Genotype first approach & familial segregation analysis help in the elucidation of disease-causing variant for fucosidosis

FUCA1 gene codes for alpha-L-fucosidase enzyme and pathogenic variations (missense, nonsense, frameshift and splice site) are known to disrupt its catalytic function, leading to an autosomal recessive disorder (lysosomal storage disorder) called fucosidosis1,2. Absence of this enzyme or its reduced activity in fucosidosis leads to impaired degradation of both fucosylated glycoproteins as well as glycolipids in lysosomes, leading to the deposition of fucosylated substrates (>20) in different tissues. Variable symptoms of fucosidosis such as psychomotor deterioration, skin and skeletal abnormalities, growth retardation and intellectual disability have been reported3. Fucosidosis is classified into two types based on phenotype. Type 1 fucosidosis is characterized by a rapid psychomotor regression with a severe neurologic involvement at an early age leading to death usually within the first decade of life, whereas type 2 fucosidosis is characterized by milder neurologic signs, psychomotor retardation and a longer survival. In severe cases, symptoms typically show up in infancy, but the affected individuals usually live into their late childhood4. Till date, 160 variants are reported in FUCA1 gene in ClinVar5, of which 23 are pathogenic and 125 are variants of uncertain significance (VOUS). There are two reports from India regarding pathogenic variations in FUCA16,7. Here, we report on genetic analysis in a case of syndromic psychomotor retardation and emphasize on importance of genotype first approach and utility of exome sequencing for detecting the copy number variants.

Two siblings, a girl of nine years and a boy of seven years of age, born out of a non-consanguineous union from Goa, India, presented to a neurologist with a history of developmental delay and language regression followed by non-verbal communication. There was no paternal or maternal family history of neurodegenerative disorders. Both patients had an uneventful perinatal history. Developmental delay was noticed from infancy in both children; however, some language in the form of single words had developed by 2.5-3 yr. However, language milestones regressed by seven years in the girl and five years in the boy. At present, both could only make sounds and point to indicated needs. There were dysmorphic facies with hypertelorism, depressed nasal bridge, low set ears, and tongue thrust (Fig. 1A). Anthropometric examination revealed stunting with failure to thrive, but head circumference was within normal range. Central nervous system examination revealed double hemiplegia, psychomotor retardation and cognitive decline in both patients; however, formal IQ testing was not done. Easy bruisability and petechiae-like rash were observed on the trunk of boy for the last 2-3 months (Fig. 1B). The girl had a fall from a high chair in 2018, which caused subluxation on C1-C2. She was treated conservatively after which she developed severe kyphosis of the spine. No seizures or cherry red spot was reported. Haematological investigations including platelet count and bleeding time were normal in both. Skeletal radiographs revealed no changes in the dysostosis multiplex. Magnetic resonance imaging of the brain exhibited hyperintense signal changes on T1-weighted imaging and showed hypointense signal changes on T2-weighted imaging in periventricular and bilateral globus pallidus regions, suggestive of hypoxic changes in both the siblings.

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Fig. 1

Genotype–phenotype correlation and Sanger sequencing and RT-PCR validation of patient. (A) Facial photograph of both siblings showing dysmorphic facies. (B) Easy bruisability and petechiae-like rash observed on the trunk of proband. (C) IGV view of the BAM file showing the coordinate of FUCA1:c.795G>C:p.Trp265Cys variant identified using BWA-GATK pipeline from the whole exome NGS data. BWA, Burrows-Wheeler Aligner; GATK, genome analysis tool kit.

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Whole blood samples (2 ml) in EDTA were collected from the affected siblings and their parents after taking a written informed consent. The genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, USA). The study protocol was approved by the Institutional Ethics Committee of Centre for DNA Fingerprinting & Diagnostics, Hyderabad (IEC 31/2019).

Whole exome sequencing of the DNA was performed using the Illumina platform (Illumina, San Diego, CA, USA) as per the manufacturer’s protocol for the male proband. The sequences were analyzed using the bioinformatics pipeline described earlier8, (details under Supplementary material online). After correlating with the clinical diagnosis, a novel homozygous NM_000147.5 (FUCA1_v001):c.795G>C, p.(Trp265Cys) (Depth: 49X) variant was identified in exon 5 of the FUCA1 gene (Fig. 1C). This homozygous missense variant of the FUCA1 gene had a combined annotation-dependent depletion (CADD) score of 31 and was predicted to be disease causing by the Mutation Taster and SIFT, in silico pathogenicity prediction programmes (

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Fig. 2

(A) Electropherograms of the siblings and both parents. The c.795G>C (p.Trp265Cys) variant is indicated by the arrows. It was present in the siblings and father but absent in mother; (B) qPCR assay showing copy number variation of patients with respect to unrelated control. GAPDH was used as the reference gene. Tukey’s multiple comparison test was employed to calculate the significance (P<0.05)ns: not significant, qPCR, quantitative PCR.

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Quantitative PCR (qPCR) assay for FUCA1 relative gene dosage for exon 5 compared to a reference gene (GAPDH) revealed heterozygous deletion in mother and both the probands (Fig. 2B). The heterozygous deletion was absent in father and two unrelated controls. The same result was verified using the read count distribution in 10 unrelated samples of whole exome sequencing data in exon 5 of FUCA1 gene along with the male proband data. Results showed significant low read count in comparison with the 10 control samples in the exome data of proband (

In our case, the phenotype in the patient was not matching any particular condition. Hence, we planned for a genotype first approach and conducted exome sequencing in one of the affected. We identified an apparent homozygous novel missense likely disease-causing variant in the FUCA1 gene that results in an amino acid change from tryptophan to cystine at the 265th position (p.Trp265Cys) of the alpha-L-fucosidase protein. The missense variant is likely to lead to change in protein structure or interfere with catalytic function of protein, which in turn will lead to deficiency or complete loss of function of fucosidase enzyme. Following this, we did reverse phenotyping and found that the clinical features in both the affected siblings were consistent with a diagnosis of fucosidosis. The phenotype was consistent with Type II fucosidosis in both siblings.

Familial segregation analysis in our case revealed the mother to be homozygous for wild-type allele. This was in contradiction to the expected heterozygosity in both parents for this autosomal recessive disorder. This phenomenon can be explained by one of the three reasons: (i) uniparental disomy resulting in homozygous variant in proband and only one parent being heterozygous for same variant; (ii) disputed paternity when father is found to be homozygous for wild type allele, or (iii) a heterozygous deletion of exon 5 in mother. Uniparental disomy and disputed paternity were unlikely in our case in view of recurrence in sibling. Hence, we hypothesized that the maternal allele carried a large deletion including exon 5, which can result in apparent homozygous wild-type allele genotype on Sanger sequencing. qPCR analysis confirmed our hypothesis and revealed normal dosage of exon 5 in father and heterozygous deletion in mother and both affected siblings. This reiterates the importance of familial segregation analysis following variant identification in exome/genome sequencing.

Our report expands the genotypic spectrum of fucosidosis and reiterates regarding need of genotype first approach in genetically heterogeneous conditions. Psychomotor retardation is a heterogenous clinical presentation, and often, no specific clinical diagnosis is apparent. Genotype first approach reduces the time to diagnosis in some cases. Family segregation studies are important even in apparently homozygous variants, as unusual phenomenon like deletion of one allele can be seen. Further genetic studies such as qPCR can help in ascertaining these rare instances. Reverse phenotyping, especially with involvement of a clinical geneticist, is important to correlate molecular findings with the phenotype.