A recurrent homozygous missense DPM3 variant leads to muscle and brain disease

Abstract Biallelic pathogenic variants in the genes encoding the dolichol‐phosphate mannose synthase subunits (DPM) which produce mannosyl donors for glycosylphosphatidylinositols, N‐glycan and protein O‐ and C‐mannosylation, are rare causes of congenital disorders of glycosylation. Pathogenic variants in DPM1 and DPM2 are associated with muscle–eye–brain (MEB) disease, whereas DPM3 variants have mostly been reported in patients with isolated muscle disease—dystroglycanopathy. Thus far, only one affected individual with compound heterozygous DPM3 variants presenting with myopathy, mild intellectual disability, seizures, and nonspecific white matter abnormalities (WMA) around the lateral ventricles has been described. Here we present five affected individuals from four unrelated families with global developmental delay/intellectual disability ranging from mild to severe, microcephaly, seizures, WMA, muscle weakness and variable cardiomyopathy. Exome sequencing of the probands revealed an ultra‐rare homozygous pathogenic missense DPM3 variant NM_018973.4:c.221A>G, p.(Tyr74Cys) which segregated with the phenotype in all families. Haplotype analysis indicated that the variant arose independently in three families. Functional analysis did not reveal any alteration in the N‐glycosylation pathway caused by the variant; however, this does not exclude its pathogenicity in the function of the DPM complex and related cellular pathways. This report provides supporting evidence that, besides DPM1 and DPM2, defects in DPM3 can also lead to a muscle and brain phenotype.

WMA, muscle weakness and variable cardiomyopathy. Exome sequencing of the probands revealed an ultra-rare homozygous pathogenic missense DPM3 variant NM_018973.4:c.221A>G, p.(Tyr74Cys) which segregated with the phenotype in all families. Haplotype analysis indicated that the variant arose independently in three families. Functional analysis did not reveal any alteration in the N-glycosylation pathway caused by the variant; however, this does not exclude its pathogenicity in the function of the DPM complex and related cellular pathways. This report provides supporting evidence that, besides DPM1 and DPM2, defects in DPM3 can also lead to a muscle and brain phenotype. The proband is a 4-year-old girl from a consanguineous Iranian Turkmen family ( Figure 1A, Family 1). She exhibited microcephaly, hypotonia, significant motor and speech delay and failure to thrive. She was able to sit unassisted at the age of 2 years, but never reached the walking milestone. She could not speak more than two words and dis-

| Patient 3
The 2.5-years-old daughter of non-consanguineous Persian parents from the same village without a family history of neurological disorders ( Figure 1A, Family 2) showed microcephaly, hypotonia, muscle weakness and epilepsy as an infant, delayed motor and speech development, and dysarthria. She reached the walking milestone (with some ataxia) at the age of 2 years and 3 months. Serum CK and TA were increased, and the EEG showed a mildly abnormal background with epileptic discharges. Echocardiography revealed a mildly dilated left ventricle with mildly decreased left ventricular systolic function.
There were no signs of eye involvement.

| Patient 4
An adult Persian man born to a consanguineous marriage ( Figure 1A,

| Patient 5
The proband is a 4-year-old Sri Lankan daughter of nonconsanguineous parents. She presented with global DD, recurrent generalised seizures since infancy, muscle weakness, hypotonia and microcephaly. She was able to sit unassisted at the age of 3 years, but An affected male sibling died at the age of 1 year and 9 months from a similar but more severe condition.  Figure S1). We also attempted to analyse the potential effects of the variant on alpha-dystroglycan (alphaDG) using the glycoconjugate specific antibody, IIH6. However, we were unable to detect alphaDG using IIH6 in patient or control fibroblast samples ( Figure S2). This is likely due to the low expression level of alphaDG in fibroblasts.  DPM3 can lead to congenital muscle and brain disease.

| DISCUSSION
The exact pathogenicity of the disorder is not well understood.
Glycosylation involves the addition of glycans to proteins and lipids through one of the eight major enzymatic pathways described in mammals. 10,11 The DPM complex plays a role in four of these pathways. 2 While DPM1 is the catalytic subunit localised in the cytoplasm, DPM2 and DPM3 subunits anchor DPM1 to the endoplasmic reticulum membrane. The coiled-coil domain of DPM3 is responsible for the anchoring of DPM1, whereas its N-terminal transmembrane domain is linked to DPM2. 2,11,12 Interestingly, the homozygous p.(Tyr74Cys) variant is located on a transmembrane domain next to p.(Pro72Ala) variant which was identified as compound het with p.(Leu115Ter) in the girl with muscle and brain presentation. 7 However, the biochemical assay could not detect any changes in the N-glycosylation pathway, which correlates with previous findings stating that the transmembrane domain is not necessary for the enzymatic activity of the complex. 12 In contrast, the highly conserved coiled-coil region was shown to be required for the enzymatic process, and variant in this domain was associated with reduced O-mannosylation of alpha-dystroglycan due to reduced binding capacity of DPM3 for DPM1. 2 The finding that the variant we identified does not seem to disturb glycosylation points to potentially other mechanisms of pathogenicity. The haplotype analysis suggests that the DPM3 variant most likely recurred in at least three families which was not unexpected given the different ethnicities of the families. This finding along with consistent clinical and genetic data across four independent families provides additional support that p.Tyr74 residue is fundamental for proper function of DPM3.
In this study, we consolidate the association of DPM3 variants with brain-muscle phenotype and further delineate the molecular and clinical spectrum associated with this new ultra-rare congenital disorder of glycosylation. T A B L E 1 (Continued)