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.

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.

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 >40× 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. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Biallelic KIF24 Variants Are Responsible for a Spectrum of Skeletal Disorders Ranging From Lethal Skeletal Ciliopathy to Severe Acromesomelic Dysplasia.

Skeletal dysplasias comprise a large spectrum of mostly monogenic disorders affecting bone growth, patterning, and homeostasis, and ranging in severity from lethal to mild phenotypes. This study aimed to underpin the genetic cause of skeletal dysplasia in three unrelated families with variable skeletal manifestations. The six affected individuals from three families had severe short stature with extreme shortening of forelimbs, short long-bones, and metatarsals, and brachydactyly (family 1); mild short stature, platyspondyly, and metaphyseal irregularities (family 2); or a prenatally lethal skeletal dysplasia with kidney features suggestive of a ciliopathy (family 3). Genetic studies by whole genome, whole exome, and ciliome panel sequencing identified in all affected individuals biallelic missense variants in KIF24, which encodes a kinesin family member controlling ciliogenesis. In families 1 and 3, with the more severe phenotype, the affected subjects harbored homozygous variants (c.1457A>G; p.(Ile486Val) and c.1565A>G; p.(Asn522Ser), respectively) in the motor domain which plays a crucial role in KIF24 function. In family 2, compound heterozygous variants (c.1697C>T; p.(Ser566Phe)/c.1811C>T; p.(Thr604Met)) were found C-terminal to the motor domain, in agreement with a genotype-phenotype correlation. In vitro experiments performed on amnioblasts of one affected fetus from family 3 showed that primary cilia assembly was severely impaired, and that cytokinesis was also affected. In conclusion, our study describes novel forms of skeletal dysplasia associated with biallelic variants in KIF24. To our knowledge this is the first report implicating KIF24 variants as the cause of a skeletal dysplasia, thereby extending the genetic heterogeneity and the phenotypic spectrum of rare bone disorders and underscoring the wide range of monogenetic skeletal ciliopathies. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

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.

Gain-of-function CEBPE mutation causes noncanonical autoinflammatory inflammasomopathy.

BACKGROUND: CCAAT enhancer-binding protein epsilon (C/EBPε) is a transcription factor involved in late myeloid lineage differentiation and cellular function. The only previously known disorder linked to C/EBPε is autosomal recessive neutrophil-specific granule deficiency leading to severely impaired neutrophil function and early mortality. OBJECTIVE: The aim of this study was to molecularly characterize the effects of C/EBPε transcription factor Arg219His mutation identified in a Finnish family with previously genetically uncharacterized autoinflammatory and immunodeficiency syndrome. METHODS: Genetic analysis, proteomics, genome-wide transcriptional profiling by means of RNA-sequencing, chromatin immunoprecipitation (ChIP) sequencing, and assessment of the inflammasome function of primary macrophages were performed. RESULTS: Studies revealed a novel mechanism of genome-wide gain-of-function that dysregulated transcription of 464 genes. Mechanisms involved dysregulated noncanonical inflammasome activation caused by decreased association with transcriptional repressors, leading to increased chromatin occupancy and considerable changes in transcriptional activity, including increased expression of NLR family, pyrin domain-containing 3 protein (NLRP3) and constitutively expressed caspase-5 in macrophages. CONCLUSION: We describe a novel autoinflammatory disease with defective neutrophil function caused by a homozygous Arg219His mutation in the transcription factor C/EBPε. Mutated C/EBPε acts as a regulator of both the inflammasome and interferome, and the Arg219His mutation causes the first human monogenic neomorphic and noncanonical inflammasomopathy/immunodeficiency. The mechanism, including widely dysregulated transcription, is likely not unique for C/EBPε. Similar multiomics approaches should also be used in studying other transcription factor-associated diseases.

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.

Differentially methylated regions in maternal and paternal uniparental disomy for chromosome 7.

DNA methylation is a hallmark of genomic imprinting and differentially methylated regions (DMRs) are found near and in imprinted genes. Imprinted genes are expressed only from the maternal or paternal allele and their normal balance can be disrupted by uniparental disomy (UPD), the inheritance of both chromosomes of a chromosome pair exclusively from only either the mother or the father. Maternal UPD for chromosome 7 (matUPD7) results in Silver-Russell syndrome (SRS) with typical features and growth retardation, but no gene has been conclusively implicated in SRS. In order to identify novel DMRs and putative imprinted genes on chromosome 7, we analyzed eight matUPD7 patients, a segmental matUPD7q31-qter, a rare patUPD7 case and ten controls on the Infinium HumanMethylation450K BeadChip with 30 017 CpG methylation probes for chromosome 7. Genome-scale analysis showed highly significant clustering of DMRs only on chromosome 7, including the known imprinted loci GRB10, SGCE/PEG10, and PEG/MEST. We found ten novel DMRs on chromosome 7, two DMRs for the predicted imprinted genes HOXA4 and GLI3 and one for the disputed imprinted gene PON1. Quantitative RT-PCR on blood RNA samples comparing matUPD7, patUPD7, and controls showed differential expression for three genes with novel DMRs, HOXA4, GLI3, and SVOPL. Allele specific expression analysis confirmed maternal only expression of SVOPL and imprinting of HOXA4 was supported by monoallelic expression. These results present the first comprehensive map of parent-of-origin specific DMRs on human chromosome 7, suggesting many new imprinted sites.

Altered Methylation of IGF2 Locus 20 Years after Preterm Birth at Very Low Birth Weight.

INTRODUCTION: People born preterm at very low birth weight (VLBW, ≤1500g) have higher rates of risk factors for adult-onset diseases, including cardiovascular diseases and type 2 diabetes. These risks may be mediated through epigenetic modification of genes that are critical to normal growth and development. METHODS: We measured the methylation level of an imprinted insulin-like-growth-factor 2 (IGF2) locus (IGF2/H19) in young adults born preterm at VLBW and in their peers born at term. We studied 158 VLBW and 161 control subjects aged 18 to 27 years from the Helsinki Study of Very Low Birth Weight Adults. Methylation fraction at two IGF2 differentially methylated regions (DMRs) - IGF2 antisense transcript (IGF2AS, also known as IGF2 DMR0) and last exon of IGF2 (IGF2_05, also known as IGF2 DMR2) - were measured with Sequenom Epityper. We used linear regression and adjustment for covariates to compare methylation fractions at these DMRs between VLBW and control subjects. RESULTS: At one IGF2AS CpG site, methylation was significantly lower in VLBW than in control subjects, mean difference -0.017 (95% CI; -0.028, -0.005), P = 0.004. Methylation at IGF2_05 was not different between the groups. CONCLUSIONS: Methylation of IGF2AS is altered 20 years after preterm birth at VLBW. Altered methylation may be a mechanism of later increased disease risk but more data are needed to indicate causality.