Gene 560 (2015) 9–14

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Gene journal homepage: www.elsevier.com/locate/gene

Review

Canavan disease: An Arab scenario Hatem Zayed Department of Health Sciences, Biomedical Program, Qatar University, Doha, Qatar

a r t i c l e

i n f o

Article history: Received 17 October 2014 Received in revised form 3 February 2015 Accepted 5 February 2015 Available online 7 February 2015 Keywords: Canavan disease Mutations Arab countries Genotype–phenotype correlation Epidemiology

a b s t r a c t The autosomal recessive Canavan disease (CD) is a neurological disorder that begins in infancy. CD is caused by mutations in the gene encoding the ASPA enzyme. It has been reported with high frequency in patients with Jewish ancestry, and with low frequency in non-Jewish patients. This review will shed light on some updates regarding CD prevalence and causative mutations across the Arab World. CD was reported in several Arab countries such as Saudi Arabia, Egypt, Jordan, Yemen, Kuwait, and Tunisia. The population with the highest risk is in Saudi Arabia due the prevalent consanguineous marriage culture. In several studies, four novel mutations were found among Arabian CD patients, including two missense mutations (p.C152R, p.C152W), a 3346 bp deletion leading to the removal of exon 3 of the ASPA gene, and an insertion mutation (698insC). Other previously reported mutations, which led to damage in the ASPA enzyme activities found among CD Arab patients are c.530 T N C (p.I177T), c.79G N A (p.G27R), IVS4 + 1G N T, and a 92 kb deletion, which is 7.16 kb upstream from the ASPA start site. This review will help in developing customized molecular diagnostic approaches and promoting CD carrier screening in the Arab world in areas where consanguineous marriage is common particularly within Saudi Arabia. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Canavan disease (CD; MIM #271900), identified in 1931 by Canavan, is an inherited autosomal recessive progressive leukodystrophy with onset in early childhood or infancy. It results from the deficiency of the aspartoacylase (ASPA; NP_000040.1) enzyme in CD patients, which leads to the accumulation of N-acetyl-L-aspartate (NAA) in the oligodendrocytes, interfering with the myelin sheath formation. The clinical presentation of CD appears after 3-months of age and is characterized by early onset macrocephaly, brain vacuolization, headlag, severe psychomotor retardation, ataxia, cerebral degeneration of the white matter, poor motor skills, seizures, sleep disturbances, visual impairment, and dysmyelination, leading to early death during childhood; though there are several reports of clinically protracted disease courses (Globus and Strauss, 1928; Canavan, 1931; Adornato et al., 1972; Adachi et al., 1973; Matalon et al., 1988, 1995; Zelnik et al., 1993; Shaag et al., 1995; Zafeiriou et al., 1999; Surendran et al., 2003; Tacke et al., 2005; Yalcinkaya et al., 2005). CD patients normally show excessive excretion of NAA in urine (Al-Dirbashi et al., 2007). The ASPA gene (Gene Bank accession # NM_000049.2) has been mapped to chromosome 17 (17p13.3), and is composed of six exons.

CD is more prevalent among individuals of Ashkenazi Jewish background, with an incidence of 1/6400 to 1/13,500 of the population (Feigenbaum et al., 2004), making 1 in every 40 to 58 Ashkenazi Jews a carrier. CD has been reported in different ethnic backgrounds with origins from Lithuania, Western Russia, Eastern Poland, and Saudi Arabia, with rates less than the Jewish population. To date, the Human Gene Mutation Database (http://www.hgmd.org) has reported more than 65 CD causative mutations in the ASPA gene. There are 71 variants (38 African American and 33 European Americans) in the ASPA gene listed in the Exome Variant Server Database through the Exome Sequencing Project (ESP). The most prevalent mutations in Ashkenazi Jewish are a nonsense mutation p.Y231* and a missense mutation p.E285A (Elpeleg et al., 1994; Kaul et al., 1994, 1996; Kronn et al., 1995; Feigenbaum et al., 2004). Together, they represent 98% of the mutant alleles among CD patients of Ashkenazi Jewish background; in non-Jewish patients they account only for 3% of CD patients (Kaul et al., 1993, 1994). Though mutations in the Jewish alleles are circulated in a conserved fashion due to the inter-Jewish marriage culture, in non-Jewish patients mutations are more diverse; though the missense mutation p.A305E was frequently seen in CD patients with non-Jewish European background (Shaag et al., 1995; Kaul et al., 1994; Yalcinkaya et al., 2005; Zeng et al., 2002, 2006). 2. Pathogenic mechanism of CD

Abbreviations: (AAV), adeno-associated viral vector; (CNS), central nervous system; SIFT, http://sift.jcvi.org/; ASPA, aspartoacylase; CD, Canavan disease; (NAA), N-acetyl-Laspartate; PolyPhen2, polymorphism phenotyping v2, http://genetics.bwh.harvard.edu; TRPV3, transient receptor potential cation channel, subfamily V, member 3. E-mail address: [email protected].

http://dx.doi.org/10.1016/j.gene.2015.02.009 0378-1119/© 2015 Elsevier B.V. All rights reserved.

The pathological features of patients with CD include lack of myelination, hypertrophy, hyperplasia, spongy degeneration, water accumulation in the brain (Matalon et al., 1995; Leone et al., 2012) and

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H. Zayed / Gene 560 (2015) 9–14

vacuolization of the white matter, predominantly in the cerebellum and brainstem (Sacks et al., 1965). The CD animal models, such as ASPA knockout (ASPA−/−) mice (Matalon et al., 2000), tremor rats (Kondo et al., 1991; Kitada et al., 2000), and the nur7 mice, which are caused by a nonsense mutation, Q193X, in the ASAP gene; leading to no detectable ASPA enzyme activity in the homozygous form of nur7/nur7 mice (Traka et al., 2008), exhibit pathology similar to patients with CD, such as white matter vacuolation, hypomyelination, and spongy degeneration of the central nervous system (CNS). ASPA enzyme hydrolyzes NAA into aspartate and acetate, and the free acetate is converted to acetyl CoA by acetyl CoA synthetase, which is used in myelin lipid synthesis. ASPA is distributed predominantly in oligodendrocytes (myelin synthesizing cells of the CNS), and NAA is primarily distributed in the neuron (Ory-Lavollee et al., 1987), which is transported from neurons to the cytoplasm of oligodendrocytes for processing by ASPA enzyme. The precise role of NAA in the pathology of CD is not yet clear-cut, and it has been debated for a long time. A number of different hypotheses have been suggested for such a role, including increased cytotoxicity (Pai and Ravindranath, 1991), metabolic imbalance (Mehta and Namboodiri, 1995), and osmotic dysregulation (Taylor et al., 1995). The degree of interplay of these mechanisms in the pathogenicity of CD is yet to be resolved, but it has been established that the rise of NAA, resulting from the lack of ASPA expression leads to the CD phenotype (Kaul et al., 1994), and is associated with the osmotic dysregulation and intramyelinic edema (Adornato et al., 1972; Leone et al., 2012). This leads to chemical imbalance, which interferes with myelin formation and the proper development of the nervous system, leading to the death of the nerve fibers and subsequently brain damage. 3. Genotype–phenotype correlation of different ethnic groups The ability to measure the activity of ASPA enzyme in fibroblasts derived from a CD patient, estimate the amount of NAA in patients' urine and brain, screen for possible variants in ASPA gene, and analyze the effect of mutations on the crystal structure of the ASPA enzyme (PDB: 2I3C) (Bitto et al., 2007), has paved the way for establishing a possible meaningful relationship between the genotype and the clinical phenotype for patients with CD. Although it is sometimes difficult to conclude such a correlation based on the underlying genetic mutations alone, and it has been believed that there was no genotype–phenotype correlation for CD (Traeger and Rapin, 1998). However, some reports indicated that such a relationship might exist for CD patients. Zeng et al. (2002) reported two British CD patients (who died with infantile severe CD) who harbored the missense variant c.746A N T (p.D249V) in a compound heterozygous with other variants, and the severe clinical course of the patients correlated well with the absence of ASPA activity and in silico predictions (Table 1). The same report also indicated possible genotype correlation for the two variants: c.640G N T

(p.E214*) and c.941A N G (p.*314 W) (Zeng et al., 2002). The two founder mutations: c.693C N A (p.Y231*) and c.854A N C (p.E285A) are responsible for most of the severe phenotype among Ashkenazi Jewish patients with CD. The p.Y231* variant is associated with infantile severe CD phenotype and completely inactivates the ASPA enzyme (Table 1), showing a direct relationship of the genotype and the severity of the clinical presentation. The p.E285A variant retains some residual enzyme activity (Table 1) (Hershfield et al., 2007; Zano et al., 2013), which is consistent with the variable severity and disease progression in patients harboring this mutation. Other studies reported different variants in the ASPA (p.F295S, p.K213E, and p.Y231C), presenting with mild and variable clinical phenotype (Tacke et al., 2005; Shaag et al., 1995; Rady et al., 1999), and with residual ASPA enzyme activities of 10%, 15%, and 24%, respectively (Table 1) (Zano et al., 2013). Two sisters diagnosed with CD, were compound heterozygous for two missense variants: c.212G N A (p.R71H) and c.914C N A (p.A305E) (Table 1). Both presented at ages 19 and 50 months, respectively (Janson et al., 2006). Though both had a mild clinical picture, their fibroblast cells showed no ASPA expression, and the cerebral and urine NAA levels in both patients were moderately high. The in vitro expression of both mutations expressed separately or in combination showed no significant ASPA enzyme activity (Table 1). The p.R71H variant occurred in homozygous form in a 28-month-old girl, who originated from Ecuador, with a mild form of CD (Velinov et al., 2008), and her NAA levels were significantly high, but lower than observed in classic cases of CD. Interestingly, the p.A305E mutation was also reported in both severe and mild forms in non-Jewish Caucasian patients (Shaag et al., 1995). It is indeed challenging to draw a clear-cut picture of the genotype– phenotype correlation for patients with CD. This might be due to several reasons: most patients with CD are compound heterozygous, the variability of the in vitro transfection assays used to measure ASPA enzyme activity of the mutant alleles, the intrinsic variability of measuring enzyme activities or the NAA concentration from patient cells, the possibility of an undiscovered epistatic mechanism, which might modulate the activity of the ASPA enzyme, and finally it seems that there are ethnicspecific mutations, and it is not possible to predict their effect on other ethnic groups with different genetic makeup. In this review, the focus is on the mutations causing CD in the Arab world. The identification of these mutations could improve CD prognosis, permit accurate carrier information and allow development of customized molecular diagnostic approaches. 4. CD epidemiology in the Arab world 4.1. Saudi Arabia Ozand et al. (1990) reported on the first study of CD in the Arab world, where they studied a group of 12 CD patients who belonged to 10 Saudi families. All cases resulted from consanguineous marriages.

Table 1 Genotype–phenotype correlation of patients with CD in diverse ethnic groups. Ethnicity

NC

AAC

CP

ASPAA⁎

PP(S,S,S)

Reference

Ashkenazi Jewish Ashkenazi Jewish Greek, Turkish Greek Turkish Ecuador/Non-Jewish Non-Jewish (pan-European) British

c.693C N A c.854A N C c.884 T N C c. 637A N G c.692A N G c.212G N A c.914C N A p.746A N T

p.Y231⁎ p.E285A p.F295Sa p.K213Eb p.Y231C p.R71H p.A305E p. D249V

Severe Mild–severe Mild Mild Mild Mild Mild Severe

Loss 2.5% 10% 15% 24% Loss Loss Loss

NA PrD (1,0,1) PrD (1,0,1) B (0.003,0.98,0.4) PrD (1,0,1) PrD (0.99,0.4,0.98) PrD (1,0,1) PrD (1,0,1)

Kaul et al. (1994) Hershfield et al. (2007) Tacke et al. (2005), Hershfield et al. (2007), Zano et al. (2013) Tacke et al. (2005), Zano et al. (2013) Rady et al. (1999), Zano et al. (2013) Velinov et al. (2008), Janson et al. (2006) Janson et al. (2006) Zeng et al. (2002), Hershfield et al. (2007)

Legend: NC: nucleotide change: AAC: nucleotide change, CP: clinical phenotype, ASPAA: ASPA activity, Loss: complete loss of ASPA activity, NA: not applicable, PrD: probably damaging, B: benign. a Found in compound heterozygous (with p.Y288C) in a Greek patient with a mild CD form (Tacke et al., 2005). b Found in compound heterozygous (p.G274R) in a Greek patient with mild phenotype (Tacke et al., 2005; Zano et al., 2013). ⁎ The activity of mutations have been validated in in vitro assays; either using patients' cells or introducing the mutations with site directed mutagenesis into ASPA gene, and test their ASPA enzyme activities in cell lines (see text for details). Note: the power of a single in silico prediction tool is very limited when expression data are available.

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All patients had macrocephaly (except for one), optic atrophy, and blindness with absent or delayed visual evoked responses. The fibroblast cells derived from these patients showed reduction of the ASPA enzyme activity ranging from zero to 13% of the normal. Ozand et al. (1990) suggested that the 12 patients are an inordinately large number considering the rarity of CD disease in general and in a non-Jewish population in particular. In 1992, Ozand et al. (1992) indicated that infantile central nervous system spongy degeneration showed definite tribal occurrence. Kaul et al. (1995), identified a novel missense mutation c.454 T N C (p.C152R) in the ASPA gene of a 2.5-year old patient of Arab descent (nationality unclear, likely Saudi). The CD symptoms were manifested in the child from the age of 5 months, and rapidly progressed within a few months. The authors have demonstrated the effect of the mutation on the ASPA activity by using in vitro culture assay of COS1 cells; they found that this mutation leads to complete loss of the ASPA activity (Table 2). This mutation is listed in the Clinvar/NCBI database as pathogenic mutation; and is predicted to be probably damaging using polyphen2 in silico prediction tool (http://genetics.bwh.harvard.edu) with a score of 0.999 (Table 2). The c.454 T N C mutation is the second missense mutation to be identified in ASPA gene and the fifth mutation of any type of the known causing mutation for CD. In 2008, Kaya et al. (2008) studied 5 Saudi patients, ages ranging from 4-months to 1 year, from unrelated families, who clinically and biochemically were diagnosed with CD using standard criteria confirming the CD. In patient 1, they identified a homozygous splice donor site mutation IVS4 + 1G N T which was previously reported by Rady et al. (2000), in a Turkish female patient. The Turkish patient was diagnosed with CD upon the finding of high level of NAA in the urine, and absence of the ASPA enzymatic activity in cultured skin fibroblasts. Patient 2 was found to harbor a homozygous missense mutation, c.79G N A (p.G27R), and this mutation was previously reported in a German CD patient by Kaul et al. (1996), and they demonstrated that this mutation was able to retain only 3% of the wild type activity of the ASPA enzyme activity using an in vitro expression of the mutant cDNA constructs in COS1 cells versus the wild type. This mutation has also been reported in a 9-month old Turkish girl in homozygous form (Eke et al., 2012); this girl met all the clinical diagnostic criteria of CD and showed an increase of NAA in the urine. Polyphen2 predicts the p.G27R mutation as probably damaging; with a score of 1.000 (Table 2), and carrier status from the associated families of patients 1

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and 2 was confirmed. In patients 3 and 4, Kaya et al. (2008) identified a large novel deletion of 3346 bp region spanning intron 2, chr17: 3,332,550 position; and ends at intron 3 chr17: 3,335,897 position (Table 2). Patient 5 was found to be homozygous for a 92 kb large deletion. The same deletion reported by Zeng et al. (2006) which is 7.16 kb upstream of the ASPA start site and the distal deletion point extended to the 3′ sequence of the adjacent TRPV3 gene resulting in deletion of exons 2–17. Zeng et al. found this deletion in two patients: an Italian patient found to be homozygous for the deletion and the other patient is a German/British who is a compound heterozygous of the 92 kb deletion and p.A305E mutation. The 92 kb large deletion results in a complete absence of the ASPA gene in the homozygous and the compound heterozygous patient, leading to the abolishing of the enzyme activity. Interestingly, the tremor rats (developed from a naturally occurring mutant) were found to harbor a genomic deletion of more than 200 kb, including the ASPA and TRPV3 genes (Kitada et al., 2000). The homozygous deletion in the tremor rat (tm/tm) exhibited absence-like seizure earlier, and was stronger in frequency than in heterozygous form (tm/+) (Higashiguchi et al., 1991). The tm/tm rat is considered to be an ideal animal model for human CD because, similar to humans, the absence of the ASPA expression leads to the NAA accumulation in the rat brain, presenting with comparable clinical neurological symptoms, such as seizure, and severe motor and cognitive impairment. Pathologically, these rats show spongiform degeneration of the white matter and gray matter (Kondo et al., 1991). Tremor rats have been used as in vivo animal model for gene therapy for CD (Seki et al., 2002; McPhee et al., 2005). 4.2. Jordan and Kuwait Masri and Hamamy, (2006) reported the first case study in Jordan. The propand was a 3-month old male infant presenting with axial and peripheral hypotonia and poor visual fixation (Table 2), born from consanguineous marriage. The family history revealed that the proband has four brothers and one sister, all manifesting the clinical symptoms consistent with the diagnostic criteria of CD, with very high level of urine NAA. Two of the proband's brothers died at the age of 10 and 5 years, while the proband himself succumbed to the condition at the age of 3-years. The two remaining affected children were alive at the ages of 13 and 9 years at the time of reporting. Molecular analysis revealed a missense mutation in exon 1 of the ASPA; c.79G N A (p.G27R) mutation

Table 2 Summary of the ASPA gene mutations, in silico prediction, and clinical presentations in Arab patients with CD. Country

NC

ASPAA⁎ CS

PN

Reference

p.C152R PrD (0.99,0.14,0.99) p.I177T PsD (0.91,0.81/0.94)

Loss

1c

Kaul et al. (1995)

2

Di Pietro et al. (2013)

p.G27R

PrD(1,0,1)

3%

2

Masri and Hamamy (2006), Kaya et al. (2008)

1c

Kaya et al. (2008), and Zeng et al. (2006) Kaya et al. (2008) Kaya et al. (2008), Rady et al. (2000)

AAC h

Arab

c.454 T N C

Egypt

c.530 T N Ch

Saudi Arabia/Jordan c.79G N Ah

PP (S,S,S)

Loss

Saudi Arabia

92 kb delh

NA

NA

Loss

Saudi Arabia Saudi Arabia

3.346 kb delh NA IVS4 + 1G N Th NA

NA NA

Loss Loss

Yemen

698insCh

FS

NA

Loss

Yemen

c.456C N Gh

C152W

PrD(1,0,1)

Loss

High pitched cry, lack of visual tracking, developmental delay, head lag, hypotonia, increased startle reflex. Neurosensory deficits, multiple tendinous retractions, severe psychomotor retardation, macrocephaly, and prominent parietal drafts. Severe psychomotor, retardation, macrocephaly, spasticity, and seizure, severe head lag, exaggerated deep tendon reflexes. Neonatal seizures, severe head-lag, central hypotonia, macrocephalic with spastic quadriparesis. Macrocephaly, severe spasticity Progressive, neuro-regression, macrocephaly, spastic quadreperesis, central hypotonia, oropharyngeal dysplasia, macrocephaly, and mild seizures. Early onset of seizures, optic atrophy macrocephaly, behavioral and development delay. Seizures, optic atrophy macrocephaly, behavioral and development delay.

2 1

1c

Zeng et al. (2002)

1nc Zeng et al. (2002), Hershfield et al. (2007)

Legend: NC: nucleotide change, AAC: amino acid change, PP: PolyPhen2 prediction (http://genetics.bwh.harvard.edu), S,S,S: score, sensitivity, specificity, PrD: probably damaging, PsD: possibly damaging, FS: frameshift, NA: not applicable, ASPAA: ASPA activity, CS: clinical symptoms, PN: number of reported Arab patients, del: deletion, c: consanguinity, nc: nonconsanguinity (mentioned only when reported), h: homozygous mutation. Note: the value of a single in silico prediction tool is limited, when expression data are available ⁎ The activity of mutations have been validated in in vitro assays; either using patients' cells or introducing the mutations with site directed mutagenesis into ASPA gene, and test their ASPA enzyme activities in cell lines (see text for details). Reference sequence: NM_000049 .2 and NP_000040.1

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(Kaul et al., 1996), as mentioned above, this mutation is a pathogenic mutation. Bin Nakhi et al. (2012) published a poster (http://adc.bmj.com/ content/97/Suppl_2/A155.3) describing a 3-month old Kuwaiti female diagnosed with Canavan disease who presented with macrocephaly, hypotonia, and global developmental delay. She was diagnosed with CD. There was no mention of the molecular analysis in the abstract for this study, and a full poster was not possible to obtain. 4.3. Egypt Recently, Di Pietro et al. (2013) have reported two CD Egyptian siblings of 4 years and of 4 months old. At four months of age, the clinical evaluation showed macrocephaly, hyperechogenicity of the white matter and thalamus, poor motility, no head control, and exophoria. Recently, Drera and Poggiani, (2014) studied the 4-month old child using neuroimaging and sonography and were able to find clinical diagnostic features unique for the 4-month CD child. The study suggested that the brain ultrasonography should be used for diagnosis of progressive macrocephaly and hypotonia as a first step, as it may be helpful to address the diagnostics features related to neuroradiological and biochemical work-up. Both the Egyptian siblings were found to harbor a homozygous c.530 T N C (p.I177T) amino acid change (Di Pietro et al., 2013). The c.530 T N C mutation was predicted by polyphen2 as possibly damaging, with a score of 0.91 (Table 1). This mutation is responsible for the severe clinical phenotype for the two Egyptian siblings, and led to the loss of the enzyme activity. Interestingly, the authors found that NAA concentration in the urine appears to be inversely correlated to the severity of the CD symptoms and progression: the 4-year old child with severe clinical phenotype showed lower NAA excretion in the urine than the 4-month old child with the less severe clinical manifestations. 4.4. Morocco and Tunisia Though there were no CD reports among Moroccan patients; it is expected that there are some Jewish Moroccans who are carriers or have the disease. In the USA, there are more than 100,000 Moroccan Jews; and also more than 1 million Israeli (15% of the population) are Moroccan in origin. Given the frequency of CD among the Jewish population (including Moroccan Jews), the Moroccans are expected to show considerable frequency of CD. In 2009, Kraoua et al. (2009) conducted a study within Tunisia over a three-year period, included 136 patients diagnosed with neurometabolic diseases, and one patient with CD was confirmed. There is no more information reported on this patient. 4.5. Yemen Zeng et al. (2002) reported two Yemenite patients with CD. One patient has an insertion in the nucleotide position 698 of exon 5 of the ASPA gene (698insC) (Table 2) with reported consanguinity. The second patient has a p.C152W mutation in homozygous form. The mutation is predicted by polyphen2 as probably damaging (Table 2). The mutation mentioned above was reported in an Arab CD patient in the same residue (p.C152R) (Kaul et al., 1995). Both mutations are predicted to be not tolerated with SIFT (http://sift.jcvi.org) (data not shown). Another Irish CD patient has been reported to have a different mutation at the same residue (p.C152Y) (Kaul et al., 1996). 4.6. Genotype–phenotype correlation of Arab patients with CD Although only 21 CD Arab patients were diagnosed across the Arab world, they demonstrated a strong correlation between the clinical severity of CD, elevated level of NAA, and diminished expression of ASPA enzyme. Of the 21 patients, 18 were originating from Saudi Arabia, predicting a high incidence of CD in Saudi Arabia. Interestingly, all the

ASPA variants, which affect the ASPA enzyme function, were found in homozygous form among 11 Arab patients (Table 2), mostly Saudis. The in silico analysis of the missense mutations detected in the 6 CD Arab patients (p.C152R, p.I177T, p.G27R, p.C152W) was predicted as probably damaging with polyphen2 (Table 2) and as not tolerated with SIFT (data not shown). These predictions together with the expression data showed a strong correlation with the clinical phenotype. The large genomic deletions, 92 kb and 3.346 kb, detected in 2 Saudi Arab patients (Table 2), showed a strong correlation between the genotype and phenotype, which is consistent with what was reported by Zeng et al. (2006) for the same two deletions and two other multiexonic deletions detected in European patients. The multiexonic deletions were 12.3 kb (including exons 4 and 5) and 56 kb (involving all exons, except exon 1 of ASPA gene) (Zeng et al., 2006), which showed a strong genotype–phenotype correlation. Using the Human Splicing Finder (a tool to predict the effects of mutations on splicing signals; http://www.umd. be/HSF), the intronic variant (IVS4 + 1G N T) found in a Saudi patient (Table 2) is predicted to have a broken splicing site at exon4–intron4 splice junction and possible creation of a new cryptic splicing site with a significant prediction score (data not shown), this in silico prediction showed a strong correlation with the ASPA expression data, and the severe clinical phenotype of the Saudi patient (Table 2). In summary, the Arab patients with CD, showed strong relationship between the severity of the clinical phenotype and the genotype, this makes the Arab scenario very interesting, to teach the medical genetics community about the molecular pathology nature, not only for CD, but also for all the monogenic and multigenic disorders, which will promote more understanding of the genotype–phenotype correlation, and help in developing therapeutics and molecular diagnostics approaches. 4.7. Treatment for Canavan disease Due to the absence of curative therapy for CD, special diets have been suggested to limit its progression and symptoms (Janson et al., 2005; Madhavarao et al., 2009). Some symptomatic treatments have been developed to alleviate the burden of the variable severe symptoms of the disease (Gordon, 2001), occupational therapy has been used to improve seating posture, motor movement, and minimizes contractures, and massage has been routinely used to help in any physical discomfort caused by CD. Protection of oligodendrocytes against damage from the rise of NAA can be controlled using biologic agents, including drugs (Baslow et al., 1999), which might be able to suppress the production of NAA. Recently, Francis et al. (2014) treated two cohorts of nur7 mice with a dietary regimen containing 35% triheptanoin (a substrate, with 98% purity, which provides ketone bodies capable of traversing the blood brain barrier). One cohort consisted of neonatal mice, which were provided with triheptanoin via nursing dams from birth onward. The second cohort was provided with dietary triheptanoin from weaning onward (28 days of age). The treatment resulted in enhancement of the survival of oligodendrocytes and reduction of the spongiform degeneration. In addition, the myelin in the brain was increased and motor functions were improved in the treated mice. The improvement in CD clinical parameters was significant in younger mice compared to older ones in which the improvement was markedly modest (Francis et al., 2014). Interestingly, there was no significant difference in NAA levels between the treated and nur7 control mice. Although this study showed a significant therapeutic benefit for treated animals, the mechanism of action of triheptanoin could not be elucidated (Francis et al., 2014). CD is considered to be an ideal candidate for gene therapy treatment due to the following criteria: the gene and protein are well identified, the window of time for treatment is well defined, the affected part is limited to the brain, and the brain can be easily monitored by imaging and scanning following treatment. Recently, Ahmed et al. (2013) have performed an encouraging gene therapy attempt in CD mice (ASPA−/−) model by infusing recombinant adeno-associated

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viruses (rAAVs) carrying the wild type ASPA gene into CD ASPA−/− mice. The treatment was able to rescue the early lethal phenotype of CD, prolong survival of CD mice, and improve the overall symptoms of the disease in the corrected mice. An auspicious in human CD gene therapy clinical trial has used an adeno-associated viral vector carrying the wild type ASPA gene (AAV2-ASPA) to infuse 6 different brain regions of 13 CD patients via intraparenchymal delivery (Leone et al., 2012). Preliminary results on the treated patients were promising showing no adverse events and marginal anti-vector immunity. Over a long period of time (5–10 years), a noticeable improvement in the patients' status was observed, and this included reduction of the NAA level, slow progression of brain atrophy, improvement on seizure frequency and on motor function, especially among the younger CD-treated patients, suggesting the significance of early therapeutic intervention (Leone et al., 2012). Considering the normative data (including the old cohort of patients), the raw scores fell within the range for patients severely affected by spastic quadriplegia (Leone et al., 2012); consistent with this, Klugmann et al. (2005) delivered AAV1/2 (a serotype superior to AAV2) to juvenile (3 week old) tremor rats resulting in major improvements in both seizure frequency and seizure length. This is an important observation, as it teaches us a great deal about the intervention time of gene therapy treatment for patients with CD. Both the gene therapy and the triheptanoin treatments are steps in the right direction. Although the progress of the gene therapy trials to treat CD raises hopes for treating a wide range of neurodegenerative disorders, there are still lingering challenges facing such treatment, including reduced efficiency of gene vector targeting to the oligodendrocytes (Chtarto et al., 2013), the unclear genotype–phenotype correlation of CD, the small number of CD patients, and the variable clinical course and progression rate of patients with CD. An administration of a non-invasive dietary regimen in CD nur7 murine model using triheptanoin is an encouraging approach, although it is proved to be efficient; the mechanism of triheptanoin in relieving the CD pathology needs to be thoroughly studied and explained. Once such mechanism is understood, a rigorous preclinical development is needed to determine a safe therapeutic dose of the drug before advancing to clinical trials in humans. Both of the treatments were very instructive in determining the window of the therapeutic intervention, which is more efficient during the developmental myelination. 4.8. Perspectives on genetic testing for CD in the Arab countries The Arab world is comprised of 22 Arab-speaking countries, an area extended from the Atlantic Ocean in the west to the Arabian Sea in the east. The Arab population is approaching 0.5 billion, and this region has been extensively exposed to many successive invaders from Turkey, Rome, and Europe, as well as traders and immigrants who contribute to mixing the ethnic demographic of the population, explaining the gene flow exchange among the Arabs and these different ethnic groups. One of the obstacles hindering a smooth and problem-free application of genetic testing, as a preventive solution for genetic diseases in Arabia, is the restrictions inherent in Arab culture, and social stigma against patients presenting with the disease. This pertains especially to the Arab women population. Although prevention is usually the best part of cure, preventing or lowering the birth of children who may most likely present with the disease, is a significant challenge for many Arabs who see it as a crime – according to the way in which religious and/or Islamic laws are interpreted – to either participate in birth control methods or to abort the fetus. An exception is found primarily when there is an eminent threat to a woman's – usually a pregnant woman's – health and well-being (Hathout, 1997). In addition, some Arabs view abortion as acceptable if performed before or within the first four months, or 120 days of pregnancy (Swinford and El-Fouly, 1987). Remedies, such as training medical personnel, increasing the level of religious and public health awareness, and emphasizing the necessity of participating in prenatal diagnostic programs, are crucial for

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educating members of the Arab population in which consanguineous marriage is a normal and prevalent part of cultural practice. CD mutations have been mainly reported in individuals of Jewish ancestry with an incidence of 1 in every 40 Jewish persons carrying CD causing mutations. Carrier screening of CD has been part of the ethnicity-based screening program mainly in the USA among Ashkenazi Jews, several molecular diagnostic companies have included the causative mutations for CD in a panel for genetic testing by sequencing the ASPA gene. Among Arab countries, Saudi Arabia is considered to have the highest incidence of CD, with different kinds of mutations, including deletion (92 kb deletion and 3.346 kb deletion), missense (p.G27R), and splice (IVS4 + 1G N T) mutations (Table 2). This high frequency of mutations are due to the prevalent culture of consanguineous marriage among the Saudi population. Although most of the mutations are detected as novel in Arab patients with CD, there is no sufficient molecular testing for well characterized Arab patients to conclude Arab founder mutations. On the contrary, mutations such as p.Y231* and p.E285A, which represent 98% of the mutated alleles among Ashkenazi Jewish population, suggest a founder effect. Understanding both the clinical picture and molecular foundation of the disease in Arabia will significantly contribute to more understanding of the molecular pathology picture, and will help in the development of customized molecular diagnostic panels for the Arab population. With the current molecular profile of Arab patients, diagnostic approaches used to test for mutations in Arab population should screen the entire ASPA gene for potential variations, including the introns and the regulatory regions, in contrast with the targeted mutation analysis for the Ashkenazi Jewish population, where the founder and causative mutations are very well known. 5. Conclusion Although CD is a rare neurological disorder with an overall small number of patients, it has been reported in many Arab countries with the highest incidence among Saudi patients, with a clinical picture consistent with the classical clinical phenotype. Several mutations have been noted to circulate among Arab patients with no specific underlying or founder mutations. Most mutations were missense mutations followed by deletion mutations. The mutation distribution among Arabs seems to be different from other ethnic groups, which gives the CD Arab patients a distinct molecular profile, believed to be mainly due to the prevalent deep-rooted intra-familiar marriage culture. Molecular studies of well-characterized Arab patients will improve our understanding of the molecular pathology of CD, contribute to the development of accurate and precise molecular diagnostics for the disease, and promote better understanding of the genotype–phenotype correlation of CD. Carrier screening of CD will be an efficient preventative measure to protect from increasing the prevalence of the disease in Arabia, which will remain screened by full gene sequencing. Both triheptanoin and gene therapy are promising treatment approaches for CD, but they are still in their infancy to establish an approved therapy for patients with CD. Conflict of interest statement I certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. References Adachi, M., Schneck, L., Cara, J., Volk, B.W., 1973. Spongy degeneration of the central nervous system (Van Bogaert and Bertrand type; Canavan's disease). Hum. Pathol. 4, 331–347. Adornato, B.T., O'Brien, J.S., Lampert, P.W., Roe, T.F., Neustein, H.B., 1972. Cerebral spongy degeneration of infancy: a biochemical and ultrastructural study of affected twins. Neurology 22, 202–210.

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