4 Analysis of CAMK4 gene

4.1 Introduction The human Ca +Z I Calmodulin dependent protein kinase 4 ( CAMK4) gene is a member of

CaM kinases (CAMK) subfamily that has been mapped onto the long arm of

chromosome 5. This gene has 11 exons, that encode 473 amino acid protein, and spans

about 270.5 kb DNA (Fig. 1). It has limited tissue distribution and highly expressed in

elongating spermatids (Wu & Means, 2000). The CAMK4 has been marked as a

candidate regulator of germ cell differentiation (Blaeser et al., 2001). In male germ cells, it

functions as a transcriptional and post-transcriptional regulator. It activates cAMP­

response element (CRE) binding (CREB) protein family of transcription factors,

including the testis specific CRE- modulator--r (CREM--r) (Blaeser et al., 2001; Matthews

et al., 1994). A study on Camk4 knockout mice has shown that CAMK4 deficient mice

were infertile and had sperm count < 4% that of wild type mice. Even few spermatozoa

produced by Camk4-/- mice were morphologically abnormal (Wu et al., 2000).

Involvement of CAMK4 in Calcium ions (Ca++) regulated sperm motility and its presence

in the flagellum of sperm has been reported in the study on human (Marin-Briggiler et

al., 2005). Therefore, it was speculated that the abnormality in CAMK4 gene might

contribute to the spermatogenic defects in human. So far, no genetic study has been

conducted to assess the role of CAMK4 gene in causing spermatogenic failure. Thus, the

possible association of human CAMK4 gene with infertility was examined by

comparative analysis between set of infertile and normal fertile Indian males. A total of

283 infertile Indian men (including 105 NOA, 103 OAT and 75 OA) and 268 ethnically

matched fertile men were chosen for this study.

4.2 Results

4.2.1 SNPs/mutations in CAMK4 gene

The exon specific intronic primers enabled us to extensively screen the sequence

variation in exons and exon-intron boundaries of CAMK4 gene. 25 variable sites were

identified, which include 20 nucleotide substitutions and five indel mutations. Among

the 20 SNPs listed in Table 4.1, one was in 5' UTR, five were in exons and remaining 14

were in introns. Of these 20 variants, 12 were novel mutations, not listed in dbSNP or

HapMap. Out of the 12 novel mutations, eight (g.-82:C>T, g.123:G>A, g.l19565:A>G,

g.150275:G>A, g.150462:T>C, g.224767:G>A, g.259618:C>A and g.259809:G>A in Table

4.1, Fig. 4.1 and 4.2) were exclusively found in infertile men. All these mutations were

always found in heterozygous condition and no man had simultaneously two mutations.

55

(Jl 0\

::J3:::;-::!! oromco :: ::J ~ • OO>Q)~ Ul ::J ::!". ..... () Q) 0 ~-< ::J G) <DNUlCD . CD ..... ::J

a.mo . ::I. 3 $: Ul - · c: ;:::;: () §:~3 - · -; Q) g :T-o Ul CD

0 CD(/)-... xz

0 Q.-u):. c: Ul ~- (/) :s: <::J"~ CDO~ o:Eco ,....:Jet> ::J"Q)::J CD o-ro - · o ::J < :E <Oro;:;: ::I. ..... ::J" - · ::J" CD<t>o 3 co ()

CD 0> <D::J!:!: ::J 0 g Q) 3 Ul ro n· o Ul 3 -::J" -OO>::J" :E ""0 CD ::J:Eco - · ro ro ::J ..... ::J :E CD 0 ::J"""O-;:::;: Q.~ CD '< CD 0"3a. ~Q(I) ·-oz -12:-u ::J"()Ul <DQ)3 ~- ::Ja. c: N ..... CD.-Q)

::J"!:!: 0 0 0 -Ul::J CD CD Ul Q) .

~~--~ - · 0 ::J" %.:E<D ..... ::J ::J 0 0" c: ::J CD n u;· o co Ul :E 8-::J" - · O:EO. < CD CD < ..... Ul ::J CD -· Q) ::J ::J .....

0 CD 7' ..... 0".... ::J Ul v c: . Q. 3 Z'< 0" 0 3 CD roQro - "0 a. ::J"::J" Q) - · ..... .-oCD :T :;· Q) CD,_~ Ul ::J" CD - · CD

~--(/) ::J 0 a.

Q) - · :f ..... Q) ,., CD ::J "'

. sNPs.repofte~ · : in·(U)SNP,butriot,

o~se[~-~~~~ -t~!~·-·

. .Novel mutations . o~s~rved iitthis study

SNPsreporJed · in;dbSNP.and • observed .·

· iri-,t~ii; • study . . ... i • g.·82:C>T .· ·rs229b6i9:C>T g.123 :G>A

~ ~ ..... 0 ::J (/)

~

~ El ~

~ ;:u

.~ .

., ... ; . , .

.-rs104210.1 :C>T . • rs68S6469 :T~G . rst 3f sr1o . . ..

rs~3 1 .75122:C;;c r~131 7.48's2 :A;:

~ 0 ::J Ul

119.4kb

'" ' ... . ;~~~~i~~~~J ; ...... _____ c.-:..

. ...... . . 1-.J>J '-' '-1 · 1 · - -

. . · ·.· .· . .. . . · . · ~ g .. 15o 1:79 ~G>G ::g : 1501~4 :G>A . · ·. 30.8kb . 9 .150264-66deiGCG

R' g.150275:G>A •·.. .

w g.150:304:A>T 1 g.1 ~o4,62 :T~~ .

~ ~~ 9 150518_.·1 ___ 9_'•_n .. sG . ·. ·_· . . ·. . ·. . : .. ·· . . · g.t52310.A?G

17.8kb g.152483:C>T .. ·

U1 ~g.170J29:C>T • .• . . . .. ~ . · . -. g.170376:C>T

51.9kb

24.1kb

~~ Skb

<0 J. . ~g.254111deiC 4.3kb g.254119_20de1AA

~+ Q_.25~240:C>T .

10kb

2:G>c ·

r LJ 9:26049L98insA

No. Mutation 1 g.-82:C>T 2 g.123:G>A 3 g.119476:C>G 4 g.119565:A>G 5 g.150179:G>C 6 g.150184:G>A 7 . g.150275;G>A 8 g.150304:A>T

Location rs No. 5' UTR Novel Exon1 Novel lntron1 rs446176 Exon 2 Novel lntron 2 · rs306067 lntron 2 Novel lntron 2. Novel lntron 2 Novel

. 9 . g.1504{)2:T>C lntrori 9 • NoveL · 10 g.152310:A>G lntron 3 rs306129

· 11·· · g.152483:c>l' · 1ritrooA •• ; .N()v~l· · 12 g.170329:C>T lntron 5 Novel

Amino Acid change

13·::;'>g':1?0.3i6:c>r· · ·lntrop K.'::·)·f~5?~$.$9.i1t:~> .:. 14 g.222093:C>A lntron 5 rs3797739 ,15( ·g,2246M:A>G intron? ·.· : ~5.21'1'§824 '··' · 16 g.224767:G>A lntron 7 Novel 17;:· g.2S8240:C>T .. :Introq9.: " rs·~t3399q'· .. 18 g.259572:G>C Exon 11 rs2228630

<19 :: g•.259618:G>A < Exon)f .. • NoVel: '':-p!His353Asp 20 g.259809:G>A Exon 11 Novel p.Met41611e

Minor Allele Frequency (Genotype Distribution) infertile Men Fertile Men 0.002 T (0, 1, 282) 0.000 (0, 0, 265) 0.002 A (0, 1, 282) 0.000 (0, 0, 265) 0.021 c (0, 12, 271) 0.013 (0, 7, 260) 0.004 G (0, 2, 281) 0.000 (0, 0, 268) 0.021 G (0, 12, 270) 0.013 (0, 7, 261) 0.046 A (3,20, 259) 0.043 (0, 23, 245) 0.002 A (0; 1, 281) 0.000 (0, 0, 268). 0.000 T (0, 0, 282) 0.002 (0, 1, 267) 0.007 c (0, 4, 278) 0.000 (0, 0, 268) . 0.023 A (0, 13, 270) 0.009 (0, 5, 261) 0.000 T(O, 0, 283) 0.002 (0, 1, 266) · 0.002 T (0, 1, 282) 0.002(0, 1, 267) o.o23 :c (o .. ~3; 2io) o.ot9 (~. $; 26or· 0.385 c (40, 138, 105) 0.368 (42, 113, 113) 0.380A (41, 133, 1 09) ' 0.364 (40, 1121 J 12) 0.004 A (0, 2, 281) 0.000 (0, 0, 268) 0.057 T(O, 32, 251) 0.060 (0, 32,.236) .· 0.409 G (42, 146, 93) 0.368 (37, 123, 108) 0.002 A(Q, 1, 280) . o.ooo (0, b; ?68) . 0.002 A (0, 1, 280) 0.000 (0, 0, 268)

Detected in NOA OAT All NOA All All OAT N NOA,OA All N NOA, N All All All NOA,OA All All NOA NOA

OR (95%CI), P-value; Minor allele as Risk Factor

NO NO 1.631 (0.637-4.174), 0.303 NO 1.643 (0.642-4.205), 0.296 1.078 (0.607-1.914), 0.798 NO NO ND 2.478 (0.877 -6.998), 0.077 NO NO

. ' 1:!23}(0;5~8~2;844), 0;617 1.078 (0.845-1.376), 0.546 1.012 (9,839-1.370), 0.579 NO 0.944 (0;570-1.564), 0.822 1.192 (0.935-1.520), 0.156 NO NO

Table 4.1 Frequency distribution of minor alleles of SN Ps and mutations in CAMK4 gene in infertile and fertile control men. Frequencies for the minor alleles and genotype distribution (in parenthesis) for all SNPs are given. The minor allele is also mentioned in column 'Infertile men'. Abbreviations: NOA = Azoospermic, OAT = Oligoasthenoteratozoospemic; N= Normal fertile; All = Indicates that SNP is detected in all categories of men.

':J/

R

(a) Q.-82:C>T (b) g.123:G>A (c) g.119565:A>G

r\ 1

(d) g.150184:G>A (e) g.150275:G>A (f) g.150304:A>T

\ I l

(g) g.150462:T>C (h) g.152483:C>T (i) g.170329:C> T

U) g.224767 G>A (k) g.259618:C>A (I) g.259809:G>A

A

(m) g.150264_66deiGCG (n) g.150518_19insG (o) g.254111deiC and g.254119_20deiAA

Fig. 4.2 Electropherograms of novel substitutions (2a to 21) and indels (2m to 2o) in the CAMK4 gene.

58

.Ana{ysis if C.A:MX4 gene ~ ~-

In S'UTR, the novel mutation g.-82:C> Twas detected in a NOA man and in exon 1 the

silent mutation g.123:G>A was detected in an OAT man. Both of these mutations were

not found in any other category of men (Table 4.1, Fig. 4.1, 4.2a and 4.2b ). In ex on 2 the

silent mutation g.l19565:A>G was detected in two NOA men (Table 4.1 and Fig. 4.2c). In

intron 2, four SNPs were identified of which, g.150179:G>C (rs306067) was listed in

dbSNP while other three were novel. The SNP g.150179:G>C did not show significant

difference between infertile and fertile men. Novel SNP g.150184:G>A, although did nol

show significant difference between infertile and fertile men, deviated from Hardy­

Weinberg equilibrium (HWE) in infertile men. This deviation was because three infertile

(two NOA and an OAT) men were homozygous mutant (Table 4.1 and Fig. 4.2d). ln

intron 2 the novel mutation g.150275:G>A was found in an OAT man, whereas

g.150304:A>T was detected in a fertile man (Table 4.1, Fig. 4.2e and 4.2f). In intron 3,

novel mutation g.150462:T>C was found in two NOA and two OA men, but not in any

other category (Table 4.1 and Fig. 4.2g). The SNP g.152310:A>G (rs306129) in intron 3 did

not show significant difference between infertile and fertile men. Novel mutation

g.152483:C>T in intron 4 was detected in a fertile man, whereas g.170329:C>T in intron 5

was found in a NOA and a fertile man (Table 4.1, Fig. 4.2h and 4.2i). The SNPs,

g.170376:C>T (rs523389) and g.222093:C>A (rs3797739) in intron 5 did not shovv

significant difference between infertile and fertile men (Table 2). The SNP g.170376:C>T

deviated from HWE in fertile men because it was homozygous in two fertile men. The

SNP g.224641:A>G (rs2116824) in intron 6 did not show significant difference between

infertile and fertile control men (Table 4.1). Novel mutation g.224767:G>A in intron 7

was detected in a NOA and an OA man (Table 4.1 and Fig. 4.2j). Finally, SNPs

g.258240:C>T (rs3733995) and g.259572:G>C (rs2228630) in intron 9 and 11, respectively,

did not show significant difference between infertile and fertile men (Table 4.1).

In exon 11 two non-synonymous novel mutations, g.259618:C>A and g.259809:G>A,

were identified (Table 4.1); these two mutations cause replacement of His353Asp and

Met416Ile respectively. Residue His353 is conserved among human, chimpanzee, rhesus

and mouse, but not between dog and rat whereas, Met416 is conserved among all

mammals analyzed (Fig. 4.3). Both of these residues are in the associative domain of the

protein and therefore expected to make it impaired to interact with other interaction

partners. The change of histidine to asparagine is expected to be more detrimental as

histidine is a basic whereas asparagine is polar amino acid. Both of these mutations were

predicted as "possibly damaging" by the PolyPhen with score 1.909 for l1is353Asp and

1.617 for Met416Ile. Moreover, SIFT also predicted them as "intolerant".

59

Human Chimp

Rhesus Mouse

Dog Rat

Mutant D

~~~);~~-= = = -- = = A A~, - - - - - - - - A·., .• ... v,..,_,. E . -- - ---- - SS AA K------- - ATN E~EEEEEEEE SIM

gene

~ ~.vA~.·~ ... L. I VAgL

I .VAI·L··· ! D p A A D ,Q

Fig. 4.3 Multiple alignment of CAMK4 protein sequences of different mammalian species. The mutations g.259618:C>A and g.259809:G>A caused p.His353Asp and p.Met416Asp substitutions, respectively. Residue His353 is conserved among human, chimpanzee, rhesus and mouse but not between dog and rat, whereas Met416 is conserved among all mammalian species analyzed.

I ' J ) II

IJ u < IJ !- < IJ !- u

" " " " " " " " " u IJ lJ < u. u <: u lJ '"' Ol v 0 '"' M .... 0 N ...... ...... "' .... ...... Ol .,. v ...... .,. ..... .... M M 0 '"' N "' Ol 0 0 N 0 N .,.

"' Ol .... "' V> "' ..... N N "' "' .... .... .... .... .... "' "' N N

do do do 0. 0. a. 0. do a. 1 2 3 4 5 6 7 8 9

• • • 100

Low D' H19h D' I Low LOD

Fig. 4.4 Pairwise Linkage Disequilibrium of SNPs in CAMK4 gene. The SNPs g.119476:C>G, g.150179:G>C, g.152310:A>G and g.170376:C>T were in complete LD (D' = 1 ). Novel SNP g.150184:G>A was not in LD with above surrounding markers. Number in the LD blocks represents the D' values multiplied by 100.

4.2.2 Linkage disequilibrium (LD) and haplotype analysis

Pairwise LD analysis showed that known SNPs g.l19476:C>G, g.150179:G>C

g.l523lO:A>G and g.1 70376:C>T were in complete LD (D' = 1). The novel SNP

g.l50184:G>A was no t in LD with the above surrounding markers (Fig. 4.4) . Moreover

known SNP pairs, g.222093:C>A- g.22464l:A>G and g.258240:C>T- g.259572:G>C, were

60

.Analysis if C.JLJ11X4 gene

in two separate LD blocks (Fig. 4.4). Haplotype based association analysis using SNPs in

LD block did not show significant difference between infertile and fertile men.

Furthermore, haplotype analysis with sliding window of 2 or 3 SNPs also did not show

any significant difference (data not shown).

4.2.3 Insertions and deletions in CAMK4 gene

In addition to the above SNPs, five novel indel mutations were observed. Four of these

indels were in intron and the other one was in 3'UTR (Table 4.2 and Fig. 4.1). As these

indels were always detected in heterozygous condition, all of them were validated,

except g.260497 _98insA, by cloning and sequencing the PCR product. Sequencing of

these clones revealed both wildtype and mutant sequences (Fig. 4.2m, 4.2n and 4.2o). Of

these five indels, g.150264_66delGCG was exclusively found in two OAT men (Table

4.2). Moreover, deletions g.254111delC and g.254119-20delAA were always found

together. No statistically significant difference was observed in the distribution of indels

between infertile men or their subcategory and fertile control men.

Number of men No. Position Location NOA OTA OA Infertile Fertile 1 g.150264,_66deiGCG intron2 0 2 0 2 0 2 ·g.150518_19insG. intron3 . 3 3 5 11 7 3 g.254111deiC intron 9 3 0 0 3 2 4 g:254119_20deiAA ·• ·)ntron 9. 3' ·• o.·· 0 3 2 5 g.260497 98insA 3'UTR 4 4 0 8 5

Table 4.2 Number of infertile and fertile men having indels. These mutations were always detected in heterozygous condition. Deletion g.150264-66deiGCG was only detected in two OAT men. Deletions g.254111deiC and g.254119-20deiAA were always found together.

4.2.4 Bioinformatics analysis of novel mutations identified in

CAMK4 gene

To evaluate whether the mutations which were exclusively found in infertile men can

alter I diminish the potential binding sites for the proteins which are responsible for

splicing, a preliminary analysis was done using various bioinformatics tools (Table 4.3).

Analysis of g.123:G>A in exon 1 using ESEfinder 3.0 did not predict binding site of any

splicing protein; however its analysis with 'Splicing Rainbow' predicted binding site of

SRp40 and ASF /SF2 in mutant sequence, although no binding site was predicted in

wildtype sequence. Analysis of g.l19565:A>G mutation in exon 2 using ESEfinder 3.0

predicted that it diminished the binding site for SRp55 (score 3.73945), but created

61

..Jlna!j;sis o/ C..Jl.MX! gene

potential binding site for the SF2/ ASF (score 2.68125) and SRp40 (score 3.50801) (Table

4.3); however, Splicing Rainbow tool predicted that both the wildtype and mutant

sequence had potential binding site for SRp40 and SRp55, but mutation created

additional binding site for the SF2/ ASF.

ESEfinder 3.0 Sglicing Rainbow No. SNP Location Normal Mutant Normal Mutant 1 ·. gJ23:G>A ExonJ SRp40,

ASFISF2

2 g.119565:A>G Exon 2 SRp55 SF2/ASF, SRp40, SRp40, SRp40 SRp55 SRp55,

SF2/ASF

3 g.150264- lntron2 *· * .SRp40, · SRp55 66qeiGCG ·SRp55

4 g.150275:G>A lntron 2 * hnRNPK, SRp20, SRp40, SRp55

5 ·g.f50462:T>C lntron 3 * * ASF/SF2, F2/ASF, SC35 SC35,

SRp40, SRp55

6 g.224767:G>A lntron 7 * * hnRNP hnRNP B1/A2, HuR B1/A2, HuR

7• g,2596-18:C>f\··· . Exori't1 -~ iSRp40, ·SRp40 SR9GB; . SR9GB, -SFVASF1 SRp55;- SRp55;. ·scJs SF2/ASF, SF2/ASF,

SC35 SC35, Tra2Beta

8 g.259809:G>A Exon 11 SC35 SRp40

-- Indicates the no Splicing protein binding site was predicted * Indicates the site was not analyzed

Table 4.3 Splicing proteins binding pattern in wildtype and mutated sequences as predicted by ESEfinder and Splicing Rainbow tools. The proteins which showed differences between wildtype and mutated sequence are shown in bold-italic letter type.

Similarly ESEfinder 3.0 predicted that the g.259618:C>A in exon 11 did not alter binding

site of the SRp40 (wildtype score 5.39988 and mutant score 3.13317), but this mutation

diminished the binding site of the SF2/ ASF (score 3.33858) and SC35 (score 2.75416);

similar analysis using Splicing rainbow predicted the potential binding site for the SR

9GB, SRp55, SF2/ ASF, SC35 in both wildtype and mutant sequences, but mutant

sequence had additional potential binding site for Tra2Beta (Table 4.3). For the

g.259809:G>A in exon 11 ESEfinder 3.0 did not predict any binding site in both wildtype

62

Ana[ysis o/ CA:M.X4 gene

and mutated sequence, however 'Splicing Rainbow' predicted binding site for SC35 in

wildype, but SRp40 in mutated sequence.

Intronic mutations were analyzed by using 'Splicing Rainbow' alone, as ESEfinder only

finds ESEs. Mutation g.150264_66delGCG in intron 2 diminished the potential binding

site of the SRp40, but did not affect the binding site of SRp55. Similarly g.150275:G>A

mutation created potential binding sites for hnRNPK, SRp20, SRp40 and SRp55, which

were not there in wildtype sequence. Mutation g.150462:T>C in intron 3 created the

potential binding site for the SRp40 and SRp55 in addition to the ASF /SF2 and SC35,

which were predicted in wildtype sequence as well. Mutation g.224767:G>A in intron 7

did not cause any change in the potential binding sites for hnRNPB1 I A2 and HuR. All

the intronic mutations were analyzed with the 'PPT analysis' and the 'BP analysis' tools,

but none of the mutation was in branching point site or causing any change in the

predicted polypyrimidine track.

4.3 Discussion

The present comparative mutation analysis of the CAMK4 gene in infertile and

normozoospermic fertile controls identified 25 variable sites in CAMK4 gene. Although

the frequency of novel mutations was low, eight mutations in Table 4.1 and

g.150264_66delGCG in Table 4.2 were exclusively observed in infertile males, whereas

only two mutations (g.150304:A>T and g.152483:C>T) were exclusively detected in

fertile men (Table 4.1). In the region of CAMK4 gene, seven more SNPs (other than found

here) have been catalogued in dbSNP (Fig. 4.1), but these were not polymorphic in

Indian men. Moreover, except for the rs2290679, frequency of these SNPs in different

HapMap populations is not available, suggesting that they are population specific rare

SNPs.

The SNP g.170376:C>T (rs523389) deviated from HWE in fertile men and many

mutations were exclusively found in infertile men. These observations were of potential

concern as these may reflect genotyping errors or population stratification. These sites

were confirmed for genotyping errors either by resequencing or cloning (for

heterozygous indels) the PCR products. The stratification along West Eurasian-related

axis was ruled out using 50 ancestry-informative markers (Chapter 11). The deviation of

g.170376:C>T from HWE might be because it's a rare SNP and hence, may not be in

equilibrium. This hypothesis was confirmed by checking HapMap samples, wherein out

of 87 Gujarati Indians in Houston, Texas (GIH) only one had C (mutant) allele.

63

.J'lna/j;sis if C.J'l.MX4 gene

Among all the SNPs identified, two mutations were non-synonymous (g.259618:C>A

and g.259809:G>A in Table 2) and both of them cause amino acid change at the

conserved position (Fig. 4.3). Both of these were observed only once in heterozygous

condition in the azoospermic men; however, as these mutations were in the associative

domain of the CAMK4, they may be antimorphic resulting in the dominant negative

mutated protein. Supporting this, in silica analysis using PolyPhen and SIFT also

predicted both of these mutations as deleterious.

Accurate and efficient pre-mRNA splicing is essential to ensure proper gene expression;

for this purpose specific consensus sequences (5' splice site, branch site and 3' donor site)

are found virtually in all exon-intron boundaries (Sun & Chasin, 2000). The splicing

enhancers (ESE and ISE) and silencers (ESS and ISS) have been demonstrated to play key

role in both alternate and constitutive splice site selection (Gabut et al., 2005). These

mutations lead to a variety of consequences, including exon skipping, activation of

cryptic splice sites, creation of new splice sites and rarely intron retention. In silica

analysis of infertile men specific intronic and exonic mutations in CAMK4 predicted that

all mutations, except g.224767:G>A, can alter I diminish the binding sites of splicing

factors (SR and hnRNPs), which will in turn affect the proper mRNA splicing and

protein translation. Further in vitro studies are required to validate the predictions in

silica splicing analysis. It has been estimated that -15% of point mutations affect

consensus splice motifs (Krawczak et al., 1992). Moreover, in few diseases as many as

half of the diseased patients have mutations that cause splicing defects (Ars et al., 2000;

Teraoka et al., 1999).

In mouse testis, CAMK4 is expressed in spermatids and associated with chromatin and

nuclear matrix (Wu & Means, 2000). Male Camk4 -I- mice were infertile with severely

reduced sperm count (<4% to that of wild type) and abnormal shape (Wu et al., 2000).

Contrary to this, another report on Camk4 -I- mice showed that it is dispensable for

spermatogenesis (Blaeser et al., 2001). CAMK4 phosphorylates protamines and

phosphorylation of protamines has been proposed to regulate their interaction with

chromatin during the assembly of nucleoprotein complexes (Green et al., 1994; Kennedy

& Davies, 1981; Louie & Dixon, 1972). Hence, any malfunctioning of CAMK4 will

directly affect (increase or decrease) the phosphorylation of the protamines and in turn

their interaction with chromatin, resulting in DNA damage and therefore, infertility

(Zini et al., 2001). Presence of CAMK4 in human sperm and its involvement in regulation

of sperm motility has been shown earlier (Marin-Briggiler et al., 2005). These

observations led us to hypothesize that defects in CAMK4 might also result in impaired

spermatogenesis in humans. Therefore, infertile men with different spermatogenetic

64

..Jlna!j;sis if C..JLM.X4 gene

defects, where the number, shape and motility of sperms were affected (OA and OAT)

and sperms altogether were absent (NOA), were included in this study.

This is the first genetic case control study evaluating the role of CAMK4 gene in male

infertility. Present results support previous reports, which show the role of CAMK4 in

mice spermatogenesis and human sperm motility. Rare mutations were observed in all

categories of men, implying that CAMK4 can be a cause of decreased semen parameters

in Indian infertile men. Observation of mutations in infertile men of different categories

supports the fact that CAMK4 is an important signaling component and it has some

other important role in spermatogenesis in addition to phosphorylation of protamines.

However, to statistically prove the association of the rare variants a very large sample

size is required, which was practically not possible for the present study. Nevertheless,

the mutations in CAMK4 gene does not seem to be a common cause of male infertility

due to their low frequency, but detection of many specific mutations in infertile men

exclusively led us to conclude that it does play significant role in spermatogenesis and

its malfunctioning can be a cause of reduced semen parameters in Indian men.

65