64 NICCD cases were confirmed genetically and/or with Western blot analysis. Among of them, 56 cases were genetically confirmed, including 21 cases with homozygous mutation, and 35 cases with compound heterozygous mutation in SLC25A13 gene. Eight cases with single heterozygous mutation were confirmed by absence of citrin protein by western blot analyses on liver sample (4 cases) or skin fibroblast (4 cases). Apart from one case born to fourth-generation cousins, no consanguineous marriages were found from parents of other cases. Up to the time of preparation of this manuscript, 54 patients have survived without liver transplant, nine cases have died, and 1 lost to follow-up. After excluding 1 case that lost to follow-up and 2 infants with insufficient follow-up data, 61 confirmed NICCD cases, including 52 cases (30 males, and 22 females) with available data in the survival group, and nine cases (seven males, and two females) in the mortality group, were included in the final analysis.
SLC25A13 gene mutation, age at referral, diagnosis, administration of LF/MCT formula, and age of death in nine deceased cases were recorded in Table
1. All cases with poor prognosis had homozygous or compound heterozygous deleterious mutations such as insertion/deletion or splice site mutations. Most cases (seven out of nine) were referred to our center after 6-months of age, while two cases presented after 1-year of age. LF/MCT formula was started after referral in 5 cases, 4 cases did not respond to dietary change, but one case suffered from unexplained death after infection while liver function was improving. Four cases did not receive LF/MCT formula due to non-adherence or atypical presentation. The youngest age of death was five-months, while the oldest child died at the age of 23 months. Eight cases died from liver failure, while one case suffered from unexplained death after infection. Five cases had evidence of infection prior to death, one infant had concomitant kidney failure, while another child had interstitial lung disease and brain MRI abnormality.
Table 1
Characteristics of deceased cases
1 | 851del4/1638ins23 | 6 | Cirrhosis, liver failure | No | 8 | Liver failure |
2 | 851del4/IVS16ins3kb | 9 | Liver failure, hepatosplenomegaly, bile sludge, ascites | No | 9.5 | Liver failure |
3 | 851del4/IVS16ins3kb | 5 | Liver failure | No | 6 | Liver failure |
4 | 851del4/851del4 | 4 | cholestasis | Yes | 5 | Infection, sudden death |
5 | 851del4/851del4 | 19 | cholestasis | Yes | 23 | Liver failure, infection |
6 | 851del4/IVS6 + 5G > A | 6 | cholestasis | Yes | 9 | Liver failure, infection |
7 | 851del4/IVS16ins3kb | 11 | Liver failure, hepato-renal syndrome | No | 11 | Liver failure, kidney failure |
8 | 851del4/1638ins23 | 6 | cholestasis | Yes | 6 | Liver failure, recurrent infection |
9 | 851del4/1638ins23 | 13 | Liver failure, hepatosplenomegaly, | Yes | 13 | Liver failure, diarrhea, suspected interstitial lung disease, brain MRI abnormality |
Risk factors associated with mortality
Sixty-one cases, including 52 children with available data in the survival group without liver transplantation, and 9 cases in the mortality group, were included in the final analysis. Gender, birth weight, age at referral, and blood test results at referral (complete blood count, serum biochemistry, blood coagulation profile, tandem mass spectrometry, and genetic test results) were compared to explore the risk factors associated with NICCD mortality (Table
2).
Table 2
Comparison of clinical and laboratory data between survival group and mortality group
General Information | Gender (Male/Female) | 30/22 | 7/2 | 0.462 |
Birth weight (g) | 3079.32 ± 694.17(44) | 2828.57 ± 360.39(7) | 0.351 |
Age at referral (months) | 3.96 ± 3.13(48) | 9.58 ± 5.03(8) |
0.000
|
Presence/absence of infection (n) | 29 /21 | 7/1 |
0.023
|
Lactose-free and/or MCT-enriched formula (Yes/No) | 47/5 | 5/4 |
0.021
|
Complete blood count | WBC (4–10 *10^9/L) | 10.34 ± 4.91(30) | 9.90 ± 2.7(5) | 0.637 |
RBC (3.5–5.5 *10^12/L) | 3.34 ± 0.66(23) | 2.94 ± 0.87(5) | 0.255 |
PLT (100–300 *10^9/L) | 387.54 ± 196.46(28) | 109.60 ± 19.26(5) |
0.010
|
Hemoglobin (110–160 g/L) | 93.81 ± 22.40(29) | 87.40 ± 22.63 (5) | 0.342 |
Serum Biochemistry | Total Biliburin (5.1–17.1 umol/L) | 117.86 ± 65.73(47) | 195.10 ± 194.99(8) | 0.503 |
Direct Bilirubin (0–6 umol/L) | 70.82 ± 39.48(47) | 113.69 ± 99.46(8) | 0.474 |
ALT (0–40 IU/L) | 44.51 ± 71.35(47) | 45.25 ± 25.44(8) | 0.148 |
AST (0–40 IU/L) | 98.41 ± 96.59(46) | 100.75 ± 60.47(8) | 0.450 |
GGT (7–50 IU/L) | 219.32 ± 127.59(47) | 80.00 ± 69.70(8) |
0.001
|
GGT (<=50 vs. > 50 IU/L) | 1/46 | 4/4 |
0.001
|
Total bile acid (0–10 ummol/L) | 185.47 ± 84.47(46) | 140.88 ± 124.80(8) | 0.206 |
Total protein (60–83 g/L) | 49.45 ± 9.56(45) | 49.84 ± 6.38(8) | 0.914 |
Albumin (35–55 g/L) | 32.74 ± 9.93(45) | 30.89 ± 6.05(8) | 0.205 |
Glucose (3.9–5.8 mmol/L) | 2.84 ± 1.28(46) | 3.36 ± 1.38(8) | 0.318 |
Total cholesterol (3.1–5.2 mmol/L) | 3.30 ± 1.06(41) | 2.25 ± 1.03(7) |
0.019
|
Triglyceride (0.56–1.70 mmol/L) | 1.48 ± 0.65(40) | 1.39 ± 0.69(8) | 0.571 |
Urea (2.5–6.5 mmol/L) | 4.02 ± 4.27(27) | 2.91 ± 1.10(5) | 0.815 |
Creatinine (20–110 umol/L) | 20.28 ± 14.00(29) | 15.51 ± 5.51(8) | 0.271 |
Lactic acid (0.7–2.1 mmol/L) | 3.63 ± 2.76(15) | 3.98 ± 1.22(5) | 0.708 |
Serum ammonia (10–47 umol/L) | 98.14 ± 45.20(34) | 142.31 ± 42.09(8) |
0.016
|
Alphafetoprotein (0–28 ng/ml) | 15,473.97 ± 25,750.21(26) | 60,476.24 ± 126,197.60(5) | 0.823 |
Blood Coagulation Profile | INR (0.8–1.2) | 1.41 ± 0.38(34) | 1.60 ± 0.58 (7) | 0.510 |
PT (12.0–14.8 s) | 17.51 ± 6.35 (37) | 18.37 ± 6.18 (7) | 0.712 |
PTA (80–120%) | 71.75 ± 25.75 (28) | 68.57 ± 34.40 (7) | 0.786 |
APTT (28.0–44.5 s) | 45.63 ± 13.29 (37) | 53.09 ± 12.56 (7) | 0.177 |
Fib (2–4 g/L) | 2.04 ± 3.34 (32) | 1.74 ± 1.05 (7) | 0.656 |
Thrombin Time (14–21 s) | 21.26 ± 4.15 (38) | 21.66 ± 3.66 (7) | 0.817 |
Blood tandem mass spectrometry | Citrulline (7–40 umol/L) | 137.06 ± 79.62 (31) | 52.34 ± 19.14 (4) |
0.010
|
Methionine (10–80 umol/L) | 157.26 ± 100.04 (23) | 231.93 ± 324.79 (5) | 0.787 |
Tyrosine (30–200 umol/L) | 130.00 ± 73.19 (21) | 250.26 ± 77.78 (3) |
0.015
|
Threonine (17–90 umol/L) | 130.67 ± 51.62 (20) | 228.30 ± 212.56 (2) | 0.732 |
SLC25A13 gene mutation allele frequency | 851del4 | 55 (104) | 11 (18) | 0.518 |
1638ins23 | 12 (104) | 3 (18) | 0.541 |
IVS6 + 5G > A | 6 (104) | 1 (18) | 1.000 |
IVS16ins3kb | 6 (104) | 3 (18) | 0.128 |
Distribution of gender and mean birth weight were not significantly different between the survival group and the mortality group (p values were 0.462 and 0.351, respectively). On the other hand, mean age at referral in the mortality group (9.58 ± 5.03 months) was significantly higher when compared to the survival group (3.96 ± 3.13 months, p < 0.000). Significantly more children (87.5%) in the mortality group had infection when compared to the survival group (58.0%, p = 0.023). 44% (4/9) of patients in the mortality group did not receive lactose-free and/or MCT-enriched formula, and this percentage was significantly higher than that of the survival group (9.6%, p = 0.021).
White blood cell (WBC) count, red blood cell (RBC) count, and hemoglobin levels were similar between the survival group and the mortality group (p values were 0.637, 0.255, and 0.342, respectively). However, PLT in the mortality group (109.60 ± 19.26*10^9/L) was significantly lower than that of the survival group (387.54 ± 196.46*10^9/L, p = 0.010).
Mean serum GGT level in the mortality group (87.43 ± 71.78 IU/L) was significantly lower than that of the survival group (223.37 ± 125.91 IU/L, p = 0.001). Mean serum Total cholesterol level in the mortality group (2.12 ± 1.19 mmol/) was significantly reduced in the mortality group, while remained within normal range (3.47 ± 0.97 mmol/L, p = 0.012) in patients from the survival group. Children with poor prognosis had similar total bilirubin (195.10 ± 194.99 umol/L) and direct bilirubin (113.69 ± 99.46 umol/L) levels when compared to those who survived (117.86 ± 65.73, and 70.82 ± 39.48 umol/L, p values were 0.503 and 0.474, respectively). Significantly more patients in the morality group had normal serum GGT levels when compared to the survival group (4/8 VS 1/47, p = 0.001). On the other hand, mean serum ammonia level in deceased children (142.31 ± 42.09 mmol/L) was significantly higher than those who survived without liver transplant (98.14 ± 45.20 mmol/L, p = 0.016). Other biochemical parameters, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bile acid, total protein, albumin, blood glucose, serum triglyceride, urea, creatinine, lactic acid, and alpha-fetoprotein levels were similar between two groups (p values were 0.148, 0.450, 0.206, 0.914, 0.205, 0.318, 0.571, 0.815, 0.271, 0.708, and 0.823, respectively).
Blood coagulation profiles, such as international normalized ratio (INR), prothrombin time (PT), prothrombin activity (PTA), activated partial thromboplastin time (APTT), fibrinogen (Fib), and thrombin time (TT) were all similar when compared between the survival group and the mortality group (p values were 0.510, 0.712, 0.786, 0.177, 0.656, and 0.817, respectively).
Blood amino-acid profiles on tandem mass spectrometry were compared between two groups. Mean level of citrulline in the mortality group was 52.34 ± 19.14 umol/L, and was significantly lower in when compared to the survival group (137.06 ± 79.62 umol/L, p = 0.010). On the contrary, mean level of tyrosine in the mortality group was 250.26 ± 77.78 umol/L, significantly higher than that of the survival group (130.00 ± 73.19 umol/L, p = 0.015). As for methionine and threonine levels, there were no statistically significant differences between two groups (p values were 0.787, and 0.072, respectively).
SLC25A13 gene mutation allele frequencies, including 851del4, 1638ins23, IVS6 + 5G > A, and IVS16ins3kb, were compared between 2 groups. However, none of these mutation allele frequencies were different between the morality group and the survival group (p values were 0.518, 0.541, 1.000, and 0.128, respectively).