Phenotypic and genetic spectra of galactose mutarotase deficiency: A nationwide survey conducted in Japan.

PURPOSE: Galactose mutarotase (GALM) deficiency was first reported in 2019 as the fourth type of galactosemia. This study aimed to investigate the clinical and genotypic spectra of GALM deficiency. METHODS: This was a questionnaire-based retrospective survey conducted in Japan between February 2022 and March 2023. RESULTS: We identified 40 patients with GALM deficiency in Japan (estimated prevalence: 1:181,835). Four of 38 patients (10.5%) developed cataracts, which resolved with lactose restriction in three out of four patients. Transient transaminitis was the most common symptom (23.1%). All the patients followed lactose restriction; discontinuation of the restriction after infancy did not cause any complications. Moreover, none of the participants experienced long-term complications. Two variants, GALM NM_138801.3: c.294del and c.424G>A, accounted for 72.5% of the identified pathogenic variants. The patients showed moderately elevated blood galactose levels with lactose intake; however, the elevation was lower than that observed in galactokinase deficiency. CONCLUSIONS: GALM deficiency is characterized by a similar but milder phenotype and lower blood galactose elevation than in galactokinase deficiency. Diagnosis and initiation of lactose restriction in early infancy should be essential for prevention of cataracts, especially in cases of irreversible opacity.

Stealthy progression of type 2 diabetes mellitus due to impaired ketone production in an adult patient with multiple acyl-CoA dehydrogenase deficiency.

Background: Multiple acyl-CoA dehydrogenase deficiency (MADD) is an inherited metabolic disorder caused by biallelic pathogenic variants in genes related to the flavoprotein complex. Dysfunction of the complex leads to impaired fatty acid oxidation and ketone body production which can cause hypoketotic hypoglycemia with prolonged fasting. Patients with fatty acid oxidation disorders (FAODs) such as MADD are treated primarily with a dietary regimen consisting of high-carbohydrate foods and avoidance of prolonged fasting. However, information on the long-term sequelae associated with this diet have not been accumulated. In general, high-carbohydrate diets can induce diseases such as type 2 diabetes mellitus (T2DM), although few patients with both MADD and T2DM have been reported. Case: We present the case of a 32-year-old man with MADD who was on a high-carbohydrate diet for >30 years and exhibited symptoms resembling diabetic ketoacidosis. He presented with polydipsia, polyuria, and weight loss with a decrease in body mass index from 31 to 25 kg/m2 over 2 months. Laboratory tests revealed a HbA1c level of 13.9%; however, the patient did not show metabolic acidosis but only mild ketosis. Discussion/conclusion: This report emphasizes the potential association between long-term adherence to high-carbohydrate dietary therapy and T2DM development. Moreover, this case underscores the difficulty of detecting diabetic ketosis in patients with FAODs such as MADD due to their inability to produce ketone bodies. These findings warrant further research of the long-term complications associated with this diet as well as warning of the potential progression of diabetes in patients with FAODs such as MADD.

Improved sensitivity and specificity for citrin deficiency using selected amino acids and acylcarnitines in the newborn screening.

Citrin deficiency is an autosomal recessive disorder caused by a defect of citrin resulting from mutations in the SLC25A13 gene. Intrahepatic cholestasis and various metabolic abnormalities, including hypoglycemia, galactosemia, citrullinemia, and hyperammonemia may be present in neonates or infants in the "neonatal intrahepatic cholestasis caused by citrin deficiency" (NICCD) form of the disease. Because at present, newborn screening (NBS) for citrin deficiency using citrulline levels in dried blood spots (DBS) can only detect some of the patients, we tried to develop a new evaluation system to more reliably detect newborns with citrin deficiency utilizing parameters already in place in present NBS methods. To achieve this goal, we re-analyzed NBS profiles of amino acids and acylcarnitines in 96 NICCD patients, who were diagnosed through selective screening or positive family history. Hereby, we identified the combined evaluation of arginine (Arg), citrulline (Cit), isoleucine+leucine (Ile + Leu), tyrosine (Tyr), free carnitine (C0) / glutarylcarnitine (C5-DC) ratio in DBS as potentially sensitive to diagnose citrin deficiency in pre-symptomatic newborns. In particular, a scoring system using threshold levels for Arg (≥9 µmol/L), Cit (≥ 39 µmol/L), Ile + Leu (≥ 99 µmol/L), Tyr (≥ 96 µmol/L) and C0/C5-DC ratio (≥327) was significantly effective to detect newborns who later developed NICCD, and could thus be implemented in existing NBS programs at no extra analytical costs whenever citrin deficiency is considered to become a novel target disease.

Case report: Young-onset large vessel ischemic stroke due to hyperhomocysteinemia associated with the C677T polymorphism on 5,10-methylenetetrahydrofolate reductase and multi-vitamin deficiency.

Hyperhomocysteinemia is an important risk factor for cerebral infarction. Herein, we report on a 30-year-old man previously diagnosed with epilepsy who presented with right hemiplegia and total aphasia. Magnetic resonance imaging showed a fronto-temporal ischemic lesion due to occlusion of the left middle cerebral artery. Clinical testing and imaging demonstrated that he had hyperhomocysteinemia induced by multiple factors including the C677T polymorphism on 5.10-methylenetetrahydrofolate reductase (MTHFR), and multiple vitamin deficiencies. The C677T polymorphism on MTHFR is closely related to hyperhomocysteinemia and folate deficiency in epileptic patients who are taking multiple anti-convulsants. Given hyperhomocysteinemia can independently cause stroke at a young age, physicians should periodically examine plasma homocysteine and serum folic acid levels in epileptic patients who are on long-term regimens of multiple anti-epileptic drugs.

A method for phenylalanine self-monitoring using phenylalanine ammonia-lyase and a pre-existing portable ammonia detection system.

Phenylketonuria is an inborn error of phenylalanine metabolism caused by a phenylalanine hydroxylase deficiency. To prevent the occurrence of neurological symptoms and maternal complications resulting from phenylketonuria, patients must adhere to a strict diet therapy, tetrahydrobiopterin supplementation, or pegvaliase injection to maintain blood phenylalanine levels within a recommended range throughout their lives. Therefore, monitoring blood phenylalanine levels is necessary to determine the recent metabolic status of phenylalanine in patients with PKU; however, there are no available instruments for individuals to monitor their own blood phenylalanine levels using whole fingertip blood. We developed a phenylalanine monitoring system (designated as PheCheck) that included a pre-existing portable ammonia detection device and phenylalanine ammonia-lyase, which converts phenylalanine to trans-cinnamic acid and ammonia. This system was able to remove 86.7% ± 0.03% of the ammonia contained in fingertip blood and successfully reduce background ammonia levels. A good correlation was found between the estimated plasma phenylalanine levels detected by PheCheck and plasma phenylalanine levels detected by high-performance liquid chromatography (R2 0.97). The entire PheCheck process for measuring blood phenylalanine takes only 20 min. PheCheck can lay the foundation for home phenylalanine monitoring with high feasibility because all the components are easily accessible. Further studies with a more user-friendly PheCheck optimized for practice are needed to improve blood phenylalanine control, reduce the burden on patients and/or caregivers, and prevent the sequelae associated with phenylketonuria.

Successful pregnancy and childbirth without metabolic abnormality in a patient with holocarboxylase synthetase deficiency.

Holocarboxylase synthetase deficiency (HSD), an autosomal recessive biotin cycle disorder, is caused by holocarboxylase synthetase (HLCS) genetic variants, resulting in multiple carboxylase deficiency. Catabolic stress can induce metabolic crises in patients with HSD. Although pharmacological doses of biotin have improved HLCS enzyme activity and HSD prognosis, the prolonged life expectancy has gradually highlighted novel issues in adult patients with HSD. To the best of our knowledge, there is only one report on a case of HSD during pregnancy and childbirth, and the metabolic profile was not well defined. In this report, we present the history and metabolic profile of a woman with HSD who had an uncomplicated pregnancy and childbirth. A high pharmacological dose of biotin, 100 mg/day, had no effect on the fetus. Even during the emergency cesarean section, the detailed metabolic assessments revealed no significant laboratory findings, such as ketolactic acidosis, hyperammonemia, and remarkable acylcarnitine change. This report suggests that a woman with HSD who regularly takes biotin can conceive and give birth safely, and biotin doses of 100 mg/day may not influence the growth and development of the fetus. Further research and case studies on pregnant women with HSD are required to determine an acceptable maximum dosage of biotin for human fetuses.

Usefulness of serum BUN or BUN/creatinine ratio as markers for citrin deficiency in positive cases of newborn screening.

Citrin deficiency belongs to a group of urea cycle disorders that can be identified during newborn screening by measuring citrulline, phenylalanine, methionine, and galactose levels. Early diagnosis of citrin deficiency is beneficial as disease-specific interventions such as permission of food preference and/or supplementation of medium-chain triglyceride can prevent metabolic decompensation. However, there are currently no laboratory tests for the diagnosis of citrin deficiency in routine clinical practice. Our retrospective study investigated the diagnostic characteristics of citrin deficiency during infancy at secondary newborn screening. The present study included 10 patients with citrin deficiency and 35 controls without the condition. The positive likelihood ratios for serum levels of blood urea nitrogen (BUN) levels were 6.8 at the first visit, 3.2 at age ≤ 60 days, and 17.5 at age ≤ 100 days. The serum BUN/creatinine ratio also showed a high positive likelihood ratio (3.9 at first visit, 16.0 at age ≤ 60 days, and 24.5 at age ≤ 100 days). Therefore, the serum BUN or BUN/creatinine ratio may help to identify patients with citrin deficiency during newborn screening. Further studies are required to confirm its diagnostic accuracy in a larger cohort and elucidate the underlying mechanisms involved.

Clinical manifestation and long-term outcome of citrin deficiency: Report from a nationwide study in Japan.

Citrin deficiency is an autosomal recessive disorder caused by mutations in the SLC25A13 gene. The disease can present with age-dependent clinical manifestations: neonatal intrahepatic cholestasis by citrin deficiency (NICCD), failure to thrive, and dyslipidemia by citrin deficiency (FTTDCD), and adult-onset type II citrullinemia (CTLN2). As a nationwide study to investigate the clinical manifestations, medical therapy, and long-term outcome in Japanese patients with citrin deficiency, we collected clinical data of 222 patients diagnosed and/or treated at various different institutions between January 2000 and December 2019. In the entire cohort, 218 patients were alive while 4 patients (1 FTTDCD and 3 CTLN2) had died. All patients <20 years were alive. Patients with citrin deficiency had an increased risk for low weight and length at birth, and CTLN2 patients had an increased risk for growth impairment during adolescence. Liver transplantation has been performed in only 4 patients (1 NICCD, 3 CTLN2) with a good response thereafter. This study reports the diagnosis and clinical course in a large cohort of patients with citrin deficiency and suggests that early intervention including a low carbohydrate diet and MCT supplementation can be associated with improved clinical course and long-term outcome.

Intravenous ketogenic diet therapy for neonatal-onset pyruvate dehydrogenase complex deficiency.

BACKGROUND: Pyruvate dehydrogenase complex (PDHC) deficiency is an inborn error of metabolism that causes lactic acidosis and neurodevelopmental changes. Five causative genes have been identified: PDHA1, PDHB, DLAT, DLD, and PDHX. Four neurological phenotypes have been reported: neonatal encephalopathy with lactic acidosis, non-progressive infantile encephalopathy, Leigh syndrome, and relapsing ataxia. Of these, neonatal encephalopathy has the worst mortality and morbidity and there is no effective treatment. SUBJECTS AND METHODS: We studied two girls who were clinically diagnosed with PDHC deficiency as neonates; they were subsequently found to have PDHA1 mutations. The clinical diagnosis was based on white matter loss and a lateral ventricular septum on fetal MRI, spasticity of the lower extremities, and lactic acidosis worsening after birth. Intravenous ketogenic diets were started within 24 h after birth. The ketogenic ratio was increased until the blood lactate level was controlled, while monitoring for side effects. RESULTS: In both cases, the lactic acidosis improved immediately with no apparent side effects. Both children had better developmental outcomes than previously reported cases; neither exhibited epilepsy. CONCLUSIONS: Intravenous ketogenic diet therapy is a treatment option for neonatal-onset PDHC deficiency. Further studies are needed to optimize this therapy.

ß-Galactosidase therapy can mitigate blood galactose elevation after an oral lactose load in galactose mutarotase deficiency.

Galactose mutarotase (GALM) deficiency (MIM# 618881), also known as type IV galactosemia, is caused by biallelic pathogenic variants of GALM. Cataracts are observed in patients with GALM deficiency as well as in other conditions associated with high levels of blood galactose and can be prevented by consuming a galactose-restricted diet or formula. Galactose restriction is the only known treatment for GALM deficiency and other types of galactosemia. We incidentally found that ß-galactosidase might reduce blood galactose levels caused by lactose loading in GALM deficiency. Consequently, we investigated the effectiveness of ß-galactosidase in decreasing the level of blood galactose in three patients with GALM deficiency. We performed two lactose loading tests per case: one with and one without ß-galactosidase. The add-on administration of ß-galactosidase significantly mitigated blood galactose elevations after lactose loading. Although urine galactitol was mildly elevated in all patients with GALM deficiency, ß-galactosidase did not prevent increased levels of urine galactitol during the loading tests. No adverse events, including cataracts, were observed during or after the tests. Therefore, ß-galactosidase could be a potential novel treatment agent for blood galactose elevation caused by lactose in patients with GALM deficiency. The effectiveness of ß-galactosidase could possibly result in loosening of the galactose dietary restrictions or treatment for patients with GALM deficiency.

The Discovery of GALM Deficiency (Type IV Galactosemia) and Newborn Screening System for Galactosemia in Japan.

The Leloir pathway, which consists of highly conserved enzymes, metabolizes galactose. Deficits in three enzymes in this pathway, namely galactose-1-phosphate uridylyltransferase (GALT), galactokinase (GALK1), and UDP-galactose-4'-epimerase (GALE), are associated with genetic galactosemia. We recently identified patients with galactosemia and biallelic variants in GALM, encoding galactose epimerase (GALM), an enzyme that is directly upstream of GALK1. GALM deficiency was subsequently designated as type IV galactosemia. Currently, all the published patients with biallelic GALM variants were found through newborn screening in Japan. Here, we review GALM deficiency and describe how we discovered this relatively mild but not rare disease through the newborn screening system in Japan.

Long-term outcome of urea cycle disorders: Report from a nationwide study in Japan.

Urea cycle disorders (UCDs) are inherited metabolic disorders with impaired nitrogen detoxification caused by defects in urea cycle enzymes. They often manifest with hyperammonemic attacks resulting in significant morbidity or death. We performed a nationwide questionnaire-based study between January 2000 and March 2018 to document all UCDs in Japan, including diagnoses, treatments, and outcomes. A total of 229 patients with UCDs were enrolled in this study: 73 males and 53 females with ornithine transcarbamylase deficiency (OTCD), 33 patients with carbamoylphosphate synthetase 1 deficiency, 48 with argininosuccinate synthetase deficiency, 14 with argininosuccinate lyase deficiency, and 8 with arginase deficiency. Survival rates at 20 years of age of male and female patients with late-onset OTCD were 100% and 97.7%, respectively. Blood ammonia levels and time of onset had a significant impact on the neurodevelopmental outcome (P < .001 and P = .028, respectively). Hemodialysis and liver transplantation did not prevent poor neurodevelopmental outcomes. While treatment including medication, hemodialysis, and liver transplantation may aid in decreasing blood ammonia and/or preventing severe hyperammonemia, a blood ammonia level ≥ 360 µmol/L was found to be a significant indicator for a poor neurodevelopmental outcome. In conclusion, although current therapy for UCDs has advanced and helped saving lives, patients with blood ammonia levels ≥ 360 µmol/L at onset often have impaired neurodevelopmental outcomes. Novel neuroprotective measures should therefore be developed to achieve better neurodevelopmental outcomes in these patients.

Hypoglycemic attacks and growth failure are the most common manifestations of citrin deficiency after 1 year of age.

Citrin deficiency develops in different symptomatic periods from the neonatal period to adulthood. Some infantile patients are diagnosed by newborn mass screening or symptoms of neonatal intrahepatic cholestasis caused by citrin deficiency, some patients in childhood may develop hepatopathy or dyslipidemia as failure to thrive and dyslipidemia caused by citrin deficiency, and some adults are diagnosed after developing adult-onset type 2 citrullinemia (CTLN2) with hyperammonemia or encephalopathy. A diagnosis is needed before the development of severe phenotypic CTLN2 but is often difficult to obtain because newborn mass screening cannot detect all patients with citrin deficiency, and undiagnosed patients often appear healthy in childhood. There are only a few reports that have described patients in childhood. To explore the clinical features of undiagnosed patients with citrin deficiency in childhood, we studied 20 patients who were diagnosed after the first year of life. Of these patients, 45% experienced hypoglycemic attacks in childhood. The acetoacetic acid level during hypoglycemic attacks was lower than expected. Growth failure at diagnosis (45%) was also noted. From the patients' history, fat- and protein-rich food preferences (80%), a low birth weight (70%), and prolonged jaundice or infantile hepatopathy (40%) were identified. To diagnose citrin deficiency in childhood, we should ask about food preferences and a history of infantile hepatopathy for all children with severe hypoglycemia or growth failure and consider the genetic test for citrin deficiency if the patient has characteristic food preferences or a history of infantile hepatopathy.

Essential oils can cause false-positive results of medium-chain acyl-CoA dehydrogenase deficiency.

Newborn screening is a public health care program worldwide to prevent patients from critical illness or conditions. Tandem mass spectrometry allows multiplex, inexpensive, and rapid newborn screening. However, mass spectrometry used for newborn screening to date is not able to separate peaks of compounds with similar m/z, which could lead to false-positive results without additional second-tier tests, such as fragmentation. We experienced three neonatal cases with high levels of markers, octanoylcarnitine and octanoylcarnitine/decanoylcarnitine ratio used to pick up possible cases of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. The babies were born consecutively in a maternity hospital. Their second acylcarnitine profiles were normal, and the genetic tests for ACADM were negative. Analysis of samples extracted from their first Guthrie cards where blood was not stained also showed peaks equivalent to octanoylcarnitine and decanoylcarnitine, indicating contamination. Environmental surveillance in the maternity ward suggested that essential oils used there might contain the contaminated compound. LC-HRMS/MS and in silico analysis revealed that false-positive results might be due to contamination with the essential oils in Guthrie cards, and causal agents were sphinganine (d17:0) and 2-[2-hydroxyethyl(pentadecyl)amino]ethanol. Thus, health care providers should be cautioned about use of essential oils when collecting blood samples on Guthrie cards. False-positive results can waste costly social resources and cause a physical and psychological burden for children and parents.

Hypoketotic hypoglycemia in citrin deficiency: a case report.

BACKGROUND: Citrin deficiency (CD) is a recessive metabolic disease caused by biallelic pathogenic variants in SLC25A13. Although previous studies have reported ketosis in CD, it was observed at the time of euglycemia or mild hypoglycemia. Blood ketone levels concomitant with symptomatic or severe hypoglycemia in CD have not been a topic of focus despite its importance in identifying the etiology of hypoglycemia and assessing the ability of fatty acid utilization. Herein, we describe a patient with CD who had repeated episodes of hypoglycemia with insufficient ketosis. CASE PRESENTATION: A 1-year-old boy with repetitive hypoglycemia was referred to us to investigate its etiology. The fasting load for 13 h led to hypoketotic hypoglycemia, indicating the possibility of partial ß-oxidation dysfunction. A genetic test led to the diagnosis of CD. The hypoglycemic episodes disappeared after switching to a medium-chain triglyceride-containing formula. CONCLUSIONS: This case report suggests that symptomatic or severe hypoglycemia in patients with CD could be associated with relatively low levels of ketone bodies, implying that ß-oxidation in these patients might possibly be partially disrupted. When encountering a patient with hypoglycemia, clinicians should check blood ketone levels and bear in mind the possibility of CD because excessive intravenous administration of glucose can cause decompensated symptoms in patients with CD as opposed to other disorders presenting with hypoketotic hypoglycemia, such as fatty acid oxidation disorders. Further studies in a large-scale cohort are warranted to confirm our speculation.

Correction: Biallelic GALM pathogenic variants cause a novel type of galactosemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Metabolic and pathologic profiles of human LSS deficiency recapitulated in mice.

Skin lesions, cataracts, and congenital anomalies have been frequently associated with inherited deficiencies in enzymes that synthesize cholesterol. Lanosterol synthase (LSS) converts (S)-2,3-epoxysqualene to lanosterol in the cholesterol biosynthesis pathway. Biallelic mutations in LSS have been reported in families with congenital cataracts and, very recently, have been reported in cases of hypotrichosis. However, it remains to be clarified whether these phenotypes are caused by LSS enzymatic deficiencies in each tissue, and disruption of LSS enzymatic activity in vivo has not yet been validated. We identified two patients with novel biallelic LSS mutations who exhibited congenital hypotrichosis and midline anomalies but did not have cataracts. We showed that the blockade of the LSS enzyme reaction occurred in the patients by measuring the (S)-2,3-epoxysqualene/lanosterol ratio in the forehead sebum, which would be a good biomarker for the diagnosis of LSS deficiency. Epidermis-specific Lss knockout mice showed neonatal lethality due to dehydration, indicating that LSS could be involved in skin barrier integrity. Tamoxifen-induced knockout of Lss in the epidermis caused hypotrichosis in adult mice. Lens-specific Lss knockout mice had cataracts. These results confirmed that LSS deficiency causes hypotrichosis and cataracts due to loss-of-function mutations in LSS in each tissue. These mouse models will lead to the elucidation of the pathophysiological mechanisms associated with disrupted LSS and to the development of therapeutic treatments for LSS deficiency.

The prevalence of GALM mutations that cause galactosemia: A database of functionally evaluated variants.

Galactosemia is a metabolic disorder that affects the appropriate metabolism of ß-D-galactose. Deficiencies in three of the enzymes of the Leloir pathway, namely, GALT, GALK1, or GALE, are characterized as type I, II, and III galactosemia, respectively. Recently, we reported a novel type of galactosemia (type IV galactosemia) due to biallelic GALM mutations. Genetic diagnosis is indispensable for diagnosing GALM deficiency because no biochemical diagnosis method has been established. Given that apparently pathogenic variants in GALM are found in public variant databases, we presumed the presence of pathogenic variants that have not been reported. In this study, we explore 67 GALM variants that are prevalent in the ExAC database, including 57 missense variants, 7 stop-gain variants, 2 frameshift variants, and 1 splice-site variant. We performed an in vitro expression assay and an enzyme activity assay. Among the 66 variants except for 1 splice-site variant, 29 produced no or faint protein expression and were judged as pathogenic variants. Furthermore, the remaining 37 variants were evaluated by enzyme activity assay. Two showed mildly reduced enzyme activity and were classified as benign. Based on our study, the estimated incidence of GALM deficiency is 1:228,411 in all populations, 1:10,388 in the African population, and 1:80,747 in the Japanese population. Our GALM mutation database is useful for the genetic diagnosis of GALM deficiency.

Biallelic GALM pathogenic variants cause a novel type of galactosemia.

PURPOSE: Galactosemia is caused by metabolic disturbances at various stages of galactose metabolism, including deficiencies in enzymes involved in the Leloir pathway (GALT, GALK1, and GALE). Nevertheless, the etiology of galactosemia has not been identified in a subset of patients. This study aimed to explore the causes of unexplained galactosemia. METHODS: Trio-based exome sequencing and/or Sanger sequencing was performed in eight patients with unexplained congenital galactosemia. In vitro enzymatic assays and immunoblot assays were performed to confirm the pathogenicity of the variants. RESULTS: The highest blood galactose levels observed in each patient were 17.3-41.9 mg/dl. Bilateral cataracts were observed in two patients. In all eight patients, we identified biallelic variants (p.Arg82*, p.Ile99Leufs*46, p.Gly142Arg, p.Arg267Gly, and p.Trp311*) in the GALM encoding galactose mutarotase, which catalyzes epimerization between ß- and α-D-galactose in the first step of the Leloir pathway. GALM enzyme activities were undetectable in lymphoblastoid cell lines established from two patients. Immunoblot analysis showed the absence of the GALM protein in the patients' peripheral blood mononuclear cells. In vitro GALM expression and protein stability assays revealed altered stabilities of the variant GALM proteins. CONCLUSION: Biallelic GALM pathogenic variants cause galactosemia, suggesting the existence of type IV galactosemia.