GENETIC ANALYSIS OF A COHORT OF DUTCH PATIENTS SUSPECTED FOR HMSN
Baas F.(1), Bienfait H.(1), Hoogendijk J.E.(2), Vreijling J.(1), Gabreëls-Festen A.(3), de Visser M.(1) (1)Department of Neurology Academic Medical Center, Amsterdam, (2)University of Utrecht, (3)University of Nijmegen, The Netherlands
Over the last three years we have collected a series of more than 1000 DNA samples from patients suspected for hereditary peripheral neuropathies. For all patients duplication of the PMP22 gene was analyzed by Southern blotting followed by hybridization with a PMP22 an VAW409 probe. Approximately 30% of the patients show a duplication of PMP22. Patients without a duplication and with clear evidence for a demyelinating neuropathy were analyzed further. SSCP was used as a prescreen for PMP22 and PO. In PMP22 five mutations were identified. The majority of patients showed mutations in the PO gene. Nine amino-acid substitutions at position 5, 30, 69, 81, 82, 91, 98, 107 and 113 were found. One patient showed two mutations, a His81Tyr and Val113Phe. Analysis of the CX32 gene in patients not showing mutations for PMP22 or PO resulted in the identification of 9 different amino-acid substitutions and two early terminations. In Summary: at least 25 different mutations were identified HMSN patients by SSCP and sequence analysis of PMP22, PO and CX32. In our group the majority of the mutations occurred in CX32 an PO and therefore these genes should be analyzed first.
AXONAL FORM OF CMT NEUROPATHY IN MORROCAN CONSANGUINEOUS FAMILIES: CLINICAL AND GENETIC HETEROGENEITY
Birouk N.(1), Bouhouche A.(1), Maisonobe T.(2), Belaïdi H.(1), Benomar A.(1), Bouslam N.(1), Kably B.(1), Mellah S.(1), Ouazzani R.(1), Le Guern E.(2), Chkili T.(1) (1)Hôpital des Spécialités, Rabat, Morocco, (2)Hôpital de la Salpêtrière, Paris, France.
The axonal form of autosomal recessive CMT disease is rare. Up to date, the genetic defect(s) of this subtype have yet to be determined. The phenotype of Charcot-Marie-Tooth neuropathy was analyzed in 11 patients belonging to 2 families of Morrocan ancestry. The parents of each affected individual were consanguineous and had normal clinical and electrophysiological examination. The clinical severity in terms of age at onset, functional disability and the existence of skeletal deformities were widely variable between families. Electrophysiological study showed characteristics of axonal form of CMT neuropathy (slightly reduced motor nerve conduction velocity, abnormal sensory nerve action potentials). Pathological study showed axonal neuropathy with the absence of any abnormal myelin proliferation. Molecular analysis consisted of exclusion of the known loci responsible for demyelinating autosomal recessive CMT and the loci responsible for axonal autosomal dominant CMT. Genome wide search using homozygosity mapping allowed to demonstrate linkage of family 1 to a new locus. This locus was excluded in family 2. Axonal form of autosomal recessive CMT disease is phenotypically heterogeneous. The corresponding genetic heterogeneity was demonstrated in two families.
UNUSUAL ELECTROPHYSIOLOGICAL FINDINGS IN A FAMILY WITH CMTX
Gutierrez A.(1), England J.D.(1), Ferer S.S.(1), Sumner A.J.(1), Garcia C.A.(1), Warner L.E.(2), Lupski J.R.(2) (1)Louisiana State University School of Medicine, New Orleans, LA USA., (2)Baylor University, Houston, TX USA.
To describe unusual electrophysiological characteristics found in a family with X-linked dominant Charcot-Marie-Tooth disease (CMTX) confirmed by DNA analysis in the family. CMTX is a hereditary neuropathy whose electrophysiology is uncertain as to whether it is a "demyelinative" or "axonal loss" polyneuropathy. We identified a three generation family with CMTX associated with a mutation in the gene coding for connexin-32. These individuals were studied by clinical neurological exam, genotyping, nerve conduction studies (NCSs) and a sural nerve biopsy in the proband. The mutation identified in our family was C105 Arg-->Trp. All affected males and obligate female carriers exhibited electrophysiological features characteristic of demyelination; however striking heterogeneity of NCSs was seen. In individuals, differential slowing of conduction velocities (CV) and temporal dispersion occurred within and between nerves. A sural nerve biopsy in the proband showed loss of large myelinated fibers and onion bulb formation. CMTX is a heterogeneous and distinctly nonuniform "demyelinating" polyneuropathy, the severity of which varies with sex and age. Such electrophysiological variability is unique among hereditary neuropathies and suggests that the connexin-32 mutation has an uneven effect upon peripheral myelin.
DISTRIBUTION OF PMP22 IN DIFFERENT CMT1A AND HNPP GENOTYPES
Hanemann C.O., D'Urso D., Gabreëls-Festen A.(1), Müller H.W. Dep. Neurology, Heinrich-Heine-Univ., Duesseldorf, Germany, 1Inst. Neurology, Univ. Hosp., Nijmegen, Netherlands
The hereditary demyelinating neuropathy Charcot-Marie-Tooth type 1A is a common inherited neurological diseases. CMT1A is caused by duplication of the gene for the peripheral myelin protein PMP22 or by point mutations in the PMP22 gene. CMT1A patients with point mutations have different pathology compared with patients with duplication. Furthermore PMP22 point mutations can lead to an HNPP phenotype. We analyzed the distribution of PMP22 in different sural nerve biopsies from CMT1A and HNPP patients with well defined point mutations using confocal laser scanning microscopy. Furthermore patients were also analyzed morphometrically to review that disease stages were comparable. We were able to use nerve biopsy from a patient with Trembler J mutation and a patient with a more C-terminal missense mutation i.e. Glyl07Val in the third transmembrane domain. From this patient sural nerve and a L5 root were available. In addition a frameshift mutation at Gly94 leading to an HNPP phenotype was analyzed. Point mutations in PMP22 leading to a CMT1A phenotype were compared with duplication of the PMP22 gene. The point mutation leading to HNPP phenotype were compared with PMP22 deletion. We show differential localization of PMP22 in human sural nerve biopsies depending on the underlying genetic defect. Thus although perturbed myelin protein synthesis emerges as a common phenomenon in hereditary neuropathies pathogenesis is most likely variable in the different genotypes.
HEREDITARY MOTOR AND SENSORY NEUROPATHY TYPE IIC (HMSN IIC) IS GENETICALLY DISTINCT FROM HMSN IIA, IIB and IID
Nagamatsu M., Jenkins R.B., Schaid D.J., Klein D.M., Dyck P.J. Mayo Clinic, Rochester, MN 55905 USA
To this time linkage analysis studies have shown that there are three genetic varieties of hereditary motor and sensory neuropathy type II (HMSN II; also called Charcot-Marie-Tooth disease type 2); HMSN IIA (linked to lp35-p36), IIB (3ql3-q22), and IID (7pl4). HMSN IIC is characterized by diaphragmatic and vocal cord paresis; its gene locus has not been mapped. We tested whether the autosomal dominant HMSN IIC phenotype in our family, previously shown not to be linked to the HMSN 11A gene locus, is linked to HMSN IIB gene locus (3ql3-q22) or HMSN IID gene locus (7pl4). Genomic DNA was obtained from the peripheral blood of thirty-three subjects, including 12 affected individuals and 11 individuals at risk, in a large family with HMSN IIC. Genotyping was performed for 8 DNA microsatellite markers spanning HMSN IIB gene locus and 7 markers spanning HMSN IIB gene locus using PCR technique. Pairwise LOD scores at q = 0 of each markers were negative. Multipoint LOD scores were less than -2 between -20.7 cM to +27.8 cM of HMSN IIB gene locus, and between -9.7 cM to +10.1 cM of HMSN IID gene locus. These results provide evidence against linkage of HMSN IIC phenotype to either the HMSN IIB or the IID gene locus. HMSN IIC is genetically distinct from HMSN IIA, IIB, and IID. At least four genetic varieties of autosomal dominant HMSN II exist.
SMALL FIBERS INVOLVEMENT IN FRIEDREICH ATAXIA
Nolano M, Crisci C, D'Addio G, Wendelschafer-Crabb G.(1), Kennedy W.R.(1), Provitera V.(2), Caruso G.(2) S. Maugeri Foundation IRCCS, Telese Terme (BN), (1)University of Minnesota, Minneapolis, MN, USA, (2)Department of Neurological Sciences, University "Federico II" of Naples, Italy
Friedreich Ataxia (FA) is a recessively inherited disease associated with the expansion of a GAA trinucleotide repeat in the first intron of the X25 gene. A sensory neuropathy involving mostly large fibers is well documented in FA, while small myelinated and unmyelinated nerve fibers have always been considered to be normal. To evaluate the possible involvement of small fibers we studied cutaneous innervation in 8 subjects with FA. Diabetic subjects were excluded. Three mm punch biopsies from hairy and glabrous skin were fixed in Zamboni solution and processed by immunohistochemical methods. Cryostat sections of 100 mm were stained using antisera to PGP 9.5, CGRP, SP, VIP, Col IV, S100, MBP and secondary antibodies labeled with cyanine 3.18, cyanine 5.18 and fluorescein. Digitized images were collected using a dedicated software (Neurolucida - MicroBrightField). In addition to alterations of Meissner corpuscles and large myelinated fibers observed in glabrous skin, in all samples we found a marked reduction of nerve fibers in epidermis, sparse fibers in the superficial dermis, a poor innervation of sweat glands and terminal arterioles. Our findings demonstrate an involvement of small myelinated and unmyelinated fibers in addition to the lack of large fibers in FA.
MYELINATED FIBERS IN CHARCOT-MARIE-TOOTH DISEASE TYPE 1B WITH ARG98HIS MUTATION OF Po
Ohnishi A., Yamamoto T., Ikeda M. University of Occup & Environ Health, Kitakyushu 807-8555 Japan
To characterize clinical, electrophysiologic and histopathologic features of the five unrelated Japanese CMT type 1B patients with the Arg98His substitution of Po protein and especially to correlate Arg98His substitution to the ultrastructural abnormalities of myelin sheath, electrophysiologic evaluation of limb nerves and morphometric analysis of sural nerves on biopsy were performed. Clinically, the obvious onset ranged from the second to the fifth decade. Both median and ulnar motor and sensory conduction velocities were in the range of 10 to 30 m/sec. Segmental demyelination and remyelination and marked loss of myelinated fibers were main findings. On electron microscopy, the widening between major dense lines was found between the paired intraperiod lines, where extramembranous portion of Po protein resides. This widening is probably directly related to Arg98His substitution. Uncompaction of major dense lines was found focally and coexisted with the widening above. This uncompaction may be caused by the secondary effect of the Arg98His substitution on the intramembranous domain of Po protein. In conclusion, myelin changes at both extracellular and cytoplasmic appositions of Schwann cell membranes were revealed in association with Arg98His substitution of Po protein.
ABNORMALITIES OF IN VITRO MYELINATION IN ORGANOTYPIC CULTURES OF DORSAL ROOT GANGLIA (DRG) FROM A TRANSGENIC RAT MODEL OF CHARCOT-MARIE-TOOTH DISEASE TYPE 1A (CMT1A)
Schenone A., Mancardi G.L., Grandis M., Nave K.(1), Sereda M.(1), Levi G.(2), Barbieri O.(2), Abbruzzese M., Windebank A.J.(3), Nobbio L. Department of Neurological Sciences, University of Genoa, 1Zentrum fur Molekulare Biologie, University of Heidelberg, Germany, 2Center for Advanced Biotechnologies, Genoa, Italy, 3Department of Molecular Neuroscience, Mayo Clinic and Mayo Foundation, Rochester, MN, USA.
CMT1A is associated with a duplication of the peripheral myelin protein 22 gene. Transgenic animal models of CMT1A have been generated. However, early myelin abnormalities are still unknown and in vitro models of the disease may be of help in evaluating therapeutic strategies. We established DRG cultures from 15 days old embryos of a CMT1A female rat mated with a normal male. After genotyping each embryo for the transgene, 10-12 DRGs were explanted onto separate dishes in groups of 2-4 DRGs/dish and coded. After 30 days, cultures were assessed by light and electron microscopy (EM) and morphometry was performed. Out of 9 cultures, 3 were correctly graded as normal and 5 as abnormally myelinated by two blind examiners; only one of the CMT1A cultures was misjudged as normal. Nodal region length was evaluated in 206 fibers from the normal cultures and 422 fibers from the CMT1A cultures. Mean nodal length was significantly longer in affected than in normal cultures (20.27m±28.9 vs 3.94m±6.16, p<0.00001) suggesting ongoing demyelination in CMT1A cultures. EM revealed, in affected cultures, a slightly higher frequency of hypomyelinated neurites and the presence of occasional neurite-Schwann cells (SC) units surrounded by concentric layers of SC basal lamina. In conclusion reproduction of a morphological phenotype similar to human CMT1A has been obtained in organotypic DRG cultures from CMT1A transgenic rats. This model may be used to study early changes in the myelination process due to PMP-22 duplication and to evaluate therapeutic strategies for CMT1A.
IDENTIFICATION OF DNA MUTATIONS IN ARCHIVAL SURAL NERVE BIOPSIES CONFIRMED BY BLOOD SAMPLES IN HMSN X: OCCASIONAL ASSOCIATION WITH BECKER'S MUSCULAR DYSTROPHY AND COLOR BLINDNESS
Schröder J.M., Senderek J., Bergmann C., Hermanns B. Institut für Neuropathologie, Universitatsklinikum der RWTH Aachen, Germany
Our previous studies on archival sural nerve biopsies revealed duplications or deletions of the PMP-22 gene in HMSN Ia and HNPP cases, and new or well known mutations of the connexin32 gene in X-linked HMSN (Acta Neuropathol 95:443; 96:13, 1998). DNA extraction from sural nerve biopsies, PCR amplification, SSCP, automated DNA sequencing, and enzyme restriction analysis resulted in the detection of four different mutations in the four unrelated index patients with HMSN X in whom a sural nerve biopsy happened to be available. Meanwhile we succeeded in identifying 20 other siblings in these four HMSN X families, showing identical mutations in each family, by using blood samples of 44 other family members. The clinical, electrophysiological, and morphological changes in these patients and families varied considerably. The neuropathy was predominantly axonal in type with usually numerous clusters of regenerated, thinly myelinated nerve fibers but only rare, incipient onion bulb formations. Several affected female patients were unconscious of their disease. It is of interest that there was one boy who was wheel chair bound at the age of 12 years already and who had X-linked, Becker's muscular dystrophy due to a sporadic deletion in the dystrophy gene in association to HMSN X. This was considered as a chance association and accounted obviously for the severe phenotype and rapid progression of both diseases in this male. In another HMSN X family, 3 males had X-linked red green color-blindness which was transmitted by two heterozygous females who had normal color vision. This excludes close linkage between the two entities. Thus molecular genetic analysis of archival sural nerve biopsies using recently available techniques can clarify the diagnosis in uncertain cases and may lead to interesting, new insights into complicated associations of hereditary or sporadic conditions.
LATE-ONSET FAMILIAL AMYLOIDOTIC POLYNEUROPATHY TYPE I (MET 30 TRANSTHYRETIN MUTATION) UNRELATED TO ENDEMIC FOCUS IN JAPAN: CLINICOPATHOLOGIC AND GENETIC FEATURES
Sobue G., Misu K., Hattori N., Nagamatsu M., Ikeda S., Ando Y., Nakazato M. Department of Neurology, Nagoya University School of Medicine, Nagoya, Shinshu University, Kumamoto University, Miyazaki Medical University, Japan
Clinicopathologic and genetic features were assessed on 35 Japanese families affected by late-onset (> 50) familial amyloidotic polyneuropathy type I (FAP I Met 30) whose siblings were unrelated to endemic Japanese foci. The most common initial symptom was paresthesias in the legs. Autonomic symptoms were generally mild and not seriously affected the daily activities. The male-to-female ratio was extremely high (11/1). A family history was evident in only 11 of 35 families, and other patients apparently were sporadic. Symptomatic siblings showed a late onset, male preponderance, and clinical features similar to those of the probands. Asymptomatic carriers, predominantly female, were detected relatively late in life. The geographic distribution of these late-onset FAP I was scattered extensively throughout Japan. In 3 autopsy cases and 20 sural nerve biopsy specimens, neurons in sympathetic and sensory ganglia were relatively preserved. Axonal degeneration was prominent in myelinated fibers, while unmyelinated fibers were relatively preserved. Amyloid deposition was markedly seen in the peripheral nervous system, but was milder than that seen in typical early-onset FAP I as observed in two Japanese endemic foci of this disease. Our cases of late-onset FAP I showed features distinct from those of typical early-onset FAP I occurred in the two Japanese endemic foci. Factors responsible for clinicopathologic differences between these two forms of FAP I need to be identified.
AXO-GLIAL INTERACTIONS IN THE PMP22-RELATED NEUROPATHIES
Yamamoto T., Ohnishi A. University of Occupational & Environmental Health, Kitakyushu 807-8555 Japan
To evaluate the axo-glial interactions in the myelinated fibers
of PMP22-related neuropathies, the morphometric studies on both
axonal and myelin factors of myelinated fibers in the sural nerve
from 2 patients of CMTlA with PMP22 duplication and 4 patients
of HNPP with PMP22 deletion were performed. In the myelin factors,
the mean G-ratio tended to be larger and the mean myelin thickness
was smaller in CMT1A than HNPP. In the axonal factors, the mean
nearest-neighbor distance (NND) between two neurofilaments was
shorter in CMTlA than HNPP. In regression analysis, there was
a positive correlation between NND and G-ratio, and a negative
correlation between NND and myelin thickness in HNPP. However,
there were tendencies of a negative correlation between NND and
G-ratio, and a positive correlation between NND and myelin thickness
in CMT1A. The results suggested that there were different characteristics
in the axo-glial interactions between CMT1A and HNPP, and that
the histopathological changes in HNPP might not be caused by a
typical demyelination process as found in CMT1A. In conclusion,
there were different neurobiological phenotypes in the alterations
of myelinated fibers between CMT1A and HNPP based on the different
genotypes of PMP22 duplication or deletion.