Dominantly inherited GAA repeat expansions in FGF14 are a common cause of spinocerebellar ataxia (GAA-FGF14 ataxia; spinocerebellar ataxia 27B). Molecular confirmation of FGF14 GAA repeat expansions ...has thus far mostly relied on long-read sequencing, a technology that is not yet widely available in clinical laboratories. We developed and validated a strategy to detect FGF14 GAA repeat expansions using long-range PCR, bidirectional repeat-primed PCRs, and Sanger sequencing. We compared this strategy to targeted nanopore sequencing in a cohort of 22 French Canadian patients and next validated it in a cohort of 53 French index patients with unsolved ataxia. Method comparison showed that capillary electrophoresis of long-range PCR amplification products significantly underestimated expansion sizes compared to nanopore sequencing (slope, 0.87 95% CI, 0.81 to 0.93; intercept, 14.58 95% CI, - 2.48 to 31.12) and gel electrophoresis (slope, 0.84 95% CI, 0.78 to 0.97; intercept, 21.34 95% CI, - 27.66 to 40.22). The latter techniques yielded similar size estimates. Following calibration with internal controls, expansion size estimates were similar between capillary electrophoresis and nanopore sequencing (slope: 0.98 95% CI, 0.92 to 1.04; intercept: 10.62 95% CI, - 7.49 to 27.71), and gel electrophoresis (slope: 0.94 95% CI, 0.88 to 1.09; intercept: 18.81 95% CI, - 41.93 to 39.15). Diagnosis was accurately confirmed for all 22 French Canadian patients using this strategy. We also identified 9 French patients (9/53; 17%) and 2 of their relatives who carried an FGF14 (GAA)
expansion. This novel strategy reliably detected and sized FGF14 GAA expansions, and compared favorably to long-read sequencing.
Cornelia de Lange syndrome (CdLS) is a clinically‐recognizable rare developmental disorder. About 70% of patients carry a missense or loss‐of‐function pathogenic variant in the NIPBL gene. We ...hypothesized that some variants in the 5′‐untranslated region (UTR) of NIPBL may create an upstream open reading frame (uORF), putatively leading to a loss of function. We searched for NIPBL 5′‐UTR variants potentially introducing uORF by (i) reannotating NGS data of 102 unsolved CdLS patients and (ii) literature and variant databases search. We set up a green fluorescent protein (GFP) reporter assay and studied NIPBL expression in a lymphoblastoid cell line (LCL). We identified two variants introducing a novel ATG codon sequence in the 5′‐UTR of NIPBL, both predicted to introduce uORF: a novel c.‐457_‐456delinsAT de novo mutation in a 15‐year‐old male with classic CdLS, and a c.‐94C>T variant in a published family. Our reporter assay showed a significant decrease of GFP levels in both mutant contexts, with similar levels of messenger RNA (mRNA) as compared to wt constructs. Assessment of LCL of one patient showed consistent results with decreased NIPBL protein and unchanged mRNA levels. 5′‐UTR uORF‐introducing NIPBL variants may represent a rare source of pathogenic variants in unsolved CdLS patients.
Start codon‐introducing 5'UTR NIPBL variants leading to repressed translation.
Constitutional epimutations are an alternative to genetic mutations in the etiology of genetic diseases. Some of these epimutations, termed secondary, correspond to the epigenetic effects of ...cis-acting genetic defects transmitted to the offspring following a Mendelian inheritance pattern. In Lynch syndrome, a few families with such apparently heritable MLH1 epimutations have been reported so far.
We designed a long-range polymerase chain reaction next-generation sequencing strategy to screen MLH1 entire gene and applied it to 4 French families with heritable epimutations and 10 additional patients with no proven transmission of their epimutations.
This strategy successfully detected the insertion of an Alu element in MLH1 coding sequence in one family. Two previously unreported MLH1 variants were also identified in other epimutation carriers: a nucleotide substitution within intron 1 and a single-nucleotide deletion in the 5′-UTR. Detection of a partial MLH1 duplication in another family required multiplex ligation-dependent probe amplification technology. We demonstrated the segregation of these variants with MLH1 methylation and studied the functional consequences of these defects on transcription.
This is the largest cohort of patients with MLH1 secondary epimutations associated with a broad spectrum of genetic defects. This study provides further insight into the complexity of molecular mechanisms leading to secondary epimutations.
Cutis laxa (CL) is a rare connective tissue disorder characterized by wrinkled, abundant and sagging skin, sometimes associated with systemic impairment. Biallelic alterations in latent transforming ...growth factor beta‐binding protein 4 gene (LTBP4) cause autosomal recessive type 1C cutis laxa (ARCL1C, MIM #613177). The present report describes the case of a 17‐months‐old girl with cutis laxa together with a literature review of previous ARCL1C cases. Based on proband main clinical signs (cutis laxa and pulmonary emphysema), clinical exome sequencing (CES) was performed and showed a new nine base‐pairs homozygous in‐frame deletion in LTBP4 gene. RT‐PCR and cDNA Sanger sequencing were performed in order to clarify its impact on RNA. This report demonstrates that a genetic alteration in the EGF‐like 14 domain calcium‐binding motif of LTBP4 gene is likely responsible for cutis laxa in our patient.
ABSTRACT
As next‐generation sequencing increases access to human genetic variation, the challenge of determining clinical significance of variants becomes ever more acute. Germline variants in the ...BRCA1 and BRCA2 genes can confer substantial lifetime risk of breast and ovarian cancer. Assessment of variant pathogenicity is a vital part of clinical genetic testing for these genes. A database of clinical observations of BRCA variants is a critical resource in that process. This article describes BRCA Share™, a database created by a unique international alliance of academic centers and commercial testing laboratories. By integrating the content of the Universal Mutation Database generated by the French Unicancer Genetic Group with the testing results of two large commercial laboratories, Quest Diagnostics and Laboratory Corporation of America (LabCorp), BRCA Share™ has assembled one of the largest publicly accessible collections of BRCA variants currently available. Although access is available to academic researchers without charge, commercial participants in the project are required to pay a support fee and contribute their data. The fees fund the ongoing curation effort, as well as planned experiments to functionally characterize variants of uncertain significance. BRCA Share™ databases can therefore be considered as models of successful data sharing between private companies and the academic world.
BRCA ShareTM is an international alliance of academic centers and commercial testing laboratories. By integrating content of the Universal Mutation Database generated by the French Unicancer Genetic Group with the testing results of Quest Diagnostics and Laboratory Corporation of America (LabCorp), BRCA ShareTM has assembled one of the largest publicly accessible collections of BRCA variants with over 6,200 variants currently available.
In addition, to funding high quality curation, BRCA ShareTM seeks to support functional studies, targeting variants of uncertain significance.
Bi‐allelic variants affecting one of the four genes encoding the AP4 subunits are responsible for the “AP4 deficiency syndrome.” Core features include hypotonia that progresses to hypertonia and ...spastic paraplegia, intellectual disability, postnatal microcephaly, epilepsy, and neuroimaging features. Namely, AP4M1 (SPG50) is involved in autosomal recessive spastic paraplegia 50 (MIM#612936). We report on three patients with core features from three unrelated consanguineous families originating from the Middle East. Exome sequencing identified the same homozygous nonsense variant: NM_004722.4(AP4M1):c.1012C>T p.Arg338* (rs146262009). So far, four patients from three other families carrying this homozygous variant have been reported worldwide. We describe their phenotype and compare it to the phenotype of patients with other variants in AP4M1. We construct a shared single‐nucleotide polymorphism (SNP) haplotype around AP4M1 in four families and suggest a probable founder effect of Arg338* AP4M1 variant with a common ancestor most likely of Turkish origin.
In this study we report three new unrelated patients from Middle‐East consanguineous families carrying the previously described NM_004722.4(AP4M1):c.1012C〉T homozygous nonsense variant. We describe their phenotype, and provide evidence that the subjects carrying this variant originate from a common ancestor.
ATL1
-related spastic paraplegia SPG3A is a pure form of hereditary spastic paraplegia. Rare complex phenotypes have been described, but few data concerning cognitive evaluation or molecular imaging ...of these patients are available. We relate a retrospective collection of patients with SPG3A from the Neurology Department of Nancy University Hospital, France. For each patient were carried out a
18
F-FDG PET (positron emission tomography), a electromyography (EMG), a sudoscan®, a cerebral and spinal cord MRI (magnetic resonance imaging) with measurement of cervical and thoracic surfaces, a neuropsychological assessment. The present report outlines standardised clinical and paraclinical data of five patients from two east-France families carrying the same missense pathogenic variation, NM_015915.4(ATL1): c.1483C > T p.(Arg495Trp) in
ATL1
. Mean age at onset was 14 ± 15.01 years. Semi-quantitatively and in comparison to healthy age-matched subjects, PET scans showed a significant cerebellar and upper or mild temporal hypometabolism in all four adult patients and hypometabolism of the prefrontal cortex or precuneus in three of them. Sudoscan® showed signs of small fibre neuropathy in three patients. Cervical and thoracic patients’ spinal cords were significantly thinner than matched-control, respectively 71 ± 6.59mm
2
(
p
= 0.01) and 35.64 ± 4.35mm
2
(
p
= 0.015). Two patients presented with a dysexecutive syndrome. While adding new clinical and paraclinical signs associated with
ATL1
pathogenic variations, we insist here on the variable penetrance and expressivity. We report small fibre neuropathy, cerebellar hypometabolism and dysexecutive syndromes associated with SPG3A. These cognitive impairments and PET findings may be related to a cortico-cerebellar bundle axonopathy described in the cerebellar cognitive affective syndrome (CCAS).
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