Uncovering the Causation of Novel Neurological Syndromes - Thesis Proposal Example

?Thesis (outline—headings and subheadings) Uncovering the Causation of Novel Neurological Syndromes I. Introduction Problem ment Research Question Statement of Purpose Aim and Objectives Hypothesis II. Biology of SUCLA2 and LMAN2-L Biological Significance of the SUCLA2 gene Neurological Disorders associated with Defects in SUCLA2 Biological Significance of the LMAN2-L gene Neurological Disorders associated with Defects in LMAN2-L III. Studies on Mutations in the SUCLA2 and LMAN2-L Studies on SUCLA2 Mutations Studies on VIPL Protein IV. Introduction to Next Generation Sequencing and Linkage Studies Next Generation Sequencing for Analysis of mtDNA defects Linkage Studies V. Methodology Justification for Using Linkage Studies Methodology followed for Next Generation Sequencing VI. Observations and Results Observations of Next Generation Sequencing Assembly and Analysis of the Sequence Conclusions from Next Generation Sequencing Observations of Linkage Analysis Conclusions from Linkage Analysis Results, Summary VII. Discussion and Conclusions VIII. Summary and Future Directions Future Directions IX. Works Cited Thesis (Text with outline—headings and subheadings) Uncovering the Causation of Novel Neurological Syndromes I. Introduction SUCLA2 and LMAN2-L are two important genes whose mutations result in several disorders including neurological syndromes. The SUCLA2 gene codes for succinate-CoA ligase (ADP forming) beta subunit. It is also referred to as the renal carcinoma antigen NY-REN-39, SCS-betaA, or Succinyl-CoA synthetase beta-A chain. The SUCLA2 gene codes for a mitochondrial matrix enzyme made of 463 amino acids, which is a part of the succinate/malate CoA ligase beta subunit family. The enzyme is responsible for the catalysis of the reaction between succinate and CoA to form succinyl-CoA in an ATP-dependant ligation mechanism. There are two isoforms of the SUCLA2 protein, which arise from alternative splicing events. The protein forms dimers with the SCS alpha subunit, resulting in the formation of the SCS-A, which is an important constituent of the Krebs cycle (Tricarboxylic acid cycle, TCA). The gene is localized in the mitochondria. Mutations in the SUCLA2 gene result in several autosomal recessive disorders. These disorders are caused because of a decrease in the copy numbers of mitochondrial (mtDNA) in the tissues that are affected. The disorders are thus called Mitochondrial DNA depletion syndromes type 5 (MTDPS5). The disorders resulting from SUCLA2 gene defects are characterized by the infantile onset of neurologic deterioration, deafness, hypotonia, external ophthalmoplegia, methylmalonic aciduria, hyperkinetic-dystonic movement disorder and dysfunction of the renal tubules (genecards.org). The LMAN2-L gene codes for an integral membrane protein – lectin mannose-binding 2-like protein, which is involved in regulating the export of several glycoproteins from the endoplasmic reticulum. The protein may also be involved in the regulation of ERGIC-53. Other names of the protein include VIP36-like protein, LMAN2-like protein, and VIPL. The protein has two isoforms that arise from alternative splicing. It is involved in vesicle-mediated glycoprotein transport. The protein is made of 348 amino acids and is localized in the membrane of the endoplasmic reticulum and partly in the Golgi apparatus. The protein has an important role in the secretory pathway. It is also involved in sorting and transportation of glycoproteins as well as high mannose type glycans. The protein also functions in binding metal ions and sugar ions. The highest expression of the gene LMAN2-L occurs in the kidney and skeletal muscle, while lower levels of expression occur in the spleen, brain, lungs and thymus. Mutation in the LMAN2-L gene results in autosomal dominant neurological disorders. The present paper aims at investigating two novel neurological disorders, one is a disease caused by a novel mutation in SUCLA2 gene and the second disease is caused by a mutation in the LMAN2-L gene mutation. These two are summarized below: Syndrome I: Autosomal recessive non-progressive dystonia-myoclonus with sensorineural hearing loss. Syndrome II: Autosomal dominant remitting childhood epilepsy, tremor, ataxia and anxiety. Neurological disorders are diseases that may target the central and peripheral nervous systems of the human body. The target organs would then be the spinal cord, brain, muscles, nerves both peripheral and cranial, and neuromuscular junctions. There are numerous types of neurological disorders depending on the cause of the disorder. This paper will discuss two types of neurological disorders caused by mutations in the genes SUCLA2 and LMAN2-L. The paper is based on research conducted through both primary and secondary methodologies. Problem Statement Although these disorders are associated with mutations of the two genes, their actual causative defects are unknown. Research Question This thesis thus aims at answering the question as to what causes these two disorders. The genetic basis of these diseases and the acquiring of the genetic disorders thus have to be investigated. Statement of Purpose Exploring a possible solution for the effect of protein mutation in humans is a breakthrough in the field of research. The solutions can only be found if the cause of the defect is understood. This could result in curing the lives of many individuals and can also lead to exploring new possibilities in the field of gene and protein synthesis. Aim and Objectives The aim of this study is to identify the cause of the defects in the two genes – SUCLA2 and LMAN2-L that result in novel neurological disorders. In order to answer the research question, the study is broken down into the following simpler objectives: Analysis of the biology of the proteins and their mutations, and discussion of the effects caused by those mutations Analysis of the causes, symptoms and possible cures of these mutations Assessment and critical analysis of the previous research done on the subject Explanation of the recessive nature of the first disorder and the dominant nature of the second disorder Hypothesis This study is based on the hypothesis that the two disorders are novel inherited Mendelian disorders. The first one is an autosomal recessive disorder caused by a mutation that leads to dysfunction in a mitochondrial protein. And the second one is an autosomal dominant disorder caused by a malfunction of a protein that has a developmental transient expression and has a key role in the process of neuronal pruning. II. Biology of SUCLA2 and LMAN2-L Biological Significance of the SUCLA2 gene The succinyl Co-A synthetase (SCS) protein whose subunit is encoded by SUCLA2 is an enzyme present in the mitochondrial matrix. The enzyme catalyzes the synthesis reaction that is involved in the reversible ligation of succinate and CoA to form succinyl CoA. The reverse of this reaction takes place in the Krebs cycle. The forward of this reaction is involved in synthesis of heme and activation of ketone bodies. Two isoforms of the SCS protein exist. One isoform is GTP-dependant while another is ATP-dependant. The protein has alpha and beta subunits that are responsible for the determination of its nucleotide specificity. The gene is recessive and is present on the chromosome 13q14.2. SUCLA2 is the beta subunit of the enzyme SCS and is involved in the catalysis of the reaction involved in the synthesis of ATP and succinate from ADP and succinyl CoA in Krebs cycle (Goldstein and Reynolds 522). It complexes with nucleoside diphosphate kinase and forms dimmers with SUCLG1. The defects in this gene lead to depletion in mtDNA. It is however not known how defects in the Krebs cycle can result in depletion of the mtDNA. Neurological Disorders associated with Defects in SUCLA2 Defects in the SUCLA2 gene lead to mitochondrial disorders and encephalomyopathy. The defect is common among people of the Faroe Islands, Southern Italy and also in Muslim families. Those with defects in the SUCLA2 gene develop early hypotonia, sensorineural deafness, and progressive dystonia. Morava et al. studied the clinical course of children having a splice site mutation in the SUCLA2 gene (438). The children studied belonged to the Faroe Islands where this defect is widespread. The mutation in these children was homozygous. The children had high 3-hydroxyisovaleric-acid levels in the urine. This is a “novel biochemical feature” of the patients with this gene defect (Morava et al. 438). Other clinical features of the disorder include increase in C4-dicarboxylic carnitine, lactate, and methylmalonic acid, apart from progressive loss in hearing. The patients are usually diagnosed by identifying these characteristic features, although direct sequence analysis of the SUCLA2 gene is often recommended in order to avoid the invasive muscle biopsies required for diagnosing the disorder. Mutations within the people of the Faroe Islands were found to have a founder effect and the patients with disorder due to the gene defect had phenotypic Leigh syndrome (Goldstein and Reynolds 522). The incidence of this syndrome among the Faroe Island population is 1 in 1700. Other mutations of the gene also exist among other ethnic groups. For instance, in some Muslim families, the gene defect comprises of a homozygous mutation by deletion of the 43 base pair and insertion of the 5 base pair (Gly118Arg; 534+1G-A and Arg284Cys; IVS4+1G-A respectively). The onset of the disorders caused by mutations in SUCLA2 is from birth to five months. Clinical symptoms of the effects of the disorder on the central nervous system include severe retardation of psychomotor development, dystonia (which develops later on), and generalized seizures. The defects in motor development are characterized by problems in feeding, severe hypotonia that develops early in life, and atrophy of the muscles. The Mitochondrial DNA Depletion (MTDPS5) syndrome associated with the disorders with this gene defect is also characterized by hearing impairment, frequent infections in the respiratory system, areflexia, contractures, ocular ptosis and strabismus, retardation in growth, and acute infections. The laboratory diagnosis involves observation through MRI showing dilation of the lateral ventricles in the brain. These symptoms are similar to Leigh syndrome. Other characteristics of the clinical diagnosis include microcytic anemia, high C4-dicarboxylic carnitine, high lactate in the cerebrospinal fluid, increased lipids in the type 1 muscle fiber in the muscles, and reduction in the I, III, and IV complexes of the oxidation system. Western blotting reveals reduced SUCLA2. There is a significant depletion of the mtDNA, resulting in a severe reduction in the ratio of mtDNA to nuclear DNA. Biological Significance of the LMAN2-L Gene The LMAN2-L gene codes for the VIP36-like protein, also called VIPL. As already stated, this protein is involved in the transportation of glycoproteins from the endoplasmic reticulum (ER). The transport of proteins from the ER is a “highly regulated and selective process” (Neve et al. 70). Once synthesized, folded and assembled in the ER, proteins are exported from the ER in transport vesicles coated with COPII. It is believed that some glycoprotein subsets require “lectin-like membrane receptors” for their appropriate transportation out of the ER (Neve et al. 70). The lectins have C-terminal trafficking signals that serve as “anterograde/retrograde transport signals for proteins recycling between the ER and the Golgi complex” (Kappeler et al. 31801). These are of the KRFY, KKFF, and KAKFY type. Two lectins, namely – VIP36 and ERGIC-53/p58 had been characterized. VIP36 is a protein that is homologous to the ERGIC-53. This protein was first isolated from vesicles that operated between the plasma membrane and the trans-Golgi network. This protein was believed to have a role in transport of glycolipids as well as glycoproteins. Studies have shown that the protein VIP36 is involved in the Golgi complex as well as the early secretory pathway, recycling between the Golgi and ER (Neve et al. 71). While earlier studies showed that the protein binds with N-acetyl-D-galactosamine, findings reported later on showed that the protein actually binded with high mannose glycans. Neve et al. sought to investigate other proteins related to VIP36 and ERGIC-53/58 through an extensively carried out database search. They identified a gene that was significantly related to VIP36 and hence named it VIPL (for VIP36-Like). They report that this gene has been evolutionarily conserved from zebrafish to man. The mRNA of VIPL was widely expressed in various tissues and localized mainly in the ER and slightly in the Golgi. The terminating sequence of the VIPL protein is KRFY, similar to that of VIP36. This motif is specifically characteristic of the proteins that recycle between ER and Golgi complex. Neve et al. also demonstrated that the VIPL protein functions as an export receptor in the ER. VIPL protein has been shown to have the maximum affinity to three residues, namely – Man-alpha1-2 Man-alpha 1-2 Man of the A branch (Mironov and Pavelka 214). Glucosylation of the protein leads to a significant decrease in its binding. The carbohydrate binding in particular of the protein is dependent on the presence of calcium and is highly efficient at a pH of 7. 5-8, which is the pH in the ER. This explains its localization in the ER. The optimum pH required by this protein along with its specificity to carbohydrates explains that the protein specifically binds to glycoproteins, specifically in the endoplasmic reticulum. It is reported that high-mannose glycoproteins are delivered by VIPL from the CNX/CRT cycle to the ERGIC53 protein for further transportation. The binding of these glycoproteins to VIPL may be required in order to protect the high-mannose glycans on these proteins from trimming down which would otherwise have led to the diversion of these glycoproteins to the Endoplasmic Reticulum Associated Protein Degradation pathway. The distribution of other lectins inside the cell is also controlled by VIPL. For instance, overexpression of this protein helps in the localization of the ERGIC53 to the endoplasmic reticulum. The short half life of the VIPL protein is only 30 minutes because of its function as an intracellular regulator. There are no direct “cargo glycoproteins” for the VIPL protein (Mironov and Pavelka 214). However, studies using siRNA knockdown of the protein have shown that its absence results in a decrease in the secretion of two unknown glycoproteins having 35 and 250 kDa molecular mass. Disorders associated with Defects in LMAN2-L Mutations in the LMAN2-L gene result in autosomal dominant disorders characterized by childhood epilepsy, tremor, ataxia and anxiety. Malfunction of the VIPL protein encoded by LMAN2-L has a developmental transient expression and has a key role in the process of neuronal pruning. There are very few studies reported on the disorders resulting from defective LMAN2-L gene. Several studies have shown the involvement of this gene in various diseases. Examples of such diseases include acne vulgaris (Paris et al. 193), adenocarcinoma (Watanabe et al. 496), arteriosclerosis (Zandberg et al. 96), cholestasis (Carreras et al. 905), encephalomyelitis (Subramanian et al. 1548), etc. inferred through ethinyl estradiol. Other such diseases include fatty liver disease, infertility, panic disorder, thrombosis, insulin resistance, hepatic failure, hyperalgesia, dyslipidemia, uternine neoplasms, obesity, etc. III. Studies on Mutations in the SUCLA2 and LMAN2-L Studies on SUCLA2 Mutations Various studies have investigated mutations in the SUCLA2 and LMAN2-L genes along with the disorders associated with these mutations. Carrozzo et al. investigated mutations in the SUCLA2 gene in 14 patients from eleven families, eight of which were from Faroe Islands, while three were from southern Italy (862). Earlier studies had shown that patients with SUCLA2 mutations develop deafness and Leigh-like encephalomyopathy, along with lactic acidosis. The reaction involved in the SCS enzyme in the Krebs cycle is distally associated with the methylmalonic pathway. Via genetic analysis, Carrozzo et al. found three novel mutations in the SUCLA2 gene. These mutations were p.Arg284Cys, p.Gly118Arg, and c.534 + 1G ?A. these mutations were also associated with mtDNA depletion and changes at the protein level as well as metabolite level. They showed that the clinical symptoms include MRI abnormalities that were Leigh-like and that primarily affected the caudate nuclei and the putamen, encephomyopathy, and early onset of deafness and dystonia. The frequency of the mutation in among the people of the Faroe Island was 2% in a 1:2500 homozygote frequency. This study by Carrozzo et al. lends insights into methylmalonic aciduria disorder resulting from genetic defects. A similar study by Ostergaard et al. investigated the SUCLA2 mutation in twelve patients having high methylmalonic acid levels along with mitochondrial encephalomyopathy that was autosomal recessive (853). All the patients belonged to the Faroe Islands. Symptoms of the disorder were hyperkinesia, retardation of growth, hypotonia, hearing impairment, and severe atrophy of the muscles. Brain imaging indicated both cortical as well as central atrophy along with demyelination. The basal ganglia also displayed atrophy. Some patients were shown to have Leigh-like symptoms along with high levels of methyl malonic acid in the plasma and urine. They identified a novel mutation f the SUCLA2 gene which resulted from a splice site mutation. The mutation was IVS4 + 1G ?A, in which the exon 4 was skipped. The first identification of mutations in the SUCLA2 gene was in two cousins that belonged to a consanguineous Israeli-Muslim family. The two cousins were presumed to have mitochondrial encephalomyopathy that was recessive. Both of them had a defect in three complexes of the respiratory chain because of mtDNA depletion. Using microsatellite markers that were spaced every 10 million bases across the genome, it was shown that there are regions having shared homozygosity with identical allelic pairs spanning a total of 20 megabases. Most of the proteins in the mitochondria are synthesized in the cytoplasm. These proteins have an additional sequence of peptides at the N-(amino)-terminal that helps the proteins in targeting the mitochondria. The number of predicted genes in the homozygous region is 113 of which only three genes code for proteins having a target for mitochondria. SUCLA2 is one of these genes important genes. The homozygous mutations in the SUCLA2 gene of the two cousins investigated by Elpeleg et al. altered the way the exonic and intronic segments of the gene are spliced for making the mature mRNA that codes for the SCS-A subunit (1081). Many more mutations in the SUCLA2 gene have been identified at different splice sites confirming the “role of SUCLA2 as an important human disease gene” (Chinnery 608). As Chinnery states: The SUCLA2 story provides further support for ongoing genetic-mapping studies of rare recessive disorders, encouraging us to think ‘outside the box’ when searching for molecular mechanisms of disease. Moreover, mutations in SUCLA2 provide key evidence that SCS-A is ‘moonlighting’—beyond its conventional role described by Hans Krebs (608). The two studies discussed already, one by Ostergaard et al. and the other by Carrozzo et al. both identified the same homozygous mutation in the SUCLA2 gene although by different approaches. The mutation (IVS4 + 1G ? A, which is the same as c.534 + 1G ? A) alters the exon splicing, resulting in the introduction of a stop codon, causing premature stopping of the mRNA transcription. This shortens the final mRNA transcript. Exon skipping is observed in the cDNA, however, different transcripts by the same mutation were observed by the two different groups (Chinnery 608). The actual molecular mechanism behind this phenomenon is yet to be investigated. Studies on VIPL protein VIPL is a monomer and is glycosylated. This protein is localized to the ER and has high-mannose-type glycans that are sensitive to endoglycosidase-H. A retention motif that is di-arginine-based is present in the cytosolic tail of the protein (Mironov and Pavelka 214). The protein also has an ER export motif, which if mutated, enables the VIPL protein to leave the ER where it is normally localized. Orthologs of the VIPL protein have been identified in several other species apart from man. These species include Xenopus, zebra fish, rat, mouse, cattle, puffer fish and chicken (Neve et al. 78). Not many mutational studies have been reported so far on the LMAN2-L gene and the disorders caused by such mutations. Nufer, Mitrovic and Hauri carried out database scanning of proteins to study novel L-type lectins in animals (15886). This study generated linear sequence motif profiles of the human L-type lectin-like proteins like ERGL, ERGIC53 and VIP36 by aligning the carbohydrate recognition domains of these proteins. Various homo and orthologous of the proteins were observed in yeasts, animals and protozoans. The VIPL was found to be a novel member among these proteins. They carried out sequence analysis of the VIPL protein and found that this protein has ubiquitous expression and appeared even before the VIP36 protein during evolution. The domain organization of VIP36 and VIPL were found to be similar. While the ERGIC53 and the VIP36 proteins are predominant in the post ER membranes and are involved in cycling in the early secretary pathway, VIPL on the other hand is a non-cycling protein of the ER. Mutagenesis experiments carried out in the same study have shown that the retention of VIPL in the ER is through a “RKR-di-arginine signal” (15886). Overexpression of this protein results in the redistribution of the ERGIC53 protein without influencing the KDEL-receptor cycling and early secretory pathway morphology. This study thus demonstrated that VIPL may be an ERGIC53 regulator in the cell. IV. Introduction to Next Generation Sequencing and Linkage Studies Next Generation Sequencing for Analysis of mtDNA defects Schuster describes next generation sequencing as follows: A new generation of non-Sanger-based sequencing technologies has delivered on its promise of sequencing DNA at unprecedented speed, thereby enabling impressive scientific achievements and novel biological applications. However, before stepping into the limelight, next-generation sequencing had to overcome the inertia of a field that relied on Sanger-sequencing for 30 years (16). Next generation sequencing has immensely revolutionized the field of biology. It enables faster and less laborious sequencing of DNA. Large volumes of sequence data can be can be produced very inexpensively using this technology (Metzker 31). While earlier, automated Sanger sequencing was the workhorse of genome sequencing, many improvements have been made in the field to this day. The automated Sanger sequencing is called the “first generation technology” and the newer, more advanced and recently developed methods are termed next generation sequencing (Metzker 31). The biggest advantage of next generation sequencing is that it enables the production of a large volume of data in a inexpensive and economical way, and in some cases, an excess of 1 billion short reads are generated per instrument run (Metzker 31). This technology helps in the determination of the order of bases in a DNA sequence. The technology involves various steps such as preparation of template, sequencing and imaging, and analysis of data. Mutations in genes can be easily identified using this technology. The clinical diagnosis of patients with mitochondrial disorders is complex (GeneDx 1). This is because disorders related to mitochondrial defects are clinically heterogeneous and may occur due to any type of mutation of the mitochondrial or nuclear genes. Dysfunction of the mitochondria may be considered in the case of any “progressive multi-system disorders” (GeneDx 1). However, in certain cases, only one symptom may exist. In people with a recognizable phenotype, the diagnosis is less complex although the clinical abnormalities associated with the mitochondrial syndrome may be complex. Usually, muscle biopsy and studies on respiratory chain complex are performed to investigate evidences of the mitochondrial disorder. Next generation sequencing, as already stated, is a sequencing with a high throughput. This technology is not only highly efficient but also highly sensitive. With the help of this technology, the entire mitochondrial genome from the genomic DNA is sequenced. The sequencing procedure is performed with the help of a “novel solid-state sequencing-by-synthesis process” using which many amplicons can be sequenced parallels (GeneDx 2). The DNA sequences are then compared with other published genome reference sequence of the mitochondria after assembling. With the help of the usual dideoxy sequence analysis or several other such methods, the variants associated with disease can be identified. Reference libraries for mitochondrial DNA may consist of more than six thousand samples belonging to people of various ethnicities and online databases containing the variations of mitochondrial DNA can be used to evaluate and compare the variants whose clinical significance is unknown. Pathogenicity can also be verified with the help of additional tests. Deletion testing can also be performed along with sequence analysis for the identification of mutations in mtDNA in approximately 40% of those tested. Around 10-20% of the patients with a mitochondrial disorder can be identified (GeneDx 1). Mutations in the mtDNA at a rate of as low as 2% heteroplasmy can be identified using next generation sequencing. Large deletions in the genome with a heterplasmy as low as 15% can also be identified by using next generation sequencing. More than 98% of the deletions and mutations in the mtDNA can be identified using this method. Linkage Studies Linkage studies are an important tool in understanding disorders associated with genes. If classical epidemiological studies suggest that genetic factors may be important in disease causation then linkage studies and gene-disease association studies may be used to try and determine the genes or genetic variants that are responsible (phgfoundation.org). Linkage studies are useful while studying disorders occurring in large families wherein the disorder runs through several generations. The aim is to identify genetic markers that are inherited by those with the disease and not by those who lack the disease. Single nucleotide polymorphisms are examples of genetic markers that are first identified for linkage study. Single nucleotide polymorphisms (SNPs) are first detected on a region of the chromosome and then the gene or its variant is narrowed down for identification. Disease genes, especially those genes that are inherited in a Mendelian manner can be studied extensively using linkage studies. These studies are helpful localizing the disease-risk associated regions of the genome. With the help of these studies, multiple genetic markers can be examined simultaneously. Linkage studies however have a few disadvantages. In order to successfully analyze a gene-associated disease risk, a large number of families with the disorder through several generations will have to be identified. Diseases that occur late in life and have a high mortality rate may make it difficult to find families with a large number of affected individuals across generations. Complex traits involving multiple genes are difficult to study through linkage studies. Linkage study is used to “map genetic loci by use of observations of related individuals” (Dawn and Barrett 17). This analysis can be used to study major disorders of the genes through parametric linkage as well as complex disorders through non-parametric linkage. The linkage is based on the odds score logarithm. Dawn and Barrett provided a framework for use in the interpretation of the odds score for linkage analysis (17). V. Methodology In this study, it is hypothesized that the two neurological syndromes associated with SUCLA2 and LMAN2-L gene mutations are Mendelian disorders. Thus, the aim is to find whether the disorders are inherited in a Mendelian manner. For this purpose, linkage analysis and next generation sequencing through whole exome sequencing is performed in order to verify whether the disorders have a Mendelian inheritance. For this research, two families having a novel neurological disorder were followed up. The disorder of the first family was characterized by dystonia with sensorineural hearing impairment. The disorder of the second family was characterized by childhood remitting epilepsy, ataxia and anxiety. After the clinical work up, genetic testing was performed and the novel mutations in the two genes were discovered. The functions of these two genes in the body will be characterized to explain the mechanism by which the mutations cause such a clinical constellation of symptoms. Justification for Using Linkage Studies Methodology followed for Next Generation Sequencing VI. Observations and Results Observations of Next Generation Sequencing Assembly and Analysis of the Sequence Conclusions from Next Generation Sequencing Observations of Linkage Analysis Conclusions from Linkage Analysis Results, Summary VII. Discussion and Conclusions VIII. Summary and Future Directions This study investigated two novel mutations in two genes, namely, SUCLA2 and LMAN2-L. The SUCLA2 gene encodes succinate-CoA ligase (ADP forming) beta subunit while the LMAN2-L gene encodes the VIPL protein. Both of the proteins associated with these genes have important roles in various physiological processes. The enzyme encoded by SUCLA2 is an important mitochondrial enzyme with two isoforms that are formed from two alternate splicing events. This enzyme catalyzes the reaction between succinate and CoA to form succinyl-CoA in an ATP-dependant ligation mechanism. This protein dimerizes with the SCS-alpha subunit. Defects in this protein due to mutations of the SUCLA2 gene result in a reduction in the copy number of mtDNA. The disorders associated with gene are thus called Mitochondrial DNA depletion syndromes type 5 (MTDPS5). The defects are characterized by infantile onset of neurologic deterioration, deafness, hypotonia, external ophthalmoplegia, methylmalonic aciduria, etc. The LMAN2-L gene also produces proteins with two isoforms that result from alternative splicing events. The VIPL protein encoded by this gene is localized in the endoplasmic membrane and is involved in the sorting and well as transportation of the glycoproteins with high mannose type glycans. The mutations in SUCLA2 are autosomal recessive while those of LMAN2-L are autosomal dominant. The neurological syndromes associated with disorders resulting from defects in these genes have complex and multi-organ symptoms. This paper investigates two families with mutations of the SUCLA2 and LMAN2-L genes. The main hypothesis is that the two disorders are Mendelian. The paper discussed the biological significance of the two genes and the neurological and other disorders associated with defects of these two genes. Various mutational studies have investigated these two genes. These studies identified various mutations in the genes and the complex array of symptoms associated with those genes. For the purpose of this study, next generation sequencing as well as linkage studies were performed. Next generation sequencing is highly sensitive and fast, and enables the processing of large amounts of sequence data. The biggest advantage of next generation sequencing is that it enables the production of a large volume of data in a inexpensive and economical way, and in some cases, an excess of 1 billion short reads are generated per instrument run. Linkage studies are helpful in studying disease genes running across families for several generations. Linkage study is useful in mapping genetic markers involved in the disease and study their inheritance patterns. In this study, which hypothesizes that the two neurological syndromes associated with SUCLA2 and LMAN2-L gene mutations are Mendelian disorders, two families with several complex symptoms were identified and the mutations in SUCLA2 and LMAN2-L genes were detected. This was done through whole exome sequencing. Linkage analysis was used to test the hypothesis. Works Cited Carreras F., G. Lehmann, D. Ferri, M. Tioni, G. Calamita, and R. A. Marinelli. “Defective hepatocyte aquaporin-8 expression and reduced canalicular membrane water permeability in estrogen-induced cholestasis.” American Journal of Physiology 292.3:7. Print. 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