Citrin deficiency (CD) is a hereditary disorder caused by SLC25A13 mutations that manifests as neonatal intrahepatic cholestasis caused by CD (NICCD), failure to thrive and dyslipidemia caused by CD (FTTDCD), and adult-onset type 2 citrullinemia (CTLN2). Citrin, an aspartate-glutamate carrier primarily expressed in the liver, is a component of the malate-aspartate shuttle, which is essential for glycolysis. Citrin-deficient hepatocytes have primary defects in glycolysis and de novo lipogenesis and exhibit secondarily downregulated PPARα, leading to impaired β-oxidation. They are unable to utilize glucose and free fatty acids as energy sources, resulting in energy deficiencies. Medium-chain triglyceride (MCT) supplements are effective for treating CD by providing energy to hepatocytes, increasing lipogenesis, and activating the malate-citrate shuttle. However, patients with CD often exhibit growth impairment and irreversible brain and/or liver damage. To improve the quality of life and prevent irreversible damage, MCT supplementation with a diet containing minimal carbohydrates is recommended promptly after the diagnosis.

Paucity of interlobular bile ducts (PILBD) is a heterogeneous disorder classified into two categories, syndromic and non-syndromic bile duct paucity. Syndromic PILBD is characterized by the presence of clinical manifestations of Alagille syndrome. Non-syndromic PILBD is caused by multiple diseases, such as metabolic and genetic disorders, infectious diseases, and inflammatory and immune disorders. We evaluated a family with a dominantly inherited PILBD, who presented with cholestasis at 1–2 months of age but spontaneously improved by 1 year of age. Next-generation sequencing analysis revealed a heterozygous CACYBP/SIP p.E177Q pathogenic variant. Calcyclin-binding protein and Siah1 interacting protein (CACYBP/SIP) form a ubiquitin ligase complex and induce proteasomal degradation of non-phosphorylated β-catenin. Immunohistochemical analysis revealed a slight decrease in CACYBP and β-catenin levels in the liver of patients in early infancy, which almost normalized by 13 months of age. The CACYBP/SIP p.E177Q pathogenic variant may form a more active or stable ubiquitin ligase complex that enhances the degradation of β-catenin and delays the maturation of intrahepatic bile ducts. Our findings indicate that accurate regulation of the β-catenin concentration is essential for the development of intrahepatic bile ducts and CACYBP/SIP pathogenic variant is a novel cause of PILDB.

The arachidonic acid (AA) cascade plays a significant role in platelet aggregation. AA released from membrane phospholipids is metabolized by cyclooxygenase (COX) pathway to thromboxane A2 (TXA2) or by 12S-lipoxygenase (ALOX12) to 12-hydroperoxyeicosatetraenoic acid (12-HPETE). In contrast to a well-known role of the COX pathway in platelet aggregation, the role of ALOX12 is not well understood. Platelets of ALOX12-deficient mice exhibit increased sensitivity for ADP-induced aggregation. However, recent evidence strongly suggests a significant role of ALOX12 in platelet aggregation and calcium signaling. 12-HPETE potentiates thrombin- and thromboxane-induced platelet aggregation, and calcium signaling. Inhibition experiments of ALOX12 demonstrated decreased platelet aggregation and calcium signaling in stimulated platelets. We studied a family with a dominantly inherited bleeding diathesis using next-generation sequencing analysis. Platelet aggregation studies revealed that the proband’s platelets had defective aggregation responses to ADP, TXA2 mimetic U46619, collagen, and AA, normal affinity of TXA2 receptor for U46619, and normal induction of GTPase activity upon stimulation with U46619. However, the production of inositol 1,4,5-triphosphate (IP3) was only increased up to 30% of the control upon U46619 stimulation, suggesting a defect in phospholipase C-β2 (PLCB2) activation downstream from TXA2 receptors. Affected family members had no mutation of PLCB2, but had a heterozygous c.1946A > G (p.Tyr649Cys) mutation of ALOX12. ALOX12 activity in platelets from the affected members was decreased to 25–35% of the control. Our data strongly suggested that a heterozygous c.1946A > G ALOX12 mutation was a disease-causing mutation; however, further experiments are required to confirm the pathogenesis of ALOX12 mutation in platelet aggregation.

Citrin deficiency is a hereditary disorder caused by SLC25A13 mutations and manifests as neonatal intrahepatic cholestasis (NICCD), failure to thrive and dyslipidemia (FTTDCD), and adult-onset type II citrullinemia (CTLN2). Citrin is a component of the malate-aspartate nicotinamide adenine dinucleotide hydrogen (NADH) shuttle, an essential shuttle for hepatic glycolysis. Hepatic glycolysis and the coupled lipogenesis are impaired in citrin deficiency. Hepatic lipogenesis plays a significant role in fat supply during growth spurt periods: the fetal period, infancy, and puberty. Growth impairment in these periods is characteristic of citrin deficiency. Hepatocytes with citrin deficiency cannot use glucose and fatty acids as energy sources due to defects in the NADH shuttle and downregulation of peroxisome proliferator-activated receptor α (PPARα), respectively. An energy deficit in hepatocytes is considered a fundamental pathogenesis of citrin deficiency. Medium-chain triglyceride (MCT) supplementation with a lactose-restricted formula and MCT supplementation under a low-carbohydrate diet are recommended for NICCD and CTLN2, respectively. MCT supplementation therapy can provide energy to hepatocytes, promote lipogenesis, correct the cytosolic NAD+ /NADH ratio via the malate-citrate shuttle and improve ammonia detoxification, and it is a reasonable therapy for citrin deficiency. It is very important to administer MCT at a dose equivalent to the liver's energy requirements in divided doses with meals. MCT supplementation therapy is certainly promising for promoting growth spurts during infancy and adolescence and for preventing CTLN2 onset. Intravenous administration of solutions containing fructose is contraindicated, and persistent hyperglycemia should be avoided due to glucose intoxication for patients receiving hyperalimentation or with complicating diabetes.

AIMS:Citrin is an aspartate/glutamate carrier that composes the malate-aspartate reduced nicotinamide adenine dinucleotide (NADH) shuttle in the liver. Citrin deficiency causes neonatal intrahepatic cholestasis (NICCD), failure to thrive and dyslipidemia (FTTDCD) and adult-onset type II citrullinemia (CTLN2). Hepatic glycolysis is essentially impaired in citrin deficiency and a low-carbohydrate diet was recommended. The lethal effect of infusion of glycerol- and fructose-containing osmotic agents was reported in these patients. Hyperalimentation was also reported to exacerbate CTLN2; however, glucose toxicity was unclear in citrin deficiency.METHODS:We studied two CTLN2 patients complicated with type 2 diabetes mellitus (DM), Case 1 presented with hyperammonemic encephalopathy accompanied with DM, while Case 2 presented with hyperammonemic encephalopathy relapse upon the onset of DM after several years' remission following supplementation with medium-chain triglycerides (MCT) and adherence to a low-carbohydrate diet.RESULTS:Insulin therapy with MCT supplementation and a low-carbohydrate diet improved hyperammonemia and liver function in Case 1. Additional insulin therapy improved hyperammonemia in Case 2.CONCLUSION:Glucose is not toxic for citrin deficiency in normoglycemia because glucose uptake and metabolism by hepatocytes are limited in normoglycemia. However, glucose becomes toxic during persistent hyperglycemia and antidiabetic therapy is indispensable for CTLN2 patients with DM.

Congenital central hypoventilation syndrome (CCHS) is caused by the paired-like homeobox 2B (PHOX2B) mutation and predominantly diagnosed during the neonatal period. Although late-onset CCHS and PHOX2B mutation carriers have been reported, the features of these disease states in adults remain uncertain. This study aimed to identify the characteristics of adult-onset CCHS and PHOX2B-mutation carriers in adult. We mainly searched the PubMed/Medline and Cochrane Databases and classified our target patients into 2 groups: group A, symptomatically diagnosed with late-onset CCHS in adulthood; group B, adult PHOX2B-mutation carriers. Then, clinical characteristics, including the onset, treatment, long-term course, and pattern of the PHOX2B mutation in both groups were analyzed. Additionally, a new adult-case of late-onset CCHS was added to the analysis. Group A was comprised of 12 patients. The onset triggers of illness included a history of respiratory compromise following general anesthesia and respiratory tract infections. All patients in group A had 20/25 polyalanine repeat mutations and required some chronic ventilatory support at least during sleep, including portable positive pressure ventilator via tracheostomy or noninvasive positive pressure ventilation. In these patients with ventilatory support during sleep, sudden death or poor prognosis was not reported. Group B was comprised of 33 adults from 24 families with PHOX2B mutations. Nine patients in group B were confirmed with the diagnosis of CCHS. Although polyalanine repeat mutations 20/25 represented the most common gene mutation, diverse mutations, including mosaicism, were observed. Hypoventilation of several cases in group B were underdiagnosed by overnight polysomnography without monitoring for CO2. Alveolar hypoventilation with unknown origin can be caused by the PHOX2B mutation even in adult cases. Both the identification of the PHOX2B mutation and the incorporation of capnography in polysomnography are important for adult cases with unexplained alveolar hypoventilation or asymptomatic mutation carriers. Hino A, Terada J, Kasai H, et al. Adult cases of late-onset congenital central hypoventilation syndrome and paired-like homeobox 2B-mutation carriers: an additional case report and pooled analysis. J Clin Sleep Med. 2020;16(11):1891–1900.

INTRODUCTION:Mutations in genes encoding nucleoporins (NUPs; components of nuclear pore complexes [NPCs]), such as NUP93, have been reported to cause steroid-resistant nephrotic syndrome (SRNS) or focal segmental glomerulosclerosis (FSGS), which often progresses to end-stage renal disease (ESRD) in childhood. The expression of NUP93 in renal or extrarenal tissues, and the mechanism by which NUP93 mutations cause this renal phenotype, remain unclear.METHODS:The expression of NUP93 in normal control kidney and in a patient with FSGS carrying NUP93 mutations was examined by immunofluorescence analysis. The expression of NUP93 in blood cells was analyzed by Western blot analysis.RESULTS:Immunofluorescence analysis detected NUP93 expression in nuclei of all glomerular and tubulointerstitial cells in human kidneys. Whole-exome sequencing identified a compound heterozygous NUP93 mutation comprising a novel missense mutation p.Arg525Trp, and a previously reported mutation, p.Tyr629Cys, in a patient with FSGS that developed ESRD at the age of 6 years. In the patient's kidney, the intensity of NUP93 immunofluorescence was significantly decreased in the nuclei of both glomerular and extraglomerular cells. The expression of CD2-associated protein (CD2AP) and nephrin in the patient's podocytes was relatively intact. The amount of NUP93 protein was not significantly altered in the peripheral blood mononuclear cells of the patient.CONCLUSION:NUP93 is expressed in the nuclei of all the cell types of the human kidney. Altered NUP93 expression in glomerular cells as well as extraglomerular cells by NUP93 mutations may underlie the pathogenic mechanism of SRNS or FSGS.

Citrin deficiency causes neonatal intrahepatic cholestasis (NICCD), failure to thrive and dyslipidemia (FTTDCD), and adult-onset type II citrullinemia (CTLN2). Owing to a defect in the NADH-shuttle, citrin deficiency impairs hepatic glycolysis and de novo lipogenesis leading to hepatic energy deficit. To investigate the physiological role of citrin, we studied the growth of 111 NICCD-affected subjects (51 males and 60 females) and 12 NICCD-unaffected subjects (five males and seven females), including the body weight, height, and genotype. We constructed growth charts using the lambda-mu-sigma (LMS) method. The NICCD-affected subjects showed statistically significant growth impairment, including low birth weight and length, low body weight until 6 to 9months of age, low height until 11 to 13 years of age, and low body weight in 7 to 12-year-old males and 8-year-old females. NICCD-unaffected subjects showed similar growth impairment, including low birth weight and height, and growth impairment during adolescence. In the third trimester, de novo lipogenesis is required for deposition of body fat and myelination of the developing central nervous system, and its impairment likely causes low birth weight and length. The growth rate is the highest during the first 6 months of life and slows down after 6 months of age, which is probably associated with the onset and recovery of NICCD. Adolescence is the second catch-up growth period, and the proportion and distribution of body fat change depending on age and sex. Characteristic growth impairment in citrin deficiency suggests a significant role of citrin in the catch-up growth via lipogenesis.

Congenital central hypoventilation syndrome is a disorder of respiratory control caused by mutations in the paired-like homeobox 2B gene. Mutations in the paired-like homeobox 2B gene are also responsible for Hirschsprung's disease. Variant Hirschsprung's disease is a rarer disorder that does not meet the diagnostic criteria of Hirschsprung's disease, although severe functional bowel obstruction persists. We present a case of an extremely low birth weight infant with congenital central hypoventilation syndrome and variant Hirschsprung's disease. A male infant who was diagnosed to have fetal growth restriction and polyhydramnios was delivered by emergency cesarean section at 30 weeks and 3 days of gestational age due to non-reassuring fetal status. The birth weight was 979 g, and intensive care was started immediately following delivery. The patient exhibited refractory apnea and was diagnosed with congenital central hypoventilation syndrome by genetic testing of the paired-like homeobox 2B gene. The patient also exhibited refractory functional bowel obstruction and was diagnosed to have variant Hirschsprung's disease through pathological examination of his intestinal specimens. The patient grew slowly but surely with intensive care including mechanical ventilation and parenteral nutrition. However, the patient repeatedly suffered from sepsis and died of fungemia at 197 days of age. This is the first congenital central hypoventilation syndrome case that was accompanied with variant Hirschsprung's disease, and the paired-like homeobox 2B mutation detected in this case (NM_003924.3: c.441G > C; p. (Gln147His)) is novel. This case suggests that the paired-like homeobox 2B mutation causes not only congenital central hypoventilation syndrome and Hirschsprung's disease, but also variant Hirschsprung's disease in humans. It also highlights the extreme difficulty in treating premature infants with severe and prolonged functional bowel obstruction.

BACKGROUND:Congenital central hypoventilation syndrome (CCHS) is caused by mutation of paird-like homeobox 2B (PHOX2B). Approximately 90% of patients were found to carry polyalanine repeat expansion mutation (PARM), and the remaining 10% had non-PARM (NPARM). In PARM, the length of the polyalanine expansion correlates with clinical disease severity. Most patients with NPARM have hypoventilation symptoms in the neonatal period and complications of Hirschsprung disease, dysregulation of autonomic nervous system, and tumors of neural crest origin. Data on the genotype-phenotype association may contribute to the clinical management of the disease.METHODS:We studied the genetic background of Japanese CCHS patients according to PHOX2B sequencing.RESULTS:Of 133 Japanese CCHS patients we identified 12 patients carrying 11 different NPARM (approx. 9% of the patients) and described the clinical manifestations in seven of them with the following novel mutations: c.941-945del5, c.678_693dup16, c.609_616del8, c.620_633del14, c.663_711del 49, c.448C>G and c.944G>C. All patients had hypoventilation in the neonatal period and also had Hirschsprung disease, with the exception of two patients carrying c.620_633del14 and c.663_711del49 mutations. The patient carrying the c.609_616del8 mutation also had a benign mediastinal tumor.CONCLUSION:Most patients carrying NPARM had severe symptoms with frequent complications, as in previous reports, and should be carefully monitored for various complications, including neural crest-derived tumor.

Citrin deficiency is a recessively inherited metabolic disorder with age-dependent clinical manifestations. It causes neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2). Patients with NICCD present with intrahepatic cholestasis in the neonatal period and usually respond to the treatment with medium-chain triglyceride (MCT) supplement and lactose-restricted formula. In adulthood, CTLN2 develops in <10 % of the patients showing hyperammonemic encephalopathy. Patients with CTLN2 required liver transplantation for the most promising prognosis; however, they were successfully treated with MCT supplement with a low carbohydrate formula. Citrin deficiency is caused by mutations in SLC25A13 on chromosome 7q21.3, with a high frequency in East Asia, including Japan. Citrin is aspartate/glutamate transporter in mitochondria, a component of malate-aspartate nicotinamide adenine dinucleotide hydrogen shuttle, and is essential for the hepatic glycolysis. Although the precise pathophysiology of citrin deficiency remains unclear, recent reports for the effective MCT supplement therapy and downregulation of peroxisome proliferator-activated receptor α suggest that citrin deficiency impairs hepatic de novo lipogenesis coupled with glycolysis leading to the energy deficit of hepatocytes. Herein, we review the current therapeutic and pathological understanding of CTLN2.

Citrin, encoded by SLC25A13, constitutes the malate-aspartate shuttle, the main NADH-shuttle in the liver. Citrin deficiency causes neonatal intrahepatic cholestasis (NICCD) and adult–onset type II citrullinemia (CTLN2). Citrin deficiency is predicted to impair hepatic glycolysis and de novo lipogenesis, resulting in hepatic energy deficit. Secondary decrease in hepatic argininosuccinate synthetase (ASS1) expression has been considered a cause of hyperammonemia in CTLN2. We previously reported that medium–chain triglyceride (MCT) supplement therapy with a low–carbohydrate formula was effective in CTLN2 to prevent a relapse of hyperammonemic encephalopathy. We present the therapy for six CTLN2 patients. All the patients’ general condition steadily improved and five patients with hyperammonemic encephalopathy recovered from unconsciousness in a few days. Before the treatment, plasma glutamine levels did not increase over the normal range and rather decreased to lower than the normal range in some patients. The treatment promptly decreased the blood ammonia level, which was accompanied by a decrease in plasma citrulline levels and an increase in plasma glutamine levels. These findings indicated that hyperammonemia was not only caused by the impairment of ureagenesis at ASS1 step, but was also associated with an impairment of glutamine synthetase (GS) ammonia-detoxification system in the hepatocytes. There was no decrease in the GS expressing hepatocytes. MCT supplement with a low–carbohydrate formula can supply the energy and/or substrates for ASS1 and GS, and enhance ammonia detoxification in hepatocytes. Histological improvement in the hepatic steatosis and ASS1-expression was also observed in a patient after long-term treatment.

Inherited GPI (glycosylphosphatidylinositol) deficiencies (IGDs), a recently defined group of diseases, show a broad spectrum of symptoms. Hyperphosphatasia mental retardation syndrome, also known as Mabry syndrome, is a type of IGDs. There are at least 26 genes involved in the biosynthesis and transport of GPI-anchored proteins; however, IGDs constitute a rare group of diseases, and correlations between the spectrum of symptoms and affected genes or the type of mutations have not been shown. Here, we report four newly identified and five previously described Japanese families with PIGO (phosphatidylinositol glycan anchor biosynthesis class O) deficiency. We show how the clinical severity of IGDs correlates with flow cytometric analysis of blood, functional analysis using a PIGO-deficient cell line, and the degree of hyperphosphatasia. The flow cytometric analysis and hyperphosphatasia are useful for IGD diagnosis, but the expression level of GPI-anchored proteins and the degree of hyperphosphatasia do not correlate, although functional studies do, with clinical severity. Compared with PIGA (phosphatidylinositol glycan anchor biosynthesis class A) deficiency, PIGO deficiency shows characteristic features, such as Hirschsprung disease, brachytelephalangy, and hyperphosphatasia. This report shows the precise spectrum of symptoms according to the severity of mutations and compares symptoms between different types of IGD.