Molecular testing in newborn screening: VUS burden among true positives and secondary reproductive limitations via expanded carrier screening panels.

PURPOSE: Expanded carrier screening (ECS) gene panels have several limitations, including variable content, current knowledge of disease-causing variants, and differing reporting policies. This study evaluated if the disease-associated variants identified in affected neonates who screened positive by California newborn screening (NBS) for an inherited metabolic disorder (IMD) by tandem mass spectrometry (MS/MS) would likely be reported by ECS gene panels. METHODS: Retrospective review of neonates referred by the California Department of Public Health for a positive NBS by multianalyte MS/MS from January 1, 2020 through June 30, 2021. RESULTS: One hundred thirty-six neonates screened positive for ≥1 NBS MS/MS indication. Nineteen neonates (14%) were ultimately diagnosed with an IMD, all of whom had abnormal biochemical testing. Eighteen of the 19 underwent molecular testing; 10 (56%) neonates had ≥1 variants of uncertain significance, 9 of whom were of non-White ancestry. ECS panels would have been negative for 56% (20/36) of parents with an affected neonate, 85% (17/20) of whom were of non-White ancestry. CONCLUSION: The number of variants of uncertain significance identified in this cohort highlights the need for more diversified variant databases. Due in part to the lack of diversity in currently sequenced populations, genomic sequencing cannot replace biochemical testing for the diagnosis of an IMD.

A homozygous Gly470Ala variant in PEX6 causes severe Zellweger spectrum disorder.

Zellweger spectrum disorder (ZSD) is a group of autosomal recessive disorders caused by biallelic pathogenic variants in any one of the 13 PEX genes essential for peroxisomal biogenesis. We report a cohort of nine infants who presented at birth with severe neonatal features suggestive of ZSD and found to be homozygous for a variant in PEX6 (NM_000287.4:c.1409G > C[p.Gly470Ala]). All were of Mixtec ancestry and identified by the California Newborn Screening (NBS) Program to have elevated C26:0-lysophosphatidylcholine but no reportable variants in ABCD1. The clinical and biochemical features of this cohort are described within. Gly470Ala may represent a founder variant in the Mixtec population of Central California. ZSD should be considered in patients who present at birth with severe hypotonia and enlarged fontanelles, especially in the setting of an abnormal NBS, Mixtec ancestry, or family history of infant death. There is a need to further characterize the natural history of ZSD, the Gly470Ala variant, and expand upon possible genotype-phenotype correlations.

Neonatal lupus is a novel cause of positive newborn screening for X-linked adrenoleukodystrophy.

We report three unrelated individuals, each exposed to maternal autoantibodies during gestation and found to have elevated very long-chain fatty acids (VLCFAs) in the newborn period after screening positive by California newborn screening (NBS) for X-linked adrenoleukodystrophy (ALD). Two probands presented with clinical and laboratory features of neonatal lupus erythematosus (NLE); the third had features suggestive of NLE and a known maternal history of Sjogren's syndrome and rheumatoid arthritis. In all three individuals, subsequent biochemical and molecular evaluation for primary and secondary peroxisomal disorders was nondiagnostic with normalization of VLCFAs by 15 months of age. These cases add to the expanding differential diagnosis to consider in newborns who screen positive for ALD via elevated C26:0-lysophosphatidylcholine. Though the pathophysiology of how transplacental maternal anti-Ro antibodies damage fetal tissue is not well-understood, we postulate that the VLCFA elevations reflect a systemic inflammatory response and secondary peroxisomal dysfunction that improves once maternal autoantibodies wane after birth. Additional evaluation of this phenomenon is warranted to better understand the intricate biochemical, clinical, and possible therapeutic overlap between autoimmunity, inflammation, peroxisomal dysfunction, and human disease.

MT-ATP6 mitochondrial disease identified by newborn screening reveals a distinct biochemical phenotype.

Although decreased citrulline is used as a newborn screening (NBS) marker to identify proximal urea cycle disorders (UCDs), it is also a feature of some mitochondrial diseases, including MT-ATP6 mitochondrial disease. Here we describe biochemical and clinical features of 11 children born to eight mothers from seven separate families who were identified with low citrulline by NBS (range 3-5 µM; screening cutoff >5) and ultimately diagnosed with MT-ATP6 mitochondrial disease. Follow-up testing revealed a pattern of hypocitrullinemia together with elevated propionyl-(C3) and 3-hydroxyisovaleryl-(C5-OH) acylcarnitines, and a homoplasmic pathogenic variant in MT-ATP6 in all cases. Single and multivariate analysis of NBS data from the 11 cases using Collaborative Laboratory Integrated Reports (CLIR; https://clir.mayo.edu) demonstrated citrulline <1st percentile, C3 > 50th percentile, and C5-OH >90th percentile when compared with reference data, as well as unequivocal separation from proximal UCD cases and false-positive low citrulline cases using dual scatter plots. Five of the eight mothers were symptomatic at the time of their child(ren)'s diagnosis, and all mothers and maternal grandmothers evaluated molecularly and biochemically had a homoplasmic pathogenic variant in MT-ATP6, low citrulline, elevated C3, and/or elevated C5-OH. All molecularly confirmed individuals (n = 17) with either no symptoms (n = 12), migraines (n = 1), or a neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) phenotype (n = 3) were found to have an A or U mitochondrial haplogroup, while one child with infantile-lethal Leigh syndrome had a B haplogroup.

Creatine Transporter Deficiency Presenting as Failure to Thrive: A Case Report of a Novel SLC6A8 Variant Causing a Treatable but Likely Underdiagnosed Genetic Disorder.

Cerebral creatine deficiency syndromes (CCDS) are a rare group of inherited metabolic disorders (IMDs) that often present with nonspecific findings including global developmental delay (GDD), intellectual disability (ID), seizures, hypotonia, and behavioral differences. Creatine transporter (CRTR) deficiency is the most common CCDS, exhibiting X-linked inheritance and an estimated prevalence as high as 2.6% in individuals with neurodevelopmental disorders. Here, we present a 20-month-old boy with worsening failure to thrive (FTT) and GDD admitted for evaluation. He was found to have persistently low serum creatinine levels and a family history notable for a mother with learning disabilities and a maternal male cousin with GDD. Urine analyses revealed a marked elevation of creatine and elevated creatine:creatinine ratio suggestive of CRTR deficiency. Molecular genetic testing of SLC6A8 identified a maternally inherited hemizygous variant and brain magnetic resonance spectroscopy (MRS) showed diffusely diminished creatine peaks, further supporting the diagnosis of CRTR deficiency. The proband was started on creatine, arginine, and glycine supplementation and has demonstrated improved development. This case highlights that CRTR deficiency should be considered in all patients presenting with FTT and abnormal neurodevelopmental features, particularly if creatinine levels are low on serum chemistry studies. The nonspecific presentation of this condition in males and females likely has resulted in CRTR deficiency being underdiagnosed. There are existing therapies for individuals affected with CRTR deficiency and other CCDS, highlighting the importance of early diagnosis and intervention for affected individuals.

Recent tPA administration can cause pseudo-hyperargininemia and may mimic arginase deficiency or arginine supplementation.

Individuals suspected of or diagnosed with a rare disorder, including inherited metabolic disorders (IMD), often need frequent and/or urgent vascular access for blood draws and treatment, making central indwelling catheters commonly used devices in this patient population. These indwelling catheters are prone to thrombosis, limiting vascular access. This complication is frequently resolved with the use of altepase, a recombinant tissue plasminogen activator (tPA). This report describes two individuals, one with a known IMD and one undergoing evaluation for an IMD, who were found to have hyperargininemia (>500 µM; reference 10-140 µM) by plasma amino acid (PAA) analysis of a specimen collected ~1.5-3 h after clearance of an indwelling catheter with tPA. In both cases, hyperargininemia resolved with repeat testing, suggesting pseudo-hyperargininemia secondary to tPA administration. Quantitative amino acid analysis of the administered tPA demonstrated an arginine level of ~200 mM, supporting tPA as the cause of pseudo-hyperargininemia. Certain formulations of tPA contain high concentrations of arginine, which if not cleared properly can result in marked elevations of arginine, mimicking arginase deficiency or suggesting arginine supplementation. Thus, the possibility of pseudohyperargininemia due to tPA administration should be considered when obtaining PAAs from an indwelling catheter in any individual being evaluated or managed for an IMD.

Aicardi-Goutières syndrome may present with positive newborn screen for X-linked adrenoleukodystrophy.

We report three unrelated probands, two male and one female, diagnosed with Aicardi-Goutières syndrome (AGS) after screening positive on California newborn screening (CA NBS) for X-linked adrenoleukodystrophy (X-ALD) due to elevated C26:0 lysophosphatidylcholine (C26:0-LPC). Follow-up evaluation was notable for elevated C26:0, C26:1, and C26:0/C22:0 ratio, and normal red blood cell plasmalogens levels in all three probands. Diagnoses were confirmed by molecular sequencing prior to 12 months of age after clinical evaluation was inconsistent with X-ALD or suggestive of AGS. For at least one proband, the early diagnosis of AGS enabled candidacy for enrollment into a therapeutic clinical trial. This report demonstrates the importance of including AGS on the differential diagnosis for individuals who screen positive for X-ALD, particularly infants with abnormal neurological features, as this age of onset would be highly unusual for X-ALD. While AGS is not included on the Recommended Universal Screening Panel, affected individuals can be identified early through state NBS programs so long as providers are aware of a broader differential that includes AGS. This report is timely, as state NBS algorithms for X-ALD are actively being established, implemented, and refined.

Diagnosis of LCHAD/TFP deficiency in an at risk newborn using umbilical cord blood acylcarnitine analysis.

Trifunctional protein deficiency/Long-chain hydroxyacyl-CoA dehydrogenase deficiency (LCHAD/TFP) deficiency is a disorder of fatty acid oxidation and ketogenesis. Severe neonatal lactic acidosis, cardiomyopathy, and hepatic dysfunction are caused by the accumulation of toxic long-chain acylcarnitines. The feasibility of umbilical cord blood use in screening for acylcarnitine analysis and free carnitine has been hypothesized but not reported in LCHAD/TFP neonates. We present a 4 week old female who was at risk of inheriting LCHAD/TFP deficiency and was diagnosed at the time of delivery using umbilical cord blood. Umbilical cord blood was collected at delivery and sent for acylcarnitine analysis. Treatment was started immediately. Acylcarnitine analysis demonstrated findings that are consistent with a biochemical diagnosis of LCHAD/TFP deficiency. Patients with LCHAD/TFP deficiency should have treatment initiated as early as possible to avoid acute decompensation and minimize the long-term complications of the disorder including cardiomyopathy. In pregnancies at risk of having a child with LCHAD/TFP deficiency, umbilical cord blood sample is an efficient method to diagnose an inborn error of metabolism such as LCHAD/TFP deficiency.

Thiamine pyrophosphokinase deficiency causes a Leigh Disease like phenotype in a sibling pair: identification through whole exome sequencing and management strategies.

We present a sibling pair with Leigh-like disease, progressive hypotonia, regression, and chronic encephalopathy. Whole exome sequencing in the younger sibling demonstrated a homozygous thiamine pyrophosphokinase (TPK) mutation. Initiation of high dose thiamine, niacin, biotin, α-lipoic acid and ketogenic diet in this child demonstrated improvement in neurologic function and re-attainment of previously lost milestones. The diagnosis of TPK deficiency was difficult due to inconsistent biochemical and diagnostic parameters, rapidity of clinical demise and would not have been made in a timely manner without the use of whole exome sequencing. Molecular diagnosis allowed for attempt at dietary modification with cofactor supplementation which resulted in an improved clinical course.