In addition to Tunisian patients, DFNB1 was shown to be responsible for NSRD in a highly inbred Bedouin family(14). Linkage to markers D13S175, D13S143, and D13S115 on chromosome 13 has also been shown in 9 out of 18 New Zealand and 1 Australian non-consanguineous kindreds with NSRD(15).  
This finding suggest that the DFNB1 locus may make an important contribution to autosomal recessive neurosensory deafness in the Caucasian population.  
Finally, DFNB1 has also been demonstrated to be responsible for hearing impairment in one large family of a group of 27 of Pakistani origin with NSRD(16). In a recent study we performed a genetic linkage study with four microsatellite markers linked to DFNB1 in a total of 48 independent Mediterranean families, of which 30 and 18 were of Italian and Spanish descent, respectively(17). A maximum two-point LOD score of 7.28 was found with marker D13S115 at a recombination frequency of 0.1.  
Significant LOD scores were also obtained for D13S143, D13S292 and D13S175. Genetic heterogeneity was confirmed using the HOMOG program which indicated absence of linkage to DFNB1 in approximately 40% of the sample.  
This study clearly demonstrated that DFNB1 plays an important role in 80% of Mediterranean families with NSRD. Furthermore, results from multipoint analysis predicted that the DFNB1 gene should has been localized between markers D13S175 and D13S115 which are separated by approximately 14.2 cM. Successively, six polymorphic markers, three linked to DFNB2, on chromosome 11, and three others to DFNB4, on chromosome 7, were analyzed in those families certainly not linked to DFNB1(18).  
Positive LOD Scores were detected with 6 families out of 48 analyzed (.12) with markers linked to DFNB4.  
The HOMOG test showed that there was linkage with heterogeneity. These findings suggested that a second locus (DFNB4) could also play an important role in our population.  
Finally, two families out of 48 (.04) showed the segregation of the alleles of markers associated to DFNB2 locus within affected and normal members of the family. In this case, the number of families most likely associated to DFNB2 was too low to reach a statistical significance and to get positive LOD Scores.  
Nevertheless, the finding of two families segregating with DFNB2 but not with the other NSRD loci so far investigated was a good suggestion for a possible implication of DFNB2 in a minority of our deaf cases.  
Considering that approximately 80% of our patients was associated to DFNB1 and that most likely an additional proportion of about 15% of cases could be associated to DFNB4 and DFNB2, it results that, despite the great genetic heterogeneity present in recessive deafness, three genes could account together for at least 95% of all cases of NSRD in our population.  
This finding has severe important implications for the genetic counselling and the prevention of such a common disease. 

La funzione della connexina-26 (GJB2)  
La connexina-26 è un componente di una grande famiglia di proteine coinvolte nella formazione delle giunzioni gap (gap-junctions) che permettono il trasferimento diretto di piccole molecole e ioni tra cellule adiacenti.  
Ogni cellula contribuisce per metà della giunzione gap, ed è composta da un insieme oligomerico di connessoni, che sono composti da un esamero di una proteina membranale integrale chiamata connexina (23,24).  
Un esempio schematico dei connessoni è riportato nella Figura 2.  
Le giunzioni gap sono rare tra i neuroni dei mammiferi, ma sono comuni nelle cellule non-neuronali, come la glia, le cellule epiteliali, le cellule lisce e le cellule del muscolo cardiaco.  
Ci sono più di 11 tipi differenti di connexine, ognuno con specificità tessutale e proprietà fisiologiche distinte in relazione alle giunzioni gap (24,25).  
Almeno altre due connexine sono implicate in malattie umane.  
Mutazioni puntiformi nel gene connexina-32 sono associate con la neuropatia Charcot-Marie-Tooth legata al cromosoma X(26), e mutazioni nel gene connexina-43 sono state trovate in pazienti con eterotassia e malformazioni cardiache.  
Il coinvolgimento delle giunzioni gap nelle risposte intercellulari al suono era stato proposto più di 20 anni fa(27,28).  
All'interno dell'organo auditivo, le  giunzioni gap sono localizzate tra le cellule pilifere esterne e le cellule di sostegno (inclusi i melanociti), fornendo una base morfologica per l'avvenimento delle risposte intracellulari al suono nelle cellule di sostegno e per l'accoppiamento elettrico delle cellule recettori.  
L'endotelio della scala media della coclea è coinvolto nella produzione di una risposta recettoriale allo stimolo uditorio ed è separato dallo spazio endolinfatico.  
Ultimamente l'analisi ultrastrutturale ed immuno-istochimica della connexina-26 nella coclea del ratto ha dimostrato che le giunzioni gap, sia nelle cellule epiteliali sia nelle cellule del tessuto connettivo, sono coinvolte nel riciclaggio degli ioni di potassio endolinfatici attraverso le cellule sensoriali durante la trasduzione del segnale elettrico in questi canali.  
L'identificazione delle mutazioni nel gene GJB2 conferma in modo chiaro il ruolo cruciale delle giunzioni gap nella trasduzione del segnale uditivo(19,29).  
La maggior parte delle mutazioni identificate nel gene GJB2 portano all'assenza completa della connexina-2619,29.  
Considerando che il GJB2 si esprime in diversi tessuti ma la sua perdita produce solamente la sordità, si deve assumere che altre connexine potrebbero sostituire la connexina-26.  
La connexina-26 e la connexina-32 possono formare eterodimeri con canali di giunzioni gap funzionanti (30,31).  
Tuttavia, le mutazioni nel gene connexina-32 determinano la neuropatia Charcot-Marie-Tooth(26), mentre le mutazioni nel gene connexina-26 causano sordità(19,29).  
Questi risultati suggeriscono che l'espressione della connexina-26 nella coclea sia indispensabile per l'udito e che le altre connexine non possano compensare la perdita della connexina-26 nelle cellule uditive.  

Connexin 26 (GJB2) function 
Connexin 26 is a member of a large family of proteins involved in formation of gap junctions which allow the direct transfer of small molecules and ions between neighboring cells.  
Each cell contributes half of the gap junction, and is composed of the oligomeric assembly of connexons, which are composed of an hexamer of an integral membrane protein named connexins(23,24).  
A schematic example of connexons is given in Figure 2. Gap junctions are rare between mammalian neurons, but are common in non-neural cells, such glia, epithelial cells and smooth and cardiac muscle cells.  
There are more than 11 different types of connexins, each with tissue specificity and distinct physiological properties to the gap junction channels(24,25).  
At least two other connexin genes have been implicated in human diseases. Point mutations in the connexin-32 gene are associated with X-linked Charcot-Marie-Tooth neuropathy(26), and mutations in the connexin-43 gene are found in patients with heterotaxia and heart malformations.  
The involvement of gap junctions in the intracellular responses to sound was suggested more than 20 years ago (27,28). Within the auditory organ, gap junctions are located between the outer hair cells and supporting cells (including melanocytes), providing a morphological basis for the occurrence of intracellular responses to sound in supporting cells and for electric coupling of receptor cells.  
The endothelium of the scala media of the cochlea is involved in the production of a receptor response to the auditory stimulus and is separated from the endolymphatic space by tight junctions in the marginal cell layer, which is coupled by gap junctions.  
Very recently, immunohistochemical and ultrastructural analysis of connexin-26 in the rat cochlea showed that gap junctions in both epithelial and connective tissue cells are involved in recycling endolymphatic potassium ions through the sensory cells during the transduction of voltage gating in these channels. The identification of mutations in the GJB2 gene clearly confirm the crucial role of gap junctions in auditory transduction(19,29). 
Most of the mutations detected in the GJB2 gene lead to complete absence of connexin-26.(19,29) Considering that GJB2 is expressed in several tissues but its loss produce only deafness, it must be assumed that other connexins could substitute for connexin-26.  
Connexin-26 and connexin-32 can form heterodimers with functional gap junction channels (30,31).  
However, mutations in the connexin-32 gene cause the Charcot-Marie-Tooth neuropathy(26) while mutations in the connexin-26 gene only cause deafness (19,29). These findings suggest that the expression of connexin-26 in the cochlea is essential for audition and that other connexins can not compensate for the loss of connexin-26 in the auditory epithelial cells.

Paolo Gasparini, Paolo Fortina*, Xavier Estivill**, Salvatore Melchionda, Lucio Vigliaroli, Leopoldo Zelante  

Servizio di Genetica Medica e Divisione di ORL, IRCCS-Osp.CSS,  
S.Giovanni Rotondo (FG), Italy  
(*) Dept. of Pediatrics, Univ. of Pennsylvania, School of Medicine, The Children's Hospital  
of Philadelphia, PA, USA  
(**) Dept. of Molecular Genetics, IRO, Crta, Castelldefels Km 2,7, Hospitalet,  
Barcelona, Spain