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PubMed2025

Exome sequencing revealed a novel homozygous variant in TRMT61 A in a multiplex family with atypical Cornelia de Lange Syndrome from Rwanda.

BACKGROUND: In 30% of patients who exhibit the clinical profile of Cornelia de Lange Syndrome (CdLS), the genetic cause remains undetermined. This proportion tends to be higher in low-resource settings including Africa. We performed a molecular characterization of CdLS in a multiplex Rwandan family. METHODS: After a clinical evaluation of two affected siblings, DNA isolated from peripheral whole blood of the affected patients and their parents underwent Exome Sequencing (ES). Sanger sequencing validated the variant segregating with CdLS. In silico predictive tools, protein modelling, and cell-based experiments using HEK293T cells were used to investigate the pathogenicity of the variant found. RESULTS: We identified a family with two parents and their two offspring (male and female), who were referred for hearing impairment. The 17-year-old female presented bilateral profound hearing impairment with moderate hypertelorism, progressive visual impairment, and secondary amenorrhea. The 14-year-old male displayed intellectual disability and a bilateral profound hearing impairment with no noticeable facial dysmorphism. Following exome sequencing (ES) of DNA samples obtained from the four family members, we found that the siblings harbored a novel likely pathogenic homozygous missense variant in the TRMT61 A gene [NM_152307.3:c.665C > T p.(Ala222Val)] inherited from both heterozygous parents. In silico analysis suggested that the variant substitutes a highly conserved amino acid, and 2-D structure modelling revealed a significant decrease in the stability of the protein. Cell-based experiment in HEK293T showed that the variant significantly affected the TRMT61 A protein localization which is thought to impact the mitochondrial and cytosolic functions. CONCLUSION: We reported a novel biallelic variant in TRMT61 A, [NM_152307.3:c.665C > T p.(Ala222Val)], which is associated with autosomal recessive atypical CdLS in a multiplex Rwandan family, the first report from Africa, and the second globally. The study emphasizes the need to expand the availability of ES for molecular characterization of rare diseases for the understudied genetically diverse population of Africa.

Humans↗
PubMed2026

Identification of a novel and a recurrent CDC45 variant in a Chinese family with Meier-Gorlin syndrome 7 and a literature review.

INTRODUCTION: Meier-Gorlin syndrome 7 (MGORS7) is a rare autosomal recessive disorder characterized by primordial dwarfism, craniosynostosis, and patellar aplasia, caused by pathogenic variants of CDC45. Here, we report a Chinese patient presenting with classic hallmarks of MGORS7 alongside atypical clinical features, including hearing and visual impairments. METHODS: Clinical and radiological data were collected. Whole-genome sequencing and Sanger sequencing were performed to identify and validate the causative variants. Their functional effects were investigated using an exon-trapping assay, and a literature review of previously reported MGORS7 cases was conducted. RESULTS: Genetic analysis identified two compound heterozygous CDC45 variants: c.1416C>T (p.H472=) and c.1559+2T>A, which are a recurrent variant in the East Asian population and a novel variant, respectively. Our exon-trapping assay indicated that c.1559+2T>A induced aberrant splicing, generating transcripts predicted to undergo nonsense-mediated mRNA decay. Additionally, growth hormone therapy was initiated in our patient, with a noted improvement in growth parameters in the initial assessment and without immediate complications. The literature review identified a total of 32 CDC45 variants in 29 patients with MGORS7, who showed high heterogeneity in clinical phenotypes. DISCUSSION: Our study further expanded the mutational spectrum of CDC45 and provided a preliminary clinical observation suggesting that growth hormone therapy may be beneficial for growth retardation in patients with MGORS7.

CDC45↗
PubMed1994

Phylogenetic relationship of Sarcocystis neurona to other members of the family Sarcocystidae based on small subunit ribosomal RNA gene sequence.

Sarcocystis neurona is a coccidial parasite that causes a neurologic disease of horses in North and South America. The natural host species are not known and classification is based on ultrastructural analysis. The small subunit ribosomal RNA (SSURNA) gene of S. neurona was amplified using polymerase chain reaction techniques and sequenced by Sanger sequencing reactions. The sequence was compared with partial sequences of S. muris, S. gigantea, S. tenella, S. cruzi, S. arieticanis, S. capracanis, Toxoplasma gondii, Eimeria tenella, and Cryptosporidium parvum. Alignments of available sites for all 10 species and alignments of the entire SSURNA sequence of S. neurona, S. muris, S. cruzi, T. gondii, and C. parvum were performed. Alignments were analyzed using maximum parsimony and maximum likelihood methods to determine relative phylogeny of these organisms. These analyses confirmed placement of S. neurona in the genus Sarcocystis and suggested a close relationship to S. muris, S. gigantea, and T. gondii. Molecular phylogeny suggests that Sarcocystis spp., which utilize the dog (Canis familiaris) as the definitive host, evolved from a common ancestor, whereas those species (including T. gondii) that utilize the cat (Felis domesticus) as the definitive host evolved from another common ancestor. This suggests a possible definitive host for S. neurona.

Animals↗
PubMed1989

Automated Sanger DNA sequencing with one label in less than four lanes on gel.

Novel Sanger dideoxy sequencing with only one fluorescent dye label for the four bases of one clone and sequence determination in two lanes on polyacrylamide gel is presented, loading A greater than G in one lane and T greater than C in the other. Sequencing reactions for the two bases in each lane are carried out in one tube. At present the ratio of ddATP:ddGTP and ddTTP:ddCPT is set to 5:1 in the two tubes. Distinction between the two bases in one lane is done by comparing the different magnitudes of the peaks. This method increases the capacity since more clones may be run simultaneously on one gel, while keeping the reliability and simplicity that comes with the use of only one fluorescent dye for the four bases of one clone. At present about 200 bases are determined with the one-dye two-lane method on the EMBL's automated fluorescent DNA sequencer, using T7 DNA polymerase. The error rate in the deduced sequence is about 1%. The technique is used for the determination of overlaps in mapping projects. In principle, it is possible to determine the sequence with one dye in only one lane on the gel by choosing the proper ddNTP ratios for all four bases, carrying out reactions in one tube and applying the product in one lane, but the error rate for this one-lane method seems too high at present and further improvements in the uniformity of peaks obtainable with the T7 DNA polymerase or other enzymes are required.

Base Sequence↗
PubMed2026

A Novel Splice Variant in the COL1A1 Gene Leads to Exon 46 Skipping and Osteogenesis Imperfecta.

BACKGROUND: Osteogenesis imperfecta (OI) is a clinical and genetic disorder characterised by bone fragility, growth deficiency and skeletal deformity. Ninety per cent of OI cases are attributable to autosomal dominant variants in the COL1A1 and COL1A2 genes. METHODS: Candidate variants were identified and verified through trio whole-exome sequencing (trio-WES), copy number variation sequencing (CNV-seq) and Sanger sequencing. Minigene splicing assays were performed in HeLa and HEK293T cells with pcDNA3.1 and pcMINI-C vectors to investigate the function of the candidate variants. A systematic review of COL1A1 splicing variants and the corresponding genotype-phenotype spectrum was performed. RESULTS: Trio-WES revealed a novel heterozygous variant in the C-terminal region of the COL1A1 gene: NM_000088.4:c.3423+5G>A. Sanger sequencing confirmed the variant in both the proband (II-2) and her foetus (III-1) who were clinically suspected of having OI. The c.3423+5G>A variant causes complete skipping of Exon 46, as demonstrated by a minigene splicing assay. We retrieved 419 COL1A1 splicing variants from PubMed, excluded 15 without phenotypic data and 2 linked to Ehlers-Danlos syndrome and stratified the remaining 402 variants into three types on the basis of splice site location: (1) Variants at canonical splicing sites (77.8%, 313/402) mostly cause mild phenotypes, whereas a minority may be severe. (2) Intron variants in other locations, such as splice region variants (17.9%, 72/402), usually cause mild clinical phenotypes, and deep intronic splice variants (0.4%, 2/402) that may result in severe phenotypes. (3) Other variants (3.7%, 15/402), such as exon variants or fragment loss, are extremely rare. We also preliminarily discuss the mechanisms underlying phenotypic variability and the characteristics of C-terminal variants. CONCLUSIONS: This intron variant in COL1A1 was classified as likely pathogenic and was confirmed to disrupt COL1A1 expression. The summary analysis results also revealed a correlation among splicing variants, C-terminal region variants and disease, suggesting that variant location provides a useful framework for prognosis prediction.

Female↗
PubMed2026

[Analysis of a Chinese pedigree affected with Townes-Brocks syndrome due to a novel variant of SALL1 gene and a literature review].

OBJECTIVE: To analyze a novel exonic variant of the SALL1 gene and its impact on the binding site of SALL protein. METHODS: Clinical data of three children diagnosed with Townes-Brocks syndrome and their family members who had presented at the First Affiliated Hospital of Shandong First Medical University in April 2022 were retrospectively collected. The pathogenic variant was identified through whole-genome sequencing (WGS) and validated by Sanger sequencing. Protein structural prediction was performed using AlphaFold and PyMOL software to construct three-dimensional models of the wild-type and mutant proteins. Additionally, previously reported cases were systematically reviewed. This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: 2023-386). RESULTS: The proband was one of triplet sisters born at 34+4 gestational weeks. All three cases had presented with anal atresia and rectovaginal fistula, and case 3 also had toe malformation of left foot. WGS revealed a novel heterozygous c.757C>T (p.Gln253*) variant in the SALL1 gene, which was predicted to be pathogenic. Sanger sequencing confirmed co-segregation of the variant with the disease within the family. Protein structural modeling demonstrated that the variant has introduced a premature stop codon at position 253, resulting in a truncated protein. CONCLUSION: Above finding has enriched the mutation spectrum of the SALL1 gene in association with Townes-Brocks syndrome, which also represented a rare case of anal atresia in triplets, and provided a basis for molecular diagnosis, genetic counseling, and further research.

Humans↗
PubMed1996

Analyzing sequencing reactions from bacteriophage M13 by matrix-assisted laser desorption/ionization mass spectrometry.

The current demand for improved DNA sequencing methodologies posed by the Human Genome Project has spurred the investigation of alternatives to gel electrophoresis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has great potential for the rapid analysis of DNA fragments. Mock Sanger sequencing mixtures have been successfully analyzed by MALDI by pooling synthesized oligonucleotides corresponding to the M13 bacteriophage sequence. More recently, analyses of Sanger sequencing fragments enzymatically generated from synthetic templates of 45 or 50 bases were reported. In the present study, these feasibility demonstrations are extended to show MALDI sequencing from the M13 bacteriophage DNA template commonly used in actual Sanger sequencing. The results show sequence determination for extension products up to 35 bases in length. Different desalting and purification procedures were investigated and it was found that salt could be efficiently reduced by removal of the template in a post-reaction step. Work in progress to stabilize DNA by chemical modification, employed in conjunction with the methods described here, should enable significant extension of the length of readable sequence.

Bacteriophage M13↗
PubMed2026

A novel missense mutation in tropomyosin 1 gene associated with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a common genetic heart disorder that can lead to heart failure or sudden death. Family-based identification of rare sarcomeric variants can support molecular diagnosis and cascade screening in inherited HCM. This study aimed to identify and evaluate a novel TPM1 variant found in a Vietnamese family with HCM. The proband, a 3-year-old boy diagnosed with HCM, and eight relatives from three generations underwent clinical and genetic evaluation. A candidate variant initially identified by targeted next-generation sequencing was validated by PCR and Sanger sequencing. Familial segregation analysis was performed, and variant pathogenicity was assessed according to ACMG guidelines with support from in silico prediction and structural modeling. Sanger sequencing confirmed a heterozygous missense variant in exon 6 of TPM1 NM_001018005.2:c.576G > C, p.(Glu192Asp), in the proband, his father, and paternal grandfather, all of whom exhibited clinical signs of HCM. The variant was absent in unaffected relatives and in public population databases. Based on ACMG criteria (PM1, PM2, PM5, and PP3), the variant was classified as likely pathogenic. This novel TPM1 variant segregated with HCM in a Vietnamese family, expands the known mutational spectrum of TPM1 in hypertrophic cardiomyopathy, and warrants further functional investigation and familial genetic evaluation.

American College of Medical Genetics and Genomics ↗
PubMed2026

MNV-aware molecular characterization of a rare homozygous TTPA complex allele in ataxia with vitamin E deficiency.

Ataxia with vitamin E deficiency (AVED) is a rare autosomal-recessive neurological disorder caused by biallelic pathogenic variants in TTPA. Early vitamin E supplementation may prevent or limit irreversible neurological damage, but diagnosis is often delayed. Multi-nucleotide variants (MNVs) in TTPA have rarely been described and may be misinterpreted when adjacent substitutions are evaluated independently. We investigated a 34-year-old woman with childhood-onset progressive ataxia using clinical, biochemical, neuroimaging, and electrophysiological assessments. Serum vitamin E levels were measured longitudinally during supplementation. Whole-exome sequencing, read-level inspection, and Sanger sequencing were used to identify and confirm a homozygous TTPA complex allele, NM_000370.3:c. 296G > A;299 A > C, predicted to result in NP_000361.1:p. Gly99_Tyr100delinsAspSer, and to assess familial segregation. Population-database review, in silico prediction, and exploratory structure-based analysis were performed to evaluate its potential clinical relevance. The patient had markedly reduced baseline serum vitamin E levels of 0.8 µg/mL, which increased to 7.5 µg/mL after 12 months of supplementation. This biochemical correction was temporally accompanied by qualitatively observed improvements in gait stability, coordination, speech, and fine motor performance. Read-level analysis supported the presence of both substitutions on the same allele, and Sanger sequencing confirmed the homozygous complex allele in the patient and heterozygous carrier status in both parents. The affected residues are conserved and located within the CRAL-TRIO domain of α-tocopherol transfer protein. Exploratory structure-based analysis suggested altered local residue interactions; however, no functional assay was performed, and effects on protein stability, α-tocopherol binding, or transfer could not be established. This report expands the molecular spectrum of AVED by describing a homozygous TTPA complex allele and highlights the importance of MNV-aware interpretation of closely spaced substitutions. Vitamin E supplementation resulted in biochemical correction and was accompanied by possible partial clinical improvement despite initiation in adulthood. Functional studies are required to determine the precise effect of this allele on α-tocopherol transfer protein function.

Humans↗
PubMed2006

454 sequencing put to the test using the complex genome of barley.

BACKGROUND: During the past decade, Sanger sequencing has been used to completely sequence hundreds of microbial and a few higher eukaryote genomes. In recent years, a number of alternative technologies became available, among them adaptations of the pyrosequencing procedure (i.e. "454 sequencing"), promising an approximately 100-fold increase in throughput over Sanger technology--an advancement which is needed to make large and complex genomes more amenable to full genome sequencing at affordable costs. Although several studies have demonstrated its potential usefulness for sequencing small and compact microbial genomes, it was unclear how the new technology would perform in large and highly repetitive genomes such as those of wheat or barley. RESULTS: To study its performance in complex genomes, we used 454 technology to sequence four barley Bacterial Artificial Chromosome (BAC) clones and compared the results to those from ABI-Sanger sequencing. All gene containing regions were covered efficiently and at high quality with 454 sequencing whereas repetitive sequences were more problematic with 454 sequencing than with ABI-Sanger sequencing. 454 sequencing provided a much more even coverage of the BAC clones than ABI-Sanger sequencing, resulting in almost complete assembly of all genic sequences even at only 9 to 10-fold coverage. To obtain highly advanced working draft sequences for the BACs, we developed a strategy to assemble large parts of the BAC sequences by combining comparative genomics, detailed repeat analysis and use of low-quality reads from 454 sequencing. Additionally, we describe an approach of including small numbers of ABI-Sanger sequences to produce hybrid assemblies to partly compensate the short read length of 454 sequences. CONCLUSION: Our data indicate that 454 pyrosequencing allows rapid and cost-effective sequencing of the gene-containing portions of large and complex genomes and that its combination with ABI-Sanger sequencing and targeted sequence analysis can result in large regions of high-quality finished genomic sequences.

Base Pairing↗
PubMed2025

Genetic analysis of three patients from two unrelated Chinese families with autosomal recessive spastic ataxia of Charlevoix-Saguenay.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare early-onset neurodegenerative disorder characterized by progressive cerebellar ataxia, spasticity, and sensorimotor peripheral neuropathy. This disorder is caused by homozygous or compound heterozygous variants in the sacsin (SACS) gene on chromosome 13q12.12. Three patients with ARSACS from two unrelated Chinese families were recruited for this study. Patient #1 was an 18-year-old male who had been walking unstably for 12 years. Patient #2, the younger sister of Patient #1, was a 5-year-old girl who had been walking unstably for 2 years. Patient #3 was a 19-year-old female who had been walking unstably and a tendency to fall for 17 years. For Patient #1, whole-exome sequencing (WES) identified a hemizygous variant c.8310_8313delAGAT (p.Asp2771fs4*) in SACS (NM_014363.6), with the father being heterozygous, the mother wild-type, and Patient #2 hemizygous, as verified by Sanger sequencing. Additional copy number variant analysis of the WES data indicated that Patient #1 had a heterozygous gross deletion of chr13q12.12 (chr13:23,808,732 - 24,890,322). Low-coverage whole-genome sequencing results revealed that Patient #2 carried a chr13q12.12 deletion (chr13:23,520,000-24,940,000). Together with Sanger sequencing results, this gross deletion was speculated to have been inherited from the mother, further explaining the hemizygous state of c.8310_8313delAGAT (p.Asp2771fs4*) in Patients #1 and #2. Through WES, Patient #3 was identified as having suspected compound heterozygous variants of c.2881 C > T (p.Arg961*) and c.6409 C > T (p.Gln2137*), inherited from the father and mother, respectively, as confirmed by Sanger sequencing. This study identified three variants in SACS. The c.8310_8313delAGAT (p.Asp2771fs4*) is novel, whereas c.2881 C > T (p.Arg961*) and c.6409 C > T (p.Gln2137*) have been reported previously. Moreover, this study highlights the growing trend that ARSACS has become increasingly prevalent worldwide rather than being localized to a specific region or race. As an increasing number of patients with ARSACS are diagnosed, the genetic spectrum of ARSACS will gradually broaden, providing an accurate genetic basis for prenatal diagnosis of mothers in the years ahead, if possible.

Adolescent↗
PubMed2026

[Genetic and phenotypic analysis of three children with Neurodevelopmental disorders due to variants of DEAF1 gene].

OBJECTIVE: To explore the genetic characteristics and clinical phenotypes of three children with novel DEAF1 gene variants. METHODS: Three children who were referred to Capital Children's Medical Center Affiliated to Capital Medical University between January 2018 and December 2025 were selected as study subjects and underwent whole exome sequencing (WES). Candidate variants were verified by Sanger sequencing, and their pathogenicity was evaluated based on the guidelines from American College of Medical Genetics and Genomics (ACMG). A systematic search of databases including PubMed and CNKI was conducted to compile previously reported cases of DEAF1 variants for clinical phenotype comparison. For the non-canonical splice site variant c.870+5G>C located in the intronic region, wild-type and mutant minigene reporter vectors were constructed and transfected into HeLa and 293T cells, respectively, and the splicing patterns were analyzed by RT-PCR and Sanger sequencing. This study was approved by the Medical Ethics Committee of Capital Institute of Pediatrics (Ethics No.: SHERLL 2020001). RESULTS: All three children were found to have carried de novo heterozygous variants of the DEAF1 gene, including two missense variants (c.764G>A, c.641T>C) in the important SAND domain and a splice site variant (c.870+5G>C) in a non-canonical splicing region. The c.764G>A and c.870+5G>C variants were unreported previously. All children had presented with intellectual developmental delay, and two were accompanied by autism spectrum disorder, and two had epilepsy and sleep disorders. In vitro minigene splicing assay showed that the c.870+5G>C variant can lead to abnormal splicing. CONCLUSIONS: This study reported three children with novel DEAF1 variants, two of which have not been previously described, thereby enriched the mutational spectrum of the DEAF1 gene. In vitro functional assay combined with the clinical manifestations of the patients confirmed the pathogenicity of the non-canonical splice site variant in the intronic region.

Humans↗
PubMed2026

Simultaneous detection of glyphosate and glufosinate target-site resistance in Eleusine indica via multiplex TaqMan qPCR.

BACKGROUND: Continuous use of glyphosate followed by glufosinate-ammonium has selected for multiple resistance to both herbicides in Eleusine indica worldwide. Managing such resistant weeds requires fast, accurate molecular detection assay. To address this critical need, we developed a robust multiplex TaqMan quantitative (q)PCR assay that simultaneously detects five well-characterized target-site resistance markers in E. indica: EPSPS copy number variation; T102I in EPSPS; P106A and P106S in EPSPS; and S59G in GS1-1. RESULTS: The multiplex qPCR assay showed analytical specificity when tested on genomic DNA from nine reference accessions: three susceptible, three glyphosate-resistant (with EPSPS CNV) and three multiple-resistant. Subsequent analysis of 56 field-collected samples demonstrated 98.2% concordance (55 of 56) with Sanger sequencing across all five resistance-associated markers: EPSPS CNV, T102I, P106A, P106S and GS1-1 S59G, confirming the reliability and practical value of the multiplex qPCR assay. Only samples 7-8 showed discordance at EPSPS position 102, where Sanger chromatograms showed overlapping peaks at this position, which is likely to be a result of heterozygous mutation distribution among amplified EPSPS gene copies. This case further underscores the advantages of the multiplex qPCR assay over Sanger sequencing in detection sensitivity and accuracy. Moreover, a strong correlation (R2 = 0.8935) in gene copy number estimation between the two methods across all samples further supports the reliability of the qPCR assay. CONCLUSIONS: In summary, this study delivers a simple, robust and high-throughput diagnostic tool for the rapid, simultaneous identification of dual herbicide target-site resistance in goosegrass, offering superior sensitivity, quantitative resolution and throughput compared with Sanger sequencing. © 2026 Society of Chemical Industry.

Herbicides↗
PubMed2006

Genomic rearrangements in MSH2, MLH1 or MSH6 are rare in HNPCC patients carrying point mutations.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disease with high penetrance, caused by germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6, PMS2 and MLH3. Most reported pathogenic mutations are point mutations, comprising single base substitutions, small insertions and deletions. In addition, genomic rearrangements, such as large deletions and duplications not detectable by PCR and Sanger sequencing, have been identified in a significant proportion of HNPCC families, which do not carry a pathogenic MMR gene point mutation. To clarify whether genomic rearrangements in MLH1, MSH2 or MSH6 also occur in patients carrying a point mutation, we subjected normal tissue DNA of 137 colorectal cancer (CRC) patients to multiplex ligation-dependent probe amplification (MLPA) analysis. Patients fulfilled the following pre-requisites: all patients met at least one criterion of the Bethesda guidelines and their tumors exhibited high microsatellite instability (MSI-H) and/or showed loss of expression of MLH1, MSH2 or MSH6 proteins. PCR amplification and Sanger sequencing of all exons of at least one MMR gene, whose protein expression had been lost in the tumor tissue, identified 52 index patients without a point mutation (Group 1), 71 index patients with a pathogenic point mutation in MLH1 (n=38) or MSH2 (n=22) or MSH6 (n=11) (Group 2) and 14 patients with an unclassified variant in MLH1 (n=9) or MSH2 (n=3) or MSH6 (n=2) (Group 3). In 13 of 52 patients of group 1 deletions of at least one exon were identified. In addition, in group 3 one EX1_15del in MLH1 was found. No genomic rearrangement was identified in group 2 patients. Genomic rearrangements represent a significant proportion of pathogenic mutations of MMR genes in HNPCC patients. However, genomic rearrangements are rare in patients carrying point mutations in MMR genes. These findings suggest the use of genomic rearrangement tests in addition to Sanger sequencing in HNPCC patients.

Adaptor Proteins, Signal Transducing↗
PubMed2024

Variants in HCFC1 and MN1 genes causing intellectual disability in two Pakistani families.

BACKGROUND: Intellectual disability (ID) is a neurodevelopmental condition affecting around 2% of children and young adults worldwide, characterized by deficits in intellectual functioning and adaptive behavior. Genetic factors contribute to the development of ID phenotypes, including mutations and structural changes in chromosomes. Pathogenic variants in the HCFC1 gene cause X-linked mental retardation syndrome, also known as Siderius type X-linked mental retardation. The MN1 gene is necessary for palate development, and mutations in this gene result in a genetic condition called CEBALID syndrome. METHODS: Exome sequencing was used to identify the disease-causing variants in two affected families, A and B, from various regions of Pakistan. Affected individuals in these two families presented ID, developmental delay, and behavioral abnormalities. The validation and co-segregation analysis of the filtered variant was carried out using Sanger sequencing. RESULTS: In an X-linked family A, a novel hemizygous missense variant (c.5705G > A; p.Ser1902Asn) in the HCFC1 gene (NM_005334.3) was identified, while in family B exome sequencing revealed a heterozygous nonsense variant (c.3680 G > A; p. Trp1227Ter) in exon-1 of the MN1 gene (NM_032581.4). Sanger sequencing confirmed the segregation of these variants with ID in each family. CONCLUSIONS: The investigation of two Pakistani families revealed pathogenic genetic variants in the HCFC1 and MN1 genes, which cause ID and expand the mutational spectrum of these genes.

Humans↗
PubMed2026

[Genetic analysis of a male with Multiple morphological abnormalities of sperm flagella combined with sperm head abnormalities due to compound heterozygous variants of DNAH1 gene and a literature review].

OBJECTIVE: To explore the clinical phenotype and genetic etiology of a male with Multiple morphological abnormalities of sperm flagella (MMAF) combined with sperm head abnormalities due to compound heterozygous variants of DNAH1 gene, with an aim to provide guidance for assisted reproductive technology in his family. METHODS: A man with MMAF combined with sperm head abnormalities who visited Women and Children's Hospital of Ningbo University in October 2024 was selected as study subject. Clinical data of the patient's family were retrospectively collected. Peripheral blood samples were collected from the patient and his spouse, and G-banding karyotyping and whole exome sequencing (WES) were carried out. Candidate variants were validated by Sanger sequencing. Conservation of the DNAH1 protein was queried on the UCSC website. The difference between wild type and variant DNAH1 proteins were analyzed using AlphaFold v3.0.1 and PyMOL v2.5.6. The pathogenicity of variant was rated based on the guidelines from American College of Medical Genetics and Genomics (ACMG). Previous literature was searched using keywords "DNAH1 gene" and "multiple morphological abnormalities of the sperm flagella" on CNKI, Wanfang Data Knowledge Service Platform, and PubMed database to identify cases of MMAF attributed to biallelic DNAH1 gene variants. The retrieval period was set from the establishment of the databases to December 31, 2025. The genotypes and clinical phenotypes of patients with biallelic DNAH1 mutations were analyzed. This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: EC2023-094). RESULTS: The 30-year-old patient and his 30-year-old wife had infertility for 2 years. Semen analysis revealed no motile sperm and a 99.0% abnormal morphology rate. Typical MMAF was observed with phase-contrast microscopy. Sperm morphology analysis revealed abnormalities of the head, neck, and tail with an approximate ratio of 9:5:1. The patient's karyotype was 46,XY, and his wife's karyotype was 45,X[4]/47,XXX[1]/46,XX[84]. WES and Sanger sequencing revealed that the patient harbored compound heterozygous variants of the DNAH1 gene, namely c.1435_1444+3del and c.12204_12206del (p.Asn4069del), but their origin remained unidentified. UCSC genome browser query results showed that the amino acid residue at position 4 069 of the DNAH1 protein is highly conserved across various species. Protein structure prediction reveals that, in the wild-type DNAH1 protein, the Asparagine at position 4 069 (Asn4069) can form hydrogen bonds with the Leucine on the main chain at position 4 086 (Leu4086) and the Serine on the side chain at position 4 087 (Ser4087). The c.12204_12206del variant, resulting in deletion of Asn4069, disrupts these hydrogen bonds and does not generate any compensatory interactions. Based on the ACMG guidelines, the c.1435_1444+3del variant was predicted to be likely pathogenic (PM2_Supporting+PVS1), and the c.12204_12206del(p.Asn4069del) variant was rated as likely pathogenic (PM2_Supporting+PM4+PM3+PP4). The couple had elected for in vitro fertilization using donor sperm. During this cycle, 12 oocytes were retrieved, 10 oocytes were successfully fertilized, 1 embryo and 6 blastocysts were obtained. Following the first transfer of a frozen-thawed blastocyst, implantation of an empty gestational sac occurred, which led to a miscarriage. After the second transfer of a high-quality blastocyst, the embryo split into twins following implantation, and the spouse had selected fetal reduction. The gestational age was 33+3 weeks on June 1, 2026. Literature review identified three studies reporting biallelic mutations of the DNAH1 gene in association with MMAF combined with sperm head abnormalities. Together with the patient from this study, a total of 20 patients were included in the analysis. The rate of sperm flagellar abnormalities in these patients was above 80.0%, while the rate of sperm head abnormalities has ranged from 12.0% to 100.0%. In four patients, the genetic basis was unknown. In the remaining 16 patients, 35 mutations were detected, with c.8626-1G>A being the most common (22.9%, 8/35). CONCLUSION: This patient showed MMAF with frequent sperm head defects. Compound heterozygous variants of the DNAH1 gene probably underlay these abnormalities, which in turn has led to his primary infertility. This study revealed the phenotypic variability of MMAF and broadened the mutational spectrum of the DNAH1 gene.

Humans↗
PubMed2024

Identification of four TTN variants in three families with fetal akinesia deformation sequence.

BACKGROUND: TTN is a complex gene with large genomic size and highly repetitive structure. Pathogenic variants in TTN have been reported to cause a range of skeletal muscle and cardiac disorders. Homozygous or compound heterozygous mutations tend to cause a wide spectrum of phenotypes with congenital or childhood onset. The onset and severity of the features were considered to be correlated with the types and location of the TTN variants. METHODS: Whole-exome sequencing was performed on three unrelated families presenting with fetal akinesia deformation sequence (FADS), mainly characterized by reduced fetal movements and limb contractures. Sanger sequencing was performed to confirm the variants. RT-PCR analysis was performed. RESULTS: TTN c.38,876-2 A > C, a meta transcript-only variant, with a second pathogenic or likely pathogenic variant in trans, was observed in five affected fetuses from the three families. Sanger sequencing showed that all the fetal variants were inherited from the parents. RT-PCR analysis showed two kinds of abnormal splicing, including intron 199 extension and skipping of 8 bases. CONCLUSIONS: Here we report on three unrelated families presenting with FADS caused by four TTN variants. In addition, our study demonstrates that pathogenic meta transcript-only TTN variant can lead to defects which is recognizable prenatally in a recessive manner.

Humans↗