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NEK1 and DYNC2H1 are both involved in short rib polydactyly Majewski type but not in Beemer Langer cases
  1. Joyce El Hokayem1,
  2. Céline Huber1,
  3. Adeline Couvé1,
  4. Jacqueline Aziza2,
  5. Geneviève Baujat1,
  6. Raymonde Bouvier3,
  7. Denise P Cavalcanti4,
  8. Felicity A Collins5,
  9. Marie-Pierre Cordier6,
  10. Anne-Lise Delezoide7,
  11. Marie Gonzales8,
  12. Diana Johnson9,
  13. Martine Le Merrer1,
  14. Annie Levy-Mozziconacci10,
  15. Philippe Loget11,
  16. Dominique Martin-Coignard12,
  17. Jelena Martinovic13,
  18. Geert R Mortier14,
  19. Marie-José Perez15,
  20. Joëlle Roume16,
  21. Gioacchino Scarano17,
  22. Arnold Munnich1,
  23. Valérie Cormier-Daire1
  1. 1Department of Genetics, INSERM U781, Université Paris Descartes, Sorbonne Paris Cité, Hôpital Necker, AP-HP, Paris, France
  2. 2Laboratoire d'anatomie et cytologie pathologiques, Hôpital Purpan, Toulouse, France
  3. 3Centre de pathologie EST, Hôpital Louis Pradel, Hôpital Pierre Wertheimer, Hôpital Femme Mère enfant, Lyon, France
  4. 4Programa de Genética Perinatal, Departamento de Genética Médica, FCM, UNICAMP, Campinas, São Paulo, Brazil
  5. 5Western Sydney Genetics Program, Department of Clinical Genetics, Children's, Hospital at Westmead, Sydney, Australia
  6. 6Service de Génétique, Groupement Hospitalier Est, HFME, Bron, France
  7. 7Service de Biologie de Développement, Université Paris Diderot, Hôpital Robert Debré, AP-HP, Paris, France
  8. 8Service de Génétique et d'Embryologie Médicales, Hôpital Armand Trousseau, (AP-HP), Université Pierre et Marie Curie - Paris 6, France
  9. 9Sheffield Clinical Genetics Service, Sheffield Children's NHS Foundations Trust, Western Bank, England
  10. 10Laboratoire de Biochimie et Biologie Moléculaire, Hôpital Nord, Marseille, France
  11. 11Centre hospitalier universitaire de Rennes, Service d'anatomie et cytologie pathologiques, Rennes, France
  12. 12Pôle de biopathologie, UF 3162, Centre hospitalier, Le Mans, France
  13. 13Unit of Fetal Pathology, Cerba Laboratory, Cergy Pontoise
  14. 14Department of Medical Genetics, Antwerp University Hospital and University of Antwerp, Antwerp, Belgium
  15. 15Département Génétique Médicale, Arnaud de Villeneuve Hospital, Montpellier, France
  16. 16Service de génétique médicale, centre hospitalier Poissy-Saint-Germain, France
  17. 17Department of Medical Genetics, Sannio University, Benevento, Italy
  1. Correspondence to Professor Valérie Cormier-Daire, Department of Genetics, INSERM U781, Hôpital Necker, Université Paris Descartes, Sorbonne Paris Cité, Paris 75015, France; valerie.cormier-daire{at}


Background The lethal short rib polydactyly syndromes (SRP type I–IV) are characterised by notably short ribs, short limbs, polydactyly, multiple anomalies of major organs, and autosomal recessive mode of inheritance. Among them, SRP type II (Majewski; MIM 263520) is characterised by short ovoid tibiae or tibial agenesis and is radiographically closely related to SRP type IV (Beemer-Langer; MIM 269860) which is distinguished by bowed radii and ulnae and relatively well tubulated tibiae. NEK1 mutations have been recently identified in SRP type II. Double heterozygosity for mutations in both NEK1 and DYNC2H1 in one SRP type II case supported possible digenic diallelic inheritance.

Methods The aim of this study was to screen DYNC2H1 and NEK1 in 13 SRP type II cases and seven SRP type IV cases. It was not possible to screen DYNC2H1 in two patients due to insufficient amount of DNA.

Results The study identified homozygous NEK1 mutations in 5/13 SRP type II and compound heterozygous DYNC2H1 mutations in 4/12 cases. Finally, NEK1 and DYNC2H1 were excluded in 3/12 SRP type II and in all SRP type IV cases. The main difference between the mutation positive SRP type II group and the mutation negative SRP type II group was the presence of holoprosencephaly and polymycrogyria in the mutation negative group.

Conclusion This study confirms that NEK1 is one gene causing SRP type II but also reports mutations in DYNC2H1, expanding the phenotypic spectrum of DYNC2H1 mutations. The exclusion of NEK1 and DYNC2H1 in 3/12 SRP type II and in all SRP type IV cases further support genetic heterogeneity.

  • SRP II
  • SRP IV
  • NEK1
  • DYNC2H1
  • ciliopathy group
  • genetic heterogeneity
  • genetics
  • aneuploidy
  • cytogenetics
  • clinical genetics
  • calcium and bone
  • chromosomal
  • molecular genetics
  • diagnosis
  • congenital heart disease
  • congenital heart disease
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The short rib polydactyly (SRPS) group belongs to the ciliopathy spectrum of diseases and includes four distinct lethal autosomal recessive conditions (type I–IV) characterised by short ribs, inconstant polydactyly, and variable malformations. Among them, Majewski syndrome or SRP type II (MIM 263520) is characterised by short tubular bones with smooth ends, pre- and postaxial polysyndactyly, and short or absent tibiae. Other features include cleft lip/palate, malformed epiglottis and larynx, renal cysts, genital, cardiac, and intestinal abnormalities.1 It is closely related to Beemer-Langer syndrome or SRP type IV (MIM 269860) which is distinguished by inconstant post-axial polydactyly, short and often bowed long bones especially in the upper limbs, shorter fibula, and high frequency of brain defects.2 Recently, mutations in NEK1 have been identified in three families with SRP type II, and compound heterozygosity for mutations in NEK1 and DYNC2H1 were found in one family supporting a possible diallelic digenic inheritance.3 NEK1 (NIMA (never in mitosis A) related protein (MIM 604588)) encodes a serine/threonine kinase with proposed function in DNA-double strand repair, neuronal development, and coordination of cell-cycle-associated ciliogenesis.4 DYNC2H1 (dynein cytoplasmic 2 heavy chain 1 (MIM 603297)) mutations have been identified in asphyxiating thoracic dysplasia (ATD; MIM 208500) and in SRP type III (Verma-Naumoff; MIM 263510),5 6 which is distinct from SRP type II by the appearance of tubular bones including round metaphyseal ends with lateral spikes. DYNC2H1 encodes a subunit of a cytoplasmic dynein complex, involved in the contact and translocation of the dynein complex along microtubules via its large motor domain, and plays a role in the generation and maintenance of mammalian cilia.5

The aim of our study was to screen NEK1 and DYNC2H1 in 13 SRP type II cases and seven SRP type IV cases either by linkage analysis in consanguineous families or by direct sequencing in sporadic cases.

Patients and methods


Thirteen SRP type II and seven SRP type IV cases were included in the study either through the French reference centre for constitutional bone disorders or through clinical geneticists from various countries (Australia, Belgium, Brazil, UK). All cases were either terminated pregnancies (15–28 week gestation) or neonatal death (32–35 week gestation). Among the SRP type II cases, five belonged to consanguineous families from Lebanon, India (with recurrent sibs), Pakistan, and France and the eight remaining cases were from Madagascar, France, Brazil (with recurrent sibs), Vietnam, Haiti, and Belgium.

They all fulfilled the diagnosis criteria for SRP type II, namely: (1) short and narrow thorax, horizontally oriented ribs; (2) short tubular bones with smooth ends; (3) ovoid tibiae that are shorter than fibula or tibia agenesis; (4) pre-and/or postaxial polysyndactyly (figure 1). Among the SRP type IV cases, two were consanguineous from Tunisia and Turkey, and five were non-consanguineous cases from France, Algeria, Italy, and UK. They all fulfilled the diagnosis criteria for SRP type IV, namely: (1) short and narrow thorax, horizontally oriented ribs and small iliac bones; (2) short tubular bones with smooth metaphyseal margins; (3) bowed radii and ulna; (4) tibiae relatively well tubulated and longer than fibulae (figure 2). Table 1 summarised the clinical details of all cases apart from two SRP type IV cases (17 and 18) for which details were not available. Appropriate written informed consents regarding human study were obtained from all subjects.

Figure 1

Skeletal manifestations of short rib polydactyly syndrome (SRP) type II cases mutated in NEK1 (A–C), mutated in DYNC2H1 (D–F), and not mutated in either NEK1 or DYNC2H1 (G–J). (A) Patient 1. Note the tibial agenesis and postaxial polydactyly of the hands and the feet. (B-1) and (B-2) Patient 3a and 3b, respectively. Note the pre- and postaxial polysyndactyly of the hands and the feet and ovoid tibiae, shorter than fibula. (C) Patient 4. Note the ovoid tibia shorter than the fibula and the postaxial polysyndactyly on the hands and the feet. (D) Patient 6. Note the tibia agenesis and hand postaxial polysyndactyly. (E) Patient 7. Note the postaxial polysyndactyly and the ovoid tibia. (F) Patient 8. Note the polysyndactyly of the hands and the feet and the tibia agenesis. (G) Patient 9. Note the pre- and postaxial polysyndactyly of the hands and feet and the tibia agenesis. DYNC2H1 was not studied because of a lack of DNA. (H) Patient 10. Note the polysyndactyly of the hands and the feet and the tibia agenesis. (I) (J) Patient 11a and 11b, respectively. Note the pre- and postaxial polysyndactyly of the hands and the feet and the tibial agenesis.

Figure 2

Skeletal manifestations of short rib polydactyly syndrome (SRP) type IV cases. (K-a) (K-b) and (L-a) (L-b): patients 13 and 14, respectively. Note bowed ulna and radius and tibia relatively well tubulated and longer than the fibula for both cases. Patient 13 has no polydactyly whereas patient 14 has postaxial polysyndactyly of the hands and postaxial polydactyly of the feet.

Table 1

Clinical and radiological features of short rib polydactyly syndrome (SRP) type II and IV cases

Microsatellite marker analysis

In all consanguineous families, microsatellite analysis of the NEK1 locus was initially performed and NEK1 was only sequenced in compatible cases. The NEK1 sequence analysis was performed in all cases from unrelated parents. Genomic DNA was extracted from peripheral blood using QIAamp DNA blood midi/maxi kit (Qiagen SA, sample and assay technology, Courtaboeuf, France). Genotyping was performed using five flanking microsatellite markers (D4S2979, D4S1597, D4S2910, D4S1545, and D4S1617) in all consanguineous families and non-consanguineous families with at least two siblings, by using markers of the ABI PRISM linkage mapping set (Applied Biosystems, Foster City, California, USA). HEX or FAM fluorescently labelled PCR products were run on an ABI 3130 sequencer and analysed using GeneMapper (Applied Biosystems).

Genomic sequencing

For mutation detection, NEK1 and DYNC2H1 exon and flanking intron sequences were amplified from patient DNA by PCR using 34 couples of primers for NEK1 and 90 couples of primers for DYNC2H1 designed with the Primer 3 software or UCSC Genome Browser Database, to amplify the 34 coding exons of NEK1 and the 90 coding exons of DYNC2H1 (sequence of primers available on request). We purified the PCR product with exonuclease I (ExoSAPIT; Fisher Scientific SAS, Illkirch, France) according to the manufacturer's instructions. Sequencing reactions were performed on both strands and run on an automatic sequencer (ABI 3130) using the BigDye Terminator Cycle Sequencing Kit v1.1 (Applied Biosystems) and then analysed by sequencing analysis (Applied Biosystems). For patients 9 and 13, we were unable to screen DYNC2H1 due to insufficient amount of DNA.


Mutations in NEK1 were identified in three consanguineous SRP type II families (2–4 with recurrence sibs in family 3), all homozygous at NEK1 locus and in one non-consanguineous SRP type II case (family 1). Four distinct mutations were identified including two missense mutations (c.433G>A; p.Gly145Arg, c.758T>C; p.Leu253Ser), one frameshift mutation (c.2846_2847insGG-2847delT; p.Asp949Glufs*6), and one nonsense mutation (c.379C>T; p.Arg127*) (table 2). They cosegregated with the disease, were present in a homozygous state in all five cases, and were not identified in 200 control chromosomes. The four NEK1 mutations were located in regions encoding conserved kinase and C domains (table 2). The prediction programme, Alamut, was queried for the missense mutations and all of them were predicted to have a damaging role.

Table 2

NEK1 and DYNC2H1 mutations identified in families with short rib polydactyly (SRP) type II syndrome

NEK1 was excluded in the eight remaining SRP type II cases. However, in four non-consanguineous SRP type II cases (families 5–8) we identified compound heterozygous mutations in DYNC2H1. Among the nine mutations, seven were missense mutations (c.1012A>G; p.Arg338Gly, c.1288C>T; p.Arg430Cys, c.4267C>T; p.Arg1423Cys, c.7985G>A; p.Arg2662Gln, c.7486C>T; p.Pro2496Ser, c.988C>T; p.Arg330Cys, and c.1483A>G; p.Lys495Arg), one was a frameshift mutation (c.8534delA; p.Asn2845Ilefs*8) and one a splicesite mutation (c.12478-2A>G). Four of nine mutations were located in regions encoding conserved dynein heavy chain (N-terminal region 1), 1/9 mutation was located in N-terminal region 2, and 1/9 mutation was located in ATP binding and hydrolysis domain (table 2). All mutations cosegregated with the disease; in family 5, three missense DYNC2H1 mutations were identified in the affected case, two inherited from the mother (c.1288C>T; p.Arg430Cys and c.1012 A>G; p.Arg338Gly) and one inherited from the father (c.4267 C>T; p.Arg1423Cys). The prediction programme, Alamut, was queried for the reported missense mutations and all of them were predicted to have a damaging role. They were not observed in 200 control chromosomes.

For family 9, only NEK1 was excluded due to insufficient amount of DNA to screen DYNC2H1. We excluded NEK1 and DYNC2H1 in families 10 and 11 (recurrent sibs, 11a and 11b). The absence of NEK1 and DYNC2H1 mutations in 3/12 SRP type II cases (16.7%) prompted us to compare their clinical and radiological features with the mutation positive SRP type II cases (figure 1 and table 1). Lingual and gingival hamartoma were frequently observed in the mutation positive group (60% in the NEK1 mutated group/25% in the DYNC2H1 mutated group) and absent in the mutation negative group, while lobulated tongue was mostly observed in the non-mutated group. Kidney cysts, intestinal malrotation, and heart defects were observed in both groups, but holoprosencephaly and polymycrogyria were only observed in the mutation negative group.

NEK1 and DYNC2H1 were excluded in six SRP type IV cases (families 12 and 14–18, figure 2). For family 13, we were unable to screen DYNC2H1 due to insufficient amount of DNA and only NEK1 was excluded. The features of the five cases for which clinical details were available (table 1) were in agreement with previously reported SRP type IV cases,2 with the observation of various cerebral abnormalities (corpus callosum agenesis and cerebellar hypoplasia), intestinal malrotation, and heart defect.

With the detailed clinical description, we also emphasised the clinical variability of SRP type II/IV cases. As previously reported, while polydactyly of fingers and toes of varying degree are mandatory features for SRP type II, they were almost absent in SRP type IV cases. Among the orofacial features of SRP type II/IV cases, the most frequent symptoms were the lingual and gingival hamartoma and retrognathia (45%). Interestingly, all the male fetuses (60%) with SRP type II/IV had genito-urethral abnormalities. Other features frequently observed in both types were renal abnormalities (50%), cerebral abnormalities (45%), intestinal malrotation (25%), common mesenterium (20%), and heart defect (25%).


We report here the identification of four distinct NEK1 mutations all present at the homozygous state in 5/13 SRP type II cases, including three novel mutations and one nonsense mutation (p.Arg127*) found in a French patient, previously reported in a patient from Turkey.3 While the previously reported mutations were either nonsense mutations or splice site mutations, we identified two missense mutations responsible for the change of highly conserved residue located in the NEK1 kinase domain, which is required for centrosomal integrity and ciliogenesis.7

In addition, we identified nine novel DYNC2H1 mutations in 4/12 SRP type II cases with 7/9 being missense mutations. The wide clinical variability, ranging from ATD to SRP type II–III, observed with DYNC2H1 mutations prompted us to look for genotype–phenotype correlation. One type II case was found to have three damaging missense mutations and was similar in severity to the three other cases. In two other type II cases, a compound heterozygosity for one missense mutation and for either a nonsense mutation (family 7) or a splice site mutation (family 8) was observed, while the fourth case harbours two missense mutations. Up until now, DYNC2H1 mutations have been reported in 10 SRP type III and in five ATD cases with, among them, six being compound heterozygous for one nonsense mutation and one missense mutation (four SRP type III and three ATD) and the nine remaining cases harbouring missense mutations.5 6 No case has been reported so far with homozygous nonsense mutation. Moreover, in all three phenotypes, namely ATD, SRP type II, and SRP type III, mutations are located throughout the gene. No correlation between the nature or the location of the mutation and the severity of the phenotype could be established.

The findings of NEK1 or DYNC2H1 mutations in a similar phenotype, namely SRP type II, also support a link between the two proteins with similar consequences on ciliogenesis; interestingly, severe reduction in cilia numbers and alteration in cilia morphology has been observed in the absence of functional full length NEK1, while shortened and abnormally shaped cilia have been observed in SRP type III chondrocytes with homozygous DYNC2H1 mutations.3 6

Finally, the absence of mutations in 3/12 SRP type II cases argue in favour of genetic heterogeneity. We did not find any significant distinctive features between the NEK1 and DYNC2H1 group, but only minor differences in the frequencies of orofacial malformations more often observed in the NEK1 mutated group. Interestingly, holoprosencephaly and polymicrogyria were only observed in the SRP type II mutation negative group. However, these minor differences might be partly explained by the small size of our cohort.

NEK1 and DYNC2H1 were excluded in all SRP type IV cases.

Our molecular findings may also lead to question whether the current clinical–radiological classification needs to evolve. While SRP type II and III are clearly distinct entities on the basis of radiological features, the findings of common molecular basis may ask whether or not they still have to be considered as separate entities. Moreover, the recent report of WDR35 mutations in so called unclassified SRP also highlights the limits of the current classification.8 On the other hand, the identification of DYNC2H1 mutations in ATD, SRP type II, and SRP type III also further illustrates the highly variable clinical expression of single gene mutations in ciliopathies (including IFT-A components), as previously reported for WDR35 and WDR19.8–10 Indeed, these latter genes are involved in phenotypes ranging from isolated nephronophtisis to lethal SRP. As previously suggested, global mutation load in ciliary genes may account for this broad clinical variability.11

We conclude that SRP type II is a genetically heterogeneous condition with NEK1 being involved in seven cases reported so far (46.7%) and DYNC2H1 being involved in five cases reported so far (35.7%). We also excluded NEK1 and DYNC2H1 in 16.7% of SRP type II and in all cases of SRP type IV, supporting the recognition of SRP type IV as an independent entity. Ongoing studies for identifying other cilia genes responsible for SRP type II/IV will further contribute to expanding the ciliopathy spectrum of diseases.

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  • Competing interests None.

  • Patient consent Obtained.

  • Ethics approval Respective participating institutions.

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

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