Connexin mutations in hearing loss, dermatological and neurological disorders

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Connexin mutations in hearing loss, dermatological and neurological disorders Raquel Rabionet, Núria López-Bigas, Maria Lourdes Arbonès and Xavier Estivill Gap junctions are important structures in cell-to-cell communication. Connexins, the protein units of gap junctions, are involved in several human disorders. Mutations in β-connexin genes cause hearing, dermatological and peripheral nerve disorders. Recessive mutations in the gene encoding connexin 26 (GJB2) are the most common cause of childhood-onset deafness. The combination of mutations in the GJB2 and GJB6 (Cx30) genes also cause childhood hearing impairment. Although both recessive and dominant connexin mutants are functionally impaired, dominant mutations might have in addition a dominantnegative effect on wild-type connexins. Some dominant mutations in β-connexin genes have a pleiotropic effect at the level of the skin, the auditory system and the peripheral nerves. Understanding the genotype–phenotype correlations in diseases caused by mutations in connexin genes might provide important insight into the mechanisms that lead to these disorders. Published online: 10 April 2002

Raquel Rabionet Núria López-Bigas Maria Lourdes Arbonès Deafness Research Group, Genes and Disease Research Program, Center of Genomic Regulation, and Medical and Molecular Genetics Center, Cancer Research Institute, Barcelona, Spain. Xavier Estivill* Deafness Research Group, Genes and Disease Research Program, Center of Genomic Regulation, Medical and Molecular Genetics Center, Cancer Research Institute, Barcelona, Spain, and Program in Genetics and Genomic Biology, Research Institute, The Hospital for Sick Children, Toronto, Canada. *e-mail: xavier.estivill @crg.es

Connexins are the protein units that form gap junctions and have crucial functions in cell-to-cell communication in vertebrates. The involvement of connexins in human disorders has become increasingly evident in recent years. Mutations in connexin genes affect many organs and systems, and the same genes are associated with different disorders. Patients with β-connexin mutations have alterations at the level of the auditory system, peripheral nerves and the skin (Table 1) [1–8]. Gap junctions are regions of contact between cells and are widely distributed among different tissues. They are very important in intercellular communication, as they allow the direct passage of ions and other small molecules (<1 kDa), such as second messengers, between the cytoplasm of both cells. They consist of aggregates of dozens to thousands of transmembrane hemichannels known as connexons, which join to similar connexons on the neighbouring cell, forming hydrophilic channels that connect the cells [9] (Fig. 1). Connexons are formed by six transmembrane proteins known as connexins. There are 20 different types of connexins, which are classified according to their molecular weight or homology into α, β, γ and an unclassified group [10]. Most connexin genes are found in clusters, like the one formed by GJB2, GJB6 and GJA3 on chromosome 13q. The genomic structure of α- and β-connexins is very similar. They have two exons, the first one non-coding and a second one containing the whole coding region. Currently, six http://tmm.trends.com

human β-connexins (Cx26, Cx30, Cx30.3, Cx31, Cx31.1 and Cx32) are known (Table 2). The six connexin subunits that form the connexon can be of the same or different types. Thus, connexons can be homomeric (all have the same connexin) or heteromeric (formed by subunits of different connexins). In addition, the two connexons that join both cells can be identical (homotypic channel) or not (heterotypic channel) (Fig. 1). These types of channels have been demonstrated for Cx26 and Cx32, amongst other connexins [11,12]. Connexins have four transmembrane domains. The carboxy- and amino-termini of connexins, as well as the loop between the second and the third transmembrane domains, are cytoplasmic (Fig. 1). Recent studies of electron crystallography have shown that the four transmembrane segments of the six connexins in the connexon form two concentric rings. The internal ring forms the porus, whereas the external ring faces the lipidic layer of the membrane [13]. It has been proposed that the wall of the porus would be formed by the third transmembrane domain, conferring the hydrophylic permeability to the channel, and the two extracellular domains, participating in the interaction between the connexons of both cells [14]. Interactions between different domains of connexins could be crucial for the permeability of the gap junction channels. The major variability in the sequence of connexins is found at the intracytoplasmic domains, which could participate in the regulation of the channel activity. Gap junctions show a selectivity in the size and charge of the molecules, which is specific to the connexin subunits that form the connexon [15]. The opening of gap junction channels can be regulated by several factors. Some connexins are regulated by phosphorylation, voltage, acidification or several other factors [9,16,17]. Modulation of gap junction permeability can be of three different types (fast, intermediate and long-term), of which the long-term seems to be most affected by mutations [18]. Gap junctions in the inner ear

Gap junctions in the inner ear are found among and between the supporting cells of the organ of Corti, and also in the cells of the lateral wall of the cochlea [19]. Expression of Cx26 has been detected in all the

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Table 1. Hearing, dermatological and peripheral nerve disorders caused by mutations in -connexins Phenotype

OMIM

Inheritance

Gene

Deafness (DFNB1)

220290

AR

Deafness (DFNB1) Deafness (DFNA2)

220290 600101

AR AD

Deafness (DFNA3) EKV

601544 133200

AD AD

EKV

605425.001

AD

PPK

148350

AD

VS

124500

AD

HED

129500

AD

GJB2 (Cx26) Non-syndromic deafness with a broad phenotype, from mild to profound hearing loss, linked to chromosome 13q GJB6 (Cx30) Non-syndromic deafness linked to chromosome 13q GJB3 (Cx31) Non-syndromic progressive neurosensory deafness, with onset in second or third decade and profound deafness within ten years of onset and linked to chromosome 1p GJB2 (Cx26) Non-syndromic neurosensory deafness linked to chromosome 13q GJB2 (Cx26) EKV, characterized by the coexistence of erythematous areas and localized or generalized hyperkeratosis GJB3 (Cx31) With erythema gyratum repens characterized by rapidly migrating figurate erythema 1–2 cm wide in an annular, garland, or spiral arrangement GJB2 (Cx26) Associated with sensorineural hearing loss: PPK, a hyperkeratosis characterized by the thickening of the skin of palms and soles, and hearing impairment GJB2 (Cx26) Vohwinkel syndrome (VS), a PPK also named mutilating keratoderma, characterized by focal or diffuse hyperkeratosis of palms and soles, constrictions on the fingers and toes (pseudoainhum) sufficient to cause autoamputation, and moderate to severe deafness GJB6 (Cx30) HED or Clouston syndrome is characterized by palmoplantar hyperkeratosis, hair defects, nail hypoplasia and nail deformities GJB3 (Cx31) Hearing impairment associated with peripheral neuropathy GJB1 (Cx32) The X-linked form of Charcot-Marie-Tooth disease, a hereditary motor and sensory neuropathy characterized by distal muscle weakness and atrophy, severely reduced motor and sensory nerve conduction velocities and absent or diminished deep tendon reflexes

Deafness and neuropathy 603324.0009 AD CMTX 302800 X-linked

Definition

Abbreviations: AD, autosomal dominant; AR, autosomal recessive; CMTX, X-linked Charcot-Marie-Tooth disease; DFNA, dominant non-syndromic sensorineural deafness loci; DFNB, recessive non-syndromic sensorineural deafness loci; EKV, erythrokeratodermia variabilis; HED, hydrotic ectodermal dysplasia; OMIM, online Mendelian inheritance in man; PPK, palmoplantar keratodermia.

inner-ear cells presenting gap junctions. These are thought to be grouped in two systems, the epithelial cell gap-junction system and the connective tissue gapjunction system [19]. It has been hypothesized that both systems contribute to the maintenance of the high concentration of K+ ions in the endolymph, which is crucial for the normal function of the hearing system. K+ ions flow into the hair cells of the organ of Corti when these cells are activated by sound, and return to the endolymph through the gap junction systems to maintain the endolymphatic potential (Fig. 2). In addition to Cx26, other connexins are expressed in the inner ear. Cx30 was found in the same supporting cells of the rat cochlea as Cx26 [20], which also express Cx43 at much lower levels [21,22]. Connexins 26 and 30 were also identified in the inner ear of human and rat embryos [23,24]. Expression of another connexin (Cx31) in the fibrocytes of the spiral ligament and the spiral limbus of the developing mouse cochlea has also been reported [25] (Fig. 2). β-connexins and hearing impairment

Deafness is associated with mutations in different connexin genes. The gene encoding Cx26 (GJB2) is mutated in a large proportion (60% in some populations) of autosomal recessive childhood-onset cases of hearing impairment. Deafness-causing mutations in GJB2 were first identified in 1997 [7,8,26], and more than 60 different mutations have been reported since, including splice, nonsense, missense and frameshift mutations, and the deletion of one amino acid (Table 3; also see http://tmm.trends.com

http://www.iro.es/deafness/). Interestingly, mutations in GJB2 are not only found in autosomal recessive cases of childhood-onset deafness, but also in a large proportion (40%) of deafness cases that are apparently sporadic in onset [27]. This is a result of a high carrier frequency for certain recessive mutations in specific populations, such as 167delT in Ashkenazi Jews (4%) [28], 235delC in Japanese populations (1%) [29] and 35delG (2–3%) in some subsets of the Caucasoid populations [27]. The overall 35delG carrier frequency for Europe is 1 in 50, but it is significantly lower in north and central Europe (1 in 75) than in the Mediterranean region (1 in 35) [30]. This correlates with the different frequencies of childhood-onset deafness caused by mutations in GJB2 in these regions. The data obtained so far do not show a clear genotype–phenotype correlation for autosomal recessive deafness as a result of mutations in GJB2. There is a wide variability of phenotype in hearing impairment even among individuals with the same mutation and from the same family, with different ages of onset and apparent progression of the hearing impairment in some cases [31,32]. Although most mutations in GJB2 are found in autosomal recessive or apparently sporadic cases of deafness, some rare mutations (mainly missense) are detected in families with autosomal dominant inheritance. Some of these mutations cause non-syndromic deafness (W44C) [33], but others lead to deafness associated with skin disorders (see below). Interestingly, most mutations leading to autosomal dominant deafness are clustered in the first

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Fig. 1. The elements that integrate gap junction channels. (a) topology of a connexin protein in the cytoplasm membrane. CL, intracellular loop; COOH, carboxy terminus; E1 and E2, extracellular domains; M1–4, transmembrane domains; NH2, N terminus. (b) Different types of channels depending on the combination of connexin proteins that compose the two hemi-channels or connexons. Colours represent different connexins. (c) Gap junction plaque formed by thousands of connexons from two adjacent cells.

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a

Table 2. Nomenclature of genes and connexin proteins

(a)

Connexon interaction

Voltage control

E1

Genes Heterotypic compatibility

E2

Extracellular

M1

M2

M3

M4

Intracellular

Control by phosphorylation

CL

NH2 Voltage control polarity

Control by pH

COOH

(b) Connexon Channel

Homomeric heterotypic

Heteromeric heterotypic

(c)

Homomeric homotypic

Cytoplasm

Membrane

Membrane Cytoplasm TRENDS in Molecular Medicine

extracellular domain, or are located near the boundaries of transmembrane domains (Table 3). Mutation M34T in GJB2 raises interesting issues regarding its relation with disease. M34T has been found as a dominant change [7], but also as a polymorphism [34] or as a recessive mutation in association with other GJB2 mutations [35]. Also, another GJB2 mutation segregates with the deafness phenotype in the same family in which M34T was first identified, thus favouring the hypothesis that it is not a dominant mutation [36]. Despite the genetic data showing that M34T is either a polymorphism or an amino acid variant with variable penetrance, functional studies in both Xenopus oocytes and in HeLa cells have reported an impaired function for the mutated protein, and the Xenopus assay supported a dominant negative effect for this mutation [37,38]. The information generated so far for the mutation M34T supports the idea that this is a mutation with a low penetrance, which could have both dominant and recessive effects depending on several factors and the presence of other GJB2 changes. A missense mutation (T5M) in GJB6 (Cx30) has been described that causes autosomal dominant http://tmm.trends.com

GJB1 GJB2 GJB3 GJB4 GJB5 GJB6 GJA1 GJA3

Gap junction  1 Gap junction  2 Gap junction  3 Gap junction  4 Gap junction  5 Gap junction  6 Gap junction  1 Gap junction  3

Proteins Cx32 Cx26 Cx31 Cx30.3 Cx31.1 Cx30 Cx43 Cx46

Connexin 32 Connexin 26 Connexin 31 Connexin 30.3 Connexin 31.1 Connexin 30 Connexin 43 Connexin 46

a

Only the connexins referred to in the main text in the article are listed.

deafness of late onset in an Italian family [5] (Table 3). Although point mutations in GJB6 are not a common cause of deafness, recent findings indicate that a large deletion (342 kb) that encompasses the GJB6 gene, but does not include GJB2, causes deafness in homozygosity or in association with mutations in GJB2 [39] in a large proportion of Spanish subjects with nonsyndromic prelingual deafness. It is possible that this large deletion is the same as the one described in Israeli families [40] and that it is also a common cause of infant-onset deafness in other populations. Other connexin genes, in addition to GJB2 and GJB6, are associated with hearing impairment. Mutations in the gene encoding connexin 31 (GJB3), cause both nonsyndromic autosomal recessive deafness and autosomal dominantly transmitted non-syndromic bilateral high-frequencies hearing impairment [6,41] (Table 3). In addition, another mutation in GJB3 has also been described, this one causing neuropathy accompanied by mild deafness [42]. Several mutations in the GJB1 (Cx32) gene lead to X-linked Charcot-Marie-Tooth (CMTX) peripheral neuropathy associated with mild hearing impairment [1]. Finally, missense mutations in an α connexin gene, GJA1 (Cx43), have been reported to cause autosomal recessive deafness in several patients of African–American origin [22]. Loss of function and dysfunction of β-connexins in deafness

It is still not well understood how the different mutations in connexin genes lead to hearing impairment. Although for the recessively transmitted cases most mutations cause the absence of a given connexin, there are mutations that change amino acids at specific locations and these might have a dominant-negative effect. GJB2 mutations leading to the absence of Cx26 should affect the normal pattern of gap junctions in the cochlea and the organ of Corti, probably leading to the presence of a different type of connexon in the gap junctions (since Cx30 would still be present and could form homotypic gap junctions). It can be postulated that the abnormal pattern of gap junctions in the cochlea (either due to the absence of one connexin type or to the presence of an abnormal connexin) affects the K+ ions recycling pathway,

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β-connexins and skin diseases Cx26, Cx30 and Cx31 expression Cx26 and Cx30 expression Cx26 and Cx30 expression

Stria vascularis Low [K+] High [K+] Spiral ligament K

+

Spiral limbus

Hair cells Supporting cells TRENDS in Molecular Medicine

Fig. 2. Expression pattern of connexins Cx26, Cx30 and Cx31 in the cochlea. Also depicted is a model for the recycling of K+ ions through the gap junctions formed by three connexins (Cx26, Cx30 and Cx31) that are known to be mutated in patients with hearing impairment. Maintenance of a high concentration of K+ ions in the endolymph is required for the normal function of the hearing system.

which would affect the endolymphatic potential, which would finally result in non-functional hair cells. Dominant mutations could alter either the composition of the connexons (by affecting the formation of heteromeric or heterotypic channels) or the permeability or regulation of the connexons. It is unknown whether the absence of expression of Cx26, Cx30, or both, during the embryonic development of the ear could also cause a malformation of the inner ear in prelingual cases of deafness. This would probably have to be addressed with murine models lacking these connexins. Mutations in GJB3 (Cx31), which has a different cochlear expression pattern to Cx26 and Cx30, and is expressed in the auditory nerve [42], possibly affects the nervous conduction of the acoustic information, in addition to its malfunctioning in the cochlea. Interestingly, a stop codon mutation in GJB3 has been reported to behave as a dominant mutation [6]. It remains to be seen whether some connexins have dosage consequences in specific tissues. It is possible that the mechanism by which Cx32 leads to hearing impairment in patients with CMTX is via the neuropathy that affects the auditory nerve, in a similar way as it has been postulated for Cx31 [42]. It is unknown how mutations in GJA1 (Cx43) would affect the gap junction composition in the cochlea and therefore lead to deafness. It has been suggested that Cx26 and Cx43 are functionally incompatible and unable to form hetero-channels [43,44]. The interaction of these two connexins needs to be functionally evaluated to understand how they lead to hearing impairment and other disorders. http://tmm.trends.com

Gap junctional communication appears to play a crucial role in the coordinate control of epidermal keratinocyte differentiation [45]. In rodent skin, these intercellular communications are mediated by at least nine different connexins, including Cx30, Cx30.3, Cx31, Cx26 and Cx31.1, and expressed in a highly regulated manner (reviewed by Richard [46]). Human skin cells also express all known β-connexin genes. Human Cx26 is present in hair follicles and eccrine sweat glands and, at lower levels, in the basal keratinocytes of the palmar and plantar epidermis, and its expression is upregulated in damaged and psoriatic epidermis [46]. Expression of other connexin genes, including GJB1, GJB3, GJB4, GJB5 and GJB6, in human cultured keratinocytes and/or human skin has been identified either by immunohistochemical studies, northern blot or RT-PCR analysis [2,47,48]. Mutations in the genes encoding connexins have been identified for several skin disorders (Tables 1 and 3). These mutations lead to amino-acid changes and segregate in an autosomal dominant fashion, with variable penetrance. GJB3 was the first connexin gene reported to cause the autosomal dominant skin disorder erythrokeratodermia variabilis (EKV) [2]. All GJB3 mutations that cause EKV are missense changes and affect several domains of Cx31 and have variable clinical consequences. Two mutations, resulting in the replacement of a phylogenetically conserved glycine residue (G12R and G12D) in the cytoplasmic N-terminal tail of Cx31, were identified in two families with different severity and type of hyperkeratosis. Another mutation affects a cysteine residue (C86S) in the conserved second transmembrane domain of the protein and was identified in two unrelated families with obvious phenotypic differences. The mutation R42P co-segregates with EKV in two independent families with EKV [49,50], and the mutation F137L was observed in a sporadic case of severe EKV with generalized, hystrix-like hyperkeratosis in the lower extremities [49]. The F137L mutation affects a conserved phenylalanine residue, positioned at the third transmembrane domain of Cx31. A mutation in GJB4, which affects the corresponding residue in Cx30.3 (Table 3), was found to co-segregate with EKV, associated with erythema gyratum repens [4]. Supporting the crucial function of this amino acid residue, a similar mutation (F141L) in the Cx32 analogous residue has been reported to cause the X-linked form of CMTX [51] (see below). These mutations appear to affect amino-acid residues that are crucial for the proper assembly or gating polarity of connexons and are supposed to have a dominant inhibitory effect on the function of wild-type connexin channels [4,49]. In addition to hearing impairment, dominant mutations in GJB2 cause palmoplantar keratoderma (PPK) associated with sensorineural hearing loss. Maestrini et al. [52] reported a missense mutation, D66H, in three unrelated families with Vohwinkel

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Table 3. Mutations in -connexins and disease

a

Mutation

Affected domain

Disorder

Refs

Mutation

Affected domain

Disorder

Refs

IC1 IC1 TM1 TM1 TM1

CMTX CMTX CMTX CMTX CMTX

[68] [70] [61] [61,70] [72]

R107W V139M R164W R164N

IC2 TM3 EC2 EC2

CMTX CMTX CMTX CMTX

[69] [1] [61] [71]

GJB2 d delE42 d G59A d R75W d D66H d W44C
3170G to A 35delG M34T V37I W44X G45E E47X 167delT 176–191del16 W77X W77R

EC1 EC1 EC1 EC1 EC1 Exon 1 IC1 TM1 TM1 EC1 EC1 EC1 EC1 EC1 TM2 TM2

PPK with deafness PPK with deafness PPK with deafness VS DFNA3 DFNB1 DFNB1 DFNB1, DFNA3 DFNB1 DFNB1 DFNB1 DFNB1 DFNB1 DFNB1 DFNB1 DFNB1

[55] [54] [53] [52] [33] [32] [8] [7,35] [77] [78] [75] [76] [8] [79] [26] [7]

235delC V84L L90P 269insT V95M 299–300delAT 310del14 312del14 314del14 333–334delAA delE120 K122I Y136X R143W R184P S199F

TM2 TM2 TM2 TM2 IC2 IC2 IC2 IC2 IC2 IC2 IC2 IC2 IC2 TM3 EC2 TM4

DFNB1 DFNB1 DFNB1 DFNB1 DFNB1 DFNB1 DFNB1 DFNB1 DFNB1 DFNB1 DFNB1 DFNB1 DFNB1 DFNB1 DFNB1 DFNB1

[29] [73] [74] [32] [73] [75] [76] [32] [73] [73] [32] [78] [29] [80] [76] [78]

GJB3 d R180X d E183K 141del Ile I141V d 66delD

EC2 EC2 TM3 TM3 EC1

DFNA2 DFNA2 Recessive deafness Recessive deafness Peripheral neuropathy deafness

[6] [6] [41] [41] [42]

G12R G12D C86S F137L R42P 652del12

IC1 IC1 TM2 TM3 EC1 IC3

EKV EKV EKV EKV EKV EKV

[2] [2] [2] [49] [49,50] [49]

GJB4 F137L

TM3

EKV with erythema [4] gyratum repens

GJB6 T5M (GJB6D13S1830)

IC1 342-kb deletion

DFNA3 DFNA3

A88V G11R

TM1 IC1

HED HED

[3] [3]

b

GJB1

R15W R15N R22X R22N S26L c

[5] [39]

a

Other mutations can be found at http://www.iro.es/deafness. Most frequent mutations in GJB1, other mutations can be found at http://molgen-www.uia.ac.be/CMTMutations. c Dominant and most frequent recessive mutations in GJB2. b

Abbreviations: CMTX, X-linked Charcot-Marie-Tooth disease; DFNA, dominant non-syndromic sensorineural deafness; DFNB, recessive non-syndromic sensorineural deafness; EC, extracellular domain; EKV, erythrokeratodermia variabilis; HED, hydrotic ectodermal dysplasia; IC, Intracellular domain; PPK, palmoplantar keratodermia; TM, transmembrane domain; VS, Vohwinkel syndrome. d These are dominant mutations. All the others are recessive.

syndrome (VS). The same mutation was found to co-segregate with the hyperkeratosis reported in another family with a mild form of VS and profound deafness [36]. Three additional dominant mutations in GJB2, R75W [53], G59A [54] and DelE42 [55], have been identified in families with PPK associated with profound deafness. The mutation R75W seems to present a variable penetrance regarding skin disease with some individuals even lacking skin disease symptoms [53,56,57]. All these mutations affect amino-acid residues positioned in the highly conserved first extracellular domain (G59A and D66H), or in the boundary of this domain with the first (DelE42) or second (R75W) transmembrane http://tmm.trends.com

domains, suggesting a correlation between the location of the mutation and the phenotype. In vitro functional analysis performed in paired Xenopus oocytes demonstrated that D66H, DelE42 and R75W mutant forms have a deleterious effect on the channel activity mediated by wild-type Cx26, and also on the channel activity mediated by Cx43, another epidermal connexin that is also mutated in some deaf patients. This trans-dominant effect was not produced by the Cx26 dominant mutation W44C that causes only deafness [33], explaining in part why mutations that are closely positioned in the same connexin cause different clinical manifestations [55]. In view of the results obtained for GJB2 mutation

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M34T in this functional assay, this type of analysis as a model for human disease should be considered with caution. It is likely that there are several amino-acid changes that have a wide variable penetrance, depending on other factors, including additional variants in other connexins. Another autosomal dominant skin disorder caused by mutations in a β-connexin gene, GJB6, is hydrotic ectodermal dysplasia [3]. Two missense mutations, G11R and A88V, affecting conserved amino-acid residues positioned in the cytoplasmatic N-terminal end and in the second transmembrane domains of Cx30 respectively, have been identified in affected unrelated families of many ethnic origins presenting an identical hydrotic ectodermal dysplasia (HED) phenotype. The importance of the mutated glycine in Cx30 for proper skin gap-junction communication is supported by the fact that an analogous mutation in the neighbouring glycine residue in Cx31 causes EKV. β-connexins and peripheral neuropathy

Acknowledgements Research of the Deafness Research Group is supported by the Fundació La Marató de TV3 and the FIS (Fondo de Investigaciones Sanitarias de la Seguridad Social, Ministerio de Sanidad). R. Rabionet and N. LópezBigas are supported by the FIS (BEFI 98/9207 and 00/9379). X. Estivill and M.L. Arbonés are supported by the Servei Català de la Salut and the DURSI (Generalitat de Catalunya).

Two connexin genes, GJB1 and GJB3, are known to be expressed in the peripheral nerves and to cause peripheral neuropathy when mutated (Table 3). There are several types of CMT caused by mutations in different genes, of which the X-linked form is caused by mutations in GJB1. The pattern of inheritance of CMTX is both dominant and recessive X-linked, with males having a severe phenotype and carrier females showing a milder form of the disease. More than 240 mutations in GJB1 have been reported in patients with CMTX since it was first reported in 1993 [1] (http://molgen-www.uia.ac.be/CMTMutations). Although most of these mutations are missense, other types of mutations have also been reported: 19 nonsense mutations, 21 frameshift mutations and some large in-frame deletions and insertions. There are also two mutations in the promoter region and one mutation in the noncoding exon 1, three cases of deletion of the complete coding region [58–60] and a case of complex rearrangement of the gene [51]. Several attempts have been made to correlate genotype with phenotype in CMT families with GJB1 mutations [60–63], and although no definitive conclusions can be extracted from these studies, it seems that the missense mutations show a milder clinical phenotype, whereas nonsense mutations and frameshift deletion/insertions show more severe phenotypes. Milder forms of the disease are also detected in carrier females. Gjb1 knockout mice revealed no signs of peripheral neuropathy at early age [64], but at a later age they developed a pathological phenotype similar to that seen in CMTX patients with GJB1 point mutations [65]. Expression studies performed by Bergoffen et al. [1] reported that human Cx32 is present in Schmidt–Lanterman incisures and nodes of Ranvier of myelinating Schwann cells. It is believed that a Schwann cell forms gap junctions with itself (reflexive gap junctions), between adjacent paranodal loops and http://tmm.trends.com

between adjacent cytoplasmic loops of the non-compact myelin at the incisures [66]. These gap junctions would greatly shorten the diffusion pathway between inner and outer parts of the Schwann cells. Experiments of radial diffusion of low-molecular-weight dyes across the myelin sheath showed no interruption of dye transfer in myelinating Schwann cells from Gjb1-null mice, indicating that at least another connexin participates in forming gap junctions in these cells [67]. This connexin might be Cx31, which we reported to be expressed in mouse auditory and sciatic nerves in a pattern similar to that of mouse Cx32 [42]. Recently, we have also reported a dominant mutation in GJB3 (D66del) present in a family with a rare type of peripheral neuropathy characterized by a wide range of disease severity, from asymptomatic cases to subjects with chronic skin ulcers in their feet and osteomyelitis leading to amputations. The neuropathy in the patients with the mutation in GJB3 was accompanied with mild, often asymmetrical, hearing impairment [42]. Conclusion

The identification of mutations in connexin genes has resulted in a surprising new area of research, diagnosis and potential therapy for several human genetic disorders affecting the skin, sensory systems and the peripheral nerves. In agreement with the multifunctional nature of connexin gap junctions, different mutations in the same connexin lead to disorders affecting different tissues. In addition, mutations in different connexins can cause the same or similar disorders. Whereas most cases of prelingual deafness are caused by a lack of specific connexins (Cx26 or Cx30), adult-onset hearing loss and skin disorders are mainly the result of missense changes in several connexins with dominant-negative effects. Several mutations causing autosomal dominant deafness or skin disorders appear to affect amino-acid residues that are crucial for the proper assembly or gating polarity of connexons and are predicted to have a dominant inhibitory effect on the function of wild-type connexin channels. Some of these amino-acid changes show a wide variability in their penetrance with regard to disease, which is probably a result of interactions with other connexins or to other factors that are capable of compensating for their detrimental effects. The development of murine models and the study of naturally occurring mutants that are being discovered in humans should provide a better understanding of the pathophysiology of these disorders. The current understanding of the molecular basis of disorders resulting from mutations in connexin genes allows the use of diagnostic tools for patients and families with these disorders. Future research should be focused on the development of treatments and should result in prevention and cure of chronic disorders for which just a few years ago no solutions could be foreseen.

Review

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Triggering caspase-independent cell death to combat cancer Ida S. Mathiasen and Marja Jäättelä Caspase-mediated apoptosis is a major hindrance to tumour growth and metastasis. Accordingly, defects in signalling pathways leading to the activation of caspases are common in tumours. Moreover, many tumour cells can unexpectedly survive the activation of caspases. As a result, caspaseindependent cell death programmes are gaining increasing interest among cancer researchers. The heterogeneity of cancer cells with respect to their sensitivity to various death stimuli further emphasizes the need for additional death pathways in the therapeutic control of cell death. An understanding of the molecular control of alternative death pathways is beginning to emerge, being comparable with that of the molecular anatomy of apoptosis at the time of the discovery of caspases less than a decade ago. Here, newly discovered triggers and molecular regulators of alternative cell death programmes are reviewed and their potential in future cancer therapy is discussed. Published online: 10 April 2002

A delicate balance between cell division and PROGRAMMED CELL DEATH (PCD) (see Glossary) is required for the

Ida S. Mathiasen Marja Jäättelä∗∗ Apoptosis Laboratory, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark. ∗e-mail: [email protected]

maintenance of tissue homeostasis. The best-defined cell death programme is known as APOPTOSIS, and it is characterized by a series of morphological changes, the most characteristic features of which are the condensation of chromatin into compact, almost geometrical, figures and the disintegration of the cell into small fragments that are engulfed by nearby cells without inciting inflammation [1–3]. The discovery of a family of cysteine proteases, the caspases, revolutionized cell death research by http://tmm.trends.com

providing the first evolutionarily conserved molecular effectors of PCD. Myriad reports have subsequently documented caspase activation in a wide array of death models and, until recently, caspase activation has been considered a hallmark not only of apoptosis but of all PCD. However, new data show that PCD can occur in the complete absence of caspases [4,5]. Furthermore, although the inhibition of caspase activation often delays cell death, it does not necessarily confer longterm survival. Instead, it might reveal, or in some cases even enhance, underlying caspase-independent death programmes [4,5]. According to the nuclear morphology of dying cells, such alternative death programmes can be divided into APOPTOSIS-LIKE PCD and NECROSIS-LIKE PCD [4,5]. Thus, PCD can certainly occur in the absence of caspase activation, whereas death fulfilling the strictest morphological criteria of apoptosis might require caspases. Caspase-dependent apoptosis

A proper understanding of alternative death pathways requires a short introduction to classic, caspase-mediated apoptosis signalling, which is discussed in more depth elsewhere [1–3]. Caspases are cysteine proteases that exist in cells as inactive zymogens that can be rapidly activated by proteolytic processing or by binding to a cofactor. As the activation of few so-called initiator caspases

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