Biochemical and molecular characteristics among infants with abnormal newborn screen for very-long-chain acyl-CoA dehydrogenase deficiency: A single center experience

Abstract

Objective

To define the biochemical and molecular characteristics and diagnostic outcomes of a large US cohort of VLCAD deficiency positive cases as detected by newborn screening (NBS) with MS:MS. This relatively common disorder of fatty acid oxidation is screened for in every state in America and often results in extensive testing of multiple samples to arrive at a diagnostic conclusion.

Materials and methods

We compared NBS dried blood spot (DBS) acylcarnitine profile (ACP) C14, C14:1, C14:2, C14:1/C12:1 ratio and plasma C14, C14:1, C14:2, C14:1/C12:1, C14:1/C16 and C14:1/C2 ratios among true positive and false positive cases. Results of VLCAD enzyme analysis, molecular testing and fibroblast fatty acid oxidation probe assay were analyzed.

Results

The presence of compound heterozygous or homozygous pathogenic variants, along with elevations of C14, C14:1 and C14:1/C12:1 ratio, identified 19 VLCAD deficiency cases. All were asymptomatic at most recent follow-up visits. The C14:1/C12:1 ratio in NBS-DBS ACP and plasma acylcarnitine profiles at follow-up (follow-up plasma ACP), is the most useful marker to differentiate between true and false positive cases. Among all cases with molecular analysis data available, approximately 56.7% had a single pathogenic mutation. Lymphocyte enzyme analysis (n = 61) was uninformative in 23% of cases studied.

Conclusion

VLCAD deficiency NBS by MS:MS is highly effective at identifying asymptomatic affected infants. Our cohort showed that elevation of C14:1/C12:1, in both NBS DBS and plasma ACP, was informative in discriminating affected from unaffected individuals and contributes to improve the accuracy of confirmatory testing of infants with presumptive positive for VLCAD deficiency.

1. Introduction

Very long chain acyl-CoA dehydrogenase (VLCAD) deficiency (OMIM 201475) is an inherited autosomal recessive long chain fatty acid beta-oxidation disorder. It is characterized by a heterogeneous phenotypic spectrum from asymptomatic to severe neonatal onset form. Clinical symptoms may include severe neonatal onset multiorgan failure, sudden death, cardiomyopathy, cardiac arrhythmia, recurrent episodes of hypoglycemia, hepatomegaly, muscle symptoms, rhabdomyolysis, and muscle weakness [[1], [2], [3]]. The incidence of VLCAD deficiency is estimated at 1:30,000 to 1:100,000 [4,5]. Newborn screening (NBS) allows prompt diagnosis and timely management of fatty acid oxidation disorders. Early diagnosis of VLCAD deficiency via NBS allows preventive measures which could prevent metabolic insufficiency and hypoglycemic events in individuals with residual VLCAD enzyme activity [3,6]. NBS also detects individuals with a mild phenotypic variant of this disease [7]. Typically, patients are diagnosed pre-symptomatically through NBS and many of them remain asymptomatic with preventive measures [7,8]. Tandem mass spectrometry (MS/MS) is a high-throughput approach which allows identification of multiple metabolites [9]. The first pilot project implementing MS/MS technology started in North Carolina from 1997 to 1999 [10]. In 2004, many states, including Louisiana, started using MS/MS technology in their newborn screening program. With expanded NBS and the development of the Recommended Universal Screening Panel (RUSP) [11], the majority of fatty acid oxidation disorders can be detected through newborn screening by quantifying acylcarnitine species using MS/MS technology.

Long chain acylcarnitines including tetradecanoylcarnitine (C14), tetradecenoylcarnitine (C14:1), tetradecadienoylcarnitine (C14:2) and dodecenoylcarnitine (C12:1) are used as biomarkers for VLCAD deficiency [12]. Elevations of C14, C14:1 and C14:1/C12:1 ratio on NBS are specific markers in screening for VLCAD deficiency. The diagnosis of VLCAD deficiency based on biochemical markers can be challenging since normalization of ACP on a second sample has been observed in individuals with VLCAD deficiency during an anabolic state [4,13]. Clinical Laboratory Integrated Reports (CLIR, formerly R4S) postanalytical testing was used in NBS programs in some states and other countries to a calculate a predictive score for VLCAD deficiency. This is done by using acylcarnitine values and ratios including C14:1, C14, C14:2, C16:1, C16OH, C12, C12:1, C14:1/C2, C14:1/C12:1, and C14:1/C16 to increase positive predictive value [14,15]. However, the use of absolute values and ratios of acylcarnitines alone are not perfectly able to distinguish affected VLCAD cases from carriers [8,16]. Elevation of C14:1 is observed in 3 groups of patients including infants with VLCAD deficiency, heterozygous carriers and healthy infants during catabolic state. The ratios of C14:1/C2, C14:1/C16 and C14:1/C12:1 have been proposed to reduce false positives and discriminate these groups [16,17]. Molecular analysis of ACADVL improves diagnostic accuracy but results often take weeks and some insurers have not historically covered molecular genetic testing. In previous studies, a single pathogenic variant was identified in rare VLCAD deficiency cases [18,19]. To avoid misdiagnoses of VLCAD deficiency, molecular analysis and/or leukocyte enzyme analysis as a second-tier testing are recommended regardless of the result of subsequent plasma ACP [4,8].

The objective of this study was to describe quantitative plasma ACP in a cohort of newborns that screened positive for VLCAD deficiency. To highlight the usefulness of these values and ratios, we also compare ACP species and ratios among true positive and false positive groups.

2. Materials and methods

2.1. Patients

This was a single-center, retrospective observational study of newborn infants with abnormal NBS for VLCAD deficiency in Louisiana from January 1, 2009 to May 31, 2023. The study was approved by the Tulane University Institutional Review Board (IRB). We analyzed results of NBS and confirmatory tests of 237 infants with abnormal NBS for VLCAD deficiency. An abnormal NBS for VLCAD deficiency was defined by elevation of C14:1 (> 0.7 μmol/L). Infants with a positive screen were referred for confirmatory testing. Newborns whose confirmatory results were not available or lost were excluded. Among 237 screen-positive newborns, a full data set data was available on 167 cases (70%).

2.2. Follow-up testing

Confirmatory tests included plasma quantitative ACP, ACADVL sequencing, lymphocyte VLCAD enzyme analysis and mitochondrial fatty acid beta-oxidation studies in cultured fibroblasts. Sample collection times for these tests varied. Sequencing of ACADVL was performed in 137 patients. Leukocyte VLCAD enzyme residual activity was performed in some cases when molecular testing was unavailable (61 patients) and in 44 cases with heterozygous pathogenic variant detected. Leukocytes VLCAD enzyme activity was sent to Children's Hospital Colorado Biochemical Genetics Laboratory. Fatty acid oxidation probe assay (cultured fibroblasts) was done in 12 patients in whom molecular testing and lymphocyte enzyme assay was unavailable and in cases with inconclusive molecular testing result.

2.3. True positive and false positive determination

True positives were determined when follow-up plasma ACP was diagnostic (e.g., persistent abnormal elevations of diagnostic acylcarnitines) and homozygous or compound heterozygous pathogenic variants were present. In cases in which follow-up plasma ACP and sequence analysis were inconclusive, leukocyte enzyme analysis and/or fibroblast fatty acid oxidation studies were performed. Of the original 167 presumptive positives, 19 were true positives.

2.4. NBS-DBS ACP analysis

We studied the differences of NBS-DBS ACP among 19 true positive cases and 81 false positive cases. Of 142 false positive cases, we analyzed the data from 81 false positive cases who had additional confirmatory testing available. Sixty-one cases were not included in this analysis since they did not have a functional studies to confirm false positive status. The analyzed cases were categorized into 2 groups according to the outcome of confirmatory testing: the first group represents true positive cases the second group represents false positive cases.

False positive cases analyzed included 1) twenty-five cases with no mutation detected on ACADVL sequencing and levels of C14 species on follow-up plasma ACP not suggestive of VLCAD deficiency, 2) nineteen cases without molecular analysis but VLCAD enzyme analysis/or fibroblast fatty acid oxidation studies reported to be normal, 3) thirty-five cases with heterozygous pathogenic variant and normal fibroblast fatty acid oxidation studies or VLCAD enzyme analysis in unaffected ranges, and 4) two cases with one pathogenic variant and one VUS but normal fibroblast fatty acid oxidation studies.

2.5. Plasma ACP analysis

Nineteen true positive cases and 31 false positive cases had abnormal plasma ACP on follow-up testing. Fifteen of 31 false positive cases had either normal fibroblast fatty acid oxidation or VLCAD enzyme analysis included in this analysis. Of the remaining 16 cases, 14 cases did not have a functional study and 2 cases had inconclusive VLCAD enzyme analysis results but abnormal, nondiagnostic plasma ACP and inability to obtain further testing. Of the fifteen false positive cases with conclusive confirmatory testing, 1) four cases had no mutation detected on ACADVL sequencing and levels of C14 species not suggestive of VLCAD deficiency, 2) three cases had heterozygous pathogenic variant and normal fibroblast fatty acid oxidation studies, and 3) Eight cases with heterozygous pathogenic variant and VLCAD enzyme activity level in the unaffected ranges.

2.6. Data collection

Demographic, clinical, and laboratory data were abstracted from paper and electronic medical records. NBS data were collected from NBS information system of the Louisiana Department of Health (LDH) Genetic Diseases Program. Information from the LDH database used for recording and tracking follow-up was exported from the LDH lab database. Confirmatory tests were collected in clinic and results were analyzed by the authors. Data were retrieved for NBS results (C14:1 (<0.7), C14 (<0.7), C14:2, C14:1/C12:1 (<5)), follow-up plasma ACP (C2, C14 (0–0.1), C14:1 (0–0.22), C14:2 (0–0.1), C12:1 (0–0.22), C14:1/C2, C14:1/C12:1, and C14:1/C16 ratio), ACADVL sequencing, VLCAD residual enzyme activity results and fatty acid oxidation studies in cultured fibroblasts.

2.7. Statistical analysis

NBS-DBS APC and follow-up plasma ACP values were summarized with means, standard deviations, and ranges (min – max). Due to the non-normally distributed nature of the data, the Wilcoxon Rank Sum Test was used to assess statistically significant differences between true positive and false positive groups. The Wilcoxon test is a nonparametric test, based on ranks of the observations within each group, and does not assume a known distribution. Box plots are presented to describe the differences in distributions of these ranks. All statistical analyses were performed using SAS9.4.

3. Results

3.1. Newborn screen outcome

Among 237 presumptive positive cases, 19 cases were diagnosed with VLCAD deficiency (true positive), 212 cases were false positive, and 6 cases were lost to follow-up, yielding a positive predictive value of 8%. There were 876,938 births between January 2009–May 2023. Hence, the incidence of VLCAD deficiency in the state of Louisiana is approximately 1:50,000. Of 212 false positive cases, complete laboratory data were available for 148 cases.

3.2. General characteristics

In this cohort, 98/237 (41%) were female. The majority of subjects, 165/237 (70%) were White. Black and Hispanic subjects made up about 16% (39/237) and 1.7% (4/237) of the remaining cohort, respectively. Of 19 true positive cases, 12/19 (63.2%) were males and 7/19 (36.8%) were female. Of the tru positive cases, around 58% were White and ages ranged from 8 months-14 years.

3.3. NBS results

Average age of NBS-DBS collection was 1.7 days. Nineteen true positive cases showed significant elevation of C14, C14:1, C14:2 and C14:1/C12:1. The mean values (SD), on the NBS for C14, C14:1, C14:2 and C14:1/C12:1 ratio, were 1.80 (1.33) μmol/L, 2.79 (1.81) μmol/L, 0.32 (0.16) μmol/L and 8.10 (3.32), respectively (Table 1). Eighteen out of nineteen true positive cases had NBS C14:1 value above 1 μmol/L. Only 6 out of 142 false positive cases had NBS C14:1 value above 1 μmol/L. NBS-DBS values for C14, C14:1 and C14:2 between 19 true positive cases and 81 false positive cases were statistically significantly different (p value <0.0001). There was some overlap in values of NBS C14, C14:1 and C14:2. NBS-DBS C14:1/C12:1 ratio showed complete discrimination between true positive cases (min C14:1/C12:1 = 3.82) and false positive cases (max C14:1/C12:1 = 3.19).

Table 1.

NBS-DBS ACP values.

NBS-DBS ACP values	True positive (19) Mean ± SD (min-max)	False positive (81) Mean ± SD (min-max)
NBS C14	1.80 ± 1.33 (0.58–4.85) n = 17	0.69 ± 0.19 (0.12–1.41) n = 77
NBS C14:1	2.63 ± 1.83 (0.79–6.7) n = 18	0.73 ± 0.13 (0.29–1.19) n = 77
NBS C14:2	0.32 ± 0.16 (0.16–0.57) n = 12	0.09 ± 0.03 (0.04–0.23) n = 71
NBS C14:1/C12:1	8.10 ± 3.32 (3.82–14.1) n = 13	1.60 ± 0.45 (0.77–3.19) n = 76

The results of C14, C14:1, C14:2 and C14:1/C12:1 ratio in NBS-DBS are illustrated in Fig. 1. Ranks of C14, C14:1, C14:2 and C14:1/C12:1 ratio were compared from true positive and false positive cases. Significant elevations for all four of these values were observed in true positive cases compared to false positive cases (p value <0.0001).

Fig. 1.

Distribution ranks of C14, C14:1, C14:2 values and C14:1/C12:1 ratio from NBS-DBS results among true positive and false positive cases. Values between groups were statistically significant (P value <0.0001).

3.4. Follow-up plasma ACP results

Average age of follow-up plasma ACP collection was 8.1 days. Follow-up plasma ACP was abnormal in all true-positive cases, with the average age of plasma ACP collection of 7.5 days. Among 19 true positive cases, 18 cases (95%) had elevation of C14, C14:1, and C14:2 on follow-up plasma ACP. Only one case had elevation of C14 and C14:2 only. On the contrary, only 31 of 142 of false positive cases (22%) were noted to have elevation of one or more C14 species on follow-up quantitative plasma ACP. Follow-up plasma ACP was normal in 111 false positive cases. Of the 31 false positive cases with abnormal follow-up quantitative plasma ACP, 24 of 31 cases (77%) were noted to have only a heterozygous pathogenic variant in ACADVL. Mean (SD), min and max of C14, C14:1, C14:2, C14:1/C12:1, and C14:1/C16 of follow-up quantitative plasma ACP in true positive and false positive cases with abnormalities on follow-up plasma ACP, are illustrated in Table 2. As depicted in Fig. 2, follow-up quantitative plasma values of C14, C14:1, C14:2, C14:1/C12:1, C14:1/C16 and C14:1/C2 among 19 true positive cases and 15 false positive cases with abnormal plasma ACP on follow-up testing were compared. Values showed statistically significant different C14, C14:1, C14:2, C14:1/C12:1 and C14:1/C2 between the 2 groups, especially C14:1/C12:1 ratio. Nevertheless, some overlapping of C14, C14:1, C14:2, and C14:1/C2 among the 2 groups was observed. C14:1/C12:1 ratio is significantly higher in true positive cases with the mean of 10.6. In cases with abnormal follow-up ACP, there is complete discrimination of true positive and false positive cases using C14:1/C12:1, using a cutoff of 3.

Table 2.

Mean (SD), min and max of C14, C14:1, C14:2, C14:1/C12:1, C14:1/C16 of follow-up quantitative plasma ACP.

Follow-up plasma ACP	True positive (N = 19) Mean ± SD (min-max)	False positive (N = 15, cases with abnormal FU ACP) Mean ± SD (min- max)	Wilcoxon Rank Sum Test P-value
C14	0.67 ± 1.12 (0.1–5.13) N = 19	0.26 ± 0.14 (0.12–0.65) N = 15	0.034
C14:1	1.52 ± 1.69 (0.11–5.54) N = 19	0.28 ± 0.21 (0.06–0.74) N = 14	0.0002
C14:2	0.40 ± 0.33 (0.04–1.54) N = 19	0.08 ± 0.05 (0.03–0.24) N = 15	<0.0001
C14:1/C12:1	10.60 ± 3.98 (4.48–20.3) N = 17	1.76 ± 0.37 (1.1–2.33) N = 14	<0.0001
C14:1/C16	3.78 ± 3.22 (0.08–9.17) N = 19	4.2 ± 2.65 (0.79–8) N = 13	0.54
C14:1/C2	0.24 ± 0.25 (0.01–0.97) N = 19	0.37 + 0.25 (0.01–0.91) N = 15	0.025

Fig. 2.

Box plot of plasma concentration of C14, C14:1, C14:2, C14:1/C12:1, C14:1/C16 and C14:1/C2 from follow-up quantitative plasma ACP in true positive and false positive cases with abnormalities on follow-up plasma ACP. Values between groups were statistically significant (P ≤0.0001).

When analyzing plasma ACP C14:1/C12:1 ratio in all false-positive cases (142 cases), there were 11 cases with C14:1/C12:1 ratio in the range of 3–6.5. However, their C14, C14:1, and C14:2 level on follow-up plasma ACP were normal. Of these, 7 cases had either normal VLCAD enzyme analysis or normal fibroblast fatty acid oxidation studies; two cases with heterozygous pathogenic variant had VLCAD enzyme activity level at 1–2 mutation range and two cases with heterozygous pathogenic variant did not have functional study.

3.5. Molecular analysis

Among 167 presumptive positive cases, molecular analysis was performed in 137 cases, 19 cases from 17 families were classified as true positive cases considering the presence of homozygous or compound heterozygous pathogenic variants in ACADVL together with significant elevation of C14 species. Clinically, 18 true positive cases are asymptomatic at most recent follow-up visit. One case was lost to follow-up. Within the group of 118 false positive cases whose ACADVL sequencing result was available, 68.6% (81/118) were noted to have one pathogenic variant in ACADVL. The majority of the changes were missense variants. Approximately 6% (7/118) false positive cases were noted to have a variant of uncertain significance (VUS) in ACADVL (p.Ala213Thr, p.Asn252Ser, p.Ser423Leu (2 cases), p.Gly523Arg, p.Arg567Trp, and c.416_431 exon 6). There was no pathogenic variant or VUS was noted in 23.7% (28/118) cases. Two cases with biallelic heterozygous pathogenic variant and VUS (p.Leu125Pro and p.Ile356Val, respectively) were reported to have normal fibroblasts fatty acid oxidation studies and were classified as false positive. The three most common pathogenic variants were c.848 T > C (p.Val283Ala), c.1376G > A (p.Arg459Gln) and c.1097G > A (p.Arg366His) with allele frequency of 19%, 16.7% and 5.8% respectively.

3.6. VLCAD enzyme analysis in lymphocytes

The measurement of VLCAD enzyme residual activity was performed in 61 cases. There are 4 reference ranges for VLCAD enzyme activity level consistent with no mutation, 1 mutation, inconclusive 1 to 2 mutations or 2 mutations. Reference ranges overlap among these categories. Seventeen cases without molecular analysis had VLCAD enzyme activity analysis. Sixteen out of seventeen cases had VLCAD enzyme activity level within unaffected range (no mutation or 1 mutation range). One case had VLCAD enzyme activity level falling into 1 to 2 mutation range (inconclusive), however, this case's plasma ACP was not suggestive of VLCAD deficiency.

Among 88 cases with one pathogenic variant or one VUS, 44 cases (50%) had VLCAD enzyme activity analysis. Of these, 31 cases were noted to have VLCAD enzyme activity level falling within unaffected range. Thirteen cases had VLCAD enzyme activity falling within the overlapping reference range for 1 mutation and 2 mutations. None of these cases had VLCAD enzyme activity in the affected range (consistent with 2 mutations).

3.7. Fibroblast fatty acid oxidation probe assay

Fibroblast fatty acid oxidation probe assay was performed in 12 cases; it was reported to be normal in all 12 cases. Sequencing in 9 of 12 cases showed 1 infant with no mutation detected in ACADVL, 4 cases with 1 pathogenic variant in ACADVL, 2 cases with 1 VUS in ACADVL and 2 cases with 1 pathogenic variant and 1 VUS in ACADVL. These 12 cases were classified as false positive NBS cases.

4. Discussion

Expanded newborn screening has resulted in ascertainment of many rare metabolic conditions in patients before they become ill. This screening test requires confirmatory testing to assign a diagnosis or determine a false positive NBS. At our institution, the combination of follow-up plasma ACP, molecular analysis, VLCAD enzyme analysis in lymphocytes, and fibroblast fatty acid oxidation probe assay were used to diagnose newborns with VLCAD deficiency. Confirmatory metabolic testing can be affected by varying metabolic states in the newborn leading to inconclusive results in some cases. Molecular testing may also fail to identify individuals with VLCAD deficiency who have pathogenic variants that are not detected by the standard sequencing method such as large deletions, duplications, promotor mutations, or deep intronic mutations [19,20]. In addition to ACADVL sequencing, functional studies including VLCAD enzyme activity study and/or fibroblast fatty acid oxidation probe assay have been used to improve the detection of VLCAD deficiency in some presumptive positive NBS cases. Fatty acid oxidation studies can only be performed on cultured fibroblasts, requiring a higher level of invasive testing (e.g., skin biopsy).

In our cohort, all 19 true positive cases were noted to have homozygous or biallelic pathogenic variants in ACADVL. The incidence of VLCAD deficiency in the state of Louisiana between January 2009–May 2023 is 1:50,000. Elevation of either C14 or C14:1 or C14:2 was not sufficient in all cases to differentiate between true positive and false positive cases. Elevation of these values is seen in VLCAD deficiency cases and may be seen in normal individuals during a catabolic state. Other ACP values such as C14:1, C14:1/C12:1, C14:1/C16, and C14:1/C2 have been used as diagnostic markers. The C14:1/C12:1 ratio was useful in a small study to differentiate individuals with VLCAD deficiency from individuals in catabolic state [21]. Additionally, the serum C14:1/C12:1 ratio has been considered to be a precise marker for detection of VLCAD deficiency [17] but has not been reported in a large cohort. Our findings agree with these prior observations. There were significant differences of C14, C14:1, C14:2, C14:1/C12:1, and C14:1/C2 among the 2 groups. We found support for the concept that C14:1/C12:1 is the most reliable discriminating measure to distinguish true positives from false positives.

VLCAD enzyme activity level in lymphocytes was a somewhat useful test to distinguish between true positive and false positive cases [16]. However, our study showed that 30% of cases with VLCAD enzyme activity level in lymphocytes performed had ambiguous results due VLCAD enzyme activity level which were “in range of 1 to 2 pathogenic mutations”.

This study showed that NBS revealed a number of cases with heterozygous pathogenic variant, which is the most common finding in false positive case, accounting for 59% of cases (81/137). Most cases with one variant identified in ACADVL had follow-up plasma ACP values and ratios which were not suggestive of VLCAD deficiency. All cases with significant elevation of C14, C14:1, C14:2, C14:1/C12:1 and C14:1/C16 suggestive of VLCAD deficiency were found to have homozygous or biallelic heterozygous pathogenic variants in ACADVL. Normalization of plasma ACP was not observed in true positive cases who had subsequent plasma ACP in different time points. With preventive measures, these 19 cases were asymptomatic at most recent follow-up visit, at the age range of 8 months to 14 years, and average age of 9 years (1 case lost to follow-up). Follow-up period ranges from 6 months to 12 years. Eighteen cases did not take medium chain triglyceride (MCT) oil or triheptanoin at most recent follow-up visit. Mildly elevated creatine kinase (CK) at 410 U/L was noted in one case at around one week old. However, subsequent CK levels were normal. There was no muscle or cardiac involvement reported in any of these cases at most recent visits. The most common variant seen in this cohort is p.Val283Ala (19%) which has been associated with residual enzyme activity. This variant, previously reported to cause milder phenotype and adult-onset forms [22], was present in 10/38 alleles in 19 cases. A missense variant, p.Arg366His, is seen in 4/38 alleles and was described in association with an adult-onset muscle form [23] and in patients who presented with neonatal decompensation [24]. No null or truncating variants, associated with severe disease and cardiomyopathy [18], were observed in our cohort. Other types of variants and clinical outcome have not been clearly defined. Ongoing research is needed to help understand the correlations of other variants.

This study has some limitations. The cohort spans a period before the use of molecular analysis was widely available. The use of enzyme assays in lymphocytes was helpful but often not informative. Fibroblast studies were informative but invasive, effortful, and time-consuming. The majority of cases had only one ACP sample result, which may not represent their average C14, C14:1, C14:2, C14:1/C12:1, C14:1/C16, C14:1/C2 ratio. This study only represents the outcome of presumptive positive NBS for VLCAD deficiency in the state of Louisiana. The ACP values and ratios may not be applied to other institutions or laboratories due to variable reagent kits/assay and cutoffs used in different laboratories. Among 237 presumptive positive cases from January 2009–April 2022, complete laboratory data were available from only 167 cases (70%).

Within these limitations, our study presents a large cohort of VLCAD NBS cases and confirms similar results in smaller previous studies. Additionally, this study correlates biochemical characteristics with molecular and enzymatic findings. The p.Val283Ala was reported as the highest frequency allele among infants with presumptive positive NBS for VLCAD deficiency [16,22] in the US. Our study demonstrated a similar result with roughly 16% of this population having one or more p.Val283Ala variant. Our findings support consideration of C14:1/C12:1 as a primary measure along with other C14 species for discrimination of false positives and true positives for VLCAD deficiency in the absence of informative molecular data.

5. Conclusions

Most common presumptive positive NBS in Louisiana for VLCAD deficiency cases are carriers of ACADVL pathogenic variants. Most true positive cases had elevation of one or more C14 species, C14:1/C12 and C14:1/C16 ratio on follow-up plasma ACP. VLCAD enzyme analysis in lymphocytes may be useful to confirm VLCAD deficiency carrier in cases with a heterozygous mutation in ACADVL. However, result of enzyme testing can be inconclusive. Thus, a combination of diagnostic methods is required. C14:1/C12:1 is a precise diagnostic marker of VLCAD deficiency, which was consistent with previous studies.

Funding

None.

Author contributions

JU, GN and HCA designed and conceptualized the study and performed clinical analysis. JU and GN performed data curation. JU drafted the manuscript. JJL performed data and statistical analysis. All authors were involved with revising the manuscript.

Institutional review board statement

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Tulane University Institutional Review Board (IRB).

Declaration of Competing Interest

The authors declare no conflict of interest.

Data availability

Data will be made available on request.