Expanding the genotypic and phenotypic spectrum of severe serine biosynthesis disorders.

Serine biosynthesis disorders comprise a spectrum of very rare autosomal recessive inborn errors of metabolism with wide phenotypic variability. Neu-Laxova syndrome represents the most severe expression and is characterized by multiple congenital anomalies and pre- or perinatal lethality. Here, we present the mutation spectrum and a detailed phenotypic analysis in 15 unrelated families with severe types of serine biosynthesis disorders. We identified likely disease-causing variants in the PHGDH and PSAT1 genes, several of which have not been reported previously. Phenotype analysis and a comprehensive review of the literature corroborates the evidence that serine biosynthesis disorders represent a continuum with varying degrees of phenotypic expression and suggest that even gradual differences at the severe end of the spectrum may be correlated with particular genotypes. We postulate that the individual residual enzyme activity of mutant proteins is the major determinant of the phenotypic variability, but further functional studies are needed to explore effects at the enzyme protein level.We are indebted to all families for participating in this study. We would like to acknowledge Dr. Natasha Laidlew, who initially suggested the diagnosis in one of the cases and provided important phenotypic information, and Dr. María-Luisa Martínez-Fernández for the critical management of biosamples in ECEMC Program of Spain. Financial assistance was received in support of the study by grants from the German Federal Ministry of Education and Research (BMBF) (GeNeRARe, FKZ: 01GM1519D) to M. Z. and from the Institute of Health Carlos III: Convenio ISCIII-ASEREMAC, and Fundación 1000 sobre Defectos Congénitos, of Spain to E. B.-S. and I. R. G.S

SRSF1 Haploinsufficiency Is Responsible for a Syndromic Developmental Disorder Associated with Intellectual Disability

SRSF1 (also known as ASF/SF2) is a non-small nuclear ribonucleoprotein (non-snRNP) that belongs to the arginine/serine (R/S) domain family. It recognizes and binds to mRNA, regulating both constitutive and alternative splicing. The complete loss of this proto-oncogene in mice is embryonically lethal. Through international data sharing, we identified 17 individuals (10 females and 7 males) with a neurodevelopmental disorder (NDD) with heterozygous germline SRSF1 variants, mostly de novo, including three frameshift variants, three nonsense variants, seven missense variants, and two microdeletions within region 17q22 encompassing SRSF1. Only in one family, the de novo origin could not be established. All individuals featured a recurrent phenotype including developmental delay and intellectual disability (DD/ID), hypotonia, neurobehavioral problems, with variable skeletal (66.7%) and cardiac (46%) anomalies. To investigate the functional consequences of SRSF1 variants, we performed in silico structural modeling, developed an in vivo splicing assay in Drosophila, and carried out episignature analysis in blood-derived DNA from affected individuals. We found that all loss-of-function and 5 out of 7 missense variants were pathogenic, leading to a loss of SRSF1 splicing activity in Drosophila, correlating with a detectable and specific DNA methylation episignature. In addition, our orthogonal in silico, in vivo, and epigenetics analyses enabled the separation of clearly pathogenic missense variants from those with uncertain significance. Overall, these results indicated that haploinsufficiency of SRSF1 is responsible for a syndromic NDD with ID due to a partial loss of SRSF1-mediated splicing activity

SRSF1 Haploinsufficiency Is Responsible for a Syndromic Developmental Disorder Associated With Intellectual Disability

SRSF1 (also known as ASF/SF2) is a non-small nuclear ribonucleoprotein (non-snRNP) that belongs to the arginine/serine (R/S) domain family. It recognizes and binds to mRNA, regulating both constitutive and alternative splicing. The complete loss of this proto-oncogene in mice is embryonically lethal. Through international data sharing, we identified 17 individuals (10 females and 7 males) with a neurodevelopmental disorder (NDD) with heterozygous germline SRSF1 variants, mostly de novo, including three frameshift variants, three nonsense variants, seven missense variants, and two microdeletions within region 17q22 encompassing SRSF1. Only in one family, the de novo origin could not be established. All individuals featured a recurrent phenotype including developmental delay and intellectual disability (DD/ID), hypotonia, neurobehavioral problems, with variable skeletal (66.7%) and cardiac (46%) anomalies. To investigate the functional consequences of SRSF1 variants, we performed in silico structural modeling, developed an in vivo splicing assay in Drosophila, and carried out episignature analysis in blood-derived DNA from affected individuals. We found that all loss-of-function and 5 out of 7 missense variants were pathogenic, leading to a loss of SRSF1 splicing activity in Drosophila, correlating with a detectable and specific DNA methylation episignature. In addition, our orthogonal in silico, in vivo, and epigenetics analyses enabled the separation of clearly pathogenic missense variants from those with uncertain significance. Overall, these results indicated that haploinsufficiency of SRSF1 is responsible for a syndromic NDD with ID due to a partial loss of SRSF1-mediated splicing activity

Search and characterization of new genes involved in albinism

L’albinisme est une affection de la pigmentation caractérisée par l’association d’anomalies oculaires à divers degrés d’hypopigmentation cutanéo-phanérienne et parfois des atteintes systémiques dans les formes syndromiques que sont les syndromes d’Hermansky-Pudlak (HPS) et le syndrome de Chediak-Higashi. Avant ce travail, 19 gènes étaient connus comme impliqués dans des formes isolées ou syndromiques d’albinisme, dont la plupart sont autosomiques récessives à l’exception de l’albinisme oculaire lié à l’X. Après étude complète de ces 19 gènes, environ 25% à 30% des patients restent sans diagnostic moléculaire. Afin de rechercher de nouveaux gènes, nous avons développé et testé un panel de 129 gènes candidats chez 230 patients non résolus sur le plan moléculaire. Ceci a permis de mettre en évidence des variants délétères dans deux nouveaux gènes. Nous rapportons d’une part deux patientes porteuses de variants perte de fonction dans le gène BLOC1S5, une délétion intragénique homozygote emportant les exons 3 et 4 et une délétion homozygote d’une paire de base (pb) de l’exon 4. BLOC1S5 code une des sous-unités du complexe BLOC-1 qui en compte huit et dont 3 sont déjà associées à des formes d’HPS (7, 8, 9). Ces patientes présentent un phénotype d’albinisme modéré et un défaut d’agrégation plaquettaire par déficit en granules denses. Les plaquettes de la patiente 1 présentent une absence de BLOC-1 montrant que la délétion entraine une dysfonction de BLOC1S5 gênant l’assemblage du complexe. Tandis que l’expression du transcrit humain sauvage de BLOC1S5, dans des mélanocytes murins avec KO de BLOC1S5, non pigmentés, restaure la pigmentation, l’assemblage de BLOC-1 et le trafic de cargo, l’expression du transcrit avec la délétion de la 1ere patiente ne le permet pas. Nous avons ainsi démontré que la perte de fonction de BLOC1S5 est responsable d’une nouvelle forme d’HPS, HPS-11. Le deuxième gène, DCT ou TYRP2 est suspecté de longue date du fait de sa collaboration à la synthèse de la mélanine avec TYR et TYRP1, gènes connus d’albinisme (OCA1 et 3, respectivement). Nous décrivons 2 patientes présentant un albinisme cutané modéré avec atteinte ophtalmologique caractéristique et porteuses de variants de DCT. Une patiente est homozygote pour un variant faux sens de l’exon 1 et l’autre hétérozygote composite pour un variant faux sens de l’exon 1 et une délétion de 14pb de l’exon 9. Les deux variants faux-sens sont situés dans des cystéines très conservées et ont été induits chez la souris par CrispR-Cas9, reproduisant l’hypopigmentation modérée et l’atteinte oculaire. Un modèle poisson zèbre a aussi été produit avec atteinte similaire. Nous concluons donc que DCT est impliqué dans une nouvelle forme d’albinisme oculo-cutané, OCA8. Enfin nous rapportons également le phénotype détaillé de trois patients porteurs de nouveaux variants dans BLOC1S3, associé à l’HPS-8, une des formes les plus rares d’albinisme syndromique. Avant ce travail seulement 2 familles et deux variants étaient décrits dans la littérature. La description détaillée du phénotype de ces patients est une aide aux praticiens dans l’accompagnement diagnostique et pronostique des patients ainsi que pour le conseil génétique. Nous avons donc rapporté deux nouveaux gènes d’albinisme, pour une forme oculocutanée et pour une forme syndromique (HPS), toutes deux apparemment modérées et décrivons de nouveaux variants pour une forme connue mais néanmoins rare d’albinisme syndromique.Albinism is a pigmentation disorder characterized by visual impairment, isolated (Ocular Albinism, OA) or associated with generalized skin and hair hypopigmentation (Oculo-Cutaneous Albinism, OCA), and sometimes with other symptoms in syndromic forms called Hermansky-Pudlak (HPS) and Chediak-Higashi (CHS) syndromes. Most are autosomal recessive affections except for X-linked OA. Prior to this work 19 genes were known to cause isolated or syndromic albinism. After extensive analysis of these genes, 25 to 30% of patients remain molecularly unsolved. To search for new genes we screened, in 230 patients without molecular diagnosis, a panel of 129 candidate genes. We identified deleterious variants in two candidate genes. First, we report two patients with predicted loss-of-function variants in BLOC1S5 in two patients. The first patient carries an intragenic homozygous deletion involving exons 3 and 4. The second patient has a homozygous one base-pair deletion in exon 4. BLOC1S5 codes for one of the 8 subunits of BLOC-1, three of which are involved HPS-7, 8 and 9. Both patients had mild hypopigmentation, classical ocular albinism and moderate bleeding diathesis, platelet aggregation deficit and a very low number of platelet dense granules, compatible with HPS. Patient 1’s platelets displayed an absence of BLOC-1, showing that the identified mutation disrupts BLOC1S5 function and impairs BLOC-1 assembly. Whereas expression of a wild-type BLOC1S5 protein in non-pigmented murine melanocytes rescued pigmentation, assembly of a functional BLOC-1, and melanosome cargo trafficking, expression of the patient's BLOC1S5 variant allele did not. These data indicate that mutation of BLOC1S5 is disease-causing, and we propose that BLOC1S5 is the gene for a new form of HPS, HPS11. The second gene, DCT or TYRP2 has been suspected for a long time, due to its collaboration with TYR (OCA1) and TYRP1 (OCA3). We describe two unrelated patients presenting with clinical diagnosis of OCA. One patient is homozygous for a missense variant in the exon 1 of DCT and the other is a compound heterozygote for a missense variant in exon 1 and a frame-shift deletion in exon 9. Both missense variants, affecting highly conserved cysteine, were introduced in mice via Crispr-Cas9, which led to mild hypopigmentation of the fur and ocular anomalies. A Zebrafish knock-down model has also been produced by morpholino injection, leading to a significant decrease in melanin levels compared to controls. We propose that DCT is the gene for a new form of OCA, OCA8. In this work we also describe three unrelated patients with novel variants in BLOC1S3 responsible for HPS-8. This rare form of HPS had been reported in only two families and we believe detailed clinical description of patients is useful for clinicians for patient’s diagnosis and prognosis evaluation and genetic counseling. In conclusion we have reported two new genes involved in isolated and syndromic forms of albinism as well as new insight in an already known yet rare form of syndromic albinism

Recherche et caractérisation de nouveaux gènes impliqués dans l’albinisme

Albinism is a pigmentation disorder characterized by visual impairment, isolated (Ocular Albinism, OA) or associated with generalized skin and hair hypopigmentation (Oculo-Cutaneous Albinism, OCA), and sometimes with other symptoms in syndromic forms called Hermansky-Pudlak (HPS) and Chediak-Higashi (CHS) syndromes. Most are autosomal recessive affections except for X-linked OA. Prior to this work 19 genes were known to cause isolated or syndromic albinism. After extensive analysis of these genes, 25 to 30% of patients remain molecularly unsolved. To search for new genes we screened, in 230 patients without molecular diagnosis, a panel of 129 candidate genes. We identified deleterious variants in two candidate genes. First, we report two patients with predicted loss-of-function variants in BLOC1S5 in two patients. The first patient carries an intragenic homozygous deletion involving exons 3 and 4. The second patient has a homozygous one base-pair deletion in exon 4. BLOC1S5 codes for one of the 8 subunits of BLOC-1, three of which are involved HPS-7, 8 and 9. Both patients had mild hypopigmentation, classical ocular albinism and moderate bleeding diathesis, platelet aggregation deficit and a very low number of platelet dense granules, compatible with HPS. Patient 1’s platelets displayed an absence of BLOC-1, showing that the identified mutation disrupts BLOC1S5 function and impairs BLOC-1 assembly. Whereas expression of a wild-type BLOC1S5 protein in non-pigmented murine melanocytes rescued pigmentation, assembly of a functional BLOC-1, and melanosome cargo trafficking, expression of the patient's BLOC1S5 variant allele did not. These data indicate that mutation of BLOC1S5 is disease-causing, and we propose that BLOC1S5 is the gene for a new form of HPS, HPS11. The second gene, DCT or TYRP2 has been suspected for a long time, due to its collaboration with TYR (OCA1) and TYRP1 (OCA3). We describe two unrelated patients presenting with clinical diagnosis of OCA. One patient is homozygous for a missense variant in the exon 1 of DCT and the other is a compound heterozygote for a missense variant in exon 1 and a frame-shift deletion in exon 9. Both missense variants, affecting highly conserved cysteine, were introduced in mice via Crispr-Cas9, which led to mild hypopigmentation of the fur and ocular anomalies. A Zebrafish knock-down model has also been produced by morpholino injection, leading to a significant decrease in melanin levels compared to controls. We propose that DCT is the gene for a new form of OCA, OCA8. In this work we also describe three unrelated patients with novel variants in BLOC1S3 responsible for HPS-8. This rare form of HPS had been reported in only two families and we believe detailed clinical description of patients is useful for clinicians for patient’s diagnosis and prognosis evaluation and genetic counseling. In conclusion we have reported two new genes involved in isolated and syndromic forms of albinism as well as new insight in an already known yet rare form of syndromic albinism.L’albinisme est une affection de la pigmentation caractérisée par l’association d’anomalies oculaires à divers degrés d’hypopigmentation cutanéo-phanérienne et parfois des atteintes systémiques dans les formes syndromiques que sont les syndromes d’Hermansky-Pudlak (HPS) et le syndrome de Chediak-Higashi. Avant ce travail, 19 gènes étaient connus comme impliqués dans des formes isolées ou syndromiques d’albinisme, dont la plupart sont autosomiques récessives à l’exception de l’albinisme oculaire lié à l’X. Après étude complète de ces 19 gènes, environ 25% à 30% des patients restent sans diagnostic moléculaire. Afin de rechercher de nouveaux gènes, nous avons développé et testé un panel de 129 gènes candidats chez 230 patients non résolus sur le plan moléculaire. Ceci a permis de mettre en évidence des variants délétères dans deux nouveaux gènes. Nous rapportons d’une part deux patientes porteuses de variants perte de fonction dans le gène BLOC1S5, une délétion intragénique homozygote emportant les exons 3 et 4 et une délétion homozygote d’une paire de base (pb) de l’exon 4. BLOC1S5 code une des sous-unités du complexe BLOC-1 qui en compte huit et dont 3 sont déjà associées à des formes d’HPS (7, 8, 9). Ces patientes présentent un phénotype d’albinisme modéré et un défaut d’agrégation plaquettaire par déficit en granules denses. Les plaquettes de la patiente 1 présentent une absence de BLOC-1 montrant que la délétion entraine une dysfonction de BLOC1S5 gênant l’assemblage du complexe. Tandis que l’expression du transcrit humain sauvage de BLOC1S5, dans des mélanocytes murins avec KO de BLOC1S5, non pigmentés, restaure la pigmentation, l’assemblage de BLOC-1 et le trafic de cargo, l’expression du transcrit avec la délétion de la 1ere patiente ne le permet pas. Nous avons ainsi démontré que la perte de fonction de BLOC1S5 est responsable d’une nouvelle forme d’HPS, HPS-11. Le deuxième gène, DCT ou TYRP2 est suspecté de longue date du fait de sa collaboration à la synthèse de la mélanine avec TYR et TYRP1, gènes connus d’albinisme (OCA1 et 3, respectivement). Nous décrivons 2 patientes présentant un albinisme cutané modéré avec atteinte ophtalmologique caractéristique et porteuses de variants de DCT. Une patiente est homozygote pour un variant faux sens de l’exon 1 et l’autre hétérozygote composite pour un variant faux sens de l’exon 1 et une délétion de 14pb de l’exon 9. Les deux variants faux-sens sont situés dans des cystéines très conservées et ont été induits chez la souris par CrispR-Cas9, reproduisant l’hypopigmentation modérée et l’atteinte oculaire. Un modèle poisson zèbre a aussi été produit avec atteinte similaire. Nous concluons donc que DCT est impliqué dans une nouvelle forme d’albinisme oculo-cutané, OCA8. Enfin nous rapportons également le phénotype détaillé de trois patients porteurs de nouveaux variants dans BLOC1S3, associé à l’HPS-8, une des formes les plus rares d’albinisme syndromique. Avant ce travail seulement 2 familles et deux variants étaient décrits dans la littérature. La description détaillée du phénotype de ces patients est une aide aux praticiens dans l’accompagnement diagnostique et pronostique des patients ainsi que pour le conseil génétique. Nous avons donc rapporté deux nouveaux gènes d’albinisme, pour une forme oculocutanée et pour une forme syndromique (HPS), toutes deux apparemment modérées et décrivons de nouveaux variants pour une forme connue mais néanmoins rare d’albinisme syndromique

Novel variants in the BLOC1S3 gene in patients presenting a mild form of Hermansky-Pudlak syndrome

Hermansky-Pudlak syndrome (HPS) associates oculocutaneous albinism and systemic affections including platelet dense granules anomalies leading to bleeding diathesis and, depending on the form, pulmonary fibrosis, immunodeficiency, and/or granulomatous colitis. So far, 11 forms of autosomal recessive HPS caused by pathogenic variants in 11 different genes have been reported. We describe three HPS-8 consanguineous families with different homozygous pathogenic variants in BLOC1S3 (NM_212550.3), one of which is novel. These comprise two deletions leading to a reading frameshift (c.385_403del, c.338_341del) and one in frame deletion (c.444_467del). All patients have moderate oculocutaneous albinism and bleeding diathesis, but other HPS symptoms are not described. One patient diagnosed with HPS-8 suffered from lymphocyte-predominant Hodgkin lymphoma. The mild severity of HPS-8 is consistent with other HPS forms caused by variants in BLOC-1 complex coding genes (HPS-7, DTNBP1; HPS-9, BLOC1S6, HPS-11, BLOC1S5)

Dopachrome tautomerase variants in patients with oculocutaneous albinism

International audiencePurpose: Albinism is a clinically and genetically heterogeneous condition. Despite analysis of the 20 known genes, ~30% patients remain unsolved. We aimed to identify new genes involved in albinism. Methods: We sequenced a panel of genes with known or predicted involvement in melanogenesis in 230 unsolved albinism patients. Results: We identified variants in the Dopachrome tautomerase (DCT) gene in two patients. One was compound heterozygous for a 14-bp deletion in exon 9 and c.118T>A p.(Cys40Ser). The second was homozygous for c.183C>G p.(Cys61Trp). Both patients had mild hair and skin hypopigmentation, and classical ocular features. CRISPR-Cas9 was used in C57BL/6J mice to create mutations identical to the missense variants carried by the patients, along with one loss-of-function indel. When bred to homozygosity the three mutations revealed hypopigmentation of the coat, milder for Cys40Ser compared with Cys61Trp or the frameshift mutation. Histological analysis identified significant hypopigmentation of the retinal pigmented epithelium (RPE) indicating that defective RPE melanogenesis could be associated with eye and vision defects. DCT loss of function in zebrafish embryos elicited hypopigmentation both in melanophores and RPE cells. Conclusion: DCT is the gene for a new type of oculocutaneous albinism that we propose to name OCA8

Evidence of mosaicism in SPAST variant carriers in four French families

International audienceHereditary spastic paraplegias (HSP) are heterogeneous disorders, with more than 70 causative genes. Variants in SPAST are the most frequent genetic etiology and are responsible for spastic paraplegia type 4 (SPG4). Age at onset can vary, even between patients from the same family, and incomplete penetrance is described. Somatic mosaicism is extremely rare with only three patients reported in the literature. We report here SPAST mosaic variants in four unrelated patients. We confirm that mosaicism in SPAST is a very rare event with only four identified cases on more than 300 patients with a SPAST variant previously described by our clinical diagnostic laboratory

Br J Ophthalmol

AIM: Oculocutaneous albinism type 1 (OCA1) is due to TYR mutations. c.1205G>A/p.Arg402Gln (R402Q) is a thermosensitive variant of the TYR gene that has been reported to be responsible for mild forms of OCA1. The aim of our study was to define the phenotype associated with this variant. METHODS: In our retrospective series, among 268 patients diagnosed with OCA1, 122 (45.5%) harboured one pathogenic variant of TYR, and the R402Q variant ensured to be in trans by segregation analysis in 69 patients (25.7%), constituting the 'R402Q-OCA1' group. 146 patients harboured two pathogenic variants of the TYR gene other than R402Q. Clinical records were available for 119 of them, constituting the 'Classical-OCA1' group. RESULTS: Most R402Q-OCA1 patients presented with white or yellow-white hair at birth (71.43%), blond hair later (46.97%), a light phototype but with residual pigmentation (69.64%), and blue eyes (76.56%). Their pigmentation was significantly higher than in the classical-OCA1 group. All patients from the R402Q-OCA1 group presented with ocular features of albinism. However the prevalence of photophobia (78.13%) and iris transillumination (83.87%) and the severity scores of iris transillumination, retinal hypopigmentation and foveal hypoplasia were lower in the R402Q-OCA1 group. Visual acuity was higher in the R402Q-OCA1 group (0.38+/-0.21 logarithm of the minimum angle of resolution vs 0.76+/-0.24). Investigations concerning a possible additive effect of the c.575C>A/p.Ser192 (S192Y) variant of TYR in cis with R402Q, suggested by others, showed no significant impact on the phenotype. CONCLUSION: The R402Q variant leads to variable but generally mild forms of albinism whose less typical presentation may lead to underdiagnosis