Co-occurrence of neurofibromatosis type 1, caused by Alu insertion, and 16p13.11 microduplication

Abstract

Background

Neurofibromatosis type 1 (NF1), an autosomal dominant disorder, characterized by a spectrum of diverse neurocutaneous manifestations, is caused by heterozygous pathogenic variants in NF1 gene. While patients with NF1 often exhibit characteristic features, atypical phenotypes can arise, necessitating consideration of differential diagnoses or concurrent pathologies.

Case presentation

A seven-year-old boy with suspected NF1 underwent clinical evaluation. He presented hallmark café-au-lait spots, axillary freckling, and neurofibromas. Neuroimaging revealed a cranial plexiform neurofibroma. Additionally, he exhibited attention-deficit hyperactivity disorder and developmental delay. Genetic testing identified an Alu insertion variant within the NF1 gene, and subsequent array comparative genomic hybridization detected a 16p13.11 duplication.

Conclusions

This case underscores the intricate molecular bases of NF1 by identifying a rare Alu insertion variant. The patient's neurocognitive challenges and dysmorphic features prompted exploration of a potential overlapping genetic condition. Coexisting genetic disorders have been documented in NF1 patients, emphasizing the necessity of discerning atypical manifestations. The observed 16p13.11 duplication likely contributes to the patient's phenotype, enhancing the precision of diagnosis, prognosis, and genetic counseling.

Background

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder characterized by neurocutaneous manifestation, as well as an increased risk of behavioral, intellectual disorders, and cancer predisposition. Common features found in patients with NF1 include café-au-lait (CAL) spots, inguinal and axillary freckling, the presence of neurofibromas, and macrocephaly. It is caused by heterozygous deleterious variants of NF1, located on chromosome 17. Diagnosis is based on clinical and familial information and molecular studies following specific criteria [1]. NF1 belongs to the spectrum of diseases caused by dysregulation in the RAS signaling pathway, known as RASopathies. Patients with NF1 often share similar physical abnormalities (dysmorphic features) with individuals affected by other RASopathies. While certain diagnostic criteria guide the recognition of typical NF1 cases, atypical phenotypes continue to challenge clinicians and should illicit the consideration of differential diagnosis or the coexistence of the two conditions, with a multilocus effect contributing to patient presentation.

Case presentation

A seven-year-old male was referred to our Medical Genetics consultation for etiological investigation, given NF1 suspicion. He is the only child of a non-consanguineous couple born at term with normal measurements after an uneventful pregnancy. His mother and maternal grandmother were clinically diagnosed with NF1. In the first year of life, he presented with several CAL spots and later axillary freckling. Brain magnetic resonance imaging (MRI) performed at four years old showed multiple areas of T2/FLAIR hyperintensity in the cerebellum, hippocampus, globus pallidus, and thalamus, typical of NF1.

At six years old, he developed facial asymmetry involving the tongue (Fig. 1) associated with redness, ptosis, and tearing of the left eye with chewing movements (Marcus Gunn phenomenon). An updated brain MRI revealed a plexiform neurofibroma in the right maxillofacial area with an infiltrative lesion involving the peri-auricular region, external auditory canal, submandibular and sublingual spaces, and right half of the tongue. Maxillofacial surgeons considered the neurofibroma inoperable.

Fig. 1

Male patient with neurofibromatosis type 1 (NF1) and 16p13.11 duplication showing facial and tongue asymmetry as a consequence of a neurofibroma plexiform, as two café-au-lait macules in the right frontal region, a broad forehead, short nose, and low-set, posteriorly rotated ears. His facial features are also coarser than expected for typical NF1/RASopathy dysmorphic features, what elicited additional studies that unravel the identification of a 16p13.11 duplication contributing to the phenotype

In our observation at the age of seven, he presented with more than six CAL spots, axillary freckling, facial and tongue asymmetry, and RASopathic dysmorphic features: a broad forehead, short nose, and low-set posteriorly rotated ears (Fig. 1). In addition, he presented with bilateral earlobe creases and coarser facial features than those expected of NF1. He presented with macrocephaly (percentile > 97th), a common feature in patients with NF1, and obesity, with a body mass index of 27 kg/m² (percentile > 97th). His behavior was hyperkinetic, with the Conners comprehensive behavior rating scale being diagnostic of attention-deficit hyperactivity disorder and the Griffith Mental Developmental Scale, revealing borderline development with a global developmental quotient of 72. Ophthalmologic, cardiac, and abdominal ultrasound evaluations were normal.

Investigation

Our patient was clinically diagnosed with NF1 following the Legius et al. criteria. The causal variant was initially investigated by NF1 next-generation single-gene DNA sequencing and multiplex ligation-dependent probe amplification (MLPA) at an external commercial laboratory, but no deleterious variant was found. Direct cDNA NF1 sequencing was performed to identify the normal transcript and an aberrant transcript with exon 6 skipping (r.587_654del), likely degraded through nonsense-mediated mRNA decay (Fig. 2A). To identify the deleterious gDNA variant, direct gDNA sequencing from exon 5 to exon 7, including exon/intron boundaries, was sequenced and compared to a negative control (Fig. 2B). This analysis revealed an Alu element insertion adjacent to the acceptor splice site of exon 6 that was absent in the negative control. The insertion was further confirmed by designing Alu insertion-specific primers, which demonstrated the formation of a new DNA copy containing the Alu insertion in the sense orientation and a target site duplication sequence of 12 bp (TTGTGTTTTTTC) after the 3’ end of the poly(A) tail of the retrotransposition element. The Alu sequence was identified as an AluYa5 subfamily consensus sequence with only three base differences. On the basis of these findings, the variant nomenclature was established as c.587-4_587-3 insAluYa5,587-15_587-4dupTTGTGTTTTTTC. This variant was maternally inherited.

Fig. 2

Molecular investigation of NF1 cDNA. A cDNA sequencing results showing exon 6 skipping in the patient. cDNA sequence NM_00104292 was used as reference. B Direct gDNA sequencing, exhibiting exon 6 acceptor splice site results from a negative control and the patient, showing a faintly perceptible background sequence corresponding to the insertion of an Alu element

Although developmental delay can be associated with NF1, the patient's dysmorphic features, obesity, and behavioral problems appear disproportionate for NF1. Therefore, we conducted array comparative genomic hybridization (aCGH) to investigate the possibility of an additional genetic disorder contributing to the neurocognitive phenotype. aCGH detected a recurrent pathogenic 1.69 Mb duplication on 16p13.11, arr[GRCh37] 16p13.11 (14835067_16525374) × 3. This variant was not present in his mother, and the father refused to be tested.

The patient initiated treatment with selumetinib for facial plexiform neurofibromas with stagnation of neurofibroma growth.

Conclusion

Our patient had a personal and familial history of NF1 caused by Alu insertion. This is an unusual mutational mechanism, first reported as causing NF1 by Wallace et al. [2], and was later shown to account for 0.4% of all NF1 pathogenic variants [3]. Similar Alu insertions causing exon 6 skipping have been previously described in two patients, supporting the hypothesis of non-random events, making this site a particularly vulnerable hotspot for Alu and long-interspersed nuclear element insertions [3].

Given the patient's neurocognitive and behavioral performance as well as his coarser dysmorphic features, it was suspected that an additional condition contributed to his phenotype. Co-occurrence of a second genetic condition with NF1 has been previously reported in several patients [4, 5]. As discussed by Muthusamy et al. [5], atypical clinical features, often interpreted as novel manifestations of NF1, could represent the phenotypes of additional genetic disorders. In this context, our patient was also heterozygous for a pathogenic duplication in 16p13.11, which likely has an additive effect on NF1 neurodevelopmental features. The 16p13.11 microduplication is a recurrent copy number variant characterized by incomplete penetrance (8.43%) and variable expression [6]. Clinical features include speech delay, learning disability, autism spectrum disorder, and abnormal brain MRI and cardiac defects [6]. Additionally, patients with 16p13.11 duplication have a higher risk of aortic aneurysm and dissection [7]. Two genes within the duplicated region, NDE1 and NTAN1, may contribute to the neurological and behavioral phenotypes, while MYH11 is the most likely candidate for predisposition to thoracic aortic aneurysm and dissection [6, 8, 9].

The identification of coexisting genetic disorders is important for the patient's family, as it allows for proper genetic counseling and follow-up.