*2.5. Cells Proliferation Assay*

The MTS cell proliferation assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega) was used to determine metabolic activity in CaCo-2, CoLo-205 cell lines. Briefly, cells were seeded into 96-well plates at a density of 2 × 103 cells/well. After 24 h, the cells were treated with free drug (5-FU), empty LPs, 5-FU/LPs and anti-FZD10/5-FU/LPs, at 5-FU concentrations ranging from 1 µmol to 10 µmol (5-50 µM, in terms of total lipid concentration for the liposomal formulations), for 24, 48 or 72 h. After cell incubation with the different samples, cells were treated with the MTS tetrazolium compound for three additional hours and the absorbance was measured at a wavelength of 490 nm using a Perkin Elmer Victor Plate Reader (Mechelen, Belgium).


**3.1 Most common genetic causes of autosomal dominant, adult onset ALS**

The three most common genetic causes of FALS, together accounting for approximately 30% of cases are mutation of the *SOD1*, *TARDBP* and fused in sarcoma (*FUS*) genes.

### **3.1.1 ALS1: Cu-Zn superoxide dismutase 1 (***SOD1***)**

The first genetic cause of familial ALS was identified by Rosen and colleagues (Rosen et al 1993) when, following analysis of FALS pedigrees demonstrating linkage to chromosome 21, mutations were identified in the *SOD1* gene. Since then, over 150 mutations have been described throughout the 5 exons encoding the gene consisting predominantly of missense mutations, although nonsense mutations, insertions and deletions have also been described (Lill et al 2011). The frequency of *SOD1* mutations is widely reported to be 20% of FALS cases, though this varies across European and North American populations, from 12% in Germany to 23.5% in USA (Andersen 2006). Whilst the majority of mutations are inherited in an autosomal dominant manner, in Scandinavia the p.D90A mutation is polymorphic, (0.5-5% of Scandinavian populations), with the disease manifesting only in individuals who are homozygous (Andersen et al 1995). However, this inheritance pattern is not attributable to the specific amino acid substitution, as p.D90A has been shown to be inherited as an autosomal dominant mutation in other populations. Mutations in *SOD1* have also been identified in sporadic ALS, albeit at lower frequencies, suggesting that some mutations have reduced penetrance. This has been shown in a family where the p.I113T mutation shows age-related penetrance (Lopate et al 2010).

Clinically, *SOD1* mutations are not associated with a distinctive phenotype. Individuals with *SOD1*-related ALS predominantly manifest with limb onset ALS, with symptoms more likely to start in the lower limbs (rather than upper limbs). However, bulbar onset is seen in approximately 7% of *SOD1*-related cases (ALSoD database: http:alsod.iop.kcl.ac.uk). Whilst duration of disease varies widely among *SOD1* mutations, even within members of the same family with the same mutation, the p.A4V mutation has been shown to be associated with a rapid disease progression and only 1-2 years survival (Andersen 2006). In contrast to the indistinguishable clinical phenotype, *SOD1*-related ALS cases appear to have a characteristic pathology distinguished by SOD1 positive, but TDP-43 negative, protein inclusions (Mackenzie et al 2007).

The mature SOD1 protein is a homodimer of 153 amino acid subunits. This free radical scavenging protein converts the superoxide anion to hydrogen peroxide; this in turn is converted to water and oxygen by glutathione peroxidise or catalase. Mutations in *SOD1* cause a toxic gain of function in the resulting mutant protein, though the mechanism(s) by which this brings about selective neurodegeneration of the motor neurones appears to be a complex interplay between multiple interacting pathomechanisms. The main hypotheses involve either an altered redox function or misfolding of the protein leading to aggregation (Rakhit & Chakrabartty 2006). Interestingly, not only have SOD1 positive aggregations been seen in SALS spinal cord, recent work has also shown that a conformation specific antibody raised against mutant SOD1 binds oxidised, but not normal, wild-type SOD1 in a subset of SALS cases thereby linking both *SOD1*-ALS and SALS (Bosco et al 2010).

Identification of *SOD1* led to the generation of many cellular and animal models which mirror aspects of the disease process and enable mechanistic insights and therapeutic approaches to be investigated. Current pathogenic mechanisms associated with mutant

Genetics of Familial Amyotrophic Lateral Sclerosis 521

that over-expression of not only mutant, but also wild-type *TARDBP* is toxic and that TDP-

Mutations in *FUS*, also referred to as translocated in sarcoma (*TLS*), were initially identified both through linkage analysis in a large Cape Verde pedigree manifesting with autosomal recessive ALS and in several autosomal dominant ALS families linked to chr16 (Kwiatkowski et al 2009; Vance et al 2009). Similarly to *TARDBP*, mutations in this second RNA binding protein gene are clustered, rather than spread across the 15 exons encoding FUS; a third occur in exons 5-6 encoding the glycine-rich region and two thirds in exon 13-14 encoding the arginine-glycine-glycine (RGG)-rich domain and the nuclear localisation signal. *FUS* mutations have been shown to account for a further 4-5% of FALS cases (Hewitt

Clinically, *FUS* mutations are associated with limb onset ALS; no bulbar onset cases with *FUS* mutations have been reported to date (ALSoD database: http:alsod.iop.kcl.ac.uk). There is a large range in the age of onset, from 26-80 years and as with *TARDBP* mutations, to date no correlations are evident between specific mutations and clinical characteristics. Pathologically, however, *FUS*-related cases have shown distinctive FUS-positive, TDP-43 negative inclusions. Specifically, those cases with basophilic inclusions and compact neuronal cytoplasmic FUS-positive inclusions had an earlier onset than those ALS cases with skein-like neuronal cytoplasmic inclusions, in whom glial cytoplasmic inclusions were

FUS is an RNA/DNA binding protein, which shuttles between the nucleus and cytoplasm. The 526 amino acid protein contains multiple protein domains, including a glutamineglycine-serine-tyrosine rich domain at the N-terminus, involved in transcriptional activation of oncogenic fusion genes involving *FUS*, a glycine-rich region, a nuclear export signal, an RNA recognition motif and two arginine-glycine-glycine motifs flanking a zinc finger motif. At the C-terminus resides the nuclear localisation signal. Mutations within this region have been shown to disrupt transportin mediated transport of FUS into the nucleus, and cause the formation of FUS containing stress granules in the cytoplasm (Dormann et al 2010; Ito et al 2011a). In contrast, mutations in the glycine-rich region of FUS have yet to demonstrate pathogenicity. The normal function of FUS is poorly understood, though there is evidence for its involvement in alternative splicing, miRNA processing and transportation of mRNA to the dendrites for localised translation. Of the animal and cellular models generated to investigate the mechanisms of mutant FUS, a rat model over-expressing human p.R521C shows progressive paralysis, axonal degeneration and loss of neurones in 1-2 months old rats, whilst rats over-expressing wild-type FUS are pre-symptomatic at 1 year, though they do show learning and memory deficits and loss of cortical neurons (Huang et al 2011). Furthermore, drosophila and yeast models demonstrate FUS toxicity is due to accumulation

of mutant protein in the cytoplasm (Kryndushkin et al 2011; Lanson et al 2011).

**3.2.1 ALS8: Vesicle associated membrane protein (VAMP) associated protein B**

ALS8 was first described in a large Brazilian kindred comprised of 28 Caucasian affected male and female family members distributed across four generations. Patients in this family had a

**3.2 Rarer genetic causes of autosomal dominant, adult onset ALS**

43 is essential for development, as knockout models show embryonic lethality.

**3.1.3 ALS6: Fused in sarcoma (***FUS***)**

et al 2010; Mackenzie et al 2010).

**(***VAPB***)**

also seen (Baumer et al 2010; Mackenzie et al 2011).

SOD1 include oxidative stress, excitotoxicity, protein aggregation, mitochondrial dysfunction, endoplasmic reticulum stress, inflammatory cascades, involvement of nonneuronal cells and dysregulation of axonal transport. Each of these mechanisms has also been shown to play a role in SALS, demonstrating the relevance of the *SOD1* models to the disease as a whole (Ferraiuolo et al 2011). Therefore, although to date therapeutic agents which have shown promising results in the SOD1 transgenic mouse models have yet to show a beneficial effect in human trials (Benatar 2007), the generation and continued use of these models has greatly extended our knowledge of ALS.

### **3.1.2 ALS10: Transactive response (TAR) DNA binding protein (***TARDBP***)**

The identification of TAR-DNA binding protein (TDP-43) as the major component of ubiquitinated cytoplasmic inclusions in ALS (and FTD) (Neumann et al 2006) led to the gene encoding this protein, *TARDBP*, to be screened in cohorts of FALS. Following the initial report of mutations being identified in exon 6 of the gene (Sreedharan et al 2008), a further 39 nucleotide substitutions have been published; the vast majority of which are in exon 6 and encode non-synonymous changes. The frequency is reported to be 4-5% of FALS cases (Kirby et al 2010; Mackenzie et al 2010), with mutations inherited in an autosomal dominant manner.

Clinically, *TARDBP*-related ALS presents as a classical adult-onset form of ALS; 73% of cases manifest with limb onset and there is a wide range in the age of onset (30-77 years) and disease duration, even in cases carrying the same mutation (e.g. p.M337V), (ALSoD database: http:alsod.iop.kcl.ac.uk). Perhaps the most distinctive feature commented upon, is the absence of dementia in these patients, despite several reports of *TARDBP* mutations in cases of FTD (Borroni et al 2009; Kovacs et al 2009). Neuropathologically, there is no distinction between *TARDBP*-related ALS and SALS cases, with both showing skein and compact ubiquitinated inclusions.

*TARDBP* encodes several isoforms of a predominantly nuclear protein, of which TDP-43 is the most prevalent. TDP-43 contains 2 RNA recognition motifs (RRM), a nuclear localisation and nuclear export signal, as well as a glycine-rich region in the C-terminus, which is encoded by exon 6. TDP-43 is involved in a variety of roles in the nucleus, including regulation of transcription, RNA splicing, microRNA (miRNA) processing and stabilisation of mRNA. Reports have recently identified RNA molecules which bind to TDP-43 in whole cell extracts using cross linking and immunoprecipitation (CLIP) methodologies (Polymenidou et al 2011; Sephton et al 2011; Tollervey et al 2011; Xiao et al 2011). This has established over 4000 TDP-43 binding targets, including ALS-related genes *FUS* and vasolin containing protein (*VCP*), as well as other RNA processing genes. One target which has been confirmed is the *TARDBP* mRNA. TDP-43 regulates its own transcription by binding to the 3'UTR region of the *TARDBP* mRNA and promoting mRNA instability (Budini & Buratti 2011). In addition, TDP-43 has been shown to interact with mutant, but not wild-type SOD1 mRNA, thereby linking the two distinct genetic pathogenic mechanisms (Higashi et al 2010). In ALS, both in *TARDBP*-related ALS and SALS, TDP-43 is seen to mislocalise to the cytoplasm and form either compact or skein like protein inclusions. It is currently unclear whether a loss of nuclear function or a gain of toxic function (or both) causes motor neuronal cell death. Numerous cellular and animal models for *TARDBP*-related ALS have been generated in multiple species, in order to investigate the mechanisms of TDP-43 associated neurodegeneration (Joyce et al 2011). What is evident from this body of work is that over-expression of not only mutant, but also wild-type *TARDBP* is toxic and that TDP-43 is essential for development, as knockout models show embryonic lethality.

#### **3.1.3 ALS6: Fused in sarcoma (***FUS***)**

**3.1.2 ALS10: Transactive response (TAR) DNA binding protein (***TARDBP***)**

these models has greatly extended our knowledge of ALS.

manner.

compact ubiquitinated inclusions.

520 Amyotrophic Lateral Sclerosis

SOD1 include oxidative stress, excitotoxicity, protein aggregation, mitochondrial dysfunction, endoplasmic reticulum stress, inflammatory cascades, involvement of nonneuronal cells and dysregulation of axonal transport. Each of these mechanisms has also been shown to play a role in SALS, demonstrating the relevance of the *SOD1* models to the disease as a whole (Ferraiuolo et al 2011). Therefore, although to date therapeutic agents which have shown promising results in the SOD1 transgenic mouse models have yet to show a beneficial effect in human trials (Benatar 2007), the generation and continued use of

The identification of TAR-DNA binding protein (TDP-43) as the major component of ubiquitinated cytoplasmic inclusions in ALS (and FTD) (Neumann et al 2006) led to the gene encoding this protein, *TARDBP*, to be screened in cohorts of FALS. Following the initial report of mutations being identified in exon 6 of the gene (Sreedharan et al 2008), a further 39 nucleotide substitutions have been published; the vast majority of which are in exon 6 and encode non-synonymous changes. The frequency is reported to be 4-5% of FALS cases (Kirby et al 2010; Mackenzie et al 2010), with mutations inherited in an autosomal dominant

Clinically, *TARDBP*-related ALS presents as a classical adult-onset form of ALS; 73% of cases manifest with limb onset and there is a wide range in the age of onset (30-77 years) and disease duration, even in cases carrying the same mutation (e.g. p.M337V), (ALSoD database: http:alsod.iop.kcl.ac.uk). Perhaps the most distinctive feature commented upon, is the absence of dementia in these patients, despite several reports of *TARDBP* mutations in cases of FTD (Borroni et al 2009; Kovacs et al 2009). Neuropathologically, there is no distinction between *TARDBP*-related ALS and SALS cases, with both showing skein and

*TARDBP* encodes several isoforms of a predominantly nuclear protein, of which TDP-43 is the most prevalent. TDP-43 contains 2 RNA recognition motifs (RRM), a nuclear localisation and nuclear export signal, as well as a glycine-rich region in the C-terminus, which is encoded by exon 6. TDP-43 is involved in a variety of roles in the nucleus, including regulation of transcription, RNA splicing, microRNA (miRNA) processing and stabilisation of mRNA. Reports have recently identified RNA molecules which bind to TDP-43 in whole cell extracts using cross linking and immunoprecipitation (CLIP) methodologies (Polymenidou et al 2011; Sephton et al 2011; Tollervey et al 2011; Xiao et al 2011). This has established over 4000 TDP-43 binding targets, including ALS-related genes *FUS* and vasolin containing protein (*VCP*), as well as other RNA processing genes. One target which has been confirmed is the *TARDBP* mRNA. TDP-43 regulates its own transcription by binding to the 3'UTR region of the *TARDBP* mRNA and promoting mRNA instability (Budini & Buratti 2011). In addition, TDP-43 has been shown to interact with mutant, but not wild-type SOD1 mRNA, thereby linking the two distinct genetic pathogenic mechanisms (Higashi et al 2010). In ALS, both in *TARDBP*-related ALS and SALS, TDP-43 is seen to mislocalise to the cytoplasm and form either compact or skein like protein inclusions. It is currently unclear whether a loss of nuclear function or a gain of toxic function (or both) causes motor neuronal cell death. Numerous cellular and animal models for *TARDBP*-related ALS have been generated in multiple species, in order to investigate the mechanisms of TDP-43 associated neurodegeneration (Joyce et al 2011). What is evident from this body of work is Mutations in *FUS*, also referred to as translocated in sarcoma (*TLS*), were initially identified both through linkage analysis in a large Cape Verde pedigree manifesting with autosomal recessive ALS and in several autosomal dominant ALS families linked to chr16 (Kwiatkowski et al 2009; Vance et al 2009). Similarly to *TARDBP*, mutations in this second RNA binding protein gene are clustered, rather than spread across the 15 exons encoding FUS; a third occur in exons 5-6 encoding the glycine-rich region and two thirds in exon 13-14 encoding the arginine-glycine-glycine (RGG)-rich domain and the nuclear localisation signal. *FUS* mutations have been shown to account for a further 4-5% of FALS cases (Hewitt et al 2010; Mackenzie et al 2010).

Clinically, *FUS* mutations are associated with limb onset ALS; no bulbar onset cases with *FUS* mutations have been reported to date (ALSoD database: http:alsod.iop.kcl.ac.uk). There is a large range in the age of onset, from 26-80 years and as with *TARDBP* mutations, to date no correlations are evident between specific mutations and clinical characteristics. Pathologically, however, *FUS*-related cases have shown distinctive FUS-positive, TDP-43 negative inclusions. Specifically, those cases with basophilic inclusions and compact neuronal cytoplasmic FUS-positive inclusions had an earlier onset than those ALS cases with skein-like neuronal cytoplasmic inclusions, in whom glial cytoplasmic inclusions were also seen (Baumer et al 2010; Mackenzie et al 2011).

FUS is an RNA/DNA binding protein, which shuttles between the nucleus and cytoplasm. The 526 amino acid protein contains multiple protein domains, including a glutamineglycine-serine-tyrosine rich domain at the N-terminus, involved in transcriptional activation of oncogenic fusion genes involving *FUS*, a glycine-rich region, a nuclear export signal, an RNA recognition motif and two arginine-glycine-glycine motifs flanking a zinc finger motif. At the C-terminus resides the nuclear localisation signal. Mutations within this region have been shown to disrupt transportin mediated transport of FUS into the nucleus, and cause the formation of FUS containing stress granules in the cytoplasm (Dormann et al 2010; Ito et al 2011a). In contrast, mutations in the glycine-rich region of FUS have yet to demonstrate pathogenicity. The normal function of FUS is poorly understood, though there is evidence for its involvement in alternative splicing, miRNA processing and transportation of mRNA to the dendrites for localised translation. Of the animal and cellular models generated to investigate the mechanisms of mutant FUS, a rat model over-expressing human p.R521C shows progressive paralysis, axonal degeneration and loss of neurones in 1-2 months old rats, whilst rats over-expressing wild-type FUS are pre-symptomatic at 1 year, though they do show learning and memory deficits and loss of cortical neurons (Huang et al 2011). Furthermore, drosophila and yeast models demonstrate FUS toxicity is due to accumulation of mutant protein in the cytoplasm (Kryndushkin et al 2011; Lanson et al 2011).

### **3.2 Rarer genetic causes of autosomal dominant, adult onset ALS 3.2.1 ALS8: Vesicle associated membrane protein (VAMP) associated protein B (***VAPB***)**

ALS8 was first described in a large Brazilian kindred comprised of 28 Caucasian affected male and female family members distributed across four generations. Patients in this family had a

Genetics of Familial Amyotrophic Lateral Sclerosis 523

deficits (Chow et al 2007). However, in one CMT4J pedigree there was a later onset of disease, with predominantly motor symptoms, similar to ALS (Zhang et al 2008). Screening of a North European cohort of FALS patients revealed 5 heterozygous mutations, resulting in either complete or significant loss of protein (Chow et al 2009). In general, cases of ALS11 are associated with a rapidly progressive disease course of approximately 1-2 years, early

*FIG4* encodes the phosphatidylinositol 3,5-bisphosphate 5-phosphatase which controls the cellular abundance of PI(3,5)P2, a signalling lipid that mediates retrograde trafficking of endosomal vesicles to the Golgi apparatus (Michell & Dove 2009). It remains unclear as to whether the deleterious variants of *FIG4* exert an effect by a dominant negative mechanism or through a partial loss of function, as seen in CMT4J patients (Chow et al 2007). Human motor neurones are considered to be particularly susceptible to disruptions in this transport network because of their high membrane component turnover demands from long axonal

Homozygosity mapping using 4 FALS cases demonstrated linkage to chr10p13 and subsequently mutations in *OPTN*, a gene previously linked to primary open-angle glaucoma (POAG), were identified in these cases (Maruyama et al 2010; Rezaie et al 2002). Mutations were initially found in both homozygous and heterozygous states. However, mutation screening of subsequent cohorts have identified only heterozygous mutations in ALS cases, occurring at a frequency of 3.4% in Japanese populations whilst this was at much lower frequencies in European and North American populations (Belzil et al 2011; Del Bo et al 2011). Interestingly, *OPTN*-related ALS is characterised by a lower limb onset with upper

The gene encodes the ubiquitously expressed optic neuropathy inducing protein which localises to the perinuclear region of the cytoplasm, where it is known to associate with the Golgi apparatus, and plays a key role in a number of biological processes, including vesicular trafficking, signal transduction and gene expression (Chalasani et al 2008). It is anticipated where mutations are inherited in an autosomal recessive manner, that reduced protein levels result in neurotoxicity through a loss of function mechanism. Conversely, the heterozygous missense substitution is predicted to exert a dominant negative effect. Examination of autopsy derived spinal cord tissue revealed extensive OPTN staining of TDP-43 positive intracytoplasmic hyaline inclusion bodies, although this was not replicated

Following linkage analysis, a rare heterozygous missense mutation in the *DAO* gene, located on chr12q22-23, has been reported to segregate with disease in a single three generational Caucasian pedigree, though there was evidence of incomplete penetrance (Mitchell et al 2010). Those affected showed a rapidly progressive form of classical ALS with a mean disease duration of 21 months. Early bulbar involvement was apparent with limited signs of cognitive impairment. Interestingly, post-mortem immunohistochemical analysis on spinal cord tissue revealed no evidence of TDP-43 positively labelled inclusion bodies within the nuclear or cytoplasmic fractions of residual motor neurones which are normally a

bulbar involvement and minimal cognitive dysfunction.

processes over many decades (Ferguson et al 2010).

motor neurone involvement and a slow clinical progression.

in a subsequent study (Hortobagyi et al 2011; Ito et al 2011b).

**3.2.4 ALS12: Optineurin (***OPTN***)**

**3.2.5 D-amino acid oxidase (***DAO***)**

distinguishing feature of ALS pathology.

characteristic clinical phenotype of a postural tremor, fasciculations and a slowly progressive upper and lower limb weakness with an unusually long duration. Linkage analysis revealed a unique locus at Chr20q13.33 and mutation screening revealed a heterozygous C>T nucleotide substitution, resulting in a p.P56S non-synonymous change within the highly conserved major sperm protein (MSP) domain of VAPB (Nishimura et al 2004).

This exon 2 variant has since been detected in 22 additional individuals from six Brazilian pedigrees in which there is evidence of a founder effect, although it has also been seen in a Japanese and European case (Funke et al 2010; Landers et al 2008; Millecamps et al 2010). The neurodegenerative phenotype associated with this mutation is variable; 3 of the families also had several confirmed cases of autosomal dominant adult onset spinal muscular atrophy (SMA). A second heterozygous point mutation (p.T46I) within the same domain of the VAPB peptide has recently been reported in a single Caucasian from the UK with classical ALS (Chen et al 2010).

VAPB is a type II integral endoplasmic reticulum (ER) membrane protein. It is involved in multiple cellular processes including intracellular trafficking, lipid transport, and the unfolded protein response (Lev et al 2008). Both mutations residing in the MSP domain result in conformational changes which lead to VAPB aggregation and an increase in ER stress (Chen et al 2010; Kim et al 2010; Suzuki et al 2009). Whilst mutations in *VAPB* are only found rarely in FALS patients, *VAPB* shows significantly decreased gene expression in SALS cases compared to age and gender matched neurologically normal controls (Anagnostou et al 2010; Mitne-Neto et al 2011).

### **3.2.2 ALS9: Angiogenin (***ANG***)**

Chr14q11.2 was first proposed as a susceptibility locus for ALS following the strong allelic association in the Irish population with a single nucleotide polymorphism (SNP) (rs11701) residing in the single exon angiogenin (*ANG*) gene (Greenway et al 2004). Mutation screening analysis of a large cohort of Irish, Scottish, English, Swedish and North American ALS cases detected 7 missense mutations in 15 patients, 4 of whom were FALS (Greenway et al 2006). Additional screening of ALS cohorts report *ANG* mutations occurring in both FALS and SALS cases, though at low frequencies (Fernandez-Santiago et al 2009; Gellera et al 2008).

ANG is a member of the pancreatic ribonuclease A (RNaseA) superfamily whose activities are known to be important in protein translation, ribosome biogenesis and cell proliferation (Crabtree et al 2007). The ribonuclease A activity has been shown to be reduced or lost in ANG mutant proteins. ANG is a potent inducer of neovascularization *in vivo* and has been shown to play a key role in neurite outgrowth and pathfinding during early embryonic development (Subramanian et al 2008). Its structure and function are partially homologous to that of vascular endothelial cell growth factor (VEGF); a previously reported genetic susceptibility and disease modifying factor in the development of neurodegeneration (Lambrechts et al 2009), and both VEGF and ANG have been shown to be neuroprotective. A proposed mechanism by which ANG prevents cell death is through inhibiting the translocation of apoptosis inducing factor into the nucleus (Li et al 2011).

### **3.2.3 ALS11: Factor-induced gene 4** *S.cerevisiae* **homolog (***FIG4***)**

The Sac1 domain containing protein 3 (SAC3) *FIG4,* located on Chr6q21, was originally identified as the causative gene of Charcot Marie-Tooth disease type 4J (CMT4J); a severe autosomal recessive childhood disorder that is characterised by both sensory and motor sperm protein (MSP) domain of VAPB (Nishimura et al 2004).

classical ALS (Chen et al 2010).

al 2010; Mitne-Neto et al 2011).

**3.2.2 ALS9: Angiogenin (***ANG***)**

522 Amyotrophic Lateral Sclerosis

characteristic clinical phenotype of a postural tremor, fasciculations and a slowly progressive upper and lower limb weakness with an unusually long duration. Linkage analysis revealed a unique locus at Chr20q13.33 and mutation screening revealed a heterozygous C>T nucleotide substitution, resulting in a p.P56S non-synonymous change within the highly conserved major

This exon 2 variant has since been detected in 22 additional individuals from six Brazilian pedigrees in which there is evidence of a founder effect, although it has also been seen in a Japanese and European case (Funke et al 2010; Landers et al 2008; Millecamps et al 2010). The neurodegenerative phenotype associated with this mutation is variable; 3 of the families also had several confirmed cases of autosomal dominant adult onset spinal muscular atrophy (SMA). A second heterozygous point mutation (p.T46I) within the same domain of the VAPB peptide has recently been reported in a single Caucasian from the UK with

VAPB is a type II integral endoplasmic reticulum (ER) membrane protein. It is involved in multiple cellular processes including intracellular trafficking, lipid transport, and the unfolded protein response (Lev et al 2008). Both mutations residing in the MSP domain result in conformational changes which lead to VAPB aggregation and an increase in ER stress (Chen et al 2010; Kim et al 2010; Suzuki et al 2009). Whilst mutations in *VAPB* are only found rarely in FALS patients, *VAPB* shows significantly decreased gene expression in SALS cases compared to age and gender matched neurologically normal controls (Anagnostou et

Chr14q11.2 was first proposed as a susceptibility locus for ALS following the strong allelic association in the Irish population with a single nucleotide polymorphism (SNP) (rs11701) residing in the single exon angiogenin (*ANG*) gene (Greenway et al 2004). Mutation screening analysis of a large cohort of Irish, Scottish, English, Swedish and North American ALS cases detected 7 missense mutations in 15 patients, 4 of whom were FALS (Greenway et al 2006). Additional screening of ALS cohorts report *ANG* mutations occurring in both FALS and SALS

ANG is a member of the pancreatic ribonuclease A (RNaseA) superfamily whose activities are known to be important in protein translation, ribosome biogenesis and cell proliferation (Crabtree et al 2007). The ribonuclease A activity has been shown to be reduced or lost in ANG mutant proteins. ANG is a potent inducer of neovascularization *in vivo* and has been shown to play a key role in neurite outgrowth and pathfinding during early embryonic development (Subramanian et al 2008). Its structure and function are partially homologous to that of vascular endothelial cell growth factor (VEGF); a previously reported genetic susceptibility and disease modifying factor in the development of neurodegeneration (Lambrechts et al 2009), and both VEGF and ANG have been shown to be neuroprotective. A proposed mechanism by which ANG prevents cell death is through inhibiting the

The Sac1 domain containing protein 3 (SAC3) *FIG4,* located on Chr6q21, was originally identified as the causative gene of Charcot Marie-Tooth disease type 4J (CMT4J); a severe autosomal recessive childhood disorder that is characterised by both sensory and motor

cases, though at low frequencies (Fernandez-Santiago et al 2009; Gellera et al 2008).

translocation of apoptosis inducing factor into the nucleus (Li et al 2011).

**3.2.3 ALS11: Factor-induced gene 4** *S.cerevisiae* **homolog (***FIG4***)**

deficits (Chow et al 2007). However, in one CMT4J pedigree there was a later onset of disease, with predominantly motor symptoms, similar to ALS (Zhang et al 2008). Screening of a North European cohort of FALS patients revealed 5 heterozygous mutations, resulting in either complete or significant loss of protein (Chow et al 2009). In general, cases of ALS11 are associated with a rapidly progressive disease course of approximately 1-2 years, early bulbar involvement and minimal cognitive dysfunction.

*FIG4* encodes the phosphatidylinositol 3,5-bisphosphate 5-phosphatase which controls the cellular abundance of PI(3,5)P2, a signalling lipid that mediates retrograde trafficking of endosomal vesicles to the Golgi apparatus (Michell & Dove 2009). It remains unclear as to whether the deleterious variants of *FIG4* exert an effect by a dominant negative mechanism or through a partial loss of function, as seen in CMT4J patients (Chow et al 2007). Human motor neurones are considered to be particularly susceptible to disruptions in this transport network because of their high membrane component turnover demands from long axonal processes over many decades (Ferguson et al 2010).

### **3.2.4 ALS12: Optineurin (***OPTN***)**

Homozygosity mapping using 4 FALS cases demonstrated linkage to chr10p13 and subsequently mutations in *OPTN*, a gene previously linked to primary open-angle glaucoma (POAG), were identified in these cases (Maruyama et al 2010; Rezaie et al 2002). Mutations were initially found in both homozygous and heterozygous states. However, mutation screening of subsequent cohorts have identified only heterozygous mutations in ALS cases, occurring at a frequency of 3.4% in Japanese populations whilst this was at much lower frequencies in European and North American populations (Belzil et al 2011; Del Bo et al 2011). Interestingly, *OPTN*-related ALS is characterised by a lower limb onset with upper motor neurone involvement and a slow clinical progression.

The gene encodes the ubiquitously expressed optic neuropathy inducing protein which localises to the perinuclear region of the cytoplasm, where it is known to associate with the Golgi apparatus, and plays a key role in a number of biological processes, including vesicular trafficking, signal transduction and gene expression (Chalasani et al 2008). It is anticipated where mutations are inherited in an autosomal recessive manner, that reduced protein levels result in neurotoxicity through a loss of function mechanism. Conversely, the heterozygous missense substitution is predicted to exert a dominant negative effect. Examination of autopsy derived spinal cord tissue revealed extensive OPTN staining of TDP-43 positive intracytoplasmic hyaline inclusion bodies, although this was not replicated in a subsequent study (Hortobagyi et al 2011; Ito et al 2011b).

#### **3.2.5 D-amino acid oxidase (***DAO***)**

Following linkage analysis, a rare heterozygous missense mutation in the *DAO* gene, located on chr12q22-23, has been reported to segregate with disease in a single three generational Caucasian pedigree, though there was evidence of incomplete penetrance (Mitchell et al 2010). Those affected showed a rapidly progressive form of classical ALS with a mean disease duration of 21 months. Early bulbar involvement was apparent with limited signs of cognitive impairment. Interestingly, post-mortem immunohistochemical analysis on spinal cord tissue revealed no evidence of TDP-43 positively labelled inclusion bodies within the nuclear or cytoplasmic fractions of residual motor neurones which are normally a distinguishing feature of ALS pathology.

Genetics of Familial Amyotrophic Lateral Sclerosis 525

Linkage analysis in a large, inbred Tunisian family mapped the gene responsible for an autosomal recessive form of ALS to chr2p33-q35 (Hentati et al 1994). Subsequent sequencing in this family, and a Saudi pedigree with juvenile primary lateral sclerosis (PLS), revealed mutations in the previously uncharacterised gene now known as *ALS2* (Yang et al 2001). Thus, mutations cause a spectrum of early onset motor neurone disorders including infantile ascending hereditary spastic paraplegia (IAHSP), PLS and ALS (Bertini et al 1993). To date, 19 *ALS2* mutations have been identified in ALS patients, which are characterised by a juvenile onset of limb and facial spasticity with subsequent lower motor neurone signs (Hentati et al 1994; Lill et al 2011). The milder phenotypes of PLS and IAHSP are characterised by isolated upper motor neurone degeneration without lower motor neurone

The *ALS2* gene is ubiquitously expressed and encodes the Alsin protein which contains three putative guanine-exchange factor (GEF) domains that activate small GTPases (Yang et al 2001). Evidence suggests that loss of Alsin is responsible for motor neurone damage and several groups have now generated *ALS2* knock-out mouse models, but only mild neurological changes have been reported in these animals to date (Cai et al 2008). The pathological mechanism of *ALS2* mutations remains unknown although evidence that Alsin has a role in endosomal transport and glutamate receptor targeting at the synapse offer interesting avenues for further study (Devon et al 2006; Hadano et al 2006; Lai et al 2006).

Following linkage analysis of a large multigenerational pedigree to chr9p, mutation screening of 34 genes in the candidate region identified a nucleotide substitution in the 3'UTR of the SIGMAR1 gene which co-segregated with the disease (Luty et al 2010). Two further FALS cases were also identified with 3'UTR substitutions, yet none of the 3 changes were present in controls. Pathological material was available from 2 individuals carrying one of the changes and both TDP-43 and FUS positive inclusions were observed. The SIGMAR1 protein functions as a subunit of the ligand-regulated potassium channel and regulates channel activity (Aydar et al 2002). It was suggested that the 3'UTR alterations alter the stability of the transcript, though how this subsequently causes motor neuronal cell

Pedigrees with clinical features of FTD, ALS and Parkinsonism have been identified with pathogenic mutations in the microtubule associated protein tau (*MAPT*) gene located on chr17 (Hutton et al 1998). Over 40 mutations have been identified to date that either affect the normal function of the tau protein to stabilise microtubules or disrupt alternative splicing leading to changes in the ratio of tau isoforms (Seelaar et al 2011). Affected individuals have a variable age of onset (25-65 years) with disease duration of 3-10 years and usually show symptoms of executive dysfunction, altered personality and behaviour. Many develop a Parkinsonism phenotype and/or other clinical features of 1 or 2 syndromes that may reflect an expansion of affected brain regions over time. Cases affected by an ALS phenotype are rare and so far

mutations in *MAPT* have not been described in pure FALS (Boeve & Hutton 2008).

**3.4 Genetic causes of autosomal recessive ALS**

**3.4.1 ALS2: Alsin (***ALS2***)**

signs (Bertini et al 1993).

**3.5 Genetic causes of ALS+FTD**

death, is unknown (Luty et al 2010).

**3.5.2 Microtubule associated protein tau (***MAPT***)**

**3.5.1 Sigma non-opioid intracellular receptor 1 (***SIGMAR1***)**

*DAO* encodes a universally expressed 39.4kDa peroxisomal flavin adenine dinucleotide (FAD)-dependent oxidase that is enriched in the neuronal and glial cell populations of the mammalian brainstem and spinal cord. The mutation results in the formation of an aberrant peptide product proposed to exert a dominant negative effect on the function of the wild type protein; *in vitro* work showed abnormal cellular morphology in cells over expressing the mutant protein, along with reduced cell viability, the presence of large intracellular ubiquitinated aggregates and an increased rate of apoptosis (Mitchell et al 2010).

### **3.2.6 Valosin containing protein (***VCP***)**

Whole exome sequencing of two affected individuals in a large Italian pedigree identified a single heterozygous missense mutation in the *VCP* gene, located on chr9p13.3 (Johnson 2010). Screening of an additional cohort of ALS cases detected further *VCP* mutations at a frequency of 1.74%. There was no distinct phenotype associated with *VCP*-related ALS, with both limb and bulbar onset and an average age of onset of 49 years. Mutations in *VCP* have previously been linked to the autosomal dominant disorder inclusion body myopathy, Paget's disease and FTD (IBMPFD), which is characterised by muscle wasting, associated with osteolytic bone lesions and FTD (Watts et al 2004).

VCP is an evolutionarily conserved AAA+-ATPase that is known to be of importance in multiple biological processes including cell signalling, protein homeostasis, organelle biogenesis and autophagy (Ritson et al 2010), through its role in identifying ubiquitinated proteins in multimeric complexes and mediating their proteasomal degradation. It has been demonstrated both *in vitro* and *in vivo* that aberrant expression of VCP results in the cellular redistribution and mislocalisation of nuclear TDP-43 within the cytoplasm where neurotoxic ubiquitinated and phosphorylated aggregates form (Custer et al 2010; Gitcho et al 2009).

### **3.3 Genetic causes of autosomal dominant, juvenile onset ALS 3.3.1 ALS4: Senataxin (***SETX***)**

The locus responsible for ALS4 was mapped to chr9q34 in an 11 generation pedigree affected by juvenile ALS and later confirmed by analysis of another two families with a similar phenotype (Chance et al 1998; De Jonghe et al 2002; Myrianthopoulos et al 1964). Sequence analysis of this region revealed disease associated mutations in the Senataxin (*SETX*) gene in all three affected families that were inherited in an autosomal dominant pattern (Chen et al 2004). *SETX* mutations are rare, with only four FALS families discovered to date, although mutations in this gene are also associated with ataxiaoculomotor apraxia-2 (AOA2), an autosomal recessive cerebellar ataxia (Anheim et al 2009). ALS4 is characterised by young onset (below the age of 25 years), distal muscle weakness and atrophy, pyramidal signs, an absence of sensory abnormalities while bulbar and respiratory muscles are spared. Disease progression is slow and patients have a normal life span (Chen et al 2004).

The *SETX* gene encodes a ubiquitously expressed DNA/RNA helicase that shares high homology to the yeast Sen1p protein and is suggested to play a role in DNA repair in response to oxidative stress (Suraweera et al 2007). SETX interacts with several RNA processing proteins, including RNA polymerase II, and is proposed to regulate transcription and pre-mRNA processing (Suraweera et al 2009).

2010).

et al 2009).

**3.3.1 ALS4: Senataxin (***SETX***)**

normal life span (Chen et al 2004).

and pre-mRNA processing (Suraweera et al 2009).

**3.2.6 Valosin containing protein (***VCP***)**

with osteolytic bone lesions and FTD (Watts et al 2004).

**3.3 Genetic causes of autosomal dominant, juvenile onset ALS**

524 Amyotrophic Lateral Sclerosis

*DAO* encodes a universally expressed 39.4kDa peroxisomal flavin adenine dinucleotide (FAD)-dependent oxidase that is enriched in the neuronal and glial cell populations of the mammalian brainstem and spinal cord. The mutation results in the formation of an aberrant peptide product proposed to exert a dominant negative effect on the function of the wild type protein; *in vitro* work showed abnormal cellular morphology in cells over expressing the mutant protein, along with reduced cell viability, the presence of large intracellular ubiquitinated aggregates and an increased rate of apoptosis (Mitchell et al

Whole exome sequencing of two affected individuals in a large Italian pedigree identified a single heterozygous missense mutation in the *VCP* gene, located on chr9p13.3 (Johnson 2010). Screening of an additional cohort of ALS cases detected further *VCP* mutations at a frequency of 1.74%. There was no distinct phenotype associated with *VCP*-related ALS, with both limb and bulbar onset and an average age of onset of 49 years. Mutations in *VCP* have previously been linked to the autosomal dominant disorder inclusion body myopathy, Paget's disease and FTD (IBMPFD), which is characterised by muscle wasting, associated

VCP is an evolutionarily conserved AAA+-ATPase that is known to be of importance in multiple biological processes including cell signalling, protein homeostasis, organelle biogenesis and autophagy (Ritson et al 2010), through its role in identifying ubiquitinated proteins in multimeric complexes and mediating their proteasomal degradation. It has been demonstrated both *in vitro* and *in vivo* that aberrant expression of VCP results in the cellular redistribution and mislocalisation of nuclear TDP-43 within the cytoplasm where neurotoxic ubiquitinated and phosphorylated aggregates form (Custer et al 2010; Gitcho

The locus responsible for ALS4 was mapped to chr9q34 in an 11 generation pedigree affected by juvenile ALS and later confirmed by analysis of another two families with a similar phenotype (Chance et al 1998; De Jonghe et al 2002; Myrianthopoulos et al 1964). Sequence analysis of this region revealed disease associated mutations in the Senataxin (*SETX*) gene in all three affected families that were inherited in an autosomal dominant pattern (Chen et al 2004). *SETX* mutations are rare, with only four FALS families discovered to date, although mutations in this gene are also associated with ataxiaoculomotor apraxia-2 (AOA2), an autosomal recessive cerebellar ataxia (Anheim et al 2009). ALS4 is characterised by young onset (below the age of 25 years), distal muscle weakness and atrophy, pyramidal signs, an absence of sensory abnormalities while bulbar and respiratory muscles are spared. Disease progression is slow and patients have a

The *SETX* gene encodes a ubiquitously expressed DNA/RNA helicase that shares high homology to the yeast Sen1p protein and is suggested to play a role in DNA repair in response to oxidative stress (Suraweera et al 2007). SETX interacts with several RNA processing proteins, including RNA polymerase II, and is proposed to regulate transcription

### **3.4 Genetic causes of autosomal recessive ALS 3.4.1 ALS2: Alsin (***ALS2***)**

Linkage analysis in a large, inbred Tunisian family mapped the gene responsible for an autosomal recessive form of ALS to chr2p33-q35 (Hentati et al 1994). Subsequent sequencing in this family, and a Saudi pedigree with juvenile primary lateral sclerosis (PLS), revealed mutations in the previously uncharacterised gene now known as *ALS2* (Yang et al 2001). Thus, mutations cause a spectrum of early onset motor neurone disorders including infantile ascending hereditary spastic paraplegia (IAHSP), PLS and ALS (Bertini et al 1993). To date, 19 *ALS2* mutations have been identified in ALS patients, which are characterised by a juvenile onset of limb and facial spasticity with subsequent lower motor neurone signs (Hentati et al 1994; Lill et al 2011). The milder phenotypes of PLS and IAHSP are characterised by isolated upper motor neurone degeneration without lower motor neurone signs (Bertini et al 1993).

The *ALS2* gene is ubiquitously expressed and encodes the Alsin protein which contains three putative guanine-exchange factor (GEF) domains that activate small GTPases (Yang et al 2001). Evidence suggests that loss of Alsin is responsible for motor neurone damage and several groups have now generated *ALS2* knock-out mouse models, but only mild neurological changes have been reported in these animals to date (Cai et al 2008). The pathological mechanism of *ALS2* mutations remains unknown although evidence that Alsin has a role in endosomal transport and glutamate receptor targeting at the synapse offer interesting avenues for further study (Devon et al 2006; Hadano et al 2006; Lai et al 2006).

### **3.5 Genetic causes of ALS+FTD**

### **3.5.1 Sigma non-opioid intracellular receptor 1 (***SIGMAR1***)**

Following linkage analysis of a large multigenerational pedigree to chr9p, mutation screening of 34 genes in the candidate region identified a nucleotide substitution in the 3'UTR of the SIGMAR1 gene which co-segregated with the disease (Luty et al 2010). Two further FALS cases were also identified with 3'UTR substitutions, yet none of the 3 changes were present in controls. Pathological material was available from 2 individuals carrying one of the changes and both TDP-43 and FUS positive inclusions were observed. The SIGMAR1 protein functions as a subunit of the ligand-regulated potassium channel and regulates channel activity (Aydar et al 2002). It was suggested that the 3'UTR alterations alter the stability of the transcript, though how this subsequently causes motor neuronal cell death, is unknown (Luty et al 2010).

#### **3.5.2 Microtubule associated protein tau (***MAPT***)**

Pedigrees with clinical features of FTD, ALS and Parkinsonism have been identified with pathogenic mutations in the microtubule associated protein tau (*MAPT*) gene located on chr17 (Hutton et al 1998). Over 40 mutations have been identified to date that either affect the normal function of the tau protein to stabilise microtubules or disrupt alternative splicing leading to changes in the ratio of tau isoforms (Seelaar et al 2011). Affected individuals have a variable age of onset (25-65 years) with disease duration of 3-10 years and usually show symptoms of executive dysfunction, altered personality and behaviour. Many develop a Parkinsonism phenotype and/or other clinical features of 1 or 2 syndromes that may reflect an expansion of affected brain regions over time. Cases affected by an ALS phenotype are rare and so far mutations in *MAPT* have not been described in pure FALS (Boeve & Hutton 2008).

Genetics of Familial Amyotrophic Lateral Sclerosis 527

unrelated FALS cases, with all mutations affecting proline amino acids in the proline-x-x repeat region near the carboxyl end of the protein. Clinically, age of onset was variable (16- 71 years) in the affected individuals, and although males were more likely to have an earlier age of onset, disease duration was similar. Some patients also showed symptoms of dementia. Post-mortem material from two unrelated FALS cases showed the classical skein like inclusions were positive for UBQLN2. The identified missense mutations lead to impairment of the protein degradation pathway in a cell model of *UBQLN2*-related ALS.

A locus for FALS that arises in conjunction with FTD has been identified in 5 American families at chr9p21-q22 (Hosler et al 2000). Affected patients had adult onset of either: ALS and FTD, ALS alone or ALS with dementia. Disease duration was typically less than 4 years although one individual had a slow progression and survived for 15 years. No pathogenic

Linkage of autosomal dominant FALS and FTD to chr9p13.2-p21.3 has been established in two pedigrees, one large Dutch kindred and a Scandinavian family (Morita et al 2006; Vance et al 2006). Clinically, all members with ALS had definite or probable ALS by the El-Escorial Criteria with mid-life onset and a typical disease course of around 3 years. In the Scandinavian family ALS and FTD occurred separately, in contrast, affected individuals in the Dutch kindred all had features of both conditions. Linkage has been narrowed down to a 12cM (11Mb) region of chr9, however the pathogenic gene mutations have yet to be

FALS accounts for 5% of ALS; an underlying mutation has been identified in approximately a third of these cases (Kiernan et al 2011). FALS causing mutations are used as a window into familial and the clinically indistinguishable sporadic disease; generating genetic models of ALS allows investigations into the mechanisms of motor neuronal degeneration, the identification of therapeutic targets and screening for candidate therapeutic agents (Van Damme & Robberecht 2009). However, the discovery of pathogenic mutations in ALS by linkage analysis is difficult because a relatively low prevalence and rapid disease course make large pedigrees difficult to obtain, therefore novel strategies to identify pathogenic

With the evolution of next generation sequencing technology, exhaustive sequencing of exonic regions of the genome has been used to identify pathogenic mutations in the *VCP* gene in ALS, and genetic mutations responsible for other diseases have also been identified from relatively few related or unrelated patients (Bowne et al 2011; Hoischen et al 2010; Johnson et al 2010a; Ng et al 2010; Ng et al 2009; Nikopoulos et al 2010; Simpson et al 2011). Exome sequencing, unlike a linkage analysis and positional cloning approach, is not targeted at a candidate region. Therefore it is likely that a large number of potential genetic variations will be discovered; the difficulty then is to determine which, if any, are pathogenic. However, next generation sequencing offers the potential for identifying at least some of the genes responsible

mutations have been identified for this region to date (Hosler et al 2000).

**3.6.5 ALS-FTD1: 9p21-q22**

**3.6.6 ALS-FTD2: 9p13.2-p21.3**

**4. Conclusion**

identified (Morita et al 2006; Vance et al 2006).

mutations are essential (Hand & Rouleau 2002).

for the remaining uncharacterised causes of FALS.

### **3.6 Genetic loci linked to ALS 3.6.1 ALS5: Spatacsin (***SPG11***)**

The study of three consanguineous Tunisian pedigrees originally established linkage of chr15q15-q21 to an autosomal recessive form of ALS (Hentati et al 1998). A more recent study of 25 unrelated FALS families revealed 10 pedigrees with linkage to the same region and disease associated mutations in the spatacsin (*SPG11)* gene (Orlacchio et al 2010). Clinically, FALS patients with linkage to this region experience a juvenile onset, slowly progressive motor neuropathy associated with both upper and lower motor neurone signs. Disease duration is typically over 10-40 years without sensory symptoms and an absence of the feature of thin corpus callosum (Hentati et al 1998; Orlacchio et al 2010).

Mutations in this gene have been previously found to be the most common cause of autosomal recessive hereditary spastic paraplegia with thin corpus callosum (HSP-TCC), a condition characterised by progressive spasticity of lower limbs, mild cognitive impairment and a thin, but otherwise normally structured, corpus callosum (Abdel Aleem et al 2011). All but one of the mutations identified in FALS are also present in HSP-TCC pedigrees and the majority of these are truncating which may suggest a loss of function and a common pathological mechanism between the two conditions (Salinas et al 2008). The *SPG11* gene has 40 exons and encodes the highly conserved Spatacsin protein, which is ubiquitously expressed in the central nervous system (Salinas et al 2008). Although the function of Spatacsin remains unknown, neuropathological studies of HSP-TCC patients with *SPG11* mutations have revealed accumulations of membranous material in non-myelinated axons which are suggestive of axonal transport disturbance (Hehr et al 2007).


**2. Case Presentation**

A 43-year-old male came into our department complaining of chest pain, dyspnea, persistent cough and weight loss; symptoms appeared during the last three months. From the patient history, we found that he was a heavy smoker, occasional consumer of ethanol as well as working with livestock as a shepherd. A routine chest x-ray showed a massive pleural effusion in the right hemithorax, with multiple round-shaped opacities within it. Blood work revealed an elevated eosinophil level (10%) with no other modified parameters. To confirm our suspicion of hydatid disease, we performed a thoracic CT scan, which revealed a large pleural effusion with multiple cystic formations of varying sizes within it (Figure 1).

**Figure 1.** Computed tomography (CT) view of primary pleural hydatid disease.

After complete investigation of the patient, we started preoperative antiparasitic treatment with Albendazole 15 mg/kg/day for 6 days and then surgery was performed by using a lateral thoracotomy through the fifth intercostal space. After opening the pleural cavity, we introduced a hypertonic saline solution as well as oxygenated water 10% in order to inactivate the scolices. After draining approximately 4 L of ivory-colored fluid from the pleural cavity we discovered several hundred hydatid cysts with sizes ranging from 1–2 mm to 5–6 cm and the right lung was collapsed in the hilum. Cysts were round, well defined with a clear liquid content. Besides the intact cysts, we also found several ruptured ones, which evacuated and further contaminated the pleural cavity (Figure 2).

**Figure 2.** Intraoperative view of primary pleural hydatidosis.

After removing all the cysts from the pleural space, we performed several more pleural lavages using a hypertonic saline solution as well as an iodine solution. Further inspection revealed no other cysts in the lung, pleura, diaphragm, mediastinum or pericardium. In addition, there was no sign of rupture or migration of the cyst from any other thoracic organ, leading us to conclude that it was a primary pleural hydatidosis. Before closing, we inflated the lung and there was no air leakage and no other visible lesions on the parenchyma. We placed a single chest drain and the thoracotomy was closed in layers.

Parasitological tests performed on the pleural liquid as well as the parasitic material removed during surgery confirmed the presence of Echinoccocus. Additionally, IGG-specific ELISA tests performed from the pleural liquid confirmed the same.

Chest x-ray performed first day postoperative showed an almost fully expanded lung with no fluid or pneumothorax and a pleural drainage of approximatively 600 mL. The patient also received antiparasitic treatment with Albendazole 15 mg/kg/day. The patient spent 2 days in the intensive care unit (ICU) and was discharged on the 13th postoperative day with the indication of continuing Albendazole treatment for 1 year with 15 mg/kg/day due to the severity of the disease.

Follow-up showed no signs of recurrence with a normal chest x-ray and an improved lung volume function at one month, 6 months and 1 year.

#### **3. Discussion**

Larvae of the Echinoccocus parasite cause hydatid disease and humans are an accidental host either from consuming contaminated meat or from unwashed fruits and vegetables [1].

Dévé published the first significant study on secondary pleural hydatid disease in 1937 and his conclusions were that all pleural presentations of hydatidosis were secondary to either a lung or liver dome cyst that ruptured into the pleural space, stating that primary pleural hydatidosis does not exist [6]. However, future case reports, such as that of Rakower published in 1964, point to the existence of this pathology [7].

Although most hydatic cysts form in the liver or the lung, due to the possibility of haematogenous dissemination, lesions can develop anywhere in the body. Most common cause of pleural hydatidosis is by either trans-diaphragmatic contamination from cysts located in the right upper hepatic lobe or from the rupture of a peripheral lung cyst [8].

Primary pleural hydatid cysts fall under the category of extra-pulmonary intrathoracic cysts, alongside those found in the parietal pleura, mediastinum, pericardium, diaphragm, fissures and chest wall. Primary pleural hydatidosis manifests as the presence of either a solitary pleural hydatid cyst or as a parasitic pleural effusion [5]. Massive pleural effusions are usually of malignant origin (58% of cases), parapneumonic (23%), tuberculosis (10%), with the remaining cases having diverse origins [9]. Parasitic pleural effusion developed in the case of Echinoccocus is more frequently found as a complication of either a pulmonary hydatid cyst that ruptures in the pleura or as a liver hydatid cyst with intrathoracic development. Parasitic pleural effusion is extremely rare and unusual in medical practice [10].

Primary pleural hydatidosis is found in less than 1% of hydatidosis cases [11]. In our case, we could not find any lesions of the pulmonary parenchyma or any other thoracic lesion. Due to this, we concluded that in our case the diagnosis was that of primary pleural hydatidosis. Although not frequent, primary pleural hydatidosis has been presented as an exceptional situation for hepatic hydatidosis [12,13]. Primary development of a hydatid cyst within the parietal pleural structures is possible and can later lead to hydatid pleural effusion. The histological structure of the cystic membranes allows the passage of calcium, magnesium, water, urea as well as other nutritional substances that may pass through by diffusion and favor the development of the cyst [14].

The symptoms presented depend on the localization of the cyst and the degree of compression of the local organs [15]. There have also been reports of primary hydatid cysts in various organs such as the heart [16], soft tissue [17], adrenal gland [18] and brain [19].

Symptoms of pleural hydatidosis are similar to those found in pleural effusions such as dyspnea, mediastinal shift and reduction in lung volume. Even so, up to 15% of cases can be asymptomatic [20]. However, these can also be present alongside chest pain and other signs of cardiac or vascular involvement or compression [21]. Hydatid disease is also a rare cause of recurrent acute pulmonary embolism. This complication may develop after invasion of the cardiovascular system or direct invasion of the inferior vena cava [22].

In diagnosing hydatid disease, the most important part is played by imaging studies. Standard chest radiography alongside computed tomography, not only helps in diagnosing the disease but it also plays a role in the planning of surgery. Some radiological signs are pathognomonic for hydatid disease such as the presence of daughter cysts, water-lily sign (for ruptured cysts), and serpent sign (ruptured and completely evacuated cysts) [23]. Other tests such as skin tests, complement fixation, blood eosinophil count and indirect hemagglutination tests can be used, but must be interpreted carefully as they have a tendency towards false-positive results [24]. In addition, even if an ultrasound would be very useful in diagnosing liver cysts, it is rarely used for cysts in the thorax, with the exception of a chest wall hydatid cyst [25,26].

However, information provided by performing an ultrasound in such cases might bring significant information, complementary to those reported by other imagistic studies such as computed tomography; moreover, it can be safely performed in pregnant patients in whom such lesions are suspected [27,28]. Therefore, in pleural hydatidosis ultrasound might reveal suggestive aspects such as complex cystic lesions presenting double line sign whenever collapsed membranes exist [27]. In this respect, ultrasound has been successfully added as part of the examination tools in cases in which intrathoracic extrapulmonary hydatidosis is suspected, Gursoy et al. reporting the successful use of this method in a case series of 14 patients [14].

Although fine needle aspiration is recommended in the diagnosing of primary liver hydatid cysts, due to the high risk of complications such as pneumothorax, haemoptysis, cyst rupture, anaphylaxis and dissemination, performing this procedure on cysts located in the lung is not advised [29]. If we were to ignore the risks associated with this procedure and perform it, the most likely result is the discovery of hydatid material in the aspirated liquid.

However, in particular cases, puncture might orientate the diagnostic; therefore, in pregnant patients in whom the imagistic methods of evaluation are restricted due to the risk of fetal irradiation, performing an ultrasound examination and an ultrasound guided puncture might serve as an important diagnostic tool; therefore, in the case presented by a French study group and published in 2011 the authors reported the case of a 23-year-old patient who was investigated during the 22nd week of pregnancy for chest pain and dyspnea in association with pneumothorax; after performing puncture and establishing the diagnostic, the patient was further submitted to surgery, the cystic mass being removed; meanwhile, two bronchial fistulas were identified which were successfully sutured while the remaining cavity was capitonnaged [28].

The most common preoperative complications that may occur in pleural or lung hydatid disease include empyema and pneumothorax [30], but there are rare cases where, due to the rupture of the cysts, the patient can suffer from anaphylactic shock. There have even been cases of patients presenting with tension pneumothorax caused by the rupture of a lung cyst [31]. Several representative studies with patients with pulmonary hydatid cysts concluded that the rupture of the cyst in the pleural cavity is the most severe complication of the disease, alongside anaphylactic shock [32].

The main treatment in hydatid disease consists of surgery followed by medical treatment with antiparasitic medication. For pulmonary hydatid disease, the most common surgical approach is by thoracotomy and resection of the cysts. However, when the disease is present bilateral a median sternotomy is preferable [24]. The main goal of surgery is complete removal of the germinative membrane after inactivation using a hypertonic saline solution [33]. Standard surgical procedure consists of cystectomy and capitonnage; however, there are cases when an extended resection is necessary when the surrounding tissue is diffusely involved or there is presence of local infection or giant cysts. In all cases, local contamination during surgery must be avoided using a hypertonic saline solution to inactivate the cysts. If this is not performed thoroughly the chances of a recurrent disease increase significantly [34]. There are however several authors that consider that capitonnage is not necessary after cystectomy and that only the bronchial openings should be sutured and the cavity should be left open, arguing that capitonnage causes lung disfigurement, prolongs the operating time and increasing morbidity [35,36]. For primary pleural hydatidosis, the same surgical principles apply when it comes to approach and case management. Removal of the cysts and pleural fluid after inactivation with hypertonic saline solution is mandatory, followed by chest drainage paired with Albendazole treatment [14].

Antiparasitic treatment plays a key role in the management of any form of hydatidosis. Most authors recommend treatment with one of two benzimidazole carbamates, Albendazole or Mebendazole, which are the only drugs that interrupt larval growth of Echinoccocus species. Some authors consider that preoperative antiparasitic treatment is very useful, especially in severe forms of illness, and that it would reduce the recurrence by up to three and a half times [37]. In our

opinion, preoperative antiparasitic treatment is extremely useful, especially in severe cases with a high risk of recurrence and intraoperative dissemination, such as the case we have presented.

The most used treatment is with Albendazole, while Mebendazole is only used as an alternative due to its adverse effects. However, antiparasitic treatment is recommended as anti-infection therapy, while the main course of treatment remains surgical [38].

As it can be observed, primary pleural hydatidosis is a rare entity, only rare cases being reported so far; experience gathered at this moment is rather related to case reports or case series presenting cases of patients diagnosed with intrathoracic extrapulmonary hydatidosis in general. Data published so far regarding this pathological entity are summarized into Table 1.


**3.7 Methodologies for addressing insights into phosphorus pollution in aquatic environments**

Various methodologies are employed to study and address phosphorus pollution in aquatic environments.

These methods allow researchers and environmental professionals to assess the extent of pollution, identify sources, and develop effective mitigation strategies.

Here are the brief details of some common methodologies used.


**FAMILY PLANNING**

#### **Family Planning**

http://dx.doi.org/10.5772/intechopen.68266 Edited by Zouhair O. Amarin

#### **Contributors**

Francis Obare, George Odwe, Wilson Liambila, Jessica Atrio, Isha Kachwala, Karina Avila, Michel Garenne, Nazli Sensoy, Yasemin Korkut, Selcuk Akturan, Canan Tuz, Bilge Tuncel, Mehmet Yilmaz, Li-Wei Chien, Heng-Kien Au, Dominic Iliescu, Stefania Tudorache, Dragusin Roxana, Iuliana Ceausu, Simona Vladareanu, Dana Oprescu, Maria Cezara Muresan, Affonso Renato Meira, Yohanes Handoko, Eka Rusdianto Gunardi, Lukman Solanke, David Schwartz, Alhaji A Aliyu, Blanca Patricia Bautista Balbás, Luis Alfredo Bautista Balbás, Alicia Pouso Rivera, Panagiotis Tsikouras, Dorelia Deuteraiou, Anna Chalkidou, Xanthoula Anthoulaki, Anastasia Bothou, Bachar Manav, Zacharoula Koukouli, Stefanos Zervoudis, George Iatrakis, Georgios Galazios, Melissa Wilde, Kajaiyaiu Hopkins, Zouhair Amarin

#### **© The Editor(s) and the Author(s) 2018**

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Individual chapters of this publication are distributed under the terms of the Creative Commons Attribution 3.0 Unported License which permits commercial use, distribution and reproduction of the individual chapters, provided the original author(s) and source publication are appropriately acknowledged. If so indicated, certain images may not be included under the Creative Commons license. In such cases users will need to obtain permission from the license holder to reproduce the material. More details and guidelines concerning content reuse and adaptation can be foundat http://www.intechopen.com/copyright-policy.html.

#### **Notice**

Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book.

First published in London, United Kingdom, 2018 by IntechOpen eBook (PDF) Published by IntechOpen, 2019 IntechOpen is the global imprint of INTECHOPEN LIMITED, registered in England and Wales, registration number: 11086078, The Shard, 25th floor, 32 London Bridge Street London, SE19SG – United Kingdom Printed in Croatia

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**2. Types of wearable biosensors**

Several human biosignals and stimuli should be concerned to display user's health conditions. For example, the assessment of biochemistry in biofluids (e.g., sweat, interstitial fluid, and blood) can provide fruitful information about personal health status and disease progression. In addition, both electrical and non-electrical biosignals such as electrooculography (EOG), mechanomyogram (MMG), electrocardiogram (ECG), electromyogram (EMG), galvanic skin response (GSR), magnetoencephalogram (MEG), and electroencephalogram (EEG) are useful indicators to offer the biosignal and info of a special tissue, organ, brain, or cellular system, such as the nervous system.

In addition to these internal vital signs, the on-skin detection of external harmful stimuli with a biosensor is also a critical issue. When it comes to the, the realm of wearable biosensing applications can be extended to motion detection, hazardous gas monitoring, disease diagnosis, and harmful UV-light detection (**Figure 2**). The sensing mechanisms of these sensors are diverse. In this Section, we will discuss several examples of chemical, optoelectronic and mechanical biosensors.

#### **2.1 Wearable chemical biosensors**

Sweat and sebum are representative human skin secretions that originate from the sweat glands in the dermal layer of the epidermis and therefore in-situ detection of these secretions on the skin is critically important for health monitoring. Sweat, a physiological aid for regulating body temperature, is secreted from the external glands. For example, sweat is a particularly useful sensing target due to the ease and presence of biomarkers associated with critical health conditions such as dehydration, physical exhaustion, mental stress and illness [13–15]. Sebum is secreted to lubricate human skin from Sebaceous glands. When sebum spreads up along the hair shaft, it is distributed over the surface of the skin, lubricating, and waterproofing the stratum corneum, the outer layer of the skin. It consists mostly of lipids. Those secretions can reflect the human health condition indirectly. Sebum provides antioxidant and antimicrobial lipids to the skin surface. Thus, proper secretion of sebum on the human skin surface increases the skin permeable barrier function. However, excess sebum frequently results in acne vulgaris. The acidic state of the skin pH is an essential factor in retaining the integrity of the skin-permeable barrier. It is reported that skin pH can be changed in skin diseases such as atopic dermatitis [16].

Wearable chemical biosensors that non-invasively analyze biofluids such as sweat, sebum, saliva, tears, and intermediate fluids provide the potential to

**Figure 2.**

*A schematic diagram of wearable biosensors. (A) Wearable chemical biosensors. Adapted from Refs. [7, 8]. (B) Wearable photodetectors. Adapted from Refs. [9, 10]. (C) Wearable electromechanical biosensors. Adapted from Refs. [11, 12].*

dramatically improve health condition evaluations by tracking changes in metabolic processes [17]. The sweat sample collection can be performed using an absorbent pad or a plastic microtube [18]. However, these methods are not compatible with remote monitoring and on-site use because they depend on cumbersome multi-step expensive benchtop hardware for sample preparation procedures and analysis. Recent advances in soft microfluidics, stretchable/flexible chemical sensing technologies form a basis of a new wearable sensor system that overcomes the limitations of this conventional approach [19]. The wearable sweat sensors are promising tools for continuous health and physiological monitoring. Among the various types of sweat sensors, optical detection utilizes photo-transmission techniques such as chromaticity, fluorescence, and light detection to provide an attractive strategy for measuring integrated chemical sensors due to cost efficiency and simplicity. Colorimetric sensors are also widely adopted in wearable sensor platforms, especially in case of the integrated microfluidic systems. Koh et al. demonstrated that a soft, flexible microfluidic platform based on silicon elastomer was recently developed, using colorimetric dyes to detect lactate, chloride, glucose, pH and loss of sweat [7]. The dye was located on top of the filter paper inserted into the micro reservoir. The lactate and glucose measurements were achieved by integrating enzymes into chromaticity-inducing reagents to enable the colorimetric readout sensing.

In terms of the detection of hazardous gases to human, Lee et al. demonstrated a flexible and transparent biosensor on polyethylene terephthalate (PET) that can detect 255 ppb NH3 using spray-deposited single wall carbon nanotube (SWCNT) with Au nanoparticles in a reproducible manner [20]. The AuNP decoration of transparent SWCNT film through the use of an electron beam (e-beam)

### *Advanced Materials and Assembly Strategies for Wearable Biosensors: A Review DOI: http://dx.doi.org/10.5772/intechopen.94451*

evaporation enhanced the performance of the gas sensor, which exhibited a high uniformity of the sensing behavior. The enhancement of this sensing performance was resulted from the carrier depletion zone control enabled by the combination structure of the AuNPs and the SWCNTs. The proposed sensor had a fast response time regarding NH3 gas but did not fully recover at room temperature.

Lee et al. developed a patch-based, integrated system that combines noninvasive sweat glucose monitoring with microneedle-assisted therapy (**Figure 3**) [21]. The patch-based wearable/strip type single-use integrated systems include large-area porous metal based electrode, minimized sensor designs for stable sweat detection from small amounts of sweat, design patchable and disposable sensors to achieve practical application, multicycle operation of the sweat measuring control and absorption layer to collect efficiently sweat samples, a porous Au nanostructure film to maximize the surface-to-volume ratio to detect a tiny amount of glucose in sweat. This sweat sensor exhibited high performances in terms of sensitivity, multimodal sensing, and accuracy.

#### **Figure 3.**

*(A) Optical camera image (top; dotted line, edges of the patch) and schematic (bottom) of the wearable sweat monitoring patch. A porous sweat-uptake layer is placed on a Nafion layer and sensors. (B) Optical camera image (top) and schematic (bottom) of the disposable sweat monitoring strip. (C) Optical camera image (top; dotted line, edges of the patch) and schematic (bottom) of the transdermal drug delivery device. Replacement-type microneedles are assembled on a three-channel thermal actuator. Adapted from Ref. [21].*

#### **2.2 Wearable optoelectronic sensors**

Photodetector is a key device attached to the front end of the optical receiver that converts input optical signals into output electrical signals [22]. Especially in medical applications, optoelectronic devices are very useful as they are able to detect biometric signals and other clinical information non-invasively. Organic optoelectronic devices including organic light emitting diodes (OLEDs), organic photodetectors (OPDs), and organic phototransistors are in the spotlight in the area of medical devices as they provide a wide absorption spectrum and high photogeneration yield with easy-to-fabricate, lightweight, and flexible features [23].

Recently, OLEDs [24–30], polymer light-emitting diodes (PLEDs) [31–34], and OPDs [35–38] were fabricated on glass or plastic substrates, implementing a muscle contraction detector and transmission mode pulse oximeter. Moreover, OLEDs and organic photovoltaics were manufactured on 1-μm-thick ultra-thin films but were operated in N2 box atmosphere [39, 40]. Realizing ultra-flexible optical sensor with an extended stability of surrounding conditions, allowing the sensors to integrate intimately and unnoticed on the skin, and to enable the application's cornucopia [10].

A representative non-invasive measurement system using pulsed oximetry characterizes peripheral oxygen saturation evaluated by oxyhemoglobin in the blood is shown in **Figure 4** [10]. An organic pulse oximeter with polymer LEDs and Si-based photodetectors were implemented based on pulse sensing and display on the human skin. The reflective pulse oxygen system used green and red LEDs to generate two wavelengths through fingers, and a light detector later measured the change in absorbance on the same side of the LED to determine peripheral oxygenation [10].

Choi et al. demonstrated an array of ultra-high-density curved MoS2-graphene light detectors using a single lens optical unit [9]. The high-density MoS2-graphene curved structure of the photodetector array was fabricated using an ultra-thin, soft material and strain-isolating/-releasing device architectures. The photodetector array and ultrathin neural-interfacing electrodes were embedded onto the soft flexible printed circuit board, accomplishing a human eye-inspired soft implantable optoelectronic device. The proposed device offered minimal mechanical deformation to the eye model, which were demonstrated by both experiments and finite element analysis (FEA) simulations. The wearable photodetector array and ultrathin neurointerference system reduced mechanical distortion of the retina and effectively stimulated the retinal nerve responded by optical input signals.

Ng et al. developed organic bulk heterojunction photodetectors having dark current as low as <1 nA cm−2 and efficient charge collection behavior. The development of a 4 μm-thick sensor layer using amorphous silicon TFTs provided a highly flexible image sensor operation with excellent performance as high as > 35% external quantum efficiency and noise equivalent power of 30 pW cm−2 at the applied reverse bias voltage of −4 V [41].

#### **Figure 4.**

*Ultraflexible organic pulse oximeter. (A) Device structure of the pulse oximeter. (B) Operation principle of the reflective pulse oximeter. Adapted from Ref. [10].*