Whole exome sequencing (WES) has been performed for all four patients described in this study (Cf Supplementary Material for details concerning method and bioinformatics analysis).

RNA study has been performed in patient 3 on blood and fibroblasts samples (Cf Supplementary Material for details concerning method).

All clinical data are summarized in Table 1.

Patient 1 is a 15 years old male. He was born from consanguineous parents and had 2 brothers among which one was also affected (patient 2). He was able to walk at 11 months of age. A Wolf Parkinson White syndrome was diagnosed and treated at 3 years. Progressive walking disorder associating peripheral neuropathy and spastic diplegia began at 4 years. In the same time, optic atrophy revealed by decrease of vision with nystagmus was present and a language delay was noticed. Cyclic vomiting (approximatively 4 days every 2 months with complete food intolerance needing fluid supplementation) began at 6 years of age associated with recurrent fever conducting to recurrent hospitalizations in emergency for IV rehydration. He needed orthopedic surgery for severe contractures of the lower limbs. Large metabolic investigations during the acute digestive episods were normal. NCS/EMG (nerve conduction study and electromyography) confirmed the axonal sensory motor neuropathy (Table 2). Cerebral MRI (except optic atrophy) was normal. Genetic investigations (micro-array DNA, Mt DNA and NGS analysis for nuclear mitochondrial gene) were normal. At age 14, he was able to walk with support. Recurrent vomiting were partly responsive to amitriptyline chlorhydrate.

Patient 2: a 14 years old male, brother of patient 1 presented the same history. He was able to walk at 12 months of age. A language delay was later noticed. At 4 years spastic diplegia with abolition of the tendon reflexes was diagnosed. Optic atrophy was diagnosed at 6 years old. Recurrent vomiting began at 6 years with weight flathering leading to undernutrition. NCS/EMG evidenced peripheral neuropathy at age 8 (Table 2). Progressive weakness resulted in loss of ambulation at age 10. Because of severe distal contractures of lower limb, he needed orthopedic surgery. Enteral nutrition and parenteral nutrition were necessary at 13 years during 6 months. Large metabolic investigations including thymidine phosphorylase activity and search for mitochondrial dysfunction were normal. Digestive explorations including enteroscan, gastric and esophageal endoscopy with biopsies were non-contributive. Muscle biopsy showed no mitochondrial dysfunction. After a long time of hospitalization because of total digestive intolerance, he was stabilized with vitamins supplementation biotin (20 mg/day), pantothenic acid (555 mg/day) and lipoic acid (300 mg/day) and amitriptyline at age 14, at 6 months of follow-up. Digestive investigations comprising oesophageal endoscopy with biopsies, rectal biopies, enteroscan, oesophagal and anorectal manometry, oeso-gastroduodenal transit, small intestin and colorectal radiography were normal.

Patient 3: this girl born of consanguineous parents experienced walking problems at 4 years of age. At age 4, she developed episodic urinary retention. Spinal cord MRI was normal and NCS/EMG (nerve conduction study and electromyography) showed mixed axonal and demyelinating sensory motor neuropathy (Table 2). Polyradiculitis was suspected at CSF analysis (proteinorachia at 0.38 g/L N: 0.15–0.6 g/L) without any autoantibodies. Large and repeated biochemical investigations were unremarkable (mitochondrial, lysosomal, acylcarnitine profile). She had pyramidal signs with severe lower limbs retractions and loss ambulation at age 5. Muscle biopsy showed a neurogenic muscular atrophy and a normal respiratory chain analysis. At 6 years she developed cyclic vomiting and multiple episodes of chronic intestinal pseudo-obstruction (CIPO). Digestive investigations comprising oesophageal endoscopy with biopsies, rectal biopies, enteroscan, oesophagal and anorectal manometry, oeso-gastroduodenal transit, small intestin and colorectal radiography were normal.

She needed nutritional support with gastrostomy and jejunostomy and at least total parenteral nutrition since few months. She died at 7 years of age after suffering quadriplegia, brainsteam dysfunction and pan digestive palsy.

Patient 4, a 23-year-old male man, was born of consanguineous parents, His oldest brother died at the age of 4 years. His two sisters were asymptomatic. At 1 year of age, he had a surgical cure of right pyelo-ureteral junction syndrome. A progressive symmetrical distal weakness of the lower limbs started at the age of 3 years, leading to clumsiness and running difficulties. At age 7 he had distal hypoesthesia, weak and amyotrophic muscles in the lower limbs, generalized osteotendinous areflexia and an axonal sensory and motor neuropathy was diagnosed. At age 8, failure to thrive was noticed, worsening at 11. At age 10, hand weakness appeared; he suffered from neuropathic pain and paresthesia treated by amitriptyline and oxcarbazepine then gabapentin; bilateral optic atrophy was diagnosed in a context of decreased visual acuity. At age 12.5, he suffered from recurrent episodes of vomiting and CIPO requiring parenteral nutrition for 2 months, associated with ileostomy, and followed by enteral nutrition through gastrostomy from age 13 to 18: oesophageal endoscopy showed Mallory Weiss syndrome during a CIPO access, proximal small intestin ileus and dyskinesia of the third lower oesophagus. Multiple biopsies, anorectal manometry, oeso-gastroduodenal transit were normal.

At age 13, he had episodic acute bladder dysfunction, and arterial hypertension treated by amlodipine and perindopril for 6 months, followed by amlodipine alone. At 17 years, bilateral cavovarus feet required a corrective surgery, after what he lost autonomous walking. NCS showed very low CMAP in lower limbs and median nerves, sensory potentials were markedly decreased in all four limbs, whereas conduction velocities were mildly diminished, overall consistent with a sensory and motor axonal neuropathy (Table 2). Brain MRI was normal, except for atrophic nerve atrophy.

Muscular biopsy showed neurogenic changes, some COX-negative muscular fibers but no specific respiratory chain complex deficit. No mitochondrial DNA pathogenic variation or deletion was identified.

Thymidine phosphorylase activity and TYMP gene sequencing were normal, as well as PDH activity and sequencing of sub-unit E2/lipoylation genes (NFU1, BOLA3, IBA57, LIAS, LIPT1). Optic atrophy panel detected no pathogenic variation in the MFN2, OPA1, OPA2, GJB1, OPA7 genes.

Other biochemical studies including transferrin electroporesis, Redox points, amino-acids and organic acids chromatographies, plasmatic homocysteine and peroxysomal dosages (VLCFA, phytanic, pristanic, pipecolic acids and plasma bile acid intermediates) were normal. But urinary measurement of 3 hydroxy valeric acid before treatment was abnormal with elevated levels: 115 μmol/mmol of creatinine (N < 18) as commonly showed in biotin deficiencies. At 23 years, he has a severe motor weakness and amyotrophic thenar muscles and wasting of lower limbs resulting in drop-foot gait. He used a wheelchair at the age of 20. He has been treated for a few months with biotin (20 mg/day), pantothenic acid (555 mg/day) and lipoic acid (300 mg/day).

NGS sequencing identified in all patients the same variation: c.460–19T>G (2: 27206553 (GRCh38)) at homozygous state in the SLC5A6 gene (NM_021095). This variation was not recorded in gnomAD database v3 nor in Clinvar before our submission (our Clinvar accession number is VCV002507024.2 VCV002507024.2 - ClinVar - NCBI (nih.gov)). In silico predicting splicing tools: SpliceAI, MaxEntscan and splice site finder concluded to a probable pathogenic effect of this variation on splicing by creating a new splicing acceptor site in intron 4 sequence (Splice AI AG score 0.80). RNA study was performed on RNA extracted from Paxgene^® (BD-Biosciences) blood tubes for patient 3 and her parents and from patient 3’s fibroblasts. It showed the retention of 18 nucleotides in the sequence of the cDNA at homozygous state in the proband and at heterozygous state in both parents, due to the use of the new cryptic acceptor-splicing site created by the variation (Figure 1). Ratio of relative expression of the SLC5A6 gene compared to the housekeeping gene ABL obtained in RT-q-PCR performed on the proband fibroblasts cDNA and on fibroblasts reference samples cDNA showed normal values and are not suggestive of changes in SLC5A6 mRNA expression levels. This suggests that the translation of the abnormal SLC5A6 mRNA results in the incorporation of 6 new amino acids (AA) in frame within the fourth transmembrane domain of the transporter (Holling et al., 2022), between AA 153 and 154.

Our study, associating family genetic and clinical data with functional mRNA tests allows to classify this variation as pathogenic according to ACMG guidelines (; on behalf of the ACMG Laboratory Quality Assurance Committee et al., 2015), with the following criteria: PP1-S, PM2, PP3, PS3.