The index patient was the youngest of five children of nonconsanguineous parents of ethnic Georgian origin. The mother was reported to be healthy; the father suffered from severe migraines. The oldest son of the family, currently 29 years old, was in good health. The 23-year-old daughter reported chronic gastrointestinal complaints but did not have neurological problems and had a healthy three-year-old son. All three younger siblings developed progressive neurological symptoms in early childhood: the first affected son developed normally until the age of five years when he suffered focal myoclonic jerks in the right hand, followed by speech arrest and right arm weakness. These symptoms resolved after two weeks, and he regained the ability to ride a bicycle. However, three months later a second episode occurred with severe headache and abdominal cramps. As appendicitis was ruled out and liver enzymes were normal he was dismissed from the hospital. On the following day, he appeared pale and stopped speaking. A few hours later, acute deteriorationwith somnolence and coma occurred; he died 24 hours later in a Russian hospital without an exact diagnosis. Imaging or lactate findings from this patient were not available. The second affected son also developed normally up to the age of five years. Then, unexpectedly, he lost consciousness and presented rightsided hemiparesis; a cranial MRI showed normal findings. A second episode occurred at seven years of age: in the course of an unspecific pneumonia he transiently lost consciousness and vision. Suspecting epileptic seizures, he was treated with carbamazepine, but his EEG was reported to be normal. An MRI showed non-specific changes of the bilateral occipital white matter. His third episode occurred at the age of 10 years. After an unspecific illness, focal convulsive status epilepticus (epilepsia partialis continua) occurred with deviation of head and eyes to the right and twitching of the right side of the face, accompanied by left-sided focal spike and slow-wave activity. Liver enzymes and all other blood tests were normal. The MRI was unchanged. After prolonged treatment with thiopentone-anaesthesia at a Georgianhospital, he was alert and responsive for three days. However, acute severe deterioration occurred, and the boy died within 10 hours due to recurrent status epilepticus, acidosis, anuria and respiratory failure.

History. The third affected son of the family was born prematurely after a normal pregnancy at the 35th week of gestation but without further complications. His development in infancy was normal: he sat up at six months, walked independently at 14 months, and spoke his first words at 10 months. A first episode of vomiting and abdominal cramps occurred at the age of eight years and four months. These symptoms ceased after two weeks, but his muscular strength remained somewhat reduced. A second episode occurred one year later. Following an onset with vomiting and abdominal pain, micturition ceased, his consciousness deteriorated, he became comatose, and he required mechanical ventilation for three weeks. Laboratory values reported from the Georgian hospital showed normal CSF-lactate and serum transaminases, borderline raised creatine kinase (CK) and hypernatraemia, low free carnitine (1.1 mg/dl, a lower limit of normal 3.5 mg/l), and significantly raised acetoacetate. He slowly recovered after this episode with residual muscle weakness, but was unable to walk for longer distances. He was unable to attend school but was educated by a private teacher.

Clinical presentation. Living in Georgia, the boy was presented at Freiburg University Medical Center (FUMC), Germany, for the first time at the age of 10 years and four months. At the initial clinical evaluation, he appeared alert and interacted adequately. However, no formal cognitive testing was performed. He was short for his age and underweight (height 118 cm (0.1 percentile), weight 16.2 kg (0.3 percentile), body mass index (BMI) 11.7 (-4.2 SDS). He looked pale, but his cardiovascular system, lungs, abdominal organs and skeleton appeared normal. With the exception of a very slight ptosis, cranial nerve function was normal, and there was no ophthalmoplegia or nystagmus. His muscle tone and strength were moderately reduced. Deep tendon reflexes (DTR) were normal at the upper limbs, whereas they were not elicitable at the lower limbs. The Babinski signwas negative. His gait was moderately ataxic, with a slightly positive Romberg sign. Standing and hopping on one leg were possible only with support, and his tandem gait was grossly abnormal. His Gowers’ sign was positive, and time to rise from supine was 25 seconds. He showed bilateral dysdiadochokinesis, but the finger-nose test was without abnormalities.

Specialist investigations. Ophthalmological investigations revealed a pale fundus with some abnormal pigmentation, but eye movements, electroretinography (ERG) and perimetry were normal. Ears, nose and throat (ENT) investigations revealed normal auditory function.

Radiological and ultrasonographic investigations. A cranial MRI at the age of 10 years and four months showed diffuse T2-hyperintense lesions in the deep white matter of both hemispheres with sparing of U-fibers. There was also a subtle increased T2-signal in the caudate heads and putamen (Figure 1). Diffusion-weighted imaging was normal and there was no abnormal contrast-enhancement. A magnetic resonance spectroscopy (MRS, PRESS 30 ms) of the basal ganglia revealed normal findings without an elevated lactate peak or other abnormal peaks. NAA/Cr and Cho/Cr ratios were within normal limits. An MRS of occipital white matter was not performed. An X-ray of the left hand showed delayed skeletal maturation (seven years and +/- 11 months at the age of 10 years and four months) and osteopenia. Ultrasonographic investigations of heart, thyroid and abdominal organs showed no abnormalities.

Further technical investigations. Spirometry was conducted without abnormalities. Electroencephalogram (EEG) background activity during waking was normal for his age, and there were no epileptic potentials or focal slowing. Nerve conduction studies gave evidence of a diffuse sensorimotor neuropathy with moderate slowing of motor and sensory nerve conduction velocities (mNCV, sNCV) and low amplitudes (A): median nerve mNCV 35 (normal [n] > 50) m/s, A 2.0 (n > 4) mV; tibial nerve mNCV 31 (n > 40) m/s, A 4.2 (n > 2) mV; median nerve sNCV 39 (n > 50) m/s, A 2.5 (n > 15) mV; sural nerve NCV 34 (n > 50) m/s, A 4.8 (n > 20) mV.

Muscle biopsy. In the index case, a muscle biopsy from the lateral vastus muscle showed very slight myopathic changes on light-microscopy (hematoxylin-eosin [HE] and trichrome stains). On histochemical staining an increase of COXnegative/ SDH-positive fibres, as well as a prominent smalldroplet

intracellular lipid accumulation (Oil Red O), were found. No neurogenic changes, no inflammatory infiltrates, and no increased glycogen accumulation were seen. Investigation of the activity of the respiratory chain complexes was performed in skeletal muscle homogenate as previously described [7] and detected normal activities of complexes I-III, but a borderline low activity of Complex IV (103.1 U/gNCP; normal range 112-351; 1.07 related to citrate synthase; normal range 1.1-5.0).

Material for genetic investigations. We were able to study muscle DNA of the index patient, as well as DNA extracted from peripheral blood of the index case, his healthy brother, and the parents. A dried blood spot of one of the deceased siblings contributed DNA for that case. In addition, we studied paternal DNA extracted from blood, saliva, buccal swab, and hair bulbs. The sister refused genetic testing in herself and her son.

Muscle DNA. In a semiquantitative analysis by a real-time polymerase chain reaction (PCR) test [8], muscle DNA of the index patient revealed reduced mtDNA copy numbers, suggestive of mtDNA depletion (20–30% of mtDNA, relative to nuclear DNA compared to controls). No mtDNA deletions were found on standard diagnostic testing with long-range PCR and Southern blot.

Polymerase-gamma-gene (POLG) analysis. Sanger sequencing of all coding regions of POLG with exon-intron boundaries in blood DNA of the index patient detected two heterozygous variants, while MLPA analysis was normal. One of these NM_002693.2: c.680G > A p.(Arg227Gln) is a missense variant that has recently been described in three infants with mitochondrial depletion syndrome (MDS) and Alpers-Huttenlocher syndrome (AHS) [9-11]. This variant was also present in blood DNA of the deceased brother but not in the parents and the healthy brother. A buccal swab, saliva and hair bulbs of the father were also negative for this mutation. Paternal blood DNA was additionally tested by deep targeted next-generation sequencing (coverage > 2500) for this POLG variant and was negative. However, paternity was confirmed by genetic markers investigated in both parents and the index case. The second variant c.3148A > Tp.Lys1050* that has not yet been described in the literature gives rise to a stop-codon; it was detected in the index patient and the deceased brother, and was also found in the mother‘s blood, but not in that of the father and the healthy eldest brother. These data suggest the presence of paternal somatic or, most probably, germline mosaicism for the c.680G > A p.(Arg227Gln) (see discussion).

The POLG gene at chromosomal location 15q26.1 codes for the catalytic alpha subunit of the polymerase-gamma. Together with additional proteins, polymerase-gamma is involved in replication of the single-stranded mitochondrial DNA (mtDNA). Pathogenic variants, if biallelic, may result in severe early onset autosomal-recessive disorders with mtDNA depletion, while heterozygous variants cause later-onset autosomal-dominant disorders with multiple mtDNA deletions [1-4].

In our index case, we found two heterozygous variants in the POLG gene, the missense variant c.680G > A p.(Arg227Gln) and the stop variant c.3148A > T p.Lys1050*. The sequence change c.3148A > T creates a premature translational stop signal (p.Lys1050*) in the POLG gene. Since this premature termination codon (PTC) is not located in the last or penultimate exon of POLG, nonsense-mediated decay (NMD) of mRNA can be assumed which is expected to result in an absent protein product. The variant c.3148A > T p.Lys1050* is not present in population databases (ExAC no frequency) or clinical databases (ClinVar). It was classified as pathogenic (class 5: evidence codes PVS1, PM2, PM3, PP3) according to the ACMG guidelines . The missense variant p.Arg227Gln results from a G to A substitution at nucleotide position 680. This nucleotide change is absent in population databases (ExAC no frequency). However, the resulting amino acid change from arginine to glutamine at codon 227, which occurs at a position that is conserved across species, has been reported in clinical databases (ClinVar) as pathogenic and as compound heterozygous in several individuals with POLGrelated disorders. Bioinformatic algorithms predict a potential impact of the missense change on protein structure and function (SIFT: ‘Deleterious’; PolyPhen-2: ‘Probably Damaging’). Based on this evidence, this variant was classified as pathogenic (class 5: evidence codes: PS1, PM2, PM3, PP1, PP2, PP4) according to the ACMG guidelines [12]. The pathogenicity of both variants is further supported by the finding of the mtDNA depletion in the muscle biopsy of our index case.

Segregation analysis in the family (Figure 3) supported pathogenicity by revealing that the index patient and one affected sibling each carried both heterozygous variants. There was no DNA available from the third affected brother. The healthy brother did not carry any of the two variants. Consistent with autosomal recessive inheritance, the healthy mother was found to be a heterozygous carrier for one of the variants (c.3148A > T p.Lys1050*). However, the variant c.680G > A p.(Arg227Gln) was absent from the father´s blood sample. Paternity of the father could be confirmed by genetic markers investigated in both parents and the index case.

Searching for somatic mosaicism of variant c.680G > A p. (Arg227Gln) in the father, additional tissues (buccal swab, saliva and hair bulbs) were investigated by targeted Sanger sequencing but were found to be negative. As Sanger sequencing is known to have a detection limit of 10% to 20% [13], low-grade somatic mosaicisms might only be detectable by more sensitive methods. It could be more frequent than previously assumed as deep sequencing of parental samples from children with de novo variants in genes causative of epileptic encephalopathy revealed low levels of somatic mosaicism ranging from 1–10% in blood in 5–6% of cases [14,15]. However, as deep sequencing was negative in our patient´s father, we rather suggest a germline mutation as the underlying cause. Germline mutations giving rise to affected siblings in apparent non-carrier parents are well known in autosomal-dominant and X-linked diseases. However, parental somatic or germline mosaicism in autosomal-recessive conditions have rarely been described [16,17].

Discussion of the multiple phenotypes in this family It is known that pathogenic variants in the POLG gene can lead to multiple phenotypes. In young infants with mtDNA depletion, myocerebrohepatopathy spectrum (MCHS) disorder is the earliest occurring and most severe phenotype. It is followed by Alpers-Huttenlocher syndrome (AHS), which leads to death by the end of the first decade [1-4,18]. In contrast, diseases associated with mtDNA deletions, such as myoclonic epilepsy myopathy sensory ataxia (MEMSA), ataxia neuropathy spectrum (ANS, SANDO) disorders, autosomal recessive (ar- CPEO), and autosomal dominant progressive external ophthalmoplegia (adCPEO), occur in older age groups [4]. It is important to note that there is a strong overlap between these phenotypes, most of which were described before the discovery of the common genetic etiology. The following additional symptoms and signs have been reported to occur in AHS and pathogenic POLG variants: failure to thrive, Leigh syndrome with dystonia and pathology of basal ganglia and brainstem, migraine attacks, stroke-like episodes with and without MRI changes (similar to MELAS), pancreatitis and gastrointestinal dysmotility and pseudo-obstruction similar to mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) [1-4]. Tang et al. found four patients with MNGIE-like disease in a cohort of 92 patients with POLG variants when reevaluating clinical data; their age at diagnosis was 15, 25, 46 and 50 years. POLG-MNGIE could be differentiated from typical TYMP-MNGIE by the lack of CNS leucoencephalopathy [19].

Several of these syndromes and symptoms were found in the family presented here, but with considerable variability between the affected brothers. A common feature was the relapsing, repetitive character of the neurological and systemic symptoms. The symptoms in the older brothers with recurring epilepsia partialis continua and encephalopathy could be well classified as Alpers disease; similar to our index case, they showed no liver involvement [1-4,18]. Their periodic episodes with coma or hemipareses appeared to be stroke-like episodes, de

Activated folate is an important cofactor in nucleic acid biosynthesis, one carbon metabolism and amino acid metabolism, and thus has an essential role in cell-proliferation and blood-cell regeneration, as well as in CNS metabolism and CNS-function. Decreased CSF 5-MTHF may be a consequence of nutritional folate deficiency or inherited intestinal folate malabsorption (for example due to pathogenic variants in the SLC46A1 gene); in both conditions low blood folate levels in serum, red blood cells and CSF are found. But low CSF 5-MTHF may also be due to impaired transport at the blood- CSF-brain barrier despite normal serum and red blood cell folate. A progressive neurological syndrome in young children has been published due to low CSF 5-MTHF as a consequence of antibody-mediated folate receptor dysfunction [20]. Steinfeld et al. first described a genetic syndrome due to pathogenic variants in the folate receptor-1 gene (FOLR1), leading to folate receptor-alpha (FRa) deficiency. Their patients suffered progressive movement disturbance, psychomotor regression, ataxia and myoclonic epilepsy. The MRI showed profound hypomyelination, and MRS indicated a combined depletion of white-matter choline and inositol [6]. The same group demonstrated that folate transport via the blood-CSF barrier is mediated by folate receptor-alpha dependent transcytosis. Fra is localised on the basolateral and apical brush border surfaces of the choroid plexus epithelium. 5-MTHF transport into the brain parenchyma is mediated by exosomes that transport 5-MTHFladen FRa molecules, traversing the cytosol of choroid plexus epithelial cells from the basolateral to the apical membranes, before being secreted into the CSF at the apical brush border. This suggests that the secretion of FRa is the major transport pathway for cerebral folate [5,21,22].

In both ‘auto-immune’ and genetic cases, improvement in neurological function and development was achieved by high dose oral and intravenous treatment with folinic acid, an activated folate that is not transporter-dependent. In genetic FOLR1 (folate receptor alpha) deficiency, treatment with oral folinic acid supplementation at doses ranging from 0.5–7 mg/kg/day has been reported to result in improvement in seizures, neuro-imaging changes and CSF 5 –MTHF levels, although the response may vary depending on the duration of symptoms and timing of treatment initiation [5].

Secondary CSF-5-MTHF deficiency in patients with mitochondrial diseases results from deficient activity of FRa due to mitochondrial dysfunction of the choroid plexus epithelium, where vesicle-mediated FRa is located (see above). Low CSF levels of folate in Kearns-Sayre syndrome (KSS) have been known since the 1980s and before the discovery of the causal mtDNA deletions [23]. Hasselmann et al. reported on a girl of three and a half with Alpers disease, a distinct CSF 5-MTHF deficiency, and raised inflammatory markers in CSF along with elevated antibodies against folate receptor [24]. The child died two years later due to disease progression, but treatment with high-dose folinic acid had by then resulted in cessation of status epilepticus and improved neurological function. Grapp et al. [22] not only showed an absence of FRa in the CSF of patients with biallelic FOLR1 variants; they also demonstrated a correlation between the reduction of FRa and a low 5-MTHF concentration in CSF in patients suffering from KSS (Figure 2). Tanji et al. studied the brains in two deceased patients with KSS and described a unique oncocytic transformation of the choroid plexus epithelium. They postulated that these abnormalities were due to the accumulation of abnormal deleted mtDNA which may affect the integrity of the choroid plexus, thereby affecting the blood-brain barrier with loss of FRa, presumably via decreased exosome-shuttling, leading to low 5-MTFH levels and raised CSF protein [23]. Elevated CSF protein has been demonstrated in 70% of patients with POLG-related epilepsies and the level of CSF protein correlates with the severity of the clinical course [25].

Our patient with POLG-related disease appears to be a further case with loss of CSF FRa, secondary to mitochondrial dysfunction. It is not possible to determine which part of his symptoms was due to the mitochondriopathy and which to the secondary folate deficiency. However, there is an overlap of symptoms between both conditions. Therefore, his neurological disability may have been aggravated by the impaired folate transport. He was treated with high dose oral and intravenous folinic acid for more than 18 months. A partial recovery of neurological symptoms under treatment with folinic acid supports the notion that cerebral folate deficiency may have contributed to his neurological symptoms. However, despite an objective improvement in neurological function, the boy eventually died because of the severe gastrointestinal manifestations and its complications.

Korinthenberg R. et al. (2020). Alpers- and MNGIE-like disease with disturbed CSF folate transport and an unusual mode of genetic transmission of POLG mutations: a case report. Journal of the International Child Neurology Association, 20(216). https://doi.org/10.17724/jicna.2020.216