J Appl Genetics (2011) 52:39–51 Table 1 Spectrum of the ultrastructural ciliary defects found in primary ciliary dyskinesia (PCD) Ultrastructural element Dynein arms Microtubules (MT) Whole cilium Defect Lack or reduction of outer dynein arms Lack or reduction of inner dynein arms Lack or reduction of outer and inner dynein arms Defects in peripheral MT number or structure Transposition, e.g., one or more peripheral MT doublets in the center of the cilium replace the central pair Discordant orientation of the central pair in adjacent cilia Swollen cilia Compound cilia Complete lack of cilia of cilia may display secondary defects of the ciliary ultrastructure (Afzelius 1981; Wisseman et al. 1981; Small- man and Gregory 1986; Pifferi et al. 2001). Ultrastructural defects of the internal ciliary anatomy can have various functional consequences, from a reduced ciliary beat frequency (CBF; below the normal range of 11–16 Hz) (Bush et al. 2007), to different changes in the pattern of ciliary movement. Nearly 20 various erratic beat patterns have been described so far (Chilvers et al. 2003); many of these patterns can be ascribed to a specific defect of the ciliary architecture (Afzelius 1979; Schidlow 1994). Absence of the dynein arms, the most severe defect, causes an eggbeater-like rotation of the cilium or movement of only the distal part of the axoneme, and a reduced CBF. Defects of radial spokes result in the increased axonemal flexibility, leading to a corkscrew rotational beat. Lack of the central MT doublet causes a shift from the whip-like to rotational beating, while transposition defects cause an increased rigidity of the proximal parts of cilia, resulting in a grabbing-like motion of their distal parts. In some cases, cilia with the normal internal architecture only quiver. On the other hand, cilia with the discordant orientation of the central MT pairs have an apparently normal beat pattern, but their beating is not synchronized (Castleman et al. 2009). Mutations in PCD Full characterization and understanding of the hereditary defects of the ciliary structure and function require identification of the causative mutations. In some cases, it is possible to link the ultrastructural and/or functional defects with the underlying protein defect(s) (Table 2). For 41 example, lack of the dynein arms is often the result of mutations in the dynein chains (Morillas et al. 2007), while abnormal localization of MTs may reflect mutations in radial spoke proteins (Castleman et al. 2009) (Table 2). In addition, defects in the ciliary ultrastructure can also be due to the mutations in proteins not directly involved in the structure, but only implicated in the assembly of the ciliary elements (Omran et al. 2008) (Table 2). The cases of immotile cilia with normal ultrastructure illustrate the problems in linking the ciliary defect with the mutation in one of the ∼200 polypeptides that constitute the cilium (Meeks and Bush 2000). Full classification, linking the molecular defect with the ultrastructural impairment, and, further, with the deficiency in the ciliary motility, is far from being complete. This would require knowledge of the mutations in the respective genes, but, so far, the genetics of PCD is not fully explained, due to the high genetic heterogeneity of the disease (Geremek and Witt 2004). Numerous linkage studies have indicated several genetic regions potentially involved in PCD pathogenesis (Geremek and Witt 2004; Blouin et al. 2000; Meeks and Bush 2000; Jeganathan et al. 2004). To date, only a few genes are confirmed to be directly associated with PCD pathogenesis (Escudier et al. 2009). Mutations in two of them, DNAI1 (9p13.3) and DNAH5 (5p15.2), are responsible for PCD in 30–40% of the affected families (Morillas et al. 2007). Both genes encode axonemal dyneins, intermediate chain 1 and heavy chain 5, respectively. Mutations in the genes which encode other proteins involved in the ciliary ultrastructure (DNAH11, DNAI2, TXNDC3, RSPH9, RSPH4A) or in the assembly of axoneme (KTU) were reported only in single PCD families (Escudier et al. 2009; Morillas et al. 2007). The genes responsible for the remaining ∼60% of PCD cases remain to be identified. In addition, mutations in the known PCD genes are characterized by the very high allelic heterogeneity—to date, approximately 80 mutations were found in DNH5 and about 20 in DNAI1 (Pennarun et al. 1999; Guichard et al. 2001; Zariwala et al. 2001, 2006; Olbrich et al. 2006; Hornef et al. 2006; Failly et al. 2008, 2009; Zietkiewicz et al. in press). In summary, the analysis of the genetic background of PCD is difficult, and often inconclusive, due to the extensive genetic and allelic heterogeneity of the disease. Diagnostic problems In light of the genetic heterogeneity of PCD, the practical diagnostic methods have to rely on the analysis of both ciliary structure and function. As detailed above, the most serious problem is that the primary and secondary defects largely overlap. The ambiguous relation between the ultrastructural defect, ciliary beat pattern, and clinical phenotype is the reason why the TEM assessment, although