Hereditary folate malabsorption due to a mutation in the external gate of the proton-coupled folate transporter SLC46A1

Hereditary folate malabsorption (HFM) is an autosomal recessive disorder characterized by impaired intestinal folate absorption and impaired folate transport across the choroid plexus due to loss of function of the proton-coupled folate transporter (PCFT-SLC46A1). We report a novel mutation, causing HFM, affecting a residue located in the 11th transmembrane helix within the external gate. The mutant N411K-PCFT was stable, trafficked to the cell membrane, and had sufficient residual activity to characterize the transport defect and the structural requirements at this site for gate function. The influx Vmax of the N411K mutant was markedly decreased, as was the affinity for most, but not all, folate/antifolate substrates. The greatest loss of activity was for 5-methyltetrahydrofolate. Substitutions with positive charged residues resulted in a loss of activity (arginine > lysine > histidine). Function was retained for the negative charged aspartate, but not the larger glutamate substitutions, whereas the bulky hydrophobic (leucine), or polar (glutamine) substitutions, were tolerated. Homology models of PCFT, in the inward and outward open conformations, based upon the mammalian Glut5 fructose transporter structures, localize Asn411 protruding into the aqueous pathway. This is most prominent when the carrier is in the inward open conformation when the external gate is closed. Mutations at this site likely result in highly specific steric and electrostatic interactions between the Asn411-substituted, and other, residues in the gate region that impede carrier function. The substrate specificity of the N411K mutant may be due to alterations of substrate flows through the external gate, downstream allosteric alterations in the folate-binding pocket, or bot

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 β-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

Identification of three novel pathogenic mutations in cystathionine beta-synthase gene of Pakistani intellectually disabled patients

Background: Classical homocystinuria (HCU) is an autosomal recessive inborn error of metabolism, which is caused by the cystathionine-β-synthase (CBS: encoded by CBS) deficiency. Symptoms of untreated classical HCU patients include intellectual disability (ID), ectopia lentis and long limbs, along with elevated plasma methionine, and homocysteine.Methods: A total of 429 ID patients (age range: 1.6-23 years) were sampled from Northern areas of Punjab, Pakistan. Biochemical and genetic analyses were performed to find classical HCU disease in ID patients.Results: Biochemically, nine patients from seven unrelated families were identified with high levels of plasma methionine and homocysteine. Targeted exonic analysis of CBS confirmed seven causative homozygous mutations; of which three were novel missense mutations (c.451G\u3eT; p.Gly151Trp, c.975G\u3eC; p.Lys325Asn and c.1039 + 1G\u3eT splicing), and four were recurrent variants (c.451 + 1G\u3eA; IVS4 + 1 splicing, c.770C\u3eT; p.Thr257Met, c.808_810del GAG; p.Glu270del and c.752T\u3eC; p.Leu251Pro). Treatment of patients was initiated without further delay with pyridoxine, folic acid, cobalamin, and betaine as well as dietary protein restriction. The immediate impact was noticed in behavioral improvement, decreased irritability, improved black hair color, and socialization. Overall, health outcomes in this disorder depend on the age and symptomatology at the time of treatment initiation.Conclusions: With personalized treatment and care, such patients can reach their full potential of living as healthy a life as possible. This screening study is one of the pioneering initiatives in Pakistan which would help to minimize the burden of such treatable inborn errors of metabolism in the intellectually disabled patients

Successful Treatment of Hereditary Folate Malabsorption With Intramuscular Folinic Acid

Background: Hereditary folate malabsorption is a multisystem disease owing to biallelic variants in the gene encoding the proton-coupled folate transporter. Hereditary folate malabsorption is treated with folinic acid, aimed to restore blood and cerebrospinal fluid folate levels. Little is known as to whether oral or intramuscular supplementation of folinic acid is most effective. Methods: Here we describe a one-year-old boy with hereditary folate malabsorption presenting with the typical features including failure to thrive, aphthous stomatitis, macrocytic anemia along with severe developmental impairment and epilepsy, as well as a magnetic resonance imaging of the brain showing bilateral occipital, cortical calcifications characteristic of hereditary folate malabsorption. We compared the effect of treatment with oral folinic acid versus intramuscular folinic acid supplementation by measuring plasma and cerebrospinal fluid folate levels. Results: Compared with oral administration, intramuscular treatment resulted in higher folate levels in blood and, most importantly, normalization of folate levels in cerebrospinal fluid. Clinically, nearly all systemic and neurological symptoms resolved. Conclusion: Normal cerebrospinal fluid folate levels can be achieved in individuals with hereditary folate malabsorption with intramuscular (but not with oral) administration of folinic acid

Identification of three novel pathogenic mutations in cystathionine beta-synthase gene of Pakistani intellectually disabled patients

Classical homocystinuria (HCU) is an autosomal recessive inborn error of metabolism, which is caused by the cystathionine-β-synthase (CBS: encoded by CBS) deficiency. Symptoms of untreated classical HCU patients include intellectual disability (ID), ectopia lentis and long limbs, along with elevated plasma methionine, and homocysteine. A total of 429 ID patients (age range: 1.6-23 years) were sampled from Northern areas of Punjab, Pakistan. Biochemical and genetic analyses were performed to find classical HCU disease in ID patients. Biochemically, nine patients from seven unrelated families were identified with high levels of plasma methionine and homocysteine. Targeted exonic analysis of CBS confirmed seven causative homozygous mutations; of which three were novel missense mutations (c.451G>T; p.Gly151Trp, c.975G>C; p.Lys325Asn and c.1039 + 1G>T splicing), and four were recurrent variants (c.451 + 1G>A; IVS4 + 1 splicing, c.770C>T; p.Thr257Met, c.808_810del GAG; p.Glu270del and c.752T>C; p.Leu251Pro). Treatment of patients was initiated without further delay with pyridoxine, folic acid, cobalamin, and betaine as well as dietary protein restriction. The immediate impact was noticed in behavioral improvement, decreased irritability, improved black hair color, and socialization. Overall, health outcomes in this disorder depend on the age and symptomatology at the time of treatment initiation. With personalized treatment and care, such patients can reach their full potential of living as healthy a life as possible. This screening study is one of the pioneering initiatives in Pakistan which would help to minimize the burden of such treatable inborn errors of metabolism in the intellectually disabled patients