Introduction

Noonan syndrome is part of a spectrum of disorders with overlapping phenotypes that include craniofacial features1 and cardiovascular abnormalities2,3 and that overlaps with cardiofaciocutaneous and Costello syndromes.4 Most of the genes mutated in patients with Noonan syndrome cause dysregulated Ras-MAPK signaling.5,6,7 Genes shown to be mutated in these disorders include PTPN11, SOS1, SOS2, RAF1, KRAS, NRAS, BRAF, SHOC2, CBL, RIT1, and LZTR1, among others. For LZTR1, no role in Ras-MAPK signaling was known prior to its association with autosomal dominant Noonan syndrome.8 Germ-line and somatic biallelic loss of function in LZTR1 is involved in schwannomatosis.9 Previously, all molecularly confirmed forms of Noonan syndrome were inherited in an autosomal dominant pattern. Here we describe the results of clinical and molecular evaluations of 12 families with Noonan syndrome inherited in an autosomal recessive pattern.

Materials and methods

The studies in Bethesda, Houston, Seattle, and Oxford were approved by their respective research ethics bodies. The work done at Utrecht and Magdeburg was not considered research. Informed consent to publish patient photos was obtained. All DNA analyses were performed using standard techniques. Linkage analyses of families 1 and 12 were performed by genotyping nuclear family members on an Illumina Human Omni Express Bead Chip or cytoSNP12v2 Bead Chip (Illumina Corp, San Diego, CA, USA). Next-generation sequencing analyses were performed at several centers. Families 1 and 3 were exome sequenced at the National Institutes of Health (NIH) Intramural Sequencing Center, as described.10 Family 2, which was in the Undiagnosed Diseases Network program, and family 6 had exome sequencing at Baylor Genetics Laboratories, as described elsewhere.11 Families 4 and 5 were sequenced in the Utrecht clinical exome laboratory following standard procedures (see the Supplementary Methods online for details). Individuals in families 7, 8, 10, and 11 had targeted next-generation sequencing of the coding sequence of 19 candidate genes (Illumina Nextera Rapid Capture): PTN11, SOS1, SOS2, RAF1, RIT1, KRAS, NRAS, RRAS, HRAS, SHOC2, LZTR1, A2ML1, BRAF, MAP2K2, MAP2K2, NF1, SPRED1, RASA1. As described in the Supplementary Methods, some probands were tested by Sanger sequencing for subsets of these 19 genes and in some cases for PPP1CB. Genome sequencing was performed on family 9 by the Mary-Claire King Laboratory at the University of Washington. Family 12 was exome sequenced at the Oxford Genomics Centre (http://www.well.ox.ac.uk/ogc/) as described.12

RNA analyses

The consequences of the splice variants in families 1 and 3 were analyzed using total lymphoblast RNA (RNeasy, Qiagen, Hilden, Germany). One microliter of RNA (300–500 ng) was amplified using the OneStep RT-PCR kit (Qiagen). Products were separated on a 1.5% agarose gel and visualized using ethidium bromide, gel-isolated (QIAquick Gel Extraction Kit, Qiagen), and sequenced (BigDye Terminator v3.1 Cycle Sequencing Kit, Thermo Fisher Scientific, Waltham, MA).

Variants were specified in Human Genome Variation Society nomenclature re: NM_006767.3, (ENST00000215739.12) and GRCh37/hg19.

Results

Clinical evaluations

Brief summaries are provided here. Table 1 lists the individual features and the Supplementary Material includes full clinical descriptions.

Table 1 Clinical features of patients with autosomal recessive LZTR1-associated Noonan syndrome
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Family 1

This family comprised four siblings and parents (Figure 1a). The parents had no facial features of Noonan syndrome (Figure 2a) and had normal echocardiograms. All four siblings had variable features of Noonan syndrome including curly hair, depressed and wide nasal bridges, low-set ears, increased posterior angulation of the ears, broad neck with low posterior hairline (Figure 2c–f), and cardiac anomalies. Individual II-3 developed acute lymphoblastic leukemia at 5 years old (y.o.), which progressed to acute myeloblastic leukemia at 7 y.o. and she died 2 years later. The maternal grandmother had onset of unilateral hearing loss in her 60s, which was associated with a tumor, assessed as a meningioma on imaging. Several individuals in this family had subtle imaging findings compatible with schwannomas.

Figure 1: Pedigrees of the 12 affected families showing 23 affected live-born offspring, 21 of whom underwent molecular analysis with mutational data on the affected children and carrier parents.
figure 1

Clinically unaffected children shown not to have two pathogenic variants are indicated with an asterisk (carrier status of minors thereby not disclosed).

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Figure 2
figure 2

Facial features of ten affected children from six families, and two unaffected parents from one family. Facial features of family 1: (a,b) unaffected parents and (c–f) four affected children. In the children note variable features of short or upturned nose; depressed bridge; low-set, posteriorly angulated, or malformed ears; midface retrusion; broad/short neck; low posterior hairline; and curly hair. (g,h) Facial features of the affected brothers from family 2 demonstrating widely spaced eyes, down-slanted palpebral fissures, midface retrusion, full cheeks, a long philtrum, and overfolded, posteriorly angulated ears. (i–k) Features of individual II-1 from family 4 at three ages. (i) At 3 years of age, (j) at 7 years of age, and (k) at 14 years of age. Notice the short, broad neck; widely spaced eyes with down-slanted palpebral fissures and bilateral ptosis; low-set and posteriorly angulated ears; and pectus excavatum. Affected individual from family 5 at (l) 4 years of age, (m) 8 years of age, and (n) 6 years of age. Her features included down-slanted palpebral fissures; bilateral epicanthus and ptosis; broad neck; low-set, small, cupped ears; and a wide thorax. (o–q) Affected individual from family 8 showing widely spaced eyes; down-slanted palpebral fissures; mild ptosis; low-set and posteriorly angulated ears; a broad, webbed neck; and curly hair. (r,s) Features of individual II-2 from family 9. Note her widely set eyes, strabismus, bilateral epicanthus, down-slanted palpebral fissures, depressed nasal bridge, short and upturned nose, short and broad neck, and thickened vermilion of the lips.

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Family 2

This family included six boys, four full siblings, and two half siblings. The proband (Figure 1b, individual II-4) had a prenatal course complicated by arthrogryposis and polyhydramnios and his postnatal features included widely spaced eyes, down-slanted palpebral fissures, midface retrusion, full cheeks, a long philtrum, overfolded ears, and increased posterior angulation of the ears (Figure 2g). He had an atrial septal defect (ASD), pulmonary artery stenosis, a dysplastic pulmonary valve, and mild left ventricular and moderate right ventricular hypertrophy. His younger brother (Figure 1a, individual II-5) had similar features (Figure 2h) but did not have arthrogryposis. Both boys had hypotonia, developmental delay, recurrent metabolic acidosis episodes with cyclic vomiting, and had clinical laboratory findings of mitochondrial complex I + III deficiency. The parents were nondysmorphic.

Family 3

The proband (Figure 1c, individual II-4) had prenatal hypertrophic cardiomyopathy (HCM) and postnatally had coarse facial features, low-set ears, depressed nasal bridge, gingival overgrowth and thickened vermilion of upper and lower lips, short neck with low posterior hairline, small nails, and widely spaced nipples. He was confirmed to have HCM and a small ASD. His older sister (Figure 1c, individual II-1) had a cystic hygroma and polyhydramnios at 6 months gestation. Postnatally she had proptosis, ptosis, a wide mouth, low-set ears, a bulbous nose, and relative macrocephaly. She was found to have septal HCM with a small ASD, short stature, and growth hormone deficiency. The father had mild short stature and ptosis. Parents were consanguineous, but nondysmorphic.

Family 4

The proband (Figure 1d, individual II-1) was found to have biventricular HCM neonatally. He had a short, broad neck, widely spaced eyes with down-slanted palpebral fissures and bilateral ptosis, low-set ears, increased posterior angulation of the ears, and pectus excavatum (Figure 2i–k). He had mild short stature. At 3 y.o., he developed acute lymphoblastic leukemia and is in remission (in his teens). His younger brother (II-2) had atrioventricular septal defect, severe biventricular HCM, and a large sacral meningomyelocele. He died at day 4 of an inoperable cardiac defect. At postmortem, several organs were enlarged, the heart showed severe hypertrophy with muscular disarray. The parents were nonconsanguineous, nondysmorphic, and had no cardiac abnormalities.

Family 5

The proband (Figure 1e, individual II-4) had prenatal findings of cystic hygroma and a small ventricular septal defect (VSD). Postnatally, she had muscular VSDs, valvular pulmonary stenosis, a type II ASD, and biventricular HCM. She had short stature; down-slanted palpebral fissures; bilateral epicanthus and ptosis; broad neck; wide thorax; low-set, small, cupped ears; a narrow palate; and pes planus (Figure 2l–n). The twins (II-1 and II-2) had prenatal cystic hygroma; they died with hydrops at midgestation. Postmortem showed a large perimembranous VSD in one twin, and the other had a VSD and a dysplastic, not completely patent tricuspid valve, and a hypoplastic right ventricle. Dysmorphic features were not assessed. The parents were nondysmorphic and had no cardiac anomalies.

Family 6

The proband (Figure 1f, individual II-6) had prenatal polyhydramnios. She had a depressed, broad nasal bridge; midface retrusion with marked frontal bossing; high anterior hairline; nevus flammeus on forehead; down-slanted palpebral fissures; bilateral epicanthus with widely spaced eyes; long philtrum; full, sagging cheeks; short neck; broad chest; and relatively short arms and legs. The mother had one stillbirth with polyhydramnios and arthrogryposis, which was not investigated. The parents were nondysmorphic.

Family 7

Prenatally, the proband (Figure 1g, individual II-1) had increased nuchal translucency. Postnatally, he had left ventricular and interventricular septum hypertrophy, a VSD, and a small ASD. He had short stature, developmental delay, curly hair, low-set ears, increased posterior angulation of the ears, widely spaced eyes, down-slanting palpebral fissures, a narrow nasal bridge and tip, a mildly pointed chin, a broad neck and chest with mild pectus excavatum, relatively short fingers, and mild hypertrichosis. A brother (II-2) of the proband had fetal hydrops and died neonatally. The parents were nondysmorphic.

Family 8

Postnatally, the proband (Figure 1h, individual II-11) had an ASD, valvular and supravalvular pulmonary stenosis, and right ventricular HCM. His facial features included widely spaced eyes, down-slanted palpebral fissures, mild ptosis, low-set ears, increased posterior angulation of the ears, a broad, webbed neck, and curly hair (Figure 2o–q). His psychomotor development was apparently delayed. Other family members were nondysmorphic. The parents are first cousins once removed.

Family 9

The proband (Figure 1i, individual II-2) is a female with an unaffected 6 y.o. sister. There was no known consanguinity. The findings noted at birth included a broad neck. She had developmental delay from an early age. Her findings at a later examination included widely spaced eyes, strabismus, bilateral epicanthus, down-slanted palpebral fissures, depressed nasal bridge, short and upturned nose, short and broad neck, thickened vermilion of the lips, broad great toes and thumbs, and hypermobility (Figure 2r). She had a thickened ventricular septum and valvular insufficiency and left axis deviation with prolonged QT interval on electrocardiogram. A head magnetic resonance image showed ventriculomegaly. Her parents were nondysmorphic.

Family 10

This female proband (Figure 1j individual II-3) was born to healthy nondysmorphic, nonconsanguineous parents. Their first pregnancy was lost with a tetralogy of Fallot and fetal hydrops. The proband came to attention for increased nuchal translucency and polyhydramnios. Postnatally, a prominent Chiari network in the right atrium was detected by ultrasound, and at age 6 weeks, HCM was diagnosed. She had mild muscular hypotonia, a broad chest with widely spaced nipples, and pes planus. She had telecanthus, epicanthus, long philtrum, broad neck, and low-set, posteriorly angulated ears. She developed mild short stature. Formal IQ tests showed a mean IQ of 95 at age 4 years and 83 at age 6 years.

Family 11

The parents were nonconsanguineous and the family history was notable for an elective termination of a fetus with cystic hygroma, hydrops, and congenital heart defect. In the proband (Figure 1k II-6), cystic hygroma and polyhydramnios were identified prenatally. Postnatal echocardiography showed left HCM, left ventricular outlet stenosis, and moderate mitral insufficiency. He had down-slanted palpebral fissures, broad nasal bridge, upturned nares, low-set ears, increased posteriorly angulated ears, broad neck with redundant nuchal tissue, wide thorax with pectus excavatum, and single left palmar crease. The parents were nondysmorphic except for a mildly broad neck in the mother.

Family 12

The proband (Figure 1l V-1) was dysmorphic at birth. At initial assessment, mild developmental delay was noted. Noonan syndrome was suspected. Echocardiography showed a small perimembranous VSD and nonobstructive HCM with mild mitral valve prolapse. Her sister (individual V-4) had a murmur neonatally. HCM and dysplastic mitral valve were diagnosed by echocardiography. There was delay in attaining motor and speech milestones. At age 3 years 9 months features of Noonan syndrome were more apparent. At age 6 years mitral valve repair with subaortic membrane resection was undertaken. The parents were consanguineous.

Molecular data

Most probands underwent DNA sequencing either to search for causative variants or to exclude genes known at the time of sequencing to cause Noonan syndrome. The sequencing analyses included genome sequencing, exome sequencing, and panel sequencing, the latter including subsets of the 20 candidate genes listed in the Materials and Methods. The LZTR1 variants were detected by a number of methods among the various centers. These included exome and genome sequencing, next-generation panel sequencing, and/or Sanger sequencing (see Materials and Methods and Supplementary Methods). All affected patients had biallelic LZTR1 variants, with heterozygous parents. None of the nine molecularly evaluated, clinically unaffected children had biallelic variants. The genotypes are presented in Figure 1 and Table 1, and detailed data are presented in the Supplementary Material. Other molecular and genetic data are summarized below.

Family 1

Analysis of the single-nucleotide polymorphism genotyping data under a model of autosomal recessive inheritance identified a chromosome 22 region with shared alleles in all four children. Further analyses showed that the 9-Mb shared region containing LZTR1 on chr22 was the only extended shared region that could explain recessive inheritance and the only shared region that achieved the maximum achievable multipoint logarithm of the odds (LOD) score of 1.8 over many consecutive markers. Surprisingly, there was only one other autosomal region that had LOD>1.3, a 3-Mb region near 8pter in which multimarker analysis showed that any possible fully shared multimarker region must be small (<1 Mb).

The effect of the c.2220-17C>A splice variant was tested using reverse-transcription polymerase chain reaction (RT-PCR) from three of the siblings, both parents, and a control. In the affected children and mother a large, unspliced product was evident, along with a modest but noticeable decrease in properly spliced complementary DNA (cDNA) (Figure 3a). This splice variant retained intron 18, which predicts p.(Tyr741Hisfs*89) and the cDNA of this intron had only the A allele of the c.2220-17C>A variant (Supplementary Figure 1A), demonstrating that retention of this intron is associated with that allele. Finally, we Sanger sequenced an exonic heterozygous single-nucleotide polymorphism (rs13054014) in the gene and showed that the cDNA is skewed toward the allele with the splice variant, with relatively reduced levels of the nonsense variant, confirming a moderate level of nonsense-mediated messenger RNA decay. In Figure 3a, a minor aberrant 386-bp splice product is an inconsistently incorporated intron 19 (84 nucleotides), with an in-frame stop codon (Supplementary Figures 1B and 2).

Figure 3
figure 3

Assays showing abnormal splice products associated with splice variants. (a) Image of reverse-transcription polymerase chain reaction (RT-PCR) products associated with the splice variant in family 1. Total lymphoblast RNA was reverse-transcribed and PCR-amplified with primers from exons 18 and 21 of LZTR1. Two products were present in all individuals: a normally spliced product of 302 bp and an alternatively spliced product of 386 bp retaining intron 19. The carrier mother (lane 4) and affected children (lanes 6–8) had an additional RT-PCR product of 885 bp retaining intron 18. This product was not seen in either the father (lane 5) or control lymphoblast RNA (lane 3). Size markers are shown in lane 1 and a no-RNA control in lane 2. (b) Image of RT-PCR products associated with the splice variant in family 3. Total lymphoblast RNA was reverse transcribed and PCR-amplified with primers from exons 15 and 18 of LZTR1. A normally spliced product of 465 bp was present in both a control (lane 3) and an affected individual (lane 4). The affected child (lane 4) had an additional RT-PCR product of 583 bp retaining a 117-bp alternate exon from within intron 16. This product was not seen at a significant level in control lymphoblast RNA (lane 3). Size markers are shown in lane 1 and a no-RNA control in lane 2.

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Family 3

RT-PCR data from lymphoblasts showed the c.1943-256C>T splice variant retained a 117-bp alternate exon that lies within intron 16 (Figure 3b).

Family 8

Homozygosity for the familial disease-associated variant was found in the (not phenotyped) abortus.

Family 12

Linkage analysis under a model of autosomal recessive inheritance identified candidate regions summing to 357 Mb of shared identity by descent in the affected siblings, not shared in the unaffecteds. Including multiple loops of consanguinity into the analysis increased the maximum multipoint LOD score from 0.82 to 2.30 and identified two regions of shared autozygosity: a 4.8-Mb region on chr22 (17,782,813 to 22,590,873) and a 3.9-Mb region on chr7 (20,461,417 to 24,349,255). No other regions achieved a LOD score >1.00.

Discussion

Here we present clinical and molecular data on 12 families with children with Noonan syndrome caused by biallelic, pathogenic LZTR1 variants. In contrast to prior molecularly confirmed occurrences of Noonan syndrome that were inherited in an autosomal dominant pattern, the inheritance in these families is compatible with autosomal recessive inheritance. Like all other forms of Noonan syndrome, the clinical phenotype was variable but included bilateral epicanthus, down-slanted palpebral fissures, ptosis, low-set ears, increased posterior angulation of the ears, prenatal cystic hygromas with broad neck and low posterior hairline, short stature, and cardiac anomalies including HCM and valvular abnormalities, abnormal cardiac septation, and dysrhythmias. The children’s parents were not affected with Noonan syndrome or known cardiac anomalies. Two parents did have short stature and one had ptosis, but we cannot distinguish coincidence from causation for these. This disorder could have a semidominant inheritance pattern (mild phenotype in heterozygotes), which necessitates further studies.

The existence of a recessive form of Noonan syndrome was suggested from clinical analysis (OMIM NS2, 605275), but to date has not been molecularly confirmed. In 1969, Abdel-Salam and Temtamy13 described a male–female sib pair, offspring of a first-cousin marriage, with “Turner syndrome.” Their features were similar to the families described here, especially the siblings in family 3, who had linear growth delay and developmental compromise. Maximilian et al.14 described three siblings with unaffected parents. The clinical features in this family were similar to the present families, with cardiac disease including pulmonic stenosis, (but not cardiomyopathy). Van der Burgt and Brunner15 described four families with apparent recessive Noonan syndrome, but molecular characterization was not done. A potential explanation for sibling recurrence of a Noonan syndrome phenotype with unaffected parents is gonadal mosaicism for a gene known to cause autosomal dominant Noonan syndrome.16 However, in the individuals described here, extensive analyses failed to identify heterozygous pathogenic variants in genes other than LZTR1 associated with Noonan syndrome.

Autosomal dominant inheritance of Noonan syndrome has been associated with LZTR1 variants.8 Three families had vertical transmission of the phenotype. They had cosegregation of c.742G>A p.(Gly248Arg), c.850C>T p.(Arg284Cys), and c.740G>A p.(Ser247Asn) in LZTR1. Two other families had a single affected individual with a de novo heterozygous LZTR1 variant (c.859C>T p.(His287Tyr) and the other c.356A>C p.(Tyr119Cys)). We could not compare the dysmorphic features in the present patients with those of Yamamoto et al.8 because they were described only as having “typical facial features.” Several did have webbed necks, short stature, and cardiac valvular disease, and two had developmental delay. Heterozygous variants (c.710G>A p.(Arg237Gln) and c.745G>C p.(Ala249Pro)) in LZTR1 were also identified in two individuals by Chen et al.,17 although they did not consider them to be pathogenic. We agree with Yamamoto et al.8 that the two families of Chen et al.17 have autosomal dominant LZTR1-associated Noonan syndrome. These data confirm that some heterozygous LZTR1 variants cause autosomal dominant Noonan syndrome.

In contrast to these papers, we present clinical and molecular data on 23 offspring from 12 families that implicate LZTR1 in an autosomal recessive form of Noonan syndrome. All 12 families have variants that are absent or rare in databases (Table 1). The most common variant (c.628C>T p.(Arg210Ter)) was found in 19/276182 alleles with a non-Finnish European minor allele frequency of 0.0001. Other variants were less common or absent in databases, compatible with a rare recessive disorder. The variants were found in the biallelic state in affected patients and cosegregated with affection status in several multiplex sibships. Five of the implicated variants are nonsense or canonical splice variants. The U12-dependent branch site variant in family 1 increased the level of unspliced cDNA (Figure 3a). This is similar to a known LZTR1 c.2220-16_2220-14delCTT variant in the same branch site.9 That variant, from a NF2/SMARCB1-negative schwannomatosis patient, did not show altered splicing by (nonquantitative) RT-PCR, and was considered likely pathogenic based on affecting evolutionary-conserved nucleotides (PhastCons 1.00 and phyloP 4.81–4.92), retention of the variant in the tumors with loss of the remaining wild-type allele, a molecular signature typical of LZTR1-associated schwannomas (L. Messiaen, personal communication). The deep intronic splice variant in families 2, 3, 10, and 11 also affected cDNA splicing (Figure 3b).

The role of this gene in Noonan syndrome is supported by linkage in families 1 and 12, the latter of which generated a maximum LOD score of 2.3, and a combined LOD score of 4.1.

The eight missense variants were predicted damaging by combined annotation dependent depletion (CADD) scores: p.(His121Asp), 29.8; p.(Arg170Trp), 34; p.(Glu217Ala), 23.2; p.(Glu563Gln), 24; p.(Arg688Gly), 34; p.(Arg697Gln), 35; p.(Arg755Gln), 35; and p.(Ile821Thr), 27.3. Family 8 had a p.(Ile205Thr) variant in cis with p.(Arg170Trp) and has a CADD score of 23.6. We suggest that p.(Arg170Trp) is more likely pathogenic, but we cannot exclude the hypothesis that the pathogenic allele/haplotype comprises both and neither would be pathogenic alone. The c.*70G>A variant was in cis with c.1943-256C>T in family 3 but was not present on the c.1943-256C>T allele in three other families. We conclude the c.*70G>A variant alone is benign. Three missense variants we detected (p.(Arg170Trp), p.(Arg697Gln), and p.(Arg688Gly)) were similar to schwannomatosis variants (p.(Arg170Gln),18 p.CArg697Trp),19 and p.(Arg688Cys)9). Two putative loss-of-function variants was identical to a schwannomatosis variant p.(Gln10Argfs*15).9

The existence of recessive and dominant forms of Noonan syndrome associated with LZTR1 variants has implications for the pathogenetic model for this disorder. To date, all variants associated with the dominant form are missense variants, although this number is small (six, including the putatively pathogenic variants of Chen et al.17). In contrast, we identified missense, nonsense, and splicing variants in the recessive form (Figure 4). There are several pathogenetic models for these observations. The first specifies a reduction of LZTR1 function below 50% resulting in Noonan syndrome, either through monoallelic dominant negative LZTR1 missense variants (autosomal dominant form) or the combination of hypomorphic and loss-of-function variants in the recessive form. Homozygous loss of function of Lztr1 in the mouse causes preweaning lethality and is probably lethal in humans, as suggested by the report of a fetus with nonimmune hydrops fetalis and a homozygous LZTR1 c.2317G>A; p.(Val773Met) variant.20 To date, no examples of biallelic germ-line loss-of-function alleles have been identified in the mouse (see web link, below) lethality (Mouse Lztr1 −/− phenotype: http://www.mousephenotype.org/data/charts?accession=MGI:1914113&allele_accession_id=MGI:4432946&zygosity=homozygote&parameter_stable_id=IMPC_VIA_001_001&pipeline_stable_id=MGP_001&phenotyping_center=WTSI.). The second model specifies that the mutational position determines whether missense LZTR1 variants cause dominant or recessive Noonan syndrome. Although the numbers of missense variants in dominant LZTR1-associated Noonan syndrome are small, they all reside between codons 119 and 287, whereas the variants we have identified in the recessive form range throughout the protein (Figure 4). A robust genotype–phenotype analysis awaits the description of additional patients with dominant and recessive LZTR1-associated Noonan syndrome.

Figure 4: Cartoon of LZTR1 mutations showing the variants identified here in autosomal recessive Noonan syndrome above the protein and those previously reported in autosomal dominant Noonan syndrome below the protein.
figure 4

The ^ symbol indicates that these two variants were in cis on this mutant allele and it is hypothesized that p.(Arg170Trp) is pathogenic; see text.

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The LZTR1 pathogenicity model must also take into account data on schwannomatosis susceptibility. There are two potential models of schwannomatosis susceptibility that involve a three-step mutational process beginning with a germ-line LZTR1 mutation, with three or four additional hits in two or three genes.21 A germ-line LZTR1 variant in combination with a somatic deletion of wild-type 22q including LZTR1, NF2, and potentially SMARCB1 in combination with one or more point mutations, can lead to tumors.21 Several family 1 members had suggestive signs of schwannomas on magnetic resonance image. As well, two variants identified here (c.27delG, p.(Gln10Argfs*15) and c.628C>T p.(Arg210*)) were also identified as a schwannomatosis susceptibility variants.9,19 This model of schwannomatosis has implications for the relatively common 22q11.2 deletion. LZTR1 lies within the 3-Mb A-D proximal region, which is found in ~85% of patients with the phenotype.22 Every patient with that deletion is haploinsufficient for LZTR1 and may be susceptible to schwannomas. Additionally, to our knowledge, there are no patients reported with both Noonan syndrome and a typical 22q11.2 deletion syndrome phenotype (one deleted allele plus a point variant in trans). We predict that such patients exist. This combined phenotype may be subtle, as has been shown for Bernard–Soulier; cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK); and van den Ende–Gupta syndromes with the typical 22q11.2 3-Mb deletion and a point mutation in trans (GP1BB, SNAP29, or SCARF2, respectively).23,24,25 We predict that patients with mild Noonan syndrome, with or without features of 22q11.2 deletion syndrome, exist and encourage physicians to be alert for this combination of phenotypic findings.

Autosomal recessive LZTR1-associated Noonan syndrome has variable expressivity and includes several mildly affected patients, but the severe end includes lethality, most commonly from cardiac disease, which can be prenatal. Several patients had significant cardiac dysfunction and while we cannot prove that hydrops and prenatal losses were attributable to LZTR1 variants, we strongly suspect that they are. Also, two of these patients developed acute lymphoblastic leukemia. Acute lymphoblastic leukemia is not the most common leukemia in known forms of Noonan syndrome.26 The possibility that these leukemias were coincidental is discounted by the role of LZTR1 in tumorigenesis.9,21 These data suggest, but do not prove, that autosomal recessive LZTR1-associated Noonan syndrome is a cancer susceptibility disorder.

We conclude that biallelic LZTR1 variants can cause Noonan syndrome inherited in an autosomal recessive pattern, based on 12 families with a total of 21 molecularly characterized affected offspring, unaffected parents, cosegregation in unaffected siblings, and rare, biallelic LZTR1 variants. These data have important implications for the molecular diagnosis of Noonan syndrome, evaluating recurrence risks for families with LZTR1 variants, the pathogenesis of familial schwannomatosis, and the heterogeneity of phenotypes associated with 22q11.2 deletions.