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Purpose Unexpected fetal abnormalities occur in 2–5% of pregnancies. While traditional cytogenetic and microarray approaches achieve diagnosis in around 40% of cases, lack of diagnosis in others impedes parental counseling, informed decision making, and pregnancy management. Postnatally exome sequencing yields high diagnostic rates, but relies on careful phenotyping to interpret genotype results. Here we used a multidisciplinary approach to explore the utility of rapid fetal exome sequencing for prenatal diagnosis using skeletal dysplasias as an exemplar. Methods Parents in pregnancies undergoing invasive testing because of sonographic fetal abnormalities, where multidisciplinary review considered skeletal dysplasia a likely etiology, were consented for exome trio sequencing (both parents and fetus). Variant interpretation focused on a virtual panel of 240 genes known to cause skeletal dysplasias. Results Definitive molecular diagnosis was made in 13/16 (81%) cases. In some cases, fetal ultrasound findings alone were of sufficient severity for parents to opt for termination. In others, molecular diagnosis informed accurate prediction of outcome, improved parental counseling, and enabled parents to terminate or continue the pregnancy with certainty. Conclusion Trio sequencing with expert multidisciplinary review for case selection and data interpretation yields timely, high diagnostic rates in fetuses presenting with unexpected skeletal abnormalities. This improves parental counseling and pregnancy management.

Bone development is a tightly regulated process. Several integrated signaling pathways including HH, PTHrP, WNT, NOTCH, TGF-β, BMP, FGF and the transcription factors SOX9, RUNX2 and OSX are essential for proper skeletal development. Misregulation of these signaling pathways can cause a large spectrum of congenital conditions categorized as skeletal dysplasia. Since the signaling pathways involved in skeletal dysplasia interact at multiple levels and have a different role depending on the time of action (early or late in chondrogenesis and osteoblastogenesis), it is still difficult to precisely explain the physiopathological mechanisms of skeletal disorders. However, in recent years, significant progress has been made in elucidating the mechanisms of these signaling pathways and genotype–phenotype correlations have helped to elucidate their role in skeletogenesis. Here, we review the principal signaling pathways involved in bone development and their associated skeletal dysplasia.

Little is known about the regulation of cortical bone. This genetic study showed that suppression of Wnt-signaling pathways by secreted frizzled-related protein 4 was critical to cortical-bone formation and strength. Osteoporosis is a skeletal disease that is characterized by low bone mass, defective bone structure, and a high risk of fracture. Cortical-bone mass is a major determinant of bone strength and therefore of susceptibility to fractures. With aging, the mass of cortical bone may decrease more than the mass of trabecular bone, and fractures occurring in older persons result mostly from cortical-bone fragility. Although progress has been made in therapeutic approaches to reducing the risk of vertebral fracture (which occurs at sites rich in trabecular bone), currently available treatments do little to reduce the risk of nonvertebral fracture, which results . . .

Wolcott-Rallison syndrome (WRS) is a rare autosomal recessive disease, characterized by neonatal/early-onset non-autoimmune insulin-requiring diabetes associated with skeletal dysplasia and growth retardation. Fewer than 60 cases have been described in the literature, although WRS is now recognised as the most frequent cause of neonatal/early-onset diabetes in patients with consanguineous parents. Typically, diabetes occurs before six months of age, and skeletal dysplasia is diagnosed within the first year or two of life. Other manifestations vary between patients in their nature and severity and include frequent episodes of acute liver failure, renal dysfunction, exocrine pancreas insufficiency, intellectual deficit, hypothyroidism, neutropenia and recurrent infections. Bone fractures may be frequent. WRS is caused by mutations in the gene encoding eukaryotic translation initiation factor 2α kinase 3 (EIF2AK3), also known as PKR-like endoplasmic reticulum kinase (PERK). PERK is an endoplasmic reticulum (ER) transmembrane protein, which plays a key role in translation control during the unfolded protein response. ER dysfunction is central to the disease processes. The disease variability appears to be independent of the nature of the EIF2AK3 mutations, with the possible exception of an older age at onset; other factors may include other genes, exposure to environmental factors and disease management. WRS should be suspected in any infant who presents with permanent neonatal diabetes associated with skeletal dysplasia and/or episodes of acute liver failure. Molecular genetic testing confirms the diagnosis. Early diagnosis is recommended, in order to ensure rapid intervention for episodes of hepatic failure, which is the most life threatening complication. WRS should be differentiated from other forms of neonatal/early-onset insulin-dependent diabetes based on clinical presentation and genetic testing. Genetic counselling and antenatal diagnosis is recommended for parents of a WRS patient with confirmed EIF2AK3 mutation. Close therapeutic monitoring of diabetes and treatment with an insulin pump are recommended because of the risk of acute episodes of hypoglycaemia and ketoacidosis. Interventions under general anaesthesia increase the risk of acute aggravation, because of the toxicity of anaesthetics, and should be avoided. Prognosis is poor and most patients die at a young age. Intervention strategies targeting ER dysfunction provide hope for future therapy and prevention.

Silver-Russell syndrome (SRS) is a congenital disorder characterized by prenatal and postnatal growth failure and craniofacial features. Hypomethylation of the H19/IGF2:IG-differential methylated region (H19LOM) is observed in 50% of SRS patients, and 15% of SRS patients with H19LOM had multilocus imprinting disturbance (MLID). Schimke immuno-osseous dysplasia (SIOD), characterized by spondyloepiphyseal dysplasia and nephropathy, is an autosomal recessive disorder caused by mutations in SMARCAL1 on chromosome 2. We report a patient with typical SRS-related features, spondyloepiphyseal dysplasia, and severe nephropathy. Molecular analyses showed H19LOM, paternal uniparental isodisomy of chromosome 2 (iUPD(2)pat), and a paternally inherited homozygous frameshift variant in SMARCAL1. Genome-wide methylation analysis showed MLID in this patient, although it showed no MLID in another patient with SIOD without SRS phenotype. These results suggest that iUPD(2)pat unmasked the recessive mutation in SMARCAL1 and that the SMARCAL1 gene mutation may have no direct effect on the patient's methylation defects.

In affected members of a consanguineous family, a syndrome, which is concurrence of set of medical signs, is often observed and commonly assumed to have arisen from pleiotropy, i.e., the phenomenon of a single gene variant affecting multiple traits. We detected six sibs afflicted with a unique combination of digit malformation that includes brachydactyly, symphalangism and zygodactyly plus infertility in males owing to azoospermia, sperm immotility or necrospermia, which we hypothesised to have arisen from a defect in a single gene. We mapped the disease locus and by exome sequencing identified in patients homozygous missense variants bone morphogenetic protein receptor type IB (BMPR1B) c.640C>T (p.(Arg214Cys)) and alpha-2 pyruvate dehydrogenase (PDHA2) c.679A>G (p.(Met227Val)). Structural protein modelling, protein sequence conservation and in silico analysis indicate that both variants affect protein function. BMPR1B is known to be responsible for autosomal dominant brachydactyly and autosomal recessive acromesomelic chondrodysplasia. Our findings show that also recessive complex digit malformation can be caused by BMPR1B variant and not all biallelic BMPR1B variants cause acromesomelic dysplasia. PDHA2 is a novel candidate gene for male infertility; the protein product is a mitochondrial enzyme with highest expression in ejaculated sperm. Our findings are a unique example of two linked variants, ~ 711 Kb apart, in different genes that together manifest as a novel syndrome. They demonstrate that exome sequencing and not candidate gene approach should be employed in disease gene hunt, defining new diseases and genetic testing, to rule out the coincidental presence of two variants contributing together to the phenotype, which may be discerned as a novel disease.

Lamin B receptor (LBR) is a polytopic membrane protein residing in the inner nuclear membrane in association with the nuclear lamina. We demonstrate that human LBR is essential for cholesterol synthesis. LBR mutant derivatives implicated in Greenberg skeletal dysplasia or Pelger-Huët anomaly fail to rescue the cholesterol auxotrophy of a LBR-deficient human cell line, consistent with a loss-of-function mechanism for these congenital disorders. These disease-causing variants fall into two classes: point mutations in the sterol reductase domain perturb enzymatic activity by reducing the affinity for the essential cofactor NADPH, while LBR truncations render the mutant protein metabolically unstable, leading to its rapid degradation at the inner nuclear membrane. Thus, metabolically unstable LBR variants may serve as long-sought-after model substrates enabling previously impossible investigations of poorly understood protein turnover mechanisms at the inner nuclear membrane of higher eukaryotes. In humans, mutations in the gene that encodes a protein called Lamin B receptor can lead to diseases ranging from harmless anomalies of blood cells to fatal developmental defects. The severity of the disease depends on the nature of the specific mutation, and whether one or both copies of the gene are affected. Lamin B receptor – or LBR for short – is found at the envelope that surrounds the cell’s nucleus and was previously proposed to anchor this envelope to an underlying scaffold to provide it with support. LBR can also catalyze a chemical reaction involved in producing cholesterol – an essential component of cell membranes. However, this enzymatic activity was assumed to be less important because a second enzyme named TM7SF2 can perform the same reaction. Thus, it was not clear – at the molecular level – why the mutations in this gene lead to a variety of diseases. All disease-causing mutations map to the part of LBR that is responsible for its enzymatic activity. This fact motivated Tsai, Zhao et al. to reassess the importance of LBR for the production of cholesterol. The experiments revealed that many human cells that can be grown in the laboratory strictly depend on LBR to produce cholesterol. As such, these findings challenge the previous assumption that TM7SF2 can compensate for the loss of LBR’s activity and sustain cholesterol synthesis. Tsai, Zhao et al. also discovered that all known disease-causing mutations strongly perturb LBR’s ability to engage in cholesterol synthesis, albeit through different mechanisms. Some mutations interfered with the enzyme ability to bind with an essential molecule or cofactor that is required to catalysis; others led to LBR rapidly degrading at the nuclear envelope. It was previously not known that proteins could be degraded at the inner membrane of the nuclear envelope of mammalian cells, and LBR mutants may turn out to be useful tools to investigate how this happens in future. Further studies could also test if other diseases caused by mutations in proteins found in the nuclear envelope act in similar ways, or if mutations in these proteins inhibit the nucleus’s protein disposal machinery.

Cantú syndrome (CS), a multisystem disease with a complex cardiovascular phenotype, is caused by gain-of-function (GoF) variants in the Kir6.1/SUR2 subunits of ATP-sensitive potassium (KATP) channels and is characterized by low systemic vascular resistance, as well as tortuous, dilated, vessels, and decreased pulse-wave velocity. Thus, CS vascular dysfunction is multifactorial, with both hypomyotonic and hyperelastic components. To dissect whether such complexities arise cell autonomously within vascular smooth muscle cells (VSMCs) or as secondary responses to the pathophysiological milieu, we assessed electrical properties and gene expression in human induced pluripotent stem cell-derived VSMCs (hiPSC-VSMCs), differentiated from control and CS patient-derived hiPSCs, and in native mouse control and CS VSMCs. Whole-cell voltage clamp of isolated aortic and mesenteric arterial VSMCs isolated from wild-type (WT) and Kir6.1[V65M] (CS) mice revealed no clear differences in voltage-gated K+ (Kv) or Ca2+ currents. Kv and Ca2+ currents were also not different between validated hiPSC-VSMCs differentiated from control and CS patient-derived hiPSCs. While pinacidil-sensitive KATP currents in control hiPSC-VSMCs were similar to those in WT mouse VSMCs, they were considerably larger in CS hiPSC-VSMCs. Under current-clamp conditions, CS hiPSC-VSMCs were also hyperpolarized, consistent with increased basal K conductance and providing an explanation for decreased tone and decreased vascular resistance in CS. Increased compliance was observed in isolated CS mouse aortae and was associated with increased elastin mRNA expression. This was consistent with higher levels of elastin mRNA in CS hiPSC-VSMCs and suggesting that the hyperelastic component of CS vasculopathy is a cell-autonomous consequence of vascular KATP GoF. The results show that hiPSC-VSMCs reiterate expression of the same major ion currents as primary VSMCs, validating the use of these cells to study vascular disease. Results in hiPSC-VSMCs derived from CS patient cells suggest that both the hypomyotonic and hyperelastic components of CS vasculopathy are cell-autonomous phenomena driven by KATP overactivity within VSMCs . Graphical Abstract Graphical Abstract

Cerebral, ocular, dental, auricular, skeletal (CODAS) syndrome is a rare autosomal recessive multisystem disorder caused by mutations in LONP1. It is characterized by intellectual disability, cataracts, delayed tooth eruption, malformed auricles and skeletal abnormalities. We performed whole-exome sequencing on a 12-year-old Japanese male with severe intellectual disability, congenital bilateral cataracts, spasticity, hypotonia with motor regression and progressive cerebellar atrophy with hyperintensity of the cerebellar cortex on T2-weighted images. We detected compound heterozygous mutation in LONP1. One allele contained a paternally inherited frameshift mutation (p.Ser100Glnfs*46). The other allele contained a maternally inherited missense mutation (p.Arg786Trp), which was predicted to be pathogenic by web-based prediction tools. The two mutations were not found in Exome Variant Server or our 575 in-house control exomes. Some features were not consistent with CODAS syndrome but overlapped with Marinesco-Sjögren syndrome, a multisystem disorder caused by a mutation in SIL1. An atypical mutation site may result in atypical presentation of the LONP1 mutation.

Stüve-Wiedemann syndrome (STWS; OMIM #610559) is a rare bent-bone dysplasia that includes radiologic bone anomalies, respiratory distress, feeding difficulties, and hyperthermic episodes. STWS usually results in infant mortality, yet some STWS patients survive into and, in some cases, beyond adolescence. STWS is caused by a mutation in the leukemia inhibitory factor receptor ( LIFR ) gene, which is inherited in an autosomally recessive pattern. Most LIFR mutations resulting in STWS are null mutations which cause instability of the mRNA and prevent the formation of LIFR, impairing the signaling pathway. LIFR signaling usually follows the JAK/STAT3 pathway, and is initiated by several interleukin-6-type cytokines. STWS is managed on a symptomatic basis since there is no treatment currently available.