Atypical phenotypes and novel OCRL variations in Southern Chinese patients with Lowe syndrome

Background: Lowe syndrome is an uncommon genetic disorder that follows an X-linked recessive inheritance pattern. It is de�ned by the occurrence of congenital cataracts, psychomotor retardation, and dysfunctional proximal renal tubules. This study examined the clinical and genetic features of eight children diagnosed with Lowe syndrome in Southern China. Methods: Whole-exome sequencing was performed on eight Lowe syndrome patients from three medical institutes in Southern China, and clinical and genetic data were collected and analyzed retrospectively. Results: In our cohort, the clinical symptoms of the eight Lowe syndrome individuals varied. One patient was diagnosed with Lowe syndrome but did not have congenital cataracts. All patients had psychomotor retardation, short stature, low molecular weight proteinuria, and albuminuria. The clinical characteristics also included elevated creatine kinase (CK)/ aspartate aminotransferase (AST)/ lactate dehydrogenase (LDH) (87.5%), cryptorchidism (66.7%), renal rickets (37.5%), renal tubular acidosis (37.5%), phosphaturia (37.5%), hypercalciuria (37.5%), nephrocalcinosis (25%) and glycosuria (25%). Eight variations in OCRLwere identi�ed in all eight patients with Lowe syndrome, involving three known and �ve novel variations. All variations are located in exons 8-23 and occur in functional domains. Three novel nonsense variations were classi�ed as pathogenic. Two patients with novel missense variations classi�ed as uncertain signi�cance showed typical severe phenotypes. Conclusion: This study describes the �rst case of an atypical Lowe syndrome patient without congenital cataracts in China and identi�es novel OCRL gene variants, which broadens the genetic and symptomatic range for Lowe syndrome.


Introduction
The oculocerebrorenal syndrome of Lowe (Lowe syndrome, OMIM #309000) is an X-linked recessive disorder that impacts multiple systems and is estimated to affect approximately 1 in 500,000 individuals [1] .The characteristic features of Lowe syndrome include congenital cataracts, developmental delays, and dysfunction of the proximal renal tubules.Various clinical and genetic research have been reported by multiple organizations since the rst description of this disease by Lowe et al. [2] .The clinical phenotype may be variable, but almost all affected individuals exhibit bilateral cataracts that are present at birth, necessitating early surgical intervention to preserve visual function.The neurological manifestations of this syndrome encompass neonatal hypotonia, stereotypic behavior, cognitive disability, and seizure disorder [3] .Magnetic resonance imaging (MRI) of the brain commonly reveals white matter abnormalities in these conditions, frequently in the form of periventricular cystic lesions [4,5] .The renal disorder is marked by a range of proximal tubular dysfunctions, which encompass low molecular weight proteinuria (LMWP), renal tubular acidosis, hypercalciuria, and aminoaciduria.These manifestations progress slowly and may eventually lead to renal failure [6] .Impaired renal function causing renal rickets can result in stunted growth, skeletal deformities, and delayed achievement of motor milestones.Lowe syndrome is primarily treated with symptomatic management, and progressive kidney failure often limits life expectancy.
The OCRL gene is located in the Xq25-26.1 region and consists of 24 exons.It encodes the enzyme known as inositol polyphosphate 5-phosphatase OCRL1 [7] .
OCRL1 is found in various compartments within the endocytic network, including the Golgi apparatus, clathrin-coated pits, and early endosomes [8,9] .Moreover, it plays a crucial part in vital cellular processes like intracellular signaling, lysosomal tra cking, and mTOR activity [10][11][12] .Around 360 variations of the OCRL gene have been identi ed, including frameshift, substitutions, gross inversions, nonsense, and missense variations, resulting in various Lowe syndrome phenotypes with varying degrees of severity [13][14][15][16] .It has been discovered that the majority of patients with missense variants in OCRL have normal mRNA expression levels in broblasts, and these variants are located in the phosphatase domain of the gene.Nonetheless, in individuals with Lowe syndrome who have nonsense, frameshift, or splicing variations, the expression of the OCRL transcript or protein is drastically reduced [14] .These results imply that decreased OCRL expression may contribute to the development of Lowe syndrome.systematic evaluation were reviewed.Samples of blood, urine, or DNA were collected from both the affected patients and their respective families.The diagnosis of Lowe syndrome was established using a combination of clinical criteria and genetic analysis.The classical diagnostic criteria for Lowe syndrome included the presence of three key features: 1) bilateral congenital cataracts, 2) dysfunction in the renal proximal tubules, and 3) psychomotor retardation.The research was authorized by local ethics committees.Written informed permission was provided by all participating parents.

Evaluation of phenotypic characteristics
All patients were evaluated by experienced clinicians.Data from the Chinese Children and Adolescents Growth Standard were utilized to acquire body mass index (BMI) and standard deviation scores (SDS) for height and weight [21][22][23] .All patients underwent renal, scrotum, and groin area ultrasound examinations by sinologists.Skeletal X-ray was performed on three patients, and MRI was performed on six patients by radiologists.An ophthalmological examination, which included a slit-lamp test, was performed by an experienced ophthalmologist.The biochemical characteristics of blood samples were discovered in the clinical laboratory of the hospital.Ion exchange chromatography was utilized for amino acid urine testing.LMWP was de ned as the presence of greater than 40% of 1-microglobulin, 2-microglobulin, or retinol-binding protein in the urine.Low serum bicarbonate levels and the need for alkali treatment were diagnostic of renal tubular acidosis.Using age-appropriate reference values [24] , greater than 0.1mg/kg(4.0mg/kg.24h)or a calcium/creatinine ratio (mmol/mmol) above the upper average level were considered to indicate hypercalciuria.Phosphate wasting was characterized by a reduction in the tubular phosphate reabsorption rate (< 80%) or the normalization of the tubular maximum for phosphate reabsorption [TmP/GFR] to the lower limit of the appropriate age-based normal range [25] or the requirement for phosphate supplementation or hypophosphatemia based on local laboratory values.

Molecular analysis
Whole-exome sequencing was performed on either the proband-parents trio or the proband-only strategy, with subsequent con rmation of all identi ed variants using Sanger sequencing.The experimental work ow strictly adhered to the manufacturer's protocol.Genomic DNA extracted from blood lymphocytes was used for exome capture, employing the SureSelect Human All Exon V6 kit from Agilent (Santa Clara, United States).Paired-end highthroughput sequencing was conducted on the Illumina Hiseq 2500 platform (Illumina, San Diego, United States), achieving an average depth of over 100 and a coverage exceeding 99% on each sample.Variant frequency annotation was performed using resources such as the 1000 Genomes Project, Exome Aggregation Consortium, HapMap, and dbSNP.Known deleterious variants were searched for in HGMD (http://www.hgmd.cf.ac.uk/) and ClinVar (http://www.ncbi.nlm.nih.gov/clinvar), while the pathogenicity of any newly discovered variants was assessed using SIFT, PolyPhen-2, PROVEN, and MutationTaster.Using the ACMG guidelines for clinical sequence interpretation, a group of molecular geneticists, bioinformaticians, and clinical molecular geneticists who are experts in hereditary diseases interpreted the variants.

Statistical analysis
SPSS Statistics 17.0 program (SPSS, Chicago, United States) was used to determine averages and dispersion parameters.DOG 2.0.1 [26] (University of Science & Technology of China) was used to draw domain structure.
All patients experienced motor and mental retardation.Two patients (2/8, 25%) had clinical and electrical seizure abnormalities that required antiepileptic therapy.Six patients had brain magnetic resonance imaging.In 5 out of 6 patients (83.3%), hyper-intense white matter abnormalities were identi ed through T2-weighted imaging.These lesions were observed in both the bilateral periventricular white matter and subcortical white matter regions (Fig. 3).

Variation analysis
DNA sequence of the OCRL gene from our study revealed eight different variations; 5 have not been reported.As outlined in

Clinical ndings
In our cohort, the clinical phenotype of most of our patients is consistent with the typical case, for example, psychomotor retardation, low molecular weight proteinuria, albuminuria, and short stature.The proportion (4/6,66.7%) of cryptorchidism documented in our study is higher than 42%, previously reported by a single Korean center [27] .We were pleasantly surprised to nd that seven patients' serum levels of muscle enzymes (CK/LDH/AST)s were elevated(7/8,87.5%).
The involvement of muscles in Lowe syndrome was indicated by the initial observation of elevated blood CK levels, often accompanied by signi cant concentrations of MB isoenzymes.Additionally, increased serum AST and LDH concentrations were noted, while liver function remained within normal ranges [28] .A biochemical study revealed mitochondrial dysfunction is one of the causes through muscle biopsy sample examinations [29] .Eujin Park et al.
conducted muscle biopsy specimens on individuals with the disease to identify primary myopathy or neurogenic atrophy [30] .Their ndings con rmed that CK/LDH/AST could serve as a signi cant biomarker in patients with OCRL variations and potentially as a predictive biomarker in genotypes associated with Dent disease.Nevertheless, their conclusion stated that serum muscle enzyme levels could not be utilized as an indicator to infer variations in either the CLCN5 or OCRL gene.
In this study, all patients exhibited LMWP and with only two of them displaying glycosuria.As we know, Fanconi syndrome is a well-known clinical diagnosis associated with Lowe syndrome, typically emerging during the early months of life.However, several studies have discovered that LMWP is always present, but glycosuria is rarely observed [6,31] .Due to the extensive variation in the degree of renal proximal tubular dysfunction seen in individuals with this disorder, some experts have questioned whether the label "Fanconi syndrome" accurately depicts the renal abnormalities observed in Lowe syndrome [32] .
Unexpectedly, patient 1 (p.Ala857Cysfs*10) was diagnosed after both lower limb exions were discovered when he was three years old, rather than congenital cataracts like others after birth.His parents did not take his foamy urine and psychomotor retardation seriously.We were surprised to discover that he had no vision issues.The occurrence of Lowe syndrome patients without cataracts is rare, with only a few documented cases.For instance, in one family, the p.Ile274Thr variant was identi ed, where the youngest sibling developed unilateral cataracts while the oldest sibling did not [14] .In another case, the intronic variant c.2257-5G > A was found in two siblings; one exhibited atypical Lowe syndrome with minimal ocular involvement, while the other presented solely with the renal phenotype [33] .Additionally, patients carrying the p.Asp523Asn variant displayed brain and renal symptoms of Lowe syndrome without congenital cataracts, with cataracts only appearing 5-10 years later [3,34] .These uncommon milder phenotypes of Lowe syndrome, without or with minimal ocular involvement, can be attributed to speci c variations, although the precise underlying mechanism remains unclear.We will continue to monitor the progress of Patient 1's eye condition.In this case, we learned that early urinalysis could be one of the most crucial and easy-to-perform tests that help us make an early clinical diagnosis of atypical Lowe syndrome.

Analyses of OCRL variations
Approximately 360 OCRL disease-causing variants have been identi ed, with missense and nonsense variations accounting for 49%, splicing variants for 12%, small deletions for 20%, and small insertions for 9% as well as extensive deletions and insertions, as reported by the Human Gene Mutation Database (HGMD) [35]   .The underlying cause of why speci c mutations in the OCRL gene lead to Lowe syndrome while others result in Dent-2 disease remains unknown, despite the fact that variations in the OCRL gene are responsible for both conditions.According to previous studies [6,14,[36][37][38][39][40] , variants related to Lowe syndrome were primarily found in exons 8 to 24, with exon 15 being the most frequently affected.In our study, all variations were identi ed in exons 9-23, which contain three signi cant functional domains: RhoGAP-like domain, ASPM-SPD-2-Hydin[ASH] domain, and 5-phosphatase domain.These domains are believed to play an essential role in the manifestation of Lowe syndrome.Speci cally, nonsense, frameshift, and splicing variations of the OCRL gene exclusively associated with Lowe syndrome were detected within exons 8-24.the ASH-RhoGAP module is responsible for regulating a majority of the currently described protein-protein interactions [14] .The proportion (6/8 alleles,75%) of variations documented in our study resulted in the generation of truncated proteins is higher than 63.6%, previously reported by Hichri et al. [14] .In comparison, 25% corresponded to missense variations.
Novel patients 1 (p.Ala857Cysfs*10) and 5 (p.Cys831*) suffered from renal hypophosphatemic rickets in our study.Both individuals exhibited reduced serum phosphate levels and a range of selective proximal tubular dysfunctions, including LMWP, aminoaciduria, and hypercalciuria.All of them were administered phosphate and alkali supplements to prevent the progression of rickets.Six of the previously published twelve Korean patients with Lowe syndrome had hypophosphatemia, and supplemental therapies were administered [27] , whereas in the study by Charnas et al. [28] , 14 of 23(61%) patients did not require phosphate supplementation.Lowe [41] indicated that kidney defects observed in Lowe syndrome are indicative of a defect in the protein tra c, including receptors, involved in reabsorption in the proximal tubule.Prior research suggested that impaired reabsorption in the proximal tubules observed in Lowe syndrome may be attributable to a change in the tra cking of the relevant transporters.Clathrin-mediated endocytosis is an essential mechanism for membrane protein control, such as the sodium-phosphate cotransporter.It has been demonstrated that OCRL1 regulates vesicular transport by interacting with clathrin, and the speci cs of this relationship have been elucidated [42][43][44] .These two novel variations were identi ed in exon23, which mapped to a region of the RhoGAP-like domain.U.Lichter-Konecki et al. discovered that variations in the RhoGAP domain of OCRL1 might impact the Arf1 signaling pathway, which plays a crucial role in Golgi function [45] , We could suggest that these two novel variations are essential in inducing renal tubulopathy.The diversity of the clinical phenotype of patients with proximal tubular dysfunction in Lowe syndrome is worthy of further study.
As part of our investigation, another novel frameshift variation c.1383delA (p.A462Lfs*58) from patient 4 and two novel missense variations c.1572C > A (p.His524Gln) and c.824G > A (p.Gly275Glu) from patient 7/8 were identi ed in exon 9/14/15 as mapping to a region of the 5-Phosphatase domain that is responsible for recognizing PI(4,5)P2 in the membrane.These three patients with variations discovered in the 5-Phosphatase domain showed typical phenotypes, i.e., ocular signs including congenital cataracts appearing after birth, neurological symptoms including neonatal hypotonia, retardation of development, epileptic seizures and proximal renal tubulopathy.It has been discovered via research that the phosphoinositide 5-phosphatase OCRL1 is located in the kidney tubular cells and the primary cilium of retinal pigment epithelial cells [46] .They are seen abundantly in the plasma membrane, which suggests their involvement in cargo sorting and selective uptake of speci c molecules [47] .OCRL1 plays a vital role as a regulator of phosphoinositide conversion, which is essential for the uncoating of clathrin and the functioning of key components involved in the endocytic machinery.Increased PI(4,5)P2 levels caused by OCRL1 loss are responsible for creating coated vesicles, which assemble numerous clathrin coat components and promote actin polymerization [48] .Hence, we infer that the variation mapped to the 5-Phosphatase domain of OCRL might have the most severe and typical clinical phenotype.Next, a meta-analysis of relevant data to con rm this hypothesis is needed.We have noticed that both missense variants in our study are inherited from their mothers.Therefore, pedigree analysis of their mother is needed to identify the sources of variation, either inherited or caused by de novo variation.
One of the missense variations is p.His524Gln which has previously been identi ed as pathogenic and submitted to ClinVar, but there are con icting interpretations.The level of mRNA expression of the OCRL gene and PI(4,5)P2 phosphatase activity should be measured in our two patients harboring missense variations to evaluate the functional effect of these novel variations in our subsequent work.
Although our study extensively examined the genotypic and phenotypic features of eight individuals with Lowe syndrome, it is important to acknowledge the limitations of our research.Firstly, the inclusion of a larger patient cohort from multiple provinces in southern China would have provided a more representative understanding of the diagnostic and heredity characteristics speci c to children with Lowe syndrome in this region.Additionally, the limited and inconsistent follow-up duration across patients hindered our ability to determine the prolonged development of Lowe Syndrome in each individual.Moreover, the lack of a standardized and consistent process for clinical evaluations may have resulted in incomplete assessments for some patients.

Conclusion
We described eight patients' phenotypic and genotypic characteristics and identi ed ve novel variants in the OCRL gene.We reported the rst case of atypical Lowe syndrome in China without congenital cataracts.The study provides valuable insights into the clinical and genetic traits of Lowe syndrome in Southern China.The identi cation of novel OCRL variations expands the understanding of the genetic spectrum of the disease.The ndings emphasize the necessity of raising awareness among clinicians to improve diagnostic accuracy and appropriate management of Lowe syndrome.Ongoing research into the underlying causes of this condition may lead to improved treatments and outcomes for those living with Lowe syndrome.

Declarations Figures
X-ray images of Osteoporosis from P1.
Slit-lamp photos showed that both lenses from P1 are normal.

Figure 3 AxisFigure 4
Figure 3 Axis brain magnetic resonance images of two patients with severe epilepsy showing white matter with high signal intensity on T2-weighted images.(A)P4, (B)P8

Table 1
Clinical and laboratory characteristics of 8 patients with Lowe syndrome.

Table 2
Variations of the OCRL gene were identi ed in 8 patients with Lowe Syndrome.
Maternal carrier status was positive in ve mothers (Patients 3,4,6,7,8).A positive family story was noted in patients 4 and 7; their elder brothers both showed bilateral congenital cataracts and severe psychomotor retardation.Patient 4's elder brother died of sepsis at 18 years old.Patient 7's elder brother was sent to the welfare house when he was two.