Neurofibromatosis (NF) Type 1 and Legius Syndrome Panel

Neurofibromatosis type 1 (NF1) is characterized by cutaneous neurofibromas, café-au-lait macules (CALMs), iris hamartoma (Lisch nodules) and freckling of axillary and inguinal regions. These features usually become apparent during puberty. Additional features include plexiform neurofibromas, central nervous system gliomas, including optic glioma, macrocephaly, scoliosis, pseudoarthritis, overgrowth and learning difficulties (Riccardi 1993). There is extensive clinical variability between individuals in age of onset, tumor burden, and disease progression. NF1 is pan-ethnic and affects 1 in 3,000 people (Rasmussen and Friedman 2000).

Neurofibromatosis-Noonan Syndrome patients (NFNS) present with clinical features characteristic of both neurofibromatosis type 1 (NF1) and Noonan syndrome (NS). The NF1 features include CALMs, freckling, neurofibromas, hamartoma of the iris, optic gliomas, and other MRI findings, such as unidentified bright objects (UBO). The NS features include facial anomalies such as hypertelorism, low-set ears, short stature, congenital heart defects, primary pulmonary valve stenosis, webbed neck and thoracic abnormalities. In NFNS patients, features common to both NF1 and NS include macrocephaly, scoliosis, mental retardation or learning difficulties (Allanson et al.1985; De Luca et al. 2005). Spinal Neurofibromatosis (SNF) is characterized by multiple bilateral spinal neurofibromas and other tumors of the central nervous system, with few or no other clinical features of NF1. Symptoms usually begin in adulthood (Kluwe et al. 2003; Quintáns et al. 2011).

Legius Syndrome, previously known as neurofibromatosis type 1-like syndrome, is a developmental disorder characterized by a mild NF1 phenotype (Brems et al. 2007; Stowe et al. 2012). The most common clinical features of Legius syndrome are multiple café-au-lait spots. Additional features may include a Noonan-like dysmorphy, axillary freckling, macrocephaly, psychomotor developmental delay, behavioral problems and learning difficulties. Although Legius syndrome resembles the Neurofibromatosis Type 1 phenotype, some typical features of NF1, such as Lisch nodules in the iris, neurofibromas and central nervous system tumors are absent (Brems et al. 2007).

Neurofibromatosis type 1 is caused by heterozygous loss-of-function pathogenic variants in the NF1 gene. It is inherited as an autosomal dominant trait in about half of cases, and is caused by de novo pathogenic variants in the other half. Over 2,300 NF1 germline variants have been reported and include all types. Large deletions account for ~ 5% of patients with NF1 and are usually associated with a severe phenotype (Friedman 2014). Gross insertions and complex rearrangements are rare. Nearly all de novo pathogenic variants occur in the paternal chromosomes (Jadayel et al. 1990), with the exception of large deletions, which occur in maternal chromosomes (Lázaro et al. 1996; Upadhyaya et al. 1998). Parental germline mosaicism has been reported (Lázaro et al. 1994).

The vast majority of NFNS cases are sporadic, although several families transmitting the trait in an autosomal dominant manner have been reported (Quattrin et al. 1987; Abuelo et al. 1988; Colley et al. 1996). Heterozygous mutations in the NF1 gene were reported in patients with NFNS, including sporadic and familial cases (Baralle et al. 2003; De Luca et al. 2005; Stevenson et al. 2006; Huffmeier et al. 2006). At least 10 different pathogenic variants in the NF1 gene were detected in patients with NFNS. About half of these are missense, the other half are small deletions or insertions that are predicted to result either in frameshift or in-frame deletions.

NF1 encodes the neurofibromin protein, a negative regulator of the RAS/MAPK pathway.

Legius syndrome is inherited in an autosomal dominant manner in most reported cases; while family medical history was not available for several patients. It is therefore not clear whether these cases are familial or sporadic. Heterozygous germline loss-of function pathogenic variants in the SPRED1 gene cause Legius syndrome. Over 70 SPRED1 pathogenic variants have been reported to date. They are distributed throughout the gene and are mostly truncating (Spencer et al. 2011).

The SPRED1 protein negatively regulates MAP kinase signaling within the RAS-MAPK pathway. It interacts with neurofibromin.

This panel will detect pathogenic variants in the:

- NF1 gene in 80-93% of patients that meet the NIH clinical diagnostic criteria for neurofibromatosis type 1 (Maruoka et al. 2014; van Minkelen et al. 2014; Pasmant et al. 2015; Zhang et al. 2015; Calì et al. 2017).

- SPRED1 gene in ~ 2 % of patients with NIH clinical diagnostic criteria for neurofibromatosis type 1 and no pathogenic variants in the NF1 gene (Messiaen et al. 2009).

- SPRED1 gene in ~ 10% of patients with Legius syndrome (mild neurofibromatosis type 1 phenotype) (Brems et al. 2012; Spurlock et al. 2009).

Deletions and duplications of NF1 represent ~5% of cases (Friedman 2014).

Large copy number variations in the genomic region of the SPRED1 gene are found in ~ 5% of patients with Legius syndrome (mild neurofibromatosis type 1 phenotype) (Brems et al. 2012).

This test is performed using Next-Gen sequencing with additional Sanger sequencing as necessary.

This panel provides 100% coverage of all coding exons of the genes plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define coverage as ≥20X NGS reads or Sanger sequencing. PGnome panels typically provide slightly increased coverage over the PGxome equivalent. PGnome sequencing panels have the added benefit of additional analysis and reporting of deep intronic regions (where applicable).

Dependent on the sequencing backbone selected for this testing, discounted reflex testing to any other similar backbone-based test is available (i.e., PGxome panel to whole PGxome; PGnome panel to whole PGnome).

Deletion and duplication testing for NF1 is performed using NGS, but CNVs detected in this gene are usually confirmed via multiplex ligation-dependent probe amplification (MLPA). Please see limitations for CNV detection via NGS.