PRIMER SEMESTRE 2011 I Clasificación de las

The sensory roles of cilia are essential for the normal functioning of many tissues, and the role of cilia is recognized in an emerging class of human genetic disorders (see Table 1 for an overview). Ciliary defects can cause a number of different phenotypes such as retinal degeneration, polycystic liver and kidney disease, situs inversus, hydrocephalus, anosmia, sinusitis, infertility and polydactyly. Many of these syndromes are often combined with global levels of cognitive impairment and developmental delay, indicating the importance of primary cilia in neurological function.

Location of cilia Properties/functions Phenotype Ciliary defect

Embryonic node 9+0; motile and non-motile/non- motile cilia senses leftward nodal flow created by motile cilia

Situs inversus Immotility

Ependymal cells in brain ventricles 9+2; motile/flow of cerebrospinal fluid Hydrocephalus Immotility Photoreceptor cells in the retina

9+0; non-motile/light perception Blindness Intraflagellar transport Apical knob on

olfactory neurons

9+2 (at the base, microtubule singlets distally); non-motile

Anosmia No cilia

Epithelium of the upper and lower airways

9+2; motile/mucus clearance Respiratory tract infections, rhinitis,

sinusitis

Immotility

Sperm 9+2; motile/sperm motility Male infertility Immotility Oviduct epithelium 9+2; motile/egg transport Female

infertility

Immotility

Epithelium of the kidney, bile, and pancreatic ducts

9+0; non-motile/mechanical sensing

Cyst formation Failure to signal

Unknown 9+0?; non-motile?/function in development and metabolism

Obesity, polydactyly, diabetes, cognitive impairment, other ailments Failure to signal?

Table 1: Cilia-related disorders - overview of the locations and properties of cilia that are known to be

Although the phenotypes of the various genetic diseases caused by ciliary defects are versatile, many of them belong to a group of retinal degenerations termed retinitis pigmentosa (RP). RP is a genetically heterogeneous form of blindness, with 33 known different gene loci that affects 1 out of 3,500 people worldwide (Berson, 1993). In a number of retinal degenerations, such as retinitis pigmentosa type 3 (RP3), Usher syndrome (USH), Bardet- Biedl syndrome (BBS) and Leber congenital amaurosis (LCA), protein complexes of the photoreceptor connecting cilium are involved.

RP3 is caused by mutations in the N-terminal domain of the X-linked gene retinitis pigmentosa GTPase regulator (RPGR) ((Roepman et al., 1996a), (Roepman et al., 1996b), (Meindl et al., 1996)). This domain shows significant homology to the regulator of chromosome condensation (RCC1) that functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Ran ((Roepman et al., 1996b), (Meindl et al., 1996)). The RCC1 homologuos domain has been identified to bind to PDEδ (Linari et al., 1999b) and to the RPGR interacting protein 1 (RPGRIP1) ((Boylan and Wright, 2000), (Roepman et al., 2000a), (Roepman et al., 2000b)). In photoreceptors, RPGR was shown to be localized to the axoneme and the basal body of connecting cilia via RPGRIP1 ((Hong et al., 2001), (Hong et al., 2004) (Khanna et al., 2005)).

Usher syndrome (USH) is the most frequent cause of combined deaf-blindness in humans. It is clinically and genetically heterogeneous and at least 11 genes are assigned to the three clinical USH types, namely USH1B-G, USH2A-C, USH3A ((Reiners et al., 2006), (Gerber et

al., 2006)). Although the colocalization of all USH1 and USH2 proteins at the photoreceptor synapse suggests a synaptic localization of the USH protein network in photoreceptors, a subset of USH proteins is also present at the ciliary apparatus of photoreceptor cells indicating a USH network here (Reiners et al., 2006).

The Bardet Biedl Syndrome (BBS) is a rare polygenetic and pleiotropic disorder associated with basal body and ciliary defects (Beales, 2005). To date, 8 genes have been identified to be associated with BBS ((Kaplan et al., 1990), (Blacque and Leroux, 2006),). Patients with this multifaceted disease can suffer from a large number of symptoms, including retinal (rod- cone) degeneration, obesity, cystic kidneys, learning disabilities, hearing loss and anosmia (Green et al., 1989), ((Beales et al., 1999), (Moore et al., 2005)).

1.3.2.1 Leber congenital amaurosis

Leber congenital amaurosis (LCA), first described by Theodore Leber in the 19th century (Leber, 1869), is the earliest and most severe form of all inherited retinal dystrophies, characterized by blindness or severe visual impairment from birth or within a few months of birth. LCA, diagnosed as bilateral congenital blindness, accounts for at least 5% of all retinal dystrophies and it is the most common congenial blindness in infants and children (Kaplan et al., 1990), (Perrault et al., 1999). Visual difficulties are usually noticed before the age of six months and the electroretinogram (ERG) is usually extinguished before the age of one year. Most of the LCA patients show nystagmus, photophobia, eye poking and sluggish pupils ((Foxman et al., 1985), (Heckenlively et al., 1988)). LCA is generally inherited in an autosomal recessive manner, although some autosomal dominant families have been described ((Heckenlively et al., 1988), (Sohocki et al., 1998)). To date, mutations in eight genes and three chromosomal loci have been associated with autosomal recessive forms of LCA (MIM 204000; RetNet): 1) Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) (Sohocki et al., 2000); 2) Crumbs homolog 1 (CRB1) (den Hollander et al., 2001); (Lotery et al., 2001); 3) cone-rod homeobox (CRX) (Freund et al., 1998); 4) guanylate cyclase 2D (GUCY2D) (Perrault et al., 1996); 5) RPE65 (Marlhens et al., 1997); 6) retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1) (Dryja et al., 2001); 7) RD3 (Friedman et al., 2006); and 8) RDH12 (Perrault et al., 2004).

Although the exact roles in eye development and homeostasis are not yet known for all LCA- associated genes, it is clear that their protein products have very diverse functions. AIPL1 and RPGRIP1 may be involved in retinal protein trafficking ((Hollander et al., 1999), (Sohocki et al., 2000), (Roepman et al., 2000a), (Boylan and Wright, 2000)). GUCY2D is essential for the recovery of the dark state after photoexitation of the photoreceptors by catalyzing the conversion of GTP to cGMP. RPE65 and RDH12 play a crucial role in the synthesis of 11- cis-retinal in the visual cycle ((Redmond et al., 1998), (Perrault et al., 2004)). CRB1 may be important for photoreceptor morphogenesis (Pellikka et al., 2002). RD3 is thought to be involved in transcription and splicing processes (Friedman et al., 2006) and CRX is a homeobox transcription factor required for the elongation of photoreceptor outer segments, and is essential for the phototransduction pathway (Furukawa et al., 1999).

1.3.2.1.1 Lebercilin

By homozygosity mapping the LCA5 gene on chromosome 6q14 was identified, which

encodes the novel ciliary protein, lebercilin (den Hollander et al., 2007). Homozygous nonsense and frameshift mutations in the LCA5 gene were identified in five LCA families. In

a sixth family the LCA5 transcript was completely absent due to a homozygous 1598-bp

deletion in the promoter region and the non-coding exon1. The patients presented a severe congenital retinal dystrophy consistent with LCA (congenital visual loss, nystagmus, high hyperopia, and a non-detectable electroretinogram) and showed normal neurological and renal function (Dharmaraj et al., 2000) or were documented with poor vision from early infancy, and were otherwise healthy as far as could be established without detailed clinical testing (Mohamed et al., 2003). All genes implicated in LCA are expressed exclusively or predominantly in the eye. An exception is LCA5 that intriguingly shows a wide expression

pattern throughout development, while the phenotype in patients is limited to the eye. Lebercilin localizes to the connecting cilia of photoreceptors and to the microtubules, centrioles and primary cilia of cultured mammalian cells. Truncation of lebercilin due to LCA5-associated null mutations abrogates the microtubule association (den Hollander et al., 2007).