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.

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.

Inactivating PTH/PTHrP Signaling Disorders.

Pseudohypoparathyroidism (PHP), pseudo-PHP, acrodysostosis, and progressive osseous heteroplasia are heterogeneous disorders characterized by physical findings, differently associated in each subtype, including short bones, short stature, a stocky build, ectopic ossifications (features associated with Albright's hereditary osteodystrophy), as well as laboratory abnormalities consistent with hormone resistance, such as hypocalcemia, hyperphosphatemia, and elevated parathyroid hormone (PTH) and thyroid-stimulating hormone levels. All these disorders are caused by impairments in the cAMP-mediated signal transduction pathway and, in particular, in the PTH/PTHrP signaling pathway: the main subtypes of PHP and related disorders are caused by de novo or autosomal dominantly inherited inactivating genetic mutations, and/or epigenetic, sporadic, or genetic-based alterations within or upstream of GNAS, PRKAR1A, PDE4D, and PDE3A. Here we will review the impressive progress that has been made over the past 30 years on the pathophysiology of these diseases and will describe the recently proposed novel nomenclature and classification. The new term "inactivating PTH/PTHrP signaling disorder," iPPSD: (1) defines the common mechanism responsible for all diseases, (2) does not require a confirmed genetic defect, (3) avoids ambiguous terms like "pseudo," and (4) eliminates the clinical or molecular overlap between diseases.

Parathyroid hormone resistance syndromes - Inactivating PTH/PTHrP signaling disorders (iPPSDs).

Metabolic disorders caused by impairments of the Gsα/cAMP/PKA pathway affecting the signaling of PTH/PTHrP lead to features caused by non-responsiveness of target organs, in turn leading to manifestations similar to the deficiency of the hormone itself. Pseudohypoparathyroidism (PHP) and related disorders derive from a defect of the α subunit of the stimulatory G protein (Gsα) or of downstream effectors of the same pathway, such as the PKA regulatory subunit 1A and the phosphodiesterase type 4D. The increasing knowledge on these diseases made the actual classification of PHP outdated as it does not include related conditions such as acrodysostosis (ACRDYS) or progressive osseous heteroplasia (POH), so that a new nomenclature and classification has been recently proposed grouping these disorders under the term "inactivating PTH/PTHrP signaling disorder" (iPPSD). This review will focus on the pathophysiology, clinical and molecular aspects of these rare, heterogeneous but closely related diseases.

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.

Modulation of signaling through GPCR-cAMP-PKA pathways by PDE4 depends on stimulus intensity: Possible implications for the pathogenesis of acrodysostosis without hormone resistance.

In acrodysostosis without hormone resistance, a disease caused by phosphodiesterase (PDE)-4D mutations, increased PDE activity leads to bone developmental defects but with normal renal responses to PTH. To identify potential mechanisms for these disparate responses, we compared the effect of PDE activity on hormone signaling through the GPCR-Gsα-cAMP-PKA pathway in cells from two lineages, HEK-293 cells stably overexpressing PTH1R (HEKpthr) and human dermal fibroblasts, including studies evaluating cAMP levels using an Epac-based BRET-sensor for cAMP (CAMYEL). For ligand-induced responses inducing strong cAMP accumulation, the inhibition of PDE4 activity resulted in relatively small further increases. In contrast, when ligand-induced cAMP accumulation was of lesser intensity, the inhibition of PDE4 had a more pronounced effect. Similar results were obtained evaluating downstream events (cellular CREB phosphorylation and CRE-luciferase activity). Thus, the ability of PDE4 to modulate signaling through GPCR-cAMP-PKA pathways can depend on the cell type and stimulus intensity.

[Genes in the cAMP pathway causing skeletal dysplasia with or without hormonal resistance]. / Gènes de régulation de la voie de l'AMPc, résistance hormonale et dysplasie squelettique.

Acrodysostosis refers to a heterogeneous group of rare skeletal dysplasia that share characteristic features including severe brachydactyly, facial dysostosis and nasal hypoplasia. The literature describing acrodysostosis cases has been confusing because some reported patients may have had other phenotypically related diseases presenting Albright Hereditary Osteodystrophy (AHO) such as pseudohypoparathyroidism type 1a (PHP1a) or pseudopseudohypoparathyroidism (PPHP). A question has been whether patients display or not abnormal mineral metabolism associated with resistance to PTH and/or resistance to other hormones that bind G-protein coupled receptors (GPCR) linked to Gsa, as observed in PHP1a. Defects in two genes, PRKAR1A and PDE4D, both important players in the GPCR-Gsa-cAMP-PKA signaling, were recently identified in patients affected with acrodysostosis. This has helped clarify some issues regarding the heterogeneity of acrodysostosis, in particular the presence of hormonal resistance. Two different genetic and phenotypic syndromes are now identified, both with a similar bone dysplasia: acrodysostosis type 1 due to PRKAR1A defects, and acrodysostosis type 2, due to PDE4D defects. The existence of hormone resistance is typical of the acrodysostosis type 1 syndrome. We discuss here the PRKAR1A and PDE4D gene defects and phenotypes identified in acrodysostosis syndromes, in particular in regard to phenotypically related diseases caused by Gsa gene defects in the same signaling pathway.

The clinical spectrum and pathophysiology of skeletal complications in lysosomal storage disorders.

Lysosomal storage disorders affect multiple organs including the skeleton. Disorders with prominent skeletal symptoms are type 1 and 3 Gaucher disease, the mucopolysaccharidoses, the glycoproteinoses and pycnodysostosis. Clinical manifestations range from asymptomatic radiographical evidence of bone pathology to overt bone crises (Gaucher), short stature with typical imaging features known as dysostosis multiplex (MPS), with spine and joint deformities (mucopolysaccharidoses, mucolipidosis), or osteopetrosis with pathological fractures (pynodysostosis). The pathophysiology of skeletal disease is only partially understood and involves direct substrate storage, inflammation and other complex alterations of cartilage and bone metabolism. Current treatments are enzyme replacement therapy, substrate reduction therapy and hematopoietic stem cell transplantation. However, effects of these interventions on skeletal disease manifestations are less well established and outcomes are highly dependent on disease burden at treatment initiation. It is now clear that adjunctive treatments that target skeletal disease are needed and should be part of future research agenda.

Expression dynamics and functions of Hes factors in development and diseases.

Hes genes, encoding basic helix-loop-helix (HLH) transcriptional repressors, are mammalian homologues of Drosophila hairy and Enhancer of split genes, both of which are required for normal neurogenesis in Drosophila. There are seven members in the human Hes family, Hes1-7, which are expressed in many tissues and play various roles mainly in development. All Hes proteins have three conserved domains: basic HLH (bHLH), Orange, and WRPW domains. The basic region binds to target DNA sequences, while the HLH region forms homo- and heterodimers with other bHLH proteins, the Orange domain is responsible for the selection of partners during heterodimer formation, and the WRPW domain recruits corepressors. Hes1, Hes5, and Hes7 are known as downstream effectors of canonical Notch signaling, which regulates cell differentiation via cell-cell interaction. Hes factors regulate many events in development by repressing the expression of target genes, many of which encode transcriptional activators that promote cell differentiation. For example, Hes1, Hes3, and Hes5 are highly expressed by neural stem cells, and inactivation of these genes results in insufficient maintenance of stem cell proliferation and prematurely promotes neuronal differentiation. Recently, it was shown that the expression dynamics of Hes1 plays crucial roles in proper developmental timings and fate-determination steps of embryonic stem cells and neural progenitor cells. Here, we discuss some key features of Hes factors in development and diseases.

Osteoimmunology in mucopolysaccharidoses type I, II, VI and VII. Immunological regulation of the osteoarticular system in the course of metabolic inflammation.

BACKGROUND: Mucopolysaccharidoses (MPSs) are rare genetic diseases caused by a deficient activity of one of the lysosomal enzymes involved in the glycosaminoglycan (GAG) breakdown pathway. These metabolic blocks lead to the accumulation of GAGs in various organs and tissues, resulting in a multisystemic clinical picture. The pathological GAG accumulation begins a cascade of interrelated responses: metabolic, inflammatory and immunological with systemic effects. Metabolic inflammation, secondary to GAG storage, is a significant cause of osteoarticular symptoms in MPS disorders. OBJECTIVE AND METHOD: The aim of this review is to present recent progress in the understanding of the role of inflammatory and immune processes in the pathophysiology of osteoarticular symptoms in MPS disorders and potential therapeutic interventions based on published reports in MPS patients and studies in animal models. RESULTS AND CONCLUSIONS: The immune and skeletal systems have a number of shared regulatory molecules and many relationships between bone disorders and aberrant immune responses in MPS can be explained by osteoimmunology. The treatment options currently available are not sufficiently effective in the prevention, inhibition and treatment of osteoarticular symptoms in MPS disease. A lot can be learnt from interactions between skeletal and immune systems in autoimmune diseases such as rheumatoid arthritis (RA) and similarities between RA and MPS point to the possibility of using the experience with RA in the treatment of MPS in the future. The use of different anti-inflammatory drugs requires further study, but it seems to be an important direction for new therapeutic options for MPS patients.

Novel mutations of the PRKAR1A gene in patients with acrodysostosis.

Acrodysostosis is characterized by a peripheral dysostosis that is accompanied by short stature, midface hypoplasia, and developmental delay. Recently, it was shown that heterozygous point mutations in the PRKAR1A gene cause acrodysostosis with hormone resistance. By mutational analysis of the PRKAR1A gene we detected four different mutations (p.Arg368Stop, p.Ala213Thr, p.Tyr373Cys, and p.Arg335Cys) in four of seven affected patients with acrodysostosis. The combination of clinical results, endocrinological parameters and in silico mutation analysis gives evidence to suppose a pathogenic effect of each mutation. This assumption is supported by the de novo origin of these mutations. Apart from typical radiological abnormalities of the hand bones, elevated thyroid stimulating hormone and parathyroid hormone values as well as short stature are the most common findings. Less frequent features are characteristic facial dysmorphisms, sensorineural hearing loss and mild intellectual disability. These results lead to the conclusion that mutations of PKRAR1A are the major molecular cause for acrodysostosis with endocrinological abnormalities. In addition, in our cohort of 44 patients affected with brachydactyly type E (BDE) we detected only one sequence variant of PRKAR1A (p.Asp227Asn) with an unclear effect on protein function. Thus, we conclude that PRKAR1A mutations may play no major role in the pathogenesis of BDE.

Recurrent PRKAR1A mutation in acrodysostosis with hormone resistance.

The skeletal dysplasia characteristic of acrodysostosis resembles the Albright's hereditary osteodystrophy seen in patients with pseudohypoparathyroidism type 1a, but defects in the α-stimulatory subunit of the G-protein (GNAS), the cause of pseudohypoparathyroidism type 1a, are not present in patients with acrodysostosis. We report a germ-line mutation in the gene encoding PRKAR1A, the cyclic AMP (cAMP)-dependent regulatory subunit of protein kinase A, in three unrelated patients with acrodysostosis and resistance to multiple hormones. The mutated subunit impairs the protein kinase A response to stimulation by cAMP; this explains our patients' hormone resistance and the similarities of their skeletal abnormalities with those observed in patients with pseudohypoparathyroidism type 1a.

Mutation of Hairy-and-Enhancer-of-Split-7 in humans causes spondylocostal dysostosis.

Spondylocostal dysostosis (SCD) is an inherited disorder that is characterized by the presence of extensive hemivertebrae, truncal shortening and abnormally aligned ribs. It arises during embryonic development by a disruption of formation of somites (the precursor tissue of the vertebrae, ribs and associated tendons and muscles). Previously, three genes causing a subset of autosomal recessive forms of this disease have been identified: DLL3 (SCDO1: MIM 277300), MESP2 (SCDO2: MIM 608681) and LFNG (SCDO3: MIM609813). These genes are all important components of the Notch signaling pathway, which has multiple roles in development and disease. Here we have used autozygosity mapping to identify a mutation in a fourth Notch pathway gene, Hairy-and-Enhancer-of-Split-7 (HES7), in an autosomal recessive SCD family. HES7 encodes a bHLH-Orange domain transcriptional repressor protein that is both a direct target of the Notch signaling pathway, and part of a negative feedback mechanism required to attenuate Notch signaling. A missense mutation was identified in the DNA-binding domain of the HES7 protein. Functional analysis revealed that the mutant HES7 was not able to repress gene expression by DNA binding or protein heterodimerization. This is the first report of mutation in the human HES7 gene, and provides further evidence for the importance of the Notch signaling pathway in the correct patterning of the axial skeleton.

The skeletal dysplasias: clinical-molecular correlations.

The skeletal dysplasias or osteochondrodysplasias are a clinically and genetically heterogeneous group of disorders of bone and/or cartilage. They are characterized by abnormalities in pattering, linear growth, differentiation, and maintenance of the human skeleton. While they have been considered to be generalized disorders of endochondral and/or membranous ossification, the extent of their clinical and molecular heterogeneity is still being elucidated. In the 2006 revision of the International Nosology and Classification of Genetic Skeletal Disorders, 372 different conditions were listed in 37 groups defined by such molecular, biochemical, and/or radiographic criteria. The evaluation of patients with chondrodysplasias mandates a multidisciplinary approach involving clinical geneticists, radiologists, molecular biologists, and biochemical geneticists for diagnosis, and a host of surgical specialists for management of their many complications. Our International Skeletal Dysplasia Registry is a worldwide referral center for the skeletal dysplasias, and we have received cases from over 3000 physicians from 50 different countries and have been involved in the identification of the molecular defect in over 40 disorders involving over 25 different genes. Instructions on accessing the Registry, using the diagnostic services provided and contributing cases for collaborative research can be found at http://www.csmc.edu/skeletaldysplasia.

Abnormal vertebral segmentation and the notch signaling pathway in man.

Abnormal vertebral segmentation (AVS) in man is a relatively common congenital malformation but cannot be subjected to the scientific analysis that is applied in animal models. Nevertheless, some spectacular advances in the cell biology and molecular genetics of somitogenesis in animal models have proved to be directly relevant to human disease. Some advances in our understanding have come through DNA linkage analysis in families demonstrating a clustering of AVS cases, as well as adopting a candidate gene approach. Only rarely do AVS phenotypes follow clear Mendelian inheritance, but three genes-DLL3, MESP2, and LNFG-have now been identified for spondylocostal dysostosis (SCD). SCD is characterized by extensive hemivertebrae, trunkal shortening, and abnormally aligned ribs with points of fusion. In familial cases clearly following a Mendelian pattern, autosomal recessive inheritance is more common than autosomal dominant and the genes identified are functional within the Notch signaling pathway. Other genes within the pathway cause diverse phenotypes such as Alagille syndrome (AGS) and CADASIL, conditions that may have their origin in defective vasculogenesis. Here, we deal mainly with SCD and AGS, and present a new classification system for AVS phenotypes, for which, hitherto, the terminology has been inconsistent and confusing.

Fucosidosis with hypothyroidism: a case report.

Fucosidosis is a rare, autosomal recessive lysosomal storage disorder caused by a severe deficiency of alpha-L-fucosidase. Here we present a 27-month-old male who was referred to us for evaluation of developmental delay, which was first detected at age six months. His past medical history was also remarkable for recurrent pulmonary infections and myoclonic seiures. His family history revealed that he was the first living child from a consanguineous marriage. He had a younger sister who died at five months of age from pneumonia who had facial resemblance to the proband, developmental delay and a congenital heart defect. Physical examination revealed length: 81 cm (25-50p), weight: 10.2 kg (25-50p), and head circumference: 49 cm (50-75p). He had a coarse face, hepatomegaly and generalized spasticity. His initial laboratory examination revealed negative urine screening column chromatography for mucopolysaccharidosis. His X-ray findings were consistent with mild form of dysostosis multiplex. Based on clinical and laboratory features, fucosidosis was suspected. Fucosidase enzyme activity was zero. In addition to fucosidosis, thyroid function tests indicated primary hypothyroidism. This is, to the best of our knowledge, the fourth case of fucosidosis diagnosed in Turkey.

Novel mutations in DLL3, a somitogenesis gene encoding a ligand for the Notch signalling pathway, cause a consistent pattern of abnormal vertebral segmentation in spondylocostal dysostosis.

The spondylocostal dysostoses (SCD) are a group of disorders characterised by multiple vertebral segmentation defects and rib anomalies. SCD can either be sporadic or familial, and can be inherited in either autosomal dominant or recessive modes. We have previously shown that recessive forms of SCD can be caused by mutations in the delta-like 3 gene, DLL3. Here, we have sequenced DLL3 in a series of SCD cases and identified 12 mutations in a further 10 families. These include 10 novel mutations in exons 4-8, comprising nonsense, missense, frameshift, splicing, and in frame insertion mutations that are predicted to result in either the truncation of the mature protein in the extracellular domain, or affect highly conserved amino acid residues in the epidermal growth factor-like repeats of the protein. The affected cases represent diverse ethnic backgrounds and six come from traditionally consanguineous communities. In all affected subjects, the radiological phenotype is abnormal segmentation throughout the entire vertebral column with smooth outlines to the vertebral bodies in childhood, for which we suggest the term "pebble beach sign". This is a very consistent phenotype-genotype correlation and we suggest the designation SCD type 1 for the AR form caused by mutations in the DLL3 gene.

A case of pycnodysostosis with growth hormone deficiency.

Pycnodysostosis is a skeletal dysplasia characterized by short stature. Treatment of pycnodysostosis with growth hormone (GH) has not been reported so far. We describe a case of pycnodysostosis with growth hormone deficiency in addition to low mean insulin-like growth factor 1 (IGF-1) concentration. Complete GH deficiency was determined by two pharmacological provocative tests (insulin and L-dopa). A good height-velocity response was obtained after GH replacement treatment. Pycnodysostosis with GH deficiency and replacement therapy have not been reported previously, to the best of our knowledge.