Splice Donor Site Of Exon Research Articles

Identification of a novel pathogenic XPC:c.2420 + 1 G>C variant in a patient with xeroderma pigmentosum.

Identification of a novel pathogenic XPC:c.2420 + 1 G>C variant in a patient with xeroderma pigmentosum.

A homozygous LAMB3 frameshift variant in junctional epidermolysis bullosa-affected Bleu du Maine sheep.

Epidermolysis bullosa (EB) is a group of inherited skin disorders characterized by skin fragility and blistering. Here, four Bleu du Maine lambs, sired by one ram, were diagnosed with EB very early in life. Due to the severity of the clinical signs, the lambs had to be euthanized. The affected lambs exhibited hoof sloughing and multiple ulcerations on the head, oral cavity, skin over the joints, and the ruminal pillars. Histopathology showed abrupt subepidermal clefts, epidermal detachment directly above the basal membrane, and ulcerations consistent with junctional EB (JEB). Two cases underwent whole-genome sequencing (WGS) to identify the genetic cause. Genomic analyses with the hypothesis of autosomal recessive inheritance identified the most likely pathogenic homozygous 1-bp deletion in the LAMB3 gene (NC_056065.1:g.73166198delG). Recessive forms of JEB in humans and dogs are caused by variants in LAMB3 gene, which encodes β3 subunit of laminin 332, a critical component of the epidermal basal membrane. The ovine frameshift variant putatively introduces a premature stop codon and disrupts the donor splice site of exon 20. The variant allele was homozygous in both sequenced cases and heterozygous in three unaffected close relatives and was absent in 1075 unrelated control sheep of various other breeds. This study highlights the importance of genetic investigation in veterinary diagnostics of and represents the first report of a LAMB3-related recessive EB in sheep. The findings enable genetic testing to inform breeding strategies and provide a second spontaneous large animal model for LAMB3-related JEB in humans.

Retinal Dystrophy Associated with Homozygous Variants in NRL.

Background/Objectives: Neural retina leucine zipper (NRL) is a transcription factor involved in the differentiation of rod photoreceptors. Pathogenic variants in the gene encoding NRL have been associated with autosomal dominant retinitis pigmentosa and autosomal recessive clumped pigmentary retinal degeneration. Only a dozen unrelated families affected by recessive NRL-related retinal dystrophy have been described. The purpose of this study was to expand the genotypic spectrum of this disease by reporting clinical and genetic findings of two unrelated families. Methods: Index patients affected by retinal dystrophy were genetically tested by whole-exome sequencing (WES) and whole-genome sequencing (WGS). Segregation analysis within the families was performed for candidate variants. A minigene assay was performed to functionally characterize a variant suspected to affect splicing. Results: Variant filtering revealed homozygous NRL variants in both families. The variant in patient A was a small deletion encompassing the donor splice site of exon 1 of transcript NM_006177.3. The minigene assay revealed that this variant led to two aberrant transcripts that used alternative cryptic donor splice sites located in intron 1. In patient B, a stop-gain variant was identified in the last exon of NRL in a homozygous state due to maternal uniparental disomy of chromosome 14. Conclusions: Our study expands the genotypic spectrum of autosomal recessive NRL-related retinal dystrophy. Moreover, it underscores the importance of actively maintaining bioinformatic pipelines for variant detection and the utility of minigene assays in functionally characterizing candidate splicing variants.

Neonatal-onset pulmonary alveolar proteinosis is a phenotype associated with poor outcomes in surfactant protein-C disorder

Neonatal-onset pulmonary alveolar proteinosis is a phenotype associated with poor outcomes in surfactant protein-C disorder

Early-Onset Aortic Dissection: Characterization of a New Pathogenic Splicing Variation in the MYH11 Gene with Several In-Frame Abnormal Transcripts.

Rare pathogenic variants in the MYH11 gene are responsible for thoracic aortic aneurysms and dissections. They are usually heterozygous missense variants or in-frame deletions of several amino acids without alteration of the reading frame and mainly affect the coiled-coil domain of the protein. Variants leading to a premature stop codon have been described in patients with another phenotype, megacystis-microcolon-intestinal hypoperistalsis syndrome, with an autosomal recessive inheritance. The physiopathological mechanisms arising from the different genetic alterations affecting the MYH11 gene are still poorly understood. Consequently, variants of unknown significance are relatively frequent in this gene. We have identified a variant affecting the consensus donor splice site of exon 29 in the MYH11 gene in a patient who suddenly died from an aortic type A dissection at the age of 23 years old. A transcript analysis on cultured fibroblasts has highlighted several abnormal transcripts including two in-frame transcripts. The first one is a deletion of the last 78 nucleotides of exon 29, corresponding to the use of a cryptic alternative donor splice site; the second one corresponds to an exon 29 skipping. Familial screening has revealed that this molecular event occurred de novo in the proband. Taken together, these experiments allowed us to classify this variant as pathogenic. This case underlines the challenging aspect of the discovery of variations in the MYH11 gene for which the consequences on splicing should be systematically studied in detail.

Prime editing strategies to mediate exon skipping in DMD gene.

Duchenne muscular dystrophy is a rare and lethal hereditary disease responsible for progressive muscle wasting due to mutations in the DMD gene. We used the CRISPR-Cas9 Prime editing technology to develop different strategies to correct frameshift mutations in DMD gene carrying the deletion of exon 52 or exons 45 to 52. With optimized epegRNAs, we were able to induce the specific substitution of the GT nucleotides of the splice donor site of exon 53 in up to 32% of HEK293T cells and 28% of patient myoblasts. We also achieved up to 44% and 29% deletion of the G nucleotide of the GT splice site of exon 53, as well as inserted 17% and 5.5% GGG between the GT splice donor site of exon 51 in HEK293T cells and human myoblasts, respectively. The modification of the splice donor site for exon 51 and exon 53 provoke their skipping and allowed exon 50 to connect to exon 53 and allowed exon 44 to connect to exon 54, respectively. These corrections restored the expression of dystrophin as demonstrated by western blot. Thus, Prime editing was used to induce specific substitutions, insertions and deletions in the splice donor sites for exons 51 and 53 to correct the frameshift mutations in DMD gene carrying deletions of exon 52 and exons 45 to 52, respectively.

An intronic variant in TBX4 in a single family with variable and severe pulmonary manifestations

A male infant presented at term with neonatal respiratory failure and pulmonary hypertension. His respiratory symptoms improved initially, but he exhibited a biphasic clinical course, re-presenting at 15 months of age with tachypnea, interstitial lung disease, and progressive pulmonary hypertension. We identified an intronic TBX4 gene variant in close proximity to the canonical donor splice site of exon 3 (hg 19; chr17:59543302; c.401 + 3 A > T), also carried by his father who had a typical TBX4-associated skeletal phenotype and mild pulmonary hypertension, and by his deceased sister who died shortly after birth of acinar dysplasia. Analysis of patient-derived cells demonstrated a significant reduction in TBX4 expression resulting from this intronic variant. Our study illustrates the variable expressivity in cardiopulmonary phenotype conferred by TBX4 mutation and the utility of genetic diagnostics in enabling accurate identification and classification of more subtly affected family members.

Identification of novel compound heterozygous variants in the SLC30A7 (ZNT7) gene in two French brothers with stunted growth, testicular hypoplasia and bone marrow failure.

Zinc is an essential trace mineral. Dietary zinc deficiency results in stunted growth, skin lesions, hypogonadism and frequent infections in humans. Mice genetically lacking Slc30a7 suffer from mild zinc deficiency and are prone to development of prostate cancer and insulin resistance. Disease-causing variants or mutations in the human SLC30A7 (ZNT7) gene have not been previously reported. Here, we describe two-boy siblings from a French family with stunted growth, testicular hypoplasia and bone marrow failure. Exome sequencing revealed compound heterozygous variants in ZNT7 consisting of NM_133496.5:c.21dup; p.Asp8ArgfsTer3 and c.842 + 15T > C inherited from their unaffected mother and father, respectively. The c.21dup variant led to a premature stop codon generated in exon 1 of the ZNT7 coding sequence. RNA-seq analysis demonstrated that the c.842 + 15T > C variant resulted in a leaky mRNA splicing event generating a premature stop codon right after the splicing donor site of exon 8. Moreover, the expression of ZNT7 protein was remarkably reduced by 80-96% in the affected brothers compared to the control cells. These findings strongly suggest that biallelic variants in SLC30A7 should be considered as a cause of growth retardation, testicular hypoplasia and syndromic bone marrow failure.

Abstract C041: FAN1 splice variant as a possible risk variant for African American hereditary breast cancer and understanding its role in tumorigenesis

Abstract Recognizing African American health and research disparities, there is a need to better understand African American breast cancer risk. Our group has established the Alabama Hereditary Cancer Cohort to aid in identifying risk variants associated with African American hereditary breast cancer. We have identified several suspected risk variants using next-generation sequencing technologies, including a low-frequency African American-specific splicing variant in FANCD2/FANCI-associated nuclease 1 (FAN1). FAN1 is an exo/endonuclease involved in DNA inter-strand crosslinking repair. There has been speculation that FAN1 could be a breast cancer susceptibility gene because FAN1 is in the Fanconi Anemia pathway with known breast cancer susceptibility genes, BRCA1 and BRCA2. This splicing variant, FAN1 c.3057+1G>A, does not interfere with the coded protein, but alters the donor splice site of exon 14, adding 34 bases to the beginning of the 3’ untranslated region. Interestingly, it has an allele frequency of 3.6% in the general African American population but was found in 5.1% of African American breast cancer cases in the Alabama Hereditary Cancer Cohort. Extremely rare in European Americans overall; it was not detected in any European American breast cancer cases. Functionalizing FAN1 c.3057+1G>A and other identified mutations is important to demonstrate pathogenicity. Our group uses prime editing to introduce variants of interest into selected cell lines. Prime editing is an innovative gene-editing technique that can introduce base substitutions, insertions, and deletions into the genome without the need for double-stranded DNA breaks or donor DNA. Upon gene-editing, cell proliferation, invasion, migration, and other cellular characteristics that contribute to tumorigenesis are investigated. Overall, it is imperative to identify African-American breast cancer risk variants and understand how they play a role in disease pathogenesis, leading to better risk assessment and novel therapeutics. Citation Format: Sheniqua R. Glover, Cierla McGuire Sams, Madison Bishop, Troy LoBue, Issac Mcneely, Nancy D. Merner. FAN1 splice variant as a possible risk variant for African American hereditary breast cancer and understanding its role in tumorigenesis [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C041.

The Protection Conferred by HSD17B13 rs72613567 on Hepatic Fibrosis Is Likely Mediated by Lowering Ballooning and Portal Inflammation

The Protection Conferred by HSD17B13 rs72613567 on Hepatic Fibrosis Is Likely Mediated by Lowering Ballooning and Portal Inflammation

PMON270 Coexistence of 46, XX Testicular Disorder of Sex Development and 11β-Hydroxylase Deficiency: In Vivo and In Vitro Studies

Abstract Background 46, XX testicular disorder of sex development and 11β-hydroxylase deficiency (11β-OHD) are two extremely rare types of disorder of sex development (DSD). No coexistence has been reported yet. Clinical Case A 22-year-old male showed small testes (volume=4ml) and gynecomastia for 2 years. At the age of 3.5, he was diagnosed as simple virilizing type of 21-hydroxylase deficiency (21-OHD) for the manifestations of the enlarged penis (length=7.5cm), hyperpigmentation, and normal blood pressure. Adrenal ultrasound suggested bilateral adrenal hyperplasia (both length * width = 2.4*0.9cm) and X-ray showed accelerated bone age (12.5 years old). Irregular cortisone acetate 6.25mg per day was administrated since then. At the age of 18, he was sent to emergency for aortic aneurysm rupture and heart failure, and hypertension urgency was diagnosed. Sanger sequencing revealed that he carried compound heterozygous variants in CYP11B1 gene: NM_000497.3: c.905_907delinsTT and NM_000497.3: c.954+7C>T, which were respectively inherited from his father and mother. Pathogenesis of the novel intron variant (c.954+7C>T) was further verified in COS7, CHO, and 273T cell lines by transfecting wild-type and mutant-type minigene, extracting RNA of cells, reversing RNA into cDNA, PCR, and Sanger sequencing. The result showed that the c.954+7C>T variant introduced a cryptic donor splice site at a site 5 bp downstream of the 3' natural splice donor site of exon 5. The level of adrenal steroids detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was consistent with the characteristics of 11β-OHD. The patient was treated by neoplasty of thoracic aortic aneurysm and added anti-hypertension drugs in addition to glucocorticoid after surgery. Unexpectedly, the abnormalities of male characteristics including small testes and gynecomastia appeared since adolescence. Laboratory tests showed an elevated level of FSH and LH and azoospermia. Scrotum Ultrasound showed bilateral testicular dysplasia (right: 1.9*1.4*0.8cm, left: 1.9*1.3*0.8cm). To figure out the unexplainable results, whole-exome sequencing was performed and revealed two copy number variants: the duplication of Xp22.33-q28 which spans 151.32Mb, and the deletion of Yp11.2-q11.23 which spans 23.34Mb. Meanwhile, the karyotype test by culturing blood lymphocytes showed 46, XX (100/100), which confirmed the diagnosis of 46, XX testicular DSD. Fluorescence in situ hybridization (FISH) revealed the presence of the SRY gene, which is translocated into the short arm of the X chromosome. Testosterone replacement is unnecessary for the normal level of testosterone (T) (4.44ng/ml, N: 1.75-7.81ng/ml), which is the result of the combined action of 11β-OHD with adrenal T accumulation and 46, XX testicular DSD with testicular T deficiency. Assisted reproduction consulting should start early. Conclusion This is the first case demonstrating the possibility of coexistence of 46, XX testicular DSD and 11β-OHD and hinting at the significance of a combination of clinical and molecular diagnostic methods. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Next-Generation Sequencing Advances the Genetic Diagnosis of Cerebral Cavernous Malformation (CCM).

Cerebral Cavernous Malformation (CCM) is a cerebrovascular disease of genetic origin that predisposes to seizures, focal neurological deficits and fatal intracerebral hemorrhage. It may occur sporadically or in familial forms, segregating as an autosomal dominant condition with incomplete penetrance and highly variable expressivity. Its pathogenesis has been associated with loss-of-function mutations in three genes, namely KRIT1 (CCM1), CCM2 and PDCD10 (CCM3), which are implicated in defense mechanisms against oxidative stress and inflammation. Herein, we screened 21 Italian CCM cases using clinical exome sequencing and found six cases (~29%) with pathogenic variants in CCM genes, including a large 145–256 kb genomic deletion spanning the KRIT1 gene and flanking regions, and the KRIT1 c.1664C>T variant, which we demonstrated to activate a donor splice site in exon 16. The segregation of this cryptic splicing mutation was studied in a large Italian family (five affected and seven unaffected cases), and showed a largely heterogeneous clinical presentation, suggesting the implication of genetic modifiers. Moreover, by analyzing ad hoc gene panels, including a virtual panel of 23 cerebrovascular disease-related genes (Cerebro panel), we found two variants in NOTCH3 and PTEN genes, which could contribute to the abnormal oxidative stress and inflammatory responses to date implicated in CCM disease pathogenesis.

First Identification of Rare Exonic and Deep Intronic Splice-Altering Variants in Patients With Beta-Sarcoglycanopathy.

BackgroundThe precise genetic diagnosis of a sarcoglycanopathy or dystrophinopathy is sometimes extremely challenging, as pathogenic non-coding variants and/or complex structural variants do exist in DMD or sarcoglycan genes. This study aimed to determine the genetic diagnosis of three patients from two unrelated families with a suspected sarcoglycanopathy or dystrophinopathy based on their clinical, radiological, and pathological features, for whom routine genomic detection approaches failed to yield a definite genetic diagnosis.MethodsMuscle-derived reverse transcription-polymerase chain reaction analysis and/or TA cloning of DMD, SGCA, SGCB, SGCD, and SGCG mRNA were performed to identify aberrant transcripts. Genomic Sanger sequencing around the aberrant transcripts was performed to detect possible splice-altering variants. Bioinformatic and segregation studies of the detected genomic variants were performed in both families.ResultsIn patients F1-II1 and F1-II2, we identified two novel pathogenic compound heterozygous variants in SGCB. One is a deep intronic splice-altering variant (DISV), c.243 + 1558C > T in intron 2 causing the activation of an 87-base pair (bp) pseudoexon, and the other one is a non-canonical splicing site variant, c.243 + 6T > A leading to the partial intron inclusion of 10-bp sequence. A novel DISV, c.243 + 1576C > G causing a 106-bp pseudoexon activation, and a nonsense variant in SGCB were identified in compound heterozygous state in patient F2-II1. Unexpectedly, the predicted nonsense variant, c.334C > T in exon 3, created a new donor splice site in exon 3 that was stronger than the natural one, resulting in a 97-bp deletion of exon 3 (r.333_429del).ConclusionThis is the first identification of rare exonic and DISVs in the SGCB gene.

Function alteration of engineered heart tissues carrying a shorter filamin C protein induced by CRISPR/Cas9

Function alteration of engineered heart tissues carrying a shorter filamin C protein induced by CRISPR/Cas9

Chronic Granulomatous Disease-Like Presentation of a Child with Autosomal Recessive PKCδ Deficiency

BackgroundAutosomal recessive (AR) PKCδ deficiency is a rare inborn error of immunity (IEI) characterized by autoimmunity and susceptibility to bacterial, fungal, and viral infections. PKCδ is involved in the intracellular production of reactive oxidative species (ROS).Material and MethodsWe studied a 5-year old girl presenting with a history of Burkholderia cepacia infection. She had no history of autoimmunity, lymphocyte counts were normal, and no auto-antibodies were detected in her plasma. We performed a targeted panel analysis of 407 immunity-related genes and immunological investigations of the underlying genetic condition in this patient.ResultsConsistent with a history suggestive of chronic granulomatous disease (CGD), oxidative burst impairment was observed in the patient’s circulating phagocytes in a dihydrorhodamine 123 (DHR) assay. However, targeted genetic panel analysis identified no candidate variants of known CGD-causing genes. Two heterozygous candidate variants were detected in PRKCD: c.285C > A (p.C95*) and c.376G > T (p.D126Y). The missense variant was also predicted to cause abnormal splicing, as it is located at the splice donor site of exon 5. TOPO-TA cloning confirmed that exon 5 was completely skipped, resulting in a truncated protein. No PKCδ protein was detected in the patient’s neutrophils and monocyte-derived macrophages. The monocyte-derived macrophages of the patient produced abnormally low levels of ROS, as shown in an Amplex Red assay.ConclusionPKCδ deficiency should be considered in young patients with CGD-like clinical manifestations and abnormal DHR assay results, even in the absence of clinical and biological manifestations of autoimmunity.

Isolated Aldosterone Deficiency Due to an Aldosterone Synthase Mutation: A Rare Cause of Life-Threatening Salt Wasting in Infancy

Purpose: Isolated hypoaldosteronism is a rare cause of salt wasting in infancy caused by the loss of activity of aldosterone synthase (AS), which is encoded by the CYP11B2 gene. This condition may be life-threatening, especially in newborns. Methods: We are reporting the case of a newborn male born to consanguineous parents, who presented at the age of 1 month with severe dehydration, vomiting, and hypotonia. Results: Lab studies showed severe hyponatremia, hyperkalemia, and metabolic acidosis. Hormonal assessment confirmed a normal corticotrope axis with severe deficiency in aldosterone. Genetic analysis revealed a novel mutation in the CYP11B2 gene in the highly conserved donor splice site of exon 3 and confirmed the isolated aldosterone deficiency. The patient was subsequently treated with fludrocortisone and sodium only, without hydrocortisone. Conclusion: AS deficiency is a rare inherited disease. There should be a high index of suspicion of cases with life-threatening salt-wasting in infancy as dramatic clinical improvements and good long-term prognosis can be achieved with timely replacement treatment.

Validation of DE50-MD dogs as a model for the brain phenotype of Duchenne muscular dystrophy.

ABSTRACTDuchenne muscular dystrophy (DMD), a fatal musculoskeletal disease, is associated with neurodevelopmental disorders and cognitive impairment caused by brain dystrophin deficiency. Dog models of DMD represent key translational tools to study dystrophin biology and to develop novel therapeutics. However, characterisation of dystrophin expression and function in the canine brain is lacking. We studied the DE50-MD canine model of DMD that has a missense mutation in the donor splice site of exon 50. Using a battery of cognitive tests, we detected a neurocognitive phenotype in DE50-MD dogs, including reduced attention, problem solving and exploration of novel objects. Through a combination of capillary immunoelectrophoresis, immunolabelling, quantitative PCR and RNAScope in situ hybridisation, we show that regional dystrophin expression in the adult canine brain reflects that of humans, and that the DE50-MD dog lacks full-length dystrophin (Dp427) protein expression but retains expression of the two shorter brain-expressed isoforms, Dp140 and Dp71. Thus, the DE50-MD dog is a translationally relevant pre-clinical model to study the consequences of Dp427 deficiency in the brain and to develop therapeutic strategies for the neurological sequelae of DMD.

Complete CSN1S2 Characterization, Novel Allele Identification and Association With Milk Fatty Acid Composition in River Buffalo.

The αs2-casein is one of the phosphoproteins secreted in all ruminants' milk, and it is the most hydrophilic of all caseins. However, this important gene (CSN1S2) has not been characterized in detail in buffaloes with only two alleles detected (reported as alleles A and B), and no association studies with milk traits have been carried out unlike what has been achieved for other species of ruminants. In this study, we sequenced the whole gene of two Mediterranean river buffalo homozygotes for the presence/absence of the nucleotide C (g.7539G>C) realized at the donor splice site of exon 7 and, therefore, responsible for the skipping of the same exon at mRNA level (allele B). A high genetic variability was found all over the two sequenced CSN1S2 alleles. In particular, 74 polymorphic sites were found in introns, six in the promoter, and three SNPs in the coding region (g.11072C>T, g.12803A>T, and g.14067A>G) with two of them responsible for amino acid replacements. Considering this genetic diversity, those found in the database and the SNP at the donor splice site of exon 7, it is possible to deduce at least eight different alleles (CSN1S2 A, B, B1, B2, C, D, E, and F) responsible for seven different possible translations of the buffalo αs2-casein. Haplotype data analysis suggests an evolutionary pathway of buffalo CSN1S2 gene consistent with our proposal that the published allele CSN1S2 A is the ancestral αs2-CN form, and the B2 probably arises from interallelic recombination (single crossing) between the alleles D and B (or B1). The allele CSN1S2 C is of new identification, while CSN1S2 B, B1, and B2 are deleted alleles because all are characterized by the mutation g.7539G>C. Two SNPs (g.7539G>C and g.14067A>G) were genotyped in 747 Italian buffaloes, and major alleles had a relative frequency of 0.83 and 0.51, respectively. An association study between these SNPs and milk traits including fatty acid composition was carried out. The SNP g.14067A>G showed a significant association (P < 0.05) on the content of palmitic acid in buffalo milk, thus suggesting its use in marker-assisted selection programs aiming for the improvement of buffalo milk fatty acid composition.

New recessive mutations in SYT2 causing severe presynaptic congenital myasthenic syndromes.

ObjectiveTo report the identification of 2 new homozygous recessive mutations in the synaptotagmin 2 (SYT2) gene as the genetic cause of severe and early presynaptic forms of congenital myasthenic syndromes (CMSs).MethodsNext-generation sequencing identified new homozygous intronic and frameshift mutations in the SYT2 gene as a likely cause of presynaptic CMS. We describe the clinical and electromyographic patient phenotypes, perform ex vivo splicing analyses to characterize the effect of the intronic mutation on exon splicing, and analyze the functional impact of this variation at the neuromuscular junction (NMJ).ResultsThe 2 infants presented a similar clinical phenotype evoking first a congenital myopathy characterized by muscle weakness and hypotonia. Next-generation sequencing allowed to the identification of 1 homozygous intronic mutation c.465+1G>A in patient 1 and another homozygous frameshift mutation c.328_331dup in patient 2, located respectively in the 5′ splice donor site of SYT2 intron 4 and in exon 3. Functional studies of the intronic mutation validated the abolition of the splice donor site of exon 4 leading to its skipping. In-frame skipping of exon 4 that encodes part of the C2A calcium-binding domain of SYT2 is associated with a loss-of-function effect resulting in a decrease of neurotransmitter release and severe pre- and postsynaptic NMJ defects.ConclusionsThis study identifies new homozygous recessive SYT2 mutations as the underlying cause of severe and early presynaptic form of CMS expanding the genetic spectrum of recessive SYT2-related CMS associated with defects in neurotransmitter release.

Comparative Functional Analysis in vitro of 2 COL4A5 Splicing Mutations at the Same Site in 2 Unrelated Alport Syndrome Chinese Families

X-linked Alport syndrome (XLAS) is a common hereditary nephropathy caused by COL4A5 gene mutations. To date, many splice site mutations have been described but few have been functionally analyzed to verify the exact splicing effects that contribute to disease pathogenesis. Here, we accidentally discovered 2 COL4A5 gene splicing mutations affecting the same residue (c.2917+1G>A and c.2917+1G>C) in 2 unrelated Chinese families. In vitro minigene assays showed that the 2 mutations produced 3 transcripts in H293T cells: one with a 96-bp deletion in exon 33, one with exon 33 skipping, and one with exon 33-34 skipping. However, fragment analysis results showed that the main splicing effects of the 2 mutations were different, the c.2917+1G>A mutation mainly activated a cryptic donor splice site in exon 33 and resulted in the deletion of 96 bp in exon 33, while the c.2917+1G>C mutation mainly caused exon 33 skipping. Our findings indicate that different nucleotide substitutions at the same residue can cause different splicing effects, which may contribute to the variable phenotype of Alport syndrome.