Laing distal myopathy with a novel mutation in exon 34 of the MYH7 gene.

We investigated a four-generation family of German ancestry with distal myopathy. Four individuals in two generations were affected. Foot and toe extensor paresis progressing very slowly over decades was the core neurological sign, reflected by fatty infiltration of the lower leg extensor muscles on muscle MRI. Additionally, finger extensor paresis was present in two patients and quadriceps muscle paresis in one. Distal sensory signs had initially given rise to the diagnosis of axonal Charcot-Marie-Tooth (CMT) disease. Two patients had extended verrucae of their foot sole, which may or may not be part of the disease spectrum. All four patients had a novel c.4645G > C mutation in exon 34 of the MYH7 gene that was not present in three clinically unaffected family members. Muscle biopsy of one patient revealed a myopathic pattern associated with type 1 muscle fibre atrophy and core-like lesions in many muscle fibres consistent with a myosin-related myopathy. We conclude that some of the typical clinical signs such as extensor weakness of the big toe and the little finger may only develop in the further course of the disease.

Novel EGR2 mutation R359Q is associated with CMT type 1 and progressive scoliosis.

Mutations in the early growth response 2 gene (EGR2) cause demyelinating neuropathies differing in severity and age of onset. We tested 46 unrelated Czech patients with dominant or sporadic demyelinating CMT neuropathy for mutations in the EGR2 gene. One novel de-novo mutation (Arg359Gln, R359Q) was identified in heterozygous state in a patient with a typical CMT1 phenotype, progressive moderate thoracolumbar scoliosis and without clinical signs of cranial nerve dysfunction. This patient is presently less affected compared to previously described Dejerine-Sottas neuropathy (DSN) patients carrying another substitution at codon 359 (Arg359Trp, R359W). This report shows that EGR2 mutations are rare in Czech patients with demyelinating type of CMT and suggests that different substitutions at codon 359 of EGR2 can cause significantly different phenotypes.

Hearing loss as the first feature of late-onset axonal CMT disease due to a novel P0 mutation.

A Czech family with three individuals carrying a novel mutation, 290 A-->T (Glu97Val), in the myelin protein zero gene (P0) is reported. The two eldest carriers developed progressive sensorineural hearing loss and abnormal pupillary reaction at age 18. These preceded the onset of the classic signs of Charcot-Marie-Tooth disease (CMT) by more than a decade. Sural nerve biopsy and nerve conduction studies were compatible with the axonal type of CMT. The authors show that progressive hearing loss can be the first symptom in P0 mutation carriers.

Early recognition of hereditary motor and sensory neuropathy type 1 can avoid life-threatening vincristine neurotoxicity.

Hereditary motor and sensory neuropathy type 1 (HMSN-1) is an autosomal dominant disorder, which is usually not associated with neoplastic diseases. The disease predisposes to severe vincristine neurotoxicity. We report a 31-year-old women with recurrent Hodgkin's lymphoma and unrecognized HMSN-1 who developed severe motor neuropathy 3 weeks after the first cycle of treatment including 2 mg of vincristine. HMSN is diagnosed in most cases retrospectively, usually suggested by the observation of foot abnormalities or family history. Recognizing early signs of HMSN, such as areflexia and pes cavus deformity, can prevent severe neurotoxicity of polychemotherapy by avoiding vincristine.

Primary congenital glaucoma: three case reports on novel mutations and combinations of mutations in the GLC3A (CYP1B1) gene.

PURPOSE: To describe three patients with congenital glaucoma homozygous and compound heterozygous for different mutations and benign sequence variants in the cytochrome P 450 1B1 (CYP1B1) gene. METHODS: All patients were examined by slit-lamp biomicroscopy, gonioscopy, measurement of the cornea and optic disc, ultrasound biometry, and automated static threshold perimetry when possible. Direct sequence analysis was performed on DNA extracted from peripheral blood from the patients and their parents. RESULTS: For patient 1, a newborn boy with buphthalmos and an opaque cornea, a novel homozygous C/T transition in codon 355 (CGA>TGA) led to a predicted nonsense codon Arg355X truncating the protein by 188 amino acids. For patient 2, a 24-year-old man, a compound heterozygous mutation 1410-1422del/1546-1555dup was found. For patient 3, a 34-year-old man, two novel heterozygous missense mutations resulting in an Ala443Gly and a Glu229Lys amino acid exchange and five benign sequence variants were found. CONCLUSION: Our results confirm the crucial role of CYP1B1 mutations for congenital glaucoma.

Transcriptional startpoints and methylation patterns in the PMP22 promoters of peripheral nerve, leukocytes and tumor cell lines.

PMP22 is a dosage sensitive gene responsible for Charcot-Marie-Tooth type 1A (CMT1A) neuropathy and hereditary neuropathy with liability to pressure palsies (HNPP). PMP22 is expressed in myelinating Schwann cells in the peripheral nerve, but also in a variety of other tissues. PMP22 expression is regulated by alternatively used promoters, the relative expression of the different PMP22 transcripts is tissue-specific. At first we analysed the transcriptional startpoints of the different PMP22 transcripts. Transcript 1A starts from a distinct nucleotide, whereas transcript 1B and the here described transcript 1C revealed multiple transcriptional startpoints in sciatic nerve as well as in the osteosarcoma and glioblastoma cell lines, RH30 and SF763. Using promoter specific primers we identified transcripts from each of the three promoters in sciatic nerve and RH30, whereas transcript 1B is absent in SF763. Leukocytes do not express PMP22 at all. Additionally, we determined the methylation pattern of CpG islands present in the PMP22 promoters 1B and 1C for leukocytes, sciatic nerve, SF763 and RH30, the latter carrying multiple copies of the PMP22 gene. We observed that there was no methylation in promoter 1B and 1C in sciatic nerve and leukocytes. However, hypermethylation of promoter 1B was discovered in SF763 and indicates a silencing effect. In RH30 most copies of promoters 1B and 1C were methylated but the few remaining hypomethylated copies were sufficient for strong expression of PMP22. These results indicate that the transcriptional control in tumor cell lines is probably different from leukocytes and sciatic nerve.

Expression analysis of the PMP22 gene in glioma and osteogenic sarcoma cell lines.

Previously we reported the amplification of the peripheral myelin protein 22 (PMP22) gene in cell lines of human osteogenic and glioma tumors. PMP22 normally is expressed at high levels in Schwann cells of the peripheral nervous system and is suggested to function as a structural protein of the myelin sheath. One of the most common inherited peripheral neuropathies, Charcot-Marie-Tooth Type 1A (CMT1A), is associated with a duplication of a 1.5-Mb DNA region on chromosome 17p11.2 - p12 containing PMP22. On the other hand, PMP22 is identical to gas3, whose expression is induced in growth-arresting NIH3T3-fibroblasts and is thought to play a role in cell proliferation. The precise role of gas3/PMP22 remains to be determined. Here we show that in the tumor cell lines RH30 and SF763 the amplified region including PMP22 comprises the whole 1.5-Mb CMT1A region. We could prove expression of PMP22 by reverse transcriptase-polymerase chain reaction (RT-PCR) and discovered an unusual PMP22 transcript in these tumor cell lines. Western blot analyses resulted in detection of a 22-kDa protein by the PMP22-specific antibody 558/2 and in exclusion of myelin protein zero (MPZ) expression in these cell lines.

Molecular diagnosis of PMP22-associated neuropathies using fluorescence in situ hybridization (FISH) on archival peripheral nerve tissue preparations.

Charcot-Marie-Tooth (CMT) syndrome type 1 and tomaculous neuropathy, also called hereditary neuropathy with liability to pressure palsies (HNPP), represent two groups of neurological disorders with different subtypes, which can be distinguished at the molecular level. It is known that a 1.5-mb region on chromosome 17p11.2-12, which includes the gene for the peripheral myelin protein 22 kDa (PMP22), is duplicated in more than 95% of patients with CMT type 1A (CMT1A; gene dosage 3) and is deleted in about 90% of subjects suffering from HNPP (gene dosage 1). This duplication/deletion can be detected reliably by interphase-two-color fluorescence in situ hybridization (FISH). We report here a technique for extraction of nuclei from paraffin-embedded and cryofixed sural nerve biopsies for precise molecular diagnosis, employing interphase-two-color FISH in clinically diagnosed CMT1 or HNPP patients. Following this technique we were able to identify six CMT1A duplications in 13 clinically diagnosed CMT1 cases and five HNPP deletions in 6 clinically diagnosed HNPP cases; 8 control persons were included in this study. This is the first report on the use of FISH in the detection of 17p11.2-12 duplication and deletion in archival biopsy material.

Clinical and morphological phenotype of HMSN 1A mosaicism.

Clinical, neurophysiological and morphological studies on a patient with mosaicism of the 17p11.2 duplication were performed in detail for the first time. Since duplication occurs during paternal meiosis, a somatic reversion is suggested, leading to mosaicism. The proportion of nuclei with duplication varied markedly between 49% in blood cells and 74% in tissue from the sural nerve. Clinically, mild symptoms of a motor and sensory neuropathy were present. However, neurophysiological changes and findings in the sural nerve biopsy were consistent with a typical hereditary motor and sensory neuropathy type 1 (HMSN 1). Differing clinical findings in patients with mosaicism of the 17p11.2 duplication may be explained by a varying degree and/or time of reversion.

The peripheral myelin protein 22 kDa (PMP22) gene is amplified in cell lines derived from glioma and osteogenic sarcoma.

Two glioma (SF188, SF763) and two osteogenic sarcoma cell lines (RH30, SA1) were examined for the presence of an amplification of the PMP22 gene by means of fluorescence in situ hybridization (FISH). In one cell line of both cell types, we found about 10 copies of the PMP22 (peripheral myelin protein 22 kDa) gene, located on different marker chromosomes within homogeneously staining regions. Surrounding chromosome 17p material was found to be coamplified, but coamplification of TP53 (17p13) and erbB2/Her2 (17q11.12) were excluded by FISH for both cell lines, SF763 and RH30. This is the first report of a PMP22 amplification in cell lines derived from human tumors.

Differential proteolytic sensitivity of yeast fatty acid synthetase subunits alpha and beta contributing to a balanced ratio of both fatty acid synthetase components.

The Saccharomyces cerevisiae genes FAS1 and FAS2 encoding the beta and alpha subunit of yeast fatty acid synthetase (FAS), respectively, were individually deleted by one-step gene disruption. Northern blot analysis of RNA from the resulting fas null allele mutants indicated that deletion of FAS2 did not influence the transcription of FAS1, while FAS2 transcription was significantly reduced in the delta fas1 strain. These data suggest an activating role of subunit beta on FAS2 gene expression or, alternatively, a repression of FAS2 by an excess of its own gene product. Compared to the intact alpha 6 beta 6 complex, the individual FAS subunits synthesized in the delta fas1 or delta fas2 strains exhibit a considerably increased sensitivity towards the proteinases present in the yeast cell homogenate. Using yeast mutants specifically defective in the vacuolar proteinases yscA (PRA1/ PEP4 gene product) and/or yscB (PRB1 gene product), it was shown that in vitro, subunit alpha is efficiently degraded by proteinase yscA while for degradation of subunit beta, the combined action of proteinases yscA and yscB is necessary. In vivo, besides the vacuolar proteinases, an additional proteolytic activity specifically affecting free FAS subunit alpha becomes increasingly apparent in cells entering the stationary growth phase. In contrast, under similar conditions uncomplexed FAS subunit beta is stable in strains lacking the vacuolar proteinases yscA and yscB. The reduced FAS subunit levels, at the stationary phase, were independent of the corresponding FAS transcript concentrations. Thus, differential degradation pathways are obviously removing an excess of either FAS subunit, at least under starvation conditions. A combination of both regulation of FAS gene expression and proteolysis of free FAS polypeptides may therefore explain the equimolar amounts of both FAS subunits observed in yeast wild-type cells.