Expanding the phenotype of biallelic RNPC3 variants associated with growth hormone deficiency

Abstract Pathogenic variants in components of the minor spliceosome have been associated with several human diseases. Recently, it was reported that biallelic RNPC3 variants lead to severe isolated growth hormone deficiency and pituitary hypoplasia. The RNPC3 gene codes for the U11/U12‐65K protein, a component of the minor spliceosome. The minor spliceosome plays a role in the splicing of minor (U12‐type) introns, which are present in ~700–800 genes in humans and represent about 0.35% of all introns. Here, we report a second family with biallelic RNPC3 variants in three siblings with a growth hormone deficiency, central congenital hypothyroidism, congenital cataract, developmental delay/intellectual deficiency and delayed puberty. These cases further confirm the association between biallelic RNPC3 variants and severe postnatal growth retardation due to growth hormone deficiency. Furthermore, these cases show that the phenotype of this minor spliceosome‐related disease might be broader than previously described.


| INTRODUCTION
Pathogenic variants in components of the minor spliceosome have been associated with several human diseases (Farach et al., 2018;Verma, Akinyi, Norppa, & Frilander, 2018). One of these diseases is isolated growth hormone (GH) deficiency with pituitary hypoplasia, caused by biallelic RNPC3 variants (Argente et al., 2014). The RNPC3 gene codes for the U11/U12-65K protein, a component of the minor spliceosome. The minor spliceosome plays a role in the splicing of precursor mRNA, during which noncoding introns are recognized and removed. Most introns are removed by the major U2-dependent spliceosome, but a small subset of introns is removed by the minor U12-dependent spliceosome. These U12-type introns are present in 700-800 genes in humans and represent about 0.35% of all introns (Turunen, Niemela, Verma, & Frilander, 2013).
After the publication by Argente et al. (2014), no other cases of biallelic RNPC3 variants have been reported in the literature, apart from one conference abstract describing two siblings that had isolated GH deficiency and overlapping RNPC3 variants (Guceva et al., 2015). Here we report novel biallelic RNPC3 variants in three siblings with GH deficiency, central congenital hypothyroidism, congenital cataract, developmental delay/intellectual deficiency, and delayed puberty.

| CLINICAL REPORT
Three affected siblings (Figure 1: II-3, II-4, and II-5) were born to healthy, nonconsanguineous Caribbean parents. The mother has two healthy children from a previous relationship. The parents have one healthy older son. Their second child (II-2) died at the age of 3.5 months, presumably due to aspiration during feeding. This girl was born at term with normal birth weight and had bilateral congenital cataract, hypotonia, hyporeflexia, and absence of sucking reflex, for which she received tube feeding.
F I G U R E 1 (a) Pedigree showing that the three affected siblings (II-3, II-4, and II-5) are compound heterozygous for the RNPC3 variants and that both parents are carrier. (b) Patient II-3, II-4, and II-5 at the age of 25, 21, and 17, respectively. Note the short stature, central adiposity, and facial features that are typical of growth hormone deficiency. (c) Amino acid positions of both variants showing complete conservation across vertebrates [Color figure can be viewed at wileyonlinelibrary.com] The three siblings were born at term after an uncomplicated pregnancy and delivery with normal birth weights. They had congenital cataract, for which they were operated. They all had severe postnatal growth retardation with the height ranging from −6.7 SD to −7.4 SD (Table 1). GH stimulation tests in patient II-3 and II-5 showed almost undetectable GH levels (patient II-4 not tested). Additionally, all three patients had almost undetectable levels of IGF-1, IGF-BP3, and prolactin. An X-ray of the hand was performed in patient II-3 and II-4, showing severely delayed bone age (bone age of 6 months at the age of 5 years and 8 months and bone age of 3 months at the age of 2 years and 2 months, respectively). They were diagnosed with central hypothyroidism and received replacement therapy with levothyroxine (patient II-3 at the age of 7 months and patient II-4 and II-5 at the age of 3 months).
In patient II-3 GH replacement therapy was started at the age of 1 year. After a short episode of treatment (with a small initial response), almost no effect on growth was noted. The patient did not attend regular follow-up visits and parents refrained from further use of GH therapy. Because of this noncompliance and since GH therapy is an intensive therapy that requires daily injections and regular monitoring of (adverse) effects, it was decided not to start GH replacement therapy in patient II-4 and II-5.
All patients had a developmental delay/intellectual deficiency. At the age of 4, patient II-3 was able to sit but not stand without support.
She had a speech delay, speaking only 2-word sentences at the age of 5. Neurocognitive examination at this age showed that her development was delayed by 3 years. Patient II-4 had a motor developmental delay, as he could walk only with support upon examination at the age of 2. During the examination, he was making sounds, although parents indicated that he was able to speak two-word sentences. At the age of 11, his development was delayed by at least 6 years.
Neurocognitive examination in patient II-5 at the age of 8 showed that his development was delayed by at least 4 years, with an IQ of <42.
Puberty was delayed and laboratory analysis in patient II-3 and II-4 was indicative of hypogonadotropic hypogonadism, patient II-5 was not tested (Table 1).
The patients were evaluated by the visiting clinical geneticist at the ages of 18, 14, and 10-years-old, respectively. Height, weight, and head circumference were all well below the third percentile. Apart from short stature, they were noted to have a depressed nasal bridge, short philtrum, and central adiposity (Figure 1).
At the moment, patient II-3 is 25 years old and she goes to a daycare center on weekdays. Although her IQ has not been formally tested, she appears to have a more severe intellectual deficiency compared to her two younger brothers (patient II-4 and II-5). They are now 22 and 18 years old and attend special education.

| Genetic testing
Trio whole-exome sequencing was performed in patient II-3 in a diagnostic setting as described previously (Houweling et al., 2019) Parents were both carriers of one of the variants in the GIF gene, however, one of the two affected siblings carried only one GIF variant.
Thus, these variants were also discarded. The RPNC3 variants were of particular interest, as biallelic variants in this gene were previously associated with growth hormone deficiency (Argente et al., 2014).
Segregation analysis demonstrated that all three affected siblings had the compound heterozygous RNPC3 variants and that parents were both carriers of one variant (Figure 1). This matches the autosomal recessive mode of inheritance that was expected based upon the pedigree (Figure 1).

| DISCUSSION
We here describe three siblings with a combination of growth hormone deficiency, central congenital hypothyroidism, congenital cataract, developmental delay/intellectual deficiency, and delayed puberty with biallelic RNPC3 variants. These cases further confirm the association between biallelic RNPC3 variants and severe postnatal growth retardation due to GH deficiency, as previously described (Argente et al., 2014;Guceva et al., 2015). However, our patients show a more extensive phenotype (Table 1). were born there was no newborn screening for congenital hypothyroidism at the Caribbean island, which resulted in a delay in diagnosis and treatment. It is known that thyroid hormone is essential for normal brain development and that untreated congenital hypothyroidism leads to neurocognitive defects (Grosse & Van Vliet, 2011;Kooistra et al., 1994). In addition, patient II-3 was diagnosed with hypothyroidism only at the age of 7 months and has a more severe intellectual deficiency compared to her younger brothers (patient II-4 and II-5), who were diagnosed with hypothyroidism at the age of 3 months.  (Farach et al., 2018). There is some evidence for genotype-phenotype associations in these RNU4ATAC -associated disorders, which could partially explain the clinical differences (Shelihan et al., 2018). Similarly, there might be a genotype-phenotype association in patients with biallelic variants in RNPC3, as the variants reported in our family differ from those reported before. However, not enough patients have yet been reported to evaluate if such an association truly exists. Additionally, modifier genes could (partly) explain the phenotypic variation between patients with RNPC3 variants.
Of further interest, there have been reports of patients with MOPD1 that had bilateral cataract, which was a feature in our patients as well (Kilic et al., 2015;Kroigard et al., 2016).
In conclusion, we show that the phenotype associated with biallelic RNPC3 variants is broader than previously described. The exact mechanisms through which pathogenic RNPC3 variants cause different phenotypes still remain to be elucidated.

ACKNOWLEDGMENTS
We would like to thank the patients and their parents for their kind cooperation. Furthermore, we would like to thank Rob Zwart for his help with the genetic analysis.

DISCLOSURE AND CONSENT
The authors declare that they have no conflict of interest. Written informed consent for publication was obtained from the mother of the three patients.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed in this study.