Sami Bizzari

Lara El-Bazzal

Pratibha Nair

Antoine Younan

Samantha Stora

Cybel Mehawej

Stephany El-Hayek

Valerie Delague

André Mégarbané
email: andre.megarbane@institutlejeune.org

Recessive marfanoid syndrome with herniation associated with a homozygous mutation in Fibulin-3

Keywords:

We have previously reported on a consanguineous family where 2 siblings, a girl and a boy, presented with tall stature, long and triangular faces, prominent forehead, telecanthus, ptosis, everted lower eyelids, downslanting palpebral fissures, large ears, high arched palate, long arm span, arachnodactyly, advanced bone age, joint laxity, pectus excavatum, inguinal hernia, and myopia, suggestive of a new

Introduction

The extracellular matrix (ECM) comprises a dynamic framework of macromolecules involved in the structural support of the organs, tissues, and cells it surrounds, as well as a multitude of essential biochemical and mechanotransduction cell signaling processes [START_REF] Hynes | The extracellular matrix: not just pretty fibrils[END_REF]. Heritable connective tissue disorders (CTD) encompass a large number of clinically and genetically heterogeneous systemic diseases, mainly caused by dysfunction in the fibrous structural and organizational components of the ECM and their regulators [START_REF] Bateman | Genetic diseases of connective tissues: cellular and extracellular effects of ECM mutations[END_REF].

Two Lebanese siblings were previously described with a constellation of symptoms resembling features of CTD [START_REF] Megarbane | Marfanoid habitus, inguinal hernia, advanced bone age, and distinctive facial features: a new collagenopathy?[END_REF]. Lack of particular features including ectopia lentis, skin bruising and hyperelasticity, tissue fragility, and cardiovascular symptoms rendered a Marfan syndrome or any subtype of Ehlers-Danlos syndrome diagnosis unlikely [START_REF] Pepe | Marfan syndrome: current perspectives[END_REF][START_REF] Joseph | Characteristics, diagnosis, and management of ehlers-danlos syndromes: a review[END_REF]. Additionally, array-CGH as well as Sanger sequencing of candidate genes implicated in CTD and overgrowth syndromes had previously failed to identify a genetic defect in this family [START_REF] Megarbane | Marfanoid habitus, inguinal hernia, advanced bone age, and distinctive facial features: a new collagenopathy?[END_REF].

In search for a genetic component, Whole Exome Sequencing (WES) was performed on these patients and possible gene associations with the phenotype were reviewed. We report on updated clinical findings as well as molecular characterization of this rare disorder with the identification of a novel homozygous missense mutation in EFEMP1 (EGF-Containing Fibulin-Like Extracellular Matrix Protein 1) (p.Cys55Arg). We additionally report on a nonsense mutation in VCPKMT (Valosin Containing Protein Lysine (K) Methyltransferase) (p.Tyr33*), as well as https://doi.org/10.1016/j.ejmg.2020.103869 Received 8 July 2019; Received in revised form 26 January 2020; Accepted 27 January 2020 suggestive of a new subtype of CTD [START_REF] Megarbane | Marfanoid habitus, inguinal hernia, advanced bone age, and distinctive facial features: a new collagenopathy?[END_REF].

The sister, patient IV.1 was reexamined at age 19. Since her last examination, she was operated for bilateral ptosis and had developed bilateral mild hearing impairment at 14 years of age. At age 15, she was operated for cystorectocele secondary to pelvic floor laxity. A year later, she had breast implants for severe breast ptosis. At age 17, she was again operated for intestinal occlusion secondary to obstruction at the level of sigmoid colon. Work-up showed acute diverticulitis with stenosis. She was operated for a diversion colostomy. At examination, her height was 180 cm (Z-score: 2.6SD); head circumference (OFC) 58 cm (Z-score > 1.8SD; >1.8SD at age 10); and weight 86 kg (Z-score: 1.8SD; 2.5SD at age 10). A month later, she was operated, by laparoscopy single port technique, for resection of the diseased sigmoid. During the operation, diffused diverticular disease that extended from the stomach, the small intestine, to the upper rectum was noted. She underwent resection of the stenotic and diverticular sigmoid with mechanical colorectal anastomosis. Post-operative course was uneventful. Ten months later she was operated for a huge ventral hernia at the single port trocar surgery site. Heart and neurological examinations were normal. Ophthalmological examination was normal except for the presence of high myopia.

The affected brother, patient IV.3, was followed up at age 14 in the clinic (Fig. 1B). At the time of examination, his height was 183 cm (Zscore: 2.4SD); OFC 56.5 cm (Z-score > 1.2SD; >1.2SD at age 5); and weight 62 kg (Z-score: 0.9SD; 1.5SD at age 7). His chest circumference was 87 cm (Z-score > 1.6SD). He presented similar, but more severe symptoms to his affected sister. Since the age of 10, he was operated for multiple inguinal, crural, and abdominal herniae, and was admitted several times for recurrent intestinal occlusions. One of the episodes needed laparoscopy exploration which showed several pseudo-diverticula extended from the stomach to the terminal ileum with intestinal pneumatosis and several diverticula that were perforated with peritoneal cavity pneumatosis. Huge diverticula on the wall of the stomach was noted as well. He also had an obstructive intestinal volvulus at the mid ileum with multiple herniae on the abdominal wall with internal obturator hernia and left inguinal hernia. Four months later he was operated for a resection of a huge diverticula of the bladder. A few months later, he was operated for a left diaphragmatic hernia, and another recurrent bilateral inguinal hernia and abdominal herniae with rectal prolapse, and then for a severe perineal hernia where a large portion of the urinary bladder was herniated through the right pelvic floor muscles into the right ischiorectal and ischioanal fossa. He exhibited recurrent episodes of diarrhea, vomiting, and abdominal pain,. Pectus excavatum was more prominent and he additionally exhibited mild right convex dorsal scoliosis, and high myopia.

Clinical examination of the parents and healthy sibling including anthropometric measurements, as well as ophthalmological and cardiac examinations were normal.

Molecular report

This study was approved by the institutional ethics review board. 3-5 ml whole blood samples were obtained from the two siblings upon informed consent and genomic DNA was extracted for WES. The boosted exome test using the TruSeq SBS v3 kit and HiSeq 2000 platform (Illumina, San Diego, CA, US) by Invitae (San Francisco, CA, US) was used, covering approximately 95% of the exome and 10bp of intron-exon boundaries at a mean depth coverage of 267x and minimum depth coverage of ≥20x per base. Variant selection processes used in filtering these variants included MAF<1%, pathogenicity, genomic position, quality, family history, and literature review; 940 genes associated with Human Phenotype Ontology clinical terms described in both siblings were additionally reviewed.

Variant filtration revealed a homozygous missense transition in the EFEMP1 gene, NM_001039348.2: c.163T > C; p.(Cys55Arg) and a homozygous nonsense transversion in the VCPKMT gene, NM_024558.2: c.99T > G; p.(Tyr33*) in both siblings. These two variants were confirmed through sanger sequencing in both patients and found to be in the heterozygous state in the healthy brother and parents (Fig. 1C andD). The former mutation replaces a phylogenetically conserved cysteine residue in the first N-terminal epidermal growth factor (EGF)-like domain of the Fibulin-3 protein and the latter mutation causes an early truncation in the lysine methyltransferase protein METTL21D (Fig. 2A,B,D). Using Combined Annotation Dependent Depletion (CADD) software, both mutations were predicted to be deleterious with high Phred-like scaled C-scores of 28.3 and 34 for the variants identified in EFEMP1 and VCPKMT respectively (https://cadd. gs.washington.edu/). Neither of these variants were reported in the heterozygous or homozygous state in public databases including dbSNP, gnomAD, ClinVar and 1000G.

A homozygous 2bp deletion resulting in frameshift and termination in the MYO3A gene NM_017433.4: c.1370_1371delGA; p. (Arg457Asnfs*25) was additionally identified in patient IV.1. CADD software predicts the latter variant to be disease causing with a Phredlike scaled C-score of 34. This variant is reported in the heterozygous state in gnomAD with an allele frequency of 0.001% (3 alleles in 3 adults of European origin out of 249394 alleles; rs760866131) and occurs at a conserved residue in the motor domain of the Myosin IIIA protein (Fig. 2C andD). This variant was not reported in ClinVar.

The EFEMP1 and MYO3A variants were checked for in the Saudi Human Genome Program database which contains over 14000 patientderived genetic variants; neither of these variants have been reported (https://genomics.saudigenomeprogram.org/en/researchers/#dbaccess) [START_REF] Abouelhoda | Revisiting the morbid genome of Mendelian disorders[END_REF]. Clinical and molecular details of the described patients were summarized in Table 1.

Discussion

We report on a consanguineous Lebanese family exhibiting a novel connective tissue syndrome. Hallmark clinical manifestations in the two affected siblings involved multiple and recurrent herniae, pelvic and rectal prolapse, huge diverticula, marfanoid habitus, joint laxity, dorsal scoliosis, advanced bone age, pectus excavatum, dysmorphic facial features, and myopia. WES and in silico analysis pointed to EFEMP1 as the probable candidate gene implicated in this autosomal recessive disorder. We additionally report on a mutation in the MYO3A gene that is likely causative of nonsyndromic progressive hearing loss.

EFEMP1 encodes Fibulin-3, a member of eight conserved secreted ECM basement membrane glycoproteins (MIM# 601548) [START_REF] Hanada | Expression and purification of recombinant fibulins in mammalian cells[END_REF]. Short fibulins including fibulin-3/-4/-5 have evolved into the specialized function of elastic fiber (EF) assembly and homeostasis. Fibulin-4 and fibulin-5 are essential in the structural and organizational assembly of EF through their interactions with ECM components that form, deposit, and merge elastin with the microfibril scaffold [START_REF] Yanagisawa | Unraveling the mechanism of elastic fiber assembly: the roles of short fibulins[END_REF]. Fibulin-3 is expressed widely in adult tissues (abundant in eye, fascia, skin and vasculature) and is predominant in basement membrane epithelial, endothelial, and fibroblast cells; it interacts with various ECM components and is thought to be involved in basement membrane integrity and EF maintenance rather than assembly [START_REF] Zhang | Focus on molecules: fibulin-3 (EFEMP1)[END_REF].

Reported heritable disease associated with EFEMP1 (Doyne's Honeycomb Retinal Dystrophy/Malattia Leventinese; MIM# 126600) involves characteristic formation of drusen (yellow-white deposits) in the posterior pole region of the eye during early adulthood, leading to progressive and severe vision loss with macular and retinal degeneration; this is attributed to a single autosomal dominant mutation p.(Arg345Trp) (rs121434491) [START_REF] Hulleman | Malattia leventinese/doyne Honeycomb retinal dystrophy: similarities to age-related macular degeneration and potential therapies[END_REF]. There is one instance of recessive transmission reported in offspring of affected heterozygous parents, which was associated with a relatively more severe ocular phenotype, corroborating the dominant toxic gain of function effect of the mutation [START_REF] Fu | The R345W mutation in EFEMP1 is pathogenic and causes AMD-like deposits in mice[END_REF]. Eye-related features reported in the patients in this study included ptosis and myopia with no evidence of drusen formation or macular degeneration. Mouse models exhibiting the EFEMP1 p.(Arg345Trp) mutation developed sub-retinal pigment epithelium (RPE) deposits and presented with cardinal features of Malattia Leventinese and age-related macular dystrophy [START_REF] Stanton | Deletion of Efemp1 is protective against the development of sub-RPE deposits in mouse eyes[END_REF][START_REF] Marmorstein | Formation and progression of sub-retinal pigment epithelium deposits in Efemp1 mutation knock-in mice: a model for the early pathogenic course of macular degeneration[END_REF], whereas knockout (EFEMP1 -/-) mice lacked these deposits and were resistant to its formation under induced conditions [START_REF] Zayas-Santiago | Mutant fibulin-3 causes proteoglycan accumulation and impaired diffusion across Bruch's membrane[END_REF].

Strikingly, multiple studies on EFEMP1 -/-mice revealed a phenotypic resemblance to the siblings reported here (Table 1). The hallmark findings in these mice involved severe herniation (in the C57BL/6 strain), in addition to early aging, reduced bone density and lordokyphosis, pelvic and rectal prolapse, reproductive impairment, generalized muscle, fat, and organ atrophy, reduced body mass, loose skin, and hair thinning [START_REF] Mclaughlin | Lack of fibulin-3 causes early aging and herniation, but not macular degeneration in mice[END_REF][START_REF] Rahn | Failure of pelvic organ support in mice deficient in fibulin-3[END_REF]. The herniated mice exhibited multiple sites of direct and indirect large inguinal hernia as well as xiphoid process protrusions; all EFEMP1 -/-mice died at a significantly earlier age relative to wild type mice, with multiple phenotypes including atrophic features, reduced body size/mass, and loose skin attributed to expected, albeit premature, consequences of aging in mice comparable to human aging. The asthenic appearance and thinness described in both patients can be compared to the atrophic (muscle and fat) features and reduced body size in the mice. In the patients' original clinical analysis 9 years prior, no skin manifestations were reported with noted absence of hyperelasticity, scarring, and redundant skin expected in CTD; however patient IV.1 has developed severe breast ptosis indicating loss of skin elasticity. CTDs including Marfan syndrome and Ehlers-Danlos syndrome have been associated with hernia predisposition [START_REF] Harrison | Collagenopathies-implications for abdominal wall reconstruction: a systematic review[END_REF]. Hypomorphic mouse models of Marfan syndrome (Fibrillin-1) were shown to exhibit diaphragmatic hernia associated with severe kyphosis, scoliosis, as well as rib -and to a lesser degree long bone-overgrowth; however pulmonary and severe vascular features lead to early death (Schwill et al., 2013). While no obvious skeletal defects or other systemic features were identified in EFEMP1 -/-mice, they exhibited severe kyphosis and reduced bone density [START_REF] Mclaughlin | Lack of fibulin-3 causes early aging and herniation, but not macular degeneration in mice[END_REF]. Our patients exhibited musculoskeletal features including advanced bone age, pectus excavatum (rib-cage deformity), scoliosis (Patient IV.3), and joint laxity indicating a role of Fibulin-3 in bone and cartilage. Fibulin-3 is expressed in condensing mesenchymal cells and is involved in skeletal and cartilaginous development at the embryonic stage [START_REF] Ehlermann | Cloning, expression and characterization of the murine Efemp1, a gene mutated in Doyne-Honeycomb retinal dystrophy[END_REF]. In studies on mouse models of osteoarthritis, severely reduced Fibulin-3 expression in articular cartilage with increasing age correlated with disrupted cartilage integrity. This is thought to be due to Fibulin-3's involvement in inhibiting chondrocyte differentiation, with healthy immature chondrocytes being a property of healthy cartilage in joints (Hasegawa et al., 2017). In EFEMP1 -/-mice, a severe reduction in EFs specifically in fascia connective tissue (e.g. transverse and superficial fascia), and much less so in other tissues, including the lung, skin (dermal EF), and aorta, was reported as the main pathology. The localized pathogenic effect in fascia tissue of EFEMP1 -/-mice may explain the prominence of various types of hernias and lack of multiple systemic CTD features in our patients, and suggests a possible loss-offunction effect of the p.(Cys55Arg) mutation. Functional studies are required to establish the deleterious effect of the reported mutation.

Fibulins exhibit tandem repeats of calcium binding EGF-like domains. These domains exhibit six conserved cysteine residues which form disulphide bond loops. The homozygous missense variant we report in EFEMP1 p.(Cys55Arg) likely disrupts the disulphide bond in the first N-terminal atypical calcium binding EGF-like domain, and is

Table 1

Summary of the Clinical and Molecular Characteristics in patient IV.1 and IV.3 as well as highlighted characteristics of EFEMP1 Null and EFEMP1 p.Arg345Trp knockin mice. predicted to be deleterious using in silico tools.

The exact molecular function of Fibulin-3 remains unclear. Fibulin-3 is thought to antagonize ECM enzyme activity by promoting expression of tissue inhibitors of metalloproteinases TIMP-3/-1 and inhibiting expression of matrix metalloproteinases MMP-2/-3/-9, leading to homeostatic protection against elastic fiber and collagen degradation [START_REF] Zhang | Focus on molecules: fibulin-3 (EFEMP1)[END_REF]. EFEMP1 along with tumor-suppressor protein WT1 were found to be most highly expressed in mouse connective tissue identical to human transversalis fascia, the protrusion site of inguinal hernias [START_REF] Jorgenson | A genome-wide association study identifies four novel susceptibility loci underlying inguinal hernia[END_REF]. Causal network analysis in the latter study highlighted common interaction between these two proteins with TIMP3; WT1 is thought to activate TIMP-3, which Fibulin-3 binds in turn to potentially amplify its inhibitory activity on proteolytic ECM enzymes. This function is further supported by studies of p.Arg345Trp EFEMP1 knockin mice which exhibited inhibited proteoglycenase (MMPs) activity in Bruch's membrane, the potential root of deposit formation located underneath the RPE [START_REF] Zayas-Santiago | Mutant fibulin-3 causes proteoglycan accumulation and impaired diffusion across Bruch's membrane[END_REF]. The retention of components in aberrant ECM is thought to elicit a complement system response which exacerbates deposit formation. Recent studies by one group have however showed conflicting results, between mouse and human RPE cell cultures, on the potential ECM enzyme inhibitory role of p.Arg345Trp Fibulin-3 and deposit formation [START_REF] Fernandez-Godino | A local complement response by RPE causes early-stage macular degeneration[END_REF], 2018). In a following study involving unaltered EFEMP1, the authors illustrate complement driven accumulation of collagen and inhibition of the ubiquitin proteasome pathway in human RPE cells; the latter is thought to impair ECM turnover with repression of TIMP-3 activity and increased aberrant MMP-2 activity compounding deposit formation (Fernandez-Godino and Pierce, 2018).

Genome wide association studies have linked various single nucleotide polymorphisms (SNPs) in EFEMP1 to a variety of phenotypes. Pertinent associations include multiple SNPs (rs2009262 and rs11899888) exhibiting the highest association to the formation of direct and indirect inguinal hernias [START_REF] Jorgenson | A genome-wide association study identifies four novel susceptibility loci underlying inguinal hernia[END_REF]. In another study, the rs3791675 SNP in EFEMP1 was one among 20 SNPs that explained approximately 3% of variation in height between two groups that expressed the least and most alleles associated with being tall respectively [START_REF] Weedon | Genome-wide association analysis identifies 20 loci that influence adult height[END_REF]. EFEMP1 SNPs also correlated with forced lung capacity, and other SNPs with opposing behavioral traits from harm avoidance to suicidal tendencies, thought to be due to a role of Fibulin-3 in neuroplasticity [START_REF] Balestri | Nine differentially expressed genes from a post mortem study and their association with suicidal status in a sample of suicide completers, attempters and controls[END_REF][START_REF] Loth | Genome-wide association analysis identifies six new loci associated with forced vital capacity[END_REF].

Patient IV.1 was additionally shown to have a homozygous mutation p.(Arg457Asnfs*25) in MYO3A, which encodes Myosin IIIA, an actin-dependent cargo transport motor protein that binds and transports actin-bundling proteins to stereocilia actin filament tips to promote elongation. This process is required for the fine-tuned height organization of the stereocilia staircase [START_REF] Ebrahim | Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like[END_REF]. Pathogenic variants in this gene were extensively reported to be the cause of bilateral progressive hearing loss that appears in the second decade or later (DFNB30; MIM# 607101) [START_REF] Dantas | Characterization of a novel MYO3A missense mutation associated with a dominant form of late onset hearing loss[END_REF][START_REF] Walsh | From flies' eyes to our ears: mutations in a human class III myosin cause progressive nonsyndromic hearing loss DFNB30[END_REF]. The variant we report potentially leads to a truncated protein devoid of its actin-binding site, and is predicted to be deleterious in silico. This mutation is highly likely to be responsible for the progressive non syndromic deafness exhibited by patient IV.1.

The siblings were also found to share a homozygous nonsense mutation p.(Tyr33*) in VCPKMT (MIM# 615260), which encodes METTL21D, a lysine methyltransferase belonging to a recently characterized family (family 16) of 10 seven-β-strand methyltransferase members [START_REF] Kernstock | Lysine methylation of VCP by a member of a novel human protein methyltransferase family[END_REF]. METTL21D was shown to uniquely tri-methylate valosin containing protein (VCP), and this modification is thought to be indirectly enhanced by the GLUT4 VCP cofactor [START_REF] Fusser | Lysine methylation of the valosin-containing protein (VCP) is dispensable for development and survival of mice[END_REF]. The variant identified here purportedly causes an early truncation in METTL21D, abolishing S-adenosyl-methionine binding sites, and is predicted in silico to be deleterious. The methylation status of VCP however reportedly seems to have no effect on health; VCPKMT knockout mice were shown to be healthy and fertile, exhibiting no pathogenic phenotype [START_REF] Fusser | Lysine methylation of the valosin-containing protein (VCP) is dispensable for development and survival of mice[END_REF]. VCP is an ATPase involved in ubiquitous cellular functions including ubiquitin-dependent protein degradation, transcription factor regulation, cell-cycle regulation and DNA damage response; dominant mutations in VCP have been associated with neurodegenerative disorders including inclusion body myopathy associated with Paget's disease (MIM# 167320), likely caused by dysregulated ATPase activity [START_REF] Halawani | Hereditary inclusion body myopathy-linked p97/VCP mutations in the NH2 domain and the D1 ring modulate p97/VCP ATPase activity and D2 ring conformation[END_REF], as well as Amyotrophic Lateral Sclerosis (MIM# 613954) [START_REF] Johnson | Exome sequencing reveals VCP mutations as a cause of familial ALS[END_REF] and axonal Charcot-Marie-Tooth disease (MIM# 616687) [START_REF] Gonzalez | A novel mutation in VCP causes Charcot-Marie-Tooth Type 2 disease[END_REF]. Although in vitro studies have showed decreased ATPase activity of VCP due to methylation of a Walker B motif of the first ATPase domain by METTL21D, in vivo studies on VCPKMT knockout mice did not show an increase in ATPase activity [START_REF] Cloutier | A newly uncovered group of distantly related lysine methyltransferases preferentially interact with molecular chaperones to regulate their activity[END_REF][START_REF] Fusser | Lysine methylation of the valosin-containing protein (VCP) is dispensable for development and survival of mice[END_REF]. VCPKMT overexpression was studied for an as of yet unclear involvement in cancer metastasis [START_REF] Kernstock | Lysine methylation of VCP by a member of a novel human protein methyltransferase family[END_REF]Thiele et al., 2011). Taking together these studies and the clinical description of our patients, the nonsense VCPKMT mutation is unlikely to contribute to the disease phenotype we describe.

In conclusion, we describe a new autosomal recessive disorder likely associated with a missense mutation in Fibulin-3, predicted to be deleterious in silico, in two Lebanese patients exhibiting a phenotype of connective tissue disease; in addition to the unrelated onset of progressive non syndromic deafness likely caused by a truncating mutation in Myosin IIIA. WES is critical in the reporting of rare disease-causing variants in the clinical scene and is especially important in consanguineous populations such as in Lebanon. Although inbred families are suited for the molecular diagnosis of rare mendelian disorders, limited segregation analysis in small families may fail to pinpoint deleterious variants when more than one potential causal mutation is identified. The identification of bi-allelic EFEMP1 variants in other patients with comparable symptoms would confirm this gene as disease causing.

Addendum

During the review and acceptance of this manuscript, an article was published in the European Journal of Human Genetics entitled "Biallelic variants in EFEMP1 in a man with a pronounced connective tissue phenotype" [START_REF] Driver | Biallelic variants in EFEMP1 in a man with a pronounced connective tissue phenotype[END_REF]. This paper describes truncating EFEMP1 variants in an individual in association with a starkly similar phenotype to the patients reported here. This further confirms the deleterious effect of loss-of-function variants in EFEMP1 and its association with a connective tissue and herniation phenotype. It is noteworthy that the clinical phenotype of this disorder was originally described by [START_REF] Megarbane | Marfanoid habitus, inguinal hernia, advanced bone age, and distinctive facial features: a new collagenopathy?[END_REF].

Funding

No funding was received.

CRediT authorship contribution statement

Declaration of competing interest

Authors declare no competing financial interests in relation to the work described in this paper.

Fig. 1 .

 1 Fig. 1. Pedigree of the studied family. Filled symbols indicate affected individuals for both EFEMP1 and VCPKMT variants. Half-filled symbols indicate healthy carriers for both EFEMP1 and VCPKMT variants. 'U' Symbol indicates untested individuals. (B) Photographs of patient IV.3 showing the marfanoid habitus, pectus excavatum, and distended herniated stomach. (C-D) Chromatograms from the patient (IV.3), one parent (III.1) and a control showing the homozygous mutations detected in the family; (C) c.163T > C (p.Cys55Arg) missense mutation in EFEMP1 (NM_001039348) and (D) c.99T > G (p.Tyr33*) nonsense mutation in the VCPKMT gene (NM_024558).

Fig. 2 .

 2 Fig. 2. Alignment of FBLN3 (NP_001034437) (B) METTL21D (NP_001035752), and (C) MYO3A (NP_059129) amino acid sequences of the protein regions comprising the mutations under investigation between different species. Colored residues are highly conserved across mammals. (D) Simplified schematic representation of Fibulin-3, METTL21D, and Myosin IIIA proteins showing protein domains and position of the variants identified in this study. EGFi: calcium binding epidermal growth factor like domain with large insertion between two Cys-Cys disulphide loops. EGF: calcium binding epidermal growth factor like domain. FBLN: Fibulin-type module. KM: Lysine methyltransferase. MH: Myosin head (motor) domain. IQ: IQ calmodulin-binding motif.

Sami

  Bizzari: Writing -original draft, Writing -review & editing, Formal analysis. Lara El-Bazzal: Writing -review & editing, Formal analysis. Antoine Younan: Writing -original draft, Writing -review & editing, Formal analysis. Stephany El-Hayek: Writing -original draft, Writing -review & editing, Formal analysis. Valerie Delague: Writingoriginal draft, Writing -review & editing, Formal analysis. André Mégarbané: Writing -original draft, Writing -review & editing.

Acknowledgements

We are thankful to the family for their cooperation despite the physical and emotional burden of the disorder they are experiencing.


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  Fig e 1.1: S ce f la ic a e i i Chi a i 2016 (ada ed f B k , Wa g, & Ja beck, 2018)

  ic a e ial , ch a ec clable a e ial . Thi ha c ea ed a e la ic a e ade e (M i a & Ha a hi, 2018). The la ic a e ade ha bee la gel di e ed S hea A ia c ie a ill a ed i Table 1.1. The g ad al i c ea e i la ic i f he c ie ha ig aled a edi ib i f he b de f he gl bal la ic a e ade (Wa g, e al., 2020)

  e f a cia ed eak ig al i a al ed ge he , eak ig al ca be i e e ed a he fi ig he ea l a i g ig f a cha ge e e ha i likel c e. Thi fi i he ce f a ici a i : a ici a i i l e ga he i g a d i e e i g a ce ai a f i f a i , a al i g i gai i igh i f e e e (Nic le c , 2018), he eak ig al i elf i i he e l a ici a i e (Le ca, llec i g a d a al i g eak ig al bec e he i a (Hil e , 2010). Acc di g Le ca a d Le ca (2014), eak ig al i he c e f a ici a i a d a a deci i -l, i ca lead a ici a ed deci i (Le ca & Le ca, 2013).

  i g e h d a d l (Bla c & Le ca, 2003). I hi d , he e ea che ill e he A ici a i F a e k b il b (Le ca, 2011). Ba ed he li e a e e ie , he e ea che ha de el ed a he e ical del a h i Fig e 1.

  Fig re 4.1

Fig

  Fig re 4.4

Fig

  Fig e 4.11-4.13 h he a f he child e life f fa ilie i l ed i he la ic a e ec cli g i d . T ic j k a d a e hei la g d, di ca ded edical eedle a d gl e a e hei . Mea hile, Fig e 4.14 i he e he li e ck li i g i a bad e i e .

Fig

  Fig e 4.17 The i i f de el ed c ie (G 3) ega di g he A ici a i F a e k

  e, Chi a ha ec g i ed a d ack ledged he ig al , he e a a g ea e e e f ge c ake ac i ec if he i a i . Ne e hele , he cha ge a c ide ed la e a d i had i ed he i f ec e a d be i a be e i i ha dle f e ible cha ge . The e ea che c lla ed a e ie f i f a i bef e he Chi e e ba a d f d ha he la ic a e ca ed e i e al ll i , he ig f a e ld a ea . F e a le, he fi he died, a d he i e beca e dd . e le' heal h i he a ea he e la ic a e i ai l ec cled a d ce ed a e e i l affec ed. Thi i a e ide ce f h eak ig al e e l ge eak, a d beca e g a i g ig al . The i a i de e i a ed f he he l cal la i h ed ig f ill e e a d he e e ge c f g ea e heal h i e a d ble . I fac , he e e e al ead ge ig al ha h ed he e i da age i c ed i he a al ec l gical e . Thi beca e a c i i i he ge e le i he a ll ed a ea failed hei h ical e a i a i f he ili a c c i i . Thi i he c e f deali g i h he ble f e a decade a d he l g e i ac e ailed.

  i bef e he i ba ca e i lace. S ch a ge ig al f he Pla ic Chi a d c e a , "J k a d la e : T a el i he ll -d lla a h ade" he bli hed b k. B k a d d c e a ie he e a e he ea l a i g ig al , c ld e i d f hi g f h ee, Chi a ha l aded acce a ce f la ic a e, h ld eek a la ic l i de eak ig al , ig al , a d ea l a i g ig al . The e e i e ga i a i a al e a d a ici a e ible e e a d hei i ac i each g f c ie b ide if i g a d c llec i g he e i i el . S e f he e e he el e a be g a i g ig . F e a le, a la ge be f la ic a e a d la ic a e ce i g la ha e el ca e hei c ie af e Chi a' la ic ba . Thi a a g a i g ig . U de he A ici a i f a e k, he e c ie i G 3 ill a e he i a i ba ed he e de e i i g fac a e li ed a bel : a) he he he e e ha e cc ed b)

  e / la ic -a d-ackagi g/ ackagi g-c e a Acce ed A il 24,2020 AP Ne (J l 25,2018) Sa F a ci c e ba la ic a , c ai e h ://a e .c /2589c9ac201f46bc9f6d255c1a891f6b/Sa -F a ci c -e --ba -la ic-a ,---c ai e Acce ed A il 26ca/ e /ca ada/b i i h-c l bia/ a c e -la ic-a -ba -18,2019) Pa lia e e f i gle-e la ic ba h ://ec.e a.e /e i e /efe/ e /e ea -a lia e -e -i glee-la ic -ba -2019-01-18_e Acce ed : A il 25,2020 E ea Pa lia e (Ma ch 27, 2019) Pa lia e eal ba h ala ia da .c /ca eg / a i /2018/07/24/ e i --i -la ic-a e-f -114-fac ie -e ked/ Acce ed A il 28,2020 Fi a cial Ti e (Oc .14,2018) Thaila d ba f eig la ic a e f 2021 h :// .f .c /c e /06b5a136-ce09-11e8-b276i d ie . a.g .a /chi a -e -lic --a e-a d01-19/ ala ia-e d -backa h-a -i --be-he-bbi h-d -f-he-ld Acce ed A il 20, 2020 Na i al Ge g a hic (N .16.2018) "Chi ai igh .c /ja a e e-e i e -i i -la -b id -f -bi la ic --e lace-e le -ba ed-la ic / Acce ed Ma 05. e e .c /a icle/ -ie a -a e/ ie a --li i -a e-i -a -hi e -b ild-.c /a icle/ -b i ai -b dge -la ic /b i ai -a --ec cled-la ic-ackagi g-idUSKCN1N32FM Acce ed A il 22,2020 Rec cli g da (Ma ch 6, 2019) I dia ba la ic c a i h :// . ec cli g da .c /a icle/i dia-ba -la ic-c a -i / Acce ed A il 28,2020 Re e (Ma 31,2019) Phili i e e d a h back Ca ada af e D e e e cala e h :// . e e .c /a icle/ -hili i e -ca ada-a e/ hili i e -e da h-back--ca ada-af e -d e e-e cala e . c .c / e /chi a/ li ic /a icle/3007280/h -chi a -ba -la ic-a e-i -beca e-ea h ake-h e Acce ed Ma 05,2020 The G a dia (Ma ch 26, 2010) Ph g ge -la icbag-a d-f a -c ai e -ba /a icle_a01d96d6-3ce0-11e8-941b-97ede2ecdcb9. h l Acce ed A il26, 2020 The Ne Y k Ti e (J e 1, 2018) Chile Ba Pla ic Bag a Re ail B i e e h :// . i e .c /2018/06/01/ ld/a e ica /chile-la ic-bag -ba .h l/ Acce ed A il 25, 2020 The Sea le Ti e (J l 1, 2018) Q&A: Sea le la ic a i e .c /Ne / ai a /a chi e /2018/10/02ia/i d e ia-ejec -ic-a alia -la ic-e c cli g-20190701-5235f.h l Acce ed A il 27,2020 V e e (Ma ch 27,2019) Vie a e d la ic c a i f 2025 h ://e. e e . e / e /b i e /ec / ie a --e d-la ic-c a -i -f -2025-3900351.h l Acce ed A il 20,2020

RESEARCH CONTEXT: CHINA S IMPORT OF PLASTIC WASTE

  Me be f he blic la ake )....................................................................................... 34 4.2.2ANTICIPATION OF BAN POLICY ON WASTE PLASTIC IMPORT....................... 46 4.2.3 GROUP 1: CHINA............................................................................................. 46 .................................................................................................................................... 52 CONCLUSION AND RECOMMENDATIONS.............................................................................52

	CHAPTER 1 (Ma he , 2016). 2019). Thi ha bee acc a egie ef a ied b a i c ea e i	lid a e, la ic a e ha
	I eak ig al e ea ch, he e ha i he ef e e l e a bec e a i a c e f lid a e (Ag ad , Se a , Sa Mig el, Ca d i a i g i e
	INTRODUCTION e i g he i e . Weak ig al e ea ch f f la ic a e a e aded e e 4.2.4GROUP 2: OTHER DEVELOPING COUNTRIES IMPORT OF PLASTIC WASTE 48 hich i a i e f & Mad id, 2007). Milli ea a d he i e ed ce l e abili h gh lea i g b c i g i h h ld a d la a i a le i e laci g la ic a a e ial . While ec cli g i he ical cha ge a d b i d ci g 4.2.5GROUP 3: DEVELOPED COUNTRIES THAT EXPORT WASTE PLASTIC............50 Fig e 4.17 The i i f de el ed c ie (G 3) ega di g he A ici a i F a e k51 1.1 INTRODUCTION ga i a i al ac ice ada he e i e e f adicall cha gi g a ell-k l i , l 9% f he ld' la ic a e ha bee ec cled, a d
	T da , i i i c a cha ge . Ma a d c e i e al i ie a d h ea . I d i g , he hel a i a e i a ed i a fa -g die ha e h ha kee e ha half f ha i e ed h d ed f c ie a l ck-i ble b e i g c e i al a i Ja beck & La , 2017; B k , Wa g & Ja beck, 2018). CHAPTER 5.5.1FROM WEAK SIGNS TO STRONG AND DANGEROUS SIGNAL-THE CHINESE i g e i e i h i h he cha gi g ld, c d he ld. (Ge e , e ail a egic ab he e l i f c a i eed be e i a i e. Ma f he e ga i a i ha e he e i e a d gge i g e i f a eg (Me d a, Ca d , & I ece decade , Chi a de a d f la ic ha elled he gl bal ec cli g a e ed EXPERIENCE (GROUP 1) 52 e i a i b i i g a d c e gi g k ledge e ai c Ca a a, 2008). Ba ed he A ici a i F a e k (Le ca, 2011), he ade. Thi i a i ha all ed Chi a achie e a l -c a fac i g e i i e. 5.2ADAPTING TO STRONG SIGNAL -THE DEVELOPING COUNTRIES EXPERIENCES (GROUP 2) 53 O ga i a i ha ca ickl ide if cha ge a d e e eak ig al i hei ga i a i f e ee e ial h ea a d i k i he e a i al e i e , i ill ad a age, hich la a c cial le i aki g Chi a he ld fac (Wa g,
	5.3ADAPTING TO STRONG SIGNAL -THE DEVELOPED COUNTRIES e a d eac he ill gai a deci i e c e i i e edge. The e eak edic he f e i e hile cla if i g f e ac i . Thi he eb c ib e he Zha , Li , Che & S he la d, 2020). Acc di g a i ic e ed b B k ,
	c	EXPERIENCES (GROUP 3) 54 c i f b b i e	del .
	ig al a fi i	d ced b A ff (1975) h clai ed ha f l g-a ge
	la i g I hi	a egic-la i g, a d ca eg i ed i f d , he e ea che e a i ed he ba f i	a i , ga i a i ed la ic a e de Chi a h ld
	e a i e Na i al S	g ig al a d eak ig al . Weak ig al ca bec e clea e d P lic a a ca e d de a e he e g h f i c	igh a i g
	di a ea eak ig al a d he a ici a i e i e. A a deci i -	l, eak ig al a e ed l e he ec ic a d e i e al ca he ble .
	e i H e e , d e a lack f i f e a d ake e	e e i i e e e gi g cha ge . U a i , hi i e ha bee e icall a al ed
	i e e a i , he ig al bec e cha ac e i ed bef e ( a Be ke i g, 2001). A ch, hi e ea ch i i el a d ac ical a eak, b a a ea l a i g
	ig al. Acc di g Hil e (2010), eak ig al ca hel de a d he de el e f he g e e Na i al S	b eak e aili g d P lic f a
	e al he e ical e ec i e. del a d e c age ga i a i	hi k diffe e l . Thi i	, e able
	he & A ff, 2018). Weak ig al a al i , a a ecific f e igh e h d l g , ca be e i a i e ab he f e (A ff, Ki le , Le i , Hel -S e e c ib e he e e ge ce f a lea i g ada i e lic -aki g b h i i a e 1.2 I ece ea , he la ic a d ela ed d c la ig ifica le i i la i g
	ga i a i he gl bal ec	a d blic ad i i a i (B k , Wa g, & Ja beck, 2018). Gl ball , la ic i idel (Me calfe, 2003).
	ed a d i add IMPORT i i ed 4.1CASE STUDY OF CHINA NATIONAL SWORD POLICY TO BAN PLASTIC WASTE he c f a d c e ie ce f de life le (Ala , Billah, & .....32 32 ha i g eak ig al ca i dica e e de el e a h i he De ec i f eak ig al e i e b h e i i i a d e e i e. C ff a (1997) Yajie, 2018; Fa aca, Ma i e -Sa che , & A , 2019).
	4.2IDENTIFICATION AND ANALYSIS THROUGH ANTICIPATION FRAMEOWRK..... 33 e. H e e , i i i a e e be ha a ici a i ha he e ial f I fac , la ic' f abili , fle ibili , d abili (E e, 2017), a d chea e ake
	ca i g f i e f he	e	ible e e a d cha ge . Gi e he i	a ce f cha gi g
	ci c	a ce , he c ce	f eak ig al i ig ifica f he de el	e	f

4.2.1WEAK SIGNAL BEFORE THE BAN POLICY............................................................ 34 a) G e hic Ph g a h OF Beiji g Wa e A ea (Oc be 2008 B Ji lia g Wa g ) (Sig al: 5.4DISCUSSION & RECOMMENDATIONS........................................................................54 APPENDIX.......................................................................................................................................66 APPENDIX 1................................................................................................................................66 idel ed a -ade a e ial , a d i j a fe decade , la ic d c i ha k cke ed (Dilke -H ff a , P a , La c ck, A h h, & La ,

Table 1

 1

	I hi	d , e di i g i h 3 g	f c	ie a de ic ed i Fig e 1.3. The fi
	ca e d , f c e	Chi a Na i al S	d P lic . The e ea ch f c e	he
	eak ig al leadi g	he adical a f	a i a d i le e a i	f he e
	lic . I he d f ca e 1, he e ea che e he A ici a i f a e	k
	de a d h	eak ig al e l ed i	g ig al ha d i e he cha ge. Wi h
	he ba f he i		Chi a, he f c	i hif ed	he ec d ca eg	f
	de el i g C	ie . Pla ic a e i	Thaila d, Vie a , a d Mala ia g e
	ig ifica l . Acc di g	he e	f G ee eace Ea A ia (2019) S hea
	A ia	ggle agai	he la ic a e ade, Mala ia	hl i	f la ic
	a e cke ed f	20,000	e i 2016 110,000	e i 2018. The ec d
	g	f c	ie	died i hi

.1: Pla ic Wa e I T e d i ASEAN c ie (S ce: T ade a h :// . ade a . g/ I da a acce ed 14 J e 2020) e ea ch i cl de I dia, Vie a , Mala ia, Tai a , Thaila d, a d I d e ia. The hi d g c i e de el ed c ie ha a e he aj c ib f la ic a e. The a e he U i ed S a e , Ja a , Ge a , he U i ed Ki gd , E e, Ca ada, A alia, a d Chile. The e e f he e a i a e died de he A ici a i f a e k i hi d . Fig e 1.3: The h ee g f ca e die i he e ea ch blic la ake , S a i ic, a d Refle . The cla ifica i b Le ca (2011) i e e ed Fig e 2.1 a h bel . F hi d , he e ea che ada ed he e cla if ig al a d ig f he a al i i ca e die i Cha e 4.

COMMON CHARACTERISTICS OF WEAK SIGNAL

	b) After the appearance of the weak signals, the ma fade awa or become stronger a i g ca be f d i a b c e da a i e i dica i g ha a e iall ig ifica 2.3 THE ANTICIPATION THEORY
	with time. The weak signals after becoming stronger can be developed into e e ha cc ed he he a i ie h ea (Le ca & Le ca, 2011). S ch
	Whe ide ifica i ca i g warning signals. he li e a e, i i f i e ible Acc di g Ha b ick (1981), Ch (2001), a d Le ca a d Le ca (2011), d ha he a h ' defi i i ble a d he e a ha a	eak ig al
	ha e he f ll i g c e g he eake a ici a i i he	cha ac e i ic f eak ig al : e i e (S i h & D b i , 2010; Hei e & Hil e , 2012). ce ha c i e f he f ll i g a ec :
	A ff (1982) defi ed eak ig al a i e al a d e e al a i g ig al ha
	a) Weak signal is the first sign of emerging phenomena/events (Hiltunen, 2010). a e ei he i c le e a d i fficie acc a el e i a e he babili f I a l , Hil e (2010) highligh ed ha he al e f a eak ig al c ld be bei g e i i e e he e a
	Sa i a a d S i h (2011) a ed ha a eak ig al c ld be a ea l ig f cc e ce a d i i ac f e e e . (Hei e , Ka jalai e , R alai e , & ible de e i ed a he e , l i h he be efi f hi d igh . I he d , eak a i g a d i agi i g a e ie f ible c e e ce a d
	cha ge b S ei lle , 2017). Hil e (2006) ide ified eak ig al a i e e , a d ha he e ea l ig c ld la e bec e i a ig al i he a ac a hi a i g f f e e e a d e d . H e e , a a i g i fl e ce f e l e e ged e e e d igh	e
	i dica ig a he a e a f f ke f ce f de el f i f eak ig al ca be a al ed a d a g ad all eake e , h ea , b i e a d ech l gical i a i ed deal i h ible cha ge i he e e c le el i fl e ci g de el e ajec b aki g ac i ili e a id a i .
	A di a ea i he f ch, he e e e he fi e de i e iga i . S ch i f ig e. Thi i , a lead f a a adig hif a i igh lead he e ible c e e ce	f	e e d, d i e , he de el e	f
	di	i . Acc di g P	a e a a d S k l a (2015), a eak ig al i
	a cia ed i h high ce ai a d i e-lagged. U de he e ci c	a ce ,
	i f	a i i f e i c	le e a d he e i	i dica i	f he fi al c e e ce f
	he a e f i e e . H e e , eak ig al	all	i	f	e cha ge a d i
	ca be aj cha ge i	da '	cie a d ec	. Ba ed	he a e f eak
	ig al , i ca h be he i dica i	f f	e de c i e e e ,	he i e e f
	e de el	e a d	ibili ie . Si ila l , Mag k (2016) al ci ed ha eak
	ig al e e ea l a d ag e ig	f	ible ig ifica e e a d cha ge ha
	affec he ajec	f f	e ce ai e e . Sa i a & S i h (2011) e i ed a
	ell ha eak ig al i a h	he ical cha ge ha c bi e l gic a d i agi a i
	a d i he fi		ig f	ible cha ge.
	Mea hile, i h ld al be eflec he igila a d e	ed ha a i gle eak ig al d e i e e he cha ge i he e i	ide a l e . Ha i g	f
	i f	a i ab ge e e i a ce, he e ga i a i he f e ible cha ge , b he cl e f ele a ill be likel eac a d a id eak ig al ible
	ca c e accide . A he ill be he fi e i f a i ab 2010; Le ca, 2011). I e i d The ea f eak ig al f i , he gai e (Daf & H be , 1986; Ma ch & Ol e , 1976). Beca e hei ea i g a e f e f ice e i a e d ig f h ea e cha ge a d e e gi g e d (Hil e , ha e h ld c llec a a a i , he ibili ha he a be ea l ig i i ead f di a e . A ch, he e d eak ig al a ible ha a e ela ed each he , a he ha a i gle eak ig al, a d a f ce ai e e , a d he effec ha he a ha e a e al ce ai a d a big bec e a ke i ee g ide (Taba abaei, 2011; R bi , 2012).
	a e i a big	i e de c . ei he e lici	e e i lici . I i ece a	gi e he	ea i g

k a d ce ai di ec i f de i ed cha ge . I li e i h hi i , Le ca (2011) gge ed ha eak ig al a d ea l a i g ig c ld be ec , i c le e i f a i ab e iall ig ifica cha ge e e i he f e. A d he e a i g ig al igh a ea e ea l i he ibili ajec . A he a ea eak a he i i ial age, e le e cei e he e ig ha e a ce ai ibili ie i he f e. Wi h he de el e f i e, he a i g ig al bec e clea e a d ge , a d e l i clea e ibili f c i g e e a d cha ge (Le ca, 2011). I he a e ec , R bi (2012) al defi ed eak ig al a e e iall a a big b e a i , a a i g f ible f e cha ge , e e . c) In the earl stages of detecting weak signals, the are often ambiguous, need anal sis to make sense. After interpreting, it can become earl warning signals. b i e e a i (Le ca, 2011). A d he ke fea e i ha eak ig al ha e diffe e i e e a i (K i & Hil e , 2011). B e lai i g he , e ha a he l i g f hei al e. S e eak a i g ig al af e i e e a i a e able fi d ible e d a d a id i k i i e (Le ca & Le ca, 2011). Le ca a d Le ca (2011) elled he idea ha eak ig al a e ef l l deci i aki g. Whe a lied i ac i , eak ig al c ld hel ga i a i a id be i fl e ced b ei e i ie f ig ifica cha ge ha igh cc i he f e. I c a , he ab e ce f eak ig al ca ha e ca a hic effec acc di g R bi (2012). The c f ig i g eak ig al ca al ake e le ha . He ce, he eak ig al a e iced, i i e e el i a a al e a d i e e he c ec l (R bi , 2012). O ga i a i ha al e eak ig al ill ac i el de ec a d a al e he . Thi A i a a ec f a egic a age e i ha a age ha e he abili ie a ici a e e ial i ie a d h ea i he e i e . Thi l i a el de e i e he c e i i e e a d ai able de el e f he ga i a i (X , e al., 2003). The a ici a i ce he ef e elie hea il i e e a i , i i i a d a e i , a age a e e i ed be e i i e eak ig al ha ca gi e a gli e f f e d a a ic cha ge e e , a d be able edic i de ail he e ial i ac , i a ce a d ge c f ible cha ge h gh eak ig al (Bla c & Le ca, 2003). T ed ce ce ai i deci i aki g (Wal e , e al. 2003), adj a egic bjec i e a d a egie (Ch , 2001).

Table 3

 3

	Categor			So rces
	Ne	a e (8 e		S he Me	li Dail
	age cie )			The P a dc ie
					The Ne Y k Ti e
					The Sea le Ti e
					Tai ei Ti e
					Fi a cial Ti e
					The S d e M i g He ald
					L A gele Ti e
	O li e Ne	e	(24	Vi i
	e	eb i e )			Te ce Ne
					S h Chi a M i g P 2
					W ld T ade O ga i a i
					ISRI
					Gl bal Ne
					CBC Ne 2
					FMT Ne
					AP Ne
					Re e 4
					Pla ic I igh
					Na i al Ge g a hic
					Rec cli g da
					Ne E	ea Pa lia e
					V e e
					BRS C e a i
					NBC Ne
					W ld Ec	ic F
					The G a dia
	G e	e P lic		E	ea C	i i 2
	D c e a i	(6		A	alia G e	e 3
	g e	e age cie )		G e	e	f S h A	alia
	D c e a Fil (2 fil )	Beiji g Be ieged b Wa e &
					Pla ic Chi a
	B k (1 b k blica i )		"J k a d la e : T a el i he
					billi -d lla a h ade"
			.1: A li f he	ce f i f	a i

hi d igh he he c e e ce cc ed. The fl cha f he a al i ce i e e ed i Fig e 3.1 a e e ed he f ll i g age.

Table 4

 4

	.1 Ti eli e f Ba / Re ic i	(Ac i	b G e	e )

a ici a e he fac i ha Chi a e i e e i l ll ed a d Chi e e e le' heal h i al affec ed. A d, Chi a, a he ld' la ge i e f la ic a e, i e i e ha bee badl affec ed. Fig e 4.15 The i i f Chi a (G 1) ega di g he A ici a i F a e k Whe he i ' he hee be f ga bage i e a d Beiji g i he Beiji g Be ieged b Wa e d c e a , he e e e ll i i e 'a c A a ed b Ka (2019), he la ic a e c ai i g ll a ha e e hel ed

  Nika de , I. O., & El a a, E.(2001). P jec a age e b ea l a i g . i h, E., & S i h J , J.(2008). U i g ec da da a i ed ca i al a d cial e ea ch. McG a -Hill Ed ca i (UK).Zha g, K., Shi, H., Pe g, J., Wa g, Y., Xi g, X., W , C., & La , P. K. (2018). Mic la ic ll i i Chi a' i la d a e e : A e ie f fi di g , e h d , cha ac e i ic , effec , a d a age e . Science of the total environment, 630, 1641-1653.

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