Silver-Russell Syndrome and Human Growth : Genetic and Epigenetic Studies

A large part of the genetic and epigenetic changes contributing to human height and growth remain unknown. Silver-Russell syndrome (SRS) is a rare human growth disorder, where maternal uniparental disomy of chromosome 7 [UPD(7)mat] and loss of methylation at 11p15 (11p15 LOM) are the two major findings. However, a substantial proportion of the SRS patients remain without a molecular diagnosis. SRS is caused by a disturbance of imprinted genes that are expressed solely or predominantly from one parent and are important for growth and development. Imprinted genes may also be especially susceptible to environmental factors, which can cause persistent changes in methylation patterns and affect development of adult-onset diseases. This thesis aims to explore new genetic and epigenetic changes in SRS, as well as epigenetic changes of imprinted genes in growth-restricted children, children of normal growth and individuals born pre-term. In Study I, genomic structural variation of 22 SRS patients was studied with the Affymetrix 250K Sty microarray. Several copy number changes were found, including a heterozygous deletion of 15q26.3 including the insulin-like growth factor 1 receptor (IGF1R) gene. In Study II, CpGs in the insulin-like growth factor 2 (IGF2) gene (located in the 11p15 region) were tested in pre-term and very low birth weight born individuals with elevated levels of cardiovascular risk factors. DNA methylation changes were found in one CpG site of the pre-term born individuals compared to controls. In Study III, genome wide comparisons between the DNA methylation levels of SRS patients that have UPD(7)mat, controls, and an individual with paternal uniparental disomy of chromosome 7 [UPD(7)pat] were done. DNA methylation was studied using the Illumina Infinium HumanMethylation450K BeadChip technology, which is capable of measuring methylation level of more than 450 000 CpG sites across the genome. The study provided new information on the DNA methylation landscape of chromosome 7, suggesting new differentially methylated regions (DMRs) and imprinted genes. In Study IV, DNA methylation levels were compared between three different subgroups of SRS: SRS with UPD(7)mat, SRS with 11p15 LOM and clinical SRS without a known molecular etiology. The promoter region of homeobox A4 (HOXA4) gene was found hypomethylated in all of the subgroups of SRS. This region was subsequently tested in other severely growth-restricted patients and was found hypomethylated. Additionally, the methylation level of the HOXA4 promoter region was found to be associated with the height of school-aged children, suggesting that HOXA4 plays a role not only in SRS but also in the regulation of height in general. These studies found molecular changes in SRS, new differentially methylated regions in chromosome 7 and epigenetic findings potentially relevant for regulation of human growth. The findings provide potential targets for further studies in human growth and development.Suurta osaa ihmisen pituuskasvua säätelevistä geneettisistä ja epigeneettisistä tekijöistä ei vielä tunneta. Silver-Russellin oireyhtymä (SRS) on harvinainen lyhytkasvuisuusoireyhtymä, jonka tunnetuimmat muutokset ovat kromosomi 7:n uniparentaalinen disomia (UPD(7)mat) eli molemman kromosomi 7:n periminen vain äidiltä ja kromosomin 11p15 -alueella esiintyvä hypometylaatio (11p15 LOM). Kuitenkin huomattavalla osalla SRS-potilaista oireyhtymän molekyyligeneettinen syy on tuntematon. SRS aiheutuu geenien leimautumisen häiriöistä. Leimautuneet geenit ilmentyvät vain joko äidiltä tai isältä periytyneestä geenikopiosta, ja ne ovat tärkeitä kasvun ja kehityksen kannalta. Leimautuneet geenit voivat myös olla erityisen alttiita ympäristötekijöille, jotka saattavat aiheuttaa pysyviä DNA:n metylaation muutoksia ja vaikuttaa myöhemmin aikuisena puhkeavien sairauksien kehittymiseen. Tämän väitöskirjan tarkoituksena on selvittää SRS:n geneettisiä ja epigeneettisiä taustatekijöitä sekä leimautuneiden geenien epigeneettisiä muutoksia kasvuhäiriöisillä lapsilla, terveillä lapsilla ja ennenaikaisesti syntyneillä henkilöillä. Ensimmäisessä osatyössä tutkittiin 22:n SRS-potilaan perimän muutoksia Affymetrix 250K Sty -mikroarrayn avulla. Tutkimuksessa löydettiin useita kopioluvun muutoksia, mukaan lukien heterotsygoottinen häviämä alueella 15q26.3, jossa sijaitsee mm. IGF1R -geeni (insuliinin kaltainen kasvutekijä 1 reseptori). Toisessa osatyössä tutkittiin 11p15 alueella sijaitsevan IGF2-geenin (insuliinin kaltainen kasvutekijä 2) CpG-kohtia ennenaikaisesti syntyneillä henkilöillä, joilla oli hyvin matala syntymäpaino ja joiden sydän- ja verisuonisaiarauksien riskitekijät olivat koholla. Yhden CpG-kohdan metylaatiotaso poikkesi kontrolleista. Kolmannessa osatyössä vertailtiin koko perimän DNA:n metylaatiotasoja SRS-potilailla, joilla on todettu UPD(7)mat, kontrolleilla ja henkilöllä, jolla on todettu paternaalinen UPD kromosomissa 7 eli UPD(7)pat. DNA:n metylaatiota tutkittiin käyttämällä Illumina Infinium HumanMethylation450K BeadChip -tekniikkaa, joka mahdollistaa yli 450 000 CpG-kohdan metylaatiotason mittauksen. Tutkimus antoi uutta tietoa siitä, miten kromosomi 7 metyloituu eri tavalla maternaalisen tai paternaalisen alkuperän mukaan, sekä osoitti uusia mahdollisia eriävästi metyloituneita alueita (DMR) sekä leimautuneita geenejä. Neljännessä osatyössä vertailtiin DNA:n metylaatiotasoja kolmen eri SRS:n alaryhmän välillä: UPD(7)mat -potilailla, 11p15-hypometyloiduilla potilailla sekä SRS-potilailla, joiden molekyyligeneettinen syy on epäselvä. HOXA4-geenin promoottorialue todettiin hypometyloituneeksi kaikissa SRS:n alaryhmissä. Sama metylaatiomuutos löytyi myös muilta vaikeasti lyhytkasvuisilta potilailta, joiden kasvuhäiriön syy oli tuntematon. Lisäksi HOXA4-promoottorialueen metylaatiotaso korreloi kouluikäisten lasten pituuden kanssa, mikä viittaa HOXA4-geenin mahdolliseen rooliin yleisesti pituuskasvun säätelyssä eikä vain SRS:ssä. Väitöskirjan osatöissä havaittiin molekyyligeneettisiä muutoksia SRS-potilailla, maternaalisen ja paternaalisen alkuperän mukaan metyloituneita alueita kromosomissa 7 ja epigeneettisiä löydöksiä, jotka mahdollisesti vaikuttavat ihmisen pituuskasvun säätelyyn. Nämä löydökset tuovat uutta tietoa ja tarjoavat lisätutkimuksen kohteita ihmisen kasvuun ja kehitykseen liittyen

New gene discoveries in skeletal diseases with short stature

In the last decade, the widespread use of massively parallel sequencing has considerably boosted the number of novel gene discoveries in monogenic skeletal diseases with short stature. Defects in genes playing a role in the maintenance and function of the growth plate, the site of longitudinal bone growth, are a well-known cause of skeletal diseases with short stature. However, several genes involved in extracellular matrix composition or maintenance as well as genes partaking in various biological processes have also been characterized. This review aims to describe the latest genetic findings in spondyloepiphyseal dysplasias, spondyloepimetaphyseal dysplasias, and some monogenic forms of isolated short stature. Some examples of novel genetic mechanisms leading to skeletal conditions with short stature will be described. Strategies on how to successfully characterize novel skeletal phenotypes with short stature and genetic approaches to detect and validate novel gene-disease correlations will be discussed in detail. In summary, we review the latest gene discoveries underlying skeletal diseases with short stature and emphasize the importance of characterizing novel molecular mechanisms for genetic counseling, for an optimal management of the disease, and for therapeutic innovations.Peer reviewe

Case report : A novel de novo IGF2 missense variant in a Finnish patient with Silver-Russell syndrome

Silver-Russell syndrome (SRS, OMIM 180860) is a rare imprinting disorder characterized by intrauterine and postnatal growth restriction, feeding difficulties in early childhood, characteristic facial features, and body asymmetry. The molecular cause most commonly relates to hypomethylation of the imprinted 11p15.5 IGF2/H19 domain but remains unknown in about 40% of the patients. Recently, heterozygous paternally inherited pathogenic variants in IGF2, the gene encoding insulin-like growth factor 2 (IGF2), have been identified in patients with SRS. We report a novel de novo missense variant in IGF2 (c.122T > G, p.Leu41Arg) on the paternally derived allele in a 16-year-old boy with a clinical diagnosis of SRS. The missense variant was identified by targeted exome sequencing and predicted pathogenic by multiple in silico tools. It affects a highly conserved residue on a domain that is important for binding of other molecules. Our finding expands the spectrum of disease-causing variants in IGF2. Targeted exome sequencing is a useful diagnostic tool in patients with negative results of common diagnostic tests for SRS.Peer reviewe

Mosaic Deletions of Known Genes Explain Skeletal Dysplasias With High and Low Bone Mass

Mosaicism, a state in which an individual has two or more genetically distinct populations of cells in the body, can be difficult to detect because of either mild or atypical clinical presentation and limitations in the commonly used detection methods. Knowledge of the role of mosaicism is limited in many skeletal disorders, including osteopathia striata with cranial sclerosis (OSCS) and cleidocranial dysplasia (CCD). We used whole-genome sequencing (WGS) with coverage >40x to identify the genetic causes of disease in two clinically diagnosed patients. In a female patient with OSCS, we identified a mosaic 7-nucleotide frameshift deletion in exon 2 of AMER1, NM_152424.4:c.855_861del:p.(His285Glnfs*7), affecting 8.3% of the WGS reads. In a male patient with CCD, approximately 34% of the WGS reads harbored a 3710-basepair mosaic deletion, NC_000006.11:g.45514471_45518181del, starting in intron 8 of RUNX2 and terminating in the 3 ' untranslated region. Droplet digital polymerase chain reaction was used to validate these deletions and quantify the absolute level of mosaicism in each patient. Although constitutional variants in AMER1 and RUNX2 are a known cause of OSCS and CCD, respectively, the mosaic changes here reported have not been described previously. Our study indicates that mosaicism should be considered in unsolved cases of skeletal dysplasia and should be investigated with comprehensive and sensitive detection methods. (c) 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.Peer reviewe

Identification of GLI1 and KIAA0825 Variants in Two Families with Postaxial Polydactyly

Polydactyly is a rare autosomal dominant or recessive appendicular patterning defect of the hands and feet, phenotypically characterized by the duplication of digits. Postaxial polydactyly (PAP) is the most common form and includes two main types: PAP type A (PAPA) and PAP type B (PAPB). Type A involves a well-established extra digit articulated with the fifth or sixth metacarpal, while type B presents a rudimentary or poorly developed superfluous digit. Pathogenic variants in several genes have been identified in isolated and syndromic forms of polydactyly. The current study presents two Pakistani families with autosomal recessive PAPA with intra- and inter-familial phenotype variability. Whole-exome sequencing and Sanger analysis revealed a novel missense variant in KIAA0825 (c.3572C>T: p.Pro1191Leu) in family A and a known nonsense variant in GLI1 (c.337C>T: p.Arg113*) in family B. In silico studies of mutant KIAA0825 and GLI1 proteins revealed considerable structural and interactional modifications that suggest an abnormal function of the proteins leading to the disease phenotype. The present study broadens the mutational spectrum of KIAA0825 and demonstrates the second case of a previously identified GLI1 variant with variable phenotypes. These findings facilitate genetic counseling in Pakistani families with a polydactyly-related phenotype

Hypomethylation of HOXA4 promoter is common in Silver-Russell syndrome and growth restriction and associates with stature in healthy children

Silver-Russell syndrome (SRS) is a growth retardation syndrome in which loss of methylation on chromosome 11p15 (11p15 LOM) and maternal uniparental disomy for chromosome 7 [UPD(7) mat] explain 20-60% and 10% of the syndrome, respectively. To search for a molecular cause for the remaining SRS cases, and to find a possible common epigenetic change, we studied DNA methylation pattern of more than 450 000 CpG sites in 44 SRS patients. Common to all three SRS subgroups, we found a hypomethylated region at the promoter region of HOXA4 in 55% of the patients. We then tested 39 patients with severe growth restriction of unknown etiology, and found hypomethylation of HOXA4 in 44% of the patients. Finally, we found that methylation at multiple CpG sites in the HOXA4 promoter region was associated with height in a cohort of 227 healthy children, suggesting that HOXA4 may play a role in regulating human growth by epigenetic mechanisms.Peer reviewe

Decreased Aerobic Capacity in ANO5-Muscular Dystrophy

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Antimicrobial use, biosecurity, herd characteristics, and antimicrobial resistance in indicator Escherichia coli in ten Finnish pig farms

We investigated connections between antimicrobial use (AMU), biosecurity, and the numbers of pigs and staff in ten Finnish farrow-to-finish herds. Data on AMU in each herd were collected for 12 months. AMU was quantified as treatment incidences per 1000 days at risk (TI) using the consensus defined daily dose calculation. Biosecurity was scored using the Biocheck.UGent T system. We also examined antimicrobial resistance patterns of indicator E. coli isolated from faeces of selected pigs. In each herd, two groups of five pigs were formed: 1) antimicrobial treatment group (ANT: at least one pig in the litter was identified as sick and treated with antimicrobials) and 2) non-antimicrobial treatment group (NON: the litter was not medicated). Faecal samples were taken from these pigs at 5 and 22 weeks of age, cultured, and indicator E. coli isolates were tested for antimicrobial susceptibilities. The AMU varied considerably between the herds. Altogether, most of the antimicrobial treatment courses were assigned to weaned piglets. When AMU was quantified as TIs, suckling piglets had the highest TI (mean 46.6), which was significantly higher (P 0.05). We found few connections: enhanced external biosecurity levels found in the large herds co-occurred with lower use of antimicrobials and herds with low biosecurity scores - especially in the internal subcategories - appeared to have higher proportions of resistant isolates. Conclusively, we suggest that enhancing internal biosecurity might contribute to a reduction in the spreading of antimicrobial resistance in pig herds.Peer reviewe