Article Text

Short report
Recessive mutations in MCM4/PRKDC cause a novel syndrome involving a primary immunodeficiency and a disorder of DNA repair
  1. Jillian P Casey1,2,
  2. Michael Nobbs3,
  3. Paul McGettigan4,
  4. SallyAnn Lynch5,
  5. Sean Ennis2,5
  1. 1National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
  2. 2School of Medicine and Medical Science, University College Dublin, Belfield, Dublin, Ireland
  3. 3Bristol Genetics Laboratory, Southmead Hospital, Westbury-on-Trym, Bristol, UK
  4. 4Animal Genomics Laboratory, UCD School of Agriculture, Food Science and Veterinary Medicine, College of Life Sciences, University College Dublin, Belfield, Dublin, Ireland
  5. 5National Centre for Medical Genetics, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
  1. Correspondence to Dr Sean Ennis, School of Medicine and Medical Sciences, Health Sciences Centre, University College Dublin, Belfield, Dublin 4, Ireland; sean.ennis{at}


Background A study is presented of 10 children with a novel syndrome born to consanguineous parents from the Irish Traveller population. The syndrome is characterised by a natural killer (NK) cell deficiency, evidence of an atypical Fanconi's type DNA breakage disorder, and features of familial glucocorticoid deficiency (FGD). The NK cell deficiency probably accounts for the patients' recurrent viral illnesses. Molecular tests support a diagnosis of mosaic Fanconi's anaemia, but the patients do not present with any of the expected clinical features of the disorder. The symptomatic presentation of FGD was delayed in onset and may be a secondary phenotype. As all three phenotypes segregate together, the authors postulated that the NK cell deficiency, DNA repair disorder and FGD were caused by a single recessive genetic event.

Methods Single-nucleotide polymorphism homozygosity mapping and targeted next-generation sequencing of 10 patients and 16 unaffected relatives.

Results A locus for the syndrome was identified at 8p11.21–q11.22. Targeted resequencing of the candidate region revealed a homozygous mutation in MCM4/PRKDC in all 10 affected individuals. Consistent with the observed DNA breakage disorder, MCM4 and PRKDC are both involved in the ATM/ATR (ataxia-telangiectasia-mutated/ATM-Rad 3-related) DNA repair pathway, which is defective in patients with Fanconi's anaemia. Deficiency of PRKDC in mice has been shown to result in an abnormal NK cell physiology similar to that observed in these patients.

Conclusion Mutations in MCM4/PRKDC represent a novel cause of DNA breakage and NK cell deficiency. These findings suggest that clinicians should consider this disorder in patients with failure to thrive who develop pigmentation or who have recurrent infections.

  • Failure to thrive
  • DNA repair defect
  • Irish Traveller
  • natural killer cell deficiency
  • familial glucocorticoid deficiency
  • MCM4

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Members of three clans from the Irish Traveller population have presented to a variety of clinical specialists, including paediatric endocrinologists, haematologists and geneticists, with intrauterine growth retardation (IUGR) and failure to thrive (FTT). All affected individuals from the first presenting family (pedigree 1) had clinodactyly, some had episodes of hypoglycaemia, and those tested had delayed bone age. Some patients had relative macrocephaly, with head circumference measurement on the 3rd centile, and height and weight below the 3rd centile. In others, all three variables were below the 3rd centile.

Members of a second clan (pedigree 2) from the Irish Traveller population presented to endocrinologists with IUGR, FTT, hypoglycaemia and clinodactyly. When investigated, the affected children were found to develop hyperpigmentation (after 2 years of age) and increased adrenocorticotropin hormone (ACTH) levels over time (mean age 5 years), with low–normal cortisol concentrations, confirming a diagnosis of familial glucocorticoid deficiency (FGD). However, symptomatic presentation of FGD was later than normal for the condition. At initial diagnosis, the patients had a tanned appearance typical of FGD. Most patients who were regularly taking their hydrocortisone had normal skin colouring, while those who were not compliant with medication continued to have a tanned appearance. Because of a history of recurrent infections, some of the children from pedigree 2 were investigated for disordered immunological function, which revealed low levels of natural killer (NK) cells and evidence of a DNA repair disorder. Clinical features of the DNA repair phenotype in members of this clan were described by Eidenschenk et al.1 Members of a third clan (pedigree 3) were referred to clinical geneticists with suspected Russell–Silver syndrome, but a diagnosis of FGD was made on the basis of the development of increased pigmentation and subsequent biochemical investigations.

As some members of the original clan (pedigree 1) were later found to have high ACTH levels, we postulated that the NK cell deficiency, DNA repair disorder and FGD were the result of a single recessive genetic event. We proceeded to test members of all the clans for the three different phenotypes and found that those with a diagnosis of FGD also had low NK cells, and some showed defective DNA repair. The DNA repair disorder was classified as mosaic Fanconi's anaemia (FA), but the patients do not have the typical mosaic FA test result or the expected clinical features of the disorder. In mosaic FA, patients have two subpopulations of cells, one of which is hypersensitive to cross-linking agents (diepoxybutane (DEB)), while the other behaves normally in response to these agents. Upon testing, patients with mosaic FA have some cells with high levels of DNA damage and others that are completely normal. However, often the patients in this study have a relatively low level of DNA damage in a minority of cells. The observed chromosome breakage is greater than that expected from a healthy individual but less than that of mosaic FA.

This study involves 10 individuals from three consanguineous Irish Traveller families who were diagnosed with a combination of an NK cell deficiency, mosaic FA and FGD (figure 1A,B,C). Many of these children would not have been investigated and diagnosed were it not for their family history. Clinical and laboratory details of the 10 patients are discussed (table 1 and online supplementary material). Details of the presentation and endocrine findings of some of these patients were previously described by O' Riordan and colleagues.2 We performed single-nucleotide polymorphism (SNP) homozygosity mapping and targeted next-generation sequencing to identify the underlying risk gene in these families.

Figure 1

Pedigrees of Irish Traveller families presenting with a novel syndrome involving a primary immunodeficiency and a disorder of DNA repair. Individuals from whom DNA was available are marked with a *. (A) Pedigree 1 includes five affected individuals initially assessed for query Russell–Silver syndrome. DNA from five affected and eight unaffected individuals was available for homozygosity mapping. Patients III:11 and IV:4 were also diagnosed as having a mild mosaic form of Fanconi's anaemia (FA). Patient IV:9 has a natural killer (NK) cell deficiency, but the results of the FA test are of unclear significance. Patient IV:10 was diagnosed as having an NK cell deficiency. (B) Pedigree 2 includes eight affected family members. One affected individual (IV:16) died aged 11 years from bronchiectasis. DNA was available for homozygosity mapping from four affected and three unaffected individuals. All of the affected individuals were diagnosed as having mosaic FA (IV:19 has classic FA), an NK cell deficiency and familial glucocorticoid deficiency (FGD). (C) Pedigree 3 includes one affected proband initially suspected of having Russell–Silver syndrome. Further investigation led to a diagnosis of an NK cell deficiency and FGD. (D) Single nucleotide polymorphism homozygosity mapping using the HomozygosityMapper programme identified a single candidate locus at 8p11.21–q11.22 (chromosome 8:40,802,495–51,349,159), which includes 34 positional candidate genes.

Table 1

Summary of the clinical features in 10 patients with a Russell–Silver-like phenotype

Subjects and methods

Patients and DNA samples

DNA was available from 10 affected and 16 unaffected members of the three Irish Traveller families. The 10 patients were diagnosed with an NK cell deficiency, mosaic FA and FGD. Clinical information for each patient is provided in the online supplementary material. Ethics approval was obtained from Our Lady's Children's Hospital Ethics Board (Dublin, Ireland), and written informed consent was obtained from all patient guardians.

SNP homozygosity mapping

DNA samples from the 10 affected and 16 unaffected individuals were genotyped for 1 million SNPs on the Illumina 1M array (Illumina, San Diego, California, USA). SNP homozygosity mapping was performed using the HomozygosityMapper programme.3

Target enrichment, exome sequencing and data analysis

DNA from six of the patients (pedigree 1, III:11, IV:4, IV:9, IV:12; pedigree 2, IV:1; pedigree 3, II:1) and four unaffected siblings (pedigree 1, III:12, IV:5, IV:11; pedigree 2, IV:2) was selected for targeted resequencing of the exons within the 8p11.21–q11.22 candidate region. Target enrichment was performed using a SureSelect XT Custom MP2 kit (Agilent, Santa Clara, California, USA), and libraries were sequenced on an Illumina HiSeq at GATC. The reads were mapped against UCSC hg18 using BWA version Duplicates were removed and the quality scores for the aligned reads were recalibrated using GATK.5 Variants and indels were detected using SAMtools.6

Analysis of MCM4 splice variant

The MCM4 c.71-2A>G variant was analysed with Human Splicing Finder version 2.4.1 ( The mutation designations are based on MCM4 transcript ENST00000262105 from Ensembl. A in the ATG start codon is defined as c.1.


Homozygosity mapping and targeted next-generation sequencing

SNP homozygosity mapping identified a single homozygous segment at 8p11.21–q11.22 that was shared by the 10 affected individuals (figure 1D). The candidate locus is 10.5 Mb in size and contains 34 candidate genes. Targeted resequencing of the 34 candidate genes was performed in six patients and four unaffected siblings (online supplementary table S1). To identify potential disease mutations, we prioritised variants that (i) are homozygous, (ii) segregate with the phenotype, and (iii) are novel or have a frequency <1% in dbSNP. The prioritisation strategy narrowed the search to two novel variants; one is located in an intron of GOLGA7 (NM_001002296.1:c.367-82C>T) and the second is located within both the 3′ acceptor splice site of MCM4 intron 1 (NM_005914.2:c.71-2A>G) and the 5′ upstream regulatory region of PRKDC (NM_006904.6:c.-57-u1331T>C) (online supplementary figure S1).

The intronic variant in GOLGA7 is located 82 bp 3′ of the acceptor splice site sequence and is of unknown significance. Analysis of the candidate MCM4 splice variant using Human Splicing Finder shows that the mutated splice site has a consensus value (CV) <70 (52.59) and a ∆CV >10% (40.46%), which is predicted to result in a broken and inactive splice site (online supplementary table S2). In addition, mutations with a CV <70 combined with a ΔCV reduction >10%, such as the MCM4 variant identified in this study, are expected to completely block the production of wild-type transcript. The MCM4 variant is also located within the 5′ upstream region of two PRKDC transcripts (NM_001081640 and NM_006904). The 5′ upstream region harbours numerous binding sites for proteins that either repress or promote transcription, and impairment of any of these features can alter transcriptional regulation leading to altered gene expression and, in some cases, susceptibility to disease.8


The 10 affected individuals are from three separate Irish Traveller clans and were initially assessed for query Russell–Silver syndrome due to IUGR, FTT and hypoglycaemia. The natural history in our patients varied, with affected patients from one clan being prone to recurrent infection and some of them developing bronchiectasis. It is possible that environmental factors, such as poor housing, are contributing to the recurrent infections in this family. Further investigations revealed that three different phenotypes (NK cell deficiency, mosaic FA and FGD) were segregating together in these families, suggesting that they may be caused by a single recessive disease gene.

SNP homozygosity mapping and targeted next-generation sequencing of the 8p11.21–q11.22 candidate locus identified two novel homozygous variants that segregated with the phenotype: (1) an intronic variant in GOLGA7 of unknown significance and (2) a variant located within the acceptor splice site of MCM4 intron 1 that also overlaps the 5′ upstream region of PRKDC. MCM4 encodes a highly conserved mini chromosome maintenance protein that is essential for DNA replication9 and important in maintaining genome stability.10 MCM4 has evolved to integrate several protein kinase regulatory signals to control progression through the S phase of mitosis. Interfering with the rate of DNA synthesis in the S phase can produce an FA-like phenotype in normal cells. The MCM4 c.71-2A>G variant is predicted to have a significant effect on splicing, with no production of wild-type transcript. This variant is also positioned within the 5′ upstream region of PRKDC, and, while the functional consequences of upstream variants are currently difficult to predict, they can affect gene expression. The presence of a single mutation affecting two genes may account for the diverse clinical features observed in the patients.

MCM4 and PRKDC are both involved in the ATM/ATR (ataxia-telangiectasia-mutated/ATM-Rad 3-related) DNA repair pathway, which is dysfunctional in patients with FA. Similar to other Fanconi genes, MCM4 and PRKDC have been shown to function in the mitochondria.11 12 Furthermore, mice homozygous for the spontaneous severe combined immunodeficiency mutation in the Prkdc gene (B6.CB17-Prkdc/ScJ) show defects in DNA repair and have an abnormal NK cell physiology characterised by markedly elevated NK cell activity in the first 10–14 weeks of life, and reduced NK cell numbers at 10–12 months. The similarity of the Prkdc mutant mouse to the phenotype of the patients in this study adds support to the involvement of PRKDC as a contributing risk factor for FA and NK cell deficiency.

Despite a diagnosis of mosaic FA, none of the patients have the typical FA phenotype. They have not developed bone marrow failure and they do not have typical Fanconi malformations. While many of them had anaemia, this was microcytic and related to poor diet. Upon breakage analysis in patients with typical mosaic FA, some cells were found to have a full mutation phenotype with high numbers of breaks and exchanges per cell, while other cells were normal with little or no chromosome damage. In this study, the patients' cells do not show a mixture of highly damaged and normal populations. Instead, in most cases, a minority of cells demonstrate a relatively low level of breaks and exchanges, not far above that of healthy controls. One of the 12 affected family members has shown a full DEB response typical of classic FA. However, similar to the 11 patients with low levels of chromosome breakage, she also shows no clinical features of FA. This generally low level and variable expressivity for an FA-type breakage syndrome has not been previously described.

The FGD phenotype in the patients is atypical in that the onset is delayed and the individuals remain small. The pigmentation improves upon treatment with hydrocortisone. However, in some families the compliance with medication has been poor. Despite lack of compliance, withdrawal of hydrocortisone has not resulted in an adrenal crisis, unlike in other patients with classic FGD. Sequence analysis of the known FGD disease genes (MC2R, MRAP and STAR) was undertaken in this study, but no causative mutations were identified. Other causes of adrenal insufficiency were also excluded. The late onset of FGD symptoms in these patients together with the absence of adrenal crisis suggests that the elevated ACTH levels may be a secondary phenotype in these patients, stemming from the NK cell deficiency and DNA repair disorder.

The biochemical and immunological results from these individuals were not necessarily present on initial testing. Many of these children were only diagnosed because the clinicians were aware of the family history. We recommend considering this diagnosis in children from consanguineous pedigrees who present with FTT, recurrent viral illnesses, and features of FGD. Assessment using ACTH with or without NK cell and DEB testing should be adequate to identify most future cases. We identified variation in the MCM4 and PRKDC genes as the most likely cause of mosaic FA, NK cell deficiency and secondary FGD in these families. Further investigation into the molecular mechanisms of MCM4/PRKDC mutations and how they result in this complex phenotype is warranted.


We sincerely thank the participating families for the use of genetic samples and clinical information.


Supplementary materials

  • Supplementary Data

    This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.


  • Funding National Children's Research Centre, Our Lady's Children's Hospital Crumlin, Health Research Board Ireland, Medical Research Charities Groups, Irish Research Council for Science, Engineering and Technology, Science Foundation Ireland. The NCRC (SAC/95/07) supported the consumable costs for SNP genotyping. The HRB (HRA_HSR/2010/3) supported the cost of targeted next-generation sequencing. JPC was supported by NCRC and HRB (MRCG/2011/17) to undertake this project. She performed some of this work while supported by an EMBARK postgraduate award from IRCSET. PM is supported by SFI (07/SRC/B1156).

  • Competing interests None.

  • Patient consent Obtained.

  • Ethics approval Ethics approval was provided by Our Lady's Children's Hospital Ethics Board.

  • Provenance and peer review Not commissioned; externally peer reviewed.