Clinical Vignette

1 Abstract

Whole-exome sequencing (WES) has revolutionized genetic diagnosis and has become a powerful tool for identifying disease-causing variants. However, WES has limitations such as uneven read coverage, which can result in multiple low-coverage regions in the exome. Here, we report two affected siblings with a phenotype consistent with Pelizaeus-Merzbacher-like disease 1 (PMLD1), a rare early-onset autosomal recessive disorder caused by biallelic variants in the GJC2 gene. WES analysis of one affected child initially revealed only a single heterozygous nonsense variant in exon 2 of the GJC2 gene. However, on reanalysis with a local de novo assembly approach using the GATK HaplotypeCaller, a second variant - a frameshift insertion - was subsequently identified. This variant was found to be present in a low coverage region which is why it was missed in the initial analysis. Analysis of the segregation pattern of the variants in the affected sibling and parents through targeted Sanger sequencing confirmed that the variants were present in compound heterozygous form in both the affected siblings. Our study emphasizes the importance of considering the limitations of exome sequencing, especially in terms of low coverage of certain exonic regions.

Keywords: Pelizaeus-Merzbacher-like disease 1, GJC2 gene, whole-exome sequencing, gnomAD, GATK HaplotypeCaller

2 Introduction

Pelizaeus-Merzbacher-like disease-1 (PMLD1), also referred to as hypomyelinating leukodystrophy type 2 (HLD2), is a rare autosomal recessive neurological disorder caused by abnormal formation or maintenance of the myelin sheath that surrounds and protects nerve fibers. This can lead to impaired nerve signal transmission and manifestation of disease symptoms such as developmental delay, progressive spasticity, motor impairment, ataxia and nystagmus, and findings of hypomyelination on magnetic resonance imaging (MRI) of the brain (Nahhas et al., 2003). Here, we report a family with Pelizaeus-Merzbacher-like disease 1 (PMLD1), where initial analysis of whole-exome sequence data identified only one heterozygous pathogenic variant, but subsequent reanalysis detected the second variant in a low-coverage region of the gene.

3 Patients and Methods

The proband (P1), an 8-year-old male child, was the fourth offspring of non-consanguineous parents (Figure 1). He was born at term gestation with a birth weight of 2.5 kg and had no history of prenatal or postnatal complications. During infancy, he had hypotonia and motor developmental delay, with delayed neck holding attained at 11 months and delayed walking without support attained at 2 years. He also had poor vision since infancy. He had regression of his motor milestones, starting from the age of around 7.5 years. There was no history of seizures or abnormal movements. The language milestones and intellectual development were normal (IQ-73 at the age of 7 year 7 months). His height was 132 cm (Z score of 0.66), weight was 31 kgs (Z score of 0.87) and head circumference was 49.5 cm (Z score of -2). On examination, he had reduced vision and nystagmus was present. Increased muscle tone was noted in all four limbs with exaggerated deep tendon reflexes, positive ankle clonus and bilateral extensor plantar reflex. Other organ systems were normal on clinical examination. Ophthalmology evaluation revealed optic atrophy with nystagmus. MRI of the brain showed homogenous T2 hyperintensities involving the central and peripheral aspects of the cerebral white matter as well as the pons and medulla regions, mild vermian atrophy, and hypoplastic corpus callosum. Based on the clinical and neuroimaging features, the diagnosis of hypomyelinating leukodystrophy was suspected in the proband.

His similarly affected 3.5-year-old male sibling (P2) presented with motor developmental delay since infancy along with nystagmus; his speech was age-appropriate.

WES analysis of the proband P1 initially revealed only a single heterozygous nonsense variant (GJC2:c.852T>A:p.Cys284Ter) in exon 2 of the GJC2 gene. Subsequent reanalysis of the WES data revealed the second variant which was a novel frameshift insertion variant (GJC2:c.907_923dup:p.Pro309fsTer168) also in exon 2 of the GJC2 gene. According to the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) guidelines, both variants are classified as ‘likely pathogenic’ as they meet the PVS1 criterion for null variants in a gene where loss-of-function is a known mechanism of disease, and the c.852T>A variant also meets the PM2 criterion for being absent in large population databases (Richards et al., 2015). Notably, the frameshift variant had low coverage (depth: 10X) and was not found in the bam file generated by GATK BaseRecalibrator but was identified in the HaplotypeCaller bam, as depicted in Figure 2.

The compound heterozygous status of the affected siblings and the heterozygous carrier status of the unaffected sibling and both parents were confirmed by targeted Sanger sequencing, supporting an autosomal recessive mode of inheritance (Figure 3).

4 Discussion

The GJC2 gene, which is highly expressed in oligodendrocytes, encodes for the connexin-47 (Cx47) protein that forms gap junction channels allowing intercellular communication between oligodendrocytes and astrocytes by heterotypic coupling (Cx47-Cx43 channels) (Kleopa et al.,2004; Qju et al., 2022). Variants in GJC2 can result in loss of function of the transmembrane protein, which may be attributed to altered channel properties or impaired protein trafficking to the cell membrane (Biancheri et al.,2013; Owczarek-Lipska et al., 2019). Exon 2 of GJC2, which codes for majority of the Cx47 protein, contains two low-coverage regions in WES data due to its GC-rich sequence. To identify such variants, it is important to examine the bam file generated by GATK HaplotypeCaller, which uses a local de novo assembly approach to detect variants, including small insertions and deletions (indels). Nonetheless, the detection sensitivity of the HaplotypeCaller may vary depending on factors such as sequencing data quality and depth of coverage.

A general low coverage of exon 2 region of GJC2 is present in the gnomAD v2.1.1 exome sequencing dataset (https://gnomad.broadinstitute.org), which may be due to the high GC content of this region (Karczewski et al., 2020). However, the gnomAD genome sequencing dataset shows uniform coverage in the same region, suggesting that the low coverage in the exome dataset may be due to limitations of exome sequencing (Figure 4). Variants within such low-coverage regions can be difficult to call, which makes the uniformly covered exome ‘extracted’ from the genome sequencing data a helpful approach in such cases. Exome sequence data ‘fetched’ from genome sequencing data facilitates the identification of such variants that may otherwise be missed in the exome regions.

The Cx47 protein is composed of two extracellular, four transmembrane and three cytoplasmic domains. The compound heterozygous variants identified in this family affect the third cytoplasmic domain of Cx47. Specifically, the nonsense variant (p.Cys284Ter) truncates the protein just before the third cytoplasmic domain, while the frameshift insertion (p.Pro309fs) is predicted to alter subsequent amino acids and produce an elongated tail of 38 additional residues in the third cytoplasmic domain (Figure 5). Hence, these variants can have a significant impact on the structure and function of the Cx47 protein.

5 Conclusion

This report highlights the limitations of exome sequencing in detecting genetic variants in certain exonic regions such as those with a high GC content and those with repeat elements or segmental duplications. Examining the bam file generated by GATK HaplotypeCaller would be helpful for identifying variants in such regions. Exome sequence data extracted from WGS data rather than through WES would be better for such regions due to the uniformity of coverage.

6 Acknowledgement

The authors thank the family for their participation in the study. This study was supported by the Indian Council of Medical Research (ICMR)-funded project titled ‘Indian Undiagnosed Diseases Program (I-UDP)’ (33/9/2019-TF/Rare/BMS).

References