UFM1 founder mutation in the Roma population causes recessive variant of H-ABC

Abstract

Objective: To identify the gene defect in patients with hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) who are negative for TUBB4A mutations.

Methods: We performed homozygosity mapping and whole exome sequencing (WES) to detect the disease-causing variant. We used a Taqman assay for population screening. We developed a luciferase reporter construct to investigate the effect of the promoter mutation on expression.

Results: Sixteen patients from 14 families from different countries fulfilling the MRI criteria for H-ABC exhibited a similar, severe clinical phenotype, including lack of development and a severe epileptic encephalopathy. The majority of patients had a known Roma ethnic background. Single nucleotide polymorphism array analysis in 5 patients identified one large overlapping homozygous region on chromosome 13. WES in 2 patients revealed a homozygous deletion in the promoter region of UFM1. Sanger sequencing confirmed homozygosity for this variant in all 16 patients. All patients shared a common haplotype, indicative of a founder effect. Screening of 1,000 controls from different European Roma panels demonstrated an overall carrier rate of the mutation of 3%-25%. Transfection assays showed that the deletion significantly reduced expression in specific CNS cell lines.

Conclusions: UFM1 encodes ubiquitin-fold modifier 1 (UFM1), a member of the ubiquitin-like family involved in posttranslational modification of proteins. Its exact biological role is unclear. This study associates a UFM1 gene defect with a disease and sheds new light on possible UFM1 functional networks.

Figure 1. MRI findings in TUBB4A (left) and UFM1 (right) mutated patient with hypomyelination with atrophy of the basal ganglia and cerebellum

Sagittal T1-weighted (A–D) and axial T2-weighted (E, F) and T1-weighted (G, H) images in a patient with the common dominant c.745 G>A TUBB4A mutation at age 3 years (A, C, E, G) and a patient with the homozygous recessive UFM1 mutation at age 13 months (B, D, F, H). The patient with the common TUBB4A mutation shows a mildly hyperintense white matter signal on T1-weighted (C, G) and T2-weighted (E) images, indicating a moderate lack of myelin. In the UFM1-mutated patient, the white matter T1 signal is hypointense, indicating a profound lack of myelin (D, H). This patient also shows mild cerebral atrophy (F, H). Both patients have a mild cerebellar atrophy (A–D), most notable at the vermis (A, B). In both patients, there is no putamen visible and there is no visible lesion in this region (arrowheads in E–H). In the TUBB4A-mutated patient, the caudate nucleus has a normal signal (arrow in E). In the UFM1-mutated patient, the caudate nucleus is atrophic and the lateral part of the head has an abnormal hyperintense T2 signal (arrow in F) and hypointense T1 signal (arrow in H).

Figure 2. Founder mutation haplotype analysis

Schematic representation of the candidate region on chromosome 13q13 containing UFM1 and part of FREM2. The area of overlapping haplotype is highlighted in light blue: section A depicts the results of the single nucleotide polymorphism (SNP) array in 5 patients: 238 overlapping homozygous SNPs starting with rs17068530 and ending with rs248423 (shown in black), flanked by rs9532135 and rs9566363 (shown in red). Section B depicts the microsatellite and SNP markers analyzed in the candidate region, which confirmed the presence of a common haplotype in all patients (markers depicted in black), flanked by microsatellite markers D13S219 and D13S1288 (shown in red).

Figure 3. c.-271_-271delTCA reduces UFM1 promoter activity in SY-5Y neuroblastoma and U373 astroglioma cell lines

(A–D) c.-273_-271delTCA in human UFM1 (hUFM1) promoter significantly reduces reporter gene expression in SY5Y (p = 0.001) and U373 (p = 0.014) but not in HeLA (cervix carcinoma) or HOG-F2 (oligodendrocytoma) cell lines. Promoter activity was measured with dual luciferase reporter assays. The nanoluciferase/firefly luciferase ratio measured in cells transfected with wildtype hUFM1 was arbitrarily set at 1 on the y-axis. delTCA indicates cells transfected with human UFM1 promoter harboring c.-273_-271delTCA. Data represent mean ± SD values obtained from 2 independent experiments testing the luciferase activity in duplo and were analyzed by 2-tailed t test.