Identification of two CUL7 variants in two Chinese families with 3‐M syndrome by whole‐exome sequencing

Abstract Background 3‐M syndrome is a rare autosomal recessive disorder characterized by primordial growth retardation, large head circumference, characteristic facial features, and mild skeletal changes, which is associated with the exclusive variants in three genes, namely CUL7, OBSL1, and CCDC8. Only a few 3‐M syndrome patients have been reported in Chinese population. Methods Children with unexplained severe short stature, facial dysmorphism, and normal intelligence in two Chinese families and their relatives were enrolled. Trio‐whole‐exome sequencing (trio‐WES) and pathogenicity prediction analysis were conducted on the recruited patients. A conservative analysis of the mutant amino acid sequences and function prediction analysis of the wild‐type (WT) and mutant CUL7 protein were performed. Results We identified a homozygous missense variant (NM_014780.4: c.4898C > T, p.Thr1633Met) in CUL7 gene in a 6‐month‐old female infant from a non‐consanguineous family, and a homozygous frameshift variant (NM_014780.4: c.3722_3749 dup GGCTGGCACAGCTGCAGCAATGCCTGCA, p. Val1252Glyfs*23) in CUL7 gene in two affected siblings from a consanguinity family. These two variants may affect the properties and structure of CUL7 protein. Conclusion These two rare variants were observed in Chinese population for the first time and have not been reported in the literature. Our findings expand the variant spectrum of 3‐M syndrome in Chinese population and provide valuable insights into the early clinical manifestations and pathogenesis of 3‐M syndrome for pediatricians and endocrinologists.

mild skeletal changes, normal intelligence, and endocrine function.
However, these characteristics are not specific and changeless. 4,5 Characteristic facial features include dolichocephaly, protruding forehead, triangular face, flat nose, long philtrum, full lips, and pointed chin. In some male patients with 3-M syndrome, hypogonadism and/ or hypospadias have occasionally been reported. 3,[6][7][8][9] Apart from intrauterine growth retardation, 3-M patients usually suffered from feeding difficulties during infancy and postpartum growth retardation. Other clinical characteristics include short neck, square shoulders, slender long tubular bones, tall vertebral bodies, winged scapulae, short thorax, transverse chest groove, pectus carinatum or excavatum, scoliosis, joint hypermobility, clinodactyly, hyperlordosis, developmental dysplasia hip and spina bifida occulta, and prominent heels. These anomalies can be clearly detected by imaging tests. 2,10 In 1975, 3-M syndrome was first described by three principal geneticists (Miller, Mukusick, and Malvaux), and the name was subsequently derived from their initials. This disease is occasionally recognized in childhood, in which its prevalence and incidence at birth are low in general populations. 2,9 To date, fewer than 100 cases of

3-M syndrome have been reported in the literature. 3-M syndrome is
classified into types I, II, and III, which have been identified to be associated with the exclusive variants in cullin 7 (CUL7; MIM *609577), obscurin-like 1 (OBSL1; MIM *610991), and coiled-coil domain-containing protein 8 (CCDC8; MIM *614145) genes, respectively. However, not all the causative variants in these three genes can account for 3-M syndrome cases. Among them, CUL7 appears to be the most predominant pathogenic gene, and the proportion of 3-M syndrome cases with CUL7 variants is 77.5%, while OBSL1 accounts for a relatively small proportion of 16.3%. 3,11 Most variants in these three genes result in a truncated protein, suggesting that loss-of-function variants are responsible for this disease. 12 It is worth noting that some patients with similar phenotypes do not carry variants in the three genes, indicating that there may be other genes that are responsible for 3-M syndrome.
Given the rarity and heterogeneity of 3-M syndrome, the patients continue to pose diagnostic and therapeutic challenges. 13 In this study, by assessing two pediatric families with unexplained short stature children through whole-exome sequencing (WES) and bioinformatics analysis, we identified two autosomal recessive ho-

| Subjects
This study was approved by the ethics committee of Guizhou Provincial People's Hospital. Informed written consent was obtained from the parents of the patients.
Family A: Patient 1 is a 6-month-old female infant who visited the pediatric outpatient department at Guizhou Provincial People's Hospital due to developmental abnormalities. She was the second child in a twin pregnancy. B-scan ultrasonography indicated the presence of a twin gestation during the fifth month of pregnancy.
At the seventh month of pregnancy, a fetus was found dead in the uterus. After half a month, there was no fetal movement, and a cesarean section was performed immediately. Her birth weight was 2700 g. Her parents were of non-consanguineous marriage, with a smoke-free wine hobby and no history of exposure to radiation and chemical substances. There were no other affected members in her family. The detailed family pedigree is shown in Figure 1A.
Family B: Two siblings, a 12-year-old female child (patient 2) and her 9-year-old brother (patient 3), were both born after a normal gestation. They were born to consanguineous parents who are second cousins. The detailed family pedigree is shown in Figure 1B. Although their birth data were not available, their birth length and weight were in normal range based on their mother's review. Growth and developmental delay were noted since their childhood. Considering that the two affected siblings were of short stature, the genetic analysis of 10 exons in growth hormone receptor (GHR) gene was initially conducted by Sanger sequencing. However, the results showed that no variant was found in GHR gene (data not shown).

| Whole-exome sequencing
Peripheral venous blood samples were collected from all the af-

| Bioinformatics analysis
Bioinformatics analysis was conducted as follows. First, low-quality and adapter reads were removed from raw data. After quality controlling, sequencing reads were aligned to GRCh37/hg19

| Conservative and in silico analysis
We did a conservative analysis of the two mutant amino acid sequences using the Clustal Omega (https://www.ebi.ac.uk/Tools / msa/clust alo/). Further, the three-dimensional structures of the wild-type (WT) and mutant CUL7 protein were predicted by using Phyre2 software (http://www.sbg.bio.ic.ac.uk/phyre 2/html/page. cgi?id=index); then, Swiss-PdbViewer 4.04 was conducted to analyze the structural diversity and physical-chemical changes between WT and mutant CUL7 proteins.    Figure 4E). When Met replaced Thr, although the complex structure did not change ( Figure 4B), one H-bonds break and disappeared. Besides, the side chain of amino acid residues also changed ( Figure 4F). Obviously, the frameshift variant resulted in a truncated protein compared with the WT one ( Figure 4D). Protein locus with mutations is highlighted with black arrows; B, the structure prediction of wild-type CUL7 protein; C, the structure prediction of missense mutation CUL7 protein; D, the structure prediction of frameshift mutation CUL7 protein; E, the partial enlargement of missense mutation CUL7 protein; F, the partial enlargement of frameshift mutation CUL7 protein long bones as well as genetic analysis of disease-causing genes may be particularly helpful. Besides, GH has been found to be a useful treatment for 3-M syndrome in few studies, 14,15 while other findings demonstrated that such treatment exerts no beneficial effect. 5,16 3-M syndrome has been causally related to CUL7, OBSL1, and  24 The disappearance of an H-bond and changes in the side chain of amino acid residue are caused by the variant, which could lead to conformational instability of CUL7 protein and change its activities. In addition, patient 1 was treated with GH for 6 months, but failed to exhibit the desired effect, which is consistent with previous findings. 16 It is possible that the variants in CUL7 gene may strongly influence the effect of GH on longitudinal growth.

| D ISCUSS I ON
As a consequence of consanguineous marriage (second cousins), two affected children in family B with CUL7 homozygous frameshift variant that may be inherited from their great-great-grandmother or great-great-grandfather. It is absent in the gnomAD and ClinVar database, but in ExAC with frequency 0.000119. Therefore, such low-frequency variant meets the classification of likely pathogenic.
This variant occurred in the cullin domain and led to an obvious truncated protein. Cullins are a family of hydrophobic proteins that act as scaffolds for ubiquitin ligases (E3). Cullin-RING ubiquitin ligases (CRLs) play an essential role in targeting proteins for ubiquitin-mediated destruction. 25 Given the above, these two variants may affect the properties and structure of the protein. Therefore, these two variants most possibility to be the pathogenesis of short stature in affected children.
Together with clinical features and genetic analysis, these three affected children in the two families were ultimately diagnosed as suffering from 3-M syndrome.
However, one of the limitations of this study is that we have not done functional experiments for these two variants. Besides, although we followed up patient 1 with six months of GH therapy and found that it was ineffective, it is a pity that we have not followed

ACK N OWLED G M ENTS
The authors would like to express their gratitude to all participants for valuable cooperation, and EditSprings (https://www.edits prings. com/) for the expert linguistic services provided.

AUTH O R CO NTR I B UTI O N S
LH, XW, XF, and SH designed the study and collected the samples; LH, TJ, YH, and JL performed experimental work, analyzed the data, and drafted the manuscript. SY, MJ, XF, BA, and SH drafted the final version of the manuscript. All authors read and approved the final manuscript.