Genotype-phenotype correlation in primary carnitine deficiency

Primary carnitine deficiency is caused by defective OCTN2 carnitine transporters encoded by the SLC22A5 gene. Lack of carnitine impairs fatty acid oxidation resulting in hypoketotic hypoglycemia, hepatic encephalopathy, skeletal and cardiac myopathy. Recently, asymptomatic mothers with primary carnitine deficiency were identified by low carnitine levels in their infant by newborn screening. Here, we evaluate mutations in the SLC22A5 gene and carnitine transport in fibroblasts from symptomatic patients and asymptomatic women. Carnitine transport was significantly reduced in fibroblasts obtained from all patients with primary carnitine deficiency, but was significantly higher in the asymptomatic women's than in the symptomatic patients' fibroblasts (P < 0.01). By contrast, ergothioneine transport (a selective substrate of the OCTN1 transporter, tested here as a control) was similar in cells from controls and patients with carnitine deficiency. DNA sequencing indicated an increased frequency of nonsense mutations in symptomatic patients (P < 0.001). Expression of the missense mutations in Chinese hamster ovary (CHO) cells indicated that many mutations retained residual carnitine transport activity, with no difference in the average activity of missense mutations identified in symptomatic versus asymptomatic patients. These results indicate that cells from asymptomatic women have on average higher levels of residual carnitine transport activity as compared to that of symptomatic patients due to the presence of at least one missense mutation.     

Functional and molecular studies in primary carnitine deficiency

Primary carnitine deficiency is caused by a defect in the OCTN2 carnitine transporter encoded by the SLC22A5 gene. It can cause hypoketotic hypoglycemia or cardiomyopathy in children, and sudden death in children and adults. Fibroblasts from affected patients have reduced carnitine transport. We evaluated carnitine transport in fibroblasts from 358 subjects referred for possible carnitine deficiency. Carnitine transport was reduced to 20% or less of normal in fibroblasts of 140 out of 358 subjects. Sequencing of the 10 exons and flanking regions of the SLC22A5 gene in 95 out of 140 subjects identified causative variants in 84% of the alleles. The missense variants identified in our patients and others previously reported (n = 92) were expressed in CHO cells. Carnitine transport was impaired by 73 out of 92 variants expressed. Prediction algorithms (Polyphen‐2, SIFT) correctly predicted the functional effects of expressed variants in about 80% of cases. These results indicate that mutations in the coding region of the SLC22A5 gene cannot be identified in about 16% of the alleles causing primary carnitine deficiency. Prediction algorithms failed to determine the functional effects of amino acid substitutions in this transmembrane protein in about 20% of cases. Therefore, functional studies in fibroblasts remain the best strategy to confirm or exclude a diagnosis of primary carnitine deficiency.     

Pharmacological rescue of carnitine transport in primary carnitine deficiency

Primary carnitine deficiency is a recessive disorder caused by heterogeneous mutations in the SLC22A5 gene encoding the OCTN2 carnitine transporter. Here we extend mutational analysis to eight new families with this disorder. To determine the mechanism by which missense mutations impaired carnitine transport, the OCTN2 transporter was tagged with the green fluorescent protein and expressed in CHO cells. Analysis by confocal microscopy indicated that several missense mutants (M1I, R169W, T232 M, G242 V, S280F, R282Q, W283R, A301D, W351R, R399Q, T440 M, E452 K, and T468R) matured normally to the plasma membrane. By contrast, other mutations (including R19P, DeltaF22, R83L, S280F, P398L, Y447C, and A142S/R488 H) caused significant retention of the mutant OCTN2 transporter in the cytoplasm. Failed maturation to the plasma membrane is a common mechanism in disorders affecting membrane transporters/ion channels, including cystic fibrosis. To correct this defect, we tested whether drugs reducing the efficiency of protein degradation in the endoplasmic reticulum (ER) (phenylbutyrate, curcumin) or capable of binding the OCTN2 carnitine transporter (verapamil, quinidine) could improve carnitine transport. Prolonged incubation with phenylbutyrate, quinidine, and verapamil partially stimulated carnitine transport, while curcumin was ineffective. These results indicate that OCTN2 mutations can affect carnitine transport by impairing maturation of transporters to the plasma membrane. Pharmacological therapy can be effective in partially restoring activity of mutant transporters.     

Retrospective review of Japanese sudden unexpected death in infancy: the importance of metabolic autopsy and expanded newborn screening

Sudden unexpected death in infancy is defined as sudden unexpected death occurring before 12 months of age. The common causes of sudden unexpected death in infancy are infection, cardiovascular anomaly, child abuse, and metabolic disorders. However, the many potential inherited metabolic disorders are difficult to diagnose at autopsy and may therefore be underdiagnosed as a cause of sudden unexpected death in infancy. In the present study we retrospectively reviewed 30 Japanese sudden unexpected death in infancy cases encountered between 2006 and 2009 at our institute. With postmortem blood acylcarnitine analysis and histological examination of the liver, we found two cases of long-chain fatty acid oxidation defects. Molecular analysis revealed that the one patient had a compound heterozygote for a novel mutation (p.L644S) and a disease-causing mutation (p.F383Y) in the carnitine palmitoyltransferase 2 gene. Furthermore, retrospective acylcarnitine analysis of the newborn screening card of this patient was consistent with carnitine palmitoyltransferase II deficiency. Metabolic autopsy and expanded newborn screening would be helpful for forensic scientists and pediatricians to diagnose fatty acid oxidation disorders and prevent sudden unexpected death in infancy.     

Maternal medium-chain acyl-CoA dehydrogenase deficiency identified by newborn screening

Prior to the advent of expanded newborn screening, sudden and unexplained death was often the first and only symptom of medium-chain acyl-CoA dehydrogenase deficiency (MCADD). With the use of tandem mass spectrometry, infants can now be identified and treated before a life threatening metabolic decompensation occurs. Newborn screening has also been shown to detect previously undiagnosed maternal inborn errors of metabolism. We have now diagnosed two women with MCADD following the identification of low free carnitine in their newborns. While one of the women reported prior symptoms of fasting intolerance, neither had a history of metabolic decompensation or other symptoms consistent with a fatty acid oxidation disorder. These cases illustrate the importance of including urine organic acid analysis and an acylcarnitine profile as part of the confirmatory testing algorithm for mothers when low free carnitine is identified in their infants.     

A mutation creating an upstream translation initiation codon in SLC22A5 5′UTR is a frequent cause of primary carnitine deficiency

Primary carnitine deficiency is caused by a defect in the active cellular uptake of carnitine by Na⁺‐dependent organic cation transporter novel 2 (OCTN2). Genetic diagnostic yield for this metabolic disorder has been relatively low, suggesting that disease‐causing variants are missed. We Sanger sequenced the 5′ untranslated region (UTR) of SLC22A5 in individuals with possible primary carnitine deficiency in whom no or only one mutant allele had been found. We identified a novel 5′‐UTR c.‐149G>A variant which we characterized by expression studies with reporter constructs in HeLa cells and by carnitine‐transport measurements in fibroblasts using a newly developed sensitive assay based on tandem mass spectrometry. This variant, which we identified in 57 of 236 individuals of our cohort, introduces a functional upstream out‐of‐frame translation initiation codon. We show that the codon suppresses translation from the wild‐type ATG of SLC22A5, resulting in reduced OCTN2 protein levels and concomitantly lower transport activity. With an allele frequency of 24.2% the c.‐149G>A variant is the most frequent cause of primary carnitine deficiency in our cohort and may explain other reported cases with an incomplete genetic diagnosis. Individuals carrying this variant should be clinically re‐evaluated and monitored to determine if this variant has clinical consequences.     

Carnitine transport by organic cation transporters and systemic carnitine deficiency

The intracellular homeostasis is controlled by different membrane transporters. Organic cation transporters function primarily in the elimination of cationic drugs, endogenous amines, and other xenobiotics in tissues such as the kidney, intestine, and liver. Among these molecules, carnitine is an endogenous amine which is an essential cofactor for mitochondrial beta-oxidation. Recently, a new family of transporters, named OCT (organic cation transporters) has been described. In this minireview, we present the recent knowledge about OCT and focus on carnitine transport, more particularly by the OCTN2. The importance of this sodium-dependent carnitine cotransporter, OCTN2, comes from various recently reported mutations in the gene which give rise to the primary systemic carnitine deficiency (SCD; OMIM 212140). The SCD is an autosomal recessive disorder of fatty acid oxidation characterized by skeletal myopathy, progressive cardiomyopathy, hypoglycemia and hyperammonemia. Most of the OCTN2 mutations identified in humans with SCD result in loss of carnitine transport function. Identifying these mutations will allow an easy targeting of the SCD syndrome. The characteristics of the juvenile visceral steatosis (jvs) mouse, an animal model of SCD showing similar symptoms as humans having this genetic disorder, are also described. These mice have a mutation in the gene encoding the mouse carnitine transporter octn2. Although various OCTN carnitine transporters have been identified and functionally characterized, their membrane localization and regulation are still unknown and must be investigated. This knowledge will also help in designing new drugs that regulate carnitine transport activity.     

Argininosuccinate lyase deficiency: Longterm outcome of 13 patients detected by newborn screening

Argininosuccinate lyase deficiency is a urea cycle disorder which can present in the neonatal period with hyperammonemic encephalopathy, or later in childhood with episodic vomiting, growth and developmental delay. Abnormal hair, hepatomegaly, and hepatic fibrosis are unique features of this disorder. Twelve patients with argininosuccinate lyase deficiency were ascertained between 4 and 6 weeks of age by urine amino acid screening. One infant in a previously identified family was diagnosed shortly after birth. Diagnosis was confirmed by enzyme assay in red blood cells and/or skin fibroblasts. At the time of last follow-up, patients had been followed for 13–33 years. All patients were asymptomatic at detection, 7 had slightly increased blood ammonia, and all were initially treated with low-protein diet. Utilization of ¹⁴C-citrulline by intact skin fibroblasts measured by ¹⁴C incorporation into macromolecules was 74–135% of the control mean for 7 of the 8 patients studied. Nine patients had normal development, 4 had learning disability, 6 had EEG abnormalities, 3 had seizure disorder. None had any episodes of hyperammonemic coma. None had hepatomegaly. Patients detected by screening had higher enzyme activity measured by the ¹⁴C-citrulline incorporation assay than comparison groups of patients with neonatal-onset and with late-onset detected by clinical disease. The ability to utilize ¹⁴C-citrulline by intact fibroblasts seems to correlate with clinical outcome and may have prognostic value. It is likely that early diagnosis and treatment contributed to the relatively mild clinical course of the study group.     

Cardiac conduction improvement in two heterozygotes for primary carnitine deficiency on l‐carnitine supplementation

Sarafoglou K, Tridgell AHC, Bentler K, Redlinger‐Grosse K, Berry SA, Schimmenti LA. Cardiac conduction improvement in two heterozygotes for primary carnitine deficiency on l ‐carnitine supplementation. Expanded newborn screening (NBS) for free carnitine levels has led to the identification of a larger number of heterozygous infants of undiagnosed mothers affected with systemic primary carnitine deficiency (PCD), which in turn leads to the identification of other undiagnosed heterozygous family members. There is an increasing recognition that individuals heterozygous for mutations of genes involved in fatty acid oxidation (FAO) may become symptomatic under environmental stress (fasting, prolonged exercise and illness). Considering the importance of carnitine in FAO, its role in heart and bowel function and in lipid metabolism, what is still little known is the phenotypic variability, biochemical parameters and clinical course of PCD heterozygotes with consistently low‐to‐normal levels to low levels of carnitine over a lifetime. We report on three generations of a family—an asymptomatic PCD heterozygous infant identified through NBS that led to the diagnosis of her asymptomatic PCD‐affected mother and the heterozygous status of the maternal grandparents who report some cardiac symptoms that overlap with PCD that improved with l ‐carnitine supplementation.     

美国新生儿筛查的现状与展望

新生儿筛查(newborn screening,NBS)是指一些目前还无法在产前作出诊断的先天性疾病,在新生儿期间取少量血液进行快速地检查,从而来判别新生儿是否患有遗传性的疾病.这些疾病多数不致死,但可以严重影响患儿的智力和体格,造成痴呆和残疾.新生儿筛查的实施,有助于早期诊断及早期治疗,从而能将新生儿出生缺陷造成的损害减低到最小,例如避免夭折、减少智能发育迟缓或永久性功能丧失等.     

Molecular spectrum of SLC22A5 (OCTN2) gene mutations detected in 143 subjects evaluated for systemic carnitine deficiency

Systemic primary carnitine deficiency (CDSP) is caused by recessive mutations in the SLC22A5 (OCTN2) gene encoding the plasmalemmal carnitine transporter and characterized by hypoketotic hypoglycemia, and skeletal and cardiac myopathy. The entire coding regions of the OCTN2 gene were sequenced in 143 unrelated subjects suspected of having CDSP. In 70 unrelated infants evaluated because of abnormal newborn screening (NBS) results, 48 were found to have at least 1 mutation/unclassified missense variant. Twenty‐eight of 33 mothers whose infants had abnormal NBS results were found to carry at least 1 mutation/unclassified missense variant, including 11 asymptomatic mothers who had 2 mutations. Therefore, sequencing of the OCTN2 gene is recommended for infants with abnormal NBS results and for their mothers. Conversely, 52 unrelated subjects were tested due to clinical indications other than abnormal NBS and only 14 of them were found to have at least one mutation/unclassified variant. Custom designed oligonucleotide array CGH analysis revealed a heterozygous ～1.6 Mb deletion encompassing the entire OCTN2 gene in one subject who was apparently homozygous for the c.680G>A (p.R227H) mutation. Thus, copy number abnormalities at the OCTN2 locus should be considered if by sequencing, an apparently homozygous mutation or only one mutant allele is identified. ©2010 Wiley‐Liss, Inc.     

新生儿G6PD缺乏症筛查及遗传咨询措施的研究

目的新生儿G6PD缺乏症筛查,计算发病率、基因频率,患儿治疗及遗传咨询。方法筛查用荧光分辨法,确诊用G6PD/6PGD比值测定法。结果2007年10月至2008年10月对我院新出生的9842例新生儿进行了G6PD缺乏症筛查,男性5286例,女性4556例,共检出确诊病例18例。发病率为1.83‰(18/9842),其中男性发病16例,为1.63‰(16/9842),女性2例,为0.2‰(2/9842)。男女比例8∶1。G6PD缺乏症基因频率为0.00303(16/5286)。18例病儿中,祖籍为上海浦东人,发病3例,占16.67%(3/18),祖籍为外地人口发生15例,占83.33%(15/18)。     

3-Methylglutaconic aciduria in carriers of primary carnitine deficiency

The etiology of secondary 3-methylglutaconic aciduria (3-MGA-uria) is not well understood although is thought to be a marker of mitochondrial dysfunction. For this reason, suspicion for a secondary 3-MGA-uria often leads to an extensive clinical and laboratory work-up for mitochondrial disease, although in many cases evidence for mitochondrial dysfunction is never found.3-methylglutaconic aciduria in healthy individuals without known metabolic disease has not been well described. Here, we describe clinical and biochemical features of 23 individuals evaluated at the Greenwood Genetic Center for low plasma free carnitine reported on newborn screening. Of the 23 individuals evaluated, four individuals were diagnosed with primary carnitine deficiency, 16 were identified as carriers for primary carnitine deficiency, and three individuals were determined to be unaffected non-carriers based on molecular and biochemical testing.Elevated 3-MGA (>20 mmol/mol of creatinine) was identified in nine carriers of primary carnitine deficiency, while all unaffected non carriers and all affected individuals with primary carnitine deficiency had a normal 3-MGA level (<20 mmol/mol of creatinine). Average 3-MGA among all carriers was 39.66 mmol/mol of creatinine. Average plasma free carnitine in among all carriers (n = 16) was 13.87 μm/L, and average plasma free carnitine was not significantly different between carriers with and those without elevated 3-MGA (p = 0.66).In summary, we describe elevated 3-MGA as a discriminatory feature in nine healthy carriers of primary carnitine deficiency. Our findings suggest that heterozygosity for pathogenic alterations on SLC22A5 should be considered in the differential for individuals with persistent 3-MGA-uria of unclear etiology.     

Population spectrum of ACADM genotypes correlated to biochemical phenotypes in newborn screening for medium-chain acyl-CoA dehydrogenase deficiency

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most frequent inherited defect of fatty acid oxidation, with a significant morbidity and mortality in undiagnosed patients. Adverse outcomes can effectively be prevented by avoiding metabolic stress and following simple dietary measures. Therefore, prospective newborn screening (NBS) is being proposed for this condition. However, technical validation of MCADD population screening and assessment of its overall benefit require broadening of the as-yet-scarce knowledge of the MCADD genetic heterogeneity unraveled by NBS and its phenotypic consequences. Here, we describe the entire spectrum of sequence variations occurring in newborns with MCADD in the population of Bavaria, Germany, in relation to the biochemical phenotype. Among 524,287 newborns, we identified 62 cases of MCADD, indicating a birth incidence of 1 in 8,456. In all of the 57 newborns available for analysis, two alterations within the MCADD gene (ACADM) were identified. The most prevalent alteration c.985A>G (Lys329Glu) occurred in 27 (47%) newborns in the homozygous and in 18 (32%) in the heterozygous state (63% of defective alleles). The mild folding variant c.199T>C (Tyr67His) was identified in nine individuals, six of them being compound heterozygous with c.985A>G (Lys329Glu). Neither of the prevalent alterations were found in the remaining nine newborns. A total of 18 sequence variations were identified; 13 of them were novel: eight missense mutations, one nonsense mutation, two splice variants, and two small deletions. The remaining five were previously reported in MCADD patients. The ACADM heterogeneity uncovered was larger as anticipated from previous c.985A>G (Lys329Glu) carrier screening data. In addition, we show that MCADD appears to occur as frequently in Turkish newborns as in the native German population. Our data validate that biochemical NBS for MCADD is a highly specific procedure for disease detection, with the identification of a significant share of milder biochemical phenotypes, such as c.199T>C (Tyr67His). These show statistically lower acylcarnitine markers, allowing us to distinguish subgroups within the spectrum of ACADM sequence variations that correlate to biochemical MCADD disease expression. Our data might provide technical and medical guidance for decision making in the worldwide efforts to introduce MCADD population screening.     

Benefits,harms, and costs of newborn genetic screening for hypertrophic cardiomyopathy: Estimates from the PreEMPT model

PurposePopulation newborn genetic screening for hypertrophic cardiomyopathy (HCM) is feasible, however its benefits, harms, and cost-effectiveness are uncertain.MethodsWe developed a microsimulation model to simulate a US birth cohort of 3.7 million newborns. Those identified with pathogenic/likely pathogenic variants associated with increased risk of HCM underwent surveillance and recommended treatment, whereas in usual care, individuals with family histories of HCM underwent surveillance.ResultsIn a cohort of 3.7 million newborns, newborn genetic screening would reduce HCM-related deaths through age 20 years by 44 (95% uncertainty interval [UI] = 10-103) however increase the numbers of children undergoing surveillance by 8127 (95% UI = 6308-9664). Compared with usual care, newborn genetic screening costs $267,000 per life year saved (95% UI, $106,000 to $919,000 per life year saved).ConclusionNewborn genetic screening for HCM could prevent deaths but at a high cost and would require many healthy children to undergo surveillance. This study shows how modeling can provide insights into the tradeoffs between benefits and costs that will need to be considered as newborn genetic screening is more widely adopted.     

Analysis of genetic mutations in Chinese patients with systemic primary carnitine deficiency

Systemic primary carnitine deficiency (CDSP) is caused by mutations in SLC22A5 gene, which encodes organic cation transporter 2(OCTN2). CDSP leads to skeletal or cardiac myopathy and hepatic encephalopathy. The present study aimed to identify SLC22A5 gene mutations and analyze the potential relationship between genotype and clinical symptoms in 20 Chinese patients with CDSP. The complete coding region of the SLC22A5 gene including intron-exon boundaries were amplified and sequenced in all patients. Eighteen different mutations were found; of which, nine were novel. The mutations clustering in exons 1 and 4 accounted for 66.7% of all mutant alleles (26/39). The c.760C>T (p. R254X) was the most frequent mutation (25.6%, 10/39), suggesting it as an ethnic founder mutation. The relationship between genotype and phenotype was investigated in patients carrying the R254X mutation. Homozygous patients with R254X were late-onset cases who presented with dilated cardiomyopathy and muscle weakness after 1 year of age. Compound heterozygous patients carrying R254X, combined with other missense mutations occurred in very specific positions, dramatically altered OCTN2 protein function. Based on the analysis of case studies, a clear relationship between free carnitine (C0) level in plasma and OCTN2 genotype was not found in the present work, however, the low plasma C0 level could not indicate disease severity or genotype. Further functional studies with a large sample size are required to understand the relationship between R254X mutation and CDSP.     

ABO血型不合溶血病合并G-6-PD缺陷症新生儿高胆11例报告

目的研究ABO血型不合溶血病合并葡萄糖-6-磷酸脱氢酶（G-6-PD）缺陷症的新生儿高胆红素血症（简称高胆）的发病特点。方法选择2007年1月至2010年12月收治母婴ABO血型不合新生儿溶血病合并G-6-PD缺陷症11例（A组）与同期单纯ABO血型不合溶血病80例（B组）新生儿高胆进行分析。结果①91例母婴ABO血型不合溶血病的新生儿中合并G-6-PD缺陷症11例（12.1%）。②新生儿高胆发病日龄：两组均以1～2 d为主（A组9例占81.82%,B组67例占83.75%）;高峰日龄：两组均以2～3d为主（A组9例占81.82%,B组73例占91.25%）;消退时间：两组均在〈6天内。发病日龄、高峰日龄、消退时间经统计学分析各组之间均无显著差异（P〉0.05）。③新生儿贫血程度：两组之间中度贫血无显著差异（P〉0.05）,但轻度贫血及血红蛋白正常者组,经统计学分析差异极显著（P〈0.01）。结论 11例母婴ABO血型不合溶血病合并G-6-PD缺陷症新生儿：①母婴ABO血型不合溶血病中合并有G-6-PD缺陷症的几率为12.1%。②高疸发病日龄基本在1～2d内,高峰日龄基本在2～3d内,消退时间均在〈6天内,与单纯ABO血型不合溶血病基本相同。③新生儿轻度贫血较单纯ABO血型不合溶血病明显,表明溶血会加重,但只要早期诊断、早期干预,预后良好。     

Factors that influence parents’ experiences with results disclosure after newborn screening identifies genetic carrier status for cystic fibrosis or sickle cell hemoglobinopathy

Objective

Newborn screening (NBS) identifies genetic carriers for sickle cell hemoglobinopathy and cystic fibrosis. We aimed to identify factors during initial NBS carrier results disclosure by primary care providers (PCPs) that influenced parents’ experiences and reactions.

Methods

Open-ended responses from telephone interviews with 270 parents of carriers were analyzed using mixed-methods. Conventional content analysis identified influential factors; chi-square tests analyzed relationships between factors and parent-reported reactions.

Results

Parents reported positive (35%) or negative (31%) reactions to results disclosure. Parents’ experiences were influenced by specific factors: content messages (72%), PCP traits (47%), and aspects of the setting (30%). Including at least one of five specific content messages was associated (p < 0.05) with positive parental reactions; omitting at least one of four specific content messages was associated (p < 0.05) with negative parental reactions. Parents reported positive reactions when PCPs avoided jargon or were perceived as calm. Parents reported negative reactions to jargon usage and results disclosure by voicemail.

Conclusion

Parents identified aspects of PCP communication which influenced their reactions and results disclosure experiences.

Practice implications

Our findings suggest ways PCPs may improve communication of carrier results. PCPs should provide specific content messages and consider how their actions, characteristics, and setting can influence parental reactions.     

Double mutation (A171T) and (D444H) is a common cause of profound biotinidase deficiency in children ascertained by newborn screening in the United States

Biotinidase deficiency is inherited as an autosomal recessive trait that, unless treated with pharmacologic doses of biotin, can result in neurologic and cutaneous symptoms. We have identified two new mutations in exon D of the biotinidase gene of children with profound biotinidase deficiency ascertained by newborn screening. Transition 511G→A near the 5′ end of exon D results in a substitution of threonine for alanine171 (A171T) and transversion 1330G→C occurs close to the 3′ end of exon D causing a substitution of histidine for aspartic acid 444 (D444H). The D444H mutation was detected in four individuals from our normal population whose mean serum biotinidase activity is 5.25 nmol/min/ml, which is significantly lower than the mean normal activity (7.1 nmol/min/ml). We calculated that this mutation causes a 52% loss of activity in the aberrant enzyme. Twenty-three individuals with the D444H mutation were found by allele specific oligonucleotide analysis of DNA from 296 randomly-selected, anonymous dried-blood spots. We estimate the frequency of this allele in the general population to be 0.039. In contrast, no individuals in 376 have the A171T mutation. Fourteen children (eleven probands and three siblings) out of 31 enzyme-deficient children have both the A171T and D444H mutations. Both mutations are inherited from a single parent as a double mutation allele. The nine families in which this allele was identified are of mostly European ancestry, although the mutation cannot be attributed to a specific nationality or ethnic group. The serum of a child who is homozygous for the double mutation allele has very little CRM and the aberrant enzyme has very low biotinyl-hydrolase activity and no biotinyl-transferase activity. This double mutation allele (A171T and D444H) is a common cause of profound biotinidase deficiency in children ascertained by newborn screening in the United States. Hum Mutat 11:410, 1998. © 1998 Wiley-Liss, Inc.