Origins of SOPH syndrome: A study of 93 Yakut patients with review of C-terminal phenotype

Since the first report of SOPH syndrome among the Yakut population in 2010, new clinical data of SOPH-like conditions continue to appear. We expand the phenotypic spectrum of SOPH syndrome and perform a comparative analysis of Yakut SOPH patients' clinical data with SOPH-like conditions reported in the world scientific literature to form a foundation for NBAS pathogenesis discussion. Clinical data from the genetic records of 93 patients with SOPH syndrome and global survey data on patients with pathogenic variants of the C-terminal in the NBAS gene were collected. A detailed phenotype description of patients is presented with a total number of 111 individuals. Underweight below the fifth centile and prone to delayed bone age in Yakut SOPH patients are retrospectively observed. We outline the short stature with optic atrophy as the leading phenotyping trait for C-terminal NBAS patients. The pathophysiology and patients management of SOPH-like conditions are discussed.

Although NBAS is known to be involved in membrane traffic from the Golgi apparatus to the endoplasmic reticulum (ER) and nonsensemediated RNA decay, the mechanism of protein realization is not enough understood to explain the various phenotypes associated with pathogenic variants, in particular ILFS2 (Infantile Liver Failure Syndrome type 2; OMIM #616483) [2][3][4] . The average age of ILFS2 manifestation is about 12 months. The syndrome starts with acute liver failure associated with an increase in body temperature. Attacks are accompanied by vomiting and increasing lethargy. Liver failure is characterized by elevated ALT and AST, liver coagulopathy and/or hepatic encephalopathy. Unlike SOPH, ILFS2 is a life threatening condition, but if the patient survives after the attack, liver function is fully restored. Interestingly, although SOPH and ILFS2 variants are in the same gene, short stature in patients with ILFS2 occurs in 70%, Pelger's anomaly in 23%, optic atrophy in less than 15% of cases, while Yakut SOPH patients have no history of liver failure whatsoever.
In an international multicenter study of NBAS-associated diseases, three main subgroups of phenotypes were identified: conditions associated with β-propeller, SEC39 domain, and C-terminal. 5 SOPHsyndrome in this study was classified to a subgroup with disorders at the C-terminal. The ambiguity and diversity of traits in these subgroups, as well as the absence of a specific domain at the C-terminal, leaves us question the pathophysiology of SOPHsyndrome. In this study, clinical data of patients with SOPH-syndrome in the Republic of Sakha (Yakutia) were analyzed to expand the phenotypic spectrum of SOPH, and a comparison was made with world scientific literature data with other SOPH-like and NBAS-associated conditions. The molecular biological nature of NBAS is discussed.

| Clinical description
To describe the phenotype, we report the proportion of individuals with given clinical symptoms (p) as well as the number of individuals in the group (n) who were also tested for this trait (p/n). To describe a particular organ system or appearance, we report the frequency of a given feature as follows: most common feature, occurring half the time and rarely. We also provide characteristics of some clinical manifestations, such as immunological studies and MRI descriptions of brain structures. Next, we compare the literature data with our patients.

| Statistics
Statistical calculations were carried out in the Python programming language (Python3). Figures were made in the statistical analysis and visualization tool R (https://www.r-project.org) using dplyr and ggplot2 packages. Smoothing when constructing centile distributions and data tables was performed using the LOWESS procedure with the parameters SmoothF = 0.3. Centile charts were taken from Yakut populational data ("Dynamics of anthropometric characteristics and blood pressure in children of the Republic of Sakha (Yakutia)," 2017).

| Molecular genetic findings
Depending on the period, from 1995 to 2022, all patients were found to be homozygous of c. 5741G>A (Arg1914His) by PCR-RFLP, realtime PCR, or targeted panel sequencing. Exceptions were one male who was found to be heterozygous for this variant and one female with the compound heterozygous variant c.2535G>T (p.W845C), c. 5741G>A (p. Arg1914His) reported earlier. 6 It was a case of osteogenesis imperfecta in a girl with SOPH-syndrome, but because of the second mutant allele localization in the SEC39 domain, she was excluded from the C-terminal comparison group.

| Demography
A total of 93 patients from 86 families (37 male and 56 female) with a confirmed case of SOPH were examined during this period. All patients were from the Yakut population, except for one from the Even population. The maximum age among the examined patients was 53 years ( Figure 2B).
It is also worth noting that one SOPH male patient died during an epileptic seizure. To date, this is the only known death of a SOPH patient under the dispensary observation in the Republic of Sakha.
At least three females from this study gave birth to one healthy heterozygous child. Data on these events are not presented. Two patients were additionally found to be heterozygous of the 3M syndrome, and Friedreich's ataxia was confirmed by genetic testing in another patient. Their phenotypes will not be described in this study.
The vast majority of SOPH patients were born at term (72/75), by physiological delivery (34/43), with normal weight (71/77) and length (69/72), and an Apgar score of at least 7 (40/43). In addition, these patients achieved their developmental milestones well, except for the age of using the first words, the median value of which crossed the threshold of the normal values by 1 month. The chief complaints of patients with SOPH are short stature compared with their peers (90%), loss of vision (72%), as well as frequent infectious diseases (71%). Complaints about the gastrointestinal tract (27%) and neurological signs (37%) also take a significant part.
The general phenotype is represented by a dysplastic body type, underweight, short stature with micromelia of the limbs ( Figure 1A,B).
In about half of the cases, anomalies of the skull (hydrocephaly, brachycephaly, microcephaly), short neck, high forehead, wide chest, brachydactyly, muscle hypotension, and senile skin are observed. Less common occurrences are dental anomalies, high-pitched voice, ear dysplasia, almond-shaped eyes, and clinodactyly.
A literature search revealed 63 articles with a genetic and clinical description of NBAS patients (n = 140). Since NBAS genotypephenotype relationship is known, patients with compound heterozygous variants on the β-propeller and SEC39 domain were excluded.
Eighteen patients were found with pathogenic variants at the C-terminal of NBAS. Out of these, two patients are presented in a correspondence on hemophagocytic lymphohistiocytosis (HLH) without detailed phenotypic data. 7 One patient is presented in a variant search study in ALF of unknown etiology, also without a detailed description. 8 The remaining 15 patients were eligible for comparative analysis. The phenotype of these patients is mainly represented by short stature; in about half of the cases, progeroid facial features with dysmorphisms, and anomalies of the skull and fingers are described; ear dysplasia, high-pitched voice, and underweight are less common.

| Cardiovascular system
Of all patients, 49 underwent a cardiovascular examination by a cardiologist using ECG and echocardiography. Almost half of the cases were found to have minor heart anomalies without clinically significant manifestations (20/49), but congenital heart disease was also found in one patient. Conduction disturbances were manifested in most cases by the bundle branch block (17/49), and rhythm disturbances were less common and manifested mainly by episodes of minor arrhythmias (15/49).
Only one case of a ventricular septal defect without clinical manifestations has been described in the literature. It should be noted that the assessment of the prevalence and frequency of ENT diseases is quite difficult in SOPH since the chief complaints of patients are already associated with frequent infectious diseases. The data presented here can be interpreted as ENT diseases that continue to recur after early childhood. The median age of the first visit to an ENT specialist was 10 years and the last visit was Infectious diseases, such as bronchitis and pneumonia, occurred in half of the cases described in the literature. In addition, one case of hearing loss of mixed etiology was identified.

| Immunological findings
A total of 29 patients were examined by immunologists. Immunodeficiency was diagnosed in eight cases. In one of those patients in an in-depth study, an inversion of CD4+/CD8+ was observed, a decrease in the absolute content of B and NK cells, an increase in the content of CD3+CD4-CD8-up to 8.8% and CD+CD16,56+ cells, as well as changes in the subpopulation of B lymphocytes, the expression F I G U R E 1 Clinical characterization of the Yakut SOPH patients: anthropometric findings with age distribution. (A) The stature-for-age chart shows that the development of short stature manifests approximately at the age of 3 years. The upper dotted lines are the 95th centile, the middle is the 50th, and the lower are the 5th centile, red lines correspond to females and blue lines-to males. (B) The BMI-for-age chart shows the development of underweight approximately at the age of 5 years.
of the BAFF receptor was sharply reduced compared with the donor.
Previously, we reported the results of an evaluation of immunological status in SOPH patients. The assessment of the immune status revealed a significant reduction in the level of serum immunoglobulins and CD16+CD56+ NK and CD19+ B cells compared with controls. 9 In more than half of the cases described in the literature, immunodeficiency was presented by a decrease in the level of IgG and hypogammaglobulinemia. In three of these cases, a decrease in B cell levels was also identified. As mentioned earlier, correspondence reported two patients suffering from HLH with an NBAS variant at C-terminal. 7

| Ophthalmological findings
All patients had partial optic atrophy at the first examination; however, there were no additional follow-ups in some of them (n = 5). Therefore, these patients were excluded from the selection based on ophthalmological features.
All described patients from the literature (n = 15) were found to have optic atrophy. In half of the cases, exophthalmos was identified, less often myopia, nystagmus, strabismus, and in one case-glaucoma.
Two patients had a color perception defect. was confirmed only in three patients. Six patients had a history of seizure episodes but without EEG confirmation.

| Skeletal system
In 33 patients, a study of the brain structure was performed.
Three of them were found to have atrophy of the cerebellar structures, and four had an empty sella turcica syndrome. Three patients showed an expansion of the periventricular zone, perivascular spaces of the posterior horns of the lateral ventricles, and subarachnoid spaces slightly expanded in the region of the poles of the frontal lobes and the region of the lateral fissures of the brain.
In half of the literature cases intellectual impairment and muscular hypotonia were identified, and episodes of seizures were less often described. Brain abnormalities in the form of a J-shaped sella turcica were found in two patients on MRI, thinning of the corpus callosum was described in another, and in the other, hypoplasia of the cerebellar vermis was also described together with thinning of the corpus callosum.

| Internal organs
Ultrasound examination of internal organs was also performed (n = 60). Gallbladder anomalies (26/60) were the most common symptom, 12 of which were clinically significant. Increased vascular patterns and changes in the parenchyma of the liver (10/60) and pancreas (12/60) were not accompanied by significant clinical manifestations, except for one confirmed case of type 2 diabetes mellitus and two cases of hepatitis B. Two episodes of a transient increase in glucose levels were described but without further examination.
Ultrasound examination also showed that consolidation of the sinuses of the kidneys occurred in 16 cases (16/60), and two cases of pyelectasis with hydrocalycosis were also described, but the sediment in the bladder was not always observed (5/60).
A literature review revealed only a few cases of hepatomegaly (n = 3) and splenomegaly (n = 2). Liver biopsy in one patient showed steatosis, in three other cases, where a biopsy was also performed, changes were not found.
Despite the absence of episodes of hepatic failure and/or other significant hepatic events in our patients, almost all described literature patients (n = 13) showed episodes of increased ALT and AST, five of them with episodes of ALF. This forced us to perform a deeper study of the laboratory data, the difficulty of which was the different standards for quantifying ALT and AST at different time periods.
Approximately, only a few SOPH patients (10/83) had an increase in ALT and AST, but without known episodes of ALF. Literature findings revealed three cases of insulin-dependent atypical diabetes mellitus. According to the literature data, more than half of the patients had a Pelger anomaly of leukocytes. Cases of anemia, thrombocytopenia and leukopenia have also been described.  We did not find any specific traits regarding the cardiovascular, endocrinological, and the hematopoietic systems. It can be said that SOPH patients do not suffer from pathologies in these systems. If such conditions occur, we cannot yet associate them with the current SOPH variant. Minor disorders in the cardiovascular system, as well as episodes of anemia, may accompany the SOPH phenotype but are not an obligatory part of it, which is consistent with the literature data. As for endocrinological signs, due to the relatively high frequency of F I G U R E 3 Yakut SOPH (R1914H) phenotype. (A) Photographs of SOPH patients. Facial features comprise the round shape of the head, short neck, round face, and long medial cleft. In some cases, we observe epicanthus, crease on the neck, rounded eyes, hypoplasia of the zygomatic bones, small nose, thin lips, senile skin, and thin hair. On the limbs: micromelia of the hands, wide feet, and a sandal-shaped gap. (B) Master table of Yakut SOPH (R1914H) clinical findings. thyroid gland disorders in Yakutia and no observed data in the literature, it is difficult for us to associate the thyroid gland abnormalities to all C-terminal patients.

| Ultimate SOPH phenotype
The neurological picture of SOPH patients includes complaints similar to residual encephalopathy. Nevertheless, we cannot say with the same certainty about the intellectual impairment, since we did not observe such a tendency in the Yakut SOPH patients. As we observe no consistent MRI findings and other neurological impairments, such as intellectual disability and seizures, we suggest moderate severity of SOPH regarding the overall C-terminal phenotype.
Internal organ abnormalities identified using ultrasound diagnostics were without significant clinical manifestations. This fact is at odds with the literature data on episodes of elevated ALT/AST and ALF in patients with variants at the C-terminal. However, we emphasize that although episodes of elevated liver enzymes occur in the SOPH Arg1914His variant, this is not the leading clinical sign. The liver phenotype in patients with SOPH is not striking, as with compound variants in the β-propeller and SEC39 domain ( Figure 4A,B).
Thus, we suggest that the carriage of the SOPH variant leads to a mild phenotype without ALF in the case of the homozygous state.
Although immunodeficiency is not diagnosed so constantly as to sound the alarm, the number of patients with frequent infectious diseases prompts us to suggest that this group of patients may be underdiagnosed. This is in line with the data of our own studies and that of other research groups. Frequent otitis and sinusitis are among the leading symptoms in the SOPH phenotype, requiring pharmacological intervention. This is one of the few symptoms that are successfully treated with a positive effect.
We outline short stature with optic atrophy as the leading phenotyping trait for C-terminal variants, especially for SOPH ( Figure 4C). These are the only symptoms found in all patients.
Although one described individual with no growth retardation, all other cases in the literature are in line with this statement. Growth retardation manifests at the age of 3 years and is the reason for applying to a medical consultation. The atrophy of the optic nerve is usually found later on further examination. There is currently no adequate treatment for these conditions.

| NBAS pathophysiology
Although the major SOPH phenotype develops through ages after birth, studies on mouse embryos and zebrafish have revealed an active role of NBAS in the developing skeletal system and brain structure progenitor cells. 17 It can be consistent with the described short stature, delayed bone age, and the presence of, for example, a highpitched voice as well as neurological manifestations in SOPH patients.
Promising studies were performed on immune cells regarding NK cells' cytotoxic degranulation. With IL-2 exposure, researchers were able to enhance the cytotoxic abilities of mutant NBAS depleted cells.
The infiltration of organs with defective immune cells is elucidated in recent studies on NBAS variants leading to HLH in some cases. 7,18 F I G U R E 4 NBAS protein phenotype. (A) 3D structure of NBAS protein and its C-terminal predicted by AlphaFold was recreated using the PYMOL molecular visualization system. Regions: orange-β-propeller, yellow-SEC39, green-C-terminal. (B) Known C-terminal variants. The red line marks "vulnerable" regions, also underlined in the C-terminal 3D model. (C) Radar plot for SOPH and C-terminal key phenotyping traits (%). Clockwise: skeletal system, general appearance, ophthalmological and immunological findings, and hepatic phenotype with neurological signs are presented.
Hence, this study approach could benefit not only to NBAS patients with primary immunodeficiency but also reduce frequent infections of SOPH individuals, especially in the younger age.
NBAS is considered to be involved in retrograde membrane traffic between the ER and the Golgi, and this role of the protein is well covered in the literature. But the exact mechanism of action is still not well understood. Recent experimental data suggest a role of NBAS in cell bulky cargo transporting machinery, such as autophagosomal secretion of collagen . [19][20][21][22][23][24][25][26][27] Collagen analysis in fibroblasts from NBAS patients revealed an accumulation of mature mCOL Iα2 and its precursor pro-COL Iα1 inside the cell; it was then considered that this was due to another variant in the P4HB gene. To test collagen secretion, Saos-2 cells were transfected in another study. A significant decrease in collagen secretion by cells with the mutant construct was identified compared with controls. It was concluded that NBAS plays a key role in the formation of large vesicles through the secretory pathway by interacting with TANGO1. 28,29 But since fibroblasts do not contain large enough structures to incorporate collagen (COPII is too small), another study showed that TGF-β1 (transforming growth factor β1) upregulated the secretion of pro-COL Iα1 in the culture supernatants and increased the colocalization of COL I with Rab8a, a marker of secretory autophagy vesicles. 30 Rab-8 known to regulate unconventional protein secretion pathway (UPS) of integral proteins bypassing the Golgi via SMGL-1 (NBAS homolog) as a guanine exchange factor in the nematode's intestine. 31 Autophagosomal secretion reduces ER stress with the release of COL I via autophagy-dependent secretion machinery. NBAS might be responsible for UPS that handle bulky cargo secretions under some steady-state processes and/or cell stress conditions. Impaired collagen metabolism may explain not only short stature but also skin abnormalities in the form of progeroid facial features. UPS pathways are conserved across species and triggered by stress events, keeping that in mind, we can guess, for example, that not just ILFS2 with its fever association, but that optic atrophy might be caused by light waves as a constant stress factor for the retina, that cells cannot cope with interrupted UPS machinery. One way or another, the role of NBAS as a protein regulating both conventional and UPS pathways remains to be discovered.

| Patients management
Currently, there is no standardized therapy for SOPH disease. We performed a wide clinical analysis of SOPH patients and their comparisons with other SOPH-like conditions. We used only genetic records of those patients who follow up in the Medical Genetic Center (MGC) of Yakutsk. Patients are administrated to MGC mostly at a young age. Therefore, in some cases, we do not have data on patients in adulthood. Information about them reaches the MGC only in case of death or other special cases. Nevertheless, the patients who do come as adults provide us with valuable information. That is how, for example, we were able to confirm one case of type 2 diabetes described in the results. Regarding the two cases with elevated glucose, we are almost certain that they also developed diabetes mellitus.
Another example is a boy, also with postnatal growth retardation in childhood, who visited MGC at the age of 29 years with a height of 162 cm. This height corresponds to the 10th centile distribution in Yakut males.
Overall, the clinical review suggests that for a group of SOPH patients, there is a window of approximately 3 years after birth in which special care can be administered to manage major symptoms as well as possible pathogenetic treatment, which remains to be discovered.

CONFLICT OF INTEREST STATEMENT
The authors declare no competing interests.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1111/cge.14319.