Introduction

Disorders of mitochondrial oxidative phosphorylation (OXPHOS) represent one of the most common groups of inherited metabolic diseases, with a combined minimum birth prevalence of 1 in 4300 live births.1, 2 Clinically, affected individuals can present with a spectrum of heterogeneous phenotypes and disease onset at any time during their life span, and often with multi-organ involvement. However organs with high energy demand, such as the brain, heart and the skeletal muscles, are the most vulnerable.3 Despite the advances in diagnostic methods, early clinical recognition of patients with mitochondrial disorders in general is still challenging.

An important subgroup amongst mitochondrial disorders comprises the mitochondrial complex III (CIII) deficiencies.4 CIII (also known as cytochrome bc1 complex or ubiquinol-cytochrome c reductase) catalyses the transfer of electrons from reduced coenzyme Q10 to cytochrome c whilst simultaneously pumping protons from the mitochondrial matrix across the inner mitochondrial membrane to the intermembrane space.5, 6 CIII is a multi-heteromeric enzyme complex consisting of 11 different subunits, of which 10 subunits (core proteins I and II, 6 small subunits, cytochrome c1 and the Rieske FeS protein) are encoded by nuclear genes, whilst the cytochrome b subunit is encoded by mtDNA.6, 7 Pathogenic variations in several nuclear genes affecting CIII structural subunits (UQCRB, UQCRQ, UQCRC2, UQCRFS1 and CYC1) or assembly factors (TTC19, BCS1L, LYRM7/MZM1L, UQCC2, UQCC3 and HCCS) have been reported to be associated with mitochondrial CIII deficiency and human disease.8, 9

The most frequent cause of mitochondrial CIII deficiencies is due to defects in the BCS1L gene encoding BCS1, a mitochondrial inner membrane protein that acts as a translocase for insertion of the Rieske FeS subunit into the precomplex of CIII to facilitate assembly of the holoenzyme complex.8, 10, 11 The two major phenotypes which are well known to be associated with disease-causing variants in BCS1L are GRACILE and Björnstad syndromes. Classical GRACILE syndrome (MIM 603358) is an autosomal recessive disease characterised by Growth Restriction, Aminoaciduria as a sign of tubulopathy, Cholestasis with Iron overload in the liver, Lactic acidosis and Early death.12 This was initially identified as a Finnish heritage disease and is mainly caused by a specific homozygous variant, c.232A>G (p.Ser78Gly), in the BCS1L gene.13, 14 The other less frequently reported phenotype is Björnstad syndrome (MIM 262000) which is characterised by brittle hair (pili torti) and sensorineural hearing loss.15, 16 Other phenotypes that have also been reported include tubulopathy, hepatopathy and encephalopathy17 and Leigh-like syndrome.18, 19

We aimed in this study to improve the clinical recognition of patients with BCS1L disease, provide better understanding of the genotype–phenotype correlations and identify reliable prognostic disease markers using data from the largest known multinational cohort of patients with confirmed biallelic pathogenic BCS1L variants.

Patients and Methods Study design and population

We conducted a multinational retrospective study of patients from 15 centres in 10 countries: Finland (Children's Hospital, Helsinki University Hospital), United Kingdom (Great Ormond Street Hospital, London), United States (Department of Clinical Genomics, Mayo Clinic, Rochester, Minnesota; Neuroscience Institute, Cleveland Clinic, Cleveland, Ohio; and Department of Pediatrics, University of Massachusetts Medical School—Baystate, Massachusetts), France (Necker-Enfants Malades Hospital, Paris), Spain (12 de Octubre University Hospital, Madrid and Hospital Sant Joan de Déu, Barcelona), Sweden (Centre for Inherited Metabolic Diseases, Karolinska University Hospital, Stockholm and The Queen Silvia Children's Hospital and Sahlgrenska University Hospital, Gothenburg), Norway (Haukeland University Hospital), Austria (University Children’s hospital, Salzburg), Italy (Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan) and Oman (Sultan Qaboos University Hospital, Oman). Previously unpublished patients with confirmed biallelic pathogenic BCS1L variants who had been diagnosed and followed up at the participating centres were considered eligible.

Detailed clinical, biochemical, muscle biopsy, neurophysiological, neuroimaging and genetic data were obtained using a standardised case report form completed by the responsible investigator(s) at each centre and reviewed by the study principal investigator (O.H). Data entry was completed in December 2020.

The date of disease onset was defined as the date of the first symptom(s) requiring medical evaluation. End of follow-up was defined as the date of the patient’s last follow-up visit or death. Available clinical and laboratory longitudinal data, both at disease onset and later during the disease course, were collected. Small for gestational age was defined as newborns with birth weight below the 10th percentile for the gestational age.20 Early death was defined as death within the first year of life.

Proximal renal tubulopathy included Fanconi syndrome and was defined as having two or more of the following: generalised aminoaciduria, glycosuria, low molecular weight proteinuria, bicarbonate loss resulting in renal tubular acidosis and renal salt wasting.21

Liver involvement was defined by the presence of two or more of the following parameters in at least two different time points: elevation of aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), gamma-glutamyltransferase (GGT), bilirubin or ammonia; low serum albumin; or pathological histological findings on liver biopsy.

Movement disorders included dystonia, athetosis and tremor.

Data analysis

Initially, detailed clinical, laboratory, neuroimaging and genetic data for the whole cohort were analysed to study the phenotypic spectrum of the disease regardless of the age of disease onset or genotype. To study the disease spectrum beyond the neonatal period, the patients were classified into those with disease onset within the first month of life and those with later onset. The study cohort was further stratified into those who were homozygous or compound heterozygous for the c.232A>G (p.Ser78Gly) variant and those with other pathogenic BCS1L variants.

Protein modelling

Models were prepared with PyRosetta22 using the cryogenic electron microscopy structures of mouse BCS1 (the protein encoded by BCS1L) PDB:6UKP and PDB:6UKS.23 The structures were energy minimised against their density maps (EMDB-20808 and EMDB-20811) and then used as a template to thread the human sequence (92% identity) of BCS1 using RosettaCM.24 The ATPγS was converted into an ATP, the 1–49 span was added as parallel helices for illustrative purposes and then further energy minimised. The difference in Gibbs free energy (∆∆G) was calculated by introducing the mutation and energy minimising in each conformation (single-point energy). The code used is openly available at https://github.com/matteoferla/BCS1_analysis. Interactive page was created using Michelaɴɢʟo.25

Statistical analysis

Detailed descriptive data analysis was performed using SPSS (Statistical Package of Social Sciences), Version 26.0. A two-sided p value less than 0.05 was considered to be statistically significant. For survival analysis, the endpoint was time to death which was defined as the time from the date of disease onset to the date of death. Univariate survival analysis was performed using the log-rank test (Kaplan–Meier) to compare differences in survival time between categories.

Ethical statement

Ethical approval for the study was obtained from the Regional Committee for Medical and Health Research Ethics, Western Norway (REK 2017/625). Each participating centre had obtained approval by the local ethical committee. The study was registered as an audit at Great Ormond Street Hospital, London, UK (Registration Number: 2224).

Results Demography

Thirty-three patients, (17 male, 16 female) with biallelic pathogenic BCS1L variants were identified. Ten were diagnosed in Finland, five in the United Kingdom, four in the United States, three each in France and Sweden, two each in Austria, Norway and Spain and one each in Italy and Oman. The majority of patients were of European ancestry (n = 23), whilst three were from the Middle East, three Turkish, two Pakistani and two Black American.

Phenotypic spectrum

The majority (73%, n = 24/33) of patients included in this study cohort had disease onset either at birth or within the first month of life. Fourteen patients were born prematurely with gestational age less than 37 completed weeks and 19 (63%, n = 19/30) patients were small for gestational age. A symptom-free period ranging from 3 months to 7 years was observed in nine patients.

Patients included in this cohort presented with a spectrum of clinical phenotypes including lactic acidosis (n = 27), failure to thrive (n = 27), proximal renal tubulopathy (n = 24), hepatopathy (n = 24), early death (n = 14), sensorineural hearing loss (n = 12), iron overload (n = 10), movement disorders (n = 9), cholestasis (n = 7) and seizures (n = 6). Other features observed in our cohort include global developmental delay (n = 9), attention deficit hyperactivity disorder (n = 3), cataract (n = 3), learning difficulties (n = 2), optic atrophy (n = 1) and hypertrophic cardiomyopathy (n = 1). A summary of the clinical phenotypes observed in our cohort is provided in Table 1. Thirteen patients fulfilled the diagnostic criteria for classical GRACILE syndrome (cases 2–10, 24, 27, 32 and 33, Table 1) and two for classical Björnstad syndrome (cases 26 and 28, Table 1).

Table 1. Phenotypic spectrum of patients with BCS1L disease included in the study cohort. Case no. Age [at death] Growth restriction Tubulo pathy Cholestasis Hepatopathy Iron overload Failure to thrive Lactic acidosis Early death Pili torti SNHL Movement disorders Seizures Other features 1 11 y Yes No ND Yes ND Yes Yes No ND Yes No Yes LD, ataxia 2 [5 d] Yes Yes Yes Yes Yes Yes Yes Yes ND ND No No 3 [3 d] Yes Yes Yes Yes Yes Yes Yes Yes ND ND No No 4 [4 d] Yes Yes Yes Yes Yes Yes Yes Yes ND ND No No 5 [21 d] Yes Yes Yes Yes Yes Yes Yes Yes ND Yes No No 6 [27 d] Yes Yes Yes Yes Yes Yes Yes Yes No Yes No No 7 [2 d] Yes Yes Yes Yes Yes Yes Yes Yes ND ND No No PDA 8 [3 m] Yes Yes ND Yes Yes Yes Yes Yes ND ND No No 9 [3 d] YES ND ND Yes Yes Yes Yes Yes ND ND No No 10 [1 d] Yes ND ND Yes ND ND Yes Yes ND ND No No PND 11 ND No Yes ND No ND No Yes ND ND No Yes No Ataxia, bulbar palsy 12 [3 y] Yes Yes ND Yes ND Yes Yes No ND No No No GDD 13 [4.5 y] No Yes ND Yes ND Yes Yes No ND No Yes Yes GDD,VI 14 11.5 y No No No No No No Yes No No Yes Yes Yes GDD, ADHD 15 9.5 y No Yes ND Yes Yes Yes Yes No No Yes Yes Yes GDD,ADHD 16 ND No Yes ND ND ND Yes Yes No ND ND Yes No Maculopathy, OA, NC 17 24 y No Yes ND No ND Yes Yes No Yes Yes Yes No Migraine, cataract 18 [1 m] Yes Yes ND Yes ND Yes Yes Yes ND ND No Yes GDD, cataract, 19 4 y Yes No ND Yes ND Yes Yes No No ND NO No GDD, LD 20 2 m ND Yes ND Yes ND Yes ND No No ND No No HCM 21 4 m Yes Yes Yes Yes ND Yes Yes No No Yes No No Cataract 22 13 y Yes Yes No No No Yes No No No No No No 23 10 y No Yes No No No Yes No No No No No No 24 [11 m] Yes Yes ND Yes ND Yes Yes Yes No No No No 25 [14 y] ND Yes No Yes ND Yes Yes No No No Yes Yes 26 13.5 y No No No No ND No No No Yes Yes No No 27 [1 m] Yes Yes ND Yes ND Yes Yes Yes ND ND No No 28 15 y No No No No ND No ND No Yes Yes No No 29 ND No ND ND Yes Yes Yes Yes No ND Yes No No 30 10 y No No ND No No No No No ND Yes Yes No GDD, anxiety, ADHD 31 [2 y] Yes Yes ND Yes ND Yes Yes Yes Yes Yes Yes No GDD 32 5 y Yes Yes ND Yes ND Yes ND ND ND ND No No AIH 33 [15 m] ND Yes ND Yes ND Yes Yes Yes No ND No No GDD Key: ADHD: attention deficit hyperactivity disorder, AIH: autoimmune hepatitis, d: days, GDD: global developmental delay, HCM: hypertrophic cardiomyopathy, LD: learning difficulties, m: months, NC: nephrocalcinosis, ND: no data available, OA: optic atrophy, SNHL: sensorineural hearing loss, VI: visual impairment, y: years.

If we stratified patients into those presenting within the first month of life (73%, n = 24/33) and those presenting later (27%, n = 9/33), we found that features consistent with GRACILE syndrome such as growth failure, lactic acidosis, tubulopathy, hepatopathy, iron overload and early death were more frequently observed in those with onset within the first month of life as compared to those with later onset. In contrast, features such as movement disorders (dystonia, athetosis and tremor) and seizures occurred across all ages but were more frequently identified in our study cohort in those with onset after the first month of life (Table 2). Of note, a paroxysmal movement disorder without evidence of magnetic resonance imag