Novel imaging findings in pyruvate dehydrogenase complex (PDHc) deficiency—Results from a nationwide population‐based study

The vast clinical and radiological spectrum of pyruvate dehydrogenase complex (PDHc) deficiency continues to pose challenges both in diagnostics and disease monitoring. Prompt diagnosis is important to enable early initiation of ketogenic diet. The patients were recruited from an ongoing population‐based study in Sweden. All patients with a genetically confirmed diagnosis who had been investigated with an MRI of the brain were included. Repeated investigations were assessed to study the evolution of the MRI changes. Sixty‐two MRI investigations had been performed in 34 patients (23 females). The genetic cause was mutations in PDHA1 in 29, PDHX and DLAT in 2 each, and PDHB in 1. The lesions were prenatal developmental in 16, prenatal clastic in 18, and postnatal clastic in 15 individuals. Leigh‐like lesions with predominant involvement of globus pallidus were present in 12, while leukoencephalopathy was present in 6 and stroke‐like lesions in 3 individuals. A combination of prenatal developmental and clastic lesions was present in 15 individuals. In addition, one male with PDHA1 also had postnatal clastic lesions. The most common lesions found in our study were agenesis or hypoplasia of corpus callosum, ventriculomegaly, or Leigh‐like lesions. Furthermore, we describe a broad spectrum of other MRI changes that include leukoencephalopathy and stroke‐like lesions. We argue that a novel important clue, suggesting the possibility of PDHc deficiency on MRI scans, is the simultaneous presence of multiple lesions on MRI that have occurred during different phases of brain development.

study in Sweden. All patients with a genetically confirmed diagnosis who had been investigated with an MRI of the brain were included. Repeated investigations were assessed to study the evolution of the MRI changes. Sixty-two MRI investigations had been performed in 34 patients (23 females). The genetic cause was mutations in PDHA1 in 29, PDHX and DLAT in 2 each, and PDHB in 1. The lesions were prenatal developmental in 16, prenatal clastic in 18, and postnatal clastic in 15 individuals. Leigh-like lesions with predominant involvement of globus pallidus were present in 12, while leukoencephalopathy was present in 6 and stroke-like lesions in 3 individuals. A combination of prenatal developmental and clastic lesions was present in 15 individuals. In addition, one male with PDHA1 also had postnatal clastic lesions. The most common lesions found in our study were agenesis or hypoplasia of corpus callosum, ventriculomegaly, or Leigh-like lesions. Furthermore, we describe a broad spectrum of other MRI changes that include leukoencephalopathy and stroke-like lesions. We argue that a novel important clue, suggesting the possibility of PDHc deficiency on MRI scans, is the simultaneous presence of multiple lesions on MRI that have occurred during different phases of brain development.

K E Y W O R D S
pyruvate dehydrogenase complex deficiency, magnetic resonance imaging, stroke-like lesions, leukoencephalopathy, Leigh-like lesions.

| INTRODUCTION
The pyruvate dehydrogenase complex (PDHc) is a multienzyme complex in the mitochondria that converts pyruvate, the end product of glycolysis to acetyl-CoA which in turn enters the Krebs cycle, and enables aerobic energy production. 1 The PDHc is composed of four subcomplexes: pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3), and E3-binding protein (E3BP also known as PDH protein X). In PDHc deficiency, pyruvate is instead metabolized to lactate, leading to impaired production of energy through the mitochondrial respiratory chain. 1,2 The brain is especially susceptible to this metabolic alteration, as glucose is the obligatory pathway for energy production under normal conditions. Consequently, PDHc deficiency predominantly affects the central nervous system. 2,3 Alternative substrates to glucose in the brain are ketone bodies, and it has been demonstrated that ketogenic diet is an effective and safe treatment in most patients with the disorder. 4 There are six known genes associated with primary PDHc deficiency: PDHA1, PDHB, PDHX, DLAT, DLD, and PDP1, where the first one is the most common, accounting for up to 90% of all known mutations. [5][6][7] PDHA1 is X-linked and one would therefore expect the disease to occur more often in males. Despite that, many studies have found equal number of males and females, with variable survival and disease course. 2,5,8 In addition, secondary PDHc deficiency has been associated with defects in branched-chain amino acid catabolism, synthesis of lipoic acid and ironsulfur complexes, transport and metabolism of thiamine, as well as disorders of fatty acid β-oxidation and the tricarboxylic acid cycle. 9 PDHc deficiency has a large phenotypical variation ranging from neonatal encephalopathy with lactic acidosis to nonprogressive infantile encephalopathy, Leigh syndrome, and relapsing ataxia. 6 The brain lesions have been attributed to two main mechanisms: (a) low energy supply during embryonic development resulting in agenesis or hypoplasia of the corpus callosum or abnormalities of cortical gyration and (b) acute energy failure either prenatally resulting in ventriculomegaly and periventricular cysts or postnatally causing acute basal ganglia or brainstem lesions. 6,10 There are few research studies exploring the MRI phenotypes in PDHc deficiency, 11,12 with the existing knowledge originating mainly from case reports and smaller case series. Ventriculomegaly is the most frequent MRI finding, ranging from 35% to 85% in different cohorts. 2,3,5 Hypoplasia or agenesis of the corpus callosum is found in over one-third of patients with in PDHA1-related disease. 3 Leigh syndrome with variable involvement of basal ganglia, brain stem, and cerebellum has been reported in 35% of patients. 5,6,12 Periventricular cysts, polymicrogyria, or other abnormalities of the cortical gyration have also been described. 6,13,14 Despite the increasing applicability of next-generation sequencing (NGS) techniques to identify patients with PDHc deficiency, the vast clinical and radiological spectrum of the disease continues to pose challenges both in diagnostics and disease monitoring. Prompt diagnosis is important to enable early initiation of treatment. The aim of this nationwide population-based study was to delineate the neuroradiological spectrum of PDHc deficiency and its evolvement over time.

| Patients
In an ongoing population-based Swedish study of PDHc deficiency, we identified 34 patients who had been investigated with MRI scans of the brain. The patients were identified from registries at the two national metabolic centers and by contacting genetics, pediatrics, and neurology departments in the country. The patients were diagnosed between 1 January 1996 and 31 December 2019. The genetic cause was determined in all the patients, with mutations in PDAH1 in 29 patients, PDHX in 2, DLAT in 2, and PDHB in 1 patient ( Figure 1). There were 11 males and 23 females. Two of them were related, a mother and daughter. Repeated MRI investigations had been performed in 16 patients, and in total, there were 62 MRI investigations.

| MRI investigations
The MRI examinations were performed at eight Swedish hospitals, either for diagnostic purposes or as part of the patient evaluation, sometimes during an acute deterioration. The examinations were carried out using a 1-Tesla magnet in two patients and 1.5-Tesla or 3-Tesla magnets in the remaining patients. The examinations consisted of sagittal spin echo (SE) T1, axial fast SE T2 in all patients; coronal fluid attenuated inversion recovery (FLAIR) images in 25 patients and diffusion-weighted images (DWI) with apparent diffusion coefficient (ADC) in 26 patients. Magnetic resonance spectroscopy (MRS) was performed in 13 patients. It was performed in the basal ganglia in all patients and in the deep white matter and cortex in occasional patients. Additional imaging sequences were occasionally obtained, including T1 with contrast, gradient echo T2, diffusion tensor imaging, proton density imaging, 3D time-of-flight angiography, and fast imaging employing steady-state acquisition.

| Study design
The MRI investigations were reevaluated by a child neuroradiologist (LI) who was blinded to the patients' phenotypes, and a pediatric neurologist (AS), using a standardized evaluation form. The patients were then subdivided into those with prenatal lesions of developmental or clastic origin and those with postnatal lesions due to suspected acute energy failure. In the patients with repeat examinations, the evolution of the imaging findings was evaluated separately for each patient and was related to significant clinical incidents and treatment. Each follow-up examination was compared to the preceding one, and the abnormalities were systematically classified as stable (no changes detected on DWI or T2 sequences), progression (new lesions present or number and/or extension of previously visualized lesions had increased), regression (complete resolution on both DWI and T2 images of lesions, previously visible), or evolution (normalization of DWI with persistent T2 changes or decreased size of the T2 signal changes as a result of encephalomalacia/atrophy). 15 We report the first abnormal MRI in all but two patients while MRS was reported if abnormal at any time. For patient #24 and #26, a later MRI that was more representative than the first one is reported, even if the first one was also abnormal.

| Terminology
The categorization used to describe the extent of the lesions was trichotomized into mild, moderate, or severe. A prenatal developmental lesion was considered in the presence of agenesis or hypoplasia of corpus callosum, abnormal cortical gyration, or gray matter heterotopias. A prenatal clastic lesion was considered in the presence of ventriculomegaly, considered to be of prenatal origin, ventricular membranes, periventricular cysts, or periventricular high T2 signal suggesting gliosis. A postnatal lesion was considered in patients with increased T2 signal, reduced diffusion, or presence of edema suggesting ongoing damage not related to gliosis. Bilateral symmetrical lesions detected in basal ganglia and/or thalamus and/or brain stem with hyperintense signal on T2-weighted and variable signal on T1-weighted imaging suggested Leigh syndrome. Leukoencephalopathy was considered if the images showed hyperintensities of the cerebral white matter in T2-weighted images with swelling, with or without diffusion changes. Reduced volume was the term used when the volume was reduced compared to what was expected for age. Atrophy was used when the volume was reduced compared to the previous radiological images. Cortical atrophy was characterized by the presence of cortical thinning leading to enlarged cortical sulci. Abnormalities of the corpus callosum were classified as follows: total agenesis, when totally absent; partial agenesis when there was absence of  at least one, but not all, of the anatomically defined regions of corpus callosum; and hypoplasia when the corpus callosum was thinner than expected but with normal anterior-posterior extent. 16 Myelination was characterized as normal, if normal compared to age-matched healthy individuals. Delayed myelination was defined as a substantial deficit in myelin deposition in the brain, with "catchup" if it was first delayed but was normal on follow-up. [17][18][19] Hypomyelination was defined as permanent, unchanged myelin deficiency in the brain, as demonstrated in a follow-up MRI while dysmyelination was defined as abnormal myelin formation. 20 High T2 signal was detected by T2-weighted images. DWI and ADC characterized the signal changes as vasogenic or cytogenic. Cytotoxic edema was considered when it appeared bright on DWI and dark on ADC, while vasogenic edema was considered when it appeared dark on DWI and bright on ADC.

| Standard protocol approvals, registrations, and patient consents
The study was approved by the Ethical Review Board at Gothenburg university (#289-17, 106-04). Oral and written consents were obtained by the patients and/or their legal guardians before undertaking any study-related procedures.

| RESULTS
We identified three patients with normal MRIs of the brain, all with postnatal onset. One male with PDHA1related disease experienced intermittent ataxia and epilepsy at the age of 3 years and 8 months. The MRI was performed at the same age. Another male with DLATrelated disease experienced ataxia and dysarthria, at the age of 2 years and 4 months. The MRI was performed 6 months later. One female with PDHA1-related disease had psychomotor delay from the age of 6 months and normal MRIs at the ages of 3 and 6 years.

| MRI changes in patients with prenatal disease onset
MRI changes of the brain considered to be of prenatal onset were observed in 19 patients ( volume of basal ganglia, thalamus, or pons in 7; and periventricular heterotopias identified in 1 of the patients. The myelination was delayed in three of them. Suspected prenatal clastic lesions were in addition identified in 13/14 of the females with developmental abnormalities, with ventriculomegaly in all and ventricular membranes in 10/14. The ventricular membranes were always associated with ventriculomegaly. Periventricular cysts were present in one of the patients. In two of the females (#7 and #9) with developmental and prenatal clastic lesions, additional postnatal lesions were also noted with generalized vasogenic edema in the supratentorial white matter (Figure 2A-H). Suspected prenatal clastic lesions without signs of developmental abnormalities or postnatal lesions were present in two of the females (#13 and #16). Widening of the sulci was identified in 11/16 of the females with PDHA1-related disease and was predominantly located to the frontotemporal area in six of them. It was always associated with ventriculomegaly. Supratentorial white-matter atrophy was present in 13/16, in 1 female without relationship to either ventriculomegaly or abnormalities in corpus callosum. One male (#17) with a hemizygous PDHA1 mutation had lesions considered to be prenatal developmental as well as pre-and postnatal clastic. The postnatal clastic lesion consisted of generalized cytotoxic edema in supratentorial and cerebellar white matter ( Figure 2I-L). One male (#18) with hemizygous PDHA1 mutation had isolated increased T2 signal in periventricular white matter. The MRI of the brain in the patient with PDHX-related disease (#19) demonstrated partial agenesis of corpus callosum and delayed myelination and atrophy of supratentorial white matter. In total, ventriculomegaly was present in 15/19 patients and was asymmetric in eight.

| MRI changes in patients with postnatal disease onset
Clinical onset was considered postnatal in 15 patients. The 12 patients with abnormal MRI findings are included in Table 2 Figure 3F-J). All but one patient with postnatal onset developed Leigh-like changes. Involvement of the basal ganglia with abnormalities in the globi pallidi was seen in all of them while involvement of putamina, nuclei caudati, and globi pallidi was seen in five patients. Involvement of the brainstem was present in four, of which two had abnormalities in midbrain, pons, and medulla oblongata and two in midbrain only. Involvement of thalamus was seen in two patients, all in combination with basal ganglia and brainstem abnormalities. Supratentorial white-matter changes were identified in eight of the patients with postnatal onset. Increased T2 signal of unspecific origin was identified in four of them.
In three of the patients, there was a swollen appearance of supratentorial white matter with generalized cytotoxic (#24) or vasogenic edema (#25 and #30). Mild ventriculomegaly was seen in three of the patients, all males. Cerebellar involvement was identified in five patients with variable involvement of cortex, white matter, or nuclei dentati. The MRI in the patient with PDHBrelated disease (#29) showed hyperintense T2 signal in posterior horns of the lateral ventricles, medulla oblongata, and the nuclei dentati. The MRI in the patient with DLAT-related disease showed Leigh-like lesions.

| Evolution of MRI changes in patients with repeated investigations
Repeat MRI investigations were done in 17 of the patients, on 43 occasions in total (Table 3)

| DISCUSSION
Even though PDHc deficiency is considered the major cause of primary lactic acidosis in children, 13 most previous studies have been small, and none have systematically reviewed the neuroimaging findings in a large population-based cohort. Similar to previous studies, 3,5,8 the most common lesions found in our study were agenesis or hypoplasia of corpus callosum, ventriculomegaly, or Leighlike lesions. In addition, we describe a broader spectrum of MRI changes that include leukoencephalopathy and strokelike lesions. An important clue from our study that should suggest the possibility of PDHc deficiency is the simultaneous presence of multiple lesions on MRI that have occurred during different phases of brain development.
The energy dependency of different neuroanatomic sites of the brain is related to the different phases of brain development. Consequently, the nature and localization of brain injury is dependent on the timing of the injury. 21 Two pathophysiological mechanisms have been proposed in PDHc deficiency, either developmental or degenerative, the latter in which cell death leads to encephaloclastic lesions. 22 Developmental lesions occur during the second trimester of pregnancy when neuronal proliferation, migration, and differentiation require large amounts of energy. It is also from this period that the development of the brain becomes dependent on the enzymatic activity of pyruvate dehydrogenase and pyruvate carboxylase. 23 Typical developmental lesions found in neuropathological studies of PDH deficiency consists of migration anomalies, corpus callosum dysgenesis, malformed nuclei dentati, internal granular cell layer and Purkinje cell layer paucity, and abnormal and ectopic inferior olivary nuclei. 24 These lesions are reminiscent of the fetal alcohol syndrome, 2 in which similar pathophysiological mechanisms to PDHc deficiency could be present. Excessive intake of alcohol is associated with deficiency of thiamine, the cofactor of PDHc, 25 and in addition, acetaldehyde, the product of alcohol oxidation, has been found to have an inhibitory effect on PDHc. 2 The second proposed mechanism is degenerative and results from acute failure of energy supply, responsible for cell death, and leading to encephaloclastic lesions which can occur either prenatally or postnatally. Prenatal degenerative neuropathological changes in PDHc deficiency consist of a variety of lesions, including cortical and white-matter atrophy, calcified migrating neurons, subependymal pseudocysts, periventricular cavitary, and noncavitary lesions surrounded by reactive gliosis, ventricular dilatation, and basal ganglia calcifications. 24 The presence of ventricular septations may also be the result of a destructive process and may represent areas of incomplete porencephaly or cystic lesions. 11 The clastic lesions in periventricular white matter and associated ventriculomegaly described in PDHc deficiency bear similarities both on neuroimaging and neuropathological studies to the hypoxic-ischemic periventricular leukomalacia/diffuse white-matter injury-like lesions typical of cerebral palsy in preterm infants. 24 It is therefore important to consider PDHc deficiency in the differential diagnosis of cerebral palsy in girls with atypical perinatal history. In contrast to a previous case report that emphasized the asymmetry of ventricular dilatation in PDHc deficiency as a distinguishing Stroke-like lesions were detected in three of our patients with PDHA1-related disease. While we have not been able to find any previous study describing strokelike episodes in PDHc deficiency, they do occur in other mitochondrial diseases, that is, POLG-related Alpers disease 19 and mitochondrial encephalopathy with lactic acidosis and stroke-like episodes. 26,27 Five patients with PDHA1-and one with PDHXrelated disease, all but one investigated in the neonatal period, had a very similar MRI pattern suggestive of leukoencephalopathy with generalized swollen appearance of supratentorial white matter due to vasogenic edema in five and cytotoxic edema in two of the patients. The different pattern of edema seen in these patients is probably reflective of the time frame of the MRI investigation in relation to the development of the underlying lesion, as brain edema could be considered as a continuum starting with cytotoxic cell swelling and ending in vasogenic edema. 28 A suggestive clue on MRI in these patients was the additional presence of either prenatal or Leigh-like lesions. This MRI pattern is similar to a previous case report 14 but seems otherwise underreported in PDH deficiency and was not mentioned in a comprehensive review of 371 published patients. 3 In addition, delayed myelination was seen in four patients with prenatal onset while four patients with postnatal onset had unspecific white-matter lesions probably related to dysmyelination or gliosis. We hypothesize that the white matter could be particularly vulnerable to disturbances in the pyruvate metabolism, especially during the fetal period and first month of life. This assumption could be of interest to study further in an animal model of the disease.
All but one of the patients with postnatal onset developed Leigh-like lesions, which is the most common presentation of mitochondrial diseases in childhood. 29 It is characterized by focal symmetrical lesions affecting the basal ganglia, diencephalon, brainstem, cerebellum, and spinal cord, typically occurring between 3 and 12 months of age when these parts of the brain are especially vulnerable to disruption in energy supply. The basal ganglia involvement in our study, with predominant involvement of the globi pallidi, is similar to previous studies 30, 31 and differs from what has been described in other mitochondrial diseases where the lesions more commonly involve the striatum. Early diagnosis is important as the deep gray-matter lesions could be reversible after introducing a ketogenic diet. 4,12 PDHA1 deficiency is an X-linked disorder leading to different manifestations between the sexes. In our cohort, there were more than twice as many females than males. This difference is derived from the group with prenatal onset and developmental lesions whereas the distribution between the sexes was equal in patients with postnatal onset. The reason for this difference could be explained by the fact that in affected males, severe PDHA1 mutations that essentially abrogate enzyme activity in the cells are thought to be embryonically lethal and only affected male fetuses with milder mutations, and significant residual enzyme activity is believed to survive until birth. 13,32 Consequently, newborn boys with PDHA1 deficiency typically do not manifest severe structural brain anomalies at birth 11 although this has been described occasionally 32 (and was found in one of our patients). In female patients, random X inactivation leads to expression of either the mutant or normal allele in neuronal cells. Therefore, even severe PDHA1 mutations result in a viable female pregnancy because cells expressing the normal allele ensure fetal survival, whereas affected cells are considered nonviable, and their death has been suggested to result in developmental abnormalities of the brain. 33 It is believed that female patients with prenatal onset PDHA1related disease, due to severe mutations and lack of residual enzyme activity, display a nonprogressive course and that interventions such as a ketogenic diet would not be effective given the lack of responsiveness on a cellular basis. 12 In our study, two of seven female patients with prenatal developmental and clastic lesions (#7, 9) showed progression on repeated MRI-s with additional postnatal lesions. It's is therefore possible that the pathophysiological mechanism could be more of continuum and that a ketogenic diet might be effective also in girls with developmental lesions as has been previously suggested. 4 Even though the majority of females with PDHA1 deficiency in our study had developmental abnormalities, we identified a more variable MRI phenotype than previously described with absence of developmental lesions in one of three and postnatal onset with stroke or Leigh-like lesions in one of five of the females. The MRI changes in females with postnatal onset appeared milder than in the males. The vast predominance of girls in our study differs from previous studies that have shown equal distribution between the sexes. 2,3,5,8,13 An explanation for this difference could be better awareness and more efficient NGS tools for diagnosis in our study compared to earlier studies. Previously, the diagnosis relied solely upon enzymatic methods which could miss the diagnosis in girls due to variable tissue distribution of the enzyme deficiency. 13 Newborn screening might be a future option to facilitate early diagnostics and treatment.
The classification into prenatal and postnatal disease onset was performed from a pragmatic perspective and should be considered as arbitrary, as the underlying disease mechanisms most likely constitute a continuum. Our cohort of patients, even though comparably large, is probably not representative of the full disease spectrum in the general population, as there is still likely a significant underdiagnosis, both in the severe and mild end of the spectrum, making solid conclusions difficult. Another limitation of our study was its retrospective design with investigations performed using different MRI scanners and protocols. The strength of our study was that we performed a systematic reevaluation of all available MRI scans, from a comparably large, nationwide, and population-based cohort of patients. Our findings add to a better understanding of the neuroradiological spectrum of PDHc deficiency and its evolvement over time, which in turn may improve timely diagnosis and enable earlier initiation of diseasemodifying treatment.