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.

Insights into the Pathophysiology of Infertility in Females with Classical Galactosaemia.

Classical galactosaemia (CG) (OMIM 230400) is a rare inborn error of galactose metabolism caused by the deficiency of the enzyme galactose-1-phosphate uridylyltransferase (GALT, EC 2.7.7.12). Primary ovarian insufficiency (POI) is the most common long-term complication experienced by females with CG, presenting with hypergonadotrophic hypoestrogenic infertility affecting at least 80% of females despite new-born screening and lifelong galactose dietary restriction. In this review, we describe the hypothesized pathophysiology of POI from CG, implications of timing of the ovarian dysfunction, and the new horizons and future prospects for treatments and fertility preservation.

Laboratory diagnosis of galactosemia: a technical standard and guideline of the American College of Medical Genetics and Genomics (ACMG).

Disclaimer: These ACMG Standards and Guidelines are developed primarily as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these Standards and Guidelines is voluntary and does not necessarily assure a successful medical outcome. These Standards and Guidelines should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own professional judgment to the specific circumstances presented by the individual patient or specimen. Clinical laboratory geneticists are encouraged to document in the patient's record the rationale for the use of a particular procedure or test, whether or not it is in conformance with these Standards and Guidelines. They also are advised to take notice of the date any particular guideline was adopted, and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.Galactosemias are inherited disorders of galactose metabolism due to deficiency in one of the three enzymes involved in the Leloir pathway: galactose-1-phosphate uridyltransferase, galactokinase, and uridine diphosphate (UDP)-galactose-4'-epimerase. Galactose-1-phosphate uridyltransferase deficiency, or classic galactosemia, is the most frequent and the most severe of the three enzyme deficiencies; it is characterized by failure to thrive, liver failure, susceptibility to sepsis, and death, if untreated. Newborn screening for classic galactosemia has been implemented in all of the United States, while screening for galactokinase deficiency and UDP-galactose-4'-epimerase deficiency is not universal. Early identification and treatment of galactosemia leads to improved outcome. This document reviews the laboratory methods and best practices for the diagnosis of galactosemia.

Misfolding of galactose 1-phosphate uridylyltransferase can result in type I galactosemia.

Type I galactosemia is a genetic disorder that is caused by the impairment of galactose-1-phosphate uridylyltransferase (GALT; EC 2.7.7.12). Although a large number of mutations have been detected through genetic screening of the human GALT (hGALT) locus, for many it is not known how they cause their effects. The majority of these mutations are missense, with predicted substitutions scattered throughout the enzyme structure and thus causing impairment by other means rather than direct alterations to the active site. To clarify the fundamental, molecular basis of hGALT impairment we studied five disease-associated variants p.D28Y, p.L74P, p.F171S, p.F194L and p.R333G using both a yeast model and purified, recombinant proteins. In a yeast expression system there was a correlation between lysate activity and the ability to rescue growth in the presence of galactose, except for p.R333G. Kinetic analysis of the purified proteins quantified each variant's level of enzymatic impairment and demonstrated that this was largely due to altered substrate binding. Increased surface hydrophobicity, altered thermal stability and changes in proteolytic sensitivity were also detected. Our results demonstrate that hGALT requires a level of flexibility to function optimally and that altered folding is the underlying reason of impairment in all the variants tested here. This indicates that misfolding is a common, molecular basis of hGALT deficiency and suggests the potential of pharmacological chaperones and proteostasis regulators as novel therapeutic approaches for type I galactosemia.

Disorders of ovarian function in childhood and adolescence: evolving needs of the growing child. An endocrine perspective.

Over the past 15 years there have been changes in the care of children and adolescents paralleling increased longevity of those with chronic illnesses and increased survival after childhood cancer and organ transplantation. A broad understanding of holistic management and long-term risks is required. Optimisation of pubertal progress and normalisation of bone and hormonal health by the end of puberty will reduce the impact of later adult bone loss in chronic disease conditions. Psychosocial issues related to both precocious and delayed puberty can have profound effects on family function.

Galactose-Induced Skin Aging: The Role of Oxidative Stress.

Skin aging has been associated with a higher dietary intake of carbohydrates, particularly glucose and galactose. In fact, the carbohydrates are capable of damaging the skin's vital components through nonenzymatic glycation, the covalent attachment of sugar to a protein, and subsequent production of advanced glycation end products (AGEs). This review is focused on the role of D-galactose in the development of skin aging and its relation to oxidative stress. The interest in this problem was dictated by recent findings that used in vitro and in vivo models. The review highlights the recent advances in the underlying molecular mechanisms of D-galactose-mediated cell senescence and cytotoxicity. We have also proposed the possible impact of galactosemia on skin aging and its clinical relevance. The understanding of molecular mechanisms of skin aging mediated by D-galactose can help dermatologists optimize methods for prevention and treatment of skin senescence and aging-related skin diseases.

Hypergalactosemia in early infancy: diagnostic strategy with an emphasis on imaging.

BACKGROUND: Portosystemic shunt is one of the main causes of persistent hypergalactosemia without enzyme deficiency, but the diagnostic imaging strategy has not yet been established. The purpose of the present study was to establish a diagnostic imaging strategy. METHODS: A retrospective investigation of the clinical and imaging findings of 10 children with persistent hypergalactosemia without enzyme deficiency detected by screening was undertaken. RESULTS: Abnormal ultrasonography (US) findings were detected in all eight patients with liver disorders. In three patients with citrin deficiency, the combination of fatty liver on US and laboratory evidence of cholestasis led to the diagnosis. In three patients with portosystemic shunt, US on sedation clearly depicted the shunt vessels. The extent was more easily understood on contrast computed tomography (CT). Per-rectal portal scintigraphy with N-isopropyl-p-I-123 iodoamphetamine and lung perfusion scintigraphy with (99m)Tc macroaggregated albumin were useful for evaluation of portal shunt index and assessment of pulmonary arteriovenous shunt. One patient underwent transarterial coil embolization. In two patients with hepatic tumor, the lesions and its vascularity were clearly demonstrated on US and dynamic CT. In one patient, small shunt index on per-rectal portal scintigraphy suggested no need for treatment. The other patient was treated with a combination of steroid, radiation, and interventional radiology. The etiology remained unknown in two children. CONCLUSIONS: In the assessment of hypergalactosemia, US is the modality of choice. CT is a useful tool for more detailed evaluation of the abnormalities found on US. Per-rectal portal scintigraphy and pulmonary perfusion scintigraphy play an important role in the evaluation of portosystemic shunt. Interventional radiology is sometimes effective.

Galactose-induced retinal microangiopathy in rats.

PURPOSE: The suitability of the galactose-fed rat as a model of diabetic retinopathy was examined in nondiabetic rats fed diets enriched with either 30% or 50% galactose for up to 2 years. METHODS: Retinal capillaries were examined by light and electron microscopy, and the prevalence or severity of diabetic-like lesions was quantitated. RESULTS: Histologic evaluation of trypsin digests of retina revealed significantly greater than normal frequencies of pericyte ghosts and acellular capillaries at both 15 and 23 months receiving a 50% galactose diet. Similar lesions were observed in rats receiving a 30% galactose diet for 23 months. Capillary basement membrane thickening, dilated hypercellular capillaries (or intra-retinal microvascular abnormalities), and foci of vascular cells appeared in rats fed 50% galactose, but saccular microaneurysms characteristic of retinopathy in diabetic patients, diabetic dogs, and experimentally galactosemic dogs were not observed. Administration of the aldose reductase inhibitor, Sorbinil, to rats fed 50% galactose resulted in a significant inhibition of cataract and of galactitol accumulation in nerve and blood (by more that 90%) and retina (by 62%), but did not inhibit development of the retinal microvascular lesions. CONCLUSIONS: Two years of galactosemia in rats seems to reproduce only a portion of the lesions characteristic of diabetic retinopathy in patients or dogs. Nevertheless, lesions characteristic of at least the early stages of retinopathy clearly do develop in this galactosemic rat model, and are not restrained by inhibition of retinal polyol accumulation by 62%.

Subnormal retinal oxygenation response precedes diabetic-like retinopathy.

PURPOSE: Determining which patients are at risk for the development of diabetic retinopathy is expected to greatly improve existing prevention and treatment options. In this study, using an animal model of diabetic retinopathy, the hypothesis was tested that magnetic resonance imaging (MRI) and a carbogen inhalation challenge provides important diagnostic information regarding the risk of developing diabetic retinopathy. METHODS: MRI was used to measure noninvasively the change in oxygen tension along the entire inner retina (i.e., from superior ora serrata to inferior ora serrata) during a carbogen (95% O2/5% CO2) inhalation challenge (IOVS 1996;37:2089). Two animal groups were examined by this MRI method at two time points: (1) rats fed either normal rat chow (n = 20) or a 50% galactose diet (n = 20) for 3.5 months (i.e., before the appearance of extensive retinal lesions) or (2) rats fed either normal rat chow (n = 3) for 15 months or a 30% galactose diet (n = 4) for 15 to 18 months (i.e., when lesions are present). Retinal biochemical and morphometric measurements were also obtained. RESULTS: After 3.5 months of galactosemia, before the appearance of extensive retinal morphologic lesions, a significant (P < 0.05) reduction in the panretinal oxygenation response was observed in the galactosemic group compared with its age-matched control. These galactose-fed animals also displayed a significantly (P < 0.05) larger oxygenation response in the inferior hemiretina than in the superior hemiretina. After 15 to 18 months of galactosemia, during the period when lesions are present, the panretinal oxygenation response remained significantly (P < 0.05) lower in the galactose-fed animals than in their age-matched controls. In contrast to the 3.5-month results, the oxygenation response in galactosemic animals at 15 to 18 months was significantly (P < 0.05) larger in the superior than in the inferior hemiretina. Hemiretinal oxygenation responses were not different in normal controls at either duration. CONCLUSIONS: MRI measurement of the retinal oxygenation response to a carbogen challenge appears to be a powerful new and noninvasive approach that may be useful for assessing aspects of pathophysiology underlying the development of diabetic retinopathy in galactosemic rats. These results support our working hypothesis and suggest that further research into the diagnostic potential of this MRI approach for predicting the development of diabetic retinopathy is warranted.

A mouse model of diabetic retinopathy.

OBJECTIVE: To obtain a model of diabetic retinopathy to which modern methods of genetic engineering may be applied, by determining the response of 2 strains of mice to long-term galactose feeding. METHODS: Both C57BL/6 mice BALB/c mice were fed each of 2 galactose-rich diets (30% and 50% galactose), and trypsin digests of their retinas were compared with those of controls at durations of up to 26 months. RESULTS: The mortality rate in galactose-fed animals was lower in C57BL/6 mice than in BALB/c mice, and both strains tolerated the 30% galactose diet significantly better than the 50% galactose diet. In C57BL/6 mice fed 30% galactose for 21 to 26 months, saccular microaneurysms were observed in the retina, together with significant increases in the thickness of capillary basement membrane and the prevalence of acellular capillaries and pericyte ghosts. The 50% galactose diet caused significantly more acellular capillaries than normal by 15 months, but excessive mortality precluded study at longer durations. The frequency of acellular capillaries also was greater than normal in BALB/c mice fed 30% galactose for 21 months. Retinal polyol levels in galactose-fed mice were found to be lower than those in galactosemic rats. CONCLUSION: The mouse may provide an inexpensive model suitable for in vivo study of the pathogenesis of diabetic retinopathy using molecular biological techniques.

Diseases of aberrant glycosylation.

Recently a defective glycosylation of glycoconjugates has been implicated in the pathogenesis of a number of heritable or acquired diseases of humans. Herein I discuss them under the name of diseases of aberrant glycosylation. These are: congenital dyserythropoietic anemia type II, carbohydrate-deficient glycoprotein syndrome, I-cell disease, galactosemia in subjects on galactose-free diet, variants of leukocyte adhesion deficiency, and of Ehlers-Danlos syndrome, paroxysmal nocturnal hemoglobinuria, and Tn syndrome. Regarding the present views on the function of glycoconjugates it is probably significant that in most instances defective or missing glycoproteins (or proteoglycans) but not glycosphingolipids, are probably involved in the pathogenesis of these diseases.

Galactose-1-phosphate is a regulator of inositol monophosphatase: a fact or a fiction?

Classic galactosemia is due to the deficiency of galactose-1-phosphate uridyl transferase and is transmitted as an autosomal recessive disorder. Patients suffering from classic galactosemia display acute symptoms such as poor growth, feeding difficulties, jaundice, hepatomegaly etc., which disappear when the individual is on galactose free diet. However, these patients continue to suffer from defects such as neurological disturbances and ovarian dysfunction, due to the accumulation of galactose-1-phosphate, which is a normal intermediate of galactose metabolism. The biochemical mechanism of galactose-1-phosphate mediated toxicity is still an enigma. Recent experiments strongly suggest that galactose-1-phosphate is also a substrate for inositol monophosphatase (IMPase). Phosphatidylinositol bisphosphate [PI(P)2] dependent signaling serves as a second messenger for several neurotransmitters in the brain. Therefore, the brain is critically dependent on IMPase for the supply of free inositol in order to sustain [PI(P)2] signaling. Circumstantial evidence strongly supports the possibility that being a substrate, galactose-1-phosphate could modulate IMPase function in vivo. The implication of this idea is discussed in relation to classic galactosemia as well as bipolar disorder, which has been thought to be due to the hyper-activation of [PI(P)2] mediated second messenger pathways(s).

[Intestinal absorption of glucose and galactose in the rat determined by blood hexose]. / L'absorption intestinale due glucose et du galactose chez le rat, mesurée par les hexosémies.

Adult rats are used to consume their diet within the space of 30 min. They are sacrified after fasting 2 hours or 30 to 90 min. after the end of meal. Blood sugars are determined. -- The administration of galactose (GAL group) at 40 p. 100 of the diet induces a high postprandial galactosemia (near 600 mg p. 100 ml) without glycemia change: the absorbed galactose is not converted into glucose. -- The consumption of glucose-galactose mixture (G-G group) don't induce postprandial hyperglycemia. However, galactosemia is about 250 mg p. 100 ml. In our conditions, glucose and galactose seem absorbed by two different systems. The galactose absorption would be favoured; otherwise, the absorbed glucose would be partly epimerized into galactose. Galatitolemia goes on 24 hours after the meal but it is not immediately modified by the galactose consumption. The galactosemia and galactitolemia variations are independent one of the others.

Galactose metabolism in male and female rats. II. Eye-ball differences.

When rats consumed a well-balanced diet containing 30% lactose or 30% glucose-galactose mixture, the biological composition of the lens was more disturbed in female: the leak of inositol and the accumulation of galactitol were higher than in the male.

[Biochemical mechanisms of the development of hereditary galactosemia in W/SSM strain rats]. / Issledovanie biokhimicheskikh mekhanizmov razvitiia nasledstvennoi galaktozemii u krys linii W/SSM.

The W/SSM rat strain with symptoms of inherited galactosemia (cataracts, hepatosplenomegaly, aminoaciduria etc.) was previously developed by selection and inbreeding of Wistar rats highly susceptible to the galactosemic effect of galactose. The decreased activity of galactose-1-phosphate uridyl transferase (Gal-1-PUT) in liver and erythrocytes is the salient biochemical feature of the strain. The crossing experiments have shown that the decrease in Gal-1-PUT activity is not a prerequisite for the expression of main galactosemia symptoms. The experiments excluded the low galactokinase activity and high susceptibility of glucoso-6-phosphate dehydrogenase and phosphoglucomutase to galactose-1-phosphate as probable causes of galactosemia. It was shown that the increased transport of 14C-galactose to erythrocytes is characteristic of the galactosemic rat strain. The intracellular accumulation of galactose concerned with its increased transport was assumed to be a major reason of the development of galactosemia symptoms in W/SSM rats. Genetic analysis has shown that lens lesions in galactosemic rats are controlled by one dominant gene. It is suggested that this gene is responsible for the enhanced transport of galactose into the rat cells and its accumulation in toxic concentrations. The main galactosemic symptoms, including cataracts, result obviously from the pleiotropic effect of this gene, while the decreased activity of Gal-1-PUT may be a consequence of its epistatic effect.

[A case of galactosemia caused by galactokinase deficiency]. / Su un caso di Galattosemia da deficit di Galattochinasi.

The authors describe an original case of galactokinase deficiency, born from a gypsies' family. He developed cataracts in the first two months of life. No other pathological features were observed. One of the brothers of the propositus was also blind for cataracts. The authors discuss the biochemical differences among the varieties of galactosaemia, and their relationship with the clinical differences.

[Elevated galactose transport into cells as the cause of development of hereditary galactosemia in rats]. / Povyshennyi transport galaktozy v kletki kak prichina razvitiia nasledstvennoi galaktozemii u krys.

A strain of rats with symptoms of inherited galactosemia (cataracts, hepatosplenomegaly, aminoaciduria etc) was produced by selection and inbreeding of Wistar rats highly susceptible to the galactosemic effect of galactose. The salient biochemical feature of these rats, like human galactosemics, is manifested as a decrease in the activity of galactose-I-phosphate uridyltransferase (Gal-I-PUT) in liver tissue and erythrocytes. However, the cross experiments have shown that the decrease in Gal-I-PUT activity was not required for expression of main galactosemia symptoms. Genetic analysis of cataract formation demonstrated that this trait was controlled by a single dominant gene. High transport rate of 14C-galactose into erythrocytes was a characteristic of galactosemic rats. Genetic analysis demonstrated that this trait was under the control of a single dominant gene, similar to the cataract formation. The intracellular accumulation of galactose ensured by its high transport, simultaneously with a decrease in Gal-I-PUT activity, were assumed to be the main reasons of galactosemic symptoms. The glucose transporter isolated from erythrocytes of the galactosemic rats, when integrated into the liposome membrane transferred more actively galactose into the liposomes than that of the control galactose resistant rats.

Oxidative stress contributes to outcome severity in a Drosophila melanogaster model of classic galactosemia.

Classic galactosemia is a genetic disorder that results from profound loss of galactose-1P-uridylyltransferase (GALT). Affected infants experience a rapid escalation of potentially lethal acute symptoms following exposure to milk. Dietary restriction of galactose prevents or resolves the acute sequelae; however, many patients experience profound long-term complications. Despite decades of research, the mechanisms that underlie pathophysiology in classic galactosemia remain unclear. Recently, we developed a Drosophila melanogaster model of classic galactosemia and demonstrated that, like patients, GALT-null Drosophila succumb in development if exposed to galactose but live if maintained on a galactose-restricted diet. Prior models of experimental galactosemia have implicated a possible association between galactose exposure and oxidative stress. Here we describe application of our fly genetic model of galactosemia to the question of whether oxidative stress contributes to the acute galactose sensitivity of GALT-null animals. Our first approach tested the impact of pro- and antioxidant food supplements on the survival of GALT-null and control larvae. We observed a clear pattern: the oxidants paraquat and DMSO each had a negative impact on the survival of mutant but not control animals exposed to galactose, and the antioxidants vitamin C and α-mangostin each had the opposite effect. Biochemical markers also confirmed that galactose and paraquat synergistically increased oxidative stress on all cohorts tested but, interestingly, the mutant animals showed a decreased response relative to controls. Finally, we tested the expression levels of two transcripts responsive to oxidative stress, GSTD6 and GSTE7, in mutant and control larvae exposed to galactose and found that both genes were induced, one by more than 40-fold. Combined, these results implicate oxidative stress and response as contributing factors in the acute galactose sensitivity of GALT-null Drosophila and, by extension, suggest that reactive oxygen species might also contribute to the acute pathophysiology in classic galactosemia.