RMRP‐related short stature: A report of six additional Japanese individuals with cartilage hair hypoplasia and literature review

Biallelic pathogenic variants in RMRP, the gene encoding the RNA component of RNase mitochondrial RNA processing enzyme complex, have been reported in individuals with cartilage hair hypoplasia (CHH). CHH is prevalent in Finnish and Amish populations due to a founder pathogenic variant, n.71A > G. Based on the manifestations in the Finnish and Amish individuals, the hallmarks of CHH are prenatal‐onset growth failure, metaphyseal dysplasia, hair hypoplasia, immunodeficiency, and other extraskeletal manifestations. Herein, we report six Japanese individuals with CHH from four families. All probands presented with moderate short stature with mild metaphyseal dysplasia or brachydactyly. One of them had hair hypoplasia and the other immunodeficiency. By contrast, the affected siblings of two families showed only mild short stature. We also reviewed all previously reported 13 Japanese individuals. No n.71A > G allele was detected. The proportions of Japanese versus Finnish individuals were 0% versus 70% for birth length < −2.0 SD, 84% versus 100% for metaphyseal dysplasia and 26% versus 88% for hair hypoplasia. Milder manifestations in the Japanese individuals may be related to the difference of genotypes. The mildest form of CHH phenotypes is mild short stature without overt skeletal alteration or extraskeletal manifestation and can be termed “RMRP‐related short stature”.


| INTRODUCTION
The Ribonuclease mitochondrial RNA processing enzyme complex (RMRP) is involved in ribosome assembly via rRNA cleavage and cell cycle control via mRNA cleavage (Martin & Li, 2007).To date, biallelic loss-of-function variants in RMRP, the gene encoding the untranslated RNA component of RMRP, have been reported in individuals with cartilage-hair hypoplasia (CHH; OMIM: 250250).CHH is a multisystem disorder that is characterized by prenatal-onset growth failure with short limbs, generalized skeletal changes including metaphyseal dysplasia and striking brachydactyly with distinctive delta-shaped epiphyses, and extraskeletal features including hair hypoplasia, variable immunodeficiency, hypoplastic anemia, and predisposition to malignancies or Hirschsprung's disease (Mäkitie & Vakkilainen, 2020).
A high incidence of CHH has been reported in Finnish and Amish populations with a prevalence of 1 per 23,000 and 1-2 per 1000, respectively (Mäkitie & Vakkilainen, 2020).A founder RMRP variant, n.71A > G (NCBI reference sequence: NR_003051.3)(Ridanpää et al., 2001(Ridanpää et al., , 2002) ) has been detected in 89% and 100% of alleles in Finnish and Amish individuals with CHH, respectively (Mäkitie & Vakkilainen, 2020;Vakkilainen et al., 2019).Thiel et al. reported that the severities of skeletal and extraskeletal manifestations depend on the residual cleavage activity of rRNA and mRNA, respectively (Thiel et al., 2007).Moderate impairment of the rRNA cleavage activity and severe interference with mRNA cleavage activity in n.71A > G give rise to the typical skeletal and extraskeletal manifestations of CHH in Finnish and Amish populations.Although interfamilial and even intrafamilial phenotypic variation exists (Mäkitie & Kaitila, 1993), pre-and postnatal growth failure and the skeletal and extraskeletal manifestations are generally consistent among Finnish and Amish CHH.
Clinical manifestations of CHH may differ among other ethnicities.
Few individuals with CHH have been reported in East Asian countries and little is known about their phenotypic characteristics and variations.
To date, 13 individuals with genetically confirmed CHH have been reported in the Japanese populations (Harada et al., 2005;Hirose et al., 2006;Nakashima et al., 2003;Ono et al., 2015;Tsuchiya et al., 2006;Yasui et al., 2017).Here, we report six additional individuals with CHH from four Japanese families and review the genotypes and the clinical spectrum in Japanese individuals.
Physical examination revealed short limbs and genu varum.Hair was dense and black.Skeletal radiographs showed metaphyseal irregularities and sclerosis of the distal femora and brachydactyly with deltashaped epiphyses of the proximal phalanges (Figure 2a), leading to a diagnosis of CHH.Hematologic and routine biochemical tests did not yield abnormal findings.However, the peripheral lymphocyte responses to in vitro stimulation by phytohemagglutinin and concanavalin A were 1860 counts per minute (reference range in adults, 47,483) (Stone et al., 2009) and 2200 counts per minute (reference range in adults, 32,768-171,901) (Stone et al., 2009), respectively.Data related to this individual had been partially reported previously (Tomomasa et al., 2022).
Physical examination revealed short limbs and genu varum.Hair was fine and light brown.Skeletal radiographs showed mild metaphyseal irregularities and sclerosis of the distal radius and to a lesser extent of the distal femora as well as mild brachydactyly with delta-shaped epiphyses of the proximal phalanges (Figure 2b).The diagnosis of CHH was made based on radiological findings.Hematologic and routine biochemical tests showed no abnormalities.He did not undergo immunological tests.

Individual 4
Subsequently, the older brother of the Individual 3 (Figure 1e, II-1) was evaluated.Birth length was 50.0 cm (+0.5 SD), and weight 3250 g (+0.6 SD).He was healthy but with mild growth restriction (Figure 1g).He did not suffer from severe infections, recurrent infections, or autoimmune diseases.At age 7 years, the height was 111.2 cm (À2.1 SD; target range, À2.8 to +0.5 SD), and weight 20.3 kg (À1.0 SD).Hair was dense and black.Skeletal radiographs showed mild brachydactyly without delta-shaped epiphyses, but the long bones were normal (Figure 2d).Hematologic and routine biochemical tests showed no abnormalities.He did not undergo immunological tests.

| Family 4
Individual 5 The proband was a Japanese boy born to nonconsanguineous parents (Figure 1h, II-3).He was a fraternal twin.His birth length was 45.5 cm (À1.2 SD), and weight 2784 g (À0.3 SD).Short-limbed short stature became evident at age 1 year.He suffered from recurrent otitis media in infancy and early childhood and underwent tympanostomy tube placement at early school age.At age 8 years, he was diagnosed with growth hormone (GH) deficiency.Peak GH levels in response to insulin tolerance and arginine stimulation testing were 2.08 ng/mL (reference range, >6.0 ng/mL) and 3.47 ng/mL (reference range, >6.0 ng/ mL), respectively.GH replacement therapy was started at a weekly dose of 0.175 mg/kg.However, the intervention was not beneficial for his growth (Figure 1i).At age 14 years, height was 139.0 cm (À3.8 SD; target range, À0.2 to +2.8 SD), and weight 39.1 kg (À1.9 SD).Individual 6 The older sister of the Individual 5 was born at term without complication (Figure 1h, II-1).Her birth length was 49.0 cm (+0.5 SD), and weight 3126 g (+1.3 SD).She was healthy except for lack of normal pubertal growth spurt (Figure 1j).She had menarche at age 15 years.Skeletal radiographs were unremarkable except for mild metaphyseal flaring of the distal femora and tibiae (Figure 2f).Hematologic and routine biochemical and immunological tests were all normal.

| Molecular analysis
After genetic counseling, we obtained informed consent from the parents of all six affected individuals and informed assent from Individuals 5 and 6.The study was approved by the Ethics Committee of the Keio University School of Medicine (#2017-0130-20).We extracted genomic DNA from the peripheral lymphocytes of the patients and their family members, excluding the father of Family 2, and analyzed the RMRP gene using polymerase chain reaction-based direct sequencing.We identified biallelic RMRP variants in all six individuals, all of which had been previously reported in individuals with CHH (Table 1).Chromatograms are shown in Figure S1.

| Genotypes and phenotypes of CHH in the Japanese population
Thirteen Japanese individuals with genetically confirmed CHH have been previously reported (Harada et al., 2005;Hirose et al., 2006;Nakashima et al., 2003;Ono et al., 2015;Tsuchiya et al., 2006;Yasui et al., 2017).We reviewed RMRP genotypes and clinical phenotypes for these 13 individuals and the 6 described in this study (Table 1).
The mean birth length SD of individuals with at least one extraskeletal manifestation did not differ significantly from those without extraskeletal manifestations (À1.1 vs. +0.1,p = 0.14 determined by t-test).

| DISCUSSION
In this report, we sought to clarify the clinical characteristics and vari- n.-14_3dup are significantly higher in the Japanese population (0.001869 and 0.000571; jMorp) than in the Finnish population (0.000 and 0.000; gnomAD).Our review also indicates that most of Japanese individuals did not exhibit the classical CHH phenotypes reported in Finnish and Amish populations.A similar observation was reported in non-Finnish Caucasian individuals (Bonafé et al., 2005) (Table S1).Our study demonstrates that the genotypes of Japanese individuals with CHH differ from those in Finnish individuals and that Japanese individuals present with milder growth failure and less frequent hair hypoplasia and other extraskeletal manifestations.
Individuals 4 and 6 in our study did not usually come to medical attention, unless they had affected siblings.In fact, they did not have obvious skeletal changes, nor did they show extraskeletal manifestations, although individual six possibly showed metaphyseal dysplasia before her growth plate closure.Their manifestations further expand the phenotypic diversity of CHH, and the non-syndromic, mildest phenotypes can be termed "RMRP-related short stature."Genetic screening might reveal that the mildest form of CHH is responsible for a certain proportion of etiology-unknown short stature, particularly in the Japanese population.
Insertion or duplication in the 5 0 end of the transcript including n.-14_3dup are rare in Finnish or non-Finnish Caucasian population.
The significance of such genomic changes has not fully understood.
Our study suggests that this type of variants is associated with mild  The severity of the extraskeletal manifestations at younger ages has been shown to correspond with the severity of prenatal growth failure and short birth length, in the Finnish and Amish cohorts (Rider et al., 2009;Vakkilainen et al., 2020).By contrast, this relationship was not recapitulated by our study, in which birth length was not different between affected individuals with and without extraskeletal symptoms.This may be due to the small sample size in our cohort.
Further studies are needed to elucidate the relationship between prenatal growth failure and occurrence of extraskeletal manifestations at younger ages.
Our study has some limitations.First, ages of affected individuals were all young (median age, 6 years; range, 1-20 years).Hence, some of them might develop clinical immunodeficiency, bone marrow failure, and malignancy later in life.Indeed, this was previously reported (Klemetti et al., 2017;Vakkilainen et al., 2020).They found that many

ACKNOWLEDGMENTS
We thank all the affected individuals and their family members for participating in this study.

FUNDING INFORMATION
This work was supported by a grant from the Foundation for Growth Science (to Noboru Uchida) and in part by a grant from Novo Nordisk Pharma Ltd. and JCR Pharmaceuticals Co., Ltd.(to Tomonobu Hasegawa).These funding sources have no role in the design of study, interpretation of data, or in the decision to submit results.
T A B L E 2 Comparison of percentage of n.71A > G and each phenotype in Japanese and Finnish individuals with cartilage hair hypoplasia.

1
Pedigrees and percentile curves for height and weight of individuals with cartilage hair hypoplasia (CHH) in the present study.(a, c, e, h) Pedigrees of the studied families.The arrows indicate the proband.Affected individuals with CHH are indicated by solid symbols, and heterozygote carriers are indicated by dotted symbols.The height SD scores of each family members are indicated.(b, d, f, g, i, j) Percentile curves for height and weight of individuals with CHH.Heights/lengths and weights are shown as black points.
pregnancy showed short femoral length.Birth length was 50.0 cm (+0.3 SD), and weight 3224 g (+1.2 SD).At birth, short limbs were not clinically evident.Mild growth restriction became apparent during infancy (Figure1f).He did not suffer from severe infections, recurrent infections, or autoimmune diseases.At age 12 months, length was 66.4 cm (À3.3 SD; target range, À2.8 to +0.5 SD), and weight 7410 g (À2.3 SD).Hair was dense and dark brown.Skeletal radiographs showed mild brachydactyly with delta-shaped epiphyses of the proximal phalanges, but not metaphyseal changes of the long bones (Figure2c).He was suspected as having CHH based on radiological findings.Hematologic and routine biochemical tests showed no F I G U R E 2 Radiographs of affected individuals with cartilage hair hypoplasia (CHH) in the present study.(a) Radiographs of Individual 1. Metaphyseal irregularities with sclerosis of the distal femora and brachydactyly with delta-shaped phalanges are observed.(b) Radiographs of Individual 2. Mild metaphyseal irregularities with sclerosis of the distal epiphysis of the distal femora, proximal tibiae, and distal radii are seen.Mild brachydactyly with delta-shaped epiphyses of the phalanges is observed.(c) Radiographs of Individual 3. Brachydactyly with delta-shaped phalanges is observed.No metaphyseal change is seen in the knee.(d) Radiographs of Individual 4. Mild brachydactyly is observed, but no metaphyseal change of the knee is seen.(e) Radiographs of Individual 5. Metaphyseal irregularities with sclerosis of the distal epiphysis of the distal femora, proximal tibiae, and distal radii are seen.Brachydactyly with cone-shaped epiphyses of the phalanges is observed.(f) Radiographs of Individual 6. Mild metaphyseal flaring of the distal femora and tibiae is seen.No brachydactyly is observed.
Short limbs were apparent.Hair was dense and black.Skeletal radiographs showed brachydactyly with delta-shaped epiphyses of the proximal phalanges and metaphyseal sclerosis of the distal radius.Retrospective review for radiographs at age 6 years revealed sclerosis and mild cupping of the distal femoral metaphyses as well as sclerosis of the proximal tibial metaphyses.(Figure2e).He was diagnosed with CHH.Results of hematologic, routine biochemical, and immunological tests (immunoglobulin profile, CD4/CD8 T lymphocyte ratio, and the responses of peripheral lymphocytes to in vitro stimulation by phytohemagglutinin and concanavalin A) were all normal.

Finnish
individuals who did not have the extraskeletal manifestations at preschool age but developed clinical immunodeficiency or malignancy later in life.Accordingly, thorough immunological evaluation and long-term follow-up studies are needed to determine whether or not the mild phenotypes of CHH in the Japanese population would develop late-onset extraskeletal manifestations.Second, we do not have auxological parameters of sitting height or arm span to assess the severity of skeletal disporportion.A comparison of auxological parameters between Finnish and Japanese individuals with CHH is needed to validate our assertions.Third, diagnostic criteria of each manifestation for previously reported Japanese individuals with CHH may not be consistent.Despite these limitations, we believe that our study demonstrates the difference between Finnish and Japanese individuals with CHH.In conclusion, CHH in the Japanese population is associated with normal birth length and few extraskeletal manifestations, due in part to the different genotypes from those of Finnish and Amish population.Individuals with the mildest form of CHH can be classified as RMRP-related short stature and may account for a certain proportion of non-syndromic short stature in the Japanese population.AUTHOR CONTRIBUTIONSNoboru Uchida performed the molecular analysis, reviewed information of the Japanese individuals with CHH, and prepared the manuscript; Gen Nishimura raised a suspicion of CHH for all probands on radiological grounds and critically reviewed and revised the manuscript; Takeshi Sato, Gen Kuratsuji, Keisuke Nagasaki, Yuki Hosokawa, Eriko Adachi, Kei Takasawa and Kenichi Kashimada followed-up the affected individuals, provided clinical information, and revised the manuscript; Yuko Tsujioka retrospectively assessed radiological findings in all individuals of the present study, summarized them in the figure, and revised the manuscript; Tomohiro Ishii and Tomonobu Hasegawa supervised this study and critically reviewed and revised the manuscript.All authors approved the final version of the mauscript submitted for publication.
Cyclic progesterone therapy had been introduced for menstrual irregularities.She did not suffer from severe infections, recurrent infections, or T A B L E 1 Pathogenic variants of RMRP and clinical phenotypes in Japanese individuals with cartilage hair hypoplasia.