Juvenile myelomonocytic leukemia-associated variants are associated with neo-natal lethal noonan syndrome

ABSTRACT Gain-of-function variants in some RAS–MAPK pathway genes, including _PTPN11_ and _NRAS_, are associated with RASopathies and/or acquired hematological malignancies, most notably

juvenile myelomonocytic leukemia (JMML). With rare exceptions, the spectrum of germline variants causing RASopathies does not overlap with the somatic variants identified in isolated JMML.

Studies comparing these variants suggest a stronger gain-of-function activity in the JMML variants. As JMML variants have not been identified as germline defects and have a greater impact on

protein function, it has been speculated that they would be embryonic lethal. Here we identified three variants, which have previously only been identified in isolated somatic JMML and

other sporadic cancers, in four cases with a severe pre- or neo-natal lethal presentation of Noonan syndrome. These cases support the hypothesis that these stronger gain-of-function variants

are rarely compatible with life. SIMILAR CONTENT BEING VIEWED BY OTHERS ADVANCES IN GERMLINE PREDISPOSITION TO ACUTE LEUKAEMIAS AND MYELOID NEOPLASMS Article 16 December 2020 GERMLINE

_ATG2B/GSKIP_-CONTAINING 14Q32 DUPLICATION PREDISPOSES TO EARLY CLONAL HEMATOPOIESIS LEADING TO MYELOID NEOPLASMS Article 25 June 2021 SYNONYMOUS _GATA2_ MUTATIONS RESULT IN SELECTIVE LOSS

OF MUTATED RNA AND ARE COMMON IN PATIENTS WITH GATA2 DEFICIENCY Article Open access 18 June 2020 INTRODUCTION Germline variants in RAS–MAPK pathway genes are associated with RASopathies, a

genetically heterogeneous set of conditions whose clinical features include short stature, cardiovascular defects, development delays, characteristic facies, and skeletal, hematologic, and

cutaneous findings.1 Prenatal findings of RASopathies are nonspecific and include increased nuchal translucency, cystic hygroma, cardiac anomalies, and hydrops fetalis. Pathogenic variants

in RAS–MAPK pathway genes have been identified in 9–17.3% of diploid fetuses with these ultrasound findings.2, 3, 4 Although germline variants in the RAS–MAPK pathway genes are associated

with RASopathies, somatic variants in _PTPN11_, _NRAS, KRAS,_ and _CBL_ are initiating drivers for isolated juvenile myelomonocytic leukemia (JMML). JMML is an aggressive myeloproliferative

neoplasm of early childhood that is commonly lethal without a hematopoietic stem cell transplant. Somatic variants in these genes are also observed, although less frequently, in other

sporadic leukemias (eg, AML, ALL, and CMML) as secondary, cooperating variants in subclones.5, 6, 7 Individuals with Noonan syndrome are at a high risk of developing a transient

myeloproliferative disorder (MPD) during infancy, which resembles JMML but normally resolves without treatment, often referred to as JMML-like MPD.1, 8 However, JMML-like MPD can result in

early lethality in a minority of individuals with RASopathies.9 Pathogenic variants in _PTPN11_ or _NRAS_ causing isolated JMML rarely overlap with those causing Noonan syndrome.10, 11, 12,

13, 14 This has been attributed to the JMML variants having a stronger gain-of-function activity than RASopathy-associated variants. There are rare observations of Noonan-associated _PTPN11_

variants identified in isolated JMML, and a few reported cases of germline inheritance of JMML variants.4, 14, 15, 16 These data have led to the suggestion that the strong gain-of-function

variants observed in isolated JMML would be embryonic lethal if inherited as a germline variant.2, 13, 14, 17 Here, we present a case series supporting this model. SUBJECTS AND METHODS CASE

1 On 15.2 weeks ultrasound, the fetus was found to have a cystic hygroma. At 17.4 weeks, a follow-up ultrasound identified a heart abnormality, pleural effusion, pericardial effusion, fetal

hydrops, and persistent cystic hygroma. Prenatal testing demonstrated a normal karyotype and microarray. The pregnancy was terminated at 19 weeks gestation. The maternal and paternal ages at

conception were 21 and 27 years, respectively. CASE 2 Prenatal ultrasonographic evaluation revealed a 9.0-mm nuchal translucency, cystic hygroma, and hydrops fetalis (Figure 1). Prenatal

testing was negative for fetal infections and showed a normal karyotype and microarray. Following testing, the pregnancy was terminated. The maternal and paternal ages at conception were 26

and 32 years, respectively. CASE 3 On 12 weeks ultrasound, the fetus was found to have cystic hygroma. Non-immune hydrops fetalis, bilateral pleural effusions, lateral ventriculomegaly (left

greater than right), polyhydramnios, absence of stomach bubble, absence of swallowing, hypertelorism, low-set ears, wide neck, mild retrognathia, and short limbs were identified on

sequential ultrasound. The pregnancy resulted in a live birth at 33 weeks gestation. The postnatal period was complicated by thrombocytopenia, hypoxemia, bilateral pneumothoraces, and

respiratory distress. A postnatal evaluation identified a normal karyotype, structurally and functionally normal heart, no evidence of esophageal atresia, and slightly below average limb

length. On day of life 2, the neonate passed away because of pulmonary hypertension as a result of pulmonary hypoplasia secondary to non-immune fetal hydrops. The maternal and paternal ages

at conception were 23 and 30 years, respectively. CASE 4 Prenatal ultrasonographic evaluation revealed a cystic hygroma. The pregnancy resulted in a live birth at 31 weeks gestation. The

neonate had a low nasal bridge, hypertelorism, low-set posteriorly rotated ears, low hairline, webbed neck, thickened eyebrows, small upturned nose, short limbs, polydactyly of the left

foot, coarseness, and scoliosis. The neonate was diagnosed with JMML by peripheral blood smear. On day of life 30, the neonate passed away from JMML and complications of necrotizing

enterocolitis. VARIANT ANALYSIS DNA was extracted from amniocytes (cases 1 and 2), blood (case 3), or fibroblasts (case 4) using either Qiagen (Valencia, CA, USA) Puregene or Perkin Elmer

(Waltham, MA, USA) Chemagen DNA extraction kits according to the manufacturers’ recommendation. For cases 1–3, sequencing of _PTPN11_, _SOS1_, _RAF1_, _KRAS_, _NRAS_, _BRAF_, _MAP2K1_,

_MAP2K2_, _HRAS_, _SHOC2_ exon 02, _CBL,_ and _SPRED1_ was performed by oligonucleotide-based target capture (SureSelect, Agilent, Santa Clara, CA, USA) and sequencing using Illumina

HiSeq2000 instrument (50-base paired end; San Diego, CA, USA). Alignment and variant calls were performed as previously described using BWA and GATK (version 1.0.4705).18 For case 4, a

microarray-based resequencing assay (GeneChip, Affymetrix, Santa Clara, CA, USA) was used, as previously described.19 For case 3, droplet digital PCR probes (ddPCR; Bio-Rad, Hercules, CA,

USA) were used to quantitate variant fraction using the manufacturer's protocol. Sanger sequencing was used to fill in failed regions or sequenced regions with insufficient coverage

(<20x), confirm clinically significant variants, and for parental testing of variants in _PTPN11_ (NM_002834.3) or _NRAS_ (NM_002524.3). Partners HealthCare Institutional Review Board

approved this study. Variants were deposited in ClinVar (http://www.ncbi.nlm.nih.gov/clinvar; SCV000204032, SCV000204031, and SCV000204071). RESULTS Sequencing of RAS–MAPK pathway genes in

four cases with a severe pre- or neo-natal presentation of RASopathy identified three variants (Supplementary Figure 1) previously only reported as somatic changes in isolated JMML and other

sporadic cancers. In case 1, c.227A>G (p.(Glu76Gly)) in exon 03 of _PTPN11_ was identified (49% alternative allele fraction). Glu76 is a hotspot for somatic changes in JMML and

p.(Glu76Gly) has been observed in >35 hematopoietic neoplasms, the majority being JMML.20 In case 2, c.214G>A (p.(Ala72Thr)) in exon 03 of _PTPN11_ was identified (51% alternative

allele fraction). Ala72 is also a hotspot for JMML and p.(Ala72Thr) has been identified in >30 hematopoietic neoplasms, of which 15 were JMML.20 In cases 3 and 4, c.34G>A

(p.(Gly12Ser)) in exon 02 of _NRAS_ was identified. In case 3, although the alternative allelic fraction from the NGS data (36%) was slightly low, mosaicism was excluded based upon ddPCR

data (50% alternative allelic fraction). However, in case 4, mosaicism cannot be excluded as the variant was identified only via Sanger sequencing. Previous reports have described

p.(Gly12Ser) in >75 hematopoietic neoplasms, the majority being AML.20 In cases 1, 2, and 4, the variants were apparently _de novo_ (paternity was not molecularly determined). Parental

samples were not available for case 3. DISCUSSION Germline-inherited JMML variants in _PTPN11_ have been hypothesized to be embryonic lethal because of their stronger gain-of-function

activity and lack of reported germline observation.2, 13, 14, 17 Supporting this, a prior report described a pregnancy with a severe presentation including a 11-mm nuchal translucency,

cystic hygroma, fetal hydrops, hydrothorax, and generalized skin edema with a _de novo_ germline variant, c.227A>T (p.(Glu76Val)), typically seen in isolated JMML.4 Similarly, another

variant, c.1520C>A (p.(Thr507Lys)), seen exclusively in other leukemias, although not JMML, was identified in two fetuses with hydrops fetalis.2, 21 Our study lends further support to

this hypothesis, given JMML variants were observed in cases 1 and 2, which both presented with severe prenatal abnormalities. However, as the pregnancy in the prior reports and two reported

here were either electively terminated or lost to follow-up, it remains suggestive that strong gain-of-function variants in _PTPN11_ are incompatible with life. Initial studies suggest that

variants in _NRAS_ have a similar spectrum as those in _PTPN11,_ with mildly activating variants causing Noonan syndrome and strong gain-of-function activity variants acting as initiating

drivers for isolated JMML.10, 11 Supporting this, most germline _NRAS_ variants associated with Noonan syndrome (c.179G>A (p.(Gly60Glu)), c.71T>A (p.(Ile24Asn)), and c.149C>T

(p.(Thr50Ile))) were found to be mildly activating when compared with the recurrent oncogenic variant, c.35G>T (p.(Gly12Val)).10, 11 In addition, embryonic expression of another oncogenic

_NRAS_ variant, p.(Gly12Asp), was embryonic lethal in mice.22 Cases 3 and 4, harboring the c.34G>A (p.(Gly12Ser)) variant and resulting in early neo-natal death, support that

germline-inherited oncogenic NRAS variants are embryonic lethal in humans. Another oncogenic _NRAS_ variant, c.38G>A (p.(Gly13Asp)), has been reported in two individuals without a severe

RASopathy presentation. The first did not have any noted RASopathy features, but presented with infantile-onset leukemia and adult-onset hematological abnormalities,15 suggesting this

presentation is likely due to tissue-specific mosaicism. The second presented with an aggressive JMML-like MPD and, upon follow-up evaluation, features of a RASopathy.16 Although further

studies are necessary to determine if oncogenic _NRAS_ variants result in early lethality, all reported cases with a germline-inherited oncogenic _NRAS_ variant had a hematological

abnormality. These observations suggest that there are variable phenotypes associated with germline inheritance of oncogenic _NRAS_ variants, but these individuals are at risk for

hematological abnormalities. This study supports the model that JMML variants with germline inheritance result in severe prenatal and/or neo-natal presentation. Additional studies are

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Medicine Laboratory of Molecular Medicine staff for excellent technical support. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Laboratory for Molecular Medicine, Partners Personalized

Medicine, Cambridge, MA, USA Heather Mason-Suares, Diana Toledo, Katherine A Lafferty, Thomas E Mullen & Matthew S Lebo * Departments of Pathology, Harvard Medical School and Brigham and

Women’s Hospital, Boston, MA, USA Heather Mason-Suares & Matthew S Lebo * Department of Medical Biology, Le CHU de Québec, Québec, Canada Jean Gekas * Section of Genetics, University of

Colorado Anschutz Medical Campus, Aurora, CO, USA Naomi Meeks * Department of Pathology, Children's Hospitals & Clinics of MN, Saint Paul, MN, USA M Cristina Pacheco * Maternal

Fetal Medicine, Riverside Methodist Hospital, Columbus, OH, USA David Sharpe Authors * Heather Mason-Suares View author publications You can also search for this author inPubMed Google

Scholar * Diana Toledo View author publications You can also search for this author inPubMed Google Scholar * Jean Gekas View author publications You can also search for this author inPubMed

 Google Scholar * Katherine A Lafferty View author publications You can also search for this author inPubMed Google Scholar * Naomi Meeks View author publications You can also search for

this author inPubMed Google Scholar * M Cristina Pacheco View author publications You can also search for this author inPubMed Google Scholar * David Sharpe View author publications You can

also search for this author inPubMed Google Scholar * Thomas E Mullen View author publications You can also search for this author inPubMed Google Scholar * Matthew S Lebo View author

publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Matthew S Lebo. ETHICS DECLARATIONS COMPETING INTERESTS HM-S, DT, KAL, TEM and

MSL are employed by non-profit, fee-for-service laboratories that offers genetic testing. ADDITIONAL INFORMATION Supplementary Information accompanies this paper on European Journal of

Human Genetics website SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 (JPG 50 KB) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Mason-Suares, H.,

Toledo, D., Gekas, J. _et al._ Juvenile myelomonocytic leukemia-associated variants are associated with neo-natal lethal Noonan syndrome. _Eur J Hum Genet_ 25, 509–511 (2017).

https://doi.org/10.1038/ejhg.2016.202 Download citation * Received: 13 April 2016 * Revised: 03 November 2016 * Accepted: 14 December 2016 * Published: 18 January 2017 * Issue Date: April

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