Kiyoshi Hayasaka

Congenital central hypoventilation syndrome (CCHS) is primarily caused by dominant PHOX2B mutations, with recessive LBX1 or MYO1H mutations being rare. Among PHOX2B mutations, polyalanine repeat expansion mutations (PARMs) are common, whereas non-PARMs (NPARMs) are less frequent. PHOX2B mutations are believed to act through loss-of-function mechanisms combined with dominant-negative and/or toxic gain-of-function effects. However, the role of PHOX2B haploinsufficiency remains unclear. We investigated the role of PHOX2B deletion and other genetic modifiers in CCHS. Among 93 patients without PHOX2B mutations, four were found to carry PHOX2B deletions via multiplex ligation-dependent probe amplification. Two had typical CCHS, whereas two siblings presented with mild sleep hypoventilation following CCHS symptoms in infancy. After ruling out pathogenic variants in LBX1 and MYO1H, we explored potential modifiers by analyzing sequence and methylation changes in the wild-type PHOX2B promoter and 3′ untranslated region (3′UTR), and the coding regions of PHOX2A and MIR204. One female patient with CCHS carried a 3′UTR haplotype predicted to reduce PHOX2B expression via MIR204 interaction. To date, 15 informative cases with PHOX2B deletions (eight males, seven females) have been reported. Respiratory phenotypes included: CCHS (n = 5), CCHS with obstructive sleep apnea (OSA) (n = 1), OSA alone (n = 2), mild central sleep apnea (n = 1), mild central sleep hypoventilation or apnea following CCHS symptoms in infancy (n = 3), and asymptomatic (n = 3). These indicate that although a heterozygous PHOX2B deficiency alone is insufficient to cause CCHS, it may delay or impair the development of respiratory control.

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.

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 9 months 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.

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 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.