a pilot expanded newborn screening programme to detect inherited metabolic disorders by means of liquid chromatography coupled with tandem mass spectrometry ( lc - ms / ms ) began in the campania region , southern italy , in 2007 . by october 2009 , > 8,800 dried blood samples on filter paper from 11 hospitals in the region had been screened in our laboratory . within this programme , we detected mitochondrial acetoacetyl - coenzyme a ( coa ) thiolase deficiency ( -kt deficiency ) in a newborn asymptomatic girl and in her older sister who had a metabolic decompensation event a few months earlier ( fig .  1 ) . in the proband , -kt deficiency was diagnosed by analysing the patient s acylcarnitine profile on a dried blood spot and in urine by using liquid chromatography coupled with tandem mass spectrometry ( lc - ms / ms ) and gas chromatography coupled with mass spectrometry ( gc / ms ) using previously described procedures ( chace 2009 ; millington et al . 1dual function of acetoacetyl - coenzyme a ( coa ) thiolase [ acat 1 ( -kt ) ] in isoleucine and ketone body metabolism . metabolite biomarkers of -kt deficiency are boxed 
  dual function of acetoacetyl - coenzyme a ( coa ) thiolase [ acat 1 ( -kt ) ] in isoleucine and ketone body metabolism . the proband , a girl , was born after 41  weeks of an uncomplicated pregnancy , the second child of healthy parents who were third cousins . birth weight was 3,390  g ( 50th centile ) , length 53  cm ( 50th75th centile ) and head circumference 36  cm ( 50th75th centile ) . the apgar score was 9 at 1  min and 10 at 5  min . the mother was 25  years old and the father 27 . the amino acid profile determined in a dried blood spot when patient 1 was 4  days old was normal , whereas acylcarnitine analysis revealed increased concentrations of tiglylcarnitine ( c5:1  =  0.34  mol / l ; upper cut - off = 0.23  mol / l ) , 3-hydroxybutyrylcarnitine ( c4oh  =  2.19  mol / l ; upper cut - off = 0.55  mol / l ) and 2-methyl-3-hydroxybutyrylcarnitine ( c5oh  =  1.17  mol / l ; upper cut - off = 0.28  mol / l ) . analysis of acylcarnitines in urine samples confirmed elevated concentrations of c5:1 ( 2.68  mmol / mol creatinine ; upper cut - off = 0.20  mmol / mol creatinine ) and c5oh ( 1.08  mmol / mol creatinine ; upper cut - off = 0.54  mmol / mol creatinine ) . c5:1 and c5oh concentrations are elevated in both -kt deficiency and 2-methyl-3-hydroxybutyryl - coa dehydrogenase deficiency ( mhbd deficiency ) ( matern et al 2003 ) , whereas increased concentrations of c4oh have been identified only in cases of -kt deficiency ( han et al 2007 and http://region4genetics.org ) . therefore , elevated c4oh concentrations were highly suggestive of -kt deficiency in patient 1 . to verify the diagnosis , we analysed urine organic acids . results showed that patient 1 had abnormal excretion of 2-methyl-3-hydroxybutyric acid ( 441  mmol / mol creatinine ; upper cut - off = 2  mmol / mol creatinine ) and tiglylglycine ( 87  mmol / mol creatinine ; upper cut - off = 2  mmol / mol creatinine ) . detection of 2-methylacetoacetic acid in the urine organic acid profile is considered a key factor for a diagnosis of -kt deficiency ; 2-methylacetoacetic acid is unstable because it undergoes spontaneous decarboxylation to 2-butanone , which is highly volatile and is therefore not easily determined ( korman 2006 ) . its absence in the urine of patient 1 can probably be attributed to her good health because of the earlier diagnosis consequent to neonatal screening . birth weight of the older sister of patient 1 was 3,240  g ( 25th centile ) . she was born after an uncomplicated pregnancy . at 18  months of age , after gastroenteritis and a vomiting event , she entered an emergency unit because of lethargy and metabolic acidosis ( ph 7.07 , hco3 5.2 ) . -kt deficiency was diagnosed when the patient was 23  months old and in an asymptomatic condition soon after the metabolic defect was discovered in her sister . serum and urine acylcarnitine analysis in patient 2 revealed elevated concentrations of both c5:1 ( serum : 0.96  mol / l with an upper cut - off = 0.12  mol / l ; urine : 12.8  mmol / mol creatinine with an upper cut - off = 0.2  mmol / mol creatinine ) and c5oh ( serum : 0.70  mol / l with an upper cut - off = 0.13  mol / l ; urine : 2.06  mmol / mol creatinine with an upper cut - off = 0.54  mmol / mol creatinine ) . urine organic acid analysis revealed massive excretion of 2-methyl-3-hydroxybutyric acid ( 1,354  mmol / mol creatinine ; upper cut - off = 2  mmol / mol creatinine ) and tiglylglycine ( 825  mmol / mol creatinine ; upper cut - off = 2  mmol / mol creatinine ) and small amounts of 2-methylacetoacetic acid ( 25  mmol / mol creatinine ) . thus , taken together , the data suggested -kt deficiency in this patient and indirectly confirmed the same diagnosis in the younger sister . to reach a more robust diagnosis , we carried out a genomic analysis in both sisters and their parents . we sequenced the amplified fragments corresponding to exons 112 of the acat1 gene and identified the c.471c > a p.s547s ( rs35188041 ) polymorphism in exon 6 and the c.1189c > g p.h397d mutation in exon 12 , both at the homozygous state in the two patients . the parents were found to be heterozygous for both the polymorphism and the mutation , thus confirming the homozygosity of the two daughters . the h397d mutation provokes a null allele in the sense that it causes the total loss of acetoacetyl - coa thiolase activity for which the potassium ion is critical ( fukao et al 2003 , 2008 ; haapalainen et al 2007 ) . such a severe three - dimensional perturbation destabilises the overall integrity of the mutant enzyme ( zhang et al 2004 ) . the proband , a girl , was born after 41  weeks of an uncomplicated pregnancy , the second child of healthy parents who were third cousins . birth weight was 3,390  g ( 50th centile ) , length 53  cm ( 50th75th centile ) and head circumference 36  cm ( 50th75th centile ) . the apgar score was 9 at 1  min and 10 at 5  min . the mother was 25  years old and the father 27 . the amino acid profile determined in a dried blood spot when patient 1 was 4  days old was normal , whereas acylcarnitine analysis revealed increased concentrations of tiglylcarnitine ( c5:1  =  0.34  mol / l ; upper cut - off = 0.23  mol / l ) , 3-hydroxybutyrylcarnitine ( c4oh  =  2.19  mol / l ; upper cut - off = 0.55  mol / l ) and 2-methyl-3-hydroxybutyrylcarnitine ( c5oh  =  1.17  mol / l ; upper cut - off = 0.28  mol / l ) . analysis of acylcarnitines in urine samples confirmed elevated concentrations of c5:1 ( 2.68  mmol / mol creatinine ; upper cut - off = 0.20  mmol / mol creatinine ) and c5oh ( 1.08  mmol / mol creatinine ; upper cut - off = 0.54  mmol / mol creatinine ) . c5:1 and c5oh concentrations are elevated in both -kt deficiency and 2-methyl-3-hydroxybutyryl - coa dehydrogenase deficiency ( mhbd deficiency ) ( matern et al 2003 ) , whereas increased concentrations of c4oh have been identified only in cases of -kt deficiency ( han et al 2007 and http://region4genetics.org ) . therefore , elevated c4oh concentrations were highly suggestive of -kt deficiency in patient 1 . to verify the diagnosis , we analysed urine organic acids . results showed that patient 1 had abnormal excretion of 2-methyl-3-hydroxybutyric acid ( 441  mmol / mol creatinine ; upper cut - off = 2  mmol / mol creatinine ) and tiglylglycine ( 87  mmol / mol creatinine ; upper cut - off = 2  mmol / mol creatinine ) . detection of 2-methylacetoacetic acid in the urine organic acid profile is considered a key factor for a diagnosis of -kt deficiency ; 2-methylacetoacetic acid is unstable because it undergoes spontaneous decarboxylation to 2-butanone , which is highly volatile and is therefore not easily determined ( korman 2006 ) . its absence in the urine of patient 1 can probably be attributed to her good health because of the earlier diagnosis consequent to neonatal screening . birth weight of the older sister of patient 1 was 3,240  g ( 25th centile ) . she was born after an uncomplicated pregnancy . at 18  months of age , after gastroenteritis and a vomiting event , she entered an emergency unit because of lethargy and metabolic acidosis ( ph 7.07 , hco3 5.2 ) . -kt deficiency was diagnosed when the patient was 23  months old and in an asymptomatic condition soon after the metabolic defect was discovered in her sister . serum and urine acylcarnitine analysis in patient 2 revealed elevated concentrations of both c5:1 ( serum : 0.96  mol / l with an upper cut - off = 0.12  mol / l ; urine : 12.8  mmol / mol creatinine with an upper cut - off = 0.2  mmol / mol creatinine ) and c5oh ( serum : 0.70  mol / l with an upper cut - off = 0.13  mol / l ; urine : 2.06  mmol / mol creatinine with an upper cut - off = 0.54  mmol / mol creatinine ) . urine organic acid analysis revealed massive excretion of 2-methyl-3-hydroxybutyric acid ( 1,354  mmol / mol creatinine ; upper cut - off = 2  mmol / mol creatinine ) and tiglylglycine ( 825  mmol / mol creatinine ; upper cut - off = 2  mmol / mol creatinine ) and small amounts of 2-methylacetoacetic acid ( 25  mmol / mol creatinine ) . thus , taken together , the data suggested -kt deficiency in this patient and indirectly confirmed the same diagnosis in the younger sister . to reach a more robust diagnosis , we carried out a genomic analysis in both sisters and their parents . we sequenced the amplified fragments corresponding to exons 112 of the acat1 gene and identified the c.471c > a p.s547s ( rs35188041 ) polymorphism in exon 6 and the c.1189c > g p.h397d mutation in exon 12 , both at the homozygous state in the two patients . the parents were found to be heterozygous for both the polymorphism and the mutation , thus confirming the homozygosity of the two daughters . the h397d mutation provokes a null allele in the sense that it causes the total loss of acetoacetyl - coa thiolase activity for which the potassium ion is critical ( fukao et al 2003 , 2008 ; haapalainen et al 2007 ) . such a severe three - dimensional perturbation destabilises the overall integrity of the mutant enzyme ( zhang et al 2004 ) . here we report the clinical and biological investigations carried out in two sisters in whom we identified -kt deficiency . using lc - ms / ms within a pilot extended metabolic screening programme , we diagnosed -kt deficiency by analysing the blood spot of patient 1 . subsequently , we identified the same metabolic defect in the patient s sister by analysing a serum sample . in the next level of diagnosis this analysis revealed abnormal excretion of 2-methyl-3-hydroxybutyric acid and tiglylglycine in both patients and a small amount of 2-methylacetoacetic acid in the older sister . familial genomic analysis confirmed that both sisters are affected by -kt deficiency and showed that they are homozygotes for the c.1189c > g ( p.h397d ) mutation in exon 12 of the acat1 gene ; the parents are heterozygotes for the same mutation . the mutation has been classified as a severe defect because it abolishes protein expression , as demonstrated in an in vitro assay ( zhang et al 2004 ) .