Diagnostic value of the lipid vacuoles in Kupffer cells
Between January 2018 and December 2020, 168 NC patients with unexplained causes underwent both liver biopsy and genetic tests. Kupffer cells with lipid vacuoles were detected in 26 patients, and 6 of whom were diagnosed as NP-C for harboring biallelic pathogenic or likely pathogenic variants in NPC1 (Table 1). No patient was found to harbor biallelic pathogenic variants in NPC2. The ratio of positive diagnosis of NP-C was 23.1% (6/26) in NC patients with vacuolar Kupffer cells. NP-C was not diagnosed among the remaining 142 NC patients who did not detect lipid vacuoles in Kupffer cells (6/26 vs. 0/142, χ2 = 33.983, P < 0.001). The sensitivity and specificity were 100.0% (6/6) and 87.7% (142/162) respectively.
Of the 26 NC patients with vacuolar Kupffer cells, 19 who had splenomegaly included the 6 NP-C patients. The ratio of positive diagnosis of NP-C was 31.6% (6/19).
Table 1
Basic information of the 168 infants with neonatal cholestasis
| With vacuolar Kupffer cells (n = 26) | Without vacuolar Kupffer cells (n = 142) |
Gender (male/female) | 14/12 | 68/35 |
Hepatomegaly | 26 (100%) | 142 (100%) |
Splenomegaly | 19 (73.1%) | 47 (33.1%) * |
Age at liver biopsy (Days) | 68 [46, 90] | 73 [59, 100] |
NP-C (NPC1) | 6 (23.1%) | 0 (0.0%) * |
NP-C, Niemann-Pick disease type C. |
* P < 0.05. |
Vacuolar Kupffer Cells Evolve Into Foam Cells
Between January 2015 and December 2017, 3 additional NC patients (P6, P8, and P9) were diagnosed as NP-C (Table 2). Of them, 1 patient (P6) underwent liver biopsy. Hence, a total of 7 NP-C patients (P1 ~ P7) underwent liver biopsy. P1 and P2 underwent liver biopsy at the age of 35 days and 36 days respectively. Lipid vacuoles were detected in a few Kupffer cells by CD68 staining (Fig. 1). Lipid vacuoles were detected in more Kupffer cells of P3 and P4 who underwent liver biopsy at the age of 49 and 63 days respectively. P5 underwent liver biopsy at 89 days of age. Lipid vacuoles were detected in almost all Kupffer cells, and a few vacuolar Kupffer cells became enlarged. Enlarged Kupffer cells with lipid vacuoles became obvious in both P6 and P7 who underwent liver biopsy at the age of 110 days and 112 days respectively.
Foam cells were not detected in hematoxylin and eosin (HE) staining sections in 5 patients (P1 ~ P5), but were typical in P6 and P7. Characteristic foam cells resided within the liver sinusoid, and were negative by periodic acid-schiff (PAS) staining.
Table 2
Molecular findings in NPC1 (NM_000271) of 9 patients with neonatal cholestasis
Patient | Gender | Variant 1 | Variant 2 | Origin |
1 | Male | c.1757 + 3_1757 + 6delGAGT | c.3254_3255delAT | M/F |
2 | Female | c.10delC | c.1211G > A (p.R404Q) | ND |
3 | Male | c.1024T > C (p.W342R) | c.2970_2971insTCCT | M/F |
4 | Male | c.3254A > C (p.Y1085S) | c.3254A > C (p.Y1085S) | F/M |
5 | Female | c.1138C > T (p.L380F) | c.1211G > A (p.R404Q) | F/M |
6 | Female | c.352_353delAG | c.2000C > T (p.S667L) | ND |
7 | Male | c.2207_2208dupTC | c.2972_2973delAG | M/F |
8 | Female | c.1421C > T (p.P474L) | c.2728G > A (p.G910S) | ND |
9 | Female | c.1301C > T (p.P434L) | c.3425T > C (p.M1142T) | ND |
ND, not done; F, father; M, mother. |
Novel pathogenic variants are shown in bold font.
Molecular Findings And Clinical Manifestations
Of the 9 NP-C patients (Table 2), 16 distinct variants were identified in NPC1, including 10 known pathogenic variants and 6 novel variants (4 frameshift indels and 2 missense variants) absented from both 1000 Genomes Project (TGP) and Exome Aggregation Consortium (ExAC). Novel missense variants, c.1024T > C (p.W342R) and c.3254A > C (p.Y1085S), were predicted to be disease causing or damaging by MutationTaster, Polyphen-2 and SIFT. According to the American College of Medical Genetics (ACMG) guideline, the 4 novel frameshift indels were rated as pathogenic variants, while the 2 novel missense variants as likely pathogenic variants.
The 9 confirmed NP-C patients came from 9 nonconsanguineous families. Jaundice and hepatosplenomegaly were identified in all 9 patients (Table 3). Six exhibited acholic stools. Cholestasis was confirmed by liver function tests (LFTs). Seven patients, but not patient (P) 3 and P7, could be classified into cholestasis with high serum γ-glutamyl transpeptidase (GGT) (GGT > 100U/L). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were elevated, and the ratios of AST/ALT ranged from 2.1 to 7.5. Blood glucose levels were lower than 3.0mmol/L in 4 patients (P5, P6, P8 and P9) after fasting for 3 hours. Bone marrow aspiration was performed in 4 patients (P1, P3, P5 and P6) at ages ranging from 42 days to 113 days (Fig. 1). No foam cell was observed in all 4 bone marrow samples.
Ursodesoxycholic acid (UDCA) and fat-soluble vitamins were given in all 9 patients. P4 and P6 lost to follow-up. P3 died at 8 months of age. For the remaining 6 NP-C patients, jaundice resolved at a median age of 4.5 months (ranging from 3 months to 9 months), but splenomegaly persisted. UDCA was stopped after jaundice resolved. At last follow-up at a median age of 15 months (ranging from 9 months to 34 months), none of the 6 patients had jaundice recurrence, but AST are still elevated in 6 patients. LFTs normalized in P9 at 17 months of age.
Table 3
Clinical information of the 9 NPC patients with neonatal cholestasis
| P1 | P2 | P3 | P4 | P5 | P6 | P7 | P8 | P9 |
First symptoms | J | J | J | J | J | J | J | J | J |
Age at first symptoms (Days) | 4 | 2 | 1 | 7 | 2 | 28 | 5 | 3 | 4 |
Other symptoms and signs | | | | | | | | | |
Acholic stools | + | + | + | - | + | + | + | - | - |
Hepatomegaly | + | + | + | + | + | + | + | + | + |
Splenomegaly | + | + | + | + | + | + | + | + | + |
Liver function tests (LFTs) | | | | | | | | | |
Age at tests (Days) | 35 | 33 | 44 | 62 | 89 | 53 | 79 | 97 | 60 |
TB (µmol/L) | 164 | 168 | 211 | 141 | 125 | 287 | 56 | 158 | 104 |
DB (µmol/L) | 136 | 102 | 119 | 108 | 104 | 229 | 46 | 117 | 58 |
ALT (U/L) | 55 | 59 | 41 | 110 | 152 | 117 | 63 | 72 | 58 |
AST (U/L) | 206 | 199 | 309 | 338 | 371 | 250 | 174 | 283 | 177 |
GGT (U/L) | 144 | 254 | 54 | 147 | 222 | 126 | 49 | 259 | 167 |
TBA (µmol/L) | 69 | 96 | 96 | 84 | 66 | 114 | 59 | 158 | 106 |
Alb (g/L) | 34.6 | 35.0 | 38.4 | 41.8 | 32.2 | 40.2 | 36.3 | 39.7 | 43.3 |
Glu (mmol/L) | 4.0 | ND | 6.0 | 4.2 | 2.9 | 2.5 | 4.1 | 2.0 | 1.9 |
P, patient; J, jaundice; ND, not done; TB, total bilirubin; DB, direct bilirubin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, γ-glutamyl transpeptidase; TBA, total bile acid; Alb, albumin; Glu, Glucose. |
-, negative; +, positive.