Kahden uuden geenin tunnistaminen ihmisen neuronaalisten seroidilipofuskinoosien taustalla

The neuronal ceroid lipofuscinoses (NCLs) are a group of mostly autosomal recessively inherited neurodegenerative disorders. The aim of this thesis was to characterize the molecular genetic bases of these, previously genetically undetermined, NCL forms. Congenital NCL is the most aggressive form of NCLs. Previously, a mutation in the cathepsin D (CTSD) gene was shown to cause congenital NCL in sheep. Based on the close resemblance of the phenotypes between congenital NCLs in sheep and human, CTSD was considered as a potential candidate gene in humans as well. When screened for mutations by sequencing, a homozygous nucleotide duplication creating a premature stop codon was identified in CTSD in one family with congenital NCL. While in vitro the overexpressed truncated mutant protein was stable although inactive, the absence of CTSD staining in brain tissue samples of patients indicated degradation of the mutant CTSD in vivo. A lack of CTSD staining was detected also in another, unrelated family with congenital NCL. These results imply that CTSD deficiency underlies congenital NCL. While initially Turkish vLINCL was considered a distinct genetic entity (CLN7), mutations in the CLN8 gene were later reported to account for the disease in a subset of Turkish patients with vLINCL. To further dissect the genetic basis of the disease, all known NCL genes were screened for homozygosity by haplotype analysis of microsatellite markers and/or sequenced in 13 mainly consanguineous, Turkish vLINCL families. Two novel, family-specific homozygous mutations were identified in the CLN6 gene. In the remaining families, all known NCL loci were excluded. To identify novel gene(s) underlying vLINCL, a genomewide single nucleotide polymorphism scan, homozygosity mapping, and positional candidate gene sequencing were performed in ten of these families. On chromosome 4q28.1-q28.2, a novel major facilitator superfamily domain containing 8 (MFSD8) gene with six family-specific homozygous mutations in vLINCL patients was identified. MFSD8 transcript was shown to be ubiquitously expressed with a complex pattern of alternative splicing. Our results suggest that MFSD8 is a novel lysosomal integral membrane protein which, as a member of the major facilitator superfamily, is predicted to function as a transporter. Identification of MFSD8 emphasizes the genetic heterogeneity of Turkish vLINCL. In families where no MFSD8 mutations were detected, additional NCL-causing genes remain to be identified. The identification of CTSD and MFSD8 increases the number of known human NCL-causing genes to eight, and is an important step towards the complete understanding of the genetic spectrum underlying NCLs. In addition, it is a starting point for dissecting the molecular mechanisms behind the associated NCLs and contributes to the challenging task of understanding the molecular pathology underlying the group of NCL disorders.Neuronaaliset seroidilipofuskinoosit (NCL-taudit) ovat ryhmä pääasiassa peittyvästi periytyviä, keskushermostoa rappeuttavia sairauksia. Niiden oireisiin kuuluvat yleensä epilepsiakohtaukset, henkinen ja motorinen taantuminen, näön heikkeneminen sekä ennenaikainen kuolema. Tämän tutkimuksen tarkoituksena oli luonnehtia kahden, aiemmin geneettisesti määrittelemättömän NCL-muodon molekyyligeneettistä taustaa. NCL-taudeista vakavin on synnynnäinen NCL. Tauti havaitaan vastasyntyneillä, ja se johtaa potilaiden kuolemaan muutaman päivän tai viikon kuluessa. Lampaalla vastaavan taudin on todettu johtuvan katepsiini D (CTSD) -geenin mutaatiosta, joten tutkimme geenin osuutta taudin syntyyn myös ihmisellä. Tunnistimme homotsygoottisen, ennenaikaisen lopetuskodonin syntymiseen johtavan mutaation CTSD-geenissä pakistanilaisella perheellä. Osoitimme soluviljelmässä ylituotetun, virheellisen proteiinin olevan vailla entsyymiaktiivisuutta. Potilaiden aivonäytteistä proteiini puuttui kokonaan, mikä viittasi virheellisen geenituotteen hajottamiseen. Tunnistimme CTSD-puutoksen myös toisella perheellä. Nämä tulokset osoittavat CTSD-puutoksen olevan synnynnäisen NCL-taudin taustalla. Turkkilaisessa väestössä esiintyvän myöhäisen lapsuusiän NCL-muodon (vLINCL) ajateltiin alun perin olevan oma geneettinen tautikokonaisuutensa (CLN7). Osalla turkkilaisista vLINCL-potilaista taudin on kuitenkin todettu johtuvan CLN8-geenin virheistä. Tutkimme kaikkien tunnettujen NCL-geenien osuutta taudin syntyyn 13 vLINCL-perheessä. Tunnistimme kaksi uutta, perhekohtaista homotsygoottista mutaatiota CLN6-geenissä. Muissa perheissä kaikki tunnetut NCL-geenit pystyttiin poissulkemaan sairauden taustalta. Löytääksemme näiden perheiden sairauden taustalla olevan geenin, jatkoimme tutkimusta genominlaajuisen kartoituksen avulla. Tunnistimme aiemmin tuntemattomassa major facilitator superfamily domain containing 8 (MFSD8) -geenissä homotsygoottiset mutaatiot kuudella perheellä. Tutkimuksemme osoittavat, että major facilitator superfamily perheeseen kuuluva MFSD8 on todennäköisimmin 12 kertaa kalvon läpäisevä lysosomiin paikantuva kuljettajaproteiini, jonka tarkempaa toimintaa ei vielä tunneta. Perheillä, joilla MFSD8-geenissä ei havaittu mutaatioita, sairauden taustalla on vielä tunnistamattomia geenejä. CTSD- ja MFSD8-geenien tunnistaminen kasvattaa ihmisen tunnettujen NCL-geenien määrän kahdeksaan, mikä on tärkeä askel kohti NCL-tautien geneettisen taustan parempaa tuntemusta. Tutkimustulos mahdollistaa molekyyligenettisen diagnostiikan ja sikiödiagnostiikan perheissä, joissa näitä tauteja esiintyy. Lisäksi se luo perustan näiden NCL-muotojen tautimekanismien tutkimukselle ja edistää koko NCL-tautiryhmän molekyylipatologian ymmärtämystä

ZNHIT3 is defective in PEHO syndrome, a severe encephalopathy with cerebellar granule neuron loss

Progressive encephalopathy with oedema, hypsarrhythmia, and optic atrophy (PEHO) syndrome is an early childhood onset, severe autosomal recessive encephalopathy characterized by extreme cerebellar atrophy due to almost total granule neuron loss. By combining homozygosity mapping in Finnish families with Sanger sequencing of positional candidate genes and with exome sequencing a homozygous missense substitution of leucine for serine at codon 31 in ZNHIT3 was identified as the primary cause of PEHO syndrome. ZNHIT3 encodes a nuclear zinc finger protein previously implicated in transcriptional regulation and in small nucleolar ribonucleoprotein particle assembly and thus possibly to pre-ribosomal RNA processing. The identified mutation affects a highly conserved amino acid residue in the zinc finger domain of ZNHIT3. Both knockdown and genome editing of znhit3 in zebrafish embryos recapitulate the patients' cerebellar defects, microcephaly and oedema. These phenotypes are rescued by wild-type, but not mutant human ZNHIT3 mRNA, suggesting that the patient missense substitution causes disease through a loss-of-function mechanism. Transfection of cell lines with ZNHIT3 expression vectors showed that the PEHO syndrome mutant protein is unstable. Immunohistochemical analysis of mouse cerebellar tissue demonstrated ZNHIT3 to be expressed in proliferating granule cell precursors, in proliferating and post-mitotic granule cells, and in Purkinje cells. Knockdown of Znhit3 in cultured mouse granule neurons and ex vivo cerebellar slices indicate that ZNHIT3 is indispensable for granule neuron survival and migration, consistent with the zebrafish findings and patient neuropathology. These results suggest that loss-of-function of a nuclear regulator protein underlies PEHO syndrome and imply that establishment of its spatiotemporal interaction targets will be the basis for developing therapeutic approaches and for improved understanding of cerebellar development.Peer reviewe

Mutations In Cln7/Mfsd8 Are A Common Cause Of Variant Late-Infantile Neuronal Ceroid Lipofuscinosis

The neuronal ceroid lipofuscinoses (NCLs), the most common neurodegenerative disorders of childhood, are characterized by the accumulation of autofluorescent storage material mainly in neurons. Although clinically rather uniform, variant late-infantile onset NCL (vLINCL) is genetically heterogeneous with four major underlying genes identified so far. We evaluated the genetic background underlying vLINCL in 119 patients, and specifically analysed the recently reported CLN7/MFSD8 gene for mutations in 80 patients. Clinical data were collected from the CLN7/MFSD8 mutation positive patients. Eight novel CLN7/MFSD8 mutations and seven novel mutations in the CLN1/PPT1, CLN2/TPP1, CLN5, CLN6 and CLN8 genes were identified in patients of various ethnic origins. A significant group of Roma patients originating from the former Czechoslovakia was shown to bear the c.881CA (p.Thr294Lys) mutation in CLN7/MFSD8, possibly due to a founder effect. With one exception, the CLN7/MFSD8 mutation positive patients present a phenotype indistinguishable from the other vLINCL forms. In one patient with an in-frame amino acid substitution mutation in CLN7/MFSD8, the disease onset was later and the disease course less aggressive than in variant late-infantile NCL. Our findings raise the total number of CLN7/MFSD8 mutations to 14 with the majority of families having private mutations. Our study confirms that CLN7/MFSD8 defects are not restricted to the Turkish population, as initially anticipated, but are a relatively common cause of NCL in different populations. CLN7/MFSD8 should be considered a diagnostic alternative not only in variant late-infantile but also later onset NCL forms with a more protracted disease course. A significant number of NCL patients in Turkey exist, in which the underlying genetic defect remains to be determined.WoSScopu

The Novel Neuronal Ceroid Lipofuscinosis Gene MFSD8 Encodes a Putative Lysosomal Transporter

The late-infantile–onset forms are the most genetically heterogeneous group among the autosomal recessively inherited neurodegenerative disorders, the neuronal ceroid lipofuscinoses (NCLs). The Turkish variant was initially considered to be a distinct genetic entity, with clinical presentation similar to that of other forms of late-infantile–onset NCL (LINCL), including age at onset from 2 to 7 years, epileptic seizures, psychomotor deterioration, myoclonus, loss of vision, and premature death. However, Turkish variant LINCL was recently found to be genetically heterogeneous, because mutations in two genes, CLN6 and CLN8, were identified to underlie the disease phenotype in a subset of patients. After a genomewide scan with single-nucleotide–polymorphism markers and homozygosity mapping in nine Turkish families and one Indian family, not linked to any of the known NCL loci, we mapped a novel variant LINCL locus to chromosome 4q28.1-q28.2 in five families. We identified six different mutations in the MFSD8 gene (previously denoted “MGC33302”), which encodes a novel polytopic 518–amino acid membrane protein that belongs to the major facilitator superfamily of transporter proteins. MFSD8 is expressed ubiquitously, with several alternatively spliced variants. Like the majority of the previously identified NCL proteins, MFSD8 localizes mainly to the lysosomal compartment. However, the function of MFSD8 remains to be elucidated. Analysis of the genome-scan data suggests the existence of at least three more genes in the remaining five families, further corroborating the great genetic heterogeneity of LINCLs