Autosomal recessive Noonan syndrome associated with biallelic LZTR1 variants

Abstract

Purpose: To characterize the molecular genetics of autosomal recessive Noonan syndrome.

Methods: Families underwent phenotyping for features of Noonan syndrome in children and their parents. Two multiplex families underwent linkage analysis. Exome, genome, or multigene panel sequencing was used to identify variants. The molecular consequences of observed splice variants were evaluated by reverse-transcription polymerase chain reaction.

Results: Twelve families with a total of 23 affected children with features of Noonan syndrome were evaluated. The phenotypic range included mildly affected patients, but it was lethal in some, with cardiac disease and leukemia. All of the parents were unaffected. Linkage analysis using a recessive model supported a candidate region in chromosome 22q11, which includes LZTR1, previously shown to harbor mutations in patients with Noonan syndrome inherited in a dominant pattern. Sequencing analyses of 21 live-born patients and a stillbirth identified biallelic pathogenic variants in LZTR1, including putative loss-of-function, missense, and canonical and noncanonical splicing variants in the affected children, with heterozygous, clinically unaffected parents and heterozygous or normal genotypes in unaffected siblings.

Conclusion: These clinical and genetic data confirm the existence of a form of Noonan syndrome that is inherited in an autosomal recessive pattern and identify biallelic mutations in LZTR1.

Keywords: Noonan syndrome; autosomal recessive inheritance; cardiomyopathy; leukemia; multiple congenital anomalies.

Figure 1

Pedigrees of the 12 affected families showing 23 affected liveborn offspring, 21 of whom underwent molecular analysis with mutational data on the affected children and carrier parents. Clinically unaffected children shown not to have two mutant alleles are indicated with an asterisk (carrier status of minors thereby not disclosed).

Figure 2

Facial features of Family 1 including unaffected parents (A, B) and four affected children (C-F). 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. Facial features of the affected brothers from Family 2 (G, H) demonstrating widely-spaced eyes, downslanted palpebral fissures, midface retrusion, full cheeks, a long philtrum, and overfolded, posteriorly angulated ears. Features of individual II-1 from Family 4 at three ages. At three years of age (I), at 7 years of age (J), and 14 years of age (K). Notice the short, broad neck, widely-spaced eyes with downslanted palpebral fissures and bilateral ptosis, low set and posteriorly angulated ears, and pectus excavatum. Affected individual from Family 5 at 4 years of age (L), 8 years of age (M) and 6 years of age (N). Her features included downslanted palpebral fissures, bilateral epicanthus and ptosis, broad neck, low-set, small, cupped ears, and a wide thorax. Affected individual from Family 8 showing widely-spaced eyes, downslanted palpebral fissures, mild ptosis, low-set and posteriorly angulated ears, a broad, webbed neck, and curly hair (O-Q). Features of individual II-2 from Family 9. Note her widely set eyes, strabismus, bilateral epicanthus, downslanted palpebral fissures, depressed nasal bridge, short and upturned nose, short and broad neck, thickened vermilion of the lips (R,S).

Figure 3

A. Image of RTPCR 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: 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 RTPCR 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.

Figure 4

Cartoon of LZTR1 mutations showing the variants identified here in autosomal recessive Noonan syndrome above the protein and the previously reported in autosomal dominant Noonan syndrome below the protein. The ˆ symbol indicates that these two variants were in cis on this mutant allele and it is hypothesized that p.(Arg170Trp) is pathogenic; see text.