Next-Generation Sequencing Verified by MLPA to Detect CNV in FH Child

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Next-Generation Sequencing Verified by Multiplex Ligation-Dependent Probe Amplification to Detect a New Copy Number Variation in a Child with Heterozygous Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) is a common autosomal dominant disorder of lipid metabolism, primarily caused by mutations in the low-density lipoprotein receptor gene (LDLR). While heterozygous FH (HeFH) typically presents with mild clinical phenotypes, homozygous FH (HoFH), compound heterozygous mutations, or double gene mutations in LDLR can lead to severe complications, including accelerated atherosclerosis and premature coronary artery disease. Accurate genetic diagnosis is critical for prognosis and treatment decisions, as lipid levels alone may not reliably distinguish HeFH from HoFH. This case report highlights the importance of integrating next-generation sequencing (NGS) with multiplex ligation-dependent probe amplification (MLPA) to identify copy number variations (CNVs), which account for approximately 10% of pathogenic LDLR variants.

Clinical Presentation and Family History

A 31-month-old female patient presented with multiple plane and tendinous xanthomas, first observed at 18 months of age. The xanthomas, ranging from 0.3 to 1.5 cm in diameter, were distributed on the fingers, wrists, elbows, buttocks, and knees. Despite normal height (95 cm) and weight (14 kg), her lipid profile revealed extreme elevations in total cholesterol (TC: 23.88 mmol/L; normal <5.18 mmol/L) and low-density lipoprotein cholesterol (LDL-C: 17.94 mmol/L; normal <3.37 mmol/L). Abdominal ultrasound showed no abnormalities.

The patient’s parents exhibited milder dyslipidemia. Her father had TC and LDL-C levels of 7.17 mmol/L and 4.1 mmol/L, respectively, while her mother’s levels were 8.15 mmol/L (TC) and 3.69 mmol/L (LDL-C). Neither parent had xanthomas or cardiovascular abnormalities. The discordance between the child’s severe phenotype and the parents’ milder presentations raised suspicion of a compound genetic defect.

Genetic Testing Strategy

Next-Generation Sequencing (NGS)

NGS analysis of the LDLR gene in the patient and her parents identified a paternal frameshift mutation in exon 4: *chr19:11216187-11216188 (NM_000527), c.606delC (p.H203Tfs3)**. This mutation, causing premature termination of translation, was classified as likely pathogenic. However, the severity of the child’s lipid abnormalities and xanthomas suggested an additional genetic contributor.

During data analysis, abnormal depth of coverage (DOC) in NGS reads indicated potential heterozygous deletions in LDLR exons 13 and 14. DOC measures the number of sequencing reads aligned to a genomic region; a 50% reduction in DOC suggests a heterozygous deletion. While NGS is less sensitive for CNV detection compared to point mutations, this finding prompted further investigation.

Multiplex Ligation-Dependent Probe Amplification (MLPA)

MLPA was performed to confirm the suspected CNV. This technique uses probe pairs hybridizing to adjacent DNA sequences; deletions or duplications alter probe ligation efficiency, detectable via capillary electrophoresis. MLPA confirmed a heterozygous deletion spanning exons 13–14 of LDLR in the patient and her mother (Figure 1). The father’s MLPA results were normal.

Genetic Diagnosis and Implications

The patient inherited two distinct LDLR mutations:

  1. Paternal mutation: Exon 4 c.606delC (p.H203Tfs*3), a frameshift variant leading to a truncated, nonfunctional protein.
  2. Maternal mutation: Exons 13–14 deletion, a large-scale CNV disrupting ligand-binding domains critical for LDL receptor function.

This compound heterozygosity reclassified the diagnosis from HeFH to HoFH, explaining the patient’s unusually severe phenotype. While HeFH patients typically develop coronary artery disease in adulthood, HoFH carries a high risk of cardiovascular events in childhood. Early initiation of aggressive lipid-lowering therapies (e.g., LDL apheresis, PCSK9 inhibitors) is crucial for HoFH, whereas HeFH management focuses on dietary modifications and statins from age 10.

Challenges in FH Genetic Diagnosis

NGS remains the primary tool for FH genetic testing, but its limitations in CNV detection underscore the need for complementary methods like MLPA. In this case, reliance on NGS alone would have missed the maternal exons 13–14 deletion, leading to misclassification as HeFH. MLPA’s exon-level resolution makes it ideal for LDLR CNV analysis, as evidenced by 100% concordance with NGS in prior studies when DOC abnormalities are flagged.

The clinical overlap between severe HeFH and HoFH further complicates diagnosis. Some HeFH patients exhibit LDL-C levels exceeding 13 mmol/L, overlapping with HoFH ranges. Genetic testing resolves this ambiguity, enabling accurate risk stratification.

Clinical Recommendations

  1. Core family trio testing: Simultaneous genetic analysis of the patient and parents enhances mutation detection, particularly for de novo or compound variants.
  2. CNV screening: MLPA or similar methods should accompany NGS in FH diagnostics. Laboratories must scrutinize NGS DOC data for regions with half-depth signals.
  3. Early intervention: HoFH patients require immediate treatment, including LDL apheresis and gene-specific therapies, to prevent childhood morbidity.

Conclusion

This case illustrates the critical role of integrating NGS with MLPA to identify CNVs in LDLR, ensuring accurate FH classification. The compound heterozygosity (paternal point mutation and maternal CNV) highlights the genetic complexity underlying severe pediatric hypercholesterolemia. Clinicians must maintain a high index of suspicion for CNVs when NGS identifies a single mutation inconsistent with the phenotypic severity. Proactive genetic counseling and early therapeutic intervention can significantly alter disease trajectories in HoFH.

doi.org/10.1097/CM9.0000000000001224

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