Mario Ćuk

S-adenosylhomocysteine hydrolase deficiency in a human: A genetic disorder of methionine metabolism

We report studies of a Croatian boy, a proven case of human S-adenosylhomocysteine (AdoHcy) hydrolase deficiency. Psychomotor development was slow until his fifth month; thereafter, virtually absent until treatment was started. He had marked hypotonia with elevated serum creatine kinase and transaminases, prolonged prothrombin time and low albumin. Electron microscopy of muscle showed numerous abnormal myelin figures; liver biopsy showed mild hepatitis with sparse rough endoplasmic reticulum. Brain MRI at 12.7 months revealed white matter atrophy and abnormally slow myelination. Hypermethioninemia was present in the initial metabolic study at age 8 months, and persisted (up to 784 μM) without tyrosine elevation. Plasma total homocysteine was very slightly elevated for an infant to 14.5–15.9 μM. In plasma, S-adenosylmethionine was 30-fold and AdoHcy 150-fold elevated. Activity of AdoHcy hydrolase was ≈3% of control in liver and was 5–10% of the control values in red blood cells and cultured fibroblasts. We found no evidence of a soluble inhibitor of the enzyme in extracts of the patients cultured fibroblasts. Additional pretreatment abnormalities in plasma included low concentrations of phosphatidylcholine and choline, with elevations of guanidinoacetate, betaine, dimethylglycine, and cystathionine. Leukocyte DNA was hypermethylated. Gene analysis revealed two mutations in exon 4: a maternally derived stop codon, and a paternally derived missense mutation. We discuss reasons for biochemical abnormalities and pathophysiological aspects of AdoHcy hydrolase deficiency.

S-Adenosylhomocysteine hydrolase deficiency: a second patient, the younger brother of the index patient, and outcomes during therapy

SummaryS-Adenosylhomocysteine (AdoHcy) hydrolase deficiency has been proven in a human only once, in a recently described Croatian boy. Here we report the clinical course and biochemical abnormalities of the younger brother of this proband. This younger brother has the same two mutations in the gene encoding AdoHcy hydrolase, and has been monitored since birth. We report, as well, outcomes during therapy for both patients. The information obtained suggests that the disease starts in utero and is characterized primarily by neuromuscular symptomatology (hypotonia, sluggishness, psychomotor delay, absent tendon reflexes, delayed myelination). The laboratory abnormalities are markedly increased creatine kinase and elevated aminotransferases, as well as specific amino acid aberrations that pinpoint the aetiology. The latter include, most importantly, markedly elevated plasma AdoHcy. Plasma S-adenosylmethionine (AdoMet) is also elevated, as is methionine (although the hypermethioninaemia may be absent or nonsignificant in the first weeks of life). The disease seems to be at least to some extent treatable, as shown by improved myelination and psychomotor development during dietary methionine restriction and supplementation with creatine and phosphatidylcholine.

Store-Operated Ca 2+ Entry Controls Induction of Lipolysis and the Transcriptional Reprogramming to Lipid Metabolism

Ca2+ signals were reported to control lipid homeostasis, but the Ca2+ channels and pathways involved are largely unknown. Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ influx pathway regulated by stromal interaction molecule 1 (STIM1), STIM2, and the Ca2+ channel ORAI1. We show that SOCE-deficient mice accumulate pathological amounts of lipid droplets in the liver, heart, and skeletal muscle. Cells from patients with loss-of-function mutations in STIM1 or ORAI1 show a similar phenotype, suggesting a cell-intrinsic role for SOCE in the regulation of lipid metabolism. SOCE is crucial to induce mobilization of fatty acids from lipid droplets, lipolysis, and mitochondrial fatty acid oxidation. SOCE regulates cyclic AMP production and the expression of neutral lipases as well as the transcriptional regulators of lipid metabolism, peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), and peroxisome proliferator-activated receptor α (PPARα). SOCE-deficient cells upregulate lipophagy, which protects them from lipotoxicity. Our data provide evidence for an important role of SOCE in lipid metabolism.

Delineation of PIGV mutation spectrum and associated phenotypes in hyperphosphatasia with mental retardation syndrome

Three different genes of the glycosylphosphatidylinositol anchor synthesis pathway, PIGV, PIGO, and PGAP2, have recently been implicated in hyperphosphatasia-mental retardation syndrome (HPMRS), also known as Mabry syndrome, a rare autosomal recessive form of intellectual disability. The aim of this study was to delineate the PIGV mutation spectrum as well as the associated phenotypic spectrum in a cohort of 16 individuals diagnosed with HPMRS on the basis of intellectual disability and elevated serum alkaline phosphate as minimal diagnostic criteria. All PIGV exons and intronic boundaries were sequenced in 16 individuals. Biallelic PIGV mutations were identified in 8 of 16 unrelated families with HPMRS. The most frequent mutation detected in about 80% of affected families including the cases reported here is the c.1022C>A PIGV mutation, which was found in both the homozygous as well as the heterozygous state. Four further mutations found in this study (c. 176T>G, c.53G>A, c.905T>C, and c.1405C>T) are novel. Our findings in the largest reported cohort to date significantly extend the range of reported manifestations associated with PIGV mutations and demonstrate that the severe end of the clinical spectrum presents as a multiple congenital malformation syndrome with a high frequency of Hirschsprung disease, vesicoureteral, and renal anomalies as well as anorectal malformations. PIGV mutations are the major cause of HPMRS, which displays a broad clinical variability regarding associated malformations and growth patterns. Severe developmental delays, particular facial anomalies, brachytelephalangy, and hyperphosphatasia are consistently found in PIGV-positive individuals.

Store-operated Ca2+ entry regulates Ca2+-activated chloride channels and eccrine sweat gland function

Eccrine sweat glands are essential for sweating and thermoregulation in humans. Loss-of-function mutations in the Ca2+ release-activated Ca2+ (CRAC) channel genes ORAI1 and STIM1 abolish store-operated Ca2+ entry (SOCE), and patients with these CRAC channel mutations suffer from anhidrosis and hyperthermia at high ambient temperatures. Here we have shown that CRAC channel-deficient patients and mice with ectodermal tissue-specific deletion of Orai1 (Orai1K14Cre) or Stim1 and Stim2 (Stim1/2K14Cre) failed to sweat despite normal sweat gland development. SOCE was absent in agonist-stimulated sweat glands from Orai1K14Cre and Stim1/2K14Cre mice and human sweat gland cells lacking ORAI1 or STIM1 expression. In Orai1K14Cre mice, abolishment of SOCE was associated with impaired chloride secretion by primary murine sweat glands. In human sweat gland cells, SOCE mediated by ORAI1 was necessary for agonist-induced chloride secretion and activation of the Ca2+-activated chloride channel (CaCC) anoctamin 1 (ANO1, also known as TMEM16A). By contrast, expression of TMEM16A, the water channel aquaporin 5 (AQP5), and other regulators of sweat gland function was normal in the absence of SOCE. Our findings demonstrate that Ca2+ influx via store-operated CRAC channels is essential for CaCC activation, chloride secretion, and sweat production in humans and mice.

A single mutation at Tyr143 of human S-adenosylhomocysteine hydrolase renders the enzyme thermosensitive and affects the oxidation state of bound cofactor nicotinamide–adenine dinucleotide

Recently, we have described the first human case of AdoHcyase (S-adenosylhomocysteine hydrolase) deficiency. Two point mutations in the AdoHcyase gene, the missense mutation p.Y143C (AdoHcyase in which Tyr143 is replaced by cysteine) and the truncation mutation p.W112stop (AdoHcyase in which Trp112 is replaced by opal stop codon) were identified [Barić, Fumić, Glenn, Cuk, Schulze, Finkelstein, James, Mejaski-Bosnjak, Pazanin, Pogribny et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 4234-4239]. To elucidate the molecular and catalytic properties of AdoHcyase, we have made recombinant wild-type and mutant p.Y143C (AdoHcyase in which Tyr143 is replaced by cysteine) enzymes for a comparative analysis. The catalytic rates of p.Y143C protein in the directions of S-adenosylhomocysteine synthesis or hydrolysis are decreased from 65% to 75%. Further, the oxidation states of coenzyme NAD differ between mutant and wild-type protein, with an increased NADH accumulation in the mutant p.Y143C enzyme of 88% NADH (wild-type contains 18% NADH). Quantitative binding of NAD is not affected. Native polyacrylamide gel electrophoresis showed, that mutant p.Y143C subunits are able to form the tetrameric complex as is the wild-type enzyme. CD analysis showed that the p.Y143C mutation renders the recombinant protein thermosensitive, with an unfolding temperature significantly reduced by 7 degrees C compared with wild-type protein. Change of Glu115 to lysine in wild-type protein causes a change in thermosensitivity almost identical with that found in the p.Y143C enzyme, indicating that the thermosensitivity is due to a missing hydrogen bond between Tyr143 and Glu115. We emphasize involvement of this particular hydrogen bond for subunit folding and/or holoenyzme stability. In summary, a single mutation in the AdoHcyase affecting both the oxidation state of bound co-factor NAD and enzyme stability is present in a human with AdoHcyase deficiency.

Functional analysis of human S-adenosylhomocysteine hydrolase isoforms SAHH-2 and SAHH-3

S-adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of AdoHcy to adenosine and homocysteine. Increased levels of AdoHcy may play a role in the development of cardiovascular diseases and numerous other conditions associated with hyperhomocysteinemia. Several polymorphic isoforms named SAHH-1 to 4 may be resolved by horizontal starch gel electrophoresis from red blood cells. We have identified the genetic background of isoforms SAHH-2 and SAHH-3. SAHH-2 represents the previously described polymorphism in exon 2 of the AdoHcyase gene (112 C>T; p.R38W). Isoform SAHH-3 is based on a new polymorphism in exon 3 (377 G>A), leading to the conversion of glycine to arginine at amino-acid position 123. To shed light on the effects of these polymorphisms on the molecular and catalytic properties of AdoHcyase, we made recombinant wild-type and polymorphic R38W and G123R enzymes for a comparative analysis. The amino-acid exchanges did not bring about major changes to the catalytic rates of the recombinant proteins. However, circular dichroism analysis showed that both polymorphisms effect the thermal stability of the recombinant protein in vitro, reducing the unfolding temperature by approximately 2.6°C (R38W) and 1.5°C (G123R) compared to wild-type protein. In view of the altered thermal stability, and slightly decreased enzymatic activity of polymorphic proteins (≤6%), one may consider the analyzed AdoHcyase isoforms as risk markers for diseases caused by irregular AdoHcyase metabolism.

Respiratory chain deficiency in nonmitochondrial disease

Objective: In this study, we report 5 patients with heterogeneous phenotypes and biochemical evidence of respiratory chain (RC) deficiency; however, the molecular diagnosis is not mitochondrial disease. Methods: The reported patients were identified from a cohort of 60 patients in whom RC enzyme deficiency suggested mitochondrial disease and underwent whole-exome sequencing. Results: Five patients had disease-causing variants in nonmitochondrial disease genes ORAI1, CAPN3, COLQ, EXOSC8, and ANO10, which would have been missed on targeted next-generation panels or on MitoExome analysis. Conclusions: Our data demonstrate that RC abnormalities may be secondary to various cellular processes, including calcium metabolism, neuromuscular transmission, and abnormal messenger RNA degradation.

Hyperinsulinism-hyperammonemia syndrome : a de novo mutation of the GLUD1 gene in twins and a review of the literature

Abstract Hyperinsulinism-hyperammonemia (HI/HA) syndrome is a rare autosomal dominant disease characterized by recurrent hypoglycemia and persistent mild elevation of plasma ammonia. HI/HA syndrome is one of the more common forms of congenital hyperinsulinism (CHI), caused by activating mutations within the GLUD1 gene that encodes the mitochondrial enzyme glutamate dehydrogenase (GDH). We report here on monozygotic twin girls presented with fasting- and protein-induced hypoglycemia and mild persistent hyperammonemia. Genetic analysis revealed that both girls were heterozygous for a novel missense mutation within exon 11 [c.1499A>T, p.(R443W)] of the GLUD1 gene. Despite early treatment with diazoxide and a low protein diet, they both developed non-hypoglycemic seizures in early childhood followed by cognitive impairment. In addition to their clinical course, a review of the literature on HI/HA syndrome is provided.

Plasma biomarker identification in S-adenosylhomocysteine hydrolase deficiency

S‐Adenosylhomocysteine hydrolase (AHCY) deficiency is a rare congenital disorder in methionine metabolism clinically characterized by white matter atrophy, delayed myelination, slowly progressive myopathy, retarded psychomotor development and mildly active chronic hepatitis. In the present study, we utilized a comparative proteomics strategy based on 2‐DE/MALDI‐MS and LC/ESI‐MS to analyze plasma proteins from three AHCY‐deficient patients prior to and after receiving dietary treatment designed to alleviate disease symptoms. Obtained results revealed candidate biomarkers for the detection of myopathy specifically associated with AHCY deficiency, such as carbonic anhydrase 3, creatine kinase, and thrombospondin 4. Several proteins mediating T‐cell activation and function were identified as well, including attractin and diacylglycerol kinase α. Further validation and functional analysis of identified proteins with clinical value would ensure that these biomarkers make their way into routine diagnosis and management of AHCY deficiency.