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FXR1 is an alternatively spliced gene that encodes RNA binding proteins (FXR1P) involved in muscle development. In contrast to other tissues, cardiac and skeletal muscle express two FXR1P isoforms that incorporate an additional exon-15. We report that recessive mutations in this particular exon of FXR1 cause congenital multi-minicore myopathy in humans and mice. Additionally, we show that while Myf5-dependent depletion of all FXR1P isoforms is neonatal lethal, mice carrying mutations in exon-15 display non-lethal myopathies which vary in severity depending on the specific effect of each mutation on the protein.

[Background]: Stüve-Wiedemann syndrome (SWS) is characterised by bowing of the lower limbs, respiratory distress and hyperthermia that are often responsible for early death. Survivors develop progressive scoliosis and spontaneous fractures. We previously identified LIFR mutations in most SWS cases, but absence of LIFR pathogenic changes in five patients led us to perform exome sequencing and to identify homozygosity for a FAM46A mutation in one case [p.Ser205Tyrfs*13]. The follow-up of this case supported a final diagnosis of osteogenesis imperfecta (OI), based on vertebral collapses and blue sclerae., [Methods and results]: This prompted us to screen FAM46A in 25 OI patients with no known mutations. We identified a homozygous deleterious variant in FAM46A in two affected sibs with typical OI [p.His127Arg]. Another homozygous variant, [p.Asp231Gly], also classed as deleterious, was detected in a patient with type III OI of consanguineous parents using homozygosity mapping and exome sequencing. FAM46A is a member of the superfamily of nucleotidyltransferase fold proteins but its exact function is presently unknown. Nevertheless, there are lines of evidence pointing to a relevant role of FAM46A in bone development. By RT-PCR analysis, we detected specific expression of FAM46A in human osteoblasts andinterestingly, a nonsense mutation in Fam46a has been recently identified in an ENU-derived (N-ethyl-N-nitrosourea) mouse model characterised by decreased body length, limb, rib, pelvis, and skull deformities and reduced cortical thickness in long bones., [Conclusion]: We conclude that FAM46A mutations are responsible for a severe form of OI with congenital bowing of the lower limbs and suggest screening this gene in unexplained OI forms.

[Background]: Osteogenesis imperfecta (OI) is a heterogeneous bone disorder characterized by recurrent fractures. Although most cases of OI have heterozygous mutations in COL1A1 or COL1A2 and show autosomal dominant inheritance, during the last years there has been an explosion in the number of genes responsible for both recessive and dominant forms of this condition. Herein, we have analyzed a cohort of patients with OI, all offspring of unaffected parents, to determine the spectrum of variants accounting for these cases. Twenty patients had nonrelated parents and were sporadic, and 21 were born to consanguineous relationships., [Methods]: Mutation analysis was performed using a next‐generation sequencing gene panel, homozygosity mapping, and whole exome sequencing (WES)., [Results]: Patients offspring of nonconsanguineous parents were mostly identified with COL1A1 or COL1A2 heterozygous changes, although there were also a few cases with IFITM5 and WNT1 heterozygous mutations. Only one sporadic patient was a compound heterozygote for two recessive mutations. Patients offspring of consanguineous parents showed homozygous changes in a variety of genes including CRTAP,FKBP10,LEPRE1,PLOD2,PPIB,SERPINF1,TMEM38B, and WNT1. In addition, two patients born to consanguineous parents were found to have de novo COL1A1 heterozygous mutations demonstrating that causative variants in the collagen I structural genes cannot be overlooked in affected children from consanguineous couples. Further to this, WES analysis in probands lacking mutations in OI genes revealed deleterious variants in SCN9A,NTRK1, and SLC2A2, which are associated with congenital indifference to pain (CIP) and Fanconi–Bickel syndrome (FBS)., [Conclusion]: This work provides useful information for clinical and genetic diagnosis of OI patients with no positive family history of this disease. Our data also indicate that CIP and FBS are conditions to be considered in the differential diagnosis of OI and suggest a positive role of SCN9A and NTRK1 in bone development.

Most patients with Ellis-van Creveld syndrome (EvC) are identified with pathogenic changes in EVC or EVC2, however further genetic heterogeneity has been suggested. In this report we describe pathogenic splicing variants in WDR35, encoding retrograde intraflagellar transport protein 121 (IFT121), in three families with a clinical diagnosis of EvC but having a distinctive phenotype. To understand why WDR35 variants result in EvC, we analysed EVC, EVC2 and Smoothened (SMO) in IFT-A deficient cells. We found that the three proteins failed to localize to Wdr35−/− cilia, but not to the cilium of the IFT retrograde motor mutant Dync2h1−/−, indicating that IFT121 is specifically required for their entry into the ciliary compartment. Furthermore expression of Wdr35 disease cDNAs in Wdr35−/− fibroblasts revealed that the newly identified variants lead to Hedgehog signalling defects resembling those of Evc−/− and Evc2−/− mutants. Together our data indicate that splicing variants in WDR35, and possibly in other IFT-A components, underlie a number of EvC cases by disrupting targeting of both the EvC complex and SMO to cilia.

Osteogenesis imperfecta or >brittle bone disease> is a collagen type I-related condition associated with osteoporosis and increased risk of bone fractures. Using a combination of homozygosity mapping and candidate gene approach we identified a homozygous single basepair deletion (c.1052delA) in SP7/Osterix (OSX) in a consanguineous patient with recessive osteogenesis imperfecta. The clinical findings of this patient include recurrent fractures, mild bone deformities, delayed tooth eruption, normal hearing, and white sclera. OSX encodes a transcription factor containing three Cys2-His2 zinc-finger DNA-binding domains at its C-terminus, which in mice has been shown to be essential for bone formation. The frameshift caused by the c.1052delA deletion removes the last 81 amino acids of the protein including the third zinc-finger motif. This finding adds a new locus to the spectrum of genes associated with osteogenesis imperfecta and reveals that SP7/OSX also plays a key role in human bone development.

Mutations in BMP1 cause osteogenesis imperfect (OI) type XIII (MIM 614856), a probably not so uncommon form of autosomal recessive OI. (Editor's Note: See Chapters 1 and 2 for discussion of OI types related to specific mutations.) We have studied an Egyptian consanguineous family with two children initially diagnosed as OI type III. They had blue sclera, severe kyphoscoliosis and large umbilical hernia. Genetic analysis of this family performed by homozygosity mapping showed lack of linkage to any of the previously known AR-OI loci but identified a homozygous 10.27. Mb DNA segment on chromosome 8p in both affected children comprising the type I collagen C-propeptide protease gene BMP1. Direct sequencing of BMP1 in the proband revealed a Phe249Leu homozygous missense change within the BMP1/mTLD astacin protease domain. Phe249 is a residue which is invariable in all invertebrate and vertebrate members of the astacin family of metalloproteases. A few months later, using a combination of whole-exome sequencing and filtering for homozygous stretches of identified variants, another mutation was reported by other authors in two affected sibs of a consanguineous family from Turkey. They showed increased bone mineral density and multiple recurrent fractures. The mutation in BMP1, a 34. G>C transversion in exon 1 resulting in a Gly12Arg (G12R) substitution within the signal peptide, impaired protein's localization to the endoplasmic reticulum, the correct post-translational glycosylation and its secretion. BMP1 expands the number of growing genes involved in AR-OI and demonstrates the high genetic complexity of the collagen I pathway disorders.

Osteogenesis imperfecta is a genetic condition characterized by bone fragility and recurrent fractures, which in the large majority of patients are caused by defects in the production of type I collagen. Mutations in the gene encoding bone morphogenetic protein 1 (BMP1, also known as procollagen C-endopeptidase) have been associated with osteogenesis imperfecta in two sib pairs. In this report, we describe an additional patient with osteogenesis imperfecta with normal bone density and a recurrent, homozygous c.34G>C mutation in BMP1. Western blot analysis of dermal fibroblasts from this patient showed decreased protein levels of the two alternatively spliced products of BMP1 and abnormal cleavage of the C-terminal propeptide of type I procollagen. In addition, fluorescence and electron microscopy showed impaired assembly of type I collagen fibrils in the extracellular matrix of cultured fibroblasts derived from two patients: the patient described here and a previously reported patient with a homozygous BMP1 c.747C>G mutation. We conclude that BMP1 is essential for human type I collagen fibrilogenesis.