Vasiliki Zouvelou

Deletion of BMP7 Affects the Development of Bones, Teeth, and Other Ectodermal Appendages of the Orofacial Complex

Sequential and reciprocal epithelial-mesenchymal interactions govern the development of most tissues and organs of the craniofacial region. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta family of secreted signaling molecules that have long been implied to have a significant contribution in this process. However, evidence for such a role during craniofacial development is largely missing. Using a lacZ reporter mouse we mapped the spatiotemporal expression of BMP7 in the developing craniofacial region. The observed pattern suggested a potential involvement of BMP7 in epithelial-mesenchymal interactions and thus a direct role for this molecule in the development of ectodermal appendages (teeth, hair follicle, lachrymal and sweat glands, taste buds) and, furthermore, palatal formation. To correlate the expression to function we analyzed germline deleted conditional BMP7-deficient embryos for malformations. We found developmental defects in many craniofacial structures such as teeth, eyes, whiskers, hair follicles, salivary glands, and palate. These findings place BMP7 as a central mediator of epithelial-mesenchymal interactions that are necessary for the correct development of structures belonging to the orofacial complex.

LRP4 antibodies in serum and CSF from amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) and myasthenia gravis (MG) are caused, respectively, by motor neuron degeneration and neuromuscular junction (NMJ) dysfunction. The membrane protein LRP4 is crucial in the development and function of motor neurons and NMJs and LRP4 autoantibodies have been recently detected in some MG patients. Because of the critical role in motor neuron function we searched for LRP4 antibodies in ALS patients.

Generation and functional characterization of mice with a conditional BMP7 allele

Bone Morphogenetic Proteins (BMPs) play multiple and important roles in embryonic development as well as in homeostasis and tissue repair in the adult. Bmp7 has been implicated in developmental disorders and in a variety of diseases, but functional studies to elucidate its role so far have been hampered, since mice deficient in BMP7 die around or just after birth. To facilitate such studies, we generated mice in which the Bmp7 gene has been rendered conditional-null by flanking its first coding exon with loxP sites. To this end, we adapted the two-loxP site strategy to Bacterial Homologous Recombination to create a Bacterial Artificial Chromosome-based vector for direct targeting in mouse embryonic stem cells. Functional analysis showed that in vivo, the conditional-null Bmp7(flx/flx) mice are phenotypically wild type, whereas post Cre-mediated recombination, the resulting Bmp7(delta/delta) mice are phenotypically null. Thus, this study validates the usefulness of the Bmp7(flx/flx) mouse which in turn should empower in vivo studies aimed at elucidating the roles of Bmp7 in postnatal development, homeostasis and disease.

Double seronegative myasthenia gravis with anti-LRP 4 antibodies

About 10% of patients with generalized myasthenia gravis do not have detectable antibodies to acetylcholine receptor or muscle specific kinase (double seronegative myasthenia). The presence of anti-low density lipoprotein receptor-related protein 4 antibodies (LRP4 Abs) has recently been reported in variable proportion of double seronegative cases. We report the presenting characteristics of two double seronegative myasthenic patients from Greece with anti-LRP4 antibodies shortly after disease onset. The first patient, a 52-year-old male, presented with a one month history of isolated neck extensor weakness; the second patient is a 52-year-old female with three months history of ocular-bulbar-cervical myasthenic weakness. Both patients presented with mild severity and responded promptly and adequately to pyridostigmine. In the female patient thymic residual tissue was detected on CT of the mediastinum. She underwent thymectomy, and histological examination revealed follicular hyperplasia. This is the first clinical report of the presenting features of newly diagnosed myasthenia with anti-LRP4 antibodies. The clinical and therapeutic implications of the anti-LRP4 antibody positivity remain to be clarified.

MRI Evidence of Early Muscle Atrophy in MuSK Positive Myasthenia Gravis

Muscle atrophy, particularly of facial and bulbar muscles, seems to be a relatively common long‐term consequence of musclespecific tyrosine kinase‐myasthenia gravis (MuSK‐MG), perhaps reflecting the chronic state of disease or long‐term therapy with corticosteroids. We performed magnetic resonance imaging (MRI) to assess muscle wasting in the facial and bulbar muscles in two female MuSK‐MG patients, with short duration of symptoms prior to diagnosis and prior to commencement of steroid therapy. The study revealed marked atrophy of temporalis, masseters, and lingual muscles with fatty replacement. MRI evidence of early muscle atrophy in MuSK‐MG may indicate that MuSK antibodies per se may predispose to muscle atrophy.

The etiology of cleft palate formation in BMP7-deficient mice

Palatogenesis is a complex process implying growth, elevation and fusion of the two lateral palatal shelves during embryogenesis. This process is tightly controlled by genetic and mechanistic cues that also coordinate the growth of other orofacial structures. Failure at any of these steps can result in cleft palate, which is a frequent craniofacial malformation in humans. To understand the etiology of cleft palate linked to the BMP signaling pathway, we studied palatogenesis in Bmp7-deficient mouse embryos. Bmp7 expression was found in several orofacial structures including the edges of the palatal shelves prior and during their fusion. Bmp7 deletion resulted in a general alteration of oral cavity morphology, unpaired palatal shelf elevation, delayed shelf approximation, and subsequent lack of fusion. Cell proliferation and expression of specific genes involved in palatogenesis were not altered in Bmp7-deficient embryos. Conditional ablation of Bmp7 with Keratin14-Cre or Wnt1-Cre revealed that neither epithelial nor neural crest-specific loss of Bmp7 alone could recapitulate the cleft palate phenotype. Palatal shelves from mutant embryos were able to fuse when cultured in vitro as isolated shelves in proximity, but not when cultured as whole upper jaw explants. Thus, deformations in the oral cavity of Bmp7-deficient embryos such as the shorter and wider mandible were not solely responsible for cleft palate formation. These findings indicate a requirement for Bmp7 for the coordination of both developmental and mechanistic aspects of palatogenesis.

Double‐seropositive myasthenia gravis

Functional status and muscle strength in people with Duchenne Muscular Dystrophy living in the community. J Rehabil Med 2004; 36:124–129. 4. Caress JB, Kothari MJ, Bauer SB, Shefner JM. Urinary dysfunction in Duchenne muscular dystrophy. Muscle Nerve 1996;19:819–822. 5. MacEwen GD, Bunnell W, Sriram K. Acute neurologic complications in the treatment of scoliosis. J Bone Joint Surg Am 1975;57:404–408. 6. Taylor PJ, Betts GA, Maroulis S. Dystrophin gene mutation location and the risk of cognitive impairment in Duchenne muscular dystrophy. PLoS One 2010;5:e8803. 7. Inoue M, Mori K, Hayabuchi Y, Tatara K, Kagami S. Autonomic function in patients with Duchenne muscular dystrophy. Pediatr Int 2009;51:33–40. 8. Hashim H, Abrams P. Overactive bladder: an update. Curr Opin Urol 2007;17:231–236.

AchR-positive myasthenia gravis with MRI evidence of early muscle atrophy.

Muscle atrophy, when it occurs in myasthenia gravis (MG), is usually associated with long-standing disease or with chronic corticosteroid treatment. Early muscle atrophy in a steroid-naive patient has been reported previously only in muscle-specific tyrosine kinase (MuSK)-MG. We report a 63-year-old male patient with acetylcholine receptor (AchR)-positive MG with a short duration of disease, no steroid treatment and MRI evidence of muscle atrophy.

MuSK-Ab positive myasthenia: Not always grave

Antibodies (Abs) to muscle-specific tyrosine kinase (MuSK) are detected in approximately 40% of generalized acetylcholine receptor antibody-negative myasthenia gravis (MG). Anti-MuSK Abs are nearly always associated with generalized symptoms, with prevalent involvement of craniobulbar, cervical and respiratory muscles and with a striking preponderance in women. The typical course of MuSK-MG is acute onset, rapid progression, brittle course in the first years, early respiratory crises and unprovoked relapses in spite of high-dose immunosuppression. Patients often require long-term management with multiple immunosuppressive (IS) agents and many of them remain dependent on IS treatment. The majority of anti-MuSK Abs are of the non-complement-binding IgG4 subclass. We report the case of a Greek female MuSK-MG patient with typical phenotype but clearly atypical clinical course during 12 years of follow-up. The patient received only corticosteroid treatment for one year and showed mild and stable MG symptoms under no treatment for the rest of the observation period, except for postpartum mild and short-lived exacerbation. In mildly symptomatic stable state, anti-MuSK Abs were of the IgG4 subclass and no other immunological peculiarity was detected.

Diabetes mellitus associated with glycogen storage disease type III.

DIABETES MELLITUS ASSOCIATED WITH GLYCOGEN STORAGE DISEASE TYPE III A 35-year-old woman developed exercise intolerance followed by progressive generalized weakness, which affected proximal and distal muscles in all limbs. Her condition deteriorated significantly until the age of 37 years, when she became unable to sit or stand from the supine or seated positions, respectively, nor to stand and walk on toes or heels. At this point the diagnosis of diabetes mellitus (DM) was also made. Although oral antidiabetic medications failed, initiation of insulin treatment achieved sufficient antidiabetic control without hypoglycemic complications. Serum creatine kinase, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase were mildly elevated on repeated measurements. Electromyography revealed small, polyphasic, early recruiting motor unit potentials and runs of waxing and waning positive waves and fibrillation potentials. Conduction studies were normal. There were no sonographic signs of hepatomegaly or cardiac involvement. Muscle biopsy demonstrated a myopathy with vacuoles that contained periodic acid–Schiff (PAS)stained material indicative of glycogenosis (Fig. 1). Histochemical staining for phosphorylase and phosphofructokinase was normal. The activity of -glucosidase in skin fibroblasts was normal, but glycogen concentration in erythrocytes was elevated. No glycogen debrancher enzyme activity was detected in leukocytes, but phosphorylase activities were within the normal range. The debrancher activity was determined by the liberation of glucose from a phosphorylase limit dextrin (Dr. M. Jackson, Institute of Child Health, London). The constellation of characteristic histological findings and enzyme activities established the diagnosis of glycogen storage disease type III (GSD-III). Although GSD-III is characterized by liver and/or muscle involvement, myopathy is not always prominent in infants and children. It might first become clinically evident in adult life. Clinical features range from exercise intolerance with hypotonia to severe generalized weakness with atrophy.1,3,4 Fasting hypoglycemia is a well-known complication that tends to resolve spontaneously during puberty.1,3 The glucose tolerance test produces a diabetic response in most patients.2,3 Hyperglycemia has been reported rarely, and clinically manifest DM is considered to be very uncommon. There are only two cases of GSD-III and DM that first presented as muscle weakness in adulthood.6,7 In our case, late-onset myopathy was the only clinical manifestation of the disease. Clinical deterioration occurred during the first 2 years, when DM was also diagnosed. Treatment with insulin was effective and, over a follow-up period of 6 years, no further progression was documented. In a previous report DM occurred on a background of preexisting liver dysfunction.6 A causal relationship was assumed, because DM is frequently seen among patients with liver cirrhosis, which has also been described in patients with GSD-III.5 In the present case, and in the absence of hepatic dysfunction, it seems rather difficult to explain the development of DM. Taking into account that GSD-III patients have a typical diabetic response to the glucose tolerance test, DM as a late clinical feature in some cases of GSD-III cannot be ruled out. Nevertheless, it seems reasonable to consider DM to be a possible, however rare, feature of GSD-III, and we recommend regular evaluation of blood glucose levels during follow-up.