Array Comparative Genomic Hybridisation and Droplet Digital PCR Uncover Recurrent Copy Number Variation of the TTN Segmental Duplication Region

Intragenic segmental duplication regions are potential hotspots for recurrent copy number variation and possible pathogenic aberrations. Two large sarcomeric genes, nebulin and titin, both contain such segmental duplication regions. Using our custom Comparative Genomic Hybridisation array, we have previously shown that a gain or loss of more than one copy of the repeated block of the nebulin triplicate region constitutes a recessive pathogenic mutation. Using targeted array-CGH, similar copy number variants can be detected in the segmental duplication region of titin. Due to the limitations of the array-CGH methodology and the repetitiveness of the region, the exact copy numbers of the blocks could not be determined. Therefore, we developed complementary custom Droplet Digital PCR assays for the titin segmental duplication region to confirm true variation. Our combined methods show that the titin segmental duplication region is subject to recurrent copy number variation. Gains and losses were detected in samples from healthy individuals as well as in samples from patients with different muscle disorders. The copy number variation observed in our cohort is likely benign, but pathogenic copy number variants in the segmental duplication region of titin cannot be excluded. Further investigations are needed, however, this region should no longer be neglected in genetic analyses

Array Comparative Genomic Hybridisation and Droplet Digital PCR Uncover Recurrent Copy Number Variation of the TTN Segmental Duplication Region

Intragenic segmental duplication regions are potential hotspots for recurrent copy number variation and possible pathogenic aberrations. Two large sarcomeric genes, nebulin and titin, both contain such segmental duplication regions. Using our custom Comparative Genomic Hybridisation array, we have previously shown that a gain or loss of more than one copy of the repeated block of the nebulin triplicate region constitutes a recessive pathogenic mutation. Using targeted array-CGH, similar copy number variants can be detected in the segmental duplication region of titin. Due to the limitations of the array-CGH methodology and the repetitiveness of the region, the exact copy numbers of the blocks could not be determined. Therefore, we developed complementary custom Droplet Digital PCR assays for the titin segmental duplication region to confirm true variation. Our combined methods show that the titin segmental duplication region is subject to recurrent copy number variation. Gains and losses were detected in samples from healthy individuals as well as in samples from patients with different muscle disorders. The copy number variation observed in our cohort is likely benign, but pathogenic copy number variants in the segmental duplication region of titin cannot be excluded. Further investigations are needed, however, this region should no longer be neglected in genetic analyses

Array Comparative Genomic Hybridisation and Droplet Digital PCR Uncover Recurrent Copy Number Variation of the TTN Segmental Duplication Region

Intragenic segmental duplication regions are potential hotspots for recurrent copy number variation and possible pathogenic aberrations. Two large sarcomeric genes, nebulin and titin, both contain such segmental duplication regions. Using our custom Comparative Genomic Hybridisation array, we have previously shown that a gain or loss of more than one copy of the repeated block of the nebulin triplicate region constitutes a recessive pathogenic mutation. Using targeted array-CGH, similar copy number variants can be detected in the segmental duplication region of titin. Due to the limitations of the array-CGH methodology and the repetitiveness of the region, the exact copy numbers of the blocks could not be determined. Therefore, we developed complementary custom Droplet Digital PCR assays for the titin segmental duplication region to confirm true variation. Our combined methods show that the titin segmental duplication region is subject to recurrent copy number variation. Gains and losses were detected in samples from healthy individuals as well as in samples from patients with different muscle disorders. The copy number variation observed in our cohort is likely benign, but pathogenic copy number variants in the segmental duplication region of titin cannot be excluded. Further investigations are needed, however, this region should no longer be neglected in genetic analyses.Peer reviewe

Nemaliinimyopatian ja sen sukuisten lihassairauksien molekyyligeneettiset tutkimukset

Nemaline myopathy (NM) is a rare muscle disorder characterised by muscle weakness and nemaline bodies in striated muscle tissue. Nemaline bodies are derived from sarcomeric Z discs and may be detected by light microscopy. The disease can be divided into six subclasses varying from very severe, in some cases lethal forms to milder forms. NM is usually the consequence of a gene mutation and the mode of inheritance varies between NM subclasses and different families. Mutations in six genes are known to cause NM; nebulin (NEB), alpha-actin, alpha-tropomyosin (TPM3), troponin T1, beta-tropomyosin (TPM2) and cofilin 2, of which nebulin and -actin are the most common. One of the main interests of my research is NEB. Nebulin is a giant muscle protein (600-900 kDa) expressed mainly in the thin filaments of striated muscle. Mutations in NEB are the main cause of autosomal recessive NM. The gene consists of 183 exons. Thus being gigantic, NEB is very challenging to investigate. NEB was screened for mutations using denaturing High Performance Liquid Chromatography (dHPLC) and sequencing. DNA samples from 44 families were included in this study, and we found and published 45 different mutations in them. To date, we have identified 115 mutations in NEB in a total of 96 families. In addition, we determined the occurrence in a world-wide sample cohort of a 2.5 kb deletion containing NEB exon 55 identified in the Ashkenazi Jewish population. In order to find the seventh putative NM gene a genome-wide linkage study was performed in a series of Turkish families. In two of these families, we identified a homozygous mutation disrupting the termination signal of the TPM3 gene, a previously known NM-causing gene. This mutation is likely a founder mutation in the Turkish population. In addition, we described a novel recessively inherited distal myopathy, named distal nebulin myopathy, caused by two different homozygous missense mutations in NEB in six Finnish patients. Both mutations, when combined in compound heterozygous form with a more disruptive mutation, are known to cause NM. This study consisted of molecular genetic mutation analyses, light and electron microscopic studies of muscle biopsies, muscle imaging and clinical examination of patients. In these patients the distribution of muscle weakness was different from NM. Nemaline bodies were not detectable with routine light microscopy, and they were inconspicuous or absent even using electron microscopy. No genetic cause was known to underlie cap myopathy, a congenital myopathy characterised by cap-like structures in the muscle fibres, until we identified a deletion of one codon of the TPM2 gene, in a 30-year-old cap myopathy patient. This mutation does not change the reading frame of the gene, but a deletion of one amino acid does affect the conformation of the protein produced. In summary, this thesis describes a novel distal myopathy caused by mutations in the nebulin gene, several novel nebulin mutations associated with nemaline myopathy, the first molecular genetic cause of cap myopathy, i.e. a mutation in the beta-tropomyosin gene, and a founder mutation in the alpha-tropomyosin gene underlying autosomal recessive nemaline myopathy in the Turkish population.Myopatiat ovat usein perinnöllisiä, kliinisesti vaihtelevia poikkijuovaisen eli luurankolihaksen sairauksia, joiden oireena on vaikeusasteeltaan eriasteinen lihasheikkous. Se voi olla yleistä lihasheikkoutta, johonkin tiettyyn lihasryhmään liittyvää tai jopa yksittäisiin lihaksiin rajautunutta. Lisäksi diagnoosin perusteena on kreatiinikinaasin määrittäminen verestä, neurofysiologiset tutkimukset ja poikkijuovaisessa lihaksessa mikroskoopilla havaittavat poikkeavuudet. Tässä väitöskirjatyössä analysoitiin yli 300 DNA-näytettä, pääasiassa ulkomaalaisista perheistä, joissa lihastaudin taustalla uskottiin tai tiedettiin olevan häiriö lihassolun supistuvan yksikön, sarkomeerin, rakenneproteiineissa. Tutkimus keskittyi näitä proteiineja koodaavien geenien analysointiin. Väitöskirjan viidestä osatyöstä kolmessa tutkittiin nemaliinimyopatiaa (NEM), joka on yleisin perinnöllisten ns. kongenitaalisten (synnynnäisten tai rakenteellisten) myopatioiden muoto. NEM-potilaiden poikkijuovaisessa lihaksessa on valomikroskoopilla havaittavia proteiinikertymiä, nemaliinikappaleita. NEM johtuu yleensä geenivirheestä ja mutaatioita on löydetty kuudesta geenistä. Geeneistä jättimäinen 183 eksonia käsittävä nebuliini (NEB) sekä beta- ja gamma-tropomyosiinit (TPM2 ja TPM3) ovat väitöskirjassa keskeisiä. Tässä työssä käyttöönotettiin nestekromatografiamenetelmä NEB-mutaatioanalyysiä varten, ja analysoitiin 44 perheen DNA-näytteet, joista löydettiin 45 uutta mutaatiota. Lisäksi määritettiin askenaasijuutalaisilla esiintyvän tunnetun mutaation yleisyys NEM-potilailla ja todettiin, että kyseessä on maailmanlaajuinen perustajamutaatio. Sekä aikaisempien että tässä työssä tehtyjen analyysien perusteella on päätelty, että on olemassa vielä ainakin seitsemäs NEM-geeni. Tämän geenin kartoitusta varten tehtiin koko genomin laajuinen analyysi 13 turkkilaisperheelle. Tämän pohjalta löydettiin jo tunnetusta NEM-geenistä, TPM3, homotsygoottinen mutaatio kahdesta perheestä. Mutaatio poistaa geenistä lopetussignaalin ja johtaa ylipitkän proteiinin tuottamiseen. Vuoteen 2007 mennessä NEB-mutaatioita oli löytynyt ainoastaan NEM-potilailta. Tässä työssä löytyi NEB-mutaatioita neljästä suomalaisperheestä, joilla oli ennestään tuntematon, pääasiassa raajojen ääreisosiin vaikuttava lihassairaus. Tämä uusi lihassairaus nimettiin distaalinebuliinimyopatiaksi (DNM). DNM-potilailla ei ole valomikroskooppitasolla havaittavia proteiinikertymiä lihassoluissaan, mutta elektronimikroskopialla voidaan havaita sarkomeerirakenteen häiriöitä. Cap-myopatia (CM) -potilailla lihassolukalvon alle muodostuu laajoja proteiinikertymiä, ikään kuin lihassoluilla olisi lakit. Tämän sairauden geneettinen tausta oli vuoteen 2007 asti selvittämättä. Tässä työssä CM-potilaalta löydettiin heterotsygoottinen mutaatio TPM2-geenistä. Mutaatio vaikuttaa proteiinituotteen rakenteeseen. Tämän väitöskirjatyön tulokset valottavat rakenteellisten myopatioiden geneettistä taustaa paljastaen molekyyligeneettisten taustojen monimuotoisuuden ja kompleksisuuden. Tämäkin työ on osoittanut, että saman taudin taustalla saattaa olla useampi eri geeni, ja toisaalta sen, että saman geenin eri mutaatiot voivat aiheuttaa erilaisia tauteja. Geenivirheiden tunnistaminen on tärkeää paitsi diagnostiikan ja mahdollisten hoitojen kehittelylle myös lihassolun toiminnan paremmalle ymmärtämiselle

Clinically variable nemaline myopathy in a three-generation family caused by mutation of the skeletal muscle alpha-actin gene

We present here a Finnish nemaline myopathy family with a dominant mutation in the skeletal muscle alpha-actin gene, p.(Glu85Lys), segregating in three generations. The index patient, a 5-year-old boy, had the typical form of nemaline myopathy with congenital muscle weakness and motor milestones delayed but reached, while his mother never had sought medical attention for her very mild muscle weakness, and his maternal grandmother had been misdiagnosed as having myotonic dystrophy. This illustrates the clinical variability in nemaline myopathy. (C) 2017 Elsevier B.V. All rights reserved.Peer reviewe

A custom ddPCR method for the detection of copy number variations in the nebulin triplicate region

The human genome contains repetitive regions, such as segmental duplications, known to be prone to copy number variation. Segmental duplications are highly identical and homologous sequences, posing a specific challenge for most mutation detection methods. The giant nebulin gene is expressed in skeletal muscle. It harbors a large segmental duplication region composed of eight exons repeated three times, the so-called triplicate region. Mutations in nebulin are known to cause nemaline myopathy and other congenital myopathies. Using our custom targeted Comparative Genomic Hybridization arrays, we have previously shown that copy number variations in the nebulin triplicate region are pathogenic when the copy number of the segmental duplication block deviates two or more copies from the normal number, which is three per allele. To complement our Comparative Genomic Hybridization arrays, we have established a custom Droplet Digital PCR method for the detection of copy number variations within the nebulin triplicate region. The custom Droplet Digital PCR assays allow sensitive, rapid, high-throughput, and cost-effective detection of copy number variations within this region and is ready for implementation a screening method for disease-causing copy number variations of the nebulin triplicate region. We suggest that Droplet Digital PCR may also be used in the study and diagnostics of other segmental duplication regions of the genome.Peer reviewe

An Extended Targeted Copy Number Variation Detection Array Including 187 Genes for the Diagnostics of Neuromuscular Disorders

Background: Our previous array, the Comparative Genomic Hybridisation design (CGH-array) for nemaline myopathy (NM), named the NM-CGH array, revealed pathogenic copy number variation (CNV) in the genes for nebulin (NEB) and tropomyosin 3 (TPM3), as well as recurrent CNVs in the segmental duplication (SD), i.e. triplicate, region of NEB (TRI, exons 82-89, 90-97, 98-105). In the light of this knowledge, we have designed and validated an extended CGH array, which includes a selection of 187 genes known to cause neuromuscular disorders (NMDs). Objective: Our aim was to develop a reliable method for CNV detection in genes related to neuromuscular disorders for routine mutation detection and analysis, as a much-needed complement to sequencing methods. Methods: We have developed a novel custom-made 4×180 k CGH array for the diagnostics of NMDs. It includes the same tiled ultra-high density coverage of the 12 known or putative NM genes as our 8×60 k NM-CGH-array but also comprises a selection of 175 additional genes associated with NMDs, including titin (TTN), at a high to very high coverage. The genes were divided into three coverage groups according to known and potential pathogenicity in neuromuscular disorders. Results: The array detected known and putative CNVs in all three gene coverage groups, including the repetitive regions of NEB and TTN. Conclusions: The targeted neuromuscular disorder 4×180 k array-CGH (NMD-CGH-array v1.0) design allows CNV detection for a broader spectrum of neuromuscular disorders at a high resolution. © 2018 - IOS Press and the authors. All rights reserved.Peer reviewe

Minderåriga och Kanta-tjänsterna - verksamhetsmodeller för hälso- och sjukvården och apoteken : version 1.2

Detta är en gammal upplaga. Den nya, ersättande upplagan finns på adressen: https://urn.fi/URN:NBN:fi-fe2022051635635</a