Tests on dry specimens prepared at 90% degree of compaction by dry tamping; + Materials tested in deionized water up to 28 days; • Completed; ◦ In progress; N/A: Not available.

#### **4. Experimental Results**

#### *4.1. Compaction Properties of GRMs*

For satisfactory performance of structures made of soil–rubber assortments, it is necessary to properly control the compaction and correctly evaluate the physical properties of compacted materials. For the GRMs investigated in this study, vibratory table tests were found to be ineffective due mainly to the low moduli and energy absorption ability of the rubber aggregates. In contrast, Proctor compaction tests resulted in a suitable testing procedure, even if the moisture content was not a controlling factor. On the other hand, the minimum unit weight was simply attained by cautiously pouring the materials in the compaction mold with nearly zero depositional height. Typical results are reported in Figure 4a in terms of dry unit weight variation with *VRC*.

**Figure 4.** Density characteristics of tested GRMs: (**a**) minimum and maximum dry unit weight; (**b**) specific gravity; and (**c**) idealized illustration of volume of solids-biased density packing (adopted from [16]).

As the rubber particles are much lighter than the gravel ones (Figure 4b), both the minimum and maximum dry unit weights of GRMs decrease almost linearly by increasing *VRC*. It is worth mentioning that, at *VRC* ≤ 40%, the dry unit weight values of G-RS were slightly above the linear trends. This is because at lower *VRC* values, small rubber particles can easily occupy the large voids between gravel grains (Figure 4c), which results in an increase in the density state of the mixtures [39–41]. In contrast, because the size of RL particles is almost similar to that of the gravel, the rubber particles cannot fit in the voids between the gravel grains, even at lower *VRC*, but rather replace the gravel grains in the mixtures. For completeness, a schematic illustration showing idealized packing density arrangements between gravel and RL and RS particles is reported in Figure 4c for *VRC* = 20% and 40%.

#### *4.2. 1-D Compressibility of GRMs*

Material compressibility (or the capability to increase/decrease in volume when subjected to an applied load) is one of the most important factors required in design considerations. For conventional granular soils with rigid particles (i.e., sand and gravel), any change in volume is due to the rotation, movement, and rearrangement of noncompressible grains [41]. The compressibility of GRMs, however, is completely different to that of granular soils, due to significant differences in the elastic modulus of the rigid particles of the host gravel and that of the soft rubber particles. Furthermore, under normal stress conditions, not much volume change is associated with individual particles of rubber (i.e., this is because the Poisson's ratio of pure rubber is ν ≈ 0.5), and distortion is the key phenomenon that takes place in pure rubber specimens.

1-D compression test results with creep loading are reported in Figure 5. In the tests, a 2-h creep (which, under the adopted testing conditions, was sufficient to attain negligible settlement under sustained vertical stress) was applied at different incremental loading stages and the measured vertical stress values were corrected for the loss of transferred load (due mainly to soil-wall friction) that was experimentally determined.

**Figure 5.** Compressibility of GRMs in 1-D compression tests with 2-h creep: (**a**) G-RL and (**b**) G-RS mixtures.

For any given vertical shear stress level, the trends reported in Figure 5 clearly show an increase in 1-D compression with increasing *VRC*. Furthermore, it can be seen that GRMs with *VRC* ≤ 10% performed better than those with *VRC* ≥ 25% (i.e., much smaller vertical strains (εv) developed under the same applied vertical effective stress (σv') level). Additionally, in the range of *VRC* tested, it can be observed that the GRMs made of small rubber particles (*AR* = 0.33) were much more compressible than those consisting of large rubber particles (*AR* = 0.67).

#### *4.3. Shear Strength of GRMs*

Alike conventional soils, shear strength is one of the most important characteristics contributing to the performance for GRMs. The shear strength is the result of friction and interlocking between particles and likely bonding at particle contacts. Due to interlocking, GRMs may contract or expand in volume as they are subject to shear strains.

The stress–strain–volumetric behavior of G-RL and G-RS attained by direct shear tests at 60 kPa normal stress (σn) is compared in Figure 6. It can be seen that, as *VRC* increased, the GRM response progressively changed from dilative with a clear peak shear state (gravel-like behavior) to contractive without a peak shear state (rubber-like behavior). Similar tendencies were observed at σn = 30 and 100 kPa.

**Figure 6.** Direct shear behavior of GRMs at 60 kPa normal stress: (**a**) G-RL; and (**b**) G-RS mixtures.

The summary plot reported in Figure 7a shows the change in maximum shear stress (τmax that was evaluated at peak state for dilative materials or at large deformation for contractive materials) with increasing *VRC* for all mixtures sheared at σn = 30, 60, and 100 kPa. It can be seen that the trends were very similar for both G-RL and G-RS, indicating that the effect of *AR* on the strength of tested GRMs was almost insignificant. Alternatively, for any given σn value, τmax decreased considerably with increasing *VRC*. Moreover, such decay was more significant at higher σn. For example, at σn = 100 kPa, τmax was approximately 137 kPa at *VRC* = 0% (gravel) and 73 kPa at *VRC* = 40%, corresponding to a reduction of 1.9 times. At σn = 30 kPa, τmax is approximately 45 kPa at *VRC* = 0% and 28 kPa at *VRC* = 40%, corresponding to a reduction of 1.6 times.

Figure 7b reports the values of the Mohr–Coulomb effective friction angle (*φ*') for all mixtures. Essentially, *φ*' decreased significantly with increasing *VRC* (and only slightly with *AR*) from about 54◦ (gravel) to 29◦ (graduated rubber). Notably, excluding G-RS with *VRC* > 85% and G-RL with *VRC* > 95%, most of the GRMs had a high strength (i.e., *φ*' > 30◦) regardless of the *VRC* and rubber particle size, making them potentially suitable structural fill materials for many geotechnical applications [17,19].

**Figure 7.** Strength characteristics of GRMs: (**a**) variation of maximum shear stress with normal stress and *VRC*; and (**b**) variation of effective friction angle with *VRC*.

The packing properties of GRMs described earlier (Section 4.1) may provide valuable information on their mechanical behavior (refer to Figures 5–7). Nonetheless, the load-transfer mechanism within the mixtures depends predominantly on the skeleton material [39–41], which is formed when particles of the same material are in contact with each other and are able to transfer loads. The material forming the skeleton becomes the matrix material that governs the general mechanical behavior of a mix. As shown by Figure 8, DEM-based micro-mechanical strong-force network analyses conducted by Chiaro et al. [43] have indicated that two matrix materials can be expected for GRMs: (i) a gravel matrix, leading to a stiffer gravel-like response of GRMs (*VRC* ≤ 30%); and (ii) a rubber matrix, producing a softer rubber-like behavior of GRMs (*VRC* ≥ 60%). In between, an intermediate gravel–rubber behavior will likely take place (30% < *VRC* < 60%). Such distinct behavioral zones provide a framework explaining the gradual change in behavior from stiff gravel-like to soft rubber-like, as observed in the direct shear tests.

**Figure 8.** Behavioral zones for GRMs in direct shear tests evaluated by DEM strong-force network analysis.

#### *4.4. Stiffness, Shear Strain Degradation and Damping Properties*

The design of geotechnical structures made of GRMs subjected to cyclic shear loading conditions (e.g., earthquake or traffic loads) will necessitate the estimation of the dynamic and cyclic behavior of the mixtures including the small-strain shear stiffness (*G*max), shear modulus (*G*) degradation, and damping ratio (*D*).

As shown in Figure 9, the results of bender element tests indicate that *G*max of GRMs decreased with increasing *VRC* and increased with confining pressure. This is essentially consistent with previous studies on sand–rubber mixtures [28]. However, for any given confining pressure value, it has been noticed that the *G*max of GRMs is generally greater than that of sand–rubber mixtures having the same *VRC* [39,41].

**Figure 9.** Variation of small-strain shear modulus (*G*max) of G-RL with: (**a**) *VRC*, and (**b**) confining pressure (adopted from [16]).

On the other hand, the results of small-strain drained cyclic triaxial tests revealed that the addition of rubber aggregates into GRMs delays the *G* degradation on one side and increases *D* on the other (Figure 10). This is an important outcome demonstrating that GRMs will perform better than gravel only under cyclic loading, maintaining their initial stiffness and dynamic properties over a larger range of shear strains. Clearly, this is due to the ability of soft rubber particles to rebound and dissipate energy.

**Figure 10.** Dynamic properties of GRMs evaluated by small-strain drained cyclic triaxial tests at 100 kPa confining pressure: (**a**) shear modulus degradation and (**b**) damping ratio.

*4.5. Leaching Characteristics of GRMs*

Tires are largely composed of carbon black, vulcanized rubber, rubberized fabric containing reinforcing textile cords, antioxidants, silica, pigments, process and extender oils, accelerators, and steel wire [4] and may contain petroleum residues acquired through use [44]. In this study, due to the recycled rubber's proposed use in geotechnical applications, clarification was needed to understand whether and to what extent metals and inorganics could leach into the environment. This is to ensure that there are no negative, long-term impacts to the environment. Factors expected to affect the rate and concentration of tire leachate include the rubber particle size and content, contact time, and the aquatic or soil environments [6].

Within this scope, in this feasibility study, six pilot 28-day batch leaching tests were conducted on GRMs with *VRC* = 40% that were placed in deionized water (additional tests on GRMs with *VRC* = 0, 10, 25% are in progress and the results will be presented elsewhere). Samples were taken on days 0, 1, 2, 4, 7, 10, 14, 17, 21, 25, and 28 to track leaching characteristics, which were determined by ICP-MS analyses. The metals and organics tested for included aluminum (Al), arsenic (As), calcium (Ca), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), manganese (Mn), phosphorus (P), lead (Pb), sodium (Na), nickel (Ni), and zinc (Zn). Note that pH, conductivity, and TOC analyses were also performed and the details are reported in Banasiak et al. [7].

Table 3 summarizes the total average mass of predominant constituents leached for G-RL and G-RS mixtures, while Table 4 provides the maximum concentrations of all the tested elements. It is expected that the Ca, K, and Na contents are attributed to the gravel and the Zn and Mg to the tires. As an example, Zn concentrations measured during the entire 28-day leaching test period are reported in Figure 11.

**Table 3.** Total mass of elements leached at 28 days (average values from 3 tests).

**Table 4.** Maximum concentration of leachate elements obtained by ICP-MS analyses (average values from three tests).

The test results were compared to New Zealand Drinking Water Standards [45] and New Zealand Landfill Waste Acceptance Criteria [46] for class A and B leachability concentration limits. Class A landfills "provide a degree of redundancy for leachate containment" to "reduce the potential for adverse environmental effects" whereas class B landfills do not have such controls. Guideline values for Zn, Mn, Fe, and Al were exceeded. These values mean that there is an increased discoloration, taste, and staining risk.

From a practical viewpoint, these results highlighted that the leachate from smaller tire particles (G-RS) had a higher content of metals, implying that particle size and surface area influence the concentration of elements in GRM leachate. Additionally, the concentration of Zn in G-RS tests exceeded the leachability limit for class B landfills. Hence, G-RS mixtures would need to undergo pre-treatment if used in geotechnical works.

**Figure 11.** Concentration of zinc from 28-day batch leaching tests on GRMs (*VRC* = 40%) placed in deionized water: (**a**) G-RL; and (**b**) G-RS mixtures.

#### **5. Design Considerations**

GRMs are essentially lightweight materials (Figure 4). As a result, one of the benefits of using GRMs would be that the in situ overburden stresses associated with the selfweight of typical geomaterials could be greatly reduced. For instance, the reduction would be 6% for *VRC* = 10%, 13% for *VRC* = 25% and 23% for *VRC* = 40%. This is expected to lead to reduced design performance requirements, in terms of bearing capacity and settlement, for natural soil deposits that underlie a GRM layer installed as part of a residential structure foundation system or a geo-structure. Moreover, it would induce less settlement underneath embankments placed on compressible soil deposits or reduce the earth pressure behind retaining structures [16].

This study has demonstrated that GRMs have an adequate strength (i.e., *φ*' > 30◦) irrespective of the *VRC* and rubber particle size, making them suitable structural fill materials for many geotechnical applications. However, the ultimate adoption of GRMs as structural fills in geotechnical application would also depend on their compressibility under sustained loads (creep). As displayed in Figure 5, it is clear that the higher the vertical effective stress applied on GRMs, the higher the vertical strain developed, and the lower the *VRC* that may be accepted in the mixtures to satisfy compressibility requirements. Moreover, *AR* also plays a key role; in fact, for any combination of *VRC* and vertical effective stress, it can be observed that the mixtures with smaller rubber particles (G-RS) experience greater vertical strain compared to those with large rubber particles (G-RL). Therefore, to have GRMs with reduced compressibility, gravel and rubber particles with similar grain size should be used.

Permeability of GRMs can be related to the porosity of the mixes (which in turns depends on the particle size of rubber and gravel) as well as the vertical stress applied. Experimental evidence from relevant studies reported in the literature have shown that usually the hydraulic conductivity coefficient of soil–rubber mixtures with large rubber particles varies between that of typical gravelly soils at low confining stress levels and that of sandy soil at higher confining stress levels [36]. For mixtures of small rubber particles and *VRC* > 10%, the hydraulic conductivity coefficient was similar to that of sandy soils regardless of the confining stress applied [36]. In the case of foundations, retaining walls, and embankments, this means that GRMs will function as a free-draining fill material ensuring the rapid dissipation of excess pore water pressure. Remarkably, Hazarika et al. [30] reported that the use of GRMs (with *VRC* < 40%) placed on the top of liquefiable sandy soils can considerably reduce the pore water pressure generation in the sandy soil deposit, the reasons being: (1) GRMs themselves are not liquefiable and, therefore, can be conveniently used to replace the top most portion of liquefiable soil

layers, and (2) having an hydraulic conductivity higher than the sand, GRMs provide a preferential pathway for water to easily flow upward and quickly dissipate pore water pressure.

The introduction of recycled ELT-derived materials in geotechnical applications may have benefits in terms of cost reductions and increased performance. However, it is essential to ensure that such innovations do not result in long-term negative impacts on the environment (e.g., through the leaching of toxic chemicals into the surrounding soil environment, groundwater, and surface water). The results of the leaching tests on GRMs have indicated that the leachate from smaller tire particles (RS) had a higher content of metals (e.g., Zn) compared to the large rubber one (RL), implying that particle size and surface area influence the concentration of elements in the GRM leachate. To minimize the leaching of metals, therefore, the use of larger rubber particle size is desirable. Otherwise, smaller rubber particles would need to undergo pre-treatment before use.

#### *5.1. Proposed Acceptance Criteria for GRM Fill Materials (Static Loads)*

In the case of most granular soils, design criteria based on frictional shear strength, bearing capacity, and permeability are used to evaluate their suitability as structural fill materials for geotechnical applications. It is often required that fills should possess a friction angle greater than or equal to 30◦ (and/or a California Bearing Ratio (CBR) > 10%), to guarantee a satisfactory shear resistance and to minimize post-construction settlement [17]. In addition, it is also recommended that fill material should have a permeability coefficient similar to that of sandy fills to ensure rapid dissipation of excess pore water pressure and to minimize internal erosion phenomena.

Because of the critical aspects identified in this study such as long-term creep compressibility and leaching of heavy metals, design criteria simply based on strength and permeability may not be sufficient to fully judge whether or not a GRM meets all the geotechnical requirements for structural fills. To overcome this issue, a modified framework with additional geotechnical design criteria including three levels of acceptance is proposed for GRMs (Figure 12):

In addition to the above proposed geotechnical criteria, GRMs must also meet environmental standards (e.g., New Zealand Drinking Water Standards and New Zealand Landfill Waste Acceptance Criteria). Alternatively, pre-treatment or confinement of leached chemicals may be required.

**Figure 12.** Proposed geo-environmental acceptance criteria for use of GRMs in geotechnical applications.

#### *5.2. Practical Application: Seismic Design of ERGSI Foundation Systems*

"Eco-rubber geotechnical seismic-isolation foundation systems" or in short "ERGSI foundation systems" are integrated systems comprising two key elements (Figure 13):

**Figure 13.** Schematic illustration of the "Eco-rubber geotechnical seismic-isolation" (ERGSI) foundation systems (adopted from [16]).

The geotechnical design of the GRM dissipative layer of a ERSGI foundation system requires considering not only the strength and compressibility criteria shown in Figure 12 (permeability is expected to be fulfilled given the fact that the GRMs are coarse granulated free-draining materials), but also the seismic performance of these synthetic materials.

Typically, in New Zealand, 1- to 2-storey timber-framed residential buildings transfer a vertical effective stress (σv') from the structure to the foundation of 7–10 kPa and 10–15 kPa [47,48], respectively. Considering only G-RL (due to its acceptable leaching characteristics, Figure 11) from Figure 7b, it is evident that mixtures with *VRC* ≤ 95% would have a friction angle much greater than 30◦, implying a good stability under static load conditions (σv' ≤ 10–15 kPa) for both 1-storey and 2-storey buildings.

On the other hand, to guarantee the serviceability during and after the superstructure construction (i.e., to avoid damage to pipelines and other essential services that may be embedded in gravel-rubber foundation layer), and meet the compressibility criteria (εv ≤ 3%), it appears that only the G-RL mixtures with *VRC* ≤ 40–45% would be suitable for 1-storey buildings and those with *VRC* ≤ 35% would be suitable for 2-storey buildings (Figure 14).

**Figure 14.** Selection of suitable GRMs for foundation applications based on maximum allowable vertical strain (εv = 3% contours are derived from the 1-D compression test results shown in Figure 5): (**a**) 1-storey building; and (**b**) 2-storey building.

The seismic performance of ERGSI foundation systems was assessed by small-scale impact load tests conducted on GRMs [49]. In such pilot tests, a foundation prototype was placed on a 60 cm thick layer (made of mixtures with *VRC* = 0, 10, 25, and 40%). As shown in Figure 15, it was found that as more rubber aggregates were added into the mixtures, the natural frequency of the foundation system shifted toward a smaller value (base-isolation effect) while the amplitude of the output peak acceleration decreased (i.e., damping effect).

**Figure 15.** Dynamic properties of G-RL mixtures from impact load tests: (**a**) decay of natural frequency with increasing *VRC*; and (**b**) reduction in output acceleration with increasing *VRC*.

It is also evident that even a small amount of rubber (i.e., *VRC* = 10%) contributes significantly in the dissipation of seismic energy. On the other hand, *VRC* of 25% and 40% seem to provide the same dissipative effects.

Based on the above, it is recommended that the range of suitable GRMs for ERGSI foundation systems should be limited to *VRC* ≤ 40% and *VRC* ≤ 35% for 1-storey and 2-storey buildings, respectively. However, to achieve the best seismic performance in terms of seismic isolation and energy dissipation, it was eventually suggested to use 25% ≤ *VRC* ≤ 35–40%.

#### **6. Conclusions**

In New Zealand, every year, approximately 3.5 million ELTs are legally or illegally disposed of through landfills and stockpiles. However, ELTs cannot be considered as a simple waste to be disposed of. Large-scale sustainable recycling initiatives are crucial to tackle this nationwide problem. A promising solution is to reuse ELTs in the form of recycled granulated rubber as construction materials in civil engineering applications. In this context, this paper has presented: (i) the results of geotechnical and environmental investigations assessing the potential use gravel–rubber mixtures (GRMs) as structural fills for geotechnical applications (including seismic-isolation foundation systems), and (ii) a framework with a set of geo-environmental criteria for the acceptance of GRMs as geo-structural fills.

The following main conclusions can be drawn from this study:

*Future works.* Note that the results reported in this paper are based on the use of uniformly-graded gravel and granulated rubber with maximum particle diameters of approximately 10 mm that could be tested in the laboratory using conventional devices. However, as at the Geotechnical Laboratory of the University of Canterbury, the capability of conducting tests on geomaterials with larger particles has become possible, the authors plan to conduct further geotechnical investigation on GRMs with larger gravel particles (i.e., *D*max = 40 mm) that are commonly used in foundation applications and other geotechnical works in New Zealand. This will make it possible to verify some of the findings reported in this paper, and if required, refine the proposed acceptance criteria for GRMs as geostructural fills.

To complement the batch leaching test results reported in this paper, additional leaching tests considering acid and alkaline solutions are intended. Moreover, a series of column tests will be conducted on 1-D consolidated GRMs with the aim of mimicking as much as possible the groundwater conditions and material properties underneath a foundation or geo-structure, and account for possible groundwater seasonal changes (i.e., repeated cycles of wet and drying) and the effect of 1-D compression on the leaching properties of GRMs.

Specifically for seismic-isolation foundation systems, the authors have planned to carry out detailed field testing on a large-scale prototype structure supported on ERGSI foundations. This is of paramount importance for several reasons: (i) to evaluate the response of the ERGSI foundation with realistic infinite boundary conditions (not feasible in the laboratory tests); (ii) to assess the effects that underlying site geology may have on the ERGSI foundation performance and design; (iii) to monitor and evaluate the longterm settlement and ensure that is within serviceable limits, and (iv) to use background seismicity to further assess the seismic performance of the foundation system.

**Author Contributions:** Conceptualization, G.C., A.P., L.B., and G.G.; Methodology, G.C. and L.B.; Investigation, A.T., G.C., A.M., and L.B.; Data curation, A.T., G.C., L.B., and G.G.; Writing—original draft preparation, A.T. and G.C.; Writing—review and editing, L.B., A.P., and G.G.; Project administration, G.C.; Funding acquisition, G.C., A.P., and L.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Business, Innovation, and Employment (MBIE) of New Zealand, MBIE Smart Ideas Endeavour Research Grant No. 56289.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The laboratory assistance of Sean Rees is greatly appreciated. Figures adopted from [16] were republished with the permission of the New Zealand Geotechnical Society.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

#### **References**


**Meet the editor**

Kenji Ikehara is a professor and director in Nara Study Center of the Open University of Japan. He is also a fellow of International Institute for Advanced Studies (IIAS) of Japan and an Emeritus Professor of Nara Women's University. He graduated from Department of Industrial Chemistry, Faculty of Engineering, Kyoto University in 1968 and received his B. Eng (1968), and

successively M. Eng. (1970) and D. Eng. (1976) degrees from Kyoto University. He engaged in studies on Sporulation Initiation of Bacillus subtilis and on the origins and evolutional processes of microbial genes, the genetic code, and proteins during around 30 years in Nara Women's University. Consequently, he has proposed GC-NSF(a) hypothesis on the origin of Genes, GNC-SNS hypothesis on the genetic code, protein 0th-order structure hypothesis on proteins and [GADV]-protein world hypothesis on the origin of life.

Contents

**Preface IX**

Kenji Ikehara

**Part 1 Background of Genetic Disorder 1**

Chapter 2 **Inbreeding and Genetic Disorder 21**

Kannan Thirumulu Ponnuraj

**Multifactorial Diseases 81**

Chapter 7 **Genomic Study in** β**-Thalassemia 149**

Paulino Goméz-Puertas and Juan Pié

Chapter 8 **HMG–CoA Lyase Deficiency 169**

Chapter 6 **Thalassemia Syndrome 101**  Tangvarasittichai Surapon

**Part 2 Unifactorial or Unigenetic Disorder 99**

Chapter 1 **Origin of the Genetic Code and Genetic Disorder 3**

Gonzalo Alvarez, Celsa Quinteiro and Francisco C. Ceballos

Chapter 3 **Cytogenetic Techniques in Diagnosing Genetic Disorders 45**

Chapter 4 **Functional Interpretation of Omics Data by Profiling Genes and Diseases Using MeSH–Controlled Vocabulary 65**  Takeru Nakazato, Hidemasa Bono and Toshihisa Takagi

Chapter 5 **Targeted Metabolomics for Clinical Biomarker Discovery in**

Saovaros Svasti, Orapan Sripichai, Manit Nuinoon, Pranee Winichagoon and Suthat Fucharoen

Beatriz Puisac, María Arnedo, Mª Concepción Gil-Rodríguez, Esperanza Teresa, Angeles Pié, Gloria Bueno, Feliciano J. Ramos,

Ulrika Lundin, Robert Modre-Osprian and Klaus M. Weinberger

## Contents

#### **Preface XIII**

	- **Part 2 Unifactorial or Unigenetic Disorder 99**

X Contents

Contents VII

Chapter 20 **Prader–Willi Syndrome, from Molecular Testing and Clinical**

Chapter 21 **Turner Syndrome and Sex Chromosomal Mosaicism 431** Eduardo Pásaro Méndez and Rosa Mª Fernández García

Chapter 22 **Microstomia: A Rare but Serious Oral Manifestation of**

**Study to Diagnostic Protocols 409**

Maria Puiu and Natalia Cucu

**Inherited Disorders 449**

Aydin Gulses

Chapter 10 **Alström Syndrome 205**  Cristina Maria Mihai, Jan D. Marshall and Ramona Mihaela Stoicescu

	- **Part 3 Multifactorial or Polygenic Disorder 319**

Chapter 20 **Prader–Willi Syndrome, from Molecular Testing and Clinical Study to Diagnostic Protocols 409**  Maria Puiu and Natalia Cucu

VI Contents

Chapter 9 **Mitochondrial HMG–CoA Synthase Deficiency 189** María Arnedo, Mónica Ramos, Beatriz Puisac, Mª Concepción Gil-Rodríguez, Esperanza Teresa, Ángeles Pié, Gloria Bueno, Feliciano J. Ramos,

Paulino Gómez-Puertas and Juan Pié

Cristina Maria Mihai, Jan D. Marshall and Ramona Mihaela Stoicescu

**A Pulmonary Genetic Disorder 227** Michael Sjoding and D. Kyle Hogarth

Jonay Poveda Nuñez, Alberto Ortiz,

Chapter 14 **Fabry Cardiomyopathy: A Global View 277**

Daniela Quaglino, Federica Boraldi, Giulia Annovi and Ivonne Ronchetti

**Part 3 Multifactorial or Polygenic Disorder 319**

Manuel J. Santos and Alfonso González

**Later After Treatment 333**

Chapter 18 **Genetic Basis of Inherited Bone Marrow Failure Syndromes 357**

Chapter 15 **The Multifaceted Complexity of Genetic Diseases:**

Yoshinari Uehara, Bo Zhang and Keijiro Saku

Chapter 13 **Fabry Disease: A Metabolic Proteinuric Nephropathy 255**

Ana Belen Sanz and Maria Dolores Sanchez Niño

Rocio Toro Cebada, Alipio Magnas and Jose Luis Zamorano

**A Lesson from Pseudoxanthoma Elasticum 289**

**Genetic Disorders Reveal the Mechanisms 321**

Chapter 17 **Repair of Impaired Host Peroxisomal Properties Cropped Up Due to Visceral Leishmaniasis May Lead to Overcome Peroxisome Related Genetic Disorder Which May Develop**

Chapter 19 **Bernard Soulier Syndrome: A Genetic Bleeding Disorder 393**  Basma Hadjkacem, Jalel Gargouri and Ali Gargouri

Salil C. Datta, Shreedhara Gupta and Bikramjit Raychaudhury

Chapter 10 **Alström Syndrome 205**

Chapter 12 **Tangier Disease 239**

Chapter 16 **Peroxisomal Biogenesis:**

Yigal Dror

Chapter 11 **Alpha One Antitrypsin Deficiency:**

Preface

were saved.

years old, respectively.

All life on the Earth, including the human race, originated from one common ancestor (comonote) which appeared on the primitive earth about 3.8~4.0 billion years ago after chemical evolutions from simple inorganic to complex organic compounds. The first life successively evolved from simple to complex organisms, such as prokaryotes, mono-cellular eukaryotes, multi-cellular micro-organisms, plants, animals and human beings. Human beings appeared on this planet between 25 and 7 million years ago and have suffered from many kinds of disease for a long time, many of which might lead to death, such as lethal viruses like smallpox and influenza and infectious bacteria like as cholera and tuberculosis. However, human beings have acquired intelligence so as to understand scientifically many concerns in various kinds of fields, including the medical sciences. Thus, human beings actually acquired the knowledge of viruses and micro-organisms to fight against diseases. Many people have seriously hoped to live as long as possible and even to get eternal life with the acquisition of intelligence. It is well-known in Asian countries that Shi Huángdì (BC259-BC210), who was an emperor

in ancient China, tried to get eternal life and took various kinds of chemicals.

Human beings were protected from infection by viruses - such as the smallpox virus by the intravenous injection of vaccines into their bodies. Owing to the medical technology of vaccines - which were first discovered by Jenner in 1796 - many lives

Furthermore, penicillin - one of the antibiotics - was first discovered by Fleming in 1881. Subsequently, many kinds of antibiotics - such as streptomycin and kanamycin were discovered. Consequently, many people were also released from diseases caused by infectious bacteria and many lives were saved, since many patients were even

In these ways, the development of medical technologies and medicines has protected human beings from many kinds of diseases caused by the infection of viruses and bacteria, resulting in extending the life span of human beings. Currently, many Japanese people can live until between 90 and 100 years old. For example, the average life spans of females and males living in Japan had reached 86.4 and 79.6 years old by 2009, respectively, while the comparative figures in 1950 were about only 62 and 58

cured of infectious diseases which lead to death through taking the antibiotics.

## Preface

All life on the Earth, including the human race, originated from one common ancestor (comonote) which appeared on the primitive earth about 3.8~4.0 billion years ago after chemical evolutions from simple inorganic to complex organic compounds. The first life successively evolved from simple to complex organisms, such as prokaryotes, mono-cellular eukaryotes, multi-cellular micro-organisms, plants, animals and human beings. Human beings appeared on this planet between 25 and 7 million years ago and have suffered from many kinds of disease for a long time, many of which might lead to death, such as lethal viruses like smallpox and influenza and infectious bacteria like as cholera and tuberculosis. However, human beings have acquired intelligence so as to understand scientifically many concerns in various kinds of fields, including the medical sciences. Thus, human beings actually acquired the knowledge of viruses and micro-organisms to fight against diseases. Many people have seriously hoped to live as long as possible and even to get eternal life with the acquisition of intelligence. It is well-known in Asian countries that Shi Huángdì (BC259-BC210), who was an emperor in ancient China, tried to get eternal life and took various kinds of chemicals.

Human beings were protected from infection by viruses - such as the smallpox virus by the intravenous injection of vaccines into their bodies. Owing to the medical technology of vaccines - which were first discovered by Jenner in 1796 - many lives were saved.

Furthermore, penicillin - one of the antibiotics - was first discovered by Fleming in 1881. Subsequently, many kinds of antibiotics - such as streptomycin and kanamycin were discovered. Consequently, many people were also released from diseases caused by infectious bacteria and many lives were saved, since many patients were even cured of infectious diseases which lead to death through taking the antibiotics.

In these ways, the development of medical technologies and medicines has protected human beings from many kinds of diseases caused by the infection of viruses and bacteria, resulting in extending the life span of human beings. Currently, many Japanese people can live until between 90 and 100 years old. For example, the average life spans of females and males living in Japan had reached 86.4 and 79.6 years old by 2009, respectively, while the comparative figures in 1950 were about only 62 and 58 years old, respectively.

#### X Preface

It is reported that the highest cause of Japanese deaths is malignant tumour or cancer. Cancers induced by genetic defects leading to deviation from the normal control of cell division can be regarded as a kind of genetic disorder. The genetic defects may occur in all organs, such as the kidney, the spleen, the stomach, the lung and the intestine etc. In addition, it is quite difficult to cure these cancers by the usual treatments such as administration of medicines (except for removal of malignant tumours by surgical operations) because at the present time it is impossible to site-specifically replace the substituted bases to the original/normal bases. This is the reason why cancers are at the top of the Japanese death causes although human beings are released from many kinds of infectious diseases.

Preface XI

**Kenji Ikehara**

Japan

The Open University of Japan, Nara Study Centre, International Institute for Advanced Studies of Japan

must recognise themselves as being a carrier of a genetic disorder as well as their impending death. However, I believe that it is important for the patient to know whether he or she is a carrier or non-carrier of even a genetic disorder resulting in death in the future, because the patient can do their best against the disease during their remaining life based on the state of knowledge regarding the genetic disorder.

Certainly, it is quite difficult or almost impossible to cure a genetic disorder fundamentally at the present time. However, our knowledge of genetic functions has rapidly accumulated since the double-stranded structure of DNA was discovered by Watson and Crick in 1956. Therefore, nowadays it is possible to understand the reasons why genetic disorders are caused. It is probable that the knowledge of genetic disorders described in this book will lead to the discovery of an epoch of new medical treatment and relieve human beings from the genetic disorders of the future, because human beings had overcome many difficulties already (such as infectious diseases through the discovery of new medical treatment using vaccines for protection against infection form viruses and of special medicines known as antibiotics for curing diseases caused by the infection of micro-organisms). As such, I have a presentiment that a new age is now dawning with respect to the overcoming of genetic disorders. The dawn may set in suddenly upon a big discovery for a new medical treatment which will be achieved by one genius in the future - because such kinds of big discoveries have always been carried out suddenly by geniuses, such as Jenner and Fleming. I hope that the descriptions in this book will contribute to such a discovery,

of a new medical treatment for genetic disorders.

Many genetic disorders are caused by base substitutions on double-stranded DNA, as with cancers. Although the mutated bases must be replaced with the original/normal bases in order to completely cure the disorders, it is quite difficult to achieve this purpose at the present time, again, as with the case of cancers as described above. Thus, genetic disorders remain diseases which are difficult to cure. In addition, mutations causing genetic disorders may occur in any cells carrying genetic elements or DNA and at anytime. Therefore, the organisms living on earth have been exposed to danger-generating base substitutions without exception, and genetic disorders may be induced in any organs because human beings are multi-cellular organisms.

There are two big problems with genetic disorders. One is that it is quite difficult to cure them, as described above. However, in addition to the knowledge about such mechanisms as DNA replication, transcription and the translation of genetic information, human beings have rapidly accumulated knowledge about the base substitutions or mutations occurring on chromosomal DNA which cause various genetic diseases, ever since Watson and Crick discovered the double-stranded structure of DNA in 1956. This knowledge is always significant because it may helpful in devising another medical treatment to cure genetic disorders. Surely, there exist several examples that the knowledge retrieved symptoms or succeeded even to save of patients suffered by genetic disorders. For example, many of the genetic disorders caused by abnormalities of metabolic enzymes could be relieved by going on a diet, which restricts the excess accumulation of the metabolite as a substrate of the enzyme and/or supplies a decreased metabolite as a product of the enzyme. In the case of a genetic disorder causing an excess accumulation of metabolites, it may be also useful to employ the intravenous administration of medicine, which can reduce the formation of toxic metabolites.

 Another one is a problem accompanied by the recent development of genetic analysis for the diagnosis of genetic disorders, because it has made it possible to judge whether a patient is a carrier or non-carrier of an incurable genetic disease, which may lead to death after several years. A patient who has been able to confirm by their diagnosis as a non-carrier of a genetic disorder can live in peace. However, a patient, who has been proven to be a carrier of a genetic disorder must live with continual uneasiness with regard to confronting their coming death during their remaining life, since the patient must recognise themselves as being a carrier of a genetic disorder as well as their impending death. However, I believe that it is important for the patient to know whether he or she is a carrier or non-carrier of even a genetic disorder resulting in death in the future, because the patient can do their best against the disease during their remaining life based on the state of knowledge regarding the genetic disorder.

X Preface

kinds of infectious diseases.

formation of toxic metabolites.

It is reported that the highest cause of Japanese deaths is malignant tumour or cancer. Cancers induced by genetic defects leading to deviation from the normal control of cell division can be regarded as a kind of genetic disorder. The genetic defects may occur in all organs, such as the kidney, the spleen, the stomach, the lung and the intestine etc. In addition, it is quite difficult to cure these cancers by the usual treatments such as administration of medicines (except for removal of malignant tumours by surgical operations) because at the present time it is impossible to site-specifically replace the substituted bases to the original/normal bases. This is the reason why cancers are at the top of the Japanese death causes although human beings are released from many

Many genetic disorders are caused by base substitutions on double-stranded DNA, as with cancers. Although the mutated bases must be replaced with the original/normal bases in order to completely cure the disorders, it is quite difficult to achieve this purpose at the present time, again, as with the case of cancers as described above. Thus, genetic disorders remain diseases which are difficult to cure. In addition, mutations causing genetic disorders may occur in any cells carrying genetic elements or DNA and at anytime. Therefore, the organisms living on earth have been exposed to danger-generating base substitutions without exception, and genetic disorders may

be induced in any organs because human beings are multi-cellular organisms.

There are two big problems with genetic disorders. One is that it is quite difficult to cure them, as described above. However, in addition to the knowledge about such mechanisms as DNA replication, transcription and the translation of genetic information, human beings have rapidly accumulated knowledge about the base substitutions or mutations occurring on chromosomal DNA which cause various genetic diseases, ever since Watson and Crick discovered the double-stranded structure of DNA in 1956. This knowledge is always significant because it may helpful in devising another medical treatment to cure genetic disorders. Surely, there exist several examples that the knowledge retrieved symptoms or succeeded even to save of patients suffered by genetic disorders. For example, many of the genetic disorders caused by abnormalities of metabolic enzymes could be relieved by going on a diet, which restricts the excess accumulation of the metabolite as a substrate of the enzyme and/or supplies a decreased metabolite as a product of the enzyme. In the case of a genetic disorder causing an excess accumulation of metabolites, it may be also useful to employ the intravenous administration of medicine, which can reduce the

 Another one is a problem accompanied by the recent development of genetic analysis for the diagnosis of genetic disorders, because it has made it possible to judge whether a patient is a carrier or non-carrier of an incurable genetic disease, which may lead to death after several years. A patient who has been able to confirm by their diagnosis as a non-carrier of a genetic disorder can live in peace. However, a patient, who has been proven to be a carrier of a genetic disorder must live with continual uneasiness with regard to confronting their coming death during their remaining life, since the patient Certainly, it is quite difficult or almost impossible to cure a genetic disorder fundamentally at the present time. However, our knowledge of genetic functions has rapidly accumulated since the double-stranded structure of DNA was discovered by Watson and Crick in 1956. Therefore, nowadays it is possible to understand the reasons why genetic disorders are caused. It is probable that the knowledge of genetic disorders described in this book will lead to the discovery of an epoch of new medical treatment and relieve human beings from the genetic disorders of the future, because human beings had overcome many difficulties already (such as infectious diseases through the discovery of new medical treatment using vaccines for protection against infection form viruses and of special medicines known as antibiotics for curing diseases caused by the infection of micro-organisms). As such, I have a presentiment that a new age is now dawning with respect to the overcoming of genetic disorders. The dawn may set in suddenly upon a big discovery for a new medical treatment which will be achieved by one genius in the future - because such kinds of big discoveries have always been carried out suddenly by geniuses, such as Jenner and Fleming. I hope that the descriptions in this book will contribute to such a discovery, of a new medical treatment for genetic disorders.

**Kenji Ikehara** The Open University of Japan, Nara Study Centre, International Institute for Advanced Studies of Japan Japan

**Part 1**

**Background of Genetic Disorder**


**6. Conclusions**

It has been shown that dulce de leche is typically produced, marketed, and consumed by small, medium, and large dairy manufacturers in South America. Dulce de leche is made via concentration and heat processing of liquid milk plus sucrose. The color and taste of dulce de leche are primarily determined by the Maillard reaction, which is enhanced by the heating processes used. Dulce de leche may be consumed as a paste or bar. Both may be manufactured on small and large scales, in open-top pans or in evaporators. The DL Technical Regulation classifies dulce de leche according to fat content and additional ingredients. It has been noted that certain production variables can influence dulce de leche's physical-chemical, compositional, and sensorial characteristics. Therefore, understanding the product's chemistry, the technology used to manufacture it is relevant in order to develop more extensive standardization practices of the products available to consumers.