Molecular Diagnostic And Clinical Phenotypic Analysis Of Citrin Deficiency

Objective:Citrin deficiency(CD)is an autosomal recessive disorder caused by biallelic SLC25A13 variants.Neonatal intrahepatic cholestasis caused by citrin deficiency(NICCD)is the most common clinical phenotype of CD at pediatric age.Due to the lack of well-recognized clinical or biochemical diagnostic criteria,NICCD diagnosis relies on SLC25A13 genetic analysis currently.The aim of this study was to identify novel SLC25A13 variants as laboratory evidences for the definite diagnosis of NICCD patients and for the genetic counseling in the affected families,and to summarize the features of SLC25A13 variants in Chinese,especially in Guangdong population,so as to provide precision medical support to the in-depth scientific understanding of the SLC25A13 variant spectrum.Methods:The subjects of the study were 103 pediatric patients,as well as their parents,who were highly suspected to have CD based on their clinical and laboratory characteristics in the Department of Pediatrics,The First Affiliated Hospital of Jinan University from the beginning of May 2017 to the end of December 2018.In this study,four prevalent SLC25A13 variants were firstly screened for by using the genomic DNA extracted from peripheral blood of the patients and their parents.Then,all 18 SLC25A13 exons and their flanking sequences were analyzed by Sanger sequencing to explore another possible pathogenic mutation in cases of only one mutation detected.Following that,when another pathogenic mutation was not found,5 large insertion/deletion variants were screened for by LA-PCR approaches.For novel missense mutations,their pathogenicity was predicted by a series of bioinformatic tools such as comparative alignment of homologous peptides,in silico prediction softwares and protein structural modeling,and minigene splicing analysis was performed to analyze the pathogenicity of novel splice-site variants.Moreover,the SLC25A13 variants detected in all NICCD patients diagnosed by our team were summarized,and their distribution was compared in Guangdong and other regions in mainland China.Result:In this study,90 new CD patients were definitely diagnosed while 8 novel mutations identified,including c.188del G,c.1620del T,c.1722del A,c.742G>A,c.762T>A,c.889G>T,c.1193T>A,and IV14-1G>A.Minigene splicing analysis confirmed that the variant IV14-1G>A led to the formation of an aberrantly-spliced variant r.1453del G,and the missense variants c.762T>A and c.742G>A were predicted to be pathogenic bioinformatically.By the end of December 2018,a total of422 CD patients have been definitely diagnosed while 64 SLC25A13 variants detected by our group.The frequency of the c.852＿855del4 mutation in the CD patients in Guangdong was as high as 71.8%,while in other provinces,only 49.3%(c~2=41.285,P<0.01).The frequencies of IVS16ins3kb and other variants were 5.4%and 5.7%,respectively,in Guangdong patients,while 12.7%and 19.2%,respectively,in patients from other regions(c~2=10.961 and 27.194,respectively,both P<0.01).Conclusions:This study diagnosed 90 new CD patients and identified 8 novel SLC25A13 variants,making the total number of CD patients diagnosed by our group reach 422,and formed a large CD patient cohort.Meanwhile,the frequency of the mutation 852＿855del4 in Guangdong was found to be significantly higher than that outside the province,while the frequency of IVS16ins3kb and other variants significantly lower than that in other regions.The above findings further enriched the SLC25A13 mutation spectrum in Chinese CD patients,providing reliable evidences for the definite diagnosis of CD patients and genetic counseling for the affected families,constituting precise medical support for deepening the scientific understanding of the SLC25A13 variant spectrum in Chinese.Objective: Neonatal intrahepatic cholestasis caused by citrin deficiency(NICCD)is the most common phenotype of Citrin deficiency(CD).This study aimed to explore the clinical features,laboratory alterations,imaging changes as well as clinical outcomes of NICCD patients,so as to provide experiences for the clinical identification and management of this condition.Methods: The clinical data,laboratory findings,imaging information and clincial outcomes of 385 NICCD patients among the 418 CD patients who were definitely diagnosed by SLC25A13 gene analysis from July 2005 to December 2018 were collected and retrospectively analyzed.The differences between the severe and non-severe CD groups were compared in terms of the birth weight,height,gestational age as well as the ages of CD onset,definite diagnosis and therapy start.Logistic multifactor retrospective analysis was further performed to explore the possible risk factors relevant to severe CD formation.Result: Among the 418 patients diagnosed by SLC25A13 gene analysis,there were 7individuals without any clinical symptoms and signs,indicating a CD penetrance of98.3%.Among the 385 NICCD Subjects,32.3%(116/359)had intrauterine growth retardation.The main clinical manifestations were jaundice(94.3%,363/385),hepatomegaly(72.0%,265/368),chubby face(37.7%,145/385),diarrhea(22.1%,85/385),anemia(18.2%,70/385),failure to thrive(13.0%,50/385),positive stool fat(62.2%,53/90),coagulopathy(75.4%,175/232),cholestasis(34.1%,98/287),hypoalbuminemia(29.2%,84/288),dyslipidemia(61.4%,173/282),vitamin D deficiency(44.4%,52/117),zinc deficiency(38.9%,112/288)and iron deficiency(10.1%,29/288).Cytomegalovirus infection(51.9%,98/189)was not uncommon in the NICCD patients.Metabolome analysis detected elevation of a variety of amino acids such as citrulline,methionine,threonine and tyrosine as well as long-chain acylcarnitines,and the urinary excretion of 4-hydroxyphenyllactate and 4-hydroxyphenylpyruvate was also increased.On imaging examination,hepatomegaly and fatty liver were common,and SPECT exhibited poor uptake and excretion of the tracer isotope.Pathological features were cholestasis,fat deposition,inflammatory cell infiltration,and fibrosis of varying degrees.Up to 2018 December,209 subjects among the 385 patients had beyond 1 year of age,and 59 had dyslipidemia,33 failure to thrive,20 typical FTTDCD,12 liver cirrhosis,6 died,and 1 hepatoblastoma,without CTLN2 cases.In severe cases,the ages for NICCD suspicion,definite diagnosis and dietary therapy were all older,while the duration from onset to NICCD suspicion,definite diagnosis and dietary therapy all longer,than those in non-severe cases.Low birth weight and late dietary therapy were independent risk factors for the development of severe cases.Conclusion:1.CD had a non-penetrance of 1.7%.2.NICCD had colorful clinical symptoms and signs as well as complicated biochemical,metabolomic,imaging and pathological changes.However,these features were all non-pathognomonic.3.NICCD patients usually demonstrated benign clinical outcomes.However,in this study,5.2%(20/385)of the NICCD patients developed into FTTDCD,3.1%(12/385)had liver cirrhosis,and 1.6%(6/385)passed away.4.Lower birth weight,and later dietary therapy led to increased risk of severe cases in NICCD patients.5.These long-term and large-cohort clinical findings deepened the understanding of the NICCD phenotype,having a certain reference value for the diagnosis and therapy of NICCD.Objective: Failure to Thrive and Dyslipidemia caused by Citrin Deficiency(FTTDCD)is a newt phenotype of Citrin deficiency(CD)proposed recently,but its clinical features and risk factors remained far from being completely understood.This study explored the clinical laboratory characteristics as well as the possible risk factors of FTTDCD and analyzed the possible related factors,so as to provide theoretical evidences for the prevention and management of this condition.Subject and Methods: The FTTDCD group in this study was composed of 30 patients definitely diagnosed via SLC25A13 analysis by our team from July 2005 to January 2019,and the control group encompassed 31 patients who had been definitely diagnosed to have NICCD during infancy,and then followed up regularly in our clinic,but neither failure to thrive nor dyslipidemia was observed on at least three follow-up examination.The differences between the two groups were compared in terms of the SLC25A13 variants,symptoms,signs,outcomes as well as the blood routine,biochemistry and metabolome indices.Logistic multifactor retrospective analysis was further performed to evaluate the possible risk factors relevant to FTTDCD formation.Results: The FTTDCD patients in this paper were definitely diagnosed at the mean age of 27.78 months,and the main clinical manifestations were stunting,underweight,hepatomegaly,splenomegaly,liver cirrhosis,hypercholesterolemia,hypertriglyceridemia,and reduced HDL-cholesterol while elevated LDL-cholesterol.Most FTTDCD subjects(20/30,66.7%)had been diagnosed to have NICCD during infancy,but NICCD history was negative in some cases(10/30,33.3%).In comparison with controls,FTTDCD patients had lower birth weight(2658.97±416.21 vs 2982.26±349.18,P<0.01)and length(48.05±1.99 vs 49.47±1.59,P=0.01)and the ages were older when CD was suspected,definitely diagnosed and dietary therapy started.Low birth weight(OR=0.998,95% CI: 0.996-0.999)and older age of definite diagnosis(OR=1.135,95% CI:1.010-1.275)were found to be independent risk factors for FTTDCD formation.At initial referral to our hospital,FTTDCD patients demonstrated higher positive rates of failure to thrive(53.3% vs 0,P<0.01),dislipidemia(60.0% vs 22.6%,P<0.01)and splenomegaly(36.7% vs 6.5%,P<0.01);The RBC counts(4.23±0.68 vs 4.71±0.36,P<0.01),Hb levels(112.44±16.25 vs123.20±9.75,P<0.01)and hematocrits(34.24±4.07 vs 37.50±2.87,P<0.01)were lower while the values of SD[40.60(37.80,49.75)vs 37.70(36.57,39.75),P=0.01] and CV[13.95(13.15,16.86)vs 13.20(12.70,14.60),P=0.03] of erythrocyte hemoglobin distribution width were higher;The plasma levels of ALT[29.15(19.75,77.25)vs 23.00(17.00,32.00),P=0.03],AST[45.50(36.00,153.25)vs 36.00(32.00,45.00),P=0.01],γ-GT[33.00(14.75,92.00)vs 16.00(13.00,23.00),P=0.01],Dbil[2.60(1.18,20.80)vs1.80(1.20,2.40),P=0.04],globulin(23.24±5.15 vs 19.55±3.17,P<0.01)and triglycerides(1.84±0.87 vs 1.19±0.58,P<0.01)were higher,while those of choline esterase(7461.94±3982.48 vs 9297.61±2075.91,P=0.03),glycosylated serum protein(128.06±26.89 vs 149.73±27.22,P=0.01),lipo A(1.34±0.51 vs 1.63±0.25,P=0.01),albumin(43.23±6.95 vs 46.79±3.20,P=0.02)and the albumin/globulin(2.16±0.55 vs2.66±0.59,P<0.01)were lower.In response to active dietary therapy,their conditions got improved or tended stable in 93.3%(28/30),but were exacerbated to death in a minority(6.7%,2/30),of the FTTDCD patients.Conclusions: In this paper,most FTTDCD cases were developed from NICCD,and the clinical features were failure to thrive,hepatosplenomegaly and dyslipidemia.At initial referral to our hospital,FTTDCD patients exhibited more serious clinical presentations,along with more marked anemia and uneven RBC size,whilst more serevely affected liver synthesis and cholestasis,as well as disturbed lipid metabolism.The ages when CD was suspected,definitely diagnosed and dietary therapy started were all older in FTTDCD cases than in controls.Lower birth weight and older age of CD definite diagnosis were two independent risk factors for FTTDCD development.Although a minority of the affected patients exhibited lethal outcomes,FTTDCD could be stabilized or improved in most cases.These findings constituted important theoretical evidences for the prevention and management of FTTDCD patients.