LEOPARD Syndrome with PTPN11 Gene Mutation in Three Family Members Presenting with Different Phenotypes

Abstract

LEOPARD syndrome (LS) is a rare autosomal dominant disorder that is characterized by multiple lentigines and various congenital anomalies. The clinical diagnosis of LS requires molecular confirmation. The most frequently reported mutations in LS patients are in the protein tyrosine phosphatase nonreceptor type 11 gene, PTPN11 . Herein, we report the cases of three family members from two generations who are affected by LS and all carry the PTPN11 mutation c.836A > G (p.Tyr279Cys), identified by next-generation sequencing, while exhibiting different phenotypes.

Introduction

LEOPARD syndrome (LS, OMIM #151100) is a rare multisystemic autosomal dominant inherited disorder with full penetrance and variable expressivity. ¹ It is also known as multiple lentigines syndrome, cardiocutaneous syndrome, lentiginosis profusa syndrome, Moynahan syndrome, Gorlin syndrome, and Noonan syndrome (NS) with multiple lentigines. The term LEOPARD is an acronym for the most common features of the syndrome, including multiple l entigines, e lectrocardiographic conduction abnormalities, o cular hypertelorism, p ulmonary stenosis, a bnormal genitalia, r etardation of growth, and sensorineural d eafness. ^{1 2} Affected patients do not usually present with all of these classical clinical features. ³

Lentigines are the most striking manifestation of the syndrome. They appear in early childhood and increase in number until puberty. Lentigines are brown to black macules of 1 to 2 mm in size and are mostly concentrated on the face, neck, and upper trunk. Black lentigines of up to 5 mm in diameter, known as café noir spots, have also been reported in patients with LS. ^{4 5} Classical histopathological findings of lentigines include an increased number of melanocytes, resulting in abundant melanin production and prominent rete ridges. ^{2 6} Less commonly reported cutaneous findings include localized hypopigmented patches, axillary freckling, and onychodystrophy.

A heterozygous missense mutation in the protein tyrosine phosphatase nonreceptor type 11 ( PTPN11 ) gene, which is located on chromosome 12q24.1, is identified in approximately 85% of patients with LS. ^{7 8} Two recurrent mutations that are highly specific for LS are located in exons 7 (p.Tyr279Cys) and 12 (p.Thr468Met). ^{3 7 8} PTPN11 encodes SRC homology 2 phosphatase 2 (SHP-2), which is a cytoplasmic protein tyrosine phosphatase that regulates the response to several growth factors, cytokines, and hormones that control various developmental processes. ⁹ Pathogenic PTPN11 variants in LS patients result in the dysregulation of downstream signaling such as Ras/MAPK and PI3K/Akt/mTOR signaling pathways. ¹⁰ Motegi et al ¹⁰ demonstrated that the pathogenesis of lentigines development in LS patients is possibly due to the activation of Akt/mTOR and STAT3 signaling pathways. Other genes reported in association with LS include RAF1 , ¹¹ BRAF , ^{12 13} and MAP2K1 . ⁵

We report the cases of three patients from two generations of a single family diagnosed with LS and harboring the same PTPN11 gene mutation but demonstrating variable phenotypes.

Patients and Methods

The affected family members were from two generations: father and two children. The father was diagnosed and followed up in an outpatient dermatology clinic at King Khalid University Hospital in Riyadh (Saudi Arabia) as a case of familial lentiginosis. Recently, the patient noticed similar skin lesions affecting both his sons. Clinical and laboratory data for the three patients were gathered from their medical records. After obtaining written informed consent from the parents, peripheral blood samples for DNA analyses were collected from both parents and the two sons ( Fig. 1 ). The molecular analysis of the patients' DNA was performed by next-generation sequencing (NGS) using TruSeq Exome Enrichment Kit (Illumina) following the manufacturer's protocol.

Fig. 1.

Family pedigree. Filled symbols represent affected members.

Cases

Case 1 : The father was a 37-year-old male. He had 12 siblings. His parents were nonconsanguineous, and by history, none of his parents or siblings appeared to be affected by LS. However, we could not examine the other family members. The patient was 168 cm tall and weighed 65.8 kg. There were numerous small, light- to dark-brown macules all over his body, predominantly on his face but not on mucous membranes, with two café au lait macules on the trunk ( Fig. 2 ). The patient's age at the initial appearance of lentigines is unknown, but the number of lentigines had progressively increased until puberty. Some dysmorphic features were noticed, including ocular hypertelorism and low-set ears, along with joint hypermobility and pectus excavatum. The patient had a history of atrial septal defect, motor developmental delay during childhood, and right cryptorchidism, for which he underwent orchidopexy. There was no history of pulmonary stenosis or deafness. We used an Alexandrite laser to treat the facial lentigines, and the desired improvement was achieved after seven sessions.

Fig. 2.

LEOPARD syndrome (case 1): multiple lentigines on the back, trunk, upper extremities, and head/neck ( A – D ), with pectus excavatum ( A ) and low-set ears ( D ).

Case 2 : The 4-year-old son presented with multiple light-brown macules scattered over his face and body ( Fig. 3 ). The first lentigo had been noticed at the age of 6 months, and the lentigines had increased in number over time. The patient had a history of undescended testes, for which he had been surgically treated, as well as gross motor and speech delay. He also exhibited skeletal abnormalities, such as pectus excavatum and in-toeing, and some facial dysmorphic features, including low-set ears and ocular hypertelorism. He had no history of pulmonary stenosis, cardiomyopathies, or sensorineural deafness.

Fig. 3.

LEOPARD syndrome (case 2): few lentigines on the trunk and thighs ( A–C ), with pectus excavatum ( A,B ) and low-set ears ( D ).

Case 3 : The 18-month-old son presented with a few brownish small macules on the neck, trunk, and lower extremities ( Fig. 4 ). The first lentigo had appeared at the age of 2 months. On general examination, he had low-set ears and ocular hypertelorism. As a neonate, he was diagnosed with a small atrial septal defect and a small posterior muscular ventricular septal defect. He had no history of pulmonary stenosis, abnormal genitalia, or sensorineural deafness.

Fig. 4.

LEOPARD syndrome (case 3): few scattered lentigines on the trunk and lower limbs ( A – C ), with low-set ears (D) .

Both the sons were a product of uneventful pregnancies and were delivered at term through spontaneous vaginal delivery with no perinatal complications.

Result of Genomic DNA Analyses

Molecular analysis of genomic DNA of the three patients by NGS revealed a heterozygous mutation in PTPN11 , c.836A > G, which was predicted to cause a change from tyrosine 279 to cysteine at the protein level (p.Y279C), in all three patients. Sanger analysis of the boys' mother DNA revealed that she carries the wild-type allele of the identified PTPN11 variant.

Discussion

In 1936, Zeisler and Becker ¹⁴ reported the case of a female patient who presented with multiple lentigines that were scattered over her body and with ocular hypertelorism, pectus carinatum, and mandibular prognathism. In 1969, Gorlin et al ¹ named this condition LEOPARD syndrome. Voron et al ¹⁵ categorized the LS manifestations as follows: cutaneous abnormalities, cardiac abnormalities, genitourinary abnormalities, endocrine findings, neurogenic defects, cephalofacial dysmorphism, short stature, skeletal anomalies, and familial history consistent with an autosomal dominant mode of inheritance. They also proposed minimal diagnostic criteria for LS: the appearance of multiple lentigines with at least two of the other characteristic features or, if lentigines are absent, three of the other cardinal features and a first-degree relative with LS. A review of data from 48 LS patients revealed that other facial anomalies were frequently reported, including ocular hypertelorism (in 83% of the cases), palpebral ptosis (in 67% of the cases), and dysmorphic ears (in 65% of the cases). Rarely reported anomalies include cryptorchidism, macrocephaly, horse kidney, hydrothorax, myelodysplasia, and umbilical hernia. ¹⁶ In our patients, initial diagnosis of LS was based on the presence of multiple lentigines with a classical distribution and morphology and on the presence of more than two other cardinal features. All the three cases meet the diagnostic criteria of LS by Voron et al. ¹⁵

Table 1 illustrated the clinical manifestations of the three family members. Cases 1 and 3 were diagnosed with mild cardiac septal defects, which closed spontaneously and did not require surgical intervention. Cases 1 and 2 had right cryptorchidism that was corrected surgically in the first year of life. Case 2 showed speech delay, and both children had physical developmental delay. Growth parameters were normal in all the cases. However, both sons were under 5 years of age, and therefore some features including growth retardation and hypertrophic cardiomyopathy (HCM) could not be conclusively assessed as it may appear with age. Our patients lacked some features associated with LS, including pulmonary stenosis and sensorineural deafness. However, pectus excavatum, which is frequently seen in patients with NS and less frequently in patients with LS, was present in cases 1 and 2. ^{15 17} In addition, cases 1 and 3 showed joint hypermobility, which is an uncommon feature of LS. ¹⁷ Case 2 had in-toeing, which, to the best of our knowledge, has never been reported in association with LS.

Table 1. Summarized clinical manifestations of LS in the three family members.

	Case 1	Case 2	Case 3
Age/sex	37-y-old male	4-y-old male	18-mo-old male
Height in cm (percentile)	163	103 (50th)	88 (>95th)
Age of first lentigines	Unknown	6 mo	2 mo
Cardiac defects	+	−	+
Ocular hypertelorism	+	+	+
Pulmonary stenosis	−	−	−
History of cryptorchidism	+	+	−
Pectus excavatum	+	+	−
Joint hypermobility	+	−	+
In-toeing	−	+	−
Motor delay	+ (history in childhood)	+	+
Deafness	−	−	−

LS and other RASopathies including NS and neurofibromatosis type 1 (NF1) shared common clinical features including psychomotor delay, facial dysmorphism, and cardiac, cutaneous, and skeletal abnormalities. LS can be distinguished from NS based on the appearance of lentigines and deafness. ^{11 18} The presence of café au lait macules in LS can be mistaken for NF1, but patients with LS do not fulfill the criteria of NF1. ¹⁸ LS features may overlap with those of other syndromes that should be considered in the differential diagnosis such as Watson syndrome, centrofacial lentiginosis, inherited patterned lentiginosis, Carney complex, and Peutz–Jeghers' syndrome. ^{17 18}

To date, 12 missense PTPN11 mutations that cause LS have been identified: Tyr279Cys/Ser, Ala461Thr/Ser, Gly464Ala, Thr468Met/Pro, Arg498Leu/Trp, Gln506Pro, and Gln510Glu/Pro. ^{3 19 20} The patients described here carry the c.836A > G missense mutation in exon 7 (p.Tyr279Cys), which is one of two most frequent mutations that account for more than 60% of cases. ^{7 8} The p.Tyr279Cys mutation has been reported in approximately 50 LS patients of German, French, Spanish, Italian, Estonian, Bosnian, Chinese, Korean, Japanese, and Australian origin. Short stature, deafness, and HCM are reportedly associated with the p.Tyr279Cys mutation. ^{8 16 20} Our patients did not develop deafness nor cardiomyopathy. However, long-term cardiac evaluation for cardiomyopathy would be advisable as it may appear later in time.

PTPN11 mutations are detected in approximately 50% of patients with NS, mostly in exons 3, 7, 8, and 13. They are recognized as gain-of-function mutations, ²¹ whereas mutations associated with LS generally have dominant negative, not activating, effects. ^{6 10} Most of the mutations (including p.Tyr279Cys) that have been previously identified in LS are clustered at the interacting portions of the amino-terminal SH2 and PTP domains. ^{21 22} The p.Tyr279Cys mutation is catalytically defective and results in decreased phosphatase activity but an increased association with upstream activators. At the functional level, the PI3K/AKT/mTOR pathway is hyperactivated, which is causally linked to the pathogenesis of LS-associated HCM, whereas the impact on Ras/MAPK (activation or inhibition) is still debated. ^{23 24} However, whether PI3K/mTOR hyperactivation is responsible for other traits of the disease and whether other signaling dysfunctions are involved in LS ontology remain to be established. ²⁵

Fundamental management items for LS patients should include the following: (1) careful cardiological assessment by echocardiography to exclude HCM, (2) surgical intervention for cryptorchidism, hypospadias, or major skeletal deformity, and (3) hearing tests to screen for hearing loss. The presence of lentigines is a cosmetic concern for most of the patients. Different laser types have been used to treat lentigines. Kontoes et al ²⁶ reported a successful use of intense pulsed light in LS, and Lee et al ²⁷ reported that Alexandrite laser treatment was effective. Case 1 was treated with an Alexandrite laser, and the results were satisfactory.

In conclusion, we reported the cases of three family members who were affected by LS in an autosomal dominant pattern and shared the p.Tyr279Cys mutation in PTPN11 , with a disparity in phenotypes expression, mainly of cardiac, genital, and skeletal anomalies.