2.1 Study Design

This was a prospective, multi-center, observational study conducted from 2012 to 2020 at six sites of the Porphyrias Consortium of the National Institute of Health Rare Diseases Clinical Research Network. It was conducted in accordance with the Declaration of Helsinki. The study was approved by the Institutional Review Board at all six participating sites. Written informed consent was obtained from participants after meeting the inclusion criteria.

2.2 Study Population and Assessments

First degree relatives of individuals with genetically confirmed AHP were contacted for enrollment. Only individuals ≥ 15 years of age, who were never screened before for the family mutation, and who have never had symptoms consistent with acute attacks were enrolled. Those with a history of alarm symptoms (gastrointestinal bleeding, anaemia with haemoglobin < 10 g/dL, hematuria, unintentional weight loss and dysphagia) were excluded. All 149 included subjects underwent genetic testing to determine their mutation status (carrier or control).

The questionnaire that was completed before genetic and biochemical testing sought information on socio-demographics, medical and surgical history, menstrual and reproductive history for female subjects and concomitant medications. Symptoms experienced by subjects that were relevant to porphyria were recorded. We collected data on specific treatments for or prophylactic measures taken to prevent acute attacks. Other covariates examined were the age of first symptoms, number of hospitalisations, precipitating factors, and quality of life. The Patient-Reported Outcomes Measurement Information System (PROMIS-57) scale was utilised to assess patient-reported health status across various domains, including physical, mental and social health. Following the completion of the questionnaires, saliva or blood samples were collected for genetic testing and urine samples were gathered for analysis of porphyrin precursors. Porphyrin precursors were normalised to urine creatinine and reported in mg/g creatinine.

The AHP clinical severity of 64 index patients, all of whom were patients in the Porphyrias Consortium Longitudinal Study [7], was examined to identify any correlation of their clinical severity to biochemical results of their corresponding first-degree relatives enrolled in the study. These 64 index patients were first-degree relatives of 99 study subjects out of 102 reported cases. Three study subjects (case 55, 63 and 66), each reported two first-degree index patients (Table S5). Index patients all had confirmed AHP diagnosis by urine biochemistry and genetic testing. The index patients were classified based on whether they reported a history of acute porphyria attacks (no attack or attacks) in the Longitudinal Study database. For index patients with a history of attacks, those with > 4 hospitalizations within 1 year, use of scheduled prophylactic hemin, or treatment with givosiran were classified as ‘Recurrent attacks’, they were classified as ‘Sporadic attack’ patients (Table S4).

3.1 Mutation Status

Mutation carrier status was analysed as a binary variable with categories, ‘carrier’ and ‘control.’

3.2 Symptom Profile and Manifestations

Symptom profile was analysed as a binary variable with categories, ‘symptomatic’ and ‘asymptomatic.’ Individual symptom manifestations were also analysed as binary (yes/no) variables.

3.3 Duration of Symptoms

Duration of symptoms was analysed as a binary variable with categories, ‘A few minutes-hours to 2 days’ and ‘Several days-weeks/months.’

3.4 Porphyrin Precursor Levels

Porphobilinogen (PBG) and delta-aminolaevulinic acid (ALA) were analysed as continuous measures (normal range of PBG and ALA: 0–2 mg/g creatinine and 0–7 mg/g creatinine respectively). All urine was analysed at the University of Texas Medical Branch Health Porphyria Center in Galveston, Texas.

3.5 High Excreters

High excreters were defined as subjects with urine PBG levels greater than four times the upper limit of normal (ULN), measured in mg/g creatinine (≥ 8) after unit conversion. In cases where unit conversion was not possible, we included one subject (case 39) with 27.6 mg/L (> 8 mg/L) and another (case 36) with 19.2 mg/24 h (> 10.8 mg/24 h), using four times the ULN values in their respective units, based on recent consensus definitions [16]. (Table S3).

3.6 Statistical Analysis

Categorical subject characteristics were summarised as counts and percentages, and continuous measures were summarised using descriptive statistics including mean (SD) and median (range). Symptom manifestations were analysed across carrier and control groups. PBG and ALA values were presented based on carrier status, sex and symptom profile. The prevalence of high excreters overall and within each sex and symptom profile subgroup was reported and represented as percentages. A subgroup analysis of PBG among high excreters was also conducted.

Differences between groups for continuous outcome measures were tested using the Welch's two sample t-test of means, and the Wilcoxon rank sum test of medians was used for comparison of subgroups within the high excreter population given the smaller sample sizes. The Fisher's exact test was used for the categorical symptom measures. All hypothesis tests were two-sided. The significance threshold was set to 0.05 for Welch's two sample t-test of means and the Wilcoxon rank sum test of medians, while a significance threshold of 0.10 was used for Fisher's exact tests in the subgroup of participants who reported non-specific symptoms. Statistical analyses were performed using R software (Version 1.4.1106 2009–2021 RStudio).

4.1 Mutation Carriers and Female Carriers Excrete Significantly Higher Urine Porphyrin Precursors Than Non-Carriers and Male Carriers

We examined the distribution of urine ALA and PBG from 62 carriers and 56 controls. Urine ALA and PBG were significantly higher in carriers than controls (mean ALA 6.98 vs. 2.21 mg/g creatine; mean PBG 9.17 vs. 1.31 mg/g creatinine Figure 2a). Twenty-three carriers (35%) had PBG levels above the ULN (5.9–89.2 mg/g creatinine) compared to only 6 (11%) controls (2.1–5.7 mg/g creatinine. Seventeen (27.4%)) carriers had ALA above the ULN (7.2–48.3 mg/g creatinine) while all controls except one case (13 mg/g creatinine) had levels within the normal range.

As women are far more likely than men to have symptomatic acute attacks [17], we examined potential sex differences in porphyrin precursor levels. Female carriers had significantly higher ALA and PBG levels compared to male carriers (mean ALA: 9.29 vs. 3.07 mg/g creatinine; mean PBG: 12.71 vs. 3.16 mg/g creatinine Figure 2b). Additionally, 66.6% of female carriers had abnormal PBG values (exceeding its ULN (2 mg/g creatinine)), compared to 39.1% of male carriers (Table S3).

4.2 Carriers Are More Likely to Report Dark Urine and Experience Longer Duration of Symptoms

We characterised symptom manifestations in undiagnosed relatives of AHP patients to see if any correlate with AHP carrier status. Of the 143 subjects that completed the questionnaire, 31 (21.7%) reported non-specific symptoms. In these subjects, 21 (68%) were carriers for the family mutation comprising 19 AIP and 2 VP subjects, indicating that 27% (21 of 77) of all carriers that completed questionnaires reported symptoms, compared to 15% (10 of 66) of non-carriers (Figure 3a). Dark urine was the only symptom significantly more likely to be reported by carriers than controls (p < 0.05 Figure 3b). AHP carriers were more likely to experience symptoms for several days to weeks or months than controls (Figure 3c). We compared the urine porphyrin precursor levels in 61 symptomatic and symptom-free carriers but did not find a statistically significant difference (Figure 3d).

4.3 Female Carriers and Symptomatic Carriers Are More Likely to Be Asymptomatic High Excreters (ASHE) With Urine PBG > 4times the ULN

A finding of considerable interest in our carrier population was the high prevalence of AHP carriers with urine PBG excretion greater than 4 times the ULN [16]. We focused on PBG levels due to the increased sensitivity and specificity in identifying not only acutely symptomatic patients but also latent carriers and those in remission [18]. Of the 65 carriers who provided urine samples for porphyrin precursor analysis, 20 (30.8%) had PBG greater than 4 times the ULN, with a median of 22 (range 8.5–89.2 mg/g creatinine) (Figure 4a). Of note, none of the controls had PBG greater than 4 times the ULN. More than 38% of female carriers were high excreters, compared to 17.39% of male carriers (Figure 4b). All male high excreters had urine PBG levels between 8.0–11.4 mg/g creatinine, while more than 70% of female high excreters had PBG > 15 mg/g creatinine (Figure 4b).

Similarly, we looked at 20 carriers who reported non-specific symptoms and found 8 (40%) to be high excreters, compared to 11 of 43 (25.6%) asymptomatic carriers (Figure 4c). We did not see a statistically significant difference in urine PBG levels between symptomatic and asymptomatic high excreters (Figure 4c), which is likely due to the presence of two asymptomatic carriers with more than a 20-fold increase in urine PBG levels (44.4 and 89.2 mg/g creatinine, respectively). Of note, only AIP carriers excreted high levels (> 8 mg/g creatinine) of PBG in urine in this study population.

4.4 No Correlation Existed Between the Genotype and Phenotype in Carriers

We asked if the increased proportion of ASHE in our study population can be attributed to the severity of disease of their corresponding index cases. We could identify first-degree index patients for 99 out of 149 study subjects, representing 64 unique index patients (Table S5). All index patients are enrolled in the US Porphyrias Consortium Longitudinal Study. We characterised their AHP disease severity to look for a potential relationship with urine PBG excretion in corresponding study subjects. Fifteen index patients have never experienced acute attacks, 37 had sporadic attacks, and 12 had frequent recurrent attacks based on recent definitions [15]. We compared the disease severity of index patients and the urine PBG in their first-degree study subjects who are mutation carriers. Twenty-two study subjects with urine PBG levels had index patients who were sporadic attack patients (Table 2), 8 study subjects had index patients who were recurrent attack patients (Table 3), and 14 study subjects had index patients who were asymptomatic (Table 4). We found no significant association between study subject urine PBG levels and the clinical severity of their index patients (Figure 5).