SUBMITTED 1 MAR 22 1 REVISIONS REQ. 18 APR, 9 JUN & 30 JUN 22; REVISIONS RECD. 20 MAY, 23 JUN & 1 2 AUG 22 3 ACCEPTED 10 AUG 22 4 ONLINE-FIRST: AUGUST 2022 5 DOI: https://doi.org/ 10.18295/squmj.8.2022.052 6 7 Severe Neonatal Presentation of Progressive Familial Intrahepatic Cholestasis Type 4 in an 8 Omani Infant 9 Samira Al Housni,1 Khalid Al-Thihli,2 Dafalla Rahmatalla,3 10 Yasser Wali,3 *Yusriya Al Rawahi3 11 12 1 Pediatric Department, Oman Medical Specialty Board, Muscat, Oman; 2 Department of Genetics, Sultan 13 Qaboos University Hospital, Sultan Qaboos University, Muscat, Oman; 3 Child Health Department, 14 Sultan Qaboos University Hospital, Sultan Qaboos University, Muscat, Oman. 15 *Corresponding Author’s e-mail: yusria@squ.edu.om 16 17 Abstract 18 Progressive familial intrahepatic cholestasis type 4 (PFIC4) is a relatively newly described 19 autosomal recessive disorder caused by biallelic mutations in the gene encoding tight junction 20 protein 2 (TJP2) which is located in chromosome 9q21. PFIC4 is characterized by cholestasis 21 with or without other extrahepatic manifestations. Bleeding tendency due to vitamin k deficiency 22 is a well-known complication of cholestasis. We present a neonate who presented with 23 cholestasis and multiple intracranial bleeds. He was found to have severe coagulopathy and his 24 genetic work up revealed a homozygous variant mutation in TJP2 gene causing PFIC4. He had 25 persistent cholestasis that necessitated an internal biliary diversion with some clinical 26 improvement. 27 Keywords: Jaundice; Intracranial haemorrhage; Progressive Familial Intrahepatic Cholestasis 28 type 4 29 30 Introduction 31 Hereditary cholestasis is a group of rare autosomal recessive liver disorders, which are caused by 32 defects in genes related to the secretion and transport of bile salts and lipids. It is characterized 33 by intrahepatic cholestasis, pruritus, jaundice and malabsorption.1 Progressive familial 34 intrahepatic cholestasis (PFIC) is one of the phenotypic manifestations of hereditary cholestasis 35 with onset in early infancy that can progress to end-stage liver disease. It accounts for 10-15% of 36 the causes of cholestasis in pediatric patients and is the cause of 10-15% of liver transplants in 37 this population.1,2 PFIC types 1 and 2 usually present in infancy as infantile cholestasis 38 characterized by low to normal gamma-glutamyl transferase (GGT). However, PFIC type 3 39 presents in older children and it is associated with high GGT.3 With advancement and increasing 40 availability of genetic testing technologies rare types of PFIC are becoming recognized over the 41 past decade. 4 PFIC type 4 is a newly described clinical entity caused by biallelic mutations in 42 TJP2. The clinical spectrum of this condition has not been fully elucidated. We report a neonate 43 who presented with jaundice and severe coagulopathy at the age of 3 weeks and was found to 44 have a homozygous NM_004817.3:c.2417G>A, p.Trp806Ter,pathogenic variant in the TJP2 45 gene. 46 47 Case Report 48 A one-month-old boy presented to the Emergency Department at a tertiary care hospital with 49 one-week history of progressive jaundice, poor feeding, dark discoloration of the urine and 2 50 days history of irritability. There was no history of acholic stools, vomiting, fever or any 51 drug/herbal medicine intake. The patient was born to apparently healthy parents related as first 52 cousins. He was delivered at 36 weeks of gestation via normal vaginal delivery with birth weight 53 of 1.9 kg (< 3rd percentile), length of 47cm and head circumference of 31cm (<3rd percentile). 54 Mother had gestational diabetes mellitus (GDM). The patient has 2 healthy older siblings (Figure 55 1). There was no family history of unexplained death, liver disease, bleeding disorders, or 56 malignancy. 57 58 Physical examination revealed an irritable, pale infant with generalized icterus. His growth 59 parameters were below the third percentile (weight 2.5 kg, Z-score -2.9, and length 48 cm, Z-60 score -2.8). He had no dysmorphic features. His anterior fontanelle was full and pulsatile. His 61 pupils were equal and reactive to light. He had no focal neurological deficit. His abdominal 62 examination revealed a firm palpable liver 2 cm below the right costal margin. There was no 63 clinical splenomegaly or ascites. He had no cutaneous findings suggestive of bleeding tendency. 64 65 Investigations revealed severe anemia with hemoglobin 3.8 g/dl (10-14), high reticulocytes 5% 66 (0.2-2) and low hematocrit of 0.12 L/L (0.33-0.39). Lactate dehydrogenase (LDH) was elevated 67 at 782 U/L (120-300). Coagulation profile showed markedly prolonged PT and APTT with high 68 INR of > 17.4 (0.9-1.12). Liver chemistry demonstrated conjugated hyperbilirubinemia with 69 raised transaminases and normal gamma-glutamyl transferase (GGT). Total bilirubin was 237 70 umol/l (0-17) and 84% of it was conjugated, alanine aminotransferase (ALT) 79 U/L (normal 71 <40), aspartate aminotransferase (AST) 261U/L (normal <41), and GGT 36 U/L (normal <200) 72 (Table 1). Metabolic workup including, newborn metabolic screen, urine reducing substances, 73 ammonia and CK level were all normal. Investigations for infective and endocrine causes were 74 all negative. Brain magnetic resonant image (MRI) showed intracranial bleed with multiple 75 parenchymal, intraventricular and extra-axial hemorrhages. The liver appeared of normal size 76 and echotexture on ultrasound examination of the abdomen, and remained so on follow up 77 examination during the neonatal period. 78 79 The patient was intubated and mechanically ventilated and kept on brain protective measures. He 80 received packed red blood cells and fresh frozen plasma. He was also commenced on 81 intravenous vitamin K. Cefotaxime and ampicillin were initiated to cover the possibility of 82 infections. He developed a generalized tonic-clonic seizure and was started on phenobarbital. He 83 did not require any surgical intervention. His coagulation profile improved the following day and 84 he was extubated after 2 days. The intracranial bleeding was clinically attributed to a late onset 85 vitamin K deficiency with superimposed cholestatic liver disease. As the patient had normal 86 GGT and the initial work up for neonatal cholestasis were negative, PFIC and bile acid synthetic 87 defects were the main differential diagnosis. He underwent ultrasound guided liver biopsy, and 88 the histopathology revealed marked cholestasis with bile plugs along with feathery degeneration 89 and rosetting (Fig 2a &b). Whole exome sequencing revealed a homozygous 90 NM_004817.3:c.2417G>A, p.Trp806Ter pathogenic variant in in the TJP2 gene, consistent with 91 a diagnosis of PFIC 4. He was also found to have a heterozygous likely pathogenic c.1642G>T 92 (p.Glu548Ter) variant in ITGB3 gene (NM_000212.3). Parental heterozygosity for the variant in 93 TJP2 was confirmed. The variant in ITGB3 was proven to be paternally inherited. Biallelic 94 pathogenic variants in this gene are related to autosomal recessive Glanzmann thrombasthenia 95 type 2. 96 97 The patient was commenced on ursodeoxycholic acid and fat-soluble vitamin supplements. After 98 discharge, he continued taking ursodeoxycholic acid, fat-soluble vitamin supplements and 99 phenobarbital. He was kept on breastfeeds and medium-chain triglyceride-based formula. He 100 remains seizure free and the repeated electroencephalogram (EEG) was normal. At age of 9 101 months he underwent internal biliary diversion. When he was last assessed at the age of 11 102 months, he was able to cruise around objects, but still unable then to stand alone. He was able to 103 drink from a cup. He had monosyllables, and he recognized his siblings by their names. He had 104 no seizures. He remained clinically jaundiced with no pruritus. His weight was 5.4 Kg (Z-score -105 5), length was 64 cm (Z-score -3 SD). His liver chemistry has improved gradually (Table 1). 106 The family consented for publication of this case report. 107 108 Discussion 109 PFIC4 is among the most recently described forms of PFIC, and it is caused by mutations in the 110 tight junction protein-2 (TJP2) gene.5 So far, a few cases of PFIC4 have been reported 111 worldwide.4,6 To the best of our knowledge, this is the first report of an Arab patient with severe 112 neonatal presentation of PFIC4. 113 114 Truncating variants, as seen in the patient we describe, are known to be causative of TJP2-115 related PFIC4.7 A total of 15 nonsense variants have been described in TJP2 so far.4,8 Patients 116 with PFIC4 present with severe progressive cholestasis during infancy or early childhood. They 117 are also at a higher risk of acquiring hepatocellular carcinoma.8 Serum GGT activity is typically 118 normal or low. In addition to cholestasis, extrahepatic features have been identified in PFIC4 119 patients, including respiratory and neurological disorders.6 The mechanism of cholestasis in 120 PFIC 4 is due inappropriate function of the tight junction’s protein at the hepatocytes. That 121 results in leakage of cytotoxic bile salts into the paracellular space, causing damage to the 122 surrounding liver cells.9 The purpose of the biliary diversion surgery is to bypass the 123 enterohepatic circulation, thereby lowering the amount of bile salts that are reabsorbed by the 124 terminal ilium. These surgeries sometimes have led to improvement in some PFIC patients.9 The 125 patient we report so far has no extra-hepatic manifestations, and although the AFP and 126 ultrasonographic appearance of the liver are not suggestive of malignancy at present, the concern 127 about future development of hepatocellular carcinoma (HCC) in this child cannot be excluded. 128 Despite the small number of patients with disorder reported so far, age-dependent penetrance of 129 some mutations and notable clinical variabilities in some families have already been 130 recognized.10 131 132 The patient we report had a severe neonatal presentation with coagulopathy and multiple 133 intracranial bleeds. This maybe explained on the basis of cholestatic liver disease and vitamin K 134 deficiency, particularly owing to the drastic improvement in coagulopathy with the supportive 135 therapy and vitamin K administration. However, the possible contribution of the heterozygous 136 likely pathogenic variant identified in the ITGB3 gene to the severity of coagulopathy arguably 137 has some legitimate ground. Both dominant and recessive phenotypes associated with 138 coagulopathy have been described in relation to this gene.11-13 Although the variant identified 139 was inherited from an asymptomatic parent the possibility of this variant being dominant with 140 variable penetrance cannot be excluded. 141 142 Given the poorly defined risk of hepatocellular carcinoma and lack of reliable clinical predictors 143 of this complication among patients with PFIC4, the patient is under close follow up and 144 monitoring with low threshold for consideration of liver transplantation when clinically merited. 145 146 Conclusion 147 In summary, our patient is the first reported patient with PFIC 4 in the Arab population. This 148 case reports highlights few important points. First, for any neonate with normal GGT cholestasis, 149 PFIC is a potential differential diagnosis and PFIC4 is among the most recently described forms 150 of PFIC. Secondly, late onset vitamin K deficiency bleeding can be secondary to fat-soluble 151 vitamin malabsorption due to neonatal cholestasis. Thirdly, TJP2 gene mutation have been 152 reported to be associated with hepatocellular carcinoma, hence it is important to closely monitor 153 PFIC4 patients from this perspective. 154 155 Author Contribution 156 This manuscript has been contributed to, seen and approved by all the authors. All the authors 157 fulfill the authorship credit requirements. Samira Al Housni, Khalid Al-Thihli, Dafalla 158 Rahmatalla, Yasser Wali and Yusriya Al Rawahi wrote the first draft of this manuscript. Khalid 159 Al-Thihli, Yasser Wali and Yusriya Al Rawahi were involve in revising the manuscript. 160 161 References 162 1. Gaur K, Sakhuja P. 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A 198 novel variant Glanzmann thrombasthenia due to co-inheritance of a loss- and a gain-of-199 function mutation of ITGB3: evidence of a dominant effect of gain-of-function mutations. 200 Haematologica. 2018 Jun;103(6):e259-e263. doi: 10.3324/haematol.2017.180927 201 Table 1: The patient blood tests over 11 months period. 202 Biochemical parameter Reference value At admission Age 2 months Age 3 months Age 4 months Age 8 months Age 11 months Total bilirubin 0-17 umol/L 269 23 132 110 302 58 Direct bilirubin 0-4 237 203 122 99 81 56 ALT 0-41 U/L 79 488 111 63 207 102 AST 0-40 U/L 261 768 130 86 309 164 GGT < 203 U/L 36 47 35 36 27 31 INR 0.9-1.1 17.4 1.1 1.06 1.06 1.17 1.2 AFP 0-7 KIU/L 1934 ND ND ND 116 20 Albumin 38-54 g/L 28 32 39 42 39 33 Hemoglobin 10-14 g/dL 3.8 9.1 10.6 11.7 11.3 10.9 ALT, alanine transaminase; AFP; Alpha-fetoprotein, AST, aspartate transaminase; GGT, Gamma 203 glutamyl transferase; INR, international normalized ratio, ND; not done. 204 205 206 https://dx.doi.org/10.3748%2Fwjg.v26.i5.550 https://doi.org/10.1182/blood-2011-07-365635 https://doi.org/10.1182/blood-2011-07-365635 https://dx.doi.org/10.3324%2Fhaematol.2017.180927 207 Figure 1: Family Pedigree of the patient. 208 209 210 211 Figure 2A: H&E stain of the liver biopsy demonstrating cholestasis with bile plugs (green 212 arrow) along with feathery degeneration (red arrow) and rosetting. 213 214 215 216 Figure 2B: H&E stain of the liver biopsy demonstrating feathery degeneration (red arrow). 217