A new polymorphic probe which defines the region of chromosome 19 containing the myotonic dystrophy locus

The region of human chromosome 19 which includes the myotonic dystrophy locus (DM) has recently been redefined by the tight linkage between it and the gene for muscle-specific creatine kinase (CKMM), which lies just proximal to DM. Utilizing human/hamster hybrid cell lines containing defined breakpoints within this region, we have assigned a number of new probes close to DM. Two of these probes, p134B and p134C, were isolated from a single cosmid clone (D19S51) and detect the same BglI RFLP; p134C detects an additional RFLP with the enzyme PstI. Analysis of these probes in the Centre d'Etude du Polymorphisme Humain families demonstrates tight linkage with a number of markers known to be proximal to DM. A two-point lod score of 6.34 at theta = .025 demonstrates the linkage of this probe to DM. Analysis of a DM individual previously shown to be recombinant for other tightly linked markers indicates that p134C is distal to DM. This result indicates that both the new probe and the existing group of proximal probes including CKMM and ERCC1 probably flank DM and define the genetic interval into which this mutation maps.     

Origin of new mutations in Duchenne muscular dystrophy

Summary Nine unrelated pedigrees in which Duchenne muscular dystrophy (DMD) was not present in more than one sibship were studied, using 6 DNA polymorphisms closely linked to the DMD gene. The reconstruction of grandparental haplotypes indicates the occurrence of at least three new mutations, two in grandpaternal chromosomes and one in a grandmaternal chromosome. Two additional (but less well documented) new mutations might have occurred respectively in a grandfather's and in a grandmother's chromosome, the latter being represented by a deletion mutation. The new mutations detected in this study therefore add to a total of either three or five out of nine apparently independent mutations present in pedigrees without recurrence of the disorder.     

Lens-derived Semaphorin3A regulates sensory innervation of the cornea

The cornea, one of the most highly innervated tissues of the body, is innervated by trigeminal sensory afferents. During development, axons are initially repelled at the corneal margin, resulting in the formation of a circumferential nerve ring. The nature and source of guidance molecules that regulate this process remain a mystery. Here, we show that the lens, which immediately underlies the cornea, repels trigeminal axons in vivo and in vitro. Lens ablation results in premature, disorganized corneal innervation and disruption of the nerve ring and ventral plexus. We show that Semaphorin3A (Sema3A) is expressed in the lens epithelium and its receptor Neuropilin-1 (Npn1) is expressed in the trigeminal ganglion during cornea development. Inhibition of Sema3A signaling abrogates axon repulsion by the lens and cornea in vitro and phenocopies lens removal in vivo. These results demonstrate that lens-derived Sema3A mediates initial repulsion of trigeminal sensory axons from the cornea and is necessary for the proper formation of the nerve ring and positioning of the ventral plexus in the choroid fissure.     

A viscoelastic anisotropic hyperelastic constitutive model of the human cornea

A constitutive model based on the continuum mechanics theory has been developed which represents interlamellar cohesion, regional variation of collagen fibril density, 3D anisotropy and both age-related viscoelastic and hyperelastic stiffening behaviour of the human cornea. Experimental data gathered from a number of previous studies on 48 ex vivo human cornea (inflation and shear tests) enabled calibration of the constitutive model by numerical analysis. Wide-angle X-ray scattering and electron microscopy provided measured data which quantify microstructural arrangements associated with stiffness. The present study measures stiffness parallel to the lamellae of the cornea which approximately doubles with an increase in strain rate from 0.5 to 5%/min, while the underlying stromal matrix provides a stiffness 2–3 orders of magnitude lower than the lamellae. The model has been simultaneously calibrated to within 3% error across three age groups ranging from 50 to 95 years and three strain rates across the two loading scenarios. Age and strain-rate-dependent material coefficients allow numerical simulation under varying loading scenarios for an individual patient with material stiffness approximated by their age. This present study addresses a significant gap in numerical representation of the cornea and has great potential in daily clinical practice for the planning and optimisation of corrective procedures and in preclinical optimisation of diagnostic procedures.     

Muscular dystrophy of mink: a new animal model.

Muscular dystrophies comprise an important group of inherited disorders of man. Although the disease has been studied extensively, little is known about the underlying primary pathomechanisms. Consequently, treatment of patients is difficult and prognosis is poor. An animal model of muscular dystrophy is a useful research tool for approaching the basic problems of pathogenesis in muscle diseases. An inherited progressive muscular dystrophy of mink which resembles the amyotonic forms of human muscular dystrophy is currently under study. Clinically, the earliest sign is progressive muscular weakness and atrophy. Muscle enzyme activities in serum are usually elevated to pathologic levels. Urinary creatine/creatinine ratio is elevated. Pathologic changes are limited to skeletal muscle and are typical of those seen in amyotonic forms of human muscular dystrophy. These changes include variation in diameter size of muscle fibers, centralized nuclei, floccular and hyaline degeneration of scattered muscle fibers, increase in connective tissue in endomysial and perimysial areas, and regenerative attempts. Both type I and type II muscle fibers are involved in the disease process. Genetic studies indicate an autosomal recessive mode of inheritance. Although the primary defect in muscular dystrophy is traditionally thought to reside in skeletal muscle, recent studies have produced theories of primary involvement of other tissues and organ systems. These theories are presented and relationships to the traditional theory are discussed.     

A study of piezoelectric and mechanical anisotropies of the human cornea

The piezoelectric and dynamic mechanical properties of human cornea have been investigated as a function of drying time. As expected, the piezoelectric coefficient, d(31), and the Young's modulus, Y, were found to be extremely sensitive to water content. d(31) decreased with dehydration of the corneal tissue and Y increased with dehydration. While these results are significant, the discovery of the unprecedented mechanical and electromechanical anisotropy exhibited by the cornea are the major findings of this study and indicate that the collagen fibrils comprising the cornea are highly oriented. The piezoelectric responses of corneas observed in this study are: diagonally cut samples starting at an average piezoelectric coefficient value of 2250 pC/N, followed by the vertically cut samples, with an average starting value of about 600 pC/N and finally the horizontally cut samples with an average starting value of about 200 pC/N.     

Innervation of the rabbit cornea. A histochemical and electron-microscopic study

The innervation of the rabbit cornea was investigated histochemically and electron-microscopically with special reference to the autonomic nerves. Both formaldehyde- and glyoxylic-acid-induced fluorescence methods revealed adrenergic nerves in the stroma; a few fibres were also observed between the basal epithelial cells near the limbus. Acetylcholinesterase- (AChE-) positive nerves were found both in the stroma and in the epithelium, whereas nonspecific cholinesterase (NsChE) activity appeared only in the stromal nerves. Under the electron microscope, both AChE and NsChE activities were observed to be located in the axon membranes. A weak NsChE reaction also appeared in the Schwann cells. When the specimens fixed with KMnO4 were examined under the electron microscope, most nerve fibres did not contain any special axoplasmic structures, although several axons contained mitochondria. Moreover, two vesicle-containing axon types were found in the stromal nerves; axons with small granular vesicles and axons containing small agranular vesicles. In the epithelium, two types of fibres were observed; one type containing only mitochondria while the other showed both agranular vesicles and mitochondria.     

A microstructurally-based finite element model of the incised human cornea.

A mechanical model of the human cornea is proposed and employed in a finite element formulation for simulating the effects of surgical procedures, such as radial keratotomy, on the cornea. The model assumes that the structural behavior of the cornea is governed by the properties of the stroma. Arguments based on the microstructural organization and properties of the stroma lead to the conclusion that the human cornea exhibits flexural and shear rigidities which are negligible compared to its membrane rigidity. Accordingly, it is proposed that to a first approximation, the structural behavior of the cornea is that of a thick membrane shell. The tensile forces in the cornea are resisted by very fine collagen fibrils embedded in the ground substance of the stromal lamellae. When the collagen fibrils are cut, as in radial keratotomy, it is argued that they become relaxed since there is negligible transfer of load between adjacent fibrils due to the low shear modulus of the ground substance. The forces in the cornea are then resisted only by the remaining uncut fibrils. The cutting of fibrils induces an anisotropy and inhomogeneity in the membrane rigidity. By assuming a uniform angular distribution of stromal lamellae through the corneal thickness, geometric arguments lead to a quantitative representation for the anisotropy and inhomogeneity. All material behavior is assumed to be in the linear elastic regime and with no time-dependency. The resulting constitutive model for the incised cornea has been employed in a geometrically non-linear finite element membrane shell formulation for small strains with moderate rotations. A number of numerical examples are presented to illustrate the effectiveness of the proposed constitutive model and finite element formulation. The dependence of the outcome of radial keratotomy, measured in terms of the immediate postoperative shift in corneal power, on a number of important factors is investigated. These factors include the value of the elastic moduli of the stromal lamellae (dependent on the patient's age), the incision depth, the optic zone size, the number of incisions and their positions, and the intraocular pressure. Results have also been compared with expected surgical corrections predicted by three expert surgeons and show an excellent correspondence.     

Differential immunogold localisation of sulphated and unsulphated keratan sulphate proteoglycans in normal and macular dystrophy cornea using sulphation motif-specific antibodies

Keratan sulphate (KS) proteoglycans (PGs) are key molecules in the corneal stroma for tissue organisation and transparency. Macular corneal dystrophy (MCD) is a rare, autosomal recessive disease characterised by disturbances in KS expression. MCD is caused by mutations in CHST6, a gene encoding the enzyme responsible for KS sulphation. Sulphated KS is absent in type I disease causing corneal opacity and loss of vision. Genetic studies have highlighted the mutational heterogeneity in MCD, but supportive immunohistochemical studies on corneal KS have previously been limited by the availability of antibodies mostly reactive only with highly sulphated KS epitopes. In this study, we employed four antibodies against specific KS sulphation patterns, including one against unsulphated KS, to investigate their reactivity in a case of MCD compared with normal cornea using high-resolution immunogold electron microscopy. Mutation analysis indicated type I MCD with deletion of the entire open reading frame of CHST6. Contrast enhanced fixation revealed larger PG structures in MCD than normal. Unlike normal cornea, MCD cornea showed positive labelling with antibody to unsulphated KSPG, but was negative with antibodies to sulphated KSPG. These antibodies will thus facilitate high-resolution investigations of phenotypic heterogeneity in support of genetic studies in this disease.