Assessment of copy number variations in the nebulin gene and other nemaline myopathy-causing genes

Nemaline myopathy (NM) and related disorders constitute a heterogeneous group of congenital myopathies. Mutations in the nebulin gene (NEB) are the main cause of the recessively inherited form. NEB is one of the largest genes in the human genome consisting of 249 kb of genomic sequence. NEB contains 183 exons and a 32 kb homologous triplicate region (TRI) where eight exons are repeated three times. The aims of this Doctoral Thesis study were to develop and implement into diagnostics new efficient variant analysis methods for NEB and other NM-causing genes. The first aim was to design and validate a custom copy number microarray targeting the NM-causing genes for the detection of copy number variations. MLPA (multiplex ligation-dependent probe amplification) and Sanger sequencing were also used. The second aim was to utilise whole-exome sequencing to search for novel disease-causing variants in the known NM genes and try to identify novel NM genes. Lastly, the aim was to collect more data in order to try to find genotype-phenotype correlations of NEB-caused NM. The design and validation of the NM-CGH microarray was successful. Of the total sample cohort of 356 NM families, 196 NM families were studied using the custom-made NM-CGH array. Nine different novel large causative variants were identified in ten NM families. The size of these variants varies greatly, covering only a part of one NEB exon on up to dozens of NEB exons (72bp - 133 kb). In addition, a novel recurrent variation of the NEB TRI region was identified in 13% of the NM families and in 10% of the studied 60 control samples. Deviations of one copy are suggested to be benign but gains of two or more copies might be pathogenic. One novel homozygous deletion was also identified in another NM gene, TPM3, in a patient with severe NM. Furthermore, ten samples were studied using exome sequencing, and for six of those samples, novel disease-causing variant(s) were identified. Two variants were identified in one family in a novel, putative NM gene that is currently under further investigation. 165 NM families from the total cohort of 356 NM families have been identified thus far with two pathogenic NEB variants. Altogether 220 different pathogenic variants were identified in these 165 families, accentuating that the patients in the majority (84%) of the families are compound heterozygous for two different NEB variants. Most of the variants are small single nucleotide changes whereas large variants are more rare. However, copy number variations are much more frequent than previously thought: pathogenic copy number variants were identified in 16% of these 356 NM-families. Genotype-phenotype correlations between the type of NEB mutation and the NM subtypes remained, however, unobtainable. The NM-CGH microarray has been implemented into molecular diagnostics of NM. Using the NM-CGH microarray followed by exome-sequencing has accelerated mutation detection. This combination has increased the coverage of the NM genes and thus improved the diagnostics of NM and NM-related disorders.Nemaliinimyopatia (NM) ja samankaltaiset taudit on monimuotoinen tautiryhmä synnynnäisten myopatioiden joukossa. Nebuliini-geenin (NEB) mutaatiot ovat yleisin resessiivisen NM:n aiheuttaja, eli taudin ilmeneminen vaatii geenivirheen sekä isältä että äidiltä perityssä alleelissa. NEB on kooltaan 249 kb, eli yksi ihmisen suurimmista geeneistä. NEB sisältää 183 eksonia ja se kattaa myös toistojaksoja, kuten ns. triplikaatioalueen (TRI), jossa kahdeksan eksonia toistuu kolme kertaa. Väitöskirjatutkimuksen tarkoituksena oli kehittää ja ottaa diagnostiseen käyttöön uusia mutaatioanalyysimenetelmiä NEB-geeniä sekä muita NM-geenejä varten. Tavoitteena oli suunnitella ja validoida NM-geeneihin kohdennettu mikrosiru, jolla voidaan tutkia kopiolukuvariaatioita näistä geeneistä. Eksomisekvensointia hyödynnettiin tautia aiheuttavien varianttien löytämiseksi tunnetuista NM-geeneistä sekä uusista geeneistä. Tavoitteena oli lisäksi kerätä lisätietoa siitä mitkä nebuliinimutaatiot aiheuttavat mitäkin nemaliinimyopatian alatyyppiä. NM-CGH-mikrosirun kehittäminen sekä validointi onnistui hyvin. Koko 356 NM-perheen näytekohortista NM-CGH-sirulla tutkittiin 196 perhettä, joista tunnistettiin yhteensä yhdeksän uutta suurta tautia aiheuttavaa varianttia kymmenestä eri NM-perheestä. Näiden mutaatioiden koko vaihtelee suuresti, kattaen vain osan yhdestä NEB-geenin eksonista aina yli puoleen koko geenistä (72 bp 133 kb). Lisäksi osoitettiin että 13 % tutkituista NM-perheistä sekä 10 % tutkituista 60 kontrollinäytteestä sisältää NEB:n triplikaatio-alueen kopiolukuvariaation. Tutkimustulosten perusteella yhden kopioluvun lisäys tai vähenemä olisi harmitonta mutta mikäli ylimääräisiä kopioita on kaksi tai enemmän, se voisi olla tautia aiheuttavaa. Lisäksi tunnistettiin homotsygoottinen suuri deleetio toisesta tunnetusta NM-geenistä, TPM3. Eksomisekvensoinnilla löydettiin puolestaan toinen tai molemmat tautia aiheuttavat variantit kuudelle kymmenestä tutkitusta NM-potilaasta. Yhdessä NM-perheessä tunnistettiin kaksi uutta varianttia potentiaalisessa uudessa NM-geenissä, ja tätä löydöstä tutkitaan parhaillaan tarkemmin. Tutkimuskohorttimme 356 NM-perheestä 165 perheelle on nyt tunnistettu kaksi tautia aiheuttavaa NEB-varianttia. Näissä perheissä esiintyi yhteensä 220 eri patogeenistä NEB-varianttia eli suurin osa potilaista (84 %) kantaa kahta eri tautia aiheuttavaa varianttia. Pääosa mutaatioista on pieniä yhden emäksen mutaatioita ja suuret kopiolukumuutokset ovat suhteellisen harvinaisia. Tautia aiheuttavat kopiolukumuutokset ovat kuitenkin paljon yleisempiä kuin oli aiemmin luultu, ja niitä todettiin 16 %:ssa tutkituista 356 NM-perheestä. Melko suuresta näytekohortista huolimatta, NEB-mutaatioiden ja niiden aiheuttaman nemaliinimyopatian alatyypin välille ei onnistuttu saamaan erityistä korrelaatiota. NM-CGH-mikrosirumenetelmä on otettu osaksi nemaliinimyopatian molekyyligeneettistä diagnostiikkaa. NM-CGH-mikrosiruanalyysin ja eksomisekvensoinnin yhdistelmä on tehostanut NM-geenien kattavuutta, edistänyt mutaatioiden löytymistä, ja näin ollen parantanut nemaliinimyopatian ja muiden samankaltaisten tautien diagnostiikkaa

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

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

A novel partial de novo duplication of JARID2 gene causing a neurodevelopmental phenotype

Publisher Copyright: © 2022 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.Background: Deletions covering the entire or partial JARID2 gene as well as pathogenic single nucleotide variants leading to haploinsufficiency of JARID2 have recently been shown to cause a clinically distinct neurodevelopmental syndrome. Here, we present a previously undescribed partial de novo duplication of the JARID2 gene in a patient displaying features similar to those of patients with JARID2 loss-of-function variants. Case report: The index patient presents with abnormalities in gross motor skills and speech development as well as neuropsychiatric disorders. The patient has markedly dark infraorbital circles and slightly prominent supraorbital ridges.Whole-genome sequencing and array comparative genomic hybridization revealed a novel disease-causing variant type, a partial tandem duplication of JARID2, covering the exons 1–7. Furthermore, RNA sequencing validated the increased expression of these exons. Expression alterations were also detected in target genes of the PRC2 complex, in which JARID2 acts as an essential member. Conclusion: Our data add to the variety of different pathogenic variants associated with JARID2 neurodevelopmental syndrome.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

Congenital asymmetric distal myopathy with hemifacial weakness caused by a heterozygous large de novo mosaic deletion in nebulin

We report the first mosaic mutation, a deletion of exons 11-107, identified in the nebulin gene in a Finnish patient presenting with a predominantly distal congenital myopathy and asymmetric muscle weakness. The female patient is ambulant and currently 26 years old. Muscle biopsies showed myopathic features with type 1 fibre predominance, strikingly hypotrophic type 2 fibres and central nuclei, but no nemaline bodies. The deletion was detected in a copy number variation analysis based on next-generation sequencing data. The parents of the patient did not carry the deletion. Mosaicism was detected using a custom, targeted comparative genomic hybridisation array. Expression of the truncated allele, less than half the size of full-length nebulin, was confirmed by Western blotting. The clinical and histological picture resembled that of a family with a slightly smaller deletion, and that in patients with recessively inherited distal forms of nebulin-caused myopathy. Asymmetry, however, was a novel feature. (c) 2021 Elsevier B.V. All rights reserved.Peer reviewe

Copy number variation analysis increases the diagnostic yield in muscle diseases

Objective: Copy number variants (CNVs) were analyzed from next-generation sequencing data, with the aim of improving diagnostic yield in skeletal muscle disorder cases.& para;& para;Methods: Four publicly available bioinformatic analytic tools were used to analyze CNVs from sequencing data from patients with muscle diseases. The patients were previously analyzed with a targeted gene panel for single nucleotide variants and small insertions and deletions, without achieving final diagnosis. Variants detected by multiple CNV analysis tools were verified with either array comparative genomic hybridization or PCR. The clinical significance of the verified CNVs was interpreted, considering previously identified variants, segregation studies, and clinical information of the patient cases.& para;& para;Results: Combining analysis of all different mutation types enabled integration of results and identified the final cause of the disease in 9 myopathy cases. Complex effects like compound heterozygosity of different mutation types and compound disease arising from variants of different genes were unraveled. We identified the first large intragenic deletion of the titin (TTN) gene implicated in the pathogenesis of a severe form of myopathy. Our work also revealed a "double-trouble" effect in a patient carrying a single heterozygous insertion/deletion mutation in the TTN gene and a Becker muscular dystrophy causing deletion in the dystrophin gene.& para;& para;Conclusions: Causative CNVs were identified proving that analysis of CNVs is essential for increasing the diagnostic yield in muscle diseases. Complex severe muscular dystrophy phenotypes can be the result of different mutation types but also of the compound effect of 2 different genetic diseases.Peer reviewe

Dominantly inherited distal nemaline/cap myopathy caused by a large deletion in the nebulin gene

We report the first family with a dominantly inherited mutation of the nebulin gene (NEB). This 100kb in-frame deletion encompasses NEB exons 14-89, causing distal nemaline/cap myopathy in a three-generation family. It is the largest deletion characterized in NEB hitherto. The mutated allele was shown to be expressed at the mRNA level and furthermore, for the first time, a deletion was shown to cause the production of a smaller mutant nebulin protein. Thus, we suggest that this novel mutant nebulin protein has a dominant-negative effect, explaining the first documented dominant inheritance of nebulin-caused myopathy. The index patient, a young man, was more severely affected than his mother and grandmother. His first symptom was foot drop at the age of three, followed by distal muscle atrophy, slight hypomimia, high-arched palate, and weakness of the neck and elbow flexors, hands, tibialis anterior and toe extensors. Muscle biopsies showed myopathic features with type 1 fibre predominance in the index patient and nemaline bodies and cap-like structures in biopsies from his mother and grandmother. The muscle biopsy findings constitute a further example of nemaline bodies and cap-like structures being part of the same spectrum of pathological changes. (C) 2019 Elsevier B.V. All rights reserved.Peer reviewe

Prenatal Diagnosis of 17p13.1p13.3 Duplication

We present here the first prenatal diagnosis of 17p13.1p13.3 duplication. 17p13.3 duplication has recently been defined as a new distinctive syndrome with several diagnosed patients. In the current case prenatal chromosome analysis (G-banding) performed on cultured amniocytes revealed additional material in chromosome 19p. This was further defined as a chromosome 17p13.1p13.3 duplication by FISH and genomic microarray analysis (GMA). In addition Prenatal BACs-on-Beads (PN_BoBs) assay was performed, which detected the duplication clearly. This enables rapid prenatal diagnosis of the duplication for this family in the future