Patients’ demographic data and clinical diagnoses
A total of 152 patients, including 94 (62%) males and 58 females, aged from 1 day to 15 year old, has been clinically diagnosed with leukodystrophy or genetic leukoencephalopathy. The distribution of the more common referred diseases among the patients was as follows: 25 patients clinically diagnosed with MLD, 13 CD, 10 PMLD, 6 PMD, 2 PMD or PMLD, 12 Tay-Sachs disease, 10 X-ALD, 8 Sandhoff disease, 8 MLC, 3 AxD, 3 KD, 4 hypomyelination and congenital cataract (HCC), 1 Sialic disease, 1 RNAse T2 deficient leukoencephalopathy, and 2 biotinidase deficiency .
Totally, 108 of 152 patient (71%) had defined MRI patterns (not available) and were clinically diagnosed with a known leukodystrophy. Measurements of lysosomal enzymes in MLD, KD, Tay-Sachs disease, Sandhoff disease were performed for diagnosis. Urinary sulfatides (for e. g. MLD), plasma very long chain fatty acids (for e. g. X-ALD) were also tested to help the diagnosis. These patients were candidates for single gene analysis.
44 of 152 patients (29%) had no definite MRI pattern and no definite biochemical or single gene test could be performed for them. They were candidates for panel gene analysis or WES.
Demographic, clinical and genetic evaluation of patients confirmed genetically
114(75%) patients were confirmed based on genetic testing. Male consist of 73 of 114(64%) of patients. The mean age of onset was 5yrs and 1m ± 18yrs and 11m.
94 of 114 (82. 5%) cases were born in a consanguineous family. The ethnicity of these patients is compared in figure-1A. The ethnical distribution showed higher incidence in Fars 32%; other ethnical distribution included 27% in Turk, Arab 13%, Lur 8%, Kurd 7%, Mazani 4%, Gilak 3%, and the rest Balooch, Afghan, Lak, and Turkeman(Figure-1A).
Based on age of onset of disease, 47 infantile (41% I), 17 late infantile (15% LI), 29 early juvenile (25% EJ), 19 late juvenile (17% LJ) and 2 adults (A) were available (supplementary table-2).
Thirty-five different leukodystrophies and genetic leukoencephalopathies were identified in this study (Table-1). The clinical characteristics of the most common genetically confirmed patients are summarized in table 1 and figure 1B. The main clinical manifestation was motor regression and neurological complaints including dystonia, hypotonia, developmental delay, ataxia, tremor, seizure, macrocephaly, nystagmus, cognition and learning impairment (Table-1 and Supplementary table-2).
Single gene analyses
Sixteen patients had mutations in the ARSA gene (MLD), 8 in ABCD1 gene (X-ALD), 12 in ASPA gene (CD), 2 in GALC(GLB), 7 in MLC1 gene (MLC), 1 in GFAP gene, 1 in PLP1 gene (PMD), 8 in GJC2 gene (PMLD), 11 in HEXA, 5 in HEXB, and 1 in SCL17A5 gene (Table 1). Totally, 74 out of 152 (49%) patients were genetically diagnosed based on single gene analysis. The diagnostic rate was 69% (74 of 108), based on the clinical diagnosis (brain MRI) and single gene analysis.
Next generation sequencing: gene-panel and WES
Gene-panel and WES identified 40 of 152 (26%) patients having leukodystrophies and leukoencephalopathies (Table-1, Supplementary Table-2). Four cases did not show any variants with multigene panel analysis of leukodystrophies.
38 of 152 (25%) patients were not genetically confirmed based on genetic analysis. Some candidates of single gene analysis were not tested for panel based analysis because the parents were not satisfied for the test performance.
Categorization of patients based on protein location
Forty nine of 114 patients were diagnosed as lysosomal disorders (28 lysosomal LD and 21 lysosomal gLE). Forty-one patients genetically were confirmed for MLD, TSD, SD and KD (Table-1).
Eleven patients were diagnosed as peroxisomal disorders which eight of them were X-ALD. One patient had peroxisomal single enzyme beta oxidation deficiency, and two patients had peroxisomal biogenesis disorders (Table-1).
Errors of intermediary metabolism and other leukoencephalopathies
Forty patients diagnosed as errors of intermediary metabolism, consisted of 12 CD, 8 PMLD and 7 MLC (Table 1). CD as the most common degenerative cerebral diseases, due to abnormal amino acid/organic acid metabolism, accounted for the second most common disease in our population. PMD and PMLD are disorders of myelin genes. 4 patients had vWM, 2 patients with hypomyelination-hypogonadotropic-hypogonadism-hypodontia, 1 hypomyalination and congenital contract, 1 PMD, 1 AD, 1 infantile neuroaxonal dystrophy/atypical neuroaconal dystrophy, 1 hypomyelination leukodystrophy 9 (HLD9), 1 Cockayene syndrome, and 1 biotinidase deficiency (Table-1 and supplementary table-2)
Thirteen patients diagnosed with mitochondrial genetic leukoencephalopathies; Leigh syndrome and L-2-HGA accounted for 4 and 3 of them, respectively. Mutations in POLG gene encoding DNA polymerase subunit gamma-1 were responsible in two patients.
Analysis of common genes and mutations
The distribution of the most common diagnosed leukodystrophies accounted for 75% (86 of 114 patients) in our studied patients included 19 of 86 (22%) MLD, 12 CD, 11 Tays-Sachs disease, 8 MLC, 8 X-ALD, 8 PMLD, 6 Sandhoff disease, 4 VWM (vanishing white matter leukoencephalopathy) and 3 L-2-hydroxyglutaric aciduria disease (Figure-1B).
12 mutations observed in at least two unrelated patients which were as follow: 6 MLD patients had Gly311Ser and 3 patients had c. 465+1G>A in ARSA gene (data under publication), two X-ALD patients had c. 1415_1416delAG in ABDC1, six CD patients showed c. 634+1G>T in ASPA, three patients (two homozygotes and one compound heterozygote) had c. 237_238insA in ASPA, each c. 118G>C(p. Ala40Pro) and c. 733T>A(p. Cys245Ser) variant in GJC2 were observed in two PMLD patients which were novel variants, c. 1528C>T in HEXA observed in 4 of 11 TSD patients (2 were from Turkish, 1 from Fars, 1 from Mazani ethnicity), and c. 509G>A in HEXA was found in two Gilak patients, two of six Sandhoff disease patients had c. 833C>T and three (two homozygotes and one heterozygote) MLC patients had c. 449_455delTCCTGCT and two MLC patients had c. 177+1G>T.
Thirty-eight novel variants were identified in 40 patients (Table-2). Each of ABCD1 and GJC2 showed four novel variants. Following genes had each two novel variants: ASPA, FUCA, GALC, HEXA, L2HGDH and MLC1 (Table-2).
The variants were classified according to ACMG guideline; 12 variants met the criteria for being pathogenic, 18 and 11 variants were likely pathogenic and VUS, respectively.
One X-ALD patient had two variants as a haplotype because his mother was heterozygous for both (case5). These two variants considered as one haplotype, although they were classified as likely pathogenic variants.
In silico analyses
Protein interaction analysis predicted that the studied proteins had physical and functional relationships except proteins encoded by PLA2G6, RARS, SLC17A5, L2HGDH and MLC1 genes (Figure-2, supplementary table-3).
Functional association was predicted among different studied proteins. In co-expression prediction analysis (Data not shown), the following protein pairs such as POLG and SUCLA, in mitochondria were expressed together.