RESUMO
PURPOSE: Mowat-Wilson syndrome (MWS) is a rare intellectual disability/multiple congenital anomalies syndrome caused by heterozygous mutation of the ZEB2 gene. It is generally underestimated because its rarity and phenotypic variability sometimes make it difficult to recognize. Here, we aimed to better delineate the phenotype, natural history, and genotype-phenotype correlations of MWS. METHODS: In a collaborative study, we analyzed clinical data for 87 patients with molecularly confirmed diagnosis. We described the prevalence of all clinical aspects, including attainment of neurodevelopmental milestones, and compared the data with the various types of underlying ZEB2 pathogenic variations. RESULTS: All anthropometric, somatic, and behavioral features reported here outline a variable but highly consistent phenotype. By presenting the most comprehensive evaluation of MWS to date, we define its clinical evolution occurring with age and derive suggestions for patient management. Furthermore, we observe that its severity correlates with the kind of ZEB2 variation involved, ranging from ZEB2 locus deletions, associated with severe phenotypes, to rare nonmissense intragenic mutations predicted to preserve some ZEB2 protein functionality, accompanying milder clinical presentations. CONCLUSION: Knowledge of the phenotypic spectrum of MWS and its correlation with the genotype will improve its detection rate and the prediction of its features, thus improving patient care.
Assuntos
Doença de Hirschsprung/diagnóstico , Doença de Hirschsprung/genética , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Microcefalia/diagnóstico , Microcefalia/genética , Anormalidades Múltiplas/genética , Adolescente , Adulto , Criança , Pré-Escolar , Fácies , Feminino , Estudos de Associação Genética/métodos , Genótipo , Humanos , Lactente , Masculino , Mutação , Fenótipo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genéticaRESUMO
PURPOSE: Mowat-Wilson syndrome (MWS) is a genetic disease characterized by distinctive facial features, moderate to severe intellectual disability, and congenital malformations, including Hirschsprung disease, genital and eye anomalies, and congenital heart defects, caused by haploinsufficiency of the ZEB2 gene. To date, no characteristic pattern of brain dysmorphology in MWS has been defined. METHODS: Through brain magnetic resonance imaging (MRI) analysis, we delineated a neuroimaging phenotype in 54 MWS patients with a proven ZEB2 defect, compared it with the features identified in a thorough review of published cases, and evaluated genotype-phenotype correlations. RESULTS: Ninety-six percent of patients had abnormal MRI results. The most common features were anomalies of corpus callosum (79.6% of cases), hippocampal abnormalities (77.8%), enlargement of cerebral ventricles (68.5%), and white matter abnormalities (reduction of thickness 40.7%, localized signal alterations 22.2%). Other consistent findings were large basal ganglia, cortical, and cerebellar malformations. Most features were underrepresented in the literature. We also found ZEB2 variations leading to synthesis of a defective protein to be favorable for psychomotor development and some epilepsy features but also associated with corpus callosum agenesis. CONCLUSION: This study delineated the spectrum of brain anomalies in MWS and provided new insights into the role of ZEB2 in neurodevelopment.Genet Med advance online publication 10 November 2016.
Assuntos
Encéfalo/diagnóstico por imagem , Doença de Hirschsprung/diagnóstico por imagem , Deficiência Intelectual/diagnóstico por imagem , Imageamento por Ressonância Magnética , Microcefalia/diagnóstico por imagem , Neuroimagem , Encéfalo/patologia , Criança , Pré-Escolar , Estudos de Coortes , Epilepsia/patologia , Fácies , Feminino , Genótipo , Haploinsuficiência , Doença de Hirschsprung/genética , Doença de Hirschsprung/patologia , Humanos , Lactente , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Estudos Longitudinais , Masculino , Microcefalia/genética , Microcefalia/patologia , Fenótipo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genéticaAssuntos
Anormalidades Cardiovasculares/genética , Deficiência Intelectual/genética , Síndrome de Sotos/genética , Adolescente , Adulto , Doenças da Aorta/complicações , Doenças da Aorta/diagnóstico , Doenças da Aorta/genética , Doenças da Aorta/patologia , Anormalidades Cardiovasculares/complicações , Anormalidades Cardiovasculares/diagnóstico , Anormalidades Cardiovasculares/patologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Deficiência Intelectual/complicações , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/patologia , Masculino , Pessoa de Meia-Idade , Síndrome de Sotos/complicações , Síndrome de Sotos/diagnóstico , Síndrome de Sotos/patologia , Adulto JovemRESUMO
The human chromosome 14q32 carries a cluster of imprinted genes which include the paternally expressed genes (PEGs) DLK1 and RTL1, as well as the maternally expressed genes (MEGs) MEG3, RTL1as, and MEG8. PEGs and MEGs expression at the 14q32.2-imprinted region are regulated by two differentially methylated regions (DMRs): the IG-DMR and the MEG3-DMR, which are respectively methylated on the paternal and unmethylated on the maternal chromosome 14 in most cells. Genetic and epigenetic abnormalities affecting these imprinted gene clusters result in two different phenotypes currently known as maternal upd(14) syndrome and paternal upd(14) syndrome. However, only few patients carrying a maternal deletion at the 14q32.2-imprinted critical region have been reported so far. Here we report on the first patient with a maternal de novo deletion of 160 kb at the 14q32.2 chromosome that does not involves the IG-DMR or the MEG3-DMR but elicits a full upd(14)pat syndrome's phenotype encompassing the three mentioned MEGs. By the analysis of this unique genotype-phenotype correlation, we further widen the spectrum of the congenital anomalies associated to this rare disorder and we propose that the paternally expressed imprinted RTL1 gene, as well as its maternally expressed RTL1as antisense transcript, may play a prominent causative role.
Assuntos
Metilação de DNA , Impressão Genômica , Deleção de Sequência , Dissomia Uniparental/genética , Proteínas de Ligação ao Cálcio , Cromossomos Humanos Par 14/genética , Hibridização Genômica Comparativa , Feminino , Genótipo , Humanos , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Fenótipo , Proteínas da Gravidez/genética , Prognóstico , RNA Longo não Codificante/genética , Síndrome , Dissomia Uniparental/patologiaRESUMO
BACKGROUND: Fragile X Syndrome (FXS), the most common cause of familiar mental retardation, is associated in over 99% of cases to an expansion over 200 repeats of a CGG sequence in the 5' UTR of the FMR1 gene (Xq27.3), leading to the hypermethylation of the promoter. Molecular diagnosis of FXS have been so far based on the use of the Southern Blot (SB) analysis, a low throughput and time consuming technique. In order to update the diagnostic approach for FXS, we evaluated the usefulness of the Methylation-Specific Multiplex-Ligation-dependent Probe Amplification assay (MS-MLPA). METHODS: The study was carried out by retrospectively analysing 44 male patients, 10 Chorionic Villus Sampling (CVS) samples and 10 females previously analyzed by SB. In addition, a prospective study on 98 male subjects, 20 females and 1 CVS sample was carried out for assessing the feasibility and the impact of MS-MLPA in a routine lab work. RESULT: Results provided by both the retrospective and the prospective parts of this study strongly demonstrate the robustness and reproducibility of the MS-MLPA assay, able to correctly detect the methylation status in all normal and full mutation male samples analyzed, including CVS male samples. On the other hand, MS-MLPA analysis on females samples produced unreliable results. CONCLUSION: Based on our results, we suggest the necessity of a separate workflow for male and female patients with suspected FXS in the routine diagnostic setting. MS-MLPA, in combination with CGG repeat sizing using a single-tube primed FMR1 PCR, represents a reliable diagnostic protocol in the molecular diagnosis of FXS male patients.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/diagnóstico , Síndrome do Cromossomo X Frágil/genética , Southern Blotting/métodos , Amostra da Vilosidade Coriônica/métodos , Metilação de DNA , Feminino , Humanos , Masculino , Mutação , Reação em Cadeia da Polimerase/métodos , Gravidez , Regiões Promotoras Genéticas , Estudos Prospectivos , Reprodutibilidade dos Testes , Estudos RetrospectivosRESUMO
Mowat-Wilson syndrome (MWS) is a genetic disease caused by heterozygous mutations or deletions of the ZEB2 gene and is characterized by distinctive facial features, epilepsy, moderate to severe intellectual disability, corpus callosum abnormalities and other congenital malformations. Epilepsy is considered a main manifestation of the syndrome, with a prevalence of about 70-75%. In order to delineate the electroclinical phenotype of epilepsy in MWS, we investigated epilepsy onset and evolution, including seizure types, EEG features, and response to anti-epileptic therapies in 22 patients with genetically confirmed MWS. Onset of seizures occurred at a median age of 14.5 months (range: 1-108 months). The main seizure types were focal and atypical absence seizures. In all patients the first seizure was a focal seizure, often precipitated by fever. The semiology was variable, including hypomotor, versive, or focal clonic manifestations; frequency ranged from daily to sporadic. Focal seizures were more frequent during drowsiness and sleep. In 13 patients, atypical absence seizures appeared later in the course of the disease, usually after the age of 4 years. Epilepsy was usually quite difficult to treat: seizure freedom was achieved in nine out of the 20 treated patients. At epilepsy onset, the EEGs were normal or showed only mild slowing of background activity. During follow-up, irregular, diffuse frontally dominant and occasionally asymmetric spike and waves discharges were seen in most patients. Sleep markedly activated these abnormalities, resulting in continuous or near-to-continuous spike and wave activity during slow wave sleep. Slowing of background activity and poverty of physiological sleep features were seen in most patients. Our data suggest that a distinct electroclinical phenotype, characterized by focal and atypical absence seizures, often preceded by febrile seizures, and age-dependent EEG changes, can be recognized in most patients with MWS.
Assuntos
Doença de Hirschsprung/fisiopatologia , Deficiência Intelectual/fisiopatologia , Microcefalia/fisiopatologia , Convulsões/fisiopatologia , Adolescente , Anticonvulsivantes/uso terapêutico , Criança , Pré-Escolar , Análise Mutacional de DNA , Eletroencefalografia , Fácies , Feminino , Doença de Hirschsprung/tratamento farmacológico , Doença de Hirschsprung/genética , Proteínas de Homeodomínio/genética , Humanos , Deficiência Intelectual/tratamento farmacológico , Deficiência Intelectual/genética , Masculino , Microcefalia/tratamento farmacológico , Microcefalia/genética , Mutação , Fenótipo , Proteínas Repressoras/genética , Estudos Retrospectivos , Convulsões/tratamento farmacológico , Convulsões/genética , Ácido Valproico/uso terapêutico , Adulto Jovem , Homeobox 2 de Ligação a E-box com Dedos de ZincoRESUMO
Sotos syndrome is a rare genetic disorder caused by haploinsufficiency of the NSD1 (nuclear receptor binding SET domain containing protein 1) gene. No clinical diagnostic consensus criteria are published yet, and molecular analysis reduces the clinical diagnostic uncertainty. We screened 1530 unrelated patients enrolled from 2003 to 2021 at Galliera Hospital and Gaslini Institute in Genoa. NSD1 variants were identified in 292 patients including nine partial gene deletions, 13 microdeletions of the entire NSD1 gene, and 115 novel intragenic variants never previously described. Thirty-two variants of uncertain significance (VUS) out of 115 identified were re-classified. Twenty-five missense NSD1 VUS (25/32, 78.1%) changed class to likely pathogenic or likely benign, showing a highly significant shift in class (p < 0.01). Apart from NSD1, we identified variants in additional genes (NFIX, PTEN, EZH2, TCF20, BRWD3, PPP2R5D) in nine patients analyzed by the NGS custom panel. We describe the evolution of diagnostic techniques in our laboratory to ascertain molecular diagnosis, the identification of 115 new variants, and the re-classification of 25 VUS in NSD1. We underline the utility of sharing variant classification and the need to improve communication between the laboratory staff and the referring physician.
Assuntos
Síndrome de Sotos , Humanos , Mutação , Histona Metiltransferases , Mutação de Sentido Incorreto , Deleção de Genes , Fatores de Transcrição/genética , Proteína Fosfatase 2/genética , Histona-Lisina N-Metiltransferase/genéticaRESUMO
Interstitial deletions involving 14q13.1q21.1 are rare. In the literature at least 10 cases involving this region have been described and all patients showed a phenotype within the holoprosencephaly (HPE) spectrum. Previous studies suggested the HPE8 region as a candidate locus for HPE at 14q13. We report an adolescent with a 14q13.1q21.1 deletion encompassing the HPE8 region associated with intellectual disability (ID), bilateral microphthalmia, and coloboma, without cerebral anomalies typical of HPE. Except for ocular defects (i.e., microphthalmia, coloboma) consistent with HPE-type anomalies, the minor facial dysmorphia was not suggestive for HPE and the absence of cerebral anomalies should rule out this diagnosis. The deletion of the potential HPE candidate genes NPAS3, EAPP, SNX6, and TULIP1, raises doubts about their pathologic role in determining HPE. It is likely that deletions of HPE genes are not sufficient to cause HPE, and that multiple genetic, chromosomal, and environmental factors interact to determine the variable clinical expression of HPE. This is the first case of a 14q deletion encompassing the HPE8 locus with the only features consistent with HPE-type anomalies affecting the ocular system (i.e., microphthalmia, coloboma), and without cerebral anomalies specific for HPE. The inclusion of potential HPE candidate genes in the deletion raises the question whether this patient is affected by a less severe form of HPE (HPE microform), or whether he has a new ID/MCA deletion syndrome.
Assuntos
Cromossomos Humanos Par 14 , Loci Gênicos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Microftalmia/diagnóstico , Microftalmia/genética , Deleção de Sequência , Adolescente , Hibridização Genômica Comparativa , Fácies , Holoprosencefalia/diagnóstico , Humanos , Hibridização in Situ Fluorescente , Masculino , FenótipoRESUMO
Most common inherited form of intellectual disability, fragile X syndrome is associated to an expansion of greater than 200 CGG repeats in the 5' untranslated region of the FMR1 gene on the X chromosome which causes transcriptional silencing and deficiency of the encoded protein FMRP. Molecular diagnosis is performed through a combination of PCR to identify fewer than 100-150 repeats and of Southern blot analysis to identify longer alleles and the methylation status of the FMR1 promoter. We present a family with one patient with mild mental retardation who showed an atypical profile at Southern analysis due to the -413C > G transversion located in the FMR1 promoter which had been described as possibly associated with mental retardation. We demonstrated this variant in other four family members along three generations, including the maternal grandfather who did not manifest any pathological feature. Though the -413C > G substitution was not found in a large control series, these findings allowed to exclude its role in determining the disease phenotype.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Idoso de 80 Anos ou mais , Sequência de Bases , Criança , Análise Mutacional de DNA , Feminino , Humanos , Recém-Nascido , Masculino , Dados de Sequência Molecular , Linhagem , Fenótipo , GravidezRESUMO
Interstitial deletions of the long arm of chromosome 14 are relatively rare. We report a 8.5-year-old girl with dysmorphic facial features and mental retardation associated with a de novo interstitial deletion of chromosome 14. The comparison between our patient and all published patients is reviewed. The genetic investigations have allowed us to define the critical chromosomal region and to start an accurate follow-up.
Assuntos
Anormalidades Múltiplas/genética , Deleção Cromossômica , Cromossomos Humanos Par 14 , Face/anormalidades , Deficiência Intelectual/genética , Transtornos Psicomotores/genética , Criança , Hibridização Genômica Comparativa , Feminino , Humanos , Hibridização in Situ Fluorescente , Análise de Sequência com Séries de OligonucleotídeosRESUMO
The molecular diagnosis of fragile X syndrome relies on the detection of the pathogenic CGG repeat expansion in the FMR1 gene. Deletions and point mutations have occasionally been reported. Rare polymorphisms might mimic a deletion by Southern blot analysis, leading to false-positive results. We describe a novel rare nucleotide substitution within the CGG repeat. The proband was a woman with a positive family history of mental retardation. Southern blot analysis showed an additional band consistent with a deletion in the region detected by the StB12.3 probe. Sequencing of this region revealed a G>C transversion that interrupts the CGG repeat and introduces an EagI site. The same variant was observed in both the healthy son and father of the proband, supporting the hypothesis that the nucleotide substitution is a silent polymorphism, the frequency of which we estimated to be less than 1% in the general population. These findings argue for a pathogenic role of nucleotide variants within the CGG repeat and suggest possible consequences of unexpected findings in the molecular diagnostics of fragile X syndrome. Thus, although the sequence context of a single nucleotide substitution may not predict possible effects on mRNA or protein function, a specific change in the higher order structures of DNA or mRNA may be functionally relevant in the pathological phenotype.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Polimorfismo de Nucleotídeo Único , Deleção de Sequência , Repetições de Trinucleotídeos/genética , Idoso , Criança , Análise Mutacional de DNA , Feminino , Síndrome do Cromossomo X Frágil/diagnóstico , Síndrome do Cromossomo X Frágil/patologia , Humanos , MasculinoRESUMO
The Italian External Quality Assessment scheme for fragile X syndrome started in 2001 as an activity funded by the National Health System and coordinated by the National Institute of Public Health. The aim of this work is to present the data of 5 years (2001--2004 and 2006) of survey. The External Quality Assessment scheme was designed to cover the following points: (a) genotyping and (b) interpretation and reporting of results. Overall, the scheme covered about 65% of all Italian public laboratories. The average reporting of results was 91.6%, with an overall success rate of 76%. The rate of diagnostic errors observed was on average 5%. Inaccuracy in sizing of CGG repeats of normal and premutated alleles was reported. During the survey the proportion of laboratories using a Southern blotting, polymerase chain reaction, and ABI sizing kit in combination rose from 36.8% to 70.6%. The reports from laboratories showed incompleteness and considerable variations in expected outcomes. For this reason, in 2004 a model for written reports was introduced. In conclusion, these data underscore the need to participate in External Quality Assessment schemes as an educational resource to ensure quality in molecular genetic testing.
Assuntos
Síndrome do Cromossomo X Frágil/genética , Testes Genéticos , Laboratórios/normas , Técnicas de Diagnóstico Molecular , Garantia da Qualidade dos Cuidados de Saúde , Coleta de Dados , Erros de Diagnóstico , Síndrome do Cromossomo X Frágil/diagnóstico , Testes Genéticos/métodos , Testes Genéticos/normas , Genótipo , Guias como Assunto , Humanos , Itália , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Controle de QualidadeRESUMO
Fragile X syndrome (FXS) is mostly caused by two distinct events that occur in the FMR1 gene (Xq27.3): an expansion above 200 repeats of a CGG triplet located in the 5'UTR of the gene, and methylation of the cytosines located in the CpG islands upstream of the CGG repeats. Here, we describe two unrelated families with one FXS child and another sibling presenting mild intellectual disability and behavioral features evocative of FXS. Genetic characterization of the undiagnosed sibling revealed mosaicism in both the CGG expansion size and the methylation levels in the different tissues analyzed. This report shows that in the same family, two siblings carrying different CGG repeats, one in the full-mutation range and the other in the premutation range, present methylation mosaicism and consequent decreased FMRP production leading to FXS and FXS-like features, respectively. Decreased FMRP levels, more than the number of repeats seem to correlate with the severity of FXS clinical phenotypes.
RESUMO
Genetic testing availability in the health care system is rapidly increasing, along with the diffusion of next-generation sequencing (NGS) into diagnostics. These issues make imperative the knowledge-drive optimization of testing in the clinical setting. Time estimations of wet laboratory procedure in Italian molecular laboratories offering genetic diagnosis were evaluated to provide data suitable to adjust efficiency and optimize health policies and costs. A survey was undertaken by the Italian Society of Human Genetics (SIGU). Forty-two laboratories participated. For most molecular techniques, the most time-consuming steps are those requiring an intensive manual intervention or in which the human bias can affect the global process time-performances. For NGS, for which the study surveyed also the interpretation time, the latter represented the step that requiring longer times. We report the first survey describing the hands-on times requested for different molecular diagnostics procedures, including NGS. The analysis of this survey suggests the need of some improvements to optimize some analytical processes, such as the implementation of laboratory information management systems to minimize manual procedures in pre-analytical steps which may affect accuracy that represents the major challenge to be faced in the future setting of molecular genetics laboratory.
Assuntos
Testes Genéticos/estatística & dados numéricos , Laboratórios/estatística & dados numéricos , Inquéritos e Questionários/estatística & dados numéricos , Carga de Trabalho/estatística & dados numéricos , Testes Genéticos/economia , Testes Genéticos/tendências , Sequenciamento de Nucleotídeos em Larga Escala/economia , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Itália , Laboratórios/economia , Laboratórios/tendências , Sistemas de Informação Administrativa , Fatores de Tempo , Carga de Trabalho/economiaAssuntos
Deleção Cromossômica , Inversão Cromossômica , Cromossomos Humanos X/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Proteínas de Membrana/genética , Proteínas Nucleares/genética , Adolescente , Adulto , Hibridização Genômica Comparativa , Feminino , Síndrome do Cromossomo X Frágil/patologia , Humanos , Hibridização in Situ Fluorescente , MasculinoRESUMO
The identification of women with a high probability of being carriers of pathogenic BRCA mutation is not straightforward and a major improvement would be the availability of markers of mutations that could be directly evaluated in individuals asking for genetic testing. The FMR1 gene testing was recently proposed as a candidate prescreening tool because an association between BRCA pathogenic mutations and FMR1 genotypes with 'low alleles' (CGG repeat number <26) was observed. To confirm this hypothesis, we evaluated the distribution of FMR1 alleles and genotypes between BRCA mutation carriers and non-carriers in a cohort of 147 Italian women, free of cancer or affected by breast and/or ovarian cancer, who were tested for the presence of BRCA mutation in a clinical setting. The distribution of FMR1 CGG repeat numbers in the two groups was similar (lower allele median/mean were 30/27.4 and 30/27.9, respectively; Mann-Whitney test P=0.997) and no difference in the FMR1 genotype distribution was present (χ(2)=0.503, d.f.=2, P=0.78). This result is in contrast with literature data and suggests that FMR1 genetic testing is not a candidate BRCA prescreening tool.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Estudos de Casos e Controles , Feminino , Triagem de Portadores Genéticos , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Heterozigoto , Humanos , Mutação , Repetições de TrinucleotídeosRESUMO
Fragile X syndrome and associated disorders are characterized by the number of CGG repeats and methylation status of the FMR1 gene for which Southern blot (SB) historically has been required for analysis. This study describes a simple PCR-only workflow (mPCR) to replace SB analysis, that incorporates novel procedural controls, treatment of the DNA in separate control and methylation-sensitive restriction endonuclease reactions, amplification with labeled primers, and two-color amplicon sizing by capillary electrophoresis. mPCR was evaluated in two independent laboratories with 76 residual clinical samples that represented typical and challenging fragile X alleles in both males and females. mPCR enabled superior size resolution and analytical sensitivity for size and methylation mosaicism compared to SB. Full mutation mosaicism was detected down to 1% in a background of 99% normal allele with 50- to 100-fold less DNA than required for SB. A low level of full mutation mosaicism in one sample was detected using mPCR but not observed using SB. Overall, the sensitivity for detection of full mutation alleles was 100% (95% CI: 89%-100%) with an accuracy of 99% (95% CI: 93%-100%). mPCR analysis of DNA from individuals with Klinefelter and Turner syndromes, and DNA from sperm and blood, were consistent with SB. As such, mPCR enables accurate, sensitive, and standardized methods of FMR1 analysis that can harmonize results across different laboratories.