Download imaging del sistema nervoso periferico

IMAGING DEL SISTEMA NERVOSO PERIFERICO
Martinoli Carlo
Cattedra “R” di Radiologia – DICMI - Università di Genova
I recenti progressi tecnologici in ecografia e risonanza magnetica (RM) hanno portato a una migliorata definizione
anatomica nello studio delle strutture di superficie, con possibilità di evidenziare la maggior parte dei nervi periferici,
delineare la loro struttura fascicolare e identificarne anormalità strutturali. In questo campo, l’ecografia, con
apparecchiature digitali e trasduttori lineari elettronici ad alta frequenza, offre una migliore risoluzione spaziale rispetto
alla RM, consente di esaminare i nervi lungo tutto il loro decorso attraverso gli arti in un tempo esame limitato e di
esplorarli durante il movimento articolare. Suo limite principale è la dipendenza dall’operatore, la necessità di una lunga
curva di apprendimento e l’impossibilità di valutare i nervi spinali, le catene del simpatico e i nervi splancnici dato il loro
decorso in profondità e/o l’interposizione di strutture ossee. La RM offre, al contrario, una migliore risoluzione di
contrasto, con la possibilità di identificare l'edema e l’iperemia intraneurale; può effettuare inoltre una vera e propria
“neurografia”, combinando tecniche di soppressione del grasso con immagini T2-pesate e ricostruzioni 3D. Il ruolo della
diagnostica per immagini nello studio del sistema nervoso periferico presenta prospettive di utilizzazione a fianco
dell’esame clinico e degli studi elettrofisiologici soprattutto nella valutazione di pazienti con quadro sintomatologico
atipico, quando si sospetti che il deficit funzionale sia correlato alla presenza di una lesione espansiva nei tessuti molli o
quando sia necessario precisare il livello della lesione. A questo proposito, la diagnostica per immagini può offrire un
parametro morfologico aggiuntivo nella valutazione del danno nervoso essendo in grado di rilevare varianti anatomiche,
malattie congenite, lesioni focali conseguenti a sindromi compressive e traumi, differenziare lesioni espansive
endoneurali da masse estrinseche, valutare l’estensione della lesione e i rapporti con le strutture contigue, come pure
l’integrità e il quadro evolutivo della neuropatia in controlli a distanza.
LONG-TERM EFFICACY OF TWO DIFFERENT CONSERVATIVE TREATMENTS
FOR CARPAL TUNNEL SYNDROME: A RANDOMIZED CONTROLLED STUDY
De Angelis MV *, F. Pierfelice*, Di Giovanni P**, Staniscia T**, Uncini A*
*Dept. of Human Motor Sciences, it, Aging Research Center, Ce.S.I., “Gabriele d’Annunzio”
University Foundation – Chieti - Pescara, **Dept. of Medicine and Aging, Sect. of Epidemiology
and Public Health, University “Gabriele d’Annunzio” - Chieti
We randomized 120 patients with carpal tunnel syndrome (CTS) into a group wearing the soft hand brace MANU® and a
group wearing the wrist splint Tielle® at night for three months. We re-evaluated the patients after three and six
months. The primary efficacy measures were changes in the Boston Carpal Tunnel Questionnaire (BCTQ) score and in
the Visual Analogical Scale (VAS) for pain and paresthesias. Secondary measures were median distal motor latency
(DML), sensory conduction velocity (SCV) and amplitude, and neurophysiological class of severity.
Both groups showed a significant reduction in BCTQ and VAS scores after three months of treatment. This benefit,
although less evident, persisted also after six months without treatment. No significant difference was found between
the two groups. After three months MANU® improved median DML and both devices were effective in increasing SCV.
This study indicates that three-month treatment with the hand brace or the splint produces symptomatic improvement
also in the long-term.
CARPAL TUNNEL SYNDROME: RELATIONSHIP BETWEEN ULTRASOUND
FINDINGS
AND
CLINICAL/NEUROPHYSIOLOGICAL/PATIENT-ORIENTED
ASSESSMENT
Pazzaglia C*, Martinoli C**, Caliandro P***, Granata G*, Foschini M*, Briani C****, Padua L***
*Ist. Neurologia, Università Cattolica “Sacro Cuore” - Roma, **Cattedra di Radiologia R, DICMI Università di Genova, *** Ist. Neurologia, Università Cattolica “Sacro Cuore” e Fond. “Don Carlo
Gnocchi” – Roma, ****Dip. Neuroscienze - Università di Padova
Objective: In order to comprehensively assess carpal tunnel syndrome (CTS), the Italian CTS study group adopts a
multidimensional protocol by using validated clinical (Hi-Ob scale), neurophysiological and patient-oriented (BCTQ)
measures and as it was demonstrated that ultrasound (US) is helpful to study compression and entrapment
neuropathies, we also adopted it in our protocol.
Methods: We performed a prospective study by using the multidimensional assessment plus US in order to study the
relationships between US results and: 1) patient’s perspective of his/her symptoms and hand function; 2) the clinical
severity of CTS; 3) neurophysiological classification; 4) hand distribution of symptoms. Moreover, we intended assess the
sensitivity of US and neurophysiological assessment utilising clinical picture as gold standard.
Results: We studied 54 dominant hands of consecutive patients (43 females, mean age 53.3, range 30-80, SD: 13.1)
with clinical CTS referred to our neurophysiological lab.
We observed striking significant relationships between cross sectional area of Median Nerve at wrist and 1) hand
function (according to BCTQ, r:0.35, p: 0.01), 2) the clinical scale (Hi-Ob scale, r: 0.51, p: <0.00007) neurophysiological
classification (r: 0.80, p: <0.0000001):) and 3) hand distribution of symptoms (p:0.017). Finally, the study confirmed
that sometimes US might show abnormal findings even if we use the most sensitive neurophysiological tests.
Conclusions: These results clearly show the crucial role that US must have in CTS assessment either for comprehensive
assessment of impairment or for diagnosis.
ULTRASONOGRAPHY BEFORE AND AFTER SURGICAL DECOMPRESSION IN
CARPAL TUNNEL SYNDROME (CTS) AND RELATIONS WITH CLINICAL AND
ELECTROPHYSIOLOGICAL FINDINGS
Mondelli M*, Filippou G**, Aretini A*, Frediani B**, Reale F***
*EMG Service, Public Health Unit 7, **Dept. Clinical Medicine and Immunological Sciences,
Rheumatology Section, Siena University, ***Neurosurgery 1, University Hospital of Siena - Siena
Objective The aims of this prospective study were: 1) to measure the cross-sectional area (CSA) of the median nerve
with ultrasonography (US) before and after surgery in subjects with CTS, 2) to determine whether there were relations
between the CSAs and clinical and electrophysiological findings and 3) to verify whether the normalization of presurgical
parameters can be predicted before surgery. Methods 67 consecutive cases (53 F, 14 M) mean age 60.5 years (31-84)
underwent surgical decompression and completed the study protocol. Before surgery, education, body mass index, work,
manual hobbies, associated pathologies, duration of symptoms, clinical and electrophysiological severity (ordinal scales)
and self-assessment of symptoms (Boston Questionnaire-BQ) were assessed. CSAs were measured proximal to the
carpal tunnel inlet (CSA-I), at mid tunnel (CSA-M) and at the tunnel outlet (CSA-O) with a 10MHz linear-array
transducer. Follow-ups of clinical, electrophysiological, BQ and US findings were performed one and six months after
surgery. Surgical complications, time to return to work and daily activities and a grading scale of patient satisfaction
were also recorded. Logistic regression was also performed with normalization of clinical and electrophysiological severity
scales, BQ and CSA findings as dependent variables. Results Before and after surgery there were correlations between
CSA-I and clinical and electrophysiological severity scales. CSA-M and CSA-O correlated only with post-surgical findings.
After one month clinical, electrophysiological and BQ findings improved and this improvement was more evident after six
months. CSA-I reduced at 1-month follow up and this reduction was more evident at 6-month follow-up. CSA-O
increased between the first and second follow-up. There was no significant variation of CSA-M. Presurgical values of
CSA-I could predict the normalization of the post-surgical clinical severity scale (OR=1.13), BQ-SYMPT (OR=1.24), BQFUNCT (OR=1.19) and full patient satisfaction with surgery (OR=1.45). Conclusions The usefulness of US in the
diagnosis of CTS is well known in the literature, but only 2 articles concerned post-operative findings. Our results show
that besides clinical and electrophysiological improvements, US findings also improved after surgery. The CSA alterations
reflect the pathogenesis of the nerve compression. In particular, the CSA-O increased its post-surgery value after six
months, while CSA-I reduced earlier, already at one month post-surgery, and was more evident after six months. The
degree of CSA improvement is related to clinical and electrophysiological improvement. The presurgical values of CSA-I
were a predictor of postsurgical normalization of clinical findings. For one mm2 of reduction in presurgical CSA-I the
probability of normalization of clinical and BQ increase was 13%, 24% and 20%, respectively. These values are low but
significant, further studies on CSA predictivity need.
ULNAR NERVE CONDUCTION ABNORMALITY IN CARPAL TUNNEL SYNDROME
AS REVEALED BY AXONS RECRUITMENT ANALYSIS.
Ginanneschi F*, Milani P*, Dominici F*, Biasella A*, Mondelli M**, Filippou G***, Reale F****,
Rossi A*
*Dip. Scienze Neurologiche e Comportamento, Unità Neurofisiologia Clinica - Università di Siena,
**Servizio EMG - ASL 7 Siena, *** Dip. Medicina Clinica e Scienze Immunologiche, Unità di
Reumatologia –- Università di Siena, ****Unità Neurochirurgia, AOUS - Siena
In a recent study conducted in CTS patients, we have observed that ulnar nerve motor axons manifests sharp changes
in recruitment property, demonstrating a motor ulnar nerve impairment at Guyon’s canal in these patients. Ulnar motor
axons manifested increasing abnormalities with increasing severity of median nerve involvement. In order to disclose a
mild sensory afferents impairment we have extended the electrophysiological examination to 144 CTS hands, analyzing
the conduction parameters of the ulnar nerve sensory branches originating below the elbow: the superficial (sensory)
branch, arising at wrist level, and the dorsal ulnar cutaneous (DUC) branch, arising approximately at the junction of the
medial and distal thirds of the forearm. The latter, not having an intra-Guyon’s canal course, will be spared in ulnar
nerve lesions localized at wrist level. Our results showed that the U5 and U4 SAPa and U5 SCV were significantly lower
with respect to the control group. Conversely, the DUC conduction was similar in both populations, demonstrating an
ulnar sensory nerve impairment at wrist. Similar to ulnar motor damage, also ulnar sensory damage was higher with the
progression of the neurophysiological median impairment. This data strongly support the idea that high pressure in the
carpal tunnel in CTS reflects on the adjacent Guyon’s canal, causing indirect compression of ulnar nerve, in a dosedependent manner. We have also examined the ultrasonographic images of Guyon’s canal and the motor axon
recruitment properties of the ulnar nerve of CTS patients, taken before and after the carpal tunnel release was done.
Finally, it’s known that a large number of CTS patients complain of sensory symptoms outside the typical median nerve
distribution.Our findings will be also discussed in order to reach a better comprehension of the pathogenesis of extramedian symptoms in CTS patients.
PERIPHERAL NEUROLOGICAL MANIFESTATIONS AND ANTIBODIES TO
RIBOSOMAL P PROTEINS IN PATIENTS WITH SLE
Briani C*, Lucchetta M*, Toffanin E*, Ruggero S*, Ghirardello A**, Zampieri S**, Scarlato M***,
Quattrini A***, Ermani M*, Battistin L****
*Dept. of Neurosciences - University of Padova, **Dept. of Rheumatology - University of Padova,
***San Raffaele Scientific Inst. – Milan, ****Dept of Neurosciences – University of Padova and
IRCCS San Camillo Hospital – Venice
Aims. Serum IgG antibodies (Abs) to phosphorilated ribosomal proteins (P ribosomal proteins) have been inconsistently
associated with nervous system involvement in systemic lupus erythematosus (SLE). The aim of our study was to assess
whether serum and cerebrospinal fluid (CSF) IgG antibodies (Abs) to ribosomal P proteins correlate with peripheral
neurological or other SLE manifestations and auto-Abs (ANA, anti-nDNA, anti-nRNP, anti-Sm, anti-SSA, anti-SSB, antiphospholipid).
Methods. A prospective cohort of 219 SLE patients (185 F, 34 M, mean age 26 yrs +/- 10) were considered. Abs to
ribosomal P proteins were investigated by immunoblotting. CSF oligoclonal bands were searched for by
immunoisoelectrofocusing. The correlations between IgG Abs to ribosomal P proteins and neurological, other SLEassociated clinical manifestations and SLE-Abs were analyzed.
Results. Abs to ribosomal P proteins were detected in 45 (20.5%) patients, 23 with neurological involvement (12 central,
4 peripheral, 7 both central and peripheral nervous system). In 6 patients Abs to ribosomal P proteins were determined
also in the CSF. In 4 of the 6 patients (2 with mononeuropathy multiplex, 1 with radiculopathy, 1 with psycosis) Abs to
ribosomal P proteins were present both in serum and CSF. The presence of Abs to ribosomal P proteins was significantly
associated with serum anti-Sm Abs, lupus anticoagulant, and the presence of radiculopathy.
Conclusion: Abs to ribosomal P proteins may be associated also with peripheral nervous system manifestations of SLE.
The CSF data indicate a passive passage of Abs through the blood-brain-barrier. The ongoing follow-up will help clarify if
the Abs to ribosomal P proteins, when present in patients without neurological involvement, might be predictive of future
neurological manifestations.
CHARACTERIZATION
NEUROPATHIES.
OF
HMGB1
EXPRESSION
IN
INFLAMMATORY
Scarlato M, Cerri F, Grassi S, Dacci P, Previtali SC, Comi GC, Quattrini A
Dip. Neurologia, Ist. Scientifico San Raffaele - Milano
High mobility group 1 protein (HMGB1), a nuclear transcription factor, is a crucial player in inflammation since it can
leaked out from necrotic cells or it can be secreted by activated monocytes and macrophages. HMGB1 acts as a
cytokine that recruits mononuclear cells to the site of tissue damage. In this study, we investigated whether HMGB1 is
expressed in the peripheral nervous system under normal and pathological conditions, in particular
inflammatory/immune-mediated neuropathies. We analyzed by immunohistochemistry its expression and localization in
sural nerve biopsies with neuropathological evidences of an inflammatory or disimmune process such as vasculits,
chronic inflammatory demyelinating neuropathy (CIDP) or anti-MAG neuropathy, and controls.
In normal human nerve, HMGB1 immunoreactivity was weakly present in some endothelial cells as well as in the
perineurium and endoneurium and was always localized in the nucleus. Sural nerve biopsies of patients with
inflammatory/immune neuropathy showed a dramatic increase of HMGB1 expression restricted to the vessel walls and
to the perivascular space where the mononuclear infiltrating cells were mostly localized. Interestingly, we found that
HMGB1 is strongly increased in S100 positive cells in the endoneurium. We correlated the number of HMGB1/S100
positive cells with the number of macrophages in order to evaluated the role of HMGB1 in sustaining the inflammatory
process. Our results suggest a close relationship between the pathogenesis of inflammatory/immune-mediated
neuropathies and HMGB1 expression
GLIAL FIBRILLARY ACIDIC PROTEIN IN CEREBROSPINAL FLUID AS A
MARKER OF ACUTE AXONAL DAMAGE IN GUILLAIN-BARRÈ SYNDROME
Notturno F, Caporale CM, Uncini A
Clinica Neurologica, Osp. SS Annunziata – Chieti
Glial fibrillary acidic protein (GFAP) is a monomeric intermediate filament protein highly expressed in cytoskeleton of
astrocytes and Schwann cells (SC). In the peripheral nerve GFAP is expressed in immature SC and in mature SC
surrounding unmyelinated axons. After nerve injury denervated myelin-forming SCs lose the contact with axons, acquire
the phenotype of non-myelin-forming SC like and increase their GFAP expression. Increased GFAP has been found in
the cerebrospinal fluid (CSF) of patients with multiple sclerosis, dementia, subaracnoid hemorrage and in few patients
with peripheral nerve diseases.
The objective of this study was evaluate whether GFAP levels in the CSF of Guillain-Barrè syndrome (GBS) patients are
associated with axonal injury and correlate to prognosis.
GFAP levels were determined by ELISA in nine controls and 33 patients with GBS within 2 weeks from onset. GBS were
classified in five subtypes: nine patients had axonal GBS, 12 patients had demyelinating GBS, five patients had
demyelinating GBS with various degrees of axonal involvement, five patients had sensory GBS and two patients had
acute motor conduction block neuropathy. GFAP levels in the acute phase were correlated with Hughes’scale scores six
months after the onset of GBS.
The mean GFAP levels, measured as optical density, were increased in axonal GBS (0.53) compared to controls (0.29;
p=0.0009), to demyelinating GBS (0.37; p=0.0196) and to the other three remaining subtypes (0.32; p=0.0008). GFAP
levels were directly correlated with Hughes’scale scores at six months (r=0.62; p=0.000).
In conclusion GFAP level in the CSF seems to be a marker of axonal damage in the acute phase of GBS and correlates
with prognosis.
DEFECTIVE T CELL FAS FUNCTION FAVORS THE SWITCH FROM GBS TO
CIDP
Comi C*, Varrasi C*, Tarletti R*, Ferretti M**, Dianzani U**, Cantello R*
*Dip. Medicina Clinica e Sperimentale, Clinica Neurologica, ** Lab. Immunologia - Università del
Piemonte Orientale – Novara
Introduction- Guillain-Barré Syndrome (GBS) and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP) are
immune-mediated neuropathies that may be variants of the same disorder. GBS is typically characterized by a remission
of symptoms over time; only a small number of GBS patients eventually develop CIDP (acute-onset CIDP). The Fas
death receptor is a molecule involved in the immune response shutting-off system, since it triggers apoptosis of Fas+
effector T cells. Defects of Fas function predispose to autoimmune diseases. In a recent study, we evaluated Fas
function in T cells from patients with GBS and CIDP and healthy controls. CIDP patients displayed lower Fas function
than both GBS patients and controls, whereas no statistically significant difference was found between GBS patients and
controls. In GBS, the Fas function defect was rare in patients with complete remission, but frequent in those with poor
outcome, and most patients with the Fas defect developed CIDP.
Aim of the study- To confirm in a longitudinal study the role of Fas function defects in predisposing GBS patients to
develop CIDP.
Patients and Methods- T cell Fas function was tested in all new cases of GBS diagnosed between January 2004 and
December 2006 (45 patients).
Results- Four patients initially diagnosed with GBS, then developed CIDP (acute-onset CIDP). All these patients
displayed resistance to Fas mediated apoptosis. The remaining 41 patients who had a complete recovery displayed a T
cell Fas function that was similar to healthy controls.
Conclusion- These findings confirm the hypothesis that Fas mediated apoptosis is one of the mechanisms for inducing a
self limitation of the autoimmune response in the inflammatory neuropathies.
T CELL RESPONSE TO GM1 GANGLIOSIDE IN GUILLAIN-BARRÉ SYNDROME
PATIENTS POST CAMPYLOBACTER JEJUNI INFECTION
Cencioni MT*, Notturno F**, Caporale CM**, Uncini A**, Battistini L*
*European Centre for Brain Research, Santa Lucia Foundation, Neuroimmunology Unit – Rome,
** Dept. of Human Motor Sciences and Neuromuscular Diseases Unit, Inst. of Aging (Ce.S.I),
Foundation University "G. d'Annunzio" – Chieti-Pescara
Guillain-Barré syndrome (GBS) is the prototype of a post-infective immune-mediated neuropathy. Patients with GBS after
Campylobacter jejuni enteritis have anti-ganglioside antibodies and Campylobacter jejuni strains isolated from such
patients have ganglioside-like structures in the lipopolysaccharide (LPS) supporting the hypothesis that anti-ganglioside
antibodies, induced through molecular mimicry by the antecedent Campylobacter jejuni infection, attack gangliosides
expressed in the human peripheral nerve.
Because anti-ganglioside IgG1 and IgG3 isotypes are frequently found in GBS, it is likely that T cell help is required for B
cell maturation and antibody switching.
Considering the potential role for T cell involvement in antibody production we investigated whether there is a
ganglioside-specific T cell response in patients with GBS.
We isolated monocytes from 4 controls and seven patients who had GBS 1-70 months before. Five patients had acute
axonal motor neuropathy (AMAN) and 2 patients had acute inflammatory demyelinating polyneuritis (AIDP). All patients
had an antecedent Campylobacter jejuni infection and 6 patients had antibodies against GM1 and/or GD1a. Immature
dendritic cells expressing CD1 molecules, cultured with autologous T cells, were stimulated with LPS of two
Campylobacter jejuni strains isolated from GBS patients and with GM1. The T cell response to LPS and to GM1 was
studied with Enzyme-Linked ImmunoSorbent Assay by TNFa and IFNg release.
All controls and patients showed T cells reactive to LPS of both Campylobacter jejuni strains. T cells of controls did not
respond to GM1. In 2 of 3 patients with AMAN and antibodies against GM1 and in one patient with AIDP T cells
responded to GM1 whereas there was no response in one patient with AMAN and monospecific antibodies to GD1a. The
T cell response to LPS and GM1 was CD1-mediated as antibodies to CD1a, CD1b and CD1c blocked T cell activation.
In conclusion, these findings provide for the first time evidence of molecular mimicry at the level of the T cell response
and suggest an important contribution of the cellular arm of the immune response in GBS.
EXPRESSION OF ENDOTHELIN B RECEPTOR IN PERIPHERAL NERVE.
IMMUNOHISTOLOGICAL AND IMMUNOELECTRONMICROSCOPY STUDY
Volpi N*, Carbotti P*, Belmonte G*, Massai L*, Alessandrini C*, Magi S**, Grasso G*, Greco G***,
Giannini F***
*Dip. Scienze Biomediche, Sez. Istologia e Anatomia - Università di Siena, ** U.O. Neurologia,
Osp. "S. Donato" – Arezzo, *** Dip. Neuroscienze- Sez. Neurologia - Università di Siena
Endothelins (ETs) are a family of multifunctional peptides, firstly identified as vasoconstrictors (1), and active in
inflammation and nociception. They are cleaved to the active form by specific ECE metalloproteases and act by linking
to receptors ETAR and ETBR in distinct tissue distribution: in neural tissue, ETBR is predominantly expressed (2).
Localization of ETRs indicates target sites of matured ETs. We previously detected ET and ECE upregulation in
inflammatory neuropathies by immunohistology and in situ hybridization. Therefore, we investigated expression of ETBR
in sural nerve from vasculitic neuropathies using immunohistology and immunoelectronmicroscopy for ET-1 and ETBR
with gold visualization. Light microscopy showed vascular ETBR localization in endothelium of epineural and endoneural
vessels and, to a lesser extent, in smooth muscle cells. Serial specific markers immunostaining revealed positivity of
lymphatic endothelium too. Perineural cells were reactive as well as many endoneural non endothelial cells. Many
inflammatory elements also displayed ETBR. TEM confirmed endothelial and smooth muscle localization of ET-1 and
ETBR, and evidentiated ETBR expression on myelinating and unmyelinating Schwann cells. In each cell type gold
particles were preferentially adjacent to cell membrane. Within a minority of axons, occasional gold particles were
observed. Immunogold reaction for ET-1 showed significant axonal and vascular reactivity. Neural localization of the ET
system is most extensively investigated in CNS. Neuronal ET expression, associated with ETBR upregulation in astrocytes
after neural injury, suggests that ET can be an inducible intercellular mediator between injured neurons and glia (3).
Experimental studies indicate that ETBR is involved in nociceptive signaling of chronic inflammatory pain (4). ETBR
expression in non myelinating Schwann cells, as already experimentally reported (5), might indicate a direct role of
peripheral glia in pain sensation which can occur in vasculitic neuropathies.
1
2
3
4
5
Yanagisawa M, Nature 1988, 332:411-415
Hori S, Endocrinology 1992, 130:1885-1895
Nakagomi S, Neuroscience 2000, 101:441-449
Piovezan AP, Br J Pharmacol 2000, 129:961-968
Pomonis JD, J Neurosci 2001, 21:999-1006
AXONAL CHARCOT-MARIE-TOOTH DISEASE: A NEUROPATHY IN MIST
Angiari C, Ferrarini M, Taioli F, Ferrari S, Cavallaro T, Rizzuto N, Fabrizi GM
Dip. Scienze Neurologiche e della Visione, Sez. Neurologia Clinica –- Università di Verona
Two years ago most of the molecular actors involved in the autosomal dominant Charcot-Marie-Tooth disease (CMT2)
were believed to be engaged (Reilly MM. The fog is slowly lifting! Neurology 2005), and in fact at least 9 genes were
discovered. The relative mutational frequencies of those genes are still unclear. We aimed at estimating the molecular
causes of several CMT2 patients using a systematic mutational analysis. The series included 90 index patients fulfilling
clinical and electrophysiological criteria and lacking mutations of P0 and Cx32; 42 patients had dominant inheritance; 48
were sporadic without laboratory features of acquired neuropathy. Molecular targets included all genes associated with
CMT2 (MFN2, RAB7, GARS, NEFL, HSP27, HSP22, GDAP1) and with autosomal dominant intermediate CMT (DI-CMT)
(DNM2, YARS). The coding exons and related exon-intron boundaries were screened by denaturing high performance
liquid chromatography (DHPLC); DHPLC-positive regions were dissected further by nucleotide sequencing. Only 21
unrelated patients were positive disclosing 17 pathogenic mutations (including 11 novel mutations) variably distributed in
NEFL (6 mutations in 8 unrelated patients), MFN2 (7 mutations in 8 unrelated patients), DNM2 (2 mutations in 2
patients), GDAP1 (1 mutation in 1 patient) and YARS (1 mutation in 1 patient). Nine patients were sporadic and 4
disclosed a predominant clinical and electrophysiological involvement of the lower limbs. Some genes or mutations still
escape genetical or molecular sieves of CMT2 so that the disease still overlaps with chronic idiopathic axonal
neuropathies. DI-CMT genes such as DNM2 and YARS should be considered also in the diagnosis of CMT2. NEFL and
MFN2 are likely the most frequent CMT2 genes, but high locus and allelic heterogeneity does not allow a definite
molecular flow-chart.
The work was supported by MIUR (grant no. 2005060584), Fondazione Mariani (grant 2005-2007) and Telethon (grant
no. GUP04009).
HEREDITARY AXONAL NEUROPATHIES IN ITALIAN POPULATION:
MOLECULAR ANALYSIS OF MITOFUSIN 2 GENE (MFN2) AND SMALL HEAT
SHOCK PROTEIN GENES (HSP27 e HSP22)
Acquaviva M*, Geroldi A*, Varese A*, Ciotti P*, Gulli R*, Doria-Lamba L**, Schenone A***, Reni
L***, Mandich P*, Bellone E*
*Dip. Neuroscienze, Oftalmologia e Genetica, Sez. Genetica,**Cattedra di Neuropsichiatria
Infantile, Ist. “G. Gaslini”, ***Sez. Neurologia - Università di Genova
Hereditary axonal peripheral neuropathies comprise a genetically heterogenous group of disorders that are clinically
known as Charcot-Marie-Tooth (CMT) disease type 2 (CMT2). Recently, several genes that are defective in patients with
the main forms of CMT2 have been identified (Inherited Peripheral Neuropathies Mutation Database,
http://www.molgen.ua.ac.be/CMTMutations). Mutations in mitofusin 2 gene (MFN2) seem to account for ~20% of
CMT2 cases. Moreover, defects in two small heat shock proteins, HSP22 and HSP27, have been shown to underlie
different forms of axonal CMT and distal hereditary motor neuropathy (dHMN).
To ascertain the role of these genes in the pathogenesis of inherited axonal neuropathies in the Italian population, we
searched for mutations of MFN2, HSP22 and HSP27 genes in a cohort of unrelated patients. The entire coding regions of
MFN2, HSP22 and HSP27 genes were amplified and screened for mutations by DHPLC. Direct sequencing of fragments
showing variant elution profiles was carried out on an automated DNA sequencer.
Up to now, molecular analysis of exons 1-4 of MFN2 gene, in a cohort of 130 CMT2 patients, identified a missense
mutation, already reported, in two unrelated patients. A cohort of 62 unrelated Italian patients affected with axonal CMT
and 33 patients with dHMN were screened for mutations in the HSP22 and HSPB27 genes. No variants in the HSP22
coding region and associated exon-intron boundaries were found. DHPLC screening of HSP27 identified six variant
elution profiles. Five variants were located in the non coding region of the gene. Direct sequencing of the latest variant
demonstrated, in a patient and his father, a heterozygous 2-bp deletion (c.476_477delCT) which predicts to cause a
frame shift, leading to a premature stop codon in exon 2 of the gene. This is the first study reporting a non sense heat
shock protein mutation.
Partially supported by grants Fondazione Mariani 2004 to P.M., Telethon 2004 and PRIN 2005 to E.B.
BSCL2 MUTATIONS IN TWO ITALIAN FAMILIES CAUSE OVERLAPPING
SPASTIC PARAPLEGIA-DISTAL HEREDITARY MOTOR NEUROPATHY
PHENOTYPES: ONE NOVEL MUTATION AND EVIDENCE FOR REDUCED
PENETRANCE AND INTERFAMILIAL PHENOTYPIC VARIABILITY
Milani M*, Balestrini MR**, Azan G***, De Lodovici ML****, Taroni F*
*Fond. IRCCS Ist. Neurologico “Carlo Besta”, U.O Biochimica e Genetica, ** U.O. Neurologia dello
Sviluppo - Milano, *** U.O. Neurologia, Ist. Auxologico Italiano - Verbania, **** U.O. Neurologia A.O. Fond. “Macchi”, Università dell’Insubria – Varese
Recessive mutations in the BSCL2 gene cause congenital lipodystrophy type 2. Recently, heterozygous BSCL2 mutations
have been associated with AD Silver spastic paraplegia syndrome (SPG17), characterized by hand muscle atrophy and
lower limb spasticity, and two forms of AD distal hereditary motor neuropathy (dHMN) predominantly involving the
hands (dHMN-V) or the legs (dHMN-II) without spasticity. BSCL2 encodes a 398-aa transmembrane protein, called
seipin, localized to the ER. To date, only two mutations have been found in the numerous BSCL2-positive families
reported thus far. We have screened for BSCL2 mutations a group of 12 unrelated patients affected by dHMN or spastic
paraplegia with predominant upper limb involvement. One novel and one previously reported mutation were found in
two patients with negative family history. Pt 1 (30-yr old) carried the previously described N88S mutation affecting the
glycosylation site in the ER-lumen domain. The disease presented at 12 yr of age with hand weakness and subsequently
progressed with hyperreflexia and distal amyotrophy of the lower limbs. Nerve conduction studies showed axonal motor
neuropathy with no sensory abnormalities. However, sural nerve biopsy demonstrated signs of axonal neuropathy, thus
indicating that the pathological process also involves the sensory system. Pt 2 was a 17-yr-old boy who presented in
infancy with lower limb spastic paraparesis, axonal neuropathy, and hand amyotrophy. Notably, this patient carried a
novel BSCL2 missense mutation (L363P) in the C-ter cytoplasmic domain of the protein. The mutation was also found in
his mother and his maternal aunt, both clinically asymptomatic, but in none of 338 control chromosomes. The results
indicate that (1) the phenotype associated with BSCL2 mutations may overlap SPG17, dHMN-II, and dHMN-V, and (2)
the disease exhibits incomplete penetrance and/or broad variability in clinical expression. (Telethon-UILDM and
Fondazione Mariani grants to FT)
INTERMEDIATE FORM OF CHARCOT-MARIE-TOOTH WITH A NEW SINGLE
NUCLEOTIDE DELETION OF PMP 22 GENE
Luigetti M*, Sabatelli M*, Zollino M**, Conte A*, Madia F*, Mereu M.L**, Marangi G*, Pomponi
M.G*, Tonali P.A*
*Ist. Neurologia, **Ist. Genetica, Policlinico A. Gemelli – Roma
We describe four Italian patients from the same kindred, affected by an autosomal dominant inherited peripheral
neuropathy disclosing an unusual combination of clinical, electrophysiological and pathological findings and in which a
new mutation of PMP22 gene was found.
The onset of the disease occurred in fifth-seventh decade and was characterized by carpal tunnel syndrome-like
symptoms in three patients and by a mild late-onset Charcot-Marie-Tooth (CMT) phenotype in the fourth patient. In all
patients pes cavus was obvious. Motor and sensory nerve conduction velocities were slowed and the pattern was
consistent with an Intermediate CMT, motor nerve velocity of the median nerve varying between 30 an 38 m/sec. No
focal slowing was detected at entrapment sites. Nerve biopsy was performed in two patients and disclosed thin myelin
sheath in near all fibres, occasional segmental demyelination, many regeneration’s clusters, focal thickening of myelin
sheath in some fibres, absence of onion bulbs. Sequence analysis of PMP22 gene showed a single nucleotide deletion
(227delG) in affected patients. This mutation, which has never been reported so far, leads to an open reading frame
(ORF) shift and probably to a truncated and unstable PMP22 protein.
It is interesting to note that in our patients, though clinical findings were consistent with Hereditary Neuropathy with
liability to Pressure Palsies (HNPP), pathological findings were not typical, since hypomyelination was a prominent
feature while tomaculae were scarce. Moreover electrophysiological examination did not show focal slowing at the sites
of entrapment. We conclude that this novel 227delG mutation of PMP22 is responsible for a peculiar phenotype whose
features are intermediate between CMT and HNPP.
TWO NOVEL MUTATIONS IN THE PERIAXIN GENE (PRX) CAUSING SEVERE
EARLY-ONSET DEMYELINATING NEUROPATHY
Milani M*, Fabrizi GM**, Cavallaro T**, Lauria G***, Taroni F*
*Fond. IRCCS Ist. Neurologico “Carlo Besta”, U.O Biochimica e Genetica – Milano, ** Dip. Scienze
Neurologiche e Visione, Sez. Neurologia Clinica - Università di Verona, *** Fond. IRCCS Ist.
Neurologico “Carlo Besta”, U.O. Malattie Neuromuscolari – Milano
Mutations in the periaxin gene (PRX, chr. 19q13.13-q13.2) have been shown to cause autosomal recessive (AR)
demyelinating Charcot-Marie-Tooth disease (CMT4F) and Déjérine-Sottas disease (DSD). The gene encodes two proteins
with PDZ-domain proteins, L(ong)-periaxin and S(hort)-periaxin, which are required for the maintenance of peripheral
nerve myelin. To date eight nonsense or frameshift mutations have been reported in <10 CMT/DSD families. We studied
20 CMT1A/MPZ/PMP22/Cx32-negative patients with early-onset severe demyelinating sensory-motor neuropathy.
DHPLC/sequencing analysis of the 4.9-kb coding region of the periaxin gene (exons 4-7 and exon-intron boundaries)
revealed two novel mutations in exon 7 in two unrelated patients but in none of 100 controls. Both mutations affect the
long (L-periaxin) but not the short (S-periaxin) variant of the protein. Patient 1 (34-yr old) was the daughter of healthy
consanguineous parents and carried a homozygous nonsense mutation (Glu682stop). The disease manifested in infancy
with distal weakness, ataxic gait, and profound deep sensory impairment. At onset, electrophysiology showed absence of
sensory responses and marked reduction of motor NCVs (median nerve: 9 m/sec; DL, 25 msec). Now, at 34 yrs of age,
she exhibits a profound motor deficit, short stature, and unevokable motor and sensory responses in both upper and
lower limbs. One sibling is reported to have a similar, albeit milder, phenotype. Patient 2, a 33-yr-old woman, carried a
homozygous 2-nt insertion predicting the premature truncation of the protein (Pro258fsX313). The disease manifested in
infancy with gait impairment and muscle weakness. With disease progression, the patient exhibited both superficial and
deep sensory deficit and unevokable motor and sensory responses in both upper and lower limbs. Sural nerve biopsy
demonstrated signs of severe hypertrophic demyelinating neuropathy. (Telethon-UILDM and Fondazione Mariani grants
to FT and GMF)
NOVEL GDAP1 MUTATIONS ASSOCIATED WITH EARLY-ONSET CHARCOTMARIE-TOOTH DISEASE
Geroldi A*, Aquaviva M*, Gulli R*, Ciotti P*, Grandis M**, Narciso E**, Schenone A**, Mandich
P*, Bellone E*
*Dip. Neuroscienze, Oftalmologia e Genetica, Sez. Genetica, ** Sez. Neurologia - Università di
Genova
Mutations in the GDAP1 gene are associated with autosomal recessive forms of Charcot-Marie-Tooth disease. Mutations
in GDAP1 can cause both axonal (AR-CMT2K) and demyelinating (CMT4A) inherited peripheral neuropathies. GDAP1 is a
component of the outer membrane of the mitochondria, regulates the mitochondrial network by promoting mitochondrial
fission and is expressed by myelinating Schwann cells and neurons of the peripheral nervous system. Patients with
GDAP1 mutations demonstrate early onset of a severe peripheral neuropathy, sometime associated with vocal cord
paralysis.
We studied 3 unrelated patients with severe peripheral neuropathy.
Neurological and electrophysiological evaluations were carried out according to standard procedures. Written consent
was obtained from all subjects included in the study.
The presence of 17p11.2 duplication and point mutation in the MPZ e GJB1 (Cx32) genes was previously ruled out in all
patients. The entire coding region of GDAP1, including exon-intron boundaries was amplified and analyzed by
Denaturing High Performance Liquid Chromatography (DHPLC). Direct sequencing of fragments showing abnormal
DHPLC chromatographic profiles was carried out on an automated DNA sequencer (ABI Prism 3100 Avant, Applied
Biosystems, CA).
In family 1, the patient was found to be compound heterozygous for two novel missense mutations (L49S, of maternal
origin and C88F of paternal origin) in exon 2 of the gene.
In family 2, sequence analysis of GDAP1 exon 2 revealed a novel homozygous missense mutation (Gly83Arg), not
present in 200 normal chromosomes.
In the last family, the patient was found to be compound heterozygous for two missense mutations
(Met116Thr/Arg120Trp) in exon 3 of the gene.
The pathophysiological role of these mutations will be discussed in the light of the clinical, neurophysiological and
pathological features.
Partially supported by grants Fondazione Mariani 2004 to P.M.,
Telethon 2004
and PRIN 2005 to E.B