Biallelic POC1A variants cause syndromic severe insulin resistance with muscle cramps

Objective To describe clinical, laboratory, and genetic characteristics of three unrelated cases from Chile, Portugal, and Saudi Arabia with severe insulin resistance, SOFT syndrome, and biallelic pathogenic POC1A variants. Design Observational study. Methods Probands’ phenotypes, including short stature, dysmorphism, and insulin resistance, were compared with previous reports. Results Cases 1 (female) and 3 (male) were homozygous for known pathogenic POC1A variants: c.649C>T, p.(Arg217Trp) and c.241C>T, p.(Arg81*), respectively. Case 2 (male) was compound heterozygous for p.(Arg217Trp) variant and the rare missense variant c.370G>A, p.(Asp124Asn). All three cases exhibited severe insulin resistance, acanthosis nigricans, elevated serum triglycerides and decreased HDL, and fatty liver, resembling three previously reported cases. All three also reported severe muscle cramps. Aggregate analysis of the six known cases with biallelic POC1A variants and insulin resistance showed decreased birth weight and length mean (s.d.): −2.8 (0.9) and −3.7 (0.9) SDS, respectively), severe short stature mean (s.d.) height: −4.9 (1.7) SDS) and moderate microcephaly (mean occipitofrontal circumference −3.0 (range: −4.7 to −1.2)). These findings were similar to those reported for patients with SOFT syndrome without insulin resistance. Muscle biopsy in Case 3 showed features of muscle involvement secondary to a neuropathic process. Conclusions Patients with SOFT syndrome can develop severe dyslipidaemic insulin resistance, independent of the exonic position of the POC1A variant. They also can develop severe muscle cramps. After diagnosis, patients should be regularly screened for insulin resistance and muscle complaints.

movements. Occasional mandibular and tongue spasms also occurred. Local warming tended to relieve pain. Nerve conduction studies were normal and concentric needle EMG showed a reduced recruitment pattern of motor units with polyphasic potentials of increased amplitude, indicating motor neuron involvement. Spontaneous muscle twitches, similar to fasciculations, were noted in limbs. The EMG needle triggered painful vastus lateralis spasms, leading to prolonged continuous muscle activity. Cramps subsided with amitriptyline.
Oligomenorrhoea persisted, associated with low serum oestradiol, high serum LH, biochemical hyperandrogenism and increased serum Anti-Mullerian Hormone concentration, which normalised on regular metformin (Supplementary Table 1). Pelvic ultrasound showed a uterine length of 5.5 cm, ovarian volumes of 12 and 4 ml, and multiple follicles of <10 mm. Adult height was 120 cm (-6.6 SDS), weight 29.6 kg, BMI 20.6 (0.3 SDS), OFC 50.0 cm (-4.0 SDS) and arm span 120 cm. Leg length was 60 cm, with a normal upper/lower segment ratio of 1.0.
Metabolic evaluation from 8.7 years old (Supplementary Table 1) showed progressive insulin resistance (IR), treated with metformin 850mg bid from 18.8 years, decreased to 425mg bid due to gastrointestinal symptoms. Serum triglycerides remained elevated from 11 years. Fatty liver was inferred from elevated aminotransferases and at 20.8 years, confirmed by ultrasonography. Blood pressure was well controlled on losartan 25 mg/day. Renal sonographic appearances were normal at 2 and 19.3 years, but at 21.5 years microalbuminuria and elevated blood urea nitrogen were recorded.
Case 2 is a 25-year-old man, the only child of unrelated Portuguese parents. His mother had a normal height (165 cm, 0.3 SDS) (6). His father was reported to be of short stature (162 cm, -2.1 SDS) (6), with a prematurely aged appearance, hearing impairment, obesity, and loss of dentition by 36 years. He was of normal intellectual ability.
The proband was born at 43 weeks' gestation with a birthweight of 2.45 kg [-3.2 SDS (7)]. A relatively large head and probably bony dysplasia were noted at birth, but skeletal surveys on two occasions during childhood failed to reveal a known dysplasia. Psychomotor development was normal. At 8.1 years height was 98 cm (-6.4 SDS), weight 21 kg (-1.6 SDS) and BMI 21.9 kg/m 2 (2.5 SDS) (8). Upper segment length was 54 cm [upper/lower segment ratio 1.23, equivalent to 3.5 SDS (4)] and arm span 93 cm [arm span minus height -1.3 SDS for age (9)]. Several small café au lait patches and joint hypermobility were noted. Brain MRI was normal. rhGH therapy from 9.5 to 10.5 years yielded no benefit, and was discontinued due to weight gain. Numerous dental procedures were required due to supernumerary teeth and dental caries, exacerbated by rapid jaw growth. Orthodontic assessment at age 12 revealed class III malocclusion. Nail growth was normal. Puberty onset was reportedly at 11 years old. At 13.6 years old health was good except for muscle cramps. Height was 125.4 cm (-4.8 SDS), BMI 24.3 kg/m 2 (1.9 SDS) and OFC 53.8 cm (-1.2 SDS for age (8). Puberty was well advanced, with Tanner stage 3 genitalia and pubic hair, and testicular volumes 8 ml. Adiposity was centripetal but there was no frank lipodystrophy. Mild acanthosis nigricans was seen. There was brachydactyly and mild fifth finger clinodactyly with broad, short nails. There were scattered depigmented patches on the abdomen, and two small, irregular café au lait patches on the lower back. Muscle tone and limb reflexes were normal and no muscular hypertrophy was observed. Oral glucose tolerance testing revealed severe IR without diabetes, and reactive hypoglycaemia.
Elevated serum ALT was consistent with fatty liver, and serum creatine kinase was mildly elevated (Supplementary Table 2 Adiposity was centripetal with pronounced acanthosis nigricans. Arms and legs were short, but hair and nails were normal. Café au lait patches were unchanged. Biochemical evaluation demonstrated extreme fasting hyperinsulinaemia (Supplementary Table 2).
At 25 years old, he reported severe muscular pains, significantly worse than in teenage years. These were spasmodic, associated with paraesthesia in the fingers, and were exacerbated by cold. They were refractory to non-steroidal anti-inflammatory drugs, and limited activity, contributing to weight gain.
No muscular hypertrophy was noted. He also described, for the first time, rapid, patchy hair loss occurring over several weeks in a non-androgenic distribution. Developmental milestones were delayed from early childhood onwards. GH deficiency was suspected and rhGH therapy given from 8 years of age for 6 years, but information on serum IGF-I, GH stimulation testing and growth response is unavailable. Currently, he is semi-independent with an IQ of 68, has no secondary sexual characteristics, and has had alopecia since adolescence.
At 22 years his height was 135 cm (-5.8 SDS), weight 51.1 kg (-2.1 SDS), and BMI 28.2 kg/m 2 . He had brachydactyly, posteriorly rotated, low set ears, small, broad hands and feet with hypoplastic distal phalanges and nails, partial alopecia, and centripetal adiposity. He exhibited a high forehead, hypotonia, joint hyperlaxity, brachycephaly, hypertelorism, broad upturned nose, long philtrum, short palpebral fissure, widely spaced first and second toes, and single palmar creases. Skeletal survey revealed short femoral neck and phalanges, short left third metacarpal and metatarsal bone, hypoplastic distal phalanges and nails, and short, thick long bones. He had nuchal and axillary acanthosis nigricans and fatty liver, confirmed ultrasonographically. DM2 was diagnosed and managed with Metformin, Sitagliptin and Pioglitazone. On this regimen serum insulin was slightly increased, with C-peptide and adiponectin within normal limits (Supplementary Table 3).
At 25-26 years, lack of secondary sexual characteristics, plasma testosterone at or below the lower limit of normal and an empty sella on imaging prompted GnRH stimulation testing, which showed a normal FSH and LH response (10) (Supplementary Table 3). Three-weekly testosterone ester (250 mg) injections were prescribed, but compliance has been poor. Other pituitary axes were normal. Symptoms abated gradually, and serum creatinine kinase concentration decreased to 300 U/L (Supplementary Table 3). Serum calcium, phosphorus and magnesium concentrations were always normal.
At 29 years severe hyperglycaemia was noted with HbA1c of 12.7%, mandating insulin therapy. Non-proliferative diabetic retinopathy was found with persisting fatty liver on ultrasonography and elevated serum aminotransferase concentrations. Hypercholesterolaemia and hypertriglyceridaemia were managed with Atorvastatin 20 mg daily.

Supplementary information on genetic analyses
POC1A variants identified are described with reference to RefSeq accession number NM_015426.4.

Case 2:
Microarray revealed no pathogenic copy number changes. Exome sequencing of genomic DNA and variant calling were performed as part of the UK10K Project, as described previously (12). Raw sequence data is available from the European Genome-Phenome Archive (https://www.ebi.ac.uk/ega/home; accession EGAN00001015634). Two POC1A variants but no other plausible causal mutations were identified and were confirmed by Sanger sequencing.

Case 3:
Woodhouse-Sakati syndrome was excluded by full sequencing of C2orf37 and autozygosity analysis. Exome sequencing was then undertaken and combined with the autozygome analysis as previously described (13,14). No candidate variants in known myopathy genes were identified.  Patient was following by home glucose monitoring for blood sugar control. 2 On metformin 500mg twice daily, sitagliptin 100mg once daily and pioglitazone 15mg once daily.   -4.0 (-5.9;-2.1) @These patients developed diabetes mellitus type 2 at 20 and 26 years, respectively *SDS of birth length, weight and OFC was calculated based on Swedish reference (7) #SDS of these measures was calculated using Dutch references (27) ^Estimated based on percentile position &The values at the oldest reported ages were used for calculating mean and SD Other SDS values are copied from the original papers. Abbreviations: F, female; M, male; OFC, occipitofrontal circumference; SDS, standard deviation score.

Supplementary Figure 1.
Muscle biopsy of patient 3. A: Haematoxylin and eosin staining reveals focal myopathic features, including variation in fibre size. Angular atrophic and hypertrophic fibres, focal prominent perimysial components and focal clumps of increased internal nuclei (black arrows) (X400). B: Cytochrome oxidase shows scattered fibres with subsarcolemmal mitochondrial accumulation (black arrows) (X200). C: Immunostaining for the fast class of myosin heavy chain highlights predominance of type II fibres (X100). D: Electron microscopy confirms nuclear abnormalities of the scattered degenerating atrophic angular fibres with nuclear clumps observed at light microscopy (X6000).