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Resumen Introduccion y objetivos TPM1 es uno de los principales genes en la miocardiopatia hipertrofica (MCH). La informacion clinica sobre portadores es relativamente escasa, lo cual limita la interpretacion de los estudios geneticos. Nuestro objetivo es establecer la correlacion genotipo-fenotipo de la variante p.Arg21Leu de TPM1 en una serie de familias. Metodos Se evaluo el TPM1 mediante secuenciacion de nueva generacion en 10.561 probandos con cardiopatias hereditarias. Se genotipifico a los familiares mediante Sanger. Se analizaron la cosegregacion, las caracteristicas clinicas y los eventos cardiovasculares. Se estimo la distribuicion geografica de las familias en Portugal y Espana. Resultados Se identifico la variente p.Arg21Leu de TPM1 en 25/4.099 (0,61%) casos con MCH y estaba ausente en 6.462 controles con otras cardiopatias familiares (p Conclusiones P.Arg21Leu es una variante patogenica de TPM1 asociada con MCH de penetrancia tardia/incompleta y pronostico generalmente favorable

© 2021 American Society of Human Genetics, EDEM3 encodes a protein that converts Man8GlcNAc2 isomer B to Man7-5GlcNAc2. It is involved in the endoplasmic reticulum-associated degradation pathway, responsible for the recognition of misfolded proteins that will be targeted and translocated to the cytosol and degraded by the proteasome. In this study, through a combination of exome sequencing and gene matching, we have identified seven independent families with 11 individuals with bi-allelic protein-truncating variants and one individual with a compound heterozygous missense variant in EDEM3. The affected individuals present with an inherited congenital disorder of glycosylation (CDG) consisting of neurodevelopmental delay and variable facial dysmorphisms. Experiments in human fibroblast cell lines, human plasma, and mouse plasma and brain tissue demonstrated decreased trimming of Man8GlcNAc2 isomer B to Man7GlcNAc2, consistent with loss of EDEM3 enzymatic activity. In human cells, Man5GlcNAc2 to Man4GlcNAc2 conversion is also diminished with an increase of Glc1Man5GlcNAc2. Furthermore, analysis of the unfolded protein response showed a reduced increase in EIF2AK3 (PERK) expression upon stimulation with tunicamycin as compared to controls, suggesting an impaired unfolded protein response. The aberrant plasma N-glycan profile provides a quick, clinically available test for validating variants of uncertain significance that may be identified by molecular genetic testing. We propose to call this deficiency EDEM3-CDG., This work was supported by the EU FP7 large-scale integrating project Genetic and Epigenetic Networks in Cognitive Dysfunction (241995) (to H.v.B.); National Institutes of Health (NIH) grants 5R01GM115730-03 (to M.H.), U54 NS115198 (to A.C.E. and M.H.), and T32GM008638 (to A.C.E.); and the Transatlantic Network of Excellence grant (10CVD03) from the Fondation Leducq and NIH NHGRI U01HG006398 (to D.J.R.). Family 4 was enrolled in the CAUSES Study; investigators include Shelin Adam, Christele Du Souich, Alison Elliott, Anna Lehman, Jill Mwenifumbo, Tanya Nelson, Clara Van Karnebeek, and Jan Friedman; it is funded by Mining for Miracles, British Columbia Children’s Hospital Foundation (grant number F15-01355) and Genome British Columbia (grant number F16-02276). D.L.P. is recipient of a CAPES Fellowship (99999.013311/2013-01). X.B. is supported by an AHA career development award (19CDA34630032).

Objective To describe the antenatal and pathological features of an immature umbilical cord teratoma associated with exomphalos, and to review the literature on this subject. Case presentation An abdominal wall defect, suspected to be an exomphalos, was identified during routine ultrasound examination performed at 13 weeks of gestation. The pregnancy was terminated. Fetopathological examination revealed an immature umbilical cord teratoma associated with exomphalos. Chromosomal microarray analysis was normal. Conclusions Umbilical cord teratomas, albeit very rare, should be emphasized as a possible differential diagnosis when abdominal wall defects are detected. Since cord teratomas may lead to adverse fetal or neonatal outcomes, close follow-up of the fetus is recommended.

Context Congenital generalized lipodystrophy, type 1 (CGL1), due to biallelic pathogenic variants in AGPAT2, is characterized by the near total loss of body fat from the face, trunk, and extremities. Patients develop premature diabetes, hypertriglyceridemia, hepatic steatosis, and polycystic ovary syndrome. However, sparing of the facial fat and precocious pubertal development has not been previously reported in CGL1. Case Description We report a 21-year-old woman of European descent with CGL1 who had sparing of the facial fat and premature thelarche at birth with premature pubarche and menstrual bleeding at age 3 years. Her serum 17-OH progesterone level rose to 1000 ng/dL (30.26 nmol/L) after cosyntropin stimulation test, suggestive of nonclassical congenital adrenal hyperplasia (NCAH) due to 21-hydroxylase deficiency. Hydrocortisone replacement therapy from age 3.5 to 10 years resulted in cessation of menstruation and growth of pubic hair, and a reduction of breast size. Sanger and whole-exome sequencing revealed compound heterozygous variants c.493-1G>C; p.(Leu165_Gln196del), and c.del366_588+534; p.(Leu123Cysfs*55) in AGPAT2 plus c.806G>C; p.(Ser269Thr) and c.844G>T; p.(Val282Leu) in CYP21A2. She developed diabetes at age 13 requiring high-dose insulin and had 7 episodes of acute pancreatitis due to extreme hypertriglyceridemia in the next 5 years. Metreleptin therapy was initiated at age 18 and after 3 years, she had remission of diabetes and hypertriglyceridemia; however, menstrual irregularity and severe hirsutism did not improve. Conclusion Concomitant NCAH in this CGL1 patient was associated with precocious pubertal development and sparing of facial fat. Metreleptin therapy drastically improved her hyperglycemia and hyperlipidemia but not menstrual irregularity and hirsutism.

Pathogenic variants in the NGLY1 gene are associated with a Congenital Disorder of Deglycosylation (CDDG) characterized by delays in reaching developmental milestones, complex hyperkinetic movement disorder, transient elevation of transaminases, and alacrima or hypolacrima. To date, only few cases of NGLY1 deficiency have been identified and reported in the literature. This report highlights a first child of non‐consanguineous parents with no relevant family history who presented with hypotonia and poor weight gain since birth. At 2 months, the child developed paroxysmal cervical dystonia, posteriorly resolving spontaneously by age of 3. Subsequently, delays in reaching developmental milestones, ataxia, dyskinesia, visual impairment due to cone rod retinal dystrophy, low triglycerides, and persistently elevated liver transaminases were observed. Extensive etiological investigation was performed, including array‐CGH and metabolic evaluation with no abnormalities to note. Trio whole exome analysis identified a homozygous pathogenic variant of the NGLY1 gene, c.1891del (p.Gln631Serfs*7), consistent with CDDG. Both parents were confirmed to be heterozygous carriers. The authors discuss in this case, the clinical presentation, the diagnostic challenges, and review other relevant NGLY1 deficiency cases previously reported in the literature. This case, along with the previous reported in the literature, indicates that pathogenic variants in NGLY1 cause a recognizable phenotype and should be considered in patients with a typical presentation. It also suggests that decreased sweating is not present universally in these patients.

Objective: To describe the ultrasonographic, pathologic and molecular findings in a fetus with TAR syndrome, and to illustrate the contribution of chromosomal microarray analysis (CMA) to the etiological investigation of fetal upper limb reduction defects. Case report: A 35-year-old woman was referred for Genetic Counseling after pregnancy termination for severe upper limb bilateral phocomelia detected in the second trimester. Fetal autopsy showed severe shortening of the arms and forearms. The fetal skeletal survey confirmed the absence of the radii, ulnae and humeri. CMA revealed an interstitial deletion in 1q21 including the RBM8A gene. Subsequent Sanger sequencing of this gene identified a hypomorphic mutant allele, c.-21G > A, confirming the diagnosis of TAR syndrome. Conclusion: The differential diagnosis of upper limb defects is broad. Identification of their cause is essential for adequate genetic counseling including prognosis and recurrence risk estimation. CMA should be considered in fetuses with upper limb reduction defects, especially when the thumbs are present. Keywords: 1q21.1 microdeletion, Limb reduction defects, Phocomelia, RBM8A, Thrombocytopenia-absent radius (TAR) syndrome

Intellectual developmental disorder with dysmorphic facies and behavioral abnormalities (IDDFBA) is a recently described autosomal dominant entity caused by pathogenic variants, mostly de novo, in the FBXO11 gene. It presents in the first years of life with highly variable clinical manifestations. The main features of IDDFBA include borderline-to-severe intellectual disability, behavioral problems, hypotonia, facial dysmorphisms, minor skeletal abnormalities, and recurrent infections. Although eye problems, such as refractive errors, eye misalignment and minor visual changes, have been described in about 48% of patients, a major ocular defect, namely, bilateral optic nerve hypoplasia, has been reported in the literature only once. We report an 8-year-old boy with a novel de novo pathogenic variant in FBXO11 gene (NM_001190274.1: c.1166dup, p.Cys390Metfs∗3) and a complex ophthalmological phenotype, consisting of right microphthalmia, very shallow anterior chamber, and persistent pupillary membrane, right dense nuclear cataract, bilateral optic nerve hypoplasia, and bilateral horizontal manifest nystagmus. info:eu-repo/semantics/publishedVersion

Hyper-IgE syndromes and chronic mucocutaneous candidiasis constitute rare primary immunodeficiency syndromes with an overlapping clinical phenotype. In recent years, a growing number of underlying genetic defects have been identified. To characterize the underlying genetic defects in a large international cohort of 275 patients, of whom 211 had been clinically diagnosed with hyper-IgE syndrome and 64 with chronic mucocutaneous candidiasis, targeted panel sequencing was performed, relying on Agilent HaloPlex and Illumina MiSeq technologies. The targeted panel sequencing approach allowed us to identify 87 (32 novel and 55 previously described) mutations in 78 patients, which generated a diagnostic success rate of 28.4%. Specifically, mutations in DOCK8 (26 patients), STAT3 (21), STAT1 (15), CARD9 (6), AIRE (3), IL17RA (2), SPINK5 (3), ZNF341 (2), CARMIL2/RLTPR (1), IL12RB1 (1), and WAS (1) have been detected. The most common clinical findings in this cohort were elevated IgE (81.5%), eczema (71.7%), and eosinophilia (62.9%). Regarding infections, 54.7% of patients had a history of radiologically proven pneumonia, and 28.3% have had other serious infections. History of fungal infection was noted in 53% of cases and skin abscesses in 52.9%. Skeletal or dental abnormalities were observed in 46.2% of patients with a characteristic face being the most commonly reported feature (23.1%), followed by retained primary teeth in 18.9% of patients. Targeted panel sequencing provides a cost-effective first-line genetic screening method which allows for the identification of mutations also in patients with atypical clinical presentations and should be routinely implemented in referral centers., Projekt DEAL; German Ministry of Education and Research (BMBF) [01E01303, 01ZX1306F, 01ZX1306A]; E-rare program of the European Commission EURO-CMC [01GM1502]; German Center for Infection Research DZIF [8000805-3, TTU-IICH_07.801]; German Research Foundation (DFG) [390939984, 39087428], Open Access funding enabled and organized by Projekt DEAL. Financial support for this research came from the German Ministry of Education and Research (BMBF, grant no. 01E01303; sys-INFLAME grant nos. 01ZX1306F and 01ZX1306A), from the E-rare program of the European Commission EURO-CMC (01GM1502), and from the German Center for Infection Research DZIF (8000805-3 and TTU-IICH_07.801). This study was supported by the German Research Foundation (DFG) under Germany's Excellence Strategy CIBSS (EXC-2189) (Project ID 390939984) and RESIST (EXC 2155) (Project ID 39087428).

Pathogenic variants in INPP5E cause Joubert syndrome (JBTS), a ciliopathy with retinal involvement. However, despite sporadic cases in large cohort sequencing studies, a clear association with non-syndromic inherited retinal degenerations (IRDs) has not been made. We validate this association by reporting 16 non-syndromic IRD patients from ten families with bi-allelic mutations in INPP5E. Additional two patients showed early onset IRD with limited JBTS features. Detailed phenotypic description for all probands is presented. We report 14 rare INPP5E variants, 12 of which have not been reported in previous studies. We present tertiary protein modeling and analyze all INPP5E variants for deleteriousness and phenotypic correlation. We observe that the combined impact of INPP5E variants in JBTS and non-syndromic IRD patients does not reveal a clear genotype–phenotype correlation, suggesting the involvement of genetic modifiers. Our study cements the wide phenotypic spectrum of INPP5E disease, adding proof that sequence defects in this gene can lead to early-onset non-syndromic IRD.

Congenital cranial dysinnervation disorders (CCDDs) are a heterogeneous group of neurodevelopmental phenotypes caused by a primary disturbance of innervation due to deficient, absent, or misguided cranial nerves. Although some CCDDs genes are known, several clinical phenotypes and their aetiologies remain to be elucidated. We describe a 12-year-old boy with hypotonia, developmental delay, sensorineural hearing loss, and keratoconjunctivitis due to lack of corneal reflex. He had a long expressionless face, severe oromotor dysfunction, bilateral agenesis/severe hypoplasia of the VIII nerve with marked atresia of the internal auditory canals and cochlear labyrinth malformation. Trio-exome sequencing identified a homozygous loss of function variant in the NEUROG1 gene (NM_006161.2: c.202G > T, p.Glu68*). NEUROG1 is considered a causal candidate for CCDDs based on (i) the previous report of a patient with a homozygous gene deletion and developmental delay, deafness due to absent bilateral VIII nerves, and severe oromotor dysfunction; (ii) a second patient with a homozygous NEUROG1 missense variant and corneal opacity, absent corneal reflex and intellectual disability; and (iii) the knockout mouse model phenotype which highly resembles the disorder observed in humans. Our findings support the growing compelling evidence that loss of NEUROG1 leads to a very distinctive disorder of cranial nerves development. info:eu-repo/semantics/publishedVersion

© The Author(s) 2021. Open AccessThis article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing,adaptation, distribution and reproduction in any medium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the CreativeCommons license, and indicate if changes were made. The images or other third partymaterial in this article are included in the article’s Creative Commons license, unlessindicated otherwise in a credit line to the material. If material is not included in thearticle’s Creative Commons license and your intended use is not permitted by statutoryregulation or exceeds the permitted use, you will need to obtain permission directly fromthe copyright holder. To view a copy of this license, visithttp://creativecommons.org/licenses/by/4.0/., Homozygosity mapping is a powerful method for identifying mutations in patients with recessive conditions, especially in consanguineous families or isolated populations. Historically, it has been used in conjunction with genotypes from highly polymorphic markers, such as DNA microsatellites or common SNPs. Traditional software performs rather poorly with data from Whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS), which are now extensively used in medical genetics. We develop AutoMap, a tool that is both web-based or downloadable, to allow performing homozygosity mapping directly on VCF (Variant Call Format) calls from WES or WGS projects. Following a training step on WES data from 26 consanguineous families and a validation procedure on a matched cohort, our method shows higher overall performances when compared with eight existing tools. Most importantly, when tested on real cases with negative molecular diagnosis from an internal set, AutoMap detects three gene-disease and multiple variant-disease associations that were previously unrecognized, projecting clear benefits for both molecular diagnosis and research activities in medical genetics.

Purpose Pseudoxanthoma elasticum (PXE) is a recessive disorder involving skin, eyes and arteries, mainly caused by ABCC6 pathogenic variants. However, almost one fifth of patients remain genetically unsolved despite extensive genetic screening of ABCC6, as illustrated in a large French PXE series of 220 cases. We searched for new PXE gene(s) to solve the ABCC6-negative patients. Methods First, family-based exome sequencing was performed, in one ABCC6-negative PXE patient with additional neurological features, and her relatives. CYP2U1, involved in hereditary spastic paraplegia type 56 (SPG56), was selected based on this complex phenotype, and the presence of two candidate variants. Second, CYP2U1 sequencing was performed in a retrospective series of 46 additional ABCC6-negative PXE probands. Third, six additional SPG56 patients were evaluated for PXE skin and eye phenotype. Additionally, plasma pyrophosphate dosage and functional analyses were performed in some of these patients. Results 6.4% of ABCC6-negative PXE patients (n=3) harbored biallelic pathogenic variants in CYP2U1. PXE skin lesions with histological confirmation, eye lesions including maculopathy or angioid streaks, and various neurological symptoms were present. CYP2U1 missense variants were confirmed to impair protein function. Plasma pyrophosphate levels were normal. Two SPG56 patients (33%) presented some phenotypic overlap with PXE. Conclusion CYP2U1 pathogenic variants are found in unsolved PXE patients with neurological findings, including spastic paraplegia, expanding the SPG56 phenotype and highlighting its overlap with PXE. The pathophysiology of ABCC6 and CYP2U1 should be explored to explain their respective role and potential interaction in ectopic mineralization.

PurposePathogenic variants in INPP5E cause Joubert syndrome, a systemic disorder that can manifest with retinal degeneration among other clinical features. We aimed to evaluate the role of INPP5E variants in non-syndromic inherited retinal degenerations (IRDs) of varying severity.MethodsTargeted or genome sequencing were performed in 12 unrelated non-syndromic IRD families from multiple research hospitals. Detailed clinical examination was conducted in all probands. The impact of new likely pathogenic variants was modeled on a tertiary INPP5E protein structure and all the new and published variants were analyzed for their deleteriousness and phenotypic correlation.ResultsFourteen INPP5E rare alleles were detected, 12 of which were novel. Retinal degeneration in all 12 probands was clinically distinguishable on the basis of onset and severity into Leber congenital amaurosis (n=4) and a milder, later-onset rod-cone dystrophy (n=8). Two probands showed mild ciliopathy features that resolved in childhood. Analysis of the combined impact of both alleles in syndromic and non-syndromic INPP5E patients did not reveal clear genotype-phenotype correlation, suggesting involvement of genetic modifiers.ConclusionsThe study expands the phenotypic spectrum of disorders due to pathogenic variants in INPP5E and describes a new disease association with previously underdiagnosed forms of early-onset non-syndromic IRD.

INTRODUCTION: Epileptic encephalopathies of childhood are characterized by early seizure-onset and adverse neurological outcomes. The development of new genetic techniques has allowed an exponential identification of the genes that are involved. Over the last years, we have observed a revolution in the diagnostic paradigm. However, there are no international guidelines regarding the diagnosis of genetic epileptic encephalopathies. We aim to discuss the current knowledge about the genetic architecture of epileptic encephalopathies of childhood. MATERIAL AND METHODS: review of the literature about infantile epileptic encephalopathies and the genetic tests currently available. A systematic approach and a diagnostic algorithm to use in clinical practice were proposed. RESULTS: Initially the patient's phenotype should be determined based on the seizure type, electroencephalogram pattern and neuroimaging. Patients with unclear etiology after brain magnetic resonance imaging should undergo an appropriate metabolic investigation to promptly exclude treatable conditions. Further studies should also include other genetic causes, mainly if associated with particular phenotypic features. Chromosomal microarray analysis should be firstly considered, particularly if dysmorphic or polymalformative abnormalities are present. If this is negative and/or there are no physical features, the next step should be next-generation sequencing multigene panels or whole-exome sequencing. Single gene study should only be considered when the patient's phenotype is highly suggestive of a specific syndrome. CONCLUSION: The revolution of the genetic knowledge about epileptic encephalopathies of childhood has led to a complex diagnostic approach. This new paradigm poses significant implications in genetic counselling, treatment and prognosis. Introdução: As encefalopatias epilépticas da infância constituem um grupo de patologias de início precoce e prognóstico neurológico reservado. O desenvolvimento das novas técnicas de estudo genético foi responsável pela identificação de novos genes implicados. Nos últimos anos, assistimos a uma revolução no seu paradigma diagnóstico. Contudo, actualmente não existem recomendações internacionais consensuais sobre a abordagem à investigação das encefalopatias epilépticas genéticas. Pretendemos discutir o conhecimento actual sobre a arquitectura genética das encefalopatias epilépticas infantis. Material e Métodos: Realizou-se uma revisão da literatura das encefalopatias epilépticas infantis genéticas e estudos utilizados no seu diagnóstico. Propomos uma abordagem sistematizada através de um algoritmo diagnóstico a utilizar na prática clínica. Resultados: Inicialmente deve-se determinar o fenótipo do doente com base no tipo de crises, padrão electroencefalográfico e neuroimagem. Nos doentes sem etiologia após resultados de ressonância magnética cranioencefálica, deve-se realizar estudo metabólico apropriado para o diagnóstico prioritário de doenças metabólicas tratáveis. A investigação de outras causas genéticas deve ser considerada, sobretudo perante características fenotípicas sugestivas. Primeiro deve-se realizar a análise de microarray cromossómico, principalmente se existirem alterações dismórficas ou polimalformativas. Se esta for negativa e/ou não existirem elementos físicos distintivos, o próximo passo deve ser realizar os painéis multigénicos ou sequenciação de exoma. Os estudos dirigidos do gene devem ser reservados para quando o fenótipo é indicativo de uma síndrome específica. Conclusão: A marcha diagnóstica das encefalopatias epilépticas tornou-se complexa com a expansão de conhecimentos genéticos. Este novo paradigma apresenta implicações terapêuticas, prognósticas e de aconselhamento familiar. info:eu-repo/semantics/publishedVersion

Copyright © 2020, American College of Medical Genetics and Genomics, Purpose: Determination of genotypic/phenotypic features of GATAD2B-associated neurodevelopmental disorder (GAND). Methods: Fifty GAND subjects were evaluated to determine consistent genotypic/phenotypic features. Immunoprecipitation assays utilizing in vitro transcription-translation products were used to evaluate GATAD2B missense variants' ability to interact with binding partners within the nucleosome remodeling and deacetylase (NuRD) complex. Results: Subjects had clinical findings that included macrocephaly, hypotonia, intellectual disability, neonatal feeding issues, polyhydramnios, apraxia of speech, epilepsy, and bicuspid aortic valves. Forty-one novelGATAD2B variants were identified with multiple variant types (nonsense, truncating frameshift, splice-site variants, deletions, and missense). Seven subjects were identified with missense variants that localized within two conserved region domains (CR1 or CR2) of the GATAD2B protein. Immunoprecipitation assays revealed several of these missense variants disrupted GATAD2B interactions with its NuRD complex binding partners. Conclusions: A consistent GAND phenotype was caused by a range of genetic variants in GATAD2B that include loss-of-function and missense subtypes. Missense variants were present in conserved region domains that disrupted assembly of NuRD complex proteins. GAND's clinical phenotype had substantial clinical overlap with other disorders associated with the NuRD complex that involve CHD3 and CHD4, with clinical features of hypotonia, intellectual disability, cardiac defects, childhood apraxia of speech, and macrocephaly., Research reported in this paper was supported by the National Institutes of Health (NIH) Common Fund, through the Office of Strategic Coordination/Office of the NIH Director under award number U01HG007672. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. GAND50’s seq+ analysis was supported by NIH National Center for Advancing Translational Science (NCATS) UCLA Clinical and Translational Science Institute (CTSI) grant number UL1TR001881. J.P.M. received funding from the National Health and Medical Research Council (APP1012161, APP1063301, APP1126357, APP1058916). T.M.P. and this research was supported by the Cedars-Sinai institutional funding program and the Cedars-Sinai Diana and Steve Marienhoff Fashion Industries Guild Endowed Fellowship in Pediatric Neuromuscular and the Fashion Industries Guild Endowed Fellowship for the Undiagnosed Diseases Program. T.M.P. is especially grateful for the wonderful and continued support from the Cedars-Sinai Fashion Industries Guild.

The 21st annual meeting of the Portuguese Society of Human Genetics (SPGH), organized by Luísa Romão, Ana Sousa and Rosário Pinto Leite, was held in Caparica, Portugal, from the 16th to the 18th of November 2017. Having entered an era in which personalized medicine is emerging as a paradigm for disease diagnosis, treatment and prevention, the program of this meeting intended to include lectures by leading national and international scientists presenting exceptional findings on the genetics of personalized medicine. Various topics were discussed, including cancer genetics, transcriptome dynamics and novel therapeutics for cancers and rare disorders that are designed to specifically target molecular alterations in individual patients. Several panel discussions were held to emphasize (ethical) issues associated with personalized medicine, including genetic cancer counseling. info:eu-repo/semantics/publishedVersion

Epileptic encephalopathies of childhood are characterized by early seizure-onset and adverse neurological outcomes. The development of new genetic techniques has allowed an exponential identification of the genes that are involved. Over the last years, we have observed a revolution in the diagnostic paradigm. However, there are no international guidelines regarding the diagnosis of genetic epileptic encephalopathies. We aim to discuss the current knowledge about the genetic architecture of epileptic encephalopathies of childhood.review of the literature about infantile epileptic encephalopathies and the genetic tests currently available. A systematic approach and a diagnostic algorithm to use in clinical practice were proposed.Initially the patient's phenotype should be determined based on the seizure type, electroencephalogram pattern and neuroimaging. Patients with unclear etiology after brain magnetic resonance imaging should undergo an appropriate metabolic investigation to promptly exclude treatable conditions. Further studies should also include other genetic causes, mainly if associated with particular phenotypic features. Chromosomal microarray analysis should be firstly considered, particularly if dysmorphic or polymalformative abnormalities are present. If this is negative and/or there are no physical features, the next step should be next-generation sequencing multigene panels or whole-exome sequencing. Single gene study should only be considered when the patient's phenotype is highly suggestive of a specific syndrome.The revolution of the genetic knowledge about epileptic encephalopathies of childhood has led to a complex diagnostic approach. This new paradigm poses significant implications in genetic counselling, treatment and prognosis.Introdução: As encefalopatias epilépticas da infância constituem um grupo de patologias de início precoce e prognóstico neurológico reservado. O desenvolvimento das novas técnicas de estudo genético foi responsável pela identificação de novos genes implicados. Nos últimos anos, assistimos a uma revolução no seu paradigma diagnóstico. Contudo, actualmente não existem recomendações internacionais consensuais sobre a abordagem à investigação das encefalopatias epilépticas genéticas. Pretendemos discutir o conhecimento actual sobre a arquitectura genética das encefalopatias epilépticas infantis.Material e Métodos: Realizou-se uma revisão da literatura das encefalopatias epilépticas infantis genéticas e estudos utilizados no seu diagnóstico. Propomos uma abordagem sistematizada através de um algoritmo diagnóstico a utilizar na prática clínica.Resultados: Inicialmente deve-se determinar o fenótipo do doente com base no tipo de crises, padrão electroencefalográfico e neuroimagem. Nos doentes sem etiologia após resultados de ressonância magnética cranioencefálica, deve-se realizar estudo metabólico apropriado para o diagnóstico prioritário de doenças metabólicas tratáveis. A investigação de outras causas genéticas deve ser considerada, sobretudo perante características fenotípicas sugestivas. Primeiro deve-se realizar a análise de microarray cromossómico, principalmente se existirem alterações dismórficas ou polimalformativas. Se esta for negativa e/ou não existirem elementos físicos distintivos, o próximo passo deve ser realizar os painéis multigénicos ou sequenciação de exoma. Os estudos dirigidos do gene devem ser reservados para quando o fenótipo é indicativo de uma síndrome específica.Conclusão: A marcha diagnóstica das encefalopatias epilépticas tornou-se complexa com a expansão de conhecimentos genéticos. Este novo paradigma apresenta implicações terapêuticas, prognósticas e de aconselhamento familiar.

Introduction: Thyroid hormones have an important role in the regulation of lipid metabolism. Higher levels of TSH could be associated with impaired lipid profile and cardiovascular disease, even with normal levels of free thyroxine (FT4). The aim of this study was to investigate the association between TSH levels and lipid profile in patients with primary hypothyroidism. Materials and methods: Retrospective evaluation of patients with primary hypothyroidism treated with levothyroxine (LT4), with normal levels of FT4. Patients with known dyslipidemia were excluded. Thyroid function, total cholesterol (TC), triglycerides (Tg), HDL-cholesterol (HDL) and LDL-cholesterol (LDL) were studied. Patients were divided according to their TSH levels: 0.05) and lower mean LDL levels (96.3mg/dL vs 106.7mg/dL, p>0.05). TSH values positively correlated with higher TC levels (r=0.319, p

Whereas the vast majority of osteogenesis imperfecta (OI) is caused by autosomal dominant defects in the genes encoding type I procollagen, mutations in a myriad of genes affecting type I procollagen biosynthesis or bone formation and homeostasis have now been associated with rare autosomal recessive OI forms. Recently, homozygous or compound heterozygous mutations in BMP1, encoding the metalloproteases bone morphogenetic protein-1 (BMP1) and its longer isoform mammalian Tolloid (mTLD), were identified in 5 children with a severe autosomal recessive form of OI and in 4 individuals with mild to moderate bone fragility. BMP1/mTLD functions as the procollagen carboxy-(C)-proteinase for types I to III procollagen but was also suggested to participate in amino-(N)-propeptide cleavage of types V and XI procollagens and in proteolytic trimming of other extracellular matrix (ECM) substrates. We report the phenotypic characteristics and natural history of 4 adults with severe, progressive OI characterized by numerous fractures, short stature with rhizomelic shortening, and deformity of the limbs and variable kyphoscoliosis, in whom we identified novel biallelic missense and frameshift mutations in BMP1. We show that BMP1/mTLD-deficiency in humans not only results in delayed cleavage of the type I procollagen C-propeptide but also hampers the processing of the small leucine-rich proteoglycan prodecorin, a regulator of collagen fibrillogenesis. Immunofluorescent staining of types I and V collagen and transmission electron microscopy of the dermis show impaired assembly of heterotypic type I/V collagen fibrils in the ECM. Our study thus highlights the severe and progressive nature of BMP1-associated OI in adults and broadens insights into the functional consequences of BMP1/mTLD-deficiency on ECM organization.

A epidermólise bolhosa juncional é uma genodermatose caracterizada por fragilidade cutânea e formação de erosões e bolhas a nível da junção dermo-epidérmica, após trauma minor. Reportamos o caso clínico de um recém-nascido, sexo masculino, caucasiano, com erupção cutânea caracterizada por bolhas tensas e erosões nas mãos, abdómen e couro cabeludo. Realizou-se uma biopsia cutânea para imunoflurescência directa com painel de anticorpos, que foi compatível com o diagnóstico de epidermólise bolhosa juncional não-Herlitz. No estudo genético identificou-se 2 mutações no gene COL17A1. Destaca-se nos antecedentes familiares, um irmão com dermatose bolhosa, falecido na terceira semana de vida por intercorrência infeciosa, sem ter sido possível efectuar o diagnóstico definitivo que possibilitasse o diagnóstico pré-natal. Salientamos a importância da referenciação rápida destes doentes a centros especializados, de forma a possibilitar o diagnóstico precoce desta patologia, uma orientação clínica adequada e um correto aconselhamento genético, incluindo o diagnóstico pré-natal em futuras gravidezes.

IntroductionPseudohypoparathyroidism is a genetically heterogeneous condition characterized by hypocalcemia and hyperphosphatemia resulting from end-organ resistance to parathyroid hormone (PTH). It is classified into several distinct entities (type Ia, Ib, Ic, type II), according to the molecular causes and clinical features of the patients.Case reportWe report a symptomatic case of a pediatric patient with hypocalcaemia, hyperphosphatemia, hypomagnesaemia and an elevated serum PTH level that presented with tetany. The molecular genetic test revealed a GNAS gene methylation defect of the A/B promoter, which confirms the diagnosis of a sporadic form of pseudohypoparathyroidism type Ib.

Variants in 11 genes of the RAS/MAPK signaling pathway have been causally linked to the neuro-cardio-facio-cutaneous syndromes group (NCFCS). Recently, A2ML1 and RIT1 were also associated with these syndromes. Because of the genetic and clinical heterogeneity of NCFCS, it is challenging to define strategies for their molecular diagnosis. The aim of this study was to develop and validate a massive parallel sequencing (MPS)-based strategy for the molecular diagnosis of NCFCS. A multiplex PCR-based strategy for the enrichment of the 13 genes and a variant prioritization pipeline was established. Two sets of genomic DNA samples were studied using the Ion PGM System: (1) training set (n =15) to optimize the strategy and (2) validation set (n = 20) to validate and evaluate the power of the new methodology. Sanger sequencing was performed to confirm all variants and low covered regions. All variants identified by Sanger sequencing were detected with our MPS approach. The methodology resulted in an experimental approach with a specificity of 99.0% and a maximum analytical sensitivity of ≥ 98.2% with a confidence of 99%. Importantly, two patients (out of 20) harbored described disease-causing variants in genes that are not routinely tested (RIT1 and SHOC2). The addition of less frequently altered genes increased in ≈ 10% the diagnostic yield of the strategy currently used. The presented workflow provides a comprehensive genetic screening strategy for patients with NCFCS in a fast and cost-efficient manner. This approach demonstrates the potential of a combined MPS-Sanger sequencing-based strategy as an effective diagnostic tool for heterogeneous diseases.

Three overlapping conditions, namely Rothmund-Thomson (RTS), Baller-Gerold (BGS) and RAPADILINO syndromes, have been attributed to RECQL4 mutations. Differential diagnoses depend on the clinical presentation, but the numbers of known genes remain low, leading to the widespread prescription of RECQL4 sequencing. The aim of our study was therefore to determine the best clinical indicators for the presence of RECQL4 mutations in a series of 39 patients referred for RECQL4 molecular analysis and belonging to the RTS (27 cases) and BGS (12 cases) spectrum. One or two deleterious RECQL4 mutations were found in 10/27 patients referred for RTS diagnosis. Clinical and molecular reevaluation led to a different diagnosis in 7/17 negative cases, including Clericuzio-type poikiloderma with neutropenia, hereditary sclerosing poikiloderma, and craniosynostosis/anal anomalies/porokeratosis. No RECQL4 mutations were found in the BGS group without poikiloderma, confirming that RECQL4 sequencing was not indicated in this phenotype. One chromosomal abnormality and one TWIST mutation was found in this cohort. This study highlights the search for differential diagnoses before the prescription of RECQL4 sequencing in this clinically heterogeneous group. The combination of clinically defined subgroups and next-generation sequencing will hopefully bring to light new molecular bases of syndromes with poikiloderma, as well as BGS without poikiloderma.

SLC26A2-related dysplasias encompass a spectrum of diseases: from lethal achondrogenesis type 1B (ACG1B; MIM #600972) and atelosteogenesis type 2 (AO2; MIM #256050) to classical diastrophic dysplasia (cDTD; MIM #222600) and recessive multiple epiphyseal dysplasia (rMED; MIM #226900). This study aimed at characterizing clinically, radiologically and molecularly 14 patients affected by non-lethal SLC26A2-related dysplasias and at evaluating genotype-phenotype correlation. Phenotypically, eight patients were classified as cDTD, four patients as rMED and two patients had an intermediate phenotype (mild DTD - mDTD, previously 'DTD variant'). The Arg279Trp mutation was present in all patients, either in homozygosity (resulting in rMED) or in compound heterozygosity with the known severe alleles Arg178Ter or Asn425Asp (resulting in DTD) or with the mutation c.727-1G>C (causing mDTD). The 'Finnish mutation', c.-26+2T>C, and the p.Cys653Ser, both frequent mutations in non-Portuguese populations, were not identified in any of the patients of our cohort and are probably very rare in the Portuguese population. A targeted mutation analysis for p.Arg279Trp and p.Arg178Ter in the Portuguese population allows the identification of approximately 90% of the pathogenic alleles.

We performed homozygosity mapping in two recently reported pedigrees from Portugal and Mexico with an autosomal-recessive autoinflammatory syndrome characterized by joint contractures, muscle atrophy, microcytic anemia, and panniculitis-induced lipodystrophy (JMP). This revealed only one homozygous region spanning 2.4 Mb (5818 SNPs) on chromosome 6p21 shared by all three affected individuals from both families. We directly sequenced genes involved in immune response located in this critical region, excluding the HLA complex genes. We found a homozygous missense mutation c.224C>T (p.Thr75Met) in the proteasome subunit, beta-type, 8 (PSMB8) gene in affected patients from both pedigrees. The mutation segregated in an autosomal-recessive fashion and was not detected in 275 unrelated ethnically matched healthy subjects. PSMB8 encodes a catalytic subunit of the 20S immunoproteasomes called β5i. Immunoproteasome-mediated proteolysis generates immunogenic epitopes presented by major histocompatibility complex (MHC) class I molecules. Threonine at position 75 is highly conserved and its substitution with methionine disrupts the tertiary structure of PSMB8. As compared to normal lymphoblasts, those from an affected patient showed significantly reduced chymotrypsin-like proteolytic activity mediated by immunoproteasomes. We conclude that mutations in PSMB8 cause JMP syndrome, most probably by affecting MHC class I antigen processing.

Context: Genetic lipodystrophies are rare disorders characterized by partial or complete loss of adipose tissue and predisposition to insulin resistance and its complications such as diabetes mellitus, hypertriglyceridemia, hepatic steatosis, acanthosis nigricans, and polycystic ovarian syndrome. Objective: The objective of the study was to report a novel autosomal recessive lipodystrophy syndrome. Results: We report the detailed phenotype of two males and one female patient, 26–34 yr old, belonging to two pedigrees with an autosomal recessive syndrome presenting with childhood-onset lipodystrophy, muscle atrophy, severe joint contractures, erythematous skin lesions, and microcytic anemia. Other variable clinical features include hypergammaglobulinemia, hepatosplenomegaly, generalized seizures, and basal ganglia calcification. None of the patients had diabetes mellitus or acanthosis nigricans. Two had mild hypertriglyceridemia and all had low levels of high-density lipoprotein cholesterol. Skin biopsy of an erythematous nodular skin lesion from one of the patients revealed evidence of panniculitis. The lipodystrophy initially affected the upper body but later became generalized involving abdomen and lower extremities as well. Conclusions: We conclude that these patients represent a novel autoinflammatory syndrome resulting in joint contractures, muscle atrophy, microcytic anemia, and panniculitis-induced lipodystrophy. The molecular genetic basis of this disorder remains to be elucidated.

Mutations in the gene that encodes Fibroblast Growth Factor Receptor 3 (FGFR3) are associated with Achondroplasia (MIM 100800), Hypochondroplasia (MIM 146000), Muenke Syndrome (MIM 602849), Thanatophoric Dysplasia (MIM 187600, MIM 187601) and Lacrimo-Auriculo-Dento-Digital Syndrome (MIM 149730).Here we report a clinical and molecular study in a large cohort of 125 Portuguese patients with these skeletal disorders. The identification of the P250R mutation allowed the confirmation of the Muenke Syndrome in 9 out of the 52 cases referred. Two known mutations were found in the Thanatophoric Dysplasia referred cases. No mutations were identified in the LADD syndrome patient. In Achondroplasia and Hypochondroplasia, genetic heterogeneity was present amongst the 70 clinically diagnosed patients with 5 different mutations identified. As in other studies, complex phenotypic heterogeneity amongst patients carrying the same gene defect was observed. In several cases, the new amino acids encoded, as a consequence of mutations, were related to the severity of patients' phenotype. The presence of 10 misdiagnosed cases emphasizes the importance of performing mutation analysis of the hotspot regions responsible for both dysplasias (Ach and Hch). For patients with an unquestionable clinical diagnosis, lacking the most common mutations, a complete screening of FGFR3 is necessary.

The Aristaless-related homeobox (ARX) gene is implicated in intellectual disability with the most frequent pathogenic mutations leading to expansions of the first two polyalanine tracts. Here, we describe analysis of the ARX gene outlining the approaches in the Australian and Portuguese setting, using an integrated clinical and molecular strategy. We report variants in the ARX gene detected in 19 patients belonging to 17 families. Seven pathogenic variants, being expansion mutations in both polyalanine tract 1 and tract 2, were identifyed, including a novel mutation in polyalanine tract 1 that expands the first tract to 20 alanines. This precise number of alanines is sufficient to cause pathogenicity when expanded in polyalanine tract 2. Five cases presented a probably non-pathogenic variant, including the novel HGVS: c.441_455del, classified as unlikely disease causing, consistent with reports that suggest that in frame deletions in polyalanine stretches of ARX rarely cause intellectual disability. In addition, we identified five cases with a variant of unclear pathogenic significance. Owing to the inconsistent ARX variants description, publications were reviewed and ARX variant classifications were standardized and detailed unambiguously according to recommendations of the Human Genome Variation Society. In the absence of a pathognomonic clinical feature, we propose that molecular analysis of the ARX gene should be included in routine diagnostic practice in individuals with either nonsyndromic or syndromic intellectual disability. A definitive diagnosis of ARX-related disorders is crucial for an adequate clinical follow-up and accurate genetic counseling of at-risk family members. Unit for Multidisciplinary Research in Biomedicine, UMIB, ICBAS-UP, Porto, Portugal was funded by FEDER funds of the Operational Program for Competitiveness Factors – COMPETE through FCT – Foundation for Science and Technology under the project: Fcomp-01-0124-FEDER-015896. The Neurogenetics research program in the Department of Paediatrics, University of Adelaide, Australia was funded by the Australian National Health and Medical Research Council (Grant No. 1063025). C. S. is supported Australian Research Council (Future Fellowship FT120100086)

Whereas the vast majority of osteogenesis imperfecta (OI) is caused by autosomal dominant defects in the genes encoding type I procollagen, mutations in a myriad of genes affecting type I procollagen biosynthesis or bone formation and homeostasis have now been associated with rare autosomal recessive OI forms. Recently, homozygous or compound heterozygous mutations in BMP1, encoding the metalloproteases bone morphogenetic protein-1 (BMP1) and its longer isoform mammalian Tolloid (mTLD), were identified in 5 children with a severe autosomal recessive form of OI and in 4 individuals with mild to moderate bone fragility. BMP1/mTLD functions as the procollagen carboxy-(C)-proteinase for types I to III procollagen but was also suggested to participate in amino-(N)-propeptide cleavage of types V and XI procollagens and in proteolytic trimming of other extracellular matrix (ECM) substrates. We report the phenotypic characteristics and natural history of 4 adults with severe, progressive OI characterized by numerous fractures, short stature with rhizomelic shortening, and deformity of the limbs and variable kyphoscoliosis, in whom we identified novel biallelic missense and frameshift mutations in BMP1. We show that BMP1/mTLD-deficiency in humans not only results in delayed cleavage of the type I procollagen C-propeptide but also hampers the processing of the small leucine-rich proteoglycan prodecorin, a regulator of collagen fibrillogenesis. Immunofluorescent staining of types I and V collagen and transmission electron microscopy of the dermis show impaired assembly of heterotypic type I/V collagen fibrils in the ECM. Our study thus highlights the severe and progressive nature of BMP1-associated OI in adults and broadens insights into the functional consequences of BMP1/mTLD-deficiency on ECM organization.

Classical Hodgkin’s disease (HD) and B-cell non-Hodgkin lymphoma (NHL) occasionally occur in the same patient. Such composite lymphomas represent interesting models to study the pathogenesis of B-cell lymphomas and the relationship between HD and B-cell NHL. We analyzed two composite lymphomas (a combination of classical HD with follicular lymphoma [FL] and a combination of classical HD with B-cell chronic lymphocytic leukemia [B-CLL]) by micromanipulation of single cells from tissue sections and amplification of immunoglobulin V region genes for the clonal relationship of the tumor cells. In both cases, clonally related variable (V) genes with both shared as well as distinct somatic mutations were obtained from the two lymphomas, showing that in each of the cases the distinct tumor cells were members of a common germinal center (GC) B-cell clone. FL cells from two different lymph nodes of patient 1 showed a similar mutation pattern, suggesting that infiltration of these lymph nodes by tumor cells was not restricted to a particular FL cell or subclone. In the FL, a single cell was identified with a mutation signature indicating that premalignant cells can persist in the tissue. The cases presented here further underline the close relationship between HD and B-cell NHL and the role of the GC in lymphomagenesis. Whereas the latter was already suggested for FL and HD, the present study indicates that also in the B-CLL subset characterized by mutated Ig genes, important steps in malignant transformation happen in the GC, and that HRS cells can derive from CD5-positive B cells.

Costello syndrome is characterized by severe failure-to-thrive, short stature, cardiac abnormalities (heart defects, tachyarrhythmia, and hypertrophic cardiomyopathy (HCM)), distinctive facial features, a predisposition to papillomata and malignant tumors, postnatal cerebellar overgrowth resulting in Chiari 1 malformation, and cognitive disabilities. De novo germline mutations in the proto-oncogene HRAS cause Costello syndrome. Most mutations affect the glycine residues in position 12 or 13, and more than 80% of patients share p.G12S. To test the hypothesis that subtle genotype-phenotype differences exist, we report the first cohort comparison between 12 Costello syndrome individuals with p.G13C and individuals with p.G12S. The individuals with p.G13C had many typical findings including polyhydramnios, failure-to-thrive, HCM, macrocephaly with posterior fossa crowding, and developmental delay. Subjectively, their facial features were less coarse. Statistically significant differences included the absence of multifocal atrial tachycardia (P-value = 0.033), ulnar deviation of the wrist (P < 0.001) and papillomata (P = 0.003), and fewer neurosurgical procedures (P = 0.024). Fewer individuals with p.G13C had short stature (height below -2 SD) without use of growth hormone (P < 0.001). The noteworthy absence of malignant tumors did not reach statistical significance. Novel ectodermal findings were noted in individuals with p.G13C, including loose anagen hair resulting in easily pluckable hair with a matted appearance, different from the tight curls typical for most Costello syndrome individuals. Unusually long eye lashes requiring trimming are a novel finding we termed dolichocilia. These distinctive ectodermal findings suggest a cell type specific effect of this particular mutation. Additional patients are needed to validate these findings.

Hepatocyte nuclear factor 1 beta (HNF1B) plays an important role in embryonic development, namely in the kidney, pancreas, liver, genital tract, and gut. Heterozygous germline mutations of HNF1B are associated with the renal cysts and diabetes syndrome (RCAD). Affected individuals may present a variety of renal developmental abnormalities and/or maturity-onset diabetes of the young (MODY). A Portuguese 19-month-old male infant was evaluated due to hypoplastic glomerulocystic kidney disease and renal dysfunction diagnosed in the neonatal period that progressed to stage 5 chronic renal disease during the first year of life. His mother was diagnosed with a solitary hypoplastic microcystic left kidney at age 20, with stage 2 chronic renal disease established at age 35, and presented bicornuate uterus, pancreatic atrophy, and gestational diabetes. DNA sequence analysis of HNF1B revealed a novel germline frameshift insertion (c.110_111insC or c.110dupC) in both the child and the mother. A review of the literature revealed a total of 106 different HNF1B mutations, in 236 mutation-positive families, comprising gross deletions (34%), missense mutations (31%), frameshift deletions or insertions (15%), nonsense mutations (11%), and splice-site mutations (8%). The study of this family with an unusual presentation of hypoplastic glomerulocystic kidney disease with neonatal renal dysfunction identified a previously unreported mutation of the HNF1B gene, thereby expanding the spectrum of known mutations associated with renal developmental disorders.

Although most congenital heart defects are isolated abnormalities of embryonic development, with little genetic contribution, a small number are components of syndromes. In such cases, an accurate diagnosis has important implications for individual prognosis and familial genetic counseling. Alagille syndrome (AGS) is a dominantly inherited multisystem developmental disorder, which primarily affects the liver, heart, eyes, skeleton, and face. In recent years, the identification of the AGS gene has drawn attention to the existence of subclinical carriers, and broadened the spectrum of phenotypical variation associated with this syndrome. The authors present a case of mother and son with benign stenosis of the pulmonary artery branches. Subtle facial aspects suggested the diagnosis of AGS, which was confirmed by molecular analysis. Relevant clinical investigations and diagnostic implications are discussed.

Introduction McCune–Albright syndrome is a rare sporadic disease characterized by fibrous bone dysplasia, café-au-lait skin spots and variable hyperfunctional endocrinopathies. McCune–Albright syndrome is caused by somatic postzygotic activating mutations in the GNAS gene that produce a broad spectrum of effects. Case presentation We report a case of McCune–Albright syndrome with multi-organ manifestations in the neonatal period. A newborn preterm black girl was referred to our Neonatal Intensive Care Unit at the age of 17 days for suspected extrahepatic cholestasis. On clinical examination she presented failure to thrive, jaundice, hypertension, marked hypotonia and café-au-lait spots on her back and lower limbs. An abdominal ultrasound excluded extrahepatic causes of cholestasis but revealed bilateral serpiginous adrenal hyperplasia. These clinical findings suggested a diagnosis of McCune–Albright syndrome with multi-organ involvement. Laboratory data confirmed adrenocorticotropic hormone-independent Cushing’s syndrome, hyperthyroidism, cholestasis and elevated transaminases. Ventricular hypertrophy was demonstrated by echocardiography. The baby girl underwent medical treatment of Cushing’s syndrome with metyrapone which was followed by a rapid recovery. A mosaic activating GNAS gene mutation was found on DNA extracted from a buccal swab sample. However, she died at 4 months due to a respiratory infection. Conclusion In the neonatal period the diagnosis of McCune–Albright syndrome depends on having a high index of suspicion and café-au-lait spots may be the clue for the diagnosis.

BACKGROUND: Classical Hodgkin's disease (HD) and B-cell non-Hodgkin lymphoma (NHL) occasionally occur in the same patient. Such composite lymphomas represent interesting models to study the pathogenesis of B-cell lymphomas and the relationship between HD and B-cell NHL. MATERIALS AND METHODS: We analyzed two composite lymphomas (a combination of classical HD with follicular lymphoma [FL] and a combination of classical HD with B-cell chronic lymphocytic leukemia [B-CLL]) by micromanipulation of single cells from tissue sections and amplification of immunoglobulin V region genes for the clonal relationship of the tumor cells. RESULTS: In both cases, clonally related variable (V) genes with both shared as well as distinct somatic mutations were obtained from the two lymphomas, showing that in each of the cases the distinct tumor cells were members of a common germinal center (GC) B-cell clone. FL cells from two different lymph nodes of patient 1 showed a similar mutation pattern, suggesting that infiltration of these lymph nodes by tumor cells was not restricted to a particular FL cell or subclone. In the FL, a single cell was identified with a mutation signature indicating that premalignant cells can persist in the tissue. CONCLUSIONS: The cases presented here further underline the close relationship between HD and B-cell NHL and the role of the GC in lymphomagenesis. Whereas the latter was already suggested for FL and HD, the present study indicates that also in the B-CLL subset characterized by mutated Ig genes, important steps in malignant transformation happen in the GC, and that HRS cells can derive from CD5-positive B cells.