Patient characteristics

A total of 49 BBS patients (aged 1.1–51.0 years, mean 17.8 years, median 16.8 years) were included in this prospective study. The sex distribution was near-balanced with 49% (24/49) male patients. The body length was normally distributed; the mean weight SDS (2.13) and mean BMI SDS (2.64) were well above the 97th percentile with higher values in children compared to adults (mean BMI z-score 2.77 vs. 2.50; Table 1).

Table 1 Demographics and selected blood laboratory data in BBS patients

Pathogenic variants in BBS genes 10 (16/49–33%) and 1 (12/49–25%) were the most common variants and accounted for 58% of the study cohort. The hot spot mutations in the BBS1 gene (BBS: c.1169T > G; p.Met390Arg) and the BBS10 gene (c.271dupT; p.Cys91Leufs*5) were prevalent in 10 patients each (BBS1: n = 7 homozygous, n = 3 compound heterozygous, 71% of all BBS1 patients; BBS10: n = 4 homozygous, n = 6 compound heterozygous, 44% of all BBS10 patients). Our cohort included patients with pathogenic variants in nine further BBS genes (Fig. 1). Homozygous variants were more frequent (26/49–53%) than compound heterozygous variants. Two truncating variants (28/49–57%) were more common than two missense variants or a combination of missense and truncating variants.

Fig. 1

Genetic findings in the BBS study cohort

Impairment of renal function was present in 27% (13/49). Three patients (6%) developed ESKD at ages 6, 10, and 46 years. Two adult patients were under dialysis treatment (hemodialysis (n = 1) and peritoneal dialysis after failure of a kidney transplant due to chronic humoral rejection (n = 1)), and one 17-year-old patient had received a kidney transplant (eGFR 63 ml/min/1.73m2, homozygous truncating variant in BBS16). Liver enzymes (GOT, GPT, GGT) were elevated in 38% (16/42; at least one parameter). The most common long-term medication was vitamin D (53%, 26/49); others included oral iron supplementation (12%, 6/49) and l-thyroxine (10%, 5/49) replacement therapy. In individual cases, medication was taken for sleep disorders, behavioral problems, seizures, high blood pressure, and steroid-free immunosuppression (cyclosporine, tacrolimus, mycophenolate mofetil) after kidney transplantation.

Technical characteristics of ultrasound examination

Ultrasound examination was overall (86%, 42/49) well tolerated (“good” cooperation) with lower rates in children compared to adults (78% [21/27] vs. 95% [21/22]). The mean duration of ultrasound examination of the liver (basic ultrasound + SWE, SWD, and ATI assessment) was 7.5 ± 2.6 min (median 7 min, range 4 to 15 min) and comparable between children and adults (7.6 vs. 7.4 min). Last food intake was more than 2 h ago in 71% (35/49). The mean abdominal wall thickness was 22.5 ± 7.5 mm (median 22, range 9 to 43; Table 2).

Table 2 Ultrasound findings including liver, spleen, and kidney in patients with BBSKidney ultrasound

In pediatric patients, the overall mean weight-adjusted total kidney volume was normal with a wide distribution (Table 2). The total kidney volume in adults with absolutely higher body weights was correspondingly higher. The mean resistance index (RI) was within the normal range, but 55% (17/31) exhibited an increased RI value. This applied to all patients with an impaired GFR. Mean renal artery peak flow velocity was within the normal range, with reduced velocity observed in 10% (28/31).

Overall, kidney structure abnormalities were present in 75% of patients (36/48). Increased renal echogenicity and decreased or increased corticomedullary differentiation, as indicators for renal abnormalities, were observed in 52% (25/48) and 63% (30/48) of the cohort, respectively (Figs. 2, 3, and 4). Renal cysts were prevalent in 21% (10/48).

Fig. 2

Increased kidney echogenicity and preserved corticomedullary differentiation without persistent fetal lobulation in an 11-year old BBS female patient (patient ID37; pathogenic homozygous variant in BBS 17 gene; c.778–3 C > T) and video representation in Supplement Fig. 2

Fig. 3

Increased kidney echogenicity and non-existent corticomedullary differentiation in a female 9-year old BBS patient (patient ID26; pathogenic compound heterozygous variant in BBS 5 gene; c.54dupC p. (ala19Argfs*14) + deletion Exon 10–12)

Fig. 4

a Left kidney with significant persistent fetal lobulation with increased kidney echogenicity and almost complete diminished corticomedullary differentiation in a 10-year old female BBS patient (patient ID5; pathogenic homozygous variant in BBS 8 gene; deletion Exon 9) and video representation in Supplement Fig. 3; b Hypoplastic right pelvic kidney with non-existent corticomedullary differentiation and incidental findings of right-sided ovarian cyst (same patient as in Fig. 4a)

Persistent fetal lobulation appeared in 44% (21/48). Urinary tract disorders were less common and presented exclusively as mild urinary tract dilatations (10.4%, 5/48). A bladder volume beyond the age-specific range (above 400 ml in adults, [years of age + 2] × 30 ml in children [40]) was detected in 20% of patients. Two cases presented with nephrocalcinosis grade 1, and one child had a left-sided pelvic kidney. Native kidneys were not detectable in one patient after kidney transplantation (Table 2).

Liver ultrasound

The sizes of the liver and spleen were age- and height-adjusted and slightly increased (mean 114% and 108%, respectively). Organ perfusion was measured in cm/s and showed no anomalies (Table 2). Increased liver echogenicity was present in 17/49 (35%). A minority of patients (10%, 5/49) exhibited a rounded lower liver margin as one major US sign for parenchymal abnormalities. In this subgroup, the mean values for BMI SDS (3.72), liver size (140%), SWE (6.8 kPa), and ATI (0.73 dB/cm/Mhz) were descriptively elevated compared to the present study cohort. According to the high percentage of patients with a fasting period > 2 h, the filling state of the gallbladder was moderate (23%, 11/47) or high (57%, 27/47) in the majority of patients.

SWE, SWD, and ATI

The results of measurements are given in Table 3. Overall, the mean ATI values were above the normal range, with descriptively slightly higher values in children than in adults. Among BBS patients, 51% (25/49) had ATI values above the 97th percentile and an additional 16% (8/49) between the 90th and 97th percentiles. The majority of BBS patients demonstrated SWE values above the 97th percentile (61%, 30/49), with higher values in adults. SWE values equal to or above 7.0 kPa, indicating hepatic fibrosis [41], were measured in 31% (15/49) of the cohort (Figs. 5, 6, 7, and 8). Regarding SWE, ROIs were placed at a mean of 4.3 ± 0.84 cm below the skin (median 4.3 cm; range 2.8 to 6.3 cm, recommended < 5 cm). The mean SWD level was in the normal range, corresponding to the 50th percentile in children (13.0 [(m/s)/kHz]) and the 75th percentile in adults (13.7 [(m/s)/kHz]) when considering BMI. However, 23% of the cohort (9/39) demonstrated SWD levels above the 97th percentile indicating elevated liver viscosity. This mainly applied to male BBS patients (89%, 8/9), patients with a pathogenic variant in the BBS10 gene (56%, 5/9), and those with elevated ATI (0.71 dB/cm/Mhz) and SWE levels (6.8 kPa).

Table 3 Ultrasound findings of liver parenchyma using ATI, SWE, and SWD in patients with BBSFig. 5

Normal liver parenchymal structure in in a 18-year old female BBS patient (patient ID34; pathogenic compound heterozygous variant in BBS 2 gene; c.823C > T; p.Arg275*) + c.1986dupT (p.Asn663*)

Fig. 6

High-grade steatosis in in a 19-year old male BBS patient (patient ID 42; pathogenic homozygous variant in BBS 10 gene; c.1269_1273del (p.Gln423fsX))

Fig. 7

High-grade liver fibrosis in a 31-year old female BBS patient (patient ID49; pathogenic homozygous variant in BBS 1 gene; c.1169 T > G p.(Met390Arg)

Fig. 8

a Same patient as in Fig. 7: Overview in the HE staining. Liver parenchyma with epitheloid cellular granulomas (objective 4× corresponding to 40× total magnification) ectatic sinusoids (objective 10× corresponding to 100× total magnification) as signs of severe chronic portal moderate active inflammation of the liver tissue with florid (non-purulent destructive) bile duct lesions, partial ductopenia of local bile ducts and septate fibrosis without complete cirrhotic remodeling (stage 2/4 according to Desmet). No fatty degeneration. No siderosis. b Same patient as in Fig. 7: Gomori staining to visualize the hepatic fibrous tissue. Fibrosis in a portal field but overall no cirrhotic remodeling (objective 10× corresponding to 100× total magnification)

Correlation analysis

A minor proportion of SWE (2%, 1/49) and SWD (2%, 1/49) measurements was winsorized prior to further analyses. This is well below a recommended threshold of 5% [35].

Exploratory bivariate analyses revealed no relationship between ATI, SWE, and SWD levels (Table 5). Concerning auxological parameters, ATI levels were positively correlated with BMI-SDS (r(47) = 0.31; p = 0.002). This also applied to abdominal wall thickness (r(47) = 0.32; p = 0.001) and confirmed the former finding. Liver size (%) and echogenicity were positively related to ATI measurements, consistent with a positive relationship with GOT (r(39) = 0.22; p = 0.05), GPT (r(40) = 0.33; p = 0.003) and GGT levels (r(39) = 0.29; p = 0.01) in a subsample of BBS patients. Also, spleen (r(43) = 0.27; p = 0.01) and kidney size (r(45) = 0.25; p = 0.02) were positively correlated with ATI levels in subsample analyses.

There was a similar relationship of SWE measurements with BMI-SDS (r(47) = 0.25; p = 0.012) and abdominal wall thickness (r(47) = 0.35; p = 0.001) as observed for ATI levels. However, apart from GGT levels (r(39) = 0.22; p = 0.045), SWE measurements were neither related to liver morphology, liver enzymes, or other organ status.

There was a statistical trend (0.05 < p < 0.10) towards higher SWD levels in males than in females (r(37) = − 0.23; p = 0.09). Moreover, SWD levels were lower in patients with pathological kidney findings (r(34) = − 0.37; p = 0.006; 0 = GFR within age range, kidney size and normal echogenicity; 1 = GFR within age range, increased echogenicity [at least one kidney] and/or reduced/increased kidney size [at least one kidney]; 2 = GFR below age range) but did not relate to any other variable. However, the power to identify bivariate correlations concerning SWD levels was just sufficient for large effect sizes (p ≥ 0.55, d = 1.32). In contrast, analyses regarding ATI and SWE levels were sufficiently powered to identify medium to large effects (p ≥ 0.4, d = 0.87).

Neither GFR nor pathological kidney function, as defined previously, demonstrated significant associations with biochemical or ultrasound tissue markers of liver structure or pathology. However, a positive correlation was observed between kidney size and increased liver echogenicity (r(45) = 0.41, p < 0.001) as well as abnormal liver configuration, specifically a rounded/pointed liver margin (r(45) = 0.33, p = 0.006), suggestive of liver steatosis and fibrosis.

Multiple regression

Choosing the most appropriate set of covariates by considering the amount of variance explained in ATI, SWE, and SWD measurements against overfitting and the minimum model size, ATI levels in BBS patients were most efficiently determined by liver size (b = 0.002, t(46) = 4.16, p < 0.001) and liver echogenicity (b = 0.02, t(46) = 5.28, p < 0.001, Supplementary Table 1). In contrast, when jointly considering multiple covariates, the only variables to significantly affect SWE and SWD levels were abdominal wall thickness (b = 0.01, t(47) = 5.50, p < 0.001) and sex (b = − 1.42, t(37) = − 2.17, p = 0.04), respectively (Supplementary Table 2 and 3). However, the latter failed to reach significance when considering a correction for multiple comparisons. These analyses were sufficiently powered to detect medium to large effect sizes (d = 0.82, assuming up to 11 covariates).

Importantly, independent sample t-tests revealed no significant difference in ATI (t(47) = 1.55, p = 0.012), SWE (t(47) = − 0.68, p = 0.25), and SWD (t(37) = 0.50, p = 0.99) levels between patients fasting less than and more than 2 h and this also applied when considering the covariates identified in the previous step of analysis by ANCOVAs (ATI: (b = − 0.145, t(44) = − 1.57, p = 0.12) | SWE: (b = 0.05, t(46) = 1.24, p = 0.22) | SWD: (b = 0.23, t(36) = 0.32, p = 0.75).

Group comparisons—BBS subtypes

Comparing the two BBS genotypes with reasonable numbers of affected patients (i.e., type 1 and type 10; Ntype1 = 12, Ntype10 = 16, Table 4) by ANCOVAs, there was no difference regarding ATI (b = 0.001, t(24) = 0.39, p = 0.70), SWE (b = − 0.001, t(25) = − 0.20, p = 0.84), and SWD levels (b = − 0.06, t(25) = − 0.60, p = 0.56) when considering covariates identified by the selection process and its results outlined above. However, ATI levels appeared descriptively higher in the BBS10 gene group, in particular compared to the BBS1 hotspot pathogenic variant c.1169T > G p.(Met390Arg) (0.71 vs. 0.56 dB/cm/Mhz). Moreover, further exploratory analyses revealed that BBS1 patients were taller (height-SDS: BBS1: 0.93 (1.22) | BBS10: − 0.09 (1.15), p = 0.03) and had higher portal vein flow (BBS1: 31.3 (8.63) cm/s | BBS10: 25.0 (4.84) cm/s, p = 0.047) than BBS10 patients. In contrast, BBS10 patients had higher BMI-SDS (BBS1: 2.02 (1.40)| BBS10: 3.19 (0.97), p = 0.02), and there was a statistical trend for increased liver echogenicity (BBS1: 2/12 (16.7%) | BBS10: 9/16 (56.3%), p = 0.05), increased kidney echogenicity (BBS1: 3/12 (25%) | BBS10: 10/16 (62.5%), p = 0.07) and a higher incidence of GFR levels below 90 ml/min (BBS1: 1/11 (9.1%) | BBS10: 7/15 (46.7%), p = 0.08) than BBS1 patients. These analyses were sufficiently powered to detect findings with effect sizes larger than d = 1.28 (assuming up to 3 covariates). The other BBS subgroups comprised between 1 and 4 patients each, rendering the dataset inadequate for robust statistical analysis. Consequently, and instead, descriptive results concerning anthropometric parameters, as well as renal and hepatic findings, in these subgroups are detailed in Table 4.

Table 4 Detailed study cohort information according to BBS genesGroup comparisons—BBS vs. norming sample

In contrast, an ANCOVA, considering liver size, liver echogenicity, age, and BMI-SDS, revealed higher ATI levels in the BBS than the norming sample (b = 0.07, t(151) = 4.60, p < 0.001) beyond the age of 2 years as investigated by a Neyman-Johnson analysis for reasons of heterogeneity of regression slopes (interaction group x age: b = 0.004, t(151) = 2.84, p = 0.005; for details, please see the Supplementary Material and Supplementary Fig. 1). In addition, SWE levels were found to be higher in the BBS than the norming sample (b = 0.07, t(155) = − 2.85, p = 0.005) when considering an analysis-specific subset of covariates (abdominal wall thickness, age, and BMI-SDS) and a heteroscedasticity consistent standard-error estimator (HC3). These results did not change when excluding a single outlier concerning the analyses of ATI (b = 0.07, t(150) = 4.54, p < 0.001) and SWE levels ((b = 0.07, t(154) = − 2.61, p = 0.01)), including sex, age, and BMI-SDS as covariates. No difference was observed in SWD measurements (b = − 0.85, t(136) = − 1.64, p = 0.10). These findings have to be interpreted against sufficient power to identify medium-sized effects (d = 0.64) (Table 5).

Table 5 Bivariate correlations of the new ultrasound techniques ATI, SWE, and SWD and potential influencing variables in BBS