A Chinese Family with Noonan Syndrome Associated with a Heterozygous LZTR1 Mutation

Background Noonan syndrome (NS) is a multisystem disorder resulting from pathogenic mutations in more than 15 genes, among which LZTR1 is newly discovered. However, its role in NS pathogenesis remains unclear, including its inheritance pattern. Methods We herein report a family with LZTR-related NS. We collected clinical data of the proband and her families. Four of them completed the whole exon sequencing. Results


Introduction
Noonan syndrome (NS, OMIM 163950) is a genetic multisystem heterogeneous disorder with a relatively high estimated incidence of every 1000-2500 live births [1] and can show autosomal dominant (AD) or autosomal recessive inheritance (AR) [2] . To date, more than fteen genes associated with NS have been reported, including PTPN11, SOS1, RAF1, BRAF, HRAS, KRAS, NRAS, SHOC2, MAP2K1, MAP2K2, CBL, RIT1, RASA2, A2ML1, and LZTR1 [3. 4] . Among them, variants of LZTR1 have been newly associated with the etiology of NS since 2014 [5][6][7][8] . LZTR1 (OMIM 600574) is the abbreviation for the leucine zipper-like transcriptional regulator 1 gene and is located on 22q11.2. At rst, LZTR1 gene loss-of-function mutations cause schwannomatosis [6. 9. 10] . Recently, no more than 50 cases of NS have been associated with pathogenic LZTR1 mutations [3. 5. 7. 11-14] , and NS can be inherited via AD or AR forms of LTZR1 mutation. The reason of this variant hereditary pattern associated with LTZR1 mutations in NS is not yet clear. To date, the factors underlying the hereditary differences among LTZR1 mutations remain unclear, but the c.1149 + 1G > T mutation of the LZTR1 gene was reported in three cases of NS with an AR mode of inheritance [11. 15] . Here, we report a family with AD NS associated with the c.1149 + 1G > T mutation of LZTR1.

Patients And Methods
Patient referral Patient 1 was the proband (Figure 1, III-3) and a 6.6-year-old female. She was admitted to our hospital because of short stature and was the rst child in her family. She was born at 41 weeks gestation to nonconsanguineous parents via vaginal delivery. Her birth weight and length were 1800 g (<P3rd) and 47 cm (<P3rd), respectively, and her psychomotor development was mildly delayed. From the newborn stage, the patient showed evident growth retardation. At the age of 6.6 years, her height, weight and body mass index (BMI) were 90.5m (-6.3 SD), 11 kg (-4.9 SD) and 13.4 kg/m2 (P10-25th), and her arm span/height and sitting height/height ratios were 0.92 (-2 SD) and 0.58 (+3 SD) according to the standard reference values [16] . Except for her short stature and developmental delay, she exhibited the following features that are typical of NS ( Figure 2): hypertelorism; downslanting palpebral ssures; epicanthal folds; low-set, oval-shaped, posteriorly rotated ears with a thick helix; a short broad nose with a depressed root and full tip; a deeply grooved and long philtrum; high and wide peaks of the vermilion; a highly arched palate; micrognathia; a short neck; cubitus valgus; scoliosis; café au lait spots; and mild hypertrichosis. She also presented with squinting, refractive errors and nystagmus. Her IGF-1 level was 61 µg/L (-1.9 SD), and the GH peak after the growth hormone stimulating test was 5.54 ng/ml. In addition, hormone levels of the adrenal gland, thyroid gland and gonad were normal. Tumor markers such as alpha-fetoprotein (AFP), human chorionic gonadotropin (HCG), and carcinoembryonic antigen (CEA) were negative. An echocardiogram revealed no congenital cardiac defect, while an electrocardiogram (ECG) showed frequent premature ventricular beats (the second and third rhythms) ( Figure 3). Ultrasounds showed no abnormalities in the liver, kidneys, uterus, or ovaries, and X-rays showed hemivertebral deformity of the third thoracic vertebra and scoliosis (Cobb's angle = 28°). The patient's bone age was 5.5 years, and magnetic resonance imaging (MRI) of the craniocerebrum and spine showed the following: 1) pituitary height of 3.3 mm, and 2) no observation of schwannomatosis or occupying lesions ( Figure 4).
Her karyotype was 46, XY. According to the clinical manifestation and laboratory tests, she was diagnosed with NS [17] with growth hormone de ciency (GHD). Whole exon sequencing WES study was completed, revealing the heterozygous mutation c.1149+1G>T of the LZTR1 gene that she inherited from her mother ( Figure 5). Patient 2 ( Figure 1, III-4) was the younger sister of the proband. She was 2.5 years old, and her birth weight and height were 3000 g and 49 cm, respectively, with a gestational age of 38 weeks. Her height and weight were 82.5 cm (-2.7 SD) and 10.7 kg (-1.8 SD). She had typical features of NS, including hypertelorism; downslanting palpebral ssures; epicanthal folds; low-set, oval-shaped, posteriorly rotated ears with a thick helix; a short broad nose with a depressed root and full tip; a deeply grooved philtrum; high and wide peaks of the vermilion; a highly arched palate; micrognathia; a short neck; and pectus carinatum. Her development and cognitive ability were normal, and she had no cardiac or genitourinary defects. The patient was diagnosed with NS, and by WES she had the same heterozygous mutation, c.1149+1G>T, of the LZTR1 gene ( Figure 5).
Suspected patient 3 ( Figure 1, II-6) was the mother of patients 1 and 2. She was 27 years old, and her height was 153 cm (-1.7 SD). She looked like a normal female except for presenting with hypertelorism; downslanting palpebral ssures; low-set, oval-shaped, posteriorly rotated ears with a thick helix; a highly arched palate; and prominent nasolabial folds. She had no cardiac, urogenital or skeletal defects and the same heterozygous mutation of the LZTR1 gene (c.1149+1G>T) ( Figure 5).
Suspected patient 4 ( Figure 1, I-3) and suspected patient 5 ( Figure 1, II-7) were the mother and younger sister of patient 3, respectively. They had appearance features similar to those of patient 3, and their heights were 151 cm (-1.9 SD) and 153 cm (-1.7 SD), respectively. Suspected patient 6 ( Figure 1, III-5) was the daughter of patient 5 and was diagnosed with presumed NS. At the age of 3 years, she showed a short stature (84.3 cm, -3.2 SD); hypertelorism; downslanting palpebral ssures; low-set, oval-shaped, posteriorly rotated ears with a thick helix; a short broad nose with a depressed root and full tip; a deeply grooved philtrum; high and wide peaks of the vermilion; a highly arched palate; and micrognathia without congenital heart, urogenital, skeletal or cognitive defects.
Unfortunately, patients 4-6 rejected the genetic analysis, and patients 2-6 did not agree to share their photos.

Molecular genetic analysis
Written informed consents for gene testing were obtained from the patients' parents. Patients 1-3 and the proband's father underwent genetic testing. Five to ten milliliters of peripheral blood was collected in disposable vacuum tubes for genetic testing. Genomic DNA was isolated using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. After construction according to the standard protocol, whole exon sequencing (100X) of the libraries was performed with the SureSelect Human All Exon V6 array on the Illumina HiSeq X Ten Platform with the PE150 strategy. A standard bioinformatics pipeline was utilized for variant identi cation with the help of Genome Analysis Toolkit (GATK) [18] software following the best practice guidelines recommended by the GATK [19. 20] . Candidate variants were retained as follows: (1) rare variants with a minor allele frequency of < 1% in the ExAC, dbSNP, 1,000 Genomes, gnomAD and local databases, and (2) functional variants including frameshift, splice, nonsense, missense and synonymous variants that can affect splicing. Then, we utilized a hypothesis-free approach to analyze all phenotype-related genes. Eventually, the intersection of the above criteria led to the identi cation of a heterozygous mutation, c.1149+1G>T, under accession number NM_006767.3 in the LZTR1 gene. The splicing mutation was ranked as "likely pathogenic" according to the American College of Medical Genetics and Genomics (ACMG) [21] . Sanger sequencing showed that the mutation was inherited from the proband's mother, who also had a mild phenotype related to NS ( Figure 5). Notably, the proband's younger sister harbored the same heterozygous mutation ( Figure 5). The proband's father did not harbor the mutation and had no symptoms of NS ( Figure 5). In previous literature, compound heterozygosity involving this variant (c.1149+1G>T) in LZTR1 has been described in three patients with AR LZTR1-linked NS [11. 12. 15] . This mutation is classi ed as frameshift and can cause abnormalities at the splice site. As a result, the variant produces truncated proteins, which may affect the function of coded proteins. In the east Asian population database (ExAC_EAS), the frequency of this mutation is 0. The patients did not harbor other mutations that have been associated with NS, GHD, skeletal dysplasia and other genetic diseases.

Discussion
NS is a genetic multisystem condition, and pathogenic genes are associated with only 75% of NS patients, thereby making the clinical diagnosis of NS very important. The phenotype of NS is variable and includes a short stature, congenital heart defects and/or cardiomyopathy, a characteristic craniofacial dysmorphism and childhood benign or malignant tumors, including leukemia and solid tumors [22][23][24] .
The diagnosis of NS depends mainly on the identi cation of typical clinical manifestations, such as a notable appearance, short stature, congenital heart disease and other dysplasia [1. 25. 26] . Our patients (III-3, III-4, III-5) were clinically diagnosed with NS based on their display of features typical of the disease. Patient 1 and patient 2 were subjected to WES, revealing the same variant, c.1149 + 1G > T, in LZTR1 that was inherited from their mother (II-6). Their maternal grandmother (I-3) and maternal aunt (II-7) had a facial appearance similar to that of their mother (II-6). From previous reports, families with AD LZTR1linked NS exhibited vertical transmission of the phenotype with differential penetrance [12] . Additionally, the phenotype of NS ranges greatly, from a normal appearance to features typical of NS. Therefore, our families displayed the AD mode of hereditary NS with incomplete penetrance.
To make accurate diagnoses quickly and effectively, we performed WES for molecular diagnoses. All the exons (including the 50 bp anking piece on either side) were captured in a single reaction, and genes related to the RASopathies were thus considered. The average sequencing depth, average coverage and 10 × coverage (coverage of sites with a depth greater than 10) in the target region were 153.02×, 99.43% and 96.28%, respectively, indicating the high con dence level of variant calling. No other variants were detected in the LZTR1 gene or in the other RASopathy genes in the proband. Nonetheless, exome sequencing is limited in detecting large deletions/duplications and deep intronic variants. Based on the fact that variant c.1149 + 1G > T in the LZTR1 gene segregated with an NS-related phenotype in multiple affected family members, we speculated that the pedigree presented as dominant inheritance.
More than 15 gene mutations are known to be involved in the etiology of NS. Pathogenic variants in the genes encoding proteins implicated in the RAS-MAPK signaling pathway are responsible for NS. These gene mutations function upstream of the RAS/MAPK cascade or its regulation and dysregulate the RAS/MAPK pathway, leading to sustained or excessive activation of ERK (which de nes RASopathies) [1] . LZTR1-related NS was recently described. LZTR1 is a highly conserved gene and encodes a protein characterized by six tandemly arranged Kelch motifs at the N-terminus and two BTB/POZ (broad complex, tramtrack and bric-a-brac/Pox virus and zinc nger) domains at the C-terminus. LZTR1 is an important regulator of the normal cell cycle and acts as a tumor suppressor. Additionally, LZTR1 is found to be a conserved regulator of RAS ubiquitination and signaling [27][28][29][30] .
Previous studies have demonstrated that LZTR1 mutations can be acquired via AR or AD inheritance [3. 5. 11. 12. 31] . In the current study, the variants of LZTR1 associated with NS were located in both the Kelch and BTB domains, and AD NS has been attributed to the Kelch motifs, especially Kelch motifs 1-4 [14. 30. 31] . A new study showed that more than one RVxF motif is located between Kelch 5 and Kelch 6 in the LZTR1 gene. RVxF is a binding location of the protein phosphatase-1 (PP1) [30. 32] , and variant c.1149 + 1G > T is located in Kelch domains 5-6. This mutation can cause splice abnormalities and produce truncated proteins and thus may in uence the binding function of the RVxF motif and PP1. To our knowledge, more than 50% of phosphoserine/threonine dephosphorylation reactions are catalyzed by PP1 in mammalian cells [33] . PP1 multifunctionally interacts with dozens of polypeptides that function as substrates, inhibitors, chaperones, anchoring/scaffolding proteins, and substrate-speci ers [32. 34] and even those associated with heart physiology [35] . The proband's arrhythmia may be associated with the dysfunction of PP1. Variant c.1149 + 1G > T has been reported in three NS patients with compound heterozygous mutation of LZTR1 [11. 12. 15] (Table 1). Our patients had the NS phenotype and the heterozygous mutation inherited in the AD form, and variant c.1149 + 1G > T of LZTR1 may thus result in NS of the AD or AR form.
The typical feature of NS is a short stature, but some NS patients have GHD, as previous studies reported.
Three NS patients diagnosed with GHD [11. 12. 36] . In our study, the proband (III-3) was the fourth NS patient identifying as having GHD (Table 2). WES revealed no gene mutations associated with GHD in this patient, and the factors underlying GHD in these four NS patients were not clear.
In conclusion, the heterozygous variant c.1149 + 1G > T of LZTR1 was transferred from maternal family members, and the manifestations of NS ranged widely. The variant c.1149 + 1G > T of LZTR1 was previously reported in autosomal recessive NS, but this is the rst report of an autosomal dominant form. Our reported cases provided evidence for a more complex inheritance pattern resulting from LZTR1 c.1149 + 1G > T mutation. These results show that variant c.1149 + 1G > T of LZTR1 may result in NS with an AD or AR form. Occasionally, NS patients can have GHD.

Consent for publication
Written consent for publication was provided by the patients' parents.

Availability of data and materials
The dataset analyzed in the current study is available from the corresponding author upon reasonable request.

Con ict of Interest Statement
We declare that we have no nancial and personal relationships with other people or organizations that can inappropriately in uence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as in uencing the position presented in, or the review of, the manuscript entitled,

Funding source statement
This research received no speci c grants from any funding agency in the public, commercial or not-forpro t sector.
Author contribution statement X Z contributed to the data collection, data interpretation and writing of the manuscript. Z S contributed to the study design and reviewed the report. L W contributed to the clinical data collection and data interpretation. FF L contributed to the imaging data collection and data interpretation. ZZ L contributed to the gene mutation interpretation. WY Z contributed to the gene-phenotype analysis.