New homozygous indel in MYORG linked to brain calcification, thyroidopathy and neuropathy

Sir, Primary familial brain calcification (PFBC) is a rare disorder characterized by bilateral brain calcifications affecting the basal ganglia, commonly associated to psychiatric, motor and cognitive impairment. This condition has been associated to pathogenic variants in five genes, four (SLC20A2, PDGFB, PDGFRB and XPR1) causing the autosomal dominant form of the condition (AD-PFBC) (Quintáns et al., 2018). The recessive form of PFBC was only recently described, when Yao et al. (2018) first reported MYORG as a causative gene for the disease. The myogenesis-regulating glycosidase (MYORG, also known as NET37 or KIAA1161) gene encodes a putative nuclear envelope transmembrane protein with a predicted glycosidase function (Datta et al., 2009). In the human brain, MYORG is expressed in several regions (Arkadir et al., 2018). Animal models have shown that MYORG is specifically expressed in astrocytes, a cell type that has not been involved in the calcification model yet, but is part of the neurovascular unit (Tadic et al., 2015; Yao et al., 2018). Since the first report of MYORG’s involvement in PFBC, 48 cases of autosomal recessive PFBC (AR-PFBC) have been described in 26 families in China, Israel, Germany, Italy and France (Arkadir et al., 2018; Chen et al., 2018; Peng et al., 2018; Yao et al., 2018; Forouhideh et al., 2019; Grangeon et al., 2019; Ramos et al., 2019). Here, we present the results of screening a Brazilian cohort, with previous negative genetic screenings for all AD-PFBC genes, for variants of MYORG. One new variant was found in MYORG, being responsible for causing PFBC in a 54-year-old female who, interestingly, also has a Hashimoto’s thyroiditis diagnosis and polyneuropathy. No other pathogenic variants were detected in the remaining patients. Twenty patients with previous negative screenings for SLC20A2, PDGFB, PDGFRB and XPR1 were screened for variants on MYORG. Genetic screening of MYORG in all patients was performed by PCR amplification of exon 2, as well as the intronic sequences near the exon’s boundaries, using primers reported previously (Yao et al., 2018), followed by direct Sanger sequencing. Informed consent was obtained from all subjects for genetic testing, processing of personal data and publication of data in scientific journals. Sanger sequencing of genomic DNA identified a homozygous duplication c.854_855dupTG at exon 2 of MYORG (NM_020702.5) in Patient 1C01BR (Fig. 1A). This variant predicts a G to W substitution in position 286 with frameshift (p.G286Wfs*149, NP_065753.2) never described before. In silico assessment by MutationTaster suggested the p.G286Wfs*149 variant to be disease causing. Unfortunately, genetic screening of her sisters and parents was not possible. The variant was not found in ExAC Browser or 1000 Genomes Project. New MYORG mutation associated with PFBC detected in Brazilian family. (A) Electropherograms show a homozygous duplication in MYORG (c.854_855dupTG, highlighted in red; NM_020702.5) in genomic DNA of the patient (below), and absent in wild-type control (above). (B) Family pedigree of Brazilian family affected. Filled, blank and slashed symbols indicate individuals with brain calcifications, without brain calcifications, and individuals who are deceased, respectively. Asterisks indicate individuals whose DNA was available, and symbols with a question mark indicate that CT scans were unavailable. Double lines indicate parental consanguinity. Patient 1C01BR is the second daughter of consanguineous parents, who were first cousins (Fig. 1B). The 54-year-old female has a medical history marked by extreme fatigue, dysarthria and movement disorder. She reports experiencing debilitating fatigue and vertigo starting at age 41, making it hard to walk especially in an erect position. In the following years the fatigue intensified, and additional hand and head tremors, reduced strength in hands and numbness affecting the hands and arms affected her fine motor skills. Additionally, dysarthria and difficulty to write developed and even though gait became more impaired with time, she was still able to walk independently for a few years. At the age of 50 the patient reports the numbness has spread to the crotch, legs and feet, rendering her unable to walk unassisted by the time she was 52. At that time, dysphagia also began to develop. As early as age 41, CT and MRI scans already showed the presence of large calcifications affecting the globus pallidus, pons, and cerebellum (Fig. 2A–C). Calcifications on the pineal gland and choroid plexus were also noted, as well as accentuated subarachnoid cisterns (Fig. 2C). A couple of years later, a new CT scan indicated the expansion of these calcifications to the thalami, centrum semiovales, subcortical white matter and to both the cerebral and cerebellar peduncles (Fig. 2F–H). Brain imaging exams also indicate she had asymmetric lateral ventricles, with the right ventricle being smaller than the left (Fig. 2C and G), accentuated cerebellar atrophy (Fig. 2A and E), and optic nerve head drusen (Fig. 2A and F). CT scan and MRI brain images of Patient 1C01BR. CT scans images showing bilateral and symmetric brain calcification on the pons (A), cerebellum (B and F), basal ganglia, choroid plexus (C and G) and subcortical white matter (D and H). Optic nerve head drusen can also be seen (A and F). MRI image showing calcification on the pons, cerebellar atrophy and enlarged posterior fossa and basal cistern (E). B–D and F–H show the same brain structures and were taken 4 years apart, allowing for the observation of lesion growth. Electromyoneurography (EMNG) of lower limbs from when she was 44 showed findings consistent with predominantly axonal sensory polyneuropathy. Haematological and biochemical screenings show she has anaemia (Hb = 10.3 g/dl; normal = 12–15.5 g/dl) and anisocytosis (red cell distribution width = 16.3%; normal = 11.9–15.5%), with a majority of microcytic (mean corpuscular volume = 63 fl; normal = 82–98 fl) and hypochromic (mean corpuscular haemoglobin = 19.7 pg; normal = 26–34 pg) red blood cells likely due to a minor form of thalassaemia. Additionally, her elevated antithyroglobulin (AbTG = 992.2 IU/ml; normal < 115 IU/ml) and anti-thyroid peroxidase antibodies (AbTPO = 96.12 IU/ml; normal < 34 IU/ml) are consistent with Hashimoto’s thyroiditis, confirmed through Doppler ultrasound. Standardized psychiatric evaluations performed when the patient was 48 resulted in a score of 30 for Mini-Mental State Examination, 124/126 for Functional Independence Measure, 13 for Beck, 11 in Unified Parkinson’s Disease Rating Scale, Stage 3 in Hoehn and Yahr, and a 90% Schwab Score. Family history shows that her father died at age 78, with a medical history of hypertension and stroke, and her mother is currently 89 years old, having been treated for Parkinson’s disease for the past 10 years; she does not walk. Her older sister, currently 71, has a medical history marked by a stroke at age 61 and brain imaging shows ventricle deviation but no calcification (images not shown). Her younger sister, currently 53 years old, is asymptomatic without complaints of any major medical issues, as are the remaining relatives. This case report brings another patient with an autosomal recessive form of PFBC, caused by a homozygous duplication on MYORG. So far, 64.5% of all reported MYORG-PFBC cases are homozygous and 35.5% are compound heterozygous. Interestingly, a high incidence of indels in MYORG can be seen in the context of PFBC: ∼48% of all confirmed AR-PFBC cases are caused (completely or partially, in the case of homozygous or compound heterozygous cases, respectively) by insertions, deletions or duplications (Supplementary Table 1). Coming from a consanguineous family, Patient 1C01BR has a severe clinical presentation marked by motor and sensorineural impairment that affects her fine motor skills, gait, ability to speak and swallow. These signs and symptoms have been widely described in patients with homozygous or compound heterozygous MYORG variants, although at different levels of severity. In fact, bulbar signs, mainly in the form of dysarthria and dysphagia, are the most commonly reported symptoms in AR-PFBC (38/48), closely followed by any sort of motor impairment (including gait ataxia, parkinsonism, and hyperkinesis; 34/48) (Supplementary Table 2). Cognitive and psychiatric impairment, however, do not seem to be as present in AR-PFBC patients (15/48 and 4/48, respectively) as they are in AD-PFBC. (Grangeon et al., 2019). Additionally, and similar to other MYORG-PFBC cases, this patient had a fast clinical deterioration, with a culmination of complete gait impairment happening <15 years from the first appearance of symptoms. Interestingly, in Patient 1C01BR the presence of optic nerve head drusen was noted; no ophthalmic abnormalities have been reported in PFBC patients so far, even though ocular calcifications have been described in PFBC animal models (Borges-Medeiros et al., 2019). The most important difference in this case is that Patient 1C01BR also has a confirmed Hashimoto’s thyroiditis diagnosis. Because brain calcifications can be both primary and secondary, the co-occurrence of any thyroid hormonal imbalances with these types of lesions usually results in the genetic screening for PFBC-related genes being considered unnecessary, and thus not being performed (Nicolas et al., 2013; Sava et al., 2013). In fact, recent literature shows at least one other case of Hashimoto’s thyroiditis with brain calcification (Lee et al., 2010). It is important to point out, however, that both the thyroidopathy and neuropathy could be either comorbidities or new clinical aspects specific to MYORG-PFBC cases. This case report highlights that patients with hormonal imbalances and bilateral brain calcifications can still be genetically diagnosed with PFBC, reinforcing the recommendation of performing genetic testing whenever possible. Understanding the patient’s family history may also be an indicative of PFBC, as many of the cases have either an autosomal dominant (SLC20A2, PDGFRB, PDGFB and XPR1) or autosomal recessive (MYORG) mode of inheritance, but sporadic cases should not be excluded from these analyses. The data that support the findings of this study are available from the corresponding author, upon reasonable request. We would like to thank the patients and their families for their participation in the study. We would also like to thank H. Leal, R. Gitirana, L. Pimentel, R. Borges-Medeiros, D. Moura and E. Cunha for technical support and helpful discussion. L.D.F. received a fellowship from Fundação de Amparo à Ciência e Tecnologia de Pernambuco (FACEPE; IBPG-0750-2.02/16). J.R.M.O. received funding from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; 310150/2016-7 and 457556/2013-7) and PROPESQ-UFPE (2018-2019). The authors report no competing interests. Supplementary material is available at Brain online.