Complex genotype-phenotype correlation of MYH11: new insights from monozygotic twins with highly variable expressivity and outcomes.

BACKGROUND: Thoracic aortic aneurysm/dissection (TAAD) and patent ductus arteriosus (PDA) are serious autosomal-dominant diseases affecting the cardiovascular system. They are mainly caused by variants in the MYH11 gene, which encodes the heavy chain of myosin 11. The aim of this study was to evaluate the genotype-phenotype correlation of MYH11 from a distinctive perspective based on a pair of monozygotic twins. METHODS: The detailed phenotypic characteristics of the monozygotic twins from the early fetal stage to the infancy stage were traced and compared with each other and with those of previously documented cases. Whole-exome and Sanger sequencing techniques were used to identify and validate the candidate variants, facilitating the analysis of the genotype-phenotype correlation of MYH11. RESULTS: The monozygotic twins were premature and presented with PDA, pulmonary hypoplasia, and pulmonary hypertension. The proband developed heart and brain abnormalities during the fetal stage and died at 18 days after birth, whereas his sibling was discharged after being cured and developed normally post follow-up. A novel variant c.766 A > G p. (Ile256Val) in MYH11 (NM_002474.2) was identified in the monozygotic twins and classified as a likely pathogenic variant according to the American College of Medical Genetics/Association for Molecular Pathology guidelines. Reviewing the reported cases (n = 102) showed that the penetrance of MYH11 was 82.35%, and the most common feature was TAAD (41.18%), followed by PDA (22.55%), compound TAAD and PDA (9.80%), and other vascular abnormalities (8.82%). The constituent ratios of null variants among the cases with TAAD (8.60%), PDA (43.8%), or compound TAAD and PDA (28.6%) were significantly different (P = 0.01). Further pairwise comparison of the ratios among these groups showed that there were significant differences between the TAAD and PDA groups (P = 0.006). CONCLUSION: This study expands the mutational spectrum of MYH11 and provides new insights into the genotype-phenotype correlation of MYH11 based on the monozygotic twins with variable clinical features and outcomes, indicating that cryptic modifiers and complex mechanisms beside the genetic variants may be involved in the condition.

[Analysis of a child with DIGFAN syndrome due to variant of MORC2 gene].

OBJECTIVE: To explore the clinical features and genetic etiology for a child with developmental delay, impaired growth, facial dysmorphism, and axonal neuropathy (DIGFAN). METHODS: A child who was admitted to the Second Affiliated Hospital of Guangxi Medical University on March 22, 2021 was selected the study subject. Clinical data of the child was collected. Following extraction of genomic DNA, the child and his parents were subjected to whole exome sequencing (WES), and candidate variant was verified by Sanger sequencing and bioinformatic analysis. RESULTS: The child, a 10-year-and-9-month-old boy, had manifested with short stature, intellectual disability, delayed speech, motor and language development, and facial dysmorphism. WES and Sanger sequencing revealed that he has harbored a novel de novo c.800T>C (p.Leu267Pro) variant of the MORC2 gene. The Leucine at position 267, which is highly conserved among various species, is located in the S5 domain of ribosome protein in the ATPase binding region of MORC2. And the Leu267Pro may affect the function of MORC2 by altering the spatial conformation and activity of ATPase. Based on the guidelines from the American College of Medical Genetics and Genomics, the c.800T>C variant was classified as likely pathogenic (PS2+PM2_Supporting+PP2+PP3). CONCLUSION: The MORC2: c.800T>C (p.Leu267Pro) variant probably underlay the pathogenesis of DIGFAN syndrome in this child.

Third generation sequencing transforms the way of the screening and diagnosis of thalassemia: a mini-review.

Thalassemia is an inherited blood disorder imposing a significant social and economic burden. Comprehensive screening strategies are essential for the prevention and management of this disease. Third-generation sequencing (TGS), a breakthrough technology, has shown great potential for screening and diagnostic applications in various diseases, while its application in thalassemia detection is still in its infancy. This review aims to understand the latest and most widespread uses, advantages of TGS technologies, as well as the challenges and solutions associated with their incorporation into routine screening and diagnosis of thalassemia. Overall, TGS has exhibited higher rates of positive detection and diagnostic accuracy compared to conventional methods and next-generation sequencing technologies, indicating that TGS will be a feasible option for clinical laboratories conducting in-house thalassemia testing. The implementation of TGS technology in thalassemia diagnosis will facilitate the development of effective prevention and management strategies, thereby reducing the burden of this disease on individuals and society.

Importance of comprehensive genetic testing for patients with suspected vascular Ehlers-Danlos syndrome: a family case report and literature review.

Vascular Ehlers-Danlos syndrome (vEDS), the most severe type of Ehlers-Danlos syndrome, is caused by an autosomal-dominant defect in the COL3A1 gene. In this report, we describe the clinical history, specific phenotype, and genetic diagnosis of a man who died of vEDS. The precise diagnosis of this case using whole-exome sequencing provided solid evidence for the cause of death, demonstrating the practical value of genetic counseling and analysis. Early diagnosis for the proband's son, who was also affected by vEDS, revealed initial complications of vEDS in early childhood, which have rarely been reported. We also reviewed the literature on COL3A1 missense mutations and related phenotypes. We identified an association between digestion tract events and non-glycine missense variants, which disproves a previous hypothesis regarding the genotype-phenotype correlation of vEDS. Our results demonstrate the necessity of offering comprehensive genetic testing for every patient suspected of having vEDS.

Case report: A novel heterozygous synonymous variant in deep exon region of NIPBL gene generating a non-canonical splice donor in a patient with cornelia de lange syndrome.

Cornelia de Lange syndrome (CdLS) is an autosomal dominant or X-linked genetic disease with significant genetic heterogeneity. Variants of the NIPBL gene are responsible for CdLS in 60% of patients. Herein, we report the case of a patient with CdLS showing distinctive facial features, microcephaly, developmental delay, and growth retardation. Whole exome sequencing was performed for the patient, and a novel de novo heterozygous synonymous variant was identified in the deep region of exon 40 in the NIPBL gene (NM_133433.4: c. 6819G > T, p. Gly2273 = ). The clinical significance of the variant was uncertain according to the ACMG/AMP guidelines; however, based on in silico analysis, it was predicted to alter mRNA splicing. To validate the prediction, a reverse transcriptase-polymerase chain reaction was conducted. The variant activated a cryptic splice donor, generating a short transcript of NIPBL. A loss of 137 bp at the 3' end of NIPBL exon 40 was detected, which potentially altered the open reading frame by inserting multiple premature termination codons. Quantitative real-time PCR analysis showed that the ratio of the transcription level of the full-length transcript to that of the altered short transcript in the patient was 5:1, instead of 1:1. These findings may explain the relatively mild phenotype of the patient, regardless of the loss of function of the truncated protein due to a frameshift in the mRNA. To the best of our knowledge, this study is the first to report a synonymous variant in the deep exon regions of the NIPBL gene responsible for CdLS. The identified variant expands the mutational spectrum of the NIPBL gene. Furthermore, synonymous variations may be pathogenic, which should not be ignored in the clinical and genetic diagnosis of the disease.

Phenotypic variability of syndromic craniosynostosis caused by c.833G > T in FGFR2: Clinical and genetic evaluation of eight patients from a five-generation family.

OBJECTIVE: Craniosynostosis is the result of the early fusion of cranial sutures. Syndromic craniosynostosis includes but not limited by Crouzon syndrome and Pfeiffer syndrome. Considerable phenotypic overlap exists among these syndromes and mutations in FGFR2 may cause different syndromes. This study aims to investigate the explanation of the phenotypic variability via clinical and genetic evaluation for eight patients in a large pedigree. METHODS: For each patient, comprehensive physical examination, cranial plain CT scan with three-dimensional CT reconstruction (3D-CT), and eye examinations were conducted. Whole exome sequencing was applied for genetic diagnosis of the proband. Variants were analyzed and interpreted following the ACMG/AMP guidelines. Sanger sequencing was performed to reveal genotypes of all the family members. RESULTS: A pathogenic variant in the FGFR2 gene, c.833G > T (p.C278F), was identified and proved to be co-segregate with the disease. Some symptoms of head, hearing, vision, mouth, teeth expressed differently by affected individuals. Nonetheless, all the eight patients manifested core symptoms of Crouzon syndrome without abnormality in the limbs, which could exclude diagnosis of Pfeiffer syndrome. CONCLUSION: We have established clinical and genetic diagnosis of Crouzon syndrome for eight patients in a five-generation Chinese family. Variability of clinical features among these familial patients was slighter than that in previously reported sporadic cases.

Whole Exome Sequencing Uncovered the Genetic Architecture of Growth Hormone Deficiency Patients.

Purpose: Congenital growth hormone deficiency (GHD) is a rare and etiologically heterogeneous disease. We aim to screen disease-causing mutations of GHD in a relatively sizable cohort and discover underlying mechanisms via a candidate gene-based mutational burden analysis. Methods: We retrospectively analyzed 109 short stature patients associated with hormone deficiency. All patients were classified into two groups: Group I (n=45) with definitive GHD and Group II (n=64) with possible GHD. We analyzed correlation consistency between clinical criteria and molecular findings by whole exome sequencing (WES) in two groups. The patients without a molecular diagnosis (n=90) were compared with 942 in-house controls for the mutational burden of rare mutations in 259 genes biologically related with the GH axis. Results: In 19 patients with molecular diagnosis, we found 5 possible GHD patients received known molecular diagnosis associated with GHD (NF1 [c.2329T>A, c.7131C>G], GHRHR [c.731G>A], STAT5B [c.1102delC], HRAS [c.187_207dup]). By mutational burden analysis of predicted deleterious variants in 90 patients without molecular diagnosis, we found that POLR3A (p = 0.005), SUFU (p = 0.006), LHX3 (p = 0.021) and CREB3L4 (p = 0.040) represented top genes enriched in GHD patients. Conclusion: Our study revealed the discrepancies between the laboratory testing and molecular diagnosis of GHD. These differences should be considered when for an accurate diagnosis of GHD. We also identified four candidate genes that might be associated with GHD.

Heterozygous Recurrent Mutations Inducing Dysfunction of ROR2 Gene in Patients With Short Stature.

PURPOSE: ROR2, a member of the ROR family, is essential for skeletal development as a receptor of Wnt5a. The present study aims to investigate the mutational spectrum of ROR2 in children with short stature and to identify the underlying molecular mechanisms. METHODS: We retrospectively analyzed clinical phenotype and whole-exome sequencing (WES) data of 426 patients with short stature through mutation screening of ROR2. We subsequently examined the changes in protein expression and subcellular location in ROR2 caused by the mutations. The mRNA expression of downstream signaling molecules of the Wnt5a-ROR2 pathway was also examined. RESULTS: We identified 12 mutations in ROR2 in 21 patients, including 10 missense, one nonsense, and one frameshift. Among all missense variants, four recurrent missense variants [c.1675G > A(p.Gly559Ser), c.2212C > T(p.Arg738Cys), c.1930G > A(p.Asp644Asn), c.2117G > A(p.Arg706Gln)] were analyzed by experiments in vitro. The c.1675G > A mutation significantly altered the expression and the cellular localization of the ROR2 protein. The c.1675G > A mutation also caused a significantly decreased expression of c-Jun. In contrast, other missense variants did not confer any disruptive effect on the biological functions of ROR2. CONCLUSION: We expanded the mutational spectrum of ROR2 in patients with short stature. Functional experiments potentially revealed a novel molecular mechanism that the c.1675G > A mutation in ROR2 might affect the expression of downstream Wnt5a-ROR2 pathway gene by disturbing the subcellular localization and expression of the protein.

Exome sequencing reveals genetic architecture in patients with isolated or syndromic short stature.

Short stature is among the most common endocrinological disease phenotypes of childhood and may occur as an isolated finding or in conjunction with other clinical manifestations. Although the diagnostic utility of clinical genetic testing in short stature has been implicated, the genetic architecture and the utility of genomic studies such as exome sequencing (ES) in a sizable cohort of patients with short stature have not been investigated systematically. In this study, we recruited 561 individuals with short stature from two centers in China during a 4-year period. We performed ES for all patients and available parents. All patients were retrospectively divided into two groups: an isolated short stature group (group I, n = 257) and an apparently syndromic short stature group (group II, n = 304). Causal variants were identified in 135 of 561 (24.1%) patients. In group I, 29 of 257 (11.3%) of the patients were solved by variants in 24 genes. In group II, 106 of 304 (34.9%) patients were solved by variants in 57 genes. Genes involved in fundamental cellular process played an important role in the genetic architecture of syndromic short stature. Distinct genetic architectures and pathophysiological processes underlie isolated and syndromic short stature.

Long-Molecule Sequencing: A New Approach for Identification of Clinically Significant DNA Variants in α-Thalassemia and ß-Thalassemia Carriers.

Multiple molecular tests are currently needed for accurate carrier testing for thalassemia. Therefore, long-molecule sequencing (LMS) was evaluated as an alternate on the PacBio Sequel platform for genotyping carriers of α-thalassemia or ß-thalassemia. Multiplex long PCR was used to generate representative amplicons for the α (HBA1/2) and ß (HBB) gene loci. Following LMS, circular consensus sequencing reads were aligned to the hg19 reference genome and variants called using FreeBayes software version 1.2.0. In a blinded study of 64 known carrier samples, all HBA1/2 and HBB variants detected by LMS were concordant with those independently assigned by targeted PCR assays. For HBA1/2 carrier samples, LMS accurately detected the common South East Asian, -α3.7, and -α4.2 deletions and four different rare single-nucleotide variants (SNVs). For HBB carrier samples, LMS accurately detected the most common Chinese insertion and deletion variant c.126_129delCTTT and 14 different SNVs/insertions and deletions and could discriminate compound heterozygous SNVs (trans configuration) and identify variants linked to benign SNPs (cis configuration). Overall, LMS displayed the hallmarks of a scalable, accurate, and cost-effective genotyping method. With further test coverage to additionally include detection of other clinically significant HBA1/2 copy number variations, such as the Thai, Mediterranean, and Filipino deletions, LMS may eventually serve as a comprehensive method for large-scale thalassemia carrier screening.

Low-pass genome sequencing versus chromosomal microarray analysis: implementation in prenatal diagnosis.

PURPOSE: Emerging studies suggest that low-pass genome sequencing (GS) provides additional diagnostic yield of clinically significant copy-number variants (CNVs) compared with chromosomal microarray analysis (CMA). However, a prospective back-to-back comparison evaluating accuracy, efficacy, and incremental yield of low-pass GS compared with CMA is warranted. METHODS: A total of 1023 women undergoing prenatal diagnosis were enrolled. Each sample was subjected to low-pass GS and CMA for CNV analysis in parallel. CNVs were classified according to guidelines of the American College of Medical Genetics and Genomics. RESULTS: Low-pass GS not only identified all 124 numerical disorders or pathogenic or likely pathogenic (P/LP) CNVs detected by CMA in 121 cases (11.8%, 121/1023), but also defined 17 additional and clinically relevant P/LP CNVs in 17 cases (1.7%, 17/1023). In addition, low-pass GS significantly reduced the technical repeat rate from 4.6% (47/1023) for CMA to 0.5% (5/1023) and required less DNA (50 ng) as input. CONCLUSION: In the context of prenatal diagnosis, low-pass GS identified additional and clinically significant information with enhanced resolution and increased sensitivity of detecting mosaicism as compared with the CMA platform used. This study provides strong evidence for applying low-pass GS as an alternative prenatal diagnostic test.

A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene.

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5' untranslated region (UTR) of the Fragile X mental retardation-1 (FMR1) gene promoter. Conventionally, capillary electrophoresis fragment analysis on a genetic analyzer is used for the sizing of the CGG repeats of FMR1, but additional Southern blot analysis is required for exact measurement when the repeat number is higher than 200. Here, we present an accurate and robust polymerase chain reaction (PCR)-based method for quantification of the CGG repeats of FMR1. The first step of this test is PCR amplification of the repeat sequences in the 5'UTR of the FMR1 promoter using a Fragile X PCR kit, followed by purification of the PCR products and fragment sizing on a microfluidic capillary electrophoresis instrument, and subsequent interpretation of the number of CGG repeats by referencing standards with known repeats using the analysis software. This PCR-based assay is reproducible and capable of identifying the full range of CGG repeats of FMR1 promoters, including those with a repeat number of more than 200 (classified as full mutation), 55 to 200 (premutation), 46 to 54 (intermediate), and 10 to 45 (normal). It is a cost-effective method that facilitates classification of the FXS and Fragile X-associated disorders with robustness and rapid reporting time.

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy.

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60-80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2-4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

A prospective study of non-invasive preimplantation genetic testing for aneuploidies (NiPGT-A) using next-generation sequencing (NGS) on spent culture media (SCM).

PURPOSE: This study was to evaluate if spent culture media (SCM) of embryos could be used as a non-invasive tool to achieve aneuploidy screening. Ploidy calls, as well as concordance rates between PGT-A results from trophectoderm (TE) and SCM, were compared. Clinical outcomes of single euploid transfers were also evaluated. METHODS: The study was conducted from March 2017 to June 2018 in a university-based ART center. SCM of day 3 to the day(s) of TE biopsy of all biopsied blastocysts were collected for testing. PGT-A results of SCM were compared with the standard results of TE, with clinical relevance and outcomes examined. RESULTS: NiPGT-A using SCM gave a sensitivity of 81.6%, specificity of 48.3%, positive predictive value of 82.6%, and negative predictive value of 46.7% in ploidy calling. The concordance rates for autosomes and sex determination were 62.1% and 82.4%, respectively. There were 14 single embryo transfer cycles of euploids as determined by TE biopsy. Clinical outcomes not only confirmed 3 false positive results from SCM but also reflected the true ploidy status of the transferred embryo in one case. If ploidy calls were dichotomized without mosaic embryos, the sensitivity and NPV would increase to 91.0% and 66.7% (p = 0.60 and p = 0.25), respectively. CONCLUSIONS: Cell-free DNA found in SCM could provide ploidy information of an embryo as in PGT-A from its TE. Given its potential to reflect the comprehensive chromosomal profile of the whole embryo, more research based on clinical outcomes is required to determine if SCM could be a reliable selection tool in PGT-A.

New insights into 5α-reductase type 2 deficiency based on a multi-centre study: regional distribution and genotype-phenotype profiling of SRD5A2 in 190 Chinese patients.

BACKGROUND: The 5α-reductase type 2 (5α-RD2) deficiency caused by mutations in the steroid 5α-reductase 2 (SRD5A2) gene results in variable degrees of undervirilisation in patients with 46,XY disorders of sex development. This study aims to profile the regional distribution and phenotype-genotype characteristics of SRD5A2 in a large Chinese 5α-RD2 deficiency cohort through multi-centre analysis. METHODS: 190 subjects diagnosed with 5α-RD2 deficiency were consecutively enrolled from eight medical centres in China. Their clinical manifestations and genetic variants were analysed. RESULTS: Hypospadias (isolated or combined with microphallus and/or cryptorchidism) was fairly common in the enrolled subjects (66.32%). 42 variants, including 13 novel variants, were identified in SRD5A2. Homozygous and compound heterozygous mutations presented in 38.42% and 61.58% of subjects, respectively, and predominated in exons 1, 4 and 5. The most prevalent variant was c.680G > A (52.37%), followed by c.16C > T, (10.79%), c.607G > A, (9.21%) and c.737G > A, (8.95%). However, their distributions were different: c.680G > A was more common in South China than in North China (62.62% vs 39.16%, p < 0.001), whereas the regional prevalence of c.16C > T was reversed (6.07% vs 16.87%, p = 0.001). Furthermore, c.680G > A prevailed in cases with normal meatus (68.75%) or distal hypospadias (66.28%), compared with those with proximal hypospadias (35.54%, p < 0.001). However, cases with proximal hypospadias showed a higher frequency of c.16C > T (20.48%) than those with normal meatus (3.13%) or distal hypospadias (3.49%, p < 0.001). CONCLUSIONS: This study profiled variable phenotypic presentation and wide mutational spectrum of SRD5A2, revealing its distinctive regional distribution in Chinese patients and further shaping the founder effect and genotype-phenotype correlation of SRD5A2.

Biparental Inheritance of Mitochondrial DNA in Humans.

Although there has been considerable debate about whether paternal mitochondrial DNA (mtDNA) transmission may coexist with maternal transmission of mtDNA, it is generally believed that mitochondria and mtDNA are exclusively maternally inherited in humans. Here, we identified three unrelated multigeneration families with a high level of mtDNA heteroplasmy (ranging from 24 to 76%) in a total of 17 individuals. Heteroplasmy of mtDNA was independently examined by high-depth whole mtDNA sequencing analysis in our research laboratory and in two Clinical Laboratory Improvement Amendments and College of American Pathologists-accredited laboratories using multiple approaches. A comprehensive exploration of mtDNA segregation in these families shows biparental mtDNA transmission with an autosomal dominantlike inheritance mode. Our results suggest that, although the central dogma of maternal inheritance of mtDNA remains valid, there are some exceptional cases where paternal mtDNA could be passed to the offspring. Elucidating the molecular mechanism for this unusual mode of inheritance will provide new insights into how mtDNA is passed on from parent to offspring and may even lead to the development of new avenues for the therapeutic treatment for pathogenic mtDNA transmission.

The current landscape for the treatment of mitochondrial disorders.

The mitochondrial organelle is crucial to the energy metabolism of the eukaryotic cell. Defects in mitochondrial function lie at the core of a wide range of disorders, including both rare primary mitochondrial disorders and more common conditions such as Parkinson's disease and diabetes. Inherited defects in mitochondrial function can be found in both the nuclear genome and the mitochondrial genome, with the latter creating unique challenges in the treatment and understanding of disease passed on through the mitochondrial genome. In this review, we will describe the limited treatment regimens currently used to alleviate primary mitochondrial disorders, as well as the potential for emerging technologies (in particular, those involving direct manipulation of the mitochondrial genome) to more decisively treat this class of disease. We will also emphasize the critical parallels between primary mitochondrial disorders and more common ailments such as cancer and diabetes.

Novel pathogenic RECQL4 variants in Chinese patients with Rothmund-Thomson syndrome.

BACKGROUND: Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder mainly characterized by cutaneous poikiloderma, sparse hair, short stature and skeletal defects. Deleterious mutations in the RecQ-like DNA helicase type 4 (RECQL4) gene have been detected in approximately two-thirds of RTS cases. METHODS: Three Chinese patients from two unrelated families were enrolled for clinical evaluation. Targeted next-generation sequencing (NGS) using a custom panel consisting of 705 short-stature-related genes was performed for the probands. Variants detected by NGS were confirmed by Sanger sequencing and examined in family members. RESULTS: The probands presented with characteristic features of severe growth delay, poikiloderma mostly on the face, buttocks and extremities, sparse or absent hair, eyelashes, and eyebrows, forearm reduction defects, small hands with hypoplasia of the middle phalanx (little finger) in one of the probands, epicanthus, hypertelorism, and dental abnormalities. In addition, novel auricle features and other rare facial features, including narrow palpebral fissure, depressed nasal bridge, and small chin were exhibited. Four novel RECQL4 variants were identified, including three pathogenic frameshift variants, c.1724_1725delAC, p.His575fs*7; c.2421dupT, p.Asp808*; c.1770_1807del, p.Pro591fs*2, and one likely pathogenic missense variant, c.691G>A, p.Gly231Ser. CONCLUSION: Our study expands the mutational spectrum of RECQL4 gene and reveals novel phenotypes observed in Chinese RTS patients.