Frameshift Variant Research Articles

Clinical characteristics and genetic analysis of a Chinese pedigree affected with tuberous sclerosis complex

To explore the genetic basis for a Chinese pedigree affected with tuberous sclerosis complex (TSC). The TSC1 and TSC2 genes were sequenced. Candidate variant was verified by Sanger sequencing of the proband and her family members. Pathogenicity of the variant was predicted based on the American College of Medical Genetics and Genomics (ACMG) guidelines. The proband was found to harbor a heterozygous c.52delC frameshift variant of the TSC2 gene, which may result in synthesis of amino acid chain starting from the 18th amino acid Leu and terminating at the 28th amino acid (p.Leu18CysfsTer28). The variant was unreported in the public database. Mutation Taster software predicted that the variant is harmful. Both parents of the proband were of the wild type, suggesting that the variant has occurred de novo. Based on the ACMG guidelines, the variant was predicted to be likely pathogenic (PVS1 +PM2). A novel pathogenic variant of the TSC2 gene c.52delC (p.Leu18CysfsTer28) was identified, which has enriched the mutational spectrum of TSC2 and provided a basis for genetic counseling for this pedigree.

Biallelic TUFT1 variants cause woolly hair, superficial skin fragility and desmosomal defects.

Desmosomes are complex cell junction structures that connect intermediate filaments providing strong cell-to-cell adhesion in tissues exposed to mechanical stress. To identify causal variants in individuals with woolly hair and skin fragility of unknown genetic cause. This research was conducted using whole-genome sequencing, whole-exome sequencing, clinical phenotyping, haplotype analysis, single-cell RNA sequencing data analysis, immunofluorescence microscopy and transmission electron microscopy. We identified homozygous predicted loss-of-function tuftelin-1 (TUFT1) variants in nine individuals, from three families, with woolly hair and skin fragility. One donor splice-site variant, c.60+1G>A, was present in two families, while a frameshift variant, p.Gln189Asnfs*49, was found in the third family. Haplotype analysis showed the c.60+1G>A substitution to be a founder variant in the Irish population that likely arose approximately 20 generations ago. Human and mouse single-cell RNA sequencing data showed TUFT1 expression to be enriched in the hair dermal sheath and keratinocytes. TUFT1 expression was highly correlated with genes encoding desmosomal components implicated in diseases with phenotypes that overlap with the cohort presented here. Immunofluorescence showed tuftelin-1 to be mainly localized to the peripheral cell membranes of keratinocytes in normal skin. Skin samples from individuals with TUFT1 variants showed markedly reduced immunoreactivity for tuftelin-1, with a loss of the keratinocyte cell membrane labelling. Light microscopy revealed keratinocyte adhesion, mild hyperkeratosis and areas of superficial peeling. Transmission electron microscopy showed panepidermal acantholysis with widening of intercellular spaces throughout the epidermis and desmosomal detachment through the inner plaques. Biallelic loss-of-function TUFT1 variants cause a new autosomal recessive skin/hair disorder characterized by woolly hair texture and early-onset skin fragility. Tuftelin-1 has a role in desmosomal integrity and function.

Molecular Genetics of GLUT1DS Italian Pediatric Cohort: 10 Novel Disease-Related Variants and Structural Analysis.

GLUT1 deficiency syndrome (GLUT1DS1; OMIM #606777) is a rare genetic metabolic disease, characterized by infantile-onset epileptic encephalopathy, global developmental delay, progressive microcephaly, and movement disorders (e.g., spasticity and dystonia). It is caused by heterozygous mutations in the SLC2A1 gene, which encodes the GLUT1 protein, a glucose transporter across the blood-brain barrier (BBB). Most commonly, these variants arise de novo resulting in sporadic cases, although several familial cases with AD inheritance pattern have been described. Twenty-seven Italian pediatric patients, clinically suspect of GLUT1DS from both sporadic and familial cases, have been enrolled. We detected by trios sequencing analysis 25 different variants causing GLUT1DS. Of these, 40% of the identified variants (10 out of 25) had never been reported before, including missense, frameshift, and splice site variants. Their structural mapping on the X-ray structure of GLUT1 strongly suggested the potential pathogenic effects of these novel disease-related mutations, broadening the genotypic spectrum heterogeneity found in the SLC2A1 gene. Moreover, 24% is located in a vulnerable region of the GLUT1 protein that involves transmembrane 4 and 5 helices encoded by exon 4, confirming a mutational hotspot in the SLC2A1 gene. Lastly, we investigated possible correlations between mutation type and clinical and biochemical data observed in our GLUT1DS cohort, revealing that splice site and frameshift variants are related to a more severe phenotype and low CSF parameters.

93. Surveying the genomic landscape of Mantle Cell Lymphoma

Mantle Cell Lymphoma (MCL) is a B-cell non-Hodgkin Lymphoma that currently has poor prognosis and few effective treatments. This project was intended to explore the variants that characterize MCL, and potentially identify targets for novel treatments. The patient cohort included 28 individuals who contributed lymph node biopsies and matched skin normal samples. All 28 patients had whole exome sequencing (WES), 10 had whole genome sequencing (WGS), and 8 had RNA sequencing. We used established in-house DNA and RNA analysis pipelines to detect single-nucleotide variants and indels, structural variants, copy number alterations, and RNA fusions. The canonical t(11;14) translocation between CCND1 and IGH was detected in 8 of 10 WGS samples via structural variant analysis. The most recurrently mutated genes include CCND1 (11/27), IGH (9/27), ATM (8/27), TP53 (6/27), IGK (5/27), and KMT2A (4/27). In addition to the t(11;14) translocation, CCND1 also had missense variants in four WES samples. Another gene of interest was ATM due to its diversity of variant types, which included missense variants, frameshift variants, deletions, and duplications. The most recurrent WGS copy number events were amplifications at 7q22.1 (9/10), and deletions at 21q22.3 (8/10) and 11q22.3 (6/10). The most common RNA fusion was a HNRNPU-CDC42EP4 fusion (2/8), and HNRNPU expression has previously been implicated in cancer progression. The results of this study will potentially identify genetic targets for MCL treatment and further integrate genomic and transcriptomic analyses of MCL.

LBODP032 Case Report Of A 13 Years Old Female With A Novel Pathogenic Variant In The Hnf1b Gene

Abstract Background MODY5 results from mutations in the gene encoding hepatocyte nuclear factor 1 homeobox B (HNF1B), a transcription factor critical in development of the pancreas, liver, intestines, and the urogenital tract (1). We report the case of a pediatric patient with a novel frameshift variant (p. Phe309SerfsTer18) in the HNF1B gene accounting for the co-occurrence of diabetes mellitus and structural renal abnormalities. Clinical case: A 13-years-old female with chronic kidney disease stage 3 (eGFR = 48 ml/min/1.73m2) secondary to congenital dysplastic-appearing left kidney and surgical removal of a cystic dysplastic right kidney was referred to our diabetes clinic for hyperglycemia. She reported long-standing polyuria and polydipsia previously attributed to her kidney disease. She required laparoscopic right nephrectomy at age 5 years due to increasing cyst size. Her left kidney also has small renal parenchymal cysts which have been monitored over time. There is no known family history of kidney disease, and grandparents have a history of diabetes. Laboratory investigation revealed a random blood glucose of 478 mg/dL and a hemoglobin A1c of 12.2%. She was not in diabetic ketoacidosis. She had a detectable insulin level of 5.1 mcIU/mL and C-peptide was 3.4 ng/mL. She was initially presumed to have Type 1 diabetes based on a relatively low insulin level and started on basal-bolus insulin (up to 0.59 units/kg of weight/day). Glycosylated hemoglobin was 8.7% one month after diagnosis. Glutamic acid decarboxylase, zinc transporter, islet cell, and insulin autoantibodies were not detected, however, and she was then suspected to have either Type 2 diabetes or MODY. Approximately one year later, she was taken off insulin due to hypoglycemia and managed only with metformin 500 mg twice daily. Next-generation sequencing revealed a de novo pathogenic variant in HNF1B causing a frameshift deletion (p. Phe309SerfsTer18). Genetic testing of parents was negative. Once her diagnosis of MODY5 was made, she was started on glipizide 2.5 mg once daily which has been titrated up to a dose of 5 mg with some success. However, she has since been re-started on insulin (up to 0.2 units/kg/day) due to hyperglycemia keeping in line with the MODY5 phenotype likely causing destruction of beta cells over time (1). Conclusion We report a case of MODY5 with a novel frameshift mutation (p. Phe309SerfsTer18) in the HNF1B gene. This case highlights the importance of screening for HNF-1B mutations in those patients with structural renal abnormalities and hyperglycemia, as diagnosis can guide management decisions and provide prognostic information. Reference Bellanné-Chantelot C, Chauveau D, Gautier JF, et al. Clinical spectrum associated with hepatocyte nuclear factor-1beta mutations. Ann Intern Med. 2004;140(7): 510-517. doi: 10.7326/0003-4819-140-7-200404060-0000 Presentation: No date and time listed

RF27 | LBSUN362 Germline CDKN1B Variants Type And Site Are Associated With Specific Phenotype In Multiple Endocrine Neoplasia 4

Abstract Background The latest of the multiple endocrine neoplasia (MEN) syndromes described is MEN4, caused by germline CDKN1B tumor suppressor gene mutation. The main clinical manifestations of MEN4 are primary hyperparathyroidism (PHPT), neuroendocrine tumors (NET) and pituitary adenomas (PitAd). CDKN1B codes for the nuclear protein p27, a key regulator of cell cycle. This protein is an intrinsically unstructured protein, that folds upon interacting a putative interactor. It is comprised of two major domains: a kinase inhibitory domain (CDKi) that binds to cyclins and cyclin dependent kinase (CDK), and a scatter domain. p27 acts by inhibiting complexes of cyclins and CDKs, thus preventing the transition between phases of the cell cycle. Less than sixty cases of MEN4 syndrome were described thus far, with clinical manifestations resembling in part to a late onset of MEN1. Hypothesis Specific alterations in p27 protein will result in distinct clinical phenotypes. Methodology Case report We collected published and unpublished data on all currently known patients with MEN4 worldwide, including newly identified kindreds from our clinic, and performed a thorough genotype-phenotype analysis of the various MEN4 manifestations. Whole exome sequencing was performed, revealing a germline CDKN1B frameshift variant (p.Q107fs*12), that was further verified by Sanger sequencing. A detailed literature review, including requests of unpublished clinical data from past studies was undertaken. Based on the data we performed a time-dependent risk analysis for the three main manifestations of MEN4 by various genotype-related parameters: variant type (single-nucleotide variant vs. indels) and site (variant- and domain-specific analyses). Major results Of 73 cases, 53.4% developed PHPT, 28.8% had PitAD and 19.2% - NET. PitAd developed earlier (age, 34.4±21.4 years) compared with PHPT (50.6±13.9 years) and NET (52.9±13.9 years). The risk to develop PHPT, PitA and NET in patients with MEN4 were 53.4%, 23.2% and 16.2% by age 60 years, respectively.CDKN1B variants clustered in genetic locations, or "hotspots" (i.e., codon 41 frameshift: n=13, codon 107 frameshift: n=11, c.-29-(-26)AGAG deletion: n=4). Although founder effect is relevant, this finding is in contrast to MEN1 genotype. We then performed time-dependent risk analysis for developing clinical manifestation by each gene domain alteration (either point mutation or upstream indel). We found a higher risk to develop PHPT or PitAd with variants affecting codons 94-96 (Log-rank, p values 0.014 and 0.047, respectively), and trend towards higher risk for PHPT for Scatter domain variants (p=0.09). Moreover, indels were associated Conclusions: MEN4 genotype is distinct than MEN1, by the presence of hotspots, and genotype-phenotype association. Further, the risk for PHPT is lower than MEN1, with a lower penetrance, and older age at diagnosis. Presentation: Monday, June 13, 2022 1:12 p.m. - 1:17 p.m.

RF05 | PSUN364 A Rare Case of Bilateral Parathyroid Carcinoma in the Setting of Multiple Endocrine Neoplasia Type 1

Abstract Background Multiple Endocrine Neoplasia type 1 (MEN1) is an autosomal dominant disorder classically characterized by parathyroid gland tumors, anterior pituitary adenomas, and tumors of the gastro-entero-pancreatic tract. The majority of MEN1 cases involving lesions of the parathyroid gland causing hyperparathyroidism are almost always benign. It is extremely rare for hyperparathyroidism to be caused by parathyroid carcinomas. We report a very rare case of parathyroid carcinoma presenting in MEN1 syndrome. Case Report We present the case of a 45 year old male with a history of untreated hypertension, nephrolithiasis, and CKD, who became suddenly unresponsive, was intubated, and found to have a severe acute intraparenchymal hemorrhage secondary to severe hypertension on Head CT. Secondary causes of hypertension were ruled out, with renin activity, aldosterone levels, and urine metanephrines unremarkable; abdominal ultrasound with doppler was also negative for renal artery stenosis. Stroke protocol was initiated, with CTA Head negative for any acute findings. However, CTA Neck showed bilateral large nodules posterior to the thyroid lobes measuring 4.0×3.2 cm on the right and 3.9×2.5 cm on the left, with mass effect on the thyroid and adjacent carotids. Routine labs showed hypercalcemia of 13.3 (8.4-10.2 mg/dL) in the setting of elevated PTH levels of 1085 (15-65 pg/mL), with repeat PTH level of 1106 confirming primary hyperparathyroidism. Other causes of hypercalcemia were investigated, with levels of Calcitonin and Calcitriol/1,25 vitamin D within normal limits, but a decreased level of 25 hydroxy vitamin D of <12.8 (30-100 pg/mL). Maxillofacial CT was negative for any ossifying fibromas, ruling out jaw tumor syndrome. CT Abdomen showed hyperdense nodular areas in the pancreas, and MRI Brain was negative for any discrete pituitary gland lesions. Thyroid US was performed and revealed mixed cystic and solid masses posterior to both lobes of the thyroid; as a result, FNA of the thyroid and posterior mass was done, confirming hypercellular parathyroid tissue and benign thyroid tissue. Due to high suspicion for parathyroid carcinoma, the patient underwent a parathyroidectomy, with pathology confirming bilateral parathyroid carcinoma. Genetic testing was positive for MEN1, with a frameshift variant mutation of c.1299_1312del (p.Val434Leufs*10). Conclusion This is a very rare case of bilateral parathyroid carcinoma presenting in MEN1. To the best of our knowledge, this genetic mutation has not been reported in online databases or published literature. The clinical presentation of parathyroid lesions and pancreatic nodules in the setting of hypercalcemia should prompt further evaluation of genetic disorders such as MEN1. In this context, it is essential that MEN1 patients be managed with a multidisciplinary approach given the disorder's complexity of diagnosis and treatment. Presentation: Saturday, June 11, 2022 1:18 p.m. - 1:23 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Genetic and Clinical Spectrum of GNE Myopathy in Russia.

GNE myopathy (GNEM) is a rare hereditary disease, but at the same time, it is the most common distal myopathy in several countries due to a founder effect of some pathogenic variants in the GNE gene. We collected the largest cohort of patients with GNEM from Russia and analyzed their mutational spectrum and clinical data. In our cohort, 10 novel variants were found, including 2 frameshift variants and 2 large deletions. One novel missense variant c.169_170delGCinsTT (p.(Ala57Phe)) was detected in 4 families in a homozygous state and in 3 unrelated patients in a compound heterozygous state. It was the second most frequent variant in our cohort. All families with this novel frequent variant were non-consanguineous and originated from the 3 neighboring areas in the European part of Russia. The clinical picture of the patients carrying this novel variant was typical, but the severity of clinical manifestation differed significantly. In our study, we reported two atypical cases expanding the phenotypic spectrum of GNEM. One female patient had severe quadriceps atrophy, hand joint contractures, keloid scars, and non-classical pattern on leg muscle magnetic resonance imaging, which was more similar to atypical collagenopathy rather than GNEM. Another patient initially had been observed with spinal muscular atrophy due to asymmetric atrophy of hand muscles and results of electromyography. The peculiar pattern of muscle involvement on magnetic resonance imaging consisted of pronounced changes in the posterior thigh muscle group with relatively spared muscles of the lower legs, apart from the soleus muscles. Different variants in the GNE gene were found in both atypical cases. Thus, our data expand the mutational and clinical spectrum of GNEM.

A novel biallelic loss-of-function variant in DAND5 causes heterotaxy syndrome.

The majority of heterotaxy cases do not obtain a molecular diagnosis, although pathogenic variants in more than 50 genes are known to cause heterotaxy. A heterozygous missense variant in DAND5, a nodal inhibitor, which functions in early development for establishment of right-left patterning, has been implicated in heterotaxy. Recently, the first case was reported of a DAND5 biallelic loss-of-function (LoF) variant in an individual with heterotaxy. Here, we describe a second unrelated individual with heterotaxy syndrome and a homozygous frameshift variant in DAND5 (NM_152654.2:c.197del [p.Leu66ArgfsTer22]). Using an in vitro assay, we demonstrate that the DAND5 c.197del variant is unable to inhibit nodal signaling when compared with the wild-type expression construct. This work strengthens the genetic and functional evidence for biallelic LoF variants in DAND5 causing an autosomal recessive heterotaxy syndrome.

A novel homozygous frameshift variant in DNAH8 causes multiple morphological abnormalities of the sperm flagella in a consanguineous Pakistani family.

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenozoospermia categorized by immotile spermatozoa with abnormal flagella in ejaculate. Whole-exome sequencing (WES) is used to detect pathogenic variants in patients with MMAF. In this study, a novel homozygous frameshift variant (c.6158_6159insT) in dynein axonemal heavy chain 8 (DNAH8) from two infertile brothers with MMAF in a consanguineous Pakistani family was identified by WES. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed DNAH8 mRNA decay in these patients with the DNAH8 mutation. Hematoxylin-eosin staining and transmission electron microscopy revealed highly divergent morphology and ultrastructure of sperm flagella in these patients. Furthermore, an immunofluorescence assay showed the absence of DNAH8 and a reduction in its associated protein DNAH17 in the patients' spermatozoa. Collectively, our study expands the phenotypic spectrum of patients with DNAH8-related MMAF worldwide.

KIF1A novel frameshift variant p.(Ser887Profs*64) exhibits clinical heterogeneity in a Pakistani family with hereditary sensory and autonomic neuropathy type IIC

Background: Hereditary sensory and autonomic neuropathies (HSANs) are rare heterogeneous group of neurological disorders caused by peripheral nerve deterioration. The HSANs sub-clinical classes have clinical and genetic overlap which often lead to misdiagnosis. In the present study a Pakistani family with five affected members suffering from severe neuropathy were genetically analyzed to identify the disease causative element in the family. Methods: Genome wide high-density single nucleotide polymorphism (SNP) microarray analysis was carried out followed by whole exome sequencing of the affected proband and another affected sibling. Shared homozygous regions in all severely affected members were identified through homozygosity mapping approach. Results: The largest homozygous region of 14.1 Mb shared by the five severely affected members of the family was identified on chromosome 2. Subsequent exome sequencing identified a novel single nucleotide deletion c.2658del; p.(Ser887Profs*64) in KIF1A. Segregation analysis revealed that this mutation was homozygous in all five affected individuals of the family with severe clinical manifestation, while members of the family that were heterozygous carriers shared abnormal skin features (scaly skin) only with the homozygous affected members. Conclusions: A novel frameshift mutation p.(Ser887Profs*64) in KIF1A is the potential cause of severe HSANIIC in a Pakistani family along with incomplete penetrance in mutation carriers. We demonstrate that using a combination of different techniques not only strengthens the gene finding approach but also helps in proper sub-clinical characterization along with identification of mutated alleles exhibiting incomplete penetrance leading to intrafamilial clinical variability in HSAN group of inherited diseases.

Whole-Exome Sequencing Reveals Migraine-Associated Novel Functional Variants in Arab Ancestry Females: A Pilot Study.

Migraine, as the seventh most disabling neurological disease with 26.9% prevalence in Saudi females, lacks studies on identifying associated genes and pathways with migraines in the Arab population. This case control study aims to identify the migraine-associated novel genes and risk variants. More than 1900 Arab ancestry young female college students were screened: 103 fulfilled the ICHD-3 criteria for migraine and 20 cases confirmed in the neurology clinic were included for the study with age-matched healthy controls. DNA from blood samples were subjected to paired-end whole-exome sequencing. After quality control, 3365343 missense, frameshift, missense splice region variants and insertion-deletion (indels) polymorphisms were tested for association with migraine. Significant variants were validated using Sanger sequencing. A total of 17 (p-value 9.091 × 10-05) functional variants in 12 genes (RETNLB, SCAI, ADH4, ESPL1, CPT2, FLG, PPP4R1, SERPINB5, ZNF66, ETAA1, EXO1 and CPA6) were associated with higher migraine risk, including a stop-gained frameshift (-13-14*SX) variant in the gene RETNLB (rs5851607; p-value 3.446 × 10-06). Gene analysis revealed that half of the significant novel migraine risk genes were expressed in the temporal lobe (p-value 0.0058) of the cerebral cortex. This is the first study exploring the migraine risk of 17 functional variants in 12 genes among Saudi female migraineurs of Arab ancestry using whole-exome sequencing. Half of the significant genes were expressed in the temporal lobe, which expands migraine pathophysiology and early identification using biomarkers for research possibilities on personalised genetics.

Identification of variants in 94 Chinese patients with hereditary spherocytosis by next-generation sequencing.

Hereditary spherocytosis (HS) is the most common type of hereditary erythrocyte membrane disease and has varied phenotypic features and genetic patterns. We herein performed a retrospective study of 94 patients with HS and aimed to investigate the genetic variations and genotype-phenotype correlations using targeted next-generation sequencing. In 79/94 (84%) patients, 83 HS variants including 67 novel variants were identified. Pathogenic variants of SPTB, ANK1, SLC4A1, SPTA1, and EPB42 were found in 32/79(41%), 22/79(28%), 15/79 (19%), 8/79 (9%), and 3/79 (4%) of the patients respectively, revealing that SPTB is the most frequently mutated HS gene in Eastern China. Most SPTB and ANK1 gene variations were nonsense and frameshift variations. Missense variants were the main variant type of SLC4A1, SPTA1, and EPB42 genes. Interestingly, one SPTA1 variant (p. Arg1757Cys) showed an autosomal dominant inheritance pattern and one EPB42 variant (p. Gln377His) was apparent as a hotspot variation. Furthermore, genotype-phenotype analysis was performed among the five mutated gene groups. Besides the finding that patients with the SLC4A1 variant had the highest mean corpuscular hemoglobin levels, no clear correlations between genotype and phenotype were observed.

Novel Synonymous and Frameshift Variants in the TRIP12 Gene Identified in 2 Chinese Patients With Intellectual Disability.

Background and ObjectivesClark-Baraitser syndrome is characterized by intellectual disability with or without autism spectrum disorders, speech delay, motor delay, behavioral abnormalities, and facial dysmorphism. It is caused by a heterozygous pathogenic variant in the thyroid hormone receptor interactor 12 (TRIP12) gene. However, loss of function and haploinsufficiency are the pathogenic mechanisms behind the TRIP12-related disorder.MethodsWe conducted an exome sequencing analysis for 2 unrelated patients with moderate intellectual disability, speech delay, and motor delay.ResultsWe identified 2 de novo TRIP12 mutations in these 2 patients. One patient had a frameshift duplication, whereas the other had a synonymous variant. Both patients presented with common features of the syndrome, but clinical heterogeneity has been also observed between them. For the synonymous variant, reverse transcription PCR in RNA extracted from leukocytes demonstrated the presence of a truncated messenger RNA (mRNA) transcript that skipped exon 12. This transcript escapes degradation at the mRNA level. To assess the effect of the synonymous substitute on TRIP12 proteolytic activity, the expression of 9 known responsive genes at the mRNA level was measured, of which 3 genes were upregulated at least 2-fold in the patient.DiscussionWe reported 2 patients with Clark-Baraitser syndrome caused by novel synonymous and frameshift variants in the TRIP12 gene, and our study expands the mutation spectrum of the TRIP12 gene. This study will help to improve our understanding of variable phenotypic presentations in TRIP12-related disorders.

Genetic screening and analysis of TUBB8 variants in females seeking ART

Research question: More than 100 variants have been identified in the TUBB8 gene, which account for approximately 30% of infertile women with oocyte maturation defects. But what is the correlation between the highly phenotypic diversity and genetic variability? Are there other variants in TUBB8 related to female infertility?Design: TUBB8 resequencing was performed in 80 female subjects who were experiencing infertility and were seeking treatment with assisted reproductive technologies (ART), or had ever experienced ART failure due to oocyte maturation defects. All variants were evaluated with pedigree analysis, population frequency, in-silico analysis and molecular modelling. The effects of the variants on oocytes/arrested embryos were assessed by morphological observations, immunostaining, embryo biopsies and chromosome euploidy analysis.Results: Nine missense variants and two frameshift variants from an additional 15 families were identified, including four novel variants and seven previously reported recurrent variants. These TUBB8 variants were related to highly variable phenotypes, including abnormalities in oocyte maturation or morphology, fertilization failure, embryonic development abnormalities and implantation failure. Also further clarified were the incomplete penetrance of heterozygous p.E108K, the likely benign significance of heterozygous p.A313V and the clinical effect of a novel variant of p.R380C.Conclusions: This study significantly expands the variant spectrum of the TUBB8 gene and, together with the available findings on TUBB8 variants and female infertility, will potentially facilitate the genetic counselling of infertile women in future.

Atypical STAT5B deficiency, severe short stature and mild immunodeficiency associated with a novel homozygous STAT5B Variant

STAT5B deficiency, a rare autosomal recessive disorder characterized by severe growth hormone insensitivity (GHI) and immunodeficiency, can manifest as fatal pulmonary complications. We describe atypical STAT5B deficiency associated with a novel homozygous frame-shift STAT5B variant [c.1453delG, p.(Asp485Thrfs*29)] identified in a young 17.6 yr old female subject who had severe postnatal growth impairment, biochemistries typical of GHI, an immune profile notable for hypergammaglobulinaemia and elevated B lymphocytes, and lack of pulmonary disease. Marked elevation of serum prolactin and pathologically diagnosed eczema were evident. In reconstitution studies, the STAT5B p.(Asp485Thrfs*29) was expressed although expression was reduced compared to wild-type STAT5B and a previously identified STAT5B p.(Gln368Profs*9) variant. Both truncated STAT5B peptides could not be activated by GH, nor mobilize to the nucleus. We conclude that an intact, functional, STAT5B is essential for normal GH-mediated growth, while expressed loss-of-function STAT5B variants may alleviate severe immune and pulmonary issues normally associated with STAT5B deficiency.

Biallelic loss of LDB3 leads to a lethal pediatric dilated cardiomyopathy

Autosomal dominant variants in LDB3 (also known as ZASP), encoding the PDZ-LIM domain-binding factor, have been linked to a late onset phenotype of cardiomyopathy and myofibrillar myopathy in humans. However, despite knockout mice displaying a much more severe phenotype with premature death, bi-allelic variants in LDB3 have not yet been reported. Here we identify biallelic loss-of-function variants in five unrelated cardiomyopathy families by next-generation sequencing. In the first family, we identified compound heterozygous LOF variants in LDB3 in a fetus with bilateral talipes and mild left cardiac ventricular enlargement. Ultra-structural examination revealed highly irregular Z-disc formation, and RNA analysis demonstrated little/no expression of LDB3 protein with a functional C-terminal LIM domain in muscle tissue from the affected fetus. In a second family, a homozygous LDB3 nonsense variant was identified in a young girl with severe early-onset dilated cardiomyopathy with left ventricular non-compaction; the same homozygous nonsense variant was identified in a third unrelated female infant with dilated cardiomyopathy. We further identified homozygous LDB3 frameshift variants in two unrelated probands diagnosed with cardiomegaly and severely reduced left ventricular ejection fraction. Our findings demonstrate that recessive LDB3 variants can lead to an early-onset severe human phenotype of cardiomyopathy and myopathy, reminiscent of the knockout mouse phenotype, and supporting a loss of function mechanism.

ACADM Frameshift Variant in Cavalier King Charles Spaniels with Medium-Chain Acyl-CoA Dehydrogenase Deficiency.

A 3-year-old, male neutered Cavalier King Charles Spaniel (CKCS) presented with complex focal seizures and prolonged lethargy. The aim of the study was to investigate the clinical signs, metabolic changes and underlying genetic defect. Blood and urine organic acid analysis revealed increased medium-chain fatty acids and together with the clinical findings suggested a diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. We sequenced the genome of the affected dog and compared the data to 923 control genomes of different dog breeds. The ACADM gene encoding MCAD was considered the top functional candidate gene. The genetic analysis revealed a single homozygous private protein-changing variant in ACADM in the affected dog. This variant, XM_038541645.1:c.444_445delinsGTTAATTCTCAATATTGTCTAAGAATTATG, introduces a premature stop codon and is predicted to result in truncation of ~63% of the wild type MCAD open reading frame, XP_038397573.1:p.(Thr150Ilefs*6). Targeted genotyping of the variant in 162 additional CKCS revealed a variant allele frequency of 23.5% and twelve additional homozygous mutant dogs. The acylcarnitine C8/C12 ratio was elevated ~43.3 fold in homozygous mutant dogs as compared to homozygous wild type dogs. Based on available clinical and biochemical data together with current knowledge in humans, we propose the ACADM frameshift variant as causative variant for the MCAD deficiency with likely contribution to the neurological phenotype in the index case. Testing the CKCS breeding population for the identified ACADM variant is recommended to prevent the unintentional breeding of dogs with MCAD deficiency. Further prospective studies are warranted to assess the clinical consequences of this enzyme defect.

The Presentation of Two Unrelated Clinical Cases from the Republic of North Ossetia-Alania with the Same Previously Undescribed Variant in the COL6A2 Gene.

Here, we described three affected boys from two unrelated families of Ossetian-Digor origin from the Republic of North Ossetia-Alania who were admitted to the Research Centre for Medical Genetics with unspecified muscular dystrophy. High-throughput sequencing was performed and revealed two novel frameshift variants in the COL6A2 gene (NM_001849.3) in a heterozygous state each in both cases: c.508_535delinsCTGTGG and c.1659_1660del (case 1) and c.1689del and c.1659_1660del (case 2). In two cases, the same nucleotide variant in the COL6A2 gene (c.1659_1660del) was observed. We have suggested that the variant c.1659_1660del may be common in the Ossetian-Digor population because two analyzed families have the same ancestry from the same subethnic group of Ossetians). The screening for an asymptomatic carriage of the nucleotide variant c.1659_1660del in 54 healthy donors from Ossetian-Digor population revealed that the estimated carrier frequency is 0.0093 (CI: 0.0002–0.0505), which is high for healthy carriers of the pathogenic variant. Molecular genetic, anamnestic data and clinical examination results allowed us to diagnose Ullrich muscular dystrophy in those affected boys. Genetic heterogeneity and phenotypic diversity of muscular dystrophies complicate diagnosis. It is important to make a differential diagnosis of such conditions and use HTS methods to determine the most accurate diagnosis.

RIPK3 dampens mitochondrial bioenergetics and lipid droplet dynamics in metabolic liver disease.

Receptor-interacting protein kinase 3 (RIPK3) mediates NAFLD progression, but its metabolic function is unclear. Here, we aimed to investigate the role of RIPK3 in modulating mitochondria function, coupled with lipid droplet (LD) architecture in NAFLD. Functional studies evaluating mitochondria and LD biology were performed in wild-type (WT) and Ripk3-/- mice fed a choline-deficient, amino acid-defined (CDAA) diet for 32 and 66 weeks and in CRISPR-Cas9 Ripk3 -null fat-loaded immortalized hepatocytes. The association between hepatic perilipin (PLIN) 1 and 5, RIPK3, and disease severity was also addressed in a cohort of patients with NAFLD and in PLIN1 -associated familial partial lipodystrophy. Ripk3 deficiency rescued impairment in mitochondrial biogenesis, bioenergetics, and function in CDAA diet-fed mice and fat-loaded hepatocytes. Ripk3 deficiency was accompanied by a strong upregulation of antioxidant systems, leading to diminished oxidative stress upon fat loading both in vivo and in vitro. Strikingly, Ripk3-/- hepatocytes displayed smaller size LD in higher numbers than WT cells after incubation with free fatty acids. Ripk3 deficiency upregulated adipocyte and hepatic levels of LD-associated proteins PLIN1 and PLIN5. PLIN1 upregulation controlled LD structure and diminished mitochondrial stress upon free fatty acid overload in Ripk3-/- hepatocytes and was associated with diminished human NAFLD severity. Conversely, a pathogenic PLIN1 frameshift variant was associated with NAFLD and fibrosis, as well as with increased hepatic RIPK3 levels in familial partial lipodystrophy. Ripk3 deficiency restores mitochondria bioenergetics and impacts LD dynamics. RIPK3 inhibition is promising in ameliorating NAFLD.