Expanding the genetic and phenotypic spectrum of skeletal dysplasias

Skeletal dysplasias constitute a large and heterogeneous group of disorders, many causing disabilities with profound effects on the quality of life of the affected individuals and their families. Each individual skeletal dysplasia is rare, however, as more than 450 different disorders have been described, skeletal dysplasias as a group affect approximately three in 10 000 individuals. The age of onset for skeletal dysplasias ranges from prenatal to adult, but most of the affected individuals are diagnosed in childhood. Skeletal dysplasias mainly affect bone and cartilage, but symptoms may involve other organs, such as sensorineural hearing loss in Stickler syndrome, nephronophthisis in Sensenbrenner syndrome, and structural heart abnormalities in acromicric dysplasia. Therefore, most skeletal dysplasias can be defined as syndromes with a significant skeletal involvement. Clinical diagnosis of skeletal dysplasias is based on meticulous phenotypic characterization, skeletal radiography (for pattern recognition) and genetic testing. Molecular diagnostics has improved significantly by massively parallel sequencing (MPS) technologies, such as whole exome and genome sequencing. However, even after extensive clinical phenotyping and advanced molecular analyses, many patients with congenital skeletal disorders still lack molecular diagnoses and many clinical entities are not well-characterized regarding their natural course and complications. Molecular diagnosis is important since it gives information about prognosis and recurrence risk, as well as, in some cases possibilities to offer targeted treatment, participation in clinical trials, and tailored medical follow-up. This thesis focuses on gene discovery, studies of previously clinically defined skeletal dysplasias with unknown genetic background and aims to improve the molecular analyses for patients with diagnoses which are difficult to solve. In study I, we identify a novel pathogenic variant in ALG9, as the cause of a lethal skeletal syndrome in two families. In study II, we show that a variant in COL2A1 causes spondyloepiphyseal dysplasia type Stanescu. Study III shows that pathogenic variants in BMPER cause ischiospinal dysostosis (ISD), which is allelic to diaphanospondylodysostosis. In study IV, we describe a novel skeletal ciliopathy in four individuals with spondylometaphyseal dysplasia and thorax hypoplasia caused by pathogenic variants in KIAA0753. Finally, study V represents a cohort of 24 unrelated patients with skeletal ciliopathies, where we solve the genetic diagnoses in 83% of them. Here, we show two rare intronic variants and two exonic synonymous variants leading to aberrant splicing, which indicates that extended RNA studies are necessary to improve molecular diagnostics in some cases. Altogether, the results of these studies expand the genetic and phenotypic spectrum of skeletal dysplasias and demonstrate that MPS technology in combination with meticulous phenotyping is a powerful method to discover disease-causing variants in patients with congenital skeletal disorders

Genome sequencing with comprehensive variant calling identifies structural variants and repeat expansions in a large fraction of individuals with ataxia and/or neuromuscular disorders

IntroductionNeuromuscular disorders (NMDs) have a heterogeneous etiology. A genetic diagnosis is key to personalized healthcare and access to targeted treatment for the affected individuals.MethodsIn this study, 861 patients with NMDs were analyzed with genome sequencing and comprehensive variant calling including single nucleotide variants, small insertions/deletions (SNVs/INDELs), and structural variants (SVs) in a panel of 895 NMD genes, as well as short tandem repeat expansions (STRs) at 28 loci. In addition, for unsolved cases with an unspecific clinical presentation, the analysis of a panel with OMIM disease genes was added.ResultsIn the cohort, 27% (232/861) of the patients harbored pathogenic variants, of which STRs and SVs accounted for one-third of the patients (71/232). The variants were found in 107 different NMD genes. Furthermore, 18 pediatric patients harbored pathogenic variants in non-NMD genes.DiscussionOur results highlight that for children with unspecific hypotonia, a genome-wide analysis rather than a disease-based gene panel should be considered as a diagnostic approach. More importantly, our results clearly show that it is crucial to include STR- and SV-analyses in the diagnostics of patients with neuromuscular disorders

Expanding the Clinical Spectrum of Phenotypes Caused by Pathogenic Variants in PLOD2

Osteogenesis imperfecta (OI) is a strikingly heterogeneous group of disorders with a broad range of phenotypic variations. It is also one of the differential diagnoses in bent bone dysplasias along with campomelic dysplasia and thanatophoric dysplasia and can usually be distinguished by decreased bone mineralization and bone fractures. Bent bone dysplasias also include syndromes such as kyphomelic dysplasia (MIM:211350) and mesomelic dysplasia Kozlowski-Reardon (MIM249710), both of which have been under debate regarding whether or not they are a real entity or simply a phenotypic manifestation of another dysplasia including OI. Bruck syndrome type 2 (BRKS2; MIM:609220) is a rare form of autosomal recessive OI caused by biallelic PLOD2 variants and is associated with congenital joint contractures with pterygia. In this report, we present six patients from four families with novel PLOD2 variants. All cases had multiple fractures. Other features ranged from prenatal lethal severe angulation of the long bones as in kyphomelic dysplasia and mesomelic dysplasia Kozlowski-Reardon through classical Bruck syndrome to moderate OI with normal joints. Two siblings with a kyphomelic dysplasia-like phenotype who were stillborn had compound heterozygous variants in PLOD2 (p.Asp585Val and p.Ser166*). One infant who succumbed at age 4 months had a bent bone phenotype phenotypically like skeletal dysplasia Kozlowski-Reardon (with mesomelic shortening, camptodactyly, retrognathia, cleft palate, skin dimples, but also with fractures). He was homozygous for the nonsense variant (p.Trp561*). Two siblings had various degrees of Bruck syndrome caused by the homozygous missense variant, p.His687Arg. Furthermore a boy with a clinical presentation of moderate OI had a possibly pathogenic homozygous variant p.Trp588Cys. Our experience of six patients with biallelic pathogenic variants in PLOD2 expands the phenotypic spectrum in the PLOD2-related phenotypes

<i>GSTM1</i> Gene Expression Correlates to Leiomyoma Volume Regression in Response to Mifepristone Treatment

<div><p></p><p>Progesterone receptor modulators, such as mifepristone are useful and well tolerated in reducing leiomyoma volume although with large individual variation. The objective of this study was to investigate the molecular basis for the observed leiomyoma volume reduction, in response to mifepristone treatment and explore a possible molecular marker for the selective usage of mifepristone in leiomyoma patients. Premenopausal women (N = 14) were treated with mifepristone 50 mg, every other day for 12 weeks prior to surgery. Women were arbitrarily sub-grouped as good (N = 4), poor (N = 4) responders to treatment or intermediate respondents (N = 3). Total RNA was extracted from leiomyoma tissue, after surgical removal of the tumour and the differential expression of genes were analysed by microarray. The results were analysed using Ingenuity Pathway Analysis software. The glutathione pathway was the most significantly altered canonical pathway in which the glutathione-s transferase mu 1 (<i>GSTM1</i>) gene was significantly over expressed (+8.03 folds) among the good responders compared to non responders. This was further confirmed by Real time PCR (p = 0.024). Correlation of immunoreactive scores (IRS) for GSTM1 accumulation in leiomyoma tissue was seen with base line volume change of leiomyoma R = −0.8 (p = 0.011). Furthermore the accumulation of protein GSTM1 analysed by Western Blot correlated significantly with the percentual leiomyoma volume change R = −0.82 (p = 0.004). Deletion of the <i>GSTM1</i> gene in leiomyoma biopsies was found in 50% of the mifepristone treated cases, with lower presence of the GSTM1 protein. The findings support a significant role for GSTM1 in leiomyoma volume reduction induced by mifepristone and explain the observed individual variation in this response. Furthermore the finding could be useful to further explore GSTM1 as a biomarker for tailoring medical treatment of uterine leiomyomas for optimizing the response to treatment.</p><p>Clinical Trials identifier</p><p><a href="http://www.clinicaltrials.gov" target="_blank">www.clinicaltrials.gov</a>: NCT00579475, Protocol date: November 2004. <a href="http://clinicaltrials.gov/ct2/show/NCT00579475" target="_blank">http://clinicaltrials.gov/ct2/show/NCT00579475</a></p></div

GSTM 1 expression leiomyoma volume reduction: Comparison of GSTM1 expression as seen by by real time PCR and Western Blot in relation to percentage of leiomyoma volume change during 3 months of mifepristone treatment.

<p>GR-good responders (>30% volume reduction); PR-poor responders (<18% volume reduction).</p>*<p>no gene expression detected.</p

Correlation of leiomyoma volume change (percentage ×0.1) and the expression levels of GSTM1 as studied by real time PCR and Western blot GR = good responders, PR = poor responders, ND = not determined (in between groups) category.

<p>Correlation of leiomyoma volume change (percentage ×0.1) and the expression levels of GSTM1 as studied by real time PCR and Western blot GR = good responders, PR = poor responders, ND = not determined (in between groups) category.</p

Glutathione pathway gene expression and leiomyoma volume reduction: Expression levels and fold changes of differentially expressed genes in glutathione pathway in good and poor responders for leiomyoma patients with 12 weeks of mifepristone treatment as studied by with 12 weeks of mifepristone treatment in leiomyoma tissue as studied by real time PCR and microarray (*The least responder did not have any detectable Ct value for GSTM1).

<p>Glutathione pathway gene expression and leiomyoma volume reduction: Expression levels and fold changes of differentially expressed genes in glutathione pathway in good and poor responders for leiomyoma patients with 12 weeks of mifepristone treatment as studied by with 12 weeks of mifepristone treatment in leiomyoma tissue as studied by real time PCR and microarray (*The least responder did not have any detectable Ct value for GSTM1).</p

Accumulation of GSTM1 protein in GR (good responders) or PR (poor responders to mifepristone treatment as demonstrated by Western Blot.

<p>Accumulation of GSTM1 protein in GR (good responders) or PR (poor responders to mifepristone treatment as demonstrated by Western Blot.</p

Differentially expressed genes, with mifepristone treatment: Genes, their cellular location and the fold changes with mifepristone treatment on leiomyoma patients as analysed by microarray followed by IPA.

<p>Differentially expressed genes, with mifepristone treatment: Genes, their cellular location and the fold changes with mifepristone treatment on leiomyoma patients as analysed by microarray followed by IPA.</p

Flow chart explaining the patient enrolment, allotment and relevent details, as per consolidated standards of reporting trials (CONSORT) in the study where leiomyoma patients were treated with mifepristone/placebo.

<p>Flow chart explaining the patient enrolment, allotment and relevent details, as per consolidated standards of reporting trials (CONSORT) in the study where leiomyoma patients were treated with mifepristone/placebo.</p