the assay employs pcr quantification of circular dna byproducts of t - cell receptor gene rearrangement , t - cell receptor excision circles ( trecs ) . although trecs have no identifiable function , their presence serves as a marker for the maturation of t - cells . insufficiency of trecs characterizes scid and other t - lymphopenic disorders including complete digeorge syndrome   and some cases of ataxia telangiectasia ( at ) . like at , nijmegen breakage syndrome ( nbs ) [ 69 ] ( formerly known as an at variant 1 ) is a disorder of dna repair associated with chromosomal instability and immune compromise that may be identified through trec screening . herein , we describe a baby with a complex perinatal history and low trecs near birth . whole exome sequencing identified heterozygous mutations in nbn , the gene that encodes nibrin , a component of a molecular complex involved in the early recognition and subsequent repair of dna damage . nibrin was absent from nuclear lysates on immunoblots , and the patient s cells were hypersensitive to ionizing radiation by colony survival assay [ 11 , 12 ] . a 41-week gestational age male ( nbs20la ) , weighing 2860 grams , was born via normal vaginal delivery to a 23-year - old , gravida 1 , paragravida 0 mother following prenatal identification of increased cerebrospinal fluid on ultrasound . postnatally , the infant was hospitalized for 1  week for meconium aspiration . by 4  weeks of age progressive hydrocephalus required a ventriculo - peritoneal shunt . at 17  weeks , he was hospitalized for a presumed urinary tract infection ; renal ultrasound showed left hydronephrosis . during the first year of life he also exhibited developmental delay and failure to thrive with weight and length for age persistently below the 3rd percentiles and head circumference at 8  months of age below the 2nd percentile . at 3  weeks of age , scid newborn screening results were interpreted as  incomplete  : trec , 13 copies / mcl ( normal > 25 ) and actin control , 9,510 copies / mcl ( normal > 10,000 ) . repeat screening was  positive  : trec , 12 copies / mcl and actin , 19,800 copies / mcl . subsequent flow cytometry at 6  weeks of life demonstrated persistent t - lymphopenia and elevated nk cell percentages and numbers ( table  1).table 1laboratory values of patient nbs20la from 1.5  months to 1  year of agepatient age1.5  month3  months5.5  months10  monthsreference rangesbirth to 2  months2  months to 4  months4  months to 1  yeartotal wbc cells / ul6800512055007880500017,000500017,000500017,000%neutrophils47445856254525452545%lymphocytes33272322466646664666%monocytes819121428  % 28  % 28  % % eosinophils121078030303cd 3 + t cells / ml ( % ) 924 ( 42)259 ( 24)289 ( 18)381 ( 22)13757129 ( 6993)25336778 ( 5579)28897995 ( 5980)cd 4 + t cells / ml ( % ) 660 ( 30)65 ( 5)224 ( 15.1)320 ( 19)11695623 ( 4875)13925210 ( 3562)17865141 ( 3861)cd 8 + t cells / ml ( % ) 242 ( 11)99 ( 8)63 ( 4)72 ( 4)2671860 ( 1333)6522449 ( 1430)9462791 ( 1434)cd19 + b - cells / ml ( % ) 88 ( 4)26 ( 2)72 ( 5)174 ( 10)1041448 ( 426)7453499 ( 1439)8583774 ( 1636)cd3-cd16/56 + nk cells /mm3 ( % ) 638 ( 29)680 ( 66)1123 ( 73)1169 ( 68)60434 ( 214)194994 ( 314)1821581 ( 313)igg ( mg / dl ) [ reference range]220 [ 198577]202 [ 165781]316 [ 2821026]igm ( mg / dl ) [ reference range]24 [ 16100]34 [ 31103]70 [ 39142]iga ( mg / dl ) [ reference range]<7 [ 152]7 [ 883]16 [ 1683]ab responses : tetanus ( iu / ml ) ( rr : 0.17)2.192.71h . influenzae b ( mcg / ml ) ( rr : 1.010.0)0.151.16lpa responses : phytohemaglutinin ( si ) [ 1:125]1734442pokeweed mitogen ( si)237523tetanus antigen ( si)21candida antigen ( si)46 laboratory values of patient nbs20la from 1.5  months to 1  year of age targeted sequencing of scid associated genes ada , ak2 , cd3d , cd3zeta , dclre1c , ilrg , il7r , jak3 , lig4 , nhej1 , pnp , ptprc , rac2 , rag1 , rag2 , rmrp , and zap70 ( genedx , gaithersburg , md ) and enzyme assays for adenosine deaminase and nucleoside phosphorylase revealed no abnormalities . informed consent was obtained for research , including cellular immune studies and whole exome sequencing ( wes ) , for the infant and both parents under approved protocols at children s hospital los angeles ( chla ) and the university of california san francisco ( ucsf ) . genomic dna from edta - anticoagulated whole blood was prepared using a gentra puregene blood kit ( qiagen usa : germantown , md ) . briefly , libraries prepared by ligating truseq adaptors ( illumina : san diego , ca ) to genomic dna fragments of 200300  bp were enriched with 10  cycles of pcr , pooled and submitted to exon capture using a roche nimblegen version 3.0 capture array . after 10 additional amplification cycles , 100  bp paired end sequence reads were generated ( hiseq2000 , llumina ) , yielding 3.8  % duplicates and a mean of > 100 reads covering the targeted regions , with > 95  % of target regions having 10 reads . reads were aligned against grch37 ( aug 2009 release ) using bwa ( v0.6.2 ) . the results were converted to bam , sorted by coordinate , indexed , and marked for pcr duplicate reads using the picard toolkit ( v 1.81 ) ( http://picard.sourceforge.net ) . local realignment was performed around known indel locations , and base quality scores were re - calibrated using gatk ( v 2.6.5 ) [ 14 , 15 ] . gatk variant quality scores were re - calibrated by vqsr ( variant quality score recalibration ) using the trio of exomes in this report , as well as 65 others sequenced at our site . hapmap v3.3 , 1000 genomes high confidence snps ( phase1 v3 2010 - 11 - 23 ) and omni chip array sets were used as training data , and to provided truth sites for snps , while the mills dataset   from the 1000 genomes was used for indels , using a truth sensitivity cutoff of 99  % . variant annotation ( including region , effect , allele frequency , disease phenotype annotation , and conservation ) was performed using our custom tool varant ( http://compbio.berkeley.edu/proj/varant/ ) . particular attention was given to high - confidence , rare , likely - damaging , protein - altering variants in genes associated with primary cellular immunodeficiency   and to those conforming to models of autosomal or x - linked recessive inheritance in the family , or de novo mutations that may be dominant . peripheral blood lymphoblastoid cell lines ( lcl ) from the patient nbs20la and controls were made with epstein - barr virus as described , and 50  g of nuclear protein lysate from each was electrophoresed on a 6  % sds - polyacrylamide gel ( page ) , blotted onto pvdf membrane ( biorad , hercules , ca ) and incubated with antibodies to nibrin ( novus , nb100 - 143 at 1:5000 , littleton , co ) overnight at 4  c . the immunoblots were subsequently incubated with an hrp - conjugated anti - rabbit secondary antibody at room temperature for 40  min for detection by enhanced chemiluminescence ( ecl ) ( amersham pharmacia , piscataway , nj ) . under the conditions of the assay p84 was used as a loading control ( genetex gtx70220 at 1:3000 ) . on the same blot , mre11 and rad50 were also measured ( novus 100142 at 1:15,000 and novus 100154 at 1:500 , respectively ) . peripheral blood lymphocytes from the patient nbs20la and controls were immortalized with epstein - barr virus as previously described , and 50  g of nuclear lysate protein from the each lymphoblastoid cell line ( lcl ) was electrophoresed on a 6  % sds - polyacrylamide gel ( page ) , blotted onto pvdf membrane ( biorad , hercules , ca ) and incubated with antibodies to nibrin and dna repair proteins mre11 , rad50 and atm ( novus , littleton , co ) overnight at 4  c . the immunoblots were subsequently incubated with an hrp - conjugated anti - rabbit secondary antibody at room temperature for 40  min for detection by enhanced chemiluminescence ( ecl ) ( amersham pharmacia , piscataway , nj ) . under the conditions of the assay , as little as 1  g of nibrin protein can be detected . lcls from wild type ( healthy controls ) , individuals with at ( radiosensitive controls ) , and nbs20la were maintained in rmpi 1640 medium with 10  % fetal bovine serum and 1  % penicillin - streptomycin - l - glutamine . after plating into paired 96-well trays , one of each pair was irradiated with 1 gray . after incubation at 37  c for 2  weeks , the trays were stained with 0.1  % 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide . individual wells with at least one colony composed of > 32 cells ( i.e. , > 5 divisions ) were scored as positive . the number of positive wells in irradiated- versus non - irradiated plates was compared to determine the survival fraction . results were compared to measurements performed by sun et al . using 104 at patients and 29 healthy controls . informed consent was obtained for research , including cellular immune studies and whole exome sequencing ( wes ) , for the infant and both parents under approved protocols at children s hospital los angeles ( chla ) and the university of california san francisco ( ucsf ) . genomic dna from edta - anticoagulated whole blood was prepared using a gentra puregene blood kit ( qiagen usa : germantown , md ) . briefly , libraries prepared by ligating truseq adaptors ( illumina : san diego , ca ) to genomic dna fragments of 200300  bp were enriched with 10  cycles of pcr , pooled and submitted to exon capture using a roche nimblegen version 3.0 capture array . after 10 additional amplification cycles , 100  bp paired end sequence reads were generated ( hiseq2000 , llumina ) , yielding 3.8  % duplicates and a mean of > 100 reads covering the targeted regions , with > 95  % of target regions having 10 reads . reads were aligned against grch37 ( aug 2009 release ) using bwa ( v0.6.2 ) . the results were converted to bam , sorted by coordinate , indexed , and marked for pcr duplicate reads using the picard toolkit ( v 1.81 ) ( http://picard.sourceforge.net ) . local realignment was performed around known indel locations , and base quality scores were re - calibrated using gatk ( v 2.6.5 ) [ 14 , 15 ] . variants were called using gatk unifiedgenotyper and freebayes ( vesion 0.9.10 ) . gatk variant quality scores were re - calibrated by vqsr ( variant quality score recalibration ) using the trio of exomes in this report , as well as 65 others sequenced at our site . hapmap v3.3 , 1000 genomes high confidence snps ( phase1 v3 2010 - 11 - 23 ) and omni chip array sets were used as training data , and to provided truth sites for snps , while the mills dataset   from the 1000 genomes was used for indels , using a truth sensitivity cutoff of 99  % . variant annotation ( including region , effect , allele frequency , disease phenotype annotation , and conservation ) was performed using our custom tool varant ( http://compbio.berkeley.edu/proj/varant/ ) . particular attention was given to high - confidence , rare , likely - damaging , protein - altering variants in genes associated with primary cellular immunodeficiency   and to those conforming to models of autosomal or x - linked recessive inheritance in the family , or de novo mutations that may be dominant . peripheral blood lymphoblastoid cell lines ( lcl ) from the patient nbs20la and controls were made with epstein - barr virus as described , and 50  g of nuclear protein lysate from each was electrophoresed on a 6  % sds - polyacrylamide gel ( page ) , blotted onto pvdf membrane ( biorad , hercules , ca ) and incubated with antibodies to nibrin ( novus , nb100 - 143 at 1:5000 , littleton , co ) overnight at 4  c . the immunoblots were subsequently incubated with an hrp - conjugated anti - rabbit secondary antibody at room temperature for 40  min for detection by enhanced chemiluminescence ( ecl ) ( amersham pharmacia , piscataway , nj ) . under the conditions of the assay , as little as 1  g of nibrin protein could be detected . on the same blot , mre11 and rad50 were also measured ( novus 100142 at 1:15,000 and novus 100154 at 1:500 , respectively ) . peripheral blood lymphocytes from the patient nbs20la and controls were immortalized with epstein - barr virus as previously described , and 50  g of nuclear lysate protein from the each lymphoblastoid cell line ( lcl ) was electrophoresed on a 6  % sds - polyacrylamide gel ( page ) , blotted onto pvdf membrane ( biorad , hercules , ca ) and incubated with antibodies to nibrin and dna repair proteins mre11 , rad50 and atm ( novus , littleton , co ) overnight at 4  c . the immunoblots were subsequently incubated with an hrp - conjugated anti - rabbit secondary antibody at room temperature for 40  min for detection by enhanced chemiluminescence ( ecl ) ( amersham pharmacia , piscataway , nj ) . under the conditions of the assay , as little as 1  g of nibrin protein can be detected . lcls from wild type ( healthy controls ) , individuals with at ( radiosensitive controls ) , and nbs20la were maintained in rmpi 1640 medium with 10  % fetal bovine serum and 1  % penicillin - streptomycin - l - glutamine . after plating into paired 96-well trays , one of each pair was irradiated with 1 gray . after incubation at 37  c for 2  weeks , the trays were stained with 0.1  % 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide . individual wells with at least one colony composed of > 32 cells ( i.e. , > 5 divisions ) were scored as positive . the number of positive wells in irradiated- versus non - irradiated plates was compared to determine the survival fraction . results were compared to measurements performed by sun et al . using 104 at patients and 29 healthy controls . wes analysis by our varant tool identified two nonsense mutations in nbn on chromosome 8q21.3 in the patient , suggesting a diagnosis of nbs . the patient and his father shared a thymine to guanine transversion cdna 842  t  >  g ( nm_002485.4(nbn):c.842  t  > g ) in exon 6 , changing leucine 281 to a stop codon ( protein l281x ) ( clinvar provisional accession : scv000196640 ) , while the infant and mother shared a cytosine to thymine transition cdna 1030c  >  t ( nm_002485.4(nbn):c.1030c  >  t ) in exon 9 , changing glutamine 344 to a stop codon ( q344x ) ( clinvar provisional  accession : scv000196641 ) . a rare x - linked cd40lg gene variant noted in our patient and mother nm_000074 ( cd40lg):c.542 g  >  c ( chrx : 135741330 , g  >  c ; r181p in cd40lg ) was predicted damaging by polyphen-2 software , but was inconsistent with the patient s phenotoype . no other high quality dna variants in the targeted exome regions were as consistent with the phenotype . neither variant had been reported in other patients with nbs nor in the 1000 genomes , espdb   or dbsnp ( v137 )   databases . the patient s paternally inherited allele carried l281x , while his maternally inherited allele carried q344x . confirmatory sanger sequencing was performed by the clia - approved molecular genetics laboratory at the university of california los angeles school of medicine . an immunoblot showed no detectable nibrin protein in nbs20la ( fig .  1 , penultimate lane ) . interestingly , mre11 and rad50 protein levels were also reduced ; this reflects the instability of the mre11-rad50-nibrin ( mrn ) complex whenever nibrin is absent . lane 2 contains lcl protein from an at patient whose cell lines were radiosensitive in fig . atm protein was absent in the atm lcl , but was present in nbs20la ( not shown ) . 1 were from healthy individuals and showed normal amounts of mrn - complex proteins and the loading control , p84 . these findings provided functional confirmation that the patient s nbn variants were both null mutations that abrogated protein expression , and that his cells scored as radiosensitive.fig . 1immunoblot allows comparison of nibrin ( nbs ) , mre11 and rad50 protein levels in an nbs cell line ( nbs20la ) to wild type and at cells . 2colony survival assay for at and nbs b lymphoblastoid cell lines showing that both the at control cells and those of patient nbs20la are in the radiosensitive range , as defined by sun et al . 2002   immunoblot allows comparison of nibrin ( nbs ) , mre11 and rad50 protein levels in an nbs cell line ( nbs20la ) to wild type and at cells . protein loading control was p84 colony survival assay for at and nbs b lymphoblastoid cell lines showing that both the at control cells and those of patient nbs20la are in the radiosensitive range , as defined by sun et al . although the primary goal of newborn screening with trecs has been the pre - symptomatic diagnosis of scid , thereby allowing early intervention , this screening has also identified individuals with non - scid t - lymphopenia . in california , the first 2  years of newborn screening for scid identified 50 infants with t - lymphopenia , defined as < 1500  t cells / mcl : 11 had typical scid , five had idiopathic t - lymphopenia or variant scid ( defined as significant t cell defects without mutation in known scid genes ) , eight had partial and one had complete digeorge syndrome , four had trisomy 21 , and three had at . , exome sequencing was utilized to identify at as the cause of t - lymphopenia in two patients with positive scid screens . in this same study retrospective analysis of older at patients suggested that approximately half of patients with at will have a positive newborn scid screen . nbs is a distinct dna breakage / chromosomal instability disorder caused by defects in nibrin , part of the same dna repair pathway as at mutated protein . the immune defects in at and nbs are similar and include impaired vdj recombination , which affects t - cell receptor and immunoglobulin rearrangement . t - lymphopenia and decreased trecs as seen in patients with at would thus also be expected in patients with nbs . in our patient , the trec newborn screening for scid was positive , but an extensive search for scid gene mutations was unrevealing . in contrast , exome sequencing revealed novel , compound heterozygous nbn mutations that were associated with absence of nibrin and radiosensitivity . a r181p missense variant in cd40lg , the gene associated with x - linked hyper - igm syndrome , was also predicted to be damaging but was inconsistent with the patient s clinical and laboratory data including absent nibrin and a total serum igg concentration in the normal range and positive igg antibody responses to tetanus and h. influenzae b. although it is not impossible for a patient to have two primary immune disorders , nbn mutations fully explain the low t cells , absent nibrin and cellular radiosensitivity that establish the diagnosis of nbs in this patient , while the features of cd40l deficient hyper - igm syndrome are absent . most patients are full term , but may have microcephaly at birth with subsequent growth failure . most patients meet early developmental milestones but have varying degrees of intellectual disability later in life [ 7 , 2527 ] . perinatal hydrocephalus as seen in our patient has been reported in two children with nbs . hydronephrosis , renal anomalies such as horseshoe or dysplastic kidneys , skeletal anomalies such as clinodactyly and partial syndactyly , caf au lait spots and vitiligo have all been seen , but are not constant features and are also found in other conditions [ 8 , 28 ] . lymphoid malignancy affects up to 40  % of patients with nbs by their 21st year . onset of malignancy can vary in these patients depending on the mutation inherited and degree of nibrin dysfunction . most patients with nbs show some degree of immunodeficiency . in a review of 57 patients from the polish nijmegen breakage syndrome registry , 19  % had normal total serum immunoglobulins , while 25  % had severe hypogammaglobulinemia . at least one igg subclass was deficient in all patients : 89  % igg4 , 73  % igg2 and 54  % igg1 . similarly , our patient had variably low total serum immunoglobulins that trended upward with age ( table  1 ) , but igg3 and igg4 remained low ( data not shown ) . specific antibody response after two h. influenza type b immunizations was negative at 6  months , but positive at 10  months following an additional immunization . our patient s absolute cd19 + b cells were reduced during the first year of life , as seen in 72  % of polish registry patients . in the polish cohort , 84  % of patients received immunoglobulin replacement therapy for frequent or prolonged respiratory tract infections , which have not occurred in our patient up to the present . lymphocyte subset analysis in nbs has shown decreased t cells , with cd4 + t cells affected most severely . this is consistent with our patient s findings ( table  1 ) . the elevated proportion of nk cells seen in our patient was also noted previously in 25 out of 40 patients . the treatment of choice for scid is most often hsct . to date , however , there is limited experience with hsct in patients with nbs . indications for attempting transplantation have included severe immunodeficiency and hematologic malignancy ; pre - emptive hsct has not been reported . of particular concern has been the use of dna - damaging chemotherapeutic conditioning regimens and the potential to induce de novo malignancies in patients with a pre - existing dna repair deficit . there has been some success with reduced intensity pre - hsct conditioning regimens , but studies to date are limited by their retrospective nature , small sample size and relatively short term follow - up . with widespread adoption of trec screening for scid , nbs may be identified prospectively , enabling the population incidence and natural history to be better understood . our case expands our appreciation of the association of nbs with hydrocephalus and hydronephrosis and illustrates the utility of wes as applied to diagnose the underlying cause of idiopathic t lymphopenia found by trec screening . there are more than 200 known genetic defects for primary immunodeficiency [ 19 , 32 ] , and testing of individual genes can be costly and time intensive . wes resulted in prompt detection of two nonsense mutations in nbn and definitive diagnosis in our patient , who presented with atypical features of nbs . establishing the diagnosis allowed avoidance of live virus vaccines and exposure to ionizing radiation , as well as anticipatory monitoring for serious infections and malignancy . trimethoprim / sulfamethoxazole was started to prevent pneumocystis jiroveci pneumonia ; however he has not received immunoglobulin infusions or hsct . exome sequencing of the parents also established the nbn mutations as inherited versus de novo and allowed for accurate and informative counseling regarding the risk of future offspring inheriting both affected genes and having nbs . the parents were informed that other family members could be carriers of these same mutations .