PURPOSE:Genetic causes of surfactant dysfunction are associated with childhood interstitial lung disease. Lysosome-associated membrane glycoprotein 3 (LAMP3) is highly expressed within lamellar bodies of alveolar epithelial type II cells, and variants in LAMP3 have recently been suggested as a novel cause of childhood interstitial lung disease. This study describes the phenotypes of participants with biallelic variants in LAMP3 and presents functional studies evaluating the role of specific LAMP3 variants. METHODS:Phenotypic data were collected through chart review and clinical evaluation. In vitro effects of LAMP3 variants were evaluated through immunohistochemistry, western blot, and flow cytometry. RESULTS:Thirteen participants were identified with biallelic variants in LAMP3. They presented with variable phenotypes ranging from neonatal respiratory distress to asymptomatic in adulthood. All symptomatic participants demonstrated ground glass opacities early in life and lung fibrosis later in life. For 1 participant, BAL analysis showed abnormal surfactant protein composition and lung biopsy revealed irregular lamellar bodies. In vitro studies in lung epithelial cells with induced expression of specific LAMP3 variants demonstrated reduced protein expression and abnormal glycosylation. CONCLUSION:Biallelic LAMP3 variants are associated with an interstitial lung disease phenotype with variable expressivity. Evaluation for LAMP3 variants should be considered in individuals with unexplained interstitial lung disease.

OBJECTIVES:To describe a cohort of fetuses with abnormal prenatal findings in whom exome sequencing (ES) identified genetic diagnoses that refined prognostic assessment and reduced prognostic uncertainty. METHODS:We retrospectively reviewed all fetal ES studies performed at our center between 2017 and 2025 for structural or biochemical abnormalities. Cases with a definitive molecular diagnosis were identified, and those with predicted mild, isolated, transient, or treatable phenotypes were classified as having comparatively favorable prognostic implications. Clinical findings and expected postnatal outcomes were analyzed. RESULTS:Among 1692 fetuses undergoing ES, a molecular diagnosis was identified in 291 (17%). Of these, five cases (∼2%) were considered reassuring: isolated postaxial polydactyly due to a GLI1 variant; X-linked ichthyosis (STS) in a fetus with low estriol; isolated situs inversus; CFAP52-related situs inversus totalis; and MAGED2-associated transient antenatal Bartter syndrome. In all cases, ES refined prognosis and reduced the likelihood of severe syndromic conditions, enabling clearer counseling. CONCLUSIONS:Beyond detecting severe disorders, ES may help distinguish mild or manageable conditions from complex syndromes. In selected cases, it provides meaningful reassurance, reduces uncertainty, and supports informed prenatal decision-making.

Exome sequencing (ES), originally developed to detect single nucleotide variants (SNVs), has been increasingly leveraged to detect copy number variants (CNVs) through read-depth analysis, enhancing diagnostic yield with minimal computational overhead. However, chromosomal microarray (CMA) testing remains widely used. To evaluate the utility of ES as a first-tier clinical diagnostic test, we compared the sensitivity of CNV detection by ES to that of CMA in individuals who underwent both tests, and developed triploidy screening based on ES data. ES identified most clinically relevant CNVs, with a 98.91% concordance for regions adequately captured. A retrospective analysis of CNVs detected from ES over a ~3-year period, comprising 1563 prenatal and 4884 postnatal cases, revealed CNVs in 3.8% of prenatal and 4.4% of postnatal samples. The relatively low percentage stems from the fact that most cases underwent CMA before ES. Pathogenic or likely pathogenic variants constituted 78.7% and 78.0% of these subgroups, with the remainder classified as variants of unknown significance. We highlight clinically relevant examples of CNVs across a range of sizes, including cases involving both CNVs and SNVs. The high consanguinity rate of the cohort allowed for systematic analysis of homozygous CNVs. Additionally, we demonstrate that ES can capture other diagnostic utilities traditionally associated with CMA, specifically uniparental disomy (UPD) and triploidy. Overall, our findings support ES as a robust, cost-effective alternative to CMA and advocate for its broader use as a first-tier diagnostic test for neurodevelopmental delay and congenital malformations, particularly until whole genome sequencing becomes more accessible and affordable.

Background LAMPS encodes a lysosomal membrane protein associated with lamellar bodies and has recently been proposed as a candidate gene for childhood interstitial lung diseases (chILD). Here, we identified two LAMPS variants in a proband with chILD and performed functional validation of these variants as well as the previously reported variants to demonstrate the role of LAMP3 in pathology. Methods LAMPS variants were identified by exome sequencing. Ex vivo studies included mRNA analysis from nasal brushing and lung tissue and immunohistochemistry from lung biopsy. In vitro functional analyses in the A549 cell line included immunofluorescence staining and expression analysis of LAMP3. Interactions between LAMP3 and the surfactant protein (SP)-B and SP-C were evaluated by co-immunoprecipitation. Findings Two heterozygous LAMPS variants (Y302Qfs*2 and T268M) were identified in a 15 year old boy with chILD. LAMPS mRNA revealed that the frameshift variant resulted in nonsense-mediated mRNA decay. Reduced LAMP3 expression was confirmed in the patient's lung tissue. Functional studies of the T268M and the previously reported G288R variant revealed reduced levels of the mutant proteins. In addition, impaired N-glycosylation and protein instability were demonstrated with the T268M variant. Finally, we provided evidence for an interaction between LAMP3 and SP-B and SP-C, revealing a direct link between LAMP3 and surfactant metabolism. Interpretation LAMPS bi-allelic variants leading to LAMP3 dysfunction emerges as a cause of chILD associated with a heterogeneous phenotype that remains to be further defined. The close links between LAMP3 and surfactant metabolism could explain the pathophysiology of this genetic disease.

Introduction: Variants in the Ras-related GTPase D (RRAGD) gene have been associated with autosomal dominant kidney hypomagnesemia (ADKH) characterized by hypokalemia, nephrocalcinosis, and dilated cardiomyopathy (DCM). RRAGD, which encodes for the RagD protein, is involved in the activation of the mechanistic target of rapamycin complex 1 (mTORC1). Owing to the limited characterization of patients’ phenotypes, the understanding of RRAGD-associated ADKH (ADKH-RRAGD) remains incomplete. Consequently, available treatment strategies are primarily symptomatic and insufficient. Methods: In the present case series, 13 new patients and 3 novel RRAGD variants, that is, p.(Ser77Phe), p.(Thr91Ile), and p.(Ile100Arg), are described. To assess the pathogenicity of the novel variants, an in vitro assay of mTORC1 activity was performed. In addition, the clinical response to diuretics (furosemide and thiazide, n = 4) and Na+-glucose cotransporter 2 (SGLT2) inhibitor, dapagliflozin (n = 6) was evaluated in patients carrying the RRAGD p.(Thr97Pro) variant during routine. Results: The patients presented with kidney tubulopathies, including hypomagnesemia, hypercalciuria, and nephrocalcinosis. Five patients also exhibited DCM. In vitro assays demonstrated constitutive activation of noncanonical mTORC1 signaling caused by the p.(Ser77Phe) and p.(Ile100Arg) variants. Clinically, patients remained sensitive to diuretic challenges, whereas dapagliflozin treatment increased serum magnesium (Mg2+) levels by 0.04 mM but exacerbated hypokalemia. Conclusion: To date, 37 patients with ADKH-RRAGD have been identified. Kidney tubulopathy is the most prominent feature within the phenotypic spectrum of ADKH-RRAGD. Molecularly, constitutive activation of noncanonical mTORC1 is present in most RRAGD variants. From a therapeutic perspective, dapagliflozin may increase serum Mg2+ levels in patients with RRAGD variants.

Synpolydactyly (SPD) is a heterogeneous distal-limb malformation syndrome, characterized by webbing and duplication of adjacent digits. SPD1, the most common type, is attributed to disease-causing variants in HOXD13, a transcription factor in the HOXD cluster that is essential for limb development. Here, we present a challenging exome-negative case of familial SPD. The case was resolved using long-read genome sequencing, which revealed a microdeletion in a conserved noncoding regulatory region upstream of the HOXD cluster. Oxford Nanopore WGS-LRS was pursued on two affected sisters, followed by CMA-Cytoscan HT validation. WGS-LRS for two affected siblings revealed a shared microdeletion of ~5.6 kb upstream of EVX2 and HOXD13 [chr2:176073523-176,079,120 (hg38)]. The deletion is predicted to affect the enhancer of several HOXD genes (EH38E2053988) and overlaps with a short stretch of high-sequence homology between human and mouse, called R1. CMA-Cytoscan HT analysis and PCR validation approved segregation of the deletion in the siblings and revealed a paternal inheritance. Our findings expand the genotype-phenotype correlation of the regulatory region upstream of HOXD13 in SPD and highlight the utility of long-read WGS for detecting noncoding variants that are undetectable by exome sequencing.

Spondylocostal dysostosis (SCDO) is a rare genetic disorder characterized by abnormal development of the axial skeleton, resulting in malformations of the vertebrae and ribs that often impair lung development and lead to significant respiratory morbidity. SCDO is thought to arise from defects in the paraxial presomitic mesoderm, an embryonic tissue that forms the vertebral column and ribs. Pathogenic variants in DLL3, MESP2, LFNG, HES7, TBX6, and RIPPLY2 have been identified in various SCDO subtypes. In addition, a single case of a lethal SCDO-like phenotype caused by a homozygous start-loss variant in DMRT2 has been reported. DMRT2 encodes a transcription factor expressed in the dermomyotome during early somite formation in mice. Here, we describe a newborn with severe costovertebral malformations and dysmorphic features, in whom exome sequencing identified a homozygous loss-of-function variant in DMRT2. The phenotype strikingly overlaps the previous report, further supporting the role of biallelic pathogenic DMRT2 variants in a severe SCDO-like disorder. Notably, our patient also exhibited thymic aplasia and immunodeficiency. A review of the exome sequencing data did not reveal any variant that could account for the immunodeficiency. These features have not been previously associated with SCDO, suggesting a potential phenotypic expansion.

BACKGROUND:Autosomal dominant (AD) inheritance often arises through haploinsufficiency, dominant-negative or gain of function (GoF) effects, while autosomal recessive (AR) inheritance generally results from partial or complete loss of function (LoF). Yet, a subset of genes demonstrates both inheritance patterns. We aimed to curate a list of such 'AD/AR' genes and to propose additional candidates. METHODS:AD/AR genes were subcategorised based on genotype-phenotype correlations and disease mechanisms. Using bioinformatic analyses, we compared genes with AD, AR and AD/AR inheritance across various metrics, including gnomAD constraint values, exon count, protein length, quaternary structure and gene ontology terms. A machine learning-based metric was used to account for interdependence among features. RESULTS:Pathogenic variants in AD/AR genes can lead to distinct or similar phenotypes, depending on the molecular mechanism. AD/AR genes exhibit unique bioinformatic properties such as intermediate constraint scores, a combination of gene ontology terms, a greater average number of exons and an elevated propensity to form homomeric/heteromeric proteins. We identified homozygous LoF or clinically reported variants in nine genes previously classified as AD only. CONCLUSION:Collectively, the data suggest that AD/AR genes possess distinctive features that likely underpin their dual inheritance modes. We propose nine candidate AD/AR genes and emphasise caution in filtering by inheritance type alone.

BACKGROUND:The DSP gene encodes the desmosomal protein desmoplakin and is located on chromosome 6. Pathogenic variants in this gene have been linked to different phenotypes that may include the skin, hair, nails, teeth, and the heart. Lethal acantholytic epidermolysis bullosa (LAEB, OMIM # 609638) is a severe and lethal form of epidermolysis bullosa, caused by biallelic pathogenic variants in the DSP gene. CASE PRESENTATION:This report describes two fetuses from the same family, both affected by LAEB. The parents were second degree cousins. Both fetuses showed multiple sonographic abnormalities antenatally. The first fetus had a lemon shaped skull, an abnormal profile with frontal bossing, flat nasal bridge, nasal hypoplasia and micrognathia. There was increased pericardial fluid, situs ambiguous (left stomach with dextrocardia) and hypoplastic aortic arch. The kidneys were suspected as dysplastic and indeed there was also oligohydramnios. Echogenic sediment was noted in the fetal stomach. The fetus was small for gestational age (SGA) with an estimated fetal weight (EFW) under the 3rd percentile. The second fetus exhibited a novel sonographic sign - constantly open eyelids. Additional notable sonographic signs were echogenic amniotic fluid, and an abnormal profile comprising of flat nasal bridge, hypoplastic nose and micrognathia. Furthermore, hypospadias was suspected as well as abnormal scrotum. The scan revealed echogenic sediment in stomach and SGA fetus with EFW under 3rd percentile similarly to the previous pregnancy. After delivery severe extensive skin and mucosal erosion and sloughing were evident and the neonate succumbed at day 2 of life. Extensive genetic workup diagnosed LAEB in both children. DISCUSSION AND CONCLUSIONS:This is the first report to describe the antenatal sonographic phenotype of LAEB. We discuss the diverse phenotypes of DSP gene pathogenic variants and review the specific biochemical and sonographic findings in the context of congenital skin denudation diseases.

Approximately 10% of children born small for gestational age (SGA) fail to achieve catch-up growth, resulting in persistent short stature and eligibility for growth hormone (GH) therapy under established guidelines. Pathogenic variants in insulin-like growth factor 1 receptor (IGF1R) are associated with SGA, syndromic short stature, neurocognitive impairment, and variable responsiveness to GH therapy. This study aimed to characterize the clinical phenotype and elucidate the molecular mechanism underlying a rare intronic variant in IGF1R identified in an affected family. Here, we performed whole-exome sequencing (WES) on a single individual, followed by segregation studies in the family and Sanger sequencing. cDNA studies were pursued to evaluate mis-spliced transcripts. WES of the proband's affected mother revealed a rare heterozygous variant in IGF1R (NM_000875.5): c.3722+5G>A. Sanger sequencing confirmed segregation of the variant with the affected status in available family members. cDNA analysis showed that the variant results in intronic retention of 134 nucleotides immediately following the penultimate exon of IGF1R. This leads to a frameshift and introduction of a premature truncation codon, supporting the classification of the variant as likely pathogenic. Our study highlights the utility of genetic testing in SGA children with persistent short stature. By characterizing a novel IGF1R intronic variant causing aberrant splicing, we expand the understanding of its clinical spectrum and molecular underpinning. The findings underscore the importance of molecular diagnostics in unexplained short stature and neurodevelopmental disorders and may inform future therapeutic strategies targeting the IGF1R signaling.

PHF20 encodes plant homeodomain finger protein 20 (PHF20), a component of the KAT8-containing nonspecific lethal (NSL) complex that deposits acetylation on histone H4 to activate gene expression. We report two unrelated individuals with developmental delay, microcephaly, and distinctive facial features, in whom exome sequencing and chromosomal microarray analysis revealed a homozygous deletion of PHF20 that segregated with the disease phenotype in their families. Breakpoint junction sequencing revealed an Alu-Alu-mediated deletion event. Western blot in cells from an affected individual showed undetectable PHF20, while levels of other NSL complex subunits were unaltered. Transcriptomic and epigenomic analysis revealed significant downregulation of gene pathways related to cell projection and neuronal development, associated with reduced histone H4K16 acetylation at these genes. In conclusion, our data suggest that homozygous deletion of PHF20 leads to a neurodevelopmental syndrome, potentially through targeted epigenetic dysregulation and altered gene expression essential for neuronal development. Identifying additional families with biallelic PHF20 variants will further delineate the phenotypic spectrum, and molecular studies in neuronal cell lines will be essential for understanding the disease mechanism.

(Abstracted from JAMA Network Open 2024;7(2):e240146 A large portion of mortality in neonatal intensive care units (NICUs) can be attributed to genetic disorders and/or birth defects; often, diagnosis of specific disorders can be difficult due to overlapping symptomology and insufficient sensitivity of tests for this age group. Next-generation sequencing has the potential to provide precision diagnosis that will assist in prognosis, lifesaving therapy, and palliative care decisions and has been shown to be highly accurate.

Importance:National implementation of rapid trio genome sequencing (rtGS) in a clinical acute setting is essential to ensure advanced and equitable care for ill neonates. Objective:To evaluate the feasibility, diagnostic efficacy, and clinical utility of rtGS in neonatal intensive care units (NICUs) throughout Israel. Design, Setting, and Participants:This prospective, public health care-based, multicenter cohort study was conducted from October 2021 to December 2022 with the Community Genetics Department of the Israeli Ministry of Health and all Israeli medical genetics institutes (n = 18) and NICUs (n = 25). Critically ill neonates suspected of having a genetic etiology were offered rtGS. All sequencing, analysis, and interpretation of data were performed in a central genomics center at Tel-Aviv Sourasky Medical Center. Rapid results were expected within 10 days. A secondary analysis report, issued within 60 days, focused mainly on cases with negative rapid results and actionable secondary findings. Pathogenic, likely pathogenic, and highly suspected variants of unknown significance (VUS) were reported. Main Outcomes and Measures:Diagnostic rate, including highly suspected disease-causing VUS, and turnaround time for rapid results. Clinical utility was assessed via questionnaires circulated to treating neonatologists. Results:A total of 130 neonates across Israel (70 [54%] male; 60 [46%] female) met inclusion criteria and were recruited. Mean (SD) age at enrollment was 12 (13) days. Mean (SD) turnaround time for rapid report was 7 (3) days. Diagnostic efficacy was 50% (65 of 130) for disease-causing variants, 11% (14 of 130) for VUS suspected to be causative, and 1 novel gene candidate (1%). Disease-causing variants included 12 chromosomal and 52 monogenic disorders as well as 1 neonate with uniparental disomy. Overall, the response rate for clinical utility questionnaires was 82% (107 of 130). Among respondents, genomic testing led to a change in medical management for 24 neonates (22%). Results led to immediate precision medicine for 6 of 65 diagnosed infants (9%), an additional 2 (3%) received palliative care, and 2 (3%) were transferred to nursing homes. Conclusions and Relevance:In this national cohort study, rtGS in critically ill neonates was feasible and diagnostically beneficial in a public health care setting. This study is a prerequisite for implementation of rtGS for ill neonates into routine care and may aid in design of similar studies in other public health care systems.

PURPOSE:Widespread application of next-generation sequencing, combined with data exchange platforms, has provided molecular diagnoses for countless families. To maximize diagnostic yield, we implemented an unbiased semi-automated genematching algorithm based on genotype and phenotype matching. METHODS:Rare homozygous variants identified in 2 or more affected individuals, but not in healthy individuals, were extracted from our local database of ∼12,000 exomes. Phenotype similarity scores (PSS), based on human phenotype ontology terms, were assigned to each pair of individuals matched at the genotype level using HPOsim. RESULTS:33,792 genotype-matched pairs were discovered, representing variants in 7567 unique genes. There was an enrichment of PSS ≥0.1 among pathogenic/likely pathogenic variant-level pairs (94.3% in pathogenic/likely pathogenic variant-level matches vs 34.75% in all matches). We highlighted founder or region-specific variants as an internal positive control and proceeded to identify candidate disease genes. Variant-level matches were particularly helpful in cases involving inframe indels and splice region variants beyond the canonical splice sites, which may otherwise have been disregarded, allowing for detection of candidate disease genes, such as KAT2A, RPAIN, and LAMP3. CONCLUSION:Semi-automated genotype matching combined with PSS is a powerful tool to resolve variants of uncertain significance and to identify candidate disease genes.

RBL2/p130 is one of three highly conserved members of the retinoblastoma (RB) protein family. It is strongly upregulated during neuronal differentiation and brain development, and is critical for survival of post-mitotic neurons. Similar to RB1, it has been implicated as a tumor suppressor gene and has been shown to be dysregulated in various types of cancer. Recent publications describe biallelic, germline loss of function variants in RBL2 in individuals with profound developmental delay. We report a child with profound developmental delay, microcephaly, and hypotonia, who developed fulminant exophthalmos at age 6 years. Brain MRI followed by a biopsy of an intra-orbital mass revealed a mesenchymal tumor. Post-surgical histopathologic examination of the resected tumor was compatible with diagnosis of nodular fasciitis. Exome sequencing from peripheral blood identified a biallelic frameshift variant (c.901dupT) in RBL2. Notably, no malignancies were reported in previous cases with RBL2 variants. This case provides a possible association between RBL2 and orbital tumors.

Consanguinity, commonplace in many regions around the globe, is associated with an increased risk of autosomal recessive (AR) genetic disorders. Consequently, consanguineous couples undergoing preimplantation genetic diagnosis (PGD) for one Mendelian disorder may be at increased risk for a child with a second, unrelated AR genetic disorder. We examined the yield of exome analysis for carrier screening of additional AR disorders, beyond the primary diagnosis, amongst consanguineous vs. non-consanguineous populations. Parental samples from trio exomes of 102 consanguineous families and 105 non-consanguineous controls were evaluated for shared carrier status, after disregarding the primary molecular diagnosis. Results were sub-classified according to disease severity. Secondary shared carrier status for pathogenic and likely pathogenic variants leading to AR disorders of moderate to profound severity was identified in 10/102 (9.8%) consanguineous couples, as compared to 1/105 (0.95%) non-consanguineous couples (χ 2 = 8.0565, p value < 0.005). Higher inbreeding coefficient values, calculated from individual exomes, correlated with secondary shared carrier status for diseases of moderate to profound severity ( r = 0.17, p value < 0.0125). Our results indicate that consanguineous couples undergoing PGD are at increased risk for a second genetic disease of moderate to profound severity. This study represents an underestimate of the rate of secondary shared carrier status due to inability to detect deep intronic variants, no assessment of copy number variants, and false negative results stemming from stringent variant interpretation. False positive results may result from inaccuracies in public databases. Additional studies in consanguineous populations will determine whether exome-based carrier screening should be recommended to all couples undergoing PGD.

AMOTL1 belongs to the Motin family of proteins that are involved in organogenesis and tumorigenesis through regulation of cellular migration, tube formation, and angiogenesis. While involvement of all AMOTs in development or suppression of cancers is relatively well described, little is known about the congenital phenotype of pathogenic variants in these genes in humans. Recently, a heterozygous variant in AMOTL1 was published in association with orofacial clefts and cardiac abnormalities in an affected father and his daughter. However, studies in mice did not recapitulate the human phenotype and the case was summarized as inconclusive. We present a female infant with cleft lip and palate, imperforate anus and dysmorphic features, in whom trio exome sequencing revealed a de novo variant in AMOTL1 affecting a highly conserved amino acid (c.479C>T; p.[Pro160Leu]). Bioinformatic predictions and in silico modeling supported pathogenicity. This case reinforces the conjecture regarding the disruptive effect of pathogenic variants in AMOTL1 on organ formation in humans. Studies of additional families will reveal the full phenotypic spectrum associated with this multiple malformation syndrome.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes responsible for charging tRNA with cognate amino acids during protein translation. Non-canonical functions are increasingly recognized, and include transcription and translation control and extracellular signaling. Monoallelic mutations in genes encoding several ARSs have been identified in axonal Charcot-Marie-Tooth (CMT2) disease, whereas biallelic mutations in ARS loci have been associated with multi-tissue syndromes, variably involving the central nervous system, lung, and liver. We report a male infant of non-consanguineous origin, presenting with successive onset of transfusion-dependent anemia, hypothyroidism, cholestasis, interstitial lung disease, and developmental delay. Whole-exome sequencing (WES) revealed compound heterozygosity for two variants (p.Tyr307Cys and p.Arg618Cys) in MARS, encoding methionyl-tRNA synthetase. Biallelic MARS mutations are associated with interstitial lung and liver disease (ILLD). Interestingly, the p.Arg618Cys variant, inherited from an unaffected father, was previously reported in a family with autosomal dominant late-onset CMT2. Yeast complementation assays confirmed pathogenicity of p.Arg618Cys, yet suggested retained function of p.Tyr307Cys. Our findings underscore the phenotypic variability associated with ARS mutations, and suggest genetic or environmental modifying factors in the onset of monoallelic MARS-associated CMT2.

Background 3-Methylcrotonyl-CoA carboxylase deficiency (3MCCD) is an inborn error of leucine catabolism. Tandem mass spectrometry newborn screening (NBS) programs worldwide confirmed 3MCCD to be the most common organic aciduria and a relatively benign disorder with favorable outcome. In addition, several asymptomatic 3MCCD mothers were initially identified following abnormal screening of their healthy babies and were appropriately termed maternal 3MCCD.Methods This is a retrospective study that summarizes all the clinical, biochemical, and genetic data collected by questionnaires of all 3MCCD individuals that were identified by the extended Israeli NBS program since its introduction in 2009 including maternal 3MCCD cases.Results A total of 36 3MCCD subjects were diagnosed within the 50-month study period; 16 were classified primary and 20 maternal cases. Four additional 3MCCD individuals were identified following sibling screening. All maternal 3MCCD cases were asymptomatic except for one mother who manifested childhood hypotonia. Most of the primary 3MCCD individuals were asymptomatic except for two whose condition was also complicated by severe prematurity. Initial dried blood spot (DBS) free carnitine was significantly lower in neonates born to 3MCCD mothers compared with newborns with primary 3MCCD (p=0.0009). Most of the mutations identified in the MCCC1 and MCCC2 genes were missense, five of them were novel.Conclusions Maternal 3MCCD is more common than previously thought and its presence may be initially indicated by low DBS free carnitine levels. Our findings provide additional confirmation of the benign nature of 3MCCD and we suggest to exclude this disorder from NBS programs.