Large exonic deletions in POLR3B gene cause POLR3-related leukodystrophy

POLR3-related (or 4H) leukodystrophy is an autosomal recessive disorder caused by mutations in POLR3A or POLR3B and is characterized by neurological and non-neurological features. In a small proportion of patients, no mutation in either gene or only one mutation is found. Analysis of the POLR3B cDNA revealed a large deletion of exons 21–22 in one case and of exons 26–27 in another case. These are the first reports of long deletions causing POLR3-related leukodystrophy, suggesting that deletions and duplications in POLR3A or POLR3B should be investigated in patients with a compatible phenotype, especially if one pathogenic variant has been identified.

Amino acid changes in the protein domains of POLR3A or POLR3B suggest a direct interference with DNA binding, a modification of the catalytic cleft structure, or a change in protein interactions of POLR3 subunits [9,10]. Current knowledge suggests that mutations are uncovered in the vast majority of 4H cases. A small percentage of cases remain negative or have only one mutation after sequencing analysis [3,8]. To date, no large deletion or duplication has been reported in 4H cases [3,9].
We here report the first large exonic deletions in patients with clinical and radiological diagnosis of 4H. The first index patient is a 15-year-old girl, first evaluated by a child neurologist at the age of 19 months for unstable stance and delayed walking. She was born from healthy non-consanguineous parents after a normal pregnancy and delivery. At initial evaluation, the patient had normal height, weight and head circumference. Neurological examination was significant for mild hypotonia as well as truncal ataxia. Patient evolution and annual examinations revealed delayed decidual teeth eruption, slowly progressive gait ataxia and halted pubertal development at Tanner Stage IV with primary amenorrhea. At the time, LHRH stimulation showed lack of LH/FSH pulsatility. The second patient, a boy, was born at term by elective caesarean section after a pregnancy sustained by progesterone in the context of having had three prior miscarriages of unknown cause. Congenital hip dislocation was first treated conservatively, and then corrected surgically. In the second year of life, delay of gross and fine motor skills became obvious. He developed frank ataxia and a mild pyramidal syndrome. Eruption of upper medial incisors was delayed. He developed myopia and short stature. Neurological deterioration led to death at age 8 years. MRI of both patients were compatible with 4H leukodystrophy.
Both patients underwent sequencing of all exons and intron-exon boundaries of POLR3A and POLR3B. Patient 1 was heterozygous for a maternally-inherited variant, c.1568 T > A (p.Val523Glu), located in exon 15 of POLR3B (Figs. 1a and 2c). The Val523Glu have been reported several times in the literature as pathogenic [1]. Patient 2 was apparently homozygous for a missense variant, c.3008A > G (p.Tyr1003Cys), located in exon 26 of POLR3B (Figs. 1b and 2c). Parental testing failed to uncover the Tyr1003Cys in the mother suggesting the presence of a large deletion on the other allele or uniparental isodisomy. No mutation in POLR3A was uncovered in either patient.
Further studies were conducted in order to uncover exonic deletion in our index patients. In patient 1, RNA analysis revealed the presence of a heterozygous deletion encompassing exons 21 and 22 (Fig. 2a and c). Subsequently, long-range genomic PCR using exonspecific primers for exons 20 and 23, confirmed a~6Kb paternally-inherited deletion in patient 1 (Figs. 2c and 3a In patient 2, RNA analysis revealed the presence of a heterozygous deletion encompassing exons 26 and 27 ( Fig. 2b and c). Subsequently, long-range genomic PCR using exon-specific primers for exons 25 and 27 respectively, confirmed a~4Kb maternally-inherited deletion. Since no homology was found at the deletion breakpoints, the POLR3B exonic deletions appear to have arisen by the simple rejoining of non-homologous DNA ends during double stranded break repair. The clinical picture in these patients is typical of 4H. The presence of a deletion together with a missense mutation reiterates the idea that complete loss of POLR3B is lethal [1]. Patients with compound heterozygous mutations have shown no significant differences in the clinical course of deterioration as compared to homozygous affected patients, except for the patients homozygous for the common POLR3B mutation (c.1568 T > A, p.Val523Glu), whose clinical symptoms are significantly milder [3,10]. This could be, at least partly, attributed to the debilitating effect of the missense mutation on the formation of multiplex complexes [9]. In summary, our findings highlight that multi-modal approaches in mutation screening of POLR3A and POLR3B genes may be required for clinically suspected 4H cases when Sanger sequencing of coding elements is negative or reveals only one mutation.

Ethics and consent statement
The project was approved by the research ethics committee of the Montreal Children Hospital (11-105-PED) and the institutional review board of the VU University Medical Center, Neuroscience Campus Amsterdam, Netherlands. Written informed consent was obtained from the patients' legal guardians.   Submit your manuscript at www.biomedcentral.com/submit