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Purpose: Postzygotic activating mutations of PIK3CA cause a wide range of mosaic disorders collectively referred to as PIK3CA -related overgrowth spectrum (PROS). We describe the diagnostic yield and characteristics of PIK3CA sequencing in PROS. Methods: We performed ultradeep next-generation sequencing (NGS) of PIK3CA in various tissues from 162 patients referred to our clinical laboratory and assessed diagnostic yield by phenotype and tissue tested. Results: We identified disease-causing mutations in 66.7% (108/162) of patients, with mutant allele levels as low as 1%. The diagnostic rate was higher (74%) in syndromic than in isolated cases (35.5%; P = 9.03 × 10 −5 ). We identified 40 different mutations and found strong oncogenic mutations more frequently in patients without brain overgrowth (50.6%) than in those with brain overgrowth (15.2%; P = 0.00055). Mutant allele levels were higher in skin and overgrown tissues than in blood and buccal samples ( P = 3.9 × 10 −25 ), regardless of the phenotype. Conclusion: Our data demonstrate the value of ultradeep NGS for molecular diagnosis of PROS, highlight its substantial allelic heterogeneity, and confirm that optimal diagnosis requires fresh skin or surgical samples from affected regions. Our findings may be of value in guiding future recommendations for genetic testing in PROS and other mosaic conditions. Genet Med advance online publication 02 February 2017

BackgroundArthrogryposis multiplex congenita (AMC) is characterised by congenital joint contractures in two or more body areas. AMC exhibits wide phenotypic and genetic heterogeneity. Our goals were to improve the genetic diagnosis rates of AMC, to evaluate the added value of whole exome sequencing (WES) compared with targeted exome sequencing (TES) and to identify new genes in 315 unrelated undiagnosed AMC families.MethodsSeveral genomic approaches were used including genetic mapping of disease loci in multiplex or consanguineous families, TES then WES. Sanger sequencing was performed to identify or validate variants.ResultsWe achieved disease gene identification in 52.7% of AMC index patients including nine recently identified genes (CNTNAP1, MAGEL2, ADGRG6, ADCY6, GLDN, LGI4, LMOD3, UNC50 and SCN1A). Moreover, we identified pathogenic variants in ASXL3 and STAC3 expanding the phenotypes associated with these genes. The most frequent cause of AMC was a primary involvement of skeletal muscle (40%) followed by brain (22%). The most frequent mode of inheritance is autosomal recessive (66.3% of patients). In sporadic patients born to non-consanguineous parents (n=60), de novo dominant autosomal or X linked variants were observed in 30 of them (50%).ConclusionNew genes recently identified in AMC represent 21% of causing genes in our cohort. A high proportion of de novo variants were observed indicating that this mechanism plays a prominent part in this developmental disease. Our data showed the added value of WES when compared with TES due to the larger clinical spectrum of some disease genes than initially described and the identification of novel genes.

DNA polymerase δ is one of the three main enzymes responsible for DNA replication. POLD1 heterozygous missense variants in the exonuclease domain result in a cancer predisposition phenotype. In contrast, heterozygous variants in POLD1 polymerase domain have more recently been shown to be the underlying basis of the distinct autosomal dominant multisystem lipodystrophy disorder, MDPL (mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome OMIM # 615381), most commonly a recurrent in-frame deletion of serine at position 604, accounting for 18 of the 21 reported cases of this condition. One patient with an unusually severe phenotype has been reported, caused by a de novo c. 3209 T > A, (p.(Ile1070Asn)) variant in the highly conserved CysB motif in the C-terminal of the POLD1 protein. This region has recently been shown to bind an iron-sulphur cluster of the 4Fe-4S type. This report concerns a novel de novo missense variant in the CysB region, c.3219 G > C, (p.(Ser1073Arg)) in a male child with a milder phenotype. Using in silico analysis in the context of the recently published structure of human Polymerase δ holoenzyme, we compared these and other variants which lie in close proximity but result in differing degrees of severity and varying features. We hypothesise that the c.3219 G > C, (p.(Ser1073Arg)) substitution likely causes reduced binding of the iron-sulphur cluster without significant disruption of protein structure, while the previously reported c.3209 T > A (p.(Ile1070Asn)) variant likely has a more profound impact on structure and folding in the region. Our analysis supports a central role for the CysB region in regulating POLD1 activity in health and disease.

PurposeWide access to clinical exome/genome sequencing (ES/GS) enables the identification of multiple molecular diagnoses (MMDs), being a long-standing but underestimated concept, defined by two or more causal loci implicated in the phenotype of an individual with a rare disease. Only few series report MMDs rates (1.8% to 7.1%). This study highlights the increasing role of MMDs in a large cohort of individuals addressed for congenital anomalies/intellectual disability (CA/ID).MethodsFrom 2014 to 2021, our diagnostic laboratory rendered 880/2658 positive ES diagnoses for CA/ID aetiology. Exhaustive search on MMDs from ES data was performed prospectively (January 2019 to December 2021) and retrospectively (March 2014 to December 2018).ResultsMMDs were identified in 31/880 individuals (3.5%), responsible for distinct (9/31) or overlapping (22/31) phenotypes, and potential MMDs in 39/880 additional individuals (4.4%).ConclusionMMDs are frequent in CA/ID and remain a strong challenge. Reanalysis of positive ES data appears essential when phenotypes are partially explained by the initial diagnosis or atypically enriched overtime. Up-to-date clinical data, clinical expertise from the referring physician, strong interactions between clinicians and biologists, and increasing gene discoveries and improved ES bioinformatics tools appear all the more fundamental to enhance chances of identifying MMDs. It is essential to provide appropriate patient care and genetic counselling.

Hypomelanosis of Ito (HI) is a skin marker of somatic mosaicism. Mosaic MTOR pathogenic variants have been reported in HI with brain overgrowth. We sought to delineate further the pigmentary skin phenotype and clinical spectrum of neurodevelopmental manifestations of MTOR-related HI. From two cohorts totaling 71 patients with pigmentary mosaicism, we identified 14 patients with Blaschko-linear and one with flag-like pigmentation abnormalities, psychomotor impairment or seizures, and a postzygotic MTOR variant in skin. Patient records, including brain magnetic resonance image (MRI) were reviewed. Immunostaining (n = 3) for melanocyte markers and ultrastructural studies (n = 2) were performed on skin biopsies. MTOR variants were present in skin, but absent from blood in half of cases. In a patient (p.[Glu2419Lys] variant), phosphorylation of p70S6K was constitutively increased. In hypopigmented skin of two patients, we found a decrease in stage 4 melanosomes in melanocytes and keratinocytes. Most patients (80%) had macrocephaly or (hemi)megalencephaly on MRI. MTOR-related HI is a recognizable neurocutaneous phenotype of patterned dyspigmentation, epilepsy, intellectual deficiency, and brain overgrowth, and a distinct subtype of hypomelanosis related to somatic mosaicism. Hypopigmentation may be due to a defect in melanogenesis, through mTORC1 activation, similar to hypochromic patches in tuberous sclerosis complex. [Display omitted]

The phenotypic spectrum of GLI3 mutations includes autosomal dominant Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS). PHS was first described as a lethal condition associating hypothalamic hamartoma, postaxial or central polydactyly, anal atresia and bifid epiglottis. Typical GCPS combines polysyndactyly of hands and feet and craniofacial features. Genotype-phenotype correlations have been found both for the location and the nature of GLI3 mutations, highlighting the bifunctional nature of GLI3 during development. Here we report on the molecular and clinical study of 76 cases from 55 families with either a GLI3 mutation (49 GCPS and 21 PHS), or a large deletion encompassing the GLI3 gene (6 GCPS cases). Most of mutations are novel and consistent with the previously reported genotype-phenotype correlation. Our results also show a correlation between the location of the mutation and abnormal corpus callosum observed in some patients with GCPS. Fetal PHS observations emphasize on the possible lethality of GLI3 mutations and extend the phenotypic spectrum of malformations such as agnathia and reductional limbs defects. GLI3 expression studied by in situ hybridization during human development confirms its early expression in target tissues.

TBR1, a T-box transcription factor expressed in the cerebral cortex, regulates the expression of several candidate genes for autism spectrum disorders (ASD). Although TBR1 has been reported as a high-confidence risk gene for ASD and intellectual disability (ID) in functional and clinical reports since 2011, TBR1 has only recently been recorded as a human disease gene in the OMIM database. Currently, the neurodevelopmental disorders and structural brain anomalies associated with TBR1 variants are not well characterized. Through international data sharing, we collected data from 25 unreported individuals and compared them with data from the literature. We evaluated structural brain anomalies in seven individuals by analysis of MRI images, and compared these with anomalies observed in TBR1 mutant mice. The phenotype included ID in all individuals, associated to autistic traits in 76% of them. No recognizable facial phenotype could be identified. MRI analysis revealed a reduction of the anterior commissure and suggested new features including dysplastic hippocampus and subtle neocortical dysgenesis. This report supports the role of TBR1 in ID associated with autistic traits and suggests new structural brain malformations in humans. We hope this work will help geneticists to interpret TBR1 variants and diagnose ASD probands.

PurposeHypomelanosis of Ito (HI) is a skin marker of somatic mosaicism. Mosaic MTOR pathogenic variants have been reported in HI with brain overgrowth. We sought to delineate further the pigmentary skin phenotype and clinical spectrum of neurodevelopmental manifestations of MTOR-related HI.MethodsFrom two cohorts totaling 71 patients with pigmentary mosaicism, we identified 14 patients with Blaschko-linear and one with flag-like pigmentation abnormalities, psychomotor impairment or seizures, and a postzygotic MTOR variant in skin. Patient records, including brain magnetic resonance image (MRI) were reviewed. Immunostaining (n = 3) for melanocyte markers and ultrastructural studies (n = 2) were performed on skin biopsies.ResultsMTOR variants were present in skin, but absent from blood in half of cases. In a patient (p.[Glu2419Lys] variant), phosphorylation of p70S6K was constitutively increased. In hypopigmented skin of two patients, we found a decrease in stage 4 melanosomes in melanocytes and keratinocytes. Most patients (80%) had macrocephaly or (hemi)megalencephaly on MRI.ConclusionMTOR-related HI is a recognizable neurocutaneous phenotype of patterned dyspigmentation, epilepsy, intellectual deficiency, and brain overgrowth, and a distinct subtype of hypomelanosis related to somatic mosaicism. Hypopigmentation may be due to a defect in melanogenesis, through mTORC1 activation, similar to hypochromic patches in tuberous sclerosis complex.