 as well as other known disease genes—and de novo variants in particular— should be re-evaluated for potential effects on splicing.

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.

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.

Mutations in the genes for thyroglobulin and thyroid peroxidase cause thyroid dyshormonogenesis and autosomal-recessive intellectual disability

We have used single-nucleotide polymorphism microarray genotyping and homozygosity-by-descent (HBD) mapping followed by Sanger sequencing or whole-exome sequencing (WES) to identify causative mutations in three consanguineous families with intellectual disability (ID) related to thyroid dyshormonogenesis (TDH). One family was found to have a shared HBD region of 12.1 Mb on 8q24.21-q24.23 containing 36 coding genes, including the thyroglobulin gene, TG. Sanger sequencing of TG identified a homozygous nonsense mutation Arg2336*, which segregated with the phenotype in the family. A second family showed several HBD regions, including 6.0 Mb on 2p25.3-p25.2. WES identified a homozygous nonsense mutation, Glu596*, in the thyroid peroxidase gene, TPO. WES of a mother/father/proband trio from a third family revealed a homozygous missense mutation, Arg412His, in TPO. Mutations in TG and TPO are very rarely associated with ID, mainly because TDH is generally detectable and treatable. However, in populations where resources for screening and detection are limited, and especially where consanguineous marriages are common, mutations in genes involved in thyroid function may also be causes of ID, and as TPO and TG mutations are the most common genetic causes of TDH, these are also likely to be relatively common causes of ID.

Investigating The Role of Treatable Genetic Diseases In Schizophrenia And Bipolar Disorder Populations And Its Clinical Diagnostic Impact

Background Many rare genetic syndromes are known to phenotypically manifest with psychiatric symptoms that can be indistinguishable from primary psychiatric disorders. While the majority of ongoing psychiatric genetic research has been dedicated to the identification and characterization of genes involved in primary psychiatric disorders, little research has been done to determine the extent to which rare genetic variants contribute to the overall psychiatric disease load. Within schizophrenia and bipolar populations, we are conducting the first study of its kind to determine the prevalence of four treatable genetic syndromes (Niemann Pick disease type C (NPC), Wilson disease, acute intermittent porphyria (AIP), and homocystinuria (HOM)) manifesting as primary psychiatric disorders. Methods We are screening 1323 schizophrenia and 1200 bipolar disorder samples, along with 980 sex- and age-matched healthy controls, all with available DNA and extensive phenotype data. We are using a matrix-type pooled targeted deep sequencing of the genes NPC1, NPC2, ATP7B, HMBS, and CBS to screen for the four genetic diseases. Pathogenic variants within the targeted genes will be identified using an in-house analytic pipeline with quality control, variant discovery designed specifically for identifying variants in the matrix pooled targeted sequencing approach, and functionality prediction programs (i.e. Polyphen, SIFT, Scone) to determine variant pathogenicity. Sanger sequencing will be used to validate identified mutations and decrease false positive calls. Results We hypothesize that a significant sub-population of patients with psychiatric disorders have underlying rare genetic conditions. Preliminary screening of 1023 schziophrenia patients for possible pathogenic variants resulted in the identification of 11 previously known pathogenic variants from the ClinVar database, with 2 pathogenic variants for NPC, 2 for HOM, and 7 for WD. Additionally, 34 variants were predicted to be pathogenic based on whether three or more pathogenicity prediction softwares (PolyPhen2, SIFT, MutationTaster and Condel) identified the variant to be damaging, of which 3 are predicted nonsense mutations. Discussion Identification of known and predicted pathogenic variants within the schizophrenia and bipolar populations strongly suggests the role of rare genetic diseases within psychiatric populations as hypothesized. Screening for treatable genetic diseases, such as NPC, WD, AIP and HOM, within schizophrenia and bipolar samples could provide a possible explanation for severe treatment resistance and treating the genetic condition can effectively “cure” patients of their otherwise difficult-to-treat psychiatric symptoms. Ultimately, this proof-of-principle study will lead to the development of molecular diagnostic tools for detection of underlying genetic disorders in psychiatric patients and will allow for precision medicine.