Molecular characterization of novel and rare DNA variants in patients with galactosemia

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TL;DR

This study identified four novel and four rare DNA variants in the GALT, GALK1, and GALE genes from five Greek newborns with galactosemia using targeted next-generation sequencing. In silico analyses predicted that all variants have deleterious or destabilizing effects on protein function and stability, supporting their pathogenic role. These findings highlight the utility of NGS for cost-effective genetic diagnosis and for elucidating the complex genetic background of galactosemia.

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**Background:** Galactosemia is an autosomal recessive disorder caused by mutations in the GALT, GALK1, or GALE genes, which encode enzymes of the Leloir pathway. Classic galactosemia (GALT deficiency) presents in neonates with feeding problems, liver dysfunction, and sepsis, while GALK deficiency primarily causes cataracts, and GALE deficiency resembles classic galactosemia. Lifelong dietary galactose restriction is the only treatment, yet long-term complications often occur. The prevalence of classic galactosemia is 1:40,000–60,000 in Europe and the US, and 1:51,000 in Greece. Over 363 GALT variants are recorded, but only nine have been reported in Greece. This study aimed to characterize novel and rare variants in five Greek newborns with elevated galactose levels identified through the national newborn screening program.

**Methods:** Five patients (two with GALT deficiency, two with GALE deficiency, one with GALK deficiency) were selected from the Greek National Newborn Screening program. Total galactose was measured from dried blood spots using enzymatic DELFIA (cutoff 20 mg/dL). GALT activity was measured via a fluorescent assay (normal >3.5 U/g Hb). Genomic DNA was extracted from whole blood, and a custom Ion AmpliSeq panel covering exons, splice regions, and parts of UTRs of GALT, GALK1, and GALE was used for next-generation sequencing on an Ion Torrent PGM. Variants were confirmed by Sanger sequencing in one family. In silico functional predictions used SIFT, Polyphen-2, PROVEAN, Mutation Taster, and CADD. Stability predictions used SDM, mCSM, MUpro, and CUPSAT. Three-dimensional protein modeling was performed with SWISS-MODEL, and structural alignments used PDBeFold to compute RMSD values.

**Key Results:** Eight nonsynonymous variants were identified: four novel (GALT p.Phe245Leu, GALK1 p.Gly193Glu, GALE p.Ile266Leu, GALE p.Ala216Thr) and four rare (GALT p.Arg204Gln, GALT p.Met298Ile, GALK1 p.Arg68Leu, GALE p.Ala180Thr). Patient D1 (female, age 4.5 years) had GALT p.Phe245Leu and Duarte-2 (mild), with initial galactose 15.8 mg/dL and normal GALT activity; she discontinued diet. Patient K1 (female, age 4.5 years) had GALT p.Arg204Gln and p.Met298Ile, initial galactose 20.4 mg/dL, GALT activity 5.9 U/g Hb, and remained on normal diet. Patient M1 (female, age 4 years) had GALE p.Ile266Leu and p.Ala180Thr, initial galactose 36.4 mg/dL, GALT activity 9.5 U/g Hb, with jaundice and cortical cataract; she remained on diet. Patient T1 (male, age 4 years) was homozygous for GALE p.Ala216Thr, initial galactose >50 mg/dL, GALT activity 3 U/g Hb (equivocal), with elevated bilirubin (16.99 mg/dL) and liver enzymes (>120 IU/dL); he remained on diet. Patient L1 (female, age 15 years) had GALK1 p.Gly193Glu and p.Arg68Leu plus GALT Duarte-2, initial galactose 64.5 mg/dL, normal GALT activity, bilateral cataract; she remained on diet. In silico analysis: GALT p.Phe245Leu was damaging by all functional tools (SIFT 0.002, Polyphen-2 0.763, PROVEAN −4.23, Mutation Taster disease-causing, CADD 24.9); stability predictions were mixed. GALT p.Met298Ile was damaging by all functional tools (SIFT 0.001, Polyphen-2 1.0, PROVEAN −3.76, CADD 29.3). GALT p.Arg204Gln was contradictory (SIFT tolerated 0.321, Polyphen-2 benign 0.007, PROVEAN neutral −0.97, but Mutation Taster disease-causing, CADD 21.7). GALE p.Ala216Thr was damaging by all functional tools (SIFT 0.0, Polyphen-2 1.0, PROVEAN −3.70, CADD 28) and destabilizing by most stability tools. GALE p.Ala180Thr was damaging by most functional tools (SIFT 0.021, Polyphen-2 0.96, CADD 25.9) and destabilizing by all stability tools. GALE p.Ile266Leu was contradictory (SIFT tolerated 0.071, Polyphen-2 benign 0.003, PROVEAN neutral −1.37, but Mutation Taster disease-causing, CADD 22.6). GALK1 p.Gly193Glu was damaging by all functional tools (SIFT 0.002, Polyphen-2 0.999, PROVEAN −6.58, CADD 23.7) and destabilizing by all stability tools. GALK1 p.Arg68Leu was neutral by all functional tools (SIFT 0.705, Polyphen-2 0.009, PROVEAN −1.48, CADD 15.41) but destabilizing by all stability tools. Structural analysis: GALE p.Ala216Thr had the highest RMSD (0.048) among GALE variants; GALK1 p.Gly193Glu and p.Arg68Leu each had RMSD ~0.44, but their combination reduced RMSD to 0.08; GALT p.Arg204Gln and p.Phe245Leu each had RMSD 0.134, but their combination with Asn314Asp reduced RMSD to 0.007.

**Clinical Implications:** The identification of four novel and four rare variants expands the mutational spectrum of galactosemia in the Greek population. In silico predictions support a pathogenic role for most variants, particularly GALT p.Phe245Leu, p.Met298Ile, GALE p.Ala216Thr, p.Ala180Thr, and GALK1 p.Gly193Glu. The mild phenotype in patient D1 (p.Phe245Leu + Duarte-2) and patient K1 (p.Arg204Gln + p.Met298Ile) suggests that these variants may have mild effects. The severe phenotype in patient T1 (homozygous p.Ala216Thr) and patient M1 (p.Ile266Leu + p.Ala180Thr) supports their pathogenicity. Patient L1’s GALK deficiency with additional GALT Duarte-2 illustrates the genetic complexity. Targeted NGS proved time- and cost-effective for comprehensive genetic diagnosis, enabling early dietary intervention and improved management. These findings underscore the importance of genetic testing in newborn screening programs to clarify genotype-phenotype correlations and guide personalized treatment.