Label free quantitative proteomic profiling of serum samples of intellectually disabled young patients revealed dysregulation of complement coagulation and cholesterol cascade systems.

Intellectual disability is a heterogeneous disorder, diagnosed using intelligence quotient (IQ) score criteria. Currently, no specific clinical test is available to diagnose the disease and its subgroups due to inadequate understanding of the pathophysiology. Therefore, current study was designed to explore the molecular mechanisms involved in disease perturbation, and to identify potential biomarkers for disease diagnosis and prognosis. A total of 250 participants were enrolled in this study, including 200 intellectually disabled (ID) subjects from the subgroups (mild, moderate, and severe) with age and gender matched healthy controls (n = 50). Initially, IQ testing score and biochemical profile of each subject was generated, followed by label-free quantitative proteomics of subgroups of IQ and healthy control group through nano-LC/MS- mass spectrometry. A total of 310 proteins were identified, among them198 proteins were common among all groups. Statistical analysis (ANOVA) of the subgroups of ID showed 142 differentially expressed proteins, in comparison to healthy control group. From these, 120 proteins were found to be common among all subgroups. The remaining 22 proteins were categorized as exclusive proteins found only in disease subgroups. Furthermore, the hierarchical cluster analysis (HCL) of common significant proteins was also performed, followed by PANTHER protein classification and GO functional enrichment analysis. Results provides that the datasets of differentially expressed proteins, belong to the categories of immune / defense proteins, transfer carrier proteins, apolipoproteins, complement proteins, protease inhibitors, hemoglobin proteins etc., they are known to involvein immune system, and complement and coagulation pathway cascade and cholesterol metabolism pathway. Exclusively expressed 22 proteins were found to be disease stage specific and strong PPI network specifically those that have significant role in platelets activation and degranulation, such as Filamin A (FLNA). Furthermore, to validate the mass spectrometric findings, four highly significant proteins (APOA4, SAP, FLNA, and SERPING) were quantified by ELISA in all the study subjects. AUROC analysis showed a significant association of APOA4 (0.830), FLNA (0.958), SAP (0.754) and SERPING (0.600) with the disease. Apolipoprotein A4 (APOA4) has a significant role in cholesterol transport, and in modulation of glucose and lipid metabolism in the CNS. Similarly, FLNA has a crucial role in the nervous system, especially in the functioning of synaptic network. Therefore, both APOA4, and FLNA proteins represent good potential for candidate biomarkers for the diagnosis and prognosis of the intellectual disability. Overall, serum proteome of ID patients provides valuable information of proteins/pathways that are altered during ID progression.

Genetic association of vitamin D receptor gene with female infertility.

BACKGROUND: Infertility is defined as failure to achieve a clinical pregnancy after 12 months of unprotected intercourse. It affects 15% of couples globally and 22% of couples within Pakistan. Female infertility can be caused by numerous genetic or environmental factors including hormone imbalances and exposure to chemicals or radiation. The prevalence of vitamin D deficiency among the adult population was reported to be 14-59% with a higher prevalence in Asian countries. Furthermore, the expression of Vitamin D receptor (VDR) can play a vital role in the reproductive organs of females. Hence, the aim of our present study was to check the association of VDR polymorphisms with infertile females. For this purpose, blood samples were collected for genotyping of four known VDR mutations [FokI (rs2228570), TaqI (rs731236), ApaI (rs7975232), and BsmI (rs1544410)] via PCR-based RFLP assay. RESULTS: Genotyping indicated that FokI, TaqI, and ApaI are associated with infertility (p = 0.004*, p = 0.013*, and p = 0.033*, respectively). However, BsmI did not show any significance. Multinomial regression analysis indicated that FokI heterozygous genotypes increase the risk of infertility by 2.5 times (hetero: OR = 2.5, 95%, p = 0.001*) as compared to wild type. Heterozygous genotypes of TaqI and ApaI were found to play a protective role and reduce the risk of infertility by 58 and 52%, respectively [TaqI: OR = 0.42, 95%, p = 0.004*, ApaI: OR = 0.48, 95%, p = 0.01*, respectively] as compared to wild type. Multinomial logistic regression analysis was also performed for allelic data as well. CONCLUSION: Thus, it could be summarized that among the studied polymorphisms of VDR, FokI SNP greatly increased the risk of infertility, while TaqI and ApaI genotypes protect from infertility. However, BsmI does not influence the risk of infertility in Pakistani females.

Development of low-cost in-house tetra-ARMS-PCR assay for the screening of five CBS mutations found in Pakistani homocystinuria patients.

BACKGROUND: Classical homocystinuria is an inborn amino acid metabolism disorder resulting from mutations in the Cystathionine-ß-Synthase (CBS) gene. These mutations lead to elevated homocysteine and methionine levels and reduced cysteine levels in the blood. Typically, diagnosis occurs after patients display symptoms, and various lab methods confirm it. DNA sequencing is the best option for early detection of genetic variants in asymptomatic suspected individuals. Unfortunately, its high cost can hinder its use, especially in low-income countries like Pakistan. OBJECTIVE: Aim of this study was to devise a robust low-cost diagnostic/screening assay based on Tetra-ARMS-PCR for five prevalent genetic variants found in Pakistani classical homocystinuria patients. MATERIALS AND METHODS: In the current study, T-ARMS-PCR assays were developed for five mutations (c.975G > C, c.770C > T, c.752T > C, c.1039 + 1G > T, c.451 + 1GG > TA), which were characterized previously in classical homocystinuria patients. These low-cost T-ARMS-PCR assays were then used to screen the affected individuals and their family members to identify their genotypes for pathogenic variations in the asymptomatic patients and carriers in their respective families. RESULTS: The outcomes were entirely consistent with those obtained from Sanger DNA sequencing, confirming the sensitivity, specificity, and reliability of the T-ARMS-PCR assay for detecting CBS mutations. CONCLUSION: T-ARMS-PCR has wide applications for low-income countries for the screening and early diagnosis of asymptomatic patients and carriers in the homocystinuria affected families as well as other inherited diseases.

Unraveling the Genetic Associations of DENND1A (rs9696009) and ERBB4 (rs2178575) with Infertile Polycystic Ovary Syndrome Females in Pakistan.

Polycystic Ovary Syndrome (PCOS) is a complex genetic disorder in reproductive-aged women which is associated with comorbidities of reproductive, metabolic, cardiovascular, endocrine, and psychological nature. PCOS is the most common cause of anovulatory infertility. Pathogenesis of PCOS involves strong interaction between environmental and genetic factors. Many Single-Nucleotide Polymorphisms (SNPs) have been associated with PCOS in different populations. Currently, very limited association studies of PCOS and infertility have been done on Pakistani population. The variants DENND1A rs9696009 and ERBB4 rs2178575 are significantly associated with PCOS in Chinese and European populations. These candidate genes regulate the production of androgen hormone, Anti-Mullerian Hormone (AMH), and luteinizing hormone. All these hormones are involved pathogenesis of PCOS and infertility. The aim of the study is to find an association of DENND1A rs9696009 and ERBB4 rs2178575 variants with PCOS in infertile Pakistani females. In this case-control study, 300 infertile females were recruited. The cases (n = 160) were infertile female diagnosed with PCOS (Rotterdam Criteria), and controls (n = 140) were infertile women with no evidence of PCOS. The genomic DNA was isolated, and genotyping was done by PCR-Restriction fragment length polymorphism and further validated by DNA Sanger Sequencing. The Chi-Square analysis showed rs2178575 (ERBB4) was significantly associated with infertility (χ2 = 10.282, p = 0.005852) while rs9696009 (DENND1A) did not show any significant association (χ2 = 3.10, p = 0.212036). Furthermore, multinomial logistic regression analysis was performed and revealed that rs2178575 (ERBB4) heterozygous genotypes (GA) and mutant genotypes (AA) decrease the risk of infertility by 0.541 times (OR = 0.541, 95% CI = 0.314-0.930, p = 0.026) and 0.416 times (OR = 0.416, 95% CI = 0.228-0.757, p = 0.004), respectively, compared to wild-type genotype (GG). The ERBB4 variant is significantly associated with PCOS infertile women and genetically indicated that ERBB4 (rs2178575) decreases the risk of infertility in females having PCOS.

Mass-spectrometric analysis of APOB polymorphism rs1042031 (G/T) and its influence on serum proteome of coronary artery disease patients: genetic-derived proteomics consequences.

Genetic polymorphisms of apolipoprotein B gene (APOB) may result into serum proteomic perturbance in Coronary Artery Disease (CAD). The current case-control cohort of Pakistani subjects was designed to analyze the genetic influence of APOB rs1042031, (G/T) genotype on serum proteome. Subjects were categorized into two groups: CAD patients (n = 480) and healthy individuals (n = 220). For genotyping, tetra ARMS-PCR was carried out and validated through sequencing, whereas LC/MS-based proteomic analysis of serum samples was performed through label-free quantification. In initial step of genotyping, the frequencies of each genotype GG, GT, and TT were 70%, 27%, and 30% in CAD patients, while in control group, the subjects were 52%, 43%, and 5%, respectively, in CAD patients. The genotypic frequencies in patients vs. control groups found significantly different (p = 0.004), and a strong association of dominant alleles GG with the CAD was observed in both dominant (OR: 2.4 (1.71-3.34), p = 0.001) and allelic genetic models (OR: 2.0 (1.45-2.86), p = 0.001). In second step of label-free quantitation, a total of 40 significant proteins were found with altered expression in CAD patients. The enriched Gene Ontology (GO) terms of molecular functions and pathways of these protein showed upregulated pathways as follows: chylomicron remodeling and assembly, complement cascade activation, plasma lipoprotein assembly, apolipoprotein-A receptor binding, and metabolism of fat-soluble vitamins in G allele carrier of rs1042031 (G > T) vs. mutant T-allele carriers. This study provides better understanding of CAD pathobiology by proteogenomics of APOB. It evidences the influence of APOB rs1042031-dominant (GG) genotype with CAD patients.

Genetic Investigation of Consanguineous Pakistani Families Segregating Rare Spinocerebellar Disorders.

Spinocerebellar disorders are a vast group of rare neurogenetic conditions, generally characterized by overlapping clinical symptoms including progressive cerebellar ataxia, spastic paraparesis, cognitive deficiencies, skeletal/muscular and ocular abnormalities. The objective of the present study is to identify the underlying genetic causes of the rare spinocerebellar disorders in the Pakistani population. Herein, nine consanguineous families presenting different spinocerebellar phenotypes have been investigated using whole exome sequencing. Sanger sequencing was performed for segregation analysis in all the available individuals of each family. The molecular analysis of these families identified six novel pathogenic/likely pathogenic variants; ZFYVE26: c.1093del, SACS: c.1201C>T, BICD2: c.2156A>T, ALS2: c.2171-3T>G, ALS2: c.3145T>A, and B4GALNT1: c.334_335dup, and three already reported pathogenic variants; FA2H: c.159_176del, APTX: c.689T>G, and SETX: c.5308_5311del. The clinical features of all patients in each family are concurrent with the already reported cases. Hence, the current study expands the mutation spectrum of rare spinocerebellar disorders and implies the usefulness of next-generation sequencing in combination with clinical investigation for better diagnosis of these overlapping phenotypes.

Need and Challenges in Establishing Newborn Screening Programs for Inherited Metabolic Disorders in Developing Countries.

Even in this post genomic era, no national level newborn screening (NBS) programs for inborn errors of metabolism (IEMs) are yet available in several developing countries including Pakistan. Through NBS, various IEMs can be screened using minute quantities of biofluids. Targeted metabolomics and genomic techniques are the main approaches used for NBS. However, lack of technical expertise and such high-end "omics" based analytical facilities, and meager funding for healthcare in developing countries are the major reasons for unavailability of NBS programs. As it is reflected by only a handful reports about IEMs from Pakistan with population of ≈220 million and consanguinity rate of about 70%, which suggests an unmet need for an NBS program owing to reasonably high prevalence of inherited diseases. Around 200 IEMs are potentially treatable if diagnosed at an earlier stage through biochemical marker and genetic screening, so such patients can get benefit from the NBS program. This overview will help to persuade the stakeholders to setup NBS programs in developing countries including Pakistan, due to multitude of benefits for IEMs; timely diagnosis and early treatment can help the patients to live a nearly healthy life, reduced suffering of the family and minimal burden on society or national healthcare system.

Tetra-ARMS-PCR assay development for genotyping of AGT rs699 T/C polymorphism, its comparison with PCR-RFLP and application in a case-control association study of cardiovascular disease patients.

Genetic variations in the AGT gene play a significant role in controlling the plasma concentration of angiotensinogen (precursor protein of bioactive octapeptide angiotensin II) and the efficacy of antihypertensive drugs. In the current study, Tetra-Amplification Refractory Mutation System-Polymerase Chain Reaction (T-ARMS-PCR) was developed for genotyping of AGT rs699 T/C polymorphism and validated through Sanger DNA sequencing. Its efficiency was also tested using 474 human DNA samples [control, n = 181; cardiovascular disease (CVD) patients, n = 293]. Results showed that T-ARMS-PCR is superior to the commonly used PCR-Restriction Fragment Length Polymorphism (PCR-RFLP). Statistical analysis revealed that the AGT rs699 CC genotype is more prevalent in the CVD patient group (37% vs. 28%) and AGT rs699 C allele and CC genotype increased the risk of CVD by 1.4 and 1.9 fold, respectively. In summary, T-ARMS-PCR is the most suitable approach for quick and efficient genotyping of AGT rs699 T/C polymorphism in a large population in resource-limited countries, Furthermore, AGT rs699 T/C polymorphism is associated with the risk of CVD in the Punjabi Pakistani population.

Association of vitamin D receptor polymorphisms with cardiometabolic conditions in Pakistani population.

Apart from bone related effects, vitamin D has roles in immune modulation, hypertension, diabetes and cardiovascular diseases. Metabolic functions of vitamin D are mediated after binding with vitamin D receptor (VDR). VDR polymorphisms affect its physiological functions. Several VDR single nucleotide polymorphisms (SNPs) are reported previously. However, VDR polymorphisms causing influence on cardiovascular and metabolic disorders have not been investigated in Pakistani population so far. Therefore, present study was conducted to evaluate the role of VDR polymorphisms (rs2228570 and rs7975232) in the pathobiology of cardiometabolic disorders. In all, 400 cardiometabolic patients and 226 healthy control human adults were enrolled from Faisalabad, Pakistan. Biochemical parameters (serum glucose, liver function test, renal function test and lipid profile) were analyzed by standard kit methods. Genetic analysis was done by ARMS-PCR assay. Data was analyzed in SPSS v20. Regression analysis revealed that GG and AG genotypes of rs2228570 A>G polymorphism significantly increased the risk of hypertension in cardiovascular patients by 5.29 and 5.94 times respectively (GG: OR=5.29, 95% CI=1.63-17.2, p=0.005; AG: OR=5.94, 95% CI=1.70-20.7, p=0.005). However, rs7975232 C>A polymorphism was not correlated with cardiometabolic conditions. In conclusion, GG and AG genotypes of VDR SNP rs2228570 significantly contribute for hypertension in cardiovascular disease patients.

High levels of blood glutamic acid and ornithine in children with intellectual disability.

Objectives: Aminoacidopathies are inborn errors of metabolism (IEMs) that cause intellectual disability in children. Luckily, aminoacidopathies are potentially treatable, if diagnosed earlier in life. The focus of this study was the screening of aminoacidopathies in a cohort of patients suspected for IEMs. Methods: Blood samples from healthy (IQ > 90; n = 391) and intellectually disabled (IQ < 70; n = 409) children (suspected for IEMs) were collected from different areas of Northern Punjab, Pakistan. An analytical HPLC assay was used for the screening of plasma amino acids. Results: All the samples (n = 800) were analyzed on HPLC and forty-three out of 409 patient samples showed abnormal amino acid profiles mainly in the levels of glutamic acid, ornithine and methionine. Plasma concentration (Mean ± SD ng/mL) were significantly high in 40 patients for glutamic acid (patients: 165 ± 38 vs. controls: 57 ± 8, p < 0.00001) and ornithine (patients: 3177 ± 937 vs. controls: 1361 ± 91, p < 0.0001). Moreover, 3 patients showed abnormally high (53.3 ± 8.6 ng/mL) plasma levels of methionine. Conclusion: In conclusion, biochemical analysis of samples from such patients at the metabolites level could reveal the underlying diseases which could be confirmed through advanced biochemical and genetic analyses. Thus, treatment to some of such patients could be offered. Thus burden of intellectual disability caused by such rare metabolic diseases could be reduced from the target populations.

Genetic Association of Beta-Myosin Heavy-Chain Gene (MYH7) with Cardiac Dysfunction.

Cardiac dysfunction accelerates the risk of heart failure, and its pathogenesis involves a complex interaction between genetic and environmental factors. Variations in myosin affect contractile abilities of cardiomyocytes and cause structural and functional abnormalities in myocardium. The study aims to find the association of MYH7 rs121913642 (c.1594 T>C) and rs121913645 (c.667G>A) variants with cardiac dysfunction in the Punjabi Pakistani population. Patients with heart failure (n = 232) and healthy controls (n = 205) were enrolled in this study. MYH7 variant genotyping was performed using tetra ARMS-PCR. MYH7 rs121913642 TC genotype was significantly more prevalent in the patient group (p < 0.001). However, MYH7 rs121913645 genotype frequencies were not significantly different between the patient and control groups (p < 0.666). Regression analysis also revealed that the rs121913642 C allele increases the risk of cardiac failure by ~2 [OR:1.98, CI: 1.31−2.98, p < 0.001] in comparison to the T allele. High levels of the cardiac enzymes cardiac troponin I (cTnI) and CK-MB were observed in patients. There was also an increase in total cholesterol, LDL cholesterol, and uric acid in patients compared to the healthy control group (p < 0.001). In conclusion, the MYH7 gene variant rs121913642 is genetically associated with cardiac dysfunction and involved in the pathogenesis of HF.

Adult GAMT deficiency: A literature review and report of two siblings.

Guanidinoacetate methyltransferase (GAMT) deficiency is a creatine deficiency disorder and an inborn error of metabolism presenting with progressive intellectual and neurological deterioration. As most cases are identified and treated in early childhood, adult phenotypes that can help in understanding the natural history of the disorder are rare. We describe two adult cases of GAMT deficiency from a consanguineous family in Pakistan that presented with a history of global developmental delay, cognitive impairments, excessive drooling, behavioral abnormalities, contractures and apparent bone deformities initially presumed to be the reason for abnormal gait. Exome sequencing identified a homozygous nonsense variant in GAMT: NM_000156.5:c.134G>A (p.Trp45*). We also performed a literature review and compiled the genetic and clinical characteristics of all adult cases of GAMT deficiency reported to date. When compared to the adult cases previously reported, the musculoskeletal phenotype and the rapidly progressive nature of neurological and motor decline seen in our patients is striking. This study presents an opportunity to gain insights into the adult presentation of GAMT deficiency and highlights the need for in-depth evaluation and reporting of clinical features to expand our understanding of the phenotypic spectrum.

Biochemical Screening of Intellectually Disabled Patients: A Stepping Stone to Initiate a Newborn Screening Program in Pakistan.

Inborn errors of metabolism (IEMs) are rare group of genetic disorders comprising of more than 1,000 different types. Around 200 of IEMs are potentially treatable through diet, pharmacological and other therapies, if diagnosed earlier in life. IEMs can be diagnosed early through newborn screening (NBS) programs, which are in place in most of the developed countries. However, establishing a NBS in a developing country is a challenging task due to scarcity of disease related data, large population size, poor economy, and burden of other common disorders. Since, not enough data is available for the prevalence of IEMs in Pakistan; therefore, in this study, we set out to find the prevalence of various treatable IEMs in a cohort of intellectually disabled patients suspected for IEMs, which will help us to initiate a NBS program for the most frequent IEMs in Pakistan. Therefore, a total of 429 intellectually disabled (IQ <70) patient samples were collected from Pakistan. A subset of 113 patient samples was selected based on the clinical information for the detailed biochemical screening. Advance analytical techniques like, Amino Acid Analyzer, GC-MS, UHPLC-MS, and MS/MS were used to screen for different treatable IEMs like aminoacidopathies, fatty acid ß-oxidation disorders and mucopolysaccharidoses (MPS) etc. A total of 14 patients were diagnosed with an IEM i.e., 9 with homocystinuria, 2 with MPS, 2 with Guanidinoacetate methyltransferase (GAMT) deficiency and 1 with sitosterolemia. These IEMs are found frequent in the collected patient samples from Pakistan. Thus, present study can help to take an initiative step to start a NBS program in Pakistan, especially for the homocystinuria having highest incidence among aminoacidopathies in the studied patients, and which is amenable to treatment. This endeavor will pave the way for a healthier life of affected patients and will lessen the burden on their families and society.

Development and application of low-cost T-ARMS-PCR assay for AGT and CYP11B1 gene polymorphisms.

Angiotensin II (Ang II: a truncated octapeptide of angiotensinogen, AGT) and 11-ß-hydroxylase influence regulation of blood pressure. Dysregulation of Ang II and 11-ß-hydroxylase can lead to hypertension and elevate aldosterone levels. Polymorphisms in AGT (encodes AGT) and CYP11B1 (encodes 11-ß-hydroxylase) shift the paradigm from physiological to pathological. Currently, various high-throughput techniques are used to genotype these polymorphisms. These techniques require expensive infrastructure and reagents. However, in developing countries, where cost is the main limiting factor, it is not feasible to use expensive techniques. So, the aim of current study was to develop efficient low-cost method for genotyping of cardiovascular disease and hypertension associated polymorphisms of AGT (rs4762, rs5051) and CYP11B1 (rs6410). For this, tetra amplification-refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method was developed and optimized for aforementioned AGT and CYP11B1 gene polymorphisms. Efficiency of T-ARMS-PCR was tested by genotyping 776 human samples. These T-ARMS-PCR assays were also validated by Sanger DNA sequencing, where 100% concordance was found, allowing the efficient use of these T-ARMS-PCR assays for polymorphism genotyping in AGT and CYP11B1 in resource limited settings. T-ARMS-PCR is low-cost, efficient and reliable assay for genotyping of AGT and CYP11B1 gene polymorphisms.

Biochemical screening of intellectually disabled and healthy children in Punjab, Pakistan: differences in liver function test and lipid profiles.

Objectives: Inborn errors of metabolism (IEMs) are rare genetic disorders. Generally, IEMs are untreatable; however, some IEMs causing intellectual disability are potentially treatable if diagnosed earlier. In this study, levels of some clinically important biochemical parameters in intellectually disabled children suspected for IEMs were tested to see their association with intellectual disability, which could be helpful in preliminary screening. Methods: This comparative cross-sectional observational study was carried out from 2014 to 2017. Blood samples from 800 boys and girls (aged 4-24 years) were collected, of which 391 were healthy (IQ >90) and 409 were intellectually disabled (IQ <70) children with unknown cause. Clinically important (Liver and kidney enzymes etc.) biochemical parameters were analyzed in sera samples using commercial kits on semi-automated clinical chemistry analyzer. Results: Serum analysis showed the levels of ALP (p < 0.00001), ASAT (p = 0.001), ALAT (p = 0.016), albumin (p < 0.001), uric acid (p < 0.001), cholesterol (p < 0.001), triglycerides (p < 0.001), and hemoglobin (p = 0.005) were significantly different between healthy and intellectually disabled children. Conclusion: Changes in the liver function test and lipid profile parameters were significantly different in children with intellectual disability; however, it requires further detailed analysis for complete characterization of these diseases.

Molecular genetic diagnosis of Wilson disease by ARMS-PCR in a Pakistani family.

Wilson disease is a rare autosomal recessive disorder caused by mutations in the ATP7B gene causing hepatic and neurological damage due to copper accumulation. Early diagnosis and treatment could lead to improved survival of patients. Patients are best treated at pre-symptomatic stages but early diagnosis of Wilson disease is challenging owing to complex diagnosis. Evidence based genetic counseling requires characterization of underlying mutations in Wilson disease families. The aim was to characterize the causative mutation(s) in a Pakistani Wilson disease family by custom developed ARMS-PCR assay. A proband (19 years old boy) having Wilson disease with evidence of K-F ring, severe neurological and psychiatric manifestations and clinical findings supported by biochemical abnormalities was followed. Following screening for 12 putative mutations in ATP7B, we identified a homozygous mutation (p.Cys271*, c.813C > A) in proband by T-ARMS-PCR assay and validated by Sanger DNA sequencing. Furthermore, on screening of his family members, a younger sister (aged 9 years) was found to have the same homozygous mutation even though she was clinically asymptomatic except for a light K-F ring. Parents were heterozygous for this mutation and an elder brother was homozygous normal. Molecular diagnosis by PCR based assays (M-ARMS-PCR and T-ARMS-PCR) is cost effective, reliable, and efficient for preliminary screening of mutations in the ATP7B gene in developing countries like Pakistan, which can be successfully applied to Wilson disease families for genetic testing and follow-up evidence based genetic counseling.

An Overview of Traditional and Novel Therapeutic Options for the Management of Phenylketonuria.

Phenylketonuria (PKU) is an autosomal recessive disorder caused by the deficiency of phenylalanine hydroxylase enzyme that catalyzes the conversion of L-phenylalanine to L-tyrosine using tetrahydrobiopterin (BH4) as a cofactor. Among aminoacidopathies, PKU is one of the most prevalent disorders in different populations. It may be caused by deficiency of BH4 or mutations in PAH. About 98% of PKU patients have mutations in the PAH, while the remaining have BH4 deficiency. If PKU is diagnosed earlier in life using advance analytical techniques (e.g., high performance liquid chromatography, mass spectrometry, and polymerase chain reaction), then it is potentially treatable by special diets (L-phenylalanine-free medical formula). However, some complications such as vitamin B12 deficiency, cardiovascular problems, and neurodevelopmental problems have been reported in PKU patients when they ate special diets for a long period. Hence, special diet alone is not a good option for proper treatment. Next generation therapies require structure-function based development. For therapies which target PAH gene (e.g., gene therapy, RNAi, gene editing), a lot of research has yet to be done. Treatment with BH4 therapy is safe and effective but only in BH4-responsive PKU patients. Therefore, research efforts should be focused on the development of more targeted pharmacological and genetic therapies especially PAH gene therapy, which can reduce the burden or deleterious effects of this disease in affected patients.

Aminoacidopathies: Prevalence, Etiology, Screening, and Treatment Options.

Inborn errors of metabolism (IEMs) are a group of inherited metabolic disorders which are caused by mutations in the specific genes that lead to impaired proteins or enzymes production. Different metabolic pathways are perturbed due to the deficiency or lack of enzymes. To date, more than 500 IEMs have been reported with most of them being untreatable. However, fortunately 91 such disorders are potentially treatable, if diagnosed at an earlier stage of life. IEMs have been classified into different categories and one class of IEMs, characterized by the physiological disturbances of amino acids is called as aminoacidopathies. Out of 91 treatable IEM, thirteen disorders are amino acid related. Aminoacidopathies can be detected by chromatography and mass spectrometry based analytical techniques (e.g., HPLC, GC-MS, LC-MS/MS) for amino acid level changes, and through genetic assays (e.g., PCR, TaqMan Genotyping, DNA sequencing) at the mutation level in the corresponding genes. Hence, this review is focused to describe thirteen common aminoacidopathies namely: Phenylketonuria (PKU), Maple Syrup Urine Disease (MSUD), Homocystinuria/Methylene Tetrahydrofolate Reductase (MTHFR) deficiency, Tyrosinemia type II, Citrullinemia type I and type II, Argininosuccinic aciduria, Carbamoyl Phosphate Synthetase I (CPS) deficiency, Argininemia (arginase deficiency), Hyperornithinemia-Hyperammonemia-Homocitrullinuria (HHH) syndrome, N-Acetylglutamate Synthase (NAGS) deficiency, Ornithine Transcarbamylase (OTC) deficiency, and Pyruvate Dehydrogenase (PDH) complex deficiency. Furthermore, the etiology, prevalence and commonly used analytical techniques for screening of aminoacidopathies are briefly described. This information would be helpful to researchers and clinicians especially from developing countries to initiate newborn screening programs for aminoacidopathies.

Role of Leptin Deficiency, Inefficiency, and Leptin Receptors in Obesity.

Leptin protein consists of 167 amino acids, which is mainly secreted from the white adipose tissue. This protein acts on the hypothalamic regions of the brain which control eating behavior, thus playing a significant role in maintaining body's metabolism. Leptin receptors belong to glycoprotein 130 (gp130) family of cytokine receptors and exist in six isoforms (LEPR a-f), and all the isoforms are encoded by LEPR gene; out of these isoforms, the LEPR-b receptor is the 'longest form,' and in most of the cases, mutations in this isoform cause severe obesity. Also, mutations in the leptin gene (LEP) or its receptors gene can lead to obesity. Some biochemical pathways affect the bioactivity of leptin and/or its receptors. To date, eleven pathogenic mutations have been reported in the LEP which are p.L72S, p.N103K, p.R105W, p.H118L, p.S141C, p.W121X c.104_106delTCA, c.135del3bp, c.398delG, c.481_482delCT, and c.163C>T. Different mutations in the LEPR have also been reported as c.2396-1 G>T, c.1675 G>A, p.P316T, etc. In some studies, where leptin was deficient, leptin replacement therapy has shown positive impact by preventing weight gain and obesity.