Integrative analysis of Lunatic Fringe variants associated with spondylocostal dysostosis type-III.

Lunatic Fringe (LFNG) is required for spinal development. Biallelic pathogenic variants cause spondylocostal dysostosis type-III (SCD3), a rare disease generally characterized by malformed, asymmetrical, and attenuated development of the vertebral column and ribs. However, a variety of SCD3 cases reported have presented with additional features such as auditory alterations and digit abnormalities. There has yet to be a single, comprehensive, functional evaluation of causative LFNG variants and such analyses could unveil molecular mechanisms for phenotypic variability in SCD3. Therefore, nine LFNG missense variants associated with SCD3, c.564C>A, c.583T>C, c.842C>A, c.467T>G, c.856C>T, c.601G>A, c.446C>T, c.521G>A, and c.766G>A, were assessed in vitro for subcellular localization and protein processing. Glycosyltransferase activity was quantified for the first time in the c.583T>C, c.842C>A, and c.446C>T variants. Primarily, our results are the first to satisfy American College of Medical Genetics and Genomics PS3 criteria (functional evidence via well-established assay) for the pathogenicity of c.583T>C, c.842C>A, and c.446C>T, and replicate this evidence for the remaining six variants. Secondly, this work indicates that all variants that prevent Golgi localization also lead to impaired protein processing. It appears that the FRINGE domain is responsible for this phenomenon. Thirdly, our data suggests that variant proximity to the catalytic residue may influence whether LFNG is improperly trafficked and/or enzymatically dysfunctional. Finally, the phenotype of the axial skeleton, but not elsewhere, may be modulated in a variant-specific fashion. More reports are needed to continue testing this hypothesis. We anticipate our data will be used as a basis for discussion of genotype-phenotype correlations in SCD3.

Noncanonical protein kinase A activation by oligomerization of regulatory subunits as revealed by inherited Carney complex mutations.

Familial mutations of the protein kinase A (PKA) R1α regulatory subunit lead to a generalized predisposition for a wide range of tumors, from pituitary adenomas to pancreatic and liver cancers, commonly referred to as Carney complex (CNC). CNC mutations are known to cause overactivation of PKA, but the molecular mechanisms underlying such kinase overactivity are not fully understood in the context of the canonical cAMP-dependent activation of PKA. Here, we show that oligomerization-induced sequestration of R1α from the catalytic subunit of PKA (C) is a viable mechanism of PKA activation that can explain the CNC phenotype. Our investigations focus on comparative analyses at the level of structure, unfolding, aggregation, and kinase inhibition profiles of wild-type (wt) PKA R1α, the A211D and G287W CNC mutants, as well as the cognate acrodysostosis type 1 (ACRDYS1) mutations A211T and G287E. The latter exhibit a phenotype opposite to CNC with suboptimal PKA activation compared with wt. Overall, our results show that CNC mutations not only perturb the classical cAMP-dependent allosteric activation pathway of PKA, but also amplify significantly more than the cognate ACRDYS1 mutations nonclassical and previously unappreciated activation pathways, such as oligomerization-induced losses of the PKA R1α inhibitory function.

A recurrent homozygous LMNA missense variant p.Thr528Met causes atypical progeroid syndrome characterized by mandibuloacral dysostosis, severe muscular dystrophy, and skeletal deformities.

Atypical progeroid syndromes (APS) are premature aging syndromes caused by pathogenic LMNA missense variants, associated with unaltered expression levels of lamins A and C, without accumulation of wild-type or deleted prelamin A isoforms, as observed in Hutchinson-Gilford progeria syndrome (HGPS) or HGPS-like syndromes. A specific LMNA missense variant, (p.Thr528Met), was previously identified in a compound heterozygous state in patients affected by APS and severe familial partial lipodystrophy, whereas heterozygosity was recently identified in patients affected by Type 2 familial partial lipodystrophy. Here, we report four unrelated boys harboring homozygosity for the p.Thr528Met, variant who presented with strikingly homogeneous APS clinical features, including osteolysis of mandibles, distal clavicles and phalanges, congenital muscular dystrophy with elevated creatine kinase levels, and major skeletal deformities. Immunofluorescence analyses of patient-derived primary fibroblasts showed a high percentage of dysmorphic nuclei with nuclear blebs and typical honeycomb patterns devoid of lamin B1. Interestingly, in some protrusions emerin or LAP2α formed aberrant aggregates, suggesting pathophysiology-associated clues. These four cases further confirm that a specific LMNA variant can lead to the development of strikingly homogeneous clinical phenotypes, in these particular cases a premature aging phenotype with major musculoskeletal involvement linked to the homozygous p.Thr528Met variant.

Diaphanospondylodysostosis: Full Case Report with Novel Pathogenic BMPER Mutation.

Diaphanospondylodysostosis is an extremely rare, recessively inherited, perinatal lethal skeletal disorder associated with BMPER gene mutations. Clinically it is characterized by defects in costovertebral ossification, absent ribs, hypertelorism, short nose with depressed nasal bridge, low-set ears, and short neck. At the extraosseous level, the most frequent pathologic finding is nephroblastomatosis with multicystic kidneys. We present the case of a child of non-consanguineous parents who died at 2 months of age in our center. Autopsy showed a marked costovertebral ossification defect, perilobar nephrogenic rests and loss of white matter with periventricular leukomalacia. After genetic study, the diagnosis of diaphanospondylodysostosis was confirmed. A previously undescribed germinal mutation in the BMPER gene (c.576 + 2dupT) was found.

Molecular diagnosis in a cohort of 114 patients with rare skeletal dysplasias.

Molecular diagnosis is important to provide accurate genetic counseling of skeletal dysplasias (SD). Although next-generation sequencing (NGS) techniques are currently the preferred methods for analyzing these conditions, some of the published results have not shown a detection rate as high as it would be expected. The present study aimed to assess the diagnostic yield of targeted NGS combined with Sanger sequencing (SS) for low-coverage exons of genes of interest and exome sequencing (ES) in a series of patients with rare SD and use two patients as an example of our strategy. This study used two different in-house panels. Of 93 variants found in 88/114 (77%) patients, 57 are novel. The pathogenic variants found in the following genes: B3GALT6, PCYT1A, INPPL1, LIFR, of four patients were only detected by SS. In conclusion, the high diagnostic yield reached in the present study can be attributed to both a good selection of patients and the utilization of the SS for the insufficiently covered regions. Additionally, the two case reports-a patient with acrodysostosis related to PRKAR1A and another with ciliopathy associated with KIAA0753, add new and relevant clinical information to the current knowledge.

Congenital cervical spine malformation due to bi-allelic RIPPLY2 variants in spondylocostal dysostosis type 6.

RIPPLY2 is an essential part of the formation of somite patterning during embryogenesis and in establishment of rostro-caudal polarity. Here, we describe three individuals from two families with compound-heterozygous variants in RIPPLY2 (NM_001009994.2): c.238A > T, p.(Arg80*) and c.240-4 T > G, p.(?), in two 15 and 20-year-old sisters, and a homozygous nonsense variant, c.238A > T, p.(Arg80*), in an 8 year old boy. All patients had multiple vertebral body malformations in the cervical and thoracic region, small or absent rib involvement, myelopathies, and common clinical features of SCDO6 including scoliosis, mild facial asymmetry, spinal spasticity and hemivertebrae. The nonsense variant can be classified as likely pathogenic based on the ACMG criteria while the splice variants must be classified as a variant of unknown significance. With this report on two further families, we confirm RIPPLY2 as the gene for SCDO6 and broaden the phenotype by adding myelopathy with or without spinal canal stenosis and spinal spasticity to the symptom spectrum.

Sleep-disordered breathing and its management in children with rare skeletal dysplasias.

Sleep-disordered breathing (SDB) is common in patients with skeletal dysplasias. The aim of our study was to analyze SDB and respiratory management in children with rare skeletal dysplasias. We performed a retrospective analysis of patients with spondyloepiphyseal dysplasia congenita (SEDC), metatropic dysplasia (MD), spondyloepimetaphyseal dysplasia (SEMD), acrodysostosis (ADO), geleophysic dysplasia (GD), acromicric dysplasia (AD), and spondylocostal dysplasia (SCD) between April 2014 and October 2020. Polygraphic data, clinical management, and patients' outcome were analyzed. Thirty-one patients were included (8 SEDC, 3 MD, 4 SEMD, 1 ADO, 4 GD, 3 AD, and 8 SCD). Sixteen patients had obstructive sleep apnea (OSA): 11 patients (2 with SEDC, 1 with SEMD, 1 with ADO, 1 with GD, 2 with AD, and 4 with SCD) had mild OSA, 2 (1 SEMD and 1 GD) had moderate OSA, and 3 (1 SEDC, 1 MD, 1 SEMD) had severe OSA. Adenotonsillectomy was performed in one patient with SCD and mild OSA, and at a later age in two other patients with ADO and AD. The two patients with moderate OSA were treated with noninvasive ventilation (NIV) because of nocturnal hypoxemia. The three patients with severe OSA were treated with adenotonsillectomy (1 SEDC), adeno-turbinectomy and continuous positive airway pressure (CPAP; 1 MD), and with NIV (1 SEMD) because of nocturnal hypoventilation. OSA and/or alveolar hypoventilation is common in patients with skeletal dysplasias, underlining the importance of systematic screening for SDB. CPAP and NIV are effective treatments for OSA and nocturnal hypoventilation/hypoxemia.

A novel variant in the PDE4D gene is the cause of Acrodysostosis type 2 in a Lithuanian patient: a case report.

BACKGROUND: Acrodysostosis is a rare hereditary disorder described as a primary bone dysplasia with or without hormonal resistance. Pathogenic variants in the PRKAR1A and PDE4D genes are known genetic causes of this condition. The latter gene variants are more frequently identified in patients with midfacial and nasal hypoplasia and neurological involvement. The aim of our study was to analyse and confirm a genetic cause of acrodysostosis in a male patient. CASE PRESENTATION: We report on a 29-year-old Lithuanian man diagnosed with acrodysostosis type 2. The characteristic phenotype includes specific skeletal abnormalities, facial dysostosis, mild intellectual disability and metabolic syndrome. Using patient's DNA extracted from peripheral blood sample, the novel, likely pathogenic, heterozygous de novo variant NM_001104631.2:c.581G > C was identified in the gene PDE4D via Sanger sequencing. This variant causes amino acid change (NP_001098101.1:p.(Arg194Pro)) in the functionally relevant upstream conserved region 1 domain of PDE4D. CONCLUSIONS: This report further expands the knowledge of the consequences of missense variants in PDE4D that affect the upstream conserved region 1 regulatory domain and indicates that pathogenic variants of the gene PDE4D play an important role in the pathogenesis mechanism of acrodysostosis type 2 without significant hormonal resistance.

Fourteen-year follow-up of a child with acroscyphodysplasia with emphasis on the need for multidisciplinary management: a case report.

BACKGROUND: Acroscyphodysplasia has been described as a phenotypic variant of acrodysostosis type 2 and pseudohypoparathyroidism. In acrodysostosis, skeletal features can include brachydactyly, facial hypoplasia, cone-shaped epiphyses, short stature, and advanced bone age. To date, reports on this disorder have focused on phenotypic findings, endocrine changes, and genetic variation. We present a 14-year overview of a patient, from birth to skeletal maturity, with acroscyphodysplasia, noting the significant orthopaedic challenges and the need for a multidisciplinary team, including specialists in genetics, orthopaedics, endocrinology, and otolaryngology, to optimize long-term outcomes. CASE PRESENTATION: The patient presented as a newborn with dysmorphic facial features, including severe midface hypoplasia, malar flattening, nasal stenosis, and feeding difficulties. Radiologic findings were initially subtle, and a skeletal survey performed at age 7 months was initially considered normal. Genetic evaluation revealed a variant in PDE4D and subsequent pseudohypoparathyroidism. The patient presented to the department of orthopaedics, at age 2 years 9 months with a leg length discrepancy, right knee contracture, and severely crouched gait. Radiographs demonstrated cone-shaped epiphyses of the right distal femur and proximal tibia, but no evidence of growth plate changes in the left leg. The child developed early posterior epiphyseal arrest on the right side and required multiple surgical interventions to achieve neutral extension. Her left distal femur developed late posterior physeal arrest and secondary contracture without evidence of schypho deformity, which improved with anterior screw epiphysiodesis. The child required numerous orthopaedic surgical interventions to achieve full knee extension bilaterally. At age 13 years 11 months, she was an independent ambulator with erect posture. The child underwent numerous otolaryngology procedures and will require significant ongoing care. She has moderate intellectual disability. DISCUSSION AND CONCLUSIONS: Key challenges in the management of this case included the subtle changes on initial skeletal survey and the marked asymmetry of her deformity. While cone-shaped epiphyses are a hallmark of acrodysostosis, posterior tethering/growth arrest of the posterior distal femur has not been previously reported. Correction of the secondary knee contracture was essential to improve ambulation. Children with acroscyphodysplasia require a multidisciplinary approach, including radiology, genetics, orthopaedics, otolaryngology, and endocrinology specialties.

Novel progressive acrodysostosis-like skeletal dysplasia, cerebellar atrophy, and ichthyosis.

Acrodysostosis refers to a rare heterogeneous group of bone dysplasias that share skeletal features, hormone resistance, and intellectual disability. Two genes have been associated with acrodysostosis with or without hormone resistance (PRKAR1A and PDE4D). Severe intellectual disability has been reported with acrodysostosis but brain malformations and ichthyosis have not been reported in these syndromes. Here we describe a female patient with acrodysostosis, intellectual disability, cerebellar hypoplasia, and lamellar ichthyosis. The patient has an evolving distinctive facial phenotype and childhood onset ataxia. X-rays showed generalized osteopenia, shortening of middle and distal phalanges, and abnormal distal epiphysis of the ulna and radius. Brain magnetic resonance imaging showed cerebellar atrophy without other brainstem abnormalities. Genetic workup included nondiagnostic chromosomal microarray and skeletal dysplasia molecular panels. These clinical findings are different from any recognized form of acrodysostosis syndrome. Whole exome sequencing did not identify rare or predicted pathogenic variants in genes associated with known acrodysostosis, lamellar ichthyosis, and other overlapping disorders. A broader search for rare alleles absent in healthy population databases and controls identified two heterozygous truncating alleles in FBNL7 and PPM1M genes, and one missense allele in the NPEPPS gene. Identification of additional patients is required to delineate the mechanism of this unique disorder.

Dysostosis Multiplex in Human Mucopolysaccharidosis Type 1 H and in Animal Models of the Disease.

Mucopolysaccharidosis type I (MPS I) is a rare autosomal recessive disorder, caused by deficiency of α-L-iduronidase, and consequent accumulation of dermatan and heparan sulfates. Severity of the disease ranges from mild (Scheie) to moderate (Hurler-Scheie) to severe (Hurler or MPS-IH). A prominent clinical manifestation of MPS-IH is dysostosis multiplex, a constellation of skeletal abnormalities. We performed a retrospective review comparing manifestations of dysostosis multiplex in patients presenting with MPSIH and relevant animal models. Dog, cat and mouse models of MPS-IH are extensively studied to better understand the pathology of the disease. While all animal models display certain characteristics of human MPSIH, species-specific manifestations must be considered when evaluating skeletal abnormalities. Moreover, some skeletal abnormalities emerge at species-specific developmental stages, e.g. thoracolumbar kyphosis is an early manifestation in humans, while it appears late in the mouse model. The choice of the appropriate diagnostic test is of importance to avoid misleading conclusions.

[Genetic analysis of a child with acrodysostosis type 2].

OBJECTIVE: To analyze the clinical characteristics and genetic variation in a child with acrodysostosis type 2. METHODS: The child has undergone history taking and physical examination. Genome DNA was extracted from peripheral blood samples from him and his parents. High-throughput sequencing was carried out. The result was verified by Sanger sequencing. RESULTS: The 8-year-old boy presented with midface hypoplasia, hypertelorism, prominent nasal bridge, small and upturned nostrils, broad thumb and great toes, and brachydactyly of remaining fingers and toes. Genetic testing revealed that the child has carried a heterozygous c.1813T>C (p.Tyr605His) missense mutation of the PDE4D gene. The same mutation was not found in either parent and was unreported previously. CONCLUSION: The child was diagnosed with acrodysostosis type 2 due to the novel mutation of the PDE4D gene.

A novel de novo PDE4D gene mutation identified in a Chinese patient with acrodysostosis.

Acrodysostosis is an extremely rare disorder at birth, that is, characterized by skeletal dysplasia with short stature and midfacial hypoplasia, which has been reported to be caused by PDE4D and PRKAR1A gene mutations. Here, a Chinese boy with acrodysostosis, ventricular septal defect, and pulmonary hypertension was recruited for our study, and his clinical and biochemical characteristics were analyzed. A novel de novo heterozygous missense mutation (NM_001104631: c.2030A>C, p.Tyr677Ser) of the PDE4D gene was detected by whole exome sequencing and confirmed by Sanger sequencing. The c.2030A>C (p.Tyr677Ser) variant was located in exon 15 of the PDE4D gene, predicted to be damaging by a functional prediction program and shown to be highly conserved among many species. Further functional analysis showed that the p.Tyr677Ser substitution changes the function of the PDE4D protein, affects its subcellular localization in transfected cells, increases PDE4 activity in the regulation of cAMP signaling and affects cell proliferation. Our study identified a novel de novo PDE4D mutation in acrodysostosis of Chinese origin that not only contributes a deeper appreciation of the phenotypic characteristics of patients with PDE4D mutations but also expands the spectrum of PDE4D mutations.

Mutations causing acrodysostosis-2 facilitate activation of phosphodiesterase 4D3.

Type 2 acrodysostosis (ACRDYS2), a rare developmental skeletal dysplasia characterized by short stature, severe brachydactyly and facial dysostosis, is caused by mutations in the phosphodiesterase (PDE) 4D (PDE4D) gene. Several arguments suggest that the mutations should result in inappropriately increased PDE4D activity, however, no direct evidence supporting this hypothesis has been presented, and the functional consequences of the mutations remain unclear. We evaluated the impact of four different PDE4D mutations causing ACRDYS2 located in different functional domains on the activity of PDE4D3 expressed in Chinese hamster ovary cells. Three independent approaches were used: the direct measurement of PDE activity in cell lysates, the evaluation of intracellular cAMP levels using an EPAC-based (exchange factor directly activated by cAMP) bioluminescence resonance energy transfer sensor , and the assessment of PDE4D3 activation based on electrophoretic mobility. Our findings indicate that PDE4D3s carrying the ACRDYS2 mutations are more easily activated by protein kinase A-induced phosphorylation than WT PDE4D3. This occurs over a wide range of intracellular cAMP concentrations, including basal conditions, and result in increased hydrolytic activity. Our results provide new information concerning the mechanism whereby the mutations identified in the ACRDYS2 dysregulate PDE4D activity, and give insights into rare diseases involving the cAMP signaling pathway. These findings may offer new perspectives into the selection of specific PDE inhibitors and possible therapeutic intervention for these patients.

Identification of novel pathogenic variants and features in patients with pseudohypoparathyroidism and acrodysostosis, subtypes of the newly classified inactivating PTH/PTHrP signaling disorders.

Albright hereditary osteodystrophy (AHO) is a complex disorder defined by the presence of a short adult stature relative to the height of an unaffected parent and brachydactyly type E, as well as a stocky build, round face, and ectopic calcifications. AHO and pseudohypoparathyroidism (PHP) have been used interchangeably in the past. The term PHP describes end-organ resistance to parathyroid hormone (PTH), occurring with or without the physical features of AHO. Conversely, pseudopseudohypoparathyroidism (PPHP) describes individuals with AHO features in the absence of PTH resistance. PHP and PPHP are etiologically linked and caused by genetic and/or epigenetic alterations in the guanine nucleotide-binding protein alpha-stimulating (Gs α) locus (GNAS) in chromosome 20q13. Another less-recognized group of skeletal dysplasias, termed acrodysostosis, partially overlap with skeletal, endocrine, and neurodevelopmental features of AHO/PHP and can be overlooked in clinical practice, causing confusion in the literature. Acrodysostosis is caused by defects in two genes, PRKAR1A and PDE4D, both encoding important components of the Gs α-cyclic adenosine monophosphate-protein kinase A signaling pathway. We describe the clinical course and genotype of two adult patients with overlapping AHO features who harbored novel pathogenic variants in GNAS (c.2273C > G, p.Pro758Arg, NM_080425.2) and PRKAR1A (c.803C > T, p.Ala268Val, NM_002734.4), respectively. We highlight the value of expert radiological opinion and molecular testing in establishing correct diagnoses and discuss phenotypic features of our patients, including the first description of subcutaneous ossification and spina bifida occulta in PRKAR1A-related acrodysostosis, in the context of the novel inactivating PTH/PTH related peptide signaling disorder classification system.

Spondylocostal Dysostosis Associated with Split Spinal Cord and Other Malformations.

Spondylocostal dysostosis is a very rare combination of complex vertebra and rib malformations, accompanied occasionally by other disorders. A 3-year-old girl presented kyphoscoliosis, foot deformities, gate disturbance, and urinary incontinence. The CT and MRI examination revealed kyphosis and scoliosis with a double curve, some absent, broadened, bifurcating and fused ribs, hemivertebrae, butterfly and cleft vertebrae in thoracic and lumbar region, sporadic cleft or absent vertebral arches or pedicles, and hypoplastic sacrum with a cleft of the S2 vertebra. Spina bifida occulta extended from T10 to T11, and from L3 to the end of the sacrum. Two hemicords, separated by a bony septum and surrounded by their own dural tubes (type I), were present from the level of T9 to the conus medullaris. Filum terminale was thick and duplicated. Syringomyelia was present in the thoracic cord from T5 to T8. Finally, a small meningocele was seen at the T10-T11 level, and a subcutaneous lipoma in the thoracolumbar region. To our knowledge, such a combination of vertebra, rib, and cord malformations, including the mentioned additional disorders, has never been reported.

Engineered stabilization and structural analysis of the autoinhibited conformation of PDE4.

Phosphodiesterase 4 (PDE4) is an essential contributor to intracellular signaling and an important drug target. The four members of this enzyme family (PDE4A to -D) are functional dimers in which each subunit contains two upstream conserved regions (UCR), UCR1 and -2, which precede the C-terminal catalytic domain. Alternative promoters, transcriptional start sites, and mRNA splicing lead to the existence of over 25 variants of PDE4, broadly classified as long, short, and supershort forms. We report the X-ray crystal structure of long form PDE4B containing UCR1, UCR2, and the catalytic domain, crystallized as a dimer in which a disulfide bond cross-links cysteines engineered into UCR2 and the catalytic domain. Biochemical and mass spectrometric analyses showed that the UCR2-catalytic domain interaction occurs in trans, and established that this interaction regulates the catalytic activity of PDE4. By elucidating the key structural determinants of dimerization, we show that only long forms of PDE4 can be regulated by this mechanism. The results also provide a structural basis for the long-standing observation of high- and low-affinity binding sites for the prototypic inhibitor rolipram.

Diaphanospondylodysostosis and ischiospinal dysostosis, evidence for one disorder with variable expression in a patient who has survived to age 9 years.

Diaphanospondylodysostosis (DSD) and ischiospinal dysostosis (ISD) are both rare skeletal dysplasias consisting of abnormal axial skeletal development but normal appendicular skeletal development. Both disorders recently have been found to result from mutations in the BMPER gene. We report a patient with one deletion and one mutation of the BMPER gene who has features most consistent with DSD but who has survived to age 9 years. Survival suggests that DSD and ISD reflect a spectrum of severity of one disease process.

Two cases of Legg-Perthes and intellectual disability in Tricho-Rhino-Phalangeal syndrome type 1 associated with novel TRPS1 mutations.

Tricho-Rhino-Phalangeal syndrome is a rare autosomal dominant genetic disorder caused by mutations in the TRPS1 gene. This malformation syndrome is characterized by distinctive craniofacial features including sparse scalp hair, bulbous tip of the nose, long flat philtrum, thin upper vermilion border, and protruding ears. Skeletal abnormalities include cone-shaped epiphyses at the phalanges, hip malformations, and short stature. In this report, we describe two patients with the physical manifestations and genotype of TRPS type I but with learning/intellectual disability not typically described as part of the syndrome. The first patient has a novel heterozygous two-base-pair deletion of nucleotides at 3198-3199 (c.3198-3199delAT) in the TRPS1 gene causing a translational frameshift and subsequent alternate stop codon. The second patient has a 3.08 million base-pair interstitial deletion at 8q23.3 (113,735,487-116,818,578), which includes the TRPS1 gene and CSMD3. Our patients have characteristic craniofacial features, Legg-Perthes syndrome, various skeletal abnormalities including cone shaped epiphyses, anxiety (first patient), and intellectual disability, presenting unusual phenotypes that add to the clinical spectrum of the disease.