Case report: Novel DGUOK variants associated with idiopathic non-cirrhotic portal hypertension in a Han Chinese child

DGUOK deficiency has primarily been associated with lethal hepatic failure with or without hypotonia, nystagmus, and psychomotor retardation, features typical of mitochondrial disease. A study in 3 Turkish children identified homozygosity for a variant in DGUOK as associated with idiopathic non-cirrhotic portal hypertension (INCPH). However, no further instances of INCPH associated with DGUOK variants have been reported. We here describe a fourth patient with DGUOK variants and childhood-onset INCPH, a 12-year-old Han Chinese boy, reporting clinical manifestations, histopathologic findings, and results of genetic studies. The child presented with hepatosplenomegaly; portal hypertension and hypersplenism were found. Vascular changes with hepatic fibrosis (Scheuer score 3) were observed on liver biopsy. Whole-exome sequencing and family analyses revealed compound heterozygosity for the DGUOK (NM_080916.3) variants c.778_781dup, (p.Thr261Serfs*28) and c.831_832del, (p.*278Thrfs*9) in the proband. These observations support ascription of instances of INCPH in children to variation in DGUOK.


Introduction
Deoxyguanosine kinase, encoded by the nuclear gene DGUOK (1), mediates the first step in the phosphorylation of purine nucleosides in the mitochondrial matrix and is essential for the purine nucleoside salvage pathway (2,3).Variants in DGUOK lead to impaired synthesis of mitochondrial dNTPs, resulting in decreased levels of mitochondrial DNA and depletion of mitochondrial DNA (2).More than 100 individuals with DGUOK-related mitochondrial DNA depletion syndrome (MDS) have been reported thus far (4).The typical characteristics of this disorder include significant hepatic failure with or without hypotonia, nystagmus, and psychomotor retardation (2,5).
Three children from 2 Turkish kindreds with the recurrent recessive homozygous p.N46S mutation in DGUOK, leading to idiopathic non-cirrhotic portal hypertension (INCPH), are described (6).INCPH is characterized by intrahepatic portal hypertension when cirrhosis and other causes of liver disease or splanchnic venous thrombosis are absent (7).Findings in the 3 Turkish children thus expanded the phenotypic spectrum of DGUOK deficiency and suggested a new cause of INCPH.However, no further cases have been reported.We here describe a fourth patient, a 12-year-old Han Chinese boy, in whom pediatric-onset INCPH is associated with DGUOK variation.

Case description Patient
The proband was a 12-year-old boy, the first child of a nonconsanguineous healthy Han Chinese couple.He was born vaginally at term (3,150 g) following an uncomplicated pregnancy.Early growth and development were unremarkable.On examination at age 4 years 9 months, occasioned by bronchopneumonia, hepatosplenomegaly was noted incidentally (liver edge 3.3 cm and spleen tip 3.2 cm below costal margin).Aside from a slightly elevated alpha-fetoprotein value [183 ng/ml; reference 0-13.6],laboratory results were within expected ranges (Table 1).Abdominal sonography and computerized tomography found only nonspecific liver heterogeneity.Laboratory findings during an episode of upper respiratory infection, aged 8 years, were within expected ranges.Aged 12 years, during another bout of respiratory-tract infection, leukopenia and thrombocytopenia were identified, with mild unconjugated hyperbilirubinemia and slight hypocoagulability.Aside from slightly elevated serum γglutamyl transpeptidase activity, values for biomarkers of hepatobiliary injury and hepatic synthetic function were within expected ranges (Table 1).Abdominal contrast-enhanced computerized tomography imaging found enlargement of the spleen, accompanied by thickening of the splenic meridians and varicose gastric veins, indicating portal hypertension (Supplementary Figure S1A).The patient's parents refused determination of portal venous system and inferior vena cava pressures.Esophageal and gastric varices were not found on endoscopy (Supplementary Figure S1B).Liver stiffness, measured using FibroScan, was 14.6 kPa (reference value: <7.3 kPa; fibrosis stage 3-4) (8).Bone marrow biopsy found hyperplasia with normal megakaryocytes.After liver biopsy (v.i.), the patient was referred in consultation.Repeat evaluation found that normal growth and development were normal, with height 165 cm, 2 standard deviations above the mean, and weight 52.9 kg, 1-2 standard deviations above the mean.Hepatosplenomegaly was found (liver edge 2 cm and spleen tip 4 cm below costal margin), without other feature of note.No abnormality was identified on specialist neurological examination.Routine tests of urine and feces found no abnormality.A reticulocyte count yielded normal results.Values for serum creatine phosphokinase activity and for biomarkers of thyroid function were within expected ranges, as were those for glucose, lactate, ketones, triglycerides, blood ammonia, cholesterol, homocysteine, ferritin, folic acid, ceruloplasmin, complement 3, complement 4 and CD series.The distribution and amounts of urinary organic acids and plasma amino acids were normal on assessment by mass spectrophotometry.No serologic evidence was found for infection by hepatotropic viruses, Epstein-Barr virus, toxoplasma, rubella, cytomegalovirus, herpes, human immunodeficiency virus, syphilis, or mycoplasma.Autoantibodies could not be demonstrated (anti-smooth muscle, anti-dsDNA, anti-liver kidney microsomal, anti-soluble liver pancreas, anti-cytosolic, anti-tissue transglutaminase) except for anti-nuclear antibody (positive at 1:160 titer).No abnormalities were found on cardiac sonography, Wechsler intelligence scale testing, brain magnetic resonance imaging, or electroencephalography.The boy's sister, aged 5 years, is without abnormality on physical examination and clinicallaboratory assessment; findings on abdominal sonography were normal.Questioning elicited no history of neurologic or hepatobiliary disease in the proband's family and relatives.

Liver biopsy
An ultrasound-guided percutaneous liver puncture was performed when the patient was 12 years old.Figure 1 and Supplementary Figure S2 show vascular changes with hepatic fibrosis (Staging 3, Scheuer's system) (9).However, cirrhosis was not present.Dilatation of hepatic sinusoids with irregular blood vessels also was seen.Some hepatocytes showed mild swelling with small fat droplets/microsteatosis. Portal tracts were not inflamed.
No other pathogenic variants consistent with the mode of inheritance were identified.

Discussion
DGUOK deficiency has primarily been associated with clinical features that typify mitochondrial diseases, with liver, neurological, and muscular systems involvement.In most cases, DGUOK related    (14).The liver stiffness value was relatively higher in our patient (14.6 kPa).The value of hepatic pressure gradient was not performed as rejected by the child's parents.Studies with more cases are still needed to evaluate the values of liver stiffness and hepatic pressure gradient in DGUOK related INCPH.Liver biopsy found architectural changes that comported with INCPH.This prompted genetic studies that identified compound heterozygosity for 2 unreported DGUOK variants, both predicted to yield frameshifts.As with the 3 Turkish children (Table 2), illness was clinically mild and progression was slow.Factors affecting penetrance of DGUOK disease remain to be identified.
Isolated liver involvement has been found in our patient as well as in the 3 Turkish children reported previously (6).Characteristic histopathologic abnormalities on liver biopsy in DGUOK-related MDS include cholestasis, microsteatosis, giant cell hepatitis, fibrosis, and cirrhosis (5, 11-13, 15-17).Unlike DGUOK-related MDS, liver biopsy in the 3 Turkish children found subtle vascular changes in the absence of significant cirrhosis (6), including portal changes with irregular portal-venule profiles, lumen narrowing, smooth-muscle proliferation, and mural fibrosis.One patient had mild focal microsteatosis and hepatocyte swelling, while another patient showed sinusoidal dilatation and mild chronic lymphocytic infiltration in portal tracts without significant interface activity.Our patient had mild focal hepatocyte swelling and microsteatosis with sinusoidal dilatation and subtle vascular changes with fibrosis but without cirrhosis (Scheuer's score 3), suggesting a chronic liver disease with concomitant signs of "INCPH".
The diagnosis of INCPH is mainly based on the presence of portal hypertension in the absence of cirrhosis or other causes of non-cirrhotic portal hypertension, and include the histologic  (7,20,21).On the limited basis of the 4 cases identified to date, DGUOK-related INCPH (MIM617068) seems to be an autosomal recessive disorder characterized by the onset of portal hypertension and hepatosplenomegaly in the first or second decades of life with no extrahepatic manifestations aside from hypersplenism.The exact pathogenesis of DGUOK-related INCPH is unknown.However, the disease is relatively benign, with slow disease progression, unlike typical MDS.
The two frameshift variants identified in the patient were novel.Each lies near the end of the coding region; each is predicted to extend the coding sequence and to produce an elongated protein, changes that may affect both protein function and mitochondrial metabolism.Only homozygosity for the c.137A > G variant in DGUOK predicted to yield the substitution p.N46S has before been associated with INCPH.However, homozygosity for the same variant was also associated with cirrhosis and liver failure in an infant aged 10 months (22).In 4 infants who were compound heterozygotes for that variant in DGUOK and another, severe liver dysfunction with variable progression was observed (5, [22][23][24], with one undergoing liver transplantation at an early age.As the variants in our patient both are novel, conclusions on their behavior in other settings and combinations are premature.It is not known whether the two novel variants might be associate with mild liver involvement or not.However, the link between genotype and phenotype in DGUOK disease appears complex.
We have above described a Han Chinese child whose INCPH/ PVSD we attribute to novel variants in DGUOK.His case provides new evidence that DGUOK variants may be associated with INCPH/PVSD in children.

FIGURE 1
FIGURE 1 The pathology of current patient at age of 12 years.(A) H&E staining indicated mild hepatocyte swelling (arrow) and areas of mild steatosis with mild sinusoidal dilatation around central vein (circle) with no inflammation or cirrhosis.(B) Masson staining showed fibrosis (Scheuer score 3).(C) Reticular fiber staining showed the exist of reticular scaffold structure without obvious collapse.(D) CD34 antibody marks the irregular outlines of the portal venules and narrow lumen.

TABLE 2
Clinical features and molecular genetics of individuals with INCPH associated with variants in DGUOK.