High diagnostic yield in skeletal ciliopathies using massively parallel genome sequencing, structural variant screening and RNA analyses.

Skeletal ciliopathies are a heterogenous group of disorders with overlapping clinical and radiographic features including bone dysplasia and internal abnormalities. To date, pathogenic variants in at least 30 genes, coding for different structural cilia proteins, are reported to cause skeletal ciliopathies. Here, we summarize genetic and phenotypic features of 34 affected individuals from 29 families with skeletal ciliopathies. Molecular diagnostic testing was performed using massively parallel sequencing (MPS) in combination with copy number variant (CNV) analyses and in silico filtering for variants in known skeletal ciliopathy genes. We identified biallelic disease-causing variants in seven genes: DYNC2H1, KIAA0753, WDR19, C2CD3, TTC21B, EVC, and EVC2. Four variants located in non-canonical splice sites of DYNC2H1, EVC, and KIAA0753 led to aberrant splicing that was shown by sequencing of cDNA. Furthermore, CNV analyses showed an intragenic deletion of DYNC2H1 in one individual and a 6.7 Mb de novo deletion on chromosome 1q24q25 in another. In five unsolved cases, MPS was performed in family setting. In one proband we identified a de novo variant in PRKACA and in another we found a homozygous intragenic deletion of IFT74, removing the first coding exon and leading to expression of a shorter message predicted to result in loss of 40 amino acids at the N-terminus. These findings establish IFT74 as a new skeletal ciliopathy gene. In conclusion, combined single nucleotide variant, CNV and cDNA analyses lead to a high yield of genetic diagnoses (90%) in a cohort of patients with skeletal ciliopathies.

Alu-Alu mediated intragenic duplications in IFT81 and MATN3 are associated with skeletal dysplasias.

Skeletal dysplasias are a diverse group of rare Mendelian disorders with clinical and genetic heterogeneity. Here, we used targeted copy number variant (CNV) screening and identified intragenic exonic duplications, formed through Alu-Alu fusion events, in two individuals with skeletal dysplasia and negative exome sequencing results. First, we detected a homozygous tandem duplication of exon 9 and 10 in IFT81 in a boy with Jeune syndrome, or short-rib thoracic dysplasia (SRTD) (MIM# 208500). Western blot analysis did not detect any wild-type IFT81 protein in fibroblasts from the patient with the IFT81 duplication, but only a shorter isoform of IFT81 that was also present in the normal control samples. Complementary zebrafish studies suggested that loss of full-length IFT81 protein but expression of a shorter form of IFT81 protein affects the phenotype while being compatible with life. Second, a de novo tandem duplication of exons 2 to 5 in MATN3 was identified in a girl with multiple epiphyseal dysplasia (MED) type 5 (MIM# 607078). Our data highlights the importance of detection and careful characterization of intragenic duplication CNVs, presenting them as a novel and very rare genetic mechanism in IFT81-related Jeune syndrome and MATN3-related MED.

Case Report: Inversion of LMX1B - A Novel Cause of Nail-Patella Syndrome in a Swedish Family and a Longtime Follow-Up.

Nail-patella syndrome (NPS, OMIM #161200) is a rare autosomal dominant disorder with symptoms from many different parts of the body, including nails, knees, elbows, pelvis, kidneys and eyes. It is caused by truncating variants in the LMX1B gene, which encodes a transcription factor with important roles during embryonic development, including dorsoventral patterning of the limbs. To our knowledge, inversions disrupting the LMX1B gene have not been reported. Here, we report a family with an inversion disrupting the LMX1B gene in five affected family members with mild but variable clinical features of NPS. Our finding demonstrates that genomic rearrangements must be considered a possible cause of NPS.

Expanding the mutation and phenotype spectrum of MYH3-associated skeletal disorders.

Pathogenic variants in MYH3 cause distal arthrogryposis type 2A and type 2B3 as well as contractures, pterygia and spondylocarpotarsal fusion syndromes types 1A and 1B. These disorders are ultra-rare and their natural course and phenotypic variability are not well described. In this study, we summarize the clinical features and genetic findings of 17 patients from 10 unrelated families with vertebral malformations caused by dominant or recessive pathogenic variants in MYH3. Twelve novel pathogenic variants in MYH3 (NM_002470.4) were identified: three of them were de novo or inherited in autosomal dominant way and nine were inherited in autosomal recessive way. The patients had vertebral segmentation anomalies accompanied with variable joint contractures, short stature and dysmorphic facial features. There was a significant phenotypic overlap between dominant and recessive MYH3-associated conditions regarding the degree of short stature as well as the number of vertebral fusions. All monoallelic variants caused significantly decreased SMAD3 phosphorylation, which is consistent with the previously proposed pathogenic mechanism of impaired canonical TGF-ß signaling. Most of the biallelic variants were predicted to be protein-truncating, while one missense variant c.4244T>G,p.(Leu1415Arg), which was inherited in an autosomal recessive way, was found to alter the phosphorylation level of p38, suggesting an inhibition of the non-canonical pathway of TGF-ß signaling. In conclusion, the identification of 12 novel pathogenic variants and overlapping phenotypes in 17 affected individuals from 10 unrelated families expands the mutation and phenotype spectrum of MYH3-associated skeletal disorders. We show that disturbances of canonical or non-canonical TGF-ß signaling pathways are involved in pathogenesis of MYH3-associated skeletal fusion (MASF) syndrome.

Skeletal ciliopathies: a pattern recognition approach.

Ciliopathy encompasses a diverse group of autosomal recessive genetic disorders caused by mutations in genes coding for components of the primary cilia. Skeletal ciliopathy forms a subset of ciliopathies characterized by distinctive skeletal changes. Common skeletal ciliopathies include Jeune asphyxiating thoracic dysplasia, Ellis-van Creveld syndrome, Sensenbrenner syndrome, and short-rib polydactyly syndromes. These disorders share common clinical and radiological features. The clinical hallmarks comprise thoracic hypoplasia with respiratory failure, body disproportion with a normal trunk length and short limbs, and severely short digits occasionally accompanied by polydactyly. Reflecting the clinical features, the radiological hallmarks consist of a narrow thorax caused by extremely short ribs, normal or only mildly affected spine, shortening of the tubular bones, and severe brachydactyly with or without polydactyly. Other radiological clues include trident ilia/pelvis and cone-shaped epiphysis. Skeletal ciliopathies are commonly associated with extraskeletal anomalies, such as progressive renal degeneration, liver disease, retinopathy, cardiac anomalies, and cerebellar abnormalities. In this article, we discuss the radiological pattern recognition approach to skeletal ciliopathies. We also describe the clinical and genetic features of skeletal ciliopathies that the radiologists should know for them to play an appropriate role in multidisciplinary care and scientific advancement of these complicated 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. © 2017 American Society for Bone and Mineral Research.

Medial Patellofemoral Ligament Injuries in Children With First-Time Lateral Patellar Dislocations: A Magnetic Resonance Imaging and Arthroscopic Study.

BACKGROUND: A lateral patellar dislocation (LPD) is the most common knee injury in children with traumatic knee hemarthrosis. The medial patellofemoral ligament (MPFL), the important passive stabilizer against LPDs, is injured in more than 90% of cases. The MPFL injury pattern is most often defined in adults or in mixed-age populations. The injury pattern in the skeletally immature patient may be different. PURPOSE: To describe MPFL injuries in the skeletally immature patient by magnetic resonance imaging (MRI), and to compare the results with the injury pattern found at arthroscopic surgery. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: This was a prospective series of patients aged 9 to 14 years with acute, first-time traumatic LPDs in whom clinical examinations, radiographs, MRI, and arthroscopic surgery were performed within 2 weeks from the index injury. The MPFL injury was divided into 3 different groups according to the location: patellar site, femoral site, or multifocal. The MPFL injury site was confirmed on MRI by soft tissue edema. The length of the MPFL injury at the patellar site was measured at arthroscopic surgery, and those ≥2 cm were defined as total ruptures. RESULTS: A total of 74 patients (40 girls and 34 boys; mean age, 13.1 years) were included; 73 patients (99%) had an MPFL injury according to MRI and arthroscopic surgery. The MRI scans showed an isolated MPFL injury at the patellar attachment site in 44 of 74 patients (60%), a multifocal injury in 26 patients (35%), an injury at the femoral site in 3 patients (4%), and no injury in 1 patient (1%). Arthroscopic surgery disclosed an isolated MPFL injury at the patellar site in 60 of 74 patients (81%) and a multifocal injury in 13 patients (18%); the MPFL injury at the patellar site was a total rupture in 49 patients (66%). Edema at the patellar attachment site on MRI was proven to be an MPFL rupture at the same site at arthroscopic surgery in 99% of the patients. A patellar-based injury, isolated or as part of a multifocal injury, was present on MRI in 95% (n = 70) of the patients, with a false-negative rate of 5% (n = 4) of patients compared with arthroscopic surgery. CONCLUSION: Skeletally immature children are more prone to sustaining an MPFL injury at the patellar attachment site. Arthroscopic surgery and MRI complement each other in the investigation of MPFL injuries.